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.

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.

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.

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.

Sotrastaurin (AEB071) alone and in combination with cyclosporine A prolongs survival times of non-human primate recipients of life-supporting kidney allografts.

BACKGROUND: Sotrastaurin (STN), a novel oral protein kinase C inhibitor that inhibits early T-cell activation, was assessed in non-human primate recipients of life-supporting kidney allografts. METHODS: Cynomolgus monkey recipients of life-supporting kidney allografts were treated orally with STN alone or in combination with cyclosporine A (CsA). RESULTS: STN monotherapy at 50 mg/kg once daily prolonged recipient survival times to the predefined endpoint of 29 days (n=2); when given at 25 mg/kg twice daily, the median survival time (MST) was 27 days (n=4). Neither once-daily monotherapy of STN 20 mg/kg nor CsA 20 mg/kg was effective (MST 6 days [n=2] and 7 days [n=5], respectively). In combination, however, STN 20 mg/kg and CsA 20 mg/kg prolonged MST to more than 100 days (n=5). By combining lower once-daily doses of STN (7 or 2 mg/kg) with CsA (20 mg/kg), MST was more than 100 (n=3) and 22 days (n=2), respectively. Neither in single-dose pharmacokinetic studies nor the transplant recipients were STN or CsA blood levels for combined treatment greater than when either drug was administered alone. STN blood levels in transplant recipients during combination therapy were dose related (20 mg/kg, 30-182 ng/mL; 7 mg/kg, 7-41 ng/mL; and 2 mg/kg, 3-5 ng/mL). STN at a daily dose of up to 20 mg/kg was relatively well tolerated. CONCLUSIONS: STN prolonged survival times of non-human primate kidney allograft recipients both as monotherapy and most effectively in combination with CsA. Pharmacokinetic interactions were not responsible for the potentiation of immunosuppressive efficacy by coadministering STN and CsA.

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.

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.

Strongly reduced alloreactivity and long-term survival times of cardiac allografts in Vav1- and Vav1/Vav2-knockout mice.

Vav proteins mediate T- and B-cell activation by functioning as GTP/GDP exchange factors for small GTPases. We have studied the role of Vav1 and Vav2 in allogeneic T-cell activation, antibody responses and allograft rejection. Alloantigen-induced proliferation of T cells from Vav1- and Vav1/Vav2-knockout (ko) mice was decreased by >90% in a mixed lymphocyte reaction. In whole-blood cultures, Vav deficiency led to markedly impaired T- and B-cell activation. Expansion of Vav1- or Vav1/Vav2-ko T cells (C57BL/6) was reduced after transfer into severe combined immune deficiency/beige recipient mice (BALB/c). After priming with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin, T-cell-dependent anti-DNP IgM and IgG antibody levels were normal in Vav1-ko mice but undetectable in Vav1/Vav2-ko mice. The median survival time of BALB/c cardiac allografts transplanted into C57BL/6 Vav1-ko mice (n = 13) or Vav1/Vav2-ko mice (n = 5) was >100 and >77 days, compared with 8-9 days in the corresponding wild-type mice. Vav1/Vav2-ko mice with <100 days graft survival developed bacterial skin infections and were prematurely killed with beating cardiac allograft. Long-term surviving transplants of single and double ko mice showed mild cellular interstitial rejection and mild to severe vascular remodeling. In conclusion, our studies show for the first time that the absence of Vav1 and Vav1/Vav2 in ko mice strongly reduces alloreactivity and results in long-term allograft survival, whereas antibody responses were only affected in Vav1/Vav2 ko mice.

The xenoantibody response and immunoglobulin gene expression profile of cynomolgus monkeys transplanted with hDAF-transgenic porcine hearts.

