Bioequivalence and the food effect of macitentan/tadalafil 10/20 fixed-dose combination tablets versus the use of single-component tablets in healthy subjects.

The primary aim was to demonstrate bioequivalence between the 10/20 mg fixed-dose combination (FDC) of macitentan/tadalafil in a single tablet and the free combination of both drugs, and to evaluate the food effect on the 10/20 mg FDC in healthy participants. In this single-center, randomized, open-label, 3-way crossover, single-dose Phase 1 study in healthy adult participants, macitentan/tadalafil was administered as a 10/20 mg FDC formulation and compared with the free combination of macitentan and tadalafil. The food effect on the FDC was also evaluated. Pharmacokinetic sampling (216 h) was conducted. The 90% confidence intervals (CIs) for the geometric mean ratios of maximum observed plasma analyte concentration (Cmax) and area under the plasma analyte concentration-time curves (AUCs) for Treatment A (FDC, fasted) versus C (free combination, fasted) were within bioequivalence limits demonstrating that the FDC formulation can be considered bioequivalent to the free combination. The 90% CIs for the geometric mean ratios of Cmax and AUC for Treatment B (FDC, fed) versus A (FDC, fasted) were contained within bioequivalence limits demonstrating that there was no food effect. The administration of the 10/20 mg FDC was generally safe and well tolerated in healthy participants. This study demonstrated bioequivalence between the FDC of macitentan/tadalafil (10/20 mg) in a single tablet and the free combination of both drugs in healthy participants, and that the FDC can be taken without regard to food, similarly to the individual components. The FDC was generally safe and well tolerated.

Bioequivalence and food effect of a fixed-dose combination of macitentan and tadalafil: Adaptive design in the COVID-19 pandemic.

The COVID-19 pandemic has forced clinical studies to accommodate imposed limitations. In this study, the bioequivalence part could not be conducted as planned. Thus, the aim was to demonstrate bioequivalence, using an adaptive study design, of tadalafil in fixed-dose combination (FDC) tablets of macitentan/tadalafil with single macitentan and tadalafil (Canadian-sourced) tablets and assess the effect of food on FDC tablets in healthy subjects. This Phase 1, single-center, open-label, single-dose, two-part, two-period, randomized, crossover study enrolled 62 subjects. Tadalafil bioequivalence as part of FDC of macitentan/tadalafil (10/40 mg) with single-component tablets of macitentan (10 mg) and tadalafil (40 mg) was determined by pharmacokinetic (PK) assessment under fasted conditions. The effect of food on FDC was evaluated under fed and fasted conditions. Fasted 90% confidence intervals (CIs) for geometric mean ratios (GMRs) were within bioequivalence limits for tadalafil and macitentan. Fed and fasted 90% CIs for area under the curve (AUC) GMR were within bioequivalence limits. However, 90% CIs for maximum plasma concentration (Cmax ) GMR for macitentan and tadalafil were outside bioequivalence limits. One FDC-treated subject experienced a serious adverse event of transient ischemic attack (bioequivalence part). To address pandemic-imposed limitations, an adaptive study design was implemented to demonstrate that the FDC tablet was bioequivalent to the free combination of macitentan and tadalafil (Canadian-sourced). No clinically significant differences in PK were determined between fed and fasted conditions; the FDC formulation could be taken irrespective of meals. The FDC formulation under fasted and fed conditions was well tolerated with no clinically relevant differences in safety profiles between the treatment groups. NCT Number: NCT04235270.

Association between sexual activity-related death and non-prescription use of phosphodiesterase type 5 inhibitors.

