Botanical supplements, herbal remedies, and plant-derived products are used globally. However, botanical dietary supplements are rarely subjected to robust safety testing unless there are adverse reports in post-market surveillance. Botanicals are complex and difficult to assess using current frameworks designed for single constituent substances (e.g. small molecules or discrete chemicals), making safety assessments costly and time-consuming. The liver is a primary organ of concern for potential botanical-induced hepatotoxicity and botanical-drug interactions as it plays a crucial role in xenobiotic metabolism. The NIH-funded Drug Induced Liver Injury Network noted that the number of botanical-induced liver injuries in 2017 nearly tripled from those observed in 2004-2005. New approach methodologies (NAMs) can aid in the rapid and cost-effective assessment of botanical supplements for potential hepatotoxicity. The Hepatotoxicity Working Group within the Botanical Safety Consortium is working to develop a screening strategy that can help reliably identify potential hepatotoxic botanicals and inform mechanisms of toxicity. This manuscript outlines the Hepatotoxicity Working Group's strategy and describes the assays selected and the rationale for the selection of botanicals used in case studies. The selected NAMs evaluated as a part of this effort are intended to be incorporated into a larger battery of assays to evaluate multiple endpoints related to botanical safety. This work will contribute to a botanical safety toolkit, providing researchers with tools to better understand hepatotoxicity associated with botanicals, prioritize and plan future testing as needed, and gain a deeper insight into the botanicals being tested.

Single-cell DNA sequencing (scDNA-seq) enables decoding somatic cancer variation. Existing methods are hampered by low throughput or cannot be combined with transcriptome sequencing in the same cell. We propose HIPSD R-seq (HIgh-throughPut Single-cell Dna and Rna-seq), a scalable yet simple and accessible assay to profile low-coverage DNA and RNA in thousands of cells in parallel. Our approach builds on a modification of the 10X Genomics platform for scATAC and multiome profiling. In applications to human cell models and primary tissue, we demonstrate the feasibility to detect rare clones and we combine the assay with combinatorial indexing to profile over 17,000 cells.

ID 19303 Poster Board 374 Evaluation of metabolic fates of drug candidates is a major activity in drug development, with results critical to the assessment of efficacy and safety. Due to the routine application of high throughput assays for hepatic clearance using in vitro systems such as human liver microsomes and human hepatocytes in the early phases of drug discovery and development, pharmaceutical companies have accumulated a collection of slowly metabolized compounds (SMC) as potential drug candidates. The metabolic stability of the SMC poses a challenge for further development as their metabolic fates cannot be readily defined using conventional approaches. To overcome this challenge, we have developed a plated hepatocyte relay assay (PHRA) with which SMC can be evaluated for intrinsic hepatic clearance as well as metabolite formation. PHRA is similar to the suspension hepatocyte relay assay developed by Di et al (2012) except that plated hepatocytes are employed in lieu of suspension hepatocytes, thereby extending each relay from 3 h to 24 h, and the elimination of the laborious centrifugation step required for medium transfer. The principle of the assay is to subject the SMC to metabolism by hepatocytes plated in a 96-well plate (4 hours after plating to allow cell attachment) on the day of evaluation, with medium transferred to freshly plated hepatocytes in subsequent days. Examples of the accumulative incubation durations in PHRA after each relay are as follows: 24 h (initial incubation), 48 h (1st relay), and 72 h (2nd relay), 96 h (3rd relay), and 120 h (4th relay). A validation study with 15 compounds with known human in vivo hepatic clearance which included the ultra low clearance compounds (CLnon-renal < 1 mL/min/kg) meloxicam, tolbutamide, warfarin, disopyramide, and tenoxicam; and the low clearance compounds (CLnon-renal 1-5.1 mL/min/kg) glimepiride, clozapine, riluzole, ibuprofen, prednisolone, voriconazole, quinidine, prednisone, dexamethasone and risperidone. Linear time-dependent disappearance of the parent compounds was observed for all compounds up to the longest incubation duration of 120 hrs. We further evaluated the potential applications of PHRA towards the derivation of enzyme kinetic parameters and metabolite formation of SMC using the model ultra low clearance compound, warfarin. Warfarin was evaluated at concentrations of 1, 2, 5, 10, 20 and 50 uM for incubation durations of 24, 48 and 72 hrs in the relay assay. Linear time-dependent warfarin clearance was observed at all concentrations, with concentration-dependent metabolic clearance consistent with Michalis Menten enzyme kinetics, allowing the derivation of apparent Km and Vmax values. Furthermore, time-dependent formation of warfarin metabolites, 7-hydroxy and 7-glucuronide warfarin, were observed. Our results suggest the PHRA can be readily applied towards to evaluation of intrinsic hepatic clearance, enzyme kinetics, as well as metabolite production of SMC.

