In the drug discovery and development setting, the ability to accurately predict the human pharmacokinetics (PK) of a candidate compound from preclinical data is critical for informing the effective design of the first-in-human trial. PK prediction is especially challenging for monoclonal antibodies exhibiting nonlinear PK attributed to target-mediated drug disposition (TMDD). Here, we present a model-based method for predicting the PK of PF-03446962, an IgG2 antibody directed against human ALK1 (activin receptor-like kinase 1) receptor. Systems parameters as determined experimentally or obtained from the literature, such as binding affinity (kon and koff), internalization of the drug-target complex (kint), target degradation rate (kdeg), and target abundance (R0), were directly integrated into the modeling and prediction. NONMEM 7 was used to model monkey PK data and simulate human PK profiles based on the construct of a TMDD model using a population-based approach. As validated by actual patient data from a phase I study, the human PK of PF-03446962 were predicted within 1- to 2-fold of observations. Whereas traditional approaches fail, this approach successfully predicted the human PK of a monoclonal antibody exhibiting nonlinearity because of TMDD.
PF-03446962 is a fully human IgG2 monoclonal antibody (mAb) raised against human ALK1 (activin receptor-like kinase 1), a cell surface type I receptor of the tumor growth factor-β receptor family. ALK1 plays an important role during vascular development and is preferentially expressed on endothelial cells as well as various solid human tumors, including melanoma, breast, lung, prostate, and hepatocellular carcinoma (Lux et al., 1999, 2006; Oh et al., 2000; Goumans et al., 2009). The expression of ALK1 is more markedly up-regulated in tumor-associated vascular endothelial cells compared with normal tissues. ALK1 has been recently proposed as a novel antiangiogenic target that complements the antivascular endothelial growth factor therapy (Hu-Lowe et al., 2011). We have also evaluated the in vivo activity of PF-03446962 in chimera mice bearing melanoma M24met tumors. Neutralization of ALK1 by PF-03446962 resulted in significant reduction of hCD31, an indicator of the antiangiogenic activity, to an extent similar to other antiangiogenesis agents such as sunitinib and bevacizumab.
The in vivo pharmacokinetics (PK) of PF-03446962 after single-dose and multiple-dose administrations to monkeys was found to be nonlinear, wherein the half-life and the total clearance decreased with increased dose. Antibodies such as PF-03446962 that have specificities for cell-surface receptors often exhibit nonlinear PK due to target-mediated drug disposition (TMDD) (Mager and Jusko, 2001; Mager et al., 2003). Several mechanism-related variables may contribute to the observed nonlinearity of the PK including binding affinity (kon and koff rates), turnover rates of the unbound receptor, and the internalization rate of the mAb-receptor complex. In the drug discovery setting, those variables may be measured in vitro using cell lines of the tested preclinical species as well as those of human. Because those data provide mechanistic links between animal and human, perhaps they should be collected routinely and incorporated into a model that allows for interspecies translation.
In the preclinical evaluation of the PK of PF-03446962, its nonlinearity in the PK poses a significant challenge in our attempt to perform traditional approaches to allometric scaling because those approaches inherently assume the PK to be linear. At the transitional phase between candidate nomination and the start of the first-in-human trial, it was recognized that developing a method for predicting the PK of PF-03446962 in human based on preclinical in vivo PK and in vitro cellular data would be crucial for informing the first-in-human study design. Therefore, the objective of this work was to evaluate the PK of PF-03446962 and develop a method for predicting human PK by incorporating the aforementioned mechanistic parameters in the model-based translational framework.
Materials and Methods
The fully human mAb PF-03446962 (IgG2, κ) was generated from human IgG2 transgenic XenoMouse (Abgenix, Fremont, CA) immunized using human ALK1 extracellular domain immunogen (Kellermann and Green, 2002). The nongermline framework mutations were fixed to be consistent with the germline sequence and the resulting antibody. Human ALK1/Fc chimera (lot 002K1721), goat anti-human κ conjugated to horseradish peroxidase (lot 032K9157), tetramethylbenzidine (TMB), and TMB stop solution were obtained from Sigma-Aldrich (St. Louis, MO).
Pharmacokinetics in Cynomolgus Monkeys.
