Improved therapy of experimental leishmaniasis by use of a liposome-encapsulated antimonial drug

The anti-leishmanial antimonial drug meglumine antimoniate (Glucantime ®) was incorporated into liposomes, and was tested for effects against experimental leishmanial infection in hamsters. When the hamsters had been infected for a short period before treatment (3 days), treatment doses of 416 mg/kg of free meglumine antimoniate, or 4 mg/kg of liposome-encapsulated drug, each gave 99.8% suppression of parasites. After 10 or 17 days of infection prior to treatment liposome-encapsulated compound was more than 300 times as effective as the antimonial drug alone. Use of liposomes containing appropriate drugs is proposed as a markedly superior means to treat certain chronic intracellular parasitic infections.     

Liposomes as drug carriers in leishmaniasis and malaria

Experimental studies suggest that liposomes could substantially improve the performance of anti-leishmanial drugs in the chemotherapy of visceral leishmaniasis. In this article, Carl Alving discusses the potential for overcoming resistance to antimonial drugs; for 'buffering' the toxicity of drugs, and for drug delivery under conditions where hospitalization is impossible or inconvenient. Liposomes can also be used experimentally to reduce the toxicity and increase the efficacy of parenterally-administered primaquine in the treatment of sporozoite-induced murine malaria.     

Oral miltefosine to treat leishmaniasis

Reduced efficacy, difficulties of administration and increasing frequency and severity of adverse events of pentavalent antimony have stimulated the quest for new anti-leishmanial drugs. Several clinical studies in Latin America testing injectable, oral and topical anti-leishmanial drugs have yielded inconsistent results. Since 1998 Indian researchers have conducted clinical trials evaluating hexadecylphosphocoline (miltefosine) in patients with visceral leishmaniasis and in 1999 clinical studies were initiated in Colombia in patients with cutaneous leishmaniasis. Up to date, more than 2,500 patients have been treated with miltefosine in India (visceral by L. donovani) and Colombia (cutaneous caused by L. panamensis) obtaining cure rates over 91% when a dose of 2.5 mg/kg/day during 28 days was used, with no difference between na?ve and relapsing patients. In Guatemala the overall cure rate for patients with cutaneous leishmaniasis was 53% (33% for L. braziliensis; 60% for L. mexicana) while in Afghanistan the cure rate of patients with L. tropica was 63%. Patients with diffuse cutaneous leishmaniasis, mucosal disease and co-infected with HIV have been treated with initial success; however these diseases have frequent relapses. Mild gastrointestinal events (i.e. nausea, vomiting and diarrhoea) were present in 35 to 60% of patients included in clinical trials and 10 to 20% had a mild increase in transaminases and creatinine levels. Miltefosine, originally an antineoplastic drug, has a potent leishmanicidal activity as consequence of its interference in parasite metabolic pathways and the induction of apoptosis, has demonstrated efficacy against L. donovani visceral disease and L. panamensis cutaneous disease. Now, miltefosine must demonstrate its efficacy against other species associated with diverse clinical presentations.     

Decrease of intestinal P-glycoprotein activity by 2n-propylquinoline,a new oral treatment for visceral leishmaniasis

Drugs currently available for visceral leishmaniasis treatment are potentially toxic, have to be administered by parenteral route and frequently give rise to drug resistance, due to the involvement of P-glycoproteins (P-gp) in Leishmania. The purpose of this study was to investigate a possible inhibitory effect of 2n-propylquinoline (2nPQ) on P-gp activity. 2nPQ is a new oral anti-leishmanial drug that has demonstrated its efficacy in BALB/c infected mice with Leishmania donovani [Antimicrob. Agents Chemother. 37 (1993) 859]. Rat everted gut sacs and human intestinal Caco-2 cell lines were used to study the effect of 2nPQ on P-gp activity. Our results demonstrate an inhibitory effect of 2nPQ on the P-gp activity with two P-gp substrates (rhodamine 123 and digoxin), two P-gp inhibitors (cyclosporin A and verapamil), and in two different species. Alone or associated with other active drugs, 2nPQ would be very useful to control Leishmania Multi-Drug-Resistance and intestinal P-gp in humans with kala-azar.     

