6^th World Congress on Bioavailability & Bioequivalence: BA/BE Studies Summit August 17-19, 2015 Chicago, USA

Andre B. Dezani has completed his Master’s degree in 2010 and since then,he is developing his PhD thesis related to permeability studies using different methods as ex vivo and in vitro models. His research field also includes solubility, biopharmaceutical classification systems, dissolution studies and ADME prediction. The studies are conducted in Faculty of Pharmaceutical Sciences of University of Sao Paulo, Brazil

An important factor for the good bioavailability of a drug is the complete absorption and studies involving this prediction have been grown in recent years. Among several studies, ex vivo methods using vertical diffusion chamber (Franz cells) experiments have gained highlight for permeation studies. This method employs an intestinal segment and it can be used for biowaiver purposes. This work aims to show a chromatographic method developed and validated for quantification of metoprolol contained in samples obtained from ex vivo assays using Franz cells. A simple, precise, specific and accurate RP-HPLC (reversed-phase high performance liquid chromatography) method was developed and validated using ICH guideline considering parameters as selectivity/specificity, linearity, precision, accuracy, limit of detection and limit of quantification. The chromatographic conditions were achieved with: Gemini C18 column (250 mm x 4.6 mm x 5 µm), fluorescence detection set at 225nm for excitation and 310nm for emission, oven set at 35°C. The mobile phase consisted of a mixture of phosphate buffer 20mM and acetonitrile (80:20%) pH4.5, flow of 1mL/min and injection volume of 25µL. The assessed parameters were in accordance with the ICH recommendations. The method presented linearity in a range of 10-1000 ng/mL (r²= 0.9993). Precision and accuracy are also adequate with intraday and interday variation no higher than 5%. During development and validation, no interferences were noticed in chromatograms, showing the selectivity and specificity of the method. The chromatographic method was developed and validated for metoprolol quantification in samples from Franz cells assay and its applicability in daily lab routine is possible for permeation studies with several kinds of biological tissues, including intestine. Also, this method can be useful for development of other chromatographic methods in similar studies. This study had financial support by FAPESP, CAPES and CNPq (Brazil).

Thaisa M Dezani has completed her Master’s degree in 2012 and since then, she is developing her PhD thesis related to permeability studies using different methods as in situ, ex vivo and in vitro models. Her research field also includes solubility, biopharmaceutical classification systems,dissolution studies and ADME prediction. The studies are conducted in Faculty of Pharmaceutical Sciences of University of Sao Paulo,Brazil.

Absorption prediction of drugs has been grown in recent years and studies involving in vivo, ex vivo and in situ methods are applied for permeability assessment of substances. Single-Pass Intestinal Perfusion (SPIP) is a technique employed for absorption studies prediction using an intestinal segment. This work aims to show a chromatographic method developed and validated for ranitidine (RNT) quantification from perfusate samples. A simple, precise, specific and accurate RP-HPLC (reversed-phase high performance liquid chromatography) method was developed and validated for RNT using ICH guideline considering parameters as selectivity/specificity, linearity, precision, accuracy, limit of detection and limit of quantification. The chromatographic conditions were achieved with: Gemini C18 column (150 mm x 4.6 mm x 5 µm), ultraviolet detection at 315nm (RNT) and 270 nm (zidovudine as internal standard), oven set at 30°C. The mobile phase consisted of phosphate buffer 10 mM, methanol and acetonitrile (65:30:5) pH 7.2, flow of 1 mL/min and injection volume of 75 µL. The assessed parameters were in accordance with the recommendations. The method presented linearity in a range of 5-150 µg/mL (r²=0.9999). Precision and accuracy are also adequate with intraday and interday variation below 5%. No interferences were noticed in chromatograms, reflecting the selectivity and specificity of the method. The chromatographic method was developed and validated for RNT quantification in perfusate samples and its applicability in daily lab routine is possible. Also, this method can be useful for development of other chromatographic methods in SPIP studies, especially in co-perfusion situation. This study had financial support by FAPESP, CAPES and CNPq (Brazil).