BACKGROUND: Recent work has indicated a role for anti-Gal alpha 1-3Gal (Gal) and anti-non-Gal xenoantibodies in the primate humoral rejection response against human-decay accelerating factor (hDAF) transgenic pig organs. Our laboratory has shown that anti-porcine xenograft antibodies in humans and non-human primates are encoded by a small number of germline IgV(H) progenitors. In this study, we extended our analysis to identify the IgV(H) genes encoding xenoantibodies in immunosuppressed cynomolgus monkeys (Macaca fascicularis) transplanted with hDAF-transgenic pig organs. METHODS: Three immunosuppressed monkeys underwent heterotopic heart transplantation with hDAF porcine heart xenografts. Two of three animals were given GAS914, a poly-L-lysine derivative shown to bind to anti-Gal xenoantibodies and neutralize them. One animal rejected its heart at post-operative day (POD) 39; a second animal rejected the transplanted heart at POD 78. The third monkey was euthanized on POD 36 but the heart was not rejected. Peripheral blood leukocytes (PBL) and serum were obtained from each animal before and at multiple time points after transplantation. We analyzed the immune response by enzyme-linked immunosorbent assay (ELISA) to confirm whether anti-Gal or anti-non-Gal xenoantibodies were induced after graft placement. Immunoglobulin heavy-chain gene (V(H)) cDNA libraries were then produced and screened. We generated soluble single-chain antibodies (scFv) to establish the binding specificity of the cloned immunoglobulin genes. RESULTS: Despite immunosuppression, which included the use of the polymer GAS914, the two animals that rejected their hearts showed elevated levels of cytotoxic anti-pig red blood cell (RBC) antibodies and anti-pig aortic endothelial cell (PAEC) antibodies. The monkey that did not reject its graft showed a decline in serum anti-RBC, anti-PAEC, and anti-Gal xenoantibodies when compared with pre-transplant levels. A V(H)3 family gene with a high level of sequence similarity to an allele of V(H)3-11, designated V(H)3-11(cyno), was expressed at elevated levels in the monkey that was not given GAS914 and whose graft was not rejected until POD 78. IgM but not IgG xenoantibodies directed at N-acetyl lactosamine (a precursor of the Gal epitope) were also induced in this animal. We produced soluble scFv from this new gene to determine whether this antibody could bind to the Gal carbohydrate, and demonstrated that this protein was capable of blocking the binding of human serum xenoantibody to Gal oligosaccharide, as had previously been shown with human V(H)3-11 scFv. CONCLUSIONS: DAF-transgenic organs transplanted into cynomolgus monkeys induce anti-Gal and anti-non-Gal xenoantibody responses mediated by both IgM and IgG xenoantibodies. Anti-non-Gal xenoantibodies are induced at high levels in animals treated with GAS914. Antibodies that bind to the Gal carbohydrate and to N-acetyl lactosamine are induced in the absence of GAS914 treatment. The animal whose heart remained beating for 78 days demonstrated increased usage of an antibody encoded by a germline progenitor that is structurally related, but distinct from IGHV311. This antibody binds to the Gal carbohydrate but does not induce the rapid rejection of the xenograft when expressed at high levels as early as day 8 post-transplantation.

Human macrophages do not require phagosome acidification to mediate fungistatic/fungicidal activity against Histoplasma capsulatum.

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mphi, also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus, human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity.

Immunosuppressive effects of surgery assessed by flow cytometry in nonhuman primates after nephrectomy.

Despite previous studies suggesting that surgery cause immune suppression, the underlying biologic mechanisms have not been studied using advanced immune function assays. Unilateral nephrectomy was performed in nonhuman primates. Blood was collected before surgery and at different time-points through 14 days after surgery. Lymphocyte proliferation (expression of proliferating cell nuclear antigen in cells in S/G(2)M-phase), production of intracellular cytokines [interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha] and expression of surface-activation antigens (CD25, CD71) on T-lymphocytes were assessed in whole blood using flow cytometry. Results were compared with nonoperated control animals. The procedure caused a decrease of 25% in absolute lymphocyte count on postoperative day 3. Inhibition of lymphocyte proliferation was maximal on postoperative day 1 (55% normalized to preoperative values) and was detectable until postoperative day 7, when it was 25%. Expression of T-cell activation antigens was decreased during the first postoperative week with a maximum on postoperative day 1 for CD71 (29%) and on postoperative day 3 for CD25 (49%). Intracellular production of cytokines by T cells was decreased only on postoperative day 1 (50% for IL-2, 29% for IFN-gamma and 22% for TNF-alpha). Immune functions returned to presurgery values by day 14. A major surgical procedure severely inhibits lymphocyte proliferation and various T-cell functions up to 1 week postoperatively.

