NP213 (Novexatin®): A unique therapy candidate for onychomycosis with a differentiated safety and efficacy profile.

NP213 (Novexatin®) is a novel antifungal peptide specifically designed for the topical treatment of onychomycosis. NP213 was designed using host defense peptides (HDP), essential components of the innate immune response to infection, as a template. NP213 is a water-soluble cyclic fungicidal peptide that effectively penetrates human nail. NP213 demonstrated a promising preclinical and clinical safety profile, with no evidence of systemic exposure following topical application to the skin and nails. NP213 was efficacious in two phase IIa human trials with 43.3% of patients having no fungi detectable by culture of fragments from NP213-treated nails after 180 days in the first study and likewise 56.5% of patients were culture negative for dermatophytes after 360 days in the second phase IIa study. In both trials, NP213 was applied daily for only 28 days in marked contrast to other topical onychomycosis treatments that require application for up to 52 weeks. Patient reported outcomes from the phase IIa studies were positive with participants recording an improved appearance of their nails after only 14 days of application. All fungi identified in these studies were Trichophyton spp. NP213 (Novexatin®) is a promising, highly differentiated peptide-based candidate for the topical treatment of onychomycosis, addressing the infectious cause and cosmetic issues of this very common condition.

Improved Methods for Assessing Therapeutic Potential of Antifungal Agents against Dermatophytes and Their Application in the Development of NP213, a Novel Onychomycosis Therapy Candidate.

Onychomycosis is a common, difficult-to-treat nail infection that is mainly caused by dermatophytes. Current therapies are not wholly effective and are associated with manifold side effects. The development of treatments for onychomycosis is challenging because standard in vitro tests are not predictive of antifungal efficacy within the nail. We have developed a new antifungal agent, NP213, for the treatment of onychomycosis. NP213 is based on endogenous host defense peptides produced within the nail. We compared the in vitro activity of NP213 and existing antifungal agents using conventional antimicrobial susceptibility test (AST) systems and more physiologically relevant models based on the human nail. We observed that the standard in vitro AST methodologies failed to predict the efficacy of antifungal agents within the nail. To address that, we present a more physiologically relevant modified AST method. This method, alongside other standard in vitro assessments of activity (including mechanism-of-action and time-of-kill studies), better reflected the activity of NP213 and other antifungal agents within the nail than standard in vitro AST methods. NP213 is a rapidly acting, fungicidal peptide that is superior to existing antifungal agents in vitro It penetrated the nail more effectively than other antifungals, as confirmed by using an optimized in vitro nail infection model. The data presented here support the current clinical development status of NP213 as a novel agent for treating onychomycosis. We propose that the modified tests developed and applied for NP213 characterization are the most relevant to use for screening any potential therapeutic candidates for onychomycosis.

Keratin hydrolysis by dermatophytes.

Dermatophytes are the most common cause of superficial fungal infections (tinea infections) and are a specialized group of filamentous fungi capable of infecting and degrading keratinised tissues, including skin, hair, and nail. Essential to their pathogenicity and virulence is the production of a broad spectrum of proteolytic enzymes and other key proteins involved in keratin biodegradation and utilization of its breakdown products. The initial stage of biodegradation of native keratin is considered to be sulfitolysis, in which the extensive disulfide bridges present in keratin are hydrolyzed, although some secreted subtilisins can degrade dye-impregnated keratin azure without prior reduction (Sub3 and Sub4). Sulfitolysis facilitates the extracellular biodegradation of keratin by the dermatophytes' extensive array of endo- and exoproteases. The importance of dermatophyte proteases in infection is widely recognized, and these enzymes have also been identified as important virulence determinants and allergens. Finally, the short peptide and amino acid breakdown products are taken up by the dermatophytes, using as yet poorly characterised transporters, and utilized for metabolism. In this review, we describe the process of keratin biodegradation by dermatophytes, with an especial focus on recent developments in cutting edge molecular biology and '-omic' studies that are helping to dissect the complex process of keratin breakdown and utilization.

A prodrug approach to the use of coumarins as potential therapeutics for superficial mycoses.

