Tamoxifen is effective against Leishmania and induces a rapid alkalinization of parasitophorous vacuoles harbouring Leishmania (Leishmania) amazonensis amastigotes

OBJECTIVES: This study was performed to investigate the activity of tamoxifen, an antioestrogen widely used in the treatment of breast cancer, against Leishmania. METHODS: Drug activity was assessed in vitro against axenically grown promastigotes and amastigotes through cell counting or by measuring the cleavage of MTT, and against intracellular amastigotes by treating infected macrophage cultures and evaluating the number of intracellular parasites. Intravacuolar pH changes induced inside parasitophorous vacuoles of Leishmania (Leishmania) amazonensis-infected macrophages were evaluated using the fluorescent probes SNAFL-calcein and Acridine Orange. RESULTS: Tamoxifen killed L. (L.) amazonensis promastigotes and amastigotes with 50% inhibitory concentrations (IC50) of 16.4 +/- 0.2 and 11.1 +/- 0.2 microM, respectively. The drug was also effective against Leishmania (Viannia) braziliensis, Leishmania (Leishmania) major, Leishmania (Leishmania) chagasi and Leishmania (Leishmania) donovani with IC(50) values ranging from 9.0 to 20.2 microM. Tamoxifen induced a rapid and long-lasting alkalinization of the vacuolar environment. We also provide evidence that tamoxifen is more effective against promastigotes and amastigotes at pH 7.5 when compared with cultures at pH 4.5. CONCLUSIONS: Tamoxifen effectively kills several Leishmania species and its activity against the parasite is increased by a modulation of the host cell intravacuolar pH induced by the drug.     

Inhibition of Leishmania (Leishmania) amazonensis growth and infectivity by aureobasidin A

OBJECTIVES: To study the effect of aureobasidin A, an inhibitor of inositol phosphorylceramide (IPC) synthase, on Leishmania growth and infectivity. METHODS: Effects of aureobasidin A were determined for: (i) promastigote growth in axenic culture; (ii) promastigote infectivity in macrophage monolayers; (iii) development of footpad lesions in BALB/c mice; (iv) differentiation of amastigotes into promastigotes. RESULTS: Aureobasidin A (20 microM) inhibited 90% of Leishmania (Leishmania) amazonensis promastigote growth in axenic culture, but the parasites remained viable, i.e. growth curves returned to normal after aureobasidin A was removed from culture medium. The aureobasidin A IC50 was determined by MTT assay as 4.1 microM for L. (L.) amazonensis promastigotes, 12.6 microM for Leishmania (Leishmania) major and 13.7 microM for Leishmania (Viannia) braziliensis. There was a significant delay in infection when L. (L.) amazonensis promastigotes pre-treated with aureobasidin A were inoculated into BALB/c mouse footpads. When aureobasidin A was added to cultured macrophages infected with amastigotes, the number of infected macrophages was reduced by >90%. CONCLUSIONS: Aureobasidin A is an interesting pharmacological tool to investigate the effect of lipid metabolism inhibition in Leishmania spp.     

In vitro antileishmanial activity of acetogenins from Annonaceae.

Twelve acetogenins from Annonaceae were evaluated in vitro for their antileishmanial activities in order to search for new lead-compounds having antileishmanial properties. The compounds were comparatively evaluated by the 50% inhibitory concentrations (IC50) determination on promastigote forms of wild-type and four drug-resistant lines of Leishmania donovani. In addition, after testing the toxicity on mouse peritoneal macrophages, the compounds were evaluated on amastigote infected macrophages and a therapeutic index was calculated. The IC50 of the acetogenins against promastigote forms of L. donovani was in a range 4.7-47.3 microM. The most active compound was Rolliniastatin 1 (IC50 at 4.7 microM). On the intramacrophage amastigote in vitro model, only seven compounds exhibited measurable antileishmanial activity with IC50 values in a range 2.5-29.7 microM. Rollinistatin 1 was the most interesting compound with IC50 of 2.5 microM and it appears as the most promising one on the basis of therapeutic index (18.08). Isoannonacin, which is active against intramacrophagic amastigotes (IC50 of 6.2 microM) with a therapeutic index of 2.05, exhibited a strong action on drug-resistant strains (IC50 from 5.1 to 9.8 microM). Acetogenins are a new chemical series with interesting in vitro antileishmanial activity and further studies will be focused on the understanding of this selectivity in regard to the membrane and mitochondrial action using specific probes.     

