Murthy Pk

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi.

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 μg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 μg/ml. The half maximal inhibitory concentration (IC(50)) of naringenin for motility of adult females was 2.5 μg/ml. Flavone immobilized female adult worms at 31.2 μg/ml (MTT>80%) and microfilariae at 62.5 μg/ml. Rutin killed microfilariae at 125 μg/ml and inhibited MTT-reduction in female worms for >65% at 500 μg/ml. Naringin had adulticidal effects at 125 μg/ml while chrysin killed microfilariae at 250 μg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin>flavone=hesperetin>rutin>naringin>chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.

Plant Products in the Treatment and Control of Filariasis and Other Helminth Infections and Assay Systems for Antifilarial/Anthelmintic Activity

Lymphatic filariasis, onchocerciasis, loaisis, and other helminth infections cause serious health problems especially in resource-limited tropical and subtropical developing countries of the world, and more than 2 billion people are infected with at least one helminth species. From times immemorial, man looked up to the plant kingdom in search of anthelmintics, antifilarials, and remedies for parasite-induced health problems. Although more than 50 % of drugs in modern medicine are derived from plants or leads from plants, a success story of plant-based anthelminthics or antifilarials is yet to be told. In the last 5 decades, more than 100 plant products were reported to be beneficial in the treatment or control of these parasitic infections but they could not be developed into viable drugs for a variety of reasons. This review focuses on the plant products reported to be useful in the control and treatment of human helminth infections with the main emphasis on filariasis and the in vitro and in vivo systems available for assaying anthelmintic activity.

Antifilarial activity of some 2H-1-benzopyran-2-ones (coumarins)

Six synthetic 2H-1-benzopyran-2-one (cournarin) derivatives (CDRI compounds # 1, 2, 3, 4, 5 and 6) were evaluated for filaricidal activity against Litomosoides carinii and Acanthocheilonema viteae infections in cotton rats (Sigmodon hispidus) and Mastomys coucha respectively. Significant effects on macrofilariae (>80% death/sterilisation) were detected with compounds #2, 3 and 6 against L. carinii and/or A. viteae. Thus detection of filaricidal activity in benzopyrones, which are so far known for anti-inflammatory activity, provides a new lead for development of better filaricidal agents for combating filariasis.

Anti-filarial activity of novel formulations of albendazole against experimental brugian filariasis.

The study was aimed at developing better orally active albendazole (ALB) formulations. Six formulations (ALB-1 to ALB-6) were prepared and tested against Brugia malayi in Mastomys coucha and jird (Meriones unguiculatus) at 200 mg/kg, orally, for 5 consecutive days. The anti-filarial efficacy was assessed against microfilariae (mf), adult worms and female reproductive potential. Three of the 6 ALB formulations showed greatly improved female worm sterilizing potential (ALB-1: 90%; ALB-3: 63%; ALB-4: 77% of untreated control) in B. malayi - M. coucha model. Sterilization efficacy of ALB-1 was also better than that shown by pure-ALB (P<0.001) or its marketed tablet formulation, Zentel (P<0.01), while that of ALB-4 was better than pure-ALB (P<0.05). The activity of ALB-3, pure-ALB and Zentel was, however, comparable. ALB-1 also showed late microfilaricidal activity with a maximum of 78% fall in microfilarial count. In contrast, neither the pure ALB nor Zentel showed any microfilaricidal activity. In the jird - B. malayi model, ALB-1 and ALB-4 showed marginal sterilizing efficacy whereas pure ALB or Zentel were ineffective. In conclusion the anti-filarial efficacy of ALB-1 was found to be superior to pure-ALB or Zentel.

Sensitization with anti-inflammatory BmAFI of Brugia malayi allows L3 development in the hostile peritoneal cavity of Mastomys coucha

