Methylsulfonylmethane (msm) for treatment of drug resistant microorganisms

ES 2 674 019 T3

DESCRIPTION

Methylsulfonylmethane (MSM) for the treatment of drug resistant microorganisms

CROSS REFERENCE TO RELATED REQUESTS

This application claims the priority of United States Provisional Applications No. 61 / 256,935, filed October 30, 2009, No. 61 / 257,751 filed November 3, 2009, No. 61 / 259,098 filed on November 6, 2009 and No. 61 / 294,437, filed on January 12, 2010.

Countryside

The present disclosure relates generally to compositions comprising methylsulfonylmethane (MSM) for treating drug resistant infectious diseases. Certain embodiments relate to the sensitization of drug resistant microbes. Some compositions disclosed herein are useful for treating MRSA, for example.

Background

Methylsulfonylmethane (MSM; (CH3) 2SÜ2), also known as dimethylsulfone, is an organosulfur compound that is a metabolite of DMSO and certain sulfur-containing amino acids. MSM has been marketed primarily as a diet supplement.

Infectious diseases are diseases produced by pathogenic microbial agents, including viruses, bacteria, fungi, parasites, and prions, among others. Despite certain improvements in the medical treatment of infectious diseases (antibiotics and vaccines), many obstacles remain in reducing mortality from infectious diseases. A major problem is the emergence and spread of drug resistant pathogens.

Methicillin-resistant Staphylococcus aureus (MRSA) is a drug-resistant bacterial pathogen that is especially problematic in hospitals where patients with open wounds, invasive devices, and weakened immune systems are at greater risk of infection than the general public. Thus, there is a need for an effective and easily administered therapy against drug resistant infectious diseases. US2006 / 0229262 discloses the use of flavonoids to enhance the efficacy of beta-lactam antibiotics. US 4 616 039 A discloses the effect of MSM against antibiotic resistant pleurisy when added to the diet of an animal.

Summary

Described herein is the unexpected discovery that MSM sensitizes drug-resistant bacterial pathogens other than Mycobacterium tuberculosis, including MRSA, to a drug to which they are resistant. Thus, surprisingly, a drug-resistant bacterial pathogen other than Mycobacterium tuberculosis can be sensitized to a drug to which the pathogen is resistant by contacting the pathogen with MSM, and can be inhibited by contacting the pathogen with both MSM and MSM. a drug to which, on the other hand, the pathogen is resistant.

Preferred embodiments of the invention are as shown in the claims.

Provided herein is a composition comprising MSM for use in a method of inhibiting a drug-resistant bacterial pathogen other than Mycobacterium tuberculosis, which method involves selecting a drug-resistant bacterial pathogen; and contacting the bacterial pathogen with a composition comprising a therapeutically effective amount of MSM and a therapeutically effective amount of an agent that inhibits the drug-sensitive form of the drug-resistant bacterial pathogen, thereby inhibiting a drug-resistant bacterial pathogen. The drug-resistant bacterial pathogen can be MRSA.

Also provided is a composition comprising MSM for use in a method of sensitizing a drug resistant bacterial pathogen other than Mycobacterium tuberculosis to a drug to which the bacterial pathogen is resistant, which method comprises selecting a drug resistant bacterial pathogen; and contacting the bacterial pathogen with a composition comprising a therapeutically effective amount of MSM, thereby sensitizing the drug-resistant bacterial pathogen to the drug to which the bacterial pathogen is resistant. The drug-resistant bacterial pathogen can be MRSA.

Also provided herein is a composition comprising MSM for use in a method of inhibiting a drug-sensitive bacterial pathogen other than Mycobacterium tuberculosis from developing drug resistance, which method comprises selecting a drug-sensitive bacterial pathogen; and contacting the drug-sensitive bacterial pathogen with a composition comprising an amount

ES 2 674 019 T3 therapeutically effective MSM and a therapeutically effective amount of an agent that inhibits the drug-sensitive bacterial pathogen, thereby inhibiting the drug-sensitive bacterial pathogen from developing drug resistance. The drug-sensitive bacterial pathogen may be Staphylococcus aureus.

In various methods described herein, the bacterial pathogen is in (or on) a subject. The composition can be administered to the subject topically or with an inhaler device.

In all embodiments, the agent is a beta-lactam (beta-lactam) antibiotic. The effective amount of MSM can be 5-20% MSM, 5-16% MSM, 5-10% MSM, 5-8% MSM, 9-16% MSM or 10-15% MSM. MSM (percent by weight of the composition comprising MSM).

In a particular composition comprising MSM for use in a method of inhibiting a drug-resistant bacterial pathogen other than Mycobacterium tuberculosis, the bacterial pathogen is MRSa, the agent is a beta-lactam antibiotic, the effective amount of MSM is 5- 10% MSM as percent by weight, and the composition is administered topically.

In a composition comprising MSM for use in a method of sensitizing a drug resistant bacterial pathogen other than Mycobacterium tuberculosis to a drug to which the bacterial pathogen is resistant, the bacterial pathogen is MRSA, the effective amount of MSM is about 5 -10% MSM in percent by weight, and the composition is administered topically.

In a particular composition comprising MSM for use in a method of inhibiting a drug-sensitive bacterial pathogen other than Mycobacterium tuberculosis from developing drug resistance, the bacterial pathogen is Staphylococcus aureus, the agent is a beta-lactam antibiotic, the amount Effective MSM is approximately 5-10% MSM, and the composition is administered topically.

It will be further understood that compositions comprising MSM for use in a method of sensitizing or inhibiting the bacterial pathogens disclosed herein are useful beyond the specific circumstances that are described in detail herein, and are expected, by example, that are useful for any of numerous conditions in which a bacterial pathogen has become drug resistant or where it is desirable to inhibit a drug-sensitive bacterial pathogen from becoming drug resistant. Brief description of the figures

Fig 1 illustrates that MSM sensitizes MRSA to oxacillin. In vitro survival of Staphylococcus aureus strain ATCC 43300 (an oxacillin and methicillin resistant MRSA strain) was studied in the presence of MSM, DMSO and oxacillin. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM and 6 pg / ml oxacillin (MA), 5-16% MSM, 6 pg / ml oxacillin and 1% DMSO (MDA), or 1% DMSO and 6 pg / ml oxacillin (DA), for 48 hours at 25 ° C. 6 pg / ml oxacillin is the MIC for this strain of MRSA (ATCC). The initial inoculation of the bacteria was 3.15 x 10⁷ cfu / ml (Log = 7.49). All conditions tested showed a decrease in cfu / ml during the 24 hour period analyzed. Positive control showed TNTC (too numerous to count) on dilution plate 10⁷ A lower survival rate was observed in the presence of 9-16% MSM with 6 pg / ml of oxacillin than in the presence of 1% DMSO and 6 pg / ml of oxacillin or in the presence of 1% DMSO, 9- 16% MSM and 6 pg / ml oxacillin. The lowest survival rate was observed in the conditions with 12 and 13% mSm with 6 pg / ml of antibiotic. These results show that specific concentrations of MSM alone can increase the sensitivity of a MRSA strain to the antibiotic more effectively than DMSO or a combination of MSM and DMSO.

Fig 2 illustrates that MSM sensitizes MRSA to oxacillin in a sham cycle of treatment. In vitro survival of Staphylococcus aureus strain ATCC 43300 (an oxacillin and methicillin resistant MRSA strain) was studied in the presence of MSM and oxacillin. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM and 6 pg / ml oxacillin (MA) for 24 hours at 25 ° C. After 24 hours, another 6 pg / ml of ml oxacillin was added to the incubations, bringing the total amount of oxacillin added to 12 pg / ml. 6 pg / ml is the MIC for this strain of MRSA. Therefore, the bacteria were under a MIC of oxacillin for 24 hours and 2x MIC for the next 24 hours. This experimental paradigm encourages the repeated application of antibiotic that a subject would receive during a treatment cycle. The initial inoculation of the bacteria was 9.14 x 10⁵ cfu / ml (Log = 5.96). Positive control showed TNTC in dilution plate 10⁵. Similar to the results shown in Fig. 1, the lowest survival rate was observed in the presence of MSM at 12 and 13%. These results confirm that MSM sensitizes MRSA to antibiotic treatment and shows that specific concentrations of MSM alone can increase the sensitivity of an MRSA strain to the antibiotic in a sham cycle of treatment.

Fig 3A-C illustrates that MSM sensitizes MRSA to multiple antibiotics. In vitro survival of Staphylococcus aureus strain ATCC 43300 (an oxacillin and methicillin resistant MRSA strain) was studied in the presence of MSM and oxacillin or MSM and methicillin. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM in combination with 6 pg / ml oxacillin or 6 pg / ml methicillin. The initial inoculation of the bacteria was

ES 2 674 019 T3

2.13 x10 ⁶/ ml (Log = 6.3). The growth periods tested were 24 hours (Fig. 3A), 48 hours (Fig. 3B) and 5 days (Fig. 3C). This is the MIC of oxacillin and methicillin for this strain of bacteria. An additional 3.5% MSM and 6 pg / ml oxacillin or 6 pg / ml methicillin were added each day. At 24 hours, MSM 5% and antibiotic showed the lowest level of bacterial survival. At 48 hours, MSM 5% and methicillin or MSM 8% and oxacillin showed the lowest level of bacterial survival. At 5 days, MSM 13-16% and antibiotic showed the lowest level of bacterial survival. Taken together, the results indicate that MSM increased the sensitivity of this strain of MRSA to oxacillin and methicillin.

Fig 4 illustrates that MSM sensitizes MRSA to multiple concentrations of oxacillin. In vitro survival of Staphylococcus aureus strain ATCC 43300 (an oxacillin and methicillin resistant MRSA strain) was studied in the presence of MSM and oxacillin. Three different concentrations of oxacillin, corresponding to 2x, 5x and 10x MIC were tested for this strain of MRSA for each antibiotic. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM and the indicated amount of antibiotic for 24 hours at 25 ° C. In this way, the bacteria were at 2x, 5x, or 10x MIC concentrations of oxacillin for 24 hours. The initial inoculation of bacteria was 3.3x10⁵/ ml (log = 5.52). At this time point, lower concentrations of MSM with antibiotics had a greater effect on bacterial survival than higher concentrations of MSM. Taken together, however, these results confirm that MSM sensitizes MRSA to oxacillin.

Fig 5 illustrates that MSM sensitizes MRSA to multiple concentrations of methicillin. In vitro survival of Staphylococcus aureus strain ATCC 43300 (an oxacillin and methicillin resistant MRSA strain) was tested in the presence of MSM and methicillin. Three different concentrations of methicillin, corresponding to 2x, 5x and 10x MIC were tested for this strain of MRSA for each antibiotic. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM and the indicated amount of antibiotic for 24 hours at 25 ° C. In this way, the bacteria were at 2x, 5x, or 10x MIC concentrations of methicillin for 24 hours. The initial inoculation of bacteria was 1.49x10⁵/ ml (log = 5.17). At this time point, lower concentrations of MSM with antibiotics had a greater effect on bacterial survival than higher concentrations of MSM. Taken together, these results confirm that MSM sensitizes MRSA to methicillin.

Detailed description

I. Terms and abbreviations

CFU colony forming units

DMEM: Dulbecco's Modified Eagle Medium

DMSO: dimethylsulfoxide

DNA: deoxyribonucleic acid

ELISA enzyme-linked immunosorbent assay

IC50: inhibitory concentration 50

LAB: lactic acid bacteria

MDSA: Staphylococcus aureus with multidrug resistance

MDR: multi-drug resistance

MIC: minimum inhibitory concentration

MRSA: Methicillin-resistant Staphylococcus aureus

MSM: methylsulfonylmethane

OSRA: Oxacillin-resistant Staphylococcus aureus

PAGE: polyacrylamide gel electrophoresis

PBP penicillin-binding protein

PBS: phosphate buffered saline

PDA: potato dextrose agar

SDS: sodium dodecyl sulfate

TNTC: too numerous to count

TSB: Tryptic Soy Broth

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art of the present disclosure. The singular forms un, uno / una, refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a bacterial cell" includes single or multiple bacterial cells and is considered equivalent to the phrase "comprising at least one bacterial cell." The term either refers to a single element or defines alternative elements, or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprise" means "includes." Therefore, "comprising A or B" means that it includes A, B, or A and B, without excluding additional elements.

Unless otherwise explained, all technical and scientific terms used herein have generally the same meaning as commonly understood by a person of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those may be used

ES 2 674 019 T3 described herein in the practice or testing of the present disclosure, suitable methods and materials are described below. Materials, methods, and examples are merely illustrative. For example, standard methods well known in the art to which the disclosed invention pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^to ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and supplements up to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999; Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.

Additional terms used in molecular genetics can be found in Benjamin Lewin, Genes V published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Additional terms used in chemistry can be found in Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.

Administration: Providing or administering to a subject, cell, or surface, a compound or agent, such as MSM, or a composition comprising a compound or agent, such as MSM, by any effective route. Illustrative routes of administration to a surface include spraying or rubbing an agent or composition containing an agent onto the surface. Illustrative routes of administration to a subject include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal (such as topical), intranasal, vaginal, and by inhalation. Specific types of administration to a subject include topical administration or administration to the nasal mucosa or lungs by inhalation administration.

Agent: Any substance or any combination of substances that is useful to achieve an end or result; for example, a substance or combination of substances useful for inhibiting bacterial growth or survival. Examples of agents include MSM, DMSO, and beta-lactam antibiotics, among others. The agents include antimicrobial agents, which are useful for inhibiting a microbe. Antibiotic agents are useful for inhibiting bacteria.

Bacterial pathogen: A disease-causing bacteria (pathogenic bacteria). Examples of pathogenic bacteria for which MSM can be used to modify include without limitation any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (Such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and uropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis, and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans,

ES 2 674 019 T3

Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica, and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari, and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsug amushi), and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus pneumoniae (for example Streptococcus pneumoniae serotype 4 resistant to chloramphenicol, Streptococcus pneumoniae serotype 6B resistant to spectinomycin, Streptococcus pneumoniae serotype 9V resistant to streptomycin, Streptococcus pneumoniae pneumoniae resistant serotype 14, Streptococcus pneumoniae resistant serotype 14 serotype 14 rifampicin-resistant serotype 18C, Streptococcus pneumoniae tetracycline-resistant serotype 19F, Streptococcus pneumoniae serotype 19F resistant to penicillin, and Streptococcus pneumoniae serotype 23F resistant to trimethoprim, Streptococcus pneumoniae serotype 4 resistant to chloramphenicol, Streptococcus pneumoniae serotype 6B resistant to spectinomycin, Streptococcus pneumoniae resistant to streptocokine serotype 9, Streptococcus pneumoniae pneumoniae resistant Streptocokine serotype 9 pneumoniae serotype 18C resistant to rifampin, Streptococcus pneumoniae serotype 19F resistant to penicillin, or Streptococcus pneumoniae serotype 23F resistant to trimethoprim), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A Streptococci, Streptococcus Streptococcus, Group C Streptococcus, Streptococcus agalactiae, Group C Streptococcus Streptococcus, Group C Streptococcus Streptococcus , Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group G Streptococci and Streptococcus anginosus Group G Streptococci, Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela, and Vibrio furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis) and Xanthomonas maltophilia among others. As used herein, bacterial pathogens do not include Mycobacterium tuberculosis, or other bacteria that can cause tuberculosis.

Beta-lactam antibiotics: A class of antibiotics that contain a beta-lactam core in their molecular structure. Examples of beta-lactam antibiotics include, penicillin derivatives, cephalosporins, penises, monobactams, carbapenenes, beta-lactamase inhibitors, and combinations thereof. Examples of derivatives of penicillin include, aminopenicillins (eg, amoxicillin, ampicillin, and epicillin); carboxypenicillins (eg, carbenicillin, ticarcillin, and thermocillin); ureidopenicillins (eg, azlocillin, piperacillin, and mezlocillin); mecillinam, sulbenicillin, benzathine penicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), penicillin O (allylmercaptomethylpenicillin), procaine penicillin, oxacillin, methicillin, nafcillin, fluoxacillin, tacampillin, hexacillin, fluoxacillin, nafcillin, fluoxacilin, cloxacillin co-amoxiclav (amoxicillin plus clavulanic acid), and piperacillin. Examples of cephalosporins include, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandeol, cefotetan, cefoxitin, ceforanide, ceftriaxone, cefotaxime, cefpodoxime proxetil, ceftazidime, cefepime, cefuroxime, cephimepyroma, and ceftizopyroma. Examples of penemos include faropenemo. Examples of monobactams include aztreonam and thygemono. Examples of carbapenems include, biapenenvdoripenem, ertapenem, imipenem, meropenem, and panipenem. Examples of beta-lactamase inhibitors include, tazobactam sodium salt of 4.4 tazobactam acid dioxide ([2S- (2alpha, 3beta, 5alpha)] - 3-methyl-7-oxo-3- (1H-1, 2,3-triazol-1-ylmethyl) -4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid), sulbactam sodium salt of 4,4-dioxide (2S, 5R) -3,3-dimethyl -7-oxo-4-thia-1azabicyclo [3.2.0] heptane-2-carboxylic acid), and clavulanic acid ((2R, 5R, Z) -3- (2-hydroxyethitidene) -7-oxo-4-oxa acid -1aza-bicyclo [3.2.0] heptane-2-carboxylic).

Biological activity: An expression that describes the beneficial or adverse effects of a substance on living matter. When the agent is a complex chemical mixture, this activity is exerted by the active principle of the substance or pharmacophore, but it can be modified by the rest of the components. Activity is generally dose dependent and it is not uncommon for it to have effects ranging from beneficial to adverse for the same substance when going from low to high doses. In one example, MSM alters, such as increases or decreases the biological activity of a microorganism, such as bacteria.

Biological sample: A biological specimen containing genomic DNA, RNA (including mRNA), protein, whole cells, cell membranes, or combinations thereof, obtained from a subject. Examples include, but are not limited to, mucus, peripheral blood, urine, saliva, tissue biopsy, needle aspirates, surgical specimens, amniocentesis specimens, and autopsy material. In one example, a sample includes a tissue biopsy obtained from a subject with a bacterial infection, such as an MRSA infection or

ES 2 674 019 T3

Staphylococcus aureus. In another example, a sample includes a mucus sample obtained from a subject with a bacterial infection of the lungs, such as a MRSA or Staphylococcus aureus infection.

Composition (or formulation): A chemical compound or mixture of compounds capable of inducing a desired effect when properly administered. Compositions usually include at least one agent. An industrial composition is a compound or chemical composition capable of inducing a desired effect when properly administered to a surface. A pharmaceutical composition is a compound or chemical composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject, or a cell. In many cases, an industrial composition could also be administered to a subject. In many cases, a pharmaceutical composition could also be delivered to a surface. In a particular example, a composition includes an agent or agents that inhibit a drug-resistant bacterial pathogen. For example, a composition can include MSM and an antimicrobial agent. A composition can include MSM and methicillin or oxacillin. Some embodiments provide compositions that include MSM without DMSO.

Putting in contact: Placement in indirect physical association; including in solid, liquid, and gaseous form. Contacting includes contact between one molecule and another molecule. Contact can occur in vitro with isolated cells or tissue or in vivo by administration to a subject.

Control: Samples considered normal (for example, representative activity or function in the absence of the variable being tested) as well as laboratory values, although they may have been arbitrarily established, taking into account that these values ​​may vary from lab to lab. A control group is practically identical to the treatment group, except for the single variable of interest whose effect is being tested, which applies only to the treatment group.

Decrease or inhibition: Reduce the quality, quantity, or concentration of something. In one example, the administration of MSM decreases or reduces one or more biological activities, such as growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action, and / or function of the microorganisms by at least 10%. , at least 20%, at least 50%, or even at least 90%, including between 10% to 95%, 20% to 80%, 30% to 70%, 40% to 50%, such as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100%. Said decrease can be measured using the methods disclosed herein as well as those known to a person of ordinary skill in the art. MSM can be used to inhibit the survival of specific microorganisms. Log scale reductions can be seen after the first 24 hours.

Dimethyl sulfoxide (DMSO): Dimethyl sulfoxide (DMSO), also known as methylsulfonylmethane or methyl sulfoxide, is an organosulfur compound with the formula (CH3) 2SO. This colorless liquid is a polar aprotic solvent that dissolves polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water. It has a distinctive property of penetrating the skin very easily, such that one can taste it after coming into contact with the skin. DMSO is well known as a nutritional supplement and as a pharmaceutical agent. One of skill in the relevant art will be familiar with these uses. Various grades of DMSO are commercially available (for example, product # 472301 from Sigma-Aldrich, Corp., St. Louis, MO) and a person skilled in the art will be familiar with the sources of DMSO.

Drug-resistant bacterial pathogen: This term refers to a bacterial pathogen that is resistant to one or more antimicrobial agents. Drug resistant refers to both partial and complete resistance. For example, MRSA is a drug-resistant bacterial pathogen that is resistant to beta-lactam antibiotics. A bacterial pathogen can be both drug resistant and drug sensitive if it is sensitive to one antimicrobial agent, but resistant to another. As used herein, a subject who comes into contact with a drug-resistant bacterial pathogen is a subject with a drug-resistant bacterial infection. As used herein, the drug-resistant bacterial pathogen does not include drug-resistant Mycobacterium tuberculosis, or other drug-resistant bacterial pathogen that can cause tuberculosis.

Drug-Sensitive Bacterial Pathogen: This term refers to a bacterial pathogen that is sensitive to one or more antimicrobial agents. A bacterial pathogen can be both drug resistant and drug sensitive if it is sensitive to one antimicrobial agent, but resistant to another. In some embodiments, a bacterial pathogen resistant to a particular antimicrobial agent can become sensitive to this agent if the bacterial pathogen is contacted with MSM. As used herein, a subject who comes into contact with a drug-sensitive bacterial pathogen is a subject with a drug-sensitive bacterial infection. As used herein, the drug-sensitive bacterial pathogen does not include Mycobacterium tuberculosis, drug-sensitive, or other drug-sensitive bacterial pathogen that causes tuberculosis. Empowerment or increase: Increase in the quality, quantity, or concentration of something.

Fungal pathogen: A fungus that causes disease. Examples of fungal pathogens for which MSM can be used to modify include, but are not limited to one or more of (or any combination of)

ES 2 674 019 T3

Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, Pityrosporum orbiculare (Malassezia furfur), Candida sp. (such as Candida albicans), Aspergillus sp. (such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor and Aspergillus clavatus), Cryptococcus sp. (such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii, and Cryptococcus albidus), Coccidioides sp., Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii), Stachybotrys sp. (such as Stachybotrys chartarum), Paracoccidioides, Blastomyce, Fusarium, Sporothrix, Trichosporon, Rhizopus, Pseudallescheria, Paecilomyces, Alternaria, Curvularia, Exophiala, Wangiella, Penicillium, and Cephalosphorium. In some embodiments, MSM is administered to inhibit or prevent an infection or disorder associated with one or more of the aforementioned fungal pathogens.

Incubation: A term that includes a sufficient period of time for an agent, such as MSM, to interact with something, such as a cell or tissue.

Inhaler Device: A device capable of delivering a composition to a subject, eg, to the lung tissue of a subject. For example, an inhaler device can be an inhaler, a nebulizer, or a ventilator. The inhaler devices described herein are constructed of a material adapted to contact DMSO and / or MSM. In some embodiments, an inhaler device is disposable or replaceable. The inhaler devices described herein are configured to deliver a composition containing DMSO or MSM to directly contact bacterial pathogens in the lung tissue of a subject. Inhaler devices are configured to generate particles of a composition that varies in size. In some embodiments, an inhaler device is configured to generate particles of a composition ranging in size from about 0.1 pm to about 10 pm or from about 0.5 pm to about 5 pm.

Inhibition or treatment of an infection or disease: Inhibiting the full development of an infection, disease or condition, for example, in a subject who is at risk of developing an infection, such as a bacterial infection. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition once it has begun to develop. As used herein, the term "ameliorate", with reference to a disease, condition, or pathological symptom, refers to any observable beneficial effect of the treatment. The beneficial effect may be evidenced, for example, by a delayed onset of the clinical symptoms of the disease / infection in a susceptible subject, a reduction in the severity of some or all of the clinical symptoms of the disease / infection, a slower progression of the disease / infection, a reduction in the number of relapses of the disease / infection, an improvement in the health or general well-being of the subject, or by other parameters well known in the art that are specific to a particular disease / infection, such as a bacterial infection.

Methicillin-resistant Staphylococcus aureus (MRSA): A Staphylococcus aureus bacteria that has complete or partial (or intermediate) resistance to one or more beta-lactam antibiotics. MRSA is also called Multi-Drug Resistance Staphylococcus aureus (MDSA), Oxacillin Resistant Staphylococcus aureus (ORsA), or Golden Staph. In some embodiments, contacting MRSA with MSM renders MRSA sensitive to a beta-lactam antibiotic that was resistant prior to contact with MSM. Using the Etest® system to determine antibiotic sensitivity, MRSA shows a MIC of at least 2 pg / ml for oxacillin (see for example, Etest® technical manual, AB bioMerieux, 2008).

Methylsulfonylmethane (MSM): An organosulfur compound with the formula (CH3) 2SO2. MSM is also known as DMSO2, dimethyl sulfone, methyl sulfone, and sulfonylbismethane. MSM has been marketed and sold primarily as a diet supplement.

MSM is structurally related to dimethyl sulfoxide (DMSO), but the behavior of the two is different. DMSO is a very polar solvent and an excellent ligand, with dissolution properties similar to water while MSM is less polar and less reactive. MSM is also a metabolite of DMSO. MSM has the following chemical structure:

, .iCH₃ * ch₃

Microorganism: A member of the prokaryotic or eukaryotic microbial species of the Archaea, Bacteria, and

ES 2 674 019 T3

Eucarya, including the latter yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms microbial cells and microbes are used interchangeably with the term microorganism.

Minimum Inhibitory Concentration (MIC): The lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm the resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents.

Modulate or modulation: Adjust, alter, regulate an activity, a degree or an index of it, including an increase or decrease in the biological activity of a molecule. In one example, MSM is administered to modulate bacterial sensitivity to antimicrobial agents.

