|Year : 2013 | Volume
| Issue : 4 | Page : 767-776
Evaluation of toxicity & therapeutic efficacy of a new liposomal formulation of amphotericin B in a mouse model
Jyotsna Mishra1, Ayan Dey2, Niti Singh3, Ramesh Somvanshi4, Sarman Singh1
1 Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India
2 Laboratory Sciences Division, International Vaccine Institute, SNU Research Park, San 4-8 Nakseongdae-dong, Kwanak-gu Seoul, Korea
3 National Reference Laboratory for Tuberculosis, LRS Institute of Tuberculosis & Respiratory Disease, New Delhi, India
4 Division of Pathology, Indian Veterinary Research Institute, Izatnagar, India
|Date of Submission||23-Dec-2011|
|Date of Web Publication||17-May-2013|
Division of Clinical Microbiology, Department of Laboratory Medicine All India Institute of Medical Sciences, New Delhi 110 029
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background & objectives: Current therapy for leishmaniasis is limited and unsatisfactory. Amphotericin B, a second-line treatment is gradually replacing antimonials, the first-line treatment and is used as the preferred treatments in some regions. Though, presently it is the only drug with highest cure rate, its use is severely restricted by its acute toxicity. In the present study novel lipid-amphotericin B formulations with lower toxicity than the parent drug were evaluated for the treatment of visceral leishmaniasis (VL) in a mouse model.
Methods: The toxicity and therapeutic efficacy of a new amphiphilic formulation of amphotericin B (Kalsome TM 10) was compared to that of amphotericin B deoxycholate (Fungizone) in a mouse model of VL using quantitative real-time PCR (qRT-PCR).
Results: The toxicity of amphotericin B was significantly less with liposomal formulation as compared to the deoxycholate form, evidenced by reduced nephrotoxicity and higher tolerated dose in BALB/c mice. The therapeutic efficacy was evaluated by quantitative real time (RT) PCR using primers highly specific for the ITS region of Leishmania donovani. There was reduction in parasite load by 2 log unit after 7 days of treatment and finally resulting in complete clearance of parasite from infected mice after 30 days of treatment with Kalsome TM 10.
Interpretation & conclusions: This new formulation showed a favourable safety profile and better efficacy when compared to conventional amphotericin B. If production cost is kept low, it may prove to be a feasible alternative to conventional amphotericin B.
Keywords: Amphotericin B - efficacy - leishmania - liposomal formulations - toxicity
|How to cite this article:|
Mishra J, Dey A, Singh N, Somvanshi R, Singh S. Evaluation of toxicity & therapeutic efficacy of a new liposomal formulation of amphotericin B in a mouse model. Indian J Med Res 2013;137:767-76
|How to cite this URL:|
Mishra J, Dey A, Singh N, Somvanshi R, Singh S. Evaluation of toxicity & therapeutic efficacy of a new liposomal formulation of amphotericin B in a mouse model. Indian J Med Res [serial online] 2013 [cited 2021 May 7];137:767-76. Available from: https://www.ijmr.org.in/text.asp?2013/137/4/767/111997
Visceral leishmaniasis (VL) or kala-azar is a chronic parasitic disease and is considered one among the ten main tropical illnesses by the World Health Organization  . It is usually a fatal disease if not treated and is characterized by irregular fever, malaise, loss of weight, hepatosplenomegaly, and anaemia with or without lymphadenopathy. Unfortunately, as yet no effective vaccine is available against leishmaniasis and control of the disease relies only on chemotherapy. Presently there are about 25 compounds and formulations showing anti-leishmanial effects but only a few have been proven worthy  . Most of the drugs have one or more limitations like unaffordable cost, difficulty in administration, toxicity or more importantly the development of resistance in the parasite. Pentavalent antimony, sodium antimony gluconate (SAG) has long been the cornerstone of anti-leishmania chemotherapy for more than 60 years. However, for the last few years outbreaks of SAG resistant kala-azar in various endemic regions of India have been reported  . As a result, the erstwhile second line treatment, amphotericin B (AmB) has now moved to the forefront for the treatment of kala-azar in India.
