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`M Editedby
`f Hans Bundgaard
`
`V
`
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 1
`
`

`
`‘ IIIKIIIY
`
`
`
`LIBRARY
`UNIVERSITY OF
`CAUFORNIA
`
`BIOLOGY LIBRARI
`
`Petitioner Amerigen Pharmaceuticals Ltd.
`
`- Exhibit 1012 - Page 2
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 2
`
`

`
`Design of prodrugs
`
`, ,_~,..%_§??
`
`_ __ #17‘
`
`Petitioner Amerigen Pharmaceuticals Ltd.
`
`_ Exhibit 1012 _ Page 3
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 3
`
`

`
`Design of prodrugs
`
`edited by
`
`Hans Bundgaard
`
`
`
`_jQmja-Q‘T
`
`
`
`Elsevier
`
`Amsterdam — New York — Oxford
`
`Petitioner Amerigen Pharmaceuticals Ltd.
`
`Exhibit 1012 - Page 4
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 4
`
`

`
`BIOLOGY
`
`© 1985, Elsevier Science Publishers B.V. (Biomedical Division)
`
`All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or
`transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise
`without the prior written permission of the publisher, Elsevier Science Publishers B.V. (Biomedical Divi-
`sion), P.O. Box 1527, 1000 BM Amsterdam, The Netherlands.
`
`Special regulations for readers in the USA:
`(CCC), Salem,
`This publication has been registered with the Copyright Clearance Center
`'
`Massachusetts.
`.
`Information can be obtained from the CCC about conditions under which the photocopying of parts of
`this publication may be made in the USA. All other copyright questions, including photocopying outside
`of the USA, should be referred to the publisher.
`
`Inc.
`
`ISBN 0-444-80675—X
`
`Published by:
`
`Elsevier Science Publishers B.V. (Biomedical Division)
`P.O. Box 211
`1000 AE Amsterdam
`The Netherlands
`
`Sole distributors for the USA and Canada:
`Elsevier Science Publishing Company, Inc.
`52 Vanderbilt Avenue
`New York, NY 10017
`USA
`
`Library of Congress Cataloging in Publication Data
`
`Main entry under title:
`
`Design of prodrugs.
`
`Includes bibliographies and index.
`1. Prodrugs.
`2. Drugs——Metabolism.
`Pharmaceutical.
`I. Bundgaard, Hans.
`pharmaceutics.
`2. Biotransformation.
`Pharmaceutical.
`QV 38 DHSTJ
`RM3o1.57.Dl+7
`1985
`615'.191
`ISBN o—I+hh—8o675—x (U.s.)
`
`3. Chemistry,
`EDNLM: 1. Bio-
`3. Chemistry,
`
`85-15926
`
`Printed in The Netherlands
`
`: Preface
`
`During the last decade it has become more obvious that the commonly used pro-
`cesses of delivering therapeutic agents to the sites of their action within the body
`are generally inefficient and unreliable. Optimization of drug delivery and, conse-
`quently, improvement in drug efficacy implies an efficient and selective delivery and
`transport of a drug substance to its site of action. Recognition of the importance
`of drug delivery for the therapeutic indices of many types of drugs has been follow-
`ed by a large increase in research activities in this area, and much attention has been
`focussed on approaches which aim at enhancing the efficacy and reducing the toxici-
`ty and unwanted effects of drugs by controlling their absorption, blood levels,
`metabolism, distribution and cellular uptake.
`Prodrug design comprises an area of drug research that is concerned with the op-
`timization of drug delivery. A prodrug is a pharmacologically inactive derivative of
`a parent drug molecule that requires spontaneous or enzymatic transformation
`within the body in order to release the active drug, and that has improved delivery
`properties over the parent drug molecule.
`A molecule with optimal structural configuration and physicochemical properties
`for eliciting the desired therapeutic response at its target site does not necessarily
`possess the best molecular form and properties for its delivery to its point of
`ultimate action. Usually, only a minor fraction of doses administered reaches the
`target area and, since most agents interact with non-target sites as well, an ineffi-
`cient delivery may result in undesirable side effects. This fact of differences in
`transport and in situ effect characteristics for many drug molecules is the basic
`reason why bioreversible chemical derivatization of drugs, i.e., prodrug formation,
`is a means by which a substantial improvement in the overall efficacy of drugs can
`often be achieved.
