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Research Article50th Anniversary Celebration Collection

The African Liver Tissue Biorepository Consortium: Capacitating Population-Appropriate Drug Metabolism, Pharmacokinetics, and Pharmacogenetics Research in Drug Discovery and Development

Collen Masimirembwa, Michele Ramsay, Jean Botha, Ewa Ellis, Harriet Etheredge, Tracey Hurrell, Comfort Ropafadzo Kanji, Nyasha Nicole Kapungu, Heather Maher, Busisiwe Mthembu, Jerolen Naidoo, Janine Scholefield, Sharan Rambarran, Francisca van der Schyff, Natalie Smyth, Bernd Strobele, Roslyn Stella Thelingwani, Jerome Loveland and June Fabian
Drug Metabolism and Disposition December 2023, 51 (12) 1551-1560; DOI: https://doi.org/10.1124/dmd.123.001400

Abstract

Pharmaceutical companies subject all new molecular entities to a series of in vitro metabolic characterizations that guide the selection and/or design of compounds predicted to have favorable pharmacokinetic properties in humans. Current drug metabolism research is based on liver tissue predominantly obtained from people of European origin, with limited access to tissue from people of African origin. Given the interindividual and interpopulation genomic variability in genes encoding drug-metabolizing enzymes, efficacy and safety of some drugs are poorly predicted for African populations. To address this gap, we have established the first comprehensive liver tissue biorepository inclusive of people of African origin. The African Liver Tissue Biorepository Consortium currently includes three institutions in South Africa and one in Zimbabwe, with plans to expand to other African countries. The program has collected 67 liver samples as of July 2023. DNA from the donors was genotyped for 120 variants in 46 pharmacogenes and revealed variants that are uniquely found in African populations, including the low-activity, African-specific CYP2C9*5 and *8 variants relevant to the metabolism of diclofenac. Larger liver tissue samples were used to isolate primary human hepatocytes. Viability of the hepatocytes and microsomal fractions was demonstrated by the activity of selected cytochrome P450s. This resource will be used to ensure the safety and efficacy of existing and new drugs in African populations. This will be done by characterizing compounds for properties such as drug clearance, metabolite and enzyme identification, and drug-drug and drug-gene interactions.

SIGNIFICANCE STATEMENT Standard optimization of the drug metabolism of new molecular entities in the pharmaceutical industry uses subcellular fractions such as microsomes and isolated primary hepatocytes, being done mainly with tissue from donors of European origin. Pharmacogenetics research has shown that variants in genes coding for drug-metabolizing enzymes have interindividual and interpopulation differences. We established an African liver tissue biorepository that will be useful in ensuring drug discovery and development research takes into account drug responses in people of African origin.

Introduction

Pharmacokinetics (PK) is a key determinant of drug safety and efficacy through determining the drug time course, exposure levels, and/or generation of active or toxic metabolites. Seminal work by Prentis et al. (1988) showed that poor PK was responsible for 40% of the failure rate of new chemical entities (NCEs) in the period of 1964–1985 (Prentis et al., 1988). This encouraged the pharmaceutical industry to invest in preclinical platforms to predict and optimize the PK processes of absorption, distribution, metabolism, and excretion (ADME). Various in silico, in vitro, and in vivo systems were developed and integrated into the drug discovery process, resulting in a drop in attrition rates due to PK liability to less than 10% by 2003 (Kola and Landis, 2004) and less than 1% by 2008 (Waring et al., 2015). ADME data generated through these systems is used to either select compounds with desirable safety and efficacy attributes or to guide molecular design and synthesis of analogs predicted to have desirable PK properties in humans (Wan, 2013). Due to the success of this approach, drug regulatory agencies such as the Food and Drug Administration and the European Medicines Agency have published guidelines for the conduct of preclinical metabolism studies (www.ema.europa.eu/contact; https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm).

The most-used tool for preclinical in vitro ADME studies is liver tissue (hepatocytes and subcellular fractions for drug transport and metabolism studies) (Krüger et al., 2019). These preclinical platforms are useful because hepatic metabolism has been observed to determine the PK of over 75% of medicines that were prescribed in the United States (Williams et al., 2004). This is consistent with the fact that most metabolic reactions include oxidation by cytochrome P450 (CYP) enzymes (70%) and glucuronidation by uridine 5′-diphospho-glucuronosyltransferases (14%), enzymes that are mainly located in the liver (Testa et al., 2012). Liver tissue is therefore used to predict NCE’s metabolic clearance, identify specific enzymes involved in the metabolism of a compound, and identify metabolites formed from the NCE. This information is used to predict dosing regimens, identify and modify metabolic hot spots in molecules, and estimate the likely extent of interindividual variation of compound clearance (Kates and Tsaioun, 2011) if compounds are metabolized by polymorphic enzymes. Such preclinical platforms are also used to evaluate NCEs for the risk of drug-drug interaction due to enzyme inhibition and induction (Masimirembwa et al., 2001).

Given the complexity of some ADME assays, most pharmaceutical companies outsource the generation of such data to contract research organizations specializing in one or more of the required evaluations. A survey of tissue biobanks of some of the major ADME contract research organizations, which sell tissue (hepatocytes and subcellular fractions) or conduct studies, showed that most tissue is derived from people of European origin, with poor representation of people from Africa and Asia (https://www.xenotech.com). Similarly, most clinical drug development is performed in European populations (Luo et al., 2017), ensuring the safety and efficacy in people of European origin. However, the same does not apply in African and other populations, raising serious concerns about the safety and efficacy of medicines used in populations in which no preclinical and clinical studies have been conducted.

