CN113423414A - Volatile organic compound formulations with antimicrobial activity - Google Patents

Abstract

The present subject matter includes novel chemical formulations having antimicrobial, antifungal, antibacterial and related effects in a wide range of applications for treating or preventing infections and treating various surfaces that may be contaminated with infectious agents. In some embodiments, the formulation further comprises isoamyl hexanoates and at least one of propionic acid and/or isobutyric acid.

Description

Volatile organic compound formulations with antimicrobial activity

Cross Reference to Related Applications

This application is a continuation-in-part of the following applications: us patent application No. 16/179,370 filed on day 11/2 in 2018, which is a continuation of us patent application No. 14/322,757 filed on day 7/2 in 2014 (and which was granted us patent 10,117,841 on day 11/6 in 2018), applications claiming the benefits of us provisional application No. 61/842,362 filed on day 7/2 in 2013 and us provisional application No. 61/948,902 filed on day 3/6 in 2014; and claim the benefit of U.S. provisional application No. 62/738,541 filed on 28/9/2018; each of which is incorporated by reference in its entirety.

Background

The importance of safely disposing of billions of pounds of human and animal waste daily to avoid the myriad of health problems associated with such waste is not exaggerated. In fact, only a small portion of this mass of material is safely disposed of, while the remaining portion is untreated and poses a threat to human and animal health. For example, it is well known that a complex of bacterial and other agents responsible for gastrointestinal disease is the single largest cause of death worldwide. It is also well known that these types of diseases primarily affect infants and children, as well as livestock. It is estimated that in the next decade, at least two million people will die of poor or inadequate sanitation facilities.

One reason for this is that about 24 million people live in areas where there are insufficient sanitary facilities. Nearly 4000 children die each day from conditions such as diarrhea. Furthermore, people suffering from water-borne diseases account for about half of hospital beds worldwide. In several asian countries, up to two times of people die from diarrhea-related diseases, such as AIDS. Poor hygienic conditions are essentially caused by or associated with the inability to adequately treat and dispose of human and animal faeces, which carry pathogenic microorganisms and promote their growth and development, at home, socially and in some cases even throughout the country.

The deleterious effects of microorganisms in industrial environments are, of course, numerous. For example, safer and more effective means for treating microorganism-laden surfaces in medical or hospital environments are needed. There is a need for safer and more effective means for treating crops for growth of harmful microorganisms. Further, means for reducing the unpleasant odor generated by the decomposition of fecal matter in industrial farming operations are highly desirable.

There is an urgent need to replace antibiotics with other types of compounds that also exhibit antimicrobial activity. The long-lasting use of most commonly used antibiotics for animals and agriculture has resulted in acquired resistance of microbial populations, particularly microorganisms capable of causing disease. Every year, at least 23,000 people die in the united states due to infections caused by drug-resistant bacteria, and the number is increasing.

Accordingly, there is a need in the art for antimicrobial compositions, and formulations and methods for human and animal fecal management, that are suitable for reducing the effects of microorganisms and microbial growth in a wide range of industrial environments. The present invention meets this need.

Summary of The Invention

In one embodiment, the present invention relates to a chemical formulation having antimicrobial activity comprising propanoic acid, isobutryic acid and at least one ester. In another embodiment, the at least one ester is isoamyl hexanoates. In another embodiment, the formulation further comprises at least one carrier selected from the group consisting of bentonite, zeolite, and perlite. In another embodiment, the ratio of propanoic acid to isobutyric acid to isoamyl hexanoates is about 3.5:3.5:2 (v/v/v). In another embodiment, the ratio of propanoic acid, isobutyric acid, and isoamyl hexanoates is about 7 parts of the two acids and 2 parts of isoamyl butyrate. In another embodiment, the formulation further comprises at least one endophyte (endophyte). In another embodiment, the endophyte is from fusarium.

In another embodiment, the invention relates to a chemical formulation consisting essentially of propanoic acid, isobutryic acid, isoamyl hexanoates and a carrier selected from the group consisting of bentonite, zeolite and perlite.

In another embodiment, the present invention relates to a chemical formulation comprising propionic acid and at least one 6-12 carbon (acid) component ester, wherein the ratio of propionic acid to ester component of the chemical formulation is about 7:2 (v/v). In another embodiment, the at least one ester is isoamyl hexanoates. In another embodiment, the formulation further comprises at least one nutritional supplement and at least one salt. In another embodiment, the formulation comprises glucose, whey protein, potassium chloride, magnesium sulfate, and sodium chloride. In another embodiment, the formulation comprises glucose, glycine, potassium chloride, sodium chloride, and magnesium acetate. In another embodiment, the formulation comprises glucose, glycine, potassium chloride, sodium chloride, magnesium acetate, and potassium dihydrogen phosphate. In another embodiment, the formulation further comprises at least one carrier. In another embodiment, the formulation consists essentially of propionic acid and isoamyl hexanoates in a ratio of propionic acid to isoamyl hexanoates of about 7:2 (v/v). In another embodiment, the formulation comprises at least one probiotic. In another embodiment, the endophyte is from fusarium.

In another embodiment, the invention relates to a method of treating an animal having a disease or condition associated with a microbial infection comprising administering to the animal an effective amount of a composition comprising at least one organic acid and at least one ester. In another embodiment, the invention relates to a composition comprising propionic acid and at least one 6-12 carbon (acid) component ester, wherein the ratio of propionic acid to ester component of the chemical is about 7:2 (v/v).

Brief Description of Drawings

The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, the embodiments shown in the drawings are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 illustrates a plate bioassay for determining bioactivity of various esters in combination with a 1:1 mixture of two organic acids, according to Table 2. The microorganisms tested were as follows-Cercospora (Cercospora) (black-bottom left) followed clockwise by Phytophthora (Phytophthora), Verticillium (Verticillium), Sclerotinia (Sclerotinia), Pythium (Pythium), fusarium, Trichoderma (Trichoderma), Rhizoctonia (Rhizoctonia) and Aspergillus (Aspergillus). The streaks are Saccharomyces (right-most bottom) followed by Candida (Candida), Escherichia coli (E coli) and Bacillus (Bacillus) (left bottom). A ═ control plates, and B ═ plates with system 1 after 30 hours incubation.

Figure 2 illustrates the effect of system 1 on bacterial growth from human feces. Approximately 5mg of fresh human feces was spread on the surface of a petri dish plate with potato dextrose agar. The plug was then removed from the center and approximately 0.5g of bentonite placed in the hole. The bentonite in the central pore had no ingredients in system 1 above (central), but the rightmost pore had system 1 at a ratio of 1ml system 1 per 10g of bentonite. The plates were incubated at 22 ℃ for 48 hours and then photographed. There was no detectable bacterial growth in the system 1 treated plates, but the control plates had abundant bacterial colonies.

Figure 3 illustrates the effect of system 2 on bacterial growth from human feces. Approximately 5mg of fresh human feces was spread on the surface of a petri dish plate with potato dextrose agar. The plug was then removed from the center and approximately 0.5g of bentonite placed in the hole. The bentonite in the central pore had no component of system 1 above (central), but the rightmost pore had system 1 at a ratio of 1ml system 1 per 10g of bentonite. The plates were incubated at 22 ℃ for 48 hours and then photographed. There was no detectable bacterial growth in the system 2 treated plates.

Fig. 4 illustrates two cat litter boxes with cat fecal material from 5 different cats at approximately 140g each. The right box has been treated with system 1 on bentonite (0.5ml/100g bentonite). After 5 days the ammonia reading of the control (left) was 14ppm, while the right treated chamber was 0 ppm. The total odor in the treated cabinet was significantly reduced.

FIG. 5 illustrates the treatment of approximately 140g of human feces with urine with Fusarium subglutinans 06-1 in the presence of system 2(1 ml on 10g zeolite). After 3 weeks fusarium oxysporum grew in large quantities (white mycelium in the right container). The ammonia level in the left control was 71.4, while the ammonia level in the right treated vessel was 12.1. There was no fungal growth nor fecal degradation in the control (left).

FIG. 6 illustrates the progressive growth of Fusarium (species) over a period of multiple days on human fecal pellets of approximately 100mg (fresh weight). The growth of the newly isolated and characterized Fusarium was each compared to P2-24 (Fusarium culmorum). The new Fusarium species, especially E06-1 and E06-5, did grow faster on feces. Growth was measured by the length of hyphae removed from an agar plug placed on a fecal mass.

FIG. 7 shows a six day old culture (top) of Fusarium gelatinatum E06-1 (a preferred fungus for treatment of human and animal feces in combination with System 2). Optical microscopic views (bottom) of spores and hyphae of fusarium oxysporum are also shown. The spores were slightly curved and 9.8-12X 2.5. mu.

FIG. 8 shows that Fusarium gelatinatum (E06-8) grew in large numbers (center) on human feces in the presence of system 2 with bentonite as the carrier. Note the inhibition of bacterial growth on the left and center of the culture plate, which is affected by the vapors emanating from the bentonite particles on the left side of the plate from system 2 that allow fungal growth. 0.5g of treated bentonite was added, approximately 100mg of human feces and plate were incubated for 12 days. See figure 6 for comparative growth measurements.

Fig. 9 depicts the biological activity of various test mixtures against a panel of test microorganisms. A small plug of each organism was placed around the PDA plate. The central well is filled with the test solution in a plastic cup holder. Control plates were also set up (A). After 30 hours, the growth of the test organisms was compared to the control and the percentage inhibition was calculated. (B) The plates contained the test mixtures. The measurement was performed 30 hours after the plate was set up.

FIG. 10 depicts the reduction of microbial contamination of ground corn by treatment with various concentrations of S-3 solution for 1 hour. Concentrations greater than 0.5% completely reduced bacterial contamination as seen by the absence of bacterial colonies in the 0.5% and 1.0% treatments (greater). Some minimal fungal contamination was observed in the latter-two fungal colonies were observed in each plate on the right. Incubate at room temperature for two days, then take pictures.

Fig. 11 depicts the use of bentonite treatment with various (S) formulations to kill escherichia coli in human feces over the course of 3 days (middle plate streaking) while allowing fusarium growth (plate top) that could otherwise break down and consume solid matter in human feces.

FIG. 12 depicts the effectiveness of the S-3 formulation in treating chicken fecal material by first plating the suspension on PDA plates and then adding 0.5g of zeolite treated with 3ml S-3 per pound. Photographs were taken after 3 days incubation at room temperature. It can be seen that the plates containing S-3 zeolite are almost free from bacterial contamination.

Fig. 13 depicts a 1 square foot plastic snap-on container filled with litter treating agent (litter treating) plus untreated bentonite in the proportions indicated by the package insert to compare the efficacy of the (S) formulation.

Figure 14 depicts ammonia levels taken at 5 minute intervals every 24 hours. Figure 14A depicts the average ammonia levels taken at 5 minute intervals every 24 hours. FIG. 14B depicts that the peak ammonia levels show a similar trend, with the S-1 treated bedding material showing significantly reduced ammonia production levels. Fig. 14B also depicts peak ammonia levels obtained from 5 minute interval testing every 24 hours.

FIG. 15 depicts the microbial activity of S-1 relative to a control sample. Fig. 15A and 15B indicate that the bentonite control (fig. 15A) has a large number of bacterial colonies growing across the plate, including those areas near the wells containing the bedding material. In contrast, the S-1 treatment (4 ml S3 per pound of carrier, FIG. 15B) had almost no bacterial colonies around the wells of the plate.

Fig. 16 depicts a 1 square foot plastic snap-on container filled with pine wood shavings and the desired dunnage treating agent in the proportions indicated by the package specifications. For these tests, S-1 applied at a rate of 15ml per pound of zeolite and an untreated zeolite control were tested.

Fig. 17 includes fig. 17A-17B, which depict ammonia levels taken at 5 minute intervals every 24 hours. Figure 17A depicts the average ammonia levels taken at 5 minute intervals every 24 hours. Figure 17B is a graph depicting peak ammonia levels showing similar trends, with Barnyard padding treated to show the lowest ammonia production levels. Figure 17B depicts peak ammonia levels obtained from 5 minute interval testing every 24 hours.

Fig. 18 depicts a 1 square foot plastic snap-on container filled with pine wood shavings (a common dunnage for large animals) and the desired dunnage treating agent in the proportions indicated by the package insert. For these tests, S-1 treated and untreated zeolite controls were tested.

Fig. 19 includes fig. 19A-19B, which depict ammonia levels taken at 5 minute intervals every 24 hours. Figure 19A depicts the average ammonia levels taken at 5 minute intervals every 24 hours. Fig. 19B is a graph depicting peak ammonia levels showing similar trends, with the S-1 treated litter showing the lowest ammonia production levels. Fig. 19B depicts peak ammonia levels obtained from 5 minute interval testing every 24 hours.

Fig. 20 includes fig. 20A-20B, depicting images of white scour calves before and after treatment. Figure 20A depicts white scoured calves prior to any treatment with S-X solution. Fig. 20B depicts the calves of fig. 20A after two rounds of treatment with S-X solution.

Fig. 21 includes fig. 21A-21B, depicting images of white scour calves before and after treatment. Figure 21A depicts white scoured calves prior to any treatment with S-X solution. Fig. 21B depicts the calves of fig. 21A 24 hours after treatment with S-X solution.

Fig. 22 is a picture depicting the condition of cows in the dairy 1.

Fig. 23 includes fig. 23A-23B, which depict images of white scour calves before and after treatment. Fig. 23A depicts a typical creamy yellow white dysentery that is exhibited on calves 919. Fig. 23B depicts fully restored calves 919 after treatment with S-X solution.

Fig. 24 includes fig. 24A-24B, which depict images of calves 166 of the pasture 9 before and after treatment. Fig. 24A depicts calf 166 suffering from white diarrhea in winter 2014. Fig. 24B depicts calves one day after treatment with S-X solution, where the animals recovered and yellow diarrhea was reduced.

Fig. 25 includes fig. 25A-25B, which depict images of a sheep afflicted with mastitis and administered a treatment. Figure 25A depicts a sheep suffering from mastitis. Figure 25B depicts administration of the S-3 formulation to the animal via a syringe.

Fig. 26 includes fig. 26A-26B, which depict images of raspberries before and after treatment. Fig. 26A depicts raspberries treated with control bentonite in the central well. FIG. 26B depicts raspberries treated with an S-31: 10 mixture. Both groups of raspberries were stored at room temperature for 1 week. The raspberries treated with S-3 were edible and not rotten.

FIG. 27 (including FIGS. 27A-27B) depicts soil treated with Pythium ultimum (P. ultimum) or S-3. FIG. 27A is a photograph of soil with red beet seeds treated with Pythium ultimum alone. Only one or two seeds were observed to germinate. FIG. 27B is a photograph of S-3 treated soil on bentonite in the presence of Pythium ultimum and red beet seeds. Many seeds were observed to germinate.

FIG. 28 (including FIGS. 28A-28D) depicts pictures of water agar plates used to test S-3 with red beet seeds. FIG. 28A is a picture of an agar plate with red beet seeds, bentonite, S-3(1 part in 10g bentonite), and Pythium ultimum. S-3 was found to control the growth of Pythium ultimum. FIG. 28B is a picture of an agar plate with red beet seeds and Pythium ultimum. FIG. 28C is a picture of an agar plate with individual red beet seeds. FIG. 28D is a picture of an agar plate demonstrating that S-3 is harmless to red beet seeds.

Detailed description of the invention

It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements found in typical antimicrobial formulations. One of ordinary skill in the art may recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein relates to all such variations and modifications of such elements and methods known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated therewith in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

As used herein, "about" when referring to a measurable value, such as an amount, time interval, etc., is intended to encompass variations from that particular value of ± 20%, ± 10%, ± 5%, ± 1% and ± 0.1%, as such variations are suitable.

As used herein, "S-1" refers to any and all formulations of system 1.

As used herein, "S-2" refers to any and all formulations of system 2.

As used herein, "S-3" refers to any and all formulations of system 3.

As used herein, "S-4" refers to any and all formulations of system 4.

As used herein, "S-5" refers to any and all formulations of system 5.

As used herein, "S-X" refers to any and all formulations of system X, which may include one or more of systems 1-5.

As used herein, the term "CLOE" refers to a formulation comprising S-1 or S-5.

As used herein, the term "Barnyard padding" refers to a formulation comprising S-1 or S-5.

As used herein, the term "pharmaceutical composition" refers to a mixture of at least one composition of the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the composition to an organism. There are a variety of techniques in the art for administering compositions, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the composition and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, involved in carrying or transporting a composition useful in the present invention in a patient or carrying or transporting the composition to a patient such that the composition performs its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation (including compositions suitable for use in the present invention) and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as groundnut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; a surfactant; alginic acid; no heat source water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and other non-toxic compatible materials employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, that are compatible with the activity of the compositions useful in the present invention and are pharmaceutically acceptable to a patient. Supplementary active compositions may also be incorporated into the composition. The "pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compositions useful in the present invention. Other additional ingredients that may be included in pharmaceutical compositions for practicing the present invention are known in the art and are described, for example, (Genaro, ed., Mack Publishing co.,1985, Easton, PA), which is incorporated herein by reference.

Throughout this disclosure, various aspects of the present invention may be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have explicitly disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, it is contemplated that the description of a range such as 1 to 6 has explicitly disclosed sub-ranges such as 1 to 3, 1 to 4,1 to 5, 2 to 4,2 to 6,3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any full and partial increments therein. This applies regardless of the breadth of the range.

The present invention relates to the discovery of effective and useful chemical formulations that have strong antimicrobial activity, either alone or in combination with certain endophytes such as fusarium, and can be particularly useful in a variety of applications, such as reducing microbial growth on surfaces or instruments in medical facilities, reducing microbial growth on crop surfaces, or decontaminating, degrading and deodorizing human and animal feces. For example, the formulations of the present invention are suitable for treating feces in any location, such as toilets, litter boxes, animal stalls (animal stalls), barns (barns), chicken facilities, piggeries, homes, pet homes in zoos, and many other locations.

In a preferred embodiment, a non-hazardous formulation containing ingredients from the FDA-GRAS list is placed in a container, such as a biodegradable plastic bag, along with a suitable combination of a suitable fungus, such as the endophytic fungus Fusarium, e.g., Fusarium collodionum. The bag also contains appropriate amount of urine absorbing polymer compatible with Fusarium endosomosum. This combination of reagents represents a safe, rapid and novel process for the recovery of components found in human and animal feces. The presence of these two components in the bag effectively kills many harmful bacteria in human feces while beginning the process of recycling the organic components of the feces to a harmless soil additive.

The present invention may be employed in connection with activities such as national emergencies, military exercises, marine related activities, natural disasters, outdoor sports (camping, hiking, canoeing, hunting, cycling, etc.), and other activities where human waste requires proper and safe disposal. It also relates to the development of safer facilities for all domestic animals and even domestic pets. For example, it has recently been noted that proper and safe disposal of human waste is an important issue for proper management of wasteland areas in the world. Aesthetic and health issues are major issues facing the managers of these areas. Thus, the present invention may be suitable for human and animal surfaces, plant surfaces, industrial surfaces, machine tools, and many other uses.

In one embodiment of the invention, the formulations of system 1(S-1), system 2(S-2), system 3(S-3), system 4(S-4) and/or system 5(S-5), and optionally bentonite, zeolite or perlite (depending on the application) are combined together in a container and the process of bacterial kill and/or fecal degradation is started immediately. In another embodiment, the invention can be used in animal litter and litter treatments where the chemical mixture (with carrier) can be applied directly to the area housing the animal, resulting in almost immediate kill of bacteria that produce noxious odors such as ammonia. In another embodiment, the present invention may be applied to surfaces, such as crop surfaces, medical facility surfaces, medical or industrial tools, and the like, to eliminate or reduce microbial counts of the treated surfaces.

Antibiotics are compounds that kill or inhibit the growth of bacteria. The common erroneous idea is that antibiotics are effective against other microorganisms, such as fungi and viruses, when antifungal and antiviral compounds are in fact required for such purposes. Antibiotics work by interfering with key steps in the metabolism and growth of bacteria and can be broadly classified into two main categories, namely bactericides and bacteriostats, respectively, depending on whether they kill bacteria or simply inhibit their growth. Antibiotics are generally safe for use in humans because the steps they target are unique to certain types of bacteria or are effective against bacteria at very low concentrations that are considered safe for humans. Other classes of chemical agents, such as certain alcohols, acids and peroxides, can have broad inhibitory and/or killing efficacy, as they affect essential elements of biochemical processes common to many life forms. These classes of compounds are classified as antibacterial (antipeptic), bactericidal (sterilant), disinfectant (disinfectitant), and bactericidal (sanitizer) agents, as well as preservatives, according to their specific effects on microbial life, mode of effective administration, and toxicity to humans. The systems of the present invention, such as S1, are mixtures composed primarily of short chain organic acids and esters, most notably propionic acid and isoamyl hexanoates. None of these molecules are classified as antibiotics, but all have antimicrobial properties and can be bactericidal or bacteriostatic depending on the concentration and length of application.

The system of the present invention does not function by the same mechanism as antibiotics. Antibiotics typically target very specific steps by identifying very specific structural motifs, whereas the system of the present invention kills bacteria and inhibits their growth by affecting the basic biochemical properties required to sustain life. Moreover, the components of the system of the present invention act synergistically such that the effect of the total mixture is greater than the sum of its fractions. The mechanism of synergy observed with these systems is not clear, but other acid/ester mixtures exhibit the same kind of extended combined action.