`
`Prodrug research matured as a branch of pharmaceutical research during the
`1970s. Over the past decade this chemical approach to optimization of drug delivery
`has undergone considerable expansion, largely as a result of an increased awareness
`and understanding of the physicochemical factors that affect the efficacy of drug
`delivery and action. Several drugs are now used clinically in the form of prodrugs,
`and as the prodrug approach is becoming an integral part of the new drug design
`
`_ Exhibit 1012 _ Page 5
`Petitioner Amerigen Pharmaceuticals Ltd.
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 5
`
`

`
`vi
`
`vii
`
`The design and utility of macromolecular prodrugs is a relatively new area which
`certainly is going to be the focus of intense research in the near future. In chapter
`10 the use of albumin as a transport group or carrier for drugs and, in particular,
`enzymes is specifically discussed. The promising properties of such conjugates for
`drug targeting are discussed, as are the many pitfalls and possible disadvantages that
`any given system might have.
`The final chapter (Ch. 11) on prodrugs versus soft drugs has been included in the
`book in order to clarify some common confusion about these two rather different
`terms. Whereas a prodrug is an inactive derivative which is activated predictably in
`vivo to the active drug, a soft drug is an active species like any other drug, but it
`is designed in such a way that it will undergo a predictable transformation or
`metabolism to an inactive metabolite. Thus, the common feature of prodrugs and
`
`soft drugs is only that a transformation in vivo is involved, it being either an activa-
`tion (prodrugs) or an inactivation (soft drugs). By definition, the two terms are just
`opposite to each other. Several examples are given to illustrate the difference be-
`tween prodrugs and soft drugs, but examples of prodrugs of soft drugs are also in-
`cluded.
`'
`
`This book presents the basic principles of prodrug design and illustrates these
`principles with many examples. In addition, it provides a comprehensive review of
`the most recent literature concerning the design and application of prodrugs.
`
`Hopefully, the book will be useful to all those concerned with drug delivery and
`drug design in universities or industry and will initiate new research for increased
`practical utilization of the prodrug concept.
`
`Hans Bundgaard
`
`process one may expect that the new drugs in many cases will appear as prodrugs.
`The purpose of the present book is to provide a comprehensive and basic source
`of information on the recent developments within the prodrug area and on the ra-
`
`tional basis for prodrug design. Admittedly, there are numerous review articles and
`a few texts devoted to one or more topics in the prodrug area, but a current and
`
`comprehensive treatment appears to be lacking.
`The book is divided into eleven chapters, each written by active scientists in the
`field. The first chapter provides a review and classification of bioreversible
`derivatives for various functional groups and chemical entities occurring in drug
`substances. For most examples discussed due attention is given to the potential
`therapeutic benefits achievable by the derivatization, e.g., improved absorption. In
`chapter 2 the design of prodrugs through consideration of enzyme-substrate
`specificities is discussed. Various enzyme classes are considered and their usefulness
`as prodrug reconversion sites is discussed in detail.
`The ideal site and rate for drug release from a prodrug depend upon the specific
`delivery problems which are meant
`to be overcome by the prodrug design.
`Therefore, the pharmacokinetic aspects are of great importance in prodrug design.
`Chapter 3 is devoted to these aspects and it provides surveys of the theory accompa-
`nying each goal achievable with a prodrug, methods for evaluating the success of
`the prodrugs and the practical limitations as evidenced by several examples, their
`successes and failures. Chapter 4 describes the use of the prodrug approach for the
`development of agents with prolonged duration of activity, including a discussion
`of polymeric prodrug sustained release delivery systems.
`Chapter 5 deals with the very important area of providing site-specific delivery
`or targeting of drugs to their site of action by the prodrug approach. Several ex-
`amples of site-specific delivery based on site-specific transport or prodrug cleavage
`are given, and the importance of the physicochemical properties of the parent drug
`to the site-specific delivery of drugs via prodrugs is stressed. The use of the prodrug
`approach to increase the therapeutic index of a drug is considered further in Chapter
`6. An extensive review is given, with most examples being taken from amongst
`steroidal and non—steroidal anti-inflammatory agents, B-stimulants and anticancer
`agents.
