Ionic-liquid/Carbowax 20 M functionalized capsule phase microextraction platform for the extraction of phosphodiesterase-5 inhibitors from human serum and urine prior to their determination by LC-MS.

Capsule phase microextraction (CPME) is an efficient bioanalytical technique that streamlines the sample preparation by integrating the filtration and stirring mechanism directly into the device. A novel composite sorbent designed to be selective towards the target analytes consisting of mixed-mode sorbent chemistry synthesized by sol-gel technology is found promising and superior to the conventional C18 sorbents. Herein we describe the encapsulation of an ionic liquid (IL)/Carbowax 20M-functionalized sol-gel sorbent (sol-gel IL/Carbowax 20 M) in the lumen of porous polypropylene tubes for the capsule phase microextraction of three phosphodiesterase-5 inhibitors namely avanafil, sildenafil, and tadalafil in human serum and urine samples. The CPME device was characterized by Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FT-IR). The experimental parameters of CPME procedure (e.g. sample pH and ionic strength, extraction time, stirring rate, elution solvent and volume) were carefully optimized to achieve the highest possible extraction efficiency for the analytes. Method validation was conducted in terms of precision, linearity, accuracy, matrix effect, lower limits of quantification, and limits of detection (LOD). The method linearity was investigated in the range of 50-1000 ng mL-1 for all analytes while the precision was less than 11.8 % in all cases. For all analytes, the LOD values were 17 ng mL-1. The IL/CW 20M-functionalized microextraction capsules could be reused at least 25 times both for urine and serum samples. The green character and the applicability of the proposed method were evaluated using the ComplexGAPI and BAGI indexes. The optimized CPME protocol exhibited reduced consumption of organic solvent and generation of waste, cost-effectiveness, and simplicity. Finally, the proposed method was successfully applied to the analysis of sildenafil in human urine after administration of drug-containing formulation.

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.

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Method development and validation of sildenafil, N-desmethylsildenafil and N1,N4-desmethylsildenafil by LC-MS/MS and its application.

This study aimed to develop an LC-MS/MS method for determining sildenafil and its metabolites N-desmethylsildenafil and N1,N4-desmethylsildenafil in human plasma and applying it to a pharmacokinetic study of sildenafil in healthy volunteers. Sildenafil-d8 was used as the internal standard. Plasma samples were pretreated via protein precipitation with acetonitrile. The extractives were then separated on an ACQUITY UPLC BEH C18 (50-mm × 2.1-mm, 1.7-µm) column using gradient elution. The aqueous and organic mobile phases were ammonium formate 2 mM supplemented with 0.1% formic acid in water and acetonitrile, respectively, and the flow rate was 0.3 mL/min. An electrospray ionization source was applied, and multiple reaction monitoring was operated in the positive mode with selective channels at m/z 475.30 â†’ 100.10, 461.20 â†’ 283.30, 483.30 â†’ 108.10, and 449.00 â†’ 283.00 for sildenafil, sildenafil-d8, N-desmethylsildenafil, and N1,N4-desmethylsildenafil, respectively. The linear calibration curves of sildenafil and its metabolites spanned 1.0-1000 ng/mL. The lower limit of quantification was 1.0 ng/mL. The extractive recovery of analytes from the biological matrix was more than 90% and the matrix effect complied with relevant provisions. The intra- and inter-day precisions of sildenafil and its metabolite were <10%. The intra- and inter-day accuracy of sildenafil, N-desmethylsildenafil, and N1,N4-desmethylsildenafil was more than 99%. The method is highly sensitive and selective, and it was successfully applied to the bioequivalence studies of 100-mg sildenafil citrate tablets in 40 healthy Chinese volunteers.

Antenatal sildenafil treatment improves neonatal pulmonary hemodynamics and gas exchange in lambs with diaphragmatic hernia.

