What Would Be The Logical Primary Metabolite Of A Phase I

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Abstracted from Levien, T. L., and Baker, D. E.: Pharmacist's Letter, December 2002, Detail-Document #150400. (Pharmacist's Letter used as sources: Hansten, P. D., and Horn, J. R.: Drug Interactions Analysis and Management. Vancouver, WA, Applied Therapeutics, 2002; and Tatro, D. S. [ed.]: Drug Interaction Facts. St. Louis, Facts & Comparisons, 2002.)

Abstracted from Levien, T. L., and Baker, D. E.: Pharmacist's Letter, December 2002, Detail-Document #150400. (Pharmacist's Letter used as sources: Hansten, P. D., and Horn, J. R.: Drug Interactions Analysis and Management. Vancouver, WA, Applied Therapeutics, 2002; and Tatro, D. S. [ed.]: Drug Interaction Facts. St. Louis, Facts & Comparisons, 2002.)

Inducers of microsomal enzymes also may enhance the metabolism of endogenous compounds, such as steroidal hormones and bilirubin. For instance, phenobarbital can increase the metabolism of cortisol, testosterone, vitamin D, and bilirubin in humans.508,509 The enhanced metabolism of vitamin D3 induced by phenobarbital and phenytoin appears to be responsible for the osteomalacia seen in patients on long-term therapy with these two anticonvulsant drugs.515 Interestingly, phenobarbital induces glucuronyltransferase enzymes, thereby enhancing the conjugation of bilirubin with glucuronic acid. Phenobarbital has been used occasionally to treat hyperbilirubinemia in neonates.516

In addition to drugs, other chemicals, such as polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene, 3-methyl-cholanthrene) and environmental pollutants (e.g., pesticides, PCBs, TCDD), may induce certain oxidative pathways and, thereby, alter drug response.508,509,511 Cigarette smoke contains minute amounts of polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, which are potent inducers of microsomal CYP enzymes. This induction increases the oxidation of some drugs in smokers. For example, theophylline is metabolized more rapidly in smokers than in nonsmokers. This difference is reflected in the marked difference in the plasma half-life of theophylline between smokers (t1/2 4.1 hours) and nonsmokers (ti/2 7.2 hours).517 Other drugs, such as phenacetin, pentazocine, and propoxyphene, also reportedly undergo more rapid metabolism in smokers than in non-smokers.518-520

Occupational and accidental exposure to chlorinated pesticides and insecticides can also stimulate drug metabolism. For instance, the half-life of antipyrine in workers occupationally exposed to the insecticides lindane and dichlorodiphenyltrichloroethane (DDT) is reportedly significantly shorter (7.7 vs. 11.7 hours) than in control subjects.521 A case was reported in which a worker exposed to chlorinated insecticides failed to respond (i.e., decreased anticoagulant effect) to a therapeutic dose of warfarin.522 As discussed previously in this chapter, multiple forms

TABLE 3.5 Potential Drug-Grapefruit Interactions Based on Grapefruit Inhibition of CYP 3A4

(isozymes) of CYP have been demonstrated.3

Many chemicals selectively induce one or more distinct forms of CYP31 (see Table 3.4.) Enzyme induction also may affect toxicity of some drugs by enhancing the metabolic formation of chemically reactive metabolites. Particularly important is the induction of CYP enzymes involved in the oxidation of drugs to reactive intermediates. For example, the oxidation of acetaminophen to a reactive imidoquinone metabolite appears to be carried out by a phenobarbital-inducible form of CYP in rats and mice. Numerous studies in these two animals indicate that phenobarbital pretreat-ment increases in vivo hepatotoxicity and covalent binding as well as increases formation of reactive metabolite in microsomal incubation mixtures.243-245,248 Induction of cy-tochrome P448 is of toxicological concern because this particular enzyme is involved in the metabolism of polycyclic aromatic hydrocarbons to reactive and carcinogenic inter-mediates.80,523 Consequently, the metabolic bioactivation of benzo[a]pyrene to its ultimate carcinogenic diol epoxide intermediate is carried out by cytochrome P448 (see section on aromatic oxidation for the bioactivation pathway of benzo[a]pyrene to its diol epoxide).523 Thus, it is becoming increasingly apparent that enzyme induction may enhance the toxicity of some xenobiotics by increasing the rate of formation of reactive metabolites.

