The use of current immunosuppressive agents to prevent solid organ allograft rejection is frequently restricted by insufficient efficacy and mechanism-based adverse effects, leading to intense interest in new agents with an improved therapeutic index (1, 2). Modulation of the T- and B-cell signaling pathways through inhibition of protein kinase C (PKC) isoforms (3–6) has recently been proposed as a novel immunosuppressive approach that could potentially reduce the need for calcineurin inhibitors (CNIs) therapy with their associated toxicities (7).
Sotrastaurin (STN) is the first in a new class of selective oral PKC inhibitors (8) that offers highly potent inhibition of early T-cell activation, with novel mechanisms of action that may complement those of CNI (9). The immunosuppressive efficacy of oral STN has been previously demonstrated in rat models of local graft versus host reaction and heart transplantation (Tx), and a synergy with other immunosuppressive agents such as cyclosporine A (CsA) or everolimus was suggested (10, 11). As an important step in the preclinical development of STN, this study aimed at examining the pharmacokinetic (PK), efficacy, and safety of orally administered STN in non-human primate (NHP) recipients of life-supporting kidney allografts. The potential pharmacologic benefits of combining STN with CsA were also explored. A part of this work was presented at the World-Transplant Congress in 2006 (12).
After a single oral dose of STN (7 mg/kg) in nontransplanted NHP, maximal blood STN concentration was observed at 2 hours post-treatment (peak drug level [Cmax]=81±39 ng/mL; 185±89 nM) and were reduced by 60% within 7 hr. A similar PK profile was previously reported for STN in naive NHP (13) and in the present study when oral CsA (20 mg/kg) was administered concomitantly (Fig. 1A).
After a single oral dose of CsA at 20 mg/kg, Tmax occurred at 5 hr (Cmax=1033±237 ng/mL; 859±97 nM) and levels decreased by 50% during the subsequent 2 hr. When STN (7 mg/kg) was given concomitantly, the mean CsA Cmax was reduced by more than 50% (420±150 ng/mL; 349±125 nM, P<0.05) but the blood STN and CsA trough levels at 24 hr postdose were unaffected by concomitant administration, ranging from 10 to 15 and 80 to 120 ng/mL, respectively (Fig. 1B).
Graft Survival Under STN Monotherapy
Two recipients were treated orally with 50 mg/kg STN once daily (QD) until euthanasia at the predefined endpoint of 29 days. Both grafts were functional at termination, with serum creatinine (SCr) less than 200 μmol/L (Fig. 2A), blood urea nitrogen (BUN) less than 10 mmol/L, and well-maintained urine output (Table 2), but ongoing acute rejection was detected by histology (Table 1). The STN trough blood levels measured at 24 hrs posttreatment (Co) varied between 9 and 229 ng/mL (21–255 nM; Fig. 3A), with mean levels of 47 and 74 ng/mL (107 and 168 nM; Table 1). Both recipients experienced episodes of emesis or diarrhea, so the group size was not expanded.
When STN was administered at a reduced dose of 20 mg/kg QD in two recipients, severe graft failure occurred at days 5 and 7 post-Tx, as indicated by SCr values more than 500 μmol/L (Fig. 2B), BUN more than 30 mmol/L, and lack of urine output (Table 2). Acute rejection was confirmed histologically in both cases (Table 1). The C0 STN levels varied between 10 and 250 ng/mL (23 and 570 nM; Fig. 3A), with mean levels of 18 and 125 ng/mL (41 and 285 nM; Table 1).
When STN was administered at 25 mg/kg twice daily (BID), severe graft failure occurred in all four cases, with termination at days 14, 18, 36, and 45 days post-Tx (Fig. 2C; Table 2). Acute rejection was confirmed by histology (Table 1). The median survival time (MST) in this group was longer than that observed in the group treated with 20 mg/kg STN QD (27 days vs. 6 days, P<0.05). The STN C0 levels in the early post-Tx period were markedly higher following 25 mg/kg BID versus 20 mg/kg QD, reaching 200 to 1000 ng/mL (456–2281 nM) before stabilization to 10 to 50 ng/mL (21–114 nM) at approximately 2 weeks (Fig. 3A). The mean C0 levels varied between 40 and 270 ng/mL (91 and 616 nM; Table 1). Overall, because of the intraindividual and interindividual variability in the STN blood levels (C0 and those measured at 4 hrs posttreatment [C4]), and because of the limited n number in each treatment group, it was not possible to draw any significant correlation between the STN doses used and the blood levels achieved.
