Role of BK Channels in Murine Carotid Body Neural Responses in vivo.

The aim of this study was to explore the role of BK channels in the hypoxic sensitivity of the in vivo murine carotid body (CB). Four strains of mice (DBA/2J, A/J, BKα1 knockout and BKα1 wild type - FVB background) were used. The mice were anesthetized, paralyzed and mechanically ventilated (PaCO(2) ~ 35 mmHg, PO(2) > 300 mmHg). We measured carotid sinus nerve (CSN) activity during three gas challenges (F(I)O(2): 0.21, 0.15 and 0.10). CSN activity was analyzed with time-variant spectral analysis with frequency domain conversion (Fast Fourier Transforms). Afferent CSN activity increased with lowering F(I)O(2) in the DBA/2J, BKKO and BKWT mice with the most robust response in 600-800 frequencies. No substantial changes were observed in the A/J mice. Although maximal neural output was similar between the BKKO and BKWT mice, the BKWT had a higher early response compared to BKKO. Thus, BK channels may play a role in the initial response of the CB to hypoxia. The contribution of BKß subunits or the importance of frequency specific responses was unable to be determined by the current study.

Inspiratory duty cycle responses to flow limitation predict nocturnal hypoventilation.

Upper airway obstruction (UAO) can elicit neuromuscular responses that mitigate and/or compensate for the obstruction. It was hypothesised that flow-limited breathing elicits specific timing responses that can preserve ventilation due to increases in inspiratory duty cycle rather than respiratory rate. By altering nasal pressure during non-rapid eye movement (non-REM) sleep, similar degrees of UAO were induced in healthy males and females (n = 10 each). Inspiratory duty cycle, respiratory rate and minute ventilation were determined for each degree of UAO during non-REM sleep and compared with the baseline nonflow-limited condition. A dose-dependent increase in the inspiratory duty cycle and respiratory rate was observed in response to increasing severity of UAO. Increases in the inspiratory duty cycle, but not respiratory rate, helped to acutely maintain ventilation. Heterogeneity in these responses was associated with variable degrees of ventilatory compensation, allowing for the segregation of individuals at risk for hypoventilation during periods of inspiratory airflow limitation. Upper airway obstruction constitutes a unique load on the respiratory system. The inspiratory duty cycle, but not the respiratory rate, determine the individual's ability to compensate for inspiratory airflow limitation during sleep, and may represent a quantitative phenotype for obstructive sleep apnoea susceptibility.

Women have a greater ventilatory responses to upper airway obstruction than men.

We examined whether gender specific differences exist in defending inspiratory tidal volumes in the face of upper airway obstruction. In normal weight- and aged-matched men (n=9) and women (n=9), we induced upper airway obstruction with inspiratory flow limitation during NREM sleep by exposing individuals to sub-atmospheric nasal pressure. The mean inspiratory airflow was used to define three distinct levels of upper airway obstruction, based on a mean inspiratory airflow of 175-225 ml/s, 125-175 ml/s and 75-125 ml/s. While duty cycle responses were similar between genders, women had a greater response in T(TOT) at all flow limited conditions. (p<0.05). However, the greater response in T(TOT) led to a more pronounced decline in tidal volume in women compared to men (p<0.05), particularly during the mild and moderate upper airway obstruction. Our data demonstrate that the respiratory rate determines the tidal volume during periods of upper airway obstruction and indicate that individuals with a higher respiratory rate are at risk to develop hypoventilation in face of upper airway obstruction. Because women have a more brisk response in the respiratory rate than men, this may explain the difference in the expression of sleep disordered breathing between genders.

Metabolism and CYP-inducer properties of astaxanthin in man and primary human hepatocytes.

