Ectopeptidases
`
`CD13/Aminopeptidase N and
`CD26/Dipeptidylpeptidase IV
`in Medicine and Biology
`
`Edited by
`
`Jiirgen Langner
`
`Martin Luther University
`
`Halle-Wmenberg, Germany
`
`and
`
`Siegfried Ansorge
`Otto von Guericke University
`Magdeburg. Germany
`
`Springer Science+Business Media, LLC
`
`Page 1 of 14
`
`Astraleneca Exhibit 2165
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`Mylan v. Astraleneca
`IPR2015-01340
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`AstraZeneca Exhibit 2165
`Mylan v. AstraZeneca
`IPR2015-01340
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`Page 1 of 14
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`ISBN 978-I-4615-0619-5 (cBook)
`ISBN 978-I-4613-S 161-0
`D01 10.1007/978-I-4615-0619-5
`
`02002 Springer Scieuoe+Business Media New York
`Originally published by Kluwer Academic/Plemnn Publishers, New York in 2002
`Softcover reprint of the hardcover lst edition 2002
`
`http1lwww.wkap.nll
`
`I0
`
`9
`
`8
`
`7
`
`6
`
`5 4 3
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`I
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`A C.l.P. record for this book is available from the Library of Congress
`
`All rights reserved
`
`No part of this book may be reproduced. stored in a retrieval system. or transmitted in any fonn
`or by any means. electronic. mechanical. photocopying. microfilming, recording. or otherwise.
`without written permission from the Publisher
`
`Page 2 of 14
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`

`

`Chapter 10
`
`Therapeutic Strategies Exploiting DP IV inhibition
`Target disease: Type 2 Diabetes.
`
`TORSTEN HOFFMANN and HANS-ULRICH DEMUTH
`
`Probiadrug Gmbfl. Weinbergweg 22. 06120 Halls (Scale). Germany
`
`1.
`
`INTRODUCTION
`
`Peptides containing proline residues have been shown to be resistant to
`proteolytic cleavage at
`their linkages. Many regulatory. neuronal and
`immune peptides contain proline residues detennining their peptide chain
`conformation and biological activity. Consequently, during evolution an
`exclusive set of proline—specific peptidases emerged capable of regulating
`the activity of such peptides. They are believed to be involved in peptide
`hormone processing and regulation. To this enzyme group belongs the
`exopeptidase dipeptidyl peptidase IV (DP IV, CD26. EC 3.4.14.5) and struc-
`turally similar DP IV-like enzymes (such as DP II) as well as other mecha-
`nistically but not structurally related enzymes (such as attractin) (chp. 7 this
`book by Abbott and Gorrell) Some of the natural substrates of DP IV-like
`enzymes turn out to be important regulators of vital mammalian functions.
`DP IV is involved in a number of different physiological regulation
`processes. On the one hand, the enzyme is a peptidase which can change the
`activity of a number of peptide hormones, neuropeptides and chemokines in
`a very specific manner (Mentlein 1999; Lambeir et al 2001: Zhang et al
`1999 and chp. 9 this book by De Meester et al), while on the other hand the
`DP IV protein molecule exerts protein-protein interactions, so mediating the
`regulation of intracellular signaling cascades independent of its peptidase
`activity (Hegen et al 1993; De Meester et al 1999).
`Frequently, the low endogenous concentration of the bioactive fomts of
`such homtones may be the cause of disorders. Hence, the pharmacological
`approach to inhibit the degradation of such endogenous peptides rather than
`
`Ectopeptidases. edited by Langner and Ansorge
`Kluwer AcademicJP|enum Publishers. New York. 2002
`
`259
`
`Page 3 of 14
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`

