GB2546534A - Culture medium - Google Patents

Abstract

A culture medium comprising a suspension of powdered nail for the growth of microbes, particularly pathogenic fungi such as Trichophyton, Microsporum or Epidermophyton. The medium may be used to test the efficacy of antimicrobial agents.

Description

CULTURE MEDIUM

Field of the Invention

The present invention relates to a culture medium for the culture of microbes causing nail infections. It further relates to use of the medium in culturing said microbes and to use of the medium in determining the efficacy of antimicrobial agents and to using the efficacy data to determine a course of treatment for a patient.

Background to the Invention

Onychomycosis (also known as tinea unguium) is a common fungal nail infection caused predominantly by dermatophyte fungi (1) belonging to the genera Trichophyton, Microsporum and Epidermophyton, of which T. rubrum is the most common causative agent globally.

Onychomycosis affects approximately 10% of the general population and upwards of one third of geriatric and diabetic populations (2). It is the most common traumatic nail disorder and affects the toe nails more frequently than finger nails (3).

However, onychomycosis is not only a significant aesthetic problem, it is a chronic disease of the nail plate and surrounding skin. Symptoms include the nail becoming thickened, yellow and/or cloudy. The surface of the nail commonly becomes rough and crumbly and the nail may separate from the nail bed. Fungal nail infections are unsightly and can be painful. They are associated with pain linked to walking, standing and from footwear (4) and also psychosocial problems, including embarrassment, low selfesteem and social withdrawal (5).

There are a limited number of antifungal therapies available in routine clinical practice to treat onychomycosis. It is a difficult condition to resolve, with 20 - 25% of patients not improving/responding to any treatment (6) and the rate of recurrence, even in those patients who do respond, is as high as 53% (7). Thus there is an obvious need for more effective, safe treatments for this condition.

The use of systemic therapies (which are generally more effective than topical therapies), is limited by toxicity (8). Topical therapies are preferred for many reasons (9), but efficacy rates in clinical use are very low and prolonged treatment is required because of poor penetration of the nail plate which is a highly effective biological barrier (10).

Despite the low clinical cure rates achieved with the use of currently approved medicines for onychomycosis (Table 1), in vitro data generated as part of the preclinical development for these compounds would suggest they are effective drugs (see MICs versus dermatophytes in Table 1).

Table 1

An inability to penetrate the nail and access the causative fungi is one reason for this disparity in data. In addition, however, standard in vitro antifungal susceptibility tests (e.g. CLSI, EUCAST) performed on dermatophytes under artificial laboratory conditions, are not predictive of clinical effectiveness of candidate treatments for nail fungus. This is because they are not reflective of the physiological conditions in which these pathogens would exist within an onychomycotic nail (metabolic state, nutrient sources and availability, cell density, etc.).

This is true for novel large compounds such as NP213 (NovaBiotics); a molecule that is able to penetrate the nail and outperform currently approved compounds in ex vivo nail model experiments and in early clinical trials, but for which in vitro susceptibility test methods (such as the broth microdilution procedure described in the Clinical and Laboratory Standards Institute (CLSI) approved standard 'Reference method for broth dilution antifungal susceptibility testing of filamentous fungi' (M38-A2)) are not appropriate.

The broth microdilution procedure uses nutrient-rich RPMI-1640 medium which contains 2 g/L glucose as the main carbon source, but no protein and only small amounts of individual amino acids (0.01 - 0.2 g/L). In contrast, protein-rich human nail predominantly comprises various keratin forms (80 - 90% 'hard' hair keratins and 10 - 20% 'soft' skin keratins) that are highly cross-linked by disulphide bonds to provide nail with its high tensile strength (18). Along with other components such as collagen and lipids (~5% in adult nails) (19, 20) these keratins provide the main source of nutrients for dermatophytes in the context of onychomycosis.

Dermatophytes specifically produce several classes of proteases, including subtilisins (serine proteases), fungalysins (metalloproteases), leucine aminopeptidases and dipeptidyl peptidase IV, that facilitate the degradation of keratin in skin, hair and nail (21-24). Indeed, the ability of dermatophytes to degrade keratin is a known virulence factor of this group of fungi (25).

Consequently, the sugar rich nutrient base of RPMI-1640 medium bears little resemblance to the nutrients available to fungi growing in/on the nail in onychomycosis.

With this in mind, the present inventors set out to develop an antifungal sensitivity testing method in an alternative media source. The aim was to provide conditions that more closely resemble the in vivo conditions in which dermatophyte fungi exist within the nail and in the context of onychomycosis treatment during exposure to antifungal treatment.

