A novel immunosuppressant, FTY720, induces peripheral lymphodepletion of both T- and B cells and immunosuppression in baboons

Transplant Immunology 1999; 7: 149-157

FTY720, A novel immunosuppressant, induces peripheral lymphodeptetion of both T- and B cells and immunosuppression in baboons Valerie Quesniauxa, Lynn Fullardb, Hiram Arendseb, Glenda Davisonb, Noel Markgraaff?, Roland Auerb, Francis Ehrhartc, Gerolf KrausC and Henk-Jan Schuurmana aNovartis Pharma Transplantation Research, Basel; bUniversity of Cape Town Medical School, Cape Town and cNovartis Pharma Drug Monitoring and Pharmacokinetic Department, Base1 Received 17 June 1999, accepted 12 July 1999

Abstract: Objective: FTY720, a new immunosuppressant active in transplantation models, modulates lymphocyte re-circulation, leading to peripheral lymphopenia and increased lymphocytes in lymph nodes and Peyer’s patches. Here, we investigated the susceptibility of baboons to FTY720 as an introductory study to transplantation protocols. Methods: FTY720 was administered orally to Chacma baboons at 0.3 or 0.1 mg/kg/day for 3 days or at 0.03 mg/kg/day for 10 days. Haematological parameters, lymphocyte phenotype (CD3, CD4, CDS, CD20), cell apoptosis, er viva blood cell proliferation in response to mitogens and drug blood levels were monitored during treatment and up to 4 weeks thereafter. M&r &ufings:FTY720 administered p.o. in baboons at 0.3 mg/kg/day caused a marked decrease in circulating lymphocytes within 4 h of treatment, reaching 6080% decrease witbin 24-48 h. The effect of FLY720 was seen both on T- and B cells, although it was slightly more rapid/pronounced on T cells. CD4+ cells were slightly more affected than CD8+ cells. The response onset was faster and the duration longer at higher dose, but the maximal peripheral lymphodepletion achieved was similar within the dose range 0.03-0.3 mgikg tested. Ex viva mitogen-induced lymphoproliferation was drastically decreased after FTY720 treatment, corresponding to the reduced blood lymphocyte counts. The blood drug levels measured after FTY720 administration correlated well with the dose applied but there was a poor correlation between FTY720 blood levels and the extent of peripheral lymphodepletion, suggestive of a high tissue distribution of the drug. When compared with cynomolgus monkeys treated in the same way, baboons had a lower initial exposure and a slightly lower response 24 h after one or two doses of PTY720 0.03-0.3 mg/kg. However, the stabilized drug blood levels and peripheral lymphodepletion achieved after 7 days of FTY720 0.03 mgkg/day were similar in both nonhuman primate species. Conclusionr: FTY720 was well tolerated and was effective in terms of peripheral T- and B lymphodepletion in baboons, indicating that it could be used in protocols of allo- and xenotransplantation. The pharmacokinetic and pharmacodynamic profiles of FI’Y720 in baboons suggest the use of high induction doses to optimize immediate response followed by a reduced dose regimen for drug maintenance.

Address for correspondence: V Quesniaux, ‘Bansplantation Research S386.155, Novartis Pharma Ltd, CH-4002 Basel, Switxerland. E-mail: [email protected] 0 Arnold 1999

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Introduction FN720, a novel immunosuppressant

produced by modification of a metabolite from Isaria sinchzirii,’ is active in transplantation models in rodentsTe9 dogs” and nonhuman primates,” and shows strong synergies with cyclosporine (CsA). FTY720 was recently shown to be active in concordant rodent xenotransplantation in combination with FK506 in a model of skin graft from Golden Syrian hamsters to Lewis rats.” The immunosuppressive effect of FTY720 is associated with a decrease in circulating lymphocytes, especially T cells.“*i3 The mode of action of FN720 on lymphocyte trafficking is still unclear. FI’Y720 seems to increase lymphocyte trafficking to Peyer’s patches mediated by lymphocyte homing receptors such as CD62L.5.‘3 The effect of FIY720 on G-protein coupled receptors such as chemokine receptors has been proposed.” Some effect of FN720 on apoptosis of blood mature T lymphocytes, especially CD4 lymphocytes, was also reported in mice and in rats at high doses.15 However, FTY720 does not cause bone marrow depression in rats15 nor in mice (Quesniaux et al., unpublished). In vitro, FN72O has no inhibitory activity on rodent mixed lymphocyte reaction comparable to that of CsA or FK506 but it induces apoptosis of lymphy and cultured cell lines at high micromolar concentrations.” 6 Baboons have long been used in nonhuman primate allotransplantation models. They are essential for pig-to-nonhuman primate xenotransplantation models, such as orthotopic cardiac transplantation, to match the donor organ size or in studies that require large blood volumes for extra-corporal blood depletion of natural antibodies. Prevention of chronic graft rejection in allo- and xenotransplantation will involve inhibiting not only the T-cell compartment but likely other cell types including B cells. Combinations of immunosuppressant drugs bearing different modes of action are thus likely to be used in transplantation. Before new immunosuppressants can be applied in transplantation protocols studies on tolerability, pharmacokinetics and pharmacodynamics have to be performed. Extrapolation across species is not directly possible and baboons have been shown to be rather insensitive to immunosuppressants such as CsA. The susceptibility of lymphocytes to in vitro immunosuppression by CsA and the oral bioavailability of CsA in vivo is much lower for baboons than for humans.‘7~‘s Here, we studied the susceptibility of baboons to the novel immunosuppressive drug FIY720.

