Patent ID: 12208075

The practical applications and benefits of this invention are illustrated further in the following Examples. Unless otherwise stated, the membrane lipids used in the Examples are delipidised and contain less than 3% conjugated extraneous lipid material as shown by HPLC analysis as described in the Methods.

EXAMPLE 1

Preparation of Adhesion Inhibitory Compositions of Membrane Lipids

The following membrane lipids were purchased from Sigma Aldrich, Poole, Dorset, UK.

TABLE 3Membrane lipidsCompound/OriginalMembrane lipidComponentsourceCodePhospholipidCrude LecithinSoya BeanLCPhospholipidPhosphatidylcholineSoya BeanPTCPhospholipidPhosphatidylethanolamineSoya BeanPTEPhospholipidPhosphatidylglycerolSoya BeanPTGPhospholipidphosphatidylinositolSoya BeanPTIPhospholipidPhosphatidylserineSoya BeanPTSSphingolipidCeramideBovine BrainCSphingolipidSphingomyelinBovine BrainSGMGlycoglycerolipidMono-galactosylWheat flourMGDGdiglycerideCholesterolLanosterolSheep woolL

Crude lecithin was purchased from GR Lane Ltd, Gloucester, UK.

Aqueous dispersions of each of the above membrane lipids were prepared by suspending 1.0 grams in 100 ml of sterile distilled water (10 mg/ml) and agitating therein for 60 minutes with the aid of a magnetic stirrer. Dispersion of ceramide, sphingomyelin and lanosterol was achieved with the aid of sonication and stirring, while dispersion of all other membrane lipids was relatively easily achieved with stirring alone. Phospholipid and glycoglycerolipid dispersions were relatively stable, but others had a tendency to separate on standing and so all dispersions were subjected to 30 seconds homogenization using a laboratory homogenizer immediately prior to use in the adhesion inhibitory assay. A suitable homogeniser is an Ultra-Turax Model 118 (IKA Works, Wilmington, NC 28405, USA) fitted with an S18N-19G dispersing tool, operating at 6,000 to 8,000 RPM.

The adhesion inhibitory properties of each phospholipid were tested as described in the methods at concentrations of 10, 5, and 2.5 mg/mi; the lower dilutions were achieved by diluting the original dispersion with sterile distilled water. Due to logistical limitations of collecting sufficient buccal epithelial cells at any one time, the assays were conducted in blocks of five individual membrane lipids on separate days, and the results shown in Table 4 are a composite of these; the Control is zero concentration of test item and Bovine Serum Albumin (BSA) is used as a protein blank.

TABLE 4Candida albicansper 100 Buccal Epithelial Cell% inhibitionatItem Code0 mg/ml2.5 mg/ml5.0 mg/ml10 mg/ml2.5 mg/mlLC5403872919228PTC54012759076PTE49616774066PTG49611462077PTI51920187061PTS5192331023955C48436328210925SGM48437927712621MGDG5402611286651L51930623613341BSA49640037930319

In preliminary evaluation of crude lecithin from various sources (egg yolk, soya and sunflower), it was noted that the adhesion inhibitory properties varied considerably, despite the reported purity being approximately the same. It was reasoned that the purchased material might contain extraneous fat or tri-glyceride and to remove this, the original material was suspended in acetone at 10% W/V and stirred for 30 minutes after which the insoluble membrane lipid was allowed to settle and the acetone decanted. The ‘de-lipidised’ lecithin (DLL) was dried in a fume hood, and its adhesion inhibitory properties evaluated as previously described. The remaining membrane lipids listed in Table 4 were delipidised in the same manner. The delipidised membrane lipids were analysed by HPLC as described in the Methods, and all were shown to contain less than 3% conjugated extraneous lipid material. The results are shown in Table 5 below, including the incremental increase in adhesion inhibitory effect achieved by de-lipidisation of individual membrane lipids.

TABLE 5Candida albicansper 100 Buccal Epithelial Cell% inhibitionatItem Code0 mg/ml2.5 mg/ml5.0 mg/ml10 mg/ml2.5 mg/mlLC5403872919228DLL48913848071 (+43)DL PTC5081320084 (+8)DL PTE5151390073 (+7)DL PTG474570088 (+11)DL PTI519680077 (+16)DL PTS51914562072 (17)DLC4933051184138 (+13)DL SGM5113011334441 (+20)DL MGDG516206116060 (+9)DL L536247914954 (+13)DL BSA51343139332916 (−3)

Solvent de-lipidisation increases the adhesion inhibitory properties of membrane lipids, especially lecithin by a factor of 2.5, bringing it in line with that of its individual components: PTC, PTE, PTG, PTI and PTS. Further de-lipidisation of the individual components of lecithin achieves further improvement in the adhesion inhibitory properties of these, although less dramatic compared to the effect achieved in de-lipidising crude lecithin. It is assumed here that the commercial process of isolation used to extract individual components of lecithin results in almost complete de-lipidisation, hence the optimal inhibitory effect of these relative to the ‘crude’ lecithin and the smaller incremental effect achieved by additional de-lipidisation.

In many of the applications for the product of this invention the adhesion inhibitory substance will be in contact with blood serum, mucus and other body fluids. It has been discovered that de-lipidised lecithin and most of its constituents lose their adhesion inhibitory properties in the presence of Bovine Serum Albumin (BSA). It has also been discovered however, that adding an organic acid salt counteracts this negative effect and restores most of the adhesion inhibitory properties to the combined membrane lipid and BSA.

In this example, 100 mM solutions of sodium lactate and sodium citrate were used. The salts were prepared first as 200 mM solutions and the pH adjusted to 4.5, and aliquots of both were used to prepare 1% solutions of bovine serum albumin (i.e. 1% BSA in 200 mM sodium salt of citrate or lactate at pH 4.5). The salt solutions were used to dilute a 1% suspension of de-lipidised lecithin (DLL) and a 1% suspension of phosphatidyl choline (PTC), to achieve preparations of 0.5% DLL and 0.5% PTC in 100 mM sodium lactate/sodium citrate at pH 4.5 with 5% BSA in each. A further dilution of the membrane lipid suspension to 0.5% with water and the use of this in halving dilutions of salt solution containing 0.5% BSA gave a composition of 0.25% membrane lipid in 100 mM salt with 0.25% BSA. Similar procedures were used to prepare 0.25% and 0.5% solutions of BSA in water and in 100 mM organic salt solution: the results are presented in Table 6.

TABLE 6Candida albicansper 100 Buccal Epithelial Cell% inhibitionItem Code0 mg/ml2.5 mg/ml5.0 mg/ml10 mg/mlat 5 mg/mlLC: water5403872919246DLL: water48913848090DLL +396320280ND30BSA waterDLL + BSA396290144ND63Sod′ CitrateDLL + BSA396340164ND58Sod′ LactatePTC: water54012759090PTC +474392344ND28BSA waterPTC + BSA474273162ND66Sod′ citratePTC + BSA474291138ND70Sod′ LactateBSA474393369ND22Sod′ citrateBSA474408372ND21Sod′ LactateBSA: water49643840036212

In this Example, with the exception of crude lecithin, all of the membrane phospholipids tested were shown to have superior inhibitory properties preventingCandida albicansadhesion to Buccal Epithelial Cells. When de-lipidised with acetone, crude lecithin is as inhibitory as its main membrane phospholipid components. In combination with bovine serum albumin, the adhesion inhibitory property is eradicated, but it can be restored in the presence of 100 mM solutions of organic acid salts at acid pH.

EXAMPLE 2

Preparation and Testing of Microbicidal Combinations of Membrane Lipid and Free Fatty Acid:

The microbicidal effect of 0.5% caprylic acid in 0.4% de-lipidised lecithin, prepared as described in the Methods, against a range of microbial species may be demonstrated using the microbicidal suspension test described in the Methods. The potency of this material is such that complete eradication of an inoculum in excess of 6 logs may be anticipated in less than 6 minutes. Gram negative species are slightly more resistant than gram positives, particularly those known to be slime producers, such asEscherichia coliandPseudomonas fluorescens, where it is thought that the slime layer protects from contact with the fatty acid for some additional minutes.

Table 7 below illustrates the reduction in viability of a range of bacteria and yeast. Because the inoculums vary, the latest time to achieve 50% in viability of the inoculum is provided in the last column (L. T. 50%), as a comparative measure of potency against that particular species.