`
`The prodrug approach has been used frequently to solve pharmaceutical formula-
`tion problems, such as stability and solubilization. Chapter 7 treats this area of pro-
`drug application in a physicochemical manner, with illustrative examples. In recent
`years the application of prodrugs to enhance the percutaneous absorption of drugs
`has received much interest, and this theme is treated in chapter 8. Besides reviewing
`the previous studies in the field, the chapter provides a rational basis for the design
`of prodrugs aiming at improving the delivery of drugs to the skin.
`Chapter 9 is concerned with anticancer prodrugs. The emphasis is put on pro-
`drugs which improve the pharmacokinetic properties of anticancer agents, and in
`particular prodrugs which are activated selectively in tumour cells to the active drug.
`
`_ Exhibit 1012 _ Page 6
`Petitioner Amerigen Pharmaceuticals Ltd.
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 6
`
`

`
`
`
`
`
`Contents
`
`‘
`
`r‘
`
`Preface
`
`Chapter
`
`1. Design of prodrugs: Bioreversible derivatives for various func-
`tional groups and chemical entities, by H. Bundgaard
`Chapter 2. Design of prodrugs based on enzyme—substrate specificity, by
`P.K. Banerjee and G.L. Amidon
`
`Chapter 3.
`
`Chapter 4.
`
`Pharmacokinetic aspects of prodrug design and evaluation, by
`R.E. Notari
`,
`Sustained drug action accomplished by the prodrug approach,
`by A.A. Sinkula
`
`Chapter 5.
`
`Site—specific drug delivery via prodrugs, by V.J. Stella and KJ.
`
`Himmelstein
`_
`Chapter 6. Decreased toxicity and adverse reactions via prodrugs, by G.
`Jones
`
`Chapter 7.
`
`Prodrugs for improved formulation properties, by B.D. Ander-
`son
`
`Prodrugs and skin absorption, by J. Hadgraft
`Chapter 8.
`Prodrugs in cancer chemotherapy, by T.A. Connors
`Chapter 9.
`Chapter 10. Albumin: A natural carrier for drug and enzyme therapy, by
`M.J. Poznansky and D. Bhardwaj
`Prodrugs versus soft drugs, by N. Bodor
`
`Chapter 11.
`Subject index
`
`v
`
`1
`
`93
`
`135
`
`157
`
`1 77
`
`199
`
`243
`
`271
`291
`
`317
`333
`355
`
`Petitioner Amerigen Pharmaceuticals Ltd. _3EXhibit 1012 _ page 7
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 7
`
`

`
`
`
`
`
`‘
`
`C
`
`g
`
`.
`
`3
`
`1:
`
`,
`
`it
`
`,7
`l
`
`Design of Prodrugs (Bundgaard, H., ed.)
`© 1985 Elsevier Science Publishers B. V. (Biomedical Division)
`
`CHAPTER 1
`
`Design of prodrugs: Bioreversible derivatives
`for various functional groups and chemical
`‘
`_
`entities
`
`HANS BUNDGAARD
`
`Royal Danish School of Pharmacy, Department of Pharmaceutical Chemistry AD,
`2 Universitetsparken, DK~2100 Copenhagen, Denmark.
`
`‘ 1.
`
`Introduction
`
`A basal requisite for the prodrug approach to be useful in solving drug delivery
`problems is the ready availability of chemical derivative types satisfying the prodrug
`requirements, the most prominent of these being reconversion of the prodrug to the
`parent drug in vivo. This prodrug~ drug conversion may take place before absorp-
`tion (e.g., in the gastrointestinal tract), during absorption, after absorption or at the
`specific site of drug action in the body, all dependent upon the specific goal for
`which the prodrug is designed. Ideally, the prodrug should be converted to the drug
`as soon as the goal is achieved. The prodrug per se is an inactive species, and
`therefore, once its job is completed, intact prodrug represents unavailable drug. For
`example, prodrugs designed to overcome solubility problems in formulating in-
`travenous injection. solutions should preferably be converted immediately to drug
`following injection so that the concentration of circulating prodrug would rapidly
`become insignificant in relation to that of the active drug. Conversely, if the obj ec~
`tive of the prodrug is to produce a sustained drug action through rate—limiting pro-
`drug conversion the rate of the conversion should not be too high.
