Drug Metabolism: Factors

What influences drug metabolism?

Enzyme concentration Enzyme induction Drug structure effects Genetic factors - Pharmacogenetics

Enzyme Activity

Function of enzyme concentration and activity If the rate of metabolism decreases

Increased intensity and duration of drug action Increased accumulation in plasma, increased toxicity risk Decreased intensity and duration of drug action In rare cases toxicity may increase - metabolites Premature and newborn babies have yet to develop maximal oxidative and conjugative enzyme capabilities

If the rate of metabolism increases

Age differences

Approaches adult levels at 1-2 months age Example: newborn jaundice or neonatal hyperbilirubinemia (kernicterus) is caused by the inability to conjugate glucuronic acid with bilirubin (Heme from hemoglobin metabolism)

Old Age may influence metabolism (underlying disease)

Enzyme Activity
Enzyme induction

Results from drug or chemical exposure Very important source of drug-drug interactions Caused by the increased rate of enzyme production Often drugs can increase their own rate of metabolism

Enzyme Activity
Enzyme induction continued

Compounds that enhance metabolism: Phenobarbital and other barbiturates, glutethimide, phenylbutazone, meprobamate, ethanol, phenytoin, rifampin, griseofulvin, carbamazepine Classical example: Phenobarbitol

If a patient starts phenobarbitol while taking warfarin, blood levels and dosage adjustment of warfarin will need be monitored and adjusted If patient stops phenobarbitol, dosage will need to be decreased Oral contraceptives are rendered ineffective by phenobarbitol and rifampin due to increased estrogen metabolism Example phenobarbitol can be used to increase conjugation of bilirubin with glucuronic acid in neonates with jaundice

Endogenous compounds can also be metabolized faster

Smokers often metabolize drugs faster due to smoke chemicals

Example: theophylline t1/2 = 4.1 vs. 7.2 hours

Enzyme Activity

Enzyme induction continued Two inducer categories:

Phenobarbitol-like inducers (P-450 enzymes) Polycyclic aromatic hydrocarbon-like inducers (P-448 enzymes)

Selective enzyme for aromatic hydrocarbons

Remember: metabolism of drugs and chemicals can result in toxic metabolites from otherwise non-toxic compounds Enzyme inhibition and inhibition of metabolism Leads to drug accumulation and toxicity Mechanisms

Substrate competition interference with protein synthesis Interference with drug metabolizing enzymes Hepatotoxicity leading to decreased metabolism Others

Structural Factors

Many drugs are racemic mixtures Typically one enantiomer is more bioactive

Receptor binding phenomenon

Selective metabolism: Substrate stereoselectivity Often enantiomers metabolized by different enzymes

H3C

Glutethimide - Halsey HO Piperidine based sedative hypnotic (+)-enantiomer O

H3C

N H

HOCH2 O N H O

(-)-enantiomer
O N H O

Structural Factors

Stereochemical aspects cont:

Preferential metabolic formation of a stereoisomer: product selectivity When a ketone is reduced to an alcohol, one stereoisomer is preferred Hydroxylation can also be stereoselective
OH

pro-R ring pro-S ring

O N H O

10% in humans
O N H O

Phenytoin

90% in humans
O N H O

OH

Structural Factors

Stereochemical aspects cont:

Regioselective metabolism

selective metabolism of one of 2 or more of the same functional groups located on a molecule

MeO MeO N OMe OMe

MeO MeO N OMe OH

Demethylation

Papaverine - Pavabid - Hoechst Marion Roussell Smooth muscle relaxer used as a peripheral vasodilator

Structural Factors

Pharmacologically active metabolites

Function of:

Plasma accumulation Rate of excretion (decreased renal function)

Metabolites no longer thought inactive Many metabolites are now marketed as drugs

Other Factors

Misc factors affecting metabolism

Dietary factors

Protein and carbohydrate consumption Indoles in brussels sprouts, cabbage and cauliflower Charcoal-broiled meats polyaromatics induce enzymes Malnutrition Starvation Vitamins and minerals

Underlying disease states

Hepatic cancer, cirrhosis, hepatitis Hyper- or hypothyroid disease

Pregnancy Circadian rhythm

Pharmacogenomics
American Journal of Health-System Pharmacy
Margaret Ma, Michael Woo, Howard Mcleod

Vogel study of the role of genetics in drug response One of the most rapidly growing areas of pharmacy Genetic makeup is responsible for a significant portion of druginduced toxicity. Eventually, pharmacogenomics may become a tool for individualizing drug therapy!

