Filaricidal efficacy of anthelmintically active cyclodepsipeptides

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis, Acanthocheilonema viteae and Brugia malayi. The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5 x 100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by > or =95%) determined 3 and 7 days after treatment were 3.125-6.25 and 6.25-12.5mg/kg, respectively. Using the spot on formulation, doses of 6.25mg/kg (L. sigmodontis), 12.5mg/kg (A. viteae) and 25mg/kg (B. malayi) were required to cause reductions of microfilaraemia levels by > or =95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100mg/kg independent of the route of administration). Repeated oral treatment (100mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi. However, single doses of 6.25 and 25mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi, respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.     

Fecundity of Litomosoides carinii (Nematoda, Filarioidea) in vivo and in vitro

Several parameters concerning the reproduction of Litomosoides carinii were assessed using quantitatively infected cotton rats (Sigmodon hispidus). The course of embryogenesis from the fertilization of eggs to the delivery of the first microfilariae was observed by daily autopsies during prepatency. The duration of embryogenesis in vivo could thus be determined as 18 +/- 2 days. The contents of embryos in the uteri of female worms had been examined at various intervals. At the onset of patency 7-8 weeks p.i. the females were 71 +/- 6 mm long and on average contained 308 X 10(3) embryos/female, of which 19% were pathologically altered. In the middle of patency 16-20 weeks p.i. the females had grown up to 100 +/- 11 mm in length and now contained 509 X 10(3) embryos/female, 25% of them were pathologically altered, the others were normally developed. A positive correlation between the body length of a female worm and its number of embryos in utero was evident. Additionally the percentage of pathologically altered embryos was increased with respect to the age of the worms. The calculated fecundity of a female L. carinii in vivo of around 20 X 10(3) microfilariae/female per day had been confirmed with worms maintained in vitro. Three combinations of media and serum supplements were used and their influence on embryogenesis evaluated.     

Experiments in vitro with Litomosoides carinii (Nematoda: Filarioidea). I. Maintenance of adult females and microfilariae as well as release of microfilariae in different culture media (author's transl)

Embryos of L. carinii continue intrauterine development to microfilariae and are totally released into the medium within 5--6 days when the latter (Tc 199) is changed daily and air is used as the gas phase. Oogenesis or further fertilization of eggs, however, does not occur in vitro in any of the media examined by us. One female releases 140 X 10(3) microfilaria/day on an average in vitro within 5--6 days. Mean initial numbers of 300 X 10(3) Mf/female/day are observed. Addition of equine serum inhibits microfilarial release in vitro; normal cotton rat serum prolongs survival of females while total numbers of released microfilariae or retained embryonic stages are not increased. The serum of post-patent animals does not influence the numbers of released microfilariae or their viability or survival of females. Microfilariae released in vitro in Tc 199 + 33% normal cotton rat serum survive for more than 8 days, when air is used as the gas phase and the medium is changed daily. Microfilariae isolated from the blood of patent animals survive for at most 6 days, at a 48-hourly change of medium survival does not even exceed 4 days.     

Notes and queries

Abstract

We investigated the renal protective effects of phophodiesterase type 5 (PDE5) inhibitors in mice with cyclosporine A (CyA; a calcineurin phosphatase inhibitor) induced nephrotoxicity. Fifty male mice were divided into five groups of 10. Group 1 received no treatment, group 2 received only saline orally, group 3 received 30 mg/kg/day CyA by subcutaneous injection, group 4 received only 30 mg/kg/day vardenafil orally, and group 5 received 30 mg/kg/day CyA by subcutaneous injection and 30 mg/kg/day vardenafil orally. At 28 days, platelet-derived growth factor A (PDGF-A) and C (PDGF-C), transforming growth factor-beta 1 (TGF-β1), cyclo-oxygenase 1 and 2 (COX-1 and COX-2), and P glycoprotein (Pgp) expression levels were measured in the renal tissues. In addition, expressions of COX-1 and COX-2 genes were determined using real-time PCR. PDE5 inhibitor administration ameliorated decreased PDGF-A and C, TGF-β1, COX-1 and -2, and Pgp expression levels by modulation of cyclic guanosine monophosphate (cGMP) activity in kidneys. The relative expressions of COX-1 and COX-2 genes to GAPDH revealed that the maximum increase was obtained in the group treated with CyA and vardenafil for both COX-1 and COX-2 genes. Our study revealed that long term oral treatment with vardenafil protects kidneys from CyA induced nephrotoxicity. We showed that long term oral treatment with PDE5 prevents pathological kidney changes caused by CyA induced nephrotoxicity.     

