A sensitive LC-MS/MS method for quantification of phenytoin and its major metabolite with application to in vivo investigations of intravenous and intranasal phenytoin delivery.

Phenytoin is a powerful antiseizure drug with complex pharmacokinetic properties, making it an interesting model drug to use in preclinical in vivo investigations, especially with regards to formulations aiming to improve drug delivery to the brain. Moreover, it has a major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, which can be simultaneously studied to achieve a better assessment of its behaviour in the body. Here, we describe the development and validation of a sensitive LCMS/MS method for quantification of phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in rat plasma and brain which can be used in such preclinical studies. Calibration curves produced covered a range of 7.81 to 250 ng/mL (plasma) and 23.4 to 750 ng/g (brain tissue) for both analytes. The method was validated for specificity, sensitivity, accuracy, and precision and found to be within the acceptable limits of ±15% over this range in both tissue types. The method when applied in two in vivo investigations: validation of a seizure model and to study the behaviour of a solution of intranasally administered phenytoin as a foundation for future studies into direct nose-to-brain delivery of phenytoin using specifically developed particulate systems, was highly sensitive for detecting phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in rat plasma and brain.

A sensitive LC-MS/MS method for the study of exogenously administered 13 C-oleoylethanolamide in rat plasma and brain tissue.

Oleoylethanolamide is an endogenous molecule with neuroprotective effects. It has been reported that exogenous oleoylethanolamide can be administered therapeutically, but the confounding presence of the endogenous molecule has led to conflicting reports regarding the mechanisms of the effects and highlights a need for an adequate methodology to differentiate them. We have developed a liquid chromatography-tandem mass spectrometry method to study oleoylethanolamide in rat plasma and brain using a 13 C-labeled isotope, 13 C-oleoylethanolamide. 13 C-oleoylethanolamide was extracted using a liquid-liquid extraction employing acetonitrile and tert-butyl methyl ether (1:4). Analysis was performed using a gradient with a total run time of 12 min. 13 C-oleoylethanolamide, d4 -oleoylethanolamide (internal standard), and 12 C-oleoylethanolamide (endogenous background) eluted simultaneously at 1.64 min. The method was validated for specificity, sensitivity, accuracy, and precision and found to be capable of quantification within acceptable limits of ±15% over the calibration range of 0.39-25 ng/mL for the plasma and 1.17-75 ng/g for the brain. It was then applied to quantify 13 C-oleoylethanolamide over 90 min after intravenous administration of a solution (1 mg/kg) in rats. Results suggest that 13 C-oleoylethanolamide does not reach therapeutic concentrations in the brain, despite a relatively prolonged plasma circulation, suggesting that rapid degradation in the brain remains an obstacle to its clinical application to neurological disease.

Cubosomes enhance drug permeability across the blood-brain barrier in zebrafish.

The potential of cubosomes to improve delivery of incorporated cargo to the brain was explored in zebrafish. Cubosomes were formulated with one of three stabilisers, Pluronic F68, Pluronic F127 or Tween 80, with the hypothesis that coating with Tween 80 will enable brain targeting of cubosomes as has been previously shown for polymeric nanoparticles. The physiochemical properties and the ability of the cubosomes to facilitate delivery of the model drug lissamine rhodamine (RhoB) into the brain was investigated. Distribution of cubosomes in the midbrain was also investigated by ultrastructural analysis via incorporation of octanethiol-functionalized gold nanoparticles. Cubosomes were typically 165-195 nm in size with a Pn3m (Pluronics) or Im3m (Tween 80) cubic phase internal structure. Cubosomes were injected intravenously into zebrafish larvae (12-14 days post fertilization) and the concentration of RhoB in the midbrain was determined by quantifying its fluorescence intensity. Uptake of RhoB was significantly greater in larvae injected with Tween 80 stabilized cubosomes as compared to a control suspension of RhoB or cubosomes stabilized with Pluronics. Collectively, we show for the first time that cubosomes can be functionalized to deliver drug across the BBB, offering new opportunities to overcome drug delivery issues across this formidable biological barrier.

Bulk and dispersed aqueous behaviour of an endogenous lipid, selachyl alcohol: Effect of Tween 80 and Pluronic F127 on nanostructure.

Tween 80 has been reported to provide a means of targeting drug nanocarriers to the blood- brain barrier. This study investigated the influence of addition of Tween 80 on the formation of different bulk and dispersed lyotropic liquid crystalline phases in selachyl alcohol-based systems. The effect of increasing concentrations of Tween 80 and Pluronic F127 (as a control) (0-25% w/w relative to SA) on the bulk phase behaviour and dispersions of selachyl alcohol (SA) were investigated using small angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy. The addition of Tween 80 to SA bulk phase samples triggered concentration-dependent phase changes with the structure sequentially evolving from a reverse hexagonal phase (H2) to a mixed H2 and inverse bicontinuous cubic (V2) then a V2 phase alone. In contrast, the addition of Pluronic F127 resulted in a phase change from H2 phase to a mixed lamellar and H2 phase system. The mean particle size of internally structured particles was 125-190 nm with low polydispersity indices (0.1-0.2). Nanoparticles retained the bulk phase internal structure in the presence of Tween 80, whereas in the presence of Pluronic F127, the additional lamellar phase that formed in bulk phase systems was not observed. Cryo-TEM revealed the formation of cubosomes and hexosomes by SA in excess water in the presence of Tween 80 and Pluronic F127 respectively. In summary, it was shown that stabilisation of SA dispersions using Tween 80 resulted in a decrease in negative curvature leading to a change in internal structure from H2 to V2 phase. The studies provide the core understanding of particle structure to progress these structured lipid nanocarriers into delivery studies with Tween 80 as a mechanism to target the blood-brain barrier.