`The necessary conversion or activation of prodrugs to the parent drug molecules
`in the body can take place by a variety of reactions. The most common prodrugs
`are those requiring a hydrolytic cleavage mediated by enzymic catalysis. Active drug
`species containing hydroxyl or carboxyl groups can often be converted to prodrug
`esters from which the active forms are regenerated by esterases within the body,
`e.g., in the blood. In other cases, active drug substances are regenerated from their
`prodrugs by biochemical reductive or oxidative processes. Sulindac, for example, is
`active only when reduced to its thioether form [1,2] and a prodrug of the pyridinium
`
`‘Z
`
`‘r
`
`6
`
`1
`
`2‘
`
`"
`
`Petitioner Amerigen Pharmaceuticals Ltd. -iExhibit 1012 _ page 8
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 8
`
`

`
`2
`
`is converted to the parent drug through an en-
`quaternary compound, 2—PAM,
`zymatic oxidation process in the body [3 — 5]. Besides usage of the various enzyme
`systems of the body to carry out the necessary activation of prodrugs, the buffered
`and relatively constant value of the physiological pH (7.4) may be useful in trigger-
`ing the release of a drug from a prodrug.
`In these cases,
`the prodrugs are
`characterized by a high degree of chemical lability at pH 7.4 while preferably ex-
`hibiting a higher stability at, for example, pH 3 — 4. As will be discussed below, ex-
`
`amples of such prodrugs include N-Mannich bases and various ring—opened
`derivatives of cyclic drugs. A serious drawback of prodrugs requiring chemical
`(non-enzymic) release of the active drug is the inherent lability of the compounds,
`raising some stability-formulation problems, at least in cases of solution prepara-
`tions. As will be shown later, such problems have, in particular cases, been over-
`
`come by using a more sophisticated approach involving pro-prodrugs or cascade
`latentiation, where use is made of an enzymatic release mechanism prior to the spon-
`
`L
`taneous reaction.
`Several types of bioreversible derivatives have been exploited for utilization in
`designing prodrugs. The purpose of the present chapter is to discuss various
`chemical approaches to obtain prodrug forms, with due attention to the potential
`therapeutic benefits achievable by the prodrug approach and with emphasis on
`recently developed types of bioreversible derivatives. In the past, esters mostly have
`been considered as prodrug types, and the best known prodrugs are in fact esters
`of drugs containing hydroxyl or carboxyl groups. Various reviews [6, 7] have dealt
`
`TABLE 1.
`
`Examples of Ester Derivatives Developed as Prodrugs for Drugs Containing a Carboxyl Group
`
`Drug
`
`Ester
`
`Prostaglandins
`‘y-Aminobutyric acid
`Acetylsalicylic acid
`
`L—Dopa
`Niflumic acid
`Non—steroida1 anti-inflammatory drugs
`Amino acids
`Carbenicillin
`Ibuprofen
`lndomethacin
`
`Glutathione
`Nicotinic acid
`
`Phenyl esters
`Aliphatic and steroid esters
`Methylsulphinylmethyl ester
`Triglycerides
`Methyl ester
`B-Morpholinoethyl ester
`Methyl esters
`Glycolic and lactic acid esters
`Aliphatic and aromatic esters
`Guiacol ester
`Triglycerides
`Phenyl esters
`Glycolic acid ester
`Ethyl ester
`Tetrapentaerythritol ester
`
`Reference
`
`39, 40
`41, 42, 43
`44,45
`46, 47
`48
`49
`50
`51
`52
`53
`54, 55
`56
`57
`57a
`58, 58a
`
`
`
`with esters as prodrug types, and therefore this important class will only be briefly
`treated herein.
`
`Some other reviews have, more or less specifically, dealt with various prodrug
`
`types [8—13] and/or paid much attention to enzyme systems available in the
`organism and their utilization for performing the necessary conversion of prodrugs
`[14—17]. Furthermore, much information on the subject can be gathered from
`reviews dealing with several other aspects of prodrugs [l8—24]; see also other
`chapters in this book.