Kyoung-Yong Chun has completed his PhD from Yonsei University and Postdoctoral studies from UC Davis and UIUC Chemical Engineering. He is the Research Professor of Korea University Mechanical Engineering. He has published more than 32 papers in reputed journals including Nature Nanotechnology and Nature Communications.

We demonstrate the thermal responsive artificial ion channel operated within the range of human body heat for the application of drug delivery. Wax-elastic copolymer, coated onto the poly carbonate track etched nanopores membrane by a controlled-vacuum filtration method, is used for building of temperature-gated nanopores. The flexible ion channel membrane can sustain reversible thermo-responsive gating in the high selective temperature range. The nanopore is actuated by the thermal expansion and the contraction of wax composite layers. The phenomenon of ionic current change according to the temperature is simulated based on analytical computation. The pad type ion channel combined with an agarose gel as an electrolyte shows an excellent sensing ability and a quick response, despite its slower mobility of ions compared to a liquid electrolyte cell. The thermal response in the bendable state of wax composite ion channel nearly has a similar behavior with the flat state. For a detection of human body heat, the current flow is effectively distinguished in the patchable ion channel membrane. Such an effective and patchable thermos-gating ion channel is promising in the regulation of bioavailability for drug delivery system.

Gulcin Tok has completed her Master’s degree in Organic Chemistry from Gazi University in 2011. She has been working in pharmaceuticals industry for 5 years as a R&D Scientist. She develops generic drugs and she is responsible for all process to come onto the market from the beginning of product. Additionally, she has published patent about generic formulation and has published the papers in reputed journals.

Colesevelam is a second generation bile acid sequestrant that is used principally for treatment of elevated LDL cholesterol. Additionally, Colesevelam is indicated to improve glycemic control in adults with type 2 diabetes mellitus. Colesevelam is an insoluble, non-absorbed polymer that binds bile acids in the intestine, impeding their reabsorption. Conventional in vivo bioequivalence (BE) study with pharmacokinetic endpoints such as Cmax and AUC is neither appropriate nor feasible for this locally acting drug. The present abstract focuses on the study of in vitro BE in tablet formulation containing 625 mg of Colesevelam HCl. Bile acid sodium salts of glycocholic acid (GCA), glycochenodeoxycholic acid (GCDA) and taurodeoxycholic acid (TDCA) were used in vitro BE studies. The binding capacity HPLC method was developed and validated for these bile acid salts. The equilibrium binding study that is the pivotal BE study and in-vitro kinetic binding study that is the support the pivotal equilibrium binding study were repeated 12 times. In the in vitro equilibrium binding studies, the Langmuir binding constants k1 (affinity) and k2 (capacity) were calculated for the three salts, individually and combined (GC+GCDC+TDC) using linear Langmuir equation for the test and reference products. The calculated capacity (k2), the more important parameter, were obtained very similar between test and reference products in the 90% confidence interval and acceptance criteria of 80% to 120%. The test/reference ratio for k2 was obtained within 0.82-1.07 from the equilibrium binding study for without acid pre-treatment and 0.96-1.08 from the equilibrium binding study for with acid pre-treatment.

Muneesh Garg has completed his MD Physician from DSMI, Russia and MD Pharmacology from BFUHS Faridkot, Punjab, India. He is the Principal Investigator of Sitec Labs Pvt. Ltd., Navi Mumbai, India, a premier contract research organization.

Nicotine Lozenges are used to aid smokers wishing to quit smoking or reduce prior to quitting. The aim of this study was to determine the bioequivalence of a test and reference formulation of Nicotine 2 mg Lozenge. This single dose, randomized, 2-period, 2 sequence, laboratory-blinded, crossover design study was conducted in 21 healthy adult Indian human male smoker subjects under fasting conditions with a washout period of 7 days. Study formulations were administered after a 10-hour overnight fast. Blood samples for pharmacokinetic profiling were taken post-dose up to 16 hours. Safety was evaluated through the assessment of adverse events, and laboratory tests. Plasma concentration of Nicotine was determined with a validated LC-MS/MS method. Bioequivalence between the products was determined by calculating 90% confidence intervals (90% CI) for the ratio of Cmax and AUC0-t values for the test and reference products, using logarithmic transformed data. The 90% confidence intervals of Cmax and AUC0-t for Nicotine were 96.16-119.10 and 92.16-111.51 respectively. Since the 90% confidence intervals for Cmax and AUC0-t were within the 80-125% interval, it was concluded that the two formulations of Nicotine 2 mg Lozenge are bioequivalent in their rate and extent of absorption.

