The novel immunosuppressant FTY720(2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride) is an analog of a natural compound derived from the fungus Isaria sinclairii(1, 2). Within 3 hr of delivery via oral gavage, a single dose of 10 mg/kg FTY720 markedly reduces the number of peripheral blood lymphocytes (PBLs*) in rats, an effect that slowly recovers during the next 14 days (3). The precise mechanism of the profound and long-lasting decrease in PBLs is unknown; FTY720 may induce lymphocyte apoptosis (3), because in vitro exposure of rat lymphocytes to high FTY720 concentrations(4×10-6 M) induced chromatin condensation, formation of apoptotic bodies, and DNA fragmentation (3, 4). Another explanation is that FTY720 alters the expression of adhesion molecules(3-5), thereby affecting the recirculation patterns of lymphocytes between the blood stream and the lymphoid organs, as evidenced by the capacity of in vitro addition of FTY720 to increase the expression of intercellular adhesion molecule-1 on human umbilical vein endothelial cells (5).
FTY720 delivered daily by oral gavage prolongs the survival of skin(6), heart (7), or liver allografts in rats, and kidney allografts in dogs, as well as prevents the development of graft-versus-host responses in rats(3, 5-10). FTY720 therapy initiated 1 day before transplantation, or on day 3 or 4 after grafting, effectively blocks the rejection of heart allografts in rats(3, 8). Although daily oral administration of 5.0 mg/kg/day FTY720 alone to dogs only slightly extended kidney allograft survival, the same FTY720 dose combined with 10.0 mg/kg/day cyclosporine (CsA;3, 8) protected kidney allografts from rejection. Similarly, the combination of individually subtherapeutic doses of FTY720(0.03 mg/kg/day) and CsA (3.0 mg/kg/day) significantly prolonged heart allograft survival in rats (7). Although these experiments in rats and dogs suggest that FTY720 potentiates the immunosuppressive effects of CsA, the limited dose range of the analysis prohibited a thorough evaluation of the exact nature of the interaction between the two drugs.
The present study examined in vitro effects of FTY720 alone and in combination with CsA or sirolimus (SRL) on the proliferative responses of human PBLs stimulated with phytohemagglutinin (PHA) or OKT3 monoclonal antibody (mAb). Although FTY720 alone failed to inhibit the in vitro proliferation of human PBLs in response to PHA or OKT3 mAb, the rigorous median effect analysis was used to document that FTY720 synergistically potentiated the inhibitory effects of CsA or SRL. Furthermore, a median effect analysis of the in vivo effects of combination therapy on cardiac allograft survival showed that FTY720 produces potent synergistic interactions with CsA or SRL.
MATERIALS AND METHODS
In vitro analysis. The median effect analysis and the proliferation assay conditions have been described in detail previously(11). Briefly, PBLs isolated from aliquots of heparinized blood obtained from 10 normal, healthy volunteers using Ficoll (Accurate Chemical & Scientific, Westbury, NY) density centrifugation were reconstituted in complete RPMI medium (Mediatech, Washington, DC), which was modified by the addition of 1% glutamine, penicillin (100 U/ml), streptomycin(100 μg/ml), L-asparagine, and 1% Hepes, as well as supplemented with 10% heat-activated fetal calf serum. PBLs were obtained from the same individuals on repeated occasions and used for each of the drug combination experiments in order to reduce the impact of inter-individual variations. Triplicate or quadruplicate cultures of PBLs (150,000/well; total volume of 150 μl/well) were activated with PHA (final concentration of 10 μg/ml) or anti-CD3 mAb(OKT3; Ortho Diagnostic Systems, Inc., Raritan, NJ; final concentration of 10 ng/ml) and maintained in 96-well round-bottomed plates (Corning) for 48 hr at 37°C in 5% CO2. In the mixed lymphocyte culture (MLC) experiments, 1×105 responder cells were cultured with irradiated (2000 rads) 1×105 stimulator cells in triplicates for 4 days. After an additional 18-hr incubation, the cultures were pulsed with 1 μCi of[3H]thymidine/well (specific activity, 6.7 Ci/mmol). After being harvested onto glass fiber filter paper, the cells were dried, and counted in liquid scintillation fluid in a Beckman LS1800 scintillation counter (Beckman Instruments, Irvine, CA). The degree of [3H]thymidine incorporation into DNA was estimated by calculating the mean count per minute (cpm) and SD. The nonstimulated values were generally less than 10% of the mean values upon PHA, OKT3 mAb, or MLC stimulation. Incubation with the vehicle for each drug consistently produced less than 15% inhibition of PBL proliferation.
