Field of invention

The invention relates to a wound cleansing spray, and in particular to a wound cleansing spray providing improved removal of a wound's biofilm.

Background

Wounds, e.g. chronic wounds, are a considerable cause for patient discomfort and morbidity. Moreover, proper wound care presents a considerable burden on the healthcare system.

Wounds offer ideal conditions for infection, i.e. microbial biofilm formation. The microbial biofilm consists of a community of microorganisms which survive as communities within a matrix of extracellular polymeric substance (EPS), which is a complex and heterogenous matrix of polymers.

Critically, bacteria in the form of biofilms have been shown to slow wound healing, particularly in chronic wounds. The ability of EPS within the biofilm to degrade therapeutic agents may be a factor in the slow healing of chronic wounds. Moreover, biofilms have been shown to provide a barrier to phagocytic cells and antimicrobial agents, such as antibiotics, which delays wound healing and increases the risk of infection.

Biofilms that grow on e.g. a wound dressing will most likely disseminate into the wound bed. The virulence of these biofilms is high, when compared with planktonic bacteria. Moreover, antibiotic resistance of biofilms can be up to 1000 times greater than that of planktonic bacteria.

The management of biofilms in wounds is a complex process principally because of the unpredictability of the biofilm's physiology. In part, this is often because of the biofilm's inherent microbiological composition.

To facilitate wound biofilm removal, cleansing to disrupt the biofilm and irrigate the wound are essential. Cleansers may be as simple as the use of physiological saline solutions.

Saline-containing wound cleansing sprays are normally used as the first step to support wound healing in clinical settings. Such wound cleansing sprays help remove bacteria and any foreign matter that is present in the wound, which may form a biofilm.

However, immersing a biofilm in saline alone is not an effective method to break down a biofilm. Hence, wound cleansing solutions may additionally comprise a surfactant to loosen e.g. biofilm, and an antimicrobial agent to help reduce the surface microbial bioburden. Wound cleansing solutions are commonly delivered by a squeeze or spray bottle, or by way of a syringe, whereby it is important to ensure that the impact pressure of solution applied does not damage newly forming tissue or cause trauma or pain to the patient.

Despite the challenges of wound cleansing and wound healing on society at large, there is still a lack of adequate wound cleansing solutions and methods.

Short description of the Figures

Figure 1 shows the average quantity of viable microorganisms recovered, following spray and immersion treatment for five seconds with four different wound cleanser jets. The horizontal line at 1 Log represents the limit of detection. Error bars represent standard deviations; CFU = colony forming units.

Description of the invention

Biofilms may be difficult to eradicate and result in persistent wound infections that are known to prevent wound healing.

As used herein, "biofilm" is defined as a community of bacteria and/or other microorganisms that form a thin but robust layer adhering to a wound, but not extending to other surfaces be they biological or biologically inert.

It is demonstrated herein that to disrupt a biofilm using a wound cleansing spray, it is advantageous to at the same time have an effective cleansing solution, and a relatively low impact pressure of the jet, whereby the biofilm is disrupted and removed while the jet at the same time does not harm newly formed tissue.

Consequently, a jet provided by an aerosol can or bag-on-valve delivery system is made use of in accordance with the present invention. Such delivery system is not provided with state of the art wound cleansing products, that merely wash the wound.

A jet is defined herein as a stream of wound cleansing solution projected from a pressure container to the outside atmosphere through a nozzle, aperture, or orifice, whereby the wound cleansing solution is atomised.

A wound cleansing spray is defined herein as the wound cleansing solution, contained in a pressure container. By using a delivery system comprising a pressure container as disclosed herein, it is possible to properly cleanse a wound while not contaminating the delivery system, by way of the distance between the pressure container and the wound to be cleaned. That is a significant advantage.

In accordance with the invention, there is provided a pressure container with a nozzle arranged to provide a jet upon manipulation of the nozzle, said pressure container containing a wound cleansing solution, the wound cleansing solution being aqueous and comprising a sequestering agent, and a surfactant, wherein the pressure in the pressure container is from 1.5 to 15 bars, e.g. from 2 to 12 bars. When releasing pressure, the wound cleansing solution exits the pressure container, through the nozzle, in the form of a jet. Thereby, the wound cleansing solution's pressure is converted into kinetic energy, thus giving the jet its velocity. E.g. the flow rate, speed, direction, shape, and pressure of the jet may all be controlled by nozzle. The skilled person is well equipped in choosing a nozzle suitable for the uses as herein described.

The sequestering agent is used to decrease the ion concentration, to thereby decrease cross-linkages and increase the water solubility of the EPS. Thereby, the biofilm structure harboring the microorganisms is disrupted.

