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Pharmacodynamic Modeling of IVIG Effects in a Murine Model of Immune Thrombocytopenia
Pharmacokinetic/Pharmacodynamic Modeling of IVIG Effects in a Murine Model of Immune Thrombocytopenia RONG DENG, JOSEPH P. BALTHASAR Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14260
Received 25 April 2006; revised 25 September 2006; accepted 28 September 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20828
ABSTRACT: IVIG may achieve its beneficial effects in immune thrombocytopenia (ITP) patients via several mechanisms; however, little is known of the relative importance of various mechanisms associated with IVIG action in ITP. The purposes of this study were to develop a pharmacokinetic/pharmacodynamic (PK/PD) model relating an anti-platelet antibody, MWReg30, to platelet counts in a mouse model of sustained ITP, to use modeling to characterize effects of IVIG on MWReg30 elimination, and to use PK/PD modeling to assess the contribution of IVIG effects on MWReg30 disposition to the effects of IVIG on MWReg30-induced thrombocytopenia in mice. A pharmacokinetic model, based on the competitive occupancy of protective FcRn receptors, was used to characterize the effects of IVIG on MWReg30 pharmacokinetics. The relationships between MWReg30 plasma concentrations to MWReg30-induced thrombocytopenia, in the presence and absence of IVIG treatment, were characterized using an indirect response model. The pharmacokinetic model well-captured MWReg30 plasma concentration-time profiles, with and without administration of IVIG. The indirect response model accurately characterized the effects of IVIG on MWReg30-induced thrombocytopenia in mice. Using these models, it was estimated that 43 5% of overall effects of IVIG on MWReg30-induced thrombocytopenia in mice could be accounted for by the IVIG-mediated increases in MWReg30 clearance. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1625–1637, 2007
Keywords: immune thrombocytopenia (ITP); intravenous immunoglobulin (IVIG); pharmacokinetics; pharmacodynamics
INTRODUCTION Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet (PLT) turnover, which is mediated by antiplatelet autoantibodies.1 In 1981, Imbach et al.2 first reported that high dose of intravenous immunoglobulin (IVIG) increased platelet counts in ITP patients. Since then, the therapeutic efficacy of IVIG in ITP and other autoimmune diseases has been well documented.1 However, the exact mechanisms of IVIG action in ITP remains controversial, and few studies have attempted to Correspondence to: Joseph P. Balthasar (Telephone: 716645-2842 ext 270; Fax: 716-645-3693; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 1625–1637 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
provide a quantitative assessment of the relative importance of proposed mechanisms of IVIG action in ITP. IVIG may increase platelet counts in ITP by (a) decreasing FcgR-mediated platelet destruction via competitive-inhibition of Fcg receptors (FcgR), (b) inhibition of complement-mediated platelet elimination, (c) increasing anti-platelet antibody clearance through saturation FcRn, (d) upregulation of the expression of FcgRIIB, leading to an indirect inhibition of FcgR-mediated PLT phagocytosis, (e) increasing platelet production, (f) decreasing antiplatelet antibody production, (g) neutralization of antiplatelet antibodies by idiotype-anti-idiotype interactions, etc.3,4 In vitro and in vivo data are available to support all of these proposed mechanisms of IVIG action; however, the contribution of each mechanism to
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the overall effects of IVIG actions in ITP is uncertain. Previous work in this laboratory led to the development of a rat model of ITP,5 and this model was found to respond to IVIG treatment.6 Pharmacokinetic studies in mice7 and rats6 demonstrated that IVIG increased the elimination of a monoclonal antiplatelet antibody (7E3). However, IVIG administration did not lead to an increase in 7E3 clearance in FcRn-knockout animals,7 supporting the hypothesis that the effects of IVIG on antiplatelet antibody clearance was mediated by saturation of FcRn, which protects IgG from elimination. Subsequent PK/PD analyses demonstrated that the effect of IVIG on 7E3 elimination was an important mechanism of IVIG action in rat model of ITP, accounting for 50 11% of the overall effects of IVIG on 7E3-induced thrombocytopenia.8 The rat model of ITP is an acute model, and IVIG effects were investigated via ‘‘pretreatment’’ with IVIG, followed by the administration of a challenge dose of 7E3. The rat model allowed sensitive assessment of IVIG effects on 7E3 elimination and on 7E3-induced thrombocytopenia; however, there has been some concern that IVIG effects in this acute model may be substantially different that IVIG effects when antibody is present at ‘‘steady-state’’ (as in the human disease).9 Recently, Deng and Balthasar10 developed a mouse model of sustained ITP, where thrombocytopenia was induced in mice by continuous infusion of an anti-platelet antibody, MWReg30. Severe thrombocytopenia was observed within 4 days from the start of the MWReg30 infusion, and PLT counts were found to remain at relatively constant values from 4 to 7 days after initiation of the infusion (i.e., approximating a condition of ‘‘steady-state’’ thrombocytopenia). Administration of IVIG to thrombocytopenic animals was found to lead to a dose-dependent increase in PLT counts, which was accompanied by a dose-dependent decrease in MWReg30 plasma concentrations. The present work has led to the development of a pharmacokinetic/pharmacodynamic (PK/PD) model relating MWReg30 plasma concentrations to platelet counts in mice, the development of a mechanism-based pharmacokinetic model to characterize the effects of IVIG on MWReg30 elimination, and to the development of an indirect response model to characterize the effect of IVIG on MWReg30-induced thrombocytopenia in mice. These models were then employed, in combination, to assess the contribution of IVIG effects on MWReg30 disposition to the effect of IVIG on JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
MWReg30-induced thrombocytopenia in mice. Similar to the results found in the rat model, the present analyses indicate that the effect of IVIG on MWReg30 disposition accounts for 43 5% of the overall benefit of IVIG in the mouse model of sustained thrombocytopenia.
METHODS IgG Pharmacokinetics The detailed description of the experiments work associated with this study has been described elsewhere.10 All animal experiments were approved by the Institutional Animal Use and Care Committee of the University at Buffalo. Briefly, MWReg30, a rat monoclonal antibody directed against a murine platelet antigen (BD Pharmingen, San Diego, CA, #553847), was continuously infused into mice through an osmotic pump implanted into the peritoneal cavity. The infusion rate was 0.5 mL/h for 168 h, and MWReg30 concentration was 82.5 mg/mL in the infusion solution. IVIG (0, 0.4, 1, or 2 g/kg) was administered by intraperitoneal injection 96 h after the start of the MWReg30 infusion. Each group consisted of 5 mice. MWReg30 and IVIG plasma concentrations were determined by ELISA, from blood samples collected up to 264 h. Mean, naı¨vepooled plasma concentration data were used for estimation of pharmacokinetic parameters. MWReg30 and IVIG disposition were characterized with a two-compartment model that allows consideration of antibody transport by FcRn. This model, which was initially presented by Hansen and Balthasar,8 is shown in Figure 1. The model assumes that IgG distributes within a central compartment, which includes the plasma, and into a peripheral compartment that includes endosomal vesicles. IgG is assumed to distribute to the peripheral compartment by linear processes (e.g., fluid phase endocytosis). Within the endosomes, IgG binds to FcRn receptors, as dictated by a standard Langmuir-type binding isotherm. Unbound antibody is eliminated (e.g., by intracellular proteolysis), and FcRn-bound IgG is recycled to the central compartment. This model, which is consistent with the accepted mechanism of FcRnmediated transport of IgG,11 is described with differential Eqs. (1–8): dXp;MWReg30 ¼ ka Xp;MWReg30 þRð1Þ dt
ð1Þ
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Figure 1. Schematic representation of pharmacokinetic model for IgG disposition. Differential equations describing the change in IgG concentrations with time for each compartment are shown in the text. The symbols are also denoted in the text.
dX1;MWReg30 ¼ ka Xp;MWReg30 kup dt X1;MWReg30 þ kret
ð2Þ
dC1;Endogenous ¼kin kup C1;Endogenous þ kret ð7Þ dt CE;Endogenous ð1 fu Þ
XE;MWReg30 ð1 fu Þ dXE;MWReg30 ¼ kup X1;MWReg30 kret dt XE;MWReg30 ð1 fu Þ
ð3Þ
dCE;Endogenous ¼ kup C1;Endogenous kret dt CE;Endogenous ð1 fu Þ
ð8Þ
kdeg CE;Endogenous fu
kdeg XE;MWReg30 fu
fu ¼ 1
dXp;IVIG ¼ ka Xp;IVIG dt
kd þ Rt þ C1;T
ð9Þ
2 C1;T
ð4Þ
dX1;IVIG ¼ka Xp;IVIG kup X1;IVIG þ kret dt XE;IVIG ð1 fu Þ
ð5Þ
dXE;IVIG ¼ kup X1;IVIG kret XE;IVIG dt ð1 fu Þ kdeg XE;IVIG fu
ð6Þ
DOI 10.1002/jps
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðkd þ Rt þ C1;T Þ2 4 C1;T Rt
C1;T ¼
X1;MWReg30 X1;IVIG þ C1;Endogenous þ V1 V1
ð10Þ
In these equations, Xp and X1 are the mass of IgG (nmole) in the peritoneal and central compartments, respectively; V1 is the volume of distribution in the central compartment (L); C1 and CE are IgG concentrations (nM) in the central and endosomal compartments, respectively, and R(1) is the infusion rate for MWReg30 into peritoneal compartment (nmole/h). The subscripts associated JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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with Xp, X1, C1, and CE refer to the species of IgG (i.e., MWReg30, IVIG, and endogenous IgG). C1,T are the total IgG concentrations in the central compartment. Model parameters include a first order absorption rate constant (ka), a first order uptake rate constant (kup), a first order transport rate constant for FcRn-bound IgG (kret), a first order degradation rate constant for unbound IgG (kdeg), a zero order production rate constant for endogenous antibody (kin), the capacity of FcRn receptors (Rt), the dissociation constant between IgG and mouse FcRn receptors (kd), and the unbound fraction of IgG in the endosomal compartment (fu). Equation (9) was derived based on a ‘‘Langmuir-type’’ binding isotherm to characterize the relationship between fu and binding capacity (Rt), binding affinity (kd), and total IgG concentrations (C1,T). As such, this model assumes that all species IgG considered in this study have the same affinity for mouse FcRn receptors, which is consistent with the report that mouse FcRn demonstrates high affinities for rat, mouse, and human IgG.12 The value of kd was fixed at 750 nM, as reported for the interaction of mouse IgG with mouse FcRn receptors.13 kret was set to be equal to kup. The steady state mouse IgG concentration was set to be 1.47 104 nM.14 kin was determined using the steady state endosomal concentrations of endogenous IgG (CE,endogenous,ss): kin ¼ kdeg fu,ss CE,endogenous,ss. CE,endogenous,ss is defined as a function of C1,endogenous,ss as follows: CE;endogenous;ss ¼
C1;endogenous;ss kup kret ð1 fu;ss Þ þ kdeg fu;ss
ð11Þ
where fu,ss is the unbound endogenous IgG fraction at steady state. All other parameters (kdeg, kup, ka, V1, Rt) were estimated by simultaneously fitting the model to all IVIG plasma concentrations and MWReg30 plasma concentrations.
