Successful Drug Discovery 3527344683, 9783527344680

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Successful Drug Discovery

­Successful Drug Discovery Volume 4

Edited by János Fischer, Christian Klein, Wayne E. Childers

Editors János Fischer

Richter Co., Plc. Gyömröi ut 19/21 1103 Budapest Hungary Christian Klein

All books published by Wiley‐VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Roche Innovation Center Zürich Cancer Immunotherapy Division Wagistrasse 10 8952 Schlieren Switzerland

Library of Congress Card No.: applied for

Wayne E. Childers

Bibliographic information published by the Deutsche Nationalbibliothek

Temple University School of Pharmacy Moulder Ctr. for Drug Discovery Res. 3307 N Broad Street Philadelphia, PA 19140 United States Cover

Supported by the international Union of Pure and Applied Chemistry (IUPAC) Chemistry and Human Health Division PO Box 13757 Research Triangle Park, NC 2770‐3757 USA

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A catalogue record for this book is available from the British Library.

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at . © 2019 Wiley‐VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Print ISBN: 978‐3‐527‐34468‐0 ePDF ISBN: 978‐3‐527‐81470‐1 ePub ISBN: 978‐3‐527‐81471‐8 oBook ISBN: 978‐3‐527‐81469‐5 Typesetting  SPi Global, Chennai, India Printing and Binding

Printed on acid‐free paper 10 9 8 7 6 5 4 3 2 1

v

Contents Advisory Board Members  xi Preface  xiii Part I 1

General Aspects 

1

Trends in Peptide Therapeutics  3 Florence M. Brunel, Fa Liu, and John P. Mayer

1.1 ­Introduction  3 1.2 ­Peptides in Metabolic Diseases  4 1.2.1 Insulins  4 1.2.2 Glucagon‐like Peptide‐1  6 1.2.3 Glucagon  8 1.2.4 Combination Therapies  8 1.3 ­Peptide Antibiotics  9 1.3.1 Non‐ribosomally Synthesized  9 1.3.2 Ribosomally Synthesized  11 1.4 ­Peptides in Cancer  12 1.4.1 Luteinizing Hormone Releasing Hormone  12 1.4.2 Somatostatin  13 1.4.3 Peptide–Drug Conjugates  14 1.4.4 Cancer Vaccines  15 1.5 ­Peptides in Bone Diseases  16 1.5.1 Calcitonin  16 1.5.2 Parathyroid Hormone (PTH) (1–34) and (1–84)  17 1.5.3 Parathyroid Hormone Related Protein  18 1.5.4 Incretin Peptides  19 1.5.5 Bone Morphogenic Protein‐Derived Peptides  19 1.6 ­Peptides in Gastrointestinal Diseases  19 1.6.1 Glucagon‐like Peptide‐2  19 1.6.2 Guanylate Cyclase‐C Agonists  20 1.7 ­Emerging Trends in Peptide Drug Discovery  22 1.7.1 Cell‐Penetrating Peptides  22 1.7.2 Macrocyclic Peptides  23 1.8 ­Summary  23

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­

Acknowledgment  24 ­List of Abbreviations  24 ­References  25 ­Biographies  34 2

Physicochemical Parameters of Recently Approved Oral Drugs  35 Andreas Ritzén and Laurent David 

2.1 ­Introduction  35 2.2 ­FDA‐Approved Drugs 2007–2017  36 2.2.1 Conclusions  40 2.3 ­Polar Surface Area in bRo5 Territory  42 2.3.1 Finding Chameleons with Molecular Dynamics  44 2.3.2 Conclusions  48 2.3.3 Methods  49 List of Abbreviations  50 ­References  51 ­Biographies  52 Part II Drug Class Studies  3

55

Antibody–Drug Conjugates: Empowering Antibodies for the Fight Against Cancer  57 Caroline Denevault‐Sabourin, Francesca Bryden, Marie‐Claude Viaud‐Massuard, and Nicolas Joubert

3.1 ­Introduction  57 3.2 ­First Generation ADCs  58 3.2.1 Molecular Design  58 3.2.2 Mechanism of Action  60 3.2.3 Therapeutic Applications  60 3.2.4 Adverse Effects  60 3.3 ­Second Generation ADCs  62 3.3.1 Molecular Design  62 3.3.2 Mechanism of Action  63 3.3.3 Therapeutic Applications  67 3.3.4 Adverse Effects  67 3.4 ­Toward Next Generation ADCs  68 3.4.1 Site‐Specific ADCs  69 3.4.2 New Formats of Immunoconjugates  70 3.4.3 ADC with New Payloads  74 3.5 ­Summary  76 ­List of Abbreviations  76 ­References  77 ­Biographies  81

Contents

4

Dopamine D2 Partial Agonists – Discovery, Evolution, and Therapeutic Potential  83 Marlene Jacobson, Wayne Childers, and Magid Abou‐Gharbia

4.1 ­Introduction  83 4.2 ­Dopamine and Dopamine Receptors  83 4.2.1 Functional Selectivity and Biased Ligand Signaling  86 4.3 ­Schizophrenia and Earlier Antipsychotic Agents  86 4.4 ­Dopamine Partial Agonism  88 4.5 ­D2 Partial Agonists  89 4.5.1 Dopamine‐like Scaffolds – The “Classical” Pharmacophore  89 4.5.2 Non‐dopamine‐like Scaffolds – The “Nonclassical” Pharmacophore  91 4.5.3 Compounds Related to Bifeprunox  92 4.5.4 Methylaminochroman Scaffold – Aplindore  94 4.5.5 D2 Partial Agonist Drug Discovery in the Wake of the Marketed Drugs  95 4.5.5.1 D2 Partial Agonists Discovered Using Traditional D2 Functional Screening  96 4.5.5.2 Bivalent Ligands  98 4.5.5.3 β‐Arrestin‐Biased D2 Partial Agonists  98 4.5.5.4 G‐protein‐Biased D2 Partial Agonists  99 4.5.6 Arylpiperazines and the Discovery of Aripiprazole and Brexpiprazole  101 4.5.7 The Road Leading to Cariprazine  102 4.6 ­Marketed D2 Partial Agonist Antipsychotics  103 4.6.1 Aripiprazole (Abilify®)  104 4.6.1.1 History 105 4.6.1.2 Synthesis 105 4.6.1.3 Drug Substance  105 4.6.1.4 Pharmacology 105 4.6.1.5 Functional Selectivity and Biased Ligand Signaling  107 4.6.1.6 Pharmacokinetics and Metabolism  107 4.6.1.7 Clinical Data  107 4.6.2 Brexpiprazole (Rexulti®)  108 4.6.2.1 History 108 4.6.2.2 Synthesis 108 4.6.2.3 Drug Substance  109 4.6.2.4 Pharmacology 109 4.6.2.5 Functional Selectivity and Biased Ligand Signaling  110 4.6.2.6 Pharmacokinetics and Metabolism  110 4.6.2.7 Clinical Data  110 4.6.3 Cariprazine (Vraylar®)  111 4.6.3.1 History 111 4.6.3.2 Synthesis 112

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4.6.3.3 Drug Substance  112 4.6.3.4 Pharmacology 112 4.6.3.5 Functional Selectivity and Biased Ligand Signaling  113 4.6.3.6 Pharmacokinetics 113 4.6.3.7 Clinical Data  114 4.7 ­Conclusions  115 ­List of Abbreviations  116 ­References  117 ­Biographies  129 Part III Case Studies  5

131

Discovery of Etelcalcetide for the Treatment of Secondary Hyperparathyroidism in Patients with Chronic Kidney Disease  133 Amos Baruch and Derek Maclean

5.1 ­Introduction  133 5.2 ­Compound Design and Structure–Activity Relationships  135 5.2.1 Optimization of the Cationic Charge  135 5.2.2 Alanine Scan of dCR6  136 5.2.3 Double Alanine Scan of dCR6  138 5.2.4 SAR of Alanine Residues in Ac‐carrrar‐NH2 and Ac‐crrarar‐NH2  138 5.2.5 Importance of the Thiol Residue  140 5.2.6 Thiol Conjugates – Selection of Etelcalcetide  140 5.3 ­Preclinical Studies  141 5.4 ­Mechanism of Action of Etelcalcetide  144 5.5 ­Clinical Studies  145 5.6 ­Summary  149 ­Acknowledgments  149 ­List of Abbreviations  149 ­References  150 ­Biographies  153 6

Development of Lenvatinib Mesylate, an Angiogenesis Inhibitor Targeting VEGF and FGF Receptors  155 Akihiko Tsuruoka, Yasuhiro Funahashi, Junji Matsui, and Tomohiro Matsushima

6.1 ­Introduction  155 6.2 ­Recent Progress in the Development of Molecular Targeted Anticancer Agents  155 6.3 ­Tumor Angiogenesis  156 6.4 ­Development of Resistance to VEGF‐Targeting Drugs  156 6.5 ­Discovery of Lenvatinib, a Drug Targeting VEGFR and FGFR  157 6.6 ­Inhibition of Kinase Activity by Lenvatinib and Discovery of the Novel Type V Kinase‐Binding Mode  158

Contents

6.7 ­Antitumor Effects of Lenvatinib in Human Thyroid Cancer Cell Lines  161 6.8 ­Antitumor Effects of Lenvatinib in Human Renal Cell Cancer Cell Lines and Its Mechanism of Action  162 6.9 ­Conclusions and Perspectives  162 ­List of Abbreviations  163 ­References  164 ­Biographies  167 7

Ocrelizumab: A New Generation of anti‐CD20 mAb for Treatment of Multiple Sclerosis  169 Andrew C. Chan, Paul Brunetta, and Peter Chin

7.1 ­Introduction: B Cells Play Critical Roles in Immunity  169 7.2 ­Role of B Cells in Autoimmunity  172 7.3 ­CD20‐Targeting Therapeutic Antibodies  173 7.4 ­Rituximab: the First Anti‐CD20 mAb Experience in Autoimmunity  176 7.5 ­Effects of Rituximab on Antibodies and Autoantibodies  178 7.6 ­Rituximab in AAV and Other Autoimmune Disorders  178 7.7 ­Beginnings of Ocrelizumab  179 7.8 ­Multiple Sclerosis  180 7.9 ­Multiple Sclerosis Disease Pathogenesis  181 7.10 ­Rituximab: The First Anti‐CD20 mAb Experience in Multiple Sclerosis  183 7.10.1 HERMES Junior  183 7.10.2 HERMES  183 7.10.3 OLYMPUS  184 7.11 ­Ocrelizumab in Multiple Sclerosis  184 7.11.1 OPERA  185 7.11.2 ORATORIO  186 7.12 ­The Conundrum of B Cells in Multiple Sclerosis  187 7.13 ­Final Comments  187 List of Abbreviations  188 ­Acknowledgments  189 ­References  189 ­Biographies  198 8

