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2. Bioequivalence of Ipratropium Bromide HFA pMDI 20 μg/actuation in Healthy Volunteers Dr Muneesh Garg, MD Pharmacology, Principal Investigator, Sitec Labs. Pvt. Ltd., Navi Mumbai, India. E-mail: muneesh.garg@siteclabs.com Sitec Labs

3. Introduction • Ipratropium is an anticholinergic drug • Used as a bronchodilator for the maintenance treatment of bronchospasm associated with chronic obstructive pulmonary disease and chronic asthma • Administered by inhalation • One metered dose contains 20 micrograms ipratropium bromide • Usual recommended dose in adults is 1 or 2 puffs three or four times daily. Up to a maximum of 4 puffs at a time • The inhaler can be used with the Aerochamber Plus™ spacer device Sitec Labs

4. Study Products • Innovator- ATROVENT CFC-free (containing ipratropium bromide 20 μg per actuation) marketed by BOEHRINGER INGELHEIM LIMITED, UK) • Generic- ipratropium bromide 20 μg per actuation Sitec Labs

5. Rationale • For marketing authorization of Ipratropium Bromide HFA pMDI 20 μg/actuation in European Union • Three bioequivalence studies were submitted to demonstrate therapeutic equivalence between test and the reference formulation of Ipratropium Bromide HFA pMDI 20 μg/actuation Sitec Labs

6. Objective • The aim of these three studies was to prove bioequivalence of test formulation of Ipratropium Bromide HFA pMDI 20 μg/actuation against the reference formulation ATROVENT CFC-free (containing ipratropium bromide 20 μg per actuation) marketed by BOEHRINGER INGELHEIM LIMITED, UK), with concurrent oral charcoal blockade, without concurrent oral charcoal blockade and with Aerochamber Plus valved holding chamber under fasting conditions Sitec Labs

7. EMA’s Stepwise Approach to Establish TE for OIDPs Sitec Labs

8. Fate of Inhaled Drug Products • Amount of drug reaching systemic circulation (with charcoal)= pulmonary • Amount of drug reaching systemic circulation (without charcoal)= pulmonary + oral Sitec Labs

9. EMA’s PK Study Requirements • European medical agency requires two studies: one with and one without charcoal blockade. • If the reference product is approved to be administered with or without a spacer, two PK studies would be required, one with and the other without the spacer device Sitec Labs

10. Inhalation PK With Charcoal Block • Study-1 was single dose, randomized, 4-period, 2‑sequence, laboratory-blinded, crossover, replicate design conducted in 90 healthy volunteers each under fasting conditions with concurrent oral charcoal blockade with a washout period of 7-14 days • The aim of study-1 was to use oral charcoal to block the gastrointestinal absorption of inhaled Ipratropium and to compare the pulmonary bioavailability of test and the reference product • PK study with charcoal was performed to prove “efficacy equivalence” Sitec Labs

11. Inhalation PK Without Charcoal Block • Study-2 was single dose, randomized, 2-period, 2‑sequence, laboratory-blinded, crossover design conducted in 24 healthy volunteers each under fasting conditions without concurrent oral charcoal blockade with a washout period of 6 days • The aim of study-2 was to compare the total systemic bioavailability of test and the reference product • PK study without charcoal was performed to prove “safety equivalence” Sitec Labs

12. Inhalation PK With Spacer • Study-3 was single dose, randomized, 2-period, 2‑sequence, laboratory-blinded, crossover design conducted in 64 healthy volunteers each under fasting conditions with Aerochamber Plus valved holding chamber with a washout period of 7-10 days • The aim of study-3 was to compare the total systemic bioavailability of test and the reference product with spacer device Sitec Labs

13. Methods - Study Protocols • The protocols were approved by an independent ethics committee prior to study initiation • The studies were conducted in accordance with the ethical principles of the Declaration of Helsinki and its amendments and the International Conference on Harmonization for Good Clinical Practice Guideline • All the three studies were conducted at Sitec Labs. Pvt. Ltd., Navi Mumbai, India Sitec Labs