Reliability and validity of the 12-item WHODAS 2.0 in patients with Kashin-Beck disease.

The purpose of this study was to check the reliability and validity of the 12-item Chinese version of the World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0) for the assessment of disability in patients with Kashin-Beck disease (KBD). We recruited 219 patients with KBD from the high-risk KBD area in the Shaanxi province, using stratified multistage random sampling. We assessed each patient using the Chinese version of the 12-item WHODAS 2.0 and the Western Ontario and McMaster Universities Index of Osteoarthritis (WOMAC). Statistical evaluations of the instruments consisted of Cronbach's alpha, intraclass correlation coefficient (ICC), confirmatory factor analysis (CFA), and Pearson's correlation coefficient. Cronbach's alpha and ICC for the six domains ranged from 0.704 to 0.906 and 0.690 to 0.852, respectively. A six-factor structure fits the data well (CFI = 0.967, TLI = 0.944, RMSEA = 0.08). Regarding convergent validity, the four domains of the 12-item WHODAS 2.0 (getting around, self-care, life activity, and participation) showed moderate-to-strong correlation for all three domains of the WOMAC (0.428 < |r| < 0.804). Regarding divergent validity, the two domains of the 12-item WHODAS 2.0 (understanding and communication, and getting along with people) showed weak correlation for the three domains of WOMAC (0.182 < |r| < 0.295). The Chinese version of 12-item WHODAS 2.0 questionnaire is a reliable and valid instrument when administered to KBD patients.

Reliability and validity of the EQ-5D-3L for Kashin-Beck disease in China.

BACKGROUND: Kashin-Beck Disease (KBD) is an endemic osteoarthropathy in areas which extend from the North-East to the South-West of China. Most of the patients with KBD suffer multiple dysfunctions in major joints causing decreased health status. However because of their low education level and unique living habits, it is hard to find tools to measure the health-related quality of life (HRQOL). European quality of life (EQ-5D-3L) patient-reported instrument is widely used to measure HRQOL. This study aimed to establish the validity and reliability of the Chinese version of the EQ-5D-3L for evaluating HRQOL of KBD individuals in rural area. METHODS: 368 individuals who were suffering from KBD were recruited through stratified multistage random sampling from Shaanxi province, China. The EQ-5D-3L and the WHOQOL-BREF were administrated in each individual by face to face interview. Test-retest reliability was assessed at 10-14 days intervals. The test-retest reliability was measured by calculating the Kappa coefficients for EQ-5D-3L five dimensions. For the EQ VAS, the intraclass correlation coefficient (ICC) was computed. Convergent and divergent analysis, construct validity was established using Spearman's rank correlation between the EQ-5D-3L and the WHOQOL-BREF. Known groups' validity was examined by comparing groups with a priori expected differences in health-related quality of life (HRQOL). RESULTS: For 362 individuals (98%), comprehensive data of all the EQ-5D-3L dimensions were available. Kappa values of the EQ-5D-3L five items ranged from 0.324 to 0.554. ICC of the EQ VAS was 0.497. For convergent validity, the three items (self-care, usual activity, and mobility) of EQ-5D-3L, index scores, and VAS showed moderate correlations with the physical health domain of the WHOQOL-BREF (r absolute value ranged from 0.339 to 0.475). For divergent validity, the 5 items of EQ-5D-3L showed weak or no correlations with environment and social relationship domains of WHOQOL-BREF. The Chinese EQ-5D-3L clearly demarcated between groups which were reporting severe disease degree, poorer general health, more number of painful joints with worse HRQOL. CONCLUSIONS: The EQ-5D-3L Chinese Version demonstrated fair to moderate levels of test-retest reliability and adequate construct validity in KBD individuals in China.

Incorporation of an Endogenous Neuromodulatory Lipid, Oleoylethanolamide, into Cubosomes: Nanostructural Characterization.