Animal experiments were carried out in accordance with Pfizer Global Research and Development Institutional Animal Care and Use Committee guidelines and with the Institute of Laboratory Animal Resources (1996). Animals were housed in compatible pairs in stainless steel cages. They were separated early in the morning for individual feeding, dosing, assessment of clinical signs, and other procedures. They remained separated throughout the day and then were paired again in the afternoon.
In the single-dose PK study, cynomolgus monkeys (3–6 kg) received a single intravenous bolus dose of PF-03446962 at 5 or 50 mg/kg. PF-03446962 formulation was prepared in a vehicle consisting of 20 mM sodium acetate, 140 mM sodium chloride, and 0.2 mg/ml polysorbate-80, pH 5.5. In the multiple-dose PK study, cynomolgus monkeys (2–8 kg) received 5 weekly intravenous doses of PF-03446962 at 2, 10, or 50 mg/kg followed by a 2-month washout period. PF-03446962 formulation was prepared in a vehicle consisting of 20 mM histidine, pH ∼5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate-80, 0.05 mg/ml disodium EDTA, and 0.1 mg/ml l-methionine. Single-dose PK study was conducted during the discovery stage while the multidose study was conducted as a part of the investigational new drug-enabling studies. At the time, the formulation used for the single-dose study was standard and was used for the majority of early-stage experiments. However, once the mAb reached a stage of “clinical candidate,” a more extensive formulation work was conducted to select the clinical formulation. Therefore, investigational new drug-enabling studies were conducted in the final formulation. It was assumed that formulation would not alter the PK of an intravenously administered mAb. Blood samples (∼1 ml) were collected via femoral venipuncture from all animals at predetermined time points. The serum was separated and stored frozen at approximately −20°C until samples were analyzed. In the single-dose study, only one animal was antidrug antibodies (ADA)-positive. In the multiple-dose study, two animals were ADA-positive. Only animals (n = 14, combination of the single- and multiple-dose studies) that were negative for ADA were selected for modeling to avoid confounding issues associated with the clearance of PF-03446962.
Preliminary PK data from an ongoing phase I study of PF-03446962 were used to validate the PK prediction based on the model. The PK data were obtained from a phase 1, multicenter, multinational trial in patients with solid tumors. The available data from the 0.5, 1, 2, 3, and 4.5 mg/kg dose cohorts (three patients per cohort) were used to verify human PK prediction. Noncompartmental analyses (NCA) were performed to compare the projected versus observed PK parameters.
Study serum samples were analyzed using a “sandwich” enzyme-linked immunosorbent assay (ELISA). Plates were coated with human ALK1/Fc chimera (50 μl/well at 2.5 μg/ml) overnight at 4°C followed by washing with PBS buffer (containing 0.05% Tween 20) and incubation with the blocking buffer (100 μl per well) for 1 h at room temperature. After the incubation, plates were washed and incubated with 50 μl per well of study samples that were diluted in the blocking buffer, standards, or quality control (QC) samples for 1 h at room temperature. After the incubation, plates were washed and incubated with 50 μl per well of secondary antibody (diluted 2000 times in PBS) for 1 h at room temperature. The plates were then washed followed by the addition of TMB (50 μl per well). The reaction was terminated by an addition of 50 μl per well of TMB stop solution at 2 to 5 min. The resulting absorbance was measured at 450 nm using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). A calibration curve was constructed by the SoftMax Pro version 5 software (Molecular Devices) using a four-parameter logistic curve. Concentrations of study and QC samples were determined by reverse prediction from their absorbance against the calibration curve. The lower limit of quantitation was 146 ng/ml. The percentage coefficient of variation for the high, low, and medium QC ranged from 3.7 to 15.8%, whereas %RE ranged from −0.8 to 2.5%.
Surface Plasmon Resonance Studies.