Objectives The objectives of the present study were (1) to determine the effects of FIY720 administered at pharmacological doses (O-03-0.3 me&day orally for 3-10 days) in baboons, in terms of tolerability and efficacy; and (2) to compare the effects of FIY720 with available data obtained in another nonhuman primate species, cynomolgus monkeys. The pharmacodynamic and pharmacokinetic read-out included: l l

haematological parameters with leucocyte differentials; immunophenotyping of CD3, CD4, CD8 and CD20 positive CdlS;

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vivo lymphoproliferative assays of whole blood stimulated with mitogens; detection of apoptotic cells by flow cytometry; and blood levels of FN720. a

Tmnsplant Immunology 1999; 7: 149-157

We show that in baboons doses of 0.03-0.3 mg/kgZday of FIY720 cause rapid and profound lymphodepletion of both T and B cells from peripheral blood with decreased er vivo lymphoproliferation but no evidence of apoptotic cells in blood.

Materials and methods (2-Amino-2-[2-(4-octyIphenyl)ethyl&opane-1,3FTY720 diol hydrochloride; MW 343.94; Novartis Pharma, Basel, Switzerland) was dissolved in sterile distilled water to be used at 0.03,O.l or 0.3 mg/kg as mentioned and applied at 2 ml&g for gastric intubation or at 1 ml/l5 kg for incorporation in food. The solution for application was prepared fresh every day. Study design

Groups of three Chacma baboons (Papio ursinus) were matched for body weight per group (range 14-20 kg). The first group received FIY720 at 0.3 or 0.1 mg/kg/day for 3 consecutive days (day 0, 1,2) and was bled on day 0, 1, 2,4, 7, 10, 14, 21 and 28. A mock treatment with vehicle was first conducted (on day -7, -6, -5) to assess the effect of the procedure on the baboon haematological parameters, immunophenotyping and w vivo whole blood proliferation. After full recovery, a 48-h pharmacokinetic study after a single administration of FI’Y720 at 0.3 mg/kg was performed on the same animals. A second group of animals received FTY720 at 0.1 mgikg/day for 3 consecutive days (day 0, 1, 2). The third group received a lo-day course of FI’Y720 at 0.03 mg/kg/day. All animals were monitored until recovery of the lymphocyte counts to pretreatment values. Drug application

Dosing was performed each morning, just after blood sampling, using a gastric catheter under ketamine hydrochloride anaesthesia [lo mgikg intramuscularly (i.m.); Brevinaxe, Intramed Ltd, Port Elisabeth, South Africa]. The animals were inspected for about 30-60 min after dosing for side-effects due to dosing. Drug administration in a ball of food was used to complete the lo-day course of FTY720 at 0.03 mg/kgZday. FIY720 was given by gastric intubation on day 0,l and 2 as well as on the day of anaesthesia for bleeding (day 7 and 9), and in food balls on the remaining days. Administration of the drug in food was first validated in three baboons receiving one administration of FN72O at a dose of 0.3 mgZkg in a ball of food. FN72O blood levels were 2.7 f 0.4 rig/ml after drug administration in food versus 2.6 f 0.3 ng/ml after administration of the same dose of FN720 by gastric gavage (n = 3). The peripheral lymphocytes were reduced by 55 f 15% after FTY720 administration in food versus 59 f 6% after gastric gavage. Blood sampling

Blood sampling was done prior to drug administration in vacutainers containing either ethlenediaminetetraacetic acid (EDTA) K3 for haematology and drug monitoring or lithium heparin for cell culture and flow cytometry. Blood sampling was done under short anaesthesia (ketamine lOmg/kg i-m.). For the 48-h pharmacokinetic study, anaesthesia was induced with ketamine hydrochloride (7-14 mg/kg i.m.) and maintained using 0.5% halothane in 30% 02, 70% N20 intratracheal to secure the airways while saline was infused i.v. (150 ml 0.09% NaCl over 8 h).

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Haematology Peripheral blood haematology parameters were determined

Results

using Technicon Hl Germany).

Baboons received oral administration of vehicle FTY720 at 0.3 or 0.1 mglkglday for 3 days or of FI’Y720 at 0.03 mg/kg/day for 10 days. The effects of the drug treatments and the recovery were studied until haematological parameters were back to normal. There was no obvious side-effect of the drug treatments in terms of, for example vomiting or body weight loss, under the conditions used.

