Prediction of clinical pharmacokinetics of AMG 181, a human anti-α4β7 monoclonal antibody for treating inflammatory bowel diseases

The purpose of this study was to predict a safe starting dose of AMG 181, a human anti-α4β7 antibody for treating inflammatory bowel diseases, based on cynomolgus monkey pharmacokinetic (PK) and pharmacodynamic (PD) data. A two-compartment model with parallel linear and target-mediated drug disposition for AMG 181 PK in cynomolgus monkey was developed. The estimated parameters were allometrically scaled to predict human PK. An Emax PD model was used to relate AMG 181 concentration and free α4β7 receptor data in cynomolgus monkey. AMG 181 clinical doses were selected based on observed exposures at the no adverse effect level of 80 mg·kg−1 in monkeys, the predicted human exposures, and AMG 181 concentration expected to produce greater than 50% α4β7 receptor occupancy in humans. The predicted human AMG 181 clearance and central volume of distribution were 144 mL·day−1 and 2900 mL, respectively. The estimated EC50 for free α4β7 receptor was 14 ng·mL−1. At the 0.7 mg starting dose in humans, the predicted exposure margins were greater than 490,000 and AMG 181 concentrations were predicted to only briefly cover the free α4β7 receptor EC10. Predictions for both Cmax and AUC matched with those observed in the first-in-human study within the 7 mg subcutaneous to 420 mg intravenous dose range. The developed model aided in selection of a safe starting dose and a pharmacological relevant dose escalation strategy for testing of AMG 181 in humans. The clinically observed human AMG 181 PK data validated the modeling approach based on cynomolgus monkey data alone.


Introduction
Crohn's disease (CD) and ulcerative colitis (UC) are the two major types of inflammatory bowel diseases (IBD). Currently, anti-tumor necrosis factor agents (anti-TNFs) are the main class of approved biologics for treating CD and UC. However, there is a need for development of new therapeutics for those patients who do not respond or have developed tolerance to anti-TNFs after initial treatment. Therapies that block the influx of pro-inflammatory cells into the intestinal mucosa through integrinmediated leukocyte tethering, rolling, and arrest (Hynes 2002) have gained attention in recent years.
Natalizumab is a humanized immunoglobulin (Ig) G 4 antibody targeting a 4 and is thus antagonistic against both a 4 b 1 for treating relapsing and remitting forms of multiple sclerosis (Polman et al. 2006) and a 4 b 7 for treating CD (Sandborn et al. 2005). Natalizumab-mediated inhibition of trafficking of a 4 b 1 -expressing leukocyte to the central nervous system through pan-a 4 inhibition may be linked to progressive multifocal leukoencephalopathy (PML) caused by reactivation of latent human John Cunningham (JC) polyomavirus infection (Berger and Koralnik 2005). Additionally, natalizumab as well as vedolizumab (IgG 1 ) are humanized antibodies and have shown greater than 10% immunogenicity in IBD patients leading to diminishing clinical efficacy (Feagan et al. , 2008Sandborn et al. 2005;Parikh et al. 2011).
AMG 181, a human monoclonal IgG 2 antibody that binds specifically to the a 4 b 7 integrin, has been designed to reduce or eliminate the immunogenic response and also avoids targeting the a 4 b 1 -expressing leukocytes implicated in the occurrence of PML reported previously in natalizumab-treated patients. Comprehensive investigational results from AMG 181 in vitro pharmacology experiments and in vivo pharmacokinetic and toxicology studies have been published previously (Pan et al. 2013).
Several recent publications have summarized methods for the first-in-human (FIH) dose estimation (Gibbs 2010;Chen et al. 2012;Zou et al. 2012). Here, a quantitative translation of AMG 181 in vitro and in vivo pharmacology from cynomolgus monkeys to humans is presented that uses 70% and 90% levels of a 4 b 7 receptor occupancy to guide the selection of a safe starting dose and pharmacologically relevant single and multiple dose escalation schemes for Phase 1 clinical trials in healthy volunteers and IBD subjects (www.clinicaltrials.gov, study identifiers NCT01164904 and NCT01290042). Comparison with the clinical PK from the single-dose Phase 1 study (Pan et al. 2014) validated our modeling predictions.

