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

and Kathryn E. Uhrich§

‡Department of Food Science and †New Jersey Institute of Food, Nutrition and Health, Rutgers University, The State University ofNew Jersey, New Brunswick, New Jersey 08901, United States§Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States

ABSTRACT: The bioaccessibility of salicylic acid (SA) can be effectively modified by incorporating the pharmacologicalcompound directly into polymers such as poly(anhydride-esters). After simulated digestion conditions, the bioaccessibility of SAwas 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 acidpolymer precursor (SADG), and 21.2 ± 3.1% for SADG-P (polymer). The release rates followed a zero-order release rate thatwas dependent on several factors, including (1) solubilization rate, (2) macroscopic erosion of the powdered polymer, (3)hydrolytic cleavage of the anhydride bonds, and (4) subsequent hydrolysis of the polymer precursor (SADG) to SA anddiglycolic acid.

■ INTRODUCTION

Salicylic acid (SA), an active metabolite of aspirin (acetylsali-cylic acid; ASA), is useful due to its anti-inflammatory,antipyretic, keratolyic and analgesic properties.1,2 While SAhas been used since the fifth century to relieve pain, recentadvances describe a new delivery system that directlyincorporates SA into a poly(anhydride-ester) (PAE) toovercome issues associated with ASA.3−6 The polymericversion of SA offers many advantages over the small moleculeof ASA; the first is the ability to formulate into variousgeometries, including powders,7 disks,8 fibers,9 and micro-spheres.10 Second, PAEs allow high SA loadings, typicallybetween 60 and 80%, because of the direct incorporation of SAinto the polymeric backbone.3 Third, PAEs enable sustainedrelease of SA; as small molecules, SA rapidly diffuses, whereasthe polymeric version delivers a sustained, controlled release ofSA over time.5,11 Thus, PAEs have great potential in variousbiomedical applications, as they have been found to be nontoxicin both in vitro12 and in vivo studies.8

In designing SA-based PAEs, both the drug release rate anddrug loading capacity can be modified by altering the chemicalcomposition of the linker molecule, enabling a tunable drugrelease profile for diverse applications.5,11,13 Upon exposure towater, PAEs undergo hydrolytic degradation; the SA releaserate is dependent upon the solution conditions (i.e., pH,temperature, etc.) and polymer composition.5 PAE’s typicallyexhibit a sustained, near zero-order rate of drug release, owingto their rate-limiting step being governed by its surface-eroding

behavior and low solubility.9,14−16 Furthermore, PAEs do notdisplay the burst release typically observed in conventionaldelivery systems, which has been associated with toxicityconcerns. While the PAEs do not demonstrate burst releasebehavior, a disadvantage of the PAEs could be the observed lagtime.3,5,14 With some PAEs, drug release could be delayed bydays, a behavior that may not be desirable if immediate painrelief, for example, is required. The lag time can be overcomeby several approaches, such as admixing small molecules,17

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

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

As numerous variables influence the polymer degradationrate, including temperature, pH, water content, and mixing, it isimportant to understand how these polymeric systems behavein the alimentary track to ensure pharmacopeial efficacy. Theinfluence of biological and formulation variables makes itessential to characterize the “release” profile from the deliveryvehicle into the luminal fluids, which is termed bioaccessibility,defined here as the cumulative percent of SA released in thejejunum and ileum (Figure 1, TIM-1 sections 5c and 5d,respectively). It is not necessary to probe the bioavailability

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

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because absorption and circulation will be only affected by therate of SA release, as it is the free SA that is absorbed. Thepurpose of this study is to observe how the chemical structuresof SA precursors and polymers influence SA release (fromSADG and SADG-P, respectively) as compared to the smallermolecules (SA) in a dynamic simulated TNO-intestinal model(TIM-1) and to determine if SA release is targeted to differentintestinal segments.

