[Show abstract][Hide abstract] ABSTRACT: Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method for the determination of intracellular unbound drug concentrations in cultured cells, and apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected HEK293 cells. Intracellular space (ICS) was calculated by subtracting [3H]-Inulin distribution volume (extracellular space, ECS) from [14C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean±SEM; μL /106 cells) of monolayers of human embryonic kidney cells (HEK-EV) and cells overexpressing human organic cation transporter 1, OCT1 (HEK-OCT1), 1.21±0.07 and 1.25±0.06, respectively, were used to determine intracellular metformin concentrations. After incubation of cells with 5 µM metformin, the intracellular concentrations were 26.4±7.8 uM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K+ buffer (140 mM KCl) compared to normal K+ buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8±3.8 μM and 198.1±11.2 μM, respectively, p<0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiological values, the positively charged metformin accumulates to particularly high levels in ER and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells.
Full-text · Article · Dec 2015 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.
No preview · Article · Oct 2015 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Liver cirrhosis is a disease characterized by the loss of functional liver mass. Physiologically based pharmacokinetic (PBPK) modeling was applied to interpret and predict how the interplay among physiological changes in cirrhosis affects pharmacokinetics. However, previous PBPK models under cirrhotic conditions were developed for permeable cytochrome P450 substrates and do not directly apply to substrates of liver transporters. This study characterizes a PBPK model for liver transporter substrates in relation to the severity of liver cirrhosis. A published PBPK model structure for liver transporter substrates under healthy conditions and the physiological changes for cirrhosis are combined to simulate pharmacokinetics of liver transporter substrates in patients with mild and moderate cirrhosis. The simulated pharmacokinetics under liver cirrhosis reasonably approximate observations. This analysis includes meta-analysis to obtain system-dependent parameters in cirrhosis patients and a top-down approach to improve understanding of the effect of cirrhosis on transporter-mediated drug disposition under cirrhotic conditions.
Preview · Article · Jun 2015 · CPT: Pharmacometrics and Systems Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Volume of distribution is one of the most important pharmacokinetic properties of a drug candidate. It is a major determinant of half-life and dosing frequency of a drug. For a similar Log P, a basic molecule will tend to exhibit higher volume of distribution than a neutral molecule. Acids often exhibit low volumes of distribution. Although a design strategy against volume of distribution can be advantageous in achieving desirable dosing regimen, it must be well-directed in order to avoid detrimental effects to other important properties. Strategies to increase volume of distribution include adding lipophilicity and introducing basic functional groups in a way that does not increase metabolic clearance.
No preview · Article · Mar 2015 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Small vessel vasculitis is a life threatening condition and patients typically present with renal and pulmonary injury. Disease pathogenesis is associated with neutrophil accumulation, activation, and oxidative damage; the latter being driven in large part by myeloperoxidase (MPO) which generates hypochlorous acid (HOCl) among other oxidants. MPO has been associated with vasculitis, disseminated vascular inflammation typically involving pulmonary and renal microvasculature, often resulting in critical consequences. MPO contributes to vascular injury by: 1) catabolizing nitric oxide (NO), impairing vasomotor function, 2) causing oxidative damage to lipoproteins and endothelial cells leading to atherosclerosis, and 3) stimulating formation of neutrophil extracellular traps (NETs), resulting in vessel occlusion and thrombosis. Here we report a selective 2-thiouracil mechanism-based MPO inhibitor (2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide), PF-1355, and demonstrate that MPO is a critical mediator of vasculitis in mouse disease models. A pharmacokinetic-pharmacodynamic (PK-PD) response model of PF-1355 exposure in relation with MPO activity was assessed in mouse peritonitis. The contribution of MPO activity to vasculitis was then examined in an immune complex model of pulmonary disease. Oral administration of PF-1355 reduced plasma MPO activity, vascular edema, neutrophil recruitment, and elevated circulating cytokines. In a model of anti-Glomerular Basement Membrane (GBM) disease, formerly known as Good Pasture's, albuminuria and chronic renal dysfunction were completely suppressed by PF-1355 treatment. These studies showed that MPO activity is critical in driving immune-complex vasculitis and provides confidence in testing the hypothesis that MPO inhibition will provide benefit in treating human vasculitic diseases.