`

`260
`
`HOFFMANN AND DEMUTH
`
`Table 1. Target diseases for DP IV inhibition
`
`
`Target disease Effect of DP IV
`inhibitors
`suppression of
`chemoltine
`
`AIDS
`
`Development
`33;
`cell culture
`
`cleavage,
`suppression of
`HIV-interaction
`
`Comments Reference
`
`mechanism (Shioda el al I998;
`not fully
`Schols et al I998;
`understood Han; et al I997;
`Olttsulti er al 2000;
`Callebaut and
`
`Hovanessian. I996)
`
`Autoimmune
`diseases
`
`general immuno-
`suppressive effects
`
`cell culture and
`animal models
`
`high doses
`necessary
`
`(Reinhold at al 2000;
`Kubola er al I992).
`
`Rheumatoid
`Arthritis
`
`suppression of
`diseue
`
`animal models
`
`Multiple
`sclerosis
`
`suppression of
`EAE
`
`Psoriasis
`
`reduction of
`
`keratinocyte
`hyperproliferation
`
`Grail rejection suppression of
`grail rejection
`
`Wound
`healing
`
`promotion of
`wound healing
`
`suppression of
`NPY cleavage.
`decrease of anxiety
`
`effective in
`animal models
`
`(Tanaltaetal I997;
`Tanalta. era! I998)
`
`(Steinbrecher er al
`2000)
`
`(Novelli at al I996:
`Reinhold ct ai I998)
`
`(Komm er al l999a;
`Korom at al I997;
`Korom er al l999b)
`
`(Praget er al I994:
`Kohl et al 1989;
`Ghersi at al 2001)
`
`unpublished results
`
`phase II studies
`
`(Hofiinann at al 200i;
`Ahr at al 200!)
`
`inhibition of
`incretin cleavage,
`improvement of
`metabolic
`
`regulation
`
`DP IV and
`fibroblast
`activation
`protein
`(FAP)are
`involved
`
`inhibition of spread
`of rnetlslases
`inhibition of
`
`angiogenesis
`
`cell culture.
`animal models
`
`(AbdelGhany eta!
`l998;PineiroSanchez
`er al I997)
`
`Anxiety
`
`Type 2
`diabetes
`
`Cancer
`
`Page 4 of 14
`
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`

`

`I 0. Therapeutic Strategies Exploiting DP IV inhibition
`
`263
`
`(GLP-l) were identified and described in the eighties (Bell et al 1983;
`Schmidt eta! 1985).
`
`GIP and GLP-l, currently known as incretins, make up the endocrine
`component of the entero-insular (gut-pancreas) axis — a concept describing
`the neural, endocrine and substrate signaling pathways between the small
`intestine and the islets of Langerhans (Unger and Eisentraut, 1969).
`Together, the incretins are responsible for over 50 % of nutrient-stimulated
`insulin release, and thus represent the most important meal-related impetus
`for insulin secretion. In addition to stimulating insulin secretion, the incretins
`share
`a
`number of non-insulin mediated
`effects
`that
`contribute
`
`synergistically towards effective glucose homeostasis. Both peptides have
`been shown to inhibit gastric motility and secretion (Schirra et al 1996;
`Pederson and Brown, 1972) to promote [3-cell glucose competence (Huypens
`et al 2000), and to stimulate insulin transcription and biosynthesis (Fehmann
`and Habener 1992; Drucker et al 1987). In addition, GIP has been shown to
`play a significant role in the regulation of fat metabolism (Pederson, I994)
`while GLP-1 has been shown to stimulate [3-cell differentiation and growth
`(I-lui et al 2001) as well as to restore islet-cell glucose responsiveness
`(Zawa|ich et al 1993).
`The incretins were tested for treatment of T2D and it was found that the
`
`main advantage over the existing antidiabetic drugs is the strong glucose
`dependence of their insulinotropic action, thus preventing hypoglycemia.
`However, there are at least two disadvantages: as polypeptides they are not
`orally available; furthennore, it was found that the natural polypeptides have
`a very short half-life. Subsequently,
`it was shown by Mentlein that the
`bioactive fonn of GLP-1, GLP-11-3., as well as GIP both containing an
`alanine in penultimate position are in vitro substrates of purified human
`placenta DP IV (Mentlein et al 1993). Thus, the fast biodegradation in vivo
`could be at least in part due to DP IV activity (Kieffer et al 1995; Pauly et a1
`l996b; Deacon et.al, 1995).
`It was Pauly and colleagues who first postulated the link between the
`possible benefits of DP IV inhibition and glycemic control due to
`enhancement of the incretin effect (Figure 2) (Pauly et al 1996a and b). The
`hypothesis that DP [V inhibition could improve glucose tolerance was later
`shown to be correct in both Wistar rats and diabetic fatty Zucker rats (Pauly
`et al 1999; Pederson et al 1998) These findings have been corroborated by
`similar studies in mouse, rat and pig (Deacon et al 1998; Balkan et a1 1999;
`Ahren. et ai 2000).
`
`Page 5 of 14
`
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`

