The biological assay of estrogenic hormones

Citation preview

THE BIOLOGICAL ASSAY of ESTP.OGEETC HQRUQIIES

By B e r n a r d P a t r i c k MclTamara

T h e s is su b m itte d to th e F a c u lty o f th e G raduate School o f t h e U n i v e r s i t y o f M aryland i n p a r t i a l f u l f i l l m e n t o f the req u irem en ts fo r th e d e g re e o f D octor o f P h ilo so p h y 19 4S

UMI Number: DP70138

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

UMI D isse rtatio n Publish«ng

UMI DP70138 Published by ProQuest LLC (2015). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code

ProQuest ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

ACiHT01712 iJG ihahl T

The a u t h o r ’ s m o s t s i n c e r e g r a t i t u d e

is

expressed

f o r th e in v a lu a b le ad v iee and a id c o n tr ib u te d tow ard t h i s w o r h b y D r. th is

C,

Chapman

u n d e r whose s u p e r v i s i o n

s t u d y was p u r s u e d .

96910

TABLE OF CONTENTS

Page INTRODUCTION.............................................................................................................

1

EXPERIMENTAL.............................................................................................................

6

A n i m a l s . . . . ........................................................................................................

6

M a te ria ls* ......

7

.............................................

P r o c e d u r e ............................................................................................................. D esign o f i n v e s t i g a t i o n

......................................................

8 8

A r r a n g e m e n t a n d p r e p a r a t i o n o f a n i m a l s ...........................

9

A d m i n i s t r a t i o n o f d o s a g e ...............................................................

10

V a g i n a l s m e a r s ......................................................................................... I S M a t h e m a t i c a l t r e a t m e n t o f r e s u l t s ...............................................

16

RESULTS.......................................................................................................................... SS DISCUSSION..................................................................................................................

44

SUMMARY.............................................

47

INTRODUCTION The " b i o l o g i c a l a s s a y o f e s t r o g e n i c "been a s u b j e c t o f d e b a t e ,

su b sta n c es has long

A l t h o u g h t h e r e a r e many m e t h o d s

o f e s tim a tin g th e a c t i v i t y o f such s u b sta n c e s , m o d if ic a tio n s o f t h e v a g i n a l sm ear m ethod on r o d e n t s a s d e s c r i b e d by A l l e n and D oisy ( 1 ) ,

a n d K a li n t a n d D o i s y ( 2 ) a r e m o s t w i d e l y em­

ployed. The p o t e n c y t h u s d e t e r m i n e d wa s o r i g i n a l l y e x p r e s s e d i n r a t o r mouse u n i t s * be in ad eq u ate

(18)

T h i s t y p e o f s t a n d a r d w as shown t o

since th e ro u te of a d m in is tra tio n ,

the

s o l v e n t , t h e number o f i n j e c t i o n s and th e i n t e r v a l o f i n ­ j e c t i o n s p ro d u c e d w ide v a r i a t i o n s i n th e e s t r u s re s p o n s e and co n seq u en tly in th e assig n e d potency. I n e f f o r t t o overcome t h e s e d i f f i c u l t i e s

two r e f e r e n c e

s t a n d a r d s w ere e s t a b l i s h e d by t h e H e a l t h O r g a n i z a t i o n o f t h e League o f N a tio n s cry stallin e

(3 ,4 ).

e s t r o n e and t h e o t h e r i s a sample o f c r y s t a l l i n e

e s tr a d io l benzoate. it

One o f t h e s e i s a s a m p l e o f

The s e c o n d s t a n d a r d w a s d e v i s e d b e c a u s e

appeared th a t th e e s t e r i f i e d

an d t h e f r e e hormone d i f f e r e d

q u a lita tiv e ly in th e ir a c tiv ity . The u n i t o f e a c h s t a n d a r d i s oestrus-producing a c tiv ity standard p re p a ra tio n

defin ed as "the s p e c if ic

contained in 0 , 1

gamma o f t h e

(3 ,4 )."

The s t a n d a r d o f r e f e r e n c e i s t o b e t r e a t e d assay in ex actly the I n t h i s way t h e

in every

same m a n n e r a s t h e e s t r o g e n u n d e r t e s t .

e f f e c t o f v a r i a t i o n s i n th e a n im a ls can be

2

overcome, The a b o v e n a m e d c o m m i t t e e d e f i n e d t h e t e r m " s p e c i f i c e s t r u s - p r o d u c i n g a c t i v i t y ” a s " th e power o f p r o d u c in g , t h e a d u l t fem ale anim al c o m p le te ly d e p riv e d o f i t s

in

ov aries,

an a c c u r a te ly re c o g n iz a b le degree o f th e changes c h a r a c te r ­ istic

o f th e norm al o e s t r u s , "

The o n l y s u c h c h a n g e r e g a r d e d

as p ro v id in g a s u ita b le b a s is fo r q u a n tita tiv e determ in atio n o f th e a c t i v i t y in com parison w ith th e s ta n d a rd p r e p a r a t io n is

the

s e r i e s of changes in th e c e l l u l a r c o n te n ts of the vag in a

o f t h e r a t a n d mouse ( 3 , 4 ) , I n f u r t h e r r e g a r d , o f t h e a s s a y m e t h o d i t w as s t a t e d

(3 ,4)

t h a t t h e c o m p a r i s o n o f a n unknown p r e p a r a t i o n w i t h t h e s t a n d a r d p r e p a r a t io n f o r such s p e c i f i c a c t i v i t y

could be a c c u r a t e l y

made o n l y i f t h e c o n d i t i o n s o f a d m i n i s t r a t i o n o f b o t h , a n d o f o b s e r v in g th e r e s u l t s p ro d u c e d i n each c a s e , were c o m p le te ly id e n tic al, (a)

p a r t i c u l a r l y in the follow ing re s p e c ts : The s t a n d a r d a n d t h e unknown p r e p a r a t i o n u n d e r t e s t

m ust be a d m i n i s t e r e d i n i d e n t i c a l (b )

solvents.

A d m i n i s t r a t i o n s h o u ld be by t r u l y s u b c u ta n e o u s and

n o t by in tr a m u s c u la r i n j e c t i o n . (c) test

The s t a n d a r d p r e a r a t i o n a n d t h e p r e p a r a t i o n u n d e r

s h o u ld be a d m i n i s t e r e d by a method e n s u r i n g slow a b s o r p t i o n

a t id en tical rates.

This c o n d itio n ,

a d m in is te rin g both d isso lv e d in th e

c o u ld be a t t a i n e d by

same o i l y

w hich c a se s i n g l e i n j e c t i o n s co u ld be u sed , bo th in w atery s o lu tio n ,

solvent,

in

o r by p r e p a r in g

and a d m i n i s t e r i n g i n an e q u a l number

3

Of f r a c t i o n s , n o t l e s s t h a n t h r e e .

In the l a t t e r

case,

the

i n j e c t i o n s sh o u ld he spaced i n an i d e n t i c a l manner f o r h o th t h e u n know n a n d t h e of not le ss (d)

s ta n d a rd p r e p a r a tio n over a t o t a l tim e

than 9 h o u rs.

The v a g i n a l

sm ears should he ta k e n a t th e

same

i n t e r v a l s a f t e r th e i n j e c t i o n from t h e a n im a ls i n j e c t e d w i t h t h e s t a n d a r d and from t h o s e i n j e c t e d w i t h t h e p r e p a r a t i o n under t e s t ,

h u t sh o u ld he c o n tin u e d a t l e a s t once d a i l y u n t i l

the anim als under t e s t

r e t u r n to a d i e s t r u s c o n d itio n .

t h e d a y i n w h i c h t h e maximum r e s p o n s e i s 2 sm ears sh o u ld he ta k e n . receiv in g th e under t e s t , 24 h o u r s , (e) stan dard,

On

expected a t l e a s t

I f t h e maximum i n t h e a n i m a l s

s ta n d a rd and i n th o se r e c e iv in g th e p r e p a r a t i o n

a r e a t t a i n e d a t i n t e r v a l s d i f f e r i n g hy more t h a n

th e com parison cannot he re g a r d e d a s v a l i d . I n com paring th e a c t i v i t y

of a p re p a r a tio n w ith th e

t h e p r o c e d u r e s h o u ld aim a t f i n d i n g a dose o f t h e

unknow n p r e p a r a t i o n p r o d u c i n g t h e c h o s e n l i m i t i n g r e a c t i o n i n t h e same p r o p o r t i o n o f a n i m a l s a s t h a t i n w h i c h i t

i s pio -

d u c e d h y a known d o s e o f t h e s t a n d a r d . T h i s r e p o r t hy t h e H e a l t h O r g a n i z a t i o n o f t h e L eague o f n a t i o n s w as a d e f i n i t e a d v a n c e i n t h e e s t i m a t i o n o f b i o l o g i c a l activ ity

in estro g en ic p re p a ra tio n s.

However,

since

estro g en ic p re p a ra tio n s are not d escribed

i n t h e U. S. P. XI a n d H. P.

V I , no o f f i c i a l

standard of

r e f e r e n c e o r no o f f i c i a l m e t h o d o f a s s a y h a s b e e n made a v a i l ­ a b l e w i t h w h ic h t h e p r e p a r a t i o n s o f d o m e s tic o r i g i n c a n he

4 compared*

The m e t h o d u s e d a t p r e s e n t h y t h e F o o d a n d D ru g

A d m inistration

(5) l i k e

t h a t u s e d hy t h e H e a l t h O r g a n i z a t i o n

o f th e League o f N a tio n s (3 ,4 ) D oisy (2) v a g i n a l

i s h a s e d on t h e A l l e n a n d

smear t e c h n i q u e .

The t e s t ,

a s conducted

hy t h e W as h in g to n l a b o r a t o r i e s employs t h e i n t e r n a t i o n a l stan d ard assayed under id e n tic a l r a tio n under t e s t .

circum stances a s the prep a­

The d e s i g n o f t h e a s s a y i s

to allow s t a t i s t i c a l

also m odified

ev alu a tio n of the r e s u lts .

The g e n e r a l a c c e p t a n c e o f t h e r e f e r e n c e

stan d ard in

t h i s a s s a y p r o c e d u r e h a s r e s u l t e d i n a g r e a t im provem ent i n t h e u n i f o r m i t y o f c o m m e r c i a l p r e p a r a t i o n s so s t a n d a r d i z e d , and i n p r o d u c t s and p r o c e d u r e s i n th e p r o c e s s o f developm ent and i n r e s e a r c h i n t h i s f i e l d . The v a r i a t i o n i n r e s u l t s

o b t a i n e d when a r e f e r e n c e

s ta n d a r d and t h e s e p r o c e d u r e s a r e n o t u sed can he found i n the l i t e r a t u r e .

In the f i r s t

o f th e League o f N a tio n s

(3)

report

o f th e H ealth O rg a n iz atio n

i t was s t a t e d t h a t

the i n t e r ­

n a t i o n a l u n i t w as a p p r o x i m a t e l y 1 / 3 o f t h e o r i g i n a l A l l e n a n d D oisy r a t u n i t .