`
`2. Esters as prodrugs for compounds containing carboxyl and hydroxyl groups
`
`The popularity of using esters as a prodrug type for drugs containing carboxyl or
`hydroxyl functions (or thiol groups) stems primarily from the fact that the organism
`
`TABLE 2
`
`Examples of Ester Derivatives Developed as Prodrugs for Drugs Containing a Hydroxyl Group
`
`Drug
`
`Salicylic acid
`Paracetamol
`
`Trichloroethanol
`
`Cymarol
`Vidarabine
`
`Thymidine
`Oxazepam, lorazepam
`
`Metronidazole
`
`,
`
`Chloramphenicol
`Various steroids
`phenols
`Lincomycin
`Epinephrine
`Etilefrine
`2-Amino—6,7-dihydroxytetrahydronaph—
`thalene (6,7-ADTN)
`Terbutaline
`Isoproterenol
`Cytarabine
`Digitoxigenin
`Acyclovir
`
`Ester
`
`Reference
`
`Carboxylate and carbonate esters
`Carbonate esters
`Phosphate ester
`Carbonate esters
`Phosphate ester
`Diacetylester
`Mono- and diesters
`Phosphate ester
`Pivaloate
`Aliphatic and aromatic esters
`Amino acid esters
`Aromatic esters
`Phosphate ester
`Amino acid esters
`
`,
`
`Palmitate and hemisuccinate
`Various esters
`Amino acid esters
`Dialkylcarbonate esters
`Dipivaloate
`Aliphatic and aromatic esters
`
`Various diesters
`Mono- and diesters
`Ditoluyl and dipivaloyl esters
`Various mono- and diesters
`Amino acid esters
`Amino acid and hemisuccinate esters
`
`59, 60
`61, 62
`63
`64, 65
`66‘
`67
`68, 69
`70
`71
`72-74
`75
`76 ~ 78
`79
`80, 81
`
`82
`19
`83
`84
`85
`86
`
`87
`88
`89
`90
`91
`92
`
`Petitioner Amerigen Pharmaceuticals Ltd.
`— Exhibit 1012 — Page 9
`
`
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 9
`
`

`
`
`
`S.
`
`©—§H-CO-NH~U f
`
`NH?
`
`’
`0/
`
`N
`
`COOR
`
`
`
`is rich in enzymes capable of hydrolyzing esters. The distribution of esterases is ubi-
`
`liver and other organs or
`quitous, and several types can be found in the blood,
`tissues. In addition, by appropriate esterification of molecules containing a hydrox-
`yl or carboxyl group it is feasible to obtain derivatives with almost any desirable ,
`hydrophilicity or lipophilicity as well as in vivo lability, the latter being dictated by
`electronic and steric factors. Accordingly, a great number of alcoholic or carboxylic
`
`acid drugs have been modified for a multitude of reasons using the ester prodrug
`approach. Several examples can be found in various reviews [6, 7, 14, 18 —20] and
`in Tables 1 and 2.
`
`Sometimes, simple aliphatic or aromatic esters may not be sufficiently labile in
`vivo to ensure a sufficiently high rate and extent of prodrug conversion. This is the
`
`case with penicillin esters. Although various simple alkyl and aryl esters of the
`thiazolidine carboxyl group are hydrolyzed rapidly to the free penicillin acid in
`animals, such as rodents,
`they proved to be far too stable in man to have any
`therapeutic potential [25]. This illustrates also — as do many other examples — the
`occurrence of marked species differences in the in vivo hydrolysis of ester prodrugs.
`A solution to the problem was found in 1965 by Jansen and Russell [26], who show-
`ed that a special double ester type (acyloxymethyl ester) of benzylpenicillin was
`hydrolyzed rapidly in the blood and tissues of several species, including man. The
`first step in the hydrolysis of such an ester is enzymatic cleavage of the terminal ester
`bond with formation of a highly unstable hydroxymethyl ester which rapidly
`dissociates to the parent penicillin and formaldehyde (Scheme 1). A reason for the
`different enzymatic stabilities of the acyloxymethyl ester and simple alkyl esters of
`penicillins is certainly that the penicillin carboxyl group is highly sterically hindered.
`The terminal ester in the acyloxymethyl derivative is less hindered, and thus should
`be more accessible to enzymatic attack.