In recent years, there has been an increase in the use of phosphodiesterase type 5 inhibitors (PDE5i) that are purchased from abroad without a doctor's diagnosis via the Internet or other means. We report six cases in which nonprescription use of PDE5i may have led to death. Among the four deceased individuals who were believed to have experienced sudden cardiac death, three (cases 1-3) had a history of cardiovascular disease, which is a contraindication, and the remaining case (case 4) involved combined use of multiple PDE5i. Sildenafil (0.063 µg/mL, 0.087 µg/mL) was detected in two of the four cases of sudden cardiac death. Tadalafil (0.096 µg/mL) was detected in one of the remaining two cases, and tadalafil (0.197 µg/mL) and vardenafil (0.011 µg/mL) were detected in the other case. Sildenafil (0.032 µg/mL), tadalafil (0.062 µg/mL), and ethanol were detected in a traffic accident case with a history of contraindications. In a case of asphyxiation by vomit aspiration, autopsy showed 90% stenosis in the anterior descending branch of the coronary artery, and sildenafil (0.063 µg/mL) was detected. To the best of our knowledge, this is the first report of postmortem blood levels of tadalafil and vardenafil likely contributing to the cause of death. Despite all the warnings about the dangers of using PDE5 inhibitors, cases of PDE5i contributing to death are still identified during autopsies. Therefore, raising public awareness of the risks of the risks associated with the imported drug use by individuals is necessary.

The Effect of Grapefruit Juice on the Pharmacokinetics of Tadalafil in Rats.

We developed and validated a novel, sensitive, selective, and inexpensive high-performance liquid chromatography (HPLC) method for the determination of tadalafil in rats plasma and to investigate the effect of grapefruit juice on the pharmacokinetics of tadalafil in rats. The ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) chromatography column can be used to separate tadalafil and carbamazepine (internal standard, IS). A mixture of acetonitrile-0.2% trifluoroacetic acid-water (48 : 10 : 42, V/V/V) was used as the mobile phase with a flow rate of 1.0 mL/min. The column temperature was set at 35.0°C. The detection wavelength was set at 286 nm. The tadalafil was extracted by ethyl acetate from plasma at the alkaline condition. 12 healthy male Sprague-Dawley (SD) rats were randomly divided into two groups, Group A (experimental group, received grapefruit juice 5 mL/kg for 7 days) and Group B (control group, received normal saline for 7 days). All the rats were given a single dose of tadalafil (5 mg/kg) after the last administration. The main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the conditions of this experiment, the plasma concentrations of tadalafil in the range of 10-2000 ng/ml had a good linear relationship. The intra- and interday precision for tadalafil in plasma were less than 15%, and the relative recovery rate was good at low, medium, and high QC levels. The C max of tadalafil in the control group and the experimental group was (725.89 ± 161.59) ng/mL and (1271.60 ± 179.31) ng/mL, t 1/2 was (9.28 ± 2.07) h and (11.70 ± 1.47) h, AUC (0-t) was (7399.61 ± 696.85) ng·h/mL and (9586.52 ± 2048.81) ng·h/mL, and AUC(0-∞) was (7995.50 ± 707.23) ng·h/mL and (10639.43 ± 2235.94) ng·h/mL, respectively. Results show that the C max of tadalafil in group A was 75.17% higher than that in group B, the Vz/F was also reduced, and the t 1/2 was increased by 2.42 h. The developed HPLC-DAD method for the determination of tadalafil in rats plasma was accurate, reproducible, specific, and it was found to be suitable for the pharmacokinetics of tadalafil and food-drug interactions. Grapefruit juice can inhibit the metabolism of tadalafil and increase the exposure of tadalafil in rats.

Maternal Blood Concentration of Tadalafil and Uterine Blood Flow in Pregnancy.

: Background and Objectives: Tadalafil for treatment of fetal growth restriction (FGR) or preeclampsia is given once a day orally. The drug kinetics of tadalafil were investigated to determine the ideal dosage to promote uterine blood flow. Materials and Methods: We recruited five pregnant women with FGR or preeclampsia before administration of tadalafil, all of which were administered tadalafil (20 mg/day, once-daily dosing). The blood concentration of tadalafil was measured 1, 2, 4, 6, 8, and 24 h after administration, and uterine blood flow was measured before tadalafil administration and 2-4 and 20-24 h after. We then analyzed the correlation between tadalafil blood concentration and uterine artery blood flow. Results: The blood concentration of tadalafil correlated with uterine artery blood flow in pregnant women. The blood concentration of tadalafil and uterine artery blood flow decreased 5 h after administration of tadalafil. Conclusions: The blood concentration of tadalafil and uterine artery blood flow fluctuate in parallel, the latter was decreased by reduced blood concentration. Thus, a study of tadalafil administered twice a day in pregnant women will be needed to stabilize uterine artery blood flow.

The prevalence of gamma-hydroxybutyrate (GHB) in motor vehicle drivers and its co-administration with amphetamine type substances (ATS) in Queensland, Australia (2011-2018).

