Pharmacognosy [9 ed.] 0812110714

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P/ia rmacognosy

Lea & Febiger 600 Washington Square Philadelphia, PA 19106-4198 U.S.A. (215) 922-133

First Edition, 1936 Second Edition, 1947 Reprinted, 1948, 1949 Third Edition, 1956 Reprinted, 1957, 1959

Fourth Edition, 1961 Reprinted, 1962 Fifth Edition, 1965 Reprinted, 1966, 1967, 1966 Sixth Edition, 1970 Reprinted, 1971, 1973, 1974

Seventh Edition, 1976 Reprinted, 1977 Eighth Edition, 1981 Reprinted, 1985

Reprinted 1991

Library of Congress Cataloging in Publication Data Tyler, Varro E. Pharmacognosy. Includes bibliographies and index. 1. Pharmacognosy 1. Brady, Lynn R., 1933— II. Robbers, James E. Ill. Title. [DNLM: 1. Pharmacognosy QV 752 T94p 86-27400 RS160.T94 1988 615'321 ISBN 0-8121-1071-4

The use of portions of the text of USP XXI and NP XVI is by permission of the US!' Convention, Inc. The convention is not responsible for any inaccuracy of quotation or for any false or misleading implication that may arise from separation of excerpts from the original context or by obsolescence resulting from publication of a supplement.

Copyright

(D

1988 by Lea & Febiger. Copyright under the International Copyright Union. All This book is protected by copyright No part of it may be reproduced In any manner

Repnnted 2003

or by any means without written permission from the Publisher. PRINTED IN THE UNIT1) sTArEs OF AMERICA

Print number: 5 4 3 2 1

Prefa :. Public interest in all things "natural and organic," including, especially, drugs and medicines, continues to increase at an unprecedented rate. This interest, coupled with the unparalleled availability of pharmacists among health professionals, places an increasing burden on them to provide their patients with detailed, accurate information on all aspects of the natural product drugs that have either been prescribed or are self-selected. Thus, the importance of pharmacognosy, "pharmacy's specific and peculiar contribution to the cause of science," continues to grow as knowledge of natural drugs—and the transmittal of that knowledge —become of ever greater significance in the practice of pharmacy. To provide such essential information to the pharmacist continues to be the objective of this text. Changes in this edition are more evolutionary than revolutionary. Material has been added to the "General introduction" to help the reader understand possible future developments in the field. New information has been added through-

out, especially ters on Antibiotics and Vitamins. Old references have been deleted and new ones incorporated wherever possible. These should be consulted by those interested in obtaining more detailed information than can he provided in a single volume of manageable size. In spite of the literally thousands of hooks and booklets devoted to natural drugs and drug products published in the last decade, we believe that Phannacognosy is the only one providing comprehensive, accurate, scientific information of a biologic and chemical nature on the truly significant drugs of plant, animal, and microbial origin currently employed in medicine. That continues to be our objective. The book's uniqueness in the field of American publishing and its utilization, often in translation, in educational institutions throughout the world, testify to our success in meeting that goal. West L afa y ette, Indiana and Seattle, Washington

V

Varro E. Tyler Lynn R. Brady James E. Robbers

Contents 1. General Introduction .................. What is Pharmacognnsy'.... ....... ..... Crude Drugs. .. . ...................... Commerce in Drugs ................... Preparation of Drugs for the Commercial Market.............................. Evaluation of Drugs ................... Classification of Drugs ................. Chemistry of Drugs ................... The Future of I'harmacognosv ........... Organization of this Text ................ 2. Carbohydrates and Related Compounds Biosy nthesis of Carbohydrates ......... Sugars and Sugar-containing Drugs ...... Drugs Containing Compounds Metabolically Related to Sugars ............... Poirsaceharides and Poly.saccharide-eontaming Drugs ....................... Cellulose ............................ Gums and Mucilages ................. Pectin .............................

Fatt-y Acids ............................96 Waxes................................ Prostaglandins .........................

I 4 6

5. Volatile Oils ...........................103 Chemistry of volatile oils .............. 107 Biosynthesis of Volatile Oil Constituents. - 109 Hydrocarbon Volatile Oils .............. III Alcohol Volatile Oils .................. 113 Aldehyde Volatile Oils ................. 119 Cinnamon ......................... 119 iKetono Volatile Oils ................... 124 Phenol Volatile Oils ................... 127 Phenolic Ether Volatile Oils ............ 130 Oxide Volatile Oils .................... 133 Ester Volatile Oils ...................... 135

10 10 13 23 26 30

6. Resins and Resin Combinations .........139 39 45•

3. Glycosides and Tannins ................37

Resins. .................................139 Oleoresins .............................. 47 Ok'o-gum-resins .......................151 Balsams ................................51

7. Steroids ...............................156

Biosynthesis of Glycosides ..............58 Anthraquinone Glycosides ...............59 Cascara Sagrada ........................bo Saponin Glycosides.. . . ..... ...... 67 Cyanophore Glycosides ................713 Isothiocyanate Glycosides ...............72 Flavonol Glycosides ....................73 Alcohol Glycosides .....................73 Aldehyde Glycosides ...................73 Lactone Glycosides .....................75 Phenol Glycosides .....................76 Tannins .............................. Tannin-containing Plant Materials ........(78

Nomenclature .......................... 156 Biosynthesis ...........................158 Sterols ................................160 Bile Acids .............................161 Cardiac Clvcosides .....................162 Steroid Hormones ......................173 Alkaloids .............................. Pyridine-Piperidine Alkaloids.,, , ....... 189 Tropane Alkaloids ...................... 191 Quinoilne Alkaloids .................... 203 Isoquinoline Alkaloids ................. 208 Indole Alkaloids ........................ 219 Irnidazole Alkaloids .................... 237 Steroidal Alkaloids ..................... 238 Alkaloidal Amines .................... 239 J'urine Bases ................... 244

4. Lipids .................................82 Biosynthesis of Lipids ..................83 Fixed Oils .............................87 Fats and Related Compounds ...........94 VII

4

Viii 9.

CONTENTS Peptide Hormones and the Endocrine System ..............................250 Historic Development .................. 250 General Physiologic Involvement and Therapeutic Philosophies ............. 250 Commercial Production ................. 252 Adrenal Glands ....................... 253 Thyroid Gland ........................ 255 Pituitary .............................. 258 Pancreas .............................. 264 Parathyroid Hormone and Calcilonin .... 269 Gastrointestinal Hormones ............. 271

10.

Antibiotics Derived from Acetate Metabolism ........................ Tetracyclines.. .. . .... .............. Macrolide Antibiotics .............. Polvenes ........................... Antibiotics Derived from Carbohydrate Metabolism .......................... 13.

Enzymes and Other Proteins ............272

Vitamins and Vitamin-containing Drugs 286 Fat-soluble Vitamins ............. 287 Vitamin A ......................... 287 Vitamin D ...................... 290 Vitamin E. . . ......................... 293 Vitamin 1

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Fig. 5-8. Menthe? piperi(a (peppermint) showing op-

posite, petiolate leaves and dense, terminal inflorescences. (Photo courtesy (if A. M. Todd Company, Kalamazoo, Michigan.)

in 1944, 867,000 lb were produced, and, in 1977, 4,400,000 lb were produced and valued at $63 million; $32 million of this total represented the value of exported oil. About 90% of the oil is obtained from Washington, Oregon, and Idaho, principally from the Willamette river valley and Madras areas of Oregon, the Columbia river basin of Washington, and portions of the Snake river valley in Idaho. The remainder of the oil is produced in Wisconsin and Indiana. A few years ago, southern Michigan was the major area of mint cultivation in the United States, but a fungus blight (V erticilliutn wilt) infected crops and

caused abandonment of thousands of acres of formerly productive land. In recent years, wilt-resistant strains of peppermint have been developed by using the technique of irradiation-induced mutations, thereby eliminating the threat to the industry of fungal blight. In 1978, approximately 17,000 acres were under cultivation in Washington and yielded an average of 70 lb of oil to the acre. Peppermint oil is a colorless or pale yellow liquid that has a strong, penetrating odor of peppermint and a pungent taste that is followed by a sensation of cold when air is drawn into the mouth. American peppermint oil contains from 50 to 78% of free (— )-menthol and from 5 to 20% combined in various esters such as the acetate. It also contains (+ )-rnenthorie, (- )-rnenthone, cineole, (-t )-isomenthone, ( -t- )-neomenthone, and ( -- )-menthofuran. Because of the commercial importance of the mint oils, the interconversions of the various terpene constituents of the oils have been studied more extensivel y than those of other volatile oil plants. Incorporation of "CO,into the various menthane derivatives that characterize different Men[ha species has allowed precursor relationships to he deduced (Fig. 5-9). Several of the proposed steps were directly demonstrated by isolating radioactively labeled monoterpenes and feeding them back to leaf slices or foliage. Cell-free extracts from Mentha leaves with pulegone-°C have confirmed the pulegone—*menthone--menthol portion of the pathway and have established that NADPH 2 is an essential cofactor in these reduction reactions. An enzyme preparation from Mentha leaves has also been shown to reduce the isopropylidene double bond of piperitenone to yield piperitone. Small amounts of menthone and menthol were also formed with piperiteflofle as the substrate, indicating that the cyclohexene double bond of the precursor was reduced. A key step in the biosynthesis of the p-menthane monoterpenes appears to he

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the dehydration of a-terpineol to terpinolene and limonene (see Fig. 5-9). The steps leading to the formation of i-terpineo1 from nievalonic acid are common to several different species of mints. The pathways then diverge where a-tcrpineol is dehydrated to limonene in spearmint and to terpinolene in peppermint. The next step in the sequence is hydroxylation and subsequent dehydrogenation to produce either the carvone series of monoterpenes found in spearmint or the piperitenone series of monoterpenes found in peppermint. Breeding experiments with the mints indicate that a single dominant gene produces the carvone series, whereas the homozygous recessive genotype produces the piperitenone series. Apparently, the gene that differentiates between these series may be the gene that governs the enzyme that deh y drates a-terpineol to either hmonene or terpinolene. The influence of environmental factors on essential oil composition has been apparent to commercial producers for many years. Plants of the same species and genotype may produce oils of different quality when grown in different areas. The long days of northern latitudes favor the production of a peppermint oil that contains relatively small amounts of menthone and menthofuran and large amounts of menthol, whereas plants subjected to short day illumination produce an oil that contains small amounts of menthol and relatively large amounts of menthofuran. Sequential studies with 14C O 2 have suggested that pulegone is the predominant terpene in young tissues of peppermint, where it is accompanied by menthofuran. Menthone, which is found in older tissues, also gradually disappears while menthol accumulates and is replaced in turn, by menthyl acetate. This sequence of biogenetic events helps to explain why peppermint oils obtained from plants containing relatively large amounts of young tissue are inferior. High concentrations (up to 30% in some cases)

menthofuran in such oils impart a disagreeable cloying odor to products in which they ma y he incorporated. The reaction pulegone -- menthofuran is apparently reversible, and the concentration of both of these compounds decreases in favor of inenthone, menthol, and menthyl acetate as the plant ages. Consequently, oils of good quality can be obtained only from plants containing a high percentage of mature tissues. The problem of obtaining mature tissues is particularly acute in the Yakima valley of central Washington where environmental conditions favor early and luxuriant flowering of mint plants with concomitant production of a large proportion of relatively young tissues. Careful control of the time of harvest enables growers in that area to produce oils of satisfactory quality. USES. Peppermint oil is a pharmaceutic aid (flavor). It has been used as a carminative, a stimulant, and a counterirritant, Its chief commercial importance is as a flavor for confections, especially for chewing gum. It was estimated in 1972 that the domestic use of peppermint oil was as follows: chewing gum. 55%; toothpaste, mouthwash, and pharmaceuticals, 34%; confectionary products, 10%; and other products, 1%. In recent years, about one third of our national production has found its way into export trade, which has increased more than 400 1 /c since the 1940s. NONPRESCRIPTION PRODUCTS. Peppermint oil is used as a flavoring agent in Listerine Mouthwash ® and as a carminative and flavoring agent in the antacid products BiSoDol ®, Gehisil®, Phillips' Milk of Magnesia ® , and Turns®. Japanese peppermint oil or mentha arvensis oil is obtained by steam distillation from Men tha arvensis Linné var. piperascens. This oil is considerably higher in menthol content but is inferior in flavor to peppermint oil. It is, therefore, solely employed as a source of menthol. The plant is indigenous to Japan and is the source of Japa-

VOLATILE OILS

nese menthol. Some years ago the plant was introduced into southern California and Brazil; both areas now produce considerable amounts of menthol. Menthol or menthan-3-ol is an alcohol obtained from diverse mint oils or prepared synthetically. Menthol may be Icvorotatoiy ( - )-menthol], from natural or synthetic sources, or racemic [( )-menthol], produced synthetically. Menthol is usually prepared from Japanese peppermint oil by refrigeration (- 22° C), during which the menthol crystallizes. The liquid portion is poured off, and the crystallized menthol is pressed between filter papers and subsequently purified by recrystallization. Synthetic racemic menthol is produced by hydrogenation of thymol. Menthol may also be prepared from pinene. Menthol occurs as colorless, hexagonal crystals that are usually needlelike, as fused masses, or as a crystalline powder. It has a pleasant, peppermintlike odor. USES. Menthol is a topical antipruritic. It has been used on the skin or mucous membranes as a counterirritant, an antiseptic, and a stimulant; internally, menthol has a depressant effect on the heart. Menthol is topically applied as 0.1 to 2% preparations for use on the skin. NONPRESCRIPTION PRODUCTS. Menthol is used as an antipruritic in such burn and sunburn preparations as Noxzema Medicated Cream®, Solarcaine® , and Unguentine®; in preparations to treat poison ivy rash, Ivy Dry Cream® and Rhulicream®; in douche powders, Zonite®; and in preparations to treat athlete's foot, NP27 Powder®. It is used as a counterirritant in external analgesic preparations that include Absorbirte Jr®, BenGay®, Mcntholatum, Minit-Rub ®, and Vicks Vaporub®. ALDEHYDE VOLATILE OILS

Aldehydes occurring in volatile oils may be divided into acyclic and cyclic, included among the former are citral, which is a 3:1

119

mixture of geranial to neral, and citronella!, the aldehyde corresponding to eltronellol. The aromatic aldehydes include cinnamaldehyde (see Fig. 5-3) and vanillin (vanilla, henzoin, tolu and Peru balsams). çH,

CH 3Cl-i3 HO

HO aHO R 3CCH1H3CCH3 Geranial Net-al

H3CCH3 (+)-CtroneIIaI

Biosynthesis of such aromatic aldehydes as benzaldehyde and vanillin takes place from phenyipropanoid precursors. Because these compounds constitute the aglycones of certain glycosides, their formation was discussed under that heading. The terpene aldehydes derive from acetate metabolism, as illustrated in Figure 5-4. Among the important drugs in this class are cinnamon, cinnamon oil, orange oil, lemon peel, lemon oil, hamamelis water, and citronella oil (Table 5-2). CINNAMON

Cinnamon or Saigon cinnamon is the dried bark of Cinnamornuni loureirii Nees (Fam. Lauraceae). An important cinnamon in U.S. commerce is Ceylon cinnamon, the dried inner bark of shoots of coppiced trees of C. zey!aruczun Nees (Pam. Lauraceae) (Fig. 5-10). Cassia cinnamon is the dried bark of C. cassia (Nees) Nees ex Blume. Cinnamon may be from the Arabic, kinnalnon; the Malay, kayu tnanis, sweet wood; or the Hebrew, ginnanion, Lourcirii is in honor of the French botanist Loureiro; zeylanicum signifies Ceylon; Cassia is from the Greek kassia, meaning to strip off the bark. Cinnamon is named as a spice in the books of Moses, by the ancient Greek and Latin historians, and in Chinese herbals as early as 2700 B.C. Its cultivation in Ceylon probably dates from 1200 A.D. The wild cinnamon trees seldom exceed

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9 meters in height. The leaves are coriaceous, green, and glossy; the flowers are in terminal panicles; and the fruit is fleshy and ovoid. Practically all commercial cinnamon is now obtained from cultivated trees in Sri Lanka (Ceylon), southeastern China, Vietnam, Laos, Indonesia, the West Indies, the Seychelles, Malagasy, and many other localities. However, cinnamon from southeastern Asia and adjacent islands is superior in quality. The bark is gathered from young trees usually less than 6 years old and, in Sri Lanka, mostly from coppice shoots 18 to 36 months old. The leaves, branches, and stem tips are distilled with steam for the volatile oil. The bark is cut transversely and longitudinally and peeled. In Sri Lanka and Indonesia, the bark is. scraped while fresh to remove epidermis and cork; in China, it is planed to remove partially the cork. In Sri Lanka, many layers of the thin inner bark are rolled into one quill; in Indonesia, several layers may be quilled together; and in China and Vietnam, each layer is quilled separately or only 2 or 3 layers are quilled together. Saigon cinnamon yields from 2 to 6% of volatile oil; cassia cinnamon, 0.5 to 1.5%; and Ceylon cinnamon, 0.5 to 1%. Other constituents are mannitol, which causes the sweetness of the bark, and tannin, which is abundant in cassia cinnamon. USES. Saigon, Ceylon, and cassia cinnamon are carminatives and flavors. The powdered cinnamon found in the grocery trade is frequently a blend of several kinds of cinnamon. The blending is done either to improve the aromatic quality or to cheapen the product. Cinnamon oil is the volatile oil distilled with steam from the leaves and twigs of Cinnamomurn cassia (Nees) Nees ex Blume (Fain. Lauraceae) and rectified by distillation. It is also known as cassia oil. Cinnamon oil is a yellowish to brownish liquid that becomes darker and thicker by age or by exposure to air; it possesses the

characteristic odor and taste of cassia cinnamon. The principal constituent of the oil is cinnamic aldehyde, 80 to 95%; the remainder consists of terpenes, such as limonene, p-cymene, (- )-linalool and -caryophyllene, and other compounds such as eugenol. USES. Cinnamon oil is used as a flavoring agent; it is also a carminative and pungent aromatic. It has antiseptic properties. Cinuamaldehyde, cinnamic aldehyde, or cinnamyl aldehyde is obtained naturally

from cassia oil or synthetically from a mixture of benzaldehyde and acetaldehyde by the action of sodium hydroxide. It should be stored in well-filled, tight, light-resistant containers and protected from excessive heat. Lemon Peel

Lemon is the fruit of Citrus linwn (Linné) Burmann filius (Fam. Rutaceae). Lemon peel is the outer yellow rind of the fresh ripe fruit of C. lirnon. Limon is from Iipnun, the name of the fruit. The plant is a small evergreen tree with shining leaves and is indigenous to northern India but cultivated to a considerable extent in such subtropical regions as southern Spain, southern Italy, Sicily, southern California, Florida, Argentina, Cyprus, Brazil, Tunisia, Israel, Mexico, India, South Africa, and Australia. The history of the lemon parallels that of the orange; it has been known since the beginning of the written history of India, its native land. The cultivation of lemon trees and the picking, selecting, and storing of lemon fruits constitute an important industry. The , ou-tèr, lemon-yellow or dark yellow layer (peel) is removed by grating or paring. It has a highly fragrant, distinctive odor and an aromatic taste. Lemon peel contains a volatile oil, a small quantity of hesperidin, bitter principles, a principle resembling tannin, and calcium oxalate. USES. Lemon peel is a flavoring agent, a

VOLATILE OILS

stimulant, and a stomachic. It is employed chiefly in combination with other drugs. Lemon oil is the volatile oil obtained by expression, without the aid of heat, from the fresh peel of the fruit of C. lirnon, with or without the previous separation of the pulp and the peel. Six processes are utilized in the recovery of oil of lemon; five of these processes yield an oil meeting pharmaceutic requirements. 1. The outer portion of the rind, which contains the volatile oil, is removed by grating; the resultant raspings are placed in canvas bags and subjected to pressure. The resultant turbid oil is allowed to stand until the sediment separates, after which the oil is decanted. 2. The sponge process is employed to a considerable extent in Sicily and along the Riviera. The lemon is peeled and pieces of the peel are pressed flat so that they flex and rupture the oil cells. The oil is absorbed by a sponge which, when it becomes saturated, is squeezed out, and the process is repeated. 3. The entire fruit is rotated in a saucershaped container that has several rows of sharp metal pins and is called an écueilé a piquer. The pins rupture the oil cells, and the exuding oil collects in a long narrow depression in the bottom of the saucer, which also serves as the handle. This method is now used in the West Indies. 4. During the machine process used in Italy, the oil is separated mechanically using a principle similar to that of the Ecuellê a piquer. Only the peel is subjected to this method. 5. Cold-pressed California oil is obtained by the application of extremely high pressure to the lemons and the rapi removal of the juice and oil. The juice-and-oil mixture is then separated by high-speed centrifugal separation at the lowest feasible temper-

123

ature and in the shortest possible time. 6. Some lemon oil is obtained by distillation. Such oil is not comparable with the expressed oil and does not conform to pharmaceutic standards. Distilled oil is usually used fo the preparation of terpeneless oil of lemon. Lemon oil contains about 90% of terpenes consisting chiefly of (+ )-limonene, the main hydrocarbon present in a range of 70 to 80%, and other monoterpene hydrocarbons, especially 3-pinene and -y-terpinene (approximately 8 to 10% of each). The most important contributors to lemon-oil flavor are neral and geranial (together called citral) Some lemon oils contain up to 13% of citral, but a range of 2 to 4% is optimum for a high-quality oil. The primary esters in lemon oil are neryl and geranyl acetates, and they are believed to be important in providing a full-bodied lemon flavor. Lemon oil that has a terebinthinate odor must not be used or dispensed; such an odor indicates decomposed terpenes or added turpentine oil. USES. Lemon oil is a flavoring agent. It has stimulant, carminative, and stomachic properties. It is a valuable commodity used not only in food flavorings but also in cosmetics and liquid cleansers because the aroma and flavor are widely accepted by consumers. In 1967, the dollar value of lemon oil consumed in the United States was $11 million, making it the number one volatile oil in dollar value and representing 2.5 million lb of oil. Turpentine oil was formerly used as an adulterant but has been replaced by terpenes obtained in the preparation of terpeneless oils. California-type lemon oil should contain between 2.2 and 3.8% of total aldehydes (principally citral) and Italian-type lemon oil between 3,0 and 5.5%. Yet, even such a citral content is no criterion of purity because citral from a cheaper source (lemongrass oil obtained from Cym-

VOLATILE OILS

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CH HC (- )•Thutone

The more important drugs in this category are camphor, spearmint, and caraway mirteae), which Contains about 80% citral) (Table 5-3). may be added. Only a careful Check of the1 i\ physical and chemical constants of the oiij,amphor can determine its purity. Camphor is a ketone obtained from CmTerpeneless oils. Lemon oil and orange oil, nainomum cam plwra (Linné) Nees et Eherby virtue of their high terpene content, maier (Fam. Lauraceae) (natural camphor) often develop a terebinthinate odor during or produced syntheticall y (synthetic camstorage. A considerable amount of these phor). Camphora is from the Arabic kathy, terpenes may be removed by distillation meaning chalk. The plant is a large everunder reduced pressure. A terpeneless green tree indigenous to eastern Asia but lemon oil with a citral content of 40 to 50% naturalized in the Mediterranean region, may be prepared. In terpeneless orange oil, Sri Lanka, Egypt, South Africa, Brazil, Jaabout 95% of the terperies have been remaica, Florida, and California. Early refmoved. Such oils are not subject to dete- erences to camphor do not pertain to the rioration and may be employed in smaller laurel camphor but rather to the Borneo quantities to obtain the same organoleptic camphor (borneol, see page 113), which effect. They are, however, considerably reached Arabia in the 6th century and Euhigher in price than the natural oils, rope in the 12th. Laurel camphor appeared

hopogon citrntus [D.C.] Staph. [Farn. Gra-

KETONE VOLATILE OILS

Ketones occurring in volatile oils may be divided into (1) monocylic terpene ketones, including menthone, carvone, piperitone, pulegone (see Fig. 5-9), and diosphenol (a crystalline ketone in buchu); and (2) dicyclic ketones, including camphor, fenchone, and thujone.

in Europe in the 17th century. When Japan annexed Taiwan, a government monopoly was created (1900). From that time until World War II, about 80% of the world's supply of natural camphor (about 4 million kg per year) was produced in Taiwan, where the tree occurs naturally in abundance and is also extensively cultivated; the remaining 20% vas produced largely in Japan and southern China. Since 1945,

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the production of synthetic camphor has pho-Phenique ® for athlete's foot and Blisgradually lessened the demand for the nat- tex® for cold sores. NOTE. Camphor must be labeled to inural product; nevertheless, Japanese and Taiwan camphors still are found on the dicate whether it is obtained from natural sources or is prepared synthetically. market. Natural camphor occurs as a crystalline product in clefts in the woody stems and Spearmint roots and, to a greater extent, dissolved in Spearmint consists of the dried leaf and the volatile oil. The wood is chipped and distilled with steam, and 1 lb of crude cam- flowering top of Mentha spicata Linné (M. phor is obtained from 20 to 40 lb of chips. viridis Linné) (common spearmint) or of M. The crude camphor is then freed of oil by cardiaca Gerard ex Baker (Scotch spearmint) centrifugation and pressing and finally re- (Fam. Labiatae). Spicata is from the Latin sublimed and pressed into the familiar spica, meaning a spike, and refers to the arrangement of the flowers. The plant is a cakes. Before World War II, about 6.5 million perennial herb that closely resembles pepkg of synthetic camphor were produced permint, is indigenous to Europe, and is annually in Europe and the United States. naturalized and cultivated in various parts During the war, the production of syn- of North America. Spearmint is extensively thetic camphor practically replaced the nat- cultivated in Washington, Idaho, Wisconural derivative. Since the war, production sin, Michigan, and Indiana. More than of natural camphor has been resumed, but 25,000 acres are presently devoted to spearit will never attain its former prominence. mint cultivation in the state of Washington, Synthetic camphor is made from pinene, and the average yield is 90 lb per acre. Total the principal constituent of turpentine oil. production of spearmint oil amounts to The starting point is the stumps of felled about 3 million lb annually. The plant appine trees previously used in turpentining. pears in many of the old herbals, and its A number of complex methods have been mention in early medieval lists demonused for producing synthetic camphor, but strates that it was cultivated in the convent all are based on (1) converting pinene into gardens of the 9th century. Spearmint closely resembles pepperbornyl esters, which are (2) hydrolyzed to isoborneol, and (3) finally oxidized to cam- mint, but the stems are usually more purple. The leaves are sessile or nearly so, inphor. The specific rotation of natural camphor florescence is either in slender, interrupted is between +4l and +430. Synthetic cam- cylindric spikes or crowded lanceolate phor is the optically inactive racemic form. spikes, and the bracts are 7 to 10 mm in length (Fig. 5-12). Odor and taste are arUSES. Camphor is a topical antipruritic, rubefacient, and anti-infective employed at omatic and characteristic; the taste is not ito 3% in preparations for use on the skin. followed by a cooling sensation. Spearmint is composed of resin, tannin, Commercially it is used in the manufacture of certain plastics. and a volatile oil (about 0.5%) that contains NONPRESCRIPTION PRODUCTS. Camphor carvone. USES. Spearmint is classed as a flavor. It is used as an ant-ipruritic in Hist-A-Balm Medicated Lotion®, Rhulicream®, and possesses carminative properties. Spearmint oil is distilled with steam Noxzema Medicated Cream ® and as a rubefacient in external analgesic prepara- from the fresh, overground parts of the tions, such as Heet ® , Mentholatum®, flowering plant of Men tha spicata or of M. Minit-Rub ®, Sloan's Liniment s, and Vicks cardiaca. It contains not less than 55%, by Vaporub® . It is also an ingredient of Cam- volume, of carvone. Most of the supply of

VOLATILE OILS

127

mouthwash, 47 chewing gum, and 3% other. PHENOL VOLATILE OILS Two kinds of phenols occur in volatile oils: those that are present naturally and those that are produced as the result of destructive distillation of certain plant products. Eugenol (see Fig. 5-3), t.hymol, and carvacrol are the most important phenols occurring in volatile oils. Eugenol occurs in clove oil, myrcia oil, and other oils; thymol and carvacrol occur in thyme oil and others; and creosol and guaiacol occur in creosote and pine tar. CH OH OH I

CH3 Thymol

H30 'CH Carvacrol

Fig. 5-12 . tictha _paita (spcarIrtint) plant hwnp typical opposite, sessile leaves.

