Acyclic pyrazolo[3,4-d]pyrimidine nucleoside as potential leishmaniostatic agent.

A new synthesis of 6-amino-1-hydroxyethoxymethyl-4 (5H)-oxopyrazolo[3, 4-d]pyrimidine (4) has been mentioned. Compound 4 exhibited inhibition of amastigotes of Leishmania donovani to the extent of 89 % at 30 microg/mL, whereas iso-guanine analogue 5 had the inhibition only to the extent of 52.8% at 100 microg/mL in vitro. In hamster model the maximum inhibitory response for compound 4 against amastigotes multiplication was observed to be 94% at 50 mg/kg single dose for 5 consecutive days.

Immunomodulatory activity of analog of muramyl dipeptide and their use as adjunct to chemotherapy of Leishmania donovani in hamster.

In search of a potent immunomodulator to be used as an immunoprophylactic agent and as adjunct to chemotherapy against Leishmania infection, two analogs of muramyl dipeptide, viz. N.Ac-norMur-MeVal-D-isoGln (86/448) and N.AcMur-Acc-D-isoGln (89/729) were evaluated for desired activity. Effect of these peptides on cell mediated and humoral immunity was studied by immunizing the peptide treated mouse with sheep red blood cells (SRBC) and determining HA-titer, plaque forming cells assay and delayed type of hypersensitivity (DTH) response after 4-5 days. Both the peptides stimulated cell mediated immunity (CMI), humoral response as well as macrophage function in terms of super oxide anion (O2-) and nitric oxide (NO) generation. Mitogen induced lymphocyte proliferation and production of IL-2 and INF-gamma increased while that of IL-4 and IL-10 decreased by both the peptides showing a typical Th1 type response. After establishing the immunostimulatory activity, these peptides were evaluated for immunoprophylactic efficacy as well as for use as adjunct to chemotherapy with stibanate (SSG) against Leishmania donovani infection in golden hamster. These peptides were found quite effective in both the modes. In adjunct use the treatment may require lower dose of SSG and thereby reduce the chances of drug toxicity.

Superior chemotherapeutic efficacy of amphotericin B in tuftsin-bearing liposomes against Leishmania donovani infection in hamsters.

Chemotherapeutic efficacy of the amphotericin B (Amp B), which is the drug of choice for treatment of the leishmanial infections (kala-azar) that become resistant to the conventional chemotherapy using antimonials, has been examined in the Leishmania donovani infected hamsters after encapsulating the drug in tuftsin-free as well as tuftsin-bearing liposomes. The activity was significantly increased (p < 0.05) by delivering Amp B in tuftsin-free liposomes. This antileishmanial effect of the liposomized Amp B was further increased (p < 0.05) by grafting the natural macrophage-activator tetrapeptide, tuftsin (Thr-Lys-Pro-Arg), on the liposome's surface. This could possibly be attributed to both the enhanced drug tolerance after liposomization as well as to the increased uptake of tuftsin-bearing Amp B-laden liposomes by the macrophages. In addition to the increased efficacy, encapsulation of Amp B in the tuftsin-bearing liposomes also enhanced the drug accessibility to areas (e.g. bone marrow) that are otherwise inaccessible to the free drug. These results further demonstrate the usefulness of tuftsin-bearing liposomes as drug vehicles in treatment of the macrophage-based infections that have been reviewed recently (Agrawal, A.K. and Gupta, C.M. (2000). Tuftsin-bearing liposomes in treatment of macrophage-based infections, Adv. Drug Deliv. Rev., 41, 135-146).

Effect of sodium stibogluconate on hepatic mixed function oxidase system and marker enzymes of golden hamsters during Leishmania donovani infection.

During L. donovani infection in golden hamsters, tremendous hepatic damage was observed as apparent from increased activities of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, succinate dehydrogenase, glucose-6-phosphatase and acid ribonuclease. The levels of cytochrome P-450 and related monooxygenases, viz. aniline hydroxylase and aminopyrine-N-demethylase registered significant decrease in infected animals. Sodium stibogluconate, a standard antileishmanial drug, though caused the removal of parasites from infected tissues, but did not help in the recovery of deranged hepatic markers. The results explain the higher mortality of stibanate treated infected animals as compared to untreated animals infected with L. donovani.

