Inhibition Of Cytochrome P450 Enzymes Research Articles

Physiological basis for differential tolerance of tomato and pepper to rimsulfuron and halosulfuron: site of action study

Tomato and pepper differ in their whole-plant tolerance to sulfonylurea (SU) herbicides rimsulfuron and halosulfuron despite both being members of the Solanaceae family. This study examined whether tomato's tolerance to SU herbicides rimsulfuron and halosulfuron was due to insensitivity of the target enzyme acetolactate synthase (ALS). Rimsulfuron and halosulfuron inhibited ALS from both tomato and pepper leaves. Enzyme inhibition and kinetic analyses showed that extractable ALS from tomato was more sensitive to rimsulfuron and halosulfuron than ALS from pepper. ALS from both species were inhibited with a mixed inhibition pattern. Thus, results indicate that enzyme insensitivity is not the reason why tomato is more tolerant than pepper to these herbicides. Tomato tolerance to rimsulfuron at the whole-plant level was reduced in the presence of terbufos, a known inhibitor of cytochrome P450 enzymes. Rimsulfuron applied at 0.018 and 0.035 kg ha−1with 1.1 kg ha−1of terbufos reduced tomato shoot weight 69 and 66%, respectively, compared with a 4 and 29% reduction when rimsulfuron was applied alone. The reduction of tomato tolerance to rimsulfuron by terbufos suggests that the sensitivity differences between these species may reflect their differences in SU herbicide metabolism.

Inhibition of cytochrome P450 enzymes participating in p-nitrophenol hydroxylation by drugs known as CYP2E1 inhibitors

p-Nitrophenol hydroxylation is widely used as a probe for microsomal CYP2E1. Several drugs are known as CYP2E1 inhibitors because of their capability to inhibit p-nitrophenol hydroxylation. Our results suggest further participation of CYP2A6 and CYP2C19 enzymes in p-nitrophenol hydroxylation. Moreover, CYP2A6 and CYP2C19 may be considered as the primary catalysts, whereas CYP2E1 can also contribute to the hydroxylation of p-nitrophenol. Further aim of our study was to evaluate the selectivity of p-nitrophenol hydroxylase inhibitors towards cytochrome P450 enzymes. The effects of antifungals: bifonazole, econazole, clotrimazole, ketoconazole, miconazole; CNS-active drugs: chlorpromazine, desipramine, fluphenazine, thioridazine; and the non-steroidal anti-inflammatory drug: diclofenac were investigated on the enzyme activities selective for CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. None of the drugs could be considered as a potent inhibitor of CYP2E1. Strong inhibition was observed for CYP3A4 by antifungals with IC 50 values in submicromolar range. However, ketoconazole was the only imidazole derivative that could be considered as a selective inhibitor of CYP3A4. The CNS-active drugs investigated were found to be weak inhibitors of CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. Diclofenac efficiently inhibited CYP2C9 and to a less extent CYP3A4 enzyme.

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC 50 of 5.9 μM and K i of 0.6 μM. In murine BV-2 microglial cells, an immortalized cell line that produces NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.

Cytochrome P-450 inhibition attenuates hypertension induced by reductions in uterine perfusion pressure in pregnant rats.

The present study tested the hypothesis that cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) are involved in mediating hypertension and renal vasoconstriction during chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats. 1-aminobenzotriazole (ABT), a CYP enzyme inhibitor (25 mg/kg per day), or vehicle (saline 0.9%) was administered for 7 days to normal pregnant (NP) rats and to pregnant rats with chronic RUPP. RUPP rats infused with vehicle showed significantly (P<0.01) higher mean arterial pressure (MAP) (130+/-2 versus 106+/-1 mm Hg), renal vascular resistance (RVR) (22.6+/-1.8 versus 16.3+/-1.1 mm Hg/mL per minute) and lower (P<0.05) glomerular filtration rate (GFR) (1.6+/-0.1 versus 2.3+/-0.1 mL/min) than NP rats. ABT decreased (P<0.01) MAP in RUPP rats (111+/-1 mm Hg), whereas it had no effect in NP rats (108+/-2 mm Hg). CYP inhibition also attenuated the differences in renal hemodynamics observed between NP and RUPP rats. After treatment with ABT, RVR and GFR were similar in RUPP rats (19.3+/-1.5 mm Hg/mL per minute and 2.0+/-0.2 mL/min, respectively) and NP rats (16.3+/-2.4 mm Hg/mL per minute and 2.4+/-0.2 mL/min). The effects of CYP enzymes inhibitor in RUPP rats were associated with a reduction (P<0.05) of 20-HETE formation (32%) and a decreased (P<0.05) expression (33%) of CYP4A protein in renal cortex. In contrast, renal epoxygenase activity did not change in these animals. These results suggest that 20-HETE contributes to hypertension and renal vasoconstriction induced by chronic RUPP in pregnant rats.