Surface plasmon resonance (SPR)-based binding studies were carried out using a Biacore 3000 instrument (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) at 25°C in 150 mM NaCl, 25 mM HEPES, pH 7.5, 2 mM MgCl2, and 0.005% P20 surfactant. PF-03446962 fragment antigen-binding(Fab) fragment was immobilized on a CM5 chip by standard 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride/N-hydrosuccinimide amine coupling chemistry at 25°C using 1 μM in 10 mM sodium acetate, pH 5.0. B and C anti-human ALK1 Fab was injected at concentrations of 97, 195, 390, and 781 pM and of 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM. A human ALK1 extracellular domain (ECD) (180 μl) was injected at concentrations of 976 pM and 1.95, 3.9, 7.81, 15.6, 31.25, 62.5, 125, 250, and 500 nM using a Kinject at a flow rate of 50 μl per min. The association and dissociation times were measured for 3.6 and 23 min, respectively. Regeneration was performed using a 1 min pulse of 10 mM H3PO4. Data analysis was performed using the Scrubber2 software (BioLogic Software, Pty., Campbell, ACT, Australia). Injections were referenced to a blank surface and by a buffer blank. Kinetic characteristics were obtained from a fit to a simple 1:1 kinetic binding model using the Scrubber2 program software (BioLogic Software, Pty.).
SPR biosensor studies were carried out to determine the direct binding properties of PF-03446962 to recombinant human and cyno-ALK1 ECD. To obtain meaningful kinetic parameters the analyte molecule in solution, we ensured that the monomeric and its concentration were accurately known. The human and cyno-ALK1 ECD were immobilized on the sensorChip surface, and the binding of the monomeric Fab form of PF-03446962 was observed. This experimental format provided the best means to compare the binding of PF-03446962 to the human and cyno-ALK1 ECD forms given that the association rate depends only on the concentration determined for the analyte molecule present in solution.
Fluorescence-Activated Cell Sorting Analysis of PF-03446962-ALK1 Complex Internalization in Human Umbilical Vein Endothelial Cell and Monkey Endothelial Cells.
Human umbilical vein endothelial cells (HUVEC) and monkey endothelial cells (smUVEC-cyno) were seeded on a 24-well plate with 300 μl of Complete HUVEC growth medium (EGM-2; Lonza Walkersville, Inc., Walkersville, MD) in each well and grown at 37°C with 5% CO2 overnight. Three microliters of PF-03446962 (1 mg/ml stock) were added to the cells and incubated at 37°C with 5% CO2 for 0, 0.5, 2, 6, or 24 h to for allow the internalization activity to take place. At the end of the time points, samples were placed on ice for 1 h. The cells were then washed with PBS and detached with 0.025% trypsin-EDTA. Cells were washed twice with ice-cold PBS-SA (0.04% sodium azide, NaN3) by centrifugation and blocked with PBS-SA containing 2% normal goat serum on ice for 15 min. The cells were then incubated with R-phycoerythrin-conjugated goat anti-human κ secondary antibody (Southern Biotechnology Associates, Birmingham, AL) for 45 min in the dark. Cells were then washed three times, resuspended in 200 μl of PBS-SA, and analyzed by GUAVA Flow Cytometer CytoSoft ExpressPro (GUAVA Technologies, Inc., Hayward, CA) using yellow fluorescent channel. PF-03446962 bound to the cell surface receptors was determined as the fluorescence intensity. The experiment was repeated to obtain replicates from which the averages of the replicates were calculated and used to determine the rate of antibody-complex internalization. The internalization rate (kint) was assumed to be the initial rate of mAb-target complex disappearance and was calculated by taking the regressed slope of percent receptor remaining values of the first three time points.
TMDD model (Mager and Jusko, 2001; Mager et al., 2003) was used to simultaneously fit the monkey single- and multiple-dose serum PK data of PF-03446962. The model diagram is shown in Fig. 1. PF-03446962 was assumed to undergo first-order elimination (kel) in the plasma compartment and distribute into the peripheral compartment as characterized by k12 and k21. The antibody was assumed to interact with the ALK1 receptor via reversible binding constants kon and koff. Because the ALK1 receptor is predominantly expressed on endothelial cells, drug-target binding was designated to take place in the central instead of the peripheral compartment. As verified by in vitro experiments, binding to target receptor leads to internalization of the antibody-receptor complex. The internalization rate of the drug-receptor complex was assumed to be first-order and was represented by kint. The turnover kinetics of the ALK1 receptor was represented by ksyn, rate of synthesis, and first-order kdeg, rate of degradation.
The PK model was described by the following set of differential equations: where Ap and At represent the drug amount in the serum and peripheral/tissue compartment, respectively; R represents the concentration of the unbound ALK1 receptor; and Cplx represents the concentration of drug-ALK1 receptor complex. Vc is the volume of distribution in the central (plasma) compartment. The receptor binding kinetics is governed by the association (kon) and dissociation (koff) rate constants. The parameters ksyn (zero-order) and kdeg (first-order) describe the intrinsic turnover kinetics of the ALK1 receptor.
