Quantitation of thiorphan in human plasma using LC-MS/MS and its application to a bioequivalence study.

Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1-200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions.

In vitro dissolution equivalence of Jordanian sildenafil generics via validated, stability-indicating HPLC method.

This study was conducted to compare dissolution profiles of four Jordanian registered sildenafil (SDF) products to the originator. Dissolution samples were analyzed utilizing a validated and stability-indicating HPLC method in human plasma. Validation was performed for specificity, linearity, limit of detection, lower limit of quantification, precision, trueness and stability. SDF was extracted from plasma samples using liquid-liquid extraction. The analysis was performed utilizing isocratic elution on C18 column with 1.0 ml/min flow rate. The regression value was ∼0.999 over 3 days with drug recovery between 86.6 to 89.8%with 10 ng/ml lower limit of quantitation. This method displayed a good selectivity of SDF with improved stability under various conditions. The method was used for SDF quantification in dissolution medium. Similarity factors for local products varied according to the used mediums, but all SDF local products passed the dissolution in vitro test since all of them showed a released of >85% after 60 min at the dissolution mediums.

[Box: see text].

Mellitin peptide quantification in seasonally collected crude bee venom and its anticancer effects on myelogenous K562 human leukaemia cell line.

BACKGROUND: Apitherapy is an emerging field in cancer research, particularly in developing communities. The potency of Melittin (MEL), a major constituent in bee venom is accounted for the cytotoxic capacity against cancer cells. It is postulated that the genotype of bees and the time of venom collection influences its specific activity against certain types of cancer. METHOD: Hereby, Jordanian crude bee venom (JCBV) was collected during different seasons of the year, specifically spring, summer and autumn and investigated for in vitro antitumour effects. Venom collected during springtime comprised the highest quantity of MEL in comparison to venom collected some other time. Springtime-collected JCBV extract and MEL were tested on an immortal myelogenous leukaemia cell line, namely K562 leukemic cells. Treated cells were examined for cell modality via flow cytometry analysis and cell death mediating gene expressions. RESULTS: Springtime-collected JCBV extract and MEL showed an IC50 of 3.7 ± 0.37 µg/ml and 1.84 ± 0.75 µg/ml, respectively. In comparison to JCBV and positive control, MEL-treated cells exhibited late apoptotic death with a moderate cellular arrest at G0/G1 and an increase of cell number at G2/M phase. Expression of NF-κB/MAPK14 axis was inhibited in MEL and JCBV-treated cells, as well as expression of c-MYC and CDK4. Moreover, marked upregulation in ABL1, JUN and TNF was observed. In conclusion, springtime-collected JCBV showed the highest content of MEL while both JCBV and pure MEL showed apoptotic, necrotic, and cell cycle arrest efficiency against K562 leukemic cells. CONCLUSION: Integration of bee venom in chemotherapy needs more investigation and should be carefully translated into clinical use. During such translation, the correlation of bee genotype, collection time and concentration of MEL in CBV should be profiled.

Lipids Alterations Associated with Metformin in Healthy Subjects: An Investigation Using Mass Spectrometry Shotgun Approach.

Metformin is an orally effective insulin-sensitizing drug widely prescribed for treating type 2 diabetes mellitus (T2DM). Metformin has been reported to alter lipid metabolism. However, the molecular mechanisms behind its impact on lipid metabolism remain partially explored and understood. In the current study, mass spectrometry-based lipid profiling was used to investigate the lipidomic changes in the serum of 26 healthy individuals after a single-dose intake of metformin. Samples were analyzed at five-time points: preadministration, before the maximum concentration of metformin (Cmax), Cmax, after Cmax, and 36 h post-administration. A total of 762 molecules were significantly altered between the five-time points. Based on a comparison between baseline level and Cmax, metformin significantly increased and decreased the level of 33 and 192 lipids, respectively (FDR ≤ 0.05 and fold change cutoff of 1.5). The altered lipids are mainly involved in arachidonic acid metabolism, steroid hormone biosynthesis, and glycerophospholipid metabolism. Furthermore, several lipids acted in an opposed or similar manner to metformin levels and included fatty acyls, sterol lipids, glycerolipids, and glycerophospholipids. The significantly altered lipid species pointed to fundamental lipid signaling pathways that could be linked to the pleiotropic effects of metformin in T2DM, insulin resistance, polycystic ovary syndrome, cancer, and cardiovascular diseases.

