Feasible TLC-Spectro-Densitometry Technique for Simultaneous Determination of Two Hepatitis C Antiviral Drugs, Sofosbuvir and Simeprevir: Application to Combined Pharmaceutical Dosage Forms and Human Plasma.

A high-performance thin-layer chromatographic method was developed and validated for the concurrent determination of simeprevir (SMV) and sofosbuvir (SOF). The chromatographic separation was attained on silica gel 60 F254 as stationary phase and ethyl acetate-hexane-methanol (5.0:4.0:1.0, v/v/v) as developing solvent with UV detection at 273 nm. The RF values were 0.67±0.02 and 0.43±0.02 for SMV and SOF, respectively. The method has been validated in respect to the guidelines of the International Conference on Harmonization. Linearity was maintained between 60-1,000 and 70-1,200 ng/band for SMV and SOF, respectively, with good correlation coefficients (0.9993-0.9997) for both drugs. The suggested method was highly sensitive as the calculated detection limits were 15 and 22 ng/band, while the quantitation limits were 44 and 66 ng/ band for SMV and SOF, respectively. The suggested methodology has been effectively employed for the determination of the mentioned drugs in their pure forms and their pharmaceutical dosage forms as well as human plasma without significant interference of the pharmaceutical excipients or plasma components.

Microenvironment improvement protocol for the sensitive spectrofluorimetric determination of an hepatitis C virus antiviral (Simeprevir): application to human plasma.

Simeprevir (SPV) is a powerful antihepatitis C virus agent that was newly introduced into the pharmaceutical market. We here established and validated an easy, simple, and sensitive spectrofluorimetric method for its estimation at λem 427 nm (λex 337 nm). The suggested procedure was based on two times enhancement in the original emission of SPV through modifying its microenvironment in buffered aqueous solution by adding Triton X-100. The relationship between the concentration of SPV and the observed fluorescence intensity was linear in the range 0.06-1.0 µg ml-1 with a correlation coefficient of 0.9997. The limits of detection and quantitation were 21 and 64 ng ml-1 , respectively. The present method was effectively applied to quantify SPV content in pharmaceutical tablets and human plasma spiked with the drug with no interference from tablet excipients or plasma components.

Estimating Efflux Transporter-Mediated Disposition of Molecules beyond the Rule of Five (bRo5) Using Transporter Gene Knockout Rats.

Transporter gene knockout models are a practical and widely used tool for pharmacokinetic studies in drug discovery. P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are major efflux transporters that control absorption and bioavailability, and are important when determining oral drug disposition. To the best of our knowledge, beyond the rule of five (bRo5) molecules launched on the market to date tend to be substrates for efflux transporters. The purpose of this study is to evaluate in vivo the impact of efflux transporters on the oral absorption process and systemic clearance using rats which lack P-gp and/or Bcrp expression. We administered five bRo5 substrates (asunaprevir, cyclosporine, danoprevir, ledipasvir, and simeprevir) intravenously or orally to wild-type and Mdr1a, Bcrp, and Mdr1a/Bcrp knockout rats, calculated the clearance, oral bioavailability, and absorption rate profile of each substrate, and compared the results. Systemic clearance of the substrates in knockout rats changed within approximately ±40% compared to wild-types, suggesting the efflux transporters do not have a significant influence on clearance in rats. On the other hand, the oral absorption of substrates in the knockout rats, especially those lacking Mdr1a, increased greatly-between 2- and 5-fold more than in wild-types. This suggests that rat efflux transporters, especially P-gp, greatly reduce the oral exposure of these substrates. Moreover, results on the absorption rate-time profile suggest that efflux transporters are constantly active during the absorption period in rats. Transporter knockout rats are a useful in vivo tool for estimating the transporter-mediated disposition of bRo5 molecules in drug discovery.

A liquid chromatography-tandem mass spectrometry method for simultaneous determination of simeprevir, daclatasvir, sofosbuvir, and GS-331007 applied to a retrospective clinical pharmacological study.

A highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of daclatasvir (DCV), simeprevir (SMV), sofosbuvir (SOF), and its major metabolite GS-331007 in human plasma using stable-isotope-labeled (SIL) analogs as internal standards (IS) to minimize a possible matrix effect. Liquid-liquid extraction (LLE) of the analytes and IS from human plasma was performed using a commercial extraction kit requiring low sample volume (50 µL). The analytes were eluted under a gradient program with mobile phase A (water + 0.1% formic acid) and mobile phase B (methanol + 0.1% formic acid) at a flow-rate of 0.6 mL/min for 10 min. The detection was performed on a Qtrap 5500 triple quadrupole tandem-mass spectrometer using multiple reaction monitoring (MRM) mode via the positive electrospray ionization interface. The method was validated according to the European Medicine Agency (EMA) guidelines over the clinically relevant concentration range of 15.6-2000 ng/mL. The high reproducibility, the low matrix effect associated with the use of SIL-IS, and the need of small sample amounts make this method particularly suited for high-throughput routine analysis. The proposed method was successfully applied to a retrospective clinical pharmacology study involving 67 HIV/HCV co-infected patients treated with a SOF-based therapy. DCV, SMV, SOF, and GS-331007 plasma levels were measured at week 4 of treatment and compared with the patients' clinical and laboratory characteristics. Higher GS-331007 plasma concentrations were observed in female patients compared to males, which can be explained by different anthropometric characteristics between genders. Importantly, patients with high plasma levels of GS-331007 also showed enhanced concentration of DCV and SMV probably due to a specific metabolic/pathological condition. Altogether, our findings indicate that the proposed method is a reliable and accurate new tool for high-throughput screening of large patient cohorts that could be readily used to optimize treatment modalities and reduce drug-related toxicities.