Graft Survival Under CsA Monotherapy
In the five recipients administered CsA 20 mg/kg QD monotherapy, histologically confirmed acute graft rejection led to termination within 7 to 11 days of Tx (MST 7 days; Tables 1 and 2). CsA C0 values varied within 10 to 100 ng/mL (8–83 nM), and mean values ranged from 25 to 55 ng/mL (21–46 nM; Table 1).
Graft Survival Under STN/CsA Combination Therapies
STN 20 or 7 mg/kg QD was administered in combination with CsA 20 mg/kg QD in six and four recipients, respectively. One recipient in each treatment group was terminated prematurely due to severe graft failure, but because acute rejection could not be confirmed histologically in either case (see below) both were excluded from further analysis. Four of the five remaining grafts in the STN 20 mg/kg group, and all three of the remaining grafts in the STN 7 mg/kg group, were functional at the predefined endpoint of 100 days (SCr<200 μmol/L and BUN≤12 mmol/L; MST>100 days; Fig. 2D and E; Table 2) but showed histologic signs of ongoing acute rejection (Table 1). For the remaining recipients treated with STN 20 mg/kg QD, it was terminated after 48 days due to histologically proven acute rejection (Table 1). Sharp but transient increases in SCr (≥300 μmol/L) occurred in a small number of recipients at 5 to 8 weeks post-Tx (Fig. 2D and E), which were associated with only mild (20 mg/kg STN) or borderline (7 mg/kg STN) rejection (Table 1). However, those events coincided with reduced drinking behavior of the recipients, and SCr values rapidly returned to less than 200 μmol/L in response to simple fluid therapy (10–70 mL of water by oral gavage or 60–180 mL NaCl by subcutaneous injections).
A combination regimen of STN 2 mg/kg QD with CsA 20 mg/kg QD was administered to two recipients, which were terminated after 18 and 27 days (MST 22 days) because of histologically confirmed acute rejection (Fig. 2F, Table 1).
The CsA drug level in blood 2 hr posttreatment (C2) and C0 levels in all three combination groups with STN were within the same range as those seen in the CsA monotherapy group, that is, within 700 to 1300 ng/mL for C2 and 25 to 125 ng/mL for C0 (Table 1; Fig. 3B). Concerning the STN C4 and C0 levels, although highly variable (Table 1, Fig. 3C), they ranged within 500 to 2000 and 20 to 180 ng/mL, respectively, under 20 mg/kg QD, given with or without concomitant CsA. In comparison, about 2- to 4-fold lower C0 and C4 STN levels were observed when STN was given at 7 mg/kg QD with concomitant CsA, and a further 2- to 3-fold reduction was observed when STN was given at 2 mg/kg QD only (Table 1).
No hematologic changes appeared as treatment related. Post-Tx recovery was associated with a transient increase in white blood cells and a reduction in peripheral blood lymphocyte counts in all treatment groups (Fig. 4). As reported in previous studies (14), few recipients in almost all treatment groups (see Appendix, SDC 1,http://links.lww.com/TP/A562) required blood transfusion (∼60 mL) or erythropoietin treatments (100–200 IU/kg for 3–4 days) within the first 2 to 3 weeks post-Tx.
All grafts that were functional at 100 days post-Tx exhibited degrees of acute cellular rejection with or without chronic allograft arteriopathy (Fig. 5), with signs of rejection being detected in graft biopsies as early as 2 weeks post-Tx (Table 1). Interstitial fibrosis and tubular atrophy were not prominent even in the long-term survivors. No C4d deposition could be detected by immunohistochemistry. There was no histopathologic evidence of CsA or STN nephrotoxicity.
