Nano-elastic liposomes as multidrug carrier of sodium stibogluconate and ketoconazole: A potential new approach for the topical treatment of cutaneous Leishmaniasis.

The present study evaluates the efficacy of sodium stibogluconate (SSG) co-loaded with ketoconazole (KTZ) in nano-elastic liposomes (NELs) for the topical treatment of cutaneous leishmaniasis (CL). SSG-KTZ co-loaded NELs were developed and assessed for various physicochemical properties and anti-leishmanial potential. The optimized nano-vesicles have an average size of 212.8 ± 3.1 nm and entrapment efficiency of 61.2 ± 2.9%. SSG-KTZ co-loaded NELs displayed 5.37-fold higher skin permeation of SSG as compared to drug solution. SSG and KTZ displayed a synergistic interaction and flow cytometry revealed enhanced killing of DsRed Leishmania mexicana in infected macrophages. In-vitro and in-vivo anti-leishmanial studies indicated a 10.67-fold lower IC50 value and a 35.33-fold reduced parasitic burden as compared with plain SSG solution, respectively. SSG-KTZ co-loaded NELs were found to be a promising approach for the topical treatment of CL.

Sodium stibogluconate loaded nano-deformable liposomes for topical treatment of leishmaniasis: macrophage as a target cell.

Topical drug delivery against cutaneous leishmaniasis (CL) signifies an effective alternate for improving the availability and reducing the toxicity associated with the parenteral administration of conventional sodium stibogluconate (SSG) injection. The basic aim of the study was to develop nano-deformable liposomes (NDLs) for the dermal delivery of SSG against CL. NDLs were formulated by a modified thin film hydration method and optimized via Box-Behnken statistical design. The physicochemical properties of SSG-NDLs were established in terms of vesicle size (195.1 nm), polydispersity index (0.158), zeta potential (-32.8 mV), and entrapment efficiency (35.26%). Moreover, deformability index, in vitro release, and macrophage uptake studies were also accomplished. SSG-NDLs were entrapped within Carbopol gel network for the ease of skin application. The ex vivo skin permeation study revealed that SSG-NDLs gel provided 10-fold higher skin retention towards the deeper skin layers, attained without use of classical permeation enhancers. Moreover, in vivo skin irritation and histopathological studies verified safety of the topically applied formulation. Interestingly, the cytotoxic potential of SSG-NDLs (1.3 mg/ml) was higher than plain SSG (1.65 mg/ml). The anti-leishmanial activity on intramacrophage amastigote model of Leishmania tropica showed that IC50 value of the SSG-NDLs was ∼ fourfold lower than the plain drug solution with marked increase in the selectivity index. The in vivo results displayed higher anti-leishmanial activity by efficiently healing lesion and successfully reducing parasite burden. Concisely, the outcomes indicated that the targeted delivery of SSG could be accomplished by using topically applied NDLs for the effective treatment of CL.

Reduction of Sb(V) in a human macrophage cell line measured by HPLC-ICP-MS.

Drugs based on pentavalent antimony are first-line treatment of the parasite disease leishmaniasis. It is generally believed that Sb(V) acts as a prodrug, which is activated by reduction to Sb(III); however, the site of reduction is not known. It has been hypothesised that the reduction takes place in the parasites' host cells, the macrophages. In this study, the human macrophage cell line Mono Mac 6 was exposed to Sb(V) in form of the drug sodium stibogluconate (Pentostam™). Cell extracts were analysed for Sb species by high-performance liquid chromatography with inductively coupled plasma-mass spectrometry detection. We found that Sb(V) is actually reduced to Sb(III) in the macrophages; up to 23% of the intracellular Sb was found as Sb(III). Transfer of the cells to Sb-free medium rapidly decreased their Sb(V) and Sb(III) content. Induction of the cell's production of reactive oxygen species did not have any marked effect on the intracellular amounts of Sb(III).

