Cytochrome P450s (P450s) are subject to inhibition/inactivation by various chemically diverse agents, which may interact directly or indirectly with either the P450 prosthetic heme or the protein moiety or with both moieties. This inhibition is clinically relevant, as it can block physiological pathways as well as result in therapeutically significant beneficial or adverse effects and clinically relevant drug–drug interactions. Herein, we discuss the various chemical agents and their chemical mechanisms that lead to such reversible, quasi-irreversible, or irreversible inhibition of these enzymes. We note that such inhibition targeted at P450-dependent physiological and/or pathological processes has been ingeniously and effectively harnessed not only in the design and development of clinically relevant therapeutic agents but also of fungicides, insecticides, and herbicides.

Inhibition of Cytochrome P450 Enzymes

Griseofulvin, an orally active antifungal drug used to treat dermatophyte infections, has a secondary effect of inducing cytochrome P450-mediated production of N-methyl protoporphyrin IX (N-MPP). N-MPP is a potent competitive inhibitor of the heme biosynthetic-enzyme ferrochelatase, and inhibits the growth of cultured erythrocyte stage Plasmodium falciparum. Novel drugs against Plasmodium are needed to achieve malaria elimination. Thus, we investigated whether griseofulvin shows anti-plasmodial activity. We observed that the intraerythrocytic growth of P. falciparum is inhibited in red blood cells pretreated with griseofulvin in vitro. Treatment with 100 μM griseofulvin was sufficient to prevent parasite growth and induce the production of N-MPP. Inclusion of the ferrochelatase substrate PPIX blocked the inhibitory activity of griseofulvin, suggesting that griseofulvin exerts its activity through the N-MPP-dependent inhibition of ferrochelatase. In an ex-vivo study, red blood cells from griseofulvin-treated subjects were refractory to the growth of cultured P. falciparum. However, in a clinical trial griseofulvin failed to show either therapeutic or prophylactic effect in subjects infected with blood stage P. falciparum. Although the development of griseofulvin as an antimalarial is not warranted, it represents a novel inhibitor of P. falciparum growth and acts via the N-MPP-dependent inhibition of ferrochelatase.

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Griseofulvin impairs intraerythrocytic growth of Plasmodium falciparum through ferrochelatase inhibition but lacks activity in an experimental human infection study

Cytochrome P450 (P450) enzymes oxidize xenobiotics into chemically reactive metabolites or intermediates as well as into stable metabolites. If the reactivity of the product is very high, it binds to a catalytic site or sites of the enzyme itself and inactivates it. This phenomenon is referred to as mechanism-based inactivation. Many clinically important drugs are mechanism-based inactivators that include macrolide antibiotics, calcium channel blockers, and selective serotonin uptake inhibitors, but are not always structurally and pharmacologically related. The inactivation of P450s during drug therapy results in serious drug interactions, since irreversibility of the binding allows enzyme inhibition to be prolonged after elimination of the causal drug. The inhibition of the metabolism of drugs with narrow therapeutic indexes, such as terfenadine and astemizole, leads to toxicities. On the other hand, the fate of P450s after the inactivation and the toxicological consequences remains to be elucidated, while it has been suggested that P450s modified and degraded are involved in some forms of tissue toxicity. Porphyrinogenic drugs, such as griseofulvin, cause mechanism-based heme inactivation, leading to formation of ferrochelatase-inhibitory N-alkylated protoporphyrins and resulting in porphyria. Involvement of P450-derived free heme in halothane-induced hepatotoxicity and catalytic iron in cisplatin-induced nephrotoxicity has also been suggested. Autoantibodies against P450s have been found in hepatitis following administration of tienilic acid and dihydralazine. Tienilic acid is activated by and covalently bound to CYP2C9, and the neoantigens thus formed activate immune systems, resulting in the formation of an autoantibodydirected against CYP2C9, named anti-liver/kidney microsomal autoantibody type 2, whereas the pathological role of the autoantibodies in drug-induced hepatitis remains largely unknown.