In the model fitting, kon and koff were fixed to values experimentally obtained from the SPR study (as described above); kint was fixed to the value obtained from the FACS experiment (as described above); and kdeg was fixed based on half-life, as estimated from the human ALK1 protein expression data from Shao et al. (2009). Because the half-life of monkey ALK1 was not available in the literature, it was assumed that it was the same as that of human. Under the initial condition, before drug administration, the free ALK1 receptor concentration, R0, was assumed constant. On the basis of eq. 3, at the initial condition, R0 = ksyn/kdeg. Because kdeg was known, the equation can be rearranged to ksyn = R0kdeg. R0 was estimated as a parameter in the model. The total initial abundance (RT) may be derived as RT = R0 + Rbound, where Rbound is the initial target-ligand complex. During model fitting, we assumed the initial Rbound to be insignificant, thus, setting it to zero at the initial condition. This inherently assumes that any initial endogenous occupation of the receptor was negligible.
Initial NCA of the monkey PK data using WinNonlin (Pharsight, Mountain View, CA) (data not shown) indicated that there was a discrepancy between the clearances of PF-03446962 when comparing single-dose data with multiple-dose data. This may have resulted from the studies being conducted at different times and with different sets of monkeys. Thus, a between-study covariate term was added to the kel parameter (Table 1). Model fitting and simulations were performed with NONMEM VII (ICON Development Solutions, Ellicott City, MD), and the first-order approximation method was used. Although the FOCE-INTERACTION method may have been preferable, attempts to use this method were not successful. The between-subject variability, assumed to be log-normally distributed, was estimated for volume of distribution (Vc) and kel. A proportional error model was used for the estimation of the residual variability. The parameter standard errors were directly obtained from the NONMEM output.
Human Pharmacokinetic Prediction.
To simulate and predict the human PK profiles of PF-03446962 from the model, physiological PK parameters (kel, k12, and k21) were allometrically scaled based on the power coefficient equation P = aBWb, where P is the human predicted parameter, a is the monkey parameter, BW is the human body weight (70 kg), and b is the power coefficient that was assumed to be 0.25 (Mahmood, 2009). However, no scaling was performed for the mechanistic parameters (kon, koff, kint, and kdeg). Instead, human values of kon, koff, and kint (such as those of monkeys) were experimentally measured as described in the previous section. The parameter kdeg was separately estimated from the human ALK1 protein expression data of Shao et al. (2009)). The human Vc was the assumed plasma volume (2.8 liters in a 70-kg human body weight) based on typical volume of IgGs (Mahmood, 2009). With the consideration that the ALK1 receptor was predominantly expressed on endothelial cells, the receptor abundance (R0), expressed in units of nanomoles (amount in molar unit normalized to liter of plasma), was assumed the same between monkey and human. This essentially assumed that total target amount of targets per plasma volume is the same between monkey and human.
On the basis of the set of human PK parameters (derived as mentioned above), population PK profiles of 100 individuals were simulated. In the absence of prior information on the variability, we arbitrarily chose 20% BSV for some model parameters. For simplicity, all subjects were assumed to have a body weight of 70 kg. Because data were available from only a limited number of subjects, we did not consider an alternative method, which was to simulate based on the distribution of the body weights of the studied subjects. For graphical comparison, the simulated individual PK profiles were overlaid with the observed clinical data in patients in an ongoing clinical trial. To compare the PK simulation against observed PK data in patients (WinNonlin 5.2; Pharsight) was used to determine the noncompartmental parameters of the mean predicted versus observed clinical data. In addition, predicted population PK profiles were graphically overlaid with observed PK profiles for comparison.