Amphotericin B, a polyene macrolide antibiotic, remained the gold standard for the treatment of fungal infection and was strongly recommended as an alternative first-line treatment for leishmaniasis. Despite its high efficacy, its inherent nephrotoxicity significantly limits its successful chemotherapeutic use. Amphotericin B (deoxycholate) induced toxicity is explained by binding of amphotericin B to lipoproteins and internalization of amphotericin B-lipoprotein complex, which is mediated by low density lipoprotein (LDL) receptors  . Amphotericin B associated with LDL exhibits toxicity for renal cells. Thus, to increase therapeutic index of the drug and to reduce toxicity, several new lipid formulations of amphotericin B have been developed by replacing deoxycholate with other lipids in which toxicity is partially ameliorated. These formulations include liposomal amphotericin B [L-AmB (Ambisome)], amphotericin B colloidal dispersion [ABCD (Amphocil)] and amphotericin B lipid complex [ABLC (abelcet) 5] . However, all presently available liposomal amphotericin B formulations are expensive, costing `21,855 per patient (per treatment as much a US $600) for complete kala-azar treatment  . This cost is far beyond the reach of poor patients and for government organizations engaged in kala-azar control programmes. Further, these formulations use sterols, such as cholesterol, cholesterol succinate and cholesterol sulphate. Cholesterol is required for the endurance of Leishmania and the parasite meets its requirement by salvaging cholesterol from these molecules. Thus, cholesterol containing liposomes nullify the advantage of carrier molecule in targeted delivery of drug. Therefore, suitable lipid constituents must be used as a carrier molecule which should adversely affect the survival of Leishmania by depriving the parasite of its cholesterol requirement. Keeping this in mind, new ionic amphiphilic formulation of amphotericin B (Kalsome TM 10) has been developed  . It is basically, a sterol enriched with mixed lammelarity amphotericin B intercalating liposomes comprising phosphatidylcholine and ergosterol in a ratio where ergosterol constitutes up to 50 molar % of the total lipids of the liposomal formulations. If production cost is kept low, the drug is expected to reduce the cost of kala-azar treatment drastically.
The aim of the present work was to evaluate, in vivo anti-leishmanial efficacy and related side effects of Kalsome TM 10 in the BALB/c mice. Conventional methods of detection and quantitation of the parasite burden in different mouse tissues and for evaluation of anti-leishmanial efficacy of drugs remains microscopic enumeration of amastigotes against host cell nuclei  . This method is time-consuming and subjective and may not be reliable if the parasites are not equally dispersed on the slides. Therefore, culture microtitration was developed  . This technique is more sensitive than the imprint method, but is labour intensive and time consuming. Other limitations include very low number of the parasites  . Since recurrences of leishmaniasis are associated with tissue loads of residual and latent parasites after treatment, non-quantitative PCR tests are of little value in indicating a positive or negative result  . Therefore, in present study, therapeutic efficacy of a new anti-leishmanial formulation was evaluated by quantitative real time PCR (qRT-PCR) using primers from the conserved sequences of internal transcriber spacer (ITS) region of L. donovani nuclear DNA (nDNA), as a proof of evidence of its anti-leishmanial activity.
| Material & Methods|| |
The study was carried out between 2005 and 2007 at the All India Institute of Medical Sciences (AIIMS), New Delhi. Amphotericin B deoxycholate (Fungizone) was purchased from Ambalal Sarabhai Enterprises Ltd, Vadodara, India. New formulation of the liposomal amphotericin B, Kalsome TM 10, was a kind gift from Lifecare Innovations Pvt. Ltd. Gurgaon, India. For use, the commercially available AmB deoxycholate was reconstituted in sterile, chilled, triple-distilled water to obtain a stock solution of 5 mg/ml. All other reagents used were of analytical grade (Sigma-Aldrich, St Louis, USA).
Experimental animals and strain of Leishmania donovani: Experiments were conducted on BALB/c male mice (5-6 wk old, 25±30 g) purchased from Laboratory Animal Facility of National Institute of Nutrition, Hyderabad, India. The animals were maintained by giving a standard pellet diet and water ad libitum. Mice were checked daily for their mortality and waning prior to commencement of the study and only healthy mice were included. The study protocol was approved by the Institute's Animal Ethics Committee.
