Mammalian Cytochrome P450 Enzymes Research Articles

Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30-40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.

The Cytochrome P450 24A1 Interaction with Adrenodoxin Modulates Substrate Binding and Relies on Species‐variable Recognition Sites

Redox partner and accessory protein interactions are known to modulate function in mammalian cytochrome P450 enzymes by exerting a level of allosteric control, most notably demonstrated by the interaction of cytochrome b5 with P450 enzymes in the endoplasmic reticulum. However, in the inner mitochondrial membrane, in which P450 electron transfer occurs solely via interaction with the ferredoxin protein adrenodoxin (Adx), the events that modulate P450 function are not clear. In this study, we report the use of combined spectroscopic techniques to demonstrate that Adx serves to modulate substrate binding in the mitochondrial vitamin D metabolizing enzyme CYP24A1. The presence of equal molar amounts of Adx potentiates substrate binding for 25(OH)D3 and 1,24,25(OH)3D3, but not for the primary substrate 1,25(OH)2D3. We follow up on the CYP24A1‐Adx interaction by utilizing solution nuclear magnetic resonance (NMR) spectroscopy to monitor the spectra of uniformly labeled 15N Adx while titrating with unlabeled CYP24A1 bound to the P450 inhibitor clotrimazole. The pattern of NMR line broadening, combined with single and double mutants of Adx, suggest the presence of multiple recognition sites between the proteins. In order to evaluate redox partner complexes for CYP24A1 isoforms displaying distinct regioselectivity for vitamin D, all of the binding data were acquired in parallel for a carbon 23 hydroxylase, a carbon 24 hydroxylase, and a combined carbon 23/24 hydroxylase. Interestingly, disruption of the CYP24A1‐Adx complex in response to Adx mutagenesis was variable between CYP24A1 isoforms, suggesting a correlation between redox partner binding and regioselectivity and consistent with the ability of Adx to modulate substrate binding. Therefore, this work provides structural evidence in support of Adx modulation of function in a mitochondrial P450 enzyme.Support or Funding InformationThis work was supported by R00GM112862 and the State University of New YorkThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural basis for the 4'-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase.

Diclofenac is a nonsteroidal anti-inflammatory drug. It undergoes hydroxylation by mammalian cytochrome P450 enzymes at 4'- and/or 5'-positions. A bacterial P450 enzyme, CYP105D7 from Streptomyces avermitilis, has been shown to catalyze hydroxylation of 1-deoxypentalenic acid and an isoflavone daidzein. Here, we demonstrated that CYP105D7 also catalyzes hydroxylation of diclofenac at the C4'-position. A spectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65 μM and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2 Å resolution. The distal pocket of CYP105D7 contains two diclofenac molecules, illustrating drug recognition with a double-ligand-binding mode. The C3' and C4' atoms of the dichlorophenyl ring of one diclofenac molecule are positioned near the heme iron, suggesting that it is positioned appropriately for aromatic hydroxylation to yield the 4'-hydroxylated product. However, recognition of diclofenac by CYP105D7 was completely different from that of rabbit CYP2C5, which binds one diclofenac molecule with a cluster of water molecules. The distal pocket of CYP105D7 contains four arginine residues, forming a wall of the substrate-binding pocket, and the arginine residues are conserved in bacterial P450s in the CYP105 family.

