Use of biomarkers to improve immunosuppressive drug development and outcomes in renal organ transplantation: A meeting report.

On September 27-28, 2018 the Food and Drug Administration (FDA) and the Critical Path Institute's Transplant Therapeutics Consortium convened a public workshop titled "Evidence-Based Treatment Decisions in Transplantation: The Right Dose & Regimen for the Right Patient/Individualized Treatment." The workshop facilitated cooperative engagement of transplant community stakeholders, including pharmaceutical industry, academic researchers, clinicians, patients, and regulators to discuss methods to advance the development of novel immunosuppressive drugs for use in solid organ transplantation. Day 1 focused on the utility of biomarkers in drug development, with considerations for seeking regulatory endorsement for use in clinical trials. Biomarkers add value to drug development by improving patient selection criteria, safety monitoring, endpoint selection, and more. Regulatory endorsement through the FDA Biomarker Qualification Program encourages the use of biomarkers in drug development by instilling confidence and consistency in biomarker interpretation across trials. Public-private partnerships or consortia allow stakeholders to share expertise, resources, and data in pursuit of biomarker qualification. Biomarkers relevant to pretransplant risk assessment, early posttransplant care, and assessment of immune response, immunosuppressive drug efficacy, and graft function as discussed on day 1 of the workshop are described.

The importance of drug safety and tolerability in the development of new immunosuppressive therapy for transplant recipients: The Transplant Therapeutics Consortium's position statement.

The Transplant Therapeutics Consortium (TTC) is a public-private partnership between the US Food and Drug Administration and the transplantation community including the transplantation societies and members of the biopharmaceutical industry. The TTC was formed to accelerate the process of developing new medical products for transplant patients. The initial goals of this collaboration are the following: (a) To define which aspects of the kidney transplant drug-development process have clear needs for improvement from an industry and regulatory perspective; (b) to define which of the unmet needs in the process could be positively impacted through the development of specific drug-development tools based on available data; and (c) to determine the most appropriate pathway to achieve regulatory acceptance of the proposed process-accelerating tools. The TTC has identified 2 major areas of emphasis: new biomarkers or endpoints for determining the efficacy of new therapies and new tools to assess the safety or tolerability of new therapies. This article presents the rationale and planned approach to develop new tools to assess safety and tolerability of therapies for transplant patients. We also discuss how similar efforts might support the continued development of patient-reported outcome measures in the future.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis.

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720.

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

Recommended Treatment for Antibody-mediated Rejection After Kidney Transplantation: The 2019 Expert Consensus From the Transplantion Society Working Group.

With the development of modern solid-phase assays to detect anti-HLA antibodies and a more precise histological classification, the diagnosis of antibody-mediated rejection (AMR) has become more common and is a major cause of kidney graft loss. Currently, there are no approved therapies and treatment guidelines are based on low-level evidence. The number of prospective randomized trials for the treatment of AMR is small, and the lack of an accepted common standard for care has been an impediment to the development of new therapies. To help alleviate this, The Transplantation Society convened a meeting of international experts to develop a consensus as to what is appropriate treatment for active and chronic active AMR. The aim was to reach a consensus for standard of care treatment against which new therapies could be evaluated. At the meeting, the underlying biology of AMR, the criteria for diagnosis, the clinical phenotypes, and outcomes were discussed. The evidence for different treatments was reviewed, and a consensus for what is acceptable standard of care for the treatment of active and chronic active AMR was presented. While it was agreed that the aims of treatment are to preserve renal function, reduce histological injury, and reduce the titer of donor-specific antibody, there was no conclusive evidence to support any specific therapy. As a result, the treatment recommendations are largely based on expert opinion. It is acknowledged that properly conducted and powered clinical trials of biologically plausible agents are urgently needed to improve patient outcomes.

Clinical Trials for Immunosuppression in Transplantation: The Case for Reform and Change in Direction.

