Kirsten Messick

Role of P-Glycoprotein on the Brain Penetration and Brain Pharmacodynamic Activity of the MEK Inhibitor Cobimetinib

Cobimetinib is a MEK inhibitor currently in clinical trials as an anticancer agent. The objectives of this study were to determine in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess the implications of efflux on cobimetinib pharmacokinetics (PK), brain penetration, and target modulation. Cell lines transfected with P-gp or Bcrp1 established that cobimetinib was a substrate of P-gp but not a substrate of Bcrp1. In vivo, after intravenous and oral administration of cobimetinib to FVB (wild-type; WT), Mdr1a/b(-/-), Bcrp1 (-/-), and Mdr1a/b(-/-)/Bcrp(-/-) knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7 mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg); oral exposure was also similar between WT and KO animals. After an oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and Bcrp1(-/-) mice, respectively. In Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor), Kp increased to 11, 6, and 7, respectively. Increased brain exposure in Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO and elacridar treated mice was accompanied by up to ∼65% suppression of the target (pErk) in brain tissue, compared to WT mice. By MALDI imaging, the cobimetinib signal intensity was relatively high and was dispersed throughout the brain of Mdr1a/1b/Bcrp1(-/-) KO mice compared to low/undetectable signal intensity in WT mice. The efflux of cobimetinib by P-gp may have implications for the treatment of patients with brain tumors/metastases.

Prediction of Drug Distribution in Subcutaneous Xenografts of Human Tumor Cell Lines and Healthy Tissues in Mouse: Application of the Tissue Composition-Based Model to Antineoplastic Drugs

Advanced tissue composition-based models can predict the tissue-plasma partition coefficient (Kp ) values of drugs under in vivo conditions on the basis of in vitro and physiological input data. These models, however, focus on healthy tissues and do not incorporate data from tumors. The objective of this study was to apply a tissue composition-based model to six marketed antineoplastic drugs (docetaxel, DOC; doxorubicin, DOX; gemcitabine, GEM; methotrexate, MTX; topotecan, TOP; and fluorouracil, 5-FU) to predict their Kp values in three human tumor xenografts (HCT-116, H2122, and PC3) as well as in healthy tissues (brain, muscle, lung, and liver) under steady-state in vivo conditions in female NCR nude mice. The mechanisms considered in the tissue/tumor composition-based model are the binding to lipids and to plasma proteins, but the transporter effect was also investigated. The method consisted of analyzing tissue composition, performing the pharmacokinetics studies in mice, and calculating the corresponding in vivo Kp values. Analyses of tumor composition indicated that the tumor xenografts contained no or low amounts of common transporters by contrast to lipids. The predicted Kp values were within twofold and threefold of the measured values in 77% and 93% of cases, respectively. However, predictions for brain for each drug, for liver for MTX, and for each tumor xenograft for GEM were disparate from the observed values, and, therefore, not well served by the model. Overall, this study is the first step toward the mechanism-based prediction of Kp values of small molecules in healthy and tumor tissues in mouse when no transporter and permeation limitation effect is evident. This approach will be useful in selecting compounds based on their abilities to penetrate human cancer xenografts with a physiologically based pharmacokinetic (PBPK) model, thereby increasing therapeutic index for chemotherapy in oncology study.

Differential effects of Rifampin and Ketoconazole on the blood and liver concentration of atorvastatin in wild-type and Cyp3a and Oatp1a/b knockout mice.

Atorvastatin is eliminated by CYP3A4 which follows carrier-mediated uptake into hepatocytes by OATP1B1, OATP1B3, and OATP2B1. Multiple clinical studies demonstrated that OATP inhibition by rifampin had a greater impact on atorvastatin systemic concentration than itraconazole-mediated CYP3A4 inhibition. If it is assumed that the blood and hepatocyte compartments are differentiated by the concentration gradient that is established by OATPs, and if the rate of uptake into the hepatocyte is rate-determining to the elimination of atorvastatin from the body, then it is hypothesized that blood concentrations may not necessarily reflect liver concentrations. In wild-type mice, rifampin had a greater effect on systemic exposure of atorvastatin than ketoconazole, as the blood area under the blood concentration-time curve increased 7- and 2-fold, respectively. In contrast, liver concentrations were affected more by ketoconazole than by rifampin, as liver levels increased 21- and 4-fold, respectively. Similarly, in Cyp3a knockout animals, 39-fold increases in liver concentrations were observed despite insignificant changes in the blood area under the blood concentration-time curve. Interestingly, blood and liver levels in Oatp1a/b knockout animals were similar to wild types, suggesting that Oatp1a/b knockout may be necessary but not sufficient to completely describe atorvastatin uptake in mice. Data presented in this work indicate that there is a substantial drug interaction when blocking atorvastatin metabolism, but the effects of this interaction are predominantly manifested in the liver and may not be captured when monitoring changes in the systemic circulation. Consequently, there may be a disconnect when trying to relate blood exposure to instances of hepatotoxicity because a pharmacokinetic-toxicity relationship may not be obvious from blood concentrations.

