Biochemistry, Biotransformation

Fundamentals

The pathways of biotransformation are divided into phase I, phase II, and phase III. These reactions may occur simultaneously or sequentially.[1][2]

Phase I: Yields a polar, water-soluble, metabolite that is often still active. Many of the products in this phase can also become substrates for phase II.

• Oxidation with cytochrome P450 (most common)
• Reduction
• Hydrolysis

Phase II: Yields a large polar metabolite by adding endogenous hydrophilic groups to form water-soluble inactive compounds that can be excreted by the body.

• Methylation
• Glucuronidation (most common)
• Acetylation
• Sulfation
• Conjugation with glutathione
• Conjugation with amino acids (glycine, taurine, and glutamic acid)

Phase III: Occurs post-phase II, where a chemical substance can undergo further metabolism and excretion. It classifies into the following superfamilies:

• ATP-binding cassette (ABC)
• Solute carrier (SLC) transporters

Cellular Level

The majority of biotransformation takes place within the liver in cells called hepatocytes. However, several of the enzymes for phase I, phase II, and phase III reactions can also occur in extrahepatic tissues, such as adipose, intestine, kidney, lung, and skin. The transformation process takes place as a result of the interaction between the substrate and enzymes found primarily in these cells' cytoplasm, endoplasmic reticulum, and mitochondria.

Molecular Level

Phase I reactions are mainly catalyzed by the cytochrome P450 system, which is a family of membrane-bound enzymes found within the endoplasmic reticulum of hepatocytes. The unique protein structures that make up the P450 enzymes will exhibit significant differences in their substrate and product selectivity. Both constitutive and inducible regulations control these enzymes. For example, there are mechanisms of transcriptional gene activation, such as those that are stimulated by drugs or other exogenous substances.

Similar gene sequences classify cytochrome P450 pathways; they are assigned a family number and a subfamily letter and then become differentiated by a number for the isoform or individual enzyme. CYP subtypes include CYP1A1/2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5/7. The most common pathways involve CYP3A4/5, CYP2C9, CYP2D6, and CYP2C19, where CYP3A4 is the most abundant isoenzyme in the human liver and accounts for about 50% of all CYP450 activity.[2][3]

Mechanism

Reactions classify as Phase I, Phase II, or Phase III. It is important to note that these reactions do not have to take place sequentially and can even take place in reverse, Phase II, and then Phase I, or as a single reaction. Phase I metabolism consists of reduction, oxidation, or hydrolysis reactions. These reactions serve to convert lipophilic drugs into more polar molecules by adding or exposing a polar functional group such as -NH2 or -OH. These reactions also often create active metabolites and which is beneficial in activating prodrugs into their active and therapeutic state. Phase I includes the use of the cytochrome P450 system found with the membrane of the endoplasmic reticulum. If the CYP450 system becomes inhibited in any way the drug level will increase, and vice versa if induced, the drug level will decrease.

Phase I reaction using the CYP450 system includes both an oxidative and reductive step using NADPH and not ATP: Drug + O2+ NADPH -> Drug*+ H20 + NADP+.

Drugs that have already have -OH, -NH2 or COOH groups can bypass Phase I and enter Phase II directly to become conjugated. Phase II reactions consist of adding hydrophilic groups to the original molecule, a toxic intermediate or a nontoxic metabolite formed in phase I, that requires further transformation to increase its polarity.  These reactions include conjugation reactions, glucuronidation, acetylation, and sulfation. The ultimate goal of phase II reactions is to form water-soluble products that can be excreted by the body. 

A drug can ultimately undergo further metabolism during Phase III. In this phase, the drug that gets transported via an ABC transporter, which will require energy (ATP) consumption to actively uptake or efflux a compound from one side of the cell membrane to another. On the other hand, it can get transported via an SLC transporter which facilitates the passage of specific solutes across the membrane and actively transports other solutes against their electrochemical gradients by merging this action with another solute or ion. The function of an uptake transporter is to transfer a molecule into the cell, and the function of an efflux transporter is to transfer a molecule outside the cell.  

