investigation of the in vitro release of gentamicin from a polyanhydride matrix

Journal of Controlled Release 63 (2000) 305–317www.elsevier.com/ locate / jconrel

Investigation of the in vitro release of gentamicin from apolyanhydride matrix

a , a a a a*Dennis Stephens , Luk Li , Dan Robinson , Shen Chen , Hung-Chih Chang ,a a a a bRong Ming Liu , Youqin Tian , Eric J. Ginsburg , Xiaoyan Gao , Timothy Stultz

aAdvanced Drug Delivery, Hospital Products Division, Abbott Laboratories, Department 97d, 100 Abbott Park Road, Abbott Park,IL 60064-3500, USA

bUniversity of Iowa, College of Pharmacy, Division of Pharmaceutics, Iowa City, IA 52240, USA

Received 26 June 1999; accepted 14 September 1999

Abstract

SeptacinE is a sustained release formulation consisting of gentamicin sulfate dispersed in a biodegradable polyanhydridematrix. The polyanhydride matrix is a copolymer of erucic acid dimer (EAD) and sebacic acid in a 1:1 weight ratio. In vitrodrug release was performed in both water and pH 7.4 phosphate buffer. The drug release in water was faster than that in thebuffer, which was the opposite of what would be expected based upon a faster polymer hydrolysis rate in the buffer.Theoretical treatment of the data using the Peppas model revealed that release in water was anomalous, while the release inpH 7.4 phosphate buffer was diffusion-controlled. Profound bead morphology differences were observed between beads inthese two in vitro release media. Cracking was observed in beads in water and swelling with no apparent cracking was seenin beads in buffer. Concurrent monitoring of drug and sebacic acid release indicated that drug release is not via surfaceerosion. Osmotic effects were found to play little role in the in vitro drug release. There was no spectroscopic evidence ofamide formation between the drug and copolymer. Sulfate release was monitored along with drug release and the resultsindicate that there is ion-exchange occurring during the pH 7.4 in vitro release. It was subsequently demonstrated thatgentamicin can form an insoluble salt with EAD. This salt formation explains the slower drug release in pH 7.4 phosphatebuffer. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Polyanhydride; Gentamicin; In vitro release; Septacin

1. Introduction centration of the antibiotic at the site of infection bysystemic administration. This can be attributed to a

Osteomyelitis is a deep bone infection that can number of factors: the short half-life of the anti-occur after hip or knee replacement surgery. Osteo- biotic; poor circulation to the infected area; andmyelitis is a particularly difficult infection to treat systemic toxicity of the antibiotic, which prohibitsbecause it is difficult to achieve a sufficient con- the use of a high systemic dose. Currently, osteo-

myelitis treatment requires large doses of antibioticsthat must be administered for long periods of time by*Corresponding author. Tel.: 11-847-938-2518; fax: 11-847-a combination of routes [1–3]. Some surgeons938-3645.

E-mail address: [email protected] (D. Stephens) currently blend antibiotics, such as aminoglycosides,

0168-3659/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 99 )00205-9

306 D. Stephens et al. / Journal of Controlled Release 63 (2000) 305 –317

with poly(methyl methacrylate) (PMMA) to deliver tured by Abbott Laboratories (North Chicago, IL).high local concentrations of drug [4]. A commercial Sebacic acid (Lot 05418CQ) was obtained fromproduct consisting of PMMA beads loaded with Aldrich Chemical (Milwaukee, WI). Gentamicingentamicin has been approved for use in Europe [5]. sulfate (Lot 94224-KA-00) was obtained from LekPMMA suffers from the disadvantage that it is not Pharmaceuticals (Czechoslovakia). Erucic acid dimerbiodegradable and must be removed at a later date. (EAD) (Lot 07904A500) was obtained from Unich-