Challenges and Opportunities of Cytochrome P450-Mediated Phytoremediation

Although environmental pollution was first realized to be a threat to public health in the latter part of the 20th century, events in the 21st century have served to increase the public awareness of the impacts of environmental pollution. Several communities, including the scientific community, initiated dialogues to help protect the planet from environmental pollution. Further, scientific community initiated basic and applied research in an effort to decontaminate soil, water, and air pollutions arising from industrial and petroleum wastes, as well as from the use of pesticides and herbicides. They utilized plant enzymes, as well as bacterial, fungal, and mammalian enzymes cloned in plants (transgenic plants), to detoxify soil and water contaminations (phytoremediation). Although there was a slow increase in research publications of phytoremediation from 1990-2000, a significant increase in their publications from 2001-2008 was observed (Figure 1). Interestingly, a decline in research publications since 2009 was observed, which suggests that there is a challenge in this type of research for further improvements in phytoremediation. A similar trend was also observed with cytochrome P450 (CYP)-mediated phytoremediation (Figure 1). In this editorial, we will discuss the challenges and opportunities of CYP-mediated phytoremediation. The discussion in this editorial is based on a recent review [1]. Figure 1 Number of publications from 1990 to 2012 using the key words PubMed (control), phytoremediation, and phytoremediation+cytochrome P450 (CYP). Left Y-axis represents number of publications for PubMed and phytoremediation and right Y-axis represents number ... CYP family of enzymes involved in the metabolism and detoxification of numerous xenobiotics, including herbicides, pesticides, and industrial pollutants, such as pharmaceuticals, petrochemicals, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated benzene (PCBs) [2]. The catalytic versatility of CYP enzymes have led to significant interest in utilizing them as biocatalysts for phytoremediation. In the last decade there has been an increase in the utilization of CYP biocatalysts for creating herbicide-resistant plants and for the detoxification of soil and water contaminants. The use of transgenic plants expressing bacterial and mammalian CYP enzymes for phytoremediation is crucial because plant CYP enzymes are poor in detoxifying these contaminants [3], whereas bacterial and mammalian CYP enzymes have the capability to detoxify herbicides and industrial pollutants [4,5]. However, the major limitation of mammalian CYP enzymes is that they have relatively low turn-over, enzyme stability, and expression in plants. In contrast, many bacterial CYP enzymes display high levels of activity and stability; however, they metabolize limited number of pollutants. These are the major challenges in the utilization of CYP enzymes for phytoremediation that may have led to a decrease in research publications for CYP-mediated phytoremediation since 2009 (Figure 1). However, these challenges also bring an opportunity to expand innovative research towards engineering of CYP enzymes for the improvement of their substrate diversity, activity, stability, and expression in plants. Since the beginning of 21st century, significant progress has been made, especially by Arnold and colleagues, in designing bacterial CYP enzymes [6,7]. For example, a bacterial CYPBM3 enzyme has been engineered for novel activity towards hydroxylation of many non-natural alkanes, such as asoctane, hexane, propane, butane, and ethane, as well as other non-alkyl substrates, such as benzene, propylbenzene, and naphthalene. Similarly, another bacterial CYPCAM enzyme has been engineered for efficient oxidation of various alkanes (butane, pentane, hexane, and heptanes), halogenated hexanes, PCBs, and PAHs. These engineered CYPBM3 and CYPCAM enzymes can potentially be used to create transgenic plants for possible phytoremediation of these toxic compounds. Further, several mammalian CYP enzymes have been engineered to improve activity, stability, and heterologous expression [8]. For examples: 1) Guengerich and colleagues have successfully engineered human CYP2A6 enzyme for increased activity with indigo compounds and cloned it into tobacco plants that produced the precursor of indole dyes; 2) Kumar and Halpert groups have engineered Rat CYP2B1 and canine CYP2B11 enzymes for enhanced activity with several compounds, including anti-cancer prodrugs cyclophosphamide and ifosfamide; 3) Gillam and colleagues have designed CYP2C enzymes for novel activity with 6′-deoxyluciferin and diclofenac. Although these mammalian CYP enzymes were not engineered for enhanced activity with herbicides, industrial contaminants, or petroleum compounds, they provide a proof-of-concept that mammalian CYP enzymes can be engineered for phytoremediation of these compounds. In conclusion, phytoremediation of soil and water contaminants arising from herbicides, pesticides, and industrial pollutants has been identified to be critically important to clean the environment and improve air and water qualities. Although generation of CYP-based transgenic plants has the potential to serve this purpose, certain limitations of CYP enzymes posed challenges in their application in phytoremediation. Thus, there is a critical need to expand the basic and applied research in designing CYP biocatalysts for phytoremediation and therefore additional effort is required from CYP enzyme and protein engineering communities towards this.