One of the main components of the system of the present invention, propionic acid, is a short chain organic acid which has a definite use as a preservative in food and agricultural production. Most organisms, including humans and many bacterial species, have metabolic pathways that favor the use of propionic acid as a nutrient, and in fact, a group of bacteria may even produce this molecule. Thus, at low concentrations, propionic acid is essentially harmless to almost all organisms, but at higher concentrations it cannot be degraded fast enough and starts to accumulate inside the cell. As its concentration within the cell increases, the acidity of the cell also increases. When the acidity within the cell is too high, the enzyme does not function properly, DNA and other biomolecules are destroyed, and the cell dies. Recent studies have shown that while the effect on intracellular acidity is the primary antimicrobial mechanism for weak organic acids, it is by no means the only mechanism. As acids dissociate and release protons within the cell, they become negatively charged. High concentrations of negatively charged molecules within cells have many adverse effects on osmolarity, nutrient storage and metabolism.

At lower concentrations, the acid can be inhibitory, but not lethal. The acidity increases when the acid dissociates and releases protons. When the acidity within the cell is too high, the cell may export protons to the outside in an attempt to maintain an appropriate pH level. This strategy, while effective, requires a large amount of energy to be expended and only occurs at low acid concentrations without being lethal. Because smaller organisms are more sensitive to smaller amounts of propionic acid, concentrations that are harmless to humans can be lethal or inhibitory to bacteria. Propionic acid is not the only organic acid in S1, but it is speculated that the antimicrobial action of other organic acids of similar size results from essentially the same mechanism.

The antimicrobial mechanism of lipids is still largely unknown. While not wishing to be bound by any particular theory, one possible clue comes from the following observation: those that are capable of more efficient incorporation into bacterial cell membranes tend to have increased antimicrobial properties for a given set of lipids. Incorporation of any molecule into the cell membrane alters the chemical and physical properties of the membrane, which leads to changes in nutrient uptake, waste output, energy production, and other essential cellular processes. While not wishing to be bound by any particular theory, this observation suggests that incorporating certain lipids into the cell membrane alters the chemical and physical properties of the membrane in a way that is harmful to the organism. Alterations in the cell membrane are also the mechanism by which longer chain organic acids are thought to act.

With the large-scale use of antibiotics beginning in the 20 th century, the problem of antibiotic resistance emerged as a major clinical problem. In the 21 st century, as the consequences of antibiotic resistance became more apparent and prevalent, the term entered public awareness and was ultimately recognized by the enormous problems it caused. In bacterial populations exposed to antibiotics, resistance is present in very few individuals or initially occurs due to natural mutations, and individuals that are resistant to antibiotics are subsequently selected for their survival advantage over non-resistant individuals. Antibiotic resistance is transmitted by vertical transmission from resistant cells to their progeny and by horizontal transmission (direct transfer of the resistance gene from resistant cells to non-resistant cells). In this way, resistance spreads rapidly and increased antibiotic use creates selective pressure to increase the survival advantage of antibiotic resistance.

Bacteria can acquire resistance to a given antibiotic by four major mechanisms: evolving enzymes that inactivate antibiotics, changing the structure of the target so that antibiotics can no longer bind, changing metabolic pathways to skip antibiotic inhibition steps, and developing efflux pumps that pump antibiotics out of the cell. Each mechanism has a genetic basis and can therefore be transferred from cells that initially develop resistance to non-resistant cells. In some cases, bacterial cells can acquire resistance to several different classes of antibiotics. This is how so-called "super-bugs" are produced, and as antibiotic use increases in the agricultural, veterinary and medical industries, the prevalence of multi-drug resistant bacterial strains also increases. In addition, combinations of small organic molecules, such as acids and esters, have been identified that act in a synergistic manner to produce nearly the same antimicrobial effect as antibiotics. Organic molecules possessing these properties may be referred to as "synergists".

The mechanisms of antibiotics and their resistance can be summarized by a word-specificity. Antibiotics work by targeting specific structural features, enzymes and macromolecules. Likewise, antibiotic resistance occurs when a bacterium develops an efflux pump specific for a given antibiotic or alters a particular structural feature, enzyme or macromolecule. If the antibiotic is specific, the components of the system of the invention are common. Organic acids and esters lack specific targets, instead they exert their antimicrobial action by altering the biochemical environment of the bacterial cells. They are effective against a very wide range of organisms and they interfere with a variety of cellular processes.

Organic acids are abundant in nature. Any given bacterial cell is always exposed to organic acids at some point in its life, and thus many bacterial species possess innate genetic mechanisms that, once induced, help bacterial species to cope with the stress brought on by natural organic acid exposure. Perhaps the best studied of these is the acid tolerance response of salmonella. Essentially when a salmonella cell is exposed to high but sub-lethal acid concentrations, it induces the expression of many genes, making it more likely to survive the next time it is exposed to acid conditions. This has been experimentally demonstrated by inoculating acidic media with previously exposed and unexposed salmonella cells. In almost every case, previously exposed cells are rendered much more resistant to acids. Coli has a well-studied acid tolerance response and it appears that this mechanism may be present in many other bacterial species. In the case of pathogenic species such as escherichia coli and salmonella, there is a great deal of concern over: induction of an acid tolerance response by exposure to sublethal concentrations of organic acid food preservatives can increase bacterial virulence as bacteria are more likely to survive exposure to acidic gastric juices from the digestive process.

However, there is a significant difference between resistance to antibiotics and resistance to organic acids. Many genes for antibiotic resistance are located on mobile genetic elements called plasmids, which can be easily transferred between bacterial cells and between bacterial species. In this way, it is a relatively simple process to obtain resistance to multiple antibiotics for any given bacterial cell. On the other hand, acid resistance mechanisms such as acid tolerance responses are encoded on chromosomal DNA. This genetic information can only be transferred to progeny cells, and therefore, as has been observed in the case of multi-drug resistant bacterial strains, the sudden appearance of "superbugs" is not applicable. Furthermore, while not wishing to be bound by any particular theory, it is possible that the synergy observed when organic acids are used in combination with organic esters may negate some of the acid resistance mechanisms.

In one embodiment, to identify microorganisms that can grow on human and animal feces, leading to their degradation, it is necessary to formulate a novel antibiotic cocktail that can kill the microbial content of the feces, which normally functions to degrade urea to ammonia and uric acid. Ammonia is lethal to most fungi, and it additionally degrades the solid components of feces. The main thing in this finding is the following known fact: propionic acid has antibacterial activity, but only at inhibitory levels, and so is isobutyric acid. Thus, these two compounds are starting components for a new effective antibiotic mixture. What is needed is another ingredient to add microbial kill to the mixture. It is then understood in a completely unexpected manner that the addition of certain esters to these small organic acids can add significantly enhanced antimicrobial activity to them.

Microorganisms living in the tropical rainforests of the world must have evolved biochemical mechanisms to cope with potential competitors in order to survive. In this regard, they have developed the ability to produce antimicrobial molecules and compounds that inhibit and destroy other microorganisms. As humans explore new antibiotics, researchers look at tropical rainforests to search for new microorganisms and the substances they produce that inhibit and destroy competitors of other microorganisms. Certain tropical rainforest microorganisms provide important chemical clues as to which compounds are selected for use in systems 1-4.

Preparation

The present invention includes, in part, a chemical formulation comprising at least one organic acid such as propionic acid, isobutyric acid, or butyric acid. In one embodiment, the chemical formulation has antibacterial activity when applied to human or animal feces. In certain embodiments, the organic acids used may contain 2-5 carbon atoms, and each acid used may vary from 0% to 80% of the biologically active mixture. In a preferred embodiment, the organic acid is propionic acid. In another embodiment, the present invention includes a chemical formulation consisting essentially of an organic acid such as propionic acid, isobutyric acid, or butyric acid. In one embodiment, the chemical consists essentially of propionic acid. In certain embodiments, the chemical formulation comprises two organic acids. In one embodiment, the two organic acids are propionic acid and isobutyric acid. In one embodiment, the chemical formulation comprises a combination of two organic acids and at least one ester. In one embodiment, the chemical formulation comprises propanoic acid, isobutryic acid and at least one ester. In another embodiment, the two organic acids are propanoic acid and isobutyric acid and the at least one ester is isoamyl butyrate. In another embodiment, the two organic acids are propanoic acid and isobutyric acid and the at least one ester is isoamyl hexanoates. In another embodiment, the two organic acids are propanoic acid and isobutyric acid and the at least one ester is isoamyl acetate.

As contemplated herein, the chemical formulation may further comprise at least one ester. As contemplated herein, the at least one ester can be any of the esters listed in table 1 or elsewhere herein. In certain embodiments, the ester may have 3 to 10 carbon atoms, and any ester or combination thereof may comprise at least 20% of the mixture. In one embodiment, the ester is isoamyl ester. As contemplated herein, embodiments of the present invention may alternatively be formulated using an entire family of isoamyl esters of various acid components (C-6 (hexanoate) to C-12 (laurate)) as well as various aromatic (acid) esters of isoamyl alcohol, such as cinnamate, benzoate, and phenyl acetate. In one embodiment, the ester is isoamyl hexanoates. As used herein, the term "hexanoate ester" may mean a single type of hexanoate ester or may include mixtures of hexanoate esters in the acid form (including branched forms). In another embodiment, the ester is isoamyl formate. In another embodiment, the ester is isoamyl butyrate. In another embodiment, the ester is isoamyl acetate. In one embodiment, the ester is isoamyl acetate. In one embodiment, the ester is selected from allyl acetate, n-decyl acetate, isoamyl acetate, and phenylethyl acetate. In one embodiment, the ester is strawberry aldehyde (3-methyl-3-phenyl-oxirane-2-carboxylic acid ethyl ester, an organic ester). In certain embodiments, the at least one ester may be any single carbon (acid) component ester. In one embodiment, the at least one ester is isoamyl formate. In certain embodiments, other compounds may be added as the ester component of the formulation. For example, the octanoate ester of isoamyl alcohol is active, as is the laurate ester. Thus, the formulations of the present invention may include the use of the entire range of 6-12 carbon (acid) components of isoamyl esters. In one embodiment, the ester is the caprylate ester of isoamyl alcohol, and in another embodiment, the ester is the laurate ester of isoamyl alcohol. In certain embodiments, a benzene component may be used along with benzoate, cinnamate, and salicylate esters. In one embodiment, the chemical formulation comprises propionic acid and at least one 6-12 carbon (acid) component ester.

In certain embodiments, the formulations of the present invention may comprise a mixture of at least one organic acid and at least one ester in any ratio. In one embodiment, the ratio of the at least one organic acid to the at least one ester is from about 6-7 to about 2-3. In a preferred embodiment, the ratio of the at least one organic acid to the at least one ester is about 7: 2. In other embodiments, the formulations of the present invention may include a mixture of two organic acids and at least one ester in any ratio. In one embodiment, the ratio of the first organic acid to the second organic acid to the at least one ester is about 3.5:3.5:2 (v/v/v). In another embodiment, the mixture of the first organic acid, the second organic acid, and the at least one ester is about 7 parts of the two acids and 2 parts of the selected ester. In one embodiment, the chemical formulation comprises propionic acid and at least one 6-12 carbon (acid) component ester, wherein the ratio of propionic acid to ester component of the chemical formulation is about 7:2 (v/v).

As contemplated herein, the present invention may include any chemical formulation plus the addition of at least one endophytic fungus. However, the invention is not limited to any particular fungus, and endophytic fungi are preferred, and fusarium fungi are more preferred. Most preferred are endophytic fungi of the species Fusarium colloidosum. In another embodiment, the endophytic fungus is a fungus of the genus Penicillium (genus Gloeosporium). As contemplated herein, the fungus may be incorporated into any formulation by inoculation of barley or other suitable carrier for the fungus, as understood by those skilled in the art. In one embodiment, the chemical formulation comprises two organic acids and at least one ester and at least one fungus, wherein the two organic acids and the at least one ester kill or reduce bacterial growth on human or animal feces, and the at least one fungus increases the rate of decomposition of the human or animal feces. In another embodiment, the formulation comprises propanoic acid, isobutryic acid, at least one ester, and at least one fungus. In certain embodiments, the formulation may additionally comprise eucalyptol (cineole), valencene (valecene), salts and any other additives, excipients or other components as required to produce a formulation with the desired characteristics.

In some embodiments, supplementation of a fungal culture with additional compounds may enhance its fungal inhibitory properties to a greater extent than the substance produced by the fungus or the compound alone. This activity is considered to be a synergistic effect. Accordingly, the present invention also provides a chemical formulation comprising at least one fungus and at least one synergist. As used herein, the term "synergist" refers to any chemical compound that, when administered in combination with another compound, exhibits greater microbe-inhibiting activity than that observed when each compound is administered alone. In one non-limiting example, when the synergist is combined with a fungal culture, the combined gas phase of the fungus and the synergist shows increased antimicrobial activity compared to the gas phase of the fungus or synergist alone.

In certain embodiments, the chemical formulations of the present invention may be used as a cat litter treatment, horse shed, cattle shed, sheep shed, or small animal litter treatment in combination with a carrier such as zeolite or bentonite. In such embodiments, the invention inhibits microorganisms that inhabit fecal material, such as e.coli, and breaks down urea in the urine to release ammonia. In another embodiment, the chemical formulation of the present invention may be added to a carrier such as, but not limited to, bentonite, zeolite, perlite or other silica-based carrier in an amount effective to kill bacteria and reduce noxious and toxic odors.

In certain embodiments, the present invention can be mixed with a foam or other dispersion solution and used as an antimicrobial spray or for surfaces contaminated with bacteria or other microorganisms, such as surfaces used to treat hospitals, home food preparation areas, contaminated areas of food processing (including all industrial food processors, fruit, meat, etc. where bacterial contamination is often an issue).

In certain embodiments, the compositions of the present invention comprise at least one chemical formulation or formula of the present invention. In one embodiment, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical composition of the invention comprises a therapeutically effective amount of the formulation of the invention and a pharmaceutically acceptable carrier. As will be appreciated by those skilled in the art, examples of pharmaceutically acceptable carriers include cremophor (cremophor) or any other biosurfactant. In one embodiment, the pharmaceutically acceptable carrier is cremophor.

In certain embodiments, the formulations of the present invention, and preferably S-3, may be used with a carrier (zeolite or bentonite or talc) to treat the area of soil that is to receive seed or seedling transplants to reduce or eliminate the risk of infection.

In certain embodiments, the formulations of the present invention may be mixed with cleaning agents to be used as carpet detergents and bacterial decontaminants for animal manure, human manure, or other biological contamination of carpet surfaces.

In certain embodiments, the formulations of the present invention may be applied as a spray to decontaminate fruits, vegetables, grains, and other agricultural products during planting, during growth, during harvesting, and/or during transportation.

In certain embodiments, the formulations of the present invention may be applied to or formulated, embedded or otherwise integrated in an infant diaper, bandage or other device requiring bacterial decontamination.

In certain embodiments, the formulations of the present invention may additionally be formulated with detergents for use as soaps for cleansing human and animal skin.

In certain embodiments, the formulations of the present invention may additionally be formulated, embedded, or otherwise integrated in a candle for use in purging an area by vapor when lit.

In particular, the chemical formulations of the present invention exhibit significant antibiotic activity against bacteria associated with human and animal feces. These mixtures can be specifically delivered to their respective target sites by inert carriers such as, but not limited to, bentonite, zeolite, perlite or other silica-based carriers. In this case, specific locations for using the combination of carrier and antibiotic include, for example, but not limited to, all domestic and zoo-related animals and bedding locations for animals used as house pets. The mixture can be applied to cots, bedding and animal habitation to reduce bacterial loads and harmful gases.

It is to be understood that the formulations of the present invention may comprise any additional salts, excipients, nutritional additives or supplements, etc., such that the final formulation is suitable for topical application, ingestion, inhalation or other desired form of administration.

System 1

As described elsewhere herein, the chemical formulation may comprise two organic acids and at least one ester. For example, in one embodiment, the formulation comprises propanoic acid, isobutyric acid, isoamyl butyrate. In one embodiment, the ratio of propanoic acid to isobutyric acid to isoamyl butyrate is about 3.5:3.5:2 (v/v/v). In another embodiment, the mixture of isoamyl propionate isobutyrate butyrate is about 7 parts of the two acids and 2 parts of the selected ester. It should be understood that the chemistry of system 1 is not limited to any particular ratio of such chemical components. In another embodiment, the chemistry of system 1 consists of only two organic acids and a single ester. In one such embodiment, the formulation consists of propanoic acid, isobutryic acid, isoamyl butyrate in the above-described ratio. In certain embodiments, the formulation may additionally comprise eucalyptol, valencene, salts or any other additives, excipients or other components as required to produce a formulation with the desired characteristics.

In another embodiment, the chemical formulation of system 1 can be added to a carrier, such as but not limited to bentonite, zeolite, perlite, or other silica-based carrier, in an amount effective to kill bacteria and reduce noxious and toxic odors. This ratio is typically 1 for a 1ml system to 224g of carrier (V/W) or other effective suitable ratio, without limitation.

System 2

As contemplated herein, the present invention may include any chemical formulation of system 1 plus the addition of at least one endophytic fungus. As demonstrated herein, endophytic fungi of the fusarium oxysporum group and the like are particularly suitable for growing on and degrading human feces. Furthermore, when another antimicrobial mixture such as system 1 is applied and the mixture maximally allows fungal growth while killing bacteria and other microorganisms, the fungus can only grow on the liquid and solid fecal combination. In one embodiment, the fungus is fusarium oxysporum. In one embodiment, the fungus is incorporated into system 2 by inoculation of barley. In certain embodiments, the formulation may additionally comprise eucalyptol, valencene, salts or any other additives, excipients or other components as required to produce a formulation with the desired characteristics.

In one embodiment, system 2 comprises the system 1 chemistry, such as propionic acid to isobutyric acid to isoamyl butyrate in a ratio of 3.5:3.5:2(V/V/V), or 7 parts of both acids and 2 parts of ester, which are then added to a dry weight carrier material, such as bentonite, perlite, or zeolite, at a ratio of 1/10(V/W) of the mixture. It should be understood that the chemistry of system 2 is not limited to any particular ratio of such chemical components. Barley inoculated with fusarium oxysporum was also added. This mixture was then added at 10g to each container, such as a plastic bag for handling and disposing of human feces. It allowed rapid growth of fusarium oxysporum compared to system 1 alone, which is not the case for system 1 alone. As will be appreciated by those skilled in the art, other items may also be added to the bag, including appropriate amounts of liquid-absorbent polymers.

System 3

As contemplated herein, the chemical formulations of the present invention may comprise at least one organic acid and at least one ester. In a preferred embodiment, the at least one organic acid is propionic acid. In one embodiment, the at least one ester is isoamyl hexanoates or a mixture of isoamyl hexanoates. In a preferred embodiment, the at least one ester is isoamyl hexanoates. In one embodiment, the chemical formulation comprises propionic acid and isoamyl acid. In certain embodiments, the formulation may additionally comprise eucalyptol, valencene, salts or any other additives, excipients or other components as required to produce a formulation with the desired characteristics.

In one embodiment, the ratio of isoamyl propionate to isoamyl hexanoate is about 7:2 (v/v). It should be understood that the chemistry of system 3 is not limited to any particular ratio of such chemical components. In another embodiment, the chemistry of system 3 consists of a single organic acid component and a single ester component. In another embodiment, the chemistry of system 3 consists of a mixture of a single organic acid component and isoamyl hexanoates. In one such embodiment, the formulation consists of isoamyl hexanoate propionate in the ratio described above. In another embodiment, the chemical formulation consists essentially of propionic acid and isoamyl hexanoates in a ratio of propionic acid to isoamyl hexanoates of about 7:2 (v/v).

In another embodiment, the chemical formulation of system 3 may be added to a carrier such as, but not limited to, bentonite, zeolite, perlite or other silica-based carrier in an amount effective to kill bacteria and reduce noxious and toxic odors. This ratio is typically 1.0 to 1.5ml system 3 to 224g support (V/W) or other suitable effective ratio, not limited to, for example, 0.1 to 5ml system 3 to 224g support (V/W), or 0.5 to 2ml system 3 to 224g support (V/W).

System 4

As contemplated herein, the chemical formulations of the present invention may comprise at least one organic acid and at least one ester. In a preferred embodiment, the at least one acid is propionic acid. In one embodiment, the at least one ester is isoamyl formate. In another embodiment, the at least one ester may be any single carbon (acid) component ester. In one embodiment, the chemical formulation comprises propionic acid and isoamyl formate. In certain embodiments, the formulation may additionally comprise eucalyptol, valencene, salts or any other additives, excipients or other components as required to produce a formulation with the desired characteristics.

In one embodiment, the ratio of propanoic acid to isoamyl formate is about 7:2 (v/v). It should be understood that the chemistry of system 4 is not limited to any particular ratio of such chemical components. In another embodiment, the chemistry of system 4 consists of a single organic acid component and a single ester component. In one such embodiment, the formulation consists of isoamyl propionate to formate in the above-described ratio. In one embodiment, the chemical formulation consists essentially of propionic acid and isoamyl formate in a ratio of propionic acid to isoamyl formate of about 7:2 (v/v).

