Oral administration of a 2-aminopyrimidine robenidine analogue (NCL195) significantly reduces Staphylococcus aureus infection and reduces Escherichia coli infection in combination with sub-inhibitory colistin concentrations in a bioluminescent mouse model.

We have previously reported promising in vivo activity of the first-generation 2-aminopyramidine robenidine analogue NCL195 against Gram-positive bacteria (GPB) when administered via the systemic route. In this study, we examined the efficacy of oral treatment with NCL195 (± low-dose colistin) in comparison to oral moxifloxacin in bioluminescent Staphylococcus aureus and Escherichia coli peritonitis-sepsis models. Four oral doses of 50 mg/kg NCL195, commencing immediately post-infection, were administered at 4 h intervals in the S. aureus peritonitis-sepsis model. We used a combination of four oral doses of 50 mg/kg NCL195 and four intraperitoneal doses of colistin at 0.125 mg/kg, 0.25 mg/kg, or 0.5 mg/kg in the E. coli peritonitis-sepsis model. Subsequently, the dose rates of four intraperitoneal doses of colistin were increased to 0.5 mg/kg, 1 mg/kg, or 2 mg/kg at 4 h intervals to treat a colistin-resistant E. coli infection. In the S. aureus infection model, oral treatment of mice with NCL195 resulted in significantly reduced S. aureus infection loads (P < 0.01) and longer survival times (P < 0.001) than vehicle-only treated mice. In the E. coli infection model, co-administration of NCL195 and graded doses of colistin resulted in a dose-dependent significant reduction in colistin-susceptible (P < 0.01) or colistin-resistant (P < 0.05) E. coli loads compared to treatment with colistin alone at similar concentrations. Our results confirm that NCL195 is a potential candidate for further preclinical development as a specific treatment for multidrug-resistant infections, either as a stand-alone antibiotic for GPB or in combination with sub-inhibitory concentrations of colistin for Gram-negative bacteria.

Robenidine Analogues Are Potent Antimalarials in Drug-Resistant Plasmodium falciparum.

Robenidine is a veterinary drug used in the poultry industry to treat coccidiosis caused by parasites in the Eimeria genus. Though this compound and related aminoguanidines have recently been studied in other pathogens, the chemotype has not been systematically explored to optimize antimalarial activity despite the close genetic relationship between Eimeria and Plasmodium (both are members of the Apicomplexa phylum of unicellular, spore-forming parasites). In this study, a series of aminoguanidine robenidine analogues was prepared and tested in vitro against Plasmodium falciparum, including multidrug-resistant strains. Selected compounds were further evaluated in vivo against murine Plasmodium yoelii in mice. Iterative structure-activity relationship studies led to the discovery of 1, an aminoguanidine with excellent activity against drug-resistant malaria in vitro and impressive in vivo efficacy with an ED50 value of 0.25 mg/kg/day in a standard 4-day test.

Herbal formulations as feed additives in the course of rabbit subclinical coccidiosis.

Two simultaneous experiments were carried out in a breeding farm of New Zealand White rabbits (Oryctolagus cuniculus f. domesticus) to determine the feasibility of replacing coccidiostats with garlic and oregano preparation. The research took place during June and July, the period of the greatest threat of coccidiosis caused by Eimeria spp. (Apicomplexa: Eimeriidae). In one investigation, 40 rabbits aged 1-3 months were divided into four groups of ten animals: Group A being a control which received no coccidiostats in feed, Group B receiving the coccidiostat Baycox in water once at weaning, Group C receiving the coccidiostat robenidine in feed, and group D receiving herbal extracts in feed. In the second trial, six mated females were allocated equally to three groups analogous to A, C, and D above during pregnancy and lactation. Bulk stool samples were collected from each group of rabbits at weekly intervals for coproscopic analysis, and the production results of the animals were recorded. In the young rabbits, both the faecal coccidia oocyst counts and body weight gains were more favourable in group D than the remaining groups. Also, the female rabbits of group D were the least infected. The results demonstrate that garlic and oregano feed additives exert a positive influence on the level and course of coccidia infection, with regard to maintaining a good level of animal productivity, and these herbal extracts appear to have potential value in coccidiosis prophylaxy.

Targeting the polyamine biosynthetic enzymes: a promising approach to therapy of African sleeping sickness, Chagas' disease, and leishmaniasis.

Trypanosomatids depend on spermidine for growth and survival. Consequently, enzymes involved in spermidine synthesis and utilization, i.e. arginase, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase, trypanothione synthetase (TryS), and trypanothione reductase (TryR), are promising targets for drug development. The ODC inhibitor alpha-difluoromethylornithine (DFMO) is about to become a first-line drug against human late-stage gambiense sleeping sickness. Another ODC inhibitor, 3-aminooxy-1-aminopropane (APA), is considerably more effective than DFMO against Leishmania promastigotes and amastigotes multiplying in macrophages. AdoMetDC inhibitors can cure animals infected with isolates from patients with rhodesiense sleeping sickness and leishmaniasis, but have not been tested on humans. The antiparasitic effects of inhibitors of polyamine and trypanothione formation, reviewed here, emphasize the relevance of these enzymes as drug targets. By taking advantage of the differences in enzyme structure between parasite and host, it should be possible to design new drugs that can selectively kill the parasites.

