Page 1 of 9
`
`Astraleneca Exhibit 2078
`Mylan v. Astraleneca
`IPR2015-01340
`
`AstraZeneca Exhibit 2078
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 9
`
`

`
`Attention before copying!
`Do you photocopy articles from this periodical? If so, have you made certain that you are not violating the legal copyright regulations and
`making yourself liable to prosecution?
`According to copyright law it is only permissible to make a few copies of individual articles for personal use. Reproduction of articles for
`commercial use by an industrial enterprise is subject to charge. Detailed information can be obtained free of charge from the VG Wissen(cid:173)
`schaft GmbH, "Copyright"', GroBer Hirschgraben 17-21, D-6000 Frankfurt/Main, this company being responsible for collection of copying
`fees.
`
`Copying in the USA
`Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Walter de
`Gruyter & Co. ·Berlin· New York for libraries and other users registered with the Copyright Clearance Center (CCC) Transactional Repor(cid:173)
`ting Service, provided that the base fee of$ 02.00 per copy is paid directly to CCC, 21 Congress St., Salem, MA01970.
`0177-3593/91$02.00.
`
`Title No. 3109006757
`Copyright© 1991 by Walter de Gruyter & Co., Berlin
`All rights reserved, including those of translations into foreign languages. No part of this journal may be reproduced in any form- by photo(cid:173)
`print, microfilm or any other means or transmitted or translated into a machine language without written permission from the publisher.
`The quotation of registered names, trade names, trade marks, etc. in this journal does not imply, even in the absence of a specific statement,
`that such names are exempt from laws and regulations protecting trade marks, etc. and therefore free for general use.
`Printed in Germany-Typesetting: Blank, Satzstudio GmbH, Mi.inchen: Printing: Grafik +Druck, Mi.inchen: Binding: Steinberger GmbH,
`Mi.inchen;Advertizing: MediendienstWolfgangFrieben,Aufdem Reeg8a, D-5307Wachtberg,Tel. (0228) 3243 74.Telefax (0228) 32 7204.
`
`ISSN: 0177-3593
`
`Page 2 of 9
`
`

`
`Bioi. Chern. Hoppe-Seyler
`Vol. 372, pp. 305-311, May 1991
`
`Dipeptidyl Peptidase IV in the Immune System
`Effects of Specific Enzyme Inhibitors on Activity of Dipeptidyl Peptidase IV and
`Proliferation of Human Lymphocytes
`
`, Ilona BoRNb, Hans-Ulrich DEMUTHb, Jtirgen FAUSTb, Klaus NEUBERTb,
`Ekkehard ScHON3
`Torsten STEINMETZERb, Alfred BARTHb and Siegfried ANSORGE3
`
`a Medizinische Akademie Magdeburg, Klinik ftir Innere Medizin, Forschungsabteilung Experimentelle Immunologie and
`b Universitat Halle-Wittenberg, Sektion Biowissenschaften, Biotechnikum
`
`(Received 2 February 1990/11 March 1991)
`
`Summary: Dipeptidyl peptidase IV(DP IV) is a mem(cid:173)
`brane peptidase playing a significant role in the pro(cid:173)
`cess of activation and proliferation of human thymus(cid:173)
`derived lymphocytes. This conclusion is drawn from
`(1) the induction of this enzyme on mitogen-acti-
`vated T lymphocytes (cf. Schon, E. &Ansorge, S.
`(1990) Biol. Chern. Hoppe-Seyler 371, 699-705)
`and
`(2) the impairment of different functions of activated
`T cells in the presence of specific inhibitors and
`antibodies against DP IV (Schon, E. & al. (1987)
`Eur. J. Immunol17, 1821-1826).
`This paper is aimed at testing new active site-specific
`peptide inhibitors for their efficiency as inhibitors of
`lymphocyte DP IV and DNA synthesis of mitogen(cid:173)
`stimulated lymphocytes.
`
`These inhibitors comprise (i) diacylhydroxylamine
`derivatives of Xaa-Pro or Xaa-Ala peptides, (ii) dif(cid:173)
`ferent oligopeptides with N-terminal Xaa-Pro-se(cid:173)
`quences, and (iii) amino-acid amides of the pyr(cid:173)
`rolidide and the thiazolidide type.
`The thiazolidides of s-( 4-nitrobenzyloxycarbonyl)-L(cid:173)
`lysine and of L-isoleucine as well as Ala-Pro-nitro(cid:173)
`benzoylhydroxylamine are the most effective in(cid:173)
`hibitors in both test systems, yielding half-maximal in(cid:173)
`hibitory concentrations in the micromolar range. Cell
`viability was not impaired in this effective concentra(cid:173)
`tion range.
`Other inhibitors of DP IV are one to two orders of
`magnitude less efficient in the suppression of lympho(cid:173)
`cyte proliferation.