TABLE 7Microbicidal Effect: 0.5% Caprylic in 0.4% DLLExposure TimeOrganismGram060120180240300360L. T. 50%StaphaureusRN4220+ve8.535.223.711.61000<120Staphepidermidis+ve8.274.962.670000<120NCTC 11047Streppyogenes+ve7.915.323.331.49000<120NCTC 8198Strepfaecalis+ve8.114.982.541.66000<120NCTC 12697E.coli−ve8.437.176.25.283.641.790<240ATCC 11698Salmonellatyphimurium−ve7.765.943.522.27000<120NCTC 74Klebsiellaaerogenes−ve6.935.153.371.99000<120NCTC 9528ProteusmirabilisC.I.−ve7.416.615.893.751.5900<240EnterobacterCloacaeC.I.−ve8.626.544.942.32000<180Pseudomanasaeruginosa−ve8.337.135.484.782.921.230<240ATCC 27853Pseudomanasfluorescens−ve7.296.425.133.631.2900<240NCTC 10038CandidaalbicansC.I.Yeast6.864.392.960000<120CandidaglabrataYeast6.744.192.230000<120NCPF 8750Crypto′neoformansC.I.Yeast5.974.112.96000<120NoteC.I = Clinical IsolateNote:L.T 50% is Latest Time to achieve 50% reduction in viability in this test

The numerical data presented are log numbers where the integer is the log and the decimal the colony count at that log: 6.3×105for example is presented as 5.63 and this convention will be used throughout this document.

For reasons of difficulty in culturing or because of special growth requirements the potency of the formulation against anaerobes is determined by the Minimum Inhibitory Concentration procedure as described in the Methods, and the results are presented in Table 8.

TABLE 8Minimum Inhibitory Concentration of 0.5% Caprylic in 0.4% DLL againstanaerobes and fungal pathogens using agar dilution technique: MIC valueis the % of Caprylic acid in the test plateOrganismNotesMICClostridium perfringensG − ve anaerobe>0.1ATCC 43150Clostridium difficileATCCG − ve anaerobe (aero intolerant)>0.143598Bacteroides fragilisATCCG − ve obligate anaerobe>0.243859Fusobacterium nucleatumG − ve anaerobe:>0.3NCTC 106523 weeks Columbia blood agarDesulfovibrioG − ve anaerobe>0.1desulfuricansPorphyromonas gingivalisG − ve obligate anaerobe>0.1Campylobacter jejuniiG − ve microaerophilic>0.075ActinobacillusG − ve microaerophilic>0.1actinomycetemcomitansCorynebacteriumG + ve facultative anaerobe>0.1diphtheriaeTreponema denticolaObligate anaerobe>0.2MycobacteriumAerobic acid fastbacillus6 weeks>0.2tuberculosison Lowenstein-jensen medium

EXAMPLE 3

Membrane Lipid Antimicrobials in Blood Contact Applications:

In common with the negative effect on adhesion inhibitory properties reported in Example 1, the presence of bovine serum albumin also impacts on the microbicidal effect. However as in Example 1, this can be overcome by incorporation of an organic acid salt, at acid pH.

An oil-in-water emulsion of 0.5% caprylic acid in 0.4% de-lipidised lecithin is used in this Example. 200 mM solutions of glycolic, acetic, lactic, and citric acids were adjusted to pH 4.5 using 200 mM sodium hydroxide. 10% W/V solutions of Bovine Serum Albumin were prepared in each of the organic acid salts and in water as a control. 5 ml aliquots of the 0.5% caprylic emulsion were added to 5 ml aliquots of each salt solution with and without BSA, and in water with and without BSA, resulting in dispersions of 0.25% caprylic emulsion in 0.2% de-lipidised lecithin dispersed in 100 mM salt solution at pH 4.5 in each test sample.

E. coliwas selected for this Example as it has been demonstrated to be one of the most resistant species. The bacterium was cultured in Brain Heart Infusion broth for 18 hours at 37° C. 10 ml fractions of each test sample were inoculated at time zero with 1.1 ml of the overnight culture, and 1.0 ml samples removed from this at 3 minute intervals for residual viability determination using serial dilution and plate counting procedures.

TABLE 9Antimicrobial effect is impaired by blood components and re-instated by combination with salts of organic acids0.25% Caprylic in 0.2% De-lipidised Lecithin +/−5% Bovine Seum Albumin036912151821242730kill timeWater8.374.972.23000000009Water + BSA8.377.516.425.985.174.933.723.332.211.49030Glycolate8.375.252.68000000009Glycolate + BSA8.376.625.353.792.411.560000018Acetate8.375.944.211.28000000012Acetate + BSA8.377.416.935.244.533.472.181.3400024Citrate8.374.92.70000000012Citrate + BSA8.376.24.51.93000000012Lactate8.375.293.19000000009Lactate + BSA8.377.14.91.8000000012

In a water diluent the effect of 5% W/V BSA is to reduce potency of 0.25% caprylic acid by a factor of 3: Kill Time is extended from 9 minutes to 27. In combination with 100 mM sodium salts of citrate and lactate acids at pH 4.5, the kill time is 12 minutes, glycolate is 18 minutes and acetate is 24. The results of combination with citrate and lactate acid salts with and without BSA are presented in Table 9 above and illustrated inFIG.1.

EXAMPLE 4

Use in Food Safety

In this Example 1 cm3sections of fresh beef were deliberately contaminated with late log phase cultures ofSalmonella entericaandEscherichia coli0157:1H7 at room temperature and allowed to adhere there for 60 minutes. Confirmation of contamination was obtained by mechanically macerating the meat sections in sterile phosphate buffered saline (PBS) and enumeration by serial dilution and plate counting.

A suitable but not optimal carcass wash was prepared using 0.5% caprylic acid in 0.4% de-lipidised lecithin and diluting that ×2 with 200 mM sodium citrate at pH 4.5 as described in Example 3. The wash was sprayed onto contaminated sections of fresh beef, and the antimicrobial effect evaluated by mechanical maceration, serial dilution and plate counting. The results are presented in Table 10 below:

TABLE 10Reduction in Viability of Pathogens on Fresh BeefTimeEscherichia coliSalmonella entericaminutesUntreatedTreatedUntreatedTreated07.146.966.916.61306.985.616.374.59606.563.896.717.73906.822.196.521.291206.69ND6.28NDNote:ND = Not detected

It should be appreciated that the rate of kill on a porous surface such as meat is extended due to the nature of the surface and the time required to permeate it.

EXAMPLE 5

Use of Membrane Lipids to Manipulate Blood Clotting Time:

As described in this Example the membrane lipid products used may be tailored to affect the rate of blood clotting in addition to contributing a significant antimicrobial effect.

Rate of blood clotting was determined using freshly aspirated sheep blood and apparatus and procedures described in the Methods section. An activated blood clotting meter was used to measure anti-clotting effects and a visual tube comparison to evaluate reduced clotting times.

It was unexpectedly discovered that an aqueous dispersion of de-lipidised lecithin (DLL) will amplify the rate of blood clotting in a concentration dependent manner. Varying concentrations of dispersions of DLL were prepared by suspending the required weight in a volume of water, stirring for 30 minutes to hydrate and homogenizing the suspension with a laboratory homogenizer.

Blood clotting and/or anti-clotting effects were assessed using a 20% ratio of test item to fresh blood: in practice 4.0 ml of fresh sheep blood was added to tubes containing 1.0 ml of test item, inverted twice to mix and then either left standing for visual assessment of reduced clotting time or a single drop was applied to the cuvette of an activated blood clotting apparatus for assessment of anti-clotting effect (extended time to clot formation).

As the concentration of DLL in the test item increases, the observed rate of blood clotting increases, i.e. time to clot formation decreases from 360 seconds for whole blood to approximately 60 seconds or less at DLL concentrations in excess of 1%.

The addition of caprylic acid by emulsification in DLL will also amplify the clotting effect up to a point where the concentration by weight equals or slightly exceeds the concentration of DLL by weight. At 0.5% DLL on its own, the clotting time is approximately 125 seconds. Addition of emulsified caprylic acid up to 0.5% does not significantly affect the clotting time. Between 0.5% and 0.75% caprylic acid there is a further depression of clotting time from 125 seconds to approximately 40 seconds (70% less). Thereafter, however, the anti-clotting effect is reversed and clotting time increases with increasing concentration of caprylic acid.

When the concentration of DLL is reduced to 0.25% with increasing concentration of caprylic acid, there is no significant reduction of clotting over and above that attributable to DLL on its own. In fact, as the concentration of caprylic increases to between 0.75% and 1.0%, the clotting time is restored to normal (360 seconds). Further increasing the relative ratio of caprylic acid in 0.25% DLL, has an anti-clotting effect.