OH > OCH

OH .00H3

CH

Creosol spearmint oil is produced in the same area as peppermint oil. Spearmint oil is a colorless, yellow or greenish yellow liquid that has the characteristic odor and taste of spearmint. Spearmint oil contains from 45 to 60% of (- )-carvone, 6 to 20% of alcohols, and 4 to 20% of esters and terpenes, chiefly (limonene and cineole. The carvone is optically isomeric with the (-4- )-carvone found in oil of caraway and oil of dill. USES. Spearmint oil is a flavor. It possesses carminative properties and is used to a considerable extent in the chewing gum industry. A 1972 government survey showed that the estimated domestic use of spearmint oil was 50% toothpaste and

Gualacol

i'he more important drugs containing phenol volatile oils are thyme, clove, myrcia oil, creosote, pine tar, and juniper tar (Table 5-4). Thymol is a phenol obtained from thyme oil (T/ti1n,.us vuRaris Linné), from horsemint oil (tvIonclrtia punctnta Linné), from Mouarda did ynia Linné oil, from ajowan oil (Carton cupticnii Renthain et Hooker), or it may be prepared synthetically from ,n-cresol or p-cvrnerle. The oil may be treated in 2 ways to obtain thymol crystals: (1) it may be subjected to freezing temperatures causing the thvmol to crystallize, or (2) it may be treated with sodium hydroxide solution, the aqueous solution of sodium thymol

VOLATILE OILS

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being separated and decomposed with acid, thus liberating the thyniol, which is subsequently purified. Thymol occurs as large colorless crystals or as a white crystalline powder. it has an aromatic thyrnelike odor and .a pungent taste. Thy mol may be readily microsublimed. USES AND DOSE. Thymol is an antifungal and antibacterial agent. It is employed topicall y in lotions, creams, and ointments in concentrations ranging from 0.1 to 1%. NONPRESCRIPTION PRODUCTS. Thyrnol is used in the feminine hygiene products PMC Hygienic Douche Powder® and Zonite®; in the otic products Auro Ear Drdps®, in the external analgesics Vicks Vaporuhr and Zemo Liquid ® ; and in Listerine mouthwash. Clove Clove or cloves is the dried flower bud of Eugenia caryophyllus (Sprengel) Bullock et Harrison (E. caryophylinta Thunherg) (Fam. Myrtaceac) (Fig. 5-13). Eugenia, which is Latin, and caryophijllus, which is Greek, mean "nut-leaf" and refer to the nutlike flower buds; "clove" is from the Latin c/a vus, meaning a nail, and refers to the shape of the whole spice. The plant is a tree that grows to 15 meters in height and is indigenous to the Molucca Islands but cultivated on the islands of Penang, Ambon, Pemba, Zanzibar, Sumatra, Madagascar, and Mauritius as well as in the Seychelles and the West Indies. The buds are gathered when they change from green to crimson and are carefully dried in the sun. The average yield of dried cloves per tree approximates 7 lb, although a large tree in a favorable year may produce as much as 40 lb. Buds are separated from their individual stems by hand and spread out on coconut mats to dry. In sunny weather, drying requires 4 or 5 days; in cloudy weather, a longer time is required. When rain threatens, the buds must be covered and pro-

19

"1 B Fig. 5-13. Eiigeizin can/opIn/lius. A, Entire flower buds in side and upper views showing the cylindric receptacle (r), the 1 calvx teeth (, and the globular closed corolla COIISIStiJ15 of 4 petals (p). B, Longitudinal section through the flower bud showing petals (17). an-

(hers (a), filaments (f), style (at), ovary (o), ovules (ou), and oil cells foci.

tected. During proper drying, the buds lose about two thirds of their original weight. The best cloves come from Pemba, but four fifths of the world's supply comes from Tanzania. Clove was known to the Chinese before 266 s.c. The Dutch, who won possession of the Spice Islands in 1605, endeavored to create a monopoly and destroyed all the trees except those on the islands of Ambon and Ternate. In 1770, however, the French succeeded in introducing the tree onto Mauritius and Reunion. From there, its cultivation spread to other clove-growing areas. Almost 65% of the world's supply of cloves is ground and mixed with tobacco in cigarettes and consequently smoked. The largest consumer is Indonesia, which imports more than half of Tanzania's cloves each year. Clove contains a volatile oil, 14 to 20%;

130

VOLATILE pits

gallotannic acid, 10 to 13%; oleanolic acid; vanillin; and the chromone, eugenin. USES. Clove is a carminative and a flavor. Clove oil is the volatile oil distilled with steam from the dried flower buds of Eugenia caryophyllus. It contains not less than 85%, by volume, of total phenolic substances, chiefly eugenol. Dove oil is a colorless or pale yellow liquid that becomes darker and thicker by age or exposure to air and has the characteristic odor and taste of clove. The oil contains free eugenol (70 to 95%), eugenol acetate, and 13-caryophyllerie. Together these constituents constitute about 99% of the oil, but they do not account for the characteristic, fresh, fruity note of clove oil. This is produced by several minor constituents, especially methyl-n-amyl ketone. USES. Clove oil is classed as a flavor. It is commonly employed as a toothache remedy that is applied topically to dental cavities as required. Clove oil also possesses antiseptic, counterirritant, and carminative properties. Oils with a particularly high content of eugenol are used in the commercial production of vanillin (see page 74). Approximately I million lb of clove oil are imported into the United States annually. NONPRESCRIPTION PRODUCTS. Clove oil is used in Noxzema Medicated Cream® and Lavoris® mouthwash. Eugenol or 4-allyl-2-methoxyphenol is a phenol obtained from clove oil and from other sources. It is usually prepared from clove oil by shaking with a 10% solution of sodium hydroxide to form sodium eugenolate. The mixture is washed with ether, and the sodium eugenolate is then decomposed with sulfuric acid. The eugenol is separated by steam distillation. It is a colorless or pale yellow, thin liquid that has a strongly aromatic odor of clove and a pungent spicy taste. USES. Eugenol is classed as a dental analgesic. It is applied topically to dental cav-

ities and is incorporated in dental protectives. NONPRESCRIPTION PRODUCTS. Eugenol is an ingredient in the toothache preparathins Benzodent®, Jiffy Toothache Drops®, and Numzident®. PHENOLIC ETHER VOLATILE OILS

A number of phenolic ethers occur in volatile oils. The following are the more important examples: anethole (see Fig. 5-3) from anise and fennel (Table 5-5), safrole from sassafras (see page 486) and nutmeg. RiCQJO

CH—CHCH. Safrole

-CHCH2 Myristicin

Derivatives of safrole are also often found in volatile oils. Notable among these is myristicin (methoxysafrole) from nutmeg. Biosynthesis of Phenolic Ethers. Studies of anethole biosynthesis in Foeniculurn vulgare have revealed that formation takes place from phenylalanine (shikimic acid-phenylpropanoid pathway, see Fig. 5-5) via a number of intermediates (Fig. 5-14). A cellfree enzyme system capable of effecting the conversion has been prepared. Methionine serves as a methyl donor for the methoxylation reaction. Other structurally related phenolic ethers are presumably formed by similar pathways. Some of the drugs containing phenolic ether volatile oil are anise, fennel, and myristica. Nutmeg Nutmeg or myristica is the dried, ripe seed of Myristica fragrans Houttuyn (Fam. Myristicaceae) deprived of its seed coat and arillode and with or without a thin coating of lime (Fig. 5-16). The tree is indigenous to the Moluccà and neighboring islands

VOLATILE OILS

131

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132 CHCN COON

CH=CH—COOH

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OCI-13 O(CH) (Methionine) OH Cinnamic acid-1-14C (from Phenylalanine) 4 . Hydroxycinnamic acid 4 .Methoxycir,namic acid 4Methoxycinnamic alcohol

CH--CH CI-1

OCH3 Anethole

Fig. 5.,-14. Biosynthesis of anetholu.

and is now extensively cultivated in other tropical regions, including the West Indies. The botanical name, Myristica fragraiis, refers to the fragrance of the nutmeg. The commercial supply is largely derived from the Malay Archipelago. The trees bear continuously. Two or three crops are collected yearly. First the fleshy pericarp is removed and then the arillode, which constitutes mace when dried, is removed. The seeds are dried, requiring from 3 to 6 weeks, and then the brittle testa is cracked off. With the exception of those from Penang, nutmegs are partially coated with lime to protect them from attack by insects. Over 5 million lb of whole nutmegs are imported into the United States annually; over 80% of this amount comes from Indonesia. Nutmegs were introduced into Europe by the Arabs about the middle of the 12th century but were not a prevailing article of commerce until the sea routes to the Indies were opened in the 16th century. They played an important part in the Dutch spice monopoly until the tree began to be cultivated in other parts of the world (1800). Nutmeg contains fixed oil, 25 to 40%,

that is solid at ordinary temperatures, sometimes occurs in prismatic crystals, and is known as "nutmeg butter"; volatile oil, 8 to 15%, that contains myristicin and safrole; proteins in considerable amounts; and starch. USES. Myristica is a flavor and a condiment. Recently, it has attracted attention as a useful agent for controlling diarrhea associated with certain carcinomas. In recent years, nutmeg has gained a reputation, especially among prison inmates, as a hallucinogenic agent. However ., the relatively large amount (up to 15 g) that must be ingested to cause the desired intoxication also produces flushing of the skin, tachycardia, absence of salivation, and other undesirable side effects. The active principle(s) responsible for the effects on the central nervous system have not been identified with certainty, but elemicin and myristicin are believed to be involved. Some theories involve the in vivo biotransformation of these nutmeg constituents into amphetaminelike, nitrogen-containing metabolites. H.CO 1OO-CH,---CHCH2 H.OU Elernicin

Fig. 5-15. Italian anise, magnified 6 times.

Nutmeg oil or myristica oil is the volatile oil distilled with steam from the dried kernels of the ripe seeds of Myristica fragrans. The oil is a colorless or pale yellow liquid that has the characteristic odor and taste of nutmeg. East Indian nutmeg oil possesses differ-

VOLATILE OILS

133

Fig. 5-16 . Nutmeg . Mi,nstjca fmniris, showing the one-seeded fruits (called nutmeg apples), the exposed seeds that are partially cm ered by the anllode (mace), and the characteu-ists leaves. The arillode has been partly peeled from a nutmeg seed (right center) to shuw how it can be removed (Photo courtesy of Di. Jolla F Morton, Director, Morton Cnllectanea, UnivcLSjt\ of Mtamc)

ent properties than those of West Indian nutmeg oil. The label of the container must indicate whether the oil is of East Indian or West Indian origin. The oil contains salroie, mvristicjn (methoxvsafrole), methoxyeugenot, camphene, -terpineol, o- and 1-pinene, myrcene, (± )-lirnortenc, and sabinene. USES. Nutmeg oil is a flavoring agent. it possesses carminative properties. OXIDE VOLATILE OILS Cineole (eucalyptol) is found in eucalyptus and several other vola tile- oil-_yielding drugs. It is also called cajuputol because it occurs in cajuput. oti,

H,C

__CH, Cineole

Eucalyptus Oil Eucalyptus

is the dried, scythe-shaped

leaf of LU((11 -11J)"Is ,lobuliis Lahillardiere or of other species of Eucalyptus (Farn. M yrtaceae) (Fig 3-17). This tree is indigenous to eastern Australia and Tasmania and is cultivated in southern Europe and in California. EuCOIIJptIIS is Greek and means well-covered, alluding to the lidlike cover of the buds, and giobulus is Latin and refers to the form of the fruit. The commercial supply of the drug is largely from Australia, Brazil, Spain, Portugal, Angola, and South Africa. The tree requires much water and has been used to dry up marsh land. Eucalyptus Contains volatile oil, 3 to 6%, several resins, and tannic acid. Eucalyptus oil is the volatile oil distilled with steam from the fresh leaf of E. globulus or from other species of Eucalyptus. 'he oil is a colorless or pale yellow liquid that has a characteristic, aromatic, somewhat cam-

1 34

Fig. 5-17. Branch of eucal y ptus

VOLATILE OILS

he

S

traits nod mcvihc-sliapcd c.,vc.

phoraceous odor and a pungent, spicy, cooling taste. Useful eucalyptus oil contains not less than 70% of cineole, It must he free from eucalyptus oils containing large amounts of phellandrene. More than 300 species of Eucalyptus are recognized by botanists, and several different chemical races of a single species may exist. For this reason, the chemistry of the various eucal yptus oils is an extremely complex subject. Eucalyptus oils intended for medicinal use contain about 70 to 85% cineole, plus lesser amounts of volatile aldehydes, terpenes, sesquiterpenes, aromatic aldehydes and alcohols, and phenols. Many of these minor components have irritant properties and are removed by redistillation of the oil. Oils intended for industrial purposes have piperitone and/or phellandrene as their principal Components. Other eucalyptus oils used in perfumery are rich in geraniol and its esters and citronellal.

Over 450,000 lb of eucalyptus oil are imported into the United States annually. USES. Eucalyptus oil is classed as a flavor. It is frequentl y used as an antiseptic, diaphoretic, and expectorant. Cineole or eucalyptol is obtained from eucalyptus oil and from other sources. It is a colorless liquid that has a characteristic, aromatic, camphoraceous odor and a pungent, cooling, spicy taste. Cineole may be obtained (1) from eucalyptus oils by fractional distillation and subsequent freezing of the distillate or by treating eucalyptus oil with phosphoric acid and subsequently decomposing the cineole-phosphoric acid with water, or (2) from terpin hydrate as a deh ydration product on treatment with acids. USE. Cineole is classed as a flavor. It has properties similar to those of eucalyptus oil. NONPRESCRIPTION PRODUCTS. Cineole and eucalyptus oil are employed in a wide variety of products, such as nasal inhalers

VOLATiLE OILS

and sprays (Dristan ®, Sine-Off Once-ADay®, Vicks Sinex ®); external analgesics (Antiphlogistine ® , Mentholatum Deep Heating® , Minit-Rub ® , and Vicks Vaporub®); and mouthwashes (Listerine' and Odara5).

ESTER VOLATILE OILS

A wide variety of esters occurs in volatile oils. The most common are the acetates of terpineol, borneo!, and geraniol. It is common practice to age perfumes to permit esterification, thus improving bouquet. Other examples of esters in volatile oils are allyl isothiocyanate in mustard oil and methyl salicylate in wintergreen oil (Table 5-6). Biosynthesis of Esters. Terpene esters are generally formed from the respective alcohols by reaction with aliphatic acid moieties (commonl y acetic acid), as was indicated for menthyl acetate. Formation of aromatic esters, at least in the case of methyl salicylate, involves the reverse process; that is, the aromatic acid reacts with an aliphatic alcohol (commonl y methanol) to form the ester. Labeled cinnamic acid has been incorporated into methyl salicylate by Gau/thria procunthcns. The reactive form of cinnamic acid is presumably an ester of coenzyme A. The biosynthetic pathway (Fig. 5-18) involves o-hydroxylation of the cinnamic acid and subsequent side-chain degradation. Among the drugs in this section are lavender oil, dwarf pine needle oil, mustard oil, and gaultheria oil. Gaultherja Oil Gaultheria, wintergreen, teaberry, or checkerberry consists of the dried leaves of Gaultheria procwnbens Linné (Fam. Encaceae), a low shrublike perennial with slender creeping or subterranean stems and branches that ascend from 5 to 15 cm in height. The leaves are alternate and evergreen, the flowers are white and axillary,

135

and the fruit is a bright red, globular, aromatic bony. The plant is common in coniferous woods throughout the eastern United States and Canada. The leaves are coriaceous, the upper surface is dark green and shining, and the under surface is pale green. The odor is distinct and aromatic, and the taste is aromatic and astringent. Methyl salicylate (see Fig. 5-3) is produced synthetically or is obtained by maceration and subsequent distillation with steam from the leaves of G. procunbens Linné (Fain. Ericaceae) or from the bark of Bet u/a fenta Lime (Fam. Betulaceae) The product must be labeled to indicate whether it was made synthetically or was distilled from either of the plants mentioned. This oil is also known as gaultheria oil, wintergreen oil, betula 0111. or Sweet birch oil. In several eastern states the oil is obtained by distilling wintergreen plants that have been chopped into small pieces and allowed to stand in water for about 12 hours. The oil may be purified b y rectification with steam. When the oil is distilled from birch bark, the process is much the same. Meth I salicylate is made syntheticallv by distilling a mixture of salicylic acid and meth y l alcohol. Methyl salicylate is a colorless, yellow or red liquid that has the characteristic odor and taste of wintergreen. Synthetic oil and that obtained from Betula are optically inactive, but the oil obtained from Gaultherja is slightly lcvorotatorv. Methyl salicylate, the chief constituent of this oil, is formed when the glycoside, gaultherin, is hydrolyzed by the naturally occurring enzyme, gaultherase, in the presence of water. In addition to methyl salicylate, wintergreen oil contains an ester that splits into enanthic alcohol and an acid. Enanthic alcohol and its ester possess the characteristic odor that distinguishes natural wintergreen oil from synthetic methyl salicylate. USES. Meth y l salicy late is a pharmaceutic

VOLATILE OILS

136

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PEPTIDE HORMONES AND THE ENDOCRINE SYSTEM

267

proteolytic enzyme in the pancreas appar- determining the potency. Insulin prepared ently cleaves peptide bonds to remove the in this manner may be subjected to further connecting polypeptide sequence and to purification by ion-exchange chromatogform the physiologically active, 2-chained raphy to yield a purified insulin product insulin. (not more than 10 parts per million promInsulin was crystallized in 1926 by the sulin Contaminant). addition of traces of zinc, and crystals of Commercial production of human insuzinc insulin formed the original reference un has been achieved using two independstandard of the USP. The isoelectric point ent approaches. Pork insulin is converted of zinc insulin is 5.1 to 5.3. Thus, it is sol- to human insulin by replacing enzymatiuble at the alkaline p!-1 of tissue fluids and cally the terminal alanine amino acid resiis rapidly absorbed from subcutaneous in- due with threonine. Recombinant DNA jection sites. Insulin is digested by proteo- techniques have also been used for microlytic enzymes (a common property of poly- bial synthesis. The genes (DNA) that direct peptide hormones), and it is ineffective the biosynthesis of the A and B polypepwhen given orally. tide chains of human insulin are connected The production of both glucagon and in- separately to DNA genetically coded for sulin involves isolation of these substances the enzyme -gaiactosidase. This step puts from pancreas glands. The procedures are the insulin genes under the control of a set relatively complex, a situation that is corn- of genes called the "lac operon"; this opmon for the isolation and purification of eron controls -galactosidase synthesis most peptide molecules. At least 20 major and secretion. The connected genes are insteps are accomplished before a form of troduced separately into plasmids. The insulin suitable for human use is finally plasmids are then taken up by Escherichia developed. Immediately following their re- co/i, producing one strain that formed the moval from slaughtered animals, the raw A chain of insulin and another strain that beef and pork pancreases are frozen to pre- formed the B chain. The addition of lactose vent enzymatic destruction of the insulin to the bacterial cultures switches on the in the gland. About 8000 lb of animal pan- "lac operon" genes. This, in turn, switches creases are needed to yield 1 lb of pure zinc on the gene for the production of either the insulin crystals. A or the B chain of human insulin because The first step in actual production in- these genes are now linked to the genes volves grinding of the frozen glands. Suc- for the formation and secretion of 13-galaccessive steps include extracting the still- tosidase, The 3-galactosidase is synthefrozen powder with acidic alcohol to obtain sized with the A and B chain of insulin the insulin and to suppress enzyme activ- attached, and these are recovered by chemity, centrifuging the crude extract to sep- ically cleaving them from the enzyme. arate the liquid from the glandular residue, Consequently, the A chain of 21 amino clarifying the liquid extract containing the acids and the B chain of 30 amino acids are insulin and impurities, evaporating in vac- purified separately, and the complete inuum to remove the alcohol, treating the sulin molecule is chemically synthesized concentrate to separate the fat, filtering to from the chains by forming 2 disulfide remove residual fat, adding salt water to bridges. precipitate the insulin, redissolving the The absolute therapeutic indication for precipitate, reprecipitat-ing by isoelectric human insulin may be rare. It produces a means, buffering to obtain a uniformly sol- lower incidence of allergic reactions than uble product, washing, drying, and pool- many of the products from animal paning the insulin obtained from other lots creas glands, and it may have special utility prepared in the same manner, and finally, in the small proportion of diabetics allergic

268

PEPTIDE HORMONES AND THE ENDOCRINE SYSTEM

to purified pork insulin. In addition, the bacterially produced hormone will relieve the American Diabetes Association's concern regarding a shortage of animal pancreas glan4sin the near future. Glucagon for injection is a mixture of the hydrochloride with one or more suitable, dry diluents. When the aqueous injection is reconstituted, it has a pH between 2.5 and 3.0 and is usually formulated to contain 1 mg in each ml. The usual parenteral dose is 500 ILg to 11 mg, repeated in 20 minutes, if necessary. Insulin injection or insulin is a sterile, neutral solution of the active principle of the pancreas that affects the metabolism of glucose. Insulin injection contains 40, 100, or 500 USP insulin units in each ml. It is a rompt-acting preparation with a peak of ::ction at 2 to 5 hours. This is the prepaation of choice when glucose tolerance fluctuates rapidly; such situations may include the presence of a severe infection, shock, surgical trauma, or unstable diabetes. Insulin preparations, including insulin injection, must be labeled to indicate the nature (beef and pork, beef, pork, or human) of the insulin; when a product meets the standards for purified insulin, this must also be indicated on the label. the usual dose, for diabetic acidosis, inravenously, is I to 2 units per kg of body weight, repeated in 2 hours as necessary; for diabetes, 10 to 20 units, subcutaneously, 3 or 4 times a day according to the needs of the patient. PROPRIETARY PRODUCTS. Beef and pork: Regular fletin I'; purified beef: Beef Regular fletin il ®; purified pork: Actrapid®, Pork Regular Iletin H ®, and Velosulin®; human: Humulin R®. Preparations of insulin are marketed in multiple-dose ampules of varying unitage. The package color of commercial products varies with the unit value. There is increasing emphasis on use of 100-unit insulin formulations to reduce dosage errors. The need for 40-unit insulins is being studied,

and the FDA may eventually decertify them. Protamine zinc insulin suspension or protamine zinc insulin is a sterile suspension, in a phosphate buffer, of insulin modified by the addition of zinc chloride and protamine. The protamine is prepared from the sperm or from the mature testes of fish belonging to the genus Oncorhynchus Suckley, or Saitno Linné (Fam. Salmonidae). Protarnine zinc insulin suspension provides 40 or 100 USP insulin units in each ml. It is a prolonged-acting insulin preparation. The usual dose, subcutaneously, is 7 to 20 units once a day. PROPRIETARY PRODUCTS. Beef and pork: Prolamine, Zinc and Iletin I®; purified beef or pork: Protamine, Zinc and Iletin ll'. Protamines are basic proteins; they combine with insulin to form protamine-insulin salts, stabilized by a trace of zinc. This complex has an isoelectric point of approximately 7.3; it is buffered to this point and dispensed in a smooth suspension. When injected subcutaneously, it is insoluble at the pH of tissue fluids and is therefore slowly absorbed to provide a prolonged action. Peak of action occurs at 14 to 20 hours, with some effect manifest over 36 hours. Isophane insulin suspension, isophane insulin, or NPH insulin is a sterile suspension, in a phosphate buffer, of insulin made from zinc-insulin crystals modified by the addition of protainine in such a manner that the solid phase of the suspension consists of crystals composed of insulin, protaniine, and zinc. It provides 40 or 100 USP insulin units in each ml. It is an intermediate-acting insulin preparation. The usual dose, subcutaneously, is 10 to 20 units, 1 or 2 times a day. PROPRIETARY PRODUCTS. Beef and pork: NPH Iletin I ®; purified beef: Beef NPH fletin Il® ; purified pork: hisulatard NPH®', Pork MPH Iletin Il ® , and Protaphane. NPH®; human: Humulin N®. Isophane insulin is insoluble at the pH of tissue fluids and therefore has a slow absorption rate. Maximum effect occurs at

PEPTIDE HORMONES AND THE ENDOCRINE SYSTEM

8 to 12 hours. A preparation containing 70% isophane insulin and 30% insulin (Mixtard) is available in a 100-unit formulation to give a rapid onset of activity and a 24-hour duration of action. Insulin zinc suspension or lente insulin is a type of intermediate-acting insulin preparation. It consists of a mixture of crystalline and amorphous materials (approximately a 7:3 ratio) suspended in an acetate buffer. It provides 40 or 100 USP insulin units in each ml. The usual dose, subcutaneously, is 10 to 20 units, once a day. PROPRIETARY PRODUCTS. Beef and pork: Lente Iletin I ®; purified beef and pork: Lentard; purified beef: Beef Lente Iletin II'; purified pork: Monotard® and Pork Lente Iletin Il®. The use of an acetate buffer provides a prolonged duration of action. Achievement of this objective without the addition of foreign proteins, such as protamine, circumvents occasional hypersensitivity problems associated with these additives. The ratio of crystalline and amorphous insulin is selected to give a convenient duration of action of approximately 24 hours. Extended insulin zinc suspension or ultralente insulin is a sterile suspension, in an acetate buffer, of insulin modified by the addition of zinc chloride in such a manner that the solid phase of the suspension is crystalline. It provides 40 or 100 USP insulin units in each ml. It is a long-acting insulin preparation; the duration of action is determined by the particle size and persists for over 36 hours. The usual dose, subcutaneously, is 7 to 20 units, once a day. PROPRIETARY PRODUCTS. Beef and pork: Ultralente Iletin 1 ® ; purified beef: Ultratard®. Prompt insulin zinc suspension or semilente insulin is a sterile suspension, in an acetate buffer, of insulin modified by the addition of zinc chloride in such a manner that the solid phase of the suspension is amorphous. It provides 40 or 100 USP insulin units in each ml. It is a rapid-acting insulin preparation; however, its duration

269

of action extends from 12 to 16 hours. The usual dose, subcutaneously, is 10 to 20 units, I or 2 times a day. PROPRIETARY PRODUCTS. Beef and pork: Semilente Iletin I ®; purified pork: Semitard®. The chief difference between prompt zinc insulin suspension and extended zinc insulin suspension is the size of the particles that make up the solid phase of the suspensions. Prompt zinc insulin suspension has a shorter duration of action. This product is not intended to replace insulin injection in combating diabetic acidosis or in emergencies when immediate action is essential. In such cases, insulin injection should he administered. PARATHYROID HORMONE AND CACITONIN

The parathyroid glands in man are usually 4 in number, oval, 5 to 6 mm in length, and situated upon or imbedded in the dorsal surface of the thyroid gland. They develop and function independently of thyroid tissue. For a number of years, after their discovery by Sandstrom in 1880, the parathyroids were considered to be remnants of embryonic thyroid tissue. The parathyroid glands exert a hormonal control over calcium metabolism. Acute deficiency results in tetany when the level of serum calcium falls from normal (10 to 11 mg/d!) to around 6 to 7 mg/dl. Fibrillary muscular twitching progresses to the convulsive state, culminating in death by tetanic spasm of the larynx and muscles of respiration. Parathyroid hyperfunction produces a condition known as Recklinghausen's disease of bone (osteitis fibrosa cystica), characterized by bone pain, marked elevation of serum calcium with a fall in serum phosphate, and cystic rarefaction of bones with spontaneous fracture and deformity. The calcium removed from bone is excreted in the urine. A similar picture may result from overdose with extracts of parathyroid

270

PEPTIDE HORMONES AND THE ENDOCRINE SYSTEM

gland. In either case, renal . stones and calcification of soft tissues occur. The parathyroid hormone (par4thyrin) is a straight-chain po1ypeptice containing 83 amino acid residues and has a.molecular. weight of approximately 9500. A portion of the molecule . that contains only 35 amino acid residues can elicit the significant physiologic activity of the hormone. The essential 35-ammo-acid subunit of human parathyroid hormone differs in 5 or 6 of its amino acid residues from the animal parathyroid hormones that are available through the meat-packing industry. Immunologicrecognition of this factor may contribute to the high incidence of tolerance noted in therapy. The hormone has a hypercalcemic action. Its principal effect involves bone resorption and calcium retease, but it also promotes absorption of calcium from the gut and renal tubules. Parathyroid hormone is inactivated in the intestinal tract, but it has been. used parenteraily in medicine for blood-calcium maintenance in cases of parathyroid tetany. Following injection, the blood calcium level rises in about 4 hours, reaching a maximum in about 16 hours and returning to the original level after 24 to .36 hours.. Repeated or prolonged administration may establish a complete tolerance with abolition of therapeutic effect. To avoid this, dihydrotachysterol may frequently be substituted; this preparation may be given orally. Adequate intake of calcium, phosphate, and vitamin D must be assured. Some authorities question the justification for using parathyroid hormone for therapeutic purposes, and its future use may be primarily in the diagnostic area. Parathyroid injection is a sterile solution in water for injection of the water-soluble principle of the parathyroid glands that has the property of increasing the calcium content of the blood. This preparation is biologically assayed and standardized so that 1 ml possesses a potency of not less than 100 USP parathyroid units, each, unit representing 1/100 of the amount required to

raise the calcium content of 100 ml of the blood serum of normal dogs 1 mg within 16 to 18 hours after administration. Calcitonin is produced by the parafollicular Or C cells of the thyroid gland, and recent clarification of its function has ig. nificantly increased. our comprehension of calcium metabolism. This hormone exerts a hypocalcemic effect by suppressing bone resorption and by inhibiting tubular reabsorptiQfl of calcium in the kidneys. Thus, calcitonin and parathyroid hormone have counterbalancing actions similar to the situations observed with glucagon-insulin and other control mediators of biologic processes. Calcitonin is a polypeptide containing 32 amino acid residues. The individual amino acid composition of calcitonin from different animal sources varies considerabl y. The kev molecular features for biologic activity appear to include a prolinamide moiety at the carboxyl terminal end of the peptide and a, cyclic subunit containing 6 amino acid residues, including the ring closing cystine, at the amino terminal end of the molecule. Reduction of the disulfide linkage causes .i loss of activity. Calcitortin can be used to treat Paget's disease (osteitis deformans) and postmenopausal osteoporosis and to control hypercalcemia secondary to other osteolytic conditions. The preparation used in therapy is a synthetic salmon calcitonin. Salmon calcitonin elicits 20-fold or more activity on a molar basis than human or porcine calcitoriins; the apparent increased potency may be caused, in part, by a greater affinity for receptor sites and thus a slower. rate of degradation and a longer duration of action. The usual maintenance dose for Paget's disease is 50 to 100 units subcutaneously per day or every other day. The usual dosage regimen in osteoporosis is 100 units subcutaneously or intramuscularly per day.. together with supplemental calcium and adequate vitamin D intake. Therapy to control secondary hypercalcemia usually starts with 4 units per kg of

PEPTIDE HORMONES AND THE ENDOCRINE SYSTEM

body weight every 12 hours, and the dose may be increased, if necessary, up to 8 units per kg every 6 hours. Intramuscular injection and multiple sites are preferred when higher doses are required. PRESCRIPTION PRODUCT, Calciinar, GASTROINTESTINAL HORMONES

The intestinal mucosa secretes such peptide hormones as cholecystrokinin, gastrin, and secretin. These hormones facilitate digestion by stimulating the release by the gastrointestinal tractor the pancreas of various enzymes and other exocrine substances. There is no therapeutic indication for these hormones, but cholecystokinin (CCK) and secretin (SecretinKabi : and Secretin-Boots ® ) from porcine duodenal mucosa are used in the diagnosis of pancreatic disorders. Cholec y stokinin is also used as a diagnostic aid in cholecystographv and cholangiographv. Cholecystokinin contains 33 amino acid residues in a linear chain, but the carboxylic acid-terminal octapeptide is fully active. Choiecystokinjri stimulates the secretion of pancreatic digestive enzymes, the flow of bile, and the contraction of the gallbladder. Ceruletide (Tymtran ® ) and sincalide (Kinevac ® ) are synthetic deca- and octapeptides, respectively, which act similarly to cholecystokinin; they are used diagnostically. Secretin normally increases the bicarbonate content and volume of secretion from the pancreas, it is a linear polypeptide containing 27 amino acid residues; the amino acid sequence has a noticeable similarity to that of glucagon. Pentgastrin (Peptavion ® ), a synthetic pentapeptide with effects similar to those of natural gastrin, is also used for diagnostic purposes. Pentagastrin increases gastrointestinal motility and stimulates the secretion of gastric acid, pepsin, and in-

271

trinsic factor; it is used to test gastric secretory function. READING REFERENCES

Anon.: Problems with Growth Hormone, Med. Letter, 27:57, 1985. Baba, S., Kaneko, T., and Yanathara, N., eds.: Promsulin, Insulin, C-Peptide, Amsterdam, Excerpla Medica, 1979. Belchetz, FE., ed.: Management of Pituitary Diseases, New York, John Wiley & Sons, inc., 1984. Bhatriagar, A.S., ed.: The A nterior Pituitary Gland, New York, Raven Press, 1983. Bloom, S.R., and Polak, J.M., eds.: Gut Hormones, 2nd ed., Edinburgh, Churchill Livingstone, 1981. Bolis, L., Verna, R., and Frati, L., eds.: Peptide Hormones, Riomonbranes, and Cell Growth, New York, Plenum Press, 1984. Bollon, A .P., ed.: Recombinant DNA Products: Insulin, interferon and Growth Hormone, Boca Raton, Flor-

ida, CRC Press, Inc., 1984.

