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Original Articles: Experimental Transplantation

Combined Use of the JAK3 Inhibitor CP-690,550 with Mycophenolate Mofetil to Prevent Kidney Allograft Rejection in Nonhuman Primates

Borie, Dominic C.1,4; Larson, Michael J.1; Flores, Mona G.1; Campbell, Andrew1; Rousvoal, Geraldine1; Zhang, Sally1; Higgins, John P.2; Ball, Douglas J.3; Kudlacz, Elizabeth M.3; Brissette, William H.3; Elliott, Eileen A.3; Reitz, Bruce A.1; Changelian, Paul S.3

Author Information
doi: 10.1097/01.tp.0000184634.25042.ea
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Abstract

Despite great progress achieved over the past 30 years in the field of immunosuppression, there is a clear and significant need for a new generation of immunosuppressive agents that are effective, yet lack the toxicity associated with current agents (1–5). Janus kinase 3 (JAK3) inhibitors are amongst the newest molecules specifically developed with the intent of achieving efficacy (i.e., potent immunosuppression), while at the same time being devoid of the nonimmune-related side effects reported with most immunosuppressive drugs currently in use (6, 7). JAK3 inhibitors block signaling downstream of γc-containing cytokine receptors and thereby blunt signals emanating from at least six T-cell growth factors (IL (interleukin)-2, IL-4, IL-7, IL-9, IL-15 and IL-21) receptors (8–10). We have recently reported the development of CP-690,550, an orally active JAK3 inhibitor with nanomolar potency against JAK3 in enzymatic assays, which has demonstrated promising initial results in allotransplantation models (11, 12). Notably, we have shown that JAK3 inhibition with CP-690,550 affords prolonged organ allograft survival and delays the onset of graft rejection in a stringent preclinical model developed in nonhuman primates (NHPs) (12, 13). As a subsequent step towards the introduction of a new JAK3 inhibitor in the clinical arena, we were interested in testing its effects in the prevention of organ allograft rejection when combined with other established immunosuppressive drugs. Because calcineurin inhibitor-free regimens are being aggressively explored (14–17), we chose to combine CP-690,550 with the inosine monophosphate dehydrogenase (IMPDH) inhibitor mycophenolate mofetil (MMF), a drug that complements JAK3 inhibition-induced signal 3 blockade by inhibiting cell cycling and immune cell proliferation (18–22). We report here, for the first time, the combined use of CP-690,550 and MMF in a preclinical model in NHPs and show that both drugs given as part of an immunosuppressive regimen afford prolonged allograft survival and delay the onset of acute cellular rejection.

MATERIALS AND METHODS

Animals and Renal Transplantations

All animals involved in this study received humane care in accordance with The Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, Revised 1996). This study was approved by the Stanford University Administrative Panel on Laboratory Animal Care. Mauritius-origin adult male cynomolgus monkeys (Macaca fascicularis) weighing between 6 and 7.5 kg were obtained from Biomedical Resource Foundation (Houston, TX). Donor and recipient pairs were based on an ABO blood group match and a stimulation index of at least 6.0 in a one-way mixed leukocyte reaction (MLR). Life-supporting kidney transplantations were performed as described in detail elsewhere (23). In brief, transplant procedures were staggered: an animal first served as a donor, was allowed to recover for 4 weeks, and was subsequently used as a recipient. After graft reperfusion, the recipient remaining native kidney was removed making the transplant life-supporting.

Study Design

Eleven NHP transplant recipients were included in this study. Two animals received the IMPDH inhibitor mycophenolate mofetil (MMF; Cellcept, Roche Laboratories Inc., Nutley, NJ) alone, dosed twice a day (BID) to produce suboptimal immunosuppression. This was achieved by targeting residual 12-hour trough levels of 2 μg/ml, as we have shown in the past that this level of exposure corresponds to suboptimal pharmacodynamic effects (20). Cellcept oral suspension was obtained from the Stanford University Central Pharmacy and reconstituted as per the manufacturer's instructions. Nine other animals received an immunosuppressive regimen consisting of the JAK3 inhibitor CP-690,550 developed at Pfizer (Groton, CT), dosed to produce various levels of exposure (see below), and Cellcept, the latter administered at suboptimal exposure, as for the monotherapy animals. By design, and on the basis of data generated earlier in monotherapy studies (13), CP-690,550 was administered to target various (either high or low) levels of exposure as reflected by the area under the time-concentration curve (see results). No steroids were used in any animals of the series.