HM/IN/KE16/1998 strain of L. donovani isolate from an untreated child from Muzaffarpur district of Bihar (eastern India) was used for infection establishment  . The parasite culture is being maintained in Medium 199 with 25mM HEPES, pH 7.4 (Hi-Media, Mumbai) supplemented with 10 per cent heat inactivated foetal bovine serum (Sigma-Aldrich, USA) at 22° C in a Biological oxygen demand (BOD) incubator.
Electron microscopic analysis of Kalsome TM 10: To increase the plasma half-life of the liposomal drug Kalsoma TM 10 (10 mg/ml of amphotericin B) for facilitating better bio-distribution in the body bath sonication was carried out for 45 min on ice. Sonicated and non-sonicated preparations of Kalsome TM 10 were observed under the scanning electron microscopy. Analysis was carried out on LEO scanning electron microscope available at electron microscopy facility of AIIMS.
In vivo single dose drug toxicity study: Groups of six mice each were injected with various concentrations of conventional Fungizone (ranging from 1-3 mg/kg of body weight) or new liposomal amphotericin B formulation: Kalsome TM 10 (range, 15-20 mg/kg) and empty liposomes i.e. liposomes without AmB (range, 20-100 μl) as control, intravenously. The toxicity of the drug was evaluated in vivo by determining the 50 per cent lethal dose (LD 50 ). For this, the mortality rate and activity levels of animals were monitored for 24 h.
Chronic toxicity study: The chronic toxicity was performed according to the Organization for Economic Co-operation and Development (OECD) test guidelines with minor modifications, as the purpose was not regulatory clearance but to provide an evidence of its efficacy  . Healthy mice were randomly assigned into four groups: A, B, C and D (6 per group). Daily administration of the LD 50 as estimated above for Fungizone and Kalsome TM 10 through the known route was given for 1 wk to group A and B, respectively. In group C normal saline was given while in group D empty Kalsome (liposomes without AmB) was given using the same route. Animals were sacrificed on day 28 after the last dose of the drug. Blood, liver, kidney and spleen tissues were collected aseptically for histopathological evaluation. Biochemistry analysis of blood urea nitrogen (BUN), creatinine and glucose level, was done using the commercially available kits in a semi-automated chemistry analyzer (Techno-168) (I.S.E Sr.t. group, Italy s.r.l.).
Establishment of infection and therapeutic protocols: To determine, the duration required to establish the L. donovani infection, group of six BALB/c mice, were infected with L. donovani (MHOM/IN/KE16/1998) strain  . For infection, the log-phase promastigotes of the parasite were washed twice using PBS buffer (pH 7.4) at 2300 g at 4 °C. Finally, the pellets were re-suspended in PBS buffer for intravenous infusion and approximately 1×10  promastigotes/ml were injected into the tail vein using 26 gauge needle. The day of infection was termed as day 0 (D0). The animals were daily observed for gross physiological and behavioural changes, if any. These included loss of hair, other signs of being unwell such as fever, loss of weight and appetite and enlargement of spleen and liver were observed for 3 wk post-infection.
After visible splenic enlargement, and other symptoms, two mice were sacrificed on every alternate day. Blood was collected and tested for the presence of antibodies against visceralising species of Leishmania using the commercially available Lc-rK39 (recombinant antigen prepared from L. chagasi) dipstick test kits (Insure TM , In-Bios Int, USA) and Ld-rKE16 (recombinant antigen prepared from L. donovani) spot test (Signal KA TM , Span Diagnostic Ltd., Surat, India). Liver and spleen samples were taken out under sterile conditions, homogenized in mortar and pestle and were subjected to PCR analysis for confirmation of infection. For DNA isolation 300 μl of homogenized sample was taken and re-suspended in two volumes of lysis buffer [50mM NaCl, 50mM Tris-HCl, 10mM EDTA (pH 7.4), 1 per cent Triton X-100, 200 μg/ml of proteinase K]. The lysed samples were than incubated in water bath for 2 h at 60 o C. Thereafter the genomic DNA of Leishmania was extracted using phenol-chloroform extraction and finally suspended in Tris-EDTA (TE) buffer or sterile water. A 1020 bp region of genomic DNA was amplified, using the following primer set  . Forward, ITSF→5'CTGGATCATTTTCCGATG-3', and Reverse, ITSR→5'ACACTCAGGTCTGTAAAC-3'.
