WEISEVIER
`
`Available online at wwwsciencedirect.com
`
`ocrENcE do,"-.""
`Biochimica et Biophysica Acta l75l (2005) 33 - 44
`
`BB
`
`http://www.elswier.com/locate/bba
`
`Review
`Type 2 diabetes-Therapy with dipeptidyl peptidase IV inhibitors
`
`Hans-Ulrich Demutha*, Christopher H.S. Mclntoshb, Raymond A, Pedersonb
`bDepartrftent "r*0r,, "^ ,rill,,ufl# lf*i#?#',{lfffiii!i;f!!ill,,f;itf'l:T:*rfffiL Marr, r,ancouve,t BC, canada
`
`Received 22 September 2004; received in revised form 14 May 20A5; accg4ted 17 May 200J
`Available online 6 June 2005
`
`Abstract
`
`The sole application of an inhibitor of the dipeptidyl peptidase DP IV (also DP 4, CD26, DPP-IVor DPP4) to a mammal subsequently
`leading to improved glucose tolerance marks a major breaktlmugh in metabolic research bearing the potential of a new revolutionary
`diabetes therapy. This was demonstrated in rat applying the specific DP IV inhibitor isoleucyl thiazolidine. It was published in 1996 forthe
`first time that a specific DP IV inhibitor in a given dose was able to completely block glucagon-like peptide-l (GLP-I) degradation in vivo
`resulting in improved insulin response acconrpanied, by accelerated peripheral glucose diqposal. Later on, these results were confirrned by
`several research teams applying DP IV inhibitors intravenously or orally. Today, the DP IV inhibition forthe teatmert ofmetabolic disorders
`is a validated principle. Now, more than l0 years after the initial animal experiments, first DP IV inhibitors as investigational drugs are tested
`in phase 3 clinical nials.
`O 2005 Published by Elsevier B.V.
`
`Keywords: Dipeptidyl peptidase; f}pe 2 diabetes; GLP-I; GIP; I:hibitor; hhibitioq Drug developm.eirt
`
`1. Introductlon
`
`The discovery of ihe blood glucose lowering potsntial
`of the incretins GLP-I and glucosedependent insulino-
`[opic peptide (GIP) and their properties as growth
`hormones opens up a totally new pathway for causal
`diabetes therapy. Since both homrones ne cham*teizedby
`an acute inzulinobopic action and a sustained effect upon
`insulin resistance and the glucose sensitivity of the cells of
`the islets of Langerhans a pleiohopic teatrnent of different
`diabetic symptoms seerns possible for tle first time.
`Since both hormones are also deactivat€d simullaneously
`by the cardiovascular exopeptidase dipeptidylpeptidase IV
`(DP IV) in contast 0o hormone montherapy, a multivalent
`method of tea;hent is opened up by the inhibition of this
`enzyme. Interestingly, other bioactive peptides are also DP
`IV substates, for example vasoactive intestinal peptide
`(VIP), somalostatin, pituitary adenylate cyclase-activating
`
`s Corresponding author. Tel.: +49 345 5559900; fax: +49 345 5559901.
`E-mail address: hans-ulrich,demruth@probiodn:g.de (H.-U. Demuth).
`
`1570-9639/$ - soe front matt€r @ 2005 Published by Elsevier B.V.
`doi: 10.1016d.bbapap.2005.05.010
`
`polypeptide (PACAP) and newopeptide Y (NPY). Con-
`sequently, additional synergy potentials may arise by the
`inhibition of DP IV activity. Unlike hormone therapy,
`however, which functions with pharmacologically ac'tve
`doses of GLP-I or its analogues, a DP IV inhibitor
`modulates "only' the endogenous released hormone con-
`centations. The following article thus gives a review of the
`development of DP IV inhibitors as potential antidiabetics.
`
`2. DP IV-a short historical perspective
`
`DP IV (EC 3.4.14.5, also DP 4,CD26, DPP ry DPP-IV
`or DPP4) was discovered in the 1960s as an amino-
`peptidase [l]. In the 1970s, the snrpe served initially as a
`model protein for the shrdy of the caalyic mechanism of
`serine peptidases and for the investigation ofthe specifics
`of proline peptide bonds [2]. In the 1980s, the potential of
`the enzyme 0o convert bioactive peptides in vibo was
`discovered, whiih intensified the search for its fimction in
`vivo [3]. The early 1990s is characterized by nurnerous
`
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`
`studies on the role of DP IV in irnmune responses, in
`particular T cell activationo signal tansduction and T cell
`proliferation [4-6].
