Costas Ioannides

Interaction of a series of nitriles with the alcohol-inducible isoform of P450: computer analysis of structure-activity relationships.

1. Structural studies are reported on a series of 20 nitriles of varying rates of P4502E-mediated oxidative metabolism. 2. Parameters of molecular and electronic structure have been calculated for the generation of quantitative structure-activity relationships (QSARs) with the rates of oxidative metabolism of the nitriles, and with their acute toxicity. 3. Correlations between molecular polarizability, excitation energy and biological activity are presented as a result of QSAR analysis.

Pharmacokinetic interactions between herbal remedies and medicinal drugs

1. The use of herbal products to treat a wide range of conditions is rising rapidly, leading to increased intake of phytochemicals. Recent studies revealed potentially fatal interactions between herbal remedies and traditional drugs. 2. In transplant patients, self-medication with St Johns wort (Hypericum perforatum) has led to a drop in plasma levels of the immunosuppressant drug cyclosporine, causing tissue rejection. 3. Intake of St Johns wort increases the expression of intestinal P-glycoprotein and the expression of CYP3A4 in the liver and intestine. The combined up-regulation in intestinal P-glycoprotein and hepatic and intestinal CYP3A4 impairs the absorption and stimulates the metabolism of cyclosporine, leading to subtherapeutic plasma levels. The St Johns wort component, hyperforin, contributes to the induction of CYP3A4. 4. St Johns wort also enhances the metabolism of other CYP3A4 substrates including the protease inhibitors indinavir and nevirapine, oral contraceptives, and tricyclic antidepressants such as amitriptyline. 5. Other herbal remedies with the potential to modulate cytochrome P450 activity and thus participate in interactions with conventional drugs include Milk thistle, Angelica dahurica, ginseng, garlic preparations, Danshen and liquorice. 6. Herbal products are currently not subject to the rigorous testing indispensable for conventional drugs. However, if potential drug interactions are to be predicted, it is essential that the ability of herbal products to interfere with drug-metabolizing enzyme systems is fully established.

Cytochromes P450 in the Bioactivation of Chemicals

The initial view that the cytochrome P450 enzyme system functions simply in the deactivation of xenobiotics is anachronistic on the face of mounting evidence that this system can also transform many innocuous chemicals to toxic products. However, not all xenobiotic-metabolising cytochrome P450 subfamilies show the same propensity in the bioactivation of chemicals. For example, the CYP2C, 2B and 2D subfamilies play virtually no role in the bioactivation of toxic and carcinogenic chemicals, whereas the CYP1A, 1B and 2E subfamilies are responsible for the bioactivation of the majority of xenobiotics. Electronic and molecular structural features of organic chemicals appear to predispose them to either bioactivation by one cytochrome P450 enzyme or deactivation by another. Consequently, the fate of a chemical in the body is largely dependent on the cytochrome P450 profile at the time of exposure. Any factor that modulates the enzymes involved in the metabolism of a certain chemical will also influence its toxicity and carcinogenicity. For example, many chemical carcinogens bioactivated by CYP1, on repeated administration, selectively induce this family, thus exacerbating their carcinogenicity. CYP1 induction potency by chemicals appears to be determined by a combination of their molecular shape and electron activation. The function of cytochromes P450 in the bioactivation of chemicals is currently being exploited to design systems that can be used clinically to facilitate the metabolic conversion of prodrugs to their biologically-active metabolites in cells that poorly express them, such as tumour cells, in the so-called gene-directed prodrug therapy.

Mechanism of induction of hepatic microsomal drug metabolizing enzymes by a series of barbiturates