Using an emulsion of 0.25% caprylic acid in 0.25% DLL as a standard (STD), it can also be observed that addition of increasing concentrations of sodium citrate salts at pH 4.5 suppresses the anti-clotting effect of DLL in combination with caprylic acid. At 0.5% concentration, the anti-clotting effect of 0.25% caprylic acid in 0.25% DLL has been suppressed completely and clotting time has been restored to above normal (400 seconds). A further increase of sodium citrate in this composition has a progressively increasing anti-clotting effect—up to 600 seconds at 2% sodium citrate in 0.25% caprylic emulsified in 0.25% DLL. The results are illustrated inFIG.2.

EXAMPLE 6

Use of Antimicrobial Membrane Lipids in Amplified Clotting for Wound Care

The combined antimicrobial and clot forming capability of the products of the invention are demonstrated in this Example. A suitable, example of an antimicrobial membrane lipid preparation with enhanced clot forming properties may be selected from the combinations prepared in Example 3. A 0.5% dispersion of DLL in sterile distilled water with 1.0% caprylic acid was used here. It will be noted that no organic acid salt is included in this example and further noted that the absence of such reduces the antimicrobial potency but does not limit the clotting effect. The use of a relatively high caprylic acid load compensates for the interference of blood components with the antimicrobial effect.

A bacterial inoculum ofE. coliwas grown in Brain Heart Infusion broth for 18 hours and a 10 ml volume of this was sedimented by centrifugation at 4,000 RPM for 10 minutes, the pellet was re-suspended in 1.0 ml of the supernatant (concentration ×10).

Two 4.0 ml samples of fresh blood were dispensed to two 15 ml Greiner centrifuge tubes and both were inoculated with 0.1 ml of concentratedE. colisuspension: a blank comprising 5.0 ml of 5% Bovine Serum Albumin in sterile distilled water was inoculated at the same time.

A 1.0 ml of volume of 400 I. U. heparin and 500 I. U. streptokinase in water was added to the first tube, and 1.0 ml of test preparation to the second. Both tubes were mixed by inversion and incubated at 37° C. for 45 minutes. The tube containing the test preparation clotted in approximately 60 seconds; no clot was detectable in the heparin/streptokinase tube after 45 minutes.

At the end of the 45 minute incubation a 1.0 ml of volume of 400 I. U. heparin and 500 I. U. streptokinase in water was added to the clotted tube with test preparation and 1.0 ml of sterile distilled water added to the second. Both samples were homogenized at 1,000 RPM for 2 minutes using an Ultra Turax homogenizer. The clot disruption procedure took approximately 15 minutes (60 minutes exposure in total), after which serial dilution and plate counting procedures were used to enumerate residual bacterial viability in all three tubes.

The BSA blank contained 6.4×107viableE. colicells per ml, the control blood (heparin/streptokinase treated) sample contained 8.3×105, and no viable bacteria were recovered from the test sample.

It will be clear to those skilled in the art that preparations such as described in this Example may be applied to wounds in the form of a liquid, spray, gel, powder, or wet-bandage. It will also be clear to those skilled in the art that the preparations described may be added to other pro-coagulants such as chitin, kaolin or alginate to enhance their pro-coagulation effect and add a microbicidal effect.

EXAMPLE 7

Use of Antimicrobial Membrane Lipids with Anti-Clotting Effect in Surgical Procedures:

Ingress of potentially infectious agents during surgical procedures is a major cause for concern among healthcare professionals. The use of irrigating fluids with antimicrobial effects facilitates prevention of this. There are also many surgical procedures where the ability to prevent blood clotting is advantageous, micro-surgery procedures for example, where pre-emptive clotting may be exacerbated by the implements used and where the use of an irrigating solution to wash out blood and body fluids to prevent occlusion of the site is desirable. A specialized application is the use of anti-clotting liquids to fill the void volume of indwelling catheters during periods when the catheter is not in use.

In this Example, a dispersion of 0.4% de-lipidised lecithin (DLL) with 0.5% caprylic acid emulsified therein is prepared as described in the Methods. The emulsion is diluted to 50% of its initial concentration with 200 mM sodium citrate at pH 4.5, the result being 0.25% caprylic acid, 0.2% DLL in 100 mM sodium citrate, or approximately 2.5% W/V sodium salt of citric acid at pH 4.5. Sodium citrate is prepared by adjusting the pH of 200 mM citric acid with 200 mM sodium hydroxide to 4.5; this is not the same as ‘tri-sodium citrate’ which is commonly used as an anti-clotting agent, because not all of the carboxylic acid residues have been salted out.

As described hereinabove, the use of a viscosity-enhancing agent to adjust the viscosity of the formulation to approximate to that of whole blood provides a distinct advantage in preventing dilution of a catheter locking solution at the catheter tip due to blood flow turbulence. Dextran 40 is used here as a viscosity-enhancing agent where it has been found that 20% W/V inclusion provides a viscosity of approximately 4 cP, the viscosity of human blood being between 3.6 and 6 cP.

The emulsified free fatty acid/membrane lipid catheter locking solution used here (ML CLS) has the following constituents: 0.2% W/V de-lipidised lecithin; 0.25% caprylic acid; 20% dextran 40, in 100 mM (2.5% W/V) sodium citrate, pH 4.5. Aliquots of this formulation were dispensed to 15 ml Greiner centrifuge tubes in the following amounts: 0, 0.25, 0.5, 0.75 and 1.0 ml amounts. To each of these 5.0, 4.75, 4.5 and 4.25 ml aliquots of fresh sheep blood were added, respectively. Each tube was evaluated consecutively with fresh blood added immediately after it was aspirated. The respective volume dilutions represent 0, 5%, 10%, 15% and 20% by volume of blood. Immediately after addition of blood, the tubes were inverted twice to mix and a single drop added to the test well of a Hemochron Signature Activated Blood Clotting Meter.

A similar procedure was adopted using a solution of 25,0001.U. of Heparin, which at 5%, 10, 15% and 20% gave sample concentrations of 1,250, 2,500, 3,750 and 5,000 units per ml of test volume.

Also tested were a commercially available Catheter Locking solution, Duralock from MedComp, which contains 47% sodium citrate only, and a composition of 0.05% Methylene Blue, 0.15% Methyl Parabens, and 0.015% Propyl Parabens in 7% (0.24M) sodium citrate, being a replica of the reported formulation for Zuragen; and Taurolock from Tauropharm AG comprising 1.35% Taurolidine in 4% sodium citrate: (see Table 2). Phosphate Buffered Saline (PBS) is used as a dilution control.

It should be noted here that the Hemochron blood clotting system relies on a clot activation process which accelerates time to clot formation; whole blood without additives clots in less than 200 seconds in this apparatus. The timelines in this experiment are not directly comparable therefore with clotting times reported in Example 5 where the baseline for normal clot time is shown as 360 seconds being the observed time for normal (non-activated) clotting. It should also be noted here that the Hemochron meter goes ‘out of range’ at 1,000 seconds of Activated Clotting Time and more extended time recordings are not available.

The results are reported in table 11 below and illustrated inFIG.3.

TABLE 11Activated Blood Clotting Times for Various Catheter Locking Solutions% Incorporation in whole blood0%5%10%15%20%ML CLS1593475377691018Heparin11592844236011023Duralock159300364484722Zuragen159219265394614Taurolock159230310380510PBS control159163165233296Note:1Heparin concentrations range from 1,250 I.U. at 5% to 5,000 I.U. per ml at 20%

In terms of metered anti-clotting effect, the inventive product of this Example is better than 25,000 I.U. of Heparin and considerably better than Duralock, Zuragen or Taurolock. It should be noted again however, that these are ‘Activated’ clotting times. In practice, none of the treated samples—apart from control PBS—showed any visual sign of clotting even after several hours.

The antimicrobial effect of the formulation in this Example was tested using procedures similar to those used in Example 6, with the exception that the clot disrupting agents, heparin and streptokinase, were used to break the clots in the control untreated bloods.

18 hour Brain Heart Infusion Broth cultures ofStaphylococcus aureus, Streptococcus epidermidis, Escherichia coliand an 18 hour culture ofCandida albicansgrown in Yeast Extract Peptone Dextrose broth were concentrated ×10 by centrifugation and re-suspension in one tenth volume of supernatant.

