Materials
Favipiravir was kindly supplied by Optrix Laboratories private Limited, India. Lamivudine, used as an internal standard, was bought from LGC GmbH, Germany. Potassium dihydrogen phosphate, sodium dihydrogen phosphate, ortho phosphoric acid, acetic acid, ammonium formate and formic acid were purchased from Scharlau, Spain. Methanol and acetonitrile (HPLC grade), Sigma Aldrich, Germany.
Pharmaceutical product
Avigan® 200 mg Tablets, manufactured by Toyama Chemical Co., Ltd., Japan.
Analytical methodology
For in-vitro testing: HPLC Alliance/e2695S separation module, Waters LC system equipped with photodiode array detector (2998 PDA), (USA) was used. Separation was achieved on Inertsil C18 (250 x 4.6 mm, 5 µm) column using acetonitrile: 10 mM phosphate buffer (pH 3) (40:60, v/v) as a mobile phase that was pumped at a flow rate of 1 mL/min. with UV detection at 320 nm.
For in-vivo testing: Waters Acquity UPLC H Class-Xevo TQD system (USA) was used for assay of favipiravir in human plasma. It was equipped with electrospray ionization. Chromatographic separation of analytes was carried out on Acquity UPLC HSS C18 (100 x 2.1 mm, 1.8 μm) column using methanol-10 mM ammonium formate + 0.1% formic acid in gradient mode as a mobile phase at a flow rate of 0.35 mL/minute. Other source dependent parameters were cone gas flow, 50 L/hr; desolvation gas flow, 800 L/hr; capillary voltage, 2.41 kV, source temperature, 120 °C; desolvation temperature, 550 °C. The optimum values for compound dependent parameters like cone voltage and collision energy were set at 33 V and 15 eV for FAV and 20 V and 15 eV for IS, respectively. Detection of the ions was performed in the multiple-reaction monitoring (MRM) mode, by monitoring the transition pairs (precursor to product ion) of m/z 156 to m/z 113 for FAV, m/z 230 to m/z 112 for IS. Mass Lynx software version 4.1 was used to control all parameters of UPLC and MS.
In-vitro dissolution assessment
In vitro dissolution studies were carried out for Avigan® 200 mg tabletsusing USP Apparatus 2 - Rotating Paddle in three different dissolution media, namely, pH 1.2, acetate buffer pH 4.5, and phosphate buffer pH 6.8. All tests were done in triplicate.
All dissolution media were kept at 37 ºC ± 0.5 ºC at 50 rpm. Samples of 5 mL were withdrawn from each dissolution medium at time intervals 5, 10, 15, 20 and 30 minutes and were replaced by 5 mL of fresh dissolution medium to keep the volume constant. Membrane filters of 0.45 µm were used to filter the withdrawn samples and the first part of the filtrate was discarded. One mL of the filtrate of each sample was placed in 10 mL volumetric flask and completed to volume with mobile phase (filtered degassed mixture of phosphate buffer (pH 3): acetonitrile (60: 40, v/v). A volume of 20 µL of each sample was injected into HPLC-UV apparatus using a validated method of analysis to detect the percent dissolvedof favipiravir at 320 nm.
Study subjects and study design
Twenty-seven male healthy Egyptian volunteers were recruited to participate in this study. Demographic data including age, height, weight and body mass index and physical examinations and vital sign were examined. All laboratory tests including biochemical, serological and urine analyses were carried out. One tablet was swallowed by each volunteer under fasting condition for 10 hours pre-dose. This clinical study was done in accordance with the Guideline for Good Clinical Practice of the International Conference on Harmonization [22] and the principles of the World Medical Association’s Declaration of Helsinki [23] and was authorized by the ethics committee of Advanced Research Center (ARC), Egypt. Each volunteer signed an informed consent form written in lay language to understand his role and rights in this study and the possible risks [24].
Blood sampling
Blood samples were obtained from the volunteers at 0.00 (pre-dose), 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 3, 3.5, 4, 5, 6, 8 and 10 hours after dose administration. The collected blood samples were centrifuged at 3500 rpm for 10 minutes and then transferred directly into 5-mL plastic tubes. The plasma samples were stored at the study site in an ultra-deep freezer at -80 ºC until measurement.
Pharmacokinetic analysis
The pharmacokinetic parameters (The maximum concentration in plasma (Cpmax), time to reach it (tmax), the area under the curve (AUC0-t and AUC0-∞) and the terminal elimination half-life (t1/2) were calculated by non-compartmental analysis using Phoenix WinNonlin® software (version 8.1; Certara USA, Princeton, NJ, USA). The AUC0–t was calculated by the linear trapezoidal rule. The elimination rate constant (k) was estimated from the slope of the terminal elimination phase of the plasma concentration-time curve and hence the elimination half-life (t1/2) was calculated by using the formula: t1/2 = 0.693/k. The AUC0-∞was calculated using the following formula:
AUC0-∞ = AUC0–t + Cp*/k
Where Cp* is the last measured concentration.
In vitro in vivo correlation (IVIVC)
The cumulative percent dissolved of favipiravir at 5, 15 and 30 minutes were correlated with partial areas under the curves of the in-vivo plasma concentration – time curves namely, AUC0-0.5, AUC0-0.75 and AUC0-1 till reaching the peak (Cpmax). This procedure was done for each dissolution medium (pH 1.2, pH 4.5 and pH 6.8). The % dissolved was depicted on x-axis (independent variable) and the calculated partial areas were depicted on y-axis. The coefficient of determination (r2) and the slope were calculated by linear regression analysis using Microsoft Excel software to develop level C correlation [25].
Statistical evaluation
The student t-test was used to determine the significance difference between the means of two sets of data for in vitro dissolution data. Microsoft Excel 365® was used to perform such a calculation. z-test is used to compare between 2 sample means [26]. These calculations were done applying special syntax using SPSS software ver. 16.0 (IBM, Armonk, NY, USA).