Abstract
The pharmacokinetics and cell trafficking dynamics of 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720), a novel immunosuppressive agent, were examined in cynomolgus monkeys (three males and three females). After single doses of 0.1 mg/kg p.o. or i.v. bolus and 1 mg/kg p.o. were administered to the animals, the concentrations of FTY720, and the numbers of lymphocytes, CD20+CD2-B cells, and CD2+CD20-T cells in blood were measured over 23 days. A linear three-compartment model characterized the time course of FTY720 concentrations with a terminal half-life of about 31 h, clearance of about 0.53 l/h/kg, and bioavailability of about 38%. The dynamic responses were not area under the curve (or dose) proportional for either males or females. An indirect response model with a distribution pool captured the cell trafficking data for all doses for each cell type, where initial blood counts (R0) were about 7650, 2100, and 5250 cells/μl; maximum fractional inhibition (Imax) about 0.88, 0.85, and 0.91; influx (kin) about 6014, 1312, and 5662 cells/μl/h; efflux (kout) about 0.798, 0.555, and 1.08 h−1; intercompartmentalkcp about 0.134, 0.192, and 0.082 h−1; and intercompartmental kpcrate constants about 3.9 × 10−4, and 0.016 and 8.9 × 10−6 h−1 for lymphocytes, B cells, and T cells, respectively. The inhibition concentration IC50 was about 0.48 μg/l for all cells, which was remarkably low. The apparent distribution volumes of peripheral pool (Vp) were markedly larger than blood volume (Vb) for all cells. TheImax for cell trafficking was achieved at doses smaller than that producing graft protection, indicating stronger central than peripheral effects of this drug. The profound cell trafficking effects of FTY720 can be readily captured and interpreted with an extended indirect response model.
2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720), a novel immunosuppressant found in Isaria sinclairii (a fungus) metabolite (Fujita et al., 1994), protects solid organ grafts with strong potency (Kahan, 1998); acts synergistically with cyclosporin, sirolimus, or tacrolimus (Kawaguchi et al., 1996; Stepkowski et al., 1998; Hoshino et al., 1999); and prevents experimental autoimmune myocarditis, autoimmune diabetes, and arthritis (Yan et al., 1998; Kitabayashi et al., 1999;Matsuura et al., 2000). Diverse pharmacological mechanisms of action were found for FTY720: it can produce cell cycle arrest of lymphocytes (Nagahara et al., 2001), and it can alter production (Yagi et al., 2000), trafficking (Chiba et al., 1998; Brinkmann et al., 2000, 2001;Pinschewer et al., 2000), infiltration (Yanagawa et al., 2000), and apoptosis (Enosawa et al., 1996; Bohler at al., 2000; Nagahara et al., 2000) of lymphocytes. Perplexities about FTY720 regarding preclinical and clinical data include that the maximum effects of FTY720 on cell trafficking were achieved at doses smaller than those producing protection against graft rejection (Yanagawa et al., 1998b); and that lymphocytes in blood attained a trough before blood FTY720 concentrations reached Cmax. Although the exact mechanism of FTY720 action is not clear (Napoli, 2000), the available fundamental literature data provide a basis for a pharmacokinetic and pharmacodynamic (PK/PD) model to quantitatively relate drug concentrations to cell trafficking responses that may assist in interpreting the above-mentioned perplexities. Kinetics and cell trafficking dynamics of FTY720 in cynomolgus monkeys (three males, three females) were studied after single intravenous and oral doses.
Materials and Methods
Animals.
Cynomolgus monkeys (three males and three females; 3–8 years old; body weights of 2–5 kg; China National Scientific Corporation, Beiging, China) were used in this study. Monkeys were quarantined for at least 3 months before treatment and were screened for tuberculosis, parasites, and any clinical pathological abnormalities. The monkeys were housed individually in stainless steel cages in a controlled environment with a 12-h light/dark cycle. Filtered tap water was available ad libitum and food was provided twice daily. On the dosing day, animals had catheters placed in the cephalic vein for dosing and in the femoral vein for blood sample collection. Animals were restrained in Plas-Lab medium restrainer chairs (Plas Labs, Inc., Lansing, MI) for up to 4 h on the dosing day.
Dosing and Sampling.