Parasite: An organism that lives inside humans or other organisms that act as hosts (for the parasite). Parasites are dependent on their hosts for at least part of their life cycle. Parasites are harmful because they consume necessary food, consume body tissues and cells, and eliminate toxic waste, making people sick. Examples of fungal pathogens for use with the disclosed methods and compositions include, but are not limited to, any one or more of (or any combination of) Malaria (Plasmodium falciparum, P. vivax, P. malariae), Schistosomes, Trypanosomes , Leishmania, Filariae type nematodes, Trichomoniasis, Sarcosporidiasis, Tapeworm (T. saginata, T. solium), Leishmania, Toxoplasma gondii, Trichinellosis (Trichinella spiralis) or Coccidiosis (Eimeria species). MSM can be used to inhibit or prevent the activity of one or more of the organisms listed above.

Pharmaceutically acceptable carriers or vehicles: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19^to Edition (1995), describes compositions and compositions suitable for the pharmaceutical administration of one or more therapeutic compounds or molecules, such as one or more peptides provided herein. In general, the nature of the carrier will depend on the particular mode of administration that is being employed. For example, parenteral compositions comprise injectable fluids including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt sera, aqueous dextrose, glycerol, or the like as a carrier. In a particular embodiment, the carrier is one that allows the therapeutic compound to cross the dermal layer. For solid compositions (eg, in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

Quantification: Determining or measuring an amount (such as a relative amount) of a molecule or the activity of a molecule, such as the amount of analyte present in a sample.

Sensitization of a drug-resistant microorganism: Any means of decreasing the drug resistance of a microorganism, including bacterial pathogens. This includes modifications to the microorganism as well as the use of an agent that increases the effectiveness of another agent to inhibit the microorganism. For example, sensitization of MRSA to methicillin includes modulating a MRSA in such a way that it is not as resistant to methicillin, as well as, contacting the MRSA with an agent that reduces the resistance of MRSA to methicillin, for example, by contacting MRSA with MSM.

Staphylococcus aureus: A round-celled, Gram-positive coconut, approximately 1 pm in diameter, that forms cluster-like clusters of grape clusters indicating the ability to divide in more than one plane. They are capable of aerobic and anaerobic respiration and most strains ferment mannitol anaerobically. On blood agar they form characteristic golden or white colonies. They produce catalase, coagulase, and an extracellular cell agglutination factor, and some strains produce capsules (see Brown et al, J. Antimicrob. Chemother., 56: 1000-1018, 2005).

Subject: Living multicellular vertebrate organisms, a category that includes human and non-human mammals. In a subject refers to substances or microorganisms (eg, bacterial pathogens) that come into contact with a subject or are in physical connection with a subject.

Subject susceptible to disease or disease: A subject capable of, prone to, or predisposed to developing a disease or disease. It is understood that a subject who already has or shows symptoms of a disease or condition is considered susceptible because he has already developed it.

Surface: The outer layer of a physical object.

ES 2 674 019 T3

Symptoms and Signs: Any subjective evidence of disease or illness in a subject, for example, such evidence as perceived by the subject; a significant change in the ailment of a subject indicative of some bodily or mental state. A sign is any abnormality indicative of disease, which can be discovered during an examination or evaluation of a subject. A sign is generally an objective indication of disease. Signs include, but are not limited to, any measurable parameters such as tests for a disorder or disease, such as a bacterial or viral infection. In one example, reducing or inhibiting one or more symptoms or signs associated with a bacterial or viral infection includes reducing or inhibiting bacterial survival or viral infection by a desired amount, for example by at least 20%, at least 50%. %, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to the bacterial survival or viral infectivity in the absence of MSM.

Therapeutically effective amount or concentration: An amount of a composition that alone, or in conjunction with additional agent (s), is sufficient to achieve a desired effect in a subject, to which the composition is administered. The effective amount of an agent or composition will depend on a number of factors, including, but not limited to, the subject, the cells or surface to which the agent or composition is administered, and the mode of administration. In one example, a therapeutically effective amount or concentration is one that is sufficient to inhibit a bacterial pathogen, eg, a drug-resistant bacterial pathogen such as MRSA.

In one example, a desired effect is to reduce or inhibit one or more symptoms associated with a disease. For example the symptoms associated with a MRSA infection. The one or more symptoms do not have to be completely eliminated for the composition or agent to be effective. For example, a composition or agent can decrease signs or symptoms by a desired amount, for example by at least 20%, at least 50%, at least 80%, at least 90%, at least 95%. , at least 98%, or even at least 100%, compared to the signs or symptoms in the absence of the composition or agent. In one example, a desired effect is to reduce or inhibit a microorganism (such as survival of the microorganism) by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least a 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to the survival of the microorganism in the absence of the composition or agent. In another example, a desired effect is to sensitize a drug resistant microorganism to the drug to which the microorganism is resistant in a desired amount, for example by at least 20%, at least 50%, at least 60%, to the less 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to the sensitivity of the microorganism to the drug in the absence of the composition or agent.

A therapeutically effective amount of a disclosed composition or agent can be administered in a single dose, or in several doses, eg, daily, during a treatment cycle. However, the therapeutically effective amount may depend on the subject or cell or surface to which the composition or agent is administered, the severity and type of the condition being treated, and the mode of administration. A therapeutically effective amount of an agent or composition can be measured as the concentration (moles per liter or molar M or weight per volume or other unit of concentration) of the agent or composition in blood (in vivo) or buffer (in vitro), among others. , which produces the desired effect (s). Alternatively, a therapeutically effective amount of an agent or composition can be measured as the amount administered to a subject per body weight of the subject, eg, mg agent / kg body weight.

Untreated cell: A cell that has not been contacted with a desired agent, such as MSM. In one example, an untreated cell is a cell that receives the vehicle in which MSM has been administered.

Virus: A microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a nucleic acid nucleus surrounded by a protein coating, and has the ability to replicate only within a living cell. Viral replication is the production of additional viruses by the appearance of at least one life cycle of the virus. A virus can disrupt the normal functions of host cells, causing the cell to behave in a way determined by the virus. For example, a viral infection can result in a cell producing a cytokine, or sensitization to a cytokine, when the uninfected cell normally does not. In some examples, a virus is a pathogen.

Specific examples of viral pathogens that could be treated in accordance with the disclosed methods and compositions include, but are not limited to, any one or more of (or any combination of); Arenavirus (such as Guanarito virus, Lassa virus, Junin virus, Machupo and Sabia virus), Arterivirus, Ronivirus, Astrovirus, Bunyavirus (such as Crimean-Congo hemorrhagic fever virus and Hantavirus), Barnavirus, Birnavirus, Bornavirus (such as Borna disease virus), Bromovirus, Calicivirus, Chrysovirus, Coronavirus (such as Coronavirus and SARS), Cistovirus, Closterovirus, Comovirus, Dicistrovirus, Flavirus (such as yellow fever virus, West Nile virus, Hepatitis C virus, and Dengue fever virus), Filovirus (such as Ebola virus and Marburg virus), Flexivirus, Hepevirus (such as Hepatitis E virus), Human adenovirus ( such as human adenoviruses AF), human astroviruses, human BK polyomaviruses, human bocaviruses, human coronaviruses (such as human coronaviruses HKU1, NL63, and OC43), human enteroviruses (such as human AD enteroviruses), human erythrovirus V9, human foam viruses, human herpesviruses

ES 2 674 019 T3 (such as human herpesvirus 1 (herpes simplex virus type 1), human herpesvirus 2 (herpes simplex virus type 2), human herpesvirus 3 (varicella zoster virus), herpesvirus 4 type Human 1 (Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barr virus type 2), human herpesvirus 5 strain AD169, human herpesvirus 5 Merlin strain, human herpesvirus 6a, herpesvirus 6B human, human herpesvirus 7, human herpesvirus 8 type M, Human P-type herpesvirus 8 and human cytomegalovirus), human immunodeficiency virus (HIV) (such as HIV 1 and HIV 2), human metapneumovirus, human papillomavirus (such as human papillomavirus-1, human papillomavirus-18, papillomavirus- 2 human, human papillomavirus-54, human papillomavirus-61, human papillomavirus-cand90, human papillomavirus RTRX7, human papillomavirus type 10, human papillomavirus type 101, human papillomavirus type 103, human papillomavirus type 107, human papillomavirus type 16, human papillomavirus type 24, human papillomavirus type 26, human papillomavirus type 32, human papillomavirus type 34, human papillomavirus type 4, human papillomavirus type 41, human papillomavirus human type 48, human type 49 papillomavirus, human type 5 papillomavirus, human type 50 papillomavirus, human type 53 papillomavirus, human type 60 papillomavirus, human type 63 papillomavirus, human papillomavirus type 6b, human papillomavirus type 7, human papillomavirus type 71, human papillomavirus type 9, human papillomavirus type 92, and human papillomavirus type 96), human parainfluenza viruses (such as parainfluenza virus 1-3 human), human paraecoviruses, human parvoviruses (such as human parvovirus 4 and human parvovirus B19), human respiratory syncytial virus, human rhinovirus (such as human rhinovirus A and human rhinovirus B), Human Foam Retroviruses, Human T Lymphotropic Viruses (such as Human T 1 Lymphotropic Virus and Human T 2 Lymphotropic Virus), Human Polyoma Virus, Hypovirus, Levivirus, Luteovirus, Lymphocytic Choriomeningitis Virus (LCM), Mamavirus, Namavirus, Nidoviral, Nodavirus , Orthomyxoviruses (such as influenza viruses), Partitivirus, Paramyxoviruses (such as mumps virus and measles virus), Picornaviruses (such as Poliovirus, the common cold virus, and Hepatitis A virus), Potivirus, Poxvirus (such as smallpox virus and cowpox virus), Sequivirus, Reovirus (such as Rotavirus), Rhabdovirus (such as rabies virus), Rhabdovirus (such as Vesicular Stomatitis Virus, Tetravirus, Togavirus (such as Rubella Virus and Ross River Virus), Tombusvirus, Totivirus, Thymovirus, and Norovirus among others.

In some aspects of the disclosure, MSM is used to inhibit the biological activity of one or more of the viruses listed above.

Yeast: A eukaryotic microorganism classified in the kingdom of fungi, with approximately 1,500 described species. Most reproduce sexually by budding, although a few reproduce by binary fission. Yeasts are generally unicellular, although some species can become multicellular by forming a ring of connected budding cells known as pseudohyphae, or false hyphae. Illustrative yeasts that can be used in the disclosed methods and compositions include, but are not limited to Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Pichia, Cryptococcus, Zygosaccharomyces, Torulopsis, Hansenula, and Debaryomyces.

II. Overview of some realizations

Disclosed herein is a method for inhibiting a drug resistant bacterial pathogen, which method involves selecting a drug resistant bacterial pathogen; and contacting the bacterial pathogen with a composition comprising a therapeutically effective amount of MSM and a therapeutically effective amount of an agent that inhibits the drug-sensitive form of the drug-resistant bacterial pathogen, thereby inhibiting a drug-resistant bacterial pathogen. In some embodiments, the drug resistant bacterial pathogen is MRSA.

Also disclosed is a method of sensitizing a drug-resistant bacterial pathogen to the drug to which the bacterial pathogen is resistant, which method comprises selecting a drug-resistant bacterial pathogen; and contacting the bacterial pathogen with a composition comprising a therapeutically effective amount of MSM, thereby sensitizing the drug-resistant bacterial pathogen to the drug to which the bacterial pathogen is resistant. In some embodiments, the drug resistant bacterial pathogen is MRSA.

Also disclosed herein is a method of inhibiting a drug-sensitive bacterial pathogen from developing drug resistance, which method comprises selecting a drug-sensitive bacterial pathogen; and contacting the drug-sensitive bacterial pathogen with a composition comprising a therapeutically effective amount of MSM and a therapeutically effective amount of an agent that inhibits the drug-sensitive bacterial pathogen, thereby inhibiting the drug-sensitive bacterial pathogen from developing drug resistance. In some embodiments, the drug-sensitive bacterial pathogen is Staphylococcus aureus.

In the methods described herein, the agent is a beta-lactam antibiotic. In some embodiments, the agent comprises penicillin derivatives, cephalosporins, penises, monobactams, carbapenenes, beta-lactamase inhibitors, or combinations thereof. In some embodiments, the agent comprises methicillin or oxacillin. In various embodiments, the therapeutically effective amount of a

ES 2 674 019 T3 agent comprises about 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or about 90 -100 MIC of a beta-lactam antibiotic. In some embodiments, the therapeutically effective amount of an agent comprises about 0.001, 0.01, 0.1, 0.5, or 1 MIC of a beta-lactam antibiotic.

In various embodiments of the methods described herein, the bacterial pathogen is in a subject. In some embodiments, the bacterial pathogen is on a surface.

In various embodiments, the effective amount of MSM is 5-20% MSM, 5-16% MSM, 5-10% MSM, 5-8% MSM, 9-16% MSM, or 10-15%. by MSM. MSM (percent by weight of the composition comprising MSM). In other embodiments, the effective amount of MSM is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16% MsM. In still other embodiments, the effective amount of MSM is 10-16% MSM.

In various embodiments, the bacterial pathogen is contacted with the composition for 24, 36, 48, 60, 72, 84, 96, 108, or 120 hours.

In some embodiments, the composition is administered topically or with an inhaler device. In some embodiments, the composition comprises 0-5% sodium chloride.

For example, in a particular embodiment of a method for inhibiting a drug-resistant bacterial pathogen, the bacterial pathogen is MRSA, the agent is a beta-lactam antibiotic, the effective amount of MSM is about 5-10% MSM in percent. in weigh.

For example, in a particular embodiment of a method for sensitizing a resistant bacterial pathogen to a drug to which the bacterial pathogen is resistant, the bacterial pathogen is MRSA, the effective amount of MSM is about 5-10% MSM by weight percent. .

For example, in a particular embodiment of a method for inhibiting a drug-sensitive bacterial pathogen from developing drug resistance, the bacterial pathogen is Staphylococcus aureus, the agent is a beta-lactam antibiotic, the effective amount of MSM is 5-10 % MSM and the composition is administered topically.

It will be further understood that the methods for sensitizing or inhibiting bacterial pathogens disclosed herein are useful beyond the specific circumstances that are described in detail herein, and are expected, for example, that are useful for any of numerous conditions in which a bacterial pathogen has become drug resistant or where it is desirable to inhibit a drug-sensitive bacterial pathogen from becoming drug resistant.

III. MSM

MSM is an organosulfur compound with the formula (CH3) 2SO2. MSM is structurally related to dimethyl sulfoxide (DMSO), but the behavior of the two is different. DMSO is a very polar solvent and an excellent ligand, with dissolution properties similar to water while MSM is less polar and less reactive. MSM is well known as a nutritional supplement and as a pharmaceutical agent (see, for example, Jacob and Appleton, MSM; the definitive guide: A comprehensive review of the science and therapeutics of Methylsulfonylmethane, Topanga, CA: Freedom Press, 2003). MSM is also known to be useful for the treatment of osteoarthritis (Kim et al., Osteoarthritis Cartilage, 14: 286-94, 2006) and hay fever (Barrager et al., J. Altern. Complement. Med., 8 : 167-74, 2002). One of skill in the relevant art will be familiar with these uses. Various grades of MSM are commercially available (eg, OptiMSM® available from Bergstrom Nutrition, Corp., Vancouver, WA); a person skilled in the art will be familiar with the source of MSM. MSM is very soluble in water. At room temperature, aqueous solutions of 20% MSM can be easily prepared. Aqueous solutions of higher MSM concentrations are possible at temperatures elevated above room temperature.

IV. DMSO

Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula (CH3) 2SO. This colorless liquid is a polar aprotic solvent that dissolves polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water. It has a distinctive property of penetrating the skin very easily, such that one can taste it after it has come into contact with the skin. DMSO is well known as a nutritional supplement and as a pharmaceutical agent. One of skill in the relevant art will be familiar with these uses. Various grades of DMSO are commercially available (eg, product # 472301 from Sigma-Aldrich, Corp., St. Louis, MO) and one of ordinary skill in the art will be familiar with the sources of DMSO.

V. Beta-lactam antibiotics

ES 2 674 019 T3

Beta-lactam antibiotics are a broad class of antibiotic agents, consisting of all antibiotic agents that contain a beta-lactam core in their molecular structure. This class of antibiotics is the most widely used group of antibiotics; a person skilled in the relevant art will understand how to select a suitable beta-lactam antibiotic for use to inhibit or treat a bacterium.

Examples of beta-lactam antibiotics include, penicillin derivatives, cephalosporins, penises, monobactams, carbapenenes, beta-lactamase inhibitors, and combinations thereof. Examples of derivatives of penicillin include, aminopenicillins (eg, amoxicillin, ampicillin, and epicillin); carboxypenicillins (eg, carbenicillin, ticarcillin, and thermocillin); ureidopenicillins (eg, azlocillin, piperacillin, and mezlocillin); mecillinam, sulbenicillin, benzathine penicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), penicillin O (allylmercaptomethylpenicillin), procaine penicillin, oxacillin, methicillin, nafcillin, fluoxacillin, tacampillin, hexacillin, fluoxacillin, nafcillin, fluoxacilin, cloxacillin co-amoxiclav (amoxicillin plus clavulanic acid), and piperacillin. Examples of cephalosporins include, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandeol, cefotetan, cefoxitin, ceforanide, ceftriaxone, cefotaxime, cefpodoxime proxetil, ceftazidime, cefepime, cefuroxime, cephimepyroma, and ceftizopyroma. Examples of penemos include faropenemo. Examples of monobactams include aztreonam and thygemono. Examples of carbapenems include, biapenenvdoripenem, ertapenem, imipenem, meropenem, and panipenem. Examples of beta-lactamase inhibitors include, tazobactam sodium salt of 4.4 tazobactam acid dioxide ([2S- (2alpha, 3beta, 5alpha)] - 3-methyl-7-oxo-3- (1H-1, 2,3-triazol-1-ylmethyl) -4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid), sulbactam sodium salt of 4,4-dioxide (2S, 5R) -3,3-dimethyl -7-oxo-4-thia-1azabicyclo [3.2.0] heptane-2-carboxylic acid), and clavulanic acid ((2R, 5R, Z) -3- (2-hydroxyethitidene) -7-oxo-4-oxa acid -1aza-bicyclo [3.2.0] heptane-2-carboxylic). These antibiotics are commercially available and one of ordinary skill in the art will be familiar with the source of each of the antibiotics disclosed herein.

Beta-lactam antibiotics are bactericidal, and work by inhibiting the synthesis of the peptidoglycan layer on the walls of bacterial cells. These antibiotics act by irreversible binding to penicillin-binding proteins (PBPs); this union disturbs the synthesis of the cell wall, preventing cell division.

All beta-lactam antibiotics have a beta-lactam ring in their structure. The efficacy of these antibiotics is based on their ability to reach intact PBPs and their ability to bind to PBPs. Thus, there are two main modes of bacterial resistance to beta-lactams.

First, bacteria sometimes develop resistance to beta-lactam antibiotics by synthesizing beta-lactamase, an enzyme that attacks the beta-lactam ring. If the bacteria produce the enzyme beta-lactamase (or the enzyme penicillinase), the enzyme will open the beta-lactam ring of the antibiotic, rendering the antibiotic ineffective. The genes encoding these enzymes can be inherently present on the bacterial chromosome or can be acquired by plasmid transfer (plasmid-mediated resistance), and beta-lactamase gene expression can be induced by exposure to beta-lactams. The production of a beta-lactamase by a bacterium does not necessarily rule out all treatment options with beta-lactam antibiotics. For example, to overcome this type of resistance, beta-lactam antibiotics are often administered with beta-lactamase inhibitors such as clavulanic acid. However, in all cases where infection with beta-lactamase-producing bacteria is suspected, the selection of an appropriate beta-lactam antibiotic should be carefully considered before treatment. In particular, the choice of a suitable beta-lactam antibiotic treatment is of utmost importance against organisms with inducible beta-lactamase expression. If beta-lactamase production is inducible, then failure to use the most appropriate beta-lactam treatment at the start of treatment will result in induction of beta-lactamase production, thus hindering further efforts with other beta-lactam antibiotics lactam.

Second, a bacterium can express an altered PBP, to which beta-lactams cannot bind as efficiently as an unaltered PBP. As a result, beta-lactams are less effective in disrupting cell wall synthesis. Notable examples of this mode of resistance include penicillin resistant Streptococcus pneumoniae and MRSA.

SAW. MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) is a Staphylococcus aureus bacteria that has resistance to one or more beta-lactam antibiotics. The level of resistance to the beta-lactam antibiotic can vary depending on the strain of MRSA. For example, a particular MRSA strain may have complete or partial resistance to a beta-lactam antibiotic.

Staphylococcus aureus bacteria resistance to the beta-lactam antibiotic is generally due to the expression of an altered PBP, such as PBP2a or PBP2 ', which has low affinity for beta-lactam antibiotics and may function in place of native PBP. PBP2a is encoded by the mecA gene (see Brown et al., J. Antimicrob. Chemother., 56: 1000-1018, 2005). Additional genes, which are also found in susceptible isolates, can alter the expression of methicillin resistance in Staphylococcus aureus, resulting in heterogeneity of resistance (see Brown et al., J. Antimicrob. Chemother., 56: 1000 -1018,

ES 2 674 019 T3

2005).

MRSA infection is caused by a Staphylococcus aureus bacteria that has complete or partial resistance to one or more beta-lactam antibiotics. Staphylococcus aureus most commonly colonizes the anterior nares (the nasal passages), although the respiratory tract, open wounds, intravenous catheters, and the urinary tract are also potential sites for infection. Healthy individuals can carry MRSA asymptomatically for periods ranging from a few weeks to many years. Patients with compromised immune systems are at significantly increased risk of symptomatic secondary infection.

MRSA can progress substantially within 24-48 hours from the initial symptoms, including the initial topical symptoms. After 72 hours, MRSA takes root in human tissues and eventually becomes resistant to treatment. Topically, the initial presentation of MRSA is small red bumps that resemble pimples, spider bites, or boils that may be accompanied by fever and occasionally rashes. Within a few days, the bumps become larger, more painful, and eventually break open into deep pus-filled boils.

Many strains of MRSA are known to exist, including hospital (or medical facility) associated MRSA (HAMRSA) and community associated MRSA (CA-MRSA). Approximately 75 percent of CAMRSA infections are localized to the skin and soft tissue and can usually be effectively treated. However, some strains of CA-MRSA show increased virulence, spreading more rapidly and causing much more serious disease than traditional HA-MRSA infections, and can affect vital organs and lead to generalized infection (septicemia), shock syndrome toxic and necrotizing (carnivorous) pneumonia. This is believed to be due to toxins carried by CA-MRSA strains. It is not known why some healthy people develop treatable cA-MRSA skin infections while others infected with the same strain develop serious infections or die.

The most common manifestations of CA-MRSA are skin infections such as necrotizing fasciitis or pyomyositis (most commonly found in the tropics), necrotizing pneumonia, infective endocarditis (affecting the heart valves), or infections of the bones or joints. CA-MRSA often results in abscess formation that requires incision and drainage. Before the spread of MRSA in the community, abscesses were not considered contagious since the infection was supposed to require breaking the integrity of the skin and the introduction of staphylococci from normal skin colonization. However, the CA-MRSA that has recently emerged is transmissible (similar, but with very important differences) from HA-MRSa. Additionally, CA-MRSA is less likely to cause cellulite than other forms of MRSA.

In some embodiments, the compositions described herein are useful for treating, inhibiting, and / or sensitizing MRSA. In some embodiments, the compositions described herein are useful for treating, inhibiting, and / or sensitizing MRSA in a subject. Methods for detecting MRSA and selecting a subject with MRSA infection are disclosed herein.

VII. Other drug-resistant bacterial pathogens

A drug-resistant bacterial pathogen is a bacterial pathogen that is resistant to one or more antimicrobial agents. A bacterial pathogen can be both drug resistant and drug sensitive if it is sensitive to one antimicrobial agent, but resistant to another.

In some embodiments, the composition disclosed herein is used to treat, inhibit, or sensitize a drug-resistant bacterial pathogen.

In certain embodiments, the compositions disclosed herein are effective in treating, inhibiting, or sensitizing various drug-resistant forms of bacterial pathogens including the drug-resistant form of one or more of Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (Such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, E.

ES 2 674 019 Enterohemorrhagic T3 coli, enteroaggregative E. coli and uropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter spinaedigelia. and Helicobacter king) (such as Klebsiella pneumoniae, Klebsiella granulomatis, and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis., Mobilcouncus spcop., Mobilcocuncus sp. . (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica, and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari, and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi), and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus pneumoniae (for example Streptococcus pneumoniae serotype 4 resistant to chloramphenicol, Streptococcus pneumoniae serotype 6B resistant to spectinomycin, Streptococcus pneumoniae serotype 9V resistant to streptomycin, Streptococcus pneumoniae pneumoniae resistant serotype 14, Streptococcus pneumoniae resistant serotype 14 serotype 14 rifampicin-resistant serotype 18C, Streptococcus pneumoniae tetracycline-resistant serotype 19F, Streptococcus pneumoniae serotype 19F resistant to penicillin, and Streptococcus pneumoniae serotype 23F resistant to trimethoprim, Streptococcus pneumoniae serotype 4 resistant to chloramphenicol, Streptococcus pneumoniae serotype 6B resistant to spectinomycin, Streptococcus pneumoniae resistant to streptocokine serotype 9 Streptococcus pneumoniae oppressive serotype 9, Streptococcus pneumoniae 14 resistant to streptocokine serotype 9 pneumoniae serotype 18C resistant to rifampin, Streptococcus pneumoniae serotype 19F resistant to penicillin, or Streptococcus pneumoniae serotype 23F resistant to trimethoprim), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A Streptococci, Streptococcus Streptococcus, Group C Streptococcus, Streptococcus agalactiae, Group C Streptococcus Streptococcus, Group C Streptococcus Streptococcus , Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group G Streptococci and Streptococcus anginosus Group G Streptococci, Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela, and Vibrio furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis) or Xanthomonas maltophilia, among others.

VIII. Drug resistant diseases

In some embodiments, a composition disclosed herein is for use in a method of treating, inhibiting, or sensitizing a drug-resistant infectious disease.

The compositions disclosed herein may be effective in treating or inhibiting drug resistant infections, measles, tetanus, malaria, upper and lower respiratory tract infections, hepatitis, typhoid fever, Staphylococcus aureus infection via vancomycin. / glycopeptide, vancomycin-resistant enterococci, MRSA, and Streptococcus pneumoniae, among others.

