Capacity of the Human Gut Microbiome in Metabolism of Drugs and Its Interaction with CYP450 Enzymes

Abstract

As a promising field of pharmaceutical sciences, gut microbiome effects on metabolism of xenobiotics, has shown great potential to be considered as a milestone. Xenobiotic chemistries are modified by some drug metabolizing enzymes in gut microbiome which are mostly unknown, however their functionality and the way they impose changes on drug structures are well known. Most of the drug metabolizing enzymes in gut microbial population have reductor effects which are in contrary to the host metabolic system with oxidative reactions. Hydrolysis and transfer of functional groups such as methyl, amine, hydroxyl and carboxyl also bring changes in the structure of xenobiotics. In this brief review, some of these changes on the structure of some important drugs and endogenous compounds have been mentioned, however, illustration of the complete picture has limitations. Furthermore, the significant regulatory role of metabolites generated from the function of gut microbiome enzymes on the expression and activity of host CYP450 enzymes are briefly discussed. Mostly, these effects are inhibitory and are imposed on the expression and activity of nuclear receptor transcription factors including Active/Androgen Receptors (CAR), Pregnane X-Receptors (PXR), Farnesoid X receptor (FXR) and Aryl Hydrocarbon Receptor (AHR).

1. Introduction

Human gut is colonized by trillions of microorganisms [1] with a total weight of ~1.8 kg [2]. Diversity of gut microorganisms is evidenced by the 500 species representing the 5 kingdoms of life. Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria and Verrucomicrobia taxa are generally the most abundant bacteria in the gut however their proportion varies significantly between individuals and different parts of the GI tract [3, 4]. The number of genes in the human gut microbiome is estimated to be as high as 100–150 times more than human genome, with an approximation of 3.3 million genes [5]. While most of the microorganisms in gut are anaerobes, there are a few species of facultative aerobes, archaea, and yeasts [6].

Based on some scientific findings, there are correlations between holistic gut microbiome composition and human health [7]. It is postulated that the gut microbiome is involved in some physiological functions such as digestion of complex dietary polysaccharides into short-chain fatty acids, metabolism of proteins and plant polyphenols, bile acids and vitamins [8]. There is evidence regarding the role of gut microbiome in biosynthesis of neurotransmitters and modulation of host neurotransmitter catabolism [9]. Also, the progression of some disease states such as Inflammatory Bowel Disease, Obesity, Diabetes, Cancers, Autism [10], Depression and Rheumatoid Arthritis, are totally dependent on alteration in gut microbial environment [7]. Gut microbiome is also involved in dynamic processing and metabolism of xenobiotics. Xenobiotic processing produces beneficial by-products in which food and energy are generated for microorganisms. Whether or not such food producing process is beneficial for host is a multiple-choice question and can involve various factors. The interaction of gut microbiome and xenobiotic is a mutual relationship that does not only affect the fate of xenobiotic agents but also the physiology of the host. Diet, age, exercise, geographic location, genetics of host and use of different medications such as antibiotics have enormous effects on the composition and diversity of gut microbiome which in turn affects the normal physiology of host [5].

Robust findings prove that gut microbial enzymatic system has an extensive effect on the kinetics and dynamics of drugs. The impact of gut microflora or their metabolites on the pharmacokinetics and pharmacodynamics of xenobiotics is still in its infancy stage and requires more attention. It is well known that all the aspects of drug disposition including absorption, distribution, metabolism, and excretion of drugs could potentially be affected by changes in the composition of the gut microbiome. Further, there is an intense relationship between the efficacy and toxicity of drugs and gut microbiome. Processing detailed biochemical mechanisms and related biomarkers derived from gut microbial metabolism would expand our understanding of intra and interindividual differences in clinical response and toxicity of drugs and may participate in the basis of personalized medicine. While the importance of the liver in the metabolism of xenobiotics is well identified, the significance of gut microbiome in the biotransformation of drugs which could have the potential to be equal to the liver [11] has not been clearly understood. In this chapter, the intent is to focus on the metabolic capacity of the gut microbiome, their interaction with the human metabolic system especially CYP450 enzymes and their effect on disposition of drugs.

2. Gut biochemistry and metabolic activity

Gut is a non-uniform environment for the residence of many different types of microorganisms. Dynamic gut ecosystems can provide a versatile genetic coding in bacteria to support a wide range of enzymatic activity. That absolutely depends on the PH, partial pressure of oxygen and the interaction of the gut microbiome with surrounding tissues. The versatility of the gut microbiome is also determined by the genetics of host and environmental factors such as diet, geography and also some intrinsic factors including circadian rhythm, gender, and hormonal status [5].

The versatile genomic structure of gut microbiome compared to the host produces a more multidisciplinary enzymatic system. Compared to humans with 57 recorded CYP450 types, bacteria in gut are predicted to present 3000 types of CYP450 enzymes [12]. Also, gut microbiota enzymatic system could apply a structural modification on a chemical molecule without respect for the source. Unlike the human enzymatic system, which is more oxidative and conjugative, the microbial enzymatic system is more reductive and hydrolytic, although many common reactions may also happen in both host and gut microbiomes. More precisely, the body facilitates the excretion of xenobiotics and toxic chemicals while gut microbiota has the intention to keep them inside in order to extract energy. Secondary metabolites produced by microbial enzymatic systems could be beneficial or harmful. Generally speaking, the products of oxidative and conjugative reactions are more water-soluble whereas reactions in the reductive and hydrolytic process produce more liposoluble chemicals [13].

3. Microbial enzymatic reactions

3.1 Reduction

Many functional groups such as alkenes, unsaturated carboxylic acid derivatives, nitro, N-oxide, azo, keto and sulfoxide groups will be reduced by gut microbial enzymes in the presence of some cofactors including NADPH, NADH and FMO. Cofactors by nature facilitate the transfer of electrons or hydride counterparts to a substrate in gut anaerobic conditions. Despite the existence of reductase enzymes in the host metabolic system, the reductive microbial systems are exclusive and are not found in any other vital organic system [14]. Reductase enzymes alter charge, hybridization and electrophilicity of compounds and affect biological activities, absorption or half-lives of xenobiotics in the body. Plenty of microbial reductase systems have been identified and will be discussed as follows.

3.2 Alkyl reductase and ring fission

Examples of drugs that undergo reductase enzymatic reactions are Digoxin, Deleobuvir, metronidazole, Zonisamide, Risperidone and Levamisole and sorivudine. Some endogenous compounds such as cholesterol could also be reduced by the gut microbial system.

3.2.1 Digoxin

Variable bioavailability following oral administration of digoxin could be attributed to the physiochemical properties of the drug such as dosage form [15] and particle size [16]. Some other factors including genetic polymorphism [17], gender differences [18] and food habits [19] may also participate in the variable pharmacokinetics of digoxin in different people [20]. Measurement of variable amounts of dihydro digoxin, as the inactive metabolite of digoxin in the urine of the patients using digoxin, suggests the interindividual differences in metabolism of the drug [21]. It is observed that the co-administration of antibiotics for 5 days with a poorly absorbable formulation of digoxin in oral form results in the excretion of a lower level of this metabolite in human urine compared with the patients using digoxin alone, suggesting that the human gut microbiome is involved in the inactivation process of this drug [22]. These findings were confirmed with the measurement of the very low level of dihydro digoxin excreted after intravenous administration of digoxin [22].

Additional findings in examining human fecal bacteria grown in digoxin-containing media led to the confirmation of the role of Eggerthella lenta in the inactivation of digoxin [23]. Therefore, it is postulated that dihydro digoxin is the product of the reduction of parent molecule digoxin by gut microorganisms and more precisely Eggerthella lenta. Further genetic studies by Haiser et al. resulted in the identification of a genetic locus in E. lenta named cardiac glycoside reductase (cgr) genes, whose expression is upregulated in the presence of digoxin. These genes encode the factors that are in charge of the inactivation of digoxin as the lack of this operon in two strains of E. lenta deprives them of such metabolic activity. The existence of cgr operons in the fecal microbial population of some individuals explains the interindividual differences in the metabolism of this drug [23].

There is a correlation between the expression of reductase enzyme in E. lenta and dietary protein content in the gut. These findings specify the role of diet on the expression of drug-metabolizing enzymes and its consecutive effects on systemic exposure to drugs [24]. Administration of digoxin to two groups of germ-free mice that underwent different food diets (Protein free and Protein fed animals) and colonizing them with E. lenta along with the measurement of serum and urinary content of digoxin and the reduced metabolite indicated that protein-fed animals excreted more amount of dihydro digoxin in their urine confirming the role of protein diet on microbial metabolism of digoxin [23].

Among different amino acids, Sperry and Wilkins elaborated on the importance of arginine in the growth of E. lenta as arginine is found to be the main source of nitrogen and carbon for their growth [25]. Along with the increase in the growth of bacteria, arginine simultaneously inhibits the digoxin inactivation process through a reduction [23]. The mechanistic procedure involved in cgr repression, and the probable clinical outcomes need more elaboration and research [23].

