Application of Pharmacokinetic/Pharmacodynamic Modeling to Bridge Mouse Antitumor Efficacy and Monkey Toxicology Data for Determining the Therapeutic Index of an Interleukin-10 Fc Fusion Protein.

Pharmacokinetic/pharmacodynamic (PK/PD) modeling was performed to quantitatively integrate preclinical pharmacology and toxicology data for determining the therapeutic index (TI) of an interleukin-10 (IL-10) fragment crystallizable (Fc) fusion protein. Mouse Fc fused with mouse IL-10 (mFc-mIL-10) was studied in mice for antitumor efficacy, and the elevation of interleukin-18 (IL-18) was examined as a PD biomarker. The in vivo mFc-mIL-10 EC50 for the IL-18 induction was estimated to be 2.4 nM, similar to the in vitro receptor binding affinity (Kd) of 3.2 nM. The IL-18 induction was further evaluated in cynomolgus monkeys, where the in vivo induction EC50 by a human IL-10 human Fc-fusion protein (hFc-hIL-10) was 0.08 nM vs. 0.3 nM measured as the in vitro Kd. The extent of the IL-18 induction correlated with mouse antitumor efficacy and was used to connect mouse efficacy to that in monkeys. The PD-based efficacious dose projected in monkeys was comparable to the results obtained using a PK-based method in which mouse efficacious exposure was targeted and corrected for affinity differences between the species. Furthermore, PK/PD relationships were developed for anemia and thrombocytopenia in monkeys treated with hFc-hIL-10, with thrombocytopenia predicted to be dose-limiting toxicity. Using quantitative pharmacology and toxicology information obtained through modeling work in the same species, the TI of hFc-hIL-10 in monkeys was determined to be 2.4 (vs. PD-based efficacy) and 1.2-3 (vs. PK-based efficacy), indicating a narrow safety margin. The model-based approaches were proven valuable to the developability assessment of the IL-10 Fc-fusion protein.

Target-Mediated Drug Disposition Affects the Pharmacokinetics of Interleukin-10 Fragment Crystallizable Fusion Proteins at Pharmacologically Active Doses.

Fragment crystallizable (Fc) fusion is commonly used for extending the half-life of biotherapeutics such as cytokines. In this work, we studied the pharmacokinetics of Fc-fused interleukin-10 (IL-10) proteins that exhibited potent antitumor activity in mouse syngeneic tumor models. At pharmacologically active doses of ≥0.1 mg/kg, both mouse Fc-mouse IL-10 and human Fc-human IL-10, constructed as the C terminus of the Fc domain fused with IL-10 via a glycine-serine polypeptide linker, exhibited nonlinear pharmacokinetics after intravenous administration to mice at the doses of 0.05, 0.5, and 5 mg/kg. With a nominal dose ratio of 1:10:100; the ratio of the area under the curve for mouse Fc-mouse IL-10 and human Fc-human IL-10 was 1:181:1830 and 1:75:633, respectively. In contrast, recombinant mouse or human IL-10 proteins exhibited linear pharmacokinetics in mice. Compartmental analysis, using the Michaelis-Menten equation with the in vitro IL-10 receptor alpha binding affinity inputted as the Km, unified the pharmacokinetic data across the dose range. Additionally, nontarget-mediated clearance estimated for fusion proteins was ∼200-fold slower than that for cytokines, causing the manifestation of target-mediated drug disposition (TMDD) in the fusion protein pharmacokinetics. The experimental data generated with a mouse IL-10 receptor alpha-blocking antibody and a human Fc-human IL-10 mutant with a reduced receptor binding affinity showed significant improvements in pharmacokinetics, supporting TMDD as the cause of nonlinearity. Target expression and its effect on pharmacokinetics must be determined when considering using Fc as a half-life extension strategy, and pharmacokinetic evaluations need to be performed at a range of doses covering pharmacological activity. SIGNIFICANCE STATEMENT: Target-mediated drug disposition can manifest to affect the pharmacokinetics of a fragment crystallizable (Fc)-fused cytokine when the nontarget-mediated clearance of the cytokine is decreased due to neonatal Fc receptor-mediated recycling and molecular weight increases that reduce the renal clearance. The phenomenon was demonstrated with interleukin-10 Fc-fusion proteins in mice at pharmacologically active doses. Future drug designs using Fc as a half-life extension approach for cytokines need to consider target expression and its effect on pharmacokinetics at relevant doses.

