Engineering: Pharmacokinetics and Pharmaceutical Technology Area

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In vitro dissolution as a tool for formulation selection: Telmisartan two-step IVIVC. Alejandro Ruiz-Picazo, Mª Teresa Martinez-Martinez, Sarin Colon-Useche, Ramon Iriarte, Barbara SanchezDengra, Marta Gonzalez-Alvarez, Alfredo Garcia-Arieta, Isabel Gonzalez Alvarez, and Marival Bermejo Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00153 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 11, 2018

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Molecular Pharmaceutics

Title: In vitro dissolution as a tool for formulation selection: Telmisartan two-step IVIVC. AUTHORS: Alejandro Ruiz Picazo†, Mª Teresa Martinez-Martinez†, Sarin Colón-Useche†∥, Ramon Iriarte†, Bárbara Sánchez-Dengra†, Marta González-Álvarez†, Alfredo García-Arietaƚ, Isabel González-Álvarez†*, Marival Bermejo† AFFILIATION: †

Engineering: Pharmacokinetics and Pharmaceutical Technology Area. Miguel

Hernandez University, Spain. ∥ ƚ

Analysis and Control Department. University of Los Andes. Venezuela.

Service on Pharmacokinetics and Generic Medicines, Division of Pharmacology and

Clinical Evaluation, Department of Human Use Medicines, Spanish Agency for Medicines and Health Care Products, Madrid, Spain.

CORRESPONDING AUTHOR FOOTNOTE *Address for correspondence: Isabel Gonzalez-Alvarez, [email protected] Edificio Muhammad Al-Shafra, Facultad de Farmacia, UMH, Carretera Alicante Valencia km 87, 03550 San Juan de Alicante, Alicante, Spain Phone +34 965 919217 Fax +34 963544911

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TABLE OF CONTENT GRAPHICS


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ABSTRACT The purpose of this investigation was to develop an exploratory two-step level A IVIVC for three Telmisartan oral immediate release formulations, the reference product Micardis and two generic formulations (X1 and X2). Correlation was validated with a third test formulation Y1. Experimental solubility and permeability data was obtained to confirm that Telmisartan is a class II compound under the Biopharmaceutic Classification System. BE studies plasma profiles were combined using a previously published reference scaling procedure. X2 demonstrated in vivo bioequivalence (BE) while X1 and Y1 failed to show bioequivalence due to the lower boundary of the 90% confidence interval for Cmax being outside the acceptance limits. Average plasma profiles, were deconvoluted by Loo-Riegelman method to obtain the oral fractions absorbed (fa). Fractions dissolved (fdiss) were obtained in several conditions in USP II and USP IV apparatus and, later, the results were compared, in order to find the most bio-predictive model, calculating the f2 similarity factor. The apparatus and conditions showing the same rank order than in vivo data were selected for further refinement of conditions. Levy plot was constructed to estimate the time scaling factor and to make both processes, dissolution and absorption, superimposable. The in vitro dissolution experiment that reflected more accurately the in vivo behaviour of the different formulations of Telmisartan employed the USP IV dissolution apparatus and a dissolution environment with a flow rate of 8 mL/min and a three-step pH change, from 1.2 to 4.5 and 6.8, with a 0.05% of Tween 80. Thus, these conditions gave rise to a biopredictive dissolution test. This new model is able to predict the formulation differences in dissolution that were previously observed in vivo which could be used as a riskanalysis tool for formulation selection in future bioequivalence trials. KEYWORDS Telmisartan, IVIVC, Bioequivalence, BCS, in vivo predictive dissolution, Telmisartan

ABBREVIATIONS Bioequivalent (BE), Non Bioequivalent (N-BE), Biopharmaceutics Classification System (BCS), high (H), low (L).



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INTRODUCTION In vivo predictive dissolution methods (iPD) are useful tools for product development since they have the ability to predict with accuracy and precision the bioequivalence of new formulations of the innovator or generic products after patent expiration. In vitro-in vivo correlations (IVIVC) are developed to confirm that in vitro dissolution methods are predictive within the design space under investigation. IVIVC corresponds with a mathematical model that allows, after the validation of its predictability by comparison with human in vivo results, to pass from an in vitro characteristic of a drug product to its in vivo biological behaviour, such as from the in vitro dissolution profiles to in vivo absorption ones.

