Compared with Cyclosporine, ISA_TX247 Significantly Prolongs Renal-Allograft Survival in a Nonhuman Primate Model

Abstract

Background. 

ISA_TX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISA_TX247 or cyclosporine.

Methods. 

Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISA_TX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISA_TX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed.

Results. 

The group receiving ISA_TX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISA_TX247 was 120±32 ng/mL and cyclosporine was 189±130 ng/mL. The average area under the curve_0–12 for ISA_TX247 was 6045±1679 ng/mL/hr and for cyclosporine was 4919±823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80±11% for ISA_TX247 and 48±12% for cyclosporine.

Conclusions. 

Allografts in monkeys treated with ISA_TX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISA_TX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

Cyclosporine (CsA) and tacrolimus are widely used as immunosuppressants to prevent the rejection of organ and tissue allografts (^1,2). Both target calcineurin (CN), which is an integral enzyme involved in the activation of T cells. Although very effective in inhibiting the rejection response, the use of both agents is associated with significant side effects, including nephrotoxicity, hypertension, hyperlipidemia, and neurotoxicity (^3–5). It is believed that these toxic side effects are, in part, a direct result of CN inhibition in nonlymphoid tissues (^6–8). However, some of the neurotoxic effects of CsA are CN independent (^9,10), and variations in immunosuppression and toxicity may also be subject to the isoform(s) of CN present in a tissue and the affinity of the drug for that isoform(s) (^11,12). Therefore, the potential exists for the development of a CN inhibitor having potent immunosuppressive activity and limited toxicity.

ISA_TX247 is a semisynthetic analog of CsA. ISA_TX247 was found to be significantly more potent than CsA in vitro in a CN inhibition assay using human whole blood and in vivo in a rat heterotopic heart-transplantation model (¹³). Toxicologic studies of ISA_TX247 in rats, rabbits, and dogs showed no decrease in renal function (¹³). No significant side effects were seen in nonhuman primates given ISA_TX247 at 150 mg/kg for 8 weeks followed by 300 mg/kg for an additional 4 weeks (¹⁴). In another study using nonhuman primates, ISA_TX247 produced greater or similar inhibition of lymphocyte proliferation, expression of T-cell surface antigens, and cytokine production when compared with CsA (¹⁵). The purpose of this study was to compare the survival times of renal allografts in nonhuman primates treated with either ISA_TX247 or CsA.

MATERIALS AND METHODS

This study was approved and conducted under the supervision of the Institutional Animal Care and Use Committee of the University of California, Davis, and the California National Primate Research Center. ISA_TX247 (90 mg/mL) was provided to the investigators by Isotechnika, Inc. (Edmonton, Alberta, Canada) as a liquid formulation for oral administration in a vehicle containing Đ-[acute]α-tocopherol polyethylene glycol 1000 succinate (vitamin E TPGS), medium chain triglyceride oil, ethanol, and Tween-40. The formulation was mixed vigorously before administration to ensure equal distribution of the drug in the vehicle. The microemulsion formulation of CsA, 100 mg/mL, (Neoral) for oral administration was purchased from Novartis Pharmaceuticals (East Hanover, NJ). Both medications were administered using a nasogastric tube.

Adult male cynomolgus monkeys, approximately 6 kg in weight, were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched (>2.5×), donor-recipient pairs. These monkeys were housed in their own ward and were tested for and proven free of simian retrovirus infection and tuberculosis. Heterotopic renal transplantation and bilateral native nephrectomies were performed on each recipient. Each recipient was placed in either an ISA_TX247 or CsA treatment group. ISA_TX247 (75 mg/kg per 12 hr orally) or CsA (15 mg/kg per 12 hr orally) were administered starting the morning of renal transplantation. The dose was adjusted to maintain 12-hour whole-blood trough levels of approximately 150 ng/mL of either drug. Animals were monitored and treated twice daily by the veterinary staff and technicians of the California National Primate Research Center. Balanced electrolyte solutions were administered as necessary to treat dehydration, electrolyte, and acid/base abnormalities. Matched blood transfusions were administered as necessary to control severe anemia (packed cell volume<20%). Complete blood counts, serum chemistry profiles, and whole-blood trough concentrations of ISA_TX247 and CsA were performed at least three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or greater mg/dL or if a serious complication or illness developed. A necropsy was performed with histopathologic examination of the allograft, spleen, heart, lung, liver, bowel, testes, and lymph nodes. A pathologist (SMG), unaware of treatment grouping or cause of death, reviewed the histologic sections. Histologic rejection was diagnosed, and the type of acute/active rejection present was described using the Banff 97 criteria for human renal-allograft histology (¹⁶). Tissue samples from all organs were examined for evidence of inflammation or toxicity.