Oleoylethanolamide (OEA) is an endogenous lipid with neuroprotective properties and the fortification of its concentration in the brain can be beneficial in the treatment of many neurodegenerative disorders. However, OEA is rapidly eliminated by hydrolysis in vivo, limiting its therapeutic potential. We hypothesize that packing OEA within a nanoparticulate system such as cubosomes, which can be used to target the blood-brain barrier (BBB), will protect it against hydrolysis and enable therapeutic concentrations to reach the brain. Cubosomes are lipid-based nanoparticles with a unique bicontinuous cubic phase internal structure. In the present study, the incorporation and chemical stability of OEA in cubosomes was investigated. Cubosomes containing OEA had a mean particle size of less than 200 nm with low polydispersity (polydispersity index <0.25). Infrared spectroscopy and high-performance liquid chromatography showed chemical stability and the encapsulation of OEA within cubosomes. Cryo-TEM and SAXS measurements were used to probe the influence of the addition of OEA on the internal structure of the cubosomes. Up to 30% w/w OEA (relative to phytantriol) could be incorporated into phytantriol cubosomes without any significant disruption of the nanostructure of the cubosomes. Combined, the results indicate that OEA-loaded cubosomes have the potential for application as a colloidal carrier for OEA, potentially preventing hydrolysis in vivo.

Serum leptin values in the healthy obese and non-obese subjects of Rawalpindi.

OBJECTIVE: To determine serum leptin concentrations from a sample of Rawalpindi population in relation to body mass index, age and gender. METHODS: The observational, comparative study was conducted at the Armed Forces Institute of Pathology, Rawalpindi, and Benazir Bhutto Hospital, Rawalpindi from August 2008 to December 2008. Subjects were 100 including healthy obese, overweight and non-obese of both genders aged between 20-50 years. Sampling was done by non-probability convenience method. Body Mass Index was calculated by formula BMI = weight in kg/height in m2: non-obese subjects were defined as 18.5-23.0 kg/m2; overweight 23.1-27.4 kg/m2; and obese 27.5-40 kg/m2. Serum glucose was measured using Glucose oxidase-phenol amino phenazone (GOD-PAP) method and serum leptin by sandwich enzyme-linked immunosorbent assay method. RESULTS: Serum leptin concentrations were higher in obese subjects (mean 52.8 +/- 24.6 ng/mL; range 28.2-77.4 ng/mL; P < 0.001) than in non-obese subjects (mean 12.7 +/- 6.1 ng/mL, range 6.6-18.8ng/mL). Mean Body Mass Index in obese group was 31.7 +/- 3.1 kg/m2 (range 28.6-34.8 kg/m2) while it was 21.2 +/- 1.5 kg/m2 (range 19.7-22.7 kg/m2) in the nonobese group. Body Mass Index was strongly positively correlated with serum leptin concentration (r = 0.59, p < 0.001) in the obese group. The mean serum leptin concentration was much higher in the healthy obese and non-obese women (64.4 ng/mL and 8.7 ng/mL respectively) than in men of both categories (40.4 ng/mL and 5.5 ng/mL respectively). Age had no significant relation with serum leptin level (p = 0.416). CONCLUSIONS: In the study sample, serum leptin concentration was positively correlated with Body Mass Index in healthy obese and non-obese subjects of both genders. The levels were higher in women than in men. Age had no significant relation with serum leptin level in this age group.

Hyperendemic pulmonary tuberculosis in peri-urban areas of Karachi, Pakistan.

BACKGROUND: Currently there are very limited empirical data available on the prevalence of pulmonary tuberculosis among residents of marginalized settings in Pakistan. This study assessed the prevalence of pulmonary tuberculosis through active case detection and evaluated predictors of pulmonary tuberculosis among residents of two peri-urban neighbourhoods of Karachi, Pakistan. METHODS: A cross-sectional study was conducted in two peri-urban neighbourhoods from May 2002 to November 2002. Systematic sampling design was used to select households for inclusion in the study. Consenting subjects aged 15 years or more from selected households were interviewed and, whenever possible, sputum samples were obtained. Sputum samples were subjected to direct microscopy by Ziehl-Neelson method, bacterial culture and antibiotic sensitivity tests. RESULTS: The prevalence (per 100,000) of pulmonary tuberculosis among the subjects aged 15 years or more, who participated in the study was 329 (95% confidence interval (CI): 195-519). The prevalence (per 100,000) of pulmonary tuberculosis adjusted for non-sampling was 438 (95% CI: 282-651). Other than cough, none of the other clinical variables was significantly associated with pulmonary tuberculosis status. Analysis of drug sensitivity pattern of 15 strains of Mycobacterium tuberculosis revealed that one strain was resistant to isoniazid alone, one to streptomycin alone and one was resistant to isoniazid and streptomycin. The remaining 12 strains were susceptible to all five drugs including streptomycin, isoniazid, rifampicin, ethambutol, and pyrazinamide. CONCLUSION: This study of previously undetected tuberculosis cases in an impoverished peri-urban setting reveals the poor operational performance of Pakistan's current approach to tuberculosis control; it also demonstrates a higher prevalence of pulmonary tuberculosis than current national estimates. Public health authorities may wish to augment health education efforts aimed at prompting health-seeking behaviour to facilitate more complete and earlier case detection. Such efforts to improve passive case-finding, if combined with more accessible DOTS infra-structure for treatment of detected cases, may help to diminish the high tuberculosis-related morbidity and mortality in marginalized populations. The economics of implementing a more active approach to case finding in resource-constrained setting also deserve further study.