14. Methods - Participants • Volunteers were screened within 21 days prior to dosing • A total of 90, 24 and 64 non-smoker adult male volunteers, between 18 and 45 years of age (inclusive), having body mass index ≥ 18.5 kg/m2 and ≤ 25.00 kg/m2, in general good health were enrolled in study-1, 2 and 3 respectively • Before inclusion into the study, the volunteers were judged to be healthy by physician based on previous medical history, physical examination, vital signs examination, ECG, chest X-ray, pulmonary function test, pulse oximetry, and clinical laboratory test results- CBC, LFT, RFT, FBS, serological tests for hepatitis B and C and HIV antibodies, Urine routine Sitec Labs

15. Demographics of 90, 24 and 64 Recruited Volunteers of Study-1, 2 and 3 Sitec Labs

16. Training on The Inhalation Technique • Volunteers were trained on the inhalation technique with the help of an in-check dial, aerosol inhalation monitor and a placebo (inactive) inhaler at least for 5 days continuously prior to dosing Sitec Labs

17. Key Points for Correct Inhalation Technique • Complete exhalation before beginning of inhalation • Ensuring a firm seal with the lips around the device mouth piece • After complete exhalation, volunteers were asked to breathe in slowly and deeply at least for 5 s • The device to be actuated while the inhalation is going on • After this the volunteers were asked to hold his breath at least for 10 s and then breath out normally through the nose Sitec Labs

18. Study Procedures on Check-in Day • Written informed consent was obtained from all willing volunteers • Breath alcohol test, drugs of abuse test, vital signs examination, and training technique • Volunteers who fulfilled all the criteria for inclusion were admitted to the study center at least 12hrs prior to dosing • In the study-1, a total of 90 were recruited. Study-1 was conducted in three batches. Batch A consisted of 42 volunteers (subject No. 01 to 42), batch B consisted of 18 volunteers (subject No. 43 to 60), and batch C consisted of 30 volunteers (subject No. 61 to 90) • In the study-2, a total of 24 were recruited • In the study-3, a total of 64 were recruited. Study-3 was conducted in two batches. Batch A consisted of 32 volunteers (subject No. 01 to 32), and batch B consisted of 32 volunteers (subject No. 33 to 64) Sitec Labs

19. Drug Administration • All the volunteers were required to fast for at least 10 h prior to dosing • The test and reference product was primed within 10 min prior to dosing by releasing 2 test sprays • After priming, test and reference product was inhaled; there was a gap of one minute between each puff inhaled by the volunteer with an allowed deviation of ± 5 sec Sitec Labs

20. Study-1 • A single dose of 4 puffs (each puff releases 20 µg of Ipratropium Bromide) of test or the reference product was inhaled by the volunteers as per the randomized sequence in standing position on four separate treatment days • 50 mL (approximately 5 gm) of activated charcoal suspension was given 2 minutes prior to the 1st puff, immediately after dosing and also at 1.00, 2.00 and 4.00 hours post-dose Sitec Labs

21. Study-2 • A single dose of 4 puffs (each puff releases 20 µg of Ipratropium Bromide) of test or the reference product was inhaled by the volunteers as per the randomized sequence in standing position on two separate treatment days Sitec Labs

22. Study-3 • A single dose of 2 puffs (each puff releases 20 µg of Ipratropium Bromide) of test or the reference product was inhaled by the volunteers with the aid of the Aerochamber Plus valved holding chamber as per the randomized sequence in standing position on two separate treatment days Sitec Labs

23. Study Restrictions • Volunteers remained seated at least for the first 2 hrs after dosing • Water was not permitted from 1 hr before dosing until 1 hr following dosing, but it was allowed at all other times • Post dosing, food was withheld for at least 4 hrs. Standardized meals were served at 4, 9, and 13 hrs after dosing Sitec Labs

24. Blood Sampling • In study-1 and 2, Blood samples (1 x 5 mL) were collected at -0.00 hrs (pre-dose) and at 0.08, 0.17, 0.25, 0.33, 0.42, 0.50, 0.75, 1.00, 1.50, 2.00, 3.00, 4.00, 6.00, 9.00, 12.00, 18.00, and 24.00 hrs post dose • In study-3, blood samples (1 x 6 mL) were collected at -0.00 hrs (pre-dose) and blood samples (1 x 5 mL) were collected at 0.017, 0.05, 0.08, 0.17, 0.25, 0.33, 0.42, 0.50, 0.75, 1.00, 1.50, 2.00, 3.00, 4.00, 6.00, 9.00, 12.00, 18.00 and 24.00 hrs post dose • Blood samples were collected with respect to start time of first puff in vacutainers containing sodium heparin anticoagulant • The blood sample tubes were centrifuged to separate plasma and plasma samples were stored at –70°C or below until sample analysis Sitec Labs

