_ on
`Balancing the immune AT for. tolerance:
`a case for Rete)a CD4 cells
`eaah aAleFaos Gao, ne eae andBar Rouse
`
`SEASUEoea
`
`ereeReite Reiners Mone Pe ate
`
`West-Ward Pharm.
`Exhibit 1036
`Page 001
`
`

`

`July 15, 1997
`Volume 64
`Number1
`
`Contents
`
`EDITORIAL
`
`Transplantation
`
`Official Journal of the Transplantation Society
`
`A Welcometo the New Editors..............
`
`a |
`
`OVERVIEW
`
`Balancing the immune system for tolerance: a case for regulatory CD4 cells.
`Dette eee eee eee
`E.H. Field, Q. Gao, N. Chen, and T.M. Rouse. .
`
`1
`
`EXPERIMENTAL TRANSPLANTATION
`
`Massive repopulation ofrat liver by transplantation of hepatocytes into specific lobes of the
`liver and ligation of portal vein branches to other lobes.
`4 Y.
`llan, N. Roy Chowdhury, R. Prakash, V. Jona, P. Attavar, C. Guha, K. Tada, and
`a. Roy Chowdhury. .... 0.00000 e eer eavesSe eee ee es
`Synthetic MHCclass | peptide prolongs cardiac survival and attenuates transplantarterio-
`sclerosis in the Lewis—Fischer 344 model of chronic allograft rejection.
`_B. Murphy, K.S. Kim, R. Buelow, M.H. Sayegh, and W.W. Hancock... 1... 2. ee esse eens
`Allo- and autotransplantation of carotid artery—a new modelof chronic graft vessel disease:
`evaluation by magnetic resonance imaging and histology,
`S. Wehr, M. Rudin, J. Joergensen, A. Hof, and R.P. Hof...2 ee _ 20
`Long-term function of fish islet xenografts in mice by alginate encapsulation.
`H. Yang, W. O’biali, H. Kearns, and J.R. Wright, Jr... eee eee
`SDZ RAD, a new rapamycin derivative: synergism with cyclosporine.
`H.-J. Schuurman, S. Cottens, S. Fuchs, J. Joergensen, T. Meerloo, R. Sedrani, M. Tanner,
`G. Zenke, and W. Schuler... . 0.2.0.2... 0.005De eee eee 32
`SDZ RAD, a new rapamycin derivative: pharmacological properties in vitro and in vivo.
`W. Schuler, R. Sedrani, S. Cottens, B. Haberlin, M. Schulz, H.-J. Schuurman, G. Zenke,
`H.-G. Zerwes, and M.H. Schreier. .......... De ee ees 36
`‘Mechanism of concordant corneal xenograft rejection in mice: synergistic effects of anti-
`leukocyte function-associated antigen-1 monoclonal antibody and FK506.
`S. Yamagami, M. Isobe, H. Yamagami, J. Hori, and T. Tsuru... 1... ee ee ees 42
`
`8°
`
`14.
`
`28
`
`CLINICAL TRANSPLANTATION
`
`Treatmentof graft-versus-host disease by extracorporeal photochemotherapy:a pilot study.
`D.P. Besnier, D. Chabannes, B. Mahé, J-M.G. Mussini, T.A.R. Baranger, J.Y. Muller, N. Milpied,
`and V.L.M. Esnault... 0.0.0.0... ee Lee eee ene eet eens
`A positive crossmatchin liver transplantation—no effect or inappropriate analysis? A pro-
`spective study.
`M. Hathaway, B.K. Gunson, A.C. Keogh, D. Briggs, P. McMaster, and J.M. Neuberger. ....
`Value of thein vitro or in vivo monoethylglycinexylidide test for predicting liver graft function.
`P. Olinga, J.K. Maring, G.M.M. Groothuis, K. Kranenburg, M. Merema,I.H. Hof, D.K.F. Meijer,
`and M.J.H. Slooff... 2. ceeSe ees 60
`
`49
`
`54
`
`
`
`Transplantation (ISSN 0041-1337) is published twenty-four times a year by
`Williams. & Wilkins, 351 West Camden Street, Baltimore, MD 21201-2436,
`Periodicals postage paid at Baltimore, MD, and at additional mailing offices.
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`Current Contents
`(Life Sciences, Science Citation Index, Research Alert,
`ISI/BioMed), BIOSIS, Excerpta Medica, Research Information Systems /Reference
`Update. Copyright © 1997 by Williams & Wilkins. This journal is printed on
`acid-free paper.
`
`West-Ward Pharm.