SeptacinE is a new product that is being de- ema (Chicago, IL).veloped for the treatment of osteomyelitis. It is acontrolled release implant that contains gentamicinsulfate dispersed into a biodegradable polyanhydride 3. Methodspolymer matrix. Polyanhydrides [6,7] are a well-studied class of bioerodible polymers [8–10]. The 3.1. In vitro dissolutiondrug loading is 20% (w/w) gentamicin sulfate. Thepolyanhydride matrix is a copolymer of erucic acid In vitro dissolution was performed by placing adimer (EAD) and sebacic acid in a 1:1 weight ratio. bead into a vial and adding 100 ml of dissolutionSebacic acid is the more hydrophilic monomer while medium. The vial was stoppered and placed into aEAD is the more hydrophobic component. Drug reciprocal shaking water bath. The bath temperaturerelease can be tailored by varying the ratio of the two was maintained at 378C and the shaking speed was amonomers [6,7]. After incorporation of gentamicin in constant 100 rev. /min. The dissolution medium wasthe copolymer by melt-mixing, the drug polymer periodically changed by decanting the dissolutionblend is injection molded into a bead (12 mm33 contents and replenishing with a fresh 100 ml ofmm) form that is suitable for use. dissolution medium. The dissolution samples were

Septacin is designed to be implanted at the stored in a refrigerator prior to analysis. Samplessurgical site when a hip or knee prosthesis is were typically collected after 1, 2, 3, 4, 5, 8, 11, 14,replaced as a result of infection. The beads slowly 18, 22, 30, and 37 days. All analyses were carriedrelease gentamicin as the polymer degrades. This out in triplicate by analyzing three beads in separateprovides a relatively high local concentration of drug vials. The percent cumulative drug released waswhile minimizing systemic exposure. The efficacy of plotted against time in days and the error barspolyanhydride-based delivery of gentamicin has been represent 61 standard deviation.shown in various osteomyelitis animal models[11,12]. 3.2. Morphological evaluation

The purpose of this paper is to summarize theinvestigation of the in vitro drug release of Septacin The morphology change of the Septacin beadsin different dissolution media. Septacin, in essence, during the course of dissolution was investigated.is an extended release dosage form per the United The beads were removed from the dissolutionStates Pharmacopeia definition [13]. In vitro drug medium and placed on filter paper. The samplesrelease, frequently run in pure water, is used for were photographed using a Sony DXC-97MD colorthese dosage forms as a quality control tool and to video camera with an 83 magnification. Remnantsdemonstrate bioequivalence [14]. Septacin also were photographed after 2, 5, 11, 18, 26, and 34 daysserves as a useful ‘model system’ for the release of a of dissolution.hydrophilic drug from an extremely hydrophobicpolyanhydride matrix. 3.3. Analysis of gentamicin in in vitro dissolution

samples

2. Materials Gentamicin sulfate concentrations of the in vitrodissolution samples were determined using flow

Septacin (Lots 15-944-DH, and 15-946-DH) and injection analysis. Briefly, the sample is derivatizedSeptacin Placebo (Lot 15-947-DH) were manufac- with o-phthalaldehyde (FluoraldehydeE Pierce

D. Stephens et al. / Journal of Controlled Release 63 (2000) 305 –317 307

Chemical). The sample fluorescence is then mea- mg/ml) were prepared in 0.1 M phosphate buffers ofsured using a flow injection system consisting of a pH 6, 7, 8, and 9. Exactly 100 ml of the drugHPLC pump (Thermo Separation Products P4000), a solution were added to a flask containing EAD. TheHPLC autosampler (Thermo Separation Products mixture was immediately stirred at 500 rev. /min.AS3000), and a fluorescence detector (Thermo Sepa- Triplicate samples were prepared at each pH. Con-ration Products FL2000). Fluorescence was moni- trols (drug solutions without EAD) were run undertored using an excitation wavelength of l 5340 nm the same conditions. After 48 h, a 0.5-ml aliquot wasex

and an emission wavelength of l 5456 nm. The filtered through a 0.45-mm Acrodisc filter forem

sample response is quantitated against a linear gentamicin analysis. The percentage of drug incalibration curve that is generated using gentamicin solution (relative to the respective control) wasstandard solutions. calculated. Upon conclusion of the interaction study,

the pH of each flask was measured and then read-3.4. Analysis of sebacic acid in in vitro dissolution justed to pH 5.0 using 5 N HCl. The amount ofsamples gentamicin in solution was determined 24 and 48 h

after pH adjustment and the amount of gentamicinSebacic acid in the dissolution samples was recovered was calculated.