Materials and Methods
In vitro pharmacology studies AMG 181 (~144 kD) was manufactured at Amgen Inc. (Thousand Oaks, CA) by expression in a Chinese hamster ovary (CHO) cell line (Hsu et al. 2010). Results from in vitro pharmacology studies including a 4 b 7 target receptor occupancy on CD4 + T-cell subsets, including central memory, effector memory, and na€ ıve T, have been reported previously (Pan et al. 2013).

In vivo studies in cynomolgus monkeys
Male and female cynomolgus monkeys (Macaca fascicularis), ranging from 2.1 to 6.4 kg, were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition (National Research Council US 2011). Animals were socially housed at an indoor, AAALAC, Intl-accredited facility in species-specific housing. All research protocols were approved by the Institutional Animal Care and Use Committee. Animals were fed a certified pelleted primate diet (PMI #5048, Richmond, IN) daily in amounts appropriate for the age and size of the animals, and had ad libitum access to water (municipality tap water processed through a reverse osmosis filter and passed through ultraviolet light treatment) via automatic watering system/water bottle. Animals were maintained on a 12:12 h light:dark cycle in rooms at 18-29°C (30-70% humidity) and had access to enrichment opportunities (small bits of fruit, cereal, or other treats). All animals were negative for simian retrovirus.
Cynomolgus monkeys were assigned to treatment groups for two single-dose PK/PD Studies A and C, a two-dose (2-weekly doses) toxicology Study B, a 3-month (13-weekly doses; Study D), and a 6-month (24-weekly doses; Study E) Good Laboratory Practice (GLP) toxicology study (Table 1). Pre-and postdose blood samples were collected for PK/PD and immunogenicity assessments. Postdose observations in toxicology studies were conducted to assess the safety of AMG 181. PK samples from the PK/PD studies and the non-GLP toxicology study were analyzed for AMG 181 using an electrochemiluminescence (ECL) immunoassay with a lower limit of quantification (LLOQ) of 2 ngÁmL À1 . The GLP toxicology study used an ECL immunoassay with a LLOQ of 20 ngÁmL À1 .
Serum samples were analyzed for anti-AMG 181-specific antibodies (antidrug antibodies; ADAs) using a validated immunoassay with a lower limit of reliable detection (LLRD) of 20 ngÁmL À1 rabbit anti-AMG 181 polyclonal antibody in neat pooled cynomolgus monkey serum. Samples with values greater than the predefined assay-specific cut point ranging from 1.08 to 1.14 based on the signal-to-noise ratio (fold difference in the anti-AMG 181-specific antibodies assay signal of samples or positive controls relative to the assay background assessed by the negative control) were defined as ADA positive.
AMG 181 PK data for modeling PK data were collected from a total of 119 monkeys (73 males and 46 females) actively treated with AMG 181 for up to 420 days after the first dose, with 86 receiving SC and 33 receiving IV dose(s). The median body weight of the monkeys was 2.8 kg (mean: 3.43 kg; range: 2.1-6.4 kg). In all five studies, anti-AMG 181 antibodies or immunogenic response was observed in a majority of the monkeys, except for those dosed 80 mgÁkg À1 SC or IV. In many cases, the presence of anti-AMG 181 antibodies clearly impacted the PK. Data point exclusion was based on a two-step process: In a first step, candidates for exclusion were identified based on ADA positivity. In the second step, only data points where PK was also visually impacted were excluded (Fig. S2). This approach resulted in removal of 120 out of the 1743 (6.88%) available PK data points. The remaining 1623 AMG 181 concentration-time values were used for compartmental PK modeling.
Compartmental modeling of AMG 181 PK data PK structural models Visual inspection of the individual cynomolgus monkey PK data showed an initial distribution phase after IV and a terminal nonlinear elimination phase after both IV and SC administration. Individual PK data were simultaneously fit to a two-compartment PK model with targetmediated drug disposition (TMDD) (Mager and Jusko 2001) using quasi-steady-state (QSS) approximation (Gibiansky et al. 2008) or quasi-equilibrium (QE) approximation (Mager and Krzyzanski 2005). A two-compartment PK model with parallel linear and nonlinear Michaelis-Menten (M-M) was also fitted, but this model was not found superior to the simplified TMDD models (data for  ), central volume of distribution (V c ), linear clearance (CL), the distribution rate constants between the central and peripheral compartments K 12 and K 21 , the total a 4 b 7 receptor concentration (R tot ), the internalization rate constant of drug-receptor complex (K int ), and the QSS constant K ss = (k int + k off )/k on , or the dissociation constant K d = k off /k on for QE model (Fig. 1). TMDD QSS/QE modeling was conducted on molar transformed AMG 181 concentration data with the free AMG 181 concentration (C f ) calculated using the equation shown in Figure 1. The K d was fixed at 0.013 nmol/L, the EC 50 value of AMG 181 binding to monkey primary T cells in vitro.