`DP IV was identified as characteristic antigen marker
`CD 26 of T cell activation and it was demonsfated that the
`protein is a component of tle T cell rcceptor complex.
`Diflerent binding partners were fomd within this cont€xt,
`for example adenosine deaminase (ADA) [7], the HIV
`mantle protein gp 120 [8], tyrosine phosphatase CD 45 [9],
`fibronectin [10] and the renal sodium proton antiportsr
`NHE3 [111.
`A contoversial debate erupted in this wea in 1993
`when the hypothesis was puX forward that CD26 is co-
`responsible for the enfy of HIV into T cells [12]. New in
`vivo functions of the enzyme were being debated whsn in
`the middle of the 1990s, the involvernent of DP IV in
`metabolism and the regulation of the cytokines, chemo-
`kines and differsnt peptide hormones tiggered prograrns
`for the development of DP IV inhibitors [13].
`It was the discovery of the role of the enzyme in energy
`homeostasis which aocelerated the desip of potsntial
`pharmaceutical agents for the heatment of that most
`impor0ant of metabolic diseases, Type 2 diabetes, and led
`to the first patent application for the use of DP IV inhibition
`in ths reduction ofblood glucose [14].
`Further enzymes with post-proline dipeptidyl amino-
`peptidase activity, which today belong to the DP IV
`subfamily of the prolyl oligopeptidases @OP), were discov-
`ered in the late 1990s [15].
`Atthis time, the crystal structurc of POR a sister enzryme
`of DP IV was elucidated t161.
`At the beginning of the new millennium, far more had
`become known of the role of cell intemalization processes
`and the comparhnentalization of DP IV during the
`association with binding paxtners in the expression of the
`immune response 117,18].
`Moreover, specific mechanisms of metastasis which
`involve DP IV were clarified [19]. The most important
`milestone in DP IV research is, however, the elucidation
`
`of its threedimensional stuctre. Sevsn stuctlres, of
`which five are human resombinant stuctres and one a
`native pig DP IV structure, have been published since
`January 2003, when resolutions of 1.8*2.6 A were
`achieved, A structure determined by electron microscopy
`based on crystalline mouse DP IV in shown in Fig. 1. All
`structures exhibit the same principal topology of domain
`organization [20-?61.
`The DP IV thrsedimensional shuctnes help not only in
`understanding the unusual protease function of the enzyme,
`but also explain additional properties whioh differentiate
`the enzyme from the 'otl4)sin' serine proteases and
`cbanctsize it as an important physiological communica-
`tion molecule.
`
`3. Dipeptidylpeptidase IV-localization, stucture and
`substates
`
`DP IV was first isolated from rat liver in 1966 [1]. Since
`theno analogous enzyme a*tivily has been detected in
`various marnmals and also in microorganisms and plants.
`In humans, ths snzyme activity is found ubiquitously in
`almost all organs and tissues with the highest local
`concenfations found renally in the proximal trbuli and
`luminally on the epithelial cells of the small intestine [27].
`With ths discovery of ths role of the enzyme in glucose
`homeostasig DP IV research has increased since the middle
`of the 1990s to such an extsnt that more than 1700 original
`papers with the keyword DP IV are registered in the
`National Library of Medicine, and more than 230 parent
`applications on protein stucare and enzyme inhibition have
`been submitt€d. Many of these resulls have been presented
`at confgrences [28-32] and recently surnmarized in an
`excellent review by Ingrid DeMeester [33].
`DP Ms a membrane-bound, homodimeric class II
`protein with a molecular weight of 110-150 kD per subunit.
`It is bound to the membxane by a transmembrme sequsnce
`of ca 22 amino acids. The 6 cytosolic amino acids play no
`
`Entry poru ln ?-memb6rGd propeller
`
`N*)
`
`Fig l. Suface representation of DP IV ob! hod by eleokon microscopy
`recopized (reproduction with kind permission of ri/. Reu$rr).
`
`slde sagecs
`[26]. fte propeller domains with an enty pore md a lage side opeiring can be
`
`MYLAN Ex. 1009, Page 2
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`

`
`DP IV/F,qP
`
`BPL1 / DPl'
`
`OP8 / DFg
`
`H.-U. Demuth et al. / Blochimica et Biophysica Acta 1751 (2005) 33-44
`
`I FP lV olasvage site
`
`*.Rffi* x x
`*r$ffix
`x'x
`
`*ubstratE lnqcwS- S
`bidnt
`
`pyryatsru S
`
`DJrdlftg
`
`I
`
`ll
`
`$t
`
`$ oyrosolic N
`tisnsrwflbran$ N[ utltno*
`Fig. 2. The human DP IV gene family.