Dacarbazine induces genotoxic and cytotoxic germ cell damage with concomitant decrease in testosterone and increase in lactate dehydrogenase concentration in the testis

Treatment of cancers with cytotoxic agents such as alkylating drugs often, but not always results in transient to permanent testicular dysfunction. The present study was planned to investigate the effects of dacarbazine [5-(3,3-dimethyltriazeno) imidazole-4-carboxamide] on testicular function in mice. Swiss albino mice (9-12 weeks old) were treated with 0, 5, 25, 50, or 100mg/kg body weight/day dacarbazine (i.p.) for 5 days at intervals of 24h between treatments. Mice were sacrificed on days 7, 14, 21, 28, 35, 49, and 70 after the last treatment (6 mice/dose/sample time), and the epididymal sperm count, sperm motility, sperm morphology, testicular histopathology (qualitative histopathology, seminiferous tubular diameter and epithelial height), and intra-testicular levels of testosterone and lactate dehydrogenase were assessed. Dacarbazine decreased the body weight only on day 28 at 25mg/kg dose-level, but increased the paired testes weights at 50mg/kg on day 7, at 25-100mg/kg on day 14, and at 25 and 50mg/kg on day 21 (P<0.05-0.01; one-way ANOVA and Bonferroni's post hoc test). The sperm count was decreased on all sampling days except at 5 and 25mg/kg dose-levels on day 70, but with severe oligospermia on days 28 and 35 (P<0.05-0.001). The sperm motility was decreased at 100mg/kg on days 14 and 21, at 5, 25, and 100mg/kg on day 28, and at all dose-levels on day 35 (P<0.05-0.001). Dacarbazine induced both head and tail abnormalities and some sperms with cytoplasmic droplets, but significant increase was seen in all dose groups on days 14 and 21, and at 100mg/kg dose-level on day 35. Drug-induced epithelial sloughing was seen on days 14-35 and other histopathological changes observed were vacuoles and abnormal cells. The STD was increased at 25-100mg/kg on day 7, at all dose-levels on day 14, at 50-100mg/kg on days 21 and 28, but without any effects on days 35-70 (P<0.05-0.001), and the tubular lumen was found dilated. The SE was increased on days 7, 21 and 28 at 100mg/kg and on day 14 at 50-100mg/kg. Dacarbazine reduced the intra-testicular testosterone level at 100mg/kg on day 7, at 5, 50 and 100mg/kg on day 14, at all dose-levels on days 21, 28, and 35, and at 50mg/kg on day 49 (P<0.05-0.001). The intra-testicular lactate dehydrogenase concentration increased at all dose-levels up to day 35, but without any effect on days 49 and 70 (P<0.05-0.001). There was no particular dose-response of dacarbazine on any parameters tested. The sperm count (except on day 7-positive correlation; Pearson product moment correlation) or sperm motility did not have any relation but increase in abnormal sperms showed negative correlation with decrease in testosterone level on days 7, 21 and 28. Decrease in sperm count was in negative correlation on days 14 and 35, and increase in abnormal sperms showed positive correlation on day 35 with increase in LDH level. Finally, the decrease in sperm motility had no correlation with increase in abnormal sperm shapes. We conclude that dacarbazine is genotoxic and cytotoxic to the mouse testis in a transient fashion, and these effects are exerted along with decrease in testosterone and increase in lactate dehydrogenase levels in the testis.     

