ARGENTUM
`Exhibit 1036
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`July 15, 1997
`Volume 64
`Number 1
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`Contents
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`EDITORIAL I
`
` Tr-ansplantatiori
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`Official Journal of the Transplantation Society
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`A Welcome to the New Editors .
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`OVERVIEW
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`Balancing the immune system for tolerance: a case for regulatory CD4 cells.
`E.H. Field, Q. Gao, N. Chen, and T.M. Rouse .
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`EXPERIMENTAL TRANSPLANTATION
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`Massive repopulationof rat liver by transplantation of hepatocytes into specific lobes of the
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`liver and ligation of portal vein branches to other lobes.
`‘E; Y.
`llan, N. Roy Chowdhury, R. Prakash, V. Jona, P. Attavar, C. Guha, K. Tada, and
`, ‘J. Roy Chowdhury .
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`Synthetic MHC class I peptide prolongs cardiac survival and attenuates transplant arterio-
`sclerosis in the Lewis+Fischer 344 model of chronic allograft rejection.
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`J ‘B. Murphy, K.S. Kim, R. Buelow, M.H. Sayegh, and W.W. Hancock. .
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`Allo- and autotransplantation of carotid artery—-a new model of chronic graft vessel disease:
`evaluation by magnetic resonance imaging and histology.
`S. Wehr, M. Rudin, J. Joergensen, A. Hof, and R.P. Hof.
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`Long-term function of fish islet xenografts in mice by alginate encapsulation.
`H. Yang, W. O’l-iali, H. Kearns, and J.R. Wright, Jr.
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`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 .
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`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.
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`‘Mechanism of concordant corneal xenograft rejection in mice: synergistic effects of anti-
`leukocyte function-associated antigen-1 monoclonal antibody and FK506.
`S. Yamagami, M. lsobe, H. Yamagami, J. Hori, and T. Tsuru .
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`CLINICAL TRANSPLANTATION
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`Treatment of 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 .
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`A positive crossmatch in 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.
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`Value of the in vitro or in vivo monoethylglycinexylidide test for predicting liver graft function.
`P. Olinga, J.K. Marlng, G.M.M. Groothuis, K. Kranenburg, M. Merema, |.H. Hof, D.K.F. Meijer,
`and M.J.H. Slooff.
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`Country of origin USA. Subscription prices subject to change. To order call
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`acid-free paper.
`000002
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`000002
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`

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`0041-1337/97/6401-36$03.00/0
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`TRANSPLANTATION
`Copyright © 1997 by Williams & Wilkins
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Vol. 64, 36-42, No. 1, July 15, 1997
`Printed in USA.
`
`SDZ RAD, A NEW RAPAMYCIN DERIVATIVE
`
`PHARMACOLOGICAL PROPERTIES IN Vrmo AND IN VIVO
`
`WALTER SCHULER,1’2 RICHARD SEDRANL1 SYLVAIN CoTTENs,1 BARBARA HABERLIN,3
`MANFRED ScHULz,1 HENK-JAN ScHUURMAN,1 GERHARD ZENKE,1 HANS-GUNTER ZERwEs,1
`AND MAX H. SCHREIER1
`
`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-O-(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 mode of action that is 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-d.erivative that is immunosuppres-
`sive and that efficiently prevents graft rejection in rat
`models of allotransplantation. SDZ RAD has therefore
`been selected for development for use in combination
`with cyclosporine A to prevent acute and chronic re-
`jection after solid organ allotransplantation.
`
`It was first reported in 1989 that the macrolide rapamycin
`(RPM*), a secondary metabolite of Streptomyces hygroscopi-
`cus, effectively suppresses the rejection of transplanted allo-
`geneic solid organs in experimental animals (1, 2). RPM is of
`particular interest as a new immunosuppressant because its
`mode of action is different from that of both cyclosporine
`
`1 Preclinical Research, Novartis Pharma AG.
`2 Address correspondence.
`to: Walter Schuler, Preclinical Re-
`search, Novartis Pharma AG, S-386.1.26, CH-4002 Basel, Switzer-
`land.
`
`3 Technical Research and Development, Novartis Pharma AG.
`* Abbreviations: CsA,
`cyclosporine; FCS,
`fetal calf serum;
`FKBP12, FK506 binding protein; GVD, graft vessel disease; IC50,
`concentration of compound needed to reach 50% inhibition; IL, in-
`terleukin; MLR, mixed lymphocyte reaction; PBMC, peripheral blood
`mononuclear cells; RPM, rapamycin; VSMC, vascular smooth muscle
`cells.