`
`Dipeptidylpeptidase IV im Immunsystem: Einfluf3 von spezifischen Inhibitoren auf die Aktivitiit der Dipeptidyl(cid:173)
`peptidase IV und die Proliferation humaner Lymphozyten
`
`Zusammenfassung: Das Membranenzym Dipeptidyl(cid:173)
`peptidase IV (DPIV) stellt ein wichtiges regulatori(cid:173)
`sches Element der Thymus-abhangigen Lymphozy(cid:173)
`ten dar. Das laBt sich ableiten a us der Induzierbarkeit
`des Enzyms nach mitogener Stimulation und aus der
`Hemmbarkeit einer Reihe von funktionellen Aktivi(cid:173)
`taten der aktivierten T-Zellen in Gegenwart spezifi(cid:173)
`scher DP IV-Inhibitoren.
`
`Neuentwickelte active-site-spezifische Inhibitoren
`wurden hinsichtlich ihrer Fahigkeit getestet, die lym(cid:173)
`phozytare DPIV-Aktivitat und die Proliferation Mi(cid:173)
`togen-stimulierter Lymphozyten zu hemmen. Dabei
`kamen folgende Gruppen von Inhibitoren zum Ein(cid:173)
`satz: (i) Diacylhydroxylamin-Derivate von Xaa-Pro(cid:173)
`und Xaa-Ala-Peptiden, (ii) verschiedene Oligopep(cid:173)
`tide mit einer N-terminalen Xaa-Pro-Sequenz und
`
`Enzyme:
`Dipeptidyl peptidase IV, dipeptidyl-peptide hydrolase (EC 3.4.14.5)
`Abbreviations:
`ACD, anticoagulant citrate dextrose; Cap, hexanoic acid or hexanoyl, DIFP, diisopropyl fluorophosphate; DP IV, Dipeptidyl
`peptidase IV, CD 26; [3H]dThd, tritiated thymidine; IL, interleukin; MNC, mononuclear cell(s) from human blood; PHA,
`phytohemagglutinin A, lectin from Phaseolus vulgaris; pNA, 4-nitroanilide; Tcell, thymus-derived lymphocyte; Z, benzyloxycar(cid:173)
`bonyl.
`
`Page 3 of 9
`
`

`
`306
`
`E. Schon, I. Born, H.-U. Demuth, J. Faust, K. Neubert, T. Steinmetzer, A. Barth and S. Ansorge
`
`Vol. 372 (1991)
`
`(iii) Aminosaureamide vom Pyrrolidid- und vomThia(cid:173)
`zolidid-Typ.
`Die Thiazolidide von c:-( 4-Nitrobenzyloxycarbonyl)(cid:173)
`L-Lysin und von L-Isoleucin
`sowie Ala-Pro(cid:173)
`Nitrobenzoylhydroxylamin erwiesen sich als starkste
`Inhibitoren in bezug auf die Hemmung der DP IV-Ak(cid:173)
`tivitat und der Proliferation humaner Lymphozyten
`
`mit halbmaximalen Hemmkonzentrationen im mi(cid:173)
`kromolaren Bereich. Unspezifische Effekte auf die
`VitaliHit der Lymphozyten konnten dabei ausge(cid:173)
`schlossen werden.
`Die wirksamen Hemmkonzentrationen der Inhibito(cid:173)
`ren unterscheiden sich zum Teil urn GroBenordnun(cid:173)
`gen in den beiden Testsystemen.
`
`Key terms: Dipeptidyl peptidase IV, Tlymphocyte, lymphocyte proliferation, active site inhibitor, peptidase.
`
`Dipeptidyl peptidase IV (EC 3.4.14.5) was described
`for the first time by Hopsu-Havu et al. in 1966[1l. This
`membrane peptidase has been found to occur in a
`variety of human organs and tissues[2·3l. As a typical
`ectoenzyme of the plasma membrane[4J, DP IV is ex(cid:173)
`pressed in high density in membrane regions organi(cid:173)
`zed as microvilli, such as brush border regions of the
`small intestinum, the kidney tubulus and the liver si(cid:173)
`nusoidal membrane[s]. In the human immune system,
`the enzyme is expressed almost exclusively in the thy(cid:173)
`•7l.
`mus-derived lymphocytes[6
`Despite many investigations on the significance of
`this enzyme as a marker in lymphoproliferative disea(cid:173)
`ses and in patients with disturbed immune func(cid:173)
`tions[8-12l, the role of this enzyme in the physiology of
`the immune system is not yet understood. In 1985 we
`were the first to show that in the presence of specific
`inhibitors of DP IV the proliferation of human lym(cid:173)
`phocytes stimulated by mitogenic lectins and alloanti(cid:173)
`gens is suppressed in a dose-dependent manner[13l.
`
`Moreover, DP IV activity was found to be raised after
`mitogenic stimulation in vitro[14.15l. This induction of
`enzyme activity with a peak in the G 1 phase is paralle(cid:173)
`led by an increasing density of epitopes reacting with
`antibodies against this enzyme[16l. In 1988, DP IV was
`classified as CD 26 under the entry lymphocyte activa(cid:173)
`tion markers at the 4th Workshop on Leukocyte Dif(cid:173)
`ferentiation Antigens[17l.