Currently trials of immunosuppression in transplantation are in decline because their objectives remain focused on improving acute rejection rates and graft survival in the first 12 months. With 1 year renal graft survival rates of greater than 90% the best that can be hoped for is noninferiority trial outcomes compared with current standard of care. Current trial design is not leading to novel therapies improving long-term outcomes and safety, and hence important unmet clinical needs in transplantation remain unanswered. Issues that need to be addressed include but are not limited to: prevention of subclinical rejection in the first year, better 5- and 10-year graft outcomes, more effective treatment for high immunological risk and sensitized (including donor-specific antibody) patients, immunosuppressive combinations that are better tolerated by patients with fewer side effects and less morbidity and mortality. In September 2015, the Transplantation Society convened a group of transplant clinical trial experts to address these problems. The aims were to substantially realign the priorities of clinical trials for renal transplant immunosuppression with the current unmet needs and to propose new designs for clinical trials for transplant immunosuppression. Moving forward, the transplant community needs to provide trial data that will identify superior treatment options for patient subgroups and allow new agents to be evaluated for efficacy and safety and achieve timely regulatory approval. Trial designs for new transplant immunosuppression must be intelligently restructured to ensure that short- and long-term clinical outcomes continue to improve.

Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates.

BACKGROUND: Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (gammac). Because mutations in genes encoding gammac or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. METHODS: Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n = 18) or its vehicle (controls, n = 3) and were euthanized at day 90 or earlier if there was allograft rejection. RESULTS: Mean survival time (+/- standard error of mean) in animals treated with CP-690,550 (53 +/- 7 days) was significantly longer than in control animals (7 +/- 1 days, P=0.0003) and was positively correlated with exposure to the drug (r = 0.79, P < 0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46 +/- 7 days from transplantation vs. 7 +/- 1 days in controls, P = 0.0003). Persistent anemia, polyoma virus-like nephritis (n = 2), and urinary calcium carbonate accretions (n = 3) were seen in animals with high exposure. Natural killer cell and CD4 and CD8 T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. CONCLUSIONS: CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

Improved assessment of graft function by echocardiography in cynomolgus monkey recipients of hDAF-transgenic pig cardiac xenografts.

BACKGROUND: The current practice of evaluating heterotopic heart xenografts by palpation allows only detection of severe graft dysfunction, which indicates terminal graft failure. Therefore, we evaluated whether echocardiography is a better method of detecting early graft dysfunction as a marker of rejection in abdominal pig heart xenografts in cynomolgus monkeys. METHODS: Six cynomolgus monkeys received heterotopic heart transplants from pig donors transgenic for human decay-accelerating factor (hDAF). Induction therapy consisted of either cyclophosphamide or rabbit anti-thymocyte globulin. Maintenance therapy consisted of cyclosporine or tacrolimus, steroids, and sodium mycophenolate or mycophenolate mofetil, GAS914 (alphaGal oligosaccharide containing glycoconjugate), and for some animals TP10 (soluble complement receptor type 1). Echocardiography was performed immediately after transplantation and 3 times a week after surgery. We scored contractility and measured left ventricular wall thickness. Impaired contractility or increased wall thickness were considered graft dysfunction and were treated with pulse steroids. Palpation score was recorded daily. We also obtained myocardial biopsy specimens. RESULTS: Palpation score remained at 4 out of 4 in all animals until 2 to 5 days before final graft failure, whereas echocardiography detected several episodes of impaired graft function, either decreased left ventricular contractility or increased left ventricular wall thickness before graft failure. Treatment with pulse steroids improved graft function only during early episodes of graft impairment. Final graft failure was steroid resistant and caused by severe vascular rejection. CONCLUSIONS: Echocardiography is a better method of assessing graft dysfunction than is palpation. Therefore, echocardiography may detect early rejection episodes of heterotopic heart xenografts in non-human primates.

In vitro evaluation of the effects of candidate immunosuppressive drugs: flow cytometry and quantitative real-time PCR as two independent and correlated read-outs.