AggScore: Prediction of aggregation-prone regions in proteins based on the distribution of surface patches.

Protein aggregation is a phenomenon that has attracted considerable attention within the pharmaceutical industry from both a developability standpoint (to ensure stability of protein formulations) and from a research perspective for neurodegenerative diseases. Experimental identification of aggregation behavior in proteins can be expensive; and hence, the development of accurate computational approaches is crucial. The existing methods for predicting protein aggregation rely mostly on the primary sequence and are typically trained on amyloid-like proteins. However, the training bias toward beta amyloid peptides may worsen prediction accuracy of such models when applied to larger protein systems. Here, we present a novel algorithm to identify aggregation-prone regions in proteins termed "AggScore" that is based entirely on three-dimensional structure input. The method uses the distribution of hydrophobic and electrostatic patches on the surface of the protein, factoring in the intensity and relative orientation of the respective surface patches into an aggregation propensity function that has been trained on a benchmark set of 31 adnectin proteins. AggScore can accurately identify aggregation-prone regions in several well-studied proteins and also reliably predict changes in aggregation behavior upon residue mutation. The method is agnostic to an amyloid-specific aggregation context and thus may be applied to globular proteins, small peptides and antibodies.

Bead-extraction and heat-dissociation (BEHD): A novel way to overcome drug and matrix interference in immunogenicity testing.

Biological therapeutics are foreign antigens and can potentially induce immune response resulting in the formation of anti-drug antibodies (ADA), which in turn may lead to a wide range of side effects. Neutralizing Ab (NAb) is a subset of ADA that can bind to the pharmacological activity regions of therapeutic to inhibit or complete neutralize its clinical efficacy. A cell-based functional NAb assay is preferred to characterize its neutralization activity. However, cell-based NAb assays are often vulnerable to drug interference, as well as interference from numerous serum factors, including but not limited to growth factors and disease-related cytokines. Bead Extraction with Acid Dissociation (BEAD) has been successfully applied to remove circulating drug and/or other interfering factors from human serum samples, thereby enriching for ADA/NAb. However, the harsh acid used in the extraction procedure can cause irreversible denaturing of NAb and lead to underestimated NAb measurement. Herein we describe a new approach when acid-dissociation is not optimal for a PEGylated domain antibody (Ab). We further demonstrate that heating at 62 °C can not only dissociate drug/ADA/NAb immune complex but also selectively and irreversibly denature domain Ab drug due to much lower thermal stability of the domain Ab, when compared to that of full antibodies. The irreversible denaturing of the drug favors the formation of an immune complex between ADA/NAb and the added biotinylated drug thus increasing the recovery of ADA/NAb from samples. We call this new procedure Bead Extraction with Heat Dissociation (BEHD), which can potentially be applied to other NAb assays that have poor compatibility with acid dissociation.

The effects of intralaboratory modifications to media composition and cell source on the expression of pharmaceutically relevant transporters and metabolizing genes in the Caco-2 cell line.

