Scaling drug biotransformation data from cDNA-expressed cytochrome P-450 to human liver: a comparison of relative activity factors and human liver abundance in studies of mirtazapine metabolism

The present study represents a comparison of three approaches to transform recombinant cytochrome P-450 (rCYP) enzyme kinetic data to human liver activity using mirtazapine (MIR) biotransformation as a model. MIR metabolite rCYP formation rates were corrected using I) relative activity factors (RAFs) determined on site, II) RAFs based on activity data provided by the rCYP manufacturer, and III) immunologically determined human liver abundance of CYP isoforms reported in the literature. For 2.5, 25, and 250 microM MIR, predictions of 1) the relative contribution of CYP isoforms to a particular reaction, 2) absolute metabolite formation rates, 3) the relative contribution of each pathway to net MIR biotransformation, and 4) the relative contribution of CYP isoforms to net MIR biotransformation were generated, and the results were compared with data obtained with human liver microsomes (HLM). We found that RAFs determined on site most accurately predict the results observed in HLM. Estimations based on liver abundance systematically underestimated CYP1A2 and overestimated CYP3A and CYP2C9 contributions to MIR metabolism and, therefore, seem less suitable to predict CYP isoform involvement in a particular reaction. Normalized RAFs calculated from the manufacturer activity data fell within the range of RAFs determined on site and lead to similar results for CYP isoform contribution to metabolic reactions and to net MIR biotransformation. Considering the time and resource-intensive step of RAF determination, manufacturer RAFs are an alternative to RAFs determined on site for the transformation of rCYP enzyme kinetic data; both of them provide more accurate estimations than immunologically determined human liver CYP isoform content.     

Suppression of hepatic cytochrome p450-mediated drug metabolism during the late stage of sepsis in rats

The effects of polymicrobial sepsis on the activity and gene expression of hepatic microsomal cytochrome P450 (CYP) were examined. Rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Liver and blood samples were taken 2, 6, and 24 h after CLP. The serum aminotransferase levels and lipid peroxidation increased 24 h after CLP. The hepatic concentrations of reduced glutathione and total CYP content decreased 24 h after CLP. The CYP1A1 activity and its protein level decreased 24 h after CLP. The CYP1A2 activity decreased 2 h and 24 h after CLP. Although the CYP2B1 mRNA expression level decreased 6 h and 24 h after CLP, the CYP2B1 activity and its protein level did not change in any of the experimental groups. The CYP2E1 activity and its protein level decreased 24 h after CLP. The CYP2E1 mRNA levels were lower at both 6 h and 24 h after CLP. The TNF-alpha mRNA expression level increased 2, 6, and 24 h after CLP. The iNOS mRNA expression level increased 24 h after CLP. These findings suggest that sepsis causes abnormalities in the microsomal drug-metabolizing function, particularly in the late stage, which is associated with higher level of oxidant stress and lipid peroxidation.     

Competition between cytochrome P-450 isozymes for NADPH-cytochrome P-450 oxidoreductase affects drug metabolism

NADPH-cytochrome P-450 oxidoreductase (CPR) is essential for the catalytic activity of cytochrome P-450 (P-450). On a molar basis, the amount of P-450 exceeds that of CPR in human liver. In this study, we investigated whether drug-drug interactions can occur as a result of competition between P-450 isozymes for this ancillary protein. For this purpose, combinations of P-450 isozymes were coexpressed together with P-450 reductase in Escherichia coli. We show that testosterone inhibited the CYP2D6-mediated bufuralol 1'-hydroxylase activity in bacterial membranes containing both CYP2D6 and CYP3A4 but not in membranes containing CYP2D6 alone. Conversely, bufuralol inhibited the CYP3A4-mediated testosterone 6beta-hydroxylase activity in bacterial membranes containing both CYP3A4 and CYP2D6 but not in membranes containing only CYP3A4. In each case, inhibition was seen even at a P-450 to P-450 reductase ratio of 1.9:1, which is more favorable than the ratio of 4 reported for human liver. The physiological significance of this mechanism was demonstrated by the observation that testosterone inhibited several prototypical P-450 enzyme activities, such as bufuralol 1'-hydroxylase, coumarin 7-hydroxylase, and 7-ethoxyresorufin O-dealkylase, in human liver microsomes, but not if tested against a panel of bacterial membranes containing the human P-450 isozymes that mainly catalyze these reactions.     