Fertilizing ability in vivo and in vitro of spermatozoa of rats and mice treated with alpha-chlorohydrin.

The fertilizing ability of epididymal spermatozoa from rats and mice treated for 3 or 4 or 9 or 10 days with various doses of alpha-chlorohydrin was tested in vitro, and in vivo by intrauterine insemination. The minimum doses (per kg/day) needed to affect fertilization significantly were: rat, in vitro, 8-8 mg for 3 or 4 days, 4-4 mg for 4 days and 2-7 mg for 9 or 10 days; in vivo, 4-4 mg for 3 or 4 days and 2-7 mg for 9 or 10 days: mouse, in vitro, 4-4 mg for 3 days and 13-3 mg for 9 days; in vivo, 44-2 mg for 3 days and 26-5 for 9 days. Rats were infertile for at least 18 days after receiving 44-2 mg alpha-chlorohydrin/kg/day for 3 days, but fertilizing ability, tested in vivo and in vitro, was restored 10-11 days and 15-18 days, respectively, after daily treatment with 11-1 mg alpha-chlorohydrin/kg for 3 days.     

Immunity to Litomosoides carinii in Mastomys natalensis. I. Effect of immunization with microfilariae and existing primary infections on the parasitaemia after microfilariae injection and challenge infection

Subcutaneous injections of intrauterine stages of Litomosoides carinii into Mastomys natalensis induced strong immunity to i.v. injected blood microfilariae. Immunity, developed after boostering with an i.p. and an i.v. injection of microfilariae, did not totally suppress the parasitaemia of a challenge infection but reduced significantly the microfilaraemia level. No effect was found on number and size of the worms of the challenge infection, the number of microfilariae or the number of leucocytes in the pleural cavity. Delayed type hypersensitivity reactions in challenged animals were similar to those in non-immunized, infected controls. Sera of immunized animals agglutinated microfilariae and mediated cell attachment to microfilariae. Challenge infections did not change this until the end of the fourth week post infection but sera taken 32 days after challenge and later failed to induce such reactions. Challenge infections performed 120 or 240 days after a primary infection did not increase the parasitaemia of recipients. Dissections carried out 130 days after the challenge showed that (a) the developmental rate of the challenge infection was reduced by about 50%; (b) the size of the challenge parasites was reduced; and (c) that these worms produced significantly less embryonic stages in comparison to worms of primary infections, of which about 90% were abnormal.     

Efficacy of mebendazole and albendazole against Trichinella spiralis in mice

Changes in the sensitivity of Trichinella spiralis to anthelmintic treatment during the first 3 days of infection in mice were studied. Oral administration of either mebendazole or albendazole at 6.25 mh/kg 2 hr after exposure to infection eliminated 95-100% of the worms as determined at necropsy on day 7 postinoculation. Beyond the first day of infection the sensitivity of the parasite to benzimidazole therapy was much reduced and an oral dose of 50 mg/kg was only partially but significantly active against the adult worms. Despite decline in drug sensitivity during the enteral phase, gavage administration of either mebendazole or albendazole at 50 mg/kg for 5 consecutive days during the invasive phase of infection significantly reduced (96 and 67%, respectively) the number of larvae subsequently recovered from host musculature on day 45 postinoculation.     

Benzofuranyl ureas with potent cardiovascular teratogenicity in rats

Studies of embryo-fetal development in rats were conducted with two 5-lipoxygenase inhibitors. SB-202235 (1,000 mg/kg/day) or SB-210661 (50, 100, or 500 mg/kg/day) was administered orally by gavage to female rats on days 6-17 postcoitus (pc) or days 7-16 pc. SB-202235 (1,000 mg/kg/day) and SB-210661 (100 mg/kg/day) reduced maternal body weight gain for the treatment period by 16% and 21%, respectively, relative to controls. SB-202235 (1,000 mg/kg/day) or SB-210661 (50 or 100 mg/kg/day), did not affect numbers of resorptions, dead or live fetuses/litter, but 500 mg/kg/day of SB-210661 caused 100% embryo lethality. SB-202235 (1,000 mg/kg/day) and SB-210661 (50 and 100 mg/kg/day) reduced fetal body weight by 15-30% and produced extensive cardiovascular malformations, as well as diaphragmatic hernias. SB-210661 also caused thymic abnormalities and cryptorchidism. Cardiovascular defects included abnormalities in aorticopulmonary septation, the aortic arch, pulmonary trunk, and ventricular septal defects are discussed relative to comparable human syndromes of cardiovascular malformation.     

