Reactive oxygen intermediates metabolizing enzymes in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Adult worms of Ancylostoma ceylanicum and Nippostronglyus brasiliensis were found to possess an active system for the detoxification of reactive oxygen intermediates. Xanthine oxidase, which is known to produce superoxide anion, was detected in both the nematode parasites in significant activities. Superoxide anion, thus produced, may quickly be eliminated by superoxide dismutase. Both parasites also exhibited the presence of catalase, peroxidase, and glutathione peroxidase for efficient removal of hydrogen peroxide. Glutathione reductase and glucose-6-phosphate dehydrogenase were, however, detected in low levels of activities. Endowment of A. ceylanicum and N. brasiliensis with these antioxidant enzymes, therefore, enables them to evade the host's effector mechanism for their survival. Superoxide dismutase of both these nematodes showed marked inhibition by KCN and, hence, the enzyme appears to be of copper-zinc type.

Synthesis and anthelmintic activity of 5(6)-(benzimidazol-2-ylcarbamoyl) and (4-substituted piperazin-1-yl)benzimidazoles.

The synthesis of alkyl 5(6)-(benzimidazol-2-ylcarbamoyl)benzimidazole-2-carbamates (6, 7), and alkyl 5(6)-(4-substituted piperazin-1-yl)benzimidazole-2-carbamates (31-40) has been carried out. When the compounds were tested for their anthelmintic activity against Ancylostoma ceylanicum in hamsters, Hymenolepis nana in rats, Litomosoides carinii in cotton rats, and Dipetalonema viteae in Mastomys natalensis, methyl 5(6)-(4-benzoylpiperazin-1-yl)benzimidazole-2-carbamate (31), methyl 5(6)-[4-(2-furoyl)piperazin-1-yl]benzimidazole-2-carbamate and methyl 5(6)-[4-[(diethylamino)carbonyl]piperazin-1-yl]benzimidazole- -2-carbamate (36) showed 100% elimination of tapeworms H. nana at three oral doses of 100-250 mg/kg. Compounds 34 and 36 also killed the microfilariae and adult worms of L. carinii in cotton rats at an intraperitoneal dose of 30 mg/kg given for 5 days.

Synthesis of substituted 1-hydroxy-2-naphthanilides as potential cestodicidal agents.

A series of substituted 1-hydroxy-2-naphthanilides 4--14 has been synthesized by treating 1-hydroxy-2-naphthoic acids 2 with substituted anilines 3. The nitronaphthanilides, on reduction and subsequent treatment with thiophosgene, gave the corresponding substituted 2-naphthanilide isothiocyanates 30--33. Substitution of the chlorine of 8 by various cyclic amines gave 3'-nitro-4'-substituted 1-hydroxy-2-naphthanilides 15--21. Various 3-aryl-4-oxo-2,3-dihydro-1,3-naphthoxazine-2-thiones 34-43 and 3 aryl-2,4-dioxo-2,3-dihydro-1,3-naphthoxazines 44--51 have been prepared by reacting the corresonding naphthanilides with thiophosgene and ethyl chloroformate, respectively. All the compounds were tested for their cestodicidal activity against Hymenolepis nana infection in rats; 30 was found to be the most active compound of the series, showing 100% clearance of infection at a single oral dose of 7.5 mg/kg.

Studies in potential filaricides. 18. Synthesis of 2,2'-disubstituted 5,5'-dibenzimidazolyl ketones and related compounds as potential anthelmintics.

A series of 2,2'-disubstituted 5,5'-dibenzimidazolyl ketones and related compounds have been synthesized of which 2,2'-bis(carbomethoxyamino)-5,5'-dibenzimidazolyl ketone exhibited a broad spectrum of anthelmintic activity in experimental animals. At doses of 10-50 mg/kg given intraperitoneally, 5 killed 100% of the adult worms of Litomosoides carinii, Dipetalonema viteae, and Brugia malayi. By the oral route the macrofilaricidal efficacy of 5 was 97-100% at 100-200 mg/kg X 5 days. The treated animals showed gradual disappearance of microfilariae and before autopsy they became amicrofilariaemic. Some of the compounds also showed 100% efficacy against the human hookworms and tapeworm, Ancylostoma ceylanicum in hamsters, and Hymenolepis nana in rats at a single oral dose of 50-250 mg/kg. Compound 5 was also effective against Syphacia obvelata in mice at a single oral dose of 100 mg/kg and was found to be well tolerated by mice up to an oral dose of 2500 mg/kg.

