In vitro synergistic activity of NCL195 in combination with colistin against Gram-negative bacterial pathogens.

In this study, the potential of using the novel antibiotic NCL195 combined with subinhibitory concentrations of colistin against infections caused by Gram-negative bacteria (GNB) was investigated. We showed synergistic activity of the combination NCL195 + colistin against clinical multidrug-resistant GNB pathogens with minimum inhibitory concentrations (MICs) for NCL195 ranging from 0.5-4 µg/mL for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, whereas NCL195 alone had no activity. Transmission electron microscopy of the membrane morphology of E. coli and P. aeruginosa after single colistin or combination drug treatment showed marked ultrastructural changes most frequently in the cell envelope. Exposure to NCL195 alone did not show any change compared with untreated control cells, whereas treatment with the NCL195 + colistin combination caused more damage than colistin alone. Direct evidence for this interaction was demonstrated by fluorescence-based membrane potential measurements. We conclude that the synergistic antimicrobial activity of the combination NCL195 + colistin against GNB pathogens warrants further exploration for specific treatment of acute GNB infections.

The antibiotic robenidine exhibits guanabenz-like cytoprotective properties by a mechanism independent of protein phosphatase PP1:PPP1R15A.

The aminoguanidine compound robenidine is widely used as an antibiotic for the control of coccidiosis, a protozoal infection in poultry and rabbits. Interestingly, robenidine is structurally similar to guanabenz (analogs), which are currently undergoing clinical trials as cytoprotective agents for the management of neurodegenerative diseases. Here we show that robenidine and guanabenz protect cells from a tunicamycin-induced unfolded protein response to a similar degree. Both compounds also reduced the tumor necrosis factor α-induced activation of NF-κB. The cytoprotective effects of guanabenz (analogs) have been explained previously by their ability to maintain eIF2α phosphorylation by allosterically inhibiting protein phosphatase PP1:PPP1R15A. However, using a novel split-luciferase-based protein-protein interaction assay, we demonstrate here that neither robenidine nor guanabenz disrupt the interaction between PPP1R15A and either PP1 or eIF2α in intact cells. Moreover, both drugs also inhibited the unfolded protein response in cells that expressed a nonphosphorylatable mutant (S51A) of eIF2α. Our results identify robenidine as a PP1:PPP1R15A-independent cytoprotective compound that holds potential for the management of protein misfolding-associated diseases.

Gram-Positive and Gram-Negative Antibiotic Activity of Asymmetric and Monomeric Robenidine Analogues.

Desymmetrisation of robenidine (1: N',2-bis((E)-4-chlorobenzylidene)hydrazine-1-carboximidhydrazide) and the introduction of imine alkyl substituents gave good antibiotic activity. Of note was the increased potency of two analogues against vancomycin-resistant Enterococci (VRE), one of which returned a MIC of 0.5 µg mL-1 . Five analogues were found to be equipotent or more potent than the lead 1. Introduction of an indole moiety resulted in the most active robenidine analogue against methicillin-resistant S. aureus (MRSA), with a MIC of 1.0 µg mL-1 . Imine C=NH isosteres (C=O/C=S) were inactive. Monomeric analogues were 16-64 µg mL-1 active against MRSA and VRE. An analogue that lacks the terminal hydrazide NH moiety showed modest Gram-negative activity at 64 µg mL-1 . A 4-tert-butyl analogue was shown to be active against both Gram-positive and -negative strains at 16-64 µg mL-1 . In general, additional modifications with aromatic moieties was poorly tolerated, except with concomitant introduction of an imine C-alkyl group. The activity of these analogues against MRSA and VRE ranged from 8 µg mL-1 to inactive (MIC>128 µg mL-1 ) with the naphthyl and indole analogues. Gram-negative activity was most promising with two compounds at 16 µg mL-1 against E. coli. Against P. aeruginosa, the highest activity observed was with MIC values of 32 µg mL-1 with another two analogues. Combined, these findings support the further development of the (E)-2-benzylidenehydrazine-1-carboximidamide scaffold as a promising scaffold for the development of antibiotics against Gram-positive and Gram-negative strains.

Evaluation of robenidine analog NCL195 as a novel broad-spectrum antibacterial agent.

