RAPAMYCIN: CLINICAL RESULTS AND FUTURE OPPORTUNITIES1

INTRODUCTION Sirolimus (rapamycin; RAPA) is a macrocyclic lactone with a novel mechanism of immunosuppressive action (1). During the past 7 years, the drug has undergone clinical trials progressing from Phase I safety, tolerability, and pharmacokinetic investigation to Phase II dose-finding studies and limited-sized, multicenter evaluations of drug combination regimens. The completion of Phase III large randomized national and international trials led to approval of the drug to achieve augmented acute rejection prophylaxis in combination with cyclosporine (CsA) and steroids by the Food and Drug Administration of the United States in September 1999. In November 2000, the drug was approved by the European Agency as an alternate to calcineurin antagonists for long-term maintenance therapy. This overview seeks to familiarize the reader with the clinical information that provided the bases for drug approval and with the single-center reports that document alternate approaches to optimize the outcomes of treatment with this immunosuppressive agent. I. Update on Preclinical Findings RAPA, via its c-7 methoxy group (2), cross-links (3) the immunophilin FK binding protein (FKBP) 12, a peptide-prolyl isomerase that acts as a folding catalyst, to the multifunctional serine-threonine kinase, the mammalian target of rapamycin (mTOR) (4). Blockade of mTOR dampens lymphocyte responses to costimulatory signal 2 during the G0 to G1 transition and to cytokine signal 3 during the G1 build-up. By blocking the costimulation signals, RAPA prevents activation of the inhibitory factor kappa kinase necessary for generation of the c-Rel transcription factors of the NF-κB complex (5), and possibly also modulates protein kinase C activity (6). During the later G1 phase, by blocking signal 3, RAPA inhibits four cytokine-driven signaling pathways: a) p27kip1 degradation (7,8) leading to cyclin activation (9,10); b) p70S6 kinase stimulation, a step necessary for the synthesis of endosomal structural proteins (11–13); c) elongation factor 4A release from its association with PHAS-I, thereby facilitating ribosomal protein synthesis (14–16); and d) transcriptional up-regulation of the anti-apoptotic proteins bcl (17,18) and p21Ras (19) (Fig. 1). Both the therapeutic and the toxic effects of RAPA are related to the same cellular actions. The drug's unique effects are complementary to calcineurin antagonists (CNAs) (20) and to interleukin-2 receptor monoclonal antibodies (anti–IL-2R mAbs); a relation that has been called the "cytokine paradigm"(21) (Fig. 2). Figure 1: Sites of enzyme action of mammalian target of rapamycin (mTOR). a) Activation of c-Rel factors downstream from reception of the costimulatory CD28 signal. b) Phosphorylation of p70S6 kinase preceding endosomal structural protein synthesis. c) Release of e-IH-4E from its association with PHAS-I, leading to the 4E activity necessary for elongation of the polypeptide chains on ribosomes. d) Dissociation of p27kip1 from cyclin C kinase, promoting cell division and up-regulated expression of bcl, an anti-apoptotic factor.Figure 2: The cytokine paradigm includes calcineurin antagonists (cyclosporine [CsA] or tacrolimus [TRL]) to block the antigen-driven signal 1 and RAPA to inhibit costimulatory signal 2, thereby mitigating transcriptional activation of cytokine synthesis during progression from G0 to G1. mAbs to IL-2R block the capacity of IL-2 to trigger signal 3 transduction events, which are inhibited by RAPA, preventing G1 progression (modified from reference 21).The preclinical development of RAPA has been extensively reviewed (22). After documentation of its immunosuppressive activity in animal models by the groups of Calne (23) and later by Morris et al. (24), RAPA was demonstrated to display a high degree of synergy with CsA both in vitro (25) and in vivo (26) by the rigorous median effect analysis. These findings provided the foundation for the drug's initial clinical development in renal transplantation, anticipating that synergy with lower doses of CsA would not only more effectively prevent rejection, but also minimize CNA-induced toxicity. Two observations suggest other unique properties of RAPA that may be exploited in future controlled clinical trials. First, high doses of RAPA block the proliferative responses to cytokines by vascular and smooth muscle cells after mechanical injury, such as balloon angioplasty, or allorejection (reviewed in 27). In a nonhuman primate model, supratherapeutic concentrations of RAPA stabilized, and possibly reversed, the intimal vascular lesion caused by the progression of immune injury in aortic allografts (28). Studies are underway to assess the potential contributions of adjunctive agents to potentiate this effect at therapeutic levels of RAPA. Second, by virtue of its inhibition of bcl-2, RAPA may produce a tolerogenic pro-apoptotic effect, in