`
`role in the binding functions. In addition to its protease
`function, DP IV has receptor properties and as extracellular
`binding protein. Fig. 1 gives an impressive zurface profile of
`DP IV.
`The enzymes fibroblast activation protein (FAP or DP 5),
`DP'8 and DP 9, and the catalytically inactive proteins, DP
`Ll (DP Q and DP L2 (DP 10), are sfuchrally relaled ro DP
`IV [15,34].
`Diffsrsnt stuctural regions of the protein are assigned
`to the primary sequence in Fig. 2. Whereas the caialytically
`active residues of the snz)me are localized in the region of
`the o/p-hydrolase domain, the protein-protein interactions
`described ocan mainly with regions of the so-called p-
`propeller domain (see also Fig. l).
`Structurally umelated but finnished with similar sub-
`strafe specificity are the monomeric en4lmes dipeptidyl-
`peptidase tr (DP tr, DP 2 or DP 7) and affactin (Atm).
`Physiological functions have thus far not been detected for
`athactin, which, like DP ry occurs in high concentations
`in the blood sbeam and is also involved functionallv in the
`immune response, nor for DP tr.
`
`Fig. 4. Charactoistics of DP IV specificity: the longer tho peptide, the lasser
`the mzyme is proline specific. Also, the longer the zubsbafe, the bsto it iB
`hydrolyzed
`
`The cellular localization of DP IV-like enzym.ss is shown
`schematically in Fig. 3.
`The initial work by Hopsu-Havu [1] and the enzymo-
`logical chansteizffion identiff DP IV as a proline-specific
`peptidase f2,31.
`However, the extensive investigation of potential phys-
`iological substates has shown that the preferential proline
`specificity is lost with increasing substale size (Fig. 4).
`The nafiral substrates of DP IV include chernokines,
`cytokines, endomorphineso hormones of the pancreatic
`polypeptide family and almost all pe,ptides of the PACAP/
`glucagon peptide family 127,351.
`Because of this substrals specificity which allows DP
`IV-analogous enzymes to be involved in numerous
`regulation processes, the modulation of DP IV activity by
`enzyme inhibitors appeas to make interesting therapeutic
`approaches a possibility. For example, the stabilization of
`neuropeptide Y by DP IV inhibitors in the CNS leads to
`
`Sardlovgsqslar
`
`PtodoHion q,l thB as$dt}
`ot
`VlP. P*rAP, F.tlM, sasadilitirr
`{.\
`.
`NPYur,rdPYY. I
`hscss.$Ne$
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`mrystrhept,t
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`usiabi$gA
`Rr$r|laiF..ot
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`by Jncrciht.n&dulBu$l
`issorldio'n
`6.gtl€ty
`P.YY u.irf GLPit
`
`qsleste.e.tss
`SiBdlBUlsECS
`t@ugt
`collqgn.'ibY$ri@tin
`plasfBlrQg€n,
`1',€tremeJiei!'a0
`
`Fig. 3. The cellular looalization og pp ryJike en2y@os.
`
`Fig. 5. Physiological procasses which are affeoted by DP IVactivity.
`
`MYLAN Ex. 1009, Page 3
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`

`
`H.-U. Dquth et al. / Biochimica et B@ttysica Acta l75I (2005) 33 -44
`op.t, oPt
`
`Dp rv
`
`Apr{
`
`frfpsin
`
`NEFfi;{1.
`
`Fig. 6. Protease attac& sites at GIP.
`
`significant reduction in the anxiety behavior of exper-
`imsntal animals f341.
`Since no tissue-specific ashvity differences have been
`found for the enzyme and unlike the enzymes of the
`coagulation cascade DP IV is contolled neither by limited
`pro0eolysis nor by endogenous inhibitors, specific DP IV
`inhibitors should possess therapeutic potential in different
`paihophysiological processes G'ig. 5).
`One such approach is examined more closely in the
`following, with the example of the development of DP IV
`inhibitors for the heatment of Type 2 diabetes.
`
`4. DP IV activity in blood glucose regulation
`
`The discoveqy ofthe insutinotropic action ofendogenous
`intestinal factors was a pivotal milestone in the investigation
`of the regulation of glucose homeostasis.
`Its considerable blood glucose-lowering activity led very
`early to speculation on a therapeutic use of tle incretins GIP
`and GLP-I as potential antidiabetics. Reviews by Kieffer
`and Habener [35] and Vilsboll and Holst [36] report in detail
`on this area of research.
