Drug metabolism

The exact compounds an organism is exposed to will be largely unpredictable, and may differ widely over time; these are major characteristics of xenobiotic toxic stress.^[3] The major challenge faced by xenobiotic detoxification systems is that they must be able to remove the almost-limitless number of xenobiotic compounds from the complex mixture of chemicals involved in normal metabolism. The solution that has evolved to address this problem is an elegant combination of physical barriers and low-specificity enzymatic systems.

All organisms use cell membranes as hydrophobic permeability barriers to control access to their internal environment. Polar compounds cannot diffuse across these cell membranes, and the uptake of useful molecules is mediated through transport proteins that specifically select substrates from the extracellular mixture. This selective uptake means that most hydrophilic molecules cannot enter cells, since they are not recognized by any specific transporters.^[4] In contrast, the diffusion of hydrophobic compounds across these barriers cannot be controlled, and organisms, therefore, cannot exclude lipid-soluble xenobiotics using membrane barriers.

However, the existence of a permeability barrier means that organisms were able to evolve detoxification systems that exploit the hydrophobicity common to membrane-permeable xenobiotics. These systems therefore solve the specificity problem by possessing such broad substrate specificities that they metabolize almost any non-polar compound.^[3] Useful metabolites are excluded since they are polar, and in general contain one or more charged groups.

The detoxification of the reactive by-products of normal metabolism cannot be achieved by the systems outlined above, because these species are derived from normal cellular constituents and usually share their polar characteristics. However, since these compounds are few in number, specific enzymes can recognize and remove them. Examples of these specific detoxification systems are the glyoxalase system, which removes the reactive aldehyde methylglyoxal,^[5] and the various antioxidant systems that eliminate reactive oxygen species.^[6]

The metabolism of xenobiotics is often divided into three phases: modification, conjugation, and excretion. These reactions act in concert to detoxify xenobiotics and remove them from cells.

In phase I, a variety of enzymes act to introduce reactive and polar groups into their substrates. One of the most common modifications is hydroxylation catalyzed by the cytochrome P-450-dependent mixed-function oxidase system. These enzyme complexes act to incorporate an atom of oxygen into nonactivated hydrocarbons, which can result in either the introduction of hydroxyl groups or N-, O- and S-dealkylation of substrates.^[7] The reaction mechanism of the P-450 oxidases proceeds through the reduction of cytochrome-bound oxygen and the generation of a highly-reactive oxyferryl species, according to the following scheme:^[8]

O₂ + NADPH + H⁺ + RH → NADP⁺ + H₂O + ROH

Phase I reactions (also termed nonsynthetic reactions) may occur by oxidation, reduction, hydrolysis, cyclization, decyclization, and addition of oxygen or removal of hydrogen, carried out by mixed function oxidases, often in the liver. These oxidative reactions typically involve a cytochrome P450 monooxygenase (often abbreviated CYP), NADPH and oxygen. The classes of pharmaceutical drugs that utilize this method for their metabolism include phenothiazines, paracetamol, and steroids. If the metabolites of phase I reactions are sufficiently polar, they may be readily excreted at this point. However, many phase I products are not eliminated rapidly and undergo a subsequent reaction in which an endogenous substrate combines with the newly incorporated functional group to form a highly polar conjugate.

A common Phase I oxidation involves conversion of a C-H bond to a C-OH. This reaction sometimes converts a pharmacologically inactive compound (prodrug) to a pharmacologically active one. By the same token, Phase I can turn a nontoxic molecule into a poisonous one (toxification). Simple hydrolysis in the stomach is normally an innocuous reaction, however there are exceptions. For example, phase I metabolism converts acetonitrile to glycolonitrile (HOCH₂CN), which rapidly dissociates into formaldehyde and hydrogen cyanide.^[9]

Phase I metabolism of drug candidates can be simulated in the laboratory using non-enzyme catalysts.^[10] This example of a biomimetic reaction tends to give products that often contains the Phase I metabolites. As an example, the major metabolite of the pharmaceutical trimebutine, desmethyltrimebutine (nor-trimebutine), can be efficiently produced by in vitro oxidation of the commercially available drug. Hydroxylation of an N-methyl group leads to expulsion of a molecule of formaldehyde, while oxidation of the O-methyl groups takes place to a lesser extent.

