Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239–243www.nanomedjournal.com

Oncology

Chemotherapeutic evaluation of alginate nanoparticle-encapsulated azoleantifungal and antitubercular drugs against murine tuberculosis

Zahoor Ahmad, PhD,a Sadhna Sharma, PhD,b Gopal K. Khuller, PhDb,⁎aCenter for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

bDepartment of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Received 3 March 2007; accepted 24 May 2007

Abstract The present study was designed to evaluate the chemotherapeutic potential of alginate nanoparticle-

No conflict of inte⁎ Corresponding

Institute of Medical160012, India.

E-mail address: gk

1549-9634/$ – see frodoi:10.1016/j.nano.20

encapsulated econazole and antitubercular drugs (ATDs) against murine tuberculosis. Alginatenanoparticles encapsulating econazole and ATDs were prepared by the cation-induced controlledgelification of alginate and were characterized. Drugs were analyzed by high-performance liquidchromatography. All the ATDs were detected above minimum inhibitory concentrations for as longas 15 days and econazole until the day 8 in organs (lungs, liver, and spleen) after administration ofencapsulated drugs, whereas free drugs remained detectable for only 12 to 24 hours. Eight doses ofalginate nanoparticle-encapsulated econazole or 112 doses of free econazole reduced bacterialburden by more than 90% in the lungs and spleen of mice infected with Mycobacterium tuberculosis.Econazole (free or encapsulated) could replace rifampicin and isoniazid during chemotherapy ofmurine tuberculosis. Alginate nanoparticles reduced the dosing frequency of azoles and ATDs by15-fold. Alginate nanoparticles are the ideal carriers of azole and antitubercular drugs, which canreduce dosing frequency of azoles as well as ATDs for the better management of tuberculosis.© 2007 Published by Elsevier Inc.

Key words: Nanoparticles; Chemotherapy; Econazole; Bioavailability

Azole antifungals have been demonstrated by variousreports to have potent antitubercular activity; these drugsdeserve close attention, because they have already beenassessed for toxicological impact and are in clinical use forhumans [1]. Earlier we have demonstrated the in vitro and exvivo potential of azoles against M. tuberculosis H37Rv aswell as the synergism of azole drugs with conventionalantitubercular drugs [2]. Antituberculosis potential of azoledrugs against latent/persistent tuberculosis has also beendemonstrated, and econazole, like rifampicin (RIF), has beenshown to prevent the formation of persistent/latent bacilli inmice [3]. In addition, econazole has been shown to be more

rest was reported by the authors of this paper.author. Department of Biochemistry, PostgraduateEducation and Research, Sector-12, Chandrigarh

khuller@yahoo.co.in (G.K. Khuller).

nt matter © 2007 Published by Elsevier Inc.07.05.001

effective than RIF against persistent bacilli [3]. Morerecently we have demonstrated that econazole could reducebacterial burden by 90% in the lungs and spleen of miceinfected with M. tuberculosis and its chemotherapeuticpotential to be equal to RIF [4]. Further, results of this studyindicated that econazole could replace RIF or isoniazid(INH), as well as both of them in chemotherapy of murinetuberculosis [4]. Although azole drugs have been shown tohave strong antimycobacterial potential against latent/persistent and active murine tuberculosis, their bioavail-ability is poor through the oral route [5]. Hence, econazolemust be administered with a high dosing frequency-twicedaily [4]. Thus, it seems that even after the incorporation ofazole drugs into a conventional antitubercular regimen, theproblem of patient noncompliance will be of more concern.Recently, we have demonstrated that bioavailability of themost potent antimycobacterial azoles (econazole andclotrimazole) could be enhanced by using alginate/poly(lactide-co-glycolide) nanoparticles as drug carriers [6].

240 Z. Ahmad et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239–243

Hence, the present study was planned with an aim to evaluatethe chemotherapeutic potential of alginate nanoparticle-encapsulated econazole alone and in combination withantitubercular drugs against murine tuberculosis.

Methods

Chemicals and drugs

Sodium alginate (medium viscosity, 3500 cps for a 2% w/vsolution), chitosan (minimum 85% deacetylated), econazole,INH, RIF, ethambutol (EMB), and pyrazinamide (PZA) wereobtained from Sigma Chemical Co. (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade sol-vents and water were obtained from Merck Ltd. (Mumbai,India). All other chemicals used in the study were ofanalytical grade.

