PHYSICIAN ASSISTANTS - Spring 2001

DRUG METABOLISM

Dr. Joseph Cohen

INTRODUCTION

Drug metabolism is the process by which biological systems introduce changes to the molecules of xenobiotics (compounds foreign to the body). Because the drug molecules undergo transformation by biological systems, the term "biotransformation" may be applied. The administered drug may be called the "parent compound." The biotransformed product is known as the "metabolite." Drugs are metabolized by enzymes - proteins that synthesize or degrade compounds. A drug that is metabolized by a particular enzyme is known as the "substrate" of that enzyme.

Generally, the nature of biotransformation of xenobiotics is such that parent compounds are converted from lipophilic to hydrophilic molecules, thereby promoting their urinary excretion. There are however, some exceptions.

The major implication of drug metabolism is termination of drug action, though this does not always occur. With some drugs, their metabolites possess pharmacological activity as well. In other cases, the parent compound is void of pharmacological activity and is converted within the body to an active metabolite. An inactive parent compound is referred to as a prodrug.

Other implications for drug metabolism are carcinogenesis and toxication as a result of bioactivation.

Factors influencing drug metabolism are age, genetic variation, state of health, diet, gender, species variations, competition between substrates for a common enzyme, enzyme induction, and route of drug administration.

The major organ of metabolism is the liver, but some degree of metabolism occurs in all organs of the body. Other sites of drug metabolism are the intestinal wall, the microbial flora of the gut, the kidneys, the lungs, and the plasma. Within the cells, drugs may be metabolized within the cytosol, the mitochondria, or within the endoplasmic reticulum.

FIRST PASS METABOLISM

The most common and convenient route of drug administration is the oral route. With oral administration, the first pass effect, in which there is extensive metabolism of the drug during its first passage through the liver, may occur with some drugs. Metabolism in the walls of the intestine also contributes to the first pass effect. As a consequence of the first pass effect, very little of the drug reaches the systemic circulation.

GUT > PORTAL VEIN > LIVER

KINETICS OF DRUG METABOLISM

The term "kinetics" refers to rates of reactions. The graph below demonstrates the relationship between drug concentration and the velocity of drug metabolism by an enzyme. As the drug concentration increases, the velocity of drug metabolism increases to a maximum rate. Three drugs, A, B, and C are metabolized at different velocities by the same enzyme.

Comparing the shape of the curves permits the determination of the relative affinity of the enzyme for each of its substrates. Although each of the drugs has the same maximal velocity of metabolism, the maximum is reached at a different concentration for each drug. The concentration at which the maximal velocity is reached is lowest for Drug A and highest for Drug C.

The affinity of an enzyme for a drug is described by a mathematical value known as the Km. The Km of an enzyme for a drug is the concentration of the drug that produces half the maximal velocity of the reaction (rate of metabolism). In the figure to the left, the concentration that produces half the maximal velocity of the reaction is lowest for Drug A. The lower the Km, the greater the affinity of an enzyme for a drug. In the graph, the Km for each of the drugs is different. For which drug does the enzyme has the lowest affinity? Determine the affinity of the enzyme for each drug in the graph found at the end of this document.

The maximal velocity at which an enzyme metabolizes a drug is described by a mathematical value known as the Vmax. At the point of Vmax, a progressive increase in drug concentration will not produce a further increase in the rate of drug metabolism. In the graph, the Vmax for all the drugs are equal.

Kinetics of drug metabolism is defined by the equation that follows in which v = rate of drug metabolism and [C] = plasma drug concentration.

v = Vmax[C]

Km + [C]

If the dose of a given drug produces a blood concentration far less than the Km of the enzyme for the drug, then Km >>> [C], and [C] contributes a very small value to the denominator. It may be dropped from the denominator in the equation above, and the equation simplifies to

v = Vmax [C]

Km

and v [C] (the velocity of the drug's metabolism is proportional to the plasma drug concentration). When v [C], a constant fraction of the remaining drug is metabolized per unit time and the drug's metabolism follows a first order process. At this point, a further increase in plasma drug concentration will increase the metabolic rate because the enzyme is not saturated. Most drugs are given at doses that produce blood concentrations much smaller than the Km, so their metabolism follows a first order process.

Conversely, when a drug is present at a concentration far exceeding the Km of the enzyme, then [C] >>> Km, and the Km contributes a very small value to the denominator and may be dropped from the denominator, in which case the equation simplifies to

v = Vmax [C],     and v = Vmax 

[C]                        

At the point of Vmax, the velocity of the reaction is maximal and constant, the enzyme is saturated, and metabolism of the drug then follows a zero order process - a constant amount of the remaining drug is metabolized per unit time. Phenytoin is an example of a drug given in doses that produce concentrations in the blood that saturate its drug metabolizing enzyme. 