Previous investigations in the rat have shown that the non-provitamin A carotenoid astaxanthin is metabolized into 3-hydroxy-4-oxo-beta-ionone and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone and, in addition, is a potent CYP1A gene inducer. Here we investigated the metabolism of this compound as well as its capacity to induce CYP genes in primary cultures of human hepatocytes. Free metabolites of 14C-astaxanthin produced in this cellular model were purified by high pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) analyses as 3-hydroxy-4-oxo-beta-ionol and 3-hydroxy-4-oxo-beta-ionone. In addition, deconjugation of polar compounds by glusulase and further analyses with HPLC and GC-MS revealed four radiolabeled metabolites including: 3-hydroxy-4-oxo-beta-ionol, 3-hydroxy-4-oxo-beta-ionone, and their reduced forms, 3-hydroxy-4-oxo-7, 8-dihydro-beta-ionol and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone. The same four metabolites were identified in human plasma from two volunteers who had orally taken 100 mg astaxanthin 24 h before blood collection. In cultured hepatocytes, astaxanthin was a significant inducer of the major cytochrome P450 enzyme, CYP3A4 as well as of CYP2B6, but not of other CYPs, including those from CYP1A and CYP2C families. The lack of autoinduction of astaxanthin metabolism in human hepatocytes suggests that neither CYP3A4 nor CYP2B6 contribute to the formation of metabolites. We conclude that metabolism of astaxanthin and its CYP-inducing capacity are different in humans and in rats. The novel methodology used in our studies could be extended to evaluating the role of metabolites of more important carotenoids such as beta-carotene in differentiation and carcinogenicity.

Long-term primary cultures of adult human hepatocytes.

In this work we have investigated a system of long-term primary cultures of adult human hepatocytes which, in contrast to those previously described, has the advantage of requiring neither the use of additive cells as in co-cultures, nor of matrix component preparations like Matrigel or collagen sandwich. This system has been used previously for long-term cultures of hepatocytes from young baboon, and some modifications have been introduced here to take into account the specificity of adult human hepatocytes. In this system, hepatocytes are plated at confluence on collagen-coated dishes and cultured in a serum-free medium consisting of Williams'E supplemented with hormones and growth factors. Proteins secreted specifically by the liver, including albumin, alpha-1 antitrypsin, plasminogen, fibrinogen, lipoproteins ApoA1 and ApoB100, have been quantified in the extracellular medium as a function of time, either by immunoblot or ELISA. In addition, the expression and inducibility of CYP proteins of the CYP1, CYP2 and CYP3 families in response to their prototypical inducers including 2,3,7,8-tetrachlorodibenzo(p)dioxin and rifampicin, have been evaluated by immunoblot analysis of microsomes or cell lysates. Moreover, the oxidative metabolism of cyclosporin A, a monoxygenase activity depending on CYP3A4, has been monitored directly on the cultured cells by HPLC analysis of extracellular medium. Our results show that, under these culture conditions, adult human hepatocytes retain these phenotypical characteristics for at least 35 days. This system meets the requirements for use as a model for screening CYP protein inducers.

Non-reflex mediated changes in plantarflexor muscles early after stroke.

The aims of this study were to determine whether changes in the non-reflex component of spastic plantarflexors had developed 2 and 4 months after stroke and to study their relationship with the level of impairment. One group of adults with hemiparesis (HPs) was tested 2 and 4 months after the onset of stroke, and data were compared with a control group (CTLs) tested once. Twenty-two patients (14 males) admitted over a 4-month period in a rehabilitation centre (mean = 62 yrs +/- 14), and 11 (6 males) non-disabled (CTLs) subjects (mean = 57yrs +/- 12.8) agreed to participate in the study. The resistive torque (RT) recorded with a myometer during slow (8-10 degrees/s) passive dorsiflexions imposed manually served as the primary outcome, whereas, the Ashworth score (spasticity), ankle ROM and Fugl-Meyer motor subscore were used as secondary measures to determine the level of impairment. The mean RT values measured at 0 degrees dorsiflexion on the affected and unaffected sides were compared with those in CTLs. As expected, the RT values 2 and 4 months post-stroke on the unaffected side did not differ from corresponding values in CTLs. Significantly higher RT values on the affected side when compared to the unaffected side were found both at 2 months (39%; p < 0.05) and at 4 months (43%; p < 0.01). No significant difference existed on the affected side between the 2nd and 4th months. A high (r = 0.80) and significant (p < 0.0001) correlation coefficient was calculated between the changes in RT values recorded at 2 and 4 months. Low and not significant correlations were computed between these RT changes and factors such as the ROM (r = -0.24), the Ashworth score (r = 0.23) and the Fugl-Meyer lower extremity motor subscore (r = -0.26). Present results indicate that: (1) changes in the non-reflex component are already present 2 months after stroke but do not increase significantly between the 2nd and 4th months; (2) these changes are not related to the level of impairment; and (3) myometry testing at 2 months could be used as a preventive measure to detect patients more at risk of developing severe passive muscle stiffness.