Expression and function of drug transporters and drug-metabolizing enzymes (DMEs) in the gastrointestinal tract are critical attributes of intestinal physiology that influence the absorption of orally administered compounds. The purpose of this study was to examine the effects of media composition and cell source on mRNA expression and function of pharmaceutically relevant drug transporters and DMEs from two different sources of Caco-2 cells. Briefly, cells were cultured in either minimum essential medium alpha or Dulbecco's modified Eagle's medium. Total RNA was isolated from each experimental group, and mRNA expression was evaluated using quantitative reverse-transcriptase polymerase chain reaction arrays. Principal component analysis was used to analyze results, which indicated variable transporter and metabolic expression attributable to differences in media composition and cell source. In addition, transport properties of paracellular markers and proton-dependent oligopeptide transporter-mediated substrates across Caco-2 cell monolayers were assessed. Transport experiments demonstrated significant differences in both paracellular and transcellular permeation resultant from differences in media composition and cell source. These studies support previous findings that media composition and cell source may significantly impact expressional and functional characteristics of Caco-2 cells. Standardization of culture-related methodology may reduce variability associated with Caco-2 cells, enabling more meaningful intralaboratory and interlaboratory data comparisons.

The effects of media on pharmaceutically relevant transporters in the human HT-29 adenocarcinoma cell line: does culture media need to be controlled?

The HT-29 cell line forms a confluent monolayer with tight junctions, but displays different phenotypes when cultured for 21 days in galactose-supplemented media (differentiated) versus glucose-supplemented media (dedifferentiated). This study is aimed at elucidating how media differences might affect selected drug transporter expression and peptide-based substrate transport toward reducing this variability. A vial of HT-29 cells was amplified and cultured over several passages in four different mediums (American Type Culture Collection recommended McCoy's 5A versus Dulbecco's modified Eagle's media containing glucose, galactose, or neither carbohydrate) with normal supplementation. Transporter mRNA expression was characterized at days 5 and 21 postseeding utilizing SABiosciences quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) drug transporter arrays. Transport studies using [H]histidine, [(3) H]glycylsarcosine, [(3) H]valacyclovir, and [(3) H]carnosine were performed to assess the functional effects of oligopeptide transporter expression changes in HT-29 cells grown in each media. qRT-PCR arrays illustrated variable, media-dependent transporter expression between both the initial and differentiated time points. Permeability studies illustrated considerable media-dependent differences in both paracellular and transcellular substrate fluxes. The results demonstrate that these cells exhibit differing monolayer characteristics and genotypic/phenotypic profile properties when cultured under different media. The results suggest a need for standardization of culture methodologies for reducing inter- and intralaboratory variability.

THE EVALUATION OF PEPTIDE/HISTIDINE TRANSPORTER 1 (PHT1) FUNCTION: UPTAKE KINETICS UTILIZING A COS-7 STABLY TRANSFECTED CELL LINE.

There have been relatively few studies focused on the proton-dependent oligopeptide transporter (POT) superfamily member, Peptide/Histidine Transporter 1 (PHT1), with respect to its contribution to the ADME of peptides and peptide-based drugs. These studies were conducted to determine hPHT1-mediated, H+-dependent uptake kinetics of histidine, carnosine, Gly-Sar and valacyclovir in stably transfected hPHT1-COS-7 cells comparative to kinetics determined in an empty vector (Mock) stably transfected cell line. The results suggest that Gly-Sar appears to be a substrate for PHT1 based on efflux from the stably transfected hPHT1 COS-7 cells. Histidine and Gly-Sar concentration- and time-dependent studies suggest mixed-uptake kinetics. These studies suggest that stably transfected hPHT1-COS-7 cells exhibit different uptake kinetics than those observed in our previous studies and illustrate the requirement for experiments to delineate the physiological role of hPHT1.

ABC and SLC transporter expression and proton oligopeptide transporter (POT) mediated permeation across the human blood--brain barrier cell line, hCMEC/D3 [corrected].