The American Society for Pharmacology and Experimental Therapeutics.
No preview · Article · Feb 2015 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: The application of modeling and simulation techniques is increasingly common in preclinical stages of the drug discovery and development process. A survey focusing on preclinical pharmacokinetic/pharmacodynamics (PK/PD) analysis was conducted across pharmaceutical companies that are members of the International Consortium for Quality and Innovation in Pharmaceutical Development. Based on survey responses, ~68% of companies use preclinical PK/PD analysis in all therapeutic areas indicating its broad application. An important goal of preclinical PK/PD analysis in all pharmaceutical companies is for the selection/optimization of doses and/or dose regimens, including prediction of human efficacious doses. Oncology was the therapeutic area with the most PK/PD analysis support and where it showed the most impact. Consistent use of more complex systems pharmacology models and hybrid physiologically based pharmacokinetic models with PK/PD components was less common compared to traditional PK/PD models. Preclinical PK/PD analysis is increasingly being included in regulatory submissions with ~73% of companies including these data to some degree. Most companies (~86%) have seen impact of preclinical PK/PD analyses in drug development. Finally, ~59% of pharmaceutical companies have plans to expand their PK/PD modeling groups over the next 2 years indicating continued growth. The growth of preclinical PK/PD modeling groups in pharmaceutical industry is necessary to establish required resources and skills to further expand use of preclinical PK/PD modeling in a meaningful and impactful manner.
No preview · Article · Jan 2015 · The AAPS Journal
[Show abstract][Hide abstract] ABSTRACT: Physiologically based pharmacokinetic (PBPK) models are increasingly being used to provide human pharmacokinetic (PK) predictions for organic anion-transporting polypeptide (OATP) substrates based on in vitro assay data. As a natural extension in the application of these models, in this study, we incorporated in vitro information of three major OATP1B1 genetic variants into a previously reported PBPK model to predict the impact of OATP1B1 polymorphisms on human PK. Using pravastatin and rosuvastatin as examples, we showed that the predicted plasma concentration-time profiles in groups carrying different OATP1B1 genetic variants reasonably matched the clinical observations from multiple studies. This modeling and simulation approach may aid decision making in early pharmaceutical research and development as well as patient-specific dose adjustment in clinical practice.
Preview · Article · Dec 2014 · CPT: Pharmacometrics and Systems Pharmacology
[Show abstract][Hide abstract] ABSTRACT: A previously developed physiologically based pharmacokinetic (PBPK) model for hepatic transporter substrates was extended to an organic anion transporting polypeptide (OATP) substrate, telmisartan. Predictions utilized in vitro data from sandwich culture human hepatocyte (SCHH) and human liver microsome (HLM) assays. We have developed a novel method to calibrate partition coefficients (Kps) between non-liver tissues and plasma based on published human positron emission tomography (PET) data in order to decrease the uncertainty in tissue distribution introduced by in silico predicted Kps. With in vitro data-predicted hepatic clearances, published empirical scaling factors (SFs), and PET-calibrated Kps, the model could accurately recapitulate telmisartan PK behavior before 2.5 hours. Reasonable predictions also depend on having a model structure that can adequately describe the drug disposition pathways. We showed that the elimination phase (2.5 to 12 hours) of telmisartan PK could be more accurately recapitulated when enterohepatic recirculation of parent compound derived from intestinal deconjugation of glucuronide metabolite was incorporated into the model. This study demonstrated the usefulness of the previously proposed PBPK modeling approach for purely predictive IV PK simulation and identified additional biological processes that can be important in prediction.