The Story of Rucaparib (Rubraca)  201 Bernard T. Golding

8.1 ­Introduction  201 8.2 ­Benzoxazole‐/Benzimidazole‐carboxamides and Quinazolinones  205 8.3 ­The Road to Rucaparib/Rubraca  212 8.4 ­The Emergence of Single‐Agent Therapy  216 8.5 ­Clinical Studies  217

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8.6 ­Conclusion  218 ­Acknowledgments  ­References  219 ­Biography  223 9

218

Discovery and Development of Venetoclax, a Selective Antagonist of BCL‐2  225 Wayne J. Fairbrother, Joel D. Leverson, Deepak Sampath, and Andrew J. Souers

9.1 ­Introduction  225 9.2 ­Discovery of Venetoclax – Structure‐Based Design  227 9.3 ­Preclinical Studies  232 9.3.1 Mechanism of Action  232 9.3.2 Predictive Biomarkers of Venetoclax Sensitivity  233 9.3.3 Efficacy of Venetoclax as Monotherapy and in Combination with Targeted Agents or Chemotherapeutics  234 9.4 ­Clinical Studies  236 ­List of Abbreviations  238 ­References  239 ­Biographies  244 Index  247

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Advisory Board Members Magid Abou‐Gharbia

(Temple University, USA) Jagath R. Junutula

(Cellerant Therapeutics, Inc., USA) Kazumi Kondo

(Otsuka, Japan) John A. Lowe

(JL3Pharma LLC, USA) Gerd Schnorrenberg

(Boehringer Ingelheim, Germany)

xiii

Preface The fourth volume of Successful Drug Discovery continues to follow the structure of the previous volumes, consisting of three parts: General Aspects, Drug Class Studies, and Case Studies. The book series focuses on new discoveries of small‐ molecule drugs and biologics. The editors thank the advisory board members: Magid Abou‐Gharbia (Temple University, USA), Jagath R. Junutula (Cellerant Therapeutics, Inc., USA), Kazumi Kondo (Otsuka), John A. Lowe (JL3Pharma LLC, USA), and Gerd Schnorrenberg (Boehringer Ingelheim, Germany), and the following reviewers who helped both the authors and the editors: Jonathan Baell, Gabriele Costantino, György Domány, John M. Beals, Stephen Hauser, Jagath R. Junutula, Béla Kiss, Paul Leeson, Gábor Mező, Tomi Sawyer, Malcolm Stevens, Michael Wagner, Peng Wu. Special thanks are due to Juergen Stohner for his review from the viewpoints of the IUPAC Interdivisional Committee on Terminology, Nomenclature and Symbols (ICTNS). Part I: General Aspects John P. Mayer and coworkers give a comprehensive survey on the recent progress in peptide therapeutics. Remarkable achievements in various therapeutic fields and several orally administered peptides are summarized in this chapter. Andreas Ritzén and coworker investigate the physicochemical parameters of recently approved drugs. A significant fraction of non‐CNS oral drugs violate two or more of the Lipinski parameters. Part II: Drug Class Studies Nicolas Joubert and coworkers give an overview on antibody–drug conjugates in cancer‐targeted chemotherapy. Over the last decade, they have been improved by the choice of better drugs, linkers, and mAb targets. Wayne E. Childers and coworkers evaluate three decades in the discovery of D2 partial agonist drugs. Progress has been made in treating the negative and cognitive symptoms of schizophrenia. Part III: Case Studies Derek Maclean and coworker report on the discovery of etelcalcetide, which affords a suitable therapy for hemodialysis patients with secondary hyperparathyroidism.

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Preface

Akihiko Tsuruoka and coworkers describe how lenvatinib mesylate was discovered to give a new drug for the treatment of differentiated thyroid cancer. Andrew C. Chan and coworkers describe the discovery and development of ocrelizumab, which represents a new generation of anti‐CD20 mAb for the treatment of multiple sclerosis. Bernard T. Golding has prepared a chapter on the discovery and development of rucaparib, a new PARP‐1 inhibitor anticancer drug for the treatment of ovarian cancer. It also represents an example of a good cooperation of academia and industry. Wayne J. Fairbrother and coworkers describe how venetoclax, a selective ­antagonist of B cell lymphoma 2 (Bcl-2), was discovered for the treatment of leukemia. The editor and authors thank Wiley‐VCH, and personally Dr. Frank Weinreich for the excellent cooperation. Budapest Philadelphia Zurich 8 November 2018

János Fischer Wayne E. Childers Christian Klein

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Part I General Aspects

3

1 Trends in Peptide Therapeutics Florence M. Brunel1, Fa Liu2, and John P. Mayer3 1

Novo‐Nordisk Research Center, 5225 Exploration Dr., Indianapolis, IN, 46241, USA Novo‐Nordisk Research Center, 530 Fairview Avenue North, Seattle, WA, 98109, USA 3 University of Colorado, MCD Biology, 1945 Colorado Avenue, Boulder, CO, 80309, USA 2

1.1 ­Introduction The growing importance of peptide drugs within the pharmacopoeia has become evident over the past several decades. Among the factors that have contributed to this trend is the recognition that peptide ligands regulate a multitude of physiological pathways and are often suitable for therapeutic applications, in either their native or modified form. In addition, certain attributes that are unique to peptides, such as their high selectivity, potency, and lack of toxicity, have ultimately become appreciated. The alternative means of drugging peptide receptors through target‐directed screening or rational design of orally available small molecules have, with few exceptions, proved unproductive. Mimicking the activity of a peptide agonist is highly challenging, particularly in the case of Class II G‐protein‐coupled receptor (GPCR) targets. Successful examples have typically involved receptor antagonists such as neurokinin, angiotensin, endothelin, and orexin. These lessons have increasingly led drug discovery scientists to consider peptides as legitimate drug candidates, rather than leads or proof‐of‐concept models for small‐molecule programs. Peptide medicinal chemists have also had to confront and overcome shortcomings such as rapid metabolism, clearance, production costs, and limited alternative delivery options. In the present chapter, we highlight the role of peptides in therapeutic areas such as metabolic disease, where peptides have been well established, as well as in areas where their impact has been minor, but now rapidly expanding. We also emphasize examples where time‐extension strategies and alternative delivery routes have helped establish and strengthen the position of peptide drugs in competitive markets. Finally, we explore two novel trends in peptide drug discovery, macrocyclic and cell‐penetrating peptides, both of which may expand future opportunities for peptide therapeutics.

Successful Drug Discovery, First Edition. Edited by János Fischer, Christian Klein, and Wayne E. Childers. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2019 by Wiley-VCH Verlag GmbH & Co. KGaA.

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1.2 ­Peptides in Metabolic Diseases The global epidemic of type 2 diabetes and obesity continues unabated, impacting quality of life, life expectancy, and economic well‐being. Health‐care organizations have devoted enormous resources toward the treatment of metabolic diseases, often dramatically improving patient outcomes [1]. Perhaps more than in any other therapeutic area, peptides have had a unique and indispensable role in treating type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) as well as obesity. This section will provide an overview of approved insulin, GLP‐1 (glucagon‐like peptide‐1), and glucagon peptide drugs as well as those in late‐stage clinical development. 1.2.1 Insulins Insulin, which was discovered by Banting and Best in 1921, became commercially available only one year after its discovery (Figure  1.1) [2]. In spite of its miraculous potential, the short duration of action of early insulin preparations (four to six hours) required multiple daily injections and prompted the search for longer acting formulations. The first of these, insulin neutral protamine Hagedorn (NPH), developed in the 1940s, consisted of an insulin suspension complexed with protamine, a cationic protein isolated from fish sperm. The slow disassociation of the NPH complex delayed absorption from the injection site, prolonging insulin action to a range of 12 to 18 hours [3]. Insulin Lente, introduced in the 1950s, involved a neutral pH suspension of insulin formulated with excess zinc, which extended the duration of action to 24 hours and beyond [3]. Between the 1920s and the early 1980s, commercial insulin production relied on extraction of pancreatic glands from cows and pigs. The advent of biotechnology enabled the production of rDNA‐derived human insulin in the early 1980s in sufficient quantity to satisfy the needs of the diabetic population, gradually

Figure 1.1  Sequences of human, porcine, bovine, and the commercially key insulin analogs.

1.2  Peptides in Metabolic Diseases

­isplacing animal sourced insulins [4]. Recombinant DNA technology also d furthered the development of short‐acting analogs to manage prandial glucose excursion, as well as long‐acting analogs to mimic basal insulin action [5, 6]. In the native state, the insulin hexamer complex consists of three non‐covalent insulin dimers, which are in turn stabilized by two zinc ions coordinating the B10 His residues. Once injected, the diffusion of the zinc ion causes a sequential breakdown of the hexamer into insulin dimers, and further into monomers, the last being the rate‐limiting step. Since physiological absorption occurs mainly via the monomeric form, research efforts have focused on weakening of the insulin dimer association. Examination of the X‐ray structure indicated that the B‐chain C‐terminal regions of two insulin molecules form the dimer interface. Weakening of this interaction in order to accelerate dimer disassociation resulted in a more rapid onset of insulin action. Three rapid‐acting insulin analogs were approved between 1996 and 2006: LysB28, ProB29, insulin lispro (Humalog®, Eli Lilly & Co.) [7]; AspB28, insulin aspart (Novolog®, Novo Nordisk A/S) [8]; and LysB3, GluB29, insulin glulisine (Apidra®, Sanofi S.A.) [9]. The onset of action for these analogs is within 5 to 15 minutes, with peak action at 30 to 90 minutes and duration of 4 to 6 hours [5]. An ultrafast, co‐formulation of insulin aspart and niacinamide (Fiasp®, Novo Nordisk A/S) was approved in late 2017. The long‐acting, or “basal” insulins, use two independent strategies: isoelectric point shift and lipidation. Insulin glargine (Lantus®, Sanofi S.A.) is a human insulin analog with a mutation of AsnA21Gly, and an additional ArgB31 and ArgB32 residues. The presence of the two arginine residues shifts the isoelectric point of the hormone from 5.6 to 6.7, reducing its solubility at physiological pH. Aqueous solubility at pH 4.0 required the GlyA21 substitution to mitigate the acidic degradation of the native AsnA21. Upon injection, insulin glargine forms an insoluble depot that gradually dissipates, releasing the drug over a 20 to 24 hour period [10]. Insulin glargine undergoes extensive metabolism in the subcutaneous depot with only its metabolites released into systemic circulation. The earliest commercial application of the lipidation strategy was insulin detemir (Levemir®, Novo Nordisk A/S), a human desB30 insulin covalently modified with myristic acid at the LysB29 side chain. This modification induces hexamer and di‐hexamer formation while also facilitating binding to serum albumin (98 % bound in plasma). The former phenomenon delays absorption from the injection site while the latter slows plasma clearance, contributing to the extended 12 to 24 hour duration of action as well as a less variable pharmacokinetic/pharmacodynamic (PK/PD) profile [11]. Insulin degludec (Tresiba®, Novo Nordisk A/S) represents the second generation of the lipidation strategy. LysB29 acylation with a γ‐glutamate linked hexadecanedioic acid results in prolonged time action promoted by higher order multi‐hexamer association at the subcutaneous injection site, and to a lesser degree, by higher affinity for albumin. Insulin degludec provides a longer duration of action (42 hours), which enables therapeutic accumulation using a once‐daily dosing regimen, thus permitting administration during the day [12]. Alternative delivery of insulin has had a mixed record of success with two previously approved pulmonary products. Exubera® (Pfizer Inc.), an inhalable, spray‐dried insulin powder administered using a reusable inhaler was approved