Toxaphene detoxification and acclimation in Daphnia magna: do cytochrome P-450 enzymes play a role?

Toxaphene is a persistent environmental contaminant that has been shown to alter male production in Daphnia magna and to induce P-450 activity in mammals. Cytochrome P-450-mediated metabolism may lead to xenobiotic detoxification resulting in acclimation. To determine if D. magna acclimate to toxaphene via P-450 pathways, chronic and acute toxicity tests were conducted with D. magna exposed to toxaphene in the presence and absence of piperonyl butoxide (PBO), an inhibitor of cytochrome P-450 enzymes. Toxaphene exposure increased male production in acute but not chronic assays, indicating that D. magna may acclimate to chronic toxaphene exposure. Upon co-administration of toxaphene and PBO in chronic tests, D. magna exhibited a decline in growth rate, fecundity and survival. The observed toxaphene acclimation in chronic tests, along with its increased toxicity in the presence of a P-450 suppressor, suggests that P-450 enzymes may contribute to detoxification and subsequent acclimation of D. magna to chronic toxaphene exposure. Additional chronic toxicity tests indicated that toxaphene acclimation occurs between 7 and 12 days following initial exposure, at which time sex determination is no longer affected. Thus, sublethal toxaphene toxicity effects such as reproductive impairments may be detectable with acute but not chronic tests, potentially due to the upregulation of P-450 isozymes.

Herbal Interactions Involving Cytochrome P450 Enzymes

The metabolism of a drug can be altered by another drug or foreign chemical, and such interactions can often be clinically significant. Cytochrome P450 (CYP) enzymes, a superfamily of enzymes found mainly in the liver, are involved in the metabolism of a plethora of xenobiotics and have been shown to be involved in numerous interactions between drugs and food, herbs and other drugs. The observed induction and inhibition of CYP enzymes by natural products in the presence of a prescribed drug has (among other reasons) led to the general acceptance that natural therapies can have adverse effects, contrary to the popular beliefs in countries where there is an active practice of ethnomedicine. Herbal medicines such as St. John's wort, garlic, piperine, ginseng, and gingko, which are freely available over the counter, have given rise to serious clinical interactions when co-administered with prescription medicines. Such adversities have spurred various pre-clinical and in vitro investigations on a series of other herbal remedies, with their clinical relevance remaining to be established. Although the presence of numerous active ingredients in herbal medicines, foods and dietary supplements complicate experimentation, the observable interactions with CYP enzymes warrant systematic studies, so that metabolism-based interactions can be predicted and avoided more readily. This article highlights the involvement of CYP enzymes in metabolism-related drug-herb interactions and the importance of gaining a mechanism-based understanding to avoid potential adverse drug reactions, in addition to outlining other contributory factors, such as pharmacogenetics and recreational habits that may compound this important health issue.

Tenofovir disoproxil fumarate: clinical pharmacology and pharmacokinetics.