Directly on the microbe, the sequestering agent may result in phospholipids of the inner membrane being exposed. Thereby, the sequestering agent may enhance the effect of other antimicrobial agents.

The sequestering agent of the solution may be chosen from the group consisting of ethylene diamine tetraacetic acid (EDTA) as a di, tri or tetra basic salt, pentetic acid, diethylene triamine pentaaceticacid, pendetide, pentetreotide, sodium phytate, tetrasodium glutamate diacetate, sodium polyitaconate, phytic acid, citric acid, and lactoferrin. Preferable salt forms of EDTA are disodium EDTA, sodium calcium EDTA, and tetrasodium EDTA. EDTA may alternatively be present in the form of an ammonium, diammonium, potassium, di-potassium, cupric disodium, magnesium disodium, or ferric sodium salt.

EDTA has been shown to dissolve biofilms, and to weaken the microbial cell through binding of cations, such as calcium, magnesium, and zinc. When used alone, EDTA has been shown to reduce the adhesion of biofilms in wounds (see: EDTA - An Antimicrobial and Anti Biofilm Agent for use in Wound Care. Simon Finnegan and Stephen L Percival).

EDTA may also prevent biofilm formation, through inhibiting the adhesion of bacteria. EDTA may moreover enhance the therapeutic effect of other antimicrobial agents

The sequestering agent of the solution is preferably present in a amount in the interval of from 0.1 to 4% by weight of the wound cleansing solution, preferably from 0.2 to 1 % by weight. Surfactants generally lower the surface tension of aqueous systems such as wounds, to facilitate removal of contaminants.

The surfactant of the solution facilitates disruption of the biofilm, thus increasing the efficacy of the jet and increasing the susceptibility of microorganisms to the immune system.

The surfactant of the solution is a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and/or an amphoteric surfactant. Preferably, only surfactant(s) with the same charge are present in the solution. The surfactant plays an important role in biofilm management.

The surfactant may facilitate biofilm dispersion and also prevent biofilm formation. This may be an effect of changes in surface tension and osmotic pressure. Moreover, the surfactant may stimulate cell migration and wound vascularization, thus facilitating wound healing.

The surfactant may also in itself have an antimicrobial effect. This effect may be effected through disruption of biological membranes, and through inhibition of certain enzymes leading to an increase of reactive oxygen species. Moreover, through interaction with microbial proteins, enzymatic stability and activity may be interfered with.

In contact with a wound, the surfactant has a role in loosening and softening debris and necrotic tissue, which proposedly promotes wound healing. Additionally, the surfactant may have a direct role in wound healing through effects on cellular repair, cell proliferation, and reduction of inflammation.

The surfactant(s) is preferably present in the wound cleansing solution in a amount in the interval of from 0.05 to 1 % by weight of the wound cleansing solution, preferably from 0.1 to 0.3 % by weight, most preferably 0.13 % by weight.

When the surfactant of the solution is cationic, it may be chosen from the group consisting of benzalkonium chloride, octenidine dihydrochloride, polyhexamethylene biguanide, ethyl lauroyl arginate, cetrimonium chloride, chlorhexidinesalts, and cetyl pyridinium chloride, chlorhexidine, and quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, polydiallyldimethylammonium chloride (polyDADMAC).

When the surfactant of the solution is anionic, it may be chosen from the group consisting of sodium dodecyl sulphate, sodium lauryl sulphate, sodium laureth sulphate, docusate sodium, sulfosuccinate, alkyl ether sulfates, alkyl sulfates, alkylbenzene sulfonates, alpha olefin sulfonates, phosphate esters, sodium cocoyl isethionate, cocoyl gluconate, sodium sarcosinate, and sodium lauryl sulphoacetate.

When the surfactant of the solution is non-ionic, it may be chosen from the group consisting of polysorbate, poloxamers, alcohol ethoxylates, alcohol ethoxylates/propoxylates, fatty acid esters, and glucosides, such as cocoyl glucosides. When the surfactant of the solution is amphoteric, it may be chosen from the group consisting of betaines, amidopropyl betaines, alkylamino dipropionates, cocamphoacetates, and diacetates.

Certain surfactants, such as benzalkonium chloride, additionally have an antimicrobial activity.

To further improve removal of bacteria and other microbes contained in a biofilm, the wound cleansing solution may comprise an antimicrobial agent, e.g. chlorhexidine or benzalkonium chloride.

In addition to being antibacterial, benzalkonium chloride is also effective against yeasts and fungi.

The addition of an antimicrobial agent can help reduce the number of viable bacteria in a wound bed and reduce wound healing time. By combining an antimicrobial agent with a surfactant, an enhancement of the antimicrobial effect may be obtained.