As contemplated herein, the present invention may include any chemical formulation of system 4 plus the addition of at least one endophytic fungus. As demonstrated herein, endophytic fungi of the fusarium oxysporum group and the like are particularly suitable for growing on and degrading human feces. Furthermore, when another antimicrobial mixture such as system 4 is applied and the mixture maximally allows fungal growth while killing bacteria and other microorganisms, the fungus can only grow on the liquid and solid fecal combination. In one embodiment, the fungus is fusarium oxysporum. In yet another embodiment, the invention comprises a 7:2 mixture comprising propionic acid and isoamyl formate, and optionally a chemical of fusarium sporotrichioides. In this embodiment, the propionic acid/isoamyl formate mixture is suitable for killing selected microorganisms without killing fusarium, which can further enhance the recovery of the stool product to which the formulation is applied. In one embodiment, the fungus is incorporated into system 4 by inoculation of barley.

In one embodiment, system 4 comprises propionic acid to isoamyl formate in a ratio of 7:2(V/V) which is then added to a carrier material such as bentonite, perlite or zeolite in a dry weight ratio of 1/10(V/W) of the mixture. Barley inoculated with fusarium oxysporum was also added. This mixture is then added to a container, such as a plastic bag for handling and disposing of human feces. This mixture allows for rapid growth of fusarium oxysporum. As will be appreciated by those skilled in the art, other items may also be added to the bag, including appropriate amounts of liquid-absorbent polymers.

System 5

As described elsewhere herein, the chemical formulation may comprise two organic acids and at least one ester. For example, in one embodiment, the formulation comprises propanoic acid, isobutyric acid, isoamyl hexanoates. In one embodiment, the ratio of propanoic acid to isobutyric acid to isoamyl hexanoates is about 3.5:3.5:2 (v/v/v). In another embodiment, the mixture of isoamyl propionate isobutyrate hexanoate is about 7 parts of both acids and 2 parts of the selected ester. It should be understood that the chemistry of system 5 is not limited to any particular ratio of such chemical components. In another embodiment, the chemistry of system 5 consists of only two organic acids and a single ester. In one such embodiment, the formulation consists of propanoic acid, isobutyric acid, isoamyl hexanoates in the above-described ratio. In certain embodiments, the formulation may additionally comprise eucalyptol, valencene, salts or any other additives, excipients or other components as required to produce a formulation with the desired characteristics.

In another embodiment, the chemical formulation of system 5 can be added to a carrier such as, but not limited to, bentonite, zeolite, perlite, or other silica-based carrier in an amount effective to kill bacteria and reduce noxious and toxic odors.

System X

As contemplated herein, the present invention may include any of the chemicals of systems 1-5 in combination with at least one of a salt, an excipient, a nutritional additive, or a supplement. In a preferred embodiment, the chemical formulation is system 3. As demonstrated herein, a chemical formulation comprising system 3, at least one nutritional supplement, and at least one salt is useful for treating diseases and conditions associated with microbial infections. Examples of nutritional supplements include, but are not limited to, sugars such as glucose, sucrose, or fructose; amino acids such as glycine; and protein sources, such as whey protein. As will be appreciated by those skilled in the art, any protein source may be used. Non-limiting examples of salts include potassium chloride, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, potassium sulfate, and magnesium acetate. Salts are suitable for use in the formulations of the invention because they enhance electrolyte balance in a subject. Any amount of salt may be used in the compositions of the present invention. Preferably the amount of salt is greater than 0%. The presence of system 3 inhibits and kills pathogenic bacteria. In one embodiment, system X comprises the system 3 chemistry, glucose, whey protein, potassium chloride, magnesium sulfate, and sodium chloride. In another embodiment, system X comprises the chemistry of system 3, glucose, glycine, potassium chloride, sodium chloride, and magnesium acetate. In another embodiment, system X comprises the chemistry of system 3, glucose, glycine, potassium chloride, sodium chloride, magnesium acetate, and potassium dihydrogen phosphate. It is to be understood that the chemistry of system X is not limited to any ratio of such chemical components. In one embodiment, the amount of organic acid is about 100% and the amount of ester is 0%. In another embodiment, the amount of organic acid is about 99% and the amount of ester is about 1%. In another embodiment, the amount of organic acid is about 1% and the amount of ester is about 99%.

In certain embodiments, system X is formulated using one or more pharmaceutically acceptable excipients or carriers. Examples of pharmaceutically acceptable carriers include cremophor or any other biosurfactant, as understood by those skilled in the art. In one embodiment, the pharmaceutically acceptable carrier is cremophor. In one embodiment, system X comprises the chemistry of system 3 and cremophor.

In certain embodiments, systems 2 and 4 can be used with or without a carrier to treat animal manure (including human manure) in the presence of fusarium oxysporum. In such embodiments, the present invention inhibits and kills bacteria while allowing the growth of fusarium oxysporum, which ultimately breaks down and putrefies solid matter in human feces.

In certain embodiments, the formulations of the present invention are used to smoke seeds contaminated with microorganisms.

In certain embodiments, the formulations of the present invention are administered as a gaseous formulation in the absence of water and any other carrier.

Method

The purification of human feces is only one problem associated with the fecal management process. Another problem solved by the present invention is the need to start the process of immediate degradation of organic matter in solid and liquid faeces. The biological and biochemical processes that occur when solid and liquid feces are combined are complex. It was demonstrated that urea in urine was immediately attacked by urease, an enzyme found in most microorganisms associated with solid feces, with the concomitant production of ammonia gas. The gas itself is harmful and produces an unpleasant smell. It is also lethal to most fungi because it causes an increase in pH. Thus, if it is desired to degrade the feces, it is necessary to stop ammonia production, which is desirable for fungal growth and ammonia remediation in the environment. Each of systems 1-4 resulted in the killing and inhibition of bacterial growth and subsequent ammonia production, and systems 2 and 4 also allowed the rapid growth of fusarium oxysporum which subsequently degraded feces. Thus, systems 1 and 3 are particularly suitable for treating animal litter, manure, etc., and reducing ammonia.

The discovery that suitable microorganisms cause rapid putrefaction of human and animal feces begins with the following considerations: viable microorganisms (i.e., endophytes) within plants would be suitable sites for initial exploration. Endophytes are the first microorganisms involved in the degradation of plants when they die from natural causes or environmental damage. They possess a group of enzymes that degrade cellulose, lignin and hemicellulose found in plant materials. These are the same composite organic materials found in human solid feces; thus, to address the problems associated with the present application, namely, the degradation of human and animal feces, the ability of a variety of endogenous microorganisms to grow on solid and liquid human feces was located and tested. In order for the microorganisms to degrade the feces, they must be insensitive to the ammonia produced, or the ammonia must be removed from the reaction. Thus, using systems 2 and/or 4 that allow fusarium growth and elimination of ammonia production, it is possible to devise useful and rational methods for handling liquid and solid manure.

In one aspect, the invention includes a method of treating human or animal waste. In one embodiment, the method comprises contacting human or animal feces with a composition of the present invention, wherein the composition kills or reduces bacterial growth on the human or animal feces. In one embodiment, the composition comprises a chemical formulation of the present invention. In one embodiment, the chemical formulation further comprises at least one fungus. In another embodiment, the at least one fungus increases the rate of breakdown of human or animal feces.

In another aspect, the invention includes a method of eliminating or reducing microbial growth at a treatment site. In one embodiment, the method comprises contacting the treatment site with a composition of the present invention, wherein the composition kills or reduces bacterial growth on human or animal feces. In one embodiment, the composition comprises a chemical formulation of the present invention. In one embodiment, the chemical formulation further comprises at least one fungus.

In another aspect, the invention includes a method of eliminating or reducing odor formation at a treatment site. In one embodiment, the method comprises contacting the treatment site with a composition of the present invention, wherein the composition eliminates or reduces odor formation on human or animal feces.

In another aspect, the invention includes a method of eliminating or reducing the amount of ammonia at a treatment site. In one embodiment, the method comprises contacting the treatment site with a composition of the present invention, wherein the composition eliminates or reduces the amount of ammonia on human or animal feces.

In another aspect, the invention includes a method of smoking a seed contaminated with a microorganism. In one embodiment, the method comprises contacting the seed with a composition of the invention, wherein the composition reduces or eliminates microbial growth on the seed, and in some embodiments, reduces or eliminates microbial growth on the seed without significantly disrupting germination.

In certain embodiments, the formulations of the present invention may be placed in a bedpan in combination with a carrier for use in hospital areas to treat human feces to prevent contamination of the area with fecal bacteria. In certain embodiments, the formulations of the present invention are useful as antimicrobial agents for treating cuts and wounds, as well as surface infections in animals and humans. For example, the present invention is useful for the treatment of bacterial and viral intestinal infections in humans and animals. It should be noted that all of the ingredients of systems 1-4 are on the GRAS list and are therefore safe. Specifically, humans consumed 10ml of S-3 without side effects. The compositions and formulations of the present invention may also be used to treat or disinfect inanimate or non-living objects, or be topically sprayed or applied to all types of plants, such as agricultural fruits, vegetables, grains, and the like, or be topically applied, ingested, or inhaled by any type of animal, such as livestock or humans.

In one aspect, the invention includes a method of preserving fruit. In one embodiment, the method comprises applying to the fruit an effective amount of a composition of the present invention. In one embodiment, the fruit is raspberry or grape.

In certain embodiments, the formulations of the present invention, and preferably S-3, can be used to disinfect corn for fermentation to alcohol.

Mastitis is an infection of cow udder tissue. Almost any bacterial or fungal organism that can opportunistically invade tissues and cause infection can cause mastitis. This represents one of the most important problems in dairy production. Most mastitis infections are caused by various species of streptococcus, staphylococcus and gram-negative corynebacteria, particularly lactose fermenting organisms of intestinal origin (commonly known as coliforms) and those including organisms such as escherichia coli and staphylococcus aureus (Staphlycoccus aureus). From an epidemic perspective, the source of the infection can be considered infectious or environmental, and dairy cows are under constant threat of being infected by these pathogens.

Infectious pathogens are spread by the hands of milking humans or the pads of milking devices during milking, in addition to mycoplasma which spread between cows by spray spread and invade the udder, subsequently causing bacteremia. The main bacterial species that take advantage of this mode of transmission include staphylococcus aureus, Streptococcus agalactiae (Streptococcus agalactiae) and Corynebacterium bovis (Corynebacterium bovis). Most other species are opportunistic invaders from the dairy cow environment, although some other streptococci and staphylococci also have infectious components.

Intra-mammary infections are commonly described as subclinical or clinical mastitis. Subclinical mastitis is the presence of an infection without significant signs of local inflammation or systemic involvement. Although transient episodes of abnormal milk production or mastitis may occur, these infections are asymptomatic for the majority of the population and are referred to as chronic if the infection persists for at least 2 months. Once established, many of these infections persist throughout lactation or throughout the life of the cow. The detection is best performed by examining the somatic cell count (mainly neutrophils) of milk using the California Mastitis Test (California mass Test) or an automated method provided by the dairy cattle improvement institution (dairy herd improvement organization). Somatic cell counts were positively correlated with the presence of infection. Although variable (especially if determined by a single assay), cows with a somatic cell count of 2:280,000 cells/mL (2: linear score of 5) have a chance of being infected > 80%. Likewise, the higher the somatic cell count in the bulk milk tank, the higher the prevalence of infection in the herd. The pathogenic bacteria must be identified by bacterial culture of milk.

Clinical mastitis is an inflammatory response to an infection that causes significant abnormal milk (e.g., color, fibrin clots). As the degree of inflammation increases, changes in the breast (swelling, heat, pain, redness) are also evident. Clinical cases that include only local signs are referred to as mild or moderate. A case is said to be severe if the inflammatory response includes a systemic involvement (fever, anorexia, shock). If the onset is very rapid, it is called an acute case of severe mastitis because it usually occurs in severe clinical cases. More severely affected cows tend to have more serous secretions in the affected cowshed.

Although any number of cowsheds may be infected with subclinical mastitis at the same time, typically only one cowshed will exhibit clinical mastitis at a time. However, it is not uncommon for a clinical episode caused by Mycoplasma (Mycoplasma) to affect multiple cowsheds. Gangrenous mastitis can also occur, particularly when subclinical, chronic infections with staphylococcus aureus become severe at the time of immunosuppression (e.g., at parturition). In the case of subclinical mastitis, culturing milk samples taken from affected cowsheds is the only reliable method of determining the etiology of clinical cases.

All dairy herds had cows with mastitis; however, the prevalence of infected cows is 15-75% and cowsheds 5-40%. Many different pathogenic bacteria can establish a chronic infection that will only occasionally show clinical signs of mastitis. The main focus of most subclinical mastitis regimens is to reduce the prevalence of the infectious pathogens Streptococcus agalactiae and staphylococcus aureus, as well as other gram-positive cocci, most notably Streptococcus dysgalactiae (which may also be infectious or environmental pathogens), Streptococcus uberis (Streptococcus uberis), enterococcus, and various other coagulase-negative staphylococci, including staphylococcus suis (S hyicus), staphylococcus epidermidis (S epidermidis), staphylococcus xylosus (S xylosus) and staphylococcus intermedius.

For infectious pathogens, adult lactating cows are mostly at risk of infection during the lactation or dry period. The major site of infection is the mammary gland; transmission occurs when milking is done with a milker's hands or milking equipment as a contaminant. Initial heifers have been reported to be infected with staphylococci and streptococci prior to calving, although prevalence rates vary greatly between herds and geographic locations. Teat-end dermatitis (Teat-end dermatitides) caused by horn flies (Haematobia irritans) carrying Staphylococcus aureus is associated with an increased risk of infection of heifers, especially in summer climates.

A common treatment involves the use of antibiotics, which pose a threat to the milk obtained from the animal, as the antibiotics will enter the udder. Milk cannot be used at least 3 days after the antibiotic is administered. The use of immunization is not possible because of the large number of possible pathogenic bacteria involved in mastitis diseases. The general recommendation is a hygienic practice to enhance cleanliness in milking parlours and in areas where animals frequently come in and go out. Currently, no available treatment has been found to be both effective and safe for treating mastitis.

In one aspect, the invention includes a method of treating an animal having a disease or condition associated with a microbial infection. In one embodiment, the method comprises administering to the animal an effective amount of a composition of the invention. In another embodiment, the method comprises administering to the animal an effective amount of a composition comprising an organic acid. Such diseases and conditions may include, but are not limited to, diarrheal diseases such as white diarrhea (scouts), food poisoning, or gastrointestinal colds; or an inflammatory infection, such as subclinical or clinical mastitis. It should be further understood that the formulations and compositions of the present invention are not limited to treating any particular type of subject. As contemplated herein, a subject may be any animal, preferably a mammal, and more preferably a livestock animal, such as a cow, sheep, or pig, or even a human. In one embodiment, the animal is a bovine, porcine, or ovine. In another embodiment, the animal is a human.

In another aspect, the invention includes a method of treating cows suffering from white diarrhea. In one embodiment, the method comprises administering to the cow an effective amount of a composition of the invention.

In another aspect, the invention includes a method of treating swine suffering from white diarrhea. In one embodiment, the method comprises administering to the pig an effective amount of a composition of the invention.

In another aspect, the invention includes a method of treating a cow suffering from mastitis. In one embodiment, the method comprises administering to the cow an effective amount of a composition of the invention.

In another aspect, the invention includes a method of treating a sheep having mastitis. In one embodiment, the method comprises administering to the sheep an effective amount of a composition of the invention.

In another aspect, the invention includes a method of treating a person suffering from a diarrhea disorder. In one embodiment, the method comprises administering to the human an effective amount of a composition of the invention. In one embodiment, the diarrheal disease is food poisoning or gastrointestinal cold.

Combination therapy

The compositions of the present invention are contemplated for use in combination with one or more additional compounds. In a non-limiting example, the compositions of the present invention can be used in combination with one or more therapeutic agents (or salts, solvents and substances or prodrugs thereof). Non-limiting examples of therapeutic agents include antibiotics such as bytecril (Baytril), sulfonamides, newfolo (Nuflor), tylosin 40-50(Tylan40-50), excelde, Noromycin LA, tulathromycin (Draxxin), and tetracycline; vaccines, such as Inforce 3; a vitamin complex; a probiotic; and toxin absorbing agents such as Toxiban; or other therapeutic agents such as Suprio.

In another embodiment, the compositions of the present invention may be used in combination with a cleaning agent. In one embodiment, the cleansing agent acts as a solubilizing agent for the composition while removing harmful bacterial-laden debris from the affected area of the subject and any other possible sources of infection such as bedding, tools, or the place where the subject resides. In non-limiting examples, the compositions of the present invention are suitable for use in the treatment of udder, bedding for the rearing of cattle that is a major source of environmental pathogens, and tools for milking procedures that have been identified as potential sources of infection, such as contaminated nipple drop (teat dip), intra-mammary infusion, water lines for breast preparation during milking, pools or mud holes, skin lesions, nipple wounds, and flies. Non-limiting examples of detergents include Sucragel CF, Chemoxide CAW, BioSoft D40, Lathanol LAL, BioTerge AS-40, Nacconol 90G, and potassium cocoate.

Pharmaceutical compositions and therapies

The application of the compositions suitable for use in the present invention can be accomplished in a number of different ways using methods known in the art. The therapeutic and prophylactic methods of the invention thus encompass the practice of the methods of the invention using pharmaceutical compositions comprising compositions useful in the invention. Pharmaceutical compositions suitable for practicing the invention can be administered to deliver a dose of 1 ng/kg/day to 100 mg/kg/day.

The relative amounts of the active ingredients, pharmaceutically acceptable carriers and any other ingredients in the pharmaceutical compositions of the invention will vary depending on the identity, size and condition of the subject being treated and further depending on the route of administration of the composition. For example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for prescribed administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to all kinds of animals. It will be readily appreciated that modifications to pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to a variety of animals may be made, and that ordinary skilled veterinary pharmacologists may design and perform such modifications using only ordinary experimentation, if any. Subjects to which the pharmaceutical compositions of the present invention are administered are contemplated to include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cows, pigs, horses, sheep, cats, and dogs.

Generally, the dosage that can be administered to an animal in the methods of the invention is preferably in an amount ranging from 0.5 μ g to about 50mg per kilogram of animal body weight. While the precise dose administered will vary depending on any number of factors, including but not limited to the type of animal and the type of disease state being treated, the age of the animal, and the route of administration, the dosage of the composition will preferably vary from about 1 μ g to about 10mg per kilogram of animal body weight. More preferably, the dosage will vary from about 3 μ g to about 1mg per kilogram of animal body weight.

Pharmaceutical compositions suitable for use in the methods of the present invention may be prepared, packaged or sold in a form suitable for oral administration, parenteral administration, topical administration, buccal administration or another route of administration. Other contemplated compositions include projected nanoparticles, liposomal formulations, red blood cells containing the release of the active ingredient, and immune-based compositions.

The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the pharmaceutical arts. Generally, such manufacturing processes include the steps of bringing into association the active ingredient with a pharmaceutically acceptable carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single or multiple dosage units.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in bulk in a single unit dosage form or in multiple single unit dosage forms. As used herein, a "unit dose" is an independent amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to the subject or a convenient fraction of such dose, such as, for example, one-half or one-third of such dose.

In one embodiment, the compositions of the present invention are formulated using one or more excipients or carriers that are acceptable for learning. In one embodiment, the pharmaceutical composition of the invention comprises a therapeutically effective amount of the composition of the invention and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, physiological saline, ethanol, and other pharmaceutically acceptable salt solutions, such as salts of phosphates and organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication co., New Jersey).

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of microbial activity can be achieved by a variety of antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferred to include isotonic agents, for example, sugars, sodium chloride or polyalcohols such as mannitol and sorbitol in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone.

The compositions may be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier materials suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral or any other suitable mode of administration known in the art. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliaries, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffers, coloring, flavoring and/or aromatizing substances, etc. They may also be mixed with other active agents (e.g., other analgesics) as desired.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: an excipient; a surfactant; a dispersant; an inert diluent; granulating and disintegrating agents; a binder; a lubricant; a sweetener; a flavoring agent; a colorant; a preservative; physiologically degradable compositions such as gelatin; an aqueous vehicle and a solvent; oily vehicles and solvents; a suspending agent; a dispersing or wetting agent; emulsifiers, demulcents; a buffering agent; salt; a thickener; a filler; an emulsifier; an antioxidant; (ii) an antibiotic; an antifungal agent; a stabilizer; and a pharmaceutically acceptable polymeric or hydrophobic material. Other "additional ingredients" that may be included in the Pharmaceutical compositions of the present invention are known in the art and are described in Genaro, eds (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference.