• Cytochrome P450 monooxygenase system
• Flavin-containing monooxygenase system
• Alcohol dehydrogenase and aldehyde dehydrogenase
• Monoamine oxidase
• Co-oxidation by peroxidases

• NADPH-cytochrome P450 reductase

Cytochrome P450 reductase, also known as NADPH:ferrihemoprotein oxidoreductase, NADPH:hemoprotein oxidoreductase, NADPH:P450 oxidoreductase, P450 reductase, POR, CPR, CYPOR, is a membrane-bound enzyme required for electron transfer to cytochrome P450 in the microsome of the eukaryotic cell from a FAD- and FMN-containing enzyme NADPH:cytochrome P450 reductase. The general scheme of electron flow in the POR/P450 system is: NADPH → FAD → FMN → P450 → O₂

• Reduced (ferrous) cytochrome P450

During reduction reactions, a chemical can enter futile cycling, in which it gains a free-radical electron, then promptly loses it to oxygen (to form a superoxide anion).

• Esterases and amidase
• Epoxide hydrolase

In subsequent phase II reactions, these activated xenobiotic metabolites are conjugated with charged species such as glutathione (GSH), sulfate, glycine, or glucuronic acid. Sites on drugs where conjugation reactions occur include carboxy (-COOH), hydroxy (-OH), amino (NH₂), and thiol (-SH) groups. Products of conjugation reactions have increased molecular weight and tend to be less active than their substrates, unlike Phase I reactions which often produce active metabolites. The addition of large anionic groups (such as GSH) detoxifies reactive electrophiles and produces more polar metabolites that cannot diffuse across membranes, and may, therefore, be actively transported.

These reactions are catalyzed by a large group of broad-specificity transferases, which in combination can metabolize almost any hydrophobic compound that contains nucleophilic or electrophilic groups.^[3] One of the most important classes of this group is that of the glutathione S-transferases (GSTs).

After phase II reactions, the xenobiotic conjugates may be further metabolized. A common example is the mercapturic acid pathway, which is the processing of glutathione (GSH) conjugates to N-acetylcysteine (mercapturic acid) conjugates.^[13][14] Here, the γ-glutamate and glycine residues in the glutathione molecule are removed by gamma-glutamyl transpeptidase and dipeptidases. In the final step, the cysteine residue in the conjugate is acetylated.

Conjugates and their metabolites can be excreted from cells in phase III of their metabolism, with the anionic groups acting as affinity tags for a variety of membrane transporters of the multidrug resistance protein (MRP) family.^[15] These proteins are members of the family of ATP-binding cassette transporters and can catalyze the ATP-dependent transport of a huge variety of hydrophobic anions,^[16] and thus act to remove phase II products to the extracellular medium, where they may be further metabolized or excreted.^[17]

The detoxification of endogenous reactive metabolites such as peroxides and reactive aldehydes often cannot be achieved by the system described above. This is the result of these species' being derived from normal cellular constituents and usually sharing their polar characteristics. However, since these compounds are few in number, it is possible for enzymatic systems to utilize specific molecular recognition to recognize and remove them. The similarity of these molecules to useful metabolites therefore means that different detoxification enzymes are usually required for the metabolism of each group of endogenous toxins. Examples of these specific detoxification systems are the glyoxalase system, which acts to dispose of the reactive aldehyde methylglyoxal, and the various antioxidant systems that remove reactive oxygen species.

Quantitatively, the smooth endoplasmic reticulum of the liver cell is the principal organ of drug metabolism, although every biological tissue has some ability to metabolize drugs. Factors responsible for the liver's contribution to drug metabolism include that it is a large organ, that it is the first organ perfused by chemicals absorbed in the gut, and that there are very high concentrations of most drug-metabolizing enzyme systems relative to other organs. If a drug is taken into the GI tract, where it enters hepatic circulation through the portal vein, it becomes well-metabolized and is said to show the first pass effect.

Other sites of drug metabolism include epithelial cells of the gastrointestinal tract, lungs, kidneys, and the skin. These sites are usually responsible for localized toxicity reactions.

The duration and intensity of pharmacological action of most lipophilic drugs are determined by the rate they are metabolized to inactive products. The Cytochrome P450 monooxygenase system (CYP) is a crucial pathway in this regard. In general, anything that increases the rate of metabolism (e.g., enzyme induction) of a pharmacologically active metabolite will decrease the duration and intensity of the drug action. The opposite is also true, as in enzyme inhibition. However, in cases where an enzyme is responsible for metabolizing a pro-drug into a drug, enzyme induction can accelerate this conversion and increase drug levels, potentially causing toxicity.^{[medical citation needed]} For example, chemotherapy prodrugs like cyclophosphamide (CPA) and ifosfamide (Ifex), which are initially inactive, become toxic as they are metabolized into cytotoxic compounds (such as phosphoramide mustard and chloroacetaldehyde) primarily from liver enzymes CYP2B6^[18] and CYP3A4. Co-administration of a strong CYP inducer, such as phenytoin or rifampicin, accelerates metabolism and increases the rate of bioactivation which causes a higher concentration of cytotoxic metabolites that may lead to higher toxicity. This drug–drug interaction may enhance the risk of adverse effects, most notably severe myelosuppression and hemorrhagic cystitis.^[19][20][21]