Many studies have demonstrated interindividual and interpopulation differences in drug metabolism that can have serious clinical implications. Such differences occur at a genetic level, where genetic variants in genes encoding drug-metabolizing enzymes and drug transporters that are unique to certain individuals or populations result in deficient, reduced, or increased metabolism of some drugs. Variability of either population-specific variants or variants with significant interpopulation frequency differences have been noted for numerous drug-metabolizing enzymes (Rajman et al., 2017; da Rocha et al., 2021), pointing to the high likelihood that preclinical studies based only on liver tissue from people of European origin for the selection and/or optimization of NCEs might not predict PK accurately in people of African origin. This explains, in part, different dose requirements and safety and efficacy levels between African and European populations for drugs such as tacrolimus (Sanghavi et al., 2017), efavirenz (Desta et al., 2019), and warfarin (Asiimwe and Pirmohamed, 2022).

Omitting African populations from drug discovery and development research is no longer justifiable given the consequent burden of associated (and preventable) morbidity and mortality. To ensure that the global research and development pharmaceutical industry prioritizes African populations in early preclinical studies, there is a need for tissue from people of African origin. In response to the need for a resource to support preclinical drug metabolism research inclusive of African populations, we report on the establishment of the African Liver Tissue Biorepository (ALTBio).

Materials and Methods

Establishment of the Consortium

The ALTBio Consortium was established as a multidisciplinary collaboration between the Faculty of Health Sciences at the University of the Witwatersrand, which includes the Wits Donald Gordon Medical Centre (WDGMC) and the Sydney Brenner Institute of Molecular Bioscience with the African Institute of Biomedical Science and Technology (AiBST), and the Council for Scientific and Industrial Research (CSIR). WDGMC is responsible for the bioethics and regulatory framework, recruiting consented participants through the living-donor liver transplant program, surgical collection of donated tissue, clinical data collection, and database management. All biospecimens are curated by the Sydney Brenner Institute of Molecular Bioscience (an approved biobanking facility within the Faculty of Health Sciences) to secure the chain of custody for all samples, which includes collection in theater at the time of surgery, DNA extraction, sample storage, aliquoting, and shipping to and from local partners for preparation of hepatocyte subcellular fractions and primary human hepatocytes. The AiBST is responsible for preparing subcellular fractions (hepatic S9, cytosol, and microsomes) and performing drug metabolism characterization and pharmacogenomic testing. Isolation of primary human hepatocytes is performed by the CSIR.

Ethical Considerations

The ALTBio Consortium conducts all procedures within a rigorous ethicolegal and research framework in accordance with the Declaration of Helsinki (approved by the University of the Witwatersrand Medical Human Research Ethics Committee, clearance number M191006). ALTBio is compliant with the South African Protection of Personal Information Act. Study data were collected and managed using REDCap (Research Electronic Data Capture) electronic data capture tools (Harris et al., 2009, 2019) hosted by the University of the Witwatersrand.

Governance

The ALTBio Consortium founding partners established a transparent governance structure addressing 1) tissue procurement, processing, and storage; 2) frameworks for access to and use of biorepository material and data; 3) terms of reference for research collaborations and service provision; 4) optimizing direct and indirect benefits for participants, their communities, and the academic community; and 5) private-public partnerships for new and existing drugs. Part of the governance structure includes a data and materials access committee, an external review board, and the ALTBio Advisory Committee (AAC). The data and materials access committee comprises one representative from each founding partner institutions, with a quorum of three needed to execute a decision. As a requirement of the University of Witwatersrand Medical Human Research Ethics Committee, the external review board ensures the safety of all research participants for any project undertaken by ALTBio, with clearly defined terms of reference and conditions. The AAC provides scientific, strategic, operational, and technical advice, including, but not limited to, financial sustainability models, funding opportunities, advice on scientific research questions, innovation opportunities, and ensuring that the services rendered are of the appropriate quality. The AAC includes a multidisciplinary group of individuals representing participants and their communities, the University of the Witwatersrand, and high-level management from founding partners. The operational framework for the ALTBio Consortium is shown in Fig. 1.

Fig. 1.
Fig. 1.

The operational framework of the ALTBio program.

Participant Consent

Most liver tissue is donated from healthy adult donors who form part of the living-donor liver transplant program at WDGMC. As part of the standard donor workup, screening is performed for detection of prior or current infections, including hepatitis A, B, and C; HIV; cytomegalovirus; and Epstein-Barr virus.

The infection status of donors is made available for those handling samples in the chain of custody. Prior to transplant surgery, members of the transplant team refer potential participants to the ALTBio coordinator, who schedules an appointment to meet. During the meeting, relevant aspects of ALTBio are discussed as per the participant information sheet, emphasizing 1) the right to refuse participation without compromising care or treatment in any way, 2) the right to withdraw participation at any point in time, 3) that there may be no direct benefit to either the participant (donor) or the recipient of the donated organ, and 4) that any future studies that would like to access participant information and/or tissue will require approval from the University of the Witwatersrand Medical Human Research Ethics.

