Joseph A. Ware

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.

Drug-drug interactions mediated through P-glycoprotein: clinical relevance and in vitro-in vivo correlation using digoxin as a probe drug.

The clinical pharmacokinetics and in vitro inhibition of digoxin were examined to predict the P‐glycoprotein (P‐gp) component of drug–drug interactions. Coadministered drugs (co‐meds) in clinical trials (N = 123) resulted in a small, ≤100% increase in digoxin pharmacokinetics. Digoxin is likely to show the highest perturbation, via inhibition of P‐gp, because of the absence of metabolic clearance. In vitro inhibitory potency data (concentration of inhibitor to inhibit 50% P‐gp activity; IC50) were generated using Caco‐2 cells for 19 P‐gp inhibitors. Maximum steady‐state inhibitor systemic concentration [I], [I]/IC50 ratios, hypothetical gut concentration ([I2], dose/250 ml), and [I2]/IC50 ratios were calculated to simulate systemic and gut‐based interactions and were compared with peak plasma concentration (Cmax),i,ss/Cmax, ss and area under the curve (AUC)i/AUC ratios from the clinical trials. [I]/IC50 < 0.1 shows high false‐negative rates (24% AUC, 41% Cmax); however, to a limited extent, [I2]/IC50 < 10 is predictive of negative digoxin interaction for AUC, and [I]/IC50 > 0.1 is predictive of clinical digoxin interactions (AUC and Cmax).

Pharmacogenetic Characterization of Sulfasalazine Disposition Based on NAT2 and ABCG2 (BCRP) Gene Polymorphisms in Humans

The role of breast cancer resistance protein (BCRP), an efflux ABC transporter, in the pharmacokinetics of substrate drugs in humans is unknown. We investigated the impact of genetic polymorphisms of ABCG2 (421C>A) and NAT2 on the pharmacokinetics of sulfasalazine (SASP), a dual substrate, in 37 healthy volunteers, taking 2,000 mg of conventional SASP tablets. In ABCG2, SASP AUC0–48 of C/C, C/A, and A/A subjects was 171 ± 85, 330 ± 194, and 592 ± 275 μg h/ml, respectively, with significant differences among groups. In contrast, AUC0–48 of sulfapyridine (SP) tended to be lower in subjects with the ABCG2‐A allele as homozygosity. In NAT2, AUCAcSP/AUCSP was significantly higher in rapid than in intermediate and slow acetylator (SA) genotypes. We successfully described the pharmacokinetics of SASP, SP, and N ‐acetylsulfapyridine (AcSP) simultaneously by nonlinear mixed‐effects modeling (NONMEM) analysis with regard to both gene polymorphisms. The data indicate that SASP is a candidate probe of BCRP, particularly in its role in intestinal absorption.

Drug absorption interactions between oral targeted anticancer agents and PPIs: is pH-dependent solubility the Achilles heel of targeted therapy?

A majority of the novel orally administered, molecularly targeted anticancer therapies are weak bases that exhibit pH‐dependent solubility, and suppression of gastric acidity with acid‐reducing agents could impair their absorption. In addition, a majority of cancer patients frequently take acid‐reducing agents to alleviate symptoms of gastroesophageal reflux disease, thereby raising the potential for a common but underappreciated drug–drug interaction (DDI) that could decrease the exposure of anticancer medication and result in subsequent failure of therapy. This article is a review of the available clinical literature describing the extent of the interaction between 15 orally administered, small‐molecule targeted anticancer therapies and acid‐reducing agents. The currently available clinical data suggest that the magnitude of this DDI is largest for compounds whose in vitro solubility varies over the pH range 1–4. This range represents the normal physiological gastric acidity (pH ~1) and gastric acidity while on an acid‐reducing agent (pH ~4).

First-in-Human Phase I Study of Pictilisib (GDC-0941), a Potent Pan–Class I Phosphatidylinositol-3-Kinase (PI3K) Inhibitor, in Patients with Advanced Solid Tumors

Purpose: This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal-tolerated dose (MTD), dose-limiting toxicities (DLT), pharmacokinetics, pharmacodynamics, and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent, and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). Patients and Methods: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily, initially on days 1 to 21 every 28 days and later, using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated 18F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. Results: Pictilisib was well tolerated. The most common toxicities were grade 1–2 nausea, rash, and fatigue, whereas the DLT was grade 3 maculopapular rash (450 mg, 2 of 3 patients; 330 mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC >20 h·μmol/L. Significant increase in plasma insulin and glucose levels, and >25% decrease in 18F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF–mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively. Conclusion: Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily. Clin Cancer Res; 21(1); 77–86. ©2014 AACR.

