Liposomes in leishmaniasis: Therapeutic effects of antimonial drugs, 8-aminoquinolines,and tetracycline

This study investigated the role of liposomes in changing the pharmacological efficacy of anti-leishmanial drugs in hamsters infected with visceral leishmaniasis. Enhanced anti-leishmanial activity could be accounted for only by liposome-encapsulated drugs. “Empty” liposomes (lacking anti-leishmanial drug) gave no therapeutic benefit by themselves, nor did they enhance the effectiveness of concurrently administered drugs. In the absence of additional drugs, empty liposomes actually resulted in a higher mortality due to endstage leishmaniasis. Mortality associated with chronic leishmaniasis, including that induced by empty liposomes, was reduced approximately 50% by orally administered unencapsulated tetracycline. Liposome-encapsulated tetracycline, given i.c., had no anti-leishmanial activity, thus indicating that tetracycline did not have inherent anti-leishmanial properties, and was beneficial because of its anti-bacterial effects. Liposomes containing an antimonial drug were effective when given i.c., i.p., or i.m., but not when given s.c. or p.o. Liposome-encapsulated antimonial drug had prophylactic activity and was effective when administered 8 days prior, but not 17 days prior, to infection. Unencapsulated antimonial drug had no prophylactic effect. In addition to antimonials, another class of compounds, 8-aminoquinolines, had marked anti-leishmanial activity in liposomes. One of these, WR 6026, was 700 to 1800 times more effective than an antimonial drug alone.     

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.     

Role of residual Sb(III) in meglumine antimoniate cytotoxicity and MRP1-mediated resistance

Despite the clinical use of pentavalent antimonials for more than half a century, their metabolism in mammals and mechanisms of action and toxicity remain poorly understood. It has been proposed that the more active and toxic trivalent antimony form Sb(III) plays a critical role in their antileishmanial activity and toxicity. The aim of this work was to investigate the role of residual Sb(III) both in the antileishmanial/antitumoral activities of the pentavalent meglumine antimoniate and in the MRP1 (multidrug resistance-associated protein 1)-mediated resistance to this drug. Samples of meglumine antimoniate differing in their amount of residual Sb(III) (meglumine antimoniate synthesized either from SbCl₅ or from KSb(OH)₆ as well as commercially-available meglumine antimoniate) were evaluated in vitro and in vivo on Leishmania amazonensis infections, as well as for their cytotoxicity to normal and MRP1-overexpressing GLC4 cell lines. Although in vitro the two most effective drugs contained the highest levels of Sb(III), no correlation was found in vivo between the antileishmanial activity of meglumine antimoniate and its residual Sb(III) content, suggesting that residual Sb(III) contributes only marginally to the drug antileishmanial activity. On the other hand, the GLC4 cells growth inhibition data strongly suggests a marked contribution of residual Sb(III). Additionally, the potassium salt of antimoniate (non-complexed form of Sb(V)) was found to be more cytotoxic than meglumine antimoniate. Although MRP1-overexpressing GLC4 cells showed a marked resistance to trivalent antimonials, cross-resistance to meglumine antimoniate was observed only for the products that contained relatively high levels of Sb(III) (at least 0.03% by weight), suggesting that MRP1 mediates resistance to Sb(III) but not to Sb(V). In conclusion, our data strongly suggest that residual Sb(III) in pentavalent antimonial drugs does not contribute significantly to their antileishmanial activity, but is responsible for their cytotoxic activity against mammalian cells and the MRP1-mediated resistance to these drugs.     

Leishmania infantum infection rates in Phlebotomus perniciosus fed on naturally infected dogs under antimonial treatment

Dogs naturally infected with Leishmania infantum Nicolle were treated with three courses of meglumine antimoniate. Changes were observed in the dogs' clinical signs, antibody titres and in infection rates of Phlebotomus perniciosus Newstead fed on the dogs. A large reduction in the sandfly infection rate was observed for 4-5 months after the first treatment. The use of antimonial drugs is advocated for the control of canine leishmaniasis and to reduce risks of L.infantum transmission.     