It has been well-established that hepatic drug metabolism may have significant impact on the efficacy and off-target toxicity of anticancer drugs. We present here an in vitro assay, the metabolism-dependent cytotoxicity assay (MDCA), for the evaluation of the roles of specific pathways on the efficacy of anticancer drugs. The assay employs a novel in vitro hepatic metabolic system, the permeabilized cofactor-supplemented human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system for the evaluation of the in vitro cytotoxicity of anticancer agents towards a designated tumor and nontumor cells. MMHH are derived from cryopreserved human hepatocytes isolated from human livers procured for but not used for transplantation provided to our laboratory from the International Institute for the Advancement of Medicine. We have previously demonstrated that MMHH retained all drug metabolizing enzyme pathways present in human hepatocytes. The advantages of MMHH include the following: 1. Drug metabolizing enzyme activities are not affected by cytotoxicity of the drug substrate, a major limitation of intact human hepatocytes; and 2. Drug metabolism pathways can be selected by cofactor specification (e.g. NADPH/NAD+ for phase 1 oxidation; Uridine 5′-diphosphoglucuronic acid (UDPGA) for glucuronidation; 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for sulfation; N-acetyl coenzyme A for N-acetylation). We present here a proof-of-concept study to evaluate the role of various hepatic metabolic pathways towards the cytotoxicity of cyclophosphamide, a commonly used drug for the treatment of renal carcinoma, towards a renal cancer cell line, HEK293 cells. We observed that NADPH enhanced the cytotoxicity, consistent with the known metabolic activation of cyclophosphamide by P450 isoforms (e.g. CYP2B6; CYP3A4) to the known metabolic activation of cyclophosphamide to 4-hydroxycloophosphamide, followed by the formation of the ultimate cytotoxic metabolites phosphoramide mustard and acrolein, which are responsible for its anticancer properties. In addition, we found that the addition of L-glutathione attenuated cyclophosphamide cytotoxicity towards the HEK293 cells, an observation consistent with the roles of this cellular detoxification system in the resistance of tumor cells to chemotherapeutic agents. Minimal effects were observed for UDPGA and PAPS, consistent with the known noninvolvement of these phase 2 conjugating pathways in cyclophosphamide metabolism. MDCA represents an in vitro experimental system that can be routinely applied towards the discovery and development of novel anticancer drugs to evaluate the roles of drug metabolism in efficacy and safety, with various cancer cells (e.g. prostate carcinoma, neuroblastoma) and cells from normal tissues (e.g. cardiomyocytes, intestinal mucosal cells), respectively, as target cells. Citation Format: Albert P. Li, Hong Wei. A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5338.

Fibroblast growth factors 15 (FGF15) and 19 (FGF19) are endocrine growth factors that play an important role in maintaining bile acid homeostasis. FGF15/19-based therapies are currently being tested in clinical trials for the treatment of nonalcoholic steatohepatitis and cholestatic liver diseases. To determine the physiologic impact of long-term elevations of FGF15/19, a transgenic mouse model with overexpression of Fgf15 (Fgf15 Tg) was used in the current study. The RNA sequencing (RNA-seq) analysis revealed elevations of the expression of several genes encoding phase I drug metabolizing enzymes (DMEs), including Cyp2b10 and Cyp3a11, in Fgf15 Tg mice. We found that the induction of several Cyp2b isoforms resulted in increased function of CYP2B in microsomal metabolism and pharmacokinetics studies. Because the CYP2B family is known to be induced by constitutive androstane receptor (CAR), to determine the role of CAR in the observed inductions, we crossed Fgf15 Tg mice with CAR knockout mice and found that CAR played a minor role in the observed alterations in DME expression. Interestingly, we found that the overexpression of Fgf15 in male mice resulted in a phenotypical switch from the male hepatic expression pattern of DMEs to that of female mice. Differences in secretion of growth hormone (GH) betweenmale and femalemice are known to drive sexually dimorphic, STAT5b-dependent expression patterns of hepatic genes. We found that male Fgf15 Tg mice presented with many features similar to GH deficiency, including lowered body length and weight, Igf-1 and Igfals expression, and STAT5 signaling. SIGNIFICANCE STATEMENT The overexpression of Fgf15 in mice causes an alteration in DMEs at the mRNA, protein, and functional levels, which is not entirely due to CAR activation but associated with lower GH signaling.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