In the determination of PF-03446962's affinity toward ALK-1 receptors, global analysis of the binding data using a simple 1:1 binding model yielded an equilibrium KD = 2.9 nM for the human ALK1 and 2.4 nM for the cyno-ALK1 ECD with kon = 31 × 104 M s−1, koff = 8.7 × 10−4 s−1 for the human, and kon = 49 × 104 M s−1, koff = 11.5 × 10−4 s−1 for the cynomolgus monkey. These values were converted to time unit of day and were used in the PK modeling and prediction. As an independent measure of the binding kinetics of the PF-03446962 antibody to the human ALK1 ECD, the antibody was also immobilized on the sensorChip surface, and binding of the human ALK1 ECD was observed. This resulted in good agreement between the kinetic parameters for the solution Fab binding and immobilized PF-03446962, indicating no gross change in the binding mode of the Fab form of the antibody.
Internalization of PF-03446962-ALK1 Complex in HUVEC and smUVEC-cyno Cells.
A quantitative flow cytometric assay was used to estimate PF-03446962-to-ALK1 complex internalization. Similar dose-dependent shifts of the ALK1-specific fluorescence were observed in HUVEC and snUVEC-cyno cells (Fig. 2, A and B). For the monkey endothelial cells and HUVEC, there were little variation between experiment 1 and experiment 2 replicates. Based on the percentage of the remaining fluorescence data, the rates of internalization were determined to be 4.10 day−1 and 4.37 day−1 for HUVEC and smUVEC-cyno cells, respectively.
Figure 3 shows the population predicted plotted against observed data. As can be seen from the figure, the specified TMDD model reasonably described the single- and multiple-dose PK profiles of PF-03446962 in monkeys. In addition, as shown in Fig. 4, the individual predicted matches well against the individual observations. The resulting parameter estimates and their associated standard errors are listed in Table 1 along with the projected parameters used for the simulation of human PK.
The simulation of 100 random subjects, assuming 20% BSV variability for some parameters along with the overlay of the observed human PK data obtained from the first five dose cohorts of patients who received PF-03446962 are shown in Fig. 5. The observed individual patient PK profiles seem to fall within the spread of the population PK prediction. However, the prediction tended to moderately overpredict the terminal half-life at the two lowest doses (0.5 and 1 mg/kg). As seen in Table 2, area under the curve (AUC), clearance, and Cmax, based on NCA conducted using WinNonlin, were generally predicted within 1-fold of observations. However, the half-life and volume tended to be under predicted for lower doses; however, the prediction is still roughly within 2-fold of observations.
Pharmacokinetic extrapolation from animal species to human remains a challenging undertaking in the drug development setting. Without a doubt, the ability to accurately predict human PK at the preclinical-to-clinical transition phase is invaluable for the effective design and efficient conduct of the first-in-human trial. Approaches to small-molecule human PK prediction from traditional allometric scaling have been broadly discussed in the literature (Mahmood, 1999a,b; Espié et al., 2009; Hosea et al., 2009). Although for therapeutic proteins, traditional allometric scaling has been shown to be feasible for the systemic clearance parameter with the scaling exponents ranging from 0.75 to 0.9, these approaches are generally not applicable for compounds exhibiting nonlinear PK (Mahmood, 2009; Ling et al., 2009; Wang and Prueksaritanont, 2010). To our knowledge, methods for accurately predicting the nonlinear PK of a monoclonal antibody exhibiting TMDD are not currently established. Furthermore, PK prediction for therapeutic proteins using mechanistic parameters derived from in vitro experiments has not been widely applied or reported in the literature. In this work, we applied a mechanistic, model-based approach to the human PK prediction of PF-03446962 mAb using experimentally derived parameters, such as mAb-target binding kinetics, mAb-target complex internalization rate, and receptor kinetics in conjunction with allometric scaling of compartmental (i.e., physiological) PK parameters.
As a comparison, in a recent article, Dong et al. (2011) evaluated 16 monoclonal antibodies using simple allometric approach to scaling in the context of empirical, nonmechanistic models and found that the prediction for mAbs exhibiting nonlinear PK were poor (Cmax was estimated up to 5.3-fold higher than the observed data) and that the prediction was best only when the PK of these compounds were at the linear, target-saturated phase. In our approach, AUC, Cmax, and clearance were predicted largely within 1-fold and half-life and volume within 2-fold. This points the robustness of the model when mechanism-related variables are taken into account. Overall, the TMDD model successfully described the single- and multiple-dose monkey PK profiles, capturing the multiphasic nature of the PK curves at the population and individual levels and predicted the human PK with good, if not practical, accuracy.
