1 Salicylic Acid (SA) Bioaccessibility from SA-Based Poly(anhydride-2 ester)3 Michael A. Rogers,*,†,‡ Yim-Fan Yan,‡ Karen Ben-Elazar,‡ Yaqi Lan,‡ Jonathan Faig,§ Kervin Smith,§

4 and Kathryn E. Uhrich§

5‡Department of Food Science and †New Jersey Institute of Food, Nutrition and Health, Rutgers University, The State University of

6 New Jersey, New Brunswick, New Jersey 08901, United States

7§Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States

8 ABSTRACT: The bioaccessibility of salicylic acid (SA) can be effectively modified by incorporating the pharmacological9 compound directly into polymers such as poly(anhydride-esters). After simulated digestion conditions, the bioaccessibility of SA10 was observed to be statistically different (p < 0.0001) in each sample: 55.5 ± 2.0% for free SA, 31.2 ± 2.4% the SA-diglycolic acid11 polymer precursor (SADG), and 21.2 ± 3.1% for SADG-P (polymer). The release rates followed a zero-order release rate that12 was dependent on several factors, including (1) solubilization rate, (2) macroscopic erosion of the powdered polymer, (3)13 hydrolytic cleavage of the anhydride bonds, and (4) subsequent hydrolysis of the polymer precursor (SADG) to SA and14 diglycolic acid.

15 ■ INTRODUCTION

16 Salicylic acid (SA), an active metabolite of aspirin (acetylsali-17 cylic acid; ASA), is useful due to its anti-inflammatory,18 antipyretic, keratolyic and analgesic properties.1,2 While SA19 has been used since the fifth century to relieve pain, recent20 advances describe a new delivery system that directly21 incorporates SA into a poly(anhydride-ester) (PAE) to22 overcome issues associated with ASA.3−6 The polymeric23 version of SA offers many advantages over the small molecule24 of ASA; the first is the ability to formulate into various25 geometries, including powders,7 disks,8 fibers,9 and micro-26 spheres.10 Second, PAEs allow high SA loadings, typically27 between 60 and 80%, because of the direct incorporation of SA28 into the polymeric backbone.3 Third, PAEs enable sustained29 release of SA; as small molecules, SA rapidly diffuses, whereas30 the polymeric version delivers a sustained, controlled release of31 SA over time.5,11 Thus, PAEs have great potential in various32 biomedical applications, as they have been found to be nontoxic33 in both in vitro12 and in vivo studies.8

34 In designing SA-based PAEs, both the drug release rate and35 drug loading capacity can be modified by altering the chemical36 composition of the linker molecule, enabling a tunable drug37 release profile for diverse applications.5,11,13 Upon exposure to38 water, PAEs undergo hydrolytic degradation; the SA release39 rate is dependent upon the solution conditions (i.e., pH,40 temperature, etc.) and polymer composition.5 PAE’s typically41 exhibit a sustained, near zero-order rate of drug release, owing42 to their rate-limiting step being governed by its surface-eroding

43behavior and low solubility.9,14−16 Furthermore, PAEs do not44display the burst release typically observed in conventional45delivery systems, which has been associated with toxicity46concerns. While the PAEs do not demonstrate burst release47behavior, a disadvantage of the PAEs could be the observed lag48time.3,5,14 With some PAEs, drug release could be delayed by49days, a behavior that may not be desirable if immediate pain50relief, for example, is required. The lag time can be overcome51by several approaches, such as admixing small molecules,17

52increasing the hydrophilicity of the linker molecule,7,11,13

53preparing copolymers7,9 and altering the pH of the degradation54environment.3 Overall, PAEs offer an effective means of55delivering drug moieties such as SA for applications requiring56both short- and long-term drug release.18