PK/PD Model of IVIG Effects on MWReg30-Induced Thrombocytopenia Platelet counts were determined from blood samples using a Cell-Dyn 1700 multiparameter hematology analyzer (Abbott Laboratories, Abbott Park, IL). Preliminary, pilot studies demonstrated that a state of sustained thrombocytopenia was achieved after 4 days of infusion of MWReg30. Due to constraints on the volume of blood that could be obtained during our study JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
period, and due to our interest in obtaining rich data to characterize the effects of IVIG in the context of sustained thrombocytopenia, blood samples were collected prior to the infusion of MWReg30, and during the time period of 96– 168 h after initiation of the infusion. No samples were obtained between 0 and 96 h. Due to large interanimal variability in initial platelet counts, platelet counts were normalized by the initial counts observed for each mouse, and data are presented as a percentage of baseline values. Parameters of the pharmacodynamic model were fitted to mean platelet data for each group. A step-wise method was used to build a pharmacodynamic model for IVIG effects on MWReg30-induced thrombocytopenia. Consistent with data demonstrating rapid rates of platelet turnover in ITP, it was assumed that MWReg30 induces thrombocytopenia by increasing the rate of platelet destruction. Additionally, based on the hypothesis that IVIG therapy leads to an inhibition in the rate of Fcg-receptor-mediated phagocytosis of antibody-opsonized platelets, our initial model assumed that IVIG inhibited platelet destruction in ITP. These effects were modeled by combining indirect response models I and III,15 where MWReg30-mediated stimulation of platelet elimination and IVIG-mediated inhibition of the platelet elimination were described by Emax functions. Several variants of this initial model were evaluated, including the investigation of linear stimulation of platelet elimination by MWReg30, the inclusion of sigmodicity parameters within the Emax functions, the inclusion of time-dependent platelet production rates, and the inclusion of inverse relationships between platelet production rate and platelet counts (with linear or Emax functions). Model variants were assessed by comparing objective and subjective measures of goodness-of-fit, including visual inspection, Akaike Information Criterion (AIC), Schwarz Criterion (SC), correlation coefficients, precision of parameter estimates, sum of residuals, and the distribution of residuals. Based on the above evaluations, a final model was selected (shown in Fig. 2). Platelet production was represented with two parameters k1in and k2in, where k2in was modeled as a function of platelet counts. MWReg30-mediated stimulation of platelet elimination and IVIG—mediated inhibition of platelet elimination were described by a linear function and by an Emax function, respectively. The effects of platelet counts on platelet production were described by a 4-compartment transduction DOI 10.1002/jps
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Figure 2. Schematic representation of an indirect response PK/PD model for IVIG effects on thrombocytopenia induced by MWR30 in mice. MWReg30 and IVIG concentrations are described by the model shown in Figure 1. MWReg30 concentration in the central compartment stimulated platelet elimination, and IVIG concentration in the central compartment inhibited platelet elimination. Differential equations describing the model are shown in the text. The symbols are also denoted in the text.
model. Plasma IVIG and MWReg30 concentrations were determined using equations 1–11 and the PD parameters were fit separately using differential Eqs. (12–15) dI1 1 PLT R0 ¼ IPLT I1 ð12Þ t dt R0 dI2 1 ¼ ðI1 I2 Þ t dt
ð13Þ
dI3 1 ¼ ðI2 I3 Þ t dt
ð14Þ
dPLT ¼ k1in þ k2in ð1 I3 Þ kout PLT dt ð1 þ SMWReg30 CMWReg30 Þ Imax;IVIG CIVIG 1 IC50;IVIG þ CIVIG
ð15Þ
In above equations, R0 is the baseline measurement for platelet counts. k1in and k2in represent the apparent zero-order constant for platelet production. IPLT is a constant that allow platelet counts to have a feed back effect on platelet production, which is written as a function 1 IPLT ðPLT DOI 10.1002/jps
R0 Þ=ðR0 Þ. As such, platelet counts above the baseline will result in a decrease in the platelet production rate, and platelet counts below the baseline will increase the platelet production rate. kout defines the first order constant for platelet elimination, which is equal to (k1in þ k2in)/R0. t is the mean transit time for each transit compartment. SMWReg30 (nM1) is a stimulation parameter that allows MWReg30 to stimulate platelet elimination. Imax, IVIG is the capacity constant for IVIG effects on platelet elimination, which is always less than or equal to 1. IC50,IVIG is the IVIG concentration that can achieve half of the maximum effects. Pharmacokinetic and pharmacodynamic parameters were fit sequentially. Pharmacokinetic parameters were estimated first, and then these parameters were fixed during the fitting of pharmacodynamic parameters. k1in was fixed at a value of 1.5 h1, a value reported for the production rate of mouse platelets.16 All other parameters (k2in, IPLT, SMWReg30, Imax,IVIG, IC50,IVIG, and t) were estimated. The time course of thrombocytopenia up to 192 h, in the absence and the presence of IVIG treatment, were simultaneously fit, according to Eqs. (12–15). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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Estimation of the Importance of IVIG Effects on MWReg30 Clearance The PK/PD model (Figs. 1 and 2) was applied to estimate the importance of IVIG effects on MWReg30 clearance relative to other effects of IVIG on platelet counts. In this assessment, platelet counts were predicted following simulated administration of MWReg30 and IVIG. In this simulation, Imax,IVIG was set to ‘‘0,’’ such that the simulation did not consider IVIG effects on the elimination of antibody-coated platelets. The effect area (i.e., area between the platelet counts v. time curve in animals following IVIG treatment and the platelet counts v. time curve in animals with MWReg30 treatment alone) was calculated. The effect area was then compared with the actual effect area observed after IVIG administration. The ratio of the predicted effect area (with Imax,IVIG set to ‘‘0’’) to the actual observed effect area was defined as a, and this ratio indicates the contribution of the pharmacokinetic effect of IVIG (i.e., the effect of IVIG on MWReg30 pharmacokinetics) to the overall effect of IVIG on MWReg30-induced thrombocytopenia in this model.
Figure 3. MWReg30 pharmacokinetics in the absence of and the presence of IVIG in mice. MWReg30 was administrated by intraperitoneal infusion (0.99 mg/ day 7 days). IVIG was given by intraperitoneal bolus injection at 96 h. MWReg30 concentrations were determined from pooled plasma samples via ELISA. Symbols represent mean plasma concentrations obtained from a sham group, a control group (saline) and IVIG treatment groups (n ¼ 5 mice/group): sham treated group (*), saline (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). Data were fit (together with the data in Figure 4) to the model shown in Figure 1. Parameter values obtained from the fit are listed in Table 1.
Data Analysis All parameters were estimated using nonlinear regression analysis with the ADAPT II computer program by the maximum likehood method.17 The variance model was defined as follows: VARi ¼ s2 Mðy; ti Þg Where VARi is the variance of the ith data point, s and g are the variance parameters, and M (y, ti) is ith predicted value from the models. Different variance parameters were used for PK and PD measures. Goodness-of-fit was evaluated by visual inspection, model convergence, AIC, SC, estimation criterion value for the maximum likelihood method in ADAPT II, correlation coefficients (R2), and examination of residuals.
RESULTS IgG Pharmacokinetics Best-fit lines charactering the effects of IVIG on MWReg30 pharmacokinetics in murine were shown in Figures 3 and 4. MWReg30 and IVIG data were fit simultaneously; however, MWReg30 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
Figure 4. IVIG pharmacokinetics in mice. MWReg30 was administrated by intraperitoneal infusion (0.99 mg/ day 7 days). IVIG was given by intraperitoneal bolus injection at 96 h. IVIG concentrations were determined by an ELISA. Symbols represent a sham treated group, a control group (saline) and IVIG treatment groups (n ¼ 5 mice/group): sham treated group (*), saline (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). Error bars represent the standard deviation. Data were fit (together with the data in Fig. 3) to the model shown in Figure 1. Parameter values obtained from the fit are listed in Table 1. DOI 10.1002/jps
PK/PD MODELING OF IVIG EFFECTS IN ITP
Table 1. Pharmacokinetic Parameters for MWReg30 and IVIG in Mice Parameter
Estimate
CV%a
kup (1/h) kdeg (1/h) Rt (nM) V (L/kg) ka/F (1/h) kd (nM)
0.064 0.088 1.71 104 0.057 0.079 750
7.15 15.06 6.01 3.52 5.10 Fixed
a CV was calculated from asymptotic standard errors resulting from a single fit to mean data.
and IVIG data were plotted separately since the concentration ranges differed greatly. Table 1 lists parameter values obtained from the fitting. As shown in Figures 3 and 4, the PK model was able to well characterize IVIG pharmacokinetics and MWReg30 pharmacokinetics in mice in the absence and in the presence of IVIG, covering a 10000-fold range in plasma concentrations of exogenous antibody. Simulations of the expected changes in endogenous IgG concentrations and the fraction of FcRn-bound IgG in the presence of IVIG are shown in Figure 5. Simulated total IgG concentrations (IVIG þ MWReg30 þ endogenous IgG) were increased following IVIG administration, and the model predicts that this increase in total IgG concentration leads to a significant decrease in endogenous IgG concentrations. Endogenous IgG concentrations are predicted to demonstrate a gradual return to baseline, where the increase in IgG concentrations lags behind the timecourse of the increase in the fraction of FcRnbound IgG. The modeling estimates that 86% of endogenous IgG binds to FcRn receptors in the absence of IVIG; however, the percentage bound drops to 51, 28, and 17% following 0.4, 1, and 2 g/kg IVIG treatment, respectively. The fractional catabolic rate (FCR) has been historically employed as the primary descriptor of the rate of elimination of IgG.18 The relationship of FCR to IgG concentration can be described by an inverted hyperbolic curve,4,18 where high IgG concentrations are associated with high values of FCR. Based on the structure of the pharmacokinetic model, FCR may be calculated with the following equation. FCR ¼
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kup kdeg fu kret ð1 fu Þ þ kdeg fu
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Following 0.4, 1, and 2 g/kg IVIG treatment, FCR was increased from 0.012 to 0.036, 0.050, and 0.056 1/h, representing increases of 2-, 3.2- and 3.7-fold, respectively.
