Effect of FTY720 on immunoregulation in concordant xenotransplantation

The present study was designed to analyze the immunosuppressive activity of FTY720 in concordant xenotransplantation. When T and B lymphocytes of human peripheral blood were incubated with FTY720, the number of viable cells decreased in a dose-dependent manner at doses higher than 4×10^?5 M. DNA fragmentation was observed at doses higher than 1×10^?5 M in T cell-rich fractions and at doses higher than 4×10^?5 M in B cell-rich fractions. These data demonstrate that FTY720 is cytotoxic to B lymphocytes as well as T lymphocytes and apoptosis may play an important role in this cytotoxicity. Golden Syrian hamsters were the donors and Lewis rats the recipients of skin grafts. The recipients were divided into the following four groups: (1) untreated recipients, (2) FTY720 (5 mg/kg per day) was administered orally for 8 days (days ?1–6), (3) FK506 (1 mg/kg per day) was injected i. m. for 7 days (days 0–6), and (4) FK506 (1 mg/kg per day) was injected i. m. for 7 days (days 0–6) and FTY720 (5 mg/kg per day) was administered orally for 8 days (days ?1–6). The mean graft survival times in groups 1–4 were 9.7 ± 0.52 days (n = 6), 12.0 ± 0.71 days (n = 6), 13.2 ± 1.6 days (n = 6), and 37.7 ± 4.3 days (n = 6), respectively. There was a significant difference in the mean survival time between groups one and four. Combined therapy with FTY720 and FK506 is a useful tool for immunoregulation in xenotransplantation.     

Effect of FTY720 on immunoregulation in concordant xenotransplantation

Abstract The present study was designed to analyze the immunosuppressive activity of FTY720 in concordant xenotransplantation. When T and B lymphocytes of human peripheral blood were incubated with FTY720, the number of viable cells decreased in a dose-dependent manner at doses higher than 4×10^-5 M. DNA fragmentation was observed at doses higher than 4×10 ^-5 M in B cell-rich fractions. These data demonstrate that FTY720 is cytotoxic to B lymphocytes as well as T lymphocytes and apoptosis may play an important role in this cytotoxicity. Golden Syrian hamsters were the donors and Lewis rats the recipients of skin grafts. The recipients were divided into the following four groups; (1) untreated recipients, (2) FTY720 (5mg/kg per day) was administered orally for 8 days (days -1–6), (3) FK 506 (1 mg/kg per day) was injected i. m. for 7 days (days 0–6), and (4) FK506 (1 mg/kg per day) was injected i.m. for 7 days (days 0–6) and FTY720 (5 mg/kg per day) was administered orally for 8 days (days-1–6). The mean graft survival times in groups 1–4 were.7 ± 0.52 days ( n = 6), 12.0 ± 0.71 days ( n = 6), 13.2 ± 1.6 days ( n = 6), and 37.7 ± 4.3 days ( n = 6), respectively. There was a significant difference in the mean survival time between groups one and four. Combined therapy with FTY720 and FK506 is a useful tool for immunoregulation in xenotransplantation.     

A new cardiac concordant xenotransplantation model

OBJECTIVE: A simplified method of heterotopic abdominal cardiac xenotransplantation and its technique problems are described. METHODS: Hamster-to-rat cardiac xenotransplantation was performed by means of "sleeve and cuff" method. The left common carotid artery of the donor heart was anastomosed to the left renal artery of the recipient with a "sleeve" anastomosis, and the "cuffed" right pulmonary artery was anastamosed to the left renal vein of the rat. The viability of the donor heart was examined daily by palpating the left abdominal wall of the rat. RESULTS: Among 105 rats that underwent heterotopic cardiac xenotransplantation, 95 were completed successfully. The xenograft survived for 3 to 4 days after the procedure suffering typical acute vascular rejection. CONCLUSIONS: A useful, easy model to investigate the mechanisms of concordant xenotransplantation was established.     