57As numerous variables influence the polymer degradation58rate, including temperature, pH, water content, and mixing, it is59important to understand how these polymeric systems behave60in the alimentary track to ensure pharmacopeial efficacy. The61influence of biological and formulation variables makes it62essential to characterize the “release” profile from the delivery63vehicle into the luminal fluids, which is termed bioaccessibility,64defined here as the cumulative percent of SA released in the65 f1jejunum and ileum (Figure 1, TIM-1 sections 5c and 5d,66respectively). It is not necessary to probe the bioavailability

Received: June 25, 2014Revised: July 31, 2014

Article

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67 because absorption and circulation will be only affected by the68 rate of SA release, as it is the free SA that is absorbed. The69 purpose of this study is to observe how the chemical structures70 of SA precursors and polymers influence SA release (from71 SADG and SADG-P, respectively) as compared to the smaller72 molecules (SA) in a dynamic simulated TNO-intestinal model73 (TIM-1) and to determine if SA release is targeted to different74 intestinal segments.

75 ■ MATERIALS AND METHODS76 Materials. All chemicals, solvents, and reagents were used as77 received, unless otherwise indicated, and purchased from Sigma-78 Aldrich (Milwaukee, WI). SA (Sigma-Aldrich) was used as a control79 and the polymer precursor (SADG) and polymer (SADG-P) were80 synthesized according to a previously published protocol.4,5,7,13,19

81 Molecular Weight Analysis. Gel permeation chromatography82 (GPC) was used to determine the molecular weight (Mw) and83 polydispersity index (PDI) of the polymer samples. A Waters system84 consisting of a 1515 isocratic high pressure liquid chromatography85 (HPLC) pump, a 717plus autosampler, and a 2414 refractive index86 (RI) detector was used. Waters Breeze 2 software running on an IBM87 ThinkCentre CPU was used for data collection and analysis. Samples88 were dissolved in dichloromethane (DCM; 10 mg/mL) and filtered89 through 0.45 μm polytetrafluoroethylene syringe filters (VWR,90 Bridgeport, NJ). A 10 μL aliquot was injected and resolved on a91 Jordi divinylbenzene mixed-bed GPC column (7.8 × 300 mm, Alltech92 Associates, Deerfield, IL) at 25 °C, with DCM as the mobile phase at a93 flow rate of 1.0 mL/min. Molecular weights were calibrated relative to94 broad polystyrene standards (Polymer Source Inc., Dorval, Canada).95 Thermal Analysis. Thermal analysis was accomplished using96 differential scanning calorimetry (DSC) to acquire the glass transition97 (Tg) temperature. DSC was performed utilizing a Thermal Advantage98 (TA, New Castle, DE) DSC Q200 running on an IBM ThinkCentre99 computer equipped with TA Universal Analysis software for data100 acquisition and processing. Samples (4−6 mg) were heated under101 nitrogen from −10 to 200 °C at a rate of 10 °C/min. A minimum of102 two heating/cooling cycles was used for each sample set. TA103 Instruments Universal Analysis 2000 software, version 4.5A, was104 used to analyze the data.