25. Bio-analysis • Ipratropium plasma concentrations were determined using validated LC-MS/MS method • The lowest limit of quantitation of method was 3.0 pg/mL for the method used for analysis “with and without charcoal blockage studies” and 1.0 pg/mL for the “with spacer study” Sitec Labs

26. Results- safety (study-1) • 26/90 (28.89%) volunteers experienced AEs • 13 AEs after test and 13 AEs after reference formulation • AEs reported: Cough (5.56%), Headache (4.44%), Injury (3.33%), Abdominal Pain (2.22%), Nasopharyngitis (2.22%), Pain (2.22%), Vomiting (1.11%), Oropharyngeal pain (1.11%), Musculoskeletal chest pain (1.11%), Wrist fracture (1.11%), Convulsion (1.11%), Syncope (1.11%), Renal colic (1.11%), Pyrexia (1.11%) • All AEs except headache were “not related” to IP • No deaths; One SAE (hospitalization), This subject experienced Seizure (convulsion) during washout period. It was considered “not related” to investigational product Sitec Labs

27. Results- safety (study-2) • There were no AEs reported during the study • No deaths/SAE Sitec Labs

28. Results- safety (study-3) • 9/64 (14.06%) volunteers experienced AEs • 5 AEs after test and 4 AEs after reference formulation • AEs reported: Pyrexia (3.13%), Electrocardiogram PR shortened (3.13%), Diarrhea (1.56%), Oropharyngeal pain (1.56%), cough (1.56%), Pain in extremity (1.56%), Mucocutaneous rash (1.56%) • All AEs were “not related” to IP • No deaths/SAE Sitec Labs

29. Results- Pharmacokinetics (study-1) • A total of 90 volunteers were recruited • 3 volunteers were discontinued from the study due to AE. 2 volunteers were dropouts • Leakage of drug was observed during dosing for 4 volunteers. Therefore, data of 4 volunteers was not considered for final pharmacokinetic and statistical analysis • Data of remaining 81 volunteers was considered for pharmacokinetic and statistical analysis who had completed at least two treatment periods Sitec Labs

30. Results- Pharmacokinetics (study-2) • A total of 24 volunteers were recruited, and all 24 volunteers completed the study • No leakage of drug was observed during dosing for all 24 volunteers • Therefore, data of all 24 volunteers was considered for final pharmacokinetic and statistical analysis Sitec Labs

31. Results- Pharmacokinetics (study-3) • A total of 64 volunteers were recruited • 1 volunteer was discontinued from the study due to AE. 5 volunteers were dropouts • No leakage of drug was observed during dosing • Data of remaining 58 volunteers was considered for pharmacokinetic and statistical analysis Sitec Labs

32. Statistical Results of Primary Pharmacokinetic Parameters Sitec Labs

33. Sitec Labs

34. Mean graph (linear) for plasma concentration vs time profile of Ipratropium for study-1 (with charcoal), study-2 (without charcoal), and study-3 (with spacer) Sitec Labs

35. Conclusion • The 90% CI of Ipratropium Bromide for Cmax and AUC0-t were within 80.00-125.00% for all the three studies, suggesting the generic formulation of Ipratropium Bromide HFA pMDI 20 μg/actuation was bioequivalent with the innovator formulation of Atrovent CFC-free 40 µg (20 μg per actuation X 2 puffs) marketed by Boehringer Ingelheim Limited, UK with and without charcoal blockade; and with spacer device according to the European Medicines Agency Guidelines on the Investigation of Bioequivalence Sitec Labs

36. Challenges in PK Studies of Inhaled Drugs • Dose selection: sometimes drug doses are too low to be detected by the standard bio-analytical methods. This either requires increasing the dose of drug or developing more sensitive methods for drug assay • Volunteer selection: healthy non-smokers • Volunteer training: Pulmonary function testing, for correct and consistent inhalation technique • Early and frequent blood sample collection in the initial 1hr Sitec Labs

37. References • http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003504.pdf Sitec Labs

38. We welcome you to our future conferences of Conference Series LLC through 8th World Congress on Bioavailability & Bioequivalence: BA/BE Studies Summit June 26-29, 2017 at San Diego, USA http://bioavailability-bioequivalence.pharmaceuticalconferences.com/ Let us meet again..