Tenofovir disoproxil fumarate (tenofovir DF) is an oral prodrug of tenofovir, a nucleotide (nucleoside monophosphate) analogue with activity against retroviruses, including HIV-1, HIV-2 and hepadnaviruses. Following absorption, tenofovir DF is rapidly converted to tenofovir, which is metabolised intracellularly to its active anabolite tenofovir diphosphate, which is a competitive inhibitor of HIV-1 reverse transcriptase and terminates the growing DNA chain. Tenofovir exerts antiviral effects in a variety of cell types, including resting cells. Tenofovir exhibits longer serum (17 hours) and intracellular (> or =60 hours) half-lives than those of nucleoside analogues, which supports a flexible once-daily administration schedule. The pharmacokinetics of tenofovir are dose-proportional and similar in healthy volunteers and HIV-infected individuals. The oral bioavailability of tenofovir is enhanced by administration with a high-fat meal, but is similar at steady state when administered with or without a typical meal. Tenofovir is not a substrate, inducer or inhibitor of human cytochrome P450 enzymes in vitro or in vivo. Tenofovir DF has been studied with 15 other antiretroviral and other concomitant medications frequently used in the HIV-1-infected population. With the exception of didanosine and atazanavir, which require dosage modifications, no clinically significant drug interactions have been observed with tenofovir DF. The recommended oral dosage of tenofovir DF in adults is 300 mg/day. Tenofovir is eliminated by renal elimination, including tubular secretion; dose-interval adjustments are necessary for tenofovir DF in patients with significant renal impairment. No dosage adjustment of tenofovir DF is necessary in patients with liver disease.

Pharmacokinetic enhancement of protease inhibitor therapy.

Combination antiretroviral therapy with two or more protease inhibitors has become the standard of care in the treatment of HIV infection. Dual protein inhibitor (PI) regimens, such as lopinavir/ritonavir, are commonly used as initial PI therapy. As viral resistance increases and the development of mechanistically novel protease inhibitors decreases, clinicians turn to ritonavir-enhanced dual PI therapy to treat salvage patients. Potency of these combination regimens is increased while pill burden, food restrictions and often, side effects are decreased. These clinical advantages result from the enhancement of their pharmacological properties, including alterations in the absorption and metabolism process. Alterations in the absorption and metabolism of protease inhibitors when co-administered with a cytochrome P450 (CYP) enzyme inhibitor, such as low dose ritonavir, are reflected by impressive changes in pharmacokinetic parameters. For example, the addition of ritonavir 100 or 200 mg to saquinavir 1200-1800 mg has been shown to increase saquinavir area under the concentration-time curve (AUC) by approximately 300-800% compared with saquinavir alone. The ability of ritonavir to increase plasma trough concentrations (C(min)) of concomitantly administered PIs is perhaps the greatest clinical benefit of dual or ritonavir-enhanced dual PI therapy since inadequate concentrations of antiretrovirals may support long term antiretroviral resistance. For example, lopinavir 400mg alone in healthy volunteers produced plasma concentrations that briefly exceeded the concentration required to inhibit 50% of viral replication (IC(50)). Yet, when low doses of ritonavir were added, C(min) values were 50- to 100-fold greater than the concentration required to produce 50% of the maximum effect for wild-type HIV (EC(50)). The following manuscript will discuss the rationale for combining protease inhibitors and will review pertinent pharmacokinetic and clinical data on these combination regimens.

QT prolongation with antimicrobial agents: understanding the significance.