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in 2006. It was withdrawn from the market after a brief time due to poor commercial performance attributed partly to the bulky device. Afrezza® (MannKind Corp.), a Technosphere® formulated human insulin delivered in a thumb‐sized device, was approved in 2014. Peak plasma insulin levels with Afrezza® are achieved within 12 to 15 minutes after administration. Its commercial fate at this time remains uncertain [13]. A survey of clinical insulin programs indicates a high level of interest in various ultrafast bolus and ultra‐long basal analogs, as well as orally administered insulins. The sponsoring companies include Adocia (ultrafast, BioChaperone formulation of insulin lispro, Phase 2; and premixed, Phase 1/2), AntriaBio (once weekly, Phase 1), Biocon/Mylan (insulin Tregopil, oral, Phase 2/3), Diasome (liver targeted, Phase 2), Eli Lilly (once‐weekly, Phase 1; and ultrafast, Phase 3), Merck (glucose‐sensitive, Phase 1), Novo Nordisk (once‐weekly, Phase 2), Oramed (ORMD‐0801, oral, Phase 2), and Sanofi S.A (ultrafast, Phase 3). 1.2.2  Glucagon‐like Peptide‐1 GLP‐1 is a peptide hormone secreted from intestinal L‐cells, which serve an essential role in glucose homeostasis (Figure 1.2). Activation of GLP‐1 receptors on pancreatic β‐cells stimulates glucose‐dependent insulin secretion, and simultaneously suppresses glucagon levels under hyperglycemic conditions. Apart from helping maintain glucose homeostasis, GLP‐1 also promotes satiety and delays gastric emptying, which together contribute to decreased food intake and lower body weight. This unique spectrum of biological and pharmacological properties has led to the development of a number of successful antidiabetic and anti‐obesity medications [14]. Endogenous GLP‐1 circulates in two equipotent bioactive isoforms, GLP‐1 (7–36)‐NH2 and GLP‐1 (7–37). Both have short half‐ lives of approximately two minutes because of dipeptidyl peptidase‐4 (DDP‐IV)‐ mediated cleavage of the N‐terminal His7‐Ala8 dipeptides. The short half‐life diminishes the pharmacological effects and has promoted the search for longer acting, DPP‐IV stabilized analogs [15]. The first approved GLP‐1 analog, exenatide (Byetta®, Eli Lilly & Co. and Amylin Pharmaceuticals), was isolated from the saliva of the Gila monster lizard. Exenatide, the active pharmaceutical ingredient in Byetta, possesses similar in vitro potency as native GLP‐1 with a half‐life of GLP-1(human): Exenatide: Lixisenatide: Albiglutide: Dulaglutide: Liraglutide:

H-HAEGT H-HGEGT H-HGEGT [ H-HGEGT H-HGEGT [ H-HAEGT

FTSDV FTSDL FTSDL FTSDV FTSDV FTSDV

SSYLE SKQME SKQME SSYLE SSYLE SSYLE

GQAAK EEAVR EEAVR GQAAK EQAAK GQAAK

EFIAW LFIEW LFIEW EFIAW EFIAW EFIAW

Semaglutide:

H-HXEGT FTSDV SSYLE GQAAK EFIAW LVRGR G-OH

Figure 1.2  Structure of GLP‐1 and marketed analogs.

LVKGR G-OH LKNGG PSSGA PPPS-NH2 LKNGG PSSGA PPSKK KKKK-NH2 LVKGR ]2 -Albumin LVKGG G(GGGGS)3 ]2-IgG4-Fc LVRGR G-OH

(X: Aib)

1.2  Peptides in Metabolic Diseases

two to four hours, attributed to resistance toward DPP‐4 cleavage. It is approved for twice daily SC injection with a dose of 5 to 10 μg [16]. Lixisenatide, an analog of exenatide modified with six lysine residues at its C‐terminus, is marketed as Adlyxin® in the United States and as Lyxumia® in the European Union by Sanofi S.A. [17]. Initial experience with GLP‐1 agonists suggested that optimal patient compliance and outcomes might require once‐daily, once‐weekly, or even less frequent dosing intervals. Efforts to extend the Byetta dosing interval involved co‐formulation with a poly(d,l‐lactide‐co‐glycolide) (PLGA) polymer, which successfully prolonged the drug’s half‐life to five to six days in humans [15]. The new formulation was approved for once‐weekly administration in 2012 (Bydureon®, AstraZeneca plc). A validated strategy for extending peptide time action involves fusion to a macromolecular carrier as a means to mitigate proteolytic degradation, slow down renal clearance, and exploit FcRn trafficking to peripheral tissues. Human serum albumin (HSA) and the Fc portion of immunoglobulin G (IgG) as molecular fusions have both been utilized to extend GLP‐1 duration of action to enable once‐weekly dosing. Albiglutide (Tanzeum®, GlaxoSmithKline plc) is a recombinant HSA construct N‐terminally extended with two tandem GLP‐1 sequences. The DPP‐IV cleavage of GLP‐1 was minimized through the substitution of Gly for Ala at the second position. Albiglutide has a half‐life of six to eight days in humans and is approved for once‐weekly injection at a dose of 30 to 50 mg [18]. Dulaglutide (Trulicity®, Eli Lilly & Co.) is a human IgG4‐Fc fusion protein N‐terminally modified with a Gly4Ser flexible linker and a GLP‐1 sequence. As in albiglutide, the DPP‐IV degradation is suppressed by Gly substitution at the second position. The half‐life of dulaglutide is approximately four days in humans, and the drug is approved for once‐weekly injection at a dose of 0.75 to 1.5 mg [19]. The lipidation strategy utilized successfully to extend insulin pharmacokinetics has also been applied to GLP‐1 peptides. It has resulted in two approved GLP‐1 analogs, both from Novo Nordisk A/S. Liraglutide (Victoza®) utilizes a GLP‐1 backbone with a palmitic acid attached to the Lys20 side chain via a γ‐­ glutamate spacer. The native Lys at position 28, replaced with Arg, facilitates site‐specific acylation at Lys20. Liraglutide’s high degree of albumin binding not only slows kidney clearance but also effectively shields the native N‐terminus from DPP‐IV cleavage. Liraglutide has a half‐life of approximately 13 hours in humans, and is approved for once‐daily dosing of 1.2 to 1.8 mg [20]. A higher dose of liraglutide, approved for the obesity indication, is marketed as Saxenda®. Semaglutide (Ozempic®) is structurally similar to liraglutide, and uses an octadecanedioic acid in place of palmitic acid with two mini‐PEG units as an additional spacer. Greater DPP‐IV stability consistent with a once‐weekly dosing requirement was achieved through substitution with 2‐aminoisobutyric acid (Aib) at the second position. The combination of high‐affinity albumin binding with greater DPP‐IV stability prolongs the half‐life of semaglutide in humans to six to seven days. It is approved for once‐weekly injection at a dose of 0.5 to 1.0 mg [21]. The commercial success of the GLP‐1 class has increased research and ­development investment in additional candidates. These include once‐weekly ­efpeglenatide (Sanofi S.A., Phase 3), a subdermal osmotic mini‐pump device, which delivers exendin‐4 (Intarcia Therapeutics Inc, ITCA‐650, food and drug

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a­dministration [FDA] complete response letter [CRL] received) and an oral ­tablet formulation of semaglutide (Novo Nordisk A/S, Phase 3). 1.2.3 Glucagon Glucagon is the primary counter‐regulatory hormone of insulin, which is secreted in response to hypoglycemic conditions [22, 23]. Its chief medicinal use is the emergency reversal of insulin‐induced hypoglycemic shock, a relatively frequent event in the treatment of type‐1 patients. Glucagon’s poor biophysical and chemical stability makes liquid formulation challenging, thus requiring reconstitution of lyophilized glucagon powder in an acidic diluent immediately prior to use. This complicates not only its current emergency use but also hinders potential additional indications, which require a stable, soluble glucagon [22]. A recent survey revealed advanced glucagon programs at Adocia (BioChaperone formulation of human glucagon, Phase 1), Eli Lilly & Co. (Nasal glucagon, Phase 3; and novel soluble analog, Phase 1), Novo Nordisk A/S (novel, soluble analog, Phase 1), Xeris Pharmaceuticals (Xerisol, Phase 3, human glucagon in dimethyl sulfoxide [DMSO]), and Zealand Pharma (Dasiglucagon, Phase 3, novel, soluble analog). 1.2.4  Combination Therapies Combination therapy, achieved through co‐administration of two agents targeting independent pathways, produced additive or synergistic efficacy and a more tolerable side‐effect profile [24]. Initiation of insulin therapy, either with a single agent or as part of a basal/bolus regimen, promotes weight gain and an increased incidence of hypoglycemia. In contrast, GLP‐1 agonists provide not only improved glycemic control but also modest body weight loss. As expected, the combination of a basal insulin and a GLP‐1 agonist consistently reduces HbA1c and body weight in most patients, while reducing the frequency of hypoglycemia [25]. Two combination products were approved by the FDA; insulin glargine plus lixisenatide (Soliqua®, Sanofi S.A.) and insulin degludec plus liraglutide (Xultophy® Novo Nordisk A/S). Glucagon and glucose‐dependent insulinotropic polypeptide (GIP) are two important metabolic hormones closely related to GLP‐1. Glucagon has been shown to stimulate lipolysis, increase energy expenditure, and complement the weight‐lowering effect of GLP‐1 [22]. GIP is an incretin that functions as a “glucose‐stat” stimulating insulin secretion under hyperglycemic conditions, while stimulating glucagon secretion in the hypoglycemic state [26]. Consequently, various combinations of GLP‐1, GIP, and glucagon pharmacology have been explored in the form of unimolecular co‐agonists [27]. A number of these programs reached the clinical development stage, including GLP‐1/glucagon dual agonists from AstraZeneca plc (MEDI0382, Phase 2), Eli Lilly & Co. (Phase 1), Johnson & Johnson (JNJ‐64565111, Phase 2), Novo Nordisk A/S (Phase 1), OPKO Health (POK88003, formerly TT401, Phase 2), Sanofi S.A. (SAR425899, Phase 2) and Zealand Pharma/Boehringer Ingelheim Gmbh (BI456906, Phase 1), GLP‐1/GIP dual agonists from Eli Lilly & Co. (LY3298176, Phase 3) and Sanofi

1.3  Peptide Antibiotics

S.A. (SAR438335, Phase 1), and a GLP‐1/glucagon/GIP tri‐agonist from Novo Nordisk A.S.(NN9423, Phase 1).

