Accepted Manuscript Title: Development of a sensitive UPLC-ESI-MS/MS method for quantification of sofosbuvir and its metabolite, GS-331007, in human plasma: Application to a bioequivalence study Author: Mamdouh R. Rezk Emad B. Basalious Iman A. Karim PII: DOI: Reference:

S0731-7085(15)00292-7 http://dx.doi.org/doi:10.1016/j.jpba.2015.05.006 PBA 10086

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

20-1-2015 7-5-2015 11-5-2015

Please cite this article as: M.R. Rezk, E.B. Basalious, I.A. Karim, Development of a sensitive UPLC-ESI-MS/MS method for quantification of sofosbuvir and its metabolite, GS-331007, in human plasma: Application to a bioequivalence study, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights  Simultaneous determination of sofosbuvir and GS-331007 in plasma was done.

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 The method was applied for a bioequivalence study.

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 A validated, highly sensitive UPLC-ESI-MS/MS method was developed.

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Development of a sensitive UPLC-ESI-MS/MS method for

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quantification of sofosbuvir and its metabolite, GS-331007, in human

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plasma: Application to a bioequivalence study

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Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, 11562, Cairo, Egypt.

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Pharmaceutics and Industrial Pharmacy Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, 11562,

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Cairo, Egypt.

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Advanced Research Center (ARC), Nasr City, Cairo, Egypt.

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Mamdouh R. Rezk1*, Emad B. Basalious2 and Iman A. Karim3

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Abstract

A rapid and simple LC-MS/MS method was developed and validated for the

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simultaneous estimation of sofosbuvir (SF) and its metabolite GS-331007 (GS)

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using famotidine as an internal standard (IS). The Xevo TQD LC-MS/MS was

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operated under the multiple-reaction monitoring mode using electrospray ionization.

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Extraction with ethyl acetate was used in sample preparation. The prepared samples

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were chromatographed on Acquity UPLC HSS C18 (50 mm x 2.1 mm, 1.8 μm)

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column by pumping 0.1% formic acid and acetonitrile (50:50, v/v) in an isocratic

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mode at a flow rate of 0.3 ml/min. Method validation was performed as per the FDA

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guidelines and the standard curves were found to be linear in the range of 10-2500

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ng/ml for both SF and its metabolite. The intra-day and inter-day precision and

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accuracy results were within the acceptable limits. A very short run time of 1.2

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minutes made it possible to analyze more than 300 human plasma samples per day.

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The developed assay method was successfully applied to a bioequivalence study in

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human volunteers.

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Keywords:

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Sofosbuvir; GS-331007; UPLC-MS/MS; Plasma; Validation; Bioequivalence.

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*Corresponding author. Tel.: +20 1224168633; Fax: +20 222738259

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E-mail

[email protected]

(Mamdouh

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Rezk)

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1. Introduction Sofosbuvir (SF), (Fig. 1a), is a phosphoramidate prodrug of β-d-2'-deoxy-2'-α-

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fluoro-2'-β-C-methyluridine nucleotide for the treatment of hepatitis C virus with

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enhanced antiviral potency compared with earlier nucleoside analogs [1].

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It shares properties with the intracellular nucleoside substrates of the target HCV

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enzymes involved in the transcription of the viral genome and, when phosphorylated

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to the nucleoside-triphosphate, lead to premature termination of the growing HCV

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RNA chain during viral replication [2]. Compared to previous treatments,

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sofosbuvir-based regimens provide a higher cure rate, fewer side effects, and a two-

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to four-fold reduced duration of therapy [3-5].

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To the best of our knowledge no published method is available for the

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simultaneous quantification of SF and its metabolite, GS-331007, in biological

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matrices.

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In the present work, development of a simple, rapid and reproducible method

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to estimate SF and GS-331007 in human plasma was achieved. This method provides

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high accuracy, sensitivity and specificity by applying simple liquid-liquid extraction

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using ultra-performance liquid chromatography and detection by electrospray tandem

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mass spectrometry (UPLC-MS/MS). In order to allow high throughput analysis,

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required for a pharmacokinetic and bioequivalence study, this method involves a very 5 Page 5 of 36

short analysis time. This method was useful to estimate the concentration of SF, and

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GS-331007 in plasma samples collected from healthy volunteers after administration

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of a single dose of sofosbuvir 400 mg.

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2. Experimental

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2.1. Chemicals and reagents

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Sofosbuvir pure standard with batch number of 201410093 was purchased

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from Virdev Intermediates Pvt Ltd., India. GS-331007 pure standard with batch

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number of SVI-ALS-14-088 was purchased from and Alsachim, Strasbourg-France.

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Famotidine (IS) pure standard was purchased from Changzhou Longcheng

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Pharmaceutical Co. Ltd., China.

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Acetonitrile and methanol were HPLC grade, J T Baker, USA. Formic acid

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and ethyl acetate were purchased from Scharlau, Spain. Double distilled water was

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obtained from Aquatron, UK. Blank plasma was obtained from National Institute of

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Urology and Nephrology (Egypt) and it was stored at -80 °C.

