Pharmacokinetic / pharmacodynamic relationships of FTY 720 in kidney transplant recipients

FTY720 is a new and effective immunosuppressive agent, which produces peripheral blood lymphopenia through a lymphocyte homing effect. We investigated the relationship between the dose of FTY720 or blood concentration (pharmacokinetics, PK) and peripheral lymphopenia (pharmacodynamics, PD) in 23 kidney transplant recipients randomized to receive FTY720 (0.25-2.5 mg/day) or mofetil mycophenolate (2 mg/day) in combination with cyclosporine and steroids. FTY720 dose, blood concentrations and lymphocyte counts were determined weekly before and 4 to 12 weeks after transplantation. The effect of PD was calculated as the absolute lymphocyte count or its reductions. PK/PD modeling was used to find the best-fit model. Mean FTY720 concentrations were 0.36 ± 0.05 (0.25 mg), 0.73 ± 0.12 (0.5 mg), 3.26 ± 0.51 (1 mg), and 7.15 ± 1.41 ng/ml (2.5 mg) between 4 and 12 weeks after transplantation. FTY720 PK was linear with dose (r2 = 0.98) and showed low interand intraindividual variability. FTY720 produced a dose-dependent increase in mean percent reduction of peripheral lymphocyte counts (38 vs 42 vs 56 vs 77, P < 0.01, respectively). The simple Emax model [E = (Emax * C)/(C + EC50)] was the best-fit PK/PD modeling for FTY720 dose (Emax = 87.8 ± 5.3% and ED50 = 0.48 ± 0.08 mg, r2 = 0.94) or concentration (Emax = 78.3 ± 2.9% and EC50 = 0.59 ± 0.09 ng/ml, r2 = 0.89) vs effect (% reduction in peripheral lymphocytes). FTY720 PK/PD is dose dependent and follows an Emax model (EC50 = 0.5 mg or 0.6 ng/ml). Using lymphopenia as an FTY720 PD surrogate marker, high % reductions (~80%) in peripheral lymphocytes are required to achieve best efficacy to prevent acute allograft rejection. Correspondence H. Tedesco-Silva Jr. Divisão de Nefrologia Hospital do Rim e Hipertensão


Introduction FTY720 (2-amino-2-[2-(4-octylphenyl)
ethyl] propane-1,3 diol hydrochloride) is a novel immunomodulator agent developed by a chemical modification of myriocine (1-3), a metabolite isolated from Iscaria sinclairii broth (4).This immunomodulator is effective in protecting solid organ grafts from acute rejection in experimental transplant models (5).FTY720 is effective even in the absence of cyclosporine, and also shows synergistic interactions with cyclosporine, tacrolimus, sirolimus, or everolimus (6)(7)(8)(9)(10)(11)(12)(13)(14).FTY720 is currently undergoing phase III clinical trials in kidney transplant recipients after FTY720 doses and combination therapy were identified in phase II studies.FTY720 produces peripheral lymphopenia in both animals and humans as a consequence of altered T-cell trafficking and redirection to secondary lymphatic organs (15,16).At therapeutic concentrations, FTY720 does not interfere with cytokine synthesis or binding, does not inhibit cell proliferation and does not cause cell death by apoptosis (17,18).Lymphopenia is fully reversible upon drug discontinuation (19).FTY720 needs to be phosphorylated by sphingosine phosphatase and the phosphorylated form (FTY720-P) appears to mediate its biological effect through the binding to sphingosine-1-phosphate, Gprotein-coupled receptors expressed in the membrane of lymphocytes and endothelial cell of lymph nodes (20)(21)(22).The proposed molecular mechanism of action is that lymphocyte homing is altered by FTY720 as a sphingosine-1-phosphate agonist targeting G-protein-coupled receptors expressed in the membrane of lymphocytes and endothelial cell of lymph nodes (23).This binding results in drug-receptor internalization, increasing intrinsic lymphocyte mobility and chemotactic response, accelerating lymphocyte homing, and trapping in secondary lymphoid tissues (21,24).FTY720 has unique pharmacokinetic properties.Its absorption is slow, reaching peak concentrations 8 to 36 h after oral administration (19).FTY720 has a large volume of distribution (1116-1737 L) and a clearance of about 123 to 383 ml/min, resulting in a long elimination half-life of about 108 h (4.5 days) (19).Since it is given once a day, presumably it takes about 4 weeks to reach steady-state concentration, with 11-to 19fold accumulation in tissues compared to first dose administration (19).
Pharmacokinetic and pharmacodynamic (PK/PD) modeling has been used increasingly in clinical pharmacology and drug development, not only to speed up the development process but, more importantly, to determine the optimal dosage of new drugs, which will deliver an appropriate effect.The relationship between FTY720 pharmacokinetics and pharmacodynamics has been studied in various experimental transplant models (13).The aim of the present study was to determine the relationship among FTY720 dose, blood concentration and surrogate biological effect in the peripheral blood compartment (lymphopenia).This information will help to choose and possibly individualize drug dose regimens, which will result in best efficacy/toxicity relationships.

