SDZ RAD, A NEW RAPAMYCIN DERIVATIVE: Pharmacological... : Transplantation (original) (raw)
It was first reported in 1989 that the macrolide rapamycin (RPM*), a secondary metabolite of Streptomyces hygroscopicus, effectively suppresses the rejection of transplanted allogeneic 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 (CsA) and FK506. The latter drugs prevent T-cell proliferation 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 hematopoietic as well as nonhematopoietic cells tested so far (5-7), including vascular smooth muscle cells (VSMC) (8). The different 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 leading 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 interesting compounds for clinical transplantation. However, development of a proper oral RPM formulation with acceptable stability, bioavailability, and predictability has proven difficult and has impeded successful clinical development. So far, the majority of published preclinical work demonstrating the potent immunosuppressive effect of RPM deals with parenteral 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 pharmacokinetic 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 (Neoral; Sandoz, Basel, Switzerland) (18-20). The 40-O-(2-hydroxyethyl)-RPM, SDZ RAD, is a new RPM analog that resulted from our efforts to overcome the formulation problems by chemical derivation, while maintaining the pharmacological 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 dosing that is at least equivalent to that of RPM.
MATERIALS AND METHODS
Reagents
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 C53H83NO14, 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
The in vitro activity of RPM analogs was assessed by determining in the various assays the concentration of the compounds that results in 50% inhibition (IC50). Serial dilutions of the test compounds, done in duplicate, were tested, and a four-parameter logistic function was applied to calculate the IC50 values. RPM was included in each individual experiment as a standard, and the inhibitory activity was expressed as relative IC50 compared with RPM (i.e., given as the ratio IC50 of test compound/IC50 of RPM). To measure in vitro cell proliferation, [3H]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 competition 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 presence of the serially diluted test compound or standard. (For technical reasons we used FK506 as the standard, as an exception only in this assay). Bound biotinylated FKBP12 was assessed by incubation with a streptavidin-alkaline phosphatase conjugate, followed 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 (IC50) reflects the affinity of a compound for FKBP12.
IL-6-driven proliferation of a B-cell hybridoma. The hybridoma B13-29-15 is a subclone of 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 IC50 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 IC50 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 hemagglutinin peptide 307-319 were derived from peripheral blood mononuclear cells (PBMC) of a normal healthy volunteer as described (21). To determine the IC50 of the compounds in this antigen-specific T-cell proliferation assay, cloned T cells (2×104) were cultured in a total volume of 200 μl 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, together 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, [3H]thymidine was added, and the cells were harvested after another 16 hr.
Proliferation of VSMC. Bovine VSMC 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 with medium, and after about 10 days the cells grew out of the explants. The cells were characterized as VSMC by morphology in culture and by immunostaining with an anti-VSMC 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) supplemented with 10% fetal calf serum (FCS) and glutamine. For the experiments, the cells were seeded in 96-well plates (1×104 or 2×105 per well) and allowed to grow to confluence (3 days). They were then growth arrested by serum deprivation for 48 hr in serum-free medium (DF10 without FCS) supplemented with insulin (0.5 mM; Boehringer Mannheim, Mannheim, Germany), transferrin (5 μg/ml; Sigma), and ascorbate (0.2 mM; Sigma). After 3 days the medium was replaced with fresh medium containing 10% FCS and [3H]thymidine (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]thymidine 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 dilutions of the SDZ RAD and RPM formulations with water were given orally by gavage. Control animals received only the administration vehicle as placebo.
Localized graft-versus-host reaction. Spleen cells (2×107) from Wistar/F rats were injected subcutaneously into the right hind footpad 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 corresponding lymph nodes were determined. The results were expressed as the inhibition of lymph node enlargement (given in percent) comparing 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 glomerulonephritis 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 HgCl2 only, start to succumb to the disease at this point.
Orthotopic kidney allotransplantation. Donor kidneys were transplanted 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 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 abdomen. The time of rejection was defined as the day on which a heartbeat was no longer palpable.