FTY720 (fingolimod) in renal transplantation

Abstract:  FTY720 (Fingolimod) is a novel immunomodulator with a mode of action that is completely different from classical immunosuppressants. FTY is a structural and functional analogue of the natural serum lipid, sphingosine, and is the first in a new class of drugs called sphingosine 1-phosphate receptor (S1P-R) modulators. This review discusses the recent findings on the mechanism of action, preclinical models and outlines the results of the ongoing clinical development program. FTY is highly effective in prolonging allograft survival in preclinical models of transplantation and in experimental models of autoimmune diseases. In clinical trials, this novel compound was investigated in de novo renal transplantation and in multiple sclerosis. Pharmacokinetics are characterized by a prolonged absorption phase, a large volume of distribution, and a long elimination half-life. FTY induces a rapid and transient decrease in lymphocyte counts, which supports the modulatory effects of the drug on lymphocyte sequestration. The most common adverse event was asymptomatic transient bradycardia, a pharmacodynamic effect modulated by atrial S1 P-R. FTY failed to show an improvement in efficacy for the prevention of renal allograft rejection in two large phase III studies. FTY treatment regimens were associated with impaired renal function and the development of macula edema. Consequently, the further development in renal transplantation was stopped. Because initial clinical studies strongly suggest that FTY is highly effective in multiple sclerosis FTY is now being explored in phase III studies for the treatment of demyelinating diseases, Ongoing studies in multiple sclerosis are eagerly awaited because they may provide novel therapeutic options for patients with autominnue diseases.     

Immunosuppressive activity of FTY720, sphingosine 1-phosphate receptor agonist: I. Prevention of allograft rejection in rats and dogs by FTY720 and FTY720-phosphate

FTY720, a new class of immunomodulator, induces lymphopenia by sequestration of circulating lymphocytes into secondary lymphoid tissues. FTY720 at 0.1 to 1 mg/kg significantly prolonged the allograft survival in a dose-dependent manner and showed a marked synergistic effect in combination with cyclosporine (CsA) in rat skin and cardiac allograft models. In addition, the canine renal allograft survival was significantly prolonged by combination therapy with FTY720 at 0.03 to 1 mg/kg and CsA at 10 mg/kg as compared with monotherapy of FTY720 or CsA. By contrast, the combination therapy with CsA and azathioprine or CsA and mycophenolate mofetil resulted in only an additive effect in rat skin allograft. When FTY720 was administered to rats, FTY720 was metabolized by omega-oxidation of the octyl side chain, and beta-oxidation subsequently, or phosphorylated by sphingosine kinase. Omega- and beta-oxidized 4 metabolities of FTY720 at 10 mg/kg i.v. showed neither lymphopenia nor immunosuppressive activity in rat skin allograft. On the other hand, (S)-enantiomer of FTY720-phosphate at 0.1 and 1 mg/kg intravenously induced a marked lymphopenia and significantly prolonged the allograft survival in the rat allotransplantation. From these results, it is suggested the lymphopenia and the immunosuppression induced by FTY720 administration is due to the agonistic activity against SIP receptors of the active metabolite, (S)-FTY720-phosphate.     

FTY720 inhibits tumor growth and angiogenesis

De novo malignancies and recurrence of tumors are some of the biggest threats to allograft recipients subjected to chronic immunosuppression. FTY720, a synthetic myriocin analogue, is an immunosuppressant that induces apoptosis of activated lymphocytes and prevents infiltration of lymphocytes into allografts, thereby prolonging allograft survival in a dose-dependent manner. Additionally, FTY720 was shown to prevent tumor growth and metastasis. Therefore, we examined the effect of FTY720 on angiogenesis in a HUVEC spheroid model. To substantiate our in vitro findings the effect of FTY720 was also tested in C57/B16 mice subcutaneously injected with Lewis Lung Carcinoma (LLC1) cells. After establishment of a palpable tumor the animals were treated daily with either saline or 1, 5, or 10 mg/kg FTY720. Subsequently, the tumor size was measured, periodically. In our experiments FTY720 showed a strong antiangiogenic effect, overcoming the stimulating effect of VEGF (20 ng/mL) even at subnanomolar concentrations. In vivo, FTY720 showed a dose-dependent inhibition of subcutaneous tumors, and the tumor size of animals treated with 10 mg/kg FTY720 was less than half of the size of tumors in control animals. In conclusion, FTY-720 demonstrated a strong antiangiogenic effect in vitro and a substantial antitumor effect in vivo. Presumably, the stabilizing effect of surrounding pericytes limits the effect of FTY720 in our mouse model. Therefore, a combination of FTY720 with an mTOR inhibitor might be the most favorable immunosuppressive drug combination for allograft recipients at risk for tumor development.     

