HEAL rn SCIENCES
`LTBRARIES
`
`Page 1 of 9
`
`AstraZeneca Exhibit 2098
`Mylan v. AstraZeneca
`IPR2015-01340
`
`

`
`journal of
`Medicinal
`Chemistry
`
`Journal o{ Medicinal Chemistry (ISSN 0022-
`2623) is published biweekly by the American
`Chemical Society at 1155 16th St., N.W.,
`Washington, DC 20036. Periodicals postage
`paid at Washington, DC, and additional
`mailing offices. POSTMASTER: Send
`address changes to Joumal of Medicinal
`Chemistry, Member & Subscriber Services,
`P.O. Bo:x 3337, Columbus, OH 43210.
`
`Ame rican Che mical Society
`1155 16th St., N.W.
`Washington, DC 20036
`(202) 872-4614
`TDD (202) 872-6076
`Fax (202) 776-8264
`
`Journal Publications
`American Chemical Society
`2540 Olentangy River Road
`P.O. Box 3330, Columbus, OH 43210
`(614) 447-3665
`Fax (614) 447-3745
`E-mail acsproof@acs.org
`
`Member & Subscriber Services
`P.O. Box 3337
`Columbus, OH 43210
`
`Members & individuals contact:
`(614) 447-3776; (800) 333-9511
`Fax (614) 447-3671
`E-mail service@acs.org
`
`Agencies & institutions contact:
`(614) 447-3674; (888) 338-0012
`Fax (614) 447-3671
`E-mailliblink@acs.org
`
`Adve rtising Office
`Centcom, Ltd.
`676 East Swedesford Road
`Suite 202, Wayne, PA 19087-1612
`(610) 964-8061
`
`Publication s Division
`Robert D. Bovenschulte, President
`
`Journal Publishing Group
`Mary E. Scanlan, Vice President
`
`Journal Publications
`Anne C. O'Melia, Assistant Director;
`Diane E. Needham, Journals Editing
`Manager; Loretta M. Yam and Lee S.
`Thurston, Associate Editors; Tracy A.
`Pritchard, Production Assistant
`
`Composition Services
`Teresa K. Lewandowski,
`Assistant Director
`
`Sales & Marke ting/Publications
`Justin R. Spence, Vice President
`Dean J. Smith, Assistant Director, Sales
`Matthew J. Price, Assistant Director,
`Product Marketing
`
`Copyright Permission: See copyright status form for certain rights (http://pubs.acs.orgl.
`Reprographic copying beyond that permitted by Section 107 or 108 of the U.S. Copyright Act is
`allowed, provided that the current per article fee is paid to the Copyright Clearance Center.
`Tel: (978) 750-8400. Republication or reproduction for sale of articles or abstracts in this jour(cid:173)
`nal is permitted only by written permission from the Copyright Office, ACS, Washington, DC.
`Tel: (202) 872-4368. Fax: (202) 776-8112. E-mail: copyright@acs.org.
`
`EDITORIAL INFoRMATION
`Instructions for Authors and Copyright Status Form: See the first issue of each volume
`or visit t he Publications Division Web site (http://pubs.acs.org) for instructions on preparing
`and submitting manuscripts and copyright forms to the Editor.
`
`Manuscript Submission: Send manuscripts to the Editor, Philip S. Portoghese, Department
`of Medicinal Chemistry, College of Pharmacy, WDH 8-114, University of Minnesota, 308
`Harvard St. SE, Minneapolis, MN 55455. European contributors may send manuscripts to Povl
`Krogsgaard-Larsen, Department of Medicinal Chemistry, The Royal Danish School of Phar·
`macy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark. Letters may be submitted over
`the Web at http://pubs.acs.org/JMC.
`Accepted Papers and Proofs: Direct correspondence to Journal Publications, Columbus, OH.
`Tel: (614) 447-3665. Fax: (614) 447-3745. E-mail: acsproof@acs.org.
`Journal Policies: The American Chemical Society and its Editors assume no responsibility
`for the statements and opinions advanced by contributors. Registered names and trademarks,
`etc., used in this publication, even without specific indication thereof, are not to be considered
`unprotected by law.
`Document Number: At the end of each document is a 9-character code that serves as a link
`between the printed and electronic products and facilitates retrieval of the document in elec·
`tronic fonn.