Inhibition of cytokine-dependent proliferative response. Interleukin (IL)-2-dependent CTLL2 cells (5×103 cells/0.05 L of complete medium supplemented with 10% fetal calf serum) were incubated with 0.1 ml of serial dilutions of recombinant IL-2, which was dissolved in complete medium at a stock concentration of 2.5 U/ml, yielding concentrations spanning the range of 0.02-1.21 U/ml. Cytokine-triggered stimulation of CTLL2 cells was induced either in the absence or in the presence of FTY720, SRL, or FTY720/SRL. The reactions were incubated in a 96-well flat-bottomed tray for 24 hr at 37°C in a 5% CO2 incubator and pulsed with 1 μCi of [3H]thymidine (5.0/nmol) for the last 4-6 hr. Similarly, monolayer mink lung cells (5×103 cells/0.05 ml of complete Dulbecco's minimum essential medium supplemented with 10% fetal calf serum) were incubated until confluence. The fresh medium without serum was added to the wells, and the cells were incubated without or with drugs in the presence of epithelial growth factor, transforming growth factor, or a combination of epithelial growth factor and transforming growth factor for 24 hr at 37°C in 5% CO2. During the last 4 hr, the cells were labeled with 1 μCi of thymidine (5.0/nmol). After 4-6 hr, the medium was aspirated and the cells were washed five times with cold phosphate-buffered saline and twice with cold 10% trichloroacetic acid, followed by a 30-min incubation at room temperature. The trays were harvested on glass fiber paper, dried, and counted in liquid scintillation cocktail in a Beckman LS1800 scintillation counter.
Cell-mediated cytotoxicity assay. PBLs (1×105) were cultured without or with drugs in a 15% AB human serum supplemented RPMI complete medium together with 1×105 irradiated (3000 rads) allogenic stimulator cells. The cytotoxic activity generated during a 5-day culture was assayed by addition to each well of 2×105 effector cells and 1×104 allogenic target lymphoblasts, which had been prepared by stimulating 10×106 cells/ml with 5 μg/ml PHA for 3 days. After incubation of plates for 4 hr at 37°C in a 5% CO2, plates were harvested using Skatron harvesting system (Skatron, Oslo, Norway) with filters and frames for counting in the Cobra II auto-gamma detector(Packard, Meriden, CT). Maximum 51Cr release was assessed by the lysis of labeled targets with 1% Triton X-100 (Sigma Chemical, St. Louis, MO), whereas spontaneous release was estimated by culture of target cells alone. The percent specific cytotoxicity was calculated as: experimental cpm - spontaneous cpm / maximum cpm - spontaneous cpm × 100.
Drugs. FTY720 (Yoshitomi Pharmaceuticals Ltd., Osaka, Japan) was diluted in phosphate-buffered saline. CsA (Novartis, Basel, Switzerland), SRL(rapamycin; Wyeth-Ayerst, Rouses Point, NY), or mycophenolic acid (MPA; Sigma Chemical) were each dissolved in 95% ethanol. Tacrolimus (TRL; FK506; Fujisawa Pharmaceutical Co., Osaka, Japan) was dissolved in 95% methanol. Stock solutions of each of the drugs (1.0 mg/ml) were stored in a freezer(-79°C). The in vitro experiments used the concentration ranges of 100-3000 ng/ml for FTY720, and 10-1000 ng/ml for each of the other drugs (CsA, SRL, TRL, or MPA). For in vivo studies, the oral formulations of FTY720, CsA, and/or SRL were obtained from each manufacturer, and administered by oral gavage according to the weight of the animal. All of the experiments included vehicle controls.