Syntheses and anthelmintic activity of alkyl 5(6)-(substituted-carbamoyl)- and 5(6)-(disubstituted-carbamoyl)benzimidazole-2-carbamates and related compounds.

A number of alkyl 5(6)-(substituted-carbamoyl)- and 5(6)-(disubstituted-carbamoyl)benzimidazole-2-carbamates and related compounds have been synthesized, and their anthelmintic activity against various intestinal helminths of experimental animals have been evaluated. A large percentage of the compounds synthesized showed noteworthy activity against Ancylostoma ceylanicum and at higher doses against Hymenolepsis nana infections. Compared to the alkyl 5(6)-(substituted-carbamoyl)benzimidazole-2-carbamates, the disubstituted carbamoyl analogues were found to exhibit better anthelmintic activity. The most active compound of the series, namely, methyl 5(6)-[(N-2-pyridylpiperazino)carbamoyl]benzimidazole-2-carbamate (90), has been screened against intestinal helminths in higher animals and as a micro- and macrofilaricidal agent. Compound 90 has been identified as a broad-spectrum anthelmintic agent. Compound 90 has been identified as a broad-spectrum anthelmintic in view of its efficacy against A. ceylanicum (hamsters and dogs), H. nana (rats), Nippostrongylus brasiliensis (rats), Syphacia obvelata (mice), A. tubaeformis (cat), Toxocara spp. (cat), and Litomosoides carinii (cotton rat).

Role of reactive oxygen species in the chemoprophylactic action of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl]-carbonyl]-1H-benzimidazol-2 yl] carbamate in hamster against Ancylostoma ceylanicum.

To delineate mechanisms involved in the prophylactic action of methyl [5-[[4-(2-pyridinyl)-1-piperazinyl]-carbonyl]-1H-benzimidazol-2 yl] carbamate (compound 81/470) in hamster against Ancylostoma ceylanicum infection, plasma level of the compound and status of reactive oxygen metabolites in jejunum at different periods of the drug treatment were examined. The compound was found to enhance the generation of both O2- and H2O2 by the jejunum possibly by activating xanthine oxidase. This stimulation was found to be both time and dose dependent. At 100 mg/kg dose the increase in O2- production could be recorded at least upto 50 days, whereas at 25 mg/kg the stimulation remained effective upto 20 days only, and at 5 mg/kg there was no change in the activity. This correlated well with the reported prophylactic pattern of the compound i.e. upto 45 and 7 days by 100 and 25 mg/kg doses, respectively. Plasma level of the compound also exhibited dose dependent variation. The compound given at 100 mg/kg dose could be detected in significant concentration upto at least 42 days while that given in 25 and 5 mg/kg doses was present in equivalent concentration upto 14 days and 1 day, respectively. It is concluded that the activation of respiratory burst in the jejunum induced by the persistent presence of compound 81/470 may represent one of the important mechanisms for the chemoprophylactic activity of this anthelmintic.

Effect of anthelmintics on the antioxidant system of Nippostrongylus brasiliensis.

To understand the mode of anthelmintic action of thiabendazole and methyl-[5-[[4-(2-pyridinyl)-l-piperazinyl]carbonyl]-1H-benzimidazole- 2-yl] carbamate (C.D.R.I. compound 81/470) against Nippostrongylus brasiliensis, their effect on the metabolism of reactive oxygen species in the parasite as well as in rat intestine was examined. Both drugs produced a significant depression in the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) of the parasite. Release of antioxidant enzymes by the drug-treated worms was also found to be appreciably lowered. Both thiabendazole and compound 81/470 induced a depression in the levels of all five constituents of the antioxidant system of rat intestine but significant alterations were detected only in the GSH content of infected and the SOD activity of normal intestine. The production of O2- by treated intestine was, on the other hand, markedly enhanced. Increased formation of O2- by the host intestine accompanied with the reduced level of SOD and GSH in N. brasiliensis appear to have a deleterious effect on the parasite. Consequently, the drug-treated worms are unable to retain themselves in situ and are ultimately expelled. The greater effect produced on these parameters by thiabendazole compared to compound 81/470 is consistent with the relative efficacy of these anthelmintics.