A single dose of liposome-encapsulated oxymorphone or morphine provides long-term analgesia in an animal model of neuropathic pain

An extended-release formulation of oxymorphone was produced by encapsulation into liposomes, using a novel technique. Liposome-encapsulated morphine was produced, using a standard technique These preparations were tested in an animal model of neuropathic pain. Male Sprague-Dawley rats (approx. 300 g) were allotted to control (non-loaded liposomes) and treatment (liposome-encapsulated oxymorphone or morphine) groups. Drugs were administered subcutaneously to all rats immediately prior to sciatic nerve ligation. Thermal withdrawal latencies were measured at baseline and daily for seven days after sciatic nerve ligation. A second experiment involved subcutaneous administration of non-loaded liposomes, morphine, or oxymorphone to rats that did not undergo sciatic nerve ligation. Thermal withdrawal latencies in sciatic nerve-ligated rats given non-loaded liposomes decreased significantly by day four, with maximal decrease at day seven after surgery, indicating development of full hyperalgesia. In contrast, ligated rats given liposome-encapsulated morphine or liposome-encapsulated oxymorphone had no decrease in thermal withdrawal latency by day four, indicating that these long-acting preparations prevented development of hyperalgesia after a single injection. This treatment effect persisted to day seven. Non-ligated rats treated with vehicle or liposome-encapsulated morphine had no change in thermal withdrawal latencies. Non-ligated rats treated with liposome-encapsulated oxymorphone had a small, but significant increase in thermal withdrawal latency from day four through day seven. One subcutaneous injection of liposome-encapsulated oxymorphone or morphine was effective in preventing hyperalgesia in this pain model for up to seven days. These results suggest that liposome-encapsulation of oxymorphone offers a novel, convenient, and effective means to provide long-term analgesia.     

Vaccination against murine cutaneous leishmaniasis by using Leishmania major antigen/liposomes. Optimization and assessment of the requirement for intravenous immunization

Efficacy of vaccination against cutaneous leishmaniasis in highly susceptible BALB/c mice was assessed comparatively by using radiation-attenuated promastigotes and colloidal Ag mixtures generated from a mixed Leishmania major (LV39) isolate (SLV39) and from a virulent cloned line (SVJ2) derived from the Jericho 2 L. major isolate. Dehydration-rehydration vesicle (DRV) liposomes were used as adjuvants. In optimization experiments phospholipid composition of DRV was varied, and the distearoyl derivative (DSPC) (liquid-crystalline phase transition temperature (Tc) 54 degrees C) of egg lecithin L-alpha-phosphatidylcholine was found to be superior to the dipalmitoyl derivative (DPPC, Tc 41.5 degrees C) and underivatized L-alpha-phosphatidylcholine (Tc -10 degrees C). The criteria studied were in vivo priming for a secondary in vitro proliferative response and the prepatency of lesion onset after L. major challenge of mice immunized once i.v. A single s.c. immunization with SLV39 either free or entrapped within DSPC liposomes primed spleen cells to produce significant levels of IL-3 when stimulated with SLV39 in vitro. In contrast, the i.v. route of immunization with the same Ag preparations led to little or no IL-3 production by the spleen cells. Despite development of significant T cell activation, both SLV39 and SVJ2 administered s.c., either free or entrapped within liposomes, were not protective. However, i.v. immunization four times with SVJ2 entrapped within DSPC liposomes induced a level of resistance comparable with that of 2 x 10(7) gamma-irradiated promastigotes in the stringent BALB/c L. major model. Although significant, protection conferred after i.v. immunization with SLV39/DSPC liposomes was less effective. These data therefore show that DSPC/DRV liposomes, although a good adjuvant for inducing protective immunity to cutaneous leishmaniasis, are not able to overcome the requirement for an i.v. route of immunization with the leishmanial Ag preparation. Additionally, they demonstrate a correlation between IL-3 secretion and non-protection. They also suggest that SVJ2 represents a better source of protective Ag than SLV39.     