Evaluation of biomarker discovery approaches to detect protein biomarkers of acute renal allograft rejection.

Management of host responses to allografts by immunosuppressive therapy is the cornerstone of transplantation medicine, but it is still deficient in one important element: biomarkers that are readily accessible and predict the fate of the transplant early, specifically, and reliably. Using a Brown Norway (BN)-to-Lewis rat renal allograft model of kidney transplantation, this study aims at evaluating two proteomic approaches to discover biomarkers for acute rejection: SELDI-MS technology and 2D gel electrophoresis combined with mass spectrometry. Several novel potential serum biomarkers have been identified for follow up. Overall, the conclusion is that apparently at the serum protein level, dramatic changes only occur at a stage where kidney function is already severely affected. Multivariate analysis of serum profiles suggests that there is an ensemble of subtle changes, comprising a proteomic signature of acute rejection at an early stage, a more detailed evaluation of which might provide novel opportunities for the diagnosis of acute rejection. Profiling of the excreted proteins indicates that urine might even present the earliest signs of the rejection process.

The novel calcineurin inhibitor ISA247: a more potent immunosuppressant than cyclosporine in vitro.

ISA247 is a novel cyclosporine analog. In this study we compare, in vitro, the effects of ISA247 on immune function with those of cyclosporine. Whole blood from cynomolgus monkeys (n = 5) was incubated with different concentrations of ISA247 or cyclosporine and stimulated with different mitogens in culture medium. Lymphocyte proliferation was assessed by [3H]-TdR incorporation assay and by flow cytometry. Flow cytometry was also used to assess production of intracellular cytokines by T cells and expression of T cell activation surface antigens. The concentration of drug necessary to attain 50% of the maximum effect (EC50) was subsequently calculated. EC50 values for ISA247 were lower than for cyclosporine, and the differences were statistically significant for lymphocyte proliferation, T cell cytokine production, and expression of all T cell activation surface antigens but one. We conclude that ISA247 suppresses diverse immune functions more potently than cyclosporine in vitro.

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.

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts.

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. RESULTS: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Pharmacodynamics in the development of new immunosuppressive drugs.

Over the past 10-20 years a number of immunosuppressive drugs, such as cyclosporine A, tacrolimus, sirolimus, or mycophenolate mofetil have been approved for clinical use and have been highly successful in preventing or delaying graft rejection. Nevertheless, there is an incessant need for better and safer drugs to improve short-term and long-term outcomes following transplantation. A number of low-molecular-weight molecules that interfere with immune cell functions are in development. These include molecules that inhibit the janus protein tyrosine kinase JAK3, compounds that alter lymphocyte trafficking (the sphingosine-1-phosphate receptor antagonist FTY720), and new malononitrilamides (FK778). All seem to show promising therapeutic potential. Among the biologic agents, there are high expectations for antibodies or recombinant chimeric molecules targeting costimulatory surface molecules or pathways involved in the migration of immune cells. The list of such targets includes the ligand pairs CD28:B7, CD154:CD40, LFA-1:ICAM-1, ICOS:B7RP-1, and VLA-4:VCAM-1. However, the clinical development of drugs for transplantation has proved to be difficult, complex, and time consuming. Therefore, newly emerging drug candidates will also demand better methods for monitoring their efficacy as well as their side effects in vivo. Pharmacokinetics (PK) and pharmacodynamics (PD) are complementary approaches used to select drugs on the basis of their in vivo efficacy as well as safety. Whereas PK monitors the handling of the drug by the body, PD focuses on the biologic effect of the drug on its target. Therefore, PD studies of in vivo efficacy are useful for clinical decisions to determine the optimal dose and type of immunosuppressant. At the preclinical stage, PD is aimed at accelerating the selection of lead compounds via PD-controlled trials in animals. Moreover, PD can help to discover new mechanisms of action for a drug or a drug candidate. However, its full potential has not been used, mainly because of laborious and time-consuming methodology. This review focuses on established and novel PD/PK approaches to assess immunosuppressive compounds in the context of new evolving drugs or drug combinations.