Journal of Antimicrobial Chemotherapy (1999) 44, 791-794
© 1999 The British Society for Antimicrobial Chemotherapy
Evaluation of the in-vivo activity and toxicity of amarogentin, an antileishmanial agent, in both liposomal and niosomal forms
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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The antileishmanial property of amarogentin, a secoiridoid glycoside isolated from the Indian medicinal plant Swertia chirata, was examined in a hamster model of experimental leishmaniasis. The therapeutic efficacy of amarogentin was evaluated in free and two different vesicular forms, liposomes and niosomes. The amarogentin in both liposomal and niosomal forms was found to be a more active leishmanicidal agent than the free amarogentin; and the niosomal form was found to be more efficacious than the liposomal form at the same membrane microviscosity level. Toxicity studies involving blood pathology, histological staining of tissues and specific enzyme levels related to normal liver function showed no toxicity. Hence, amarogentin incorporated in liposomes or niosomes may have clinical application in the treatment of leishmaniasis.
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Introduction |
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The therapy of leishmaniasis, which is estimated to affect around 10 million people worldwide,1 poses problems because the toxicity of drugs that are often used in treatment. Clinical reports2 indicate that a large proportion of cases are becoming unresponsive to chemotherapy. In view of the present clinical scenario it is desirable that new drugs as well as new macrophage-specific delivery systems are developed. Our laboratory has reported the use of plant glycosides in liposomes as delivery systems for the purpose of targeting specific cellular sites.3,4 The interesting structural feature of the plant glycosides in general is that a hydrophilic sugar moiety is attached to a hydrophobic aglycone. The hydrophilic sugar moiety remains at the surface of liposomes, facilitating adsorption and attachment to the tissues. The novelty of amarogentin (Figure 1), a glycoside isolated from the indigenous plant Swertia chirata, is that it could be used both for targeting specific cellular sites and for chemotherapy. The antileishmanial activity of amarogentin originates from its capacity to inhibit DNA topoisomerase I,5 a vital enzyme for the survival of the parasite Leishmania donovani, the causative agent of visceral leishmaniasis. In this paper, we describe the effective use of amarogentin as an antileishmanial agent both free and incorporated in liposomes or niosomes. Non-ionic surfactant vesicles (niosomes) were used as an alternative to liposomes.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chemicals
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 chloroform–methanol 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 chloroform–methanol 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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Leishmanicidal activity of amarogentin in vivo
The single dose–response 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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The effects of chemotherapy on hamsters infected for 30 days with L. donovani are shown in Table I. Experiments were performed with the same equivalent drug concentration for free, liposomal and niosomal forms. The dose given to each animal was 2.5 mg/kg every 3 days. The sc injection of free amarogentin reduced the parasite load by 34%, whereas the intercalation of free drug in the liposomal and niosomal forms reduced the parasite load by 69 and 90%, respectively. Thus niosomal amarogentin showed better efficacy than liposomal amarogentin. Reduction of spleen parasite load with empty liposomes or niosomes was 12–14%.
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Toxicity of amarogentin
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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To check kidney function, the concentrations of urea and creatinine were examined in the sera of hamsters undergoing chemotherapy. Although the concentrations of both increased initially on infection and further on free-drug treatment, they came down to normal when amarogentin was applied in either liposomes or niosomes. The change in haemoglobin levels was not significant both before and after chemotherapy.
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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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Although the leishmanicidal properties of crude extracts of the Indian medicinal plant S. chirata have long been known,18 the mechanism of its action was not clear. Recently the active component, amarogentin, a secoiridoid glycoside isolated from a methanol extract of S. chirata, has been reported5 to inhibit the activity of DNA-topoisomerase I of L. donovani. The compound exerts its inhibitory effect by binding to the enzyme and thus preventing binary complex formation between topoisomerase I and DNA, resulting in the killing of the parasites. This potent inhibitory effect may provide a lead for the design of more-effective drugs against leishmaniasis. In our studies with experimental leishmaniasis in hamsters we observed 90% parasite killing with only six doses. However, at the same membrane fluidity and at an equivalent drug concentration, the niosomal form was found to be more effective than the liposomal form. At comparable toxicity levels, amarogentin was found to be very active in either of the two vesicular forms. Hence, amarogentin could possibly have clinical application in the treatment of leishmaniasis.
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Acknowledgments |
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We thank the Department of Science and Technology (Grant DST[SP/SO/D-24/93]), and the Department of Biotechnology (Grant [MKB/BT/Pro 493/Med/09/096]), New Delhi, for financial assistance, and Professor B. R. Maity of Calcutta University for histological studies.