Also disclosed herein are compositions that can effectively treat, inhibit, or sensitize various drug-resistant forms of infectious diseases, including the drug-resistant form of one or more than one Acinetobacter infection, actinomycosis, adenovirus infection, sleeping sickness. from Africa (African trypanosomiasis), AIDS, amoebiasis, anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis (BV), bacteroides infection, balantidiasis, Baylisascaris infection, BK virus infection, black stone, Blastocystis hominis infection, blastomycosis, Bolivian hemorrhagic fever , Borrelia infection, botulism, Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Calicivirus infection, campylobacteriosis, candidiasis (moniliasis; Vaginal yeast infection, cat scratch disease, cellulitis, Chagas disease, chancroid, fowl pox, chlamydia, Chlamydophila pneumoniae infection, cholera, chromoblastomycosis, clonorchiasis, Clostridium difficile infection, coccidioidomycosis, tick fever, Colorado, common cold Creutzfeld-Jakob disease, Crimean-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous larva migrans (CLM), cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever, dientamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (roundworm infection), infection

ES 2 674 019 T3 due to Enterococci, enterovirus infection, epidemic typhus, erythema infectiosum, sudden rash, fasciolopsiasis, fasciolosis, fatal familial insomnia (FFI), filariasis, food poisoning, free-living amoeba infection, Fusobacterium infection, gas gangrene (clostridial myonecrosis), geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, inguinal granuloma (donovanosis), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand, foot and mouth disease (HMFD), Hantavirus, Helicobacter pylori infection, hemolytic uremic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), Hepatitis A, B, C, D, or E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human Ehrlichiosis ewingii, Human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human monocytic erlichiosis, human papillomavirus (HPV) infection, human parainfluenza virus infection, or hymenolepiasis, among others.

Also disclosed herein are compositions that can effectively treat, inhibit, or sensitize various drug-resistant forms of infectious diseases, including the drug-resistant form of one or more Epstein-Barr virus (Mono) infectious mononucleosis, influenza ( flu), Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis, Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease, Lymphatic filariasis, Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever (MHF), Measles, Melioidosis (Whitmore's disease). Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis Microsporidia, Molluscum contagiosum (MC), Mumps, Murine typhus, Mycoplasma pneumoniae, Mycetoma, Myiasis, Neonatal conjunctivitis, Onchocerciasis (river blindness), Paracoccidioidomycosis, South American paracoccidioidomycosis, Blastomycosis, Paracoccidioidomycosis (Blastomycosis) capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic area lice, nits), Pelvic inflammatory disease (piD), Pertussis (Pertussis), Plague, Pneumococcal infection, Pneumocystis pneumoniae (PCP), Pneumonia, poliomyelitis, poliovirus, Primary amoebic meningoencephalitis (pAm), Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Fever due to rabies, respiratory bite Rhinosporidiosis, Rhinovirus infection, Rickettsia infection, Rickettsia smallpox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Septicemia, Shigellosis, Herpes (Herpes zoster), Smallpox, Sporotrichosis, Staphylococcal Food Poisoning, Staphylococcal Infection, Strongyloidiasis, Syphilis, Lockiasis, Syphilis) Tinea barbae (beard folliculitis), Tinea capitis (ringworm of the scalp), Tinea corporis (Ringworm of the body), Tinea cruris (Jock itch), Tinea manuum (Ringworm of the hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans (OLM)), Toxocariasis (Visceral Larva Migrans (VLM)), Toxoplasmosis, Trichinellosis, Trichuria, Infection by whipworms), Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia, West Nile fever, White stone, Yersiniosis, Yellow fever, or zygomycosis, among others.

VIII. Methods of treatment, inhibition or sensitization of a bacterial pathogen

The embodiments disclosed herein include compositions for use in methods of inhibiting a drug-resistant bacterial pathogen, methods of sensitizing a drug-resistant bacterial pathogen to a drug to which the bacterial pathogen is resistant, and methods of inhibiting a bacterial pathogen. drug-sensitive develop drug resistance, among others.

Some embodiments include compositions for use in methods of inhibiting a bacterial pathogen other than Mycobacterium tuberculosis by selecting for a drug resistant pathogen and contacting the pathogen with a composition as described herein, for example, a composition comprising MSM. and an antimicrobial agent. For example, contacting a selected drug resistant bacterial pathogen with a composition comprising 10-16% MSM and a MIC level of an antimicrobial agent. In some embodiments, the bacterial pathogen is MRSA and the microbial agent is a beta-lactam antibiotic, eg, methicillin or oxacillin. In some embodiments, the bacterial pathogen is on a subject or on a surface.

Some embodiments include compositions for use in methods of sensitizing a drug-resistant bacterial pathogen other than Mycobacterium tuberculosis to a drug to which the bacterial pathogen is resistant, selecting a drug-resistant bacterial pathogen and contacting the pathogen with a composition comprising MSM. For example, by contacting a selected bacterial pathogen with a composition comprising 10-16% MSM. In some embodiments, the selected bacterial pathogen is MRSA. In some embodiments, the bacterial pathogen is on a subject or on a surface.

Some embodiments include compositions for use in methods of inhibiting a drug-sensitive bacterial pathogen other than Mycobacterium tuberculosis from developing drug resistance by selecting for a drug-sensitive bacterial pathogen and contacting the pathogen with a composition, as described herein. , for example, a composition comprising MSM and an antimicrobial agent. For example, contacting a selected drug-sensitive bacterial pathogen with 10-16% MSM and a MIC level of an antimicrobial agent. In some embodiments, the bacterial pathogen is Staphylococcus aureus.

ES 2 674 019 T3 and the anti-microbial agent is a beta-lactam antibiotic, for example, methyl or oxacillin. In some embodiments, the bacterial pathogen is on a subject or on a surface.

In some embodiments, a composition comprising MSM and a sub-MIC concentration of an antibiotic is equally or more effective in reducing or killing certain drug-resistant bacteria compared to the antibiotic alone at MIC levels. In other embodiments, a composition comprising MSM and an antibiotic is more effective in reducing or killing bacteria from an infection site compared to the antibiotic alone. In some embodiments, the compositions disclosed herein enhance MRSA treatment.

Selection of a drug-sensitive bacterial pathogen

Methods of selecting a drug-sensitive bacterial pathogen are well known to those of skill in the art. For example, in some embodiments, a drug-sensitive bacterial pathogen is detected as described herein, and subsequently selected, thereby selecting for a drug-sensitive bacterial pathogen. Methods of detecting a bacterial pathogen as described herein are well known to those of skill in the art. For example, such detection may be based on the determination of the MIC of an antibiotic for a drug-sensitive bacterial pathogen. Methods of determining a MIC are well known to those skilled in the art (see, for example, Andrews, J. of Antimicrobial Chemotherapy, 48: 5-16, 2001). For example, MICs can be determined by agar or broth dilution methods usually following the guidelines of a reference body such as CLSI, BSAC or EUCAST. There are a few commercial methods available, including the well established Etest® strips (bioMerieux SA, France), and the Oxoid MICEvaluator method. The Etest® system comprises a predefined and continuous concentration gradient of different antimicrobial agents, which when applied to inoculated agar plates and incubated, creates ellipsis of microbial inhibition. MIC is determined when the ellipsis of inhibition intersects with the strip, and the MIC reading scale is easily read on the strip (see, for example, Andrews, J. of Antimicrobial Chemotherapy, 48: 5-16, 2001).

In some embodiments, a Staphylococcus aureus bacterium is detected as described herein, and subsequently selected, thereby selecting for a drug-sensitive bacterial pathogen. Staphylococcus aureus is a Gram-positive coconut in which rounded cells, approximately 1 pm in diameter, form cluster-like clusters indicating the ability to divide in more than one plane. This bacterium is capable of aerobic and anaerobic respiration and most strains ferment mannitol anaerobically. On blood agar they form characteristic golden or white colonies. They produce catalase, coagulase, and an extracellular cell agglutination factor, and some strains produce capsules (see Brown et al., J. Antimicrob. Chemother., 56: 1000-1018, 2005).

Methods for detecting Staphylococcus aureus are well known to those of skill in the art. Non-limiting detection methods include tube coagulase test, slide coagulase test, latex agglutination test, DNase and thermostable nuclease tests, commercial biochemical tests such as the VITEK system. 2 (bioMérieux), Staphychrom II (International Microbio, Signes, France), the Phoenix system (Becton Dickinson Microbiology Systems, Sparks, MD) and molecular assays, including PCR- and DNA probe-based assays. For a review, see Brown et al., J. Antimicrob. Chemother., 56: 1000-1018, 2005). Using the Etest® system, a drug-sensitive bacterial pathogen Staphylococcus aureus will generally have a MIC for oxacillin of 2 pg / ml or less (see, for example, the Etest® technical manual, AB bioMérieux, 2008).

Some embodiments comprise selecting a drug-sensitive bacterial pathogen, wherein the pathogen is on the surface. These embodiments comprise determining whether a drug-sensitive bacterial pathogen is on a surface, and selecting said surface, thereby selecting a drug-sensitive pathogen on a surface. Alternatively, some embodiments include selecting a surface with a drug-sensitive bacterial pathogen. A person skilled in the relevant art will understand how to carry out such methods. Non-limiting examples include observing the surface or obtaining a sample from the surface and detecting a drug-sensitive bacterial pathogen in the sample. Methods for detecting a drug-sensitive bacterial pathogen are known to those of skill in the art and are described herein. Examples of surface observation include recognizing when a surface has come into contact with a drug-sensitive bacterial pathogen, for example, if a drug-sensitive bacterial pathogen is known to have come into contact with the surface.

Some embodiments comprise selecting a drug-sensitive bacterial pathogen, wherein the pathogen is in a subject. These embodiments comprise determining whether a drug-sensitive bacterial pathogen is in a subject, and selecting said subject, thereby selecting a drug-sensitive pathogen in a subject. Alternatively, some embodiments include selecting a subject with a drug-sensitive bacterial pathogen. A person skilled in the relevant art will understand how to carry out such methods. Non-limiting examples include observing the subject or obtaining a biological sample from the subject and detecting a drug-sensitive bacterial pathogen in the sample. Methods for detecting a bacterial pathogen are known to those of skill in the art.

ES 2 674 019 T3 sensitive to the drug and are described herein. Examples of observing the subject include recognizing the symptoms exhibited by a subject having a drug-sensitive bacterial pathogen therein as well as observing the success of drug treatment of the bacterial pathogen in the subject.

Selection of a drug-resistant bacterial pathogen

Methods of selecting a drug resistant bacterial pathogen are well known to those of skill in the art. For example, in some embodiments a drug resistant bacterial pathogen is detected as described herein, and subsequently selected, thereby selecting for a drug resistant bacterial pathogen. Methods of detecting a drug resistant bacterial pathogen as described herein are well known to those of skill in the art. For example, Said selection may be based on the MIC of an antibiotic for a bacterial pathogen. Methods of determining a MIC are well known to those skilled in the art and are described herein.

In some embodiments, selecting a drug resistant bacterial pathogen comprises selecting a MRSA. Methods of selecting a pathogen in which the drug-resistant bacterial pathogen is MRSA are known to those of skill in the art. For example, in some embodiments an MRSA bacterium is detected as described herein, and subsequently selected, thereby selecting for a MRSA. Methods for detecting MRSA are known to those of skill in the art.

For example, non-limiting examples of MRSA detection include the Etest® system (bioMérieux SA, France) in combination with a finding that the bacterium in question is a Staphylococcus aureus bacterium, Disk diffusion, an agglutination test of the latex with antibodies against PBP2a or PBP2 ', and molecular assays including PCR and DNA probe-based assays. For a review, see Brown et al., J. Antimicrob. Chemother., 56: 1000-1018, 2005 and Sturenburg, Ger. Med. Sci., 6: Doc 06, 2009. More methods are described in US Patents 7,449,289, 7,297,517, 5,496,706, 5,776,712 and 6,197,504 and in patent application publications of United States numbers 2003/0165953, 2006/0040871, 2007/0082340, 2008/0220428, 2008/0227087, 2009/0081663, 2009/0130115, 2009/0181395, 2009/0203013, 2009/0325147, 2010/0197649. Using the Etest® system, a MRSA bacterial pathogen will generally have a MIC for oxacillin of 2 pg / ml or more (see eg Etest® technical manual, AB bioMerieux, 2008).

Some embodiments comprise selecting a drug resistant bacterial pathogen, wherein the pathogen is on the surface. These embodiments comprise determining whether a drug-resistant bacterial pathogen is on a surface, and selecting said surface, thereby selecting a drug-resistant pathogen on a surface. Alternatively, some embodiments include selecting a surface with a drug resistant bacterial pathogen. A person skilled in the relevant art will understand how to carry out such methods. Non-limiting examples include observing the surface or obtaining a sample from the surface and detecting a drug resistant bacterial pathogen in the sample. Methods for detecting a drug resistant bacterial pathogen are known to those of skill in the art and are described herein. Examples of surface observation include recognizing when a surface has come into contact with a drug-resistant bacterial pathogen, for example, if a drug-resistant bacterial pathogen is known to have come into contact with the surface.

Some embodiments comprise selecting a drug resistant bacterial pathogen, wherein the pathogen is in a subject. These embodiments comprise determining whether a drug-resistant bacterial pathogen is in a subject, and selecting said subject, thereby selecting for a drug-resistant bacterial pathogen in a subject. Alternatively, some embodiments include selecting a subject with a drug resistant bacterial pathogen. A person skilled in the relevant art will understand how to carry out such methods. Non-limiting examples include observing the subject or obtaining a biological sample from the subject and detecting a drug resistant bacterial pathogen in the sample. Methods for detecting a drug resistant bacterial pathogen are known to those of skill in the art and are described herein. Examples of observing the subject include recognizing symptoms exhibited by a subject having a drug resistant bacterial pathogen therein as well as observing failure of drug treatment of the bacterial pathogen in the subject.

Some embodiments comprise selecting a drug resistant bacterial pathogen in a subject, wherein the drug resistant pathogen is MRSA. These embodiments comprise determining whether MRSA is in a subject, and selecting said subject, thereby selecting an MRSA in a subject. Alternatively, some embodiments include selecting a subject with MRSA. An expert in the field will know how to carry out said methods. Non-limiting examples include observing a subject or obtaining a biological sample from the subject and detecting the presence of MRSA in the sample.

Examples of observing the subject include recognizing the symptoms of MRSA infection in the subject, observing the failure of treatment with a beta-lactam antibiotic of a subject having an infection with bacterial pathogens, for example, a Staphylococcus aureus infection. , as well as, to observe the failure of treatment with a beta-lactam antibiotic of a subject who has a symptom of MRSA. The experts in

ES 2 674 019 T3 subject matter knows other methods of observing a subject to determine if the subject is a subject with MRSA.

Methods for obtaining a biological sample from the subject and testing the sample for the presence of MRSA are well known to those of skill in the art. For example, MRSa can be detected by cleaning the nostrils of patients and isolating the bacteria found inside. Methods for detecting a drug resistant bacterial pathogen are known to those of skill in the art and are described herein. Examples of observing the subject include recognizing the symptoms exhibited by a subject having a bacterial pathogen therein as well as observing failure of drug treatment of the bacterial pathogen in the subject.

Contact with a bacterial pathogen

Methods of contacting a bacterial pathogen with a composition are well known to those of skill in the relevant art. Said methods will depend on the type and location of the bacterial pathogen and the type of composition used. Described herein are methods of contacting a bacterial pathogen, including methods of contacting a bacterial pathogen on a subject, and methods of contacting a bacterial pathogen on a surface, among others. An expert in the field will know which method to apply.

A method of contacting a bacterial pathogen on a surface is provided with a composition as described herein. For example, spraying a composition as provided herein onto the surface, or wiping a surface with a cloth comprising a composition provided herein. One skilled in the art will understand how to apply compositions described herein to a surface.

A method of contacting a bacterial pathogen in a subject with a composition is disclosed, as described herein. Such methods include administering a compound as described herein to a subject such that the compound comes into contact with a bacterial pathogen. For example, a compound or agent, such as MSM, can be administered to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal (such as topical), intranasal, vaginal and by inhalation. Specific types of administration include topical administration or administration to the nasal mucosa or lungs by inhalation administration.

In some embodiments, a composition formulated for topical administration can be used to come into contact with a bacterial pathogen located on or on the skin of a subject. A composition formulated for administration by inhalation is to be used to come into contact with a bacterial pathogen localized to the airways, mucosal membranes or lungs of a subject, among others.

In some embodiments, a composition comprising MSM, or MSM and an antimicrobial agent, is formulated for topical administration and used to contact a drug-resistant bacterial pathogen localized on or in a subject. One skilled in the art will understand when topical administration is appropriate. For example, topical administration is suitable when the bacterial pathogen is localized on the skin of a subject or on the surface of the subject's body.

In other embodiments, a composition comprising MSM or MSM and an antimicrobial agent is formulated for administration by an inhaler device and is used to contact a drug-resistant bacterial pathogen localized on or in a subject. One skilled in the art will understand when administration via an inhaler device is appropriate ... For example, administration by an inhaler device is suitable when the bacterial pathogen is localized on the mucosal membranes of the subjects or in the lungs of the subjects.

Methods of coming into contact with a bacterial pathogen may employ specialized devices, for example, an inhaler device. Inhaler devices are useful for administering a composition described herein to a bacterial pathogen located on the mucosal membranes, airways, and / or lungs of a subject.

Inhalers, according to some embodiments, provide direct access of MSM and / or other agents to infected lung tissue to sensitize bacterial pathogens to the antibiotic. Alternatively, inhalers provide direct access of MSM and an antimicrobial agent to infected lung tissue to inhibit or treat the bacterial pathogen. According to some embodiments, inhalers are useful for treating MRSA-infected lung tissue.

In one embodiment, an inhaler is provided to the target of the infection site (eg, lungs) of some infectious diseases, such as MRSA infection. In some such embodiments, the inhaler device comprises a nebulizer. In other embodiments, an inhaler is used. In some embodiments, it is used

ES 2 674 019 T3 is a pressurized metered dose inhaler, and the composition is inhaled as a liquid aerosol. In other embodiments, dry powder inhalers are used and the composition is inhaled as a powder aerosol. In some embodiments, oral, intravenous, intramuscular, or subcutaneous administration is used in addition to or instead of inhalation therapy.

In certain embodiments, the inhaler device delivers droplets or particles of the inhaled formulation of a size capable of reaching the bronchioles of the patient's lungs. In some embodiments, the inhaler device is synchronized with the patient's respiratory rate to transport the formulation to the bronchioles. Inhalation therapy according to one embodiment allows more direct administration of the inhaled formulation to infected lung target tissues. Direct targeting is advantageous in some embodiments because it allows reduction of the amount of antimicrobial compounds incorporated into the formulation while maintaining or improving the efficacy of the formulation against infectious microorganisms. In other embodiments, direct administration increases the efficacy of a given antimicrobial regimen against one or more drug resistant strains of microorganism. Direct targeting, in accordance with other embodiments, minimizes side effects by minimizing undirected tissue contact.

The direct droplet or particle size provided according to some embodiments reduces the volume of MSM that is delivered compared to traditional ventilation therapy.

The ability to deliver antimicrobial agents as an inhaler (eg, as a powdered aerosol) with MSM, is especially advantageous in some embodiments because it allows for increased shelf stability and prepackaged dosages. These are of particular use to individuals in underdeveloped or developing nations who do not have regular access to sanitary facilities. Full cycles of treatment can be provided to an affected subject in a single visit to a healthcare specialist without the need for hospital stays or repeat visits. In some embodiments, the compositions disclosed herein are suitable for self-administration (eg, via inhaler devices) and are therefore especially suitable for patients with access to medical care.

In certain embodiments, the total volume of inhaled composition comprising MSM and / or other agents is about 2-8 ml. In some embodiments, the total volume of inhaled composition comprising MSM and / or other agents is from about 2 ml to about 4 ml. In some embodiments, the total volume of MSM and / or other agents is from about 6 ml to about 8 ml. In still other embodiments, the total volume of inhaled composition comprising MSM and / or other agents is from about 3 ml to about 7 ml, including 4, 5, and 6 ml. Thus, in some embodiments, the concentration of MSM administered by inhalation ranges from about 0.01% to about 20%, including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%.

In various embodiments, contacting a bacterial pathogen includes contacting the bacterial pathogen with a therapeutically effective amount of an antimicrobial agent. Such contacting may involve administration in a single dose, or in several doses, eg, daily, during a treatment cycle.

In some embodiments, contacting a bacterial pathogen with a composition, as described herein, includes contacting the pathogen with the composition for about 1 hour to about 10 days, or any time in between. In some embodiments, the bacterial pathogen is contacted for about 12, 24, 36, 48, 60, 72, 84, 96, 108, or about 120 hours, with a composition, as described herein. In some embodiments, the bacterial pathogen is contacted for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, or about 24 hours, with a composition, as described herein. In some embodiments, the bacterial pathogen is contacted with a composition, as described herein, for about 24, 36, 48, 60, 72, 84, 96, 108, or about 120 hours. In some embodiments, longer or shorter contact times can be used.

X. Compositions

The agents (eg, MSM and beta-lactam antibiotics) described herein can be formulated in various ways depending on the intended use. Various types of compositions are disclosed herein, including compositions for use on a subject and compositions for use in an industrial setting. The person skilled in the art will know when to use a particular composition.

Compositions for use in a subject can be formulated in various ways depending on the location and type of disease to be treated or avoided in the subject. Such compositions are therefore provided for local use in or near an affected area and for systemic use (where the agent is administered in a manner that is widely disseminated by the cardiovascular system). Other types of compositions are also disclosed herein, eg, compositions for use on a surface.

ES 2 674 019 T3

This disclosure includes within its scope compositions that include DMSO, SMSO without MSM, MSM, MSM without DMSO and / or antimicrobial agents, or combinations thereof that are formulated for use in human medicine or veterinary medicine.

For example, the compositions provided can include compositions comprising MSM in the ranges of about 0.01% by weight to about 20% by weight. In other embodiments, the composition contains between about 0.01% and 5% MSM by weight. Other embodiments contain between about 5-10% MSM, about 10-15% MSM, about 15-20% MSM. Another composition comprises about 5-20% MSM, about 5-16% MSM, about 5-10% MSM, about 5-8% MSM, about 9-16% MSM, or 10-15% MSM. other embodiments include about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% MSM. Some embodiments include about 10-16% MSM, about 10-14% MSM, or 10-12% MSM. In some embodiments, higher or lower percentages can be used. Although compositions comprising MSM will normally be used to treat human subjects, they can also be used to treat similar or identical diseases in other vertebrates, such as other primates, dogs, cats, horses, and cows.

Compositions provided herein also include compositions containing a beta-lactam antibiotic. For example, the compositions as described herein can include penicillin derivatives, cephalosporins, penems, monobactams, carbapenenes, beta-lactamase inhibitors, and combinations thereof. Examples of derivatives of penicillin include, aminopenicillins (eg, amoxicillin, ampicillin, and epicillin); carboxypenicillins (eg, carbenicillin, ticarcillin, and temocillin); ureidopenicillins (eg, azlocillin, piperacillin, and mezlocillin); mecillinam, sulbenicillin, benzathine penicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), penicillin O (allylmercaptomethylpenicillin), procaine penicillin, oxacillin, methicillin, nafcillin, fluoxacillin, tacampillin, hexacillin, fluoxacillin, nafcillin, fluoxacilin, cloxacillin co-amoxiclav (amoxicillin plus clavulanic acid), and piperacillin. Examples of cephalosporins include, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandeol, cefotetan, cefoxitin, ceforanide, ceftriaxone, cefotaxime, cefpodoxime proxetil, ceftazidime, cefepime, cefuroxime, cephimepyroma, and ceftizopyroma. Examples of penemos include faropenemo. Examples of monobactams include aztreonam and thygemono. Examples of carbapenems include, biapenenvdoripenem, ertapenem, imipenem, meropenem, and panipenem. Examples of beta-lactamase inhibitors include, tazobactam sodium salt of 4.4 tazobactam acid dioxide ([2S- (2alpha, 3beta, 5alpha)] - 3-methyl-7-oxo-3- (1H-1, 2,3-triazol-1-ylmethyl) -4-thia1-azabicyclo [3.2.0] heptane-2-carboxylic acid), (2S, 5R) -3,3-dimethyl- acid sulbactam 4,4-sodium dioxide 7-oxo-4-thia-1azabicyclo [3.2.0] heptane-2-carboxylic), and clavulanic acid ido ((2R, 5R, Z) -3- (2-hydroxyethylidene) -7-oxo-4-oxa- 1-azabicyclo [3.2.0] heptane-2-carboxylic), or another beta-lactam antibiotic.

Many antibiotics have a set minimum inhibitory concentration (MIC) at which they are effective in reducing or killing certain bacteria. Such compositions provided herein comprise an amount of a beta-lactam antibiotic equal to about 0.001 to 100 MIC of the particular bacterial pathogens disclosed herein. In some embodiments, the composition comprises about 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or about 90 -100 MIC of a beta-lactam antibiotic. In some embodiments, the composition comprises about 0.001,0,01,0,1,0.5, or 1 MIC of a beta-lactam antibiotic. A person skilled in the art will know the MIC of an antibiotic for a specific bacterial pathogen, or the skilled technician will know how to determine the MIC of an antibiotic for a specific bacterial pathogen. Disclosed herein are methods of determining the MIC of a particular antibiotic for a particular bacterial pathogen, as disclosed herein, eg, using the Etest® system. it is conventional for the skilled technician to calculate a MIC of a particular antibiotic for a particular bacterial pathogen.

Compositions provided herein also include combinations of MSM and a beta-lactam antibiotic. Such combinations can include any amount of MSM and / or beta-lactam antibiotic as a composition that includes only MSM or a beta-lactam antibiotic. In some embodiments, the compositions provided herein include 10-16% MSM and an amount of beta-lactam antibiotic equal to 1 MIC for a bacterial pathogen to come into contact with the composition.

The compositions provided herein containing MSM are water-based compositions. Compositions provided herein containing MSM preferably contain about 0% to about 5% sodium chloride by weight. In some embodiments, the compositions comprise about 0.0001, 0.001, 0.01, 0.1, 0.5, 1.2, 3, 4, or 5% sodium chloride.

