Other Forms of Immunosuppression

Chemical Structure and Pharmacology

Leflunomide ( N -(4)) trifluoro-methylphenyl-5-methylisoxawol-4-carboximide) is a prodrug and is rapidly converted to its biologically active metabolite teriflunomide (A771703). Serum levels of teriflunomide are referred to as leflunomide levels. The half-life of teriflunomide is long in humans (approximately 15 days). The drug enters the enterohepatic recirculation and is excreted by the intestinal and urinary systems in equal proportions. Leflunomide is insoluble in water and is suspended in 1% carboxymethylcellulose for oral administration.

The MNAs are designed to be structurally similar to A771726. Oral bioavailability of FK778 is not substantially affected by food, and no gender effect on pharmacokinetics was observed in phase I studies.

Mechanism of Action

Leflunomide and its analogs have strong antiproliferative effects on both T lymphocytes and B lymphocytes, thus limiting the formation of antibodies. Inhibition of pyrimidine synthesis is the most important mechanism of action as leflunomide directly inhibits the enzyme dihydroorotate dehydrogenase. Lymphocytes rely entirely on the de novo pathway of pyrimidine biosynthesis and cannot use another, the so-called “pyrimidine salvage pathway.” Dihydroorotate dehydrogenase inhibition leads to depletion of the nucleotide precursors uridine triphosphate and cytidine triphosphate, which are necessary for the synthesis of RNA and DNA, and hence strongly suppress DNA and RNA synthesis.

The in vivo mechanism of action of leflunomide may depend on factors such as drug levels, disposable uridine pools, and the immune activation pathway involved. Studies have indicated that, in addition to inhibition of dihydroorotate dehydrogenase, leflunomide and the MNAs may act through inhibition of tyrosine kinases. Phosphorylation of the epidermal growth factor receptor of human fibroblasts has been shown to be inhibited by leflunomide. It was shown that leflunomide directly inhibited the interleukin (IL)-2-stimulated protein tyrosine kinase activity of p56lck and p59fyn, which is associated with activation through the T cell receptor/CD3 complex. At higher concentrations, A771726 also inhibited IL-2-induced tyrosine phosphorylation of Janus kinase (JAK)1 and JAK3 protein tyrosine kinases. Leflunomide analogs have also been shown to possess strong inhibitory activity on the antiapoptotic tyrosine kinase Bruton’s tyrosine kinase, a key factor for T cell-independent antibody formation. The hypothesis that leflunomide may exhibit more than one mechanism of action in vivo was further illustrated in mice where uridine restored proliferation and IgM production by lipopolysaccharide-stimulated B cells, whereas suppression of IgG production was not reversed. This phenomenon correlated in a dose-dependent manner with tyrosine phosphorylation of JAK3 and STAT6 proteins, known to be involved in IL-4-induced signal transduction pathways. This double in vivo mechanism of action was confirmed in rats, in which xenoreactivity was counteracted by the administration of uridine, whereas alloreactivity was not.

Inhibition of various macrophage functions by leflunomide and MNAs has also been described; in particular, inhibition of the production of oxygen radicals, the inhibition of IgE-mediated hypersensitivity responses, the expression of IL-8 receptor type A, as well as tumor necrosis factor (TNF)-mediated nuclear factor kappa B (NF-κB) activation. Tacrolimus also inhibits maturation of dendritic cells by preventing upregulation of activation markers and IL-12 production, and this phenomenon was not reversible by exogenous uridine. FK778 has equivalent or stronger immunosuppressive activity than leflunomide, both in vitro and in vivo. The immunosuppressive effect is synergistic with that of calcineurin inhibitors (CNI) and mycophenolate mofetil (MMF).

Interestingly, FK778 and leflunomide have been shown to possess antiviral effects, although the precise mechanism is unclear: inhibition of viral replication of members of the herpesvirus family by preventing tegument acquisition by viral nucleocapsids during the late stage of virion assembly has been implicated. Leflunomide is effective against multidrug-resistant cytomegalovirus (CMV) in vitro, although this in vitro activity is modest and the selectivity index is low. This anti-CMV effect of leflunomide and FK778 was confirmed in a rat model of heterotopic heart transplantation. Another interesting feature is that both leflunomide and FK778 have vasculoprotective effects, independent of the inhibition of dihydroorotate dehydrogenase.