3.2.2 Cholesterol

The cholesterol pool in the body that originates from both endogenous and exogenous sources could be affected by gut microbial diversity. These effects could participate in cholesterol metabolism and homeostasis in the body. Endogenous cholesterol in the form of bile acids enters enterohepatic circulation in which the gut microbiome has a significant role. For exogenous cholesterol, the gut microbiome directly participates in its biodegradation in the small intestine. The latter pathway is involved in the generation of a nonabsorbable, and inactive metabolite called coprostanol in a three-step reductive process. The rate-limiting enzyme involved in this process is called cholesterol dehydrogenase. Individuals harboring coprostanol-forming microorganisms are prevalent in geographically human cohorts and have significantly lower fecal cholesterol levels as well as lower serum total cholesterol [26]. Despite this fact, the bacteria involved in this process in human gut microbiome are still uncharacterized. Studies of Xavier and Balskus at Harvard University resulted in finding a correlation between the existence of IsmA (Intestinal Sterol Metabolism A)-encoding genes in uncultured bacteria of cluster IV Clostridium in the human stool and cholesterol dehydrogenase [26]. Although the isolation of these beneficial bacteria from human stool was not successful, some strains of Lactobacillus were isolated from food with a high level of expression of such enzyme. Lactobacillus rhamnosus Strain BFE 5264 was isolated from Kimchi and has shown positive outcomes in reducing cholesterol levels in a murine model of dyslipidemia after oral administration as probiotics [27, 28]. The clinical outcome of probiotics in decreasing serum cholesterol in humans needs more elaboration.

3.3 Azoreducatse

Bacterial azoreductase enzymes are flavin-dependent NADPH, flavin-dependent NADH or flavin-independent NADPH. These enzymes metabolize many drugs and xenobiotics including azo dyes in food and textiles. Azoreducatse is produced mostly in aerobic and facultative aerobic bacteria except for Clostridium perfringens. Studies by Morrison et al. indicated that despite the existence of this system in Clostridium perfringens, Azo dyes such as tartrazine, Methyl Red, Trypan Blue, Direct Blue, and Methyl Orange slow down the growth of bacteria suggesting that there is a resistance to azo dyes in Clostridium perfringens. Therefore, there is a theory that different types of azoreductase may participate in the reduction of xenobiotics [29]. Studies by Rafii and coworkers confirmed that every single azoreductase-producing microorganism in the gut generates its own specific isoenzymes which might show different activities from others [30].

3.3.1 Azo dyes

Some of the aniline metabolites of food dyes are known to be nontoxic however some of the textile dyes are known to have significant toxicity and are responsible for genotoxicity, mutagenicity, and carcinogenicity in live organisms as well as enviro-polluting activities in nature [31]. It is well characterized that workers in textile industries dealing with Azo dyes have higher rates of bladder and lung cancers [32]. Also, feeding mice with textile azo dyes results in urinal excretion of a microbial by-product of azo dyes called bis-aniline benzidine, which is an aromatic amine compound with significant carcinogenic activities.

The toxicity of azo dyes is not only dependent on their backbone chemical structure but also on the types of gut bacteria involved in azoreductase metabolism [30].

3.3.2 Prontosil and neoprontosil

Azoreductase is found to be important in the activation of some prodrugs or increasing the biological activities of some other drugs. An example is the formation of active sulfonamide metabolite from its prodrug prontosil [3]. Prontosil is a synthetic dye with no biological activity, however, following its metabolism by the gut azoreductase system to sulfonamide, antibiotic activities will emerge [33]. The percentage of bioconversion of prontosil by the gut microbial system is only 20% means that only 20% of oral doses of prontosil can be metabolized by the gut microbiome. One water-soluble derivative of prontosil called neoprontosil is completely reduced to sulfonamide after oral absorption by microbial azoreductase system in the gut. This compound is completely nonabsorbable in the gut unless its structure is changed to sulfonamide following microbial bioconversion [34].

3.3.3 Sulfasalazine, olsalazine and balsalazide

Biological activities of some other drugs such as sulfasalazine used in the treatment of Ulcerative Colitis (UC) and Chron’s Disease (CD) are dependent upon their pro-activation following oral administration. Basically, sulfasalazine has poor absorption in the gut with an estimated bioavailability of ~3–12% [35]. This poor bioavailability is attributed to the low solubility and permeability of the drug, however, the role of ATP-Binding Cassette efflux transporter-G2 (ABCG2) in limiting the absorption of the drug cannot be ruled out [35]. The existence of the Azo bond in the molecule makes it a target for microbial azoreductase system in the colon [36]. The product of such microbial metabolism would be 5-Aminosalicylic acid and sulfapyridine. While sulfapyridine is well absorbed, 5-Aminosalicylic acid has no absorption and distributes in the colon [35]. As observed, the primary microbial metabolism of this drug is necessary for tissue accumulation and efficacy of the drug. This observation was confirmed with the administration of the drug to germ-free animals and the collection of the unchanged drug from feces. These findings signify the role of the gut microbiome in the cleavage of the Azo bond and the effectiveness of sulfasalazine at the site of inflammation where bacterial breakdown is more likely to occur. Several studies confirmed that the genus Clostridium produces the highest level of Azo reductase and plays a significant role in the microbial metabolism of sulfasalazine [37]. Due to the systemic absorption of sulfapyridine, some side effects such as anorexia, nausea, skin rash and blood dyscrasias may be observed in patients using this medication [11]. Similar drugs such as olsalazine and balsalazide have the likewise metabolic procedure and are used for the same therapeutic purposes [11]. The product of the balsalazide metabolic procedure by the Azo reductase of the gut microbiome is two molecules of 5-Aminosalicylic acid, with the expected fewer adverse effects than sulfasalazine. Similarly, olsalazine is synthesized as a prodrug that generates 5-Aminosalicylic acid when it is metabolized by azo reductase system in the colon. The carrier molecule of 4-aminobenzyoyl ß-alanine is expected to have no absorption and therefore produces fewer adverse effects compared to sulfasalazine [11].

3.4 Nitroreductase

The bacterial nitroreductase system is among the most complex enzymatic systems available in gut bacteria, as they may interact with both aromatic or aliphatic nitro groups and may be oxygen sensitive or insensitive. Type I nitroreductase is a flavoenzyme that catalyzes NADPH or NADH-dependent reduction of the nitro groups on aromatic or heterocyclic compounds without the presence of oxygen. Type II nitroreductase however is oxygen sensitive and catalyzes the single electron reduction of the nitro group to produce nitro anion radical [38, 39]. Most gut bacteria may contain several types of nitroreductase systems. For instance, Bacteroid fragilis, one of the symbiotic gut bacteria has four different types of nitroreductase systems [40].

The bacterial nitroreductase system can significantly impact the pharmacological and/or toxicological effects of drugs. Drugs such as chloramphenicol [39], nitrofurazone, nitrazepam, clonazepam [41], GW9662 and CB1954 are substrates of the gut bacterial nitroreductase system [42]. Also, some pesticides such as parathion and some explosives such as 2,4,6-Trinitrotoluene (TNT) and picric acid are the substrate of the nitroreductase system in the gut microbiome [43].

3.4.1 Carcinogenicity and toxicity of compounds undergo nitro reduction by the gut microbiome

Gut bacteria nitroreductase system transforms many of the compounds into carcinogenic metabolites due to the reduction of nitro groups into arylhydroxylamines and acetoxyarylamines. These compounds are electrophilic nitrenium ions with the possibility of interacting with DNA which in turn may have carcinogenic and mutagenic effects [40]. For instance, Trinitrotoluene (TNT) amino derivatives can abduct hemoglobin and bring toxicity and carcinogenicity [40]. Also, many environmental pollutants such as 1-Nitropyrene, 2,4, Dinitrotoluene and Nitro polycyclic aromatic hydrocarbons (e.g., 2-Nitroflurene) are being reduced to absorbable metabolites with significant mutagenicity, genotoxicity, and carcinogenicity [44]. In this regard, the cooperation of microbial (nitroreduction) and host metabolism (subsequent oxidation in the liver) play a significant role [44].

3.4.2 Nitrobenzodiazepines

Nitrazepam, Flunitrazepam and clonazepam are benzodiazepines with nitro group used in the treatment of sleep and anxiety disorders. Among them, nitrazepam, a short-term benzodiazepine is reduced to 7-Aminonitrazepam (3–20% of the dose) when it is incubated with microbial suspension from rat cecal contents. Clonazepam is also converted to 7-Aminoclonazepam after being incubated with rat intestinal lumen content [45]. Administration of 300 mg/kg of nitrazepam to pregnant rats resulted in the excretion of 7-Aminonitrazepam and 7-acetylamino nitrazepam in urine and feces [46]. Pre-treatment of rats with antibiotics resulted in the reduction of their fecal and renal metabolite excretion (From 30% of the dose before antibiotic treatment to 2% of the dose after antibiotic treatment) [46]. This finding has a robust approval of the role of gut microbiome in metabolism of this drug.