A Metabolic Pattern in Healthy Subjects Given a Single Dose of Metformin: A Metabolomics Approach.

Metformin is a widely prescribed medication for the treatment of type 2 diabetes mellitus (T2DM). It possesses effective roles in various disorders, including cancer, dyslipidemia, and obesity. However, the underlying mechanisms of metformin's multiple benefits are not fully understood. Herein, a mass spectrometry-based untargeted metabolomics approach was used to investigate the metabolic changes associated with the administration of a single dose of metformin in the plasma of 26 healthy subjects at five-time points; pre-dose, before the maximum concentration of metformin (Cmax), Cmax, after Cmax, and 36 h post-dose. A total of 111 metabolites involved in various biochemical processes were perturbed, with branched-chain amino acid (BCAA) being the most significantly altered pathway. Additionally, the Pearson similarity test revealed that 63 metabolites showed a change in their levels dependent on metformin level. Out of these 63, the level of 36 metabolites was significantly altered by metformin. Significantly altered metformin-dependent metabolites, including hydroxymethyl uracil, propionic acid, glycerophospholipids, and eicosanoids, pointed to fundamental biochemical processes such as lipid network signaling, energy homeostasis, DNA lesion repair mechanisms, and gut microbiota functions that could be linked to the multiple beneficial roles of metformin. Thus, the distinctive metabolic pattern linked to metformin administration can be used as a metabolic signature to predict the potential effect and mechanism of actions of new chemical entities during drug development.

The Prevalence of Overweight and Obesity Among Women in Jordan: A Risk Factor for Developing Chronic Diseases.

OBJECTIVE: The study aimed to investigate the prevalence of obesity among Jordanian women and its association with a wide range of chronic diseases. METHODS: Subjects were enrolled in the present cross-sectional study based on a random drop-off technique at the Obstetrics and Gynecology clinics at Jordan University Hospital. Initially, any female 18 years of age and older was asked to enroll in the study. Relevant data were gathered using a questionnaire composed of 30 questions, and body mass index (BMI) was determined from each participant's weight and height. The following variables were collected: socio-demographic, chronic diseases, and health status. Each variable's frequencies were reported, and the 95% confidence interval (95% CI) for each variable was calculated. For association analysis, Chi-square analysis was performed with an odds ratio (OR) and 95% CI. Multinomial logistic regression analysis was applied to a combination of independent variables and a dependent condition with covariate factors. RESULTS: The age-standardized prevalence of overweight/obese Jordanian women was 70.6% (95% CI 66.0-74.8%). On the other hand, the age-standardized prevalence of only obese women was 36.4 (95% Cl 31.9-41.2%). Furthermore, the association between age and overweight/obesity was significant (p<0.0001). The percentage of overweight and obesity started to be significant in the 30-39 year age group. Moreover, the OR for obesity ranged from 2.7 to 7.0 (p<0.05-0.01) for those women with only elementary education. Besides, high parity was significantly associated with obesity and elementary education. For chronic conditions, the percentages of hypertension, diabetes, hypertriglyceridemia, osteoporosis, and rheumatoid arthritis were significantly correlated with increased BMI in Jordanian women. With age adjustment, however, only hypertension was associated with obese level 3 with OR of 7.2 and 95% CI of 2.1-25.1 (p<0.01). CONCLUSION: There is a high prevalence of overweight/obesity among women in Jordan, which was related to high parity and low education level. This high prevalence of obesity increased the incidence of chronic diseases, such as hypertension. Therefore, community-based multiple strategies are required to combat obesity in Jordanian women.

