Joseph P. Balthasar

Monoclonal Antibody Pharmacokinetics and Pharmacodynamics

More than 20 monoclonal antibodies have been approved as therapeutic drugs by the US Food and Drug Administration, and it is quite likely that the number of approved antibodies will double in the next 7–10 years. Antibody drugs show several desirable characteristics, including good solubility and stability, long persistence in the body, high selectivity and specificity, and low risk for bioconversion to toxic metabolites. However, many antibody drugs demonstrate attributes that complicate drug development, including very poor oral bioavailability, incomplete absorption following intramuscular or subcutaneous administration, nonlinear distribution, and nonlinear elimination. In addition, antibody administration often leads to an endogenous antibody response, which may alter the pharmacokinetics and efficacy of the therapeutic antibody. Antibodies have been developed for a wide range of disease conditions, with effects produced through a complex array of mechanisms. This article attempts to provide a brief overview of the main determinants of antibody pharmacokinetics and pharmacodynamics.

Pharmacodynamic modeling of chemotherapeutic effects: Application of a transit compartment model to characterize methotrexate effects in vitro

The time course of chemotherapeutic effect is often delayed relative to the time course of chemotherapeutic exposure. In many cases, this delay is difficult to characterize mathematically through the use of standard pharmacodynamic models. In the present work, we investigated the relationship between methotrexate (MTX) exposure and the time course of MTX effects on tumor cell growth in culture. Two cancer cell lines, Ehrlich ascites cells and sarcoma 180 cells, were exposed for 24 hours to MTX concentrations that varied more than 700-fold (0.19–140 μg/mL). Viable cells were counted on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 20, 22, and 24 for Ehrlich ascites cells and on days 1, 2, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, and 21 for sarcoma 180 cells, through the use of a tetrazolium assay. Although MTX was removed 24 hours after application, cell numbers reached nadir values more than 100 hours after MTX exposure. Data from each cell line were fitted to 3 pharmacodynamic models of chemotherapeutic cell killing: a cell cycle phase-specific model, a phase-nonspecific model, and a transit compartment model (based on the general model recently reported by Mager and Jusko, Clin Pharmacol Ther. 70:210–216, 2001). The transit compartment model captured the data much more accurately than the standard pharmacodynamic models, with correlation coefficients ranging from 0.86 to 0.999. This report shows the successful application of a transit compartment model for characterization of the complex time course of chemotherapeutic effects; such models may be very useful in the development of optimization strategies for cancer chemotherapy.

Evaluation of a Catenary PBPK Model for Predicting the In Vivo Disposition of mAbs Engineered for High-Affinity Binding to FcRn

Efforts have been made to extend the biological half-life of monoclonal antibody drugs (mAbs) by increasing the affinity of mAb–neonatal Fc receptor (FcRn) binding; however, mixed results have been reported. One possible reason for a poor correlation between the equilibrium affinity of mAb–FcRn binding and mAb systemic pharmacokinetics is that the timecourse of endosomal transit is too brief to allow binding to reach equilibrium. In the present work, a new physiologically based pharmacokinetic (PBPK) model has been developed to approximate the pH and time-dependent endosomal trafficking of immunoglobulin G (IgG). In this model, a catenary sub-model was utilized to describe the endosomal transit of IgG and the time dependencies in IgG–FcRn association and dissociation. The model performs as well as a previously published PBPK model, with assumed equilibrium kinetics of mAb–FcRn binding, in capturing the disposition profile of murine mAb from wild-type and FcRn knockout mice (catenary vs. equilibrium model: r2, 0.971 vs. 0.978; median prediction error, 3.38% vs. 3.79%). Compared to the PBPK model with equilibrium binding, the present catenary PBPK model predicts much more moderate changes in half-life with altered FcRn binding. For example, for a 10-fold increase in binding affinity, the catenary model predicts <2.5-fold change in half-life compared to an ∼8-fold increase as predicted by the equilibrium model; for a 100-fold increase in binding affinity, the catenary model predicts ∼7-fold change in half-life compared to >70-fold increase as predicted by the equilibrium model. Predictions of the new catenary PBPK model are more consistent with experimental results in the published literature.