Animals

Laca mice of either sex weighing 20-25 g obtained fromthe Central Animal House, Postgraduate Institute of MedicalEducation and Research (Chandigarh, India) were used inthe study. Animals were housed in biosafety cabinets (NuaireInstruments, NU 605-600E, Series 6) and provided withpellet diet/water ad libitum. The study was approved by theInstitute's Animal Ethics Committee.

Preparation of ATD-loaded alginate nanoparticles

Alginate nanoparticles were prepared by the cation-induced controlled gelification of alginate [7] with slightmodifications [8]. Briefly, calcium chloride (0.5 mL, 18 mM)was added to 9.5 mL of sodium alginate solution (0.06%)containing drugs (ratio of drug to polymer was 7.5:1 w/w %).Addition of 2 mL of chitosan solution (0.05%) was followedby stirring for 30 minutes and maintenance at roomtemperature (23-25°C) overnight. Drug-loaded nanoparticleswere recovered by centrifugation at 19,000 rpm for35 minutes and washed thrice with distilled water. Drug-free nanoparticles were also prepared in the same manner.

Characterization of alginate nanoparticles

Alginate nanoparticles were characterized for their sizeand polydispersity index on Zetasizer 1000 HS (MalvernInstruments, Malvern, UK) as described earlier [8]. The drugencapsulation efficiency was determined as described earlier[8]. The drugs were analyzed by HPLC; in the case of RIF/INH/PZA, the three drugs were separated and quantitated byusing a USP gradient program as described earlier [9].Because of the ultraviolet transparency of EMB, it wasanalyzed by using a USP isocratic program as describedearlier [9]. The sensitivity of the methods was RIF 0.4 μg/mL, INH 0.2 μg/mL, PZA 1.0 μg/mL, and EMB 0.5 μg/mL[9]. The econazole was analyzed by using a USP isocraticprogram as described earlier with an analytical sensitivity of0.2 μg/mL [6].

In vivo drug disposition studies

The drug doses used throughout the study were RIF12 mg/kg, INH 10 mg/kg, PZA 25 mg/kg, EMB 16 mg/kg,and econazole 3.3 mg/kg body weight according to thestandard adult human doses. Although higher drug doses aregenerally recommended for mice (considering that micepossess a higher surface area-to-body weight ratio ascompared with humans), we have recently demonstratedthe two doses to be equipotent [10]. The dose being differentfor each drug, the initial amount of drug taken to prepare theformulations was calculated as previously described [10].

For the single-dose drug disposition studies, mice weregrouped as follows with six animals in each group: group 1,oral free econazole; group 2, oral free ATDs; group 3, oralfree econazole + ATDs; group 4, oral econazole-loadedalginate nanoparticles; group 5, oral ATD-loaded alginatenanoparticles; group 6, oral econazole + ATD-loadedalginate nanoparticles; group 7, oral drug-free alginatenanoparticles (a positive control to explore the influence ofalginate nanoparticles on drug estimation). The animals werebled at several time points. The plasma obtained from eachmouse was analyzed for the estimation of drug presence asdescribed earlier [9]. The drugs were analyzed by HPLC andcompared with calibration graphs (obtained by analyzingpooled blank mice plasma spiked with known drug amounts)to obtain the plasma drug concentration versus time profile.The area under the curve for plasma drug concentration inrelation to time was calculated using data analysis tools inSigmaPlot software (Version 8.0) and further used tocompute the relative bioavailability of each drug.

The animals were sacrificed at different time points. Druglevels were determined in 20% w/v of tissue homogenates(lungs, liver, and spleen) by following the same analyticalprocedure as described for plasma [9]. It should be noted thatthe HPLC analysis in plasma/tissues in the case of micedosed with alginate nanoparticles was restricted to free drugsonly (i.e., no attempt was made to lyse the nanoparticles).