The inducing effect of certain barbiturates (secobarbitone, thiopentone, pentobarbitone, allobarbitone, phenobarbitone and barbitone) on the levels of the hepatic microsomal drug‐metabolizing enzymes has been studied in the rat both in vivo and in vitro. The extent of induction was related to the plasma half‐lives of the barbiturates; compounds with low rates of metabolism and long half‐lives were the most potent inducing agents. The latter (phenobarbitone, pentobarbitone and allobarbitone) were shown by spectral technique to interact with cytochrome P‐450 suggesting that their mechanism of enzyme induction was ‘substrate induction’ in type. Barbiturates containing an allyl group (secobarbitone and allobarbitone) had a weaker inducing effect than expected, possibly due to their destruction of cytochrome P‐450. Despite its short plasma half‐life of 0·5 h thiopentone was a relatively potent inducer probably due to its metabolism to pentobarbitone, which has a much longer plasma half‐life (1·3 h). Barbitone is an effective inducer of the drug‐metabolizing enzymes, yet does not interact spectrally with cytochrome P‐450; this is in accord with the observations that although there are increases in NADPH‐cytochrome c reductase and cytochrome b5, following administration of barbitone there is no increase in cytochrome P‐450. Barbiturate pretreatment does not affect the activities of glucose‐6‐phosphatase, glucose‐6‐phosphate dehydrogenase or 6‐phosphogluconate dehydrogenase.

Interactions of imidazole antifungal agents with purified cytochrome P-450 proteins.

The imidazole N-substituted antifungal agents ketoconazole, miconazole and clotrimazole have been shown to be potent inhibitors of oxidative metabolism by both a phenobarbital-induced cytochrome P-450 (P-450b) and a 3-methylcholanthrene-induced cytochrome P-448-protein (P-450c) in reconstituted systems. All three compounds inhibited the cytochrome P-450b-dependent 7-pentoxyresorufin-O-dealkylase and the cytochrome P-450c-dependent 7-ethoxyresorufin-O-deethylase activities. When 7-benzyloxyresorufin and 7-ethoxycoumarin were employed as substrates with both cytochrome preparations, all three antifungal compounds exhibited selective inhibition of the cytochrome P-450b preparation; ketoconazole was always the weakest inhibitor. The three antifungal agents were also shown to elicit a type II difference spectral interaction with both isoenzymes, the magnitude of the spectral interaction being greater with the cytochrome P-450b preparation.

Molecular dimensions of the substrate binding site of cytochrome P-448.

The molecular geometries of specific substrates, inhibitors and inducers of cytochrome P-448 activity were determined using computer-graphic techniques for use in defining the molecular dimensions of the substrate binding site of this enzyme. Specific substrates of cytochrome P-448 are essentially planar molecules characterised by a small depth and a large area/depth ratio. In contrast, compounds that do not serve as substrates of cytochrome P-448 are bulky, non-planar molecules characterised by small area/depth ratios and greater flexibility in molecular conformation. Specific inhibitors of cytochrome P-448 whose effect is mediated through interaction with the haem still meet the dimensional criteria for substrates indicating that they must also interact with the substrate binding-site, which is probably located in proximity to the haem. Inducers of cytochrome P-448 activity exhibit similar molecular geometries to the substrates from which it may be inferred that the cytosolic receptor associated with the induction of cytochrome P-448 activity is structurally related to the active site of the cytochrome.

Cytochrome P-448 and the activation of toxic chemicals and carcinogens.

The metabolic activation of carcinogens and some toxic chemicals appears to involve oxygenation in conformationally hindered positions in the chemical molecules. Oxygenation of xenobiotics in hindered positions is effected by cytochrome P-448 (LM4) but not by cytochrome P-450 (LM2). Substrate-interaction spectra show that cytochrome P-448 has an active site with a conformation different from that of cytochrome P-450. Induction of cytochrome P-448, as specifically measured by ethoxyresorufin O-deethylase activity, occurs in rat liver, kidney and lung after administration of the carcinogens, 3-methylcholanthrene, Aroclor 1254, 2-anthramine, safrole, 7,12-dimethylbenz[a]anthracene, MNNG and 2-acetamidofluorene. The doubtful carcinogens, saccharin, DDT and aldrin, resulted in no significant induction. The drugs paracetamol, antipyrine, imipramine and rifampicin resulted in diminished enzyme activity, indicating the absence of any induction of cytochrome P-448. In studies with the matched pairs of carcinogens and non-carcinogens, benzo[a]pyrene and benzo[e]pyrene, and 1,2,5,6-dibenzanthracene and anthracene, only the carcinogenic analogue resulted in induction of cytochrome P-448. With alpha- and beta-naphthylamine, both resulted in marked induction of cytochrome P-448 in liver, kidney and lung, indicating that both isomers might be carcinogenic.

