CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This international application filed under the Paris Convention Treaty (PCT) claims
priority to U.S. Provisional Patent Application Serial No. 61/740,396 filed December 20,
2012 and titled ANTIMICROBIAL COMPOSITIONS, the contents of which are
incorporated herein in their entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention relates generally to the field of antimicrobial compositions. More
particularly, it relates to antimicrobial agents and methods of eliminating biofilm and
planktonic cells using these antimicrobial agents. In particular, the invention relates to
antimicrobial compositions containing a transport enhancer and a chelating agent. In one
exemplary embodiment, it relates to such compositions which contain MSM and EDTA.
BACKGROUND OF THE INVENTION
[0003] It is believed that all wounds are colonized by microbes. If the microbes reach a level
of clinical infection, their presence is believed to impair healing and may be a contributing
factor to wound chronicity. It has recently been estimated that hospital-acquired (nosocomial)
infections are the fourth-leading cause of death in the United States, affecting 2 million
patients per year and causing over 100,000 annual deaths, with a total annual cost of over $30
billion. Staphylococcal species such as S. epidermidis and S. aureus are responsible for the
majority of nosocomial infections; treatment of these infections is often made much more
challenging by the tendency of staphylococci to form biofilms.
[0004] Recently researchers have proposed that it may not be planktonic but rather biofilm
communities which contribute to wound chronicity. Biofilms are polymicrobial groupings of
bacteria which are held together in an extracellular polymeric substance consisting of protein,
DNA, and polysaccarhides and are not totally susceptible to antibiotic treatment. It has been
shown that 60% of the chronic wounds tested contained biofilm. (James et al., Wound Repair
Regen., 16(l):37-44, 2008.)
[0005] Biofilms are populations of bacteria or fungi growing attached to an inert or living
surface. Mounting evidence has shown that biofilms constitute a significant threat to human
health. The Public Health Service estimates that biofilms are responsible for more than 80%
of bacterial infections in humans (National Institutes of Health, 1998 RFA# DE-98-006).
Examples of diseases caused by biofilms include dental caries, periodontitis, cystic fibrosis
pneumonia, native valve endocarditis, and otitis media (Costerton et al. Science 1999
284:1318-1322), as well as infection of various medical devices such as urinary catheters,
mechanical heart valves, cardiac pacemakers, prosthetic joints, and contact lenses (Donlan, R.
M. 2001 Emerging Infect. Dis. 7:277-281). Fungi also form biofilms of clinical significance,
for example Candida infections. Biofilm infections afflict tens of millions of patients in the
U.S. annually and require a significant expenditure of health care dollars (Costerton et al.
Science 1999 284:1318-1322). Bacteria growing in biofilms exhibit increased resistance to
antimicrobial agents and are nearly impossible to eradicate. New methods for treating biofilm
infections are needed.
[0006] Bacterial biofilms are sources of contamination that are difficult to eliminate in a
variety of industrial, environmental and clinical settings. Biofilms are polymer structures
secreted by bacteria to protect bacteria from various environmental attacks, and thus result
also in protection of the bacteria from disinfectants and antibiotics. Biofilms may be found on
any environmental surface where sufficient moisture and nutrients are present. Bacterial
biofilms are associated with many human and animal health and environmental problems. For
instance, bacteria form biofilms on implanted medical devices, e.g., catheters, heart valves,
joint replacements, and damaged tissue, such as the lungs of cystic fibrosis patients. Biofilms
also contaminate surfaces such as water pipes and the like, and render also other industrial
surfaces hard to disinfect.
[0007] Biofilm is commonly known as the primary cause of many diseases and infections in
biology. Biofilms also play a detrimental role on many other non biological surfaces. These
biofilms, which exists not only on biological surfaces but also on all manner of surfaces, can
be defined as a diverse community of microorganisms. The microorganisms bind tightly to
one another, in addition to the solid surface, by means of an extracellular matrix consisting of
polymers of both host and microbial origin.