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2.2. Pharmaceutical formulation ®

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Sovaldi 400 mg tablets, (reference product), batch no. PMPW, was

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manufactured by Gilead Sciences, Inc. Foster City, USA for Gilead Sciences

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Canada, Inc. 6 Page 6 of 36

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Mpiviropack 400 mg tablets, (test product), batch no. 1432512, was manufactured

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by Marcyrl Pharmaceutical Industries, Egypt.

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Each tablet from test or reference product is claimed to contain 400 mg of

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sofosbuvir.

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2.3. Instrumentation

Quantitative analysis was performed on a Waters Acquity UPLC H-Class-

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Xevo TQD system (MA, USA) equipped with electrospray ionization operated in the

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positive ionization mode. Chromatographic separation of analytes was carried out on

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Acquity UPLC HSS C18 (50 x 2.1 mm, 1.8 μm) column using acetonitrile-0.1%

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formic acid (50: 50, v/v) as a mobile phase at a flow rate of 0.3 ml/minutes,

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isocraticaly. The column was maintained at 25 °C and the pressure of the system was

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6500 psi. The source dependent parameters maintained for both the analytes and the

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internal standard (IS) were: cone gas flow, 50 L/hr; desolvation gas flow, 800 L/hr;

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capillary voltage, 3.5 kV, source temperature, 120 °C; desolvation temperature, 350

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°

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collision energy were set at 40 V and 20 eV for SF; 25 V and 12 eV for GS-331007;

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40 V and 20 eV for IS, respectively. Unit mass resolution was employed and the

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dwell time was set at 100 ms. Detection of the ions was performed in the multiple-

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reaction monitoring (MRM) mode, by monitoring the transition pairs (precursor to

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C. The optimum values for compound dependent parameters like cone voltage and

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product ion) of m/z 530.21 to m/z 243.03 for SF, m/z 261.13 to m/z 112.95 for GS-

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331007, m/z 338.20 to m/z 188.76 for IS. Mass Lynx software version 4.1 was used

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to control all parameters of UPLC and MS.

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2.4. Calibrators and quality control samples

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Primary stock solutions, (200 µg/ml), of SF, GS-331007 and IS for

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preparation of standard and quality control samples were prepared from separate

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stock solutions. All the primary stock solutions were prepared in methanol and

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stored at -20 °C; they were found to be stable for one month. Appropriate dilutions

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were made in methanol for the primary stock solutions to produce working stock

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solutions of 20 µg/ml for both SF and GS-331007, while for IS it was 750 ng/ml on

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the day of analysis and these stocks were used to prepare the calibration curves.

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Calibration curves and quality control samples were prepared every time before

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sample analysis. Eight different working standard solutions of SF and GS-331007

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were prepared by accurately taking different volumes from its primary, secondary

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stock solutions with appropriate dilution into 10 ml with methanol to prepare the

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calibration and quality control samples. Calibration and QC samples were prepared

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by spiking 950 µl of control human plasma with 25 µl of each; SF and GS-331007

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on the day of analysis as illustrated in table 1.

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2.5. Sample preparation A volume of 50 µl of famotidine, 750 ng/ml, (IS) was added to 0.5 ml plasma. Extraction was done by adding 3 ml of ethyl acetate to the spiked plasma

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samples then vortex was performed for 60 seconds. Finally, centrifugation was

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applied at 3500 rpm for 10 minutes to allow separation of the organic phase. A

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volume of 2.5 ml of the upper organic layer was transferred into another dry

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clean tube. The organic solvent was evaporated at 60 ºC using Eppindorf

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sample concentrator till dryness. The residue was reconstituted with 150 µl of

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mobile phase then a volume of 5 μl of this solution was injected into the UPLC-

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MS/MS system. The peaks were detected by Acquity UPLC H-Class-Xevo

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TQD and were interpreted in the form of reported peak areas. Concentrations of

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SF and GS-331007 in unknown samples were calculated by referring to the

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prepared calibration curves.

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2.6. Method validation

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The method was validated to meet the acceptance criteria of industrial

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guidance for bioanalytical method validation [6, 7].

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2.6.1. Specificity and selectivity

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The specificity of the method was determined by analyzing six different

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batches of human plasma to demonstrate the lack of chromatographic interference

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from endogenous plasma components. 9 Page 9 of 36

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2.6.2. Calibration curve Calibration curves were acquired by plotting the peak area ratio of the

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transition pair of analytes to that of IS against the nominal concentration of

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calibration standards. The concentrations used for both SF and GS-331007

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calibration curves were 10, 20, 100, 500, 800, 1200, 1800 and 2500 ng/ml, while 30,

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1000 and 2000 ng/ml were used for LQC, MQC and HQC, respectively. Blank

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sample (without IS) and zero samples (with IS) were run with each calibration curve.

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The acceptance criterion for each back-calculated standard concentration was ±15%

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deviation from the nominal value except at LLOQ, which was set at ±20%.