Since amplicon size of our product was 1000 bp (ideal 100-500 bp real time PCR), the RT-PCR was standardized with low salt concentration and adjusted reaction time. PCR was performed in 50 μl reaction volumes containing 100 ng of genomic DNA, 10 pmol each of the gene-specific forward and reverse primers, 10 μM of each dNTP, 2 mM MgCl2 and 5 units of Pfu Taq DNA polymerase (Bangalore Genei, India). The conditions for PCR were as follows: 95°C for 2 min, then 34 cycles at 95°C for 20 sec, 53°C for 30 sec and 72°C for 1 min. Final extension was carried out at 72°C for 1 min.
Leishmania infected mice were randomly assigned to three groups; groups A, B and C (6 mice per groups) and treatment was started one week after establishment of infection. Groups A and B mice were treated on each alternate day with Fungizone (2 mg/kg of body weight) and 0.75X dose of Kalsome TM (7.5 mg/kg of body weight, half of the LD 50 ), respectively. Group C including six healthy mice was used as negative control group [Figure 1]. Drugs were injected by the intravenous route in 0.1 ml volume. Three mice were sacrificed by cervical dislocation at day 7, and the remaining three at the termination of the study i.e. day 28 post-treatment.
Assessment of efficacy
Determination of parasite burden: Detection of parasitic load in liver was carried out by blinded microscopic enumeration with Giemsa-stained liver impression smear in three groups. Leishman-Donovan units (LDU) were calculated per organ for the liver by using the formula [LDU= number of amastigotes per 1,000 nucleated cells x organ weight (in g) x 2x10  , according to Stauber's formula  .
(ii) Quantitative real-time PCR for quantitative analysis of parasite load: Both liver and spleen are well accepted system for assessment of L. donovani infection, however, due to larger volume of the tissue liver was used for quantitation of parasite load. A real-time hot-start PCR was performed with the Smart Cycler DNA Master SYBR Green Kit (Cepheid, USA) in a Smart Cycler (Cepheid, USA) to determine the parasitic load in liver tissues of control group as well as in Fungizone and Kalsome -treated mice. The 25 μl reaction mixture contained 12.5 μl DNA Master SYBR Green, 0.5 μM each primer and 1 μl of template. Time and temperatures were same as used in normal PCR reaction. For fluorescence signal acquisition, channel F1 was used and standard threshold of 30 fluorescence was set. The fractional cycle number reflecting a positive PCR result is called the cycle threshold (C T).
Statistical analysis: To determine the variability of the assays, intra- and inter-assay (repeatability) precision was measured. Three replicates of eight different concentrations of L. donovani DNA were tested simultaneously in the same run. Variability is shown as the mean ± standard deviation (SD) and reported as the coefficient of variation (CV). Statistical procedures were performed using Microsoft Excel and STATA Statistics Package 9 (Stata Corp. College Station, Tx, USA). Statistical analyses were performed by using an unpaired t-test (two-tailed) followed by Mann-Whitney test using, GraphPad Prism5 version (GraphPad Software Inc., La Jolla, CA).
| Results|| |
Electron microscopic features of the new formulation: The sonicated and non-sonicated drug samples revealed typical micro sphere structure of approximately 1.0 μm in diameter. These micro spheres tend to aggregate in non-sonicated samples, while after sonication these got separated [Figure 2]A & B). This finding is crucial for the clinical use of this formulation.
|Figure 2. Amphotericin B loaded liposomal microspheres (KalsomeTM10) analyzed by scanning electron microscopy. (A) sonicated (A) nonsonicated preparation of KalsomeTM10.|
Click here to view
LD 50 evaluation: The LD 50 for standard drug Fungizone was estimated to be 2 mg/kg of body weight of the mice. The LD 50 for Kalsome TM 10 (10 mg/ml) was 15 mg/kg of body weight of the mice. Hence, Kalsome TM 10 (10 mg/ml) was markedly less toxic to mice. Study on empty Kalsome revealed that the carrier molecule used was non-lethal to the animals [Figure 3].