The routine analysis of driver specimens for gamma-hydroxybutyrate (GHB) is rarely performed by toxicology laboratories as the physical and chemical properties of GHB make it unamenable to the screening methods usually employed. The prevalence of the drug in driver populations has therefore only rarely been reported. This study outlines the results of the routine analysis for GHB in the blood of motor vehicle drivers in Queensland, Australia, over an eight-year period (2011-2018). The methodology for GHB analysis was updated over the course of the study; screening for GHB was conducted using GC/FID or GC/MS between 2011 and 2016 and by LC/MS/MS from 2017 onwards. Due to the endogenous nature of GHB, any specimens containing greater than 5mg/kg GHB were subjected to quantitative analysis by either; GC/MS after liquid-liquid extraction and derivatisation with BSTFA+1%TMCS (2011-2016), or by LC/MS/MS analysis after solvent precipitation from 2017 onwards. Of the 15,061 specimens analysed, 160 were positive for GHB (1.1% of all cases, range 0.4-1.8%). GHB positive drivers were 66.9% male (33.1% female) and had an average age of 32 years. The mean GHB concentration identified was 89mg/kg (range 6-354mg/kg). GHB was found to be closely associated with amphetamine type substances (ATS), particularly methylamphetamine. Though GHB was present in only 2.2% of all ATS positive specimens submitted to the laboratory, 91.2% of all GHB positive cases contained an ATS. Other drugs commonly co-administered with GHB were THC, cocaine, benzodiazepines and erectile dysfunction drugs. GHB was found to be more commonly identified in drivers from city areas and a geographical localisation of the use of the drug was identified in the Gold Coast region of Queensland.

Pharmacokinetic interaction between linagliptin and tadalafil in healthy Egyptian males using a novel LC-MS method.

Aim: Assessment of pharmacokinetic interaction between linagliptin (LNG) and tadalafil (TDL) in healthy males. Methods: First, a novel LC-MS method was developed; second, a Phase IV, open-label, cross-over study was performed. Volunteers took single 20-mg TDL dose on day 1 followed by wash out period of 2 weeks then multiple oral dosing of 5-mg/day LNG for 13 days. On day 13, volunteers were co-administered 20-mg TDL. Results: LNG and TDL single doses did not affect QTc interval. Smoking did not alter pharmacokinetics/pharmacodynamics of LNG and TDL. Co-administration of LNG with TDL resulted in TDL longer time to reach maximum plasma concentration (Tmax), decreased oral clearance (Cl/F) and oral volume of distribution (Vd/F), increased its maximum plasma concentration (Cmax), area under concentration-time curve (AUC), muscle pain and QTc prolongation. Conclusion: LNG and TDL co-administration warrants monitoring and/or TDL dose adjustment.

A Phase I Study to Show the Relative Bioavailability and Bioequivalence of Fixed-Dose Combinations of Ambrisentan and Tadalafil in Healthy Subjects.

PURPOSE: Pulmonary arterial hypertension (PAH) is a life-threatening disease that typically causes shortness of breath and exercise intolerance. Combination therapy with ambrisentan and tadalafil has proven to be more effective at preventing clinical failure events in patients with PAH than either drug alone. The aim of this study was to evaluate the bioequivalence of an ambrisentan/tadalafil fixed-dose combination (FDC) compared with co-administration of the 2 monotherapies. METHODS: This 3-part, randomized, single-dose, open-label crossover study was conducted in healthy volunteers. The first part of the study consisted of a 5-way crossover that compared the relative bioavailability of 4 FDC formulations (10-mg ambrisentan + 40-mg tadalafil) with co-administered reference monotherapies. One formulation was selected and its relative bioavailability was assessed when produced in 3 different granulation sizes during the second part of the study. In the third part of the study, the bioequivalence of the candidate FDC with the reference monotherapies was evaluated for the 10-mg/40-mg dose strength, in addition to 2 other dose strengths (5 mg/20 mg and 5 mg/40 mg). For all parts of the study, blood samples were taken at regular intervals after each dose, ambrisentan and tadalafil concentrations determined, and pharmacokinetic (PK) parameters (Cmax, AUC0-∞, and AUC0-t) obtained. Test/reference ratios of the geometric means of PK parameters were used to evaluate bioequivalence. Safety and tolerability were assessed by recording adverse events and monitoring vital signs, ECGs, and clinical laboratory data. FINDINGS: Of the 174 subjects screened for eligibility, 112 were allocated to a randomized treatment sequence across all study parts, and 100 completed their full assigned treatments. All 4 FDC formulations tested during part 1 of the study yielded PK parameters similar those of the reference treatments. In part 2, granulation size was found to not affect the relative bioavailability of the selected formulation. In part 3, the selected FDC was found to be bioequivalent to co-administration of the monotherapies in both the fasted and fed states. The FDC was also found to be bioequivalent to the reference treatments at the 2 additional dose strengths. All but one of the adverse events was mild to moderate in intensity, and no serious adverse events were reported. IMPLICATIONS: An ambrisentan/tadalafil FDC was bioequivalent to concurrently administered monotherapies and therefore represents a viable alternative treatment to co-administration. Use of an FDC is likely to be associated with reduced costs and improved patient compliance. ClinicalTrials.gov identifier: NCT02688387.