Tuftsin-bearing liposomes as rifampin vehicles in treatment of tuberculosis in mice.

The antitubercular activity of rifampin was considerably increased when it was encapsulated in egg phosphatidylcholine liposomes. A further increase in the activity was observed when the macrophage activator tetrapeptide tuftsin was grafted on the surface of the drug-loaded liposomes. Intermittent treatments (twice weekly) with these preparations were significantly more effective than the continuous treatments. Rifampin delivered twice weekly for 2 weeks in tuftsin-bearing liposomes was at least 2,000 times more effective than the free drug in lowering the load of lung bacilli in infected animals. However, pretreatment with drug-free tuftsin-bearing liposomes did not render the pretreated animals resistant to the Mycobacterium tuberculosis infections, neither did it appreciably increase the chemotherapeutic efficacy of the liposomized rifampin. These results clearly demonstrate that liposome targeting to macrophages could considerably increase the antitubercular activity of liposomized drugs such as rifampin. Also, it shows that immunoprophylactic treatment with macrophage activators such as tuftsin does not afford any advantage in treatment of tuberculosis infections, presumably because of inactivation of the primed macrophages by the mycobacterial sulfatides.     

In vitro antileishmanial properties of tri- and pentavalent antimonial preparations.

To better understand the antileishmanial effects of antimonial agents we synthesized complexes of tri- and pentavalent antimony with mannan. The 50% inhibitory concentrations (IC50s) of these agents, along with those of potassium antimony tartrate [Sb(III)] and sodium stibogluconate [Sb(V)], were determined for promastigotes and intramacrophage amastigotes. The trivalent antimonial agents were more potent than the pentavalent agents. Although the IC50s were 60- to more-than-600-fold higher for promastigotes than for amastigotes, similar intracellular antimony concentrations in both life forms were measured after incubation with all four drugs at their respective IC50s. Macrophages accumulated antimony during a 4-h exposure that was retained intracellularly for at least 3 days. Amastigotes inside macrophages had a higher antimony content 6 days after a single 4-h treatment than they did immediately after treatment, suggesting that macrophages serve as a reservoir for antimonial agents and prolong parasite exposure. Macrophages concentrated antimony from the medium with potassium antimony tartrate, trivalent antimony-mannan, and pentavalent antimony-mannan treatments. N-Acetylcysteine antagonized the antileishmanial effects of these three drugs against intracellular amastigotes; in contrast, it had minimal effects on the action of sodium stibogluconate.     

Platelet hyperreactivity, scavenger receptors and atherothrombosis

Summary.  Scavenger receptors (SRs) were initially identified as macrophage receptors that recognize modified lipoproteins. The lists of SRs, their ligands and cells expressing SRs have been significantly extended during the last two decades. What has become clear is that many ligands of SRs are present in vivo only in pathologic conditions. Several SRs have been identified on platelets with the best studied being scavenger receptors CD36 and SR-BI. Platelet SRs are multiligand receptors with properties of pattern recognition receptors. CD36 and SR-BI are exposed on resting platelets, while other SRs are rapidly expressed upon platelet activation. Thus, platelets may serve as sensors of 'pathologic ligands' in circulation. The role of platelet SRs in platelet physiology is still poorly understood. However, the data are accumulating that SR ligands, present in the circulation under pathologic conditions, interact with platelet SR and modulate platelet reactivity, thereby contributing to thrombosis and cardiovascular pathology.     

In vitro effects of four macrolides (roxithromycin, spiramycin, azithromycin [CP-62,993], and A-56268) on Toxoplasma gondii.