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2.6.3. Precision and accuracy

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Inter- and intra-assay precision and accuracy were determined by analyzing

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six replicates at the lower level of quantification (LLOQ) in addition to three

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different QC levels as described above on different days. The criteria for

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acceptability of the data included accuracy ±15% standard deviation (SD) from the

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nominal values and a precision ≤ 15% relative standard deviation (RSD).

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2.6.4. Recovery

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The recovery of SF and GS-331007 was determined by comparing the responses of

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the analytes extracted from replicate QC samples at LQC, MQC and HQC with the

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response of analytes from post-extracted plasma standard sample at equivalent

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concentrations [8].

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2.6.5. Matrix effect

The effect of plasma constituents over the ionization of analytes and IS was

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determined by comparing the responses of the post extracted plasma standard QC

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samples (n=4) with the response of analytes from neat samples at equivalent

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concentrations[9]. Matrix effect was determined at same concentrations for each

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analyte as in recovery experiment.

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2.6.6. Dilution accuracy

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Dilution accuracy was investigated to ensure that samples could be diluted

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with blank matrix without affecting the final concentration. SF and GS-331007

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separately spiked human plasma samples prepared at concentrations 4000 ng/ml for

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both, and were diluted with pooled human plasma two and four folds in six

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replicates and analyzed. The six replicates should have precision ≤ 15% and

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accuracy of 100 ± 15%.

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2.6.7. Stability experiments

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The stability of analytes and IS in the injection solvent was determined

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periodically by injecting replicate preparations of processed samples up to 24 hours 11 Page 11 of 36

(in auto-sampler) after the initial injection. The peak-areas of the analytes and IS

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obtained at initial cycle were used as the reference to determine the relative stability

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of the analytes at subsequent points. Stability of analytes in the plasma after 8 hours

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exposure in an ice bath (bench top) was determined at three concentrations in six

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replicates. Freezer stability of the analytes in plasma was assessed by analyzing the

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QC samples stored at -80 ± 10 °C for at least 6 weeks. The stability of analytes in

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plasma following repeated three freeze-thaw cycles (stored at -80 °C) was assessed

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using QC samples spiked with analytes. Samples were processed as described above.

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Samples were considered to be stable if assay values were within the acceptable

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limits of accuracy (i.e. ±15% SD) and precision (i.e. ≤ 15% RSD) [7].

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2.7. Pharmacokinetic/bioequivalence study and statistical analysis The purpose of the study was to investigate the bioequivalence of one tablet of

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Mpiviropack 400 mg (Marcyrl Pharmaceutical Industries, Egypt) and one tablet of

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Sovaldi® 400 mg (Sciences, Inc. Foster City, USA), manufactured for Gilead

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Sciences, Canada, after a single oral dose administration of each to healthy adult

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volunteers under fasting conditions. The design of the study was an open label,

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balanced, randomized two-treatment, two-period, two-sequence, crossover, single-

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dose bioequivalence study in 24 healthy adult Egyptian subjects under fast

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conditions. The primary target variables of the study were Cmax, AUC0-72

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AUC0-inf, which were analyzed using the confidence interval approach. The

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secondary end points of the study included AUC0-72 hrs/AUC0-inf, Tmax, Kel and t1/2. The concerned subjects were informed about the objectives and possible risks

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involved in the study and a written consent was obtained. The study was conducted

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as per International Conference on Harmonization and US-FDA guidelines. A

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cannula was inserted into each subject’s forearm vein before drug administration.

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The subjects were orally administered a single dose of test and reference

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formulations with 240 ml of water after a recommended washout period of two

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weeks. Blood samples were collected, into heparinized tubes, at 0.00 (pre-dose), 0.5,

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1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, 24, 48 & 72

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administration of the dose for test and reference formulations. The number of blood

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collections for drug analysis was 18 samples in each study period. The collected

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blood samples were centrifuged at 3500 rpm for 10 minutes at 4 oC and then the

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plasma was transferred directly into 5-ml plastic tubes. The plasma samples were

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stored at the study site in an ultra deep freezer at -80 oC till the analysis time.

hours after oral

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Sampling was truncated to 72 hours due to relatively long half life of the

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metabolite (GS-331007). During the study, subjects had a standard diet while water

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intake was unmonitored. The pharmacokinetic parameters for SF and GS-331007

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were estimated by non-compartmental analysis using in-house validated excel

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software. 13 Page 13 of 36

The 90% confidence interval for the difference of means between the two

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formulations least square means was calculated for the target variable using log-

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transformed data. Similarly, power and ratio analysis was performed on the log

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transformed data. The terminal end points for the elimination rate constant were

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automatically selected using the software using the best fit model. To determine

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whether the test and reference formulations were pharmacokinetically equivalent,

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Cmax, AUC0-72hrs and AUC0-inf and their ratios (test/reference) using log transformed

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data were assessed. The drug formulations were considered equivalent if the

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difference between the compared parameters was statistically non significant (p ≥

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0.05) and the 90% confidence intervals for these parameters were within 0.8-1.25.