[0008] Biofilms, exhibit an open architecture. The open architecture, which consists of
channels and voids, helps to achieve the flow of nutrients, waste products, metabolites,
enzymes, and oxygen through the biofilm. Because of this structure, a variety of microbial
organisms can make up biofilms, including both aerobic and anaerobic bacteria. The
microbial composition of biofilms includes a multitude of species of bacteria, archaea, fungi
and viruses, which all exist in a relatively stable environment called microbial homeostasis.
Biofilms are responsible for many of the diseases common in the body including dental
diseases, non healing wounds and sores. Biofilms also are the cause of undesirable body odor
resulting from biofilms on the body surfaces. Further biofilms are common on many
engineering surfaces, and lead to material erosion, and to subpar engineering performance of
these surfaces.
[0009] Bacterial biofilms are ubiquitous in nature and are usually defined as matrix-enclosed
bacterial populations which adhere to each other and/or to surfaces or interfaces. Bacterial
biofilm formation is an extremely common phenomenon with a major economic impact in
different industrial, medical and environmental fields. Biofilms can comprise a single species
or multiple species and can form on a wide range of abiotic and biotic surfaces and interfaces.
Although polymicrobial biofilms predominate in most situations single species biofilms can
occur under certain circumstances and are an increasing problem on the surface of medical
implants. Growth as a biofilm offers a number of significant advantages to the bacterium over
planktonic growth not the least of which is the attachment to the surface that enables the
bacterium to localize itself in a favorable environment. In polymicrobial biofilms metabolic
activities can be integrated and the presence of a variety of species allows for greater
flexibility in metabolic and catabolic activities as the genome of the biofilm population
increases with increasing species diversity. The Centers for. Disease Control and Prevention
estimate that 65% of human bacterial infections involve biofilms. Biofilms often complicate
treatment of chronic infections by protecting bacteria from the immune system, decreasing
antibiotic efficacy and dispersing planktonic cells to distant sites that can promote r e
infection. Bacterial cells within a biofilm have been shown to be up to 500 times more
resistant to certain antimicrobial agents than planktonic cells which is achieved by a number
of processes including, the slowing of penetration of some antimicrobial agents into the
biofilm matrix, the slowing of the growth rate of bacteria in the deeper layers of the biofilm
and the binding of some antimicrobial agents to extracellular polymers thereby reducing the
effective concentration. In addition, microbial biofilms have been described as microbial
landscapes, which have a topography that protects against shear stress whilst allowing mass
transfer. Most importantly in the oral cavity failure to attach and grow as a biofilm will
rapidly result in clearance.
[0010] The oral cavity is a fertile environment for the growth of bacteria with a range of hard
and soft tissue surfaces that provide a variety of distinctly different microhabitats. The
stability of oral microbial biofilms requires dynamic balances by a range of synergistic and
antagonistic interactions among species and the environment they create. Minor adjustments
in the oral environment can affect these natural balances potentially leading to shifts in the
ecology and changes in the species composition of oral microbial biofilms. For example,
increased dental caries incidence is often caused by increased consumption of dietary
carbohydrates, which is linked to the acidification of fluids at the tooth surface due to the
bacterial fermentation of these carbohydrates. Experts agree that most forms of periodontal
disease are caused by specific pathogens, particularly gram-negative bacteria. The microbial
composition of dental biofilms includes over 700 species of bacteria and archaea, which all
exist in a relatively stable environment called microbial homeostasis. (Kroes I, Lepp PW,
Reiman DA Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci
USA 1999; 96(25): 14547-14552.)
[0011] Bacterial biofilms develop in variety of bodily cavities, including those of the ear,
such as the middle ear, and of the nose, such as the frontal or maxillary sinuses, for example.
Once bacterial growth has been established, the bacteria will often aggregate, stop dividing,
and begin forming protective bacterial biofilm layers, or "slime layers," comprised of
polysaccharide matrices.
[0012] The protective bacterial biofilm interferes with the body's natural immune response as
well as traditional methods of treatment. In particular, the bacteria emit exotoxins, which
incite the body's immune system to respond with white cells. However, the bacterial biofilm
interferes with the efficacy of the white cells' ability to attack the bacteria. The biofilm can
also act as a barrier against topical administration of antibiotics and other medicaments.