PK/PD Modeling of IVIG Effects on MWReg30-Induced Thrombocytopenia in Mice Different models were tested during model building. The sigmoidicity parameter (g) was fitted to 0.9989; consequently, gamma was taken to be 1.00, and removed from the Emax functions. IVIG and MWReg30 each demonstrate effects on platelet elimination, and the interaction of these effects may be antagonistic, additive, or synergetic. Additive () and synergistic () interaction functions were examined, and the synergetic function was selected for use in the final model based on selection criteria. The effects of IVIG on platelet counts in mice are shown in Figure 6, with the lines representing the best-fit of the data. Table 2 lists the pharmacodynamic parameter values obtained from the fitting. As shown, the model was found to provide an adequate characterization of the time course of MWReg30-induced thrombocytopenia, in animals that did or did not receive treatment with IVIG.
Estimation of the Importance of IVIG Effects on MWReg30 Clearance The final PK/PD model assumes that IVIG can increase platelet counts in ITP by increasing the elimination of anti-platelet antibodies (i.e., functionally decreasing the driving force for MWReg30-mediated platelet elimination) and by direct inhibition of MWReg30-stimulated platelet elimination (e.g., consistent with IVIG-mediated inhibition of Fcg-receptor-mediated elimination of antibody-coated platelets). With Imax,IVIG set to ‘‘0,’’ where the model considers the former kinetic mechanism and not the latter, effect areas were calculated as: 776, 1824, and 2612% h for 0.4, 1, and 2 g/kg IVIG, respectively (Fig. 7, panel A). In contrast, the observed overall effect areas were: 3561 1829, 4093 933, 6150 1849% h for 0.4, 1, and 2 g/kg IVIG, respectively (Fig. 7, panel B). As such, the model suggests that the effect of IVIG on MWReg30 clearance accounts for 37 38%, 46 9%, and 45 11% of the overall effect of 0.4, 1, and 2 g/kg IVIG on MWReg30induced thrombocytopenia. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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Figure 5. IVIG effects on endogenous mouse IgG concentrations, fraction of FcRnbound IgG and fraction of catabolic rate (FCR) in mice. Simulations were performed, using parameter values listed in Table 1 and the model shown in Figure 3 predict the effects of IVIG on endogenous mouse IgG concentration (Panel A), fraction of FcRn-bound IgG (Panel B) and FCR (Panel C) in mice. The lines represent predicted endogenous mouse IgG concentration (Panel A) and fraction of FcRn-bound IgG (Panel B) in MWReg30 alone (top line), 0.4 g/kg IVIG (2nd line), 1 g/kg IVIG (3rd line) or 2 g/kg IVIG (bottom line). The lines in panel C represent predicted FCR in MWReg30 alone (bottom line), 0.4 g/kg IVIG (2nd line), 1 g/kg IVIG (3rd line) or 2 g/kg IVIG (top line).
DISCUSSION In the present study, we have developed a new PK/PD model allowing characterization of the disposition of MWReg30, an antiplatelet antibody, the relationship between MWReg30 plasma concentration and the time-course of MWReg30induced thrombocytopenia, the disposition of exogenous immunoglobulin following IVIG therapy, and the effects of IVIG on MWReg30 disposition and on MWReg30-induced thromboJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
cytopenia. The model is mechanistic in that it attempts to mathematically describe the kinetic mechanisms associated with the effect of IVIG on MWReg30 disposition (i.e., competitive inhibition of MWReg30 transport by FcRn), the kinetics of MWReg30-induced stimulation of platelet elimination, and the kinetics of IVIG-mediated inhibition of the elimination of MWReg30-opsonized platelets (e.g., via blockade of Fcg-receptors). The model, which well-characterized the pharmacokinetics and pharmacodynamics of MWReg30 and DOI 10.1002/jps
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Figure 6. IVIG effects on MWReg30-induced thrombocytopenia in mice. MWReg30 was administrated by intraperitoneal infusion (0.99 mg/day 7 days). IVIG was given by intraperitoneal bolus injection at 96 h. Platelet counts were obtained using Cell-Dyn 1700 multiparameter hematology analyzer. Symbols represent the sham control group and IVIG treatment groups (n ¼ 5 mice/group): sham treated group (*), saline (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). Error bars represent the standard deviation. An indirect response model was developed to characterize IVIG effects on MWReg30 mediated thrombocytopenia. Parameter values obtained from the fit are listed in Table 2.
IVIG, was applied to assess the contribution of IVIG-induced enhancement in MWReg30 elimination (i.e., the ‘‘PK effect’’ of IVIG therapy) to the overall effect of IVIG in attenuating MWReg30induced thrombocytopenia. Model simulations suggest that the ‘‘PK effect’’ accounts for 43% of the overall effect of IVIG in this mouse model of ITP.
Table 2. Pharmacodynamic Parameters for IVIG Effects on MWReg30-Induced Thrombocytopenia in Mice Parameter k1in (%a1/h) k2in (%a1/h) IPLT SMWReg30 (1/nM) Imax,IVIG IC50,IVIG (nM) t (h)
Estimate
CV%a
1.5 0.1213 95.9 1.175 0.9037 2.51 104 40.12
Fixed 40.77 N/A 32.72 26.67 51.89 10.40
a CV was calculated from asymptotic standard errors resulting from a single fit to mean data.
DOI 10.1002/jps
Figure 7. Importance of increased MWReg30 clearance relative to the overall IVIG effect on MWReg30induced thrombocytopenia in mice. Simulations to determine the relative importance of IVIG effects on MWReg30 clearance were performed. The models shown in Figures 1 and 2 and the parameters shown in Tables 1 and 2 were used to simulate the expected time courses of thrombocytopenia following IVIG treatment with I ¼ 0 (Panel A: MWReg30 alone (bottom line), 0.4 g/kg IVIG (2nd line), 1 g/kg IVIG (3rd line), 2 g/kg IVIG (top line)). Actual time courses of thrombocytopenia following IVIG treatment are shown in panel B. Symbols represent a MWReg30 treatment alone group (saline) and IVIG treatment groups (n ¼ 5 mice/group): MWReg30 alone (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). IVIG effects on MWReg30 clearance can account for 43 5% of the overall IVIG effects in this model.
The model-building process considered a number of factors that may be important determinants of the PK/PD of MWReg30 and IVIG, most notably: target-mediated elimination of MWReg30, the role JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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of FcRn in MWReg30 and IVIG pharmacokinetics, the relationship of thrombocytopenia to the rate of platelet production, and the time-course of platelet production. Target-mediated disposition refers to the phenomenon where the interaction of an agent with its biological target plays a significant role in the agent’s distribution and elimination.19 MWReg30 is a monoclonal antibody with high affinity for mouse glycoprotein IIb, an antigen present on mouse platelets. It is quite possible that the high-affinity, capacity-limited interaction between MWReg30 and mouse platelet glycoprotein IIb influences the distribution of MWReg30, and it is likely that platelet-bound MWReg30 is eliminated by cells of the RES during the process of RES-mediated phagocytosis and destruction of MWReg30-opsonized platelets. Indeed in previous work conducted with rats, this laboratory has found that a murine monoclonal IgG1 antiplatelet antibody (7E3) was eliminated more rapidly than a murine monoclonal IgG1 antibody directed against a soluble antigen (anti-methotrexate IgG), with clearance values of 0.78 0.09 mL/h/ kg and 0.44 0.05 mL/h/kg, respectively.6 The differences between clearance values observed for these two antibodies suggest that antiplatelet antibodies may be subject to target-mediated elimination. During the model-building process, models were developed that incorporated the possible effects of platelets count on MWReg30 pharmacokinetics. However, our present data evaluated a single MWReg30 dosing protocol, and insufficient data were available to justify the characterization of possible target-mediated MWReg30 elimination. Nonetheless, the final model, which does not consider target-mediated MWReg30 elimination, does provide an adequate characterization of MWReg30 plasma pharmacokinetics (Fig. 3). Recent reports have demonstrated that the FcRn receptor is a prime determinant of the disposition of IgG antibodies.11,14,20 FcRn, which protects IgG from catabolism and contributes to the long plasma half-life of IgG, was first postulated by Brambell et al. in 196421 and cloned in late 1980s.22,23 FcRn is a heterodimer comprising of a b2m light chain and a MHC class-I like heavy chain, and the receptor is ubiquitously expressed in cells and tissues.11 Several studies have shown that IgG clearance in b2m knockout mice14,20 and FcRn-heavy chain knockout mice24 is increased 10- to 15-fold. Recent work conducted in this laboratory has shown that high dose of IVIG enhanced the elimination of antiplatelet antiboJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
dies in vivo in ‘‘wild-type’’ rats in mice, but not in FcRn-knockout mice, suggesting that this pharmacokinetic effect is due to competitive inhibition of FcRn.6,7 Based on these experimental findings, Hansen and Balthasar8 proposed a two-compartment pharmacokinetic model of IgG disposition that incorporates capacity-limited binding and transport of IgG by FcRn. This mechanism-based pharmacokinetic model was incorporated into the present PK/PD model to allow characterization of the effect of IVIG on MWReg30 disposition. During the process of model-building, we also considered the possible relationship of platelet counts and the rate of platelet production. Previous reports have demonstrated that an acute loss of platelets leads to the stimulation of thrombopoiesis. Thrombopoietin (TPO) plays a critical role in this process, stimulating all stages of megakaryocytopoiesis and thrombopoiesis.25–27 Plasma TPO concentration is inversely correlated to the total body quantity of platelets and megakaryocytes, which internalize and degrade TPO via specific TPO receptors.25–27 As such, thrombocytopenia effectively decreases the rate of TPO binding and elimination, increasing plasma concentrations of TPO, which leads to increased platelet production. In the current mathematical model, two parameters were used to describe platelet production, k1in and k2in, where k1in is independent of platelet count, and where k2in was inversely related to platelet count. This modeling approach is consistent with previous publications, which reported that platelet production is controlled by two independent process,28 and that platelet production rate is inversely related to platelet number.29 Of note, based on the secondary parameter kout, the half-life of PLT elimination may be estimated to be 42 h (i.e., ln(2)/kout). This estimate is similar to, but somewhat lower than, the reported life span of platelets in C57Bl/6/ 129SV mice (4 days, as reported by Berger et al.30). The model that we have proposed relates k2in to platelet count through the use of a transduction function.31 The transduction function was incorporated to mathematically characterize the cascade of events that is known to be involved in thrombopoiesis. That is, multipotent progenitor cells are continuously generated from hemopoietic stem cells in the bone marrow, and then step by step divided into committed megakaryocyte-progenitor cells, megakaryoblasts, mature megakaryocytes, proplatelets and finally into platelets.25 Transit compartments have been DOI 10.1002/jps