|Figure 3. Single dose drug toxicity study. Values are expressed as mean of three seperate experiments ± SEM.|
Click here to view
Toxicity in animal models: Treatment with new liposomal formulation was found to be associated with less nephrotoxicity compared with treatment with fungizone. The level of serum creatinine and blood urea nitrogen increased significantly in mice treated with Fungizone. Creatinine value increased >2-fold over base line values (0.7±0.04 and 0.28±0.02 mg/dl; P>0.001) and blood urea nitrogen level increased by > 2.5 fold (64.8 ± 1.04 and 22.4 ± 1.74 P<0.001). Mice treated with Kalsome showed minimal renal function impairment (creatinine value 0.40±0.04 mg/dl; and blood urea nitrogen level 24.1 ± 0.9 mg/dl) [Table 1]. Serum glucose levels were almost similar in all experimental groups. The analysis of kidney tissue revealed normal histopathology in all treatment groups except the 2 mg/kg Fungizone group, which showed focal lesions consisting of inflammatory cells. Some dilation and engorgement were seen in blood vessels. Mild hemosiderosis and a few megakaryocytes were also seen showing degenerative features. The histology assessment in the spleen and liver did not reveal any vascular changes (data not shown). Empty liposome did not impart any untoward characteristic to amphotericin B liposomes. Mice injected with new liposomal formulation showed more tolerance to the drug as shown by the high tolerated dose of AmB.
|Table 1. Changes in serum glucose, serum creatinine and blood urea nitrogen (BUN) level in BALB/c mice treated with Fungizone, KalsomeTM10, normal saline and empty kalsome|
Click here to view
Assessment of infection establishment: Serological tests (both rK39 and rKE16) were found positive for antibodies against Leishmania in all the six mice in group I on day 29. Further establishment of infection was confirmed by conventional PCR using ITS region primers of L. donovani [Figure 4].
|Figure 4. PCR detection of Leishmania infection in mice. BALB/c mice were infected with ~1×107 promastigotes/ml of L. donovani. After 3 wk post-infection liver and spleen samples were taken out under sterile conditions, DNA was isolated and subjected to PCR analysis for confirmation of infection. Mice injected with normal|
saline were served as negative control. Lane 1 - Positive control (KE-16 strain DNA), lane 2 - PCR product from liver tissue sample of saline infected mice (healthy mice), lane 3 - PCR product from liver tissue sample of Leishmania infected mice (challenged mice), lane 4 - PCR product from spleen tissue sample of Leishmania infected mice (challenged mice), lane 5 - Blank (distilled water), and lane 6 - 1 Kb molecular weight marker
Click here to view
qRT-PCR for detection of visceral leishmaniasis from mouse tissue: Quantitative real-time PCR was standardized using the same ITS region primers of L. donovani and a standard curve having C T value on X-axis and number of parasitic genomes on Y axis were plotted [Figure 5]. To determine the detection limit of assay and establish a standard curve that could be used for quantitation, serial dilutions of L. donovani DNA with final concentrations ranging from 10,000 parasites to about 1 parasite per reaction were used to a real-time PCR analysis. As little as 0.6 parasite could be detected, corresponding to approximately 600 fg, per reaction in a 20-μl reaction volume. The mean standard curve, calculated from three independent experiments, was linear over 8-log range of DNA concentrations, with a correlation coefficient of 0.756 [Figure 5]. A negative control consisting of the reaction mixture and water instead of template DNA was added in each run. A melting curve analysis of PCR products showed that the melting temperature of the DNA amplicons was ca. 88.5°C. Real-time PCR efficiency was calculated from the slope. The corresponding real-time PCR efficiency (E) of one cycle in the exponential phase was calculated according to the equation: E = 10 [-/slope] as described by Pfaffl  [Figure 5]. Investigated transcripts showed 112 per cent real-time PCR efficiency.
|Figure 5. Sensitivity of qRT-PCR. A plot of mean cycle threshold (CT) value against the logarithmic concentration of parasite DNA (10-1000 parasites per reaction).|
Click here to view
Three replicates of eight numbers of 4-fold DNA concentrations, from 10  parasites per reaction to 0.6 parasite per reaction, were assessed in a single run. Three replicates of 4-fold L. donovanith DNA dilutions were performed on different days. The inter-assay variations of C T values for the number of parasitic genome ranging from 10  to 0.6 were 2.89, 0.82, 0.84, 2.94, 2.62, 2.48, 1.26 and 1.24 per cent, respectively [Figure 5].