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3. Results and discussion

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3.1. Sample preparation and chromatographic conditions

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Sample preparation is an important step for the determination of SF and its

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metabolite, GS-331007, in human plasma. Different approaches were tried as liquid-

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liquid extraction technique (using ethyl acetate, diethyl ether, dichloromethane, and

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n-hexane) and precipitation technique (using methanol and acetonitrile) for

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simultaneous determination of SF and its metabolite GS-331007. Extraction of SF

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and GS-331007 from human plasma was best achieved using ethyl acetate as an

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extracting solvent. The organic extract, containing SF, GS-331007 and IS was 14 Page 14 of 36

evaporated using Eppindorf sample concentrator then the residue was reconstituted

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with 150 µL of mobile phase and a volume of 5 µL was injected into the UPLC-

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MS/MS system.

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SF and its metabolite, GS-331007, can be easily protonated under acidic

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chromatographic conditions. Therefore, electrospray ionization in the positive ion

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mode was used for MRM analysis. The Q1 full-scan mass spectra of SF,

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GS-331007 and IS showed predominant protonated precursor [M+H]+ ions at m/z

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530.21, 261.13 and 338.20, respectively. Detection of ions was performed in MRM

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mode by monitoring the transition pairs as described under the experimental section.

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To optimize the proposed UPLC-MS/MS method, the effects of several

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chromatographic parameters were investigated. These included the type of organic

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modifier, pH of aqueous solution, and organic modifier-aqueous ratio. These

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parameters were optimized based on the peak shape, peak intensity/area, peak

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resolution and retention time for the analytes on ACQuity UPLC HSS C18 (50 mm x

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2.1 mm, 1.8 μm) column.

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Initially acetonitrile-methanol was used as an organic modifier along with

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mobile phase additives like ammonium formate, ammonium acetate, and 0.1%

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aqueous formic acid. It was observed that the composition and pH of the mobile

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phase had a significant impact on separation selectivity and sensitivity of the

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method. The sensitivity was significantly increased with the use of 0.1% aqueous 15 Page 15 of 36

formic acid compared with ammonium acetate or ammonium formate at pH 3.0,

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using acetonitrile as the organic modifier. Various acetonitrile to 0.1% formic acid

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ratios were also studied by varying the organic modifier ratio. Finally, the best

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chromatographic conditions were achieved using an isocratic mode of acetonitrile:

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0.1% formic acid (50:50, v/v) at a flow rate of 0.3 ml/min. Increased lifetime of

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smaller columns, reduction of instrument time and less eluent consumption together

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with a cleaner mass source thanks to a smaller injection volume make this analytical

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approach even more attractive [10]. All the analytes and IS are eluted in the narrow

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range of retention times (0.30-0.67 min.) which is advantageous for the

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compensation of matrix effects (Fig.2). The reproducibility of retention times for the

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analytes, expressed as CV, was ≤ 0.67% for 100 injections on the same column.

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3.2. Method validation

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3.2.1. Selectivity

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The selectivity of the proposed method was demonstrated by its ability to

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differentiate and quantify the analytes from endogenous components in the plasma

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matrix. Fig. 2 shows the chromatograms of (a) drug-free human plasma; (b) blank

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plasma spiked with IS and analytes at LLOQ; (c) plasma sample from a subject 1.5 h

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after administration of one tablet containing 400 mg sofosbuvir and (d) plasma

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sample from a subject 6 h after administration of one tablet containing 400 mg

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sofosbuvir. Additionally, none of the commonly used medications by human

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volunteers interfered at the retention of analytes and IS. The method selectivity was

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demonstrated on six blank plasma samples obtained from healthy volunteers: the

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chromatograms were found to be free of interfering peaks.

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3.2.2. Linearity and limit of quantification

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The calibration curves were linear in the studied range. The calibration curve

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equation is y = bx + c, where y represents analyte/internal standard peak area ratio

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and x represents the analyte concentration in ng/ml. The mean equations of the

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calibration curve (n=6) obtained from 6 points were

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y = 0.0105404 x + 0.019829, r = 0.9998, for SF and

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y = 0.001237 x + 0.00186, r = 0.9996, for GS-331007.

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The limit of quantitation was 10 ng/ml for both SF and GS-331007.

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3.2.3. Precision and accuracy

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The precision, characterized by the relative standard deviation, was 9.1%, and 8.4%

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at LLOQ for SF and GS-331007 in order, while the accuracy, defined as the

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deviation between the true and the measured value expressed as percentages, was

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9.5%, and 11.2% for the two analytes at these concentration (n=6).

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The intra-assay precision and accuracy results across three QC levels are shown in

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Table 2. The precision (RSD) ranged from 3.1 to 5.2% and the accuracy was within

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95.6-110.3% for the analytes. Similarly for inter-assay experiments, Table 3, the

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precision varied from 3.9 to 6.8% and the accuracy was within 108.5 -112.8 %.

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3.2.4. Extraction recovery

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The mean extraction recovery for SF and GS-331007 was calculated at all QC

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levels. It varied from 90.2 to 92.4 %, and 89.3 to 93.1 %, for SF and GS-331007, in

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order. The mean extraction recovery for famotidine (IS) was calculated and found to

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be 95.5%.