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Whole blood mononuclear cells were tested for cell surface expression of CD3 and CD20 or of CD4 and CD8 by flow cytometry using dual colour analysis. The antibodies used were antiRhesus CD3 Ab [clone FN18 coupled to fluorescein isothiocyanate (FITC) or biotin] from Biosource Europe SA (Fleurus, Belgium) and anti-human CD4 [clone SFC/12T4Dll coupled to phycoerythrin (PE)], CD8 (clone B9-11 coupled to FITC) and CD20 (clone H299 Bl-RDl coupled to PE) from Coulter Corporation (Miami, Florida). Briefly, whole blood (40 fl) was incubated together with antibody [lo pl of pretitrated dilution made in 1% bovine serum albumin (BSA) 0.1% NaN3 containing phosphate-buffered saline (PBS) (PBS-BSA)] for 30 min on ice. Red cell lysis of stained cells was then done by adding 2 ml of lysis buffer (Becton Dickinson, Mountain View, CA, USA) for exactly 10 min at room temperature. The cells were then washed twice in PBS-BSA and resuspended in PBS-BSA for flow cytometry analysis using a Becton Dickinson FACScan with CellQuest software. Whole blood proliferation assay Whole blood cultures (at a 1:40 final dilution in RPM1 medium, 3-4 replicates per point) were stimulated with purified phytohaemagglutinin (PHA, 8&d, Murex Diagnostics Cat# HA16) or concanavalin A (ConA 4 @ml; Sigma Cat# CS275) for 5 days at 37°C. 3H-thymidine (ICN Cat# 2406605; 6.7 Ci/mmole) was added during the last 18 h of culture (at 1 l.KX/well) and cell proliferation was measured by 3H-thymidine incorporation. Detection of apoptotic cello

Baboon mononuclear cells (PBMC) were isolated by ficoll gradient separation (Histopaque”-1077, Sigma) of peripheral blood diluted one-quarter in PBS (centrifugation 20 min at 1800 rpm, RT). The cells were washed once in PBS and counted. The Annexin V-FITC, together with propidium iodide, detection system (Boehringer-Mannheim, Mannheim, Germany) was used for determination of baboon PBMC apoptotic cells by flow cytometry as described by the manufacturer. In control experiments in vitro FlY720-induced apoptosis could be detected by the Annexin V detection system after incubation of human PBMC for 4 h in the presence of 3 PM FI”y720. Drus monitoring Blood samples were kept frozen until completion of each group study and all samples were tested in parallel per group. Blood levels of FTY720 were determined by liquid chromatography/mass spectroscopy on a HP Series 1100 HPLC system interfaced with a Finnigan Mat TSQ 7000 mass spectrometer. The analytical performance was estimated from the calibration curves and, tirn the results obtained with the quality control samples, analysed along with the samples. The limit of quantification (LOQ), defined by the lowest concentration of the calibration samples that could be quantified with an accuracy of 100% f 20%, was 0.38 ng/ml. The test of the samples from the low dose FTY720 0.03 m@g/day group was done with a LOQ of 0.072 ng/ml. The area under the curve (AUC) was calculated using the linear trapezoidal rule. Transplant Immunology 1999; 7: 149-157

Decrease in peripheral blood lymphocyte counte

Figure la shows the decrease in lymphocyte counts in three baboons during a FTY720 0.3 mg/kg/day treatment (on day 0, 1,2) and the recovery phase over 4 weeks. Within one day of FTY720 treatment at 0.3 mg/kg p.o. there was a 59 f 6 % decrease in circulating lymphocytes in the three baboons and a 70 f 7 % decrease 24 h a&r the second dose. The lymphocyte counts were already recovering on day 4, i.e. 48 h after the last FTY720 0.3 mg/kg administration. Recovery was steady in one baboon and rather variable in the remaining two animals. There was no systematic effect of the ketamine anaesthesia and gastric intubation with vehicle given on 3 consecutive days on the lymphocyte counts (variability f 20%; not shown). There was also no systematic effect of the FTY720 treatments on the haematological parameters beside the effect seen on lymphocyte counts (not shown). The early kinetics of peripheral lymphocyte depletion by FTY720 were investigated after a single dose of FTY720 at 0.3 mg/kg (Figure la inset). A decrease in circulating lymphocytes was already observed after l-2 h, it reached -50% by 4 h and was at a maximum of 77 f 3% decrease at 24 h post-treatment (n = 3). No apoptosis of peripheral blood mononuclear cells (PBMC) could be detected in the blood 1, 2, 4, 8 or 24 h after administration of FTY720 0.3 mgikg p.o. (not shown). The decrease in lymphocyte was still maximum 48 h after dosing. A dose-effect response study was next performed: A dose of FTY720 of 0.1 mgIkg/day induced a slight decrease in circulating lymphocytes after 1 day, reaching 49 f 7% decrease 24 h a&r the second dose (Figure 1b). There was still a 58 f 7% decrease 2 days after the third and last FTY720 0.1 mg/kg dose on day 4, indicative of some drug accumulation. This effect was very transient, as all three baboons were essentially back to pretreatment lymphocyte counts 3 days later. A low dose treatment of FTY720 at 0.03 mg/kdday p.o. was then administered for 10 days to achieve stabilization of the drug levels. Two out of three baboons had a decrease in circulating lymphocytes of 42 and 56% within 1 day of treatment (Figure lc). The lymphocyte decrease reached 60-80% on day 7-9 of treatment in these animals. Both baboon lymphocyte counts were back to pretmatment values on day 22, i.e. 13 days after the end of the treatment. The third baboon showed essentially no lymphocyte decrease in response to FTY720 at 0.03 mgIkg/day. Thus, FTY720 caused a profound and transient peripheral lymphodepletion in baboons, with very little inter-animal variability at high doses (0.3 mg/kg/day). Intersnimal di!&ences were more pronounced a&r low FTY720 dose treatments and during the recovery phase. Effect on both T- and B-cell peripheral blood counts FTY720reducedboth T- and B-cell counts in peripheral blood, with a slightly more pronounced effect on T cells than on B cells at high dose (Figure 2). Within 24 h of FTY720 0.3 mg/kg treatment CD3+ T cells decreased by 64 f 8 % whereas CD20+ B cells decreased by 39 f 6 % in the three treated baboons. After 2 days of treatment, CD3+ T cells were reduced by 75 f 7% and CD20+ B cells by 5 1 f 9 % (Figure 2a).

