chemotherapy is a non-toxic macrofilaricidal agent or at least an agent which can sterilize filariae perma- nently, thus ... and antiviral activities (Gulati et al. 1994a ...
Tropical Medicine and International Health volume 2 no. 6 pp 535–543 june 1997
Antifilarial activity of a synthetic marine alkaloid, aplysinopsin (CDRI Compound 92/138)* Som Nath Singh1, Sunita Bhatnagar1, Nigar Fatma1, P. M. S. Chauhan2 and R. K. Chatterjee1 1 2
Division of Parasitology, Central Drug Research Institute, Lucknow, India Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow, India
Summary
CDRI Compound 92/138, a synthetic analogue of aplysinopsin, was evaluated in experimental filarial infections, Litomosoides carinii in cotton rats (Sigmodon hispidus) and Acanthocheilonema viteae in Mastomys coucha. The compound killed 63.8 and 90% of adult L. carinii and A. viteae at doses of 30 and 50 mg/kg (i.p.) respectively given for 5 days. By the oral route, at 100 mg/kg for 5 days the compound caused 50.9 and 57% mortality of adult L. carinii and A. viteae, respectively. At 200 mg/kg administered orally on days 0, 10 and 25 post-infection, it reduced establishment of adult A. viteae by 68.5%. We also found 43.7 and 37.8% effect in vivo respectively on L3 and L4 stages of A. viteae at a single dose of 250 mg/kg, p.o. The compound was active in vitro at 100 ìg/ml concentration and caused a significant decline in MTT reduction and 14C-glucose uptake by adult filariids. Thus synthetic marine aplysinopsin could provide a new pharmacophore for the development of antifilarial agents.
keywords aplysinopsin, antifilarial, L. carinii, A. viteae correspondence Dr R. K. Chatterjee, Division of Parasitology, Central Drug Research Institute, Lucknow 226001, India
Introduction Lymphatic filariasis is a major health problem in tropical and subtropical countries (Ottesen & Ramachandran 1995) and control of this infection is still dependent on the application of diethylcarbamazine (DEC), which is mainly a microfilaricidal agent with poor activity on adult parasites. The candidate drug ivermectin is also microfilaricidal (Campbell 1993, Schares et al. 1994) with side-effects similar to those of DEC (Kumaraswami et al. 1988; Fan 1992). The present-day requirement for filarial chemotherapy is a non-toxic macrofilaricidal agent or at least an agent which can sterilize filariae permanently, thus blocking further transmission of infection. Compounds acting against developing larvae *CDRI Communication No. 5386.
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would also be important for the control of infection in endemic areas where exposure to infective larvae through mosquito bites is a continuous process. In search of a new chemical lead for the development of a potent macrofilaricide, studies were carried out to explore the availability of materials from marine organisms, as marine flora and fauna have chemicals which differ greatly from terrestrial materials and constitute a valuable source for drug development (de Vries & Beart 1995). Synthetic analogues of aplysinopsin, isolated from the extracts of Australian barrier reef sponges, Thorecta aplysinopsis, Fascaplysinopsis reticulata and the Caribbian sponge, Verogia spongelli, are reported to have antileishmanial and antiviral activities (Gulati et al. 1994a,b). Here we report antifilarial activity of CDRI Compound 92/138, a synthetic derivative of aplysinopsin, against experimental filarial infections.
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Antifilarial activity of a synthetic analogue of aplysinopsin
Figure 1 Structure of CDRI Compound 92/138.
Materials and methods Experimental filarial infections Two rodent filarial species were used for assessing antifilarial efficacy. Six-weeks-old cotton rats (Sigmodon hispidus) were infected with Litomosoides carinii through exposure to infected mites (Liponyssus bacoti) (Lämmler et al. 1968). Six-week-old male Mastomys coucha were infected with Acanthocheilonema viteae by subcutaneous inoculation of 50 infective larvae (L3) isolated from experimentally infected ticks, Ornithodoros moubata (Sänger & Lämmler 1979; Singh et al. 1989).
days, against L. carinii in cotton rat and A. viteae in mastomys respectively. DEC citrate was administered only intraperitoneally against L. carinii and A. viteae at 6 and 50 mg/kg for 5 consecutive days respectively. The micro and macro-filaricidal activities of the agents were evaluated according to the method of Fatma et al. (1989). Five ìl of tail blood of animals were examined for microfilaraemia just before treatment and thereafter at weekly intervals up to day 42. Microfilaricidal action was calculated as the percentage reduction in microfilariae counts in comparison to pretreatment levels. To assess adulticidal (macrofilaricidal) action, treated and control animals were sacrificed on day 42 after the start of medication for recovery of adult parasites. The percentage recovery of live worms in treated animals was calculated from the number of worms recovered in control animals and formed the basis of assessment of macrofilaricidal action (Lämmler 1977). Parasites were also examined under the microscope for cell adhesion and surface damage if any. The condition of developing stages in uteri was examined by teasing all recovered worms individually in saline (0.9% NaCl) on glass slides. Female worms containing dead/degenerated stages of larvae or empty uteri were considered sterilized.