`Recently, we found that various functions of T lym(cid:173)
`phocytes, such as production of cytokines, interleu(cid:173)
`kin-2-augmented proliferation and as helpers of
`B lymphocytes, are dependent on the activity of this
`membrane peptidase, either directly or indirectly[18.19l.
`
`These investigations on the role of DP IV in the phy(cid:173)
`siology of lymphocytes were backed by the develop(cid:173)
`ment of specific inhibitors serving as key tools in our
`studies. These active site inhibitors are potential im(cid:173)
`munomodulatory drugs. To be selected for applica(cid:173)
`tion in animal models, these compounds have to meet
`several criteria in vitro. The present paper describes
`our attempt to select DP IV inhibitors on the basis of
`
`their efficiency at low doses, sufficient stability under
`cell-culture conditions and lack of cytotoxic side ef(cid:173)
`fects in vitro. A series of diacyl hydroxylamines of
`Xaa-Pro- and Xaa-Ala-peptides, oligopeptides with
`N-terminal Xaa-Pro as well as amino acyl pyrrolidi(cid:173)
`des and thiazolidides have been tested for their ability
`to inhibit lymphocyte DP IV activity and DNA synthe(cid:173)
`sis of mitogen-stimulated lymphocytes. Aminoacyl
`amides with heterocyclic amide structures were found
`to be the most efficient inhibitors oflymphocyte proli- ·
`feration in vitro.
`
`Materials and Methods
`
`1) Cell preparation
`Mononuclear cells of human venous blood drawn from healthy vo(cid:173)
`lunteers were separated using Boyum's method of density gradient
`centrifugation with minor modifications as described earlier[ISJ. For
`cell cultures, only heparinized blood samples were used as a lym(cid:173)
`phocyte source. For biochemical tests, ACD-treated blood dona(cid:173)
`tions obtained as buffy coats from the blood bank served as a lym(cid:173)
`phocyte source.
`
`2) Test system D P IV activity
`Enzyme source: MNC at a cell density of approximately 5 x 107 lml
`were agitated for 30 min at 4 oc with 0.1% Triton X-100. The cell ly(cid:173)
`sate was centrifuged at 1.7 x 104 x g for 30 min. The supernatant
`was used for determination ofDP IV activity in lymphocyte lysates.
`Substrate: Gly-Pro-pNA, final concentration lmM.
`Buffer: O.lM KH2P04/K2HP04 buffer pH 7.6.
`Assay: 0.025 ml enzyme source ~as preincubated with an equal vo(cid:173)
`lume of inhibitor for 60 min at room temperature. After adding
`0.05 ml substrate solution, the reaction was allowed to proceed at
`37 oc for 120 min and stopped by adding 0.4 ml of 1M sodium ace(cid:173)
`tate buffer pH 4.5. The absorbance of liberated 4-nitroaniline was
`read in a spectrophotometer at 390 nm. Using this approach, less
`than 2% of Gly-Pro-pNA-degrading activity in lymphocytes can be
`attributed to enzymes other than DP IV[20l.
`
`3) Test system lymphocyte proliferation
`The lymphocyte culture system used to evaluate the effect of DP IV
`inhibitors on mitogen-induced lymphocyte proliferation was des(cid:173)
`cribed previously in detail[ISJ and is only summarized in the follo(cid:173)
`wing.
`Culture medium: based on RPMI 1640, supplemented as descri(cid:173)
`bed[18l, and with the addition of 10% pooled inactivated human
`serum.
`
`Page 4 of 9
`
`

`
`Vol. 372 (1991)
`
`Action of Dipeptidyl Peptidase IV Inhibitors on Human Lymphocytes
`
`307
`
`Culture vessels: polystyrene 96-well, flat-bottom microtiter plates;
`0.2-ml cultures containing 2 x 105 MNC, were prepared in quadru(cid:173)
`plicate for each condition.
`Mitogen: phythaemagglutininA(Wellcome, Beckenham, Kent) at
`final concentrations of 0.3 and 0.03%.
`Cell cultures were kept at 37 oc in a humidified 5% C02/ air atmo(cid:173)
`sphere. Total culture time was 96 h, including the incorporation of
`37 kBq Cl~-tCi) [3H]thymidine per well for the last 6 h. Cell suspen(cid:173)
`sion (0.1 ml) was harvested on filter paper and prepared for liquid
`scintillation counting in a beta counter. DP IV inhibitors were ad(cid:173)
`ded at the beginning of the culture in concentrations ranging from
`0.5~-tM to 0.5mM.
`
`4) Test system cell viability
`To exclude unspecific toxic side effects of the inhibitors on human
`lymphocytes, the viability of cells cultured in the presence and ab(cid:173)
`sence of mitogen under conditions as described above was tested
`after 0, 2, 24, 48, 72 and 96 h of culture. In brief, 0.01 ml cell suspen(cid:173)
`sion was put on glass slides and incubated with 0.01 ml 0.01% ethi(cid:173)
`dium bromide for 30 s followed by addition of 0.01 ml 0.01% acri(cid:173)
`dine orange solution. The cell viability was evaluated using a fluor(cid:173)
`escence microscope, where viable cells show a green nucleus and
`dead cells appear red.