PK variability models
Values for interindividual variability (IIV) of K A , CL, V c , and R tot were estimated assuming lognormal distribution. The residual variability was assumed to have a mixture of proportional plus additive distribution.

Estimation method, diagnosis, and validation
PK datasets and results were constructed, tabulated, and plotted using S-PLUS â (Version 8.2, November 2010; TIBCO Software Inc., Palo Alto, CA) or SigmaPlot (Version 12.5; Systat Software, Inc., Chicago, IL, USA). Modeling of the monkey PK data and simulation of the human PK profiles were performed using nonlinear mixed-effects modeling using NONMEM â , Version 7.2 with gfortran FORTRAN compiler (Beal et al. 2009).
The first-order conditional estimation method with interaction (FOCE INTERACTION) was used to estimate the population PK parameters. Importance sampling method, with EONLY = 1 ("evaluation only"), was applied to facilitate obtaining the standard error of model parameters.
Model selection was guided by visual inspection of goodness-of-fit plots, parameter estimate precision/plausibility, and comparison of objective function values. All parameter estimates were reported with the relative standard error of the estimates (%SE). Internal model validation was carried out by visual predictive check (VPC) plots; four hundred datasets were simulated based on final parameter estimates and the estimated 10th, 50th, and 90th percentiles of the simulated AMG 181 concentration-time profiles were plotted against the observed data.

AMG 181 PD data and modeling
Study A was a dedicated PK/PD study in cynomolgus monkeys, where AMG 181 occupancy of free a 4 b 7 on CD4+ T cell and its subsets were evaluated ex vivo. Mean fluorescence intensity (MFI) readings of free a 4 b 7 and the time-matched AMG 181 concentrations in serum were used for curve fitting using an E max model: where the effect (E or MFI) was estimated through baseline (E 0 ), maximum effect (E max ), AMG 181 concentration at half-maximal effect (EC 50 ), and AMG 181 concentration (C). Subsequent calculations for EC 75 , EC 90 , and EC 99 and the AMG 181 concentrations at 75%, 90%, and 99% maximal effect, respectively, were carried out based on the E max model. Results from Studies B-E were not used for PD modeling due to high data variability and limited availability (especially GLP studies).

Allometric scaling and simulation of human PK
For the prediction and simulation of human PK, the structural PK models developed for cynomolgus monkeys (median observed body weight of 2.8 kg) were assumed to also describe AMG 181 disposition in humans (assumed median body weight of 70 kg) with parameters allometrically scaled (Dong et al. 2011). The AMG 181 CL and V c values in human were determined from those in cynomolgus monkey by scaling by body weight ratio with exponents of 0.75 and 1, respectively. The distribution rate constants (K 12 and K 21 ) were scaled with an exponent of À0.25. The K d constant was fixed at 0.031 nmol/L, the EC 50 value of AMG 181 in vitro binding potencies to human primary T cells. Other PK parameters including K A , F1, R tot , and K int were assumed to be equivalent for cynomolgus monkeys and humans.

Exposure margins and FIH dose selection
The exposure margins were calculated based on the observed AMG 181 C max (4850 lgÁmL À1 ) and area under the concentration-time curve (AUC, 485,000 hÁlgÁmL À1 ) values in cynomolgus monkeys at the no observed adverse effect level (NOAEL; Study D) of 80 mgÁkg À1 IV divided by the predicted human C max and AUC values.
AMG 181 doses for the FIH study were selected based on: the exposure margins; the duration of serum AMG 181 concentration above 50-90% a 4 b 7 receptor occupancy; and the maximum recommended starting dose (MRSD) in humans according to the United States Food and Drug Administration (US FDA) guidance (FDA 2005).

Comparison of predicted versus observed AMG 181 PK in humans
The predicted AMG 181 AUC and C max in humans were compared to the AMG 181 PK parameters observed in the FIH study with doses ranging from 7 mg SC to 420 mg IV. Data from the FIH study of AMG 181 have been reported separately (Pan et al. 2014).