Overview

Genetic differences

Man vs. Monkey vs. Rabbit vs. Rat vs. Guinea pig Differences can be where in the drug metabolism occurs

Meta vs. para in aromatic rings and which of two aromatic rings Cant conjugate phenols by glucuronic acid Sulfate conjugate instead ASA BAD for kitty! Lack sulfotransferase but very efficient glucoronic acid conjugation

Example: Cats

Example: Pigs

Ex: Rabbits

Cottontail met. hexobarbitol 10X faster than New Zealand

Humans: Genetic/hereditary differences account for huge differences seen in the rate of enzyme metabolism

Overview

Differences between the sexes

Appears to be species dependent

Huge difference between male and female rats No differences in rabbits and mice

May also be a function of what drug is being metabolized Sex hormones: androgens tend to increase metabolism Humans

Examples: nicotine and aspirin

Genetics Review

Allele: Any one of a series of two or more different genes that occupy the same position (locus) on a chromosome. Alleles determine genotype Genotype displayed as a phenotype (eye color) Two identical alleles result in a homozygous dominant or recessive trait of that gene.

Blue eyes, blonde hair, Nonsynonymous or synonymors (silent)

Single nucleotide polymorphism: SNP

Variations in human genome often SNPs

Glucose-6-Phosphate Dehydrogenase

Early 1950s discovery (one of the first!) Anti-malarials causing hemolytic anemia in people with G6PD deficiency More than 400 known variants

All seem to produce decreased G6PD activity. Reduced GSH concentrations in RBCs Hemolytic anemia

Affects 400 million people worldwide. Most are asymptomatic Deficiency is an X-linked recessive trait G6PD deficiency varies among ethnic groups

Most common in males of Mediterranean/African heritage

Glucose-6-Phosphate Dehydrogenase

G-6-PD expressed in all body tissues

Carbon flow through pentose phosphate shunt Production of NADPH Glutathione reduction (GSSG GSH)

Absence of GSH allows oxidation of Hb sulfhydroxyl groups hemolysis Now over 20 drugs known!

Primaquine, sulfones, sulfonamides, nitrofurans, Vitamin K analogues, cefotetan, chloramphenicol

N-Acetyl Transferase (NAT)

Phase II conjugating enzyme (Liver) N-acetylation (deactivation): arylamines (carcinogens) O-acetylation (activation): hererocyclic amines Most work on NAT2 locus 27 alleles reported Possible link to cancer risk? Acetylation with Acetyl-CoA is either fast or slow

Genetic differences in NAT activity Caucasions & African Americans 40-70% Slow

Japanese & Canadian Eskimo 10-20% Slow Egyptians > 80% - Slow Asia: Further N, less chance of Slow. Eskimo & Asians often Fast

SLOW : more likely to show toxicity or adverse reactions to drugs

FAST: more likely to show an inadequate therapeutic response to standard doses of drugs

Isoniazid and Hydralazine are key drugs linked to this enzyme system

N-Acetyl Transferase (NAT)

Example: Isoniazid used for tuberculosis

SLOW: t1/2 = 140-200 minutes

Higher plasma accumulation and higher cure rate More adverse side effects and drug-drug interactions Example of drug interaction: phenytoin use with isoniazid

Isoniazid inhibits phenytoin metabolism leading to accumulation of high and toxic plasma levels of phenytoin

Fast t1/2 = 45-80 minutes

Lower plasma accumulation and lower cure rate More associated liver damage and hepatitis with isoniazid due to the more rapid formation of more acetylhydrazine
O N-Acetylisoniazid H3C NHNH2 Covalent binding in liver cell macromolecules leading to liver damage

Isoniazid

Cytochrome P450
Inducers and Inhibitors:

An overwhelming subject information overload! Primary method to eliminate drugs CYP mainly the liver; also GI epithelium and other tissues Pharmacogenetic factors large number CYP isoenzymes

Most arise due to single nucelotide differences or polymorphisms (SNP) in genes encoding drug metabolism enzymes May result in altered activity, altered stability of the enzyme, or introduction of a premature stop codon leading to a truncated protein SNP errors can lead to mis-splicing of genes, complete gene deletion or gene amplification Changes can lead to drug accumulation (toxicity), increased rates of drug elimination, and changes in activity / toxicity profiles due to altered formation of active metabolites

Cytochrome P450
Overview continued: At LEAST 50 (57) isoenzymes, grouped based on their a.a. sequences Example: CYP3A4: Cytochrome P450, family 3, subfamily A and the 4th enzyme in the subfamily Most CYP-450 enzymes involved in drug metabolism belong to the three distinct families, CYP1, CYP2 and CYP3 (50% of all drugs)

Some drugs processed by several CYP450 isoenzymes

CYP450
Relative Importance of P450s in Drug Metabolism
CYP2E1 CYP1A2

Relative Quantities of P450s in Liver

CYP2C

CYP1A2

CYP2C CYP3A CYP2D6 CYP2E1 CYP3A CYP2D6

Shimada T et al. J Pharmacol Exp Ther 1994;270(1):414.