Lymphoid neogenesis (i.e., formation of follicle-like structures containing germinal centers in nonlymphoid tissues) was observed in four of the grafts that survived 100 days, all in recipients treated with STN 20 or 7 mg/kg in combination with CsA 20 mg/kg (Table 1).
Tubulointerstitial nephritis and glomerular abscesses because of bacterial infection were present in two grafts that failed at days 17 and 18. Similar infections (with gram-positive Staphylococcus or Streptococcus species) were found in animals from the same NHP shipment during other unrelated studies. Severe acute tubulointerstitial cellular rejection (grade IIA or III and vascular rejection) was present in all other grafts that failed between day 5 and 45.
All recipients treated with STN 50 mg/kg QD or 25 mg/kg BID had diarrhea, had one to three episodes of nausea/emesis (mostly within the first 1–4 hr postdose), and exhibited 10% to 15% weight loss during the first 2 weeks post-Tx. Such adverse events were not observed in the two recipients given the lower dose of STN monotherapy at 20 mg/kg QD.
In the STN/CsA combination therapy groups, all recipients who reached 100 days experienced one to three emesis and diarrhea episodes, generally during the first 2 to 3 weeks of treatment. Two of the four recipients given STN 7 mg/kg with CsA experienced a single emesis episode; one recipient had also sporadic diarrhea. Of the two recipients treated with STN 2 mg/kg and CsA, one experienced emesis and diarrhea during the first 2 weeks.
No treatment-related liver liability could be suspected in all treatment groups using as indicator the postTx monitoring of blood alanine transaminase and aspartate transaminase (Table 2). Most recipients exhibited higher blood pressure (BP) and heart rate (HR) at termination versus pre-Tx baselines (Table 2) but, as this was observed in all treatment groups, hemodynamic consequences of ongoing graft rejection can be suspected rather than treatment-related effects.
Follicular hyperplasia was observed in the spleens and lymph nodes in four of the six recipients treated with STN at 20 mg/kg and CsA and in three of the four recipients given STN at 7 mg/kg with CsA. Necrotizing arteritis was detected in several organs (primarily in the gastrointestinal tract) of one single recipient, the one terminated after 48 days under STN at 20 mg/kg and CsA. The monitoring of this recipient took place shortly after 2 cases presenting glomerular abscesses because of bacterial infection, and as it also showed signs of ongoing infection (elevated lymphocyte and platelet counts) at 7 days post-Tx, it received antibiotic (amoxicillin) and aspirin until termination.
Results from this study demonstrate that STN monotherapy prolongs, in a dose-dependent manner, the survival of life-supporting kidney allografts in NHP and support previous work in rodents (9), to establish STN as a new orally bio available immunosuppressive agent. The short-lived blood exposure previously described for oral STN in naive NHP (13) was also confirmed by this study, but no clear difference could be observed when comparing efficacy of STN at BID versus QD dosing.
With those results, STN at 20 mg/kg QD appeared as nontherapeutic in NHP, and this dose was considered suitable for use in combination with a nontherapeutic dose of CsA, previously set at also 20 mg/kg QD (15). The objective of testing such a combination was not to establish an optimal immunosuppressive treatment to achieve infinite graft survival but, in the context of logistic constrains limiting maximal post-Tx monitoring to 100 days, rather to assess the potential pharmacologic benefit of combining both drugs. Hence, combining STN and CsA at the nontherapeutic doses of 20 mg/kg QD each succeeded in increasing graft survival to the 100 days target in four of six grafts, although acute rejection was clearly ongoing in some grafts. A 3-fold reduction in STN dose (7 mg/kg QD) did not diminish the apparent efficacy of the combination treatment, so a further 3.5-fold dose reduction (to 2 mg/kg QD) was necessary for complete loss of efficacy. Consistent with previous data from rodent models of heart Tx (10), these results suggest that the immunosuppressive efficacy of STN and CsA could be potentiated as a result of their co-administration. Importantly, this cannot be explained in terms of drug–drug interactions as the PK profiles for both drugs in naïve NHP were either similar (STN) or altered (CsA) when administered in combination. In addition, no major difference could be seen when comparing the STN and CsA blood levels in recipients under combination treatment versus monotherapy.