Czech, M.P., ed.: Molecular Basis of Insulin Action, New

York. Plenum Press, 1985. DeGrot, L.J., Larsen 1'.R., Refetoff, S., and Stanbury, J.B.: The Thyroid and Its Diseases, 5th ed., New York, John Wiley & Sons, Inc., 1984. Griffiths, E.C., and Bennett, G.W., eds.: ThyrotropiriReleasin,y Hormone, New York, Raven Press, 1983. lmura, If., ed.: The Pituitary Cl,id, New York, Raven Press, 1985. Jagiello, C.. and Vogel, H.J., eds.: Bioregulators of Reproduction, New York, Academic Press, Inc., 1981. James, V.H.T., ed.: The A drenal Gland, New York, Raven Press, 1979, Li, CH., ed.: Hormonal Proteins and Peptide, Vols. l—Xfl, New York, Academic Press, Inc., 1973-1984. Martin, C. Endocrine Physiology, New York, Oxford University Press, 1985. McKerns, K. 'A'., and Naor, Z., eds.: Hormonal Control of the Ilypothalamo-Piloitary-Gonadaj A xis, New York, Plenum Press, 1984. Oppenheimer, J.H., and Samuels, H.H., eds.. Molecular Basis of Th yroid Hormone A ction, New York, Academic Press, Inc., 1983, Pedile, A ., ed.: Calcitonin 1980: Chemistry, Physiology, Phartnaeolo,ci-y, and Clinical A spects, Amsterdam,

Excrpta Medica, 1981.

Segal, 5 . 1. , ed.: Chorionic Gonadotropin, New York,

Plenum Press, 1980. Tolls, C., Labrie, F., Martin, J.B, and Naflolin, F., eds.: Neuroendocr-inology, New York, Raven Press, 1979, Unger, RH., and Ord, L., eds.: Glucagon, New York, Elsevier North Holland, Inc., 1981. Vander, A.J., Sherman, J.H., and Luciano, D.S,: Human Physiology, 4th ed., New York, McGrawHill Book Co., 1985. Wilson, J.D., and Foster, D.W., eds,: Williams Textbook of Endocrinology, 7th ed., Philadelphia, W.B. Saunders Co., 1985.

10 Enzymes and Other Proteins enzyme is assigned a systematic code number (E.C.) composed of 4 digits. The major Enzymes are organic catalysts produced classes in this system are: b y living organisms. They make possible I. Oxidoreductases---catalyzing oxidothe many complex chemical reactions that reductions between 2 substances. make up life processes. Although pro2. Transferases----catalvzing a transfer of duced by living organisms enzymes are a group, other than hydrogen, belifeless. When isolated, they still exert their tween a pair of substrates. characteristic catalytic effect. Although Hydrolases—catalyzing hydrolysis of 3. their chemical composition varies, they do ester, ether, peptide, glycosyl, acidexhibit several common properties: (1) enanhydride, C-C, C-halide,, or P-N zymes are colloids and are soluble in water bonds. and dilute alcohol but are precipitated by 4. Lyases—catalyzing removal of concentrated alcohol; (2) most enzymes act groups from substrates by mechabest at temperatures between 35 and 400 C; nisms other than hydrolysis, leaving temperatures above 65'C, especially in the double bonds. presence of moisture, usually completely 5. Isomerases--catalyzing interconverdestroy them, whereas their activity is negsion of optic, geometric, or positional ligible at 0 C; (3) certain heavy metals, forisomers. maldehyde, and free iodine retard the en6. Ligases—catalyzing linkage of 2 comzyme's activity. Their activity is markedly pounds coupled to the breaking of a affected by the pH of the medium in which pyrophosphate bond in ATP or a simthey act or by the presence of other subilar compound. stances in this medium. They are usually Because the nomenclature of enzymes highly selective in their action. The enzymes are proteins that range in was rather well established prior to the promolecular weight from about 13,000 to as mulgation of this system, the well-known much as 840,000. At present they are class- trivial names are still ordinarily employed ified according to their action by a complex in the pharmaceutic literature. Those ensystem established by the Commission on countered with some frequency are: 1. Esterases, including lipase, phosphoEnzymes of the International Union of Biolipase, acetyicholinesterase, and othchemistry. Six major classes are recogers. nized; each has 4 to 13 subclasses, and each ENZYMES

272

ENZYMES ANO . OTI-IER PROTEINS

2. Carbohydrases, including diastase, lactase, maltase, invertase, cellulase, hyaluronidase, glucuronidase, lysozyme, and others. Nucleases, including ribonuclease, 3. desoxyribonuclease, nucleophosphatase, and others. 4. Nuclein deaminases, including adenase, adenosine deaminase, and others. 3. Amidases, including arginase, urease, and others. 6. Proteolytic enzymes, including pepsin, trypsin, chymotrypsin, papain, fibrinolysin, streptokinase, urokinase, and others. Enzymes often occur in combination with inorganic or organic substances that have an important part in the catalytic action. If these are nonprotein organic compounds, they are known as coenzymes. If they are inorganic ions, they are referred to as activators. Coenzy mes are integral components of a large number of enzyme systems. Several vitamins (thiamine, riboflavin, nicotinic acid) are recognized as having a coenzymatic function. Because enzymes may be recovered from plant and animal cells and because many have been purified, they are utilized as therapeutic agents in addition to their use as controlling factors in certain chemical reactionsin industry. Pepsin, pancreatin, and papain are employed therapeutically as digestants; hyaluronidase facilitates the diffusion of injected fluids; streptokinase and streptodornase dissolve clotted blood and purulent accumulations; zymase and rennin are used extensively in the fermentation and cheese industries; and penicillinase inactivates the various penicillins. Although the terminology is variable, the names used to designate enzymes usually end in -ase or -in. Some of the more common enzymes are listed in the following classification.

273

I. The Amylolytic Enzymes or Carbohydrases Diastase and amylase are terms applied to 2 well-known amylolytic enzymes. Salivary diastase or ptyalin and pancreatic diastase or amylopsin are found in the digestive tract of animals; they are sometimes called "animal diastase." Malt diastase is formed during the germination of barley grains and converts starch into maltose. It is most active in solutions that are approximately neutral; acidity of pH 4 destroys the enzyme. Invertase or sucrase is found in yeast and in the intestinal juices. It brings about the hydrolysis of sucrose into glucose and fructose. Maltase, which causes the conversion of maltose into glucose, is also found in yeast and the intestinal juices. Zymase is a fermenting enzyme causing the conversion of monosaccharides (glucose, fructose) into alcohol and carbon dioxide. Emulsin is an enzyme found in almonds. It causes the hydrolysis of p-glucosides, thus, amvgdalin is h ydrolyzed into glucose, benzaldehyde, and hydrogen cyanide. Myrosin is found in white and black mustard; it hydrolyzes sinalbin, sinigrm, and other glycosides. II. The Esterases Lipase is a lipolytic enz yme widely distributed in the animal and vegetable kingdoms. It is found in the pancreatic juice of animals and in oily seeds. Lipase causes the hydrolysis of fats into glycerin and fatty acids. Pectase splits pectin into pectic acid and methyl alcohol. Steapsin is a lipolytic enzyme capable of digesting dietary fat. Urease is obtained from soybeans and is used as a laboratory reagent for converting urea to ammonia.

274

ENZYMES AND OTHER PROTEINS

III. The Proteolytic Enzymes Pepsin is a proteolvtic enzyme found in the gastric juice. It is most active at a pH of about 1.8, but in neutral or alkaline media, pepsin is entirely inactive. It converts proteins into proteoses and peptones. Trypsin is formed when the proenzyme, trypsinogen, is acted on by the enterokinase of the intestinal juices. Trypsin is a proteolytic enzyme that is considerably more active than pepsin, converting proteoses and peptones into polypeptides and amino acids. It acts best in an alkaline medium of about pH 8 and may thus be distinguished from pepsin, which acts onl y in acid media. Erepsin is a proteolytic enzyme also found in the intestinal juices. It converts proteoses and peptones into amino acids. Rennin is a coagulating enzyme present in the mucous membrane of the stomach of mammals. It curdles the soluble casein of milk. Papain is a mixture of active proteolytic enzymes found in the unripe fruit of the papaya tree. It is a meat tenderizer. The rationale for oral or parenteral use of proteolytic enzymes in the treatment of traumatically induced inflammationand edema of soft tissues is questionable. Evidence of therapeutic usefulness in such conditions is based solely on the subjective interpretation of results and is, at best, inconclusive. IV. The Oxidizing Enzymes Peroxidases are widely distributed in plants. They bring about the oxidation reactions that cause the discoloration of bruised fruits. Thrombin converts the fibrinogen of the circulating blood into the insoluble fibrin of the blood clot. Zymase, although splitting monosaccharides, is essentially an oxidizing enzyme because the monosaccharide is split by oxidation.

Malt Extract Barley is the dried grain of one or more

varieties of Hordeurn z'ule,are Linné (Fam. Gramineae). Barley is grown throughout the world wherever, the climate is favorable. Malt or malted barley is dried, artificially germinated barley grain. To prepare malt, heaps of barley grain are kept wet with water in a warm room and allowed to germinate until the caulicle protrudes. The grain is then quickly dried. The enzyme diastase in the moist warm grains converts the starch to maltose, thereby stimulating the embryo to growth. The embryo is killed when the grain is dried. Dry malt resembles barley but is more crisp, has an agreeable odor, and has a sweet taste. It contains 50 to 70 1/c of the sugar, maltose; 2 to 15% of dextrins; 8 11c of proteins; diastase; and a peptase enzyme. Malt is used extensively in the brewing and alcohol industries. Malt extract is the product obtained by extracting malt, the partially and artificially germinated grain of one or more varieties of Horileum vulgare. The malt is infused with water at 600 C, and the expressed liquid is concentrated at a temperature not exceeding 600 C, preferably under reduced pressure. Malt extract may be mixed with 10%, by weight, of glycerin. It contains dextrin, maltose, a small amount of glucose, and amylolytic enzymes. It can convert not less than 5 times its weight of starch into watersoluble sugars. USES AND DOSE. Malt extract is used as an easily digested nutritive and as an aid in digesting starch. The usual dose is 15 g. Many commercial extracts of malt do not contain diastase, which is destroyed by the heat used for their Sterilization. Such extracts should not be confused with this product. They are used as bulk-producing laxatives. An example is Ma1tsupex. Diastase is a yellowish white, amorphous powder obtained from an infusion

NLYMES AND OTHER PROTEINS

of malt. It can convert 50 times its weight of potato starch into sugars. Lactase is an enzyme that hydrolyzes lactose to galactose and glucose. It is obtained commercially from the yeast, Saccharoniyces lactis, and is used as LactAid ® Powder to help patients with lactose intolerance to digest the lactose in milk or milk products. Pepsin Pepsin is a substance containing a proteolytic enzyme obtained from the glandular layer of the fresh stomach of the hog, Sus scrofa Linné var. domesticus Gray (Fam. Suidae). The generic name Sus is from the Greek Us, meaning hog; scrofa is Latin and means breeding sow; and domesticus is Latin and means the household. Pepsin is prepared by digesting the minced stomach linings with hydrochloric acid. This solution is clarified, partially evaporated, dial y zed, concentrated, and either poured on glass plates to dry, thus forming scale pepsin, or carefully evaporated in a vacuum, forming spongy pepsin. Pepsin occurs as lustrous, transparent, or translucent scales, as granular or spongy masses ranging in color from light yellow to light brown, or as fine white or creamcolored amorphous powder. It is free from offensive odor and has a slightly acid or saline taste. Pepsin digests not less than 3000 and not more than 3500 times its weight of coagulated egg albumin. A pepsin of higher digestive power may be reduced to the standard by admixture with a pepsin of lower power or with lactose. NOTE: Pepsin produced commercially, especially spongy pepsin, often is 4 to 5 times as active as that used medicinally. Pepsin is administered to assist gastric digestion. It is a proteolytic enzyme and should preferably be given after meals and followed by a dose of hydrochloric acid. The usual dose is 500 mg. It is often combined with pancreatiri in product formulations. Pepsin has a long histor y of use in

275

medicine, but its actual beneficial contribution is poorly documented. Panc reatin Pancreatin is a substance containing enzymes, principally amylase, lipase, and protease. It is obtained from the pancreas of the hog, Sus scrofa Lirirté var. do,nesticus Gray (Fam. Suidae), or of the ox, Bos taurus Linne (Fam. Bovidae). The pancreas is a gland that lies directly inside the posterior wall of the abdomen. The fresh glands are minced and extracted by methods similar to those employed in the manufacture of pepsin. Pancreatin is a cream-colored ainorphous powder with a faint, characteristic, but not offensive, odor. Its greatest activity is in neutral or faintl y alkaline solution. More than traces of mineral acids or large amounts of alkali hydroxides render pancreatin inert, and an excess of alkali carbonates inhibits its action. Pancreatin contains, in each mg, not less than 25 USP units of amylase activity, not less than 2 USP units of lipase activity, and not less than 25 USP units of protease activity. Pancreatin of a higher digestivc power may be labeled to indicate it strength in whole-number multiples of the 3 minimum activities or may be diluted by appropriate admixture to conform to aforementioned specifications. One LiSP unit of am y lase activity is contained in the amount of pancreatin that digests 1 mg of dry USP Potato Starch Reference Standard, 1 USP unit of lipase activity liberates 1 i.Eq of acid 370 C, and per minute at a pH of 9 and at 1 USP unit of protease activity digests 1 mg of casein, all under specified conditions. • Pancreatin is a digestive aid and is also used in the preparation of predigested foods for invalids. Enteric-coated granules of pancreatin have been used to treat infants with celiac disease and related pancreatic deficiencies. The usual dose is 325 mg to I g as tablets, capsules, or granules. PROPRIETARY PRODUCTS. Elzyme, Panteric ® , Viokase ® . Products containing both

276

ENZYMES AND OTHER PROTEINS

pepsin and pancrea tin in combination with bile salts include Digestozyme ®, Donnazvme, Enzobile® , Entozyme, Gastroenterase®, Gourmase®, and Nu'Leven'. Bilogen, Co-Bile 5', Digestalin, Digestex®, Digolase ® , Enzymet®, Enzypari, and Zypan® are related products. Pancrelipase Pancrelipase is essentially a more concentrated form of pancreatin. Ineach mg it contains not less than 24 USP units of lipase activity, 100 LiSP units of amylase activity, and 100 LiSP units of protease activity. Thus the lipase activity is increased 12-fold, but the activity of amylase and protease only 4-fold when compared with panCreahn. Employed as a digestive aid, pancrelipase increases the intestinal absorption of tat, thus aiding in the control of steatorrhea. It is available in the form of capsules, powder packets, and tablets. The usual dose range is 8000 to 24,000 USP units of lipolytic activity prior to each meal or snack, to he determined by the practitioner according to the needs of the patient suffering from pancreatic insufficiency. PROPRIETARY PRODUCTS. Accelerase, Cotazvm, Ilozyme 5, Ku-Zyrne HP, Pancrease s , Viokase®. Papain

Papain is the dried and purified latex of the fruit of Carica papaya Linné (Fam. Caricaceae). The papaya tree is indigenous to tropical America and is cultivated in Sri Lanka, Tanzania, Hawaii, and Florida. It attains a height of about 5 to 6 meters. The fruit (Fig. 10-1) grows to a length of about 30 cm and a weight of 5 kg. The epicarp adheres to the orange-colored, fleshy sarcoârp, which surrounds the central cavity. This cavity contains a mass of nearly black seeds. The full-grown but unripe fruit is subjected to shallow incisions on the 4 sides. The latex flows freely for a few seconds but soon coagulates. After collection, the co-

agulated lumps are shredded and dried by the sun or by the use of artificial heat, the latter method yielding the better grade of crude papain. Incisions and collections are made at weekly intervals as long as the fruit exudes the latex. The crude papain is purified by dissolving in water and precipitating with alcohol. Papain has been referred to as "vegetable pepsin" because it contains enzymes somewhat similar to pepsin; however, unlike pepsin, papain acts in acid, neutral, or alkaline media, Papain contains several enzymes: one or more proteolytic enzymes, among which is peptidase I, capable of converting proteins into dipeptides and polvpeptides; a renninlike, coagulating enzyme that acts on the casein of milk an amylolytic enzyme; a clotting enzyme similar to pectase; and an enzyme that has a feeble activity on fats. It is quite apparent that more than one proteolytic enzyme is present because a single sample of papain yields variable results, depending on the protein used. Although differing in strength in accordance with the method of manufacture, papain can digest about 35 times its own weight of lean meat. For this reason, it is used to tenderize meats. The best grade of papain digests 300 times its own weight of egg albumin. Papain is used as a digestant for proteins because it has an action much like that of pepsin. It is employ ed to relieve the symptoms of episiotomy (surgical incision of the vulva for obstetric purposes). Another use of the enzyme is as an ingredient in cleansing solutions for soft contact lenses. in the meat packing industry, papain is used extensively for tenderizing beef. PROPRIETARY PRODUCTS. Panafj l®, Papase®, and .Softlens Enzymatic Contact Lens Cleaner®. Chymopapain

Chymopapain is a nonpyrogenic proteo. lyric enzyme obtained from the latex of earica papaya Linné (Fain. Caricaceae). It is a sulfhydryl enzyme similar to papain with respect to substrate specificities but differ-

ENZYMES AND OTHER PROTEINS

277

Fig. 10-1. Fruits of Carva ?'apnm.

ing in electrophoretic mobility, stability, and solubility. Employed in the treatment of herniated lumbar intervertebral discs, chymopapain is injected into the nucleus pulposus to hydrolyze the noncollagenous polypeptides or proteins that maintain the tertiary structure of the chondromucoprotein. This relieves the compressive symptoms of lower back pain by lessening osmotic activity and thereby decreasing fluid absorption and reducing intradiscal pressure. About 75% df the patients so treated respond favorably. The unit of chymopapain activity is the nanoKatal (nKat), and I mg of the enzyme contains at least 0.52 n•Kat units. The dosage is 2 to 5 nKatunits per disc or a volume of injection of I The maximum dose in a single patient requiring treatment of multiple discs is 10 nKat units. Chymopapain is marketed in 5-nil sterile vials con-

to ml.

taming 10 to 12.5 nKat units. This is equivalent to 2 or 2.5 units per ml following reconstitution. PROPRIETARY PRODUCTS. Chymodiactin e , Discase®. Bromelains Bromelains, bromelain, or bromelin is a mixture of protein-digesting and milkclotting enzymes obtained from thAg juice of the pineapple plant, A nanas cotno.cus (Linné) Merr. (Farn. Bromeliaceae). Although this enzyme can appear in the juice of the fruit, it can also occur in the stem of the plant. It differs from papain because it is obtained from both the ripe and unripe fruits. Bromelain is used as adjunctive therapy to reduce inflammation and edema and to accelerate tissue repair, especially follow-

278

ENZYMES AND OTHER PROTEINS

ing episiotomy. Its effectiveness in such conditions is apparently owing to depolymerization and permeability modifications that they induce following oral administration. Bromelains are also employed in the production of protein hydrolysates, in tenderizing meats, and in the leather industry. PRESCRIPTION PRODUCT. Ananase.

in solution to the posterior chamber of the eye, under the iris, to achieve zonal lysis. One to two ml of a solution containing 75 to 150 units per ml are ordinarily applied. Products, usually in combination with trypsin, are available for oral use. PRESCRIPTION PRODUCTS. Alpha Chymar®, Avazyme°, Catarase ®, Zolyse. COMBINATION PRODUCTS. Chymoral®, Orenzyme®.

Trypsn Crystallized trypsin is a proteolytic enzyme crystallized from an extract of the 1-lyaluronidase • Hyaluronidase for injection is a sterile, pancreas gland of the ox, Bos taurus Lirtné (Fam. Bovidae). When assayed as directed, dry, soluble, enzyme product prepared it contains not less than 2500 USP trypsin from mammalian testes and capable of hyunits in each mg. It occurs as a white to drolyzing mucopolysaccharides of the type y ellowish white, odorless, cr ystalline or of hyaluronic acid. its potency is expressed -imorphous powder. The assay involves a in USP hyaluronidase units. I-lyaluronipectrophotometric comparison of the so- dase for injection contains not more than -ution to be tested to known solutions of 0.25 .g of tyrosine for each USP hyaluronmeasured USP trypsin crystallized refer- idase unit. Hyaluronidase is a mucolyfic enzyme caence standard. Similar to other enzymes, y cr stallized trypsin is stable in the dry state pable of depolymerizing and catalyzing hybut rapidly deteriorates in solution form. -aluronic acid and similar hexosamine-conThus, it should be stored in tight containers taming polysaccharides. It is also a spreading and a diffusing factor. It occurs away from excessive heat. Crystallized trypsin is a proteolvtic en- in human testes, in various bacterial culzvme. It has been employed orally, topi- tures as a metabolic product, in heads of cally, or by inhalation or local injection for leeches, and in snake venoms. Because of Jebridement of necrotic and pyogenic sur- its action on hyaluronic acid, this enzyme face lesions. Proof of efficacy of oral and promotes diffusion and hastens absorption parenteral administration of proteolytic en- of subcutaneous infusions. 1-lyaluronidase for injection is a spreadzymes in such conditions is lacking. The ing agent. The usual dose, hypodermocurrent use of trypsin is primarily topical clysis, is 150 USP units. by aerosol application for wound and ulcer PRESCRIPTION PRODUCTS.. Alidase5', Wycleansing. dase. PRESCRIPTION PRODUCT. Granulex®. Chymotrypsin Chymotrypsin is a proteolytic enzyme crystallized from an extract of the pancreas gland of the ox, Bos taurus Linné (Fam. Bovidae). It contains not less than 1000 USP chymotrypsin units in each mg. The enzyme occurs as a white to yellowish white, odorless, crystalline or amorphous powder. Chymotrypsin is available as chymotrypsin for ophthalmic solution. This proteolytic enzyme is administered

Streptokinase Streptokinase is a purified bacterial protein elaborated by group C p-hemolytic streptococci. It is supplied as a lyophilized powder. The compound acts to convert plasminogen to the proteolytic enzyme plasmin. Plasmin degrades not only fibrin clots but also fibrinogen and other plasma proteins. Use of streptokinase is indicated in the treatment of pulmonary embolism, deep

ENZYMES AND OTHER PROTEINS

vein thrombosis, arterial thrombosis and embolism, arteriovenous cannula occlusion, and coronary artery thrombosis. At present, it is particularly widely used for the last condition, often producing a prompt recanalization of the involved vessel. The route of administration, dosage, and duration of treatment vary for each of the above conditions. Streptokinase is marketed in sterile vials containing 250,000 to 750,000 IU. PRESCRIPTION PRODUCTS. Kabikinase5, Streptase®. Urokinase Urokinase is an enzyme isolated from human urine or obtained from human kidney cells by tissue culture techniques. There are two forms; both have similar clinical effects, but they differ in molecular weight. The product now available commercially derives from tissue culture and is primarily the low-molecular-weight form. It is marketed as a sterile, lyophilized white powder. The enzyme acts on the endogenous fibrinolytic system, converting plasminogen to the enzyme plasmin. Plasmin degrades fibrin clots as well as fibrinogen and other plasma proteins. Use of urokinase is indicated in the treatment of pulmonary embolism, coronary artery thrombosis, and in restoring the patency of intravenous catheters. It appears to have a reduced probability of serious allergic reactions, presumably owing to its human origin but should be used with appropriate caution. The usual dosage regimen is a priming dose followed by administration of 4,400 units per kg of body weight per hour for 12 hours by intravenous infusion. PRESCRIPTION PRODUCT. Abbokinase®. Fibrinolysin and Desoxyribonuclease Fibrinolysin is in the blood serum as a protease and in plasma as the inactive precursor, profibrinolysin (or plasminogen). It is prepared commercially by activating a human blood plasma fraction with strep-

279

tokinase. In the dried form, fibrinolysin retains its proteolytic activity almost indefinitely; however, in solution form, it rapidly deteriorates. Its enzymatic activity is lost completely when it is exposed to rcomtemperature for 6 to 8 hours. It can attack the protein portions of dead tissues, exudates, and blood clots found in wounds, ulcers, and burns. Fibrinolysin is used primarily in the treatment of blood clots within the cardiovascular system, exclusive of thrombi of the coronary and cerebral arteries. Desoxyribonuclease or deoxyribonuclease is a nucleolytic enzyme that is obtained in a highly purified state from pancreatic glands of bovine origin. Like fibrinolysin, it is stable in dry form but rapidly loses its activity in solution form. It can catalyze cleavage of the giant molecules of desoxyribonucleic acid into numerous fragments of smaller size (polynucleotides); thus, it acts against devitalized tissues in purulent states. It is available as a combination product with bovine fibrinolysin. COMBINATION PRODUCT. Elase®. Sutilains Sutilains is a substance containing pro teolytic enzymes derived from the bacterium Bacillus subtilis. It contains not less than 2.5 million USP casein units of proteolytic activity per g. This cream-colored powder is applied topically, in ointment form, 2 to 4 times daily, for wound debridement. PRESCRIPTION PRODUCT. Travase® Ointment contains 82,000 USP casein units of proteolytic activity per g. Collagenase Collagenase is an enzyme preparation obtained from fermentative cultures of Clostridium histolyticum. It cleaves collagen and is used topically to debride dermal ulcers and severely burned areas. Care should be exercised to avoid heavy metal inactivation of the enzyme; Burow's solu-

280

ENZYMES AND OTHER PROTEINS

tiori can be used to stop the enzyme's action if the risk of bacteremia develops. Available ointments contain 250 units of collagenase activity per g. PROPRIETARY PRODUCTS. Collagenase ABC'S, Santyl®. i -Asparaginase

L-Asparaginase, an enzyme obtained from cultures of certain strains of Escherichia coli, induces hematologic and clinical remissions of short duration in a significant percentage of children with acute leukemia. The antitumor effect may be attributed to degradation by the enzyme of circulating L-asparagine, which results in the eath of cells that require exogenous •ources of this amino acid for survival. The ' otable absence of toxicity to normal mar.)W elements suggests that the effectiveess of the drug is related to a difference n the requirement for I.asparagine beiween normal cells and some neoplastic -ells. A number of serious adverse reactions are noted with asparaginase, including alergic reactions and fatal anaphylaxis. It is ised primarily in combination with other hemotherapeutic agents, such as predniane and vincristine. Administration is inravenous, usually 1,000 units per kg of body weight daily, or intramuscular, 6,000 units per square meter of body surface at 3-day intervals. PRESCRIPTION PRODUCT. E1spar. OTHER PROTEINS Proteins are nitrogenous organic substances produced by and associated with living matter. They occur in both pl ants and animals; those from plants are more easily isolated in crystalline form. Plants usually store proteins in the form of aleurone grains. In animals, proteins occur as living matter, thus making them difficult to obtain in the individual state. Proteins may be classified into 3 groups: simple, conjugated, and derived. The sim-

ple proteins hydrolyze entirely into amino acids; the conjugated proteins are combinations of a protein and a nonprotein group (the latter is called the prosthetic group); and the derived proteins are degradation products of the proteins. Each of these groups has several subdivisions. Because they are present in all living matter, proteins are of great importance in biochemistry. They form an important class of food and are equally as essential as carbohydrates and fats. Meat, fish, and eggs are important sources of animal protein foods. Cereal grains, particularly wheat and soybeans, are sources of plant protein foods. Although proteins are important in metabolism, relatively few isolated proteins are employed as therapeutic agents. Whole glandular products, oil-bearing plant seeds, antitoxins, serums, and globulins contain proteins in combination with other biochemical substances—all these substances possess therapeutic activity, but they are classified in other chapters of the text. Allergens are usually proteinaceous by nature; however, carbohydrates and fats may also produce allergic reactions. Allergens are described in Chapter 14. Certain proteins are highly poisonous: the plant lectins (formerly known as toxalbumins), ricin from castor beans, robin from locust bark, and abrin from jequirity seeds. Among the poisonous animal proteins are hemolysins from salamanders (Trituru spp.) and the various toxins, neurotoxoids, from snake venom (see page 408). The following drugs are composed of proteins, modified proteins, and amino acids; their therapeutic applications are extremely varied. They are grouped together according to homogeneity of origin rather than similarity of function. Gelatin

Gelatin is a product obtained by the partial hydrolysis of collagen derived from the skin, white connective tissue, and bones of

ENZYMES AND OTHER PROTEINS

animals. Commercially, gelatin is prepared from the suitable by-products of slaughtered cattle, sheep, and hogs. Bones are first decalcified by treatment with hydrochloric acid. The materials are extracted with boiling water and steam under pressure until the collagen is hydrol yzed. The solution is then filtered by electro-osmosis, concentrated under reduced pressure, allowed to gel, and rapidly dried on netting in currents of warm air. Gelatin occurs in sheets, flakes, shreds, or as a coarse or. fine powder. It is faintly yellow or amber and has a slight, characteristic odor and taste. When dry, gelatin is stable in the air, but when moist or in solution, it is subject to bacterial decomposition. Gelatin is insoluble in cold water but swells and softens when immersed in cold water, gradually absorbing from 5 to 10 times its weight of water. It is soluble in hot water and insoluble in most immiscible solutions and in volatile and fixed oils. Commercially, gelatin is available as 2 types: A and B. Type A exhibits an isoelectric point between pH 7 and 9 and is incompatible with anionic compounds such as acacia, tragacanth, and agar. Type B, on the other hand, should be used when such mixtures are desired because it exhibits an isoelectric point between pH 4.7 and 5. If gelatin is intended for use in the manufacture of capsules to contain medication or for the coating of tablets, it may be colored with a certified color, may contain various additives, and may have any suitable gel strength. Gelatin contains amino acids: alanine, argiriine, aspartic acid, c y stine, cysteine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, threonine, tyrosine, and valine. Because only traces of other important amino acids are present and hyptophan is absent, gelatin is an incomplete nutritional protein. The gelatinizing constituent is known as

281

chondrin, and the adhesive substance is known as glutin. Gelatin is a pharmaceutic aid (encapsulating agent, suspending agent, tablet binder, and coating agent). Combined with glycerin, it forms glycerinated gelatin; as such, it is employed as a vehicle and also for the manufacture of suppositories. Zinc oxide is added to form zinc gelatin, which is used as a topical protectant. In addition, gelatin is a nutrient and is extensively used for the preparation of commercial food products and for bacteriologic culture media. Absorbable Gelatin Sponge Absorbable gelatin sponge is a sterile, absorbable, water-insoluble, gelatin-base spong.'. It consists of a light, nearly white, porous, pliable, nonantigenic matrix prepared from purified, specially treated gelatin, and is sterilized by heat. Even when handled roughly, this product shows little tendency to disintegrate. It absorbs about 50 times its weight of water and about 45 times its weight in blood. Control of capillary oozing and of bleeding from veins is effected through the use of absorbable gelatin sponge applied in the dry form or saturated with sterile, isotonic sodium chloride solution or sterile thrombin solution. The sponge is applied to the blecdmg area and held for 10 to 15 seconds, alter which it is left in place. Absorbable gelatin sponge is a local hemostatic. It is applied topically in operative wounds. COMMERCIAL PRODUCT. Gelfoam. This is supplied as individual sponges, dental packs, prostatectomy cones, and powder intended for a variety of uses (Fig. 10-2). Absorbable Gelatin Film Absorbable gelatin film is a specially prepared gelatin product used in neurosurgery and in thoracic and ocular surgery. It Consists of a thin, pliable, nonantigenic absorbable film of purified gelatin. In the dry state, it resembles cellophane in ap-

282

ENZYMES AND OTHER PROTEINS

Li Fig. 10-2. Complete operation of the .......................... . :. chtin (foreground), filling molds, baking, cutting the finished product into desired shapes, packaging, and sterilizing. (Photo courtesy of The Upjohn Company.)

pearance and stiffness and occurs in pieces about 25 x 50 mm or 100 x 125 mm in size and about 0,075 mm in thickness. When moistened by immersion in salt solution, it is easily cut into the shape needed to fit into the Contours of the incision. COMMERCIAL PRODUCT. Gelfilm'. Microfibrillar Collagen Microfibrillar collagen is a fibrous, water-insoluble material prepared from purified bovine corium collagen. It is an absorbable, topical hemostatic agent that is used in surgical procedures when control of bleeding by ligature or other conventional means is ineffective or impractical.