`
`5) Synthesis of amino-acid and peptide derivatives
`The compounds listed in the table were synthesized by known stan(cid:173)
`dard methods for peptide synthesis. Phe-Pro-NHO-CO-C6H 4-
`-N02 and Ala-Pro-NHO-CO-C6H4-N02 were prepared according
`to the method described[21l.
`For the synthesis of the amino-acid pyrrolidides and thiazolidi(cid:173)
`des[22l the corresponding Boc-amino-acid was coupled with pyrroli(cid:173)
`dine and thiazolidine, respectively, using the mixed anhydride pro(cid:173)
`cedure. After removal of the Boc-group with hydrogen chloride in
`acetic acid (in case of the Boc-amino-acid thiazolidides in presence
`of thioanisole under nitrogen atmosphere) the resulting amino(cid:173)
`acid derivatives were isolated as hydrochlorides.
`The dipeptides Ile-Pro, Lys [Z(N02)]-Pro and Lys(Cap)-Pro were
`obtained by coupling of the pentafluorophenyl or N -hydroxysucci(cid:173)
`nimide ester of the Bee-protected N-terminal amino-acids or
`amino-acid derivatives with L-proline followed by removal of the
`Boc-group with hydrogen chloride in acetic acid.
`In the case of the tripeptides diprotin A (Ile-Pro-Ile) and diprotin B
`(Val-Pro-Leu) the Bee-protected N-terminal dipeptides (synthesi(cid:173)
`zed as described above) were coupled with L-isoleucin-tert-butyl
`ester and L-leucin-tert-butyl ester, respectively, by the method of
`mixed anhydrides followed by simultaneous removal of the protec(cid:173)
`ting groups with hydrogen chloride in acetic acid. DiprotinAand di(cid:173)
`protin B were recrystallized from ethanoVether.
`j3-Casomorphin-(1-5)-peptide (Tyr-Pro-Phe-Pro-Gly) and mor(cid:173)
`phiceptin (Tyr-Pro-Phe-Pro-NH2) were synthesized by stepwise
`elongation from the C-terminal end as described previously[23l.
`For the synthesis of the lymphotoxin-(1-5)-peptide (Leu-Pro-Gly(cid:173)
`-Val-Gly) a (2+3)-segment condensation was successful. Boc-Leu-
`-Pro was coupled with Gly-Val-Gly-OMe using the mixed anhy-
`dride procedure. After alkaline hydrolysis and removal of the Bee(cid:173)
`group with hydrogen chloride in acetic acid the pentapeptide was
`purified by crystallization from methanoVether. The homogeneity
`of the amino-acid derivatives and the peptides, usually characteri(cid:173)
`zed as hydrochlorides, was checked by TLC and HPLC (purity
`>98%).
`
`Results and Discussion
`
`The starting point for the present study was the evi(cid:173)
`dence that dipeptidyl peptidase IV is involved in the
`
`physiology ofT lymphocytes. This was revealed by
`previous studies showing suppression of mitogen-or
`alloantigen-induced proliferation, lymphokine pro(cid:173)
`duction and B cell differentiation in cultures of
`human MNC in the presence of specific inhibitors or
`antibodies to DP IV[l3JSJ9l. Thus, DP IV inhibitors are
`regarded as immunomodulating substances with po(cid:173)
`tential therapeutical significance. Before any in vivo
`application, the following in vitro criteria should be
`met by these compounds: (i) high selectivity with re(cid:173)
`gard to the enzyme, (ii) high efficiency at low concen(cid:173)
`trations, (iii) no cytotoxic action in the effective con(cid:173)
`centration range, and (iv) sufficient stability under
`cell culture conditions.
`From these considerations a test hierarchy was de(cid:173)
`veloped to answer the following questions.
`(1) Is the compound to be tested specific and effec(cid:173)
`tive against lymphocyte DP IV activity?
`(2) Is the compound toxic for lymphocytes in vitro?
`(3) How does the compound influence the mitogen(cid:173)
`induced lymphocyte proliferation?
`Three groups of DP IV inhibitors were screened with
`regard to the above questions:
`diacylhydroxylamine derivatives of Xaa-Pro(cid:173)
`(i)
`and Xaa-Ala-peptides;
`oligopeptides comprising Xaa-Pro and substi(cid:173)
`tuted derivatives thereof, the microbiological
`tripeptides diprotinAand B, theN-terminal pen(cid:173)
`,8-casomorphine,
`tapeptides of lymphotoxin,
`morphiceptin;
`amino acyl amides of the pyrrolidide and the
`thiazolidide type.