2.5 ml aliquots of the bacterial concentrates were used to inoculate 22.5 ml volumes of freshly aspirated sheep blood, mixed and the blood dispensed as 5.0 ml, 4.75 ml, 4.5 ml, 4.25 ml and 4.0 ml volumes to Greiner centrifuge tubes containing 0, 0.25 ml, 0.5 ml, 0.75 ml and 1.0 ml volumes of the test formulation of this Example.

The inoculated tubes were incubated for 45 minutes at 37° C. following which time, a 1.0 ml of volume of 400 I. U. heparin and 500 I. U. Streptokinase in water was added to all tubes and each was subjected to slow speed homogenization at 1,000 RPM for 2 minutes: the only visible clotting was in the control tubes. Immediately after 60 minutes had elapsed, serial dilution and plate count methods were used to assess residual viability in all samples. For comparative purposes, the same procedure was repeated with Taurolock, Duralock, Zuragen and Heparin at the maximum dose loading of 1.0 ml in 4.0 ml of blood only. The results are presented in Table 12 and illustrated inFIG.4.

TABLE 12Comparative Microbicidal Effect of Catheter Locking Solutions in wholebloodEscherichiaStaphylococcusStreptococcusCandidacoliaureusepidermidisalbicansTime zero7.698.718.126.67Blank Time8.198.98.516.3360 minML CLS 5%4.833.624.173.9ML CLS 10%2.581.441.970ML CLS 15%1.62000ML CLS 20%0000Taurolock 20%6.534.154.725.72Duralock 20%6.976.497.786.54Zuragen 20%5.564.915.734.28Heparin7.928.398.226.9425,000 I.U.20%

In this test the 20% by volume ML CLS according to the invention achieved complete eradication ofE. coli(8 logs), Staphaureus(8 logs), Strepepidermidis(8 logs) andCandida albicans(6 logs) in one hour in whole blood: a 5% volume achieved approximately 4 log reduction of the test inoculums in the same time. Of the four comparative preparations (Duralock, Taurolock, Zuragen or Heparin), only Zuragen and Taurolock had an appreciable microbicidal effect achieving a reduction of between 3 and 4 logs in viability of the test inoculum in one hour; Duralock appears to have a microbistatic effect while no microbial inhibition could be attributed to Heparin.

Thus, the above catheter locking solution according to the invention exhibits significantly better anti-clotting effects and much greater microbicidal effect than existing conventional products.

EXAMPLE 8

Use of Membrane Lipids in Antimicrobial Surface Coating:

A suitable method of applying a persistent coating of a product according to the invention involves emulsification of 1% caprylic acid in 0.8% de-lipidised lecithin prepared as described in the Methods. An equivalent volume of 100 mM sodium citrate buffer pH 4.5 is added to the emulsion to obtain a final concentration of 0.5% caprylic acid, 0.4% de-lipidised lecithin in 50 mM sodium citrate. Eight volumes of absolute ethanol are then added slowly to two volumes of the emulsion with constant vigorous stirring to make the final coating material in 80% ethanol.

In order to coat a plastic surface it is preferable to use some form of surface conditioning which may include a process known as corona discharge wherein an electrical field is generated across the surface imparting a residual charge which facilitates adhesion of the applied coat. Following corona treatment, the ethanol solution described above is sprayed on the surface and dried under forced air conditions at 60° C. Several coats may be applied to construct a layer of antimicrobial coating.

A base layer of membrane lipid may first be applied to an inert surface, and once dried and annealed it is used to ‘anchor’ a second layer of de-lipidised membrane lipid emulsified free fatty acid according to the invention.

In this Example an organic solvent solution of a membrane lipid is applied to the surface of a microtitre plate well, dried and fixed by heating at 60° C. followed by a further application of an aqueous suspension of caprylic acid emulsified in de-lipidised lecithin prepared as described in the Methods. The de-lipidised lecithin emulsion was dried and annealed to the first lecithin coating by heating at 60° C. for 3 hours. Plates treated with de-lipidised lecithin, without caprylic acid were used as a control and untreated plates were used to determine optimum biofilm formation.

A base layer of de-lipidised lecithin (DLL) was prepared by suspending 5% by weight DLL in 80% ethanol: water, and dispensing 100 μl aliquots of this to the test wells.

Wells for determination of optimum biofilm were left untreated. The ethanol fractions were dried in a fume hood and annealed at 60° C. for one hour in an oven. 1% W/V dispersion of de-lipidised lecithin was prepared in sterile distilled water as described in Example 1, and a volume of this used to emulsify 0.25% caprylic acid. 100 μl aliquots of DLL or DLL+0.25% caprylic were transferred to DLL, pre-coated wells and dried in an oven at 60° C. for 3 hours.

A 10 hour culture ofStaphylococcus aureusRN 4220 grown in Brain Heart Infusion broth (BHI), supplemented with 4% sodium chloride was used as inoculum. The mid log phase culture was diluted to 1% with fresh sodium chloride supplemented BHI and 200 μl of this added to the microtitre plate wells, covered and incubated.

At each sample point, 100 μl from each well was transferred to a fresh plate for assessment of growth by Optical Density at 570 nM and the plate was then decanted and washed with copious volumes of sterile distilled water, dried and annealed in an oven at 60° C.

When dry, 100 μl of 0.4% crystal violet was added to each well—including untreated controls. After 4 minutes, excess crystal was drained off, and the plates were again washed with copious volumes of water to remove excess dye and again dried with the aid of an oven at 60° C. The results are presented in table 13 and illustrated inFIG.5.

TABLE 13Inhibition of Biofilm Formation: 24 hour cultureOptical Density 570 nMControl growth2.8Coated planktonic growth2.4Uncoated biofilm0.9DLL coated biofilm0.7DLL + Cap Inhibited biofilm0.1

Uncoated planktonic growth is largely unaffected by the coating of inhibitory DLL+caprylic acid. Wells coated with DLL alone (DLL coated biofilm) are slightly reduced, but in comparison, wells coated with the product of this invention (DLL+Cap Inhibited Biofilm) are essentially free of any biofilm: the coating itself takes up some of the crystal violet dye which accounts for a small increase in Optical Density in the DLL+Cap wells.

EXAMPLE 9

The Use of Membrane Lipids to Achieve Sustained Release of Microbicidal Free Fatty Acids

In therapeutic applications considerable advantage may be gained from using combinations of membrane lipid emulsions with ‘tailored’ release characteristics, which facilitates sustained microbicidal effect at the epithelial surface.

In this Example individual membrane lipids were selected from each of the four classes presented in Table 1 and were the same as those used in adhesion inhibitory studies in Example 1. 0.4% Aqueous dispersions of each were prepared and 0.2% caprylic emulsified in each using procedures described in the Methods for de-lipidised lecithin (DLL). A 0.4% sample of DLL with 0.2% caprylic was also prepared. Each of the membrane lipid preparations was diluted to 50% of its volume with 200 mM sodium citrate at pH 4.5, and therefore each preparation then consisted of 0.1% caprylic acid and 0.2% membrane lipid in 100 mM sodium citrate at pH 4.5. It should be noted here that this is less than half of the microbicidal caprylic acid content of the test item used in Example 3, Table 9.

The yeastCandida albicanswas used in this Example, and the inoculum prepared as an 18 hour YEPD broth culture as described in the Methods. The late log phase culture was centrifuged at 4,000 RPM for 5 minutes and re-suspended in one tenth volume of supernatant to concentrate ×10.

12.0 gram samples of each test preparation were dispensed to Sterilin tubes and each of these was inoculated with 1.2 ml aliquots of concentrated yeast culture. Samples of 1.0 ml volume were withdrawn form these inoculated tubes at timed intervals over the course of one hour and added to 9.0 ml of diluting buffer containing 3% Tween 80 to neutralize. Serial dilutions and plate counting was undertaken to enumerate residual viability as described in the Methods. The results are presented in Table 14 below and illustrated inFIG.6.

TABLE 14Variable Release Characteristics of Membrane Lipids: Kill time for >6 logs;Candida albicansMinutes051015202530354045505560Blank7.136.926.866.697.196.676.937.216.737.176.896.786.84DLL6.395.644.533.211.960PTC6.54.842.571.2200PTE7.325.413.671.880PTG6.765.935.174.533.682.821.951.140PTI7.416.114.833.792.321.160PTS6.86.365.855.234.533.793.132.611.881.10C7.247.286.896.546.145.965.324.974.654.293.873.743.32SGM6.846.556.125.855.424.874.223.763.132.792.221.841.33MGDG7.36.666.15.164.343.321.840L7.396.455.865.344.784.213.672.982.331.741.170

It is evident from the results that the slowest acting emulsions are Ceramide and Sphingomyelin followed by Lanosterol, Phosphatidylserine and Phosphatidylglycerol; the fastest acting emulsions are Phosphatidylcholine, Phosphatidylethanolamine and the combination of phospholipids in de-lipidised lecithin (DLL).