All animals were sedated twice daily with an intramuscular injection of 5–10 mg/kg ketamine hydrochloride (Ketaset, Fort Dodge Lab, Fort Dodge, IA). Blood samples for 12-hour trough level measurement were collected three times weekly prior to morning dosing. Blood samples were also obtained 3 and 12 hr after the morning dose every Friday for subsequent determination of exposure to CP-690,550 (area under the time-concentration curve; AUC0–12). Immunosuppressive drug dosage adjustments were made as appropriate based upon levels measurements obtained with an Agilent Technologies LC/MS, 1100 series (Agilent Technologies, Palo Alto, CA) as described in detail elsewhere (24, 25).

When appropriate, results observed in animals of the current series were compared to those observed in a series of animals operated under identical conditions and that received either CP-690,550 as monotherapy (n=18) or its vehicle (n=3) and that have been reported in detail elsewhere (13).

Posttransplantation Monitoring

Clinical Follow-up and Laboratory Tests

Urine output and fluid intake were recorded every 12 hours. Vital signs, appetite, attitude and bowel movements were recorded daily. Euthanasia was performed for rejection-induced oliguria or anuria, serum creatinine > 8 mg/dl, illness or severe complication or, at day 90 posttransplantation (study termination). When weight loss exceeded 10% compared to the animal's weight at the time of surgery, caloric supplementation was given in the form of a calorie-dense gruel. Laboratory tests were performed at least twice a week. Reticulocyte counts were performed once a week. Finally, urinalysis was performed on all animals approximately 7 days postsurgery to screen for urinary tract infection.

Lymphocyte Subsets

Blood was drawn weekly and stained with fluorochrome-conjugated anti-CD3ε, anti-CD4, anti-CD8, anti-CD16, anti-CD20 and appropriate isotype control monoclonal antibodies (BD Biosciences Pharmigen, San Diego, CA). Lymphocyte subsets were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) using the software CellQuest Pro. To permit group analysis of animals with different survival times and outcomes, two individual posttransplantation time-points were considered and compared to pretransplantation values: a) two weeks after transplantation and initiation of drug therapy, and b) at terminal rejection or completion of study.

Biopsies and Histology

Percutaneous renal transplant biopsies were obtained on postoperative days 27, 41 and 69. Renal allograft tissue obtained at the time of necropsy was fixed, sectioned and stained with hematoxylin and eosin. Pathology specimens were reviewed by a single experienced renal pathologist (J.P.H.) blinded to the clinical course of the recipient and scored according to the modified Banff criteria for acute renal allograft rejection (26).

Statistics

Statistical analyses were performed using the statistical program SPSS for Windows, version 10.0 or SigmaStat version 2.03 (both from SPSS Inc., Chicago, IL). Values are shown as mean ± standard deviation unless otherwise indicated. Univariate analysis was performed with the unpaired, independent samples, two-tailed Student's t test. Cell subsets were analyzed with a one-way analysis of variance run on ranks when appropriate. Subsequent pairwise multiple comparison procedures (Dunn's method) were run when significant differences where found between groups. Animal survival was analyzed by the Kaplan-Meier method and groups compared with the Log rank test. P less than or equal to 0.05 was considered as significant.

RESULTS

Treatment with the combination therapy regimen CP-690,550/MMF significantly prolongs survival and delays the onset of acute allograft rejection following renal transplantation.

To determine the efficacy of the combination therapy regimen CP-690,550/MMF in preventing acute allograft rejection, we evaluated these agents in a model of life-supporting renal allotransplantation developed in NHPs (23). Survival times and pathology at necropsy for each of the 11 treated animals are summarized in Table 1. One animal in the combination therapy group experienced primary graft nonfunction, was euthanized on day 3 and was excluded from subsequent analyses. The MMF (n=2) and combination therapy groups (n=8) were similar in MLR stimulation indices (43±14 (SD), vs. 28±1, P=NS), early graft function as reflected by urine output on day 0 (581±436 vs. 452±155 ml, P=NS) and mean duration of graft cold ischemia (47±4 vs. 50±5 min., P=NS). For those same criteria, the MMF and combination therapy animals were not significantly different from previously reported (13) vehicle-treated animals (n=3) and CP-690,550 monotherapy-treated animals (n=18), with the exception of the duration of cold ischemia which was slightly longer in the vehicle-treated group when compared to either MMF alone or the combination therapy group (64.0±6.2 min., P<0.05 vs. either group). We compared survival times for respective groups of animals and performed additional analyses after combination therapy animals were divided according to exposure to CP-690,550 (group combo high: median postoperative area under the time-concentration curve (AUC0–12); 550< AUC0–12<2500; n=5; group combo low: 350< AUC0–12<550; n=3, see below).