1.3 ­Peptide Antibiotics While cyclic peptides such as gramicidin were among the earliest antibiotics discovered, their clinical application has been limited by their lack of oral availability, short half‐life, and systemic toxicity. As a result, their use has been restricted to topical application in ophthalmology and dermatology. However, their inherent advantages, including their broad‐spectrum activity, lesser susceptibility to microbial resistance, as well as the potential for broader indications, have combined to revive interest in this peptide category. The peptide antibiotics are structurally diverse and frequently highly complex natural products produced by either ribosomal or non‐ribosomal biogenic pathways. Peptides such as the mammalian defensins, the insect‐derived cecropins, and the amphibian antimicrobial peptides (AMPs), assembled by ribosomal synthesis, are occasionally posttranslationally modified. Peptides of microbial origin, assembled by non‐ ribosomal synthesis, incorporate a wide selection of non‐native amino acids and are represented by the bacitracins, polymyxins, gramicidins, and vancomycin. 1.3.1  Non‐ribosomally Synthesized The isolation of tyrothricin (a mixture of gramicidins and tyrocidines) by R. Dubos in the late 1930s from Bacillus brevis provided the first clinically useful antibiotic for skin and throat infections [28]. Gramicidin S is mainly used for ophthalmic indications and treatment of surface wounds infected by gram‐positive and gram‐negative bacteria [29]. The polymyxins, discovered in the late 1940s, were used clinically for a number of years against gram‐negative bacterial infections. As a result of reported systemic toxicity the polymyxins were all discontinued by the 1970s, but have recently re‐emerged as a last resort treatment against resistant gram‐negative bacteria. Recent reports have also described atypical synthetic polymyxin analogs with high antibiotic activity and minimal toxicity [30]. Bacitracin, first isolated in 1945 from B. brevis and used in combination with other antibiotics to treat skin and eye infections, is marketed as Neosporin®. A more recently developed lipidated cyclic depsipeptide, daptomycin (Cubicin®, Cubist Pharmaceuticals), was approved by the FDA in 2003 for complicated skin and skin‐structure infections caused by Staphylococcus aureus. Daptomycin is also approved for systemic use in the treatment of bacteremia associated with right‐sided endocarditis [31]. The glycopeptides (lipoglycopeptides) are an important class of antibiotics that has remained relevant 60  years after the discovery of vancomycin (Figure 1.3a), (Vancocin®, Eli Lilly & Co.). A group led by E.C. Kornfeldt isolated the molecule from soil samples collected in Borneo in 1953 [32]. Vancomycin moved quickly through development, with approval in 1958, and was used primarily against gram‐positive strains. Its use declined with the introduction of newer antibiotics through the 1980s; however, increasing resistance to these

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HO OH O NH

O

NH2 H N

O

H N

OH

N H

O N H

O

HO

O Cl

N H

Cl

O

HO

O OH

(a)

O

H2N

OH

HO O

OH P HN

HO OH

OH

NH2 O

HN HN

O H N

O N H

HO

O N H Cl

O

NH O

O OH

O

O O

Cl

O

OH OH

O OH

O

(b)

HO2C H N

OH

N H

O

OH

O

O

HO

OH N

O

H N

Figure 1.3  Structures of vancomycin (a) and telavancin (b).

1.3  Peptide Antibiotics

newer agents led to vancomycin’s re‐introduction particularly for use against S. aureus [33]. Its complex structure was not elucidated until 1982 [34], which effectively limited exploration of its structure–activity relationships prior to that time. The unique efficacy of vancomycin stimulated extensive research into other glycopeptide antibiotics [32], leading to the discoveries of other naturally occurring analogs such as teicoplanin (Targocid®, Sanofi S.A.). A number of successful second generation semisynthetic glycopeptides based on vancomycin were subsequently introduced [35]. For example, telavancin (Vibativ®, Theravance Biopharma) introduced in 2009 demonstrates enhanced pharmacokinetic properties as well as efficacy against S. aureus strains (Figure 1.3b). The echinocandins are semisynthetic lipidated, cyclic hexapeptides, which present an important option for combating invasive systemic fungal infections. All are structurally based on the parent echinocandin B natural product discovered in 1974 [36, 37]. The echinocandins exert their antifungal activity through a unique mechanism that involves potent inhibition of the (1  →  3)‐β‐d‐glucan enzyme synthesis complex. Their main therapeutic indication is the treatment of Candida fungal infections, particularly those resistant to fluconazole and amphotericin B. They are also highly efficacious against a number of Aspergillus species [38]. Marketed echinocandins include caspofungin (Cancidas®, Merck & Co., Inc.), anidulafungin (Eraxis®, Pfizer Inc.), and micafungin (Mycamine, Astellas Pharma Inc.). 1.3.2  Ribosomally Synthesized Lantibiotics (or lanthipeptides) are representative ribosomally synthesized peptides, which undergo posttranslational modification. The lantibiotics are characterized by thioether amino acid residues lanthionine and methyllanthionine [39]. The prototypical and oldest lantibiotic is nisin, originally isolated from Lactococcus lactis in the 1930s, which has been used for decades as a food preservative. More recently discovered analogs have attracted considerable interest for their multiple modes of action and high in vitro potency against a number of problematic organisms such as methicillin‐resistant S. aureus and vancomycin resistant Streptococcus pneumoniae [40]. The clinical and commercial development of lantibiotics has been complicated by inefficient production and poor chemical and biophysical properties. Nevertheless, their unique therapeutic potential will likely help maintain interest in the lantibiotics class in the future. Our understanding of the role and potential utility of peptide antibiotics was transformed in the 1980s by seminal discoveries of the cecropins, the magainins, and the defensins. The cecropins, isolated by Boman’s group from the hemolymph of the cecropia moth, are 30 to 37 residue peptides that mediate cell‐free immunity for the insect and are active against both gram‐positive and gram‐negative bacteria [41] with similar peptides identified in related insect species. Selsted et al. isolated homologous AMPs termed “defensins” from human and rabbit leukocytes with potent activity against bacterial and fungal organisms, suggesting that these peptides play a key role in mammalian host defense [42]. The defensins were subsequently also detected in mammalian respiratory secretions where they are thought to provide a first line of defense against microbial invasion [43].

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Skin secretions from frogs and toads are a rich source of bioactive peptides. The serendipitous discovery of the magainins in the skin of the African clawed frog by Zasloff [44] was the first of many subsequently discovered ribosomally synthesized AMPs of amphibian origin. Other AMP families include dermaseptins, bombinins, brevinins, esculentins, and ranalexins with additional examples frequently reported. An updated database can found at http://aps.unmc.edu/AP. Many of these peptides have been shown to have potent, broad‐spectrum activity against a variety of gram‐positive and gram‐negative bacterial as well as fungal pathogens. Despite their structural heterogeneity, most AMPs share a cationic, amphiphilic a‐helical structure, which enables them to penetrate and disrupt bacterial cell membranes, their presumed mode of action [45, 46]. Their cationic nature, however, contributes to their hemolytic properties, which have thus far limited their use as systemic antibiotics. Despite this shortcoming, the use of AMPs in the clinical setting is being explored in wound healing [47] as well as skin and oral infections [47]. A number of AMPs have entered clinical trials: CLS001 (omniganan) in rosacea and acne vulgaris, AB‐103 (reltecimod) in necrotizing soft tissue infections, SGX942 (dusquetide) and brilacidin each in oral mucositis [48]. A number of other candidates are either in early or preclinical stages of development.

1.4 ­Peptides in Cancer Until recently, peptides had only limited application in the treatment of cancer; specifically, they were used to induce hormonal deprivation as a means of slowing tumor growth and disease progression. Nevertheless, luteinizing hormone releasing hormone (LHRH) and somatostatin (SST) analogs remain the standard of care for numerous cancer indications, and currently provide a versatile platform for diagnostic and therapeutic innovation, which helps advance cancer treatment. 1.4.1  Luteinizing Hormone Releasing Hormone The history of the LHRH peptide family was recently reviewed by Schally, whose group was principally involved in its discovery and subsequent introduction into reproductive medicine and oncology (see Figure  1.4) [49]. The demonstration that continuous administration of LHRH hormone (Figure 1.4a) downregulated LH and follicle‐stimulating hormone (FSH) secretion was central to the clinical application of LHRH agonists to cancers of the prostate, breast, and endometrium [50]. The earliest approved examples of this class were short‐acting agonists leuprolide (Lupron®, Abbott Laboratories), triptorelin (Trelstar®, Allergan, Inc.), buserelin (Suprecur®, Sanofi S.A.), and goserelin (Zoladex® AstraZeneca plc), which were all subsequently developed as depot formulations, providing continuous exposure for up to several months (Figure  1.4b–d respectively). Exploration of LHRH structure–activity relationships resulted in the discovery of LHRH antagonists such as degarelix (Figure  1.4e) (Firmagon®, Ferring Pharmaceuticals, Inc.) capable of inducing competitive LHRH receptor blockade

1.4  Peptides in Cancer (a) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (b) pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt (c) pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 (d) pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt OH

O

HN

H N

N H NH N

HO O

O N H

O

O

H N

O N H

NH2

O NH

O

HN

NH2

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NH

NH (e)

O

NH O NH2

O

H N O

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H N O

OH H N

O N H N

O

O

O

H N

N H

N H

O NH

(f)

HN

NH

Cl

O

O N

O N H

H N

NH2 O

O NH

O

Figure 1.4  Structures of (a) LHRH, (b) leuprolide, (c) triptorelin, (d) buserelin, (e) goserelin, and (f ) degarelix.