The pharmaceutical compositions of the present invention may comprise from about 0.005% to 2.0% by total weight of the composition of a preservative. Preservatives are used to prevent decay in the event of exposure to contaminants in the environment. Examples of preservatives suitable for use according to the present invention include, but are not limited to, those selected from the group consisting of: benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The pharmaceutical composition preferably comprises an antioxidant and a chelating agent that inhibits degradation of the formulation. Preferred antioxidants for some formulations are BHT, BHA, alpha-tocopherol, and ascorbic acid in the preferred range of about 0.01% to 0.3%, and more preferably BHT in the range of 0.03% to 0.1% by total weight of the composition. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by total weight of the composition. Particularly preferred chelating agents include edetate (e.g., disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by total weight of the composition. Chelating agents are useful for chelating metal ions in compositions that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are particularly preferred antioxidants and chelating agents, respectively, for some formulations, other suitable and equivalent antioxidants and chelating agents may be substituted therefor as will be known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily nature, ethanol, vegetable oils (such as arachis oil, olive oil, sesame oil or coconut oil), fractionated vegetable oils and mineral oils (such as liquid paraffin). Liquid suspensions may further include one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweetening agents. The oily suspension may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinyl pyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phospholipids, such as lecithin; condensation products of alkylene oxides with fatty acids, with long chain aliphatic alcohols, with partial esters derived from fatty acids and hexitol, or with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and gum arabic. Known preservatives include, but are not limited to, methylparaben, ethylparaben, n-propylparaben, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerin, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, with the primary difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, an "oily" liquid is a liquid that contains carbon-containing liquid molecules and exhibits less polar character than water. The liquid solutions of the pharmaceutical compositions of the present invention may comprise each of the components described in relation to the liquid suspensions, it being understood that suspending agents will not necessarily aid in the dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethanol, vegetable oils (such as arachis oil, olive oil, sesame oil or coconut oil), fractionated vegetable oils and mineral oils (such as liquid paraffin).

Powdered and granular formulations of the pharmaceutical formulations of the present invention can be prepared using known methods. Such formulations may be administered directly to a subject for, for example, forming tablets, filling capsules, or preparing aqueous or oily suspensions or solutions by adding aqueous or oily vehicles thereto. Each of these formulations may further comprise one or more of a dispersing or wetting agent, a suspending agent and a preservative. Additional excipients, such as fillers and sweeteners, flavoring or coloring agents, may also be included in these formulations.

The pharmaceutical compositions of the present invention may also be prepared, packaged or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil; mineral oils, such as liquid paraffin; or a combination of these. Such compositions may also comprise one or more emulsifying agents, such as naturally-occurring gums, for example gum arabic or tragacanth; naturally occurring phosphatides, such as soy bean or lecithin, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods of depositing or bonding the chemical compositions onto a surface, methods of incorporating the chemical compositions into the structure of the material during synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing aqueous or oily solutions or suspensions to an absorbent material, followed or not followed by drying.

In addition to the disclosure set forth elsewhere herein, controlled or sustained release formulations of the compositions of the present invention can be prepared using conventional techniques. In some cases, the dosage form to be used may be provided with sustained or controlled release of one or more of the active ingredients therein using, for example, hydroxypropylmethylcellulose (hydroxypropyl cellulose), other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes or microspheres, or combinations thereof, to provide the desired release profile in varying proportions. Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use in the compositions of the present invention.

Controlled release of the active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water or other physiological conditions or compounds. The term "controlled release component" in the context of the present invention is defined herein as one or more compounds including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, nanoparticles or nanospheres or combinations thereof that facilitate controlled release of the active ingredient.

Administration/administration

The administration regimen may affect what constitutes an effective amount. The therapeutic agent can be administered to the subject before or after diagnosis of the disease. In addition, several divided and staggered doses may be administered daily or sequentially, or the doses may be continuously infused, or may be bolus injections. In addition, the dosage of the therapeutic agent may be proportionally increased or decreased as indicated by the treatment or prevention of the emergency.

Administration of the compositions of the present invention to a subject (preferably a mammal, more preferably a human) can be carried out using known procedures at a dose and for a period of time effective to prevent or treat the disease. The effective amount of the therapeutic composition necessary to achieve a therapeutic effect can vary depending on factors such as the activity of the particular composition employed; the time of administration; the rate of excretion of the composition; the duration of the treatment; other drugs, compositions or materials used in combination with the composition; the state, age, sex, weight, condition, general health and prior medical history of the disease or condition of the subject being treated, and similar factors well known in the medical arts. The dosing regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Non-limiting examples of effective dosage ranges for the therapeutic compositions of the present invention are about 1 and 5,000mg/kg body weight/day. One of ordinary skill in the art will be able to study the relevant factors and make decisions regarding the effective amount of the therapeutic composition without undue experimentation.

The composition may be administered to the animal as frequently as several times per day, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of administration will be readily apparent to the skilled artisan and will depend on any number of factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like. The compositions of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the pharmaceutical arts. Generally, such methods of preparation include the steps of bringing into association the active ingredient with the carrier or one or more other auxiliary ingredients and then, if necessary or desired, shaping or packaging the product into the desired single or multiple dosage units.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied so as to obtain an amount, composition and mode of administration of the active ingredient effective to achieve the desired therapeutic response for a particular subject, without toxicity to the subject.

A physician, such as a physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of the composition of the invention employed in a pharmaceutical composition below a desired level in order to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

In particular embodiments, it is particularly advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined amount of the therapeutic composition calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical vehicle. The dosage unit forms of the invention are indicated by and directly dependent on the following: (a) the unique characteristics of the therapeutic composition and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of compounding/formulating such therapeutic compositions for the treatment of a disease in a subject.

In one embodiment, the compositions of the present invention are administered to a subject in a dose of from once to five times daily or more. In another embodiment, the compositions of the present invention are administered to a subject in a dosage range including, but not limited to, once daily, once every two days, once every three days to once a week, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the invention will vary between subjects depending on a number of factors including, but not limited to, age, the disease or condition to be treated, sex, general health and other factors. Thus, the invention should not be construed as limited to any particular dosage regimen and precise dosage, and the composition to be administered to any subject will be determined by the attending physician considering all other factors with respect to the subject.

The composition of the invention for administration may be in the following ranges: about 0.1mg to about 1,000mg, about 0.2mg to about 950mg, about 0.4mg to about 900mg, about 1mg to about 850mg, about 5mg to about 750mg, about 20mg to about 700mg, about 30mg to about 600mg, about 50mg to about 500mg, about 75mg to about 400mg, about 100mg to about 300mg, about 120mg to about 250mg, and any and all full or partial increments therein.

In some embodiments, the dosage of the compositions of the invention is from about 1mg to about 2,500 mg. In some embodiments, the dosage of the compositions of the invention for use in the compositions described herein is less than about 10,000mg, or less than about 8,000mg, or less than about 6,000mg, or less than about 5,000mg, or less than about 3,000mg, or less than about 2,000mg, or less than about 1,000mg, or less than about 500mg, or less than about 200mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second composition as described herein (i.e., the drug used to treat the same disease as the composition of the invention treats or another disease) is less than about 1,000mg, or less than about 800mg, or less than about 600mg, or less than about 500mg, or less than about 400mg, or less than about 300mg, or less than about 200mg, or less than about 100mg, or less than about 50mg, or less than about 40mg, or less than about 30mg, or less than about 25mg, or less than about 20mg, or less than about 15mg, or less than about 10mg, or less than about 5mg, or less than about 2mg, or less than about 1mg, or less than about 0.5mg and any and all full or partial increments thereof.

In one embodiment, the present invention relates to a packaged pharmaceutical composition comprising a container holding a pharmaceutically effective amount of the composition of the present invention alone or in combination with a second agent; and instructions for using the composition to treat, prevent, or reduce one or more symptoms of a disease in a subject.

Route of administration

Routes of administration of any of the compositions of the invention include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (buccal), (transurethral), vaginal (e.g., vaginal and perivaginal), (intra) nasal and (transrectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, soft capsules (gel caps), lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, syrups, dragees (lozenes), creams, pastes, plasters, lotions, crenellations (disco), suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like. It is to be understood that the formulations and compositions suitable for use in the present invention are not limited to the specific formulations and compositions described herein.

Oral administration

For oral administration, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and soft capsules. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, mouthrinses, or emulsions. Compositions intended for oral use may be prepared according to any method known to the art, and such compositions may contain one or more agents selected from inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents, such as corn starch; binders, such as starch; and lubricating agents, such as magnesium stearate.

The tablets may be uncoated or they may be coated by known methods to effect delayed disintegration in the gastrointestinal tract of a subject to provide sustained release and absorption of the active ingredient. For example, materials such as glyceryl monostearate or glyceryl distearate may be employed to coat the tablets. Additionally, for example, tablets can be prepared using U.S. Pat. nos. 4,256,108; 4,160,452, respectively; and 4,265,874 to form osmotic controlled release tablets. Tablets may also contain sweetening agents, flavoring agents, coloring agents, preserving agents or some combination of these in order to provide pharmaceutically elegant and palatable preparations.

Physiologically degradable compositions such as gelatin can be used to make hard capsules comprising the active ingredient. Such hard capsules comprise the active ingredient and may also comprise additional ingredients including, for example, inert diluents such as calcium carbonate, calcium phosphate or kaolin.

Physiologically degradable compositions such as gelatin can be used to prepare soft gelatin capsules containing the active ingredient. Such soft capsules comprise the active ingredient in admixture with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the compositions of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders; a filler; a lubricant; a disintegrant; or a humectant. If desired, the tablets may be coated using suitable methods and coating materials, such as OPADRY available from Colorcon, West Point, Pa.^TMFilm coating systems (e.g. OPADRY)^TMOY type, OYC type, organic enteric OY-P type, aqueous enteric OY-A type, OY-PM type and OPADRY type^TM White,32K18400)。

Liquid formulations for oral administration may be in the form of solutions, syrups or suspensions. Liquid formulations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl parabens or sorbic acid). Liquid formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged and sold in liquid form or in the form of a dry product intended to be reconstituted with water or another suitable vehicle prior to use.

Tablets containing the active ingredient may be prepared, for example, by compressing or molding the active ingredient optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing in a suitable apparatus the active ingredient in a free-flowing form such as a powder or granular formulation optionally mixed with one or more of a binder, lubricant, excipient, surfactant and dispersant. Molded tablets may be prepared by molding in a suitable apparatus a mixture of the active ingredient, the pharmaceutically acceptable carrier, and at least sufficient solution to wet the mixture. Pharmaceutically acceptable excipients used to make tablets include, but are not limited to, inert diluents, granulating or disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized corn starch, polyvinyl pyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, and talc.

Granulation techniques are well known in the pharmaceutical art for modifying the starting powder or other particulate material of an active ingredient. The powder is typically mixed with a binder material into larger permanent free-flowing aggregates or granules, known as "prilling". For example, a "wet" granulation process using a solvent is generally characterized by combining a powder with a binder material and wetting with water or an organic solvent under conditions that result in the formation of a wet granulation mass from which the solvent must evaporate.

Melt granulation is generally composed of a material that is solid or semi-solid at room temperature (i.e., has a relatively low softening or melting range) in use to facilitate granulation of the powder or other material substantially without the addition of water or other liquid solvent. When heated to a temperature within the melting point range, the low melting solid liquefies to act as a binder or granulation medium. The liquefied solid spreads itself over the surface of the powdery material with which it comes into contact and, on cooling, forms a solid particle mass in which the starting materials are bound together. The resulting molten granules are then provided to a tablet press or packaged for preparing oral dosage forms. Melt granulation improves the dissolution rate and bioavailability of the active ingredient (i.e., drug) by forming a solid dispersion or solution.

U.S. patent No. 5,169,645 discloses directly compressible wax-containing particles with improved flow characteristics. When the wax is mixed in the form of a melt with certain flow-improving additives, and then cooled and the mixture granulated, granules are obtained. In certain embodiments, only the wax itself is melted in the molten combination of the wax and the additive, and in other cases, both the wax and the additive will melt.

The invention also includes a multilayer tablet comprising a layer providing delayed release of one or more of the compositions of the invention and another layer providing immediate release of a drug for treating a disease. With the wax/pH sensitive polymer mixture, a gastric soluble composition can be obtained in which the active ingredient is embedded, ensuring its delayed release.

Parenteral administration

As used herein, "parenteral administration" of a pharmaceutical composition includes physical conduits characterized by the tissue of the subject and any route of administration by which the pharmaceutical composition is administered through a gap in the tissue. Thus parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, intratumoral, and renal dialysis infusion techniques.

Formulations of pharmaceutical compositions suitable for parenteral administration comprise the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable preparations may be prepared, packaged or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable formulations. Such formulations may further include one or more additional ingredients, including but not limited to suspending, stabilizing or dispersing agents. In one embodiment of the formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged or sold in the form of sterile injectable aqueous or oleaginous suspensions or solutions. Such suspensions or solutions may be formulated according to known techniques and may contain additional ingredients in addition to the active ingredient, such as dispersing, wetting or suspending agents as described herein. Such sterile injectable preparations may be prepared using non-toxic parenterally-acceptable diluents or solvents, for example, such as water or 1, 3-butanediol. Other acceptable diluents and solvents include, but are not limited to, ringer's solution, isotonic sodium chloride solution, and non-volatile oils such as synthetic mono-or diglycerides. Other parenterally administrable formulations suitable for use include those comprising active ingredients in microcrystalline form, in the form of liposomal formulations, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

Topical application

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for topical administration. There are a number of advantages to delivering compositions, including drugs or other therapeutic agents, to the skin (dermal drug delivery) or through the skin to the body (transdermal drug delivery). Transdermal composition delivery offers an attractive alternative to injections and oral drugs. Dermal composition delivery provides an effective means of delivering the composition to the skin of a mammal, and preferably a human, and provides a method of treating the skin, or otherwise affecting the skin without destroying or damaging the outer layers of the skin. In the present invention, dermal delivery of the compositions of the invention via dermal action provides these advantages for the treatment of skin-related disorders, conditions or diseases.

Many compounds, including some drugs, will penetrate the skin effectively and simply because the molecules are relatively small and potent at small doses of 0.1mg to 15 mg/day (Kanikkannin et al, 2000, Curr. Med. chem.7: 593-. Many other compounds and drugs can only be delivered if an additional enhancement system is provided to "force" them through the skin. Several methods of transdermal drug delivery are electroporation, sonophoresis, iontophoresis, penetration enhancers (cyclodextrins), and liposomes. Liposomes represent the preferred dermal delivery method, although the methods described above also include dermal delivery of the compositions used in the present invention.

The compositions of the present invention may consist of the individual active ingredients in a form suitable for administration to a subject, or the composition may comprise at least one active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. As is well known in the art, the active ingredient may be present in the composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion. It will also be understood that the compositions of the present invention encompass pharmaceutical compositions suitable for treating other disorders, conditions and diseases associated with the skin.

In one aspect, the dermal delivery vehicle of the present invention is a composition comprising at least one first compound that facilitates dermal delivery of at least one second compound associated with or in close physical proximity to the composition comprising the first compound. As the skilled artisan will appreciate when provided with the disclosure set forth herein, such delivery vehicles include, but are not limited to, liposomes, nanocapsules (nanosomes), phospholipid-based non-liposomal compositions (e.g., selected helical shells (cochleates)).

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid formulations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically applicable formulations may, for example, contain from about 0.001% to about 90% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

In one aspect of the invention, the dermal delivery system comprises a liposome delivery system, and the invention should not be construed as being limited to any particular liposome delivery system. Based on the disclosure set forth herein, the skilled artisan will appreciate how to identify liposome delivery systems suitable for use in the present invention.

The present invention also encompasses improving dermal and transdermal drug delivery through the use of permeation enhancers (also known as adsorption enhancers or accelerators) that permeate into the skin to reversibly reduce barrier resistance. For permeation enhancing activity, a number of compounds are known in the art, including sulfoxides (such as dimethyl sulfoxide, DMSO), azones (e.g., laurone), pyrrolidones (e.g., 2-pyrrolidone, 2P), alcohols and alkanols (ethanol or decanol), glycols (e.g., Propylene Glycol (PG), excipients commonly used in topical dosage forms), surfactants (also commonly used in dosage forms), and terpenes. Other enhancers include oleic acid, oleyl alcohol, ethoxyglycol, laurocapram, alkanecarboxylic acids, dimethyl sulfoxide, polar lipids or N-methyl-2-pyrrolidone.

In alternative embodiments, the topically active pharmaceutical or cosmetic composition may optionally be combined with other ingredients, such as humectants, cosmetic adjuvants, antioxidants, chelating agents, surfactants, foaming agents, conditioning agents, wetting agents, humectants, emulsifiers, fragrances, viscosity increasing agents, buffering agents, preservatives, sunscreens, and the like. In another embodiment, a permeation or penetration enhancer is included in the composition and is effective to improve transdermal penetration of the active ingredient into or through the stratum corneum relative to a composition lacking the permeation enhancer. A variety of permeation enhancers are known to those skilled in the art, including oleic acid, oleyl alcohol, ethoxyglycol, laurocapram, alkane carboxylic acids, dimethyl sulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

In another aspect, the composition may further comprise a water-solubilizing agent for increasing disorganization of the stratum corneum and thus allowing increased transport through the stratum corneum. A variety of water solubilizers are known to those skilled in the art, such as isopropanol, propylene glycol or sodium xylene sulfonate. The compositions of the present invention may also contain an active amount of retinoids (i.e., compounds that bind to any member of the retinoic acid receptor family), including, for example, tretinoin, retinol, esters of tretinoin and/or retinol, and the like.

The compositions of the present invention may comprise from about 0.005% to 2.0% by total weight of the composition of a preservative. Preservatives are used to prevent spoilage in the case of hydrogels upon exposure to environmental contaminants, such as exposure to the air or to the skin of a patient (including contact with the fingers used to apply the compositions of the present invention, such as therapeutic gels or creams), due to repeated patient use. Examples of preservatives suitable for use according to the present invention include, but are not limited to, those selected from the group consisting of: benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The composition preferably comprises an antioxidant and a chelating agent that inhibit degradation of the composition used in the present invention in the hydrogel formulation. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid, preferably in the range of about 0.01% to 5% and BHT in the range of 0.01% to 1% by total weight of the composition. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by total weight of the composition. Particularly preferred chelating agents include edetate (e.g., disodium edetate) and citric acid in the range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by weight of the total weight of the composition. Chelating agents are suitable for use with metal ions in chelating compositions that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are particularly preferred antioxidants and chelating agents for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefor as will be known to those skilled in the art.

Additional components may include, but should not be limited to, components including: water, oils (e.g., olive oil/PEG 7), aloe vera oil (biovera oil), waxes (e.g., jojoba wax), squalene, myristate (e.g., isopropyl myristate), triglycerides (e.g., caprylic triglyceride), Solulan 98, cocoa butter, shea butter (shea butter), alcohols (e.g., behenyl alcohol), stearates (e.g., glyceryl monostearate), chelating agents (e.g., EDTA), propylene glycol, SEPIGEL (Seppic, inc., Fairfield, NJ), silicones and silicone derivatives (e.g., dimethicone, cyclomethicone), vitamins (e.g., vitamin E), and the like.

Buccal administration

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for buccal administration. Such formulations may, for example, be in the form of tablets or dragees prepared using conventional methods and may, for example, be 0.1 to 20% (w/w) of the active ingredient(s), the remainder comprising the orally soluble or degradable composition and optionally one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder or an aerosolized solution or suspension comprising the active ingredient. Such powdered, atomized formulations, when dispersed, preferably have an average particle size or droplet size in the range of about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

Rectal administration

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for rectal administration. Such compositions may be, for example, suppositories, retention enema preparations and solutions for rectal or colonic lavage.

Suppository formulations may be prepared by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20 ℃) and liquid at the rectal temperature of the subject (i.e., about 37 ℃ in healthy humans). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols and various glycerides. The suppository formulation may further comprise a variety of additional ingredients including, but not limited to, antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonic lavage can be prepared by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using a delivery device that conforms to the rectal anatomy of the subject or may be packaged within such a delivery device. The enema preparation may further comprise a variety of additional ingredients including, but not limited to, antioxidants and preservatives.

Additional forms of administration

Additional dosage forms of the present invention include, for example, those described in U.S. Pat. nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837 and 5,007,790. Additional dosage forms of the invention also include dosage forms as described in U.S. patent application nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688 and 20020020051820. Additional dosage forms of the invention also include dosage forms as described in PCT application nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755 and WO 90/11757.

Controlled release formulations and drug delivery systems

Controlled or sustained release formulations of the pharmaceutical compositions of the invention can be prepared using conventional techniques using, for example, proteins having a pH sensitive domain or protease cleavable segment. In some cases, the dosage form to be used may be provided with sustained or controlled release of one or more of the active ingredients therein using, for example, hydroxypropylmethylcellulose (hydroxypropyl cellulose), other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes or microspheres, or combinations thereof, to provide the desired release profile in varying proportions. Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use in the compositions of the present invention. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps and caplets suitable for controlled release, are encompassed by the present invention.

Most controlled release drug products have a common goal of improving drug therapy relative to that achieved by their non-controlled counterparts. Ideally, the use of optimally designed controlled release formulations in medical treatment is characterized by the use of the least amount of drug substance to cure or control the condition in the least amount of time. Advantages of controlled release formulations include prolonged activity of the drug, reduced dosing frequency, and increased subject compliance. In addition, controlled release formulations can be used to affect the time of onset of action and other characteristics, such as blood levels of the drug, and thus can affect the occurrence of side effects.

Most controlled release formulations are designed to initially release an amount of drug that immediately produces the desired therapeutic effect, and gradually and continuously release other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug that is metabolized and excreted from the body.

Controlled release of the active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water or other physiological conditions or compounds. The term "controlled release component" in the context of the present invention is defined herein as one or more compounds including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or nanospheres or combinations thereof that facilitate controlled release of the active ingredient.