T1-21
TABLE 1:
Inclusion and pharmacokinetic characteristics of cynomolgus monkeys recipients of life-supporting kidney allografts immunosuppressed with MMF in the presence or absence of CP-690,550

Historical vehicle-treated control animals had a mean survival time (±SEM) of 7.0±0.6 days (95% confidence interval (CI): 5.9, 8.1 days) and were euthanized on postoperative day 6, 7 and 8 due to severe (Banff type III) acute rejection. Animals treated with MMF alone had significantly prolonged survival as compared to control animals, resulting in a mean survival time of 23.0±1.0 days (95% CI 21, 25 days; P=0.04) (Fig. 1A). Addition of CP-690,550 to suboptimal MMF resulted in a significant prolongation of the survival time of treated animals to 59.5±9.8 days (95% CI: 40, 79 days, P=0.02 vs. MMF alone animals). In the combination therapy group, animals that were exposed to higher levels of CP-690,550 had a significantly better survival (75.2±8.7 days, 95% CI:58, 92 days) than animals that received less CP-690,550 (33.3±12.6 days, 95% CI:8, 58 days, P=0.02). Survival time in high-exposure combination therapy animals was significantly longer than that in MMF alone animals (P=0.008) whereas that in low-exposure combination therapy animals was not (P=0.36).

F1-21
FIGURE 1.:
Survival curves and main clinical and biological parameters following kidney allograft transplantation in nonhuman primates. (A) Kaplan-Meier survival curves for transplanted animals. Survival plots are shown according to the immunosuppressive regimen received: MMF alone (n=2, dotted line), and CP-690,550/MMF combination therapy group (n=8, solid line). For comparison, survival curves of animals treated with vehicle for CP-690,550 (n=3, dashed line) and animals treated with CP-690,550 as monotherapy (n=18, interrupted line), as reported in details elsewhere (13), are plotted. Combination therapy significantly improved survival over MMF alone (Log Rank, P=0.02, see text for details). Survival in the combination therapy group is not significantly different from that previously observed in a series of animals treated with CP-690,550 at various levels of exposure (Log Rank, P=0.6, see text for details). (B) Trends in serum creatinine levels in combination therapy animals. The horizontal superimposed dotted line represents the higher limit of normal for that variable at our laboratory. (C) Evolution of MMF trough levels (closed triangles) in a kidney transplant recipient (49537) dosed with MMF at monotherapy (closed circles). Compound accumulation concurrent with elevation of serum creatinine levels (open circles) results in subsequent amelioration of serum creatinine levels and forces drastic dose reduction to maintain target trough levels. (D) Average CP-690,550 AUC0–12 (12-hour area under the time-concentration curve) in consecutive animals treated with CP-690,550 as monotherapy (CP5-CP17) or as part of the combination regimen with MMF. Starting with CP-10, animals were exposed to CP-690,550 at levels similar to that later used in combination therapy animals and were used as a comparator group. (E) Trends in hemoglobin (Hgb) levels in combination therapy animals (closed circles) and animals treated with MMF alone (open circles). Hgb levels plateau after an initial drop after transplantation. Percentage reticulocytes (closed triangles) in combination therapy animals stay on average under 5% throughout the postoperative course. (F) Averaged absolute cell numbers in combination therapy animals. No significant changes are seen for T and B cells at the time-points considered. To the contrary, a trend towards reduced NK cell numbers is seen with a significant reduction of NK cell numbers at necropsy when compared to pretransplantation (PreTx) numbers (P=0.02).

Survival time in the combination therapy group of eight animals was not significantly different from that observed in a historic group of animals (n=18) treated with CP-690,550 as monotherapy (53.2±6.8 days, 95% CI: 40,66 days, P=0.6) (13). The latter group, however, included animals with a wide range of drug exposure (13). We therefore subsequently selected a subgroup of 7 consecutively transplanted animals (CP10, 11, 12, 13, 14, 16, 17) that represented our most recent experience with CP-690,550 as monotherapy at efficacious exposure. Daily dosing (data not shown) and average exposure (Fig. 1D) for these animals were similar to that achieved in the combination therapy animals (CMB1-9). For comparison, the average AUCs for earlier animals who were dosed at much higher exposures (CP5-9) and therefore not selected for the current analysis are also shown. Survival time for this CP-690,550 monotherapy subgroup (53.1±10.8 days, 95% CI: 32, 74 days), although slightly shorter, was not significantly different from that of the combination therapy animals (59.5±9.8 days, Log Rank; P=0.69).