The story of CGP 40 215: studies on its efficacy and pharmacokinetics in African green monkey infected with Trypanosoma brucei rhodesiense.

CGP 40 215 is an inhibitor of S-adenosylmethionine decarboxylase, a key enzyme in trypanosomal polyamine biosynthesis. It is highly active against Trypanosoma brucei rhodesiense and T. b. gambiense in vitro and in the corresponding rodent models, and therefore was a promising candidate for further development as a new drug against human African trypanosomiasis. We conducted initial pharmacokinetic and efficacy studies in African green monkeys: based on two dose-finding studies, an infection-treatment and a pharmacokinetic study in eight monkeys infected with T. b. rhodesiense in the 1st stage of infection. PK analysis revealed curative drug levels in the serum but complete absence of the drug in the cerebrospinal fluid. No adverse effects of the drug were observed, although in rats CGP 40 215 had caused hypotension. The following PK parameters were calculated using a two-compartment model: t1/2=1.8 h, VSS/f=0.4 l/kg, CL/f=3.0 ml/min x kg and AUC=21 900 ng x h/ml. Six of the eight monkeys were cured, one animal relapsed on day 222 and one animal died of unknown reasons, but was aparasitaemic. The study confirmed the curative potential of CGP 40 215 for 1st stage T. b. rhodesiense infection. Unfortunately, it was also found that the compound did not pass the blood-brain barrier, a pre-requisite for cure of 2nd stage (CNS) infection. As the majority of sleeping sickness patients seeking treatment are in the 2nd stage of the disease, further development of the compound was stopped.

In vivo trypanocidal activities of new S-adenosylmethionine decarboxylase inhibitors.

A series of novel aromatic derivatives based on the structure of methylglyoxal bis(guanylhydrazone) (MGBG) was examined for trypanocidal activities in human and veterinary trypanosomes of African origin. One agent, CGP 40215A, a bicyclic analog of MGBG which also resembles the diamidines diminazene (Berenil) and pentamidine, was curative of infections by 19 isolates of Trypanosoma brucei subspecies as well as a Trypanosoma congolense isolate. Several of these isolates were resistant to standard trypanocides. Curative doses were < or = 25 mg/kg of body weight/day for 3 days in these acute laboratory model infections. In addition, CGP 40215A also cured a model central nervous system infection in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl, eflornithine). Curative combinations were 14 days of oral 2% DFMO (approximately 5 g/kg/day) plus 5, 10, or 25 mg/kg/day for 3 or 7 days given by intraperitoneal injection or with a miniosmotic pump. Combinations were most effective if CGP 40215A was given in the second half or at the end of the DFMO regimen. MGBG has modest activity as an inhibitor of trypanosome S-adenosylmethionine decarboxylase (50% inhibitory concentration [IC50]. 130 microM), while CGP 40215A was a more active inhibitor (IC50, 20 microM). Preincubation of trypanosomes with CGP 40215A for 1 h caused a reduction in spermidine content (36%) and an increase in putrescine content (20%), indicating that one possible mechanism of its action may be inhibition of polyamine biosynthesis.

Efficacy of diclazuril in the prevention and cure of intestinal and hepatic coccidiosis in rabbits.

The efficacy of diclazuril against intestinal and hepatic coccidiosis was studied in artificially infected rabbits. Prophylaxis against intestinal coccidiosis was evaluated using a mixed infection of Eimeria intestinalis, Eimeria magna and Eimeria perforans. Continuous medication in the feed at 1 p.p.m. was 100% effective in reducing oocyst output and faecal scores, and weight gain and feed efficiency were normal. Hepatic coccidiosis induced by Eimeria stiedai was prevented at 0.5 and 1 p.p.m. as shown by negative oocyst counts, normal liver weight, absence of liver lesions, and normal body-weight gain and feed efficiency. Medication at 1 p.p.m. for 7 consecutive days during the prepatent phase of hepatic coccidiosis resulted in large reductions in oocyst counts and lesion scores with a normal liver weight and growth performance. Diclazuril at 1 p.p.m. in the feed prevented both intestinal and hepatic coccidiosis in rabbits and can be advocated for safe mass medication.

Evolution of coccidial infection in commercial and domestic rabbits between 1982 and 1986.

