Methacrylate-copolymer Eudragit EPO as solubility-enabling excipient

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Methacrylate-copolymer Eudragit EPO as solubilityenabling excipient for anionic drugs: Investigation of drug solubility, intestinal permeability, and their interplay Noa Fine-Shamir, and Arik Dahan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00057 • Publication Date (Web): 23 May 2019 Downloaded from http://pubs.acs.org on May 26, 2019

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Molecular Pharmaceutics

Methacrylate-copolymer Eudragit EPO as solubility-enabling excipient for anionic drugs: Investigation of drug solubility, intestinal permeability, and their interplay

Noa Fine-Shamir1 and Arik Dahan1,*

1

Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

*

Corresponding Author: Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.Box 653, Beer-Sheva 84105, Israel. Tel: +972-8-6479483. Fax: +972-86479303. E-mail address: [email protected]

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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

ABSTRACT: The purpose of this work was to investigate the use of the dimethylaminoethyl methacrylate-copolymer Eudragit EPO (EPO) in oral solubility- enabling formulations for anionic lipophilic drugs, aiming to guide optional formulation design, and maximize oral bioavailability. We have studied the solubility, the permeability, and their interplay, using the low solubility non-steroidal anti-inflammatory drug mefenamic acid as a model drug. Then, we studied the biorelevant solubility enhancement of mefenamic acid from EPO-based formulations throughout the gastrointestinal tract (GIT), using the pH-dilution dissolution method. EPO allowed profound and linear solubility increase of mefenamic acid, from 10 µg/mL without EPO to 9.41 mg/mL in the presence of 7.5% EPO (∼940-fold; 37 °C); however, concomitant decrease of the drug permeability was obtained, both in-vitro and invivo in rats, indicating a solubility-permeability tradeoff. In the absence of excipient, the unstirred water layer (UWL) adjacent to the GI membrane was found to hinder the permeability of the drug; accounting for this UWL effect and revealing the true membrane permeability allowed good prediction of the solubility-permeability tradeoff as a function of EPO level using direct relationship between the increased solubility afforded by a given EPO level and the consequent decreased permeability. Biorelevant dissolution studies revealed that EPO levels of 0.05% and 0.1% were insufficient to dissolve mefenamic acid dose during the entire dissolution time course, while 0.5% and 1% EPO allowed complete solubility with no drug precipitation. In conclusion, EPO may serve as a potent solubility-enabling excipient for BCS Class II/IV acidic drugs, however it should be used carefully; it is prudent to


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use the minimal EPO amounts just sufficient to dissolve the drug dose throughout the GIT, and not more than that. Excess amounts of EPO provide no solubility gain and cause further permeability loss, jeopardizing the overall success of the formulation. This work may help the formulator to hit the optimal solubilitypermeability balance, maximizing the oral bioavailability afforded by the formulation.

KEYWORDS: Eudragit EPO, low solubility, solubility-enabling formulations, intestinal permeability, oral drug delivery, solubility-permeability interplay. 3 ACS Paragon Plus Environment


1. INTRODUCTION

Drug solubility/dissolution in the gastrointestinal (GI) milieu and the effective permeability through the GI membrane are the two key processes of oral drug absorption 1-4. Low aqueous solubility of new oral drug candidates is a significant challenge in today's drug development, and various approaches may be used to tackle this obstacle 5-7. Yet, overall drug bioavailability may fail to increase even when the formulation increases the apparent solubility significantly 8-10. We have explained this failure by the solubility-permeability tradeoff phenomenon, showing that increased solubility using formulation is often accompanied by apparent permeability decrease, hence the overall absorption is not improved. This tradeoff was shown for formulations containing cyclodextrin 11-13, surfactant 14, cosolvent 15, and hydrotrope 16. On the other hand, other solubility-enabling approaches exhibited different, favorable, types of solubility-permeability interplay; amorphous solid dispersions (ASD) allow significant apparent solubility increase (via supersaturation) while the permeability remains unchanged 17. When the excipient exhibits P-gp inhibitory characteristics (e.g. vitamin E TPGS), and the absorption of the drug is limited by this efflux mechanism, the formulation may concomitantly increase both the solubility and the permeability 18-20. Hence, accounting for both the solubility and the permeability, and defining their interplay, when developing a solubility-enabling formulation for a given oral lipophilic drug, is essential. The dimethylaminoethyl methacrylate-copolymer Eudragit EPO (EPO) has broad pharmaceutical applications 21, including taste masking, moisture-protective coating 22,

immediate- and extended-release drug dispenser 23, 24, colon targeting 25, 26, 4 ACS Paragon Plus Environment