Jaswanth S Bhandaru has completed his Master’s degree in Pharmacy from Panjab University and has a 1.5 yr of industrial experience. He is currently doing his Doctoral research work on multi- component systems and supramolecular chemistry, particularly pharmaceutical cocrystals. He developed cocrystals of few antihypertensive drugs and proved their improved physicochemical properties. He is very enthusiastic in developing systems for improving the oral bioavailability of the molecules and studies their industrial applicability. He also has good publications to his credit.

Co-crystallization is a useful technique for the enhancement of the physicochemical properties of the molecules. The poor physiochemical properties of the API’s tend to show low oral bioavailability which leads to enhanced dosage and consequent adverse profiles of the drugs to the patients. Eprosartan mesylate (EM), an angiotensin II antagonist, is used in the treatment of hypertension exhibits poor bioavailability (13%) due to low absorption window. Hence we recently reported pharmaceutical co-crystals of EM (EM-SUC, EM-SAL & EM- PABA) with improved solubility and dissolution profiles. The present abstract focuses on the study of enhanced oral bioavailability of the EM cocrystals. Pharmacokinetic studies of EM cocrystals were performed using animal models (male Wister rats) to evaluate bioavailability of the drug. Four groups (n=6) of rodents were orally administered with pure EM (API suspended in water) and EM cocrystals (12.3 mg/ kg body weight in aq. solution) respectively. Serial blood samples were collected at predetermined time points and were analyzed for EM plasma concentration using a validated HPLC assay method. Pharmacokinetic and statistical data analysis was performed using Kinetica 5.0 and GraphPad Prism software. The results suggested that the EM cocrystals showed enhanced AUC and Cmax than the pure drug. Mean retention time of the cocrystals was observed to be high with low clearance values which suggest the enhanced absorption window of the prepared cocrystals. EM-SUC showed more than 3 times (3.4 fold) enhancement in the relative oral bioavailability than the pure EM suggesting that the succinic acid as preferred coformer for the preparation of the EM cocrystals.

Introduction: Asthma and other pulmonary diseases are currently treated in millions of people over the world with orally inhaled drugs (OIDs). The traditional pharmacokinetic approaches (AUC and Cmax determination) are currently not accepted by the FDA for evaluating the Inhalations drugs equivalence. The goal of the FDA solicitation (Solicitation Number: 10-223-SOL-00277, part 4) is that pharmacokinetic studies provide an indications and information's about deposition of inhaled drugs at different regional of the lung. for slowly dissolving drugs (e.g fluticasone propionate) a smaller AUC and Cmax will be observed, if the drug is deposited more centrally, as the mucociliary clearance present in the central parts of the lung will remove a larger portion of the more centrally deposited dose, thus leading to a reduction in absorbed dose, AUC and Cmax. Moreover, fast dissolving drugs or drugs in solution differences in Cmax will indicate differences in the c/p ratio. Methods: The in-vitro performance of delivering Tiotropium Bromide from Tiova® were compared with results obtained from Spiriva by using Next Generation Impactor (NGI) operated at different flow rate (30,40,50 and 60 L/min). The method described in the US pharamacopoeia. Results: The Emitted dose of Spiriva at flow rate of 30, 40, 50 and 60 L/min as percentage nominal dose (SD) were 100.4 (0.2), 100.4 (0.4), 100.2 (0.2) and 100.2 (0.2) respectively. In addition, the fine particle fraction of Spiriva at same flow rate (SD) were 50.3 (2.2), 55.6 (0.7), 60.4 (1.5) and 63.3 (6.7) respectively. The mass median aerodynamic diameter of Spiriva at flow rate of 30, 40, 50 and 60 L/min were 3.3 (0.4), 2.9 (0.1), 2.4 (0.1) and 2.4 (0.1) respectively. In contrast, the Emitted dose of Tiova at flow rate of 30, 40, 50 and 60 L/min as percentage nominal dose (SD) were 100.4 (0.4), 100.2 (0.1), 100.1 (0.1) and 100.1 (0.1) respectively. The fine particle fraction of Tiova at flow rate of 30, 40, 50 and 60 L/min (SD) were 44.9 (0.7), 49.2 (1.2), 52.2 (0.7) and 49.1 (2.7) respectively. In addition, the mass median aerodynamic diameter of Spiriva at the same flow rate (SD) were 3.4 (0.1), 3.3 (0.1), 3.2 (0.1) and 3.5 (0.2) respectively.