`
`(iii)
`
`(ii)
`
`Effects of DP IV inhibitors on lymphocyte DP IV
`activity
`
`The table summarizes the effect of inhibitors toward
`DP IV activity in lymphocyte extracts. The order of
`the compounds corresponds to their inhibitory effi(cid:173)
`ciency in the biochemical test system. Fig. 1 shows a
`selection of three typical inhibition curves obtained in
`these studies.
`a
`represent
`Peptidyl-N, 0-diacylhydroxylamines
`group of inhibitors of serine and cysteine proteinases
`that act in an irreversible manner[21
`271. The Phe(cid:173)
`24
`•
`-
`Pro- and Ala-Pro-derivatives were equally effective
`against lymphocyte DP IV, whereas the Ala-Ala-de(cid:173)
`rivative (not included in the table) expectedly showed
`only minor effects on DP IV with Gly-Pro-pNA as a
`substrate.
`The
`N-(Phe-Pro)-0-(nitrobenzoyl)(cid:173)
`hydroxylamine, however, was strongly toxic for
`human lymphocytes at concentrations of 10p,M and
`was therefore excluded from cell culture experi(cid:173)
`ments.
`
`Page 5 of 9
`
`

`
`308
`
`E. Schon, I. Born, H.-U. Demuth, J. Faust, K. Neubert, T. Steinmetzer, A. Barth andS. Ansorge
`
`Vol. 372 (1991)
`
`Table. Effect of compounds on DP IV activity and mitogen-induced
`DNA synthesis of human lymphocytes.
`IC50 values represent the concentration of a compound causing
`50% inhibition in the test system. IC50 values were interpolated
`from the dose response curves; data represent arithmetic means
`± standard error of means from 4 to 5 separate experiments. In
`cases where less than 50% inhibition was observed, the residual ac(cid:173)
`tivity at the highest test concentration is indicated (last column).
`For further details see Methods.
`
`Compound
`
`DPIV
`activity
`ICso
`
`ICso
`
`DNA synthesis
`
`Residual
`activity at
`5 X 10-4
`moVl
`[% ofcontr.]
`
`[JLmoVl]
`
`[JLmoVl]
`
`2.0± 0.2
`
`2.7± 0.3
`
`2.8± 0.2
`6.0± 0.3
`
`8.0± 0.8
`
`10 ± 2
`1
`17 ±
`
`20 ±
`
`2
`
`30 ± 3
`
`38 ± 3
`4
`54 ±
`87 ± 6
`
`320 ± 60
`
`1 Lys[Z(N02)]-
`pyrrolidide
`2 Lys[Z(N02)]-
`thiazolidide
`3 Ile-thiazolidide
`4 Val-pyrrolidide
`5 Ile-Pro-Ile
`(diprotinA)
`6 DIFP
`7 Ile-pyrrolidide
`8 Phe-Pro-NHO-CO-
`-C6H4-NOz
`9 Ala-Pro-NHO-CO-
`-C6H4-NOz
`10 Leu-pyrrolidide
`11 Phe-pyrrolidide
`12 Ala-thiazolidide
`13 Leu-Pro-Gly-Val-Gly
`[lymphotoxin-(1-5)-
`peptide]
`14 Tyr-Pro-Phe-Pro-NH2 500 ± 60
`(morphiceptin) .
`15 Tyr-Pro-Phe-Pro-Gly
`[{3-casomorphin-
`(1-5)-peptide]
`16 Ile-Pro
`
`530 ± 70
`
`620 ± 110
`
`17 Lys[Z(N02)]-Pro
`18 Lys[Cap]-Pro
`19 Val-Pro-Leu
`( diprotin B)
`20 Ala-Ala
`
`700 ± 60
`
`820 ± 50
`
`920 ± 160
`f
`
`a
`
`8.0± 1.5
`
`47 ± 4
`b
`
`b
`
`c
`400 ± 190
`a
`
`50 ± 23d
`370 ± 50
`b
`260 ± 48
`320 ± 150
`
`b
`
`e
`
`e
`
`e
`200 ± 90d
`250 ± 40
`e
`
`e
`
`b
`
`54± 14
`
`69±20
`
`61 ± 11
`
`61 ± 18
`
`69 ± 8
`
`a Toxic to lymphocyte cell cultures.
`b Less than 50% inhibition at highest concentration tested
`(5 X 10-4M).
`c Not applicable in cell culture.
`ct Concanavalin A used as mitogen (10 f.Lglml).
`e No effect (evaluation limit 80% of the DNA synthesis rate of
`the control culture) up to a concentration of 500f.LM.
`f No effect up to lmM.
`
`In the group of oligopeptides, the tripeptide Ile-Pro(cid:173)
`-Ile (diprotin A) was the most effective inhibitor of
`lymphocyte DP IV activity with IC50 values in the
`micromolar range. Tetrapeptides and pentapeptides
`with N-terminal Xaa-Pro-sequences comprising parts
`of or full length biologically active peptides like lym(cid:173)
`photoxin-(1-5)-peptide, ,B-casomorphin-(1-5)-pep(cid:173)
`tide and morphiceptin were only weak competetive
`
`(0)
`
`-7
`
`-5
`-6
`lg[l] [mol//]
`
`-4
`
`Fig. 1. Effect of selected compounds on the DP IV activity of
`human lymphocytes.