Preview · Article · Aug 2014 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: In this work, we leverage a mathematical model of the underlying physiochemical properties of tissues and physicochemical properties of molecules in order to support the development of hepatoselective glucokinase activators. Passive distribution is modeled via a Fick-Nernst-Planck approach, using in vitro experimental data to estimate the permeability of both ionized and neutral species. The model accounts for pH and electrochemical potential across cellular membranes, ionization according to Henderson-Hasselbalch, passive permeation of the neutral species using Fick's law, and passive permeation of the ionized species using the Nernst-Planck equation. The mathematical model of the physiochemical system allows derivation of a single set of parameters governing the distribution of drug molecules across multiple conditions both in vitro and in vivo. A case study using this approach in the development of hepatoselective glucokinase activators (GKA) via Organic Anion Transporting Polypeptide (OATP) mediated hepatic uptake and impaired passive distribution to the pancreas is described. The results for these molecules indicate the permeability penalty of the ionized form is offset by its relative abundance, leading to passive pancreatic exclusion according to the Nernst-Planck extension of Fickian passive permeation. Generally, this model serves as a useful construct for drug discovery scientists to understand subcellular exposure of acids or bases using specific physiochemical properties.
Full-text · Article · Jul 2014 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: Physiologically based pharmacokinetic (PBPK) models provide a framework useful for generating credible human pharmacokinetic predictions from data available at the earliest, preclinical stages of pharmaceutical research. With this approach, the pharmacokinetic implications of in vitro data are contextualized via scaling according to independent physiological information. However, in many cases these models also require model-based estimation of additional empirical scaling factors (SFs) in order to accurately recapitulate known human pharmacokinetic behavior. While this practice clearly improves data characterization, the introduction of empirically derived SFs may belie the extrapolative power commonly attributed to PBPK. This is particularly true when such SFs are compound dependent and/or when there are issues with regard to identifiability. As such, when empirically-derived SFs are necessary, a critical evaluation of parameter estimation and model structure are prudent. In this study, we applied a global optimization method to support model-based estimation of a single set of empirical SFs from intravenous clinical data on seven OATP substrates within the context of a previously published PBPK model as well as a revised PBPK model. The revised model with experimentally measured unbound fraction in liver, permeability between liver compartments, and permeability limited distribution to selected tissues improved data characterization. We utilized large-sample approximation and resampling approaches to estimate confidence intervals for the revised model in support of forward predictions that reflect the derived uncertainty. This work illustrates an objective approach to estimating empirically-derived SFs, systematically refining PBPK model performance and conveying the associated confidence in subsequent forward predictions.
No preview · Article · Apr 2014 · Journal of Pharmacokinetics and Biopharmaceutics
[Show abstract][Hide abstract] ABSTRACT: In this work, we provide a unified theoretical framework describing how drug molecules can permeate across membranes in neutral and ionized forms for unstirred in vitro systems. The analysis provides a self-consistent basis for the origin of the unstirred water layer (UWL) within the Nernst-Planck framework in the fully unstirred limit and further provides an accounting mechanism based simply on the bulk aqueous solvent diffusion constant of the drug molecule. Our framework makes no new assumptions about the underlying physics of molecular permeation. We hold simply that Nernst-Planck is a reasonable approximation at low concentrations and all physical systems must conserve mass. The applicability of the derived framework has been examined both with respect to the effect of stirring and externally applied voltages to measured permeability. The analysis contains data for 9 compounds extracted from the literature representing a range of permeabilities and aqueous diffusion coefficients. Applicability with respect to ionized permeation is examined using literature data for the permanently charged cation, crystal violet, providing a basis for the underlying mechanism for ionized drug permeation for this molecule as being due to mobile counter-current flow.