`Unfortrmately, the short biological half-life of these active
`peptides impair their sustained therapeutic use. Although Xhe
`hormones are still detecta.ble in senm 2-4 h aftrr prandial
`secretion by means of C-0emdnus specific antibodies their
`biological activrty is of very limited duration. Thus, the half-
`life of GLP-I is of the order of I min and that of GIP of the
`order of 10 min [36].
`
`In a work published in 1993, Mentlein et al. [13]
`describe the DP tl-catzlyznd hydmlysis of a series of
`neuropeptides and the gasfo-intestinal peptide homtones
`GIP and GLP-I in vino. It had been shown previously
`by Pederson and Brown tn 1,976 l37l that only the intact
`N-terrninal hormone GIPr-az (or GIPl-3e) possesses
`insulinotopic action and could be deactivated by amino-
`peptidases or dipepxidyl aminopeptidases (F'ig. 6). Exo-
`peptidases such as inter alia aminopeptidase N and
`different dipeptidyl peptidases (I, tr and IV) come into
`consideration for the proteolysis of the peptides in vivo as
`do endopeptidases such as trypsin and neutal endopepti-
`dase (NEP 22.11).
`In 1996. the in vivo use of DP ry inhibitors and their
`potent glucose-lowering activity confirmed the pivotal
`role of DP IV in the carabolism of incretins U4,38,391
`and initiated considerable interest within the pharmaceut-
`ical industy for the development of DP IV inhibitors.
`Interestingly, it was recently shown that overweight is
`associaled with increased DP IV ac[v$. The authors
`infsr here a connsction betwesn adiposity-induced Type 2
`diabetes and increase incrstin degradation in these patients
`[40]. Meanwhile, it was dernonsrabd that DP lV-negative
`rats and mice have extemely good glucose tolerance and,
`unlike wild type animals, do not tend 0owaxds overweight
`and diabetic symptoms with a fat-rich diet [41,42]. Thus,
`as a principle for the therapzutic beatusnt of hyper-
`
`GlP sod GLP: I ,actlon by
`gtnnda$on ot
`e lnsuluiislsa6q
`t In6iJlh.h|owntheeig
`t lles.il Frolff@ralion
`
`lnfrbldoc? of
`'I Gaqirtc grurtvir,g
`.I Gh$agorrdeas$ (GLP-{J
`:f Foodup{she tSLP-l)
`
`Fig. 7. Schematic representation of DP lV-modulated incretin action.
`
`Fig. 8. lrcretin-nediated therapy-a multivalent thetqeutic approach.
`
`$li.!.\{r$lr
`$SluEs*e
`
`'t$o therEiycon$spts srs pFuFlhfe
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`f;;iYF*l
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`DF lV lnhlbhorn
`
`MYLAN Ex. 1009, Page 4
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`

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`'H.-U. Demuth et aI. / Biochlmica et Biopttysica Acta 1751 QA0, 33-44
`
`JI
`
`glycaemia and therewith, fhe associated metabolic disease
`states and subsequent consequsnces DP Mnhibition is a
`recognized current development arca 143,M], The action
`of DP IV inhibitors is presented in a highly simplified
`form in Fig. 7.
`
`5. DP Mnhibitors
`
`The possibility of the exploitation of multiple synergetic
`effects of different peptidic signals in the post-prandial
`phase appears especially atfactive for the potential ther-
`apeutic use of DP Mnhibitors. The properties of the
`incretins are summarized in Fig. 8.
`Cunently, more than 30 phamraceutical and biotech-
`nology companies have programs on the development of
`DP IV inhibitors for the heatmsnt of Type 2 diabetes
`(Fig. e).
`DP IV inhibitors have been prepared since 1977.
`Their synthesis and the different stucfural classes have,
`in the meantimeo bee,n the subject of differEnt reviews
`145,46), Initial revsrsible and ireversible compounds
`were chemically modified product aaalogues whose
`inhibitory potency extends into the sub-nanomolar
`regton. More rscentlyo compounds of non-peptidic
`stuchre have become increasingly known from scresn-
`ing procedures [46]. In additioq the three-dimensional
`stuctures of the enzyme published since 2003 have
`made a rationally motivaled inhibitor stuchre design
`possible, which, in tle case of the aminopyrimidines, has
`led to increases in activity of about five orders of
`magnitrde [46,471.
`In respect of current clinical developments of the
`thousands of individual compounds prepared in the msan-
`
`10
`0ts
`
`3S&
`
`38
`80
`zfi
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`n-csv
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`)cr***rr'
`H3C-C.H
`%r"
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`F3e€8
`
`Fig. 10, Reversible product analogue inhibitors (e.g. pyrrolidines and
`thiazolidines).