The dosage form of the composition will be influenced by the selected mode of administration. For example, in addition to injectable fluids, inhalation, topical, ophthalmic, peritoneal, and oral formulations may be employed. Inhalation preparations can include aerosols, particles. In general, the target for inhalation particle size is about 1 pm or less, in order for the agent to reach the alveolar region of the lung for absorption.

Compositions including MSM, DMSO, an antimicrobial agent, or a therapeutic compound, as described

ES 2 674 019 T3 described herein as active ingredient, or including a mixture of two or more agents thereof, with or without additional agent (s) as active ingredients, can be formulated with a suitable solid or liquid carrier, depending on the specific mode of administration selected. Oral formulations can be liquid (eg, syrups, solutions, or suspensions), or solid (eg, powders, pills, tablets, or capsules). For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. The actual methods for preparing such dosage forms are known or will be apparent to those of ordinary skill in the art.

For oral administration, the compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinized cornstarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, talc, or silica); disintegrants (eg potato starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (eg, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (eg, lecithin or acacia); non-aqueous vehicles (eg, almond oil, fatty esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (eg, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffering salts, flavoring, coloring agents, and sweetening agents, as appropriate.

For administration by inhalation, the agents and compositions for use in accordance with the present disclosure are conveniently administered in the form of an aerosol spray preparation from pressurized containers or a nebulizer, with the use of a suitable propellant, by example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or any other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mixture of the compound and a suitable powder base, such as lactose or starch.

For topical administration, the compounds can, for example, be mixed with a liquid delivery agent for local administration. Agents used therapeutically (such as DMSO, MSM, and / or a beta-lactam antibiotic other than the inhibitor or therapeutic compound described herein) are readily soluble or suspended in water, and as such, this would be useful for administration because water does not produce adverse effects on biological tissues. This allows sufficiently high doses to be administered locally or systemically, without secondary toxicity from the delivery vehicle. Those of skill in the art will understand suitable buffering conditions for MSM and the beta-lactam antibiotic.

Compositions comprising at least one agent described herein as an active ingredient will normally be formulated with a suitable solid or liquid carrier, depending on the particular mode of administration selected. The pharmaceutically acceptable carriers and excipients useful in this disclosure are conventional. For example, parenteral formulations typically comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid carriers such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol, or the like. Excipients that may be included are, for example, proteins, such as human serum albumin or plasma preparations. If desired, the composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art.

For example, for parenteral administration, the therapeutic agent (s) can generally be formulated by mixing it to a desired degree of purity, in a unit injectable dosage form (solution, suspension or emulsion), with a pharmaceutically acceptable carrier, for example, one that is non-toxic to receptors at the dosages and concentrations employed and is compatible with other ingredients in the formulation. A pharmaceutically acceptable carrier is a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or auxiliary formulation of any kind.

In general, the formulations are prepared by contacting the agent (s) each uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Optionally, the carrier is a parenteral carrier, and, in some embodiments, is a solution that is isotonic with the recipient's blood. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. The

Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

Compositions comprising at least one agent will, in some embodiments, be formulated in a unit dosage form, suitable for individual administration of precise dosages. The amount of active compound (s) administered will depend on the subject being treated, the severity of the condition, and the manner of administration and is best left to the discretion of the treating physician. Within these limits, the formulation to be administered will contain an amount of the active component (s) in amounts effective to achieve the desired effect in the subject being treated.

Preparations for oral administration can be suitably formulated to provide controlled release of the therapeutic agent (s) (eg, DMSO, MSM, beta-lactam antibiotic, and so on). For example, the compositions may be in the form of particles comprising a biodegradable polymer and / or a gelatinizing polysaccharide and / or a bioadhesive polymer, an amphiphilic polymer, a modifying agent for the properties of the interface of the particles, and a pharmacological substance. active. These compositions have certain biocompatibility characteristics that allow a controlled release of the active substance. See, for example, US Patent No. 5,700,486.

Polymers can be used for controlled release. Various degradable and non-degradable polymeric matrices are known in the art for use in controlled drug delivery (Langer, Accounts Chem. Res. 26: 537, 1993). For example, the block copolymer, Polaxamer 407 exists as a more viscous mobile liquid at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained administration of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425, 1992; Pec, J. Parent. Sci. Tech. 44 (2 ): 58, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled protein release (Intema et al., Int. J. Pharm. 112: 215, 1994). In yet another aspect, liposomes are used for controlled release as well as pharmacological targeting of lipid capsulated compounds (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Numerous additional systems are known for the controlled administration of therapeutic proteins (eg, US Patent No. 5,055,303; US Patent No. 5,188,837; US Patent No. 4,235,871; United States Patent No. 4,501,728; United States Patent No. 4,837,028; United States Patent No. 4,957,735; and United States Patent No. 5,019,369; United States Patent No. 5,055,303, US Patent No. 5,514,670; US Patent No. 5,413,797; US Patent No. 5,268,164; US Patent No. 5,004,697; US Patent No. 4,902,505; US Patent No. 5,506,206; US Patent No. 5,271,961; US Patent No. 5,254,342; and US Patent No. 5,534,496).

Compositions for use in industrial settings can be formulated in various ways depending on the location and type of surface to be treated. Such compositions can be formulated according to any means by which a pharmaceutical composition can be formulated, but additionally, they can be formulated in additional means that would not normally be acceptable for administration to a subject. Compositions for use in an industrial setting are water based and contain 0-5% sodium chloride. In some embodiments, such compositions are formulated to be applied to a surface by wiping. In some embodiments such compositions are formulated to be sprayed onto a surface. One skilled in the art will understand how to prepare such formulations.

X. Other achievements

As described herein, formulations comprising DMSO and / or MSM are provided. Despite the increasing prevalence of drug resistant pathogenic microbes, some of the formulations disclosed herein are unexpectedly effective in treating drug resistant bacteria or other microbes. Other drug resistant pathogens are also treated by DMSO alone, MSM alone, or a combination of DMSO and MSM, along with a therapeutic agent.

The combination of DMSO and MSM may allow a lower concentration of DMSO and / or MSM to be used. Alternatively, the use of DMSO and / or MSM reduces the minimum effective concentration of the different constituents of the formulation, thereby also reducing the side effects of those constituents. For example, in one embodiment, the addition of DMSO, MSM or DMSO and MSM will allow a reduced dosage of antibiotics to achieve comparable or enhanced therapeutic effects.

Formulations comprising DMSO and / or MSM can sensitize drug resistant pathogens to drugs or partially or completely reverse the drug resistant nature of bacterial strains.

In some embodiments, formulations comprising DMSO and / or MSM and at least one therapeutic agent result in decreases in concentration of DMSO, MSM, and / or the therapeutic agent necessary to effectively treat one or more types of infection. A formulation comprising DMSO, mSm, or a combination of the two can sensitize bacteria (whether or not they are drug resistant) to antibiotics. Therefore, said formulation: (i) reduces the necessary dose of antibiotic; (ii) reduces treatment time; (iii) reduce the number of

ES 2 674 019 T3 different antibiotics needed, and / or (iv) makes an effective antibiotic. Accordingly, the unwanted side effects associated with antibiotics can be reduced in some embodiments, including liver damage, kidney damage, eye defects, hyperuricemia, thrombocytopenia, leukopenia, and neutropenia. A DMSO and / or MSM formulation can sensitize drug resistant pathogens to isoniazid, rifampin, pyrazinamide, and / or ethambutol. Alternatively, DMSO and / or MSM can potentiate the effects of isoniazid, rifampin, pyrazinamide, and / or ethambutol on non-drug resistant tuberculosis.

Many infections lead to local inflammation (or even inflammation of a large area of ​​tissue around the site of infection). DMSO and / or MSM can work synergistically with therapeutic agents to reduce inflammation to a greater degree than DMSO, MSM, or the agent alone.

In some examples disclosed herein, DMSO and MSM in a single formulation with a therapeutic agent act synergistically to reduce the amount of DMSO necessary to achieve effective delivery amounts of a therapeutic agent to a target site of infection. MSM enhances the penetrating effect of DMSO, allowing a therapeutic agent to reach a target area of ​​infection at an increased concentration (or a reduced time frame). Thus, the synergy between MSM and DMSO reduces the side effects associated with the administration of DMSO, including subsequent administration of unpleasant odor, nausea, diarrhea, and skin / throat irritation, among others.

Alternatively, DMSO and MSM in a single formulation with a therapeutic agent act synergistically to reduce the amount of therapeutic agent necessary to effectively treat an infection. For example, many antibiotics have a set minimum inhibitory concentration (MIC) at which they are effective in reducing or killing certain bacteria. a formulation comprising DMSO and MSM and a sub-MIC concentration of an antibiotic is equally or more effective in reducing or killing certain resistant bacteria compared to the antibiotic alone at MIC levels. Alternatively, a formulation comprising DMSO, MSM, and an antibiotic is more effective in reducing or killing bacteria from an infection site compared to the antibiotic alone. Some formulations disclosed herein enhance the treatment of multiple drug resistant bacterial pathogens.

Also disclosed herein is a formulation comprising DMSO and ethambutol that sensitizes bacteria to drugs other than ethambutol. Formulations comprising DMSO enhance sensitivity or susceptibility to drugs such as sub-MIC concentrations of ethambutol, isoniazid, rifampin, and streptomycin by about 2-fold to about 100-fold. Unlike previous reports, DMSO concentrations greater than 50% can be very particularly advantageous (Jagannath et al. J. Antimicrobial Chemotherapy 35, 381-390, 1995).

In some aspects of the disclosure, DMSO and / or MSM allow antibiotics (or other therapeutic agents) to penetrate lung tissue infected with a bacterial pathogen, including a drug-resistant bacterial pathogen. In one aspect of the disclosure, DMSO and / or MSM: (i) allows antibiotics to reach deeper levels of infected tissue; (ii) allows direct contact of the infected tissue; (iii) lengthens the exposure time of the antibiotic to the infected tissue; and / or (iiv) decreases the time to achieve a desired antibiotic effect. In one aspect of the disclosure, DMSO and / or MSM achieve one or more of these desired effects through the use of an inhaler, wherein the inhaler further comprises one or more antibiotics or other therapeutic agents.

In some aspects of the disclosures, the combined use of MSM reduces or eliminates the odor normally associated with DMSO. This is surprisingly beneficial because specialists in charge of treatment have avoided using DMSO in high concentrations (or in any amount) due to its unpleasant odor.

In some aspects of the disclosure, the DMSO and / or MSM formulations comprise antiparasitic agents that are important in treating infections caused by parasites, such as nematodes, cestodes, trematodes, protozoa, or amoebae.

In some aspects of the disclosure, DMSO and / or MSM formulations comprise antifungal agents that are effective in treating fungal infections, such as those caused by ringworm, candidiasis, and Cryptococcus (eg, cryptococcal meningitis).

In some aspects of the disclosure, the DMSO and / or MSM formulations comprise antiviral agents that are effective in treating viral infections. In some aspects of the disclosure, specific classes of antiviral agents are used to treat infections caused by a particular type of virus. In some aspects of the disclosure, agents are used that target HIV, herpes viruses, hepatitis B or C viruses, and influenza viruses.

In some aspects of the disclosure, DMSO and / or MSM formulations comprise antibiotics that are effective in treating bacterial infections by inhibiting, for example, bacterial growth, metabolism, proliferation, activity, and / or function. In some aspects of the disclosure, bacteriostatic antibiotics are used, while in others, bactericidal antibiotics are used. Bacteriostatic antibiotics and

ES 2 674 019 T3 bactericides can be incorporated into a single formulation comprising DMSO and / or MSM. Antibiotics of one or more classes can be incorporated into a formulation comprising DMSO and / or MSM. The formulation may include one or more than one: aminoglycoside, ansamycin, carbazephenes, carbapenenes, cephalosporins (1a, 2^to, 3^to, 4^to, or 5^togeneration), glycopeptides, macrolides, monobactams, penicillin, polypeptide, quinolone, sulfonamide, tetracycline.

In some aspects of the disclosure, specific diseases are addressed by incorporating specific antibiotics in a formulation comprising DMSO and / or MSM. For example, macrolides such as azithromycin or erythromycin are incorporated into formulations used to treat respiratory or mycoplasma infections. Also, penicillins such as amoxicillin or oxacillin are incorporated into formulations used to treat a wide range of streptococcal infections.

In still other aspects of the disclosure, specific disease-causing microorganisms are targeted by specific antibiotics incorporated into a formulation comprising DMSO and / or MSM. For example, aminoglycosides, such as neomycin, are incorporated into formulations used to treat Escherichia coli infections. Antibiotics normally used to fight microbial infections can be used. Antibiotics including, but not limited to, isoniazid, rifampin, pyrazinamide, and ethambutol can be incorporated into formulations comprising one or more of DMSO and MSM and used to treat bacterial pathogens, including drug resistant bacteria.

Formulations comprising DMSO, MSM, and one or more of the following therapeutic agents are also disclosed: rifampin, isoniazid, and ethambutol. Formulations comprising DMSO and at least one of rifampicin, isoniazid, pyrazinamide, and ethambutol are also disclosed. Formulations comprising MSM and at least one of rifampin, isoniazid, pyrazinamide and ethambutol are also disclosed. Formulations comprising DMSO and / or MSM in combination rifampin, isoniazid, pyrazinamide, and ethambutol are further disclosed to treat bacterial pathogens, including drug-resistant bacteria.

Rifampicin can be given in a daily dose ranging from about 400 mg to about 800 mg per day, or in a total daily dose range ranging from about 500 mg to about 700 mg per day, or in a total daily dose that varies. from about 550 to about 650 mg per day, including 560, 570, 580, 590, 600, 610, 620, 630, and 640 mg per day.

Isoniazid can be given in a total daily dose ranging from about 100 mg to about 500 mg per day, or in a total daily dose ranging from about 200 mg to about 400 mg per day, or in a total daily dose ranging from about 250 mg to about 350 mg per day, including 260, 270, 280, 290, 300, 310, 320, 330, and 340 mg per day.

Pyrazinamide can be provided in a total daily dose ranging from about 1.0 to about 4.0 g per day, or in a total daily dose ranging from about 2.0 to about 3.0 g per day, or in a total daily dose ranging from approximately 2.0 to 2.5 g per day, including 2.1, 2.2, 2.3, and 2.4 g.

Ethambutol is provided in a total daily dose ranging from about 0.5 to about 2.5 g per day, or in a total daily dose ranging from about 1.0 to 2.0 g per day, or in a daily dose. total ranging from about 1.0 to about 1.5 g per day, including 1.1, 1.2, 1.3, and 1.4 g.

In some aspects of the disclosure, DMSO and / or MSM is used to pretreat a patient suffering from an infectious disease. The dose of DMSO and / or MSM used to pretreat patients can range from about 10% to 50% by weight to volume, or from about 20% to about 40%, from about 25% to 35%, including 26, 27, 28 , 29, 30, 31, 32, 33, and 34%. In some aspects of the disclosure, about 50% to about 100% DMSO and / or MSM is used. Pretreatment with DMSO and / or MSM can enhance the ability of an antibiotic to inhibit bacterial activity and / or sensitize a drug resistant strain to a drug that was previously ineffective.

In some aspects of the disclosure, a formulation is prepared in which antimicrobial agents are dissolved in DMSO and / or MSM prior to administration. This is particularly advantageous since the antimicrobial agent and DMSO (and optionally MSM) can be administered to a subject by inhalation. Inhalers provide direct access of DMSO and / or MSM to the infected lung.

The use of MSM reduces the amount of DMSO needed to achieve a comparable effect and / or enhances the effectiveness of DMSO by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5 times, 10 times. , 50 times, or 100 times. In other embodiments, the use of MSM reduces the amount of a therapeutic agent necessary to achieve a comparable effect and / or enhances the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2-fold, 3-fold. , 5 times, 10 times, 50 times, or 100 times. It is also disclosed herein, that the use of DMSO reduces the amount of a therapeutic agent necessary to achieve a comparable effect and / or enhances the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 50 times, or 100 times. In other embodiments, the use of DMSO and MSM reduces the amount of a therapeutic agent needed to

ES 2 674 019 T3 achieve a comparable effect and / or enhance the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 50 times, or 100 times compared to DMSO or MSM alone and / or the therapeutic agent alone.

It is also disclosed herein, that a pretreatment formulation comprising DMSO, alone or in combination with MSM, is administered to a subject intravenously, intramuscularly, topically or orally to potentiate the effects of an inhaler therapy comprising DMSO and / or or MSM with therapeutic agents, such as antibiotics. Pretreatment with DMSO, alone or in combination with MSM, enhances the therapeutic effects of the inhaler by at least 10%, 25%, 50%, 10%, 2 times, 3 times, 5 times, 10 times, 50 times, or 100 times. .

It is also disclosed herein, that subjects having a drug-resistant infectious disease are treated with a formulation comprising, consisting or consisting essentially of DMSO, alone or in combination with MSM and one or more therapeutic agents, such as antibiotics. The formulation additionally includes other therapeutic agents, carriers, or excipients. The formulation additionally includes arginine, vitamin D, antioxidants, macrolides, linezolid, thioacetazone, thioridazine, or combinations thereof.

DMSO easily disturbs the integrity of many materials (particularly the plastics and polymers used in the manufacture of disposable medical equipment). Accordingly, devices to facilitate the storage and administration of DMSO are disclosed herein. DMSO can be stored in glass bottles and administered through a non-reactive tubing. Inhaler devices can be specially designed to be resistant to DMSO. The parts of the inhaler devices can be disposable or replaceable. Formulations comprising DMSO can be manufactured, stored, and / or administered using the materials and devices disclosed in U.S. Patent Application No. 12 / 066,480, which is the national phase entry for International Application No. : PCT / US06 / 35499, filed September 11, 2006.

In some embodiments, the addition of MSM unexpectedly reduces the unpleasant odor normally experienced with the use of DMSO. For example, in certain embodiments, the DMSO and MSM formulations do not produce a noticeable odor after use. In some different embodiments that have DMSO concentrations approaching or exceeding 50%, the combination with MSM in the formulation reduces or eliminates the DMSO-based odor. Such a result is unexpected, since the use of DMSO is normally associated with a strong unpleasant odor.

The use of DMSO and / or MSM with therapeutic agents (such as antibiotics) allows the manufacture and / or delivery of small droplets or particle sizes, thereby reducing irritation of the mucosa of the mouth and throat, as droplets or particles travel deeper into the patient's lungs. The depth of travel of the droplets or particles increases the concentration of the dissolved antibiotics in the lungs of the patient.

DMSO and / or MSM formulations can be combined with therapeutic agents (such as antibiotics) and provided as an aerosol to deliver locally active drugs to the respiratory system to treat infectious or other respiratory diseases. The lower airways can be contacted (or exclusively contacted) with the formulation. The formulation can be used to treat diseases systemically. For systemically active drugs, the aerosol particles are sized to reach the alveolar surface in peripheral areas of the lung.

The use of DMSO and / or MSM formulations comprising a therapeutic agent (such as an antibiotic) is particularly advantageous because it provides a rapid onset of action. Administration by inhalation provides a large absorption area of ​​the lung. For drugs that act locally, the onset of action can be immediate. Systemically active inhaled formulations, according to some embodiments, quickly reach the bloodstream. Inhalation therapy can provide a therapeutic effect in about 1-90 minutes. DMSO and / or MSM can enhance the bioavailability of the therapeutic agent. DMSO and / or MSM can reduce the degradation of the therapeutic agent. The aerosol formulations disclosed herein can reduce gastrointestinal side effects or skin irritation that can occur with oral or topical treatment.

Inhalant particles can be sized to minimize the deposition of those particles by inertial impact in the upper airways without reaching the site of action. The particles can be sized to minimize deposit in the mouth and throat, thereby minimizing swelling and unwanted local or systemic side effects. The particles can be smaller than 2, 5 or 10 pm. In one embodiment, the particles are about 3-5 pm and are transported in the bifurcations and smaller airways of the bronchi and bronchioles. In another embodiment, the particles are less than 3 pm and follow the air flow in the alveoli. The use of DMSO and / or MSM allows to optimize the particle size of the therapeutic agent. Additionally, the use of DMSO and / or MSM can sensitize drug-resistant bacterial pathogens to antibiotics.

DMSO and / or MSM can form a solution, mixture, emulsion, suspension, or other suitable combination with the therapeutic agent. Homogenization, sonication, high shear fluid processing or

ES 2 674 019 T3 other mechanical methods to combine the therapeutic agent with DMSO and / or MSM. The therapeutic agent can be easily dissolved in DMSO. Unlike other strong solvents, DMSO is not harmful to lung tissue. Therefore, DMSO is especially advantageous because it can dissolve the therapeutic agent and deliver said agent without damaging the lung tissue. DMSO can dissolve at least 50%, 75%, 90%, 95%, or 99% of the therapeutic agent, and can prevent unwanted precipitation of the therapeutic agent.

The following examples are provided to illustrate certain features and / or specific embodiments. Examples

Example 1. MSM alone does not affect the survival of MRSA.

This example describes in vitro experiments testing the survival of Staphylococcus aureus strain ATCC 43300 (a methicillin and oxacillin resistant strain) in the presence of MSM alone. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM for 24 and 48 hours. 3.5% more MSM was added each day. The results show that MSM does not affect the survival of this bacterial strain.

Methods:

The USP <51> antimicrobial efficacy protocol was used as the template for the experimental paradigm. The initial concentrations of MSM tested were 5, 8, 10, 12, 13, 14, 15, and 16% MSM. All concentrations were plated with dilutions of 10^-7 to discern cfu / ml.

The materials used were Flake OptiMSM® MSM (lot number 0604751), Staphylococcus aureus strain ATCC 43300, 30 ml borosilicate glass culture tubes, lactose broth (LB; Alpha Biosciences; Lot: L07-03), cal o Modified Letheen (MLB, Alpha Biosciences, lot 108-09), Trypto soy agar with lecithin and Tween 80 (TSA; Alpha Biosciences, lot: F08-42), USP grade sodium oxacillin lot J and sodium methicillin (AS ; Cat. # 1410002, Batch KOH338).

Flake OptiMSM® MSM was weighed on a certified Mettler Toledo AG245 SN: 1115210833 balance and distributed in aliquots for each concentration. MSM was introduced into 30 ml borosilicate glass culture tubes. MSM was added to the tubes as follows: 5% (0.5g), 8% (0.8g), 10% (1.0g), 12% (1.2g), 13% (1, 3 g), 14% (1.4 g), 15% (1.5 g), and 16% (1.6 g). The material was calculated for a volume of 10 ml.

All tubes were inoculated with a MRSA dilution that provides a final level of colony forming units of 2.0 x10.⁵ / ml (Log = 5.23). The tubes were incubated at 25 ° C and periodically mixed. Plating was carried out at 24 and 48 hours. Each condition was plated by diluting 1 ml of growth material in 9 ml of MLB dilution broth. This mixture was serially diluted to 10^-7, and 1 ml was placed in a sterile petri dish for each dilution point. 20 ml of TSA was added to each dilution, centrifuged and allowed to solidify. All dilutions were placed in an incubator at 35 ° C for 24 hours. Bacterial colonies on each plate were counted and colony counts transformed to logarithmic format. A positive and negative control were elucidated.

Results:

The results showed that none of the MSM concentrations tested had any effect on the survival of Staphylococcus aureus strain ATCC 43300 for any 24 or 48 hour time point.

Example 2. MSM sensitizes MRSA to oxacillin.

This example describes in vitro experiments studying the survival of Staphylococcus aureus strain ATCC 43300 (a strain of MRSA resistant to oxacillin and methicillin) in the presence of MSM, DMSO and oxacillin. The results are shown in Figure 1. A lower survival rate was observed in the presence of 9-16% MSM with 6 pg / ml oxacillin than in the presence of 1% DMSO and 6 pg / ml oxacillin or in presence of 1% DMSO, 9-16% MSM and 6 pg / ml of oxacillin. The lowest survival rate was observed in the 12 and 13% MSM conditions with 6 pg / ml of antibiotic. These results show that specific concentrations of MSM alone can increase the sensitivity of a MRSA strain to the antibiotic more effectively than DMSO or a combination of MSM and DMSO.

Methods

The USP <51> antimicrobial efficacy protocol was used as the template for the experimental paradigm. MSM concentrations tested were 5-16% in increments of one. All concentrations were plated with dilutions of 10^-7 to discern cfu / ml. The initial oxacillin concentration used was 6 pg / ml, which is the MIC for oxacillin. This concentration is the industry standard for determining MRSA resistance in clinical applications.

ES 2 674 019 T3

Materials used were Flake OptiMSM® MSM (lot number 0604751), DMSO (Jacob Labs, lot number 48074), Staphylococcus aureus strain ATCC 43300, 30 ml borosilicate glass culture tubes, lactose broth (LB; Alpha Biosciences; Lot: L07-03), Modified Letheen Broth (MLB, Alpha Biosciences, Lot 108-09), Trypto Soy Agar with Lecithin and Tween 80 (TSA; Alpha Biosciences, Lot: F08-42) and Grade Sodium Oxacillin USP lot J.

Flake OptiMSM® MSM was weighed on a certified Mettler Toledo AG245 SN: 1115210833 balance and distributed in aliquots for each concentration. MSM was introduced into 30 ml borosilicate glass culture tubes. The material was calculated for a volume of 10 ml. MSM was added to the tubes as follows: 5% (0.5g), 6% (0.6g), 7% (0.7g), 8% (0.8g), 9% (0, 9 g), 10% (1.0 g), 11% (1.1 g), 12% (1.2 g), 13% (1.3 g), 14% (1.4 g), 15 % (1.5 g), and 16% (1.6 g). Sterile DMSO was added to the appropriate tubes. Sterile oxacillin was added to the appropriate tubes in 300 microliters of 30 mg / 10 ml concentration giving it a final concentration of 6 pg / ml. The control conditions were lactose broth inoculated with Staphylococcus aureus strain ATCC 43300 with and without 6 pg / ml of oxacillin, as well as uninoculated negative controls that show no signs of contamination.

The appropriate tubes were inoculated at a dilution of Staphylococcus aureus strain ATCC 43300 that provides a final concentration of colony-forming units of 3.15x10.⁷/ ml (Log = 7.49). The tubes were incubated at 25 ° C and periodically mixed. Plating was carried out at 48 hours. The 5-16% tubes were plated by diluting 1 ml of material in 9 ml of MLB dilution broth. They were serially diluted up to 10^-7 with 1 ml placed in a sterile petri dish for each dilution point. 20 ml of TSA was then added to each dilution and centrifuged and allowed to solidify. All dilutions were placed in an incubator at 35 ° C for 24 hours. Plates were counted and colony counts were changed to logarithmic format. A positive and negative control were elucidated.