`Digital Object Identifier (DOl): The DOl identification system for digital media has been
`designed to provide persistent and reliable identification of digital objects. Information on the
`DOl and its governing body, the International DOl Foundation, can be found at hUp:l/
`www.doi.org. In the Web editions of ACS journals, the DOl appears at the top of the HnfL
`version of an article and at the bottom of the ftrst page in its PDF version; in the print editions,
`the DOl appears in the same location as in the PDF version.
`
`2004 SUBSCRIPTION AND ORDERING INFORMATION
`
`Printed Edition
`
`Members
`Student Members
`Institutional
`
`North
`America
`s 150
`s 113
`$1697
`
`Outside
`North America*
`
`$309
`s 272
`$1856
`
`Web Edition•
`
`$ 75
`$ 75
`s 384
`s 225
`Print/Web Combination•
`Members
`*Air service included. • For Institutional rates, call M&SS Agency & Institutional Customer
`Service.
`
`Members
`
`Web Edition : This journal is available to subscribers via the Internet. Members may contact
`Customer Service. Tel: (614) 447-3776 or (800) 333-9511. E-mail: service@acs.org. Institutional
`subscribers may contact Agency & lnstitutional Customer Service. Tel: (614) 447-3674 or (888l
`338-0012. Fa:": (614) 447-3671. E-mail: liblink@acs.org. For additional details, visit the Publi(cid:173)
`cations -Division Web site (http://pubs.acs.org).
`New and Renewal Subscript ions: Send with payment to American Chemical Society,
`P.O. Box 182426, Columbus, OH 43218-2426. Institutional subscribers in Japan must enter
`subscription orders with Maruzen Company Ltd., 3-10 Nihonbashi 2-chome, Chuo-ku, Tokyo
`103, Japan. 'fel: (03) 272-7211.
`Subscription Donations: Members may donate/share their personal subscriptions tolwitb
`libraries but only after 5 years from publication.
`Change of Address: Notify Member & Subscriber Services, ACS, Columbus, OH. Tel: (6141
`447-3776 or (800) 333-9511. Fax: (614) 447-3671. E-mail: address@acs.org. Include both old and
`new addresses and a mailing label from a recent issue.
`Microfilm, Microfiche, Ba ck Issue, and Printed Edition Single Issu e Orders: Send
`requests to Publications Support Services, ACS, Washington, DC. Tel: (202) 872-4376. Fu:
`(202) 872-6325. E-mail: pss@acs.org. Printed edition not available prior to 2000.
`Bulk Reprint Orders: For quotes and information, contact Cadmus Reprints. Tel: (888) 257-
`2134 or (410) 8 19-3995. Fax: (410) 820-9765.
`Claims for Issues Not Received : Claims will be honored only if submitted within 90 days af
`the issue date (subscribers in North America) or within 180 days of the issue date (all other
`subscribers). Members and individuals may contact Customer Service. Tel: (614) 447-3776 or
`(800) 333-9511. E-mail: service@acs.org. Institutional subscribers may contact Agency & Insti(cid:173)
`tutional Customer Service. Tel: (614) 447-3674 or (888) 338-0012. Fax: (614) 447-3671. E-mail;
`liblink@acs.org.
`Supporting Information (SD: SI from 1995 to the present is available free of charge via the
`Internet http://pubs.acs.org/jmc from the journal's home page. For information on electronic
`access, send e-mail to journalhelp@acs.org. SI prior to 1995 is available, for a fee, from Publi(cid:173)
`cations Support Services. Tel: (202) 872-4376. Fax: (202) 872-6325. E-mail: pss@acs.org.
`
`Canadian GS'r Reg. No. 127571347
`Printed in the USA
`
`©Copyright 2004 American Chemical Society
`
`Page 2 of 9
`
`

`
`J. Med. Chem. 2004, 47, 4135- 4141
`
`4135
`
`Dipeptidyl P eptidase IV Inhibitors for the Treatment of Diabet e s
`
`Ann E. Webert
`
`Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
`
`- r:~'"'
`©;!mpO"g ~
`1 i'
`~
`!~ !