: Asthma and other pulmonary diseases are currently treated in millions of people over the world with orally inhaled drugs (OIDs). From a regulatory prospective, assessing the bioequivalence of OIDs differs from other orally delivered drugs. The traditional pharmacokinetic approaches (AUC and Cmax determination) are currently not accepted by the FDA for evaluating the Inhalations drugs equivalence. Nowadays, In-vitro Bioequivalence studies are the commonly accepted methods applying a weight of evidence approach that compares between two products. The goal of the FDA solicitation (Solicitation Number: 10-223-SOL-00277, part 4) is that pharmacokinetic studies provide an indications and information's about deposition of inhaled drugs at different regional of the lung. for slowly dissolving drugs (e.g fluticasone propionate) a smaller AUC and Cmax will be observed, if the drug is deposited more centrally, as the mucociliary clearance present in the central parts of the lung will remove a larger portion of the more centrally deposited dose, thus leading to a reduction in absorbed dose, AUC and Cmax. Moreover, fast dissolving drugs or drugs in solution differences in Cmax will indicate differences in the c/p ratio. As the testing of both hypothesis (fast and slowly dissolving drugs) would exceed the volume of these preliminary studies (Goyal and Hochhaus 2010). Methods: The In-vitro performance of delivering Tiotropium Bromide from Tiova® were compared with results obtained from Spiriva by using Next Generation Impactor (NGI) operated at different flow rate (30,40,50 and 60 L/min). The method described in the US pharamacopoeia. Results: The Emitted dose of Spiriva at flow rate of 30, 40, 50 and 60 L/min as percentage nominal dose (SD) were 100.4 (0.2), 100.4 (0.4), 100.2 (0.2) and 100.2 (0.2) respectively. In addition, the fine particle fraction of Spiriva at same flow rate (SD) were 50.3 (2.2), 55.6 (0.7), 60.4 (1.5) and 63.3 (6.7) respectively. The mass median aerodynamic diameter of Spiriva at flow rate of 30, 40, 50 and 60 L/min were 3.3 (0.4), 2.9 (0.1), 2.4 (0.1) and 2.4 (0.1) respectively. In contrast, the Emitted dose of Tiova at flow rate of 30, 40, 50 and 60 L/min as percentage nominal dose (SD) were 100.4 (0.4), 100.2 (0.1), 100.1 (0.1) and 100.1 (0.1) respectively. The fine particle fraction of Tiova at flow rate of 30, 40, 50 and 60 L/min (SD) were 44.9 (0.7), 49.2 (1.2), 52.2 (0.7) and 49.1 (2.7) respectively. In addition, the mass median aerodynamic diameter of Spiriva at the same flow rate (SD) were 3.4 (0.1), 3.3 (0.1), 3.2 (0.1) and 3.5 (0.2) respectively Future work: In-vivo study should be carried out by using a 12 healthy volunteer. The applicants hypothesized recently that pharmacokinetics are able to answer the main questions concerning the local bioequivalence of inhalation drugs, namely whether generic and innovator product are equivalent (1) in the dose available to the lung (AUC), (2) the pulmonary residence time (Cmax) and the central to peripheral (c/p) deposition ratio. It is recognized that differences in the dose available to the lung will be reflected in the area under the concentration time profile for drugs with negligible oral bioavailability or under condition where the oral absorption pathway is blocked (e.g. with charcoal). It is also generally accepted that the pulmonary residence time, a factor important for pulmonary targeting (Hochhaus, Möllmann et al. 1997) can be assessed by pharmacokinetic methods, as long as the oral absorption is negligible or blocked.