V arious o th e r l a b o r a t o r i e s

(6-17) have

p l a c e d t h i s v a lu e from 1 /5 to 1 /3 8 o f t h e r a t u n i t . v arious la b o ra to rie s , estrogen

That i s ,

i n d e t e r m i n i n g t h e p o t e n c y o f one

( in te r n a tio n a l stan d ard estrone)

ap p ro x im ately 8 tim e s in term s of

found i t

rat u n its.

to vary

T h i s i s a much

g r e a t e r e r r o r t h a n would be e x p e c te d f o r t h i s p a r t i c u l a r p r o ­ cedure,

s i n c e B u l b r i n g and Burn ( l l )

t o h e n o t g r e a t e r t h a n 30 p e r c e n t . great v ariatio n

found th e T herefore,

e rro r of assay t h e r e m ust be

from l a b o r a t o r y t o l a b o r a t o r y i n d e t e r m i n i n g

5 th e

rat u n it.

This d iv e rg e n cy can to

t r o l l e d hy a s s a y i n g th e r e f e r e n c e under th e in

e x t e n t he con­

s t a n d a r d and t e s t p r o d u c t

same c i r c u m s t a n c e s .

o b j e c t i o n o f t h i s mode o f a s s a y c o n c e r n s t h e m e t h o d

of ad m in istratio n .

T h i s f a c t o r i s a c c e p t e d a s one o f t h e

v a r i a b l e s w hich i n f l u e n c e s th e m ercial estrogens re g a rd le ss gen erally

e stro g en ic response.

e s t r o n e a n d u s u a l l y by s u b ­

I n v e s t i g a t i o n b y Howe a n d Si m o n d

( 1 9 ,2 0 ) , and Sondem a n d Sealey

(21) have i n d i c a t e d t h a t t h e

ra tio s of b io lo g ical a c tiv ity for whe n c o m p a r e d t o

estrone,

and o r a l a d m in is tr a tio n .

Com­

o f t h e i r chem ical n a tu re a re

standardized a g ain st

cutaneous a d m in is tr a tio n .

ex ist,

a great

these d if f e r e n t

are not the T herefore,

estrogens,

same b y s u b c u t a n e o u s i f t h i s c o n d itio n does

a p r o d u c t h a v i n g a c e r t a i n e s t r o n e v a l u e when g i v e n

s u b c u t a n e o u s l y may h a v e a n e n t i r e l y d i f f e r e n t w he n g i v e n b y m o u t h .

C onsequently,

e stro n e value

an o r a l a s s a y f o r p ro ­

d u c ts in te n d e d f o r o r a l use should give a t r u e r v alue o f th e c l i n i c a l a c t i v i t y as e x e r t e d by t h i s

route of adm inis­

tratio n . The p r i m e o b j e c t

of t h i s research is

to determ ine th e

r e l a t i v e p o t e n c y e v a l u a t i o n o f t h e two m e t h o d s a s a p p l i e d to

com m ercial p r o d u c ts in te n d e d fo r o r a l th e ra p y .

6 EXPERIMENTAL A nim als The a n i m a l s u s e d i n t h i s o b t a i n e d f r o m two s o u r c e s .

s tu d y were fe m a le ,

albino

rats

One w a s t h e L a b o r a t o r y S u p p l y

C orporation, P h ila d e lp h ia ,

Pa.

d a y s o f a g e wh en p u r c h a s e d .

T h e s e a n i m a l s w e r e 2 1 t o 30

A f t e r r e c e i p t t h e y were m a in ­

t a i n e d on a u n i f o r m d i e t

c o n s i s t i n g o f " f o x b l o c k s " ma nu ­

f a c t u r e d by A l l i e d H i l l s

Co. o f Wayne, I n d i a n a .

V a g in a l sm ears were the presence of th e

made d a i l y on t h e s e r a t s

estru s

cycle.

r e g u l a r c y c l e was d i s c a r d e d .

Any

anim al not

to e s t a b l i s h showinga

Those g i v i n g t h e p r o p e r r e s p o n s e

were o v a r ie c to m iz e d u n d er l i g h t

e t h e r a n e s t h e s i a a c c o rd in g to

th e te c h n i q u e d e s c r i b e d by B um ( 4 1 ) .

C a r e w as e x e r c i s e d i n

t h i s o p e r a t i o n t o remove a good p o r t i o n o f t h e f a l l o p i a n t u b e s and t h e

surrounding f a t in o rd e r to

o varian tis s u e .

remove a n y a c c e s s o r y

The a n i m a l s w e r e a l l o w e d t o

com pletely r e ­

c o v e r f r o m t h e o p e r a t i o n a n d a g a i n v a g i n a l s m e a r s v /e r e t a k e n fo r a p e rio d o f 2 weeks.

A nim als n o t showing a p erm an en t

d i e s t r u s w ere t h e n d i s c a r d e d . The s e c o n d s o u r c e o f a n i m a l s was t h e R e s e a r c h L a b o r a t o r i e s o f A y e r s t , McKenna a n d H a r r i s o n , M o n t r e a l , were r e c e iv e d a l r e a d y c a s t r a t e d .

the

These r a t s

V a g i n a l s m e a r s w e r e made f o r

2 to 3 weeks to c o n firm th e c a s t r a t i o n . m aintained under the

Canada.

These a n i m a ls were

same l a b o r a t o r y c o n d i t i o n s a s t h o s e

source f i r s t m entioned.

fiom

The a v e r a g e w e i g h t o f t h e a n i n a l s

7 from b o th s o u r c e s d u r in g t h e s e This e n t i r e

e x p e r i m e n t s w a s 150 gm.

group o f a n im a ls w ould s i m u l a t e t h e popu­

l a t i o n u sed i n most com m ercial a s s a y s a s c a r e and d i e t a r e

far as s e le c tio n ,

conoem ed, M aterials

S tandard O estro n e.

I n t e r n a t i o n a l Standard o f th e

E y d r o x y - K e t o n i c Form o f t h e O e s t r u s - p r o d u c i n g H o rm o n e , O b ta in e d from L a b o r a to r y o f H ygiene D epartm ent o f P e n s io n s and H e a lth , O ttaw a, A nm iotin. S.

Canada,

S o l u b l e g e l a t i n c a p s u l e s m a n u f a c t u r e d by

B, S q u i b b a n d S o n s , l a b e l l e d

u n its p er capsule,

t o c o n t a i n 2000 i n t e r n a t i o n a l

F u r th e r claim ed to

contain E s tr in ,

The

S s t r u s - P r o d u c i n g H or mo ne , F o lestrin ,

4 minim g l a n u l e s

(so lu b le g e la tin capsules)

m a n u f a c t u r e d b y The A rm o u r L a b o r a t o r i e s . la b e lle d to co n tain p u r if ie d The p o t e n c y i s

This p ro d u c t i s

e x tra c t of pregnant m ares1 u rin e .

e x p r e s s e d a s 2000 i n t e r n a t i o n a l

estrogenic

u n its per glan u le. E s t r o g e n i c Hormones. C am iek.

T a b l e t s m a n u f a c t u r e d b y Heed a n d

A c c o rd in g t o l a b e l e a c h t a b l e t c o n t a i n s m ixed

e s tr o g e n s from p r e n a t a l m a r e s 1 u r i n e in tern atio n al

estro n e u n its .

Brogynon-DH.

T a b l e t s m a n u f a c tu r e d by t h e

C o rp o ra tio n and l a b e l l e d It

i s also

eq u iv alen t to 1000

to

S hering

co n tain E stro g e n ic Substance.

e t a t e a t h a t e a c h c o n t a i n s 1 / 1 0 mg.

a - e s t r a d i o l o r 1200 Bat U n its

(A lle n -D o isy ).

of c ry sta llin e

8 P rem arin,

An e x p e r i m e n t a l s a m p l e o f t a b l e t s o f e s t r o n e

s u l f a t e m a n u f a c t u r e d b y a n d o b t a i n e d f r o m A y e r s t , McKenna and H a rris o n o f M o n tre a l, M eth y lstilb estro l

Canada.

( M e r c k 1s ) .

An e x p e r i m e n t a l s a m p l e

o f c r y s t a l l i n e 4 * 4 l - d i h y d r o x y - a l p h a - b e t a - d i e t h y l s t i l b e n e ob­ t a i n e d f r o m A y e r s t , McKenna a n d H a r r i s o n , M o n t r e a l ,

Canada.

The p r o b l e m o f b i o - a s s a y d o e s n o t a p p l y t o t h i s e stro g en since i t

synthetic

i s a c r y s t a l l i n e p r o d u c t and g e n e r a l l y

p r e s c r i b e d a s a c h e m i c a l e n t i t y on a w e i g h t b a s i s .

I t has

b e e n i n c l u d e d i n t h i s work f o r c o m p a r a t i v e p u r p o s e s .

I t was

a s s a y e d by o r a l and s u b c u ta n e o u s a d m i n i s t r a t i o n i n b o t h o i l and a q u e o u s mediums.

These t e s t s w ere c o n d u c te d e x a c t l y a s

th o se to be d e s c rib e d . I n a d d i t i o n t h e p o t e n c y o f t h i s p r e p a r a t i o n was d e ­ t e r m i n e d i n a q u e o u s me dium u s i n g a t o t a l o f drug.

of 5 ad m in istratio n s

T h e s e d o s e s w e r e s p a c e d a t 24 h o u r i n t e r v a l s .

A l l o f th e s e sam ples m entioned ex cep t th e Standard O estrone,

d ieth y lstilb estro l,

In tern atio n al

and P rem arin T a b le ts

w ere p u r c h a s e d on t h e open m a r k e t . Procedure D esign o f I n v e s t i g a t i o n . g a tio n i s such t h a t

The d e s i g n o f t h i s

in v esti­

com m ercial p r e p a r a t i o n o f e s t r o g e n i c

s u b s t a n c e s s o l d f o r o r a l t h e r a p y can be compared t o

the

i n t e r n a t i o n a l s t a n d a r d e s t r o n e b o th by o r a l and s u b c u ta n e o u s assay,

and a l s o t o

determ ine any d if f e r e n c e s in r a t i o

of

d i f f e r e n t t y p e s o f e s t r o g e n s w he n c o m p a r e d t o i n t e r n a t i o n a l

9 s t a n d a r d e s t r o n e "by t h e two m e t h o d s o f a d m i n i s t r a t i o n * F u rth er,

th e experim ents a re a rra n g e d fo r a s t a t i s t i c a l

e v alu a tio n of the

resu lts.

A rrangem ent and P r e p a r a t i o n o f A nim als. under t e s t

The a n i m a l s

f o r e a c h p r o d u c t w e r e d i v i d e d i n t o two g r o u p s .

One o f t h e s e g r o u p s r e c e i v e d t h e

e s t r o g e n by s u b c u ta n e o u s

i n j e c t i o n and th e o th e r by o r a l a d m i n i s t r a t i o n . These g ro u p s were f u r t h e r d i v i d e d i n

such a manner to

perm it a t l e a s t th r e e dosage l e v e l s f o r each ro u te o f m edication.

The v a l u e o f s u c h g r a d i e n t s i n d o s a g e l e v e l

h a s b e e n p o i n t e d o u t by v a r i o u s i n v e s t i g a t o r s

(5 ,2 2 ,2 5 ).

