The use of cultured hepatocytes to investigate the metabolism of drugs and mechanisms of drug hepatotoxicity

Hepatotoxins can be classified as intrinsic when they exert their effects on all individuals in a dose-dependent manner, and as idiosyncratic when their effects are the consequence of an abnormal metabolism of the drug by susceptible individuals (metabolic idiosyncrasy) or of an immune-mediated injury to hepatocytes (allergic hepatitis). Some xenobiotics are electrophilic, and others are biotransformed by the liver into highly reactive metabolites that are usually more toxic than the parent compound. This activation process is the key to many hepatotoxic phenomena. Mitochondria are a frequent target of hepatotoxic drugs, and the alteration of their function has immediate effects on the energy balance of cells (depletion of ATP). Lipid peroxidation, oxidative stress, alteration of Ca(2+) homeostasis, and covalent binding to cell macromolecules are the molecular mechanisms that are frequently involved in the toxicity of xenobiotics. Against these potential hazards, cells have their own defence mechanisms (for example, glutathione, DNA repair, suicide inactivation). Ultimately, toxicity is the balance between bioactivation and detoxification, which determines whether a reactive metabolite elicits a toxic effect. The ultimate goal of in vitro experiments is to generate the type of scientific information needed to identify compounds that are potentially toxic to man. For this purpose, both the design of the experiments and the interpretation of the results are critical.]     

Metabolism Comparative Cytotoxicity Assay (MCCA) and Cytotoxic Metabolic Pathway Identification Assay (CMPIA) with cryopreserved human hepatocytes for the evaluation of metabolism-based cytotoxicity in vitro: proof-of-concept study with aflatoxin B1

Recently, we have improved the cryopreservation procedures for human hepatocytes, leading to cells that can be cultured after thawing (“plateable” cryopreserved human hepatocytes). The ability to culture cryopreserved human hepatocytes allows application of the cells for prolonged incubations such as long-term (days) metabolism studies, enzyme induction studies, and cytotoxicity studies. We report here the application of the plateable cryopreserved human hepatocytes to evaluate the relationship between xenobiotic metabolism and toxicity. Two assays were developed: The Metabolism Comparative Cytotoxicity Assay (MCCA) and the Cytotoxic Metabolic Pathway Identification Assay (CMPIA). The MCCA was designed for the initial identification of the role of metabolism in cytotoxicity by comparing the cytotoxic potential of a toxicant in a metabolically competent (primary human hepatocytes) and a metabolically incompetent (Chinese hamster ovary (CHO)) cell type, as well as the evaluation of the role of P450 metabolism by comparing the cytotoxicity of the toxicant in question in human hepatocytes in the presence and absence of a nonspecific, irreversible P450 inhibitor, 1-aminobenzotriazole (ABT). The CMPIA was designed for the identification of the P450 isoforms involved in metabolic activation via the evaluation of the cytotoxicity of the toxicant in the presence and absence of isoform-selective P450 inhibitors. Results of a proof-of-concept study with the MCCA and CMPIA with a known hepatotoxicant, aflatoxin B1 (AFB1), are reported. AFB1 is known to require P450 metabolism for its toxicity. In the MCCA, AFB1 was found to have significantly higher cytotoxicity in human hepatocytes than CHO cells, therefore confirming its requirement for biotransformation to be toxic. ABT was found to effectively attenuate AFB1 cytotoxicity, confirming that P450 metabolism was involved in its metabolic activation. In the CMPIA, AFB1 cytotoxicity was found to be attenuated by ketoconazole and diethyldithiocarbamate, but not by furafylline, quinidine, and sulfaphenazole. Results with the isoform-selective inhibitors suggest that the isoforms inhibited by ketoconazole (mainly CYP3A4) and diethyldithiocarbamate (mainly CYP2A6, and CYP2E1), but not the isoforms inhibited by furafylline (mainly CYP1A2), sulfaphenazole (mainly CYP2C9) and quinidine (mainly CYP2D6) are involved in the metabolic activation of AFB1. This proof-of-concept study suggests that MCCA and CMPIA with cryopreserved human hepatocytes are potentially useful for the evaluation of the relationship between human xenobiotic metabolism and toxicity.     