Class II drugs are characterized by low solubility and high permeability, and in this case, dissolution is the limiting process in the absorption rate. For low solubility drugs, bioequivalence should in principle be demonstrated by means of human pharmacokinetics studies unless a class A IVIVC is developed and validated. Level A correlation is a point to point relationship between the in vitro dissolution and the in vivo absorption rate of a drug from the dosage form.1 The main objective of this investigation was to develop an exploratory two-step level A IVIVC for three Telmisartan oral immediate release formulations, the reference product Micardis and two generic formulations (X1 and X2). X2 demonstrated in vivo bioequivalence (BE) while X1 failed to show bioequivalence due to the lower boundary of the 90% confidence interval for Cmax being outside the acceptance limits. Telmisartan belong to class II Biopharmaceutic Classification System. Is a low solubility high permeability compound (log P of 7.232) with pH-dependent solubility.3 Telmisartan is ionizable substance with pKa values of 3.5, 4.1, and 6.0. Its solubility is extremely low between pH 3 and 9 (intrinsic solubility 0.00469 mcg/ml)4. In the present work Telmisartan BCS classification was experimentally confirmed through solubility-pH profile characterization and permeability estimation in vitro. The work included the in vivo BE assay provided by a pharmaceutical company, the development of the in vitro dissolution method, the analysis of the potential reasons of the BE failure on the bases of drug and formulation characteristics and the development of the in vitro-in vivo correlation. 4 ACS Paragon Plus Environment

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EXPERIMENTAL SECTION 1. Drug and formulations Telmisartan (MW=514.64 g/mol; logP= 7.232) was given by a pharmaceutical company. Test formulations and reference product were kindly provided by two pharmaceutical companies. All of them consist of immediate release Telmisartan formulations with conventional excipients in customary amounts. Metoprolol and HPLC liquids were purchased from Sigma (Barcelona, Spain).

2. In vivo studies Study 1 was a single-blind, controlled, balanced, randomized, two-period crossover BE study using 71 healthy subjects. Study 2 was an open label, balanced, randomised, two-treatment, three-period, three sequence, single dose, reference-replicated, crossover, BE study in 55 healthy subjects. In each study the volunteers received two formulations, one immediate release (IR) dose of the test formulation (X1 or X2, 80 mg) and one dose of the reference formulation (Micardis 80 mg) in the sequence determined by randomization. A washout period of 14 and 10 days, respectively, was set between periods in each study. Blood samples were taken up to 72 hours. Telmisartan concentration in blood samples was determined by a validated HPLC method in both studies. Cmax and AUC were calculated from the average or individual plasma concentration time profiles. AUC were estimated individually by noncompartmental methods from the in vivo observations. On the other hand, public data from the 90% confidence interval as the outcome of a third BE study was available as well as the employed formulation Y1 that was also kindly provided by the manufacturing pharmaceutical company. The results obtained in all the cross-over BE studies are reported in Table 1. The non-bioequivalent formulations X1 and Y1 (NBE) were not bioequivalent formulation in Cmax as their 90% confidence interval was not inside the acceptance limits (0.81.25). In addition, the 90% confidence intervals of X1 and Y1 Cmax did not include the 100% value, showing that there was a statistical significant difference with the reference products at the significance level employed. Table 1 should be here



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3. Experimental techniques Solubility assays: Saturation Shake-Flask Procedure The solubility of Telmisartan was estimated by adding an excess of solid in a standard buffer solution at 37ºC (pH 1.2, 4.5 and 6.8) and taking samples until saturated conditions were reached, when concentration remained unchanged. Flasks were shaken for 4, 8, 24 and 48 h. The determination of sample concentration was done by fluorescence detection using a validated HPLC method.