T h is m ethod f u r n i s h e s th e f o ll o w i n g i n f o r m a t io n : 1.

Changes i n t h e a b s o l u t e s e n s i t i v i t y o f t h e a n i m a l s .

2*

The s t a n d a r d e r r o r o f t h e a s s a y .

3.

The s i g n i f i c a n c e

of d ifferen c es in th e

r e s p o n s e by

t h e two r o u t e s o f a d m i n i s t r a t i o n . 4.

The h o m o g e n e i t y o f a g r o u p o f a n i m a l s u s e d on a n y

sin g le assay. The p a t t e r n o f t h e s e t e s t s w as s u c h t h a t r e g r e s s i o n l i n e s c o u ld be c a l c u l a t e d f o r each p ro d u c t by o r a l and sub­ c u ta n e o u s a d m i n i s t r a t i o n an d com pared t o t h o s e computed f o r th e stan d ard under th e

same e x p e r i m e n t a l c o n d i t i o n s .

One w e e k b e f o r e a t e s t

e ach a n im a l was " p r im e d ” by

s u b c u t a n e o u s i n j e c t i o n o n c e d a i l y o n two s u c c e s s i v e d a y s w i t h 3 gamma o f e s t r o n e i n 0 . 2 c c . a q u e o u s s o l u t i o n . p ro c e d u re h a s lo n g been used to response

(1).

This

in crease the u n ifo rm ity o f

V a g in al sm ears were th e n ta k e n and any an im al

10 n o t r e s p o n d i n g t o t h i s p r i m i n g d o se was d i s c a r d e d .

Seven

days a f t e r th e f i r s t prim ing dose a l l resp o n d in g anim als t h a t h ad r e t u r n e d t o a d i e s t r u s c o n d i t i o n were re a d y f o r assay. F o llo w in g one a s s a y th e a n im a ls were r e s t e d

f o r one

week b e f o r e p r i m i n g f o r a n o t h e r t e s t . The same r a t s w e r e u s e d t h r o u g h o u t t h e s e e x p e r i m e n t s . Those r e c e i v i n g a n e s t r o g e n hy m outh on one t e s t w e re i n ­ je c te d subcutaneously in th e n e x t.

M oreover,

the anim als

w e r e s h i f t e d f r o m o n e d o s a g e l e v e l t o a n o t h e r on s u c c e e d i n g assays.

There i s

evidence

(l)

th a t the

extent of g e n ita l

a t r o p h y i n c a s t r a t e s i n f l u e n c e s t h e amount o f e s t r o g e n r e ­ quired.

For t h i s re a so n prim ing and th e c ro s s -o v e r tech n iq u e

was e m ploye d. A d m i n i s t r a t i o n o f D osa ’g e .

D iffe re n t o p inions a re held

a s to th e number o f i n j e c t i o n s to he g iv e n i n assays.

estrogenic

T h e s e i n c l u d e t h e s i n g l e i n j e c t i o n m e t h o d o f Co w ar d

and Bum (2 4 ),

th e 4 d iv id e d doses o f H a rriso n and P arkes

(25),

and t h e 12 d i v i d e d do se m ethod a s d e s c r i b e d hy H ain and Robson ( 1 6 ) .

I n t h e p r o c e d u r e a d o p t e d i n t h i s work d o s e s

w ere a d m i n i s t e r e d tw ic e a day f o r 3 s u c c e s s i v e days m aking a to ta l o f 6 doses.

T h i s s y s t e m w as f o l l o w e d i n b o t h o r a l

and subcutaneous t e s t s

in order th a t

th e r e s u l t s would be

com parable. D i l u t i o n s f o r s u b c u ta n e o u s a d m i n i s t r a t i o n were e i t h e r i n c o t t o n s e e d o i l o r i n w a te r a c c o rd in g to t h e n a t u r e o f th e product being te s t e d .

S o l u t i o n s i n o i l w e r e p r e p a r e d on t h e

11 day o f th e f i r s t

i n j e c t i o n and k ep t in a r e f r i g e r a t o r d u rin g

th e rem ain d er of th e i n j e c t i o n period#

D ilu tio n s in w ater

were p r e p a r e d eac h day j u s t p r e v i o u s to i n j e c t i o n . s a m p l e u n d e r t e s t was a l w a y s c o m p ared t o t h e in the in 0 .2

same s o l v e n t . co.

The

stan d a rd given

O n e - s i x t h t h e t o t a l dose was c o n t a i n e d

a n d t h i s v o l u m e wa s a d m i n i s t e r e d t o

g a r d l e s s o f "body w e i g h t .

each r a t

re­

The i n j e c t i o n s w e r e made f r o m a

1 . 0 o c . L u e r s y r i n g e t h r o u g h a 1 /E i n c h ,

24 g a u g e n e e d l e

(B ecton, D ick in so n & C o .). S o l u t i o n s f o r o r a l a d m i n i s t r a t i o n were p r e p a r e d a t t h e same t i m e a n d i n t h e sam e m a n n e r a s t h o s e u s e d f o r s u b c u t a n ­ eous a d m in is tr a tio n .

These d i l u t i o n s were a l s o p r e p a r e d in

co tto n see d o i l o r w ater according to the n a tu re of th e p ro ­ duct.

A gain,

t h e p r o d u c t was compared to t h e

m in iste re d in the dose.

same s o l v e n t a n d i n t h e

standard ad­

same v o l u m e p e r

P r e p a r a t i o n s were d i l u t e d i n o i l t o c o n t a i n 1 / 6

t o t a l dose in 0 .3 cc.

T h o s e made i n a q u e o u s me diu m w e r e

d i l u t e d to c o n ta in 1 /6 the t o t a l dose in 1 .0 cc. volum es were g iv e n p e r r a t

irresp ectiv e

These

o f body w e ig h t.

The o r a l d o s e s w e r e p l a c e d d i r e c t l y i n t o t h e to p re v e n t th e l o s s o f any o f t h e com plished i n th e needle curved.

(B ecton,

estro g en .

fo llo w in g manner.

stom ach

T h i s w as a c ­

A 2 inch,

24 g a u g e

D i c k i n s o n & C o .) was b l u n t e d a n d s l i g h t l y

The e n t i r e

s h a f t w a s c o v e r e d by j u s t e n o u g h v e r y

sm all c a t h e t e r tu b in g to s l i g h t l y o v e rla p th e t i p need le.

the

T h i s n e e d l e when a t t a c h e d t o a 1 . 0 c c .

was v e r y e a s i l y i n s e r t e d d i r e c t l y i n t o t h e r a t ' s

of the

Luer S yringe stom ach.

12 V ag in al Sm ears*

The " b a s i s o f t h e v a g i n a l s m e a r a s a

o r i t e r i o n f o r e s t i m a t i o n o f e s t r o g e n i c a c t i v i t y grew out o f t h e w o r k o f S t o c k a r d a n d P a p a n i c o l a o u ( 2 6 ) who s h ow e d t h a t d if f e r e n t stag e s of th e

e s t r u s cyole o f th e guin ea p ig a re

c h a r a c t e r i z e d by c l e a r - c u t c h a n g e s i n t h e o f th e vagina.

c e llu la r contents

They d e f i n e d t h e p e r i o d s i n t h e g u i n e a p i g

e s t r u s c y c l e a c c o r d i n g t o t h e t e r m i n o l o g y o f Heape (27) a n d c o r r e la te d th e s e p e rio d s w ith the a c t i v i t y

of the o v aries

and th e corpus luteum . I n 1922 s i m i l a r v a g i n a l c h a n g e s c o r r e s p o n d i n g to t h e changes in th e e s t r a l

c y c l e were d e m o n s tr a t e d i n t h e

Lo ng a n d E v a n s ( 2 8 ) a n d i n t h e m o u s e b y A l l e n ( 2 9 ) . f o r m e r a u t h o r s showed t h a t t h e l e n g t h o f t h i s rat

cycle

r a t by The in the

i s a b o u t 4 to 5 days and t h a t c y c le s o c cu r i n c o n s ta n t

succession.

They d e f i n e d t h e p e r i o d s i n t h e

e s tr u s cycle

o f a r a t as follow s: F irst cycle.

Stage

(P ro estru s).

p a r t of the

sexual

The o v a r i a n f o l l i c l e s a r e b e g i n n i n g t o d e v e l o p .

nucleated e p ith e lia l c e lls tim e.

The f i r s t

a p p e a r i n th e v a g i n a l smear a t

Only th is

T h i s s t a g e l a s t s a b o u t 12 h o u r s . Second S tag e

(E stru s).

The p e r i o d o f h e a t w h e r e i n t h e

o v a r i e s e x h i b i t l a r g e m ature f o l l i c l e s . T h ird Stage fo llicles

(O vulation).

The s t a g e d u r i n g w h i c h t h e

r u p tu r e and r e l e a s e th e ova.

w ith stage 2 l a s t s

a b o u t 27 t o 30 h o u r s a n d t h e v a g i n a l s m e a r

c o n ta in s only c o m i f i e d F o u rth Stage

This sta g e to g e th e r

cells.

(M etaestrus).

Ova a r e

in th e f a l l o p i a n tube

13 and young c o rp o ra l u t e a a r e to be found,

The s m e a r d u r i n g

th is p erio d

shows l e u c o c y t e s a n d c o r n i f i e d c e l l s a n d t h i s

stage l a s t s

about

F i f t h Stage

6

hours#

(D iestru s).

i n g a b o u t 57 h o u r s .

This i s th e r e s t i n g

stage l a s t ­

H e re in the smear e x h i b i t s p r e d o m in a te ly

le u c o c y te s and o c c a s io n a lly e p i t h e l i a l c e l l s . These r h y th n io changes i n th e r a t o ccu r s p o n ta n e o u s ly u n d e r norm al c o n d i t i o n s b u t d i s a p p e a r im m e d ia te ly on c a s t r a t i o n . When t h e o v a r i e s a r e

re m o v e d * a p e r m a n e n t d i e s t r u s r e s u l t s .

The a c t i o n o f e s t r o g e n i c th e anim al in t o

substances in the c a s tr a te b rin g s

estru s.

A f t e r t h e i n t r o d u c t i o n o f t h e v a g i n a l sm ear a s a method o f e stim atin g th e a c ti v it y of estro g en ic m a te ria ls , began to ap p ear in th e l i t e r a t u r e

on t h e m e t h o d o f m a k i n g

s m e a r s a n d on w h a t c o n s t i t u t e s a p o s i t i v e A l l e n and D oisy (33)

d iscu ssio n

considered p o s itiv e

reactio n

(3 0 ,3 1,3 2).

o n ly sm ears showing

c o n s i d e r a b l e number o f c o r n i f i e d c e l l s a n d no l e u c o c y t e s , l a q u e u r ( 3 0 ) , Loewe ( 3 1 ) , a n d L i p s c h u t z proestrus end-point.