`

`264
`
`HOFFMANN AND DEMUTH
`
`
`
`Figure 2: MALDI-TOP-mas: spawn of GLP-l incubated with DP IV. Upper spectrum: DPlV
`caulyud degradation ofGLP-1 in presenceof20uM isoleucyl thiunlidinr-_ Lower spectrum:
`DPIV-eualyaeddegndnlonofGLP-I inabeenceofinhibitor.(adqatedtromPuilyctal
`I996)
`
`Based on the initial results applying the specific DP IV inhibitors to rats.
`they enhanced insulin secretion and improved glucose tolerance and such
`improvements were shown to be much more profound in the diabetic. "fatty"
`animals than in their lean littermates (Figure 3) (Pederson er al i998). As a
`consequence a drug discovery and candidate selection program was started.
`Balkan et al. confirmed these findings by using the DP IV inhibitor NVP-
`DPP728. and. importantly provided additional experimental evidence for the
`previously postulated and demonstrated stabilization of, and rise in. plasma
`active GLP-17.3. (GLP-la) afier inhibitor treatment (Balkan er al 1999).
`reflecting an appropriate dmg discovery program.
`
`
`
`Figure 3: Glucose tolerance improvement by application of I DP IV inhibitor to Wists: rls
`l0uunpriortoanmalglueuetolermcetea(l3/kgglucoaebyoralgavage)
`
`Page 6 of 14
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`

`

`10. Therapeutic Strategies Exploiting DP IV inhibition
`
`265
`
`2.2
`
`Selection of DP IV inhibitor
`
`Three therapeutic approaches for enhancement of postprandial incretin
`actions are possible: I) application of pharmaceutical
`incretin doses, 2)
`application of DP IV resistant incretin analogs or 3) application of DP IV-
`inhibitors. For incretin analogs, two structural peptide modifications can be
`anticipated: a) stabilization of the scissile peptide bond, b) modification of
`one or both of the two N-tenninal amino acids thereby preventing DP IV
`recognition. This has been reported to result in successful stabilization of
`both peptides, GIP and GLP-1,
`in the circulation (Deacon et al 1998;
`0'Harte er al 1999; Knudsen et al 2000). In case of GLP-1-analoga at least
`thm such developments are in clinical trials. Peptides acting as agonists at
`the GLP-l receptor, the so called exendins, were found in the Gila monster
`(fleloderma horridum) (Eng 1992). These exendins exhibit a prolonged half-
`life caused by the substitution of alanine to serine or glycine residues in their
`penultimate position, resulting in a diminished turnover by DP lV (Young et
`al 1999). However, the major drawback of all peptide-based approaches is
`their limited oral availability. Since DP IV inhibitors are small, such delivery
`and absorption problems could possibly be overcome. For DP IV, a large
`number of inhibitors representing different chemical classes has been
`described. For summaries see (Demuth l990; Augustyns er al 1999;
`Villhauer er al 2001). From a drug development perspective one could
`envisage a number of properties of a DP IV-inhibitor:
`
`- efficacy
`
`blocking the vast amount of DP IV activity in the
`circulation
`
`- reversibility
`- no reactive groups
`- stability/kinetics
`- oral availability
`
`no long lasting DP IV inhibition
`avoiding side reactions
`enabling a once daily or twice daily application
`maximizing patients compliance
`
`Based on initial data, amino acid pyrrolidides (Pyr) and thiazolidides
`(Thia) were considered. Such compounds were known to be effective as
`competitive DP [V inhibitors with a fast on- and a fast offset and K.-values
`in the low micromolar to nanomolar range (Table 2). They represent a
`minimal
`recognition structure necessary for
`inhibition of DP IV
`characterized by a free N-terrninus, the ct-carboxyl function of the peptide
`bond and a C-tenninal proline analog (Demuth and Heins, 1995; Villhauer er
`al 2001).
`
`Page 7 of 14
`
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`