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes.

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.

Biochemical changes in heat stressed promastigotes of Leishmania donovani.

On exposing promastigotes of L. donovani (Dd-8) to 34 degrees C for 30 hr, the flagella were shed, and size was decreased with 10% viability loss. The in vitro and in vivo infectivity of two forms was more or less similar. The 45Ca2+ uptake by the transformed cells was increased as compared to normal cells. Activity of 5'-nucleotidase was increased while activity of Mg(2+)-ATPase remained same. Parasite antioxidant enzymes were also significantly altered by heat shock. There was significant increase in superoxide dismutase, glutathione reductase and glutathione peroxidase. It was accompanied by decrease in ratio of reduced glutathione to oxidized glutathione.

Status of glutathione in lymphoid tissues of golden hamster during Leishmania donovani infection.

During Leishmania donovani infection of golden hamsters, changes in the glutathione levels of lymphoid tissues were observed. While in liver and thymus there was significant decrease in the levels of reduced and oxidised glutathione, that in spleen, bone marrow cells, peritoneal exudate cells and lymph nodes increased, indicating a role for host glutathione in establishing the infection.

Immunostimulant activity of a novel lipopeptide and its protective action against Leishmania donovani.

Novel lipopeptides 84/201 and 86/450 synthesized in this laboratory stimulated antibody and delayed type hypersensitivity (DTH) response to ovalbumin in guinea pigs. Lipopeptide 86/450 also stimulated antibody and DTH responses in albino mice and enhanced nonspecifically macrophage migration index (MMI), phagocytic activity and incorporation of [14C] glucosamine in peritoneal macrophages of the treated animals. Proliferative response of splenocytes from lipopeptide 86/450 treated animals was significantly higher than that from untreated controls. Peritoneal macrophages from lipopeptide 86/450 treated mice were less susceptible to Leishmania donovani promastigote invasion when co-cultured in vitro. The treated animals on challenge with L. donovani promastigote/amastigote showed 80 to 90% lower intake of infection than the control animals.

Effect of new diamidines against Leishmania donovani infection.

The impact of interamidine distance on antileishmanial activity of new aryldiamidines have been evaluated against amastigotes of L. donovani in hamster. Of the 20 compounds tested, only four (2,8-diamidino-9,10-dihydrodibenzoxepin; 2,7-diamidinoxanthone; 2,7-diamidinothioxanthone and 2,7-diamidinoxanthene) showed significant inhibition (more than 80%) of multiplication of amastigotes in spleen. The interamidine distance in the structure appears to have bearing on antileishmanial activity. The observations made are likely to evoke new understanding on the structure activity relationship of diarylamidines.

Immunostimulant activity of Picroliv, the iridoid glycoside fraction of Picrorhiza kurroa, and its protective action against Leishmania donovani infection in hamsters.

Picroliv, a standardised fraction from root and rhizome of Picrorhiza kurroa, consisting of iridoid glycosides and shown to be responsible for its hepatoprotective activity, was studied for immunostimulant activity. Oral administration of Picroliv (10 mg/kg x 7 days) in mice prior to immunization with sheep red blood cells (SRBC) resulted in a significant increase in haemagglutinating antibody (HA) titre, plaque forming cells (PFC), and delayed hypersensitivity (DTH) response to SRBC. Picroliv enhanced the non-specific immune response characterized by an increase in macrophage migration index (MMI), [14C]-glucosamine uptake, phagocytosis of [14C]-leucine labelled Escherichia coli, chemiluminescence of peritoneal macrophages, and higher uptake of [3H]-thymidine in the lymphocytes of treated mice. It also induced a high degree of protection in golden hamsters against challenge infection with Leishmania donovani promastigotes.