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.     

Responses of Leishmania (Viannia) braziliensis cutaneous infection to N-methylglucamine antimoniate in the rhesus monkey (Macaca mulatta) model

The antileishmanial efficacy of the reference drug N-methylglucamine antimoniate (Glucantime) was evaluated in groups of rhesus monkeys with acute and chronic Leishmania (Viannia) braziliensis cutaneous infection. The therapeutic responses in experimental animals to either a low dose (5 mg/kg body wt/day for 28 days) or a routine dose (20 mg/kg/day for 28 days) of pentavalent antimony were similar to those reported in the human disease. Primates were cured of their lesions after treatment, but with cryptic parasitism and/or relapse. The rhesus model of L. (V.) braziliensis cutaneous leishmaniasis therefore provides an additional resource for preclinical trials with newer drugs.     

Efficacy and toxicity of pentavalent antimonials (Glucantime and Pentostam) in an American cutaneous leishmaniasis animal model: luminometry application

The pentavalent antimonial compounds Glucantime and Pentostam are the first line drugs used in anti-Leishmania treatment. However, no in vivo studies have compared the efficacy and toxicity of these drugs where host variability has been controlled. Biochemical studies of Leishmania have detected differences between the two drugs with regard to DNA topoisomerase I inhibition, a phenomenon that possibly impacts treatment efficacy. To evaluate the clinical efficacy, hamsters were infected intradermally in the right hind foot with 10(6) promastigotes of a wild type or luciferase-transfected Leishmania panamensis. At three weeks post-inoculation, the animals were treated intramuscularly with either Glucantime or Pentostam (30, 60 or 120 mg SbV/kg per day for 20 days). To evaluate parasitological efficacy a luminometry assay was standardized for quantitation of amastigotes in hamster tissues. To evaluate toxicity, hamsters were treated intramuscularly with Glucantime or Pentostam (120, 160 or 240 mg SbV/kg per day for 20 days). Animals inoculated with either of the parasite strains and treated with either drug, showed a similar rate of lesion reduction, as compared to untreated controls (p<0.01). Parasite burden was also comparable, and no significant differences were found in the cure rate. No renal or hepatic alterations occurred as evidenced by normal serum levels of creatinine, aspartate aminotransferase, alanine aminotransferase and amylase. Hamsters treated with 120 mg SbV/kg per day for 20 days or higher doses of Pentostam showed macro- and microscopic signs of inflammation at the site of injection. These effects were absent in the animals treated with Glucantime. The results confirmed clinical observations regarding the similar efficacy of the two drugs, as well as the higher local toxicity of Pentostam.     

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)     

Development of Leishmania (Viannia) panamensis lesions and relationship of numbers of amastigotes to lesion area on antimony-treated and untreated hamsters

Young adult (60-70-g) male golden hamsters (Mesocricetus auratus) each were injected intradermally at the dorsal base of the tail with 15 x 10(6) promastigotes of Leishmania (Viannia) panamensis (MHOM/PA/83/WR539), and progression and regression of subsequent lesions were evaluated for up to 17 wk postinfection (PI) as to area, weight, and number of amastigotes within lesions in untreated hamsters and in hamsters treated with meglumine antimoniate (Glucantime). In untreated hamsters total area of lesion, weight, and numbers of amastigotes generally increased rapidly and concomitantly up to 3-4 wk PI. Amastigote numbers tended to decrease from 4 to 11 wk PI and subsequently the numbers of amastigotes within the lesions decreased rapidly, whereas relatively little change occurred in the area and weight of the lesions. Meglumine antimoniate treatment of cutaneous hamster lesions resulted in marked concomitant decrease in size of the lesions and numbers of amastigotes within the lesions examined 1 wk after treatment. Measurement of the area of cutaneous leishmanial lesions thus would appear to be a valid method of evaluating the efficacy of promising compounds against L. panamensis in hamsters when measurements are taken 3-5 wk after experimental infection and reflects the number of amastigotes present in the lesion.     