quantitated using a reversed phase HPLC with a In a separate experiment, 2.26 g (|4.7 mmol) ofWaters Symmetry C-18 (5 mm) 2.1 mm3150 mm gentamicin free base [15] were dissolved in 100 mlcolumn, and UV detection at 204 nm. The mobile of methanol; 3.70 g (5.5 mmol) of EAD werephase was 50% methanol /50% 37 mM phosphate dissolved in 100 ml of methanol; 30 ml of EADbuffer (pH53) maintained at a flow rate of 0.3 solution were added to 15 ml gentamicin free baseml /min. The injection volume was 20 ml. Linearity solution resulting in a molar ratio of 2.3 mol EADwas established from 4 to 220 ppm sebacic acid. per mol gentamicin, equivalent to the ratio of sulfateQuantitation was performed using a single standard to gentamicin in gentamicin sulfate. The solutionwhose concentration was 13 ppm. The HPLC system immediately became cloudy. The methanol was thenconsisted of a pump (Waters 410), autosampler removed under vacuum. In vitro release of gen-(Waters 717plus), and a UV detector (Applied Bio- tamicin from the precipitate was determined in watersystems 785). using the same in vitro dissolution procedure used

for Septacin.3.5. Sulfate determination in in vitro dissolutionsamples 3.7. Gentamicin /polysebacic acid amide formation

The release of sulfate from Septacin was moni- In order to search for any evidence of amidetored by determining sulfate content of the dissolu- formation between gentamicin and polyanhydride, antion medium. Sulfate was measured as sulfur using a attempt was first made to force amide formationPerkin-Elmer 3000XL ICP fitted with a P-E cyclonic between polysebacic acid and gentamicin. This wasspray chamber. The sulfur was determined at l5 accomplished by mixing 159 mg of gentamicin180.669 nm using high purge and two point back- sulfate and 116 mg of polysebacic into 50 ml ofground correction (60.017 nm). A four-point cali- 0.1 M pH 7.4 phosphate buffer and shaking at 100bration was used with the sample matrix as zero rev. /min at 378C for 3 weeks. The mixture wasconcentration and sulfur at 10, 30, and 50 mg/ml. filtered, lyophilized, extracted with methanol, and the

methanol extract was concentrated to a solid. The3.6. Gentamicin /EAD salt formation residue was subjected to spectroscopic analysis.

The ionic interaction between gentamicin and 3.8. Spectroscopic analysisEAD was investigated as a function of pH. Portionsof EAD (100 mg) were placed in respective 125-ml Several spectroscopic methods were used to detectErlenmeyer flasks. Gentamicin sulfate solutions (125 formation of amide between gentamicin and polymer


in various samples. Infrared spectroscopy was per-formed using a Nicolet Magna 750 FT-IR micro-scope. Mass spectroscopy was performed usingseveral different techniques. Electrospray LC/MSwas performed using a Finnigan TSQ 7000 instru-ment. Fast atom bombardment (FAB) mass spec-troscopy was performed using a JEOL SX102 withthioglycerol as the FAB matrix. Desorption chemicalionization (DCI) was performed on a Finnigan SSQusing ammonia as the chemical ionization reagent.Atmospheric pressure chemical ionization (APCI)

Fig. 1. In vitro release of Septacin in both water (♦) andmass spectrometry was performed using a Finniganphosphate buffer at pH57.4 (j).SSQ. Matrix assisted laser desorption ionization

(MALDI) mass spectrometry was performed using aby a pure surface erosion mechanism, one wouldBruker Reflex unit with 2-(4-hydroxyphenylazo)-expect that release at pH 7.4 would be faster thanbenzoic acid (HABA) as the matrix. A liquid / liquidthat in more acidic, unbuffered water.extraction was performed on Septacin remnant sam-

ples prior to mass spectral analysis. Remnants weredissolved in 20 ml of methylene chloride and

4.2. Theoretical treatment of release dataextracted with an equal volume of 0.1 N H SO .2 4

After extraction, there were also insoluble remnantsModeling of the controlled release of drugs fromobserved.

polymeric devices has been the subject of consider-Nuclear magnetic resonance (NMR) spectroscopyable research over the past 25 years. Most of thewas also performed on various samples. Samplesmodels have been based on solutions of the Fickianwere dissolved in either perdeuterated DMSO or

13 1 diffusion published in the classic book of Crank [18].D O. C, H, and heterocorrelation spectra were2Higuchi derived a simple relationship that describedrecorded using a Varian FT-NMR spectrometer.drug release from a matrix as a function of time [19](Eq. (1)).