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3.2.5. Matrix effects

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The effect of plasma constituents over the ionization of analytes and IS was

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determined by comparing the responses of the post extracted plasma standard QC

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samples at the three levels, QCL, QCM and QCH, (n=4) with the response of

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analytes from neat samples at equivalent concentrations. Matrix effect was

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determined by comparing analyte peak area counts from plasma samples fortified

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with both analytes at three concentration levels covering the linearity range (30,

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1000 and 2000 ng/mL) as well as IS at 750 ng/mL post extraction, to samples from

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neat solutions at the same concentrations for analyte and IS. Numerical values (%)

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for each concentration level were calculated by dividing the area of plasma extracted

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sample spiked with analyte and IS, by the area of the respective neat solution. Matrix

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effect profiles for the whole chromatographic run were investigated by the

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application of the post-column infusion protocol [11]. The relative standard

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deviation of peak area ratios (analyte/IS), was lower than 2% and the relative

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standard deviation of peak areas of individual compounds was lower than 4%,

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indicating no significant matrix effects.

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3.2.6. Dilution accuracy

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Spiked human plasma samples prepared at concentrations 4000 ng/ml for both SF

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and GS-331007 were diluted with pooled human plasma two and four folds in six

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replicates and analyzed. The precisions (CV) for dilution integrity were between

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3.44 and 6.15%, while the accuracy results were within 93.1-102.2% for the analytes

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in order.

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3.2.7. Sample stability

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Stability was concluded if the concentration change was less than 15% of the

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nominal concentration. All the primary stock solutions were prepared in methanol

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and stored at -20 °C; they were found to be stable for one month. Stock solution

19 Page 19 of 36

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stability was studied at two concentration levels and it was found to be 97.1 - 99.4

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%, 97.3 - 98.2 % and 98.6 - 99.7 % for SF, GS-331007 and IS, respectively.

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3.2.7.1. Short-term stability

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The short term stability of analytes in plasma samples (with a low, medium and high

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quality control samples) was studied for period of 24 h at room temperature (25 °C)

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and ambient light. The results are shown in table 4, where the samples were stable

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under the studied conditions.

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3.2.7.2. Post-preparative stability

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Three sets of spiked samples with low, medium and high concentrations of the

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analytes were analyzed and left in the autosampler at 25 °C for one day. The samples

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were analyzed using a freshly prepared calibration samples. The processed samples

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were stable at room temperature for this period. The results are shown in table 4.

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3.2.7.3. Long-term stability

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The long-term stability of frozen plasma samples was examined after 6 weeks

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storage at -80 °C. The samples were stable under studied conditions and the results

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are shown in table 4.

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3.2.7.4. Freeze and thaw stability 20 Page 20 of 36

Plasma samples with low, medium and high concentrations of the three analytes

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were prepared. The samples were stored at -80 °C and subjected for 3 freeze/thaw

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cycles. During each cycle triplicate one ml aliquots was processed, analyzed and the

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results averaged. No significant substance loss during repeated thawing and freezing

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was observed as shown in table 4.

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3.3. Application to biological samples

387

The proposed method was applied to the determination of SF and GS-331007 in

388

plasma samples from a bioequivalence study, which was approved by the ethical

389

committee. An open-label, randomized, single-dose study with two way cross-over

390

design was performed to compare the bioavailability of sofosbuvir between two

391

products, in 24 healthy adults volunteers mean age of the group was 39 years (range

392

24-53), mean weight was 73 kg (range 55-90). Each subject received a tablet from

393

the test product (Mpiviropack 400 mg) and a tablet from reference product (Sovaldi®

394

400 mg) under fasting conditions, in a randomized fashion with a washout period of

395

two weeks. Twenty-four healthy volunteers completed the cross-over process and

396

blood samples of the 24 volunteers were analyzed.

397

Nausea and gastric upset as adverse events (3.8%) were reported during the study

398

but they were transient and mild. SF is extensively metabolized in the liver to form

399

the pharmacologically active nucleoside analog triphosphate. The metabolic

Ac ce p

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386

21 Page 21 of 36

activation pathway involves sequential hydrolysis of the carboxyl ester moiety

401

catalyzed by human cathepsin A or carboxylesterase 1 and phosphoramidate

402

cleavage by histidine triad nucleotide-binding protein 1 followed by phosphorylation

403

by the pyrimidine nucleotide biosysthesis pathway. Dephosphorylation results in the

404

formation of nucleoside metabolite GS-331007 that cannot be efficiently

405

rephosphorylated and lacks anti-HCV activity in vitro [12]. There is no evidence for

406

back-conversion of SF to GS-331007 during plasma collection, sample extraction,

407

on bench top or long term storage condition.

408

SF has a very rapid elimination half life (0.4 h) compared with its metabolite, GS-

409

331007, as shown in figure 2 (d). Evaluation of truncated area for GS-331007 in the

410

assessment of its bioequivalence was done [13, 14].

