Pathways of Biotransformation:

Biotransformation of drugs has been divided into two major phases:

1. Phase-I reactions
2. Phase-II reactions

1. Phase-I:

• These includes reactions which catalyze oxidation, reduction and hydrolysis of drugs.
• In this phase, small polar functional groups like –OH, -NH₂, -SH, -COOH, etc. are either added or unmasked so that resulting products may either undergo phase II reactions or are directly excreted.
• This phase includes following reactions:

a. Oxidation:

• Oxidation of drugs enhance the hydrophilicity of drugs by introducing polar functional groups such as –OH.
• Oxidation of drugs is non-specifically catalyzed by a number of enzymes located in microsomes.
• Some are also catalyzed by non-microsomal enzyme.
• Most important group of oxidative enzymes are microsomal mixed function oxidases (MFO) or monooxygenases.
• These enzymes are primarily located in E.R of hepatocytes and require both molecular oxygen (O₂) and reduced NADPH to effect the chemical reaction.
• Most important component of mixed function oxidases is the cytochrome P-450 (CYP-450) enzymes that catalyze the phase I oxidation of many xenobiotic.
• CYP-450 enzymes can be induced or inhibited by many drugs and substances causing several drug interactions in which one drug either enhances the toxicity or reduces the therapeutic effect of another drug.
• Drug that are metabolized by CYP-450 system attain higher concentrations and have prolonged half-lives in neonates and older animals.

b. Reduction:

• Reduction is the acceptance of one or more electrons or their equivalent from another substrate.
• It usually involves the addition of hydrogen molecules which occurs less frequently than the oxidative reactions.
• Biotransformation by reduction is also capable of generating polar functional group such as hydroxyl and amino groups, which can undergo further biotransformation.
• These reactions usually lead to conversion of inactive metabolites into active drugs, thereby delaying drug removal from the body.

c. Hydrolysis:

• It donot involve the change in oxidation state of substrate, but involves the cleavage of drug molecules by taking up a molecule of water.
• A number of drugs with ester, ether, amide and hydrazide linkages undergo hydrolysis.
• Ex; cholinesters, procaine, procainamide and pethidine.

2. Phase II Reaction:

• Phase II reactions or conjugation reactions involve combination of the drug or its phase I metabolite with an endogenous substance to form a highly polar product which can be efficiently excreted from the body.
• In biotransformation of drugs, such products or metabolites carry two parts; exocon the portion derived from exogenous compound and endocon, the portion derived from endogenous substances.
• Endogenous substances for conjugation reaction are derived mainly from carbohydrates or amino acids and they generally possess large molecular size.
• They are strongly polar or ionic in nature in order to make the substrate water-soluble.
• High molecular weight conjugates are excreted from bile. While low molecular weight conjugates are excreted mainly in urine.
• The unconjugated drug/metabolite may precipitate toxicity.

a. Conjugation with glucuronic acid/glucuronidation:

• It is the most common and most important phase II reactions in vertebrates except cats and fish.
• It occurs in liver microsomal enzyme system which is only reaction.
• It makes drug water soluble so that they are easily secreted in bile and excreted in urine.
• Biochemical factor of glucuronic acid is uridine diphosphate α-D-glucuronic acid (UDPGA) and the reaction is carried out by enzyme uridine diphosphate-glucuronyl transferase.
• Glucuronidation can take place in most body tissues because the donor is present in abundant quantity in body.
• In cats there is reduced glucuronyl transferase activity while in fish there is deficiency of endogenous glucuronic acid donor. This limited capacity of this metabolic pathway in cats may increase the duration of action, pharmacological response, and potential toxicity of several lipid soluble drugs.

b. Conjugate with sulphate/sulphation:

• Conjugation with sulphate is similar to glucuronidation but is catalyzed by non-microsomal enzyme and occurs less commonly.
• Endogenous donor of the sulphate group is 3’ phosphodenosine-5’ phosphosulphate (PAPS) and enzyme catalyzing the reaction is sulphotransferase.
• Sulphate esters are polar and readily excreted in urine.
• In pigs, it is minimum because the donor PAPS get depleted easily.
• Functional groups capable of forming sulphate conjugates includes phenols, alcohols, arylamines, N-hydroxylamines and N-hydroxyamides.
• Drugs undergoing sulphate conjugation include chloramphenicol, phenols, and adrenal and sex steroids. Aging doesnot affect the process.

c. Conjugation with methyl group/Methylation:

• It involves a transfer of methyl group (-CH₃) from the co-factor s-adenosyl methionine to the acceptor substrate by various methyl transferase enzymes.
• It is more important for biosynthesis and inactivation of endogenous amines.
• Occasionally, the metabolites formed by methylation are not polar or water-soluble and may possess equal or greater activity than the parent compounds.

d. Conjugation with glutathione and mercapturic acid formation:

• It is minor but important metabolic pathways in animals.
• Glutathione is tripeptide having glutamic acid, cysteine, and glycine.
• It has strong nucleophilic character due to the presence of –SH (thiol) group in its structure.
• Thus, it conjugates with electrophilic substrates.
• The interaction between substrate and the GSH is catalyzed by enzyme glutathione-s-transferase, which is located in the soluble fraction of liver homogenates.
• Glutathione is either excreted as such in bile or further metabolized to form mercapturic acid conjugate that is excreted in urine.

e. Conjugation with acetyl group/acetylation:

• It is an important metabolic pathway for drugs containing the amino groups.
• Co-factor for these reactions is acetyl coenzyme A and enzymes are non-microsomal N-acetyl transferases.
• It is not a true detoxification process, because it sometimes results in decrease in water solubility of an amine and thus increase in toxicity.

f. Conjugation with amino acid:

• It occurs in limited extent in animals because of limited availability of amino acids in the body.
• Most important reactions involve conjugation with glycine.
• Eg; salicylic acid, nicotinic acid and cholic acid forms glycine or glutamine conjugates.

g. Conjugation with thiosulphate:

• It is an important reaction in the detoxification of cyanide.
• Conjugation of cyanide ion involves transfer of Sulphur atoms from thiosulphate to cyanide in presence of rhodanese to form inactive thiocyanate.
• Thiocyanate is less toxic than cyanide and excreted in urine.