Committee

Potential participants are given the opportunity to read consent forms in their preferred language (translated from English) and highlight questions for discussion and are invited to join the study. If participation is declined, we acknowledge appreciation for their time and note the reason for nonparticipation (if stated). For each consented participant, the coordinator alerts the ALTBio team, ensuring all partners can perform the necessary procedures at the time of transplantation. A few days after the surgery, the study coordinator visits participants prior to discharge to advise them of which samples were collected and thank them for their participation. The process for liver collection and processing is shown in Fig. 2.

Fig. 2.
Fig. 2.

Flowchart for liver tissue collection and processing.

Surgical Collection of Liver Tissue

Once under general anesthesia, suitable lines and monitoring devices are placed according to standard protocol, the abdomen is prepared and draped in a sterile fashion, and a timeout procedure is performed to identify the patient, confirm the nature of the surgery, and confirm that all consents have been signed. The abdomen is entered via a midline laparotomy incision, and the peritoneal cavity is examined to confirm that there are no contraindications to proceeding with living-donor hepatectomy. The left lobe of the liver is mobilized by dividing the left triangular ligament as well as the gastro-hepatic ligament, preserving the replaced left hepatic artery if present. Attention is then turned to the porta hepatis, where the left hepatic artery is dissected out followed by dissection of the left portal vein, which usually includes division of branches to the caudate lobe.

The potential site of division of the left hepatic duct is then identified and marked with a metal clip, whereafter a cholecystectomy is performed, and a catheter is inserted into the cystic duct so that an intraoperative cholangiogram can be performed to exactly identify the intended site of division of the left hepatic duct. Using intraoperative ultrasound, the middle hepatic vein is identified so that the intended line of parenchymal transection is kept to the left of the middle hepatic vein. Parenchymal transection is then performed with a combination of ultrasonic dissector, electrocautery, and radiofrequency devices until the left lateral segment of the liver remains attached by only the left portal structures and the left hepatic vein. Between 2000 and 3000 IU of heparin is administered to the patient systemically and allowed to circulate for 3 minutes, following which the left hepatic artery is ligated and divided, the left portal vein clamped and divided, and, finally, the left hepatic vein clamped and divided and the segment of liver removed. The left portal vein and left hepatic vein orifices are sutured closed.

The amount of liver mass that the child requires is calculated according to their weight, and the estimated weight of the donor liver is calculated preoperatively on the computed tomography scan. In an adult-to-child liver transplant, the donated weight is usually 100% more than necessary. This allowed us to safely excise the portion of liver tissue (approximately 2 × 2 cm) from the cut surface of the donor liver without compromising either the donor or recipient in any way.

On the back table in a bath of ice-cold slush; the liver is perfused with chilled preservation solution until the effluent from the hepatic vein is clear and most of the blood has been flushed out of the liver. A small wedge of tissue is then cut from the edge of the liver and placed in 1.15% ice-cold potassium chloride (KCl) solution before being snap frozen in liquid nitrogen.

Genotyping for Pharmacogenes

Single Nucleotide Polymorphism Genotyping

GenoPharm, an analytically validated 120-assay custom pharmacogenetic open array chip that showed 99.5% concordance and 98.9% reproducibility with Coriell reference samples (Kanji et al., 2023), was used for single nucleotide polymorphism (SNP) genotyping. SNP genotyping was carried out as reported by Mbavha et al. (2022). The assay identifications (IDs) for the SNPs under investigation are listed in Supplemental Table 1. In brief, a mixture of TaqMan open array genotyping master mix (Applied Biosystems, Waltham, MA) and sample DNA in the ratio of 1:1 was loaded onto the custom open array using the AccuFill system. The QuantStudio 12K Flex real-time polymerase chain reaction (PCR) instrument (Applied Biosystems, Life Technologies Holding, Singapore) was used to amplify the genomic DNA samples on a custom OpenArray plate. Universal PCR conditions were used as per the manufacturer’s guidelines. The characterized SNPs were chosen based on the likelihood that they would affect enzyme functionality.

CYP2D6 Copy Number Variation

The CYP2D6 gene copy number was determined using the TaqMan copy number assay for exon 9 (Life Technologies, California) according to the manufacturer’s instructions. The QuantStudio 12K Flex real-time PCR machine was used to amplify the genomic DNA samples in 96-well plates using the comparative computed tomography method. Primer pairs for CYP2D6 exon 9 (TaqMan Copy Number Assay ID: Hs00010001_cn) were used. RNase P was employed as the internal control (Life Technologies). Each sample was tested in triplicate. The reaction mix was a total volume of 10 uL, which consisted of TaqPath ProAmp master mix, copy number assay, RnaseP reference assay, and the template DNA.

Prediction of Phenotype from Genotype

As pharmacogenomics dosing guidelines issue recommendations based on the genotype translated to a phenotype, genotype-phenotype translation was conducted. Based on gene-specific information tables obtained from PharmGKB, the phenotypes for these genes were determined using the observed genotype diplotype. Two haplotypes for each gene correspond to a diplotype, which is then translated into a phenotype. The typical phenotype designations are poor metabolizers (PMs), intermediate metabolizers (IMs), normal metabolizers, rapid metabolizers, and ultra-rapid metabolizers. CYP2D6 phenotypes were determined using the recently published consensus terms (Caudle et al., 2020). The genotype-phenotype translation used the allele activity values. The sum of the diplotype combination of allele activity value determined the activity score (AS). The AS was translated to a phenotype based on the contiguous AS scale.