It is applied dry and directly Onto the bleeding surface; the microfibrillar collagen attracts platelets that adhere to the fibrils and trigger the formation of thrombi. PROPRIETARY PRODUCT. Avitene®. Absorbable Surgical Suture Absorbable surgical suture is a sterile strand prepared from collagen derived from healthy mammals or from a synthetic polymer. It can be absorbed by living mammalian tissue but ma y be treated to modify its resistance to absorption. it may be impregnated with a suitable antimicrobial agent and may be colored by a color ad-

ENZYMES AND OTHER PR01ftis

ditive approved by the federal Food and Drug Administration. The USP lists specifications for labeling, length, diameter, tensile strength, and other requirements. This product is also known as catgut suture, surgical catgut, and surgical gut. Nonabsorbable Surgical Suture Nonabsorbable surgical suture is a strand of material that is suitably resistant to the action of living mammalian tissue. It may be composed of either natural or synthetic fibers; in some cases, metal wire is employed. The label must contain detailed information about the product.. Penicillamine Penicillamine is D-3-mercaptovahne or 0,0-climethylcysfine. it is a degradation product of penicillin-type antibiotics. This substance is a metal-chelating agent employed in Wilson's disease (hepatolcnticular degeneration) to promote urinary excretion of excess copper. It is also useful in treating lead poisoning, and for reasons unknown, it is sometimes useful in cases of severe active rheumatoid arthritis that are refractory to conventional therapy. The usual dose in Wilson's disease is 250 mg, 4 times a day; a single daily dose of up to 1.5 g is used in rheumatoid arthritis. PRESCRIPTION PRODUCT. Cuprimine. Heparin Sodium Heparin sodium is the sodium salt of forms of a sulfated glycosaminoglycan of mixed mucopolysaccharide nature varying in molecular weights. It is usually obtained from the intestinal mucosa or other suitable tissues of domestic animals used for food by humans. Heparin sodium is a mixture of active principles that prolong the clotting time of blood, mainly through formation of a complex with the plasma protein antithrombin and by inhibition of other coagulation proteases. Used for their anticoagulant activity, salts of heparin are the drugs of choice

283

when an immediate effect is desired. Strengths oi heparin sodium are labeled in liSP units per ml, but the USP unit is not equivalent to the international unit (IU). Administration is is by deep subcutaneous injection, direct intravenous injection, or intravenous infusion. The usual intravenous dose is 10,000 USP units initially, then 5000 to 10,000 USP units every 4 to 6 hours. Heparin sodium isavailable commercially in concentrations ranging from 1000 to 40,000 USP units per ml. PRESCRIPTION PRODUCTS. Hepathrom®,. Heprinar®; Liquaernin 10 Sodium, and LipoHepin". Heparin Calcium Heparin calcium is very similar to the product just described, except for the substitution of calcium for sodium in the preparation of the salt. It is said to he superior to heparin sodium in reducing the incidence of bleeding, hematoma formation, and discomfort at the site of injection. These claims require substantiation. PROPRIETARY PRODUCT. CaIciparine. Protamine Sulfate Protamine sulfate is a purified mixture of simple protein principles obtained from the sperm or testes of suitable species of fish, usually those belonging to the genera Oncorhynchus Suckley, Salmo Linné, or Truttn Jordan et Evermann (Earn. Salmonidae). It has the property of neutralizing heparin. Each mg of protamine sulfate neutralizes not less than 80 USP units of heparin activity derived from lung tissue and not less than 100 USP units of heparin activity derived from intestinal mucosa. Protamine sulfate is a fine, white or offwhite, amorphous or crystalline powder that is sparingly soluble in water. It is an antidote to heparin and is administered intravenously. The usual dose, intravenously, is 1 mg for each 90 or 115 USP units of heparin activity, derived from beef lung tissue or porcine intestinal mucosa, re-

284

ENZYMES AND OTHER PROTEINS

spectively, in I to 3 minutes, up to a max' imum of 50 mg in any 10-minute period, repeated as necessary. Protamine sulfate for injection is a sterile mixture of protamine sulfate with one or more suitable dry diluents. Protamine sulfate injection is a sterile isotonic solution of protamine sulfate. Protein Hydrolysate Injection Protein hydrolysate injection is a sterile solution of amino acids and short-chain peptides that represents the approximate nutritive equivalent of the casein, lactalbumin, p1asa, fibrin, or other suitable protein from which it is derived by acid, enzymatic, or other method of hydrolysisThis preparation must have not less than 50% of the total nitrogen present in the form of ce-amino nitrogen; for this reason, it may be modified by partial removal and restoration of the amino acids or by addition of one or more amino acids. This drug is intended for use when the patient is unable to ingest or digest food to supply the nitrogen necessary to replace that amount lost through tissue metabolism.

Protein hydrol ysate injection is a parenteral nutrient. The usual dose, intravenously, is 2 to 3 liters of a 5% solution daily. PRESCRIPTION PRODUCTS. Amigence, Aminogen ® , Lacotein® , Travamin®, and Virex®.

HO

NH, Levodopa

High oral doses of levodopa have been effective in relieving parkinsonism; improvement in patients receiving up to 8 g daily ranges from modest to dramatic. Most symptoms are relieved to some degree, but akinesia and rigidity respond more readily than tremor. The major adverse effects noted include transitory nausea and vomiting, orthostatic faintness, and transient depression of granulocytes. Symptoms of parkinsonisni appear to be related to the depletion of striatal dopamine. Exogenously administered dopamine is either destroyed or does not cross the blood-brain barrier, but levodopa, the biosynthetic precursor of doparnine, does cross the blood-brain barrier. The action of levodopa presumably involves the decarhoxylation in the neural ganglia of the amino acid to give the amine. The usual dose, initiall y , is 250 mg. 2 to 4 times a day, gradually increasing the total dail y dose in increments of 100 to 750 mg every 3 to 7 days as tolerated. The usual dose range is 500 mg to 8 g daily. Administration is in the form of capsules or tablets. PRESCRIPTION PRODUCTS. Bio/Dopa®, Dopar, Larodopaa , Levopa® , Parda®, Rio-Dopa®.

Levodopa Levodopa or 3-hydroxy-L-tyrosine is an amino acid that occurs in the seeds of Viola faba Urine (Fam. Leguthinosae), commonly referred to as the horse bean, the velvet bean, or the broad bean. Isolation of the compound from protein hydrolysate is somewhat difficult owing to its tendency to become oxidized; therefore, synthetic methods of production are employed. An efficient microbial conversion of L-tyrosine to levodopa has been reported.

READING REFERENCES Bohak, Z., and Sharon. N., eds. Biotechnical Applications of Proteins and Enzymes. New York, Academic Press, Inc., 1977. Boyer, P.D., ed.: The Enzymes, 3rd ed., Vols. l-X111, New York, Academic Press, Inc., 1970.-1976. Colowick, S.P., and Kaplan, NO., eds.: Methods in Enzzpnoloi1, Vols. 1-107, New York, Academic Press, Inc., 1955-1984. Dixon, M., and Webb, E.C. Enzymes, 3rd ed., London, Longman Group Ltd., 1979. Ferdinand, W : The Enzyme Molecule, New York, John Wiley & Sons, Inc., 1976. Magnusson, S., Oflesen, M., Foltmann, B., Dane, K., and Neurath, H, eds.: Regulaton, Proteolytic Eu-

ENZYMES AND OTHER PROTEINS

zymes and Their lithibitws, New York, l'ergarnn Press, 1978. Neureth, H., and 11111, R.L., eds.: Proiins, 3rd Vols. I-1V, New York, Academic Press, Inc., 1975-1979. Nord, F.F., and Meister, E., eds.: Advances in nioloy, Vats. 1-56, New York ., John Wiley & Sons, Inc., 1941-1984. Ory, R.L., and St. Angelo, AL., eds.: Enzymes in Food

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and Beocra, e Processi, Washington, D.C., American Chemical Socety, 1977. Ruyssen, R. and Lauwers. A., ads.: Pharmaceutical En:iwes,' Cent, E. Story-Scientia, 1978. Schulz, G. E., and Schirmer, I'LH.: Principles of Protein Strutnrc. New York, Springer-Verlag 1979. Scnmgeour, KG.: Chemistry and Control of Enzyme Reactions, New York, Academic Press, Inc., 1977. Wolf, M., and Ransberger, K.: Enzyme Therapy, New York, Vantage Press, 1972.

Vitamins and Vitamincontaining Drugs

3 11

Vitamins are organic substances, not synthesized within the bod y, that are essential in small amounts for the maintenance of normal metabolic functions. They do not furnish energy and are not utilized as building units for cellular structure. The jack of specific vitamins leads to distinctive deficiency states such as beriberi, rickets, scurvy, and xerophthalmia, or to conditions without definitive symptoms. The tern, vitamin was derived in 1911 when an amine thought to prevent beriberi was isolated from rice bran; this essential or vital amine was called a vitamin. The term has been retained even though it is technically incongruous. Not all vitamins are amines; vitamins A, C, D, E, K, and inositol lack a nitrogen function of any type. The vitamins are diverse chemically, ranging from a simple molecule such as niacin to a complex molecule such as cyanocobalamin. Biologic diversity is also noted with the vitamins. Vitamin B,, niacin, and pantothenic acid function as coenzymes. Vitamin B 12 and folic acid are involved in the biosynthetic transfer of 1-carbon units, and vitamin C is required for the biosynthesis of hvdroxyproline, an essential component of collagen. Vitamins B 1 and B6 are involved in the metabolism of carbohydrates and amino acids, respectively, and biotin has a

function in metabolic carboxylation. Vitamins D and E selectivel y influence membrane transport. A number of vitamins (A, B-,, B 12, C, E, and niacin) are involved, directly or indirectly, in metabolic oxidationreduction reactions. Vitamins are distributed widely and are normally ingested as constituents of various food substances. Fresh fruits, leafy vegetables, whole grains, eggs, and liver are rich dietar y sources of vitamins Standardized, partially purified concentrates and isolated vitamins can be obtained for commercial purposes from a variety of animal, microbial, and plant sources; however, chemical synthesis is more feasible for many of the vitamins. Vitamins obtained from natural sources and those prepared synthetically are indistinguishable biochemically, nutritionally, and therapeutically. Vitamins may be used as special dietary supplements or as drugs. Vitamin supplements are technically foods for special dietary needs and are unnecessary in most cases in which there is a balanced diet. Vitamins are considered drugs if they are taken to treat a condition of vitamin deficiency or to prevent imminent development of a disease. Deficiency conditions are the classic therapeutic uses for vitamins, but recognition of their utility in a

286

VITAMINS AND VITAMIN-CONTAINING DRUGS

variet y of hereditary conditions inborn errors of metabolism) is increasing. Situations in which vitamin supplementation is frequently indicated to prevent development of deficiency states can be grouped into four general categories. Inadequate dietary intake is encountered among alcoholics, the aged, and the impoverished. Increased metabolic requirements are associated with pregnancy, lactation, major surgery, and severe infections. Impaired intestinal absorption creates a problem in severe Gl disorder, surgical resection, old age, obstructive jaundice, and cystic fibrosis. latrogenic situations, such as prolonged use of broadspectrum antibiotics, isoniazid, total parenteral nutrition, or even oral contraceptives, sometimes require supplemental vitamin intake. Accurate assessment of vitamin nutritional status can be very difficult, and selfdiagnosis is the rule rather than the exception. These factors have contributed to the common daily ingestion of a multivitamin product by a large percentage of the American population. The risks to health associated with daily ingestion of unnecessary vitamins is undoubtedly less than with over ingestion of caloric foods, but the pharmacist and other health professionals must he alert to the need for detailed diagnostic assessment if an actual deficiency is suspected. The U.S. Food and Drug Administration requires that vitamin products he labeled to show the percentage of the U.S. recommended daily allowance (US RDA) for each ingredient. The actual dietary needs depend on a number of variables, including age, illness, sex, stress, and weight. The US RDA guidelines are set sufficiently high to compensate for individual variations in the normal person. Extra strength formulations are frequently employed in situations of special need, such as pregnancy. When individual vitamins are used to treat inborn errors of metabolism, the dosage re mens are not related to the US

287

Rl)A estimate. The US RDA values, which must he used for official labeling purposes, should not he confused with the recommended dietary allowances (RDA), values determined b y the Food and Nutrition Board of the National Research Council. RDA values are considered to be good indicators of dietary needs for adequate nutrition in most healthy persons and are used widely by nutritionists, but they differ somewhat from the US RDA figures. It is convenient for a number of purposes to classify the vitamins as fat soluble or water soluble. _______ FAT-SOLUBLE VITAMINS Vitamins A, D. E, and K are fat soluble. Their absorption from the intestinal tract is a5soidled witti [hat of lipids, and a deficiency state ma y he caused by conditions that impair fat absorption. These conditions include pathologic situations such as biliary cirrhosis, cholecystitis, and sprue, and therapeutic situations such as cholestyramine regimens and excessive use of mineral oil laxatives. Vitamin A Vitamin A is a term applied to all derivatives of -iononc, other than the carotenoids, that possess the biologic activity of all-trans retinol. Retinol is the major natural form of the vitamin, but known forms include the acetate and palmitate esters of the alcohol and such oxidation products as retinal, retinoic acid, and 3-dehydroretinol. The ester forms have good stability characteristics, and the acetate and palmitate esters of synthetically prepared all-trans retinol are the major commercial forms of vitamin A.

0H3 L. Vitamin A Alcohol (Retinal)

Retinol is readily absorbed (80 to 90%)

288

VITAMINS AND VITAMIN-CONTAINING DRUGS e

min A -c

ornin E

th

&4J

-

DOINTMENT,

Fig. 11-1. Representative fat-soluble vitamli

MINOR

I 

/M

roducts.

.from the normal intestinal tract and is '—stored in body tissues, especially the liver. An estimated one third of the ingested vit-'--nin A is stored under normal circumtances. Fish liver oils are the richest town natural sources of the vitamin and formerly were its primary commercial sources. Common dietary sources of vitamin A are animal organs (heart, kidney, liver), eggs, dairy products, and fish Vitamin A activity is also derived from some plant carotenoids that occur in carrots and green leafy vegetables. Only carotenoids that possess at least one unhydroxylated 13-ionone ring (a-, 13-, and -y-carotene and cryptoxanthin) can be converted to vitamin A. Beta-carotene and related carotenoids (provitamin A substances) are cleaved by 13-carotene oxygenase in rnucosal cells of the intestine to yield retinal, most of which is promptly reduced in the presence of NADH to retinol. Vitamin A is involved in vision, growth, and tissue differentiation. A deficienc y of this vitamin can result in a variety of conditions, including nyctalopia (night blindness), xerophthalmia, hyperkeratosis of the skin, growth retardation, and decreased resistance to infection. Activation of the visual pigment rhodopsin involves

retinal (retinol is probably oxidized in situ). The function of vitamin A in tissue differentiation may relate to the synthesis of specific glycoproteins (cell receptors or regulators); retinyl phosphate plays an essential role in transferring sugars to glycoprotein. Evidence suggests that vitamin A is biochemically involved in many other essential processes, but details of its molecular involvement need clarification. For example, observations suggest that a deficiency of vitamin A prompts a decline in the plasma level of a macroglobulin that is an inhibitor of collagenases and other proteinases in the cornea; the consequence can be the development of cornea! lesions. Vitamin A is indicated specifically for the treatment of a deficiency of this vitamin, a situation rarely encountered in practice. The vitamin is used primarily for prophylactic purposes when normal dietary intake is inadequate or when normal absorption is compromised. This vitamin has not been implicated in any genetic errors of metabolism, but high doses of some analogs, especially 13-cis retinoic acid, have shown interesting promise in retarding the development of certain preneoplast-ic lesions. The particular advantage of 13-cis retinoic acid relates to the fact that it is not stored in the body, thus avoiding some of

VITAMINS AND VITAMIN-CONTAINING DRUGS

the toxicity potential of high doses of retinol. The efficacy of high doses of vitamin A in the treatment of acne has not been established and should be avoided. Acute toxicity, usually manifest as increased intracranial pressure (hydrocephalus) within 8 to 12 hours, has been observed with high doses of vitamin A, and a hypervitaminosis A syndrome has occurred with chronic overuse of the vitamin. Symptoms of this syndrome include fatigue, night sweats, abdominal discomfort, anorexia, and vomiting. Competent medical supervision and caution should he exercised with extended usage of daily adult doses of 25,000 units or more. The usual US RDA of vitamin A for adults and children over 4 years of age is 5000 units (sometimes expressed as 1000 retinol equivalents). The US RDAs are 1500 units for infants, 2500 for children under the age of 4, and 8000 units for pregnant and lactating women. Units and equivalents originally represented biologic standards, but a spectrophotometric assay is employed currently; 1 unit is equal to 0.3 Vg of retinol. The usual adult dose of vitamin A for treatment of a deficiency is 10,000 to 25,000 units per day for I to 2 weeks or until improvement occurs, in cases of xerophthalmia, the usual daily adult dose is 25,000 to 50,000 units, and up to 100,000 units may be used with close medical supervision in the treatment of corneal lesions caused by hvpovitaminosis A. The usual pediatric dose for treatment of a deficiency is 5000 Units per kg of body weight per day. Vitamin A is usually taken orally, but it may be administered intramuscularly. Therapy may be discontinued when liver storage of the vitamin is determined to be

adequate. Vitamin A equivalent to an adult requirement for 2 years is normally stored in the hepatic tissues of persons receiving a balanced diet. Vitamin A is a yellow to red oily liquid that may solidify when refrigerated and that should be protected from air and light. It may be nearly odorless or may have a slightly fishy odor. It has no rancid odor or taste. It may he diluted with edible Oils, or it may be incorporated in solid, edible carriers or excipients, and product formulations may contain suitable antimicrobial agents, dispersants, and antioxidants. Capsule, tablet, oral solution, and injection dosage forms are available. PROPRIETARY AND PRESCRIPTION PROD-

UCTS. Alphahin and Aquasol A®. Tretinoin or all-trans retinojc acid is available in several formulations (cream, gel, and solution) for topical purposes. It appears to increase epidermal cell mitosis and epidermal cell turnover and is used to treat acne vulgaris. PRESCRIPTION PRODUCT. Retin-A®. Isotretinoin or 13-cis retinoic acid is available for oral use in special situations. It is approved for use in severe recalcitrant cystic acne. It is also used sometimes in keratinization disorders of the skin, which are frequently preneoplastic. Use of the drug must be closely monitored since adverse reactions are common and may require its discontinuation. It should not be used by women who are pregnant, and contraception should be continued for at least one month after termination of therapy. The recommended dosage regimen for cystic acne is I to 2 mg per kg of body weight per day, in two divided doses, for 15 to 20 weeks. A second course of therapy, ,

CH

CH, CHI CH I CHI

I

t'CH

289

CHI CHI 13-Carotene

CHI CH,

290

VITAMINS AND VITAMIN-CONTAINING DRUGS

if needed, may be initiated after a vacation period of at least 8 weeks. PRESCRIPTION PRODUCT. Accutane. Beta-carotene, in addition to being a provitamin A substance, is effective in reducing photosensitivity in individuals with erythropoietic protoporphyria. It does not act as a sunscreen in normal individuals and should not be used for that purpose. However 3-carotene does provide a novel and safe approach to the treatment of a specific type of photosensitivity. When ingested over a period of several weeks, the drug produces carotenemia, a yellowing of the skin often first observed in the palms of the hands or on the soles of the feet. The mode of action has not been established, but it is no doubt related to the pigmentation of the skin. During the course of therapy, elevated blood carotene levels are observed, but vitamin A levels do not rise above normal. The usual adult dose of 3-carotene is 30 to 300 mg per day, adjusted according to the severity of the symptoms and to the response of the patient. Increased exposure to sunlight should not occur until the patient first appears carotenemic, 2 to 6 weeks after initiation of therapy. The protective effect is not total and varies with each individual. PRESCRIPTION PRODUCT. Solatene.

Vitamin 0

Vitamin D is a term that is used for several related steroids and their metabolites that are essential for the absorption and utilization of calcium. Cholecalciferol, or vitamin D, is the primary form of the vitamin encountered in zoologic species; it is stored in a number of tissues, including the liver and skin. Fish liver oils are a rich natural source of this material. Ergocalciferol, or vitamin D 2 , is derived from ergosterol, a plant steroid. It is the form of this vitamin normally used to fortify such foods as milk, bread, and cereals.

H3C, CH, CR3

Vitamin D3 (Cholecalciferol)

Vitamin D 2 (Ergocalciferol) Vitamin D has been called the sunshine vitamin since ultraviolet light is involved in the conversion of provitamin substances to vitamins D. and D-,. 7-Dchydrocholesterol is converted to cholecalciferol in the skin upon exposure to the ultraviolet rays in sunlight, and ergosterol is converted to ergocalciferol in vitro b y controlled exposure to ultraviolet irradiation. If the irradiation process is not adequately contro l led, Cr go c a IC if Cr o I be c o mc s contaminated with undesirable reaction products, including lumisterol, tachysterol, toxisterol, and supresterols. Cholecalciferol and ergocalciferol undergo metabolic hydroxylations in the body to yield molecular forms with greater physiologic activity . The initial activation reaction occurs in the liver and involves formation of 25-hydroxy l derivatives (calcifediol and 25-hydroxyergoca]ciferol, respectively). The second hydroxylation reaction occurs in the kidney and involves the 1-position; the resulting calcitriol and 1,25-dihvdroxvergocalciferol are considered to be the most active molecular forms of this vitamin. Dihydrotachysterol, a substance prepared by synthetic reduction of tachysterol, is closel y related to ergocalciferol and possesses useful vitamin D activity. This

VFIAMINS AND VITAMIN-CONTAINING DRUGS

compound undergoes hydroxylation in the liver to give an active 25-hydroxyl metabolite; no kidney metabolism occurs or is required for full activity. Vitamin D is abborbed readily from the small intestine of normal individuals, but deficiencies causd by malabsorption are known. Cholec*lcifcrol is absorbed with somewhat greater efficiency than ergocalciferol. Bile salts are required for absorption of the latter material. The body's requirements for vitamin U are normally satisfied by dietary sources and by the activating action of sunlight (ultraviolet component) on the skin. Butter, cream, and liver are good natural sources of vitamin D, and milk and cereals are usually fortified with this vitamin. Vitamin I) aids in the utilization of calcium and phosphate and is essential to the development and niamtenance of strong teeth and bones. Deficiency states lead to rickets in children and osteomalacia in adults. Calcitonin and parathyroid hormone (see page 269) are also involved in calcium homeostasis. Vitamin D increases serum calcium and phosphate concentrations by stimulating absorption of these ions from the small intestine and b y mobilizing calcium resorption front The enhanced serum levels of calcium and phosphate normally promote bone mineralization, and the vitamin effect on bone resorption of calcium becomes significant only in hypocalcemic conditions, in which it helps prevent muscular tetany. Vitamin ft the antirachitic vitamin, is indicated specifically for the prevention and treatment of deficiency states. Such conditions are rarely encountered in persons receiving a balanced diet and some exposure to sunlight. However, deficiencies are sometimes encountered in patients with ii,tstinal malabsorption of various etiologies, in those on strict vegetarian diets (no dairy products), in which cholesterol and vitamin intake is inadequate, and in those with renal impairment, in which activation of the vitamin is precluded. Vitamin D is

291

also used to treat familial hypophosphatemia and hypoparathyroidism and to supplement the diet in therapeutic regimens involving long-term use of cholestyramine or anticonvulsant drugs. Calcitriol has special utility in patients with kidney failure. Individuals vary in their sensitivity to vitamin D, and some infants may show hyperactivity even with low doses. However, most toxicities are associated with prolonged ingestion of high doses and can be serious. Chronic vitamin U—induced hypercalcemia may result in soft-tissue calcification, including lethal vascular calcification and nephrocalcinosis. Common symptoms may include gastrointestinal disturbances and hypertension. The serum calcium levels of patients receiving 50,000 units of ergocalciterol per day should be monitored closel y ; serum calcium concentrations should he maintained at 8 to 9 mg per 100 ml. The US RDA for vitamin D is 400 units per day; there is no variation for age and no other special considerations. One unit is equal to 0.025 pg of cholecalciferol or ergocakiferol. The usual dose of cholecalciferol and ergocalciferol for treatment of a deficiency is 1000 to 4000 units per day, the dose being reduced to 400 units per day when appropriate. Much higher doses of ergocalcifc'rol are employed in the treatment of familial hypophosphatemia (50,000 to 100,000 units per day), hypoparathyroidism (50,000 to 200,000 units per day), and vitamin D—dependent rickets (3000 to 10,000 units per day, up to 50,000 units per day in resistant cases, for infants; 10,000 to 60,000 units per day, up to 500,000 units per day in resistant cases, for children and adults). Vitamin D substances are usually taken orally, but ergocalcifcrol is available for intramuscular injection in cases of intestinal malabsorption. Cholecalciferol, vitamin D 1, or 9,10-secocholesta-5,7,10(19)-trien-3-o1 is a white, water-insoluble substance that is affected by air and light. It is available as 400- and 1000-unit tablets.

292

VITAMINS AND VITAMIN-CONTAINING DRUGS

PROPRIETARY PRODUCT.  Delta-D®.