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The early kinetics of the response to FTY720 0.3 n&kg is shown in Figure 2a inset. At lower doses of FTY720, namely 0.1 mg/kg&y for 3 days (Figure 2b) or 0.03 mg&gMay for 10 days (Figure 2c), the response was more variable and the decrease of peripheral T- and B cells was essentially equivalent (9, 75, 88% T-cell decrease and 21, 74,56% B-cell decrease after 7 days of FTY720 0.03 mg@Aiay). The recovery of B cells after cessation of the PTY720 treatments was usually slower than that of T cells. Thus, not only peripheral T cells, but also B cells ate a&cted after FTY720 treatments in baboons. Effect on CD4+ and CDS+ peripheral blood T cells Peripheral blood CIM+ cells were slightly more aflbcted than CD8+ cells by all FTY720 tmatments (Figure 3). Within 24 h of FTY720 0.3 mg/kg treatment blood CD4+ cell counts were reduced by 73 f 6% and CDS+ cells by 57 f 10% (Figure 3a). After 2 days of treatment CD4+ cell counts were reduced by 84 f 7% and CD8+ cells by 66 f 10%.

Tmnsplant Immunology 1999; 7: 149-157

Figure 2 Decrease in peripheral CD3+ and CD2O+ cells after FI’Y720 treatments. PTY720 was administered on day 0,l and 2 at 0.3 m@g/day (a) or 0.1 mg/kg/day (b) and for 10 days at 0.03 mgIkg/day (c). The insert in (a) shows the early kinetics profile after a single administration of FTY720 0.3 mg&. The results are expressed as the decrease in peripheral CD3+ or CD20+ absolute cell counts as compared to pretreatment values on day 0. The mean f SD of three baboons per group is shown.

The early kinetics of this response is shown in Figure 3a insert. Doses of FTY720 0.1 mg/kgIday also affected CD4+ cells slightly more than CDS+ cells (Figure 3b). A similar trend was seen a&r treatment with FTY720 0.03 mg&g/day for 10 days (Figure 3c), especially on days 7-9 of treatment, in the two baboons showing peripheral lymphocyte depletion (8693% CD4+ decrease and 52-82% CDS’ decrease after 7 days of FTY720 0.03 mgikg/day). The third baboon in this group, which showed no overall decrease of peripheral lymphocyte counts, nevertheless had a strong imbalance of the CD4+ and CDS+ T cells: CD4+ T cells were decreased by up to 36% on day 7, whereas CDS+ T-cell counts were more than doubled on day 7-12. All FTY720-treated baboons were back to essentially normal CD4+ and CD8+ cell counts l-4 weeks post-treatment. The CD8+ T-cell counts recovered quicker and were overshooting more than CD4+ cells.

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There were some circulating CD4KD8 double positive cells in the peripheral blood of the baboons and this percentage essentially doubled l-7 days after FTY720 administration in nine out of the 12 FTY720 treatments. Five animals showed up to l&11% CD4+CD8+ in their lymphocyte population. The cells were shown to be CD3+CD4+CD8+ triple positive by three-colour FACScan analysis (not shown). Thus, the effect of FTY720 was slightly more rapid and more prolonged on CD4+ than on CD@ cells. Decreased ex viuo whole blood proliferation The response of whole blood cells to ConA decreased dramatical-

ly after treatment with FTY720 0.3 mg/kg and recovered within 2 weeks (Figure 4a). The nadir for ex vivo proliferation was on day 4 in two out of three baboons, e.g. 2 days after the last FTY720 dose. The pretreatment stimulation index was 1OO-300-fold and dropped to 0.5-5-fold on day 4, to return to pretreatment values on day 14. Transplant Immunology 1999; 7: 149-157

treatment with bTY720. FTY720 was administered on day 0,l and 2 at 0.3 mg/k@day (a) or 0.1 mgkg/day (c). The early kinetics protile after a single administration of FTY720 0.3 mgikg is shown in (b). Whole blood was either nonstimulated (open symbol) or stimulated with ConA (4 ugknl; closed symbol), incubated for 5 days, and 3H-thymidine incorporation measured over the last 18 h of culture.

