What influences drug metabolism? Enzyme concentration Enzyme induction Drug structure effects Genetic factors - Pharmacogenetics

Drug Metabolism: Factors

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

If the rate of metabolism increases Decreased intensity and duration of drug action In rare cases toxicity may increase - metabolites

Age differences Premature and newborn babies have yet to develop

maximal oxidative and conjugative enzyme capabilities 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 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 Endogenous compounds can also be metabolized faster

Example – phenobarbitol can be used to increase conjugation of bilirubin with glucuronic acid in neonates with jaundice

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

Enzyme Activity

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

NH

OO

CH3

NH

OO

CH3

OH

NH

OO

HOCH2

(+)-enantiomer

(-)-enantiomer

Glutethimide - HalseyPiperidine basedsedative hypnotic

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

NH

O O

pro-Rring

pro-Sring

Phenytoin

NH

O O

OH

NH

O O

OH

90% inhumans

10% inhumans

Structural Factors

Stereochemical aspects cont: Regioselective metabolism

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

Papaverine - Pavabid® - Hoechst Marion RoussellSmooth muscle relaxer used as a peripheral vasodilator

N

OMe

OMe

MeO

MeON

OH

OMe

MeO

MeO

Demethylation

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

Structural Factors

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 drug-

induced 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 Example: Cats

Can’t conjugate phenols by glucuronic acid Sulfate conjugate instead ASA BAD for kitty!

Example: Pigs Lack sulfotransferase but very efficient glucoronic acid conjugation

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

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

Overview

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,

Single nucleotide polymorphism: SNP Nonsynonymous or synonymors (silent)

Variations in human genome often SNP’s

Glucose-6-Phosphate Dehydrogenase Early 1950’s 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 RBC’s 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

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

Isoniazid N-AcetylisoniazidCH3 NHNH2

O Covalent binding in liver cellmacromolecules leading toliver damage

N-Acetyl Transferase (NAT)

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

Cytochrome P450

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

CYP3ACYP2D6

CYP2C

CYP1A2CYP2E1

Relative Importance ofP450s in Drug Metabolism

CYP3A

CYP2C

CYP1A2

CYP2E1

?

CYP2D6

Relative Quantities of P450s in Liver

CYP450

CYP1A1 Multiple PAH

CYP2A6 Liver aflatoxins

CYP2B6 Liver nicotine

CYP2C8 Liver taxol

CYP2E1 Liver, GI tract ethanol, benzene

CYP3A4 Liver, small intest. paracetamol

Isoenzyme Organ Typical substrate

Cytochrome P450 Summary

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

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!

CYP3A Family

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. John’s 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)

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

CYP2D6 Absent in 7% of Caucasians,

1–2% 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

CYP2C9 Linked to impaired metabolism

Phenytoin S-Warfarin Tolbutamide (diabetes) Losartan (antihypertensive) NSAID’s including COX-2

Biggest problems: warfarin and phenytoin Poor metabolism – increased effects! Warfarin bleeding out

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 20–30% of Asians, 3–5% Caucasians Inhibited by:

Omeprazole Isoniazid Ketoconazole

CYP1A2 Induced by smoking tobacco Catalyzes primary metabolism of:

Theophylline Imipramine Propranolol Clozapine

Inhibited by: Many fluoroquinolone antibiotics Fluvoxamine Cimetidine

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