PHASE I METABOLISM OF DRUGS

Drugs may undergo phase I metabolism. Polar groups on the molecule are exposed or introduced to the molecule. Phase I reactions are hydrolysis, oxidation, and reduction. Functional groups added or exposed are -OH, -SH, -NH₂, and -COOH. The metabolites are ionized at the physiological pH of the body, promoting decreased absorption from the urinary tubules and increased urinary excretion.

PHASE II METABOLISM OF DRUGS

Drugs may undergo phase II metabolism, a biosynthetic process. The product of the process is given the general term "conjugate." Conjugates are formed between the parent compound or a phase I metabolite and an endogenous molecule formed in the body. The endogenous molecule donates a portion of itself in the reaction. Thus, if ENDOX represents the endogenous molecule and S is the substrate, then

ENDOX + S SX + ENDO.

Phase II metabolism therefore involves the linkage of two molecules. The net result is a polar, more water soluble product that is more readily excreted by the kidney. Phase II metabolism does not always produce metabolites that are inactive, and some conjugates are known to possess pharmacological activity.

Phase II metabolism includes glucuronide conjugation, sulfate conjugation, glycine conjugation, methylation, acetylation, transulfuration, glutathione conjugation, and mercapturic acid synthesis.

PATTERNS OF DRUG METABOLISM

The parent molecule may be active and undergo phase I metabolism to an inactive or active metabolite. The phase I metabolite may undergo further transformation by either a phase I or phase II pathway.

The parent molecule may undergo metabolism to a phase II metabolite which may then undergo further transformation by a phase I pathway.

A prodrug (pharmacologically inactive) undergoes phase I metabolism to the active metabolite. An example is the conversion of L-dopa, used in the therapy of Parkinson's disease, to dopamine.

The anti-glaucoma agent dipivirine (dipivalic epinephrine) is converted to epinephrine within the cornea of the eyes after topical administration.

MICROSOMAL METABOLISM

The term "microsomal metabolism" refers to drug metabolism by the endoplasmic reticulum. When a tissue, for example the liver, is homogenized, the cells are broken and the organelles are released. The organelles are separated by differential centrifugation, and the endoplasmic reticulum is pelleted at 100,000 x g. Upon microscopic examination, the endoplasmic reticulum is observed to no longer have the channel-like appearance as in the intact cell but instead, the appearance of small round vesicles referred to as "microsomes."

Many drugs are metabolized by the microsomal enzyme system, and endogenous compounds such as the steroids, bilirubin, and thyroxin are also subjected to such metabolism.

In order to have access to microsomal enzymes, drugs must have the ability to dissolve within the membrane of the endoplasmic reticulum. Thus, only lipophilic compounds are substrates.

Microsomal metabolism includes oxidation by a family of isoenzymes known as cytochrome P450. Cytochrome P450 conducts many types of oxidations. The general reaction is summarized as follows: SH + O₂ + 2H⁺ + 2e^- SOH + H₂O, where SH represents the substrate.

Nitro and azo reduction and hydrolysis of esters and amides also occur within microsomes. The phase II glucuronide conjugation reaction occurs at the microsomal level.

NONMICROSOMAL METABOLISM

Drug metabolism is not mediated solely by microsomal enzymes. Oxidative reactions occurring in the cytosol involve alcohol and aldehyde dehydrogenases and xanthine oxidase. Several monoamines are oxidized by the mitochondrial enzyme monoamine oxidase. Reduction of substrates is conducted in the cytosol by alcohol and aldehyde dehydrogenases. Hydrolysis of acyl esters by esterases occurs in the cytosol and in the plasma. Hydrolysis of amide esters by amidases occurs in the cytosol of hepatic cells.

Phase II reactions not associated with the microsomes include sulfate conjugation, acetylation, methylation, glycine conjugation, and glutathione conjugation (a small amount of glutathione conjugation is reported to be mediated by the microsomes). Detoxification of cyanide occurs through two phase II pathways involving the transfer of sulfur from the donor molecules thiosulfate or ß-mercaptopyruvate by the enzymes mitochondrial sulfurtransferase and cytosolic sulfurtransferase respectively.

AZO AND NITRO AND REDUCTIONS

These enzymatic reactions may occur in the cytosol, plasma, and microsomes.

1. Azo Reduction RN=NR' RNH₂ + R'NH₂

2. Nitro Reduction RNO₂ RNH₂

ESTER AND AMIDE HYDROLYSIS

These enzymatic reactions may occur in the cytosol, plasma, and microsomes.

1. Ester hydrolysis RCOOR' RCOOH + R'OH

2. Amide hydrolysis RCONR'R" RCOOH + HNR'R"

ENZYME INHIBITION

Competitive and noncompetitive antagonism may prevent the metabolism of a drug. The result is an increase in the half-life of the drug. Cimetidine, an H₂ histamine antagonist, inhibits the metabolism of many drugs.

