Oral Delivery of Meglumine Antimoniate-{beta}-Cyclodextrin Complex for Treatment of Leishmaniasis

The need for daily parenteral administration represents oneof the most serious limitations in the clinical use of pentavalentantimonials against leishmaniasis. In this work, we investigatedthe ability of ß-cyclodextrin to enhance the oralabsorption of antimony and to promote the oral efficacy of meglumineantimoniate against experimental cutaneous leishmaniasis. Theoccurrence of interactions between ß-cyclodextrinand meglumine antimoniate was demonstrated through the changesinduced in the spin lattice relaxation times of protons in bothcompounds. When free and complexed meglumine antimoniate weregiven orally to Swiss mice, plasma antimony levels were foundto be about three times higher for the meglumine antimoniate-ß-cyclodextrincomplex than for the free drug. Antileishmanial efficacy wasevaluated in BALB/c mice experimentally infected with Leishmaniaamazonensis. Animals treated daily with the complex (32 mg ofSb/kg of body weight) by the oral route developed significantlysmaller lesions than those treated with meglumine antimoniate(120 mg of Sb/kg) and control animals (treated with saline).The effectiveness of the complex given orally was equivalentto that of meglumine antimoniate given intraperitoneally ata twofold-higher antimony dose. The antileishmanial efficacyof the complex was confirmed by the significantly lower parasiteload in the lesions of treated animals than in saline-treatedcontrols. This work reports for the first time the effectivenessof an oral formulation for pentavalent antimonials.

INTRODUCTION

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Introduction

Pentavalent antimonials, including meglumine antimoniate, arethe main drugs used in the treatment of all forms of leishmaniasis(1, 2, 9). These highly water-soluble compounds are consideredinactive when given enterally and are subject to rapid renalclearance after parenteral administration, requiring a multiple-dosingregimen. Antimony (Sb) therapy is often accompanied by localpain during and just after intramuscular injections and by severesystemic side effects requiring very careful medical supervision.All these factors contribute to high cost and compliance difficultiesthat may ultimately lead to treatment failures. This also explainswhy so much effort is being devoted to the search for orallyactive antileishmanial drugs (4, 7, 13).

The association of drugs with carrier systems is a feasiblestrategy to improve oral absorption. Among drug carrier systems,cyclodextrins, which are cyclic oligosaccharides composed ofglucose units joined through ${alpha}$ -1,4 glucosidic bonds, have beenone of the most successful drug absorption enhancers for oraldelivery (5, 14).

We show here that ß-cyclodextrin forms a complex withmeglumine antimoniate and report the impact of this associationon the oral absorption of antimony in mice and on the efficacyof meglumine antimoniate in an experimental model of cutaneousleishmaniasis.

MATERIALS AND METHODS

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Materials and Methods

Materials. N-methyl-D-glucamine and SbCl₅ were obtained from Aldrich ChemicalCo. (Milwaukee, Wis). ß-Cyclodextrin and Dulbeccomodified Eagle medium were obtained from Sigma Chemical Co.(St. Louis, Mo.). Heat-inactivated fetal calf serum was obtainedfrom Cultilab (Campinas, Brazil).

Parasites. Leishmania amazonensis (MHOM/BR/75/Josefa strain) promastigotestransfected with green fluorescent protein (L. amazonensis-GFPpromastigotes) were used. Parasites were routinely isolatedfrom mouse lesions and maintained as promastigotes in Dulbeccomodified Eagle medium containing 10% heat-inactivated fetalcalf serum and 150 µg of Geneticin/ml at 26°C.

Preparation and characterization of meglumine antimoniate-ß-cyclodextrin complex. Meglumine antimoniate was synthesized according to the methodof Demicheli et al. (3) from an equimolar mixture in water ofN-methyl-D-glucamine and freshly precipitated and hydrated antimonypentoxide, obtained from SbCl₅ hydrolyzed in water. The resultingproduct contained 29% Sb by weight, as determined by inductivelycoupled plasma optical emission spectrometry.

