summary of Lecture IV

PHASE II (CONJUGATION REACTIONS)

Or Synthetic reaction

• Capable of converting parent xenobiotics or phase I metabolites into polar and water soluble products.
• Conjugated products are relatively excretable, biologically inactive and non toxic.
• Some phase II reactions such as methylation and acetylation only terminate the
  pharmacological activity.but does not not increase solubility.
• Phase II reactions is the truly detoxifying pathway in drug metabolism (few exceptions).

Drug + Conjugating Agent → Conjugating Product

• The conjugated agent (e.g. glucuronic acid, sulfate, methyl & acetyl) is activated
  to give coenzyme with the appropriate transferase enzyme leads to the attachment to the accepting substrate.
• In case of glycine and glutamine → substrate is initially activated.
• Glutathione does not require initial formation of an activated coenzyme or substrate.

I- Glucuronic acid conjugation

• Most common conjugative pathway in drug metabolism

1. Readily available supply of D- glucuronic acid in the body (from D- glucose).
2. A large number of functional groups can combine enzymatically with glucuronic acid e.g. hydroxyl groups (phenols, alcohols, enols, N-hydroxylamines, N- hydroxylamides) carboxyl groups (aryl acids, arylalkyl acids). nitrogen groups (arylamines, alkylamines, amides, sulfonamides, tertiary amines) sulfhydryl groups.
3. The conjugated products are water soluble and are eliminated very fast.

• Involves two steps
  a-Synthesis of the activated coenzyme, uridine-5'-diphospho –α-D- glucuronic
  acid (UDPGA).
  b-Transfer of glucuronyl group from UDPGA to an appropriate substrate.
  catalyzed by microsomal enzymes called UDP-glucuronyltransferases.

α-D-Glucose will be transformed to α-D-Glucose-1-phosphate which is then converted to Uridine-5'-diphospho-α-D-glucose (UDPG) then by UDP-Glucoronyl-transferase will produce-B-Glucoronide.

II- Sulfate conjugation

• The amount of sulfate available is limited.
• Utilized to conjugate endogenous compounds e.g. steroids, catecholamines, and thyroxin.
• Phenols and alcohols, aromatic amines, and N- hydroxyl compounds can combine
  enzymatically with sulfate.
• The sulfate conjugation process involves:
  a-Activation of inorganic sulfate into the coenzyme 3-phosphoadenosine -5'-
  phosphosulfate (PAPS).
  b-Sulfate group by sulfotransferases (in liver, kidney and intestine) is transferred to the substrate.

III-Conjugation with glycine, glutamine and other amino acids

• Availability of amino limited acids in the body.
• Glycine and glutamine are conjugated to carboxylic acids (aromatic acids and arylalkyl acids).
• Glycine and glutamine are not activated to coenzymes. the carboxylic acid substrate is activated.
• The conjugation process involves:
  a-The carboxylic substrate in presence of ATP and coenzyme A is converted to acyl coenzyme A complex
  b- Acyl coenzyme A complex react with glycine or glutamine in presence of N- acyl transferase enzymes is transferred onto glycine or glutamine causing acylation(conjugation).

IV- Glutathione or mercapturic acid conjugation.

• Covalent interaction of electrophilic metabolites with cellular nucleophiles causes toxicity.
• Glutathione conjugation (nucleophilic sulfhydryl group) is a pathway to detoxifiy chemically reactive electrophilic compounds.
• No initial formation of an activated coenzyme or substrate is required.
• The inherent reactivity of the nucleophilic GSH towards an electrophilic substrate will sufficient driving force to cause conjugation.

• The conjugation process involves:

1. The substrate conjugates with glutathione (γ- glutamylcysteinylglycine) by enzymes known as glutathione S- transferases gives (glutathione adduct).
2. Glutathione adduct undergoes sequential enzymatic cleavage of two amino acids (glutamic acid and glycine).
3. N- Acetylation of the S- substituted cysteine residue will produce mercapturic acid.

V-Acetylation

• Important metabolic route for drugs containing primary amino groups (e.g. primary
  aromatic amines sulfonamides, hydrazines, hydrazides, and primary aliphatic
  amines).
• The produc is inactive and non toxic but water solubility is not enhanced.
• The conjugation process involves:

1. Formation of Acetylcoenzyme A conjugating agent (active form).
2. Transfer of the acetyl group from accetyl coenzyme A to the accepting amino substrate by N- acetyltransferases enzyme.

VI-Methylation

• Methylation does notproduce polar or water soluble metabolites except when it produces ammonium derivative.
• Methylated products are mostly pharmacologically inactive.
• Methylation plays an important role in the biosynthesis of many endogenous
  compounds (e.g. epinephrine) in addition to the inactivation of physiologically active biogenic amines (e.g. norepinpherine, serotonin and histamine).
• Groups which under go methylation are: catechols, phenols, amines, N-heterocyclic and thiol compounds.
• The conjugation process involves:

1. The methyl group is converted into its active form (S- adenosylmethionine (SAM)).
2. The activated methyl group is transferred onto accepting substrate by methyltransferases
   enzyme.