`
`time, three substance classes stand out whose representatives
`are under investigation in man (Figs. 10-12).
`
`- Reversible product analogue inhibitors (e.g. pyrrolidines,
`thiazolidines).
`- Covalently modi$ing product analogue inhibiton (e.g.
`cyanopyrrolidines).
`- Reversible non-peptidic heterocyclic inhibitors (e.g.
`xanthines and aminomethylpyrimidines).
`
`Unlike the proteolytic snzymes which occur in the blood
`stream in only very small amounts, DP lVoccurs in very high
`
`Fig. 9. Estimared number of DP IV inhibitor patmts since 1995 (as of l2l20H). (Probiodrug AG sold its DPIV patent estate for mohbolic diseases in 2004 to
`Prosidion Ltd., UK).
`
`MYLAN Ex. 1009, Page 5
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`

`
`38
`
`H.-U. Demuth et al. / Bioehimica et Btophyslca Acta 175] (2005) 33 -44
`
`NVP OPP ??S (Novartls)
`
`(BM$..4rfl$J
`LAF e$7 (Novartls)
`Fig. ll. Covalenfly modifing product aralogue inhibiton (e.g. cyanopynolidines).
`
`concsntrations. On the basis ofthe specific activity of the
`wzyme and the concentration in different tissues [48], a
`lower concenhation limit of 3 pM/l blood can be estimated
`for the enzyme protein. If at a given time poinl the DP IV in
`the blood sheam is to be blocked by 700o/o, a dose ofat least
`3 pM/l drug must be present in the blood steam. With a
`theoretical molecular weight of an active substrate ofroughly
`500 D, this means a.bout 10 mg in an adult penon.
`
`Since pharmaco-kinetic fac0ors such as intestinal absorp-
`tion rates, disribufion, metabolism and excrexion also play a
`role in the build-up of a corresponding level of activity, a
`still higher active dose is to be expected. Correspondingly,
`active doses of 30 and 100 mg have been described for
`differenl tlpes of inhibitor, although the in vifro effective-
`ness of these inhibiton can lie several orders of mapitude
`apafi 1491.
`
`Xtilhlns({$oVO}
`
`Xanihine (El)
`
`Aml nom€thyl pyrldlne (Rmhel
`
`MK;0i131{[n6rckl
`
`Fig. 12. Reversible noa-peptidio hoterocyclic inhibitors.
`
`MYLAN Ex. 1009, Page 6
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`

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`H.-U. Dem*h et al. / Biochlmlca et Blophysica Acta I75l (2005) 33-44
`
`."*. Cdnrol {n*€}
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`-.B eo,
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`Io ao,
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`b.
`
`Fig. 13. Daily pharmacokinetic profile of the DP IV inhibitor P32/98 in
`VDF rats [52].
`
`by a reduction of the normally leptin-stimulated producxion
`and secretion of satiation factors, nernopeptides such as
`CRH, on the one hand, and by stimularion of orexigenic
`peptides, such as NPY on the other. The leptin signal
`cascade in oblob mice is similarly impaired when the same
`symptoms as in the db (or the fa) mutation are obsenred,
`except that m the oblob defect, the mutaJion is localized in
`the leptin gene.
`Lr addition to the /o mutation" ZDF rats haveo in
`conbas! furthsr genetic changes whicho particularly
`through an elevated triglyceride toxicity, lead to accelerated
`p-cell loss and thus in a falal progression to manifest
`diabetes. The IIFD mouse and the DIO rat are being
`increasingly used in the investigation of potential anti-
`diabetics. Commoil to all these animal models is that they
`become over-weight through hyperphagia and also inqreas-
`ingly glucose intolerant. In the course of their dwelopment
`the animals are also characterized by an increasing insulin
`resistance and elevated fasting and postpraadial blood
`glucose levels.
`Meanwhilg investigations into the activity of DP IV
`inhibitors as antidiabetics have been canied out in pigs,
`dogs and monkeys [38,53,54]. Here, the active compounds
`are administered orally and, depending upon their pharrna-
`cokinetic properties in the respective animal model, require
`single or multiple doses (F'ig. 13),
`
`10
`
`Fr.csngul {n*6}
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`
`Et
`
`r6so(
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`t
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`0ecr0 ir:gG 14.{0 17$0, ef.00 e&00 ot',00 0.sr00
`llms{hanrs}
`
`Fig. I 4. Daily gluoose profile after 6 weeks of subolmnic teabnent of VDF
`rats with P32198 152,63J.