Pediatric Visceral Leishmaniasis in Albania: A Retrospective Analysis of 1,210 Consecutive Hospitalized Patients (1995–2009)

Background

Little information is available about infantile visceral leishmaniasis (VL) in Albania as regards incidence, diagnosis and management of the disease.

Methodology/Principal Findings

Demographic data, clinical and laboratory features and therapeutic findings were considered in children admitted to University Hospital of Tirana from 1995 to 2009, and diagnosed as having VL. The diagnosis was based on bone-marrow microscopy/culture in 77.5% of patients, serology in 16.1%, and ex juvantibus in 6.4%. A total of 1,210 children were considered, of whom 74% came from urbanized areas. All patients were in the age range 0–14 years, with a median of 4 years. Hepatosplenomegaly was recorded in 100%, fever in 95.4% and moderate to severe anemia in 88% of cases. Concomitant conditions were frequent: 84% had bronchopneumonia; diarrhea was present in 27%, with acute manifestations in 5%; 3% had salmonellosis. First-line therapy was meglumine antimoniate for all patients, given at the standard Sb^v dosage of 20 mg/kg/day for 21 to 28 days. Two children died under treatment, one of sepsis, the other of acute renal impairment. There were no cases of primary unresponsiveness to treatment, and only 8 (0.67%) relapsed within 6–12 months after therapy. These patients have been re-treated with liposomal amphotericin B, with successful cure.

Conclusions

Visceral leishmaniasis in pediatric age is relatively frequent in Albania; therefore an improvement is warranted of a disease-specific surveillance system in this country, especially as regards diagnosis. Despite recent reports on decreased responses to antimonial drugs of patients with Mediterranean VL, meglumine antimoniate treatment appears to be still highly effective in Albania.     

Potential of Artesunate in the treatment of visceral leishmaniasis in dogs naturally infected by Leishmania infantum: Efficacy evidence from a randomized field trial

Leishmaniasis is among the world’s most neglected diseases. Dogs are the main reservoirs/hosts of Leishmania infantum, causative agent of both canine and human visceral leishmaniosis. Canine leishmaniasis (CanL) represents a public health problem as one of the most prevalent zoonotic diseases worldwide. Current therapeutics present drawbacks; thus, there is a need for more effective, safer, and cheaper drugs. The aim of this study was to evaluate and to compare the efficacy of oral administration of artesunate or meglumine antimoniate/allopurinol in dogs with clinical leishmaniasis. Forty-two dogs with naturally occurring clinical leishmaniasis were included in this open-label, simple randomized positive-control clinical field trial with 6 months of follow-up. Dogs received meglumine antimoniate 100 mg/kg/day and allopurinol 30 mg/kg/day for 28 days (control group, n = 26) or artesunate 25 mg/kg/day for 6 days (test group, n = 16). The animals were evaluated for their clinical evolution, parasite load (by qPCR) and humoral response at different time points: 0, 30, 90, and 180 days after treatment. Data analyses showed a significant improvement in both groups in clinical scores, parasitemia and antibody titers after treatment. Compared to the control group, the artesunate group showed significantly lower clinical score (P = 0.0001), lower parasitemia (P = 0.0001) and antibody titers after 6 months of follow-up. Compared to baseline values, a rapid, significant reduction (P < 0.012) in antibody levels, 2.28- versus 3.04-fold for the control versus artesunate groups, respectively, was observed 30 days after treatment. Antibody levels continued to decrease further in the artesunate group, where 58% of cases became seronegative at the 6-month follow-up. All qPCR-positive dogs were negative after treatment with artesunate, while 14.3% remained positive with the appearance of two new cases in the control group. Artesunate was well tolerated, and no side effects were recorded. Treatment failures were similar in both groups with 27.27% (6/22), including 18.18% (4/22) mortality in the control group, versus 26.66% (4/15), including 13.33% (2/15) mortality in the artesunate group. This is the first report showing the potential of artesunate in the treatment of dogs with clinical leishmaniasis. Artesunate showed higher efficacy than the current first-line treatment for CanL without any adverse effects. It could be a good alternative chemotherapy for CanL, and may be considered for further studies in human leishmaniases. Further clinical trials are needed to confirm these findings, to determine if there are relapses after treatment and if dogs remain infective to sandflies, to define the ideal therapeutic dosage and duration of treatment with artesunate.     