411

Fig. 3 (a) shows the mean plasma concentrations of GS-331007, while Fig. 3 (b)

412

shows the mean plasma concentrations of SF; the error bars indicate standard

413

deviations at individual time points. It is clear that SF is rapidly metabolized in

414

human body so its mean plasma concentration will not give accurate data for its

415

pharmacokinetics and it is not suitable to estimate the bioequivalence of the two

416

pharmaceutical formulations. Instead, the metabolite pharmacokinetic data can

417

provide sufficient data to compare the bioequivalence of one tablet of Mpiviropack

418

400 mg (test product), and one tablet of Sovaldi® 400 mg (reference product).

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cr

ip t

400

22 Page 22 of 36

Table 5 shows the pharmacokinetic parameters of SF and GS-331007 following oral

420

administration of one tablet of Mpiviropack 400 mg (test product), and one tablet of

421

Sovaldi® 400 mg (reference product).

422

From log-transformed data, at a 90% confidence interval, the study revealed that

423

AUC0-t, AUC0-inf and Cmax were found to be 96.52% (85.76%-108.64%), 96.41%

424

(85.72%-108.43%) and 100.19% (83.10%-120.78) %, respectively, for SF.The values

425

were found to be 101.47 % (91.80%-112.16%), 100.94% (92.35%-110.33%) and

426

99.63% (93.23%-106.48) %, respectively, for its metabolite, GS-331007.

427

The parametric 90% confidence intervals of the mean values for the test/reference

428

ratio were, in each case, within the bioequivalence acceptable boundaries of 80.00%

429

to 125.00% for the pharmacokinetic parameters AUC0-t, AUC0-inf and Cmax. The results

430

of this bioequivalence study showed the equivalence of the two studied products in

431

terms of the rate of absorption as indicated by Tmax and Cmax and in terms of the extent

432

of absorption as indicated by AUC0-t and AUC0-inf.

433

In conclusion, the two formulations can be considered bioequivalent in regard to the

434

extent and rate of absorption and therefore interchangeable.

436

cr

us

an

M

d

te

Ac ce p

435

ip t

419

4. Conclusions

437

The developed and validated UPLC-MS/MS method allows determination of

438

sofosbuvir and its metabolite, GS-331007, in human plasma. The precision and 23 Page 23 of 36

accuracy of the method are well within the limits required for bioanalytical assays.

440

The low limit of quantification permits the use of the method for pharmacokinetic

441

studies.

442

It is possible to analyze more than 300 human plasma samples per day. The

443

developed assay method was successfully applied to a bioequivalence study in

444

human volunteers.

us

cr

ip t

439

445

Acknowledgement

447

Authors would like to acknowledge Marcyrl for Pharmaceutical Industries (MPI),

448

Egypt, for sponsoring this research and providing the facilities for completing this

449

work.

450

te

d

M

an

446

References

452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468

[1] M.J. Sofia, D. Bao, W. Chang, J. Du, D. Nagarathnam, S. Rachakonda, P.G. Reddy, B.S. Ross, P. Wang, H.R. Zhang, S. Bansal, C. Espiritu, M. Keilman, A.M. Lam, H.M. Steuer, C. Niu, M.J. Otto, P.A. Furman, Discovery of a beta-d-2'-deoxy-2'-alpha-fluoro-2'-beta-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus, J Med Chem 53 (2010) 7202-18. [2] A.B. Eldrup, M. Prhavc, J. Brooks, B. Bhat, T.P. Prakash, Q. Song, S. Bera, N. Bhat, P. Dande, P.D. Cook, C.F. Bennett, S.S. Carroll, R.G. Ball, M. Bosserman, C. Burlein, L.F. Colwell, J.F. Fay, O.A. Flores, K. Getty, R.L. LaFemina, J. Leone, M. MacCoss, D.R. McMasters, J.E. Tomassini, D. Von Langen, B. Wolanski, D.B. Olsen, Structure-activity relationship of heterobase-modified 2'-C-methyl ribonucleosides as inhibitors of hepatitis C virus RNA replication, J Med Chem 47 (2004) 5284-97. [3] F.A. Berden, W. Kievit, L.C. Baak, C.M. Bakker, U. Beuers, C.A. Boucher, J.T. Brouwer, D.M. Burger, K.J. van Erpecum, B. van Hoek, A.I. Hoepelman, P. Honkoop, M.J. Kerbert-Dreteler, R.J. de Knegt, G.H. Koek, C.M. van Nieuwkerk, H. van Soest, A.C. Tan, J.M. Vrolijk, J.P. Drenth, Dutch guidance for the treatment of chronic hepatitis C virus infection in a new therapeutic era, Neth J Med 72 (2014) 388-400. [4] E. Cholongitas, G.V. Papatheodoridis, Sofosbuvir: a novel oral agent for chronic hepatitis C, Ann Gastroenterol 27 (2014) 331-7. [5] A. Cha, A. Budovich, Sofosbuvir: a new oral once-daily agent for the treatment of hepatitis C virus infection, Pharmacy & Therapeutics 39 (2014) 345-52.