105Thermogravimetric analysis (TGA) was used to acquire the106decomposition temperature (Td) of polymer samples. TGA analysis107was performed using a PerkinElmer TGA7 analyzer with TAC7/DX108controller equipped with a Dell OptiPlex Gx 110 computer running109PerkinElmer Pyris software (PerkinElmer, Waltham, MA). Polymer110samples (10 mg) were heated under nitrogen at a rate of 10 °C/min111from 25 to 400 °C. Td was defined as the onset of decomposition,112represented by the beginning of a sharp slope on the thermogram.113Simulated Digestion. Pancreatin was obtained from Sigma-114Aldrich. Fresh pig bile was obtained from Farm-to-Pharm (Warren,115NJ, U.S.A.). The bile was collected, standardized from a slaughter-116house, and pooled together before an aliquot for individual TIM117experiments was taken and stored at −20 °C until use. Rhizopus lipase118(150000 units/mg F-AP-15) was obtained from Amano Enzyme Inc.119(Nagoya, Japan). Trypsin from bovine pancreas (7500 N-α-benzoyl-L-120arginine ethyl ester (BAEE) units/mg, T9201) was obtained from121Sigma-Aldrich.122TNO Intestinal Model (TIM-1). A dynamic, in vitro gastro-123intestinal (GI) model, TIM-1, developed by TNO (Zeist, The124Netherlands), was utilized to simulate digestion. The TIM-1 system125models the human digestive tract utilizing four compartments126mimicking the stomach, duodenum, jejunum and ileum, peristaltic127movements, nutrient/drug and water absorption, gastric emptying, and128transit time, as would be observed in vivo (Figure 1). Compartments129are infused with formulated gastric secretions, bile, and pancreatic130secretions to modify pH and reproduce digestive conditions, respective131of a fed or fasted state. In the fasted state, the pH of the gastric132compartment is 2.2 upon administration of the pharmacological agent133and decreases to 1.5 over 90 min, and the gastric emptying rate has a134half-life of 20 min.135A 1.4% pancreatin solution (Pepsin from porcine gastric mucosa,136lyophilized powder, >2500 units/mg protein, Sigma-Aldrich) and137small intestinal electrolyte solution (SIES: NaCl 5 g/L, KCl 0.6 g/L,138CaCl2 0.25 g/L) were prepared. Duodenal start residue (60 g; 15 g139SIES, 30 g diluted fresh porcine bile (20% bile), 2 mg trypsin solution,14015 g pancreatin solution), jejunal start residue (160 g: 40 g SIES, 80 g141fresh porcine bile, 40 g pancreatin solution), and ileal start residue142(160 g SIES) were injected into respective compartments prior to143heating the system to physiological temperature (37 °C) in144preparation for feeding.

Figure 1. Schematic diagram of the in vitro gastrointestinal model, TIM-1: (1) food inlet, (2) jejunum filtrate, (3) ileum filtrate, (4) ileal colorectalvalve, (5a) gastric compartment, (5b) duodenal compartment, (5c) jejunum compartment, (5d) ileal compartment, (6a) hollow fiber membranefrom jejunum, (6b) hollow fiber membrane from ileum, (7a) and (7b) secretion pumps. Reprinted with permission from ref 20. Copyright 2013American Chemical Society.

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145 Different formulations were tested during 5 h experiments in the146 TIM-1 model, simulating fasted-state physiological conditions147 following ingestion of SA, SADG, or SADG-P. The test sample was148 placed in a mesh teabag located in between the two glass149 compartments of the gastric unit, so that the formulation was exposed150 to physiologically relevant waves of peristalsis mixing, but not to direct151 pressure forces. To simulate the initial amount of gastric juice, 5 g152 gastric enzyme (NaCl 4.8 g/L, KCl 2.2 g/L, CaCl2 0.22 g/L, and) was153 added to the gastric compartment. Formulations were standardized so154 a total of 249.3 mg of SA could be hydrolytically generated and each155 formulation was prepared as a fine powder, loaded into a tea bag and156 suspended in the gastric compartment. Then, 45 g gastric electrolyte157 solution, 100 g of water, and 2.5 mg amylase were immediately added158 into the gastric compartment, followed by a 50 g of water rinse.159 Secretion of HCl (1 M) into the gastric compartment during digestion160 was controlled to follow a preprogrammed computer protocol, which161 regulates gastric emptying (half-life = 20 min), intestinal transit times,162 pH (pH 2.2 to 1.5), and secretion fluid amounts.21