Animals. Adult male inbred Wistar Furth (WF; RT1u), ACI(RT1a), Buffalo (BUF; RT1b), and Brown Norway (RT1n) rats weighing 160-250 g were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and cared for under the treatment guidelines of the institutional Animal Welfare Committee. Rats were housed in wire-bottomed cages with controlled temperature and light/dark cycles. The rats received water and chow ad libitum. All operations were performed under aseptic conditions, and each rat's postoperative condition was monitored daily.
Organ transplantation. The detailed techniques of heterotopic cardiac and orthotopic liver or small bowel transplantation have been described previously (12-14).
Median effect analysis. The median effect principle of Chou(15) states that: Equation (1) wherefa and fu represent the fractions of the system that are affected (% inhibition) and unaffected (1 - fa), respectively, by a drug at dose (concentration) D. For [3H]thymidine incorporation assays, percent inhibition refers to the difference in cpm value between cells cultured without and with the immunosuppressive drug. Greater than 60-day survival for heart allografts was defined as 100% protection(fa=1). Dm is the dose required for 50% inhibition(ED50), the median effect; m is a coefficient that describes the sigmoidicity of the dose-effect curve. The interaction between FTY720 and another drug was assessed by the combination index (CI) analysis of the doses necessary to achieve x% inhibition. Equation (2) When FTY720 was combined with CsA and SRL, CsA and SRL were considered a single agent. This expression, which represents the mutually nonexclusive case where each drug has a distinct mode of action, was applicable to SRL, CsA, and presumably FTY720. CI values less than 1 reflect synergistic, those equal to 1, additive, and those greater than 1, antagonistic interactions. A computer software program was used to determine the median effect parameters(Dm, m, and r) and the CI values(15).
Effect of FTY720 alone or in combination with CsA, SRL, TRL, or MPA on the proliferation of human PBLs. The inhibitory effects of FTY720(100-3000 ng/ml) alone or in combination with CsA (10-1000 ng/ml), SRL(10-1000 ng/ml), TRL (10-1000 ng/ml), or MPA (10-1000 ng/ml) on the response of purified human PBLs to stimulation with PHA or OKT3 mAb are shown inTable 1. The concentrations of FTY720 necessary to achieve 50% inhibition of the control (Dm) were 31.2×10-6 M for PHA stimulation and 37.2×10-6 M for OKT3 mAb stimulation(Table 1). The Dm values for FTY720 were significantly higher than those for CsA (0.07×10-6 M), SRL(0.21×10-6 M), TRL (0.02×10-6 M), and MPA(0.13×10-6 M) when PBLs were stimulated with OKT3 mAb. When PBLs were stimulated with PHA the Dm values for CsA, SRL, TRL, and MPA were 0.18×10-6 M, 1.16×10-6 M, 0.07×10-6 M, and 0.12×10-6 M, respectively.
A median effect analysis documented that FTY720 acted synergistically with CsA or SRL, but not with TRL or MPA, to inhibit PHA- and OKT3-stimulated human PBLs in vitro (Table 1). The CI value for 95% inhibition by FTY720 and CsA was 0.32 after PHA stimulation and 0.55 after OKT3 stimulation. Even greater in vitro synergism was observed between FTY720 and SRL, namely, CI values of 0.15 for inhibition of PHA-stimulated and 0.07 for inhibition of OKT3-stimulated human PBLs. The values for the interaction between FTY720 and TRL or MPA suggested pharmacologic antagonism or additivity, namely, 1.0 and 4.5 or 0.9 and 1.0 for PHA and OKT3, respectively(Table 1). Thus, FTY720 displays selective synergistic immunosuppressive effects. FTY720 alone showed variable and non-dose-dependent inhibition of MLC response only at concentrations of 2000-3000 ng/ml, which may represent toxic FTY720 concentrations (data not shown). In cell-mediated lympholysis assay, FTY720 failed to inhibit the generation of alloantigen-specific T cells (data not shown). Finally, FTY720 had no effect at nontoxic doses (<2000 ng/ml) on cytokine-derived proliferative responses induced either by epithelial growth factor and transforming growth factor in mink lung cells, or IL-2 in CTLL2 as well as HT-2 T-cell clones (data not shown).