Summary of the Invention

According to a first aspect there is provided a culture medium for culturing microbes, such as fungi, comprising a suspension of powdered nail in a suitable buffer wherein the powdered nail has a concentration of at least 0.1% w/v.

In a second aspect there is provided use of a culture medium comprising a suspension of powdered nail in a suitable buffer wherein the powdered nail has a concentration of at least 0.1% w/v for the culture of microbes.

In a third aspect there is provided a method of culturing microbes comprising the steps: a. preparing a culture medium comprising a suspension of powdered nail in a suitable buffer wherein the powdered nail has a concentration of at least 0.1% w/v, b. inoculating the medium of step a with a microbe, and c. incubating the inoculated medium of step b under conditions suitable for culture of the microbe.

In a fourth aspect there is provided a method of determining the efficacy of an antimicrobial agent comprising culturing microbes according to the method described in the third aspect wherein the inoculated medium of step b is further provided with one or more antimicrobial agents, and measuring the metabolic activity of the inoculated medium before and after the incubation according to step c and optionally at one or more time points during the incubation period

In a fifth aspect there is provided a method of identifying the most efficacious antimicrobial agent or combination of antimicrobial agents comprising the steps: a. obtaining a sample of the microbial infection and culturing the microbe with a series of antimicrobial agents according to the method of the fourth aspect, and b. identifying the most efficacious antimicrobial agent or combination of antimicrobial agents.

And in a sixth aspect there is provided use of the most efficacious antimicrobial agent or combination of antimicrobial agents identified by the method of the fifth aspect for use in the treatment of the microbial infection thereof.

Advantageously the use of powdered nail as the only source of carbon and nitrogen available in the culture medium provides a more realistic growing environment for the microbes. That is, an environment which more closely replicates the environment of the microbe when colonising/infecting a nail.

In contrast to known methods of testing compounds which are potentially therapeutic in the treatment or prevention of fungal nail infections, the methods of the present invention are accurate, repeatable, physiologically relevant and may be used in connection with all types of nail infections. The environment used to culture microbes and to test the efficacy of potentially therapeutic compounds according to the methods of the present invention is similar to the environment in which the compounds will be used. As such, the medium and the methods of the present invention provide a more accurate indication of how the potentially therapeutic compounds will behave in vivo.

Brief Description of the Drawings

The present invention will now be described by way of example only with reference to the accompanying Figures in which:

Figure 1 shows: Effect of incubation with different concentrations of powdered human nail (% (w/v)) on metabolic activity of T. rubrum NCPF0118 at 37°C. Metabolic activity was determined by monitoring changes in the fluorescence of alamarBlue® (excitation = 530 nm; emission = 590 nm) every 24 h for 216 h. All results represent the mean of triplicate samples from experiments carried out 3 times. Error bars represent the standard error of the mean.

Detailed Description

As employed herein culture medium means any medium, liquid or solid, for the purpose of culturing microbes, that is for the growth and replication of microbes.

As employed herein liquid culture medium means a liquid medium (suspension or solution) for the purpose of culturing microbes, that is for the growth and replication of microbes.

In one embodiment the culture medium comprises powdered nail and buffer. In one embodiment the culture medium consists of powdered nail and buffer.

In one embodiment the culture medium is a liquid culture medium.

Microbes as employed herein encompasses fungi, bacteria, viruses and protozoa, such as fungi and bacteria, for example fungi. In one embodiment the microbes are fungi.

The microbes of particular interest are those that colonise the nail and/or nail bed of mammals, particularly primates such as humans. Generally such microbes are pathogenic, that is they cause a pathology (a disease or infection).

The fungus may be any which may cause a fungal nail infection. The fungus may be a dermatophyte or a non-dermatophyte mould or a yeast.

In one embodiment the microbes are pathogenic.

In one embodiment the pathology caused by the pathogenic microbe is onychomycosis.

The fungus is generally a dermatophyte, such as those isolated from a tinea infection, such as a tinea unguium, tinea corporis, tinea capitis, tinea cruris, tinea faciae and tinea pedis infection. The dermatophyte may be an isolate of Trichophyton spp. In particular the dermatophyte may be Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton violaceum, Trichophyton interdigitale, Trichophyton tonsurans, Trichophyton soudanense or Trichophyton verrucosum, Trichophyton schoenleinii, Epidermophyton floccosum, Microsporum gypseum, Microsporum audouinii or Microsporum canis.

Alternatively the fungus may be a yeast such as Candida spp, typically Candida albicans, Candida krusei, C. glabrata, C.famata, C. parapsilosis, C. tropicalis, C. sake, Malassezia furfur and Trichosporon spp.