`
`Because of the different modes of action (non-covalent
`reversible or hansiently covale,ntly modifying inhibitors),
`two beatrnent principles cunently evolve; pharmaceuticals,
`which are taksn with meals and whose concentations
`accompany the physiological occurrence of tle inoetins,
`and active compounds, which have to be taken independent
`of meals and are permanently present in tle body ('Iable 1).
`Consequently, the respective side effect profile will be
`critical for the medical success of compounds under
`investigation.
`Concgrns about potential side effects of DP IV inhibitors
`resuft from the multiplicity of biological functions of DP IV-
`related euymes.
`DP ry acts as a multifunctional Fotein. It conhols the
`bioactivity of regulatory and nernonal peptides by limited
`pro0eolysis and may serye as binding prctein for numerous
`biologically important binding proteins (see Irig. 5). So far,
`litrle was published concEining the saious side effects of the
`compounds under investigation as potential drugs; however,
`recsnt investigation suggest tlat compounds which not or
`purely discriminate between DP IV-related enzymes such as
`DP 4, DP 8 and DP 9 might cause severe immunotoxicity
`ts0l.
`Whether long-temr or long-lasting DP IV inhibition will
`intsrfere with physiological pathways refened to above (sse
`also Section 3) must be left to firture long-term studies or
`even to dala gathered after the approval sn6 mar!6fing of
`the first drugs, as recently observed with some pain
`medication.
`
`6. Preclinical lnvestlgafion results
`
`The first aetivrty shrdies on DP JV inhibitors were
`carried out in rats and later in pigs l5ll. Since 1998, the
`antidiabetic activity of DP IV inhibitors has been tested in
`differsnt diabetic animal models such as oblob nic.e, dbldb
`mrce, falfa rats, Vancouver diabetic fatty (VDF) rats,
`Zucker diabetic fafiy (ZDF), Goto Kakizaki (GK) rats, high
`faf diet (IIFD) mice and diet-induced obese (DIO) rats.
`Although, for example, the fa (and db) gme defect occurs
`less than once in 10,000 people in the hrman popuktio4
`this animal model is suitable for the investigation of
`potential antidiabetics. The leptin rcceptor mutation under-
`lying the defect leads to hyperphagrq which is charactsdzed
`
`Table I
`Activ8 profiIes of oral DP IV fuhibitors mder developm€nt
`Active type
`Non-covalent
`Covaleirtly
`modi$ing
`reversible
`
`Dose regimo
`
`Rate of absorytion
`Metabolism (half life)
`Activity level
`Enzyme ir-hibition
`
`to sev€dzl
`Onc€
`tinas per day
`Rapid
`2-7 h
`Highly variabl€
`Short-fived
`
`Once to several
`times per day
`Slow
`>4h
`Constaatly high
`Long-lasting
`
`MYLAN Ex. 1009, Page 7
`
`€
`

`
`H.-U. Demuth et al. / Biochtmica et Biophysica Acte l75l (2005) 33*44
`
`400
`
`..r,-.cotwol (neo).
`*'ow F3f,ES{il-.d}
`
`rIti
`
`5 .
`
`s50gI
`
`tEst
`
`{
`IL
`
`r0 ?0 40 E0 80 lAA 190
`.tins (mirr)
`
`Fig. 17. Insulin kinetias during a stmdrd OGT"I after 12 weeks of
`teabnent of VDF rats nith the DP IV inlibitor P3U98 [52,65].
`
`[66]. This sffect is affibutable to the growth factor fimction
`of the incretins.
`Meanwhile, it was also dernonstrated that the "old"
`antidiabstic me$omrin has an effect upon incretin-medialed
`glucose homeostasis not through DP IV inhibition but
`through an increased GLP secretion 167-711, This finding
`makes an interesting combination therapy of metfomtin
`and DP IV inhibitors possible. The GLP secretion
`stimulated by metformin and hence, the impnovement in
`glucose tolerance, which because of the DP IV aotivlty
`towards GLP-I is only partially effective, are synergeti-
`cally reinforced by the concomitant presence of a DP IV
`inhibitor l70,7ll (Fig. 18).
`Such findings and the toxicological safety of DP IV
`inhibitors proven by animal experimentalion have moti-
`vaied several pharmaceutical companies to commencs
`Phase 3 studies last year.
`
`7. Results of cllnlcal investlgatlons
`
`Thus far, only sparse results from human studies with DP
`IV inhibitors have been published. Some of the currently
`known clinical development candidates are listed in Table 2.