4′,7-dihydroxyflavone conjugated carbon nanotube formulation demonstrates improved efficacy against Leishmania parasite

One of the global problems of rising concern is the spread of the neglected tropical disease, leishmaniasis. There are several drugs used for the treatment of the disease but the repertoire of drugs has drawbacks like toxicity and low therapeutic value. Considering the need for new drugs, we studied the synthesis of 4′,7-dihydroxyflavone conjugated multi-walled carbon nanotubes (47DHF-MWCNTs) and evaluated their anti-leishmanial activity against Leishmania donovani. The compound 47DHF was conjugated to the acid oxidized MWCTNs by Steglich esterification. The synthesized 47DHF-MWCNTs were characterized by UV spectroscopy, and, from the zeta value of 35 mV, they were found to be stable. 47DHF-MWCNTs possessed 84% drug loading efficiency and 63% cumulative drug release at intra-macrophage pH of 5.8. Moreover, the evaluation of 47DHF-MWCNTs for activity showed an IC₅₀ value of 0.051 ± 0.01 μg/ml and 0.072 ± 0.01 μg/ml against the promastigote and amastigote form, respectively. 47DHF-MWCNTs exhibited an infectivity index of 42 and selectivity index of 95, suggesting the activity of 47DHF-MWCNTs against intracellular amastigotes in the study. The 47DHF-MWCNTs also had low cytotoxicity towards macrophage cells. Fascinatingly, the 47DHF-MWCNTs treatment causes a high accumulation of ROS in the promastigotes suggesting the mechanism of anti-leishmanial activity to be ROS mediated. Summarizing from our results, we propose for the first time a novel 47DHF conjugated MWCNTs capable of anti-leishmanial activity with lower cytotoxicity that has a huge potential to be a formulation against leishmaniasis.     

Liposome-mediated therapy of human immunodeficiency virus type-1 and mycobacterium infections

Abstract

We review our recent work on the use of liposomes for the delivery of antiviral agents to human immunodeficiency virus type-1 (HIV-1) infected cells, and antimycobactcrial drugs to cells harboring Mycobacterium avium complex or Mycobacterium tuberculosis. Soluble CD4 has been used to target liposomes to HIV-1-infected cells. Antisense oligodeoxynucleotides have been effectively delivered into HIV-1-infected macrophages using pH-sensitive liposomes. pH-sensitive liposomes with serum stability are being developed as in vivo delivery vehicles. Liposomes encapsulating an HIV-1 protease inhibitor were more effective in inhibiting virus production in infected macrophages than the free drug. Anionic liposomes were found to inhibit HIV-1 infectivity, while cationic liposomes had a differential toxicity for HIV-1-infected macrophages. Lipophilic sulfated cyclodextrins have been synthesized as novel antiviral agents. Liposome-encapsulated ciprofloxacin treatment reduced the number of viable M. avium in macrophages more than the free antibiotic. Liposome-encapsulated paromomycin and sparfloxacin were effective against M. tuberculosis inside macrophages, including multi-drug-resistant strains. Streptomycin encapsulated in liposomes and delivered intravenously or subcutaneously reduced the number of viable M. tuberculosis in infected mice and prevented mortality.     

Water-soluble polymer–drug conjugates for combination chemotherapy against visceral leishmaniasis

There is a need for new safe, effective and short-course treatments for leishmaniasis; one strategy is to use combination chemotherapy. Polymer–drug conjugates have shown promise for the delivery of anti-leishmanial agents such as amphotericin B. In this paper, we report on the preparation and biological evaluation of polymer–drug conjugates of N-(2-hydroxypropyl)methacrylamide (HPMA), amphotericin B and alendronic acid. The combinatorial polymer–drug conjugates were effective anti-leishmanial agents in vitro and in vivo, but offered no advantage over the single poly(HPMA)–amphotericin B conjugates.     

Preparation,Biodistribution and Neurotoxicity of Liposomal Cisplatin following Convection Enhanced Delivery in Normal and F98 Glioma Bearing Rats