NADH oxidase and fumarate reductase of Ancylostoma ceylanicum.

Ancylostoma ceylanicum, the hookworm parasite of cat, dog and man, was found to contain NADH and/or NADPH oxidase as well as fumarate reductase activities. Both the enzyme systems were predominantly located in the membranes of mitochondrial-rich preparations. The membranes also exhibited the presence of a reduced pyridine nucleotide transhydrogenase activity which transferred hydrogen from NADPH to NAD. Amongst respiratory inhibitors, rotenone (Site I inhibitor) markedly depressed both NADH oxidase and fumarate reductase while others, namely antimycin-A, KCN and azide, had a lesser effect.

On the potential of amino acids to support survival and energy status of Nippostrongylus brasiliensis in vitro.

Aerobically but not anaerobically, amino acids can sustain motility as well as glycogen and ATP levels of N. brasiliensis as effectively as glucose can. Proline is the most active amino acid and in combination with lysine, cysteine and phenylalanine, can completely replace glucose.

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.

Molecular basis of hyperlipidemia in golden hamsters during experimental infection with Ancylostoma ceylanicum (Nematoda:Strongylidae).

An infection of golden hamsters with Ancylostoma ceylanicum, a hookworm parasite, induced profound hyperlipidemia, particularly hypertriglyceridemia, and the effect was directly related to the degree of infection. A significant increase was also noticed in serum cholesterol and phospholipid levels. The appearance of lipoprotein-X, an abnormal low density lipoprotein, was detected in the serum of hookworm-infected animals. The hyperlipidemia was further characterized by an increase in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) with a concomitant decline in high density lipoproteins (HDL). Decreased lipolytic activities, especially triglyceride lipase, in hepatic tissue and induction of lipolytic activities in intestine and adipose tissues indicated mobilization of fats from adipose and jejunum with a defective removal of triglyceride-rich lipoproteins in hepatic tissues. Accumulation of lipids in liver and depletion in adipose tissue supported these results. The derangement may have a significant effect on host parasite interaction and is an important pathophysiological feature occurring during experimental ancylostomiasis.

Stimulation of nonspecific resistance by thymopentin and its analogs against Leishmania donovani infection in hamsters.

Thymopentin and its analogs have been synthesized by the solution phase method of peptide synthesis and evaluated for their prophylactic efficacy against L. donovani infection in hamsters. Thymopentin and some of the analogs were found to stimulate nonspecific resistance of the host against Leishmania donovani infection in hamsters.

Leishmania donovani: cellular and humoral immune responses in Indian langur monkeys, Presbytis entellus.

We have previously reported that disease mimicking human visceral leishmaniasis can be established in Presbytis entellus, the Indian langur monkey, following a single intravenous challenge of 10(8) Leishmania donovani amastigotes. In the present report, infection was assessed in monkeys infected intravenously with a single dose of 10(8) amastigotes (HDA group), three weekly doses of 10(7) amastigotes (LDA group) and three weekly doses of 5 x 10(7) promastigotes (HDP group). Typical clinical infection was established in all three groups with significant parasite load. There was a gradual and sustained rise in anti-leishmania specific immunoglobulin G response, and a severe fall in the lymphoproliferative response to the T cell mitogens PHA and Con A by day 80 post infection (p.i.). The antibody level remained elevated until death in monkeys of the HDA and HDP groups; the T-cell responses showed a recovery prior to death. T-cell responses to leishmania antigen, however, could not be demonstrated in any of these monkeys prior to death. One monkey of the LDA group survived for 155 days and two monkeys spontaneously eradicated the infection. Surprisingly, one monkey of the HDA group also achieved spontaneous cure. In the three monkeys which eradicated infection spontaneously, the antibody level declined to baseline levels on day 180 p.i. with a well demonstrable antigen specific lymphoproliferative response; no parasites could be demonstrated in splenic aspirates by direct examination of culture. These data demonstrate that disease severity may be the function of the total inoculum dose rather than the stage of the parasite and that the immunological responses in the Indian langur model parallel the reported changes in human visceral leishmaniasis. This makes the langur a potentially useful model for the evaluation of candidate anti-leishmanial drugs and vaccines.