A 3 × 3 crossover experiment was performed using the six monkeys with a dosing interval of 28 days. Single doses of 0.1 mg/kg p.o. or i.v. bolus and 1 mg/kg p.o. were administered to the animals; blood samples (0.2 ml) collected at 0 (predose), 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h were obtained for FTY720 concentration measurements; and blood samples (0.5 ml) collected at 0 (predose), 1, 4, 6, 8, 24, 48, 72, 96, 120, 312, and 552 h were obtained for lymphocyte, CD20+CD2-B cell, and CD2+CD20-T cell measurements. The samples were analyzed within 24 h after collection.
Drug Assay.
The FTY720 concentrations in blood were analyzed by liquid-liquid extraction and liquid chromatography/mass spectrometry/mass spectrometry measurement. To 0.1 ml of blood was added 0.1 ml of internal standard solution (100 ng/ml Y-32919 in methanol) and vortexed for 2 to 3 s, pH was adjusted using 0.5 ml of 0.1 N NaOH, and a 5-ml mixture of tertbutyl-methyl ether/dichloromethane (75:25, v/v) was added. Tubes were shaken for 30 min, centrifuged at 2010g for 10 min, and the organic layer was transferred and evaporated under pure N2 gas stream. The dry extract was reconstituted with 200 μl of 0.02 M ammonium acetate/methanol (50:50) with 2- to 3-s vortexing and 3-min sonication, and centrifuged at 11,400g for 5 min. The supernatant was diluted 1:20 with the reconstitution solvent, and 100 μl was injected onto a 3.5-μm Symmetry Shield RP8 high-performance liquid chromatography column (50 × 4.6 mm) at 40°C, eluted at 1 ml/min using a gradient mobile phase consisting of methanol and 0.02 M CH3COONH4. The concentrations were determined by mass-spectrometry using atmospheric pressure ionization as an interface. The calibration curve ranged from 1.16 to 1010 ng/ml FTY720, and the recovery was 98% for drug and 91% for internal standard at 5 ng/sample. The limit of quantification was 0.55 ng/ml for 0.5 ml of blood.
Cell Counting.
A flow cytometer (EPICS XL-MCL; Beckman Coulter, Inc., Fullerton, CA) was used for counting cells in blood. Lymphocyte count was determined by lymphocyte gating. The T and B cells were counted by two-color flow cytometry, where cells in samples were stained by fluorescein isothiocyanate-labeled mouse anti-human CD2 monoclonal antibody (Clone T11; Beckman Coulter, Inc.), and by phycoerythrin-labeled mouse anti-human CD20 monoclonal antibody (Clone B1; Beckman Coulter, Inc.).
Pharmacokinetics.
The PK model for FTY720 was a linear three-compartment model depicted in the upper part of Fig.1. Triexponential fittings of mean blood FTY720 concentrations, Civ(t), versus time t for males and females were performed by weighted (1/Ct2) nonlinear regression using WinNonlin Professional, version 2.1 (Pharsight, Apex, NC):
Three-compartmental pharmacokinetic model for the time course of FTY720 concentrations and the indirect response model with a distribution compartment for the dynamics of lymphocytes, B cells, and T cells. Parameters are defined in the text or in the abbreviation list.
Pharmacodynamics.
In normal conditions, the number of any kind of lymphocyte in blood is relatively constant as controlled by the balance of influx and efflux of the recirculating cells. FTY720 inhibits thymocyte emigration (Yagi at al., 2000) and induces lymphopenia in the thoracic duct (Chiba et al., 1998) and in the whole body system (Luo et al., 1999), showing that the input of lymphocytes to blood was altered. Considering that some lymphocytes are recirculating in blood to lymph and some are resident in peripheral tissues (Smith and Ford, 1983; Butcher, 1986; Picker and Butcher, 1992;Butcher and Picker, 1996; Pinschewer et al., 2000), the transfer of cells between blood and distribution pools is requisite. Therefore, the PD model for FTY720 was established as depicted in the lower part of Fig. 1 as indirect response model 1 with a distribution compartment (Krzyzanski and Jusko, 2001). It can be described by the following equations:
Before FTY720 was given, the lymphocyte amounts in blood and peripheral pools (baselines) are at homeostasis, which means eqs. 7, 8, and 9= 0, based on which the initial condition forRp(t) was set as eq. 8b, as well as the following:
If multiple-doses of FTY720 were given daily for a long period, the blood cell concentrations should come to a steady-stateRss, which means eqs. 7 and 8 = 0 again but eq. 9 > 0, and therefore
After the PD fitting, the area between the baseline and the effect curve (ABEC), the ratio of ABEC/AUC, and the mean reduction percentage of peripheral counts were calculated using the trapezoidal method for each dose, gender, and cell type.
Results
Pharmacokinetics.