`
`fl i Glucose-dependent
`insulin biosynthesis
`and secretion
`~
`Active GLP-1
`.1. Glucagon
`~ Improved ~-cell function
`
`/
`
`Other potential
`substrates
`(e.g. GIP, GLP-2,
`PACAP)
`
`Received December 24, 2003
`
`Introduction
`Ingestion of food results in the release of peptide
`hormones in the gut, termed incretins, that regulate
`insulin in a glucose-dependent manner. 1 When blood
`glucose levels are high, the incretin hormone glucagon(cid:173)
`like peptide 1 (GLP-1[7- 36) amide or GLP-1) stimulates
`insulin secretion and biosynthesis and inhibits glucagon
`release. In addition, it serves as an "ileal brake", slowing
`gastric emptying a nd reducing appetite. GLP-1 also
`appears to regulate the growth and differentiation of
`the insulin-producing f3 cells in pancreatic islets in
`rodents. Thus, GLP-1 therapy for the treatment of type
`2 diabetes is an area of active research.2
`GLP-1 is rapidly degraded in vivo through the action
`of dipeptidyl peptidase IV (DPP-IV), which cleaves the
`N-terminal two amino acids to give the inactive GLP-
`1[9- 36) amide (Figure 1).3 Thus, GLP-1 must be
`administered via chronic infusion in order to achieve
`sustained elevated plasma levels. DPP-IV resistant
`GLP-1 analogues represent one means to circumvent
`this issue, but like GLP-1, these are peptides that must
`be administered parenterally. Orally bioavailable, small(cid:173)
`molecule agonists of the GLP-1 receptor have yet to be
`reported, though several patents claim low molecular
`weight GLP-1 agonists and potentiators.4 Inhibition of
`DPP-IV, which leads to an increase in circulating levels
`of endogenous GLP-1, is an alternative approach that
`appears highly amenable to drug discovery.s
`
`DPP -IV Substrates
`A cell sw-face serine protease, DPP-IV6 is ubiquitously
`expressed, with the highest levels found in the kidney
`and the lower levels in liver, pancreas, placenta, thy(cid:173)
`mus, spleen, epithelial cells, vascular endothelium, and
`lymphoid and myeloid cells. A soluble form is shed into
`the circulation. Substrate specificity studies point to
`DPP-IV's strong preference for cleavage of peptides
`containing a proline residue in P 1,7 though interestingly
`GLP-1 and related glucagon family members contain
`alanine at this position. A wide range of substituents
`are allowed at P2, and there is also little preference for
`specific residues on the prime side except that proline
`and hydroxyproline are disfavored at P1' .
`While a large number of pep tides are cleaved by DPP(cid:173)
`IV in vitro,8 very few have been shown to be endogenous
`substrates based on the following stringent criteria: (i)
`cleavage occurs in vitro at the penultimate residue; (ii)
`cleavage products are observed in vivo but are absent
`in the presence of a selective inhibitor or in DPP-rv-t(cid:173)
`mice; (iii) cleavage is the major route of clearance of the
`
`1 Phone: 732-594-5796. Fax: 732-594-5850. E-mail: ann webe.<gl
`mer ck.com.
`-
`
`Inactive GLP-1
`
`.1. Food intake
`Figure 1. DPP-IV regulates glucose homeostasis via inactiva(cid:173)
`tion of GLP-1 and other incretin hormones.
`
`peptide. GLP-1 meets these criteria, as does the incretin
`hormone glucose-dependent insulinotropic polypeptide
`(GIP, also known as gastric inhibitory peptide).9 GIP,
`which is secreted in the proximal gut in response to food
`stimulates insulin secretion in a glucose-dependent
`manner and is believed to account for approximately
`half the incretin response in healthy humans.9c Unlike
`GLP-1, the insulinotropic effects of GIP are reduced in
`type 2 diabetics, and this may contribute to the reduced
`incretin effects in these patients.
`DPP-IV inhibitors evoke decreases in glucose excur(cid:173)
`sion following an oral glucose challenge. Recent studies
`demonstrate efficacy of inhibitors in mice lacking one
`or both of the receptors for GLP-1 and GIP.1° Clearly
`there are other substrates in addition to these incretins
`that contribute to the biological activity of DPP-IV
`inhibitors. One potential candidate is pituitary adeny(cid:173)
`late cyclase-activating polypeptide (PACAP), a pancre(cid:173)
`atic neuropeptide that regulates lipid and carbohydrate
`metabolism. Intravenous administration of this peptide
`to mice results in rapid cleavage at the penultimate
`residue. The DPP-IV cleavage product is absent in DPP(cid:173)
`rv-/- mice, suggesting a potential role for the enzyme
`in in vivo processing ofPACAP.ll
`
`DPP-IV Inhibitor SAR
`In light of DPP-IV's substrate specificity, it is not
`surprising that o.-aminoacylpyrrolidine derivatives have
`been widely explored as DPP-IV inhibitors. The most
`potent of these contain an electrophile at the 2-position
`of the pyrrolidine ring (Figure 2), which forms an adduct
`with the active site serine. Irreversible inhibitors con-
`
`10.1021/jm030628v CCC: $27.50 © 2004 American Chemical Society
`Published on Web 07/20/2004
`
`Page 3 of 9
`
`

`
`4136 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 17
`
`R orR' = H,
`bulky, lipophilic preferred,
`.