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Notes |
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* Corresponding author. Tel: +91-33-4736797/4733493; Fax: +91-33-4735197/4730284; E-mail: iichbio{at}giasc101.vsn1.net.in
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References |
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1 . Chance, M. L. (1981). The six diseases of WHO: leishmaniasis. British Medical Journal, 283, 1245–7.
2 . Antimonials: large scale failure in leishmaniasis ‘alarming’. In TDR News, No. 34, UNDP/World Bank/WHO, Special Programme for Research and Training in Tropical Diseases, UNDP–World Bank–WHO, Geneva, 1990.
3 . Das, N., Bachhawat, B. K., Mahato, S. B.& Basu, M. K. (1987). Plant glycosides in a liposomal drug delivery system. Biochemical Journal 247, 359–61.[Medline]
4 . Medda, S., Mukherjee, S., Das, N., Naskar, K., Mahato, S. B. & Basu, M. K. (1993). Sugar-coated liposomes: a novel delivery system for increased drug efficacy and reduced drug toxicity. Biotechnology and Applied Biochemistry 17, 37–47.
5 . Ray, S., Majumder, H. K., Chakraborty, A. K., Mukhopadhyay, S., Gil, R. R. & Cordell, G. A. (1996). Amarogentin, a naturally occurring secoiridoid glycoside, and a newly recognised inhibitor of topoisomerase I from Leishmania donovani. Journal of Natural Products 59, 27–9.[Medline]
6 . Gregoriadis, G. & Ryman, B. E. (1972). Lysosomal localization of fructofuranosidase containing liposomes injected into rats. Biochemical Journal 129, 123–33.[Web of Science][Medline]
7 . Yoshioka, T., Sternberg, B. & Florence, A. T. (1994). Preparation and properties of vesicles (niosomes) of sorbitan monoesters (span 20, 40, 60 and 80) and a sorbitan triester (span 85). International Journal of Pharmaceutics 105, 1–6.
8
.
Shinitzky, M. & Berhenholz, Y. (1974). Dynamics of the hydrocarbon layer in liposomes of lecithin and sphingomyelin
containing
dicetylphosphate. Journal of Biological Chemistry 249, 2652–7.
9 . Mukherjee, S., Ghosh, C. & Basu, M. K. (1988). Leishmania donovani. Role of microviscosity of macrophage membrane in the process of parasite attachment and internalization. Experimental Parasitology 66, 18–26.[Medline]
10 . Dutta, M., Bandyopadhyay, R., Ghosh, C. & Basu, M. K. (1990). Fluidity-dependent Mg2+-ATPase activity in membrane from L. donovani promastigotes. Biochemical Journal265 , 923–6.[Medline]
11 . Basu, M. K., Flamm, M., Schachter, D., Bertles, J. F. & Maniatis, A. (1980). Effect of modulating erythrocyte membrane cholesterol on Rh0(D) antigen expression. Biochemical and Biophysical Research Communications 95, 887–93.[Medline]
12 . Schachter, D. & Shinitzky, M. (1977). Fluorescence polarization studies of rat intestinal microvillus membranes. Journal of Clinical Investigation 95, 536–48.
13 . Looker, D. L., Bercus, R. L. & Marr, J. J. (1983). Purine metabolism in L. donovani amastigotes and promastigotes. Molecular and Biochemical Parasitology 9, 15–28.[Medline]
14 . Das, N., Mahato, S. B., Naskar, K., Ghosh, D. K. & Basu, M. K. (1990). Targeting of urea stibamine encapsulated in liposomes to reticuloendothelial system for the treatment of experimental leishmaniasis. Biochemical Medicine and Metabolic Biology 43, 133–9.[Medline]
15 . Gurr, E. (1962). Staining: Animal Tissues, Practical and Theoretical. Leonard Hill, London.
16 . Reitman, S. & Frankel, S. (1957). A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminase. American Journal of Clinical Pathology 28, 56–63.[Web of Science][Medline]
17 . Kind, P. R. N. & King, E. J. (1954). Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-antipyrine. Journal of Clinical Pathology 7, 322–6.
18 . Singha, U. K., Guru, P. Y., Sen, A. B. & Tandon, T. S. (1992). Antileishmanial activity of traditional plants against Leishmania donovani in golden hamsters. International Journal of Pharmacology 30, 289–95.
Received 30 June 1998; returned 21 February 1999; revised 12 April 1999; accepted 7 July 1999
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