The PK profiles of mean FTY720 concentrations for male and female monkeys for the 1-mg/kg oral dose and the 0.1-mg/kg oral and i.v. bolus doses with the fitted curves are shown in Fig. 2. The relevant PK parameters for a three-compartment model are depicted in Table1. For the i.v. bolus dose, the disposition kinetics were triexponential, with a brief first distribution phase (λ1), a longer second distribution phase (λ2), a longest terminal phase (λ3), and a corresponding long mean residence time (MRT). For males, the half-lives for the three phases were t1/2, λ1 = 0.07 h; t1/2, λ2 = 3.46 h; and t1/2, λ3 = 30.82 h, consisting 10.2, 28.9, and 60.9% of the total AUC, with an MRT of 28.54 h. For females, t1/2, λ1 = 0.10 h; t1/2, λ2 = 4.24 h; and t1/2, λ3 = 31.55 h, consisting 9.2, 25.6, and 65.2% of the total AUC, and MRT was 31.26 h.
Pharmacokinetic parameters (CV%) for FTY720 in cynomolgus monkeys
The blood clearance of FTY720 of about 0.52 l/h/kg can be compared with hepatic blood flow in monkey of 2.6 l/h/kg (Davies and Morris, 1993), indicating that the drug is of low-to-moderate clearance. Thus, the incomplete bioavailability (32–45%) is not likely due to hepatic first-pass. The Vss of 15 l/kg is very large, indicative of appreciable tissue binding. Furthermore, the long terminal t1/2 is probably determined by this extensive distribution and binding in tissues.
For males or females, the parameters obtained from the i.v. data characterized the oral data well as shown in Fig. 2. For the oral doses, the absorption was slow and obviously delayed, and bioavailabilities were moderate. The absorption half-life (t1/2, ka) and the time for maximum blood concentration (Tmax) were 13.22 and 12.01 h for males, and 11.38 and 12.12 h for females. The fittedF and Tmax values were almost equal to those calculated by PK analysis. The ratio AUC/dose for 0.1 and 1 mg/kg were 0.655 and 0.670 kg × h/l for males, and 0.866 and 0.940 kg × h/l for females. Analysis of variance for CL, Vss, andVc showed no significant difference between the two oral dose levels (p > 0.05); and the CL/F, Vss/F,Vc/F, and MRT of mean data were almost equal for the two dose levels for each gender. Thus, FTY720 exhibited linear PK within the oral dose range of 0.1 to 1 mg/kg in monkeys.
The CV% for the PK parameters of the mean data were 0.5 to 18.5% for males, and 2.0 to 11.5% for females, with most of them below 10%. The analysis of variance for each PK parameter showed no significant difference between males and females (p > 0.05).
Pharmacodynamics.
As shown in Figs.3, 4, and5, the mean lymphocyte, B cell, and T cell numbers in blood for all monkeys decreased quickly after the doses were given, attained a trough at about 4 to 24 h, and then returned slowly to the baselines at 312 to 552 h. As seen from the fitted lines in these figures, the indirect response model with a distribution compartment captured all the PD data well, with characteristics of response slopes and troughs for different i.v./p.o. doses being caught simultaneously. The fitted lines went down quickly to a trough at 6 to 14 h, and rose back through the data points to baselines when blood drug concentrations approached zero (Figs. 3-5).
Lymphocyte counts in blood and the lymphocyte count fluctuation percentages in the peripheral compartment (PC%) after dosing of 0.1 mg/kg i.v. bolus (triangles), 0.1 mg/kg p.o. (squares), and 1 mg/kg p.o. (circles) for male and female monkeys. The lines in the panels are fittings based on eqs. 7 to 9.
CD20+CD2-B cell counts in blood and the lymphocyte count fluctuation percentages in the peripheral compartment (PC%) after dosing of 0.1 mg/kg i.v. bolus (triangles), 0.1 mg/kg p.o. (squares), and 1 mg/kg p.o. (circles) for male and female monkeys. The lines in the panels are fittings based on eqs. 7 to 9.
CD2+CD20-T cell counts in blood and the lymphocyte count fluctuation percentages in the peripheral compartment (PC%) after dosing of 0.1 mg/kg i.v. bolus (triangles), 0.1 mg/kg p.o. (squares), and 1 mg/kg p.o. (circles) for male and female monkeys. The lines in the panels are fittings based on eqs. 7 to 9.