EXAMPLE 10

The Use of Combinations of Antimicrobial Membrane Lipid Emulsions to Fortify Mucus and Achieve a ‘Tailored’ Microbicidal Effect at the Mucosal Surface:

Mucosal fortification involves complementary hydration, lubrication and enhanced antimicrobial effect of mucosal secretions of the eye, nose, mouth, naso-pharyngeal surfaces, the gastro-intestinal tract and the genitalia. This Example describes a preparation suitable for fortification of the mucosal secretions of the mouth and vagina and most particularly suitable for use by individuals susceptible to recurring oral and/or vaginal thrush and other common bacterial and viral infections responsive to the products of this invention.

Examples of Mucosal Fortificants:

Part A: A fast acting membrane lipid emulsion of caprylic acid (Cap) is based on 0.2% W/V Phosphatidylcholine (PTC) dispersed in sterile distilled water, hydrated and homogenized as described in the Methods and used to emulsify 0.25% W/V caprylic acid by the procedure described.Part B: A slow acting membrane lipid emulsion of caprylic acid (Cap) is based on 0.2% Sphingomyelin (SGM), dispersed, hydrated and homogenized as described and then used to emulsify 0.25% W/V caprylic acid as described in the methods.Part C: A 200 mM solution of citric acid is adjusted to pH 4.5 with 200 mM sodium hydroxide. A cellulose based viscosity-enhancing agent (hydroxypropylmethylcellulose, Methocel E4M from Dow Gmbh, Germany) is added at 1% W/V: the polymer is sifted in while vigorously stirring the sodium citrate solution and allowed to hydrate for 30 minutes.

A test preparation of PTC+Caprylic acid (PTC+Cap) was prepared by mixing equal amounts of Part A and Part C, yielding an emulsion of 0.125% caprylic acid with 0.1% PTC in 100 mM sodium citrate containing 0.5% W/V Methocel.

A test preparation of SGM+Caprylic acid (SGM+Cap) was prepared by mixing equal amounts of part B and Part C, yielding an emulsion of 0.125% caprylic acid with 0.1% SGM in 100 mM sodium citrate containing 0.5% W/V Methocel.

A test combination preparation comprising fast and slow acting emulsions was prepared by mixing 30% PTC+Cap with 70% SGM+Cap and combining this with an equal volume of part C (30:70 blend), The 30:70 blend is an emulsion of 0.03% caprylic acid in 0.075% PTC combined with 0.07% caprylic acid in 0.0875% SGM in 100 mM sodium citrate containing 0.5% Methocel.

The microbicidal and adhesion inhibitory properties of all three test preparations were assessed using the standard viability and adhesion inhibition assay described in the Methods. The inoculum for both assays was an 18 hour broth culture ofCandida albicans, grown in YEPD medium and concentrated ×10 by centrifugation and re-suspension in one tenth volume of supernatant. 12.0 gram samples of each test preparation were dispensed to Sterilin tubes and each of these was inoculated with 1.2 ml aliquots of concentrated yeast culture. Samples of 1.0 ml volume were withdrawn form these inoculated tubes at timed intervals over the course of one hour and added to 9.0 ml of diluting buffer containing 3% Tween 80 to neutralize. Serial dilutions and plate counting was undertaken to enumerate residual viability as described in the Methods. The results are presented in Table 15 and illustrated inFIG.7.

TABLE 15Mucosal Fortification: Viability ofCandida albicansin Tailored Release PreparationsMinutes0510152025303540455055Blank6.816.926.796.856.976.766.846.996.696.956.786.9PTC +6.54.843.131.720CapSGM +6.846.556.125.855.424.874.223.763.132.792.221.84Cap30:706.735.214.143.563.122.82.422.161.891.551.330.89blend

The fast acting PTC+Cap behaved as expected reducing viability to zero detectable cells in 20 minutes. The slow acting SGM+Cap was also as expected with viability being reduced by 5 logs in 55 minutes. The combination 30:70 blend gives a good example of a ‘tailored release’ profile, viability was reduced by more than 2 logs in 10 minutes, a rate which paralleled the fast acting PTC+Cap, thereafter the microbicidal rate slowed considerably, and approximated to that of the slow acting SGM+Cap. The blend however had achieved 1 log greater reduction in viability at 55 minutes compared to SGM+Cap alone.

Assessment of adhesion inhibitory properties of the three test preparations was undertaken using the Buccal Epithelial Cell model described in the Methods and used in Example 1. For comparison, preparations of the two membrane lipids, PTC and SGM, at 0.1% in 100 mM sodium citrate buffer pH 4.5 with 0.5% Methocel were also prepared and included in the test procedure.

Washed yeast cell pellets were re-suspended in 2.5 ml volumes of the test preparations (PTC, PTC+Cap, SGM, SGM+Cap, 30:70 blend described above and BSA blank). 100 mM sodium citrate at pH 4.5 was used for determination of control adhesion. After 10 minutes pre-exposure, the yeast suspensions were used to re-suspend washed Buccal Epithelial Cell pellets which had been harvested and prepared as described in the Methods. The combined yeast and Buccal Epithelial cell in test, blank or control were incubated with gentle agitation for 60 minutes at 37° C., after which direct microscopic counts using a Hemocytometer slide were used to enumerate the numbers of yeast adhering to 100 Buccal Epithelial cells: The results are presented in Table 16 and illustrated inFIG.8.

TABLE 16Mucosal fortification: Inhibition of Adhesion in Tailoredrelease Preparations:Candida albicansto Buccal Epithelial CellCount/100 BEC% Adhesion% InhibitionBlank5091000PTC2394753SGM3747327PTC + Cap20496SGM + Cap266524830:70 Blend8817830.5% BSA4258317

The adhesion inhibitory properties of 0.1% PTC on its own and with 0.125% caprylic acid are considerably better than equivalent preparations of SGM: as might be expected the 30:70 blend lies midway between the two.

From the data in Table 16 it is also evident that the emulsified fatty acid has a synergistic effect on the adhesion inhibitory properties of the membrane lipid. The percentage reduction in adhesion achieved with PTC alone (53%), is reduced by a further 43% in combination with caprylic acid.

It should be noted that according to the viability data in Table 16 and as illustrated inFIG.7, none of the yeast cells in PTC+Cap is viable after 20 minutes exposure, and approximately 50% to 60% of those in the other two samples are dead. Under the microscope however, yeast cells appear to be intact and while greatly reduced there are still a few apparent that are adhering to Buccal Epithelial cells, suggesting that dead cells are capable of adhesion and biofilm formation.

EXAMPLE 11

The Use of Membrane Lipids in Skin Antisepsis and Prevention of Cross Contamination in Hospitals and Patient Care Establishments:

Methods of evaluating skin antiseptic agents in wash and gel formulations are well established and are fully described in the official procedures of the EU for ISO Certification under EN 1500 (hand gel) and EN 1499 (liquid soap).

In this Example, the relatively non-pathogenicEscherichia coliK12 NCTC 10538 was used (The National Collections of Industrial and Marine Bacteria Ltd, UK: Catalogue of Type Strains ISBN No 0 9510269 3 3). The bacterium is routinely cultured and maintained on tryptone soya agar or broth (TSB), which may be acquired commercially from Oxoid, UK and has the following constituents: 1.5% W/W tryptone (pancreatic digest of casein), 0.5% W/W peptone (papaic digest of soybean meal), 0.5% W/W sodium chloride, and agar at 1.5% W/W when required as a solid medium.

Prior to the test volunteers wash their hands with a mild non-antiseptic soap; a suitable product is E45 Emollient Wash Cream from Boots Healthcare, UK. After washing and drying, the hands are dipped into a 2 litre beaker containing 1 litre of 24 hour culture ofE. coligrown in TSB and containing not less than 2×108viable cells per ml as confirmed by serial dilution and plate counting. Both hands are immersed in the contaminating suspension up to the mid-metacarpals and held there for 5 seconds, and then removed. Excess contaminating fluid is allowed to run-off and then the hands are air dried in the horizontal position for 3 minutes.