It has been shown that nitrazepam has a teratogenic effect in rats with various malformations, however, mice fetuses did not show any significant symptoms of malformations. The difference between these two species is attributed to the degree of acetylation of 7-Aminonitrazepam in rat and mouse livers. The degree of acetylation in rat liver cytosol is 8.5-fold greater than that of mice. Therefore, it seems that 7-Aminonitrazepam needs one more step of acetylation in the liver before being a teratogenic compound [47]. This is a good example of sequential metabolism by gut microbial enzymes followed by host metabolism in which a teratogenic compound is being produced.

Teratogenic effects of nitrazepam were confirmed in 43 pregnant women who attempted suicide with very large doses of nitrazepam. Although the interaction of gut microbiome and host hepatic metabolism was identified in rats as the source of congenital abnormalities, this toxicity in humans was attributed to the disruption of protein metabolism in the fetal mesenchyme [48]. Whether or not microbial metabolism is involved in this process needs more clarification.

It has been demonstrated that under anaerobic conditions, nitroreduction system of E. coli significantly reduces flunitrazepam [41]. Anaerobic bacteria such as C. perfringens and B. fragilis can catalyze the nitroreduction of Nitrobenzodiazepines at a rate 50–70 times higher than E. coli [49]. Benzodiazepine nitroreduction in E. coli is under the control of Type I nitroreductase (NfsB) gene. Kinetic studies using purified recombinant NfsB indicated that clonazepam has a lower K_cat/K_m ratio than nitrazepam and flunitrazepam suggesting that clonazepam is more prone to be reduced by gut microbial nitroreductase system compared to the other two drugs [41].

3.4.3 Nitroreductase is responsible for bacterial antibiotic resistance

Activity of the Bacterial nitroreductase system in gut is controlled by a gene called NfsB as previously indicated. It has been demonstrated that the transfer of the NfsB gene from Hemophilus influenza to Escherichia coli increases its ability to express nitroreductase system. As a matter of fact, this system is known as the main reason for bacterial resistance to chloramphenicol [39], Nitrofurantoin [50] and metronidazole [51]. It is important to know that Lactococcus lactis, a homolog of E. coli that carries the NfsA gene is unable to reduce chloramphenicol in in-vitro scale [39]. Therefore, between the bacteria that express NfsA or NfsB, the latter is more important for antibiotic resistance. Escherichia coli is part of a normal gut bacterial population but Lactococcus lactis is not. In Hemophilus influenza [52] chloramphenicol is changed to 4-aminophenyl allylic alcohol by its nitroreductase system.

3.4.4 Nitrofurantoin

Some of the nitrofuran antibiotics such as nitrofurantoin are the substrate of bacterial nitroreductase. This molecule is a prodrug, and its nitro group is reduced to a reactive hydroxylamine group for producing a pharmacological activity. These intermediates attack bacterial ribosomal protein non-specifically and cause inhibition of protein synthesis. Robust studies on E. coli indicate that the existence of some strains with microbial resistance is due to reduced activities in reducing nitrofurantoin [50].

3.4.5 Metronidazole

Metronidazole, is an antiprotozoal antibiotic that undergoes metabolism by bacterial nitroreductase system under anaerobic conditions. Similar to nitrofurantoin, metronidazole is a prodrug and is inactive until taken orally. Reduction of the nitro group of metronidazole occurs in two ways [53]. In the first pathway, the imidazole ring in the metronidazole structure is reduced to N-(2-hydroxyethyl)-oxamic acid and acetamide. Production of these two metabolites is compromised in germ-free rats. The presence of a glucuronidated form of this metabolite in urine but not feces of rats indicates the interaction of microbial and host metabolic systems in the metabolism of metronidazole [54]. N-(2-hydroxyethyl)-oxamic acid has no biological activity, however, the generated acetamide is proven to have liver carcinogenic effects in rats and mice. No epidemiological data are available to support the carcinogenicity of this product in humans [55]. In the alternative second pathway, the nitro group is reduced to a nontoxic amino derivative. The inactivation process by either one of the mechanisms could result in microbial resistance to metronidazole [53].

3.5 Sulfoxide reductase

Sulfoxide reductase is one of the enzymatic microbial systems available in the gut. The role of this system on drug disposition has been studied for some of the sulfur-containing drugs such as sulphinpyrazone, sulindac [31] and flosequinan.

3.5.1 Sulindac

Sulindac, an inhibitor of COX-1 and COX-2 systems with anti-inflammatory properties, is a prodrug that is metabolized to its active metabolite, sulindac sulfide [56] by both intestinal bacterial and liver reductase system [31, 56] Both aerobic and anaerobic gut bacteria have the ability to metabolize this drug by their own methionine sulfoxide reductase system [31], however from six known members of this reductase system in E.coli, only methionine sulfoxide reductase type A and a membrane-associated methionine sulfoxide reductase system can metabolize this prodrug to its active metabolite [56].

3.5.2 Sulfinpyrazone

For sulfinpyrazone, which is used to prevent gout and its related arthritis, the sulfoxide reductase system in the gut produces the active metabolite of the drug, sulfinpyrazone sulfide, similar to that of sulindac. Likewise, both aerobic and anaerobic microbial populations in both human and rabbit guts are involved in such process however, relative to sulindac, the extent of metabolism is much smaller. Administration of some antibiotics such as metronidazole in rabbits could decrease the extent of metabolism of sulfinpyrazone, however after the administration of tetracycline, the extent of metabolism of sulfinpyrazone was not affected [31].

In one study, the administration of a single 200 mg of sulfinpyrazone to 11 healthy volunteers, resulted in an increase in peak plasma concentrations of sulfinpyrazone after 2 hours, however, the peak concentration of sulfide metabolite did not occur till 15 hours after the administration of the drug. Concurrent administration of metoclopramide with sulfinpyrazone resulted in a 4.3-fold decrease in T_max and a 5.3-fold increase in C_max of the active metabolite. The AUC of active metabolite was also increased 2.8-fold. Administration of slow-release formulation of metoclopramide increased the ratio of AUC of metabolite to parent drug by 3.5-fold. That is mostly due to the increase in the absorption of controlled-release medications in large intestine which results in their augmented interaction with gut microflora. Administration of the drug in patients with resection of the distal part of the intestine, resulted in a negligible amount of metabolite in plasma after oral administration, however, the plasma concentration of the parent drug in these patients was comparable with normal subjects. These data overall support the role of gut microbiome in the metabolism of sulfinpyrazone [57].

3.5.3 Flosequinan

Flosequinan, is an antibiotic comprised of a chiral mixture of R (+) and S (−) enantiomers. This drug is the substrate of the sulfoxide reductase system in the gut due to the presence of the sulfoxide group in its structure. The chiral metabolism of this quinolone-related antibiotic is completely dependent upon the type of residential bacteria in the gut. Many of the facultative anaerobic bacteria including E. coli, Klebsiella oxytoca and Klebsiella pneumonia are involved in the reduction of R (+) enantiomer to its sulfide derivative, however for S (−) flosequinan, Enterobacter aerogenes and Micrococcus agilis show more susceptibility. Both sulfide enantiomers are readily absorbed in the intestine and oxidize to their sulfone forms [58].

3.6 Keto reductase

The Existence of Aldo, keto and carbonyl reductases in the host are well characterized, however, there is no equivalent for aldo or carbonyl reductase in gut microbiome. The gut microbiome uses a keto reductase enzymatic system for the bioconversion of some xenobiotics including Nabumetone and Tacrolimus.

3.6.1 Nabumetone

Nabumetone or (4-(6-methoxy-2-naphthyl)-2-butanone), is a nonsteroidal anti-inflammatory prodrug that undergoes oxidative cleavage of its side chain by liver enzymes following oral administration. The formed metabolite, 6-methoxy-2-naphthylacetic acid which accounts for ~35% of the administered dose, has strong COX-2 inhibitory effects and is responsible for the anti-inflammatory actions of the drug. In the incubation of this prodrug with the suspension of commensal E. coli ATCC 25922, an inactive metabolite was identified which is responsible for ~1% of the administered dose. This inactivation process is the result of the reduction of molecules and formation of 4-(6-methoxy-2-naphthyl)-butane-2-ol. This microbial metabolite was also identified following hepatic microsomal and cytosolic incubation of the drug. This metabolite is excreted in urine however the percentage was low as previously indicated.

The administration of a broad-spectrum antibiotic, imipenem to rats could inhibit the bacterial keto reductase system and formation of inactive metabolite, however, the plasma concentration of active metabolite by the liver was not affected [59].

