HK1031839B - Methods of inactivating bacteria including bacterial spores - Google Patents

Description

The present invention relates to methods of inactivating bacteria including spores by contacting the bacteria with an oil-in-water emulsion which inactivates bacteria upon contact.

BACKGROUND OF THE INVENTION

It is known that if a water-immiscible lipid phase is mixed into an aqueous phase by mechanical agitation, for example, by means of an ultra-disperser, a dispersion, such as an oil-in-water emulsion, will develop. The stability of the resulting dispersion may require the addition of an emulsifying agent, the molecules of which are adsorbed onto the oil/water interface to form a kind of continuous membrane which prevents direct contact between two adjacent droplets. One advantage of oil-in-water emulsions is that they may readily be diluted with water to a desired composition.

In addition to discrete oil droplets dispersed in an aqueous phase, oil-in-water emulsions can also contain other lipid structures, such as small lipid vesicles (i.e., lipid spheres which often consist of several substantially concentric lipid bilayers separated from each other by layers of aqueous phase), micelles (i.e., amphiphile molecules in small clusters of 50-200 molecules arranged so that the polar head groups face outward toward the aqueous phase and the apolar tails are sequestered inward away from the aqueous phase), or lamellar phases (lipid dispersions in which each particle consists of parallel amphiphile bilayers separated by thin films of water). These lipid structures are formed as a result of hydrophobic forces which drive apolar residues (i.e., long hydrocarbon chains) away from water.

WO 96/33725 discloses a method of inactivating an envelope virus that comprises the step of providing a virus-inactivating solution, which lacks a compound which enhances uptake of said virus-inactivating solution.

WO 95/31966 discloses a method of inhibiting the growth of an infection pathogen comprising providing an antimicrobial oil-in-water emulsion, which comprises a cationic halogen-containing compound having a C₁₂ - C₁₆ chain.

The portals of entry of pathogenic bacteria are predominantly the skin and mucus membranes. The first step in many infections is attachment or colonization on skin or mucus membranes, followed by subsequent invasion and dissemination of the infectious pathogen. Accordingly, it is desirable to provide a bacteria-inactivating formulation and methods of using such formulations to inactivate bacteria.

In addition, many types of bacteria form highly resistant, thick-walled endospores also referred to as spores, in response to unfavorable conditions, which resume their metabolic activities when conditions improve. These dehydrated bodies contain the cellular components held in a state ofdormancy, ready to absorb water and resume their activities. It would thus be desirable to provide bacterial spore-inactivating formulations and methods of using the formulations to inactivate bacterial spores.

Bacteria, including spores, can be inactivated by heat, pressure and the use of chemical agents often referred to as bacteriocides. For example, corrosive compositions, e.g., formaldehyde and sodium hypochlorite (bleach), have been used to inactivate spores. Unfortunately, such compositions are toxic or irritating to skin and mucus membranes. It would therefore be desirable to provide compositions and methods for inactivating bacteria including bacterial spores, which are non-toxic to skin and mucus membranes. It would also be desirable to provide compositions and methods for inactivating bacteria and bacterial spores which are effective in vivo.

Accordingly, an object of the present invention is to provide a method of inactivating bacteria, including spores, by contacting the bacteria with a bacteria-inactivating emulsion.

It is a further object of the invention to provide a non-toxic, non-irritating preparation and method of using same that inactivates bacteria including spores, upon contact.

Another object of the present invention is to provide a method of preventing bacterial infection in an affected subject by administering a bacteria-inactivating emulsion to the subject.

SUMMARY OF THE INVENTION

The present invention provides a method of inactivating bacteria, where the method includes the steps of providing a bacteria-inactivating emulsion and contacting the bacteria with the emulsion. The emulsion is an oil-in-water emulsion comprising a surfactant, an organic phosphate based solvent, and a carrier oil. In one embodiment, the bacteria is a gram positive bacteria, i.e., bacteria with dense peptidoglycan walls which readily absorb a purple dye (crystal violet) in a process referred to as Gram's stain. In certain preferred embodiments, the gram positive bacteria or bacterial spores are Bacillus. In a particularly preferred embodiment, the bacteria or spores are Bacillus anthracis.

In another embodiment, the bacteria is a gram negative bacteria, i.e., bacteria which do not readily absorb the purple dye in a Gram's stain. In this embodiment, the bacteria-inactivating emulsion is premixed with a compound capable of increasing the uptake of the emulsion by the cell wall. In certain preferred embodiments, the compound is a chelating agent, e.g., ethylenediaminetetraacefic acid (EDTA), a solvent e.g., dimethyl sulfoxide (DMSO), a detergent, e.g., sodium dodecyl sulfate (SDS), and combinations thereof. In other preferred embodiments, the compounds in combination with peptides are used to increase the uptake of the emulsions by the cell wall, e.g., dipeptide and oligopeptide permeases, diglycine, triglycine, mixtures thereof, or other oligopeptides.