The purpose of this study was to evaluate two novel liposomal formulations of cisplatin as potential therapeutic agents for treatment of the F98 rat glioma. The first was a commercially produced agent, Lipoplatin™, which currently is in a Phase III clinical trial for treatment of non-small cell lung cancer (NSCLC). The second, produced in our laboratory, was based on the ability of cisplatin to form coordination complexes with lipid cholesteryl hemisuccinate (CHEMS). The in vitro tumoricidal activity of the former previously has been described in detail by other investigators. The CHEMS liposomal formulation had a Pt loading efficiency of 25% and showed more potent in vitro cytotoxicity against F98 glioma cells than free cisplatin at 24 h. In vivo CHEMS liposomes showed high retention at 24 h after intracerebral (i.c.) convection enhanced delivery (CED) to F98 glioma bearing rats. Neurotoxicologic studies were carried out in non-tumor bearing Fischer rats following i.c. CED of Lipoplatin™ or CHEMS liposomes or their “hollow” counterparts. Unexpectedly, Lipoplatin™ was highly neurotoxic when given i.c. by CED and resulted in death immediately following or within a few days after administration. Similarly “hollow” Lipoplatin™ liposomes showed similar neurotoxicity indicating that this was due to the liposomes themselves rather than the cisplatin. This was particularly surprising since Lipoplatin™ has been well tolerated when administered intravenously. In contrast, CHEMS liposomes and their “hollow” counterparts were clinically well tolerated. However, a variety of dose dependent neuropathologic changes from none to severe were seen at either 10 or 14 d following their administration. These findings suggest that further refinements in the design and formulation of cisplatin containing liposomes will be required before they can be administered i.c. by CED for the treatment of brain tumors and that a formulation that may be safe when given systemically may be highly neurotoxic when administered directly into the brain.     

Evaluation of target-specific natural compounds for drug discovery against Leishmaniasis

Leishmaniasis is a parasitic disease with no effective vaccine still now. Globally, it has affected millions of people, precisely in the undeveloped and developing countries. The control strategy for leishmaniasis depends only on chemotherapeutic methods that are associated with several side effects. Therefore, to overcome these negative impacts natural products are the best alternative for developing effective herbal-based drugs, which can act as one of the safest and effective alternative options to treat this particular disease. Leishmania, the causative agent of this disease possesses unique enzymes and metabolic pathways that are different from its mammalian host. Moreover, these unique enzymes, along with the signaling molecules and metabolic pathways that are crucial for its survival, serve as a suitable drug target for the evaluation of specific natural inhibitors to overcome leishmaniasis. Hence, in this review, we have discussed various specific targets of Leishmania, along with their natural inhibitors which can play a significant role in anti-leishmanial drug discovery.     

Sensitivity of Leishmania braziliensis promastigotes to meglumine antimoniate (glucantime) is higher than that of other Leishmania species and correlates with response to therapy in American tegumentary leishmaniasis

The first line drugs for the treatment of leishmaniasis are antimonial derivatives. Poor clinical response may be credited to factors linked to the host, the drug, or the parasite. We determined the sensitivity of Leishmania sp. promastigotes and amastigotes by counting parasites exposed to increasing concentrations of meglumine antimoniate (Glucantime). Leishmania braziliensis promastigotes were significantly more sensitive than those belonging to other species. The sensitivity of L. braziliensis isolates from patients with unfavorable clinical outcome, such as therapeutic failure or relapse, was significantly lower than those from patients who had clinical cure. Poor clinical response to therapy (therapeutic failure or relapse) was also associated with inadequate antimonial therapy. We also found a significant and positive correlation between promastigotes and intracellular amastigotes with regard to their in vitro susceptibilities to meglumine antimoniate. Our data provide evidence for an association between the sensitivity of promastigotes to antimonials in vitro and clinical response to therapy in American tegumentary leishmaniasis. The high sensitivity of the local L. braziliensis to meglumine antimoniate in vitro provides an explanation for the good clinical response of cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil, even when low-dose regimens are employed.     

Liposome-encapsulated oxymorphone hydrochloride provides prolonged relief of postsurgical visceral pain in rats

Adequate pain control is necessary for optimal postsurgical recovery and humane treatment of laboratory and companion animals. Opioid drugs are currently the most potent analgesic agents available in human and veterinary medicine. Long-acting formulations of opioid drugs confer several important advantages over standard pharmaceutical preparations, especially for use in animals. A long-acting formulation of oxymorphone hydrochloride was produced by encapsulation into liposomes. Liposome-encapsulated (LE) oxymorphone was tested in a rat model of visceral postoperative pain. Rats were given one subcutaneous injection of LE oxymorphone (1.2 or 1.6 mg/kg of body weight) or standard oxymorphone (0.3 mg/kg) at the time of intestinal transection or resection. A single administration of LE oxymorphone hydrochloride was as effective for relief of postoperative pain in rats (P = 0.18), as were multiple (q4 h or q8 h) injections of 0.3 mg/kg of the standard pharmaceutical preparation. The rats given LE oxymorphone prior to intestinal resection also had significantly higher body weight at three and seven days after surgery than did rats that were given standard oxymorphone. In conclusion, LE oxymorphone was effective in treating visceral pain associated with intestinal surgery in rats. On the basis of body weight gain, rats treated with LE oxymorphone had improved recovery outcome, compared with rats treated with repeated injections of standard oxymorphone.     