Experimental Experience

In various rodent transplantation studies, leflunomide was shown to be at least equally potent as cyclosporine, and able to synergize with cyclosporine to induce tolerance. Specific characteristics of leflunomide-mediated immunosuppression in rats were its ability to interrupt ongoing acute rejections, and its efficacy in preventing and treating chronic vascular rejection.

One of the most attractive characteristics of leflunomide and the MNAs is their strong capacity to delay xenograft rejection and to induce partial xenograft tolerance. This may be related to the strong suppressive effects of leflunomide on T cell-independent xenoantibody formation, and on its capacity to induce natural killer (NK) cell nonresponsiveness and to modulate xenoantigen expression.

Monotherapy with FK778 in rats, and its combination with microemulsified cyclosporine in dogs or tacrolimus in nonhuman primates, reduced chronic allograft nephropathy and significantly prolonged renal allograft survival.

Clinical Experience

The main role of leflunomide in renal transplantation nowadays is the treatment of BK virus nephropathy (BKVN), although its efficacy has never been documented in trials. Based on the in vitro effective anti-BK concentration, an in vivo target level of 50 to 100 mg/mL has been proposed. In a prospective study, 26 renal transplant recipients with biopsy-proven BKVN were treated with leflunomide in combination with discontinuation of MMF and reduction of tacrolimus to a 4 to 6 ng/mL range. Although the leflunomide levels were in the lower range (on average 50 mg/mL), a significant reduction in serum and urine BK virus (BKV) titers was obtained, allograft function stabilized, and the overall graft loss rates because of BKV were only 15%. Less encouraging results were obtained in another prospective open-label study in which viral clearance was only obtained in 40% of patients with significant toxicity, resulting in discontinuation of the drug in 17% of patients. The contribution of reduction of immunosuppressive agents and leflunomide to the efficacy of BKVN treatment is unclear at this moment. Based on recent in vitro data, it has been suggested that the combination of mammalian target of rapamycin (mTOR) inhibition with leflunomide might be an effective treatment approach. Treatment with leflunomide in kidney transplant recipients with BK viremia was able to prevent the development of BKVN. A study from Jaw et al. reported on three kidney transplant recipients with BKVN who were treated with a combination of leflunomide and everolimus; all patients experienced significant reductions in viral loads (one with complete resolution) and two patients had preserved allograft function at the end of follow-up. In case reports, patients with resistant/refractory CMV infections, extensive cutaneous warts, and a patient with Kaposi’s sarcoma have been successfully treated with leflunomide. FK778 has also been studied in the context of BKVN, but although it was able to decrease BK viral load, FK778 treatment was associated with more acute rejections, decreased renal function, and more adverse events compared with reduction of immunosuppression.

In animal studies, leflunomide was able to reverse acute and chronic rejection. Two clinical studies reported that leflunomide was capable of stabilizing allograft function in patients with worsening allograft function due to chronic allograft dysfunction.

A phase II multicenter study was performed with FK778 involving 149 renal transplant patients, where FK778 was combined with tacrolimus and corticosteroids. The patients receiving FK778 experienced a reduced number of acute rejections, but there was no effect on graft survival at week 16. The reduction of acute rejection episodes was most pronounced in the subgroup in which target levels were obtained in the second week. Of note, mean total and low-density lipoprotein cholesterol levels were 20% lower in the FK778 group versus the placebo group. The validity of these results was hampered by the design of the study, and, at this time, the development of FK778 in the field of organ transplantation has ceased.

Toxicity

Although rats tolerate leflunomide well, dogs readily develop anemia and gastrointestinal ulcerations. Reportedly, the most frequent side effects in arthritis patients receiving long-term leflunomide treatment were diarrhea (17%), nausea (10%), alopecia (8%), and rash (10%), leading to a dropout rate of ± 5%. Recently, thrombotic microangiopathy attributed to leflunomide was reported in patients treated for BKVN. In the phase II study mentioned previously, involving FK778, there was a dose-dependent increase in side effects, including anemia, hypokalemia, symptomatic myocardial ischemia, and esophagitis. Other reported side effects are pneumonitis and peripheral neuropathy. Leflunomide has teratogenic effects in both animals and humans, and a washout period with cholestyramine is advised for both women and men before considering conception. Combining leflunomide with methotrexate might increase the risk for bone marrow suppression and liver toxicity. Furthermore, rifampin accelerates the conversion of leflunomide to teriflunomide, and might increase the levels. Combination with warfarin potentially increases the international normalized ratio.