Use of Transgenic Mouse Models to Understand the Oral Disposition and Drug-Drug Interaction Potential of Cobimetinib, a MEK Inhibitor

Data from the clinical absolute bioavailability (F) study with cobimetinib suggested that F was lower than predicted based on its low hepatic extraction and good absorption. The CYP3A4 transgenic (Tg) mouse model with differential expression of CYP3A4 in the liver (Cyp3a−/−Tg-3A4Hep) or intestine (Cyp3a−/−Tg-3A4Int) and both liver and intestine (Cyp3a−/−Tg-3A4Hep/Int) were used to study the contribution of intestinal metabolism to the F of cobimetinib. In addition, the effect of CYP3A4 inhibition and induction on cobimetinib exposures was tested in the Cyp3a−/−Tg-3A4Hep/Int and PXR-CAR-CYP3A4/CYP3A7 mouse models, respectively. After i.v. administration of 1 mg/kg cobimetinib to wild-type [(WT) FVB], Cyp3a−/−Tg-3A4Hep, Cyp3a−/−Tg-3A4Int, or Cyp3a−/−Tg-3A4Hep/Int mice, clearance (CL) (26-35 ml/min/kg) was similar in the CYP3A4 transgenic and WT mice. After oral administration of 5 mg/kg cobimetinib, the area under the curve (AUC) values of cobimetinib in WT, Cyp3a−/−Tg-3A4Hep, Cyp3a−/−Tg-3A4Int, or Cyp3a−/−Tg-3A4Hep/Int mice were 1.35, 3.39, 1.04, and 0.701 μM⋅h, respectively. The approximately 80% lower AUC of cobimetinib in transgenic mice when intestinal CYP3A4 was present suggested that the intestinal first pass contributed to the oral CL of cobimetinib. Oxidative metabolites observed in human circulation were also observed in the transgenic mice. In drug-drug interaction (DDI) studies using Cyp3a−/−Tg-3A4Hep/Int mice, 8- and 4-fold increases in oral and i.v. cobimetinib exposure, respectively, were observed with itraconazole co-administration. In PXR-CAR-CYP3A4/CYP3A7 mice, rifampin induction decreased cobimetinib oral exposure by approximately 80%. Collectively, these data support the conclusion that CYP3A4 intestinal metabolism contributes to the oral disposition of cobimetinib and suggest that under certain circumstances the transgenic model may be useful in predicting clinical DDIs.

Solvent-based formulations for intravenous mouse pharmacokinetic studies: tolerability and recommended solvent dose limits

Abstract 1. Modern high-throughput small molecule drug discovery requires rapid screening of the pharmacokinetic parameters of multiple candidate molecules in parallel. The mouse is often used for such screening, as are solvent-based intravenous formulations. Despite this, the intravenous toxicity of many commonly used solvents is unknown. The purpose of this investigation is to establish recommended no-observed-effect level (NOEL) and maximum tolerated dose (MTD) for several commonly used intravenous solvents in the CD-1 mouse. 2. The acute tolerability of polyethylene glycol 400, N-methylpyrrolidone, dimethyl sulfoxide, ethanol, dimethylacetamide and propylene glycol was established, along with combinations of polyethylene glycol 400 and/or ethanol and DMSO. Based on these data, an acute NOEL and recommended MTD is reported for each solvent or solvent combination. 3. These data can guide the use of these solvents to support single-dose intravenous pharmacokinetic studies in mice. By establishing a defined dose tolerability range for the most commonly used intravenous solvents, undue pain and distress in animals can be avoided while maximizing the generation of critical pharmacokinetic data for project teams.