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widely utilized to characterize transduction processes,31–33 where a transit time parameter (t) and one or more transit compartments are incorporated to provide a delay between a driving force (e.g., thrombocytopenia) and a physiological function (e.g., increased platelet release into blood). The use of a single fit parameter (t) and four transit compartments, combined with the indirect response modeling of MWReg30 and IVIG effects on platelet elimination, allowed for good characterization of the time-course of MWReg30-induced thrombocytopenia and for characterization of the magnitude and time-course of IVIG-mediated increases in platelet counts (Fig. 6). The parameter t was estimated to be about 40 h in the current model; therefore, the 3-compartment transduction model predicts a mean transduction time of 120 h. This estimate is in line with the report of Grossmann et al.34 who found that platelet counts begin to increase approximately 5 days after mice are treated with thrombopoietin. Although IVIG has been in clinical use for treatment of ITP for over 20 years, very little has been done to characterize IVIG pharmacokinetics, the effects of IVIG on antiplatelet antibody disposition, or the effects of IVIG on platelet counts in human ITP or in animal models of ITP. Bleeker et al.35 developed a pharmacokinetic model to describe IVIG effects on IgG disposition, where a two-compartment mammillary model was employed and where IgG elimination was assumed to occur from the central compartment. This model did not directly incorporate IgG-FcRn binding kinetics, but it did model a concentration-dependence in IgG elimination through the use of a hyperbolic function that related IgG concentration to the FCR of IgG. As discussed by Jin and Balthasar,4 the model presented by Bleeker et al. is based on steady-state relationships between FCR and IgG concentration and, owing to its mammillary nature, the model may underpredict the influence of IVIG dosing on IgG elimination (i.e., because IVIG dosing leads to a rapid, nonsteady state increase in plasma IgG concentrations). More recently, Hansen and Balthasar8 presented a more mechanistic model that directly incorporated a Langmuir-type binding function to describe the relationship of capacity limited FcRn binding on IgG elimination, and this model was successfully applied to characterize the pharmacokinetic effect of IVIG on the elimination of antiplatelet antibody in rats, in wild-type mice, and in b2m-knockout mice that lack expression of functional FcRn. DOI 10.1002/jps
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The pharmacokinetic model presented by Hansen and Balthasar was incorporated within the present PK/PD model, which provided good characterization of the effects of IVIG on MWReg30 disposition (Fig. 3) and good characterization of IVIG disposition (Fig. 4). In comparison to model of Hansen and Balthasar, the present model utilized a much higher value of the FcRn-IgG dissociation constant, kd (i.e., 750 nM vs. 4.8 nM). The higher kd value was reported in a study13 that was published after submission of the Hansen and Balthasar8 model. Although the kd values differ substantially, the model is not very sensitive to this kd range, due to the much higher value of the endogenous IgG concentration (14700 nM). Consequently, comparable PK parameters were obtained in the present work and in the prior publication.8 It is not possible to obtain reliable, independent estimates for kup and kret with the present data; consequently, kup and kret were set to be equivalent to simplify the model. Current work in the laboratory is assessing the tissue disposition of IgG in wild-type and FcRn-knockout mice, and these data may allow the independent characterization of rate processes associated with IgG uptake into endosomes and IgG recycling (i.e., defining kret and kup more precisely). Of note, a single kd value was used to describe the binding of mouse FcRn to IVIG, MWReg30, and endogenous mouse IgG. The assumption of equivalent FcRn binding affinity for MWReg30, IVIG, and endogenous mouse IgG is consistent with the report that mouse FcRn binds rat, human, and mouse IgG with high affinity.12 As discussed above, some published reports have presented mathematical models to describe the pharmacokinetic effects of IVIG therapy on endogenous antibody concentrations and on the disposition of exogenous, antiplatelet antibodies; however, to our knowledge, no previous reports have presented a mathematical model relating IVIG administration to the time-course of platelet counts in human ITP or in animal models of ITP. Clearly, the pharmacokinetic effect of IVIG (i.e., increasing the elimination of antiplatelet antibody) may explain part of the beneficial effect of IVIG; however, substantial literature exists that suggests that IVIG leads to a direct inhibition in the elimination of antibody-coated platelets.3,4 These effects may be achieved by IVIG-mediated blockade of Fcg receptors or complement receptors, although other mechanisms have been proposed.3,4 In the current PD model, an effect of IVIG on the elimination of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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antibody-coated platelets was incorporated into the PD model through the use of a mathematical function allowing an IVIG-mediated inhibition of MWReg30-mediated stimulation of platelet elimination. As such, the structure of the model is consistent with the accepted conceptual understanding of IVIG-mediated inhibition of platelet elimination. That is, IVIG is expected to alter the elimination of antibody-coated platelets, but IVIG is not expected to have any effect on the elimination of platelets in the absence of antiplatelet antibodies. The present PK/PD model was applied to assess the contribution of the pharmacokinetic effect of IVIG to the overall effect of IVIG on MWReg30induced thrombocytopenia. As shown in Figure 6, the modeling suggests that the pharmacokinetic effect of IVIG (i.e., increasing MWReg30 elimination) is responsible for 43% of the overall effect of IVIG in this model. This finding is very similar to the earlier report of Hansen and Balthasar,8 which reported that the effect of IVIG on antiplatelet antibody elimination accounted for 50% of the overall effect in a rat model of acute ITP. Additionally, the present results are consistent with the recent report of Akilesh et al.36 where IVIG effects were assessed in humoral immune models using FcRn-knockout animals and wildtype animals. Although mathematical modeling was not applied to quantify pathways of IVIG effect, inspection of their data from FcRn-knockout animals allowed Akilesh et al. to conclude that the majority of IVIG efficacy was related to the effect of IVIG on autoantibody disposition. In summary, this report presents a new PK/PD model that allows characterization of antibody disposition, the relationship of antiplatelet antibodies to ITP, the effect of IVIG on antiplatelet antibody disposition, and the effect of IVIG on the time-course of ITP in a mouse model of ITP. The PK/PD model was applied to assess the contribution of the effect of IVIG on antiplatelet antibody disposition to the overall effect of IVIG on antiplatelet antibody-induced thrombocytopenia in this animal model, it was found that the pharmacokinetic effect was responsible for 43% of the effect of IVIG on MWReg30-induced thrombocytopenia. The apparent importance of this mechanism for IVIG effects, and the broad utility of IVIG in the treatment of autoimmunity, suggests that more specific, selective, and effective inhibitors of FcRn may be useful for the treatment of ITP and other humoral autoimmune diseases. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
ACKNOWLEDGMENTS This research was supported by grants HL67437 and AI60687 from the National Institutes of Health.
REFERENCES 1. George JN, Woolf SH, Raskob GE, Wasser JS, Aledort LM, Ballem PJ, Blanchette VS, Bussel JB, Cines DB, Kelton JG, Lichtin AE, McMillan R, Okerbloom JA, Regan DH, Warrier I. 1996. Idiopathic thrombocytopenic purpura: A practice guideline developed by explicit methods for the American Society of Hematology. Blood 88:3–40. 2. Imbach P, Barandun S, d’Apuzzo V, Baumgartner C, Hirt A, Morell A, Rossi E, Schoni M, Vest M, Wagner HP. 1981. High-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood. Lancet 1:1228–1231. 3. Hansen RJ, Balthasar JP. 2004. Mechanisms of IVIG action in immune thrombocytopenic purpura. Clin Lab 50:133–140. 4. Jin F, Balthasar JP. 2005. Mechanisms of intravenous immunoglobulin action in immune thrombocytopenic purpura. Hum Immunol 66:403–410. 5. Hansen RJ, Balthasar JP. 2001. Pharmacokinetics, pharmacodynamics, and platelet binding of an antiglycoprotein IIb/IIIa monoclonal antibody (7E3) in the rat: A quantitative rat model of immune thrombocytopenic purpura. J Pharmacol Exp Ther 298:165–171. 6. Hansen RJ, Balthasar JP. 2002. Effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombocytopenia. Blood 100:2087–2093. 7. Hansen RJ, Balthasar JP. 2002. Intravenous immunoglobulin mediates an increase in antiplatelet antibody clearance via the FcRn receptor. Thromb Haemost 88:898–899. 8. Hansen RJ, Balthasar JP. 2003. Pharmacokinetic/ pharmacodynamic modeling of the effects of intravenous immunoglobulin on the disposition of antiplatelet antibodies in a rat model of immune thrombocytopenia. J Pharm Sci 92:1206–1215. 9. Kelton JG. 2002. The mechanism of action of highdose IgG in idiopathic thrombocytopenic purpura. Blood 100:1933. 10. Deng R, Balthasar JP. 2006. Comparison of the effects of antibody-coated liposomes, IVIG and antiRBC immunotherapy in a murine model of passive chronic immune thrombocytopenia. Epub ahead of print DOI 10.1182/blood-2006-04-018093. 11. Junghans RP. 1997. Finally! The Brambell receptor (FcRB). Mediator of transmission of immunity and protection from catabolism for IgG. Immunol Res 16:29–57. DOI 10.1002/jps