In a study of the early kinetics after a single administration of 0.3 mg/kg FTY720, the proliferative response to ConA was already decreased by 50 f 15% at 4 h postgavage, 73 f 20% at 8 h and was minimal at 24-48 h post-treatment (95 f 4% decrease; Figure 4b). Treatment of baboons with FTY720 0.1 mg/kg/day for 3 days also reduced the proliferative response to ConA on days 14 (Figure 4~). The stimulation index obtained after incubation of baboon whole blood with PHA was usually about IO-fold lower than with ConA, but the general trend and the kinetics of reduced response after FTY720 treatment were similar (not shown). Thus there was rapid and profound decrease of the lymphoproliferative capacity in response to FTY720, essentially reflecting the decrease in peripheral lymphocytes. Fn720 blood levels The concentrations of FI’Y720 found in peripheral whole blood after treatment with FTy720 0.3 mg/kg/day for 3 days are shown in Figure 5a. All animals were exposed to FTY72Q and there was

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(0-4g h) was 77.9 f 23.6 h ng/ml and the half-life (t& estimated to 36 f 12 h. Administration of FIT720 at 0.1 mg/kg/day for 3 days resulted in a mean blood concentration of -0.6ng/ml after day 1 which remained within this concentration range for the period observed, e.g. up to day 7 (not shown). Administration of 0.03 mgkg/day FIT720 for 10 days resulted in a mean blood concentration of 0.161 f 0.139 @ml after 1 day, increasing to 0.317 f 0.167 ng/ml after 2 days and reaching a maximum of 0.718 f 0.025 @ml after 7 days of treatment, afterward it remained at this value in two out of three animals (Figure 5~). Inter-animal variability was rather low, especially after 7 days of treatment, although the concentrations measured were very low. Thus, there was a clear drug accumulation, in line with the estimated tm>24 h but the concentrations of FN720 stabilized on days 7-9 for low dose lTY720 0.03 mgikg/day administration. FTY720 dose-exposureandblooddrugIevelsversus

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There was a good correlation between the 24-h trough blood levels of FIT720 measured and the dose of FIT’720 administered, in the range of 0.03,O.Ol and 0.3 mgkgklay. The correlation coefficient was 0.947 after a single administration (Figure 6a) and 0.964 after two daily doses of FTY720 (not shown). We then tried to correlate the FIT720 blood levels with the peripheral lymphodepletion obtained in these studies (Figure 6b). Only FIY720 blood levels measured 24 h postdosing, a time point when maximal lymphocyte peripheral depletion is seen

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a relatively low inter-animal variability in the concentration-time profiles. FTY720levels of 2.6f 0.3 @ml were found in circulation 24 h after the first dose and of 4.6f 0.8&ml 24 h after the second dose. Although increased blood levels of FIT720 paralleled the peripheral lymphodepletion on day 1 and 2

(Figure la), there was no clear correlation between FIT720 blood levels and lymphodepletion in the wash-out period. On day 7, FTY720 blood levels were down to 0.9 f 0.4 @ml (individual values 0.6, 1.3 and 0.8 @ml), namely -20% of the FIT720 level observed on day 2, whereas lymphocyte count decrease was 43, 73 and -4% of pretreatment values, respectively. The pharmacokinetic study after a single oral administration of FIT720 0.3 mgikg to three baboons showed that the 24-h profile of blood FTY720 values was rather flat, with peak blood concentrations of unchanged drug of 2.16 f 0.58 ng/ml observed between 2 h and 24 h postdose (Figure 5b). AUC Transplant Immunology 1999; 7: 14S157

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after a single dose of FN720 0.3 mg/kg (Figure la inset), were considered. There was little correlation between the 24-h FTY72O trough blood levels and the lymphocyte peripheral depletion (Figure 6b; correlation coefficient 0.426). The correlation was somewhat better at very low (O-O.7 ng/ml) FIY720 blood levels (correlation coefficient 0.784) but above that concentration, and especially above 1 ng/ml, the lymphocyte depletion was usually in the maximal range, i.e. 60-80%, with no correlation within a range of FTY720 concentrations of l-5 @ml. Thus the oral bioavailability of FI’Y720 seems reproducible and linear within the dose-range studied and a maximal peripheral lymphodepletion was usually observed for FTY720 blood levels above 1 n&nl. Swcepeibility to FTY720 of baboons versus cynomol~ monkeys