Enzyme Inhibition

Several drugs, other xenobiotics including grapefruit, and possibly other foods can inhibit drug metabolism (Table 3.5).32,483-486 With decreased metabolism, a drug often accumulates, leading to prolonged drug action and serious adverse effects. Enzyme inhibition can occur by diverse mechanisms, including substrate competition, interference with protein synthesis, inactivation of drug-metabolizing enzymes, and hepatotoxicity leading to impairment of enzyme activity. Some drug interactions resulting from enzyme inhibition have been reported in hu-mans.524,525 For example, phenylbutazone (limited to veterinary use) stereoselectively inhibits the metabolism of the more potent (S)(—) enantiomer of warfarin. This inhibition may explain the excessive hypoprothrombinemia (increased anticoagulant effect) and many instances of hemorrhaging seen in patients on both warfarin and phenylbutazone ther-apy.56 The metabolism of phenytoin is inhibited by drugs such as chloramphenicol, disulfiram, and isoniazid.512 Interestingly, phenytoin toxicity as a result of enzyme inhibition by isoniazid appears to occur primarily in slow acety-lators.464 Several drugs, such as dicumarol, chlorampheni-

TABLE 3.5 Potential Drug-Grapefruit Interactions Based on Grapefruit Inhibition of CYP 3A4





Increased bioavailability


Increased AUC


Increased AUC, peak and trough plasma concentrations


Increased AUC

Cyclosporine, tacrolimus

Increased AUC and serum concentrations

Atorvastatin, simvastatin

Increased absorption and plasma concentrations


Increased absorption and plasma concentrations

Abstracted from Kehoe, W. A.: Pharmacist's Letter, 18, September 2002, Detail Document #180905. AUC, area under the curve.

Abstracted from Kehoe, W. A.: Pharmacist's Letter, 18, September 2002, Detail Document #180905. AUC, area under the curve.

col, and phenylbutazone,512 inhibit the biotransformation of tolbutamide, which may lead to a hypoglycemic response.

The grapefruit-drug interaction is complex. It may be caused by the bioflavonoids or the furanocoumarins. Grapefruit's main bioflavonoid, naringin, is a weak CYP inhibitor, but the product of the intestinal flora, naringenin, does inhibit CYP3A4. The literature is very confusing because many of the studies were done in vitro, and they cannot always be substantiated under in vivo conditions. In addition, components in grapefruit also activate P-glyco-protein, which would activate the efflux pump in the gastric mucosa and thus interfere with oral absorption of the certain drugs. The combination of CYP enzyme inhibition and P-glycoprotein activation can lead to inconclusive re-sults.526 The general recommendation when a drug interaction is suspected is that the patient avoid grapefruit and its juice.

Miscellaneous Factors Affecting Drug


Other factors also may influence drug metabolism. Dietary factors, such as the protein-to-carbohydrate ratio, affect the metabolism of a few drugs. Indoles present in vegetables such as Brussels sprouts, cabbage, and cauliflower, and polycyclic aromatic hydrocarbons present in charcoal-broiled beef induce enzymes and stimulate the metabolism of some drugs. Vitamins, minerals, starvation, and malnutrition also apparently influence drug metabolism. Finally, physiological factors, such as the pathological state of the liver (e.g., hepatic cancer, cirrhosis, hepatitis), pregnancy, hormonal disturbances (e.g., thyroxine, steroids), and circa-dian rhythm, may markedly affect drug metabolism.

Stereochemical Aspects of Drug Metabolism

Many drugs (e.g., warfarin, propranolol, hexobarbital, glutethimide, cyclophosphamide, ketamine, and ibuprofen) often are administered as racemic mixtures in humans. The two enantiomers present in a racemic mixture may differ in pharmacological activity. Usually, one enantiomer tends to be much more active than the other. For example, the (S)(—)

enantiomer of warfarin is 5 times more potent as an oral anticoagulant than the (R)(+) enantiomer.527 In some instances, the two enantiomers may have totally different pharmacological activities. For example, (+)-a-propoxyphene (Darvon) is an analgesic, whereas (—)-a-propoxyphene (Novrad) is an antitussive.528 Such differences in activity between stereoisomers should not be surprising, because Chapter 2 explains that stereochemical factors generally have a dramatic influence on how the drug molecule interacts with the target receptors to elicit its pharmacological response. By the same token, the preferential interaction of one stereoisomer with drug-metabolizing enzymes may lead one to anticipate differences in metabolism for the two enan-tiomers of a racemic mixture. Indeed, individual enan-tiomers of a racemic drug often are metabolized at different rates. For instance, studies in humans indicate that the less active (+) enantiomer of propranolol undergoes more rapid metabolism than the corresponding (—) enantiomer.529 Allylic hydroxylation of hexobarbital occurs more rapidly with the R(—) enantiomer in humans.530 The term substrate stereoselectivity is used frequently to denote a preference for one stereoisomer as a substrate for a metabolizing enzyme or metabolic process.291

