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This scientific symposium took place in September 2019, under the auspices of the Delaware Valley Drug Metabolism Discussion Group (DVDMDG).
Andy Goodman’s (Yale) title was “Microbiome Contributions to Drug Metabolism.” Gut bacterial metabolism can reduce drug efficacy and either increase or reduce toxicity; bacterial drug metabolites can be absorbed, distributed systemically in the host, and excreted in the urine; and the variability between individuals (and even within the same individual over time) makes it almost impossible to predict the impact of the gut microbiome on the metabolism of a given drug in a given patient at a given time. Can the gut microbiome account for interpersonal differences in drug response? Maybe so…in humans, there is little overlap in genes across individuals. Bacteria can metabolize a large number of drugs across a wide range of therapeutic areas, but in most cases it is challenging to estimate, quantitatively, how much gut bacteria contribute to the overall metabolism of a given drug in vivo, especially for metabolites that can be produced by the host as well. The question was approached by studying the metabolism of 271 FDA-approved drugs by 76 gene-sequenced gut bacterial isolates, using high-throughput LC-MS. The common features of metabolized drugs (~2/3 of the total) and non-metabolized drugs were identified. Candidate drug metabolites were identified by untargeted mass spectrometry, and the change in structure deduced based on the mass shift vs. the parent drug. Although many bacteria may encode a given enzyme, not all bacteria use it for drug metabolism. Bacterial drug-metabolizing enzymes were identified using a gain-of-function strategy by cloning DNA fragments into E. coli. Enzyme expression was associated with drug-metabolizing activity across human gut species, and complex network diagrams of drugs/enzymes/metabolites were created. Using the drug brivudine as a probe, a loss-of-function strategy was also used, by colonizing the gut of germ-free mice with a wild-type strain of a bacterial species or a mutant strain missing a particular enzyme, to quantify the contribution of gut bacteria to the formation of a toxic drug metabolite via a pathway (dihydropyrimidine dehydrogenase) that is also expressed by the host. This led to the development of a PBPK model of microbiome-metabolized drugs to estimate and predict the gut microbiome’s contribution to systemic exposure of drug metabolites. Much of this monumental work was published recently in Nature (Zimmermann, et al., Nature. 2019; 570:462-467). In my humble opinion, this paper will be the foundation for much of the future research in this area.
Jason Boer’s (Incyte Corporation) title was “Integrating Drug Metabolism via the Gut Microbiome into the Drug Discovery and Development Paradigm.” He discussed two examples of drugs where major circulating human metabolites were not identified in vitro due to the involvement of gut bacteria in vivo. One drug candidate, an HIV drug co-dosed with ritonavir, produced a disproportionate human metabolite, which is the product of host aldehyde oxidase (AO) activity on a reduced intermediate produced by gut bacteria. Because the drug is co-dosed with the CYP3A4 inhibitor ritonavir, the drug is shunted through the reduced intermediate, which would otherwise be oxidized back to the parent by CYP3A4. The metabolite is not seen in preclinical tox species because their AO activity is very low. Another drug, epacadostat, has multiple metabolites, including M11 and M12, which is seen only in vivo. M11 is produced in vitro by human feces, and M12, a secondary metabolite produced from M11 by CYPs, is not seen when the parent drug is incubated in vitro because under those conditions there is no M11 (produced by gut bacteria). The question is whether examples such as these will, or should, motivate drug companies to incorporate gut bacterial drug metabolism into their drug discovery and development workflow. I think they should, because now that medicinal chemists have largely figured out how to avoid CYP-mediated drug metabolism, gut bacterial drug metabolism will become more common.