Conclusion

The role of leflunomide in renal transplantation is limited to the treatment of patients with BKVN and some promising results have been reported in this respect. The MNAs, because of their shorter half-life, were considered a promising class of immunosuppressants but results in randomized clinical trials have been disappointing, and the development of these agents in organ transplantation has been halted.

Chemical Structure and Pharmacology

FTY720 or 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride is a synthetic structural analog of myriocin, a metabolite of the ascomycete Isaria sinclairii, a type of vegetative wasp. Maximal concentration and area under the curve are proportional to the dose, indicating that the pharmacokinetic profile of FTY720 is linear. The volume of distribution is largely superior to the blood volume, indicating a widespread tissue penetration. FTY720 undergoes hepatic metabolism and has a long half-life (around 100 hours). ASP0028 is a newly developed S1P ₁ /S1P ₅ -selective agonist in Astellas Pharma Inc.

Mechanism of Action

FTY720 has a unique mechanism of action as it mainly affects lymphocyte trafficking. FTY720 acts as a high-affinity agonist of the sphingosine 1-phosphate receptor-1 (S1PR1 or Edg1). Binding of its receptor results in internalization of S1PR1, rendering lymphocytes unable to respond to the naturally occurring gradient of S1P (low concentrations in thymus and secondary lymphoid organs, high concentrations in lymph and plasma) retaining lymphocytes in the low-S1P environment of lymphoid organs. After FTY720 administration in mice, B and T cells immediately leave the peripheral blood and migrate to the peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches. The cells return to the peripheral blood after withdrawal of the drug without undergoing apoptotic death. This altered cell trafficking is accompanied by a reduction of lymphocyte infiltration into grafted organs. Interestingly, lymphocytes treated ex vivo with FTY720 and reintroduced in vivo similarly migrate to the peripheral lymphoid tissues, indicating that FTY720 acts directly on lymphocytes. This process of accelerated homing was completely blocked in vivo by coadministration of anti-CD62L, anti-CD49d, and anti-CD11a monoclonal antibody. In vitro, FTY720 in the presence of TNF-α increases the expression of certain intercellular adhesion molecules on human endothelial cells. Thus alteration of cell trafficking by FTY720 may result not only from its direct action on lymphocytes, but also from an effect on endothelial cells.

Interestingly, it has been suggested that CD4 ⁺ CD25 ⁺ regulatory T cells are differently affected by FTY720 compared with T-effector cells. CD4 ⁺ CD25 ⁺ regulatory T cells express lower levels of S1P ₁ and S1P ₄ receptors and, hence, show reduced response to FTY720. Furthermore, in vitro FTY720-treated CD4 ⁺ CD25 ⁺ T-regulatory cells possess an increased suppressive activity in an antigen-specific proliferation assay.

Unlike CNI, FTY720 is a poor inhibitor of T cell function in vitro. In particular, FTY720 does not influence antigen-induced IL-2 production. In vitro exposure to high FTY720 concentrations (4 × 10 ^–6 ) induces chromatin condensation, typical DNA fragmentation, and formation of apoptotic bodies. Whether administration of FTY720 in vivo is also associated with significant apoptosis is a matter of debate.

S1PR are also present on murine dendritic cells. Upon administration of FTY720, dendritic cells in lymph nodes and spleen are reduced, the expression of CD11b, CD31/PECAM-1, CD54/ICAM-1, and CCR-7 is downregulated, and transendothelial migration to CCL19 is diminished. In a recent study it was demonstrated that FTY720 inhibited lymphangiogenesis and thus prolonged allogeneic islet survival in mice.

Experimental Experience

FTY720 given daily by oral gavage has marked antirejection properties in mice, rats, dogs, and monkeys. FTY720 (0.1–10 mg/kg) prolongs survival of corneal and skin allografts in highly allogeneic rodent models. In a DA to LEW rat combination, a short course of peritransplant oral FTY720 (5 mg/kg; days −1 and 0) prolongs cardiac allograft survival and is as efficient as a 10-day posttransplant treatment with tacrolimus at 1 mg/kg. Cardiac and liver allograft survival is prolonged in the August and Copenhagen Irish (ACI) rat to Lew rat model by either induction or maintenance treatment with FTY720. Even delayed administration of FTY720 interrupts an ongoing allograft rejection, suggesting a role for FTY720 as a rescue agent. FTY720 blocks not only rejection but also graft-versus-host disease after rat intestinal transplantation. FTY720 may also protect from ischemia-reperfusion injury, partially through its cytoprotective actions.