Superficial mycoses are fungal infections of the outer layers of the skin, hair and nails that affect 20-25% of the world's population, with increasing incidence. Treatment of superficial mycoses, predominantly caused by dermatophytes, is by topical and/or oral regimens. New therapeutic options with improved efficacy and/or safety profiles are desirable. There is renewed interest in natural product-based antimicrobials as alternatives to conventional treatments, including the treatment of superficial mycoses. We investigated the potential of coumarins as dermatophyte-specific antifungal agents and describe for the first time their potential utility as topical antifungals for superficial mycoses using a prodrug approach. Here we demonstrate that an inactive coumarin glycone, esculin, is hydrolysed to the antifungal coumarin aglycone, esculetin by dermatophytes. Esculin is hydrolysed to esculetin ß-glucosidases. We demonstrate that ß-glucosidases are produced by dermatophytes as well as members of the dermal microbiota, and that this activity is sufficient to hydrolyse esculin to esculetin with concomitant antifungal activity. A ß-glucosidase inhibitor (conduritol B epoxide), inhibited antifungal activity by preventing esculin hydrolysis. Esculin demonstrates good aqueous solubility (<6 g/l) and could be readily formulated and delivered topically as an inactive prodrug in a water-based gel or cream. This work demonstrates proof-of-principle for a therapeutic application of glycosylated coumarins as inactive prodrugs that could be converted to an active antifungal in situ. It is anticipated that this approach will be applicable to other coumarin glycones.

Anti-inflammatory implications of the microbial transformation of dietary phenolic compounds.

Due to the success of therapeutic anti-inflammatory compounds to inhibit, retard, and reverse the development of colon cancer, the identification of dietary compounds as chemopreventives is being vigorously pursued. However, an important factor often overlooked is the metabolic transformation of the food-derived compounds in the gut that may affect their bioactivity. Commonly consumed dietary phenolics (esterified ferulic acid and its 5-5'-linked dimer), which have the potential to undergo predominant microbial transformations (de-esterification, hydrogenation, demethylation, dehydroxylation, and dimer cleavage), were incubated with human microbiota. The metabolites were identified (high-performance liquid chromatography and nuclear magnetic resonance) and confirmed to be present in fresh fecal samples from 4 human volunteers. The potential anti-inflammatory properties were compared by measuring the ability of the parent compounds and their metabolites to modulate prostanoid production in a cell line in which the inflammatory pathways were stimulated following a cytokine-induced insult. The compounds were readily de-esterified and hydrogenated, but no dimer cleavage occurred. Only the monomer underwent demethylation and selective de-hydroxylation. The resultant metabolites had differing effects on prostanoid production ranging from a slight increase to a significant reduction in magnitude. This suggests that the microbial transformation of dietary compounds will have important inflammatory implications in the chemoprevention of colon cancer.

Contribution of acetate to butyrate formation by human faecal bacteria.

Acetate is normally regarded as an endproduct of anaerobic fermentation, but butyrate-producing bacteria found in the human colon can be net utilisers of acetate. The butyrate formed provides a fuel for epithelial cells of the large intestine and influences colonic health. [1-(13)C]Acetate was used to investigate the contribution of exogenous acetate to butyrate formation. Faecalibacterium prausnitzii and Roseburia spp. grown in the presence of 60 mm-acetate and 10 mm-glucose derived 85-90 % butyrate-C from external acetate. This was due to rapid interchange between extracellular acetate and intracellular acetyl-CoA, plus net acetate uptake. In contrast, a Coprococcus-related strain that is a net acetate producer derived only 28 % butyrate-C from external acetate. Different carbohydrate-derived energy sources affected butyrate formation by mixed human faecal bacteria growing in continuous or batch cultures. The ranking order of butyrate production rates was amylopectin > oat xylan > shredded wheat > inulin > pectin (continuous cultures), and inulin > amylopectin > oat xylan > shredded wheat > pectin (batch cultures). The contribution of external acetate to butyrate formation in these experiments ranged from 56 (pectin) to 90 % (xylan) in continuous cultures, and from 72 to 91 % in the batch cultures. This is consistent with a major role for bacteria related to F. prausnitzii and Roseburia spp. in butyrate formation from a range of substrates that are fermented in the large intestine. Variations in the dominant metabolic type of butyrate producer between individuals or with variations in diet are not ruled out, however, and could influence butyrate supply in the large intestine.

Effects of esculin and esculetin on the survival of Escherichia coli O157 in human faecal slurries, continuous-flow simulations of the rumen and colon and in calves.

The human pathogen Escherichia coli O157:H7 is thought to be spread by direct or indirect contact with infected animal or human faeces. The present study investigated the effects of the plant coumarin esculin and its aglycone esculetin on the survival of a strain of E. coli O157 under gut conditions. The addition of these compounds to human faecal slurries and in vitro continuous-flow fermenter models simulating conditions in the human colon and rumen caused marked decreases in the survival of an introduced strain of E. coli O157. When four calves were experimentally infected with E. coli O157 and fed esculin, the pathogen was detected in five of twenty-eight (18 %) of faecal samples examined post-inoculation, compared with thirteen of thirty-five (37 %) of faecal samples examined from five control calves not fed esculin. Coumarin compounds that occur naturally in dietary plants or when supplemented in the diet probably inhibit the survival of E. coli O157 in the gut.