Aaron Wright’s (Pacific Northwest National Lab and Washington State University) title was “Functional Characterization of Drug Metabolizing Enzymes at the Host-Gut Microbiome Interface.” His approach is activity-based protein profiling (ABPP), which provides a direct measure of protein function and enables identification of the enzymes responsible for a given function. Custom-designed activity-based probes bind irreversibly to functionally active protein targets, followed by click chemistry (N3 attached to a fluorophore or biotin), enrichment, and either proteomics (by LC-MS), SDS-PAGE, fluorescence microscopy, or flow cytometry. The most abundant human bacteria (gut, skin) vary by geographical region, and even within a region, which partially invalidates the general assumption that people are going to metabolize a given drug similarly, regardless of where they live. This is also true in mice: different litters in the same environment differ significantly in gut microbiome. Individual susceptibility to PAHs such as benzo[a]pyrene (activated to toxic species by CYPs, followed by covalent binding to DNA or elimination by glucuronidation or sulfation) was investigated. A study of enterohepatic recirculation, in which gut bacteria convert glucuronides (excreted via bile) to the corresponding aglycone via beta-glucuronidase, revealed that phylogenetically disparate populations can reconstitute that enzyme activity following antibiotic treatment. Therefore, microbial composition (in terms of phylogeny) is a poor predictor of gut metabolic activity; what’s really relevant are the bacterial genes, which is consistent with several of the other presentations from this meeting. And activity-based protein profiling is a really powerful approach.
View Part II Here >>
Peter Spanogiannopoulos’s (UCSF) title was “A Prevalent Operon from the Human Gut Microbiome is Responsible for the Inactivation of Fluoropyrimidine Anticancer Drugs.” Host-targeted drugs impact the growth of gut bacteria and the composition of the gut microbiome. 5-Fluorouracil (5FU) is a thymidylate synthase inhibitor and anticancer drug that acts by interfering with pyrimidine (thymidine) synthesis. 5FU is inactivated by reduction to dihydrofluorouracil (DHFU). 5FU has antibacterial activity by the same mechanism as the anti-cancer activity in the host (pyrimidine metabolism is highly conserved from bacteria through mammals). Closely related bacteria have very different susceptibility to 5-FU; one mechanism of 5FU resistance is inactivation of the drug. Proteobacteria (e.g., E. coli, Salmonella enterica) and Firmicutes can inactivate 5FU to DHFU. It was demonstrated conclusively and convincingly that a specific E. coli operon is responsible for 5FU inactivation, but I will not go into detail because the work is unpublished. This is just one of many examples of unintended consequences involving gut bacteria…an anticancer drug that kills some gut bacteria and is inactivated by others (thereby shifting the balance toward the latter), which in turn impacts its efficacy.
Joe Dempsey’s (University of Washington) title was “A Multi-omic Approach to Understand the Development of the Gut-Liver Axis.” The neonatal human gut microbiome is relatively aerobic and high in Lactobacillus, Difidobacterium, Staphylococcus, and Enterococcus; in adults it is more diverse, anaerobic, and high in Firmicutes (with increases in Proteobacteria and Bacteroidetes after the age of 70). Multi-omic approach: genomics, metabolomics, transcriptomics, and proteomics. Age-related changes in the gut microbiome modulate the metabolome within the gut-liver axis, altering the expression and function of xenobiotic-processing genes. Primary bile acids are synthesized (including conjugation with taurine or glycine) in the liver, and deconjugated and converted to secondary bile acids in the gut, and age-related changes in bile acid composition correlate with changes in the function and composition of the gut microbiome. PXR in the host is activated by the secondary bile acid lithocholic acid (LCA), which is a product of the gut microbiome. Not surprisingly, the expression and activity of Cyp3a11 are much lower in germ-free mice (no gut bacteria, no LCA) than in conventional mice. Products of host metabolism (bile acids) impact the gut bacteria (see David Shen’s talk), and products of gut bacterial metabolism (LCA and others) impact the host (PXR activation, leading to induction of CYPs, etc.). It’s a recurring theme, and it can no longer be ignored. Based on work from this lab (Julia Cui’s group), I’m pretty sure that variations in gut bacteria account for the tremendous variation in CYP3A4 activity among humans.