Age of Natural Menopause Among Jordanian Women and Factors Related to Premature and Early Menopause.

OBJECTIVE: The aim of this study was to assess factors related to the onset of premature/early natural menopause among Jordanian women. METHODS: A cross-sectional study was conducted in early 2016. Subjects were enrolled based on random drop-off technique to the Obstetrics and Gynecology clinics at the Jordan University Hospital. Women 18 years of age and above were initially eligible to enroll, and women who had surgically induced menopause or specific disease were excluded from the analysis. Relevant data were collected using a questionnaire that included 30 questions. The following variables were collected: socio-demographic, body mass index, chronic conditions, diseases, reproductive characteristics, and health status. Hormone indicators of menopause were tested by measuring estrogen (E2) and follicle-stimulating hormone (FSH) levels. Age at natural menopause (ANM) was self-reported retrospectively and considered an independent variable against BMI, smoking, hormone therapy, and concomitant diseases. Association analysis and multinomial logistic regression were used to examine the associated factors of ANM with adjusted odds ratios (ORs), and their 95% confidence intervals (CIs) were reported. RESULTS: A total of 409 women were included in the analysis, aged between 20-75 years. The mean ANM in our sample was 48.5±5.0, with 2.7% of the women experienced premature menopause (ANM <40) and 7.8% early menopause (ANM 40-44). Within the menopause women (n=242), the percentage of women who had premature menopause was 4.5%, 13.6% with early menopause, and 21.1% with late menopause (ANM >52). Smoking was the major risk factor for premature/early menopausal age among Jordanian women with an OR of 2.46 (95% CI: 1.08-5.59, p<0.05). On the other hand, women with occasional arthritis symptoms and diseases such as hypertension, diabetes, dyslipidemia, and their combination were associated with average (45-52 years) or late menopause (>52 years). CONCLUSION: Smoking is the main contributor of premature/early menopause in Jordanian women. Increased awareness and public health policy about the adverse effects of smoking on women's reproductive health are needed.

The effect of grape seed and green tea extracts on the pharmacokinetics of imatinib and its main metabolite, N-desmethyl imatinib, in rats.

BACKGROUND: Imatinib is mainly metabolized by CYP3A4 and to a lesser extent by other isoenzymes, with N-desmethyl imatinib being its major equipotent metabolite. Being a CYP3A4 substrate, imatinib co-administration with CYP3A4 modulators would change its pharmacokinetic profile. The cancer chemoprevention potential and anticancer efficacy of many herbal products such as grape seed (GS) and green tea (GT) extracts had led to an increase in their concomitant use with anticancer agents. GS and GT extracts were demonstrated to be potent inhibitors of CYP3A4. The aim of this study is to investigate the effect of standardized GS and/or GT extracts at two different doses on the pharmacokinetics of imatinib and its metabolite, N-desmethyl imatinib, in SD-rats. METHODS: Standardized GS and/or GT extracts were administered orally once daily for 21 days, at low (l) and high (h) doses, 50 and 100 mg/kg, respectively, before the administration of a single intragastric dose of imatinib. Plasma samples were collected and analyzed for imatinib and N-desmethyl imatinib concentrations using LC-MS/MS method, then their non-compartmental pharmacokinetic parameters were determined. RESULTS: h-GS dose significantly decreased imatinib's Cmax and the [Formula: see text] by 61.1 and 72.2%, respectively. Similar effects on N-desmethyl imatinib's exposure were observed as well, in addition to a significant increase in its clearance by 3.7-fold. l-GT caused a significant decrease in imatinib's Cmax and [Formula: see text] by 53.6 and 63.5%, respectively, with more significant effects on N-desmethyl imatinib's exposure, which exhibited a significant decrease by 79.2 and 81.1%, respectively. h-GT showed similar effects as those of l-GT on the kinetics of imatinib and its metabolite. However, when these extracts were co-administered at low doses, no significant effects were shown on the pharmacokinetics of imatinib and its metabolite. Nevertheless, increasing the dose caused a significant decrease in Cmax of N-desmethyl imatinib by 71.5%. CONCLUSIONS: These results demonstrated that the pharmacokinetics of imatinib and N-desmethyl imatinib had been significantly affected by GS and/or GT extracts, which could be partially explained by the inhibition of CYP3A-mediated metabolism. However, the involvement of other kinetic pathways such as other isoenzymes, efflux and uptake transporters could be involved and should be characterized.