Determination of Phosphodiesterase Inhibitors Tadalafil, Roflumilast and Roflumilast N-Oxide Using LC-MS in Guinea Pig Plasma.

Phosphodiesterases are known as a super-family of 11 isoenzymes, which can exert various functions based on their organ distribution. In this work, a rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed for quantification of tadalafil (phosphodiesterase five inhibitor), roflumilast (RF) (phosphodiesterase four inhibitor) and its active metabolite, RF N-oxide in guinea pig plasma. Chromatographic separation was carried out on UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) at a flow rate 0.5 mL/min, using 0.2% formic acid in acetonitrile and 0.2% formic acid in water as mobile phases within 4 min. Detection was performed using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive mode using multiple reaction monitoring mode. The method utilized deuterium labeled internal standards, and was validated according to European Medicines Agency guidelines. It showed excellent linearity in the range of 0.5-500.0 ng/mL for all analytes with coefficient of determination >0.99. The intra- and inter-day precisions (relative standard deviation %) were within 6.7%, and the recoveries were greater than 73.4%. Using this method, plasma samples from experiments of phosphodiesterase four, and five inhibitors in a model of ovalbumin-induced allergic inflammation in guinea pigs were analyzed.

Comparison of tadalafil pharmacokinetics after administration of a new orodispersible film versus a film-coated tablet.

BACKGROUND: An orodispersible film (ODF) of tadalafil may provide increased convenience for erectile dysfunction (ED) patients as compared to conventional tablet formulations. In this study, we aimed to compare the pharmacokinetic, safety, and tolerability profiles of a newly developed ODF formulation of tadalafil to those of a film-coated tablet (FCT) of tadalafil. MATERIALS AND METHODS: This study was conducted in healthy male subjects using an open-label, randomized sequence, two-period, two-formulation, single-dose, crossover design. The subjects were randomly assigned to one of two sequences of the two formulations: both the test drug (ODF) and the reference drug (FCT) contained 20 mg of tadalafil. Blood samples were collected up to 72 h after administration. Plasma concentrations of tadalafil were analyzed using liquid chromatography-tandem mass spectrometry. Geometric mean ratios (GMRs) of the ODF to FCT formulations and their 90% CIs for the pharmacokinetic parameters were estimated. Safety and tolerability were assessed throughout the study. RESULTS: Forty healthy male subjects were enrolled, and 36 of these completed the study. The GMRs (90% CIs) of the maximum plasma concentration and the area under the plasma concentration-time curve from time zero to the time of the last quantifiable concentration for tadalafil were 0.927 (0.882-0.974) and 0.972 (0.918-1.029), respectively. Both ODF and FCT formulations were well tolerated, and no clinically significant changes from the baseline were observed after dosing. CONCLUSION: The pharmacokinetics of the tadalafil ODF formulation did not differ significantly from those of the FCT formulation. Furthermore, the safety and tolerability profiles of the ODF formulation were comparable to those of the FCT formulation. Therefore, this tadalafil ODF formulation offers a convenient treatment option for patients with erectile dysfunction.

Selective and rapid determination of tadalafil and finasteride using solid phase extraction by high performance liquid chromatography and tandem mass spectrometry.