in the absence of an intrinsic agonist effect. In contrast to the earlier agonists, administration of LHRH antagonists produces immediate androgen deprivation, and importantly does not provoke the characteristic LH release or “flare” [51]. This was confirmed in a comparative study of the antagonist degarelix versus the agonist leuprolide, which demonstrated equally durable testosterone suppression for both agents. However, testosterone suppression occurred faster and with no reported “flare” in the degarelix arm [52]. 1.4.2 Somatostatin SST analogs represent the other major class of peptide therapeutics with application to cancer treatment. The initial observations noting that hypothalamic

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extracts inhibited secretion of growth hormone (GH) from the pituitary led to the isolation and characterization of the 14 amino acid peptide, SST‐14, by Vale and Guillemin [53, 54]. Early research demonstrated that in addition to the known inhibition of GH, somatostatin peptides are also potent suppressors of a number of other hormones, including insulin, TSH, and glucagon [55]. The potential application of SST ligands to cancer treatment was suggested by the finding that SST receptors are overexpressed in tumors originating within SST target tissues. This prompted Vale and coworkers, as well as investigators at the former Sandoz laboratories starting in the 1970s to find more potent and stable SST analogs [56]. The latter’s efforts culminated in the development and approval of the potent SST agonist octreotide (Sandostatin® Novartis Pharmaceuticals Corporation) (Figure 1.5a). Octreotide demonstrated a three‐fold higher potency in insulin inhibition as well as a nearly 20‐fold higher potency in GH inhibition relative to the native hormone [57]. Following its launch in 1983, it proved a highly valuable treatment option for a number of conditions such as carcinoid syndrome, intestinal, pancreatic, and pituitary tumors [58]. Octreotide’s ability to suppress pituitary secretion of GH also made it invaluable for the treatment of acromegaly. In addition to octreotide, a structurally similar SST analog lanreotide (Somatuline®, Ipsen S.A.S.) (Figure 1.5b) was approved in 2007 and is used to treat various gastro‐enteropancreatic‐neuroendocrine tumors (GEP‐NETs). Both octreotide and lanreotide are available as extended time‐action depot formulations: Sandostatin LAR Depot from Novartis and Somatuline Depot from Ipsen. 1.4.3  Peptide–Drug Conjugates The clinical success of antibody–drug conjugates (ADCs) has validated Paul Ehrlich’s century‐old concept of a targeted “magic bullet” cancer therapy. The exquisite selectivity of peptide ligands has been successfully harnessed for the design of peptide–drug conjugates (PDCs), utilizing the same receptor targeting

NH2 O

HO HO

O N H

S

HN O

(a)

NH

S

O

H2N

O OH O O

N H H

H2N

O

H N

NH NH

H N

OH

O O

NH

NH

NH2

HN S O

N H

(b)

Figure 1.5  Structures of octreotide (a) and lanreotide (b).

NH2

O

S

O HN H

H N O

N H

O NH

OH

1.4  Peptides in Cancer

strategy as the ADCs to deliver a cytotoxic payload directly to a tumor while minimizing systemic toxicity [59]. Substitution of an antibody with a peptide offers several advantages such as a reduction in immunogenicity, and a potential increase in cell and tissue penetration. From a technical standpoint, the installation of a linker and drug conjugation may be more straightforward in the case of a PDC. In addition, the peptide’s lower molecular weight also permits higher drug loading relative to an ADC. In the case of LHRH‐based PDCs, several cytotoxic analogs have been evaluated clinically, including AEZS‐152, a doxorubicin conjugate of [d‐Lys6]‐LHRH [60]. The octreotide platform also continues to be of great utility for both diagnostic and therapeutic applications [61]. The imaging agent 111 In‐DPTA‐octreotide (diethylene triamine pentaacetic acid) (OctreoScan® Mallinckrodt Pharmaceuticals) has been used extensively to localize somatostatin‐expressing neuroendocrine tumors (NET) while 99mTc‐depreotide (NeoTect® Diatide, Inc.) is used as a diagnostic for small cell lung cancer. The octreotide scaffold also serves as a targeting ligand in peptide radionuclide therapy. 177Lu octreotate (Lutathera® Advanced Accelerator Applications) was recently approved by the FDA for the treatment of somatostatin receptor‐positive GEP‐NETs [62]. Integrin motifs, such as ArgGlyAsp (RGD) and AsnGlyArg (NGR), are used to target tumor vasculature. NGR‐hTNF is a fusion protein consisting of the CNGRCG tumor homing peptide and tumor necrosis factor (hTNF) cytokine [63]. NGR‐hTNF is being investigated for the treatment of malignant pleural mesothelioma, either as a stand‐alone therapy or in combination with standard chemotherapeutic regimens. Mipsagargin is a conjugate of a prostate‐specific membrane antigen (PSMA) homing peptide fused with the cytotoxic sesquiterpene lactone thapsigargin, which is in clinical trials for hepatocellular carcinoma [64]. ANG 1005 is a conjugate formed with paclitaxel and angiopep‐2, a peptide targeting the low density lipoprotein receptor‐related protein 1 (LRP‐1), being developed by Angiochem, Inc. for a glioblastoma indication [65]. 1.4.4  Cancer Vaccines Cancer vaccines have been previously tested in late‐stage or metastatic settings and have shown only modest results [66]. Recently, however, this therapeutic approach has made enormous progress and for the first time gained substantial clinical validation [67]. The two basic peptide‐based immunization strategies can be classified as utilizing either self‐antigens or neo‐antigens and are discussed briefly below. Successful vaccination with a self‐antigen, typically directed at an overexpressed receptor, has been demonstrated in animal models and recently in the clinic. A breast cancer vaccine, Neuvax™, based on a human epidermal growth factor receptor‐2 (HER‐2) immuno‐dominant epitope administered with granulocyte colony‐stimulating factor (GMCSF) as an adjuvant has been evaluated extensively in a number of clinical trials designed to reduce breast cancer recurrence [68]. A meta‐analysis has shown that Neuvax either as a single agent or in combination with Herceptin reduces risk of recurrence and prolongs both disease‐free and overall survival [69]. Despite these promising results, the self‐antigen strategy is based on a single epitope that may diminish the magni-

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tude of the immune response [70]. The strategy is further limited by major histocompatibility complex (MHC) restriction to HLA‐A2 and HLA‐A3 positive patients [71]. An emerging vaccine paradigm involves amplification of tumor‐specific T‐cell response through immunization with multiple neo‐antigens, unique sequences arising from cancer‐specific somatic mutations. In contrast to self‐antigens, neo‐ antigens bypass central thymic tolerance and are therefore more likely to produce a strong T‐cell response [72]. The sequences of the neo‐antigen peptides are predicted from algorithms utilizing genomic, proteomic, and predicted MHC binding data from an individual patient’s tumor, synthesized and assembled into a personalized cancer vaccine. The strategy has been successful in small clinical trials, particularly in cases of tumors with a high mutagenic load. Of six patients diagnosed with advanced melanoma and treated with personalized neo‐antigen vaccines, four were recurrence free 25 months post vaccination [73]. The two patients who experienced recurrence responded favorably to anti‐programmed cell death protein‐1 (anti‐PD‐1) therapy, an effect attributed to stimulation of their tumor‐specific T cells. In another study, late‐stage melanoma patients were treated with a poly‐epitope vaccine constructed from two synthetic ribonucleic acids (RNAs) encoding linker connected antigens. Of the 13 patients in the study, eight remained recurrence free for 12 to 23 months. Of the five who relapsed, one achieved complete response to an anti‐PD‐1 antibody [74].

1.5 ­Peptides in Bone Diseases As a molecular class, peptides are underrepresented in the treatment of bone disease. With the singular exception of calcitonin, used for the treatment of osteoporosis, most drugs approved for bone diseases have been orally administered  –  small molecules such as estrogen, the selective estrogen receptor modulator, raloxifene (Evista®, Eli Lilly & Co.), and the bisphosphonates. The competitive landscape changed dramatically with the introduction of teriparatide and later abaloparatide, peptides, which have demonstrated restoration of osteopenic bone and a reduction in fracture rates. 1.5.1 Calcitonin A 32 amino acid peptide first described in the early 1960s, calcitonin (Figure 1.6a,b), is secreted by the parafollicular cells of the thyroid gland and is responsible for maintaining calcium homeostasis and regulation of bone turnover [75]. Because of its greater pharmacological potency, salmon calcitonin has been utilized in place of human calcitonin for most therapeutic applications. Initially approved in 1984 for the treatment of postmenopausal osteoporosis, calcitonin is currently also prescribed for Paget’s disease and hypercalcemia. Its anti‐resorptive activity at bone is mediated primarily through inhibition of osteoclast formation, although its precise mechanism is still not entirely clear. Clinical studies established that persistent parenteral administration of salmon calcitonin prevented postmenopausal bone loss and increased bone mineral

1.5  Peptides in Bone Diseases (a) C − G − N − L − S − T − C − M − L − G − T − Y − T − Q − D − F − N − K − F − H − T − F − P − Q − T − A − I − G − V − G − A − P — NH2 (b) C − S − N − L − S − T − C − V − L − G − K − L − S − Q − E − L − H − K − L − Q − T − Y − P − R − T − N − T − G − S − G − T − P — NH2 (c) S − V − S − E − I − Q − L − M − H − N − L − G − K − H − L − N − S − M − E − R − V − E − W − L − R − K − K − L − Q − D − V − H − N − F — OH (d) A − V − S − E − H − Q − L − L − H − D − K − G − L − S − I − Q − D − L − R − R − R − E − L − L − E − K − L − L − X − K − L − H − T − A — OH X = Amino isobutyric acid (Aib)

Figure 1.6  Structures of (a) human calcitonin, (b) salmon calcitonin, (c) teriparatide, and (d) abaloparatide.