Cardiac toxicity has been relatively uncommon within the antimicrobial class of drugs, but well described for antiarrhythmic agents and certain antihistamines. Macrolides, pentamidine and certain antimalarials were traditionally known to cause QT-interval prolongation, and now azole antifungals, fluoroquinolones and ketolides can be added to the list. Over time, advances in preclinical testing methods for QT-interval prolongation and a better understanding of its sequelae, most notably torsades de pointes (TdP), have occurred. This, combined with the fact that five drugs have been removed from the market over the last several years, in part because of QT-interval prolongation-related toxicity, has elevated the urgency surrounding early detection and characterisation methods for evaluating non-antiarrhythmic drug classes. With technological advances and accumulating literature regarding QT prolongation, it is currently difficult or overwhelming for the practising clinician to interpret these data for purposes of formulary review or for individual patient treatment decisions. Certain patients are susceptible to the effects of QT-prolonging drugs. For example, co-variates such as gender, age, electrolyte derangements, structural heart disease, end organ impairment and, perhaps most important, genetic predisposition, underlie most if not all cases of TdP. Between and within classes of drugs there are important differences that contribute to delayed repolarisation (e.g. intrinsic potency to inhibit certain cardiac ion currents or channels, and pharmacokinetics). To this end, a risk stratification scheme may be useful to rank and compare the potential for cardiotoxicity of each drug. It appears that in most published cases of antimicrobial-associated TdP, multiple risk factors are present. Macrolides in general are associated with a greater potential than other antimicrobials for causing TdP from both a pharmacodynamic and pharmacokinetic perspective. The azole antifungal agents also can be viewed as drugs that must be weighed carefully before use since they also have both pharmacodynamic and pharmacokinetic characteristics that may trigger TdP. The fluoroquinolones appear less likely to be associated with TdP from a pharmacokinetic perspective since they do not rely on cytochrome P450 (CYP) metabolism nor do they inhibit CYP enzyme isoforms, with the exception of grepafloxacin and ciprofloxacin. Nonetheless, patient selection must be carefully made for all of these drugs. For clinicians, certain responsibilities are assumed when prescribing antimicrobial therapy: (i) appropriate use to minimise resistance; and (ii) appropriate patient and drug selection to minimise adverse event potential. Incorporating information learned regarding QT interval-related adverse effects into the drug selection process may serve to minimise collateral iatrogenic toxicity.

Toxicant-induced hypospadias in the male rat.

Prenatal exposure to endocrine disrupting chemicals that interfere with the androgen or insulin like factor 3 signaling pathways during sexual differentiation can induce malformations of the reproductive tract of the male rodent offspring. The pattern of malformations in the male depends upon the specific mechanism of action of the toxicant, the dosage level administered and the timing of administration during pregnancy. Hypospadias occurs in male rats or mice after maternal treatment with 1). potent estrogens or estrogenic drugs, 2). drugs that inhibit 5 alpha reductase, 3). drugs, herbicides and dicarboximide and conazole fungicides that act as androgen receptor (AR) antagonists, and 4). drugs, herbicides and conazole fungicides that inhibit cytochrome P450 enzymes involved in steroid hormone synthesis. In addition, 5). several phthalate diesters including di-n-butyl phthalate (DBP), di-n-ethylhexyl phthalate (DEHP), and benzylbutyl phthalate(BBP) also induce hypospadias and by altering fetal testis Leydig cell differentiation, resulting in reduced steroid and peptide hormone production.

An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies.

Herbals and other natural remedies could affect the disposition of conventional pharmaceuticals through inhibition of human cytochrome P-450 (CYP) enzymes. We have developed an approach to the problem of determining a critical potential for CYP enzyme inhibition by commercial herbal remedies etcetera using standardised extraction procedures in combination with commercially available human CYP enzyme 3A4, 2D6 and 2C19 inhibition assays. We present a survey of commercially available natural remedies on the local market using this approach together with a discussion on how to decide when further in vivo interaction studies may be warranted. We confirmed earlier findings on Hypericum (St. John's wort) and Echinacea purpurea activities, and report that extracts of Valeriana as well as a fish oil preparation were potent inhibitors of all tested enzymes. As a first estimate to assess the relevance of in vitro findings, we have chosen to express the inhibitory potency as the volume that the extractable inhibitory activity for a recommended human dose could be diluted into to yield a half-maximal inhibition--litres per dose unit. We propose that preparations for which this measure approaches four litres/dose unit, i.e. corresponding to the human blood volume, should be investigated further for potential enzyme interaction with pharmaceuticals.

