Use of healthy male volunteers in bioequivalence studies

Use of healthy male volunteers in bioequivalence studies of antineoplastic drugs: a pivotal study with capecitabine Gilberto De Nucci Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil

Capecitabine • Capecitabine is an adjuvant treatment for colon cancer and for the treatment of metastatic breast cancer in patients whose pathology did not improve during treatment with otherapeutic agents. • Capecitabine is a prodrug, and it is selectively activated by tumor cells to its cytotoxic moiety, 5 -fluorouracil, by thymidine phosphorylase, which is generally expressed at high levels in tumors.

Capecitabine • Clinical and pharmacokinetics studies for capecitabine are performed in patients with cancer. • Standard dosing is 1, 250 mg/m 2 orally twice daily, morning and evening, for 14 consecutive days in 3 -week cycles. • For an average healthy male volunteer weight (70 kg) and height (170 cm) this would mean 2150 mg per dose (4300 mg per day). Corporal Area = 1, 809 m 2

Metabolic activation of capacitabine in humans Reiner et al, 1998

Adverse effects • Asthenia/ fatigue • Diarrhea • Lymphopenia • Leukopenia • Mucositis • Nausea • Neutropenia • Thrombocytopenia • Vomit • Hand Foot Syndrome

April 10 th, 2010 Committee of Ethics in Research approval the pilot study protocol Item V- CER evaluation The Committee of Ethics in Research of the Faculty of Medical Sciences of UNICAMP, obeying the evaluations of the previously designated members for the present case and attending all of dispositions of Resolutions 196/96 and complementary, decided to approve without restrictions the Research Protocol, the informed consent, as well as all the attached files included in the research proposal.

June 6 th 2011 2 nd World Congress on Bioavailability & Bioequivalence 2011 (BABE 2011) Presentation of the results of the two pilot studies of capecitabine on 16 healthy male volunteers

National Commission on Ethics in Research (CONEP) August 10 th 2011 CONEP had received from the National Agency of Sanitary Vigilance (ANVISA) a note warning about the approval from the Committee of Ethics in Research of clinical studies that violated the ethical concepts of resolution CNS 196/96. In response, CONEP released a note stating the following: “Healthy individuals should not enroll on bioequivalence studies of drugs with high toxic potential (chemotherapeutics); Studies with these agents should only be performed on patients that may benefit from the studied drug. ”

- Evaluated the safety of a capecitabine bioequivalence study (150 mg tablet) using 8 healthy male volunteers under fasting and non-fasting conditions. - The study was initially conducted with an open, randomized, two-period crossover design in a 2 -week washout with fasted volunteers. - After the fasted study a new protocol was submitted to the Ethics Committee to evaluate the non-fasted study. - The volunteers were selected for the study after having their health status previously assessed by a clinical evaluation and laboratory tests. - A single capecitabine tablet (150 mg) was given in each interment. - The drug was well tolerated by the volunteers, and they presented no adverse reactions. The biochemical and hematological parameters presented no clinically relevant alterations. - Results indicate that it is safe to perform capecitabine bioequivalence studies in healthy male volunteers.

Technical Note 05/2012 Item 2 – Relative bioavailability/ bioequivalence studies with drugs present on attachment I must respect the general measures described on this note as well as specific measures to each drug described on the same attachment. Item 3. 1 – Based on the toxic potential of these drugs the investigators must decide on the participation of patients or healthy volunteers, as well as the most adequate dose to use in the study. Attachment I – Drugs that must have bioavailability/ bioequivalence studies conducted accordingly to this note Capecitabine Study population Oral dose Male volunteers Female volunteers with definitive sterilization 150 mg

Technical Note 06/2012 Item 2 – Relative bioavailability/ bioequivalence studies with drugs present on attachment I must be conducted on patients of the target population of the studied drug; patients must be receiving treatment to a pathology to which the reference drug is indicated. These studies must respect the technical aspects depicted on this note. Attachment I – Drugs that must have bioavailability/ bioequivalence studies conducted on patients. Azacitidine Ganciclovir Temozolomide Chlorambucil Goserelin Testosterone Clozapine Leuprorelin Topotecan Doxorubicin Melphalan Triptorelin Etoposide Mercaptopurine Vorinostat Everolimus Nabilone Felbamate Nilutamide

European Medicines Agency December 13 th, 2012 6. 2. 1. Phase I, single agent dose and schedule finding trials – Non-clinical data and, when available, data from healthy volunteers should be used to design the studies to be conducted in patients – Based on preclinical tolerability and toxicology findings and the assumed pharmacology of the compound, early trials may sometimes be conducted in healthy volunteers.