For each type of cell, the count reductions in blood and peripheral pools (Figs. 3-5) were driven by blood FTY720 concentrations in the nonlinear inhibition function of eq. 9. The fittings for B cells were better than for other cells, and those for females were better than for males. These were shown by AIC values of the fittings: for B cells, lymphocytes, and T cells, the AIC values were 186.0, 280.4, and 252.1 for males, and 109.5, 233.9, and 240.4 for females. The net PD effects of the three doses were 1 mg/kg p.o. > 0.1 mg/kg i.v. > 0.1 mg/kg p.o. in both blood and peripheral compartments. Blood ABEC values were not proportional to AUC values for each cell or gender, nor were the mean reduction percentages of peripheral cell counts (Figs. 3-5; Table2). Peripheral B cells were more susceptible to FTY720 than T cells and lymphocytes for each dose or gender shown by the higher kpcvalue (Table 3) and the higher mean reduction percentages (Table 2).
Pharmacodynamic secondary parameters for FTY720 in cynomolgus monkeys
Pharmacodynamic modeling parameters (CV%) for FTY720 in cynomolgus monkeys
For each gender, the R0 andkin values for lymphocytes were close to the sum of those for B and T cells, and other parameters for lymphocytes were almost the average of those for B and T cells. This reflected the fact that CD20+CD2-B cells and CD2+CD20-T cells were the majority subtypes of lymphocytes in these monkeys. The CV% for PD parameters was greater than PK parameters, especially when the parameter was very small (Table 3). It was particularly difficult to estimate kpc as indicated by the large CV% values.
Discussion
Pharmacokinetics.
As shown in Fig. 2, the three-compartment model reasonably characterized the PK data in monkeys for the three doses. The PK model was chosen based on AIC values: they were −40.9 and −49.8 for three-compartment, and −5.8 and −8.8 for two-compartment models for males and females, respectively. Because simultaneous fitting of p.o. and i.v. data could not capture the i.v. data over 0 to 2 h representing the distribution phase of the drug, data for the two oral doses were fitted simultaneous to get onlyka, F, andTlag after other parameters were obtained from i.v. data. Those parameters originating from i.v. data well characterized the terminal phases of the oral data for the two doses. There were three reasons why simultaneous fitting of p.o. and i.v. data did not capture the early i.v. data: First, theTlag and theka made the p.o. data during this period much lower than i.v. data, thereby requiring much higher weight (1/C2) than i.v. data. Second, theTlag and the lowka made the distribution phase of the two oral doses less obvious and occur later than that for the i.v. dose. Third, there were two oral doses, but only one i.v. dose, which means that oral PK data have more influence than the i.v. PK.
Overall, none of the PK parameters showed significant gender differences. The PK for males and females was handled separately because the PD data, especially B-cell data, showed significant differences between gender, and it was consistent to use gender-specific PK parameters.
Pharmacodynamics.
Lymphocyte homing and recirculation through conduits of blood and lymph comprise the physiological processes by which lymphocytes seek out and localize to particular tissues and to specific microenvironments (Smith and Ford, 1983; Butcher, 1986; Picker and Butcher, 1992; Butcher and Picker, 1996). This underlies the proposed two-compartment PD model (Krzyzanski and Jusko, 2001) with FTY720 assumed to inhibit lymphocyte influx to blood (Chiba et al., 1998; Luo et al., 1999; Yagi at al., 2000).
The Imax values in the model were calculated using eq. 14 instead of fitting. This was necessary owing to the large number of parameters and initial conditions that created difficulties in fitting. The Imaxvalues of 0.82 to 0.92 for all cells (Table 3) were consistent with the literature values of 80 to 90% in cynomolgus monkeys (Quesniaux et al., 2000). Both the R0 andImax values for females were lower than for males, showing that it was worthwhile to pool data for each gender.
Another similarity to literature findings (Enosawa et al., 1996;Nagahara et al., 2000) was that theImax for T cells was greater, showing more susceptibility than B cells. On the contrary, peripheral T cells were much less susceptible than B cells (Figs. 3-5; Table 2), especially for females, owing to the larger apparent distribution volume (Vp) of T cells than B cells (Table 3).
This model justifies why the lymphocyte responses in blood attained a trough before blood FTY720 reachedCmax. According to the PD fittings, the cell counts in blood attained a trough at 12.3 h, where 98.6% reduction occurs during the first 6 h for an oral dose of 1 mg/kg and 94.5% for 0.1 mg/kg, whereas according to the PK fittings,Tmax is 10.9 h for males and 12.1 h for females. This indicates that the maximum effect of FTY720 is actually attained at 6 h, much earlier thanTmax. Also, the higher the dose, the earlier the maximum effect. This phenomenon is due to the low IC50 values in Table 3 (0.45 μg/l). This phenomenon was also seen in human FTY720 data (V. Brinkman, L. Chodoff, M. Figlimeni, P. Heining, J. Jaffe, T. Sabinski, R. Schmouder, unpublished observations), indicating FTY720 has a remarkably strong effect on trafficking of blood lymphocytes.