Both small- and large-animal models provide evidence that FTY720 acts in synergy with CNI, and that this benefit does not result from pharmacokinetic interactions. An induction course with FTY720 acts in synergy with posttransplant tacrolimus in prolonging cardiac allograft survival in rats. A similar phenomenon has been observed when FTY720 is used posttransplant in combination with cyclosporine in rat skin and heart allografts. FTY720 shows synergistic effect with CNI in heart and liver transplant in the ACI to Lew rat model. FTY720 shows synergy with cyclosporine in dog kidney (0.1–5 mg/kg/day) and monkey kidney (0.1–1 mg/kg/day) transplantation. FTY720 (0.1 mg/kg) synergizes with CNI in dog liver transplantation. Synergy between FTY720 and rapamycin was also observed in rat cardiac transplantation. In a murine lung transplant model, FTY720 attenuated ischemia-reperfusion injury. In a sensitized murine cardiac transplant model, FTY720 in combination with CTLA4-Ig resulted in inhibition of alloantibody production, reduction of donor-specific IFN-γ-producing T cells and prolonged allograft survival.

KRP-203 or 2-amino-2-(2-[4-3(-benzyloxyphenylthio)-2-cholorophenyl]ethyl)-1,3-propanediol hydrochloride has a similar molecular structure as FTY720. KRP-203 alone or in combination with low-dose cyclosporine or MMF prolonged skin, heart, and renal allograft survival. A short course of KRP203 in BALB/c mice receiving C57BL/10 islet allografts resulted in significantly prolonged islet allograft survival. Additional injection of intragraft regulatory T cells allowed for prolonged drug-free graft survival, suggesting tolerogenic effects.

In rats ASP0028 at a dose of 1.0 mg/kg level significantly decreased the number of peripheral lymphocytes. In addition, heart transplant studies in rats indicated that ASP0028 combined with suboptimal-dose of tacrolimus significantly prolonged allograft survival, comparable to that of FTY720 in combination with suboptimal-dose of tacrolimus with a wider margin of safety than FTY720, in terms of side effects of macular edema and bradycardia. In a study using a cynomolgus monkey renal transplantation model, ASP0028 was evaluated in combination with suboptimal-dose of tacrolimus. In the animals receiving ASP0028 allograft median survival time was significantly prolonged.

Clinical Experience

Stable renal transplant patients maintained on cyclosporine tolerate well one oral dose of FTY720 (0.25–3.5 mg). Similarly to its effect in animals, single doses of FTY720 cause a lymphopenia that is dose-dependent in intensity and duration, and that equally affects CD4 cells, CD8 cells, memory T cells, naïve T cells, and B cells.

In phase II and III studies in de novo renal transplantation, it was shown that 2.5 mg FTY720 in combination with full-dose cyclosporine and steroids is as effective as MMF in combination with full-dose cyclosporine and steroids, although the FTY720-treated patients had lower creatinine clearance at 12 months. FTY720 5 mg did not allow a 50% reduction in cyclosporine exposure. FTY720 2.5 mg in combination with reduced-dose cyclosporine resulted in underimmunosuppression. Also in combination with tacrolimus, FTY720 2.5 mg was not superior to MMF in a recent study in de novo renal transplant recipients. Recently, it was reported that FTY720 in combination with everolimus was not beneficial with regard to prevention of acute rejection and preservation of allograft function in renal transplant recipients at high risk for delayed graft function.

Toxicity

The side effects of FTY720 are in general similar to those of other immunosuppressants, with hypertension, anemia, constipation, and nausea most commonly reported. Side effects specific for FTY720 are bradycardia, macular/retinal edema, dyspnea, and a transient rise in liver function tests. Although it is considered a main impediment of further clinical development, reduction of heart rate after the first dose of FTY720 is transient and does not persist in the maintenance phase. Importantly, typical side effects of CNI, such as nephrotoxicity, neurotoxicity, and diabetogenicity, have not been observed with FTY720.

Conclusion

FTY720 has a unique mechanism of action. The available clinical studies show that FTY720 is not superior to standard care and therefore its future in organ transplantation is uncertain.

Chemical Structure and Pharmacology

Bredinin, 4-carbamoyl-1-β- d -ribofurano-syhmidazolium-5-olate, is a nucleoside analog that is structurally similar to ribavirin. It was first isolated from the culture media of the ascomycetes Eupenicillium brefeldianum harvested from the soil of Hachijo Island (Japan, 1971). It has weak antibiotic activity against Candida albicans.