Drug Targeting to Macrophages With Solid Lipid Nanoparticles Harboring Paromomycin: an <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation Against <Emphasis Type="Italic">L. major</Emphasis> and <Emphasis Type="Italic">L. tropica</Emphasis>

Leishmaniasis is a worldwide disease that leads to high mortality and morbidity in human populations. Today, leishmaniasis is managed via drug therapy. The drugs that are already in clinical use are limited to a number of toxic chemical compounds and their parasite drug resistance is increasing. It is therefore essential, in order to circumvent the current difficulties, to design a new anti-leishmanial drug treatment strategy. Besides producing new, active anti-leishmanial entities, another promising strategy could be developing novel delivery systems and formulations of the existing pharmaceutical ingredients to improve drug efficacy. In the present study, paromomycin sulfate (PM), as one of the promising anti-leishmanial drugs, was formulated in solid lipid nanoparticles (SLN), and its in vitro efficacy was investigated against different strains of Leishmania using a MTT test, Parasite-Rescue-Transformation-Assay, SYTO Green staining, and fluorescent microscope imaging. The results show that PM-loaded SLN is significantly more effective than PM in inhibiting parasite propagation (P?<?0.05) and that cytotoxicity of PM-SLN formulations is size dependent. According to our results, delivery of the drugs to the macrophages via nanoparticle utilization seems to be an accessible and practical approach.     

Investigation into in vitro anti-leishmanial combinations of calcium channel blockers and current anti-leishmanial drugs

The need for drug combinations to treat visceral leishmaniasis (VL) arose because of resistance to antimonials, the toxicity of current treatments and the length of the course of therapy. Calcium channel blockers (CCBs) have shown anti-leishmanial activity; therefore their use in combination with standard drugs could provide new alternatives for the treatment of VL. In this work, in vitro isobolograms of Leishmania (Leishmania) chagasi using promastigotes or intracellular amastigotes were utilised to identify the interactions between five CCBs and the standard drugs pentamidine, amphotericin B and glucantime. The drug interactions were assessed with a fixed ratio isobologram method and the fractional inhibitory concentrations (FICs), sum of FICs (ΣFICs) and the overall mean ΣFIC were calculated for each combination. Graphical isobologram analysis showed that the combination of nimodipine and glucantime was the most promising in amastigotes with an overall mean ΣFIC value of 0.79. Interactions between CCBs and the anti-leishmanial drugs were classified as indifferent according to the overall mean ΣFIC and the isobologram graphic analysis.     

Liposomes with entrapped doxorubicin exhibit extended blood residence times

The blood residence time of liposomes with entrapped doxorubicin is shown to be significantly longer than for identically prepared empty liposomes. Liposomal doxorubicin systems with a drug-to-lipid ratio of 0.2 (w/w) were administered at a dose of 100 mg lipid/kg. Both doxorubicin and liposomal lipid were quantified in order to assess in vivo stability and blood residence times. For empty vesicles composed of phosphatidylcholine (PC)/cholesterol (55:45, mole ratio) and sized through filters of 100 nm pore size, 15-25% of the administered lipid dose was recovered in the blood 24 h after i.v. injection. The percentage of the dose retained in the circulation at 24 h increased 2-3-fold when the liposomes contain entrapped doxorubicin. For 100 nm distearoyl PC/chol liposomal doxorubicin systems, as much as 80% of the injected dose of lipid and drug remain within the blood compartment 24 h after i.v. administration.     