The effect of four macrolides against intracellular Toxoplasma gondii was determined in three different in vitro systems. Unactivated murine peritoneal macrophages were infected with the virulent RH strain of T. gondii. The activity of the macrolides was first measured with [3H]uracil, which is incorporated by the parasite but not the host cell. The 50% inhibitory concentrations (IC50s) and 95% confidence limits were calculated at 54 (38 to 73), 140 (98 to 201), 147 (101 to 214), and 246 (187 to 325) micron for roxithromycin, azithromycin (CP-62,993), A-56268, and spiramycin, respectively. Inhibition of Toxoplasma growth was confirmed by microscopic examination of the infected macrophages after treatment with roxithromycin. Compared with untreated controls, roxithromycin concentrations near the IC50s decreased the number of infected cells, the number of tachyzoites per vacuole, and the number of cells containing rosettes (i.e., clusters of more than eight tachyzoites). After treatment with the four macrolides, tachyzoites were released from the macrophages and subcultured in HeLa cells, which are nonprofessional phagocytes, to assess the viability of the remaining parasites. This showed that the macrolides at concentrations corresponding to four times their 90% inhibitory concentrations (IC90s) had no significant killing effect. At 8 times the IC90, roxithromycin showed an incomplete killing effect, similar to that of the combination of pyrimethamine (0.41 microM)-sulfadiazine (99.42 microM). All macrolides tested showed inhibitory effects against intracellular T. gondii, but amounts of azithromycin and A-56268 corresponding to the IC90 appeared to be toxic against the host macrophages, which might have had nonspecific activity against Toxoplasma metabolism.     

A trial of immunotherapy against Leishmania amazonensis infection in vitro and in vivo with Z-100, a polysaccharide obtained from Mycobacterium tuberculosis, alone or combined with meglumine antimoniate

OBJECTIVES: To determine the efficacy and the immunomodulatory function of Z-100 alone or combined with meglumine antimoniate on Leishmania amazonensis infection. METHODS: The effect of the compounds was evaluated by microscopic counting of intracellular amastigotes in macrophages stained with Giemsa, or axenic promastigotes, and IC(50) was determined by linear regression. The antileishmanial effect of the compounds was assessed in infected BALB/c mice by a limiting dilution analysis and the production of gamma interferon (IFN-gamma), interleukin 10 (IL-10), IL-4, IgG1 and IgG2a was measured by ELISA. RESULTS: In vitro, Z-100 showed antileishmanial activity against intracellular amastigotes of L. amazonensis with an IC(50) of 13 mg/L. Moreover, infected macrophages treated with Z-100 (12 mg/L) showed smaller parasitophorous vacuoles with fewer parasites than the control. In addition, the efficacy of Z-100 plus meglumine antimoniate [14 mg/L pentavalent antimony (Sb(v))] was higher (46% inhibition) than either Z-100 or meglumine antimoniate alone. Nevertheless, no effect of Z-100 on axenic promastigotes was observed. Infected BALB/c mice treated with Z-100 (100 microg/kg) alone did not show any antileishmanial effects in comparison with the control group, and IFN-gamma, as well as IL-10 and IL-4, was up-regulated by the treatment. In addition, both IgG1 and IgG2a were also increased by the Z-100 treatment. Although Z-100 plus meglumine antimoniate (14 or 28 mg/kg Sb(v)) controlled both the parasite load and the footpad swelling in comparison with control mice, no significant differences were found with meglumine antimoniate alone. CONCLUSIONS: In vitro, Z-100 alone or combined with meglumine antimoniate showed an antileishmanial effect on L. amazonensis. However, no effect was observed in infected BALB/c mice treated with Z-100, suggesting that the up-regulation of IL-10 and IL-4 production by the treatment could be interfering with the development of a protective Th1-type response. For further understanding of the effects of Z-100 in vivo, another strain of mice such as C57BL/6 should be tested in future.     