4. Results and discussionMt 1 / 2]5 kt (1)M4.1. In vitro drug release in water and pH 7.4 `

bufferwhere M and M are the respective masses of drugt `

released at time equals t and `. It can be seen thatThe in vitro release of Septacin has been evaluatedin both water (pH |4 to 5) and phosphate buffer(pH57.4) and the release profiles are shown in Fig.1. The observation that gentamicin was releasedmore quickly in the medium of lower pH (water) wasunexpected. Literature data suggest that the erosionof polyanhydride copolymer (EAD:SA51:1 w/w) isfaster at higher pH [16,17]. In order to study thecopolymer erosion, degradation of the placebo beads(copolymer without drug) was studied as a functionof pH. Sebacic acid release was monitored and theresults are summarized in Fig. 2. It is clear that therelease of sebacic acid is faster under more alkaline Fig. 2. Polymer erosion as a function of pH performed on theconditions. Therefore, if drug release was controlled EAD:SA copolymer placebo.


the relative release of the drug is linear with the monitored in both water and pH 7.4 phosphatesquare root of time. buffer. The results are summarized in Fig. 3. In

Peppas extended the Higuchi model to a more water, the bead was observed to crack over thegeneralized form: course of the drug release experiment. The cracking

may be attributable to the faster drug release inMt n water. When the bead cracks, more surface area is]5 kt (2)M exposed to water which facilitates faster drug release`

through water-filled channels. This corresponds towhere n is the diffusional exponent. Information the anomalous release mechanism described by theabout the release mechanism can be gained by fitting fit to Eq. (2). The beads in phosphate buffer do notthe drug-release data (for the first 60% dissolved) crack and appear to swell over the course of drugand comparing the values of n to the semi-empirical release. This indicates that the matrix may serve as avalues for various geometries reported by Peppas diffusion barrier and drug release is mainly achieved[20,21]. For a cylindrical geometry, values of n of by diffusion through the matrix. This result agrees0.45 (or less) correspond to a purely Fickian diffu- with the diffusion model as predicted by the fit tosion mechanism. Values of n greater than 0.89 Eq. (2).indicate a relaxation controlled-release mechanism, The placebo bead in water (Fig. 3) shows no signsand n values between 0.45 and 0.89 indicate an of cracking indicating that the cracking of Septacinanomalous release mechanism. in water is related to the presence of the gentamicin.

The above equations are (at best) only approxi- The placebo bead in phosphate buffer was found tomations of a very difficult moving boundary-diffu- rapidly degrade. Based on this observation, it appearssion problem. Assumptions made in the derivation of that the gentamicin is slowing down the erosion ofthese equations include pseudo-steady state approxi- the copolymer in the Septacin bead at pH 7.4. This ismations of Fick’s law, one-dimensional diffusion, based upon the fact that the Septacin bead remainsand no alteration of the polymer matrix. Despite the intact in pH 7.4 buffer while the placebo polymerapproximations, it has been found that these relation- undergoes substantial erosion.ships can be applied to release data, indicating thatthe diffusion-based models of Higuchi and Peppas 4.4. Osmotic effectsdescribe drug release from a polymeric system quitewell. Osmotic forces have been shown in the literature

The data from the release curves of Fig. 1 were to substantially affect drug release for a variety offitted to the Peppas equation. The diffusional expo- different controlled release devices [23]. One couldnent for the drug-release curve in water was 0.723, theorize that the release of gentamicin would beindicating that the release mechanism was anomal- faster in pure water than in buffer because there is aous. The release data in phosphate buffer, on the greater osmotic force driving water into the matrix asother hand, yielded a diffusional exponent of 0.456, the drug is dissolved locally in the polymer matrix.indicating that Fickian diffusion plays an important This would create a relatively high local concen-role. If drug release occurred purely by surface tration of drug within the matrix. This effect woulderosion, as has been achieved for some poly- be less in the phosphate buffer because the dissolvedanhydride /drug combinations [22], diffusion would electrolytes in the buffer would result in a lowerplay little role. These results indicate that matrix osmotic pressure differential between the mediumdiffusion (not surface erosion) may play a significant and the matrix.role in the release of gentamicin sulfate from Sep- In order to investigate this effect, in vitro drugtacin in phosphate buffer at pH 7.4. release studies were carried out in 0.9% and 0.45%