Fibroblast growth factors 15 (FGF15) and 19 (FGF19) are endocrine growth factors that play an important role in maintaining bile acid homeostasis. FGF15/19-based therapies are currently being tested in clinical trials for the treatment of nonalcoholic steatohepatitis and cholestatic liver diseases. To determine the physiologic impact of long-term elevations of FGF15/19, a transgenic mouse model with overexpression of Fgf15 ( Fgf15 Tg) was used in the current study. The RNA sequencing (RNA-seq) analysis revealed elevations of the expression of several genes encoding phase I drug metabolizing enzymes (DMEs), including Cyp2b10 and Cyp3a11 , in Fgf15 Tg mice. We found that the induction of several Cyp2b isoforms resulted in increased function of CYP2B in microsomal metabolism and pharmacokinetics studies. Because the CYP2B family is known to be induced by constitutive androstane receptor (CAR), to determine the role of CAR in the observed inductions, we crossed Fgf15 Tg mice with CAR knockout mice and found that CAR played a minor role in the observed alterations in DME expression. Interestingly, we found that the overexpression of Fgf15 in male mice resulted in a phenotypical switch from the male hepatic expression pattern of DMEs to that of female mice. Differences in secretion of growth hormone (GH) between male and female mice are known to drive sexually dimorphic, STAT5b-dependent expression patterns of hepatic genes. We found that male Fgf15 Tg mice presented with many features similar to GH deficiency, including lowered body length and weight, Igf-1 and Igfals expression, and STAT5 signaling.

In this chapter, drug-metabolizing enzymes known to play key roles in the enhancement of drug toxicity via metabolic activation, and in the reduction of drug toxicity via metabolic detoxification, as well as toxic drug–drug interactions are reviewed. The drug-metabolizing enzymes reviewed include both P450 and non-P450 pathways. Each pathway is reviewed in terms of substrates, inducers, inhibitors, individual variations, and involvement in drug toxicity.

The reported roles of drug metabolism and transporter-mediated uptake and efflux in the toxicity of 12 marketed drugs with severe, sometimes fatal toxicity including acetaminophen, cerivastatin, felbamate, flucloxacillin, nefazodone, obeticholic acid, sitaxentan, sorivudine, tacrine, terfenadine, troglitazone, and trovafloxacin are reviewed. Based on the information on drug metabolism and transport, key risk factors that may exacerbate the toxicity of these drugs are proposed. The information provides a scientific basis for the Multiple Determinant Hypothesis of idiosyncratic drug toxicity.

In drug development, it is important to identify drug candidates where drug metabolism plays key roles in their toxicity. Major reasons for the identification of drug candidates with metabolism-dependent toxicity are as follows: 1. Safety profiles determined in preclinical animal species may not accurately predict human effects due to species difference in drug metabolism; and 2. idiosyncratic drug toxicity may occur in individuals due to enhanced metabolic activation or compromised detoxification. We recently developed a novel in vitro hepatocyte system, the MetMax Human Hepatocyte (MMHH) system with permeabilized human hepatocytes supplemented with metabolic cofactors, for the evaluation of drug metabolism. The permeabilization treatment removes the plasma membrane as a barrier for drug diffusion into the hepatocytes, and the diffusion of the metabolites of the hepatocytes. A further advantage of MMHH is that cytotoxicity of the intended substrate would not affect drug metabolism, a challenge often encountered in the evaluation of metabolism of toxic drugs in intact hepatocytes. We report here the development of the MMHH/HEK assay for the evaluation of metabolic activation and detoxification of toxic drugs. In this assay, drug toxicity is quantified in a drug metabolism incompetent target cell line (HEK293) in the presence of absence of MMHH as an exogenous metabolic activating system, and in the presence of selective cofactors for the evaluation of key metabolic activation and detoxification pathways. As a proof-of-concept study, the MMHH/HEK assay was applied in the the evaluation of the model metabolically-activated drugs that are known to be associated with severe drug induced liver injuries (DILI) - acetaminophen, cyclophosphamide, and troglitazone. Our results showed that the cytotoxicity of these model DILI drugs was enhanced in the presence of MMHH supplemented with NADPH, thereby confirming the role of oxidative metabolism in metabolic activation. Furthermore, supplementation of NADPH with reduced glutathione (GSH), a physiologically relevant detoxifying conjugating cofactor for reactive metabolites, led to significant attenuation of cytotoxicity, demonstrating that these model drugs were metabolized by hepatocytes to to reactive, cytotoxic metabolites. Supplementation with UDPGA and PAPS also attenuated the in vitro cytotoxicity of these model drugs in the MMHH/HEK assay, demonstrating the involvement of glucuronidation and sulfation conjugating pathways in toxification. Troglitazone cytotoxicity in the MMHH/HEK assay was found to be higher than that observed for the known less hepatotoxic analogs, rosiglitazone and pioglitazone. Our results suggest that MMHH/HEK assay can be applied towards the evaluation of metabolic activation and detoxification and the identification of drugs with idiosyncratic DILI potential.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