Experimental infection and chemotherapy

Mice were infected via the lateral tail vein with either 105

to 107 bacilli of M. tuberculosis H37Rv as described earlier[4]. The confirmation of infection and basal bacterial loadwere determined as described earlier [4]. Subsequently, micewere grouped as follows (eight animals per group): group I,untreated controls (received phosphate-buffered saline[PBS]); group II, econazole; group III, RIF; group IV,INH, PZA, EMB, and RIF; group V, econazole, INH, PZA,and EMB; group VI, econazole, PZA, EMB, and RIF; groupVII, econazole, PZA, and EMB; group VIII, nanoparticlescontaining ,econazole; group IX, nanoparticles containingRIF; group X, nanoparticles containing INH, PZA, EMB,and RIF; group XI, nanoparticles containing econazole, INH,EMB, and PZA; group XII, nanoparticles containingeconazole, RIF, EMB, and PZA; group XII, nanoparticlescontaining econazole, EMB, and PZA. Control animals

Table 1Comparison of organ drug levels following oral administration of alginate-encapsulated azoles with and without antitubercular drugs to mice

Druganalyzed

Time(days)

ATDs or econazole(μg/mL)

ATDs + econazole(μg/mL)

Lungs

Econazole 7 0.31 ± .006 0.31 ± 0.01

Rifampicin 15 0.69 ± 0.07 0.66 ± 0.04

Isoniazid 15 0.24 ± 0.03 0.25 ± 0.01

Pyrazinamide 15 12.61 ± 2.14 10.66 ± 0.5

Ethambutol 15 1.48 ± 0.15 1.51 ± 11

Liver

Econazole 7 0.61 ± 0.01 0.29 ± 0.07

Rifampicin 15 0.77 ± 0.04 0.77 ± 0.015

Isoniazid 15 0.277 ± 0.006 0.27 ± 0.01

Pyrazinamide 15 11.43 ± 0.57 10.85 ± 0.41

Ethambutol 15 1.57 ± 0.04 1.64 ± 0.16

Spleen

Econazole 7 0.29 ± 0.070 0.21 ± 0.0

Rifampicin 15 0.72 ± 0.025 0.74 ± 0.05

Isoniazid 15 0.26 ± 0.015 0.26 ± 0.01

Pyrazinamide 15 11.13 ± 0.380 11.53 ± 0.09

Ethambutol 15 1.52 ± 0.100 1.62 ± 0.04

Values are mean ± SD of six animals.

Table 2Chemotherapeutic efficacy of econazole in free or alginate nanoparticle-encapsulated form against tuberculosis in mice infected with a high dose(107 bacilli) of M. tuberculosis H37Rv

Groups Log10 CFU

Lung Spleen

Untreated controls 6.88 ± 0.035 6.90 ± 0.025

Encapsulated econazole weekly (six doses) 4.85 ± 0.05 4.90 ± 0.04

Free econazole twice daily (90 doses) 4.87 ± 0.04 4.89 ± 0.02

Values are mean ± SD of six animals.P b .01 as compared with untreated controls.

241Z. Ahmad et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 239–243

received PBS only, whereas other groups were administeredthe drugs mentioned through the oral route at therapeuticdoses (INH 10 mg/kg, RIF 12 mg/kg, PZA 25 mg/kg, EMB16 mg/kg, and econazole 3.3 mg/kg body weight). Freedrugs (INH, RIF, PZA, and EMB) were administered oncedaily, however, econazole and EMB (in the presence ofeconazole) were administered twice daily. All encapsulatedATDs were administered every two weeks, and encapsulatedeconazole was administered weekly. Animals were killed onday 31, 46, and 58 of chemotherapy; lungs and spleen wereisolated under sterile conditions and homogenized in 3 mL ofisotonic saline. Homogenates (100 μL of undiluted, 1:10,and 1:1000 dilutions) were plated on Middlebrook 7H11agar plates supplemented with oleic acid/albumin/dextrosecatalase (OADC) for enumeration of colony-forming units(CFU), and colonies were counted on day 28 afterinoculation. The CFU data were analyzed by one-wayanalysis of variance followed by Student's unpaired t-test.

Results

Physicochemical characterization of alginate nanoparticles

The alginate nanoparticles had an average size of 229 nmwith a polydispersity index of 0.44. The drug encapsulationefficiency of alginate nanoparticles for econazole was foundto be 92% to 97.5%, and that of ATDs were 80% to 90% for

RIF, 88% to 95% for EMB, and 70% to 90% for INH andPZA. The drug loading capacity of alginate nanoparticlesranged from 525 to 730 mg per 100 mg of alginate. Thecoefficient of variation was found to be less than 4% withbatches prepared on the same day and less than 7% inbatches prepared on different days. Furthermore, thevariation remained within the narrow limits irrespective ofthe batch size.