The meglumine antimoniate-ß-cyclodextrin complex wasprepared by mixing ß-cyclodextrin and meglumine antimoniatein distilled water at a 1:1 cyclodextrin/Sb molar ratio, heatingthe mixture for 48 h at 55°C, and finally freeze-dryingthe resulting solution. The resulting compound was characterizedby C,H,N elemental analysis, inductively coupled plasma opticalemission spectrometry (Sb), thermogravimetry, and nuclear magneticresonance (NMR) spectroscopy. ¹H NMR studies were performedin D₂O on a Bruker DRX 400-Advance spectrometer operating at400 MHz at 27°C using trimethylsilyl as an internal standard.Typical spectrometer conditions were 65,536 data points, 11.19-kHzspectral width, and 10.0-s recycle time. Proton resonances wereassigned using data from the literature for ß-cyclodextrin(11) and from previous work for meglumine antimoniate (3). ¹Hinversion recovery (T₁) peak intensity data (I[t]) were fittedto a three-parameter exponential equation: I[t] = I[0] + P ·exp(-t/T₁), where I[t] is the signal intensity at t = ${tau}$ , I[0]is the maximum measurable signal intensity, P is a constantwhose value depends on the initial conditions (typically, P= 2I[0]), and T₁ is the spin lattice relaxation time; 10 ${tau}$ values,the time between the 180 and 90° pulses, of 10 to 15,000ms were used.

Elemental and Sb analyses showed the following composition:C, 33.51%; H, 6.57%; N, 0.78%; and Sb, 7.94%. Thermogravimetryindicated the presence of nine water molecules. According tothis data, the resulting product consisted of a 1:1 meglumineantimoniate-ß-cyclodextrin complex (calculated: C,33.89%; H, 6.57%; N, 0.81%; Sb, 7.02%).

Oral absorption of antimony from meglumine antimoniate and its complex with ß-cyclodextrin. Swiss mice (female; weighing 25 ± 3 g) received throughoral inoculum, with the aid of a needle gauge, 0.3 ml of anaqueous solution of either meglumine antimoniate or its complexwith ß-cyclodextrin at 100 mg of Sb/kg of body weight.Three mice from each group were sacrificed at the followingtimes: 0.5, 1, 2, 3, 4, 6, 12, and 24 h after inoculation. Bloodsamples were obtained, and the plasma was recovered and frozen.

The plasma was assayed for antimony by graphite furnace atomicabsorption spectrometry without digestion of the sample, usingzirconium (Zr) and rhodium (Rh) as permanent modifiers. Briefly,samples were diluted five times with 1% (vol/vol) nitric acidcontaining 0.02% tricetyl methyl ammonium. Pyrolytic-graphite-coatedtubes were initially treated, as previously described (12),with 10 µl of a mixture containing 500 µg of Zrand 250 µg of Rh; 20 µl of the diluted samples wasthen added to the graphite tube. All measurements were carriedout with a Hitachi (Mitorika, Ibaraki, Japan) Z-8200 atomicabsorption spectrometer equipped with a graphite furnace andan autosampler (SSC-300; Hitachi) and with polarized Zeemaneffect background correction. The hollow cathode lamp for Sbwas operated at 12.5 mA with a slit of 0.4 nm. Pyrolysis andatomization temperatures of 900 and 1,900°C, respectively,which gave symmetric absorption peaks and Sb recuperation efficienciesclose to 100% for Sb-contaminated samples, were selected.

Antileishmanial activity of meglumine antimoniate and its complex with ß-cyclodextrin. Five groups of BALB/c mice (five mice per group) were infectedin the ear with 2 x 10⁶ L. amazonensis-GFP promastigotes. Thelesion sizes were periodically measured using a dial caliperand were expressed as the difference between the thicknessesof the infected and uninfected ears. Treatment was initiated10 days after infection with the following preparations: meglumineantimoniate-ß-cyclodextrin compound by the oral route(8 mg in 200 µl of saline, equivalent to 32 mg of Sb/kg,for each daily dose), meglumine antimoniate by the oral route(8 mg in 200 µl of saline, equivalent to 120 mg of Sb/kg),and meglumine antimoniate by the intraperitoneal (i.p.) route(4 mg in 100 µl of saline, equivalent to 60 mg of Sb/kg).The controls received saline (phosphate buffered) by the i.p.route. The animals were treated daily on days 10 through 16and 31 through 36 of infection. On day 80 of infection, theanimals were sacrificed, and the parasite loads in the lesionswere quantitated by fluorimetry as previously described (10).Briefly, each infected ear was cut off and homogenized in 2ml of phosphate-buffered saline with a tissue grinder. Afterthe removal of tissue debris by gravity sedimentation for 10min, 200 µl of twofold dilutions of the cell suspensionswas transferred in triplicate to black microplates, and thefluorescence was read in a plate reader fluorimeter (Fluoroskan;LabSystems) at 435-nm excitation and 538-nm emission. The specificfluorescence was expressed as the difference between the infectedand uninfected ears. The data were analyzed by one-way analysisof variance followed by a Tukey posttest.