`The administration of 60 mg P32l98 led to a significant
`pos@ardial improvement in glucose tolerance in diabetics
`[49]. As in tle aforementioned animal models, the glucose-
`
`N6("lr+?x$f
`EEOEl'tr
`
`(5
`
`35
`
`9r0
`
`?,5
`2fi
`
`1;.S
`i.o
`$.5
`
`0:o
`
`Cofltrot Md,tlo{Tnln Pg8&1 M9I}FSSOJ
`
`Fig. 18. Effeot of meformin, tho DP IV inhibitor P93/01 anda oombination
`ofboth aotive oorpounds on glucnso toleranoe in a standard OGTI in DIO
`rats.
`
`r8@
`
`1?m
`
`g*:I
`
`-*! contYol (rF6)
`-{uF08/9g'tn.o}
`
`rt00
`
`o
`
`g3nE t
`
`0,
`
`E0
`
`)sg
`
`f*.-.lW'-*t
`@100 t1:@ 't4:W 17rW. €9100 A*00 0A:00 05t0O
`ttfirB {hoUtc}
`Fig. 15. Reduction in endogenous insulin secretion afto 6 weeks of
`subchronic Eeatm€nt of VDF rab wifh P3298 [52,65].
`
`Essentially all studies confirm that the acutc adminis-
`tation of DP IV inhibitors leads to an improvement of
`immediare glucose tolerance by acute increase in insulin
`secretiorq whilst long-term studies show tlat subchronic
`inhibitor doses lead to a reduction in daily glucose load and
`to an improvemsnt in the glycaemic index (F'igs. 14 and l5).
`For more ds0ailed discussions on the outcome of many
`studies which have been perforrred since the first applica-
`tion of an DP IV inhibitor to a mammal [14], the interested
`rsader is refsrred to special reviews which appeared after the
`submission of this article [55-64].
`Furthermore, both the fasting and the postprandial blood
`glucose could be significantly lowered by chronic DP IV
`inhibitor adminishation (Fig. 16).
`This notable improvement in glucose tolerance in
`experimental animals is accompanied by an increase in
`glucose sensitivity of the islet, which likewise leads 0o
`reestablishment of 'normal" insulin kinetics. that is the
`increased secretion of so*alled first phase insulin (Fig. U).
`A significant lowering of IIbAlc is also associafsd with
`these long-lerm effects. In the meantime, it was also possible
`to show that in a type I diabetes animal model, the SDZ-
`teated WistBr rat in which toxin-mediated p-cell apoptosis
`leads to diabetes development, DP W inhibitor adminisha-
`tion, both preventativd and parallel to toxicity, can lead to
`almost complete or at least pmtial pancreas regeneration
`
`"-s!-" P,34l68 {rF.S}
`."r..ccinltot {n,€}
`
`.F
`
`9eo
`$rso
`{t
`Bru
`Eo9
`&
`
`,.{:"""""""**'-{
`
`t..r:t,0 g0 40 m $0 iso 1?o
`$nrs (min)
`
`Fig 16. Oral glucose lolemnce lsst (OGT"I) after 12 weeks ofsubchronic
`treabneirr of VDF rafs with P32/98 L52,65J.
`
`MYLAN Ex. 1009, Page 8
`
`

`
`H,-U. Demuth et al. / Biochimica et Biophysica Acta 1751 (2005) 33-44
`
`41
`
`e00
`
`1$S
`
`rEq
`
`sEG
`
`t
`.N
`
`6g
`
`1eo {8n t40 300
`$iW{ynln)
`
`Fig. 20. Inorease in active GIP in 20 pafiorts tlmugh the astion of 60 mg
`P32198 dudng a glucose tolerarcs test [49].
`
`The DP IV inhibitors are in competition with the DP IV-
`resistanx incretin analogues [75]. Whereas the adminishation
`of peptidic GLP-I analogues must currently be carried out
`subcutareously and dailg the low molecular weight DP IV
`inhibiton are charac0erized by their oral availability. On the
`basis of data from cunsnt sfirdies, both teatmsnt stategies
`achieve comparable therapeutic endpoints.
`Therefore, in addition to the therapy behavior ofpatients,
`the possible immunogenicity and the tachyphylactic poten-
`tial of these receptor agonists, which are administered in
`pharmacological doses, will be of relevance for successful
`diabetes tr€ahnent with incretin analogues.
`Since DP IV inhibitors modulate ooonly" the biologically
`active life-time of the endogenous concentraiion of the
`incretins and other GI hormones, their success as anti-
`diabetics wiil be judged primarily by their side effect
`profile.