In one specific embodiment, the solution comprises a cationic surfactant in the form of benzalkonium chloride, and a sequestering agent in the form of a salt of EDTA. The salt of EDTA may be as a di, tri or tetra basic salt. The benzalkonium chloride may be present in of in an amount of 0.13 % by weight of the solution, and EDTA, as a salt, may be present in an amount of 0.1 % by weight of the solution.

To provide a more user-friendly and less painful experience for the person whose wound is being cleaned, the wound cleansing solution may further comprises NaCI in an amount of from 0.5 to 3 % by weight of the solution, preferably in an amount of 0.9% by weight of the solution.

The pressure in the pressure container comprising the wound cleansing solution is of utmost importance for the invention as disclosed herein. The pressure container's pressure has been chosen such that the impact pressure on the wound is adequate, when using the jet at a normal distance of between 10 and 40 cm from the wound to be cleansed. Only through choosing a suitable container pressure is it possible to obtain adequate wound biofilm removal, while at the same time not disturbing the healing wound. Disturbance of the healing wound would prolong the healing process, and might constitute the starting point of deleterious long-lasting or chronic biological processes. Accordingly, the pressure in the container is in the interval of from 1.5 to 15 bars, preferably 2 to 12 bars, more preferably from 3 to 8 bars, and most preferably from 3 to 5 bars.

The interval of pressure in the container may be created from any two end-points chosen from 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5; 9; 9.5; 10; 10.5; 11; 11.5; 12; 12.5; 13; 13.5; 14; 14.5; and 15 bars, whereby the lower pressure value is the lower end-point and the higher pressure value is the higher end-point of the pressure interval.

The upper pressure limits of 15 bars, and 12 bars, have been chosen such that the atomised cleansing solution does not splash back on the person cleansing the wound. The pressure container used in accordance with the invention is a bag-on-valve or an aerosol can. A bag- on-valve provides the cleansing solution with a long shelf life, while at the same time providing a controlled spraying pattern. Moreover, as shown herein, the nozzle of a bag-on-valve prevented external contamination from reaching the cleansing solution inside the bag. This would help reducing the risk of transferring infection(s) to a patient, or indeed re-infecting a patient.

An aerosol can, on the other hand, provides administration of the same amount of active ingredients, throughout its time of use until it is empty. Moreover, the nozzle of an aerosol can may prevent external contaminants from reaching the inside of the can.

In addition to cleansing a wound, the wound cleansing solution, in atomised form, may be used to spray moisturise a wound, in particular a chronic wound. This is important since moist wounds heal approximately 50% faster than dry wounds. Slow wound healing in dry wounds is due to slower cells migration, since cells only find moisture deeper in the wound bed.

Using the wound cleansing spray as disclosed herein, a biofilm in the wound is disrupted. This means that the biofilm is broken down, whereby removal of the bacteria and/or other microorganisms from the wound's surface is enabled.

Throughout this disclosure, all aspects and embodiments may be combined. The person skilled in the art is well equipped to make such combinations.

The invention shall now be described in more detail, with reference to the accompanied figure. The person skilled in the art realizes that there are alternatives to the embodiments disclosed herein that, while not expressly disclosed, are still part of the scope of the invention.

Examples

Biofilm disruption

This example shows the biofilm disruption capabilities of various wound cleanser jets against a mixed species 24 hour pre-formed biofilm.

To assess the biofilm disruption capabilities of the wound cleansing jets, at different pressures, a Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans mixed species biofilm was provided, using a CDC reactor biofilm model.

Staphylococcus aureus NCTC 8325, Pseudomonas aeruginosa NCIMB 10434, and Candida albicans ATCC® MYA 2876™ were being used for the test. This is an accredited method ISO17025 (Perfectus) using species that are known to form biofilms commonly found in wounds. The test agents used in this study are described in Table 1.

Table 1: test agents used Equipment and media

Equipment:

UKAS calibrated multichannel pipettes (P20 and P300) - Gilson®, UK

UKAS calibrated pipettes (0.5 - 1000 pL range) - Proline® Plus, UK

Microbiological orbital incubator - Innova 4300, Eppendorf UK Limited Polycarbonate coupon, RD128-PC - Biosurface Technologies, USA

CDC Biofilm Reactor® - Biosurface Technologies, USA

Sonicating water bath - VWR, UK

96 well plates - Scientific Laboratory Supplies (SLS), UK Media:

Tryptone Soya Agar (TSA) - Southern Group Laboratories, UK Tryptone Soya Broth (TSB), Acumedia® - SLS, UK