apparent contradistinction to high doses of CNAs. RAPA treatment concomitant with mAb blockade of the costimulatory signal by anti-CD154 in mice induces tolerance (29,30), and the combination of RAPA and anti-B7 in nonhuman primates seems to facilitate graft survival (31). Thus, RAPA may mitigate the vasculopathic response to immune or mechanical injury and facilitate tolerance induction. A compelling aspect of RAPA therapy is the absence of the vasomotor renal side effects exhibited by the CNAs CsA and tacrolimus (TRL). Treatment with RAPA preserves glomerular filtration rates (GFR) and renal blood flow in normal (32), salt-depleted (33), and spontaneously hypertensive (34) rats, as well as in micropuncture preparations (35). Although initial studies in salt-depleted rats suggested that high doses of RAPA potentiate CsA-induced nephrotoxicity (36), recent experiments demonstrate that these adverse effects are caused by pharmacokinetic (PK) interactions that elevate renal tissue CsA concentrations disproportionately to whole blood drug levels (37). Indeed, a median effect analysis based upon renal tissue CsA concentrations suggests that RAPA displays a protective effect, which has been postulated to be related to inhibition of the intrarenal angiotensin II cascade (37). However, RAPA does produce a dose-dependent tubular toxicity in rats, which seems to be caused by delayed recovery of tubular epithelial function after injury (36). II. Clinical Pharmacology An understanding of the PK behavior of immunosuppressants is critical to guide the selection of doses and administration schedules, to predict food and drug interactions, and to assess the impact of ethnicity, age, gender, and organ function on drug exposure. The RAPA data presented in Table 1 were derived from complete concentration-time profiles in 690 subjects, and trough (Cminss) measurements from nearly 1000 patients in 40 clinical studies. The subjects included healthy volunteers, stable and de novo renal transplant recipients, children and adults on dialysis therapy, and patients with hepatic impairment or psoriasis. Table 1: Steady state pharmacokinetic parameters of RAPA in various patient populationsRAPA has been detected in whole blood samples by two high performance liquid chromatography (HPLC) methods specific for parent compound; ultraviolet wavelength (UV) (38,39) and mass spectroscopy (MS) (40). A third automated immunoassay (IMx, Abbott, N. Chicago, IL) is less selective for parent compound because it displays a 42.5% cross-reactivity with metabolites (41). Because the parent compound, not metabolite, concentrations determine biologic activity (42), HPLC/UV and HPLC/MS are the reference measurement methods used at present. RAPA systemic bioavailability (F) is approximately 14%, and the drug shows dose proportionality (43) with a maximal concentration at about 1 hr. RAPA is manufactured as an oral solution and a tablet, which are bioequivalent (44,45). RAPA is widely distributed in tissues (19 L/kg) (46) and more extensively partitions into blood cells (B) compared with plasma (P), with B/P ratios ranging from 36 in renal transplant recipients to 79 in healthy volunteers. The results of in vitro experiments using human liver microsomes suggest that cytochrome P450 3A4 is the major biotransformation system (47), generating inactive hydroxy, di-hydroxy, hydroxy-demethyl, didemethyl, 7-0 demethyl, and 41-0 demethyl metabolites (48). More than 90% of drug-associated radioactivity has been recovered in feces. Urine represents a minor route of elimination (2.2%). The average elimination half-life (t1/2) of 60 hr, albeit dose-independent, shows the greatest interpatient variation, particularly among individuals with hepatic impairment (110 hr) or in the pediatric age group (as low as 10 hr), but not among subjects of African-American versus Caucasian ethnicity. Adult stable and de novo renal transplant recipients display 38% intersubject and 45% intrasubject coefficients of variation in steady-state oral clearance (unpublished data on file, Wyeth-Ayerst Research). Because of this variability, therapeutic drug monitoring is recommended. Cminss determinations provide an adequate index of RAPA exposure for clinical use, displaying a robust correlation with AUC values (r2=0.95) (43,49) (Fig. 3). Figure 3: Correlation between trough level (Cminss) with area under the concentration-time curve (AUC) in de novo renal transplant patients. The solid line shows the equation (Cminss = −0.081 + 0.0294 * AUC), which fits observed values with r2=0.95. Each open circle is a paired observation of AUC and Cminss values. The dotted lines show the 95% prediction interval (reprinted from reference 91 with permission).Although high-fat meals slow the rate of but slightly increase the extent of RAPA absorption (50), administration with either orange juice or water produces equivalent exposures. Prominent interactions occur with other drugs