`Exhibit 1036
`Page 002
`
`West-Ward Pharm.
`Exhibit 1036
`Page 002
`
`

`

`0041-1837/97/6401-36$03,00/0
`.
`TRANSPLANTATION
`Copyright © 1997 by Williams & Wilkins
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Vol. 64, 836-—42,.No. 1, July 15, 1997
`Printed in U.S.A.
`
`SDZ RAD, A NEW RAPAMYCIN DERIVATIVE
`
`PHARMACOLOGICAL PROPERTIES IN VITRO AND IN VIVO
`
`WALTER SCHULER,”” RICHARD SEDRANI,’ SYLVAIN CoTTENs,' BARBARA HABERLIN,®
`MANFRED SCHULZ,’ HENK-JAN ScHUURMAN,! GERHARD ZENKE,’ Hans-Gtrer ZERwes,!
`AND Max H. Scrrerer?!
`
`Preclinical Research, and Technical Research and Development, Novartis Pharma AG, CH-4002 Basel, Switzerland
`
`Background. This report describes the preclinical
`pharmacological profile of the new rapamycin analog,
`SDZ RAD,i.e., 40-O0-(2-hydroxyethyl)-rapamycin.
`Methods. The pharmacological effects of SDZ RAD
`were assessed in a variety of in vitro and in vivo mod-
`els, which included an autoimmune disease model as
`well as kidney and heart allotransplantation models
`using different rat strain combinations.
`Results. SDZ RAD has a modeofaction thatis differ-
`ent from that of cyclosporine or FK506. In contrast to
`the latter, SDZ RAD inhibits growth factor-driven cell
`proliferation in general, as demonstrated for the in
`vitro cell proliferation of a lymphoid cell line and of
`vascular smooth muscle cells. SDZ RAD is immunosup-
`pressive in vitro as demonstrated by the inhibition of
`mouse and human mixed lymphocyte reactions and
`the inhibition of antigen-driven proliferation of hu-
`man T-cell clones. The concentrations needed to
`achieve 50% inhibition in all of these assays fall into
`the subnanomolar range. SDZ RAD is effective in the
`in vivo models when given by the oral route in doses
`ranging between 1 mg/kg/day and 5 mg/kg/day. When
`compared with rapamycin, the in vitro activity of SDZ
`RAD is generally about two to three times lower; how-
`ever, when administered orally, SDZ RAD is at least as
`active in vivo as rapamycin.
`Conclusions. In conclusion, SDZ RAD is a new,orally
`active rapamycin-derivative that is immunosuppres-
`sive and thatefficiently prevents graft rejection in rat
`models of allotransplantation. SDZ RAD has therefore
`beenselected for development for use in combination
`with cyclosporine A to prevent acute and chronic re-
`jection after solid organ allotransplantation.
`
`It wasfirst reported in 1989 that the macrolide rapamycin
`(RPM*), a secondary metabolite of Streptomyces Ahygroscopi-
`cus, effectively suppresses the rejection of transplanted allo-
`geneicsolid organs in experimental animals (1, 2). RPM isof
`particular interest as a new immunosuppressant because its
`mode of action is different from that of both cyclosporine
`
`1 Preclinical Research, Novartis Pharma AG.
`? Address correspondence. to: Walter Schuler, Preclinical Re-
`search, Novartis Pharma AG, 8-386,1.26, CH-4002 Basel, Switzer-
`land.
`° Technical Research and Development, Novartis Pharma AG.
`* Abbreviations: CsA,
`cyclosporine; FCS,
`fetal calf serum;
`FKBP12, FK506 binding protein; GVD, graft vessel diseage; IC50,
`concentration of compound needed to reach 50% inhibition: IL, in-
`terleukin; MLR, mixed lymphocyte reaction; PBMC, peripheral blood
`mononuclearcells; RPM, rapamycin; VSMC,vascular smooth muscle
`cells,
`
`(CsA) and FK506. The latter drugs prevent T-cell prolifera-
`tion by blocking transcriptional activation of early T cell-
`specific genes, thus inhibiting the production of T-cell growth
`factors, like interleukin (IL)-2. RPM, in contrast, acts at a
`later stage of the cell cycle, blocking not the production of
`growth factors but rather the proliferative signal that is
`provided by these factors; RPM arrests the cells at the late
`G1stageofthe cell cycle, preventing them from entering the
`S phase. (For a review on RPM and its mechanism of action
`see 3, 4), It is of note that this effect of RPM is not restricted
`to IL-2-driven proliferation of T cells; RPM inhibits growth
`factor-dependentproliferation in general of any hematopoi-
`etic as well as nonhematopoietic cells tested so far (6-7),
`including vascular smooth muscle cells (VSMC) (8). The dif-
`ferent modes of action of RPM and CsA provide a rationale
`for synergistic interaction of the two compounds, and this
`synergism has indeed been demonstrated (9-11). Further,
`the ability to inhibit growth factor-driven cell proliferation
`makes RPM a potential compoundfor the prevention of late
`graft loss due to graft vessel disease (GVD); growth factor-
`driven proliferation of VSMC lc ading to intimal thickening
`and eventually vessel obstruction seemsto play a crucial role
`in the development of GVD (for a review see 12). RPM has
`indeed been shown to inhibit arterial intimal thickening in
`rat recipients of orthotopic femoral artery allografts (13) as
`well as such thickening produced by mechanical injury where
`no immunological mechanism is involved (14).