3.6.2 Tacrolimus

Tacrolimus is an immunosuppressive agent administered to patients subsequent to organ transplantation and decreases the rejection of the donated organs by the host immune system. Oral administration of tacrolimus is associated with low and variable absorption and hence bioavailability (4–89% with an average of approximately 25%). Tacrolimus is the substrate of both CYP3A4 and P-gp in both the liver and intestine. Administration of ketoconazole, a strong inhibitor of both CYP3A4 and P-gp to the patients using tacrolimus intravenously, was associated with a significant increase in AUC of drug (42%). This drug is a low extraction ratio drug. Therefore, it is not anticipated to see a significant change in oral AUC of drug after administration of ketoconazole. In the absence of ketoconazole, the first-pass metabolism of drug was calculated to be 8%, however, the administration of ketoconazole decreased the first-pass metabolism of drug to about 6.2%. Administration of ketoconazole to the regiment of the patients using tacrolimus orally, disproportionally increased the AUC of tacrolimus by about 109%, which is about 3-fold increase in the AUC of tacrolimus after IV administration. These results indicate that either P-gp or gut microbiome has significant role in the first-pass metabolism of drug [60].

In a separate study, ketoconazole was administered to the patients approximately 10 hours after the administration of a single oral dose of tacrolimus. This drug regiment did not affect the total body clearance or volume of distribution of tacrolimus however, the oral bioavailibity of drug increased more than 2-fold indicating the significant role of intestinal microorganisms in metabolism of drug [61].

To magnify the importance of the gut microbiome in the metabolism of tacrolimus, the effect of oral administration of some antibiotics such as erythromycin, clarithromycin, chloramphenicol, and clindamycin on systemic exposure to tacrolimus was studied. While the increase in blood concentration of tacrolimus by some of these drugs such as clarithromycin and erythromycin could be attributed to their inhibitory effects on intestinal CYP3A4, the effect of others on gut microbial metabolism of the drug is inevitable [62]. A positive correlation between low absorption of drug and microbial metabolism in kidney transplant patients is in place due to the high fecal abundance of Faecalibacterium prausnitzii in these patients. Incubation of tacrolimus and Faecalibacterium prausnitzii in an in-vitro state resulted in the formation of two metabolites with the hypothetical names of M1 and M2 [63] Faecalibacterium prausnitzii belongs to the Clostridial order taxonomically and is the most abundant bacteria in the human gut. The formation of these two metabolites was not confirmed following liver microsomal incubation of this drug, indicating the role of the gut microbiome in the metabolism of tacrolimus [63]. Elucidation of their structures revealed that M1 metabolite is a product of keto reduction in tacrolimus structure and is called 9-Hydroxytacrolimus [63]. The pharmacological activity of M1 using human peripheral blood mononuclear cells indicated that the parent drug has 15-fold more immunosuppressive activity than its metabolite [63].

4. Hydrolysis

Catalysis of molecules by microbial hydrolytic enzymes results in the formation of a hydrated molecule followed by bond cleavage. Hydrolytic activities of the microbiome may change the clinical activities and physical properties of the substrate. For instance, an increase or decrease in the polarity of the molecule may result in alteration of absorption, toxicity, or biological activity of the molecule [31]. Hydrolysis is often essential for the further transformation of molecules. The most abundant hydrolytic enzymes in the gut are protease, sulfatase, glycosidase and glucuronidase.

4.1 Protease

Protease enzymes are available in both host enzymatic system as well as gut microbial system. They are responsible for the cleavage of peptide bonds present in polypeptides and proteins. The nature of the protease enzymes of host and gut microbiomes is site-specific. The small intestine, the location of host protease, is mostly dominated by pancreatic serine proteases whereas cysteine and metalloprotease microbial protease are abundant in the colon [14].

Hydrolysis by metalloproteinase is facilitated by the presence of a carboxylic group in the active site of the enzyme. This group can draw a proton from a water molecule and make this group nucleophilic. These nucleophile groups are attacked by polarized water on the carbonyl carbon of the peptide bond and break down the peptide molecule. It is believed that the conformational and configurational structure of peptides can play a significant role in their stability in colon. If the hydrophilic structure of a hydrophilic peptide is buried deep down inside the folded protein, the availability of this group for being exposed to metalloproteinase is compromised, therefore the peptide would be more stable. It is also believed that the hydrophobic parts of the molecules considerably participate in conformational structure and exposure of hydrophilic structure to the active site of the enzyme. On the other hand, the conformation of amino acids is also an important factor in the stability of linear peptide molecules. It has been demonstrated that L-amino acids are more prone to the catalytic effects of peptidase than D-amino acids. This procedure explains the less stability of vasopressin and oxytocin compared to desmopressin [64].

The log p-value of a molecule which is defined as the ratio of hydrophobicity to the hydrophilicity of a molecule is another important factor in the stability of a peptide molecule in the colon. An example is cyclosporine which is a highly lipophilic molecule with a unique cyclic structure and substantially N-terminated nature. This drug is shown to be more stable than peptide drugs with disulfide bonds such as desmopressin and oxytocin. Breakage of disulfide bridges in desmopressin and oxytocin in the anaerobic environment of the colon results in detangling of molecular structure and making linear unstable formats [64].

Other than structure and lipophilicity, the size, and weight of the molecule could also be another important determinant in the stability of the molecule. Large peptide drugs such as insulin, glucagon and calcitonin are metabolized rapidly by the gut microbiome, however, the metabolism of smaller peptides is more variable and depends on their conformational complexity. Comparison in degradation of calcitonin and insulin when incubated with the caecal contents of rat specifies the faster level of degradation for calcitonin than that of insulin. Presence of the inhibitors such as camostat and aprotinin (serine protease inhibitors) could escalate their oral bioavailability and increase their systemic circulation [65].

Most of the dietary proteins are susceptible to both host and microbial proteases. While host proteases are normally involved in the regular cleavage of peptide bonds within the proximal small intestine, in the colon, microbial proteases are involved in some hydrolytic processes that produce free amino acids (FAAs). FAAs are taken up by bacteria and produce microbial proteins with the ability to enter a catabolic pathway equipped with highly specific enzymes with deamination and/or decarboxylation capacity [66].

4.2 Sulphatase

A variety of roles are attributed to the microbial sulfatase in the body. Sulfatases catalyze the hydrolysis of sulfate groups from small organic xenobiotic molecules to large endobiotic macromolecules such as Mucin, Glycosaminoglycans (GAG), neurotransmitters and bile acids including chenodeoxycholic acid. Metabolism of highly sulfated glycans not only helps the colonization of the intestinal mucosal layer [67], but also plays the most essential role in the adaptation of Bacteroides thetaiotaomicron, the major human commensal bacterium, to its host [68]. Sulfate esters could also provide sulfur for the growth of bacteria when they are not able to use carbon sources for energy [69]. Metabolism of some other endogenous compounds such as bile acids is also attributed to the effect of bacterial sulfatase.

4.2.1 Role of sulfatase in the enterohepatic circulation

Enterohepatic circulation (EHC) is a physiological procedure that clearly demonstrates the interaction between host and microbial metabolic system. In EHC, liver produces the conjugated forms of an exogenous or endogenous compound using phase II drug metabolizing enzymes, either in the form of sulfur, glucuronic acid, glycoside, or alkyl conjugates which supposedly should be excretion into bile and feces by passing through colon. In the colon, however, the residing microorganisms produce a deconjugated form of these compounds and turn them back into the upper part of intestine which circulates back into the blood and liver again.

Bacterial sulfatase plays a significant role in the EHC of some endogenic substances and xenobiotics. In this procedure, the transfer of a sulfo group from a donor molecule to an acceptor is facilitated by the function of hepatic sulfotransferase. After this step and the transfer of the sulfated metabolite to the colon, microorganisms in the colon use their own sulfatase system to turn sulfate conjugates back into their original molecule. This step is the prerequisite for the reabsorption of drug molecules to the systemic circulation. Many drugs that undergo the sulfotransferase system in the liver, will be cleaved by the bacterial sulfatase system in the gut. The best examples, in this case, would be the enterohepatic circulation of hormones such as estrogens, and DHEA (Dehydroepiandrosterone [70]. Also, EHC of bile acids is controlled by microbial sulfatase in gut.

4.2.1.1 Estrogens

Estrogens undergo enterohepatic circulation by SULT1E1 and sulfatase system between the liver, bile, and gut [71]. It has been shown that enterohepatic circulation of estrogens can be modified by antibiotic treatment (especially rifampin). Elevated fecal excretion of glucuronide and sulfate conjugates of these drugs following antibiotic therapy is mostly attributed to the decrease in the expression of hydrolyzing intestinal flora [72]. Failure of the contraceptive effect of estrogen in rifampin-treated women is the net effect of such interaction. It seems that antibiotic therapy can disrupt the normal enterohepatic circulations of estrogens present in oral contraceptives and affect plasma, bile, urinal and fecal concentrations of both parent drugs and their metabolites [72] In Gnotobiotic rats selectively associated with estrone-desulphated bacteria, the excretion of estrone 3-sulfate is shown to be more rapid than that of germ-free and conventional rats. The elimination half-life of estrone 3-sulfate was 22 h in conventional rats, 32 h in germ-free rats and 13 h in gnotobiotic rats [73].