Effect of the lysophospholipid analogues edelfosine, ilmofosine and miltefosine against Leishmania amazonensis

OBJECTIVES: Analysis of the effect of edelfosine, ilmofosine and miltefosine on Leishmania amazonensis and of potential targets of these lysophospholipid analogues. METHODS: Quantification and ultrastructural analysis of the effect of lysophospholipid analogues on promastigote forms and on infected peritoneal macrophages, and flow cytometry analysis of treated promastigotes labelled with propidium iodide and rhodamine 123 (Rh123). RESULTS: The lysophospholipid analogues presented potent antiproliferative activity with IC50/3 days of 1.9-3.4 microM for promastigotes and 4.2-9.0 microM for intracellular amastigotes. Treatment with these analogues in Schneider medium for 1 day led to a dose-dependent decrease in Rh123 fluorescence, an effect more accentuated in edelfosine-treated parasites, suggesting interference with the potential of the mitochondrial membrane. In both forms of L. amazonensis, edelfosine induced extensive mitochondrial damage, multinucleation and, in promastigotes, also led to plasma membrane alterations, formation of autophagic structures and membranous arrangements inside the flagellar pocket. CONCLUSIONS: The alkylglycerophosphocholines edelfosine and ilmofosine were more active than the alkylphosphocholine miltefosine against promastigotes and intracellular amastigotes of L. amazonensis, and ultrastructural and flow cytometry data indicate the mitochondrion as a target of edelfosine.     

PEGylated cationic liposomes robustly augment vaccine-induced immune responses: Role of lymphatic trafficking and biodistribution

Lymph nodes (LNs) are peripheral lymphoid organs essential for vaccine-induced immune responses. Although cationic liposomes have been documented as a novel adjuvant and vaccine delivery system, whether enhancing LN targeting would improve the efficiency of cationic liposome-formulated vaccines has not been elucidated yet. In the present study we investigated the effect of PEGylation on LN targeting and the immunogenicity of cationic liposome-formulated vaccines. DOTAP cationic liposomes were incorporated with 1 or 5mol% of DSPE-PEG2000 and labeled with near infrared fluorescent dyes. The lymphatic trafficking and biodistribution of different liposomes after subcutaneous (s.c.) injection were recorded using an in-vivo imaging system. The results showed that incorporation of 1mol% DSPE-PEG2000 not only accelerated the drainage of DOTAP liposomes into draining LNs, but also prolonged their LN retention and enhanced liposome uptake by resident antigen-presenting cells. On the other hand, although incorporating 5mol% of DSPE-PEG2000 into DOTAP liposomes enhanced their LN retention and uptake to a lesser extent, it prolonged blood circulation of DOTAP liposomes and increased their splenic accumulation. In addition, PEGylated DOTAP liposomes augmented primary and secondary anti-OVA antibody responses more potently than nonPEGylated DOTAP liposomes did. Hence, incorporating a small amount of DSPE-PEG2000 into DOTAP liposomes not only increased the passive LN targeting of DOTAP-formulated vaccines but also modulated their biodistribution in vivo, which consequently improved the efficiency of cationic liposome-formulated vaccines.     

Identification of a small-molecule, nonpeptide macrophage scavenger receptor antagonist.

Class A scavenger receptor (SR-A) antagonists may prevent the initiation of atherosclerosis, because a recent report found that SR-A/apolipoprotein E (apoE) double-knockout mice had 60% smaller lesions than apoE single-knockout littermates. We transfected human embryonic kidney (HEK) 293 cells with SR-A type I or II receptors to find small-molecule antagonists. Uptake of 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL) showed that among common polyanionic ligands, polyinosine was the most potent, with an IC50 of 0.74 microgram/ml, whereas the novel compound (E)-methyl 4-chloro-alpha-[4-(4-chlorophenyl)-1, 5-dihydro-3-hydroxy-5-oxo-1-(2-thiazolyl)-2H-pyrrol-2-ylidene]benzene acetate gave an IC50 of 6.1 microgram/ml (13 microM). The novel antagonist also inhibited DiI-AcLDL uptake in cultured human peripheral and rat peritoneal macrophages with IC50 values of 21 microM and 17 microM, respectively. With [125I]AcLDL as ligand for transfected HEK 293 cells, binding/uptake and degradation at 37 degrees C for 5 h was saturable and selective. In a comparison of both types of receptor, we found no difference between the capacity of SR-AI or SR-AII for either binding or degradation. Polyinosine competed both [125I]AcLDL binding and degradation with a Ki of 1 microgram/ml, whereas the novel antagonist competed with a Ki of 19 microgram/ml (40 microM) and 8.6 microgram/ml (18 microM), respectively, for binding and degradation. Saturation binding in the presence of the ionophore monensin indicated that the novel compound behaved as a noncompetitive antagonist and perhaps as an allosteric effector. This is the first report to describe a small-molecule macrophage scavenger receptor antagonist. Utilization of this permanently transfected HEK 293 cell line will allow the identification of more potent macrophage scavenger receptor antagonists, so that their utility as therapeutics for atherosclerosis can be determined.     