Three of the eight combination therapy animals completed the 90-day study and were euthanized with a subnormal renal function and acute cellular rejection (Banff type IA; n=1, type IB; n=2) on final pathology evaluation. All other animals experienced renal failure due to varying degrees of allograft rejection at necropsy (Table 1). In several instances, rejection was delayed by treatment as evidenced by limited changes (e.g., no evidence of rejection, borderline changes) found on staged protocol biopsies (Table 2).

T2-21
TABLE 2:
Pathology results on protocol kidney biopsies performed in NHPs recipient of life-supporting kidney allografts immunosuppressed with CP-690,550 and MMF

Renal Function in Treated Animals

All animals but one recovered normal serum creatinine (Fig. 1B) and blood urea nitrogen (BUN) levels (data not shown) shortly after graft implantation. With the exception of the three long-term survivors, most combination therapy animals then developed progressive increases in serum creatinine levels that ultimately mandated sacrifice. Two of the long-term animals sacrificed with near normal renal function (121642, 120329) had, however, experienced abnormal serum creatinine levels earlier in their postoperative course that had subsequently and spontaneously returned to normal. In this regard, these two animals paralleled the postoperative course of two animals treated with MMF alone in which initial renal failure episodes manifesting as increased serum creatinine levels resolved as the immunosuppressive drug accumulated concurrent with impairment of renal excretion (Fig. 1C). None of the animals in the current series developed urinary accretions. In three cases, final pathology evaluation found indication of past episodes of pyelonephritis in addition to acute cellular rejection changes. No evidence of polyoma virus nephritis was found in this series.

Laboratory Parameters in Transplanted Animals Treated with CP-690,550

Serum chemistry results are listed in Table 3. All results were within normal range, with the exception of lower than normal total protein levels and slightly increased triglyceride levels. Because of the potential for blockade of the erythropoietin (EPO) receptor by transient inhibition of JAK2 (9), and of the known potential for anemia associated with MMF therapy, hemoglobin (Hgb) levels and reticulocyte counts were monitored in treated animals. Initial reduction of Hgb levels after transplantation was observed in both MMF alone and combination therapy animals (Fig. 1E). This was accompanied in the combination therapy-treated animals by a slight increase in reticulocyte counts (average of 2.4±2.3%) which, however, generally stayed under 5% and did not allow long-term survivors to recover normal hemoglobin levels at completion of study. None of the animals in this series received recombinant human erythropoietin (EPO) postoperatively.

T3-21
TABLE 3:
Laboratory tests in nonhuman primate recipients of renal allografts immunosuppressed with CP-690,550 and MMF

FACS Analysis of Lymphocyte Subsets in Animals Dosed with CP-690,550

Analysis of complete blood cell counts in combination therapy animals revealed reduced lymphocyte numbers at the time of necropsy that were, however, not different from numbers observed prior to transplantation (median counts of 846 vs. 2592 cells/μl, respectively, P=0.09). Neither CD4+, CD8+ T cells, nor CD20+ B cell numbers at the time-points tested after surgery were different from pretransplantation numbers (Fig. 1F). To the contrary, a progressive reduction of NK cell numbers was observed postoperatively with significantly reduced numbers at necropsy as compared to pretransplantation numbers (median counts of 29 vs. 266 cells/μl, respectively, P=0.02).

Pharmacokinetics of Oral Dosing with MMF and CP-690,550

Results of MMF and CP-690,550 blood levels at trough (Cmin) concentration, and exposure to CP-690,550 as reflected by 12-hour area under the concentration-time curves (AUC0-12's) are summarized on Table 1. Although the average MMF daily dosing in MMF alone animals was significantly lower than that used in combination therapy animals (15.9±13.2 vs. 22.2±8.3 mg/kg, P<0.001), the average trough levels were not significantly different (2.7±2.0 vs. 2.1±1.5 μg/ml, respectively, P=NS). Although survival time of CP-690,550 “high-exposure” combination therapy animals was significantly longer than that of “low exposure” animals, no correlation was found between CP-690,550 median AUC0–12's values and survival time (Pearson product moment coefficient, r=0.6).

Tolerability of Oral Dosing with CP-690,550

To the exception of the one animal with early graft failure, postoperative recoveries were smooth and uneventful, followed by a return of normal behavior and appetite within several days. Urine production and serum creatinine values were normal in eight of the nine cases by postoperative day 3. All surgical wounds healed normally and without complications. The combination regimen was, however, in general, poorly tolerated in NHPs mostly because of gastro-intestinal intolerance (vomiting or constipation and loss of appetite), weight loss, and anemia that, in one instance, required whole blood transfusion immediately prior to sacrifice. Temporary enteral nutritional support was intermittently provided for five of eight animals and the average weight loss observed in the group of combination therapy animals was 8.5% over the term of the study. Some animals experienced symptoms compatible with septic phenomenon (e.g., hypothermia in 121611, persistent leukocytosis in 121642) in the absence of any documented pathogen, while moderate bacteriuria was diagnosed in another animal (41321). Aside from that latter case, there was no other documented case of either bacterial or viral infection.