`

`266
`
`HOFFMANN AND DEMUTH
`
`Inhibitory and transport properties of selected DP lV inhibitors: K;-values of
`Table 2.
`competitive inhibition of DP IV and transport data (effective tlux on flog isolated oocytes
`Daniel. I996) mediated by PepTl are listed
`
`
`Compound
`
`K.-value (pmol/l)
`
`Asn-Pyr
`Asn-This
`His-Pyr
`His-Thin
`lle-Pyr
`lie-This
`lle-Azetidin
`lle-Piperldin
`Pro-Pyr
`Pro-Thia
`Asp-Pyr
`Asp-This
`Glu-Pyr
`Glu-Thin
`L-allo-lle-Thia
`D-threo-lle-Thia
`D-allo-lle-Thia
`Val-Thia
`Glu-Pyr
`Glu-This
`Glu(Gly)-Thia
`G|u(GI_\Q3-Thia
`
`l2 1 0.50
`3.5 1 0.0.4!
`3.510.l6
`0.84 1 0.044
`0.21 10.016
`0.08 1 0.014
`3.! 1 0.23
`6.0 1 0.44
`4.! ‘1 0.23
`l.2 1 0.078
`I4 1 0.95
`2.9 1 0. l6
`2.2 1 0.25
`0.6! 1 0.070
`0.09 1 0.0l4
`no inhibition
`no inhibition
`0.08
`2.2 1 0.25
`0.61 1 0.070
`0.107 1 0.007
`0.l92 1 0.016
`
`Transport by PEPTI
`1“, gm)
`30 1 5
`83 1 ll
`715
`I2 1 6
`1416
`25 1 8
`not determined
`not determined
`not determined
`not determined
`0
`0
`9 1 6
`35 1 I3
`0
`0
`0
`I00
`9 1 6
`35 1 13
`0
`0
`
`Furthermore, amino acid pyrrolidides and thiazolidides harbor no
`chemical reactive group and they were shown to be stable for weeks in
`aqueous solutions.
`Kinetic characterization and uptake measurements (binding to the
`mucosal peptide transporter PepT-I, active flux measurements) revealed
`thiazolidides as compared to pyrrolidides to be more effective towards
`DP IV and also better transported (Table 2) (Daniel, 1996; Brandsch et al
`1999). lsoleucyl thiazolidine (lle-Thia) was found to be the compound with
`the best transport and inhibitory characteristics and was therefore chosen as
`the first candidate for further development.
`
`2.3
`
`Kinetics and pharmacology of isoleucyl thiazolidine
`in animals
`
`Pharmacokinetic behavior of lle-Thia was de tennined in mice, rats, dogs
`and monkeys, and confirmed the good oral availability expected from
`transport studies mentioned above. The substance exhibited a fast absorption
`
`Page 8 of 14
`
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`

`

`I 0. Therapeutic Strategies Exploiting DP II’ inhibition
`
`267
`
`reaching the maximal plasma concentrations within approximately I hour in
`all species (Figure 4). Elimination of the drug was found to be mainly by
`metabolization and renal excretion of the parent dmg and its metabolites.
`The main metabolite found was isoleucyl thiazolidine sulfoxide formed by
`the P450 enzyme Cyp3A4. Interestingly. species differences were found
`with respect to elimination half-life and metabolite fonnation. In dog the
`shortest half-life (~50 min) and the highest rate of metabolite formation was
`observed. Increasing elimination half-life of the compound was found in
`mice. monkeys and rats.
`
`
`
`Figure 4: Pharmacokinetics of lle-This (cu) and the main metabolite lle-This-sulfoxide (0) in
`rats (dose: 360 mglkg. oral)
`
`In an ADMB (administration. distribution. metabolism. excretion) study
`in rats using a "C-Ile labeled Ile-'I‘hia it was demonstrated that more than
`97 % of the applied radioactivity was recovered within 96 h. whereas the
`main part was recovered in the first 12 h after application. The remaining
`3 % shows a similar distribution over all organs and tissues.
`Intensive pharmacological
`investigations of DP IV inhibitors were
`performed in diabetic rat models and in monkeys. In the first studies it could
`be shown that the fat Zucker rat (fa/fa rat). an animal model of type 2
`diabetes. shows a more pronounced glucose tolerance improvement as
`compared with nomial
`rats.
`In single dose escalation studies of oral
`application of Ile-Thia a dose dependent decrease of plasma DP IV activity
`was found. resulting in an increase of early phase insulin and an enhanced
`glucose tolerance. For rats an optimal dosing regimen applying the drug 5 -
`30 min before starting an oral glucose tolerance test (OG'l'I') for the
`enhancement of glucose tolerance was found.
`In a study designed to relate inhibitory efficacy and transport efiiciency
`to in viva potency for glucose tolerance improvement. three structurally
`
`Page 9 of 14
`
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`
`