`
`enzymatic
`Drug—COO-CH2-OCOR ———-——> Drug—COO~CH2OH + R—COOH
`
`fast
`
`Drug-COOH + CHZO
`
`Scheme 1
`
`The principle has been used successfully to improve the oral bioavailability of am-
`picillin (1), and no fewer than three ampicillin prodrug forms are now on the
`market, namely, the pivaloyloxymethyl ester (2) (pivampicillin) [27], the phthalidyl
`ester (3) (talampicillin) [28, 29] and the ethoxycarbonyloxyethyl ester (4) (bacampi-
`cillin) [30], the latter containing a terminal carbonate ester moiety. The properties
`of these prodrugs as well as of other similar acyloxyalkyl esters of B—lactam an-
`
`i R=H
`_2_ R: cH,-ooc—c(cH3)3
`
`3 R=$H-O'E’o'C2H5
`CH3
`-0
`/o\
`g R: CH
`c=o
`
`tibiotics, such as mecillinam and cephalosporins, have been reviewed extensively
`
`.
`‘
`[24].
`In more recent years the applicability of this double ester concept in prodrug
`design has been expanded further. Thus, similar esters have been prepared from in-
`domethacin and other non—steroidal anti—inflammatory agents [31] as well as from
`cromoglycic acid [32], and found to be useful as prodrugs for enhancement of the
`dermal delivery of these acidic drugs. Other carboxylic acid agents where acyloxy-
`alkyl esters have been developed as prodrugs include isoguvacine [33], methyldopa
`[34 — 36] and tyrosine [37]. Whereas methyldopa (5) is variably and incompletely ab-
`sorbed its pivaloyloxyethyl ester (6) is almost completely and more uniformly ab-
`sorbed in man following oral administration and is hydrolyzed rapidly on the first
`pass to the parent drug [35, 36]. A different ester type of methyldopa, a
`(5-methyl—2-oxo—l,3-dioxol—4—yl)methy1 derivative (7), was recently. reported to be
`another potentially useful prodrug for improving the oral bioavailability [38]. A
`similar ester type of ampicillin has been described recently and shown to be an orally
`well absorbed prodrug [38a].
`
`CIH3
`H0j:\)rCH2-E-COOR
`H0
`NH;
`
`§ R=-H
`g R=—eH—ooc—cicH3i3
`CH3 0/‘.3.\0
`
`1
`
`= -CH2 ' CH3
`
`The applicability of acyloxyalkyl esters as biologically reversible transport forms
`has been extended to include phenolic drugs, the derivatives being acyloxyalkyl
`ethers (8). Bodor and co—workers [93, 94] have recently prepared such acyloxyalkyl
`ethers of various phenols‘ (e.g., B—estradiol and phenylephrine),
`thiophenol and
`catechols (e.g., dopamine and epinephrine). The derivatives are hydrolyzed by a. se-
`quential reaction involving the formation of an unstable hemiacetal intermediate
`(Scheme 2) and they are as susceptible as normal phenol esters to undergo enzymatic
`hydrolysis by, e.g., human plasma enzymes. However, the acyloxyalkyl ethers ap-
`
`
`
`
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1012 — Page 10
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 10
`
`

`
` 1
`
`bis-N,N-dimethylcarbamate of terbutaline (11) was also examined in the work by
`Olsson and Svensson [96] referred to above. It showed a half—life of hydrolysis in
`human plasma of about 10 hours, and this relatively high stability was partly due
`to the fact that the compound inhibited its own hydrolysis by reversible binding to
`plasma esterases. As a result of the improved hydrolytic stability, the prodrug sur-
`vived the first-pass hydrolysis in the dog to a substantial degree and produced sus-
`tained blood levels of the parent drug in the dog following a single oral dose.
`The enzymatic hydrolytic behaviour of carbamate esters has been examined by
`
`Digenis and Swintosky [6]. N—Unsubstituted or -monosubstituted carbamates deriv-
`ed from phenols showed high lability and strong enzymatic catalysis whereas most
`N-disubstituted carbamates proved highly stable, as did carbamates of aliphatic
`
`hydroxy compounds. The kinetics and mechanism of the non—enzymatic hydrolysis
`of carbamates have been studied thoroughly [97 —-102].
`
`lCH3l2N"’@'0
`
`(CH3)2N-‘C-0
`I
`0
`
` H0H~Cw-NH-C(Cw)3
`
`.11
`
`0
`
`@0CHR’-0-E-R
`
`Q
`
`(C:[6‘-cHR’—"o‘ +RcooH
`
`@011 + R’cH =0
`
`Scheme 2
`
`pear to be more stable against chemical (hydroxide ion-catalyzed) hydrolysis than
`phenolate esters, and this may make them more favourable in prodrug design [93].