density (BMD) [76]. Later studies with daily injectable salmon calcitonin also demonstrated an ability to reduce the risk of both vertebral [77] and hip fractures [78]. In addition to its potent in vivo activity, salmon calcitonin is reported to exhibit high bioavailability by nasal administration, estimated at 10 to 25 %. This offers an important option, which has facilitated greater patient acceptance and compliance. Clinical results with nasal calcitonin (Miacalcin®, Mylan), however, have demonstrated only a modest 1.7 % increase in BMD in osteoporotic women after one year [79] and also appeared inferior in a 12 month study versus alendronate [80]. The efficacy of nasal calcitonin in prevention of fractures is also less convincing, despite numerous clinical studies. A closer analysis of clinical trial data suggests that calcitonin pharmacology is, generally, more pronounced in patients with high bone turnover and established osteoporosis. The potential clinical utility of calcitonin is not limited to existing bone indications; it has also been investigated for its analgesic properties in the treatment of acute vertebral fracture pain [81] and osteoarthritis [82]. 1.5.2  Parathyroid Hormone (PTH) (1–34) and (1–84) Originally isolated by Collip in the 1920s [83], parathyroid hormone (PTH) was noted for its effects on calcium levels in the 1930s, but not fully investigated for its therapeutic potential until the 1970s. An early, small clinical study of 21 patients with osteoporosis confirmed that PTH (1–34) dosed daily at 100 μg for 6 to 24 months resulted in significant increases in new bone formation, particularly in trabecular bone [84]. This proved to be a seminal finding supporting therapeutic use of the hormone, since chronic overexposure to PTH as observed in hyperparathyroidism leads to osteoporosis. Subsequently, a number of clinical trials established the efficacy of PTH (1–34) in the management of severe osteoporosis in postmenopausal women, particularly those who had suffered a previous vertebral fracture. These studies revealed improvements in a number of clinical end points such as trabecular and cortical bone mass, mineral content, density, and fracture healing [85]. Additional studies also confirmed the importance of intermittent exposure to PTH (1–34) to achieve optimal anabolic activity, rather than continuous dosing with persistent plasma elevation of the hormone [86]. PTH (1–34) offers unprecedented therapeutic benefits relative to calcitonin, the bisphosphonates, and estrogen, which achieve their effect primarily through inhibition of osteoclast‐mediated bone resorption. These clinical results demonstrated the seminal point that pharmacology differs from

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physiology and pathology [87]. Through careful selection of clinical dosing and intermittent administration, the biological outcome derived from PTH is completely reversed from bone loss to bone growth. PTH (1–34) pharmacological effects are largely the result of its anabolic properties, which include bone‐lining cell activation, osteoblast cell differentiation, and proliferation [88]. PTH (1–34) was approved in 2002 by the FDA for the treatment of osteoporosis in postmenopausal women and men with high risk of fracture as teriparatide (Forteo®, Eli Lilly & Co.) (Figure 1.6c). Clinical data collected prior to and post FDA approval support Forteo as the first truly regenerative medicine that stimulates active bone formation and restores osteopenic bone to near normal health. Despite some initial controversy, which pertained to the prospect of irreversible effects of bisphosphonates, teriparatide has been shown to work well with their concurrent administration, presumably because of the additive anti‐resorptive effect of the oral agents [89]. The risks of teriparatide include increased blood calcium levels and osteosarcoma. The latter effect, which was specific to rodent studies, resulted in a restricted label with a clinical use limit of two years. The full‐length PTH (1–84) protein was marketed for osteoporosis indication in the European Union as Preotact® starting in 2006, until its withdrawal for commercial reasons in 2014. Currently, it is approved for the treatment of chronic hypoparathyroidism in the European Union (as Natpar®, Shire Pharmaceuticals) and the United States (as Natpara®). 1.5.3  Parathyroid Hormone Related Protein Parathyroid Related Protein (PTHrP) and PTH signal through a common receptor; however, there are notable differences in their respective modes of receptor interaction. A recent in vitro study examined the binding preference of PTH and PTHrP peptides to the R0 (G‐protein‐independent) and RG (G‐protein‐dependent) conformations of the PTHr1 [90]. The findings indicated that while both PTHrP and PTH peptides bind to the RG conformation of PTHr1, the PTHrP ligands bind the R0 conformation with much lower affinity. The high RG selectivity of PTHrP is characteristic of a transient signaling response and consistent with a pronounced anabolic effect relative to PTH. The recently approved 36 amino acid fragment of PTHrP, abaloparatide (Figure  1.6d) (Tymlos®, Radius Health, Inc.), exerts qualitatively similar pharmacological effects as teriparatide, with several notable therapeutically significant differences. Studies in human osteoblast cells indicated that abaloparatide exerts a lesser effect on the expression of bone‐resorptive factors compared to teriparatide, supporting an overall greater net anabolic character than the latter [91]. Other clinical studies, notably a pivotal 18‐month Phase 3 study (ACTIVE) in 2463 postmenopausal women using abaloparatide, teriparatide, and placebo revealed respective vertebral fracture rates of 0.6 %, 0.8 %, and 4.2 %, and non‐vertebral fracture rates of 2.7 %, 3.3 %, and 4.7 %. In addition, the study noted lower hypercalcemia rates for abaloparatide than for teriparatide (3.4 % versus 6.4 %), consistent with the hypothesis that the former agent exerts a lesser bone‐resorptive effect than the latter. While the overall results were supportive of greater efficacy of abaloparatide, the study noted slightly higher rates of adverse events for this agent [92].

1.6  Peptides in Gastrointestinal Diseases

1.5.4  Incretin Peptides Recently, a number of peptides have been investigated for their bone remodeling/ healing potential, and while these have not advanced into clinical trials, they nevertheless show promise. The growing interest in the extra‐pancreatic actions of GLP‐1 and GIP has revealed a strong link between incretin activity, bone strength, and fracture reduction [93]. A bone‐densitometry study by Yamada [94] of GLP‐1 receptor knockout mice and their littermate controls revealed cortical osteopenia and bone fragility in the receptor‐deficient animals, due to increased osteoclast resorption. GLP‐1 and exendin‐4 also reversed osteopenia in hyperlipidic and hypercaloric rat models [95, 96]. Clinical data, however, appear mixed as a meta‐analysis conducted by Su [97] found a significant decrease in bone fractures in patients treated with liraglutide, but an increase in patients treated with exenatide. Preclinical evidence for a bone‐related benefit seems more compelling in the case of GIP. GIP overexpressing mice exhibited increased bone formation and decreased bone resorption [98], while GIP receptor deficient mice exhibited bone weakening including decreased cortical thickness, increased resorption, and decreased bone mineralization [99]. An important link to human biology comes from association of functional GIP receptor polymorphism Glu354Gln and fracture risk conducted by Torekov et al. [100]. The Glu354Gln substitution attenuates GIP signaling, resulting in lower insulin secretion and higher glucose levels. Women with this allele in a 10 year period were found to have lower bone density and greater fracture risk. 1.5.5  Bone Morphogenic Protein‐Derived Peptides Bone morphogenic proteins (BMPs), members of the TGF‐β (transforming growth factor) family, play an important role in bone formation and development [101, 102]. A number of peptide sequences derived from BMP proteins have shown potent osteogenic activity in animal models. Two peptides derived from BMP‐7, bone‐forming peptide‐1 and 2, stimulated differentiation of bone marrow stem cells in vitro and in vivo [103, 104]. Work with BMP‐9 identified a peptide that promoted the differentiation of pre‐osteoblasts and deposition of calcium, conditions required for bone mineralization [105, 106].

1.6 ­Peptides in Gastrointestinal Diseases The treatment of gastrointestinal (GI) disorders has been one therapeutic area where peptide drugs played virtually no role until very recently. The low pH and presence of proteolytic enzymes have made the GI track particularly unsuitable for peptide drugs. Nevertheless, in the last six years three new important peptide drugs have been launched, each addressing unmet needs in GI disease states. 1.6.1  Glucagon‐like Peptide‐2 A 33 amino acid peptide secreted by entero‐endocrine L‐cells, glucagon‐like peptide‐2 (GLP‐2) exerts a number of effects on the GI tract. Most notably, it

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increases intestinal nutrient absorption and the stimulation of intestinal growth mediated by the release of insulin‐like growth factor‐1, epidermal growth factor, and keratinocyte growth factor [107]. Much like GLP‐1, GLP‐2 has a short biological half‐life of approximately seven minutes, due to DPP‐IV inactivation. Teduglutide (Figure 1.7) is a DPP‐IV‐stabilized GLP‐2 analog where the native Ala at the second position is replaced by Gly. The clinical development of teduglutide sponsored by NPS Pharmaceuticals focused on short bowel syndrome (SBS), a condition characterized by significant loss of bowel mass and function. Patients afflicted by SBS may lose sufficient absorptive function to the extent that they need to rely on parenteral support (PS) to maintain nutrient intake and electrolyte balance. A 21 day Phase II open label clinical study of teduglutide demonstrated a statistically significant increase in intestinal wet weight absorption in 15 of 16 patients [108]. The trial also noted favorable histological changes in most patients, specifically increases in villus height, crypt depth, and mitotic index. A subsequent Phase III study in SBS patients who had suffered intestinal failure met its primary end point, demonstrating a statistically significant reduction in PS requirements, as well as a secondary end point of allowing patients to gain additional days off PS, or entirely eliminating the need for it [109]. Teduglutide was approved by the FDA in 2012 for adults with SBS requiring PS support and marketed as Gattex® by NPS Pharmaceuticals. Zealand Pharma recently announced initiation of a Phase 3 trial of their GLP‐2 analog glepaglutide in SBS. 1.6.2  Guanylate Cyclase‐C Agonists The natriuretic peptide hormones guanylin and uroguanylin (Figure  1.8a,b respectively) are produced by the entero‐endocrine cells of the GI tract and sigHN

N

NH2 H2N

O O

N H

O

N H O

O HO O O

O

OH

N H O

HO

OH O N H

HO

O N H N H

OH HN

O

N H

N H

HN

HO

N H HN

N H

N H

O O

HN O O OO

Figure 1.7  Structure of teduglutide.

H N O

N H HO NH O O H2N NH

H N

O

NH2 OO

O

O O O

HN NH2

HN O

S O

NH

HN

O

H2N

H N

HN

O N H

O

O

O NH NH2

N H HO

O

O

N H

O N HO H N N H

OH O OO N H HO

NH HO

O

1.6  Peptides in Gastrointestinal Diseases

nal through the guanylate cyclase (GC‐C) receptors located on the intestinal enterocytes. Activation of the GC‐C pathway is essential for maintaining fluid as well as chloride and bicarbonate ion homeostasis within the GI tract, which are supportive of healthy intestinal transit [110]. Linaclotide (Figure 1.8c), a peptide closely related to guanylin and uroguanylin, was developed by Ironwood Pharmaceuticals for symptoms related to constipation‐predominant irritable bowel syndrome (IBS‐C). Oral administration of linaclotide to healthy volunteers was shown to be safe and efficacious with a dose‐dependent increase in stool frequency and weight [111]. Remarkably, the study also found no evidence of systemic absorption following oral dosing, suggesting that linaclotide works locally in the GI tract. In a subsequent placebo‐controlled Phase II study in patients suffering from IBS‐C, linaclotide improved bowel function in terms of frequency, severity of straining, stool consistency, and abdominal pain [112]. In a 26 week, double‐blind, placebo‐controlled Phase III trial in 804 patients, daily oral administration of 290  μg of linaclotide showed statistically significant improvements in the frequency of complete spontaneous bowel movements (CSBM) and a reduction of abdominal pain episodes [113]. Based on this as well as an additional Phase III clinical study linaclotide was approved by the FDA for adults suffering from IBS‐C and chronic idiopathic constipation (CIC) in 2012 and marketed as Linzess® by Allergan and Ironwood Pharmaceuticals. Plecanatide (Figure 1.8d) is a GC‐C agonist peptide structurally related to uroguanylin, differing only in the substitution of Glu3 for the native Asp3. Plecanatide reversed symptoms of acute and chronic ulcerative colitis in several animal models [114], suggesting that uroguanylin and possibly guanylin deficiency may be a causative factor in GI inflammation. Synergy Pharmaceuticals undertook the development of plecanatide for various gastrointestinal disorders. A Phase I clinical study found plecanatide to be safe, and similarly to linaclotide, showed no evidence of systemic absorption [115]. A larger 12 week trial tested 3 and 6 mg doses of plecanatide or placebo in 1346 patients suffering from CIC. Both dose cohorts met the primary and secondary end points of significantly increasing CSBM and spontaneous bowel movements (SBM) [116]. The favorable results of

(a)

P−G−T−C−E−I−C−A−Y−A−A−C−T−G−C

(b)

N−D−D−C−E−L−C−V−N−V−A−C−T−G−C−L

(c)

C−C−E−Y−C−C−N−P−A−C−T−G−C−Y

(d)

N−D−E−C−E−L−C−V−N−V−A−C−T−G−C−L

Figure 1.8  Structures of (a) guanylin, (b) uroguanylin, (c) linaclotide, and (d) plecanatide.