Oxalobacter formigenes and its role in oxalate metabolism in the human gut.

Oxalate is ingested in a wide range of animal feeds and human foods and beverages and is formed endogenously as a waste product of metabolism. Bacterial, rather than host, enzymes are required for the intestinal degradation of oxalate in man and mammals. The bacterium primarily responsible is the strict anaerobe Oxalobacter formigenes. In humans, this organism is found in the colon. O. formigenes has an obligate requirement for oxalate as a source of energy and cell carbon. In O. formigenes, the proton motive force for energy conservation is generated by the electrogenic antiport of oxalate(2-) and formate(1-) by the oxalate-formate exchanger, OxlT. The coupling of oxalate-formate exchange to the reductive decarboxylation of oxalyl CoA forms an 'indirect' proton pump. Oxalate is voided in the urine and the loss of O. formigenes may be accompanied by elevated concentrations of urinary oxalate, increasing the risk of recurrent calcium oxalate kidney stone formation. Links between the occurrence of nephrolithiasis and the presence of Oxalobacter have led to the suggestion that antibiotic therapy may contribute to the loss of this organism from the colonic microbiota. Studies in animals and human volunteers have indicated that, when administered therapeutically, O. formigenes can establish in the gut and reduce the urinary oxalate concentration following an oxalate load, hence reducing the likely incidence of calcium oxalate kidney stone formation. The findings to date suggest that anaerobic, colonic bacteria such as O. formigenes, that are able to degrade toxic compounds in the gut, may, in future, find application for therapeutic use, with substantial benefit for human health and well-being.

Isolation, growth on prebiotics and probiotic potential of novel bifidobacteria from pigs.

Bifidobacteria were isolated from the faeces of pigs of various ages and examined for their potential use as probiotics in combination with di- and oligosaccharides. Ninty-six per cent of the isolates were found to have characteristics in common with Bifidobacterium boum, B. thermophilum and B. choerinum. B. thermophilum was most commonly isolated from sows, whereas most of the other strains were isolated from piglets. A few strains of each species were able to grow in the presence of air. A microplate assay was modified to allow comparison of growth on different substrates. Di- and oligosaccharides considered to promote bifidobacteria were screened for their ability to support growth of selected isolates in vitro. Growth on these substrates varied within and between species. Of the fructose oligosaccharides tested, Actilight P supported the best growth of the widest range of strains. The strains which grew best on the disaccharide lactulose were related to B. choerinum and some of these strains grew on xylo-oligosaccharides. It seems that prebiotic di- and oligosaccharides may have both a species and intra-species/strain selective effect. B. choerinum appeared to be well adapted to the gut of pre-weaned piglets.

Effects of alternative dietary substrates on competition between human colonic bacteria in an anaerobic fermentor system.

Duplicate anaerobic fermentor systems were used to examine changes in a community of human fecal bacteria supplied with different carbohydrate energy sources. A panel of group-specific fluorescent in situ hybridization probes targeting 16S rRNA sequences revealed that the fermentors supported growth of a greater proportion of Bacteroides and a lower proportion of gram-positive anaerobes related to Faecalibacterium prausnitzii, Ruminococcus flavefaciens-Ruminococcus bromii, Eubacterium rectale-Clostridium coccoides, and Eubacterium cylindroides than the proportions in the starting fecal inoculum. Nevertheless, certain substrates, such as dahlia inulin, caused a pronounced increase in the number of bacteria related to R. flavefaciens-R. bromii and E. cylindroides. The ability of three strictly anaerobic, gram-positive bacteria to compete with the complete human fecal flora was tested in the same experiment by using selective plating to enumerate the introduced strains. The Roseburia-related strain A2-183(F) was able to grow on all substrates despite the fact that it was unable to utilize complex carbohydrates in pure culture, and it was assumed that this organism survived by cross-feeding. In contrast, Roseburia intestinalis L1-82(R) and Eubacterium sp. strain A2-194(R) survived less well despite the fact that they were able to utilize polysaccharides in pure culture, except that A2-194(R) was stimulated 100-fold by inulin. These results suggest that many low-G+C-content gram-positive obligate anaerobes may be selected against during in vitro incubation, although several groups were stimulated by inulin. Thus, considerable caution is necessary when workers attempt to predict the in vivo effects of probiotics and prebiotics from their effects in vitro.

