Aims: The effect of transdermal nicotine on stress reactivity was investigated in currently smoking, detoxified, substance-dependent individuals (65% alcohol dependent, n = 51; 31 male) following a psychosocial stressor. Methods: Using a randomized, double-blind, placebo-controlled design, subjects were assigned to receive either active transdermal nicotine (low or high dose) or placebo. Six hours following nicotine administration, subjects performed a laboratory psychosocial stressor consisting of two 4-min public-speaking sessions. Results: Consistent with prior reports, substance-dependent individuals displayed a blunted stress response. However, a review of the cortisol distribution data encouraged additional analyses. Notably, a significant minority of the substance-dependent individuals (33%) demonstrated elevated poststress cortisol levels. This group of responders was more likely to be alcohol dependent and to have received the high dose of nicotine [χ2(2) = 32, P < 0.0001], [χ2(2) = 18.66, P < 0.0001]. Differences in salivary cortisol responses between responders and nonresponders could not be accounted for by the length of sobriety, nicotine withdrawal levels, anxiety or depressive symptomatology at the time of the psychosocial stressor. Conclusion: These results suggest that nicotine administration may support a normalization of the salivary cortisol response following psychosocial stress in subgroups of substance-dependent individuals, particularly those who are alcohol dependent. Given the association between blunted cortisol levels and relapse, and the complex actions of nicotine at central and peripheral sites, these findings support the systematic study of factors including nicotine, which may influence stress reactivity and the recovery process in alcohol-dependent individuals.

A rapid method to determine fexofenadine concentrations in human plasma using protein precipitation in 96-well plates and liquid chromatography-tandem mass spectrometry was validated. Plasma proteins were precipitated with acetonitrile containing the internal standard fexofenadine-d6, mixed briefly, and then filtered into a collection plate. The resulting filtrate was diluted and injected onto a Phenomenex Gemini C18 (50 × 2.0 mm, 5 micron) analytical column. The mobile phase consisted of 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (35:65, v/v). The flow rate was 0.2 ml/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization and high resolution multiple reaction monitoring mode (H-SRM). The linear standard curve ranged from 1 to 500 ng/ml and the precision and accuracy (intra- and inter-run) were within 4.3% and 8.0%, respectively. The method has been applied successfully to determine fexofenadine concentrations in human plasma samples obtained from subjects administered a single oral dose of fexofenadine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving fexofenadine.

Study Objective

To evaluate whether the level of systemic exposure to atenolol explains observed interindividual differences in adverse metabolic responses.

Design

Open-label, prospective, pharmacokinetic pilot substudy of the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study.

Setting

General clinical research center.

Patients

Fifteen hypertensive adults (mean age 46 ± 8.9 yrs) who were enrolled in the PEAR study.

Intervention

Patients received atenolol therapy for at least 8 weeks, with 5 of those weeks at a dosage of 100 mg/day, and then underwent a 2-hour oral glucose tolerance test during a pharmacokinetic study visit.

Measurements and Main Results

Twenty-hour plasma atenolol concentrations were measured during the pharmacokinetic visit. Glucose and insulin levels were measured during the 2-hour oral glucose tolerance test, and fasting plasma lipid, glucose, and insulin levels were measured at baseline and after 8 weeks of atenolol treatment. A significant association was noted between atenolol area under the concentration-time curve (AUC) and change in fasting glucose level when adjusted for covariates (p=0.0025); the effect was strongest in women. No significant relationship was noted between plasma atenolol concentration and glucose AUC during oral glucose tolerance testing (r=0.08, p=0.78), nor between atenolol AUC and change in triglyceride levels (r=0.13, p=0.63).

Conclusion

Higher plasma atenolol exposure may be a risk factor for an increase in fasting plasma glucose level during atenolol treatment. These findings require confirmation in a larger sample.

Objective

To examine the phenotypic expression of CYP2E1 in liver transplant patients, as measured by the in vivo probe chlorzoxazone, and to evaluate CYP2E1 activity over time after transplantation.

Methods

Thirty-three stable liver transplant patients were given 250 mg chlorzoxazone within 1 year after transplantation as part of a multiprobe CYP cocktail; urine and blood were collected for 8 hours. Chlorzoxazone and 6-hydroxychlorzoxazone concentrations were determined by HPLC. Twenty-eight healthy control subjects, eight patients with moderate to severe liver disease, and four patients who had not received liver transplants were also studied for comparison. The chlorzoxazone metabolic ratio, calculated as the plasma concentration of 6-hydroxychlorzoxazone/chlorzoxazone at 4 hours after chlorzoxazone administration, was used as the phenotypic index. In a subgroup of patients and control subjects, additional blood samples were obtained to allow for the calculation of chlorzoxazone pharmacokinetic parameters by noncompartmental methods.