that serve as substrates for CYP450 3A4. RAPA exposure is increased by diltiazem and ketoconazole and decreased by rifamycin and anticonvulsants (Table 1). Adding RAPA to the regimen of patients treated with CsA-Prednisone (Pred) produced a modest increase in steroid concentrations (51), which were not significantly different between patients on RAPA versus CsA base therapy (52). Furthermore, concomitant therapy with RAPA resulted in higher mycophenolate mofetil (MMF) exposure than did CsA and the pharmacokinetic interaction probably explains the exaggerated myelosuppressive side effects (53) that are shared by the two agents. Of greatest interest is the interaction between RAPA and CsA. RAPA concentrations are increased by concomitant versus spaced administration of Neoral, the microemulsion formulation of CsA (54) (Fig. 4) but not by concomitant administration of Sandimmune, a finding of particular benefit in the early posttransplantation period when adequate drug exposure is critical for effective immunosuppression (for review, see 55). Conversely, RAPA increases CsA exposure approximately 2-fold, presumably because of competition for metabolism by CYP 3A4 (56) and possibly drug extrusion by p-glycoprotein. Figure 4: Effect on RAPA AUC of CsA administration Neoral microemulsion either concomitant with (simultaneous, A ▪) or 4 hours after (staggered, B □) CsA using a crossover design in 20 patients. The difference between the two regimens was significant, P <0.001 by two-tailed Wilcoxon signed rank test (reprinted from reference 54 with permission).III. Clinical Development A. Phase I and II studies. The first clinical study employed a blinded randomized design to examine the safety of RAPA (1 to 13 mg/m2) versus placebo added to the CsA-Pred regimen of quiescent renal transplant patients (57) (Table 2). A dose-dependent reduction in mean platelet number and, to a far lesser extent, leukocyte count, was accompanied by increased serum cholesterol and triglyceride values. There were no changes in GFR, blood pressure, or liver function test results. Table 2: Phase I, II, and III clinical trials of RAPA in renal transplantationIn the Phase I/II trial, 40 recipients of living-donor renal transplants were treated de novo with ascending doses of RAPA (0.5, 1.0, 2.0, 3.0, and 5.0 mg/m2 per day; n=4 per group) added to a baseline regimen of full concentration-controlled exposure to CsA and tapering doses of Pred (58). An African-American male recipient of a spousal kidney who was treated in the lowest dose group experienced the only acute rejection episode. Because of the potent immunosuppression displayed by the other 19 patients (5% acute rejection rate), two subsequent cohorts of 10 recipients each were treated with 7 mg/m2 RAPA and full exposure to CsA accompanied by steroid withdrawal at 1 week or 1 month after transplantation. The one acute rejection episode in each group produced an overall acute rejection rate of 7.5% (3/40) among the CsA-RAPA group in comparison to 35% in a historical CsA-Pred cohort, suggesting that early withdrawal of corticosteroids may be feasible in RAPA-treated patients. A multicenter Phase IIB study demonstrated that, despite the protocol-mandated administration of Sandimmune at doses producing reduced drug exposure, the addition of RAPA to the regimen achieved rejection rates among non–African-American recipients of cadaveric renal transplants as low as those displayed by patients who received full CsA exposure with RAPA (59). Two additional Phase II studies explored the use of RAPA as base therapy. Although RAPA-Azathioprine (Aza)-Pred (Phase IIC1) (60) or RAPA-MMF-Pred (Phase IIC2) (53) combinations produced similar acute rejection rates to CsA-Aza-Pred or CsA-MMF-Pred regimens (about 40%), renal transplant function at 12 and 24 months was significantly better among the RAPA groups in both studies. To capture the initial immunosuppressive potency of a CsA-RAPA combination without the risk of long-term nephrotoxic complications, two large open-label trials were conducted in which CsA was withdrawn at 3 months from the regimen of patients who had experienced neither delayed graft function nor an acute rejection episode. As early as 6 months after transplantation, the renal function among patients from whom CsA was withdrawn was significantly better than those remaining on CNA therapy; however, the incidence of acute rejection episodes was numerically but not significantly greater, and all episodes responded to augmented steroid therapy (61,62). In aggregate, these studies suggest a variety of strategies for RAPA use: a RAPA-CsA combination to permit steroid withdrawal despite reduced CsA exposure; CNA avoidance using a RAPA nucleoside-inhibitor-steroid combination; or a 3-month window of RAPA-CsA-steroid therapy followed by CNA withdrawal. Open-label, long-term Phase IV studies are underway to assess the risks and benefits of these alternate