Compartmental modeling of AMG 181 PK in cynomolgus monkeys
In the final model, the individual AMG 181 concentrationtime data for all cynomolgus monkeys from Studies A-E were simultaneously fit using a two-compartment TMDD QE PK model ( Fig. 1). This model performed well for single and multiple dose administration with the exception of lowest single-dose cohort (0.01 mgÁkg À1 IV), where the model underpredicted the observed concentrations (Fig. 2). The diagnostic plots presented in Figure 3 suggest that the PK model characterized the observed concentration-time data well. Some divergence from the line of identity was observed at higher concentrations in the DV versus PRED plot. This deviation may have been due, in part, to the attempt to reconcile the PK from the 80 mgÁkg À1 IV and SC doses, where SC dosing resulted in higher exposure compared to the IV dose (Fig. S1A). Table 2 summarizes the AMG 181 population parameters and IIV in cynomolgus monkeys estimated in the final model. The population PK parameter estimates as well as the IIVs were generally well estimated with relative standard error (%SE) of less than 30%. In Studies A, C, and D, where AMG 181 was administered both SC and IV, relative bioavailability after SC was estimated to be 80-96% via noncompartmental analysis. In our first PK modeling attempt, the SC bioavailability (F1) was initially estimated to be close to 1 and was subsequently set to 1.0 (or 100%) to stabilize the model and to provide a conservative approach when the same assumption was also applied in prediction of human PK. No IIV on F1 was estimated.
The estimated population central volume of distribution (V c ) was 116 mL, which is physiologically plausible for a typical 2.8 kg cynomolgus monkey (Davies and Morris 1993). Based on the typical PK parameters, the linear elimination half-life of AMG 181 was estimated to be 12 days. Since the target (a 4 b 7 receptor) concentrations were not available and few drug concentrations were measured at the terminal phase, R tot and K d could not be simultaneously estimated. Therefore, K d was fixed to 0.013 nmol/L, the EC 50 value of AMG 181 binding to a 4 b 7 receptors on monkey primary T cells in vitro, in the final model and R tot was estimated relative to this assumption (Table 2). Figure 4 shows the internal model validation carried out using visual predictive checks (VPC). With 400 datasets simulated based on final parameter estimates, the estimated medians and the two-sided 80% prediction intervals (10th, 50th, and 90th percentiles) of the simulated AMG 181 concentration-time profiles contained the observed PK data well, especially in light of the small sample sizes per dose group and over an 8000-fold dose range (0.01-80 mgÁkg À1 ).

Modeling of AMG 181 PD in cynomolgus monkeys
For Study A, individual observed free a 4 b 7 receptor on total CD4 + T cells (measured as MFI) versus AMG 181 concentration data is compared to the model fit in Figure 5. The estimated PD model parameters were 531 (MFI), 0.922, and 0.0140 AE 0.0082 lgÁmL À1 for E 0 , E max , and EC 50 , respectively. Based on the model, the calculated values for EC 75 , EC 90 , and EC 99 are 0.042, 0.126, and 1.38 lgÁmL À1 , respectively.
As a pharmacologic consequence of a 4 b 7 occupancy, counts of total CD4 + T cell and its subsets, including na€ ıve and central memory cell counts, were elevated due to the inhibition of their trafficking into the intestinal tissues. Because the CD4 + T cell and its subset count data were sparse and highly variable in all studies, PD modeling attempts using cell count measures were not successful: qualitative assessments have been reported elsewhere (Pan et al. 2013).

Allometric scaling, simulation, and dose selection in humans
The estimated AMG 181 PK parameters in humans, determined through allometric scaling from parameters determined in cynomolgus monkeys, are presented in Table 3. These PK parameters (e.g., CL and V c ) are physiologically reasonable for kinetics of monoclonal antibodies in human (Davies and Morris 1993). Using these parameters, the AMG 181 concentration-time profiles were simulated under various single SC or IV dosing regimens or multiple SC dosing regimens (Fig. 6). The EC 50 , EC 75 , EC 90 , and EC 99 values for AMG 181 occupancy of free a 4 b 7 receptors on CD4 + T cell in humans were assumed to be the same as those estimated in monkeys and are given as reference concentration levels for the pharmacologic effect in Figure 6A. These levels were used to assess the duration of human AMG 181 concentration over various target saturation levels in designing the subsequent FIH study.
Based on our simulations, the estimated C max at the starting dose of 0.7 mg SC would be associated with approximately 10% of the maximum PD effect (EC 10 ), while the 21, 70, 210, and 420 mg doses were predicted to cover EC 90 for approximately 2, 3, 5, and 6 months, respectively (Fig. 6A). All four multiple dosing regimens were predicted to maintain AMG 181 concentration above EC 90 for 3-8 months (Fig. 6B).
The model-predicted AMG 181 C max and AUC values in humans, based on the simulated PK profiles, are presented in Figure 6, and the estimated exposure margins relative to the NOAEL of 80 mgÁkg À1 in cynomolgus  Table 4. Based on these exposure ratios, a greater than 490,000-fold safety margin at the starting dose of 0.7 mg SC in healthy subjects was anticipated. This low starting dose of 0.7 mg was selected because it was predicted to only briefly cover the predicted EC 10  The MRSD was calculated to be 2.58 mgÁkg À1 based on the body surface scaling with exponent 0.67, with an average body weight of 3 kg for monkeys and 60 kg (per MRSD FDA guidance) for humans, a NOAEL of 80 mgÁkg À1 in monkeys, and a default safety factor of 10.