Population
Between June 1st and September 30, 2000, 23 kidney transplant recipients were enrolled at our center as part of an international multicenter prospective, open-label, randomized, dose-finding, and exploratory trial, which included 208 patients.The primary objective was to evaluate the safety, tolerability and preliminary efficacy of increasing doses of FTY720 versus mofetil mycophenolate (MMF, CellCept ® , Roche Laboratories, New Jersey, NJ, USA) in combination with a cyclosporine microemulsion and prednisone.
The local Medical Ethics Committee approved the protocol and the study was performed in accordance with the Declaration of Helsinki and US Food and Drug Administration guidelines for good clinical practice.All patients signed an informed consent term after being informed of the details of this study, and were enrolled according to study-specific inclusion and exclusion criteria.The present report describes a retrospective analysis of data obtained from 23 patients enrolled at our transplant center during the clinical trial.

Study design
The study involved 24 weeks of observation divided into 12 weeks of treatment and 12 weeks of follow-up.Patients were randomized 24 h after renal transplantation to one of four FTY720 groups (0.25 mg (N = 4), 0.5 mg (N = 4), 1 mg (N = 5), 2.5 mg (N = 5)) or to one MMF group (N = 5).To be enrolled in the study patients had to be between 18 and 65 years old and to be receiving their first living or cadaver donor allografts.All patients received initial doses of cyclosporine of 8 to 10 mg kg -1 day -1 twice a day adjusted to achieve therapeutic concentrations of 200-400 ng/ml during the first month, 150-250 ng/ml during the second and third months, and 100-200 ng/ml thereafter.Prednisone was introduced two days after the transplant, with initial doses of 0.5 mg kg -1 day -1 followed by a gradual reduction to 0.2 mg kg -1 day -1 at 12 weeks post-transplant.Within 24 h after transplant surgery, a loading dose of 1, 2, 4, and 4 mg was administered to patients randomized to receive fixed maintenance doses of 0.25, 0.5, 1, or 2.5 mg/day of FTY720.Patients randomized to the MMF group received 2 g twice a day.FTY720 treatment lasted 12 weeks with the patients being converted to azathioprine, 2 mg kg -1 day -1 , or MMF, 2 g/ day thereafter at the discretion of the investigator.Drug doses were also adjusted for safety and tolerance.

Pharmacokinetics/pharmacodynamics
There were 14 study visits, namely pretransplant and at 1, 2, 4 to 12 weeks during the treatment phase and 16, 20 and 24 weeks during the follow-up phase.On the occasion of each study visit, blood samples were obtained to measure blood concentrations of cyclosporine and FTY720 and to perform lymphocyte counts.
Pharmacokinetic analysis.Whole blood cyclosporine concentrations were measured daily during the first week and doses were adjusted until therapeutic concentrations (>200 ng/ml) were achieved.Subsequent monitoring was performed twice a week during the first month, once a week during the second month, and every other week thereafter.Whole blood cyclosporine concentrations were measured by a fluorescence polarization immunoassay (Abbott Laboratories, Chicago, IL, USA) according to the manufacturer instructions.Since FTY720 has a long terminal half-live of about 100 h, steady-state concentrations are achieved only after 4 weeks of treatment.Therefore, blood samples were collected to measure whole blood FTY720 concentrations between 4 and 12 weeks.Blood concentrations of FTY720 were determined using a validated HPLC/mass spectrometry/ mass spectrometry method from Novartis (East Hanover, NJ, USA).
Pharmacodynamics study.The surrogate marker of the pharmacodynamic effect of FTY720 used in this study was the peripheral lymphocyte count in the blood compartment.Lymphocyte counts were performed on the occasion of each study visit (pretransplant and 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, and 24 weeks after transplant) in all 5 groups using the CELL-DYN 3200 automatic counter (Abbott Park, Chicago, IL, USA) whose methods of determinations are based on flow cytometry.Since MMF has no effect on peripheral lymphocytes (25), lymphocyte counts obtained from patients receiving MMF were used to calculate the basal pharmacodynamic effect when FTY720 doses or exposures were equal to zero.The pharmacodynamic effect of FTY720 was determined either as absolute reduction in peripheral lymphocyte counts or as percent reduction compared to the lymphocyte count obtained pre-transplant and before the administration of the first dose of FTY720 or MMF.
PK/PD correlations.Peripheral lymphocyte counts were correlated with FTY720 doses and blood concentrations.PK/PD modeling was used to find the best-fit model of the correlation between absolute or percent reduction in peripheral lymphocyte count and increasing doses or blood concentrations of FTY720.For these analysis we used the scientific software WinNonlin (SCI Software) to find the best fit PK/PD model that explains the relationship between FTY720 dose or blood concentration and the chosen pharmacodynamic surrogate marker.

