Metabolism of Xenobiotics in Gut
Small intestine has been predominantly considered as the main site for absorption of water, nutrients, and various xenobiotics. However, the role of the intestinal tract has enormously expanded by discovering its huge metabolic capacity and potential implication in metabolism of xenobiotics. Metabolism of drugs and other orally ingested xenobiotics at intestinal level may largely affect bioavailability of certain drugs (Kaminsky and Zhang, 2003). Gastrointestinal enzymes are classified as luminal enzymes, gut wall/mucosal enzymes and bacterial enzymes (Stojancevic et ah, 2014).
Luminal and Cell Wall Metabolism of Drugs
The lumen and mucosa of the gastrointestinal tract are sites of high enzymatic activity, thus can affect the faith of orally administered compounds prior to their entry into the blood.
Luminal enzymes present in the gut fluids include enzymes from pancreatic and intestinal secretions. Peptidases present in gastrointestinal and pancreatic secretions split the amide linkages contributing to inactivation of some protein and polypeptide drugs such as insulin, calcitonin, thyrotropin releasing hormone. Ester type drugs are hydrolyzed by esterases that are also present in the intestinal tract and mucosa playing an important role in activation of many drugs administered in the form of pro drugs (Gavhane and Yadav, 2012).
Intestinal mucosa contains both phase I and phase II enzymes. Although the intestine is rich in enzymatic activity along its entire length, enzymes of the proximal small intestine is much more active compared to the colonic mucosa (Thelen and Dressman, 2009). Interindividual variations in expression of metabolic enzymes, are more manifested in the intestine compared to the same enzymes in the liver (Kaminsky and Zhang, 2003). Among various cytochromes identified in human intestinal tract such as CYP1A1, CYP2D6, CYP2E1, CYP3A4 and CYP3A5, the most important is CYP3A4 which concentration reaches 80-100% of the CYP3A4 concentration in the liver (Gavhane and Yadav, 2012; Stojancevic et al., 2014; Wacher et al., 2001). Synergistic action of P-glycoprotein (P-gp) and CYP3A4 may significantly limit the absorption, thus affecting the bioavailability of various drugs and other xenobiotics (Martinez and Amidon, 2002). Additionally, various phase II enzymes such as UDP-glucuronosyltransferases, glutathione S-transferases, sulfotransferases, /V-acetyl transferases and methyltransferases are also found in the human intestinal mucosa, with even higher concentrations for some of them compared to liver (Wacher et al., 2001).
Gut Microflora Implication in Xenobiotic Metabolism
Gut microflora (or intestinal microbiota) represents a complex ecosystem consisted of a various microorganisms residing in or passing through the gastrointestinal tract, which exert a significant influence on the host in homeostasis and disease. The number of microorganisms in gastrointestinal tract has been estimated to exceed 1014 containing at least 100-fold more genes than the complete human genome (Thursby and Juge, 2017; Stojancevic et al., 2014]. The majority of the human intestinal bacteria belongs to obligate anaerobes including species of the genera Bacteroides, Clostridium, Bifidobacterium, Lactobacillus, Escherichia (Wilson and Nicholson, 2009).
Although the intestinal microflora has been mostly studied in the context of host health benefit for the host, it has recently become clear that this microbial community has an important role in metabolism of many endogenous and exogenous compounds by providing additional enzymatic activities involved in the biotransformation (Stojancevic et al., 2014). The involvement of gut flora in the metabolism of some endogenous compounds has been well known for a long time. For example, the major modifications of bile acids including 7a-dehydroxylation, deconjugation, and oxidation/epimerization of hydroxyl groups at C-3, C-7 and C-12 are carried out by a broad spectrum of intestinal anaerobic bacteria in human colon (Banic et al., 2018; Djanic et al., 2016; Lazarevic et al., 2019; Monte et al., 2009; Pavlovic et al., 2018; Ridlon et al., 2006; Stojancevic et al., 2013). However, the role of intestinal bacteria in drug metabolism has been still insufficiently explored. Some researchers consider gut microflora as a special organ with huge metabolic potential that complements the enzymatic activity of liver, which may bring about the alteration in the drug disposition and toxicity (Wilson and Nicholson, 2009). The composition of intestinal bacteria may be affected by a variety of endogenous and exogenous factors such as diseases, antibiotic treatment, diet and the maternal environment (Thursby and Juge, 2017). As a consequence, bacterial composition is specific for each individual, thus being the cause of interindividual differences in therapeutic response to drugs (Stojancevic et al., 2014). Therefore, the screening of microbial metabolism should be included in drug development process.