Microsomal enzymes can undergo induction (increased levels in the cell). The consequence of such induction is increased rate of metabolism of their substrates and a faster decline of drug blood levels which in turn reduces the half-life (T1/2) of the drugs. Implications for enzyme induction are important in drug therapy. As a consequence of reduced half-life, the duration of therapeutic effectiveness of a drug is reduced.

Many drugs induce specific enzymes. Phenobarbital, clofibrate, and the polycyclic hydrocarbon compounds benzopyrene and 3-methylcholanthrene are examples.

CARCINOGENESIS

Some xenobiotics are metabolized to products that are carcinogenic. For example, benzopyrene is metabolized by cytochrome P450 to a product that interacts with cellular DNA. The result is cancer.

METABOLISM OF ACETAMINOPHEN

Acetaminophen is an example of a drug whose disposition in the body involves several metabolic routes. Both phase I and phase II metabolisms are involved. Also involved in its metabolism are bioactivation (conversion of the drug by biological systems to a toxic agent), toxication (the adverse interaction of the agent with tissue macromolecules), and detoxication (the process by which physiological systems oppose toxication). Glutathione is important for detoxication in the metabolism of acetaminophen.

Acetaminophen-O-glucuronide is produced by the microsomal enzyme glucuronyl transferase. The endogenous donor, uridine-5'-diphospho--D-glucurinic acid (UDPGA), donates glucuronic acid to the formation of the glucuronide conjugate.

Acetaminophen-O-sulfate is produced by the soluble enzyme sulfotransferase. The endogenous donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), donates sulfate to the formation of the sulfate conjugate. Glucuronide and sulfate conjugations are the major pathways of acetaminophen metabolism.

A small amount of acetaminophen (approximately 4%) is metabolized by cytochrome P450 to the metabolite N-hydroxyacetaminophen which rearranges to the toxic intermediate N-acetyl-p-benzoquinoneimine. N-acetyl-p-benzoquinoneimine produces toxicity by interacting with tissue macromolecules. Normally, glutathione, a tripeptide and normal body constituent, conjugates to the toxic metabolite by the enzyme glutathione transferase so that tissue macromolecules are spared. If the glutathione supply is exhausted, the metabolite instead interacts with tissue macromolecules, for example proteins. The result is toxicity to the liver. Chronic ingestion of ethanol induces cytochrome P450 and increases the level of this metabolite. Chronic ethanol consumption combined with regular use of acetaminophen can result in liver necrosis.

Therapy of acetaminophen toxicity involves gastric lavage followed by oral administration of N-acetylcysteine.

EXAMPLES OF DRUG INTERACTIONS RELATED TO DRUG METABOLISM

Ketoconazole is an imidazole antifungal agent. It binds to the heme iron of cytochrome P450. Co-administration of oral azoles with terfenadine or astemizole may increase the plasma concentration of terfenadine (discontinued by the manufacturer in December, 1997) and astemizole by inhibiting their metabolism. This may result in severe cardiac arrhythmias including ventricular tachycardia and death. Therefore, co-administration of the oral azoles with either terfenadine or astemizole is absolutely contraindicated.

Cimetidine is an H₂ histamine antagonist. It binds to the heme iron of cytochrome P450. It inhibits the cytochrome P450-catalyzed oxidative drug metabolism pathway. The drug also reduces hepatic blood flow, which may further reduce clearance of other drugs. Co-administration of cimetidine with any of the following drugs may result in increased pharmacologic effects or toxicity:

Warfarin, phenytoin, propranolol, metoprolol, labetalol, quinidine, caffeine, lidocaine, theophylline, alprazolam, diazepam, flurazepam, triazolam chlordiazepoxide, carbamazepine, ethanol, tricyclic antidepressants, metronidazole, calcium channel blockers, sulfonylureas

Phenobarbital is an inducer of cytochrome P450 isoenzyme 2C9 of which the oral anticoagulant warfarin is a substrate. The blood level of warfarin declines faster in the presence of phenobarbital. Thus, the half-life of warfarin is reduced by phenobarbital.

1. Consider the previous equation that expresses the rate of metabolism of a drug. If Km = 20 mM, drug concentration = 60 mM, and v = 15 nmoles of drug metabolized per gram of tissue per minute, the Vmax equals

a. 15 nm/g/min

b. 60 nm/g/min

c. 20 nm/g/min

d. 1200 nm/g/min

e. 10 nm/g/min

[DRUG]mM expresses the millimolar concentration of the drug.

(ng/g tissue/min = nanograms of drug metabolized per gram of tissue per minute)