`These features make RPM and RPM analogsvery interest-
`ing compoundsforclinical transplantation. However, devel-
`opment of a proper oral RPM formulation with acceptable
`stability, bioavailability, and predictability has proven diffi-
`cult and has impeded successful clinical development. Sofar,
`the majority of published preclinical work demonstrating the
`potent immunosuppressive effect of RPM deals with paren-
`teral administration of the compound(for references see 15);
`efficacy of an oral RPM formulation was shown only very
`recently in a pig and a rat modelofallotransplantation (15,
`16). However, wide interindividual variation in the pharma-
`cokinetic parameters was noted in the pig study as well as in
`a recent report on first clinical experience with an oral RPM
`formulation (17).
`The formulation of a compound can have a marked effect
`on clinical outcomes in transplantation, as seen with the
`introduction of the microemulsion preconcentrate of CsA (Ne-
`oral; Sandoz, Basel, Switzerland) (18-20). The 40-O-(2-hy-
`droxyethyl)-RPM, SDZ RAD, is a new RPM analog that re-
`sulted from our efforts to overcome the formulation problems
`by chemical derivation, while maintaining the pharmacolog-
`
`West-Ward Pharm.
`Exhibit 1036
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`West-Ward Pharm.
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`Page 003
`
`

`

`July 15, 1997
`
`
`
`Ficurr 1. Chemical structure of SDZ RAD, 40-0-(2-hydroxyethyl)-
`RPM.
`
`SDZ RAD
`
`jcal benefits of RPM. In this study we report on the in vitro
`and in vivo pharmacological characteristics of SDZ RAD, We
`show that. despite a slightly reduced in vitro activity, SDZ
`RAD hasanefficacy after oral dosing that is at least. equiv-
`alent to that of RPM.
`
`MATERIALS AND METHODS
`
`Ri-agents
`RPM was obtained by fermentation of the actinomycetes strain
`A91-259211. SDZ RAD, 40-0-(2-hydroxyethyl)-RPM (Fig. 1), was
`derived by chemical derivation of RPM; the molecular formula is
`CgalIggNOj,, and it has a molecular weight of 958.25. For the in vitro
`experiments, 10-4 M stock solutions of the compounds in ethanol
`were used. Stock solutions were stored at —20°C; samples to be
`tested were diluted on the day of the experiment in phosphate-
`buffered saline or culture medium.For the in vivo experiments SDZ
`RAD and RPM were formulated as liquid formulations, which kept
`the compounds dissolved even after dilution with aqueous vehicles.
`These formulations were adapted for both compounds with respect to
`their physicochemical properties. Stored at 4°C,the formulationsare
`stable for >3 months.
`
`In Vitro Assays
`
`The in vitro activity of RPM analogs was assessed by determining
`in the various assays the concentration of the compoundsthat re-
`sults in 50% inhibition (IC;). Serial dilutionsof the test compounds,
`done in duplicate, were tested, and a four-parameterlogistic function
`was applied to calculate the IC; values. RPM was included in each
`individual experiment as a standard, and the inhibitory activity was
`expressed as relative IC; compared with RPM (e., given as the
`ratio ICgq of test compound/IC,;5 of RPM). To measure in vitro cell
`proliferation, [?H]thymidine incorporation into DNA was determined
`following standard procedures.