The emulsion used in the methods of the present invention consists primarily of droplets of an oily discontinuous phase dispersed in an aqueous continuous phase, such as water. The discontinuous phase is prepared from a surfactant, an oil carrier, and an organic phosphate-based solvent such as tri-n-butyl phosphate. The emulsions are highly stable, and are not decomposed even after long storage periods.

The bacteria-inactivating emulsions are non-toxic and safe when swallowed, inhaled, or applied to the skin. This is in contrast to chemical microbicides which are known irritants. The bacteria-inactivating emulsions also appear to be non-toxic to plants.

Oils useful in forming oil-in-water emulsions include a broad spectrum of water-immiscible materials, such as soybean oil, avocado oil, squalene oil, other fish oils, squalane oil, sesame oil, olive oil, canola oil, com oil, rapeseed oil, safflower oil, sunflower oil, flavor oils, and mixtures thereof.

Surfactants useful in forming the emulsions used in the methods of the present invention include a variety of anionic and nonionic surfactants, as well as other emulsifiers capable of promoting the formation of oil-in-water emulsions. In general, the emulsifier will be relatively hydrophilic, and blends of emulsifiers can be used to achieve the necessary qualities. Nonionic surfactants have advantages over ionic emulsifiers: they are compatible with a broad pH range and often form more stable emulsions than do ionic (e.g., soap-type) emulsifiers. Particularly useful surfactants include the detergents sold under the trademarks Tween 20, Tween 80, and the phenoxypolyethoxyethanols like Triton (i.e., X-100). A most preferred surfactant is Triton X-100 (t-octylphenoxypolyethoxyethanol).

Organic phosphate-based solvents useful in forming the oil-in-water emulsions include dialkyl and trialkyl phosphates. In a preferred embodiment, each alkyl group of the di- or trialkyl phosphate has one to ten carbon atoms, more preferably two to eight carbon atoms. The alkyl groups of the di- or trialkyl phosphate can all be the same or can be different. A particularly preferred trialkyl phosphate is tri-n-butyl phosphate, which is a plasticizer. Mixtures of different dialkyl and trialkyl phosphates can be employed. in addition, alcohols may be employed as a solvent, e.g., octanol.

In another embodiment of the invention, at least a portion of the emulsion may be in the form of lipid structures including, but not limited to, unilamellar, multilamellar, and paucilamellar lipid vesicles, micelles, and lamellar phases.

The present invention also provides methods of treating a subject by applying a bacteria-inactivating preparation suitable for pharmaceutical administration, which may also include a pharmaceutically acceptable carrier. The preparation can be applied topically to skin surface areas, mucus membranes, or oral surfaces, for example, as a cream, gel, spray, or mouthwash, to treat or prevent bacterial infections. The preparation can also be applied to wounds caused by bacterial infection. Accordingly, the present invention further provides a method for inactivating a bacteria, including bacterial spores, by topical application of the emulsions described herein.

In a further embodiment, the invention includes methods of preventing bacterial infection in a subject by applying the emulsion described herein to the skin or mucous membrane of the subject to inactivate the bacteria or spores. By inactivating bacteria or spores before attachment or colonization, subsequent invasion and dissemination of the infectious pathogen may be prevented.

In an additional embodiment, the invention includes methods of decontamination, i.e., inactivating bacteria and particularly spores found on any surface. Surfaces which will likely come in contact with a human, e.g., vehicles, equipment, instruments, etc., may thus be decontaminated by applying the emulsions described herein to the surfaces.

Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and subjoined claims and by referencing the following drawings in which:

• Figure 1 is a graph showing the bactericidal efficacy of an emulsion of the present invention on B. cereus spores; and
• Figures 2A-2C are photographs of bacterial smears showing the bactericidal efficacy of an emulsion of the present invention on B. cereus spores.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of inactivating bacteria including spores by contacting the bacteria with oil-in-water emulsions made up of droplets of an oily discontinuous phase containing an organic phosphate-based solvent dispersed in an aqueous continuous phase, and a surfactant. The emulsions are stable, non-toxic, and simple and inexpensive to formulate.

The term "bacteria-inactivating," as used herein, means having the ability to kill bacteria or spores on contact. It appears that inactivation is achieved by surfactant and solvent interactions with bacteria cell membranes, thereby disrupting the cell membrane and causing cell death. Accordingly, one aspect of the present invention provides a method of applying a bacteria-inactivating oil-in-water emulsion which contains materials capable of interacting with the bacterial membrane or spore and disrupting the structure so that the bacteria or spore is inactivated.

As described in more detail in Specific Example 2, infra, the methods of the present invention can rapidly inactivate gram positive bacteria. In preferred embodiments, the inactivation of bacteria occurs after no more than six hours, more preferably after no more than two hours, and even more preferably in less than one hour after the bacteria is contacted with an emulsion according to the present invention.