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producing and stabilizing acidic drugs in solid dispersions form 27, 28; however, only recently it has raised interest in terms of solubility and bioavailability enhancement. Recently, Saal et al showed excellent in-vitro impact of EPO as solubility-enabling excipient, for several drugs 29, 30. EPO copolymer belongs to methacrylic acid copolymers family, composed of dimethylaminoethyl methacrylate, butyl methacrylate, and methyl methacrylate (molar ratio 2:1:1). The exact mechanism by which EPO improves drug apparent solubility is not fully discovered yet. Since EPO is protonated at pH lower than eight and positively charged at physiological environment, it was assumed that ionic interactions between the protonated amino alkyl group of EPO and the deprotonated drug are involved in the solubility enhancement of anionic drugs by this excipient 29. Such interactions are not relevant between EPO and basic drugs, however, increased solubility of basic drugs by EPO was also reported 30. Additional hydrophobic molecular interactions between the drug and the polymer were demonstrated 31, likely contributing to the overall molecular polymer-drug interactions and drug solubility enhancement. At any rate, neither micellization (like with surfactants) nor drug entrapment (like with cyclodextrins) occurs with EPO, and hence, how this excipient effects the permeability is unknown; revealing the type of solubility-permeability interplay when using EPO is essential for developing successful EPO-based solubility-enabling formulations. The aim of this work was to study the key factors dictating the success/failure of EPO-based oral formulation, including its effects on drug solubility, intestinal permeability, and the solubility-permeability interplay. The role of the unstirred water layer (UWL) adjacent to the GI membrane was accounted for, as well as 5 ACS Paragon Plus Environment


biorelevant dissolution aspects throughout the GIT. This all-inclusive approach is aimed to guide the development of a successful solubility-enabling EPO-based formulation. The BCS class II nonsteroidal anti-inflammatory drug (NSAID) mefenamic acid was chosen as the model drug 32; its acidic and lipophilic characteristics favor solubility enhancement with EPO, due to both ionic and hydrophobic interactions. Decreased diffusion coefficient of mefenamic acid in the presence of 0.5% EPO was reported, highlighting that this decreased diffusion coefficient indicates a strong molecular association between the polymer and the drug, supporting the increased drug solubility enhancement by EPO 29. The concentration-dependent effects of EPO on the solubility, in-vitro and invivo permeability, and biorelevant dissolution of mefenamic acid were studied, and successful predictive model of the solubility-permeability interplay was developed. Since the formulation development process is often empirical and the use of EPO as a solubility-enabling excipient is relatively new, this work aimed to facilitate the mechanistic development of successful EPO based formulations for oral drug delivery.


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2. MATERIALS AND METHODS

2.1.

Materials. Mefenamic acid, sodium phosphate monobasic, sodium

phosphate dibasic dehydrate, trifluoroacetic acid (TFA), DMSO and HCL 1M were purchased from Sigma Chemical Co (St. Louis, MO USA). NaCl was purchased from Fisher Scientific Inc. (Pittsburgh, PA). SIF powder for preparation of FaSSIF was obtained from Biorelevant.com Ltd. (London, UK). EUDRAGIT® EPO was generously donated by Evonic (Essen, Germany). Acetonitrile and water (Merck KGaA, Darmstadt, Germany) were UPLC grade. All other chemicals were of analytical reagent grade.

2.2.

Solubility Studies. The solubility of mefenamic acid in solutions

containing increasing concentrations of EPO was tested according to a previously described method 33. Briefly, EPO solutions (0, 0.1, 0.5, 1, 2.5, 5 and 7.5% w/w) in 10 mM MES buffer pH 6.5 were prepared and were placed in glass test tubes with excess amounts of mefenamic acid (n=4 for each EPO concentration); pH was measured at the start and end-point of the experiment. The tubes were placed in a 100 rpm shaking water bath at 37 °C or at room temperature (25 °C) for 72 hours. The vials were then centrifuged (14,000 rpm), and immediately assayed for drug content by UPLC.



2.3.

Parallel Artificial Membrane Permeability Assay (PAMPA). In-vitro

drug permeability experiments were carried out in in Millipore (Danvers, MA) 96well MultiScreen-Permeability filter plates with 0.3 cm² polycarbonate filter support (0.45 mm), at room temperature (25 ˚C), as previously reported 34, 35. Fifteen (15) μL of hexadecane solution in hexane (5% v/v) were added to each well, and the plate was left for one hour to ensure complete evaporation of the hexane. Then, the donor wells were filed with 0.2 mL of the different mefenamic acid-EPO solutions and the receiver wells were filed with 0.3 mL of blank matched buffer. In order to maintain equivalent thermodynamic activity in all groups, mefenamic acid concentrations were chosen according to the room temperature solubility studies, to achieve 50% saturation in all experimental groups. The donor plate was then placed upon the 96-well receiver plate, creating the PAMPA sandwich, without rotation. Samples were taken from the receiver plate every half-hour over two hours (30, 60, 90 and 120 min) and drug content was immediately determined by UPLC. Each experiment (i.e. EPO concentration) was repeated 4 times (n=4). In order to reveal the true membrane Papp of mefenamic acid, similar PAMPA experiment was carried out in different plate rotation speeds (0, 3×103, 7×103 and 9×103 rpm) in the absence of EPO. Each experiment was repeated 6 times (n=6). In all PAMPA studies, apparent permeability coefficient (Papp) was calculated from the linear plot of drug accumulated in the receiver side versus time, according to the following equation:

Papp =

dQ/dt A × C0


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Where dQ/dt is the steady-state appearance rate of mefenamic acid on the receiver side, C0 is the initial concentration of mefenamic acid in the donor side, and A is the hexadecane membrane surface area (0.048 cm²). Linear regression was carried out to obtain the steady-state appearance rate of drug on the receiver side.