■ MATERIALS AND METHODSMaterials. All chemicals, solvents, and reagents were used as

received, unless otherwise indicated, and purchased from Sigma-Aldrich (Milwaukee, WI). SA (Sigma-Aldrich) was used as a controland the polymer precursor (SADG) and polymer (SADG-P) weresynthesized according to a previously published protocol.4,5,7,13,19

Molecular Weight Analysis. Gel permeation chromatography(GPC) was used to determine the molecular weight (Mw) andpolydispersity index (PDI) of the polymer samples. A Waters systemconsisting of a 1515 isocratic high pressure liquid chromatography(HPLC) pump, a 717plus autosampler, and a 2414 refractive index(RI) detector was used. Waters Breeze 2 software running on an IBMThinkCentre CPU was used for data collection and analysis. Sampleswere dissolved in dichloromethane (DCM; 10 mg/mL) and filteredthrough 0.45 μm polytetrafluoroethylene syringe filters (VWR,Bridgeport, NJ). A 10 μL aliquot was injected and resolved on aJordi divinylbenzene mixed-bed GPC column (7.8 × 300 mm, AlltechAssociates, Deerfield, IL) at 25 °C, with DCM as the mobile phase at aflow rate of 1.0 mL/min. Molecular weights were calibrated relative tobroad polystyrene standards (Polymer Source Inc., Dorval, Canada).Thermal Analysis. Thermal analysis was accomplished using

differential scanning calorimetry (DSC) to acquire the glass transition(Tg) temperature. DSC was performed utilizing a Thermal Advantage(TA, New Castle, DE) DSC Q200 running on an IBM ThinkCentrecomputer equipped with TA Universal Analysis software for dataacquisition and processing. Samples (4−6 mg) were heated undernitrogen from −10 to 200 °C at a rate of 10 °C/min. A minimum oftwo heating/cooling cycles was used for each sample set. TAInstruments Universal Analysis 2000 software, version 4.5A, wasused to analyze the data.

Thermogravimetric analysis (TGA) was used to acquire thedecomposition temperature (Td) of polymer samples. TGA analysiswas performed using a PerkinElmer TGA7 analyzer with TAC7/DXcontroller equipped with a Dell OptiPlex Gx 110 computer runningPerkinElmer Pyris software (PerkinElmer, Waltham, MA). Polymersamples (10 mg) were heated under nitrogen at a rate of 10 °C/minfrom 25 to 400 °C. Td was defined as the onset of decomposition,represented by the beginning of a sharp slope on the thermogram.

Simulated Digestion. Pancreatin was obtained from Sigma-Aldrich. Fresh pig bile was obtained from Farm-to-Pharm (Warren,NJ, U.S.A.). The bile was collected, standardized from a slaughter-house, and pooled together before an aliquot for individual TIMexperiments was taken and stored at −20 °C until use. Rhizopus lipase(150000 units/mg F-AP-15) was obtained from Amano Enzyme Inc.(Nagoya, Japan). Trypsin from bovine pancreas (7500 N-α-benzoyl-L-arginine ethyl ester (BAEE) units/mg, T9201) was obtained fromSigma-Aldrich.

TNO Intestinal Model (TIM-1). A dynamic, in vitro gastro-intestinal (GI) model, TIM-1, developed by TNO (Zeist, TheNetherlands), was utilized to simulate digestion. The TIM-1 systemmodels the human digestive tract utilizing four compartmentsmimicking the stomach, duodenum, jejunum and ileum, peristalticmovements, nutrient/drug and water absorption, gastric emptying, andtransit time, as would be observed in vivo (Figure 1). Compartmentsare infused with formulated gastric secretions, bile, and pancreaticsecretions to modify pH and reproduce digestive conditions, respectiveof a fed or fasted state. In the fasted state, the pH of the gastriccompartment is 2.2 upon administration of the pharmacological agentand decreases to 1.5 over 90 min, and the gastric emptying rate has ahalf-life of 20 min.