Taken together, these findings suggest the existence of a synergistic pharmacodynamic effect between STN and CsA in NHP. This is fully in line with the demonstration that the selective inhibition of PKC isoforms (mainly PKCθ and PKCα) by STN and of calcineurin by CsA are two different and complementary molecular mechanisms to inhibit T-cell activation (8, 9). The role of PKC isoforms in promoting T-cell activation through a calcineurin-independent pathway was nicely reviewed elsewhere (5). Hence, if translated clinically, the STN/CsA synergy could be considered as a strategy for CNI dose lowering and reduction of related side effects. In addition, other recent studies have demonstrated the lack of PK interaction between STN and tacrolimus (16) or everolimus (17), suggesting the possibility for other combination regimens. Significant prolongation of NHP kidney allograft survival was indeed achieved by combining nontherapeutic doses of STN and of everolimus (18).
STN at a daily dose of 20 mg/kg was relatively well tolerated, consistent with clinical data showing good overall tolerability with gastrointestinal symptoms such as nausea and vomiting representing the most frequently reported adverse events (19–21). The necrotizing arteritis observed in one recipient is most likely unspecific as infection was suspected and antibiotic/aspirin were concomitant treatments. In addition, necrotizing arteritis in NHP under standard immunosuppressants is not understood and has not been predictive for humans (22).
In conclusion, this study demonstrates that (1) STN prolongs survival times of NHP recipients of life-supporting kidney allografts; (2) combination of STN with CsA increased further survival times compared with STN monotherapy; and (3) improved efficacy of STN/CsA combination is not attributable to PK interactions and is likely to result from complementary mechanisms of T-cell inhibition. Further exploration of immunosuppressive regimens that combine STN and CsA therapy will most likely help to identify optimal dose minimization strategies and expand the respective therapeutic indexes.
MATERIALS AND METHODS
Animal handling, care, and use was in line with the Swiss Federal Law for animal protection. The cynomolgus monkeys (Macaca fascicularis) were captivity-bred young adults (SICONBREC Inc., Manila, Philippines) with normal hematology and serum or urine chemistry and were negative for tuberculosis, Salmonella or Shigella, viral infections (herpes B, simian T-cell leukemia virus, SIV, simian retrovirus type D, and hepatitis B), and ectoparasites and endoparasites. They had free access to food and water, and for the majority of the study, they were housed in the colony.
Donor/recipient pairs for Tx were selected according to ABO match, full DRB exon 2 mismatch (23) and responses in one-way mixed lymphocyte reaction (stimulation indexes >9 and <50) (19). The outcomes of this selection were trios (one donor for two recipients) or pairs (kidney transfer between two animals) (see Appendix, SDC 1,http://links.lww.com/TP/A562). Each recipient was implanted with a telemetric probe (Data Sciences Inc, St Paul, MN) for monitoring arterial BP, HR, and motor activity.
Heterotopic, life-supporting kidney Tx was performed using standard microvascular techniques (20), with less than or equal to 4 hr cold ischemia and less than or equal to 40 min warm ischemia. Postoperative care included analgesia (0.01–0.02 mg/kg intramuscular buprenorphine three times a day) and antibiotherapy (25 mg/kg intramuscular cefotaxime three times a day) for 5 days post-Tx. Under these conditions, untreated recipients showed typically a MST of 8 days (14).
Postoperative Care and Monitoring
The recipients were single housed for 2 weeks post-Tx and thereafter transferred for group housing at least twice per week for a 24-hr period to permit monitoring of general health, BP, HR, body weight, food or water intake (supplemented to a maximum of 100–150 mL/kg/day), urine output, and hematology (using Technicon H-1 analyzer, Bayer Diagnostics, Zurich, Switzerland). The serum or urine chemistry was also monitored (using a Synchron CX5 analyzer, Beckmann Coulter, Nyon, Switzerland) and included SCr, BUN, glucose, Na+, K+, aspartate transaminase, and alanine transaminase. Graft ultrasonography (21) was performed at least twice a week, and ultrasonography-guided biopsies were performed under general anesthesia every 2 to 4 weeks (24).