To ensure adequate contamination and to establish a pre-value for enumerating reduction, the hands are sampled by dipping the tips of the fingers and thumb of each hand into 10 ml of sterile PBS in a Petri-dish, and rubbed against the base of the plate for 1 minute. After sampling, the hands are treated with either the product of this invention or Spirigel; 4 ml of either preparation is applied to the hands and manipulated over the entire surface area of both hands. The hands are then rinsed under clean (potable) running water, which is lukewarm at approximately 37° C. for a timed period of 20 seconds. After rinsing, the hands are held in an upright position while an assistant dries the palms and wrists with a paper towel. The finger tips and thumb are then sampled by immersion in 10 ml of PBS as described above.

Immediately after sampling, prior to or after washing, 1 ml of the sampling fluid was aseptically transferred to and spread on the surface of a TSB agar plate and another 1 ml is transferred aseptically to 9 ml of sterile PBS and mixed and the process of serial dilution and plate counting proceeds as described previously.

A test preparation of the product of this invention was prepared, being a combination of caprylic acid in phosphatidyl choline at 30% and caprylic acid in sphingomyelin at 70% prepared as described in the Methods. In this Example the membrane lipids were prepared as 1% concentrates with 1% caprylic acid and blended at 30:70 ratio.

A viscosity-enhancing agent is employed to improve the rheology of the test preparation. In this case a Carbopol co-polymer, Pemulen TR-1 from Noveon Inc, Cleveland Ohio was used at 0.45%. The polymer was added to a volume of absolute ethanol equivalent to 70% of final preparation volume and allowed to disperse therein for 30 minutes. A 30% volume of 30:70 blend of the product of this invention as described above was then added to the ethanol and polymer with constant stirring to facilitate rapid dispersion.

The Final Test Product Contains:

0.045% Caprylic acid in 0.045% Phosphatidylcholine: 0.105% Caprylic acid in 0.105% Sphingomyelin: 0.45% Carbopol polymer; 29.25% water; 70% ethanol.

Spirigel is reported to contain 70% ethanol, 30% water and an unknown amount of an unknown viscosity-enhancing agent.

In this Example both test product and Spirigel were used immediately after experimental contamination of volunteers' hands to evaluate de-contamination, and both were used on experimentally re-contaminated hands 10 minutes after application of Spirigel and test preparation according to the invention; the results are presented in Table 17.

TABLE 17Persistent Effect of tailored release membrane lipids in skinantisepsisSpirigelTest itemPre-treatment contamination6.426.69Post treatment contamination, i.e.3.23 (−3.19)3.42 (−3.27)residual viabilityPre-application 10 min pre-Not ApplicableNot Applicablecontamination (treatment 10 minpre-contamination)Contamination: 10 min post6.426.59treatmentContamination: 10 min Post5.972.67 (−3.3)application, i.e. viable cellsrecovered for contamination10 mins post application

As might be anticipated from the alcohol content, when used immediately after contamination both products achieved greater than 3 log reductions of the applied contamination. When used on hands 10 minutes prior to contamination however the alcohol content of both products had evaporated by the time the contamination was applied, and the results show that Spirigel had no significant residual effect (0.45 log reduction). The persistent nature of the membrane lipid emulsion of fatty acid was still present on the hands in the test item, and achieved greater than 3 log reduction.

Spirigel has an immediate but no persistent antimicrobial effect, while the test item according to this invention is both immediate and persistent microbicidal effects.

EXAMPLE 12

Use of Membrane Lipid Emulsions in Wound Care

To illustrate the potential utility of the product of this invention in wound care an ex-vivo model employing freshly slaughtered sections of beef brisket is used. Brisket has an optimal distribution of lean muscle, fat and collagen, and is consequently considered to be representative of all potentially infected wound surfaces. Brisket sections are excised immediately post slaughter, without chilling and with the external fascia membrane intact, these are divided under aseptic conditions into cubes of approximately 1 cm square. Prepared cubes are ‘infected’ by submerging them in late log phase cultures of bacteria for one minute, dried and suspended in a 37° C. environment for one hour to facilitate bacterial adherence and colonization of the meat surfaces.

A suitable example of a wound care formulation is the “standard formulation” of this invention (containing 0.5% caprylic acid in 0.4% DLL) diluted 1:1 in 200 mM sodium citrate buffer at pH 4.5, which then consists of 0.25% caprylic acid in 0.2% DLL in 100 mM sodium citrate buffer at pH4.5 Test sections of infected meat are treated by spraying the wound care formulation directly onto the infected surface and evaluating the test samples for residual bio-burden at predetermined exposure times. Infection and its eradication are confirmed by mechanical maceration of treated and untreated sections in sterile phosphate buffered saline (PBS) and enumeration of the bio-burden by standard microbiological serial dilution and plate counting techniques.

Typical results are presented inFIG.9, where greater than 7 logs of the common wound infecting bacteria,Staphylococcus aureusandStreptococcus pyogenesare shown to be eradicated in less than 120 minutes. It should be appreciated that the rate of kill on a fissured surface such as a wound is extended due to the nature of the surface and the time required for the formulation to permeate to the seat of the infection. A comparison of the relative potency of the standard formulation in citrate buffer as above with the alternative and commonly used antimicrobials, chlorhexidine gluconate and silver sulphadiazine is presented inFIG.10. At 100 minutes exposure the product of this invention has virtually eradicated 7 logs ofStaphylococcus aureus:1 log remains under chlorhexidine treatment and 3.5 logs with silver sulphadiazine.

EXAMPLE 13

The Surgical Use of Membrane Lipids in Prevention and Disruption of Biofilm

Staphylococcus aureusRN4220 is noted for its ability to form tenacious biofilm under laboratory conditions when stress cultured in the presence of sodium chloride. The organism was cultured to mid log phase (10 hours) in Brain Heart Infusion broth and 20 micro-liter volumes used to inoculate wells of a 96 well microtitre plate containing 180 μl of BHI supplemented with 4% sodium chloride. When incubated at 37° C. under these conditions for 6 hours an appreciable biofilm is formed at the base of each well. The biofilm is quantified by decanting the culture and washing the wells with sterile distilled water, after which the plates are dried and stained with 0.4% crystal violet, re-washed and dried; the Optical Density of the stained biofilm is measured at 570 nm.

From previous Examples it will be clear that incorporation of the membrane lipid emulsified product of this invention will have a microbicidal effect, preventing growth and biofilm formation. As illustrated here where biofilm already exists, however, contact with an emulsion of membrane lipid and free fatty acid will effectively kill all viable bacteria in the biofilm and disrupt the film itself.

Assessment of reduction of viability of an established biofilm prepared as described above was achieved by incorporating Alamar Blue at 10% by volume in fresh BHI broth and re-charging the wells of a 96 well microtitre plate with pre-formed biofilm. Alamar Blue is a redox indicator from Invitrogen Ltd, Paisley, UK. It imparts a deep blue color to the media, and when reduced by microbial metabolic activity the color changes from non-fluorescent blue to a highly fluorescent red; absorbance and emerging fluorescence may be measured at 570 nm and 600 nm.

Microtitre plate wells containing pre-formed biofilm were treated with the membrane lipid CLS (ML CLS) used in Example 7 and similar wells were also treated with Zuragen, Duralock and Taurosept for time periods ranging from zero to 60 minutes. At the end of each exposure period, the plates were decanted and washed once with phosphate buffered saline containing 3% Tween 80 and twice with sterile distilled water. 200 μl of BHI containing 10% Alamar Blue was added to the wells and the plates incubated for 60 minutes. There was no detectable color change in any of the wells treated with the membrane lipid CLS of this invention indicating complete eradication of viability within the biofilm in less than one hour. All of the wells treated with Zuragen, Duralock or Taurosept had changed from blue to red demonstrating little or no reduction in viability in the established biofilm.

The efficacy of the membrane lipid CLS of this invention, and the comparable ineffectiveness of the other three formulations in reducing the actual amount of pre-formed biofilm may be demonstrated using similar procedures.

Microtitre plate wells containing pre-formed biofilm were treated with the four formulations for time periods from zero to 60 minutes, decanted and washed as described previously. The treated plates were dried and stained with 0.4% crystal violet, dried and the intensity of stain being a measure of residual biofilm was recorded at 570 nm. The results are illustrated inFIG.11wherein it is evident that although some reduction in biofilm was achieved with Zuragen, Duralock and Taurosept, it is inconsequential in comparison to the near total eradication of biofilm achieved with the membrane lipid CLS (ML CLS) of this invention.