Development of an <Emphasis Type="Italic">in vitro</Emphasis> assay for the investigation of metabolism-induced drug hepatotoxicity

In a number of adverse drug reactions leading to hepatotoxicity drug metabolism is thought to be involved by generation of reactive metabolites from nontoxic drugs. In this study, an in vitro assay was developed for measurement of the impact of metabolic activation of compound on the cytotoxicity toward a human hepatic cell line. HepG2 cells were treated for 6 h with compound in the presence or absence of rat liver S9-mix, and the viability was measured using the MTT test. The cytotoxicity of cyclophosphamide was substantially increased by S9-mix in the presence of NADPH. Three NADPH sources were tested: NADPH (1 mmol/L) or NADPH regenerating system with either NADP⁺/glucose 6-phosphate (G6P) or NADP⁺/isocitrate. All three NADPH sources increased the cytotoxicity of cyclophosphamide to a similar extent. Eight test compounds known to cause hepatotoxicity were tested. For these, only the cytotoxicity of diclofenac was increased by S9 enzymes when an NADPH regenerating system was used. The increased toxicity was NADPH dependent. Reactive drug metabolites of diclofenac, formed by NADPH-dependent metabolism, were identified by LC-MS. Furthermore, an increase in toxicity, not related to enzymatic activity but to G6P, was observed for diclofenac and minocycline. Tacrine and amodiaquine displayed decreased toxicity with S9-mix, and carbamazepine, phenytoin, bromfenac and troglitazone were nontoxic at all tested concentrations, with or without S9-mix. The results show that this method, with measurement of the cytotoxicity of a compound in the presence of an extracellular metabolizing system, may be useful in the study of cytotoxicity of drug metabolites.     

Accurate prediction of human drug toxicity: a major challenge in drug development

Over the past decades, a number of drugs have been withdrawn or have required special labeling due to adverse effects observed post-marketing. Species differences in drug toxicity in preclinical safety tests and the lack of sensitive biomarkers and nonrepresentative patient population in clinical trials are probable reasons for the failures in predicting human drug toxicity. It is proposed that toxicology should evolve from an empirical practice to an investigative discipline. Accurate prediction of human drug toxicity requires resources and time to be spent in clearly defining key toxic pathways and corresponding risk factors, which hopefully, will be compensated by the benefits of a lower percentage of clinical failure due to toxicity and a decreased frequency of market withdrawal due to unacceptable adverse drug effects.     

Drug Metabolism for the Perplexed Medicinal Chemist

Two related and significant issues may elicit perplexity in medicinal chemists and are discussed here. First, a broad presentation of the pharmacological and toxicological consequences of drug metabolism should justify the significance of drug metabolism and serve as an incentive to further study. When comparing the pharmacological activities of a drug and its metabolite(s), a continuum is found which ranges from soft drugs (no active metabolites) to prodrugs (inactive per se, as illustrated here with clopidogrel and prasugrel). Innumerable intermediate cases document drugs whose activity is shared by one or more metabolites, as exemplified with tamoxifen. The toxicological consequences of metabolism at the molecular, macromolecular, and macroscopic levels are manyfold. A brief overview is offered together with a summary of the reactions of toxification and detoxification of the antiepileptic valproic acid. The second issue discussed in the review is a comparison of the relative significance of cytochromes P450 and other oxidoreductases (EC 1), hydrolases (EC 3), and transferases (EC 2) in drug metabolism, based on a ‘guesstimate’ of the number of drug metabolites that are known to be produced by them. The conclusion is that oxidoreductases are the main enzymes responsible for the formation of toxic or active metabolites, whereas transferases play the major role in producing inactive and nontoxic metabolites.     

A comprehensive approach for drug safety assessment

A comprehensive, multidisciplinary approach is proposed here for the development of a drug with an acceptable safety profile. Key parameters to be considered for drug safety evaluation based on this comprehensive approach include the following: (1) Pharmacology: Possible toxicity due to drug-target interactions, including interactions with unintended molecular targets, or with molecular targets in unintended organs. (2) Chemistry: Chemical scaffolding and side-chains with safety concerns. (3) Toxicology: Toxicity in animals in vivo, and in relevant animal and human cells in culture. (4) Drug metabolism and pharmacokinetics: Safety concerns due to toxification or detoxification, organ distribution, clearance and pharmacokinetic drug-drug interactions. (5) Risk factors: Physiological, environmental and genetic factors that may enhance a patient's susceptibility. It is proposed that this integrated, multidisciplinary approach to safety evaluation may enhance the accuracy of the prediction of drug safety and thereby the efficiency of drug development.     

Human hepatocytes: isolation, cryopreservation and applications in drug development

The recent developments in the isolation, culturing, and cryopreservation of human hepatocytes, and the application of the cells in drug development are reviewed. Recent advances include the improvement of cryopreservation procedures to allow cell attachment, thereby extending the use of the cells to assays that requires prolong culturing such as enzyme induction studies. Applications of human hepatocytes in drug development include the evaluation of metabolic stability, metabolite profiling and identification, drug-drug interaction potential, and hepatotoxic potential. The use of intact human hepatocytes, because of the complete, undisrupted metabolic pathways and cofactors, allows the development of data more relevant to humans in vivo than tissue fractions such as human liver microsomes. Incorporation of key in vivo factors with the intact hepatocytes in vitro may help predictive human in vivo drug properties. For instance, evaluation of drug metabolism and drug-drug interactions with intact human hepatocytes in 100% human serum may eliminate the need to determine in vivo intracellular concentrations for the extrapolation of in vitro data to in vivo. Co-culturing of hepatocytes and nonhepatic primary cells from other organs in the integrated discrete multiple organ co-culture (IdMOC) may allow the evaluation of multiple organ interactions in drug metabolism and drug toxicity. In conclusion, human hepatocytes represent a critical experimental model for drug development, allowing early evaluation of human drug properties to guide the design and selection of drug candidates with a high probability of clinical success.     

Detection of fungal metabolites showing toxic activity through Artemia salina bioassay

The aim of this study was to detect toxic metabolites from fungi contaminating food and medicinal herbs by applying the toxicity assay to Artemia salina. According to toxicity percentages, the extracts were classified as nontoxic (NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified as T and HT were assayed for mycotoxins. Only 6 out of 71 strains were found to be T (8.5%) for A. salina. Penicillium brevicompactum Dierckx, isolated from sausages, was found to be HT, mainly due to the presence of ochratoxin A and two other unidentified metabolites.     

Metabolism and excretion of acetylchromenes by the migratory grasshopper

The extent of metabolism and excretion of three acetylchromenes (two toxic, one relatively nontoxic) were examined in adult migratory grasshoppers (Melanoplus sanguinipes) following topical administration. Both the total amount excreted (parent plus metabolites) and the proportion of parent compound in the excreta were inversely correlated with contact toxicity. Both toxic and nontoxic acetylchromenes are rapidly absorbed from the cuticle, with maximum excretion of parent and metabolite chromenes from 4 to 8 h posttreatment in each case. Much of the applied compounds (60–80%) apparently remains within the insect, and cannot be recovered by extraction of the insect. Metabolites formed result from simple oxidative and reductive transformations. For all of the compounds tested (including the allatocidin precocene II), the major mode of metabolism results from aliphatic hydroxylation of one of the geminal methyl groups on the chromene. No conjugated metabolites were found in the excreta.     

Effets sur le spermatozoïde humain du Diuron (3-(3,4-dichlorophényl)-1,1-diméthyl-urée) et de l’un de ses produits de transformation, la 3,4-dichloroaniline (3,4-DCA) (Etude préliminaire)

Diuron belongs to the family of halogenophenylureas, one of the main groups of herbicides used for more than 40 years. These herbicides absorb sunlight and can be photochemically transformed in the environment (herbicides are transformed on the soil surface exposed to sunlight) or biotransformed by microorganisms present in soil or in water. The metabolites (chlorohydroxyphenylurea, chlorophenylaniline, respectively) are more toxic than the parent compound, as demonstrated by a bioluminescence inhibition assay performed with a marine bacterium (Vibrio fischeri toxicity test). The lipophilicity of these pesticides makes the cell membrane a target for their action, especially the spermatozoa cell membrane. The aim of this study is to use human spermatozoa to evaluate the effect of this urea pesticide and its biotransformed product on the spermatozoa membrane. We investigated the structural and functional effects of these environmental pollutants on spermatozoa. Three million spermatozoa purified on a 95/47.5% Percoll gradient were suspended in 250 μl of modified Earle’s medium (without phenol red) supplemented with 7.5% of human decomplemented serum. Pesticides (Diuron or 3,4-dichloroaniline (3,4-DCA)) were added at a final concentration of 0.1; 1 and 5 mM. Samples were incubated at room temperature for 24 hours. We show that both Diuron and 3,4-DCA decrease motility and vitality of spermatozoa incubated with the highest concentration of pesticides. Our preliminary results show that the effects are more rapid and more intense with the biotransformed product (3,4-DCA) than with Diuron. Addition of herbicide to human spermatozoa increases membrane fluidity, assessed by measuring the fluorescence polarisation anisotropy with a fluorescent probe: 1,6-diphenyl-1,3,5-hexatriene (DPH). Changes in membrane fluidity may be a primary toxic effect of these herbicides. These results suggest that human spermatozoa may constitute a valuable indicator of the toxic effects of pesticides.     

肿瘤化疗与药物代谢酶

药物代谢酶(DME)在药物代谢解毒和药物代谢活化中起着重要的作用,对组织器官的药物效应和毒性的易感性产生重要影响。DME在肿瘤组织和非肿瘤组织表达和活性存在差异。与常用化疗药物有关的药物代谢酶主要有细胞色素P450(CYP)、谷胱甘肽S-转移酶(GST)、尿苷二磷酸-葡萄糖醛酸转移酶(UGT)、巯嘌呤甲基转移酶(TPMT)和二氢嘧啶脱氢酶(DPD),这些酶均具有遗传多态性,在一定条件下可以被诱导,具有个体差异。     

Thermodynamically based profiling of drug metabolism and drug-drug metabolic interactions: a case study of acetaminophen and ethanol toxic interaction

Drug-drug metabolic interactions can result in unwanted side effects, including reduced drug efficacy and formation of toxic metabolic intermediates. In this work, thermodynamic constraints on non-equilibrium metabolite concentrations are used to reveal the biochemical interactions between the metabolic pathways of ethanol and acetaminophen (N-acetyl-p-aminophenol), two drugs known to interact unfavorably. It is known that many reactions of these pathways are coupled to the central energy metabolic reactions through a number of metabolites and the cellular redox potential. Based on these observations, a metabolic network model has been constructed and a database of thermodynamic properties for all participating metabolites and reactions has been compiled. Constraint-based computational analysis of the feasible metabolite concentrations reveals that the non-toxic pathways for APAP metabolism and the pathway for detoxifying N-acetyl-p-benzoquinoneimine (NAPQI) are inhibited by network interactions with ethanol metabolism. These results point to the potential utility of thermodynamically based profiling of metabolic network interactions in screening of drug candidates and analysis of potential toxicity.     

Structural Alerts,Reactive Metabolites,and Protein Covalent Binding: How Reliable Are These Attributes as Predictors of Drug Toxicity?

In an increasing number of cases, a deeper understanding of the biochemical basis for idiosyncratic adverse drug reactions (IADRs) has aided to replace a vague perception of a chemical class effect with a sharper picture of individual molecular peculiarity. Considering that IADRs are too complex to duplicate in a test tube, and their idiosyncratic nature precludes prospective clinical studies, it is currently impossible to predict which new drugs will be associated with a significant incidence of toxicity. Because it is now widely appreciated that reactive metabolites, as opposed to the parent molecules from which they are derived, are responsible for the pathogenesis of some IADRs, the propensity of drug candidates to form reactive metabolites is generally considered a liability. Procedures have been implemented to monitor reactive‐metabolite formation in discovery with the ultimate goal of eliminating or minimizing the liability via rational structural modification of the problematic chemical series. While such mechanistic studies have provided retrospective insight into the metabolic pathways which lead to reactive metabolite formation with toxic compounds, their ability to accurately predict the IADR potential of new drug candidates has been challenged. There are several instances of drugs that form reactive metabolites, but only a fraction thereof cause toxicity. This review article will outline current approaches to evaluate bioactivation potential of new compounds with particular emphasis on the advantages and limitation of these assays. Plausible reason(s) for the excellent safety record of certain drugs susceptible to bioactivation will also be explored and should provide valuable guidance in the use of reactive‐metabolite assessments when nominating drug candidates for development.     

Some distinctions between flavin-containing and cytochrome P450 monooxygenases

This minireview summarizes information concerning the differences and similarities of the human flavin-containing- (FMO, E.C. 1.14.13.8) and the cytochrome P450-monooxygenases (CYP, E.C. 1.14.14.1). Human FMO oxygenates soft nucleophiles. CYP mainly catalyzes C-H abstraction but also oxidizes nitrogen- and sulfur-containing compounds. Both FMO and CYP generally convert lipophilic compounds into more hydrophilic metabolites. The mechanism by which each monooxygenase operates is quite distinct. Sometimes, CYP or FMO bioactivate chemicals to reactive metabolites but to date, drug toxicity thus far observed in the clinic is mainly the result of CYP-dependent oxidation. Both FMO and CYP possess genetic variability that may contribute to inter-individual variability observed for drug metabolism. In contrast to CYP, FMO is not induced or readily inhibited and potential adverse drug-drug interactions are minimized for drugs prominently metabolized by FMO. These properties may provide advantages in drug design, and by incorporating FMO detoxication pathways into drug candidates, more drug-like materials may emerge.     

The relevance of xenobiotic metabolism in the interindividual susceptibility to chemicals

Biotransformation enzymes may catalyze either detoxication or bioactivation reactions; indeed, many xenobiotics exert their toxic effects after metabolic activation to electrophilic chemicals, interacting with nucleophilic sites on cellular macromolecules. On the other hand, by increasing xenobiotic hydrophilicity, the drug-metabolizing enzymes favors excretion of lipophilic chemicals, not allowing their bioaccumulation up to toxic levels. The expression of the enzymes of the drug-metabolizing system is modulated by genetic, pathological, developmental, environmental and dietary factors. Genetic polymorphism resulting in interindividual and interethnic variation in xenobiotic metabolism is responsible for differences in the susceptibility to chemical-induced toxicity and carcinogenicity, allowing the identification of people at increased risk. Moreover, differences in drug metabolism may correspond to variability in drug response during pharmacological therapy, which can be manifest either as adverse reactions or as a lack of benefit.     

Potential drug targets on insomnia and intervention effects of Jujuboside A through metabolic pathway analysis as revealed by UPLC/ESI-SYNAPT-HDMS coupled with pattern recognition approach

Potential metabolites from the metabolic pathways could be therapeutic targets and useful for the discovery of broad spectrum drugs. UPLC/ESI-SYNAPT-HDMS coupled with pattern recognition methods including PCA, PLS-DA, OPLS-DA and Heatmap were integrated to examine the global metabolic signature of insomnia and intervention effects of Jujuboside A (JuA). Six unique pathways of the insomnia were identified using Ingenuity Pathway Analysis (IPA) software. The VIP-value threshold cutoff of the metabolites was set to 10, above this threshold, were filtered out as potential target biomarkers. Sixteen distinct metabolites were identified from these pathways, and 6 of them can be considered for rational drug design. It was further experimental validation that the changes in metabolic profiling were restored to their baseline values after JuA treatment according to the multivariate data analysis. Potential metabolite network of the insomnia was preliminarily predicted JuA-target interaction networks, and could be further explored for in silico docking studies with suitable drugs. Thus, our method is an efficient procedure for drug target identification through metabolic analysis. It can guide testable predictions, provide insights into drug action mechanisms and enable us to increase research productivity toward metabolomic drug discovery.     

Use of an antiarrhythmic drug against acute selenium toxicity

ObjectiveSelenium is an essential trace element. But, selenium may have toxic effects in high doses. There are no proven antidotes or curative treatments for acut selenium toxicity. Treatment involves stopping the exposure and providing supportive care for symptoms. Therefore, it is necessary to find more effective substances in the treatment of selenium toxicity. The aim of this study was to increase the survival rate of animals by supporting the heart with amiodarone and to determine the effect of amiodarone on the pathological, hematological and biochemical parameters in acute selenium intoxication.Methods64 Wistar-Albino rats were divided into four groups. Group I was given only distilled water, Group II was given 18 mg/kg dose of amiodarone, Group III was given 18 mg/kg amiodarone and 10 mg/kg sodium selenite and Group IV was given sodium selenite 10 mg/kg (LD₅₀ dose)orally.Results11 of the 16 animals in Group IV died within the first 48 h of drug administration. However, no deaths were observed in the rats in Group III. No hematological changes were observed. Biochemically, CK, CK-MB and LDH levels of Group IV were higher than the other groups on both the 2nd and 10th days. In Groups II and III, this serum level decreased, and vitamin B12 levels increased. In macroscopic inspections of the organs of Groups III and IV, slight paleness was detected. Histopathologically, degenerative changes in tissue were observed, especially in Group IV.ConclusionThis study shows that amiodarone application has a reducing effect on selenium toxicity. This was because amiodarone protected the heart by reducing CK and CK-MB levels and increased vitamin B12 levels, which play a role in the synthesis of S-adenosyl methionine that converts selenium into a nontoxic form.     

Background and overview of current sediment toxicity identification evaluation procedures

Laboratory bioassays can provide an integrated assessment of the potential toxicity of contaminated sediments to aquatic organisms; however, toxicity as a sole endpoint is not particularly useful in terms of identifying remedial options. To focus possible remediation (e.g., source control), it is essential to know which contaminants are responsible for toxicity. Unfortunately, contaminated sediments can contain literally thousands of potentially toxic compounds. Methods which rely solely on correlation to identify contaminants responsible for toxicity are limited in several aspects: (a) actual compounds causing toxicity might not be measured, (b) concentrations of potentially toxic compounds may covary, (c) it may be difficult to assess the bioavailability of contaminants measured in a sediment, and (d) interactions may not be accounted for among potential toxicants (e.g., additivity). Toxicity identification evaluation (TIE) procedures attempt to circumvent these problems by using toxicity-based fractionation procedures to implicate specific contaminants as causative toxicants. Phase I of TIE characterizes the general physio-chemical nature of sample toxicants. Phase II employs methods to measure toxicants via different analytical methods, and Phase III consists of techniques to confirm that the suspect toxicants identified in Phases I and II of the TIE actually are responsible for toxicity. These TIE procedures have been used to investigate the toxicity of a variety of samples, including sediments. Herein we present a brief conceptual overview of the TIE process, and discuss specific considerations associated with sediment TIE research. Points addressed include: (a) selection and preparation of appropriate test fractions, (b) use of benthic organisms for sediment TIE work, and (c) methods for the identification of common sediment contaminants.     