4.2.1.2 Bile acids

Conjugated bile acids are produced in liver due to the combination of some primary bile acids such as chenodeoxycholic acid, with some amino acids such as taurine. It is well known that following conjugation process of amino acids with primary bile acids, their excretion to gut lumen through bile duct is facilitated however, this process will be interrupted by the deconjugation process in gut through microbial enzymes. This process results in production of primary bile acids which undergo another microbial enzymatic process called dehydroxylation. The generated secondary bile acids are more hydrophilic and can be excreted in feces. Bacterial sulfatase is involved in deconjugation process of primary bile acids with sulfur in their structures. It is well known that only small part of the conjugated form of the primary bile acids undergo deconjugation process by sulfatase. Interruption of desulfation process in some disease states such as IBD in which the normal population of commensal bacteria in gut is reduced, results in accumulation of sulfated conjugates of bile acid. This fact is compromised by knowing that bile acids by themselves have anti-inflammatory effects and may take part in restoring the impaired gut microbiome in IBD [74].

4.2.2 Role of sulfatase in toxicity of xenobiotics

Bacterial sulfatase could potentiate the toxicity of some of the chemicals that seem to be nontoxic when they are used as food additives or drugs.

4.2.2.1 Cyclamate

Cyclamate was synthesized in 1937 by Audrieth and Sveda as a food sweetener. Thereafter, it was used extensively in the food and drug industry after being approved by FDA in 1951 as GRAS (Generally Recognized as Safe). In the beginning, cyclamate was thought to be eliminated from the body as an unchanged drug, however further investigation resulted in the recognition of a gut microbial metabolite called cyclohexylamine which is being excreted in urine. Cyclohexylamine which is the result of the hydrolysis of cyclamate by bacterial sulfatase in the gut was found to have the potential to generate bladder cancer in rodents. FDA immediately removed cyclamate from the GRAS list and banned its usage in the food and drug industry [75].

4.2.2.2 β-Glycosidase

Microbial β-glycosidases are produced by a wide range of gut bacterial populations and are involved in the cleavage of the bond between the sugar and aglycon part of a molecule. Glycosidases are not only involved in the metabolism of carbohydrates but also in many drugs and xenobiotics. The products of such metabolic procedures may have different biological activity, altered toxicity and or diverse absorption.

4.2.3 Effect of bacterial β-glycosidase on the toxicity of xenobiotics

4.2.3.1 Amygdaline

The toxicity of Amygdalin, a plant-base chemical found in some stone fruits and berries, is high when it is metabolized by the gut microbiome. Amygdalin, (Cyanogenic diglycoside) is present in its conjugated form with two molecules of glucose and one molecule of mandelonitrile. Two steps of deglycosidation happen in molecules following microbial β-glycosidase function in the gut. In each step one sugar molecule is cleaved from the molecule. The final formed molecule after sugar cleavage is unstable and is converted to hydrogen cyanide (HCN) and benzaldehyde. HCN along with the oxidized form of benzaldehyde called benzoic acid is the source of toxicity in cells. These molecules and especially HCN could inhibit the cytochrome oxidase of the Electron Transport Chain (ETC) in the mitochondria and prevent the cellular utilization of oxygen [76].

4.2.3.2 Cycasin

Like the former example, the toxicity of cycad plants may increase after being deglycosidated to an aglycon by the function of microbial β-glycosidases in the gut. Cycad plants contain cycasin which is the β- glucoside form of methyl-azoxymethane. Cycasin shows no toxicity after IV injection or oral consumption in germ-free animals, however following the oral administration in normal animals, it causes hepatotoxicity and carcinogenicity. That is due to the metabolism of the drug by the β- glucosidase system of some S. faecalis strains in the gut [77].

4.2.3.3 Doxorubicin

Doxorubicin is an anticancer antibiotic that belongs to the family of anthracyclines. From structure point of view is a glycoside anthracycline. In intravenous administration of this drug, about 50% of the dose is excreted unchanged in bile and feces and about 5% is excreted in urine [78]. Both host organs and the gut microbial population take part in the metabolism of this drug. A cytoplasmic NADPH-dependent aldoketoreductase system present in red blood cells, kidney and liver is involved in the organ metabolism of doxorubicin. The product of the aldoketoreductase system would be a water-soluble, hydroxy metabolite called doxorubicinol. This metabolite has less pharmacological activity and is excreted in the urine (30%) and bile (20%). Also, it can be deglycosidated by gut microbiome β-glycosidases system and forms doxorubicinol -aglycone.

Excretion of about 50% of the administered intravenous dose of doxorubicin in bile and feces, increases the exposure of drug molecules with gut microbial enzymatic system during the process of enterohepatic circulation. The end products of this biotransformation are poorly water-soluble aglycones called 7-hydroxydoxorubicinone and 7-deoxydoxorubicinone. It is clearly shown the microbial biotransformation of the drug passes through two different stages of deglycosidation and keto reduction. Incubation of human fecal content with doxorubicin resulted in the identification of a strain called Raoultella planticola involved in the inactivation of doxorubicin under anaerobic conditions. Raoultella planticola has a shorter life span in aerobic conditions compared to anaerobic conditions and cannot metabolize the drug. Under anaerobic conditions, Raoultella planticola converts doxorubicin to its aglycon metabolite by the deglycosidation process. It is further demonstrated that doxorubicin is also degraded by Klebsiella pneumonia and E. coli BW25113 under anaerobic conditions. The formed aglycon metabolite passes through another keto-reduction process. Whether or not this keto reduction step is a microbial process, needs to be determined. It is believed that the end products of microbial metabolism have less toxicity than their parent molecule.

4.2.4 Role of intestinal bacterial β-glycosidase on activation of prodrugs

4.2.4.1 Isoflavones

Most of the soy isoflavones including daidzein are not biologically active due to their structure. Metabolism of daidzein by microbial β-glycosidase in the gut modifies its structure to a more pharmacologically active compound called s-equal. Conversion of daidzein to s-equal in the human gut is subjective and depends on the availability of the microbes responsible for such conversion in the gut of the host. Therefore, a limited number of individuals who has an available microbial population for such conversion benefit from the full pharmacological actions of soy isoflavones. S-equal is more absorbable and has lower clearance than daidzein [79, 80].

4.2.4.2 Sennosides

Metabolism of sennosides by the human gut microbial population passes through two different pathways. In the first pathway, sennosides (A and B) may be hydrolyzed directly and generate 8-glucosyl rhein anthrone which is converted to rhein anthrone by a β-glycosidation process. In the second pathway, two consecutive deglycosidation steps produce sennidin A or B monoglycoside and then sennidin A or B, which are converted to rhein anthrone. Regardless of the pathway, the generated end-product, rhein anthrone, has a strong purgative effect. There are nine species of anaerobic Bifidobacteria with β-glycosidase activity, however, only two types of Bifidobacterium dentium and Bifidobacterium adolescnti have a specific type of β-glycosidase with the ability to generate rhein anthrone [81].

4.3 β-Glucuronidase

β-Glucuronidase is produced by gut microbiome in the procedure of EHC. The most well-known endogenous compounds in body that undergoes the EHC using microbial β-Glucuronidase is bilirubin. Some other exogenous compounds such as estrogen, NSAIDs and irinotecan are also the substrate of this enzyme.

4.3.1 Estrogens

It is postulated that microbial β-Glucuronidase is involved in the reactivation of estrone and estradiol and their recirculation in blood stream after being deconjugated during EHC. This procedure contributes to a variety of hormonal disorders in women including breast cancer and endometriosis.

During phase II metabolism of estrogens in liver, β-Glucuronic acid is appended to estrogen molecules by UDP-glucuronosyltransferase enzymes (UGTs), which are pharmacologically inactive and should be excreted in bile and feces. Bacterial β-Glucuronidase in the colon reverses this enzymatic procedure and estrogens are reabsorbed back into blood stream. Repeated iterative rounds of EHC increase the unbound blood and tissue concentration of estrogen which predispose the susceptible patients to cancer and endometriosis [82].

4.3.2 Irinotecan

Irinotecan (CPT-11) is an anticancer agent used in various types of cancers including colorectal, pancreatic and lung cancers. Mechanistically, it inhibits topoisomerase I in cancer cells and is derived from camptothecin alkaloid. Irinotecan is a prodrug that activates to SN-38 in liver using carboxylesterase converting enzymes (CCE). SN-38 has 100–1000-fold more potency than the parent drug. SN-38 is conjugated in liver by UGT1A1 and undergoes EHC. Deconjugation of SN-38-glucorunide by gut microbial β-Glucuronidase and the release of SN-38 in the intestinal lumen is associated with mucosal damage in intestinal epithelia causing diarrhea as a sign of toxicity [83]. Studies of Brandi et al. indicated the significant role of gut microbiome in this procedure. They showed the lethal dose of CPT-11 is increased by ~2.5 fold (Decrease in toxicity) in GF mice indicating the role of gut microbiome in this procedure [84].