Breast cancer resistance protein (ABCG2) and drug disposition : intestinal expression, polymorphisms and sulfasalazine as an in vivo probe

Breast cancer resistance protein (BCRP) is an efflux transporter expressed in tissues that act as barriers to drug entry. Given that single nucleotide polymorphisms (SNPs) in the ABCG2 gene encoding BCRP are common, the possibility exists that these genetic variants may be a determinant of interindividual variability in drug response. The objective of this study is to confirm the human BCRP-mediated transport of sulfasalazine in vitro, evaluate interindividual variation in BCRP expression in human intestine and to determine the role of ABCG2 SNPs to drug disposition in healthy patients using sulfasalazine as a novel in vivo probe. To evaluate these objectives, pinch biopsies were obtained from 18 patients undergoing esophagogastro–duodenoscopy or colonoscopy for determination of BCRP expression in relation to genotype. Wild-type and variant BCRP were expressed in a heterologous expression system to evaluate the effect of SNPs on cell-surface trafficking. A total of 17 healthy individuals participated in a clinical investigation to determine the effect of BCRP SNPs on sulfasalazine pharmacokinetics. In vitro, the cell surface protein expression of the common BCRP 421 C>A variant was reduced in comparison with the wild-type control. Intestinal biopsy samples revealed that BCRP protein and mRNA expression did not significantly differ between patients with 34GG/421CC versus patients with 34GG/421CA genotypes. Remarkably, in subjects with 34GG/421CA genotype, sulfasalazine area under the concentration–time curve was 2.4-fold greater compared with 34GG/421CC subjects (P<0.05). This study links commonly occurring SNPs in BCRP with significantly increased oral sulfasalazine plasma exposure in humans. Accordingly, sulfasalazine may prove to have utility as in vivo probe for assessing the clinical impact of BCRP for the disposition and efficacy of drugs.

Targeted Disruption of Murine Organic Anion-Transporting Polypeptide 1b2 (oatp1b2/ Slco1b2) Significantly Alters Disposition of Prototypical Drug Substrates Pravastatin and Rifampin

Organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are widely acknowledged as important and rate-limiting to the hepatic uptake of many drugs in clinical use. Accordingly, to better understand the in vivo relevance of OATP1B transporters, targeted disruption of murine Slco1b2 gene was carried out. It is noteworthy that Slco1b2(-/-) mice were fertile, developed normally, and exhibited no overt phenotypic abnormalities. We confirmed the loss of Oatp1b2 expression in liver using real-time polymerase chain reaction, Western Blot analysis, and immunohistochemistry. Expression of Oatp1a4 and Oatp2b1 but not Oatp1a1 was greater in female Slco1b2(-/-) mice, but expression of other non-OATP transporters did not significantly differ between wild-type and Slco1b2(-/-) male mice. Total bilirubin level was elevated by 2-fold in the Slco1b2(-/-) mice despite the fact that liver enzymes ALT and AST were normal. Pharmacological characterization was carried out using two prototypical substrates of human OATP1B1 and -1B3, rifampin and pravastatin. After a single intravenous dose of rifampin (1 mg/kg), a 1.7-fold increase in plasma area under the concentration-time curve (AUC) was observed, whereas the liver-to-plasma ratio was reduced by 5-fold, and nearly 8-fold when assessed at steady-state conditions after 24 h of continuous subcutaneous infusion in Slco1b2(-/-) mice. Likewise, continuous subcutaneous infusion at low (8 μg/h) or high (32 μg/h) dose rates of pravastatin resulted in a 4-fold lower liver-plasma ratio in the in Slco1b2(-/-) mice. This is the first report of altered drug disposition profile in the Slco1b2 knockout mice and suggests the utility of this model for understanding the in vivo role of hepatic OATP transporters in drug disposition.

Curcumin inhibits the activity of ABCG2/BCRP1, a multidrug resistance-linked ABC drug transporter in mice.

PurposeTo evaluate the in vivo efficacy of curcumin as an inhibitor of the multidrug-resistance-linked ATP Binding Cassette (ABC) drug transporter, ABCG2.MethodsPhotoaffinity labeling with [125I]-iodoarylazidoprazosin was used to characterize the interaction of sulfasalazine, a substrate of the mouse ABCG2, with human ABCG2. In addition, the inhibitory effect of curcumin on ABCG2 was evaluated in brain capillaries from rats. Furthermore, the effect of curcumin on absorption of orally administered sulfasalazine in wild-type and abcg2−/− mice was also determined.ResultsSulfasalazine interacted at the drug-substrate site(s) of human ABCG2. Curcumin inhibited ABCG2 activity at nanomolar concentrations at the rat blood-brain barrier in the ex vivo assay. Based on studies in wild type and abcg2−/− mice, we observed that oral curcumin increased Cmax and relative bioavailability of sulfasalazine by selectively inhibiting ABCG2 function.ConclusionsThis study validates our previous in vitro results with human ABCG2 (Chearwae et al., Mol. Cancer Ther. 5:1995–2006, 2006) and provides the first in vivo evidence for the inhibition by curcumin of ABCG2-mediated efflux of sulfasalazine in mice. Based on these studies, we propose that non-toxic concentrations of curcumin may be used to enhance drug exposure when the rate-limiting step of drug absorption and/or tissue distribution is impacted by ABCG2.