`FKBP12 binding assay. Binding to the FK506 binding protein
`(FKBP12) was indirectly assessed by means of an ELISA-type com-
`petition assay. Microtiter plate wells were coated with FK506 that
`was covalently coupled to bovine serum albumin. Coupling of F'K506
`to bovine serum albumin was performed by reacting bovine serum
`albumin
`with
`N-succinimidyl-oxycarbonyl]-3’-propionyloxy-33-
`FK506, Biotinylated recombinant human FKBP12 was allowed to
`bind to the immobilized FK506 in the absence(as a control) and the
`presence of the serially diluted test compound or standard, (For
`technical reasons we used FK506 as the standard, as an exception
`
`SCHULER ET AL.
`
`37
`
`only in this assay). Bound biotinylated FKBP12 was assessed by
`incubation with a streptavidin-alkaline phosphatase conjugate, fol-
`lowed by incubation with p-nitrophenol phosphate as a substrate.
`Readout wasthe optical density at 405 nm. Bindingof a compoundto
`the biotinylated FKBP12 resulted in a decrease in the amount of
`FKBP12 available for binding to the immobilized FK506, ie., the
`magnitude of this inhibition (ICs) reflects the affinity of a compound
`for FKBP12.
`IL-6-driven proliferation of a B-cell hybridoma. The hybridoma
`B13-29-15 is a subcloneof the hybridoma B13-29, which was kindly
`provided by L. Aarden (Central Laboratory of the Netherlands Red
`Cross-Blood Transfusion Service, Amsterdam, The Netherlands);
`this clone is strictly dependent on IL-6, To determine the ICx5o of a
`compoundin this assay, 10* cells per microtiter well (supplemented
`to contain 0.3 ng of IL-6 per ml) were incubated for 72 hr with serial
`dilutions of the compounds. [°H]thymidine was added at the end of
`the incubation period, 5 hr before harvesting the cells for measuring
`the [?H]thymidine incorporation into DNA.
`Mixed lymphocyte reaction (MLR). To determine the ICs) values
`of the compoundsin a two-way MLR, 10° spleen cells per well each
`of BALB/c and CBA mice were incubated in flat-bottom microtiter
`plates, either in the absence or the presence of the serially diluted
`compounds. Serum-free tissue culture medium supplemented with
`serum replacement factors (CG medium, Camon GmbH, Wiesbaden,
`Germany) was used. After 4 days of incubation [*Hithymidine was
`added, and thecells were harvested after another 16-hr incubation
`period.
`-
`Proliferative response of antigen-specific human T-cell clones.
`CD4-positive (helper type) T-cell clones specific for the hemaggluti-
`nin peptide 307-819 were derived from peripheral blood mononu-
`clear cells (PBMC)of a normal healthy volunteer as described (27).
`To determine the IC,, of the compounds in this antigen-specific
`T-cell proliferation assay, cloned T cells (2X 10*) were cultured in a
`total volume of 200 yl of RPMI medium (supplemented to contain 5%
`human AB serum) in 96-well round-bottom microtiter plates with
`10° irradiated PBMC from normal HLA-DR matched donors,
`to-
`gether with the peptide antigen (hemagglutinin) and the serially ..
`diluted test compounds. As a control, T cells plus PBMC in the
`absence of peptide antigen or T cells in medium alone were included.
`Cultures were set up in duplicate. After 48 hr of incubation, [?H]thy-
`midine was added, andthe cells were harvested after another 16 hr.
`Proliferation ofVSMC. Bovine VSMC were derivedby the explant
`technique from small pieces of media (dissected free of adventitia
`and intima) from fresh bovineaortae. Explants of about 1 mm® were
`placed in culture dishes, covered with medium, and after about 10
`daysthecells grew out of the explants. The cells were characterized
`as VSMCby morphology in culture and by immunostaining with an
`anti-VSMCactin antibody (clone 144; Sigma, St. Louis, MO). They
`were used at passages 2 through 10. The cells were grown in DF10
`medium consisting of equal volumes of Dulbecco’s modified Hagle’s
`medium and Ham’s F12 (Life Technologies, Gaithersburg, MD) sup-
`plemented with 10% fetal calf serum (FCS) and glutamine. For the
`experiments, the cells were seeded in 96-well plates (1X 104 or 2105
`per well) and allowed to grow to confluence (3 days). They were then
`growth arrested by serum deprivation for 48 hr in serum-free me-
`dium (DF10: without FCS) supplemented with insulin (0.5 mM;
`Boehringer Mannheim, Mannheim, Germany), transferrin (5 pe/ml;
`Sigma), and ascorbate (0.2 mM; Sigma). After 3 days the medium
`was replaced with fresh medium containing 10% FCS and [PHthy-
`midine (1 mCi/ml), together with serial dilutions of the compoundsto
`be tested (three to four replicate wells for each concentration), The
`cells were harvested after a 24-hr incubation period and the [*Hithy-
`midine incorporation into the DNA was measured.