The microbiology of butyrate formation in the human colon.

Butyrate arising from microbial fermentation is important for the energy metabolism and normal development of colonic epithelial cells and has a mainly protective role in relation to colonic disease. While certain dietary substrates such as resistant starch appear to be butyrogenic in the colon, it is not known to what extent these stimulate butyrate production directly, e.g. by promoting amylolytic species, or indirectly, e.g. through cross-feeding of fermentation products. Cultural and molecular studies indicate that the most numerous butyrate-producing bacteria found in human faeces are highly oxygen-sensitive anaerobes belonging to the Clostridial clusters IV and XIVa. These include many previously undescribed species related to Eubacterium, Roseburia, Faecalibacterium and Coprococcus whose distribution and metabolic characteristics are under investigation. A better understanding of the microbial ecology of colonic butyrate-producing bacteria will help to explain the influence of diet upon butyrate supply, and to suggest new approaches for optimising microbial activity in the large intestine.

Roseburia intestinalis sp. nov., a novel saccharolytic, butyrate-producing bacterium from human faeces.

Five strains of butyrate-producing, anaerobic, gram-positive bacteria were isolated from human faecal material. These strains were slightly curved rods that showed motility by means of multiple subterminal flagella. The DNA G + C content of the strains was 29-31 mol%. A detailed investigation of the phenotypic and phylogenetic characteristics of the strains revealed that they represent a novel species of anaerobic, low-G+C-content, butyrate-producing bacterium that shows net acetate utilization during growth on media containing carbohydrates and short-chain fatty acids. The 16S rRNA gene sequences of the five isolates were determined and they confirmed that these strains were closely related to each other. Phylogenetic analysis indicated that the most closely related species are Eubacterium rectale, Eubacterium oxidoreducens and Roseburia cecicola, members of cluster XIVa of the Clostridium subphylum of gram-positive bacteria, although they share less than 95% sequence identity with the novel strains. It is proposed that a novel species, Roseburia intestinalis sp. nov., be created, with strain L1-82T (= DSM 14610T = NCIMB 13810T) as the type strain.

Acetate utilization and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate-producing bacteria from the human large intestine.

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.

Oxalobacter formigenes and its potential role in human health.

Oxalate degradation by the anaerobic bacterium Oxalobacter formigenes is important for human health, helping to prevent hyperoxaluria and disorders such as the development of kidney stones. Oxalate-degrading activity cannot be detected in the gut flora of some individuals, possibly because Oxalobacter is susceptible to commonly used antimicrobials. Here, clarithromycin, doxycycline, and some other antibiotics inhibited oxalate degradation by two human strains of O. formigenes. These strains varied in their response to gut environmental factors, including exposure to gastric acidity and bile salts. O. formigenes strains established oxalate breakdown in fermentors which were preinoculated with fecal bacteria from individuals lacking oxalate-degrading activity. Reducing the concentration of oxalate in the medium reduced the numbers of O. formigenes bacteria. Oxalate degradation was established and maintained at dilution rates comparable to colonic transit times in healthy individuals. A single oral ingestion of O. formigenes by adult volunteers was, for the first time, shown to result in (i) reduced urinary oxalate excretion following administration of an oxalate load, (ii) the recovery of oxalate-degrading activity in feces, and (iii) prolonged retention of colonization.

Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov.

Two newly isolated strains of obligately anaerobic bacteria from human faeces are shown here to be related to Fusobacterium prausnitzii, which is regarded as one of the most abundant colonizers of the human colon. These strains, along with Fusobacterium prausnitzii ATCC 27768(T) and 27766, are non-motile and produce butyrate, formate and lactate, but not hydrogen as fermentation products. A new finding is that all four strains produce D-lactate, but not L-lactate. The strains have a requirement for acetate in the growth medium and this may account for the previously reported requirement for rumen fluid. The DNA G+C content of the four strains is 47-57 mol%. Together with phylogenetic analysis based on 16S rRNA sequencing, this establishes that Fusobacterium prausnitzii strains are only distantly related to Fusobacterium sensu stricto and are more closely related to members of Clostridium cluster IV (the Clostridium leptum group). It is proposed that a new genus, Faecalibacterium gen. nov. be created; this genus should include Faecalibacterium prausnitzii gen. nov., comb. nov. ATCC 27768(T) (= NCIMB 13872(T)) (formerly Fusobacterium prausnitzii) as the type species together with ATCC 27766 and the newly isolated strains A2-165 and L2-6.