(34) a c c e p te d th e

smear (s m a ll n u c l e a t e d e p i t h e l i a l c e l l s )

a s an

K ahnt and D oisy (2) a d o p te d f o r t h e i r p o s i t i v e

r e a c t i o n na p r e d o m i n a n c e o f c o r n i f i e d c e l l s w i t h some nucleated e p ith e lia l

c e l l s a n d a v e r y fe w l e u c o c y t e s . "

L a q u e u r a n d DeJongh (3 5 )

considered th e disappearance o f

leu co cy tes as a su ita b le

en d -p o in t w hile B um

th at th is c rite rio n is d i e s t r u s sm ears.

too

(36)

states

e a s ily confused w ith v a r ia n tz o f

Kennedy (37)

d e c la r e d com plete d i s a p p e a r ­

ance of le u c o c y te s and c o m i f i c a t i o n o f a t l e a s t h a l f o f th e

14 ep ith elial

c e l l s to he a p o s i t i v e re s p o n s e ,

The r e p l a c e m e n t

o f l e u c o c y t e s an d predom inance o f o o r n i f i e d c e l l s i s a ls o a c o e p t e d h y D'Am our a n d G u s t a v s o n ( 1 7 ) a n d S o n d e m a n d Sealey

(S i).

M arrian

(58) s t a t e d t h a t f a u l t y i n t e r p r e t a t i o n

o f s m e a r s c o n s t i t u t e d one o f t h e m o s t f r e q u e n t s o u r c e s o f e rro r in

the b io lo g ic a l a ssa y o f th e e stro g en s.

Th e f r e q u e n c y o f t a k i n g t h e v a g i n a l s m e a r s i s a n o t h e r p o i n t o f d i s c u s s i o n among h i o - a s s a y i s t . took t h e i r re a d in g s 45, o f the

experim ent.

A lle n and D oisy ( 5 3 )

56 a n d 72 h o u r s f r o m t h e b e g i n n i n g

A lien ,

s m e a r in g once on t h e f i r s t

D i c k e n s a n d Dodds ( 3 9 ) rec om m en d day,

second d ay , t h i r d day and

f o u r t h d a y ; a n d 3 r e a d i n g s on t h e f i f t h d a y .

Among o t h e r

s u g g e s t i o n s a r e 3 0 , 4 2 , a n d 54 h o u r i n t e r v a l s

(40);

daily

(36);

54,

60 a n d 70 h o u r s a f t e r t h e o r i g i n a l i n j e c t i o n

(21); and 8 hour i n t e r v a l s in je c tio n

3 sm ears

s t a r t i n g 48 h o u r s a f t e r t h e f i r s t

(17).

The f r e q u e n c y o f s m e a r i n g i n t h i s p a r t i c u l a r B t u d y m e e t s th e re q u ire m e n ts s e t f o r t h hy th e H e a lth O rg a n iz a tio n o f th e leag u e of n a tio n s

(3 ,4 ).

s ta te d in t h i s re p o rt tak en a t the

As p r e v i o u s l y m e n t i o n e d i t wa s

t h a t t h e v a g i n a l sm ea rs s h o u l d he

same i n t e r v a l s f r o m t h e a n i m a l s u s e d f o r t h e

sta n d a rd p r e p a r a t io n and f o r th o se used f o r th e p r e p a r a tio n under t e s t .

I t was f u r t h e r s p e c i f i e d t h a t t h e s e

sm ears

s h o u l d h e t a k e n a t l e a s t o n c e d a i l y a n d t w i c e on t h e d a y wh en t h e maximum r e s p o n s e i s e x p e c t e d . I n o r d e r to fo llo w t h e s e s p e c i f i c a t i o n s and a t th e same t i m e t o

s im u la te g e n e r a l com m ercial p r o c e d u r e s ,

the

15 f o l l o w i n g sm ear i n t e r v a l was a d o p te d * startin g

hours a f t e r the l a s t

8

Smears w ere t a k e n

in jectio n .

A sec o n d smear

w a s t a k e n o n t h e same d a y 1 8 h o u r s a f t e r l a s t

in jectio n .

One s m e a r w a s t a k e n on e a c h o f t h e t w o s u c c e e d i n g d a y s a t in terv als

o f 12 h o u r s e a c h .

I t had been found by p r e l i m i n ­

a r y w o r k t h a t t h e g r e a t e s t r e s p o n s e o c c u r r e d on t h e d a y follow ing the l a s t

in jectio n .

M oreover, a f t e r 3 days th e

anim als had re tu rn e d to a d i e s t r u s The m e t h o d o f t a k i n g , i s as follow s: by f l u s h i n g

condition.

reading and reco rd in g th e

The v a g i n a l

sm ears

c o n t e n t s o f an a n im a l was removed

se v e ra l tim es w ith about 0 . 2

s o l u t i o n by means o f a p i p e t t e

co.

o f norm al s a l i n e

(m edicine-dropper).

p i p e t t e was u s e d t h r o u g h o u t th e t e s t s .

This

Any r e s i d u e f r o m a

p r e c e d i n g r a t was rem oved b e f o r e e a c h s u c c e e d i n g sm ear b y r i n s i n g i n c l e a n t a p w a t e r , t h e n i n 70

p er cent

a lc o h o land

f i n a l l y in the

of cells

wasexamined

salin e.

The s u s p e n s i o n

u n s t a i n e d and w h ile s t i l l w et u n d e r t h e low pow er o f t h e m ic r o ­ scope. The c e l l s o b s e r v e d i n t h e s e

sm ears a r e le u c o c y t e s ,

n u c l e a t e d e p i t h e l i a l c e l l s and squamous o r c o r n i f i e d For th e purpose o f t h i s o f squamous c e l l s

cells.

s tu d y a smear showing a predom inance

a n d no m o re t h a n a f e w l e u c o c y t e s a n d / o r

nucleated e p ith e lia l

c e l l s was ju d g e d p o s i t i v e .

In re c o rd in g the d a ta th e follow ing d e sig n a tio n fo r t h e d i f f e r e n t ty p e s o f sm ears o b s e rv e d

were u s e d ;

— - no c e l l B p r e s e n t 0

* occasional:

1

to 1 0 p e r

field

16 2

- f a i r l y num erous: of c e lls p resent.

a b o u t h a l f t h e number

3 s num erous: t h i s ty p e com prised most o f the c e ll s p re se n t. 4 ** t h e s m e a r c o n s i s t e d o f a l a r g e m a s s o f t h i s p a r t i c u l a r type o f c e l l . M a th e m a tic a l T reatm ent o f R e s u l t s P h a rm a c o lo g ic a l s t u d i e s upon a l a r g e v a r i e t y o f o rg a n ­ i s m s b y man y b i o l o g i s t s h a v e e s t a b l i s h e d t h e s i g m o i d c h a r a c ­ t e r o f th e ty p ic a l dosage-response curve, c e l l u l a r form s.

I t h a s b e e n s hown t h a t

e s p e c ia lly in m u lti-

s u c h c u r v e s c a n be

e a s i l y p l o t t e d a s s t r a i g h t l i n e s and t h e i r l a t e r a n a l y s i s thereby f a c i l i t a t e d

(4 2 ,4 3 ,4 4 ,4 5 ).

These s t r a i g h t l i n e s a r e

g i v e n t h e name " r e g r e s s i o n l i n e s . ” The f i t t i n g o f a d o s a g e - r e s p o n s e c u r v e t o a s e r i e s o f e x p e r im e n ta l o b s e r v a t i o n s , however cru d e o r r e f i n e d , t h e tech n iq u e i s an a tte m p t to i n f e r , in d iv id u als,

from a l i m i t e d number o f

th e e m p ir ic a l r e l a t i o n s h i p of dosage and r e s ­

ponse f o r a g iv en ag en t in an i n f i n i t e l y l a r g e r p o p u la tio n from w hich t h e y r e p r e s e n t o n ly a sam ple. The r e g r e s s i o n l i n e may be c o n s t r u c t e d g r a p h i c a l l y or c a lc u la te d m athem atically. w ill be very n e a r th e

Sometimes t h e g r a p h i c l i n e

c a lc u l a t e d one.

However, t h e c a l c u ­

l a t e d l i n e v r lll o f t e n r e p r e s e n t an im p o r ta n t c o r r e c t i o n to a graphic lin e . In th e se

experim ents the a r ith m e tic a l procedure o f f i t ­

t i n g h a s b e e n a d o p t e d and t h e m ethods and sym bols employed b y P i s h e r (4 6 ) a n d d e s c r i b e d by B l i s s

(42) have been u s e d .

17 The f o r m u l a f o r t h e r e g r e s s i o n l i n e may h e e x p r e s s e d as: Y a a - h (X - x ) . where t h e

sym bols h ave t h e

follow ing d e sig n a tio n s:

Y * r e s p o n s e i n p r o h i t s on t h e lin e. 2

reg ressio n

« any g iv e n dosage {in l o g a r ith m s ) c o r ­ r e s p o n d i n g t o t h e r e s p o n s e p r o h i t Y.

a a y * n um erically the average p ro h it fo r a l l d eterm in atio n s in th a t p a rt of th e ex p erim en t w hich i s b e in g f i t t e d . x a av erages of th e dosages a d m in is te re d ( i n l o g a r i t h m s ) f o r t h e same s e c t i o n of d ata. b - th e re g re s s io n c o e f f ic ie n t o r the slo p e o f th e line* It is,

th e re fo re , necessary to c alc u la te

perim en tal data th e

q u an tities x,

y, and h.

from t h e ex­ The f o r m u l a e

a r e aB f o l l o w s :

~ s S(wx) „ STwT-

- - S(wy) 7 S(w) * *b = S(wxy) - x S(wy) f A

A * S(wx2 ) - x S(wx) These sym bols a r e d e f i n e d a s : S ■ " t h e sum o f " w * w eight of a given o b s e r v a tio n , the product of the w eighting c o e f f ic ie n t m u l t i p l i e d hy t h e number o f a n i m a l s used. x = a f u n c tio n o f th e dose a d m in is te r e d ex p erim en tally , u s u a lly i t s lo garithm .

y e th e p r o h i t co rre sp o n d in g to th e ob­ served percentage response. The b a s i s

f o r t h i s m ethod o f c a l c u l a t i o n i s t h e n orm al

frequency d i s t r i b u t i o n curve

(42 ,4 5 * 4 7 ).

The p r o b i t v a l u e s

co rresp o n d in g to each p e rc en ta g e resp o n se and th e w eig h tin g c o e f f i c i e n t s a r e d e r i v e d from t h i s

curve m a th e m a tic a lly .

These v a l u e s can be fo u n d i n t a b l e s p r o p o s e d by B l i s s

(42).