`r = B(OH)2. CN > H
`hydrophilic allowed
`0
`,· R~N-"
`X = S > CH > SO 0
`.
`II
`sma nng:
`NH (I 1 ' /
`2
`•·
`allowed\
`'1;(
`'· R'
`.
`-F allowed
`n = 1 > 2. 3
`requlfed
`Figure 2. SAR of reversible a-aminoacylproline-derived DPP(cid:173)
`IV inhibitors.
`
`taining diphenylphosphonate esters12 and 0-acylhy(cid:173)
`droxamic acids 13 have been identified. A boronic acid
`moiety provides highly potent inhibitors that are slowly
`reversible, 14 but the most extensively studied agents are
`those containing a nitrile at this position. Replacement
`of the pyrrolidine with thiazolidine gives derivatives
`with increased potency; however, larger rings (e.g.,
`piperidine, homopiperidine) or those containing other
`heteroatoms (e.g., oxazolidine) are less potent.15 With
`the exception of fluorine, substituents on the pyrrolidine
`ring are not well-tolerated. In the thiazolidine series,
`oxidation of the sulfur to sulfoxide or sulfone leads to a
`decrease in activity.
`A basic amine at Pz is strictly required for inhibition.
`Consistent with the substrate specificity studies, a wide
`range of side chains at P2 are tolerated, including bulky,
`lipophilic groups, and those containing polar functional(cid:173)
`ity. Branching at this position is preferred, and of the
`simple amino acid substituents, isoleucyl, and cyclo(cid:173)
`pentylglycyl, and cyclohexylglycyl provide the most
`potent inhibitors.16
`Peptides containing sarcosine at P2 are a lso sub(cid:173)
`strates for DPP-IV, and this knowledge led to the
`exploration ofN-substituted glycine derivatives as DPP(cid:173)
`IV inhibitors. 17 Like their a.-substituted amino acid
`counterparts, these inhibitors tolerate both straight(cid:173)
`chain and cyclic substituents at this position, with polar
`and lipophilic side chains including the very bulky
`adamantyl group. Two derivatives from this class have
`been studied in the clinic: DPP728 (1a) and LAF237
`(2, ICso = 22 and 3.5 nM, respectively; Chart 1).
`Substituents on nitrogen appear to fill the same Sz
`site as those on the a.-carbon. Indeed N ,a.-bis-substituted
`analogues show greatly decreased potency.17c Small
`rings bridging carbon and nitrogen are tolerated, in(cid:173)
`cluding proline at Pz. A series oftetrahydroisoquinoline-
`3-carbonylcyanopyrrolidine derivatives are also reported
`to have good DPP-IV inhibitory activity (e.g., 3 ; ICso =
`4 nM).18
`Because of the presence of the required basic amine,
`electrophile-containing inhibitors generally exhibit a
`high degree of solution instability. This may contribute
`in part to the relatively short half-life of these deriva(cid:173)
`tives in vivo. Product-like inhibitors, those lacking an
`electrophile, have also been developed. While more
`stable, they typically are much less potent than the
`corresponding nitriles. One of these, threo-isoleucylthi(cid:173)
`azolidide or P32/98 (4 , K; = 126 nM),19 was advanced
`to clinical trials. Cyclohexylglycylpyrrolidide (5, K; = 64
`nM) is among the most potent DPP-IV inhibitors lacking
`an electrophilic serine trap.16 Recently, derivatives with
`substitution at the 4-position ofthe cyclohexyl ring were
`reported to have increased potency. The 4-(2,2,2-tri(cid:173)
`fluoroethyl)sulfonamidophenylsulfonylamino derivative
`6 has an IC50 of2.6 nM and is > 1000-fold selective over
`
`the related prolyl peptidase QPP (quiescent cell proline
`dipeptidase).20
`The amide bond is not strictly required for potency,
`and inhibitors such as 7 containing a fluoroolefin amide
`bond replacement have been reported.21 A number of
`heterocyclic structures devoid of peptide-like character
`have also been shown to inhibit DPP-IV. These include
`xanthine derivatives such as 8 (ICso = 5 nM)22 and
`isoquinoline23 and isoquinolone24 derivatives 9 and 10
`(IC5o = 320 and 250 nM, respectively).