Ergocaiciferol, vitamin D2, or 9,10-secoergosta-5,7,1O(19),22- tetra en-33-o1 is a white, water-insoluble substance that is affected by air and light. Capsule (25,000 and 50,000 units), tablet (50,000 units), oral solution (8000 units per ml), and injection (100,000 and 500,000 units per ml) dosage forms are available. PROPRIETARY AND PRESCRIPTION PROD-

UCTS. Calciferol ® , Deltalin ® , and Drisdol®. Calcifediol or 25-hydroxycholecalciferol is a normal metabolite of cholecalciferoj that is used in the management of metabolic bone diseases and hypocalcemia in patients on chronic renal dialysis. The usual initial adult dose is 300 to 350 V g per week administered on a once-a-day or alternate-day schedule, the dosage being increased; if necessar y , at monthly intervals. It is available as 20- and 50-kg capsules. PRESCRIPTION PRODUCT. Calderoj®. Calcitriol or 1,25-dihydroxycholecalcjferol is the most active normal metabolite of cholecalcjferol. It is used in the management of hypocalcemia in patients on chronic renal dialysis. The usual initial dose is 0.25 g per day, the dosage being increased in increments of 0.25 p.g every 2 to 4 weeks, if necessar y up to a maximum of 3 g per day, depending on the condition being treated. Calcitriol is available as 0.25- and 05- .g capsules. PRESCRIPTION PRODUCT. Rocaltrol®. Dihydrotachysterol or 9,10-secoergosta5,7,22-tTien-3-ol is a white, water-insoluble substance with vitamin D activity; 1 mg of dihydrotachysterol is approximately equivalent to 3 mg of ergocalciferol. It is used in the treatment of acute, latent, and chronic forms of postoperative tetany, idiopathic tetany, and hypoparathyroidism. The usual initial dose is 0.8 to 24 mg daily for several days, then a daily maintenance dose of 0.2 to 1.0 mg, as required for normal serum calcium levels. Capsule (0.125 mg), tablet (0.125, 0.2, and 0.4 mg), and oral solution (0.25 mg per ml) dosage forms are available.

PRESCRIPTION PRODUCTS. DHT® and Hytakerol®. Oleovitamin A and 0 is a solution of vitamin A and vitamin D in fish liver oil or in an edible vegetable oil. The product label must indicate the content of vitamins A and D in mg per g and Kg per g, respectively; the content of these vitamins may also be expressed in units per g. The label must further state whether the product contains cholecalciferol, ergocalciferol, or vita nun D from a natural source. Oleovitamin A and D is available as a capsule, usually containing 10,000 units of vitamin A and 400 units of vitamin D (e.g., Super D Perles ® ). It is used as a dietary supplement. Vitamins A and D are also available in a number of topical emollients for temporary relief of minor burns, sunburn, windburn, abrasions, chapped skin, and other skin irritations (e.g., Andoin5', Balmex® , Caldeserie Medicated ® , Clocream®, Comfortine ®, Desitin®, Lobana®', and Prirnaderm®). I liver oil is the partially desteariiated, fixed oil obtained from the fresh livers of Cadus morrhua Linné and other species in the family Gadidae. The generic name Gadus is from the Greek gados, meaning codfish, and morrhua is the Latin name of the codfish. Codfish inhabit the northern Atlantic Ocean, and cod liver oil is a byL product of the fishing industry. The livers are removed during the fish processing, taking care to exclude the gallbladders, and frozen until subjected to a steaming process to remove the oil. The steaming takes place in closed containers in a carbon dioxide atmosphere to prevent oxidation. The separated oil is chilled to a temperature below 50 C; the precipitated stearin is separated from the lighter vitamin-containing oil by decantation and filtration. Finally, the oil is adjusted to a definite vitamin content by admixture, if necessary, of different lots with higher or lower vitamin levels. Cod liver oil is a thin, oily liquid that has a distinctive, slightly fishy but not rancid

VITAMINS AND VITAMIN.CONTAININ DRUGS

odor and a fishy taste. Medicinal grade cod liver oil must contain, in each gram, not less than 255 g of vitamin A (850 units) and not less than 2.125 g (85 units) o vitamin D. It may be flavored by the addition of not more than 1% of a suitable flavoring agent or a mixture of such substances. The oil also contains glyceryl esters of unsaturated (about 857) and saturated (about 15%) fatty acids (A sterile solution of the sodium salts of the fatty acids, sodium morrhuate injection, is employed as a sclerosing agent; see page 97.) The unsaturated acids include oleic, unoleic, gadoleic, and paintitoicic. Myristic and palmitic acids are the major saturated acids; only traces of stearic acid remain in the oil. Bile salts and the alkaloids, morrhuine and aselline, should he absent; presence of the former indicates contarnination of the livers with gallbladders, and presence of the latter indicates decomposition. Cod liver oil was introduced into medicine during the middle of the 18th century. Fish liver oils were the predominant therapeutic sources of vitamins A and D for y ears. Pure, laboratory-prepr red vitamins A and D have substantially replaced the fish liver oils but cod liver oil still finds some use as a dietary supplement. The usual daily dose is 5 ml containing 1170 l.Lg (3900 units) of vitamin A and 9.7 pg (386 units) of vitamin D. The oil may also be used in formulating oleovitarnin A and D products and topical emollients containing these vitamins.

293

possess only low levels of vitamin E activit)'. Vitamin Ii requires bile salts and dietary• lipids for efficient absorption from the intestinal tract. The vitamin is stored extensivelv in adipose tissues of the body; the stored vitamin E in normal situations represents up to 4 years' requirements. Thus, it is not surprising that no defined adult deficiency state is known. Severe deficiency conditions characterized by muscular dystrophy, coronary disease, and sterility can he induced experimentally in animals, and many of the more common human uses are prompted by such studies. Vitamin E is widely distributed in nature, and the body's requirements are normally satisfied b y dietar y sources. Plant oils, green vegetables, whole grains, egg yolks, and meats are common dietary sources of this vitamin. Wheat germ oil is a traditional natural source of vitamin E for therapeutic purposes.

CO. CH

CH, Ch, CH, ., I CH, CH

a.Tocopherol

The exact biochemical function of vitamin E is unknown, but the antioxidant properties of the vitamin have been implicated. The vitamin may help preserve the integrity of cellular membranes, including those of red blood cells, by preventing freeradical attack and peroxidase cleavage of unsaturated bonds of lipid membrane comVitamin F ponents. It may also act as a cofactor in Vitamin E is a term that refers to various some enzyme systems. Vitamin F is used primarily for prophyforms of .x-tocopheiol, including the dcxlactic purposes to prevent the development trorotatory isomer, the racemic mixture, of deficiency states in conditions in which and their acetate and acid succiriate esters. reduced absorption is likely. Such condiThe free alcohol forms are readily subject tions include diseases of the small intesto oxidation, but the esters are more stable. tine, pancreatic insufficiency, postgastrecSeveral structurally related tocopherol antomy states, and prolonged 'therapeutic alogs also occur in nature, including -, y-, and -tocopherols, but these substances regimens involving cholestyramine or mm-

294

VITAMINS AND VITAMIN-CONTAINING DRUGS

eral oil. It is also used to treat a syndrome usually expressed in units, but it may be sometimes encountered in premature in- expressed in milligrams. [ants that may actually. represent a defiThe usual adult dose of vitamin E for ciency state. The beneficial effect of vitamin prophylactic purposes is 30 units per day; E in cancer, fibrocystic disease of the 60 to 75 units per day are used to treat a breast, intermittent claudication, sickle-cell deficiency. The usual pediatric dose to treat disease, and pollution-associated lung tox- a deficiency is 1 unit per kg of body weight icity has been suggested, but further evi- per day. Vitamin E is usually administered dence is required to establish efficacy. A orally, but it may be given intramuscularly large number of unsubstantiated claims in cases of severe absorption problems. have been made for vitamin E; the pro- Capsule (30 to 1000 units), tablet (100, 200, motion of high doses of vitamin E to in- and 400 units), chewable tablet (200 and crease physical endurance or sexual ability, 400 units), oral solution (50 units per ml), to prevent arteriosclerosis or aging, and to and injection (200 units per ml) dosage treat loss of hair or the menopausal syn- forms are available. Vitamin F is also indrome must be considered fraudulent. corporated into a number of topical emolVitamin E is generally considered to lack lient products for control of dry or chapped significant acute or chronic toxicity when skin or for temporary relief of minor skin ingested in normal therapeutic doses. disorders. PROPRIETARY AND PRESCRIPTION PRODHowever, very high doses of the vitamin (higher than 800 units per day) may cause UCTS. Aquasol fi ® , CEN-E, E-Ferol®, Eadverse reactions, including increased Vital®, Epsilan-M®, Pheryl-E®, and Viterra bleeding tendencies in vitamin-K—deficient E®. patients. The US RDA for vitamin E is 30 units for Vitamin K Vitamin K is a term that refers to 2adults and children over 4 years of age, 10 units for children under 4 years of age, and methyl-1,4-naphthoquinone and deriva5 units for infants, it should be noted that tives of this compound that exhibit an anthese figures are significantl y higher than tihemorrhagic activity. The naturally octhe actual daily dietary requirements. One curring forms of vitamin K possess large unit is equal to I mg of d1--tocopheryl ace- aliphatic substituents at position 3. Phytate. The equivalents for 1-mg quantities of tonadione (vitamin K 1 ) occurs in green the other forms of a-tocopherol are dl-ct- leafy vegetables; synthetically prepared tocopheryl acid succinate, 0.89 units; dl-a- phytonadione is used for therapeutic purtocopherol, 1.1 units; d-a-tocopheryl ace- poses. Vitamin K 2 (prenylmenaquinone-7) tate, 1.36 units; d-a-tocopheryl acid succi- has a larger aliphatic side chain than phynate, 1.21 units; and d-a-tocopherol, 1.49 tonadione (35 carbons compared to 20 carunits. Products containing vitamin E must bons). It has been isolated from putrefied be labeled to indicate the form and amount fish meal and other natural sources, but of the vitamin they contain; the amount is vitamin K7 and its related naturally occur-

0 CHI

CH CH3 CH

(CH2),CH(CH,)3CH(Cf42)3CHCHI CH 2CH 0

Phytonadione

VITAMINS AND  V ITAMIN-CONT1ING DRUGS

ring analogs are not commercially available for medicinal use. Menadione or 2-methyl1,4-naphthoquinone, sometimes called vitamin K3 , is a synthetic material with vitamin K activity. Menadiol (vitamin K 4), a reduced form (hydroquinone) of menadione, is another synthetic form of this vitamin that is used therapeutically. Menadiol is oxidized to menadione in the body. Menadione is alkylated metabolically to yield menaquinone-4 or other physiologically active molecules; the alkylation process may involve geranyl p yrophosphate, farnesyl p yrophosphate, or geranylgeranyl pyrophosphate P

CH3

0 Menadone

Phvtonadione is absorbed from the intestinal tract only in the presence of bile salts. Menadione and menadiol are absorbed directly. The lymphatic system is the major means of transporting vitamin K to the liver where it tends to concentrate. This vitamin is metabolized hepatically and eliminated readil y . There is no significant storage of vitamin K in the body tissues, in distinct contrast with the other fatsoluble vitamins. Vitamin K is distributed widel y in dairy products and many fruits and vegetables, green leafy vegetables being especially good dietary sources. The intestinal microflora also provide a significant portion of the normal human supply of this vitamin. The latter factor presumably explains the infrequent occurrence of deficiency conditions; no recommended daily or dietary allowances are recognized for vitamin K. Intestinal microbial synthesis of vitamin K is particularly important in poultr y . It has been observed that when low levels of antibiotics are added to chicken feed to enhance growth efficiency, dietary supplementation with substances possessing

295

vitamin l activity is often necessary to prevent h em.rrhagic conditions. Vitamin K is necessary for normal clotting of blood. The vitamin promotes the hepatic formation of prothrombin (factor II), other essential clotting factors (VII, IX, X), and sone proteins of unknown physiologic significance. Details of the action of vitamin K need considerable clarification, but a vita nin . K_dependenf carboxylase seems to be rivolved. Hemorrhageis the most common symptom in vitamin K deficiency, and this vitamin is used hi coagulation disorders characterized by in'paired formation of factors II, VII, IX, and X. Disruption in the formation of the clotting factors may result from a deficiency of vitamin K, usually related to intestinal malabsorption, or from interference with vitamin K activity, including hvpo prothronibinemia caused by oral anticoagulants, salic y lates, and some antibiotics. Allergic or h ypersensitive reactions are encountered occasional] y upon administration of vitamin K. Serious hyperbilirubinemia has been associated with its use in premature infants who often lack adequately developed hepatic function. Erythrocyte hemolysis may occur in patients with a deficiency of glucose-6- phosphate dehydrogenase who receive menadione and menadiol. Neither of these forms of the vitamin should he given to women during the last few weeks of pregnancy or to newborn infants.

Phytonadione, phylloquinone, phytylmenaquinone, 2 - m ethyl-3phytyl-1,4naphthoquinone, or vitamin K1 is administered by subcutaneous or intramuscular injection when possible. The usual dose to treat hypoprothrombinemia attributable to anticoagulant drugs, malabsorption, or other causes is 2 to 25 mg for adults, 2 to 10 mg for children, and 1 to 2 mg for infants; the response of prothrombin time should he monitored, and the dose may be repeated after 6 to 8 hours if necessary. A 1-mg dose is frequentl y given to neonates

296

VITAMINS AND  V ITAMIN-CONTAINING DRUGS

to prevent hemorrhagic disease since placental transfer of vitamin K is low and an intestinal microflora has not yet been acquired. Tablets (5 mg) are available for oral use. The usual dose is 2.5 to 10 mg and may be repeated after 12 to 48 hours if necessary. Phytonadione decomposes when exposed to sunlight and must be properly protected. PRESCRIPTION PRODUCTS, aquaMEPHYTON®, Kon i kio&C , and Mephyton®. Menadione, menaquinone, 2-methyl1,4-naphthoquinone, or vitamin K is a yellow, crystalline, synthetic material with prothrombogenic properties. It is affected by sunlight and has irritating properties. Tablets (5 mg) of menadione are available for oral use, the usual dose being 5 to 10 mg daily. Administration to infants is not recommended Menadiol or 2-methyl-1,4-naphthalene diol is available as the water-soluble his (dihydrogen phosphate) tetrasodium salt. Tablet (5 mg) and injection (5, 10, and 37.5 mg per ml) dosage forms are used. [he usual oral dose for treatment of hypoprothrombinernia secondar y to obstructje jaundice, biliary fistulas, or administration of certain drugs is 5 to 10 mg per da y. The usual intramuscular or subcutaneous dose is 5 to 15 mg once or twice daily for adults Or 5 to 10 Mt, once or twice daily for children; intravenous administration is used occasionally for a fa ster response. PRESCRIPTION P RODUCT, Synknyvitei. WATER-SOLUBLE WAMINS

The water-soluble itamins are dominated by the vitamin b complex, but this solubiii*y classification aso includes ascortc acid (vitamin C), bio tin,and such physiologically quesonab1e substances as paminobenzoic acid, the ajoflavonoids (see page 73), choline, ind ifl ojtol The vitamin B complex includ4 thiame (B 1 ), riboflavin (B 2 ), niacin pantothnjccid (B.),

pyridoxine (B 9 ), folic acid (B 9 ), and cyanocohalamin (B 1 ). Liver and yeast are recognized as rich sources of the B vitamins. The water-soluble vitamins range from simple to complex chemical molecules, play diverse physiologic roles, and have a wide scope of therapeutic applications. Vitamin B Complex Vitamin B,

Thiamine or vitamin B has substituted pyrimidine and thiazole rings linked by a methylene bridge. Final steps in both the biosynthesis and chemical s ynthesis of this vitamin involve linkage of the two ring systems. Commercial supplies of thiamine are prepared b y chemical synthesis, and it is usuall y used as the h y drochloride salt. The vitamin is stable in an acidic environment but decomposes readily above pH 5.0. It is e .,tjrnated that about 50% of the vitamin in foods is destroyed during cooking. H 3C.N. .NH-S, CHCHOH . 2 CtJ N, Thiamine

Whole grains, legumes, and meats are good dietar y sources of thiamine. Although the substance is absorbed readily from the small intestine, alcohol inhibits its absorption. Beriberi is the classic dietary deficiency state; however, most of the commonly observed deficiency conditions (sy mptoms include emotional hypersensitivity , loss of appetite, fatigue, and muscular weakness) involve malabsorption in alcoholics. Fhiamjne is associated with several infrequently encountered geneticbased deficiencies or inborn errors of metabolism, including lactic acidosis due to pyruvat-c carboxylase deficiency, branched-chain aminoacidopathv, and the Wernicke-Korsakoff syndrome. There is no appreciable storage of the vitamin in the body; it is metabolized hepaically and eliminated renally. Thiamine is required for carbohydrate metabolism (approximately 0.2 to 0.3 mg

VITAMINS AND  VIT AMIN-CONTAINING DRUGS

AS

297

•

TROPFIIIE. Y•,S a,

•

. GkW

E&I* N.. 2.40

BETALIWS 7HtMMt

owl 0

ca

ee

- 1.1-. .-,C TABLE 75 USe

IS Fl. 0..

250 ng

1 Pt.1 473 1t4

Fig. 11-2. Rcprescritativ, 'ter -oIuHe vitamin  products.

P er 1000 calories) and for some neurologic functions. It is phosphorylated in the body to give thiamine diphosphate or cucarboxv!ase, its active form. Cocarhoxvjase functions biochemically as a coenvzme for c-ketoacid clecarhoxvlases (acetylcoenzvme A formation and acetate metabolism) and transketo[asc (hexose monophosphate shunt). The US RDA of thiamine is 1.5 mg for adults and children over 4 years of age, 0.7 mg for children under 4 years of age, 0.5 mg for infants, and 1.7 mg for pregnant and lactating women. The actual requiremerits vary somewhat with body weight, caloric intake, and carbohydrate content of the diet. The maximum daily absorption of thiamine upon oral ingestion is usually 5. to 15 ing, so megadose oral regimens of this vitamin are rarely justified. Hypersensitivity is encountered occasionally with exogenously administered thiamine, but the vitamin is considered to possess a very ow risk of toxicity, especially with oral ingestion.

Thiamine is used to supplement an in-adequate diet (rare) and to treat deficiency conditions resulting from intestinal lila! absorption of various etiologies, and frrv certain genetic errors. There is no substantive evidence to support its use as an insect repellant or an appetite stimulant or to treat dermatitis, chronic diarrhea, fatigue, mental disorders, multiple sclerosis, or ulcerative colitis. Thiamine is used as the hydrochloride and mononitrate salts and is available in tablet (5 to 500 mg), elixir (usually 1 mg per 5 ml), and injection (usually 100 mg per ml) dosage forms. The usual adult oral doses are 1 to 2 mg per day for dietary supplementation, 5 to 10 mg . three times a day until improvement occurs in general deficiency conditions, 40 mg per day in alcohol-induced deficiency, and 10 to 20 rng per da y in genetic enzyme-deficiency diseases. Pai-enterai administration is reserved for critical deficienc y conditions; the adult dose is 5 to 100 mg thie&' times a day

I-

VITAMINS AND VITAMIN-CONTAINING DRUGS

298

by intramuscular or slow intravenous injection. Pediatric doses are proportionally lower.

verted to FAD. The heart, kidneys, and especially the liver are the primary storage sites for bound flain (flavoproteins). PROPRIETARY AND PRESCRIPTION PROD- Free riboflavin is.apidly eliminated by the UCTS. Betalin S and Biamine. kidneys; peak Hood concentrations occur within 2 hours follin . ingestion of large Vitamin B. doses of the vitamin Riboflavin or vitamin B 2 is a yellow, FMN and FAt' ftinilion as coenzymes in heat-stable substance that is slightly solu- the transfer of &echons in a number of ble in water. It is sensitive to light and will Important biokJk.pxidation reduction rechange into lumichrome or lumiflavin, de- actions. These enzyme systems cover both pending on whether the irradiated solution aerobic and anaerobic conditions, and are is acidic or alkaline; neither lumichrome usually designated oxidases and dehydronor lurniflavin possesses physiologic activ- genases, respectively. Key riboflavinity. Riboflavin can be synthesized chemi- containing enzyme systems include glucally , but bacterial synthesis is more con- cose oxidase (Warburg's old yellow envenient and economical for commercial zyme), -aminoacid oxidases, xanthine purposes. oxidase, cvtuchroni.e: eductase, succinic dehydrogenase, acyl-coenzyme A deh y -drogeias,nythcluioe reductase. The list enzyme is a useful biochemical indicator of riboflavin deficiency. Riboflavin deficiency is rarel y encountered in healthy persons receiving a balH anced diet, but symptoms of deficiency may occur in cases of inadequate nutrition, intestinal malabsorption, and a few specific H drug regimens (phenothiazines, tricyclic Riboflavin antidepressants, and probenecid). It Yeast is the richest natural source of ri- should be noted that alcohol inhibits inboflavin. Dairy produds, eggs, legumes, testinal absorption of riboflavin. Deficiency and meats are the main dietary sources of symptoms are usually dermatologic in nathis vitamin. Small amounts are provided ture, including cheilosis, glossitis, seborby cereal grains, fruits, and green vegeta- rheic dermatitis, and corneal vascularizables. Riboflavin is stable during cooking in tiori. Although riboflavin seldom causes toxthe absence of light. icity in those with normal renal function, Riboflavin occurs in foods in the fretyellow discoloration of the urine may cause form and as riboflavin 5'-phosphate (flavin concern in patients taking high doses of the monoriucleotide or FMN) and flavin advitamin. Hemod ialysis removes riboflavin enine dinucleotide (FAD). The nucleotides but more slowly than normal renal excreare hydrolyzed to riboflavin in the upper tion. gastrointestinal tract. Free riboflavin is abThe US RDA of riboflavin is 1.7 mg for sorbed readily into cells of the intestinal mucosa by an active transport system that adults and children over 4 years of age, 0.8 is enhanced by bile salts. The riboflavin is mg for children under 4 years of age, 0.6 phosphorylated by mucosa] ulavokinase, mg for infants, and 2.0 mg for pregnant and the FMN is bound to plasma albumin and lactating women. The actual requireand transported to the liver where it is con- ments for riboflavin vary somewhat with S.

VITAMINS AND VITAMIN-CONTAINING DRUGS

caloric intake. There is no evidence to justify ingestion of megadoses of this vitamin. Riboflavin is used to supplement an inadequate diet and to treat symptoms of riboflavin deficiency of any cause. It has not been proved effective for treatment of acne, migraine headaches, or muscle cramps. The usual dose to treat a deficiency is 3 to 30 mg per day orally, in divided doses for several days, followed by a I to 4 mg per day dietary supplemental dose. The usual initial pediatric dose is 3 to 10 mg orally for several days, then 0.6 mg per 1000 calories ingested for dietary supplementation. Intramuscular administration is reserved for deficiency situations in which oral administration is not acceptable; the usual adult parenteral dose is 50 mg. Riboflavin is available in tablet (5, 10, 25, 50, and 100 mg) and injection (50 mg per ml) dosage forms. It is usually formulated as the free riboflavin, but niacinamide or some other suitable solubilizing agent must be added to the injectable product. The more soluble riboflavin 5'-phosphate sodium is also available commercially. A number of proprietary products are marketed under the generic designations riboflavin and vitamin B. PRESCRIPTION PRODUCT. Riobin-50.

Niacin Niacin, nicotinic acid, or vitamin B 3 is a simple, naturall y occurring pyridine derivative that prevents pellagra. Niacinamide or nicotinamide also occurs naturally, has antipeflagra activity, and is used for dietary and therapeutic purposes. The terms niacin and niacinamide are usually employed, except in the chemical literature, since they do not have the phonetic similarity to nicotine. These compounds can be prepared easily by chemical synthesis. They are readily absorbed from the gastrointestinal tract under normal circumstances.

299

. COOH  CO- NHr .-. N Niacin Niaonamide

Lean meats, fish, and dairy products are good dietary sources of niacin; the vitamin is stable during cooking. Cereal grains and a number of other foods contain appreciable quantities of niacin that is present in a bound form and thus is not readily bioavailable. The roasting of coffee beans results in the release of a significant quantity of niacin as well as in the development of a characteristic flavor. Tryptophan is also converted to niacin in the body (Fig. 11-3). It is estimated that 60 mg of tryptophan is approximately equivalent to 1 mg of niacin and that such metabolic synthesis may account for up to one half of the body's requirement for this vitamin. Niacin is metabolized to niacinarnide which is a component of nicotinarnide adenine dirtucleotide (NAD), or coenzyme I, and nicotinamide adenine dinucleotide phosphate (NADP), or coenzyme II. These coenzymes are involved in electron transport in a large number of essential enzyme systems associated with glycogenolysis, lipid metabolism, and tissue respiration. Pellagra is the classic niacin-deficiency condition. Symptoms of the deficiency involve the nervous system, the skin, and the gastrointestinal tract and are sometimes summarized as the 3D's—dementia, dermatitis, and diarrhea. Oral lesions, especially angular stomatitis, cheilosis, and red tongue, are more distinctive than the other symptoms. Several genetic errors have been associated with niacin. The best documentation is found with Hartnup's disease, an inherited condition that is characterized by a defective intestinal absorption of tryptophan and thus an impaired in-vivo synthesis 01 niacin. A NJAD-dependent schizophrenia and a niacin-responsive familial hypercholesterolemia have also been recognized; the

VITAMINS AND VITAMIN-CONTAINI NG DRUGS

300

CO--CH 2 - CH-cOOt .CH-000H  NH .- N H

Tryptophafl

NH2 NH

NHCHO

Kynurenine

N.FOrtTlYIkYflUreflifle - COOH

COOH

CHCH—COOH U

.CO.--CH,--CH-000H (.

1coo J-.

NH,

I

NH2 CDOH OH

OH

3.Hydroxyaflthraflilic acid 3.HydroXykYflUrefl0

Nicotinic acid Quinolinic acid Fig. 11-3. BiosynthetiC pathway from tryptophan  10 nicotinic

ormer appears to involve an enzymatic block between tr y ptophari and NAD. Niacin COUSCS direct peripheral vasodiation. It ma y cause cutaneous flushing and a sensation of warmth, especially in the area of the face, neck, and ears. Because gastrointestinal upset is sometimes encountered with this vitamin, it should be taken with meals or milk. Niacinamide lacks the vasodilating property, a distinction of therapeutic sigriittcance. The US RDA of niacin is 20 mg for adults and children over 4 years of age. 9 mg for children under 4 years of age, and 8 mg for infants. There is no justification for ingestion of megadoses of this vitamin, except for limited therapeutic use in hyperlipidemia Niacin and niadriamide are used to prevent or treat deficiency conditions. Such conditions may result from inadequate nutrition, intestinal malabsorption, or genetic errors of metabolism. Niacin, but not niacinamide, is also used as adjuncti ve therapy in some patients with primary hyperlipidemia. Niacin is not useful in the treatment of mental disorders, including schizophrenia or for the prevention of heart attacks; it has not proved effective for the treatment of acne, motion sickness, or peripheral vascular disease.

acid.

[he usual oral doses of macin and niacinamide are 10 to 20 mg per day for dietary supplementation; up to 500 mg per day, usuall y in divided doses, for treatment of pellagra; and 50 to 200 mg per day in l-lartnup's disease. Antihyperlipidemic use of niacin involves a usual adult oral dosage of 1 to 2 g, 3 times per day. Farenteral administration of niacin and niacinamide is reserved for cases in which oral administration is not acceptable; the usual dose by slow intravenous infusion (not exceeding 2 mg per minute) is 25 to 100 rng, 2 or more times per day. Niacin is available in elixir (10 mg per ml), tablet (25, 50, 100, and 500 mg), sustained release (125, 150, 250, 300, 400, and 500 mg), and injection (50 and 100 mg per ml) dosage forms. PROPRIETARY AND PRESCRIPTION PROD-

UCTS. Niac, Nico-400, Nicobid ® , Nicolar, Nico-Span, NicotineX ® , Span-Niacin-150® , and Tega-Span®. Niacinamide is available in tablet (50, 100, and 500 mg) and injection (100 mg per MI) dosage forms. Pantothenic Acid

Pantothenic acid or vitamin B 5 is a component of the vitamin B complex that is sometimes known as the "chick antider-

VITAMINS AND VITAMIN-CONTAINING DRUGS

matitis factor" (based on a prior bioassay procedure). Pantothenic acid is a naturally occurring compound that on hydrolysis yields 13-alanine and pantoic acid, a substituted butyric acid derivative. Bios ynthetic evidence reveals that pantothenic acid is derived from -alanine and -ketoisovaleric acid. CH 3 OR HOCH 2 C -CONH(CH,)2CO CH, H Pantothenic Acid

C

Pantothenic acid is a viscous, oily liquid, so it is usually used as the calcium salt. It is readily s ynthesized. Both the racemic mixture and the dextrorotatory isomer are available commercially, the former having approximately one half the physiologic activity of the latter. Panthenol, a synthetic racemic mixture of the alcohol analog of -pantothenic acid, is also available as a crystalline substitute for the acid; presumably panthenol is oxid'ized in vivo to pantothenic acid. Pantothenic acid is a precursor of coenzyme A, a cofactor that is essential for roetabolism of carbohydrates, lipids, and proteins Coenz y me A is involved in the synthesis of fatty acids and sterols, the oxidation of fatty acids, pyruvic acid, and cketoglutaric acid, as well as in direct biologic acetylations. Animal organs (heart, kidney, and liver) and cereal grains are rich dietary sources of pantothenic acid, but this vitamin is so widely distributed that deficiency conditions are encountered only following deprivation or experimental induction. The most distinctive of the deficiency symptoms are paresthesias of the extremities or "burning foot" syndrome. The US RDA for pantothenic acid is 10 mg for adults and children over 4 years of age, 5 mg for children under 4 years of age, and 3 mg for infants. However, it is recognized that there are no reliable data on

301

the body's actual requirements for this vitamin. Adverse reactions are unknown upon ingestion of the usual dose (5 to 10 mg per day). There is no known justification for use of rnegadoses. Tablets of pantothenic acid (50 mg to I g) and calcium pantothenate (10 to 250 mg) are available, but this vitamin is more cornmonly taken for dietary supplementation as a component of multivitamin preparations. Pantothenic acid, calcium pantothenate, and panthenol are used in various multivitamin products. This vitamin has not been proved effective for treating diabetic neuropathy, preventing arthritis, gray hair, or stress-related diseases, or improving mental processes. Dexpanthenol, the alcohol analog of d-pantothenic acid, is available as an intran-iusuclar injection (250 to 500 mg; llopan and Panol 5 ) for prevention or treatment of postoperative adynarnic ileus and as an oral tablet with choline bitartrate (50 and 25 mg, respectively; llopan-Choline) to help relieve intestinal gas retention of various etiologies. Dexpanthenol is also available in emollient preparations (Panthoderm) for relief of itching and minor skin irritations.