High performance liquid chromatography - Mass spectrometric bioanalytical method for the determination of dapoxetine in human plasma: Application for bioequivalence study.

Dapoxetine is an oral medication used for treatment of premature ejaculation (PE) in men aged (18-64 years). In this study, we present a validated, precise and sensitive method for determination of dapoxetine in human plasma by liquid chromatography/ electrospray ionization-tandem mass spectrometry. Dapoxetine and the internal standard (Dapoxetine- d6) were extracted from plasma via liquid-liquid extraction (LLE). The LC separation was performed utilizing ACE C8 (4.6 X50) mm, 5 µm column. The mobile phase was composed of acetonitrile and buffer (0.01 M Ammonium acetate +0.02% Formic acid solution) (85:15, v/v). The method was linear within the concentration range of 5.0-600 ng/mL for Dapoxetine in human plasma. Short analytical run was achieved with 1.6 min run time. Intra-day and inter-day accuracy was between 97 and 106% with precision (CV, %) of ≤ 5% achieved across all the quality control samples. Dapoxetine was stable in several conditions with recovery rates > 90%. This method was utilized successfully in clinical pharmacokinetic study following oral administration of 60 mg Dapoxetine tablets in 36 healthy male subjects. The result for all 90% confidence intervals were within the preset ranges. The method proved to be highly reproducible and sensitive and thus can be employed in bioequivalence studies and large scale sample analysis of Dapoxetine.

Development and Validation of Liquid Chromatography Method for Determination of Glimepiride in Presence of (Vimto®) Soft Drinks in Rats: Application to Pharmacokinetics Studies.

CONTEXT: Diet and beverages are thought to have notable effects on drugs. Recently, this relationship has received significant consideration. AIMS: To develop and validate a simple, rapid, and sensitive method for the determination of glimepiride in rat serum. This will be performed using high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Potential pharmacokinetic interactions between glimepiride and the soft drink, Vimto, will also be investigated in the serum of experimental rats. MATERIALS AND METHODS: HPLC-MS/MS was constructed and clarithromycin was used as an internal standard. RESULTS: The method was validated in terms of linearity, precision, accuracy, stability, and system suitability parameters. The method was found to be satisfactory and suitable for the determination of glimepiride. The precision of glimepiride was high (coefficient of variation, CV% <15%), the accuracy over all 3 days of validation was within the accepted criteria. Glimepiride peak serum concentration (C max) was 126.01 ng/mL and was reached within 1 h (T max) of administration. Mean area under curve (AUC) was 964.70 ng/mL and was reached within 24 h of administration. The Vimto soft drink significantly (P < 0.050) reduced glimepiride peak serum concentration to 57.87 ng/mL and was reached within 2 h of administration. AUC was significantly reduced to 335.04 ng*h/mL (P < 0.050). CONCLUSION: Glimepiride pharmacokinetic parameters such as C max and AUC were significantly affected by the Vimto soft drink. Therefore, this study developed a simple, rapid, and sensitive method for validation and determination of the effects of soft drinks on drugs using the LC-MS/MS method.

Tablet fragmentation without a disintegrant: A novel design approach for accelerating disintegration and drug release from 3D printed cellulosic tablets.