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12. Ober RJ, Radu CG, Ghetie V, Ward ES. 2001. Differences in promiscuity for antibody-FcRn interactions across species: Implications for therapeutic antibodies. Int Immunol 13:1551–1559. 13. Zhou J, Johnson JE, Ghetie V, Ober RJ, Ward ES. 2003. Generation of mutated variants of the human form of the MHC class I-related receptor, FcRn, with increased affinity for mouse immunoglobulin G. J Mol Biol 332:901–913. 14. Junghans RP, Anderson CL. 1996. The protectionreceptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor. Proc Natl Acad Sci USA 93:5512–5516. 15. Dayneka NL, Garg V, Jusko WJ. 1993. Comparison of four basic models of indirect pharmacodynamic responses. J Pharmacokinet Biopharm 21:457– 478. 16. Piguet PF, Vesin C. 2002. Modulation of platelet caspases and life-span by anti-platelet antibodies in mice. Eur J Haematol 68:253–261. 17. D’Argenio D, Schumitzky A. 1997. ADAPI II user’s guide. Los Angeles, Biomedical Simulations Resource. 18. Waldmann TA, Strober W. 1969. Metabolism of immunoglobulins. Prog Allergy 13:1–110. 19. Mager DE, Jusko WJ. 2001. General pharmacokinetic model for drugs exhibiting target-mediated drug disposition. J Pharmacokinet Pharmacodyn 28:507–532. 20. Ghetie V, Hubbard JG, Kim JK, Tsen MF, Lee Y, Ward ES. 1996. Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur J Immunol 26:690–696. 21. Brambell F, Hemmings W, Morris I. 1964. A theoretical model of gamma-globulin catabloism. Nature 203:1352–1355. 22. Simister NE, Mostov KE. 1989. An Fc receptor structurally related to MHC class I antigens. Nature 337:184–187. 23. Simister NE, Mostov KE. 1989. Cloning and expression of the neonatal rat intestinal Fc receptor, a major histocompatibility complex class I antigen homolog. Cold Spring Harb Symp Quant Biol 54:571–580. 24. Roopenian DC, Christianson GJ, Sproule TJ, Brown AC, Akilesh S, Jung N, Petkova S, Avanessian L, Choi EY, Shaffer DJ, Eden PA, Anderson CL. 2003. The MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis,
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and fate of IgG-Fc-coupled drugs. J Immunol 170: 3528–3533. Kaushansky K. 1998. Thrombopoietin. N Engl J Med 339:746–754. Wolber EM, Jelkmann W. 2002. Thrombopoietin: The novel hepatic hormone. News Physiol Sci 17: 6–10. Kuter DJ, Begley CG. 2002. Recombinant human thrombopoietin: Basic biology and evaluation of clinical studies. Blood 100:3457–3469. Harker LA, Roskos LK, Marzec UM, Carter RA, Cherry JK, Sundell B, Cheung EN, Terry D, Sheridan W. 2000. Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers. Blood 95:2514–2522. Shen S, DeNardo GL, Jones TD, Wilder RB, O’Donnell RT, DeNardo SJ. 1998. A preliminary cell kinetics model of thrombocytopenia after radioimmunotherapy. J Nucl Med 39:1223–1229. Berger G, Hartwell DW, Wagner DD. 1998. PSelectin and platelet clearance. Blood 92:4446– 4452. Mager DE, Jusko WJ. 2001. Pharmacodynamic modeling of time-dependent transduction systems. Clin Pharmacol Ther 70:210–216. Sun YN, Jusko WJ. 1998. Transit compartments versus gamma distribution function to model signal transduction processes in pharmacodynamics. J Pharm Sci 87:732–737. Lobo ED, Balthasar JP. 2002. Pharmacodynamic modeling of chemotherapeutic effects: Application of a transit compartment model to characterize methotrexate effects in vitro. AAPS Pharm Sci 4:42. Grossmann A, Lenox J, Ren HP, Humes JM, Forstrom JW, Kaushansky K, Sprugel KH. 1996. Thrombopoietin accelerates platelet, red blood cell, and neutrophil recovery in myelosuppressed mice. Exp Hematol 24:1238–1246. Bleeker WK, Teeling JL, Hack CE. 2001. Accelerated autoantibody clearance by intravenous immunoglobulin therapy: Studies in experimental models to determine the magnitude and time course of the effect. Blood 98:3136–3142. Akilesh S, Petkova S, Sproule TJ, Shaffer DJ, Christianson GJ, Roopenian D. 2004. The MHC class I-like Fc receptor promotes humorally mediated autoimmune disease. J Clin Invest 113: 1328–1333.
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Received 25 April 2006; revised 25 September 2006; accepted 28 September 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20828
ABSTRACT: IVIG may achieve its beneficial effects in immune thrombocytopenia (ITP) patients via several mechanisms; however, little is known of the relative importance of various mechanisms associated with IVIG action in ITP. The purposes of this study were to develop a pharmacokinetic/pharmacodynamic (PK/PD) model relating an anti-platelet antibody, MWReg30, to platelet counts in a mouse model of sustained ITP, to use modeling to characterize effects of IVIG on MWReg30 elimination, and to use PK/PD modeling to assess the contribution of IVIG effects on MWReg30 disposition to the effects of IVIG on MWReg30-induced thrombocytopenia in mice. A pharmacokinetic model, based on the competitive occupancy of protective FcRn receptors, was used to characterize the effects of IVIG on MWReg30 pharmacokinetics. The relationships between MWReg30 plasma concentrations to MWReg30-induced thrombocytopenia, in the presence and absence of IVIG treatment, were characterized using an indirect response model. The pharmacokinetic model well-captured MWReg30 plasma concentration-time profiles, with and without administration of IVIG. The indirect response model accurately characterized the effects of IVIG on MWReg30-induced thrombocytopenia in mice. Using these models, it was estimated that 43 5% of overall effects of IVIG on MWReg30-induced thrombocytopenia in mice could be accounted for by the IVIG-mediated increases in MWReg30 clearance. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1625–1637, 2007
Keywords: immune thrombocytopenia (ITP); intravenous immunoglobulin (IVIG); pharmacokinetics; pharmacodynamics
INTRODUCTION Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet (PLT) turnover, which is mediated by antiplatelet autoantibodies.1 In 1981, Imbach et al.2 first reported that high dose of intravenous immunoglobulin (IVIG) increased platelet counts in ITP patients. Since then, the therapeutic efficacy of IVIG in ITP and other autoimmune diseases has been well documented.1 However, the exact mechanisms of IVIG action in ITP remains controversial, and few studies have attempted to Correspondence to: Joseph P. Balthasar (Telephone: 716645-2842 ext 270; Fax: 716-645-3693; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 1625–1637 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
provide a quantitative assessment of the relative importance of proposed mechanisms of IVIG action in ITP. IVIG may increase platelet counts in ITP by (a) decreasing FcgR-mediated platelet destruction via competitive-inhibition of Fcg receptors (FcgR), (b) inhibition of complement-mediated platelet elimination, (c) increasing anti-platelet antibody clearance through saturation FcRn, (d) upregulation of the expression of FcgRIIB, leading to an indirect inhibition of FcgR-mediated PLT phagocytosis, (e) increasing platelet production, (f) decreasing antiplatelet antibody production, (g) neutralization of antiplatelet antibodies by idiotype-anti-idiotype interactions, etc.3,4 In vitro and in vivo data are available to support all of these proposed mechanisms of IVIG action; however, the contribution of each mechanism to
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the overall effects of IVIG actions in ITP is uncertain. Previous work in this laboratory led to the development of a rat model of ITP,5 and this model was found to respond to IVIG treatment.6 Pharmacokinetic studies in mice7 and rats6 demonstrated that IVIG increased the elimination of a monoclonal antiplatelet antibody (7E3). However, IVIG administration did not lead to an increase in 7E3 clearance in FcRn-knockout animals,7 supporting the hypothesis that the effects of IVIG on antiplatelet antibody clearance was mediated by saturation of FcRn, which protects IgG from elimination. Subsequent PK/PD analyses demonstrated that the effect of IVIG on 7E3 elimination was an important mechanism of IVIG action in rat model of ITP, accounting for 50 11% of the overall effects of IVIG on 7E3-induced thrombocytopenia.8 The rat model of ITP is an acute model, and IVIG effects were investigated via ‘‘pretreatment’’ with IVIG, followed by the administration of a challenge dose of 7E3. The rat model allowed sensitive assessment of IVIG effects on 7E3 elimination and on 7E3-induced thrombocytopenia; however, there has been some concern that IVIG effects in this acute model may be substantially different that IVIG effects when antibody is present at ‘‘steady-state’’ (as in the human disease).9 Recently, Deng and Balthasar10 developed a mouse model of sustained ITP, where thrombocytopenia was induced in mice by continuous infusion of an anti-platelet antibody, MWReg30. Severe thrombocytopenia was observed within 4 days from the start of the MWReg30 infusion, and PLT counts were found to remain at relatively constant values from 4 to 7 days after initiation of the infusion (i.e., approximating a condition of ‘‘steady-state’’ thrombocytopenia). Administration of IVIG to thrombocytopenic animals was found to lead to a dose-dependent increase in PLT counts, which was accompanied by a dose-dependent decrease in MWReg30 plasma concentrations. The present work has led to the development of a pharmacokinetic/pharmacodynamic (PK/PD) model relating MWReg30 plasma concentrations to platelet counts in mice, the development of a mechanism-based pharmacokinetic model to characterize the effects of IVIG on MWReg30 elimination, and to the development of an indirect response model to characterize the effect of IVIG on MWReg30-induced thrombocytopenia in mice. These models were then employed, in combination, to assess the contribution of IVIG effects on MWReg30 disposition to the effect of IVIG on JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
MWReg30-induced thrombocytopenia in mice. Similar to the results found in the rat model, the present analyses indicate that the effect of IVIG on MWReg30 disposition accounts for 43 5% of the overall benefit of IVIG in the mouse model of sustained thrombocytopenia.