Studies with Brugia pahangi 17. The anthelmintic effects of diethylcarbamazine.

Diethylcarbamazine (DEC) was active in vitro against infective larvae and microfilariae of Brugia pahangi but only at high concentrations. When fed to mosquitoes which were infected with B. pahangi it had little or no activity. In jirds it was inactive against B. pahangi microfilariae and adults when administered at 300 mg/kg for 5 days either by the intraperitoneal or oral route. In cats given 25 or 50 mg DEC/kg intraperitoneally on 3 or 5 occasions it was not microfilaricidal, but most of the adult worms died within 30 days of the end of treatment. Although most microfilariae disappeared from the blood of cats immediately (i.e., within an hour) after treatment, they reappeared within a few hours in the same numbers. Microfilarial levels were reduced after treatment but there was no precipitate decline as occurs in human B. malayi patients.     

Prolongation of canine pancreas allograft survival with cyclosporin A: preliminary report.

Studies were conducted on dogs to test the efficacy of cyclosporin A (CyA) in prolonging normoglycaemia and graft survival after whole-organ pancreas allograft transplantation. Five dogs subjected to pancreatectomy alone served as controls. Withholding immunosuppression after transplantation (five animals) resulted in the same median duration of survival as occurred in the controls (13 days). Azathioprine and steroids (seven animals) produced median durations of normoglycaemia and survival of 9 and 23 days respectively. Animals given CyA 18 mg/kg/day (five) and 25 mg/kg/day (10), however, showed median durations of normoglycaemia of 18 and 55 days (p less than 0.05 and p less than 0.02) respectively and median survival times of 36 and 85 days (NS and p less than 0.02). If CyA proved effective in controlling rejection of pancreas allografts in man it would offer unstable diabetics in renal failure a more hopeful outlook than conventional immunosuppression.     

Azithromycin: antimalarial profile against blood- and sporozoite-induced infections in mice and monkeys

The spectrum of antimalarial activity of the new macrolide antibiotic azithromycin was evaluated against blood- and sporozoite-induced infections with a chloroquine-resistant strain of Plasmodium yoelii nigeriensis (N-67) in Swiss mice and with simian parasite Plasmodium cynomolgi B in rhesus monkeys. Against experimental rodent malaria, a 70 mg/kg/day dose showed curative blood-schizontocidal activity in a four-dose regimen administered orally from day 0 to day 3 or from day 2 to day 5 to mice harboring established infection. The curative response was also obtained with a 40 mg/kg/day dose administered in an extended seven-dose (days 0-6) regimen. Azithromycin was also effective in the causal prophylactic test, since a 50 mg/kg dose from day -1 to day +2 protected mice against P. y. nigeriensis (N-67) sporozoite challenge. In comparison, erythromycin did not show either of the above activities up to a 405 mg/kg/day dose in identical regimens. Comparison of the ED(90) values showed that azithromycin was 31-fold more effective than erythromycin as a blood schizontocide. In the simian model, trophozoite-induced infections of P. cynomolgi B were cured with 25 mg/kg/day azithromycin administered for 7 days. In the causal prophylactic test, the prepatent period was significantly extended in monkeys challenged with P. cynomolgi B sporozoites, presumably because of the growth inhibition of preerythrocytic schizonts in hepatocytes. Azithromycin did not exhibit any hypnozoitocidal (dormant exoerythrocytic stages) activity at 25 mg/kg/day in a seven-dose regimen.     