approaches. B. Phase III pivotal trials. Large-scale studies using randomized and blinded designs were performed to document the therapeutic efficacy of RAPA. Thirty-eight U.S. transplant centers randomized 719 patients after transplantation once the renal graft displayed initial function. The stratification scheme was based upon ethnicity (63) because of the overwhelming impact of the African-American demographic factor on outcomes (64). Thirty-four centers in Canada, Europe, U.S., and Australia randomized 576 patients before transplantation, stratifying patients based upon living versus cadaveric donor source into a Global trial (65). Both randomization programs enrolled two patients at each dose of RAPA (2 or 5 mg daily) versus one patient for the control treatment, namely Aza (U.S. trial) or placebo (Global trial), in combination with a Cminss-controlled regimen of CsA and a stipulated protocol for tapering of steroids. Routine antimicrobial prophylaxis was mandated for Pneumocystis carinii infection and for cytomegalovirus only in cases of donor-positive to recipient-negative mismatches; otherwise, centers were stipulated to follow their customary policy. Antibody induction therapy was prohibited. Both trials demonstrated that addition of RAPA to CsA-Pred regimens reduced the incidences at 6 and 12 months posttransplantation of the clinical composite endpoint of efficacy failure: a biopsy-proven acute rejection episode, graft failure, loss to follow-up, or death (Fig. 5). The primary component of benefit was the reduced incidence of rejection episodes, which was also significant at 24 months (Table 3). Furthermore, both RAPA groups showed a significant reduction in the occurrence of moderate and severe grades of rejection, as well as in the use of antilymphocyte antibody preparations to treat rejection episodes. The incidences and of graft loss and of death were similar among the groups in the U.S. multicenter Phase III trial at 12 (63) and 24 months (Table As has been observed in the renal transplantation the of patient death in the Phase III trials were and or The 12 months at the rate among the placebo group in the Global study was no significant difference among the Because the pivotal trials that led to approval a rigorous blinded the effects of a reduction in CNA exposure not be CsA had to be in to achieve rejection prophylaxis in the However, a interaction between CsA and RAPA was by a analysis of whole blood concentrations in samples at 2, 3, and and at months 2, 3, and 12 that RAPA Cminss concentrations of approximately 10 in combination with low CsA (Cminss approximately reduced rejection rates to during the first posttransplantation when of all such episodes In the of RAPA, CsA exposure (Cminss approximately produced increase in immunosuppressive potency (Fig. of the median effect equation that the RAPA-CsA combination 90% of patients of acute rejection episodes at CsA lower than those for the or and at RAPA concentrations lower than those with These findings therapeutic synergy and the results of the preclinical studies. Figure RAPA the incidences of composite efficacy and of acute rejection episodes at 12 months in both the U.S. and the Global pivotal trials. The shows the incidences of acute rejection to graft loss and death among patients in each treatment The number of patients randomized is by at the bases of the P values by test are the for the composite efficacy rates and the for the acute rejection 3: patient and acute rejection rates using an 4: trial analysis of graft loss and in the U.S. multicenter Phase III of the of an acute rejection episode during the first as a function of the concentrations of CsA and RAPA. A was used to the of an acute rejection episode as a function of RAPA Cminss values by the antibody in the and the CsA Cminss values with a selective mAb in the selective The line at shows the impact on the occurrence of acute rejection episodes of increases in CsA Cminss exposure among patients in the placebo and Aza groups of the Phase III pivotal trials. The line shows the impact on the occurrence of acute rejection episodes of RAPA Cminss exposure among patients in the RAPA 2 and RAPA 5 groups at CsA Cminss for and to more than patients received at one dose of RAPA. In the Phase III trials patients received RAPA for 6 for 12 and for 24 from therapy 1 an of tolerability, at similar in the 2 mg RAPA in the placebo in the 5 mg RAPA and in the Aza rates for efficacy were higher in the placebo and Aza lower in the 2 mg RAPA and lowest in the 5 mg RAPA the rate for adverse was among the 5 mg RAPA and about for all of the other Thus, the higher dose was more but less well for the increased incidence in the 5 mg RAPA group of to (Table the Phase III data showed no significant difference in among all groups at 12 In the rates of cytomegalovirus and were similar in all dose The cases of Pneumocystis carinii all in patients in whom prophylaxis had