Results:

Table 1 and Figure 1 show the results of this study.

Table 1: Survival of Staphylococcus aureus strain ATCC 43300 in 5-16% MSM and oxacillin for 48 hours.

<td>% MSM (if added)</td><td>(MA) 6 pg / ml Oxacillin MSM (Log cfu / ml)</td><td>(MDA) 6 pg / ml Oxacillin MSM 1% DMSO (Log cfu / ml)</td><td>(DA) 6 pg / ml oxacillin without MSM 1% DMSO (Log cfu / ml)</td>

<td> 16</td><td> 5,1</td><td> 5,5</td><td> 5,8</td>

<td> 15</td><td> 4,9</td><td> 5,7</td><td> 5,7</td>

<td> 14</td><td> 4,8</td><td> 5,7</td><td> 5,7</td>

<td> 13</td><td> 4,6</td><td> 5,8</td><td> 5,4</td>

<td> 12</td><td> 4,6</td><td> 5,8</td><td> 5,8</td>

<td> 11</td><td> 4,8</td><td> 5,8</td><td> 5,8</td>

<td> 10</td><td> 5,1</td><td> 5,8</td><td> 5,7</td>

<td> 9</td><td> 5,5</td><td> 5,9</td><td> 5,9</td>

<td> 8</td><td> 5,9</td><td> 5,8</td><td> 5,9</td>

<td> 7</td><td> 6,8</td><td> 5,9</td><td> 5,9</td>

<td> 6</td><td> 6,6</td><td> 6,1</td><td> 6,3</td>

<td> 5</td><td> 6,7</td><td> 6,2</td><td> 6,2</td>

These results show that 9-16% MSM increased the sensitivity of Staphylococcus aureus strain ATCC 43300 to oxacillin for at least 48 hours. A log reduction was observed. minimum in cfu / ml under MA, DA, and MDA conditions for the 5-8% MSM concentration range. Log reduction in cfu / ml refers to a decrease (reduction) in the cfu / ml of a bacterial culture compared to the cfu / ml that were initially inoculated from the bacterial culture. For the 9% MSM concentration, the MA condition showed a 2.0 log reduction in cfu / ml compared to the DA and MDA conditions, which show a log reduction. 1.6 in cfu / ml. For the 10-16% MSM concentration, the MA condition showed a maximum log reduction of 2.9 in cfu / ml compared to the DA and MDA conditions, which showed a log reduction. in cfu / ml of 1.69 and 2.0, respectively. For the 5-7% concentration of MSM, the MA condition showed an increase in bacterial growth compared to the DA and MDA conditions. DMSO may have some inhibitory effect on the ability of 9-16% MSM / oxacillin to inhibit the growth of Staphylococcus aureus strain ATCC 43300.

Positive control showed TNTC in dilution plate 10⁶ The negative control showed no signs of contamination. Taken together, these results indicate that MSM can sensitize a strain of MRSA to antibiotic treatment.

Example 3. MSM sensitizes MRSA to oxacillin in a sham cycle of treatment.

ES 2 674 019 T3

This example describes in vitro experiments testing the survival of Staphylococcus aureus strain ATCC 43300 (a methicillin and oxacillin resistant strain) in the presence of MSM and oxacillin. The results are shown in Figure 2. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM and 6 pg / ml oxacillin (MA) for 24 hours at 25 ° C. After 24 hours, another 6 pg / ml of ml oxacillin was added to the incubations, bringing the total amount of oxacillin added to 12 pg / ml. 6 pg / ml is the MIC for this strain of MRSA. Therefore, the bacteria were under a MIC of oxacillin for 24 hours and 2x MIC for the next 24 hours. This experimental paradigm encourages the repeated application of antibiotic that a subject would receive during a treatment cycle. Analogously to the results shown in Example 2, the lowest survival rate was observed in the presence of MSM at 12 and 13%. These results confirm that MSM sensitizes MRSA to antibiotic treatment and shows that specific concentrations of MSM alone can increase the sensitivity of an MRSA strain to the antibiotic in a sham treatment cycle.

Methods:

The USP &lt;51&gt; antimicrobial efficacy protocol was used as the template for the experimental paradigm. The MSM concentrations tested were 5, 8, 10, 12, 13, 14, 15, and 16%. All concentrations were plated with dilutions of 10^-7 to discern the cfu / ml. The initial concentration of oxacillin used was 6 pg / ml, which is the MIC of oxacillin for this strain of bacteria. This concentration is the industry standard for determining MRSA resistance in clinical applications. An additional 6 pg / ml oxacillin was added daily.

Materials used were Flake OptiMSM® MSM (lot number 0604751), Staphylococcus aureus strain ATCC 43300, 30 ml borosilicate glass culture tubes, lactose broth (LB; Alpha Biosciences; Lot: L07-03), broth Modified Letheen (MLB, Alpha Biosciences, lot 108-09), Trypto soy agar with lecithin and Tween 80 (TSA; Alpha Biosciences, lot: F08-42) and sodium oxacillin, USP grade lot J. Flake OptiMSM® MSM was weighed on a Mettler Toledo AG245 SN: 1115210833 balance and aliquoted for each concentration. MSM was introduced into 30 ml borosilicate glass culture tubes. MSM was added to the tubes as follows: 5% (0.5g), 8% (0.8g), 10% (1.0g), 12% (1.2g), 13% (1, 3g), 14% (1.4g), 15% (1.5g), 16% (1.6g). The material was calculated for a volume of 10 ml. Sterile oxacillin was added to each experimental condition daily at 300 µl of a concentration of 30 mg / 10 ml giving in each case a final concentration of 6 µg / ml.

All tubes were inoculated at a MRSA dilution that provides a final concentration of colony-forming units of 9.14x10.⁵/ ml (Log = 5.96). The tubes were incubated at 25 ° C and periodically mixed. Plating was carried out at 48 hours and 7 days. The 5-16% MSM tubes were plated by diluting 1 ml of material in 9 ml of MLB broth. They were serially diluted up to 10^-7 with 1 ml placed in a sterile petri dish for each dilution point. 20 ml of TSA was added to each dilution and centrifuged and allowed to solidify. All dilutions were placed in an incubator at 35 ° C for 24 hours. Plates were counted and colony counts were changed to logarithmic format. A positive and negative control were elucidated.

Table 2 and Figure 2 show the results of this study.

Table 2: Survival of Staphylococcus aureus strain ATCC 43300 in 5-16% MSM and oxacillin at 48 and 168 hours:

<td></td><td>48 hours</td><td>7 days</td>

<td>% MSM</td><td>6 pg / ml Oxacillin MSM (Log cfu / ml)</td><td>6 pg / ml Oxacillin MSM (Log cfu / ml)</td>

<td> 16</td><td> 5,3</td><td> 1,0</td>

<td> 15</td><td> 4,1</td><td> 6,9</td>

<td> 14</td><td> 4,0</td><td> 7,0</td>

<td> 13</td><td> 3,8</td><td> 7,2</td>

<td> 12</td><td> 3,8</td><td> 7,1</td>

<td> 10</td><td> 4,6</td><td> 7,2</td>

<td> 8</td><td> 4,0</td><td> 7,6</td>

<td> 5</td><td> 7,0</td><td> 7,9</td>

These results show that MSM increased the sensitivity of Staphylococcus aureus strain ATCC 43300 to oxacillin for at least 48 hours. At the 48 hour time point, 12-15% MSM increased the sensitivity of Staphylococcus aureus strain ATCC 43300 to oxacillin more than any other concentration of MSM studied. At the seven day time point, except for the highest concentration of MSM tested (16%),

ES 2 674 019 T3

MSM did not increase the sensitivity of Staphylococcus aureus strain ATCC 43300 to oxacillin. 16% MSM with the addition of 6 pg / ml each day showed no surviving bacteria on day 7. This result shows that, except at the highest MSM concentration tested (16%), any initial MSM-induced oxacillin sensitization of Staphylococcus aureus strain ATCC 43300 was reduced after prolonged exposure to MSM and antibiotic. Controls were terminated after 48 hours to minimize the hazard associated with uncontrolled growth of the MRSA organism.

Example 4. MSM sensitizes MRSA to multiple antibiotics.

This example describes in vitro experiments testing the survival of Staphylococcus aureus strain ATCC 43300 (a methicillin and oxacillin resistant strain) in the presence of MSM and oxacillin or MSM and methicillin. The growth periods tested were 24 hours, 48 ​​hours and 5 days. 5-16% MSM was tested along with 6 pg / ml oxacillin or 6 pg / ml methicillin. This is the MIC of oxacillin and methicillin for this strain of bacteria. An additional 3.5% MSM and 6 pg / ml oxacillin or 6 pg / ml methicillin were added each day. The results are shown in Figures 3A-3C. At 24 hours, MSM 5% and antibiotic showed the lowest level of bacterial survival. At 48 hours, 5% MSM and methicillin or 8% mSm and oxacillin showed the lowest level of bacterial survival. At 5 days, MSM 13-16% and antibiotic showed the lowest level of bacterial survival. Taken together, the results indicate that MSM increased the sensitivity of this strain of MRSA to oxacillin and methicillin.

Methods:

The USP &lt;51&gt; antimicrobial efficacy protocol was used as the template for the experimental paradigm. The initial concentrations of MSM tested were 5, 8, 10, 12, 13, 14, 15, and 16% MSM. All concentrations were plated with dilutions of 10^-7 to discern the cfu / ml. The initial concentration of oxacillin methicillin used was 6 pg / ml, which is the MIC for oxacillin. This concentration is the industry standard for determining MRSA resistance in clinical applications. An additional 3.5% MSM and 6 pg / ml oxacillin or 6 pg / ml methicillin were added daily.

Materials used were Flake OptiMSM® MSM (lot number 0604751), Staphylococcus aureus strain ATCC 43300, 30 ml borosilicate glass culture tubes, lactose broth (LB; Alpha Biosciences; Lot: L07-03), broth Modified Letheen (MLB, Alpha Biosciences, lot 108-09), Trypto Soy Agar with lecithin and Tween 80 (TSA; Alpha Biosciences, lot: F08-42), USP grade sodium oxacillin lot J and sodium methicillin (AS; Cat # 1410002, Lot KOH338).

Flake OptiMSM® MSM was weighed on a certified Mettler Toledo AG245 SN: 1115210833 balance and distributed in aliquots for each concentration. MSM was introduced into 30 ml borosilicate glass culture tubes. MSM was added to the tubes as follows: 5% (0.5g), 8% (0.8g), 10% (1.0g), 12% (1.2g), 13% (1.3g), 14% (1.4g), 15% (1.5g), 16% (1.6g). The material was calculated for a volume of 10 ml. Sterile oxacillin or methicillin was added to each experimental condition daily at 300 µl of a concentration of 30 mg / 10 ml giving in each case a final concentration of 6 µg / ml. An additional 3.5% MSM and 6 pg / ml were added each day.

All tubes were inoculated with a MRSA dilution that provides a final level of colony forming units of 2.13 x10.⁶ / ml (Log = 6.3). The tubes were incubated at 25 ° C and periodically mixed. Plating was carried out at 24 hours, 48 ​​hours and 5 days. Each condition was plated by diluting 1 ml of growth material in 9 ml of MLB dilution broth. This mixture was serially diluted to 10^-7, and 1 ml was placed in a sterile petri dish for each dilution point. 20 ml of TSA was added to each dilution, centrifuged and allowed to solidify. All dilutions were placed in an incubator at 35 ° C for 24 hours. Bacterial colonies on each plate were counted and colony counts transformed to logarithmic format. A positive and negative control were elucidated.

Results:

The results of this study are shown in Table 3.

Table 3: Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and oxacillin or methicillin for 24, 48 and 120 hours.

<td colspan="3">24 hours</td>

<td>% MSM</td><td>6 pg / ml Methicillin MSM (Log cfu / ml)</td><td>6 pg / ml Oxacillin MSM (Log cfu / ml)</td>

<td> 16</td><td> 5,00</td><td> 5,25</td>

<td> 15</td><td> 5,00</td><td> 5,23</td>

<td> 14</td><td> 4,90</td><td> 5,20</td>

ES 2 674 019 T3

<td> 13</td><td> 4,90</td><td> 5,00</td>

<td> 12</td><td> 4,60</td><td> 5,20</td>

<td> 10</td><td> 4,40</td><td> 5,00</td>

<td> 8</td><td> 4,50</td><td> 5,0</td>

<td> 5</td><td> 4,30</td><td> 4,90</td>

<td colspan="3"></td>

<td colspan="3">48 hours</td>

<td>% MSM</td><td>6 pg / ml Methicillin MSM (Log cfu / ml)</td><td>6 pg / ml Oxacillin MSM (Log cfu / ml)</td>

<td> 16</td><td> 4,17</td><td> 4,17</td>

<td> 15</td><td> 4,00</td><td> 4,40</td>

<td> 14</td><td> 3,98</td><td> 4,40</td>

<td> 13</td><td> 3,90</td><td> 4,20</td>

<td> 12</td><td> 3,70</td><td> 4,30</td>

<td> 10</td><td> 3,70</td><td> 4,20</td>

<td> 8</td><td> 3,60</td><td> 3,60</td>

<td> 5</td><td> 3,60</td><td> 5,20</td>

<td colspan="3"></td>

<td colspan="3">120 hours</td>

<td>% MSM</td><td>6 pg / ml Methicillin MSM (Log cfu / ml)</td><td>6 pg / ml Oxacillin MSM (Log cfu / ml)</td>

<td> 16</td><td> 2,80</td><td> 3,00</td>

<td> 15</td><td> 2,80</td><td> 3,00</td>

<td> 14</td><td> 2,90</td><td> 3,00</td>

<td> 13</td><td> 2,80</td><td>TNTC</td>

<td> 12</td><td> 4,20</td><td>TNTC</td>

<td> 10</td><td>TNTC</td><td>TNTC</td>

<td> 8</td><td>TNTC</td><td>TNTC</td>

<td> 5</td><td>TNTC</td><td>TNTC</td>

The results of this study indicate that MSM increased the sensitivity of a strain of MRSA to oxacillin and methicillin. At 24 hours, less cfu / ml were observed with MSM and methicillin than with MSM and oxacillin, for all MSM concentrations tested. Since the MIC for methicillin and oxacillin is the same for this strain of

MRSA (6 pg / ml), this result suggests that MSM increases methicillin sensitivity more than oxacillin sensitivity. At 24 hours, MSM 5% and antibiotic showed the lowest level of bacterial survival.

Similar results were seen at 48 hours. At this time point, the mean log reduction in cfu / ml observed for methicillin ranged from 2.7 (initial 5% MSM) to 2.1 (16% initial MSM) and for oxacillin 2.7 (8% initial MSM) to 2.1 (16% initial MSM). At 48 hours, 5% MsM and methicillin or 8% mSM and oxacillin showed the lowest level of bacterial survival. At 48 hours, bacterial survival was the same as for the initial 8% MSM and under either oxacillin or methicillin conditions.

At 120 hours, the initial conditions of MSM 14-16% and oxacillin and the initial conditions of MSM 13-16% and methicillin showed a logarithmic reduction in cfu / ml of -3.3-3.5 respectively. The initial 5-10% MSM and methicillin and the initial 13% MSM and oxacillin showed colony counts that were too numerous to count (TNTC). 13-16% of MSM and antibiotic showed the lowest level of bacterial survival.

Taken together, these results show that MSM can increase the sensitivity of a MRSA strain of bacteria to methicillin and oxacillin. These results also suggest that a combination of methicillin and MSM is

ES 2 674 019 T3 more effective in sensitizing the MRSA strain than a combination of MSM and oxacillin. A purely hypothetical explanation for these results is that the MSM molecule is somehow overcoming resistance to antibiotics, possibly by penetration, or by transporting the antibiotic into the cell. Another purely hypothetical conclusion is that methicillin can bind to MSM and be transported into the cell from MRSA directly due to a greater number of MSM-binding sites compared to oxacillin. The inventors observed that, for the 24 and 48 hour time points, lower concentrations of MSM with antibiotic had a greater effect on bacterial survival than at the day 5 time point. A purely hypothetical and non-limiting explanation for this is that the concentration of MSM in the test tube decreases over time, possibly due to the action of bacteria.

Example 5. MSM sensitizes MRSA to multiple antibiotics.

This example describes preliminary in vitro experiments studying the survival of Staphylococcus aureus strain ATCC 43300 (a strain of MRSA resistant to oxacillin and methicillin), in the presence of MSM and oxacillin or MSM and methicillin. Three different concentrations of these two antibiotics were tested: 12, 30 and 60 pg / ml, corresponding to 2x, 5x and 10x MIC for this strain of MRSA for each antibiotic. Staphylococcus aureus strain ATCC 43300 was incubated with 5-16% MSM and the indicated amount of antibiotic for 24, 48 or 96 hours at 25 ° C.

Methods:

The USP &lt;51&gt; antimicrobial efficacy protocol was used as the template for the experimental paradigm. The initial MSM concentrations tested were 5-16% in increments of one. All concentrations were plated with dilutions of 10^-7 to discern the cfu / ml. The initial concentration of oxacillin and methicillin used was 12, 30 or 60 pg / ml, which corresponds to 2x, 5x and 10x MIC for oxacillin. This concentration is the industry standard for determining MRSA resistance in clinical applications.

Materials used were Flake OptiMSM® MSM (lot number 0604751), Staphylococcus aureus strain ATCC 43300, 30 ml borosilicate glass culture tubes, lactose broth (LB; Alpha Biosciences; Lot: L07-03), broth Modified Letheen (MLB, Alpha Biosciences, Lot I08-09, Trypto Soy Agar with lecithin and Tween 80 (TSA; Alpha Biosciences, lot: F08-42), USP grade sodium oxacillin lot J and sodium methicillin (AS; n Cat. # 1410002, Lot KOH338).

Staphylococcus aureus strain ATCC 43300 was streaked for isolates and 25 clones were spiked and diluted to a McFarland standard of one. Subsequently, the estimated value was taken to inoculate the tubes containing growth medium and 12, 30 or 60 pg / ml of methicillin or oxacillin with around 10^-7cfu / tube. For the remainder of the experiment, a clone that demonstrated cloudy growth at 24 hours was selected for all antibiotic concentrations.

Flake OptiMSM® MSM was weighed on a certified Mettler Toledo AG245 SN: 1115210833 balance and distributed in aliquots for each concentration. MSM was introduced into 30 ml borosilicate glass culture tubes. MSM was added to the tubes as follows: 5% (0.5g), 6% (0.6g), 7% (0.7g), 8% (0.8g), 9% (0.9g), 10 % (1.0g), 11% (1.1g), 12% (1.2g), 13% (1.3g), 14% (1.4g), 15% (1.5g), and 16% (1.6 g). The material was calculated for a volume of 10 ml. Sterile oxacillin or methicillin were added to each experimental condition to a final concentration of 12, 30 or 60 pg / ml of antibiotic. Control conditions of MSM 5-16% and no antibiotics were tested to determine MRSA survival. Control conditions with 12, 30 or 60 pg / ml of antibiotic, but not with MSM were tested for the growth of MRSA.

All tubes were then inoculated with a dilution of the selected clone of Staphylococcus aureus strain ATCC 43300 that provides a final level of colony-forming units of 9.4x10.⁷/ ml (Log = 7.97). The tubes were incubated at 25 ° C and periodically mixed. An additional 3.5% MSM was added to the tubes daily as well as the initial amount of antibiotic. Plating was carried out at 24 hours. Each condition was plated by diluting 1 ml of growth material in 9 ml of MLB dilution broth. This mixture was serially diluted to 10^-7, and 1 ml was placed in a sterile petri dish for each dilution point. 20 ml of TSA was added to each dilution, centrifuged and allowed to solidify. All dilutions were placed in an incubator at 35 ° C for 24 hours. Bacterial colonies on each plate were counted and colony counts transformed to logarithmic format. A positive and negative control were elucidated.

Results:

Table 4 and Figure 3A-C show the results of this study.

Table 4: Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30 or 60 pg / ml oxacillin or methicillin for 24 hours.

<td>% MSM</td><td>12 pg / ml Methicillin MSM (Log cfu / ml)</td><td>12 pg / ml Oxacillin MSM (Log cfu / ml)</td>

ES 2 674 019 T3

<td> 16</td><td> 4,56</td><td> 4,77</td>

<td> 15</td><td> 4,7</td><td> 4,93</td>

<td> 14</td><td> 4,7</td><td> 4,88</td>

<td> 13</td><td> 4,77</td><td> 4,9</td>

<td> 12</td><td> 4,89</td><td> 4,8</td>

<td> 11</td><td> 4,87</td><td> 4,8</td>

<td> 10</td><td> 4,8</td><td> 4,96</td>

<td> 9</td><td> 4,99</td><td> 5,16</td>

<td> 8</td><td> 5,0</td><td> 5,18</td>

<td> 7</td><td> 5,46</td><td> 5,18</td>

<td> 6</td><td> 5,2</td><td> 5,26</td>

<td> 5</td><td> 5,43</td><td> 5,28</td>

<td colspan="3"></td>

<td>% MSM</td><td>30 pg / ml Methicillin MSM (Log cfu / ml)</td><td>30 pg / ml Oxacillin MSM (Log cfu / ml)</td>

<td> 16</td><td> 4,47</td><td> 4,59</td>

<td> 15</td><td> 4,54</td><td> 4,74</td>

<td> 14</td><td> 4,6</td><td> 4,82</td>

<td> 13</td><td> 4,7</td><td> 4,9</td>

<td> 12</td><td> 4,7</td><td> 4,87</td>

<td> 11</td><td> 4,8</td><td> 4,94</td>

<td> 10</td><td> 4,8</td><td> 4,99</td>

<td> 9</td><td> 5,07</td><td> 5,02</td>

<td> 8</td><td> 5,09</td><td> 5,2</td>

<td> 7</td><td> 5,13</td><td> 5,12</td>

<td> 6</td><td> 5,18</td><td> 5,15</td>

<td> 5</td><td> 5,3</td><td> 5,17</td>

<td colspan="3"></td>

<td>% MSM</td><td>60 pg / ml Methicillin MSM (Log cfu / ml)</td><td>60 pg / ml Oxacillin MSM (Log cfu / ml)</td>

<td> 16</td><td> 4,8</td><td> 4,6</td>

<td> 15</td><td> 4,65</td><td> 4,75</td>

<td> 14</td><td> 4,77</td><td> 4,8</td>

<td> 13</td><td> 4,77</td><td> 4,83</td>

<td></td><td></td><td></td>

<td> 12</td><td> 4,68</td><td> 4,77</td>

<td> 11</td><td> 4,66</td><td> 4,8</td>

<td> 10</td><td> 4,66</td><td> 4,87</td>

<td> 9</td><td> 4,9</td><td> 4,95</td>

<td> 8</td><td> 5,1</td><td> 5,0</td>

<td> 7</td><td> 5,0</td><td> 5,14</td>

<td> 6</td><td> 5,2</td><td> 5,12</td>

<td> 5</td><td> 5,2</td><td> 5,2</td>

The logarithmic reduction observed in cfu / ml was similar for all antibiotic concentrations. The mean log reduction in cfu / ml for 5% MSM with antibiotic was 2.2, while the mean log reduction in cfu / ml for 16% MSM with antibiotic was 3.3 (excluding methicillin 60 pg / ml as an outlier). The overall efficacy of methicillin was slightly better than that of oxacillin. Overall, a greater sensitivity to the antibiotic was observed the higher the MSM concentrations were. The antibiotic control condition without MSM presented a greater number of colonies than those that were inoculated initially. Control conditions with antibiotic, but without MSM, showed TNTC, demonstrating that there was no inhibition with the antibiotic alone.

Results from the 48 and 96 hour time points showed that all the cultures at these time points were too numerous to count. This can be explained by the use of a clone that had a high growth for all three antibiotic concentrations, which selects for a more resistant strain of MRSA than that obtained naturally. Added to this, there would be a higher inoculation value of 9.4x10⁷/ ml (log = 7.97);

normally, the inoculation should be between 10⁴ and 10⁶ cfu / ml. A merely hypothetical, non-limiting explanation for this is that for these experimental conditions, any effect of MSM and antibiotics on bacterial survival is masked by other factors. Alternatively, the antibiotic could also have degraded since the inventors used it in the last experiment for a long period of time.

These results show that MSM increased the sensitivity of Staphylococcus aureus strain ATCC 43300 to

ES 2 674 019 T3 oxacillin and methicillin for at least a 24 hour time point; however, the results are not conclusive at other time points. At the 24 hour time point, greater sensitivity was observed for all antibiotic concentrations tested, and for all MSM concentrations tested. Higher concentrations of MSM increased antibiotic sensitivity to a greater extent than lower concentrations of MSM. One difference between this experiment and previous experiments is that a clone was selected that met the growth requirements for all three antibiotic concentrations.

Example 6. Effect of MSM and 12, 30, or 60 pg / ml of oxacillin on MRSA.

This example describes an in vitro experiment that studies the survival of Staphylococcus aureus strain ATCC 43300 in the presence of MSM 5-16% and an initial concentration of 12, 30 or 60 pg / ml of oxacillin. This corresponds to 2x, 5x and 10x MICs for oxacillin. More antibiotic was added every 24 hours. The growth periods tested were 24 (see Fig. 4), 48, 72, 96 and 120 hours. The results show that MSM sensitized a strain of MRSA to oxacillin. At the earliest starting points (24 and 48 hours), the results show a lower bacterial survival, indicating a higher sensitivity to oxacillin, for all the antibiotic concentrations tested, and for all the MSM concentrations tested. However, at the later time points (96 and 120 hours), only decreased bacterial survival was observed at higher MSM concentrations (10-16% MSM), indicating that MSM-induced sensitivity to oxacillin is time dependent. The results also show that, in the later time points, a high oxacillin concentration was correlated with decreased bacterial survival.

Methods:

The USP &lt;51&gt; antimicrobial efficacy protocol was used as the template for the experimental paradigm. The initial MSM concentrations tested were 5-16% in increments of one. All concentrations were plated with dilutions of 10^-7 to discern the cfu / ml. The initial oxacillin concentration used was 12, 30 or 60 pg / ml, which corresponds to 2x, 5x and 10x MIC for oxacillin. More antibiotic was added to the initial concentration every 24 hours.