As described in more detail in Specific Example 3, infra, the methods of the invention can also rapidly inactivate certain gram negative bacteria. In such methods, the bacteria-inactivating emulsions are premixed with a compound which increases the uptake of the emulsion by the cell wall. For example, compounds such as EDTA, DMSO and SDS are effective when mixed with the emulsions in increasing the uptake of the emulsions by the cell wall. Oligopeptides such as diglycine and triglycine may also be employed as cell wall uptake enhancers. It should be noted that the emulsion and cell wall uptake enhancer are effective against certain Gram positive and negative bacteria but are not effective against all Gram negative bacteria and thus may be administered orally where they will come in contact with necessary gut bacteria, without unacceptable adverse effects to the subject's colonic microflora (i.e., E. coli).

As described in more detail in Specific Example 4, infra, the methods of the present invention can also inactivate a bacterial spore. In preferred embodiments, the inactivation occurs no more than six hours, more preferably no more than four hours, after the spore is contacted with the emulsion.

As set forth in detail in Specific Example 5, infra, the methods of the present invention are effective in inactivating bacteria including spores in vivo, without significant toxicity.

Also, as further described in Specific Examples 6 and 7, infra the bacteria-inactivating methods of the present invention are non-toxic, e.g., the emulsions may be applied topically and orally and have an acceptable toxicity profile.

The term "emulsion," as used herein, includes classic oil-in-water dispersions or droplets, as well as other lipid structures which can form as a result of hydrophobic forces which drive apolar residues (i.e., long hydrocarbon chains) away from water and drive polar head groups toward water, when a water immiscible oily phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.

The bacteria-inactivating oil-in-water emulsions used in the methods of the present invention can be formed using classic emulsion forming techniques known in the art. In brief, the oily phase is mixed with the aqueous phase under relatively high shear forces to obtain an oil-in-water emulsion containing oil droplets which are approximately 1-2 microns in diameter. The oily discontinuous phase is formed by blending (a) an oil carrier; (b) a surfactant; and (c) an organic phosphate-based solvent. The emulsion is formed by blending the oily phase with an aqueous phase (e.g., water) on a volume-to-volume basis ranging from about 1:4 to 4:1, preferably about 1:4 oily phase to aqueous phase. The oil and aqueous phases can be blended using any apparatus capable of producing shear forces sufficient to form an emulsion (e.g., French Press or commercial high shear mixers).

The bacteria-inactivating oil-in-water emulsions used in the methods of the present invention can be used to inactivate a variety of bacteria and bacterial spores upon contact. For example, the presently disclosed emulsions can be used to inactivate Bacillus including B. cereus, B. circulans, B. megaterium and B. subtilis, also including Clostridium, e.g., C. botulinum and C. tetani. The methods of the present invention may be particularly useful in inactivating certain biological warfare agents, e.g., B. anthracis.

The bacteria-inactivating emulsion described herein may be used as a preparation suitable for pharmaceutical administration. Such preparation may comprise an oil-in-water emulsion of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier," as used herein, refers to any physiologically compatible carrier for stabilizing emulsions of the present invention for pharmaceutical administration. Use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the emulsions of the present invention, use thereof in a pharmaceutical preparation is contemplated.

The present invention further provides methods for inactivating bacteria by topical and/or oral administration of an oil-in-water emulsion of the present invention, preferably in the form of a pharmaceutical preparation. The term "topical," as used herein, includes, without limitation, application to mucous membranes, oral surfaces, skin, including wounds, and the surfaces of any bodily orifice, such as the nasal cavity, vagina or rectum. The term "oral," as used herein includes, without limitation, application by swallowing by the subject. It will be appreciated that the emulsions may be combined with other edible substances for swallowing by the subject.

The specific examples below further describe the compositions and methods of the present invention.

SPECIFIC EXAMPLE 1

In this example, a bacteria-inactivating oil-in-water emulsion containing a surfactant and a trialkyl phosphate, was formed and characterized.

The emulsion was formed as follows: an oil phase was made by blending tributyl phosphate, soybean oil, and a surfactant (e.g., Triton X-1 00) and then heating the resulting mixture at 86°C for one hour. An emulsion was then produced by injecting water into the oil phase at a volume/volume ratio of one part oil phase to four parts water. The emulsion can be produced manually, with reciprocating syringe instrumentation, or with batch or continuous flow instrumentation. Table 1 shows the proportions of each component , the pH , and the size of the emulsion as measured on a Coulter LS 130 laser sizing instrument equipped with a circulating water bath.

BCTP
Triton X-100	2%
Tributyl phosphate	2%	5.16	1.074	0.758-1.428
Oil (ex. Soy bean)	16%
Water	80%
BCTP 0.1†
Triton X-100	0.20%	5.37	0.944	0.625-1.333
Tributyl phosphate	0.20%
Oil (ex. Soy bean)	1.60%
Water	98%

† This emulsion was obtained by diluting the BCTP emulsion with water in a ratio of 1:9.

The emulsions of the present invention are highly stable. The BCTP and BCTP 0.1, emulsions have been found to be substantially unchanged after storage at room temperature for at least 24 months.