Role of simeprevir plasma concentrations in HCV treated patients with dermatological manifestations.

BACKGROUND AND AIMS: Up till now the role of simeprevir plasma concentrations has not been described in treated patients affected by chronic hepatitis C and with dermatological side-effects. In this study, we have evaluated a possible relationship between plasma levels and the onset of skin complaints for the first time. METHODS: We report a clinical and pharmacokinetic analysis of 56 patients treated with simeprevir-based therapies. RESULTS: Simeprevir plasma concentrations were significantly related to dermatological side-effects at early time-points (P<0.001). In logistic regression, simeprevir concentrations at 1 week was the best predictive factor forskin symptoms (OR=1.901, 95%IC: 1.001-2.304; P=0.007). CONCLUSION: Simeprevir plasma measurements could be a useful tool in a real-life clinical setting for prevention of dermatological symptoms.

Elucidating the Plasma and Liver Pharmacokinetics of Simeprevir in Special Populations Using Physiologically Based Pharmacokinetic Modelling.

The disposition of simeprevir (SMV) in humans is characterised by cytochrome P450 3A4 metabolism and hepatic uptake by organic anion transporting polypeptide 1B1/3 (OATP1B1/3). This study was designed to investigate SMV plasma and liver exposure upon oral administration in subjects infected with hepatitis C virus (HCV), in subjects of Japanese or Chinese origin, subjects with organ impairment and subjects with OATP genetic polymorphisms, using physiologically based pharmacokinetic modelling. Simulations showed that compared with healthy Caucasian subjects, SMV plasma exposure was 2.4-, 1.7-, 2.2- and 2.0-fold higher, respectively, in HCV-infected Caucasian subjects, in healthy Japanese, healthy Chinese and subjects with severe renal impairment. Further simulations showed that compared with HCV-infected Caucasian subjects, SMV plasma exposure was 1.6-fold higher in HCV-infected Japanese subjects. In subjects with OATP1B1 genetic polymorphisms, no noteworthy changes in SMV pharmacokinetics were observed. Simulations suggested that liver concentrations in Caucasians with HCV are 18 times higher than plasma concentrations.

Development of a simple HPLC-UV method for the determination of the hepatitis C virus inhibitor simeprevir in human plasma.

A simple high-performance liquid chromatography method for the determination of the hepatitis C virus protease inhibitor simeprevir in human plasma was developed and validated. The method involved a rapid and simple solid-phase extraction of simeprevir using Oasis HLB 1cc cartridges, isocratic reversed-phase liquid chromatography on an XTerra RP18 (150 mm×4.6 mm, 3.5 µm) column, and ultraviolet detection at 225 nm. The mobile phase consisted of phosphate buffer (pH 6, 52.5 mM) and acetonitrile (30:70, v/v). This assay proved to be sensitive (lower limit of quantification of 0.05 µg/mL), linear (correlation coefficients ≥0.99), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.3%), and accurate (deviations ranged from -8.0 to 1.2% and from -3.3 to 6.0% for intra-day and inter-day analysis, respectively). The method was applied to therapeutic monitoring of patients undergoing simeprevir treatment for hepatitis C and proved to be robust and reliable. Thus, this method provides a simple, sensitive, precise and reproducible assay for dosing simeprevir that can be readily adaptable to routine use by clinical laboratories with standard equipment.

Evaluation of the Pharmacokinetics and Renal Excretion of Simeprevir in Subjects with Renal Impairment.

BACKGROUND: Simeprevir is a N3/4 protease inhibitor approved for the treatment of hepatitis C virus (HCV) infection. HCV prevalence is higher in patients with chronic kidney disease compared with the general population; safe and efficacious therapies in renal impairment are needed. OBJECTIVES: To evaluate simeprevir renal excretion in healthy subjects and to compare the simeprevir steady-state pharmacokinetics between subjects with severe renal impairment and healthy subjects. METHODS: In the mass balance study, healthy adults received a single 200-mg dose of (14)C-simeprevir; radioactivity in the urine and feces was quantified until concentrations were <2% of the administered dose and seven or more stools were produced. In the pharmacokinetic study, non-HCV-infected adults with severe renal impairment (estimated glomerular filtration rate ≤29 mL/min/1.73 m(2)) and matched healthy subjects (estimated glomerular filtration rate ≥80 mL/min/1.73 m(2)) received 150 mg simeprevir for 7 days. Pharmacokinetic analysis was performed post-dose on Day 7. RESULTS: (14)C-simeprevir recovery from the urine was low (0.009-0.138% of total dose). The minimum plasma concentration, maximum plasma concentration, and area under the plasma concentration-time curve at 24 h were 71, 34, and 62% higher, respectively, in subjects with severe renal impairment compared with healthy subjects. The mean fraction of simeprevir unbound to protein was <0.0001 (all subjects). Most adverse events were grade I or II; one subject with renal impairment who was receiving fenofibrate presented with grade 3 rhabdomyolysis. CONCLUSIONS: Simeprevir plasma concentrations were mildly elevated in subjects with severe renal impairment. The results suggest that simeprevir may be administered without dose adjustment in patients with renal impairment.