In vivo drug disposition studies

Econazole was cleared from plasma within 4 hours afteradministration as free drug. In contrast, all the ATDs with orwithout econazole were cleared from the circulation within24 hours. In comparison to free drugs, alginate-encapsulateddrugs were detected in plasma from 3 hours onward;econazole, EMB, RIF, and INH/PZA were observed for aslong as 5, 7, 9, and 11 days, respectively, both alone or incombination. The bioavailabilities of drugs encapsulated inalginate nanoparticles were increased significantly (P b .001)in comparison with free counterparts.

All the ATDs (RIF, INH, EMB, and PZA) were detectedin tissues (i.e., lungs, liver, and spleen) up to day 1 followingthe administration of the free-ATD combination. However,free EMB was detected only up to 12 hours in all tissueswhen administered together with econazole, whereas tissuedistribution of the other three ATDs remained unchanged inthe presence of econazole. Free econazole was detected up to12 hours in tissues after administration alone as well as incombination with ATDs. All the ATDs were detected intissues up to 15 days at above the minimum inhibitoryconcentration [11] following the administration of encapsu-lated drugs alone or in combination with econazole (Table 1).Econazole was detected as late as day 7 in tissues at abovethe minimum inhibitory concentration following the admin-istration of alginate nanoparticles in the presence or absenceof encapsulated ATDs (Table 1).

Chemotherapeutic efficacy

Based on tissue drug distribution, free drugs (INH, RIF,PZA, and EMB) were administered once daily; however,econazole and EMB (in the presence of econazole) wereadministered twice daily. All encapsulated ATDs were

Table 3Chemotherapeutic efficacy of azoles with or without antitubercular drugs infree or alginate nanoparticle-encapsulated form against tuberculosis in miceinfected with low dose (105 bacilli) of M. tuberculosis H37Rv

Groups Log10 CFU*

4 weeks 6 weeks

Lung Spleen Lung Spleen

INH, PZA, EMB,and RIF

b1.0 b1.0 b1.0♦ b1.0♦

Eco, INH, EMB, andPZA

b1.0 b1.0 b1.0♦ b1.0♦

Eco, RIF, EMB, andPZA

b1.0 b1.0 b1.0♦ b1.0♦

Eco, EMB, and PZA 2.3 ± 0.03 2.32 ± 0.05 b1.0♦ b1.0♦

Encap. INH, PZA,EMB, and RIF

b1.0♦ b1.0♦ b1.0♦ b1.0♦

Encap. Eco, INH,EMB, and PZA

b1.0♦ b1.0♦ b1.0♦ b1.0♦

Encap. Eco, RIF,EMB, and PZA

b1.0♦ b1.0♦ b1.0♦ b1.0♦

Encap. Eco, EMB,and PZA

2.3 ± 0.06 2.33 ± 0.03 b1.0♦ b1.0♦

Untreated controls 4.02 ± 0.03 4.1 ± 0.04 4.71 ± 0.04 4.73 ± 0.03

Eco = econazole; Encap. = nanoparticle-encapsulated drugs.*b1.0, ♦ indicates no detectable bacilli. Values are mean ± SD of six animals.

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administered every two weeks, and encapsulated econazolewas administered weekly. Eight weeks of chemotherapywith econazole alone either in free form (112 doses,administered twice daily) or in encapsulated form (8 doses,administered weekly) resulted in approximately 90%clearance of bacilli from lungs and spleen of animalsinfected with 1 × 107 cells of M. tuberculosis as comparedwith untreated controls (Table 2). Further chemotherapeuticpotential of econazole was comparable to that of RIF,because both these drugs (free or encapsulated) decreasedbacterial burden from 6.88 log CFU and 6.9 log CFU toapproximately 4.87 log CFU and 4.89 log CFU in thelungs and spleen, respectively.

The administration of four drug combinations (INH +PZA + EMB + RIF or econazole + INH + EMB + PZA, oreconazole + RIF + EMB + PZA) either free or encapsulated(administered as per tissue distribution described above) for30 days to M. tuberculosis-infected mice resulted inundetectable CFU of tubercle bacilli in undiluted homo-genates of lungs and spleen as compared with ~4 log CFU inuntreated controls (Table 3). However, it took 45 days for thethree-drug combination (econazole, EMB, and PZA) in freeor encapsulated form (dosing strategy same as above) toyield the same results (i.e., undetectable CFU).