A 1.4% pancreatin solution (Pepsin from porcine gastric mucosa,lyophilized powder, >2500 units/mg protein, Sigma-Aldrich) andsmall intestinal electrolyte solution (SIES: NaCl 5 g/L, KCl 0.6 g/L,CaCl2 0.25 g/L) were prepared. Duodenal start residue (60 g; 15 gSIES, 30 g diluted fresh porcine bile (20% bile), 2 mg trypsin solution,15 g pancreatin solution), jejunal start residue (160 g: 40 g SIES, 80 gfresh porcine bile, 40 g pancreatin solution), and ileal start residue(160 g SIES) were injected into respective compartments prior toheating the system to physiological temperature (37 °C) inpreparation 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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Different formulations were tested during 5 h experiments in theTIM-1 model, simulating fasted-state physiological conditionsfollowing ingestion of SA, SADG, or SADG-P. The test sample wasplaced in a mesh teabag located in between the two glasscompartments of the gastric unit, so that the formulation was exposedto physiologically relevant waves of peristalsis mixing, but not to directpressure forces. To simulate the initial amount of gastric juice, 5 ggastric enzyme (NaCl 4.8 g/L, KCl 2.2 g/L, CaCl2 0.22 g/L, and) wasadded to the gastric compartment. Formulations were standardized soa total of 249.3 mg of SA could be hydrolytically generated and eachformulation was prepared as a fine powder, loaded into a tea bag andsuspended in the gastric compartment. Then, 45 g gastric electrolytesolution, 100 g of water, and 2.5 mg amylase were immediately addedinto the gastric compartment, followed by a 50 g of water rinse.Secretion of HCl (1 M) into the gastric compartment during digestionwas controlled to follow a preprogrammed computer protocol, whichregulates gastric emptying (half-life = 20 min), intestinal transit times,pH (pH 2.2 to 1.5), and secretion fluid amounts.21

Secretion of digestive fluids were setup based on the following:duodenal secretion consisting of fresh porcine bile at a flow rate of 0.5mL/min, a 1.4% pancreatin solution at 0.25 mL/min, and SIES at 3.2mL/min. Jejunal secretion consisting of SIES and fresh porcine bilewere introduced at a flow rate of 3.2 mL/min. Ileal secretion consistingof SIES was pumped at a flow rate of 3.0 mL/min. Secretion of HCl (1M) into the gastric compartment during digestion was monitored via apreprogrammed computer protocol that regulates gastric emptying,intestinal transit times, pH values, and secretion fluid amounts. ThepH of duodenal, jejunal, and ileal compartments was maintained at 6.5,6.8, and 7.2, respectively, by controlled secretion of sodiumbicarbonate solution (1 M).The available SA fraction was observed by collection and analysis of

dialysate fluids, which were passed through semipermeable capillarymembranes (Spectrum Milikros modules M80S-300−01P, Irving, TX)with 0.05 μm pores at the ileal and jejunal compartments. Jejunal andileal filtrates as well as ileal efflux were cooled on ice to reduce residualenzyme activity once the samples passed through the capillarymembranes. Samples were gathered at 30, 60, 90, 120, 180, 240,and 300 min and immediately analyzed on an HPLC (see parametersbelow). This process allows the individual compartments of the upperGI to have their isolated effects on hydrolytic release of SA. Residueswere not collected for analysis following experiment termination at300 min. SA bioaccessibility was evaluated for each formulation intriplicate and each run was analyzed in duplicate, providing threesample triplicates and two technical duplicates for each variable.