Atazanavir: profile report

Current recommendations for first-line therapy in patients with HIV infection include combinations of a sole (or boosted) protease inhibitor together with a dual nucleoside reverse transcriptase inhibitor backbone among the options for highly active antiretroviral therapy.[1] Such triple therapy, while producing durable suppression of viral replication (a measure of a regimen’s success is achieving a viral load of <50 HIV RNA copies/mL within 6–9 months of starting treatment), has been associated with the emergence of metabolic, tolerability, adherence, resistance and drug interaction concerns, particularly with protease inhibitor-based regimens.[4,5] Atazanavir (ReyatazTM1) is a new azapeptide protease inhibitor with a high specificity for, and activity against, HIV-1 protease.[6] The resistance profile of atazanavir is distinct, with an I50L protease substitution appearing to be the signature mutation.[1,7] Atazanavir has a pharmacokinetic profile that allows for once-daily oral administration.[1] It is a moderate inhibitor of hepatic cytochrome P450 enzymes and interacts with several drugs. Atazanavir was not associated with increases in total cholesterol, low density lipoprotein-cholesterol or triglyceride levels after 108 weeks.[4,8-11] In combination with stavudine plus didanosine, atazanavir 200, 400 or 500mg once daily produced a rapid and sustained reduction from baseline in viral load of 2.57, 2.42 and 2.53 log10 copies/mL, respectively, in treatment-naive patients after 48 weeks, compared with a decrease of 2.33 log10 copies/mL with nelfinavir 750mg three times daily.[4] Nausea was the most clinically relevant adverse event reported in patients receiving atazanavir-based regimens in clinical trials.[1,4]

Antiandrogenic activities of diesel exhaust particle extracts in PC3/AR human prostate carcinoma cells.

We collected diesel exhaust particles (DEPs) emitted from three diesel-engine vehicles--a car, a bus, and a truck--in daily use, and prepared DEP extracts (DEPEs), designated as EC, EB, or ET, respectively. The androgenic and antiandrogenic effects of the DEPE samples were examined by a luciferase reporter assay in human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen gene promoter-driven luciferase expression vector pGLPSA5.8. PC3/AR is a subline of human prostate carcinoma PC3 transformed to stably express wild-type human androgen receptor (AR). While DEPE samples did not exhibit any androgenic effect, they exerted antiandrogenic effect, inhibiting dihydrotestosterone (10 pM) -induced luciferase activity by 24 to 52% at an extract concentration of 10 microg/ml. The antiandrogenic effect was greater in the following order: ET > EB > EC. Co-treatment of PC3/AR cells with SKF-525A, a nonselective inhibitor of cytochrome P450 (CYP) enzymes, enhanced the antiandrogenic effect, indicating that the antiandrogenic effect is caused by intact species of DEPE constituents. The antiandrogenic effect of DEPE samples was reversed by alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist. The antiandrogenic activity of a DEPE sample correlated with its AhR agonist activity assayed in PC3/AR cells transiently transfected with CYP1A1 gene promoter-driven luciferase expression vector pLUC1A1. Equimolar mixtures of ten polycyclic aromatic hydrocarbons (PAHs) having four or more rings, structures found in the DEPEs, showed significant antiandrogenic effects and AhR agonist activity at concentrations equivalent to those found in DEPE samples. Further, DEPE samples elicited only antiandrogenic effects in recombinant yeast cells, which express beta-galactosidase in response to androgen. A competitive AR binding assay showed that AR-binding constituents exist in DEPE samples, indicating that greater part of AR-binding constituents in DEPEs are AR antagonists. All these findings show that DEPE samples exhibit significant antiandrogenic effect in cell-based transcription assay and that this effect is due in part to the constituents with AhR agonist activity including PAHs and to the constituents with AR antagonist activity.

Possibility of the involvement of 9H-pyrido[3,4-b]indole (norharman) in carcinogenesis via inhibition of cytochrome P450-related activities and intercalation to DNA