Measuring NAD+ levels in mouse blood and tissue samples via a surrogate matrix approach using LC–MS/MS

BACKGROUND NAD(+) is an endogenous analyte and is unstable during blood sample collection, both of which present obstacles for quantitation. Moreover, current procedures for NAD(+) sample collection require onsite treatment with strong acid to stabilize the NAD(+) in mouse blood cells. RESULTS NAD(+) can be stabilized by addition of acid before the frozen mouse blood sample was thawed. A simple sample collection procedure was proposed to facilitate the analysis of NAD(+) in mouse blood and tissue samples. A LC-MS/MS method was developed for quantifying NAD(+) in mouse blood and various tissue samples. The described method was used to measure endogenous NAD(+) levels in mouse blood following oral administration of the nicotinamide phosphoribosyltransferase inhibitor GNE-617. CONCLUSION This study presents a suitable assay and sample collection procedure for high throughput screening of NAD(+) samples in preclinical discovery studies.

Rifampin-Mediated Induction of Tamoxifen Metabolism in a Humanized PXR-CAR-CYP3A4/3A7-CYP2D6 Mouse Model

Animals are not commonly used to assess drug-drug interactions due to poor clinical translatability arising from species differences that may exist in drug-metabolizing enzymes and transporters, and their regulation pathways. In this study, a transgenic mouse model expressing human pregnane X receptor (PXR), constitutive androstane receptor (CAR), CYP3A4/CYP3A7, and CYP2D6 (Tg-composite) was used to investigate the effect of induction mediated by rifampin on the pharmacokinetics of tamoxifen and its metabolites. In humans, tamoxifen is metabolized primarily by CYP3A4 and CYP2D6, and multiple-day treatment with rifampin decreased tamoxifen exposure by 6.2-fold. Interestingly, exposure of tamoxifen metabolites 4-hydroxytamoxifen (4OHT), N-desmethyltamoxifen (NDM), and endoxifen also decreased. In the Tg-composite model, pretreatment with rifampin decreased tamoxifen area under the time-concentration curve between 0 and 8 hours (AUC0-8) from 0.82 to 0.20 µM*h, whereas AUC0-8 of 4OHT, NDM, and endoxifen decreased by 3.4-, 4.7-, and 1.3-fold, respectively, mirroring the clinic observations. In the humanized PXR-CAR (hPXR-CAR) model, rifampin decreased AUC0-8 of tamoxifen and its metabolites by approximately 2-fold. In contrast, no significant modulation by rifampin was observed in the nonhumanized C57BL/6 (wild-type) animals. In vitro kinetics determined in microsomes prepared from livers of the Tg-composite animals showed that, although Km values were not different between vehicle- and rifampin-treated groups, rifampin increased the Vmax for the CYP3A4-mediated pathways. These data demonstrate that, although the hPXR-CAR model is responsive to rifampin, the extent of the clinical rifampin-tamoxifen interaction is better represented by the Tg-composite model. Consequently, the Tg-composite model may be a suitable tool to examine the extent of rifampin-mediated induction for other compounds whose metabolism is mediated by CYP3A4 and/or CYP2D6.

Assessment of the hepatic CYP reductase null mouse model and its potential application in drug discovery.

CYP Oxidoreductase (Por) is the essential electron donor for all CYP enzymes and is responsible for the activation of CYP. The Taconic Hepatic CYP Reductase Null (HRN) mouse model possesses a targeted mutation that results in liver-specific deletion of the Por gene thereby resulting in a disruption of CYP metabolism in the liver. The objectives of these studies were to further characterize the HRN mouse using probe drugs metabolized by CYP. In addition, tumor exposure in xenograft tumor bearing HRN immune-compromised (nude) mice was also determined. In HRN mice following intravenous (iv) administration of midazolam, clearance (CL) was reduced by ∼ 80% compared to wild-type mice (WT). After oral administration, the AUC of midazolam was increased by ∼ 20-fold in HRN mice compared to WT mice; this greater effect suggests that hepatic first pass plays a role in the oral CL of midazolam. A 50% and an 80% decrease in CL were also observed in HRN mice following iv administration of docetaxel and theophylline, respectively, compared to WT mice. In addition, a 2- to 3-fold increase in tumor concentrations of G4222, a tool compound, were observed in tumor bearing HRN nude mice compared to tumor bearing nude WT mice. The observations from these experiments demonstrate that, for compounds that are extensively metabolized by hepatic CYP, the HRN mouse model could potentially be valuable for evaluating in vivo efficacy of tool compounds in drug discovery where high hepatic CL and low exposure may prevent in vivo evaluation of a new chemical entity.