Drugs that may be suitable candidates for exposure and metabolism by gut microflora are orally administered drugs with low solubility and/or permeability which results in prolonged gastrointestinal residence time and therefore a greater probability of microbial interactions (Banic et al., 2019; Enright et al., 2016). In addition, drugs that are subjected to enterohepatic circulation may be also exposed to bacterial enzymes (Sousa et al., 2008). Interactions of intestinal bacteria with drugs may be achieved also at the level of bacterial transport proteins since all prokaryotic cells possess the homologous transporters as in eukaryotic cells, thus influencing additionally the bioavailability, efficacy and safety of drugs (Djanic et al., 2016). Since newly emerging drug candidates have a tendency toward low solubility and/or permeability, it may be concluded that wide array of substrates are possible candidates for metabolism by gut microflora (Enright et al., 2016). The most common reactions catalyzed by intestinal bacteria are reductive and hydrolytic reactions (particularly on conjugates) (Klaassen and Cui, 2015; Wilson and Nicholson, 2009). Contrary to highly polar and high-molecular weight metabolites produced by hepatic enzymes, the results of microbial transformation are usually non-polar low molecular weight byproducts (Sousa et al., 2008). Microbial transformation leads to activation of drugs to varying degrees, inactivation or even a formation of toxic metabolites (Stojancevic et al., 2014).
The initial knowledge on gut microflora involvement in drug metabolism dates back to the early nineties after recognition of potentially fatal drug-drug interaction mediated by bacterial metabolism in gut. Namely, in a tragic event in Japan, 18 patients died due to acute toxicity after the concomitant administration of an antiviral sorivudine with an anticancer drug 5-fluorouracil in patients suffering from cancer and viral disease. It was found that sorivudine was metabolized in the intestine to a metabolite (E)-5-(2-bromovinyl) uracil by bacterial enzyme pyrimidine nucleoside phosphorylase. This reactive metabolite binds irreversibly to hepatic dehydrogenase, an enzyme required for the catabolism of 5-fluorouracil in rats and humans, thus resulting in a significant increase in the 5-fluorouracil concentration and toxic conditions (Watabe et al., 1997; Diasio, 1998; Okuda et al., 1998). Therefore, sorivudine was withdrawn from the market within one month of its launch.
Reduction of drugs by microbiota
Due to strictly anaerobic conditions, particularly in lower parts of gut, reductive reactions are the most predominant reactions catalyzed by gut microflora. A variety of enzymes reducing azo- and nitro group to amino group, and keto/aldehyde group to hydroxyl group are well-known.
Bacterial azo-reductases are plying the crucial role in activation of some azo-containing drugs for the treatment of inflammatory bowel disease and some antibacterial drugs (Ryan,
2017). Azo-antibacterial prodrug prontosil was among the first drugs for which the importance of the gut microflora was recognized. The structure of this drug is based on sulfanilamide and the reaction of activation by intestinal bacteria is shown in Fig. 6.19 (Gingell etal., 1971).
Figure 6.19 Azo reduction of prontosil and release of active drug, sulfanilamide.
In addition, gut microflora is of great importance for the activation of azo-drugs used for the treatment of inflammatory bowel disease. In these prodrugs such as sulfasalazine, 5-aminosalicylic acid (5-ASA) is covalently linked via an azo bond to an inert carrier to prevent rapid absorption from the intestinal tract and is released upon reduction by intestinal bacteria and cleavage of azo bond by azo-reductases secreted by the gut microflora (Fig. 6.20) (Ryan, 2017; Mikov et al., 2006).
The common targets for bacterial enzymes are nitro groups, which are particularly susceptible to bacterial reductases. Examples of drugs with nitro functional groups are nitrazepam, bromezepam and clonazepam, which belong to benzodiazepine class, and some nitroimidazole antibiotics such as metronidazole and misonidazole, used to treat various parasitic and bacterial infections (Wilson and Nicholson, 2017; Roldan et ah, 2008). Nitroreduction results in the production of amino metabolites from the parent drugs, as shown in Fig. 6.21 on the example of nitrazepam that is reduced to 7-aminonitrazepam. Reductive metabolism of nitro groups may lead to unwanted toxicological consequences such as nitrazepam-related teratogenicity (Takeno and Sakai, 1991) or the formation of metronidazole amino metabolite which is a known rat carcinogen (Koch and Goldman, 1979; Koch et al., 1979).