The spread of multidrug resistance among bacterial pathogens poses a serious threat to public health worldwide. Recent approaches towards combating antimicrobial resistance include repurposing old compounds with known safety and development pathways as new antibacterial classes with novel mechanisms of action. Here we show that an analog of the anticoccidial drug robenidine (4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine; NCL195) displays potent bactericidal activity against Streptococcus pneumoniae and Staphylococcus aureus by disrupting the cell membrane potential. NCL195 was less cytotoxic to mammalian cell lines than the parent compound, showed low metabolic degradation rates by human and mouse liver microsomes, and exhibited high plasma concentration and low plasma clearance rates in mice. NCL195 was bactericidal against Acinetobacter spp and Neisseria meningitidis and also demonstrated potent activity against A. baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. in the presence of sub-inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA) and polymyxin B. These findings demonstrate that NCL195 represents a new chemical lead for further medicinal chemistry and pharmaceutical development to enhance potency, solubility and selectivity against serious bacterial pathogens.

Robenidine Analogues as Gram-Positive Antibacterial Agents.

Robenidine, 1 (2,2'-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC's of 8.1 and 4.7 µM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), and 4-C(CH3)3 (27) (23.7-71 µM) and with 3-Cl (3), 4-CH3 (21), and 4-CH(CH3)2 (26) (8.1-13.0 µM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH2OH moiety (75; 2,2'-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2'-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2'-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC's of 4.2-21.6 µM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T1/2 > 247 min, CLint < 7 µL/min/mg protein, and EH < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75.

Influence of three coccidiostats on the pharmacokinetics of florfenicol in rabbits.

In-feed Medication has been used for a long time to prevent coccidiosis, a worldwide protozoal disease in rabbits. Florfenicol (FFC) has been widely used in veterinary clinics for bacterial diseases treatment. Therefore, the use of combinations of coccidiostats with FFC in rabbits is common. In the present study, we aimed to evaluate the effect of three coccidiostats, sulfaquinoxaline (SUL), robenidine (ROB), and toltrazuril (TOL), as feed additives on the pharmacokinetic profile of FFC in rabbits. The disposition kinetics of FFC in rabbits were investigated after a single intravenous injection (25 mg/kg) in rabbits fed anticoccidial-free diets or feeds containing SUL (250 ppm), ROB (66 ppm), or TOL (2 ppm), respectively, for 20 days. Plasma FFC concentrations were determined by the high performance liquid chromatography (HPLC) method. The pharmacokinetic parameters of FFC were analyzed using a non-compartmental analysis based on the statistical moment theory. The results demonstrated that ROB feeding resulted in an obvious decrease in plasma FFC level as compared with anticoccidial-free feeding. The terminal elimination half-life (t1/2z), area under the concentration-time curve (AUC), area under the first moment curve (AUMC), and mean residence time (MRT) significantly decreased, whereas the elimination rate constant (λz) and total body clearance (CLz) obviously increased in rabbits pretreated with ROB. However, we did not find that SUL or TOL feeding had any effect on the pharmacokinetic profile of FFC. Our findings suggested that more attention should be paid to the use of FFC in rabbits supplemented with ROB.

In vitro destruction of Eimeria oocysts by essential oils.

This study aims to assess the ability of essential oils (EOs) to destroy Eimeria oocyst in vitro using microscopic counting and 273 nm absorbing material release. A screening for the ability of ten EOs to destroy Eimeria oocyst was carried out in liquid medium. Out of these ten, artemisia, tea tree, thyme and clove EOs were identified as being the most effective. The treatment of Eimeria oocyst with these EOs leads to their lysis as shown by the release of substances absorbing at 273 nm. These results were obtained after approximately three hours contact. Four EOs were proven to destroy Eimeria oocysts in a few hours at low concentration. This destructive effect is a consequence of their lysis. This work is a preliminary contribution aiming to develop a new generation of natural efficient agents for destroying Eimeria oocyst to fight coccidiosis in broiler chicken.

Fate and antibacterial potency of anticoccidial drugs and their main abiotic degradation products.