Efficacy of Kalsome TM 10 in experimental VL: Microscopic enumeration for the assessment of the amastigote burden one week after the completion of therapy showed that in untreated animals the amastigote burden (expressed in millions of LDU) was 200 ±18 LDU but in Fungizone treated mice it decreased to 2.3 ± 2 LDU [Table 2]. However, the decrease in parasite burden was more in Kalsome TM 10 (7.5 mg/kg)-treated group (1.4 ±.14 LDU). The amastigote suppression efficacy of Kalsome TM 10 was 98.85 per cent as compared to 99.3 per cent for Fungizone which is a standard drug. After one month, no LDU was detected in any of the treated groups resulting in complete clearance of parasite.
Results were also confirmed by qRT- PCR. Of total DNA eluted from 100 mg of tissue, ~100 ng of DNA was used for qPCR experiment. The number of Leishmania genomes found in 100 ng is further calculated in per gram of liver. The mean ± SD C T value for the control group was 12.23 ± 0.127. At day 7, the mean C T value decreased to 14.15 ± 0.22 and 14.58 ± 0.21 in the Fungizone and Kalsome treated groups, respectively. The amastigote suppression efficacy of fungizone was 98.79 per cent and that of Kalsome was 99.55 per cent in these experimental conditions. The C T value >35 was observed at day 30 for both the treated groups [Table 3]. In qRT-PCR evaluation the mice treated with Kalsome TM 10 reduced parasite burden by about 2.34 log units within one week and finally resulted in complete clearance of parasites. In all of the assays, the C T values of negative controls were always >35.
|Table 2. Efficacy of new amphiphilic formulation of amphotericin B (KalsomeTM10) against established L. donovani infection in BALB/c mice|
Click here to view
|Table 3. Results of real-time and conventional PCR assays for Leishmania DNA in mouse tissue samples|
Click here to view
| Discussion|| |
Efficient and cost effective short course treatment of leishmaniasis remains a much desired research question  . The major limitations of the existing treatment options for VL are the emergence of unresponsive strains , and cost of the treatment , . Amphotericin B is the current second line drug of choice with 97 per cent cure rate and no reported resistance , . The limitation is infusion related toxicities, especially the renal toxicity , and other adverse effects restrict its use. However, the systemic side effects are reported to be considerably reduced by the particulate forms of liposomes which are highly effective as well as require a shorter course of 3-5 days therapy and facilitate targeted delivery of liposomal amphotericin B but, the stability and cost of these formulations seriously limit their widespread use.
Choice of sterols is a critical factor in designing of liposomal formulation of a drug specifically to prevent binding of liposomal amphotericin B to mammalian host cells. The therapeutic antimicrobial action as well as toxicity of amphotericin B to the host cells result from its binding with sterols of the cell membranes, resulting in pore formation and finally leading to cytolytic action of the drug. Ergosterol in combination with certain lipids in liposomes appears to be a better choice since the drug has stronger affinity for the ergosterol or their precursor in Leishmania than to cholesterol, present in most mammalian cell membranes. Also, cholesterol is an essential requirement for the endurance of Leishmania, and cholesterol containing liposomes may serve as a source of cholesterol for Leishmania.
In the present study the toxicity as well as efficacy of the new liposomal formulation of amphotericin B were evaluated against established L. donovani infection in BALB/c mice. The study demonstrated a major reduction in the toxicity of amphotericin B upon incorporation in liposomes compared with amphotericin B deoxycholate. The evaluation of the toxicity of liposomal amphotericin B (Kalsome TM 10) in mouse model involved the study of LD 50 profile of new drug. Further, the safety study of lipid-based carrier i.e. empty Kalsome (liposome without amphotericin B) showed it to be safe for application. In vivo single dose acute toxicity study showed that amphotericin B upon encapsulation in liposomes induced less toxicity in comparison with amphotericin B deoxycholate which would allow accommodation of much higher doses of the drug for in vivo use. Mice treated with new liposomal formulations of amphotericin B showed a maximum tolerated dose that was 7-fold greater than amphotericin B deoxycholate which would be advantageous to less susceptible or drug-resistant isolates.