HPLC-Evaporative Light Scattering Detector (ELSD) Anal-ysis. Separations were carried out by using Waters e2695 AllianceHPLC system (Waters, Milford, MA, U.S.A.) equipped with a freefatty acid HP column (4 μm, 3.9 × 150 mm; Waters, Milford, MA,U.S.A.) set at 30 °C. The injection volume was 50 μL, the flow ratewas 0.5 mg/min, and run time was 6 min. The isocratic eluting systemconsisted of 50% water and 50% tetrahydrofuran (THF). The effluentwas monitored with Waters 2424 Evaporative Light ScatteringDetector (ELSD; Waters, Milford, MA, U.S.A.) with the followingsettings: drift tube temperature for ELSD set at 65 °C and nebulizerfor nitrogen gas adjusted to 40 psi. Comparing the retention time withthe reference compound identified the chromatographic peaks. Thecorresponding retention times are 2.4 for SA and 1.8 min for SADG. Atotal of 10 concentrations of SA (≥99.0%; Sigma-Aldrich, St. Louis,MO, U.S.A.) solutions were injected in triplicate to generate thecalibration curve constructed by plotting the peak areas versus theconcentration of the SA. Quantification was carried out from theintegrated peak area and corresponding calibration curve.

■ RESULTS AND DISCUSSION

SADG and SADG-P were successfully synthesized according topublished procedures.4,5,7,13,19 Following SADG-P isolation, Mwand PDI were determined to be 16.0 kDa and 1.4, respectively.Furthermore, thermal properties were assessed to ensure thepolymer morphology would not be altered during the course ofthe study conducted at 37 °C. SADG-P was found to possessfavorable thermal properties with a Tg of 72 °C and Td of 330°C. In vitro evaluation of SA, SADG, and SADG-P wasperformed using the TIM-1 model under fasted conditions.The TIM-1 system was chosen as the simulated digestionmodel because of the dynamic nature of the apparatus andbecause it has been previously reported to have strong in vitro/in vivo correlation (IVIVC) for orally administered drugs in thehuman GI tract.22−24 Previous research demonstrated that thehydrolytic degradation of a similar PAE, using sebacic acidrather than diglycolic acid was pH-dependent.3 As shown forpolyanhydrides, in general, the relative rates of anhydride bondhydrolysis increases when subjected to more basic condi-tions.3,25−27 This data suggests that SA-derived PAEs might bean effective delivery system for releasing SA into the lowerintestine.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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hydrolytic cleavage of the PAE anhydride and ester bonds, it ispossible to determine the SA release rate from the SADG andSADG-P and subsequently infer SA bioaccessibility from eachformulation in the different intestinal segments (Figure 2).Very little SA is released during the first 60 min of digestion

(Figure 2). This result is consistent with the cyclicinterdigestive pattern, also known as the migrating motility/myoelectricity complex (MMC), for the fasted state. MMCconsists of three phases: a resting period of approximately 60−70 min, a phase of 20−30 min during which the frequency ofmuscular contractions increases, and a final phase ofapproximately 5−10 min of forceful contractions.28 Given theknown lag time for polyanhydrides and PAEs, we anticipatedthat SA release from the polymer formulation would bedelayed. Yet, for all test samples (SA, SADG, and SADG-P), SAconcentrations increased with transit time between 180 and240 min and declined after 240 min. The sharpest increase inconcentrations was observed between the 60−120 min and120−180 min time intervals. In the jejunum, section 5c inFigure 1, SA bioaccessibility increases linearly during the first

120 min after which there is a 2-fold increase in the release perunit time (Figure 2A). SA bioaccessibility is not only limited bythe solubility, but also by the rate of surface erosion of thepowder or rate of dispersion. As expected, the SA concentrationreleased by SA was the highest (138 mg), followed by SADG(78 mg) and SADG-P (53 mg), respectively.Although no initial lag phase was observed for any drug

formulation, differences in the total SA released by the SA,SADG, and SADG-P indicate a delay in the hydrolyticdegradation of the SADG and SADG-P. This divergencebecame even more pronounced between 90 and 120 min(Figure 2A−C). Further, the amount of SA released by SADG-P was lower compared to SADG. This difference is attributed tothe fact that SADG-P must first hydrolyze to SADG viaanhydride bond cleavage, and then SADG undergoes hydrolysisfurther to the SA and diglycolic acid (DG) via ester bondcleavage (Scheme 1). As in the jejunum, a slower hydrolysisrate was observed with the SADG and SADG-P in the ileum(Figure 2B). Overall, the fraction of SA available in the ileum,section 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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the jejunum, indicating that the majority of hydrolysis occurredin the jejunum. This trend was also observed at the ileal efflux(Figure 2D, section 4 in Figure 1), which represents the SAdelivered to the colon.Although the hydrolysis of ASA is a second-order reaction,