19 Example o f C a l c u l a t i o n o f the re g re ssio n lin e I n te r n a tio n a l S tandard E strone

T o t a l Dose i n gamma

Humber o f P e r c e n t A nim als R esponse

in W ater— O ral Assay

X

*2

Y

101

22

36.6

2.0043

4.016

4.6575

126

22

66. 6

2.1004

4.41

5.4289

167

22

59.0

2.1959

4.8220

5.2275

196

22

86.36

2.2923

5.2446

6.0939

YJY

rrv HIJX-2

m

Y/XY

w eighting C oefficient

1/

• 609

13.40

26.8

53.8

62.5

124.9

.615

13.50

28.4

59.6

73.3

154.0

.623

13.70

30.1

66.0

71.6

157.0

.412

9.05

20.7

4 7.4

55.2

126.0

Sums

4 9 . 65

106.0

226.8

262*6

561.9

20

Example o f C a l c u l a t i o n o f th e R eg ressio n Line (co n tin u ed ) I n t e r n a t i o n a l S ta n d a rd E s tro n e i n T/ater— O ra l Assay

- _ S( m c) 106.0 _ p . x " S{w) B 4 9 .6 5 “ 2 *1 3

W

“

* ” STwl

-

2 6 2 « 6

“

C

p p r

4 9 . 6 5 " 5 *225

SS(wx) = 2 . 1 3 Z 1 0 6 .0 = 2 2 6 .0 xS(wy) - 2 .1 3 Z 2 6 2 .6 - 5 5 8 .0 A = Sf w x 2 ) - Srsfv/x) - 2 2 6 . 8 - 2 2 6 . 0 = 0 . 8 - S(v?xy - x S ( w y ) -

561.9 - 558.0 = 4 ^ 3 3 0.8

21

E x a m p l e o f C a l c u l a t i o n o f RD 50 I n t e r n a t i o n a l S ta n d a rd E stro n e i n W ater— O ral A ssay

Y = y * 13 (X - x ) 5 = 5.225 + 4 .88 X = 2.082

(X - 2 . 1 3 )

(logarithm )

KD 50 = 1 2 0 . 3

22 RESULTS

A ssay o f I n t e r n a t io n a l Standard E stron e in Water

T o t a l Do SQ i n gamma 0.50

Number o f P e r c e n t A nim als R esponse 10

30.0 TABLE I ,

0.625

10

2 0 .0

0.78

22

4 0 .9

0.97

22

68.18

1 .2 1

22

68.18

1.51

22

90.9

T o t a l Bose i n gamma

ITumber o f P e r c e n t Response A nim als

101

22

36.6

126

22

6 6 .6

157

22

59.0

196

22

86.36

Two s u b c u t a n e o u s d o s e s p e r day f o r 3 days

RD 50 = 0 * 9 0 6 8 gamma 5 value o f r e g r e s s io n l i n e - 9.08

TABLE I I .

Two o r a l d o s e s p e r day f o r 3 days

RB 50 s 1 2 0 . 3 gamma b value o f re g r e s s io n l i n e s 4.88

23 A ssay o f I n t e r n a t io n a l Standard E stron e in O il

T o t a l Dose i n gamma

Dumber o f P e r o e n t Response A nim als

0.78

15

6.6

0.97

25

36.0

1.21

35

31.1

RD 50 « 2 . 1 7 8 gamma

1.51

30

33.3

b value o f re g re s s io n l i n e s 1.795

1.84

20

45.0

2.25

20

55.0

T o t a l Dose i n gamma

Humber o f P e r c e n t Response A nim als

101

15

33.3

126

15

60.0

157

15

80.0

22 4

15

93.3

TABLE I I I .

TABLE IV.

Two s u b c u t a n e o u s d o s e s p e r day f o r 3 days

Two o r a l d o s e s p e r day f o r 3 days

RD 50 = 1 1 3 . 7 gamma b value o f r e g r e s s i o n l i n e s 4.986

24 P o ten cy o f I n t e r n a t i o n a l Standard E strone

S u b c u t a n e o u s RD 50 ( w a t e r ) = 0 * 9 0 6 8 gamma » 9 . 0 6 8 I.IT . O ral

RD 50 ( w a t e r )

■ 1 2 0 .3

gamma ■ 1 2 0 2

I.U .

S u b c u t a n e o u s RD 50 ( o i l )

a 2 . 1 7 8 gamma ■ 2 1 . 7 8 I . U .

O ral

a 1 1 3 . 7 gamma ■ 1 1 3 7

RD 50 ( o i l )

I.U .

R a t io b e tw e e n am ount o f s t a n d a r d e s t r o n e n e c e s s a r y to p r o d u c e a 5 0 ^ r e s p o n s e b y o r a l a n d s u b c u t a n e o u s m e th o d

O ra l ( w a te r ) - 1203 - 1 3 2 .7 9 .0 6 8 1 s . c . (w a te r) O ral S .C .

(o il) (o il)

= 1 1 3 .7 = 5 2 .3 2 .1 7 8 1

O ral O ral

(w a te r) (o il)

- 1203 - 1 .0 6 1137 1

S .C . S. C.

(w a te r) (o il)

- 9 .0 6 8 = .4 1 7 2 1 .7 8 1

25 A ssay o f E s tr o g e n ic Hormone T a b le t s i n Water TABLE V.

Two s u b c u t a n e o u s d o s e s p e r day f o r 3 days

T o t a l Lose in p a rts of ta b le ts

H umber of A n im a ls

P er oent R esponse

0 .0 0 7 8

20

25

0 .0 0 9 7

20

45

0 .0 1 2 1

20

85

RL 50 s 0 . 0 0 9 5 6 t a b l e t s b v a lu e o f r e g r e s s i o n l i n e b 9 .4 7 5

TABLE V I.

Two o r a l d o s e s p e r day f o r 3 days

T o t a l L o s e , Humber in p a rts of A n im a ls o f ta b le ts

Per cent R esponse

0 .5 4

10

20

1 .1 0

20

55

1 .5 7

20

80

RD 50 a 0.98 t a b l e t s b v a lu e o f r e g r e s s i o n l i n e

*

4 .9

26 P o ten cy o f E s tr o g e n ic Hormone T a b le ts

S u b c u t a n e o u s RD 50 E s t r o g e n i c Hormone T a b l e t s a 0 . 0 0 9 5 6 t a b l e t s S u b c u t a n e o u s RD 50 E s t r o n e i n w a t e r

* 0 . 9 0 6 8 gamma

O r a l RD 50 E s t r o g e n i c Hormone T a b l e t s « 0 . 9 8 t a b l e t s O r a l RD 50 E s t r o n e i n w a t e r

= 1 2 0 . 3 gamma

L a b e lle d P o te n c y

1 T a b le t ■ 1000 I.U .

By S .C . A s s a y

1 T a b l e t = 9 5 . 0 gamma e s t r o n e * 950 I . U .

By O r a l A s s a y

1 T a b l e t - 1 2 5 . 4 gamma e s t r o n e « 1 2 5 4 I . U .

O ra l:S u b c u ta n e o u s r a t i o

ex p ressed in

I.U .

O ral 1254 - 1 .3 2 S7U7 s 950 “ " 1

O ral:Subcutaneous r a t i o e x p r e ss e d i n p a r t s o f t a b l e t s O ral 0 .9 8 8 1 0 2 .5 STU7 ” 0 . 0 0 9 5 6 I

27 A ssay o f Progynon-2H T a b le t s i n Water TABLE V I I .

T o t a l D ose . in p a rts of ta b le ts

Two s u b c u t a n e o u s d o s e s p e r day f o r 3 days

Dumbe r of A n im a ls

Per cent R esponse

0 .0 0 3 2 1

10

50

0 .0 0 5 0

20

65

0 .0 0 7 8

20

85

RD 50 3 0 . 0 0 3 6 1 t a b l e t s b v a lu e o f r e g r e s s io n l i n e TABLE V I I I .

= 3 .2 1

Two o r a l d o s e s p e r day f o r 3 days

T o t a l Do s< i Dumber in p a rts of A n im als of ta b le ts

Per cent R esponse

0 .3 7 9

10

4 0 .0

0 .5 4

19

6 3 .1 5

0 .7 7

20

8 0 .0

1 .1 0

10

1 0 0 .0 ____,

ED 50 = 0 .4 6 t a b l e t s b v a lu e o f r e g r e s s i o n l i n e = 4 .5 8

28

P o te n c y o f Progynon-DH T a b le ts

S u b c u t a n e o u s HD 50 P ro g y n o n -D H T a b l e t s - 0 . 0 0 5 6 1 t a b l e t s S u b c u t a n e o u s RD 50 E s t r o n e i n w a t e r a 0 . 9 0 6 8 gamma O r a l RD 50 P r o g y n o n - D E T a b l e t s « 0 . 4 6 t a b l e t s O r a l RD 50 E s t r o n e i n w a t e r a 1 2 0 . 3 gamma

L a b e lle d P o te n c y

1

T a b l e t « 0 . 1 mg. a e s t r a d i o l

By S .C . A s s a y

1

T a b l e t = 251*0

gamma e s t r o n e = 2 ^5 1 0 I.U *

By O r a l A s s a y

1

T a b le t • 2 6 2 .0

gamma e s t r o n e =■ 2 , 6 2 0 I . U .

O ra l:S u b c u ta n e o u s r a t i o O r a l ________ - 2 6 2 0 S u b c u t a n e o u s “ 251 0

e x p re sse d i n I.U .

s 1 .0 4 2 " I

O ra l:S u b c u ta n e o u s r a t i o

ex p ressed in p a r ts o f t a b l e t s

O ral _ 0 .4 6 _ 1 2 7 .5 S u b c u ta n e o u s “ 0 .6 0 3 6 1 " T

29 Assay o f Premarin T a b le ts i n Water

TABLE IX ,

Two s u b c u t a n e o u s d o s e s p e r day f o r 3 days

T o t a l Lose in p a rts o f ta b le ts

H um ber of A n im a ls

P er oent R esponse

0 .0 0 1 6 8

41

1 4 .6

0 .0 0 2 5 2

41

3 6 .7

0 .0 0 3 7 8

44

8 4 .2

RL 50 - 0 . 0 0 2 7 6 t a b l e t s b v a lu e o f r e g r e s s io n l i n e

TABLE X.

- 4 .6 9

Two o r a l d o s e s p e r day f o r 3 days

T o t a l L ose H um ber of in p a rts o f t a b l e t £ A n im a ls

Per cent R esponse

0 .0 0 7 2

43

1 6 .2

0 .0 2 0 8

44

3 6 .5

0 ,0 3 0

45

6 9 .0

,

RD 50 » 0 .0 2 3 t a b l e t s b v a lu e o f r e g r e s s i o n l i n e = 4 . 8

30

P o ten cy o f Premarin T a b le ts

S u b c u t a n e o u s RD 50 P r e m a r i n T a b l e t s » 0 . 0 0 S 7 6 t a b l e t s S u b c u t a n e o u s RD 50 s t a n d a r d e s t r o n e i n w a t e r - 0 . 9 0 6 8 gamma O r a l RD 50 P r e m a r i n T a b l e t s - 0 . 0 2 3 t a b l e t s O r a l RD 50 s t a n d a r d e s t r o n e i n w a t e r = 1 2 0 * 3 gamma

L a b e l l e d P o t e n c y - unknow n By S*C. A s s a y

- 1 T a b l e t * 3 2 8 , 9 gamma e s t r o n e ■ 3 * 2 8 9 I . U .

By O r a l A s s a y

- 1 T a b l e t a 5 2 3 0 . 0 gamma e s t r o n e a 523 00 I . U .

O ra l:S u b c u ta n e o u s r a t i o

e x p re s s e d i n I.U .

O r a l _ 5230 - 1 5 . 9 S .C . “ 3 2 8 . 9 ~ 1

O ral:Subcutaneous r a t i o e x p r e sse d i n p a r t s o f t a b l e t s O ra l _ 0 .0 2 3

- 8 .3 4

SmTT - o'.TOWff ~ “I

31 A ssay o f Am niotin C apsules i n O il

T o t a l Dos* j H um ber in p a rts of o f c a p s u le s A n im a ls

P er cent R esponse TABLE X I .