According to a further aspect of the present invention the fungus may be a non-dermatophyte mould such as Acremonium spp (for example A. roseogriseum), Alternaria spp., Arthrographis kalrae, Aspergillus spp. (including A. flavus, A. fumigatus, A. terreus, A. ustus, A. sydowii, A. versicolor), Arthroderma tuberculatum, Bipolaris spp., Botryodiplodia theobromae, Chrysosporium (Geomyces) pannorum, Cladosporium spp., Fusarium spp (including F. oxysporum, F. proliferatum, F. solani), Geotrichium candidum, Nattrassia spp., Onychocola canadensis, Paecilomyces spp., Penicillium spp., Phyllostricta sydowii, Pyrenochaeta unguis-hominis, Scopulariopsis brevicaulis, Scytalidium spp. (including S. didmidiatum, S. hyalinum), Synchephalastrum racemosum, Trichoderma spp. and Ulocladium spp.

The bacteria may be any of which may cause a bacterial nail infection. The bacteria may be Gram negative or Gram positive bacteria such as Pseudomonas aeruginosa, Klebsiella spp., Proteus spp. and Staphylococcus aureus.

In one embodiment the microbe is a fungus.

In one embodiment the fungus is a dermatophyte fungus. Such as those belonging to the genera Trichophyton, Microsporum or Epidermophyton.

In one embodiment the fungus is Trichophyton rubrum

In one embodiment the microbe is a bacterium.

Suspension as employed herein means a heterogeneous mixture containing solid particles that are sufficiently large for sedimentation. Usually they must be larger than one micrometer. Generally, the internal phase (solid) is dispersed throughout the external phase (fluid) through mechanical agitation. In some embodiment the suspension is solidified.

Powdered nail as employed herein means a complete nail or nail fragment from a human or animal body. Typically the nail to be powdered is disease or infection free. In one embodiment the powdered nail is prepared by cutting the nail into small fragments and then grinding the fragments into a fine powder in liquid nitrogen using a pestle and mortar. Typically the powdered nail is passed through a fine mesh sieve to remove any larger pieces. In one embodiment the powdered nail is sterilised, for example by autoclaving. Advantageously using powdered nail increases the surface area of the nail accessible to be broken down by the microbe, thereby releasing nutrients for microbe growth and reproduction.

Alternatively, the powdered nail comprises the constituents of nail, that is the nutrients of nail. In one embodiment the nail is artificial powdered nail. That is, comprises man made analogues of the nutrients, such as keratins, found in nail. In one embodiment the nail comprises of the constituents of nail isolated from alternate natural sources, for example keratin from wool, such as sheep's wool.

Advantageously using powdered nail as the nutrient source for the microbes provides a more realistic environment for growth which better mimics the in vivo environment. In turn, this provides for more realistic activity of the antimicrobial agent, thereby providing more robust data for antimicrobial activity.

It is clear from Table 2 that the culture medium of the present disclosure results in MICs that are more reflective of the in vivo clinical results seen when treating fungal nail infections with antifungal agents. Advantageously, antimicrobial agents that are clinically more efficacious show lower MICioo than those which are less efficacious when grown using the culture medium comprising powdered nail compared to those grown using RPMI-1640 or those grown in the absence of powdered nail. Further advantageously, the MICioo of more clinically efficacious antimicrobial agents was decreased using the culture medium comprising powdered nail compared to using RMPI-1640 (or in the absence of powdered nail) whilst the MICioo of less clinically efficacious antimicrobial agents was increased using the culture medium comprising powdered nail compared to using RMPI-1640 (or in the absence of powdered nail).The use of culture medium comprising powdered nail has greater distinguishing power for clinically efficacious antimicrobial agents than culture medium lacking powdered nail, such as RMPI-1640. That is, the culture medium of the present disclosure provides a more accurate assessment/prediction of the clinical efficacy of antimicrobial agents than media lacking powdered nail.

In one embodiment the powdered nail is prepared from a human nail or nail fragment. In one embodiment the powdered nail is prepared from one or more subjects, such as 2, 3,4,5 or more different subjects.

In one embodiment the culture medium comprises at least 0.1% w/v of powdered nail. Such as approximately 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95,1.0, 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5% w/v or more. For example approximately 0.75% w/v powdered nail.

In one embodiment the powdered nail is prepared from finger nail. In one embodiment the powdered nail is prepared from toe nail. In one embodiment the powdered nail is prepared from both finger and toe nail.

In one embodiment powdered nail is the sole source of carbon and nitrogen available in the medium.

Suitable buffer as employed herein is any buffer capable of maintaining the suspension at the desired pH.

In one embodiment the buffer is selected from Potassium phosphate buffer, Phosphate buffer, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, MOBS, DIPSO, TAPSO, Trizma[Tris], HEPPSO, POPSO, TEA, EPPS [HEPPS], Tricine, GLY-GLY, BICINE, TAPS, AM PD and sodium phosphate.