A challenging aspect of our approach to predicting human PK within the construct of a TMDD model was determining how to best scale the target density (R0) parameter from monkey to human. Because this parameter contributes significantly to the extent of nonlinearity in the PK, the decision on how to scale this parameter affects the outcome of the prediction. In this work, we assumed R0 for ALK1, expressed in units of nanomolars, to be the same for monkey and human. This essentially means that the target amount is proportional to the central volume of distribution or that the target concentration is the same. This assumption is reasonable for ALK1 receptor since it is known to be predominantly expressed in the vascular endothelial cells, which are in direct contact with the plasma. Although we feel that this qualitative assumption is valid based on the mechanism, it may have possibly contributed the moderate underestimation of the terminal half-lives and volume of distribution of PF-03446962. Whether this parameter should be scaled depends on what is known about the specific receptor in question.
An alternative approach to the above assumption was to experimentally determine R0. However, there are experimental limitations to being able to accurately determine total target abundance. For example, methods, such as flow-cytometric analysis of cell-surface receptor expression, gives arbitrary light intensity unit, which is not easily convertible to target concentration units (Ng et al., 2005). In addition, in many cases, the total number of cells that are expressing the target and are available for mAb binding is unknown and, thus, the estimation of total number of cells carrying a particular receptor is not possible (Ng et al., 2005). In addition, it has been our experience from a number of cases that semiqualitative estimation of the receptor abundance by multiplying the cell-surface density to the total number of cells has yielded unrealistic values, which were uninformative to TMDD model, causing nonconvergence during the optimization. In this work, the R0 of ALK1 in monkey was conveniently estimated as a parameter, avoiding the need to experimentally determine this value. Whether this value reflects the “true” target concentration is certainly a valid question. However, it remains useful to understand the drug exposure relative to this model-estimated target level. For example, as can be seen from Fig. 3, concentrations of PF-03446962 at or near the R0 of 1.74 nM occur at the rapid phase of the elimination. In addition, based on the figure, an exposure of at least 10-fold above the R0 is needed to be in the longer phase of the elimination.
On the basis of these results, it seems that kint and kdeg contributed little to the difference in the PK of PF-03446962 between monkey and human. From the FACS results, the internalization rates of PF-03446962-ALK1 complex in monkey and human endothelial cells were similar (4.10 day−1 for monkey and 4.37 day−1 for human). In addition, because there currently is no literature reported value for the turnover rate of the ALK1 receptor in monkeys, the value of kdeg was assumed to be the same between the two species. Thus, the parameters that may largely contribute to the species difference in the PK of PF-03446962 may be the physiological parameters (kel, k12, k21) and binding (kon, koff) and receptor abundance (R0) parameters. Beyond PF-03446962, it might be valuable to apply this approach to tease out important parameters governing the interspecies differences in the PK of a range of monoclonal antibodies exhibiting TMDD.
In summary, we successfully predicted the human PK of a monoclonal antibody, PF-03446962, exhibiting TMDD from preclinical data. Unlike empirical approaches such as simple allometric scaling, which are generally not applicable for compounds exhibiting nonlinear PK, this approach incorporates mechanistic parameters relating to target kinetics to inform the model-based translation. The mechanistic approach has greater simulative power relative to empirical approaches and, thus, may be more preferentially used to inform the clinical development and continued preclinical evaluation of the antibodies exhibiting TMDD.
Participated in research design: Luu, Hu-Lowe, and Kraynov.
Conducted experiments: Bergqvist and Chen.
Contributed new reagents or analytic tools: Bergqvist and Chen.
Performed data analysis: Luu, Bergqvist, and Chen.
Wrote or contributed to the writing of the manuscript: Luu, Bergqvist, Chen, and Kraynov.
We thank Dr. Erjian Wang for gathering the clinical data to validate the human PK prediction portion of the manuscript and Dr. Scott Fountain for supporting the overall publication effort.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- monoclonal antibody
- antidrug antibodies
- phosphate-buffered saline
- quality control
- surface plasmon resonance
- target-mediated drug disposition
- fragment antigen-binding
- activin receptor-like kinase 1
- extracellular domain
- human umbilical vein endothelial cells
- monkey endothelial cells
- sodium azide
- fluorescence-activated cell sorting
- noncompartmental analysis
- area under the curve
- between-subject variability.
- Received January 15, 2012.
- Accepted March 12, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics