Biotransformation: Impact and Application of Metabolism in Drug Discovery

Abstract

Biotransformation has a huge impact on the efficacy and safety of drugs. Ultimately the effects of metabolism can be the lynchpin in the discovery and development cycle of a new drug. This article discusses the impact and application of biotransformation of drugs by mammalian systems, microorganisms, and recombinant enzymes, covering active and reactive metabolites, the impact of the gut microbiome on metabolism, and how insights gained from biotransformation studies can influence drug design from the combined perspectives of a CRO specializing in a range of biotransformation techniques and pharma biotransformation scientists. We include a commentary on how biology-driven approaches can complement medicinal chemistry strategies in drug optimization and the in vitro and surrogate systems available to explore and exploit biotransformation.

Figure 1

Late-occurring and long-circulating CYP-derived plasma metabolites of AZD7325 formed via metabolic cyclization and aromatization following repeat dosing in humans and preclinical animals. Adapted from Gu et al., 2018, under the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

Figure 2

Microbial biotransformation of tolterodine to form the active metabolite 5-hydroxymethyl tolterodine, a pro-drug of which, fesoterodine, replaced the original drug to circumvent metabolism by the polymorphic CYP2D6 and eliminate undesirable side effects.

Figure 3

Biotransformation of regorafenib to multiple phase I and phase II metabolites, resulting in an increase in complexity of its disposition. The main circulating active metabolites at steady-state in human plasma are M2 (N-oxide) and M5 (demethylated N-oxide). Adapted from Figure 4 in Gerisch et al., 2018, under the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

Figure 4

Comparison of the different pharmacological properties of midostaurin and active metabolites CGP62221 and CGP52421 produced by CYP3A4.^,

Figure 5

Biotransformation of the drug valsartan by microbial whole cell biotransformation and recombinant microbial enzymes to yield hydroxylated metabolites, one of which is the product of human liver microsomal biotransformation of valsartan by CYP2C9.

Figure 6

Metabolism inspired design of ezetimibe from SCH48461, and ezetimibe’s major metabolic route to a major O-glucuronide by UGTs 1A1, 1A3, and 2B15.

Figure 7

Metabolism of drugs approved to treat tardive dyskinesia through inhibition of VMAT2 (vesicular monoamine 2 transporter). Austedo uses a deuterated form of tetrabenazine (3R, 11bR enantiomer shown) to improve exposure of hydroxylated active metabolites. In contrast Ingrezza uses a prodrug approach involving hydrolysis of the valine ester to a specific hydroxylated active metabolite.^,

Figure 8

Major metabolic pathways of ketamine (HNK= Hydroxynorketamine). Major circulating metabolites of ketamine in plasma are boxed in blue. Adapted from Figure 1 with permission from Zanos P. et al. Ketamine and ketamine metabolite pharmacology. Pharmacol. Rev.2018, 70 (3), 630. Copyright 2018 ASPET.

Figure 9

Production of the major N-glucuronide metabolite of moludistat by microbial fermentation.

Figure 10

Late stage oxidation of a phosphodiesterase 2A (PDE2) inhibitor lead compound using a biotransformation approach to generate lead candidate PF06815189, which possessed “exquisite drug-like attributes”.

Figure 11

Late-stage oxidation of a protein kinase inhibitor through microbial biotransformation resulting in a hydroxylated derivative with improved potency, as well as achieving the desired reduction in lipophilicity.

Figure 12

(a) Gram scale production of the main human metabolite of the epothilone analogue, sagopilone, by human CYP2C19 expressed in E. coli. A 100 L scale biotransformation of sagopilone resulted in 5 g of metabolite after 23 h at an isolated yield of 54%. (b) Selection of human CYP, AO, and FMO3 derived metabolites and other oxidized analogues synthesized by recombinant enzymes in the PolyCYPs+ screening kit. Reactions catalyzed by recombinant microbial PolyCYPs isoforms include aliphatic and aromatic hydroxylation, carboxylation, epoxidation, and dealkylation.

Figure 13

Biotransformation of the HIV-1 protease inhibitor ritonavir to oxidized metabolites by recombinant microbial CYP enzymes. In humans the main metabolite formed is ε-HO-N-methylisopropylthiazoloylmethyl-ritonavir (M2) by the action of CYP3A4 and CYP2D6.

Figure 14

Provision of human CYP and non-CYP metabolites M2 (20.1 mg), M4 (66.2 mg), and M12 (18.4 mg) of Samatolisib at multi-milligram scale utilizing a mixed approach of microbial biotransformation, chemical synthesis, and liver S9 incubations.

Figure 15

Biotransformation of ingenol disoxate to its main human metabolite (M27), using a microbial strain. M27 is a metabolite that was more prevalent in humans than in preclinical species, i.e. a disproportionate metabolite, and thus subject to the FDA’s MIST guidelines. Yield of M27 was optimized by early harvest of the reaction to prevent onward oxidation to dihydroxylated derivatives, resulting in the purification of 100s of milligrams of the metabolite for further evaluation. M27 was shown to have similar pharmacological effects to the parent compound but was less potent.

Figure 16

Hydroxylated metabolites of ruxolitinib produced by a single bacterial species, following a panel screen consisting of multiple microbial species. Keto derivatives were also produced and identified.

Figure 17

Proposed pathway for the CYP/AOX dependent metabolism of momelotinib. Reprinted in part with permission from Zheng, J.; et al. Drug Metab. Dispos., 2018, 46, 237–247. Copyright 2018, ASPET.

Figure 18

Proposed pathway for the CYP3A dependent metabolism and bioactivation of lapatinib. Reprinted in part with permission from Bissada, J.E. et al. Drug Metab. Dispos., 2019, 47, 1257–1269. Copyright 2019, ASPET.

Figure 19

Proposed pathway for the CYP and ADH/ALDH dependent metabolism of NBP to its major circulating metabolite, M5-2. Reprinted in part with permission from Diao, X. et al. Drug Metab. Dispos., 2013, 41, 430–444. Copyright 2013, ASPET.

Figure 20

Primary route of bromfenac bioactivation is initiated by glucuronidation and culminates in CYP-dependent quinone imine/methide reactive metabolite formation.

Figure 21

Major metabolites of epacadostat produced by microbial biotransformation via mixed metabolic pathways. Scale-up of the most productive biotransforming microbes for M9 and M11 enabled the supply of 112 mg of the glucuronide (M9) and 69 mg of the gut metabolite (M11).

Figure 22

Metabolism of BILR 355 involves a complex interplay between CYP-, AO-, and gut microbiota-mediated processes.