In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term release formulations, rapid-offset (rapid-offset) release formulations, and controlled release formulations, such as sustained release formulations, delayed release formulations, and pulsed release formulations.

The term sustained release is used in its conventional sense to refer to a pharmaceutical formulation that provides a gradual release of the drug over an extended period of time and, although not necessarily, may result in substantially constant blood levels of the drug over an extended period of time. This period of time can be as long as a month or more and should be a longer release than the same amount of agent administered as a bolus.

For sustained release, the composition can be formulated with suitable polymeric or hydrophobic materials that provide the composition with sustained release characteristics. Likewise, the compositions for use in the methods of the invention may be administered in particulate form, for example by injection or by implantation in the form of wafers or castellations.

In a preferred embodiment of the invention, the composition of the invention is administered to a subject alone or in combination with another agent using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a pharmaceutical formulation that provides for the initial release of the drug after some delay following administration of the drug, and may include delays of about 10 minutes up to about 12 hours, although not necessarily.

The term pulsatile release is used herein in its conventional sense to refer to pharmaceutical formulations that provide release of a drug in a manner that results in a pulsatile profile of the drug after administration of the drug.

The term immediate release is used in its conventional sense to refer to a pharmaceutical formulation that provides for release of the drug immediately after administration of the drug.

As used herein, short term refers to any period of time up to and including the following after drug administration: about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all full or partial increments thereof.

As used herein, a rapid-deviation refers to any period of time up to and including the following after administration of a drug: about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all full or partial increments thereof.

Thus, in one embodiment, the present invention relates to a chemical formulation comprising propanoic acid, isobutyric acid, at least one ester, and at least one carrier, wherein the chemical formulation has antibacterial activity when applied to human or animal feces. In another embodiment, the at least one ester is isoamyl butyrate. In another embodiment, at least one support is a silica-based support. In another embodiment, the at least one carrier is selected from bentonite, zeolite and perlite. In another embodiment, the ratio of propanoic acid to isobutryic acid to isoamyl butyrate is about 3.5:3.5:2 (v/v/v). In another embodiment, the ratio of propanoic acid, isobutyric acid, and isoamyl butyrate is about 7 parts of the two acids and 2 parts of isoamyl butyrate. In another embodiment, the chemical formulation consists essentially of propanoic acid, isobutryic acid, isoamyl butyrate, and a carrier. In another embodiment, the carrier is selected from bentonite, zeolite and perlite. In another embodiment, the chemical formulation has antibacterial activity when applied to human or animal feces.

In another embodiment, the present invention relates to a chemical formulation comprising propanoic acid, isobutryic acid, at least one ester, at least one carrier, and at least one fungus. In another embodiment, the at least one ester is isoamyl butyrate. In another embodiment, at least one support is a silica-based support. In another embodiment, the at least one carrier is selected from bentonite, zeolite and perlite. In another embodiment, the ratio of propanoic acid to isobutryic acid to isoamyl butyrate is about 3.5:3.5:2 (v/v/v). In another embodiment, the ratio of propanoic acid, isobutyric acid, and isoamyl butyrate is about 7 parts of the two acids and 2 parts of isoamyl butyrate. In another embodiment, at least one fungus is an endophyte. In another embodiment, the endophyte is fusarium. In another embodiment, the endophyte is fusarium oxysporum.

In another embodiment, the present invention relates to a chemical formulation comprising propionic acid and at least one ester of a 6-12 carbon (acid) component, wherein the ratio of propionic acid to ester component of the chemical formulation is about 7:2 (v/v). In another embodiment, the at least one ester is isoamyl hexanoates. In another embodiment, the formulation further comprises at least one nutritional supplement and at least one salt. In another embodiment, the formulation comprises glucose, whey protein, potassium chloride, magnesium sulfate, and sodium chloride. In another embodiment, the formulation comprises glucose, glycine, potassium chloride, sodium chloride, and magnesium acetate. In another embodiment, the formulation comprises glucose, glycine, potassium chloride, sodium chloride, magnesium acetate, and potassium dihydrogen phosphate. In another embodiment, the formulation comprises at least one pharmaceutically acceptable carrier. In another embodiment, the carrier is cremophor. In another embodiment, the formulation consists essentially of propionic acid and isoamyl hexanoates in a ratio of propionic acid to isoamyl hexanoates of about 7:2 (v/v). In another embodiment, the invention relates to a chemical formulation consisting essentially of propionic acid, isoamyl hexanoates, and a carrier.

In another embodiment, the invention relates to a chemical formulation comprising propionic acid and an ester of a single carbon (acid) component, wherein the ratio of propionic acid to ester component of the chemical formulation is about 7:2 (v/v). In another embodiment, the at least one ester is isoamyl formate. In another embodiment, the formulation consists essentially of propionic acid and isoamyl formate in a ratio of propionic acid to isoamyl formate of about 7:2 (v/v). In another embodiment, the formulation comprises at least one carrier. In another embodiment, at least one support is a silica-based support. In another embodiment, the at least one carrier is selected from bentonite, zeolite and perlite. In another embodiment, the chemical formulation consists essentially of propionic acid, isoamyl formate, and a carrier.

In another embodiment, the invention includes a chemical formulation comprising propanoic acid, isoamyl formate, and at least one fungus. In another embodiment, the ratio of propanoic acid to isoamyl formate is about 7:2 (v/v). In another embodiment, at least one fungus is an endophyte. In another embodiment, the endophyte is from fusarium. In another embodiment, the endophyte is fusarium oxysporum.

In another embodiment, the present invention relates to a method of treating human or animal waste comprising contacting human or animal waste with a composition comprising propanoic acid, isobutryic acid and at least one ester, wherein the composition kills or reduces bacterial growth on the human or animal waste. In another embodiment, the present invention relates to a method of treating human or animal waste comprising contacting human or animal waste with a composition comprising propanoic acid, isobutryic acid, at least one ester, and at least one fungus, wherein the propanoic acid, isobutryic acid, and at least one ester kill or reduce bacterial growth on the human or animal waste, and the at least one fungus increases the rate of decomposition of the human or animal waste. In another embodiment, the present invention relates to a method of eliminating or reducing microbial growth at a treatment site comprising contacting the treatment site with a composition comprising propionic acid and at least one ester having a ratio of propionic acid to ester of about 7:2, wherein the ester is isoamyl formate or isoamyl hexanoate, and the composition kills or reduces bacterial growth on human or animal feces. In another embodiment, the present invention relates to a method of treating human or animal feces comprising contacting the human or animal feces with a composition comprising propanoic acid, isoamyl formate in a ratio of propanoic acid to isoamyl formate of about 7:2, and at least one fungus, wherein the propanoic acid and isoamyl formate mixture kills or reduces microbial growth on the human or animal feces and the at least one fungus increases the rate of decomposition of the human or animal feces. In another embodiment, the invention relates to a method of treating an animal having a disease or condition associated with a microbial infection comprising administering to the animal an effective amount of a composition comprising at least one organic acid. In another embodiment, the composition consists essentially of an organic acid. In another embodiment, the composition consists of an organic acid. In another embodiment, the at least one organic acid is propionic acid. In another embodiment, the at least one organic acid is isobutyric acid. In another embodiment, the animal is a human. In another embodiment, the disease or condition is a diarrheal disease. In another embodiment, the animal is a bovine, porcine, or ovine. In another embodiment, the disease or condition is selected from diarrheal diseases and intramammary infections. In another embodiment, the diarrheal disease is white diarrhea. In another embodiment, the intramammary infection is subclinical mastitis or clinical mastitis. In another embodiment, the composition further comprises at least one ester. In another embodiment, the at least one ester is isoamyl hexanoates. In another embodiment, the composition further comprises at least one nutritional supplement and at least one salt. In another embodiment, the composition comprises glucose, whey protein, potassium chloride, magnesium sulfate, and sodium chloride. In another embodiment, the composition comprises glucose, glycine, potassium chloride, sodium chloride, and magnesium acetate. In another embodiment, the composition comprises glucose, glycine, potassium chloride, sodium chloride, magnesium acetate, and potassium dihydrogen phosphate. In another embodiment, the composition further comprises at least one pharmaceutically acceptable carrier. In another embodiment, the carrier is cremophor. In another embodiment, the composition comprises propionic acid and isoamyl hexanoates in a ratio of propionic acid to isoamyl hexanoates of about 7:2 (v/v). In another embodiment, the at least one ester is isoamyl hexanoates. In another embodiment, the ratio of propanoic acid to isobutyric acid to isoamyl hexanoates is about 3.5:3.5:2 (v/v/v). In another embodiment, the ratio of propanoic acid, isobutyric acid, and isoamyl hexanoates is about 7 parts of the two acids and 2 parts of isoamyl butyrate.

In another embodiment, the invention relates to a chemical formulation consisting essentially of propanoic acid, isobutryic acid, isoamyl hexanoates, and a carrier. In another embodiment, the carrier is selected from bentonite, zeolite and perlite. In another embodiment, the chemical formulation has antibacterial activity when applied to human or animal feces.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the invention and are encompassed by the following claims. For example, it is understood that reaction conditions (including but not limited to reaction time, reaction size/volume) and experimental reagents (such as solvents, catalysts), pressure, atmospheric conditions (e.g., nitrogen atmosphere) and reducing/oxidizing agents as well as art-recognized alternatives and modifications using only routine experimentation are within the scope of the present application.

It is to be understood that whatever values and ranges are provided herein, all values and ranges encompassed by such values and ranges are intended to be encompassed within the scope of the present invention. Moreover, all values falling within these ranges, as well as the upper and lower limits of the ranges of values, are also encompassed by the application.

The following examples further illustrate aspects of the invention. They are, however, by no means limiting of the teachings or disclosures of the invention set forth herein.

Experimental examples

The invention will now be described with reference to the following examples. These embodiments are provided for illustrative purposes only, and the present invention is not limited to these embodiments, but encompasses all variations apparent from the teachings provided herein.

Example 1

Discussed below are a number of experiments leading to the discovery of fusarium oxysporum and systems 1 and 2 that can be used in a variety of human and animal waste management devices, along with animal bedding, cots, and the like.

Table 1 shows the inhibitory activity of various esters (used in combination with a 1:1(v/v) mixture of propionic and isobutyric acids, and thus the ratio of the two acids to the esters is 7:2(v/v)) on a series of test fungi and bacteria that are common organisms for screening for antibiotic activity. From these data, system 1 and system 2 were selected for use in the present invention (see highlighted area on the table).

Table 2 summary of molecular genetic data for a number of new fusarium isolates grown on human faeces in the presence of system 2 mixture (with carrier bentonite or zeolite) as described above. From this it is evident that any of these organisms is almost identical to or in most cases superior to Fusarium flavum (P2-24, the subject of the earlier patent). The data set of P2-24 is also included.

Table 3. growth of various fusarium species on human feces during the 7 week experiment caused a reduction in the dry mass of the pellet. The experimental set-up contained 0.5g of bentonite on a water agar plate with human feces having a wet weight of about 100mg and a small agar plug with the test fusarium with system 2. The incubation period was 7 weeks at 22 ℃. The remainder of the human feces was physically removed and dried at 80 ℃ for 4 hours, and then weighed.

FIG. 1 indicates how the assays were performed to generate the data set in Table 1. Various esters were combined with a 1:1 mixture of propionic and isobutyric acids, and these were added at 7:2(V/V) with the ester to be tested. Then 9 μ l were placed in the central well with a plug of agar of a single test organism around it as shown in the figure.

Figure 2 demonstrates the efficacy of system 1 (above) in killing and inhibiting human fecal-associated bacteria. Fresh feces were collected and then approximately 5mg was spread evenly on the surface of a potato dextrose agar plate. Plates were incubated 2 and photographed after incubation. The right panel is a no treatment control with 0.5g bentonite without antibiotic in the middle and 0.5g bentonite with system 1 on the left.

Figure 3 demonstrates the efficacy of system 2 (above) in killing and inhibiting human fecal-related bacteria. Fresh feces were collected and then approximately 5mg was spread evenly on the surface of a potato dextrose agar plate. Plates were incubated 2 and photographed after incubation. The right panel is a no treatment control with 0.5g bentonite without antibiotic in the middle and 0.5g bentonite with system 2 on the left.

Figure 4 illustrates how odor can be eliminated by treatment with system 1. Two cat litter boxes with cat fecal material, each box having fecal material from about 140g of 5 different cats. The left box had been treated with system 1 on bentonite (0.5ml/100g bentonite). After 5 days the ammonia reading of the control (left) was 14ppm, while the right treated chamber was 0 ppm. The total odor in the treated cabinet was significantly reduced.

Figure 5 illustrates how fungi can grow on fresh human feces and reduce odor levels. Approximately 140g of human feces in the presence of urine were treated with Fusarium oxysporum 06-1 in the presence of system 2(1 ml on 10g zeolite). After 3 weeks there was a massive growth of fusarium oxysporum (white hyphae in the right container). The ammonia level in the left control was 71.4, while the ammonia level in the right treated vessel was 12.1.

FIG. 6 shows here the growth of a new isolate of a number of Fusarium species compared to the growth of Fusarium flavum (P-2-24) on human feces. Here is shown the progressive growth of fusarium over a period of many days on a small piece of human stool of approximately 100mg (fresh weight). The growth of newly isolated and characterized fusarium was each compared to P2-24 (fusarium flavum, which was the subject of a previous patent on this topic). The new Fusarium species, especially E06-1 and E06-5, did grow faster on feces. Growth was measured by the extent of hyphae removed from agar plugs placed on the fecal mass.

FIG. 7. upper panel-a six day old culture of Fusarium oxysporum E06-1 (a preferred fungus for treatment of human and animal feces in combination with System 2). Bottom panel-optical microscopic view of spores and hyphae of fusarium oxysporum. Spores were slightly curved and 9.8-12X 2.5. mu.

FIG. 8 Fusarium oxysporum (E06-8) grew in large numbers on human feces in the presence of system 2. Note the inhibition of bacterial growth on the right side of the culture plate, which is affected by the vapors emanating from the system 2 allowing fungal growth from the bentonite particles on the left side of the plate. 0.5g of treated bentonite was added, approximately 100mg of human feces and plate were incubated for 12 days. See figure 6 for comparative growth measurements.

Method 1-Experimental procedure for isolating Fusarium endophytes

Isolates of Fusarium can be collected according to standard protocols understood by those skilled in the art. Briefly, shoots (twig pieces) were soaked in 70% aqueous ethanol sufficiently for surface disinfection and then the skin/epidermis was removed with a sterile scalpel. Pieces of endothelium were aseptically transferred to the surface of Water Agar (WA) and glycerol-arginine medium (GAM). After several days of incubation at 25 ℃, the tips of the developing fungi mycelia should be removed aseptically and placed on Potato Dextrose Agar (PDA). In this way a pure fungal culture is obtained. In particular, isolates having pink to reddish staining and having fusarium spores are likely to be fusarium endophytes. Further characterization can be performed by molecular techniques, as understood by those skilled in the art. This procedure was used to find each organism used and described herein.

Table 1 shows the inhibitory and killing effects of propionic acid and isobutyric acid together and alone, as well as with various esters. These tests were carried out at 22 ℃ over a period of 30 hours. Appropriate controls were measured and therefore percent inhibition of growth on the treated versus control organisms (untreated) can be calculated. After 30 hours the bacterial and fungal-like organisms were evaluated according to the relative growth rate. Those highlighted areas on the table show those compounds (esters) that have the greatest compatibility with the 1:1(v/v) mixture of propionic and isobutyric acids and the appropriate esters (systems 1 and 2 above) at a 7:2 ratio. From this test, system 1 and system 2 were found. In the plate assay, acid was added alone at 7 μ l, while the combination of ester and acid was added at a level of 9 μ l.

TABLE 1 test organisms

Note that: when no growth or 100% inhibition was observed, the organism died and failed to revitalize.

Table 2 shows a description of the molecular genetic data (below) obtained on new isolates of fusarium tested for their ability to degrade human feces. Each of these isolates is so named under the heading. Details of data acquisition are provided at the end of the table.

Table 2.

E06-05 Fusarium oxysporum

Sequence (480 bases):

AACATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCAGCTTGGTGTTGGGACTCGCGAGTCAAATCGCGTTCCCCAAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTAAAACCCTCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAA(SEQ ID NO:1)

NCBI BLAST matching:

e06-08 Fusarium oxysporum

Sequence (478 bases):

CATACCAATTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCAGCTTGGTGTTGGGACTCGCGAGTCAAATCGCGTTCCCCAAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTAAAACCCTCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAA(SEQ ID NO:2)

NCBI BLAST matching:

fusarium E4-5

Sequence (488 bases):

CTTAATGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAACGGGACGGCCCGCCAGAGGACCCAAACTCTAATGTTTCTTATTGTAACTTCTGAGTAAAACAAACAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCTGGTATTCCGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCCCGGGTTTGGTGTTGGGGATCGGCTCTGCCTTCTGGCGGTGCCGCCCCCGAAATACATTGGCGGTCTCGCTGCAGCCTCCATTGCGTAGTAGCTAACACCTCGCAACTGGAACGCGGCGCGGCCATGCCGTAAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGT AGGAATACCCGCTGAACTTAAGCATATCAATAG(SEQ ID NO:3)

NCBI BLAST matching:

PC-2-24 (control) Fusarium flavum

Sequence (477 bases):

CATACCTTATGTTGCCTCGGCGGATCAGCCCGCGCCCCGTAAAAAGGGACGGCCCGCCGCAGGAACCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTATAAAAAACAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCCAGCTTGGTGTTGGGAGCTGCAGTCCTGCTGCACTCCCCAAATACATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAATTTACATATCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAG(SEQ ID NO:4)

NCBI BLAST matching:

e30-14 Fusarium avenaceum

Sequence (485 bases):

CAGAAGTTGGGGTTTTACGGCATGGCCGCGCCGCGTTCCAGTTGCGAGGTGTTAGCTACTACGCAATGGAGGCTGCAGCGAGACCGCCAATGTATTTCGGGGGCGGCACCGCCAGAAGGCAGAGCCGATCCCCAACACCAAACCCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCGGAATACCAGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTGTTTGTTTTACTCAGAAGTTACAATAAGAAACATTAGAGTTTGGGTCCTCTGGCGGGCCGTCCCGTTTTACGGGGCGCGGGCTGATCCGCCGAGGCAACATTAAGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCA(SEQ ID NO:5)

NCBI BLAST matching:

e06-7 Fusarium oxysporum

Sequence (469 bases):

CAGAAGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAACGAGGGTTTTACTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGGGGAACGCGATTTGACTCGCGAGTCCCAACACCAAGCTGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGTCCGCCGAGGCAACAATTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGC(SEQ ID NO:6)

NCBI BLAST matching:

e-30-7 Fusarium avenaceum

Sequence (480 bases):

GAAGTTGGGGTTTTACGGCATGGCCGCGCCGCGTTCCAGTTGCGAGGTGTTAGCTACTACGCAATGGAGGCTGCAGCGAGACCGCCAATGTATTTCGGGGGCGGCACCGCCAGAAGGCAGAGCCGATCCCCAACACCAAACCCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCGGAATACCAGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTGTTTGTTTTACTCAGAAGTTACAATAAGAAACATTAGAGTTTGGGTCCTCTGGCGGGCCGTCCCGTTTTACGGGGCGCGGGCTGATCCGCCGAGGCAACATTAAGGTATGTTCACAGGGGTTTGGGA GTTGTAAACTCGGTAATGATCCCTCC(SEQ ID NO:7)

NCBI BLAST matching:

e06-01 Fusarium oxysporum

Sequence (468 bases):

GAAGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAACGAGGGTTTTACTACTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGGGGAACGCGATTTGACTCGCGAGTCCCAACACCAAGCTGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAATTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATG ATCCCTCCGCA(SEQ ID NO:8)

NCBI BLAST matching:

ITS-based phylogenetic analysis

Phylogenetic analysis of these organisms was performed by obtaining the ITS-5.8S ribosomal gene sequences. The fungi were grown on PDA for 7 days and DNA templates were prepared by using a Prepman Ultra Sample Preparation Reagent (Applied Biosystems, USA) according to the manufacturer's instructions. The ITS regions of the fungus were amplified using Polymerase Chain Reaction (PCR) with the universal ITS primers ITS1(5 'TCCGTAGGTGAACCTGCGG 3'; SEQ ID NO:9) and ITS4(5 'TCCTCCGCTTATTGATATGC 3'; SEQ ID NO: 10). The PCR conditions used were as follows: initial denaturation at 94 ℃ for 3 min, followed by 30 cycles of 94 ℃ for 15 sec, 50 ℃ for 30 sec, 72 ℃ for 45 sec, and final extension at 72 ℃ for 5 min. A50. mu.l reaction mixture contained 1 х PCR buffer, 200 dNTPs each, 1.5mM MgCl₂10pmol of each primer, 1-5 ng of DNA and 2.5U of Taq DNA polymerase. The amplification products (5. mu.l) were visualized on a 1% (w/v) agarose gel to confirm the presence of a single amplification band. The amplification products were purified by Amicon Ultra column (Millipore, USA) and 20-40ng were used for 10. mu.l sequencing reactions using the Big Dye Terminator sequencing kit (Big Dye Terminator sequencing kit) (v.3.1) with 2 picomoles of forward or reverse primer in the cycle sequencing reaction. Twenty cycles of 10s at 96 ℃, 5s at 50 ℃ and 4 minutes at 60 ℃ were performed and the extension product was purified by ethanol precipitation, dissolved in 10. mu.l of HiDi formamide, incubated for 1 minute at 95 ℃ and then loaded onto an ABI Prism 377 genetic Analyzer (Perkin-El)mer, USA). All reagents used for sequencing were from Applied Biosystems, USA. The amplified products were sequenced and aligned to sequences in GenBank by the BLASTN program (Altschul et al 1997). Sequencing was performed in U Calif, Berkeley.