dependent upon the initial ASA concentration and pH, it isoften assumed to be a near zero-order reaction when the pH isconstant.29 Using the absolute concentrations obtained fromFigure 2, a cumulative bioaccessible concentration was obtainedand plotted as a function of time (Figure 3A−C) to facilitatethe calculation of the rate kinetics. The zeroth-order reactionrate becomes prevalent when the total SA bioaccessibility isobserved as a function of time; as all test samples (SA, SADGand SADG-P) display near zero-order release profiles. It isplausible that the near zero-order release of SADG-P is due topredominantly surface eroding characteristics, as this iscommon in PAE systems.14 The cumulative release rates ofSA were determined to be 0.42 mg/min (R2 = 0.98) for SA,0.28 mg/min (R2 = 0.95) for SADG, and 0.19 mg/min (R2 =0.93) for the SADG-P (Table 1).As expected, free SA had a consistently higher release rate in

each of the intestinal segments compared to SADG and SADG-P. The bioaccessibility of free SA appears to be dependent onlyon the solubilization rate and power erosion. Very little SA wasdetected in the ileal efflux (i.e., delivered to the colon),suggesting that very little of the drug would be metabolized bythe gut microflora. The SA release rate from the SADG was 1/3more slow than the SA, illustrating that SADG is not onlyaffected by the solubilization rate but must also undergohydrolysis to dissociate into the SA and diglycolic acid. Asexpected, the SA release rate was slowest for the polymer,

SADG-P. For SA to be released from the polymer, several stepsmust occur: the polymer surface is hydrolyzed, the anhydridebonds are cleaved, then both ester bonds must be hydrolyzedbefore the SA is solubilized (Scheme 1). Interestingly, themajority of ASA was released in the jejunum compared toeither the ileum or the efflux (Figures 2 and 3). Since thepowder was partially confined in the stomach, only the soluble/dispersed fraction reaches the jejunum, and upon reaching thejejunum, the pH changes from 1.5 to 6.5, which facilitates thehydrolytic cleavage of the ASA monomers.After 5 h of simulated digestion, the bioaccessibility of SA

was observed to be 55.5 ± 2.0% for free SA, 31.2 ± 2.4% forSADG, and 21.2 ± 3.1% for SADG-P (Figure 4). Thestatistically significant (p < 0.001) differences in SAbioaccessibility correlate with the differences in drug for-mulation/composition. A polymeric version of SA ultimatelytranslates to decreased SA bioaccessibility throughout the entireGI tract.

■ CONCLUSIONS

The bioaccessibility of SA, found to be a zero-order hydrolysisreaction, can effectively be modified by incorporating SA into aPAE backbone. After simulated digestion using the TIM-1, theSA bioaccessibility was 55.5 ± 2.0% for SA, 31.2 ± 2.4% forSADG, and 21.2 ± 3.1% for SADG-P. The SA release rateswere dependent on (1) solubilization rate, (2) macroscopicerosion of the powder, (3) hydrolytic cleavage of the polymer’sanhydride bonds, and (4) hydrolytic cleavage of the ester bondsto 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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■ AUTHOR INFORMATIONCorresponding Author*E-mail: rogers@aesop.rutgers.edu.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe would like to acknowledge the technical support fromTNO and TNO Triskelion and Susann Bellmann on theoperation and technical support for the TIM-1 GI model.M.A.R. and K.E.U. also gratefully acknowledge support for thisproject supplied from the New Jersey Institute of Food,Nutrition and Health (IFNH) seed grant program.

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