Leishmania infantum: stage-specific activity of pentavalent antimony related with the assay conditions

The determination of the intrinsic sensitivity of Leishmania strains to pentavalent antimonials in clinical trials, before treatment is begun, is essential in order to avoid failures and to allow alternative drugs to be chosen. A comparative study of SbV activity on promastigotes, axenic amastigote-like cells, and intracellular amastigotes of Leishmania infantum, when administered in the form of meglumine antimoniate and free, in hydrochloric solution, was performed. Results indicate that the conditions under which the promastigotes were cultured affect the IC(50) obtained, although results were homogeneous when the products were assayed on axenic-like and intracellular amastigotes. The IC(50) obtained for SbV in the form of meglumine antimoniate or in hydrochloric solution on promastigotes cultured in Schneider's medium depends on the growth rate of the culture and therefore could be regulated by modifying the fetal calf serum concentration in the medium. The pH of the culture medium strongly affected the activity of meglumine antimoniate but not that of the SbV hydrochloric solution on promastigotes cultured in Schneider's medium. This influence of pH was observed to a much lesser extent when promastigotes were cultured on M199 or RPMI media. In homogeneous culture conditions, which included the regulation of the promastigote growth rate through the heat-inactivated fetal calf serum concentration in the medium and the dilution of the meglumine antimoniate with Schneider's medium at pH 6.5, the activity of SbV, free or in the form of meglumine antimoniate, was the same in promastigotes, intracellular amastigotes, and axenic amastigote-like cells.     

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.     

Humoral factors and nonspecific immune suppression in Syrian hamsters infected with Leishmania donovani

Leishmania donovani produces progressive wasting and ultimately fatal visceral leishmaniasis in Syrian hamsters and provides an excellent model of progressive disease in humans. Experimentally infected hamsters were used to investigate the development of nonspecific immune suppression during visceral leishmaniasis and its association with humoral factors and wasting. At 2 wk all infected hamsters had developed antibody against a 59-kDa parasite antigen not recognized by sera of control hamsters. By 4 wk, strong antibody responses were noted against antigens of 26, 35, 46, 69, 107, and 120 kDa. No additional antigen was recognized at 6 or 8 wk or in hamsters treated with high doses of a pentavalent antimonial (stibogluconate sodium, 100 mg/kg/day for 5 days). Weight loss was first noted in infected hamsters at 8 wk. No difference in splenic lymphocyte proliferation in response to concanavalin A (Con A) was noted at 2 wk, but by 6 wk infected animals had only 20% of the Con A response of controls, and by 8 wk only 13%. Furthermore, incubation of splenic lymphocytes from uninfected control animals with 5% fetal calf serum and 5% serum from infected hamsters obtained at 4, 6, or 8 wk suppressed Con A responses by 50%, 99%, and 100%, respectively. Spleen cells from drug-treated animals exhibited no suppression of Con A responses when incubated with 5% autologous serum, but there was profound suppression when they were incubated with 5% autologous serum obtained during the acute phase of infection. Humoral factors, but not wasting, contributed to the suppression of lymphocyte responses.     

