Joseph W. Polli

Membrane transporters in drug development

Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.

An Unexpected Synergist Role of P-glycoprotein and Breast Cancer Resistance Protein on the CNS Penetration of the Tyrosine Kinase Inhibitor Lapatinib (GW572016)

1 An Unexpected Synergist Role of P-glycoprotein and Breast Cancer Resistance Protein on the CNS Penetration of the Tyrosine Kinase Inhibitor Lapatinib (GW572016). Joseph W. Polli, Katie L. Olson, John P. Chism, Lisa St. John-Williams, Russell L. Yeager, Sesha M. Woodard, Vicky Otto, Stephen Castellino, and Victoria E. Demby Preclinical Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Research Triangle Park, North Carolina (J.W.P., K.L.O, J.P.C., L.S.J.W., R.L.Y., S.M.W, V.O., S.C., V.E.D.) DMD Fast Forward. Published on December 4, 2008 as doi:10.1124/dmd.108.024646Lapatinib is a tyrosine kinase inhibitor approved for use in combination with capecitabine to treat advanced or metastatic breast cancers overexpressing human epidermal receptor 2 (ErbB2). This work investigated the role of P-glycoprotein (Pgp; the protein from the Mdr1a/b gene) and breast cancer resistance protein (Bcrp; the protein from the Bcrp1 gene) in modulating the central nervous system penetration of lapatinib at steady-state conditions in FVBn mice (wild-type), Mdr1a/b(–/–), Bcrp1(–/–), and Mdr1a/b(–/–)/Bcrp1(–/–) knockout mice. After an intravenous infusion of lapatinib for 24 h to a targeted steady-state plasma concentration of 700 ng/ml (0.3 mg/kg/h) or 7000 ng/ml (3 mg/kg/h), lapatinib brain-to-plasma ratios were approximately 3- to 4-fold higher in Mdr1a/b(–/–) knockout mice (ratio range from 0.09 to 0.16) compared with wild-type mice (ratio range from 0.03 to 0.04). There was no difference in the brain-to-plasma ratio in the Bcrp1(–/–) knockout mice (ratio range from 0.03 to 0.04) compared with wild-type mice. In contrast, Mdr1a/b(–/–)/Bcrp1(–/–) triple knockout mice had a 40-fold higher brain-to-plasma ratio (ratio range from 1.2 to 1.7), suggesting that Pgp and Bcrp work in concert to limit the brain-to-plasma ratio of lapatinib in mice. This finding has important potential consequences for the treatment of brain tumors in breast cancer patients treated with tyrosine kinase inhibitors as well as the basic understanding of ATP binding cassette transporters expressed in the blood-brain barrier on the central nervous system disposition of drugs.

Lapatinib Distribution in HER2 Overexpressing Experimental Brain Metastases of Breast Cancer

ABSTRACTPurposeLapatinib, a small molecule EGFR/HER2 inhibitor, partially inhibits the outgrowth of HER2+ brain metastases in preclinical models and in a subset of CNS lesions in clinical trials of HER2+ breast cancer. We investigated the ability of lapatinib to reach therapeutic concentrations in the CNS following 14C-lapatinib administration (100xa0mg/kg p.o. or 10xa0mg/kg, i.v.) to mice with MDA-MD-231-BR-HER2 brain metastases of breast cancer.MethodsDrug concentrations were determined at differing times after administration by quantitative autoradiography and chromatography.Results14C-Lapatinib concentration varied among brain metastases and correlated with altered blood-tumor barrier permeability. On average, brain metastasis concentration was 7–9-fold greater than surrounding brain tissue at 2 and 12xa0h after oral administration. However, average lapatinib concentration in brain metastases was still only 10–20% of those in peripheral metastases. Only in a subset of brain lesions (17%) did lapatinib concentration approach that of systemic metastases. No evidence was found of lapatinib resistance in tumor cells cultured ex vivo from treated brains.ConclusionsResults show that lapatinib distribution to brain metastases of breast cancer is partially restricted and blood-tumor barrier permeability is a key component of lapatinib therapeutic efficacy which varies between tumors.