EXAMPLE 14

Comparative Adhesion Inhibitory Properties

De-lipidised lecithin is a more potent adhesion inhibitory substance compared to milk serum apo-proteins in WO 03/018049, wherein the apo-proteins are generated by lipase hydrolysis of whey proteins. For adhesion inhibitory comparison, a whey protein hydrolysate was prepared as described in WO 03/018049 using Carbelac 80 whey protein concentrate. A whey protein isolate (Provon 190 from Glanbia PLC) was also tested contemporaneously. For full comparison purposes a formulation of 0.5% W/V caprylic acid in 0.4% W/V de-lipidised lecithin was prepared in 100 mM sodium citrate pH 4.5 as described in the Methods and included in the test sequence. All test items were dispersed in 100 mM sodium lactate buffer at pH 4.5. The results are presented in Table 18 below.

TABLE 18Candida albicansper 100 Buccal Epithelial Cells%inhibition02.55.01015at 5mg/mlmg/mlmg/mlmg/mlmg/mlmg/mlCarbelac 8048339532026018933Provon 1905143132771135346Lipase digest of49133015448068Carbelac 80De-lipidised504130330093lecithin (DLL)0.5% Caprylic51746000100in 0.4% DLL

While de-lipidised lecithin on its own is significantly better than the three dairy based preparation, the emulsified combination of de-lipidised lecithin and caprylic acid is the most potent.

EXAMPLE 15

Comparative Microbicidal Effect

The MIC by agar dilution technique is also used here to demonstrate the superior potency of the “standard formulation” described hereinabove over the product disclosed in WO2009/072097 comprising a blend of free fatty acids emulsified in the whey protein isolate, Provon 190 from Glanbia PLC. The product of WO2009/072097 contains 28% by weight of free fatty acid blend, while the standard formulation of this inventions contains just 0.5% by weight. In order to make a suitable comparison between the two products the product of WO2009/072097 was diluted by 5/28 in sterile distilled water to obtain a dispersion comprising 5.0% free fatty acid which was then diluted further and used to prepare agar plates ranging from 1.0% free fatty acid to 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.075%, 0.05% and 0.025% (of fatty acid content).

For further comparison an emulsion was constructed using 0.5% caprylic acid in 0.4% Provon 190, using the emulsification agent from WO2009/072097 instead of de-lipidised lecithin. The results are shown in Table 19 below.

TABLE 19Comparative MIC valuesFormulation ofinvention0.5%containingProduct ofCaprylic in0.5% caprylicWO/0.4%acid inTest organism2009/072097Provon 1900.4% DLLStaph aureusRN4220>0.4%>0.7%>0.1%Staph epidermidis>0.4%>0.7%>0.1%NCTC 11047Strep pyogenes>0.4%>0.7%>0.075%NCTC 8198Strep faecalis>0.4%>0.7%>0.075%NCTC 12697E. coli>1.0%>1.0%>0.4%ATCC 11698Salmonella typhimurium>0.8%>0.8%>0.3%NCTC 74Pseudomanas>1.0%>1.0%>0.5%aeruginosaATCC27853Pseudomanas>1.0%>1.0%>0.5%fluorescensNCTC10038Candida albicansC.I.>0.4%>0.7%>0.075%Candida glabrata>0.4%>0.7%>0.1%NCPF 8750

For the two Staphylococci, the two Streptococci and theCandidasp, the measured MIC for the standard formulation is one quarter or less than that of the product of WO2009/072097, indicating a four times greater potency. In the case of the twoPseudomonassp,E. coliandSalmonella, the measured MIC is one half to one third less than the product of WO2009/072097 again clearly demonstrating significantly greater potency. The measured MIC's for the emulsion of caprylic acid in Provon 190 are significantly greater (significantly less potent) than the standard formulation of this invention.

EXAMPLE 16

Comparative Microbicidal and Adhesion Inhibitory Effect of Different Free Fatty Acids in Both Emulsified and Free Form

Fatty acids in free form (not emulsified) have relatively little microbicidal effect primarily because of their insolubility in aqueous medium. Emulsification in membrane lipids as described herein expands the relative surface area of the fatty acid and facilitates its dispersal in aqueous medium. The membrane lipid emulsification agent also facilitates contact and transfer of the fatty acid to a microbial cell surface. As illustrated in previous Examples, variable lipophylicity between different membrane lipids affects the rate of microbicidal effect. In general, membrane lipids are superior emulsification agents, exhibiting superior microbicidal effect as demonstrated in Example 15. As demonstrated here the superior potency of membrane lipid emulsified free fatty acid extends across a range of microbicidal fatty acids.

Seven separate emulsions of seven different free fatty acids at 0.5% W/V were prepared in 0.4% W/V de-lipidised lecithin as described in the methods. Emulsions were prepared at temperatures above the melting points of the individual free fatty acids: caproic, caprylic and pelargonic at room temperature (20° C.); capric and undecylenic acid were prepared at 37° C., lauric acid was emulsified at 50° C. and myristic at 60° C. A blend of 50% caproic and 50% lauric acid has a melting point of less than 28° C. as does a blend of 40% lauric acid in oil of lemon balm, and these were emulsified at 37° C.

The comparative microbicidal effect of each individual fatty acid and blend thereof in free non-emulsified form and emulsified in de-lipidised lecithin was evaluated using the microbicidal suspension test described in the methods. Evaluation of the microbicidal effect of free non-emulsified fatty acids is frustrated by their insolubility. Inclusion of the non-emulsified form was considered necessary to illustrate the exponential increase in potency in the emulsified form. Each free fatty acid was prepared at 0.5% W/V in water and agitated vigorously to disperse followed by immediate pipetting of 1.0 ml aliquots to test containers, prior to inoculation with the test organism. The test organism wasStaphylococcus aureusRN 4220 grown to late log phase on Brain Heart Infusion Broth.

The results are presented in Table 20 below.

TABLE 20Comparative Microbicidal EffectFree fatty acids or blends thereof at 0.5% W/Vnon-emulsified in the test and at 0.5% W/Vemulsified in 0.4% W/V de-lipidised lecithin in the test.Exposure Time in Seconds at 37° C.Fatty acid or blend060120180240300360Free caproic acid7.877.176.827.576.936.476.73Emulsified caproic acid7.876.214.892.53000Free caprylic acid6.946.796.836.216.335.896.17Emulsified caprylic acid6.945.323.710000Free pelargonic acid6.946.556.315.965.675.835.27Emulsified pelargonic acid6.944.632.991.81000Free capric acid6.946.326.486.736.365.976.11Emulsified capric acid6.944.793.442.190Free undecylenic acid7.877.196.996.576.836.676.31Emulsified7.874.972.690000undecylenicFree lauric acid7.877.357.777.417.987.237.65Emulsified lauric acid7.875.794.633.862.5500Free myristic acid7.387.277.846.937.116.266.84Emulsified myristic7.386.465.815.214.983.692.99Free lauric caproic 50:507.386.997.186.746.376.956.81Emulsified Lauric: caproic7.386.765.323.172.9800Free 40% lauric in oil7.387.217.767.396.956.876.49of lemon balmEmulsified 40% Lauric7.385.923.132.77000in Oil of Lemon BalmDe-lipidised Lecithin 0.4% W/V7.337.927.416.837.147.666.95Blank determinations were conducted in all assays by suspending the inoculum in phosphate buffered saline at 37° C. and no reduction in inoculum viability was detected over the six minute exposure period in any test sequence.

Free fatty acids in non-emulsified form achieve at best 1 log reduction in viability over the test period of 6 minutes. The membrane lipid emulsification agent (de-lipidised lecithin) equally exerts little or no microbicidal effect. In comparison, with the exception of emulsified myristic acid, equivalent weight emulsions of all other fatty acids and blends thereof in de-lipidised lecithin reduce residual viability in a test inoculum of 6.94 to 7.87 logs to zero in less than 4 minutes.

Neither free fatty acids on their own nor the de-lipidised membrane lipid on its own have any detectable microbicidal effect within the time-frame of this test. An emulsified combination of the two enables the microbicidal effect of the fatty acid synergistically.

The same test items used in microbicidal evaluation above were also tested for their adhesion inhibitory properties using the Buccal Epithelial Cell assay described in the Methods.