Diversity and Substrate Specificity in the Structures of Steroidogenic Cytochrome P450 Enzymes

Mammalian cytochrome P450 (CYP) comprise a large group of enzymes that play many important roles in the biosynthesis of steroid hormones and vitamins. In addition, they participate in the metabolism of drugs and xenobiotics. All known mammalian CYP enzymes are membrane-associated proteins, which complicated their X-ray crystallographic analysis. In recent years, however, significant progress has been made in the X-ray crystallographic analysis of mammalian CYP enzymes involved in the steroid hormone and vitamin D(3) metabolism. The knowledge from three-dimensional structures of mammalian CYP enzymes will benefit drug discovery and development.

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes.

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation.

In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil, erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes

Inhibition of mammalian cytochrome P450 enzymes (CYPs) is well characterized; major hepatic CYPs can be inhibited by drugs and other environmental contaminants. CYP function and inhibition has not yet been well established in fish yet these studies are important for several reasons. First, such studies will provide functional information for non-mammalian CYPs. Second, specific inhibitors can be used as a diagnostic tool for studying CYP-mediated reactions. Lastly, pharmaceutical mixtures are found in the aquatic environment and adverse effects associated with drug–drug interactions, including CYP inhibition by pharmaceuticals may be of concern. Using liver microsomes from untreated and β-naphthoflavone (BNF)-treated rainbow trout, eight fluorescent CYP-mediated catalytic assays were used to assess in vitro CYP inhibition by four pharmaceuticals: fluoxetine, ciprofloxacin, gemfibrozil and erythromycin. Expressed zebrafish CYP1 proteins (CYP1A, CYP1B1, CYP1C1 and CYP1C2) were assessed for inhibition with selected substrates. All pharmaceuticals decreased the metabolism of a number of substrates. Fluoxetine was the strongest and most broad inhibitor of CYP-mediated reactions in liver microsomes. Zebrafish CYP1s were strongly inhibited by erythromycin and fluoxetine. Although the pharmaceuticals are selective CYP inhibitors in mammals, inhibition across a number of substrates suggests they are broad inhibitors in fish. These data demonstrate that in vitro hepatic CYP inhibition by pharmaceuticals is possible in fish and the patterns seen here are different than what would be expected based on CYP inhibition in mammals.

Endogenous Functions of the Aryl Hydrocarbon Receptor (AHR): Intersection of Cytochrome P450 1 (CYP1)-metabolized Eicosanoids and AHR Biology

The AHR,2 a ligand-activated transcription factor, was first identified indirectly in the 1970s; the mouse and human genes were cloned in the early 1990s. Molecular mechanisms and biological consequences of AHR-mediated regulation of mammalian cytochrome P450 enzymes by foreign chemicals (e.g. polycyclic aromatic hydrocarbons and dioxins) have been studied extensively. Binding of such ligands to AHR leads to transcriptional activation of CYP1A1, CYP1A2, and CYP1B1; in turn, these enzymes catalyze oxidative detoxication or activation of most ligands. From the start, AHR endogenous ligands and functions were postulated; although this was initially controversial, the robust phenotype of Ahr–/– knock-out mice provided clear evidence of physiological roles (and endogenous ligands) for AHR. AHR has numerous important endogenous functions: during conception and embryonic and fetal development; in the immune, cardiovascular, neural, and reproductive systems; and in hepatocytes, skin cells, and adipocytes. These myriad AHR-mediated processes mirror the vast universe of action of the eicosanoids, lipid mediators known to undergo cytochrome P450-dependent oxidation. We propose that many endogenous and exogenous cellular stimuli lead to (i) AHR-dependent CYP1-dependent eicosanoid synthesis and degradation and (ii) AHR-dependent CYP1-independent (eicosanoid-dependent and -independent) responses. These two pathways can be delineated from one another in genetic models: the former is absent in the recently characterized Cyp1a1/1a2/1b1–/– triple-knock-out mouse, and the latter is absent in the Ahr–/– knock-out mouse. Identification of specific eicosanoids whose synthesis or degradation is carried out by each CYP1 enzyme should allow for identification of physiological endogenous AHR ligands. (For “History and Background,” see supplemental material (Box 1).)