Significant scientific efforts have been done to decrease the toxicity of SN-38 by inhibiting microbial β-Glucuronidase and erupting EHC of this drug. Most of the related efforts were unsuccessful although the damage of mucosal damage in intestinal epithelia was decreased substantially. That was mostly due to the negative effect of these drugs on plasma concentration of CPT-11 and decreasing its bioavailability which mostly compromise its clinical efficacy [84].

4.3.3 Bilirubin

Bilirubin is a yellow-orange pigment of bile that is resulted from the degradation of heme in hemoglobin. Degradation of heme produces biliverdin which is converted into unconjugated bilirubin (UCB), a water insoluble form of bilirubin that enters blood circulation after binding to albumin. UCB in liver renders to its conjugated form by adding β-Glucuronic acid to its structure using UGT1A1. The conjugated form of UCB is water soluble and excreted in bile or recirculated back into blood stream after the deconjugation process by microbial β-Glucuronidase. Anaerobic intestinal bacterial flora in gut also forms a group of three tetrapyrroles (stercobilinogen, urobilinogen, or mesobilinogen), that are collectively called urobilinogens. Urobilinogens are colorless and after oxidization generate brownish yellow color pigments responsible for the distinctive color of feces. About 20% of these pigments are reabsorbed by deconjugation process of gut microbiome and the 80% is excreted in feces [85].

It has been demonstrated that impaired function or inhibition of UGT1A1 in liver causes Jaundice, however, there are some studies indicating the role of gut microbiome in the pathogenesis of this disease. It has been shown that imbalance of gut microbiota such as Firmicutes, Proteobacteria, Bifidobacteria and Escherichia coli affect the EHC of bilirubin and participate in the pathogenesis of Jaundice [86].

5. Functional group transfer reactions

Transfer of some functional groups such as acetyl, hydroxyl, acyl, amine, carboxyl, and methyl groups between two substrates through nucleophilic substrate reactions is facilitated by transferase enzymes that are abundant in the gut microbiome. These functional groups can be transferred to or from xenobiotics in the presence of activated co-substrates such as acetyl coenzymes, Adenosine triphosphate (ATP), S-Adenosyl methionine (SAM) or cobalamin (COB) and tetrahydrofolate (THF). The choice between co-substrates is completely dependent upon the reactions. Mostly, adding substrates to a chemical scaffold needs acetyl coenzyme, Adenosine triphosphate (ATP) and S-Adenosyl methionine (SAM), however for removal of functional groups, the presence of COB or THF would be enough [14].

5.1 Demethylation

Demethylation is a shared biological process between both host and gut microbiome. While the products of the demethylation process in the host are mostly excreted outside the body, the demethylated by-product of the gut microbial population are kept inside the body and act as an energy source used for their growth and division. Furthermore, gut microbial populations have a significant role in the demethylation of xenobiotics. The produced metabolite may have a different profile of activity or toxicity from the parent molecule [14].

5.1.1 Methylmercury

Methylmercury is one of the most toxic environmental pollutants that causes neurotoxicity in both humans and animals. Methylmercury is formed following the exposure of inorganic mercury to the aquatic bacteria living in lakes, seas and stagnant waters and is found to be accumulated in the body of fish and other sea creatures. Methylmercury is found to be absorbed at a higher rate than mercury in human and wildlife GI tracts however, the gut microbial population can transform the methylmercury to inorganic mercury by a demethylation process and decrease its absorption and toxicity [87]. Methylmercury can challenge intestinal microbial diversity which in turn modifies the neuronal activity of intestinal neurotransmitters [14].

5.1.2 Methamphetamine

Methamphetamine is a racemic mixture of levo and dextro methamphetamine with sympathomimetic effects that is used as a CNS stimulant in ADHD. In-vitro incubation of methamphetamine with rectal contents of a female Dunkin-Hartley guinea pig for 72 h at 37°C indicated that methamphetamine is being N-demethylated to amphetamine by intestinal microbiome. Further investigation approved the participation of Lactobacilli, Enterococci, and Clostridia in the intestinal metabolism of methamphetamine. Whether or not this method of methamphetamine metabolism has a significant role in the disposition of this drug in humans is not clear. The reason must be sought in the high level of absorption of the parent drug in the lower gut [88].

5.2 Deamination, decarboxylation, dehydroxylation and hydroxylation

Deamination is one of the most important processes in the gut microbiome that involves the metabolism of food components and xenobiotics. Deamination of amino acids as part of routine biological processes is important in the production of bioactive components that play a critical role in maintaining intestinal epithelial cell homeostasis, immune cell responses and neuronal excitability. On the other hand, drug deamination by gut microbiome can generate metabolites with different biological activities or clinical adverse effects than the parent molecule. Although deamination could happen independently but mostly is associated with, decarboxylation, dehydroxylation and/or hydroxylation by the gut microbial system. These processes are responsible for the generation of some biologically active amino acid derivatives that could modulate the activity of different organs including the brain.

5.2.1 Melamine

Melamine is an additive used for increasing the apparent protein concentration in cat and dog food as well as children’s milk. Also, it is used in the production of various plastics in the industry. From the chemical point of view, melamine is a s-triazine derivate which could be deaminated by gut microbial enzymes and generate ammonia and cyanuric acid. Melamine is excreted in the urine unchanged, however, cyanuric acid can co-crystallize with the parent drug and make an insoluble complex in kidney tubules, which is responsible for kidney degeneration and the death of thousands of cats and dogs used food containing melamine [20]. Melamine-contaminated infant formula in China is responsible for causing kidney stones in 300,000 kids and at least 6 deaths. It is postulated that other chemically related compounds such as atrazine could also be metabolized by the deamination microbial system in the gut. This compound is an herbicide and the potential to cause toxicity is high if it enters the human body as residue in herbs [14].

5.2.2 5-Flurocytosine

5-Flurocytosine or Flucytosine is an active antifungal medication used in the treatment of systemic candidiasis, chromoblastomycosis and cryptococcosis in combination with other medications including amphotericin B. Mechanistically, it enters the fungal cells by an enzymatic pathway called permease which is not found in mammalian cells. Inside the fungal cells, it will be converted to 5-Flurouracil by fungal deaminase enzyme and then to 5-fluorodeoxyuridilic acid, which is a false nucleotide and inhibits the synthesis of DNA. In serum concentrations of more than 100 μg/ml, some side effects are associated with using 5-Flurocytosine. These side effects are very similar to the ones that are seen during 5-Flurouracil chemotherapy and include some bone marrow suppression and hematological adverse effects such as anemia, and leukopenia. Also, a trace of 5-Flurouracil was measured in the urine of patients using 5-Flurocytosine indicating the existence of a metabolic pathway similar to what is observed in fungal cells. On a semicontinuous in-vitro culture system using human fecal contents incubated with 5-Flurocytosine, the formation of 5-Flurouracil was confirmed indicating that anaerobic bacteria in the gut are involved in such conversion. Certain microorganisms in the intestine including Lactobacillus and Bacteroides may not constitutively possess deaminase enzymes, however, enterobacteria including E. coli express a high level of deaminase constitutively. In the incubation of 5-Flurocytosine, two types of chronic and acute exposure resulted in two different yields for the formation of 5-Fluorouracil. In acute exposure to a high dose of 5-Flurocytosine, a lag time of 8 hours existed between the beginning of the exposure time and the measurement of small quantities of 5-Flurouracil. In the chronic exposure, however, no lag time was observed for the generation of metabolite. Also, the level of metabolite was much higher than in acute exposure indicating that there is a microbial enzyme induction process involved in such a reaction [89].

5.2.3 Levodopa

Oral levodopa (L-dopa) is used in the treatment of Parkinson’s disease, a condition characterized by dopaminergic-neuronal death. L-dopa crosses the blood-brain barrier, where it is decarboxylated by host enzymes (Amino acid decarboxylase AADC) to restore dopamine. AADC is not only expressed in brain but also in some other peripheral tissues including gut. Expression of this enzyme in gut in not limited to the gut tissue but also gut microbiome. Extensive metabolism within the gut by both host and microbial enzymes affects the concentration of L-dopa reaching the brain. In other words, bioavailability of L-dopa could be compromised by microbial and peripheral metabolism of drug. Different microbial population in gut have encoded decarboxylate gene. For instance, Enterococcus faecalis in the gut has this enzymatic system which decarboxylates L-dopa to dopamine. The produced dopamine undergoes a further dehydroxylation process in some other microbial strains in gut such as Eggerthella lenta which converts dopamine to m-tyramine. The latter is further oxidized to m-hydroxyphenyl acetic acid. Differences in these activities may contribute to the substantial variation observed in patient response to L-dopa. Capacity of Eggerthella lenta in dehydroxylation of dopamine to tyrosine by dopamine dehydroxylating enzyme (Dadh) is attributed to a gene with a same name that exist in about 10 different bacterial species in gut, however only Eggerthella lenta has such capacity to dehydroxylate dopamine. Rekdal and colleagues at the Harvard university found a single nucleotide differences (SNAP) in the dadh gene responsible for such activity.