Novel mediators of FTY720 in human lymphocytes

FTY720 (FTY), a novel immunosuppressive drug, can be distinguished from other immunosuppressive drugs by a completely different mechanism of action. FTY induces altered lymphocyte trafficking, leading to peripheral blood lymphopenia and to increased lymphocyte counts in lymph nodes. FTY mediates its immune-modulating effects by binding to sphingosine 1-phosphate receptors expressed on lymphocytes. In an attempt to identify mediators of the FTY-induced signal transduction, we used a proteomic approach. FTY-treated peripheral blood lymphocytes (PBLs) were investigated for the expression of 622 proteins. We identified 15 differentially expressed proteins in PBLs possibly related to FTY action. As indicated by protein function, several identified proteins could be linked to the cytoskeleton/cell motility, to cell adhesion, and vesicle trafficking. No changes were found concerning the expression of various apoptosis regulators as well as the immunophilins FKBP12 and calcineurin. Our data suggest that FTY affects cytoskeleton rearrangements, cell adhesion, and vesicle trafficking/sorting in human PBLs.     

Effects of FTY720 on rat lymphoid organs

INTRODUCTION: In this study, we examined the in vivo effect of FTY720 on rat thymus and spleen. MATERIALS AND METHODS: Inbred male Lewis (RT-I(l)) rats, 5-weeks-old, received FTY720 (0.1, 1, or 10 mg/kg) by intramuscular injection into 1 of 4 limbs daily for 7 days. The rats were humanely killed at 1, 3, 5, or 7 days after starting administration of FTY720. The thymus and spleen were obtained for hematoxylin and eosin (HE) as well as immunoperoxidase staining using the antibodies OX8 (CD8), W3/25 (CD4), and OX6 (major histocompatibility complex class II). RESULTS: HE staining revealed marked atrophy in the cortical region of the thymus among rats administered FTY720 at the dose of 10 mg/kg. The atrophy extended to the whole cortex. On day 7 of administration of FTY 720 (10 mg/kg), the medulla of the thymus showed relative expansion due to cells accumulation. Also, the spleens of FTY720-treated rats revealed an obvious reduction in the T-cell-dependent areas around the central artery. In conclusion, the immunosuppressive effect of FTY720 may be due to an inhibitory effect on T-cell emigration from the thymus to the periphery.     

FTY720 impairs necrosis development after ischemia-reperfusion injury

Ischemia-reperfusion (IR) injury is a common early feature that contributes to graft damage by impairing resident cell function. Our previous results showed that IR injury impaired renal function, by causing extensive tubular necrosis and increasing MHC class II and ICAM-1 molecule expression by mesangial cells (MC). MCs are likely candidates to come into close contact with immune cells such as monocytes or lymphocytes. It has been suggested that under inflammatory circumstances, there is increased MC expression of MHC class II, of adhesion molecules (such as ICAM-1), of cytokines receptors, and of molecules associated with cellular death (apoptosis). The immunosuppressive properties of FTY720 have been shown in clinical and experimental situations. It has also been shown to be protective against IR injury in rats. We sought to evaluate the role of FTY720 in a murine IR model by measuring renal function, tubular necrosis, and surface molecule expression by cultured mesangial cells. Intravenous administration of FTY720 (1 mg/kg) immediately before IR induction did not improve the short-term (24 hours) outcome of renal function or reduced MHC class II and ICAM-1 surface molecule expression. However, there was a decreased percentage of tubular necrosis in mice treated with FTY720 (51.3% +/- 1.6%) compared with vehicle-treated mice (66% +/- 5.5%). These results suggest a protective role of FTY720 in an IR injury model. More studies are required to identify the mechanisms involved in the protective activity of FTY720 in the IR injury model.     