163 Secretion of digestive fluids were setup based on the following:164 duodenal secretion consisting of fresh porcine bile at a flow rate of 0.5165 mL/min, a 1.4% pancreatin solution at 0.25 mL/min, and SIES at 3.2166 mL/min. Jejunal secretion consisting of SIES and fresh porcine bile167 were introduced at a flow rate of 3.2 mL/min. Ileal secretion consisting168 of SIES was pumped at a flow rate of 3.0 mL/min. Secretion of HCl (1169 M) into the gastric compartment during digestion was monitored via a170 preprogrammed computer protocol that regulates gastric emptying,171 intestinal transit times, pH values, and secretion fluid amounts. The172 pH of duodenal, jejunal, and ileal compartments was maintained at 6.5,173 6.8, and 7.2, respectively, by controlled secretion of sodium174 bicarbonate solution (1 M).175 The available SA fraction was observed by collection and analysis of176 dialysate fluids, which were passed through semipermeable capillary177 membranes (Spectrum Milikros modules M80S-300−01P, Irving, TX)178 with 0.05 μm pores at the ileal and jejunal compartments. Jejunal and179 ileal filtrates as well as ileal efflux were cooled on ice to reduce residual180 enzyme activity once the samples passed through the capillary181 membranes. Samples were gathered at 30, 60, 90, 120, 180, 240,182 and 300 min and immediately analyzed on an HPLC (see parameters183 below). This process allows the individual compartments of the upper184 GI to have their isolated effects on hydrolytic release of SA. Residues185 were not collected for analysis following experiment termination at186 300 min. SA bioaccessibility was evaluated for each formulation in187 triplicate and each run was analyzed in duplicate, providing three188 sample triplicates and two technical duplicates for each variable.

189HPLC-Evaporative Light Scattering Detector (ELSD) Anal-190ysis. Separations were carried out by using Waters e2695 Alliance191HPLC system (Waters, Milford, MA, U.S.A.) equipped with a free192fatty acid HP column (4 μm, 3.9 × 150 mm; Waters, Milford, MA,193U.S.A.) set at 30 °C. The injection volume was 50 μL, the flow rate194was 0.5 mg/min, and run time was 6 min. The isocratic eluting system195consisted of 50% water and 50% tetrahydrofuran (THF). The effluent196was monitored with Waters 2424 Evaporative Light Scattering197Detector (ELSD; Waters, Milford, MA, U.S.A.) with the following198settings: drift tube temperature for ELSD set at 65 °C and nebulizer199for nitrogen gas adjusted to 40 psi. Comparing the retention time with200the reference compound identified the chromatographic peaks. The201corresponding retention times are 2.4 for SA and 1.8 min for SADG. A202total of 10 concentrations of SA (≥99.0%; Sigma-Aldrich, St. Louis,203MO, U.S.A.) solutions were injected in triplicate to generate the204calibration curve constructed by plotting the peak areas versus the205concentration of the SA. Quantification was carried out from the206integrated peak area and corresponding calibration curve.

207■ RESULTS AND DISCUSSION

208SADG and SADG-P were successfully synthesized according to209published procedures.4,5,7,13,19 Following SADG-P isolation, Mw210and PDI were determined to be 16.0 kDa and 1.4, respectively.211Furthermore, thermal properties were assessed to ensure the212polymer morphology would not be altered during the course of213the study conducted at 37 °C. SADG-P was found to possess214favorable thermal properties with a Tg of 72 °C and Td of 330215°C. In vitro evaluation of SA, SADG, and SADG-P was216performed using the TIM-1 model under fasted conditions.217The TIM-1 system was chosen as the simulated digestion218model because of the dynamic nature of the apparatus and219because it has been previously reported to have strong in vitro/220in vivo correlation (IVIVC) for orally administered drugs in the221human GI tract.22−24 Previous research demonstrated that the222hydrolytic degradation of a similar PAE, using sebacic acid223rather than diglycolic acid was pH-dependent.3 As shown for224polyanhydrides, in general, the relative rates of anhydride bond225hydrolysis increases when subjected to more basic condi-226tions.3,25−27 This data suggests that SA-derived PAEs might be227an effective delivery system for releasing SA into the lower228intestine.3 As the SA release rate is a direct result of the

Figure 2. SA release from free SA powder, SADG, and SADG-P at 30, 60, 90, 120, 180, 240, and 300 min in the jejunum (A), ileum (B), jejunum +ileum (C), and at the ileum efflux (D).