This study investigated the inhibitory effect of 9H-pyrido[3,4-b]indole (norharman), one of the naturally occurring β-carbolines, on cytochrome P450 (CYP)-related activities and the relationship between its inhibitory effect, its intercalation to DNA, and its comutagenic effect. Norharman reduced the mutagenicities of heterocyclic amines (HCAs) containing 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1), aflatoxin B 1, benzo[a]pyrene (BP), and some nitrosamines in the presence of 10 μl liver S9 (20.9 μg protein/ml) from polychlorinated biphenyl-treated rats. Norharman inhibited microsomal CYP-related enzyme activities and CO-binding to the CYP heme (50% inhibitory concentration (IC50), 0.07–6.4 μg/ml). It also inhibited the formation of 3-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (N-OH-Glu-P-1) and was a noncompetitive-inhibitor of CYP1A-related activities, while it enhanced the direct mutagenicity of N-OH-Glu-P-1 (50% effective concentration, 25.0 μg/ml) and inhibited topo I activity (IC50, 31.0 μg/ml). In the presence of norharman, S9 up to 100 μl incrementally enhanced the mutagenicities of HCAs, BP and dimethylnitrosamine. These data clarified that norharman acts as an inhibitor of the CYP-mediated biotransformation of Glu-P-1 via inhibition of O 2-binding to CYP heme, and its inhibition of CYP enzymes occurs at much lower concentration than that for its intercalation to DNA. It is indicated that norharman’s inhibitory effect on CYP results in the inhibition of excess metabolism by S9 and this is more likely the mechanism for comutagenic action than the intercalation. Norharman’s inhibition of CYP and its enhancement of the N-OH-Glu-P-1 mutagenicity suggest that β-carbolines modulate chemical carcinogenesis by controlling the xenobiotic metabolism and by intercalating to DNA.

Contribution of cytochrome P450 4A isoforms to renal functional response to inhibition of nitric oxide production in the rat

Many studies have clearly indicated that products of the ω/ω-1 hydroxylase pathway of cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolism are synthesized in the kidney and exert profound effects therein (McGiff & Quilley, 1999). The major ω-hydroxylation product of AA in tubular and vascular structures of the renal cortex and outer medulla of the rat is 20-hydroxyeicosatetraenoic acid (20-HETE) (Omata et al. 1992a 1992b; Imig et al. 1996), an important regulator of renal vascular tone, tubular reabsorption and the control of arterial pressure (see McGiff & Quilley, 1999). ω-Hydroxylation of fatty acids, including AA has been characterized and shown to be catalysed by enzymes of the CYP4A family. In the rat, four isoforms have been identified: CYP4A1, −4A2, −4A3 and −4A8, and mRNA for all four have been identified in the kidney (Kimura et al. 1989a 1989b; Stromstedt et al. 1990). These isoforms, although sharing 66–98 % homology and a common unique catalytic activity, i.e. hydroxylation at the ω-carbon, are localized to different renal structures. For example, CYP4A1, −4A3, and −4A8 are highly expressed in proximal tubules (Stromstedt et al. 1990; Hardwick, 1991; Omata et al. 1992a 1992b) and the renal microvasculature (Wang et al. 1999). On the other hand, CYP4A2, the constitutively expressed isoform especially in male rats (Kimura et al. 1989a 1989b; Sundseth & Waxman, 1993), is preferentially expressed in the outer medulla and thick ascending limb of the loop of Henle and is believed to be the major isoform in the kidney (Kimura et al. 1989a 1989b; Sundseth & Waxman, 1993). The differential expression of the AA ω-hydroxylating enzymes along the nephron may therefore contribute to the selective effects of 20-HETE on tubular function. The regulation of CYP4A is the subject of active interest and hypolipidaemic agents have been shown to selectively increase the expression of CYP4A1 and −4A3 isoforms (Hardwick, 1991). A potential exists also for a differential regulation of renal CYP4A by nitric oxide (NO) but this has not been actively explored. It has been demonstrated that NO inhibits CYP enzymes including the 4A family (Oyekan et al. 1999) by forming stable iron-nitrosyl complexes at the catalytic haeme binding site in this enzyme (Minamiyama et al. 1997; Mehl et al. 1999). This inhibition is corroborated by the observations that NO donors inhibit the synthesis of 20-HETE by renal microsomes (Alonso-Galicia et al. 1997; Oyekan et al. 1999) and that inhibition of NO production increased CYP4A expression and renal efflux of 20-HETE in the perfused rat kidney (Oyekan et al. 1999) and in the isolated proximal tubule of the normal rat (Escalante et al. 2002) and in the renal microvessels of the pregnant rat (Wang et al. 2002). In addition, incubation of recombinant CYP4A protein with NO donors revealed a differential formation of iron-nitrosyl complexes between different CYP4A isoforms (Wang et al. 2002). Since the capacity for 20-HETE production and therefore its renal effect are determined by the expression of specific CYP4A isoforms, for which expression differs between vascular and tubular sites in the kidney, we therefore hypothesize that the renal effect of NO inhibition (to increase CYP4A expression) will depend on the extent of NO regulation of specific CYP4A isoform(s). The availability of antisensense technology in the form of molecular probes has facilitated a definition of the functional role of each of the isoforms of the CYP4A family, permitting recognition of their separate and overlapping spheres of activity and, therefore, of the physiological significance of each isoform. Antisense technology has been used in other studies to demonstrate the roles of CYP4A1 versus -4A2 isoforms in the regulation of blood pressure in normotensive and spontaneously hypertensive rats (Wang et al. 1999, 2001). In the present study, we evaluated changes in renal haemodynamics and excretory function in rats that were treated with antisense oligonucleotides directed against CYP4A1, -A2 and -A3.