Some pharmacokinetic and pharmacodynamic studies of TTY720 in cynomolgus monkeys (Macaccu fasciculati) have been conducted as an introduction to transplantation studies and will be reported elsewhere (H-J.S., manuscript in preparation). The FIY720-induced peripheral lymphodepletion was slightly less pronounced 24 h after the first or second dose of TTY720 at 0.3 or 0.1 mg/kg in baboons as compared with cynomolgus monkeys treated in the same way (see lhble 1). However, after 7 days of treatment with FE720 at 0.03 mg/kg/day, the decrease of lymphocytes in peripheral blood was -5,72,80% in the three baboons (49 f 47%) as compared with 59 f 9% in cynomolgus monkeys (n = 4; H-J& manuscript in preparation). The lower initial susceptibility of baboons to PTY720 seemed due to a lower drug exposure. The initial exposure of baboons to FTY720 was indeed roughly half that of cynomolgus monkeys 24 h after the first or second dose of TTY720 at 0.3 or 0.1 mg/kg (‘lhble 2; H-J.S., manuscript in preparation). However, the concentration reached after 7 days of daily treatment with 0.03 mg/kg/day FTY720 were slightly higher in baboons than in cynomolgus monkeys (0.72 f 0.03 versus 0.48 i 0.22 @ml). Thus, baboons seem to have a lower initial exposure to FTY720 than cynomolgus monkeys but, when drug levels were stabilized, the blooddrug concentrations and the response to FN720 0.03 mg/kg/day in terms of peripheral lymphodepletion were similar in both species.

‘lable 1 FI’Y720 dose-response in baboons and cynomolgus monkeys: peripheral lymphocyte count decrease 24 h after administration FTY720 0.3,O.l or 0.03 mg/kg

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Discussion Pharmacokinetic and pharmacodynamic studies of new immunosuppressants are essential to establish dose-response and design new drug regimens for transplantation experiments, especially in nonhuman primates. Depending upon the drug under study, haematological parameters or immunophenotyping of the different lymphocyte populations by flow cytometry can be used as a ‘read-out’ of the drug effect. This is the case here with FT’Y72f.lthat causes a rapid and profound decrease in lymphocyte counts in peripheral blood. However, other immunosuppressants, such as cyclosporine, FK506, rapamycin or RAD do not affect the number of lymphocytes in circulation, but rather their responsiveness to activation stimuli. Ex vivo lymphoproliferation assays may be useful to assess the pharmacodynamics of such immunosuppressive drugs. Whole blood assays are preferred for pharmacodymumc evaluations since they avoid drug redistribution during lymphocyte separation and require only small quantities of blood, allowing repeated sampling for kinetic studies. Here, we performed pharmacodynamic studies of FTY720 in baboons based on whole blood lymphophenotyping and ex vivo lymphoproliferation as an introductory study for the use of FTY720 in baboon allo- or xenotransplantation. All baboons treated with FJY720 were exposed to the drug. The main effect of FTY observed was a rapid and profound decrease in peripheral lymphocyte counts. Both T- and B cells were reduced and although the effect was slightly more rapid/pronounced on T cells, it was more persistent on B-cell counts that took longer to recover. Thus, we could not see any predominant effect of FTY720 on T cells as reported.“*r3 CD4+ cells were slightly more affected than CD8+ cells and this effect was more persistent as CD8+ cell counts recovered quicker and were overshooting more than CD4+ cells. Thus, there was a decrease of T/B-cell and CD4/CD8 ratios, as seen after steroid administration, but there was no parallel increase of neutrophil counts, excluding a steroid-mediated effect. FPY720 treatments reproducibly induced a strong and rapid decrease of a vivo whole blood proliferation in response to mitogens. Carry-over of FrlY720 in the er vivo assay could not explain these results, since the maximal FN720 concentrations seen in blood after treatment were in the range of 0.015 p.M (5 ng/ml), whereas FTY720 did not inhibit lymphocyte proliferation in baboon mixed lymphocyte cultures at sub-micromolar concentrations (not shown). The decrease in whole blood pro-

lhble 2 Comparison of FlY720 blood levels in baboons and cynomolgus monkeys 24 h after administration of FTY720 at 0.3, 0.1 or 0.03 mgflrg/day

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Cynomolgus

FTY720 treatment

Baboons

Cynomolgus

One day at: 0.3 mg/kg 0.1 mgkg 0.03 mgkg

60f6 17f12 27f37

77f6 56+13 29f9

One day at: 0.3 mgikg 0.1 mg/kg 0.03 mgkg

2.55 f 0.33 0.54 f 0.48 0.16 f 0.14

4.74 f 1.17 1.33 * 0.21 0.21 f 0.04

‘ho days at: 0.3 mg/kg 0.1 mg/kg 0.03 ma/ha

70f7 49f7 42f26

86f6 59flO 46f13

‘Kvodays at: 0.3 mg/kg 0.1 mg/kg 0.03 mgkg

4.56 f 0.77 0.63 f 0.22 0.32 f 0.17

5.73 f 1.28 2.02 * 0.24 0.47 f 0.16

Results expressed in % decrease in lymphocyte counts as compared to day Oofthegiventreatment(meanfSD,n=3inbakonsandn=4incynos).

Transplant Immunology 1999; 7: 14%157

Results expressed in ngM FN720 in whole blood (mean rt SD; n = 3 in baboons and n = 4 in cynomolgus monkeys).