Pyridoxine Vitamin B6 is a term that is applied to pyridoxol, pyridoxal, and pyridoxamine, three closely related, naturally occurring, highly substituted pyridine derivatives with comparable physiologic activity. Pyridoxine is the term that is usually used for pyridoxol in pharmacy and medicine. This alcohol is the predominant form of the vitamin in plant materials. Pyridoxal and pyridoxamine occur in animal tissues. Because pyridoxine is the most stable of these substances, synthetically prepared pyridoxine is the material usually used for exogenous dietary supplementation and therapeutic purposes.

302

VITAMINS AND VITAMIN-CONTAINING DRUGS

CH2OH HO.-..CH20H

Pyridoxol (Pyridoxine)

CHO CH2OFHO H1CN Pyridoxal

Pvridoxiric is readily absorbed in thejejunum, but the vitamin synthesized by the rnicroflora of the colon is largely excreted in feces. Pyridoxine is converted to pyriy y doxal in er throc tes; pyridoxal 5-phosphate or codecarhoxv!ase is the main physiologically active form of the vitamin. l'yridoxamine 5-phosphate also functions to a lesser extent as an enzy me cofactor. Although pyridoxal 5-phosphate is protein hound, it is ultimately metabolized in the liver. The estimated biologic hall-life of the vitamin is 15 to 20 days. Codecarhoxvlase is involved in man y reactions of carbohydrate, lipid, and protein metabolism. As a cofactor for aminotransferases, clehvdratases, and decarboxylases, it is particularl y important in amino acid metabolism. It is involved in a number of reactions, including the onvcrsion of tryptophan to niacin and the biosynthesis of porphyrins. Meats, bananas, whole cereal grains, nuts, and potatoes are good dietary sources of vitamin B. Up to 40% of its activity may he lost during cooking, but deficiency conditions are encountered in persons receiving a balanced diet only in such special situations as intestinal malabsorption, drug-induced (iatrogeiiic) requirements, and inborn errors of metabolism. A number of drugs, including chloramphunicol, cvcloserine, hydralazine isoniazid, and oral contraceptives, act as pyridoxine antagonists or increase its renal excretion. When they are used, intake of this vitamin must be increased. Congenital metabolic dysfunctions associated with vitamin B ( include pyridoxine-dependent infantile convulsions, siderohlastc anemia, xanthuk

CH NH HO,

Pyridox amine

renic aciduria, homocystinuria, and cystathioninurea. S y mptoms of vitamin B deficiency somewhat resemble those of niacin and riboflavin deficiencies. The y include neurologic abnormalities (confusion, irritability, and convulsive seizures), skin lesions (giossitis, sehorrheic dermatitis, and stomatitis), and hypochromic microcytic anemia. An oral loading dose of trvptophan can he used for diagnostic purposes; high urinary elimination of xanthurenic add, normall y a minor tryptophan metabolite, is indicative of a deficiency state, The US RDA for pyridoxine is 2 mg for adults and children over 4 years of age, 0.7 in for children under 4 years of age, 0.4 rng for infants, and 2.5 mg for pregnant and lactating women. Actual dietar y requirements depend on the protein coniponent of the diet as well as on genetic and iatrogenic factors. Pvridoxine is used to supplement dietary intake and to treat deficiency states, congenital metabolic dysfunctions, and poisonings caused by cycloserine and isoniazid. There is no substantive evidence to support the beneficial use of pyridoxine in acne, alcohol intoxication, mental disorders, migraine headaches, morning sickness, or premenstrual tension. The usual dose of pyridoxine is 10 to 20 mg per da y for dietary supplementation, 10 to 300 mg per day for drug-induced deficiencies, and 100 to 600 mg per day for hereditary deficiencies. When a pyridoxine dependency sy ndrome is encountered, it is treated with an initial adult dosage regimen of 30 to 600 mg per day, followed by a 50-mg daily maintenance dose for.. life.

VITAMINS AND VITAMIN-CONTAINING DRUGS

Poisoning cases may justify doses of I g or more. Pyridoxine is usuall y administered orally; parenteral administration is reserved for infantile convulsions, poisoning cases, and patients in whom oral administration is not acceptable. Adverse reactions are rarely encountered with pyridoxine at doses of 25 mg per day or less. Ataxia and paresthesia have been noted in patients taking higher doses, and a pyridoxine dependency syndrome is sometimes induced in patients consuming 200 mg or more daily for periods exceeding a month. Because doses as small as 5 mg per day will stimulate decarboxylation of levodopa in peripheral tissues, patients taking this drug for parkinsonism should use a pyridoxine-free vitamin product. Pyridoxine is available as the h y drochloride salt in tablet (5 to 500 mg), sustained release (5 to 500 mg), and injection (50 and 100 mg per ml) dosage forms. Pyridoxine should he protected against exposure to light. PROPRIETARY AND PRESCRIPTION PROD-

UCTS. Beesix ®, Hexa-Betajjn', Nestrex, and Vitabee-6®. Folk Acid Folic acid, folacin, pteroylglutamic acid, and occasionally vitamin B 9 are terms that refer to a material with antiariemia properties; it is a conjugate of a pteridine derivative, p-arninoberizojc acid, and glutamic acid (Fig. 11-4). A number of other related metabolites with antianemia properties also exist in nature, but synthetically prepared folic acid is the most common commercial form of this vitamin. Dihydrofolate reductase in the liver and plasma reduces folic acid to tetrahydrofolic acid, the physiologically active form of the vitamin. N "- Form y ltetrah ydrofolic acid (folinic acid Or leucovorin) is a naturally occurring biochemical metabolite of folic acid that finds special use in an anticancer drug regimen emplo y ing folate antagonists. Leaf y vegetables (origin of the term

303

"folic") and liver are good dietary sources of pterovlpo]yglutamates (folates), the primary dietary forms of this vitamin. Up to 90% of dietary folates are lost during the cooking process; raw liver has been recommended in deficiency conditions, and a raw spinach salad is superior to cooked spinach as a dietary source. The pteroyipolyglutamates do not cross mammalian cell membranes but are hydrolyzed to pteroylmonoglutamate by -y-gl utamylhydrolase prior to absorption in the jejunum. Polyglutamate metabolites that are resynthesized within mammalian cells are the in-vivo storage form of this vitamin. Fluman tissue storage of folates is estimated to be 5 to 10 mg. About one half of this amount is normall y stored in the liver. Tetrahydrofolic acid functions via a series of lolate coenzymes that are acceptors and donors of 1-carbon units in amino acid (e.g., methionine) and nucleotide (e.g., purinc and pynmidine) metabolism. These coenzymes are essential in processes, such as erythropoiesis, leukopoiesis, and nucleoprotein synthesis, which are characterized b y a high rate of cell turnover and metabolism. Deficiency of folic acid is rarely encountered in the absence of intestinal malabsorption or impaired hepatic function (including alcoholism). Clinical symptoms of a deficiency state include megaloblastic and macrocytic anemias and glossitis. Folic acid is used to treat deficiency states and to supplement dietary intake. Increased dietary supplementation may he required in chronic diseases of the liver and small intestine, prolonged stress and infection, a number of inborn errors of metabolism, and long-term use of medications such as anticonvulsants, oral contraceptives, and glucocorticosteroids Folic acid has not been proved effective for relief of stress or prevention of mental disorders. Although folic acid will correct the megaloblastjc anemia associated with vitamin B 12 deficiency, it will not stop the progressive neurologic lesions. Thus, pernicious

304

VITAMINS AND VITAMIN-CONTAINING DRUGS COOH H2N--H2 H

Purifle (equivalent)

H2NN-N, NNO (—CH20H)

HO}C0OH CH1

COOH HO Glutamic acid Shikimic acid

1.

H 2 N flCOOH

'OH

2-Amino-4-hydryPteridifl' 6carboxaldehyde (or 6-hydroxymethyl)

p-Arninobenzoic acid

COOH r 'N1

OH

"----co-----NH-----cH -NH - / C. OOH Folic acid

Fig. 11-4. Bie;ynth'sis of toiic acid.

anemia should be excluded before more than 0.4 mg per day of folic acid is taken; this precaution will avoid masking a key hematologic indication of pernicious anemia. Folic acid is generally considered to he free of other serious adverse reactions, but there is no justification for use of megadoses of this vitamin. The US RDA of folic acid is 0.4 mg for adults and children over 4 years of age, 0.2 mg for children under 4 years of age, 0.1 mg for infants, and 0.8 for pregnant and lactating women. The usual dose of folic acid for treating a deficiency condition is 0.25 to 1.0 mg per day until a hematologic response occurs. The usual doses for dietary supplementation parallel the US RDA values, but these doses may be increased slightly, if necessary. Parenteral (intramuscular, intravenous, or deep subcutaneous) administration is reserved for cases of intestinal malabsorption or other situations in which oral administration is not acceptable.

Folic acid is available as tablet (0.1, 0.4, 0.8, and I mg) and injection (5 mg and 10 mg per ml) dosage forms. It must be packaged in well-closed, light-resistant containers. Most folic acid products are available by prescription onl y , but products containing 0.4 mg or less (0.8 mg or less if intended for pregnant and lactating women) may be sold over-the-counter. PRESCRIPTION PRODUCT. Fo1vite. Folinic acid or leucovorin is available as the calcium salt in tablet (5 and 25 mg) and injection (3 and 5 m per ml) dosage forms. It is used as the "folinic acid rescue" antidote in chemotherapeutic regimens employing a potent folate antagonist such as methotrexate. PRESCRIPTION PRODUCT. Welicovorin®. Vitamin 812

Vitamin 812 and cobalamins are terms that refer to a series of porphyrin-related

VITAMINS AND VITAMIN-CONTAINING DRUGS

corrinoid derivatives that function as extrinsic factors to prevent pernicious anemia. These macromolecules contain cobalt, and the corrin nucleus is derived biosynthetically via a pathway that is similar to that for the porphyrin nucleus of heme. Cyanocobalamin, a red crystalline material, is the most stable of the cobalamins; consequently, it is the form of vitamin B2 most frequently utilized in therapy. Hydroxocobalamin also finds some therapeutic use; in it the c y ano group is replaced with a hydroxyl substituent. The physiologically active in vivo forms of the vitamin are 5'-deox y adenos y lcobalarnin (coenzyme B 2 ) and methylcobalarnin Cy anocobalamin has been synthesized in a monumental chemical effort, but coinmercial supplies of the vitamin are obtained semisvnthetically using microorganisms, especially S(repfonnices griseus. The total cohalamin fraction obtained by fermentation is readily converted to cyanocobalamin by controlled treatment with cyanide. Cvanocohajamjn can be converted, if desired, to hydroxocohalamin. Vitamin B2 is stored in the liver (the liver half-life is estimated to be 400 days), and liver extract has been the classic source of this vitamin. Meat, seafood, eggs, dairy products, and fermented foods, such as soy sauce, are good dietary sources of the vitamin. Because vegetables are a poor dietary source of vitamin 1312, deficiency conditions have been associated with some H N H2COCH2 cH,

strict vegetarian diets (no soy sauce or other fermented foods). Vitamin B 1 , is absorbed in the lower half of the ileum. Two distinct mechanisms of absorption exist for this vitamin. A diffusion-type mechanism is operative when the amount of vitamin is large, but an active absorption process involving a glycoprotein (intrinsic factor) is more significant. Intrinsic factor is secreted by the parietal cells of the gastric mucosa. A vitamin B 1 intrinsic factor complex is formed in the stomach and passes into the intestine where it binds to receptor sites on the ileal mucosa...Calciuni ion appears to be involved in breaking the vitamin-intrinsic factor complex at the receptor site. The absorbed vitamin is then transported in the plasma bound to transcobalamin I or transcobalamin II. Although the normal excretion process is hiliary, with 65 to 75% reabsorption, nutritionally excessive quantities of cobalamins are excreted renlly, The cobalarnins are essential to cell reproduction, growth, hematopoiesis, and synthesis of myelin and nucleoprotein. These cofactors participate in folate rec y -cling,pdmetabos hy]in reactions. The adenos ylcobalamin coenzyme is involved in a number of rearrangement reactions mediated by mutase and dehydrase enzymes; methylcobalamin is essential for the methyltransferase enzyme that catalyzes tetrahydrofo.latc regeneration-

N N /

am --

0-

NH2COCH A I CH1 _-2 CH3 H2CHCONH E I;1 CH 2CH 200NHCH, C-

305

-I

CH 3 0 Cyanocobalamin

306

VITAMINS AND VITAMIN-CONTAINING DRUGS

Deficiency of vitamin B12 is usually associated with intestinal malabsorption or increased requirements for the vitamin in certain iatrogefliC situations or in some pathologic states. Nutritional deficiency is rare, developing very slowly as a result of high liver storage and efficient enterohepatic recycling. Symptoms of vitamin B12 deficiency usually involve rapidly dividing cells of the hematopoietic system (e.g., megaloblastic anemia) and irreversible neurologic damage (e.g., defective myelin nerve sheaths); they include irritability, weakness, memory loss, mood swings, and a sensation of tingling oc numbness of the arms and legs. A number of drugs, including the aminoglycoside antibiotics, piirunosahcylic acid, many anticonvulsarits, ilolestyramine, and colchicine, interfere vith intestinal absorption of the cobalamins. These medications may require diota2y supplementation with the vitamin. The need for cobalamin supplementation may also be associated with a number of chronic clinical conditions, including hyperthyroidism, stress, malignancy of the pancreas, hepatic-bilary disease, and diseases of the small intestine. A deficiency in transcobalamin II and a failure to synthesize one or both of the active coenzyme forms of the vitamin are recognized as rare inborn errors of metabolism. The US RDA of vitamin B 12 is 6 p.g for adults and children over 4 years of age, 3 p..g for children under 4 years of age, 2 g for infants, and 8 vg for pregnant and lactating women. Oral cyanocobalamin (I to 25 pg per day for adults) is taken for nutritional supplementation when intestinal absorption is normal. intramuscular or subcutaneous administration of cyanocobalamin or hydroxocobalamiri is employed in cases of intestinal malabsorption and in the treatment of pernicious anemia. The initial parenteral dosage regimen is 30 to 100 p.g of either cy anocobalamin or hydroxocobalamin per da y for 5 to 10 days, followed by a main-

tenance dose of 100 to 200 pg once a month for life. Serious adverse reactions are uncommon with the therapeutic use of vitamin B 12 . Hypokalemia can occur when megaloblastic anemia is converted to normal eiythropoiesis under the influence of the vitamin. Patients with hereditary optic atrophy (Leber's disease) suffer an accelerated rate of atrophy when treated with cyanocobalamin. Hydroxocobalamin is the preferred therapeutic agent in the latter situation, but some patients develop antibodies to the hydroxocoba]amin-transcobalamin II complex. Hydroxocobalamin has also been used to prevent and treat cyanide toxicity associated with sodium nitroprusside. The cobalamins have no proven efficacy in the treatment of a number of conditions, including acute viral hepatitis, aging, mental disorders, multiple sclerosis, sterility, tngeminal neuralgia, and other rleuropathies. Cyanocobalarnin is available as tablet (10 p.g to I mg) and injection (30 rig, 100 g. and 1 mg per ml) dosage forms. It must be packaged and stored in light-resistant containers. PROPRIETARY AND PRESCRIPTION PROD-

UCTS. Bay Bee-12 1 , Berubigen', Betalin 12, Cabadon-M 5 , Cobex®, Crystimin1000 ®, Cyanoject ® , Cyomin ® , Kavbovite lOUD®, Pernavit ® , Redisol ®, Rubesol lOUD®, Rubramin PC® , Sytobex® , and Vibal®. Hydroxocobalamin is available only as an injection dosage form (100 ig and 1 mg per ml). It also must be packaged and stored in light-resistant containers. PRESCRIPTION PRODUCTS. Alphamirt®, AlphaRedisol®, Codroxomin ® , Droxom i n®, Hybalamin®, Flydrobexan®, Hydro-Cobex® , Hydroxo-12 19, LA-12® , and Vibal LA®. Although products containing cr ystalline cvanocobalamin are preferred for treating vitamin 13 12 deficiency, preparations containing a partially purified liver extract are available for oral and parenteral use.

VITAMINS AND VITAMIN-CONTAINING DRUGS

The liver extract contains a number of vitamins, including folic and folinic acids; its potency, however, is expressed on the basis of vitamin B 12 activity. An oral product (Biopar Forte®) also contains intrinsic factor concentrate, a concentrate prepared from the mucosal glands of porcine stomach walls. Parenteral products are usually fortified with crystalline cyanocobalamin plus either thiamine (Reticulogen ® and Reticulogen Fortified® ) or folic acid (CrystiLiver®, Folabee® , Fol-Li-Bee Forte ®, Hepfomin ® , Hyliver Plus ® , Lifolbex Improved®, Li folex ®, Lifomin ®, Livroben®, Sanguis ®, and Vifex®). Parenteral cobalamins are preferred for the treatment of pernicious anemia, but a number of oral products containing cyanocohalamin and intrinsic factor concentrate, as well as iron and other vitamins (ascorbic acid, folic acid, niacin, riboflavin, and thiamine), are still available for dietary supplementation. Such products include Chromagen ® , Contrin® , Eviron ®, Ferotrinsic ® , Heptuna Plus®, Lextron® , Omnihemin® , Perihemin® , Pronemia® , Reticulex® , TriHemic 600®, h-insicon®, and TnTinic®. Cyanocobalamin Co 57 and cyanocobalamin Co 60 are substances that contain radioactive cobalt in their molecular structures. ihe specific activity of these materials is not less than 0.5 Ci per vLg of cy anocobalamm. They are used as diagnostic aids in determining pernicious anemia. Absorption of the radioactive c-yanocohalamin following oral ingestion is less than normal in patients with pernicious anemia. However, it may be normalized by concurrent administration of intrinsic factor, thus differentiating pernicious anemia from other causes of cyanocobalamin malabsorption. Cyanocobalamin Co 57 and canocobalarnin Co 60 are available as capsules and oral solutions. The usual test dose is 0.5 to 1 jCi at least 24 hours after ingestion of any other cobalamin-containing preparation. When calculating dosage, a correction

307

should be made for radioactive decay because the radioactive half-life of 57Co is 270 days and that of 6OCo is 5.27 years. The expiration date of these products is not later than 6 months after the date of standardization. PRESCRIPTION PRODUCTS. Rubratope-571-' and Rubratope-60®. Dried Yeast

Yeasts are unicellular organisms that are well-known for their ability to metabolize sugar into alcohol and carbon dioxide. They have a long history of use in the baking and fermentation industries. Yeast cells contain a number of enzymes, other proteins, and B vitamins, so dried yeast was used medicinally at an early date as a source of the vitamin B complex. Pure vitamin substances are normally used at the present time although dried yeast is still used on occasion, especially by individuals with a strong preference for natural products. Brewer's yeast, Sacchnroinyces cerez'isiae Hansen (Farn. Saccharomycetaceae), or torula yeast, Candida uti/is (Henneberg) Loder and Kreger-Van RiJ (Earn. Crvptococcaceae), is grown in suitable nutrient media in large fermentation tanks, and the cell mass is recovered. If the cell mass is a by-product of beer production, the cells must be washed thoroughly, usuall y using one or more alkaline solutions, to remove the insoluble acidic bitter resins from the hops. The yeast cells are dried and marketed as a granular powder or compressed tablets. Representative dried yeast contains approximately 45% protein and, in each gram, 120 g of thiamine, 40 jig of riboflavin, and 300 jig of niacin, plus detectable quantities of various other vitamins. When dried yeast is used as a dietary supplement, the usual dose is 10 g, 4 times a day. Vitamin C Vitamin C or L-ascorbic acid is a naturally occurring vitamin substance that pre-

308

VITAMINS AND VITAMIN-CONTAINING DRUGS

vents scurvy and has useful antioxidant properties. It occurs in equilibrium with dehydro-L-ascorbic acid, an oxidized form, which also has antiscorbutic properties. Vitamin C is the least stable of all the vitamins. Dehydro-L-ascorbic acid is irreversibly oxidized to the inactive 2,3-diketoL-gulOruic acid; alkaline conditions enhance this oxidation. HO. / CH 2 OH HO,,CH2OH

HO'OH 1-Ascorbic Acid

Dehydro-L-Ascorbic Acid

L-Ascorbic acid is derived biosynthetically from 1)-glucose .. D-galactose, or other sugars with the correct chiral centers at C2 and C-3 to yield the appropriate stereochemistry at C-4 and C-5 of ascorbic acid. L-AScorbic acid can he isolated, using appropriate precautions to prevent oxidative inactivation of the molecule, from citrus fruits, rose hips, or other natural sources. Nevertheless, most commercial supplies of this vitamin are obtained b y chemical synthesis starting with n-glucose. The physiologic properties of naturally occurring and synthetically prepared L-ascorhic acid are indistinguishable. Exogenous vitamin C is required for primates (including humans), guinea pigs, fruit bats, some birds, and certain fish that lack the ability to form the material biosynthetically. This vitamin is essential for proper formation of collagen and other intercellular materials in tissues, especially in bones, skin, and teeth; it has been implicated in the hydroxylation of such collagen components as proline and lysine. It also appears to be associated with hydroxylases that act on cholesterol and tyrosine to produce corticosteroids and catecholamines, respectively. Vitamin C likewise is involved in biologic oxidation-reduction reactions (hydrogen and electron transport). Good dietary sources of ascorbic acid in-

dude citrus fruits, tomatoes, strawberries, and other fresh fruits and vegetables. Although the vitamin content is preserved on freezing, up to 50% of the vitamin C content is lost upon cooking. Intestinal absorption involves a Na t -dependent transport mechanism; the maximum intestinal absorption capacity is about 1200 mg per 24 hours. A normal healthy adult has an ascorbate body pool in excess of 1500 mg. l'his pool must drop below 300 mg before clinical signs of scurvy appear, a depletion process that usually takes several months with a vitamin C—deficient diet. Deficiency symptoms include fatigue, muscular pain, increased susceptibility to infection and stress, skin lesions, bleeding gums, and other signs of collagen degeneration. The US RDA for ascorbic acid is 60 mg for adults and children over 4 years of age, 40 mg for children under 4 years of age, and 35 mg for infants. It is estimated that pregnant and lactating women may require 100 mg per day. Medications such as barbiturates, salicylates, and tetracycline may increase the requirements for this vitamin. Patients who smoke or who have gastrointestinal disease, cancer, peptic ulcer, hyperthyroidism, stress, or severe burns also may require increased vitamin C supplementation. The need for enhanced dietary supplementation is usually related to increased metabolic turnover or to reduced efficiency of intestinal absorption; for example, smokers have a daily turnover of approximatel y 100 mg (60 mg is normal) and intestinal absorption in the 63 to 80% range (78 to 88% of ingested ascorbic acid is normal). Ascorbic acid is used to prevent or treat deficiency conditions and to enhance wound healing. High doses of the vitamin have been used as a urinary acidifier to enhance the effectiveness of methenamine, and it has been used with deferoxamine in iron toxicities to increase the excretion of the iron. Ascorbic acid is predominantly metabolized to carbon dioxide and oxalic acid prior to renal elimination, except upon

VITAMINS AND VITAMIN-CONTAINING DRUGS

ingestion of high doses. The vitamin is generally considered free of serious adverse reactions or toxicities. When problems are encountered, they usually involve high doses and relate to undesirable urinary acidification or interference with diagnostic tests (e.g., serum lactic dehydrogenase and transaminase, occult blood in the stools, and urinary glucose). The usual doses of ascorbic acid for dietary supplementation are 50 to 100 Tog per day for adults and 20 to 50 mg per day for infants. For treatment of a deficiency, the usual doses are 100 to 250 mg, 1 to times a day for adults and 100 to 300 mg per day in divided doses for infants. The usual dosage regimen to enhance wound healing is 300 to 500 mg daily for 7 to 10 days. Doses of 4 to 12 g per day, in divided doses every 4 hours, are used in adults for urinary acidification. A potential role for vitamin C in prevention and treatment of cancer has been suggested, but efficacy has not been proved. Claims for the benefit of megadoses of vitamin C for the amelioration or prevention of the common cold, for the prevention of vascular thrombosis, and for the treatment of atherosclerosis, hypercholesterolemia, infertility, mental depression, and ulcers remain unsubstantiated. Ascorbic acid is available in tablet (25 mg to I g), chewable tablet (100 mg to I g), effervescent tablet (1 g), extended-release tablet (250 mg to I g), extended-release capsule (250 and 500 mg), oral solution (35 and 100 mg per ml), syrup (20 and 100 mg per ml), and injection (50 to 500 mg per ml) dosage forms. Calcium ascorbate and sodium ascorbate are available as sugar-free powders and in tablet (equivalent to 500 mg of ascorbic acid) and injection (calcium salt: equivalent to 100 mg-Per ml; sodium salt: equivalent to 200, 250, and 500 mg per ml) dosage forms. PROPRIETARY AND PRESCRIPTION PROD-

UCTS. Arco—Cee ® , Ascorbicap ® , C-Caps 5000 , C-Span ® , Ce-Vi-Sol® , Cecon® , Ceehater, Cemjll® , Cenotate ® . Cetarie® , Ce-

309

valin® , CeviBid® , DullC ®, Flavorcee', and Vita-C®. Biotin

Biotin, D-biotin, or vitamin H is a physiologically active substance that Consists of fused imidazolidone and tetrahydrothiophene rings and a valeric acid side chain. Egg yolk, liver, cereal grains, and milk are good dietary sources of biotin, but commercial supplies are prepared by chemical synthesis. H  -NH H (C H 2 )COO H Biotin

Even though biotin is produced by th. intestinal microflora, evidence of the nutritional significance of enterically produced biotin is equivocal. It is known that biotin deficiency is rarely encountered and that biotin excreted in the feces is 2 to 5 times greater than the dietary intake. When biotin deficiency is encountered, it is usually caused by avidin, a glycoproteft' in raw egg whites that forms a nonabsorh able avidin-biotin complex, or by inborn metabolic errors. This vitamin functions as a carboxyl-carrying cofactor in several carboxylase and decarboxylase enzyme systems. Propionic acidemia, lactic acidosis, and 3-methyicrotonyl glycinuria encountered in some infants represent inborn errors in the metabolism of propionyl-CoA carboxylase, pyruvate carboxylase, and 3-meth y lcrotonv]-CoA carboxylase, all of which are biotin-dependent carboxylases. Symptoms of biotin deficiency include alopecia, erythroderma desquamativa (Leincr's disease), and seborrheic dermatitis. Circumstantial evidence has linked biotin deficiency with the sudden infant death syndrome, but conclusive documentation is lacking. The US RDA for biotin is 300 pg for

310

VITAMINS AND"^^^^^ ""' DRUGS

adults and children over 4 years of age, 150 hg for children under 4 years of age, and 50 ig for infants. Biotin is generally considered to be well tolerated by humans and free of side effects, even at high doses. It is used to prevent or treat deficiency conditions and is available as an ingredient (usually 30 to 300 p.g) in some multiple vitamin products.

VITAMIN-RELATED FACTORS Para-aminobenzoic Acid

Para-aminobenzoic acid or PABA, a precursor of folic acid, occurs in nature. Although sometimes grouped with the vitamin B complex, it is not an essential nutrient for humans and should not he conidered a vitamin. It is added as an accessory food factor (usually 15 to 100 mg) to a few multiple vitamin and mineral products. Because PABA may increase salicylate serum levels when taken concomitantly, it is sometimes used (500 mg, 4 to 6 times a da y) for this purpose in rheumatic fever and other conditions benefiting from salicylate therapy. PABA interferes with the antibacterial action of sulfonamides, and they should not he taken concurrently. PABA is also used, either as the acid or various esters, as an ultraviolet sunscreen in a number of topical preparations. Choline

Choline or 3hydroxyethyltrimethylafl1monium hydroxide is a component of lecithin, a phospholipid, and a precursor of acetylcholine, a neurotransmitter. Choline is widely distributed in nature, occurring in egg yolk, animal organs (brains, heart, liver, and thymus), fish, milk, cereal grains, fruits, and root vegetables. It is known to be an essential nutrient in certain higher animals, but no proof exists for a dietary need in humans. Thus, it is technically not a vitamin, even though it is frequently associated with them.