A first-in-human phase I study to evaluate the pan-PI3K inhibitor GDC-0941 administered QD or BID in patients with advanced solid tumors.

3501 Background: The PI3K-PTEN-AKT signaling pathway is deregulated in a wide variety of cancers. GDC-0941 is a potent and selective oral inhibitor of the class I PI3K with 3 nM IC50 for the p110-alpha subunit in vitro and 28 nM IC50 in a cell-based pAKT assay and demonstrates broad activity in breast, ovarian, lung, and prostate cancer models. METHODS A Phase I dose escalation study using a 3+3 design was initiated in patients (pts) with solid tumors. GDC-0941 was given on d1, followed by 1 wk washout to study single-dose PK and PD markers. GDC-0941 was then administered qd on a 3 wk on, 1 wk off, schedule. Steady-state PK and PD were evaluated after 1 wk of continuous dosing. A separate concurrent dose-escalation arm with bid dosing was initiated after the third qd cohort. RESULTS Nineteen pts have been enrolled in 5 successive dose-escalation cohorts in the qd arm with dose levels up to 80 mg daily. Seven pts were enrolled in 2 cohorts in the bid arm at total daily doses of 60 and 80 mg. The most frequently reported drug-related AEs were Grade 1/2 nausea, fatigue, diarrhea, peripheral edema, and dysgeusia; no drug related grade >3 events have been reported. PK data suggest dose-proportional increases in Cmax and AUC. Potential signs of anti-tumor activity have been observed with a soft tissue sarcoma pt on-study for >176 days with stable disease (30 mg qd), an ovarian cancer pt with an on-study 2.8-fold decrease in CA-125 response to normal levels (30 mg bid) and a pt with endometrial cancer with a decrease in tumor FDG-PET uptake (80 mg qd). CONCLUSIONS GDC-0941 is generally well-tolerated with potential signs of anti-tumor activity. Preliminary PK data suggest dose-proportional increases in exposure over the dose levels evaluated. Dose-escalation on both the qd and bid schedules continues with updated data to be presented. [Table: see text].

Antibody-Mediated Inhibition of Fibroblast Growth Factor 19 Results in Increased Bile Acids Synthesis and Ileal Malabsorption of Bile Acids in Cynomolgus Monkeys

Fibroblast growth factor 19 (FGF19) represses cholesterol 7α-hydroxylase (Cyp7α1) and inhibits bile acid synthesis in vitro and in vivo. Previous studies have shown that anti-FGF19 antibody treatment reduces growth of colon tumor xenografts and prevents hepatocellular carcinomas in FGF19 transgenic mice and thus may be a useful cancer target. In a repeat dose safety study in cynomolgus monkeys, anti-FGF19 treatment (3-100 mg/kg) demonstrated dose-related liver toxicity accompanied by severe diarrhea and low food consumption. The mechanism of anti-FGF19 toxicity was investigated using in vitro and in vivo approaches. Our results show that anti-FGF19 antibody had no direct cytotoxic effect on monkey hepatocytes. Anti-FGF19 increased Cyp7α1, as expected, but also increased bile acid efflux transporter gene (bile salt export pump, multidrug resistant protein 2 [MRP2], and MRP3) expression and reduced sodium taurocholate cotransporting polypeptide and organic anion transporter 2 expression in liver tissues from treated monkeys and in primary hepatocytes. In addition, anti-FGF19 treatment increased solute transporter gene (ileal bile acid-binding protein, organic solute transporter α [OST-α], and OST-β) expression in ileal tissues from treated monkeys but not in Caco-2 cells. However, deoxycholic acid (a secondary bile acid) increased expression of FGF19 and these solute transporter genes in Caco-2 cells. Gas chromatography-mass spectrometry analysis of monkey feces showed an increase in total bile acids and cholic acid derivatives. These findings suggest that high doses of anti-FGF19 increase Cyp7α1 expression and bile acid synthesis and alter the expression of bile transporters in the liver resulting in enhanced bile acid efflux and reduced uptake. Increased bile acids alter expression of solute transporters in the ileum causing diarrhea and the enhanced enterohepatic recirculation of bile acids leading to liver toxicity.