The binding of aristolochic acid I to the active site of human cytochromes P450 1A1 and 1A2 explains their potential to reductively activate this human carcinogen

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Using the 32P-postlabeling assay we showed that AAI is activated by human recombinant cytochrome P450 (CYP) 1A1, CYP1A2 and NADPH:CYP reductase to species generating DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N 6-yl)aristolactam I, 7-(deoxyguanosin-N 2-yl)aristolactam I and 7-(deoxyadenosin-N 6-yl)aristolactam II were identified as AA-DNA adducts formed from AAI by the enzymes. The formation of these AA-derived DNA adducts indicates that all the human enzymes reduce the nitro group of AAI to the putative reactive cyclic nitrenium ion responsible for adduct formation. The concentrations of AAI required for its half-maximum DNA binding were 38, 65 and 126 μM AAI for reductive activation by human CYP1A2, CYP1A1 and NADPH:CYP reductase, respectively. CYP1A1 and 1A2 homology modeling followed by docking of AAI to the CYP1A1 and 1A2 active centers was utilized to explain the potential of these enzymes to reduce AAI. Models of human CYP1A1 and 1A2 were constructed on the basis of the crystallographic structure of truncated mammalian CYP enzymes, CYP2B4, 2C5, 2C8, 2C9 and 3A4. The in silico docking of AAI to the active sites of CYP1A1 and 1A2 indicates that AAI binds as an axial ligand of the heme iron and that the nitro group of AAI is in close vicinity to the heme iron of CYP1A2 in an orientation allowing the efficient reduction of this group observed experimentally. The orientation of AAI in the active centre of CYP1A1 however causes an interaction of the heme iron with both the nitro- and the carboxylic groups of AAI. This observation explains the lower reductive potential of CYP1A1 for AAI than CYP1A2, detected experimentally.

Electron transfer in human cytochrome P450 reductase.

Cytochrome P450 reductase (CPR) is a diflavin enzyme responsible for electron donation to mammalian cytochrome P450 enzymes in the endoplasmic reticulum. Dissection of the enzyme into functional domains and studies by site-directed mutagenesis have enabled detailed characterization of the mechanism of electron transfer using stopped-flow and equilibrium-perturbation methods, and redox potentiometry. These studies and the mechanism of electron transfer in CPR are reported herein.

Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons.

Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environmental contaminants. Wild-type CYP102 (P450(BM-3)) from Bacillus megaterium has low activity for the oxidation of the PAHs phenanthrene, fluoranthene and pyrene. The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40-fold. Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity. Both these mutations increased the NADPH turnover rate, but the A264G mutation increased the coupling efficiency while the F87A mutation had dominant effects in product selectivity. Fast NADPH oxidation rates were observed (2250 min-1 for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies were relatively low (< 13%), resulting in a highest substrate oxidation rate of 110 min-1 for fluoranthene oxidation by the R47L/Y51F/A264G mutant. Mutation of M354 and L437 inside the substrate access channel reduced PAH oxidation activity. The PAHs were oxidized to a mixture of phenols and quinones. Notably mutants containing the A264G mutation showed some similarity to mammalian CYP enzymes in that some 9,10-phenanthrenequinone, the K-region oxidation product from phenanthrene, was formed. The results suggest that CYP102 mutants could be useful models for PAH oxidation by mammalian CYP enzymes, and also potentially for the preparation of novel PAH bioremediation systems.