High-Throughput Method Development for Sensitive, Accurate, and Reproducible Quantification of Therapeutic Monoclonal Antibodies in Tissues Using Orthogonal Array Optimization and Nano Liquid Chromatography/Selected Reaction Monitoring Mass Spectrometry

Although liquid chromatography/mass spectrometry using selected reaction monitoring (LC/SRM-MS) holds great promise for targeted protein analysis, quantification of therapeutic monoclonal antibody (mAb) in tissues represents a daunting challenge due to the extremely low tissue levels, complexity of tissue matrixes, and the absence of an efficient strategy to develop an optimal LC/SRM-MS method. Here we describe a high-throughput, streamlined strategy for the development of sensitive, selective, and reliable quantitative methods of mAb in tissue matrixes. A sensitive nano-LC/nanospray-MS method was employed to achieve a low lower limit of quantification (LOQ). For selection of signature peptides (SP), the SP candidates were identified by a high-resolution Orbitrap and then optimal SRM conditions for each candidate were obtained using a high-throughput, on-the-fly orthogonal array optimization (OAO) strategy, which is capable of optimizing a large set of SP candidates within a single nano-LC/SRM-MS run. Using the optimized conditions, the candidates were experimentally evaluated for both sensitivity and stability in the target matrixes, and SP selection was based on the results of the evaluation. Two unique SP, respectively from the light and heavy chain, were chosen for quantification of each mAb. The use of two SP improves the quantitative reliability by gauging possible degradation/modification of the mAb. Standard mAb proteins with verified purities were utilized for calibration curves, to prevent the quantitative biases that may otherwise occur when synthesized peptides were used as calibrators. We showed a proof of concept by rapidly developing sensitive nano-LC/SRM-MS methods for quantifying two mAb (8c2 and cT84.66) in multiple preclinical tissues. High sensitivity was achieved for both mAb with LOQ ranged from 0.156 to 0.312 μg/g across different tissues, and the overall procedure showed a wide dynamic range (≥500-fold) and good accuracy [relative error (RE) < 18.8%] and precision [interbatch relative standard deviation (RSD) < 18.1%, intrabatch RSD < 17.2%]. The quantitative method was applied to a comprehensive investigation of the steady-state tissue distribution of 8c2 in wild-type mice versus those deficient in FcRn α-chain, FcγIIb, and FcγRI/FcγRIII, following a chronic dosing regimen. This work represents the first extensive quantification of mAb in tissues by an LC/MS-based method.

Mechanistic considerations for the use of monoclonal antibodies for cancer therapy.

Since the approval of rituximab in 1997, monoclonal antibodies (mAbs) have become an increasingly important component of therapeutic regimens in oncology. The success of mAbs as a therapeutic class is a result of great strides that have been made in molecular biology and in biotechnology over the past several decades. Currently, there are 14 approved mAb products for oncology indications, and there are ten additional mAbs in late stages of clinical trials. Compared to traditional chemotherapeutic agents, mAbs have several advantages, including a long circulating half-life and high target specificity. Antibodies can serve as cytotoxic agents when administered alone, exerting a pharmacologic effect through several mechanisms involving the antigen binding (Fab) and/or Fc domains of the molecule, and mAbs may also be utilized as drug carriers, targeting a toxic payload to cancer cells. The extremely high affinity of mAbs for their targets, which is desirable with respect to pharmacodynamics (i.e., contributing to the high therapeutic selectivity of mAb), often leads to complex, non-linear, target-mediated pharmacokinetics. In this report, we summarize the pharmacokinetic and pharmacodynamics of mAbs that have been approved and of mAbs that are near approval for oncology indications, with particular focus on the molecular and cellular mechanisms responsible for their disposition and efficacy.

Sensitive high performance liquid chromatographic assay for assessment of doxorubicin pharmacokinetics in mouse plasma and tissues.