Table 3 shows that the growth of various fusarium species on human feces causes a reduction in the dry mass of the pellet during the 7 week experiment. The experimental set-up contained 0.5g of bentonite on a water agar plate with human feces having a wet weight of about 100mg and a small agar plug with the test fusarium with system 2. The incubation period was 7 weeks at 22 ℃. The remainder of the human feces was physically removed and dried at 80 ℃ for 4 hours, and then weighed.

Table 3.

Fusarium isolate name	Dry weight of human feces remaining after 7 weeks. mg of
		control-Fusarium free	43
EC-4-5	14
		E06-7	23
E 06-7	25
		P2-24 Fusarium flavum primary control	24
E30-2	18
		E 30-7	27
E 06-1	16
		E 30-14	15
E06-8	14

Example 2

Establishment of a mixture of S-3 and S-4

Tests were performed in a similar manner as described for systems 1 and 2 above (fig. 9) to determine the biological activity of various test mixtures against a panel of test microorganisms. A small plug of each organism was placed around the PDA plate. The test solution in the plastic cup holder was placed in the central well. Control plates were also set up (A). After 30 hours, the growth of the test organisms was compared with the growth of the control and the percentage inhibition was calculated. (B) The plates contained the test mixtures. The measurement was performed 30 hours after the plate was set up.

It should be noted that the system 1 and 2 mixtures described herein contain about 3.5 parts propionic acid and 3.5 parts isobutyric acid and a final second ester, isoamyl butyrate (system 1) or isoamyl isobutyrate (system 2). It will be appreciated that while these mixtures are effective in a variety of applications, there may be other mixtures that are even more effective depending on the range of their biological activity, their utility and efficacy at their low doses. For this purpose, studies were carried out using standard propionic acid as starting point while omitting isobutyric acid (because of its pungent odor) and now incorporating larger molecular weight esters as ester components. Relatively surprisingly and unexpectedly, it was found that the use of parts of propionic acid (7) and parts of Isoamyl hexanoates (Isoamyl Hexonate) (2) produced a volatile mixture with biological activity exceeding that of B-23 (see below) and S-1, as shown in Table 4. This new mixture is designated system 3 and the formulation comprising propionic acid (7) and formate (2) is designated system 4. It should be noted that system 4 was less active than system 3 against most of the tested organisms, but system 4 did kill e.coli while allowing fusarium growth. For this purpose, system 4 together with fusarium is an effective mixture for use as a human faecal management agent.

Table 4. effect of various esters and propionic acids on growth of test organisms measured at room temperature for 30 hours. The effect is expressed as percent inhibition of growth when compared directly to the uninoculated control. The measurement (average of two) was measured as the growth of hyphae from the edge of the inoculated plugs.

Activity against yeasts and bacteria

Is as grown

Moderate to some growth

No growth

S-3 testing was performed at room temperature for 30 hours, then measured and photographed. Measurements were taken from the edge of the inoculum cake to the edge of the colony. Two measurements were made and then averaged. The test was performed at room temperature. The results show that S-3 is the most biologically active mixture in the tested solutions. It is also noted that S3-inhibits Erwinia carotovora (Erwinia carotovora): 80-90% and inhibits Lactobacillus about 50%.

The B-23 formulation tested was as follows: 1.39 parts of acetaldehyde; 2.83 parts of 2-butanone; 30.56 parts of propionic acid, 2-methyl-, methyl ester; 2.29 parts of acetic acid, 2-methylpropyl ester; 1.09 parts of propionic acid, 2-methyl-, 2-methylpropyl ester; 1.78 parts of 1-propanol, 2-methyl-; 1.51 parts of 2-butenal, 2-methyl-, (E) -; 4.79 parts of 1-butanol, 3-methyl-acetate; 4.78 parts of propionic acid, 2-methyl-2-methylbutyl ester; 5.38 parts of 1-butanol and 3-methyl-; 351.18 parts of propionic acid and 2-methyl-; 1.31 parts of acetic acid and 2-phenylethyl ester.

It should be noted that the S-3 mixture had 100% inhibition of many organisms tested (Table 4). The effect is greater than with isoamyl hexanoate or propionic acid alone. Thus in some cases it appears to be strongly synergistic, i.e. 66% inhibition of sclerotinia sclerotiorum (sclerotiorum) with acetate, 0% with propionic acid and 100% when the two are combined. Some other organisms also react in a similar manner, such as rhizoctonia solani. In addition, it appears that S-3 is more active than S-1 as well as B-23 and of course isoamyl hexanoates or propionic acid alone (Table 4). Other combinations of propionic acid and other esters or combinations of esters and terpenes such as eucalyptol or valencene are not as effective (table 4). The S-4 preparation, although not having the same activity as S-3, does not exert such a large effect on fusarium, but is inhibitory to other microorganisms, and therefore it may be most suitable for use in combination with fusarium as a useful agent for treating human feces. In the test, S-3 did kill both bacterial test organisms (Table 4). S-3 also affected Erwinia and Lactobacillus (Table 4).

Establishment of appropriate ratios of S-3 Components

The mixture of propionic acid and isoamyl hexanoates was varied and subsequently tested according to the procedure outlined above. The most advantageous mixture proved to be the two components in a ratio of 7:2 (table 5). All other combinations gave lower inhibition values (table 5). Addition of terpenes such as valencene does not promote biological activity. Thus, for practical applications, a ratio of 7:2(v/v) of the two components is most preferred.

Table 5. effect of various ratios of propionic acid to isoamyl hexanoates on a panel of test organisms. All tests were performed as described in table 4.

Is as grown

Moderate to some growth

No growth

C. Testing of other esters with propionic acid

Alternative ester to propionic acid mixtures with an ester ratio of 7:2 were tested as shown in table 6 below. These formulations are in addition to formulations S-1, S-2, S-3 and S-4 and thus form part of the formulations of the present invention. It is also to be understood that the present invention may include a combination of multiple esters or any of the esters described throughout this document along with propionic acid, preferably in a ratio of 7:2 propionic acid to ester mixture.

TABLE 6 testing of esters

Is unavailable to living body

	F. Fusarium solani	G. Trichoderma viride	H. Rhizoctonia solani	I. Aspergillus flavus
					Propionic acid and isoamyl benzoate (7:2)	0	77	74	92
Propionic acid and isoamyl phenylacetate (7:2)	0	60	54	92
					Propionic acid and isoamyl cinnamate	74	66	64	96
Propionic acid and isoamyl octanoate (7:2)	66	64	62	96
					Propionic acid and isoamyl salicylate (7:2)	0	64	43	92
Propionic acid and isoamyl laurate (7:2)	0	47	62	20

Is as grown

Moderate to some growth

No growth

All tests were performed according to the method of Table 4

Example 3

Corn clean-up test

Fermenting corn to produce ethanol. It is ground, heated to a paste and treated with enzymes, then yeast cells are added to make the final formulation. One or more antibiotic agents which tend to inhibit other competing microorganisms which may contaminate the fermentation process are also added. As such antibiotics are being withdrawn from the market, other antimicrobial treatment processes are needed. To determine whether the S-3 formulation is efficacious against corn contaminating microorganisms, the following operations were performed:

about 5g of ground corn (cracked corn) was treated with 10ml of S-3 prepared as a 0% (control), 0.25%, 0.5% and 1% solution at 7:2(v/v) plus 10. mu.l of Triton-x 100 (per 10ml) for 1 hour. The treatment was continued for 1 hour and the product was semi-dried on paper towels to remove excess liquid. Approximately 2 grams of material was placed directly on the PDA plate and incubated for 2 days, then photographed. In another case, the crushed seeds were dried in a fume hood (hood) and then spread on a PDA.

The results demonstrate that treatment levels of 0.5% and 1.0% S-3 completely removed bacterial contamination of the crushed corn particles for 1 hour (fig. 10). When the corn particles were blotted dry and further dried and tested in the same manner, the results were nearly identical.

In summary, the results indicate that the S-3 solution can be used to decontaminate agricultural and food-based products and materials. This can be equally applied to instruments, equipment, garments and processing food for consumption.

Example 4

Human feces with Fusarium oxysporum were treated with S-4.

Tests on formulations S-3 and S-4 were conducted to understand their efficacy in treating human fecal bacteria. The test was carried out using 0.5g of bentonite in a ratio of 1ml of S-1, S-2, S-3 or S-4 or B-23 per 10g of bentonite. The control had no mixture of compounds. Barley seeds contaminated with fusarium oxysporum (the same as used before) were placed on PDA plates. The intermediate plate was streaked with a pure culture of E.coli obtained from human feces. Cover and seal with plastic wrap, then incubate for 3 days and take pictures. S-3 and S-4 kill E.coli, but with B-23 the effect is less. Fusarium grows in the presence of S-3 and better in the case of S-4. Coli was grown in the control and slightly in the presence of B-23, all as shown in FIG. 11. It should be noted that S-4 in bentonite results in complete killing of E.coli and Fusarium growth does occur. The same is true for the S-1 and S-3 treatments, but the Fusarium is more inhibited. Control B-23 was also effective in killing E.coli, but not to the same extent. The results show that S-4 is a good and reasonable human excrement treating agent for killing intestinal bacteria when used together with fusarium, so that human excrement substances are degraded.

Example 5

Treatment of animal faeces with S-3

S-3 was applied at a rate of 3ml per pound of zeolite. To test their efficacy in treating animal feces and controlling bacterial growth, the following experiments were conducted. The treated zeolite (0.5g) was placed in the central well of a PDA plate (cut). The plates have been completely streaked and covered with a suspension of bacterial cells produced by chicken, goat, cat and horse manure. Plates were covered and sealed with preservative film and then incubated for 3-4 days before observation and photographing. The results in all cases show that S-3 is an effective antimicrobial mixture by virtue of the zone of inhibition created on the plate. This effect was also observed in the case of horse, goat and cat fecal matter bacteria spread on a flat plate (fig. 12). The results indicate that the S-3 zeolite combination has potential for use as a cat litter treatment agent or a coop treatment agent or as an animal litter application.

Example 6

S-3 was further tested for efficacy as a cat litter treatment and as an animal litter treatment.

Comparative efficacy test of cat mat litter treatment agent

A 1 square foot plastic snap-on container was filled with the desired nesting treatment plus untreated bentonite in the proportions indicated by the package insert (fig. 13). For these tests, the CLOE (used at a rate of 4ml per pound) and untreated bentonite control were tested. A constant temperature is maintained in the test facility for the duration of the test (e.g., 70 ° f). Approximately 50g of cat feces and 5mL of urine were added to the treated container. The ammonia level in each tank was measured using a Z-800 ammonia meter at 24 hour intervals. The measurements were made by placing the meter into each bin at respective 5 minute intervals, thereby minimizing the amount of time it takes for the container to open. The ammonia meter produced an average ammonia level over 5 minutes and a peak ammonia level reached over the course of 5 minutes. After the measurement was completed, 1 day old fecal material was removed and about 50g of fresh fecal material and 5mL of urine were added. The container is sealed again. These steps were continued daily for 1 week to determine the relative efficacy of the product. Ammonia readings were taken once per vessel at one hour intervals.

After 8 days, the average ammonia level readings taken every 24 hours at 5 minute intervals showed much lower ammonia production on the CLOE-treated bedding compared to the untreated control (see fig. 14A and 14B).

Example 7

Microbiological analysis

Microbiological studies were performed to demonstrate that the process of odor formation is directly related to the microbial activity in feces and the feces environment. Inhibition or control of microbial activity should have a beneficial effect on reducing odor. The CLOE had a direct effect on this microbial activity rather than improving it by absorbing odors as other cat litter treatment products do. To demonstrate this property, approximately 0.5g of CLOE was placed in the central well of a Potato Dextrose Agar (PDA) plate, which had been covered with a bacterial lawn from fresh solid cat feces. Plates were incubated at 23 ℃ for 1 week and photographed. The series of photographs shown in fig. 15A and 15B indicate that the bentonite control (15A) has a large number of bacterial colonies growing throughout the plate, including in those areas adjacent to the wells containing the bedding material. In contrast, the CLOE treatment (4 ml S3, 15B per pound of carrier) had few bacterial colonies around the wells of the plate, but it did support some growth of penicillium, which is inherent to the carrier material and does not contribute to odor production. Note also penicillium in control wells.

Clearly, the antimicrobial activity of the CLOE product is directly related to its efficacy as a cat litter treatment agent and its ability to reduce ammonia and other odors emanating from animal (especially cat) feces, as evidenced by many users. This is also demonstrated by the ability of the product to inhibit and kill fecal related microorganisms such as E.coli. The CLOE formulation is absorbed by the carrier material, but it is also slowly released over time and may equally effectively act at a distance from a point source of bentonite or zeolite carrier particles.

Example 8

Comparative efficacy test of chicken litter improver

A 1 square foot plastic snap-on container was filled with pine wood shavings and the desired dunnage treatment in the proportions indicated by the package specifications (fig. 16). For these tests, Barnyard padding and untreated zeolite controls at a rate of 15ml per pound of zeolite were tested, as these products are the more popular items in the market. A constant temperature is maintained in the test facility for the duration of the test (e.g., 70 ° f). About 50g of chicken droppings were added to the treated container and dispersed with about 2mL of water. The ammonia level in each tank was measured using a Z-800 ammonia meter at 24 hour intervals. The measurements were made by placing an ammonia meter into each tank at respective 5 minute intervals, thereby minimizing the amount of time it takes for the container to open. The ammonia meter produced an average ammonia level over 5 minutes and a peak ammonia level reached over the course of 5 minutes. After the measurement was completed each day, about 50g of fresh fecal material and 2mL of water were added and the container was sealed again. These steps were continued for 3 weeks to determine the relative efficacy of the formulations. Ammonia readings were taken once per vessel at one hour intervals.

After 8 days, the average ammonia level readings taken every 24 hours at 5 minute intervals showed the highest ammonia production on the zeolite control and the lowest on the baryard Bedding treated litter (fig. 17A and 17B).

Example 9

Comparative efficacy test of nest pad material improver for large animal litter

A 1 square foot plastic snap-on container was filled with pine wood shavings (a litter commonly used for large animals) and the desired litter treating agent (fig. 18) in the proportions indicated by the package insert. For these tests Barnyard padding and untreated zeolite controls were tested. A constant temperature is maintained in the test facility for the duration of the test (e.g., 70 ° f). About 100g of fresh horse manure and about 10mL of urine were added to the treated container. After 24 hours, the Z-800 ammonia meter was placed in each tank and measurements were taken at respective 5 minute intervals to minimize the amount of time the container was opened. The ammonia meter produced an average ammonia level over 5 minutes and a peak ammonia level reached over the course of 5 minutes. After the measurement was completed, the 1 day old manure and the urine-soaked pine bedding were removed. The recommended ratio of the litter treatment for wet contaminants (wet spots) plus about 100g of fresh manure and about 10mL of urine was added and each container was resealed. These steps were continued for 1 week to determine the relative efficacy of the product. Ammonia readings were taken once per vessel at one hour intervals.

After 8 days, the average ammonia level readings taken every 24 hours at 5 minute intervals showed the highest ammonia production on the control litter and the lowest on the Barnyard padding treated litter (fig. 19A and 19B).

Example 10

Formula S-3 for treating white scour of calves

Calf white dysentery is a disease caused by viral and bacterial infection of calf. In some cases, diarrhea can occur in up to 70% of calves in a herd and result in the death of 50% of infected calves. Although these events have a viral etiology, the most common cause is one of the more pathogenic bacterial strains of E.coli, followed by strains of the genera Cryptosporidia (Cryptosporidia) and Salmonella (Salmonella).

S-3 Minimal Inhibitory Concentration (MIC) for E.coli < 0.00125%. See also table 5 above. Thus, 50ml of about 1% S-3 solution is effective in treating calves suffering from white diarrhea. Solutions (S-X) were prepared for testing of calves diagnosed with classic white diarrhea symptoms.

The S-X formula contains the following components:

per 100ml:

1g glucose

1g whey protein

0.25g KCl

0.25g MgSO₄

0.5g NaCl

1ml S-3

Glucose and whey protein are added to provide a nutritional supplement to the treated animal, while other salts are added to enhance the electrolyte balance of the animal. The S-3 component is present to inhibit and kill pathogenic bacteria.

The 1 st test was performed on 5 newborn Holstein calves without white diarrhea to see if the S-X solution was toxic or producing any side effects. 50ml of S-X solution was administered to each healthy calf, and after 1 day to several weeks, no adverse side effects, and in particular, no signs of chemical side effects or abnormal behavior, were observed.

Then arranged for in vivo animal testing with the S-X mixture at a pasture in Bozeman, Montana. Animals with white diarrhea (Angus rearing) were first reported in cold, humid climates several weeks ago. Each white diarrhea animal has all symptoms associated with this disease. The dose for each animal was set at 50ml per animal per treatment.

Fifteen calves identified as having white diarrhea were treated orally with 50ml of the solution through a tube. Thirteen calves that had been treated with 1 dose recovered overnight. Two animals required a second dose and recovered overnight after the second dose. Figure 20A shows one of the two white diarrhea calves (label 166) that had to receive a second 50ml S-X solution treatment (picture taken before S-X solution administration). Note the lower right corner with large piles of excreta and the head and ears drooping downward (fig. 20A). One day after the second treatment with S-X solution, the calves were able to ambulate without white diarrhea (FIG. 20B). The day after the second S-X treatment, the calves suckled by their mother.

No mortality was reported in this experiment. Pasture owners report that S-X solutions are far superior to all other treatments they have used to date. Thus, S-X solutions represent a safe, rapid and effective treatment for white diarrhea.

Example 11

Treatment of white scour of livestock

The formulation for treating white dysentery containing S-3 preparation, sugar, amino acids, sodium chloride, potassium chloride and magnesium acetate is developed. Many animals are treated for infectious white diarrhea (caused by pathogenic bacteria). Typically, if infectious white dysentery is involved, the stool is yellow to brown to somewhat green. Also, if the parasite causes white diarrhea, the fecal material contains blood, and this is evident. If non-infectious diarrhea (milky white dysentery) is involved, the fecal material is whitish. In this study, at least two animals had milky white diarrhea and did not recover. Also, it appears that one animal suffers from parasitic white diarrhea, and that it is not recovered. Essentially all other animals (suffering from viral or bacterial white diarrhea) do recover when given S-X treatment. Recovery occurs within 12-24 hours in about 90% of cases, with symptom recovery occurring within 3-4 hours. In a few cases, two days are required for recovery in case a second treatment is required. This is different from any other available treatment. The material is delivered orally via a gastric tube or syringe. Other treatments with antibiotics and nutrient electrolyte solutions, as in most cases treated with S-3, have sought to help the animal, but recovery is uncertain.

Exemplary formulations and treatment of white scour in calves

Per 90ml of water:

1g glucose

1g Glycine

0.5g NaCl

0.25g KCl

0.25g magnesium acetate

1ml S-3 containing 0.7ml propionic acid and 0.2ml isoamyl hexanoate.

Each animal was administered 50ml per treatment via syringe or gastric tube, and some re-treatment was necessary if the animals did not recover within 24 hours.

Exemplary formulations and treatment of white scour in piglets

Per 90ml of water:

1g glucose

1g Glycine

0.5g NaCl

0.25g KCl

0.25g magnesium acetate

0.1g KH₂PO₄

2ml S-3 containing 0.7ml propionic acid and 0.2ml isoamyl hexanoate.

1-2ml was administered per piglet via syringe.

Research on the treatment of S-X white dysentery

Case 1-pasture 1

Day 5 and 16 in 2014-one calf falls due to white diarrhea on day 5 and 6, and has been treated with Banomine and given two needles LA-2009 (anti-infective). The electrolyte solution was also administered daily at a dose of 1 pint; however, the animals did not recover and were associated with a loss of vitality for 9 to 10 days of chronic white diarrhea. S-X solution was delivered in the morning of 17 days 5 months and 50ml was orally administered to white diarrhea calves by syringe. A significant improvement in the condition was observed by the calves in the evening and a complete recovery in the morning of day 5 and 18. Calves did not show additional signs of white diarrhea from 5 months and 22 days 2014. These results demonstrate that almost all white diarrhea treatments are ineffective in this animal and that the convalescent phase is not reached until S-3 treatment is given.

Case 2-pasture 2

Ranch 2 experienced invasion of white diarrhea calves and death at the end of the winter in 2014. They are provided with an S-X technical solution and the rancher owner uses this solution to treat calves via a gastric tube. Three calves are treated in this way. These calves recovered from white diarrhea within 24 hours after treatment and were not administered other known drugs while the S-X treatment helped to recover. Sick animals were taken to the barn and photographed during their recovery. One of these animals is depicted in figure 21.