BACKGROUND: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan RT-PCR). METHODS: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-gamma, IL-2, IL-4, and TNF-alpha and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan RT-PCR. RESULTS: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC(50)'s) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3(+) gate to a lymphocyte gate improved correlation (r = 0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r = 0.97) was observed when drug IC(50)'s were considered. CONCLUSIONS: Flow cytometry and TaqMan RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

Immunosuppression with a combination of the leflunomide analog, FK778, and microemulsified cyclosporine for renal transplantation in mongrel dogs.

BACKGROUND: The leflunomide analog, FK778, is a selective pyrimidine synthesis inhibitor. In rodent models, FK778 is efficacious in the prevention of allograft and xenograft rejection, and a combination of FK778 and cyclosporine has synergistic immunosuppressive efficacy. METHODS: Heterotopic renal transplantation was performed in 20 dogs. Dogs were randomly assigned to three treatment groups: Neoral alone (n=6), FK778 alone (n=7), or a combination of Neoral and FK778 (n=7). Dogs were killed when the plasma creatinine concentration exceeded 7 mg/dL. A complete postmortem examination was performed and the type of acute-active allograft rejection described. RESULTS: A combination of Neoral and FK778 significantly prolonged allograft survival (P=0.0007), with median survival times of 14.5 days for Neoral alone, 7 days for FK778 alone, and 36 days for Neoral and FK778. Allograft histologic changes were consistent with acute-active allograft rejection in 19 of 20 dogs: in the Neoral-alone group, four dogs were type IB, and two were type IIA; in the FK778-alone group, five dogs were type IB, and one was type IIB; and in the Neoral and FK778 group, three dogs were type IB, three were IIA, and one dog was type III. The dog with type III rejection appeared to experience acute sepsis before rejection. Vomiting, diarrhea, and histologic gastritis and enteritis were commonly observed in dogs treated with the combination of Neoral and FK778. CONCLUSIONS: A combination of Neoral and FK778 prolonged allograft survival in a robust rejection model. Further investigation of FK778 in organ transplantation is warranted.

Treatment with humanized monoclonal antibodies against CD80 and CD86 combined with sirolimus prolongs renal allograft survival in cynomolgus monkeys.

BACKGROUND: Co-stimulatory blockade has been shown to prolong allograft survival in different transplant models. We investigated the effect of combining humanized anti-CD80 and anti-CD86 monoclonal antibodies (mAb) with sirolimus in cynomolgus monkey renal transplant recipients. METHODS: After renal transplantation, groups of four animals were treated daily with sirolimus, sirolimus and nine weekly doses of mAb, two weekly doses of mAb, or sirolimus and two weekly doses of mAb. RESULTS: Survival was significantly better in monkeys treated with the combination of sirolimus and mAb when compared with treatment with either agent alone (P=0.0067 by log-rank analysis). When combined with sirolimus, nine weekly doses of mAb did not result in an additional survival benefit compared with only two mAb doses (P=0.74). None of the treatment regimens used in this study resulted in development of transplantation tolerance. CONCLUSIONS: Sirolimus can be successfully combined with humanized mAb against CD80 and CD86. Induction with a short course of mAb is effective in prolonging allograft survival in combination with sirolimus.

Effects of AGI-1096, a novel antioxidant compound with anti-inflammatory and antiproliferative properties, on rodent allograft arteriosclerosis.