In vivo renal toxicity was assessed by estimating serum creatinine, blood urea nitrogen and glucose level in mice. Though the levels of serum creatinine and blood urea nitrogen were elevated in mice treated with amphotericin B deoxycholate, no renal or hepatic alterations occurred, as evidenced by normal levels of creatinine and blood urea nitrogen levels in liposomal amphotericin B treated mice.
The present study showed in vivo efficacy of this new liposomal formulation of amphotericin B using the L. donovani infected BALB/c mouse model by real time PCR. The conventional PCR and gel based visualization of amplified products has several limitations such as time, low sensitivity, short dynamic range < 2 logs, low resolution, non-automated, only size-based discrimination and non-quantitative results  . To overcome these limitations, highly sensitive and specific quantitative real time PCR method was used for anti-leishmanial efficacy. Though, 100-500 bp is optimal size for amplicons of real time PCR, but up to 1000 bp was possible with low salt concentration and adjustment in reaction time. This method was used not only to detect the L. donovani infection in the experimentally infected animals but also to quantify the parasite load accurately up to a minimum detection level of 1 parasite per reaction. Treatment of infected mice with Kalsome TM 10 revealed that it reduced parasite burdens more efficiently than amphotericin B deoxycholate. Though, potential limitation of this study included the fact that daily dose- sub acute toxicity tests and subchronic toxicological evaluations could not be done as per OECD guidelines. Hence, these results cannot be used for regulatory purposes, as the purpose of this study was only collecting the evidence of efficacy and diagnostic modalities to assess the efficacy. Single dose acute toxicity experiments and chronic toxicity experiments were performed according to the OECD test guidelines  and the results showed that the new formulation was efficacious and displayed no major toxic effects in the animals.
Conflicts of interest: Authors declare no conflicts of interests.
| Acknowledgment|| |
The authors thank Lifecare Innovations Pvt. Ltd. for providing new formulation of the Liposomal Amphotericin B. The technical assistance of Electron Microscopy Facility of All India Institute of Medical Sciences, New Delhi is gratefully acknowledged. Part of this study was presented at the HUGO's 13 th Human Genome Meeting, Hyderabad, India during September 27-30, 2008. The study was financially supported by the Government of India, Department of Science and Technology, New Delhi (Drugs and Pharmaceuticals Research Programme) vide grant number: VII-PRDSF/16/2004-05/TT.
| References|| |
|1.||Singh S. New developments in diagnosis of leishmaniasis. Indian J Med Res 2006; 123 : 311-30. |
|2.||Mishra J, Saxena A, Singh S. Chemotherapy of leishmaniasis: past, present and future. Curr Med Chem 2007; 14 : 1153-69. |
|3.||Berman J. Visceral leishmaniasis in the New World & Africa. Indian J Med Res 2006; 123 : 289-94. |
|4.||Wasan KM, Morton RE, Rosenblum MG, Lopez-Berestein G. Decreased toxicity of liposomal amphotericin B due to association of amphotericin B with high-density lipoproteins: role of lipid transfer protein. J Pharm Sci 1994; 83 : 1006-10. |
|5.||Bodhe PV, Kotwani RN, Kirodian BG, Pathare AV, Pandey AK, Thakur CP, et al. Dose-ranging studies on liposomal amphotericin B (L-AMP-LRC-1) in the treatment of visceral leishmaniasis. Trans R Soc Trop Med Hyg 1999; 93 : 314-8. |
|6.||Thakur CP, Kumar A, Mitra DK, Roy A, Sinha AK, Ranjan A. Improving outcome of treatment of kala-azar by supplimentation of amphotericin B with physiologic saline and potassium chloride. Am J Trop Med Hyg 2010; 83 : 1040-3. |