2.4.

Rat Single-Pass Intestinal Perfusions (SPIP). The EPO concentration-

dependent effects on the rat intestinal permeability of mefenamic acid were studied using the SPIP method. All in-vivo experiments were approved by Ben-Gurion University of the Negev Animal Use and Care Committee (Protocol IL-08-01-2015), and the animals were housed and handled according to Ben-Gurion University of the Negev Unit for Laboratory Animal Medicine Guidelines. Wistar Rats (Harlan, Israel) were fasted overnight (12 h) before each experiment, with free access to water. SPIP studies were performed according to previously reported protocol 36. Briefly, after inducing general anesthesia by intra-muscular injection (1 mL/kg ketamine:xylazine), 3 cm midline abdominal incision was made in order to expose the small intestine. Ten (10) cm long proximal jejunal segment (starting 2 cm below the ligament of Treitz) was cannulated on two ends, rinsed with physiological saline solution, and perfused with the different mefenamic acid MES buffer (pH 6.5) solutions (consisting EPO levels of 0, 0.0005, 0.005, 0.1, 0.5 and 1% w/w) at a perfusion flow rate of 0.2 mL/min (Watson-Marlow 205S, Wilmington, MA). Samples were collected at 10 min intervals for 2 hours, centrifuged, and immediately assayed for mefenamic acid content by UPLC. Similar studies with higher flow rates (2 and 4 mL/min) were also performed to evaluate the unstirred water layer (UWL) effect on 9 ACS Paragon Plus Environment


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mefenamic acid permeability 37. The effective permeability (Peff) through the rat jejunum was determined by the following equation:

Peff =

C′out ―Qln( ) C′in 2πRL

where Q is the perfusion flow rate, C'out/C'in is the ratio of mefenamic acid outlet vs. inlet concentrations (C'in is the initial mefenamic acid concentration in the perfused solution, and C'out is the drug concentration in the outlet solution after the intestinal perfusion, determined by UPLC analysis). R is the jejunal radius (0.2 cm), and L is the length of the perfused jejunal segment as was accurately measured at the end of the experiment.

2.5.

Biorelevant pH-Dilution Dissolution Studies. A set of biorelevant in-

vitro pH-dilution dissolution experiments were carried out, aiming to simulate drug dissolution from a given formulation, while traveling along the rat GIT; this method was shown before to exhibit very good correlation to in-vivo data

15, 38, 39.

Mefenamic acid dose (60 µg) solubilized in 600 µL of different EPO formulations were first diluted into 1×10-4 M HCl (pH 4.0) at a dilution factor of 1:0.66 and agitated for 15 min (100 rpm at 37 °C), to mimic the rat stomach compartment. Then, samples were further diluted with FaSSIF (Biorelevant.com Ltd., London, UK) at a dilution factor of 1:0.5 and continued to be agitated for additional 15 min. Further FaSSIF dilutions were carried out with a dilution factor of 1:1 (agitation time of 30 min), and then another dilution of 1:1 with agitation time of 60 min, for a total 10 ACS Paragon Plus Environment

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experiment time of 2 h. Throughout the experiment, samples are taken at set time points, centrifuged, filtrated and immediately assayed for drug content by UPLC. Comparison between the solubilized drug content (obtained from the UPLC results) and the total drug content (calculated from the initial dose and the subsequent dilutions) allows to evaluate the solubility increase allowed by each formulation during the experiment.

2.6.

Ultra-Performance Liquid Chromatography (UPLC). UPLC analyses of

all samples were performed on a Waters (Milford, MA) Acquity UPLC H-Class system equipped with a photodiode array detector and controlled by Empower software. A Waters Bridge C8 3.5 μm 4.6 × 150 mm column was used. A gradient mobile phase, consisted of 15:85 going to 85:15 (v/v) water:acetonitrile (both containing 0.1% TFA) over 8 min was run at a flow rate of 0.5 mL/min. Injection volumes ranged 5-20 µL, and the detection wavelength was 352 nm.

2.7.

Statistical Analysis. All in-vitro experiments were n=4-6, while each

group of the animal studies consisted of 3-4 rats. Values are expressed as mean ± standard deviation (SD). To determine statistically significant differences among the experimental groups, the nonparametric Kruskal-Wallis test was used for multiple comparisons, and the two-tailed nonparametric Mann-Whitney U-test for two-group comparison, where appropriate. p

Methacrylate-copolymer Eudragit EPO as solubility-enabling excipient

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