SPECIFIC EXAMPLE 2 In Vitro Bactericidal Efficacy Study I - Gram Positive Bacteria

In order to study the bactericidal efficacy of the emulsions of the present invention, the emulsions were mixed with various bacteria for 10 minutes and then plated on standard microbiological media at varying dilutions. Colony counts were then compared to untreated cultures to determine the percent of bacteria killed by the treatment. Table 2 summarizes the results of the experiment.

	1.3 x 10°	100	BCTP
	5.1 x 10°	100	BCTP
	4.0 x 10'	98-100	BCTP

In order to study the bactericidal effect of the emulsions of the present invention on various vegetative forms of Bacillus species, an emulsion at three dilutions was mixed with four Bacillus species for 10 minutes and then plated on microbiological medium. Colony counts were then compared with untreated cultures to determine the percent of bacteria killed by the treatment. Table 3 contains a summary of the bactericidal results from several experiments with the mean percentage kill in parenthesis.

1:10	99%	95-99%	99%	99%
	(99)	(97%)	(99)	(99)
1:100	97-99%	74-93%	96-97%	99%
	(98%)	(84%)	(96%)	(99)
1:1000	0%	45-60%	0-32%	0-39%
	(0)	(52%)	(16%)	(20%)

SPECIFIC EXAMPLE 3 In Vitro Bactericidal Efficacy Study II - Gram Negative Bacteria

To increase the uptake of the bacteria-inactivating emulsions by the cell walls of gram negative bacteria, thereby enhancing the microbicidal effect of the emulsions on the resistant gram negative bacteria, EDTA (ethylenediamine-tetraacetic acid) was premixed with the emulsions. The EDTA was used in low concentration (50-250µM) and the mix was incubated with the various gram negative bacteria for 15 minutes. The microbicidal effect of the mix was then measured on Trypticase soy broth. The results are set forth in Table 4 below. There was over 99% reduction of the bacterial count using BCTP in 1/100 dilutions. This reduction of count was not due to the killing effect of EDTA alone as shown from the control group in which 250µM of EDTA alone could not reduce the bacterial count in 15 minutes.

	1,830,000	1,370,000	40	790,000
	910,000	690,000	0	320,000

The addition of very small amounts of other substances such as dimethyl sulfoxide (DMSO) or sodium dodecyl sulfate (SDS) also increases the uptake of the emulsions into the cells, thereby enhancing the microbicidal effect.

Studies are performed to show the enhanced microbicidal effect of mixtures of the emulsions and diglycine or triglycine, to increase the uptake of the emulsions by the cell wall using the bacterial enzymes dipeptide and polypeptide permeases.

SPECIFIC EXAMPLE 4 In Vitro Bactericidal Efficacy Study III - Vegetative And Spore Forms

Bacillus cereus (B. cereus, ATCC #14579), was utilized as a model system for Bacillus anthracis. Experiments with BCTP diluted preparations to study the bactericidal effect of the compounds of the present invention on the vegetative form (actively growing) of B. cereus were performed. Treatment in medium for 10 minutes at 37°C was evaluated. As summarized in Table 5, the BCTP emulsion is efficacious against the vegetative form of B. cereus. A 10 minute exposure with this preparation is sufficient for virtually complete killing of vegetative forms of B. cereus at all concentrations tested including dilutions as high as 1:100.

BCTP	>99%	>99%	59 - >99%
	Avg = >99%	Avg = >99%	Avg = 82%

Number of experiments = 4

The spore form of B. anthracis is one of the most likely organisms to be used as a biological weapon. Spores are well known to be highly resistant to most disinfectants. As describe above, effective killing of spores usually requires the use of toxic and irritating chemicals such as formaldehyde or sodium hypochlorite (i.e., bleach). The same experiment was therefore performed with the spore form of B. cereus. As shown in Table 6, treatment in both medium for 10 minutes at 37°C was not sufficient to kill B. cereus spores.

BCTP	0%-12%	0%	0%
	Avg = 6%	Avg = 0%	Avg = 0%

Number of experiments = 2

To evaluate the efficacy of the compounds of the present invention on the spore form of B. cereus over a period of time, BCTP was incorporated into solid agar medium at 1:100 dilution and the spores spread uniformly on the surface and incubated for 96 hours at 37°C. No growth occurred on solid agar medium wherein BCTP had been incorporated, out to 96 hours (i.e., >99% killing, average >99% killing, 3 experiments).

