Cyrus Khojasteh

GDC―0449―A potent inhibitor of the hedgehog pathway

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.

Comparison of Metabolic Soft Spot Predictions of CYP3A4, CYP2C9 and CYP2D6 Substrates Using MetaSite and StarDrop

Metabolite identification study plays an important role in determining the sites of metabolic liability of new chemical entities (NCEs) in drug discovery for lead optimization. Here we compare the two predictive software, MetaSite and StarDrop, available for this purpose. They work very differently but are used to predict the site of oxidation by major human cytochrome P450 (CYP) isoforms. Neither software can predict non-CYP catalyzed metabolism nor the rates of metabolism. For the purpose of comparing the two software packages, we tested known probe substrate for these enzymes, which included 12 substrates of CYP3A4 and 18 substrates of CYP2C9 and CYP2D6 were analyzed by each software and the results were compared. It is possible that these known substrates were part of the training set but we are not aware of it. To assess the performance of each software we assigned a point system for each correct prediction. The total points assigned for each CYP isoform experimentally were compared as a percentage of the total points assigned theoretically for the first choice prediction for all substrates for each isoform. Our results show that MetaSite and StarDrop are similar in predicting the correct site of metabolism by CYP3A4 (78% vs 83%, respectively). StarDrop appears to do slightly better in predicting the correct site of metabolism by CYP2C9 and CYP2D6 metabolism (89% and 93%, respectively) compared to MetaSite (63% and 70%, respectively). The sites of metabolism (SOM) from 34 in-house NCEs incubated in human liver microsomes or human hepatocytes were also evaluated using two prediction software packages and the results showed comparable SOM predictions. What makes this comparison challenging is that the contribution of each isoform to the intrinsic clearance (Clint) is not known. Overall the software were comparable except for MetaSite performing better for CYP2D6 and that MetaSite has a liver model that is absent in StarDrop that predicted with 82% accuracy.

Second generation 2-pyridyl biphenyl amide inhibitors of the hedgehog pathway

Potent and efficacious inhibitors of the hedgehog pathway for the treatment of cancer have been prepared using the 2-pyridyl biphenyl amide scaffold common to the clinical lead GDC-0449. Analogs with polar groups in the para-position of the aryl amide ring optimized potency, had minimal CYP inhibition, and possessed good exposure in rats. Compounds 9d and 14f potently inhibited hedgehog signaling as measured by Gli1 mRNA and were found to be equivalent or more potent than GDC-0449, respectively, when studied in a Ptch(+/-) medulloblastoma allograft model, that is, highly dependent on hedgehog signaling.

Evaluation of Time Dependent Inhibition Assays for Marketed Oncology Drugs: Comparison of Human Hepatocytes and Liver Microsomes in the Presence and Absence of Human Plasma

PurposeTo evaluate an alternative in vitro system which can provide more quantitatively accurate drug drug interaction (DDI) prediction for 10 protein kinase inhibitors for which DDI risk was over-predicted by inhibition data generated in human liver microsomes (HLM).MethodsThree cryopreserved human hepatocyte (hHEP) systems: 1) plated hHEPs; 2) hHEPs suspended in Dulbecco’s Modified Eagle Medium (DMEM) and 3) hHEPs suspended in human plasma (plasma hHEPs) were developed to detect CYP3A time dependent inhibition, and the static mechanistic model was used to predict clinical outcomes.ResultsA general trend was observed in the CYP3A inactivation potency (kinact/KI, app) as HLM > plated > DMEM ≥ plasma hHEPs. Using the static mechanistic model, DDIs predicted using parameters estimated from plated, DMEM and plasma hHEPs had 84, 74 and 95% accuracy (out of 19 clinical interaction studies) within 2-fold of the reported interaction, respectively. They demonstrated significant improvement compared to the DDIs predicted using parameters estimated from HLMs where 58% accuracy was obtained.ConclusionsBased on 19 DDIs, plasma hHEPs demonstrate a more reliable clinical DDI prediction for 10 protein kinase inhibitors and prototypical CYP3A time dependent inhibitors.