At the time of necropsy, an enlarged mesenteric lymph node was found in animal 2211, which at pathology was found to consist in a developing lymphosarcoma. No other focus of malignant proliferation was found in this animal or in any of the other animals of the series.

DISCUSSION

Following the demonstration of the potency of JAK3 inhibition with CP-690,550 in the prevention of acute allograft rejection in various animal models (11–13), our most recent aim was to test, in a preclinical model, an immunosuppressive regimen combining CP-690,550 with another clinically used immunosuppressive drug, MMF. In this series, we found that combining CP-690,550 with suboptimal MMF dosing contributed to significantly improved graft survival in comparison to animals that received only MMF. Moreover, three of eight animals (38%) that received the combination therapy completed the 3-month dosing period and were sacrificed with near normal renal function. A dose-effect response was observed in the combination therapy group in which animals that received CP-690,550 at high exposure survived significantly longer than others that received less CP-690,550. The observed effects resulted mostly from the exposure to CP-690,550 as, when combination therapy animals were compared to a historic group of animals immunosuppressed with CP-690,550 at similar levels of exposure, the addition of suboptimal MMF did not improve survival nor graft pathology. We have no explanation for the fact that MMF monotherapy animals required less MMF than combination therapy animals to achieve identical trough levels. A direct drug-drug interaction is unlikely as extensive PK studies performed in cynos prior to the current efficacy study did not show any alteration of either drug PK induced by the combination (unpublished data). As graft outcome may not necessarily correlate well with MPA plasma level (20) future studies might benefit from MPA AUC monitoring. To determine precisely whether combination of both drugs might reveal additive or synergistic effects, additional studies combining both drugs, each delivered at suboptimal exposure are warranted. Until results of such studies become available, data from the present study suggests that both drugs can be combined without overtly compromising respective efficacies, a fact that needed to be verified with this yet untested immunosuppressive combination regimen.

Combination therapy produced long-term (90 days) surviving animals that reached the study endpoint with normal or near normal renal function and produced results similar to those seen in our previous experience with CP-690,550 administered as monotherapy. Similarly, protocol staged biopsies showed that occurrence of acute cellular rejection was delayed in many animals. Despite the combination therapy, we could not see a clear improvement in the grading of biopsies and most 3-month surviving animals displayed, at necropsy, inflammatory changes consistent with the diagnosis of acute cellular rejection. In human kidney transplant recipients, a 30% prevalence of subclinical rejection has also been reported in the first 3 months posttransplantation (27, 28). The prevalence of type I or greater rejection at 3 months was 25% and 13% in cyclosporine and tacrolimus treated patients, respectively, and both groups had a prevalence of borderline changes of about 30% (29, 30). In NHPs, we (31, 32) and others (33) have also observed fluctuant subclinical inflammation of varying degrees in the early posttransplantation period. Because of the limited follow-up we cannot therefore conclude as to whether those changes were consistent with upcoming rejection. The inclusion of additional immunosuppression (e.g., induction therapy and/or steroids) should therefore be considered in future clinical trials testing the combination regimen.

The potential for overwhelming sepsis is a concern when combining two potent immunosuppressive drugs. Although pathological evaluation of the graft of three animals displayed changes that suggested past episodes of pyelonephritis, mild bacteriuria was diagnosed in only one case and furthermore resolved with antibiotic treatment. Although no changes consistent with past pyelonephritis (i.e., segmental atrophy and fibrosis) were seen in animals treated with CP-690,550 as monotherapy, such changes were, on occasion, encountered in animals treated with other immunosuppressive regimen in this species (our unpublished observations). Whereas 11% incidence of polyoma virus (BK) nephritis cases was reported in animals receiving CP-690,550 as monotherapy (13), and with the caveat of a relatively short follow-up, neither BK nephritis nor any other viral infections were documented in the current series. This is interesting considering that incidence of BK nephritis of 5–10% and 21% have been reported in human patients treated with high-dose immunosuppressive regimens containing tacrolimus and/or mycophenolate mofetil (34–36) and in NHP (37) transplant recipients treated with other major immunosuppressants, respectively. It should be noted that the two animals on CP-690,550 monotherapy who developed evidence of BK virus infection were dosed at extremely high exposures (CP6 and CP7, average AUC0–12 hr, 4,000 ng-hr/ml). In contrast, none of the seven monotherapy animals dosed at exposures comparable to the combination group developed viral infections.