[cH33__cH_cHioHi OHO

choline is available as the bitartratc, chloride, and dihydrogen citrate salts. As such, It is added (20 to 240 mg) to a number of multiple vitamin products. Choline has been associated, at least by implication, with fatty infiltration of the liver; it is sometimes used (usually 250 mg to I g per day) as a lipotropic agent in conditions such as liver cirrhosis, Huntington's chorea, presenile dementia, and tardive dyskinesia. However, its beneficial effect as a lipotropic agent has not been unequivocally established. Inositol Irmositol, meso-iriositol, mvoinositol, or cis-1,2,3,5-trans-4,6-cyclohexaneheX Ol is a sweet-tasting, optically inactive substance that is sometimes associated with the B vitamins. However, because dietar y deliciencv is unknown in humans, it is technically not a vitamin. Sources of inositol include endogenous synthesis and the normal American diet, which is estimated to provide 900 mg per day of exogenous inositol. It is distributed ubiquitously in food substances; particularly rich dietary sources are legumes, cereal grains, nuts, and animal organs, including liver and kidney. Inositol occurs free and as a component of phospholipids in animals but is bound to phosphate as phytate in plants. OH H OH HHH OH H lnositol

The biologic function of inositol appears to be related to its role as a phospholipid component of membranes and lipoproteins. It has been implicated in membrane

VITAMINS AND VITAMIN-CONTAINING DRUGS

transport of amino acids and potassium and sodium ions. Inositol is added 5 to 250 mg) to a numberof multiple Vitamin products. It is also sometimes used (1 to 3 g daily in divided doses) as an unproven lipotropic agent. The compound is metabolized to glucose in the kidney. Although inositol is free of serious adverse reactions, large doses may cause moderate diarrhea. MULTIVITAMIN THERAPY

Diets deficient in one vitamin are likely to be deficient in several, and conditions that hinder the absorption of one vitamin may likewise interfere with the absorption of others. Inadequate vitamin intake can result not only from a poor diet but also from alcoholism, increased needs during pregnancy and lactation, prolonged broadspectrum antibiotic therap y , and the course of parenteral nutrition. Poor absorption of ingested vitamins occurs frequently in elderly persons, chronically ill persons, and others who suffer from infections, reduced bile flow, intestinal disease, diarrhea, arid the like. For these reasons, the supplementation of diets with multivitamin preparations does have a rational basis in certain circumstances. Decavitamin capsules and decavitamin tablets serve as model multivitamin preparations, providing 10 vitamins for which recommended dietary allowances (RDA) have been established and one vitamin for which there is no such recommendation. Each capsule or tablet contains the labeled amounts of vitamins A, B, B 21 B6, 131 ,, C, D, E, calcium pantothente, folic add, and niacinarnide. The usual dose is I capsule or tablet daily. PROPRIETARY PRODUCTS. (Some of these ma y differ slightly from the composition of Decavitarnin) Dava1ets, Multicehrin, Onc-ADav 1 , Sigtabv , Tht'racebrin ® , Theragran", Unicap, Vigran ®, and Zymacap. Hexavitamin capsules and hexavitamin

311

tablets each Contain the following specified amounts of 6 vitamins: vitamin A, 1.5 mg; 131 , 2 mg; 132 , 3 mg; C, 75 mg; D, 10 g; niacinamide, 20 mg. PROPRIETARY PRODUCT. Hepicebrin®. A wide variety of other combination proprietary products is available. Such preparations may be grouped, for purposes of summation, into the vitamin B complex (Betalin Compound®, Becotin, Lederplex®, and Surbex® ), the vitamin B complex with C (Ailbee with C ®, Cebenase®, and Stresscaps® ), multivitamins with iron (Dayalets Plus Iron ® , Poly-Vi-Sol with Iron® , LJriicap Plus Iron ®, and Vi-Daylin Plus Tron®), multivitamins with calcium and iron (EnCebrin ® , Natabec® , Natafort® , and Natalins), and multiple vitamins with iron and other minerals (Eldec®, MiCebrin ®, Myadec®, and TheragranM®). READING REFERENCES

Baker, B.M., and Bender, DA.: Vitamins in Medicine, 4th ed., V ols. 1-2, London, Heinemann Medical Books Ltd., 1980-1982. Botez, Mi., and Re ynolds, E.H,, eds.: Folio A cid in Neurology, Pschiotnj, and l,t,'rn/ Medicine. New York, Raven Press, 1979. Dolphin, Li, ed.: B, New York, John Wiley & Sons, Inc., 1982. Ever®, J .. Anderson, B., and Y ou, K.-B., eds.: The Piridjue Nucleotide Coenzymes, New York, Academic Press, Inc., 1982. Goodhart, R.5,, and Shils, ME., eds.: Modern Nutrition in Health and Disease, 5th ed., Philadelphia, Lea & Febiger, 1980. Kumar, R., ed.: Vitanoo D, The Hague, M. Nijoff Publishing, 1984. Lubin, B., and MachIm, B., eds.: Vitamin F: 8ioclwrr run, Heron! okegical. and Clinical Aspects, New York, New York Academy of Sciences, 1982. Machim, M. ed.: Handbook of Vitamins, New York, Marcel Dekker, inc., 1984. Nohite, S., and Woodhiti, J.M.: Vitamin C, Hingham, Massachusetts, MTI' Press, 1981. Sable, HZ., and Guhier, CT, eds.: Thiamin: Twent:j Y ears of Progress. New York, New York Academy of Sciences, 1982. Silverman, H.M., Romano, J.A., and Elmer, C.: The Vitamin Book, New York, Bantam Books, 1985. Sporn, M.B., Roberts. A.B., and Goodman, D.S., eds. The Retinoids, Vols. 1-2, Orlando, Academic Press, Inc., 1984.

Try fiates, G.P.. ed.: Vitamin B,— Metabolism and Role iii Growth, Westport, Connecticut, Food & Nutrition Pre5s, Inc., 18O.

12 Antibiotics Antibiotics probably represent the greatest single contribution of drug therapy in the past half-century, a period characterized by unprecedented advancements in health care, This group of drugs provides 'lfective control of many human microbial pathogens that previously caused prolonged incapacitation or death without appreciable regard for age, economic status, or physical fitness, The word "antibiotic" is derived from the term antibiosis, which literally means "against life" (anti—against, bios— life). A measure of the significant and spectacular contribution of antibiotics to therapy is indicated b y the common inclusion of the word in the layman's vocabulary. Most people have an accurate, or at least a functional, general concept of the word, but workers intimately involved in the antibiotic field find considerable difficulty in drafting a precise definition. The varied scientific concepts of this word reflect the viewpoints of scientific specialists, a rapidly expanding field of knowledge about all aspects of antibiotics and their applications, and such factors as a recognition of the lack of definitive separation for conditions previously considered etiologically distinct (e.g., certain neoplastic conditions and viral infections). The most widely accepted concept defines an antibiotic as a chemical substance produced by a microorganism that has the capacity , in low concentration, to inhibit

selectively or even to destroy bacteria and other microorganisms through an antimetabohc mechanism. Essentially all definitions limit antibiotics to biologic constituents that exert their action in low concentrations. This definition excludes microbial metabolites, such as ethanol, that are active against protoplasmic functions at higher concentrations. The definition of the term may be expanded by including higher plants as a source and tumors as a site of action. The concept of antibiotics as used by health-care professionals, exclusive of some individuals practicing in experimental clinics and hospitals, is limited for practical purposes to commercially available substances. Fortunately, this reduces confusion resulting from special research objectives or "antibiotics" that are too toxic for feasible therapy. A logical case can be made for including the antiplasmodial activity of quinine under an antibiotic designation, but the arbitrary exclusion of quinine from most antibiotic concepts has caused little confusion for the practitioner. The selective action of some naturally occurring compounds on the abnormal metabolism and cells of neoplasms may create greater problems in the future. Practitioners must maintain a flexible approach toward the scope of antibiotics to accommodate the applications of scientific advances. DEVELOPMENTAL HISTORY

The history and development of antibiotics as therapeutic agents are similar to

312

ANTIBOTIC5 the patterns noted for other t y pes of drugs. Relatively ineffective attempts to use materials that are now recognized as having antibiotic associations can be detected in folk medicine and in prepenicillin scientific literature. Development in the antibiotic field since 1940 is characterized by a practical blending of empiric observation and increasingly sophisticated manipulations of biologic and chemical factors. This familiar pattern is frequently overlooked because an aura of 20th-century miracle drugs has surrounded the antibiotics. Reports, some dating back 2500 years, indicate that various ancient and primitive peoples applied moldy bread, soybean curds, and other materials to boils and wounds liable to infection; this can be considered a folk-medicine type of antibiotic therapy. Pasteur demonstrated bacterial antagonism shortly after he established the bacterial etiology of infectious disease. During the 1880s, attempts were made to utilize antagonism to achieve an ecologic control of the human microbial flora by introducing selected nonpathogenic organisms. Pyocyanase, a crude mixture of metabolites extracted from Pseudomonas aeruginosa, became available around the turn of the century and could he considered the first commercial antibiotic. Pyocyanase, at best, was a poor antibiotic by modern standards, but its failure to achieve wide acceptance as a therapeutic agent can be related, in part, to the variable composition of the crude mixture and the resultant lack of reproducible or predictable therapeutic responses. Establishment of the therapeutic feasibility of penicillin antagonism in the early 1940s stimulated the intensive efforts that have culminated in the high level of current antibiotic development. Numerous approaches to the production and use of antibiotics have been used concurrently in the past, and practical considerations of biologic, chemical, and economic factors will undoubtedly dictate a similar situation in the predictable future.

313

The progressive trend in the logistic aspects of antibiotic development can be illustrated by the following sequence of objectives: (1) Screen diverse sources of microorganisms for detection of useful antagonism. (2) Select improved microbial mutants, determine optimal environmental and nutritional conditions, and develop suitable procedures for recovering antibiotics from cultures. (3) Direct or induce the formation of specific, desired metabolites. (4) Modify the fermentative metabolites by biologic or chemical manipulations to yield more useful antibiotic substances. (5) Develop procedures for total synthesis of antibiotics for possible economic advantage. (6) Use an adjunct agent to modify the availability or i mpact of an antibiotic. Initially, antibiotic therapy was commonly employed in a wide range of microbial infections with only limited logic or design. However, with the accumulation of experience and the availability of a greater variety of antibiotics, the trend has moved toward a more precise diagnosis of the pathologic organism, including a consideration of nsitivity variations with certain pathogen, and a more conservative use of these valuable therapeutic agents. Production of Commercial quantities of the various antibiotics involves many different approaches and procedures to accommodate the individual biologic idiosyncrasies of the producing organisms and the chemical characteristics of the individual antibiotics. A detailed consideration of antibiotic production is obvioü1y a subject for specialized study: Forti.nately, the health-science practitioner O$ly needs a general knowledge of the proluction procedures and of the significance of key manipulations. This background provides a basis for understanding the scientific limits and economic com.ppnents of these therapeutic agents and for comprehending readily the types of research developments that will lead to future. advances and change. SCREENING FOR ANT(BIOTrCS

In searching for new antibiotics, relatively simple and rapidmethds have been

ANTIBIOTICS

314

developed for screening microorganisms for antibiotic-producing ability. Soil samples are commonly employed in the screen because the y are a rich source of antibioticproducing organisms (Fig. 12-1). Most of these organisms are members of a group of branching, procaryotic microorganisms that occupy a position in their morphologic characteristics between fungi and bacteria. They are placed in the taxonomic order Actinomycetales and are given the common name actinomycetes. A compilation of the microbial sources of antibiotics discovered in the United States and Japan between 1953 and 1970 reveals that approximately 85% are produced by aclinomycetes, 11% by fungi, and 4% by bacteria. These facts do not detract from the significance of antibiotics from other organisms and sources, hut they do suggest a greater probability for the discovery of new useful antibiotics from soil microorganisms. The antibiotics currently used in therapy are produced by surprisingly few groups of ditantiv related organisms. The important genera and their taxonomic relations are as follows:

Phylum Schizomycophyta Class SchizomyeteS Order Eubaceria1eS (bacteria) Family Bcillaceae

Fig 2— COUTtCSV oi

iiI san- ke Lilly Ca.)

Genus Bacillus Order Actinomycetales (actinomvcetes) Family Streptomycetaceae Genus Micro uonos porn Genus Streptomces Phylum Eumycophvta (fungi) Class Ascomycetes Order Aspergillales Family Aspergillaceae Genus I'euicilliuni Form-Class Deuteromycetes (Fungi Imperfecti) Form-Order Moniliales Form-Family Moniliaceae

Form-Genus Cepha/osporium A general method for screening first involves treating the soil sample with chemicals that inhibit the growth of interfering bacteria and fungi but do not affect actinomycetes. Cycloheximide is an antifungal antibiotic often emplo yed for this purpose, and a 1:140 dilution ot phenol is used as an antibacterial agent. Varying dilutions of the treated soil sample are streaked on agar plates containing medium that supports the growth of actinomvcetes. After incubation for 3 to 7 days at 25 to 30° C, the plates are examined for characteristic colonies of actinomycetes These colonies are

various pal k or th :% oi id arct ar p!o 'hiwing coIoiies o soil flora. IPhota

ANTIBIOTICS

then selectively transferred onto fresh medium. Giant colonies of the selected organisms are grown, and plugs are cut from the colonies that include not onl y the organism but also the underlying agar. If the organism produces an antibiotic, it should diffuse into the agar medium. The plugs are placed on an agar plate that has been seeded with a test organism that gives an indication of the potential usefulness of the antibiotic. For example, activity against gram-positive bacteria can be determined with Staphylococcus aureus or Bacillus subthis, activity against gram-negative bacteria with Lsciwricl, ha coli or Salmonella ti/phi, and antifungal activity with Neurospina crassa. The test plates are incubated under conditions appropriate for maximum growth of the test organism, and if after incubation there is a clear zone around the plug of the actmomycete, it can he assthried that an antibiotic in the plug inhibited the growth of the test organism (Fig. 1.2-2). The next step in the screening procedure is to determine whether the chemical substance that produced the inhibition is a new antibiotic or a known compound. A rapid method that has been developed for this determination is termed bioautography. This assay emplo y s paper chromatography or thin-layer chromatography and a biologic assay. An extract containing the newly discovered antibiotic is chromatographed along with reference, known antibiotics using several different solvent systems, Because each antibiotic would possess a characteristic mobility on the chrornatogram in a given solvent system, a comparison of the mobilities of the unknown antibiotic with those of known antibiotics in several solvent systems would indicate whether the newly discovered antibiotic was a known compound. The detection of the antibiotics on the developed chromatogram using chemical detection methods is difficult because the antibiotics are widely diverse chemically; consequently, a biologic method is used to detect the antibiotics. By placing the developed

315

Fig. 12-2. A method for the detection of antibioticproducing organisms: a, giant colonies ,qr()wllg on an agar plate; 1 giiiit colonies with plugs removed; e. plugs from giant colonies showing zones of inhibition clear zone around plug) on test plate seeded with aureiis, indicating antibiotic activity against gram-positive bacteria; d, plugs on test plate seeded with Esciwrieh(a col, showing fewer zones of inhib, hon , indicating little antibiotic activit y against gram-negative bacilli, (Photo courtesy of Eli Lilly &

Co.)

chromatogram on an agar medium that has been seeded with an appropriate test organism, the antibiotics diffuse from the chromatogram into the agar, and after incubation, clear zones on the agar owing to inhibition of growth of the test organism indicate the position of the antibiotics on the chromatogram (Fig. 12-3). After it is established that a microorganism has been isolated that produces a new antibiotic, quantitative assays must be employed to monitor the antibiotic titer through the various processes of production and isolation. The 2 most commonly employed assays, the turhidimetric (tube dilution) assay and the plate (agar diffusion) assa y , require the use of a test organism as in bioautography. In the turhidimetric assay, the test organism is grown in test tubes that contain different concentrations of the antibiotic. There is a direct

316

ANTIBIOTICS

clear zone of growth inhibition around the filter paper disc is related to the concentration of antibiotic (Fig. 12--5). F.

COMMERCIAL PRODUCTION

C

12 toe test organism, the zmi'', Of inhibition iidicate he following antibiotics were separated on the paper chromatogram; U, cephilexin; /', cephatoridiiie; c, desacetvlcepha]othin; . oephalot Nm, (Ihoh, courtes y of Fig.

Eli Lilly & Co.)

relationship of the concentration of antibiotic to the growth of the test organism, and by measuring the growth of the organism, which is indicated by the turbidity of the contents of the test tube, the antibiotic titer can be determined; Clear tubes indicate a higher antibiotic concentration than turbid tubes, and the lowest concentration of antibiotic that completely prevents the appearance of turbidity is known as the minimum inhibitor y concentration (lvHC) (Fig. 12-4). In the plate assay, filter paper discs are impregnated with solutions of antibiotic of varying concentrations, allowed to dry, placed on agar media seeded with an appropriate test organism, and incubated. As the concentration of the antibiotic increases, its diffusion through the agar medium increases; therefore, the size of the

When a new antibiotic has been discovered, investigations into the chemical, physical, and biologic properties of the antibiotic are required before the decision to produce the antibiotic commercially can be made. Two important requirements for production are; (1) the organism must produce the antibiotic in submerged culture as opposed to surface culture, and (2) the organism must excrete the antibiotic into the culture medium. However, some antibiotics, such as those of the polvene group, are retained in the cells of the organism and require special extraction procedures for recovery. These requirements are important considerations in production costs which, in turn, determine whether the antibiotic can compete with other antibiotics for a portion of the market. Other considerations are chemical stabilit y, the minimum inhibitor y concentration against strains of pathogenic organisms, toxic manifestations in mammals, and activity In vivo. The commercial production of antibiotics is an excellent example of the benefits that can be achieved from a multidisciplinary approach to solving a technologic problem. One must be impressed when one thinks, on the one hand, of an obscure microorganism growing in soil and, on the other hand, of the product of that microorganism-----a pure crystalline chemical substance used to save a human life. The transition from one to the other has required the most diligent application of the sciences of microbiology, chemistry, and engineering. Commercial fermentative production of an antibiotic almost alwa y s involves growth of the producing organism in aerated tanks holding thousands of gallons of nutrient medium. Spores or occasionally vegetative growth from a stock culture of

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3l7

Fig. 12-4. Turbidinietric assa y tubes with curve illustrating the relationship of incrcasing prccnt of light transmittance on they axis and increasing concentration of antibiotic on the x axis. (Photo courtesy of Eli Lill y & Co.)

the organism is used to start the fermentation process it is important to maintain stock cultures (e.g., by lyophilizatjcjn) that require transfer as infrequently as possible because repeated transfer may select for those cells of the organism that are poor producers of antibiotic (Fig. 12-6) The several hundred gallons of vegetative growth that are necessary for inoculating the large fermentation tanks are obtained by successively transferring the organism to increasingly larger volumes of nutrient (Fig. 127). The use of a large standard inoculum reduces the incubation time required for production of the antibiotic, lessens the chance for costly contamination by foreign microorganisms, and provides the best possible opportunity for control of subtle environmental and nutritional factors that influence the antibiotic yield. In the production of antibiotics there are

often distinct phases in the fermentative process. These phases can he divided into the growth phase of the organism, which is also termed the trophophase, and the antibiotic production phase, also termed the idiophase. Figure 12-8 illustrates these phases in the course of a typical penicillin fermentation carried out in a culture medium containing glucose and lactose as the sources of carbon nutrition, corn steep liquor for nitrogen sources, and phosphate buffer. During the growth phase, the culture becomes thick owing to the formation of aggregates of fungal cells called mycelium Growth is indicated in the figure by the curve showing an increase in mycelia] nitrogen and lasts from the beginning of the culture period to a pproximately I day later (0 to 24 hours). During the growth phase, glucose rather than lactose is preferentjallv utilized because it can be used

319

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t Fig. 12-5. A microbiologic assay pla e sheng zones of inhibition of var ying size owin g to different concentrations of antibiotic on the filter paper discs. The disc on the right contains the greatest concentration of antibiotic. (Photo courtesy of Eli Lilly & Co.)

directl y as a source of carbon. In the growth process, ammonia is liberated by deaminatiori of amino acids of the corn steep liquor. This liberation raises the pH of the medium to 7, the optimum pH for penicillin stability, and buffers in the medium maintain the ph I close to neutrality. Penicillin production increases rapidly between 24 to 80 hours. At the start of the antibiotic production phase, glucose has been used up, and the fungus then uses lactose for a carbon source. Little additional growth occurs because the lactose cannot be utilized until it is hydrolyzed to glucose and galactose. The decreased availability of a carbon source is thought to be the triggering mechanism for penicillin production.

INCREASING COMMERCIAL VIEW

Considerable effort is devoted to determining the optimal environmental and nutritional conditions for antibiotic production- Optimal conditions for antibiotic formation are frequently quite different from those for maximum vegetative

growth. Factors that are often observed to have qualitative or quantitative importance for antibiotic production include sources of nutritional carbon and nitrogen, ratio of carbon-nitrogen nutrients, mineral composition of medium, incubation tempera-. ture, initial pH and control of pH during the fermentation period, rate and method of aeration, and addition and timing of addition of special growth- and antibioticpromoting substances. Selection of optimal fermentation conditions is usually based on empiric observations, but careful attention to such factors is often critical. For example, some strains of Bacillus subtiiis produce optimal yields of bacitracin when the C/N ratio is about 15; at lower ratios the yield is less, and when the ratio is reduced to approximately 6, licheniformin, a related but commercially undesired antibiotic, is produced. The practical benefit of adding special chemicals to the fermentation cultures has probably achieved only a small fraction of its ultimate potential, but some examples. will show the practical utility of this general approach. It was observed at a fairl y early stage in the development of penicillin production that the addition of phenylacetamide or related compounds to the culture medium had a minor beneficial effect on the y ield of penicillin substances and had a major influence on the composition of the penicillin mixture. The presence of phenylacetic acid derivatives in the nutrient mixture favored the formation of penicillin G; this reduced the problems of using a mixture of unknown or variable composition and the cost of separating the individual antibiotic substances. Use of various acyl moieties to direct the fermentative formation of other penicillins (e.g., penicillin V) achieved limited commercial success, but semisynthetic techniques have superseded this approach to the production of specialized perticillins. The use of mercaptothiazole in cultures of Streptomyces aureofnciens emphasizes that additives can he beneficial without being

Fig. 12-6. in stock culture maintenance, the Ivophilized cultuL-s

ut antibiotic-producing organisms are preserved in small, sealed, glass tubes. The freeze-dried pellets in the small glass tubes will be used to start antibiotic production. (Photo courtesy of Eli Lill y & Cu.)

Stock Culture .1, Liquid Medium Cultures (in one-liter flasks) Several flasks Bazooka (a special device used to transfer aseptically into fermentation tanks) .1 Inoculum Tank (also called Seed Tank or Bump Tank) Production Tank (usually lOx the volume of Inoculum Tank— some tanks are as large 100,000 gallons and six stories high) Fig. 12-7. The scale-up procedure in the commercial production of antibiotics.

incorporated into the antibiotic molecule. Strains of this actinomycete usually produce both chlortetracycline and tetracycline; the proportions depend to some degree on the availability of chloride ion in the culture medium. Tetracycline has the greater therapeutic utility, but the resolution of mixtures of these 2 tetracyclines is costly. Because the organism tends to be a chloride scavenger and because chloride ion is one of the most difficult ions to ex-

dude quantitativel y from water and nutrients, control of the presence of this ion in the nutrient medium to favor the production of tetracy cline is not commercially feasible. However, the addition to the fermentation mixture of mercaptothiazole or any other compound that presumably inhibits chlorination favors tetracycline production. Some additives may increase antibiotic production through an enzyme induction effect. For example, the addition of methjonine to a cephalosporin C fermentation during the trophophase stimulates the production of the antibiotic. Because methionine does not serve as a biosynthetic precursor to the antibiotic, as compared to the role of phenylacetic acid in penicillin G biosynthesis, it is assumed that methionine stimulates the production of the cephalosporin C biosynthetic enzymes. Conversely, it has been demonstrated that in penicillin fermentation, lysine in the culture medium inhibits antibiotic production. Penicillin and lysine are end products of a branched biosynthetic pathway in which a-aminoadipic acid is a common

320

I C Z

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4

8-1600 pH

6-1200

L I ,.

renIcIiIt/

2}-

---.-

C - =

,

-'-....  ,Sugar

4-800

0 C 5' I - 0 I I

LMyceiiaiN/ _—

2 —I 400

IF- /E...

N H3N

-- -

20 0 40 60 80 Time, hours

I 10 100

Fig. 12-8. Chemical changes, myceliel growth, and penicillin production in a typical penicillin fermentation. (Reprinted with permission from Brown and Petersen, 1950. Industrial and Engineering Chemistry, 42:17691774. Copyright by the American Chemical Society.)

precursor. Lysine production is regulated either by inhibition or repression of the enzymes required for the production of u-aminoadipic acid, which ultimately results in a decrease in penicillin formation. sugar

-.penicillin a-pinoadipic aci NH,'  lysine 1' I Feedback Inhibition or - I Repression

Another important approach to increasing the yield of antibiotic is mutation and Strain selection. Mutation induced by exposing the parent strain to ultraviolet light, x rays, or various mutagenic chemicals, such as nitrogen mustards and analogs of purines and pyrirnidiries, is the major approach for selecting improved strains, but a search of natural sources for new wildtype or different species that produce the antibiotic in higher yield than the original producing organism is also employed. In the case of induced mutations, lethal levels of the mutagen are adjusted so that approximately 90 to 99% of the cells of the organism are killed. Mutants that produce

a higher yield of antibiotic are selected from the surviving cells. Penicillin production offers a good illustration of the potential success of these approaches. Penicillin antagonism was observed originally with a culture of Penicillium iwtutum Westling, which produced in surface culture 4 mg of penicillin per liter of culture medium. No mutants of P. notaturn were found in the early selection process that would give a satisfactory yield of penicillin in submerged fermentation; however, in 1944, through natural selection, a strain of P. chrysagenum Thom was discovered that yielded penicillin in the amount of 40 mg per liter. Subsequently, b y utilizing procedures of mutation and strain selection, the yield has been increased to 21,000 mg per liter, RECOVERY AND ISOLATION

Most of the commercially important antibiotics are excreted readily into the nutrient medium where they accumulate. In cases such as certain of the peptide antibiotics, in which the antibiotic is retained

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endollularly until the cells reach an advanced physiologic age, the ferrnerttation periof is terminated when most of the cell membranes have undergone lysis or have lost their selective retention property. Thus, isolation of antibiotic substances is basically recovery from the culture broth. ThfundamentaI approaches that are usually considered are selective precipitation, selective adsorption, or selective extraction with an immiscible solvent. The chemical characteristics of various antibiotics and their accompanying metabolites govern the manipuJtions that will be effective in any given situation. Ideally, the initial isolation procedure should he as efficient and selective as possible to give the best yield and to facilitate subsequent purification, but economic considerations commonly dictate a compromise procedure. Precipitation is thoretica1ly one of the best ways to recover a substance from a large volume of an aqueous mixture, but this approach has not proved commercially satisfactory for any of the therapeutically important antibiotics. The most nearly feasible application of this approach probably involves polymyxin. Polymyxin forms an insoluble helianthate complex when helianthine (methyl orange) is added to the culture broth, but this antibiotic can be recovered more economically by using an adsorption procedure. Lack of selectivity and recoverability from the precipitated complex is the most commonly cited technical disadvantage to the practical utility of this general method. Liquid-liquid extraction using some water-immiscible organic solvent is the approach utilized for most antibiotics. This procedure lacks a high degree of selectivity with most solvents that are sufficiently inexpensive to be employed on a commercial scale. It is also relatively inefficient because antibiotic substances tend to be fairly polar molecules. However, the economic advantage of easy adaptation to a chemical engineering flow process more than offsets these limitations in most cases unless the

321

antibiotic molecule is so polar that the partition coefficient favors the aqueous phase. Highly polar antibiotics, such as neomycin and other arninog]ycoside antibiotics, are usually recovered from the culture broth by adsorption on some suitable adsorbent. Many adsorbents remove antibiotics of this type from culture broths with varying degrees of selectivity. The major limitation to sel€ct'ing adsorbents is the need to recover the antibiotics by reversing the adsorption process without using extreme conditions that would be destructive, Use of charcoal of controlled activity grades and elution of the antibiotic with dilute acid is a typical example of this isolation approach. Once the crude antibiotic has been recovered from the nutrient broth, it is subjected to chromatography, recrystallization, or other standard manipulations to effect all appropriate degree of purification. It should he noted that attainment of chemical purity is usually considered impractical and unnecessary for therapeutic purposes. Extraneous metabolites, such as foreign proteins that cause undesirable side effects, are routinely excluded during purification, but separation of closely related antibiotic molecules is often unfeasible. Most fermentatively produced antibiotics used in therapy are actually mixtures of closely related compounds with one of the metabolites constituting the majority of the mixture. This practical approach permits reproducible therapeutic responses because a given antibiotic molecule always accounts for most of the mixture; it also provides the most economic materials for drug formulations because the inefficiency and expense of total separation of similar chemical molecules, the relative concentrations of which are unequal, can be avoided. The presence of up to 6% chlortetracycline in commercial tetracycline represents a practical application Of such purification considerations. Accepted standards of purity for antibiotics and antibiotic preparations are controlled

322

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by the United States Pharmacopeia. Qualitative and quantitative evaluations of antibiotic preparations for adherence to established standards utilize both biologically and chemically based tests. Colorimetric and spectrophotometric approaches and definitive measurements have largely replaced microbiologic assay and arbitrary units for quantitative purposes. However, biologic tests are still employed to detect the presence of pyrogens in parenteral antibiotic formulations. The objectives and approaches of most tests for evaluation of antibiotic preparations are not significantly different from those used to ensure the standards of other drugs. The one unusual aspect of evaluating antibiotics is associated with the need to guarantee sterility in parenteral preparations. Masking of the presence of microbial contaminants through bacteriosta tic action of the antibiotic must be precluded. Three basic approaches can be used to eliminate the antibiotic masking of microbial contaminants. Preparations containing antibiotics that are inactivated readily by biologic or chemical means ma y he subjected to the appropriate treatment before testing for sterility. Penicillinase inactivation of penicillin G and hydroxylamine hydrochloride inactivation of streptomycin illustrate this approach. Parenteral solutions of all antibiotics, especially those containing the more stable ones, can be evaluated by diluting the preparation such that the antibiotic level is below the minimum threshold concentration for activity or by initially removing any microorganisms with a sterile Millipore filter in a manipulation that separates the organisms from the antibiotic. MANIPULATIVE FORMULATIONS

Effective use of many drug substances can be enhanced through various manipulations in pharmaceutic formulations. Antibiotics are no exception. Three approaches for the protection of labile

antibiotic molecules, the use of insoluble derivatives to eliminate objectionable tastes and thus gain patient acceptance for certain oral formulations, and the use of either soluble or insoluble salts to facilitate the desired delivery of the therapeutic agent illustrate the practical utilization of manipulative formulations for various antibiotics. Buffers in oral penicillin G preparations reduce the destructive effect of gastric acidity, and enteric coatings of some oral erythromycirl formulations protect the macrolactone ring of this antibiotic until it passes through the acidic environment of the stomach and into the small intestine where it is absorbed. Er y thromycin estolate (the dodecyl sulfate salt of the propionyl ester) and triacetyloleandomycin are much more insoluble than the parent macrolide antibiotics. This property makes a dual contribution to oral suspensions of these antibiotic substances. The insolubility helps to avoid the extremely hitter taste of these drugs and to protect them until they reach the lower intestine. Fhe glucoheptonate and lactobionate salts of erythromycin are used to increase the solubility of the antibiotic sufficiently to permit intravenous administration. The relatively insoluble henzathine and procaine salts of some penidllins are used intramuscularly for repository effects. When benzathine penicillin G is used in oral suspensions, this insolubility characteristic contributes a stability factor. The use of an adjunct agent is another sophisticated approach to modifying the therapeutic availability or impact of an antibiotic. The classic example is probenecid, which inhibits the tubular excretion of penicillins. Concurrent administration of penicillins and probénecid is used to achieve prolonged blood levels of these antibiotics. Recent examples are the addition of ciavulanic acid, a 3-lactarnase inhibitor without significant antibiotic activity per se, to a formulation of amoxicillin or ticarcillin;

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the result of the combinations is an expanded therapeutic spectrum.