CDRI compound 92/138 The compound was prepared in the Medicinal Chemistry Division of this Institute (Figure 1) (Gulati et al. 1994a,b). In vivo activity evaluation Compound 92/138, 3[(2*-imino-3*-methyl-5*-oxo-4*imidazolidinylidene methyl)]-1-benzoyl-1H indole was administered through intraperitoneal (i.p.) or oral routes in the form of a fine suspension made in distilled water with 0.1% Tween-80. Experiments with each dose level were conducted 3 times using 3 animals on each occasion. The same numbers of identically infected animals were used as untreated controls. DEC citrate was used as the control drug in the study. By the oral route, compound 92/138 was used at 100 mg/kg for 5 consecutive days against both species of filaria whereas by the intraperitoneal route, it was administered at 30 and 50 mg/kg for 5
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Assessment of effect on larval stages of A. viteae (establishment of infection) The effect of the compound was also evaluated on developing stages of A. viteae broadly following the method of Lämmler (1977). In each set of experiments, 6-week-old males of Mastomys coucha were divided into 6 groups (5 animals in each group). Group 1 was treated with Compound 92/138 at a dose of 50 mg/kg i.p. for 3 days and Group 2 with 200 mg/kg p.o. also for 3 days. The first dose was administered before exposure to 50 infective larvae (L3) and the second and third doses were given on days 10 and 25. Group 3 was treated with a single dose of 250 mg/kg p.o. of the compound on day 0 to study the effect on L3, and Groups 4 and 5 were treated with a single dose of 250 mg/kg p.o. on days 10 and 25 of L3 exposure to study the effect on developing L4 and pre-adult stages. Animals of Group 6 exposed identically to 50 L3 remained untreated and served as the control group. After
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Antifilarial activity of a synthetic analogue of aplysinopsin
2 months, animals were sacrificed for worm recovery. The percentage change in parasite establishment was calculated over control. Three sets of the above experiments were performed.
In vitro effect of compound 92/138 on adult filariae Effect on motility Adult parasites (L. carinii and A. viteae) recovered from their respective hosts were incubated at 37)C with different concentrations of Compound 92/138 in RPMI-1640 medium containing 5.5 mm glucose and antibiotics (100 ìg streptomycin sulphate and 100 units of penicillin G per ml). Observations for motility of parasites were recorded at 30 minuteintervals up to 6 hours, and thereafter at hourlyintervals up to 12 hours and finally at 24 hours. Paralysed parasites were placed in fresh drug-free medium and observed for 1 hour. If the parasites did not revive, the condition was considered irreversible and the concentration lethal. For each concentration, 3 replicates each using 2 male and 2 female parasites were used and the process was repeated three times under identical conditions. MTT reduction assay The effect on viability of adult A. viteae and L. carinii was studied by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay following the method described by Comley et al. (1989). Briefly, after 6 hours of exposure to different concentrations of Compound 92/138, the parasites were further incubated for 30 minutes individually in 1 ml phosphate buffered saline (pH 7.4) containing 0.25 mg/ml MTT. Thereafter the parasites were washed and MTT formazan was solubilized in dimethyl sulphoxide (DMSO); optical density (OD) of purple blue DMSO after 2 hours was recorded against DMSO blank at 510 nm. Changes in OD compared to control parasites not exposed to drugs were calculated. Glucose uptake In vitro drug-exposed (6 hours) parasites were incubated for 10 minutes at 37)C in Hank’s balanced salt solution (HBSS) with pH 7.4 contain-
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ing 5.5 nm glucose, antibiotics (100 ìg streptomycin sulphate and 100 units of penicillin G/ml) and 1 ìCi 14 C-glucose. Thereafter the parasites were washed with ice-chilled saline containing 5.5 mm glucose and solubilized in 30% KOH. Radioactivity in alkali extracts was determined (Srivastava & Ghatak 1983) using a Rock Beta Liquid Scintillation Counter. Statistical analysis The Mann–Whitney U-test was used for statistical analysis. The test is applicable for n1 =3 and n2 =3 (Sidney 1956).
Results Compound 92/138 was effective on both microfilariae and adults of L. carinii in the cotton rat. At 30 mg/kg for 5 consecutive days (i.p.) and 100 mg/kg for 5 consecutive days (p.o.), the compound reduced the microfilarial count by 98.3 and 84.4% on day 8 since the start of treatment and reduced live worm recovery by 63.8% and 50.9% on day 42. Microfilarial counts increased later but remained below pretreatment levels. In the case of DEC, there were 98.7 and 57.3% reductions in mf count on days 8 and 21 respectively at 6 mg/kg i.p. for 5 days and no death of adult filariae occurred (Table 1). Against A. viteae, the compound caused 90 and 57% reductions in live worm recovery respectively at doses of 50 mg/kg for 5 days (i.p.) and 100 mg/kg for 5 days (p.o.). However, it did not show any microfilaricidal action, although a gradual reduction in microfilaraemia was detected (Table 2). DEC at 50 mg/kg for 5 days (i.p.) showed only a 35.7% reduction in microfilarial count against this infection (A. viteae) in M. coucha. Compound 92/138 caused a 68.5% reduction (P
