Simultaneous determination of FL118 and W34 in rat Blood by LC-MS/MS: Application to pharmacokinetic studies.

W34 is a prodrug of FL118, and it can be converted to FL118 via a hydrolysis reaction. In this report, a highly sensitive LC-MS/MS method using a C18 column was validated and used for the simultaneous determination of W34 and FL118 in rat blood. A stepwise gradient elution with 0.1% formic acid in water and acetonitrile was employed. The assays were linear over a concentration range of 0.50-50.0 ng/ml for both W34 and FL118. The accuracy of the validation method ranged from 89.74 to 98.94% for W34 and from 88.61 to 94.60% for FL118. The precision was within 7.15% for W34 and 9.63% for FL118. Extraction recoveries of W34 were 94.56-100.49 and 87.67-106.32% for FL118. No significant matrix effects for both W34 and FL118 were observed in blood. The assay has been successfully applied to biological samples obtained from a stability and pharmacokinetic study of W34 and FL118.

Analysis of Lycobetaine in Rat Plasma by LC-ESI-MS/MS.

In this study, a selective and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric method was developed and validated for the determination of lycobetaine in rat plasma. Berberine was selected as the internal standard, and rat plasma samples were pretreated via protein precipitation and further separated on a diamonsil octadecyl-silylated silica column using 0.2% (v/v) aqueous formic acid and methanol as the mobile phase. Selected reaction monitoring was performed using the transitions m/z 266.1 → 208.1 and m/z 336.1 → 320.0 to determine the concentrations of lycobetaine and internal standard, respectively. The injection volume was 1 µL, and the calibration curve was linear (R2 = 0.9998), while the validated lower limit of quantification was 25 ng/mL. Precision varied from 3.4% to 9.9%, and accuracy varied from -2.6% to 8.7%. Lycobetaine remained stable under all relevant analytical conditions tested in the study. The method was successfully applied to determine the plasma concentration of lycobetaine in a pharmacokinetic study. After intravenous administration of 10 mg/kg and oral administration of 200 mg/kg lycobetaine in rats, the pharmacokinetic parameters were calculated and the oral bioavailability of lycobetaine was determined as 7.30% ± 1.44%.

Extended Evaluation of Virological, Immunological and Pharmacokinetic Endpoints of CELADEN: A Randomized, Placebo-Controlled Trial of Celgosivir in Dengue Fever Patients.

UNLABELLED: CELADEN was a randomized placebo-controlled trial of 50 patients with confirmed dengue fever to evaluate the efficacy and safety of celgosivir (A study registered at ClinicalTrials.gov, number NCT01619969). Celgosivir was given as a 400 mg loading dose and 200 mg bid (twice a day) over 5 days. Replication competent virus was measured by plaque assay and compared to reverse transcription quantitative PCR (qPCR) of viral RNA. Pharmacokinetics (PK) correlations with viremia, immunological profiling, next generation sequence (NGS) analysis and hematological data were evaluated as exploratory endpoints here to identify possible signals of pharmacological activity. Viremia by plaque assay strongly correlated with qPCR during the first four days. Immunological profiling demonstrated a qualitative shift in T helper cell profile during the course of infection. NGS analysis did not reveal any prominent signature that could be associated with drug treatment; however the phylogenetic spread of patients' isolates underlines the importance of strain variability that may potentially confound interpretation of dengue drug trials conducted during different outbreaks and in different countries. Celgosivir rapidly converted to castanospermine (Cast) with mean peak and trough concentrations of 5727 ng/mL (30.2 µM) and 430 ng/mL (2.3 µM), respectively and cleared with a half-life of 2.5 (± 0.6) hr. Mean viral log reduction between day 2 and 4 (VLR2-4) was significantly greater in secondary dengue than primary dengue (p = 0.002). VLR2-4 did not correlate with drug AUC but showed a trend of greater response with increasing Cmin. PK modeling identified dosing regimens predicted to achieve 2.4 to 4.5 times higher Cmin. than in the CELADEN trial for only 13% to 33% increase in overall dose. A small, non-statistical trend towards better outcome on platelet nadir and difference between maximum and minimum hematocrit was observed in celgosivir-treated patients with secondary dengue infection. Optimization of the dosing regimen and patient stratification may enhance the ability of a clinical trial to demonstrate celgosivir activity in treating dengue fever based on hematological endpoints. A new clinical trial with a revised dosing regimen is slated to start in 2016 (NCT02569827). Furthermore celgosivir's potential value for treatment of other flaviruses such as Zika virus should be investigated urgently. TRIAL REGISTRATION: ClinicalTrials.gov NCT01619969.