Absolute bioavailability and dose-dependent pharmacokinetic behaviour of dietary doses of the chemopreventive isothiocyanate sulforaphane in rat

Sulforaphane is a naturally occurring isothiocyanate with promising chemopreventive activity. An analytical method, utilising liquid chromatography-MS/MS, which allows the determination of sulforaphane in small volumes of rat plasma following exposure to low dietary doses, was developed and validated, and employed to determine its absolute bioavailability and pharmacokinetic characteristics. Rats were treated with either a single intravenous dose of sulforaphane (2.8 micromol/kg) or single oral doses of 2.8, 5.6 and 28 mumol/kg. Sulforaphane plasma concentrations were determined in blood samples withdrawn from the rat tail at regular time intervals. Following intravenous administration, the plasma profile of sulforaphane was best described by a two-compartment pharmacokinetic model, with a prolonged terminal phase. Sulforaphane was very well and rapidly absorbed and displayed an absolute bioavailability of 82 %, which, however, decreased at the higher doses, indicating a dose-dependent pharmacokinetic behaviour; similarly, Cmax values did not rise proportionately to the dose. At the highest dose used, the rate of absorption constant k(ab), biological half-life t(1/2) and apparent volume of distribution decreased significantly. It is concluded that in the rat orally administered sulforaphane is rapidly absorbed, achieving high absolute bioavailability at low dietary doses, but dose-dependent pharmacokinetics was evident, with bioavailability decreasing with increasing dose.

SAFROLE - ITS METABOLISM, CARCINOGENICITY AND INTERACTIONS WITH CYTOCHROME-P-450

Abstract A review of studies on safrole metabolism shows that the compound gives rise to a large number of metabolites by two major pathways, oxidation of the allyl side chain and oxidation of the methylenedioxy group with subsequent cleavage to form a catechol. The mechanism by which safrole exerts the weak hepatocarcinogenicity that has been demonstrated in rats and mice is considered on the basis of published work and recent studies by the authors. Metabolic conversion of the allyl group gives rise to intermediates capable of covalent binding with DNA and protein, and recent findings are compatible with conversion of the methylenedioxy group to a carbene, which forms ligand complexes with the haem moiety of cytochromes P -450 and P -448. It is suggested that while the allyl group is responsible for the mutagenic potential of safrole, the methylenedioxy moiety may be associated with epigenetic aspects of carcinogenicity.

Structural and functional integrity of precision-cut liver slices in xenobiotic metabolism: a comparison of the dynamic organ and multiwell plate culture procedures.

1. Objectives were two-fold: (1) to compare the viability of precision-cut liver slices in two culture systems, namely the dynamic organ and the multiwell plate; and (2) to evaluate whether increasing the number of slices per incubation results in a proportional increase in the extent of metabolism. 2. With both culturing systems, the major products of 7-ethoxycoumarin metabolism were the sulphate and glucuronide conjugates of 7-hydroxycoumarin with very low levels of the free compound. When the multiwell plate procedure was used, metabolism increased linearly for at least 10 h, whereas it tended to plateau after 6 h in the dynamic organ culture system. At preincubations > 10 h, significantly more metabolism of 7-ethoxycoumarin was seen in the slices cultured using the multiwell system compared with the dynamic organ system. 3. Morphological evaluation employing light and electron microscopy revealed that liver slices incubated using the multiwell system were structurally better preserved compared with those incubated using the dynamic organ system. 4. Using the multiwell system, increasing the number of slices per incubation from one to two resulted in only a modest increase in the metabolism of 7-ethoxycoumarin. The rate of metabolism of this substrate was much higher with one liver slice when expressed per mg homogenate protein. 5. It is concluded that (1) the multiwell plate culture system for culturing slices is superior to the dynamic organ system in studying the metabolism of xenobiotics following long-term incubations, (2) increasing the number of slices per incubation does not result in a corresponding increase in the rate of metabolism, and (3) in both culture systems optimal viability appears to be within 24 h of incubation.