In an attempt to more closely define the time at which killing of spores by BCTP occurred, the following experiment was performed. Briefly, a spore preparation was treated with BCTP at a dilution of 1:100 and compared to an untreated control. The number of colony forming units per milliliter (CFU/ml) was quantitated after 0.5, 1, 2, 4, 6, and 8 hours. As shown in Figure 1, CFU/ml in the untreated control increased over the first 4 hours of incubation and then reached a plateau. Bacterial smears prepared at time zero, 1, 2, 4 and 6 hours, and stained for spore structures, revealed that by 2 hours no spore structures remained (Figures 2A-2C). Thus, 100% germination of spores occurred in the untreated control by the 2 hour time point. In the spore preparation treated with BCTP, CFU/ml showed no increase over the first 2 hours and then declined rapidly over the time period from 2-4 hours. The decline from baseline CFU/ml over 2-4 hours was approximately 1000-fold. Bacterial smears prepared at the same time points and stained for spore structures revealed that spore structures remained to the end of the experiment at 8 hours. Hence, germination of spores did not occur in the BCTP treated culture due to either inhibition of the germination process or because the spores were damaged and unable to germinate.

In order to determine whether the emulsions were effective in killing other Bacillus species in addition to B. cereus, a similar experiment was performed as described above, wherein spore preparations were treated with emulsions and compared to an untreated control after four hours of incubation. The following table shows the results wherein the numbers represent a range of results from several experiments with the average in parenthesis.

1:10	71-93%	30-77%	80-99%	5-90%
	(82)	(61%)	(93%)	(31%)
1:100	87-95%	23-82%	74-99%	0-87%
	(96)	(61 %)	(92%)	(39%)
1:1000	0-94%	20-79%	90-97%	0-87%
	(47)	(55%)	(94%)	(22%)

SPECIFIC EXAMPLE 5 In Vivo Bactericidal Efficacy Study

Bacillus cereus was passed three times on blood agar (TSA with 5% sheep blood, REMEL). B. cereus was scraped from the third passage plate and resuspended in Trypticase soy broth (TSB) (available from BBL). The B. cereus suspension was divided into two tubes. An equal volume of sterile saline was added to one tube and mixed. 0.1 ml of the B. cereus suspension/saline was injected subcutaneously into 5 CD-1 mice. An equal volume of BCTP (diluted 1:5 in sterile saline) was added to one tube and mixed, giving a final dilution of BCTP at 1:10. The B. cereus suspension/BCTP was incubated at 37°C for 10 minutes while being mixed. 0.1 ml of the B. cereus suspension/BCTP was injected subcutaneously into 5 CD-1 mice. Equal volumes of BCTP (diluted 1:5 in sterile saline) and TSB were mixed, giving a final dilution of BCTP at 1:10. 0.1 cc of the BCTP/TSB was injected subcutaneously into 5 CD-1 mice.

The number of colony forming units (cfu) of B. cereus in the inocula were quantitated as follows: 10-fold serial dilutions of the B. cereus and B. cereus/BCTP suspensions were made in distilled H₂O. Duplicate plates of TSA were inoculated from each dilution (10 ul per plate). The TSA plates were incubated overnight at 37°C. Colony counts were made and the number of cfu/ml was calculated. Necrotic lesions appears to be smaller in mice which were inoculated with B. cereus which was pretreated with BCTP. The following table shows the results of the experiment.

	1528	necrosis at injection site
	1529	necrosis at injection site
cfu/mouse	1530	dead
	1531	dead
	1532	necrosis at injection site
	1348	necrosis at injection site
	1349	no reaction
cfu/mouse	1360	no reaction
(BCTP treated)	1526	necrosis at injection site
	1527	necrosis at injection site
BCTP/TSB	1326	no reaction
	1400	no reaction
	1375	no reaction
	1346	no reaction
	1347	no reaction

Bacillus cereus was grown on Nutrient Agar (Difco) with 0.1 % Yeast Extract (Difco) and 50 ug/ml MnSO₄ for induction of spore formation. The plate was scraped and suspended in sterile 50% ethanol and incubated at room temperature for 2 hours with agitation in order to lyse remaining vegetative bacteria. The suspension was centrifuged at 2,500 x g for 20 minutes and the supernatant discarded. The pellet was resuspended in diH₂O, centrifuged at 2,500 X g for 20 minutes, and the supernatant discarded. The spore suspension was divided. The pellet was resuspended in TSB. 0.1 ml of the B. cereus spore suspension diluted 1:2 with saline was injected subcutaneously into 3 CD-1 mice. Equal volumes of BCTP (diluted 1:5 in sterile saline) and B. cereus spore suspension were mixed, giving a final dilution of BCTP at 1:10 (preincubation time). 0.1 ml of the BCTP/B. cereus spore suspension was injected subcutaneously into 3 CD-1 mice.

The number of colony forming units (cfu) of B. cereus in the inoculum was quanitated as follows. 10-fold serial dilutions of the B. cereus and B. cereus/BCTP suspensions were made in distilled H₂O. Duplicate plates of TSA were inoculated from each dilution (10ul per plate). The TSA plates were incubated overnight at 37°C. Colony counts were made and the number of cfu/ml was calculated. Necrotic lesions appeared to be smaller in mice which were inoculated with B. cereus spores which were pretreated with BCTP.