NaCl. Sodium chloride solutions yield the same pH4.3. Changes in bead morphology as the water dissolution medium (pH55). The 0.1 M

pH 7.4 phosphate buffer was found to have anThe morphology of the Septacin remnant was osmolarity of 225 mOsm; 0.9% NaCl has a calcu-


Fig. 3. Morphological evaluation of bead remnants after 34 days in vitro dissolution: (a) Septacin in H O, (b) Septacin in 0.1 M phosphate2

buffer (pH57.4), (c) placebo polymer in H O, (d) placebo polymer in 0.1 M phosphate buffer (pH57.4). Each square on the grid represents2

a 333-mm area.

lated osmolarity of 303 mOsm, and 0.45% NaCl is154 mOsm. The in vitro drug release of Septacin wascarried out in 0.9% and 0.45% NaCl and the releasecurves (along with the water and pH 7.4 phosphatebuffer data) are plotted as Fig. 4.

It can be seen that the osmolarity of the dissolu-tion medium has a relatively small effect on the drugrelease rate. The most significant effect was realizedduring the initial release. After |5 days, the releasecurves for Septacin in 0.45% and 0.9% NaCl areessentially parallel to the release curve in water. Itwould appear that osmotic force plays a minor role, Fig. 4. Drug release of Septacin in pH 7.4 phosphate buffer (0.1and it is not sufficient to account for the difference M), 0.9% saline, 0.45% saline, and water.


seen in the drug release curves in phosphate buffer tamicin is relatively hydrophilic. Shah et al. demon-and water. However, in comparison with the placebo strated that the relationship between the drug releasebeads in water, the cracking of the Septacin beads in rate and copolymer erosion rate is dependent on thewater is likely attributed to the osmotic pressure hydrophilic /hydrophobic nature of the loaded drugbuild-up by the dissolved gentamicin in the bead. [24]. They demonstrated that hydrophilic compounds

(such as mannitol) were released faster than the4.5. Release of sebacic acid polyanhydride erosion.

The release of sebacic acid is plotted along withThe polyanhydride utilized in Septacin is a co- the release of gentamicin in pH 7.4 phosphate as

polymer of erucic acid dimer (EAD) and sebacic shown in Fig. 6. The release curves for the drug andacid. The sebacic acid is the more hydrophilic sebacic acid do not overlap, confirming that the drugmonomer. It would be useful to compare the release release in pH 7.4 buffer is also not via surfaceof this monomer with drug release in order to gain erosion. The data are consistent with the results ofsome additional insight into the drug release mecha- the theoretical treatment above. It is also noteworthynism. In order to ensure that the sebacic acid release that the sebacic acid release is apparently faster thanprofiles are obtained under sink conditions, the the drug release in pH 7.4 buffer.solubility of sebacic acid in water and phosphate The release profiles of sebacic acid from Septacinbuffer (pH57.4) at 378C was determined. The in water and pH 7.4 buffer are comparable (Fig. 7).solubility was determined to be 0.37 mg/ml in water This is very different from the results obtained withand 9.41 mg/ml in pH57.4 phosphate buffer. In all the placebo beads, from which sebacic acid iscases the sebacic acid levels measured in the dissolu- released approximately three times faster at pH 7.4tion solutions were well below the measured solu- than at pH 5 (Fig. 2). This result indicates that thebility limits. Gentamicin sulfate was also found to be release of sebacic acid is suppressed in the presencefreely soluble (.2.8 mg/ml) in both water and of gentamicin. This could be due to a chemicalpH57.4 buffer. interaction between the drug and the copolymer and/

The release of sebacic acid (in water) is plotted or its respective monomers.along with the release of gentamicin in Fig. 5. It canbe clearly seen that the drug release and polymer 4.6. Amide formationerosion (as shown by release of sebacic acid) curvesdo not overlap. This indicates that the drug is not One theory for the suppressed drug release ofreleased via a pure surface erosion mechanism and Septacin in phosphate buffer is amide formationthat diffusion through the matrix is occurring. Itwould appear that the drug is released faster than thepolymer erodes. This is due to the fact that gen-

Fig. 6. Release of drug and sebacic acid from Septacin in pH 7.4Fig. 5. Release of drug and sebacic acid from Septacin in water. phosphate buffer.


the anhydride bond of the copolymer to form anamide. The published pK value for gentamicin is 7.9a

[26]. Therefore, at pH 7.4 one can calculate (fromthe Henderson–Hasselbach equation) that about 24%of the available amines are deprotonated. Once theamide is formed it may be insoluble, causing gen-tamicin to be entrapped in the bead. Alternatively, ifa soluble amide is formed, the amine is blocked andwill no longer react with the o-phthalaldehyde. Thisprevents its detection in the in vitro dissolutionsamples.