Materials used were Flake OptiMSM® MSM (lot number 0604751), Staphylococcus aureus strain ATCC 43300, 30 ml borosilicate glass culture tubes, lactose broth (LB; Alpha Biosciences; Lot: L07-03), broth Modified Letheen (MLB, Alpha Biosciences, lot 108-09), Trypto soy agar with lecithin and Tween 80 (TSA; Alpha Biosciences, lot: F08-42) and USP grade sodium oxacillin lot J.

Staphylococcus aureus strain ATCC 43300 spread in streaks from the isolates. An isolated colony was selected, streaked again, and incubated. The isolate was selected and then diluted to a McFarland standard of one. Subsequently, the estimated value was taken to inoculate the tubes containing growth medium and 12, 30 or 60 pg / ml of methicillin or oxacillin with around 10⁵ cfu / ml. These tubes were incubated and plated to determine the cfu count. A clone for which no log reduction of cfu was observed was selected for the remainder of the experiment.

Flake OptiMSM® MSM was weighed on a certified Mettler Toledo AG245 SN: 1115210833 balance and distributed in aliquots for each concentration. MSM was introduced into 30 ml borosilicate glass culture tubes. MSM was added to the tubes as follows: 5% (0.5g), 6% (0.6g), 7% (0.7g), 8% (0.8g), 9% (0.9g), 10% (1.0g), 11% (1.1g), 12% (1.2g), 13% (1.3g), 14% (1.4g), 15% (1.5g), and 16% (1.6 g). The material was calculated for a volume of 10 ml. Sterile oxacillin was added to each experimental condition to a final concentration of 12, 30 or 60 pg / ml of antibiotic. Control conditions were tested with 5, 10 and 16% MSM and without antibiotic, or without MSM and 12, 30 or 60 pg / ml of antibiotic.

All tubes were then inoculated with a dilution of Staphylococcus aureus strain ATCC 43300 to provide clones at a final level of 3.3x10 colony-forming units.⁵/ ml (log = 5.52). The tubes were incubated at 25 ° C and periodically mixed. The antibiotic was reoccupied every 24 hours at the initial concentration to maintain pressure on the body. Plating was carried out at 24, 48, 72, and 120 hours. Each condition was plated by diluting 1 ml of growth material in 9 ml of MLB dilution broth. This was carried out every time. 1 ml of growth medium containing MSM was subsequently added to the tube to maintain a constant volume and concentration of MSM. This mixture was serially diluted to 10^-7, and 1 ml was placed in a sterile petri dish at each dilution point, in triplicate. 20 ml of TSA was added to each dilution, centrifuged and allowed to solidify. All dilutions were placed in an incubator at 35 ° C for 24 to 72 hours. Bacterial colonies on each plate were counted and the average taken was converted to logarithmic format. Any cfu / ml greater than 3.3x10⁵ (log = 5.5) were recorded as&gt; 3.3x10⁵.

Results:

In this experiment the positive controls (those inoculated with MRSA) used for the three concentrations of oxacillin alone (12 pg / ml, 30 pg / ml and 60 pg / ml) did not show significant decrease in colony-forming units from the value of the initial inoculums. Tables 5-9 show the results of this study.

ES 2 674 019 T3

Table 5: Survival of Staphylococcus aureus strain ATCC 43300 in 5-16% MSM and 12, 30 or 60 pg / ml oxacillin for 24 hours.

<td>% MSM</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>24 hour average log</td>

<td> 16</td><td>7.6x10³</td><td>7.4x10³</td><td>7.6x10³</td><td> 3,88</td>

<td> 15</td><td>1.35x10⁴</td><td>1.30x10⁴</td><td>1.29x10⁴</td><td> 4,11</td>

<td> 14</td><td>1.00x10⁴</td><td>1.02x10⁴</td><td>1.01x10⁴</td><td> 4,00</td>

<td> 13</td><td>9.0x10³</td><td>8.7x10³</td><td>9.2x10³</td><td> 3,95</td>

<td> 12</td><td>7.0x10³</td><td>7.0x10³</td><td>7.3x10³</td><td> 3,85</td>

<td> 11</td><td>8.2x10³</td><td>8.5x10³</td><td>8.0x10³</td><td> 3,91</td>

<td> 10</td><td>5.8x10³</td><td>5.6x10³</td><td>6.0x10³</td><td> 3,76</td>

<td> 9</td><td>5.6x10³</td><td>5.2x10³</td><td>5.0x10³</td><td> 3,72</td>

<td> 8</td><td>6.6x10³</td><td>6.6x10³</td><td>6.9x10³</td><td> 3,82</td>

<td> 7</td><td>8.2x10³</td><td>8.5x10³</td><td>8.3x10³</td><td> 3,91</td>

<td> 6</td><td>8.6x10³</td><td>8.5x10³</td><td>8.0x10³</td><td> 3,92</td>

<td> 5</td><td>8.4x10³</td><td>8.0x10³</td><td>8.0x10³</td><td> 3,91</td>

<td> 0</td><td>3.3x10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td>% MSM</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>24 hour average log</td>

<td> 16</td><td>8.3x10³</td><td>8.3x10³</td><td>8.0x10³</td><td> 3,91</td>

<td> 15</td><td>8.7x10³</td><td>8.9x10³</td><td>8.8x10³</td><td> 3,94</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 14</td><td>6.1x10³</td><td>6.0x10³</td><td>6.0x10³</td><td> 3,78</td>

<td> 13</td><td>4.4x10³</td><td>4.1x10³</td><td>4.2x10³</td><td> 3,62</td>

<td> 12</td><td>3.9x10³</td><td>3.9x10³</td><td>3.9x10³</td><td> 3,59</td>

<td> 11</td><td>3.9x10³</td><td>3.7x10³</td><td>3.7x10³</td><td> 3,57</td>

<td> 10</td><td>2.2x10³</td><td>2.2x10³</td><td>2.0x10³</td><td> 3,32</td>

<td> 9</td><td>1.06x10³</td><td>1.05x10³</td><td>1.02x10³</td><td> 3,01</td>

<td> 8</td><td>1. 18x10³</td><td>1.18x10³</td><td>1.16x10³</td><td> 3,07</td>

<td> 7</td><td>9.7x10²</td><td>9.6x10²</td><td>9.2x10²</td><td> 2,98</td>

<td> 6</td><td>2.9x10²</td><td>2.9x10²</td><td>2.9x10²</td><td> 2,46</td>

<td> 5</td><td>3.1x10²</td><td>3.3x10²</td><td>3.3x10²</td><td> 2,51</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td>% MSM</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>24 hour average log</td>

<td> 16</td><td>6.9x10³</td><td>6.9x10³</td><td>6.9x10³</td><td> 3,83</td>

<td> 15</td><td>6.6x10³</td><td>6.2x10³</td><td>6.4x10³</td><td> 3,81</td>

<td> 14</td><td>1.8x10²</td><td>1.7x10²</td><td>1.8x10²</td><td> 2,03</td>

ES 2 674 019 T3

<td> 13</td><td>6.3x10²</td><td>6.0x10²</td><td>6.0x10²</td><td> 2,80</td>

<td> 12</td><td>7.3x10²</td><td>7.1x10²</td><td>7.5x10²</td><td> 2,86</td>

<td> 11</td><td>6.7x10²</td><td>6.7x10²</td><td>6.9x10²</td><td> 2,83</td>

<td> 10</td><td>4.2x10²</td><td>4.4x10²</td><td>4.4x10²</td><td> 2,63</td>

<td> 9</td><td>3.2x10²</td><td>3.2x10²</td><td>3.2x10²</td><td> 2,50</td>

<td> 8</td><td>4.6x10²</td><td>4.6x 10²</td><td>5.0x10²</td><td> 2,67</td>

<td> 7</td><td>2.1x10²</td><td>2.3x10²</td><td>2.1x10²</td><td> 2,34</td>

<td> 6</td><td>2.4x10²</td><td>2.4x10²</td><td>2.4x10²</td><td> 2,38</td>

<td> 5</td><td>3.5x10²</td><td>3.3x10²</td><td>3.6x10²</td><td> 2,54</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

As shown in Table 5 and Figure 4, the number of cfu / ml observed at the 24 hour time point was similar for all oxacillin concentrations. Lower MSM concentrations had a greater log reduction in colony count than higher MSM concentrations. The average log reduction in the number of cfu / ml for 12 pg / ml of oxacillin is 1.5 logs which is carried over for all concentrations with little or no change. The 30 pg / ml and 60 pg / ml conditions had a logarithmic reduction in the number of cfu / ml at the lower end of the MSM concentrations, averaging a 3 log difference. The 60 pg / ml condition demonstrated the largest log reduction in the number of cfu / ml in the first 24 hours of the three oxacillin concentrations. The 60 pg / ml oxacillin condition for 14% MSM had a 3.5 log reduction in the number of cfu / ml. Negative LB controls showed no signs of contamination. Positive LB controls showed cloudy growth. The 5%, 10% and 16% negative MSM controls had no signs of contamination. The 5%, 10%, and 16% positive MSM controls had signs of cloudy growth. The oxacillin controls did not show signs of significant reduction from the initial inoculums, and the negative controls showed no signs of contamination.

Table 6: Survival of Staphylococcus aureus strain ATCC 43300 in 5-16% MSM and 12, 30 or 60 pg / ml oxacillin for 48 hours.

<td>% MSM</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>48 hour average log</td>

<td> 16</td><td>4.2x10³</td><td>5.0x10³</td><td>4.3x10³</td><td> 3,7</td>

<td> 15</td><td>5.0x10³</td><td>4.5x10³</td><td>4.6x10³</td><td> 3,7</td>

<td> 14</td><td>1.0x10¹</td><td>1.2x10¹</td><td>1.1x10¹</td><td> 1,0</td>

<td> 13</td><td>6.8x10²</td><td>6.8x10²</td><td>5.8x10²</td><td> 2,8</td>

<td> 12</td><td>3.6x10²</td><td>4.0x10²</td><td>4.2x10²</td><td> 2,8</td>

<td> 11</td><td>6.4x10²</td><td>5.8x10²</td><td>6.5x10²</td><td> 2,8</td>

<td> 10</td><td>5.6x10²</td><td>5.4x10²</td><td>5.4x10²</td><td> 2,7</td>

<td> 9</td><td>2.4x10²</td><td>2.5x10³</td><td>2.3x10²</td><td> 3,4</td>

<td> 8</td><td>4.9x10³</td><td>4.9x10³</td><td>5.4x10³</td><td> 3,7</td>

<td> 7</td><td>9.0x10³</td><td>8.9x10³</td><td>8.5x10³</td><td> 3,9</td>

<td> 6</td><td>1.7x10³</td><td>1.4x10³</td><td>1.7x10³</td><td> 3,2</td>

<td> 5</td><td>4.5x10³</td><td>4.0x10³</td><td>4.2x10³</td><td> 3,6</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td>% MSM</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>48 hour average log</td>

<td> 16</td><td>4.6x10²</td><td>4.0x10²</td><td>4.1x10²</td><td> 2,6</td>

ES 2 674 019 T3

<td> 15</td><td>6.2x10²</td><td>6.4x10²</td><td>6.4x10²</td><td> 2,8</td>

<td> 14</td><td>3.6x10²</td><td>3.2x10²</td><td>3.5x10²</td><td> 2,5</td>

<td> 13</td><td>3.0x10²</td><td>3.1x10²</td><td>3.1x10²</td><td> 2,5</td>

<td> 12</td><td>3.9x10²</td><td>3.6x10²</td><td>4.0x10²</td><td> 2,6</td>

<td> 11</td><td>2.5x10²</td><td>2.7x10²</td><td>2.8x10²</td><td> 2,4</td>

<td> 10</td><td>4.3x10²</td><td>4.3x10²</td><td>4.0x10²</td><td> 2,6</td>

<td> 9</td><td>2.8x10²</td><td>2.6x10²</td><td>2.2x10²</td><td> 2,4</td>

<td> 8</td><td>1.23x10³</td><td>1.24x10³</td><td>1.20x10³</td><td> 3,1</td>

<td> 7</td><td>2.0x10²</td><td>2.1x10²</td><td>2.2x10²</td><td> 2,3</td>

<td> 6</td><td>5.5x10³</td><td>5.5x10³</td><td>5.9x10³</td><td> 3,7</td>

<td> 5</td><td>5.8x10³</td><td>6.0x10³</td><td>5.9x10³</td><td> 3,8</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td>% MSM</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>48 hour average log</td>

<td> 16</td><td>9.0x10¹</td><td>9.0x10¹</td><td>6.0x10¹</td><td> 1,9</td>

<td> 15</td><td>1.2x10²</td><td>1.3x10²</td><td>1.2x10²</td><td> 2,1</td>

<td> 14</td><td>1.5x10²</td><td>1.7x10²</td><td>1.2x10²</td><td> 2,2</td>

<td> 13</td><td>6.0x10¹</td><td>4.0x10¹</td><td>5.0x10¹</td><td> 1,7</td>

<td> 12</td><td>1.1x10²</td><td>1.0x10²</td><td>1.0x10²</td><td> 2,0</td>

<td> 11</td><td>1.3x10²</td><td>1.7x10²</td><td>1.2x10²</td><td> 2,1</td>

<td> 10</td><td>1.6x10²</td><td>1.4x10²</td><td>1.6x10²</td><td> 2,2</td>

<td> 9</td><td>1.8x10²</td><td>1.7x10²</td><td>1.5x10²</td><td> 2,2</td>

<td> 8</td><td>7.5x10²</td><td>7.3x10²</td><td>7.0x10²</td><td> 2,8</td>

<td> 7</td><td>1.0x10¹</td><td>1.1x10⁴</td><td>1.1x10⁴</td><td> 4,0</td>

<td> 6</td><td>6.0x10¹</td><td>5.0x10¹</td><td>6.0x10¹</td><td> 1,8</td>

<td> 5</td><td>3.0x10³</td><td>2.7x10²</td><td>3.3x10³</td><td> 3,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

As shown in Table 6, the number of cfu / ml observed at the 48 hour time point was similar for all oxacillin concentrations. A trend was observed for most lower MSM concentrations to have a smaller log reduction in the number of cfu / ml than higher MSM concentrations across all oxacillin concentrations. The 30 pg / ml and 60 pg / ml oxacillin conditions showed a greater log reduction in the number of cfu / ml at the 48-hour time point than at the 24-hour time point for concentrations of 8% -16 % of MSM. The 60 pg / ml oxacillin condition maintained a greater mean log reduction in the number of cfu / ml compared to the corresponding 24 hour time points for the three oxacillin concentrations. Two points of interest are that 14% MSM at 12 pg / ml oxacillin had a 4.5 log reduction in the number of cfu / ml and 14% MSM at 60 pg / ml oxacillin showed only a reduction slightly higher logarithmic in number of cfu / ml than corresponding 24-hour time point. This could represent a sweet spot for treatment with oxacillin and MSM. Negative LB controls showed no signs of contamination. Positive LB controls showed cloudy growth. The 5%, 10% and 16% negative MSM controls had no signs of contamination. The oxacillin controls did not show signs of significant reduction from the initial inoculums, and the negative controls showed no signs of contamination.

Table 7. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30, or 60 pg / ml oxacillin for 72 hours.

ES 2 674 019 T3

<td>% MSM</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>72 hour average log</td>

<td> 16</td><td>1.1x10²</td><td>1.2x10²</td><td>1.6x10²</td><td> 2,1</td>

<td> 15</td><td>2.3x10²</td><td>2.2x10²</td><td>1.2x10²</td><td> 2,3</td>

<td> 14</td><td>1.1x10²</td><td>1.2x10²</td><td>1.1x10²</td><td> 2,0</td>

<td> 13</td><td>5.2x10²</td><td>5.1x10²</td><td>5.4x10²</td><td> 2,7</td>

<td> 12</td><td>1.5x10³</td><td>3.1x10¹</td><td>1.3x10³</td><td> 3,1</td>

<td> 11</td><td>4.4x10³</td><td>4.5x10³</td><td>4.0x10³</td><td> 3,6</td>

<td> 10</td><td>1.2x10⁴</td><td>1.1x10⁴</td><td>1.0x10¹</td><td> 4,0</td>

<td> 9</td><td>4.7x10⁴</td><td>4.2x10⁴</td><td>4.5x10⁴</td><td> 4,5</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td></td><td>30 pg / ml</td><td>30 pg / ml</td><td>30 pg / ml</td><td>72 hours</td>

<td>% MSM</td><td>oxacillin MSM (cfu / ml)</td><td>oxacillin MSM (cfu / ml)</td><td>oxacillin MSM (cfu / ml)</td><td>Average log</td>

<td> 16</td><td>9x10¹</td><td>8.0x10¹</td><td>8.0x10¹</td><td> 1,9</td>

<td> 15</td><td>2.3x10²</td><td>2.2x10²</td><td>2.8x10²</td><td> 2,4</td>

<td> 14</td><td>1.4x10²</td><td>1.7x10²</td><td>1.8x10²</td><td> 2,2</td>

<td> 13</td><td>9.0x10¹</td><td>8.0x10¹</td><td>4.0x10¹</td><td> 1,8</td>

<td> 12</td><td>4.8x10²</td><td>3.9x10²</td><td>4.9x10²</td><td> 2,6</td>

<td> 11</td><td>3.2x10¹</td><td>1.7x10³</td><td>1.5x10³</td><td> 3,2</td>

<td> 10</td><td>8.9x10³</td><td>9.0x10³</td><td>8.5x10³</td><td> 3,9</td>

<td> 9</td><td>8.8x10²</td><td>8.1x10²</td><td>8.5x10²</td><td> 2,9</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.6x10⁴</td><td>3.6x10⁴</td><td>3.2x10⁴</td><td> 4,5</td>

<td> 6</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td>% MSM</td><td>60 pg / ml Oxacillin MSM ((cfu / ml))</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>72 hour average log</td>

<td> 16</td><td>6.0x10¹</td><td>6.0x10¹</td><td>5.0x10¹</td><td> 1,8</td>

<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td></td>

<td> 14</td><td>2.0x10¹</td><td>2.0x10¹</td><td>4.0x10¹</td><td> 1,5</td>

<td> 13</td><td>1.0x10¹</td><td>1.0x10¹</td><td>1.0x10¹</td><td> 1</td>

<td> 12</td><td>2.8x10²</td><td>2.1x10²</td><td>2.5x10²</td><td> 2,4</td>

<td> 11</td><td>1.0x10¹</td><td>1.0x10¹</td><td>2.0x10¹</td><td> 1</td>

ES 2 674 019 T3

<td> 10</td><td>3.9x10²</td><td>4.0x10²</td><td>3.4x10²</td><td> 2,6</td>

<td> 9</td><td>3.3x10³</td><td>3.3x10³</td><td>3.2x10³</td><td> 2,5</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>6.5x10²</td><td>6.4x10²</td><td>6.1x10²</td><td> 2,8</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

As shown in Table 7, 15% MSM with an oxacillin condition of 60 pg / ml at 72 hours showed no signs of colonies. The tube was removed from the experiment and tested for growth of Staphylococcus strain ATCC 43300. The tube was centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted into 90 ml of Mueller-Hinton broth with 6 pg / ml oxacillin. This is for the enrichment and recovery of any MRSA cells that may be present both healthy and stressed. The pellet had 10 ml of Mueller-Hinton broth with addition of 6 pg / ml of oxacillin added thereto to recover any cells that may be present. Both vessels were incubated at 35 ° C for 48 hours followed by plating every 24 hours. Each container was plated at 10 dilutions to obtain a detection limit of 1 cfu. No colonies were observed, indicating total destruction. This time point for graphical representation purposes was reported as 0 logs.

As shown in Table 7, the number of cfu / ml observed at the 72 hour time point was similar for all oxacillin concentrations. The 5-12% MSM conditions showed a lower mean log reduction in the number of cfu / ml at this time point than for the initial time points. This may demonstrate a reduction in the sensitivity of MRSA to oxacillin. If so, then the reduction is correlated with the MSM concentration (i.e., conc. lower MSM = higher oxacillin resistance). The 12 pg / ml oxacillin condition has a 1 log reduction in the number of cfu / ml for MSM concentrations of 12% -16%. The 60 pg / ml oxacillin condition maintains a greater mean log reduction in the number of cfu / ml between the three oxacillin concentrations that differs compared to the first 24 h period. Two points of interest are that 14% of MSM at a condition of 12 pg / ml of oxacillin showed a logarithmic reduction of 4.5 in the number of cfu / ml and 12% of MSM at a condition of 60 pg / ml of oxacillin showed only a slightly greater log reduction in the number of cfu / ml than the previous time point. This could represent a sweet spot for oxacillin treatment. Negative LB controls showed no signs of contamination. The 5% 10%, and 16% MSM negative controls had no signs of contamination. The oxacillin controls did not show signs of significant reduction from the initial inoculums, and the negative controls showed no signs of contamination.

Table 8. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30, or 60 pg / ml oxacillin for 96 hours.

<td>% MSM</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>96 hour average log</td>

<td> 16</td><td>1.4x10²</td><td>1.4x10²</td><td>1.6x10²</td><td> 2,2</td>

<td> 15</td><td>1.2x10²</td><td>1.3x10²</td><td>1.2x10²</td><td> 2,1</td>

<td> 14</td><td>6.0x10¹</td><td>6.0x10¹</td><td>5.0x10¹</td><td> 1,8</td>

<td> 13</td><td>1.6x10³</td><td>1.7x10³</td><td>3.2x10¹</td><td> 3,2</td>

<td> 12</td><td>6.8x10³</td><td>7.0x10³</td><td>6.8x10³</td><td> 3,8</td>

<td> 11</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 10</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 9</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

ES 2 674 019 T3

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td>% MSM</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>96 hour average log</td>

<td> 16</td><td>3.0x10¹</td><td>3.0x10¹</td><td>1.0x10¹</td><td> 1,3</td>

<td> 15</td><td>8.0x10¹</td><td>8.0x10¹</td><td>8.0x10¹</td><td> 1,9</td>

<td> 14</td><td>5.0x10¹</td><td>5.0x10¹</td><td>6.0x10¹</td><td> 1,7</td>

<td> 13</td><td>3.3x10²</td><td>3.1x10²</td><td>3.2x10²</td><td> 2,5</td>

<td> 12</td><td>6.9x10³</td><td>3.9x10³</td><td>3.9x10³</td><td> 3,7</td>

<td> 11</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 10</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 9</td><td>1.0x10¹</td><td>1.02x10⁴</td><td>1.01x10⁴</td><td> 4,0</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td> 16</td><td>2.0x10¹</td><td>3.0x10¹</td><td>2.0x10¹</td><td> 1,3</td>

<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td></td>

<td> 14</td><td> 0</td><td> 0</td><td> 0</td><td></td>

<td> 13</td><td>4.0x10¹</td><td>4.0x10¹</td><td>5.0x10¹</td><td> 1,6</td>

<td> 12</td><td>3.0x10³</td><td>3.5x10³</td><td>3.1x10³</td><td> 3,5</td>

<td> 11</td><td> 0</td><td> 0</td><td> 0</td><td></td>

<td> 10</td><td>3.9x10³</td><td>4.0x10³</td><td>3.5x10³</td><td> 3,6</td>

<td> 9</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>4.4x10³</td><td>4.5x10³</td><td>4.4x10³</td><td> 3,6</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

As shown in Table 8, the conditions of 11% and 14% MSM with 60 pg / ml oxacillin at 96 hours showed no signs of colonies. The tube was removed from the experiment and tested for growth of Staphylococcus strain ATCC 43300. The tube was centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted into 90 ml of Mueller-Hinton broth with 6 pg / ml oxacillin. This is for the enrichment and recovery of any MRSA cells that may be present both healthy and stressed. The pellet had 10 ml of Mueller-Hinton broth with addition of 6 pg / ml of oxacillin added thereto to recover any cells that may be present. Both vessels were incubated at 35 ° C for 48 hours followed by plating every 24 hours. Each container was plated at 10 dilutions to obtain a detection limit of 1 cfu. No colonies were observed, indicating total destruction. This time point for graphical representation purposes was reported as 0 logs.

As shown in Table 8, the 12 pg / ml oxacillin conditions with 12 and 13% MSM exhibited higher colony counts compared to the initial time points. The condition of 12 pg / ml of

ES 2 674 019 T3 oxacillin under 14-16% conditions exhibited the same colony counts as the previous 72 hour time point. The 30 pg / ml oxacillin condition showed a greater reversion to oxacillin resistance than for the lower MSM percentages of 5-13%. The logarithmic reduction in cfu / ml observed was small for 14-16% compared to the previous time point and appears to remain constant. The 60 pg / ml oxacillin showed no colonies at the 11 and 14% mSm concentrations. For 60 pg / ml of oxacillin with MSM concentrations of 5-10% and 12%, the observed cfu / ml were greater than the detection limit. The data at 11-12% may possibly show that the amount of MSM addition is crucial for the complete depletion of the organism as the higher amounts (14 and 15%) show signs of reduced total kill.

Table 9. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30 or 60 pg / ml of oxacillin for 120 hours.