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 hybridoma 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±0.7). The relative IC50 of SDZ RAD for inhibition of bovine VSMC was 1.9±0.75 (Table 2); however, 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-stimulated 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 lymphocytes 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 immunosuppressive activity of SDZ RAD is about two- and fivefold lower, respectively, in these assays.
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 vigorously 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 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). Inhibition was statistically significant with respect to the placebo-treated control; no statistically significant difference was found between SDZ RAD and RPM.
Mercuric chloride-induced glomerulonephritis. Low doses of mercuric chloride (HgCl2), repeatedly injected into rats, induce an autoimmune disease that is characterized by a T-dependent polyclonal B-cell activation (22,24). This polyclonal 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 glomerular damage; the animals develop a severe proteinuria within 2 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 HgCl2-induced 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 compound led to partial inhibition. In conclusion, SDZ RAD is effective in an animal model for autoimmune glomerulonephritis, 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 CsA results in the indefinite survival of the graft, a phenomenon that is restricted to rats and is not seen with any other species.
Table 6 shows the results for SDZ RAD and RPM in experiments 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. Untreated 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 prolongation of graft survival times was achieved, with one animal in the SDZ RAD group not rejecting its graft during the observation 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
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 considered 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 termination of the experiments 3 to 4 weeks after transplantation. Only moderate signs of rejection were found histologically in all groups, with the exception of the 1 mg/kg SDZ RAD group, in which rejection was severe. Although we did not achieve long-term survival in this strain combination with SDZ RAD or RPM given alone, we did with a combination of low doses of SDZ RAD and CsA, indicating synergy of the two compounds (25).
DISCUSSION
The immunosuppressant SDZ RAD is a novel RPM derivative 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 physicochemical 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 be susceptible to hydrolytic cleavage under physiological conditions. The strategy we pursued was aimed at modifications that are resistant to hydrolytic and metabolic 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 representative, both in vitro and in vivo. SDZ RAD binds with high affinity to FKBP12, which is a prerequisite for the inhibitory activity of immunosuppressants belonging to the RPM class (23). The binding of SDZ RAD to FKBP12 is about threefold weaker than that of RPM. This rather small loss of affinity can be attributed to the fact that C40-O-alkylation disrupts the hydrogen bond existing in the FKBP12/RPM complex between the C40 hydroxyl and the Gln-53 main chain carbonyl (27, 28). Steric interaction between the hydroxyethyl side chain of SDZ RAD and FKBP12 might also contribute to the slightly reduced binding. SDZ RAD has the same mode of action as RPM. At the molecular level SDZ RAD blocks, as RPM, activation of p70 S6 kinase (Schuler, unpublished results); blocking the activation of this kinase is one of the discussed explanations for the growth-inhibitory effect(s) of RPM (4). Further, like RPM, SDZ RAD at subnanomolar concentrations inhibits growth factor-driven cell proliferation, like that of a B-cell hybridoma or that of VSMC. Likewise, it inhibits mouse MLR and antigen-driven proliferation of antigen-specific human T-cell clones. In general, the in vitro activity of SDZ RAD is about two to three times lower when compared with RPM. The degree to which the in vitro activity is reduced is consistent with the observed decrease of affinity for FKBP12 and the demonstrated requirement of FKBP12 binding for immunosuppressive activity (23, 29).