Methyl 5(6)-4-2-pyridyl piperazino carbamoyl benzimidazole-2-carbamate--a new broad spectrum anthelmintic.

Methyl 5(6)-4-2-pyridyl piperazino carbamoyl benzimidazole-2-carbamate (CDRI Comp. 81-470) was tested against various nematode and cestode infections in different experimental and domestic animals. The compound showed 100% effectivity against adult of Ancylostoma ceylanicum (hookworm) in hamsters (6.25 mg/kg p.o. X 1), Nippostrongylus brasiliensis (trichostrongylid) in rats (100 mg/kg p.o. X 3), Hymenolepis nana (cestode) in rats (25 mg/kg p.o. X 1) and Syphacia obvelata (oxyurid) in mice (12.5 mg/kg p.o. X 3). It was also found highly effective against artificial and natural helminth parasites of higher animals. The compound removed all, A. caninum and A. ceylanicum (hookworms) and Toxocara sp. (ascarid) from dogs at a dose of 10 mg/kg p.o. X 3; A. tubaeformis (hookworm) and Toxocara sp. at 25 mg/kg p.o. X 3 and 2.5 mg/kg p.o. X 3 respectively from cats and Ascaridia galli (ascarid) at 10 mg/kg p.o. X 3 from fowl. The compound in doses of 1500 mg/kg by oral route and 1000 mg/kg by i.p. route did not cause any mortality or produce adverse effect in mice. The expanded anthelmintic action and large therapeutic index indicate compound's great anthelmintic potentiality.

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.

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.

Effect of thiabendazole & mebendazole on in vitro metabolism of Nippostrongylus brasiliensis adults.

Mebendazole and thiabendazole were found to inhibit glucose uptake and its metabolism in the adult rat hookworm (N. brasiliensis) in vitro. Rates of endogenous glycogen utilisation, and excretion of one of the end products of glycolysis viz., lactic acid, were found to be increased, when the intact N. brasiliensis adults were incubated for 60 min with mebendazole and thiabendazole, respectively.

Evaluation of enzyme-linked immunosorbent assay in the diagnosis of kala-azar in Malda district (West Bengal).

An immunological test based on indirect (plate) ELISA has been successfully standardized and modified using promastigote soluble antigen. The test carried out on 813 subjects from a kala-azar endemic area (including parasitologically confirmed patients, subjects presenting with clinical symptoms of visceral leishmaniasis and endemic controls) and a non-endemic area (with diseases other than kala-azar and apparently normal subjects) was found to detect, specifically, antileishmanial antibodies. The plate ELISA has been simplified to a more sensitive dot-ELISA where the results are read within 2-3 h. The antigen requirement is 250 ng per test. No cross-reactivity with sera from patients of malaria, tuberculosis, leprosy, amoebiasis and filariasis was observed. The follow up monitoring of antibodies in successfully treated kala-azar patients showed a decline of antibodies. A drop of blood taken on filter paper is sufficient to conduct the test. Dot ELISA therefore is a simple, inexpensive and stable test in serodiagnosis of visceral leishmaniasis.

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.

Evaluation of filarial skin test with Brugia malayi larval antigen in the field.

To obtain comparable and reproducible results by filaria skin test with B. malayi larval antigen, standard procedure has been evolved. Antigen protein of 2 micrograms per test, injected intracutaneously, was found optimum for positive skin reaction. The reaction ratio based on increase of wheal area by 2 times or more was found to be statistically significant for interpreting positive results. However, in larger field trials, the simpler measurement of increase of wheal diameter by 1 1/2 times or more was found to be equally reliable. Patients treated with diethylcarbamazine citrate, antihistaminics and anti-inflammatory drugs are likely to be unresponsive to filarial antigen and will yield incorrect information.