Abstract A136: Nonclinical disposition, metabolism, and in vitro drug-drug interaction assessment of DM1, a component of trastuzumab emtansine (T-DM1).

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. T-DM1 is composed of trastuzumab, a stable linker, and the microtubule polymerization inhibitor DM1, a derivative of maytansine. After binding to HER2 on tumor cells, T-DM1 undergoes receptor-mediated internalization and is expected to go through proteolytic degradation and catabolism, resulting in the directed delivery of DM1 to the tumor cells overexpressing HER2. Although catabolism and elimination of T-DM1 have been reported previously, little is known about the metabolism and elimination of the DM1 component. This abstract summarizes aspects of the metabolism and distribution of DM1 to further the understanding of its disposition and potential for T-DM1 drug-drug interactions. In vivo disposition and metabolism in rats: [3H]-DM1 at 91 μCi/kg (200 μg/kg) was administered to rats via a bolus intravenous injection. Blood, various tissues, urine, bile and feces were collected over the following 3 to 5 days and analyzed for radioactivity. DM1 distributed rapidly and extensively to the lungs, liver, kidneys, spleen, heart, gastrointestinal tract, and adrenal glands. The major route of excretion of DM1 was through the bile/feces, with a minimal amount excreted in the urine. A good mass balance (∼100% recovery of the injected dose) was achieved over 5 days. DM1 was extensively metabolized to multiple metabolites through oxidation, hydrolysis, methylation, and glutathione conjugations. In vitro metabolism and potential for drug-drug interactions: To identify the major human cytochrome P450 (CYP) isoforms responsible for the metabolism of DM1 and determine the metabolites formed, DM1 was incubated in human liver microsomes (HLMs) in the presence of selective chemical inhibitors and with human recombinant CYP. The results of these studies demonstrated that DM1 was metabolized mainly by CYP3A4 and, to a lesser extent, by CYP3A5. The oxidative and hydrolysis metabolites of DM1 were detected in the in vitro incubation. The induction and inhibitory properties of DM1 on CYP were evaluated using cryopreserved human hepatocytes and HLMs, respectively. DM1 was found to be neither an inducer nor an inhibitor of major CYP isoforms up to the highest concentrations tested at 1 μM (738 ng/mL) and 0.7 μM (500 ng/mL), respectively. In addition, a monolayer assay using MDCKII-MDR1 cells assessed the potential of DM1 as a substrate and inhibitor of the P-glycoprotein (P-gp) transporter. DM1 was found to be a substrate, but not an inhibitor, of P-gp when tested at 0.5 M (369 ng/mL). Conclusion: Collectively, the in vitro data suggest that the DM1 component of T-DM1 has little potential for drug-drug interactions as a perpetrator, especially considering the low systemic exposure of DM1 (averaging 5 ng/mL or 0.007 μM) achieved in clinical studies and its lack of inhibition of CYP catalytic activities and P-gp activity at clinically relevant concentrations. This is consistent with the lack of evidence of drug-drug interaction observed in phase Ib/II clinical studies of T-DM1 combined with pertuzumab and taxanes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A136.

Mixed Matrix Method Provides A Reliable Metabolite Exposure Comparison for Assessment of Metabolites in Safety Testing (MIST)

BACKGROUND The regulatory guidances on metabolites in safety testing (MIST) by US Food and Drug Administration (FDA) and International Conference on Harmonisation (ICH) describe the necessity to assess exposures of major circulating metabolites in humans at steady state relative to exposures achieved in nonclinical safety studies prior to the initiation of large scale clinical trials. This comparison can be accomplished by measuring metabolite concentrations in animals and humans with validated bioanalytical methods. However, bioanalysis of metabolites in multiple species and multiple studies is resource intensive and may impact the timelines of clinical studies. METHOD A simple, reliable and accurate method has been developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volume of human plasma with blank plasma of animal species and vice versa followed by an analysis using LC-SRM or LC-HRMS. Here, we explored the reliability and accuracy of this method in several development projects at Genentech and compared the results to those obtained from validated bioanalytical methods. RESULTS The mixed-matrix method provided comparable accuracy (within ±20%) to those obtained from validated bioanalysis but does not require authentic standards or radiolabeled compounds, which could translate to time and resource savings in drug development. CONCLUSION Quantitative assessment of metabolite coverage in safety species can be made using mixed matrix method with similar accuracy and scientific rigor to those obtained from validated bioanalytical methods. Moving forward, we are encouraging the industry and regulators to consider accepting the mixed matrix method for assessing metabolite exposure comparisons between humans and animal species used in toxicology studies.