A sensitive high performance liquid chromatography method (HPLC) has been developed for the quantification of doxorubicin in mouse plasma and tissues. Samples of serum or tissue homogenates, 20 microl, were analyzed following a single step protein precipitation using perchloric acid (35%, v/v). Doxorubicin was separated from the internal standard, daunorubicin, on a Zorbax 300SB C(18) column at 35 degrees C. Mobile phase was comprised of acetonitrile and water (25:75) containing 0.1% triethylamine, and was adjusted to pH 3 with phosphoric acid. Peaks eluting from the column were detected with a fluorescence detector with excitation and emission wavelengths of 480 and 560 nm, respectively. Standard curves were linear in the range 5-1000 ng/ml, and correlation coefficients were typically greater than 0.999. Intra-assay recoveries ranged from 94.7 to 99.9%, and inter-assay recoveries were in the range of 95.2-101%. The associated coefficient of variation (CV) was less than 10% in all cases. The method was successfully applied to investigate doxorubicin plasma pharmacokinetics and tissue distribution in athymic Fox(nu) mice.

AAPS Workshop Report: Strategies to Address Therapeutic Protein–Drug Interactions during Clinical Development

Therapeutic proteins (TPs) are increasingly combined with small molecules and/or with other TPs. However preclinical tools and in vitro test systems for assessing drug interaction potential of TPs such as monoclonal antibodies, cytokines and cytokine modulators are limited. Published data suggests that clinically relevant TP-drug interactions (TP-DI) are likely from overlap in mechanisms of action, alteration in target and/or drug-disease interaction. Clinical drug interaction studies are not routinely conducted for TPs because of the logistical constraints in study design to address pharmacokinetic (PK)- and pharmacodynamic (PD)-based interactions. Different pharmaceutical companies have developed their respective question- and/or risk-based approaches for TP-DI based on the TP mechanism of action as well as patient population. During the workshop both company strategies and regulatory perspectives were discussed in depth using case studies; knowledge gaps and best practices were subsequently identified and discussed. Understanding the functional role of target, target expression and their downstream consequences were identified as important for assessing the potential for a TP-DI. Therefore, a question-and/or risk-based approach based upon the mechanism of action and patient population was proposed as a reasonable TP-DI strategy. This field continues to evolve as companies generate additional preclinical and clinical data to improve their understanding of possible mechanisms for drug interactions. Regulatory agencies are in the process of updating their recommendations to sponsors regarding the conduct of in vitro and in vivo interaction studies for new drug applications (NDAs) and biologics license applications (BLAs).

Nano-scale Liquid Chromatography/Mass Spectrometry and On-the-fly Orthogonal Array Optimization for Quantification of Therapeutic Monoclonal Antibodies and the Application in Preclinical Analysis

Therapeutic monoclonal antibodies (mAbs) constitute a group of highly effective agents for treating various refractory diseases. Nonetheless it is challenging to achieve selective and accurate quantification of mAb in pharmaceutical matrices, which is required by PK studies. Liquid chromatography/mass spectrometry under selected reaction monitoring mode (LC/SRM-MS) is emerging as an attractive alternative to immunoassays because of the high specificity and multiplexing capacity it provides, but may fall short in terms of sensitivity, reliability and quantitative accuracy. Moreover, the strategy for optimization of the MS conditions for many candidates of signature peptides (SP) and the selection of the optimal SP for quantification remains elusive. In this study, we employed a suite of technical advances to overcome these difficulties, which include: (i) a nano-LC/SRM-MS approach to achieve high analytical sensitivity, (ii) a high-resolution nano-LC/LTQ/Orbitrap for confident identification of candidate peptides, (iii) an on-the-fly orthogonal array optimization (OAO) method for the high-throughput, accurate and reproducible optimization for numerous candidate peptides in a single LC/MS run without using synthesized peptides, (iv) a comprehensive evaluation of stability of candidates in matrix using the optimized SRM parameters, (v) the use of two unique SP for quantification of one mAb to gauge possible degradation/modification in biological system and thus enhancing data reliability (e.g. rejection of data if the deviation between the two SP is greater than 25%) and (vi) the utilization of purified target protein as the calibrator to eliminate the risk of severe negative biases that could occur when a synthesized peptide is used as calibrator. To show a proof of concept, this strategy is applied in the quantification of cT84.66, a chimeric, anti-CEA antibody, in preclinical mouse models. A low detection limit of the mAb down to 3.2 ng/mL was achieved, which is substantially more sensitive than established immunoassay methods for anti-CEA antibodies. The quantitative method showed good linearity (within the range of 12.9 ng/mL to 32.3 μg/mL in plasma), accuracy and precision. Additionally, the ultra-low sample consumption (2 μL plasma per preparation) permits the acquisition of an entire set of time course data from the same mouse, which represents a prominent advantage for PK study using small-animal models. The developed method enabled an accurate PK investigation of cT84.66 in mice following intravenous and subcutaneous administrations at relatively low doses over an extended period of time. The strategy employed in this study can be easily adapted to the sensitive and accurate analysis of other mAb and therapeutic proteins.