Studies of potential filaricides: Part 14--Activity of 1-iso-butoxycarbonyl-4-methylpiperazine against experimental filariasis

The synthesis and filaricidal activity of 1-iso-butoxycarbonyl-4-methylpiperazine against Litomosoides carinii in Sigmodon hispidus and Dipetalonema viteae in Mastomys natalensis is reported. At an intraperitoneal or oral dose of 3 mg/kg given for 6 days, the compound removed 91% of the circulating microfilariae but had no effect on adult L. carinii. However, it killed all microfilariae and adults of D. viteae at a subcutaneous dose of 50 mg/kg given for 6 days. The compound also possessed chemoprophylactic activity against the larvae of L. carinii and D. viteae at a dose of 30 and 50 mg/kg respectively.     

Teratogenic action of platinum thymine blue

The teratogenic activity of the antitumor agent cisplatinum-2-thymine (platinum thymine blue) was investigated in rats. Pregnant Wistar-derived albino rats were given single ip injections of an aqueous solution of platinum thymine blue (PTB) at one day of pregnancy from day 5 through day 14 (sperm day=day 0). The dosages used ranged from 20 to 80 mg/kg maternal body weight. At autopsy (day 20) fetuses were recovered and subsequently examined for skeletal and soft-tissue abnormalities. PTB was embryolethal and teratogenic at several stages during rat gestation. Embryonic deat occured following all doses, and was dose dependent, except at day 5. The majority of malformed fetuses, however, were observed only after treatment at day 6 or 7 following injection with 50, 60, or 80 mg/kg. Eye defects were the predominant abnormality followed by hydrocephalus, gastroschisis, and ectopia cordis. The skeleton was only slightly affected. PTB is a potent inhibitor of DNA synthesis, but its mechanism of teratogenic action is unknown.     

Activity of natural and synthetic naphthoquinones against Toxoplasma gondii,in vitro and in murine models of infection

The effect of 14 natural and synthetic naphthoquinones in the replication of Toxoplasma gondii was evaluated. In vitro studies were accomplished in cultures of 2C4 fibroblasts infected with RH-strain. Enzyme-linked immunosorbent assay was used to quantify parasite growth. For the studies in vivo, mice were infected with tachyzoites of the RH strain or cysts of the T. gondii EGS strain. In vitro, seven naphthoquinones demonstrated significant inhibition of intracellular T. gondii growth at concentrations of 1 and 5 micrograms/ml. Only three compounds were significantly protective when tested in animals: 2-hydroxy-3'-(3'-pentenyl)-1,4-naphthoquinone (PHNQ4), 2-hydroxy-3-(1'-vinylphenyl)-1,4-naphthoquinone (PHNQ5), and 2-hydroxy-3-(1'-propen-3'-phenyl)-1,4-naphthoquinone (PHNQ6). In animals infected with the EGS strain and treated with PHNQ4 (50 mg/kg/day orally), a 7-day prolongation of the time to death was observed. Treatment with 100 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ5 resulted in a 5-day and 16-day prolongation of the time to death, respectively. Treatment with 50 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ6 resulted in a 4-day prolongation of the time to death or up to 30 days after treatment, respectively. Our results suggest that the naphthoquinones may be important therapeutic agents for the treatment of toxoplasmosis.     

Valproic acid developmental toxicity and pharmacokinetics in the rhesus monkey: an interspecies comparison

This study was undertaken to assess the developmental toxicity and drug distributional and metabolic characteristics of prenatal valproic acid (VPA) exposure in rhesus monkeys. Oral administration of 20-600 mg/kg/day VPA (approximately 1-15 X human therapeutic dose) to 33 animals on variable gestational days (GD) during organogenesis resulted in dose-dependent developmental toxicity manifested as increased embryo/fetal mortality, intrauterine growth retardation, and craniofacial and skeletal defects. Biphasic plasma elimination curves were observed for total and free VPA on the first (GD 21) and last (GD 50) days of treatment in the 100- and 200-mg/kg/day dose groups. VPA exhibited dose-independent elimination kinetics at the plasma concentrations observed in this study. There was no significant change in pharmacokinetic parameters (maternal plasma elimination rate, area under the curve, peak plasma concentration) between the first and last days of treatment at either dose level. Placental transfer studies indicated that embryos were exposed to half the free VPA concentrations present in maternal plasma on GD 37. Comparisons of interspecies sensitivity to VPA-induced developmental toxicity in the mouse, rat, monkey, and man are made.     