As with CP-690,550 monotherapy, combination therapy with MMF did not induce radical changes of serum chemistry results. Notably, blood glucose and lipid profile abnormalities reported in NHPs undergoing tacrolimus and sirolimus therapy (38) were not observed in our series. These observations will obviously need to be expanded to be able to formally conclude with regard to the putative metabolic effects—or lack thereof—of the tested combination regimen. We were particularly interested in assessing the results of the combination therapy on red cells and immune cell subsets. Because in enzymatic assays the potency of CP-690,550 at inhibiting JAK3 was only 20 times higher than at inhibiting JAK2 (12), and because approximately 25% of patients treated with MMF experience anemia (39, 40), red blood cell parameters were closely monitored. In both MMF alone and combination therapy groups, a drop in hemoglobin levels was seen early after transplantation and plateaued thereafter in long-term survivors. Reticulocyte counts remained low throughout the postoperative course in contrast with what was seen in our previous experience with CP-690,550-treated long-term survivors (13). In those latter animals, reticulocytosis rates in excess of 12%, either spontaneous or promoted by the injection of exogenous recombinant human EPO—which was not used postoperatively in the present study—contributed to the recovery of normal hemoglobin levels at study completion time. Hence, in this model, the combination of both drugs appeared to have more impact on hemoglobin levels than CP-690,550 administered as monotherapy. It is possible to envision that adapting dosing of respective drugs, with the potential to use exogenous EPO, will minimize the effects of the combination regimen on red blood cell parameters.

A typical signature pattern that reflects the effects of JAK3 inhibition on immune cells was observed in the current series. More particularly, as observed in previous studies reporting the use of the compound in NHPs (13, 41, 42) we observed a significant reduction of NK cell numbers in treated animals at the time of necropsy. NK cell homeostasis heavily relies on the presence of IL-15 and IL-21, two cytokines whose actions are expected to (43), and actually are (41), blunted in the presence of JAK3 inhibition. Because of the overall lesser exposure to CP-690,550 in the current study compared to that in our previous series of CP-690,550 monotherapy (13, 41, 42), the effects on immune cells were less marked and, notably we did not observe a significant reduction in either CD4+ or CD8+ T cell numbers.

From a clinical standpoint, combination therapy animals experienced episodes of constipation or vomiting—but no diarrhea. These symptoms were not seen in the two animals of this series dosed with MMF alone, nor in the animals previously dosed with CP-690,550 as monotherapy (13). These phenomenon resulted in an 8.5% weight loss, which was higher than that seen in animals that received CP-690,550 as monotherapy [average of 2.7% (13)], but which compared favorably with our previous experience (31). Because gastrointestinal (GI) side effects were seen at rather low MMF dosing (i.e., 11 mg/kg BID), it is likely that those phenomenon reflect a particular susceptibility of the species to the combination of MMF with CP-690,550. GI side effects are notorious in humans dosed with Cellcept and we have previously reported on identical GI symptoms in NHPs dosed with Cellcept, albeit at higher doses (20). In the current series, we observed one case of lymphosarcoma. The tumoral proliferation was found at the routine necropsy check-up and appeared isolated and restricted to a single mesenteric lymph node. NHPs, and particularly cynomolgus monkeys, may be prone to develop lymphomas (44, 45) as early as 36 days after renal transplantation upon treatment with cyclosporine (46). Despite the fact that there were no such findings in our previous series of 18 NHP transplant recipients immunosuppressed with CP-690,550 as monotherapy, the current finding obviously warrants caution in the clinical development of this immunosuppressive combination regimen.

In conclusion, we report here the first preclinical use of a calcineurin inhibitor-free immunosuppressive regimen combining the JAK3 inhibitor CP-690,550 with the IMPDH inhibitor MMF. This regimen, even though not clearly superior to CP-690,550 monotherapy, afforded prolonged allograft survival and contributed to delayed graft rejection. In NHPs, tolerability of the combination regimen was not as good as that of CP-690,550 administered alone but it is possible to envision that adjustments in dosing strategies and potential use of adjuvant therapies might contribute to minimize some of the side effects encountered. Because the combination therapy afforded significant prolongation of graft survival, we believe that such a regimen, potentially combined with an induction immunosuppression protocol, could form the basis of a calcineurin inhibitor-free regimen to be tested in clinical trials.