	2/3 (66%) mice exhibited necrosis at injection site
spores/mouse
	1/3 (33%) mice exhibited necrosis at injection site
spores/mouse
(BCTP treated)
	3/3 (100%) mice exhibited necrosis at injection site
vegetative
forms/mouse
	3/3 (100%) mice did not exhibit symptoms
ctu/mouse
BCTP/TSB	1/3 (33%) mice appeared to have some skin necrosis

Bacillus cereus was grown on Nutrient Agar (Difco) with 0.1 % Yeast Extract (Difco) and 50 µg/ml MnSO₄ for induction of spore formation. The plate was scraped and suspended in sterile 50% ethanol and incubated at room temperature for 2 hours with agitation in order to lyse remaining vegetative bacteria. The suspension was centrifuged at 2,500 X g for 20 minutes and the supernatant discarded. The pellet was resuspended in distilled H₂O, centrifuged at 2,500 X g for 20 minutes, and the supernatant discarded. The pellet was resuspended in TSB. The B. cereus spore suspension was divided into three tubes. An equal volume of sterile saline was added to one tube and mixed. 0.1 ml of the B. cereus suspension/saline was injected subcutaneously into 10 CD-1 mice. An equal volume of BCTP (diluted 1:5 in sterile saline) was added to the second tube and mixed, giving a final dilution of BCTP at 1:10. The B. cereus spore suspension/BCTP (1:10) was incubated at 37°C for 4 hours while being mixed. 0.1 ml of the B. cereus spore suspension/BCTP (1:10) was injected subcutaneously into 10 CD-1 mice. An equal volume of BCTP (diluted 1:50 in sterile saline) was added to the third tube and mixed, giving a final dilution of BCTP at 1:100. The B. cereus spore suspension/BCTP (1:100) was incubated at 37°C for 4 hours while being mixed. 0.1 ml of the B. cereus spore suspension/BCTP (1:100) was injected subcutaneously into 10 CD-1 mice. Equal volumes of BCTP (diluted 1:5 in sterile saline) and TSB were mixed, giving a final dilution of BCTP at 1:10. 0.1 ml of the BCTP/TSB was injected subcutaneously into 10 CD-1 mice. Equal volumes of BCTP (diluted 1:50 in sterile saline) and TSB were mixed, giving a final dilution of BCTP at 1:100. 0.1 ml of the BCTP/TSB was injected subcutaneously into 10 CD-1 mice.

	1
5.5 x
	2	no abnormalities observed
No treatment group	3
	4
	5
	6
	7
	8
	9
	10

Note: Skin lesions grey in color with edema, necrotic areas red/dry.

	41	no abnormalities observed
	42	no abnormalities observed
spores/mouse	43
in the		slight edema
BCTP 1:10	44
treated group	45
	46
	47
		area in center
	48
	49
	50	no abnormalities observed
	51
	52
spores/mouse	53
in the	54
BCTP 1:100	55
treated group	56
	57
	58
	59
		1 cm diameter
	60
BCTP 1:10 alone	11
	12
	13	no abnormalities observed
	14
	15
	16	no abnormalities observed
	17
	18	no abnormalities observed
	19
	20
BCTP 1:100 alone	21-	no abnormalities observed
	30
TSB	31-	no abnormalities observed
alone	40

Re-isolation of B. cereus was attempted from skin lesions, blood, liver, and spleen. Skin lesions were cleansed with betadine followed by 70% sterile isopropyl alcohol. An incision was made at the margin of the lesion and swabbed. The chest was cleansed with betadine followed by 70% sterile isopropyl alcohol. Blood was drawn by cardiac puncture. The abdomen was cleansed with betadine followed by 70% sterile isopropyl alcohol. The skin and abdominal muscles were opened with separate sterile instruments. Samples of liver and spleen were removed using separate sterile instruments. Liver and spleen samples were passed briefly through a flame and cut using sterile instruments. The freshly exposed surface was used for culture. BHI agar (Difco) was inoculated and incubated aerobically at 37°C overnight.

	3	24 hours	skin lesion >300 cfu
	6	48 hours	skin lesion >300 cfu
spores/mouse	7	48 hours	skin lesion >300 cfu
in the	8	72 hours	skin lesion 100 cfu
Untreated group	9	72 hours	skin lesion 25 cfu
	10	72 hours	skin lesion 100
	1	96 hours	skin lesion >300 cfu
	4	96 hours	skin lesion >300 cfu
	5	96 hours	skin lesion >300 cfu
	48	48 hours	skin lesion 17 cfu
	50	48 hours	skin lesion >300 cfu
spores/mouse	46	72 hours	skin lesion >200 cfu
in the	47	72 hours	skin lesion 100 cfu
BCTP 1:10	49	72 hours	skin lesion >300 cfu
treated group	41 **	96 hours	skin lesion >300 cfu
	42**	96 hours	skin lesion 20 cfu
	43		cultures not done
	44	96 hours	skin lesion >300 cfu
	45		cultures not done
	46		cultures not done
	48	48 hours	skin lesion 18 cfu
	50**	48 hours	skin lesion >300 cfu
spores/mouse	52	72 hours	skin lesion 1 cfu
in the	54	72 hours	re-isolation negative
BCTP 1:100	56	72 hours	skin lesion >300 cfu
treated group	58	96 hours	skin lesion 173 cfu
	59	96 hours	skin lesion 4 cfu
	60	96 hours	skin lesion 6 cfu