Fused deposition modelling (FDM) 3D printing has shown the most immediate potential for on-demand dose personalisation to suit particular patient's needs. However, FDM 3D printing often involves employing a relatively large molecular weight thermoplastic polymer and results in extended release pattern. It is therefore essential to fast-track drug release from the 3D printed objects. This work employed an innovative design approach of tablets with unique built-in gaps (Gaplets) with the aim of accelerating drug release. The novel tablet design is composed of 9 repeating units (blocks) connected with 3 bridges to allow the generation of 8 gaps. The impact of size of the block, the number of bridges and the spacing between different blocks was investigated. Increasing the inter-block space reduced mechanical resistance of the unit, however, tablets continued to meet pharmacopeial standards for friability. Upon introduction into gastric medium, the 1 mm spaces gaplet broke into mini-structures within 4 min and met the USP criteria of immediate release products (86.7% drug release at 30 min). Real-time ultraviolet (UV) imaging indicated that the cellulosic matrix expanded due to swelling of hydroxypropyl cellulose (HPC) upon introduction to the dissolution medium. This was followed by a steady erosion of the polymeric matrix at a rate of 8 µm/min. The design approach was more efficient than a comparison conventional formulation approach of adding disintegrants to accelerate tablet disintegration and drug release. This work provides a novel example where computer-aided design was instrumental at modifying the performance of solid dosage forms. Such an example may serve as the foundation for a new generation of dosage forms with complicated geometric structures to achieve functionality that is usually achieved by a sophisticated formulation approach.

Saliva versus Plasma Bioequivalence of Valsartan/Hydrochlorothiazide in Humans: Validation of Classes II and IV Drugs of the Salivary Excretion Classification System.

AIM: The aim of this study is to investigate the robustness of using non-invasive saliva instead of plasma for bioequivalence of valsartan and hydrochlorothiazide (HCT) in humans based on Salivary Excretion Classification System (SECS). METHODS: Plasma and resting saliva samples were collected over 24 h after oral administration of single dose 160 mg valsartan and 12.5 mg HCT to 12 healthy male volunteers after 10 h overnight fasting. Plasma and saliva concentrations were determined by validated liquid chromatography-mass spectrometry. WinNonlin program V5.2 was used to determine pharmacokinetic parameters and bioequivalence metrics. Moreover, optimized effective intestinal permeability was estimated using PK-Sim/Mobi program V5.6. RESULTS AND DISCUSSION: Valsartan is SECS class IV drug due of low permeability and high protein binding and hence didn't appear in saliva. However, HCT is SECS class II drug due to low permeability and low protein binding. No significant differences were observed in the pharmacokinetic parameters in both plasma matrix and saliva matrix (P˃0.05). The 90% confidence intervals did not pass in all parameters due to the high intra-subject variability and small sample size used in this study. Saliva to plasma ratios of HCT were low, yet with high correlation coefficient of 0.96-0.98. So saliva can be used as alternative to plasma sample in pharmacokinetic studies and in bioequivalence when adequate sample size is used.

The influence of Eruca sativa (Arugula) on pharmacokinetics of Sildenafil in rats.

OBJECTIVES: A drug like Sildenafil is commonly used for the treatment of erectile dysfunction. Eruca sativa is known as a garden plant used in folk medicine to enhance the sexual desire in males. Nevertheless, the interaction of Sildenafil and Eruca sativa was not studied. In the current study, we aimed to examine the influence of Eruca sativa on Sildenafil pharmacokinetics in rats. STUDY DESIGN: A crossover experiment with washout period of two weeks was conducted. To one group of animals, Eruca sativa was given as food and a drinking solution to rats for 12 hours before the day of the experiment. On the day of the experiment, the same group received 5 ml (50 mg/ml) orally and a half an hour later animals received 1 ml Sildenafil citrate (2.85 mg/kg) oral administrated to the study group. The other group of rats only received Sildenafil. Two-weeks later a cross-over design on the same animals was conducted. Blood samples were collected from optical vein on different time intervals, samples were analyzed using validated (HPLC-UV) method. RESULTS AND CONCLUSION: Pre-administration of Eruca sativa has increased Sildenafil Cmax from 226.72 to 345.25 ng/ml, (p<0.05). In addition, the AUC of Sildenafil has significantly increased when it was pre-administered with Eruca sativa (550.59 vs. 916.48 ng/ml*hr). Our findings suggest that co-administration of Eruca sativa with Sildenafil enhances the pharmacokinetics of Sildenafil in rats plasma.