Case 3-pasture 3

The pasture 3 experiences a particularly cold winter season during the calving season, including high winds and extensive snowfall. These conditions make white diarrhea more prevalent in calves.

S-X treatment was administered to white diarrhea calves in the pasture from 4 months 2 days to 4 months 24 days in 2014. Eleven animals were treated with a 50ml dose using the gastric tube method. The results were successful, with nine doses; while one animal must be treated a second time and the other only three separate treatments. In some cases, S-X is not the only treatment administered. Some animals also present symptoms of pneumonia and do require baypren, sulfa-pills or nefferlo. All animals treated with the S-X solution recovered, while most (9) recovered within twenty-four hours after treatment.

Case 4-Dairy farm 1

5/15/2014-dairy 1 is a holstein cow farm. The dairy was housed in 300 animals, which had to meet their health and daily needs. This dairy was shown to have a rotavirus of origin in white diarrhea, which was found by veterinary centers located near their dairy. S-X treatment was administered to 7 young animals with white diarrhea by oral syringe application. All animals given S-X treatment recovered. All animals recovered within 24 hours except one, and one recovered after the second S-X treatment.

According to dairies 1, in 12 months 2013 they lost 30 calves due to white diarrhea, although these calves have been given multiple electrolyte treatments and antibiotics. Multiple treatments of these calves generally take 5-7 days compared to 1-2 doses of S-X treatment on 1 day. Treatment via oral drench administration is preferred by the dairy 1 and has proven effective.

The operator of the dairy 1 says "more needed" because this is really effective. "she means S-X solution and success with these treatments. The dairy will continue to treat white scour in the calves with S-X technology and provide information about the animals subsequently tested with the S-X formula.

Case 5-pasture 4

The S-X technique was also tested in the Hutterite swine production facility (Hutterite swine production facility) where hundreds of piglets exhibited white diarrhea for the week 4, month 14, 2014. The presence of PED virus in this herd was confirmed by Newport Labs, Worthington, MN. According to the producers, over 850 piglets died within the previous three weeks, representing almost 100% of the deaths of infected piglets.

The producer was asked about his test results on piglets with S-X technology for white diarrhea pigs 4 month 19 in 2014. The manufacturer noted that 6ml of the S-X formulation was orally administered via syringe to 8-day-old piglets suffering from white diarrhea at or about 4 months and 12 days 2014. The piglets improved after 5 hours and showed no signs of white diarrhea the following day. Furthermore, 10 piglets, 14 days old, showed signs of white diarrhea at 4 months and 15 days, with typical symptoms of dark yellow thin stools. To these piglets 4ml of S-X solution was administered orally by syringe and within 24 hours the faeces of each animal became completely "dry". The producer also indicates that he will have a number of fatalities to expect from his previous experience with this disease.

In addition, 10 piglets, which were 3 days old, showed signs of white diarrhea at or about 19 days 4 months, and were given 2ml of S-X solution via syringe. After several weeks, all of these piglets survived. Another treatment was administered to 30 piglets at 3 days of age showing signs of white diarrhea. These 30 piglets were given 3ml of S-X solution and all piglets survived. Based on previous experience with the producer, he would expect almost all of these animals to die if the treatment was not administered. In this study, the producers also administered tylosin 40-50 to each 3-day-old piglet, the treatment being on days 15 and 19 at 4 months. Tylosin is an antibiotic used to treat pneumonia. The producers felt that tylosin-40 in combination with S-X treatment was responsible for the survival of these young animals; however, according to the expert in the field, antibiotics are generally not effective against enteroviral and bacterial infections. While not wishing to be bound by any particular theory, in this case tylosin-40 may have no effect on survival of piglets.

Because his piglets were treated with S-X solution, the producer did not lose any animals due to white diarrhea. In addition, samples from piglets sent to the Norwalk laboratory after addition of S-X treatment were confirmed to be no longer present with PED since 5 months and 27 days 2014. This finding indicates that S-X solution successfully treated white diarrhea in Harlowton pigs. Furthermore, the disappearance of PED virus from this area may be attributed to S-X treatment, as all piglets with white diarrhea were treated and recovered with this technique.

Case 6-pasture 5

Pastures 5 near the canadian border are involved in S-X treatment of their beef cattle ranch for white diarrhea. In winter with strong wind and heavy snowfall in the last year, the temperature during calving is very low. The S-X solution was provided at the beginning of 4 months and the first date of subsequent treatment was 4 months and 6 days 2014. Twenty one animals were treated for white diarrhea with S-X solution only, and the animals were treated by gastric tube administration of 50ml of S-X solution. After 24 hours, 18 calves treated with the S-X solution recovered from white diarrhea; however, 3 animals required a dose re-administration. These animals recovered quickly after treatment. Of the 21 animals treated, 8 showed symptoms of pneumonia and were given nefferlo. Recovery in animals treated with S-X was not associated with treatment for incidence of pneumonia. The last date of treatment was 4 months and 18 days 2014. In summary, approximately 85% of animals treated with the S-X technique recovered with one dose of solution. These results demonstrate that the S-X technique requires less time and is more effective than traditional forms of treatment for animals with white diarrhea.

Case 7-pasture 6

Pasture 6 is a dairy farm that uses S-X technology as a treatment for white diarrhea in mid 4 months of 2014. The dairy is owned by about 100 Holstein cows and a variety of other animals, including beef cattle. The incidence of white diarrhea in this pasture is particularly high and most animals develop white diarrhea shortly after birth. The S-X formulation was given to the dairy and administered to beef cattle and Holstein calves in 50ml doses by oral syringes.

Initial treatment of the S-X formulation 8 calves were given via syringe at a dose of 50ml, of which 7 had exhibited a typical creamy yellow white dysentery and 1 calf (No. 80) had a white pasty white dysentery and developed a watery white dysentery after 2 days. The condition of this calf has a typical milk dysentery (milk). This particular calf was treated with S-X after its symptoms had developed and died after 4 days. Due to the nature of white diarrhea, this particular calf is suspected of having a nutritional cause of white diarrhea, in which case the S-X technique is ineffective. Most animals treated with S-X solution recovered and recovered completely within 24 hours; however, one holstein calf took 48 hours to recover completely. In addition, one calf of the farm (No. 34 as untreated control) was noted to have white diarrhea without administering a dose of S-X solution with other animals and died within two weeks. Figure 23 is a series of pictures showing calves before and after treatment with S-X.

Case 8-pasture 7

Owners of the ranch 7 were interested in treating new born calves that develop white diarrhea using S-X technology in early 2014. The S-X solution was administered to the rancher and, over the course of several weeks, calves showing signs of white diarrhea were immediately treated on the turf with 50ml of S-X solution in an oral syringe in two doses of 25 ml. At least 15 calves were treated with the S-X solution and recovered within 24 hours per head except one. This calf, who did not recover within 24 hours, had white faeces, which could be attributed to "milk dysentery". The calves also suffer from pneumonia, so they are also treated with neffersonia and drench (drench). Two additional treatments of S-X solution were also administered thereto. The calves do recover from white diarrhea and are now in a normal state. The rancher noted that most of the time calves recovered within 3 to 4 hours after S-X solution treatment. The rancher also commented on the recognition that calves stopped "grinding teeth" and generally improved alertness. The rancher instructions that the oral syringe method of S-X solution treatment would be very easy for her to administer a dose to 100 pound calves. These results demonstrate that S-X administration can be performed using either gastric tube or syringe methods.

Case 9-pasture 8

From 5/4/2013 to 5/21/2014, experiments on beef cattle with white diarrhea were conducted at a pasture 8 at an altitude of 4,459 feet, focusing on the spring of 2013 and the winter and spring of 2014. On the pasture, 300 calves live in spring and 300 in autumn. One hundred forty-two different calves were treated with S-X solution or a combination of S-X solution and other drugs. The initial S-X formulation was used throughout 1 month in 2014, followed by a modified S-X technical white diarrhea treatment formulation. Temperature changes from-20F up to 50F at 2 and 3 months. During this period a lot of snowfall and wind were observed and severe temperature fluctuations were reported.

A large number of calves exhibit clinical signs of white diarrhea (ear drop, deep eye and white diarrhea). These calves were given 50mL S-X solution through a gastric tube system and examined after approximately six hours to determine if they recovered or if they required re-administration of the drug. Of the 243 calves treated, 36 were given either the solution alone or the solution plus a vitamin supplement for the first or second dose, while the remaining animals were given standard antibiotics and S-X white diarrhea treatment. Only twelve of the S-X treatments were administered the second or third dose on the same day. In total, 243 treatments included S-X solutions, and of these 29 treatments 2 or 3 times. The total individual calf count treated with both formulas was 142. Drugs commonly administered with S-X therapy include: excede (for respiratory infections), probiotics, Toxiban (absorption of toxins in charcoal), Noromycin LA (antibiotic for pinkeye, foot rot and other infections), multivitamins, Inforce 3 (three-way respiratory vaccine), tulathromycin (antibiotic for pinkeye, foot rot or respiratory disease) and sulfonamide tablets (sulfonamides for antibacterial treatment).

Owners of the pasture 8 were asked about their experience with S-X treatment of white diarrhea 3 months and 3 days 2014. White dysentery was first reported in the calving phase of 2 months and 27 days 2014 and 40 doses were provided at 50mL each time. From 2 months and 27 days until 3 months and 3 days, 15 calves with signs of white diarrhea have been treated with the S-X technique. Thirteen of these 15 calves recovered overnight after one dose, while 2 required a second dose and recovered overnight, with no mortality in either case. Half of the calves were treated on grass, of which only 6 heads were brought to the barn. Conventional treatments for white diarrhea include electrolytes, intravenous administration of fluids, and some antibiotics such as tetracycline.

The producers observed that some of the calves treated had pneumonia and administered other drugs; however, it is also noted that S-X treatment is far superior to other drugs and relies on their use for rapid and complete recovery. It is estimated that one third or more of the calves with white diarrhea die if S-X is absent. An additional observation of S-X treatment is that it is easy to use and carry when applied to herds, is a rapid treatment option and if white diarrhea is treated fast enough, no antibiotic treatment is required. Figure 24 is a series of pictures showing calves before and after treatment with S-X.

Table 7 depicts S-X treatment administered at pastures 3, 5 and 8 without any additional medication other than vitamin supplements.

Table 7: S-X therapy without other drugs

Receiving a multivitamin supplement

Table 8 depicts all S-X treatments given for pastures 3, 5 and 8, regardless of whether any additional drugs were administered.

Table 8: all S-X treatments

Indicate S-X treatment through nasal passages (2cc)

Example 12

Treatment of animal mastitis

Mastitis problems in the mammary glands of animals are often caused by infections caused by escherichia coli or staphylococcus aureus and other bacterial pathogens. The nipple becomes inflamed and eventually the disorder can spread to all other parts of the breast. Lactation is stopped. If left untreated, the animal may die. Most antibiotic treatments are expensive and ineffective. During the past three months, each teat of one sweater lamb (yew lamb) and two cows with mastitis was treated with 15mL of mastitis treatment solution. Fig. 25A depicts teats of a sheep suffering from mastitis. The treated animals developed well in terms of disease and did not die but remained healthy. The mammary gland has ceased to function. The S-3 formulation was administered via syringe (fig. 25B). Two cows with mastitis are in the early stages of the disease. Each infected teat of each cow was treated with 15ml and complete recovery was observed within 24 hours.

Exemplary formulations and treatments for mastitis in farm animals

Per 90ml of water:

5mg cremophor or other suitable surfactant

0.7ml propionic acid and 0.2ml isoamyl hexanoate

The formulation was shaken well and administered to cows with a syringe with up to 15ml per teat. Cremophor is used to bring the ingredients of the S-3 formulation into solution.

Example 13

MIC testing of S-3 and S-4 formulations

MIC protocol for testing S-3 and S-4

Adjusting the OD of a turbid bacterial culture grown in a suitable nutrient broth culture₆₅₀0.4 and subsequent dilution in broth at 1:100, expressed as a concentration of 1 × 10⁶CFU/ml. 50 μ l of this culture was added to each well, except for the negative control, where 50 μ l of broth was added. The final bacterial load in each well was 5X 10⁶CFU。

Mu.l of stock B-23 antibiotic solution was added to 480. mu.l of broth. 250 μ l of this solution was diluted 1: 2. This was repeated twice to form four gradually diluted antibiotic solutions. Dilution was done so that the final concentration of antibiotic in the appropriate wells was equal to 1%, 0.5%, 0.25% and 0.125% of the stock B-23 antibiotic solution.

96-well microtiter plates were used. Spread total 6 treatments: 1%, 0.5%, 0.25%, 0.125%, 0.061%, 0.03%, and 0% antibiotics with bacterial inoculation; and bacteria-free inoculation. Each treatment was plated in triplicate.

Broth was added to each well to reach a final volume of 200 μ Ι. In wells without bacterial inoculation or antibiotic solution, an additional 50 μ l of broth was added.

MIC plates were incubated under appropriate growth conditions to the time points recorded in the results table. Endpoint was selected when the positive control wells were turbid.

The MIC point was taken as the lowest concentration at which there was no significant growth.

Results

The MICs for the following organisms were as follows:

b, bacillus subtilis: 0.06125 percent

Vibrio cholerae (Vibrio cholerae): 0.06125 percent

Pseudomonas aeruginosa (Pseudomonas aeruginosa): 0.125 percent

Salmonella enterica serovar Typhimurium (Salmonella enterica serovar Typhimurium) 0.06125%

Coli: 0.125 percent

Methicillin-resistant staphylococcus aureus: 0.06125 percent

For other MIC tests-potato dextrose broth was used instead of nutrient broth and the test was performed in the same manner. The results were:

erwinia amyloliquefaciens (Erwinia amylovora): 0.0612%

And (3) lactobacillus: 0.0625%

Erwinia carotovora: 0.125 percent

The results demonstrate that S-3 and S-4 can be used for the treatment of diseases caused by microorganisms in plants, animals and humans. These diseases include plant diseases caused by erwinia and the problem of ethanol production from grain fermentation by lactobacillus biofilms produced by pseudomonas. Additional diseases include food disorders caused by salmonella, escherichia coli and the general major disease caused by MRSA.

Example 14

Raspberry processing

The results described herein demonstrate that the S-X technique can be used to preserve fruits and vegetables during transportation and storage. The S-3 formulation was mixed to form two formulations: 1ml of S-3(1:10 mixture) per 10g of bentonite; 1ml of S-3 was added to 20g of bentonite (1:20 mixture) or other carrier. Place 1 gram of the mixture into a small plastic cup with a raspberry purchased at a store. The material was placed in a small clear plastic box, sealed and kept at room temperature for 1 week, and then checked for the presence of contaminating fungi. The results demonstrate that the normal flora of fruit quickly rot it after 1 week at room temperature (fig. 26A). However, the use of the 1:10 mixture resulted in no decay (FIG. 26B). However, the 1:20 mixture does not perform as well as the 1:10 mixture because at least 1 berry shows decay. Nevertheless, the 1:20 mixture may be used to prevent spoilage of berries, and the treated berries may be consumed. Similar experiments were performed with Thompson delicious grapes purchased at the store with similar results, in which it was observed that the control grapes showed decay, while the treated grapes did not. The grapes were also edible because 4 individuals consumed them and provided an assessment of their acceptability.

Example 15

Use of S-X for treating food poisoning and/or gastrointestinal colds in humans

The symptoms and conditions of food poisoning and/or gastrointestinal colds in humans are similar to those occurring in animals suffering from white diarrhea. For example, possible symptoms include: abdominal cramps, diarrhea (with possible blood), fever and chills, headache, nausea, vomiting, and weakness (which may be severe). Most people suffer from this experience (12-48 hours) only by trying to rest, and drink replacement fluids and minerals lost through diarrhea and vomiting. There appears to be no product available that provides immediate relief.

However, in ten volunteers suffering from one or more of these symptoms, at least 10-15ml of the 1% S-3 formulation was orally administered at the onset of symptoms or within hours of symptom appearance. In all cases, the patient described a sense of improvement within one to two hours after treatment. Fever, stomach ache, diarrhea and vomiting all ceased and the patient was fully recovering. All patients were adults, caucasian and represented a male and female category. However, one patient reported no detectable difference in gastric condition after taking a 10ml dose of the 1% S-3 formulation. While not wishing to be bound by any particular theory, it is suspected that the patient is experiencing a virus-induced gastric infection that is not responsive to S-X therapy. Nevertheless, the fact that 90% of the treated people recovered so rapidly and completely, combined with all animal studies on white diarrhea, supports the following hypothesis: s-3 can be used for treating people suffering from gastrointestinal colds and stomach poisoning caused by bacteria. This hypothesis is also supported by the significant MIC values of S-3 against e.coli and s.aureus, two known causes of food poisoning in humans (example 13).

Example 16

Mastitis and S-X technology of cow

Treatment:

formulations containing 2% S-3 in purified water in the presence of 5mg cremophor (a non-ionic solubilizer) were mixed thoroughly and used as therapeutic agents. Eight cows with pre-clinical to sub-clinical mastitis were treated with 12ml of the formulation per teat. In seven cases, the treatment was repeated over the course of one day. In all cases, the animals recovered completely the following day. While not wishing to be bound by any particular theory, recovery of the animal may be due to the fact that: common bacterial causes of mastitis, such as E.coli and Staphylococcus aureus, are organisms that are extremely sensitive to the S-X formulations described herein (see example 13).

Example 17

S-3 detergent test

Several cleaners obtained by sample ordering were tested for efficacy against surfaces heavily contaminated with various pathogenic bacteria using the S-3 solution. These surfaces include laboratory and feminine floors, toilets and door handles. For floor testing, approximately 5ml of each cleaner solution (1 ml S-3 per 100ml deionized water) was poured onto different portions of the floor and wiped dry with a paper towel. When this portion of the floor was dry, it was then wiped with a Kimwipe, which was then wiped across the surface of the potato dextrose broth petri dish plate. For toilet testing, a paper towel is wetted with a detergent solution and a portion of the surface is wiped. Once the surface was dried, it was wiped with a Kimwipe and re-streaked through a potato dextrose broth plate. The procedure for the door handle was the same as for the sink, except that only one detergent and control were tested. The results are described in tables 9 and 10 below.

Table 9: laboratory floor results

		Experiment 1	Experiment 2
					Amount of detergent	Number of colonies	Number of colonies
Control		22	23
				Sucragel CF	1ml of	1	0
Chemoxide CAW	2ml of	1	2
				BioSoft D40	0.5ml	0	3
Lathanol LAL	1g		2					1
				BioTerge AS-40	1ml of	1	2
Nacconol 90G	1g	1	4
				Coconut acid potassium salt	2ml of	1	1

Table 9 shows the number of bacterial or fungal colonies grown on potato dextrose broth plates streaked with various detergents or samples wiped with Kimwipe alone as a control after 48 hours. One milliliter S-3 per 100 milliliters deionized water was used.

Table 10: detergent testing in a female toilet

Table 10 shows the results from testing various surfaces (floor, toilet and door handles) in a female toilet, as well as the number of bacterial or fungal colonies after 48 hours of growth on potato dextrose broth plates wiped from the surfaces with a Kimwipe.

Example 18

Verticillium experiments

Thirty pea seeds were inoculated with verticillium after being placed on a petri dish for fungal growth. The seeds were allowed to roll freely around, then fungal samples were collected and placed in petri dishes sealed with plastic wrap along with the pea seeds, and then left for three days. After three days had elapsed, potato dextrose agar plates with a sterile lid placed in the center of the plate were filled with 50 microliters of S-3, 20 microliters of S-3, or empty as controls. Ten pea seeds of the inoculated group were placed in each of three petri dishes containing potato dextrose agar and a filled or unfilled lid. Peas were allowed to stand for two days and then examined for fungal growth and germination. The results of the experiment are described in table 11.

Table 11: verticillium inoculated pea seeds

Treatment of	Percentage of fungal growth
		Control	100
20 microliter S-3	0
		50 microliter S-3	0

Percentage of verticillium inoculated pea seeds that germinated and showed fungal growth after 48 hours in the case of controls (no S-3), with 20 microliters S-3 and with 50 microliters S-3.

Example 19

Experiment of camelina sativa

Camelina sativa seeds, known to be contaminated with various fungal and bacterial pathogens, are taken and placed with S3 to see if fungal and bacterial growth has ceased. Several potato dextrose broth plates were taken along with a lid for placing S-3. About forty seeds were placed on one of the plates and an empty sterile lid was placed in the center as a control group. The plates were sealed with cling film and left for two days to determine germination and fungal and bacterial growth. More than one hundred seeds were placed on another petri dish with a sterile lid filled with 50 microliters of S-3. The seeds were left with S-3 in a petri dish tightly sealed with a preservative film for the following interval of hours (twenty to thirty seeds were removed at these points and individually plated on one petri dish): 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, and 48 hours. For each interval, the plates were left for 48 hours and then checked for germination and pathogen growth.

Table 12: capsella bursa-pastoris seed germination and pathogen growth

Table 12 shows the percentage values of infected camelina sativa seeds germinated in percentage values and with pathogenic bacteria growth at hourly intervals, with no S-3 (control) or 50 microliters of S-3. All samples were recorded 48 hours after placing on potato dextrose broth plates.

The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. It is intended that the following claims be interpreted to embrace all such embodiments and equivalent variations.

