Bioequivalence Study of Easyrect 100 Mg Tablets Versus Spedra® 100 mg Tablets after a Single Oral Dose Administration of Each to Healthy Volunteers Male Adults under Fasting Conditions

Dosage Forms and Strengths

The submission proposes registration of the following dosage forms and strengths:

• Spedra (avanafil) 50 mg tablets blister package
• Spedra (avanafil) 100 mg tablets blister package
• Spedra (avanafil) 200 mg tablets blister package

Dietary Restrictions, Standardized Diet and Fluid Intake

No consumption of alcohol was permitted for the subjects 48 hours prior to the study’s drugs administration until the collection of the last sample of the respective study period. No consumption of any beverages or foods containing methylxanthines, e.g., caffeine (coffee, tea, cola, cocoa, chocolate, etc.) was Hesham AEliwa and AA Alaa Eldin. Bioequivalence Study of Easyrect 100 Mg Tablets Versus Spedra® 100 mg Tablets after a Single Oral Dose Administration of Each to Healthy Volunteers Male Adults under Fasting Conditions. Bioequiv & Bioavailab Int J 2018, 2(3): 000134.

permitted for the subjects 48 hours prior to the study’s drugs administration until the collection of last blood sample of the respective study period [8].

In addition, the consumption of any beverages or foods containing grapefruit was prohibited one week before the first study’s drugs administration and throughout the entire study.

Food and fluid intake were identical in both study periods, starting from the dinner served 10 hours before study’s drugs administration on study day 1until the end of confinement. Meals were standardized in composition and amount in both periods. The subjects were not allowed to consume any additional beverages or foodstuffs other than those provided throughout the period of confinement [9].

No excessive fluid intake (>150 mL of water per hour) was allowed from 1to10 hours prior to dosing. From one hour before study’s drugs administration to two hours after, no fluid intake was allowed apart from the 240 mL of water used for the administration. Following the four hours, subjects were allowed to drink water but not exceeding 150 mL per hour.

Collection and Handling of Blood Samples for Analysis

In the morning of study day 1of each study period and before study’s drugs administration, a cannula was inserted into the subject’s forearm vein and it remained there until the last blood sample was collected [10].

The volume of blood taken for the determination of Avanafil in plasma was 5 mL per sample. The following blood samples for the analysis of Avanafil in plasma were collected: at the following intervals: 0, 5 min, 10 min, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 & 48 hours after dosing. The number of blood collections for drug analysis was 17 samples in each study period [11].

Blood samples were collected into tubes containing heparin as an anticoagulant slightly shaken, and centrifuged at approximately 3500 rpm for 10 minutes [12]. After centrifugation, plasma samples were transferred directly into a 5-mL plastic tube. These samples were immediately stored at the study site in an ultradeep freezer at a nominal temperature of -80 oC.

The label of the collecting tubes had the study’s code number, subject number, study period, and the Copyright© Hesham AEliwa and AA Alaa Eldin.

designated sample number. It did not contain information that would allow identifying the given treatment. This assured that the analysts at MRC analyzed the samples blindly. The total amount of blood loss during the whole study (including blood for laboratory tests) did not exceed 350 mL in a period of one month. All procedures involving handling of blood samples will be documented [13].

Bio-analytical Drug Determination Methodology

A high performance liquid chromatographic (Shimadzu Prominence with rack changer) method coupled with mass spectrometric detection (LC-MS/MS) was developed, optimized and validated at MRC laboratories for the determination of Avanafil in human plasma. The method was fully validated according to the "FDA Bioanalytical Method Validation Guidelines 2003". Linearity of the assay method was verified within the concentration range of 65 - 7500 ng/mL. All results were within the acceptance criteria as stated in the recommended guidelines. The mean recovery of Avanafil was 102.82% at 65 ng/mL, and 91.91% at 7500 ng/ml. The described method is proved to be sensitive, accurate and reproducible with lower limit of quantification of 65 ng/mL for Avanafil [14].

**Data Quality Assurance**

The MRC’s quality assurance procedures were implemented to assure the built-in quality system. All data entry was done by the trained staff of MRC and checked by the QAU personnel. All procedures were performed according to the internal MRC-approved SOPs with the results being documented and reported. Deliberately, all in-use manuals were archived by the QAU.

All sheets used to document results were issued and approved by the QAU serially, and ultimately reserved in the QAU.

Logbooks were audited internally by the MRC QAU personnel during the internal audit of both the clinical part and the analytical part of the study. All laboratory (clinical and analytical) results were checked and their source documents retained by the QAU [15]. Source document verification was done by the QAU after each data entry. Instrumental outputs after calculations were checked by the QAU personnel. Necessary actions were taken and corrective and/or preventive measures were recommended. A report after each audit period was delivered to the MRC management. Report of audits were followed up and reserved by the QAU. The QAU implements an internal quality system to keep all essential records related to the study guaranteeing the appropriate authorized direct access and traceability of data with utmost confidentiality [16].

All audit trails were enabled within the operated software. After the study report preparation, the QAU audited the report and released its quality assurance statement, which evidenced each audit task [17].

**Pharmacokinetic Calculations**

The pharmacokinetic parameters of Avanafil were estimated using standard non-compartmental methods. The maximal plasma concentration was taken directly from the measured data. The area under the plasma concentration-time curve (AUCt) was calculated from measured datapoints from the time of administration to the time of last quantifiable concentration (Clast) by the linear trapezoidal rule.

The area under the plasma concentration–time curve extrapolated to infinity (AUC∞) was calculated according to the following formula:

$$\text{AUC0-∞} = \text{AUC0-t} + \text{Clast} / [\ln(2)/t_{1/2}],$$ where Clast is the last quantifiable concentration. The ratio AUC0-t/AUC0/-∞ as a percent was determined as an indicator for the adequacy of sampling time.

The elimination half-life $t_{1/2}$ was calculated as $t_{1/2} = \ln(2)/(-b)$ where $b$ was obtained as the slope of the linear regression of the ln-transformed plasma concentrations versus time in the terminal period of the plasma curve.

**Statistical Analysis**

Statistical analysis was performed using a Kinetica version 5.1 (Thermo Scientific, USA).

**Reagents, Chemicals & Standards**

• Avanafil working standard
• Ezogabine working standard
• Water for chromatography (Sharlau, Spain)
• Acetonitrile, HPLC grade (Sigma Aldrich Chemie GmbH, Steinheim-Germany)
• Ammonium Formate, Dichloromethane, (Sigma Aldrich Chemie GmbH, Steinheim-Germany)
• Blank plasma obtained from the Holding Company for Biological Products & Vaccines (VACSERA), Giza, Egypt.

Plasma Concentration-Time Profiles for Each Volunteer

Hesham AEliwa and AA Alaa Eldin. Bioequivalence Study of Easyrect 100 Mg Tablets Versus Spedra® 100 mg Tablets after a Single Oral Dose Administration of Each to Healthy Volunteers Male Adults under Fasting Conditions. Bioequiv & Bioavailab Int J 2018, 2(3): 000134.

Copyright© Hesham AEliwa and AA Alaa Eldin.

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Statistical Program

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Hesham AEliwa and AA Alaa Eldin. Bioequivalence Study of Easyrect 100 Mg Tablets Versus Spedra® 100 mg Tablets after a Single Oral Dose Administration of Each to Healthy Volunteers Male Adults under Fasting Conditions. Bioequiv & Bioavailab Int J 2018, 2(3): 000134.

Copyright© Hesham AEliwa and AA Alaa Eldin.