`According to their cleavage mechanism, acyloxyalkyl esters and ethers can be con-
`sidered as double prodrugs (pro-prodrugs). An interesting variant of the double
`ester prodrug concept is provided in the work by Olsson and co-workers [95, 96]
`on terbutaline (9).
`In order to achieve increased absorption, reduced first—pass
`metabolism and prolonged duration of action, a p—pivaloyloxybenzoate double ester
`prodrug (10) was made. Since the pivaloyl ester group is the most susceptible to
`undergo enzymatic hydrolysis, it was expected that the prodrug would undergo first-
`pass hydrolysis preferentially at the p—pivaloyloxy bond, followed by conjugation
`reactions with sulphuric and glucuronic acid at the resulting p—hydroxybenzoyl
`moiety (Scheme 3). In this way the active resorcinol moiety in terbutaline would be
`protected during first—pass and free terbutaline may be generated from hydrolysis
`of the conjugated or free p—hydroxybenzoate during and after the distribution
`phase. Experimental support for the cascade ester to function in this way was ob-
`tained and prolonged terbutaline plasma profiles were observed in dogs with this
`prodrug [96].
`
`Carbamate esters may be promising prodrug candidates for phenolic drugs. The
`
`9
`o
`icH3i3c—t—o©3—o
`'
`@cHoH—cH2—NH—cicH3i3
`
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`0
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`
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`
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`
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`
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`
`HOH-CH2-NH-C(Ci-Q3
`
`9
`
`Whereas carbamates of alcohols in general appear to be of no value in prodrug
`design due to their high stability, certain activated carbamates may be useful.
`Imidazole-l-carboxylic acid esters belong to this category and such derivatives of
`hydrocortisone (l2) and testosterone (13) have been shown recently to undergo a
`relatively facile hydrolysis in aqueous buffer solutions [103]. At pH 7.4 and 37°C
`the half-life of hydrolysis of the hydrocortisone derivative was found to be 8
`minutes and that for the testosterone derivative 65 hours, the different reactivity be-
`ing ascribed to the different steric hindrance in the alcohol portions of the steroids.
`No enzymatic catalysis by human plasma was observed. Due to protonation of the
`imidazole group (pKa z 3.5) the derivatives showed increased solubility in acidic
`aqueous solution relative to the parent steroids [103].
`
`CH N/:N
`I
`2
`\=l
`c=o
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`
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`
`Ester formation has long been recognized as an effective means of increasing the
`aqueous solubility of drugs containing a hydroxyl group, with the aim of developing
`prodrug preparations suitable for parenteral administraton. Two physicochemical
`
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1012 — Page 11
`
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 11
`
`

`
`
`
`8 s
`
`vivo half-life in dogs (5 minutes) to that observed in vitro in dog plasma (25
`minutes). A disadvantage of this prodrug is that it is not sufficiently stable for for-
`mulation as a ready-to—use solution [81] and must be used as a formulation to be
`reconstituted as a solution prior to use. Recently, some kinds of water-soluble
`amino acid 21-esters of corticosteroids possessing both a high in vitro stability and
`a high susceptibility of undergoing enzymatic hydrolysis have been developed by
`Anderson et al. [119— 121].
`
`TABLE 3
`Half-lives for the Hydrolysis of Various Amino Acid Esters of Metronidazole in 80% Human Plasma
`(pH 7.4) and 0.05 M Phosphate Buffer (pH 7.40) at 37°C 3
`
`Ester
`
`1% in human plasma
`(minutes)
`
`t,/2 in buffer
`(minutes)
`
`N,N—Dimethylglycinate
`Glycinate
`N-Propylglycinate
`3-Aminopropionate
`3-Dimethylaminopropionate
`3—Dimethylaminobutyrate
`4-Morpholinoacetate
`4-Methyl-1-piperazinoacetate
`
`a. From Bundgaard et al. [80].
`
`12
`41
`8
`207
`46
`334
`30
`523
`
`250
`115
`90
`315
`52
`S80
`1880
`1720
`
` z 5 min) and completely to metronidazole [81]. It is of interest to compare the in
`
`trategies can be employed to increase aqueous solubility: (i) introduction of an
`ionic or ionizable group by the pro—moiety and (ii) derivatization in such a manner
`that the prodrug shows a decreased melting point [104].