Statistical Analysis and Allele Population Frequency Comparison

The collected data were used to estimate allele and genotype frequencies for the genes investigated. The observed genotypes were matched to the Hardy-Weinberg equilibrium expectation. The χ2 test was used to calculate the difference between the observed and expected genotype frequencies, with a P value of 0.05 indicating deviation from the Hardy-Weinberg equilibrium. The χ2 test was used to determine the variations in allele frequency between populations, with a P value of less than 0.05 highlighting a statistically significant difference.

Isolation and Characterization of Human Liver Subcellular Fractions

Human liver subcellular fractions were isolated using the method from Hogeboom et al. (1946) as depicted in Fig. 3. Human liver tissue was labeled with the ALTBio sample ID chronologically from ALT00001T, where T is for tissue. Isolated fractions were labeled with the respective ALT ID plus the type of fraction; for example, the S9 fraction for ALT00001T was labeled ALT00001S9.

Fig. 3.
Fig. 3.

Summary of the procedure for the isolation of human liver subcellular fractions. Adapted from “Subcellular fractions isolation workflow,” by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates.

Protein Concentration Determination

Protein concentration was determined using the Bradford method adapted from Thermo Scientific Bradford assay kit instructions (https://tools.thermofisher.com/content/sfs/manuals/MAN0011203_CoomassiePlus_Bradford_Asy_UG.pdf). Bovine serum albumin (BSA) standards (Sigma-Aldrich, St Louis, MO) were prepared by diluting a stock solution of 2 mg/mL BSA to give the concentrations 0, 0.025, 0.125, 0.250, 0.750, 1.000, 1.500, and 2 mg/mL. The Bradford reagent (Sigma-Aldrich) was equilibrated to room temperature and gently mixed. To 0.05 mL of each standard or subcellular fraction sample, 1.5 mL of the Bradford reagent was added, and the solutions were mixed and incubated at room temperature in the dark for 10 minutes. The absorbance of the samples was measured at 595 nm, and the protein concentration in subcellular fractions was determined from the BSA standard curve.

Linearity of Enzyme Activity with Time and Protein Concentration

Experiments were performed to determine the appropriate initial rate conditions with respect to time and microsomal protein concentration. To determine the microsomal protein concentration, incubations were done at eight different enzyme concentrations of 0, 0.05, 0.1, 0.5, 1, 3, and 5 mg/mL. The optimum enzyme concentration of 0.5 mg/mL was then used to establish the incubation time. To determine the incubation time for each reaction, incubations were done at 0, 5, 10, 15, 20, 30, and 60 minutes. The optimum time, 15 minutes, was when the enzyme did not exhaust more than 20% of the substrate while still producing an adequate metabolite for quantification.

Microsomal Incubation

The method for microsomal incubations was adapted from Wang et al. (2014). Microsomes (0.5 mg/mL) were preincubated with substrate, 0.1 M phosphate buffer (pH 7.4), and water for 5 minutes in a water bath at 37°C. The incubation concentrations of the probe substrates in a cocktail manner were around the reported Michaelis-Menten constant (Km) values, i.e., 40, 2, 5, 40, 5, and 2 μM for phenacetin, amodiaquine, diclofenac, dextromethorphan, and midazolam for CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A4, respectively (Merck Life Science, Modderfontein, South Africa). The final reaction volume was 200 µL. The final concentration of solvent in the incubation was kept between 0.5 and 1% (v/v). Reactions were started by the addition of 10 μL of 20 mM NADPH. The incubation was terminated with 100 µL ice-cold acetonitrile with 100 ng/mL verapamil as the internal standard. Samples were allowed to stand for protein precipitation at 4°C for 15 minutes, then vortexed and centrifuged at 3000g for 10 minutes. The supernatant was transferred to an high perfomance liquid chromatography (HPLC) vial, and 5 μL was injected into the liquid chromatography tandem-mass spectrometry/mass spectrometry (LC-MS/MS) analysis.

In addition to the individual donor human liver microsome (HLM), a pooled sample of 17-donor HLM was also made and characterized for activity and used for the determination of Km and Vmax for selected P450s. To evaluate the effect of CYP2C9 genetic variants on 4-hydroxyaltion of diclofenac, incubations of diclofenac (100 uM) with 16 individual HLMs with different CYP2C9 genotypes and the pooled HLM were conducted.

Km/Vmax Determination

The kinetic constants for each substrate were determined using 10 concentrations ranging from 0 to 200 μM for amodiaquine (CYP2C8), diclofenac (CYP2C9), dextromethorphan (CYP2D6), and midazolam (CYP3A4) and 0–400 μM for phenacetin (CYP1A2). The specific metabolites, i.e., acetaminophen, dextrorphan, desethylamodiaquine, 4-hydroxydiclofenac, and 1-hydroxymidazolam, were detected and quantified using liquid chromatography tandem-mass spectrometry/mass spectrometry (LC-MS/MS).

The kinetic data were analyzed using SigmaPlot (version 14.5; Systat Software, San Jose, CA) according to the enzyme kinetic equation, v = (Vmax[S])/(Km + [S]), where v is the initial velocity of the reaction, Vmax is the maximal reaction velocity, [S] is the substrate concentration, and Km is the Michaelis-Menten constant.