Characterization of K+ channel-dependent as well as -independent components of pinacidil-induced vasodilation

The mechanisms of pinacidil-induced direct vasodilation were studied in vitro in RMA and RAO. In RMA, pinacidil produced dose-dependent relaxations of norepinephrine (5 microM)-induced contractions with an IC50 of 0.2 microM. This component of pinacidil relaxation appeared to be dependent on K+ conductance because pretreatment with tetraethylammonium (10 mM), Ba++ (0.5 mM), glyburide (1 microM) and 20 mM K+ all caused a rightward shift of the pinacidil dose-response curve (DRC) and a corresponding increase in the pinacidil IC50. However, additional relaxation effects of pinacidil were still evident in the presence of various K+ channel blockers. Pinacidil also showed a relaxation DRC under the condition of 80 mM K+ contraction in both RMA and RAO with IC50 values of 27 and 50 microM, respectively. Pinacidil could also produce maximal relaxation in RMA and RAO remained unaffected in 145 mM K+ (zero Na+) depolarizing solution suggesting a lack of dependence on Na(+)-Ca++ exchange mechanism for this action of pinacidil. Studies using 1 or 3 min pulse labeling with 45Ca showed an absence of an inhibitory effect of pinacidil (at 50 and 100 microM) on unidirectional 45Ca influx stimulated by high-K+. Net 45Ca uptake studies showed that pinacidil inhibited high-K+ stimulated 45Ca uptake at 100 but not at 50 microM. Ryanodine (10-100 microM) was used as a tool to investigate the role of sarcoplasmic reticulum (SR) in this action of pinacidil. Under the condition in which ryanodine (10-100 microM) treatment was found to cause the SR to be nonfunctional, pinacidil relaxation DRC remained unaltered, suggesting a lack of a stimulatory effect of pinacidil on SR Ca++ accumulation. These data thus show that the K+ channel-independent effect of pinacidil does not involve to any significant degree an effect of pinacidil on plasmalemmal voltage-sensitive Ca++ channels, SR Ca++ stores, Na(+)-Ca++ exchange or membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyaluronan-modified and regular multilamellar liposomes provide sub-cellular targeting to macrophages, without eliciting a pro-inflammatory response

Macrophages, pivotal cells in onset and progression of inflammation, can benefit from sub-cellular drug targeting to the molecular loci of drug action, whether cell membrane or cell interior. Postulating manipulation of liposome size and surface properties can provide sub-cellular targeting, we studied: thermodynamics of liposome-macrophage binding; liposome cellular localizations; liposome safety including pro-inflammatory cytokine production. We aimed at extending the body of knowledge on interactions of regular unilamellar (RL-ULV) and multilamellar (RL-MLV) liposomes with macrophages. We investigated, for the first time, the interactions of hyaluronan (HA) surface-modified liposomes (HA-ULV and HA-MLV) with macrophages, with respect to multiple equilibria binding combined with cellular localization. Macrophages bound all four liposome types, substantially-favoring the two MLV species over the two ULV species, and internalizing only RL-MLV. Three macrophage-internalization inhibitors (2-deoxyglucose, LY294002 and Wortmannin) reduced RL-MLV internalization but not binding affinity nor binding capacity. Both MLV types were not detrimental to cell proliferation, nor did they elicit TNF-α production in resting and in LPS-activated macrophages. Moreover, a 24-hour exposure of LPS-activated macrophages to HA-MLV reduced TNF-α production by 40%, indicating potential for anti-inflammatory activity. In conclusion RL-MLV and HA-MLV are the liposomes of choice for delivering anti-inflammatory drugs to the macrophage surface or its interior, according to the loci of drug action.     