Pharmacokinetic-pharmacodynamic model for fantofarone cardiac and brachial haemodynamic effects in healthy volunteers.

AIMS: To investigate the pharmacokinetics of SR 33671, the main active metabolite of the calcium antagonist fantofarone, and the relationships between its concentrations and pharmacodynamic effects after a single oral administration of two doses (100 and 300 mg) of fantofarone. METHODS: A placebo-controlled, randomized, double-blind and crossover study was performed in six healthy volunteers. SR 33671 plasma concentrations (C, ng ml-1 ) and effects (E) on heart rate (HR, beats min-1 ), PR interval duration (ms), brachial artery flow (BAF, ml min-1 ) and brachial vascular resistance (BVR, mmHg s ml-1 ) were determined repeatedly after drug intake. Haemodynamic effects were expressed as percent changes from initial values. Bi-exponential (pharmacokinetics), and linear [E=S.C+E0, for cardiac effects] or sigmoid [E=Emax.Cgamma/(CEgamma50+Cgamma ), for haemodynamic effects] models were fitted to individual data. RESULTS: Peak plasma concentrations and areas under the curve up to 24 h were (mean+/-s.d.) 16+/-10 ng ml-1 and 157.50+/-89.13 ng ml-1 h, and 63+/-11 ng ml-1 and 535.50+/-135.11 ng ml-1 h, after 100 and 300 mg, respectively. Terminal half-life was approximately 4 h. For pharmacodynamics, we obtained: S=-0.201+/-0.057 beats min-1/ng ml-1 for HR, S=0.526+/-0.114 ms/ng ml-1 for PR interval duration, Emax=42+/-6%, CE50=8.8+/-7.2 ng ml-1 and gamma=2.2+/-1.5 for BAF, and Emax=-28+/-4%, CE50=5.8+/-5.1 ng ml-1 and gamma=3.4+/-1.8 for BVR. At a SR 33671 concentration of 15 ng ml-1, BVR is decreased by 27% whereas HR is reduced by less than 3 beats min-1 and PR interval duration is increased by less than 8 ms. CONCLUSIONS: Fantofarone is able to induce submaximal peripheral vasodilating effects at doses that are devoid of any clinically significant cardiac effect.

The lesions of locoweed (Astragalus mollissimus), swainsonine, and castanospermine in rats.

To better characterize and compare the toxicity of and lesions produced by locoweed (Astragalus mollissimus) with those of swainsonine and a related glycoside inhibitor, castanospermine, 55 Sprague-Dawley rats were randomly divided into 11 groups of five animals each. The first eight groups were dosed via subcutaneous osmotic minipumps with swainsonine at 0, 0.1, 0.7, 3.0, 7.4, or 14.9 mg/kg/day or with castanospermine at 12.4 or 143.6 mg/kg/day for 28 days. The last three groups were fed alfalfa or locoweed pellets with swainsonine doses of 0, 0.9, or 7.2 mg/kg/day for 28 days. Swainsonine- and locoweed-treated rats gained less weight, ate less, and showed more signs of nervousness than did controls. Histologically, these animals developed vacuolar degeneration of the renal tubular epithelium, the thyroid follicular cells, and the macrophage-phagocytic cells of the lymph nodes, spleen, lung, liver, and thymus. Some rats also developed vacuolation of neurons, ependyma, adrenal cortex, exocrine pancreas, myocardial epicytes, interstitial cells, and gastric parietal cells. No differences in lesion severity or distribution were detected between animals dosed with swainsonine and those dosed with locoweed. Rats dosed with castanospermine were clinically normal; however, they developed mild vacuolation of the renal tubular epithelium, the thyroid follicular epithelium, hepatocytes, and skeletal myocytes. Special stains and lectin histochemical evaluation showed that swainsonine- and castanospermine-induced vacuoles contained mannose-rich oligosaccharides. Castanospermine-induced vacuoles also contained glycogen. These results suggest that 1) swainsonine causes lesions similar to those caused by locoweed and is probably the primary locoweed toxin; 2) castanospermine at high doses causes vacuolar changes in the kidney and thyroid gland; and 3) castanospermine intoxication results in degenerative vacuolation of hepatocytes and skeletal myocytes, similar to genetic glycogenosis.