`
`36
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`(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
`G1 stage of the 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-dependent proliferation in general of any hematopoi-
`etic as well as nonhematopoietic cells tested so far (5-7),
`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 compound for the prevention of late
`graft loss due to graft vessel disease (GVD); growth factor-
`driven proliferation of VSMC le ading to intimal thickening
`and eventually vessel obstruction seems to 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 analogs very interest-
`ing compounds for clinical transplantation. However, devel-
`opment of a proper oral RPM formulation with cceptable
`stability, bioavailability, and predictability has profyen diffi-
`cult and has impeded successful clinical development. So far,
`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 model of allotransplantation (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-
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`SCHULER ET AL.
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`37
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`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 was the optical density at 405 nm. Binding of a compound to
`the biotinylated FKBP12 resulted in a decrease in the amount of
`FKBP12 available for binding to the immobilized FK506, i.e., the
`magnitude of this inhibition (I050) reflects the affinity of a compound
`for FKBP12.
`
`SDZ RAD
`
`FIGURE 1., Chemical structure of SDZ RAD, 40-O-(2-hydroxyethyl)—
`RPM.
`
`si
`
`ical 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 has an efficacy after oral closing that is at least equiv-
`alent to that of RPM.
`
`MATERIALS AND METHODS
`
`Ra agents
`
`RPM was obtained by fermentation of the actinomycetes strain
`A91—259211. SDZ RAD, 40-O-(2-hydroxyethyl)-RPM (Fig. 1), was
`derived by chemical derivation of RPM; the molecular formula is
`053H00NO14, and it has a molecular weight of 958.25. For the in vitro
`experiments, 10‘3 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 formulations are
`stable for >3 months‘.
`
`In Vitro Assays
`
`IL-6-driven proliferation of a B-cell hybridoma. The hybridoma
`B18—29-15 is a subclone of the hybridoma Bl3—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 I050 of a
`compound in this assay, 104 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. [3H]thymidine was added at the end of
`the incubation period, 5 hr before harvesting the cells for measuring
`the [3H]thymidine incorporation into DNA.
`'
`Mixed lymphocyte reaction (MLR). To determine the I050 values
`of the compounds in a two-way MLR, 105 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 [3H]thymidine was
`added, and the cells 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 307-319 were derived from peripheral blood mononu-
`clear cells (PBMC) of a normal healthy volunteer as described (21).
`To determine the I050 of the compounds in this antigen-specific
`T-cell proliferation assay, cloned T cells (2><104) were cultured in a
`total volume of 200 pl of RPMI medium (supplemented to contain 5%
`human AB serum) in 96-well round—bottom microtiter plates with
`105 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 PBMO 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, [3I-I]thy-
`midine was added, and the cells were harvested after another 16 hr.
`Proliferation ofVSM0. Bovine VSMO were derived by the explant
`technique from small pieces of media (dissected free of adventitia
`and intima) from fresh bovine aortae. Explants of about 1 mm3 were
`placed in culture dishes, covered withmedium, and after about 10
`days the cells grew out of the explants. The cells were characterized
`as VSMO by morphology in culture and by immunostaining with an
`anti-VSMO actin antibody (clone 1A4; 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 Eagle’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 (IX 104 or 2X 105
`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 ug/ml;
`Sigma), and ascorbate (0.2 mM; Sigma). After 3 days the medium
`was replaced with fresh medium containing 10% FCS and [3H]thy-
`midine (1 mCi/ml), together with serial dilutions of the compounds to
`be tested (three to four replicate wells for each concentration). The
`cells were harvested after a 24-hr incubation period and the [3H]thy-
`midine incorporation into the DNA was measured.
`
`The in vitro activity of RPM analogs was assessed by determining
`in the various assays the concentration of the compounds that re-
`sults in 50% inhibition (1050). Serial dilutions of the test compounds,
`done in duplicate, were tested, and a four-parameter logistic function
`was applied to calculate the 1050 values. RPM was included in each
`individual experiment as a standard, and the inhibitory activity was
`expressed as relative I050 compared with RPM (i.e., given as the
`ratio I050 of test compound/I050 of RPM). To measure in vitro cell
`proliferation, [3I'I]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 FK506
`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
`Throughout all of the in vivo experiments the compounds were
`presence of the serially diluted test compound or standard. (For
`technical reasons we used FK506 as the standard, as an exception
`given once daily, for the indicated period of time. Freshly prepared
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`TRANSPLANTATION
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`Vol. 64, No. 1
`
`dilutions of the SDZ RAD and RPM formulations with water were
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`given orally by gavage. Control animals received only the adminis-
`tration vehicle as placebo.