METHODS IgG Pharmacokinetics The detailed description of the experiments work associated with this study has been described elsewhere.10 All animal experiments were approved by the Institutional Animal Use and Care Committee of the University at Buffalo. Briefly, MWReg30, a rat monoclonal antibody directed against a murine platelet antigen (BD Pharmingen, San Diego, CA, #553847), was continuously infused into mice through an osmotic pump implanted into the peritoneal cavity. The infusion rate was 0.5 mL/h for 168 h, and MWReg30 concentration was 82.5 mg/mL in the infusion solution. IVIG (0, 0.4, 1, or 2 g/kg) was administered by intraperitoneal injection 96 h after the start of the MWReg30 infusion. Each group consisted of 5 mice. MWReg30 and IVIG plasma concentrations were determined by ELISA, from blood samples collected up to 264 h. Mean, naı¨vepooled plasma concentration data were used for estimation of pharmacokinetic parameters. MWReg30 and IVIG disposition were characterized with a two-compartment model that allows consideration of antibody transport by FcRn. This model, which was initially presented by Hansen and Balthasar,8 is shown in Figure 1. The model assumes that IgG distributes within a central compartment, which includes the plasma, and into a peripheral compartment that includes endosomal vesicles. IgG is assumed to distribute to the peripheral compartment by linear processes (e.g., fluid phase endocytosis). Within the endosomes, IgG binds to FcRn receptors, as dictated by a standard Langmuir-type binding isotherm. Unbound antibody is eliminated (e.g., by intracellular proteolysis), and FcRn-bound IgG is recycled to the central compartment. This model, which is consistent with the accepted mechanism of FcRnmediated transport of IgG,11 is described with differential Eqs. (1–8): dXp;MWReg30 ¼ ka Xp;MWReg30 þRð1Þ dt
ð1Þ
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Figure 1. Schematic representation of pharmacokinetic model for IgG disposition. Differential equations describing the change in IgG concentrations with time for each compartment are shown in the text. The symbols are also denoted in the text.
dX1;MWReg30 ¼ ka Xp;MWReg30 kup dt X1;MWReg30 þ kret
ð2Þ
dC1;Endogenous ¼kin kup C1;Endogenous þ kret ð7Þ dt CE;Endogenous ð1 fu Þ
XE;MWReg30 ð1 fu Þ dXE;MWReg30 ¼ kup X1;MWReg30 kret dt XE;MWReg30 ð1 fu Þ
ð3Þ
dCE;Endogenous ¼ kup C1;Endogenous kret dt CE;Endogenous ð1 fu Þ
ð8Þ
kdeg CE;Endogenous fu
kdeg XE;MWReg30 fu
fu ¼ 1
dXp;IVIG ¼ ka Xp;IVIG dt
kd þ Rt þ C1;T
ð9Þ
2 C1;T
ð4Þ
dX1;IVIG ¼ka Xp;IVIG kup X1;IVIG þ kret dt XE;IVIG ð1 fu Þ
ð5Þ
dXE;IVIG ¼ kup X1;IVIG kret XE;IVIG dt ð1 fu Þ kdeg XE;IVIG fu
ð6Þ
DOI 10.1002/jps
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðkd þ Rt þ C1;T Þ2 4 C1;T Rt
C1;T ¼
X1;MWReg30 X1;IVIG þ C1;Endogenous þ V1 V1
ð10Þ
In these equations, Xp and X1 are the mass of IgG (nmole) in the peritoneal and central compartments, respectively; V1 is the volume of distribution in the central compartment (L); C1 and CE are IgG concentrations (nM) in the central and endosomal compartments, respectively, and R(1) is the infusion rate for MWReg30 into peritoneal compartment (nmole/h). The subscripts associated JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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with Xp, X1, C1, and CE refer to the species of IgG (i.e., MWReg30, IVIG, and endogenous IgG). C1,T are the total IgG concentrations in the central compartment. Model parameters include a first order absorption rate constant (ka), a first order uptake rate constant (kup), a first order transport rate constant for FcRn-bound IgG (kret), a first order degradation rate constant for unbound IgG (kdeg), a zero order production rate constant for endogenous antibody (kin), the capacity of FcRn receptors (Rt), the dissociation constant between IgG and mouse FcRn receptors (kd), and the unbound fraction of IgG in the endosomal compartment (fu). Equation (9) was derived based on a ‘‘Langmuir-type’’ binding isotherm to characterize the relationship between fu and binding capacity (Rt), binding affinity (kd), and total IgG concentrations (C1,T). As such, this model assumes that all species IgG considered in this study have the same affinity for mouse FcRn receptors, which is consistent with the report that mouse FcRn demonstrates high affinities for rat, mouse, and human IgG.12 The value of kd was fixed at 750 nM, as reported for the interaction of mouse IgG with mouse FcRn receptors.13 kret was set to be equal to kup. The steady state mouse IgG concentration was set to be 1.47 104 nM.14 kin was determined using the steady state endosomal concentrations of endogenous IgG (CE,endogenous,ss): kin ¼ kdeg fu,ss CE,endogenous,ss. CE,endogenous,ss is defined as a function of C1,endogenous,ss as follows: CE;endogenous;ss ¼
C1;endogenous;ss kup kret ð1 fu;ss Þ þ kdeg fu;ss
ð11Þ
where fu,ss is the unbound endogenous IgG fraction at steady state. All other parameters (kdeg, kup, ka, V1, Rt) were estimated by simultaneously fitting the model to all IVIG plasma concentrations and MWReg30 plasma concentrations.
PK/PD Model of IVIG Effects on MWReg30-Induced Thrombocytopenia Platelet counts were determined from blood samples using a Cell-Dyn 1700 multiparameter hematology analyzer (Abbott Laboratories, Abbott Park, IL). Preliminary, pilot studies demonstrated that a state of sustained thrombocytopenia was achieved after 4 days of infusion of MWReg30. Due to constraints on the volume of blood that could be obtained during our study JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
period, and due to our interest in obtaining rich data to characterize the effects of IVIG in the context of sustained thrombocytopenia, blood samples were collected prior to the infusion of MWReg30, and during the time period of 96– 168 h after initiation of the infusion. No samples were obtained between 0 and 96 h. Due to large interanimal variability in initial platelet counts, platelet counts were normalized by the initial counts observed for each mouse, and data are presented as a percentage of baseline values. Parameters of the pharmacodynamic model were fitted to mean platelet data for each group. A step-wise method was used to build a pharmacodynamic model for IVIG effects on MWReg30-induced thrombocytopenia. Consistent with data demonstrating rapid rates of platelet turnover in ITP, it was assumed that MWReg30 induces thrombocytopenia by increasing the rate of platelet destruction. Additionally, based on the hypothesis that IVIG therapy leads to an inhibition in the rate of Fcg-receptor-mediated phagocytosis of antibody-opsonized platelets, our initial model assumed that IVIG inhibited platelet destruction in ITP. These effects were modeled by combining indirect response models I and III,15 where MWReg30-mediated stimulation of platelet elimination and IVIG-mediated inhibition of the platelet elimination were described by Emax functions. Several variants of this initial model were evaluated, including the investigation of linear stimulation of platelet elimination by MWReg30, the inclusion of sigmodicity parameters within the Emax functions, the inclusion of time-dependent platelet production rates, and the inclusion of inverse relationships between platelet production rate and platelet counts (with linear or Emax functions). Model variants were assessed by comparing objective and subjective measures of goodness-of-fit, including visual inspection, Akaike Information Criterion (AIC), Schwarz Criterion (SC), correlation coefficients, precision of parameter estimates, sum of residuals, and the distribution of residuals. Based on the above evaluations, a final model was selected (shown in Fig. 2). Platelet production was represented with two parameters k1in and k2in, where k2in was modeled as a function of platelet counts. MWReg30-mediated stimulation of platelet elimination and IVIG—mediated inhibition of platelet elimination were described by a linear function and by an Emax function, respectively. The effects of platelet counts on platelet production were described by a 4-compartment transduction DOI 10.1002/jps
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Figure 2. Schematic representation of an indirect response PK/PD model for IVIG effects on thrombocytopenia induced by MWR30 in mice. MWReg30 and IVIG concentrations are described by the model shown in Figure 1. MWReg30 concentration in the central compartment stimulated platelet elimination, and IVIG concentration in the central compartment inhibited platelet elimination. Differential equations describing the model are shown in the text. The symbols are also denoted in the text.
model. Plasma IVIG and MWReg30 concentrations were determined using equations 1–11 and the PD parameters were fit separately using differential Eqs. (12–15) dI1 1 PLT R0 ¼ IPLT I1 ð12Þ t dt R0 dI2 1 ¼ ðI1 I2 Þ t dt
ð13Þ
dI3 1 ¼ ðI2 I3 Þ t dt
ð14Þ
dPLT ¼ k1in þ k2in ð1 I3 Þ kout PLT dt ð1 þ SMWReg30 CMWReg30 Þ Imax;IVIG CIVIG 1 IC50;IVIG þ CIVIG
ð15Þ
In above equations, R0 is the baseline measurement for platelet counts. k1in and k2in represent the apparent zero-order constant for platelet production. IPLT is a constant that allow platelet counts to have a feed back effect on platelet production, which is written as a function 1 IPLT ðPLT DOI 10.1002/jps
R0 Þ=ðR0 Þ. As such, platelet counts above the baseline will result in a decrease in the platelet production rate, and platelet counts below the baseline will increase the platelet production rate. kout defines the first order constant for platelet elimination, which is equal to (k1in þ k2in)/R0. t is the mean transit time for each transit compartment. SMWReg30 (nM1) is a stimulation parameter that allows MWReg30 to stimulate platelet elimination. Imax, IVIG is the capacity constant for IVIG effects on platelet elimination, which is always less than or equal to 1. IC50,IVIG is the IVIG concentration that can achieve half of the maximum effects. Pharmacokinetic and pharmacodynamic parameters were fit sequentially. Pharmacokinetic parameters were estimated first, and then these parameters were fixed during the fitting of pharmacodynamic parameters. k1in was fixed at a value of 1.5 h1, a value reported for the production rate of mouse platelets.16 All other parameters (k2in, IPLT, SMWReg30, Imax,IVIG, IC50,IVIG, and t) were estimated. The time course of thrombocytopenia up to 192 h, in the absence and the presence of IVIG treatment, were simultaneously fit, according to Eqs. (12–15). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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Estimation of the Importance of IVIG Effects on MWReg30 Clearance The PK/PD model (Figs. 1 and 2) was applied to estimate the importance of IVIG effects on MWReg30 clearance relative to other effects of IVIG on platelet counts. In this assessment, platelet counts were predicted following simulated administration of MWReg30 and IVIG. In this simulation, Imax,IVIG was set to ‘‘0,’’ such that the simulation did not consider IVIG effects on the elimination of antibody-coated platelets. The effect area (i.e., area between the platelet counts v. time curve in animals following IVIG treatment and the platelet counts v. time curve in animals with MWReg30 treatment alone) was calculated. The effect area was then compared with the actual effect area observed after IVIG administration. The ratio of the predicted effect area (with Imax,IVIG set to ‘‘0’’) to the actual observed effect area was defined as a, and this ratio indicates the contribution of the pharmacokinetic effect of IVIG (i.e., the effect of IVIG on MWReg30 pharmacokinetics) to the overall effect of IVIG on MWReg30-induced thrombocytopenia in this model.
Figure 3. MWReg30 pharmacokinetics in the absence of and the presence of IVIG in mice. MWReg30 was administrated by intraperitoneal infusion (0.99 mg/ day 7 days). IVIG was given by intraperitoneal bolus injection at 96 h. MWReg30 concentrations were determined from pooled plasma samples via ELISA. Symbols represent mean plasma concentrations obtained from a sham group, a control group (saline) and IVIG treatment groups (n ¼ 5 mice/group): sham treated group (*), saline (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). Data were fit (together with the data in Figure 4) to the model shown in Figure 1. Parameter values obtained from the fit are listed in Table 1.