BACKGROUND: AGI-1096 is a novel phenolic intracellular antioxidant with anti-inflammatory and antiproliferative properties. In vitro, AGI-1096 inhibited the inducible expression of vascular cell adhesion molecule (VCAM)-1, E-selectin, and monocyte chemoattractant protein (MCP)-1 in endothelial cells and tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta secretion from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells. It also inhibited serum-stimulated proliferation of aortic smooth-muscle cells. In vivo, AGI-1096 demonstrated anti-inflammatory properties in a murine delayed-type hypersensitivity model. Given these antioxidant, anti-inflammatory and antiproliferative properties, we reasoned that AGI-1096 may be able to prevent chronic allograft arteriosclerosis. This hypothesis was tested in a rodent aortic transplantation model. METHODS: Donor descending aortas from August-Copenhagen-Irish rats were heterotopically transplanted into Lewis rat abdomens in end-to-end fashion. Animals were assigned to six groups as follows: AGI-1096 0 mg/kg per day (vehicle, n = 10), 10 mg/kg per day (n = 10), 20 mg/kg per day (n = 10), 40 mg/kg per day (n = 10), positive control (cyclosporine A 10 mg/kg per day by oral gavage, n = 10), and isograft negative control (Lewis-to-Lewis, n = 5). AGI-1096 was administrated subcutaneously to recipient animals three days before the surgery and for 90 days thereafter. On day 90, the paraffin-embedded allograft sections were stained with Elastin-van Gieson's stain, and the intima/media (I/M) ratio and luminal narrowing (1%LN) was assessed by digital morphometry. RESULTS: AGI-1096 demonstrated dose-dependent lowering of the I/M ratio and %LN when compared with vehicle controls. CONCLUSION: This is the first study to show that treatment of allograft recipients with AGI-1096 decreases the incidence of transplant arteriosclerosis. These data suggest that AGI-1096 may be a promising new therapeutic agent for use in clinical transplantation.

Compared with cyclosporine, ISATX247 significantly prolongs renal-allograft survival in a nonhuman primate model.

BACKGROUND: ISATX247 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 ISATX247 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 ISATX247 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 ISATX247 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 ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120 +/- 32 ng/mL and cyclosporine was 189 +/- 130 ng/mL. The average area under the curve 0-12 for ISATX247 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 ISATX247 and 48 +/- 12% for cyclosporine. CONCLUSIONS: Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

JAK3 inhibition as a new concept for immune suppression.

Although current immunosuppressive drugs are effective, they have numerous severe side effects that mandate the search for new agents. Mutations in the gene for janus kinase (JAK)3 result in severe combined immune deficiency with severely impaired humoral and cellular immunity, an observation that has prompted the development of JAK3 inhibitors. Due to its central role in lymphocyte activation, proliferation and homeostasis, targeting the JAK/signal transducer and activator of transcription (STAT) pathway may provide the required efficacy, without the toxicities associated with current therapies. Several studies conducted in rodents have validated the proof-of-concept, with a variety of JAK3 inhibitors demonstrating efficacy for immune suppression. In addition, the selective JAK3 inhibitor CP-690550 (Pfizer Inc) significantly improved allograft survival in a stringent preclinical model in primates and exhibited a good safety profile in non-human primates. This, along with studies of protein kinase inhibitors for cancer treatment, could demonstrate that development of effective, safe and selective kinase inhibitors for immunosuppression is possible.

In vivo evaluation of the novel calcineurin inhibitor ISATX247 in non-human primates.