Highly selective and fast liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for simultaneous determination of tadalafil (TDL) and finasteride (FNS) in human plasma. The method was successfully applied for analysis of TDL and FNS samples in clinical study. The method was validated as per USFDA (United States Food and Drug Administration), EMA (European Medicines Agency), and ANVISA (Agência Nacional de Vigilância Sanitária-Brazil) bio analytical method validation guidelines. Glyburide (GLB) was used as common internal standard (ISTD) for both analytes. The selected multiple reaction monitoring (MRM) transitions for mass spectrometric analysis were m/z 390.2/268.2, m/z 373.3/305.4 and m/z 494.2/369.1 for TDL, FNS and ISTD respectively. The extraction of analytes and ISTD was accomplished by a simple solid phase extraction (SPE) procedure. Rapid analysis time was achieved on Zorbax Eclipse C18 column (50 × 4.6 mm, 5 µm). The calibration ranges for TDL and FNS were 5-800 ng/ml and 0.2-30 ng/ml respectively. The results of precision and accuracy, linearity, recovery and matrix effect of the method are acceptable. The accuracy was in the range of 92.9%-106.4% and method precision was also good; %CV was less than 8.1%.

Plasma Drug Concentrations in Patients with Pulmonary Arterial Hypertension on Combination Treatment.

BACKGROUND: Combination therapy with the phosphodiesterase type 5 inhibitors (PDE-5i) sildenafil or tadalafil and the endothelin receptor antagonists (ERA) bosentan, ambrisentan, or macitentan may cause mutual pharmacokinetic interactions in patients with pulmonary arterial hypertension (PAH). OBJECTIVE: The objective of this study was to analyze plasma drug concentrations in PAH patients receiving different combination treatments. METHODS: PAH patients receiving a stable combination treatment with ERA and PDE-5i with targeted dosage for at least 1 month were routinely assessed, including clinical parameters and plasma drug concentrations. Concentrations were normalized considering dose and time from last medication intake and presented as multiples of the expected mean (MoM) of the respective monotherapies. RESULTS: A total of 125 PAH patients (84 female, 41 male, 57% idiopathic/heritable) were included. Sildenafil and tadalafil concentrations were lowest in combination with bosentan (MoM 0.44 ± 0.42, 95% confidence interval [CI] 0.30-0.57, and MoM 0.89 ± 0.53, 95% CI 0.50-1.28, respectively) compared to the combination with ambrisentan (MoM 1.3 ± 0.97, 95% CI 0.86-1.73, and MoM 1.67 ± 0.63, 95% CI 1.40-1.94, respectively) and macitentan (MoM 1.16 ± 0.87, 95% CI 0.86-1.46, and MoM 1.59 ± 0.99, 95% CI 0.80-2.38, respectively). The combination of sildenafil and bosentan led to more than twice the expected bosentan concentrations in 53.8%. Patients switching from sildenafil-bosentan to macitentan showed a significant increase in sildenafil concentrations (p < 0.001). CONCLUSIONS: Only the combination with macitentan or ambrisentan led to targeted mean PDE-5i plasma concentrations and should therefore be preferred to combination with bosentan. Sildenafil-bosentan showed the strongest interaction, with low sildenafil and high bosentan concentrations. The study was not powered to analyze whether lower PDE-5i concentrations cause unsatisfying clinical response. However, plasma concentrations within a targeted range are desirable and may become of increasing importance.

A comparison study of macrocyclic hosts functionalized reduced graphene oxide for electrochemical recognition of tadalafil.

The present work described the comparison of ß-cyclodextrin (ß-CD) and p-sulfonated calix[6]arene (SCX6) functionalized reduced graphene oxide (RGO) for recognition of tadalafil. In this study, tadalafil and two macrocycles (ß-CD and SCX6) were selected as the guest and host molecules, respectively. The inclusion complexes of ß-CD/tadalafil and SCX6/tadalafil were studied by UV spectroscopy and molecular simulation calculations, proving the higher supermolecular recognition capability of SCX6 than ß-CD towards tadalafil. The ß-CD@RGO and SCX6@RGO composites were prepared by a wet-chemical route. The obtained composites were characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, atomic force microscopy, and zeta potential. The SCX6@RGO showed a higher electrochemical response than ß-CD@RGO, which was caused by the higher recognition capability of SCX6 than ß-CD. By combining the merits of SCX6 and the RGO, a sensitive electrochemical sensing platform was developed based on the SCX6@RGO nanohybrids. A linear response range of 0.1-50 µM and 50-1000 µM for tadalafil with a low detection limit of 0.045 µM (S/N=3) was obtained by using this method. The constructed sensing platform was successfully used to determine tadalafil in herbal sexual health products and spiked human serum samples, suggesting its promising analytical applications for the trace level determination of tadalafil.