Figure 6.20 Bacterial azoreduction of sulfasalazine and release of active drug, 5-aminosalicilic acid.
Figure 6.21 Bacterial nitroreduction of nitrazepam to teratogenic metabolite, 7-aminonitrazepam.
An important example of a drug affected by gut microflora reductases is digoxin, a drug used to treat heart failure and atrial fibrillation. Reduction of lactone ring brings about the formation of cardioinactive metabolite, dihydrodigoxin (Fig. 6.22)
(Lindenbaum et al., 1981). A specific intestinal bacteria Eubacterium lentum (or Eggerthella lenta) was found to be responsible for this reduction (Saha et al., 1983). Increased amount of amino acid arginine was shown to act inhibitory on the conversion of digoxin by E. lenta; thus high-protein diet has been proposed to prevent digoxin deactivation and lack of therapeutic effect (Haiser etal., 2014).
Figure 6.22 Bacterial reduction of digoxin to inactive metabolite dihydrodigoxin.
The intestinal microfora plays a role in the reduction of some drugs which contain the sulfoxide groups such as non-steroidal anti-inflammatory drugs, sulindac and sulfinpyrazone, catalyzing their conversion to respective sulfite metabolites with increased activity compared to parent drugs (Strong et al., 1987). The activation of sulindac (sulfoxide) to a non-selective COX inhibitor sulindac sulfide, which is responsible for the anti-inflammatory properties of sulindac, by anaerobic intestinal bacteria (Gurpinar etal., 2013), is presented in Fig. 6.23.
Figure 6.23 Bacterial reduction of sulindac to active metabolite sulindac oxide.
Microbial metabolism of drugs by hydrolysis
Hydrolysis is one of the most common types of microbial reactions, p-glucuronidase, (3-glucosidase, esterases, peptidases, sulfatases and phosphorylases are the major types of hydrolytic drug- metabolizing enzymes produced by gut microbes.
3-Glucuronidase of bacterial origin is among the most examined bacterial enzymes due to its role in the deconjugation of glucuronidated metabolites from liver (for example morphine- 6-glucuronide). This may result in subsequent enterohepatic circulation of drugs that may delay drugs excretion and prolong the action of drugs or even contribute to toxicity. Various bacterial genera express this enzyme, including Lactobacillus, Bifidobacterium, Clostridium, Streptococcus, Ruminococcus (Walsh et al., 2018; Wang and Roy, 2017). Similarly, (3-glucosidase hydrolyze the glycosidic bond thus releases the parent compound making a significant contribution to conversion of dietary plant substances into bioactive molecules (Michlmayr and Kneifel, 2014). Dietary plant substances that are the most susceptible to microbial degradation in the human intestinal tract are the phytochemicals (phenolics and flavonoids) (Swanson, 2015). Considering the popular use of traditional medicines and herbal supplements, more attention should be paid to microbially derived plant metabolites and their safety profiles.
Furthermore, the glutathione conjugates formed in the liver are subsequently excreted in the bile where the gut microflora acts on them through bacterial C-S-lyases leading to regeneration of the parent compound itself in the form of free thiol metabolites (Wilson and Nicholson, 2017). In vivo studies demonstrated the implication of gut microflora and C-S-lyases in paracetamol- glutathione conjugates metabolism demonstrating a significant reduction in the excretion of 3-methylthioparacetamol by urine compared to germ-free mice (Bojic et al., 2014).
Phenacetin was a popular analgesic drug which exhibited good absorption and rapid metabolism to paracetamol in liver. However, phenacetin (Fig. 6.24) is also a substrate for deacetylating enzymes under the anaerobic conditions of intestine resulting in the formation of p-phenetidine which was proposed as the main agent responsible for nephritis following chronic used of phenacetin (Smith and Griffiths, 1976). Therefore, phenacetin was withdrawn from market and replaced by its active metabolite, i.e., paracetamol.
Figure 6.24 Bacterial deacetylation of phenacetin to toxic metabolite, p-phenatidine.
Bacterial esterases are important group of enzymes involved in the hydrolysis of ester, thioester, amide or carbamate containing drugs to corresponding free acids that may have a crucial role in the activation of some drugs which are administered in the forms of esters (Foti and Dalvie, 2016; Parkinson etal., 2001).
Based on presented examples of gut microflora implication in drug metabolism, it may be concluded that the list of potential substrates are huge and that is continuously expanding. Due to large differences in drug response among patients and the role in drug efficacy and toxicity, the effects of gut microflora must be taken into consideration during drug development process.