Effects of the genetic variants of organic cation transporters 1 and 3 on the pharmacokinetics of metformin in Jordanians.

BACKGROUND: Human response to the antidiabetic metformin is influenced by some factors, such as genetic variants in the SLC22A genes. This study aimed to determine the frequency of main SLC22A1 and SLC22A3 genetic variants and their influence on metformin pharmacokinetics among healthy unrelated Arab Jordanians. PATIENTS AND METHODS: The SLC22A1 and SLC22A3 genes were genotyped by DNA sequencing of exons 1, 3, 7, and 9 in the SLC22A1 gene and exons 6, 7, and 9 in the SLC22A3 gene. Then, a clinical pharmacokinetic study was conducted on 26 healthy volunteers. The pharmacokinetic parameters were calculated using non-compartmental model analysis. The study was an open-label, randomized study with single 1000 mg metformin administration. RESULTS: Results showed that volunteers with SLC22A3 rs8187722 variant had higher (χ2, p<0.05) metformin Cmax and AUC values than the wild SLC22A3 volunteers, whereas T½ and Kel were not affected. In addition, volunteers with the heterozygote SLC22A3 rs2292334 variant had significantly higher (χ2, p<0.05) metformin Cmax and AUC and lower Kel values than the wild-type SLC22A3 genotype. CONCLUSIONS: The SLC22A3 rs8187722 and rs2292334 genetic variants affected metformin pharmacokinetics among a clinical sample of Jordanians. The findings may increase our understanding of the inter-individual and inter-ethnic variations in metformin response.

A Liquid Chromatography-Tandem Mass Spectrometry Method for Evaluation of Two Brands of Enalapril 20 mg Tablets in Healthy Human Volunteers.

Enalapril is an angiotensin-converting enzyme inhibitor used for treatment of hypertension and chronic heart disease. Enalaprilat is its active metabolite responsible for the activity. This study aimed to develop and validate a method for enalapril and enalaprilat analysis and to determine the bioequivalence of two tablet formulae of enalapril. LC-MS/MS bioanalytical method was developed and validated and then applied to evaluate the bioavailability of two enalapril formulae. Antihyperglycemic sitagliptin was used as internal standard (IS). The method was accurate for the within- and between-days analysis, and precise CV% was <5%, being linear over the calibration range 1.0-200.0 ng/ml. Stability was >85% and the LOD was 0.907 and 0.910 ng/ml for enalapril and enalaprilat, respectively, and LLOQ was 1 ng/ml. The pharmacokinetic parameters Cmax, tmax, AUC0-72, and AUC0-∞ values of enalapril and enalaprilat of the two formulae were calculated and nonsignificant differences were found. A linearity, specific, accurate, and precise method was developed and applied for the analysis of enalapril and enalaprilat in human plasma after oral administration of two formulae of enalapril 20 mg tablets in healthy volunteers. Depending on the statistical analysis it was concluded that the two enalapril formulae were bioequivalent.