In one embodiment a balanced salts solution is employed as the buffer, for example Phosphate buffered saline (PBS), Dulbecco's phosphate buffered salines (DPBS), Hanks' balanced salts solutions (HBSS), Earle's balanced salts solutions (EBSS), Gey's balanced salts solutions (GBSS), Alsever's solution, Puck's balanced salts solutions (PBSS), Ringer's balanced salts solutions (RBSS), Singer's balanced salts solutions (SBSS), TRIS buffered saline (TBS)

In one embodiment the buffer is approximately 5-25 mM sodium phosphate buffer, such as approximately 10,15, 20mM sodium phosphate. In one embodiment the sodium phosphate buffer is 10 mM sodium phosphate buffer.

In one embodiment the desired pH is in the range approximately 6.0 to 8.0, such as approximately 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9 or any other pH capable of sustaining microbial growth. For example, approximately 7.0.

In one embodiment a microbe sample is used to inoculate the culture medium to employ the method of culturing the microbe. A microbe sample, such as a fungal sample, as used for inoculating the culture medium may include one or more specified microbial cultures, such as fungal cultures, and may also comprise pharmaceutically acceptable carriers or excipients. The mixture of microbial cultures in the microbe sample is generally known.

In one embodiment the microbe sample is a fungal sample.

In one embodiment the fungal sample is a one or more dermatophytes.

The microbe sample, such as a fungal sample, may comprise a single microbe culture, or a mixture of more than one microbe cultures. The identity of the microbe cultures contained in the sample would generally be known prior to inoculation. According to one embodiment, the microbe sample may comprise more than one microbe cultures, typically two or three microbe cultures.

In one embodiment, the fungal sample may comprise more than one dermatophyte. The fungal sample may comprise at least one dermatophyte and one or more non-dermatophyte moulds or more than one yeast.

According to one embodiment the fungal sample may comprise more than one yeast.

Typically the fungal sample is a cultured fungal sample obtainable according to the steps of: obtaining a fungal isolate; growing the fungal isolate generally for 1 to 14 days at 30 to 37 degrees Celsius, typically on an appropriate sterile agar such as Sabouraud dextrose agar or potato dextrose agar until the fungal isolate has grown substantially, generally to cover the entire surface of the agar and has produced aerial hyphae where appropriate, and subsequently following the instructions for preparation of a standard inoculum described in the Clinical and Laboratory Standards Institute (CLSI) Approved Standard (M38-A2); Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi (2nd Edition) or CLSI Approved Standard (M27-A3); Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts (3rd Edition).

Typically, an inoculum may be prepared by taking a culture stock, prepared by combining the fungal isolate with a cryoprotectant, such as dimethyl sulfoxide (DMSO) at typically 1 to5 % v/v in Sabouraud Dextrose broth and stored frozen at minus70 - minus 85°C. Aliquots (typically 50 microlitres ) of the culture stock may be transferred to slopes of sterile Agar medium, normally Potato Dextrose Agar or Oatmeal Agar and incubated at elevated temperature, typically more than 25 degrees Celsius, suitably 28 to 35 degrees Celsius, more suitably approximately 30 degrees Celsius. Incubation is typically for up to 14 days, suitably 5 to 7 days or until the isolate forms a clearly visible mat on the surface of the agar. A spore suspension is prepared by adding water (typically around 3 millilitres) to the culture tube and resuspending the fungal mat.

In one embodiment there is provided a method of culturing microbes, such as fungi, employing the culture medium described herein.

In one embodiment the culture medium is prepared by suspending an amount of powdered nail (prepared as described herein) in a buffer.

As employed herein inoculate means to introduce microbes to a culture medium.

In one embodiment the culture medium is inoculated with a microbe to be cultured. In one embodiment the microbe to be cultured is a fungus, such as a dermatophyte. For example, belonging to the genera Trichophyton, Microsporum or Epidermophyton, such as Trichophyton rubrum.

The duration and conditions applicable for the incubation are dependent on many variables including the number of cfu in the microbe sample and the type of microbe(s) in the microbe sample. The incubation period is also dependent on the desired number of cfu in the final culture. The incubation period is sufficient to allow a microbial growth to become established in the medium and this may be determined, at least in part, through monitoring metabolic activity using fluorescence.

The incubation period is typically up to 5 to 70 days, generally up to 50 days, suitably 20 days or less, more suitably up to 14 days. For example the incubation period is up to 168 hours or more, such as approximately 12, 24, 36,48,60, 72, 84, 96,108,120,132,144,156 or 168 hours. Typically growth, for example metabolic activity, is monitored every 24 hours.