ACKNOWLEDGMENTS

The authors would like to thank Alfredo Green and Zack Nakao for their excellent technical and veterinary care of the animals on study and Ms. Kathy Richards for her editorial assistance in the preparation of the manuscript.

REFERENCES

1. Serkova N, Jacobsen W, Niemann CU, et al. Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain. Br J Pharmacol 2001; 133: 875–885.
2. Gourishankar S, Turner P, Halloran P. New developments in immunosuppressive therapy in renal transplantation. Expert Opin Biol Ther 2002; 2: 483–501.
3. Kahan BD, Camardo JS. Rapamycin: clinical results and future opportunities. Transplantation 2001; 72: 1181–1193.
4. Ojo AO, Hanson JA, Wolfe RA, et al. Long-term survival in renal transplant recipients with graft function. Kidney Int 2000; 57: 307–313.
5. Hariharan S. Case 4: cardiovascular risk in renal transplantation. Transplantation 2003; 75: 1610–1614.
6. Borie D, Si MS, Morris RE, et al. JAK3 inhibition as a new concept for immune suppression. Curr Opin Invest Drugs 2003; 4: 1297–1303.
7. O'Shea JJ, Pesu M, Borie DC, Changelian PS. A new modality for immunosuppression: targeting the JAK/STAT pathway. Nat Rev Drug Disc 2004; 3: 555–564.
8. Hofmann SR, Ettinger R, Zhou YJ, et al. Cytokines and their role in lymphoid development, differentiation and homeostasis. Curr Op Allergy and Clin Immunol 2002; 2: 495–506.
9. Leonard WJ, O'Shea JJ. Jaks and STATs: Biological implications. Annu Rev Immunol 1998; 16: 293–322.
10. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 2002; 109 Suppl: S121–S131.
11. Kudlacz E, Perry B, Sawyer P, et al. The novel JAK-3 inhibitor CP-690550 is a potent immunosuppressive agent in various murine models. Am J Transplant 2004; 4: 51–57.
12. Changelian PS, Flanagan ME, Ball DJ, et al. Prevention of organ allograft rejection by a specific Janus kinase 3 inhibitor. Science 2003; 302: 875–878.
13. Borie DC, Changelian PS, Larson MJ, et al. Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates. Transplantation 2005; 79: 791–801.
14. Braun WE. Renal transplantation: basic concepts and evolution of therapy. J Clin Apheresis 2003; 18: 141–152.
15. Pescovitz MD, Govani M. Sirolimus and mycophenolate mofetil for calcineurin-free immunosuppression in renal transplant recipients. Am J Kidney Dis 2001; 38: S16–S21.
16. Flechner SM, Kurian SM, Solez K, et al. De novo kidney transplantation without use of calcineurin inhibitors preserves renal structure and function at two years. Am J Transplant 2004; 4: 1776–1785.
17. Flechner SM, Goldfarb D, Modlin C, et al. Kidney transplantation without calcineurin inhibitor drugs: a prospective, randomized trial of sirolimus versus cyclosporine. Transplantation 2002; 74: 1070–1076.
18. Barten MJ, van Gelder T, Gummert JF, et al. Pharmacodynamics of mycophenolate mofetil after heart transplantation: new mechanisms of action and correlations with histologic severity of graft rejection. Am J Transplant 2002; 2: 719–732.
19. Barten MJ, van Gelder T, Gummert JF, et al. Novel assays of multiple lymphocyte functions in whole blood measure: new mechanisms of action of mycophenolate mofetil in vivo. Transpl Immunol 2002; 10: 1–14.
20. Klupp J, Dambrin C, Hibi K, et al. Treatment by mycophenolate mofetil of advanced graft vascular disease in non-human primate recipients of orthotopic aortic allografts. Am J Transplant 2003; 3: 817–829.
21. Morris RE, Hoyt EG, Eugui EM, Allison A. Prolongation of rat heart allograft survival by RS-61443. Surg Forum 1989; 40: 337–338.
22. Morris RE, Meiser B. A new pharmacologic action for an old compound. Med Sci Res 1989; 17: 877–878.
23. Borie D, Hausen B, Larson M, et al. A life-supporting technique of renal allotransplantation in Macaca fascicularis to evaluate novel immunosuppressive drugs in nonhuman primates. J Surg Res 2002; 107: 64–74.
24. Gummert JF, Christians U, Barten M, et al. High-performance liquid chromatographic assay with a simple extraction procedure for sensitive quantification of mycophenolic acid in rat and human plasma. J Chromatogr B Biomed Sci Appl 1999; 721: 321–326.