Effect of cell density and epidermal growth factor on the inducible expression of CYP3A and CYP1A genes in human hepatocytes in primary culture.

The influence of cell density and epidermal growth factor (EGF) on the expression and inducibility of cytochrome P450 (CYP) genes of the CYP3A and CYP1A families in adult human hepatocytes in primary culture has been evaluated. Only when cultured at subconfluence and in the presence of EGF did hepatocytes exhibit a proliferative response, assessed by measuring DNA synthesis and cyclin A accumulation. In the absence of EGF, the accumulation of CYP3A4 and CYP1A2 messenger RNAs (mRNAs) in response to their respective inducers (rifampicin and dioxin) was dramatically decreased in subconfluent culture with respect to confluent cultures. The presence of EGF only slightly decreased the accumulation of these mRNAs in both confluent and subconfluent cultures. The accumulation of CYP2D6 and CYP2E1 proteins, which are constitutively expressed in confluent cultures, and the production of fibrinogen and apolipoprotein (Apo) B100 exhibited similar behavior, while nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity was affected neither by cell density nor by EGF. In contrast, the accumulation of CYP1A1 mRNA in response to dioxin was similar in confluent and subconfluent cultures, irrespective of the presence of EGF. Interestingly, CYP3A7, a gene that is preferentially expressed in the fetal liver, was expressed constitutively neither in confluent nor in subconfluent cultures, irrespective of the presence of EGF. It is concluded that the loss of cell-cell contacts rather than the proliferative status of cells per se is responsible for the dramatic decrease in the expression of CYP genes, normally expressed in the adult human liver.

The fetal specific gene CYP3A7 is inducible by rifampicin in adult human hepatocytes in primary culture.

We investigated by RNase protection the differential expression of CYP3A4 and CYP3A7 mRNAs in fetal and adult human livers and in adult hepatocytes in primary culture. Our results show that CYP3A7 is expressed in the liver of 8 of 9 adult Caucasians examined, at an average level of 1.7% of the level found in a fetal liver. CYP3A4 mRNA appeared to be expressed in this fetal liver. In adult hepatocytes in primary culture, the constitutive level of CYP3A4 mRNA was low but detectable after 96 hours in untreated cells, but CYP3A7 mRNA remained undetectable. However, when the cells were treated for 48 hours with 25 microM rifampicin, both CYP3A4 and CYP3A7 mRNAs were strongly induced in the 3 and in 2 of 3 cultures examined, respectively. Using an isoelectric focusing immunoblotting the two proteins were resolved. Protein CYP3A4 was detectable in induced cells but CYP3A7 was not. These results show for the first time that CYP3A7 and CYP3A4 mRNAs, but not the proteins, are co-inducible by rifampicin in adult human hepatocytes in culture.

Metabolism of the new immunosuppressor cyclosporin G by human liver cytochromes P450.

Cyclosporin G is a new immunosuppressor structurally similar to cyclosporin A. Although this drug is pharmacologically as active as cyclosporin A, it is less toxic, in particular at the kidney level. The aim of this work was to identify the enzyme system(s) involved in the oxidative metabolism of cyclosporin G in man: (1) in a bank of human liver microsomes (n = 22), cyclosporin G oxidase activity correlated significantly with cyclosporin A oxidase activity (P < 0.0001) and with the level of CYP3A4 (P < 0.002), determined by immunoblot; (2) specific inhibitors of CYP3A4, troleandomycin, and ketoconazole, inhibited cyclosporin G oxidase activity by more than 80%; (3) antiCYP3A4 antibodies specifically inhibited this activity by nearly 90%; (4) cyclosporin A was a competitive inhibitor of cyclosporin G oxidase and vice versa; (5). Among a battery of cDNA-expressed CYPs, only CYP3A4 was able to generate detectable amounts of metabolites of cyclosporin G and cyclosporin A with a turnover number close to that calculated from experiments with liver microsomes; (6) in human hepatocytes in culture, pretreatment of cells with rifampicin and phenobarbital, 2 inducers of CYP3A4, produced a great increase in cyclosporin G oxidase activity, while beta-naphthoflavone, an inducer of CYP1As, did not. We conclude that CYP3A4 is the major enzyme involved in the oxidative metabolism of cyclosporin G in human liver.