Carbamoyl phosphate synthase-1: a marker of mitochondrial damage and depletion in the liver during sepsis

OBJECTIVE: Mitochondrial damage and dysfunction are thought to play an important role in the pathogenesis of sepsis-induced organ failures. Unfortunately, specific markers of mitochondrial damage in vital organs do not currently exist. Recently, carbomyl phosphate synthase (CPS)-1, a protein primarily localized to liver mitochondria, was found to be present in high concentrations in the plasma of septic humans. Thus, we hypothesized that the circulatory release of CPS-1 would correlate with mitochondrial damage or impaired mitochondrial function in the liver in a clinically relevant model of sepsis. DESIGN: Prospective, randomized, controlled animal study. SETTING: University medical center research laboratory. SUBJECTS: Male, Balb/C mice, aged 10-12 wks. INTERVENTIONS: Animals were assigned to receive cecal ligation and puncture (CLP sepsis) or sham operation and compared with untreated controls. Plasma CPS-1 levels and liver mitochondrial variables, including morphology, respiratory activity, mass (i.e., cardiolipin content), and protein carbonylation, were assessed at various time points (8, 24, and 48 hrs and 6 days) after surgery. MEASUREMENTS AND MAIN RESULTS: Oxidant stress (i.e., carbonylation) was detected within 8 hrs of CLP and persisted through 48 hrs. Plasma CPS-1 levels increased dramatically at 24 hrs, remained significantly elevated at 48 hrs, and normalized by 6 days in the sepsis group. Abnormalities of liver mitochondrial morphology and function coincided with increased plasma CPS-1 levels. Mitochondrial depletion in the liver was not due to cell death but was associated with increased lysosomal clearance. Increased expression of mitochondrial biogenesis factors preceded restoration of mitochondrial variables and normalization of CPS-1 levels by day 6. CONCLUSIONS: Circulating CPS-1 is a marker of mitochondrial damage and depletion in the liver during the subacute phase of CLP sepsis. From a mechanistic standpoint, mitochondrial depletion is not due to cell death but is apparently related to the removal of damaged mitochondria by lysosomes (i.e., autophagy), followed by repletion of mitochondrial populations. Further studies are needed to determine the clinical utility of CPS-1 as a marker of sepsis severity.     

The anti-inflammatory effect of curcumin in an experimental model of sepsis is mediated by up-regulation of peroxisome proliferator-activated receptor-gamma

OBJECTIVE: Although phytochemical curcumin has been shown to possess anti-inflammatory properties, it remains unknown whether this agent has any beneficial effects in sepsis. The purpose of this study was to demonstrate whether curcumin protects septic animals and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR)-gamma, an anti-inflammatory nuclear receptor, plays any role. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: A bolus injection of 0.2 micromol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 micromol/day) for 3 days via a primed 2-mL mini-pump. The rats were then subjected to sepsis by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS: Serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase), lactate, albumin, and tumor necrosis factor (TNF)-alpha were measured at 20 hrs after CLP (i.e., late stage of sepsis). In addition, a 10-day survival curve was conducted following CLP and cecal excision with or without curcumin treatment. Furthermore, macrophages cell line RAW 264.7 cells were treated with curcumin followed by stimulation with endotoxin. TNF-alpha and PPAR-gamma expression were then measured. The results indicate that intravenous administration of curcumin before the onset of sepsis attenuated tissue injury, reduced mortality, and decreased the expression of TNF-alpha in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-gamma in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-gamma antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-alpha expression and markedly up-regulated PPAR-gamma expression without affecting cell viability. Curcumin also prevented morphologic alterations in macrophages induced by endotoxin. CONCLUSIONS: The protective effect of curcumin makes it or its analogues strong candidates as a novel therapy for sepsis. The beneficial effect of curcumin appears to be mediated by up-regulation of nuclear receptor PPAR-gamma.     