`A few containdicative phenomena are reiterated: DP IV
`is involved both as a peptide-cleavage enzyme and as a
`receptor molecule in different regulation cascades. For
`exaurple, the protein is involved in the maturation of blood
`cells in the bone mrurow. DP IV inhibition influences the
`actwity ofthe tubularNa/proton antiporterNH3 [11]. DP IV
`is involved in the expression of the T cell-mediated immune
`response through the regulation of the activity of cytokines
`and chemokines [76]. The endocrine intestinal L cells
`secrete not only GLP-I but also the pos@randial peptide
`hormone PYY [77]. In contast to the DP lV-mediat€d
`deactivation of GLP-1 and GIP, the conve$ion of PYYI-36
`into the tuncaled PYY3-36 by DP IV sesms not to
`deact:vatn the hormone. It appears thal ths subcutaneous
`administation of PYY3-35 in physiological doses has led to
`a reduction in food intake in experimental subjects of ca"
`30% [78]. It might be possible thar DP IV here even
`activates by limited proteolysiso which is known, for
`instance, from some of the chemokines [33]. These and
`other pmperties of the target e,nzyme DP Mas thus led to
`the developmsnt of different inhibitor qrpes and admin-
`istration paradigms. Which of these will be successfrrl in the
`long terrn will possibly be fnst deducible from the phase 4
`data- However,. should DP IV inhibitors become safe and
`thus chronically adminisfable pharmaceuticals, they have
`
`Table 2
`Clinioaf pbaso II/Itr progrmrs with DP fV inhibiton as mtidiabetics
`Aotive corryound coda Producer
`hhibitm type
`NVP DPP-728
`Novartis
`LAI.237
`Novartis
`MK-0431
`Merck and Co.
`BMS477l18
`Bristol-Myers Squibb
`P3U98
`kobiodrug
`P93l01=PSN930l
`Probiodrug/Prosidion
`GW-229A
`GlaxoSmithKline
`
`Covalently modi$ing
`Covalently modi$ing
`Non-covalent reversible
`Covaleartly modi$ing
`Non-covalat revelsible
`Non-covalent reversible
`Covalently modiffing
`
`lowering action of DP IV inhibiton is attributable to the pro-
`0ecdon ofpostprandial GLP-I degradation (Fig. 19). In our
`investigations with P3?98 and P93/01, we were also able to
`confirm mambiguously thaf the prctection of the second
`incretin, GIP, can be achieved in patients with DP IV inhi-
`bitors administered within tle activity range (Fig. 20) [49].
`Since the fint administration to man with P32l98,
`several drugs have been investigated in humans and
`clinical data published L72-741. In 93 diabetics, a dose
`of 100 mg NVP DPP 728 over 4 weeks led to a significant
`reduction in fasting glucose and postprandial glucose load
`as well as to a reduction in HbAlc by about 05% 1721, For
`instance, the 4-week administation of the DP IV inhibitor
`LAF 237 to 40 drug-naive patients resulted in the lowering
`of fasting blood glucose by about 18 mg/dl p3l. The same
`drug administered to pafients who received in parallel the
`generic drug metformin for up to I year caused a reduction
`of the long.term marker of glucose homeostasis IIbAlc by
`l,lYo us compared to a placebo-grcup receiving only
`metforrnin [74].
`
`8. Summary and conclusions
`
`The discovery of the regulatory fimction of the enzyme
`DP IV in glucose homeostasis in the middle ofthe 1990s has
`allowed the developmsnt of novel oral antidiabetics. In
`contrast to former diabetic pharmaceuticals, this therapy is
`causal and pleiotropic sincs it utilizes multiple functionq
`notably the small intestine homrones GLP-I and GIP.
`
`oEE
`
`t
`ot
`Er!
`T.rl
`€t
`
`0
`
`.r00 @
`q{ng (tt*n}
`
`3G}
`
`Fig. 19. Incroase in active GLP-I in 20 patienb by the action of 60 mg P3Z
`98 during a glucose toleremce test.
`
`MYLAN Ex. 1009, Page 9
`
`

`
`H.-U. Denuth et al. / Biochlmica et Biopltyslca Acta l75l (2005) 33-44
`
`considera.ble potential to revolutionize decisively cursnt
`diabetes thsntpy.
`
`Acknowledgements
`
`The authors gt:eatly acknowledge ths work and efforts by
`their co-workers and their students, specifically by RP.
`Pauly, J.A. Pospisilik, S.A. Hinkq L. Wagner, J.-U.
`Rahfeld, U. Heissr and T. Hoffrnann.
`
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