In Vitro Investigations into the Roles of Drug Transporters and Metabolizing Enzymes in the Disposition and Drug Interactions of Dolutegravir, a HIV Integrase Inhibitor

Dolutegravir (DTG; S/GSK1349572) is a potent HIV-1 integrase inhibitor with a distinct resistance profile and a once-daily dose regimen that does not require pharmacokinetic boosting. This work investigated the in vitro drug transport and metabolism of DTG and assessed the potential for clinical drug-drug interactions. DTG is a substrate for the efflux transporters P-glycoprotein (Pgp) and human breast cancer resistance protein (BCRP). Its high intrinsic membrane permeability limits the impact these transporters have on DTG’s intestinal absorption. UDP-glucuronosyltransferase (UGT) 1A1 is the main enzyme responsible for the metabolism of DTG in vivo, with cytochrome P450 (P450) 3A4 being a notable pathway and UGT1A3 and UGT1A9 being only minor pathways. DTG demonstrated little or no inhibition (IC50 values > 30 μM) in vitro of the transporters Pgp, BCRP, multidrug resistance protein 2, organic anion transporting polypeptide 1B1/3, organic cation transporter (OCT) 1, or the drug metabolizing enzymes CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, UGT1A1, or 2B7. Further, DTG did not induce CYP1A2, 2B6, or 3A4 mRNA in vitro using human hepatocytes. DTG does inhibit the renal OCT2 (IC50 = 1.9 μM) transporter, which provides a mechanistic basis for the mild increases in serum creatinine observed in clinical studies. These in vitro studies demonstrate a low propensity for DTG to be a perpetrator of clinical drug interactions and provide a basis for predicting when other drugs could result in a drug interaction with DTG.

The Steady-State Michaelis–Menten Analysis of P-Glycoprotein Mediated Transport Through a Confluent Cell Monolayer Cannot Predict the Correct Michaelis Constant Km

PurposeTypically, the kinetics of membrane transport is analyzed using the steady-state Michaelis–Menten (or Eadie–Hofstee or Hanes) equations. This approach has been successful when the substrate is picked up from the aqueous phase, like a water-soluble enzyme, for which the Michaelis–Menten steady-state analysis was developed. For membrane transporters whose substrate resides in the lipid bilayer of the plasma membrane, like P-glycoprotein (P-gp), there has been no validation of the accuracy of the steady-state analysis because the elementary rate constants for transport were not known.MethodsRecently, we fitted the mass action elementary kinetic rate constants of P-gp transport of three different drugs through a confluent monolayer of MDCKII-hMDR1 cells. With these elementary rate constants in hand, we use computer simulations to assess the accuracy of the steady-state Michaelis–Menten parameters. This limits the simulation to parameter ranges known to be physiologically relevant.ResultsUsing over 2,300 different vectors of initial elementary parameters spanning the space bounded by the three drugs, which defines 2,300 “virtual substrates”, the concentrations of substrate transported were calculated and fitted to Eadie–Hofstee plots. Acceptable plots were obtained for 1,338 cases.ConclusionThe fitted steady-state Vmax values from the analysis correlated to within a factor of 2–3 with the values predicted from the elementary parameters. However, the fitted Km value could be generated by a wide range of underlying “molecular” Km values. This is because of the convolution of the drug passive permeability kinetics into the fitted Km. This implies that Km values measured in simpler systems, e.g., microsomes or proteoliposomes, even if accurate, would not predict the Km values for the confluent monolayer system or, by logical extension, in vivo. Reliable in vitro–in vivo extrapolation seems to require using the elementary rate constants rather than the Michaelis–Menten steady-state parameters.

Breast Cancer Resistance Protein (ABCG2) in Clinical Pharmacokinetics and Drug Interactions: Practical Recommendations for Clinical Victim and Perpetrator Drug-Drug Interaction Study Design

Breast cancer resistance protein (BCRP; ABCG2) limits intestinal absorption of low-permeability substrate drugs and mediates biliary excretion of drugs and metabolites. Based on clinical evidence of BCRP-mediated drug-drug interactions (DDIs) and the c.421C>A functional polymorphism affecting drug efficacy and safety, both the US Food and Drug Administration and European Medicines Agency recommend preclinical evaluation and, when appropriate, clinical assessment of BCRP-mediated DDIs. Although many BCRP substrates and inhibitors have been identified in vitro, clinical translation has been confounded by overlap with other transporters and metabolic enzymes. Regulatory recommendations for BCRP-mediated clinical DDI studies are challenging, as consensus is lacking on the choice of the most robust and specific human BCRP substrates and inhibitors and optimal study design. This review proposes a path forward based on a comprehensive analysis of available data. Oral sulfasalazine (1000 mg, immediate-release tablet) is the best available clinical substrate for intestinal BCRP, oral rosuvastatin (20 mg) for both intestinal and hepatic BCRP, and intravenous rosuvastatin (4 mg) for hepatic BCRP. Oral curcumin (2000 mg) and lapatinib (250 mg) are the best available clinical BCRP inhibitors. To interrogate the worst-case clinical BCRP DDI scenario, study subjects harboring the BCRP c.421C/C reference genotype are recommended. In addition, if sulfasalazine is selected as the substrate, subjects having the rapid acetylator phenotype are recommended. In the case of rosuvastatin, subjects with the organic anion–transporting polypeptide 1B1 c.521T/T genotype are recommended, together with monitoring of rosuvastatins cholesterol-lowering effect at baseline and DDI phase. A proof-of-concept clinical study is being planned by a collaborative consortium to evaluate the proposed BCRP DDI study design.

