This article reviews the information available to assist pharmacokineticists in the prediction of metabolic drug interactions. Significant advances in this area have been made in the last decade, permitting the identification in early drug development of dominant cytochrome P450 (CYP) isoform(s) metabolising a particular drug as well as the ability of a drug to inhibit a specific CYP isoform. The major isoforms involved in human drug metabolism are CYP3A, CYP2D6, CYP2C, CYP1A2 and CYP2E1. Often patients are taking multiple concurrent medications, and thus an assessment of potential drug-drug interactions is imperative. A database containing information about the clearance routes for over 300 drugs from multiple therapeutic classes, including analgesics, anti-infectives, psychotropics, anticonvulsants, cancer chemotherapeutics, gastrointestinal agents, cardiovascular agents and others, was constructed to assist in the semiquantitative prediction of the magnitude of potential interactions with drugs under development. With knowledge of the in vitro inhibition constant of a drug (Ki) for a particular CYP isoform, it is theoretically possible to assess the likelihood of interactions for a drug cleared through CYP-mediated metabolism. For many agents, the CYP isoform involved in metabolism has not been identified and there is substantial uncertainty given the current knowledge base. The mathematical concepts for prediction based on competitive enzyme inhibition are reviewed in this article. These relationships become more complex if the inhibition is of a mixed competitive/noncompetitive nature. Sources of uncertainty and inaccuracy in predicting the magnitude of in vivo inhibition includes the nature and design of in vitro experiments to determine Ki, inhibitor concentration in the hepatic cytosol compared with that in plasma, prehepatic metabolism, presence of active metabolites and enzyme induction. The accurate prospective prediction of drug interactions requires rigorous attention to the details of the in vitro results, and detailed information about the pharmacokinetics and metabolism of the inhibitor and inhibited drug. With the discussion of principles and accompanying tabulation of literature data concerning the clearance of various drugs, a framework for reasonable semiquantitative predictions is offered in this article.

Use of In Vitro and In Vivo Data to Estimate the Likelihood of Metabolic Pharmacokinetic Interactions

The sedative drug thalidomide ([+]-alpha-phthalimidoglutarimide), once abandoned for causing birth defects in humans, has found new therapeutic license in leprosy and other diseases, with renewed teratological consequences. Although the mechanism of teratogenesis and determinants of risk remain unclear, related teratogenic xenobiotics are bioactivated by embryonic prostaglandin H synthase (PHS) to a free-radical intermediates that produce reactive oxygen species (ROS), which cause oxidative damage to DNA and other cellular macromolecules. Similarly, thalidomide is bioactivated by horseradish peroxidase, and oxidizes DNA and glutathione, indicating free radical-mediated oxidative stress. Furthermore, thalidomide teratogenicity in rabbits is reduced by the PHS inhibitor acetylsalicylic acid, indicating PHS-catalyzed bioactivation. Here, we show in rabbits that thalidomide initiates embryonic DNA oxidation and teratogenicity, both of which are abolished by pre-treatment with the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). In contrast, in mice, a species resistant to thalidomide teratogenicity, thalidomide does not enhance DNA oxidation, even at a dose 300% higher than that used in rabbits, providing insight into an embryonic determinant of species-dependent susceptibility. In addition to their therapeutic implications, these results constitute direct evidence that the teratogenicity of thalidomide may involve free radical-mediated oxidative damage to embryonic cellular macromolecules.

Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity

Thalidomide, a hypnosedative drug introduced in the 1950s, has been used in a variety of dermatologic conditions during the past few decades. Although originally withdrawn from the world market on discovery of its teratogenic effect, it has since been selectively reintroduced for use in various disorders thought to have an autoimmune or inflammatory basis. A review of the literature focused on clinical uses of thalidomide in the treatment of dermatologic diseases was performed. Diseases for which thalidomide has been found effective include erythema nodosum leprosum, prurigo nodularis, actinic prurigo, discoid lupus erythematosus, aphthous stomatitis, Behcet's syndrome, and graft-versus-host disease. Side effects such as teratogenicity and peripheral neuropathy remain its limiting factor. Thalidomide is a useful addition to the therapeutic armamentarium for treatment-resistant dermatoses as long as proper vigilance for adverse effects is maintained.

Rediscovering thalidomide: A review of its mechanism of action, side effects, and potential uses

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targets of reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