`Each point on the diagram represents the arithmetic mean
`± standard error of mean from 4 separate experiments. _8,
`Compd. 2, Lys[Z(N02)]-thiazolidide; D, Compd. 3, Ile-thia(cid:173)
`zolidide; 0, Compd. 11, Phe-pyrrolidide. For further details,
`see Methods section.
`
`inhibitors, being effective in a concentration range
`about two orders of magnitude higher than the effec(cid:173)
`tive concentration range of diprotin A. A series of
`other dipeptides not listed in the table did not inhibit
`DP IV at concentrations up to 1mM.
`A new strategy for developing highly specific DP IV
`inhibitors based on a detailed knowledge of the
`catalytic mechanism of dipeptidyl peptidase IV led us
`to the synthesis of amino acyl-pyrrolidides and
`thiazolidides[23'25l. Three of
`these
`inhibitors,
`Lys[Z(N02)]-pyrrolidide, Lys[Z(N02)]-thiazolidide
`and Ile-thiazolidide, are listed at the top of the table
`with halfmaximal inhibitor concentrations of 2 to
`3J:LM, well below that .of the irreversible serine en(cid:173)
`zyme inhibitor DIFP. Lys[Z(N02)]-pyrrolidide, how(cid:173)
`ever, was highly toxic for lymphocytes, and was there(cid:173)
`fore excluded from cell-culture studies.
`Our reaction conditions for comparing the inhibitor
`efficiency of human lymphocyte DP IV deserve some
`comments. The compounds listed in the table act by
`different mechanisms on the target enzyme dipep(cid:173)
`tidyl peptidase IV. The oligopeptides are slowly
`hydrolysed substrates[26l and act like the dipeptides,
`thiazolidides and pyrrolidides as competetive in(cid:173)
`hibitors under conditions chosen, whereas DIFP and
`the diacylhydroxylamines modify the enzyme irrever(cid:173)
`sibly[25l. To achieve comparable results, with deriva-
`
`Page 6 of 9
`
`

`
`Vol. 372 (1991)
`
`Action of Dipeptidyl Peptidase IV Inhibitors on Human Lymphocytes
`
`309
`
`tives of such different reactivities, we decided to
`preincubate the enzyme for 60 min in accordance with
`previous studies where the time-dependence of DP
`IV inhibition by DIFP[Z?J and by the diacylhydroxyl(cid:173)
`amines[Zl] was evaluated.
`The IC50 values listed in the table, expressing the rela(cid:173)
`tive efficiency of the inhibitors against lymphocyte
`DP IV, are not directly comparable to kinetic data
`using the purified enzyme from pig kidney. The deter(cid:173)
`gent extract of lymphocytes contains various pep(cid:173)
`tidases that possibly degrade inhibitors or interfere
`with DP IV in binding inhibitors to some extent. Since
`the aim of this study was to select the most potent in(cid:173)
`hibitors for cell physiology experiments, the test sys(cid:173)
`tem chosen by us reflects more the situation at the cel(cid:173)
`lular level. Here, interference between different pep(cid:173)
`tidases is also likely. Despite this, the relative effi(cid:173)
`ciency of the inhibitors in each group ( diacylhydroxyl(cid:173)
`amines, oligopeptides, thiazolidides, pyrrolidides)
`compares well with data obtained with the purified
`enzyme. Thus, the use of a detergent extract of cells as
`the enzyme source seems to be applicable within cer(cid:173)
`tain limits, in order to answer the above question.
`
`Effects of DP IV inhibitors on mitogen-induced DNA
`synthesis in lymphocytes
`
`Most of the compounds that were inhibitory to lym(cid:173)
`phocyte DP IV were also added to lymphocyte cul(cid:173)
`tures. As a well established standard system, the
`DNA synthesis of lymphocytes was studied after acti(cid:173)
`vation by PHAas a mitogenic lectin[18l. The incorpora(cid:173)
`tion of tritiated thymidine served as a measure of the
`proliferating activity of the cells. The results of these
`studies are summarized in the table. Fig. 2 shows
`selected examples of the dose-dependent effect of D P
`IV inhibitors on DNA synthesis.
`Comparing the half maximal inhibitor concentrations
`in both test systems, a rough correlation can be ob(cid:173)
`served between both rows. Again, the Lys[Z(N 0 2)]
`thiazolidide was a highly potent inhibitor, acting in
`the same concentration range in the cell culture sys(cid:173)
`tem, followed by Ile-thiazolidide and N-(Ala-Pro )-0-
`(nitrobenzoyl)hydroxylamine, which were one order
`of magnitude less powerful.