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this trial, as well as additional Phase III trials in CIC and IBS‐C patients, supported the FDA approval of plecanatide (Trulance®, Synergy Pharmaceuticals). There are currently a number of peptide drugs in development for GI disorders and are summarized in a recent review [117]. These include larazotide, a peptide based on zonula occludent toxin, which is being advanced by Innovate Pharmaceuticals for celiac disease in patients who are symptomatic despite adhering to a gluten‐free diet [118]. Relamorelin, a ghrelin agonist, is being investigated for several GI conditions including CIC and gastroparesis, the latter a currently an unmet medical condition [119].

1.7 ­Emerging Trends in Peptide Drug Discovery 1.7.1  Cell‐Penetrating Peptides The lack of membrane permeability, which effectively prevents access of various drugs to intracellular targets, is a common problem in drug discovery. Thirty years ago, novel peptides capable of transporting cargo across biological membranes were termed cell‐penetrating peptides (CPPs). Members of this group range from 5 to 40 amino acids and have been successfully used to facilitate intracellular uptake of various cargoes including proteins, quantum dots, and siRNAs [120–122]. Recently, confirmation of the ability of CPPs to transport drug cargo safely into cells was demonstrated in preclinical and clinical studies. CPPs can be utilized in the form of a non‐covalent complex or as a covalently bound drug conjugate. CPPs fall into three major categories and they include cationic, amphipathic, or hydrophobic. A representative of the cationic class, which was also the first CPP family member identified, is the trans‐activating transcriptional (TAT) activator from human immunodeficiency virus 1 (HIV‐1) (see Figure 1.9 for the sequences of various CPPs) [123, 124]. The minimal HIV‐ TAT derived sequences required for transduction were characterized several years later [125, 126]. Another important CPP is penetratin, a minimized 16 amino acid cationic sequence derived from Antennapedia, a homeoprotein of Drosophila melanogaster [127, 128]. Two examples of amphipathic CPPs containing both hydrophilic and hydrophobic amino acids, are transportan [129] and the model amphipathic peptide (MAP) [130]. Hydrophobic CPPs such as the Pep‐7 peptide [131] are less common but are of particular interest as a result of their energy‐independent mechanism. The list of CPPs is growing and their use is being explored for the treatment of infectious diseases [132], inflammation [133], and cancer [134] with several candidates in late‐stage clinical development HIV-TAT (48–60) Penetratin Transportan MAP Pep-7

GRKKRRQRRRPPQ RQIKIWFQNRRMKWKK GWTLNSAGYLLGKINLKALAALAKKIL KLALKLALKALKAALKLA SDLWEMMMVSLACQY

Figure 1.9  Structures of various CPPs.

1.8 Summary

[135]. The preferred CPP peptides used in preclinical and clinical trials today are generally based on the shorter TAT or penetratin sequences. Kai Pharmaceuticals (acquired by Amgen in 2012) had advanced at least two compounds containing TAT derivatives into clinical trials (KAI‐9803 [136], KAI‐1678 [137]). Sarepta Therapeutics, a company with multiple CPP‐based clinical candidates, has developed eteplirsen, a phosphorodiamidate morpholino oligomer (PMO) conjugated to a proprietary arginine‐rich CPP, to treat Duchenne muscular dystrophy, which received approval as Exondys 51™ [138]. Brimapitide, an arginine‐rich sequence combined with a c‐Jun‐N‐terminal kinase (JNK) inhibitor developed by Xigen, has received FDA fast track status for hearing loss treatment [139]. 1.7.2  Macrocyclic Peptides Natural product‐derived macrocyclic peptides, such as vancomycin and cyclosporine, serve as an important historical precedent for peptide medicinal chemists. As a class, macrocyclic peptides possess properties that are atypical of those of conventional peptides, particularly protease stability, and in the rare case of cyclosporine, even oral availability [140]. A wide variety of chemistries can be used to construct macrocyclic peptides including disulfide, thioether, head‐to‐ tail, and depsi‐peptide bonds [141, 142]. In general, as the size of the macrocycle increases, the structure becomes more flexible and susceptible to proteolytic degradation. Additional constraint in the form of a second ring not only enhances proteolytic stability but can also increase affinity and selectivity, an approach pioneered by Bicycle Therapeutics. The company is advancing BT1718, a bicyclic macrocyclic peptide, for the treatment of advanced solid tumors. A second clinical candidate based on this technology, THR‐149, a novel plasma kallikrein inhibitor for the treatment of diabetic macular edema, is being developed by Thrombogenics. PeptiDream has combined genetic code reprogramming to incorporate non‐proteinogenic amino acids into vast libraries of macrocycles. The company’s proprietary Peptide Discovery Platform System (PDPS) uses Flexizymes, artificial ribozymes capable of charging tRNAs with unnatural amino acids [143]. Aileron Therapeutics has applied the Grubbs olefin metathesis to construct “stapled peptides,” which exhibit remarkable protease stability as well as unique ability to engage intracellular targets such as BCL‐2 and MDMX and MDM2, by virtue of their covalently stabilized α‐helical structure  [144]. Aileron has used this platform to deliver their ALRN‐6924 clinical candidate, an MDMX/MDM2 inhibitor currently in multiple Phase 1 and 2a clinical trials for the treatment of advanced solid tumors, peripheral T cell lymphoma (PTCL), acute myeloid leukemia, and advanced myelodysplastic syndrome [145]. Other companies focusing on the development of therapeutics based on cyclic peptides include Ra Pharma and Polyphor.

1.8 ­Summary The peptide therapeutic class has experienced significant growth in recent years, as is evident from the approved and late‐stage clinical programs highlighted in this

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chapter. However, a comprehensive survey of earlier clinical and preclinical efforts suggests that this growth will accelerate in the future as peptides impact multiple therapeutic areas. A key reason for this trend is the inherent efficacy of peptide drugs in restoring normal physiological function to patients suffering from diabetes, osteoporosis, and other chronic diseases. This attribute has often enabled peptides to compete successfully against less efficacious oral agents. One example is the commercial success of injectable GLP‐1 agonists when competing against orally administered DPP‐4 inhibitors. Another is the superior performance of injectable PTH (1–34), relative to the oral bisphosphonates. The traditional advantages of oral agents over injectable drugs have recently narrowed through introduction of extended duration peptide analogs, which prolong the dosing interval from days to weeks or even longer. Additionally, a number of recently introduced peptide drugs such as the GC‐C agonists can be delivered orally, and several more orally administered peptide candidates are in late stage clinical trials. Innovative concepts such as cell‐penetrating peptides, stapled peptides, and peptide–drug conjugates promise to enable engagement of diverse, previously inaccessible targets. We anticipate that the above trends will expand the niche for peptide therapeutics in the pharmaceutical armamentarium of the future.

­Acknowledgment The authors would like to thank Prof. Richard D. DiMarchi of Indiana University, Bloomington, for his contribution to the bone disease section and encouragement during the preparation of the manuscript.

­List of Abbreviations ADC Aib AMP BMD BMP CPP CRL CSBM DDP‐IV CIC DMSO DNA DTPA FDA FSH GC‐C GEP‐NET GH

antibody–drug conjugates 2‐aminoisobutyric acid antimicrobial peptide bone mineral density bone morphogenic protein cell‐penetrating peptide complete response letter complete spontaneous bowel movement dipeptidyl peptidase‐4 chronic idiopathic constipation dimethyl sulfoxide deoxyribonucleic acid diethylene triamine‐pentaacetic acid food and drug administration follicle stimulation hormone guanylate cyclase C gastro‐enteropancreatic neuroendocrine tumors growth hormone

­  References

GI gastrointestinal GIP glucose‐dependent insulinotropic polypeptide GLP‐1 glucagon‐like peptide‐1 GLP‐2 glucagon‐like peptide‐2 GPCR G‐protein‐coupled receptor GMCSF granulocyte colony‐stimulating factor HER‐2 human epidermal growth factor receptor‐2 HIV human immunodeficiency virus HSA human serum albumin hTNF human tumor necrosis factor IBS irritable bowel syndrome IgG immunoglobulin G JNK c‐Jun‐N‐terminal kinase LHRH luteinizing hormone releasing hormone LRP‐1 low‐density lipoprotein receptor‐related protein 1 MAP model amphipathic peptide MHC major histocompatibility complex NET neuroendocrine tumor NPH neutral protamine Hagedorn PD‐1 programmed cell death protein‐1 PDC peptide‐drug conjugate PDPS Peptide Discovery Platform System PEG polyethylene glycol PK/PD pharmacokinetic/pharmacodynamic PLG poly(d,l‐lactide‐co‐glycolide) PMO phosphorodiamidate morpholino oligomer PS parenteral support PSMA prostate‐specific membrane antigen PTCL peripheral T cell lymphoma PTH parathyroid hormone PTHrP parathyroid hormone related protein RNA ribonucleic acid SBM spontaneous bowel movement SBS short bowel syndrome SST somatostatin TAT trans‐activating transcriptional activator TGF transforming growth factor