The reaction between gentamicin and polysebacicFig. 7. Copolymer erosion of Septacin (as monitored by sebacicacid was studied as a model system. A desorptionacid release) in pH 7.4 phosphate buffer and water.chemical ionization mass spectral (DCI-MS) profile

between the polyanhydride and the drug. Domb et al. (using ammonia as the CI reagent) of the extract isdemonstrated that phenyl alkyl amines formed shown in Fig. 8. It can be seen that the base ion inamides with poly(sebacic acid) at pH 7.4 [25]. At pH the spectrum is at m /z of 220, which corresponds to

17.4 the amine groups on the gentamicin are suffi- a [M1NH ] for sebacic acid. The structure of4

ciently deprotonated enabling nucleophilic attack at gentamicin is given in Fig. 9. Gentamicin is a

Fig. 8. Desorption chemical ionization-mass spectrum (DCI-MS) of the methanol extract of mixing gentamicin sulfate and polysebacic acidat pH 7.4. The spectrum is expanded 203 above m /z of 400.


Fig. 9. The structure of gentamicin.

mixture of four components with molecular weights After the liquid / liquid extraction of the remnantof 477 (C1), 449 (C1a), and 463 (both C2 and C2a). samples, the methylene chloride layer, aqueous layer,The three expected protonated molecular ions [M1 and the insoluble portion were all analyzed using a

1H] are observed for the four components of gen- variety of mass spectroscopic techniques (includingtamicin. In addition, ions are observed at m /z of 662, MALDI, FAB, and ESI) in both positive and nega-

1648, and 635 corresponding to [M1H] of the tive ion modes. These analyses proved inconclusive,amides of gentamicin and sebacic acid. as no amide was observed in any of the samples.

The reaction mixture was also analyzed by NMR. Infrared spectroscopy was also used to evaluateIn the proton spectrum acquired in DMSO-d , the the remnant samples. The infrared spectrum of the6

amide protons are observed from 7.5 to 8.5 ppm insoluble portion of the remnant is presented in Fig.21indicating that some amide bonds are formed from 10. The bands at 1642 and 1547 cm could be

the reaction of one of the amine groups in gen- attributed to vibrations as a result of secondarytamicin with polysebacic acid. The amide protons amide. However, the reference spectrum of gen-disappear when two drops of D O are added to the tamicin sulfate (Fig. 11) also contains these bands.2

DMSO-d solution. The amide carbonyl groups are Therefore, it could simply be that bands observed in6

also observed in the carbon spectrum at 186.8 ppm. the remnant are from the residual drug. It was notedHowever, the proton–carbon long range (2–3 bonds) that the prominent sulfate band in the gentamicin

21correlation spectrum did not reveal any correlation sulfate reference spectrum at 1050 cm was absentbetween any proton in gentamicin and the carbonyl in the insoluble remnant. This could possibly be thegroups. This result could be caused by the fact that a result of ion-exchange of sulfate from the matrix tosmall amount of amide bond is randomly formed at the medium during the in vitro drug release. Thisthe five possible sites in the gentamicin molecule so was further investigated by monitoring sulfate re-that the concentration of each component is too low lease in the medium.to be detected.