OBJECTIVES: Infants with congenital diaphragmatic hernia (CDH) are predisposed to pulmonary hypertension after birth, owing to lung hypoplasia that impairs fetal pulmonary vascular development. Antenatal sildenafil treatment attenuates abnormal pulmonary vascular and alveolar development in rabbit and rodent CDH models, but whether this translates to functional improvements after birth remains unknown. We aimed to evaluate the effect of antenatal sildenafil on neonatal pulmonary hemodynamics and lung function in lambs with diaphragmatic hernia (DH). METHODS: DH was surgically induced at approximately 80 days' gestation in 16 lamb fetuses (term in lambs is approximately 147 days). From 105 days' gestation, ewes received either sildenafil (0.21 mg/kg/h intravenously) or saline infusion until delivery (n = 8 fetuses in each group). At approximately 138 days' gestation, all lambs were instrumented and then delivered via Cesarean section. The lambs were ventilated for 120 min with continuous recording of physiological (pulmonary and carotid artery blood flow and pressure; cerebral oxygenation) and ventilatory parameters, and regular assessment of arterial blood gas tensions. Only lambs that survived until delivery and with a confirmed diaphragmatic defect at postmortem examination were included in the analysis; these comprised six DH-sildenafil lambs and six DH-saline control lambs. RESULTS: Lung-to-body-weight ratio (0.016 ± 0.001 vs 0.013 ± 0.001; P = 0.06) and dynamic lung compliance (0.8 ± 0.2 vs 0.7 ± 0.2 mL/cmH2 O; P = 0.72) were similar in DH-sildenafil lambs and controls. Pulmonary vascular resistance decreased following lung aeration to a greater degree in DH-sildenafil lambs, and was 4-fold lower by 120 min after cord clamping than in controls (0.6 ± 0.1 vs 2.2 ± 0.6 mmHg/(mL/min); P = 0.002). Pulmonary arterial pressure was also lower (46 ± 2 vs 59 ± 2 mmHg; P = 0.048) and pulmonary blood flow higher (25 ± 3 vs 8 ± 2 mL/min/kg; P = 0.02) in DH-sildenafil than in DH-saline lambs at 120 min. Throughout the 120-min ventilation period, the partial pressure of arterial carbon dioxide tended to be lower in DH-sildenafil lambs than in controls (63 ± 8 vs 87 ± 8 mmHg; P = 0.057), and there was no significant difference in partial pressure of arterial oxygen between the two groups. CONCLUSIONS: Sustained maternal antenatal sildenafil infusion reduced pulmonary arterial pressure and increased pulmonary blood flow in DH lambs for the first 120 min after birth. These findings of improved pulmonary vascular function are consistent with improved pulmonary vascular structure seen in two previous animal models. The data support the rationale for a clinical trial investigating the effect of antenatal sildenafil in reducing the risk of neonatal pulmonary hypertension in infants with CDH. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Effects of Maternal Sildenafil Treatment on Vascular Function in Growth-Restricted Fetal Sheep.

Objective- The objective of this study was to investigate the effect of intravenous maternal sildenafil citrate (SC) administration on vascular function in growth-restricted fetal sheep. Approach and Results- Fetal growth restriction (FGR) results in cardiovascular adaptations that redistribute cardiac output to optimize suboptimal intrauterine conditions. These adaptations result in structural and functional cardiovascular changes, which may underlie postnatal neurological and cardiovascular sequelae. Evidence suggests SC, a potent vasodilator, may improve FGR. In contrast, recent clinical evidence suggests potential for adverse fetal consequence. Currently, there is limited data on SC effects in the developing fetus. We hypothesized that SC in utero would improve vascular development and function in an ovine model of FGR. Preterm lambs (0.6 gestation) underwent sterile surgery for single umbilical artery ligation or sham (control, appropriately grown) surgery to replicate FGR. Ewes received continuous intravenous SC (36 mg/24 h) or saline from surgery until 0.83 gestation. Fetuses were delivered and immediately euthanized for collection of femoral and middle cerebral artery vessels. Vessel function was assessed via in vitro wire myography. SC exacerbated growth restriction in growth-restricted fetuses and resulted in endothelial dysfunction in the cerebral and femoral vasculature, irrespective of growth status. Dysfunction in the cerebral circulation is endothelial, whereas smooth muscle in the periphery is the origin of the deficit. Conclusions- SC crosses the placenta and alters key fetal vascular development. Extensive studies are required to investigate the effects of SC on fetal development to address safety before additional use of SC as a treatment.