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­Biographies Dr. Florence M. Brunel is currently Senior Research Scientist at the Novo‐Nordisk Research Center in Indianapolis, Indiana. Over the previous decade she had held positions as a research scientist successively at Amylin, Pfizer, and Vertex working in the area of medicinal chemistry. She obtained her MS degree in organic chemistry (French Engineer Degree) from the Ecole Nationale Superieure de Chimie de Montpellier, followed by a PhD degree with Prof. Arno F. Spatola at the University of Louisville specializing in peptide chemistry. She received additional peptide training under the direction of Prof. Philip Dawson at The Scripps Research Institute. Her current interests focus on peptides and proteins polypharmacy in the field of diabetes, obesity, and NASH. Dr. Fa Liu is currently the Director of Discovery Chemistry at Novo Nordisk Research Center in Seattle, WA. Prior to joining Novo‐Nordisk he was the Director of Chemistry at Calibrium LLC (2014– 2015) and a Senior Research Scientist at Eli Lilly & Co. (2009–2014). Dr. Liu received his PhD from the Shanghai Institute of Organic Chemistry in 2004 followed by a five year appointment at the National Cancer Institute as a Postdoctoral Fellow (2004– 2007) and a Staff Scientist (2007–2009). His research focuses on peptide/protein therapeutics and the development of novel peptide/ protein chemistries. Dr. John Mayer is a Research Scientist in the Molecular, Cellular, and Developmental Biology Department at the University of Colorado, Boulder. Prior to this, he had been employed in the biopharmaceutical industry as a peptide chemist having held positions at Amgen, Eli Lilly, Calibrium LLC, and Novo‐Nordisk. He received his undergraduate degree in Biochemistry from Northwestern University (1979) and a PhD degree in Medicinal Chemistry from Purdue University (1987). This was followed by two years of postdoctoral training in peptide chemistry in Dr. Richard DiMarchi’s group at Eli Lilly. He has published extensively on peptide chemistry and served on the American Peptide Society board as an elected council member (2009–2015).

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2 Physicochemical Parameters of Recently Approved Oral Drugs Andreas Ritzén1 and Laurent David2 1 2

Drug Design, LEO Pharma A/S, Industriparken 55, 2750 Ballerup, Denmark Medicinal Chemistry, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark

2.1 ­Introduction Guidelines, e.g. the rule of five (Ro5) [1] and rules for polar surface area (PSA) [2,  3] and rotatable bonds [3], are widely used to guide the design of new ­molecules into drug‐like chemical space. But how well do recently approved drugs comply with these rules? In this chapter, we present an analysis of the distribution of six key properties for all 175 orally dosed drugs with systemic action that were approved by the United States Food and Drug Administration (FDA) in 2007–2017. The properties we analyze are molecular weight (MW), topological polar surface area (TPSA), [4] predicted log P, number of hydrogen bond acceptors (HBAs), number of hydrogen bond donors (HBDs), and number of rotatable bonds. We find that a significant fraction (19%) of the drugs do not comply with the Ro5 (i.e. have more than one violation) and are thus classified as “beyond rule of five” (bRo5) compounds. In this set of drugs, 29% of all compounds have MW > 500, and in the period 2013–2017, this fraction was 34%, showing a slight trend toward increasing MW in recent years. While Ro5 violations of MW, log P, and HBA count are fairly common, very few drugs have more than five HBDs. PSA is considered an important parameter for oral absorption, but nevertheless, 15% of the drugs have a TPSA (N + O atoms) [4] greater than the commonly cited [2, 3] cutoff of 140 Å2. We discuss the limitations of the TPSA parameter and explore some recent developments of more advanced metrics of polarity. Furthermore, drugs were classified according to whether they are approved to treat central nervous system (CNS) disorders or non‐CNS disorders. Property space for the CNS drugs is found to be more restrictive compared with non‐CNS drugs, with only a few CNS drugs having any Ro5 violation and only four compounds having a TPSA significantly greater than 80 Å2. It has been suggested that large, polar bRo5 molecules must be chameleonic [5–9] to be orally bioavailable: They expose polarity in aqueous environments to aid solubility, but they bury polar groups by intramolecular hydrogen bonding in the lipid environment of biological membranes to achieve good permeability. Successful Drug Discovery, First Edition. Edited by János Fischer, Christian Klein, and Wayne E. Childers. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2019 by Wiley-VCH Verlag GmbH & Co. KGaA.

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To explore this phenomenon, we undertook molecular dynamics simulations of selected examples in explicit solvents. Water and n‐octane were chosen to mimic the aqueous and membrane environments a drug molecule must traverse to reach its target after oral administration. Our results support the notion of chameleonic behavior for all seven representative high MW drugs analyzed.

2.2 ­FDA‐Approved Drugs 2007–2017 Since their introduction, the Ro5 and the TPSA  600 has increased in recent years. None were approved in 2007 and 2008, one was approved each of the following three years, and two to four such high MW drugs have followed every year since for a total of 20 during 2007–2017. This trend is driven by hepatitis C virus (HCV) NS3/4A and NS5 inhibitors (NS, nonstructural protein), e.g. pibrentasvir (1), that account for 13 of the 20 drugs, and protein–protein interaction (PPI) inhibitors, e.g. everolimus (2), that account for another two drugs (see Figure  2.2 for structures). On average, CNS drugs have lower MW than non‐CNS drugs, and only one CNS drug, the antiemetic neurokinin 1 (NK1) receptor antagonist netupitant (3), has a MW significantly above 500. The six fluorine atoms in 3 contribute over 100 Da to the MW of this compound. The median lipophilicity appears to be fairly constant in the 2.0 to 4.5 range of predicted log P although several recently approved drugs show very high numbers and the spread is quite large. No difference in predicted log  P between non‐CNS and CNS drugs was found, although the spread in this parameter is smaller for the CNS

2.2  FDA‐Approved Drugs 2007–2017 1200

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Figure 2.1  (Continued )

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2  Physicochemical Parameters of Recently Approved Oral Drugs

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Figure 2.1  Distribution of five key molecular descriptors by year and class (non‐CNS and CNS), and total distributions for each parameter. Non‐CNS drugs are shown as blue circles and CNS drugs as orange circles. Box plots show the first and third quartile of the data as the top and bottom of the solid box and the median as a horizontal line inside the box. Whiskers correspond to the highest and lowest point that is not an outlier, where outliers are defined as those points that are outside the median ±1.5× the interquartile range, i.e. the height of the box. The labels refer to the compound numbers in the text (see Figures 2.2 and 2.6 for structures). (a) Molecular weight, (b) log P predicted using Simulations Plus Medchem Designer (S+logP), (c) TPSA, (d) HBA, (e) HBD.

set than for the non‐CNS set. Outliers in the CNS set include the very lipophilic drug 3 discussed above, but also the very polar drugs vigabatrin (4) and droxidopa (5). Active transport mechanisms probably play a role in the absorption from the gut and the crossing of the blood–brain barrier for these amino acids. PSA calculated as TPSA of N + O atoms [4] shows a very large variability but no significant trend over time. It is notable that 15% of drugs have TPSA > 140 Å2 and some as high as 200 Å2. This observation prompted us to revisit the foundation on which the commonly used cutoff of 140 Å2 rests, and that

2.2  FDA‐Approved Drugs 2007–2017 F

O O

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Figure 2.2  Structures of selected drugs.

investigation is the subject of Section 2.3. The CNS drugs fall in a much more limited range with only four compounds significantly above 80 Å2: Compound 5 was discussed above, macimorelin (6) is a growth hormone secretagogue receptor agonist that is used to diagnose (not treat) adult growth hormone deficiency, vilazodone (7) is a 5‐HT (serotonin) reuptake inhibitor and 5‐HT1A partial agonist, and gabapentin enacarbil (8) is a prodrug that is hydrolyzed to gabapentin prior to crossing the blood–brain barrier. HBA count, herein defined as the sum of the number of O and N atoms, shows no significant trend over time. Although some of the larger drugs have up to

39

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2  Physicochemical Parameters of Recently Approved Oral Drugs

16 heteroatoms, the median for the period was 7 HBAs for the non‐CNS set and 4 HBAs for the CNS set. Similar to what was observed for TPSA above, the CNS set has less spread in this parameter, and the compounds with the highest number of O + N atoms include 6 discussed above and the orexin receptor antagonist suvorexant (9) used to treat insomnia. A similar pattern is seen for the HBD count, herein defined as the number of H atoms directly bonded to O or N atoms, but it is noteworthy that very few drugs, only 5 out of 175, have more than five donors. Among the CNS drugs, 28 out of 35 compounds have two or fewer donors, and only three CNS drugs have five or more HBDs. These comprise compounds 5 and 6 discussed above and the amphetamine prodrug lisdexamfetamine (13), which is hydrolyzed prior to crossing the blood–brain barrier. The non‐CNS drugs with HBD > 5 are the natural co‐factor sapropterin (10), the renin inhibitor aliskiren (11), and the peripheral mixed opioid receptor ligand eluxadoline (12). Active uptake mechanisms probably operate for 10, while 11 and 12 may be able to adopt conformations with intramolecular hydrogen bonds (IMHBs) that mask some of the polarity. Given the very few exceptions noted above, it appears that the maximum of five HBDs is a hard cutoff while the other three Ro5 parameters are more tolerant of violations. Our findings are consistent with those reported in a recent analysis of a set of 1116 proprietary bRo5 compounds from AbbVie [15]. Although high MW (≤1132) and TPSA (≤229) were tolerated, no compounds with HBD > 6, HBA > 14, or number of rotatable bonds > 19 were orally bioavailable (defined as fraction absorbed ≥5% in the rat). In another recent analysis of bRo5 drugs and clinical candidates, Kihlberg found the limits of “possible to be orally absorbed” space to be MW ≤ 1000, −2 ≤ ClogP ≤ 10, HBD ≤ 6, HBA ≤ 15, PSA ≤ 250 Å2, and number of rotatable bonds ≤20 [16]. To keep a reasonably low log P, TPSA would be expected to increase with increasing MW, and this is indeed observed (see Figure 2.3). The combined criteria of TPSA  550 and the highest MW was 770. Shortly after the Veber rule was published, it was found not to be predictive for other datasets, and its applicability was called into question [17, 18]. In Figure 2.4, we show how the oral drugs approved in 2007–2017 compare with the Veber rule. Clearly, recently approved drugs extend far beyond the limits of polarity and flexibility suggested in the 2002 publication by Veber et al. [3] but their properties are still mostly within the broader “possible to be orally absorbed” space found by Kihlberg [16]. 2.2.1 Conclusions A significant fraction of non‐CNS oral drugs approved in 2007–2017 violate two or more of the Lipinski parameters (22% of drugs) and the TPSA  600 has increased from none in 2007 and 2008 to two to four each year since 2012. The quest for drugging more difficult targets drives medicinal chemists far beyond the Ro5 territory. This is still largely uncharted land, and the Ro5 and TPSA