`

`10. Therapeutic Strategies Exgplairing DP IV inhibition
`
`271
`
`DP IV activity (Figure 9). where t...,of DP IV activity is exactly at the t... of
`drug concentration. Thi
`clearly confimis
`the fast and reversible
`interconnection of the drug with its target enzyme DP IV.
`
`
`
`Figure 9: Time course of plasma DP IV activity alter application of different om doses of [le-
`Thia. (study I. n = 9)
`
`As a first proof of principle in man we measured in healthy volunteers a
`dose dependent impact on stabilization of active GLP-l up to a dose of
`60mg. Higher doses seem to have no additional effects on GLP-1
`stabilization. indicating that the DP IV activity in vivo is sufficiently blocked
`by the drug. Stabilization of GLP-l could also be shown in type 2 diabetes
`patients whereas the total concentrations of GLP-1 (active and inactive) were
`similarly effected as in the volunteers. resulting in an up to seven-fold
`increase of peak concentration of active GLP-l in comparison to untreated
`control (Figure I0).
`
`
`
`Figure I0: Stabilization of active OLP-I in plains of diabetic patient: (study 3. M24) by
`oral applicationof60m3lle-Thia lsminpriorutonlzlucoaetolenneeteat
`
`Page 10 of 14
`
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`

`

`272
`
`H0!-‘FMANN AND DEMUTH
`
`increase of insulin levels could be seen
`In patients, a subsequent
`(Figure ll) which was also reflected in an increase of proinsulin and
`C-peptide levels. No increase of insulin was observed in healthy subjects.
`
`250300
`
`than (min)
`
`Figure ll: Enhancanent of insulin response afla OGTT by a single oral dose (60 mg) of ile-
`Thia in diabetes type ll patients (n = 24)
`
`the drug application leads in man to an enhanced glucose
`Finally,
`tolerance. For healthy subjects a dose dependent effect of the inhibitor was
`discovered as known for DP IV resistant GLP-1 analogs (Deacon er al 1998;
`Knudsen er al 2000). but the enhancement was more pronounced in the
`patients (Hoffmann et al 2001).
`Because of the pleiotropic action of GLP-1 and GIP seen after chronic
`treatment with DP IV inhibitors in animals on insulin release, beta-cell
`growth, sensitization of the target organs (liver, muscle) and effects on lipid
`metabolism, one might speculate about
`the consequences of chronic
`treatment in man (see below).
`
`2.5
`
`DP IV inhibitors in clinical investigations
`
`The use of DP IV inhibitors to afl’ect the entero-insular axis has attracted
`
`much recent interest as a potential therapeutic strategy in the treatment of
`diabetes. Several
`recent studies have established the efficacy of such
`inhibitors on an acute scale (Balkan et al 1999; Pauly er al 1996; Pederson et
`al 1998).
`There have been several inhibitor classes developed and biochemically
`tested (Dernuth, 1990; Augustyns er al 1999; Villhauer er al 2001). So far,
`only lie-Thia (P32/98 = Isoleucyl thiazolidine hemifumarate) a reversible,
`competitive inhibitor and the slow-tight binding DP IV inhibitor NV?-
`DPP728 (l-[[[2-[(5-Cyanopyridin-2-yl)arnino]ethyl]amino]acetyl]-2-cyano-
`(S)-pyrrolidine) have been tested in patits (Ahren et al 2001) (Figure 12).
`
`Page 11 of 14
`
`150
`
`200
`
`100
`
`E 3 i
`
`Page 11 of 14
`
`

`

`I0. Therapeutic Strategies Exploiting DP IV inhibition
`
`273
`
`Using P32/98 in initial clinical studies the prove of principle for the
`application of DP IV inhibitors as glucose lowering agents could be achieved
`(Hoffmann et al 2001). This was confirmed by recently published results of
`a 4-week placebo-controlled
`double-blind subchronic application to 93
`diabetic patients of NVP-DPP728, a DP IV inhibitor of a different structural
`class and mode of action (Ahren et al 2001). In the study a significant
`reduction in fasting glucose and maximum prandial peak glucose excursion
`was achieved. As already observed in the animal studies, an insulin saving
`effect can be interpreted as a break of insulin resistance. Moreover, the
`important clinical parameter HbA;¢ (used to monitor long-term glucose
`burden in the circulation) was reduced by 0.5 %.
`In summary,
`these results of initial hmnan studies demonstrate that
`modulation of circulating DP IV activity is a feasible approach in the
`management of type 2 diabetes.
`In contrast
`to all known antidiabetic
`medications
`it makes use of the pleiotropic action of endogenous
`polypeptide hormones which control energy homeostasis at different tissues
`and organs at the same time.
`
`
`
`Figure I2: DP IV-inhibitors tested in volunteers and T2D-patients
`
`3.
`
`SUMMARY
`
`The polypeptide substrates of DP IV, GI!’ and GLP-1, also known as
`incretins, are the endocrine component of the entero-insular (gut-pancreas)
`axis - a concept describing the neural, endocrine and substrate signaling
`pathways between the small intestine and the islets of Langerhans. Together,
`GIP and GLP-1 comprise the hormonal component of the entero-insular axis
`and account for approximately 50 % of nutrient-stimulated insulin secretion.
`In addition to stimulating insulin secretion, the incretins share a number of
`non-insulin mediated effects that contribute synergistically towards effective
`glucose homeostasis. In recent years, it has been demonstrated by ourselves
`and others that the dipeptidyl peptidase IV rapidly cleaves the N-terminal
`dipeptides from these incretins both in vitro and in viva. yielding truncated.
`biologically inactive GIPM and GLP-1,“. The physiological role played by
`
`Page 12 of 14
`
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`
`