This model can explain why the maximum effects of FTY720 on cell trafficking are achieved at doses smaller than that producing protection against graft rejection in FTY720 therapy (Yanagawa et al., 1998b). For the oral dose of 1 mg/kg, when T cell counts in blood are already reduced by 90%, counts in the peripheral compartment were reduced no more than 0.01% (Figs. 3-5), thus almost nothing would happen to prevent these cells from infiltrating to the graft (if accessible). Only after a long period of larger doses of FTY720 administration could the peripheral T-cell counts be reduced to a level that is low enough to prevent infiltration to the graft. Of further complexity, the model assumes that numbers of cells in the general pool are not perceptibly altered by FTY720. The significance of timing of FTY720 administration on graft survival is also justified by this model: FTY720 should be given before or soon after graft implantation to prevent T cells from infiltrating to the graft. This necessity for FTY720 was reported recently (Yanagawa et al., 2000).
Although there was an absorption delay of about 0.4 h for oral doses, both PD nadir time and nadir level for 0.1 mg/kg p.o. were almost equal to those for 1 mg/kg p.o. and 0.1 mg/kg i.v. The nonlinear ABEC/AUC ratio values in Table 2 may account for the approximate nadir level values: the lower the AUC (exposure) value, the stronger the PD effect for per unit exposure. The consistency in nadir time indicated that the PD effect might occur before FTY720 was absorbed into systematic blood for oral doses. It was indeed found that Peyer's patches gathered lymphocytes before lymph nodes for oral FTY720 doses in rats (Yanagawa et al., 1998a).
Limitations of the experimental design are that a placebo group was not studied for baseline PD data, and no samples were collected between 8 to 24 h. This does not allow consideration of possible circadian changes in cell trafficking.
Naive lymphocytes probably traffic continuously between the different secondary lymphoid organs until they die or respond to their cognate antigen (Sprent et al., 1991; Picker and Butcher, 1992). Memory and effector lymphocytes, generated in secondary lymphoid tissues in response to antigen and then exported back to circulation, display migratory properties that are different from those of naive cells (Mackay 1992; Picker and Butcher, 1992; Butcher and Picker, 1996). This study measured only the naive lymphocyte counts, which may not correlate to survival of a graft.
In summary, FTY720 has profound effects on trafficking of lymphocytes, B cells, and T cells, and a mechanism-based pharmacodynamic model captured these patterns and facilitated the quantitative comparison of effects for the three administration modes.
Acknowledgments
We thank Jin Yan, Feng Jin, and Dr. Donald E. Mager (SUNY at Buffalo) for discussion and help.
Footnotes
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This research was supported by Novartis Pharma AG, Basel, Switzerland, and by Grant GM24211 from the National Institute of General Medical Sciences, National Institutes of Health.
- Abbreviations:
- PK
- pharmacokinetics
- PD
- pharmacodynamics
- k10
- systemic elimination rate constant
- F
- bioavailability
- ka
- first-order absorption rate constant of drug
- Tlag
- lag time of drug being absorbed
- AGI
- drug amount in gastrointestinal tract
- Ai (i = 1–3)
- drug amount in compartment i
- Vi (i = 1–3)
- distribution volume of drug in compartment i
- C1
- drug concentration in compartment 1
- kij (i
- j = 1, 2, 3), transfer rate constant of drug between compartments i and j
- λi (i = 1–3)
- exponential disposition slopes
- kin
- zero order input constant of cells into central compartment
- kout
- first-order output constant of cells
- kcp and kpc
- transfer rate constants of cells between compartments
- R0
- initial blood counts
- R
- lymphocyte concentration in blood
- Rp
- total cell content in peripheral pool
- Vb
- blood volume
- Vp
- apparent distribution volume of cells in peripheral pool
- Imax
- maximal fractional inhibition
- ABEC
- area between baseline and effect curve
- AUC
- area under the curve
- MRT
- mean residence time
- CL
- clearance
- AIC
- Akaike's information criterion
- Y-32919
- 2-amino-2-[2-(4-octyloxyphenyl)ethyl]propane-1,3-diolhydrochloride
- Received September 25, 2001.
- Accepted December 27, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