25. Paniagua R, Campbell AC, Changelian PS, et al. Quantitative analysis of the immunosuppressant CP-690,550 in whole blood by column switching high-performance liquid chromatography with mass spectrometry detection. Ther Drug Monit 2005; 27(5):608–616.
26. Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999; 55: 713–723.
27. Baylis C, Corman B. The aging kidney: insights from experimental studies. J Am Soc Nephrol 1998; 9: 699–709.
28. Segoloni GP, Messina M, Triolo G, et al. Impact of donor age in kidney transplantation. Transplant Proc 1991; 23: 2620–2621.
29. Jurewicz WA. Immunological and nonimmunological risk factors with tacrolimus and Neoral in renal transplant recipients: an interim report. Transplant Proc 1999; 31: 64S–66S.
30. Rush D, Nickerson P, Jeffery J. Protocol biopsies in the management of renal allograft recipients. Curr Opin Nephrol Hypertens 2000; 9: 615–619.
31. Hausen B, Klupp J, Christians U, et al. Coadministration of either cyclosporine or steroids with humanized monoclonal antibodies against CD80 and CD86 successfully prolong allograft survival after life supporting renal transplantation in cynomolgus monkeys. Transplantation 2001; 72: 1128–1137.
32. Birsan T, Hausen B, Higgins JP, et al. Treatment with humanized monoclonal antibodies against CD80 and CD86 combined with sirolimus prolongs renal allograft survival in cynomolgus monkeys. Transplantation 2003; 75: 2106–2113.
33. Cho CS, Burkly LC, Fechner JH, Jr., et al. Successful conversion from conventional immunosuppression to anti-CD154 monoclonal antibody costimulatory molecule blockade in rhesus renal allograft recipients. Transplantation 2001; 72: 587–597.
34. Nickeleit V, Hirsch HH, Binet IF, et al. Polyomavirus infection of renal allograft recipients: from latent infection to manifest disease. J Am Soc Nephrol 1999; 10: 1080–1089.
35. Nickeleit V, Klimkait T, Binet IF et al. Testing for polyomavirus type BK DNA in plasma to identify renal-allograft recipients with viral nephropathy. N Engl J Med 2000; 342: 1309–1315.
36. Fishman JA. BK virus nephropathy–polyomavirus adding insult to injury. N Engl J Med 2002; 347: 527–530.
37. van Gorder MA, Della PP, Henson JW, et al. Cynomolgus polyoma virus infection: a new member of the polyoma virus family causes interstitial nephritis, ureteritis, and enteritis in immunosuppressed cynomolgus monkeys. Am J Pathol 1999; 154: 1273–1284.
38. Qi S, Xu D, Peng J, et al. Effect of tacrolimus (FK506) and sirolimus (rapamycin) mono- and combination therapy in prolongation of renal allograft survival in the monkey. Transplantation 2000; 69: 1275–1283.
39. Mycophenolate mofetil in renal transplantation: 3-year results from the placebo-controlled trial. European Mycophenolate Mofetil Cooperative Study Group. Transplantation 1999; 68: 391–396.
40. Vanrenterghem Y, Ponticelli C, Morales JM, et al. Prevalence and management of anemia in renal transplant recipients: a European survey. Am J Transplant 2003; 3: 835–845.
41. Conklyn M, Andresen C, Changelian P, Kudlacz E. The JAK3 inhibitor CP-690550 selectively reduces NK and CD8+ cell numbers in cynomolgus monkey blood following chronic oral dosing. J Leukoc Biol 2004; 76: 1248–1255.
42. Borie DC, Changelian PS, Larson MJ, et al. Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates. Transplantation 2005; 79: 791–801.
43. Borie DC, O'Shea JJ, Changelian PS. JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants. Trends Mol Med 2004; 10: 532–541.
44. McInnes EF, Jarrett RF, Langford G, et al. Posttransplant lymphoproliferative disorder associated with primate gamma-herpesvirus in cynomolgus monkeys used in pig-to-primate renal xenotransplantation and primate renal allotransplantation. Transplantation 2002; 73: 44–52.
45. Schmidtko J, Wang R, Wu CL, et al. Posttransplant lymphoproliferative disorder associated with an Epstein-Barr-related virus in cynomolgus monkeys. Transplantation 2002; 73: 1431–1439.
46. Gaschen L, Schuurman HJ. Ultrasound detection of non-Hodgkin's lymphoma in three cynomolgus monkeys after renal transplantation and cyclosporine immunosuppression. J Med Primatol 2001; 30: 88–93.
Keywords:

Transplantation; Immunosuppression; Primates; JAK3; JAK/STAT; CP690; 550; Mycophenolate mofetil

© 2005 Lippincott Williams & Wilkins, Inc.