Development and validation of an ultra performance liquid chromatography tandem mass method for sildenafil and N-desmethyl sildenafil plasma determination and quantification.

Sildenafil is a selective inhibitor of cGMP-specific type 5 phosphodiesterase (PDE5) used for the treatment of masculine erectile dysfunction and Pulmonary Arterial Hypertension (PAH). Sildenafil causes vasodilatation; relax of the smooth muscle and reduction of pulmonary arterial pressure. In the liver cytocrome P450 metabolizes sildenafil into its active metabolite, N-desmethyl sildenafil. The determination of plasma levels of sildenafil and N-desmethyl sildenafil could be useful for therapy optimization and pharmacokinetic studies. We have developed and validated a new method for the quantification of sildenafil and its metabolite in human plasma by rapid protein precipitation extraction, using an UPLC system, coupled with a tandem mass spectrometric detector (UPLC-MS/MS). The calibration range was fitted at least square model (r(2)≥0.999), with an accuracy and an intra- and inter-day RSD% (Relative Standard Deviation), both for sildenafil and N-desmethyl sildenafil, lower than 15%, as required by the FDA guidelines; LLOQ, LLOD, ULOQ were 3.9ng/mL, 1.95ng/mL and 1000ng/mL, respectively, for both analytes. Matrix effect, expressed as mean percent deviation of peak areas, was in the range between 2.6% and 5.8%, lower than 15% as required by guidelines. The mean recovery was 83.2 % for sildenafil and 84.5% for N-desmethyl sildenafil. This method has successfully been applied to a clinical pharmacokinetic study of sildenafil and N-desmethyl sildenafil in patients with PAH undergoing cardiac surgery.

Pharmacokinetic interactions among imatinib, bosentan and sildenafil, and their clinical implications in severe pulmonary arterial hypertension.

AIMS: This study characterized the population pharmacokinetics (PK) of imatinib in patients with severe pulmonary arterial hypertension (PAH), investigated drug-drug interactions (DDI) among imatinib, sildenafil and bosentan, and evaluated their clinical implications. METHODS: Plasma concentrations of imatinib, bosentan and sildenafil were collected in a phase III study and were used to characterize the PK of imatinib in this population. DDIs among the three drugs were quantified using a linear mixed model and log-transformed drug concentrations. RESULTS: The population mean estimates of apparent clearance (CL/F) and volume (V/F) were 10.8 l h(-1) (95% CI 9.2, 12.4 l h(-1) ) and 267 l (95% CI 208, 326 l), respectively. It was estimated that sildenafil concentrations increased, on average, by 64% (95% CI 32%, 103%) and bosentan concentrations by 51% (95% CI 12%, 104%), in the presence of imatinib. Despite increased concentrations of co-medications, treatment differences between imatinib and placebo for change in 6 min walk distance and pulmonary vascular resistance were relatively constant across the entire concentration range for sildenafil and bosentan. Overall, higher concentrations of imatinib and bosentan were not associated with increasing liver enzymes (serum glutamic oxaloacetic transaminases [SGOT]/serum glutamic-pyruvic transaminase [SGPT]). CONCLUSIONS: Population PKs of imatinib in patients with severe PAH were found comparable with those of patients with chronic myeloid leukemia. Imatinib was found effective regardless of the co-medications and showed intrinsic efficacy beyond merely elevating the concentrations of the co-medications due to DDIs. There was no evidence of increased risk of liver toxicity upon co-administration with bosentan.