The antibacterial potency of eight anticoccidial drugs was tested in a soil bacteria bioassay (pour plate method), EC(50)-values between 2.4 and 19.6 microM were obtained; however, one compound, nicarbazin exhibited an EC(50)-value above the maximum tested concentration (21 microM, 9.1 mg L(-1)). The potency of mixtures of two of the compounds, narasin and nicarbazin, was synergistic (more than additive) with 10-fold greater antibacterial potency of the mixture than can be explained by their individual EC(50)-values. The influence of pH, temperature, oxygen concentration and light on the transformation of robenidine and salinomycin was investigated. Robenidine was transformed by photolysis (DT(50) of 4.1 days) and was unstable at low pH (DT(50) of approximately 4 days); salinomycin was merely transformed at low pH, the latter into an unknown number of products. The antibacterial potency of the mixtures of transformation products of robenidine after photolysis and at low pH was comparable with that of the parent compound. Finally five photo-transformation products of robenidine were structural elucidated by accurate mass measurements, i-FIT values (isotopic pattern fit) and MS/MS fragmentation patterns.

Effect of subcutaneously administered diclazuril on the output of Eimeria species oocysts by experimentally infected rabbits.

The effect of subcutaneously injected diclazuril on the output of Eimeria species oocysts was studied in experimentally infected rabbits. Diclazuril was administered either prophylactically at 0.5, 1 or 2 mg/kg bodyweight two days before each rabbit was inoculated with 20,000 oocysts of a mixed-species field isolate of Eimeria or therapeutically at 1, 2 or 4 mg/kg bodyweight five days after they were inoculated. The prophylactic treatments significantly reduced (P<0.05) the output of oocysts in faeces and the numbers in the rabbits' livers at all doses. The therapeutic treatment at 4 mg/kg also caused a significant reduction (P<0.05) in oocyst shedding, but the lower doses resulted in only a moderate reduction. The shedding of the pathogenic species Eimeria stiedae, Eimeria magna, Eimeria irresidua, Eimeria flavescens, Eimeria piriformis and Eimeria intestinalis was significantly reduced (P<0.05) in all the treated groups. The burden of oocysts in the livers of the therapeutic groups (4000 to 9000) were significantly lower (P<0.05) than in the inoculated but untreated control group (23,000), but higher than in the prophylactic groups (around 1000).

Mechanisms of allosteric regulation of Trypanosoma cruzi S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.

3-Aminooxy-1-aminopropane and derivatives have an antiproliferative effect on cultured Plasmodium falciparum by decreasing intracellular polyamine concentrations.

The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with K(i) values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 microM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.

Strong binding in the DNA minor groove by an aromatic diamidine with a shape that does not match the curvature of the groove.

A combination of biophysical techniques has been used to characterize the interaction of an antitrypanosomal agent, CGP 40215A, with DNA. The results from a broad array of methods (DNase I footprinting, surface plasmon resonance, X-ray crystallography, and molecular dynamics) indicate that this compound binds to the minor groove of AT DNA sequences. Despite its unusual linear shape that is not complementary to that of the DNA groove, a high binding affinity was observed in comparison with other similar but more curved diamidine compounds. The amidine groups at both ends of the ligand and the -NH groups on the linker are involved in extensive and dynamic H-bonds to the DNA bases. Complementary and consistent results were obtained from both the X-ray and molecular dynamics studies; both of these methods reveal direct and water-mediated H-bonds between the ligand and the DNA.

Studies of resistance to anticoccidials in Eimeria field isolates and pure Eimeria strains.

Ten Eimeria field isolates from North Germany were studied in battery tests for sensitivity to selected anticoccidials. A high percentage of the Eimeria field isolates (9 out of 10) showed resistance to anticoccidials, mostly multiple resistance. Partial or complete resistance to maduramicin was found in 7 field isolates, to monensin in 6, to salinomycin in 5, to nicarbazin in 8, to halofuginone in 7, to robenidine and toltrazuril in 1, and to diclazuril in 2 field isolates. Multiple resistance had developed in 7 of the 10 isolates. Cross-resistance between maduramicin, monensin, and salinomycin occurred in 5 Eimeria isolates. One isolate showed cross-resistance between diclazuril and toltrazuril. From the resistant isolates 15 pure E. acerculina and 5 pure E. brunetti strains were obtained by single oocyst infections. Seven of the E. acerculina and 4 of the E. brunetti strains showed resistance or partial resistance that was also present in the original isolate. Ten of 11 resistant strains were multiply resistant.

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.

Antileishmanial effect of a potent S-adenosylmethionine decarboxylase inhibitor: CGP 40215A.