Atazanavir.

Atazanavir is a novel azapeptide protease inhibitor with high specificity for, and activity against, HIV-1 protease. The resistance profile of atazanavir is distinct, with an I50 L protease substitution appearing to be the signature mutation. Atazanavir was not associated with increases in total cholesterol, low density lipoprotein-cholesterol or triglyceride levels after 108 weeks. Atazanavir has a pharmacokinetic profile that allows for once-daily oral administration. It is a moderate inhibitor of hepatic cytochrome P450 enzymes and interacts with several drugs. In combination with stavudine plus didanosine, atazanavir 200, 400 or 500 mg once daily produced a rapid and sustained reduction from baseline in viral load of 2.57, 2.42 and 2.53 log(10) copies/mL, respectively, in treatment-naive patients after 48 weeks, compared with a decrease of 2.33 log(10) copies/mL with nelfinavir 750 mg three times daily. Nausea was the most clinically relevant adverse event reported in patients receiving atazanavir-based regimens.

Arachidonic acid-induced vasodilation of rat small mesenteric arteries is lipoxygenase-dependent.

We examined the mechanism of arachidonic acid-induced vasodilation in rat small mesenteric arteries and determined the primary arachidonic acid metabolites produced by these arteries. Responses to arachidonic acid in small mesenteric arteries from Sprague-Dawley rats were investigated in vitro in the presence or absence of endothelium or after pretreatment with inhibitors of nitric oxide (NO), cyclooxygenase, cytochrome P450, lipoxygenase, or K+ channels. In addition, the metabolism of arachidonic acid was examined by incubating arteries with [3H]arachidonic acid in the presence and absence of cyclooxygenase, cytochrome P450, or lipoxygenase inhibitors. Finally, the vascular response to both 12(S)-hydroxyeicosatetraenoic acid (HETE) and 12(S)-hydroperoxyeicosatetraenoic acid (HPETE) was determined. Arachidonic acid induced an endothelium-dependent vasodilation that was abolished by lipoxygenase inhibitors [cin-namyl-3,4-dihydroxy-cyanocinnamate (CDC) or 5,8,11-eicosatriynoic acid (ETI)] and KCl, whereas it was partially inhibited by either tetraethylammonium or iberiotoxin. In contrast, neither NO nor cytochrome P450 enzyme inhibitors affected arachidonic acid-mediated dilation, whereas inhibition of cyclooxygenase enhanced dilation. Biochemical analysis revealed that small mesenteric arteries primarily produce 12-HETE, a lipoxygenase metabolite. Moreover, CDC and ETI inhibited the production of 12-HETE. Finally, both 12(S)-HETE and 12(S)-HPETE induced a concentration-dependent vasodilation in mesenteric arteries. These findings provide functional and biochemical evidence that the lipoxygenase pathway mediates arachidonic acid-induced vasodilation in rat small mesenteric arteries through a K+ channel-dependent mechanism.

Rosuvastatin: a highly effective new HMG-CoA reductase inhibitor.