`The X-ray crystal structure of DPP-IV bound to an
`inhibitor has recently been solved by severallaborato(cid:173)
`ries.25 The enzyme is a homodimer. Each subunit
`comprises an a/fJ-hydrolase domain and an eight-bladed
`fJ-propeller domain. A large cavity, roughly 30- 45 A
`wide, is located between the two domains, and inhibitors
`bind to a small pocket in this cavity, with key residues
`from both domains making up the binding site. The
`basic amine forms a salt bridge with Glu205 and, in
`some cases, Glu206 from the fJ-propeller domain. Argl25,
`also from that domain, stabilizes the amide carbonyl
`moiety. The proline binding pocket is formed by a group
`of hydrophobic, primarily aromatic residues from the
`a/fJ-hydrolase domain, leaving little room to accom(cid:173)
`modate larger substituents at that position. The active
`site serine, Ser630, forms an imidate with the nitrile
`as predicted. The S 2 site is bounded by Ser209, Phe357,
`and Arg358. This pocket readily accommodates the
`5-iodopyrid-2-ylaminoethyl side chain of glycine deriva(cid:173)
`tive 1b and the 4-iodobenzyl side chain of phenylalanine
`derivative 11. Thus, the crystal structures provide ready
`explanations for the structural requirements of the basic
`amine and pyrrolidine residues, the increased potency
`of the nitrile-containing analogues, and the less strin(cid:173)
`gent requirements for moieties at P2. It remains to be
`seen how this information will be used to design the
`next generation of DPP-IV inhibitors.
`Related Enzymes
`In the current inhibitor designs, selectivity over a
`wide range of proteases is generally possible. The
`stringent requirement for a basic amine provides inhibi(cid:173)
`tors with selectivity over proline endopeptidases, while
`the preference for pyrrolidine at P1 provides selectivity
`over other aminopeptidases.178 However, inhibitors that
`are thought to be specific for DPP-IV may in fact inhibit
`other enzymes in the "DPP-IV activity and/or structural
`homologue" (DASH) family.26 Several family members
`have only recently been described, and thus, selectivity
`data are not generally available.
`Fibroblast activation protein a. (FAP-a, also known
`as seprase) shares the highest sequence homology to
`DPP-IV. This enzyme, which is found in tissue remodel(cid:173)
`ing sites and tumors but not in healthy adult tissue,
`may be important in wound healing and cancer inva(cid:173)
`sion. Recently the DPP-IV inhibitor Val-boro-Pro (12a)
`was shown to inhibit both DPP-IV and FAP.27 This
`compound's ability to stimulate regeneration ofneutro(cid:173)
`phils following cyclophosphamide treatment, originally
`attributed to DPP-IV inhibition, was noted in both wild(cid:173)
`type and DPP-IV-deficient mice, suggesting that another
`prolyl peptidase such as FAP-a. might be responsible for
`the observed biological activity.
`Two other closely related DPP-IV-like proteins, DPPS28
`and DPP9,29 are soluble, cytoplasmic enzymes that are
`
`Page 4 of 9
`
`

`
`Journal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4137
`
`Chart 1. Structures of DPP-IV Inhibitors
`9N
`~~N.,_)l~L)
`
`Xn
`
`,_N
`
`H
`
`0
`
`18, X= CN (DPP728)
`1b, X= I
`
`Me 0
`
`MeJ0N""'s
`NH2 l_j
`4 (P32/98)
`
`CN
`
`F
`Me~
`
`NH2
`
`Me';rMe
`
`O
`
`r
`PhCO......,._N) l _N G
`', .. )L )/-NJ
`0 -:::::>--N N
`I
`Me
`8
`
`~~"T'Me
`AcNH~Me
`Ph NH2
`10
`
`0
`
`CN
`
`R0No;
`
`NH2
`138, R = CMe3 (FE999011)
`13b, R=Me
`
`ubiquitously expressed. DPP8 is up-regulated on acti(cid:173)
`vated T-cells, while high levels of DPP9 are found in
`skeletal muscle, heart, and liver. The latter enzyme was
`originally reported to lack peptidase activity, but that
`has recently been refuted.29b The biological function of
`both proteins is currently unknown, as is the degree to
`which "selective" DPP-IV compounds inhibit them.