Objective To evaluate bioequivalence between two formulations of capecitabine (test and reference) in healthy male volunteers

Why evaluate on healthy volunteers? Capecitabine is employed for the treatment of metastatic cancer, therefore the prognosis of these patients is usually reserved. To expose these critical patients to either ineffective or toxic doses of the drug (due to unforeseen infra or supra-bioavailability of the test formulation), to experimental procedures such as internment, venous puncture and blood collection to evaluate bioequivalence should be ethically re-evaluated. Furthermore, studies performed in patients are with therapeutic doses, using several tablets for each administration (generally a combination of both 150 and 500 mg tablets), a procedure that limits the discrimination between dosage forms.

Methods • The clinical protocols were approved by the university IRB. • The studies were conducted using an open, randomized, twoperiod (150 mg single administration of Xeloda; F. Hoffmann-La Roche Ltd. , vs a test formulation) crossover design with a oneweek washout interval, in two groups, with food. • All male subjects were negative for HIV, HCV and HBV. The laboratory tests (biochemical and hematological parameters) were performed on average three days after the first confinement, and 7 days after the second confinement.

Methods • Subjects: Seventy-two healthy male volunteers were recruted • Hematological evaluation: Hemoglobin, Hematocrit, Erythrocyte Count, VCM, Hb. CM, White Cell and Platelet Count • Biochemistry: Total Cholesterol, Triglycerides, Total Proteins, Albumin, Uric Acid, Total Bilirubins (Direct and Indirect), Alkaline Phosphatase, SGOT (AST), SGPT (ALT), Urea, Creatinine, Fasting Blood Glucose • Physical exam and EKG.

Methods • Dose: 150 mg single-dose on each treatment with a one-week washout period between treatments. • Volunteers had a standardized breakfast 30 minutes before drug administration. • Blood samples were collected at 0: 10, 0: 15, 0: 20, 0: 30, 0: 40, 0: 50, 1: 00, 1: 10, 1: 20, 1: 30, 1: 40, 1: 50, 2: 00, 2: 30, 3: 00, 3: 30, 4: 00, 5: 00, 6: 00 and 7: 00 h after drug administration

Determination of Capecitabine • HPLC coupled to tandem mass spectrometry • Deuterated capecitabine Capecitabine M. W. 359. 93 g/mol Capecitabine-d 11 M. W. 371. 08 g/mol

Results • Three drop-outs due to time unavailability. • The drug was well tolerated by the 69 volunteers that concluded the study. • One volunteer had a mild headache. • Three individuals presented hypertriglyceridemia.

Results • To determinate if hypertriglyceridemia was due to capecitabine, plasma samples remaining from the analitical study were evaluated for triglycerides. • Two out of the 3 volunteers had hypertriglyceridemia before the first and the second dose of capecitabine, and did not have significant variation through out the treatment day. • The volunteer that had normal triglycerides before treatments only presented mildly elevated levels (273 mg/d. L) 8 days after the second dose (discharge evaluation). It resolved without treatment, as evaluated 30 days after the last dose.

Conclusion It is safe to perform capicetabine (150 mg) bioequivalence study in healthy male volunteers.

Use of male volunteers in bioequivalence studies of antineoplastic drugs: a pivotal study with capecitabine. Acknowledgments to the Galeno Analytical Unit Team • • • André Borges – Quality Assurance Antonio Sergio Silveira – Analyst Guilherme Pellegatta – Administrative Supervisor Gustavo Mendes - Bio. Statistical Manager Jaime O. Ilha – Coordinator Lu Shi Chen – Analytical Manager Marinalva Sampaio – Clinical Manager Tainah Babadópulos Magalhães – Quality Manager Thiago Gagliano – Clinical investigator