Isolation of Primary Human Hepatocytes

Primary human hepatocytes (PHHs) were isolated using a three-step collagenase perfusion protocol provided from the Karolinska Institutet (Jorns et al., 2014) as summarized in Fig. 4. Briefly, liver transplant recipients undergoing transplantation for indications where the liver parenchyma is unaffected, such as metabolic diseases, were approached for participation in the study. After the recipient hepatectomy is completed, on the back table in a bath of ice-cold slush the explanted liver is flushed with chilled histidine-tryptophan-ketoglutarate solution until almost all the blood has been removed from the liver. The vascular anatomy was then dissected, identifying the right and left branches of the hepatic artery as well as the right and left branches of the portal vein. The left hepatic vein was similarly dissected out. The left lateral segment was then separated from the rest of the liver by sharply dividing the parenchyma and diving the left hepatic vein, left portal vein, and left hepatic artery. The two lobes of the liver were each cannulated via the right and left portal vein and further perfused with histidine-tryptophan-ketoglutarate solution. The flow of perfusate through the portal cannula was assessed, and, where required, the cut surface was sealed. Both left and right liver lobes were then weighed, placed on ice under sterile conditions, and then packaged and transported on ice from WDGMC to the CSIR. Liver lobes were placed into a sterile bag and submerged in a circulating water bath (37°C). Perfusion tubing was connected and liver lobes were perfused for ∼15 minutes with Hanks’ balanced salt solution (no Ca2+/Mg2+; Thermo Fisher Scientific, 14170088) supplemented with EGTA (5 mM; Sigma Aldrich, E0396-25G) and HEPES (10 mM; Sigma-Aldrich, H0887-100ML). This was followed by perfusion for 10–12 minutes with Hanks’ balanced salt solution (with Ca2+/Mg2+; Thermo Fisher Scientific, 24020091) supplemented with HEPES (10 mM). Digestion was conducted by perfusing for 25–30 minutes with Minimum Essential Medium (Thermo Fisher Scientific) supplemented with Collagenase XI (125 mg/500 ml; Sigma Aldrich, C7657-500MG) and DNase I (25 mg/500 ml; Sigma Aldrich, DN25-100MG). Digested liver was placed in Kreb’s buffer (Sigma Aldrich, K3753), and cells were released into suspension. Cells were filtered twice, through a gauze filter, and pelleted by centrifugation (100g, 5 minutes at 4°C). Cell pellets were washed twice, with Kreb’s buffer, and resuspended in William’s E medium (Thermo Fisher Scientific, A1217601) supplemented with GlutaMax (2 mM; Thermo Fisher Scientific, A1286001), HEPES (10 mM), Insulin-Transferrin-Selenium (100 nM; Thermo Fisher Scientific, 41400045), dexamethasone (100 nM; Sigma-Aldrich, D1756-1G), gentamicin (10 µM; Thermo Fisher Scientific, 15-750-045), and amphotericin B (55 nM; Sigma-Aldrich, A9528-100MG). The number of PHHs recovered was determined using a hemocytometer and trypan blue exclusion and cryopreserved at 5–10 × 106 cells per vial in Cryostor cell cryopreservation media (Sigma Aldrich, #C2874-100ML).

Fig. 4.
Fig. 4.

Isolation and characterization of primary human liver hepatocytes. Adapted from “Hepatocyte isolation workflow,” by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates.

Plating and Thawing of Cryopreserved Primary Human Hepatocytes

Fresh or cryopreserved PHHs were seeded at 2 × 105 cells/cm2 on plates coated with 5 µg/cm2 rat-tail collagen I (Thermo Fisher Scientific, A1048301) in William’s E medium (supplemented as above). Cryopreserved cells were briefly thawed at 37°C, pipetted onto a 27% Percoll gradient (Sigma-Aldrich, P1644-100ML), and centrifuged for 10 minutes at 1000g. Cell pellets were washed with William’s E medium supplemented with 10% fetal bovine serum (Gibco, 10500064), pelleted (100g, 3 minutes) and resuspended in FBS-containing William’s E medium for plating.

P450-Glo CYP2C9 and CYP3A4 Assay

CYP2C9 and CYP3A4 activity were assessed using nonlytic P450-Glo CYP2C9 (Promega, V8792) and P450-Glo CYP3A4 (Promega, V9002) assays, respectively, with minor modifications from the manufacturer’s instructions. Adherent PHHs were washed with PBS and incubated with either Luciferin H (100 µM) or Luciferin-IPA (3 µM) in Kreb’s buffer for 2 hours at 37°C. Substrate was mixed with equal parts detection reagent and incubated for 10 minutes at room temperature, and luminescence was measured on a Tecan INFINITE F500 microplate reader.

CellTiter-Glo Luminescent Cell Viability Assay

The ATP content of PHHs was measured for normalization of P450 activity using a CellTiter Glo Luminescent Cell Viability Assay kit (Promega), with minor modifications from the manufacturer’s instructions. Adherent PHHs were washed with PBS and incubated with equal parts Kreb’s buffer and CellTiter-Glo reagent for 10 minutes at room temperature. Cell supernatant was transferred, and luminescence was measure on a Tecan INFINITE F500 microplate reader.