ISA_TX247 and CsA levels were quantified in whole blood using a validated liquid chromatography/mass spectrometry assay (¹⁷). For evaluation of the total drug exposure, an area under the concentration-time curve (AUC) from 0 to 12 hours was calculated using the parallel trapezoids rule (¹⁸). CN activity assays were performed on whole-blood samples collected at the same time as those collected for the 12-hour trough CsA levels. These blood samples were sent to and analyzed by Isotechnika, Inc. (Edmonton, Alberta, Canada) (¹⁹). Whole blood was prepared by directly extracting 60 μL of blood into 225 μL of lysis buffer. The reaction mixture contained 30 μL of the whole-blood extract and 60 μL of assay buffer (20 mm Tris [pH 8], 100 mm NaCL, 6 mm MgCl₂, 0.1 mm CaCl₂, 0.5 mm dithiothreitol, 500 nm okadaic acid, 0.1 mg/mL bovine serum albumin). Samples were assayed for the ability of CN to dephosphorylate a 19 amino acid peptide (DLDVPIPGRFD-RRV[[32]PSVAAE). Okadaic acid was present in the assay to inhibit PP1 and PP2a. After a 15 minute incubation at 30°C, the hydrolyzed inorganic phosphate was separated on a Dowex column and counted in a scintillation counter. CN activity, measured as (32)P released, was determined as the phosphatase activity sensitive to the removal of Ca⁺⁺ and the addition of 200 μm EGTA to the assay buffer. CN activity is expressed as the percent ³²P-labeled peptide substrate hydrolyzed. The coefficient of variation (sd±mean) was used to describe the variation between the mean CN activities.

Statistical analysis of survival was performed using the Kaplan-Meier survival curves of cumulative survival against time. The groups were compared using a log-rank test and a value of P<0.05 was considered significant.

RESULTS

Survival Times

The monkeys in the group receiving ISA_TX247 (n=8) survived significantly (P=0.0036) longer than the monkeys in the group receiving CsA (n=7) (Fig. 1). In each group, one monkey was killed at either postoperative day 3 (CsA) or postoperative day 4 (ISA_TX247) because of delayed graft function. One monkey in the ISA_TX247 group died of aspiration pneumonia. These three monkeys were not included in the statistical analysis. The mean survival time for the monkeys in the ISA_TX247 group was 50.43±28.93 days. The mean survival time for the monkeys in the CsA group was 13.83±5.78 days.

FIGURE 1.: Kaplan-Meier cumulative survival plot for monkeys treated with ISA_TX247 or cyclosporine (CsA).

Whole-Blood Drug Concentrations

In the ISA_TX247 group, the mean (±sd) trough whole-blood concentration was 120±32 ng/mL for the study period (Figure 2). In the CsA group, the mean (±sd) trough whole-blood concentration was 189±131 ng/mL for the study period (Figure 3). The mean (±sd) AUC_0–12 for the ISA_TX247 group was 6045±1679 ng/mL.hr and for the CsA group was 4919±823 ng/mL.hr; this difference was not statistically significant (P=0.4). The average percent CN inhibition at trough whole-blood concentrations was 66% for the ISA_TX247 group and 48% for the CsA group.

FIGURE 2.: ISA_TX247 whole-blood concentration levels. Each data point represents the mean and standard deviation of the samples collected over a 7-day time period. The number in parentheses represents the number of samples used to calculate each data point.

FIGURE 3.: CsA whole-blood concentration levels. Each data point represents the mean and standard deviation of the samples collected over a 7-day time period. The number in parentheses represents the number of samples used to calculate each data point.

Allograft Histopathology

In the CsA group, all monkeys were killed when the plasma creatinine concentration was 7 or greater mg/dL. Six of the seven had histologic changes in the renal allograft consistent with acute/active rejection and were typed according to the Banff 97 criteria (Ia, Ia, IB, IIa, IIa, III). One monkey was killed on postoperative day 3 because of delayed graft function. A Banff 97 score of nonrejection was given; there were no significant changes seen on histopathologic examination of the allograft.

