Molecular and Cellular Characterization of Human Embryonic Stem Cell Derived Hepatocytes

Thesis (Ph.D.)--University of Washington, 2015-12Primary human hepatocytes are commonly used to evaluate liver drug metabolism and toxicity. Pluripotent stem cell derived hepatocytes (SCDHs) have the potential to overcome access and function-related limitations associated with primary hepatocytes. However, in order for SCDHs to become routinely used in preclinical drug metabolism and toxicity screening, they must demonstrate reproducible activity of drug metabolism proteins, particularly the oxidative CYP enzymes, and at a level comparable to that of primary human hepatocytes. The work presented in this dissertation explores the potential of SCDHs to be used as an in vitro pre-clinical model for drug metabolism studies. Namely, we focus on further identifying how similar SCDHs are to primary human hepatocytes with regard to drug-metabolizing enzymes. Moreover, we performed genotype profiling on the commonly used human embryonic stem cell lines (hESCs) to further identify their utility in pharmacogenetic screening. In addition, we investigated the miRNA expression profile of SCDHs to identify miRNA candidates that could improve the maturity of SCDHs. Our data showed that SCDHs are immature compared to primary human hepatocytes in terms of the expression and activity of drug-metabolizing enzymes, notably the CYP enzymes. We found that SCDHs more closely resemble fetal hepatocytes regarding expression of hepatocyte markers, drug-metabolizing enzymes, transporters, and transcription factors. This translated into minimal CYP activity for only CYPs 1A and 3A when examining metabolite formation. In addition, we show that the miRNA expression profile of SCDHs compared to stem cells, cryopreserved hepatocytes and human liver tissue indicates global changes associated with immaturity in SCDHs. Taken together, our findings provide a more thorough characterization of SCDHs with regards to their use in drug metabolism studies, and provide insight into a possible mechanism to enhance the maturity and functionality of SCDHs. Additional studies are warranted to further evaluate the effect of the miRNAs identified here on the differentiation of hepatocytes

Pharmacogenetic profiling and metabolic activity of human embryonic stem cell derived hepatocytes: focus on CYP450-mediated oxidation

Primary human hepatocytes are commonly used to evaluate liver drug metabolism and toxicity. Pluripotent stem cell derived hepatocytes (SCDHs) have the potential to overcome access and function-related limitations associated with primary hepatocytes. SCDHs may also provide an improved system for evaluating genotype-phenotype relationships, e.g. cytochrome P450 (CYP) gene polymorphisms and their impact on drug metabolism and toxicity. However, in order for SCDHs to become routinely used in preclinical drug metabolism and toxicity screening, they must demonstrate reproducible activity of drug metabolism proteins, particularly the oxidative CYP enzymes. We characterized the differentiation status of SCDHs, with emphasis on the expression and functional capacity of drug-metabolizing enzymes, and genetically profiled the commonly used WiCell® hESC lines, focusing on CYP2D6 in our analysis. An assay of enzymatic function in SCDHs using a CYP cocktail assay designed to measure 8 different isozymes revealed only minimal activity for CYP3A, below that observed in primary hepatocytes. With regard to CYP2D6 gene copy number variation, we found the H1 line has only one gene copy, which also harbors the CYP2D6*41 splicing defect, predictive of a CYP2D6 poor/intermediate metabolizer. We identified no CYP2D6 gene duplications, indicating no representative ultra-rapid metabolizer. The H7 and H14 lines are heterozygous for the non-functional CYP2D6*4 variant resulting in a predicted intermediate metabolizer phenotype. In addition, we compared the penetrance of the CYP2D6*41 splicing defect in SCDHs and liver tissue via reverse-transcription PCR assay. We found incomplete penetrance of the CYP2D6*41 allele in liver tissue and variable penetrance in SCDHs. Based on gene expression profiling, SCDHs most closely resemble fetal hepatocytes, especially with regards to AFP, CYP3A7 and FMO1 expression. Finally, these studies indicate a low degree of genetic diversity of pharmacogenetically-relevant genes in the WiCell® hESC lines

Exposure‐safety and exposure‐efficacy analyses for tisotumab vedotin for patients with locally advanced or metastatic solid tumors

Abstract The antibody‐drug conjugate (ADC) tisotumab vedotin (TV) received accelerated approval from the US Food and Drug Administration for treatment of adults with recurrent or metastatic cervical cancer (r/mCC) with disease progression on or after chemotherapy. A population pharmacokinetic (PK) model, developed using dosing data from four clinical TV studies, was used to estimate individual exposure and explore safety and efficacy exposure‐response (ER) relationships. Because PK analysis showed no appreciable accumulation of TV and monomethyl auristatin E (MMAE) with repeated dosing, cycle 1 exposure metrics and predicted average concentrations from time zero until end of the cycle in which an event occurred (CavgLast) were used for ER analyses. The probability of achieving objective response increased significantly as the ADC cycle 1 maximum serum concentration (Cmax) increased. The probability of treatment‐related adverse events (AEs) leading to dose modification increased significantly as ADC cycle 1 area under the concentration‐time curve (AUC) increased. Number of grade 2+ ocular AEs increased significantly as ADC cycle 1 AUC, Cmax, and ADC CavgLast increased. MMAE cycle 1 AUC predicted risk of serious treatment‐related AEs. The relationship between ADC exposure and efficacy end points suggests ADC treatment was associated with clinically meaningful response across the observed exposures; greater exposure was associated with increased efficacy. The relationship between ADC and MMAE exposure and safety end points suggests increased exposure was associated with increased AE risk. These results align with clinical findings showing TV 2 mg/kg (≤200 mg for patients ≥100 kg) every 3 weeks is efficacious and tolerable for patients with r/mCC