Investigations into the Mechanisms of Pyridine Ring Cleavage in Vismodegib

Vismodegib (Erivedge, GDC-0449) is a first-in-class, orally administered small-molecule Hedgehog pathway inhibitor that is approved for the treatment of advanced basal cell carcinoma. Previously, we reported results from preclinical and clinical radiolabeled mass balance studies in which we determined that metabolism is the main route of vismodegib elimination. The metabolites of vismodegib are primarily the result of oxidation followed by glucuronidation. The focus of the current work is to probe the mechanisms of formation of three pyridine ring-cleaved metabolites of vismodegib, mainly M9, M13, and M18, using in vitro, ex vivo liver perfusion and in vivo rat studies. The use of stable-labeled (13C2,15N)vismodegib on the pyridine ring exhibited that the loss of carbon observed in both M9 and M13 was from the C-6 position of pyridine. Interestingly, the source of the nitrogen atom in the amide of M9 was from the pyridine. Evidence for the formation of aldehyde intermediates was observed using trapping agents as well as 18O-water. Finally, we conclude that cytochrome P450 is involved in the formation of M9, M13, and M18 and that M3 (the major mono-oxidative metabolite) is not the precursor for the formation of these cleaved products; rather, M18 is the primary cleaved metabolite.

Utility of CYP3A4 and PXR-CAR-CYP3A4/3A7 Transgenic Mouse Models To Assess the Magnitude of CYP3A4 Mediated Drug–Drug Interactions

Species differences in the expression, activity, regulation, and substrate specificity of metabolizing enzymes preclude the use of animal models to predict clinical drug-drug interactions (DDIs). The objective of this work is to determine if the transgenic (Tg) Cyp3a-/-Tg-3A4Hep/Int and Nr1i2/Nr1i3-/--Cyp3a-/-Tg-PXR-CAR-3A4/3A7Hep/Int (PXR-CAR-CYP3A4/3A7) mouse models could be used to predict in vivo DDI of 10 drugs; alprazolam, bosutinib, crizotinib, dasatinib, gefitinib, ibrutinib, regorafenib, sorafenib, triazolam, and vandetinib (as victims); with varying magnitudes of reported CYP3A4 clinical DDI. As an assessment of the effect of CYP3A4 inhibition, these drugs were coadministered to Cyp3a-/-Tg-3A4Hep/Int mice with the CYP3A inhibitor, itraconazole. For crizotinib, regorafenib, sorafenib, and vandetanib, there was no significant increase of AUC observed; with alprazolam, bosutinib, ibrutinib, dasatinib, and triazolam, pretreatment with itraconazole resulted in a 2-, 4-, 17-, 7-, and 15-fold increase in AUC, respectively. With the exception of gefinitib for which the DDI effect was overpredicted (12-fold in Tg-mice vs 2-fold in the clinic), the magnitude of AUC increase observed in this study was consistent (within 2-fold) with the clinical DDI observed following administration with itraconazole/ketoconazole. As an assessment of CYP3A4 induction, following rifampin pretreatment to PXR-CAR-3A4/3A7Hep/Int mice, an 8% decrease in vandetanib mean AUC was observed; 39-52% reduction in AUC were observed for dasatinib, ibrutinib, regorafenib, and sorafenib compared to vehicle treated mice. The greatest effect of rifampin induction was observed with alprazolam, bosutinib, crizotinib, gefitinib, and triazolam where 72-91% decrease in AUC were observed. With the exception of vandetanib for which rifampin induction was under-predicted, the magnitude of induction observed in this study was consistent (within 2-fold) with clinical observations. These data sets suggest that, with two exceptions, these transgenic mice models were able to exclude or capture the magnitude of CYP3A4 clinical inhibition and induction. Data generated in transgenic mice may be used to gain confidence and complement in vitro and in silico methods for assessing DDI potential/liability.