`
`In Vivo Experiments
`Throughout all of the in vivo experiments the compounds were
`given once daily, for the indicated period of time. Freshly prepared
`West-Ward Pharm.
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`West-Ward Pharm.
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`Page 004
`
`

`

`38
`
`TRANSPLANTATION
`
`Vol. 64, No, 1
`
`dilutions of the SDZ RAD and RPM formulations with water were
`given orally by gavage. Control animals received only the adminis-
`tration vehicle as placebo.
`Localized graft-versus-host reaction. Spleen cells (2107) from
`Wistar/F rats were injected subcutaneously into the right hind foot-
`pad of (Wistar/F x Fisher 344)F, hybrid rats. The left footpad was
`left untreated. The animals were treated with SDZ RAD or RPM on
`4 consecutive days (days 0-3). The popliteal lymph nodes were
`removed on day 7, and the weight differences between two corre-
`sponding lymph nodes were determined. The results were expressed
`as the inhibition of lymph node enlargement(given in percent) com-
`paring the lymph node weight differences in the experimental groups
`to the weight difference between the corresponding lymph nodes
`from a group of animals left untreated with a test compound.
`Mercuric chloride-induced glomerulonephritis. Autoimmuneglo-
`merulonephritis was induced by treatment with HgCl, (22). Female
`Brown Norway rats, 9 weeks of age, were injected subcutaneously
`during a 3-weekperiod, three times per week, with 1 mg ofHgCl, per
`kg body weight (10 injections in total), SDZ RAD and RPM were
`given on 5 consecutive days per week. On days 0, 7, 14, and 21, urine
`was taken and the protein concentration was determined by means
`of bromophenol blue staining and colorimetric detection using a TCL
`scanner. The detection limit of this method is 1 mg protein/ml; the
`upper threshold of this method is 16 mg protein/ml. The experiment
`is normally terminated between day 21 and day 24 because the
`control animals, treated with HgCl, only, start to succumb to the
`disease at this point.
`Orthotopic kidney allotransplantation. Donorkidneys weretrans-
`planted orthotopically into recipient rats. The left kidney of the
`recipient animal was removed and replaced with the donor kidney,
`with end-to-end anastomoses of blood vessels and ureter. After 1
`week, contralateral nephrectomy was performed, leaving the animal
`fully dependent on the grafted kidney. Recipients were treated with
`the immunosuppressive compounds or the placebo for the initial 2
`weeks after transplantation.
`Vascular heterotopic heart allotransplantation. Donor hearts
`were transplanted heterotopically inte the abdomenofrecipient rats
`by making end-to-side anastomoses of the donor’s aorta with the
`recipient’s infrarenal abdominal aorta as well as with the donor’s
`right pulmonary to the recipient’s inferior vena cava. Recipients
`received the immunosuppressive compounds or the placebo once
`daily for the entire course of the experiment. The heartbeat of the
`transplanted heart was monitored daily by palpation of the abdo-
`men, The time of rejection was defined as the day. on which a
`heartbeat was no longerpalpable.
`
`RESULTS
`
`Binding to FKBP12
`Binding to FKBP12, the abundant intracellular binding
`protein of FK506, is a prerequisite for the biological activity
`of RPM-type macrolides (23). Therefore, we determined the
`ability of SDZ RAD to bind to FKBP12. As shownin Table 1,
`binding of SDZ RAD to FKBP12 is about threefold weaker
`than that of RPM.
`
`Inhibition of Growth Factor-Driven Proliferation
`The immunosuppressive activity of RPM is explainedbyits
`ability to inhibit growth factor-driven cell proliferation. We
`assessed SDZ RAD for this effect in two in vitro systems:
`IL-6-stimulated cell proliferation of the IL-6-dependent hy-
`bridoma clone B13-29-15, and FCS-stimulated proliferation
`of bovine VSMC. Table 2 shows that the ability of SDZ RAD
`to inhibit the IL-6-driven proliferation of the hybridoma cells
`is about two- to threefold less compared with that of RPM
`(ie., relative IC,, of 2.5+0.7). The relative IC,, of SDZ RAD
`
`
`TABLE 1. Binding to FKBP12°
`Com
`d
`Relative IC59+8D°
`
`poun
`(range, absolute IC,o)
`FK506
`1
`(0.8~1,.2 nM)
`0.640.2* (n=5)
`(0.4—0.9 nM)
`2,0+0.4*** (n=8)
`SDZ RAD
`(1.8-2.6 nM)
`
`RPM
`
`*The ability of the compounds to compete with immobilized
`FK506 for binding to biotinylated FKBP12 was determined in a
`competitive binding assay.