`Quiescent cell proline dipeptidase (QPP), renamed
`DPP7, appears to be identical to DPP n so and is located
`in intracellular vesicles. This enzyme shares sequence
`homology to prolyl carboxypeptidase but has DPP-IV(cid:173)
`like, prolyl aminodipeptidase activity. Val-boro-Pro
`(12a), which inhibits DPP-IV with a Ki of 2 nM, is a
`125 nM inhibitor of QPP.31 Treatment of peripheral
`blood monocytes with this inhibitor induces apoptosis
`in quiescent but not activated lymphocytes. This effect
`is seen in T-cells lacking DPP-IV and has thus been
`attributed to the compound's ability to inhibit QPP in
`these cells. Selective inhibitors of QPP have been
`reported and may be useful in determining the biological
`role of this enzyme. 32
`Selectivity over these related enzymes may prove to
`be important for identifying safe and well tolerated
`inhibitors. In addition, caution must be used in inter(cid:173)
`preting studies with DPP-IV inhibitors because it is
`clear that in some cases, biological effects have been
`incorrectly attributed to DPP-IV inhibition .
`
`Preclinical Proof of Concept Studies
`There is a growing body of evidence to suggest that
`inhibition of DPP-IV will have therapeutic effects in
`treating diabetes. DPP-rv-1- mice show decreased blood
`glucose levels accompanied by an increase in insulin
`following an oral glucose challenge.33 In addition, Fis(cid:173)
`cher F344/DuCrj rats, which have a natural point
`mutation in DPP-IV affecting trafficking of the enzyme,
`
`0
`
`CN
`
`R
`
`0
`
`{3(0H)2
`
`11
`
`NH2
`128, R =Me
`12b, R = H
`
`,~0 RvN0,
`J
`\_~H t)
`
`f0(0Ph)2
`
`15
`
`have greatly reduced plasma DPP-IV activity and show
`improved glucose tolerance.34
`Acute inhibition ofDPP-IV by small-molecule inhibi(cid:173)
`tors leads to increases in plasma GLP-1 levels and
`decreases in glucose excursion following an oral glucose
`challenge in both normal mice and rats and in animal
`models of diabetes and impaired glucose tolerance,
`including diet-induced obese (DIO) mice35 and Zucker
`fatty rats. as An increase in insulin precedes the decrease
`in blood glucose, suggesting that the mechanism of
`glucose lowering is increased insulin secretion. The
`indirect effect of these compounds is also supported by
`the observation that DPP-IV inhibitors have no effect
`on glucose-stimulated insulin secretion in isolated is(cid:173)
`lets.35 In db/db mice, DPP-IV inhibition reduces glucose
`excursion in young animals but not in older animals
`with impaired ,8-cell function and pronounced insulin
`resistance.37 Data from this acute study thus suggest
`that DPP-IV inhibitors may not prove to be efficacious
`in advanced diabetics but rather in patients with early
`stages of the disease.
`A number of chronic animal studies provide support
`for the use of DPP-IV inhibitors in the long-term
`treatment of diabetes. Chronic administration of DPP(cid:173)
`IV inhibitor isoleucylthiazolidide (4) to VDF Zucker rats,
`a model characterized by mild hyperglycemia, hyper(cid:173)
`insulinemia, and insulin resistance, resulted in a de(cid:173)
`crease in the 24 h glucose profile and a progressive
`decrease in both fasting and peak blood glucose levels. as
`Following 12 weeks of treatment, an increase in glucose
`uptake in soleus muscle was evident as was an increase
`in the rate of insulin secretion in perfused pancreases
`from treated animals. The first-phase insulin response,
`which was absent in controls, was restored in the
`treated animals. Euglycemic- hyperinsulinemic clamp
`
`Page 5 of 9
`
`

`
`4188 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 17
`
`studies showed an increase in glucose disposal and a
`decrease in hepatic glucose output. 39
`Chronic studies in Zucker diabetic fatty (ZDF) rats,
`which become overtly diabetic at about 8 weeks of age,
`suggest that DPP-IV inhibition may delay the develop(cid:173)
`ment of disease.40 Treatment of6 week old animals with
`the potent DPP-IV inhibitor FE 999011 (13a, ICso = 7
`nM) delayed the onset of hyperglycemia from day 8 in
`vehicle-treated animals to day 15 in rats dosed with 10
`mg/kg FE 999011 QD. In ZDF rats dosed with 10 mg/
`kg b.i.d., the onset was delayed to day 24, suggesting
`that near-complete, 24 h inhibition ofDPP-IV is neces(cid:173)
`sary to obtain maximal efficacy. Free fatty acids and
`triglycerides were maintained below levels considered
`toxic to P-cells in the b.i.d.-treated animals; thus,
`preservation of islet function is a possible mechanism
`for the delayed onset of diabetes.