<td>% MSM</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>12 pg / ml Oxacillin MSM (cfu / ml)</td><td>120 hour average log</td>

<td> 16</td><td>3.0x10¹</td><td>3.0x10¹</td><td>2.0x10¹</td><td> 1,5</td>

<td> 15</td><td>1.1x10²</td><td>1.3x10²</td><td>1.2x10²</td><td> 2,0</td>

<td> 14</td><td>1.1x10²</td><td>1.2x10²</td><td>1.1x10²</td><td> 2,0</td>

<td> 13</td><td>6.2x10³</td><td>6.7x10³</td><td>6.2x10³</td><td> 3,8</td>

<td> 12</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 11</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 10</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 9</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

<td>% MSM</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>30 pg / ml Oxacillin MSM (cfu / ml)</td><td>120 hour average log</td>

<td> 16</td><td>2.0x10¹</td><td>3.0x10¹</td><td>1.0x10²</td><td> 1,3</td>

<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 14</td><td>3.0x10¹</td><td>2.0x10¹</td><td>2.0x10¹</td><td> 1,3</td>

<td> 13</td><td>1.02x10³</td><td>1.01x10³</td><td>1.02x10³</td><td> 3,0</td>

<td> 12</td><td>6.8x10⁴</td><td>3.9x10⁴</td><td>6.9x10⁴</td><td> 4,7</td>

<td> 11</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 10</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 9</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td colspan="5"></td>

ES 2 674 019 T3

<td>% MSM</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>60 pg / ml Oxacillin MSM (cfu / ml)</td><td>120 hour average log</td>

<td> 16</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 14</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 13</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 12</td><td>1.72x10⁴</td><td>1.6x10⁴</td><td>1.5x10⁴</td><td> 4,2</td>

<td> 11</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 10</td><td>1.6x10⁴</td><td>1.5x10⁴</td><td>1.7x10⁴</td><td> 4,2</td>

<td> 9</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 8</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 7</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 6</td><td>3.7x10⁴</td><td>3.4x10⁴</td><td>2.4x10²</td><td> 4,5</td>

<td> 5</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

<td> 0</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td>3.3X10⁵</td><td> 5,5</td>

As shown in Table 9, the 15% MSM condition with 30 pg / ml oxacillin, and the 13 and 16% MSM condition with 60 pg / ml oxacillin, at 120 hours showed no signs of colonies. . The tube was removed from the experiment and tested for growth of Staphylococcus aureus strain ATCC 43300. The tube was centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted into 90 ml of Mueller-Hinton broth with 6 pg / ml oxacillin. This is for the enrichment and recovery of any cells of the ATCC 43300 Staphylococcus aureus strain that may be present both healthy and stressed. The agglomerate had 10 ml of Mueller-Hinton broth (Mueller &amp; Hinton, Proc. Soc. Exp. Diol. And Med .; 48: 330-333, 1941); with addition of 6 pg / ml of oxacillin added thereto to recover any cells that may be present. Both vessels were incubated at 35 ° C for 48 hours followed by plating every 24 hours. Each container was plated at 10 dilutions to obtain a detection limit of 1 cfu. No colonies were observed, indicating total destruction. This time point for graphical representation purposes was reported as 0 logs.

Concentrations of 12 pg / ml show that there is a slight decrease or increase for MSM at 14-16%. For 30pg / ml, the 15% MSM tipping point had true death with no observed recovery. The 14% and 16% concentration points had no significant value changes from the previous time point. The MSM concentration points at 13% and 16% of 60 pg / ml had true death without recovery of the organism. The 12% concentration point shows a continued increase in log from the previous time point.

Taken together, the data presented in Tables 5-9 show that in the first 24 hours there was no significant reduction in the log for the lower concentrations (5-10%) in each of the three oxacillin levels (the trend lines of MSM concentration of 16-5% have a negative slope). The next 48 hour time point shows that the reduction rates are beginning to move from the low end of 5-10% to the high end of 11-16%) (the trend lines of the 16-5% MSM concentration have a positive slope). The 72 hour milestone shows a log reduction in cfu / ml for higher MSM concentrations where lower MSM concentrations show little or no reduction in cfu / ml. The 96 and 120 hour time points show reduced bacterial survival at higher MSM concentrations, but no effect on bacterial survival at lower MSM concentrations.

The results of this experiment raise some intriguing questions. The data at 24 hours show a greater reduction to the lower percentages of MSM for the three oxacillin concentrations evaluated. This trend changes slightly at 48 hours, observing a maximum reduction in the mean to higher concentrations of MSM. By 72 hours, the trend has definitely changed to a greater log reduction at the higher MSM concentrations. MSM was added only when additional growth medium (LB containing the same original concentration of MSM as the growth vial) was added to maintain the original volume in the vial. For example, if a 1 ml aliquot of the 12 pg / ml oxacillin was removed with 5% MSM for plating, then 1 ml of LB containing 5% MSM was added again.

The following are offered as hypothetical explanations for some of the effects noted above;

ES 2 674 019 T3 however, these explanations are not intended to be limiting. A hypothetical and non-limiting possibility for the observed results is that MSM is consumed by organisms. Another hypothetical and non-limiting possibility for the observed results is that the ability of MSM to cause reversal of sensitivity could be due to the ability of MSM to transport oxacillin to MRSA cells. Another hypothetical and non-limiting possibility for the observed results is that too high a level of MSM is less effective at certain time points. For example, this may be due to competition from an overabundance of free MSM molecules that compete with MSM that binds with oxacillin at higher concentrations. This would cause more free MSM to penetrate cells than MSM-bound oxycillin. Lower concentrations have a higher overall percentage of total MSM molecules in solution that bind oxacillin, allowing more bound oxacillin to penetrate the cell as there is less free MSM to compete. Under this hypothesis, for a given number of MSM molecules penetrating the MRSA cell, the ratio of MSM to oxacillin is important to obtain the maximum effect. If an overabundance of MSM is present, then the possibility of non-oxacillin-bound MSM molecules penetrating the cell is increased.

Example 8. Treatment of a drug-resistant bacterial pathogen in a subject

This example describes a representative method of treating a subject with a drug-resistant bacterial infection (eg, MRSA infection) by selecting a subject with a drug-resistant bacterial infection and administering to the subject a therapeutically effective amount of MSM and an agent that inhibits. the drug-sensitive form of the bacterial pathogen that produces the drug-resistant bacterial infection. In this example, the subject has an MRSA infection on his skin and the treatment comprises topical administration of a composition comprising 12% MSM and 30 pg / ml oxacillin.

First, a subject having a MRSA infection is selected. The subject is selected by obtaining a biological sample from the subject and analyzing the biological sample for the presence of Staphylococcus aureus bacteria followed by detecting the MIC of the bacteria for oxacillin. If the MIC of oxacillin for Staphylococcus aureus bacteria in the subject's biological sample is greater than 2 pg / ml, then the bacteria is a strain of MRSA bacteria. The sample contains Staphylococcus aureus bacteria and the MIC detected for oxacillin in bacteria is more than 2 pg / ml for oxacillin using the Etest® system (AB bioMérieux, SA France). Therefore, the subject is selected.

Following selection of the subject with MRSA, a composition comprising 12% MSM and 30 pg / ml oxacillin is administered topically to the subject's skin in the area surrounding the MRSA infection. The composition is water-based, formulated for topical administration, and comprises 1% NaCl. The composition is administered twice a day, for 10 days. Following this course of treatment, the subject's MRSA infection symptoms were observed to have decreased by at least 50% relative to MRSa infection symptoms present prior to treatment with MSM and oxacillin, indicating that the subject had treaty.

Example 9: The absorption of MSM in the topical formulation is within recognized safety levels

This example shows that the absorption of MSM in topical formulations is within recognized safety levels.

New Zealand White rabbits, which are an accepted animal model for dermal absorption studies, were used to assess the absorption and resulting blood levels of mSm. Rabbits were obtained from Charles River Canada (Saint-Constant, Quebec). Five male rabbits, aged 12-13 weeks and weights ranging from 2.6 kg to 2.7 kg were used for dermal absorption studies. Rabbits were used because their skin permeability is higher compared to rats, pigs, or humans. Therefore, testing in rabbits is a more conservative strategy for the safety of topical products for human use. The size of the rabbit was based on the ethical restrictions of extracting more than 6 ml / kg of body weight of blood in a period of two weeks. The total volume of blood to be drawn during this study was 10 ml in a single day. One animal per group was used to minimize the number of animals required. Animals were housed individually in stainless steel cages with 12 hour light / dark cycles. The environment of the animal house was monitored daily (ranges sought: 18-26 ° C and relative humidity 25-50%). Fresh air was supplied to the room at a rate sufficient to provide approximately 15 to 17 room air changes per hour. Clinical observations were carried out on all animals to ensure that the animals were in good health prior to dosing. Morbidity and mortality observations were also made during the study period.

The treatment groups were as shown in Table 10:

ES 2 674 019 T3

Table 10. Study design

<td>Group</td><td>Experimental article</td><td>Exposed surface area</td><td>Volume Applied</td><td>Number of animals</td><td>Blood collection times (min)</td>

<td>TO</td><td>10% MSM + 90% Water</td><td>6 cm²</td><td>0.5 ml</td><td> 1</td><td>0 (previous dose), 10, 30, 120,480 minutes</td>

<td>B</td><td>50% DMSO + 50% Water</td><td>6 cm²</td><td>0.5 ml</td><td> 1</td><td>0 (previous dose), 10, 30, 120,480 minutes</td>

<td>C</td><td>70% DMSO + 30% Water</td><td>6 cm²</td><td>0.5 ml</td><td> 1</td><td>0 (previous dose), 10, 30, 120, 480 minutes</td>

<td>D</td><td>10% MSM + 50% DMSO 40% Water</td><td>6 cm²</td><td>0.5 ml</td><td> 1</td><td>0 (previous dose), 10, 30, 120,480 minutes</td>

<td>AND</td><td>10% MSM + 70% DMSO 20% Water</td><td>6 cm²</td><td>0.5 ml</td><td> 1</td><td>0 (previous dose), 10, 30, 120,480 minutes</td>

One day before the study, the rump of each rabbit was clamped tightly with hairpins. An area of ​​6 cm was measured²and was marked to ensure equivalence in the application of the various compositions. Each product was applied by pipetting 0.5 ml of each composition into the center of the test area and spread to cover the entire test area. After a 5 minute exposure period, the compositions were removed by wiping, rinsing and drying the test area.

Before blood collection, animals were tranquilized with Accepromazine (1 mg / kg) by intramuscular injection into the muscle of the right hind paw after which an EMLA cream (lidocaine / prilocaine) was applied to both ears lengthwise. of the artery of the ear. Blood was drawn by inserting a 21G needle (connector removed) into the artery of the ear. Approximately 2 ml of whole blood was collected in 4 ml Vacutainer tubes (Becton Dickinson, Mississauga, ON) containing K2EDTA. The tubes were inverted to mix with the anticoagulant and stored refrigerated until the plasma was removed by centrifugation. Plasma was separated from whole blood by centrifugation at 3000 xg for 10 minutes. Plasma was collected, transferred and stored in a cryovial at -70 ° C until further processing for MSM analysis.

Following the 5 minute exposure period to the various test products (see Table 1), blood was drawn after 10 minutes, 30 minutes, 2 hours and 8 hours. Before the 2 and 8 hour blood draws, EMLA cream was applied to the ears (approximately 30 minutes before each blood draw) as the anesthetic effects of EMLA cream last approximately 1 to 2 hours. EMLA cream and Accepromazine were used for ethical considerations and to take care of the welfare of the animals used in this study.

MSM concentrations in plasma were quantified by gas chromatography-mass spectrometry (GC / MS) according to established methods. Briefly, 450 µl of plasma sample was mixed with 50 µl of physiological saline and vortexed for 30 seconds. Following this, 1 ml of acetonitrile (Fisher, HPLC grade) was added to the mixture. The solution was vortexed intensively for 60 seconds and centrifuged at 2000 rpm for 5 minutes. One microliter of clear supernatant was introduced into the GC / MS system (GC / MS QP20108 EI, Shimadzu, Kyoto, Japan). Analysis was carried out on a Shimadzu SHR5XLB column (0.25mm ID X 30m length, 0.25um film, Kyoto, Japan). MSM retention time was 6.1-6.3 minutes. MSM was detected with MS and m / z 79 (M + -15) was used to monitor the profiles of the MSM SIM ions. Helium gas was used as the carrier gas, the overhead pressure was 0.25 kg / cm2, the recovery gas was 30 ml / min, the column temperature was 80 ° C, the injector temperature was 120 ° C, the separator temperature was 200 ° C, and the ion source temperature was 250 ° C. the ionization energy was 70eV. An external normalized graph was prepared with the MSM dissolved in acetonitrile at the following concentrations: 62.5 pg / ml, 31.3 pg / ml, 15.6 pg / ml, 7.8 pg / ml, 3.9 pg / ml, 1.9 pg / ml, 0.98 pg / ml and 0.49 pg / ml. The concentration of MSM in plasma samples was calculated from the slope of the standard curve. The best fit plot was linear with an R2 value of 0.998.

All animals were observed before the start of the study and all showed good health. During the study cycle and after the study, all animals demonstrated good health. Morbidity, mortality, and lesions were assessed twice daily. The animals did not demonstrate any morbidity, mortality, or injury.

Table 11. Concentration of MSM in plasma after exposure to MSM and DMSO

<td>Treatment</td><td>Time point (minute)</td><td>MSM concentration (pg / ml)</td>

<td rowspan="5">10% MSM + 90% water</td><td> 0</td><td> 25,6</td>

<td> 10</td><td> 17,6</td>

<td> 30</td><td> 16,3</td>

<td> 120</td><td> 14,0</td>

<td> 480</td><td> 15,4</td>

<td></td><td> 0</td><td> 4,2</td>

ES 2 674 019 T3

<td></td><td> 10</td><td> 6,9</td>

<td rowspan="3">50% DMSO + 50% water</td><td> 30</td><td> 6,9</td>

<td> 120</td><td> 7,4</td>

<td> 480</td><td> 12,6</td>

<td rowspan="5">70% DMSO + 30% water</td><td> 0</td><td> 56,7</td>

<td> 10</td><td> 89,0</td>

<td> 30</td><td> 98,9</td>

<td> 120</td><td> 128,7</td>

<td> 480</td><td> 120,2</td>

<td rowspan="5">10% MSM M + 50% DMSO + 40% water</td><td> 0</td><td> 104,2</td>

<td> 10</td><td> 116,5</td>

<td> 30</td><td> 127,9</td>

<td> 120</td><td> 128,4</td>

<td> 480</td><td> 140,4</td>

<td rowspan="5">10% MSM + 70% DMSO + 20% water</td><td> 0</td><td> 26,8</td>

<td> 10</td><td> 37,3</td>

<td> 30</td><td> 30,9</td>

<td> 120</td><td> 33,9</td>

<td> 480</td><td> 44,4</td>

The results of the absorption study are summarized in Table 11. Initial plasma concentrations of MSM (before exposure to the experimental articles) ranged from 4.2 pg / ml to 104.2 pg / ml. The variation from baseline is within the normal range of natural MSM concentrations that have been established in previous studies. Upon exposure to the various experimental items, the highest plasma concentrations of the measured MSMs were less than or equal to about 140 pg / ml. This maximum concentration is the result of exposure to 10% MSM + 70% DMSO + 20% water. When corrected for natural variation in concentrations from baseline MSM, the largest variation in plasma MSM was detected in the 70% DMSO + 30% water group. These data suggest that variations in MSM, due either to absorption or due to DMSO metabolism, are within the natural range of MSM concentrations.

Example 10. Effect of MSM on MRSA sensitivity to 12, 30 or 60 pg / ml of methicillin.

This example describes in vitro experiments studying the survival of Staphylococcus aureus strain ATCC 43300 in the presence of 5-16% MSM and an initial concentration of 12, 30 or 60 pg / ml of methicillin. This corresponds to 2x, 5x and 10x MICs for methicillin. More antibiotic was added every 24 hours. The growth periods tested were 24 (Figure 5), 48, 72, 96 and 120 hours. The results show that MSM sensitized a strain of MRSA to methicillin.

Methods:

The USP &lt;51&gt; antimicrobial efficacy protocol was used as the template for the experimental paradigm. The initial MSM concentrations tested were 5-16% in increments of one. All concentrations were plated with dilutions of 10^-7 to discern the cfu / ml. The initial concentration of methicillin used was 12, 30 or 60 pg / ml, which corresponds to 2x, 5x and 10x MIC for methicillin. More antibiotic was added to the initial concentration every 24 hours.

Materials used were Flake OptiMSM® MSM (lot number 0604751), Staphylococcus aureus strain ATCC 43300, 30 ml borosilicate glass culture tubes, lactose broth (LB; Alpha Biosciences; Lot: L07-03), broth Modified Letheen (MLB, Alpha Biosciences, Lot I08-09), Trypto soy agar with lecithin and Tween 80 (tSa; Alpha Biosciences, lot: F08-42) and methicillin sodium grade uSp lot KOH338.

Staphylococcus aureus strain ATCC 43300 spread in streaks from the isolates. An isolated colony was selected, streaked again, and incubated. The isolate was selected and then diluted to a McFarland standard of one. Subsequently, the estimated value was taken to inoculate the tubes containing growth medium and 12, 30 or 60 pg / ml of methicillin with around 10⁵ cfu / ml. These tubes were incubated and plated to determine the cfu count. A clone for which no log reduction of cfu was observed was selected for the remainder of the experiment.

Flake OptiMSM® MSM was weighed on a certified Mettler Toledo AG245 SN: 1115210833 balance and distributed in aliquots for each concentration. MSM was introduced into 30 ml borosilicate glass culture tubes. MSM was added to the tubes as follows: 5% (0.5g), 6% (0.6g), 7% (0.7g), 8% (0.8g), 9% (0.9g), 10 % (1.0g), 11% (1.1g), 12% (1.2g), 13% (1.3g), 14% (1.4g), 15% (1.5g), and 16% (1.6 g). The material was calculated for a volume of 10 ml. Sterile methicillin was added to each experimental condition to a final concentration of 12, 30 or 60 pg / ml of antibiotic. Lactose broth positive controls were inoculated with and without the 12 pg / ml, 30 pg / ml, 60 pg / ml

ES 2 674 019 T3 of antibiotic with MRSA. the lactose broth also had a set of negative controls. MSM controls (no antibiotic) at concentrations of 5%, 10% and 16% were also tested with and without MRSA. The tubes were uniquely configured, but plated in triplicate, as described below.

All tubes were then inoculated with a dilution of Staphylococcus aureus strain ATCC 43300 that provides clones at a final level of 1.49x10 colony-forming units.⁵/ ml (log = 5.17). The tubes were incubated at 25 ° C and periodically mixed. The antibiotic was reoccupied every 24 hours at the initial concentration to maintain pressure on the body. Plating was carried out at 24, 48, 72, and 120 hours. Each condition was plated by diluting 1 ml of growth material in 9 ml of MLB dilution broth. This was carried out every time. 1 ml of growth medium containing MSM was subsequently added to the tube to maintain a constant volume and concentration of MSM. This mixture was serially diluted to 10^-7, and 1 ml was placed in a sterile petri dish at each dilution point, in triplicate. 20 ml of TSA was added to each dilution, centrifuged and allowed to solidify. All dilutions were placed in an incubator at 35 ° C for 24 to 72 hours. Bacterial colonies on each plate were counted and the average taken was converted to logarithmic format. Any cfu / ml greater than 1.49x10⁵/ ml (log = 5.17) were recorded as TNTC. Plots were recorded as 5.2 log for TNTC to show the comparison between the different data points.

Results:

In this experiment the positive controls (those inoculated with MRSA) used for the three concentrations of methicillin alone (12 pg / ml, 30 pg / ml and 60 pg / ml) did not show significant decrease in colony-forming units from the value of the initial inoculums. Tables 12-16 show the results of this study.

Table 12. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30 or 60 pg / ml of methicillin for 24 hours.

<td>Concentration as% MSM</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>24 hour average log</td>

<td> 16</td><td>1.01x10³</td><td>1.03x10³</td><td>1.04x10³</td><td> 3,0</td>

<td> 15</td><td>6.4x10³</td><td>6.6x10³</td><td>6.5x10³</td><td> 3,8</td>

<td> 14</td><td>8.8X10³</td><td>8.5X10³</td><td>8.2X10³</td><td> 3,9</td>

<td> 13</td><td>3.3x10³</td><td>3.5x10³</td><td>3.4x10³</td><td> 3,5</td>

<td> 12</td><td>2.9x10³</td><td>3.0x10³</td><td>3.8x10²</td><td> 3,5</td>

<td> 11</td><td>2.2x10³</td><td>2.3x10²</td><td>2.3x10²</td><td> 3,3</td>

<td> 10</td><td>1.4x10³</td><td>1.4x10³</td><td>1.4x10³</td><td> 3,1</td>

<td> 9</td><td>9.0x10²</td><td>9.4x10²</td><td>9.2x10²</td><td> 3,0</td>

<td> 8</td><td>9.6x10²</td><td>9.5x10²</td><td>9.3x10²</td><td> 3,0</td>

<td> 7</td><td>4.4x10²</td><td>4.4x10²</td><td>5.0x10²</td><td> 2,7</td>

<td> 6</td><td>4.0x10²</td><td>4.4x10²</td><td>4.5x10²</td><td> 2,6</td>

<td> 5</td><td>7.8x10²</td><td>8.0x10²</td><td>8.2x10²</td><td> 2,9</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

<td colspan="5"></td>

<td>% MSM</td><td>30 pg / ml methicillin /</td><td>30 pg / ml methicillin /</td><td>30 pg / ml methicillin /</td><td rowspan="2">24 hour average log</td>

<td>Concentration</td><td>MSM (cfu / ml)</td><td>MSM (cfu / ml)</td><td>MSM (cfu / ml)</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 16</td><td>9.6x10³</td><td>9.8x10³</td><td>9.7x10³</td><td> 4,0</td>

<td> 15</td><td>7.8x10³</td><td>7.6x10³</td><td>8.0x10³</td><td> 3,9</td>

<td> 14</td><td>6.3X10³</td><td>6.7X10³</td><td>6.9X10³</td><td> 3,8</td>

ES 2 674 019 T3

<td> 13</td><td>6.5x10³</td><td>6.5x10³</td><td>6.4x10³</td><td> 3,8</td>

<td> 12</td><td>3.3x10³</td><td>3.3x10³</td><td>3.3x10³</td><td> 3,5</td>

<td> 11</td><td>3.3x10³</td><td>3.8x10²</td><td>3.1x10³</td><td> 3,5</td>

<td> 10</td><td>3.2x10¹</td><td>1.3x10³</td><td>3.1x10¹</td><td> 3,1</td>

<td> 9</td><td>3.1x10¹</td><td>1.3x10³</td><td>3.2x10¹</td><td> 3,1</td>

<td> 8</td><td>9.4x10²</td><td>9.6x10²</td><td>9.6x10²</td><td> 3,0</td>

<td> 7</td><td>3.4x (102)</td><td>3.5x10²</td><td>3.3x10²</td><td> 2,5</td>

<td> 6</td><td>2.2x10²</td><td>2.2x10²</td><td>2.5x10²</td><td> 2,4</td>

<td> 5</td><td>3.8x10²</td><td>4.1x10²</td><td>4.0x10²</td><td> 2,6</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

<td colspan="5"></td>

<td>% MSM</td><td>60 pg / ml methicillin /</td><td>60 pg / ml methicillin /</td><td>60 pg / ml methicillin /</td><td rowspan="2">24 hour average log</td>

<td>Concentration</td><td>MSM (cfu / ml)</td><td>MSM (cfu / ml)</td><td>MSM (cfu / ml)</td>

<td> 16</td><td>8.4x10³</td><td>8.4x10³</td><td>8.5x10³</td><td> 3,9</td>

<td> 15</td><td>9.3x10³</td><td>8.5x10³</td><td>8.4x10³</td><td> 3,9</td>

<td> 14</td><td>8.3X10³</td><td>8.8X10³</td><td>8.5X10³</td><td> 3,9</td>

<td> 13</td><td>7.0x10³</td><td>6.8x10³</td><td>6.5x10³</td><td> 3,8</td>

<td> 12</td><td>4.5x10³</td><td>4.1x10³</td><td>4.7x10⁴</td><td> 3,6</td>

<td> 11</td><td>3.5x10³</td><td>3.8x10³</td><td>3.9x10³</td><td> 3,6</td>

<td> 10</td><td>2.2x10³</td><td>2.1x10³</td><td>2.0x10³</td><td> 3,3</td>

<td> 9</td><td>2.0x10³</td><td>1.6x10³</td><td>1.8x10³</td><td> 3,2</td>

<td> 8</td><td>9.4x10²</td><td>9.8x10²</td><td>9.7x10²</td><td> 3,0</td>

<td> 7</td><td>6.1x10²</td><td>6.3x10²</td><td>6.2x10²</td><td> 2,8</td>

<td> 6</td><td>4.2x10⁴</td><td>4.2x10²</td><td>4.8x10²</td><td> 2,6</td>

<td> 5</td><td>8.3x10²</td><td>8.1x10²</td><td>8.1x10²</td><td> 2,9</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

As shown in Table 12 and Figure 5, the number of cfu / ml observed at the 24 hour time point was similar for all concentrations. The lower end of MSM concentrations tends to have a greater log reduction in colony count than higher MSM concentrations. The average log reduction in colony count for 12 pg / ml methicillin is 1.5 logs which is carried over for all MSM concentrations with little or no change. The 30 pg / ml methicillin conditions had a significant reduction in the lower end of MSM concentrations with an average difference of 3 log. The 60 pg / ml methicillin condition showed a slightly lower log reduction in colony count over the first 24 hour period for all three antibiotic concentrations. One point of interest is that the 12 pg / ml with 16% MSM had a log reduction of 2.2, which is the most observed at the higher concentrations. A hypothetical possibility is that this is an outlier or optimal point for antibiotic uptake for this concentration. The 30 pg / ml had the highest global log reduction of 2.8 in colony count for the 6% concentration. Negative LB controls showed no signs of contamination. Positive LB controls showed cloudy growth and were terminated at this point for safety reasons. The 5%, 10% and 16% negative MSM controls had no signs of contamination. The 5%, 10%, and 16% MSM positive controls had signs of cloudy growth, were TNTC for all plating dilutions, and were terminated at this time point for safety reasons. The antibiotic controls did not show signs of significant reduction from the initial inoculums, and the negative controls did not show signs of contamination.

ES 2 674 019 T3

Table 13. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30, or 60 pg / ml of methicillin for 48 hours.