`The most commonly used esters for increasing aqueous solubility of alcoholic
`drugs are hemisuccinates, phosphates, dialkylaminoacetates and amino acid esters.
`However, their use is not without problems, considering the ideal properties of such
`prodrugs: they should possess adequate aqueous solubility, sufficient aqueous solu-
`tion stability to allow long—term storage of its solution (i.e., 2 years at room
`temperature) and yet they should be converted rapidly in vivo to the active parent
`drug. For example, succinate esters are not good substrates for hydrolytic enzymes
`[15] and often show relatively slow and incomplete cleavage in vivo, as has been
`described for such esters of various corticosteroids [105, 106] and chloramphenicol
`[82, 107 — 109]. Besides, their solution stability is limited due to intramolecular reac-
`tions (e.g., catalysis of ester hydrolysis or 0-acyl migration in corticosteroids) of the
`terminal succinate carboxyl group [110, 111]. Phosphate esters as sodium salts are
`freely water-soluble and are so stable in vitro that solutions with practical shelf-lives
`often can be formulated [112 — 115]. Thus, a shelf-life of more than 10 years for an
`aqueous solution of vidarabine-5 ’—phosphate at pH 6.8 and 25°C has been predicted
`[113]. They are also rapidly hydrolyzed enzymatically in vivo (e.g., Refs. 116 — 118,
`122), although exceptions exist. Thus, the phosphate ester (15) of metronidazole (14)
`shows a rather slow rate of conversion in human serum, the hydrolysis exhibiting
`apparent zero—order kinetics
`[79]. For
`the third type of water—soluble ester
`derivatives, i.e., esters with an ionizable amino function in the acid portion, only
`sparse information is available on their enzymatic hydrolysis. Bundgaard et al. [80,
`81] have prepared eight amino acid esters of metronidazole (14) and evaluated their
`potentiality as water-soluble parenteral delivery forms of the parent drug whose
`solubility in water is limited (z 1% w/v). Hydrochloride salts of all the esters ex-
`hibited a water solubility greater than 20% w/v but their susceptibility to undergo
`enzymatic hydrolysis varied widely, as seen from the data in Table 3. Due to its
`facile cleavage in plasma, excellent solubility properties (> 50% w/v in water) and
`ease of synthesis and purification, the hydrochloride salt of metronidazole N,N-
`dimethylglycinate (16) appeared to be the most promising prodrug candidate [80].
`Following intravenous administration to dogs the ester was converted rapidly (t./Z
`
`
`
`Sulphate esters of alcohols and phenols have long been considered as prodrug
`forms useful for obtaining injectable preparations [14]. However, recent studies in-
`dicate that such esters may be very resistant to undergoing hydrolysis in vivo and,
`accordingly, would not be suitable prodrugs. Thus, Miyabo et al. [122] found that
`dexamethasone—21—sulphate produced virtually no free dexamethasone in plasma
`and urine following intravenous injection in man, but was excreted largely unchang-
`ed in urine. Similarly, Williams et al. [123] have found that the sulphate esters of
`paracetamol and 3—hydroxymethyl-phenytoin do not generate the parent drugs when
`administered parenterally to mice or rats.
`A high crystal lattice energy of solid compounds, as manifested in a high melting
`point, results in poor solubility (in all solvents). Therefore, an approach to reduce
`this energy may result in improved aqueous solubility. An example of the usefulness
`of this approach in prodrug design concerns vidarabine (17). It has a low water
`solubility (0.5 mg ml‘ 1), primarily due to the occurrence of intermolecular
`hydrogen bonding in the crystalline state, as reflected in its melting point of 260°C.
`By esterification of the 5’-hydroxyl group this bonding is reduced, and, further, by
`choosing an only slightly lipophilic acyl group such as formyl a vidarabine ester with
`
`Petitioner Mylan Pharmaceuticals Inc. — Exhibit 1012 — Page 12
`
`
`
`H c
`
`1 1
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`N
`CHZCHZOH
`5
`
`N
`
`H3C
`
`1 we
`2
`N
`CHZCHZO-R
`-;R=— -(om;
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1012 - Page 12
`
`

`
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`greatly increased aqueous solubility has been obtained [68]. The 5’—formate ester
`(18) is hydrolyzed rapidly in human b