Risk assessment and mitigation strategies for reactive metabolites in drug discovery and development

Drug toxicity is a leading cause of attrition of candidate drugs during drug development as well as of withdrawal of drugs post-licensing due to adverse drug reactions in man. These adverse drug reactions cause a broad range of clinically severe conditions including both highly reproducible and dose dependent toxicities as well as relatively infrequent and idiosyncratic adverse events. The underlying risk factors can be split into two groups: (1) drug-related and (2) patient-related. The drug-related risk factors include metabolic factors that determine the propensity of a molecule to form toxic reactive metabolites (RMs), and the RM and non-RM mediated mechanisms which cause cell and tissue injury. Patient related risk factors may vary markedly between individuals, and encompass genetic and non-genetic processes, e.g. environmental, that influence the disposition of drugs and their metabolites, the nature of the adverse responses elicited and the resulting biological consequences. We describe a new strategy, which builds upon the strategies used currently within numerous pharmaceutical companies to avoid and minimize RM formation during drug discovery, and that is intended to reduce the likelihood that candidate drugs will cause toxicity in the human population. The new strategy addresses drug-related safety hazards, but not patient-related risk factors. A common target organ of toxicity is the liver and to decrease the likelihood that candidate drugs will cause liver toxicity (both non-idiosyncratic and idiosyncratic), we propose use of an in vitro Hepatic Liability Panel alongside in vitro methods for the detection of RMs. This will enable design and selection of compounds in discovery that have reduced propensity to cause liver toxicity. In vitro Hepatic Liability is assessed using toxicity assays that quantify: CYP 450 dependent and CYP 450 independent cell toxicity; mitochondrial impairment; and inhibition of the Bile Salt Export Pump. Prior to progression into development, a Hepatotoxicity Hazard Matrix combines data from the Hepatic Liability Panel with the Estimated RM Body Burden. The latter is defined as the level of covalent binding of radiolabelled drug to human hepatocyte proteins in vitro adjusted for the predicted human dose. We exemplify the potential value of this approach by consideration of the thiazolidinedione class of drugs.     

Filamentous fungal biofilm for production of human drug metabolites

In drug development, access to drug metabolites is essential for assessment of toxicity and pharmacokinetic studies. Metabolites are usually acquired via chemical synthesis, although biological production is potentially more efficient with fewer waste management issues. A significant problem with the biological approach is the effective half-life of the biocatalyst, which can be resolved by immobilisation. The fungus Cunninghamella elegans is well established as a model of mammalian metabolism, although it has not yet been used to produce metabolites on a large scale. Here, we describe immobilisation of C. elegans as a biofilm, which can transform drugs to important human metabolites. The biofilm was cultivated on hydrophilic microtiter plates and in shake flasks containing a steel spring in contact with the glass. Fluorescence and confocal scanning laser microscopy revealed that the biofilm was composed of a dense network of hyphae, and biochemical analysis demonstrated that the matrix was predominantly polysaccharide. The medium composition was crucial for both biofilm formation and biotransformation of flurbiprofen. In shake flasks, the biofilm transformed 86 % of the flurbiprofen added to hydroxylated metabolites within 24 h, which was slightly more than planktonic cultures (76 %). The biofilm had a longer effective lifetime than the planktonic cells, which underwent lysis after 2?×?72 h cycles, and diluting the Sabouraud dextrose broth enabled the thickness of the biofilm to be controlled while retaining transformation efficiency. Thus, C. elegans biofilm has the potential to be applied as a robust biocatalyst for the production of human drug metabolites required for drug development.