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24 Page 24 of 36

ip t

cr

us

an

M

d

te

496

[6] FDA, Guidance for industry: bioanalytical method validation. US Department of Health and Human Services, Guidance for industry: bioanalytical method validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CV) (2001). [7] D. Zimmer, New US FDA draft guidance on bioanalytical method validation versus current FDA and EMA guidelines: chromatographic methods and ISR, Bioanal 6 (2014) 13-9. [8] R. Dams, M.A. Huestis, W.E. Lambert, C.M. Murphy, Matrix effect in bio-analysis of illicit drugs with LCMS/MS: influence of ionization type, sample preparation, and biofluid, J Am Soc Mass Spectrom 14 (2003) 1290-4. [9] A. Van Eeckhaut, K. Lanckmans, S. Sarre, I. Smolders, Y. Michotte, Validation of bioanalytical LC-MS/MS assays: evaluation of matrix effects, J Chromatogr B Analyt Technol Biomed Life Sci 877 (2009) 2198-207. [10] C. De Nardi, F. Bonelli, Moving from fast to ballistic gradient in liquid chromatography/tandem mass spectrometry pharmaceutical bioanalysis: Matrix effect and chromatographic evaluations, Rapid Commun Mass Spectrom 20 (2006) 2709-16. [11] F.L. Sauvage, J.M. Gaulier, G. Lachatre, P. Marquet, A fully automated turbulent-flow liquid chromatography-tandem mass spectrometry technique for monitoring antidepressants in human serum, Ther Drug Monit 28 (2006) 123-30. [12] A. Alberti, S. Piovesan, The evolution of the therapeutic strategy in hepatitis C: features of sofosbuvir and indications, Dig Liver Dis 46 Suppl 5 S174-8. [13] P. Sathe, J. Venitz, L. Lesko, Evaluation of truncated areas in the assessment of bioequivalence of immediate release formulations of drugs with long half-lives and of Cmax with different dissolution rates, Pharm Res 16 (1999) 939-43. [14] M.R. Rezk, K.A. Badr, Development, optimization and validation of a highly sensitive UPLC-ESI-MS/MS method for simultaneous quantification of amlodipine, benazeprile and benazeprilat in human plasma: application to a bioequivalence study, J Pharm Biomed Anal 98 (2014) 1-8.

Ac ce p

469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

25 Page 25 of 36

496

Table 1

497

Preparation of calibrators and quality control samples for SF and GS-331007 Adding 25 µL of each working standard solution (ng/ml) GS-331007

400

400

800

800

4000

4000

20000

20000

32000

32000

48000

48000

72000

72000

100000

100000

QCL

1200

QCM QCH

GS-331007

10

10

20

20

100

100

500

500

800

800

1200

1200

1800

1800

2500

2500

1200

30

30

40000

40000

1000

1000

80000

80000

2000

2000

1000 µl

an

950 µl

SF

M

Calibrators

Final plasma concentration (ng/ml)

cr

SF

Final volume

ip t

Plasma volume

us

Prepared samples

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498

26 Page 26 of 36

499

Table 2

500

Intra-assay precision and accuracy

GS-331007

Concentration (ng/ml) Measured

Bias (%)

RSD (%)

6

30

32.91

9.7

4.5

6

1000

1094

9.4

4.1

6

2000

2206

10.3

3.8

6

30

28.68

-4.4

5.2

6

1000

1066

6.6

3.5

6

2000

2204

10.2

3.1

M

501 502

506 507

te Ac ce p

505

d

503 504

cr

Added

us

SF

N

an

Compound

ip t

498

27 Page 27 of 36

508

Table 3

509

Inter-assay precision and accuracy

GS-331007

Concentration (ng/ml) Measured

Bias (%)

RSD (%)

18

30

32.64

8.8

6.8

18

1000

1106

10.6

5.2

18

2000

2256

12.8

3.9

18

30

32.91

9.7

6.3

18

1000

1085

8.5

4.8

18

2000

2228

11.4

6.4

M

510

cr

Added

us

SF

N

an

Compound

ip t

507

Ac ce p

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d

511

28 Page 28 of 36

511

Table 4 Stability of SF and GS-331007 in matrix by the proposed method GS-331007 Bias (%), RSD (%)

-3.2, 2.8 -4.8, 3.4 -3.9, 1.2

-2.7, 3.9 -3.3, 2.7 -3.1, 2.3

-1.4, 4.7 -2.3, 2.3 -1.6, 3.2

-3.8, 5.1 -4.9, 3.5 -2.4, 3.7

-6.1, 2.4 -7.5, 1.5 -6.4, 1.9

-7.3, 5.3 -5.4, 3.3 -4.1, 2.2

-4.7, 2.8 -5.9, 3.3 -4.7, 2.5

-5.8, 4.5 -4.5, 3.3 -5.6, 3.9

us an

te

d

Spiked concentration for SF and GS-331007: level 1= 30 ng/ml; level 2= 1000 ng/ml; level 3=2000 ng/ml. a n=6