Oxidative metabolism of zolpidem by human liver cytochrome P450S.

The aim of this study was to identify the form(s) of cytochrome P450 (CYP) responsible for the biotransformation of zolpidem to its alcohol derivatives which, after rapid conversion to carboxylic acids, represents the main way of metabolism in humans. In human liver microsomes, zolpidem was converted to alcohol derivatives. Production of these correlated with the level of CYP3A4 and with cyclosporin oxidation and erythromycin N-demethylation activities, but not with the level of CYP1A2 nor with ethoxyresorufin O-deethylation or S-mephenytoin 4'-hydroxylation activities. Liver microsomes from CYP2D6-deficient patients exhibited normal activity. Production of alcohol derivatives was significantly inhibited by anti-CYP3A antibodies and by ketoconazole. Antibodies directed against other CYP forms (including CYP1A1, CYP1A2, CYP2A6, CYP2B4, and CYP2C8), and CYP-specific substrates or inhibitors (including propranolol, coumarin, mephenytoin, sulfaphenazole, quinidine, aniline, and lauric acid) produced a moderate or no inhibitory effect. cDNA-expressed CYP3A4 and CYP1A2 generated significant amounts of one of the alcohol derivatives, whereas CYP2D6 generated both of them in similar amounts. In human hepatocytes in primary culture, zolpidem was extensively and almost exclusively converted to one of the carboxylic acid derivatives, the main species identified in vivo. Treatment of cells with inducers of CYP1A (beta-naphthoflavone) and CYP3A (rifampicin and phenobarbital) greatly increased the rate of production of this metabolite. We conclude that the formation of alcohol derivatives of zolpidem is rate-limiting and principally mediated by CYP3A4. Both CYP1A2 and CYP2D6 participate in alcohol formation; but, because of their low relative level of expression in the human liver, their contribution is minor.

Oxidative metabolism of lansoprazole by human liver cytochromes P450.

The aim of this work was to identify the form(s) of human cytochrome P450 (P450) involved in the hepatic biotransformation of lansoprazole to its two main metabolites, i.e., the sulfone and the hydroxy derivative. In liver microsomes, the production of the sulfone of lansoprazole correlated with the level of P450 3A4, cyclosporin oxidase, and the production of the hydroxy derivative, as well as of omeprazole sulfone. The production of hydroxylansoprazole moderately correlated with the level of P450 3A4, cyclosporin oxidase, and (S)-mephenytoin 4'-hydroxylase. The production of the sulfone and of the hydroxy derivative of lansoprazole was significantly inhibited by anti-P450 3A4 antibodies, by cyclosporin and ketoconazole, and by tolbutamide. Anti-P450 2C8 and 2C3 antibodies moderately inhibited the biotransformation of lansoprazole, whereas they completely inhibited (S)-mephenytoin 4'-hydroxylase activity under the same conditions. In primary cultures of human hepatocytes, the biotransformation of lansoprazole and the oxidation of cyclosporin were strongly increased by rifampicin and phenobarbital, whereas (S)-mephenytoin 4'-hydroxylation was not. beta-Naphthoflavone did not induce the formation of the sulfones but stimulated the production of hydroxylansoprazole. Among several forms of cDNA-expressed human P450s, 3A4 generated significant amounts of the sulfones of lansoprazole and omeprazole and 2C18 was active for the production of hydroxylansoprazole but inactive in the 4'-hydroxylation of (S)-mephenytoin. We conclude that P450 3A4 is the major enzyme involved in the production of the sulfone of lansoprazole and that this P450, as well as P450 2C18 and/or another 2C-related form, could contribute to the production of hydroxylansoprazole.

FK 506 renal toxicity and lack of detectable cytochrome P-450 3A in the liver graft of a patient undergoing liver transplantation.