Filarial parasites survive by inducing tolerance in host but the antigens and mechanisms involved are not clear. Recently we found that BmAFI, a Sephadex G-200 eluted fraction of Brugia malayi adult worm extract, stimulates IL-10 release from THP-1 cells. In the present study, we determined the SDS-PAGE profile of BmAFI and infective 3rd stage larva (L3), investigated the effect of pre-sensitization of host with BmAFI on the survival and development of L3 in the non-permissive peritoneal cavity (p.c.) of the permissive host Mastomys coucha and in the p.c. of non-permissive Swiss mice, and studied immunological correlates for the observed effects. The parasite development and burden in p.c., was determined in sensitized infected M. coucha and Swiss mice and the release of TGF-β, IL-4, IL-10, IL-13, IFN-γ and NO, cellular proliferative response to Con A and BmAFI and levels of IgG subclasses and IgE were determined in sensitized infected M. coucha. Cellular proliferative response to Con A and BmAFI, mRNA expression of GATA-3, CTLA-4 and T-bet were determined in sensitized Swiss mice. In addition, the parasitological parameter was also studied in BmAFI-sensitized M. coucha exposed to the infection by standard subcutaneous (s.c.) route to assess whether sensitization enhances the intensity of infection. BmAFI-sensitization permitted survival of L3 and their development to adult stage by day 60 p.i. in the p.c. of M. coucha; in non-sensitized animals L3 could molt to L4 only and no parasite could be recovered beyond day 30 p.i. In M. coucha that received infection by s.c. route, pre-sensitization with BmAFI enhanced the microfilaraemia and adult worm recovery. In sensitized Swiss mice L3 could successfully molt to L4 in p.c. with improved recovery of parasite. BmAFI sensitization upregulated TGF-β and IL-10 release, IgG1 and IgG2b levels, GATA-3 and CTLA-4 mRNA expression, suppressed the cellular proliferative response and downregulated Con A stimulated response, IgE, IL-13, IFN-γ and NO responses. Immunoblot analysis showed that the BmAFI antiserum also strongly reacts with some L3 molecules. The results show, for the first time, that sensitization with the anti-inflammatory BmAFI which shares some of its molecules with those in L3, facilitates parasite survival in the non-permissive p.c. of the permissive host M. coucha, render a non-permissive Swiss mouse partially permissive to infection and enhances parasite load in M. coucha receiving the infection through permissive s.c. route by evoking a modified Th2 type of response and anti-inflammatory milieu. In conclusion, the findings suggest that the anti-inflammatory BmAFI fraction facilitates survival of B. malayi infection even in non-permissive environment.

Influence of Brugia malayi life stages and BmAFII fraction on experimental Leishmania donovani infection in hamsters.

The influence of live Brugia malayi parasites and a Sephadex G-200 fraction of the adult parasite extract (BmAFII) on the progression of Leishmania donovani infection was studied. Inbred hamsters were first infected with B. malayi infective 3rd stage larvae (L3), adult worms or microfilariae (mf), and then with L. donovani amastigotes (Ld), or vice versa or received both the infections simultaneously; a group of animals were first immunized with BmAFII and then infected with Ld. L. donovani parasite burden was determined between 17 and 19 days post amastigote challenge (p.a.c.) and, in case of immunized animals, between 32 and 35 days p.a.c also. Nitric oxide (NO) release from peritoneal macrophages and cellular proliferative responses of lymphnode cells were assessed in BmAFII-immunized animals given leishmania infection or no infection. Leishmanial parasite burden was significantly reduced in animals exposed to filarial L3 before amastigote inoculation and in animals given filarial adult worms after or together with amastigotes. Prior immunization of leishmania-infected animals with BmAFII also reduced the leishmanial parasite burden (17-19 days p.a.c.: >90%; 32-35 days p.a.c.: 60%). These animals showed upregulation of NO release and cellular proliferative responses to promastigote antigen or BmAFII stimulation in vitro. The findings show, for the first time, that B. malayi L3/adult worms or immunization with BmAFII inhibits progression of L. donovani infection in hamsters and this is associated with upregulation of NO and lymphocyte proliferative responses indicating that Th1 response might be responsible for this.

Efficacy of a substituted methyl benzimidazole carbamate against developing and adult helminth parasites

The efficacy of a substituted methyl benzimidazole carbamate, methyl 5(6)-[4-N-(2-pyridyl)] piperazino carbamoyl benzimidazole-2-carbamate, was assessed against larval and adult forms of Ancylostoma ceylanicum (hookworm), Nippostrongylus brasiliensis (trichostrongylid), Hymenolepis nana (tapeworm) and Brugia malayi (filariid) in experimentally-infected animals. The compound was found to have high efficacy against the developing stages (L3, L4, L5) of A. ceylanicum in hamsters at a single dose of 12.5 mg kg-1, against larvae of N. brasiliensis at 17.5 mg kg-1 and against cysticercoids of Hymenolepis nana at 100 mg kg-1 daily for 3 days given per os (p.o.) or intraperitoneally (i.p.). All the stages of B. malayi in Mastomys were killed when the compound was given i.p. at a dose of 6.25 mg kg-1 for 5 consecutive days. A dose of 6.25 mg kg-1 eliminated all adult A. ceylanicum from infected hamsters, 100 mg kg-1 resulted in complete removal of Syphacia obvelata adults from 63.6% of infected mice, 25 mg kg-1 X 5 dose eliminated 100% of adult B. malayi from infected Mastomys and a single 50 mg kg-1 dose expelled all H. nana adults from infected rats.