Effect of FTY720 on organ allograft survival in rats. Untreated WF (RT1u) rats rejected BUF (RT1b) heart allografts at a mean survival time (MST) of 6.4±0.6 days. Daily treatment with FTY720 delivered by oral gavage prolonged MSTs in dose-dependent fashion: 0.05 mg/kg/day FTY720 extended survivals to 9.2±3.8 days (NS); 0.1 mg/kg/day to 16.2±6.1 days (P<0.01); 0.5 mg/kg/day to 25.4±2.9 days (P<0.01); 1.0 mg/kg/day to 29.4±1.7 days (P<0.01); 2.0 mg/kg/day to 40.2±7.6 days(P<0.01); 4.0 mg/kg/day to 40.5±4.8 days(P<0.01); and 8.0 mg/kg/day to 40.2±7.6 days(P<0.01; Fig. 1).
Untreated Lewis (LEW; RT11) recipients rejected orthotopic Brown Norway (RT1n) small bowel allografts at 10.6±1.9 days(Table 2). Although a 14-day course of oral treatment with 2.0 mg/kg/day FTY720 only slightly prolonged small bowel allograft survival(12.2±2.3 days; NS), 8.0 mg/kg/day FTY720 extended the MST to 23.5±4.0 days (P<0.01). Furthermore, untreated LEW recipients rejected ACI (RT1a) liver allografts at 12.8±0.8 days(Table 2), whereas LEW recipients treated daily for 14 days with 1.0 mg/kg/day FTY720 via oral gavage displayed prolonged liver allograft survival to 57.6±29.8 days, which was extended to 74.4±35.1 days (with 3/5 hosts surviving more than 100 days) when the dose of FTY720 was increased to 2.0 mg/kg/day (both P<0.001). Thus, FTY720 blocks the rejection of a variety of organ allografts in the rat model.
Interaction of FTY720 with CsA or SRL to block allograft rejection in rats. A 14-day course of 0.5 or 1.0 mg/kg/day CsA alone delivered by oral gavage did not affect heart allograft survival in WF recipients(6.6±0.6 days and 7.0±0.7 days, respectively; NS;Fig. 2), whereas 2.5 mg/kg/day prolonged the MST to 20.6±7.6 days (P<0.005). The addition to a 14-day CsA course (0.5 mg/kg/day) of 0.05, 0.1, or 0.5 mg/kg/day FTY720 extended BUF heart allograft survival to 18.8±2.6 days (P<0.01; CI=0.55), 32.6±16.2 days (P<0.01; CI=0.30), or 69.0±30.1 days (P<0.01; CI=0.12), respectively(Figs. 2 and 3). Similarly, addition of 0.01 mg/kg/day FTY720 to a 14-day course of 1.0 mg/kg/day CsA had no effect(8.8±3.0 days; NS; CI=1.4), whereas addition of 0.05 mg/kg/day prolonged the MST to 51.0±32.4 days (P<0.01; CI=0.35), with 1/5 grafts functioning for more than 100 days, and addition of 0.1 mg/kg/day FTY720 extended the MST to 87±29 days with 4/5 recipients surviving more than 100 days (P<0.001; CI=0.15;Figs. 2 and 3). Although a 14-day course of oral 0.08 mg/kg/day SRL alone was ineffective (6.8±0.8 days; NS), addition of 0.05, 0.1, or 0.5 mg/kg/day FTY720 prolonged the MSTs to 15.5±5.5 days(P<0.01; CI=0.5), 30.2±20.2 days (P<0.05; CI=0.22), or 38.4±12.9 days (P<0.001; CI=0.28), respectively (Figs. 3 and 4). Similarly, although a 14-day oral course of 0.8 mg/kg/day SRL alone only slightly extended graft survival (12.2±2.4 days; P<0.01), combined therapy with 0.01, 0.05, or 0.1 mg/kg/day FTY720 produced MSTs of 16.4±5.5 days(P<0.01; CI=0.83), 32.0±12.0 days (P<0.01; CI=0.15), or 56.0±19 days (P<0.001; CI=0.01;Figs. 3 and 4), respectively. Thus, the median effect analysis documented an in vivo synergistic interaction between FTY720 and CsA(CI=0.15-0.37), as well as between FTY720 and SRL (CI=0.22-0.53).