Use of an Anti-Vascular Endothelial Growth Factor Antibody in a Pharmacokinetic Strategy to Increase the Efficacy of Intraperitoneal Chemotherapy

The efficacy of intraperitoneal chemotherapy for ovarian cancers is limited by poor penetration of drug into peritoneal tumors. Based on pharmacokinetic theory that suggests that penetration depth is primarily determined by the rate of drug removal via tumor capillaries, we have hypothesized that co-administration of antiangiogenic therapy will allow for decreased drug removal, increased drug concentrations in tumor, and increased efficacy of intraperitoneal chemotherapy. Pharmacokinetic modeling was conducted to simulate the effect of tumor blood flow on tumor concentrations of topotecan. Simulations predicted that tumor blood flow reductions, as potentially achieved by antiangiogenic therapy, would lead to substantial increases in tumor concentrations after intraperitoneal chemotherapy but would lead to a slight decrease after systemic chemotherapy. Pharmacokinetic studies performed using the A2780 xenograft tumor model showed that animals receiving combined intraperitoneal topotecan and an anti-vascular endothelial growth factor (VEGF) monoclonal antibody had ∼6.5-fold higher (p = 0.0015) tumor topotecan concentrations compared with animals receiving intraperitoneal topotecan alone, whereas there was no significant (p = 0.16) difference for systemic topotecan. Therapeutic studies conducted with two different drugs, topotecan and cisplatin, showed that animals receiving combined intraperitoneal chemotherapy and anti-VEGF therapy displayed superior survival relative to animals treated with chemotherapy alone (i.e., cisplatin or topotecan), anti-VEGF alone, or intravenous chemotherapy with concomitant anti-VEGF therapy. Combined intraperitoneal topotecan and anti-VEGF resulted in the complete cure of four of 11 mice. The proposed combination of antiangiogenic therapy and intraperitoneal chemotherapy, which was predicted to be beneficial by pharmacokinetic simulations, may provide substantial benefit to patients with peritoneal malignancies.

Application of Pharmacodynamic Modeling for Designing Time-Variant Dosing Regimens to Overcome Nitroglycerin Tolerance in Experimental Heart Failure

AbstractPurpose. Prolonged continuous administration of nitroglycerin (NTG) leads to hemodynamic tolerance. We used a previously developed pharmacokinetic-pharmacodynamic (PK/PD) model of NTG tolerance in experimental heart failure to test whether dosage regimens, designed from this model, may allow avoidance of tolerance development upon continuous NTG inftision. Methods. Simulation experiments (using ADAPT II) were performed to evolve a time-variant infusion regimen that would theoretically provide sustained hemodynamic effect (30% reduction in left ventricular end-diastolic pressure, LVEDP) throughout 10 hours of drug dosing. A computer controlled infusion pump was utilized to deliver this time-variant input. Infusion experiments were then conducted in CHF rats to challenge the predictability of the applied PK/PD model. Results. Simulations showed that exponentially increasing input functions provided more sustained LVEDP effects when compared to linear or hyperbolic input functions delivering the same total NTG dose. A computer-selected infusion regimen of 6.56e0.00156×minutes μg/min was anticipated to provide the desired hemodynamic profile in our animal model. Experiments conducted in rats with congestive heart failure (n = 4) confirmed the prediction of sustained hemodynamic effect without tolerance (28 ± 4% reduction in LVEDP at 10 hrs). Conclusions. These findings support the utility of our PK/PD model of NTG tolerance in predicting NTG action, and serve as an example of therapeutic optimization through PK/PD considerations.