Rosuvastatin, a new statin, has been shown to possess a number of advantageous pharmacological properties, including enhanced HMG-CoA reductase binding characteristics, relative hydrophilicity, and selective uptake into/activity in hepatic cells. Cytochrome p450 (CYP) metabolism of rosuvastatin appears to be minimal and is principally mediated by the 2C9 enzyme, with little involvement of 3A4; this finding is consistent with the absence of clinically significant pharmacokinetic drug-drug interactions between rosuvastatin and other drugs known to inhibit CYP enzymes. Dose-ranging studies in hypercholesterolemic patients demonstrated dose-dependent effects in reducing low-density lipoprotein cholesterol (LDL-C) (up to 63%), total cholesterol, and apolipoprotein (apo) B across a 1- to 40-mg dose range and a significant 8.4% additional reduction in LDL-C, compared with atorvastatin, across the dose ranges of the two agents. Rosuvastatin has also been shown to be highly effective in reducing LDL-C, increasing high-density lipoprotein cholesterol (HDL-C), and producing favorable modifications of other elements of the atherogenic lipid profile in a wide range of dyslipidemic patients. In patients with mild to moderate hypercholesterolemia, rosuvastatin has been shown to produce large decreases in LDL-C at starting doses, thus reducing the need for subsequent dose titration, and to allow greater percentages of patients to attain lipid goals, compared with available statins. The substantial LDL-C reductions and improvements in other lipid measures with rosuvastatin treatment should facilitate achievement of lipid goals and reduce the requirement for combination therapy in patients with severe hypercholesterolemia. In addition, rosuvastatin's effects in reducing triglycerides, triglyceride-containing lipoproteins, non-HDL-C, and LDL-C and increasing HDL-C in patients with mixed dyslipidemia or elevated triglycerides should be of considerable value in enabling achievement of LDL-C and non-HDL-C goals in the numerous patients with combined dyslipidemias or metabolic syndrome who require lipid-lowering therapy. Rosuvastatin is well tolerated alone, and in combination with fenofibrate, extended-release niacin, and cholestyramine, and has a safety profile similar to that of currently marketed statins. A large, long-term clinical trials program is under way to investigate the effects of rosuvastatin on atherosclerosis and cardiovascular morbidity and mortality.

Cushing's syndrome due to pharmacological interaction in a cystic fibrosis patient

Treatment of allergic bronchopulmonary aspergillosis with itraconazole is becoming more widespread in chronic lung diseases. A considerable number of patients is concomitantly treated with topical or systemic glucocorticoids for anti‐inflammatory effect. As azole compounds inhibit cytochrome P450 enzymes such as CYP3A isoforms, they may compromise the metabolic clearance of glucocorticoids, thereby causing serious adverse effects. A patient with cystic fibrosis is reported who developed iatrogenic Cushing's syndrome after long‐term treatment with daily doses of 800 mg itraconazole and 1600 μg budesonide. The patient experienced symptoms of striae, moon‐face, increased facial hair growth, mood swings, headaches, weight gain, irregular menstruation despite oral contraceptives and increasing insulin requirement for diabetes mellitus. Endocrine investigations revealed total suppression of spontaneous and stimulated plasma cortisol and adrenocorticotropin. Discontinuation of both drugs led to an improvement in clinical symptoms and recovery of the pituitary‐adrenal axis after 3 mo. Conclusion: This observation suggests that the metabolic clearance of budesonide was compromised by itraconazole's inhibition of cytochrome P450 enzymes, especially the CYP3A isoforms, causing an elevation in systemic budesonide concentration. This provoked a complete suppression of the endogenous adrenal function, as well as iatrogenic Cushing's syndrome. Patients on combination therapy of itraconazole and budesonide inhalation should be monitored regularly for adrenal insufficiency. This may be the first indicator of increased systemic exogenous steroid concentration, before clinical signs of Cushing's syndrome emerge.

Inhibition of cytochrome P450 enzymes by enrofloxacin in the sea bass ( Dicentrarchus labrax)

Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome P450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic P450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6β-testosterone hydroxylase. In vitro experiments showed that EF at 10 μM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6β-testosterone-hydroxylase, likely dependant on a P450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K i of 3.7 μM and a K inact of 0.045 min −1. An immunoblot analysis with anti P450 3A27 of trout showed that the P450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the P450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the P450 enzymes. The mechanism by which EF or CF inactivate the P450 enzymes has not been studied but an attack of P450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of P450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.