Is prostacyclin responsible for producing the hyperdynamic response during early sepsis?

OBJECTIVE: Although polymicrobial sepsis is characterized by an early hyperdynamic phase (2-10 hrs after cecal ligation and puncture [CLP]), followed by a late hypodynamic phase (20 hrs after CLP), it remains unknown whether prostacyclin or prostaglandin I2 (PGI2) plays a significant role in modulating the hyperdynamic state during early sepsis. The aim of this study was to determine whether inhibition of PGI2 synthesis prevents the occurrence of the hyperdynamic response during early sepsis. DESIGN: Prospective, controlled animal study. SETTING: A university research laboratory. SUBJECTS: Adult male Sprague-Dawley rats were subjected to sepsis by CLP. INTERVENTIONS AND MEASUREMENTS: Blood samples were collected at 2, 5, 10, or 20 hrs after CLP, and plasma concentrations of PGI2, in the form of its stable product 6-keto-PGF1alpha, were measured by radioimmunoassay. In additional studies, a PGI2 synthase inhibitor, tranylcypromine, was administered subcutaneously at the time of CLP and again at 3 hrs after CLP. At 5 hrs after the onset of sepsis, the maximal rates of the left ventricular pressure rise (+dP/dtmax) and fall (-dP/dtmax) were determined by an in vivo heart performance analyzer. Microvascular blood flow in the liver, small intestine, and spleen was assessed by laser Doppler flowmetry. MAIN RESULTS: Plasma concentrations of 6-keto-PGF1alpha increased significantly at 2-20 hrs after CLP. At 5 hrs after the onset of sepsis, +/-dP/dt(max) and microvascular blood flow in the tested tissues increased significantly. Inhibition of PGI2 synthase activity did not prevent the occurrence of hypercardiovascular responses under such conditions. Moreover, the administration of tranylcypromine significantly reduced circulating concentrations of 6-keto-PGF1alpha at 5 hrs after CLP. CONCLUSIONS: Because inhibition of PGI2 production did not prevent the occurrence of the hyperdynamic and hypercardiovascular response during the early stage of sepsis, mediators other than PGI2 appear to play a major role in producing the hyperdynamic response under such conditions.     

Latent overexpression of hepatic CYP2C7 in adult male and female rats neonatally exposed to phenobarbital: a developmental profile of gender-dependent P450s

For more than 20 years it has been known that neonatal exposure to phenobarbital results in a delayed, but permanent overexpression of drug-metabolizing enzymes in adult male and female rats. Accordingly, to identify the specific isoform(s) of P450 responsible for the imprinted overexpression of hepatic monooxygenases, we have monitored the developmental profile of some dozen hepatic P450 isoforms in 4- to 150-day-old male and female rats neonatally treated with the barbiturate. Some of the cytochrome P450s (CYP), i. e., CYP2A1, 2A2, 2C6, 3A1, and 3A2, exhibit the typical transient response in which isoform levels (mRNA, protein, and/or specific catalytic activity) rise precipitously at the time of phenobarbital administration and rapidly decline to preinduction levels after withdrawal of the barbiturate. Other isoforms, i.e., CYP1A1, 1A2, 2C7, 2C11, 2C12, and 2C13, were neither constitutively expressed nor phenobarbital inducible in the neonate. Only one of these isoforms, female predominant (M:F, approximately 1:2) CYP2C7, exhibited a barbiturate-induced delayed, but persistent approximately 30 to 50% overexpression from puberty through adulthood. We propose that at the time of exposure, neonatally administered phenobarbital produces a "silent" programming defect resulting in a delayed, but persistent overexpression of the isoform, contributing, at least in part, to a permanent elevation of hepatic drug-metabolizing enzyme activities.     

Activation of human cytochrome P-450 3A4-catalyzed meloxicam 5'-methylhydroxylation by quinidine and hydroquinidine in vitro.

In humans, meloxicam is metabolized mainly by cytochrome P-450 (CYP)-dependent hydroxylation of the 5'-methyl group. The predominant P-450 enzyme involved in meloxicam metabolism is CYP 2C9, with a minor contribution of CYP 3A4. Quinidine, a CYP 3A4 substrate commonly used as a selective in vitro inhibitor of CYP 2D6, was found to markedly increase the rate of meloxicam hydroxylation during in vitro experiments with human liver microsomes. A similar activation was observed with other compounds that are structurally related to quinidine. Besides quinidine, quinine and hydroquinidine were the most potent activators of meloxicam hydroxylation. Using expressed cytochrome P-450 enzymes and selective chemical inhibitors of CYP 2C9 and CYP 3A4, it was found that quinidine markedly increased the rate of CYP 3A4-mediated meloxicam hydroxylation but was virtually without effect on CYP 2C9. Kinetic analysis was performed to obtain insight into the possible mechanism of activation of CYP 3A4 and into the mutual interaction of quinidine/hydroquinidine and meloxicam. Quinidine and hydroquinidine decreased Km and increased Vmax of meloxicam hydroxylation, which was consistent with a mixed-type nonessential activation. Meloxicam, in turn, decreased both Km and Vmax of quinidine metabolism by CYP 3A4, indicating an uncompetitive inhibition mechanism. These results support the assumption that CYP 3A4 possesses at least two different substrate-binding sites. A clinically relevant effect on meloxicam drug therapy is not expected, because the most likely outcome in practice is moderately decreased meloxicam plasma concentrations.     

Effect of calcium channel antagonists nifedipine and nicardipine on rat cytochrome P-450 2B and 3A forms.

Calcium channel antagonists are widely prescribed for treatment of hypertension. In this study, we examined whether treatment with the calcium channel antagonists, nicardipine, nifedipine or diltiazem, alters cytochrome P-450 2B or 3A (CYP2B or CYP3A, respectively) expression in rat liver. Western blot analyses were undertaken using antibodies specific for one or several members of these cytochrome P-450 subfamilies. Nicardipine was found to be an effective inducer of CYP3A; in particular, CYP3A23 was increased approximately 36-fold following treatment with 100 mg of nicardipine/kg/day. Nicardipine induced CYP2B forms up to approximately 3.1-fold. Nifedipine did not alter CYP3A expression but did increase CYP2B expression such that total CYP2B, CYP2B1, and CYP2B2v (a splice variant of CYP2B2) were increased approximately 5- to 15-fold after treatment with 100 mg of nifedipine/kg/day, with increases in benzyloxyresorufin O-dealkylase and erythromycin N-demethylase activities, respectively. The distinct differences in cytochrome P-450 induction profile induced by nicardipine and nifedipine suggest that they may enhance cytochrome P-450 expression by different mechanisms unrelated to their effects on calcium channels.     

Hepatic and central nervous system cytochrome P450 are down-regulated during lipopolysaccharide-evoked localized inflammation in brain

The effect of central nervous system inflammation on the levels and activity of hepatic and brain cytochrome P450 were examined in the rat. Brain ethoxyresorufin dealkylkase (EROD) was depressed during localized inflammatory responses evoked by lipopolysaccharide (LPS) injected into the lateral ventricle. This loss was accompanied by a concomitant loss of EROD activity and cytochrome P450 in liver. Similar losses in hepatic enzyme were observed for benzyloxy-resorufin and pentoxy-resorufin dealkylase (CYP2B) and chlorzoxazone hydroxylation (CYP2E). Protein levels of CYP2D and CYP2E1 but not CYP1A also were depressed. Similar i.p. doses of LPS had no effect on hepatic cytochrome P450, indicating that the hepatic effect was not caused by LPS leakage from the central nervous system. Also in support of this contention is that heat shock protein 27 was expressed throughout the brain by LPS given i.c. v. but was undetectable in the liver. Tumor necrosis factor-alpha given i.c.v. depressed EROD activity in the brain but this was not accompanied by a concomitant loss in the liver. Hepatic EROD did respond to the i.p. injection of tumor necrosis factor-alpha. The LPS-evoked loss in hepatic cytochrome P450 could not be prevented by blocking beta-receptor-mediated sympathetic nerve activity. This study demonstrates that localized inflammatory responses in the brain cause a concomitant down-regulation of cytochrome P450 and drug-metabolizing activity in the liver and the brain. The effect on brain cytochrome P450 may be regulated via cytokine-mediated pathways but signaling to the liver does not involve a cytokine-mediated pathway nor a beta-receptor-mediated sympathetic nerve pathway.     

Modulation of cytochrome P-450 gene expression in endotoxemic mice is tissue specific and peroxisome proliferator-activated receptor-alpha dependent.

Administration of the bacterial endotoxin lipopolysaccharide (LPS) causes induction of cytochrome P-450 (CYP) 4A mRNAs in rat liver and kidney. Because induction of the CYP4A subfamily by chemicals requires peroxisome proliferator-activated receptor-alpha (PPARalpha), we determined whether CYP4A induction by LPS also requires PPARalpha by comparing the responses of PPARalpha-null (-/-) and wild-type (+/+) mice. Renal expression of CYP4A10, CYP4A14, and acyl-CoA oxidase was induced by LPS treatment in (+/+) mice, and these effects were absent in the (-/-) mice. In contrast, hepatic expression of CYP4A10 was down-regulated in the (+/+) animals, and no significant induction of acyl-CoA oxidase or CYP4A14 was detected in liver. Expression of the peroxisomal bifunctional enzyme was not significantly affected by LPS treatment. These results indicate that PPARalpha is activated in mouse kidney after LPS treatment and that this leads to modulation of some PPARalpha-regulated genes. However, the species and tissue specificity of these effects suggest that inflammatory pathways may modulate the induction via PPARalpha. Mice pair fed with LPS-treated mice showed no induction of renal CYP4A10 or CYP4A14, indicating that renal CYP4A induction during endotoxemia is not due to hypophagia. Down-regulation of CYP2A5, CYP2C29, and CYP3A11 by LPS was attenuated or blocked in the (-/-) mice, suggesting a role for PPARalpha in CYP down-regulation as well. Finally, we found that clofibrate caused an acute induction of two hepatic acute-phase mRNAs that was only partially dependent on PPARalpha.     

Delayed ascorbate bolus protects against maldistribution of microvascular blood flow in septic rat skeletal muscle

OBJECTIVE: Although early administration of ascorbate has been shown to protect against the microvascular dysfunction in sepsis, it is not clear if a delayed introduction of ascorbate also yields beneficial effects. The main objective was to determine the therapeutic window for treatment of an animal model of sepsis with bolus injection of ascorbate. We also determined if sepsis per se affects urinary excretion of ascorbate. DESIGN: Prospective, controlled laboratory study. SETTING: Animal laboratory in a university-affiliated research institute. SUBJECTS: Male Sprague-Dawley rats, 300-400 g of body weight. INTERVENTIONS: Rats were made septic by cecal ligation and perforation (CLP) and volume resuscitated by continuous saline infusion. Ascorbate bolus (7.6 mg/100 g of body weight) or saline vehicle was injected intravenously at 1, 6, or 24 hrs after CLP. MEASUREMENTS AND MAIN RESULTS: At 24 hrs post-CLP, sepsis caused antidiuresis and decreased plasma ascorbate concentration, but it did not affect urinary excretion of ascorbate in rats that received only saline. Sepsis also caused maldistribution of capillary blood flow in skeletal muscle. This maldistribution of flow was prevented by ascorbate injected at 6 hrs post-CLP. At 48 hrs post-CLP, in addition to the flow maldistribution, sepsis caused systemic arterial hypotension and fever that were prevented by both immediate (1 hr post-CLP) and delayed injections of ascorbate (24 hrs post-CLP). CONCLUSION: Despite volume resuscitation, the present model of sepsis resulted in maldistribution of capillary blood flow within 24 hrs and hypotension within 48 hrs. Our finding that intravenous bolus of ascorbate can protect against these deficits even if delayed 6-24 hrs after the septic insult shows, for the first time, that ascorbate can reverse microcirculatory dysfunction after the onset of sepsis.     

Lipopolysaccharide and cecal ligation/puncture differentially affect the subcellular distribution of the pregnane X receptor but consistently cause suppression of its target genes CYP3A

The repressed expression of cytochrome P450 (CYP) enzymes in septic patients contributes significantly to therapeutic failures. Mice treated with sepsis-inducing agent lipopolysaccharide (LPS) sequentially express reduced mRNA levels of the pregnane X receptor (PXR) and its target genes Cyp3a(s), suggesting that reduction of Cyp expression is associated with the repression of PXR. The present study was undertaken to determine whether septic rats induced by LPS and cecal ligation/puncture (CLP) express reduced levels of rat PXR protein and whether the subcellular distribution of PXR is altered in septic conditions. Rats were treated with LPS (55 vs. 1 mg/kg) or underwent CLP, and the expression of CYP3A and PXR was determined. In LPS-treated rats, the expression of CYP3A enzymes was consistently decreased regardless of the doses used. In contrast, high dose and repeated low dose of LPS caused significant decreases on the nuclear PXR, whereas the opposite was true with the cytosolic PXR. When rats were administered with only a single low dose of LPS, both nuclear and cytosolic PXR levels were significantly increased. In the CLP model, rats undergoing CLP for 30 h expressed significantly lower levels of CYP3A but the PXR levels were not significantly altered. In addition, when rats were treated with dexamethasone, a significant induction of CYP3A was detected. However, such an induction was markedly antagonized by the treatment with LPS. The differential changes on the levels of the nuclear PXR and CYP3A between LPS and CLP models suggest that PXR plays negligible roles in the constitutive expression of CYP3A. The antagonism of LPS against dexamethasone-mediated CYP3A induction suggests that endotoxemia minimizes the inducibility of PXR target genes.     

Important role of prodromal viral infections responsible for inhibition of xenobiotic metabolizing enzymes in the pathomechanism of idiopathic Reye's syndrome, Stevens-Johnson syndrome, autoimmune hepatitis, and hepatotoxicity of the therapeutic doses of acetaminophen used in genetically predisposed persons

Upper respiratory tract febrile illnesses caused by various viruses, mycoplasma, chlamydia infections, and/or inflammatory diseases are usually observed a few days to a few (several) weeks before the onset of Reye's syndrome, Stevens-Johnson syndrome, autoimmune hepatitis (hepatotropic virus infections), or hepatotoxicity associated with therapeutic administration of acetaminophen in persons with varying degrees of deficits of important enzymatic activity. Activation of systemic host defense mechanisms by inflammatory component(s) results in depression of various induced and constitutive isoforms of cytochrome P-450 mixed-function oxidase system superfamily enzymes in the liver and most other tissues of the body. Because several cytochrome P-450 enzymes activities important for biotransformation of many endogenous and egzogenous substances show considerable variability between individuals, in some genetically predisposed persons, even the administration of therapeutic doses of a drug may result in serious clinical mishaps, if an important concomitant risk factor (eg, acute viral infection) is involved. Several inflammatory cytokines, such as interleukins, transforming growth factor beta1, human hepatocyte growth factor, and lymphotoxin, downregulate gene expression of major cytochrome P-450 enzymes with the specific effects on mRNA levels, protein expression, and enzyme activity observed with a given cytokine varying for each P-450 studied, thus eventually leading to metabolite-mediated adverse drug reactions and immunometallic diseases which sometimes result in tissue injury beyond the site(s) where metabolic bioactivation takes place. On the other hand, it must be emphasized that inhibition of metabolism of several drugs, as well as influence on the concentration and/or ratio of various cytokines in inflamed tissues, may exert beneficial effects in patients with different diseases, thus opening new therapeutic possibilities. Clinically relevant interactions may be exemplified by the effects of some fluoroquinolone antibiotics, such as pefloxacin and ciprofloxacin, which probably have a steroid-sparing effect in some patients with frequently relapsing nephrotic syndrome, and an increased bioavailability of several drugs following concomitant intake with freshly pressed grapefruit juice, eventually caused by inhibition of their metabolism, mediated mainly by CYP3A and specifically inhibited by naturally occurring flavonoids.