`There is additional evidence to suggest that chronic
`DPP-IV inhibition may preserve or restore islet function.
`In isolated islets from DIO mice treated with DPP728
`(la), an increase in insulin response at medium glucose
`concentrations was noted while maximal glucose(cid:173)
`stimulated insulin secretion was not effected.41 This was
`accompanied by an increase in GLUT-2, a P-cell glucose
`transporter. While there was no effect on body weight
`in the treated animals, islet size was normal; thus, DPP(cid:173)
`IV inhibition appears to counteract the increase in islet
`size that is typically seen in animals fed a high-fat diet.
`In a recent report,42 Wistar rats were treated chroni(cid:173)
`cally with isoleucylthiazolidide (4) beginning 1 week
`before or 1 week after administration of streptazotocin
`(STZ), a toxin that destroys pancreatic P-cells. In the
`early treatment group, postprandial glucose levels were
`less than both the late treatment and control STZ(cid:173)
`treated groups and plasma insulin levels were higher.
`The early treatment group showed an increase in
`glucose-stimulated insulin secretion in perfused pan(cid:173)
`creas studies and an increase in P-cell number, indicat(cid:173)
`ing a cytoprotective effect of DPP-IV inhibition. After 6
`weeks, the late treatment group also showed a progres(cid:173)
`sive decrease in glucose and an increase in insulin. Both
`early and late treatment groups had increases in the
`smallest size subset of islets relative to STZ-treated
`controls, with near-normal P-cell fractions, suggesting
`P-cell regeneration or islet neogenesis.
`Taken together, data from preclinical studies indicate
`that treatment with a DPP-IV inhibitor may provide
`improved efficacy in the early stages of diabetes and
`may delay progression of the disease. The potential for
`preservation and regeneration of P-cells suggests a role
`for DPP-IV inhibition even in the late stages of diabetes.
`
`Clinical Studies
`Preliminary clinical results have been disclosed on
`three DPP-IV inhibitors:
`isoleucylthiazolidide (4),
`DPP728 (la), and LAF237 (2). Isoleucylthiazolidide was
`reported to be safe and well-tolerated in normal volun(cid:173)
`teers at doses up to 240 mg.43 DPP-IV was inhibited in
`a dose-dependent manner . In an open label study in
`diabetic patients a decrease in glucose excursion fol(cid:173)
`lowing an OGTT was seen at a dose of 60 mg.44
`Increases in both active GLP-1 and GIP were noted.
`Patients with mild diabetes were treated for 4 weeks
`with DPP728.45 Because of the short half-life of this
`
`compound in humans (t112 = 50 min), the total daily dose
`of 300 mg was divided (150 mg b. i.d. and 100 mg t.i.d.).
`Both dosing regimens led to decreases in fasting and
`prandial glucose and mean 24 h glucose relative to
`placebo. While the drug was generally well-tolerated,
`transient pruritus localized to the palms was noted in
`treated subjects, perhaps due to potentiation of an
`unknown bioactive peptide such as substance P.
`Development of DPP728 has been discontinued in
`favor of LAF237, which has a profile suitable for once
`daily dosing. Following administration to diabetic pa·
`tients at a dose of 100 mg qd for 4 weeks, decreases in
`fasting glucose, postprandial glucose, and postprandial
`glucagon levels were seen.46
`While these initial clinical studies appear promising,
`the long-term safety, efficacy, and durability of DPP(cid:173)
`IV inhibitor treatment remain to be established.
`
`Additional Opportunities and Potential Pitfalls
`With its mechanism of glucose-dependent insulin
`biosynthesis and secretion, DPP-IV inhibition provides
`the potential opportunity for excellent synergy with
`existing diabetes treatments. Combination therapy with
`insulin sensitizing agents such as PPARy agonists and
`agents that control hepatic glucose output such as
`biguanides may prove to be particularly effective. The
`former combination has been explored in obese Zucker
`rats.47 Following a 10 day treatment, a synergistic effect
`was observed with DPP-IV inhibitor LAF237 (2) and
`PPARy agonist pioglitazone, in particular, on the in(cid:173)
`crease in rate of glucose disposal.