Data Analysis All parameters were estimated using nonlinear regression analysis with the ADAPT II computer program by the maximum likehood method.17 The variance model was defined as follows: VARi ¼ s2 Mðy; ti Þg Where VARi is the variance of the ith data point, s and g are the variance parameters, and M (y, ti) is ith predicted value from the models. Different variance parameters were used for PK and PD measures. Goodness-of-fit was evaluated by visual inspection, model convergence, AIC, SC, estimation criterion value for the maximum likelihood method in ADAPT II, correlation coefficients (R2), and examination of residuals.
RESULTS IgG Pharmacokinetics Best-fit lines charactering the effects of IVIG on MWReg30 pharmacokinetics in murine were shown in Figures 3 and 4. MWReg30 and IVIG data were fit simultaneously; however, MWReg30 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
Figure 4. IVIG pharmacokinetics in mice. MWReg30 was administrated by intraperitoneal infusion (0.99 mg/ day 7 days). IVIG was given by intraperitoneal bolus injection at 96 h. IVIG concentrations were determined by an ELISA. Symbols represent a sham treated group, a control group (saline) and IVIG treatment groups (n ¼ 5 mice/group): sham treated group (*), saline (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). Error bars represent the standard deviation. Data were fit (together with the data in Fig. 3) to the model shown in Figure 1. Parameter values obtained from the fit are listed in Table 1. DOI 10.1002/jps
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Table 1. Pharmacokinetic Parameters for MWReg30 and IVIG in Mice Parameter
Estimate
CV%a
kup (1/h) kdeg (1/h) Rt (nM) V (L/kg) ka/F (1/h) kd (nM)
0.064 0.088 1.71 104 0.057 0.079 750
7.15 15.06 6.01 3.52 5.10 Fixed
a CV was calculated from asymptotic standard errors resulting from a single fit to mean data.
and IVIG data were plotted separately since the concentration ranges differed greatly. Table 1 lists parameter values obtained from the fitting. As shown in Figures 3 and 4, the PK model was able to well characterize IVIG pharmacokinetics and MWReg30 pharmacokinetics in mice in the absence and in the presence of IVIG, covering a 10000-fold range in plasma concentrations of exogenous antibody. Simulations of the expected changes in endogenous IgG concentrations and the fraction of FcRn-bound IgG in the presence of IVIG are shown in Figure 5. Simulated total IgG concentrations (IVIG þ MWReg30 þ endogenous IgG) were increased following IVIG administration, and the model predicts that this increase in total IgG concentration leads to a significant decrease in endogenous IgG concentrations. Endogenous IgG concentrations are predicted to demonstrate a gradual return to baseline, where the increase in IgG concentrations lags behind the timecourse of the increase in the fraction of FcRnbound IgG. The modeling estimates that 86% of endogenous IgG binds to FcRn receptors in the absence of IVIG; however, the percentage bound drops to 51, 28, and 17% following 0.4, 1, and 2 g/kg IVIG treatment, respectively. The fractional catabolic rate (FCR) has been historically employed as the primary descriptor of the rate of elimination of IgG.18 The relationship of FCR to IgG concentration can be described by an inverted hyperbolic curve,4,18 where high IgG concentrations are associated with high values of FCR. Based on the structure of the pharmacokinetic model, FCR may be calculated with the following equation. FCR ¼
DOI 10.1002/jps
kup kdeg fu kret ð1 fu Þ þ kdeg fu
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Following 0.4, 1, and 2 g/kg IVIG treatment, FCR was increased from 0.012 to 0.036, 0.050, and 0.056 1/h, representing increases of 2-, 3.2- and 3.7-fold, respectively.
PK/PD Modeling of IVIG Effects on MWReg30-Induced Thrombocytopenia in Mice Different models were tested during model building. The sigmoidicity parameter (g) was fitted to 0.9989; consequently, gamma was taken to be 1.00, and removed from the Emax functions. IVIG and MWReg30 each demonstrate effects on platelet elimination, and the interaction of these effects may be antagonistic, additive, or synergetic. Additive () and synergistic () interaction functions were examined, and the synergetic function was selected for use in the final model based on selection criteria. The effects of IVIG on platelet counts in mice are shown in Figure 6, with the lines representing the best-fit of the data. Table 2 lists the pharmacodynamic parameter values obtained from the fitting. As shown, the model was found to provide an adequate characterization of the time course of MWReg30-induced thrombocytopenia, in animals that did or did not receive treatment with IVIG.
Estimation of the Importance of IVIG Effects on MWReg30 Clearance The final PK/PD model assumes that IVIG can increase platelet counts in ITP by increasing the elimination of anti-platelet antibodies (i.e., functionally decreasing the driving force for MWReg30-mediated platelet elimination) and by direct inhibition of MWReg30-stimulated platelet elimination (e.g., consistent with IVIG-mediated inhibition of Fcg-receptor-mediated elimination of antibody-coated platelets). With Imax,IVIG set to ‘‘0,’’ where the model considers the former kinetic mechanism and not the latter, effect areas were calculated as: 776, 1824, and 2612% h for 0.4, 1, and 2 g/kg IVIG, respectively (Fig. 7, panel A). In contrast, the observed overall effect areas were: 3561 1829, 4093 933, 6150 1849% h for 0.4, 1, and 2 g/kg IVIG, respectively (Fig. 7, panel B). As such, the model suggests that the effect of IVIG on MWReg30 clearance accounts for 37 38%, 46 9%, and 45 11% of the overall effect of 0.4, 1, and 2 g/kg IVIG on MWReg30induced thrombocytopenia. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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Figure 5. IVIG effects on endogenous mouse IgG concentrations, fraction of FcRnbound IgG and fraction of catabolic rate (FCR) in mice. Simulations were performed, using parameter values listed in Table 1 and the model shown in Figure 3 predict the effects of IVIG on endogenous mouse IgG concentration (Panel A), fraction of FcRn-bound IgG (Panel B) and FCR (Panel C) in mice. The lines represent predicted endogenous mouse IgG concentration (Panel A) and fraction of FcRn-bound IgG (Panel B) in MWReg30 alone (top line), 0.4 g/kg IVIG (2nd line), 1 g/kg IVIG (3rd line) or 2 g/kg IVIG (bottom line). The lines in panel C represent predicted FCR in MWReg30 alone (bottom line), 0.4 g/kg IVIG (2nd line), 1 g/kg IVIG (3rd line) or 2 g/kg IVIG (top line).
DISCUSSION In the present study, we have developed a new PK/PD model allowing characterization of the disposition of MWReg30, an antiplatelet antibody, the relationship between MWReg30 plasma concentration and the time-course of MWReg30induced thrombocytopenia, the disposition of exogenous immunoglobulin following IVIG therapy, and the effects of IVIG on MWReg30 disposition and on MWReg30-induced thromboJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
cytopenia. The model is mechanistic in that it attempts to mathematically describe the kinetic mechanisms associated with the effect of IVIG on MWReg30 disposition (i.e., competitive inhibition of MWReg30 transport by FcRn), the kinetics of MWReg30-induced stimulation of platelet elimination, and the kinetics of IVIG-mediated inhibition of the elimination of MWReg30-opsonized platelets (e.g., via blockade of Fcg-receptors). The model, which well-characterized the pharmacokinetics and pharmacodynamics of MWReg30 and DOI 10.1002/jps
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Figure 6. IVIG effects on MWReg30-induced thrombocytopenia in mice. MWReg30 was administrated by intraperitoneal infusion (0.99 mg/day 7 days). IVIG was given by intraperitoneal bolus injection at 96 h. Platelet counts were obtained using Cell-Dyn 1700 multiparameter hematology analyzer. Symbols represent the sham control group and IVIG treatment groups (n ¼ 5 mice/group): sham treated group (*), saline (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). Error bars represent the standard deviation. An indirect response model was developed to characterize IVIG effects on MWReg30 mediated thrombocytopenia. Parameter values obtained from the fit are listed in Table 2.
IVIG, was applied to assess the contribution of IVIG-induced enhancement in MWReg30 elimination (i.e., the ‘‘PK effect’’ of IVIG therapy) to the overall effect of IVIG in attenuating MWReg30induced thrombocytopenia. Model simulations suggest that the ‘‘PK effect’’ accounts for 43% of the overall effect of IVIG in this mouse model of ITP.
Table 2. Pharmacodynamic Parameters for IVIG Effects on MWReg30-Induced Thrombocytopenia in Mice Parameter k1in (%a1/h) k2in (%a1/h) IPLT SMWReg30 (1/nM) Imax,IVIG IC50,IVIG (nM) t (h)
Estimate
CV%a
1.5 0.1213 95.9 1.175 0.9037 2.51 104 40.12
Fixed 40.77 N/A 32.72 26.67 51.89 10.40
a CV was calculated from asymptotic standard errors resulting from a single fit to mean data.
DOI 10.1002/jps
Figure 7. Importance of increased MWReg30 clearance relative to the overall IVIG effect on MWReg30induced thrombocytopenia in mice. Simulations to determine the relative importance of IVIG effects on MWReg30 clearance were performed. The models shown in Figures 1 and 2 and the parameters shown in Tables 1 and 2 were used to simulate the expected time courses of thrombocytopenia following IVIG treatment with I ¼ 0 (Panel A: MWReg30 alone (bottom line), 0.4 g/kg IVIG (2nd line), 1 g/kg IVIG (3rd line), 2 g/kg IVIG (top line)). Actual time courses of thrombocytopenia following IVIG treatment are shown in panel B. Symbols represent a MWReg30 treatment alone group (saline) and IVIG treatment groups (n ¼ 5 mice/group): MWReg30 alone (!), 0.4 g/kg (&), 1 g/kg (^), 2 g/kg (~). IVIG effects on MWReg30 clearance can account for 43 5% of the overall IVIG effects in this model.