TABLE 21Comparative Adhesion Inhibitory EffectFree fatty acids or blends thereof at 0.5% W/V non-emulsified in the testand at 0.5% W/V emulsified in 0.4% W/V de-lipidised lecithin in the test.BlankTestCount/Count/%%Fatty acid or blend100 BEC100 BECAdhesioninhibitionFree caproic acid4293808911Emulsified caproic acid50300100Free caprylic acid429400937Emulsified caprylic acid50300100Free pelargonic acid4293608416Emulsified pelargonic50320.399.7acidFree capric acid4293869010Emulsified capric acid50300100Free undecylenic acid5214488614Emulsified undecylenic50800100Free lauric acid474450955Emulsified lauric acid508561189Free myristic acid474436928Emulsified myristic508681387Free lauric caproic 50:50474450955Emulsified Lauric: caproic50800100Free 40% lauric in oil of5264608713lemon balmEmulsified 40% Lauric in52600100Oil of Lemon BalmDe-lipidised Lecithin4979619810.4% W/V

De-lipidised lecithin on its own in the test achieves 81% inhibition of adhesion. Emulsions of caproic, caprylic, pelargonic, capric and undecylenic achieve greater than 99% inhibition, and emulsions of lauric and myristic achieve greater than 86% inhibition. All non-emulsified free fatty acids achieve less than 17% inhibition. The adhesion inhibitory effect of caprylic acid for example is amplified by a factor of 14 in emulsified form.

At the concentrations of test item used in Table 21, the adhesion inhibitory effect is essentially swamped by the intrinsic microbicidal effect. It has been demonstrated that the majority of microbial cells will be dead as a result of exposure to the microbicidal effect of the emulsified fatty acid, and it must be assumed that this will influence adhesion.

A more appropriate measure of the amplified adhesion inhibitory effect attributable to emulsions vs free acid or non-emulsified membrane lipids may be obtained using a concentration below the Minimum Inhibitory Concentration (MIC) of the emulsified free fatty acid. The MIC of caprylic acid in the formulation of this invention is greater than 0.1%.

A formulation of 0.5% caprylic in 0.4% de-lipidised lecithin (as used above) is diluted by a factor of 5 in sterile distilled water to achieve a concentration of 0.1% caprylic in 0.08% de-lipidised lecithin and further diluted by half to achieve 0.05% caprylic in 0.04% DLL. These dilutions together with the same concentrations of DLL and non-emulsified free caprylic acid were tested in the Buccal Epithelial Cell assay as above. The results are presented in Table 22.

TABLE 22Low Dose Adhesion Inhibitory EffectCaprylic acid at 0.1% W/V and 0.05% W/V non-emulsified inthe test and at 0.1% W/V and 0.05% W/V emulsified in0.08% W/V and 0.04%% W/V DLL respectively in the test,together with 0.08% and 0.04% non-emulsified DLL.BlankTestCount/Count/%%100 BEC100 BECAdhesioninhibitionFree caprylic acid 0.1%489466955Free caprylic acid 0.05%487459946DLL 0.08%5334218515DLL 0.04%50945387130.1% caprylic in49925551490.08% DLL0.05% caprylic in51330359410.04% DLL

As illustrated in Table 22, the addition of caprylic acid at concentrations below its MIC (0.1% and 0.05%) amplifies the adhesion inhibitory effect of 0.08% DLL and 0.04% DLL by more than a factor of three in both cases.

EXAMPLE 17

Reduction of Antagonistic Effect on Mammalian Cells

Mammalian cell membranes are susceptible to disruption by free fatty acids in a manner not dissimilar to their effect on microbial cell membranes. The effect is not classical cell toxicity as it relates to superficial cell surface damage and not interference with metabolic process or nucleic acid replication. It is ameliorated significantly by body fluids in vivo. Protection against mammalian cell membrane damage can be enhanced by adding additional amounts of free membrane lipid to a membrane lipid emulsion of a free fatty acid. The protective effect is not confined to the use of membrane lipids. As illustrated here, milk serum whey protein isolate also serves as a suitable, although not optimal barrier against mammalian cell damage.

The test item is an emulsion of 0.5% caprylic acid in 0.4% de-lipidised lecithin prepared as described in the Methods, and combined with an equal volume of 200 mM sodium citrate at pH 4.5 also prepared as described in the Methods. Dispersions of 0.4% and 0.8% de-lipidised lecithin were prepared in 200 mM sodium citrate at pH 4.5, and combined in equal volumes with aliquots of the emulsion of caprylic acid in de-lipidised lecithin to achieve emulsions of 0.25% caprylic acid in 0.2% de-lipidised lecithin suspended in an aqueous solution of 100 mM sodium citrate at pH 4.5 in which further amounts of either 0.2% or 0.4% de-lipidised lecithin were dispersed, these are described as Test+0.2% DLL or Test+0.4% DLL

Similar suspensions of the same emulsion were prepared in sodium citrate dispersions of a Whey Protein Isolate (WPI) (Provon 190 from Glanbia PLC) and with Bovine Serum Albumin for comparison purposes. These are described as Test+0.2% or Test+0.4% WPI or BSA.

Raji B lymphocytes were grown as described in the Methods and viability after a 60 minute exposure to the various test solutions was assessed using an Invitrogen Countess Cell Viability meter as described in the Methods. The results are presented in Table 23 below.

TABLE 23Reduction in cell viability at 60 minute exposure to test itemsRaji B LymphocytesTest +Test +Test +Test +Test +Test +Buffer0.2%0.4%0.2%0.4%0.2%0.4%controlblankTestDLLDLLWPIWPIBSABSA% Viable787275777376797571T zero% Viable726912596848531921T 60 min% cell929616779363672530survival

The % cell survival in the test item is just 16% after 60 minutes. By comparison, the control consisting of Raji B cells in cell culture media lost just 8% viability and the buffer blank being 100 mM sodium citrate at pH 4.5 was even less at 4% reduction. With the addition of 0.2% and 0.4% free membrane lipid (de-lipidised lecithin) % cell survival in the test was increased from 16% to 77% and 93%, and although not quite as effective, the addition of free WPI increased cell survival from 16% to 63% and 67%. Bovine serum albumin was considerably less effective in protecting against cell damage.

The inclusion of free membrane lipid in an aqueous dispersion of membrane lipid emulsion has no significant effect on the microbicidal properties of the membrane lipid emulsion. A late log phase culture of the yeastCandida albicansgrown as described in the Methods contained 1.33×107viable cells per ml. This was used to inoculate aliquots of each test item from above at a dilution of 1:10 such that each ml of test item contained in excess of 6 logs of yeast cells. Samples were withdrawn over time periods of up to 10 minutes and assessed for residual viability using the procedures described in the Methods. As illustrated in Table 24 below, detectable viability was eradicated in less than 5 minutes by all test items.

TABLE 24Microbicidal Effect of Free Membrane Lipid in Membrane Lipid EmulsionsTime to kill greater than 6 logsCandida albicans.Test +Test +Test +Test +Test +Test +Buffer0.2%0.4%0.2%0.4%0.2%0.4%controlblankTestDLLDLLWPIWPIBSABSATimeNANA<5<5<5<5<5>5>5Mins

EXAMPLE 18

Use in a Medical Food

Separate emulsions of caprylic, capric and lauric acid were prepared as 5.0% W/V fatty acid emulsified in 4.0% de-lipidised lecithin as described in the Methods. The individual emulsions were mixed in a ratio of 1:1:1.

Marvel skim milk powder from Premier International Foods (UK) Ltd, Spalding, Lincolnshire, England was re-constituted using 90% of the water volume according to the manufacturer's instructions. Once fully hydrated 10% by volume of the combined mix of separately emulsified free fatty acids was added and mixed by stirring, bringing the total volume to 100%.

Helicobacter pyloriwas grown on Columbia Blood Agar supplemented with 5% de-fibrinated sheep blood in an anaerobic jar using Anaerogen low oxygen gas packs from Oxoid UK.Salmonella typhimuriumandE. coliK12 were grown on Brain Heart Infusion agar as described in the Methods.

The microbicidal efficacy of the re-constituted milk supplemented with the three individually emulsified free fatty acids was determined using the Minimum Inhibitory Concentration (MIC) method of agar dilution described in the methods. Dilutions of the re-constituted skim milk were prepared in sterile distilled water such that further dilutions of aliquots of these in cooled agar provided an agar with combined free fatty acid concentrations ranging from 1% to 0.1% in 0.1% increments and from 0.1% to 0.01% in increments of 0.01%. Cultures of the three test organisms were inoculated onto these plates and incubated according to culture requirements:Helicobacterunder low oxygen tension,SalmonellaandE. coliunder aerobic conditions all at 37° C.

The minimum Inhibitory Concentration, being the lowest dilution where no growth was observed was greater than 0.5% forHelicobacterand greater than 0.1% for bothSalmonellaandE. coli.