Detection of High-Risk Human Papillomavirus Genotypes 16 and 18 in Head and Neck Squamous Cell Carcinomas in Jordan

Background: Recently, associations of the human papillomavirus (HPV) with head and neck cancer have become well established. Of particular concern, the severity and pathological outcomes of squamous cell carcinomas are remarkably affected by the genotypes of HPV present in such lesions. This study was conducted to investigate the occurrence of HPV genotypes, particularly high risk 16 and 18, among oral and laryngeal squamous cell carcinomas in Jordan. Methods: During the period of May 2015 to March 2016, we evaluated a total of 108 paraffin-embedded tissue samples, histologically confirmed as SCC, of both oral and laryngeal tumors for the presence of HPV DNA. DNA was extracted using a Zymogen commercial kit. HPV genotypes were detected by nested PCR using consensus primers followed by primer-specific PCR for HPV-16 and HPV-18 genotypes. The genotypes were confirmed by DNA sequencing methods. Results: Sixteen samples were positive for HPV DNA (14.8%) with higher rates in oral tumors compared to their laryngeal counterparts (20% and 6% respectively). The HPV-16 genotype predominated, being detected in 81.3% of the cases as a single infection and in 18.7% in combination with HPV-18. A significant association between the anatomical location and the HPV-16 genotype was observed (p < 0.05). In contrast, no significant associations could be established with tumor grade and gender or age. Conclusions: A relatively high rate of high-risk HPV genotypes, especially HPV 16, is evident in head and neck cancers SCCs in Jordan. Genotyping of HPV might be of considerable value for evaluation of progression.

Combination of RAD001 (everolimus) and docetaxel reduces prostate and breast cancer cell VEGF production and tumour vascularisation independently of sphingosine-kinase-1.

Resistance to docetaxel is a key problem in current prostate and breast cancer management. We have recently discovered a new molecular mechanism of prostate cancer docetaxel chemoresistance mediated by the mammalian target of rapamycin (mTOR)/sphingosine-kinase-1 (SK1) pathway. Here we investigated the influence of this pathway on vascular endothelial growth factor (VEGF) production and tumour vascularisation in hormone resistant prostate and breast cancer models. Immunofluorescent staining of tumour sections from human oestrogen receptor (ER)-negative breast cancer patients showed a strong correlation between phosphorylated P70S6 kinase (mTOR downstream target), VEGF and SK1 protein expression. In hormone-insensitive prostate (PC3) and breast (MDA-MB-231 and BT-549) cancer cell lines the mTOR inhibitor RAD001 (everolimus) has significantly inhibited SK1 and VEGF expression, while low dose (5 nM) docetaxel had no significant effect. In these cell lines, SK1 overexpression slightly increased the basal levels of VEGF, but did not block the inhibitory effect of RAD001 on VEGF. In a human prostate xenograft model established in nude mice, RAD001 alone or in combination with docetaxel has suppressed tumour growth, VEGF expression and decreased tumour vasculature. Overall, our data demonstrate a new mechanism of an independent regulation of SK1 and VEGF by mTOR in hormone-insensitive prostate and breast cancers.

Saliva Versus Plasma Bioequivalence of Azithromycin in Humans: Validation of Class I Drugs of the Salivary Excretion Classification System.

AIM: The aim of this study was to compare human pharmacokinetics and bioequivalence metrics in saliva versus plasma for azithromycin as a model class I drug of the Salivary Excretion Classification System (SECS). METHODS: A pilot, open-label, two-way crossover bioequivalence study was done, and involved a single 500-mg oral dose of azithromycin given to eight healthy subjects under fasting conditions, followed by a 3-week washout period. Blood and unstimulated saliva samples were collected over 72 h and deep frozen until analysis by a validated liquid chromatography with mass spectroscopy method. The pharmacokinetic parameters and bioequivalence metrics of azithromycin were calculated by non-compartment analysis using WinNonlin V5.2. Descriptive statistics and dimensional analysis of the pharmacokinetic parameters of azithromycin were performed using Microsoft Excel. PK-Sim V5.6 was used to estimate the effective intestinal permeability of azithromycin. RESULTS AND DISCUSSION: No statistical differences were shown in area under the concentration curves to 72 h (AUC0-72), maximum measured concentration (C max) and time to maximum concentration (T max) between test and reference azithromycin products (P > 0.05) in the saliva matrix and in the plasma matrix. Due to the high intra-subject variability and low sample size of this pilot study, the 90% confidence intervals of AUC0-72 and C max did not fall within the acceptance range (80-125%). However, saliva levels were higher than that of plasma, with a longer salivary T max. The mean saliva/plasma concentration of test and reference were 2.29 and 2.33, respectively. The mean ± standard deviation ratios of saliva/plasma of AUC0-72, C max and T max for test were 2.65 ± 1.59, 1.51 ± 0.49 and 1.85 ± 1.4, while for the reference product they were 3.37 ± 2.20, 1.57 ± 0.77 and 2.6 ± 1.27, respectively. A good correlation of R = 0.87 between plasma and saliva concentrations for both test and reference products was also observed. Azithromycin is considered a class I drug based on the SECS, since it has a high permeability and high fraction unbound, and saliva sampling could be used as an alternative to plasma sampling to characterize its pharmacokinetics and bioequivalence in humans when adequate sample size is used.