[0013] Biofilm-forming bacteria also present obstacles to traditional, antibiotic treatments
that act to kill dividing bacteria. In particular, the bacteria in a biofilm-forming state may
have already ceased cell division, rendering such antibiotics largely ineffective. Antibiotic
doses that kill free-floating bacteria, for example, need to be increased as much as 1,500
times to kill biofilm bacteria. At these high doses, the antibiotic is more likely to kill the
patient before the biofilm bacteria. (Elder MJ, at al. Biofilm-related infections in
ophthalmology. Eye 1995; vol. 9 (Pt. 1): 102-109.)
[0014] Methods of inhibiting biofilm formation in medical and industrial settings have
previously been developed using metal chelators, specifically iron chelators. For example,
U.S. Pat. No. 6,267,979, issued Jul. 31, 2001, to Raad et al, discloses the use of metal
chelators in combination with antifungal or antibiotic compositions for the prevention of
biofouling in water treatment, pulp and paper manufacturing and oil field water flooding.
U.S. Pat. No. 7,314,857, issued Jan. 1, 2008, to Madhyastha, discloses synergistic
antimicrobial compositions for inhibiting biofilm formation using combinations of an ironsequestering
glycoprotein, a cationic peptide, and an iron chelating agent. U.S. Pat. No.
7,446,089, issued Nov. 4, 2008, to Singh et al, is also directed to methods of inhibiting
biofilm formation by limiting the amount of iron available to a population of bacteria, such
that biofilm formation can be inhibited. These disclosures generally target iron, a higher
affinity metal ion.
[0015] Given the serious medical, industrial, and environmental problems associated with
bacterial biofilms, the need persists to develop targeted approaches to inhibit biofilm
formation. Therefore, there it is desirable to develop an agent that efficiently controls and
inhibits biofilm formation in medical and industrial applications.
SUMMARY OF THE INVENTION
[0016] MSM and EDTA (chelators in general) were not known to have an antimicrobial
effect. MSM also does not have any anti-microbial properties. However, treatment with a
combination of a transport enhancer (e.g., MSM) and chelator (e.g., EDTA) surprisingly and
unexpectedly showed dramatic reductions in levels of bacteria and fungi.
[0017] In some embodiments, the present invention relates to methods for use of the
formulations comprising a transport enhancer (such as MSM) and a chelating agent (EDTA)
for reduction of microbial levels on a surface.
[0018] In particular embodiments, the chelating agents are selected from the tetrasodium salt
of iminodisuccinic acid (Baypure® CXI 00; LANXESS GMBH (previously Bayer
Chemicals) Leverkusen, DE) or salts of poly-asparatic acid (Baypure® DS100; LANXESS
GMBH, Leverkusen, DE).
[0019] In some embodiments, the chelating agents are tetra sodium salts of L-glutamic acid
N,N-diacetic acid (GLDA - Dissolvine®, AkzoNobel, Netherlands).
[0020] In one aspect of the invention, methods are provided for prevention or treatment of
dental plaque or calculus in a subject.
[0021] The method involves administering to the subject an effective amount of a
formulation composed of a therapeutically effective amount of a chelating agent and an
effective transport-enhancing amount of a transport enhancer having the formula (I)
(I)
wherein R1 and R2 are independently selected from C2-C alkyl, Ci-C heteroalkyl, C -Ci4
aralkyl, and C2-Ci2 heteroaralkyl, any of which may be substituted, and Q is S or P.
[0022] The transport enhancing agent can be, for example, methylsulfonylmethane (MSM;
also referred to as methylsulfone, dimethylsulfone, and DMS0 2), and the chelating agent can
be ethylene diamine tetra-acetic acid (EDTA) and the like.
[0023] The formulation may be administered in any form suitable including liquid, paste, gel,
solid and particulate solid state compositions. Additionally, in a preferred embodiment, the
formulation is entirely composed of components that are naturally occurring and/or as GRAS
("Generally Regarded as Safe") by the U.S. Food and Drug Administration.
[0024] Accordingly, the present invention provides a method for inhibiting formation of a
biofilm comprising bacteria, the method comprising contacting the bacteria with an effective
amount of a formulation comprising a transport enhancer (such as MSM) and a chelating
agent (such as EDTA), whereby formation of the biofilm is inhibited.