Individual enantiomers of a racemic mixture also may be metabolized by different pathways. For instance, in dogs, the (+) enantiomer of the sedative-hypnotic glutethimide (Doriden) is hydroxylated primarily a to the carbonyl to yield 4-hydroxyglutethimide, whereas the ( —) enantiomer undergoes aliphatic w-1 hydroxylation of its C-2 ethyl group.140,141 Dramatic differences in the metabolic profile of two enantiomers of warfarin also have been noted. In humans, the more active (S)( — )-isomer is 7-hydroxylated (aromatic hydroxylation), whereas the (R)(+)-isomer undergoes keto reduction to yield primarily the (R,S) warfarin alcohol as the major plasma metabolite.56,296 Although numerous other examples of substrate stereoselectivity or enantiose-lectivity in drug metabolism exist, the examples presented should suffice to emphasize the point.291,531

Riser Diagram

Drug biotransformation processes often lead to the creation of a new asymmetric center in the metabolite (i.e., stereoisomeric or enantiomeric products). The preferential

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metabolic formation of a stereoisomer^ product is called product stereoselectivity.291 Thus, bioreduction of ketone xenobiotics, as a general rule, produces predominantly one stereoisomeric alcohol (see "Reduction of Ketone Carbonyls").116,291 The preferential formation of (S)(-)-hydroxyhexamide from the hypoglycemic agent acetohex-amide293,294 and the exclusive generation of 6^-naltrexol from naltrexone297,298 (see "Reduction of Ketone Carbonyls" for structure) are two examples of highly stereoselective bioreduction processes in humans.

Oxidative biotransformations display product stereoselectivity, too. For example, phenytoin contains two phenyl rings in its structure, both of which a priori should be susceptible to aromatic hydroxylation. In humans, however, p-hydroxylation occurs preferentially (—90%) at the pro-(S)-phenyl ring to give primarily (S)(-)-5-(4-hydroxyphenyl)-5-phenylhydantoin. Although the other phenyl ring also is p-hydroxylated, it occurs only to a minor extent (10%).496 Microsomal hydroxylation of the C-3 carbon of diazepam and desmethyldiazepam (using mouse liver preparations) has been reported to proceed with remarkable stereoselectivity to yield optically active metabolites with the 3(S) absolute configuration.139 Interestingly, these two metabolites are pharmacologically active and one of them, oxazepam, is marketed as a drug (Serax). The al-lylic hydroxylation of the N-butenyl side group of the analgesic pentazocine (Talwin) leads to two possible alcohols (cis and trans alcohols). In human, mouse, and monkey, pentazocine is metabolized predominantly to the trans alcohol metabolite, whereas the rat primarily tends to form the cis alcohol.129 130 The product stereoselectivity observed in this biotransformation involves cis and trans geometric stereoisomers.

The term regioselectivity532 has been introduced in drug metabolism to denote the selective metabolism of two or more similar functional groups (e.g., OCH3, OH, NO2) or two or more similar atoms that are positioned in different regions of a molecule. For example, of the four methoxy groups present in papaverine, the 4-OCH3 group is regioselectively O-demethylated in several species

(e.g., rat, guinea pig, rabbit, and dog).533 Trimethoprim (Trimpex, Proloprim) has two heterocyclic sp2 nitrogen atoms (N1 and N3) in its structure. In dogs, it appears that oxidation occurs regioselectively at N3 to give the corresponding 3-N-oxide.232 Nitroreduction of the 7-nitro group in 5,7-dinitroindazole to yield the 7-amino derivative in the mouse and rat occurs with high regiose-lectivity.534 Substrates amenable to O-methylation by COMT appear to proceed with remarkable regioselectiv-ity, as typified by the cardiotonic agent dobutamine (Dobutrex). O-methylation occurs exclusively with the phenolic hydroxy group at C-3.474

Pharmacologically Active Metabolites

The traditional notion that drug metabolites are inactive and insignificant in drug therapy has changed dramatically in recent years. Increasing evidence indicates that many drugs are biotransformed to pharmacologically active metabolites that contribute to the therapeutic as well as toxic effects of the parent compound. Metabolites shown to have significant therapeutic activity in humans are listed in Table 3.4.2,535 The parent drug from which the metabolite is derived and the biotransformation process involved also are given.