Results

The chlorzoxazone metabolic ratio for the liver transplant patients in the first month after transplantation (mean ± SD, 6.4 ± 5.1) was significantly higher than that after 1 month after surgery (2.1 ± 2.0), when the chlorzoxazone metabolic ratio was not different from control subjects (0.8 ± 0.5). The chlorzoxazone metabolic ratios in the patients who had not received liver transplants (1.1 ± 0.7) were equivalent to those of healthy control subjects. The maximum observed 6-hydroxychlorzoxazone plasma concentration was 3046 ± 1848 ng/ml in seven liver transplant patients in the first month after surgery compared with 1618 ± 320 ng/ml in 16 healthy control subjects (p < 0.05). The maximum observed concentration of chlorzoxazone, the chlorzoxazone apparent oral clearance, and the formation clearance of 6-hydroxychlorzoxazone were also significantly different between the groups.

Conclusions

We conclude that significant induction of CYP2E1, as indicated by the chlorzoxazone metabolic ratio, occurs in the first month after sugery in liver transplant patients and that drugs that are substrates for CYP2E1 may require dosage alteration during that period. Contrary to expectations, drug metabolism is not uniformly depressed after liver transplantation.

Uric acid (UA) is known to be a major biological antioxidant in plasma. However, there is a strong correlation between UA levels and cardiovascular risk. Recent studies suggest that in the intracellular environment, UA can become a prooxidant that causes endothelial dysfunction. For conducting detailed studies of UA’s role in human pathogenesis, there is a critical need for a sensitive and specific method for the determination of intracellular UA levels. We therefore developed a simple, sensitive method for determination of trace amounts of intracellular UA, as well as comparatively large amounts of UA in plasma and urine (for the determination of extracellular concentrations of UA), based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). UA was separated from interferences by HPLC and quantified by mass spectrometry in the negative ESI mode using single reaction monitoring (SRM). For the identification and quantification of UA, the parent ions selected were m/z 167.0, which corresponds to the urate anion, and m/z 169.0, which corresponds to the 1,3-15N–UA anion. 1,3-15N–UA is used as an internal standard to ensure accuracy of the measurement. After precipitation of proteins with 10% TCA solution, UA was subjected to LC-MS/MS analysis. The correlation coefficient was 0.9998 to 1.0000 based on the calibration curve. The intra- and inter-day precision (C.V. %) ranged from 0.01 to 3.07 and 0.01 to 3.68 in vivo and in vitro systems. Recovery tests of added standards have been successfully performed and the values ranged from 90.10 to 103.59 % and 98.74 to 106.12 % for in vivo and in vitro analyses, respectively. This study demonstrates that intracellular levels of UA can be measured using LC-MS/MS with isotope labeled UA as an internal standard.

Background

Coenzyme Q10 (CoQ10) is a provitamin synthesized via the HMG-CoA reductase pathway, and thus may serve as a potential marker of intrinsic HMG-CoA reductase activity. HMG-CoA reductase inhibitors (statins) decrease CoQ10, although it is unclear whether this is due to reductions in lipoproteins, which transport CoQ10.

Objectives

We evaluated whether baseline plasma CoQ10 concentrations predict the lipid-lowering response to high-dose atorvastatin, and to what extent CoQ10 changes following atorvastatin therapy depend on lipoprotein changes.

Methods

Individuals without dyslipidemia or known cardiovascular disease (n=84) received atorvastatin 80 mg daily for 16 weeks. Blood samples collected at baseline and after 4, 8, and 16 weeks of treatment were assayed for CoQ10.

Results

Individuals with higher baseline CoQ10:LDL-C ratios displayed diminished absolute and percent LDL-C reductions at 8 and 16 weeks of atorvastatin treatment (P<0.001 to 0.01). After 16 weeks of atorvastatin, plasma CoQ10 decreased 45% from 762±301 ng/ml to 374±150 ng/ml (P<0.001). CoQ10 changes were correlated with LDL-C and apolipoprotein B changes (r=0.27-0.38, P=0.001-0.02), but remained significant when normalized to all lipoproteins. CoQ10 changes were not associated with adverse drug reactions.