Understanding the transport properties of metabolites: case studies and considerations for drug development

Recent analyses demonstrated that metabolites are unlikely to contribute significantly to clinical inhibition of cytochrome P450 (P450)–mediated drug metabolism, and that only ∼2% of this type of drug interaction could not be predicted from the parent drug alone. Due to generally increased polarity and decreased permeability, metabolites are less likely to interact with P450s, but their disposition is instead more likely to involve transporters. This commentary presents case studies illustrating the potential importance of transporters as determinants of metabolite disposition, and as sites of drug interactions, which may alter drug efficacy and safety. Many of these examples are hydrophilic phase II conjugates involved in enterohepatic cycling, where modulation of transporter-dependent disposition may alter pharmacokinetics/pharmacodynamics. The case studies suggest that characterization of metabolite disposition, toxicology, and pharmacology should not focus solely on metabolites with appreciable systemic exposure, but should take into consideration major excretory metabolites. A more thorough understanding of metabolite (phase I and II; circulating and excreted) transport properties during drug development may provide an improved understanding of complex drug-drug interactions (DDIs) that can alter drug and/or metabolite systemic and intracellular exposure. Knowledge and capability gaps remain in clinical translation of in vitro and animal data regarding metabolite disposition. To this end, useful experimental and modeling approaches are highlighted. Application of these tools may lead to a better understanding of metabolite victim and perpetrator DDI potential, and ultimately the establishment of approaches for prediction of pharmacodynamic and toxicodynamic consequences of metabolite transport modulation.

Safety, pharmacokinetics and pharmacodynamics of remogliflozin etabonate, a novel SGLT2 inhibitor, and metformin when co-administered in subjects with type 2 diabetes mellitus

BackgroundThe sodium-dependent glucose co-transporter-2 (SGLT2) is expressed in absorptive epithelia of the renal tubules. Remogliflozin etabonate (RE) is the prodrug of remogliflozin, the active entity that inhibits SGLT2. An inhibitor of this pathway would enhance urinary glucose excretion (UGE), and potentially improve plasma glucose concentrations in diabetic patients. RE is intended for use for the treatment of type 2 diabetes mellitus (T2DM) as monotherapy and in combination with existing therapies. Metformin, a dimethylbiguanide, is an effective oral antihyperglycemic agent widely used for the treatment of T2DM.MethodsThis was a randomized, open-label, repeat-dose, two-sequence, cross-over study in 13 subjects with T2DM. Subjects were randomized to one of two treatment sequences in which they received either metformin alone, RE alone, or both over three, 3-day treatment periods separated by two non-treatment intervals of variable duration. On the evening before each treatment period, subjects were admitted and confined to the clinical site for the duration of the 3-day treatment period. Pharmacokinetic, pharmacodynamic (urine glucose and fasting plasma glucose), and safety (adverse events, vital signs, ECG, clinical laboratory parameters including lactic acid) assessments were performed at check-in and throughout the treatment periods. Pharmacokinetic sampling occurred on Day 3 of each treatment period.ResultsThis study demonstrated the lack of effect of RE on steady state metformin pharmacokinetics. Metformin did not affect the AUC of RE, remogliflozin, or its active metabolite, GSK279782, although Cmax values were slightly lower for remogliflozin and its metabolite after co-administration with metformin compared with administration of RE alone. Metformin did not alter the pharmacodynamic effects (UGE) of RE. Concomitant administration of metformin and RE was well tolerated with minimal hypoglycemia, no serious adverse events, and no increase in lactic acid.ConclusionsCoadministration of metformin and RE was well tolerated in this study. The results support continued development of RE as a treatment for T2DM.Trial registrationClinicalTrials.gov, NCT00376038

New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity

A scientific session entitled “New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity” focused on novel biomarkers to monitor kidney injury both preclinically and clinically, as well as providing mechanistic insight of the induced injury. Further, the role and impact of kidney membrane transporters in drug-induced kidney toxicity provided additional considerations when understanding kidney injury and the complex role of drug transporters in either sensitivity or resistance to drug-induced injury. The onset of nephropathy in diabetic patients was also presented, focusing on the quest to discover novel biomarkers that would differentiate diabetic populations more susceptible to nephropathy and renal failure. The session highlighted exciting new research areas and novel biomarkers that will enhance our understanding of kidney injury and provide tools for ensuring patient safety clinically.