Antibacterial activity of liposome-entrapped ampicillin in vitro and in vivo in relation to the lipid composition

The antibacterial activity of liposome-entrapped ampicillin against Listeria monocytogenes was investigated in relation to the lipid composition of the liposomes. This was studied in vitro in mouse peritoneal macrophages infected with L. monocytogenes, as well as in vivo in experimental L. monocytogenes infection in mice. Two types of liposomes, a relatively fluid type, consisting of cholesterol-phosphatidylcholine-phosphatidylserine (5:4:1), and a less fluid type, consisting of cholesterol-distearoylphosphatidylcholine-dipalmitoylphosphatidylglyc erol (10:10:1), were used. The uptake of both types of liposomes by macrophages in vitro was similar. However, the rate of intracellular degradation appeared to be dependent on the lipid composition. A correlation was found between the relatively slow degradation of the less fluid liposomes and a delayed intracellular release of the encapsulated ampicillin, as reflected in absent or delayed intracellular killing of L. monocytogenes in vitro. The results obtained in vitro were confirmed by the observations in vivo. Slow degradation of the less fluid liposomes in vivo resulted in a decrease in the therapeutic effect of the antibiotic.     

Antileishmanial effect of 3-aminooxy-1-aminopropane is due to polyamine depletion

The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, catalyzes the conversion of ornithine to putrescine. As the polyamine biosynthetic pathway is essential for the growth and survival of Leishmania donovani, the causative agent of visceral leishmaniasis, inhibition of the pathway is an important leishmaniacidal strategy. In the present study, we examined for the first time the effects of 3-aminooxy-1-aminopropane (APA), an ODC inhibitor, on the growth of L. donovani. APA inhibited the growth of both promastigotes in vitro and amastigotes in the macrophage model, with the 50% inhibitory concentrations being 42 and 5 microM, respectively. However, concentrations of APA up to 200 microM did not affect the viability of macrophages. The effects of APA were completely abolished by the addition of putrescine or spermidine. APA induced a significant decrease in ODC activity and putrescine, spermidine, and trypanothione levels in L. donovani promastigotes. Parasites were transfected with an episomal ODC construct, and these ODC overexpressers exhibited significant resistance to APA and were concomitantly resistant to sodium antimony gluconate (Pentostam), indicating a role for ODC overexpression in antimonial drug resistance. Clinical isolates with sodium antimony gluconate resistance were also found to overexpress ODC and to have significant increases in putrescine and spermidine levels. However, no increase in trypanothione levels was observed. The ODC overexpression in these clinical isolates alleviated the antiproliferative effects of APA. Collectively, our results demonstrate that APA is a potent inhibitor of L. donovani growth and that its leishmaniacidal effect is due to inhibition of ODC.     

Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC(50)s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC(50)s of these three quinazoline compounds (2.4 +/- 0.4, 3.4 +/- 0.6, and 3.9 +/- 1.1 microM, respectively) were in the range of the IC(50) of ganciclovir (1.2 +/- 0.2 microM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.     

Antileishmanial activity of liposome-encapsulated amphotericin B in hamsters and monkeys.

Visceral leishmaniasis (kala-azar) results from parasitization of the macrophages of the liver, spleen, and the rest of the visceral reticuloendothelial system with Leishmania donovani. Pentavalent antimony is the drug of choice for leishmaniasis chemotherapy; amphotericin B (AmB) is active but is rarely used, because of drug toxicity. AmB encapsulated within macrophage-directed carriers (liposomes) has been used to treat humans with systemic mycoses complicating neoplastic diseases; dosages of up to 5 mg of encapsulated AmB per kg per day for greater than 14 days are without apparent kidney or liver toxicity. In the present work, greater than 99% of L. donovani parasites were eliminated from the liver and spleen of infected hamsters by one administration of 1.5 to 11 mg of liposome-encapsulated AmB (L-Amb) per kg. A total of 98 to 99% of hepatosplenic parasites were eliminated from squirrel monkeys by three administrations of 4 mg of L-AmB per kg. L-AmB was 170 to 750 times as active as antimony in hamsters, and approximately 60 times as active as antimony in monkeys. The demonstration that apparently nontoxic human dosages of L-AmB eliminate essentially all hepatosplenic parasites in hamster and primate models suggests that this preparation should be considered for clinical trial against kala-azar.     