RESULTS

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Results

Physicochemical characteristics of meglumine antimoniate-ß-cyclodextrin complex. The meglumine antimoniate-ß-cyclodextrin complex wasobtained following reaction of an equimolar mixture of meglumineantimoniate and ß-cyclodextrin in water and freeze-dryingof the resulting solution. Thermogravimetry analysis indicatedthe presence of nine water molecules in the association compound,suggesting that ß-cyclodextrin did not release originallyincluded water molecules (8) into the bulk water upon associationwith meglumine antimoniate.

The occurrence of interactions between meglumine antimoniateand ß-cyclodextrin was investigated by ¹H NMR in D₂O.The proton NMR spectrum for the association compound was registeredand compared to the spectra of individual solutions of meglumineantimoniate and ß-cyclodextrin. In the case of thecomplex, the proton resonances for meglumine antimoniate andß-cyclodextrin were not significantly altered, showingchanges of <0.02 parts per million (data not shown). Sincethe proton resonances of meglumine antimoniate differ markedlyfrom those of free meglumine (3), one can infer that the antimonialdrug did not suffer dissociation in the association compound.

Evidence for the association of meglumine antimoniate with ß-cyclodextrinwas obtained through analysis of the spin lattice relaxationtimes of protons in both compounds: H_1a, H_1b, H_2b, H_3a, H_3b,and H_4b for meglumine antimoniate and H₁, H₂, H₃, and H₄ forß-cyclodextrin (Table 1). The other protons couldnot be analyzed because of the overlay between proton resonancesof ß-cyclodextrin and meglumine antimoniate. Markedalterations of the spin lattice relaxation times were observedfor H₃ in ß-cyclodextrin and for H_3a and H_3b in meglumineantimoniate, indicating a change in the environment of theseprotons. These results, in light of the truncated cone-shapedstructure of ß-cyclodextrin, which exhibits a hydrophilicouter surface and a hydrophobic central cavity, suggest thatmeglumine antimoniate interacts with ß-cyclodextrinthrough hydrogen bonds with the hydrophilic outer surface ofthe cyclodextrin molecule, presumably at the largest rim's torus,where OH-3 (hydroxyls at the 3 position) are located.

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TABLE 1. Spin lattice relaxation time T₁-¹H measurements for meglumine antimoniate, ß-cyclodextrin, and their association compound

Oral absorption of antimony from meglumine antimoniate and its complex with ß-cyclodextrin. Figure 1 displays the levels of antimony in plasma when meglumineantimoniate or its complex with ß-cyclodextrin wereorally administered to mice at 100 mg of Sb/kg. The antimonyconcentrations were found to be about three times higher forthe association compound than for meglumine antimoniate. Thesedata clearly established that the association of meglumine antimoniatewith ß-cyclodextrin strongly improved the bioavailabilityof antimony by the oral route.

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FIG. 1. Levels of antimony in plasma following oral administration of meglumine antimoniate (MA) or its complex with ß-cyclodextrin (MA/CD) in Swiss mice at 100 mg of Sb/kg. The results are expressed as means ± standard deviations (error bars) (n = 3).

Efficacy by oral route of meglumine antimoniate and its complex with ß-cyclodextrin against experimental cutaneous leishmaniasis. Figure 2 shows the course of infection in BALB/c mice upon treatmentwith meglumine antimoniate or its complex with ß-cyclodextrin.As expected, the i.p. treatment with meglumine antimoniate effectivelycontrolled lesion growth, whereas the oral treatment was ineffective,despite the double amount of antimony given orally. In agreementwith the toxicological data obtained for ß-cyclodextrin(6) showing that it is not absorbed across the gastrointestinaltract, no significant effect on the course of cutaneous leishmaniasiswas found using ß-cyclodextrin alone (data not shown).On the other hand, complexation of meglumine antimoniate withß-cyclodextrin rendered it highly effective by theoral route. The effectiveness of the complex by the oral routewas equivalent to that of meglumine antimoniate given parenterallyat a twofold-higher antimony dose.

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FIG. 2. Lesion growth in mice treated with meglumine antimoniate (MA) or its complex with ß-cyclodextrin (MA/CD). The mice were infected in the ear with L. amazonensis-GFP and then treated daily with meglumine antimoniate-ß-cyclodextrin compound (32 mg of Sb/kg) or meglumine antimoniate (120 mg of Sb/kg) on days 10 to 16 and 31 to 36 of infection. The controls received saline or meglumine antimoniate (60 mg of Sb/kg) by the i.p. route. The ear thickness was measured on the indicated days and expressed as means ± standard deviations (n = 5).