Comparison of predicted versus observed AMG 181 PK in humans
The observed AMG 181 C max and AUC inf values in the FIH study were well predicted based on scaling of cynomolgus monkey PK (Fig. 7). With the exception of lower doses (0.7 and 2.1 mg AMG 181), where C max and AUC inf were underpredicted, the observed human AMG 181 C max and AUC inf values were within twofold of the predicted values, consistent with what has previously been reported for allometric scaling of monoclonal antibody PK from cynomolgus monkey to human (Dong et al. 2011). Given the large exposure margins at the proposed FIH starting dose of 0.7 mg, this slight underprediction was not expected to have consequences for patient safety.   (14) CV, coefficient of variation; IIV, interindividual variability; NE, not estimated.

Discussion and Conclusions
AMG 181 is a human monoclonal antibody that specifically targets intestinal-homing T cells and has been tested for pharmacological activity in vitro as well as for safety and PK/PD in vivo in the pharmacologically relevant species M. fascicularis (cynomolgus monkey) (Pan et al. 2014). This report presents a quantitative translational approach for the selection of a safe starting dose and pharmacologically relevant single and multiple dose esca-    Table 3. Estimated human AMG 181 pharmacokinetic parameters. The PK parameters CL, V c , K 12 , and K 21 were allometric scaled from cynomolgus monkeys. F1, K d , K int , and R tot were assumed to be the same as the cynomolgus monkeys' parameters.
PK parameter (unit) Human estimate lation schemes for Phase 1 clinical trials in healthy volunteers and IBD subjects. Based on visual inspection of the PK profiles in monkeys, AMG 181 disposition was dose-and concentrationdependent with typical linear clearance dominating at higher concentrations and nonlinear clearance dominating at low concentrations (<1 lgÁmL À1 ,~7 nmol/L), likely due to saturable binding of AMG 181 to the target T-cell surface a 4 b 7 receptors. To describe the parallel linear and nonlinear disposition, a TMDD model (Mager and Jusko 2001) was utilized and all individual monkey AMG 181 concentration-time data were fitted simultaneously.
AMG 181 binds to a 4 b 7 receptor with high affinity with a dissociation constant (K D = K off /K on ) of 13 pmol* L À1 , suggesting that the drug-target association is faster than drug dissociation/distribution/elimination as well as elimination of the target and drug-target complex. In this case, the QSS approximation to TMDD, which assumes that drug-target complex rapidly approaches a quasisteady state, is reasonable (Gibiansky and Gibiansky 2009). To overcome the difficulties with model parameter identifiability during model development, QSS model was further simplified to a QE model with K ss = K d = k off / k on .
During the model development, fitting of an empirical two-compartment PK model with parallel linear and nonlinear Michaelis-Menten (M-M) (Dong et al. 2011) elimination was also attempted. The M-M model fit similarly well to the high concentration data as did the TMDD models, but underpredicted concentrations at the terminal elimination phase where AMG 181 concentration were <0.1 lg*mL À1 (Fig. S3A and B); consistent with what has been demonstrated by Gibiansky (2009), Yan et al. (2010). The exception to this was for the lowest dose (0.01 mgÁkg À1 IV) where, unexpectedly, the M-M model provided a better model fit than QE TMDD model. Three factors posed challenges for better estimation of the AMG 181 PK across all concentrations and dose routes. First, AMG 181 is a human antibody and would unavoidably be immunogenic in monkeys, especially at lower concentrations. Second, only a limited number of monkeys were assigned to the recovery phase of the GLP studies. Both factors reduced the potential amount of PK data available for modeling the terminal elimination phase. Third, the relatively higher exposures observed after SC administration of 80 mgÁkg À1 AMG 181 than after IV administration of the same dose (Studies B and D vs. Study D, Fig. 2B) was undoubtedly a challenge for the model to reconcile. While this could potentially by explained by PK variability and small sample sizes, the observed differences could also have stemmed from nonneutralizing AMG 181 immune complex formation, which might have served as a reservoir (Chirmule et al. 2012) for AMG 181 through gradual disassociation and slow release of AMG 181 during the terminal elimination phase. The estimated slow half-life of~32 days (= 0.693/ K int ) for a 4 b 7 receptor internalization may support this hypothesis and thus K int might be a measure of both target receptor internalization and immune complex elimination.