Minimal physiologically-based pharmacokinetic modeling of DSTA4637A, A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus, in a mouse model

ABSTRACT DSTA4637A, a THIOMAB™ antibody-antibiotic conjugate targeting Staphylococcus aureus, has shown promising bactericidal activity in a mouse model. DSTA4637A consists of a monoclonal anti-S. aureus antibody with an average of two rifalogue antibiotic molecules, dmDNA31, linked to its light chains. The goal of this study was to develop a minimal physiologically-based pharmacokinetic (mPBPK) model to characterize the pharmacokinetic (PK) properties of three analytes of DSTA4637A (i.e., total antibody, antibody-conjugated dmDNA31, and unconjugated dmDNA31) in mice, and to predict pharmacokinetics of DSTA4637A analytes in humans, as well as to provide an initial assessment for potential PK drug-drug interactions (DDI) in clinical trials via cross-species scaling of the mPBPK model. In the proposed model, selected organs, including heart, liver, and kidney, were connected anatomically with plasma and lymph flows. Mouse plasma and tissue concentrations of the three analytes of DSTA4637A were fitted simultaneously to estimate the PK parameters. Cross-species scaling of the model was performed by integrating allometric scaling and human physiological parameters. The final mPBPK model was able to successfully capture PK profiles of three DSTA4637A analytes in mouse plasma and in investigated organs. The model predicted a steady-state peak unbound dmDNA31 concentration lower than 5% of the IC50 of dmDNA31 towards cytochrome P450 following 100 mg/kg weekly intravenous dose, which suggests a low risk of PK DDI in humans for DSTA4637A with co-administered cytochrome P450 substrates. The proposed mPBPK modeling and cross-species scaling approaches provide valuable tools that facilitate the understanding and translation of DSTA4637A disposition from preclinical species to humans.

Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies

Subcellular fractionation of tissue homogenate provides enriched in vitro models (e.g., microsomes, cytosol, or membranes), which are routinely used in the drug metabolism or transporter activity and protein abundance studies. However, batch-to-batch or interlaboratory variability in the recovery, enrichment, and purity of the subcellular fractions can affect performance of in vitro models leading to inaccurate in vitro to in vivo extrapolation (IVIVE) of drug clearance. To evaluate the quality of subcellular fractions, we developed a simple, targeted, and sensitive LC-MS/MS proteomics-based strategy, which relies on determination of protein markers of various cellular organelles, i.e., plasma membrane, cytosol, nuclei, mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, cytoskeleton, and exosomes. Application of the quantitative proteomics method confirmed a significant effect of processing variables (i.e., homogenization method and centrifugation speed) on the recovery, enrichment, and purity of isolated proteins in microsomes and cytosol. Particularly, markers of endoplasmic reticulum lumen and mitochondrial lumen were enriched in the cytosolic fractions as a result of their release during homogenization. Similarly, the relative recovery and composition of the total membrane fraction isolated from cell vs tissue samples was quantitatively different and should be considered in IVIVE. Further, analysis of exosomes isolated from sandwich-cultured hepatocyte media showed the effect of culture duration on compositions of purified exosomes. Therefore, the quantitative proteomics-based strategy developed here can be applied for efficient and simultaneous determination of multiple protein markers of various cellular organelles when compared to antibody- or activity-based assays and can be used for quality control of subcellular fractionation procedures including in vitro model development for drug metabolism and transport studies.