**Although no lesions were present in these mice, organisms were removed from the injection site.

Pretreatment of both vegetative B. cereus and B. cereus spores reduce their ability to cause disease symptoms when introduced into experimental animals. This is reflected in the smaller size of skin lesions and the generally lower numbers of B. cereus recovered from the lesions. In addition, less frequent re-isolation of B. cereus from blood, liver, and spleen occurs suggesting that septicemia may be preventable.

SPECIFIC EXAMPLE 6 In Vivo Toxicity Study

CD-1 mice were injected subcutaneously with 0.1 cc of the compounds of the present invention and observed for 4 days for signs of inflammation and/or necrosis. Dilutions of the compounds were made in sterile saline.

Tissue samples from mice were preserved in 10% neutral buffered formalin for histopathologic examination. Samples of skin and muscle (from mice which were injected with undiluted compounds) sent for histological review were reported to show indications of tissue necrosis. Tissue samples from mice which were injected with diluted compounds were not histologically examined. The following two tables show the results of two individual experiments.

BCTP	1326	undiluted	necrosis
	1327	undiluted	no reaction
	1328	1:10	no reaction
	1329	1:10	no reaction
	1324	1:100	no reaction
	1331	1:100	no reaction
Saline	1344		no reaction
	1345		no reaction

BCTP	1376	undiluted	necrosis
	1377	undiluted	no reaction
	1378	1:10	no reaction
	1379	1:10	no reaction
	1380	1:100	no reaction
	1381	1:100	no reaction
Saline	1394		no reaction
	1395		no reaction

Guinea pigs were injected intramuscularly (in both hind legs) with 1.0 ml of compounds of the present invention per site and observed for 4 days for signs of inflammation and/or necrosis. Dilutions of the compounds were made in sterile saline.

Tissue samples from guinea pigs were preserved in 10% neutral buffered formalin for histological examination. Tissue samples were not histologically examined.

BCTP	1023-1	undiluted	no reaction
	1023-2	1:10	no reaction
	1023-3	1:100	no reaction
Saline	1023-10		no reaction

The results of In vivo Toxicity Study I show that subcutaneous and intramuscular injection of the compounds tested did not result in grossly observable tissue damage and did not appear to cause distress in the experimental animals.

SPECIFIC EXAMPLE 7 In vivo Toxicity Study II

One group of Sprague-Dawley rats each consisting of five males and five females were placed in individual cages and acclimated for five days before dosing. Rats were dosed daily for 14 days. On day 0-13, for 14 consecutive days each rat in Group 1 received by gavage three milliliters of BCTP, 1:100 concentration, respectively. The three milliliter volume was determined to be the maximum allowable oral dose for rats. Prior to dosing on Day 0 and Day 7, each rat was weighed. Thereafter rats were weighed weekly for the duration of the study. Animals were observed daily for sickness or mortality. Animals were allowed to rest for 14 days. On Day 28 the rats were weighed and euthanized.

The mean weight results of the oral toxicity study are shown in Table 16. Mean weights for males and females on Days 0, 7, and 14, 21 and 28 and the mean weight gains from Day 0 - Day 28, are also shown in Table 16. One rat died due to mechanical trauma from manipulation of the gavage tubing during dosing on Day 14. All surviving rats gained weight over the 28-day course of the study and there was no illness reported.

Thus, although tributyl phosphate alone is known to be toxic and irritating to mucous membranes, when incorporated into the emulsions of the present invention, these characteristics are not in evidence.

The BCTP emulsion, 1:100 concentration, was also tested for dermal toxicity in rabbits according to the protocols provided in 16 CFR §1500.3 (data not shown). The emulsion was not irritating to skin in the animals tested.

9028	M	3	332.01	356.52	388.66	429.9	394.07	62.06
9029	M	3	278.62	294.65	296.23	310.7	392.6	113.98
9030	M	3	329.02	360.67	325.26	403.43	443.16	114.14
9031	M	3	334.64	297.04	338.82	357.5	416.89	82.25
9032	M	3	339.03	394.39	347.9	331.38	357.53	18.5
9063	F	3	302	298.08	388.66	338.41	347.98	45.98
9064	F	3	254.54	247.97	256.78	278.17	279.2	24.66
9065	F	3	225.99	253.81	273.38	290.54	308.68	82.69
9066	F	3	246.56	260.38	266.21	235.12	272.6	26.04
9067	F	3	279.39	250.97	deceased

Claims (37)