A liposomal form of diamidine: reduced toxicity]

The cultures of Nuttalia eque mainly develop in the reticuloendothelial organs and so in treatment of nuttaliosis in horses and the Nuttalia carriers diamidine, an analog of imidocarb or imidozoline, was used encapsulated in liposomes. The liposomes were prepared with a modification of the phase inversion method (the lipids were dissolved in a mixture of freon-11 and chloroform). The content of the organic solvents in the preparation, as evidenced by gas liquid chromatography, was less than 0.2 per cent. The main fraction consisted of particles 1.5 to 2.5 microns in diameter. The tests on animals of various species revealed a significant decrease in the toxicity of diamidine when used encapsulated in liposomes as compared to the use of the free diamidine. The LD50 of the liposome encapsulated diamidine administered intravenously and intramuscularly was for albino mice 52 and 6000 mg/kg, respectively whereas that of the free diamidine was 0.8 and 84 mg/kg, respectively. In a dose of 10 mg/kg administered intramuscularly the free diamidine induced death in 100 per cent of the horses while in a dose of 10 mg/kg the liposome encapsulated diamidine was satisfactorily tolerated by the animals. The liposome encapsulated diamidine had no unfavourable effect on hepatic antitoxic and metabolic functions. One should hope that the low toxicity of the liposome-encapsulated diamidine will provide its higher chemotherapeutic index.     

Ex Vivo Host and Parasite Response to Antileishmanial Drugs and Immunomodulators

Background

Therapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses.

Methodology

To achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 μg Sb^V/mL) or miltefosine (2, 4, 8, 16 and 32 μM HePC). Concentrations of 4 μM of miltefosine and 8 μg Sb^V/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays.

Principal Findings

Anti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL -10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine.

Conclusions and Significance

Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.     

Mechanisms of delivery of liposome-encapsulated cytosine arabinoside to CV-1 cells in vitro. Fluorescence-microscopic and cytotoxicity studies

Fluorescence microscopy and assays of the cytotoxicity of liposome-encapsulated cytosine arabinoside (araC) have been used to examine the interactions of CV-1 cells with pH-sensitive liposomes, combining phosphatidylethanolamine (PE) with oleic acid or with double-chain protonatable amphiphiles, and with pH-insensitive liposomes combining phosphatidylcholine (PC) and phosphatidylglycerol (PG). Fluorescence-microscopic observations indicate that double-chain protonatable amphiphiles remain tightly associated with pH-sensitive liposomes during incubations with CV-1 cell monolayers, and that cellular uptake of liposomes is strongly promoted by transferrin coupled to the liposome surface. Liposome-encapsulated araC showed much greater cytotoxicity toward CV-1 cells than did the free drug at equivalent concentrations under the same conditions. The cytotoxicity of encapsulated araC was strongly enhanced by liposome-conjugated transferrin and was maximal using pH-sensitive liposomes combining PE with the double-chain protonatable amphiphile N-(N'-oleoyl-2-aminopalmitoyl)serine. However, the drug was also markedly more cytotoxic when encapsulated in other types of transferrin-conjugated liposomes, including pH-insensitive PC/PG/cholesterol liposomes, than in the free form. The cytotoxicity of liposome-encapsulated araC is significantly attenuated by the nucleoside transport inhibitor nitrobenzothioinosine, and fluorescence microscopy using calcein-containing liposomes provides no evidence for efficient fusion between cellular membranes and any of the types of liposomes examined here. Based on these observations, we suggest that the major mechanism for cytoplasmic delivery of liposome-encapsulated araC is the carrier-mediated transport of drug that has been released from liposomes into the endosomal and/or the lysosomal compartments.     

Leishmania braziliensis in the squirrel monkey: development of primary and satellite lesions and lack of cross-immunity with Leishmania donovani