In one embodiment the incubation period is 168 hours.

In one embodiment the metabolic activity is monitored approximately every 24 hours.

The inoculated medium may be incubated at temperatures elevated from room temperature. The elevated temperature may be 25 to 40 degrees Celsius, such as 26, 27, 28, 29,30, 31, 32,33,34,35,36, 37, 38 or 39 degrees Celcius, such as approximately 30 to 37 degrees Celsius, for example around 30 degrees Celsius +/-1 degree Celsius.

In one embodiment the inoculated medium is incubated under conditions suitable for culture of the microbe.

Growth of the microbe can be monitored by employing a detection reagent in the method of culturing the microbe.

As employed herein a detection reagent is any reagent which enables changes in the growth and cell viability of the microbe to be identified. For example a fluorescent reagent which fluoresces when the microbe is metabolising. Thus metabolic activity is a measurable indicator of microbial growth and reproduction. The skilled person will appreciate that methods other than fluorescence are equally envisioned by the inventors.

In one embodiment the method of detecting growth of the microbe is fluorescence.

Examples of fluorescent reagents include, but are not limited to: alamarBlue (resazurin; IUPAC name 7-hydroxy-10-oxidophenoxazin-10-ium-3-one) (alamarBlue/resazurin fluoresces at Ex 530 nm/Em 590 nm), Fluorescein diacetate (FDA), CellTiterBlue (Promega) and PrestoBlue (ThermoFisher).

In one embodiment the method of detecting growth is luminescence- based, for example using CellTiterGlo (Promega).

In one embodiment the method of detecting growth employs colorimetric dyes, for example MTT or XTT.

In one embodiment the fluorescent reagent is alamarBlue (resazurin or resazurin-based).

In one embodiment the liquid culture medium comprises approximately 0.0025% w/v alamarBlue.

AlamarBlue is a brand name for rezasurin. The terms are used interchangeably throughout.

In one embodiment there is provided the use of a fluorescent reagent, such as alamarBlue in an assay to determine the metabolic activity of microbes in a culture medium comprising powdered nail.

In one embodiment there is provided the use of a fluorescent reagent, such as alamarBlue, in an assay to determine the efficacy of antimicrobial agents against microbes in a culture medium comprising powdered nail.

In one embodiment the liquid culture medium consists 0.75% w/v powdered nail, 0.0025% w/v alamarBlue and lOmM sodium phosphate buffer at pH 7.0. Optionally the liquid culture medium further comprises an antimicrobial agent, such as an antifungal agent.

In one embodiment the culture medium further comprises an antimicrobial agent, such as an antifungal agent.

As disclosed herein there is provided a method of determining the efficacy of an antimicrobial agent by culturing the microbe in the culture medium in the presence of a detection reagent. The method entails adding one or more antimicrobial agents, such as antifungal agents, to the inoculated culture medium and obtaining a starting measurement from the resulting medium. In one embodiment the measurement is a measurement of metabolic activity. The resulting medium is then incubated and the measurement is repeated at one or more chosen time point, for example every 24 hours.

As employed herein an antimicrobial agent is a compound with known or potential activity that results in the inhibition or retardation of growth or the death of a microbe. Antifungal agents have particular activity against one or more fungi.

As used herein the term "compound" encompasses the pharmaceutically acceptable salts thereof.

Potentially antifungal agents or compositions are generally tested through broth dilution antifungal susceptibility assays at the preclinical stage. Such assays do not provide an environment similar to that in which the compositions will be used in vivo. In contrast, the method of the present invention provides an accurate reliable test of the potentially antimicrobial compositions in conditions similar to which they would be used. Surprisingly it has been found that some compounds exhibit far higher efficacy in the method of the present invention and in vivo than would be expected from the results of broth dilution antifungal susceptibility assays. The method of the present invention thus provides a useful, accurate indication of how a potentially antimicrobial agent is likely to behave in vivo, in particular when compared to current assays.

The potentially antimicrobial agent may be potentially useful in the prevention and/or treatment of microbial nail infections.

The potentially antimicrobial agent may be any compound of interest in the treatment or prevention of a fungal nail infection. According to one embodiment, the potentially active compound may be a peptide, small molecule, allylamine (such as amorolfine, butenafine, naftifine and terbinafine), abafungin, azole (such as bifonazole, butaconazole, clotrimazole, econazole, efinaconazole, fenticonazole, fluconazole, isaconazole, isavuconazole, itraconazole, ketoconazole, luliconazole, miconazole, oxiconazole, posaconazole, ravuconazole, sertaconazole, sulconazole, terconazole, tioconazole and voriconazole), polyene (such as amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin and rimocidin), echinocandin (such as anidulafungin, caspofungin and micafungin), benzoic acid, ciclopirox olamine, flucytosine, griseofulvin, haloprogin, piroctone olamine, selenium sulphide, sodium bicarbonate, tavaborole, tolnaftate, undecylenic acid, zinc pyrithione or other alternative therapies. The active compound may further comprise a pharmaceutically acceptable excipient. Additionally, different formulations of potentially active compounds can be used in this model.