Pharmacokinetics and pharmacology of terbinafine and itraconazole

Background: Two new systemic antifungal agents, terbinafine and itraconazole, have expanded the choices for treatment of onychomycosis. The pharmacokinetic and pharmacologic properties provide the basis of their activity and are related to their efficacy and safety in dermatophyte infections. Objective: We describe the pharmacodynamics, pharmacokinetics, and pharmacology of terbinafine and itraconazole and the features that form a framework for comparing their efficacy. Pharmacodynamics: Both terbinafine and itraconazole ultimately block ergosterol synthesis; terbinafine disrupts fungal cell wall synthesis earlier (squalene to squalene epoxide) than does itraconazole (lanosterol to ergosterol). In vitro, terbinafine exposure results in a toxic accumulation of squalene and decreased production of ergosterol. Minimal inhibitory concentrations (MICs) of terbinafine for dermatophytes are essentially equal to minimal fungicidal concentrations (MFCs). However, the MFCs of itraconazole are much higher than the MICs. Pharmacologic Profile: Both itraconazole and terbinafine penetrate keratinizing tissue; levels reached in nail plate exceed those in plasma. Therapeutic levels of the itraconazole persist in nails for up to 6 months after discontinuation of 3 months of therapy (200 mg/day) and during various pulsed cycles. After discontinuation of 1 month of therapy, terbinafine persists at therapeutic levels in the nail. Itraconazole has an affinity for mammalian cytochrome P-450 enzymes as well as for fungal P-450–dependent enzyme, and thus has the potential for clinically important interactions (e.g., astemizole, terfenadine, rifampin, oral contraceptives, H 2 receptor antagonists, warfarin, cyclosporine). Terbinafine is not metabolized through this system and has little potential for drug-drug interactions. Conclusion: The low MFCs exhibited by terbinafine for dermatophytes may be important in its clinical efficacy and low relapse rates. The safety profile of terbinafine directly reflects its mechanism of action. (J Am Acad Dermatol 1998;38:S42-7.)

Chromatographic separation and behavior of microsomal cytochrome P450 and cytochrome b5

The methods used for separation of the multiple mammalian cytochrome P450 enzymes by liquid chromatography are reviewed. In addition to the chromatographic techniques, preparation and handling of samples and prefractionation procedures are considered. Conditions that affect stability and chromatographic resolution of cytochromes P450 are also discussed. Special emphasis is put on useful methods which are not routinely used for P450 separation, such as immobilized metal affinity or hydrophobic-interaction chromatography. Applications of low- and high-pressure methods with regard to preparative and analytical separations are compared. It is shown that high- and medium-pressure ion-exchange chromatography are suitable tools for separation of closely related P450 enzymes, especially when specific detection methods are available. In addition to fractionation of cytochromes P450, the isolation and chromatographic behavior of cytochrome b5 is discussed.

Cytochrome P450 proteins and potential utilization in biodegradation.

The cytochrome P450 enzymes are major catalysts involved in the oxidations of xenobiotic chemicals in microorganisms as well as higher animals and plants. Because of their functional roles, they offer potential in biodegradation technology. A number of microbial P450s have already been characterized and offer advantages in terms of their high catalytic rates and facile expression in microorganisms. One approach to extending the catalytic selectivity to more compounds in the environment is rational design. In three cases, the three-dimensional structures of bacterial cytochrome P450 enzymes are available and can be further understood through studies with molecular dynamics. Many mammalian cytochrome P450 enzymes have been studied extensively and have potential for biodegradation because of their broad catalytic selectivities (e.g., P450 2E1). Several advances have been made in the heterologous expression of these proteins in microorganisms. Improvements under development include electron transfer from flavodoxin and the use of cytochrome P450:NADPH-cytochrome P450 reductase fusion proteins. Random mutagenesis offers the potential of improving the catalytic activities of some of these proteins. Future challenges include the use of cytochrome P450 expression vectors in microorganisms capable of thriving in the environment; recent success in expression of vectors in Salmonella genotoxicity tester strains may be encouraging in this regard.