CGP 40215A, specific S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor was found to inhibit the growth of Leishmania donovani promastigotes (strain UR6) in a dose-dependent manner with an IC50 of 18 microM. The growth inhibition was reversed with 100 microM of spermidine and spermine. The growth inhibition in vitro by this inhibitor was accompanied by a significant decrease in AdoMetDC activity and spermidine levels. CGP 40215A was more potent than other AdoMetDC inhibitors, Berenil or methyl glyoxal (bis) guanyl hydrazone. The combination of CGP 40215A with other polyamine biosynthetic inhibitors like DL-alpha-difluoromethylornithine or the bis (benzyl) polyamine analogue (MDL 27695) shows an accentuated inhibitory effect on leishmanial growth.

Sensitivity of Escherichia coli O157:H7 strain 932 to selected anticoccidial drugs in broiler chicks.

The ability of selected anticoccidial drugs to inhibit the colonization of day-old male broiler chicks (Cornish Rocks) by Escherichia coli O157:H7, strain 932 was examined. Chicks were challenged with 1.8 x 10(9) E. coli O157:H7 on Day 1, and fed diets supplemented with three selected anticoccidial drugs; monensin, nicarbazin, or robenidine. The cecal and colon fecal contents of the chicks were removed on Day 7, 14, and 21 postinoculation and examined for the concentration of E. coli O157: H7 per gram of contents. Monensin effectively reduced cecal and colon colonization of the chicks by E. coli O157:H7. By Day 7, there were significant reductions in the bacterial population of the cecal contents of chicks receiving the monensin-medicated feed, and by Day 21 no E. coli O157:H7 was recovered from the cecal and colon contents. The bacterial counts in the colon contents of the nicarbazin- and robenidine-medicated and unmedicated chicks were significantly higher than the monensin-treated chicks. Bacterial populations in the colon contents were high only when there were high bacterial concentrations in the cecal contents.

Action of robenidine on the intestinal transport and digestion of nutrients in rabbit.

Robenidine is an anticoccidial guanidine used as an additive in rabbit fodder. Because its action is restricted to the small intestine, the present work addresses the question whether robenidine affects the growth of the animals, sugar and amino acid intestinal transport and membrane-bound intestinal digestion. For this purpose we have determined the intestinal transport of the substrates, and the enzymatic activity of neutral aminopeptidase and sucrase. We have found that robenidine diminishes the tissue accumulation of L-leucine and D-galactose at long incubation times, and increases the transepithelial mucosal to serosal flux of both substrates. These results suggest that robenidine may stimulate the enterocyte basolateral membrane flux of sugars and neutral amino acids. These results have been corroborated by means of isolated brush border and basolateral membrane vesicles. Apart from these effects, robenidine has also been shown to increase the enzymatic activity of neutral aminopeptidase and sucrase and thus resulting in a better digestion of nutrients.

The effects of shuttle programs upon the growth of broilers and the development of immunity to eimeria species.

A floor-pen trial was conducted to investigate the effects of different shuttle programs upon the growth of broilers to 8 wk of age. Nicarbazin, halofuginone, and robenidine, when included in the starter feed for 3 wk, were effective in preventing lesions due to Eimeria. The effects of medication upon performance were apparent, medicated groups gaining more weight by 6 wk and having a lower feed conversion at 6, 7, and 8 wk than the unmedicated controls. There were no significant differences in body weight at 6, 7, or 8 wk or feed conversion at 6 or 7 wk among the medicated groups, whether medication was withdrawn for 7 or 14 days. A decrease in the number of small and medium oocysts in the litter was observed as the trial progressed. Few large oocysts (Eimeria maxima) were seen in the medicated groups. Numbers of oocysts did not increase following withdrawal of medication. Birds from all medicated groups were challenged at 6 wk with oocysts of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. Weight gains were similar to that of the unchallenged controls, indicating that they had acquired immunity to these species of Eimeria.

Sensitivity of field isolates of Eimeria tenella to anticoccidial drugs in the chicken.

Thirty isolates of Eimeria tenella obtained from broiler and breeder farms were examined for their sensitivity to anticoccidial drugs. All were sensitive to robenidine, 28 were sensitive to methyl benzoquate, 25 to clopidol and 21 to nicarbazin. Most isolates were resistant or partly resistant to amprolium and dinitolmide.