Suitable pharmaceutical excipients include anti-adherents, binders, coatings, disintegrants, fillers and diluents, flavours, colours, glidants, lubricants, preservatives, sorbents, solvents, stabilisers, and sweeteners, such as polyethylene glycol and urea, and as described in the US Food and Drug Administration "Inactive Ingredient Approved Drug Products" database.

The concentration of potentially therapeutic compound in the potentially therapeutic composition is dependent on the fungal nail infection to be treated. The concentration is typically 1 to 5% w/v, such as 2, 3 or 4% w/v such as 3% w/v or less. Alternatively, the concentration may be 20 % w/v or less, generally 5 to 15% w/v, typically 5 to 10% w/v. The concentration may be 1, 5 or 10 % w/v.

The therapeutic compositions tested are generally potentially effective against any fungal nail infection. Examples of typical fungal nail infections include distal subungual onychomycosis, white superficial onychomycosis, proximal subungual onychomycosis and/or candidal onychomycosis.

According to one aspect of the present invention, the method of assessing efficacy may be repeated using one or more different potential antimicrobial agents. The metabolic activity following addition of the different potential antimicrobial agent may be assessed. This may be compared with the metabolic activity following addition of a control and/or the metabolic activity following addition of the first potential antimicrobial agent. In this way the most efficacious antimicrobial agent or combination of agents against a particular microbe can be identified.

Typically the method of comparing the efficacy of potential antimicrobial agents is carried out with a single potential antimicrobial agent per microbial culture. That is, the microbes are not treated with two antimicrobial agents consecutively. In some embodiments the efficacy of combinations of potential antimicrobial agents is tested. Typically these antimicrobial agents are administered simultaneously or essentially simultaneously.

Advantageously, once the most efficacious antimicrobial agent or combination of agent has been identified, the agent(s) can be administered to a patient with an infection caused by the microbe.

In the context of this specification "comprising" is to be interpreted as "including".

Aspects of the invention comprising certain elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements.

Where technically appropriate, embodiments of the invention may be combined.

Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.

Technical references such as patents and applications are incorporated herein by reference.

Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.

EXAMPLES

Taking into account the importance of keratin degradation to dermatophyte virulence, an alternative growth medium was developed for a broth microdilution antifungal susceptibility assay with a sole carbon and nitrogen source of powdered human nail in 10 mM sodium phosphate buffer, pH 7.0. As this medium was a suspension, rather than a transparent solution like RPMI-1640 medium, the method of determining antifungal susceptibility was also altered from measurement of growth by changes in turbidity (530 nm) to assessment of viability by metabolic activity determination by fluorescence using, alamarBlue® (26).

In this study, we describe the development and application of this novel method for the determination of the antifungal susceptibility of T. rubrum NCPF0118 against Novexatin®, ciclopirox olamine and terbinafine hydrochloride.

Materials and Methods

Chemicals and Growth Media

All chemicals and growth media were purchased from Sigma-Aldrich (UK), unless otherwise stated. RPMI-1640 growth medium was supplemented with 2.0 g/L glucose and 0.3 g/L L-glutamine and phenol red as a pH indicator, but without sodium bicarbonate and was buffered to pH 7.0 ± 0.1 with 0.165 mol/L MOPS (3-[N-morpholino]propanesulfonic acid) and sterilised by filter-sterilisation (0.2 pm). Terbinafine hydrochloride and ciclopirox olamine were prepared aseptically as 1.6 mg/mL stock solutions in dimethyl sulfoxide (DMSO) and subsequently diluted with sterile aqueous solutions and sterilised by filter-sterilisation (0.2 pm). Human nails were obtained from a NHS podiatrist (NHS

Grampian), following appropriate ethical approval (REC reference: 05/S0801/115), from donors following nail avulsion due to ingrown toenails. All nails were disease-free, based on the podiatrists diagnosis. Nails were cut into small fragments and ground to a fine powder in liquid nitrogen in a mortar and pestle. Following this, powdered human nail was passed through a fine-meshed sieve and sieved nail powder was used to prepare 0.2,0.5,1.0,1.5 and 2.0% (w/v) suspensions in 20 mM sodium phosphate buffer, pH 7.0. Nail suspensions were sterilised by autoclaving for 20 min at 121°C. Trichophyton rubrum NCPF0118, originally isolated from human nail, was obtained from the National Collection of Pathogenic Fungi (Public Health England, UK) and maintained on potato dextrose agar at 30°C. For long-term storage, cultures were maintained at -85°C in sterile Sabouraud liquid medium containing 5% (v/v) dimethyl sulfoxide. CLSI Antifungal Susceptibility Testing Procedure

Antifungal susceptibility testing was carried out using the broth microdilution procedure described in the Clinical and Laboratory Standards Institute (CLSI) Approved Standard 'Reference method for broth dilution antifungal susceptibility testing of filamentous fungi' (M38-A2) (17). All experiments were conducted a minimum of three times, each containing three technical replicates.

