Biomembrane Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700032, India
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Abstract |
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Introduction |
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Materials and methods |
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Egg phosphatidyl choline (PC), cholesterol (Chol), phosphatidic acid (PA), p-nitrophenol phosphate and 6-carboxyfluorescein were obtained from Sigma (St Louis, MO,
USA; DL-alanine from SRL (Bombay, India); -oxoglutaric acid, aniline and
citric acid from Merck (Darmstadt, Germany); eosin and haematoxylin from Loba Chemie
(Bombay, India); 2,4-dinitrophenyl hydrazine from Aldrich (Milwaukee, WI, USA; sorbitan
monolaurate (Span 20) from Fluka Chemie AG (Switzerland). Amarogentin was isolated and
purified from a methanol extract of S. chirata.
Preparation of liposomal and niosomal amarogentin
Liposomes were prepared essentially by the method of Gregoriadis and Ryman.6 For preparation of liposomal amarogentin, the lipids in the proportions PC:Chol:PA 7:4:1 were dissolved in a chloroformmethanol mixture (2:1 v/v). To the resulting solution amarogentin (500 µg in methanol) was added. The thin dry film that formed after evaporating the solvents was swelled in phosphate-buffered saline for 1 h and sonicated for 30 s. For the preparation of niosomes, the non-ionic surfactant of the span series, e.g. sorbitan monolaurate (Span 20), was used as a substitute for phospholipids.7 For niosomal amarogentin, the reagents, e.g. Span 20:Chol:PA 1.0:0.5:0.1, were dissolved as before in a chloroformmethanol mixture and amarogentin (500 µg in methanol) was added. The dry film was swelled, sonicated and centrifuged (100,000g) for 30 s to remove excess amarogentin.
The intercalation of amarogentin in liposomes and niosomes was around 33 and 24%, respectively. The stability of liposomes and niosomes, as judged from the leakage of 6-carboxyfluorescein when incubated in vitro with hamster blood plasma, was 3 and 5 h, respectively. The composition of the two vesicle types was adjusted so that the membrane microviscosity, measured with a fluorescence depolarization technique with diphenyl hexatriene as the fluorescent probe,8 ,9 ,10 ,1112 was similar.
Efficacy of amarogentin in liposomes and niosomes in a hamster experimental leishmaniasis model
Our colony of golden hamsters (Mesocricatus auratas), originally from the Haffkine Research Institute (Bombay, India), was used to maintain L. donovani isolate AG83, from an Indian kala-azar patient, by intracardial passage every 6 weeks. Amastigotes were isolated from the infected spleen by the method of Looker et al.13 with some modifications.14 Amastigote numbers were determined by use of a haemocytometer. Each animal was infected intracardially with 2 x 106 amastigotes. After 30 days the hamsters were selected for chemotherapy and were distributed in groups of four for the testing of amarogentin in free, liposomal and niosomal forms at the same equivalent concentration. The dose given to each animal was 2.5 mg/kg bodyweight. In practice, 2 mg of amarogentin intercalated in 0.5 mL liposomal or niosomal suspension was injected into each hamster subcutaneously every 3 days for a total of six doses over 15 days. Free amarogentin was also administered to one group. The animals were killed 5 days after the last injection. Parasite burden in the spleen was assessed microscopically from stained impression smears taken on slides after fixing in absolute methanol and Giemsa staining. The total number of amastigotes was determined by using Stauber's formula.9
Investigation of drug toxicity
Various factors including tissue histology, blood pathology and specific enzyme levels
related
to normal liver function were examined to determine the toxic effects of the drug delivered both
free and in liposomal and niosomal forms. Fresh blood was taken from both untreated and treated
hamsters just before they were killed and was collected in EDTA tubes. The red blood cells,
white
blood cells and haemoglobin were examined by established clinical procedures. The spleens of
the
animals were removed and processed for histological examination after staining with eosin and
haematoxylin.15 The sera of the animals undergoing drug
treatment were assayed for serum glutamate pyruvate transaminase (SGPT) and alkaline
phosphatase by published procedures.16,17 In brief, for assay of alkaline phosphatase, the serum was incubated with p-nitrophenol phosphate for 30 min at 37°C. The release of p-nitrophenol
was measured by absorbance at 410 nm. For SGPT, the serum was incubated with 10 mL
phosphate-buffered saline (pH 7.5) containing 0.5 g DL-alanine and 2 mg
-oxoglutaric acid. The mixture was further treated with aniline citrate for 20 min at
37°C followed by the incubation with 2,4-dinitrophenyl hydrazine hydrochloride solution
for
20 min. The reaction was stopped by addition of 0.02 M NaOH and the absorbance was
measured
at 520 nm.
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Results |
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The single doseresponse curve for survival of hamsters infected for 30 days with L. donovani and treated with free amarogentin (1.25 mg/kg, 2.5 mg/kg, 5.5 mg/kg and 11.0 mg/kg bodyweight) was examined. No death occurred over a period of 15 days with any dose. The optimum dose, as judged from reduction in parasite burden in the spleen (Figure 2), was 2.5 mg/kg bodyweight.
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Liver function was tested by examination of specific enzyme activity in sera of hamsters with experimental leishmaniasis. Both the SGPT and alkaline phosphatase activity increased on free-drug treatment but were close to normal levels when liposomal or niosomal amarogentin was used, indicating no apparent toxicity in either of the two vesicular forms (Table II).
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Histological examination of the spleen was made after staining with eosin and haematoxylin.15 Although some changes were noticed in comparison with the untreated controls, the cells looked healthy and no toxic effects were evident.
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Discussion |
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Acknowledgments |
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Notes |
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References |
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Received 30 June 1998; returned 21 February 1999; revised 12 April 1999; accepted 7 July 1999