Albumin ELISA and Bicinchoninic Acid Assay

The Human Serum Albumin Human ELISA Kit (Themo Fisher Scientific; EHALB) was used to quantify albumin secreted from primary human hepatocytes. Culture media was harvested, debris was pelleted (1000g; 5 minutes), and the supernatant was transferred for storage at −80°C. Reagent and samples were thawed at 4°C overnight, 100 µl of samples was loaded onto a precoated 96-well strip plate, and the protocol was conducted per the manufacturer’s instructions. Plates were read on a Tecan INFINITE F500 microplate reader at wavelength of 450 nm. For normalization, cells were harvested directly from the well using RIPA buffer (Sigma-Aldrich, R0278-50ML) containing EDTA-free protease inhibitor cocktail (Sigma Aldrich, 4693132001), clarified by centrifugation (16,000 g; 10 minute), and quantified using the bicinchoninic acid assay (BCA). Briefly, BCA solution was made by mixing a 1:50 ratio of reagent B [4% copper II sulfate pentahydrate (Sigma-Aldrich, 209198-100G)] to reagent A [2% sodium carbonate (Fluka Chemika, 71351), 0.9% sodium bicarbonate (Sigma-Aldrich, S5761-1KG), and 1% bicinchoninic acid disodium salt hydrate (Sigma-Aldrich, D8284-25G)]. Five microliters of standard or samples were mixed with 145 µl BCA solution and incubated at 60°C in the dark for 30 minutes.

Plates were read on a Tecan INFINITE F500 microplate reader at wavelength of 620 nm.

Results

Establishment and Operationalization of CONSORTIUM

The four founding institutes successfully established the ALTBio Consortium over a period of 3 years (Fig. 1). During this time, additional human research ethics approval was obtained for biospecimens from whole (diseased) explanted livers from adult recipients who received a transplanted liver and to collect liver tissue samples from adults undergoing liver resection procedures. Out of the 80 prospective participants, seven refused consent, either for no stated reason, anxiety about the transplant, or religious reasons (Fig. 2). Additionally, six samples from those who had provided consent could not be collected at the time of surgery due to logistical issues. A total of 67 liver specimens were biobanked as of July 2023, and of these, 34 are Black South Africans, 17 are White, 10 are Colored, 5 are Indian, and 1 is Asian (Table 1).

View this table:
TABLE 1

Baseline characteristics for 67 liver donors

Pharmacogenomic Characterization Sample Donor DNA

Observed genotypes and phenotypes for selected genes are summarized in Supplemental Table 2. The frequencies of CYP450 gene alleles among the 27 black South African liver donors that have been genotyped to date in the study cohort are summarized in Table 1, together with allele frequencies in the continental superpopulations from the 1000 Genomes Project. The African, European, American, and Asian superpopulations show significant allele frequency differences. Although the 27 samples from this study are a small number to obtain accurate allele frequencies, it is noteworthy that they had more similar frequencies when compared with the African superpopulation than with the other continental superpopulations. Actionable pharmacogenetic variants were observed among the polymorphic drug-metabolizing enzyme genes. Notable observations include population variability in prevalence of predicted phenotypes within the CYP2B6, CYP2C9, CYP2C19, and CYP3A5 genotypes (Fig. 5A). For example, the reduced-activity CYP2C9 variants CYP2C9 *5 and *8 were observed at 3% and 9%, respectively, and are found mainly in people of African origin. Each donor had at least one actionable drug-gene interaction and up to five, with an average of three actionable drug-gene interactions per donor (Fig. 5B).

Fig. 5.
Fig. 5.

Phenotype frequencies of clinically important pharmacogenes in the liver tissue donor population. The phenotypes are derived from the translation of a person’s genotype to an activity score, which in turn is used to assign the phenotype status of PM, IM, normal metabolizer (NM), ultra-rapid metabolizer (UM), and rapid metabolizer (RM) and distribution of actionable pharmacogenetic variants within the samples in the cohort.

Subcellular Fractionation and Enzyme Kinetic Characterization of Microsomes

Subcellular fractions were successfully isolated (Fig. 3). The Km and Vmax for CYP1A2, 2B6, 2C8, 2D6, and 3A4 were determined using pooled liver microsomes, and the results obtained were within the ranges reported in the literature (Table 3). A full Km/Vmax representative is shown for CYP2D6-catalyzed dextromethorphan metabolism (Fig. 6). Individual donor samples were evaluated for CYP2C9 metabolism of diclofenac to 4-OH- diclofenac. In general, HLM with CYP2C9 *1/*1 genotypes had a rate of diclofenac 4-hydroxylation twice that of either *1/*8 or *1/*5 genotypes, with mean activities of 1.34, 0.67, and 0.61 µmol/min per mg protein, respectively. The CYP2C9 *5/*8 genotype had the lowest activity. Some individuals, however, showed unexpected genotype-activity correlation such as ALT00004 and ALT00035 of CYP2C9*1/*1, which had very low activity when we expected them to have the highest activity (Fig. 7).

Fig. 6.
Fig. 6.

Representative Michaelis-Menten plot for the 0-demethylation reaction of dextromethorphan by the CYP2D6 enzyme.

Fig. 7.
Fig. 7.

The activity of CYP2C9 across a panel of HLM from donors with different CYP2C9 genetic variants.