|7.||Verma JN, Verma L, Tripathi KK. Sterol enriched mixed lammelarity amphotericin intercalating liposomes in saline and the process for their preparation. Patent Number WO/2005/120460. 13-6-2005. |
|8.||Leslie A. An eight-day method for screening compounds against Leishmania donovani in the golden hamster. J Eukaryot Microbiol 1958; 5 : 269-73. |
|9.||Buffet PA, Sulahian A, Garin YJ, Nassar N, Derouin F. Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice. Antimicrob Agents Chemother 1995; 39 : 2167-8. |
|10.||Titus RG, Marchand M, Boon T, Louis JA. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol 1985; 7 : 545-55. |
|11.||Lachaud L, Dereure J, Chabbert E, Reynes J, Mauboussin JM, Oziol E, et al. Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. J Clin Microbiol 2000; 38 : 236-40. |
|12.||Sivakumar R, Dey A, Sharma P, Singh S. Expression and characterization of a recombinant kinesin antigen from an old Indian strain (DD8) of Leishmania donovani and comparing it with a commercially available antigen from a newly isolated (KE16) strain of L. donovani. Infect Genet Evol 2008; 8 : 313-22. |
|13.||Organisation for Economic Co-Operation and Development (OECD), Guidelines for Testing of Chemicals, Repeated Dose 28-day Oral Toxicity Study in Rodents #407, Paris, France. 1995. |
|14.||El Tai NO, El FM, Mauricio I, Miles MA, Oskam L, El Safi SH, et al. Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol 2001; 97 : 35-44. |
|15.||Stauber LA. Host resistance to the Khartoum strain of Leishmania donovani. Rice Institute Pamphlet 1958; 45 : 80-96. |
|16.||Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29 : e45. |
|17.||Gradoni L, Bryceson A, Desjeux P. Treatment of Mediterranean visceral leishmaniasis. Bull World Health Organ 1995; 73 : 191-7. |
|18.||Sundar S, Murray HW. Cure of antimony-unresponsive Indian visceral leishmaniasis with amphotericin B lipid complex. J Infect Dis 1996; 173 : 762-5. |
|19.||Sundar S, More DK, Singh MK, Singh VP, Sharma S, Makharia A, et al. Failure of pentavalent antimony in visceral leishmaniasis in India: report from the center of the Indian epidemic. Clin Infect Dis 2000; 31 : 1104-7. |
|20.||Faraut-Gambarelli F, Piarroux R, Deniau M, Giusiano B, Marty P, Michel G, et al. In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral leishmaniasis. Antimicrob Agents Chemother 1997; 41 : 827-30. |
|21.||Thakur CP, Narayan S, Ranjan A. Epidemiological, clinical & pharmacological study of antimony-resistant visceral leishmaniasis in Bihar, India. Indian J Med Res 2004; 120 : 166-72. |
|22.||Davidson RN, di ML, Gradoni L, Giacchino R, Gaeta GB, Pempinello R, et al. Short-course treatment of visceral leishmaniasis with liposomal amphotericin B (AmBisome). Clin Infect Dis 1996; 22 : 938-43. |
|23.||Davidson RN, di ML, Gradoni L, Giacchino R, Russo R, Gaeta GB, et al. Liposomal amphotericin B (AmBisome) in Mediterranean visceral leishmaniasis: a multi-centre trial. Q J Med 1994; 87 : 75-81. |
|24.||Dietze R, Milan EP, Berman JD, Grogl M, Falqueto A, Feitosa TF, et al. Treatment of Brazilian kala-azar with a short course of amphocil (amphotericin B cholesterol dispersion). Clin Infect Dis 1993; 17 : 981-6. |
|25.||Dietze R, Fagundes SM, Brito EF, Milan EP, Feitosa TF, Suassuna FA, et al. Treatment of kala-azar in Brazil with Amphocil (amphotericin B cholesterol dispersion) for 5 days. Trans R Soc Trop Med Hyg 1995; 89 : 309-11. |
|26.||Meyerhoff A. U.S. Food and Drug Administration approval of AmBisome (liposomal amphotericin B) for treatment of visceral leishmaniasis. Clin Infect Dis 1999; 28 : 42-8. |
|27.||Paiva-Cavalcanti M, Regis-da-Silva CG, Gomes YM. Comparison of real-time PCR and conventional PCR for detection of Leishmania (Leishmania) infantum infection: a minireview. J Venom Anim Toxins Trop Dis 2010; 16 : 537-42. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]