The effectiveness of the association compound by the oral routeagainst cutaneous leishmaniasis was further evaluated by measuringthe parasite load in the lesions on day 80 of infection, asexpressed by the fluorescence in the ear tissues infected withL. amazonensis-GFP. The results displayed in Fig. 3 show thatthe complex, given orally as daily doses of 32 mg of Sb/kg,significantly reduced the number of parasites in the lesionscompared to saline (P < 0.001). The parasite load was similarto that achieved with meglumine antimoniate given i.p. at atwofold-higher antimony dose (P > 0.05), confirming the greateffectiveness of the association compound in killing the parasiteswhen administered by the oral route. Although oral meglumineantimoniate partially reduced the parasite burden compared tosaline controls (P < 0.001), the burden in animals treatedwith the complex was significantly lower than that after treatmentwith meglumine antimoniate (P < 0.001). These data clearlyestablish that the association of meglumine antimoniate withß-cyclodextrin renders the antimonial drug orallyactive, most probably through enhancement of its oral absorption.

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FIG. 3. Parasite loads in mice treated with meglumine antimoniate (MA) or its complex with ß-cyclodextrin (MA/CD). The mice were infected and treated as described in the legend to Fig. 2. On day 80 of infection, the fluorescence in the ear homogenate was measured and expressed as the mean plus standard deviation (n = 5).

DISCUSSION

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Discussion

In the present study, we showed that ß-cyclodextrinforms a complex with meglumine antimoniate and that this associationenhanced the oral absorption of antimony in mice. The resultingformulation was found to be effective by the oral route in amouse model of cutaneous leishmaniasis.

Cyclodextrins are well known in recognition chemistry as molecularhosts capable of including, with a degree of selectivity, water-insolubleguest molecules via noncovalent interactions within their hydrophobiccavities. Taking into account that meglumine antimoniate ishighly soluble in water and most probably interacts with theexternal surface of ß-cyclodextrin, the associationcompound obtained in the present study differs physicochemicallyfrom conventional inclusion compounds. The higher oral absorptionof antimony observed with the association compound stronglysuggests enhanced permeability of antimony across the intestinalbarrier. This interpretation is consistent with previous findingsthat ß-cyclodextrin increases the percutaneous absorptionof antimony across isolated mouse skin (R. Ochoa, L. A. Ferreira,F. Frézard, J. B. da Silva, R. D. Sinisterra, and C.Demicheli, Proc. 30th Annu. Meet. Exposition Control. ReleaseSoc., abstr. 864, 2003). In that study, a twofold increase inthe flux of antimony across the skin was observed when meglumineantimoniate was applied onto the skin in the form of the associationcompound with ß-cyclodextrin. Moreover, the partitionof antimony into the skin was also found to be enhanced significantlywith the association compound. Strikingly, the increase in antimonyflux promoted by the association of meglumine antimoniate withß-cyclodextrin was even higher ( $~$ 8-fold) when the skinwas used without the stratum corneum. These data are thereforeconsistent with the model in which ß-cyclodextrinincreases the lipophilicity and permeability of antimony acrossbiological membranes, presumably through its free internal cavity.This hypothesis is further supported by the observation of Uekamaet al. (14) that free ß-cyclodextrin can remove componentsof the membrane surface, thereby modifying the transport propertiesof the membranes and facilitating drug absorption, especiallyfor water-soluble drugs.

The fact that pentavalent antimonials have to be given parenterallyrepresents one of the most serious limitations of the presentchemotherapy for leishmaniasis. The recent introduction of theorally active drug miltefosine (13) illustrates well the greateffort devoted to the search for orally active antileishmanialagents. However, this drug showed some adverse side effects,and its use will probably remain limited to visceral leishmaniasis.Allopurinol is another example of an orally active antileishmanialcompound, but its efficacy is quite low in comparison to conventionalantimonial therapy (2). In this context, the demonstration ofthe oral efficacy of meglumine antimoniate when given in associationwith ß-cyclodextrin opens promising new perspectivesfor the treatment of leishmaniases. The importance of theseresults is reinforced by the fact that oral efficacy was achievedwith a low dose of antimony.

ACKNOWLEDGMENTS

This work was supported by grants from the Brazilian agenciesCNPq (521010/97-7 and Brazilian Nanobiotechnology Network),CAPES, and FAPEMIG.

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Ave. Antônio Carlos 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brazil. Phone: 55-31-34992940. Fax: 55-31-34992924. E-mail: frezard{at}mono.icb.ufmg.br.

REFERENCES

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