Isolation and Characterization of Hepatocytes

Four primary human hepatocyte isolations have been performed to date, each utilizing the left liver lobe, which ranged in weight between 110–500 g, with a median weight of 235 g. Successful isolations yielded a maximum cell number of 4 billion cells. Freshly plated primary human hepatocytes were functionally validated for CYP3A4 enzyme activity and/or albumin secretion at ∼18 hours postisolation (Fig. 4). Adherent cells, with a post-thawing cell viability ranging between 65%–82%, were assessed for CYP2C9 enzyme activity. Tissue from the first isolation was genotyped as CYP2C9*1/*1 (normal metabolizer) and had approximately sixfold higher enzyme activity compared with a subsequent tissue donor carrying the CYP2C9*5/*8 (reduced function) genotype, thereby highlighting the potential functional impact of these variants on drugs that rely on CYP2C9 metabolism.

Discussion

To our knowledge, the ALTBio Consortium is the first comprehensive initiative to establish a liver tissue biorepository for drug metabolism, pharmacokinetics, and pharmacogenomics research in Africa. The living-donor liver transplant program at WDGMC, on which this resource is based, is well established, hence sustainable on a long-term basis. Together, the core partners consolidated a critical constellation of exceptional skill sets that enabled this unique platform. It is a timely development as African researchers embark on drug discovery and development programs, which all require support with drug metabolism and pharmacokinetics for the selection and/or design of hits and lead compounds predicted to be safe and efficacious in humans (Winks et al., 2022). The program also offers a practical, affordable, and population-appropriate solution that prioritizes the genomic diversity of African populations in precision medicine (Rajman et al., 2017).

Pharmacogenetic studies across different populations have shown important interindividual and interpopulation differences, with potential clinical implications for the safe and efficacious use of some medicines (Rajman et al., 2017; da Rocha et al., 2021; Mbavha et al., 2022). The genotyping of 27 Black South African samples made available through the ALTBio Consortium indicate allele frequency similarities with the 1000 Genomes Project African superpopulation but important differences from the European, American, and Asian superpopulations (Table 2).

View this table:
TABLE 2

Comparison of population allele frequencies for P450 genes observed in this study (N = 27 Black South Africans genotyped to date) with those observed in the 1000 Genomes Project continental superpopulations labeled African, Eastern Asian, South Asian, European, and American

The letters a–e highlighting statistical significance (P < 0.05) for allele frequency comparison for those obtained in this study versus the world population groups.

View this table:
TABLE 3

Probe substrate reactions and Km and Vmax values expressed as mean of a single experiment run in duplicate

To highlight the clinical relevance of our findings, we confirmed the findings from a previous population study (Mbavha et al., 2022), showing a high frequency (40%) of the IM phenotype for the CYP2C19 gene. In 2022, the Clinical Pharmacogenetics Implementation Consortium published guidelines for the use of clopidogrel, a life-saving antiplatelet drug used to treat those with acute myocardial infarction and peripheral vascular disease and indicated for invasive percutaneous coronary interventions. After administration, clopidogrel is converted from an inactive to active form by CYP2C19; thus, those with one IM or two PM nonfunctional alleles will have lower than expected serum concentrations. Consequently, the Food and Drug Administration–approved drug label for clopidogrel contains a boxed warning regarding the risk of reduced antiplatelet effects of those who are IM or PM, with recommendations for using non-CYP2C19 substrate alternatives, prasugrel or ticagrelor (Lee et al., 2022). In a small observational study from Zimbabwe, a 23% prevalence of CYP2C19 IM was observed in stroke patients taking clopidogrel and was associated with poor treatment outcome (Jayeoba et al., manuscript submitted). This shows the potential utility of the ALTBio resource in early drug discovery and development phases to identify important drug-gene interactions.

Another example pertains to the anticoagulant warfarin, which is metabolized by CYP2C9, where there are African population–specific, low-activity variants such as CYP2C9*5, *8, and *11. These variants have been included in the Clinical Pharmacogenetics Implementation Consortium guidelines to ensure informative and appropriate dosing for people of African origin (Johnson et al., 2017). In samples characterized to date, CYP2C9 variants that lead to low enzyme activity (*5 and *8) were observed (Table 2). These two enzyme variants are almost absent in the other populations, which instead have a higher frequency of the reduced-activity variants CYP2C9*2 and *3. The CYP2C9*8 variant results in a 30% reduction in warfarin clearance (Liu et al., 2012). Furthermore, in vivo studies have shown decreased activity of the *5 variant with losartan metabolism, a frequently prescribed antihypertensive medication (Allabi et al., 2004). Thus far, we have found that CYP2C9*5 and *8 have highly reduced enzyme activity in the metabolism of diclofenac (Fig. 6). Inclusion of these variants in the warfarin dosing algorithm was shown to predict dose optimization more accurately in people of African origin (Hernandez et al., 2014). For CYP2C9, the potential functional effects of African-specific variants have been demonstrated as shown in Fig. 7.

This study reported a high frequency of the low-activity CYP2D6*17 variant of 19% in the study population, which is comparable to what has been observed in previous studies (Mbavha et al., 2022). According to our previous report on tamoxifen therapy, the CYP2D6*17 allele has been predicted to have an activity score of 0.3 (Kanji et al., 2023). Since tamoxifen is a prodrug, homozygous carriers may require dose adjustment to attain therapeutic concentrations of the metabolite. CYP2D6 copy number variation has profound effects on the CYP2D6 phenotype, which might result in ultra-rapid metabolizers. In our study, three of the samples had three copies of the CYP2D6 gene, which are predictive of the ultra-rapid metabolizer phenotype (Table 2).