Drug Bioactivation Covalent Binding to Target Proteins and Toxicity Relevance

Although exposure during pregnancy to many drugs and environmental chemicals is known to cause in utero death of the embryo or fetus, or initiate birth defects (teratogenesis) in the surviving offspring, surprisingly little is known about the underlying biochemical and molecular mechanisms, or the determinants of teratological susceptibility, particularly in hu- mans. In vim and in vivo studies based primarily on rodent models suggest that many potential embryotoxic xenobiotics are actually proteratogens that must be bioactivated by enzymes such as the cytochromes P450 and peroxidases such as prostaglandin H synthase to teratogenic reactive intermediary metabolites. These reactive intermediates generally are electrophiles or free radicals that bind covalently (irreversibly) to, or directly or indirectly oxidize, embryonic cellular macromolecules such as DNA, protein, and lipid, irreversibly altering cellular function. Target oxidation, known as oxidative stress, often appears to be mediated by reactive oxygen species (ROS) such as hydroxyl radicals. The precise nature of the teratologically relevant molecular targets remains to be established, as do the relative contributions of the various types of macromolecular lesions. Teratological susceptibility appears to be determined in part by a balance among pathways of maternal xenobiotic elimination, embryonic xenobiotic bioactivation and detoxification of the xenobiotic reactive intermediate, direct and indirect pathways for the detoxification of ROS (cytoprotection), and repair of macromolecular lesions. Due largely to immature or otherwise compromised embryonic pathways for detoxification, cytoprotection, and repair, the embryo is relatively susceptible to reactive intermediates, and teratogenesis via this mechanism can occur from exposure to therapeutic concentrations of drugs, or supposedly safe concentrations of environmental chemicals. Greater insight into the mechanisms involved in human reactive intermediate-mediated teratogenicity, and the determinants of individual terato- logical susceptibility, will be necessary to reduce the unwarranted embryonic attrition from xenobiotic exposure.

Biochemical Toxicology of Chemical Teratogenesis

Teratogen metabolism: thalidomide activation is mediated by cytochrome P-450.

Thalidomide and two analogues, EM87 and EM12, inhibited the attachment of tumor cells to concanavalin A-coated surfaces only if the drugs were first incubated with hepatic microsomes and cofactors. Most agents that inhibit attachment are demonstrated teratogens. Thalidomide undergoes spontaneous hydrolysis to at least 12 products in saline buffered to a pH of greater than 7. These hydrolysis products did not inhibit attachment nor could they be activated to inhibitory products with hepatic microsomes. Similarly EM12 and EM 87 hydrolysis products were neither inhibitory nor substrates for activation. If the three drugs were incubated in buffered saline, there was a progressive decline in their ability to act as substrates for activation to an inhibitory product. It was possible to remove microsomes from the incubation mixture following drug activation by centrifugation. This microsome-free mixture inhibited cell attachment. When mouse ovarian tumor (MOT) cells were added to the microsome-free mixture, attachment was inhibited. However, if the activated drugs were incubated in saline, there was a progressive decline in their ability to inhibit attachment. Decay rates differed for the three compounds. At a pH of 7.4, thalidomide, EM87, and EM12 required 3 h, 1h and 6h, respectively, to decay to control levels. These relative rates of decay are consistent with the relative teratogenicity of the three drugs.

Teratogen metabolism: spontaneous decay products of thalidomide and thalidomide analogues are not bioactivated by liver microsomes.

Commercial hickory-smoke flavouring is a human lymphoblast mutagen but does not induce lung adenomas in newborn mice

Four types of chromatographic materials were evaluated for degree of recovery of mutagenic components during column chromatography. Materials tested were silica, alumina, Florisil, and cyanopropyl-bonded silica. A combustion-generated complex mixture that was highly characterized and known to contain mutagenic components was used as the reference sample. The cyanopropyl material was found to be the most efficient material for mutagen recovery and alumina proved to be the least efficient. 15 references, 3 figures, 5 tables.

Preserving toxicologic activity during chromatographic fractionation of bioactive complex mixtures.

The polycyclic aromatic compounds (PAC) produced from the pyrolysis of a bituminous coal at temperatures of 1125 to 1425 degrees K prove to be mutagenic to S. typhimurium, both in the presence and in the absence of postmitochondrial supernatant (PMS) prepared from Aroclor 1254-induced rat liver. Mutagenicity of the PAC samples measured in the absence of PMS exhibits little dependence on pyrolysis temperature; that measured in its presence is higher at the higher pyrolysis temperatures. However, because of the decrease in PAC yield as the temperature is raised, mutagenicity per mass of coal consumed falls with an increase in temperature if measured without PMS (-PMS) and peaks at an intermediate temperature of 1378 degrees K if measured with PMS (+PMS). Using a new chromatographic technique, we have split each coal-derived PAC sample into two fractions: LC1, containing PAC with alkyl and O-containing substitutions and LC2, consisting of unsubstituted PAC. Substituted (LC1) fractions show no significant +PMS mutagenicity, indicating that, as a whole, the alkylated PAC in our coal pyrolysis products are not mutagenic. Only at the higher temperatures do the substituted fractions exhibit significant -PMS mutagenicity, attributed to PAC with carbonyl or etheric functionalities. The extremely low yields of the substituted PAC under the conditions where they show some activity, however, ensure that they contribute little to overall mutagenicity. In contrast to the substituted fractions, the unsubstituted (LC2) fractions display significant mutagenicity under all conditions and appear to be responsible for virtually all of the mutagenicity in these coal-derived PAC samples. In this fraction, -PMS activity is attributed to nitrogen-containing heterocyclic aromatics.

Andrew G. Braun

Papers

Teratogen metabolism: thalidomide activation is mediated by cytochrome P-450.

Teratogen metabolism: spontaneous decay products of thalidomide and thalidomide analogues are not bioactivated by liver microsomes.

Commercial hickory-smoke flavouring is a human lymphoblast mutagen but does not induce lung adenomas in newborn mice

Preserving toxicologic activity during chromatographic fractionation of bioactive complex mixtures.

The relationship between mutagenicity and chemical composition of polycyclic aromatic compounds from coal pyrolysis.