How significantly an active metabolite contributes to the therapeutic or toxic effects ascribed to the parent drug depend on its relative activity and quantitative importance (e.g., plasma concentration). In addition, whether the metabolite accumulates after repeated administration (e.g., desmethyldiazepam in geriatric patients) or in patients with renal failure is determinant.

From a clinical standpoint, active metabolites are especially important in patients with decreased renal function. If renal excretion is the major pathway for elimination of the active metabolite, then accumulation is likely to occur in patients with renal failure. Especially with drugs such as procainamide, clofibrate, and digitoxin, caution should be exercised in treating patients with renal failure.2,413

Many of the toxic effects seen for these drugs have been attributed to high-plasma levels of their active metabolites. For example, the combination of severe muscle weakness and tenderness (myopathy) seen with clofibrate in renal failure patients is believed to be caused by high levels of the active metabolite chlorophenoxyisobutyric acid.536,537 Cardiovascular toxicity owing to digitoxin and procainamide in anephric subjects has been attributed to high plasma levels of digoxin and N-acetylprocainamide, respectively. In such situations, appropriate reduction in dosage and careful monitoring of plasma levels of the parent drug and its active metabolite often are recommended.

The pharmacological activity of some metabolites has led many manufacturers to synthesize these metabolites and to market them as separate drug entities (Table 3.6). For example, oxyphenbutazone (Tandearil, Oxalid) is the p-hydroxyl-ated metabolite of the anti-inflammatory agent phenylbuta-zone (Butazolidin, Azolid), nortriptyline (Aventyl) is the N-demethylated metabolite of the tricyclic antidepressant amitriptyline (Elavil), oxazepam (Serax) is the N-demethyl-ated and 3-hydroxylated metabolite of diazepam (Valium), and mesoridazine (Serentil) is the sulfoxide metabolite of the antipsychotic agent thioridazine (Mellaril).

Antivirals that are used in treating herpes simplex virus, varicella-zoster virus, and/or human cytomegalovirus must be bioactivated.538 These include acyclovir, valacyclovir, penciclovir, famciclovir, and ganciclovir, which must be phosphorylated on the pentoselike side chain to the triphos-phate derivative to be effective in inhibiting the enzyme DNA polymerase. The antiviral cidovir is dispensed as a monophosphate and only needs to be diphosphylated for conversion to the active triphosphate metabolite. The nucleoside antivirals that are used in treating acquired immunodeficiency syndrome/human immunodeficiency virus (AIDS/ HIV) must also undergo a similar metabolic conversion to the triphosphate metabolite.539 The triphosphate derivative acts as a competitive inhibitor of the enzyme, reverse transcrip-tase, which normally uses the triphosphorylated form of

TABLE 3.6 Pharmacologically Active Metabolites in Humans

Parent Drug


Biotransformation Process



Ketone reduction









Glutathione conjugation




Chloral hydrate


Aldehyde reduction



Aromatic hydroxylation


Chlorophenoxyisobutyric acid

Ester hydrolysis



Ketone reduction


Desmethyldiazepam and oxazepam

N-Demethylation and 3-hydroxylation



Alicyclic hydroxylation


Diphenoxylic acid

Ester hydrolysis









Benzylic hydroxylation






Aromatic hydroxylation



Ketone reduction



Hydroxylation and oxidation to ketone






Aromatic hydroxylation



Allylic hydroxylation


Sulfide metabolite of sulindac

Sulfoxide reduction





Warfarin alcohols

Ketone reduction

nucleic acids. Examples include zidovudine, stavudine, zal-citabine, lamivudine, and didanosine.

One of the more recent uses of drug metabolism in the development of a novel agent includes the example of oseltamivir, a neuraminidase inhibitor used in treating influenza. Ro-64-0802, the lead drug, showed promise against both influenza A and B viruses in vitro but was not very ef fective when used in vivo. To improve the oral bioavailabil-ity, the ethyl ester, oseltamivir, was developed as a prodrug. Administration of the more lipophilic oseltamivir allowed good penetration of the active metabolite in various tissues, especially in the lower respiratory tract. The metabolism proceeds via a simple ester hydrolysis to yield the active free car-

boxylic acid.540

review questions

1. What would be the logical primary metabolite of a phase I reaction for the following drug molecule?

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  • Eliza
    What would be the logical primary metabolite of a phase I?
    3 years ago
  • jan-erik
    What would be the logical primary metabolite for a phase 1 reaction for the following drug molecule?
    2 years ago

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