Elements of key enteric drug metabolism and disposition pathways are reviewed to aid the assessment of the applicability of current cell-based enteric experimental systems for the evaluation of enteric metabolism and drug interaction potential. Enteric nuclear receptors include vitamin D receptor, constitutive androstane receptor, pregnane X receptor, farnesoid X receptor, liver X receptor, aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor. Enteric drug metabolizing enzyme pathways include both cytochrome P450 (P450) and non-P450 drug metabolizing enzymes based on gene expression, proteomics, and activity. Both uptake and efflux transporters are present in the small intestine, with P-glycoprotein found to be responsible for most drug-drug and food-drug interactions. The cell-based in vitro enteric systems reviewed are 1) immortalized cell line model: the human colon adenocarcinoma (Caco-2) cells; 2) human stem cell-derived enterocyte models: stem cell enteric systems, either from intestinal crypt cells or induced pluripotent stem cells; and 3) primary cell models: human intestinal slices, cryopreserved human enterocytes, permeabilized cofactor-supplemented (MetMax) cryopreserved human enterocytes, and cryopreserved human intestinal mucosa. The major deficiency with both immortalized cell lines and stem cell-derived enterocytes is that drug metabolizing enzyme activities, although they are detectable, are substantially lower than those for the intestinal mucosa in vivo. Human intestine slices, cryopreserved human enterocytes, MetMax cryopreserved human enterocytes, and cryopreserved human intestinal mucosa retain robust enteric drug metabolizing enzyme activity and represent appropriate models for the evaluation of metabolism and metabolism-dependent drug interaction potential of orally administered xenobiotics including drugs, botanical products, and dietary supplements. SIGNIFICANCE STATEMENT: Enteric drug metabolism plays an important role in the bioavailability and metabolic fate of orally administered drugs as well as in enteric drug-drug and food-drug interactions. The current status of key enteric drug metabolism and disposition pathways and in vitro human cell-based enteric experimental systems for the evaluation of the metabolism and drug interaction potential of orally administered substances is reviewed.

Commercial formulations of 29 commonly used herbal supplements (HSs) and grapefruit juice were evaluated for drug interaction potential via quantification of their CYP3A inhibitory potential in two in vitro experimental models of human small intestine, cryopreserved human intestinal mucosa (CHIM), and cryopreserved human enterocytes (CHEs). Two CYP3A substrates were used-in the studies with CHIM, CYP3A activity was quantified via liquid chromatography tandem mass spectrometry quantification of midazolam 1'-hydroxylation, whereas in CHE, luciferin-IPA metabolism to luciferin was quantified by luminescence. Upon treatment of CHIM with the estimated lumen concentration of the HS upon each oral administration (manufacturers' recommended dosage dissolved in 200 ml of culture medium), >80% CYP3A inhibition was observed for green tea extract, St. John's wort, valerian root, horehound, and grapefruit juice. Less than 50% inhibition was observed for fenugreek, aloe vera, guarana, soy isoflavone, maca, echinacea, spirulina, evening primrose, milk thistle, cranberry, red yeast rice, rhodiola, ginkgo biloba, turmeric, curcumin, white kidney bean, garlic, cinnamon, saw palmetto berries, panax ginseng, black elderberry, wheat grass juice, flaxseed oil, black cohosh, and ginger root. The results were confirmed in a a dose-response study with HSs obtained from three suppliers for the four inhibitory HSs (green tea extract, horehound, St. John's wort, valerian root) and three representative noninhibitory HSs (black cohosh, black elderberry, echinacea). Similar results were obtained with the inhibitory HSs in CHE. The results illustrate that CHIM and CHE represent physiologically relevant in vitro experimental models for the evaluation of drug interaction potential of herbal supplements. Based on the results, green tea extract, horehound, St. John's wort, and valerian root may cause drug interactions with orally administered drugs that are CYP3A substrates, as was observed for grapefruit juice. SIGNIFICANCE STATEMENT: In vitro evaluation of 29 popular herbal supplements in cryopreserved human intestinal mucosa identified green tea extract, horehound, St. John's wort, and valerian root to have CYP3A inhibitory potential similar to that for grapefruit juice, suggesting their potential to have clinically significant pharmacokinetic interaction with orally administered drugs that are CYP3A substrates. The results suggest that cryopreserved human intestinal mucosa can be used for in vitro evaluation of drug interactions involving enteric drug metabolism.

We have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12-inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non-P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway-selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12-inch segments of the proximal small intestine shows that the segments immediately after the first 12-inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.