The model-building process considered a number of factors that may be important determinants of the PK/PD of MWReg30 and IVIG, most notably: target-mediated elimination of MWReg30, the role JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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of FcRn in MWReg30 and IVIG pharmacokinetics, the relationship of thrombocytopenia to the rate of platelet production, and the time-course of platelet production. Target-mediated disposition refers to the phenomenon where the interaction of an agent with its biological target plays a significant role in the agent’s distribution and elimination.19 MWReg30 is a monoclonal antibody with high affinity for mouse glycoprotein IIb, an antigen present on mouse platelets. It is quite possible that the high-affinity, capacity-limited interaction between MWReg30 and mouse platelet glycoprotein IIb influences the distribution of MWReg30, and it is likely that platelet-bound MWReg30 is eliminated by cells of the RES during the process of RES-mediated phagocytosis and destruction of MWReg30-opsonized platelets. Indeed in previous work conducted with rats, this laboratory has found that a murine monoclonal IgG1 antiplatelet antibody (7E3) was eliminated more rapidly than a murine monoclonal IgG1 antibody directed against a soluble antigen (anti-methotrexate IgG), with clearance values of 0.78 0.09 mL/h/ kg and 0.44 0.05 mL/h/kg, respectively.6 The differences between clearance values observed for these two antibodies suggest that antiplatelet antibodies may be subject to target-mediated elimination. During the model-building process, models were developed that incorporated the possible effects of platelets count on MWReg30 pharmacokinetics. However, our present data evaluated a single MWReg30 dosing protocol, and insufficient data were available to justify the characterization of possible target-mediated MWReg30 elimination. Nonetheless, the final model, which does not consider target-mediated MWReg30 elimination, does provide an adequate characterization of MWReg30 plasma pharmacokinetics (Fig. 3). Recent reports have demonstrated that the FcRn receptor is a prime determinant of the disposition of IgG antibodies.11,14,20 FcRn, which protects IgG from catabolism and contributes to the long plasma half-life of IgG, was first postulated by Brambell et al. in 196421 and cloned in late 1980s.22,23 FcRn is a heterodimer comprising of a b2m light chain and a MHC class-I like heavy chain, and the receptor is ubiquitously expressed in cells and tissues.11 Several studies have shown that IgG clearance in b2m knockout mice14,20 and FcRn-heavy chain knockout mice24 is increased 10- to 15-fold. Recent work conducted in this laboratory has shown that high dose of IVIG enhanced the elimination of antiplatelet antiboJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
dies in vivo in ‘‘wild-type’’ rats in mice, but not in FcRn-knockout mice, suggesting that this pharmacokinetic effect is due to competitive inhibition of FcRn.6,7 Based on these experimental findings, Hansen and Balthasar8 proposed a two-compartment pharmacokinetic model of IgG disposition that incorporates capacity-limited binding and transport of IgG by FcRn. This mechanism-based pharmacokinetic model was incorporated into the present PK/PD model to allow characterization of the effect of IVIG on MWReg30 disposition. During the process of model-building, we also considered the possible relationship of platelet counts and the rate of platelet production. Previous reports have demonstrated that an acute loss of platelets leads to the stimulation of thrombopoiesis. Thrombopoietin (TPO) plays a critical role in this process, stimulating all stages of megakaryocytopoiesis and thrombopoiesis.25–27 Plasma TPO concentration is inversely correlated to the total body quantity of platelets and megakaryocytes, which internalize and degrade TPO via specific TPO receptors.25–27 As such, thrombocytopenia effectively decreases the rate of TPO binding and elimination, increasing plasma concentrations of TPO, which leads to increased platelet production. In the current mathematical model, two parameters were used to describe platelet production, k1in and k2in, where k1in is independent of platelet count, and where k2in was inversely related to platelet count. This modeling approach is consistent with previous publications, which reported that platelet production is controlled by two independent process,28 and that platelet production rate is inversely related to platelet number.29 Of note, based on the secondary parameter kout, the half-life of PLT elimination may be estimated to be 42 h (i.e., ln(2)/kout). This estimate is similar to, but somewhat lower than, the reported life span of platelets in C57Bl/6/ 129SV mice (4 days, as reported by Berger et al.30). The model that we have proposed relates k2in to platelet count through the use of a transduction function.31 The transduction function was incorporated to mathematically characterize the cascade of events that is known to be involved in thrombopoiesis. That is, multipotent progenitor cells are continuously generated from hemopoietic stem cells in the bone marrow, and then step by step divided into committed megakaryocyte-progenitor cells, megakaryoblasts, mature megakaryocytes, proplatelets and finally into platelets.25 Transit compartments have been DOI 10.1002/jps
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widely utilized to characterize transduction processes,31–33 where a transit time parameter (t) and one or more transit compartments are incorporated to provide a delay between a driving force (e.g., thrombocytopenia) and a physiological function (e.g., increased platelet release into blood). The use of a single fit parameter (t) and four transit compartments, combined with the indirect response modeling of MWReg30 and IVIG effects on platelet elimination, allowed for good characterization of the time-course of MWReg30-induced thrombocytopenia and for characterization of the magnitude and time-course of IVIG-mediated increases in platelet counts (Fig. 6). The parameter t was estimated to be about 40 h in the current model; therefore, the 3-compartment transduction model predicts a mean transduction time of 120 h. This estimate is in line with the report of Grossmann et al.34 who found that platelet counts begin to increase approximately 5 days after mice are treated with thrombopoietin. Although IVIG has been in clinical use for treatment of ITP for over 20 years, very little has been done to characterize IVIG pharmacokinetics, the effects of IVIG on antiplatelet antibody disposition, or the effects of IVIG on platelet counts in human ITP or in animal models of ITP. Bleeker et al.35 developed a pharmacokinetic model to describe IVIG effects on IgG disposition, where a two-compartment mammillary model was employed and where IgG elimination was assumed to occur from the central compartment. This model did not directly incorporate IgG-FcRn binding kinetics, but it did model a concentration-dependence in IgG elimination through the use of a hyperbolic function that related IgG concentration to the FCR of IgG. As discussed by Jin and Balthasar,4 the model presented by Bleeker et al. is based on steady-state relationships between FCR and IgG concentration and, owing to its mammillary nature, the model may underpredict the influence of IVIG dosing on IgG elimination (i.e., because IVIG dosing leads to a rapid, nonsteady state increase in plasma IgG concentrations). More recently, Hansen and Balthasar8 presented a more mechanistic model that directly incorporated a Langmuir-type binding function to describe the relationship of capacity limited FcRn binding on IgG elimination, and this model was successfully applied to characterize the pharmacokinetic effect of IVIG on the elimination of antiplatelet antibody in rats, in wild-type mice, and in b2m-knockout mice that lack expression of functional FcRn. DOI 10.1002/jps
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The pharmacokinetic model presented by Hansen and Balthasar was incorporated within the present PK/PD model, which provided good characterization of the effects of IVIG on MWReg30 disposition (Fig. 3) and good characterization of IVIG disposition (Fig. 4). In comparison to model of Hansen and Balthasar, the present model utilized a much higher value of the FcRn-IgG dissociation constant, kd (i.e., 750 nM vs. 4.8 nM). The higher kd value was reported in a study13 that was published after submission of the Hansen and Balthasar8 model. Although the kd values differ substantially, the model is not very sensitive to this kd range, due to the much higher value of the endogenous IgG concentration (14700 nM). Consequently, comparable PK parameters were obtained in the present work and in the prior publication.8 It is not possible to obtain reliable, independent estimates for kup and kret with the present data; consequently, kup and kret were set to be equivalent to simplify the model. Current work in the laboratory is assessing the tissue disposition of IgG in wild-type and FcRn-knockout mice, and these data may allow the independent characterization of rate processes associated with IgG uptake into endosomes and IgG recycling (i.e., defining kret and kup more precisely). Of note, a single kd value was used to describe the binding of mouse FcRn to IVIG, MWReg30, and endogenous mouse IgG. The assumption of equivalent FcRn binding affinity for MWReg30, IVIG, and endogenous mouse IgG is consistent with the report that mouse FcRn binds rat, human, and mouse IgG with high affinity.12 As discussed above, some published reports have presented mathematical models to describe the pharmacokinetic effects of IVIG therapy on endogenous antibody concentrations and on the disposition of exogenous, antiplatelet antibodies; however, to our knowledge, no previous reports have presented a mathematical model relating IVIG administration to the time-course of platelet counts in human ITP or in animal models of ITP. Clearly, the pharmacokinetic effect of IVIG (i.e., increasing the elimination of antiplatelet antibody) may explain part of the beneficial effect of IVIG; however, substantial literature exists that suggests that IVIG leads to a direct inhibition in the elimination of antibody-coated platelets.3,4 These effects may be achieved by IVIG-mediated blockade of Fcg receptors or complement receptors, although other mechanisms have been proposed.3,4 In the current PD model, an effect of IVIG on the elimination of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
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antibody-coated platelets was incorporated into the PD model through the use of a mathematical function allowing an IVIG-mediated inhibition of MWReg30-mediated stimulation of platelet elimination. As such, the structure of the model is consistent with the accepted conceptual understanding of IVIG-mediated inhibition of platelet elimination. That is, IVIG is expected to alter the elimination of antibody-coated platelets, but IVIG is not expected to have any effect on the elimination of platelets in the absence of antiplatelet antibodies. The present PK/PD model was applied to assess the contribution of the pharmacokinetic effect of IVIG to the overall effect of IVIG on MWReg30induced thrombocytopenia. As shown in Figure 6, the modeling suggests that the pharmacokinetic effect of IVIG (i.e., increasing MWReg30 elimination) is responsible for 43% of the overall effect of IVIG in this model. This finding is very similar to the earlier report of Hansen and Balthasar,8 which reported that the effect of IVIG on antiplatelet antibody elimination accounted for 50% of the overall effect in a rat model of acute ITP. Additionally, the present results are consistent with the recent report of Akilesh et al.36 where IVIG effects were assessed in humoral immune models using FcRn-knockout animals and wildtype animals. Although mathematical modeling was not applied to quantify pathways of IVIG effect, inspection of their data from FcRn-knockout animals allowed Akilesh et al. to conclude that the majority of IVIG efficacy was related to the effect of IVIG on autoantibody disposition. In summary, this report presents a new PK/PD model that allows characterization of antibody disposition, the relationship of antiplatelet antibodies to ITP, the effect of IVIG on antiplatelet antibody disposition, and the effect of IVIG on the time-course of ITP in a mouse model of ITP. The PK/PD model was applied to assess the contribution of the effect of IVIG on antiplatelet antibody disposition to the overall effect of IVIG on antiplatelet antibody-induced thrombocytopenia in this animal model, it was found that the pharmacokinetic effect was responsible for 43% of the effect of IVIG on MWReg30-induced thrombocytopenia. The apparent importance of this mechanism for IVIG effects, and the broad utility of IVIG in the treatment of autoimmunity, suggests that more specific, selective, and effective inhibitors of FcRn may be useful for the treatment of ITP and other humoral autoimmune diseases. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 6, JUNE 2007
ACKNOWLEDGMENTS This research was supported by grants HL67437 and AI60687 from the National Institutes of Health.
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