0 .0 0 3 1 2 5

15

3 3 .3

0 .0 0 3 9

25

2 4 .0

0 .0 0 4 8 5

25

2 4 .0

0 .0 0 6 0 5

15

6 6 .6

T o t a l Lose Humber in p a rts of o f c a p s u le s A n im a ls 0 .5 0 5

10

Per cent R esponse

Two s u b c u t a n e o u s d o ses p e r day f o r 3 days

RL 50 ® 0 . 0 0 6 3 4 c a p s u l e s b v a lu e o f r e g r e s s io n l i n e = 2 .5 9 3

TABLE X I I .

Two o r a l d o s e s p e r day f o r 3 days

2 0 .0 RD 50 ® 1 . 0 5 c a p s u l e s

0 .5 5

14

7 .1 4

0 .6 3

10

3 0 .0

0 .7 8 5

24

4 1 .7

1 .1 2

15

7 3 .3

b v a lu e o f r e g r e s s i o n l i n e * 1 . 82

32 P o ten cy o f Amniotin Capsules

S u b c u t a n e o u s HD 50 A m n i o t i n C a p s u l e s * 0 . 0 0 6 3 4 c a p s u l e s S u b c u t a n e o u s HD 50 s t a n d a r d e s t r o n e i n o i l = 2 * 1 7 8 gamma O r a l RD 50 A m n i o t i n C a p s u l e s = 1 . 1 5 c a p s u l e s O r a l HD 50 s t a n d a r d e s t r o n e = 1 1 3 . 7 gamma

l a b e l l e d P o te n c y

1 c a p su le

» 2 0 00 I . U .

By S .C . A s s a y

1 c a p su le

= 3 4 3 gammae s t r o n e

By O r a l A s s a y

1 c a p su le

» 9 8 . 9 gamma e s t r o n e =

O r a l : S u b c u ta n eo u s r a t i o

«3 4 3 0

989 I . U .

e x p re s s e d i n I.U .

O ra l . 9 8 .9 « 0 .2 8 8 STST * “ 34 3 I O ra l:S u b c u ta n e o u s r a t i o O ra l « 1 .1 5 ... 1 8 1 . 5 STS7 “ C.00'631' " 1

I.U .

ex p ressed in p a r ts o f c a p su le s

33

A ssa y o f F o l e s t r i n G lan ules i n O il

T o t a l D o se in p a rts o f g la n u le s

Number of A n im a l s

P er oent R esponse TABLE X I I I ,

Two s u b c u t a n e o u s d o se s p e r day f o r 3 days

0 .0 0 3 1 2 5

15

6. 6

0 .0 0 3 9

24

3 3 .3

RD 50 = 0 . 0 0 8 7 7 g l a n u l e s

0 .0 0 4 8 5

24

2 0 .8

b v a lu e of re g re s s io n lin e = 2 .1 3

0 .0 0 6 0 5

15

4 0 .0

T o t a l D ose in p a rts o f g la n u le s

Hum ber of A n im a l s

P er cent R esponse

0 .3 8 5

5

4 0 .0

0 .5 0 5

10

2 0 .0

TABLK XIV.

Two o r a l d o s e s p e r day f o r 3 days

RD 50 s 0 . 6 3 g l a n u l e s 0 .5 5

31

2 7 .3

0 .6 3

10

5 0 .0

0 .7 8 5

25

3 2 .0

1 .1 2

15

8 0 .0

b v a lu e o f r e g r e s s i o n l i n e = 1 .7 9

34 P o ten cy o f F o l e s t r i n G lan ules

S u b c u t a n e o u s RD 50

F o l e s t r i n G la n u le s = 0 .0 0 8 7 7 g l a n u l e s

S u b c u t a n e o u s RD 50

sta n d a rd e stro n e

O r a l HP 50 P o l e s t r i n

in o il

= 2 , 1 7 8 gamma

G la n u le s ■ 0 ,6 3 g la n u le s

O r a l HP 50 s t a n d a r d e s t r o n e i n o i l = 1 1 3 . 7 gamma

l a b e l l e d P o te n c y

1 g l a n u l e = 2000 I . U ,

By S IC . A s s a y

1 g la n u le

s 2 5 9 . 0 gamma e s t r o n e = 2590 I . U .

By O r a l A s s a y

1 g la n u le

■ 1 7 3 . 6 gamma e s t r o n e « 1 7 3 6 I . U .

O ra l:S u b c u ta n e o u s r a t i o S S a ’ 1"

= 1 W 8590 "

e x p re s s e d i n I.U .

- 0 .6 7 8 1

O r a l: Subcutaneous r a t i o e x p r e sse d i n p a r t s o f g l a n u le s O ral _ STTTT *

0 .6 3 _ 0 . 0 0 8 7 7 “ 1±±5L 1

35 A ssay o f D i e t h y l s t i l b e s t r o l i n Water

T o t a l D ose HumBer o f P e r c e n t i n gamma A n im a ls R espo nse 0*060

34

9 7 .1

0 .0 7 5

35

9 1 .4

0 .1 7 1

34

5 2 .9

0 .2 4 0

13

4 6 .2

0 .4 2 0

25

2 8 .0

0 . 600

10

1 0 .0

TABLE XV.

One s u b c u t a n e o u s d ose p e r day f o r 3 days

RD 50 » 0 . 1 5 4 4 gamma

T o t a l D ose n u m b e r o f P e r c e n t A n im a ls R espo nse i n gamma

0 .6 0

11

1 8 .2

0 .9 0

13

5 3 .8

1 .2 0

24

4 1 .7

1 .8 0

37

6 2 .2

2 .7 0

13

8 4 .6

“b v a l u e o f r e g r e s s i o n l i n e = 1*72

TABLE XVI.

One o r a l d o s e p e r day f o r 3 days

RD 50 s 1 . 2 5 9 4 gamma

b v a lu e o f r e g r e s s i o n l i n e = 2 .5 8

36 A ssa y o f D i e t h y l s t i l b e s t r o l i n Water

TABLS X V II,

Two s u b c u t a n e o u s d o s e s p e r day f o r 3 days

T o t a l D ose Dumber o f P e r c e n t i n gamma A n im a ls R esp on se 0 .0 7 5

12

1 6 .7

0 .2 4

12

3 3 .3

0 .4 2

12

7 5 .0

HD 50 * 0 , 2 4 gamma b v a lu e o f re g re s s io n l i n e

TASLK X V I I I .

~ 2 .5 8

Two o r a l d o s e s p e r day f o r 3 days

T o t a l D ose Dumber o f P e r c e n t A n im a ls R esponse i n gamma 0 .5 2

12

2 5 .0

1 .2 0

13

6 1 .5

2 .7 0

12

9 1 .6

RD 50 ■ 1 .0 3 3 0 gamma b v a lu e o f r e g r e s s i o n l i n e = 3 .9 2

37 A ssay o f L i e t h y l s t i l b e s t r o l i n O il

TABLE XIX.

Two s u b c u t a n e o u s d o s e s p e r day f o r 3 d ay s

T o t a l L os 4Bumber o f P e r c e n t i n gamma A n im a ls R esp o n se 0 .0 7 5

12

8 .3

0 .2 4

12

2 5 .0

0 .4 2

11

6 3 .6

RL 50 - 0 . 3 3 9 gamma b v a lu e o f re g r e s s io n lin e

TABLE XX.

= 2 .4 2

Two o r a l d o s e s p e r day f o r 3 days

T o t a l L o s e Rum ber o f P e r c e n t A n im als R esp o n se i n gamma 0 .5 2

12

8 .3

1 .2 0

12

2 5 .0

2 .7 0

12

7 5 .0

RL 50 » 1 .7 5 4 4 gamma b v a lu e o f r e g r e s s i o n l i n e * 4 .4 7

38

P o ten cy o f D i e t h y l s t 1113e s t r o l

A ssa y by s i x d o se m eth o d i n w a t e r S u b c u t a n e o u s RD 50 d i e t h y l s t i l b e s t r o l = 0 . 2 4 gamma S u b c u t a n e o u s RD 50 s t a n d a r d e s t r o n e

- 0 . 9 0 68 gamma

O r a l RD 50 d i e t h y l s t i l b e s t r o l = 1 . 0 3 3 gamma O r a l RD 50 s t a n d a r d e s t r o n e « 1 2 0 * 3 gamma

P o te n c y by s u b c u ta n e o u s a s s a y 0 . 1 gamma d i e t h y l s t i l b e s t r o l = 0 . 3 7 8 gamma e s t r o n e = 3 . 7 8 I . U . P o te n c y by o r a l a s s a y 0 . 1 gamma d i e t h y l s t i l b e s t r o l « 1 1 . 9 3 gamma e s t r o n e ** 1 1 9 . 3 I . U

O ra l:S u b c u ta n e o u s e x p re sse d in I.U . O ral 1 1 9 .3 . 3 1 .6 0 S .C . = 3 . 7 6 "* 1 O r a l : S u b c u ta n eo u s r a t i o O ra l - 1 .0 3 3 _ 4 .5 S. C. ” 0 . 2 4 1

e x p r e s s e d i n gamma

39

P o te n c y o f D i e t h y l s t i l b e s t r o l

A ssay by s i x d o s e m eth o d i n o i l S u b c u t a n e o u s HD 50 d i e t h y l s t i l b e s t r o l = 0 . 3 3 9 gamma S u b c u t a n e o u s RD 50 s t a n d a r d e s t r o n e = 2 . 1 7 8 gamma O r a l HD 50 d i e t h y l s t i l b e s t r o l »1 . 7 5 4 4 O r a l HD 50 s t a n d a r d

gamma

e s t r o n e » 1 1 3 . 7 gamma

P o te n c y by su b c u ta n e o u s a s s a y 0 . 1 gamma d i e t h y l s t i l b e s t r o l - 0 . 6 4 1 gamma e s t r o n e a 6 . 4 1 I . U . P o t e n c y by o r a l a s s a y 0 . 1 gamma d i e t h y l s t i l b e s t r o l O ra l:S u b c u ta n eo u s r a t i o

= 6 . 4 7 gamma e s t r o n e « 6 4 . 7 I . U .

ex pressed in

I.U .

O ra l - 6 4 .7 _ 1 0 .0 9

S7U7 “ £741 "

1

O ra l:S u b c u ta n e o u s r a t i o

e x p r e s s e d i n gamma

O r a l _ 1 . 7 5 4 4 _ 5 .1 7 S7TTT ” 0 . 3 3 9 1

40

P o ten cy o f D i e t h y l s t i l b e s t r o l

A s s a y b y t h r e e d o s e m e th o d i n w a t e r S u b c u t a n e o u s RD 50 d i e t h y l s t i l b e s t r o l = 0 . 1 5 4 4 gamma O r a l HP 50 d i e t h y l s t i l b e s t r o l s 1 . 2 5 9 4 gamma

O ra l:S u b c u ta n eo u s r a t i o

e x p r e s s e d i n gamma o f d i e t h y l s t i l b e s t r o l

O ra l _ 1 .2 5 9 4 _ 8 .2 4 S .C . “ 0 . 1 5 4 4 " T~

41 S UMMARY

^ b I£

OF

RESULTS

1

RD 50 i n parts o f a unit (c a p s ,, t a b ., e t c .) Pro du c t Oral

S.C.