`Localized graft-versus-host reaction. Spleen cells (2><107) from
`Wistar/F rats were injected subcutaneously into the right hind foot-
`pad of (Wistar/F >< Fisher 344)F1 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. Autoimmune glo-
`merulonephritis was induced by treatment with HgCl2 (22). Female
`Brown Norway rats, 9 weeks of age, were injected subcutaneously
`during a 3-week period, three times per week, with 1 mg of HgCl2 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 I-IgCl2 only, start to succumb to the
`disease at this point.
`Orthotopic kidney allotransplantation. Donor kidneys were trans-
`planted orthotopicpally 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 into the abdomen of recipient 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 shown in 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 explained by its
`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
`(i.e., relative IC50 of 2.5 i'O.7). The relative IC50 of SDZ RAD
`
`000005
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`Compound
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`FK506
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`RPM
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`SDZ RAD
`
`TABLE 1. Binding to FKBP12“
`Relative IC50:SDb
`(range, absolute IC50)
`1
`(O.8—1.2 nM)
`0.6i0.2* (n=5)
`(0.4—O.9 nM)
`2.0i0.4*** (n=3)
`(1.8~2.6 nM)
`
`“The ability of the compounds to compete with immobilized
`FK506 for binding to biotinylated FKBP12 was determined in a
`competitive binding assay.
`1’ FK506 was included as a standard in each individual experi-
`ment. Results are expressed as means:SD of the relative IC50 values
`(i.e., ratio of IC5o test compound to IC50 of FK506). The range of
`absolute IC50 values is given in parenthesis; n=number of individual
`experiments. Statistical analysis, t test: *P<0.05; ***P<0.001.
`
`TABLE 2. Inhibition of growth factor-stimulated cell proliferation
`Relative IC50iSD“
`(range, absolute I050)
`I-Iybridoma B13-29-15/IL-6
`Bovine VSMC/FCS
`
`Compound
`
`RPM
`
`SDZ RAD
`
`1
`<o.o7—0.5 nM)
`2.5:o.7** (n=5)
`(0.2—1.4. nM)
`
`L
`
`1
`<0.4—3.5 nM)
`1.94.-0.75“ (n=3)
`(0.9—3.6 nM)
`
`“ RPM was included as a standard in each individual experiment.
`Results are expressed as means:SD of the relative ICED values (i.e.,
`ratio of IC50 test compound to IC50 of RPM). The range of absolute
`ICE-,0 values is given in parenthesis; n=number of individual exper-
`iments. Statistical analysis, t test: **P<0.01; ’"’‘not significant.
`
`for inhibition of bovine VSMC was 1.9i0.’75 (Table 2); how-
`ever, this was not statistically significant when compared
`with inhibition by RPM. The absolute IC50 values found here
`for RPM are in agreement with those reported for platelet-
`derived growth factor or basic fibroblast growth factor-stim-
`ulated VSMC proliferation [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 lymphocyte of mouse
`origin as well as in experiments with antigen-spedlfic 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 3. Immunosuppressive activity in vitro“
`Relative IC50iSD”
`(range, absolute I050)
`T-cell clone
`
`MLR
`
`Compound
`
`RPM
`
`SDZ RAD
`
`1
`(0.06—0.9 nM),
`2.1:0.4* (n=4)
`(0.2—1.6 nM)
`
`‘
`
`1
`(0.014—0.037 nM)
`5.4:3.5* (n=3)
`(0.05—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, t test:" *P<0.05.
`
`000005
`
`

`

`July 15, 1997
`
`SCHULER ET AL.
`
`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
`spleen cells into one hind footpad of F1 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 of the popliteal lymph node from
`the site of injection with that of the untreated contralateral
`lymph node gives an indication of the severity of the“ reaction.
`SDZ RAD effectively inhibited lymph node swelling elicited
`by this localized graft-versus-host reaction. This is shown in
`Table 4. Maximal inhibition of about 7 0—80% was achieved
`with an oral dose of 8 mg/kg per day of either SDZ RAD or
`RPM. (Increasing the doses of SDZ RAD or RPM did not lead
`to stron er inhibition of lymph node swelling; data not
`
`shown). fiiiihibition was statistically significant with respect
`
`to the pla ebo-treated control; no statistically significant dif-
`ference was found between SDZ RAD and RPM.