2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (1110U81) potently inhibits human cytomegalovirus replication and potentiates the antiviral effects of ganciclovir.

We studied the effects of 2-acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (1110U81 or A1110U), a potent inhibitor of the ribonucleotide reductases encoded by herpes simplex virus types 1 and 2 and by varicella-zoster virus, against human cytomegalovirus (HCMV) replication in infected MRC-5 cells. We show that 1110U81 is a potent inhibitor of HCMV DNA replication (50% inhibitory concentration [IC50], 3.6 microM; IC90, 5.6 microM) and also potentiates the effects of ganciclovir (GCV) against HCMV. The IC90 of GCV is reduced from 65 microM when GCV alone is given to 2.8 microM when GCV is combined with 1110U81 at a molar ratio of 1:1.     

Stereoselective modulation of ryanodine-sensitive calcium channels by the delta isomer of hexachlorocyclohexane (delta-HCH).

delta-Hexachlorocyclohexane (delta-HCH) is shown to be 30-fold more potent as a positive inotropic agent with rat atrial strips compared with lindane (gamma-HCH). Threshold and ED50 values for enhanced contractile force at a pacing frequency of 0.5 Hz are less than 1 microM and 2.2 microM for delta-HCH and 40 microM and 63 microM for gamma-HCH, respectively. Contracture developed in atria exposed to greater than 4 microM delta-HCH (ED50 = 11 microM) but not in atria exposed to gamma-HCH. Uptake and release of Ca++ measured from actively loaded cardiac sarcoplasmic reticulum (SR) vesicles is measured with antipyrylazo III. Although delta-HCH (30 microM) decreases Ca(++)-dependent ATPase by 20%, it does not significantly alter Ca++ loading in the presence of ruthenium red. Addition of delta-HCH (5-50 microM) after loading is complete causes rapid, dose-dependent release of Ca++ from SR. Ca++ release induced by delta-HCH is markedly stereoselective. Compared with gamma-HCH (50 microM), delta-HCH (50 microM) induces a nearly 20-fold higher initial rate of Ca++ release (4.3 nmol of Ca++/mg/sec). Studies with [3H]ryanodine demonstrate that delta-HCH sharply inhibits Ca(++)- or daunorubicin-activated radioligand binding (IC50 = 37 and 25 microM, respectively, logit slope = 2). Inhibition of [3H]ryanodine-binding by delta-HCH is stereoselective inasmuch as IC50 values for alpha, beta and gamma isomers are greater than 100 microM. The delta-HCH modified Ca++ channel appears to proceed by a noncompetitive mechanism (reducing Bmax in equilibrium experiments) with respect to the conformationally sensitive binding site for [3H]ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipid composition on activity of liposome-entrapped ampicillin against intracellular Listeria monocytogenes.

The effect of lipid composition on the intracellular antibacterial activity of ampicillin-containing liposomes was studied in vitro by using mouse peritoneal macrophages infected with Listeria monocytogenes. Two types of liposomes, a fluid type, consisting of cholesterol-phosphatidylcholine-phosphatidylserine (5:4:1), and a solid type, consisting of cholesterol-distearoylphosphatidylcholine-dipalmitoylphosphatidylglyc ero l (10:10:1), were used. Although the cellular uptake of both types of liposomes was similar, they differed with respect to the rate of intracellular degradation. A correlation was found between the relatively slow degradation of the solid liposomes and a delayed intracellular release of the encapsulated ampicillin, as reflected in absent or delayed intracellular killing of L. monocytogenes.