Effects on the murine mononuclear phagocyte system of chronic administration of liposomes containing cytotoxic drug or lipid A compared with empty liposomes

In experiments designed to examine the adverse effects of chronic liposome administration in vivo on the mononuclear phagocyte system (reticuloendothelial system), the presence of drug entrapped in the liposomes may increase the level of reticuloendothelial impairment. We have compared the effects on the mononuclear phagocyte system in mice of chronic administration of empty liposomes with the effects of liposomes containing the anti-leishmanial drug meglumine antimoniate. We have also examined the effect on the mononuclear phagocyte system of continued injections of liposomes containing lipid A, a component of bacterial lipopolysaccharide, which is responsible for macrophage activation. Ten intravenous injections of multilamellar liposomes composed of dipalmitoylphosphatidylcholine and cholesterol (1:0.75 M ratio) were given to ICR mice over a 25-day period. Two individual groups of mice received endotoxin-free liposomes in which meglumine antimoniate was either present or absent. One addition group received liposomes containing lipid A derived from Escherichia coli lipopolysaccharide. A control group received sterile saline injections. In each group, a depression of the phagocytic index, as measured by reduction of uptake of particulate carbon, was observed among some of the individual animals 24 h after the first injection. In many mice a marked splenomegaly was observed. A depressed phagocytic index and splenomegaly were most marked for mice receiving lipid A liposomes. However, there was a large individual variability among mice receiving these preparations and some mice in each group had normal spleen size and a nearly normal phagocytic index. Tissue distribution of liposomes containing [14C]dipalmitoylphosphatidylcholine as a phospholipid marker was examined in all groups in mice 24 h after the last injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeting of urea stibamine encapsulated in liposomes to reticuloendothelial system for the treatment of experimental leishmaniasis

The antileishmanial antimonial drug urea stibamine was encapsulated in mannosylated and nonmannosylated liposomes and was tested against experimental leishmanial infection in hamsters. The study demonstrated that liposome encapsulation of urea stibamine enhanced its effectiveness, an effect which was greater when mannosylated liposomes were used.     

Mycobacterium indicus pranii (Mw) re-establishes host protective immune response in Leishmania donovani infected macrophages: critical role of IL-12

Leishmania donovani, a protozoan parasite, causes a strong immunosuppression in a susceptible host and inflicts the fatal disease visceral leishmaniasis. Relatively high toxicity, low therapeutic index, and failure in reinstating host-protective anti-leishmanial immune responses have made anti-leishmanial drugs patient non-compliant and an immuno-modulatory treatment a necessity. Therefore, we have tested the anti-leishmanial efficacy of a combination of a novel immunomodulator, Mycobacterium indicus pranii (Mw), and an anti-leishmanial drug, Amphotericin B (AmpB). We observe that Mw alone or with a suboptimal dose of AmpB offers significant protection against L. donovani infection by activating the macrophages. Our experiments examining the anti-leishmanial activity of Mw alone or with AmpB also indicate a p38MAPK and ERK-1/2 regulated pro-inflammatory responses. The Mw-AmpB combination induced nitric oxide production, restored Th1 response, and significantly reduced parasite burden in wild type macrophages but not in IL-12-deficient macrophages indicating a pivotal role for IL-12 in the induction of host-protection by Mw and AmpB treatments. In addition, we observed that Mw alone or in combination with suboptimal dose of AmpB render protection against L. donovani infection in susceptible BALB/c mice. However, these treatments failed to render protection in IL-12-deficient mice in vivo which added further support that IL-12 played a central role in this chemo immunotherapeutic approach. Thus, we demonstrate a novel chemo-immunotherapeutic approach- Mw and AmpB crosstalk eliminating the parasite-induced immunosuppression and inducing collateral host-protective effects.     

Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes

The abundant Leishmania promastigote surface Ag gp63 and Leishmania promastigote lipophosphoglycan were reconstituted into liposomes and used as a vaccine against the agent of New World cutaneous leishmaniasis, Leishmania mexicana. The Ag were inoculated s.c., i.p., and i.v. into CBA/ca and BALB/c mice. Even at low Ag dosages, 8 to 10 micrograms/mouse, the Ag induced appreciable levels of protection. In CBA/ca mice complete protection was obtained by s.c. inoculation of antigen-containing liposomes. Protection could be transferred with T cells to naive mice. Interestingly, the Ag-containing liposomes did not cause the disease exacerbation observed in previous vaccine studies with crude parasite extracts.