Statistical analysis
Demographic characteristics were analyzed by analysis of variance (ANOVA) for continuous variables and by the chi-square test for categorical variables.Summary statistics were expressed as means ± SD, or frequencies or median and range, respectively.The observed and calculated PK/PD parameters were presented as means ± SD and as dose/response and exposure/response relationships.Linear regression analyses were used to correlate FTY720 dose and blood concentration.Nonlinear regressions were used to correlate the dose and drug concentration in blood with biological response.All statistical analyses were performed using the SPSS 7.5 software (SPSS Inc., Chicago, IL, USA), with the level of significance set at P < 0.05.

Demographics and baseline characteristics
Demographic characteristics of the allograft recipients were similar across treatment groups (Table 1).Mean age was 40.1 ± 11.0 years, weight 62.2 ± 8.8 kg, and mean body mass index was 22.8 ± 2.6 kg/m 2 .Most patients were males (61%) and white (61%), with 4 (17%) blacks and 5 (22%) with various degrees of miscegenation.The primary etiology leading to renal failure was chronic glomerulonephritis (22%), followed by diabetes (17%) and nephrosclerosis (13%).Mean number of HLA mismatches was 2.9 ± 0.5 and 100% showed no preformed anti-HLA antibodies.All recipients tested negative for hepatitis B and C viruses and 50% of the recipients were positive for cytomegalovirus IgG.No significant differences in demographic characteristics were observed between the 5 groups.
Mean FTY720 blood concentrations according to dose level and study visit are shown in Figure 2.There were no significant differences in mean blood concentration between study visits (General Linear Model for repeated measurements) within each dose level, confirming that at week 4 steady-state FTY720 concentrations had been achieved (Table 2).Interindividual variability (% coefficients of variation, %CV) increased from 24 to 41% with increasing doses of FTY720 (P = 0.05) but no significant differences were observed between study visits.Aver-  Figure 1.Time course of mean cyclosporine doses (A) and blood concentrations (B).There were no significant differences for mean cyclosporine doses or blood concentrations among study groups at each visit (independent Student t-test).MMF = mofetil mycophenolate.
age intra-individual variability (%CV) ranged from 25 to 28 and was not dose dependent.
In the 10-fold dose range studied in this trial (0.25 to 2.5 mg/day), FTY720 pharmacokinetics was linear with dose (r 2 = 0.679 and 0.982), as can be observed in Figure 3.

Pharmacodynamics
The time course of the FTY720-induced reduction of peripheral lymphocyte count is shown in Figure 4.There were no significant differences in pre-transplant values between groups and lymphopenia was not observed in the MMF group throughout the study.The    Table 3. Mean peripheral blood lymphocyte counts and percent lymphocyte reduction between weeks 4 and 12 in the 5 groups of patients.