Brain Heart Infusion Agar (BHIA), Acumedia® - SLS, UK

Phosphate Buffered Saline (PBS), Acumedia® - SLS, UK

Sabouraud Dextrose Agar (SDA) - Trafalgar, UK

Sabouraud Dextrose Broth (SDB) - Trafalgar, UK

Glucose - Sigma-Aldrich, UK

Methodology

Development of a CPC reactor biofilm

24 or 48 hour cultures of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans cans were harvested from a Tryptone Soya Agar (TSA) or Sabouraud dextrose agar (SDA) plates and used to prepare a 1 x 107 ± 5 x 106, 1 x 104 ± 5 x 103 and 1 x 105 ± 5 x 105

CFUmL-1 suspension, respectively. The working inoculum was confirmed by serial dilution and spread plating. The prepared inoculum was used to inoculate a sterile CDC reactor containing polycarbonate coupons. The reactor was incubated for 24 hours at 37 ± 2°C and 50 ± 5 rpm to encourage biofilm growth.

Treatment of pre-formed biofilms

Following 24 hours of incubation, pre-formed biofilms attached to the polycarbonate coupons were washed three times in sterile phosphate buffered saline (PBS) in order to remove planktonic organisms. Test agent aerosol cans were shaken for 5 seconds and sprayed for one second to prime the nozzle thereof. The washed biofilms were exposed to test agent jets (Table 1) for 5 seconds at a distance of 10 cm on each side of the coupon.

Coupons were then incubated at room temperature for 5 minutes before recovery. Negative control, Positive control and sodium chloride were tested by immersion in 2 mL aliquots for 5 minutes. All tests were performed in triplicate. Recovery and quantification of remaining viable organisms

Following the above treatment, coupons with biofilm were transferred to 2 mL of PBS and placed into a sonicating water bath for 5 minutes in order to recover remaining attached organisms. The resultant suspensions were serially diluted and 100 pL samples of each dilution were spread onto brain heart infusion agar (BHIA) in duplicate. BHIA plates were incubated for 24 hours at 37 ± 2°C, and species counted individually to determine the total microbial load. The number of colonies were counted and expressed as average Log recoveries (LoglOCFUmL ¹ ).

Data was presented as the mean ± standard deviation (SD) from three independent replicates with duplicate repeats for each independent replicate.

Statistical analysis

An F-Test was used to assess variance of data. A Student's two sample t-test (two-tailed) was used to assess statistical differences between the test samples and the negative control.

Data was considered statistically significant when p-value < 0.05. The minimum limit of detection for this study was 1 Log.

Results

The mixed species biofilm disruption capabilities of four wound cleanser products against the 24 hour pre-formed Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans mixed species biofilm was assessed.

An average total viable recovery of 6.47 ± 0.22 LoglOCFUmL-1 was observed following treatment with the negative control. Following treatment with the positive control, no viable organisms were recovered. This was a reduction of 6.47 ± 0.00 LoglOCFUmL-1 compared to the negative control treatment, (p < 0.001) (see Table 2 below, and Figure 1).

Table 2: Average quantity of viable microorganisms recovered, and average reductions compared to the negative control following spray and immersion treatment for five seconds with four wound cleanser jets, and three wound cleanser liquids. SD = standard deviation, CFU = colony forming units. - = not statistically significant, ** = < 0.01 and *** = < 0.001.

Prevention of contamination from a pressure container nozzle

A bag-on-valve sample nozzle was inoculated (i.e. contaminated) with methicillin resistant Staphylococcus aureus (MRSA) and total viable count was recovered from the sprayed product, 0.9 % saline, at days 0, 3, and 5, respectively, post-inoculation. Following the contamination of the nozzle, a significant reduction in MRSA was observed after 3 days of incubation, and no visible organisms were recovered after 5 days (see Table 3 below).

Table 3: Quantity of methicillin resistant Staphylococcus aureus recovered from a wound cleanser jet following nozzle contamination.

Conclusion

Treatment with all wound cleanser jets as disclosed herein resulted in biofilm reductions, in comparison to the negative control, showing that the mechanical effect of the jet was sufficient to significantly reduce biofilm attachment. Interestingly, treatment with a jet of increasing pressure resulted in increased levels of reduction.

The results show that a wound cleansing solution according to the invention described herein, when spray administered in atomised form from a pressure container having a pressure in the interval of 2 to 15 bars, can be used successfully to reduce formed biofilm, and thus to support wound healing. Moreover, use of a pressure container, such as a bag-on-valve or aerosol can, has been shown to additionally reduce the microbial burden in wound care, by way of prevention of contamination of the wound cleansing solution contained in the bag or can.