Permeability assays: Cell culture and transport studies Dubelcco’s Modified Eagle’s Media supplemented with L-glutamine, fetal bovine serum (10%) and penicillin/streptomycin (5%) was the media adequated to grow Cace-2 cells. They were maintained in an incubator at 37ºC, 90% relative humidity and 5% CO2 during the growth of cells in flasks and they were seeded on polycarbonate membranes 250,000 cells/cm2 and maintained during 20 days until the experiment to ensure transporters were expresed. Afterwards, the trans-epithelial electrical resistance (TEER) was measured to evaluate the integrity of each cell monolayer. Standard operating procedures (SOPs) were described and validated previously in our laboratory.5-9 To fill the receiver chamber and to prepare the drug solution (API or formulation) that was placed in the donor chamber, Hank’s balanced salt solution (HBSS) supplemented with HEPES was used. The transport studies in presence of formulation excipients were carried out crushing and dissolving a tablet of each formulation in 250 mL of transport buffer and drug. An orbital environmental shaker at constant temperature (37ºC) and at agitation rate of 50 rpm was used to conduct the transport studies, which were performed in apical-to-basal (A-to-B) direction and pH=7 in both chambers. At 15, 30, 45 and 90 minutes, samples were taken from the receiver side and replaced each time with fresh buffer. Permeability assays have been carried out in Metoprolol (100µM), Telmisartan (API) and four pharmaceuticals formulations (micardis, X1, X2 and Y1).

The apparent permeability coefficient was calculated according the following equation:


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where Creceiver,t is the drug concentration in the receiver chamber at time t, Qtotal is the total amount of drug, Vreceiver and Vdonor are the volumes in receiver and donor chamber, Creceiver,t−1 is the drug concentration in receiver chamber at previous time, f is the sample replacement dilution factor, Peff is the permeability coefficient, S is the surface area and ∆t is the time interval as were described by Mangas-Sanjuan et al7.

Disintegration The tablet disintegration rate was measured with a tablet disintegration tester (PharmaTest) in water at 37°C and the experiment was done by six times (Ph. Eur. Method 2.9.1). Mean of values and standard deviation were reported.

Dissolution assays Dissolution tests. Media composition Media used for dissolutions assays were the standard buffers described in European Pharmacopeia10-12. The same media at pH 4,5 and 6,8 but containing Tween 80 at 0.05% and fasted state simulating media, Fassif, were also tested 10-12.

An apparatus 2 (Paddle method) (Pharma-Test PT-DT70) with 900mL of different media (Table 2) at 37±0.5ºC and 50 rpm was used to perform the dissolution assays. Samples (5mL) at different times were taken and filtered in line through a 10 µM (Pharma test) filter. The same volume of buffer was replaced in order to keep the test volume constant throughout the entire test. These experiments were performed in 6 replicates. Telmisartan dissolution profiles were assayed also using USP IV apparatus. These dissolution profiles were obtained with an automated flow-through cell system, USP IV apparatus (Erweka, Germany) with 22.6 mm cells (i.d.) and a piston pump (Erweka, Germany).

The experiments using USP IV apparatus (without recycling) were performed at 37.0 ±0.5°C, and with a flow rate of 8 ml/min. Sequential sampling using 0.45 µm nitrocellulose membranes (Millipore) were taken at different intervals of time using 6 7 ACS Paragon Plus Environment


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replicates. Table 2 and 3 show, respectively, the experimental conditions using Apparatus 2 (paddle model) and Apparatus 4 (automated flow-through cell system). The amount of dissolved Telmisartan was determined by HPLC.

Table 2 should be here Table 3 should be here To compare the dissolution profiles between test and reference formulations in each condition F2 similarity factor was used. F2 values greater than or equal to 50 indicates that the dissolution profiles are similar. When more than 85% of the drug is dissolved within 15 minutes, dissolution profiles may be accepted as similar without further mathematical evaluation10, 13.