156

V Quesniaux et al.

liferation is likely to be caused by a numerical deficit rather than a functional deficit in the responder T-lymphocyte population after FTY720 treatment. We showed here that whole blood ex vivo proliferation in response to ConA is sufficiently reproducible and uses little material to be suitable for longitudinal efficacy studies of immunosuppressive drugs. FTY720 induces cell apoptosis at high micromolar concentmtions in vitro and apoptosis has been proposed as a possible mechanism for FTY’IZO-induced peripheral lymphodepletion. However, this is still controversial; apoptotic cells in thymus, spleen and lymph nodes were reported in mice after FTY720 treatment.” However, no apoptotic thymocytes or splenocytes could he detected 4-12 h post-FTY720 in mice treated with 1 mgZkg i.p.,* whereas these mice showed up to 40% of apoptotic peripheral blood lymphocytes 4 h after injection of FTY720. In the present studies in baboons, no apoptotic PBMC could be detected after FTY720 treatment using a read-out that detects in vitro FTY7204nduced apoptosis. No

attempt was made to analyse the lymphoid organs in terms of cell populations or immunohistochemistry during the FTY720 treatment period, since the protocol included follow-up of the post-treatment recovery phase and only post-mortem histology. The circulating CD4/CD8 double positive T cells found in baboon peripheral blood essentially doubled l-7 days after FTY720 administration. Increase of CD4/CD8 double positive cells in periphery has also been reported in rats l-8 days after a single FTY720 treatment. ls This could be a sign of accelerated T-cell production, as a response to the massive disappearance of T cells, should massive T-cell apoptosis occur. However, we could not detect any apoptotic cells in the peripheral blood. It was recently shown that the percentage of CD4CD8 double positive thymocytes decrease (from 74 to 57%) 16 h after FTY720 1 mgkg in mice.* This was interpreted

as FTY720 inhibiting negative selection of immature thymocytes, although there was no data on CD4CD8 double positive cells in peripheral blood in these animals. It could be that the effect of FTY720 on lymphocyte re-circulation also affects the trafficking of thymocytes to the periphery. There was a good correlation between the FI’Y720 24-h trough blood levels and the FIY720 dose applied in the range 0.03-0.3 mg/kg/day. The quickest onset of peripheral T-cell lymphodepletion was clearly obtained with the highest drug dose and the inter-individual variability of the response seemed to increase with lower dosage. At high FTY720 dosage, there was very little inter-baboon variability in the FlY720-induced reduction of lymphocyte counts, be it T cells, B cells or CD4+ and CD@ cells, whereas a much stronger inter-individual variation of lymphocyte counts was seen during the recovery phase. The duration of the effect was clearly longer after higher dose, as seen after 3 days of treatment with FI’Y720 at 0.3 or 0.1 mg/kg/day. However, the maximal effect in terms of lymphocyte count decrease in peripheral blood was similar after treatments with 0.3 or 0.03 mg/kg/day of FTY720 (up to 80% decrease). Therefore there was no clear dose-response effect. However, the data suggest that high induction doses (e.g. 0.3 mgikglday here) should be used to optimize immediate response and that a reduced dose regimen should be sufficient for drug maintenance (e.g. 0.03 mg/kg/day). There was little correlation between the 24-h FIY720 trough blood levels and the lymphocyte peripheral depletion. The correlation was somewhat better at very low (O-O.7 ng/ml) FT”y720 blood levels but above a concentration of 1 @ml, the lymphocyte depletion was usually in the maximal range. It seemed that there was a threshold of FI’Y720 levels above which cells were affected and left the blood circulation. The effect of Transplant Immunology 1999; 7: 149-157

FTY720 on the expression of lymphocyte homing receptor such as CD62L,r3 G-protein coupled receptors14 or apoptosis8~‘9~20 have been proposed but the mode of action of FIY720 on lymphocyte trafficking is still unclear. Discrepancies between FTY720 blood levels and lymphocyte blood counts were also noted in the single dose pharmawkinetic study where the circulating blood levels of FTY720 decreased by 30% between 24 and 48 h post-dose, whereas the lymphocyte counts were stable. These results suggest that FIY720 accumulates in the tissues and/or that it has a long duration of action. The data obtained in baboons therefore question the predictive value of FTY720 peripheral blood levels for drug effectiveness. In conclusion, we show here that baboons are susceptible to FIY720 and that not only peripheral blood T cells but also B cells are profoundly affected by FTY720 treatment. We document that the effect is more profound and persistent on CD4+ than on CDS+ T cells and that it translates into reduced ex tivo T-cell proliferation. The mechanism of FI’Y720 peripheral lymphodepletion, which was originally reported as preferentially exerted on T cells, is still controversial and the relevance of this phenomenon for the actual immunosuppressive activity of FN720 is still under debate. We also show that, although baboons have a lower initial exposure to FIY720 than cynomolgus monkeys, the blood-drug concentrations and the response to FIY720 in terms of peripheral lymphodepletion were similar in both species when drug levels were stabilized. It will now be interesting, especially in the context of chronic rejection and xenotransplantation, to assess whether the FTY720induced decrease in peripheral B cells also translates in some B cell immunosuppression in T-cell-independent B-cell models.