Further development of

The combinations of acid(s) and ester discussed above, including, but not specifically limited to, various combinations of propionic acid and/or isobutyric acid in combination with isoamyl hexanoate, provide an unexpected degree of antibacterial activity. In particular, five specific exemplary formulations designated S-1 through S-5 or generally designated S-X demonstrate varying degrees of efficacy for various purposes and/or in various products. The inventors refer to these various formulations (including but not limited to S-1 to S-5 formulations) generally as "Sx" formulations or embodying "Sx technology".

Sx technology involves the combination of specific small molecular weight organic acid(s) with more complex esters or ester mixtures that, taken together, exhibit impressive antimicrobial activity. Also, as the ingredients are produced from natural sources, they are likely to be prepared and registered as "organic" for an increasingly expanding market.

The initial use of Sx technology was to treat white diarrhea in cattle and other animals. It has now also been found that products including Sx technology can also be used in a variety of other ways, for methods and products designed to control a wide range of microorganisms that may be present in various human endeavors or otherwise associated with various human and animal activities and health.

Many of these uses are applicable to pre-post harvest and other food and feed for humans, livestock, pets, various other mammals, birds and fish, as well as vegetables, fruits and crops. Such uses include antifungal, antibacterial, antiprotozoal, and antiparasitic effects, including for the treatment or prevention of pathogen and wound infections, as an antibacterial or sterilization method, including pre-and/or post-operative wounds, cuts, abrasions, and the like. Treatment and prevention of diarrhea; pulmonary infection; chronic disorders of the gastrointestinal tract or intestine; vaginal infections; infection of the reproductive tract; ear, eye, nose and oral infections; fungal infections of the fingers or toes; whelk; aphtha.

The formulation may include any form commonly used for treatment including, but not limited to, creams, lotions, oils, scrubs, sprays, ointments, shampoos, gels, plasters, liquids, infusions, salves, ear washes, powders, eye washes, mouthwashes, gases, and the like.

Pathogens to be treated include, but are not limited to, escherichia coli, salmonella, staphylococcus aureus, listeria, clostridium, cryptosporidium, giardia, helminths, roundworms, and the like. Helminths that can be treated include, for example: the following were used:

-small living red worms (Cyathostomin spp.)

Roundworm (horse roundworm)

Pinworm (pinworm)

Nematodes (roundworm species Wechsler)

Tapeworm (naked head tapeworm)

Pneumocystis (Nephilus andersoni)

-fly maggot (stomach fly)

Human and related uses

The antimicrobial compositions discussed so far can be used in humans in place of commonly used antibiotics. As previously discussed, the present antimicrobial compositions provide the benefits of commonly used antibiotics without negative effects, such as bacterial resistance.

Mouthwash

The mouth of a person has many periodontal pockets (pockets) and crevices between the teeth and gums that can trap food particles and become a breeding ground for harmful microorganisms. The moist nature of the mouth, combined with constant exposure to food, can lead to overgrowth of harmful microorganisms. As a result, microorganisms can cause gum infections, plaque build-up, dental caries and many other problems.

The antimicrobial composition of the present invention may be provided in a mouth rinse to combat harmful microorganisms in a human mouth. The wash may be a liquid containing the microbial composition of the invention and a flavouring such as artificial mint or fruit. In some embodiments, the mouthwash product can be formulated for administration by a dentist in a professional setting. Alternatively, the mouthwash product can be formulated as an over-the-counter consumer rinse, which can be administered similar to currently known mouthwashes. In some embodiments, the lotion can include a base liquid, such as water, one or more of the presently discussed antimicrobial compositions, and a flavoring composition.

Antibacterial wound treatment

One or more of the antimicrobial compositions of the present invention may be provided in the form of a cream, serum (serum) or other topical formulation for application to a wound, sore or other lesion of the human skin or mucosa. The cream may be an emulsion comprising a visceral (viscus) base, the antimicrobial composition of the invention, additional pharmaceutical compositions such as anaesthetics and other biologically active substances such as vitamins, minerals and proteins. In some embodiments, the cream may be used to treat drug-resistant bacterial strains, such as staphylococcus aureus, that are unresponsive to conventional antibacterial therapy. The antimicrobial compositions of the present invention may be applied to bone or other tissue within the body to disrupt bacterial biofilms that may have formed, for the prevention or treatment of sepsis.

Fungal infections

The antimicrobial compositions can be used as antifungal therapy. The nail, foot and crotch areas are the areas of the body most susceptible to fungal growth. These areas are also often sensitive and poorly respond to heavy pharmaceutical ointments. Ointments containing concentrated amounts of the antimicrobial compositions of the present invention may be provided to treat these areas with less irritation.

The antimicrobial composition can be used for treating infection caused by Candida albicans. AIDS patients often experience fungal infections caused by such fungal strains, which infections can be combated by treatment with antimicrobial compositions containing the present invention. In addition, the uterus of women is also susceptible to infections caused by candida albicans, which may be treated using the antimicrobial compositions of the present invention.

It is within the scope of the present disclosure to provide the antimicrobial compositions of the present invention as sprays, gels, pads, liquids, and bandages. In addition, the composition can be used on implants. For example, a coating on a durable implant may include the composition, or a biodegradable implant may have the composition of the invention dispersed throughout. The present antimicrobial compositions may be provided in the form of a medicament for treating the nose, mouth, eyes, ears, and/or rectum of a human.

In addition to use on the human body, the antimicrobial compositions of the present invention can also be used to provide an antimicrobial environment around the human body. Thus, the present antimicrobial formulations can be incorporated into cleaning products not designed for human use. For example, the compositions of the present invention may be used to treat human feces in hospitals and homes. Litter that may have been in contact with human feces may be cleaned using a cleaning product containing the antimicrobial composition of the invention. Feces may also be treated with the antimicrobial compositions of the present invention to help prevent microbial contamination of the surrounding area.

Animal use

Mastitis

Mastitis is a major problem in dairy cows caused by salmonella and other bacterial pathogens. Since Sx formulations are effective against these organisms, an effective treatment is to apply a buffered saline Sx solution to the affected area (affected nozzle segment). This has been successful, for example, in a dairy plant in idaho. Alternatively, uterine irrigation and/or teat dip treatment may also be applied to infected cows, or even as a routine preventative measure. Many uses are also applicable to sheep and goats.

Infection of skin and feet

Treatment of such infections includes, for example, the use of Sx formulations to treat foot rot, rain blotch, verruca, and various bacterial and other fungal infections of the skin, hooves, ears, mouths, and other body parts. For example, Sx products may be designed for initial clean wound application by developing a solution of Sx salts, and then or alternatively, applied daily or in other conventional ways as a spray solution to maintain a proper environment and treat or avoid such infections.

Garbage and excrement treatment

As noted above, Sx technology may be used to treat bedding and fecal sites including, but not limited to, cat litter or cat litter containers. Of course, the same basic formulation can also be used for human fecal control as well as for other human and animal environments.

Animal feed applications

Particularly in view of the recent FDA requirements for the removal of standard antibiotics from animal feed, there is a need for a viable alternative to antibiotic products. It is speculated that if the standard animal diet contains one or more antibiotics, infectious microorganisms will be prevented from causing disease and may cause the animal to grow at a higher than average rate. The problem with such practices means that there are a large number of drug-resistant pathogenic microorganisms in our environment, resulting in a large number of drug-resistant infections in human and animal populations, which are not easily treated, resulting in thousands of accidental deaths.

Sx techniques and formulations may be applied directly to animal feed, whether in pellet feed or other form (e.g., crumb), and include, but are not limited to, use in dairy and beef cattle, as well as poultry and other animals (e.g., pets, farms, zoos, or other wild animals). The problem of Sx preparations not developing resistant microorganisms-Sx preparation resistant microorganisms have never been observed. Indeed, for example, administration of Sx formulations to calves results in healthier animals with faster weight gain and less evidence of disease. Such Sx products can be formulated in many ways, for example, with a carrier such as zeolite, or as small soluble beads, or as a top dressing in a feeding tray. Many alternatives are readily available for successful delivery of Sx formulations.

Treatment of animal feed to avoid spoilage is another major application of this Sx technology. This is because Sx preparations are very effective against almost all major decay-causing fungal organisms (e.g., Fusarium, Aspergillus, etc.). Sx formulations can be applied to stored animal feed, for example, in the gas phase, to control contaminating fungi and other microorganisms. Optionally, Sx preparations may be included in the feed to prevent spoilage by decay causing organisms.

Food preparation, storage and preservation

Since the Sx formulation is very effective in controlling bacterial and fungal growth, and since it is usually edible in dilute solutions, it has applications in many aspects of human food use, including as a prophylactic, therapeutic or preservative, as shown below:

-treatment of animal carcasses to prevent escherichia coli and salmonella infestation. This may be done in the form of sprays or other formulations, including but not limited to those used for poultry, cattle, pigs, goats, sheep and horses;

-treatment of industrial surfaces, floors, walls, ceilings, gutters instead of touch (handles) and packaging of food (plant and animal products) with Sx preparations for control of harmful bacterial and fungal microorganisms;

-treating household drain boards, bathroom surfaces and other places where harmful microorganisms may be contained, including drains, floors, etc. This would require special Sx formulations to provide appropriate product odor, color, etc.;

-treatment of the soil or other growing medium required for the production of food products such as fruits, vegetables, mushrooms, etc. Sx formulations will be used as detergents for harmful microorganisms (including pathogens);

-treating the harvested fruits, vegetables and cereals to prevent the development of decay causing microorganisms. This has a wide range of applications, including the use of Sx formulations in large barns and in cabins carrying large quantities of international wheat and other grains being transported. Sx formulations can be used, for example, as gases to kill harmful pathogens and pests; and

-treating the tools and equipment used in food preparation to decontaminate these items.

Industrial applications

The occurrence of harmful bacterial and fungal growth in and on industrial machinery encounters a number of problems and difficulties. Some of which include the growth of harmful bacteria in the lubricating oil of heavy machinery. In addition, bacterial contamination of the water in cooling towers has led to the death of humans from legionnaires' disease, a disease caused by bacteria. In addition, there are many bacterial problems associated with fouling surfaces in naval operations. Sx formulations may be used to help solve or remedy these problems.

Biofilms and their effects need to be controlled in almost all aspects of human health and human effort

Microorganisms are ubiquitously attached to surfaces and produce extracellular polysaccharides, thereby forming biofilms. Biofilms constitute a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the possibility that these organisms cause infections in patients who leave medical devices. Knowledge of the role of biofilms in infection should enhance the clinical decision making process. Many biofilms are also associated with biofouling issues on industrial surfaces. Many aspects of biofilms are associated with problems encountered by the U.S. navy and its operations. Sx preparations are effective against bacteria that produce biofilms, such as Pseudomonas.

Other applications

Sx technology has been applied to control protozoan parasites as well as viruses.

Variants of the Sx formulations have anthelmintic and insect-resistant properties.

Further possible products and treatments

Anti-fungal & anti-bacterial

-treatment or prevention of rain blotch/brown spot (scald)

Foot rot

-scratch injury

Infections of the ears (yeasts and fungi) in livestock or pets

Genital cleansing

Wound care

Mild and severe laceration

Scratch and mar resistance

-dermatitis of the skin

-stopping diarrhea

-horse electrolyte (electrolyte)

Treatment of fleas, ticks and other ectoparasites

Helminths and endoparasites

-biosafety products

Human health

-oral electrolytes

-feed additives

-dog and cat antidiarrheal

The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

Although the present invention has been disclosed with reference to particular embodiments, it is apparent that other embodiments and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. It is intended that the following claims be interpreted to embrace all such embodiments and equivalent variations.

Sequence listing

<110> G.A. stonebob

B. Brad's hand

<120> formulations of volatile organic compounds with antimicrobial activity

<130> 206054-0001-00-US.603296

<140> US 14/322,757

<141> 2014-07-02

<150> US 61/842,362

<151> 2013-07-02

<150> US 61/948,902

<151> 2014-03-06

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 480

<212> DNA

<213> Fusarium oxysporum

<400> 1

aacataccaa ttgttgcctc ggcggatcag cccgctcccg gtaaaacggg acggcccgcc 60

agaggacccc taaactctgt ttctatatgt aacttctgag taaaaccata aataaatcaa 120

aactttcaac aacggatctc ttggttctgg catcgatgaa gaacgcagca aaatgcgata 180

agtaatgtga attgcagaat tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc 240

cagtattctg gcgggcatgc ctgttcgagc gtcatttcaa ccctcaagcc cagcttggtg 300

ttgggactcg cgagtcaaat cgcgttcccc aaattgattg gcggtcacgt cgagcttcca 360

tagcgtagta gtaaaaccct cgttactggt aatcgtcgcg gccacgccgt taaaccccaa 420

cttctgaatg ttgacctcgg atcaggtagg aatacccgct gaacttaagc atatcaataa 480

<210> 2

<211> 478

<212> DNA

<213> Fusarium oxysporum

<400> 2

cataccaatt gttgcctcgg cggatcagcc cgctcccggt aaaacgggac ggcccgccag 60

aggaccccta aactctgttt ctatatgtaa cttctgagta aaaccataaa taaatcaaaa 120

ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag 180

taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca 240

gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagccca gcttggtgtt 300

gggactcgcg agtcaaatcg cgttccccaa attgattggc ggtcacgtcg agcttccata 360

gcgtagtagt aaaaccctcg ttactggtaa tcgtcgcggc cacgccgtta aaccccaact 420

tctgaatgtt gacctcggat caggtaggaa tacccgctga acttaagcat atcaataa 478

<210> 3

<211> 488

<212> DNA

<213> Fusarium

<400> 3

cttaatgttg cctcggcgga tcagcccgcg ccccgtaaaa cgggacggcc cgccagagga 60

cccaaactct aatgtttctt attgtaactt ctgagtaaaa caaacaaata aatcaaaact 120

ttcaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcaaaat gcgataagta 180

atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcacattgc gcccgctggt 240

attccggcgg gcatgcctgt tcgagcgtca tttcaaccct caagcccccg ggtttggtgt 300

tggggatcgg ctctgccttc tggcggtgcc gcccccgaaa tacattggcg gtctcgctgc 360

agcctccatt gcgtagtagc taacacctcg caactggaac gcggcgcggc catgccgtaa 420

aaccccaact tctgaatgtt gacctcggat caggtaggaa tacccgctga acttaagcat 480

atcaatag 488

<210> 4

<211> 477

<212> DNA

<213> Fusarium flavum

<400> 4

cataccttat gttgcctcgg cggatcagcc cgcgccccgt aaaaagggac ggcccgccgc 60

aggaacccta aactctgttt ttagtggaac ttctgagtat aaaaaacaaa taaatcaaaa 120

ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag 180

taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca 240

gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagccca gcttggtgtt 300

gggagctgca gtcctgctgc actccccaaa tacattggcg gtcacgtcga gcttccatag 360

cgtagtaatt tacatatcgt tactggtaat cgtcgcggcc acgccgttaa accccaactt 420

ctgaatgttg acctcggatc aggtaggaat acccgctgaa cttaagcata tcaatag 477

<210> 5

<211> 485

<212> DNA

<213> Fusarium avenaceum

<400> 5

cagaagttgg ggttttacgg catggccgcg ccgcgttcca gttgcgaggt gttagctact 60

acgcaatgga ggctgcagcg agaccgccaa tgtatttcgg gggcggcacc gccagaaggc 120

agagccgatc cccaacacca aacccggggg cttgagggtt gaaatgacgc tcgaacaggc 180

atgcccgccg gaataccagc gggcgcaatg tgcgttcaaa gattcgatga ttcactgaat 240

tctgcaattc acattactta tcgcattttg ctgcgttctt catcgatgcc agaaccaaga 300

gatccgttgt tgaaagtttt gatttatttg tttgttttac tcagaagtta caataagaaa 360

cattagagtt tgggtcctct ggcgggccgt cccgttttac ggggcgcggg ctgatccgcc 420

gaggcaacat taaggtatgt tcacaggggt ttgggagttg taaactcggt aatgatccct 480

ccgca 485

<210> 6

<211> 469

<212> DNA

<213> Fusarium oxysporum

<400> 6

cagaagttgg ggtttaacgg cgtggccgcg acgattacca gtaacgaggg ttttactact 60

acgctatgga agctcgacgt gaccgccaat caatttgggg aacgcgattt gactcgcgag 120

tcccaacacc aagctgggct tgagggttga aatgacgctc gaacaggcat gcccgccaga 180

atactggcgg gcgcaatgtg cgttcaaaga ttcgatgatt cactgaattc tgcaattcac 240

attacttatc gcattttgct gcgttcttca tcgatgccag aaccaagaga tccgttgttg 300

aaagttttga tttatttatg gttttactca gaagttacat atagaaacag agtttagggg 360

tcctctggcg ggccgtcccg ttttaccggg agcgggctga tccgccgagg caacaattgg 420

tatgttcaca ggggtttggg agttgtaaac tcggtaatga tccctccgc 469

<210> 7

<211> 480

<212> DNA

<213> Fusarium avenaceum

<400> 7

gaagttgggg ttttacggca tggccgcgcc gcgttccagt tgcgaggtgt tagctactac 60

gcaatggagg ctgcagcgag accgccaatg tatttcgggg gcggcaccgc cagaaggcag 120

agccgatccc caacaccaaa cccgggggct tgagggttga aatgacgctc gaacaggcat 180

gcccgccgga ataccagcgg gcgcaatgtg cgttcaaaga ttcgatgatt cactgaattc 240

tgcaattcac attacttatc gcattttgct gcgttcttca tcgatgccag aaccaagaga 300

tccgttgttg aaagttttga tttatttgtt tgttttactc agaagttaca ataagaaaca 360

ttagagtttg ggtcctctgg cgggccgtcc cgttttacgg ggcgcgggct gatccgccga 420

ggcaacatta aggtatgttc acaggggttt gggagttgta aactcggtaa tgatccctcc 480

<210> 8

<211> 468

<212> DNA

<213> Fusarium oxysporum

<400> 8

gaagttgggg tttaacggcg tggccgcgac gattaccagt aacgagggtt ttactactac 60

gctatggaag ctcgacgtga ccgccaatca atttggggaa cgcgatttga ctcgcgagtc 120

ccaacaccaa gctgggcttg agggttgaaa tgacgctcga acaggcatgc ccgccagaat 180

actggcgggc gcaatgtgcg ttcaaagatt cgatgattca ctgaattctg caattcacat 240

tacttatcgc attttgctgc gttcttcatc gatgccagaa ccaagagatc cgttgttgaa 300

agttttgatt tatttatggt tttactcaga agttacatat agaaacagag tttaggggtc 360

ctctggcggg ccgtcccgtt ttaccgggag cgggctgatc cgccgaggca acaattggta 420

tgttcacagg ggtttgggag ttgtaaactc ggtaatgatc cctccgca 468

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> chemical Synthesis

<400> 9

tccgtaggtg aacctgcgg 19

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> chemical Synthesis

<400> 10

tcctccgctt attgatatgc 20

Claims (20)

1. An agent having antimicrobial, antifungal, antibacterial, antiprotozoal or antiparasitic action comprising isoamyl hexanoates with at least one acid selected from propionic acid and isobutyric acid.

2. The formulation of claim 1, wherein the formulation is in the form of a cream, ointment, lotion, oil, scrub, spray, shampoo, gel, plaster, solution, suspension, infusion, salve, ear wash, powder, eye wash, mouthwash, nail polish, or gas.

3. The formulation of claim 2, wherein the formulation is in the form of a mouthwash.

4. The formulation of claim 1, wherein the formulation is in a form suitable for treating a wound to treat or prevent infection.

5. The formulation of claim 4, wherein the formulation is in the form of a cream or ointment.

6. The formulation of claim 1, wherein the formulation is in the form of a nail polish or a skin treatment agent.

7. The formulation of claim 1, wherein the formulation is in a form suitable for treating human or animal feces.

8. The preparation of claim 1, wherein the preparation is in a form suitable for inclusion in an animal feed or feed supplement.

9. A method of treating or preventing pathogenic or wound infections comprising administering to a subject or surface in need thereof an effective amount of the formulation of claim 1.

10. A method of treating or preventing pathogenic or wound infections comprising administering to a subject or surface in need thereof an effective amount of the formulation of claim 2.

11. A method of treating or preventing an oral infection comprising administering to a subject in need thereof an effective amount of the formulation of claim 3.

12. A method of treating or preventing an infection during or after a medical procedure comprising administering an effective amount of the formulation of claim 2 at an appropriate time before, during and/or after the medical procedure.

13. A method of treating or preventing a fungal infection comprising administering to a subject in need of such treatment or prevention an effective amount of the formulation of claim 1.

14. The method of claim 12, wherein the formulation is administered to the subject's nail bed or skin.

15. The method of claim 13, wherein the formulation is administered to treat or prevent a fungal infection of the uterus.

16. A method of applying the formulation of claim 7 to litter or feces in a hospital or home.

17. A method of maintaining or improving the health of an animal comprising administering an effective amount of the formulation of claim 8.

18. The method of claim 17, wherein the administering comprises periodic or cyclical administration of the formulation of claim 8.

19. A method of disinfecting a surface comprising applying to the surface an effective amount of the formulation of claim 1.

20. The method of claim 19, wherein the surface is located at a home, hospital, or food processing center or location.

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