No preview · Article · Nov 2013 · European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences
[Show abstract][Hide abstract] ABSTRACT: The mineralocorticoid receptor (MR) antagonists PF-03882845 and eplerenone were evaluated for renal protection against aldosterone-mediated renal disease in uninephrectomized Sprague-Dawley (SD) rats maintained on a high salt diet and receiving aldosterone by osmotic mini-pump for 27 days. Serum K(+) and the urinary albumin to creatinine ratio (UACR) were assessed following 14 and 27 days of treatment. Aldosterone induced renal fibrosis as evidenced by increases in UACR, collagen IV staining in kidney cortex, and expression of pro-fibrotic genes relative to sham-operated controls not receiving aldosterone. While both PF-03882845 and eplerenone elevated serum K(+) levels with similar potencies, PF-03882845 was more potent than eplerenone in suppressing the rise in UACR. PF-03882845 prevented the increase in collagen IV staining at 5, 15 and 50 mg/kg BID while eplerenone was effective only at the highest dose tested (450 mg/kg BID). All doses of PF-03882845 suppressed aldosterone-induced increases in collagen IV, transforming growth factor-β 1 (Tgf-β 1), interleukin-6 (Il-6), intermolecular adhesion molecule-1 (Icam-1) and osteopontin gene expression in kidney while eplerenone was only effective at the highest dose. The therapeutic index (TI), calculated as the ratio of the EC50 for increasing serum K(+) to the EC50 for UACR lowering, was 83.8 for PF-03882845 and 1.47 for eplerenone. Thus, the TI of PF-03882845 against hyperkalemia was 57-fold superior to that of eplerenone indicating that PF-03882845 may present significantly less risk for hyperkalemia compared to eplerenone.
Full-text · Article · Oct 2013 · Frontiers in Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The discovery of antidiabetic agent ertugliflozin is described. The compound belongs to a new class of SGLT2 inhibitors bearing a dioxa-bicyclo[3.2.1]octane motif. This article describes the critical role that organic synthesis played in both influencing our medicinal chemistry strategy and speeding up the progression of our program.
Full-text · Article · Dec 2012 · Medicinal Chemistry Communication
[Show abstract][Hide abstract] ABSTRACT: Historically, recovery had been used to evaluate the data quality of plasma protein binding or tissue binding obtained from equilibrium dialysis assays. Low recovery was often indicative of high nonspecific binding, instability, or low solubility. This study showed that, when equilibrium was fully established in the dialysis assay, low recovery due to nonspecific binding had no impact on the determination of fraction unbound. The conclusion was supported by the principles of the equilibrium dialysis assay, experimental data, and mathematic simulations. The results suggested that the use of recovery as an acceptance criterion for the equilibrium dialysis assay in drug discovery was too restrictive, and introduced the additional burden of repeating studies unnecessarily.
No preview · Article · Mar 2012 · Journal of Pharmaceutical Sciences
[Show abstract][Hide abstract] ABSTRACT: Sodium-glucose co-transporter-2 (SGLT2) inhibitors are an emerging class of agents for use in the treatment of type 2 diabetes mellitus (T2DM). Inhibition of SGLT2 leads to improved glycemic control through increased urinary glucose excretion (UGE). In this study, a biologically based pharmacokinetic/pharmacodynamic (PK/PD) model of SGLT2 inhibitor-mediated UGE was developed. The derived model was used to characterize the acute PK/PD relationship of the SGLT2 inhibitor, dapagliflozin, in rats. The quantitative translational pharmacology of dapagliflozin was examined through both prospective simulation and direct modeling of mean literature data obtained for dapagliflozin in healthy subjects. Prospective simulations provided time courses of UGE that were of consistent shape to clinical observations, but were modestly biased toward under prediction. Direct modeling provided an improved characterization of the data and precise parameter estimates which were reasonably consistent with those predicted from preclinical data. Overall, these results indicate that the acute clinical pharmacology of SGLT2 inhibitors in healthy subjects can be reasonably well predicted from preclinical data through rational accounting of species differences in pharmacokinetics, physiology, and SGLT2 pharmacology. Because these data can be generated at the earliest stages of drug discovery, the proposed model is useful in the design and development of novel SGLT2 inhibitors. In addition, this model is expected to serve as a useful foundation for future efforts to understand and predict the effects of SGLT2 inhibition under chronic administration and in other patient populations.