Leishmania donovani: effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Indian clinical isolates to sodium stibogluconate.

Although pentavalent antimonials are the first-line drug for treatment of visceral leishmaniasis all over the world, yet, in India, increasing number of patients are being reported to be unresponsive to sodium stibogluconate. Verapamil, a calcium channel blocker, affects drug uptake by preventing its efflux and thereby accumulation within the cell. In the present study, effect of verapamil on in vitro susceptibility of both promastigote and amastigote stages of 15 clinical isolates and standard strain of Leishmania donovani to sodium stibogluconate was evaluated by detection of acid phosphatase. Amastigotes were found more susceptible to sodium stibogluconate than the promastigotes (p<0.05) and in the presence of verapamil, IC(50) value of sodium stibogluconate was reduced only for those isolates, which had a higher IC(50). Verapamil alone did not have any effect on the parasites. The results indicate that amastigotes are more susceptible to sodium stibogluconate than promastigotes and verapamil can reverse the in vitro drug resistance of L. donovani clinical isolates to sodium stibogluconate.

Reduction of anti-leishmanial pentavalent antimonial drugs by a parasite-specific thiol-dependent reductase, TDR1.

The reason why Leishmania parasites are susceptible to organic antimonial drugs, the standard chemotherapeutic agents for over 50 years, apparently lies in the fact that the mammalian stage of the parasite reduces the pentavalent form of the administered drug to a trivalent form that causes parasite death. We have identified and characterized a parasite-specific enzyme that can catalyse the reduction of pentavalent antimonials and may therefore be central to the anti-parasite activity of the drug. The unusual protein, a trimer of two-domain monomers in which each domain has some similarity to the Omega class glutathione S-transferases, is a thiol-dependent reductase (designated TDR1) that converts pentavalent antimonials into trivalent antimonials using glutathione as the reductant. The higher abundance of the enzyme in the mammalian stage of the parasite could explain why this parasite form is more susceptible to the drug.

Visceral leishmaniasis in the BALB/c mouse: antimony tissue disposition and parasite suppression after the administration of free stibogluconate.

BALB/c mice with an acute or chronic Leishmania donovani infection were treated with intravenous sodium stibogluconate solution and the parasite suppressions determined in the spleen, liver and femur bone marrow. Antimony concentrations in these and other tissues were determined by hydride generation-atomic absorption spectrophotometry. There was little correlation between tissue antimony levels one hour after treatment and drug efficacy. It would appear that the peak tissue antimony concentration achieved soon after dosing, rather than the lower concentrations which are readily sustained in most tissues, is the most important factor in the antileishmanial activity of stibogluconate. A high peak antimony concentration occurred in the liver, where parasites were significantly suppressed, and was not observed in the two other sites of infection, where the parasites were apparently less susceptible to stibogluconate therapy.

Biochemistry of Pentostam resistant Leishmania.

Promastigotes of Leishmania mexicana amazonensis WR 669 clone 4 were made resistant to antimony in the form of Pentostam (sodium stibogluconate) by exposure to media containing increasing concentrations of Sb. The dose of Sb expected to kill 50% of promastigotes and amastigotes of the parent sensitive clone (WR 669) and the resistant clone (WR 669R) was determined by exposure of suspensions in physiologic salt solution for 3 hr. The approximate Ed50s in microgram Sb/ml were: 10,000 for WR 669R promastigotes; 7,000 for WR 669R amastigotes; 200 for WR 669 promastigotes; and 150 for WR 669 amastigotes. Thus, Sb resistance and Sb sensitivity expressed by promastigote clones are also expressed by their respective amastigotes. Studies with 125Sb-Pentostam showed that WR 669R amastigote resistance was not due to altered Sb uptake over 1 hr. When amastigotes pretreated with Pentostam were incubated with 14C labeled metabolic precursors, susceptibility to Sb was correlated with inhibition of glycolytic enzymes and of fatty acid beta-oxidation.

