|4. Drug metabolism|
3′-Phosphoadenosine-5′- phosphosulfate (PAPS)
Glutathione-S-transferases / spontaneous
N-, S-, and O-methyl-
Amino acid transferases
Free amino acids/ATP
Phase II metabolism consists of conjugation reactions. Some of these reactions have already been illustrated; others will be shown below.
|4.4.1||Morphine skips phase I and is conjugated directly|
Phase I reactions are not necessary if a drug molecule already contains functional groups suitable for conjugation. An example is morphine, which has two hydroxyl groups. The conjugation of either, or both, with glucuronic acid is sufficient for excretion. Interestingly, one of the two single glucuronides—the one not shown in this slide—retains pharmacological activity.
The morphine metabolite shown in this slide is the major one found in the urine. In addition to conjugation, N-demethylation of morphine by cytochrome P450 may also occur, but it makes little difference for urinary excretion.
|4.4.2||Epoxides of aromatic hydrocarbons can intercalate and react covalently with DNA|
Benzopyrene and related aromatic compounds are contained in tobacco smoke and in industrial
emissions. Their activation by cytochrome P450 enzymes, notably CYP1A1, is an essential
step in their carcinogenic action. The study that reported the structure of this DNA adduct
also discusses how it interacts with DNA polymerase to induce mutations Author: Bauer, Jacob et al.
Title: A structural gap in Dpo4 supports mutagenic bypass of a major benzo[a]pyrene dG adduct in DNA through template misalignment
Journal: Proc Natl Acad Sci U S A
Like CYP3A4, CYP1A1 is also subject to transcriptional induction, but this occurs downstream of a different nuclear receptor (the aromatic hydrocarbon receptor, AHR).
|4.4.3||Enzymatic detoxification of benzopyrene epoxy-derivatives|
While reactive benzopyrene metabolites are harmful, most such molecules will actually be captured and inactivated before reacting with DNA—otherwise, famous smokers like Winston Churchill and Deng Xiaoping would not have had enough time to become famous …
The major detoxification pathways are hydrolysis, which is catalyzed by epoxide hydrolase, and glutathione conjugation, which is catalyzed by glutathione-S-transferase (GST).
|4.4.4||Hepatic metabolism of acetaminophen|
An oxidative reaction that we haven’t seen so far is the conversion of acetaminophen to N-acetyl-p-benzoquinone imine (NAPQI). This metabolite is readily conjugated with glutathione and excreted. If acetaminophen is overdosed, this metabolic pathway can deplete glutathione in the liver cells and thereby cause severe, even fatal, liver toxicity.*Interestingly, the major cytochrome P450 isoform responsible for acetaminophen oxidation is 2E1, which is induced by ethanol and acetone and also degrades both these inducers.
Acetaminophen is used for its inhibitory effect on cyclooxygenase. NAPQI is also formed in the reaction of acetaminophen with cyclooxygenase, and this forms the basis of its inhibitory activity (see slide 9.3.4).
|4.4.5||Activation of the anticancer drug canfosfamide by glutathione-S-transferase|
Glutathione reacts readily not only with epoxides but with many other cytotoxic compounds that are reactive toward cellular nucleophiles. Many such compounds are used as anticancer drugs, and cancer cells often acquire resistance to these through mutations that increase the expression of glutathione-S-transferase (GST).
The drug canfosfamide was designed to turn this resistance mechanism on its head. The molecule, which contains a moiety that resembles glutathione, is cleaved rather than alkylated by GST. It is activated by this cleavage, and it should therefore be more active in tumor cells that overexpress the enzyme.
The cytotoxicity of canfosfamide is due to its reactive chloroethylamine groups; slide 2.3.5 shows how these groups will react with cellular nucleophiles. These groups are already present in canfosfamide, and they are not involved in GST-mediated cleavage; therefore, how does this cleavage increase the drug’s activity?
I have seen no experimental evidence for or against the following hypothesis, but it seems possible that the difference in activity between the prodrug and the cleaved product is due to the extrusion of the prodrug. Glutathione conjugates make good substrates for ABC transporters. Canfosfamide resembles such a conjugate and thus may also be subject to extrusion. Cleavage by GST may allow the drug to avoid extrusion, stay in the cell and exert its cytotoxic effect.
|4.4.6||Acetylation of INH by N-acetyltransferase 2 (NAT 2)|
Isoniazid (isonicotinic acid hydrazide, INH) is the classical example of drug metabolism through acetylation. The acetylated product may undergo hydrolysis to release acetylhydrazide, which is reactive and may cause cytotoxicity. Nevertheless, overall, N-acetylation reduces toxicity, since metabolites with even greater toxicity would otherwise arise through N-hydroxylation by cytochrome P450.
This potential metabolic toxicity notwithstanding, INH is tolerated better than most other tuberculostatic drugs and is the most commonly prescribed one.
|4.4.7||Bimodal distribution of INH acetylation speed|
In the depicted experiment, the speed of acetylation was measured in a group of patients.
A fixed test dosage was applied at t=0, and the amount of drug still remaining in the blood
plasma after six hours was measured. There clearly are two separate peaks, which represent
the fast and slow acetylators, respectively. Figure prepared from original data in Author: Goedde, H W et al.
Title: Studies on pharmacogenetics. I. The enzymatic acetylation of isonicotinic acid hydrazide (INH)
Journal: Biochem Pharmacol
Among Caucasians, about 50% express an inactive NAT2 allele, which causes the
slow-acetylator phenotype. The percentage of slow acetylators is lower among Asians (but
was higher in a small study done on Kenyans Author: Rashid, J R et al.
Title: Acetylation status using hydralazine in African hypertensives at Kenyatta National Hospital
Journal: East Afr Med J
ISBN: 0012-835X; I don’t know how representative that study is).
Apart from isoniazid, the NAT2 enzyme and its polymorphism also affect the inactivation
rates of some other drugs, such as procainamide and hydralazine, which are more likely to
cause toxicity in slow acetylators. NAT2 has also been implicated in the susceptibility to
bladder cancer caused by aromatic amines, which in Europeans was found to correlate with
slow acetylator status. Surprisingly, the same correlation was not observed in Chinese
Author: Golka, Klaus et al.
Title: The enhanced bladder cancer susceptibility of NAT2 slow acetylators towards aromatic amines: a review considering ethnic differences
Journal: Toxicol Lett
ISBN: 0378-4274. The reason for this discrepancy is unknown.
|4.4.8||Metabolic activation of arylamine carcinogens|
N-Acetyltransferases (NAT), cytochrome P450 (CYP) and sulfotransferases (ST)
cooperate in the metabolic activation of arylamine carcinogens such as benzidine or
2-naphthylamine. The acetoxy and sulfohydroxamate products decay spontaneously to reactive
electrophiles, which can then react with cellular macromolecules including DNA. Figure
drawn after a scheme shown in Author: Mattano, S S et al.
Title: Purification and biochemical characterization of hepatic arylamine N-acetyltransferase from rapid and slow acetylator mice: identity with arylhydroxamic acid N,O-acyltransferase and N-hydroxyarylamine O-acetyltransferase
Journal: Mol Pharmacol
|4.4.9||Glutamine conjugation of phenylacetate|
Glutamine, glycine and taurine can be conjugated to various xenobiotics that are organic acids; the figure shows glutamine conjugation of phenylacetic acid as an example.
An interesting application of glycine and glutamine conjugation is for alternate pathway therapy in enzyme defects of the urea cycle (see slide 10.2.6).
|Lecture notes on biochemical pharmacology|