`® FK506 was included as a standard in each individual experi-
`ment, Results are expressed as means+SDofthe relative IC;, values
`(i.e., ratio of ICsq test compound to IC;9 of FK506). The range of
`absolute ICg, values is given in parenthesis; n=numberof individual
`experiments. Statistical analysis, ¢ test: *P<0.05; ***P<0,001.
`
`RPM
`
`
`TaBLe 2. Inhibition of growth factor-stimulated cell proliferation
`Relative IC,9+SD*
`
`(range, absolute IC5,)
`Compound
`
`Hybridoma B18-29-15/1L-6
`Bovine VSMC/FCS
`1
`1
`(0.07-0.5 nM)
`(0.4-3.5 nM)
`2.540.7** (n=5)
`1.90.75" (n=8)
`SDZ RAD
`
`(0.2-1.4 nM) (0.9-3,6 nM)
`“ RPM wasincluded as a standard in each individual experiment.
`Results are expressed as means+SDof the relative IC,, values (ié.,
`ratio of ICs, test compound to ICs, of RPM). The range of absolute
`ICgq values is given in parenthesis; n=numberof individual exper-
`iments. Statistical analysis, ¢ test: **P<0.01; "*not significant.
`
`for inhibition of bovine VSMC was 1.90.75 (Table 2); how-
`ever, this was not statistically significant when compared
`with inhibition by RPM. The absolute IC;, values found here
`for RPM are in agreement with those reported for platelet-
`derived growth factor or basic fibroblast growth factor-stim-
`ulated VSMCproliferation [5 nM and 0.8 nM,respectively
`(8)].
`
`Immunosuppressive Activity In Vitro
`The immunosuppressive activity of SDZ RAD was assessed
`in two-way MLR experiments with lymphocytel, of mouse
`origin as well as in experiments with antigen-specific human
`helper T-cell clones. The results are shown in Table 3. The
`data show that, compared with RPM,the in vitro immuno-
`suppressive activity of SDZ RAD is about two- and fivefold
`lower, respectively, in these assays.
`
`
`TABLE 8, Immunosuppressive activity in vitro”
`Relative ICs9+SD°
`
`Compound
`(range, absolute IC;o)
`
`
`
` MLR T-cell clone
`
`RPM
`
`1
`1
`(0.014-0.0387 nM)
`(0.06-0.9 nM).
`5.4£3,5* (n=8)
`2.1+0.4* (n=4)
`SDZ RAD
`
`(0.2—1.6 nM) (0.056-0.17 nM) ©
`
`“ The effect on two-way MLR performed with mouse spleen cells,
`as well as on the antigen-specific (hemagglutinin peptide 307-319)
`proliferation of a human T-cell clone, was tested.
`» As in Table 2, Statistical analysis, ¢ test: *P<0,05.
`
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`
`
`
`
`
`Compound
` Vehicle O.3mg/kgp.oc. O6me/kgp.o. 1.25 mg/kg p.o:
`
`Control
`10/10
`o/o***
`T/11t*
`8/9
`SDZ RAD
`
`
`
`5/5 a/5**RPM 1/5**
`“ Numberof animals on day 21 with proteinuria(i.e., urine protein
`levels >2 mg/ml) of total number of animals in each dosage group.
`Statistical analysis (chi-square) with respect to control: **P<0.01;
`EP<O,001.
`.
`,
`
`Inhibition of Localized Graft-Versus-Host Reaction
`In this experimental model of cell-mediated immunity, a
`strong local T-cell reaction is induced by injecting parental
`spleencells into one hind footpad of F, hybrid recipients. The
`injected immunocompetent donor spleen cells home to the
`local draining popliteal lymph node, where they react vigor-
`ously to alloantigens present on the host’s cells: they become
`activated, secrete cytokines, and proliferate. This reaction
`manifests itself in an enlargement of the respective lymph
`node. Comparing the weight ofthe popliteal lymph node from
`the site of injection with that of the untreated contralateral
`lymph nodegives an indication of the severity of the'reaction.
`SDZ RAD effectively inhibited lymph nede swelling elicited
`by this localized graft-versus-host reaction. This is shown in
`Table 4. Maximal inhibition of about 70-80% was achieved
`with an oral dose of 3 mg/kg per day of either SDZ RAD or
`RPM.(Increasing the doses of SDZ RAD or RPM did not lead
`to stronger inhibition of lymph node swelling; data not
`
`shown). Hpbition was statistically significant with respect
`
`to the placebo-treated control; no statistically significant dif-
`ference was found between SDZ RAD and RPM.