Discussion

Azole drugs have proven their antimycobacterial potentialunder in vitro, ex vivo and in vivo conditions against murine

tuberculosis caused by susceptible, resistant, and latentbacilli. However, the bioavailability of azole antifungaldrugs through the oral route is a staggering problem. Drugdelivery technology has shown its potential to enhance thebioavailability of otherwise poor orally bioavailable drugs,and azole drugs are noexception to this fact. The presentstudy aimed to explore the chemotherapeutic potential ofnanoencapsulated azoles against murine tuberculosis.

The size of alginate nanoparticles observed in this presentstudy is favorable, because particles bearing size less than500 nm are known to be suitable for oral drug delivery. Thedrug encapsulation efficiency of alginate nanoparticles wasbetter than that previously obtained for alginate micro-spheres or poly(lactide-co-glycolide) micro/nanoparticles. Asingle oral administration of drug-loaded alginate nanopar-ticles to mice maintained therapeutic drug levels in plasmafrom 5 to 11 days as compared with less than 1 day the incase of free drugs. Econazole and ATDs were detected in thetissues until day 7 and 15, respectively, in the case of alginatenanoparticles (Table 1), whereas free drugs were detectableonly up to 12 or 24 hours. This formed the basis of thechemotherapeutic schedule wherein the alginate formulationwas administered to M. tuberculosis-infected mice on everyeighth/fifteenth day as compared with free drugs adminis-tered once or twice daily.

Eight doses of encapsulated econazole (administeredweekly) or 112 doses of free econazole (administered twicedaily) proved to bear equal therapeutic potential (Table 2).These data clearly demonstrate the sustained-releasepotential of alginate nanoparticles (which can reduce dosingfrequency of azole drugs 15-fold without compromisingtherapeutic efficacy) as well as the antimycobacterialpotential of azole drugs. The equipotency of four differentdrug combinations (INH, PZA, EMB, and RIF/econazole;INH, EMB, and PZA/econazole; RIF, EMB, and PZA) infree or in alginate nanoparticle-encapsulated form reflectsthe potential of alginate nanoparticles to be suitable carriersfor azoles as well as ATDs. This also highlights thepotential of econazole to be a good substitute for RIF/INHor both of these drugs in free or encapsulated form duringmurine tuberculosis chemotherapy. The role of econazolein reducing bacterial burden from lungs and spleens ofinfected mice in the presence of other ATDs (INH + PZA +EMB; RIF + PZA + EMB; PZA + EMB) is supported bythe observation that all of these combinations withouteconazole cleared half the bacterial burden in comparison-with untreated controls (Table 3). Furthermore, thateconazole played a significant role in reducing bacterialburden from lungs and spleens of infected mice in thepresence of other ATDs is also supported by the previousstudies wherein the more potent ATD combination (INH,PZA, and RIF) failed to yield undetectable levels ofcolony-forming units in 4 weeks [12]. The observed potentantimycobacterial potential of econazole can be explainedon the basis of multiple targets of econazole in M.tuberculosis [13]. Furthermore, encapsulated as well as

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free econazole alone or in combination with antituberculardrugs did not produce hepatotoxicity in normal or M.tuberculosis-infected mice based on biochemical para-meters, indicating their safe use.

So far, attempts to enhance the oral bioavailability ofazoles have focused on the use of cyclodextrins, which hadlimited success [5]. Additionally, alginate poly-1-lysinemicroparticles have been used to encapsulate azoleantifungal ketoconazole with encapsulation efficiency of71.5% and the particle size being 80 to 130 μm [14]. Ourresults clearly demonstrate the superiority of alginatenanoparticles over cyclodextrins as well as alginate poly-1-lysine microparticles, as azole carriers with respect toencapsulation, formulation size, polymer consumption, anduse of chitosan in place of poly-1-lysine. The present studyalso emphasizes that nanotechnology is a powerful tool thatcan be used not only to improve the oral bioavailability ofazole drugs but to retain their full chemotherapeuticpotential. Not only ATDs and azoles have been success-fully encapsulated in alginate, but a large number of drugssuch as indomethacin [15], sodium diclofenac [16],nicardipine [17], dicoumarol [18], gentamicin [19], vitaminC [14], and amoxicillin [20] have been successfullyincorporated in alginate. This study reports for the firsttime the in vivo potential of alginate nanoparticle-encapsulated econazole alone or in combination withATDs against experimental tuberculosis.

Acknowledgments

Z. A. P. thanks the Council of Scientific and IndustrialResearch, New Delhi, India, for the award of a SeniorResearch Fellowship.