`Mercuric chloride-induced glomerulonephritis. Low doses
`of mercuric chloride (I-IgCl2), 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 of a variety of
`autoantibodies. Antibodies directed against the glomerular
`basement membrane cause infiltration of polymorphonuclear
`granulocytes and glomerulé. 2‘ damage; the animals develop a
`severe proteinuria withinlz to 3 weeks of treatment with
`HgCl2. As can be seen from Table 5, a dose of 1.25 mg/kg/day
`of SDZ RAD or RPM completely prevented this I-IgCl2-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 HgCl2 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 glomerulone-
`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
`
`TABLE 5. Mercuric chloride-induced glomerulonephritis
`Development of proteinuria
`
`Compound Vehicle
`Control
`10/10“
`SDZ RAD
`RPM
`
`0.3 mg/kg p.o.
`
`0.6 mg/kg p.o.
`
`1.25 mg/kg p.o.
`
`8/9
`5/5
`
`7/11**
`2/5**
`
`0/9***
`1/5**
`
`“ Number of 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;
`***P<0.001.
`’
`
`CsA results in the indefinite survival of the graft, a phenom-
`enon that is restrictedpto rats and is not seen with any other
`species.
`,
`.
`Table 6 shows the results for SDZ RAD and RPM in exper-
`iments transplanting kidneys from (Wistar/F >< Fisher
`344)F1 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 thus be 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, i.e.—, 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 most of
`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 not rejecting 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 showed severe cellular
`rejection.
`
`Vascular Heterotopic Heart Allotransplantation
`
`TABLE 4. Inhibition of localized graft-versus-host reaction
`
`Compound
`
`N0_ of
`animalsb
`
`SDZ RAD
`RPM
`
`5
`5
`
`' Percent inhibition of lymph node swelling“
`3.0 mg/kg/day p.o.
`1.0 mg/kg/day p.o.
`58:23**
`77—_i:9>i<:i<*
`66i11***
`61t22**
`
`“ Lymph node weight differences were determined on day 7. Re-
`sults are given as mean v‘alues:SD of inhibition compared with a
`control group of five animals that received the vehicle only. The
`weight differences between the respective lymph nodes from animals
`in the control group was 34:2 mg. Statistical analysis (analysis of
`variance) with respect to the control group: **P<0.01; ***P<0.001.
`1’ Number of animals in each dosage group.
`
`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
`combination represents a very strong mismatch and is con-
`sidered the most stringent rat transplantation model. As
`Table 8 shows, we were unable to achieve long-term graft
`survival with any dose of SDZ RAD or RPM tested, even
`though we treated the animals daily until rejection occurred.
`Increasing the dose from 2.5 to 5 mg/kg of either compound
`did not improve graft survival times; rather, the higher doses
`led to severe weight loss under these conditions, forcing ter-
`mination of the experiments 3 to 4 weeks after transplanta-
`tion. Only moderate signs of rejection were found histologi-
`cally in all groups, withthe exception of the 1 mg/kg SDZ
`RAD group, in which rejection was severe. Although we did
`000006
`
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`
`

`

`TABLE 6. Suppression of allograft rejection of (Wistar/F><Fisher344)F1 rat donor kidneys in Wistar/F recipients“
`Individual survival times (days after transplantation) after treatment
`
`TRANSPLANTATION
`
`Vol. 64, No. 1
`
`Compound
`
`SDZ RAD
`RPM
`
`7, 7, 7, 7, 7, 7, 2151,2100, 2100"
`7, 7, 7, 7, 7, 7, 12
`
`2100, 2100, 2100, 2100, 2100
`7, 2100, 2100, 2100, 2100
`
`0.25 mg/kg/day
`
`0.6 mg/kg/day
`
`1.0 mg/kg/day
`
`2100, 2100, 2100
`2100, 2100, 2100
`
`“ The immunosuppressive compounds were administered p.o. daily from day 0 through day 13 at the indicated dose. Placebo-treated
`animals showed severe cellular rejection by day 7.
`1’ Each figure indicates the survival time after transplantation of an individual animal. More than or equal-to sign (2) 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
`treatment
`
`Compound
`
`1.0 mg/kg/day p.o.
`
`2.5 mg/kg/day p.o.
`
`snz RAD
`RPM
`
`20,20, 25, 37, 278“
`18, 22, 26
`
`26, 280, 2100, 2100,2100
`35, 283, 283
`
`“ 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
`2.5 mg/kg/day
`
`1.0 mg/kg/day
`
`5.0 mg/kg/day
`
`Compound
`
`SDZ RAD
`RPM
`
`12,14,145
`1215,27
`
`18,22, 25, 27, 228
`25,31,33,281
`
`22,33
`22,32
`
`“The immunosuppressive compounds were administered p.o.
`daily continuously throughout
`the entire experiment. Placebo-
`treated animals showed severe cellular rejection between day 7 and 10.
`I’ 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, i.e., 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 beflsusceptible to hydrolytic cleavage under
`physiological conditions. The strategy we pursued was aimed
`at