<td>Concentration as% MSM</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>48 hour average log</td>

<td> 16</td><td>8.1x10²</td><td>8.3x10²</td><td>8.4x10²</td><td> 2,9</td>

<td> 15</td><td>5.1x10²</td><td>5.3x10²</td><td>5.6x10²</td><td> 2,7</td>

<td> 14</td><td>5.1x10²</td><td>5.4x10²</td><td>5.2x10²</td><td> 2,7</td>

<td> 13</td><td>3.6x10²</td><td>3.3x10²</td><td>3.4x10²</td><td> 2,5</td>

<td> 12</td><td>6.3x10²</td><td>6.4x10²</td><td>6.2x10²</td><td> 2,8</td>

<td> 11</td><td>5.9x10²</td><td>5.7x10²</td><td>6.0x10²</td><td> 2,8</td>

<td> 10</td><td>4.1x10²</td><td>4.6x10²</td><td>2.7x10⁴</td><td> 2,6</td>

<td> 9</td><td>2.5x10²</td><td>2.8x10²</td><td>3.2x10²</td><td> 2,4</td>

<td> 8</td><td>1.0x10²</td><td>1.0x10²</td><td>1.1x10²</td><td> 2,0</td>

<td> 7</td><td>4.0x10¹</td><td>3.0x10¹</td><td>4.0x10¹</td><td> 1,6</td>

<td> 6</td><td>1.0x10¹</td><td>1.0x10¹</td><td>1.0x10²</td><td> 1,1</td>

<td> 5</td><td>2.0x10²</td><td>1.8x10²</td><td>2.1x10²</td><td> 2,3</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

<td colspan="5"></td>

<td>Concentration as% MSM</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>48 hour average log</td>

<td> 16</td><td>4.5x10²</td><td>2.7x10⁴</td><td>4.6x10²</td><td> 2,7</td>

<td> 15</td><td>5.3x10²</td><td>5.2x10²</td><td>5.6x10²</td><td> 2,7</td>

<td> 14</td><td>7.0x10²</td><td>6.8x10²</td><td>7.4x10²</td><td> 2,8</td>

<td> 13</td><td>4.0x10²</td><td>4.0x10²</td><td>4.0x10²</td><td> 2,6</td>

<td> 12</td><td>4.8x10²</td><td>5.0x10²</td><td>5.1x10²</td><td> 2,7</td>

<td> 11</td><td>1.9x10²</td><td>1.8x10²</td><td>2.1x10²</td><td> 2,3</td>

<td> 10</td><td>1.2x10²</td><td>1.8x10²</td><td>1.6x10²</td><td> 2,2</td>

<td> 9</td><td>2.1x10²</td><td>2.3x10²</td><td>2.4x10²</td><td> 2,3</td>

<td> 8</td><td>4.1x10²</td><td>4.0x10²</td><td>3.4x10²</td><td> 2,6</td>

<td> 7</td><td>1.8x10²</td><td>1.5x10²</td><td>1.6x10²</td><td> 2,2</td>

<td> 6</td><td>2.9x10²</td><td>2.2x10²</td><td>2.5x10²</td><td> 2,4</td>

<td> 5</td><td>4.6x10²</td><td>4.8x10²</td><td>4.4x10²</td><td> 2,7</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

<td>Concentration as% MSM</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>48 hour average log</td>

<td> 16</td><td>2.1x10²</td><td>2.2x10²</td><td>1.8x10²</td><td> 2,3</td>

<td> 15</td><td>3.5x10²</td><td>3.8x10²</td><td>3.8x10²</td><td> 2,6</td>

<td> 14</td><td>2.9x10²</td><td>2.9x10²</td><td>3.1x10²</td><td> 2,5</td>

<td> 13</td><td>4.0x10²</td><td>3.7x10²</td><td>3.6x10²</td><td> 2,6</td>

ES 2 674 019 T3

<td> 12</td><td>3.3x10²</td><td>3.8x10²</td><td>3.7x10²</td><td> 2,6</td>

<td> 11</td><td>5.0x10¹</td><td>5.0x10¹</td><td>4.0x10¹</td><td> 1,7</td>

<td> 10</td><td>2.1x10²</td><td>2.4x10²</td><td>2.5x10²</td><td> 2,4</td>

<td> 9</td><td>1.3x10²</td><td>1.2x10²</td><td>1.3x10²</td><td> 2,1</td>

<td> 8</td><td>8.0x10¹</td><td>6.0x10¹</td><td>7.0x10¹</td><td> 1,8</td>

<td> 7</td><td>2.4x10²</td><td>2.1x10²</td><td>2.3x10²</td><td> 2,4</td>

<td> 6</td><td>2.0x10²</td><td>2.0x10²</td><td>3.0x10²</td><td> 2,4</td>

<td> 5</td><td>1.0x10¹</td><td>1.0x10²</td><td>1.0x10¹</td><td> 1,1</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

As shown in Table 13, the log reduction in colony count is beginning to be more similar across all concentrations. The trend begins to show the upper end of the MSM percent conditions becoming similar in log reduction as the lower end of the MSM percent conditions. The 12 pg / ml methicillin with 6% -7% MSM conditions show a greater log reduction in the overall colony count except for the 5% MSM and the 60 pg / ml and 6% MSM condition and the condition of 12 pg / ml of methicillin, which have the highest log reduction in colony count. A hypothetical explanation for this result is that these could possibly show an optimal concentration for this time period.

The 30 and 60 pg / ml methicillin had an overall reduction better than the 24 hour results under the 8% -12% MSM conditions. The 60 pg / ml maintains a greater mean log reduction in colony count compared to the first 24 hour period for all three antibiotic concentrations. Two points of interest are (1) the 6% MSM condition at 12 pg / ml methicillin having a 4.1 log reduction in colony count and (2) the 5% MSM condition and 60 pg. / ml methicillin having a 4.1 log reduction in colony count. A merely hypothetical explanation for this is that methicillin has additional polar binding sites for MSM thus providing an optimal concentration for possible uptake into cells. The overall graph shows an even greater reduction for MSM concentrations from 8% to 16%. Negative LB controls showed no signs of contamination. Positive LB controls showed cloudy growth having a TNTC on plating at 10⁵. The 5%, 10% and 16% negative MSM controls had no signs of contamination. The antibiotic controls did not show signs of significant reduction from the initial inoculums, and the negative controls did not show signs of contamination.

Table 14. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30, or 60 pg / ml of methicillin for 72 hours.

<td>Concentration as% MSM</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>72 hour average log</td>

<td> 16</td><td>3.3x10²</td><td>3.1x10²</td><td>3.4x10²</td><td> 2,5</td>

<td> 15</td><td>1.6x10²</td><td>2.1x10²</td><td>2.2x10²</td><td> 2,3</td>

<td> 14</td><td>2.2x10²</td><td>2.5x10²</td><td>2.0x10²</td><td> 2,3</td>

<td> 13</td><td>1.4x10²</td><td>1.7x10²</td><td>1.6x10²</td><td> 2,2</td>

<td> 12</td><td>2.3x10²</td><td>2.5x10²</td><td>2.6x10²</td><td> 2,4</td>

<td> 11</td><td>5.1x10²</td><td>4.9x10²</td><td>5.0x10²</td><td> 2,7</td>

<td> 10</td><td>2.1x10²</td><td>1.9x10²</td><td>1.8x10²</td><td> 2,3</td>

<td> 9</td><td>1.1x10²</td><td>1.3x10²</td><td>1.4x10²</td><td> 2,1</td>

<td> 8</td><td>4x10¹</td><td>5x10¹</td><td>5x10¹</td><td> 1,7</td>

<td> 7</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td>2.4x10²</td><td>2.7x10²</td><td>2.6x10²</td><td> 2,4</td>

ES 2 674 019 T3

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

<td colspan="5"></td>

<td>Concentration as% MSM</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>72 hour average log</td>

<td> 16</td><td>1.0x10²</td><td>1.2x10²</td><td>1.1x10²</td><td> 2,0</td>

<td> 15</td><td>2.7x10²</td><td>2.7x10²</td><td>2.9x10²</td><td> 2,4</td>

<td> 14</td><td>1.9x10²</td><td>2.0x10²</td><td>2.2x10²</td><td> 2,3</td>

<td> 13</td><td>1.7x10²</td><td>1.8x10²</td><td>1.7x10²</td><td> 2,2</td>

<td> 12</td><td>1.6x10²</td><td>1.9x10²</td><td>1.9x10²</td><td> 2,3</td>

<td> 11</td><td>1.1x10²</td><td>1.3x10²</td><td>1.1x10²</td><td> 2,1</td>

<td> 10</td><td>1.0x10²</td><td>1.0x10²</td><td>1.0x10²</td><td> 2,0</td>

<td> 9</td><td>1.2x10²</td><td>1.1x10²</td><td>1.0x10²</td><td> 2,0</td>

<td> 8</td><td>2.2x10²</td><td>2.5x10²</td><td>2.4x10²</td><td> 2,4</td>

<td> 7</td><td>2.0x10¹</td><td>2.0x10¹</td><td>2.0x10¹</td><td> 1,3</td>

<td> 6</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 5</td><td>6x10¹</td><td>8x10¹</td><td>7x10¹</td><td> 1,8</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

<td colspan="5"></td>

<td>Concentration as% MSM</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>72 hour average log</td>

<td> 16</td><td>2.0x10¹</td><td>4.0x10¹</td><td>2.0x10¹</td><td> 1,4</td>

<td> 15</td><td>1.3x10²</td><td>1.3x10²</td><td>1.3x10²</td><td> 2,1</td>

<td> 14</td><td>1.1x10²</td><td>1.4x10²</td><td>1.4x10²</td><td> 2,1</td>

<td> 13</td><td>1.7x10²</td><td>1.5x10²</td><td>1.8x10²</td><td> 2,2</td>

<td> 12</td><td>1.2x10²</td><td>1.3x10²</td><td>1.2x10²</td><td> 2,1</td>

<td> 11</td><td>1.0x10²</td><td>1.0x10²</td><td>1.1x10²</td><td> 2,0</td>

<td> 10</td><td>8.0x10¹</td><td>9.0x10¹</td><td>8.0x10¹</td><td> 1,9</td>

<td> 9</td><td>8.0x10¹</td><td>8.0x10¹</td><td>8.0x10¹</td><td> 1,9</td>

<td> 8</td><td>1.0x10²</td><td>1.1x10²</td><td>1.1x10²</td><td> 2,0</td>

<td> 7</td><td>5.0x10¹</td><td>4.0x10¹</td><td>6.0x10¹</td><td> 1,7</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

As shown in Table 14, the 6% and 7% MSM conditions with 12 pg / ml methicillin at 72 hours showed no signs of colonies in all plating. The 6% and 7% MSM tubes were removed after the experiment and analyzed for MRSA. The tubes were centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted into 90 ml of Mueller-Hinton broth with 6 pg / ml of methicillin. This is for the enrichment and recovery of any MRSA cells that may be present both healthy and stressed. The pellet had 10 ml of Mueller-Hinton broth with the addition of 6 pg / ml of methicillin added thereto to recover any cells that may be present. Both vessels were incubated at 35 ° C for 48 hours and plated every 24 hours. Each container was plated at

ES 2 674 019 T3 dilutions to obtain a detection limit of 1 cfu. Since all vessels were at 1/10 dilution in the enrichment solution, plating each vessel in this manner gave the inventors a detection limit of 1 cfu / ml. This was considered to be a true total death as recovery of the organism could not be achieved. This time point for graphical representation purposes was reported as 0 logs.

As shown in Table 14, the 5% and 6% MSM conditions with 60 pg / ml methicillin at 72 hours showed no signs of colonies in all plating. The 5% and 6% MSM tubes were removed after the experiment and analyzed for MRSA. The tubes were centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted into 90 ml of Mueller-Hinton broth with 6 pg / ml of methicillin. This is for the enrichment and recovery of any MRSA cells that may be present both healthy and stressed. The pellet had 10 ml of Mueller-Hinton broth with the addition of 6 pg / ml of methicillin added thereto to recover any cells that may be present. Both vessels were incubated at 35 ° C for 48 hours and plated every 24 hours. Each container was plated at 10 dilutions to obtain a detection limit of 1 cfu. Since all vessels were at 1/10 dilution in the enrichment solution, plating each vessel in this manner gave the inventors a detection limit of 1 cfu / ml. This was considered to be a true total death as recovery of the organism could not be achieved. This time point for graphical representation purposes was reported as 0 logs.

When compared to the oxacillin experiment shown in Example 6, the results at the 72 hour time point show a greater log reduction in colony count except for the 6% MSM condition at 30 pg / ml of methicillin. A merely hypothetical explanation for this is that methicillin has more binding sites for MSM and tends to be more uniform in reduction across all concentrations. The 72 hour milestone trend is beginning to show that the lower and upper concentration have a more uniform reduction, with the lower end having a greater impact on non-recovery of viable organisms at 5-7% MSM concentrations. The methicillin test shows a lower rate of reversion of antibiotic resistance than the oxacillin test in the previous experiment. A purely hypothetical explanation for this is that MSM enters cells more efficiently due to the increase in potential methicillin binding sites. The 72-hour methicillin time point shows more true deaths from MRSA than previously observed. One thing to note is that MSM was substituted at the same concentration and was not substituted at a fixed concentration as carried out in previous experiments.

Table 15. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30, or 60 pg / ml of methicillin for 96 hours.

<td>Concentration as% MSM</td><td>12 g / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>96 hour average log</td>

<td> 16</td><td>1.1x10²</td><td>1.3x10²</td><td>1.2x10²</td><td> 2,1</td>

<td> 15</td><td>1.4x10²</td><td>1.3x10²</td><td>1.6x10²</td><td> 2,2</td>

<td> 14</td><td>1.2x10²</td><td>1.2x10²</td><td>1.3x10²</td><td> 2,1</td>

<td> 13</td><td>1.1x10²</td><td>1.0x10²</td><td>1.0x10²</td><td> 2,0</td>

<td> 12</td><td>1.6x10²</td><td>1.8x10²</td><td>1.8x10²</td><td> 2,2</td>

<td> 11</td><td>1.0x10²</td><td>1.1x10²</td><td>1.2x10²</td><td> 2,0</td>

<td> 10</td><td>1.1x10²</td><td>1.2x10²</td><td>1.2x10²</td><td> 2,1</td>

<td> 9</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 8</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 7</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td>1.33x10³</td><td>1.40x10³</td><td>1.47x10³</td><td> 3,1</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td></td>

<td colspan="5"></td>

ES 2 674 019 T3

<td>Concentration as% MSM</td><td>30 pg / ml methicillin / MSM M (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>96 hour average log</td>

<td> 16</td><td>2.0x10¹</td><td>1.8x10¹</td><td>1.6x10¹</td><td> 2,3</td>

<td> 15</td><td>1.1x10²</td><td>1.1x10²</td><td>1.3x10²</td><td> 2,1</td>

<td> 14</td><td>1.2x10²</td><td>1.3x10²</td><td>1.1x10²</td><td> 2,1</td>

<td> 13</td><td>1.0x10²</td><td>1.5x10²</td><td>1.0x10²</td><td> 2,1</td>

<td> 12</td><td>4.0x10¹</td><td>4.0x10¹</td><td>4.0x10¹</td><td> 1,6</td>

<td> 11</td><td>3.0x10¹</td><td>4.0x10¹</td><td>3.0x10¹</td><td> 1,5</td>

<td> 10</td><td>2.0x10¹</td><td>2.0x10¹</td><td>1.0x10¹</td><td> 1,2</td>

<td> 9</td><td>2.5x10²</td><td>2.7x10²</td><td>2.8x10^-2</td><td> 2,4</td>

<td> 8</td><td>4.5x10²</td><td>4.5x10²</td><td>4.0x10²</td><td> 2,6</td>

<td> 7</td><td>1.1x10²</td><td>13x10²</td><td>1.5x10²</td><td> 2,1</td>

<td> 6</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 5</td><td>4.2x10²</td><td>4.1x10²</td><td>4.5x10²</td><td> 2,6</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td colspan="5"></td>

<td> 16</td><td>4.0x10¹</td><td>7.0x10¹</td><td>6.0x10¹</td><td> 1,75</td>

<td> 15</td><td>1.0x10¹</td><td>1.0x10¹</td><td>1.0x10¹</td><td> 1</td>

<td> 14</td><td>1.0x10¹</td><td>1.0x10¹</td><td>1.0x10¹</td><td> 1</td>

<td> 13</td><td>6.0x10¹</td><td>3.0x10¹</td><td>3.0x10¹</td><td> 1,6</td>

<td> 12</td><td>3.0x10¹</td><td>2.0x10¹</td><td>2.0x10¹</td><td> 1,4</td>

<td> 11</td><td>1.0x10¹</td><td>2.0x10¹</td><td>1.0x10¹</td><td> 1,1</td>

<td> 10</td><td>6.0x10¹</td><td>6.0x10¹</td><td>6.0x10¹</td><td> 1,8</td>

<td> 9</td><td>2.0x10¹</td><td>3.0x10¹</td><td>1.0x10¹</td><td> 1,3</td>

<td> 8</td><td>1.39x10⁴</td><td>1.41x10⁴</td><td>1.36x10⁴</td><td> 4,1</td>

<td> 7</td><td>1.2x10⁴</td><td>1.2x10⁴</td><td>1.5x10⁴</td><td> 4,1</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

As shown in Table 15, at 96 hours and at 12 pg / ml of methicillin, the concentration of MSM at 9% showed higher survival of bacteria. A purely hypothetical explanation for this is that the bacteria have reversed resistance to the antibiotic. The MSM condition at 5% MSM also shows higher survival of bacteria.

The 8% MSM concentration showed no signs of colonies in all plating. The 8% tube was removed from the experiment and tested for MRSA. The tube was centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted into 90 ml of Mueller-Hinton broth with 6 pg / ml of methicillin. This is for the enrichment and recovery of any MRSA cells that may be present both healthy and stressed. The remaining test tube had 10 ml of Mueller-Hinton broth added with 6 pg / ml of methicillin to recover any cells that may be present. Both vessels were incubated at 35 ° C for 48 hours and plated every 24 hours. Each container was plated at 10 dilutions to obtain a detection limit of 1 cfu. Since all vessels were at 1/10 dilution in the enrichment solution, plating each vessel in this manner gave the inventors a detection limit of 1 cfu / ml. This was considered to be a true total death as recovery of the organism could not be achieved. This time point for graphical representation purposes was reported as 0 logs.

ES 2 674 019 T3

As shown in Table 15, at 96 hours and at 30 pg / ml of methicillin, higher MSM concentrations show a greater log reduction in cfu / ml. Concentrations from 5% to 9% MSM (except for 6% MSM) show less reduction in cfu / ml. At 96 hours and 60 pg / ml of methicillin, MSM concentrations of 9% to 16% showed a greater log reduction in colony count than at the 72 hour time point. The 7% and 8% MSM conditions show less reduction in cfu / ml.

the condition of MSM at 8% and 12 pg / ml does not show survival of viable organisms. TNTC was observed under the conditions of 7% and 8% MSM and 60 pg / ml of methicillin and MSM at 5% and 12 pg / ml of methicillin. Taken as a whole, at 96 hours, the 10-16% MSM conditions showed lower or similar colony counts compared to the previous time point of 72 hours.

The 30 pg / ml showed higher colony counts for the 5 to 9% MSM conditions. The log reduction in colony counts is slightly less or remains the same for 10-16% compared to the previous time point and appears to remain constant.

Table 16. Survival of Staphylococcus aureus strain ATCC 43300 in MSM 5-16% and 12, 30, or 60 pg / ml of methicillin for 120 hours.

<td>Concentration as% MSM</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>12 pg / ml methicillin / MSM (cfu / ml)</td><td>120 hour average log</td>

<td> 16</td><td>2.0x10²</td><td>2.3 x10²</td><td>2.4 x10²</td><td> 2,3</td>

<td> 15</td><td>2.9 x10²</td><td>3.4 x10²</td><td>3.1 x10²</td><td> 2,5</td>

<td> 14</td><td>3.5 x10²</td><td>4.0 x10²</td><td>3.8 x10²</td><td> 2,6</td>

<td> 13</td><td>2.0 x10²</td><td>1.8 x10²</td><td>2.1 x10²</td><td> 2,3</td>

<td> 12</td><td>1.21 x10³</td><td>1.21 x10³</td><td>1.21 x10³</td><td> 4,1</td>

<td> 11</td><td>1.24 x10⁴</td><td>1.22 x10⁴</td><td>1.23 x10⁴</td><td> 4,1</td>

<td> 10</td><td>1.10 x10⁴</td><td>1.10 x10⁴</td><td>1.13 x10⁴</td><td> 4,0</td>

<td> 9</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 8</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 7</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td colspan="5"></td>

<td>Concentration as% MSM</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>30 pg / ml methicillin / MSM (cfu / ml)</td><td>120 hour average log</td>

<td> 16</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 15</td><td>3.0 x10¹</td><td>4.0 x10¹</td><td>3.0 x10¹</td><td> 1,5</td>

<td> 14</td><td>5.0 x10¹</td><td>7.0 x10¹</td><td>6.0 x10¹</td><td> 1,8</td>

<td> 13</td><td>5.0 x10¹</td><td>4.0 x10¹</td><td>4.0 x10¹</td><td> 1,6</td>

<td> 12</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 11</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 10</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 9</td><td>1.3 x10³</td><td>1.1 x10³</td><td>1.2 x10³</td><td> 3,1</td>

<td> 8</td><td>9.0 x10²</td><td>9.1 x10²</td><td>9.5 x10²</td><td> 3,0</td>

<td> 7</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

ES 2 674 019 T3

<td> 6</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 5</td><td>1.21 x10³</td><td>1.21 x10³</td><td>1.21 x10³</td><td> 3,0</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td colspan="5"></td>

<td>Concentration as% MSM</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>60 pg / ml methicillin / MSM (cfu / ml)</td><td>120 hour average log</td>

<td> 16</td><td>1.0 x10¹</td><td>2.0 x10¹</td><td>2.0 x10¹</td><td> 1,2</td>

<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 14</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 13</td><td>3.0 x10¹</td><td>3.0 x10¹</td><td>3.0 x 10¹</td><td> 1,5</td>

<td> 12</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 11</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td></td><td></td><td></td><td></td><td></td>

<td> 10</td><td>1.0 x10¹</td><td>1.0 x10¹</td><td>1.0 x10¹</td><td> 1</td>

<td> 9</td><td>1.0 x10¹</td><td>1.0 x10¹</td><td>1.0 x10¹</td><td> 1</td>

<td> 8</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 7</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 0</td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 5,2</td>

As shown in Table 16, at 120 hours and 30 pg / ml of methicillin, the 10%, 11%, 12% and 16% concentrations of MSM showed no signs of colonies; and at 120 hours and 60 pg / ml of methicillin, the concentrations of 11%, 12%, 14% and 15% of MSM did not show signs of colonies. Tubes corresponding to these conditions were removed from the experiment and tested for MRSA. The tubes were centrifuged at 5000 rpm for 10 minutes. The supernatant was then decanted into 90 ml of Mueller-Hinton broth with 6 pg / ml of methicillin. This is for the enrichment and recovery of any MRSA cells that may be present both healthy and stressed. The remaining test tubes had 10 ml of Mueller-Hinton broth with the addition of 6 pg / ml of methicillin to recover any cells that may be present. Both vessels were incubated at 35 ° C for 48 hours and plated every 24 hours. Each well was plated 10 times to obtain a detection limit of 1 cfu. Since both vessels were at 1/10 dilution in the enrichment solution, plating each vessel in this manner gave the inventors a detection limit of 1 cfu. This was considered to be a true total death as recovery of the organism could not be achieved. This time point for graphical representation purposes was reported as 0 logs.

As a whole, at 120 hours and at 12 pg / ml of methicillin, there is a slight increase in cfu / ml in 13-16% of MSM and a greater increase at concentrations of 10% -12% of MSM compared to the previous time point. The 5% MSM condition and 12 pg / ml methicillin showed TNTC cfu / ml. MSM concentrations higher than 13-16% showed only a slight increase in cfu / ml. A purely hypothetical explanation for this is that bacteria revert to antibiotic resistance, but that higher concentrations of MSM had a significant impact on slowing down the reversion process as the inventors observed a slight increase in cfu / ml at higher concentrations of MSM while the lower concentrations had a significant increase in cfu / ml.

At 30 pg / ml of methicillin, the 16%, 12%, 11% and 10% MSM conditions showed true death with no observed recovery. The 15%, 14% and 13% MSM conditions showed a slight reduction in cfu / ml from the previous time point. The 5%, 8% and 9% MSM conditions showed slightly higher cfu / ml than the previous time point.

At 60 pg / ml of methicillin, the 15%, 14%, 12% and 11% MSM conditions showed true death without recovery of the organism. The 10% and 9% concentration points show a continued decrease in

ES 2 674 019 T3 the cfu / ml from the previous time point. One possibility is that these concentrations of MSM and antibiotic have a greater impact on MRSA.

Taken together, the combination of MSM and methicillin demonstrated the ability to significantly decrease the colony-forming units of MRSA at all time points evaluated. Based on the results of this experiment, MSM appears to have the ability to restore MRSA's sensitivity to methicillin in vitro. When compared to oxacillin in previous experiments, the rate of reduction in log value was even greater across concentrations. A hypothetical explanation for this is that methicillin has more binding sites when compared in structure to oxacillin than is shown in general experiments. There are more significant true deaths and the reduction over all concentrations is greater with methicillin than with oxacillin.

Taken together, the data presented in Tables 12-16 show that in the first 24 hours there was a greater reduction in the lower percentages of MSM for the three methicillin concentrations evaluated. This trend changes slightly at 48 hours, observing a maximum reduction in the mean to higher concentrations of MSM. By 72 hours, the trend has definitely changed to a greater log reduction at the higher MSM concentrations. MSM was added only when additional growth medium (LB containing the same original concentration of MSM as the growth vial) was added to maintain the original volume in the vial. For example, if a 1 ml aliquot of the 12 pg / ml methicillin was removed with 5% MSM for plating, then 1 ml of LB containing 5% MSM was added again. Antibiotics were added at the appropriate concentrations every 24 hours as described above.

The following are offered as hypothetical explanations for some of the effects noted above; however, these explanations are not intended to be limiting. A hypothetical and non-limiting possibility for the observed results is that MSM is consumed by organisms. Another hypothetical and non-limiting possibility for the observed results is that the ability of MSM to cause reversal of sensitivity could be due to the ability of MSM to transport methicillin to MRSA cells. Another hypothetical and non-limiting possibility for the observed results is that too high a level of MSM is less effective at certain time points. For example, this may be due to competition from an overabundance of free MSM molecules that compete with MSM that binds with methicillin at higher concentrations. This would cause more free MSM to penetrate cells than MSM-bound methicillin. Lower concentrations have a higher overall percentage of total MSM molecules in solution that bind to methicillin, allowing more bound methicillin to penetrate the cell as there is less free MSM to compete. Under this hypothesis, for a given number of MSM molecules penetrating the MRSA cell, the ratio of MSM to methicillin is important to obtain the maximum effect. If an overabundance of MSM is present, then the possibility of non-methicillin-bound MSM molecules penetrating the cell is increased.