While the host decarboxylation process of L-dopa in the gut can be inhibited by the concomitant administration of carbidopa, the decarboxylation process of the gut microbial population cannot be affected by this drug. Instead, a drug called S- α- fluoromethyltyrosine (AFMT) has been developed for inhibiting the gut microbial decarboxylation process. This drug is a good candidate to be used in clinic for increasing the oral bioavailability of levodopa [90, 91].

Microbial metabolism of levodopa not only results in the production of dopamine, m-Tyrosine and m-Tyramine due to a decarboxylation and dehydroxylation process, but also m-hydroxyphenyl propionic acid and m-hydroxyphenyl acetic acid by a deamination process on its metabolites (m-Tyrosine and m-Tyramine) [89]. Pre-treatment of Parkinson’s patients with neomycin results in a decrease in the urinal excretion of m-hydroxyphenyl acetic and more pronounced clinical outcomes [92]. Deamination of an aromatic amino acid such as Tyrosine which is the metabolite of levodopa in the lower part of the gastrointestinal tract by anaerobic microbial populations (e.g., Clostridium sporogenes) is responsible for decrease in ileal motility of patients using this medication. As a matter of fact, 3-(3,4-hydroxyphenyl) propionic acid was detected in fecal samples of Parkinson’s disease patients using levodopa medication. This metabolite elicits an inhibitory effect on ileal motility in an ex-vivo model and is responsible for the constipation side effect associated with using levodopa in 90% of the patients [93].

5.2.4 Tryptophan

Tryptophan is an essential amino acid found in many foods such as red meat, fish, and egg. It is comprised of two isomers of L and D- tryptophan. L-tryptophan is used as a xenobiotic in the treatment of sleep disorders with an effect on decreasing total wakefulness or increasing sleep time [94], whereas D-tryptophan does not pass the blood-brain barrier (BBB) and shows no biological activity. L-tryptophan passes BBB and has a significant role in the production of antidepressive amines such as 5- Hydroxy tryptamine (5-HT) or serotonin and 5-Hydroxyindol acetic acid in the brain [95]. While the production of serotonin and 5-Hydroxyindol acetic acid in brain are controlled by host enzymatic system, enzymatic system of gut microbial also has significant role in production of 5-HT and indoles by both direct and indirect pathways. As a matter of fact, serotonin synthesis in brain by host enzymatic system comprises only 1% of serotonin pool in body. The rest of serotonin is generated in enterochromaffin cells in gut with some other biological functions such as stimulation of GI motility [96]. There are some evidence indicating the direct and indirect effects of gut microbiome in the production of serotonin in both gut and brain [97].

Gut microbiome has also a significant role in bioconversion of tryptophan using decarboxylase. Following this bioconversion step, the produced metabolite, which is called tryptamine, is a precursor of the synthesis of serotonin using another microbial enzyme called eukaryote-like aromatic amino acid hydroxylase-1 (Tph-1). Tryptophane decarboxylase (TrpD) is a pyridoxal phosphate (PLP)-dependent enzyme and along with Tph-1 expressed in several gut bacteria including E. coli K-12, Lactiplantibacillus plantarum, Lactococcus lactis, Streptococcus spp, Candia spp. Clostridium sporogenes, Ruminococcus gnavus and Enterococcus spp. [98].

Tryptophane hydroxylase-1 (Tph1), a rate limiting enzyme in the synthesis of serotonin in gut enterochromaffin cells, is encoded by Tph-1 gene. Enterochromaffin cells start producing serotonin once they receive a signal through gut microbiome-produced metabolites that upregulate the expression of Tph-1 gene. Studies of Sjogren et al. indicated that germ-free mice have substantially reduced colonic Tph1 mRNA expression, serum serotonin levels, and increased serotonin-selective reuptake transporter mRNA expression compared to control mice [99].

Tryptophan is metabolized by different populations of gut microbiome. Lactobacillus species convert tryptophan to I3A (Indol 3-Aldehyde) by deamination and a decarboxylation process, whereas Clostridium sporogenes modifies its structure to IPA (Indol 3-propionic acid) by deamination process. Some other forms of intestinal bacteria such as Bifidobacterium longum, Bacteroides fragilis and Parabacteroides distasonis convert tryptophan to Indol.

5.3 Interplay between gut microbiome and host CYP-450 enzymes

First pass metabolism of xenobiotics is an upgraded modified term due to the significant role of gut microbiome enzymatic system in drug disposition. As proven, gut microbiome has significant role on absorption, distribution and elimination of drugs. In between, elimination & metabolism of drugs are the most important procedures affected by gut microbiome. In its novel meaning, elimination & metabolism of drugs are the product of the collaboration between gut microbial and host enzymatic systems [100]. An indirect regulatory mechanism explains this relationship in which some remote sensing signals send from gut microbiome produced metabolites, control the expression of genes responsible for host metabolic system including CYP-450 enzymes in liver, gut and other organs.

In order to study the regulatory role of gut microbiome on the host drug metabolizing system, several models of germ-free mice (GF), probiotic treated mice and specific pathogen free mice (SPF) have been extensively used. Germ-free (GF) mice are great tool in understanding the regulatory role of gut microbiome on CYP-450 enzymes, although the unphysiological enlarged cecum in GF mice makes the interpretation of scientific results more challenging [101]. Other than GF mouse model, probiotic-treated mouse model (PT) is also used in clarification of gut microbiome on the expression of host drug metabolizing enzymes in liver.

Different studies have demonstrated the importance of gut microbiome on the expression of different drug metabolizing enzymes. Jourova et al. demonstrated that colonization of non-pathologic bacteria alters the mRNA expression of different CYP-450 enzymes in the liver of GF mice. While the mRNA expression of CYP1a2 and CYP2e1 were higher in GF mice compared to specific pathogen-free (SPF) mice, the expression of CYP3a11 mRNA level was lower. In their study, they monocolonized GF mice with non-pathogenic bacteria to see its effect on the expression of these enzymes. Colonization of either Lactobacillus plantarum or Escherichia coli Nissle 1917 into GF mice colon, normalized Cyp1a2 mRNA level in liver, however, they had no effect on the mRNA expression of CYP3a11 in GF mice (remained decreased). In terms of CYP2e1, the elevated mRNA expression of this enzyme in GF mice was not affected by monoclonization of L. plantarum, however monoclonization of probiotic E. coli Nissle 1917 caused a decrease in CYP2e1 level and partially restored to the conditions of Specific SPF mice [102].

Metabolism of xenobiotics is largely regulated by Nuclear Receptors (NR), primarily the Constitutive Active/Androgen Receptors (CAR) and Pregnane X-Receptors (PXR). Studies of Pettersson et al. on hepatic gene expression of these NRs in GF mouse model compared to conventionally raised SPF animals demonstrated that gut microbiome has extensive effects on the expression of CAR genes. Gene expression studies conducted on microarrays from these two groups of animals demonstrated the differential expression of genes, affecting the liver metabolic functions. In GF mice, genes regulated by CAR showed higher expression. Other than liver, the expression of CAR-regulated genes is at higher levels in GF colonial epithelium compared to SPF mice. In addition, the CAR-regulated gene POR, coding for the P450 oxidoreductase, the unique electron donor for all type II cytochrome P450 enzymes, is more expressed in the GF animals. The speculation is that signals leading to the higher expression of CAR genes is related to the absence of gut microbiome, however the role of bile acids, steroid hormones, cholesterol and bilirubin which are also regulated by gut microbiome, cannot be ruled out. Bile acids, Bilirubin and steroid hormones in liver activate CAR and have positive effects on expression of CYP-450 enzymes. It has also been shown that GF animals have lower level of fecal secretion of bile acids and higher level of cholesterol in liver. Therefore, it can be concluded that the higher expression of CAR in GF mice could be related to the higher levels of bile acids, steroid hormones and possibly cholesterol [103].

Bile acids have regulatory effect on intestinal microbiome. In other words, they may decrease the population of some gut microbial residence such as Bacteroidetes from 37% to almost none in rats. They may also increase the population of Firmicutes from 54 to 95%. The result of such regulatory function of microbial population would be the controlling of Bile acid flow and synthesis. With increase in gut microbial population, formation of secondary bile acids is augmented which is similar to what happens after the consumption of high-fat diet. Bile acids (BAs) are potent ligands for nuclear receptors including Farnesoid X receptor (FXR), Pregnane X receptor (PXR), and Vitamin D receptor (VDR). In between FXR also could regulate synthesis and release of BAs in liver through the release of fibroblast growth factor 15 (Fgf15) in intestine. There is substantial evidence regarding the role of FXR in regulating certain drug-metabolizing enzymes. FXR has a potent induction effect on PXR which regulates so many phase I and II drug metabolizing enzymes as well as transporters (CYP3A4, Sulfotransferase 2A1, MRP2 and MRP3). As a matter of fact, FXR role in regulating the expression of phase I drug-metabolizing enzyme genes development in early stages of life in liver is well established [104].