`Additional opportunities may exist in the treatment
`of diseases beyond diabetes. When fed a high-fat diet,
`DPP-JV-1- mice and DPP-IV-deficient Fischer F344/
`DuCrj rats are resistant to weight gain, suggesting a
`role for DPP-IV inhibition in the treatment of obesity.48
`Food intake in both the DPP-JV-1- mice and Fischer rats
`is reduced. Pair-fed wild-type mice weigh more than the
`DPP-JV-1- animals; thus, a metabolic component may
`exist. In animal studies with DPP-IV inhibitors, how(cid:173)
`ever, weight loss is typically minimal or not seen at all.
`While it is unclear how these results will translate into
`clinical findings, DPP-IV inhibitors are not likely to
`cause the weight gain that is often associated with
`current diabetes medications.
`Potentiation of endogenous substrates beyond GLP-1
`and GlP may provide further therapeutic opportunities.
`For example, GLP-2, an intestinal growth factor re(cid:173)
`leased in the gut in response to nutrient ingestion, is
`inactivated by DPP-IV in vivo in rats.49 Thus, inhibition
`of DPP-IV may prove to be useful in the treatment of
`intestinal injury and disease. This indication remains
`to be fully explored. DPP-IV is also thought to regulate
`endomorphin-2, a tetrapeptide (Tyr-Pro-Phe-Phe-NH2>
`with high affinity for the fl opioid receptor.50 The icv
`administration ofDPP-IV inhibitors alanylpyrrolidine-
`2-nitrile50a (13b) and DiprotinA50b (Ile-Pro-lle) evoked
`dose-dependent potentiation of endomorphln-2 induced
`analgesia in the mouse paw withdrawal and tail flick
`models, respectively.
`There are a variety of other substrates that DPP-N
`cleaves in vitro.8 These include chemokines such as
`RANTES, eotaxin, IP-10, and SDF-la., neuropeptides
`such as substance P, ,8-casomorphin, NPY, and PYY,
`
`Page 6 of 9
`
`

`
`and growth factors such as GRH. In many cases, the
`DPP-IV cleavage product is inactive or has altered
`receptor specificity. Additional studies are needed to
`determine whether these are in vivo substrates and, if
`so, whether DPP-IV inhibition will lead to desired
`therapeutic benefits in other diseases or will r esult in
`potentially toxic side effects.
`DPP-IV has a number of proposed functions in addi(cid:173)
`tion to its role in metabolic control.6 It binds adenosine
`deaminase, an enzyme important in the normal devel(cid:173)
`opment and function of the immune system, and likely
`modulates local concentrations of adenosine. It also has
`a binding site for the extracellular matrix proteins
`collagen and fibronection, though its role as an adhesion
`molecule remains unclear. DPP-IV is identical to the
`cell surface marker CD26 and serves as a costimulatory
`molecule in T-cell activation. While DPP-IV inhibitors
`have been shown to inhibit T-cell activation in vitro,
`concentrations required for this activity are well above
`their reported K ; values; thus, a role for enzymatic
`activity in this function appears unlikely.
`Because of the potential role ofDPP-IV in the immune
`system, inhibitors have been studied in a number of
`immunological models. DPP-IV inhibitors Ala-boro-Pro
`(12b) and Lys(Z-N02) thiazolidide (14) as well as two
`natural product DPP-IV inhibitors have been shown to
`inhibit hind paw swelling in collagen- and alkyldiamine(cid:173)
`induced models of arthritis in rats.51 The irreversible
`inhibitor Pro-Pro-diphenyl phosphonate (15) prolonged
`aJlograph survival in a rat cardiac transplant model.52
`With the discovery of additional enzymes possessing
`DPP-IV-Hke activity, these results remain to be con(cid:173)
`fii·med with more selective DPP-IV inhibitors.
`
`Conclusions
`
`Inhibition of DPP-IV is an attractive new approach
`to the treatment of type 2 diabetes. Because DPP-IV
`inhibitors stimulate insulin secretion in a glucose(cid:173)
`dependent fashion, the potential for hypoglycemic side
`effects is minimal. The lack of weight gain, and potential
`for weight loss, with DPP-IV inhibitor treatment pro(cid:173)
`vides another potential benefit to diabetics, the vast
`majority of whom are obese. Finally, recent data sug(cid:173)
`gest