been Table and among patients in both pivotal trials 12 the Global study the incidence of posttransplantation in the 5 mg RAPA group was a numerically higher than in other groups but neither significantly higher nor of the observed in studies of other immunosuppressive agents (Table 5). the of of patients had RAPA and to treatment with other immunosuppressants such as or a recent suggests that a RAPA has an inhibitory effect on infection In the incidence of was among patients in the placebo and the RAPA 5 groups in the Global trial and higher in the Aza than the RAPA groups for the U.S. multicenter Because of its effects in vitro on a variety of cell RAPA has been to be for patients transplantation as treatment for liver However, are necessary before one that the effects of RAPA be to the induction or the progression of function. As suggested by findings in preclinical RAPA does not To the factors with transplantation, renal function was in patients with and treated with 3, or 5 mg/m2 of RAPA for 12 These groups showed no difference in mean serum values (Fig. Furthermore, the Phase IIB (60) and (53) studies (Table significantly better mean values at 12 and 24 months among RAPA-treated patients compared with patients (Fig. However, renal tubular been observed among patients treated with a and Figure of RAPA on renal function in Phase I, II, and III clinical trials. The at the of the are the mean values and those at the are the of patients in the a) serum levels after 3 months treatment of patients with 3, or 5 mg/m2 RAPA Phase II data on file, Research). b) of mean serum values among patients treated with or RAPA-MMF-Pred versus CsA-Aza-Pred or CsA-MMF-Pred P values by one analysis of c) of mean serum values of patients treated with CsA-Pred in combination with placebo Aza RAPA 2 mg or RAPA 5 The difference between the RAPA and the other groups as by one analysis of was significant at 6 and 12 months P d) serum values among patients as a function of CsA treated with either were at 3 months or on CsA therapy P values by analysis of P P and the mean serum levels displayed by patients in both Phase III studies were significantly higher than those of the or cohorts (Fig. Because RAPA seems to not glomerular toxicity by it seems that these higher values CsA than RAPA toxicity and a clinical of the PK interaction observed in the (37). Indeed, recent studies suggest that cadaveric kidney function be albeit by reduction or elimination of CsA using RAPA as base therapy In a Phase II study of renal transplant recipients and a subsequent Phase III trial of subjects who were randomized at 3 months to CsA from a regimen displayed significantly better renal function at and 12 months and had significant in and blood compared with patients who on maintenance doses of CsA (Fig. These results are with the that CsA exposure an in the nephrotoxic effects of the RAPA-CsA Thus, RAPA a robust for baseline therapy to long-term patient exposure to CNAs with their risk of renal the for approval by the European RAPA has been observed to elevate blood both in the absence and in the of CsA. CsA regimens are also to produce increases in Although RAPA dampens activity in cell a similar effect was not in In RAPA significantly the of in however, it produces increases in and studies suggest that RAPA treatment increases and caused by delayed clearance of The pivotal trials that, during the first 2 months after transplantation, increased mean values of serum cholesterol among both RAPA groups and the and patients. groups showed an in values possibly related to decreased CsA steroid exposure, to in and to an increase in However, of the RAPA cohorts showed a dose-dependent in the of their values during the first after transplantation, RAPA-treated patients more adjunctive treatment with therapy in and versus and and versus for RAPA 5 mg and RAPA 2 mg versus groups in the U.S. and Global 24 months the were not significant for the U.S. trial, and versus but to be significant for the Global trial, namely and versus therapy for was less and versus and and versus Both and were well and no increases in side effects of these drugs were observed in patients on RAPA versus control therapy. Of and show that the of 2 years, the difference between treatment groups was the of a of of RAPA-treated patients who displayed serum cholesterol values (Fig. or triglyceride values (Fig. therapy, the number of patients and their values decreased by 12 and particularly by 24 months Figure serum levels among patients in the Phase III U.S. multicenter a) values. b) values. The and shows the mean as the + the median as the the and as the of the the and 90% as the on the and patients as for Phase II trials RAPA without CsA (Phase and the mean values of serum cholesterol and were similar among versus patients at 2 years, therapy was in versus of Although the for RAPA and were not with

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