Subcellular localization and characterization of neuronal nitric oxide synthase.

In contrast to the predominantly particulate, Ca2+/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca2+ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca2+/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC50 = 90 microM) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.

Comparison of rainbow trout and mammalian cytochrome P450 enzymes: Evidence for structural similarity between trout P450 LMC5 and human P450111A4

Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450IIIA enzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450IIIA1 in dexamethasone-induced rat liver microsomes and with P450IIIA4 in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450IIIA1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450IIIA4 IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6 beta-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by anti-P450IIIA4 and by gestodene, a mechanism-based inactivator of P450IIIA4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450IIIA4.

Fluconazole: A New Antifungal Agent

The chemistry, activity, pharmacokinetics, clinical efficacy, adverse effects, drug interactions, and administration of fluconazole are reviewed. Fluconazole is a triazole antifungal agent labeled for use in the treatment of oropharyngeal and esophageal candidiasis and cryptococcal meningitis. Like the imidazoles, fluconazole inhibits the C-14 demethylation of lanosterol, but it has little or no effect on mammalian cytochrome P-450 enzymes. Fluconazole's activity in vitro does not reflect its effectiveness in vivo. Pharmacokinetic characteristics include water solubility and excellent bioavailability, low protein binding, extensive tissue distribution, and penetration into the cerebrospinal fluid. Fluconazole is eliminated primarily by the kidneys; dosage adjustments are necessary in patients with renal insufficiency. Studies have shown fluconazole to be effective in patients with cryptococcal meningitis and candidiasis, but the superiority of fluconazole over such agents as amphotericin B, ketoconazole, clotrimazole, and itraconazole remains to be proved. Fluconazole has shown varying degrees of success in the treatment of other systemic mycoses. The adverse effects of fluconazole are relatively infrequent and are primarily gastrointestinal. Tolbutamide, warfarin, rifampin, cyclosporine, and phenytoin interact with fluconazole. The drug is available in both oral and intravenous formulations. Fluconazole is a broad-spectrum antifungal drug that appears to be similar in efficacy to other antifungals in the treatment of some systemic mycoses. More studies must be completed before fluconazole can be recommended as a first-line antifungal therapy.

Toxicological Profile and Safety Evaluation of Antifungal Azole Derivatives

For the development of new systemically acting, oral antifungal azoles, it is of key importance to compare them with ketoconazole, the first available drug in this therapeutic class. Ketoconazole is a major breakthrough although hepatic side-effects as well as interactions with mammalian steroids might rarely occur during prolonged treatment. The prediction of these side-effects is difficult but the potential to interact with mammalian cytochrome P-450 enzymes is considered to be important. Therefore, for the selection of itraconazole a multidisciplinary approach was applied to study this potential. The present paper deals with the toxicological profile of itraconazole and its safety evaluation. In addition, a further comparison with ketoconazole and also with fluconazole is provided, in so far sufficient information is available. For the liver as a potential target organ, the available data indicate that itraconazole is not a predictable hepatotoxic drug in man. The major endocrine targets for overdosing with antifungal azoles are the adrenal cortex and the gonads. Endocrine studies show that itraconazole is not bearing a potential to interfere with steroid hormones in patients, which is a major improvement when compared to ketoconazole. In rats, elevation of serum cholesterol is observed especially after chronic exposure to itraconazole. This species-specific phenomenon leads at toxic dose levels to secondary events, especially in the long-term toxicity studies. In man, including those with existing hypercholesterolemia, serum cholesterol is not adversely affected by itraconazole. In pregnant rats, ketoconazole was shown to be teratogenic at high, toxic doses. The same observation has been made for itraconazole and this also might be true for fluconazole.(ABSTRACT TRUNCATED AT 250 WORDS)