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229 hydrolytic cleavage of the PAE anhydride and ester bonds, it is230 possible to determine the SA release rate from the SADG and231 SADG-P and subsequently infer SA bioaccessibility from each

f2 232 formulation in the different intestinal segments (Figure 2).233 Very little SA is released during the first 60 min of digestion234 (Figure 2). This result is consistent with the cyclic235 interdigestive pattern, also known as the migrating motility/236 myoelectricity complex (MMC), for the fasted state. MMC237 consists of three phases: a resting period of approximately 60−238 70 min, a phase of 20−30 min during which the frequency of239 muscular contractions increases, and a final phase of240 approximately 5−10 min of forceful contractions.28 Given the241 known lag time for polyanhydrides and PAEs, we anticipated242 that SA release from the polymer formulation would be243 delayed. Yet, for all test samples (SA, SADG, and SADG-P), SA244 concentrations increased with transit time between 180 and245 240 min and declined after 240 min. The sharpest increase in246 concentrations was observed between the 60−120 min and247 120−180 min time intervals. In the jejunum, section 5c in248 Figure 1, SA bioaccessibility increases linearly during the first

249120 min after which there is a 2-fold increase in the release per250unit time (Figure 2A). SA bioaccessibility is not only limited by251the solubility, but also by the rate of surface erosion of the252powder or rate of dispersion. As expected, the SA concentration253released by SA was the highest (138 mg), followed by SADG254(78 mg) and SADG-P (53 mg), respectively.255Although no initial lag phase was observed for any drug256formulation, differences in the total SA released by the SA,257SADG, and SADG-P indicate a delay in the hydrolytic258degradation of the SADG and SADG-P. This divergence259became even more pronounced between 90 and 120 min260(Figure 2A−C). Further, the amount of SA released by SADG-261P was lower compared to SADG. This difference is attributed to262the fact that SADG-P must first hydrolyze to SADG via263anhydride bond cleavage, and then SADG undergoes hydrolysis264further to the SA and diglycolic acid (DG) via ester bond265 s1cleavage (Scheme 1). As in the jejunum, a slower hydrolysis266rate was observed with the SADG and SADG-P in the ileum267(Figure 2B). Overall, the fraction of SA available in the ileum,268section 5d in Figure 1, was lower than the fraction available in

Scheme 1. Hydrolytic Degradation of SA-Based Polymers (SADG-P): Anhydride Linkages are First Hydrolyzed to Form theIntermediate (SADG) and Then the Ester Bonds Further Hydrolyze to Yield SA and DG

Figure 3. Cumulative SA release from free SA, SADG and SADG-P concentrations in jejunal filtrate (A), ileal filtrate (B), combined jejunal and ilealfiltrates (C), and ileal efflux (D).

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269 the jejunum, indicating that the majority of hydrolysis occurred270 in the jejunum. This trend was also observed at the ileal efflux271 (Figure 2D, section 4 in Figure 1), which represents the SA272 delivered to the colon.273 Although the hydrolysis of ASA is a second-order reaction,274 dependent upon the initial ASA concentration and pH, it is275 often assumed to be a near zero-order reaction when the pH is276 constant.29 Using the absolute concentrations obtained from277 Figure 2, a cumulative bioaccessible concentration was obtained

f3 278 and plotted as a function of time (Figure 3A−C) to facilitate279 the calculation of the rate kinetics. The zeroth-order reaction280 rate becomes prevalent when the total SA bioaccessibility is281 observed as a function of time; as all test samples (SA, SADG282 and SADG-P) display near zero-order release profiles. It is283 plausible that the near zero-order release of SADG-P is due to284 predominantly surface eroding characteristics, as this is285 common in PAE systems.14 The cumulative release rates of286 SA were determined to be 0.42 mg/min (R2 = 0.98) for SA,287 0.28 mg/min (R2 = 0.95) for SADG, and 0.19 mg/min (R2 =