Estrogenic Hormone Tablets Premarin Tablets Progynon-DH Tablets

0.9068

S.C.

Oral

ii

u

Estrone fin water)

Potency per unit in I.U.

0.00361

Amniotin Capsules Folestrin Glanules

2.178

"

0.00634

0*24

4.88

9.475

4.9

0.023

3,289

52,300

15.9 1

8.34 1

4.69

4.8

0.46

2,510

2,620

1.042 1

127.3 1

3.21

4.58

1.795

4.986

113.7

»r

3,430

989

0.288 1

181.5 1

2.593

1.82

2,950

1,736

0.678 1

71.8 1

2.13

1.79

31.60 1

4.3 1

2.58

3.92

10.09 1

5.17 1

2.42

4.47

8.24 1

1.72

2.58

0.63

”

1.033”

D ie th y lstilb e str o l fin o i l)

0.339

n

1.7 544 ”

D ie th y lstilb e str o l (in water ) '

0.1544 ”

1.2594 ”

" “ gamma 1 b 3 dose method

9.08 102.5 1

0.1 gamma 0.1 gamma s 3.78 I.U. s 119.3 I.U. D ie th y lstilb e s tr o l (in water)

Oral

1.32 1

1.05

0.00877

S.C.

1,254

52.3 1 Estrone fin o i l )

b values

950

0.98

0.00276

in I.U.

Oral _ , . Ratio in parts o f unit

132.7 1

120.3

0.00956

Oral _ . . S.C. Ratio

0.1 gamma 0.1 gamma b 6.41 I.U. s 64.7 I.U.

42 DISCUSSION liany commercial e s t r o g e n s in g e n e r a l u se today are o f v a r i a b l e and unknown c o m p o sitio n .

The U .S .P . XI or P.P. VI

do n o t s e t f o r t h any o f f i c i a l a s s a y method, hut th e m a jo r ity o f such e s t r o g e n i c p r o d u cts ‘are a ss a y e d su b c u ta n ec u sly a g a in s t i n t e r n a t i o n a l standard e s t r o n e , and t h e p o te n c y i s e x p r e ss e d in in te r n a tio n a l u n it s . ’Then an e s t r o g e n i c product i s a ssa yed a g a in s t a sta n d a r d t th e p o te n c y found should alw ays he th e same when e x p r e sse d in term s o f th e standard r e g a r d l e s s o f t h e method o f a s s a y . T h is i s based on th e assum ption th a t i f both th e sample and th e stan dard are handled i n t h e same way the t e s t anim als w i l l vary to both i n t h e sarne manner.

C on sequ en tly, the

same p o te n c y sh ou ld he o b tain ed r e g a r d l e s s o f t h e a s s a y method. S in ce many o f t h e commercial e s tr o g e n s are in te n d ed f o r o r a l th erap y and a ssayed by th e subcutaneous method t h i s i n v e s t i g a t i o n was performed to t e s t the above mentioned c o n c e p t. The data s e t f o r th under " R e s u lts 11 i n t h i s paper show th a t t h i s th e o r y does not h o ld tru e even fo r c r y s t a l ­ l i n e standard e s t r o n e i t s e l f . \7hen standard e s t r o n e i s t e s t e d in an aqueous medium the subcutaneous RD 50 i s 0 .9 0 6 8 gamma w h ile th e o r a l RD 50 i n the same medium i s 1 2 0 .3 gamma (s e e t a b l e I and I I ).

43 T he r a t i o

b e t w e e n t h e s e tw o

When t h i s

sam e c o m p a r i s o n i s made u s i n g a n o i l y s o l v e n t

t h e sam e r a t i o tru e . is

doses,

th e re fo re ,

i s 1 :1 3 2 .7 .

s h o u ld h o ld i f t h e p r e v io u s a ss u m p tio n i s

H o w e v e r, t h e

s u b c u t a n e o u s RD 50 f o u n d i n t h i s c a s e

2 . 1 7 8 gamma w h i l e t h e o r a l RD 50 r e m a i n s p r a c t i c a l l y t h e

sam e ( 1 1 5 . 7 gamma) a n d t h e r a t i o d o s e s i s now 1 : 5 2 . 3 T h is i s

(see ta b le

e x i s t i n g b e t w e e n t h e s e tw o

I I I and IV ).

an i n d i c a t i o n t h a t an e s tr o g e n ic r e f e r e n c e s ta n d ­

a rd a lo n e i s n o t a d eq u a te to in s u re u n if o r m ity o f p o te n c y . It

s u g g e s t s t h a t a s t a n d a r d a s s a y m e th o d i s a l s o n e c e s s a r y

i f u n i f o r m i t y o f r e s u l t s a r e t o be o b t a i n e d . fu rth e r

e x a m in a tio n o f th e

The p r o d u c t s t e s t e d

d a ta b e a rs out t h i s

s ta te m e n t.

show g r e a t v a r i a t i o n i n p o t e n c y b y s u b ­

c u t a n e o u s a n d o r a l a s s a y when c o m p a r e d t o t h e m s e l v e s b y t h e s e tw o p r o c e d u r e s o r w h e n c o m p a r e d t o t h e i n t e r n a t i o n a l s t a n d a r d . The d o s a g e n e c e s s a r y t o p r o d u c e a 50'^ r e s p o n s e w as g r e a t e r by th e o r a l th a n by t h e p ro d u ct te s te d

( s e e t a b l e X X I) .

su b cu ta n e o u s a ss a y f o r ev ery The o r a l - s u b c u t a n e o u s r a t i o

b e t w e e n t h e s e tw o d o s e s v a r i e d f o r t h e d i f f e r e n t p r o d u c t s and th e v a r i a t i o n s t i l l

e x i s t e d w hen t h e RD 50 v a l u e s w e re

com pared to t h e c o r r e s p o n d i n g v a lu e s f o r s t a n d a r d and e x p re ssed in in te r n a tio n a l u n its .

e stro n e

The e x t r e m e s o f

v a r i a t i o n w e r e show n b y d i e t h y l s t i l b e s t r o l a n d A m n i o t i n ( s e e t a b l e XX).

The f o r m e r r e q u i r e d 4 . 3 t i m e s t h e c o r r e s ­

p o n d i n g s u b c u t a n e o u s d o s e t o p r o d u c e a 5 0 fa o r a l r e s p o n s e and th e l a t t e r

r e q u ir e d 1 8 1 .5 tim e s i t s

co rre sp o n d in g sub-

44 c u t a n e o u s d o s e t o p r o d u c e a 50fo o r a l r e s p o n s e # ed in i n t e r n a t i o n a l u n i t s , 0#288 A m n io tin ) t o

When e x p r e s s ­

th e o r a l p o te n c ie s v a rie d

51#6 ( d i e t h y l s t i l b e s t r o l )

from

tim e s t h e i r c o r r e s ­

p o n d in g su b c u ta n e o u s p o te n c ie s # Two o f t h e m a t e r i a l s i n c l u d e d and d i e t h y l s t i l b e s t r o l ) p o te n c y i s c ia lly

( T a b l e t s o f P ro g y n o n -D H

w ere c r y s t a l l i n e

e x p r e s s e d by w e ig h t#

e s t r o g e n s w hose

N e i t h e r i s a s s a y e d commer­

a g a i n s t s t a n d a r d e s t r o n e b u t th e y w ere i n c l u d e d to

show w h a t v a r i a t i o n i s p o s s i b l e w hen tw o d i f f e r e n t a s s a y m e th o d s a r e used# The p r o d u c t s a s s a y e d i n o i l y m edium g a v e l o w e r v a l u e s b y o r a l a d m i n i s t r a t i o n t h a n b y t h e s u b c u t a n e o u s r o u t e when e x p ressed in in te r n a tio n a l U n its

( s e e t a b l e X X I) .

On t h e

o th e r hand p ro d u c ts t e s t e d in w a te r had h ig h e r e s tro n e v a l u e s b y t h e o r a l m e t h o d t h a n by t h e s u b c u t a n e o u s t e c h n i q u e # W h eth e r o r n o t t h i s w as a f u n c t i o n of t h e s ta te d

sin c e a l l

s o lv e n t can n o t be

e x c e p t d i e t h y l s t i l b e s t r o l w ere c o m m e rcial

p ro d u c ts and th e t r u e n a tu r e

o f t h e a c t i v e p r i n c i p l e s c o u ld

n o t be a s c e r t a i n e d . The p o t e n c y o f e a c h o f t h e s e e s t r o g e n s w h ic h w as s t a t e d on t h e l a b e l i n te r m s o f e s t r o n e w ere c o n firm e d by t h e su b ­ c u t a n e o u s m e th o d o f a s s a y . p r o d u c ts w ere a s s a y e d o r a l l y

C o n v e r s e l y , when t h e s e

same

some w e r e b e lo w l a b e l p o t e n c y

and o t h e r s w ere c o n s i d e r a b l y s t r o n g e r th a n d e p i c t e d

(see

t a b l e X X I). P r o g y n o n -D H T a b l e t s and E s t r o g e n i c Hormone T a b l e t s

( r & C)

45 w hose o r a l : s u b c u t a n e o u s r a t i o

ap proaches th a t o f sta n d a rd

e s t r o n e gave s i m i l a r v a lu e s by b o th m e th o d s, t h e

su b c u ta n e o u s

v a lu e b e in g s l i g h t l y lo w e r in each c a s e . A m n io tin C a p s u le s a n d F o l e s t r i n G la n u le s w ere w e ll w i t h i n t h e a d v e r t i s e d p o t e n c y by s u b c u t a n e o u s a s s a y a n d b e l o w t h e l a b e l l e d v a l u e when t e s t e d o r a l l y

(see page 32).

T he p o t e n c y o f P r e m a r i n T a b l e t s w as n o t e x p r e s s e d s i n c e t h i s w as an e x p e r im e n ta l sam p le.

H o w e v e r, i t s

su b cu ta n e o u s

p o t e n c y w as g o o d i n c o m p a r i s o n w i t h t h e o t h e r l i k e p r o d u c t s and i t s

o r a l p o te n c y f a r exceeded th e su b c u ta n e o u s v a lu e

( s e e t a b l e X X I). A d i f f e r e n c e b e t w e e n t h e two a s s a y m e t h o d s i s a l s o sho w n b y t h e r e l a t i v e b v a l u e s o b t a i n e d f r o m t h e r e g r e s s i o n lin e s

( s e e t a b l e X X I).