Immunostimulant Activity of Picroliv, the Iridoid Glycoside Fraction of Picrorhiza kurroa, and its Protective Action against Leishmania donovani Infection in Hamsters1.

Picroliv, a standardised fraction from root and rhizome of PICRORHIZA KURROA, consisting of iridoid glycosides and shown to be responsible for its hepatoprotective activity, was studied for immunostimulant activity. Oral administration of Picroliv (10 mg/kg x 7 days) in mice prior to immunization with sheep red blood cells (SRBC) resulted in a significant increase in haemagglutinating antibody (HA) titre, plaque forming cells (PFC), and delayed hypersensitivity (DTH) response to SRBC. Picroliv enhanced the non-specific immune response characterized by an increase in macrophage migration index (MMI), [ (14)C]-glucosamine uptake, phagocytosis of [ (14)C]-leucine labelled ESCHERICHIA COLI, chemiluminescence of peritoneal macrophages, and higher uptake of [ (3)H]-thymidine in the lymphocytes of treated mice. It also induced a high degree of protection in golden hamsters against challenge infection with LEISHMANIA DONOVANI promastigotes.

Effect of five-membered heterocycles against Leishmania donovani infection.

Impact of change of heteroatom in pentavalent heterocycles, viz., pyrroles, isoxazoles, imidazoles and crotonates on the profile of antileishmanial activity against amastigotes of L. donovani using in vivo test system and macrophage-amastigote culture system has been studied. Sixty-three compounds were tested. Nine imidazoles showed marginal activity in vivo, whereas 3 out of 10 compounds of isoxazolone series and 2 out of 4 substituted aminocrotonates exhibited antileishmanial activity. Of the 30 substituted pyrroles, except 8 all showed antileishmanial activity in vivo on day 7 post treatment.

An indirect fluorescent antibody (IFA) test for the serodiagnosis of Kala-Azar.

The IFA test developed using Leishmania donovani promastigote and amastigote antigens showed very high antibody titre in clinically and parasitologically established cases (30) of kala-azar, the geometrical mean reciprocal titre (GMRT) being 870 +/- 5.4 and 5370 +/- 1.80 respectively with the two antigens. In contrast, the GMRT of normal subjects of endemic area was only 12.44 +/- 6.19 and 80.35 +/- 1.66 respectively with these antigens. None of the subjects from non-endemic area suffering with other parasitic diseases, such as malaria, filaria, amoebiasis, leprosy or tuberculosis (20 cases each) gave a positive response to any of the antigen. The test holds promise in the diagnosis of Kala-azar.

Iridoids: a new class of leishmanicidal agents from Nyctanthes arbortristis.

Iridoid glucosides (arbortristosides A [I], B [2], C [3], and 6beta-hydroxy-loganin [4] isolated from the traditional plant Nyctanthes arbortristis show antileishmanial activity in both in vitro (against amastigotes in macrophage cultures) and in vivo (in hamsters) test systems.

Ultrastructural study on the antileishmanial effect of 1,2-dimethyl-3-methoxy carbonyl-4-(2-nitro-4,5-dimethoxyphenyl) pyrrole in hamsters.

An ultrastructure study was carried out on the effect of a standard drug sodium stibogluconate and a newly identified active CDRI compound 87/305 [1,2-dimethyl-3-methoxy carbonyl-4-(2-nitro-4,5-dimethoxyphenyl) pyrrole] on amastigotes of Leishmania donovani in macrophage cells of spleen of infected hamsters. While Na-stibogluconate exerted its effect by activating the digestion capacity of host macrophages, compound 87/305 was directly lethal to the parasites without, exerting any effect on the host cells.

Enzyme-linked immunosorbent assay in the diagnosis of kala-azar in Bhadohi (Varanasi), India.