PK/PD analysis
The observed linear correlation between FTY720 dose and steady-state blood concentrations did not translate into linear correlations between dose or blood concentrations and lymphopenia.In the 10-fold dose range studied in this trial (0.25 to 2.5 mg/day) FTY720 pharmacodynamics was not doselinear (r 2 = 0.57 and 0.53), as can be observed in Figure 5A and B. Searching for a best-fit model we identified the inhibitory E max model as the one with best prediction of the relationship between both FTY720 dose or blood concentrations and absolute peripheral lymphocyte count.This model applies the following formula: E = E max -(E max -E 0 ) * [C/(C + EC 50 )], where E is the pharmacodynamic effect at a given drug dose or concentration (C), E max is the maximum pharmacodynamic effect attributed to the drug, E 0 is the effect when drug dose or concentration is equal to zero, and EC 50 is the drug dose or concentration which produces 50% of the maximum pharmacodynamic effect.The last parameter determines the drug potency.Using this model, the correlation between individual FTY720 doses or steadystate blood concentrations and absolute peripheral lymphocyte counts produced coefficients of determination of r 2 = 0.96 and r 2 = 0.87, respectively.For FTY720 dose and concentration correlations, E 0 and E max were very similar, with EC 50 of 0.08 mg or 0.13 ng/ml, respectively (Figure 6A,B).Conversely, the simple E max model was the one that best predicted the relationship between either FTY720 dose or blood concentration and percent reduction in peripheral lymphocyte count.This model applies the following formula: E = (E max * C)/(C + EC 50 ), where E is the pharmacodynamic effect at a given drug dose or concentration (C), E max is the maximum pharmacodynamic effect attributed to the drug, and EC 50 is the drug dose or concentration which produces 50% of the maximum pharmacodynamic effect.Again, using this model, the correlation between individual FTY720 doses or steady-state blood concentrations and percent reduction in peripheral lymphocyte counts produced coefficients of determination of r 2 = 0.94 and r 2 = 0.89, respectively.For FTY720 dose and concentration correlations, E max was 87.8% and 78.3%, with EC 50 of 0.48 mg or 0.59 ng/ ml, respectively (Figure 7A,B).

Discussion
FTY720 is a novel immunomodulating drug with a unique mechanism of action, which is different from the currently used immunosuppressive agents.Results from phase II clinical trials have demonstrated that the combination of FTY720 with cyclosporine produces a very low incidence of acute rejection (26).Furthermore, except for a first dose-associated mild negative chronotropic effect, FTY720 is not nephrotoxic, does not potentiate cyclosporine nephrotoxicity and lacks other specific drugrelated negative side effects (27).
In the present study, we set out to determine FTY720 PK/PD relationships in a cohort of patients undergoing a dose-finding phase II clinical trial (28).The demographic characteristics revealed a low risk transplant population since this was an exploratory study, but no significant differences were observed among the study groups (Table 1).All patients received comparable initial cyclosporine doses and underwent similar dose reductions resulting in virtually equal cyclosporine concentrations throughout the first 24 post-transplant weeks.Moreover FTY720 did not produce any significant drug interaction with cyclosporine since blood cyclosporine concentrations were similar for all FTY720 dose groups and for the MMF group (29).
The pharmacokinetic properties of FTY720 have been described previously (30).The long terminal half-life required a loading dose to achieve steady-state concentrations more rapidly.Except for the higher dose level, whereby mean blood concentrations were still increasing after week 4 (Table 2, Figure 2), it appears that steady-state concentrations were achieved after 4 weeks of daily administration.In this cohort of patients, we observed a relatively low inter-and intra-individual variability in FTY720 trough blood concentrations, regardless of FTY720 dose or time post-transplant.Moreover, FTY720 showed dose-linear pharmacokinetics (13).Taken together, these characteristics may result in less need for dose adjustments, and fast and more predictable achievement of target concentrations when dose adjustments are needed.
As shown in Table 3, MMF did not produce lymphopenia during the study period.FTY720-induced lymphopenia began early and could be detected as early as 1 week after transplantation (Figure 4).Actually, in more detailed studies, lymphopenia was already noticed after the loading dose and paralleled the decrease in heartbeat (28).This has been attributed to the physiological mechanism of action of FTY720, which, after oral administration, is phosphorylated (FTY720-P) and binds to sphingosine-1-phosphate receptors present in the plasma membrane of lymphocytes and cardiac myocytes, respectively (31,32).Nadir lymphopenia is reached between 2 and 4 weeks and after drug discontinuation lymphocyte counts return to baseline values within 4 to 8 weeks depending on the magnitude of the FTY720 dose.This indicates that FTY720-induced lymphopenia is reversible and therefore safer, since it does not produce prolonged apoptosis-related lymphopenia and does not produce long-term effects like those observed using depleting monoclonal or polyclonal antibodies (33).Inter-and intra-individual variability of the peripheral absolute lymphocyte count is apparently higher than that seen for the FTY720 pharmacokinetics.Interand intra-individual variability of percent reduction of lymphocyte counts decreases with increasing FTY720 doses, perhaps due to achievement of maximal pharmacodynamic effect.
Visual observation of the relationship between FTY720 doses or blood concentration absolute lymphocyte count or percent reduction in lymphocyte count revealed a nonlinear pattern (Figure 3), supported by the low coefficient of determination (r 2 ) of the linear regression analysis (Figure 4).Searching for a best-fit model that would explain this relationship we found two PK/PD models.For the absolute lymphocyte count, the inhibitory E max model was selected since there was a decrease (inhibition) in the number of lymphocytes in peripheral blood with increasing drug dose or concentration, showing high coefficients of determinations (Figure 6).The E 0 term was included to describe the effect when drug dose or concentrations were equal to zero (in this study, lymphocyte counts from patients receiving MMF).For the percent reduction in lymphocyte counts, the simple E max model was selected since there was an increase in percent reduction of the number of lymphocytes in peripheral blood with increasing drug dose or concentration, also showing high coefficients of determination (Figure 7).There was an evident difference in EC 50 between the two PK/ PD models for both FTY720 dose (0.08 vs 0.48 mg) and blood concentration (0.13 vs 0.59 ng/ml).A simple explanation for this finding is the large but frequently observed variability of the peripheral lymphocyte counts when drug dose or concentrations were equal to zero (E 0 term), suggesting that the use of percent reduction, which naturally corrects for baseline differences, is more appropriate as a surrogate marker of the pharmacodynamic effect of FTY720 in the blood compartment.
Finally, relationships between pharmacokinetics and pharmacodynamics are useful to find ideal doses to produce the desired effect.However, it is still important to determine the relationship between the pharmacodynamic effect and efficacy and safety, which is the ultimate goal of pharmacology.In a phase II clinical trial, the 2.5-mg dose of FTY720 showed good efficacy for the prevention of acute rejection after kidney transplantation.This dose produced a reduction of about 80% in lymphocyte count, close to the maximum effect (E max ).Since FTY720 accumulates 11-to 19-fold in various tissues, perhaps in the secondary lymphoid organs, which are rich in lymphocytes, and therefore in sphingosine-1-phosphate receptors, the EC 50 required for drug-receptor complex internalization might be higher.Moreover, data from the same phase II clinical trial demonstrated that FTY720 at doses of 5 mg/day, which probably produce maximum percent reduction of lymphocyte count, is still effective and safe.Recently, it has been demonstrated that the active form of FTY720 is the phosphorylated form (FTY720-P) and studies are needed to assess the correlation between FTY720-P and the degree of lymphopenia.Of course, the lacks of similar dose-response curves for toxicity impair the establishment of a therapeutic window.
In conclusion, the PK/PD relationship of FTY720 follows an E max model.Using peripheral lymphopenia as an FTY720 pharmacodynamic surrogate marker, high pharmacodynamic effects (percent reduction in peripheral lymphocytes) are required to achieve best efficacy, similar to what is seen in various physiological enzymatic systems.Further prospective studies are required to test the clinical applicability of this strategy.
Data are reported as means ± SD (range).MMF = mofetil mycophenolate; BMI = body mass index; HLA = human leukocyte antigen; ESRD = end-stage renal disease; HCV = hepatitis C virus; CMV = cytomegalovirus.Panel reactive antibody between donor and recipients greater or less than 5%.