Recipients were killed in the event of (1) severe graft failure as indicated by SCr more than 500 to 800 μmol/L, BUN more than 20 mmol/L, if available, reduced ultrasonography score, and characteristic histopathologic changes on biopsy; (2) general health problems and overt clinical signs of distress; and (3) study completion at 29 or 100 days post-Tx, those time limits being defined by the amount of STN available for treatments and logistic considerations for optimal animal housing and care, respectively. After each necropsy, the graft and all other major organs were processed for subsequent histologic analysis.
In total, this study included seven treatment groups:
- Three groups with STN alone at 50 mg/kg QD (n=2), 20 mg/kg QD (n=2), or 25 mg/kg BID (n=4).
- One group with CsA alone at 20 mg/kg QD (n=5).
- Three groups with STN at 20, 7, or 2 mg/kg QD combined with CsA at 20 mg/kg QD (n=6, 4, or 2, respectively).
All treatments were administered by oral gavage starting on the day before Tx. STN was dissolved in acidic buffer (PEG400-HCl/distilled water or acetic acid 0.1 M/distilled water, both 1/8 v/v, pH 3.1–4.3) and dosed at 2.5 to 5 mL/kg. For BID administrations, treatments were given at 8-hr intervals.
CsA 100 mg/mL (Neoral®, Novartis Pharma AG, Basel, Switzerland) was administered at a daily dose of 2 mL/kg orally. On the day of Tx, CsA 8 mg/kg was given intravenously using Sandimmun® concentrate (Novartis Pharma AG) for infusion 50 mg/mL.
Pharmacokinetic Interaction Study
Four treatment-naïve animals were given a single oral CsA dose of 20 mg/kg, followed by oral STN at a dose of 7 mg/kg 2 weeks later. After a further 2 weeks, the two doses were administered concomitantly. Blood samples were collected over a 24-hr period and stored at −20°C until assessment of STN and CsA levels.
Monitoring of Drug Exposures
Whole-blood samples were collected to assess drug concentrations at 2 or 4 hr postdosing (C2 or C4) and at trough (C0), that is, either 16 or 24 hr after BID or QD dosing and stored at −20°C. STN concentrations were measured using reversed-phase micro high-performance liquid chromatography directly coupled to an electrospray ionization source of an iontrap mass spectrometer applying multiple reaction monitoring for detection. CsA concentrations were measured using the CYCLO-Trac® SP Whole-Blood radioimmunoassay kit (DiaSorin Inc., Stillwater, MN). The results were expressed as mean±standard error of the mean.
Histology and Immunohistochemistry
When NHP were killed, collected organs were fixed in 4% buffered formalin and embedded in paraffin wax. The sections (3-μm thick) were cut from paraffin blocks and stained with hematoxylin-eosin, periodic acid-Schiff, trichrome, and Verhoeff stains and immunostained for C4d (25). The stainings were examined by an experienced pathologist and scored according to the Banff '97 working classification (26) with Banff '07 modifications (27).
Survival times of renal allograft recipients were compared between groups using the Wilcoxon-Gehan test (exact P values, StatXact 6; Cytel Corp, Cambridge, MA). Blood drug concentrations were compared by analysis of variance for repeated measurements (SAS Institute Inc, Cary, NC). Differences were considered statistically significant if P less than 0.05.
The authors thank K. Menninger for coordinating the in-life logistics; C. Jean and I. Barbet for contributing to the pharmacokinetic analysis; S. Riesen, M. Aeberhard, R. Apolloni, M. Bernhard, J.M. Blum, C. Cannet, A. Cattini, M. Erard, P. Gfeller, A. Harnist, P. Horne, A. Kunkler, M. Künzli, G. Litzler, A. Marcantonio, C. Maurer, J. Peter, N. Stuber, S. Storni, C. Vedrine, G. Vogt, and T. Weitz for their excellent technical contributions; D. Mazumder for editorial support; and E. André (BioPRIM, France) for in-life logistic support.
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