In the ISA_TX247 group, five of the eight monkeys were killed when the plasma creatinine concentration was 7 or greater mg/dL. One monkey was killed on postoperative day 4 for delayed graft function. A Banff 97 score of nonrejection was given; there were no significant changes seen on histopathologic examination of the allograft. Two of the five allografts had histologic changes consistent with acute/active rejection and were typed according to the Banff 97 criteria (Ia, IIa). One additional allograft had early changes associated with acute/active rejection, but the lesions did not reach a rejection grade. The plasma creatinine level of this animal at the time of euthanasia was 7.5 mg/dL on postoperative day 19. Histologically, the last allograft was described as having lymphoma/posttransplant lymphoproliferative disorder (PTLD). No rejection grade could be given.

Three monkeys in the ISA_TX247 group were killed before the plasma creatinine concentration attained 7 mg/dL. On postoperative day 26, one animal aspirated medications given at treatment and developed a fatal aspiration pneumonia. The plasma creatinine concentration was 2.1 mg/dL on the day of death, and upon histopathologic examination of the renal allograft, there were changes consistent with acute/active rejection (Banff 97 IIa). One animal was killed on postoperative day 66 because of the development of labored breathing and progressive respiratory distress. The retropharyngeal lymph nodes and tonsils were markedly enlarged, obstructing the airway. The plasma creatinine concentration was 0.9 mg/dL on the day of death, and histopathologic examination of the renal allograft showed no evidence of rejection. One animal was killed on postoperative day 85 because of weakness and an easily palpable abdominal mass. The plasma creatinine concentration was 5.7 mg/dL on the day of death. Histopathologic examination of the allograft revealed PTLD. No rejection grade could be given.

Adverse Affects

Three monkeys in the ISA_TX247 group developed PTLD. One monkey was killed on postoperative day 66 because of severe respiratory distress. Necropsy revealed markedly enlarged retropharyngeal lymph nodes, tonsils, and spleen. On histopathologic examination, a neoplastic infiltration of medium-sized lymphocytes and lymphoblasts were found in the tonsils, lymph nodes, spleen, and liver. The allograft was normal; the plasma creatinine concentration was 0.9 mg/dL on the day of death. The average whole-blood trough concentration of ISA_TX247 of this animal was 150±69 ng/dL, and the average CN inhibition at trough whole-blood concentration was 69%. A second animal was again killed because of respiratory distress on postoperative day 59; the plasma creatinine concentration was 7 mg/dL. Necropsy revealed enlarged retropharyngeal lymph nodes, tonsils, and spleen. On histopathologic examination, a neoplastic infiltration of medium-sized lymphocytes and lymphoblasts was found in the liver, spleen, tonsils, and allograft. The average whole blood-trough concentration of ISA_TX247 of this animal was 158±78 ng/dL, and the average CN inhibition at trough whole-blood concentration was 87%. One animal was killed on day 85 because of weakness and an easily palpable abdominal mass. Necropsy revealed generalized lymphadenopathy, enlarged tonsils, and a mesenteric mass invading the stomach wall. On histopathologic examination, a neoplastic infiltration of medium-sized lymphocytes and lymphoblasts was found in the lymph nodes, tonsils, stomach, small intestine, liver, pancreas, spleen, renal allograft, the mesenteric mass, and bone marrow. On flow cytometric analysis, the majority of the cells in the peripheral blood and the bone marrow were CD 20⁺ and CD 38⁺, indicative of a B-cell lineage. The plasma creatinine concentration was 5.7 mg/dL on the day of death. The average whole-blood concentration of ISA_TX247of this animal was 119±49 ng/mL, and the average CN inhibition at trough whole-blood concentration was 73%.

One animal in the ISA_TX247 group survived 80 days and did not develop PTLD. The average whole-blood concentration of ISA_TX247 of this animal was 123±72 ng/mL, and the average CN inhibition at trough whole-blood concentration was 46%.