[Show abstract][Hide abstract] ABSTRACT: Pharmacokinetic–pharmacodynamic (PK–PD) modeling greatly enables quantitative implementation of the “learn and confirm” paradigm across different stages of drug discovery and development. This work describes the successful prospective application of this concept in the discovery and early development of a novel κ-opioid receptor (KOR) antagonist, PF-04455242, where PK–PD understanding from preclinical biomarker responses enabled successful prediction of the clinical response in a proof of mechanism study. Preclinical data obtained in rats included time course measures of the KOR antagonist (PF-04455242), a KOR agonist (spiradoline), and a KOR-mediated biomarker response (prolactin secretion) in plasma. Clinical data included time course measures of PF-04455242 and prolactin in 24 healthy volunteers following a spiradoline challenge and single oral doses of PF-04455242 (18 and 30 mg). In both species, PF-04455242 successfully reversed spiradoline-induced prolactin response. A competitive antagonism model was developed and implemented within NONMEM to describe the effect of PF-04455242 on spiradoline-induced prolactin elevation in rats and humans. The PK–PD model-based estimate of K
for PF-04455242 in rats was 414 ng/mL. Accounting for species differences in unbound fraction, in vitro K
and brain penetration provided a predicted human K
of 44.4 ng/mL. This prediction was in good agreement with that estimated via the application of the proposed PK–PD model to the clinical data (i.e., 39.2 ng/mL). These results illustrate the utility of the proposed PK–PD model in supporting the quantitative translation of preclinical studies into an accurate clinical expectation. As such, the proposed PK–PD model is useful for supporting the design, selection, and early development of novel KOR antagonists.
[Show abstract][Hide abstract] ABSTRACT: A novel series of pyrazolo[1,5-a]pyrimidine derivatives was synthesized and evaluated as NPY Y1R antagonists. High binding affinity and selectivity were achieved with C3 trisubstituted aryl groups and C7 substituted 2-(tetrahydro-2H-pyran-4-ylamino)ethylamine moieties. Efforts to find close analogs with low plasma clearance in the rat and minimal p-glycoprotein efflux in the mouse were unsuccessful. Compound 2f (CP-671906) inhibited NPY-induced increases in blood pressure and food intake after iv and icv administration, respectively, in Sprague-Dawley (SD) rat models. Oral administration of compound 2f resulted in a modest, but statistically significant, reduction in food intake in a Wistar rat model of feeding behavior. Small inhibitions of food intake were also observed in an overnight fasting/refeeding model in SD rats. These data suggest a potential role for Y1R in the regulation of food intake in rodents.
No preview · Article · May 2011 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract] ABSTRACT: In this study, a pharmacodynamic model is developed, based on calcium–parathyroid hormone (PTH) homeostasis, which describes the concentration–effect relationship of a negative allosteric modulator of the calcium-sensing receptor (CaR) in rats. Plasma concentrations of drug and PTH were determined from plasma samples obtained via serial jugular vein sampling following single subcutaneous doses of 1, 5, 45, and 150 mg/kg to male Sprague–Dawley rats (n = 5/dose). Drug pharmacokinetics was described by a one-compartment model with first-order absorption and linear elimination. Concentration-time profiles of PTH were characterized using a model in which the compound allosterically modulates Ca+2 binding to the CaR that, in turn, modulates PTH through a precursor-pool indirect response model. Additionally, negative feedback was incorporated to account for tolerance observed at higher dose levels. Model fitting and parameter estimation were conducted using the maximum likelihood algorithm. The proposed model well characterized the data and provided compound specific estimates of the K
and cooperativity constant (α) of 1.47 ng/mL and 0.406, respectively. In addition, the estimated model parameters for PTH turnover were comparable to that previously reported. The final generalized model is capable of characterizing both PTH–Ca+2 homeostasis and the pharmacokinetics and pharmacodynamics associated with the negative allosteric CaR modulator. As such, the model provides a simple platform for analysis of drugs targeting the PTH–Ca+2 system.