4. Analysis of the samples Samples were analyzed by HPLC with fluorescence detector (excitation wavelength 305 nm and emission wavelength 365 nm) in following conditions: the column was a Nova-Pak C18 column (4 µM, 3.9 x 150 mm), mobile phase was 6.5 mM trifluoroacetic, acid solution and acetonitrile (54:1:45) and flow-rate of 1.0 mL/min. The accuracy of the method was calculated using 5 standards and analyzed by triplicate. Precision was calculated as the coefficient of variation of five determinations over the same standards (values less than 5%). Linearity was established over the range of concentrations present in the samples (r2> 0.999). The limit of quantification for Telmisartan was 3.4 µg/ml. Statistical analysis Two-tailed Student t-tests were used to compare mean values of two groups and analysis of variance (ANOVA) and Scheffé post hoc test were used to compare mean values of more than two groups with a significance level of 0.05. The statistical analyses were made with the statistical package SPSS, V.20.00.

In vitro-in vivo correlation

To develop the IVIVC with three formulations of different release rate the data from both BE studies were combined. Test formulations plasma profiles were normalized based on the reference’s ratios.14 Normalization was carried out using the average concentration time profiles from each reference formulation. At each sampling 8 ACS Paragon Plus Environment

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time, reference ratios were calculated to obtain the normalization factor for correcting the test average concentration profiles of the second study. So finally reference profile, test 1 and test 2 normalized curves were used for the deconvolution analysis. As sampling times differed slightly in both BE studies for the non-matching sampling times the normalization factor was estimated as the average of the previous and next matching sampling time.

Telmisartan intravenous pharmacokinetic parameters were obtained by curve fitting a two compartment open model to the plasma profiles of a 40 mg intravenous 30 min infusion15. Data was captured from Figure 1 of the reference with the aid of GetData Graph Digitizer V.2.26. Curve fitting was performed in Microsoft Excel® (Redmond, WA) with PKsolver add-in macro.16 Distribution microconstants (k12 and k21) and elimination rate constant from central compartment (k13) were estimated from alpha and beta disposition rate constants. In vivo oral fractions absorbed were estimated by Loo-Riegelmann deconvolution method from the average plasma profiles. In vitro fractions dissolved and in vivo fraction absorbed profiles were made superimposable by a time scale correction based on a previously constructed Levy plot. Levy plot consist of a relationship between the time required in vivo and in vitro for the dissolution/absorption of a given fraction.

An extent correction factor was also

necessary to account for the incomplete in vitro release of Telmisartan. In vitro profiles were scaled in extent and the scaled in vitro dissolved fraction were used for construction of levy plot. In order to estimate the time needed for in vitro dissolution of a particular fraction, a first order dissolution model was previously fitted to the experimental values of each formulation.

The internal validation was done by estimating the prediction error percentage (PE%) on Cmax and AUC values with the three formulations used for developing the correlation i. e, Reference, X1 and X2. The external validation was done by calculating the prediction error of Cmax of Y1, the non-bioequivalent formulation not included in the IVIVC development. RESULTS The solubility values of Telmisartan were 0.229 ± 0.009, 1.05·10-3± 3.55·10-5 and 1.56·10-3 ± 1.18·10-4 mg/mL, at pH 1.2, 4.5 and 6.8 respectively, therefore the lowest solubility of Telmisartan was 1.05·10-3± 3.55·10-5 at pH 4.5. The dose numbers 9 ACS Paragon Plus Environment


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(Do=80 mg/250mL/solubility mg/mL) were 1.40, 304.76 and 205.13, respectively at pH 1.2, 4.5 and 6.8. In all the conditions Do is higher than 1 confirming the low solubility classification for Telmisartan.

The permeability of Telmisartan was studied at the clinical concentration (80 mg administered with 250 mL of water: 185,7 µM) and to determine BCS classification the permeability of Telmisartan value was compared with metoprolol permeability value (Figure 1). In this figure the permeability of different formulations of Telmisartan was showed too. Figure 1 should be here

Mean values of Telmisartan and Metoprolol were compared with two-tailed Student t-tests and differences among both compounds were detected (p

Engineering: Pharmacokinetics and Pharmaceutical Technology Area

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