Cytochrome P450 Summary

Isoenzyme
CYP1A1

Organ
Multiple

Typical substrate
PAH

CYP2A6
CYP2B6 CYP2C8 CYP2E1 CYP3A4

Liver
Liver Liver Liver, GI tract Liver, small intest.

aflatoxins
nicotine taxol ethanol, benzene paracetamol

CYP3A Family

Predominant subfamily of CYP enzymes Expressed primarily in liver & small intestine Involves metabolism of

HIV protease inhibitors Benzodiazepines Calcium channel blockers HMG CoA Reductase inhibitors - Statins Antineoplastic drugs Nonsedating antihistamines Immunosupressants

Variation creates efficacy & toxicity differences Common types: CYP3A4, CYP3A5, & CYP3A7

CYP3A Family

CYP3A4 ~ 50% of drug/corticosteroid metabolism Major contributor of first-pass metabolism Individual variance as much as 50-fold CYP3A5 Present in only 10 30% of livers tested May play a significant role in CYP3A metabolism Important contributor to racial CYP variation Accounts for at least 50% of CYP3A in those with the wild type allele (CYP3A5*1) People with at least one wild type allele express large amounts of CYP3A5 2.5 x increase in midazolam (Versed) met. More frequently expressed in non-caucasions

30% - Caucasions, Japanese, Mexicans 40% - Chinese 60% - African Americans, SE Asians, Pacific Is., SW Native Am.

People with mutations in both 4 & 5: show lack of efficacy!

For the CYP3A family, think: INCREASE

CYP3A Inhibitors

Antifungals

Ketoconazole Itraconazole Fluconazole

Cimetidine Macrolide antibiotics

Clarithromycin Erythromycin Troleandomycin

Grapefruit juice

CYP3A Inducers

Carbamazepine Rifampin Rifabutin Ritonavir St. Johns wort

CYP2D6

Metabolizes 25 30% of clinically key medications

Dextromethorphan Beta-blockers Antiarrythmics Anti-depressants Antipsychotics Morphine derivatives codeine, oxycodone, etc. Others

Most genetic variation (75 variants so far) Linked more commonly to slow/poor metabolizers

1% - Asians 2-5% - African Americans 6-10% Caucasions

Slower on average
Lower frequency of nonfunctional alleles Higher frequency of reduced activity alleles

Diversity of CYP2D6

Metabolism of the drug debrisoquine (antihypertensive)

CYP2D6
Absent in 7% of Caucasians, 12% non-Caucasians Hyperactive in up to 30% of East Africans Catalyzes primary metabolism of:

Codeine Many -blockers Many tricyclic antidepressants

Inhibited by:
Fluoxetine Haloperidol Paroxetine Quinidine

Aklillu E et al. J Pharmacol Exp Ther 1996;278(1):441 446

CYP2C9

Linked to impaired metabolism

Phenytoin S-Warfarin Tolbutamide (diabetes) Losartan (antihypertensive) NSAIDs including COX-2 Poor metabolism increased effects! Warfarin bleeding out

Biggest problems: warfarin and phenytoin

Absent in 1% Caucasians and African-Americans Inhibited by Fluconazole

CYP2C19

Mutations mostly lead to slow metabolizers Responsible for metabolism of relatively few drugs Important drugs affected:

S-mephenytoin Proton-pump inhibitors (omeprazole - Prilosec) Diazepam - Valium Propanolol (-blocker) Imipramine Tofranil (antidepressant) Amitryptiline Elavil (antidepressant)

Absent in 2030% of Asians, 35% Caucasians Inhibited by:

Omeprazole Isoniazid Ketoconazole

CYP1A2

Induced by smoking tobacco Catalyzes primary metabolism of:

Theophylline Imipramine Propranolol Clozapine

Many fluoroquinolone antibiotics Fluvoxamine Cimetidine

Inhibited by:

Reasons for In vitro Assays

Speed Expense Ability to select specific enzymes Ability to control reaction conditions

Differences in human versus animal isozymes

New Technology - AmpliChip

July 2003 Roche Pharm. CYP2D6 & CYP2C19 $350 - $400 Roughly 10% of Caucasians and 20% of Asians are poor metabolizers

100,000 Deaths in US alone

25 million people affected