`

`274
`
`HOFFMANN AND DEMUTH
`
`DP IV in the regulation of incretin activity, and thus in the regulation of
`blood glucose, was established by studies showing enhanced insulin
`secretion and improved glucose tolerance resulting from the administration
`of specific DP IV inhibitors.
`incretin analogues or incretin receptor
`in contrast to DP IV-resistant
`agonists such as exendin, the low-molecular weight DP IV inhibitors are
`readily orally available and provide an alternate approach to the classical
`peptide hormone therapy.
`However, dipeptidyl peptidase IV and the structurally and functionally
`related DP lV-like enzymes are involved also in the regulation of the
`mammalian immune functions. Hence, long-tenn treatment of T2D patients
`with inhibitors of these enzymes might bear the risk of unwanted immune
`modulation. Consequently, there is still a longer way to go until the first
`DP IV inhibitors applied to man have proven there safety in long-tenn
`applications to diabetics. Thus, designing DP IV inhibitors for chronic
`treatment of hyperglycemia need to orientate on pharmacokinetic objectives.
`
`ACKNOWLEDGMENTS
`
`We would like to thank all the colleagues who have helped in many
`invaluable ways in performing the studies reviewed here, in particular H.
`Daniel, E.-J. Freyse, K. Glund, S. Hinke, S. Kruber, A. Meyer, U. Heiser, C.
`H. S. Mclntosh, R. A. Pederson , A. Pospisilik, D. Schlenzig.
`Some of the results presented here were supported in part by grants from
`the BMBF Gennany, grant # 0312302, the Arbeitsgemeinschaft industrieller
`Forschungsvereinigungen “Otto von Guerieke” e.V., Berlin, grant # 28/00,
`and by LFI Sachsen-Anhalt, Magdeburg, Gennany, grant # 9704/00l l5.
`
`REFERENCES
`
`AbdelGhany, M.. Cheng. H. C., Levine, R. A., and Pauli, B. U., I998, Truncated dipeptidyl
`peptidase IV is a potent anti-adhesion and anti- rnetastuis peptide for rat brent cancer
`cells lnvas. Metast. I8: 35-43.
`
`Ahren. B... Simonsson, N.E.. Efendic, S.. Eriksson, 1., Venholm, P.B., Jannson, P.A.. Landin-
`Olsson, M.. Torgeirason, H., Rask, E., Sandquist. M., Dickinson, S.. and Holmes. D..
`200l, inhibition of DPPIV by NVP DPP728 improves metabolic control over a 4 week
`period in type 2 diabetes. Diabetes 50 Supp 2:, M04. Abstract.
`Ahren, B., Holst, .l.J., Martensson, H., and Balkan, B., 2000, improved glucose tolerance and
`insulin secretion by inhibition of dipeptidyl peptidase IV in mice. Eur. J. Pharmacol. 404:
`239-245.
`
`Zhang,X. M..Bollaert, W.,Larnbeir,A.M.,
`Austlslylls. K., Bal, G.. Thonus. G.. Belyaev. A.,
`.
`.1 2.-_u-_-1».
`
`I999 The un'ue -4;-in ‘cs ot'di'd I-
`
`Page 13 of 14
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`

`10. Therapeutic Strategies Exploiting DP IV inhibition
`
`275
`
`peptidue IV (DPP IVlCD26) and the therapeutic potential of DPI’ IV inhibitors. Curr.
`Medicinal. Chem. 6: 3 I I-327.
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