An enzyme-linked immunosorbent assay (ELISA) developed using Leishmania donovani promastigote soluble antigen gave positive responses in practically all the clinically and parasitologically diagnosed patients with kala-azar in a preliminary study. Healthy control subjects from non-endemic and endemic areas gave negative results. No cross-reaction was observed with patients having leprosy, filariasis, malaria, tuberculosis, or amoebiasis. Blood samples collected on filter paper were adequate for the test. The test appears promising in the diagnosis of kala-azar.

Impact of seasonal variation on Leishmania donovani infection in hamsters.

The impact of seasonal variation on the course of L. donovani infection in hamsters was investigated. Though the animals were maintained in controlled climatic conditions (25 degrees C +/- 2), parasites exhibited seasonal rhythm. During summer (April-July) when the atmospheric temperature ranged from 20.5 degrees C to 41.8 degrees C, the parasite load from an inoculum of 1 x 10(7) amastigotes/animal was found to be less than 1 to 9 per 100 cell nuclei (based on spleen biopsy) on day 25-35 post infection. An escalation in count was observed from August onwards, which reached the peak (approximately 30/100 cell nuclei) in February-March (temp. range 11.3 degrees C to 31.4 degrees C). The multiplication rate monitored 15 days after the initial assessment also showed a similar pattern. The secondary organs examined showed no parasites. The study revealed that despite the non-involvement of the vector in experimental infection in hamster, the parasites retained its periodic character as in man, corresponding to cyclicity of vector.

In vitro culture of erythrocytic stages, including gametocytes of Plasmodium berghei (NK 65 strain) using candle jar method and a newly designed continuous medium flow apparatus.

Three methods have been described for cultivation of erythrocytic stages, specially gametocytes of P. berghei NK65 strain, (1) by using vial candle jar where the cultures were subcultured by addition of fresh erythrocytes, (2) a newly designed simple and compact continuous medium flow apparatus, where medium was continuously perfused but fresh erythrocytes were not added and (3) where the subcultures were also done using simple and compact continuous medium flow apparatus for comparison. The maximum percentage of parasitized erythrocytes obtained by these methods was 24.3, 27.1 and 26.4% respectively. Parasites in vials could survive for more than 10 days with 3 to 4 subcultures with maximum 1.96% gametocytaemia. However, the gametocytaemia in continuous medium flow apparatus, where subcultures were not made reached 2.2% compared to that of 2.7% in this apparatus where subcultures were done. The asexual as well as sexual stages of this parasite survived for about 16-18 days in compact continuous medium flow apparatus, where at least 7 subcultures were done.

Exploration of antileishmanial activity in heterocycles; results of their in vivo & in vitro bioevaluations.

A total of 51 imidazoles, pyrroles, quinolines and isoxazolines compounds were screened for antileishmanial activity in vivo and in vitro, using Leishmania donovani as the test parasite. The screening revealed hitherto unknown antileishmanial activity in these heterocycles. Three of the compounds screened (one belonging to isoxazoline series and two from pyrrole series) showed significant anti-leishmanial activity, ranging from 86-91 per cent inhibition in hamsters. When tested in vitro, using macrophage amastigote culture system, these compounds showed inhibition of 62-78 per cent at 30 micrograms/ml concentration.

Suppression of the hepatic microsomal cytochrome P-450 dependent mixed function oxidase activities in golden hamster during Leishmania donovani infection.

Experimental infection of golden hamsters with Leishmania donovani caused significant alterations in the hepatic microsomal mixed function oxidase system. Gross examination of liver indicated hepatomegaly. Microsomal protein contents were only marginally elevated. Cytochrome P-450 as well as haem contents were significantly decreased and it directly correlated with the degree of infection. Cytochrome b5 exhibited elevation at lower degrees of infection which came down to control levels at the peak infection. Concomitant suppression was also noticed in cytochrome P-450 dependent monooxygenase activities, viz. aniline hydroxylase, benzo[a]pyrene hydroxylase and aminopyrine N-demethylase. No significant change was observed in NADH-cytochrome b5 reductase and NADPH-cytochrome c reductase. The results indicate suppression of hepatic microsomal MFO activities during visceral leishmaniasis.