The therapeutic index (TI) of a drug is the measurement of its efficacy, calculated as the ratio of the median toxic dose (TD₅₀) to the median effective dose (ED₅₀).^[22] Various Cytochrome P450 metabolic enzymes are inhibited or induced by many drugs. For example, chronic alcohol consumption will induce Cytochrome P450 enzymes, like CYP2E1, which enhances the metabolism of ethanol.^[23] As a consequence, the induction of CYP2E1 will increase a person's tolerance levels and reduce the toxicity of ethanol. Additionally, CYP2E1 is involved with the metabolism of acetaldehyde (CH₃CHO), a metabolite of alcohol that is highly reactive and toxic, which can contribute to an alcohol-induced liver injury along with overoxidation.^[24][25]

Various physiological and pathological factors can also affect drug metabolism. Physiological factors that can influence drug metabolism include age, individual variation (e.g., pharmacogenetics), enterohepatic circulation, nutrition, sex differences or gut microbiota.^{[medical citation needed]} This last factor has significance because gut microorganisms are able to chemically modify the structure of drugs through degradation and biotransformation processes, thus altering the activity and toxicity of drugs. These processes can decrease the efficacy of drugs, as is the case of digoxin in the presence of Eggerthella lenta in the microbiota.^[26] Genetic variation (polymorphism) accounts for some of the variability in the effect of drugs.^[26] An example of polymorphism affecting drug metabolism is the alcohol flush reaction caused by the ALDH2 genetic mutation. The ALDH2 genetic mutation is prevalent among east Asians and causes a reduced activity of aldehyde dehydrogenase (ALDH), which assists in breaking down acetaldehyde (CH₃CHO).^[27] Approximately 560 million people (8% of the world's current population) have this genetic mutation, which poses various health risks like metabolic disorders or an increase cancer risk.^[28]

In general, drugs are metabolized more slowly in fetal, neonatal and elderly humans and animals than in adults. Inherited genetic variations in drug-metabolizing enzymes result in different catalytic activity levels. For example, N-acetyltransferases (involved in Phase II reactions), individual variation creates a group of people who acetylate slowly (slow acetylators) and those who acetylate quickly (rapid acetylators), split roughly 50:50 in the population of Canada. However, variability in NAT2 alleles distribution across different populations is high, and some ethnicities have a higher proportion of slow acetylators.^[29] This variation in metabolizing capacity may have dramatic consequences, as the slow acetylators are more prone to dose-dependent toxicity. NAT2 enzyme is a primary metabolizer of antituberculosis (isoniazid), some antihypertensive (hydralazine), anti-arrhythmic drugs (procainamide), antidepressants (phenelzine) and many more ^[30] and increased toxicity as well as drug adverse reactions in slow acetylators have been widely reported. Similar phenomena of altered metabolism due to inherited variations have been described for other drug-metabolizing enzymes, like CYP2D6, CYP3A4, DPYD, UGT1A1. DPYD and UGT1A1 genotyping is now required before administration of the corresponding substrate compounds (5-FU and capecitabine for DPYD and irinotecan for UGT1A1) to determine the activity of DPYD and UGT1A1 enzyme and reduce the dose of the drug in order to avoid severe adverse reactions.^[31]

Dose, frequency, route of administration, tissue distribution, and protein binding of the drug affect its metabolism.^[32] Pathological factors can also influence drug metabolism, including liver, kidney, or heart disease.^[33][34][35]

In silico modelling and simulation methods allow drug metabolism to be predicted in virtual patient populations prior to performing clinical studies in human subjects.^[36] This can be used to identify individuals most at risk from adverse reaction.

Studies on how people transform the substances that they ingest began in the mid-nineteenth century, with chemists discovering that organic chemicals such as benzaldehyde could be oxidized and conjugated to amino acids in the human body.^[37] During the remainder of the nineteenth century, several other basic detoxification reactions were discovered, such as methylation, acetylation, and sulfonation

In the early twentieth century, work moved on to the investigation of the enzymes and pathways that were responsible for the production of these metabolites. This field became defined as a separate area of study with the publication by Richard Williams of the book Detoxication mechanisms in 1947.^[38] This modern biochemical research resulted in the identification of glutathione S-transferases in 1961,^[39] followed by the discovery of cytochrome P450s in 1962,^[40] and the realization of their central role in xenobiotic metabolism in 1963.^[41][42]

• Biodegradation
• Microbial biodegradation