The ubiquitous role of CAR on the expression CYP enzymes is mixed with other NR such as PXR, Farnesoid X-Receptors (FXR), peroxisome proliferator activator receptors (PPAR) and Aryl hydrocarbon receptors (AHR). AHR is mostly not identified as a member of NR superfamily but has very similar function with NR [105]. In between, there is a specific overlap between CAR and PXR in regulating CYP-450 expressions. CAR was first linked to the induction of CYP2B gene superfamily by phenobarbital and controls the expression of CYP3A4, CYP2A6 and CYP2Cs as well. PXR is the mediator of CYP3A gene and PPAR-α regulates CYP3A4 and CYP2C8 transcription [106].

Comparing the microarray of GF and SPF mice resulted in identification of 112 genes with inhibitory effects on PXR. Also, another FXR is expressed more in GF mice compared to SPF mice. Expression of FXR in the SPF mice is 1.3-fold lower compared to GF mice. On the other hand, CAR is expressed 1.6-fold lower in SPF mice compared to GF mice. Increased expression level of CAR in GF mice has significant effects on metabolism of xenobiotics. This is observed in the higher level of pentobarbital metabolite which results in 35% shorter time of barbiturate induced anesthesia (53 min) in GF mice compared to SPF animals (81 min).

Increased expression of regulatory nuclear receptors (NR) in GF mice are associated with increased expression of Cyp2c9, Cyp2a4, Cyp2b13 and Cyp4a14 in these mice compared to SPF mice. Cyp2c9, Cyp2a4, Cyp2b13 and Cyp4a14 are 46.5-fold, 6.8-fold, 3.5-fold and 8.4-fold less expressed in SPF mice compared to GF mice respectively [103].

Using different mouse strain is associated with different results on the expression of CAR. Although in NRMI and C57BL/6 mice liver and colon, the condition of GF induced the CAR expression, there was no differences in expression of CAR genes between SPF and GF groups using C3H/Orl mice. Expression of other NR showed conflicting results on different strains of animals. While lower level of AHR, FXR and PXR are observed in GF model using IOI/Jic mouse strain, the expression of AHR and PXR were higher in C57BL/6 mice. No differences between GF and SPF mice were observed in NMRI model in terms of the expression of PXR [3]. Other than strain, age of mouse is also a big factor. Selwyn et al. observed that the difference between the expression of AHR and PXR in GF and SPF groups starts after 90 days [107].

AHR may also have an important effect on the cross-talk between gut microbiome and hepatic drug metabolism. AHR is a transcription factor and mostly found in cytosol of many cells including hepatocytes. AHR in cytosol is found in its inactive form as a complex with heat shock protein 90 (HSP90), aryl hydrocarbon receptor associated 9 (ARA9) and P23. Upon binding to its ligands, which could be synthetic, semisynthetic or natural compounds, conformational changes occur in the complex leading to the translocation of AHR complex to the nucleolus. Upon this translocation, AHR complex binds to its heterodimeric partners Aryl hydrocarbon receptor nuclear translocator (ARNT) and Aryl hydrocarbon response element (AHRE) which promote the transcription of downstream genes including CYP1A1, CYP1A2 and CYP1B1 [106, 108]. Some fermented products of naturally occurring elements found in food could act as a ligand of AHR and promote the transcription of genes involved in the synthesis of CYP450 enzymes. For instance, butyrate, a short chain fatty acid which is a product of fermentation process of nondigestible polysaccharides by colon microbiome, has an indirect regulatory function on AHR. Such impact in turn controls the gene expression of drug metabolism enzymes in liver. Butyrate as an epigenic factor and ligand of AHR can regulate AHR expression in both human primary hepatocytes and HepG2-C3 cell line. AHR and its target genes are upregulated in these cells by butyrate in a dose dependent manner. This increase is translated in increasing the expression of CYP1A1 and CYP1A2 as well as AHR repressor (AHRR) [109].

5.4 Correlation between drug administration and gut microbiome on the expression of drug metabolizing enzymes

Recent studied have found a correlation between drug administration and gut microbiome modification. Change in the composition of gut microbiome due to the drug administration can affect the expression of drug metabolizing enzymes in both liver and intestine of host.

5.4.1 Nabemutone

Jourova et al. found that plasma concentration of the active metabolite of Nabumetone is higher in GF mice compared to SPF mice. Nabumetone is a nonsteroidal anti-inflammatory drug which is metabolized by the contribution of Cyp1a2 and Cyp3a4 in liver to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). On the other hand, gut microbiome under both aerobic and anaerobic conditions forms an inactive metabolite of drug (Hydroxylated form of Nabemutone). Therefore, the increase in the bioavailability of this drug could be attributed to the role of gut microbiome in the inactivation of the drug [59]. Moreover, they found that orally administered Nabumetone by itself increases the expression of Cyp1a2, Cyp2c38, Cyp2d22, Cyp3a11, and Cyp3a13 in the small intestine and liver of GF mic. They also found a modification in the expression of four different transcription factors of Hepatocyte nuclear factor 4 (HnF4), PXT, CAR and AHR in the small intestine and liver of Nabumetone treated mice in both groups of GF and SPF mice. Decrease in the expression of Hnf4 along with increase in the expression of PXR in the small intestine of SPF mice which is associated with decrease in the mRNA expression of CAR and AHR in the liver of GF mice proves that both drug administration and gut microbiome contribute in the fate of drug [110].

5.4.2 Metronidazole

Treatment of HepaRG cells and cryopreserved human hepatocytes with metronidazole decreases the expression of some drug metabolizing enzymes including CYP2C8, CYP2C9 and CYP 3A4 as well as transcription factor CAR in a concentration dependent manner [111]. On the other hand, the fate of metronidazole in mice is affected by the presence or absence of gut microbiome. It has been observed that the plasma concentration of metronidazole in GF mice is ~30% higher compared to SPF mice. Interaction of the presence or absence of gut microbiome and the effect of metronidazole produced further changes in the mRNA expression of hepatic CYP enzymes as well as CAR, PPAR-α, AHR and PXR. While the mRNA expression of PPAR-α was three times higher in GF mice, the administration of metronidazole resulted in a time course difference in mRNA expression. The interaction between the presence or absence of gut microbiome and metronidazole administration on mRNA expression of CAR was a little different. Similarly, mRNA expression of CAR was higher in GF mice, however, the administration of metronidazole did not change the time course difference in mRNA expression. Similar trend was observed for mRNA expression of PXR. For AHR, no significant difference was observed between the two groups of GF and SPF mice in terms of mRNA expression. Also, metronidazole administration did not affect this trend.

Presence or absence of gut microbiome only affected the hepatic mRNA expression of cyp3a11, however in their combined effect with metronidazole, the hepatic mRNA expression of cyp2b10 and cyp2c38 was affected. Metronidazole by itself influenced the mRNA expression of cyp2c38, cyp2b10, cyp1a2 and cyp2d22.

Absence of gut microbiome increased the protein expression of CYP enzyme for cyp2B10 which reflects the increase in the expression of corresponding mRNA. This expression was decreased after 24 h of metronidazole administration. Protein expression of cyp1a2, cyp2d1 and cyp3a4 did not change significantly in both groups of mice by metronidazole administration [112].

6. Conclusion

It is well known that host genetics influence the composition of commensal intestinal flora. Fate of used drugs in GI tract is not only affected by the host enzymatic system but also microbial metabolic capacity. Regardless of the type of microbial enzymes involve in modification of drugs, the formed metabolites may show altered pharmacological or toxicological effects. That is mostly due to the different chemistry involved in the formation of metabolites by gut microbiome compared to host metabolic system. Even though liver has the dominant role in the metabolism of drugs in host, the role of gut microbiome on regulation of host metabolism cannot be ignored. One unique aspect of these studies could be related to the bidirectional relationship between administered drugs and gut microbiome. In other word, not only commensal intestinal flora can modify the structure of drugs but also, drugs could modify the microbial composition. Examples have been observed in administration of antibiotics, proton pump inhibitors (Omeprazole and Pantoprazole), levodopa and metformin.

In this manuscript, we have opened a new gate for identification of the importance of gut microbiome on the kinetic and more specifically metabolism of xenobiotics. Also, we briefly mentioned about the important regulatory role of gut microbial population on host metabolic system and more specifically CYP450 enzymes. Unfortunately, we had limitations for expanding the topics to the pharmacodynamic aspects of the formed metabolites. Hopefully, this manuscript and many other similar papers in this field could show the importance of gut microbiome in DMPK studies and persuade the authorities to include this exciting field as part of pharmacy curriculum.

Disclosure

The author declares no conflict of interest.