David Shen’s (Penn) title was “Physiologic Implications of Co-metabolism Between the Gut Microbiome and its Host.” The host produces urea and primary bile acids, which are metabolized to ammonia and secondary bile acids, respectively, by gut bacteria. Bacterial products of bile acid metabolism impact the host, for example, as FXR agonists, and FXR activation, in turn, modulates the small intestinal microbiome. Bile acids are toxic to gram-positive bacteria. Obeticholic acid (OCA), derived from chenodeoxycholic acid (CDCA) is a potent, selective FXR agonist. In clinical trials of OCA in primary biliary cholangitis, increases in Gram-positive bacteria (due to decreased bile acids) were seen in the stool. This bacterial taxonomic signature in response to OCA in humans may be due to changes in the small intestine (especially the proximal small intestine), which has a different population of bacteria than the large intestine. Manipulation of gut bacteria to treat disease: bacteria in the colon convert urea to ammonia via urease, which is not expressed by mammals. In a properly prepared mouse host, inoculation with a urease-deficient consortium of bacteria results in long-term reduction in fecal ammonia, and reduces morbidity and mortality in a mouse model of liver disease. Another example of bilateral gut bacteria↔host interactions. It also makes me wonder about the impact of bacteria in the small intestine (as opposed to the large intestine, which is where we normally think of gut bacteria because they are more abundant there) on drug metabolism.
Julia Cui’s (University of Washington) title was “Gut-Liver Axis and Environmental Chemical Exposures.” Polybrominated diphenyl ethers (PBDEs) are fat-soluble, highly persistent, and bioaccumulative. Very high levels of PBDEs are found in human breast milk and in the blood of adult humans. Neonatal exposure to PBDEs is associated with an increased risk of disease (hypothyroid, low IQ, reproductive tox, hepatotox, cancer, diabetes) later in life. The gut microbiome may inactivate PBDEs (conventional vs. germ-free mice). Persistent environmental chemicals such as bisphenol A (BPA), PBDEs, and polychlorinated biphenyls (PCBs) alter the gut microbiome with corresponding changes in the levels of bacterial metabolites such as succinate, acetate, and lactate in the liver, resulting in persistent epigenetic and transcriptomic changes in adult mice that were exposed as neonates. The results suggest that the hepatic reprogramming amounts to microbial metabolite-mediated epigenetic regulation of the host. More on the theme of bilateral gut bacteria↔host interactions.
Seth Walk’s (Montana State University) title was “Microbiome Determinants of Arsenic Toxicity,” and he also presented some recently published results on gut bacterial residency. Arsenic in drinking water is a major global health threat (ranked #1 on the Toxic Substance and Disease Registry since 1997). Gut microbiome has a highly beneficial effect with regard to arsenic toxicity, due to expression of arsenic (As, +3 oxidation state) methyltransferase (AS3MT). With a small set of human stool donors, there was a correlation between bacterial species and protection vs. arsenic toxicity in mice, and the effect of individual bacterial species is detectable in the hypersensitive AS3MT knockout mouse model. Note: some bacteria cannot colonize the gut of germ-free mice by themselves and require a partner such as E. coli. Bioaccumulation of arsenic in gut bacteria appears to reduce toxicity. Gut bacterial residency: The stability of resident gut bacteria depends on how you look at it. In the human gut, some bacterial clones are extremely stable, others are changing all the time. The E. coli genome is highly diverse; only 20% of genes are present in all strains. The resolution of 16S RNA is low; therefore, at the level of 16S RNA sequencing (relevant to higher taxonomic levels), resident gut bacteria appear to be extremely stable. However, at the clonal level of one common gut bacterial family, there is very little stability. I have to admit that the terminology and computational methods used to evaluate gut bacterial residency is well outside of my comfort zone. But one conclusion is that you shouldn’t have to take probiotics every day, as the manufacturers recommend…if you do, they’re not working the way they’re supposed to.
Wednesday, November 20, 2019
Analytical Development Summit
Presenter: Karen Doucette, MBA
Director of Operations, ACF Bioservices
What you will learn: