Alfentanil-induced miosis as a surrogate measure of alfentanil pharmacokinetics in patients with mild and moderate liver cirrhosis.

OBJECTIVES: (i) To evaluate the pupillary response to alfentanil as a surrogate measure of alfentanil pharmacokinetics in cirrhotic patients and to compare the data observed in cirrhotic patients with those found in healthy volunteers (historical control group); and (ii) to compare this test with other liver function tests in cirrhotic patients. METHODS: Six patients with mild cirrhosis (Child-Pugh grade A) and six patients with moderate cirrhosis (Child-Pugh grade B) were studied after a single 15 microg/kg bolus of alfentanil. Alfentanil plasma concentrations were measured by liquid chromatography-tandem mass spectrometry, and pupillary responses were measured with a Pupilscan II pupillometer. Alfentanil pharmacokinetics (plasma concentration, area under the plasma concentration-time curve from time zero to infinity [AUC(infinity(p))] and from time zero to 2 hours [AUC(2(p))], apparent volume of distribution at steady state, clearance and terminal elimination half-life [t((1/2)(p))]) and miosis pseudo-kinetic parameters [AUC(infinity)((miosis)), AUC(2)((miosis)), t((1/2))((miosis))] were determined using a noncompartmental analysis method. In six patients (three Child-Pugh grade A and three Child-Pugh grade B), antipyrine (measure of liver intrinsic activity) and D-sorbitol (measure of liver blood flow) tests were performed. RESULTS: A significant correlation was found between the alfentanil AUC(infinity(p)) and AUC(infinity)((miosis)) (r = 0.6, p < 0.05) in cirrhotic patients. This correlation was even more significant if AUC determinations were limited to the first 2 hours after alfentanil administration (r = 0.9, p < 0.01). Statistically significant differences in pharmacokinetics and miosis pseudo-kinetic parameters were observed between cirrhotic patients and healthy volunteers from our previous experiment (historical control group). The correlations were significant between alfentanil clearance and antipyrine clearance (n = 6, r = 0.9, p < 0.05), alfentanil clearance and steady-state hepatic blood clearance [CL(H(b))] measured by the D-sorbitol test (n = 6, r = 0.9, p < 0.05). CONCLUSION: Alfentanil pharmacokinetic parameters were correlated with miosis pseudo-kinetic parameters in cirrhotic patients. There was a significant decrease in pharmacokinetics and miosis pseudo-kinetics in cirrhotic patients compared with volunteers from the historical control group. Alfentanil-induced miosis has the advantage of being noninvasive and can be limited to miosis measurements during the first 2 hours after alfentanil administration in cirrhotic patients. We thus propose to substitute the AUC(2(miosis)) for alfentanil pharmacokinetics in cirrhosis.

CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination.

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95 +/- 0.24 microM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 +/- 0.21 microM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p = 0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75 micromol/mg protein/min) with lower P450 content (0.3 nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC(50) approximately 21.6 microM) compared to DM induced (IC(50) approximately 32.3 microM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10-30 microM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1,000 microM) a nearly similar inhibition (76%) was observed than that at 50 microM concentration (68.2%).

Alfentanil-induced miosis clearance as a liver CYP3A4 and 3A5 activity measure in healthy volunteers: improvement of experimental conditions.

The aims of this study were to demonstrate the correlation between alfentanil-induced miosis evaluation and alfentanil pharmacokinetics (PK) as a CYP3A4 and 3A5 activity probe in volunteers and to explain the variability in pupilar response and in alfentanil PK. In ambient light, the miosis kinetic parameters were significantly correlated with PK (CLs: r = 0.9, P = .00; AUCs: r = 0.8, P = .01). In dark, a similar correlation was observed between miosis and alfentanil clearances (r = 0.85, P = .03). In 6 volunteers, the sigmoid E(max) model was applicable (average E(max) = 2.5 +/- 0.7 mm, gamma = 2.5 +/- 1.6 and EC(50) = 76.8 +/- 22.3 ng/mL), and in 3, the simple E(max) model was applicable (average E(max) = 2.8 +/- 0.3 mm and EC(50) = 19.9 +/- 8.5 ng/mL). There was a large interindividual variability in PK parameters (coefficient of variation = 19.7%-31.2%). Free drug fraction concentrations were negatively correlated with plasma alpha(1)-AGP (r = -0.9, P = .04) and albumin levels (r = -0.94, P = .02). Alfentanil-induced miosis clearance as a noninvasive CYP3A4 and 3A5 activity measure can be done in both ambient and dark conditions. Drug free fraction may be responsible for large intersubject variability in alfentanil PK.

Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection.

Immune-based therapy is the mainstay treatment for chronic hepatitis C virus (HCV) infection but causes multiple side effects and achieves durable viral clearance in only approximately 50% of patients. Most new investigational anti-HCV compounds are direct-acting antivirals for which durability of response and risk of viral mutations and resistance are not yet known. Therefore, continuing discovery and development of new immune-based treatments is desirable. Toll-like receptors (TLRs) are pathogen recognition receptors that initiate the innate immune response. The responsiveness of HCV or other ongoing chronic systemic infections to treatment with a selective TLR agonist has not been reported. Isatoribine is a selective agonist of TLR7. In a proof-of-concept study, we found that once-daily 7-day treatment with intravenous isatoribine 800 mg caused a significant (P = .001) reduction of plasma HCV RNA (mean, -0.76; range, -2.85 to +0.21 log(10) units) in otherwise untreated patients (n = 12) who were chronically infected with HCV. Viral load reduction occurred in patients infected with genotype 1 as well as non-genotype 1 HCV. The reduction of viral load was correlated with induction of markers of a heightened immune antiviral state, including 2'-, 5'- oligoadenylate synthetase levels in whole blood. This treatment was well tolerated, with a low frequency of mild to moderate adverse events. In conclusion, systemic administration of the selective TLR7 agonist isatoribine resulted in dose-dependent changes in immunologic biomarkers and a statistically significant antiviral effect with relatively few and mild side effects.

Genetic polymorphisms of CYP2C9 and CYP2C19 in the Beninese and Belgian populations.

AIMS: To investigate the distribution of cytochrome P450 2C9 (CYP2C9) and 2C19 (CYP2C19) genotype frequencies in the Beninese and Belgian Caucasian populations. METHODS: Beninese (n = 111) and Belgian (n = 121) were genotyped for CYP2C9*2, *3, *4, *5, and *11 as well as for CYP2C19*2 and*3. RESULTS: The distribution of alleles was: CYP2C9*1: 95.5 vs. 82.2% (P < 0.001); CYP2C9*2: 0 vs. 10% (P < 0.001); CYP2C9*3: 0 vs. 7.4% (P < 0.01); CYP2C9*4: both 0%; CYP2C9*5: 1.8 vs. 0% (P = 0.05); and CYP2C9*11: 2.7 vs. 0.4% (P < 0.05). The frequencies of the CYP2C19*2 allele were 13 vs. 9.1%, respectively. CYP2C19*3 was not detected in either population. The 95% confidence intervals for the differences of frequencies of CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C9*4, CYP2C9*5, CYP2C9*11, CYP2C19*1, CYP2C19*2 and CYP2C19*3 between Belgian and Beninese were 7%, 19%; - 14%, - 6%; - 11%, - 4%; - 1%, 1%; 0%, 4%; 0%, 5%; - 10%, 2%; - 2%, 10%; - 1%; respectively. The distributions of CYP2C9 genotypes in the Beninese and Belgian individuals were: CYP2C9*1/*1: 91 vs. 67% (P < 0.00001); CYP2C9*1/*2: 0 vs. 18.2% (P < 0.0001); CYP2C9*1/*3: 0 vs. 11.6% (P < 0.001); CYP2C9*1/*5: 3.6 vs. 0% (P = 0.05); CYP2C9*1/*11: 5.4 vs. 0.8% (P = 0.05); CYP2C9*2/*3: 0 vs. 1.6% (NS); CYP2C9*3/*3: 0 vs. 0.8% (NS). The distributions of CYP2C19 genotypes between these ethnic groups were: CYP2C19*1/*1: 73.9 vs. 83.5% (NS); CYP2C19*1/*2: 26.1 vs. 14.9% (P < 0.05); CYP2C9*2/*2: 0 vs. 1.6% (NS). CONCLUSIONS: Differences of allele frequencies between Beninese and Belgian populations were statistically significant for CYP2C9*2, *3, *5 and *11, but not for CYP2C9*4 or for CYP2C19*2 and *3.

Oxidative stress, KLF6 and transforming growth factor-beta up-regulation differentiate non-alcoholic steatohepatitis progressing to fibrosis from uncomplicated steatosis in rats.

BACKGROUND/AIMS: Pathogenesis of non-alcoholic steatohepatitis (NASH) remains poorly understood. Cytochrome P450 2E1 (CYP 2E1), cytokines, oxidative stress and activation of hepatic stellate cells seem to play a role in this process. The aim was to determine the potential implication of these factors in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis. METHODS: Animals were fed a standard diet, a 5% orotic acid-diet (OA) developing hepatic steatosis, or the methionine-choline deficient (MCD) diet inducing steatohepatitis for 2 and 6 weeks. Lipid peroxidation, CYP 2E1 expression and activity, expression of UCP-2, interleukin (IL)-6, transforming growth factor (TGF)beta1, KLF6 mRNAs, and activation of hepatic stellate cells were examined by gas chromatography, high-performance liquid chromatography, Western blotting, quantitative polymerase chain reaction and immunohistochemistry. RESULTS: Lipid peroxidation increased in the MCD model whereas only minor changes occurred in the OA model. KLF6 and TGFbeta1 mRNAs were selectively up-regulated in MCD animals. Stellate cell activation, inflammation and collagen deposition only occurred in the MCD group. CYP 2E1 expression and activity increased in the OA group while both decreased in MCD animals. UCP-2 and IL-6 mRNA increased in both groups. CONCLUSIONS: In the context of steatosis, lipid peroxidation is associated with inflammation and stellate cell activation with concomitant increase in TGFbeta1 production, possibly through up-regulation of KLF6.

Rosuvastatin decreases caveolin-1 and improves nitric oxide-dependent heart rate and blood pressure variability in apolipoprotein E-/- mice in vivo.

BACKGROUND: Decreased heart rate variability (HRV) and increased blood pressure variability (BPV), determined in part by nitric oxide (NO)-dependent endothelial dysfunction, are correlated with adverse prognosis in cardiovascular diseases. We examined potential alterations in BPV and HRV in genetically dyslipidemic, apolipoprotein (apo) E-/-, and control mice and the effect of chronic statin treatment on these parameters in relation to their NO synthase (NOS)-modifying properties. METHODS AND RESULTS: BP and HR were recorded in unrestrained, nonanesthetized mice with implanted telemetry devices with or without rosuvastatin. Cardiac and aortic expression of endothelial NOS and caveolin-1 were measured by immunoblotting. Both systolic BP and HR were elevated in apoE-/- mice, with abolition of their circadian cycles. Spectral analysis showed an increase in their systolic BPV in the very-low-frequency (+17%) band and a decrease in HRV in the high-frequency (-57%) band, reflecting neurohumoral and autonomic dysfunction. Decreased sensitivity to acute injection of atropine or an NOS inhibitor indicated basal alterations in both parasympathetic and NOS regulatory systems in apoE-/- mice. Aortic caveolin-1 protein, an inhibitor of endothelial NOS, was also increased in these mice by 2.0-fold and correlated positively with systolic BPV in the very-low-frequency band. Rosuvastatin treatment corrected the hemodynamic and caveolin-1 expression changes despite persisting elevated plasma cholesterol levels. CONCLUSIONS: Rosuvastatin decreases caveolin-1 expression and promotes NOS function in apoE-/-, dyslipidemic mice in vivo, with concurrent improvements in BPV and HRV. This highlights the beneficial effects of rosuvastatin on cardiovascular function beyond those attributed to lipid lowering.

Effect of hepatic impairment on the pharmacokinetics of atomoxetine and its metabolites.

BACKGROUND AND OBJECTIVES: Atomoxetine is a treatment for attention-deficit/hyperactivity disorder and is primarily eliminated via cytochrome P4502D6 (CYP2D6). The pharmacokinetics of atomoxetine and its primary metabolites were investigated in 10 adults with hepatic impairment (6 moderate, 4 severe) and 10 age- and sex-matched control subjects, all being genotyped as CYP2D6 extensive metabolizers. METHODS: A single oral 20-mg dose of atomoxetine was given. Multiple blood samples were collected for 48 hours in healthy subjects and for 120 hours in patients. Urine was collected up to 24 hours. Before atomoxetine administration (10-20 days), sorbitol clearance and debrisoquin (INN, debrisoquine) metabolic ratio were determined as markers of hepatic blood flow and CYP2D6 activity, respectively. RESULTS: The systemic clearance of atomoxetine was significantly reduced in those with hepatic impairment compared with controls, thereby resulting in increased exposure (area under the concentration-time curve from time 0 to infinity, 1.58 versus 0.85 microg. h(-1). mL(-1); P =.035) but no change in maximum concentration. Mean 4-hydroxyatomoxetine area under the concentration-time curve from time 0 to time t and maximum concentration were increased approximately 7-fold and 2-fold, respectively (P =.0001 and P =.0056, respectively). For the glucuronide conjugate of 4-hydroxyatomoxetine, the mean half-life was longer and the mean area under the concentration-time curve from time 0 to infinity and the maximum concentration were lower (P =.0028, P =.003, and P =.0001, respectively). The sorbitol clearance was lower and the debrisoquin metabolic ratio was higher, reflecting reduced hepatic blood flow and decreased CYP2D6 activity, respectively. Decreased atomoxetine clearance in patients with hepatic impairment was clearly correlated with decreased CYP2D6 activity and decreased hepatic blood flow. Mean atomoxetine plasma protein binding was lower in patients with hepatic impairment compared with controls (96.5% versus 98.7%, P =.0008). Atomoxetine was well tolerated in the 2 populations. CONCLUSION: For patients with attention-deficit/hyperactivity disorder who have hepatic impairment, dosage adjustment is recommended. Initial target doses should be reduced to 25% and 50% of the normal dose for patients with severe and moderate hepatic impairment, respectively.

Watercress has no Importance for the elimination of ethanol by CYP2E1 inhibition.

Watercress, a cruciferous vegetable, is known to inhibit the metabolism of several CYP2E1 substrates such as paracetamol and chlorzoxazone. Since ethanol and its metabolite, acetaldehyde, are CYP2E1 substrates, the influence of watercress on ethanol and acetaldehyde was investigated in healthy human volunteers. According to a randomized cross-over design, ethanol and acetaldehyde pharmacokinetic parameters were determined in 9 persons at 3 occasions: without watercress and after watercress ingestion preceding ethanol consumption from 1 or 10.5 hr, respectively. Ethanol tmax occurred significantly later when watercress was ingested 1 hr before ethanol ingestion. Likewise, acetaldehyde Cmax was significantly higher whereas acetaldehyde AUCs were increased by watercress but not significantly. All other ethanol and acetaldehyde pharmacokinetic parameters were similar between the 3 treatments. In healthy volunteers, no major watercress effect was observed on ethanol clearance but a weak inhibiting effect on acetaldehyde metabolism is possible. Ethanol absorption is also delayed by single ingestion of watercress immediately preceding ethanol consumption.