Four of the eight animals in the ISA_TX247 group developed a severe nonregenerative anemia (hematocrit≤20%) 19 to 53 days after transplantation that persisted until the day of death (26–85 days). One monkey died on posttransplantation day 26 because of the development of aspiration pneumonia and had received no treatment for the anemia. The other three monkeys survived 59 to 85 days. Two of the 3 received three to four blood transfusions to maintain the HCT at greater than 20%. Two of the three had PTLD, but the bone marrow was affected in only one. Bone-marrow biopsies procured from two of the monkeys revealed marked erythroid hypoplasia with a normal or hypercellular and immature myeloid series. The bone-marrow biopsy of one monkey revealed erythroid hypoplasia with marked lymphoid hyperplasia consistent with B-cell lymphoma (CD20⁺/CD38⁺ cells). The erythropoietin (EPO) concentrations were assayed on two of the anemic monkeys and were compared with those of healthy, nonanemic monkeys. The values in the anemic monkeys were 1.4 and 1.6 mU/mL. The EPO concentrations in all three healthy monkeys were all less than 1 uU/mL.

Gingival hyperplasia was observed in one monkey in the ISA_TX247 group (80-day survival). There was no trend toward hypercholesterolemia or hypertriglyceridemia in either group.

DISCUSSION

On the basis of significantly prolonged allograft survival times and a higher average percent CN inhibition at trough, ISA_TX247 has demonstrated to be a more potent immunosuppressive agent than CsA at equal exposure levels in this nonhuman primate model of renal-allograft transplantation. The degree of CN inhibition was considerable in both groups, with an average percent inhibition at trough drug levels of 66% for the ISA_TX247 group and 48% for the CsA group. In a stable group of human renal-transplant patients, the average percent CN inhibition at 1 to 2 hours after oral administration of CsA and maximum drug concentrations was 64% to 66% (¹⁹). The 12-hour trough whole-blood levels averaged 219±119 μg/mL, with an average CN inhibition of 30%.

The development of PTLD in three of the monkeys in the ISA_TX247 group may have been a result of the degree of CN inhibition and the longer duration of survival of the monkeys in that group. PTLD in cynomolgus monkeys has been reported after transplantation and immunosuppression and has been associated with a primate gamma-herpes virus that induces B-cell proliferation (²⁰). There was no relationship between the use of a particular immunosuppressive agent, combination of agents, or the severity of immunosuppression and the development of PTLD. Although the incidence of PTLD was similar after both allotransplantation and xenotransplantation (4%), the disorder occurred much sooner in the xenotransplant groups; median of 40 versus 104 days. In the ISA_TX247 group, PTLD was the cause of euthanasia of 37% of the monkeys at 59 to 85 days in this study. Potentially, the potent immunosuppressive effect of ISA_TX247 resulted in the rapid onset and high incidence of PTLD in this group.

A severe, nonregenerative anemia occurred in 50% of the monkeys in the ISA_TX247 group despite excellent renal-allograft function and serum EPO concentrations higher than healthy, nonanemic monkeys. Simian parvovirus infection can produce a severe, nonregenerative anemia in cynomolgus monkeys (^21–23). However, there was no evidence of intranuclear inclusion bodies or dyserythropoiesis that are characteristic of parvovirus infection in the bone-marrow biopsies obtained from the monkeys in this study.

There are two signaling pathways that can act independently to control hemoglobin production in differentiating cells (^24,25). EPO regulates the production of circulating erythrocytes by controlling proliferation, differentiation, and survival of erythroid progenitor cells. Early down-regulation of c-myb expression in erythroid cells is a common feature of the action of EPO. Agents that increase the cytoplasmic free calcium concentration can mimic the effect of EPO on c-myb expression and activate nuclear signal transduction processes involved in the induction of hemoglobin synthesis (²⁵). This calcium-induced stimulation of hemoglobin synthesis is mediated by CN. There is evidence that the two pathways can have an additive effect in the early stages of erythroid differentiation and that EPO may act by both Ca²⁺-independent and Ca²⁺-dependent pathways (²⁵).

CsA has been shown to inhibit Ca²⁺-induced hemoglobin synthesis, and this effect is attributed to the inhibition of CN (²⁴). The monkeys in the ISA_TX247 group had a pronounced suppression of CN activity, with a longer survival period than the monkeys in the CsA group. This degree of CN suppression over a longer period of time may have been attributed to the severe nonregenerative anemia that developed in this group.

In summary, renal allografts in monkeys treated with ISA_TX247 survived significantly longer than those treated with CsA. On the basis of survival times and the degree of CN inhibition, ISA_TX247 has proven to be a more potent immunosuppressive agent than CsA at equal exposure levels (no significant difference in trough drug concentrations or AUC_0–12) in this nonhuman primate model. The more potent immunosuppression, produced by this CN inhibitor, was associated with PTLD and anemia. Careful monitoring of CN inhibition and toxicity is warranted in clinical trials.