References 1 Ada&i, K, Kohara, T, Nakao, N et al. Design, synthesis, and struc-

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ture-activity relationships of 2-substituted-2-amino-1,3-propanediols: discovery of a novel immunosuppressant, FIY 720. Bioo~Med Chem Lett 1995; 5: 853-56. Suzuki S, Enosawa SN, Kakefuda T et al. A novel immunosuppressant, FIY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation. Tmnsplanturion 1996; 61: 200-205. Suzuki S, Enosawa S, Kakefuda T et al. Immunosuppressive effect of a new drug, FTY720, on lymphocyte responses in vitro and cardiac allograft survival in rats. Tmnsplant Immunol19%, 4: 252-55. Chiba K, Hoshino Y, Suzuki C et al. FTY720, a novel immunosuppressant possessing unique mechanisms. I. Prolongation of skin agograft survival and synergistic effect in combination with cyclosporine in rats. TransplantPmc 1996; 28: 10X-59. Chiha K, Yanagawa Y, Masubuchi Yet al. FIY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. I. PTY72O selectively decreases the number of circulating mature lymphocytes by acceleration of lymphocyte homing. J Zmmunol 1998; 160: 5037-44. Hoshino, Y, Suzuki, C, Ohtsuki, M, Masubuchi, Y, Amano, Y, Cbiba, K. FTY720, a novel immunosuppressant possessing unique mechanisms. II. Long-term graft survival induction in rat heterotopic cardiac allografts and synergistic effect in combination with cyclosporin A. Tmnspknt Pnx 1996; 28: 1060-61. Yanagawa Y, Sugahara K, Kataoka H, Kawagucbi T Masubuchi Y, Chiba K. FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. II. FIY720 prolongs skin agograft survival by decreasing T-cell infiltration into grafts but not cytokine production in vivo. JZmmunol1998; 160: 5493-99.

JTY720, a novel immunosuppressont

8 Shimizu C, Li X, Kimura M et al. A novel immunosuppressant, FIY720, increases the efficiency of a superantigen-induced peripheral T-cell deletion whilst inhibiting negative selection in the thymus. Immunology 1998; 94: 503-12. 9 Wang ME, ‘Ibjpal N, Qu X ei al. Immunosuppressive effects of FIY720 alone or in combination with cyclosporine and/or sirolimus. Tmnsphttation 1998; 65: 899-905. 10 Kawaguchi T, Hoshino Y, Rahman Fetal. FTY720, a novel immunosuppressant possessing unique mechanisms. III. Synergistic prolongation of canine renal allograft survival in combination with cyclosporin A. TransplantPmc 1996; 28: 1062-63. 11 ‘Roncosco P, Kahan B. Preclinical evaluation of a new immunosuppressive agent, FTY720. Clin Biochem 1998; 31: 369-73. 12 Fukuda Y, Ohdan H, Miyata Y, Shintaku S, Dohi K. Effect of FIY720 on immunoregulation in concordant xenotransplantation. TransplZnt 1998; 11: S461-64. 13 Yanagawa Y, Masubuchi Y, Chiba K. FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocy-tes by acceleration of lymphocyte homing in rats, III. Increase in frequency of CD62L-positive T cells in Peyer’s patches by FTY720induced lymphocyte homing. Immunology 1998; 95: 591-94. 14 Brinkmann V, Xue C, Feng L, Chiba K, Pinschewe D. FTY720: a novel drug that prevents organ graft rejection by modulating T- and B-cell recirculation. TiPS 1999, in press.

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15 Enosawa S, Suzuki S, Kakefuda T, Li XK, Amemiya H. Induction of selective cell death targeting on mature T lymphocytes in rats by a novel immunosuppressant, FN72O. ImmunophannacorogY 1996; 34: 171-79. 16 Shinomiya T, Li XK, Amemiya H, Suzuki S. An immunosuppressive agent, FTY720, increases intracellular concentration of calcium ion and induces apoptosis in I-IL-60. Immune logy 1997; 91: 594-600. 17 Smit JA, Drielsma RF, Myburgh JA, Laupacis A, Stiller CR. Renal allograft survival in the baboon using a pretreatment protocol with cyclosporine. lhpiantation 1983; 36: 121-24. 18 Stark JH, Smit JA and Gridelli B. Sensitivity of baboon lymphocytes to cyclosporin A and FK 506: relative resistance of alloactivated cells to CyA. Tmnspl Int 1994; 7: 372-78. 19 Suzuki S, Li XK, Enosawa S, Shinomiya T A new immunosuppressant, FT”y720, induces bcl-Zassociated apoptotic cell death in human lymphocytes. Immunology 1996,89: 518-23. 20 Suzuki S, Li XK, Shinomiya T et al. The in vivo induction of lymphocyte apoptosis in MRL-lprflpr mice treated with FTY720. Clin Exp Immunol1997; 107: 103-11.