When administered orally at doses between 1 mg/kg/day and 5 mg/kg/day, SDZ RAD is effective in relevant animal models comprising an autoimmune disease model and several allotransplantation models. It should be emphasized that it is impossible to compare directly our results obtained with SDZ RAD with results published by others on the in vivo efficacy of RPM because different galenical formulations and, in most cases, only parenteral administration routes were used in the studies reported in the literature. We therefore compared the oral activity of the two compounds by using our own galenical formulation of RPM, which was developed following the same principle as for SDZ RAD. These formulations were adapted for both compounds with respect to their physiochemical properties. Using this type of formulation, we have shown that SDZ RAD in vivo is at least equipotent to RPM when given orally. This is in contrast to the lower in vitro activity of SDZ RAD. This observed difference between the relative in vitro and the relative in vivo activity cannot simply be explained by differences in the exposure to the two compounds: treatment with the same doses of our RPM and SDZ RAD formulations led to comparable exposure to the two compounds. Daily treatment with 1.5, 5, and 15 mg/kg SDZ RAD resulted in area under the concentration-time curve values on day 28 of 435, 1468, and 6076 ng·h/ml, respectively. The same treatment with RPM led to area under the concentration-time curve values of 228, 1104, and 4071 ng·h/ml, respectively. Also, formation of RPM or pharmacologically active RPM metabolites cannot serve as a possible explanation. Any biotransformation pathway leading to the formation of RPM or its metabolites would require the cleavage of the hydroxyethyl side-chain by initial hydroxylation. From metabolism studies in vitro, as well as in animals, we do not have evidence that this happens to any significant degree, i.e., SDZ RAD is not a prodrug of RPM. Rather, we suggest as an explanation that the chemical modification in SDZ RAD, which altered its physicochemical properties, led to more favorable pharmacokinetic properties (e.g., disposition of compound), which compensate in vivo for the observed lower in vitro activity. Considering the relatively narrow therapeutic window of these drugs, more favorable pharmacokinetic properties promise to provide a clinical advantage, i.e., it should be easier to handle and to monitor such a drug in clinical practice.
Finally, a major unsolved problem in clinical allotransplantation is long-term graft loss due to chronic rejection. The pathogenesis of chronic rejection is complex and multifactorial in nature. A major factor contributing to late graft loss is intimal thickening of the graft vessels, which eventually leads to vessel occlusion (GVD). Intimal thickening is due to growth factor-driven proliferation and migration of VSMC after injury of the endothelium (for a review see 12). Clinical observations have shown a close correlation between the frequency as well as the intensity of acute rejection episodes and the incidence of chronic rejection (30-33). It can thus be inferred that one triggering event that eventually leads to chronic rejection is an alloimmune injury of the graft vessels. On the other hand, vascular damage resulting from nonimmunological events, e.g., from ischemia or reperfusion injury, also can lead to intimal thickening and may contribute to late graft loss (12). Taking these considerations into account, a two-pronged approach seems most promising for improving the long-term prospects in allotransplantation. This approach would provide improved immunosuppression, to prevent an alloimmune insult in the first place, combined with measures to prevent VSMC proliferation and/or migration. With respect to the first consideration, we have shown here that SDZ RAD efficiently prevents allograft rejection in the rat; in an accompanying publication (25) we report that in animal models for allotransplantation, SDZ RAD and CsA act in a synergistic manner. This synergism, if proven in humans, offers the chance to increase the efficacy of the immunosuppressive regimen by combining the two drugs, with the prospect of mitigating their respective side effects. We have further shown here that SDZ RAD efficiently inhibits the growth factor-stimulated in vitro proliferation of VSMC cells. Elsewhere we will report (H. Schuurman et al., manuscript in preparation) that SDZ RAD also inhibits in vivo vascular changes that were induced either by mechanical damage (balloon catheter injury) or by an alloimmune reaction (aorta transplantation in an allogeneic setting). We believe that the increased immunosuppressive efficacy of a drug combination composed of CsA and SDZ RAD, combined with the ability of SDZ RAD to inhibit VSMC proliferation, bears the potential for improving the prospects for long-term graft acceptance.
Acknowledgments. The authors thank Dr. J. Borel for critical review of the manuscript and comments, and Y. Hartmann, H. Jundt, J. Joergensen. T. Meerloo, U. Strittmatter, M. Tanner, B. Thai, C. Wilt, and C. Wioland for their expert technical assistance.
Chemical structure of SDZ RAD, 40-O-(2-hydroxyethyl)-RPM.
Footnotes
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, interleukin; MLR, mixed lymphocyte reaction; PBMC, peripheral blood mononuclear cells; RPM, rapamycin; VSMC, vascular smooth muscle cells.
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