Many commonly used drugs are substrates for hepatic cytochrome P-450 3A in human beings, and its role in the metabolism of potentially toxic immunosuppressants has been highlighted (cyclosporine, FK 506). One hundred fifty human liver grafts were biopsied before and after liver transplantation, and levels of cytochromes P-450 3A, 1A2, 2D6 and 2C were estimated by means of the Western-blot technique and correlated with histological appearance, glycogen content and clinical course. In 15 of the grafts, cyclosporine oxidase was also measured, and in 12 of 15 recipients, urinary 6 beta-hydroxycortisol excretion was assayed. A wide range of cytochrome P-450 3A values were observed (25 to 366 arbitrary units/mg; mean, 105 +/- 63 arbitrary units/mg). In one graft (no. 730) no cytochrome P-450 3A was detectable on immunoblotting, despite increasing homogenate concentrations. In this sample, cytochromes P-450 1A2, 2D6, and 2C were present in normal ranges. Levels of cyclosporine oxidase and 6 beta-hydroxycortisol in the urine specimens of the recipient were found to be low. The recipient of graft 730 experienced reversible complications of FK 506 therapy despite adherence to the administration protocol and drug plasma level in the normal range. The subsequent appearance of the cytochrome P-450 3A was associated with the consequent tolerance of oral FK 506. The absence of detectable amounts of P-450 3A in one biopsy from a donated human liver graft dramatically emphasizes the extreme range of this enzyme levels and has important clinical implications.

Evaluating motor recovery early after stroke: comparison of the Fugl-Meyer Assessment and the Motor Assessment Scale.

This study compared the measurements of the Motor Assessment Scale (MAS) to that of the Fugl-Meyer Assessment (FMA), a reliable and valid test for motor function in stroke patients. Thirty-two patients (20 men, 12 women) with a mean age of 60 years, and a mean time since stroke of 64.5 days, were tested with the FMA and MAS on two consecutive days. The Spearman correlation coefficient for total FMA and total MAS scores was 0.96. For selected items, significant (p < 0.001) correlations ranged from 0.65 to 0.93, except for sitting balance (-0.10). Low negative correlations between sitting balance scores and other items (motor and sensation) were found only for the FMA test, suggesting that the FMA sitting balance test is not valid for measuring balance and is likely responsible for the low correlation. Comparison of scores (normalized in percent of maximal value) for corresponding items of the two instruments also indicated that the FMA measured a higher (Wilcoxon = p < 0.0001) level of motor recovery, (especially in more disabled patients), for both the upper (15.7%) and lower extremities (27.5%). Lastly, a cumulative frequency distribution analysis indicated that a larger proportion of patients was found in the lower class interval scores of the MAS in comparison to the FMA. These results (1) support the concurrent validity of the MAS for measuring motor recovery in acute stroke patients; (2) demonstrate the poor validity of the FMA sitting balance test, and (3) suggest that the FMA scale can better discriminate the level of motor recovery than the MAS in the early stage of recovery or in the more disabled subjects.

Omeprazole and lansoprazole are mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture.

The ability of several gastric antiulcer drugs including lansoprazole, cimetidine and ranitidine to affect the expression of human liver microsomal cytochromes P450 comparatively to omeprazole, reported previously to be a CYP1A inducer, was evaluated in primary cultures of human hepatocytes. Poly (A)+ RNA and microsomes extracted from the cells were analyzed in Northern and Western blots with specific cDNA probes and antibodies, and assayed for form-specific monoxygenase activities. Lansoprazole induced both CYP1A1 and CYP1A2 as omeprazole and did not apparently bind to the aryl hydrocarbon receptor with high affinity. Omeprazole sulfone was not an inducer of CYP1A. Omeprazole, omeprazole sulfone and lansoprazole induced CYP3A in approximately 50% of tested cultures, whereas 100% of tested cultures responded to omeprazole and to rifampicin in terms of CYP1A and CYP3A induction, respectively. Finally, cimetidine and ranitidine were not inducers. We conclude that omeprazole and lansoprazole constitute a new class of mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture and that the induction of CYP3A in response to these molecules could be polymorphic in humans.

What changes drug metabolism in critically ill patients? Two preliminary studies in isolated human hepatocytes.