323

or organism against properly selected antibiotics known to be potentially active against the organism. Antibiotics with antibacterial activity are often classified into THERAPY AND UNDERLYING 2 broad categories on the basis of inhibiting BIOLOGIC FACTORS predominantly gram-negative or gramVarious antibiotics are widely employed positive bacteria. Knowledge that a given for the effective control of most serious in- antibiotic or group of antibiotics is charfections, but prophylactic administration of acterized by a gram-negative or a gramantibiotics to individuals is rarely justified, positive spectrum has some therapeutic Effective antibiotic therapy involves the utility, especially for selecting an antibiotic correct diagnosis of the pathogen and the for initiating therapy in the absence of deproper selection of an antibiotic. Diagnos- finitive bacteriologic data and for considtic bacteriologic examination is usually a ering alternate antibiotic approaches. minimum basis for rational therapy. Excep- When the pathogen is known or strongly tions include diseases such as scarlet fever, suspected, selection of an effective antityphoid fever, or other conditions charac- biotic can frequent]'' be based on the terized by clinical symptoms that are in- knowledge that the spectrum of an antidicative of a specific microbial etiology. In- biotic includes a specific microorganism. terim antibiotic therapy is usually initiated However, judicious selection of an effecon a calculated judgment basis in acute tive antibiotic for control of Escherjchja coli cases of meningitis, pneumonia, urinary and many pathogenic species of Kiebsielia, tract infections, and similar conditions Proteus, Pseudomonas, and Staphylococcus with multiple possible causes pending bacnecessitates individual determination of teriologic diagnosis; the therapeutic ap- susceptibility because various Strains of proach is modified as necessary upon conthese pathogens have different antibiotic firmation of the causative organism. sensitivities, In order for the physician to exercise clinMany pathogens are susceptible to more ical judgment properly, he must have a than one commercially available antibiotic. knowledge of the bacteriologic statistics of The choice of antibiotic for any given therinfection, i.e., he must know what orga- apeutic situation must be based on comnisms most often produce a certain type of posite considerations of a number of facinfection in particular areas of the body and tors and is rarely unequivocal. Properties in patients at a particular age. For example, frequently cited for a clinically ideal antiin cases of bacterial meningitis in adults, biotic include a complete freedom from the most common causative organisms are acute and chronic toxicities;an optimal acNeisseria meningitidis and Streptococcus pneu- tivity near pH 7 that is not influenced by inouiae. In children under 10 years of age, serum, other body fluids, or pus; sufficient I-Iaernophilus influenzae is also a common solubility in a . queous fluids to facilitate causative agent; however, in infants less good distribution to all body tissues; chemthan 1 month of age, coliforn-i bacteria such ical stability; efficient absorption following as species of  Escherichia, Kiebsiella, and Enoral administration; no tendency to induce terobacter are added to the list of common the development of resistant strains of causative agents (Table 12-1). pathogens, and a low expense factor. No It should be emphasized, however, that known antibiotic possesses all of these rational antibiotic therapy depends first on ideal characteristics. The naturally occurisolating and identifying the pathogenic or ring penicillins probably most nearly apganism from the focus of infection and then proach many of these properties for theron determining the sensitivity of that strain apeutic situations in which their spectrum

Table 12-1. A Summary of Common Pathogens That Cause Infections

Pathogenic Organism

Treatable with Antibiotics Disease Produced -

Gram-positive cocci Staphylococcus aureus

Streptococcus faccalis Streptococcus pneumoviae Streptococcus pi ogeues

-hemolytic group A

cellulitis, impetigo, septicemia, endocarditis, meningitis, osteomVetUts, pneumonia, 1000 poisoning, turunculoSiS subacute bacterial endocarditis, urinar y tract infection pneumonia, meningitis, OtitiS scarlet fever, rheumatic fever, er y sipelas, pharyngitis, impetigo

Gram-positive bacilli Bacillus anthracis Clostridium botulin urn Clostridium difficile Clostridium perfriugens Clostridium tetani Corvnebacteriurn diplithr'riac'

anthrax food poisoning (botulism) pseudomernhranous colitis gas gangrene tetanus diphtheria

Gram-negative cocci Neisseria conorrhocae Neisseria nicningitidis

Gram-negative bacilli Bacteroides fra il/s Bordetella pertussis Llrucella ahortzis, B nic'htcrisit, and B. .rii Enterobuctc'r acrogenes Eschc'ric)iia co/i Hoerno phi/us influen:ac Klc'bsiella pnewlwninc Leyiinella pnei' ioploia I','oti'us z':ilgaris Psc,.idornonas a.'rllgiiiii-sl Salmonella species Salmonella ti/phi Shigdla dysenterine Vibrio chalerne Y ersinia pestis

gonorrhea meningitis abscesses of abdomen, lung brew whooping cough brucellosis pneumonia wound infcciions, urinary tract injection urinary tract inject-ion, septicemia, respiratory infections, peritonitis respiratory infections, meningitis, Outs pneumonia, urinar y tract infection, septicemia I egionnairc"s disease urina cv tract injection, septicemia urinar y tract infection, pneuf'.Oiva, burn-wound infection sc' pIlCc'mia food poisoning (silmoncilosis) typhoid fever bacillary dysentery Asiatic dysentery bubonic plague

Acid-fast bacilli Mycobacteriurn leprae Mycobcterhini titherciilosL4

Spirochetes Treponeina liallidion

leprosy tuberculosis s yphilis

Fungi Biastrnriyces dermat it/it /s Condida aibjcans Coccidioides 1mm/f Is Cryptococcus neofoi-uu-ms H/stop/name capsulation £pidermophylon, Microsporuui, and Trichophytoii (various species)

North AmePcan hlastomvcosis carididiasis (honiliasis) coccidioidomvcosis (Sin Joaquin fever) OrYptOCOCCOSiS histoplasniosis dermatomycoses (ringworm, athlete's foot).

-Miscellaneous, Rickettsiae, Large Viruses .Mycaplasma pneuoaooiae Rickettsia prowazekil Rickettsia rickettsii Rickettsia typhi Chlairnldie psiftaci Chiarnydia trachomuitis 324

respiratory infectious epidemic typhus- Rocky Mountain spotted fever endemic typhus psittacosis (parrot /ever) triehuma, posigonococcal urcthrttis

ANTIBIOTICS

is adequate because they tend to have a rapid onset of systemic activity when orally administered, cause a low incidence of toxicity, and are inexpensive. However, serious penicillin hypersensitivities contraindicate the use of these antibiotics in some individuals, and the development of resistance by some pathogens is a definite therapeutic concern. Cost is never a major or exclusive criterion for selection of a firstchoice antibiotic for therapeutic purposes, but if all other factors are equal, the least expensive theMpeutic approach (not necessarily the least expensive unit formulation) serves the best interests of the patient. Properties of the antibiotic per se are not the only considerations in selecting the best therapeutic agent. Such factors as age and secondary debilitating conditions may influence the use or choice of antibiotics in specific situations. The following examples illustrate generally the situations that may be encountered. Gradual development of normal renal function during the neonatal period necessitates adjustment in the dosage and administration interval when employing antibiotics that are eliminated by the kidneys. An antibiotic that is excreted in the urine also must be used cautiously for systemic purposes in adult patients with renal complications, and chioramphenicol is usually considered an antibiotic of last resort when an infection is accompanied by hematopoietic abnormalities. When serious gastrointestinal complications would contribute to erratic absorption upon oral administration, the pareriteral features of an antibiotic become dominant. Data are being accumulated on the modes and mechanisms of action of various antibiotics, on the bases for toxicities in antibiotic therapy, and on the details of resistance. The available information is sufficient to rationalize scientifically many developments that may be observed during therapy. A consideration of these factors will undoubtedly provide a basis for more effective and precise antibiotic therapy in

325

the future when more complete knowjçdge becomes available. MODES AND MECHANISMS OF ACTION

A number of different classification schemes could be used to categorize the selective toxicity of antibiotics for susceptible microorganisms. The recognition of 4 general modes of action, namely, inhibition of microbial cell-wall formation or biosynthesis of some essential protein, disruption of deoxyribonucleic acid metabolism, and alteration of normal function of the cellular membrane, is satisfactory pending the accumulation of more data. It is frequently difficult to distinguish primary from referred responses in preliminary attempts to determine the mode of antibiotic action. When more detailed information becomes available, current concepts on the mode of action of a few antibiotics (Table 12-2) may be altered, and the relative therapeutic importance of alternate modes of action will be clarified for antibiotics that give experimental indications for more than one general basis for their antagonistic effects. The mechanism of action of an antibiotic, as contrasted with the general mode of action, is frequently an individualistic feature, and distinctive mechanisms of action are often observed for 2 antibiotics with the same mode of action. Precise knowledge of the mechanism of action offers tremendous potential for sophisticated developments in antibiotic therapy. Sufficient information is available on the mechanism of action of certain antibiotics that interfere with cell-wall formation and protein biosynthesis to show representative patterns of biologic involvement. Inhibition of cell-wall formation involves the disruption of mucopeptide synthesis. Gram-positive bacteria are particularly susceptible to antibiotics that inhibit mticopeptide formation because they possess a cell wall that contains a relatively thick mu-

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326

Table 12-2. General Mode of Antibiotic Action Inhibition of cell wall fornsation 8aiitracin Cephalosporins Cycloserine Penicillins Vancomycin Other B-Ictam a,ttibiotics Disruption of deoiyribonucleic acid metabo11am Actinomycin 0 Doxorubicin Mitomycin C Novobiocin Plicamycin Rifompin Bleomycin

Inhibition of protein bius y rithesis Amikacin Chlorarnphenicot (Jindamvcin Erythromycin Centamtcin Kanarnycin Lincomydn Neomycin Netilmicin Parornomydri Spectthomycin Streptomycin Tetrcvc!iries Tobramyin Froleandomvcin

Alteration in cellular [oembeans function Amphotricin ll Coils tin Nvstatiri Polvinvxin

copeptide layer to provide structural support of the cytoplasm. The mucopeptide layer is known variousl y as murein, glycopeptide, or peptidoglvcan, and because of the nature of its chemical structure, it is tough and fibrous. Support is required because gram-positive bacteria concentrate low-molecular-weight metabolites such as amino acids and nucleotides, which impart a high internal osmotic pressure. On the other hand, gram-negative bacteria have a relatively low internal osmotic pressure with a thin layer of mucopeptide. The synthesis of mucopeptide occurs in distinct steps. The first step is a series of reactions inside the cell that result in the production of the basic building units (undine diphospho-N-acetyl-muramvlpentapeptide) Cycloserme inhibits the formation of the pentapeptide portion of the building block. In the next step, the building units are carried to the outside of the cell membrane. During this process, the units are linked covalently to the preexisting cell wall. Vancomycin and bacitracin

inhibit this step of the biosynthesis. The final stage of the biosynthesis is the crosslinking of linear molecules to form the highly cross-linked, 3-dimensional mucopeptide. The last reaction in mucopeptide formation is catalyzed by a transpeptidase that splits the terminal D-alanine iesidues of the pentapeptide of the building unit arid, in the case of Staphylococcus aureus, forms a peptide bond between the terminal glycine of a pentaglycirie bridge and the penultimate D-alanine of a mucopeptide strand (Fig. 12-9). Therefore, each polypeptide side chain of each repeating building unit becomes covalently linked to the side chains of neighboring mucopeptide strands. The cross-linking process has 2 steps; carhoxvpeptidation, followed by transpeptidation (Fig. 12-9). The penicillins and the cephalosponins are competitive inhibitors of this cross-linking. Although the precise mechanism is not known, it appears that the penicillin or cephalosponin molecule occupies the D-alanyl-D-alanlne substrate site of the DD-carhoxypeptidase and/or the peptidoglvcan transpeptidase, forming a covalent adduct that is stable to subsequent hydrolysis and, therefore, irreversibly inactivates the enzyme. These penicillin-sensitive enzymes are also known as penicillin-binding proteins (PBPs).  The PBPs are found in the cell membrane of all bacteria examined to date. Bacterial membranes yield multiple PBPs ranging in number from 3 in gonococci to 10 or more in Escherichia coli. On sodium dodec ylsulfate-polyacrylamide gels, PBPs have molecular weights usually ranging from 40,000 to 120,000 and are numbered in order of decreasing molecular weight. The currently used nomenclature involves assigning numbers to the protein, with 1 being the highest molecular weight; therefore, the numerical connotation of PBPs is strictly a reference to their relative molecular size within the group of PBPs detected in a microorganism. Thus, PBP-1 of Esch-

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327

(giy)5

(gly)—L-iys O1 L-ala -(N-acetyl gJucosamlrieNacetyl murarnic acid•N-acelyl glucos3mine.NcetyI muramic acid)L-ala peptidoglycan 0-glu transpeptidase (91y)5--L-lys 0-ala / , DD-carboxypeptjdase -. 0-ala glygly-gly-gly•gly L-lyS 0-ala oglu Lab -(Nacetyl rnuramic acidN -acetyf glucosarnneracetyl mJrarnc acid-N-acetyl gtucosamjne). D.glu [-lys -' (gIy).-.-.> 0-a (gly)5

Fig. 1.2-9. Partial structure of bacteria] cell wall rnucopepude showing polvsaccharides cornposedoipolvmerjc chains of alternating units of N-acetylglucosam i ne and N-acetvhyiui-arnjc add. The peptide chains cross-link the polvsacchandc chains to make a rigid 3-dimensional structure. The last steps in mucopeptide formation are catalyzed by a DD-carboxypeptidase that splits off the terminal D-a!enine and peptidoglyn transpeptidase that forms a peptide bond between the penultimate D-Ianine and, in the case of Stnphi,1c5 aureus, the glycrne of a pentaglydne bridge.

erichia coil need not have anything in common with PBP-i of gonococci. The physiologic function of PBPs in the formation of cell wall has been assigned so far in onl y a limited number of bacteria. In Esclierichja coli, PBP-1, PBP-2, and PBP-3 all have transpeptidase activity and are involved, respectively, in elongation, shape, and septation during growth. 13-Lactam antibiotics that bind to PBP-1 cause cell lysis, whereas binding to PBP-2 produces giant

spherical shaped cells; those binding to PBP-3 produce filamentation. Most -lactam antibiotics bind to PBP-1 and PBP-3, whereas amdinocillin binds preferentially to PBP-2. Formation of an essential protein may be blocked at any of the basic stages of protein biosynthesis. The antibiotic could adversely influence the replication and synthesis of DNA , the transcription of the genetic code and the specific sequential

328

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synthesis of DNA, the transcription of the genetic code and the specific sequential synthesis of mRNA, or the synthesis and assembly of the ribosomes. All of these biologic processes are fundamental for the eventual synthesis of a protein, but many constituents that act at these levels tend to be relatively toxic. Most of the therapeutically useful (more selectively toxic) antibiotics that act on protein biosynthesis influence in some manner the normal assembly of the amino acids into proteins at the surface of the mRNA-ribosome complex. The ribosomes found in bacteria have a sedimentation coefficient of 70S, and they are composed of 2 particles of different size, the 50S and the 30S ribosomal subunits, Each subunit is composed of ribosomal RNA and a number of different proteins. Antibiotic action to inhibit protein biosynthesis can be focused on the events that take place on the ribosomes. These are initiation, binding of aminoacyl-tRNA, peptide bond formation, translocation, and termination (Fig. 12-10). Streptomycin, an aminoglycoside antibiotic, affects initiation as well as elongation and termination of protein synthesis. The antibiotic binds to the 30S subunit and causes a breakdown of the initiation complex, resulting in the release of f-mettRNA. Other aminoglycoside antibiotics such as kanamycin, geritamicin, and neomycin interfere with initiation of protein synthesis; however, these antibiotics affect elongation of the peptide chain more markedly through inhibition of trarislocation than through initiation. In addition, the aminoglycoside antibiotics with a streptarnine or 2-deoxystreptamine moiety provoke codon misreading or induce the uptake of incorrect amino acids that do not correspond to the codon. The tetracyclines interfere with the binding of aminoacyltRNA to the acceptor site of the 70S ribosome. Experimental evidence points to a single strong binding site for tetracycline located on the 30S subunit; however, it has

not been completely ruled out that the 505 subunit might also be involved in the binding. Chloramphenicol binds to the 50S subunit where it disrupts the function of peptidyl transferase. Erythromycin also binds to the 50S subunit. It does not inhibit peptide bond formation, but it does block translocation. The antitumor antibiotics and others disrupt DNA metabolism. Actinomycin D and plicamycin bind through hydrogen bonding to guanine residues of the DNA double helix. Mitomycin C covalently cross-links between the complementary strands of the DNA double helix. These complexes of the drug with the DNA template block the transcription of RNA by DNA-dependent RNA polymerase which, in turn, is responsible for the antitumor effect. Other antibiotics affect the permeability of the cell membrane in a way that causes leakage of cytoplasmic solutes. The 2 most important groups of these drugs are the polvene antibiotics, amphotericin B and nystatin, and the peptide antibiotics, such as the polyrnyxins. The polyene antibiotics are antifungal agents that affect the membranes of eucaryotic cells but have no activity on bacteria. This difference in sensitivity of different organisms to these antibiotics is determined by the presence of sterols in the cell membrane of eucaryotic cells. The polyenes bind to the membrane and the extent of binding is proportional to the amount of sterol present. Molecular models show the polyenes to have a rodlike structure held rigid by an all-trans extended conjugated system that is equal in length to a sterol molecule. One surface is lipophilic and the opposite, studded with hydroxyl groups, has a hydrophilic face (see page 366). There is evidence that there is a packing of alternating sterol and polyene molecules, which creates a pore through the cell membrane. The pore is thought to be a hollow cylinder with a polar interior surface caused by the hydrophilic hydroxyl groups of the polyene molecules and an exterior surface corn-

329

ANTIBIOTICS RIBOSOMAL SUBUNIT 30S

peptidyl donor site

=

m-RNA codon = -

ominoocyl or acceptor site m-RNA codon

messenger-RNA codon ,--'-Er

IliAC

C G C Ucu I

I

f-met-(C-N-)  oiJ® 11 H

org /

RIBOSOMAL SUBUNIT SOS Fig. 12-10, Sequence of event q of protein yntheis on the 705 ribosome: I. Formation of initiation complex. Involves the binding of the first aniinoacyl-tRNA to the ribosorne lii bacteria, the first amino acid hound is formylmethonine ([-met). II. Binding of the next aminoacyI-tRNA to aminoacyl site. III. Formation of peptide bond catal y zed by a ribosome-bound peptdyi transfease. IV. Release of fomylmethionine-specific tRNA. V. The peptidyl-tRNA (f-met-ala-tRNA) moves to the peptidyl site. This is called the translocation step, and the mRNA shifts to the next codon. VI. The aminoacyl site is free and available for the next addition of aminoacyl-tRNA which, in this case, is argtnine-tRNA.

posed of sterols being attracted to the Iipophilic side of the polyenes (Fig. 12-11). Ions from the c ytoplasm such as K would leak through the polar pore, causing damage to the cell by the upset of the ion balance. The peptide antibiotics also bind to the cell membrane and disturb membrane function, however, these antibiotics are active against bacteria since sterols are not required for binding.

BASES OF TOXICITY

One limitation to the therapeutic use of an antibiotic substance is mammalian toxicity. The manifestation of such adverse reactions varies greatly with different antibiotic molecules. Basically, these side effects are an extension to mammalian biologic processes of the mechanisms of antibiotic action, hypersensitivity, or a

330

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'   O :tl Li Pore

OH HO

—OH

Membrane layer

HO—'

0:

OH HO_j

I

Polyene

Pore

- Sterol

Cross section of pore

Fig. 12—I1. The possible interaction 01 polyene antibiotics in a eucaryohc cell membrane. The polvenes complex with membrane sterols to form a pore through the membrane through shich ions ma y pass.

pharmacologic action that is independent Of the antibiotic activity of the molecule. An antibiotic that acts by inhibiting protein synthesis in susceptible microorganisms is potentially toxic to mammalian systems involving the same or related essential proteins. Theoretically, the safest antibiotic inhibits an essential process, such as cell-wall formation, that is unique to the microorganism. Actual situations usually follow the theoretical considerations, as illustrated by the relative safety of the penicillins and the relative toxicity of chioramphenicol. However, some degree of deviation from the ideal is probably universal because antibiotic molecules normally lack absolute specificity or the ability to influence only one biochemical reaction. In the case of penicillins, hypersensitization with serious consequences precludes the use of these antibiotics in some indi-

viduals. Many antibiotic molecules are characterized by reactive functional groups, and hypersensitivity may be a problem with molecules containing functional oxygen groups that can react with proteins to yield a potentially antigenic hapten-protein molecule. Toxicity caused by some independent pharmacologic property of the antibiotic is usually difficult to predict; this type of complication must be evaluated individually for each antibiotic. In addition to any adverse pharmacologic action of-an antibiotic per se, indirect toxicities can be observed with these therapeutic agents. The most common type of indirect antibiotic-induced toxicity is associated with an alteration in the ecologic balance of the intestinal flora. This problem is greatest with the broad-spectrum antibiotics because a major portion of the intestinal flora may be suppressed. Candida

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albicans is an example of the slow-growing, unsusceptible microorganism that may become a dominant component of the intestinal flora following the administration of antibiotics. The body frequently has no prior adaptation or tolerance to the level of foreign metabolites resulting from the unusual proliferation of such organisms; the toxicity is usually manifest as gastrointestinal disturbances rather than as acute toxicities The pseudomembranous colitis caused by Clostridium difficile in some patients receiving clindamycin is another example of such clinical problems. MODES OF RESISTANCE

Antibiotic resistance is a major therapeutic concern. One practical way to circumvent this problem, at least for short-term purposes, is to develop and use new antibiotics, but experts are concerned justifiably about the practicality of long-term developmental aspects of this approach. Resistance to antibiotics may result through spontaneous or induced genetic mutation. However, many of the practical problems have developed via the process of selection or, in other words, favoring through the use of antibiotics the low frequency of organisms of antibiotic-resistant genotype that exists naturally in the antibiotic-sensitive, wild population. Spontaneous mutation is believed to make only a minor contribution to the total problem of antibiotic resistance. Bacterial cells can acquire genetic material from other bacterial cells through the processes of transformation, transduction, and conjugation. Transformation, which is a process by which DNA from a lysed bacterial cell is inserted directly into a recipient cell, makes no substantial contribution to the clinical problem of drug resistance. Transduction, or the phage-induced transfer of resistant determinant sections of bacterial DNA, is believed to be an important factor in the emergence of drug-resistant strains of S!aphylococcus. Conjugation is a widely rec-

331

ognized mechanism for transmitting resistance among gram-negative bacilli of clinical concern. Conjugation of compatible cells (which may represent different species or even genera) provides a means for direct transfer of R-factor genes residing on bacterial episomes, and great danger lies in the fact that bacterial episomes may contain genetic information for multiple resistance. Multiple mechanisms of resistance to many antibiotics appear to exist,, and the lack of precise information in many cases makes general categorization difficult. However, some modes of resistance that can be noted include: 1. Enzymatic inactivation of the anti2. Altered permeability of the pathogen to the antibiotic; 3. Development of altered, less sensitive enzymes or of alternate metabolic pathways in the pathogen. The 1-lactamase inactivation of penicillins and cephalosporins is by far the best documented mechanism leading to antibiotic resistance. The significance of penicillinase was recognized at an earl y date in antibiotic therap y, and the semisynthetic penicillins are a direct result of efforts to avoid the specificity of the enzyme. A penicillin amidase also occurs in some microorganisms; this amidase yields the inactive 6-aniinopenicillanic acid, but this type of penicillin inactivation does not appear to contribute significantly as a means of pathogenic resistance in any actual therapeutic problem. Gram-negative bacteria bearing any of several R-factors for multiple resistance can enzymatically inactivate aminoglycoside antibiotics by forming either phosphoryl, adenyl, or acetyl derivatives of these antibiotics. Altered permeability is a frequently mentioned mode of resistance. Actual substantiation of this type of involvement is limited. Tetracycline resistance in Escherichia co/i appears to be related to a decrease in the ability of the bacterial cells to take

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up the antibiotic. Another possible exampie is chioramphenicol. One biochemical basis of resistance to this antibiotic is an acquired selective impermeability of cellular membranes of some organisms; however, it is also known that certain strains of E . coil that are resistant to chioramphenicol enzymatically inactivate the antibiotic by acetylation. Resistance caused by the development of altered enzymes or metabolic pathways is poorly documented in the current scientific literature. This general mode of resistance is recognized, e.g., certain resistant strains of Bacillus subtilis that fail to bind erythromycin at the 50S subunits on the ribosonies, but the overall therapeutic significance of this type of resistance is relatively unknown. ANTIBIOTICS DERIVED FROM AMINO ACID METABOLISM

The commercially available and therapeutically useful antibiotics can he classified on the basis of the biosynthetic origin of the antibiotic molecules. These useful microbial metabolites are products of amino acid, acetate, and carbohydrate metabolism. Only one of the basic groups of metabolites is involved in the formation of most medicinally important antibiotics, but in the case of some, such as the macrolides, precursors from diverse metabolic origins are combined to yield the antibiotic molecule. Antibiotics derived from amino acids include the penicillins, the cephalosporins chioramphenicol, cycloserine, dactinomycm, and the polypeptide antibiotics (e.g., bacitracin, polymyxin). Considerations of chronology, sophisticated state of current development, and significance suggest the penicillins for initial monographic coverage. PeniciHins

Penicillin antagonism attracted the attention of Sir Alexander Fleming in .1928.

Fleming was studying staphylococci at St. Mary's College in London when he noticed a zone of inhibition surrounding a Pen jcühum contaminant in one of his cultures. The Penicihlium was initially identified as P. ribru,n but was later determined to be P. notatuin. Interest in this antagonism remained largely academic until after 1940. In 1938, Florey and Chain at Oxford University first isolated a crude penicillin mixture from the mold, and during the early 1940s, the therapeutic potential of penicillin was demonstrated. Conditions in England during the first half of World War II were such that efforts to determine suitable procedures for producing commercial quantities of the antibiotic were conducted primarily in the United States. Significant early discoveries included the influence of nutrient composition on penicillin production and the discovery of a strain of P. chrysogenum that would produce the antibiotic in submerged fermentation. The presence of phenyipropanoid or phenvlacetvl derivatives in the nutrient medium favored the formation of benzvlpenicillin (penicillin C). Other penicilliris could be formed by adding the appropriate precursor moieties to the fermentation cultures; penicillin V is an example of a therapeutically useful penicillin that was prepared initially by this type of manipulated biologic process (Fig. 12-12). Discovery in the late 1950s of a strain of P. chrysogenuin that accumulated high yields of 6-aminopenicilanic acid provided an alternate approach to preparing unusual penicillins, such as penicillin V, and provided an opportunity for even greater modification, in the antibiotic molecules. 6-Aminopenicillanic acid has no significant antibiotic activity per se, but this biologically prepared substance can be chemically acylated to give a wide variety of active molecules. Amdinocillin, ampidim, amoxicillin, azlodllin, bacampicillin, carbenicillin, cloxacillin, cyclacihin, dicloxacillin, methicillin, mezlocuilin, nafdillin,

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333

Fig. 12 12. ri tihiiic l eimentation area. Final fermentation takes place in these 18,000-gallon tanks 'the tanks are 30 feet high but are buried to within a few feet of their tops. A rnae of pipes carries water, steam, and air into he area. Storage tanks of 30,000-gallon capacity are located nearb y . (Photo courtesy of Eli Lilly & Co.l

oxacilhn, piperacillin, and ticarcillin are therapeutically utilized sernisvnthetic penicillins that have been selected for Various advantages offered by their chemical, physical, or spectral properties. Structures of the commercially available penicillins are shown in Figure 12-13. BIOSYNTHESIS OF PENICIWNS. The amino acids c y steine and valine are incorporated into the 6-aminopenicillanic acid portion of penicillin molecules, and the acyl group of penicillin G is derived from phenviacetic acid. Many of the details of the biosynthetic pathway require further clarification. It is generall\' believed that terminal steps in the pathway involve introduction of the characteristic acyl group and the action of an acvl transferase on isopenicillin N is sus-

pected (Fig. 12-14). The tripeplide, (-aminoadipyl)-cysteinylvaline, is the presumed precursor of isopenicillin N, and dehydrogenation involving the mercapto function and one of the meth yl groups of the valine residue of some metabolite of this tripeptide appears to yield the nucleus of cephalosporin C. PROPERTIES OF THE PENICILLINS. The chemical structure of the penicillin nucleus is unusual and is characterized by a 4-membered -lactam ring fused to a thiazolidine ring. This ring s y stem contains 3 asymmetric carbon atoms in a fixed spatial arrangement, and an y disruption of this arrangement b y rupturing either the 3lactam ring or the thiazolidine ring results in a complete loss of antimicrobial activity.

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