Conclusion

Baseline CoQ10:LDL-C ratio was associated with the degree of LDL-C response to atorvastatin. Atorvastatin decreased CoQ10 concentrations in a manner that was not completely dependent on lipoprotein changes. The utility of CoQ10 as a predictor of atorvastatin response should be further explored in patients with dyslipidemia.

Uric acid (UA) can be directly converted to allantoin enzymatically by uricase in most mammals except humans or by reaction with superoxide. UA can react directly with nitric oxide to generate 6-aminouracil and with peroxynitrite to yield triuret; both of these metabolites have been identified in biological samples. We now report a validated high-performance liquid chromatography and tandem mass spectrometry method for the determination of these urinary UA metabolites. Urine samples were diluted 10-fold, filtered and directly injected onto HPLC for LC–MS/MS analysis. The urinary metabolites of UA were separated using gradient HPLC. Identification and quantification of UA urinary metabolites was performed with electrospray in positive ion mode by selected-reaction monitoring (SRM). Correlation coefficients were 0.991–0.999 from the calibration curve. The intra-and inter-day precision (R.S.D., %) of the metabolites ranged from 0.5% to 13.4% and 2.5–12.2%, respectively. In normal individuals (n = 21), urinary allantoin, 6-aminouracil and triuret, were 15.30 (±8.96), 0.22 (±0.12), and 0.12 (±0.10) μg/mg of urinary creatinine (mean (±S.D.)), respectively. The new method was used to show that smoking, which can induce oxidative stress, is associated with elevated triuret levels in urine. Thus, the method may be helpful in identifying pathways of oxidative stress in biological samples.

Objectives

To establish and assess the effectiveness of a 10-week summer research program on increasing doctor of pharmacy (PharmD) students' interest in research, particularly as it related to future career choices.

Design

Survey instruments were sent to 25 participants who had completed the research program in the summer of 2004, 2005, or 2006 to assess their satisfaction with the program and its influence on their career choices after graduation.

Assessment

Respondents reported a high degree of satisfaction with the program, indicating that the program allowed them to determine their suitability for a career in research, and 55% reported their intention to pursue additional research training.

Conclusion

A brief introduction to the clinical research environment helped pharmacy students understand the clinical sciences and careers in research. The introduction increased the likelihood of students pursuing a research career path after obtaining their PharmD degree.

The effect of a single dose of ceftazidime on circulating concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in a rat model of sepsis was studied. IL-6 concentrations were significantly elevated (100 to 200 times the baseline) 6 h after ceftazidime administration in both septic and nonseptic (control) rats. TNF-α concentrations increased significantly in nonseptic (∼40 times the baseline) rats but not septic (∼2 to 3 times the baseline) rats. Ceftazidime administration was not associated with an increase in endotoxin concentrations. These findings suggest that ceftazidime modulation of proinflammatory cytokine concentrations may be independent of its antimicrobial properties.

Although several dosage adjustment regimens have been proposed, there is little quantitative information to guide the initiation of ceftazidime therapy in patients who are receiving continuous renal replacement therapy. To determine the clearance of ceftazidime by continuous venovenous hemofiltration (CVVH) and continuous venovenous hemodialysis (CVVHD), we performed controlled clearance studies with stable hemodialysis patients with three hemofilters: a 0.6-m2 acrylonitrile copolymer (AN69; Hospal) filter, a 2.1-m2 polymethylmethacrylate filter (PMMA; Toray) filter and a 0.65-m2 polysulfone (PS; Fresenius) filter. Subjects received 1,000 mg of ceftazidime intravenously prior to the start of a clearance study. The concentration of ceftazidime in multiple plasma and dialysate or ultrafiltrate samples was determined by high-performance liquid chromatography. The diffusional clearances (CIdiffusion) and sieving coefficients of ceftazidime were compared by a mixed-model repeated-measures analysis of variance with filter and blood, dialysate inflow, or ultrafiltration rate as the main effect and the patient as a random effect. The fraction of ceftazidime bound to plasma proteins was 17% ± 7% (range, 10 to 25%). The clearances of ceftazidime, urea, and creatinine by CVVHD were essentially constant at blood flow rates of 75 to 250 ml/min for all three filters. Significant linear relationships (P < 0.0001) were observed between CIdiffusion of ceftazidime and clearance of urea for all three filters: AN69 (slope = 0.83), PMMA (slope = 0.89), and PS (slope = 1.03). Ceftazidime clearance was membrane independent during CVVH and CVVHD. CVVH and CVVHD can significantly augment the clearance of ceftazidime. Dosing strategies for initiation of ceftazidime therapy in patients receiving CVVH and CVVHD are proposed.