To demonstrate the functional status of the human liver microsomes, enzyme kinetic studies were done for selected P450s (CYP1A2, 2C9, 2C8, 2D6, and CYP3A). Km and Vmax values obtained in this study are comparable to those in the literature (Li et al., 2002; Spaggiari et al., 2014; Gao et al., 2017); this validates our laboratory protocols for microsome preparation and enzyme kinetic analysis. We therefore believe that the data we will generate from this biorepository will feed in and improve population-based in vitro to in vivo extrapolation algorithms such as those found in SimCYP software (Jamei et al., 2013; Ezuruike et al., 2022). In the hierarchy of in vitro systems for drug metabolism research, PHHs are considered the most physiologically representative of integrated metabolic process, hence likely to give better predictions of in vivo outcomes. They are now considered a standard tool for metabolic clearance, especially for low-clearance compounds, enzyme induction, hepatic uptake, and drug-induced liver injury studies (Sahi et al., 2010; Bell et al., 2016). Successful isolation of PHHs is, however, relatively challenging compared with preparation of subcellular fractions such as S9, cytosol, and microsomes. To the best of our knowledge, our project represents the first successful isolation of functional primary human hepatocytes for metabolism research in Africa. Ongoing work in our program involves the establishment of 3D spheroids as these have been shown to better predict physiologic processes, including drug metabolism in the liver, than the standard PHHs in suspension (Bell et al., 2018; Kanebratt et al., 2021; Xu, 2021).

We are initially encouraging an access model where we carry out the metabolic characterizations for clients instead of providing them with the subcellular fractions or hepatocytes. The ALTBio program therefore includes an industry standard preclinical ADME-PK platform (Masimirembwa and Thelingwani, 2012; Kapungu et al., 2020) with high capacity to support different drug discovery projects with standard assays, such as solubility, permeability, plasma protein binding, and metabolic studies (metabolic clearance, enzyme identification, metabolite identification, drug-drug interactions, reactive metabolite trapping, and drug-induced liver injury).

Conclusion

The ALTBio program is a significant milestone in efforts to improve the understanding of drug metabolism, pharmacokinetics, and pharmacogenomics in people of African origin. The ongoing sample collection framework is sustainable and meets national and international ethical requirements and laboratory best-practice standards. The ongoing biochemical, enzyme kinetic, and pharmacogenetic characterization is already showing important results of value to the drug discovery and development research community.

Acknowledgments

The authors acknowledge the volunteers who donated liver tissue to this program and the scientific and technical input of Emeritus Professor Tommy Andersson.

Note Added in Proof: An incorrect version of Figure 4 was published in the Fast Forward version on September 26, 2023. Figure 4 is now correct.

Data Availability

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Authorship Contributions

Participated in research design: Masimirembwa, Ramsay, Botha, Ellis, Etheredge, Maher, Scholefield, Fabian.

Conducted experiments: Botha, Hurrell, Kanji, Kapungu, Maher, Mthembu, Naidoo, Scholefield, Rambarran, van der Schyff, Smyth, Strobele, Thelingwani, Loveland, Fabian.

Contributed new reagents or analytic tools: Masimirembwa, Ramsay, Ellis, Scholefield.

Performed data analysis: Masimirembwa, Hurrell, Kanji, Kapungu, Maher, Scholefield, Thelingwani, Loveland, Fabian.

Wrote or contributed to the writing of the manuscript: Masimirembwa, Ramsay, Botha, Ellis, Etheredge, Hurrell, Kanji, Kapungu, Maher, Mthembu, Naidoo, Scholefield, Rambarran, van der Schyff, Smyth, Strobele, Thelingwani, Loveland, Fabian.

Footnotes

    • Received May 24, 2023.
    • Accepted September 13, 2023.

  • ↵1J.L. and J.F. contributed equally to this work as joint last authors.

  • This project was self-funded by contributions from the founding four institutions: WDGMC, SBIMB, CSIR, and AiBST.

  • No author has an actual or perceived conflict of interest with the contents of this article.

  • dx.doi.org/10.1124/dmd.123.001400.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

AAC
ALTBio Advisory Committee
ADME
absorption, distribution, metabolism, and excretion
ALTBio
African Liver Tissue Biorepository
AS
activity score
BCA
bicinchoninic acid assay
BSA
bovine serum albumin
CSIR
Council for Scientific and Industrial Research
HLM
human liver microsome
ID
identification
IM
intermediate metabolizer
Km
Michaelis-Menten constant
NCE
new chemical entity
P450
cytochrome P450
PCR
polymerase chain reaction
PHH
primary human hepatocyte
PK
pharmacokinetic
PM
poor metabolizer
[S]
substrate concentration
SNP
Single Nucleotide Polymorphism
WDGMC
Wits Donald Gordon Medical Centre

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Research Article50th Anniversary Celebration Collection

ALTBio Consortium Developed for Drug Metabolism Research

Collen Masimirembwa, Michele Ramsay, Jean Botha, Ewa Ellis, Harriet Etheredge, Tracey Hurrell, Comfort Ropafadzo Kanji, Nyasha Nicole Kapungu, Heather Maher, Busisiwe Mthembu, Jerolen Naidoo, Janine Scholefield, Sharan Rambarran, Francisca van der Schyff, Natalie Smyth, Bernd Strobele, Roslyn Stella Thelingwani, Jerome Loveland and June Fabian
Drug Metabolism and Disposition December 1, 2023, 51 (12) 1551-1560; DOI: https://doi.org/10.1124/dmd.123.001400
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