The influence of intracerebroventricular administration of (+/-) propranolol and (+/-) verapamil on experimental myocardial ischemia and necrosis in rats

In albino rats, infarctoid myocardial lesions were produced by intraperitoneal (i.p.) administration of isoproterenol (75 mg/kg, during 3 days). In other groups, the descending anterior left coronary artery was ligated. In both experimental settings, the intracerebroventricular (i.c.v.) administration of (+/-) propranolol (100-200-300 microg/animal/day, during 7 days) or (+/-) verapamil (40-80-160 microg/animal/day, during 7 days) afforded a significant protection (with the exception of the lowest dose) on the investigated parameters: arrhythmias, ischemic zone (in coronary ligated rats), lactate dehydrogenase and aspartate aminotransferase activity of the serum, focal necrosis (in isoproterenol treated rats). This protective activity is lower than that afforded by i.p. administered (+/-) propranolol (5 mg/kg, during seven days) or (+/-) verapamil (5 mg/kg, during seven days). From these data it may be concluded that (+/-) propranolol and (+/-) verapamil have a protective action on the experimental myocardial ischemia and necrosis in rats, not only when the drugs come in direct contact with the heart, but also acting upon the central nervous system.     

Therapy of filariasis in Tamarins.

A regimen of thiacetarsamide sodium (0.22 ml/kg twice daily for 2 days) plus levamisole phosphate (11 mg/kg/day for 10 days) was effective in eliminating unidentified microfilariae from the blood in seven of eight tamarins. No serious side effects resulted from the treatment. All of the animals were initially freed of circulating microfilariae after treatment, and five have remained microfilaria-negative for 1 year. Two of the tamarins died of causes unrelated to filariasis and were microfilaria-negative before death. One tamarin remained microfilaria-positive after two courses of this treatment.     

Use of coconut coir fibers as an inert solid support for production of cyclosporin A

In the present study, coconut coir was evaluated as an inert support for the production of cyclosporin A (CyA) using Tolypocladium inflatum MTCC 557 by solid state fermentation. Initially, four different inert supports such as coconut coir, polyurethane foam, polystyrene beads, and sugarcane baggase were screened using different production media as moistening agents for the maximum production of CyA. Different parameters such as fermentation time, carbon sources, moisture content, pH, and inoculum size were optimized. It was observed that coconut coir impregnated with medium modified with glycerol as carbon source, pH 6, at 80% moisture content, and inoculum size of 2.5 mL/2.5 g support produced 2641 mg/kg of CyA after 12 days as compared to 998 mg/kg before optimization. The yields were further increased to 3597 mg/kg substrate with addition of combination of amino acids after 48 h of fermentation.     

The use of 125I-HIPDM for studying tissue response due to toxic effects of cyclosporin-A in rats

¹²⁵I-HIPDM was used to study the response of various tissues in cyclosporin-A, CyA, treated and control rats. The rats were given 50 mg/kg of CyA for 7 consecutive days. The liver, kidney and heart showed significant increase while the spleen had a pronounced decrease in the uptake of ¹²⁵I-HIPDM in CyA treated compared to control rats. This difference in the uptake of ¹²⁵I-HIPDM between CyA treated and control rats is assumed to be the tissue response to toxic effects of CyA. The results indicate that CyA is toxic to liver, kidney, spleen and probably heart. There was no difference in the uptake of ¹²⁵I-HIPDM in the lung and brain of CyA treated and control rats. This lack of difference is assumed to indicate that CyA does not adversely affect the lung and brain.