Ac ce p

515 516

M

a) Short term stability of analyte in matrix at room temperature Spiked concentration level 1a Spiked concentration level 2a Spiked concentration level 3a b) Post-preparative stability at 4°C Spiked concentration level 1a Spiked concentration level 2a Spiked concentration level 3a c) Long term stability of analyte in matrix at -80°C Spiked concentration level 1a Spiked concentration level 2a Spiked concentration level 3a d) Freeze and thaw stability Spiked concentration level 1a Spiked concentration level 2a Spiked concentration level 3a

SF Bias (%), RSD (%)

cr

Parameter

ip t

512 513 514

29 Page 29 of 36

529 530 531 532 533 534 535 536

ip t

Reference

1823.7± 639.1 624.2-2500

1860.1 ±739.1 777.6-2495

1471.8± 517.8 735.6-2428.7

1455.5 ± 444.1 822.1-2397.9

0.75 0.5-2

0.75 0.5-4

3.5 2-6

4 2-6

1941.8±699.3 752.6-3426.3

2062.5±898.8 941.8-4169.8

19558.8±7647.7 10369.2-39246.9

19293.8±7926.6 7865.7-41032.4 21194.3 ± 8290.9 10122.6-43252.9

us

cr

Test

an

528

GS-331007

Reference

21352.8 ± 7814.5 12220.4-40400.1

1.4278±0.4015 0.6095-2.3565

1.3901 ± 0.4445 0.4296-2.2438

0.0300 ± 0.0078 0.0176-0.0483

0.0311 ± 0.0105 0.0144-0.0625

0.53 ± 0.20 0.29-1.14

0.54 ±0.26 0.31-1.61

24.51 ± 5.94 14.35-39.31

24.68 ± 8.16 11.10-47.94

1957.7 ± 706.3 767.4-3510.9

2078.8 ± 898.0 949.4-4240.2

M

527

Cmax (ng/ml) Mean Range Tmax (hr) Median Range AUC0-t (ng hr/ml) Mean Range AUC0-inf (ng hr/ml) Mean Range k(hr-1) Mean Range t1/2(hr) Mean Range

SF Test

d

526

Parameter

te

525

Table 5 Pharmacokinetic parameters of sofosbuvir and GS-331007 following oral administration of one tablet of Mpiviropack 400 mg tablets (test product), and one tablet of Sovaldi® 400 mg tablets (reference product).

Ac ce p

517 518 519 520 521 522 523 524

30 Page 30 of 36

536 537 538 (a)

ip t

539 540

cr

541

us

542 543 (b)

an

544 545

M

546

550 551 552 553 554 555

te

549

Ac ce p

548

d

547

Fig. 1. Chemical structure of (a) sofosbuvir; (b) sofosbuvir metabolite (GS-331007)

31 Page 31 of 36

555 556

a

559

ip t

558

Intensity

557

cr

560 561

an

564

Intensity

563

us

562

565

M

566

570 571 572 573

d te

569

Ac ce p

568

Intensity

567

Time (min)

574

Fig. 2. Chromatograms of (a) drug-free human plasma; (b) spiked plasma at lower limit of

575

quantitation; (c) plasma sample from a subject at 1.5 h after administration of one tablet containing

576

400 mg sofosbuvir. (d) plasma sample from a subject at 6 h after administration of one tablet

577

containing 400 mg sofosbuvir.

578 579 32 Page 32 of 36

580 581 582

b

cr

585

Intensity

584

ip t

583

586

an

589

Intensity

588

us

587

M

590

597 598 599 600

Intensity

596

Ac ce p

594 595

d

593

te

592

Intensity

591

Time (min)

601 602 603 604 605

Fig.2. (Continued) 33 Page 33 of 36

606 607

c

610

ip t

609

Intensity

608

cr

611

614

Intensity

613

us

612

an

615

d

619

te

620

622 623 624 625 626

Intensity

621

Ac ce p

618

Intensity

617

M

616

Time (min)

627 628 629 630

Fig.2. (Continued)

631 34 Page 34 of 36

632 633 634

d

ip t

635

638

cr

637

Intensity

636

us

639 640

M

643

Intensity

642

an

641

644

d

645

648 649 650 651 652

Intensity

647

Ac ce p

te

646

Time (min)

653 654 655 656 657

Fig.2. (Continued) 35 Page 35 of 36

658 659 660 (a)

ip t

661 662

cr

663 664

us

665

671 672 673 674 675 676

an

Test Mpiviropack

1600.0

Reference Sovaldi®

1400.0

M

670

1800.0

1200.0 1000.0 800.0

d

669

(b)

600.0 400.0 200.0 0.0 0

te

668

2000.0

10

Ac ce p

667

M ean P lasm a C o n c.o f S o fo sb u vir ‘s m etab o lite (GS-331007) , n g/m l

666

20

30

40

50

60

70

80

Time

677

Fig.3. Mean plasma concentration (±SD) after a single 400 mg sofosbuvir oral dose administered

678

to 24 healthy subjects, (a) SF; (b) sofosuvir metabolite (GS-331007)

679

37 Page 36 of 36