Interaction of FTY720 with CsA and SRL to block heart allograft rejection. Our previous studies showed that combinations of CsA and SRL delivered by oral gavage act synergistically to block allograft rejection in rats (11). Although 0.5 mg/kg/day CsA or 0.08 mg/kg/day SRL delivered individually by oral gavage was ineffective(Table 3, lines B and C), the combination of CsA and SRL extended the MST to 13.2±3.5 days (P<0.01;Table 3, line D). Addition of 0.01 mg/kg/day FTY720 to this CsA/SRL combination (0.5:0.08 mg/kg/day) prolonged BUF heart allograft survival in WF recipients in synergistic fashion to 23.8±6.3 days, with 1/5 surviving more than 100 days (P<0.01; CI=0.43;Table 3, line E); addition of 0.05 mg/kg/day to 55.5±27.0 days, with 2/6 surviving more than 100 days(P<0.01; CI=0.20; Table 3, line F); and addition of 0.1 mg/kg/day to more than 100 days (P<0.001; CI=0.18; Table 3, line G), respectively. By comparison, combination of 0.1 mg/kg/day FTY720 and 0.5 mg/kg/day CsA extended survivals to 32.6±16.2 days (P<0.01; CI=0.3), whereas combination of 0.1 mg/kg/day FTY720 and 0.08 mg/kg/day SRL extended survival to 30.2±20.2 days (P<0.05; CI=0.22). Thus, FTY720 displays substantially more synergism when used in combination with CsA and SRL together than when used with either drug alone (Fig. 3).
The present experiments examined the effects of FTY720 when delivered alone versus in combination with CsA and/or SRL on the proliferative responses of human PBLs in vitro as well as on organ allograft rejection in rats in vivo. In vitro studies showed that FTY720 is a poor inhibitor of T-cell proliferation, as evidenced by Dm values (50% inhibition) greater than 30×106 M after PBLs were stimulated with PHA or OKT3 mAb. In contrast, 50% inhibition of the same responses was readily obtained with 30- to 3000-fold lower doses of CsA, SRL, TRL, or MPA. Previous in vitro studies suggested that >4×10-6 M FTY720 was needed to induce apoptosis, as documented by the fragmentation of chromosomal DNA(3). However, it is unlikely that these concentrations are observed in vivo. For example, dogs treated with 5 mg/kg/day FTY720 had blood trough levels of approximately 200 ng/ml, which would produce a concentration of only 0.58×10-6 M (3). If the immune mechanism is dependent on parent compound concentrations, it is unlikely that apoptosis is the most important mechanism of FTY720 action. Indeed, our recent studies show that the lymph nodes harvested from rat heart allograft recipients treated for 7 days with therapeutic doses of FTY720 (2.0 mg/kg) had a frequency of alloantigen-specific T cytotoxic cells that was similar to that observed in untreated recipients (L. Tian, manuscript in preparation). We have also confirmed the finding that a single 2 mg/kg/day FTY720 dose delivered to rats by oral gavage decreased the levels of PBLs within 3 hr(3). Thus, the synergistic effect of FTY720 in vitro probably occurs via a different mechanism than the in vivo effect.