FTY720诱导大鼠心脏移植物长期存活

目的 观察FTY720对大鼠同种异体心脏移植物存活时间的影响。方法 进行SD Wistar大鼠的腹部异位心脏移植，将受者随机分为对照组、甲泼尼龙(MP)组、环孢素A((SsA)组、FTY720组、FTY720与CsA二药联用组和FTY720、CsA及MP三药联用组，各组按分组要求分别于术前3d至术后14d通过灌胃给予FTY720和CsA，术前1d至术后2d腹腔注射给予MP，观察各组动物术后外周血淋巴细胞数量变化和移植物存活时间。结果 FTY720组、二药联用组和三药联用组的大鼠外周血淋巴细胞在给药后3h开始明显下降，停药后开始回升，至停药14d后恢复正常；移植心脏的存活时间，对照组平均为7．8d，CsA组为16．0d，MP组为27．6d，三药联用组为16．8d，而FTY720组和二药联用组分别超过了150d和124d。结论 FTY720可诱导同种异体大鼠心脏移植物长期存活。     

Effect of FTY720 on chronic cyclosporine nephropathy in rats

BACKGROUND: Long-term treatment with cyclosporine A (CsA) causes tubulointerstitial inflammation and fibrosis in the kidney. To define the role of lymphocytes in this process, the novel lymphocyte-specific inhibitor FTY720 was administered to rats with experimental model of chronic CsA nephropathy. METHODS: Sprague-Dawley rats were treated daily for 4 weeks with CsA (7.5 mg/kg), or both CsA and FTY720 (0.125 mg/kg). The effects of FTY720 on CsA-induced renal injury were evaluated using renal function tests and histopathology, and the expression of mediators of CsA-induced renal injury (osteopontin, transforming growth factor-beta1 [TGF-beta1], betaig-h3, and angiotensin II). RESULTS: FTY720 treatment significantly decreased T-lymphocyte accumulation in kidneys compared with CsA treatment alone. FTY720 treatment improved not only CsA-induced renal dysfunction but also renal histopathology, demonstrated by decreased macrophage infiltration and interstitial fibrosis. Increased osteopontin, TGF-beta1, betaig-h3, and angiotensin II expression in CsA-treated rat kidneys were decreased with FTY720 treatment. CONCLUSIONS: FTY720 treatment prevents CsA-induced renal injury.     

FTY720对小鼠外周血淋巴细胞的影响

目的　观察FTY72 0对近交系小鼠外周血淋巴细胞的影响。方法　选用近交系雄性C5 7BL/ 6小鼠 ,连续口服FTY72 0 (3mg·kg-1·d-1) 14d。每只动物于服药后 2、4、6、8、12、2 4h以及停药后 1、2、3、4、5、6周自尾静脉采血 ,计数淋巴细胞数。结果　服用FTY72 0后 ,小鼠外周血淋巴细胞迅速减少 ,2h后即由服药前的 (870 0± 6 5 6 ) / μl降至 (3783± 176 ) / μl(P <0 .0 1) ,并于 4h降至最低值 (2 4 6 7± 2 5 2 ) / μl,之后基本保持稳定。停用FTY72 0后小鼠外周血淋巴细胞没有立刻开始恢复 ,在低水平徘徊 2周后 ,第 3周淋巴细胞数开始出现明显回升 ,停药后第 6周恢复到 (7833± 76 4 ) / μl,与服药前相比 ,差异无显著性 (P >0 .0 5 )。结论　FTY72 0起效迅速 ,特异性强 ,且作用可逆。     

Evidence that FTY720 induces rat thymocyte apoptosis

FTY720, a novel immunomodulator with the potential to improve immunosuppressive therapy after organ transplantation, is currently under clinical investigation. FTY720 drastically decreases blood lymphocytes, especially T cells, accelerating lymphocyte homing to secondary lymphoid organs. However, its immunosuppressive effects remain unknown. We investigated these effects in rat thymocytes. Rats were intramuscularly injected with 10mg/kg/day FTY720 or saline for 7days. Thymuses were removed on days 0, 1, 3, 5, 7 and 14 after treatment. Three-color analysis was performed with a flow cytofluorometer. Apoptotic nuclei in the tissue sections were identified by TUNEL. Genomic DNA was then extracted and samples were electrophoresed on 2.0% agarose gel. FTY720 reduced the total number of thymocytes and, with time, significantly reduced the percentage of CD4+8+ TCRalphabeta(negative/low) thymocytes. Light microscopy of thymuses of FTY720-treated rats revealed obvious reductions in the size of the cortical region. TUNEL analysis showed that FTY720 induced thymocyte apoptosis in the cortical region. Furthermore, DNA fragmentation was observed in thymocytes treated with FTY720, indicating thymocyte apoptosis. FTY720 reduced the number of CD4+8+ thymocytes before TCRalphabeta expression resulting in impaired thymocyte differentiation and maturation. This might be an immunosuppressive effect of FTY720.