Study the influence of licorice and pomegranate drinks on nicotine metabolism in human urine by LC-orbitrap MS.

Nicotine-diet interactions have a particular importance on human health. Some food substances are subject to change hepatic CYP2A6 metabolism rate for nicotine and its levels in smokers consequently. This study investigates the effect of pomegranate and licorice drinks on nicotine metabolism, by a new developed and validated method for simultaneous determination of nicotine with its major metabolites (cotinine and nicotine N-oxide) in human urine, utilizing LC ESI-orbitrap-MS. Twenty-four Jordanian healthy and smoker volunteers were participated in two equal groups, crossover design for each of pomegranate and licorice test drink. In the study periods each group assigned either to drink test juice three times a day or to be avoided from test drink for 7 successive days, and then both groups switched their drink treatment in subsequent period. Early morning urine samples were collected from all volunteers after each period. Nicotine metabolism rate was evaluated from nicotine/cotinine and nicotine/nicotine N-oxide ratios in urine. A consistent trend of increase in metabolism rate for nicotine was observed from urine analysis under pomegranate or licorice drink conditions compared to control conditions. Pomegranate and licorice drinks are increasing the metabolism rate for nicotine in terms of induction effect for hepatic cytochrome p450 enzymes.

Everolimus (RAD001) sensitizes prostate cancer cells to docetaxel by down-regulation of HIF-1α and sphingosine kinase 1.

Resistance to docetaxel is a key problem in current prostate cancer management. Sphingosine kinase 1 (SK1) and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways have been implicated in prostate cancer chemoresistance. Here we investigated whether their combined targeting may re-sensitize prostate cancer cells to docetaxel.In hormone-insensitive PC-3 and DU145 prostate cancer cells the mTOR inhibitor everolimus (RAD001) alone did not lead to significant cell death, however, it strongly sensitized cells to low levels (5 nM) of docetaxel. We show that mTOR inhibition has led to a decrease in hypoxia-inducible factor-1α (HIF-1α) protein levels and SK1 mRNA. HIF-1α accumulation induced by CoCl2 has led to a partial chemoresistance to RAD001/docetaxel combination. SK1 overexpression has completely protected prostate cancer cells from RAD001/docetaxel effects. Using gene knockdown and CoCl2 treatment we showed that SK1 mRNA expression is downstream of HIF-1α. In a human xenograft model in nude mice single RAD001 and docetaxel therapies induced 23% and 15% reduction in prostate tumor volume, respectively, while their combination led to a 58% reduction. RAD001 alone or in combination with docetaxel has suppressed intratumoral mTOR and SK1 signaling, however as evidenced by tumor size, it required docetaxel for clinical efficacy. Combination therapy was well tolerated and had similar levels of toxicity to docetaxel alone.Overall, our data demonstrate a new mechanism of docetaxel sensitization in prostate cancer. This provides a mechanistic basis for further clinical application of RAD001/docetaxel combination in prostate cancer therapy.