Toxicological significance of mechanism-based inactivation of cytochrome p450 enzymes by drugs.

Changes in levels of hepatic biotransformation enzymes and haemoglobin levels in female plaice (Pleuronectes platessa) after oral administration of a technical polychlorinated biphenyl mixture (Clophen A40).

1. Multiple forms of cytochrome P450 (CYP) catalyse the oxidation of chemicals of endogenous and exogenous origin, including drugs, carcinogens, steroids and eicosanoids. However, this unusual low substrate specificity also makes CYP susceptible to inhibition by a wide range of drugs, leading to pharmacokinetic interactions of potential clinical significance. 2. Some drugs are converted by CYP to reactive metabolites that bind covalently to sites within the active centre of the same CYP. Such mechanism-based inhibition leads to CYP inactivation or complexation. These processes give rise to long-term effects on drug pharmacokinetics, as the inactivated or complexed CYP must be replaced by newly synthesized CYP protein. 3. Drugs that inactivate CYP generally possess recognizable functional groups that are oxidized to reactive products. Thus, drugs with side chains containing unsaturated carbon-carbon bonds and furan ring systems are associated with CYP inactivation. Nitrogen-containing systems may also inactivate CYP. 4. Metabolites formed from drugs containing alkylamino and methylenedioxy functionalities can trap CYP as inert complexes without eliciting inactivation. However, the functional effects of inactivation and complexation on drug pharmacokinetics are indistinguishable. Drugs that elicit CYP complexation include the first generation macrolide antibiotics, but newer analogues appear much safer. Some antidepressants, antiepileptics and tuberculostatic agents have been associated with CYP complexation.

Drug‐mediated inactivation of cytochrome p450

1. A hepatic green pigment with inhibitory properties towards the enzyme ferrochelatase has been isolated from the liver of mice treated with griseofulvin and identified as N-methylprotoporphyrin. 2. All four structural isomers of N-methylprotoporphyrin have been demonstrated to be present, NA, where ring A of protoporphyrin IX is N-methylated, being the predominant isomer. 3. In addition to N-methylprotoporphyrin, a second green pigment, present in far greater amounts, was also isolated from the liver of griseofulvin-treated mice. This second green pigment is also an N-monosubstituted protoporphyrin, but in this case the substituent on the pyrrole nitrogen atom appears to be intact griseofulvin rather than a methyl group. 4. The fragmentation of this adduct in tandem m.s. studies suggests that griseofulvin is bound to the pyrrole nitrogen through one of its carbon atoms and further suggests that N-methylprotoporphyrin may arise as a secondary product from the major griseofulvin pigment.

Isolation of two N-monosubstituted protoporphyrins, bearing either the whole drug or a methyl group on the pyrrole nitrogen atom, from liver of mice given griseofulvin.

Two distinct types of drug toxicity are illustrated, both with liver haem as the main target. Drugs with unsaturated side chains and certain dihydropyridines both convert liver haem into N -alkylated porphyrins but the underlying mechanisms and biological properties of the resulting pigments differ in the two cases. A monooxygenated derivative of the unsaturated drugs becomes itself bound onto one of the pyrrole nitrogen atoms of the haem of cytochrome P-450 and the N -alkylated porphyrin which is produced does not inhibit the last enzyme of haem biosynthesis, protohaem ferro-lyase. In contrast, with 3,5-diethoxycarbonyl-l,4-dihydrocollidine only the 4-methyl substituent of the drug is transferred onto the pyrrole nitrogen atom and the resulting porphyrin is a powerful inhibitor of protohaem ferro-lyase. The latter pathway involves transfer of the intact 4-alkyl group onto the pyrrole nitrogen.

J.H. Lamb

Papers

Isolation of two N-monosubstituted protoporphyrins, bearing either the whole drug or a methyl group on the pyrrole nitrogen atom, from liver of mice given griseofulvin.

Liver Haem as a Target for Drug Toxicity