`Some of the inhibitors affecting DP IV activity in the
`range of 0.1-1mM show no significant influence on
`lymphocyte proliferation, e.g.,
`,B-casomorphin(cid:173)
`(1-5)-peptide, Ile-Pro or Lys(Cap)-Pro. This may be
`explained, at least in part, by limited stability in the
`cell culture system, due to spontaneous degradation
`or enzymatic hydrolysis. Only systematic studies of
`the half life of these compounds under cell culture
`conditions can resolve this question.
`.
`
`100
`
`:g 80
`
`c
`0
`0
`0
`:::R
`~ 60
`0
`~
`Q. 8 40
`
`0
`
`-~
`'"0
`..c
`'o
`I' 20
`"2.....
`
`0
`
`(0)
`
`5 X 10-?
`
`5 X 10-5
`
`5 X 10-4
`
`5 X 10-6
`[I] [mo11n
`
`Fig. 2. Effect of selected compounds on PHA-induced DNA
`synthesis of human lymphocytes.
`Each point on the diagram represents the arithmetic mean
`±standard error of the mean from 4 separate experiments. D,
`Compd. 2, Lys[Z(N02)]-thiazolidide; •, Compd. 3, Ile-thia(cid:173)
`zolidide; 0, Compd. 11, Phe-pyrrolidide. For further details;
`see Methods section.
`
`Special attention was payed to the exclusion of
`cytotoxic side effects of inhibitors, which could
`mimick an antiproliferative effect in the cell culture.
`Before any application to the cell proliferation sys(cid:173)
`tem, the viability of cultured lymphocytes was tested
`as indicated in the Methods section. Only those in(cid:173)
`hibitors were used in the lymphocyte proliferation
`test that showed no apparent influence on cell viabil(cid:173)
`ity up to a concentration of 0.5mM. We cannot explain
`the cytotoxic effect. of two inhibitors mentioned
`above (see table).
`As previously shown, DP IV inhibitors do not cause a
`selective depletion of cells expressing DP IV, or the
`CD 4 antigen which is. characteristic forT helper
`cells[lsJ. Taking into consideration the high selectivity
`of active site-directed DP IV inhibitors as discussed
`20l, the observed dose-dependent suppres(cid:173)
`earlier[18
`'
`sion of DNA synthesis in the presence of specific DP
`IV inhibitors is obviously a consequence of inhibition
`of DP IV on the cell membrane of lymphocytes.
`The time kinetic of the inhibitor action deserves com(cid:173)
`ment. As evident from the biochemical studies, a
`preincubation period of 1 h at room temperature is
`sufficient for the inhibitors to be bound to their
`target. Regarding their action on lectin-activated lym(cid:173)
`phocytes, several attemps have been made to deter-
`
`Page 7 of 9
`
`

`
`310
`
`E. Schon, I. Born, H.-U. Demuth, J. Faust, K. Neubert, T. Steinmetzer, A. Barth and S. Ansorge
`
`Vol. 372 (1991)
`
`mine the time course of inhibitor action. From these
`studies, we can conclude that the strongest effect on
`lymphocyte proliferation is achieved if the inhibitor is
`added at the beginning of the culture[14l. Any time
`delay between setting up the culture and adding in(cid:173)
`hibitor results in a reduced effect. Thus the test system
`as described in the Methods section should provide
`for the optimal record of inhibitor effects on lympho(cid:173)
`cyte proliferation.
`In the studies reported here, PHA was used as a broad
`range mitogen for human lymphocytes. However,
`some evidence exists that the extent of inhibition of
`proliferation in the presence of DP IV inhibitors also
`depends on the kind of mitogenic lectin. Thus we ob(cid:173)
`served a stronger suppression of DNA synthesis after
`stimulation with concanacvalin A as a mitogen than
`with PHA (see table) and refJ28l). Obviously, differ(cid:173)
`ent cell populations vary in their sensitivity towards
`DP IV inhibitors. This phenomenon also deserves
`further systematic study.
`
`Conclusions
`
`The present paper provides confirmatory evidence
`that the membrane peptidase DP IV plays a signifi(cid:173)
`cant role in the regulation of the immune response in
`man. Results of other investigators obtained by differ(cid:173)
`ent methodological approaches indicate that this en(cid:173)
`zyme is involved either directly or indirectly in differ(cid:173)
`ent functions of T lymphocytes such as lymphokine
`induction[29l, triggering and proliferation of cytotoxic
`31l, maintaining long-term growth of T cell
`T cells[30
`'
`33l or activation of memory T cells[34l.
`32
`clones[30
`'
`'
`Thus the question arises of what the specific function
`of DP IV in the immune system is. The mechanisms of
`action of DP IV in the lymphocyte, at the cellular and
`at the molecular level, are not yet understood. Ac(cid:173)
`cording to our hypothesis[lSJ, the enzyme might be in(cid:173)
`volved in the modulation of cytokine activity and/or
`dipeptide-mediated signalling through limited pep(cid:173)
`tidolytic processing.
`The development of active site-specific agents as in(cid:173)
`hibitors of D P IV enabled us to use these as tools tore(cid:173)
`veal the mechanism of action of D P IV in the immune
`system, at the same time providing a theoretical basis
`for a potential application of these inhibitors as im(cid:173)
`munomodulatory drugs.