7-O-[4-methyl piperazine-1-(2-acetyl)]-2H-1-benzopyran-2-one: a novel antifilarial lead compound

In preliminary studies we found that benzopyrones (coumarins), which are known to exert many biological activities including anti-inflammatory effect, possess promising macrofilaricidal action as well. In order to explore the possibility of combining such a macrofilaricidal activity with the microfilaricidal potential of the known piperazine pharmacophore, we synthesized a series of compounds and evaluated their antifilarial effect. In the present study, one of these compounds, 7-O-[4-methyl piperazine-1-(2-acetyl)]-2H-1-benzopyran-2-one (2), which has shown promising macrofilaricidal action against rodent filariid Litomosoides carinii in cotton rats, was evaluated against infection with Brugia malayi in Mastomys coucha and jird (Meriones unguiculatus). In the B. malayi-M. coucha system, the compound at a dose of 300 mg/kg, oral (p.o.) x5 days showed 53.6% adulticidal and 46.0% microfilaricidal activity along with 46.3% sterilization effect on the female worms. In addition, the compound interfered with the establishment of infective larvae (L(3))-induced infection to an extent of 50% at the same dose level. At 1 microM concentration it inhibited protease activity of B. malayi to 82%. The compound thus provides a novel lead for further synthesis and development of antifilarial agents with macrofilaricidal, microfilaricidal, female-sterilizing and possible larvicidal efficacy.

Longitudinal humoral immune responses of Indian leaf monkey (Presbytis entellus) to Brugia malayi infection.

Humoral immune responses of the Indian leaf monkey (Presbytis entellus) experimentally infected with Brugia malayi and exhibiting disease manifestations were studied. Microfilaraemia, filaria-specific IgG and circulating immune complexes (CICs) were determined in the monkeys at different time-points after inoculation of B. malayi 3rd-stage larvae. Sera were analysed for recognition pattern of adult parasite antigen molecules by immunoblotting. More than 60% of the infected monkeys developed episodic or persistent limb oedema with or without fever and with low or no microfilaraemia. While both CIC and filaria specific IgG levels were comparable in animals showing no disease symptoms (asymptomatics) and some animals showing symptoms (symptomatics), IgG levels peaked during pre-patent stage in symptomatics and during latent stage in asymptomatic animals. However, some of the symptomatic animals showed a low level of filaria-specific IgG as compared to asymptomatic and other symptomatic animals. The immunoblot analysis showed non-reactivity of 17 and 55 kDa antigens with sera of symptomatic animals. The results thus suggest that humoral immune responses as measured in the present study do not precede the development of the manifestations. However, 2 non-reactive antigen molecules identified by symptomatic sera need further study to establish their possible involvement, if any, in the development of acute disease manifestations in this model.

Brugia malayi in Mastomys coucha: establishment in immunosuppressed animals

Investigations on various aspects of human filariasis using target filarial parasite, Brugia malayi is jeopardised to a great extent due to its prolonged incubation period and poor harvest from the existing experimental animal models. To obviate these difficulties it was contemplated to establish B. malayi infection in immunosuppressed Mastomys coucha. Cortisone, a well known immunosuppressant, was used at 10 mg/kg dose level subcutaneously in two courses each of 5 days duration. The first course was administered 1 week before and the second, a week after infective exposure. Mastomys were exposed either with 100 or 200 infective larvae (L3) each. Untreated age-matched animals were also exposed simultaneously. The minimum prepatent period was observed to be 90.7 days in immunosuppressed animals exposed to 200 L3. The course of microfilaraemia in immunosuppressed and control animals was identical up to 180 days of observation period. However, the adult worm recovery from the former group of Mastomys was higher. It is surmised that exposure with B. malayi L3 in immunosuppressed Mastomys would be of great advantage in getting larger harvests of adult worms of B. malayi.