Caecal samples were collected from 751 domestic rabbits of various origin and from 1229 diarrhoeic rabbits issued from 61 commercial rabbitries. They were screened for coccidiosis. In 1982, the year of introduction of the anticoccidial robenidine in commercial rabbit feeds, a dramatic decrease of coccidial infection ratio was detected in commercial rabbitries: only 6% of samples contained greater than 100 oocysts per gram against 85% in 1979, when sulphaquinoxaline/pyrimethamine was used. Only Eimeria magna, E. media and E. perforans were detected, whereas the highly pathogenic species E. flavescens and E. intestinalis had disappeared from commercial units. After 4 years of continuous use of robenidine, infection ratio rose progressively, although still far below the 1979 levels. Most of the other species reappeared, but only in very low proportions (1-4% of samples). The percentage occurrence of E. magna, E. media and E. perforans on the contrary rose progressively to 25, 26 and 34%, respectively, suggesting drug resistance. In domestic rabbitries, the incidence of coccidial infection was markedly higher and all nine species of Eimeria were detected. Eimeria magna, E. media and E. perforans were very common, E. flavescens, E. intestinalis, E. piriformis and E. stiedai were less common, whereas E. irresidua and E. coecicola were relatively rare. Notwithstanding the lower activity of robenidine against E. stiedai, no rise of hepatic coccidiosis became evident.

Chemotherapy of experimental toxoplasmosis with special reference to robenidine.

Robenidine as an anticoccidial agent alone (in doses of 50 and 100 mg/kg) or in association with two other antiprotozoal compounds--pyrimethamine and sulfadoxine (in doses of 2.5 and 250 mg/kg, respectively) were tested in the treatment of chronically infected mice with an avirulent cyst-forming HF strain of Toxoplasma gondii. The efficacy of the used drugs was evaluated by the cysts number in the brains of treated mice versus control group, antibody level and viability of survived cysts by the consecutive infection of mice. According to these criteria none of the tested drugs alone or combined exhibited to be able to produce an effective cure of cyst-stage of toxoplasmosis in experimental mice.

Eimeria stiedai in rabbits: the demonstration of responses to chemotherapy.

The activity of sulphaquinoxaline, robenidine, methyl benzoquate, clopidol and a mixture of methyl benzoquate and clopidol (Lerbek; Dow), was studied in rabbits infected with hepatic coccidiosis due to Eimeria stiedai. Growth inhibition, oocyst production and the activity in the serum of glutamate dehydrogenase and gamma glutamyltransferase were studied as indicators of parasite development. Only sulphaquinoxaline and Lerbek gave satisfactory control of this parasite. The latter formulation was more effective than either of its constituents used alone.

Robenidine treatment of rabbits naturally infected with coccidia.

New Zealand White rabbits naturally infected with Eimeria intestinalis, E. magna, E. media and E. perforans and fed 100 parts/10(6) robenidine in their diet for 70 days ceased to excrete oocysts in their faeces after 19 days of treatment.

Field trial with the coccidiostatics metichlorpindol and robenidine in a rabbit farm.

The effect of metichlorpindol and robenidine in broiler rabbits was examined. Administration of 125 ppm metichlorpindol did not reduce mortality and oocyst output significantly. In comparison with unmedicated rabbits, weight gain of metichlorpindol medicated rabbits was 4.76% lower after seven seeks of treatment, whereas feed conversion was improved by 1.87%. There was no effect on the production index. Medication with 33 and 66 ppm robenidine resulted in a significant reduction of mortality and oocyst output (0.05 > P > 0.01). The effect on oocyst reduction became already significant after two weeks of treatment. After four weeks both robenidine medicated groups gained significantly (0.05 > P > 0.01) more weight than metichlorpindol medicated and non medicated rabbits. After seven weeks of medication only the 66 ppm medicated group gained significantly (0.05 > P > 0.01) more weight than the 125 ppm metichlorpindol medicated group. In comparison with non medicated rabbits weight gain was 3.46% better. Feed conversion was improved by 6.76 and 6.55% at 33 and 66 ppm levels respectively, while the production index was improved by 19% and 22% respectively in comparison with non medicated and metichlorpindol medicated rabbits.

[Effectiveness of a number of anticoccidial agents. A brief survey taken in the field (author's transl)]. / De werkzaamheid van een aantal anticoccidiosismiddelen. Een momentopname in de praktijk.

A floor pen trial was carried out on broilers in 1975 to compare the anticoccidial efficacy of monensin1), 3,5 dinitro-o-toluamide2), clopidol3), amprolium + ethopabacte4), amprolium + ethopabate + sulfaquinoxaline + pyrimethamine5) and robenidine6) in the presence of untreated controls. 4,200 Hybro chicks were distributed over twenty-eight pens, each initially housing 150 birds. Four pen replicates were allocated to each of the seven treatments. With the exception of robenidine, all agents used resulted in a statistically significant (P less than or equal to 0,05) gain in final weight compared with the untreated controls. The feed conversion rate showed significant (P less than or equal to 0,05) improvement in the groups treated with monensin, robenidine, amprolium + ethopabate.

Robenidine resistance in Eimeria tenella.

Robenidine protected chickens against cecal coccidiosis infections initiated by a strain of the parasite that had no previous exposure to drugs. No cross resistance was found with 13 strains resistant to other anticoccidials. A strain of Eimeria tenella that was serially propagated in chickens fed mash containing robenidine became resistant to the chemical. No cross resistance was detected when this experimental strain was tested against 12 other anticoccidials.