`Mercuric chloride-induced glomerulonephritis. Low doses
`of mercuric chloride (HgCl,), repeatedly injected into rats,
`induce an-autoimmune disease ‘that is characterized by a
`T-dependent polyclonal B-cell activation (22, 24), This poly-
`clonal B-cell activation leads to the production ofa variety of
`autoantibodies. Antibodies directed against the glomerular
`basement membranecauseinfiltration of polymorphonuclear
`granulocytes and glomerulsr damage; the animals develop a
`severe proteinuria within z to 8 weeks of treatment with
`HgCl,. As can be seen from Table 5, a dose of 1.25 mg/kg/day
`of SDZ RAD or RPM completely prevented this HgCl,-in-
`duced development of proteinuria. (One animal in the RPM
`group showed proteinuria already on day 7, but this was
`most likely not related to the HgCl, treatment). The 0.3
`mg/kg dose was ineffective, whereas 0.6 mg/kg of either com-
`pound led to partial inhibition. In conclusion, SDZ RAD is
`effective in an animal model for autoimmune glomeruione-
`phritis, with the same dose-response relationship as RPM.
`
`Orthotopic Kidney Allotransplantation in the Rat
`SDZ RAD was tested in rat kidney allotransplantation.
`using several donor-recipient strain combinations. Grafted
`recipients underwent contralateral nephrectomy 7 days after
`transplantation so that the survival of an animal depended
`fully on the function of the grafted allogeneic kidney. A
`peculiarity of this rat model is that a 2-week treatment with
`
`July 18, 1997
`
`SCHULER ET AL.
`
`39
`
`TaBLe 5. Mercuric chloride-induced glomerulonephritis
`Developmentof proteinuria
`
`CsA results in the indefinite survival of the graft, a phenom-
`enon that is restrictedto rats and is not seen with any other
`species.
`Table 6 showsthe results for SDZ RAD and RPM in exper-
`iments transplanting kidneys from (Wistar/F x Fisher
`344)F, donors into Wistar/F recipients. Untreated control
`animals showed severe cellular rejection on day 7. In this
`strain combination, donor and recipient are partly matched;
`prolonged graft survival can thusbe obtained with rather low
`levels of immunosuppression. Survival times of more than
`100 days were obtained with 0.5 mg/kg of either SDZ RAD or
`RPM.At this dose no histological signs of rejection were seen
`with SDZ RAD, whereas one animal in the RPM group
`showed moderate signs of chronic rejection. A dose of 0.25
`mg/kg SDZ RAD led to a substantial prolongation of the graft
`survival time in three of nine recipients; no histological signs
`of rejection were found in these long-term survivors.
`The results of kidney allograft experiments using a strain
`combination with a strong mismatch, ie., Brown Norway rat
`donor and Lewis rat recipient, are shown in Table 7. Un-
`treated control animals showed severe cellular rejection on
`day 7. A dose of 2.5 mg/kg of either SDZ RAD or RPM .
`prolonged the survival of Brown Norway kidneys in mostof
`the Lewis recipients for more than 80 days. The long-term
`survivors in the SDZ RAD group showed marginal signs of
`chronic rejection. Even with 1 mg/kg, substantial prolonga-
`tion of graft survival times was achieved, with one animal in
`the SDZ RAD group notrejecting its graft during the obser-
`vation period (78 days). This animal showed histologically
`moderate chronic rejection. The other animals in this group
`showed moderate to severe cellular rejection, whereas all
`animals in the respective RPM group showedseverecellular
`rejection.
`
`Vascular Heterotopic Heart Allotransplantation
`SDZ RAD was further tested in the model of vascular
`heterotopic heart allotransplantation in the rat using DA
`rats as donors and Lewis rats as recipients, This strain
`TABLE 4. Inhibition of localized graft-versus-host reaction
`combination represents a very strong mismatch and is con-
`
`sidered the most stringent rat transplantation model. As
`No. of Percent inhibition of lymph node swelling®
`Compound—snimals?
`Table 8 shows, we were unable to achieve long-term graft
`
`1.0 mg/kg/day p.o.
`3,0 mg/kg/day p.o.
`survival with any dose of SDZ RAD or RPM tested, even
`58+23**
`TTagees
`5
`SDZ RAD
`though wetreated the animals daily until rejection occurred.