Non-ionic surfactant vesicles, niosomes, as a delivery system for the anti-leishmanial drug, sodium stibogluconate.

Liver and serum concentrations of antimony in the mouse have been determined after administration of sodium stibogluconate in the free, liposomal and niosomal form. High liver and low serum values were attained by the use of both vesicular formulations. Niosomal sodium stibogluconate was shown to be more active than free drug against experimental murine visceral leishmaniasis, an effect apparently dependent on maintaining high drug levels in the infected reticuloendothelial system.

Electrocardiographic changes during treatment of leishmaniasis with pentavalent antimony (sodium stibogluconate).

Serial electrocardiograms (ECGs) were obtained during 65 courses of sodium stibogluconate treatment in 59 Kenyan patients with leishmaniasis (56 visceral and 3 cutaneous). ECG abnormalities developed during 54% of the treatment courses. The frequency with which abnormalities occurred was related to the total daily dose of antimony (Sb), increasing from 2/9 patients treated with 10 mg Sb/kg/d to 25/48 treated with 20-30 mg Sb/kg/d and 8/8 treated with 40-60 mg Sb/kg/d. The frequency with which ECG abnormalities developed was also related to the duration of treatment, increasing from 11/65 patients after 7 days to 18/44 after 15 days, 26/39 after 30 days and 11/12 after 60 days. ECG abnormalities were similar to those previously described during treatment with trivalent antimonial drugs, the most common being flattening and/or inversion of T waves. Prolongation of the corrected QT interval occurred in 13 patients, all of whom were treated for more than 30 days or with more than 20 mg Sb/kg/d. One patient died suddenly during the fourth week of treatment with 60 mg Sb/kg/d, and 2 patients died of measles after 9 or 10 days of treatment with 30 mg Sb/kg/d. QT prolongation and a concave ST segment developed in all 3 patients who died. We conclude that minor ECG abnormalities are common when sodium stibogluconate is used at doses above 20 mg Sb/kg/d for more than 15 days, and that life-threatening arrhythmias may occur if very high doses are used.

Quantitative and ultrastructural studies on the uptake of drug loaded liposomes by mononuclear phagocytes infected with Leishmania donovani.

This study compared splenic and hepatic uptake of free and liposome-entrapped sodium antimony gluconate after i.v. administration to mice infected with Leishmania donovani. It was demonstrated that entrapment within liposomes greatly altered the kinetics of uptake of the drug. We were also able to show that liposomes composed of sphingomyelin, stearylamine and cholesterol were marginally better than any other preparation in delivering entrapped drug to liver and spleen. X-ray microanalytical studies on the uptake of liposomes by Kupffer cells infected with L. donovani have indicated that internalised liposomes probably fuse with parasitophorous vacuoles, transferring their contents into the immediate locality of the leishmanial parasites. It is proposed that this is the way in which liposome entrapped antileishmanial agents have an enhanced therapeutic effect over free drug therapy.

Renal clearance of pentavalent antimony (sodium stibogluconate).

Kenyan kala-azar is sometimes unresponsive to a standard course of sodium stibogluconate. The renal excretion of sodium stibogluconate was therefore studied in patients with kala azar and in volunteers; both urine and serum levels of sodium stibogluconate were measured. After intravenous injection sodium stibogluconate seemed to be distributed throughout the extracellular fluid and to have a renal clearance similar to that of inulin. At 6 h blood levels had fallen to less than 1% of peak values. After intramuscular injection, peak blood levels were lower and more sustained. However, more than 80% was excreted in the first 6 h, and blood levels fell to around 1% of peak values in 16 h. The dangers of cumulative toxicity may be exaggerated, and restriction of courses of sodium stibogluconate to 30 days or even to 10 days (in the U.S.A.) may not be necessary. If shorter courses are ineffective prolonged and continued courses may be given provided that renal function is assessed and the dosage is adjusted when indicated.