Treatment with rilpivirine does not alter plasma concentrations of the CYP3A substrates tadalafil and midazolam in humans.

OBJECTIVES: Antiretroviral combination therapy of patients infected with HIV has greatly increased their life expectancy. Hence, the treatment of HIV-related long-term complications and age-related comorbidities has become more important. Reported incidence rates of erectile dysfunction (ED) and pulmonary arterial hypertension (PAH) are increasing in HIV-positive patients, potentially requiring treatment with phosphodiesterase 5 inhibitors such as sildenafil or tadalafil. In vitro, the NNRTI rilpivirine is both a pregnane X receptor agonist and cytochrome P450 (CYP) 3A inhibitor. Clinical data concerning the potential effects of rilpivirine coadministration on the pharmacokinetics of the CYP3A substrate tadalafil are lacking. METHODS: We enrolled 20 healthy volunteers in an open-label, two-part, one-arm Phase I clinical trial to investigate acute and chronic effects of multiple doses of 25 mg of oral rilpivirine on single-dose and steady-state pharmacokinetics of multiple oral 20 mg doses of tadalafil. CYP3A activity was measured simultaneously with the oral midazolam microdose test. RESULTS: We did not observe a change of tadalafil single-dose and steady-state exposure or of CYP3A activity measured at initiation, during maintenance and upon discontinuation of rilpivirine treatment after single-dose and chronic administration of rilpivirine. CONCLUSIONS: Tadalafil can be combined with rilpivirine without dose adjustment or drug monitoring in HIV patients with ED or PAH. Rilpivirine at daily therapeutic doses of 25 mg does not induce or inhibit CYP3A-dependent drug metabolism.

Validated spectrofluorimetric method for determination of two phosphodiesterase inhibitors tadalafil and vardenafil in pharmaceutical preparations and spiked human plasma.

A valid, sensitive and rapid spectrofluorimetric method has been developed and validated for determination of both tadalafil (TAD) and vardenafil (VAR) either in their pure form, in their tablet dosage forms or spiked in human plasma. This method is based on measurement of the native fluorescence of both drugs in acetonitrile at λem 330 and 470 nm after excitation at 280 and 275 nm for tadalafil and vardenafil, respectively. Linear relationships were obtained over the concentration range 4-40 and 10-250 ng/mL with a minimum detection of 1 and 3 ng/mL for tadalafil and vardenafil, respectively. Various experimental parameters affecting the fluorescence intensity were carefully studied and optimized. The developed method was applied successfully for the determination of tadalafil and vardenafil in bulk drugs and tablet dosage forms. Moreover, the high sensitivity of the proposed method permitted their determination in spiked human plasma. The developed method was validated in terms of specificity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision and accuracy. The mean recoveries of the analytes in pharmaceutical preparations were in agreement with those obtained from the comparison methods, as revealed by statistical analysis of the obtained results using Student's t-test and the variance ratio F-test.

A novel liquid chromatographic method with fluorescence detection for quantitation of tadalafil and dapoxetine hydrochloride in pharmaceutical dosage form and human plasma.

Tadalafil (TAD) and dapoxetine HCl (DAP) are recently co-formulated and both show native fluorescence. Therefore, a novel, accurate, specific and sensitive reversed-phase high performance liquid chromatographic method with fluorescence detection was developed and validated for their separation and quantitation in dosage form and human plasma using avanafil as an internal standard (IS). Separation was achieved using isocratic elution within 7.0 min on C18 column and acetonitrile-0.15% triethylamine (40:60, v/v; pH 4) as a mobile phase. The flow rate was 1.0 mL/min and the detection was time-programmed at 330, 410 and 370 nm for TAD, DAP and IS, respectively, after excitation at 236 nm. The linear ranges from 0.01 to 30.00 µg/mL for each drug with the limits of detection of 4.20 and 7.20 ng/mL for TAD and DAP, respectively. The method was validated in accordance to the International Conference on Harmonization (ICH) guidelines and was successfully applied to spiked human plasma with mean recoveries of 98.17% and 98.83% for TAD and DAP respectively.