BACKGROUND: ISA(TX)247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first in vivo study of the effects of ISA(TX)247 on lymphocyte functions in non-human primates. METHODS: Groups of cynomolgus monkeys were treated orally twice daily for 7 days, each dose consisting of 25 mg/kg cyclosporine (n = 5), 25 mg/kg ISA(TX)247 (n = 6) or 50 mg/kg ISA(TX)247 (n = 6). Levels of cyclosporine and ISA(TX)247 in whole blood were measured by liquid chromatography/mass spectrometry. After mitogen stimulation, lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow cytometry (expression of proliferating cell nuclear antigen in cells in S/G(2)M phase). Flow cytometry was also used to assess production of intracellular cytokines by T cells (interleukin-2, interferon-gamma, tumor necrosis factor-alpha) and expression of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154). RESULTS: Trough (C(14 hr)) and peak (C(3 hr)) drug levels, as well as area under the concentration-time curve, were significantly higher for cyclosporine than ISA(TX)247 (370 ng/ml vs 70 ng/ml, 877 ng/ml vs 303 ng/ml and 6,262 ng. h/ml vs 1,979 ng. h/ml, respectively). On Day 7 at C(14 hr), lymphocyte proliferation had been suppressed by approximately 50% in all groups compared with proliferation before treatment. Three hours after dosing, lymphocyte proliferation was inhibited significantly more by ISA(TX)247 (approximately 80%, with no differences between the two ISA(TX)247 dose levels) than by cyclosporine (65% inhibition). Similar differences between the immunosuppressive effects of ISA(TX)247 and cyclosporine were found for inhibition of expression of T-cell surface activation antigens. Despite lower ISA(TX)247 exposures compared with cyclosporine, the cyclosporine treatment only rarely suppressed cytokine production more than treatment with ISA(TX)247. CONCLUSIONS: In non-human primates, ISA(TX)247 produces a greater or similar inhibition of lymphocyte proliferation, expression of T-cell activation surface antigens, and cytokine production when compared with cyclosporine, despite ISA(TX)247's lower blood levels and total exposure. We conclude that ISA(TX)247 suppresses diverse T-cell functions more potently than cyclosporine in non-human primates in vivo.

Weight gain after lung transplantation.

BACKGROUND: Weight gain is frequently observed after lung transplantation, but the magnitude, predictors and implications of weight gain after lung transplant are unknown. METHODS: This retrospective cohort study included 826 lung transplant recipients randomly selected from 12 international transplant centers. We included adult patients with available weight data at baseline and 1 year post-transplant. We examined demographic and clinical predictors of first year weight gain using a multiple linear regression model (n = 579) with percent weight change as the dependent variable. To study the association between first year weight gain and subsequent survival, we performed a Cox proportional hazards analysis. p < 0.05 was considered statistically significant. RESULTS: The median weight change was 10% (range -32% to 84%). On multi-variate analysis, increasing age and prolonged mechanical ventilation were inversely associated with weight gain; obstructive disease, interstitial disease and increasing ischemic time were positively associated with weight gain. Increasing baseline weight was negatively associated with weight gain in patients with obstructive and interstitial disease. The model accounted for 14% of the variance in weight gain. Patients with weight gain above the median had better subsequent survival (adjusted hazard ratio 0.61, 95% confidence interval 0.41 to 0.90). Infection was a more common cause of death in these patients, whereas malignant deaths were more frequent in patients with below-median weight gain. CONCLUSIONS: Substantial weight gain occurs in the first year after lung transplantation. The predictors of weight gain may be used to target high-risk patients for early intervention. Higher weight gain is associated with better subsequent survival.

Sustained suppression of peripheral blood immune functions by treatment with mycophenolate mofetil correlates with reduced severity of cardiac allograft rejection.

BACKGROUND: We tested the hypothesis that sustained suppression of immune functions by mycophenolate mofetil (MMF) throughout the dosing interval reduces the severity of rejection. METHODS: Four groups of rat heart allograft recipients were treated orally daily through Day 5 with either: "low-dose" MMF, 10 mg/kg once daily (QD) or 5 mg/kg twice daily (BID); or "high-dose" MMF, 20 mg/kg QD or 10 mg/kg BID. The following were determined for all animals on Day 6: pharmacokinetics (PK, using high-performance liquid chromatography) of mycophenolic acid (MPA); pharmacodynamics (PD, by flow cytometry quantitation of whole blood mitogen-stimulated lymphocyte proliferation and expression of diverse T-cell surface activation molecules); and histologic graft rejection scores (RS). RESULTS: RS correlated with PD for suppression of both lymphocyte proliferation and transferrin receptor expression (r2 = 0.85 and 0.81, respectively) more highly than with MPA plasma levels (r2 = 0.45), which shows the validity of PD as surrogate markers of MMF efficacy. MMF 5 mg/kg BID produced greater (p < 0.001) suppression of lymphocyte proliferation (area under the PD effect-time curve, AUE = 2,010% inhibition. hour) and sustained trough (E0) PD effect (86% suppression) than MMF 10 mg/kg QD (AUE = 1,436% inhibition. hour, E0 = 55%). RS did not differ between the 20 mg/kg QD and 10 mg/kg BID "high-dose" groups, because PD was maximally suppressed. CONCLUSIONS: PD were surrogate markers for MMF immunosuppressive efficacy. MMF 5 mg/kg BID produced more sustained suppression of both PD and rejection than MMF 10 mg/kg QD.