Both NMR and mass spectroscopic analysis indi-cate the gentamicin amide can be formed using 4.7. Sulfate releasepolysebacic acid in aqueous media. In Septacin,gentamicin could be acylated with either EAD or The release of sulfate as a function of dissolutionsebacic acid. Acylation by EAD should yield a medium is shown in Fig. 12 along with the gen-relatively insoluble amide. Remnants obtained after tamicin release data. It can be seen that the gen-in vitro drug release experiments were examined for tamicin and sulfate release profiles are very similarthe presence of such amides. in water. In pH 7.4 phosphate buffer, at early time


Fig. 10. Infrared spectrum of insoluble remnant from phosphate in vitro dissolution.

points, sulfate release is significantly faster than 4.8. Gentamicin EAD salt formationgentamicin suggesting that there is ion-exchangeoccurring during the in vitro experiment [27]. In order to further investigate the possibility of salt

These data are consistent with the hypothesis of formation between EAD and gentamicin, EAD wasthe formation of an insoluble salt of monomeric or added to a series of drug solutions at different pH.oligomeric carboxylate anion and protonated gen- The solutions were allowed to stir overnight in thetamicin. As the polymer is hydrolyzed in pH 7.4 presence of the EAD and the gentamicin concen-buffer, the resulting carboxylate anions can form an tration was measured. At pH greater than 5, theionic network with the multiply charged cationic equilibrium concentration of the gentamicin in thedrug. The carboxylate exchanges for sulfate, which is aqueous phase decreases. This experiment essentiallyreleased into solution. In water the polymer hy- is an indirect titration of the EAD and the resultsdrolysis is slower. This minimizes the amount of indicate that the pK of EAD is |6.5. It was alsoa

carboxylate. In addition, the pH of the unbuffered demonstrated that when the pH was readjusted towater is lower (approximately pH 5) so that the pH55, the gentamicin concentrations in the solu-carboxylate groups are protonated. This inhibits the tions increased to the initial values.salt formation with gentamicin. In a separate experiment, the salt of gentamicin


Fig. 11. Infrared spectrum of gentamicin sulfate.

free base and EAD was prepared. The salt was tested lent to 2.3 mol H SO per mol of gentamicin was2 4

for gentamicin release in water using the same added to the dissolution solution. This ratio wasprocedure used for the Septacin beads, except that on chosen because gentamicin sulfate contains 2.3 molthe 21st day, a sufficient amount of H SO equiva- of sulfate per mol of gentamicin. This lowered the2 4

pH from |5.5 to 3.0. The results are summarized inFig. 13. It can be seen that before addition of H SO ,2 4

the drug release profile is quite similar to that forSeptacin in pH 7.4 buffer. In addition, the loweringof the pH after day 21 results in an increase in therelease rate. This is consistent with the carboxylateion becoming protonated, causing the dissociation ofthe EAD and gentamicin, and release of solublegentamicin sulfate.

There is literature precedent for such salt forma-tion. EAD salt formation in the absence of any drug

¨has been investigated by Gopferich et al. [28]. Theyshowed DSC and IR evidence for the formation ofFig. 12. In vitro release of gentamicin and sulfate from Septacin

1 2in both water and pH 7.4 phosphate buffer. Na EAD salts during the dissolution of EAD/SA


sebacic acid p(SA). However, no amide formationwas observed between gentamicin and the EAD:SAcopolymer. Sulfate ion exchange was observed dur-ing drug release at pH 7.4 indicating that a salt maybe formed between gentamicin and monomer (oroligomers) in the degraded copolymer matrix. Addi-tional data on the salt formation between gentamicinand EAD further support the conclusion that the slowrelease of gentamicin from Septacin in pH 7.4 bufferis attributed to the formation of insoluble salt ofgentamicin and EAD monomer (or oligomers) in theFig. 13. In vitro release of gentamicin from the insoluble gen-copolymer matrix.tamicin:EAD salt in water. On day 21, H SO was added to lower2 4

These results indicate that the relationship betweenthe pH from 5 to 3.

the drug and the polymer can play a critical role inthe in vitro release characteristics. In addition,polyanhydrides. In addition, salt formation betweencationic drugs have the potential to form hydro-gentamicin and oligolatic acid was reported byphobic salts with monomeric (or oligomeric) hy-Mauduit et al. [29]. Spectroscopic and release datadrolysis products of polyanhydrides. These salts canwere accounted for by considering salt formationradically affect the in vitro release profile.between oligomer carboxylate chain ends and the

cationic form of the drug.

References5. Conclusions

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and monomer solubilities in these two different paedic Infection, W.B. Saunders, Philadelphia, 1989.dissolution media. Fitting the in vitro drug release [5] D. Seligson, S.L. Henry, Newest knowledge of treatment fordata to the Peppas model indicated anomalous bone infection: antibiotic-impregnated beads, Clin. Orthop.

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