FTY720 administered by oral gavage delays the rejection of heart, liver, or small bowel allografts in rats. In fact, 0.5 mg/kg/day FTY720 completely inhibited heart allograft rejection and 1.0 mg/kg/day significantly prolonged liver allograft survival. In contrast, 8.0 mg/kg/day FTY720 produced only modest prolongation of the survival of small bowel allografts. Although previous experiments suggested that FTY720 was 30-fold more effective than CsA in blocking the graft-versus-host immune response induced by the injection of allogenic spleen cells into the foot pads of F1 recipients(9), it appears that the small bowel transplant model is refractory to FTY720.
The rigorous median effect analysis was employed to evaluate the in vivo interactions between FTY720 with CsA and/or SRL in a rat heart transplant model. The CI values documented potent synergistic interactions between FTY720 and CsA (0.01-0.47), and between FTY720 and SRL (0.01-0.83). In addition, the dose-reduction index values calculated for the synergistic interaction between CsA and FTY720 documented that CsA doses may be reduced by 3- to 7-fold and FTY doses by 14- to 22-fold. Similarly, SRL/FTY720 synergistic interaction reduced in combination SRL doses by 0.6- to 3-fold and FTY720 doses by 2- to 14-fold. Although previous transplant experiments in rats and dogs suggested that addition of FTY720 potentiated the immunosuppressive effects of CsA, the limited number of doses used and the lack of mathematical modeling prevented definitive conclusions regarding the nature of this interaction(3, 6). We have previously used the median effect analysis to document that CsA and SRL act synergistically to prolong heart or kidney allograft survivals in rats (11) and mice(16). Furthermore, in vitro experiments showed that the combination of CsA and SRL synergistically blocked proliferation of human PBLs, after stimulation with PHA or anti-CD3 mAb, as well as with alloantigens in MLCs (17). Thus, in vitro analyses using human PBLs, as well as in vivo experiments using rat organ transplant models to block allograft rejection, were used to predict a synergistic interaction between CsA and SRL, a favorable interaction that has been confirmed in clinical practice. Addition of low-dose SRL therapy to a dual-drug CsA/prednisone regimen reduced the incidence of acute graft rejection episodes from 35% to 7% for living-related transplants, and from 40% to only 10% for cadaveric transplants (18). Addition of SRL in the former setting allowed withdrawal of corticosteroids, and in the latter setting, a substantial CsA dose reduction in nonblack recipients.
The optimization of CsA-based immunosuppression has been a focus of clinical investigation since the first reports of CsA-induced renal dysfunction and other side effects (19). Indeed, the pleiotropic array of toxicities attributed to CsA has created a compelling need for regimens that permit significant CsA dose reduction (especially during the induction phase of immunosuppression) without sacrificing the effect of rejection prophylaxis (20, 21). Although the combination of CsA and SRL proffers such an alternative, because SRL itself does not produce nephrotoxicity (22), these two drugs have overlapping lipidogenic toxicities and thus even the reduced doses of CsA still cause toxic side effects. Addition of low FTY720 doses to rat recipients treated with the synergistic CsA/SRL combination produced potent beneficial effects (CI values of 0.01 to 0.46), allowing CsA doses to be further reduced by 3- to 7-fold, SRL doses by 9- to 34-fold, and FTY720 doses by 15- to 188-fold. Thus, the addition of FTY720 may improve the efficacy of CsA- or CsA/SRL-based immunosuppressive regimens.
Although FTY720 alone is not effective in vitro to block the proliferative responses of human PBLs stimulated with PHA or OKT3, it potently inhibits in vivo organ allograft rejection in rats. Combinations of FTY720 and CsA or SRL are synergistic both in vitro to block PBL proliferative responses, and in vivo to prevent allograft rejection. Furthermore, FTY720 displays synergistic effects when added to the combination of CsA and SRL.
*Abbreviations: BUF, Buffalo; CI, combination index; CsA, cyclosporine; IL, interleukin; LEW, Lewis; mAb, monoclonal antibody; MLC, mixed lymphocyte culture; MPA, mycophenolic acid; MST, mean survival time; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin; WF, Wistar-Furth; SRL, sirolimus; TRL, tacrolimus.
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