Modified Antifungal Susceptibility Testing Procedure

Antifungal susceptibility testing was carried out as described above, but with the following modifications. RPMI-1640 medium was substituted for 0.1, 0.25, 0.5, 0.75 and 1.0% (w/v) powdered human nail suspensions in 10 mM sodium phosphate buffer, pH 7.0 containing 0.0025% (w/v) alamarBlue* (ThermoFisher Scientific, UK). Metabolic activity was monitored by fluorescence (Ex 530 nm/Em 590 nm) every 24 h for up to 168 h, or until high metabolic activity (100000 U) was observed in inoculated controls in the absence of any antifungal agent. Sodium phosphate buffer, pH 7.0, alone did not support dermatophyte growth (data not shown). All experiments were conducted a minimum of three times, each containing three technical replicates.

Results

The antifungal susceptibility of T. rubrum NCPF0118 versus Novexatin® (NP213), ciclopirox olamine and terbinafine hydrochloride was determined by the CLSI broth microdilution procedure (Table 2). As can be seen in Table 2 the MICs of ciclopirox and terbinafine fall within the susceptible MIC range described in CLSI M38-A2 (17). The MICioo of Novexatin® (NP213), by contrast, is very high, at least 1000-fold higher than the ciclopirox and terbinafine MICs using RPMI-1640 as the growth medium.

Changes in metabolic activity of T. rubrum NCPF0118 incubated at 37°C with different concentrations of powdered human nail was determined (Figure 1) and rapid increases in metabolic activity were observed following incubation with 0.5, 0.75 & 1.0% (w/v) powdered human nail, whereas increases in metabolic activity were much slower when 0.1 & 0.25% (w/v) powdered human nail was used. When grown on RPMI-1640 medium, T. rubrum NCPF0118 reached maximum metabolic activity after 72 h, 24 h earlier than achieved when incubated with 0.75% or 1.0% (w/v) powdered human nail. On the basis of these results, 0.75% (w/v) powdered human nail was selected for antifungal susceptibility testing as it demonstrated similar metabolic activity increases to growth in RPMI-1640 medium and was less particulate than 1.0% (w/v) powdered human nail, resulting in smaller error bars (Figure 1).

When the MICioo of Novexatin® (NP213) was determined using 0.75% (w/v) powdered human nail there was a dramatic reduction in the MIC when compared to the MIC determined by the CLSI broth microdilution procedure using RPMI-1640 medium (Table 2), 8-32 pg/ml and 1000 pg/ml, respectively. However, this was not the case for ciclopirox olamine and terbinafine hydrochloride, for which there was a concomitant increase in MICioo of 16 and 4 - 8-fold, respectively, when incubated with powdered human nail. This is not unexpected as both ciclopirox and terbinafine are known to bind to nail keratin, resulting in reduced antifungal efficacy (27-29).

Table 2: MICioo (pg/ml) of Novexatin* (NP213), ciclopirox olamine and terbinafine hydrochloride versus T. rubrum NCPF0118 following incubation with 0.75% (w/v) powdered human nail and fold change in MICioo relative to MICioo determined using RPMI-1640 medium. Metabolic activity was determined by monitoring changes in the fluorescence of alamar Blue® (excitation = 530 nm; emission = 590 nm) every 24 h for up to 216 h. All results represent the mean of triplicate samples from experiments carried out 3 times.

Tables 3-5 provide a more detailed analysis of the comparison of powdered nail versus RPMI-1640.