Novel assays of multiple lymphocyte functions in whole blood measure: new mechanisms of action of mycophenolate mofetil in vivo.

The antiproliferative effects of MMF are believed to be the mechanism of its immunosuppressive action. We further investigated the mechanisms of action by assessing the pharmacodynamics (PD) of MMF in treated animals using whole blood assays not only of lymphocyte proliferation but also of activation. In vitro, different MPA concentrations were added to rat whole blood. In vivo, Lewis rats were treated with single doses of 5, 10 or 20 mg/kg MMF (n = 6 rats/dose group). Blood was obtained before and at different times after drug administration. For both in vitro and in vivo studies, different mitogens with calcium-dependent (TCR) or -independent (co-stimulatory) pathways of lymphocyte activation were added to the blood for stimulation. Proliferation was measured by [3H]TdR incorporation and by flow cytometric detection of DNA content. Activation was measured by changes in T cell surface expression of CD25, CD134, CD71, CD11a and CD54. In vitro and in vivo studies showed a dose-dependent inhibition by MPA and MMF, respectively, of lymphocyte proliferation and surface antigen expression. We observed high correlations between MMF PD effects over time with both MMF dose and MPA plasma concentrations in vivo. We show that MMF, apart from its antiproliferative effect, induced a dose-dependent suppression of calcium-dependent and -independent stimulated expression of important lymphocyte cell surface antigens. These data suggest that the ex vivo assessment of immune function in whole blood can uncover new mechanisms of MMF action. Our results demonstrated that the measurement of the PD is a means to assess the functional effects of MMF after its administration in vivo.

Effects of the malononitrilamide FK778 on immune functions in vitro in whole blood from non-human primates and healthy human volunteers.

BACKGROUND: FK778, a malononitrilamide analog of leflunomide, is currently being investigated for use in clinical transplantation. METHODS: Whole blood from cynomolgus monkeys (n=4) and healthy human volunteers (n=4) was incubated with different concentrations of FK778 and stimulated with mitogens in culture medium. Lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow-cytometric analysis of expression of proliferating cell nuclear antigen on cells in S/G(2)M phase. Flow cytometry was also used to assess expression of T and B lymphocyte activation surface antigens and production of intracellular cytokines by T cells. RESULTS: Not only lymphocyte proliferation, but also expression of various T cell surface antigens (CD25, CD11a, CD95, CD154) was suppressed by FK778. Fifty percent effective concentration values for the different immune functions were lower in human blood than in blood from cynomolgus monkeys. CONCLUSIONS: FK778 inhibits multiple immune functions. Their flow cytometric evaluation can be used to assess the effects of the drug in vivo.

Modified noble plication for the prevention of intestinal intussusception after renal transplantation in dogs.

Intestinal intussusception is a frequent problem after experimental transplantation in dogs. This report describes the safety and efficacy of performing a modified Noble plication for the prevention of intussusception. Heterotopic renal transplantation and plication was performed in 20 dogs. Dogs were killed when the serum creatinine concentration exceeded 7 mg/dL because of acute rejection (19 dogs) or venous occlusion (1 dog). Gastrointestinal signs were commonly observed, but no dog experienced an intestinal intussusception, compared to 3 of 14 dogs (21%) previously treated using a similar immunosuppressive regimen. This study supports the routine use of enteroplication in dogs undergoing renal transplantation.