t1 288 0.93) for the SADG-P (Table 1).289 As expected, free SA had a consistently higher release rate in290 each of the intestinal segments compared to SADG and SADG-291 P. The bioaccessibility of free SA appears to be dependent only292 on the solubilization rate and power erosion. Very little SA was293 detected in the ileal efflux (i.e., delivered to the colon),294 suggesting that very little of the drug would be metabolized by295 the gut microflora. The SA release rate from the SADG was 1/3296 more slow than the SA, illustrating that SADG is not only297 affected by the solubilization rate but must also undergo298 hydrolysis to dissociate into the SA and diglycolic acid. As299 expected, the SA release rate was slowest for the polymer,

300SADG-P. For SA to be released from the polymer, several steps301must occur: the polymer surface is hydrolyzed, the anhydride302bonds are cleaved, then both ester bonds must be hydrolyzed303before the SA is solubilized (Scheme 1). Interestingly, the304majority of ASA was released in the jejunum compared to305either the ileum or the efflux (Figures 2 and 3). Since the306powder was partially confined in the stomach, only the soluble/307dispersed fraction reaches the jejunum, and upon reaching the308jejunum, the pH changes from 1.5 to 6.5, which facilitates the309hydrolytic cleavage of the ASA monomers.310After 5 h of simulated digestion, the bioaccessibility of SA311was observed to be 55.5 ± 2.0% for free SA, 31.2 ± 2.4% for312 f4SADG, and 21.2 ± 3.1% for SADG-P (Figure 4). The313statistically significant (p < 0.001) differences in SA314bioaccessibility correlate with the differences in drug for-315mulation/composition. A polymeric version of SA ultimately316translates to decreased SA bioaccessibility throughout the entire317GI tract.

318■ CONCLUSIONS

319The bioaccessibility of SA, found to be a zero-order hydrolysis320reaction, can effectively be modified by incorporating SA into a321PAE backbone. After simulated digestion using the TIM-1, the322SA bioaccessibility was 55.5 ± 2.0% for SA, 31.2 ± 2.4% for323SADG, and 21.2 ± 3.1% for SADG-P. The SA release rates324were dependent on (1) solubilization rate, (2) macroscopic325erosion of the powder, (3) hydrolytic cleavage of the polymer’s326anhydride bonds, and (4) hydrolytic cleavage of the ester bonds327to SA and diglycolic acid.

Table 1. SA Release Rates Calculated As Zeroth-Order Rate from the Combined Ileum and Jejunum Bioaccessible Fractions

jejunum ileum jejunum + ileum efflux

sample release rate (mg/min) R2 release rate (mg/min) R2 release rate (mg/min) R2 release rate (mg/min) R2

SA 0.29 0.96 0.16 0.99 0.42 0.98 0.03 0.51SADG 0.21 0.94 0.06 0.92 0.27 0.95 0.02 0.26SADG-P 0.16 0.96 0.04 0.96 0.19 0.93 0.02 0.33

Figure 4. Total SA bioaccessibility (i.e., combined jejunal and ileal filtrate concentrations) after 5 h of simulated digestion. Letters denote significantdifferences based on triplicate digestions using a two-way ANOVA and Tukey’s multiple comparison test (p < 0.0001).

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328 ■ AUTHOR INFORMATION329 Corresponding Author330 *E-mail: rogers@aesop.rutgers.edu.331 Notes332 The authors declare no competing financial interest.

333 ■ ACKNOWLEDGMENTS334 We would like to acknowledge the technical support from335 TNO and TNO Triskelion and Susann Bellman on the336 operation and technical support for the TIM-1 GI model.337 M.A.R. and K.E.U. also gratefully acknowledge support for this338 project supplied from the New Jersey Institute of Food,339 Nutrition and Health (IFNH) seed grant program.

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Biomacromolecules Article

dx.doi.org/10.1021/bm500927r | Biomacromolecules XXXX, XXX, XXX−XXXF