FACTORS AFFECTING DRUG METABOLISM

• A drug is not metabolized by a single metabolic pathway.
• A single drug may be metabolized by different phase I and phase II metabolic pathways giving several metabolites from the same parent drug.
• Relative amount of metabolites depends on the concentration and the activity of the enzymes metabolizing them.

The following factors may affect the metabolic rate of the drug:

1.Age-related differences:

• Newborn suffer from underdevelopment or deficiency of oxidative and conjugative enzymes leading to reduction of metabolic capacity. e.g.oxidative (cytochrome P-450) metabolism of tolbutamide is reduced in newborn leading to increased t ½ (40 hours Vs 8 hours in adults).
• Glucuronyltransferase activity reductuin leading to the reduction inchloramphenicol conjugation with glucuronic causing the accumulation of toxic levels of the drug causing gray baby syndrome.
• Elderly patients show evidence of inefficientdrug metabolism due to compromised liver functions.

2-Genetic or hereditary Factors:

• There are differences in the rate of metabolism observed of some drugs in hum.
• Acetylation shows rate differences in human e.g. procainamide undergoes conjugation of amino groups with acetyl group.
• Marked difference among individuals in the rate of acetylation of these drugs leads to it's being either slowly or rapidly metabolized (bimodal distribution).
• Rapid acetylators (have more acetyl transferase in their liver).
• The rate of acetylation affects the therapeutic response and toxicity of these drugs.
• Rapid acetylators undergo fast elimination leading to inadequate therapeutic response to the drug.
• Slow acetylators are subject to toxicity due to accumulation of the drug.

3-Species differences:

• A particular drug may be metabolized differently by different species.
• Strain differences in metabolism may also take place e.g. oxidative deamination or aromatic hydroxylationare two metabolic pathways of amphetamine.
• In human, guinea pig and rabbit oxidative deamination predominates.
• In rats aromatic hydroxylation is the predominates.
• There is also some difference due to presence or absence of the particular transferase enzyme e.g. cats lack glucuronyl transferase while pigs lack sulfotransferase enzyme and thus unable to conjugate phenols .

4-Sex differences:

• Sex differences is species dependent.
• Rabbits and mice do not show sex differences in human there arevery few reports of
  sex differences in metabolism e.g. the metabolism of nicotine and aspirin seem to differ between men and women.

5-Enzyme induction:

• Exposure to certain drugs leads to markedly increased activity of hepatic microsomal enzymes e.g. cytochrome P450 (enzyme induction), E.g pesticides and polycyclic aromatic hydrocarbons and other environmental pollutants.
• Due to the increase in the synthesis of more enzymes in the liver.
• Enzyme induction leads to increase in the rate of metabolism of drugs including the the those drugs that induced the enzyme activity in the first place.
• Drug induction leads to serious drug interaction if a known enzyme-inducing drug is coadministered with one or two drugs which are known to be largely metabolized by liver enzymes.
• This will lead to marked decrease in the bioavailability of the drugs leading to decrease in activity.
• Coadministration of warfarine (anticoagulant) ((largely eliminated through liver metabolism)) and the hypnotic phenobarbitone (efficient enzyme enducer) leads to marked decrease in anticoagulant activity of warfarine.
• Dosage readjustment of warfarin is very important when it is coadministered with phenobarbital and also when the patient suddenly stops taking the barbiturate.
• Poycyclic hydrocarbons e.g. benzo[α] pyrine and environmental pollutants e.g. polychlorinated biphenyls induce certain oxidative pathways and consequently the metabolism of drugs.
• Enzyme induction increases toxicity of some drugs by increasing the production of a certain chemically reactive metabolite. E.g oxidation of acetophenone → reactive imidoquinones is catalyzed by phenobarbital-inducible form of cytochrome P450 enzymes in rats.
• Phenobarbital pretreatment leads to in vivo hepatotoxicity and covalent binding of phenacetine.

6-Enzyme inhibition:

• Certain drugs are capable of inhibiting drug metabolizing enzymes.
• Through substrate competition, inhibition of protein synthesis, inactivation of drug-metabolizing enzyme and hepatotoxicity leads to impairment of drug metabolism causing accumulation of the drug in the body.
• Phenylbutazone stereoselectively inhibits the metabolism of the more potent (S)(-)- enantiomer of warfarine.This explains the increased hypoprothrombinemia and hemorrhaging in patients taking both warfarine and phenylbutazone.
  

7-Miscellaneous factors affecting drug metabolism:

• Dietary factors e.g. protein/carbohydrate ratio, indoles present in some vegetables e.g. cabbage, vitamins and minerals, starvation and malnutrition will all influence drug metabolism.
• Physiologic state of the liver (Hepatic cancer, cirrhosis, hepatitis).
• Pregnancy, hormonal disturbances (thyroxin, steroids).
• Circadian rhythm may all influence the metabolism of drugs.