The metabolism of many drugs is abnormal in the critically ill patient. The causes of this are unknown, and investigation in patients is difficult. We therefore used isolated, cultured human hepatocytes to study the effects of hypoxia and induction on cytochrome P450 3A, the cytochrome responsible for the metabolism of many drugs. When hepatocytes were exposed to 5% oxygen, the amount of 3A produced, after induction with rifampicin, was five to 10 times less than the amount produced in 21% oxygen. In another study, we exposed isolated hepatocytes for 4 days to serum from five critically ill patients or from volunteers. At the end of this time, the functional ability of the hepatocytes to glucuronidate 14C progesterone was measured. Four of the five patients had a substance in their plasma that reduced the ability of the hepatocytes to glucuronidate progesterone. The nature of this substance and the reason that the serum from the fifth patient did not affect metabolism are unknown. This model is able to simulate the abnormalities which occur in critically ill patients. Further studies are needed to explain our observations and to identify the substances in the serum from critically ill patients that alter drug metabolism.

Metabolism of cyclosporine after orthotopic liver transplantation.

The aim of this work was to determine whether the extensive metabolism of cyclosporine, acquired in a donor by treatment with an inducer of cytochrome P450 3A (P450 3A) (cyclosporine oxidase), was transmissible to the recipient by orthotopic liver transplantation. For this purpose, male Wistar rats were divided into five groups including: control animals (group C), animals treated with dexamethasone (an inducer of P450 3A, 50 or 300 mg/kg/day, for 4 days, group D), animals transplanted with the livers of control rats (group G) or with the livers of dexamethasone-induced rats (group GD), and animals treated with beta-naphthoflavone (an inducer of P450 1A, group B). All animals received a single i.v. dose of 10 mg/kg cyclosporine 24 hr after either the last dose of inducer or the transplantation. For each group of animals, the area under the curve (AUC) of cyclosporine was calculated from the curves of blood cyclosporine levels (by radioimmunoassay) against time; liver microsomes were assayed for cyclosporine oxidase activity by HPLC, erythromycin demethylase and P450 3A level by western blot with specific anti-P450 3A antibodies. The decrease in the AUC in groups D and GD with respect to C and G was correlated with increased level of P450 3A (4-5-fold with respect to control) as well as of microsomal cyclosporine oxidase. In addition, cyclosporine oxidase activity of liver microsomes was specifically inhibited by anti-P450 3A antibodies and troleandomycin. The animals in group B did not exhibit increased metabolism of cyclosporine either in vivo or in vitro. We conclude that: (1) cyclosporine is predominantly oxidized in the rat liver by a form of P450 from the 3A subfamily; (2) the extensive metabolism of cyclosporine acquired by donor rats after treatment with dexamethasone is transmissible to the recipients through orthotopic liver transplantation.

Effect of corticosteroids on the expression of cytochromes P450 and on cyclosporin A oxidase activity in primary cultures of human hepatocytes.

Prednisone, prednisolone, and methylprednisolone are currently administered in association with cyclosporin A in the postoperative treatment of transplant patients. The aim of this work was to evaluate the effects of these corticosteroids on the expression of several forms of cytochromes P450 (P450), including P450 1A2, 2D6, 2E1, and 3A, and on cyclosporin A oxidase activity in human liver. For this purpose, human hepatocytes prepared from lobectomies were maintained in culture in a serum-free medium, in collagen-coated dishes, for 96-144 hr, in the absence or presence of 50-100 microM corticosteroids, rifampicin, or dexamethasone. To mimic more closely the current clinical protocol, hepatocyte cultures were also co-treated with corticosteroids and cyclosporin A or ketoconazole (a selective inhibitor of P450 3A). Cyclosporin A oxidase activity, intracellular retention of cyclosporin A oxidized metabolites within hepatocytes, accumulation of P450 proteins and corresponding messages, and de novo synthesis and half-lives of these P450 were measured in parallel in these cultures. Our results, obtained from seven different hepatocyte cultures, showed that 1) dexamethasone and prednisone, but not prednisolone or methylprednisolone, were inducers of P450 3A, at the level of protein and mRNA accumulation, as well as of cyclosporin A oxidase activity, known to be predominantly catalyzed by these P450; 2) although corticosteroids are known to be metabolized in human liver, notably by P450 3A, partial or total inhibition of this P450 by cyclosporin or ketoconazole, respectively, did not affect the inducing efficiency of these molecules; 3) corticosteroids did not affect the half-life of P450 3A or the accumulation of other forms of P450, including 1A2, 2D6, and 2E1; 4) chronic treatment of cells with cyclosporin did not affect P450 3A accumulation; 5) corticosteroids were all competitive inhibitors of cyclosporin A oxidase in human liver microsomes, with Ki values of 61 +/- 12, 125 +/- 25, 190 +/- 38, and 210 +/- 42 microM for dexamethasone, prednisolone, prednisone, and methylprednisolone, respectively; and 6) chronic treatment of cells with corticosteroids did not influence the excretion of oxidized metabolites of cyclosporin from the cells. These results support most of clinical reports dealing with mutual interactions between cyclosporin A and corticosteroids.

Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture.

The expression of several forms of cytochrome P450 including P450 1A2, 2D6, 2E1, and 3A was investigated in human hepatocytes maintained in primary culture for 96 h in the absence or presence of 50 microM of various imidazole derivatives. These included ketoconazole, clotrimazole, miconazole, fluconazole, secnidazole and metronidazole. In addition, the typical inducers rifampicin and beta-naphthoflavone were used for comparison. Western and Northern blot analysis of microsomes and RNA prepared from these cultures as well as de novo synthesis experiments revealed that, among the imidazole derivatives tested, only clotrimazole was a strong rifampicin-like inducer of P450 3A. The expression of the other forms of P450 tested was not affected by the treatments. Analysis of the inhibition of 13 monoxygenase activities, including ethoxyresorufin and phenacetin O-deethylases, coumarin 7 alpha-, lauric acid 11- and 12-, mephenytoin 4-, debrisoquin 4-, and aniline hydroxylases, benzphetamine, aminopyrine, mephenytoin and erythromycin demethylases, and cyclosporin oxidase (representative of 10 different forms of P450 in human liver microsomes) revealed that ketoconazole was a strong and selective in vitro inhibitor of P450 3A (cyclosporin oxidase) with a Ki less than 1 microM. Clotrimazole and miconazole were also strong inhibitors of P450 3A-mediated activities in contrast to the other imidazole derivatives.

[Cyclosporin A metabolism and induction of cytochrome P-450 in orthoptic hepatic transplantation in rats]. / Métabolisme de la ciclosporine A, et induction du cytochrome P450 dans la transplantation hépatique orthotopique chez le rat.

The aim of our study were 1) to establish that cyclosporin (CsA) metabolism was correlated with the rate of cytochrome P4503A (cyt.) in Wistar rats induced with dexamethasone (Dex.), 2) to demonstrate that the induction of cyt. with Dex. in liver "rat donor" was transmissible to "recipient rat" after liver transplantation. Sixty rats were divided in 5 groups. In group T, a single dose of CsA (10 mg/kg) was administered intravenously in 10 rats; in group D, 10 rats were treated with Dex (300 mg/kg daily for 4 days) and then received CsA as above; in group BN 5 rats were treated with beta-naphthoflavone. Thirty five rats underwent a liver transplantation either from "non induced donors" (group G, n = 11) or from "induced donors with Dex." (group GD, n = 24) followed by CsA injection the next day. For each rat, CsA plasma levels were determined by radioimmunoassay in 6 samples. Liver microsomes cyt. from samples of the liver of donor rats (group G and GD) or after sacrifice (group T, D, BN) were quantitated by immunoblot analysis and estimated from densitometric analysis of the blot. Mean maximal plasma concentration (Cmax) were 2,822 +/- 997 ng/ml in group T, 1,447 +/- -458 ng/ml in group D, 2,685 +/- 1,383 ng/ml in group G, 1,337 +/- 713 ng/ml in group GD and 3,094 +/- 685 ng/ml in group BN. Considering the Cmax and the ASC (area under curve), there was a significant difference between all groups and separately between groups T and D, G and GD.(ABSTRACT TRUNCATED AT 250 WORDS)