Biotransformation Xenobiotic metabolism “Essentials of Toxicology”

Biotransformation Biotransformation means chemical alteration of chemicals such as nutrients, amino acids, toxins, xenobiotics or drugs in the body. It is also needed to render nonpolar compounds polar so that they are not reabsorbed in renal tubules and are excreted. Biotransformation may results into- Active Inactive form Active Active or toxic form Inactive Active form Unexcretable Excretable form

Biotransformation Results of biotransformation more potent active TOXIC less potent active active Drug or Poison biotransformed Drug or Poison inactive inactive In general - nonsynthetic precede synthetic reactions nonsynthetic reactions can produce active metabolites synthetic reactions produce inactive metabolites

Biotransformation

Biotransformation Why is Biotransformation necessary? • Most drugs are excreted by the kidneys • For renal excretion drugs should: – have small molecular mass – be polar in nature – should be fully ionized at body pH • Most drugs are complex and do not have these properties and thus have to be broken down to simpler products.

Biotransformation Pharmacologically active organic molecules tend to- Ctd… Pharmacologically active organic molecules tend to- Be highly lipophilic & remain unionized or partially ionized at physiologic PH. • Thus readily pass across biological barriers –membranes • Strongly bound to plasma proteins Such substances are not readily filtered at the glomerulus. Their lipophilicity also facilitates to be reabsorbed through lipophilic renal tubular membranes. • This property also stops them from getting eliminated • They have to be converted to simpler hydrophilic compounds so that they are eliminated and their action is terminated.

Biotransformation Ctd… • Biotransformation can also result in bioactivation, which involves the production of reactive metabolites that are more toxic, mutagenic, or carcinogenic than their parent compound(s). • Drugs may converted to- – less toxic materials – more toxic materials – materials with different type of effect or toxicity Beside these, biotransformation is called a biochemical defense mechanism as it handles different xenobiotics, drugs, toxicants, body wastes (hemoglobin) or other unwanted substances to those we get exposed.

Biotransformation Where do biotransformations occur? Liver is the principal organ of drug metabolism although every tissue has some ability to metabolize drugs. Other tissues that display considerable activity include the GIT, the lungs, the skin, and the kidneys. Following oral administration, many drugs (e.g. isoproterenol,morphine) absorbed intact from the small intestine and transported first via the portal system to the liver, where they undergo extensive metabolism ( first-pass metabolism). Some orally administered drugs (e.g.clonazepam, chlorpromazine) are extensively metabolized in the intestine than in the liver.

Biotransformation Ctd… Thus intestinal metabolism may contribute to the overall first-pass effect. First pass effects may so greatly limit the bioavailability of orally administered drugs. The lower gut harbors intestinal microorganisms that are capable of many biotransformation reactions. Although drug biotransformation in vivo can occur by spontaneous, noncatalyzed chemical reactions, the vast majority are catalyzed by specific cellular enzymes.At the cellular level, these enzymes may be located in the – i) Endoplasmic reticulum ii) mitochondria iii) cytosol iii) lysosomes iv) even the nuclear envelope or v) plasma membrane.

Biotransformation Water soluble xenobiotics are easier to eliminate in urine, feces but not exhalation as t1/2 is low. Lipophilic barbiturates such as thiopental & phenobarbital would have half-lives greater than 100 years if they were not converted to water-soluble compounds. Multiple enzymes (families) Constitutively expressed Inducible Broad specificity Polymorphic Stereo-isomer specificity

Biotransformation Relatively harmless Potentially toxic xenobiotic Metabolic activation Detoxification Inactive metabolite Reactive intermediate

Converting lipophilic to water soluble compounds (non-polar) Xenobiotic Phase I - Activation Reactive intermediate Phase II - Conjugation Conjugate Water soluble (polar) Excretion

Phase I introduction of functional group hydrophilicity increases slightly may inactivate or activate original compound major player is CYP or mixed function oxygenase (MFO) system in conjunction with NAD(P)H location of reactions is smooth endoplasmic reticulum

Phase II conjugation with endogenous molecules (GSH, glycine, cystein, glucuronic acid) hydrophilicity increases substantially neutralization of active metabolic intermediates facilitation of elimination location of reactions is cytoplasm

Phase I reactions epoxide Oxidation Reduction Hydrolysis C O Hydroxylation (addition of -OH group) N- and O- Dealkylation (removal of -CH side chains) Deamination (removal of -NH side chains) Epoxidation (formation of epoxides) Oxygen addition (sulfoxidation, N-oxidation) Hydrogen removal Reduction Hydrogen addition (unsaturated bonds to saturated) Donor molecules include GSH, FAD, NAD(P)H Oxygen removal Hydrolysis Splitting of C-N-C (amide) and C-O-C (ester) bonds epoxide O C C C O

Biotransformation Activation of xenobiotics is a key element (e.g. benzene, vinyl chloride) Reactive intermediates include epoxides and free radical species (unpaired electrons) that are short-lived and hence highly reactive Protection is provided by endogenous antioxidant substances, e.g. GSH vitamins C and E antioxidant enzymes Antioxidant molecules are oxidized in the process but have the capacity to regenerate the reduced form from the oxidized - NAD(P)H is a key player

Cytochrome P450 (CYP) Mixed Function Oxidases (MFO) Located in many tissues but highly in liver ER Human: 16 gene families CYP 1,2,3 perform drug metabolism >48 genes sequenced Key forms: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 Highly inducible Alcohol CYP2E1 Barbiturates CYP2B

Proportion of drugs metabolized by different CYPs CYPs are the major enzymes involved in drug metabolism, accounting for ~75% of the total metabolism.Most drugs undergo deactivation by CYPs, either directly or by facilitated excretion from the body. Also, many substances are bioactivated by CYPs to form their active compounds. Proportion of drugs metabolized by different CYPs

Figure CYP450 Reaction Sequence OH DRUG DRUG CYP450 Fe3+ CYP450 Fe3+ CYP450 Fe3+ DRUG DRUG NADPH + H+ OH e- CYP450 reductase CYP450 Fe3+ CYP450 Fe2+ NADPH + H+ DRUG DRUG : .. O H+ e- H2O O2 CYP450 Fe2+ CYP450 Fe2+ DRUG DRUG O21- O2

Oxidation of vinyl chloride to an epoxide

Metabolic enzymes Microsomal: Non-microsomal Both CYP450 monooxygenases Flavin monooxygenase Non-microsomal Alcohol dehydrogenase Aldehyde dehydrogenase Monoamine and diamine oxidases Both Esterases and Amidases Prostaglandin synthase Peroxidases

Cooxidation of acetaminophen by prostaglandin endoperoxide synthetase

Hydrolysis of esters and amides

Hydrolysis of organophosphates

Hydrolysis of epoxides

Stereoselective hydroxylation

Metabolism of benzo(a)pyrene to 9,10 epoxide: Potent mutagen that binds DNA