1. A method of inactivating bacteria selected from the group consisting of Gram positive bacteria and bacterial spores comprising contacting said bacteria with a Gram positive bacteria-inactivating emulsion or a bacterial spores-inactivating emulsion, wherein said Gram positive bacteria-inactivating emulsion or bacterial spores-inactivating emulsion comprises an oil-in-water emulsion in the form of a discontinuous oil phase distributed in an aqueous phase with a surfactant stabilizer, said oil phase including an organic phosphate-based solvent and a carrier oil.
2. The method of claim 1, wherein said oil phase in said emulsion is composed of droplets having a mean particle size in the range from 0.5 to 5 microns.
3. The method of claim 1, wherein said surfactant is selected from the group consisting of Tween 20, Tween 80 and Triton X-100.
4. The method of claim 1, wherein said organic phosphate-based solvent is selected from the group consisting of dialkyl phosphates and trialkyl phosphates.
5. The method of claim 4, wherein said trialkyl phosphate comprises tri-n-butyl phosphate.
6. The method of claim 1, wherein said carrier oil is selected from the group consisting of soybean oil, avocado oil, squalane oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, sunflower oil, fish oils, flavor oils, water insoluble vitamins and mixtures thereof.
7. The method of claim 6, wherein said carrier oil comprises soybean oil.
8. The method of claim 1, wherein said Gram positive bacteria is Bacillus.
9. The method of claim 1, wherein said Gram positive bacteria is Bacillus anthracis.
10. The method of claim 1, wherein said bacterial spore is Bacillus.
11. The method of claim 1, wherein said bacterial spore is Bacillus anthracis.
12. The method of inactivating Gram negative bacteria comprising contacting said bacteria with a Gram negative bacteria-inactivating emulsion, wherein said Gram negative bacteria-inactivating emulsion comprises an oil-in-water emulsion in the form of a discontinuous oil phase distributed in an aqueous phase, with a surfactant stabilizer, said oil phase including an organic-phosphate based solvent and carrier oil, and wherein said contacting further comprises contacting with a compound which enhances uptake of said emulsion into said bacteria's cells.
13. The method of claim 12, wherein said oil phase in said emulsion is composed of droplets having a mean particle size in the range of about 0.5 to about 5 microns.
14. The method of claim 12, wherein said surfactant is selected from the group consisting of Tween 20, Tween 80 and Triton X-100.
15. The method of claim 12, wherein said organic phosphate-based solvent is selected from the group consisting of dialkyl phosphates and trialkyl phosphates.
16. The method of claim 15, wherein said trialkyl phosphate comprises tri-n-butyl phosphate.
17. The method of claim 12, wherein said carrier oil is selected from the group consisting of soybean oil, avocado oil, squalane oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, sunflower oil, fish oils, flavor oils, water insoluble vitamins and mixtures thereof.
18. The method of claim 17, wherein said carrier oil comprises soybean oil.
19. The method of claim 12, wherein said Gram negative bacteria are selected from the group consisting ofVibrio, Salmonella, Shigella and Pseudomonas.
20. The method of claim 12, wherein said compound which enhances uptake of said emulsion into said bacteria's cells is selected from the group consisting of EDTA, DMSO and SDS.
21. The method of claim 20, wherein said compound is EDTA.
22. The method of claim 21, wherein said EDTA is at a concentration of 50 to 250 µM.
23. Use of a bacteria-inactivating emulsion comprising an oil-in-water emulsion in the form of an oil phase distributed in an aqueous phase with a surfactant stabilizer, said oil phase including an organic phosphate-based solvent and a carrier oil, for the manufacture of a medicament for treating or preventing bacterial infection caused by a bacteria selected from the group consisting of Gram positive bacteria and bacterial spores in a subject.
24. The use of claim 23, wherein said medicament is formulated for topical administration.
25. The use of claim 23, wherein said medicament is formulated for oral administration.
26. The use of claim 23, wherein said medicament is formulated for administration by means of a porous pad.
27. The use of claim 23, wherein said oil phase in said emulsion is composed of droplets having mean particle size in the range of 0.5 to 5 microns.
28. The use of claim 23, wherein said surfactant is selected from the group consisting of non-ionic and anionic surfactants.
29. The use of claim 28, wherein said surfactant is Triton X-100 .
30. The use of claim 23, wherein said organic phosphate-based solvent comprises a trialkyl phosphate.
31. The use of claim 30, wherein said trialkyl phosphate comprises tri-n-butyl phosphate.
32. The use of claim 23, wherein said carrier oil consists essentially of a vegetable oil.
33. The use of claim 32, wherein said vegetable oil is soybean oil.
34. The use of claim 23, wherein the Gram positive bacteria are Bacillus species.
35. The use of claim 34, wherein the Gram positive bacteria is Bacillus anthracis.
36. The use of claim 23, wherein said bacteria-inactivating emulsion is non-toxic to the affected subject.
37. The use of claim 23, wherein said bacteria-inactivating emulsion is not irritating to the affected subject.