Dried Blood Spot Technique for the Monitoring of Ambrisentan, Bosentan, Sildenafil, and Tadalafil in Patients with Pulmonary Arterial Hypertension.

Endothelin receptor antagonists (ERA) and phosphodiesterase 5 inhibitors (PDE5I) are long-term therapeutics for the treatment of pulmonary arterial hypertension (PAH). Their interindividual pharmacokinetic variability is remarkably large, and despite the seriousness of the disease, nonadherence is occurring. Therefore, methods to monitor sufficient circulating drug levels are essential. The objectives of this study were to develop and validate dried blood spot (DBS) assays for the quantification of ambrisentan, bosentan, sildenafil, tadalafil, and their main metabolites. We also quantified the influence of different hematocrit levels and assessed the correlation of simultaneously taken capillary whole blood (DBS) and venous plasma samples. The aliquot punches were extracted by liquid/liquid extraction followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantification methods. All assays fulfilled the requirements of the FDA and EMA guidelines for assay validation with a lower limit of quantification of 2.5 ng/mL for the ERAs, 5 ng/mL for sildenafil, and 10 ng/mL for tadalafil. All analytes were stable for at least 147 days when stored on DBS filter paper cards at room temperature in the dark. Due to poor distribution into erythrocytes, drug concentrations in DBS were always lower than in plasma, resulting in conversion factors of 1.58 for ambrisentan and sildenafil and 1.52 for bosentan and tadalafil.

Ambrisentan and Tadalafil Up-front Combination Therapy in Scleroderma-associated Pulmonary Arterial Hypertension.

BACKGROUND: Scleroderma-associated pulmonary arterial hypertension (SSc-PAH) is a rare disease characterized by a very dismal response to therapy and poor survival. We assessed the effects of up-front combination PAH therapy in patients with SSc-PAH. METHODS: In this prospective, multicenter, open-label trial, 24 treatment-naive patients with SSc-PAH received ambrisentan 10 mg and tadalafil 40 mg daily for 36 weeks. Functional, hemodynamic, and imaging (cardiac magnetic resonance imaging and echocardiography) assessments at baseline and 36 weeks included changes in right ventricular (RV) mass and pulmonary vascular resistance as co-primary endpoints and stroke volume/pulmonary pulse pressure ratio, tricuspid annular plane systolic excursion, 6-minute walk distance, and N-terminal pro-brain natriuretic peptide as secondary endpoints. RESULTS: At 36 weeks, we found that treatment had resulted in significant reductions in median (interquartile range [IQR]) RV mass (28.0 g [IQR, 20.6-32.9] vs. 32.5 g [IQR, 23.2-41.4]; P < 0.05) and median pulmonary vascular resistance (3.1 Wood units [IQR, 2.0-5.7] vs. 6.9 Wood units [IQR, 4.0-12.9]; P < 0.0001) and in improvements in median stroke volume/pulmonary pulse pressure ratio (2.6 ml/mm Hg [IQR, 1.8-3.5] vs. 1.4 ml/mm Hg [IQR 8.9-2.4]; P < 0.0001) and mean ( ± SD) tricuspid annular plane systolic excursion (2.2 ± 0.12 cm vs. 1.65 ± 0.11 cm; P < 0.0001), 6-minute walk distance (395 ± 99 m vs. 343 ± 131 m; P = 0.001), and serum N-terminal pro-brain natriuretic peptide (647 ± 1,127 pg/ml vs. 1,578 ± 2,647 pg/ml; P < 0.05). CONCLUSIONS: Up-front combination therapy with ambrisentan and tadalafil significantly improved hemodynamics, RV structure and function, and functional status in treatment-naive patients with SSc-PAH and may represent a very effective therapy for this patient population. In addition, we identified novel hemodynamic and imaging biomarkers that could have potential value in future clinical trials. Clinical trial registered with www.clinicaltrials.gov (NCT01042158).