Initial studies indicate that the newly developed hCMEC/D3 cell line may prove to be a useful model for studying the physiology of the human blood-brain barrier (BBB) endothelium. The purpose of this study was to assess the mRNA expression of several ABC and SLC transporters, with an emphasis on the proton-coupled oligopeptide transporter superfamily (POT) transporters in this immortalized BBB cell model. The transport kinetics of POT-substrates was also evaluated. The hCMEC/D3 cell line was maintained in a modified EGM-2 medium in collagenated culture flasks and passaged every 3-4 days at approximately 85%-95% confluence. Messenger RNA (mRNA) expression of a variety of ABC and SLC transporters was evaluated using qRT-PCR arrays, while additional qRT-PCR primers were designed to assess the expression of POT members. The transport kinetics of mannitol and urea were utilized to quantitatively estimate the intercellular pore radius, while POT substrate transport was also determined to assess the suitability of the cell model from a drug screening perspective. Optimization of the cell line was attempted by culturing with on laminin and fibronectin enhanced collagen and in the presence of excess Ca(2+). hCMEC/D3 cells express both hPHT1 and hPHT2, while little to no expression of either hPepT1 or hPepT2 was observed. The relative expression of other ABC and SLC transporters is discussed. While POT substrate transport does suggest suitability for BBB drug permeation screening, the relative intercellular pore radius was estimated at 19 A, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. These studies characterized this relevant human hCMEC/D3 BBB cell line with respect to both the relative mRNA expression of various ABC and SLC transporters and its potential utility as an in vitro screening tool for brain permeation. Additional studies are required to adequately determine the potential to establish an in vivo correlation.

The effects of excipients on transporter mediated absorption.

Traditionally most pharmaceutical excipients used for peroral dosage forms have been considered to be inert, although they have been known to play an important role in governing the release of the active pharmaceutical ingredient (API) required for the desired therapeutic effect. Of considerable interest is the emerging data demonstrating that many of these "inert" excipients may produce subtle changes that could directly or indirectly alter the activity of membrane-spanning proteins such as transporters. In this way, excipients could be altering the overall ADMET properties of an incorporated drug thereby affecting its intended therapeutic efficacy and/or enhancing adverse side effects. Therefore, given this recent evidence, it seems necessary to review what has been reported in the literature on interactions of excipients with human physiological entities, particularly transporters. As of today, safety/toxicity evaluations are typically based on the appearance of gross morphological changes rather than the effects on a cellular level, the ability of excipients in modifying the pharmacological activity of an active drug could lead to toxicity evaluation in routine for each additive used in oral formulations. Further knowledge on this subject will enable formulators to make more rational decisions in dosage form design and will help answer the question of whether certain excipients should be considered active pharmaceutical components of formulations.

The pharmacodynamic effects of sirolimus and sirolimus-calcineurin inhibitor combinations on macrophage scavenger and nuclear hormone receptors.

BACKGROUND: Sirolimus (SIR) alone or in combination with cyclosporine (CsA) or tacrolimus (TAC) are used in solid organ transplantation, but uncertainty remains regarding their respective atherogenic potentials. METHODS: THP-1 cells were cultured as macrophages and then treated with plasma trough and peak concentration doses of SIR, SIR/CsA or SIR/TAC to assess the time- and dose-dependent mRNA or protein expression of selected atherogenic genes. The selected atherogenic genes included: the macrophage scavenger receptors (MSRs) CD36, CD68, scavenger receptor (SR)-A, SR-BII, and LOX-1; the nuclear hormone receptors peroxisome proliferator activated receptor gamma (PPARgamma) and liver-X-receptor alpha (LXRalpha); and the cholesterol efflux transporter (ABCA-1). RESULTS: SIR-mediated changes in mRNA included the upregulation of ABCA1, downregulation of CD68, SR-A and SR-BII, and concentration- and/or time-dependent effects on CD36, LOX-1, PPARgamma, and LXRalpha that did not translate into significant protein changes. With SIR/CsA, the protein expressions of PPARgamma and ABCA-1 were downregulated at 8 h. In contrast, with SIR/TAC, PPARgamma, and ABCA-1 protein expressions were upregulated at 8 h. CONCLUSIONS: Combination results differed from findings with SIR alone, supporting the observed clinical phenotype with calcineurin inhibitors. These findings may provide a rationale for the development of novel drug delivery strategies to mitigate adverse pharmacodynamic responses.