Three female and 2 male adult laboratory-reared squirrel monkeys (Saimiri sciureus) that previously had been inoculated with Leishmania (Leishmania) donovani and had recovered from experimental visceral leishmaniasis were each inoculated intradermally at the dorsal base of the tail with 2.2 x 10(7) culture-derived promastigotes of Leishmania (Viannia) panamensis. The progression and regression of subsequent lesions were examined for 36 wk in all 5 monkeys after which 3 of the monkeys were killed (1 with a primary lesion and all with satellite lesions) and the 2 surviving monkeys (1 with primary lesion and both with satellite lesions) were treated with 104 mg/kg/day of meglumine antimoniate for 10 days. All of the monkeys developed a primary lesions at the site of injection of the parasite and later developed satellite lesions peripheral to the primary nodule. The primary lesions had disappeared from 3 of the 5 monkeys by 36 wk, whereas satellite lesions persisted on all at this time. Satellite lesions were present at 52 wk after treatment and persisted for 169 wk in the 2 surviving monkeys. The histopathologic appearance of the lesions was characterized as granulomatous inflammation. Our results indicated that squirrel monkeys that had recovered from visceral leishmaniasis remained susceptible to infection with L. (V). panamensis.     

Characterization of reactions of antimoniate and meglumine antimoniate with a guanine ribonucleoside at different pH

It has been shown previously that Sb^V forms mono- and bis-adducts with adenine and guanine ribonucleosides, suggesting that ribonucleosides may be a target for pentavalent antimonial drugs in the treatment of leishmaniasis. In the present work, the reactions of antimoniate (KSb(OH)₆) and meglumine antimoniate (MA) with guanosine 5′-monophosphate (GMP) have been characterized at 37 °C in aqueous solution and two different pH (5 and 6.5), using ESI(−)-MS and ¹H NMR. Acid and base species for both 1:1 and 1:2 Sb^V–GMP complexes were identified by ESI(−)-MS. The ¹H NMR anomeric region was explored for determining the concentrations of mono- and bis-adducts. This allows for the determination of stability constants for these complexes (5900 L mol⁻¹ for 1:1 complex and 370 L mol⁻¹ for 1:2 complex, at pD 5 and 37 °C). Kinetic studies at different pH indicated that formation and dissociation of both 1:1 and 1:2 Sb–GMP complexes are slow processes and favored at acidic pH (2150 L mol⁻¹ h⁻¹ for the rate constant of 1:1 complex formation and 0.25 h⁻¹ for the rate constant of 1:1 complex dissociation, at pD 5 and 37 °C). When MA was used, instead of antimoniate, formation of 1:1 Sb–GMP complex occurred, but with a slower rate constant. Assuming that MA consists essentially of a 1:1 Sb–meglumine complex, a stability constant for MA could also be estimated (8600 L mol⁻¹ at pD 5 and 37 °C). Thermodynamic and kinetic data are consistent with the formation of 1:1 Sb–ribonucleoside complexes in vertebrate hosts, following treatment with pentavalent antimonial drugs.     

Activity of quinfamide against natural infections of Entamoeba criceti in hamsters: a new potent agent for intestinal amoebiasis

A novel tetrahydroquinolinyl ester, quinfamide, administered orally in multiple doses for 3 days had an ED50 of 0.25 mg/kg/day (total dose 0.75 mg/kg) for eradicating Entamoeba criceti in hamsters in several tests. It was significantly more active by direct comparison than 3 commercially available amoebicides and at least as active as 2 other esters of the parent compound, 1-(dichloroacety)-1,2,3,4-tetrahydro-6-quinolinol. After administration of a single dose, ED50 calculations for quinfamide averaged 0.9 mg/kg. Quinfamide was considerably more active than the other tetrahydroquinolinols, diloxanide furoate and teclozan, and it was approximately 1.5 times more active than etofamide; a statistical significance between the latter 2 drugs could be demonstrated in one of 4 tests. Administered prophylactically, quinfamide was shown to protect hamsters from re-infection with E. criceti. It also inhibited propagation of E. histolytica in vitro at a concentration of 20 microgram/ml. No adverse effects were noted in rodents after a single dose as high as 10 g/kg. Daily administration to monkeys of doses up to 500 mg/kg for as long as 37 days produced no pharmacological aberrations during or after medication; haematological studies and urine analyses were normal and no gross or microscopical tissue changes attributable to quinfamide were observed. No toxicity was revealed following acute (2 g/kg) and chronic (500 mg/kg/day x 31 days) administration of the drug to dogs and rats, respectively.