References

[1] Guardiola-Diaz HM, Foster LA, Mushrush D, Vaz AND. Azole-antifungal binding to a novel cytochrome P450 from Mycobacteriumtuberculosis. Biochem Pharmacol 2001;61:1463-70.

[2] Zahoor A, Sharma S, Khuller GK. In vitro and ex vivo antimyco-bacterial potential of azole drugs againstM. tuberculosisH37RV. FEMSMicrobiol Lett 2005;251:19-22.

[3] Zahoor A, Pandey R, Sharma S, Khuller GK. The potential of azoleantifungals against latent/ persistent tuberculosis. FEMS MicrobiolLett 2006;258:200-3.

[4] Zahoor A, Pandey R, Sharma S, Khuller GK. Azole antifungals asnovel chemotherapeutic agents against murine tuberculosis. FEMSMicrobiol Lett 2006;261:181-6.

[5] Hostetler JS, Hanson LH, Stevens DA. Effect of cyclodextrin on thepharmacology of antifungal oral azoles. Antimicrob Agents Che-mother 1992;36:477-80.

[6] Pandey R, Zahoor A, Sharma S, Khuller GK. Nano-encapsulation ofazole antifungals: potential applications to improve oral drugbioavailability. Int J Pharm 2005;301:268-76.

[7] Rajaonarivony M, Vauthier C, Couarraze G, Puisieux F, Couvreur P.Development of a new drug carrier made from alginate. J Pharm Sci1993;82:912-7.

[8] Zahoor A, Pandey R, Sharma S, Khuller GK. Evaluation ofantitubercular drug loaded alginate nanoparticles against experimentaltuberculosis. J Nanosci 2006;1:81-5.

[9] Pandey R, Sharma S, Khuller GK. Chemotherapeutic efficacy ofnanoparticle encapsulated antitubercular drugs. Drug Deliv 2006;13:287-94.

[10] Zahoor A, Pandey R, Sharma S, Khuller GK. Pharmacokinetic andpharmacodynamic behaviour of antitubercular drugs encapsulated inalginate nanoparticles at two doses. Int J Antimicrob Agents 2006;27:420-7.

[11] Rattan A. Development of new drugs for tuberculosis. In: GuptaS, Sood OP, editors. Tuberculosis: Proceedings of the 7th RoundTable Conference, Ranbaxy Research Foundations, New Delhi; 2000.p. 113-22.

[12] Pandey R, Zahoor A, Sharma S, Khuller GK. Nanoparticleencapsulated antitubercular drugs as a potential oral drug deliverysystem against murine tuberculosis. Tuberculosis 2003;83:373-8.

[13] Leys D, Mowat CG, McLean KJ, Richmond A, Chapman SK,Walkinshaw MD, et al. Atomic structure of Mycobacterium tubercu-losis CYP121 to 1.06 Å reveals novel features of cytochrome P450.J Biol Chem 2003;278:5141-7.

[14] Cui JH, Goh JS, Park SY, Kim PH, Lee BJ. Preparation and physicalcharacterization of alginate microparticles using air atomizationmethod. Drug Dev Ind Pharm 2001;27:309-19.

[15] Joseph I, Venkataram S. Indomethacin sustained release from alginate-gelatin or pectin-gelatin coacervates. Int J Pharm 1995;126:161-8.

[16] Gonzalez-Rodriguez ML, Holgado MA, Sanchez-Lafuente C, RabascoAM, Fini A. Alginate-chitosan particulate systems for sodiumdiclofenac release. Int J Pharm 2002;232:225-34.

[17] Takka S, Acarturk F. Calcium alginate microparticles for oraladministration: I: effect of sodium alginate type on drug release anddrug entrapment efficiency. J Microencapsulation 1999;16:275-90.

[18] Chickering DE, Jacob JS, Desai TA, Harrison M, Harris WP, MorrellCN, et al. Bioadhesive microspheres: III. An in vivo transit andbioavailability study of drug loaded alginate and poly (fumaric-co-sebacic anhydride) microspheres. J Control Release 1997;48:35-46.

[19] Lannuccelli V, Coppi G, Cameroni R. Biodegradable intraoperativesystem for bone infection treatment. I. The drug/polymer interaction.Int J Pharm 1996;143:195-201.

[20] Whitehead L, Collett JH, Fell JT. Amoxicillin release from a floatingdosage form based on alginates. Int J Pharm 2000;210:45-9.