`
`
`
`5 61+22**RPM 66+11***
`Increasing the dose from 2.5 to 5 mg/kg ofeither compound
`“ Lymph node weight differences were determined on day 7. Re-
`did not improve graft survival times; rather, the higher doses
`sults are given as mean values+SD of inhibition compared with a
`led to severe weight loss under these conditions, forcing ter-
`control group of five animals that received the vehicle only, The
`mination of the experiments 3 to 4 weeks after transplanta-
`weight differences between the respective lymph nodes from animals
`tion. Only moderate signs of rejection were found histologi-
`in the control group was 84+2 mg. Statistical analysis (analysis of
`cally in all groups, with the exception of the 1 mg/kg SDZ
`variance) with respect to the control group: **P<0.01; ***P<0.001.
`RAD group, in which rejection was severe. Although we did
`’ Number of animals in each dosage group.
`West-Ward Pharm.
`Exhibit 1036
`Page 006
`
`West-Ward Pharm.
`Exhibit 1036
`Page 006
`
`

`

`40
`TRANSPLANTATION
`Vol, 64, No. 1
`TABLE 6, Suppression of allograft rejection of (Wistar/Fx Fisher344)F, rat donor kidneys in Wistar/F recipionts®
`Individual survival times (days after transplantation) after treatment
`Compound
`
`0.25 mg/kg/day
`0.5 mg/kg/day
`1.0 mg/kg/day
`7,7,7, 7,7, 7, 261, 2100, =100°
`2100, =100, =100, =100, =100
`=100, =100, 2100 -
`SDZ RAD
`
`RPM 2100, =100, 2100 7,7, 7, 7,7, 7, 12 7, =100, =100, =100, =100
`
`
`“The immunosuppressive compounds were administered p.o, daily from day 0 through day 18 at the indicated dose. Placebo-treated
`animals showed severe cellular rejection by day 7.
`» Hach figure indicates the survival time after transplantation of an individual animal, More than or equal-to sign (=) indicates that the
`animal was killed for histology while the animal was in good health and the graft. was still functioning,
`
`TABLE 7. Suppression of allograft rejection of Brown Norway rat
`donor kidneys in Lewis recipients
`Individual survival times (days after transplantation) after
`
`Compound
`treatment
`
`1.0 mg/kg/day p.o.
`2.5. mg/kg/day p.o.
`SDZ RAD
`20, 20, 25, 37, =78°
`26, =80, =100, 2100, =100
`
`RPM
`18, 22, 26
`35, =83, =83
`* As in Table 6.
`.
`
`TABLE 8, Prolongation of DA rat heart. allograft survival in Lewis
`rat recipients®
`Individual survival times (days after transplantation)
`after treatment
`Compound
`
`1.0 mg/kg/day
`2.5 mg/kg/day
`5,0 mg/kg/day
`12,14,14°
`18, 22, 25, 27, =28
`22, 33
`SDZ RAD
`
`RPM 22, 382 12, 15, 27 25, 31, 338, =81
`
`
`*The immunosuppressive compounds were administered p.o.
`daily continuously throughout
`the entire experiment. Placebo-
`treated animals showed severecellular rejection between day 7 and 10.
`> As in Table 6.
`
`not achieve long-term survival in this strain combination
`with SDZ RAD or RPM given alone, we did with a combina-
`tion of low doses of SDZ RAD and CsA,indicating synergy of
`the two compounds(25).
`
`DISCUSSION
`
`The immunosuppressant SDZ RAD is a novel RPM deriv-
`ative in which the hydroxyl at position 40 of RPM has been
`alkylated with a 2-hydroxyethyl group. The introduction of
`this functionalized side chain results in altered physicochem-
`ical properties with respect to RPM,ie., the solubility in
`several organic solvents and galenic excipients is markedly
`increased. Several C40-modified analogs of RPM,like esters,
`carbonates, and carbamates have been previously described
`in the patent literature. These derivatives can be viewed as
`prodrugs of RPM,as the newly introduced functional groups
`are known to besusceptible to hydrolytic cleavage under
`physiological conditions. The strategy we pursued was aimed
`at modifications that are resistant to hydrolytic and meta-
`bolic degradation. Therefore a series of 40-O-alkylated RPM
`analogs was prepared (26), of which the 40-O-hydroxyethyl-
`derivative, SDZ RAD, proved to be the most active represen-
`tative, both in vitro and in vivo. SDZ RAD binds with high
`affinity to FKBP12, which is a prerequisite for the inhibitory
`activity