The v a l u e s o b t a i n e d b y s u b c u t a n e o u s

a s s a y s ra n g e from 1 .7 9 5 to 9 .4 7 5 f o r s i m i l a r a s s a y s . e ra lly ,

th e

Gen­

f i g u r e s o b t a i n e d w hen a n o i l y s o l v e n t w a s e m p lo y e d

w as lo w e r th a n t h e v a lu e s f o r w a te ry d i l u t i o n s . The b v a l u e s f o r t h e 4 .9 8 6 f o r th e th a n 3 .9 2 .

o r a l a s s a y s r a n g e d from 1 .7 9 t o

s i x d o s e t e c h n i q u e w i t h o n l y tw o v a l u e s l o w e r

B o th o f t h e s e lo w v a l u e s w e r e o b t a i n e d fro m t e s t s

c o n d u cte d u s in g o i l y In th e t e s t

so lv e n ts.

o f d i e t h y l s t i l b e s t r o l w hen 3 d o s e s w e r e

a d m i n i s t e r e d t h e s u b c u ta n e o u s and o r a l b v a l u e s w ere 1 .7 2 and 2 .5 8 r e s p e c t i v e l y . The d i f f e r e n c e

i n r a n g e o f t h e s e tw o s e r i e s o f b v a l u e s

shows t h a t t h e u n i f o r m i t y o f r e s p o n s e i s g r e a t e r f o r th e

46 o r a l m eth o d o f a s s a y .

M o reo v er, t h e lo w e r v a lu e s o b ta in e d

w hen o i l y s o l v e n t s a r e u s e d i n d i c a t e s a g r e a t e r animal v a r i a t i o n t o t h i s m ed iu m t h a n t o w a t e r . T he r e l a t i v e l y lo w v a l u e s o b t a i n e d f o r d i e t h y l s t i l b e s t r o l w hen t e s t e d b y t h e t h r e e

d o s e m e th o d p o i n t s o u t t h a t t h i s

m e th o d i s l e s s u n if o r m and s e n s i t i v e

t h a n a m e th o d i n v o l v i n g

six d o ses. Prom t h e f o r e g o i n g , n a tio n a l

it

is

c o n clu d e d t h a t th e i n t e r ­

s ta n d a r d e s tr o n e i s n o t enough i n i t s e l f to

u n ifo rm p o te n c y o f e s tr o g e n ic p r e p a r a t i o n s . v is a b le th a t fic ia lly

see m s a d ­

a s ta n d a r d a s s a y te c h n iq u e sh o u ld a ls o be o f ­

a d o p ted .

g iv e a b e t t e r

It

in su re

In t h i s r e g a r d , an o r a l a s s a y sh o u ld

e v a lu a tio n o f th e p o te n cy a s e x e r te d c l i n i c a l l y

by o r a l t h e r a p y .

47

SUMMARY A stu d y o f th e p o te n c y o f e s tr o g e n ic p r e p a r a t io n s , a s ex p ressed in in te r n a tio n a l u n its ,

when tofe* a s s a y e d h y

o r a l a n d s u b c u ta n e o u s a d m i n i s t r a t i o n , s h o w s w id e v a r i a t i o n i n t h e p o t e n c i e s o b t a i n e d b y t h e tw o m e t h o d s .

The r a t i o s

b e t w e e n t h e s e tw o p o t e n c i e s a s w e l l a s t h e o r a l : s u b o u t a n e o u s ra tio

ex p ressed in p a r ts

o f th e p r e p a r a t i o n s th e m s e lv e s

d i f f e r m a rk e d ly from one p r o d u c t t o a n o t h e r . C o m m e r c ia l p r e p a r a t i o n s p u r c h a s e d o n t h e o p e n m a r k e t w ere fo u n d t o

co n fo rm w ith t h e s t r e n g t h a s e x p r e s s e d on

t h e l a b e l w hen t e s t e d by s u b c u t a n e o u s a s s a y . same p r o d u c t s w e r e t e s t e d by i n t e r n a t i o n a l u n i t s

When t h e s e

o ra lly th e p o te n c ie s a s

ex p ressed

v a r i e d c o n s i d e r a b l y from t h o s e a d v e r ­

tise d . The r e s u l t s

o b t a i n e d b y t h e o r a l a s s a y m e th o d v /e re

m o re u n i f o r m t h a n t h o s e p r o d u c e d b y s u b c u t a n e o u s t e s t s . I n t h i s r e s p e c t t h e r e s p o n s e fr o m p r e p a r a t i o n s a d m i n i s t e r e d i n a q u e o u s m edium w e r e m o re p r e d i c t a b l e t h a n t h e r e s p o n s e from p r o d u c t s a d m in is te r e d i n o i l . d i e t h y l s t i l b e s t r o l in w a te r r e s u lte d

C o m p a rativ e t e s t s

in b e t t e r u n ifo rm ity

o f r e s p o n s e w hen t h e e s t r o g e n w a s a d m i n i s t e r e d i n t h a n w hen t h r e e

s ix doses

d o sag e s w ere g iv e n .

The o r a l a s s a y p r o c e d u r e i s to

on

reco m m en ded a s s u p e r i o r

th e su b cu ta n e o u s te c h n iq u e f o r s ta n d a r d iz in g e s tr o g e n ic

p r e p a r a tio n s in te n d e d fo r o r a l th e ra p y .

A lth o u g h t h e r e

n o e v i d e n c e t h a t r e s u l t s o b t a i n e d on l a b o r a t o r y a n i m a l s

is

48

w i l l ag ree w ith o l i n i e a l r e s u l t s , th a t

it

is

r e a s o n a b l e t o assum e

o r a l a s s a y p r o c e d u r e o n l a b o r a t o r y a n i m a l s w i l l m o re

n e a r l y p a r a l l e l t h e c l i n i c a l r e s p o n s e ev o k ed by o r a l t h e r a p y th a n w ill

s u b c u t a n e o u s a s s a y m e th o d s *

BIBLIOGRAPHY

A l l e n , E . a n d E . A. D o i s y ; 81: 8 1 9 , 1923

J . Am* Hed* A s s ' n .

E a h n t , L . 0 . a n d E . A. D o i s y ; E n d o c r i n . 1 2 :

760, 1928

Q u a r t* B u ll* H e a l t h O rgan* L e a g u e o f R a t i o n s 4: 1 2 1 , 1935 Q u a r t* B u ll* H e a l t h O rgan* L e a g u e o f R a t i o n s 4: 622, 1935 C u rtis , 1:

J . L. and E. W i tt; 3 6 3 , 1 94 1

J.

C lin .

E n d o c rin .

G i r a r d , A; J . P h a r m a c i e 1 7 : 3 6 3 , 1941 C o u r r i e r , R. a n d R. R a y n a u d ; C om pt. r e n d . 115: 229, 1934 H i n g l a i d , H* a n d II. H i n g l a i s ; 1 1 7 : 1 0 0 5 , 1934

Soc. de B io l.

C om pt. r e n d .

L a q u e u r , E.; K L in .: Y /c h n sc h r. 1 4 :

Soc* de B i o l .

3 3 9 , 1 93 5

S e h o e l l e r , TJ . , II. D o h rn a n d YiT. E. H o lw eg ; E l i n . U c h n s c h r . 1 4 : 8 2 6 , 1 93 5 B u llrin g ,

E. and J .

11. B u r n ; J .

F ra ttin i,

B . ; A rch. 1 s t h io c h im .

P h y sio l. ita l.

8 5 : 3 2 0 , 1 93 5

7: 1 , 1935

C a r t l a n d , G. F . , R* E . L e y e r , L. C. a n d 1.1. H* R u t z ; J . B i o l . Chem* 1 0 9 : 2 1 3 , 1 9 3 5 L e y e r , R. E . , L. C. L i l l e r a n d C. F* C a r t l a n d ; J . B i o l . Chem. 1 1 2 : 5 9 7 , 1 9 3 6 E a i n , A. L . a n d J . H. R o b s o n ; T h e ra p . 57: 3 3 7 , 1936

J . P h a rm . a n d E x p e r .

D*Amour, F . E . a n d R. G. G u s t a v s o n ; J . P h a rm . a n d E x p e r. T h e ra p . 57: 4 7 2 , 1936 Bum ,

J . H. ; B i o l o g i c a l S t a n d a r d i z a t i o n U n i v e r s i t y P r e s s , 1 9 3 7 ) p . 140

(L ondon, O x fo rd

Rowe, L. U. a n d A. E . S im o n d ; J . Am. P h a rm . A s s n . 25: 201, 1936

.96910

20)

Rowe, L. V/* and A* E. Simond; J. Am# Pharm, Assn. 26: 378, 1937

21)

Sondern, C. V/. and J . L. S e a le y ; Endoorin. 27: 670, 1940

22)

H i l l e r , L .C ., C .I , B l i s s and H, A. Braun; J, Am. Pharm. A ssn . 28: 644, 1939

25)

Barlow, 0. 17. and K. D# Sprague; Endoorin. 28: 203, 1941

24)

Ooward, H. and J. H. Bum; J. P h y s i o l . 53: 270, 1927

25)

ila r r ia n , G. E. and A. S. Parkas; J. P h y s i o l ,

26)

Stockard, C. R. and G. P. P ap an icolaou ; Am. J. Anat. 22: 225, 1917

27)

Heape, Q, J . ; H ie . S c i . 44: 1900

28)

Long, J. A. and E. 11. Evans; liem oirs, Univ. ITo. 6, 1922

29)

A l l e n , E. ; Am. J. Anat. 30: 297, 1922

30)

Laoueur, E. ; Deutseh. lied. TTchnschr. 52: 4 ,

31)

67: 389, 1929

C a lif.,

1926

Loewe, S. and E. Lange; Deutseh. lied. Uchnschr. 52: 1 286 , 1926

32)

Zondek, B. and S. Aschlieim; K lin . Uchnschr.

5: 979,

1926

33)

A l l e n , E. and E. A. Doisy; P h y s i o l . Rev. 7: 600, 1927

34)

L ip s c h u tz , A .; Compt. rend. Soc. de B i o l . 94: 738, 1926

35)

Laqueur, E. and S. E. DeJongh; J. Pharm. and Exp. Therap. 36: 1 , 1929

36)

Bum , J.H. ; P h y s i o l . Rev. 10: 146, 1930

37)

Kennedy, ’7 . P .; Quart. J. Exp. P h y s i o l . 20:

38)

Ila r ria n , G. P .; P h y s i o l . Rev. 13: 185, 1933

39)

A l l e n , H ., P. Dickens and E. C. Dodds; J. P h y s i o l . 68: 348, 1930

71, 1930

40)

D1Amour, E. E. and R. G. Gustavson; J. Pharm. and Exper. Therap. 40: 475, 1930

41)

B um , J. H. ; B i o l o g i c a l S ta n d a r d iz a tio n (London, Oxford U n iv e r s i t y P r e s s , 1937) page 156

B l i s s , C. I . ; Annals o f A pplied B io lo g y 2 2 ; 1 3 4 , 1935 B l i s s , 0 . I« ; S c i e n c e , 7 4 , 38 and 4 0 9 , 1934 G-addum, J* H . j Med. R e s . C ou n cil S p ec. Rep. No. 183 H is M a j e s t y f s S t a t i o n e r y O f f ic e Hemmingsen, A. M.j Quart, J . Pharm. 6 , 1 8 7 , 1 9 3 3 F i s h e r , R. A . ; S t a t i s t i c a l Methods f o r Research Workers, I V , (Edinburgh, O liv e r and Boyd, 1 9 2 5 ) page 117 Burn, J , H.j B i o l o g i c a l S t a n d a r d is a t io n (London, Oxford U n i v e r s i t y P r e s s , 1937) page 40