The immunogenic response against AMG 181 clearly impacted specific PK data points, as illustrated for two representative animals in Figure S2. For this reason, 120 ADA-positive concentration-time points (representing less than 7% of total data) were excluded from model. This approach is conservative from a safety perspective as it resulted in higher estimated exposures in monkey and consequently in human. Additionally, since AMG 181 is a human IgG2, it is anticipated to carry less immunogenic potential in humans. Indeed, no ADAs were reported in the Ph1a study (Pan et al. 2014). Dong et al. (2011) suggest that translation of nonlinear elimination to human might be improved by accounting for the between-species difference in target expression and binding. The lack of knowledge of a4b7 receptor abundance in cynomolgus monkeys and human necessitated the assumption that AMG 181 has the same in vivo pharmacology characteristics and a 4 b 7 receptor abundance in cynomolgus monkeys and humans. This assumption was justified based on similar in vitro binding to primary T cells and blocking of a 4 b 7 :MAdCAM-1 binding on primary T cells and T-cell adhesion for cynomolgus monkey and human (Pan et al. 2013). Additionally, potential differences are not expected to affect patient safety due to the large observed safety margins.
The 75% and 90% receptor occupancy levels for natalizumab (anti-a 4 ) and vedolizumab (anti-a 4 b 7 ) have been reported to be related to the clinical efficacy in multiple sclerosis (FDA 2003) and IBD (Feagan et al. , 2008. Therefore, the AMG 181 EC 75 and EC 90 values for a 4 b 7 receptor occupancy were used as guides to aid the selection of AMG 181 dosing regimens in UC and CD clinical trials: 7, 21, 70, or 210 mg SC once every 1, 2, 3, or Table 4. Predicted AMG 181 pharmacokinetic exposure parameters in humans based on the two-compartment model with target-mediated drug disposition (TMDD) and quasi-equilibrium (QE) approximation. 4 months, respectively, would maintain AMG 181 concentration above EC 90 within each dosing interval (Fig. 6B) and thus should be efficacious.
Analysis of the available clinical PK, safety and efficacy data from the ongoing studies have shown consistency with our prediction results as shown in Figure 7 (Pan et al. 2014). The predictions of C max and AUC were within twofold for single doses between 7 and 420 mg which is well accepted for large and small molecules (Hosea et al. 2009;Dong et al. 2011).
In conclusion, AMG 181 in vivo pharmacology from cynomolgus monkeys was successfully translated to humans using the described quantitative translation approach. The developed model was successfully employed to support the selection of a safe starting dose and pharmacologically as well as clinically relevant single and multiple dose escalation schemes for AMG 181 clinical trials in healthy volunteers and IBD subjects.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Diagnostic plots for the two-compartment TMDD QE PK model by study and cohort: (A) Observed versus population predicted concentrations. (B) Observed versus individual predicted concentrations. For study A, B, and C cohorts were combined for plotting purposes. Figure S2. Concentration-time profiles in two representative animals to illustrate the ADA effect on PK. Red symbols and lines represent the mean (AESD) of the same dose group, black symbols indicate data of an individual subject; ADA-positive data points are depicted by x. Figure S3. Simultaneous fit of the individual AMG 181 concentration-time data from all cynomolgus monkeys using the two-compartment MM PK model: (A) Single IV or SC dose and (B) 2-weekly, 13-weekly, or 24-weekly IV or SC doses. Symbols and dotted lines represent mean (AESD) observations. Solid lines represent the mean of model predicted individual concentration-time profiles in the respective cohort.