`
`The authors are indepted to Ms. Karin Klemm, Ms. Astrid
`Becker, Ms. Ute Thiel and Ms. Barbel Roesler for their skillful
`technical assistance throughout these studies.
`
`Supported in part by the Bundesministerium fUr Forschung und
`Technologie and the Verband der chemischen Industrie der Bun(cid:173)
`desrepublik Deutschland.
`
`References
`
`8
`
`1 Hopsu-Havu, V.K. & Sarimo, S.R. (1967) Hoppe-Seyler's
`Z. Physiol. Chern. 348, 1540-1550.
`2 Gossrau, R. (1979) Histochemistry 60, 231-248.
`3 Walter, R., Simmons, W.H. & Yoshimoto, T. (1980) Mol.
`Cell. Biochem. 30, 111-127.
`4 Kenny, A.J., Booth, A.G., George, S.G., Ingram, J.,
`Kershaw, D., Wood, E.J. & Young, A.R. (1976) Bio(cid:173)
`chem. J. 157, 169-182.
`5 Kenny, A.J. & Booth, A.G. (1977) Essays Biochem. 14,
`1-44.
`6 Lojda, Z. (1977) Histochemistry 54, 299-309.
`7
`Feller,A.J., Heijnen, C.J., Ballieux, R.E. & Parwaresch,
`M.R. (1982) British J. Haematol. 51,227-234.
`Scott, C.S., Stark, A.N., Minowada, J. & Drexler, H.-G.
`(1988) Leukemia Res. 12, 129-134.
`9 Chilosi, M., Pizzolo, G., Semenzato, G., De Rossi, G. &
`Pandolfi, F. (1984)Acta Haematol. 71,277-281.
`10 Ansorge, S., Bosselmann, P., Franke, A., Gaede, B.,
`Gotze, D., Ittenson, A., Mansfeld, H.-W., Schon, E. &
`Mey, U. (1984) Dt. Gesundh.-Wesen39, 1458-1460.
`11 Schon, E., Essbach, U., Machura, R., Frabke, A. & An(cid:173)
`sorge, S. (1986)Acta histochem., Suppl. 33, 131-138.
`12 Harland, C., Shah, T., Webster, A.D. & Peters, T.J. (1988)
`Clin. Exp. Immunol. 74,201-205.
`13 Schon, E., Mansfeld, H.-W., Demuth, H.-U., Barth, A. &
`Ansorge, S. (1985) Biomed. Biochim. Acta 44, K9-K15 ..
`14 Schon, E., Eichmann, E., Jahn, S., Kopp, J., Volk, H.-D.
`&Ansorge, S. (1988) Bioi. Zentbl. 107, 141-149.
`15 Schon, E., Eichmann, E., Grunow, R., Jahn, S., Kiessig,
`S.T.,Volk, H.-D. &Ansorge, S. (1986) Biomed. Biochim.
`Acta 45, 1523-1528.
`16 Schon, E. &Ansorge, S. (1990) Bioi. Chern. Hoppe-Seyler
`371,699-705.
`17 Knapp, W., Rieber, P., Darken, B., Schmidt, R.E., Stein,
`H. & van den Borne, A.E.G.K. (1989) Immunol. Today
`10,253-258.
`18 Schon, E., Jahn, S., Kiessig, S.T., Demuth, H.-U., Neu(cid:173)
`bert, K., Barth, A., von Baehr, R. & Ansorge, S. (1987)
`Ew: J. Immunol. 17, 1821-1826.
`19 Schon, E., Demuth, H.-U., Eichmann, E., Horst, H.-J.,
`Korner, I.-J., Kopp, J., Mattern,T.,Neubert, K., Noll, F.,
`Ulmer,A.J., Barth, A. &Ansorge, S. (1989) Scand. J. Im(cid:173)
`munol. 29, 127-132.
`20 Schon, E., Demuth, H.U., Barth, A. &Ansorge, S. (1984)
`Biochem. J. 223,255-258.
`21 Demuth, H.U., Baumgrass, R., Schaper, C., Fischer, G.
`& Barth, A. (1988) J. Enzyme Inhibition 2, 129-142.
`22 Demuth, H.-U. (1990) J. Enzyme Inhibition 4, 249-278.
`23 Hartrodt, B., Neubert, K., Matthies, H., Rekowski, P.,
`Kupryszewski, G. & Barth, A. (1985) Pharmazie 40,
`201-204.
`24 Demuth, H.U., Neumann, U. & Barth, A. (1989) J. En(cid:173)
`zyme Inhibition 2, 239-248.
`25 Demuth, H.U. (1989) in Schwerpunkte und Trends der bio(cid:173)
`technologischen Forschung an der Martin- Luther- Universi(cid:173)
`tiit Halle- Wittenberg (M. Luckner, ed.) pp. 194-219, Mar(cid:173)
`tin-Luth