Figure 2 .
Figure 2. Time course of mean FTY720 blood concentrations between 4 and 12 weeks post-transplant.P < 0.01 for 1.0 and 2.5 mg FTY720 compared to 0.25 and 0.5 mg FTY720 during administration time (ANOVA).

Figure 3 .
Figure 3. Linear regression analysis correlating individual (A) or mean (B) FTY720 blood concentrations and dose levels.

Figure 4 .
Figure 4. Time course of FTY720-induced lymphopenia during the 12-week treatment phase and return to baseline values upon drug discontinuation.MMF = mofetil mycophenolate.

Figure 6 .
Figure 6.Pharmacokinetic and pharmacodynamic inhibitory E max model correlating FTY720 dose (A) or blood concentration (B) with absolute lymphocyte counts in peripheral blood.

Figure 5 .
Figure 5. Linear regression analysis correlating mean FTY720-induced reduction of peripheral lymphocyte counts and drug dose (A) or concentrations (B).

Figure 7 .
Figure 7. Pharmacokinetic and pharmacodynamic simple E max model correlating FTY720 dose (A) or blood concentration (B) with percent reduction in lymphocyte counts in peripheral blood.

Table 1 .
Demographic characteristics of the transplant population.

Table 2 .
FTY720 blood concentrations and percent coefficient of variation (%CV) between weeks 4 and 12 according to FTY720 dose level.