Table 3: Antifungal susceptibility of T. rubrum NCPF0118 versus Novexatin® (NP213) grown on different amounts of powdered human nail

Table 4: Antifungal susceptibility of T. rubrum NCPF0118 versus Ciclopirox olamine grown on different amounts of powdered human nail

Table 5: Antifungal susceptibility of T. rubrum NCPF0118 versus Terbinafine hydrochloride grown on different amounts of powdered human nail

The MIC range, as stated by CLSI (17), for ciclopirox versus Trichophyton spp. is 0.5-2.0 pg/ml, so the use of powdered human nail as nutrient source takes the MIC versus T. rubrum NPCF0118 to the upper limit of the MIC range and beyond, whereas the CLSI MIC range for terbinafine versus Trichophyton spp. is 0.002 - 0.008 pg/ml, so the use of powdered human nail takes the MIC versus T. rubrum NPCF0118 beyond the upper limit of the stated MIC range (17). Ciclopirox concentrations in excess of 1000 pg/g are achievable in human nail (30), so the increased ciclopirox MIC observed here may not affect antifungal efficacy. However, the studies cited measured the concentration of ciclopirox in the nail and not how much is bioavailable. Additionally, the effective concentration of nail in an infected nail would be much higher than in this assay system and therefore even greater inhibition of antifungal efficacy may occur. Terbinafine concentrations of 0.52 - 1.01 pg/g, 18 weeks after 6 or 12 weeks of systemic treatment are achievable in human nail (31), which is in excess of the elevated MICs determined in this study and therefore unlikely to affect antifungal efficacy. Again, this study measured the concentration of terbinafine in the nail and not how much is bioavailable and able to contribute to antifungal activity. The increases in ciclopirox and terbinafine MICs in the presence of human nail observed in this study and others (27-29) may contribute to the difficulties observed clinically in the resolution of onychomycosis and in the frequently observed recurrence of infection (6, 7).

Discussion/Conclusions

The use of powdered human nail as nutrient source for the determination of antifungal efficacy of agents for the treatment of onychomycosis is a closer approximation of the nutrients available in vivo than the use of RPMI-1640 medium and therefore the concentration of Novexatin® (NP213) required to achieve the MICioo may more accurately reflect the amount of drug candidate that must be bioavailable in the nail for the successful clinical treatment of onychomycosis.

In summary, this study demonstrates a more clinically/physiologically relevant method to assess the efficacy of antifungal agents intended for use in the treatment of onychomycosis with an adaptation of the CLSI broth microdilution procedure that more closely resembles conditions within the nail and can therefore generate more robust data than antifungal efficacy determination under standard conditions.

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Claims (19)

Claims

1. A culture medium for culturing microbes comprising a suspension of powdered nail in a suitable buffer.

2. A culture medium according to claim 1 wherein the buffer is sodium phosphate.

3. A culture medium according to claim 1 or 2 wherein the pH is approximately 7.0.

4. A culture medium according to any one of claims 1 to 3 wherein the powdered nail has at concentration of 0.75%.

5. A culture medium according to any one of claims 1 to 3 wherein the powdered nail is human toe or finger nail.

6. A culture medium according to any one of claims 1 to 5 wherein the microbes are pathogenic.

7. A culture medium according to claim 6 wherein the pathogenic microbes are fungi.

8. A culture medium according to claim 7 wherein pathogenic fungi cause onychomycosis.

9. A culture medium according to claim 7 or 8 wherein pathogenic fungi are dermatophyte fungi belonging to the genera Trichophyton, Microsporum or Epidermophyton.

10. A culture medium according to claim 9 wherein the dermatophyte fungi are Trichophyton rub rum.

11. A culture medium according to any one of claims 1 to 10 wherein the culture medium is a liquid culture medium.

12. A culture medium according to any one of claims 1 to 11 further comprising a detection reagent.

13. Use of the culture medium according to any one of claim 1 to 12 for the culture of microbes.

14. A method of culturing microbes comprising the steps: a. preparing a culture medium according to any one of claims 1 to 12, b. inoculating the medium of step a with a microbe, and c. incubating the inoculated medium of step b under conditions suitable for culture of the microbe.

15. A method according to claim 14 further comprising the step of detecting growth of the microbe by providing a detection reagent with the inoculated medium of step b.

16. A method according to claim 15 wherein the culture medium is a liquid culture medium, the detection reagent is a fluorescent reagent such as alamarBlue and the growth of the microbe is detected by fluorescence.

17. A method of determining the efficacy of an antimicrobial agent comprising: culturing microbes according to the method of any one of claims 15 or 16 wherein the inoculated medium of step b is further provided with one or more antimicrobial agents, and measuring the metabolic activity of the inoculated medium before and after the incubation according to step c and optionally at one or more time points during the incubation period.

18. A method of identifying the most efficacious antimicrobial agent or combination of antimicrobial agents against a microbe causing a microbial infection in a patient, comprising the steps: a. obtaining a sample of the microbe causing a microbial infection and culturing the microbe with a series of antimicrobial agents according to the method of claim 17, and b. identifying the most efficacious antimicrobial agent or combination of antimicrobial agents.

19. The most efficacious antimicrobial agent or combination of antimicrobial agents identified in claim 18 for use in the treatment of the microbial infection of claim 18.