In general, these reactions serve to deactivate substances. However, they may also bioactivate some substances by transforming them from nontoxic and/or inactive to the toxic and/or active form. If the process produces a toxic intermediate, its effect on the body is dependent on a few different factors. Some of these factors include how rapidly the intermediate can undergo further metabolism to a less toxic metabolite, the amount of toxic byproduct that is produced and then accumulated in cells, the type and amount of damage caused by the toxic intermediate, and the factors that would prolong excretion of the toxic metabolite.[2][3][4]

Clinical Significance

Biotransformation is affected by age, sex, nutritional status, disease state, medications, and genetics of a patient. There is variance in expression and activity level of the CYP450 system throughout prenatal development and from the neonatal period to adulthood. A limited amount of studies have shown that cytochrome P450, specifically the activity of CYP3A4 is higher in females than in males, in comparison to the activity of many other systems involved in drug metabolism which may be higher in males than in females. A balanced diet, as compared to a protein-deficient diet, will provide the necessary protein as well as essential metals and minerals such as copper, zinc, and calcium needed for normal cellular enzymatic activities. Also of note, patients with liver disease, such as cirrhosis, have damage to hepatocytes that can then be replaced by fibrous connective tissue that is unable to carry out metabolic processes.[5][6]

Most importantly, drug interactions should undergo checking for modulators of the CYP450 systems. Drugs with CYP activity may be inhibitors, inducers, or substrates for a specific CYP enzymatic pathway which can lead to alteration of the metabolism of concurrently administered agents. Some common substrates for the cytochrome P450 system include anti-epileptics, theophylline, warfarin, oral contraceptive pills, and vitamin D. Therefore when exposed to inhibitors drug toxicity can ensue due to the decreased metabolism of the substrate, whereas inducers would speed up the process by which these substrates become metabolized.[7][8][9]

Common Inducers:

• Carbamazepine
• Rifampin
• Alcohol (chronic use)
• Phenytoin
• Griseofulvin
• Phenobarbital
• Sulfonylureas
• Modafinil
• St John’s wort
• Ginseng
• Tobacco (smoking)

Common Inhibitors:

• Valproic Acid
• Alcohol (acute use)
• Azole antifungals
• Isoniazid
• Sulfonamides
• Chloramphenicol
• Amiodarone
• Non-dihydropyridine calcium channel blockers 
• Erythromycin/clarithromycin
• Quinidine
• Cimetidine
• Ciprofloxacin
• Omeprazole
• Metronidazole
• Ritonavir
• Fluoxetine
• Grapefruit juice
• Vitamin E
• Thyroid hormone

Specific inducers and inhibitors of the CYP450 system include:

CYP3A4

Inhibitors: Aprepitant, clarithromycin, erythromycin, fluconazole, grapefruit juice, isoniazid, itraconazole, ketoconazole, metronidazole, telithromycin, valproic acid, voriconazole

Inducers: Carbamazepine, dexamethasone, oxcarbazepine, phenobarbital, phenytoin, rifampin

CYP2C9/8

Inhibitors: Fluconazole, ibuprofen, indomethacin, isoniazid, ketoconazole, sulfamethoxazole, trimethoprim

Inducers: Carbamazepine, phenobarbital, phenytoin, rifampin

CYP2D6

Inhibitors: Isoniazid, ketoconazole, methadone, nicardipine

CYP2C19

Inhibitors: Fluconazole, ketoconazole, isoniazid, omeprazole

Inducers: Carbamazepine, phenytoin, rifampin 

Metabolic reactions also may vary from one individual to another. Consequently, this affects drug dosing and frequency to achieve safe and effective levels of drug within the body.[10]

Phase II reactions can unergo alteration in patients due to acetylation polymorphisms. A person's phenotype as a slow acetylator can be homozygous for the slow acetylator gene, or in rapid acetylators, they can be either heterozygous or homozygous for the rapid acetylator gene. Slow acetylators have a genetically mediated decrease in hepatic N-acetyltransferase and can lead to higher concentrations of a drug in the blood, which can then lead to an increase in toxic reactions or fatal toxicity. The hereditary acetylator status of patients provides valuable information about their potential therapeutic, pharmacologic, and toxicologic responses. It also serves as a good prognostic indicator of susceptibility to toxicity from commonly used drugs for the treatment of a wide range of diseases.[11]

Phase III reactions can also undergo alteration with the consumption of certain medications. P-glycoprotein (P-gp) is one of the efflux transporters located in the small intestine that will transfer drugs from the cytoplasm into the intestinal lumen for excretion during Phase III. Specific substrates, inducers, and inhibitors of P-gp include:

Substrates: Digoxin, loperamide, vinblastine

Inducers: Rifampin, phenytoin, phenobarbital

Inhibitors: Amiodarone, cyclosporine, clarithromycin