[0025] In another embodiment, the present invention provides a method for inhibiting
formation of a biofilm on a device, the method comprising contacting the bacteria with an
effective amount of a formulation comprising a transport enhancer (such as MSM) and a
chelating agent (such as EDTA), whereby formation of a biofilm on the device is inhibited.
[0026] In another embodiment, the present invention provides a topical pharmaceutical
composition for inhibiting formation of a biofilm on or within a mammal, comprising an
effective amount of a formulation comprising a transport enhancer (such as MSM) and a
chelating agent (such as EDTA), and at least one pharmaceutically acceptable carrier.
[0027] In a further embodiment, the present invention provides a surgical rinse for inhibiting
formation of a biofilm comprising bacteria, wherein the surgical rinse comprises an effective
amount of a formulation comprising a transport enhancer (such as MSM) and a chelating
agent (such as EDTA).
[0028] In yet another embodiment, the present invention provides a method for inhibiting
formation of a biofilm comprising bacteria, the method comprising contacting the bacteria
with an effective amount of a formulation comprising a transport enhancer (such as MSM)
and a chelating agent (such as EDTA), wherein the bacteria are selected from the group
consisting of Acidothermus cellulyticus, Actinomyces odontolyticus, Alkaliphilus
metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens,
Bacillus clausii, Bacillus halodurans, Bacillus licheniformis, Bacillus pumilus, Bacillus
subtilis, Bifidobacterium adolescentis, Bifidiobacterium longum, Caldicellulosiruptor
saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium acetobutylicum,
Clostridium beijerinckii, Clostridium botulinum, Clostridium cellulolyticum, Clostridium
difficile, Clostridium kluyveri, Clostridium leptum, Clostridium novyi, Clostridium
perfringens, Clostridium tetani, Clostridium thermocellum, Corynebacterium diphtheriae,
Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium jeikeium,
Corynebacterium urealyticum, Desulfitobacterium hafniense, Desulfotomaculum reducens,
Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna, Geobacillus
kaustophilus, Geobacillus thermodenitrificans, Janibacter sp., Kineococcus radiotolerans,
Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua, Listeria welshimeri,
Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis, Mycobacterium
gilvum, Mycobacterium leprae, Mycobacterium paratuberculosis, Mycobacterium smegmatis,
Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii,
Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis, Pelotomaculum
thermopropionicum, Rhodococcus sp., Saccharopolyspora erythraea, coagulase-negative
Staphylococcus species, Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant Staphylococcus epidermidis
(MRSE), Streptococcus agalactiae, Streptococcus gordonii, Streptococcus mitis,
Streptococcus oralis, Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, Streptomyces avermitilis, Streptomyces coelicolor, Thermoanaerobacter ethanolicus,
Thermoanaerobacter tengcongensis, and combinations thereof, whereby formation of the
biofilm is inhibited.
[0029] A further embodiment of the present invention provides a bandage impregnated with a
safe and effective amount of a formulation comprising a transport enhancer (such as MSM)
and a chelating agent (such as EDTA), wherein the bandage inhibits the formation of a
biofilm on the skin.
[0030] Yet another embodiment of the present invention provides a personal cleansing
formulation comprising a transport enhancer (such as MSM) and a chelating agent (such as
EDTA), wherein the personal cleansing composition inhibits formation of a biofilm on the
skin.
[0031] A further embodiment of the present invention provides a hard surface cleaning
formulation comprising a transport enhancer (such as MSM) and a chelating agent (such as
EDTA), wherein the composition inhibits formation of a biofilm on a hard surface.
[0032] A further embodiment of the present invention provides a dental rinse for inhibiting
formation of a biofilm, the dental rinse comprising a formulation comprising a transport
enhancer (such as MSM) and a chelating agent (such as EDTA).
[0033] These and other aspects will become apparent from the following description of the
preferred embodiment taken in conjunction with the following drawings, although variations
and modifications therein may be affected without departing from the spirit and scope of the
novel concepts of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The following drawings form part of the present specification and are included to
further demonstrate certain aspects of the present disclosure, the inventions of which can be
better understood by reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
[0035] The patent or application contains at least one drawing executed in color. Copies of
this patent or patent application publication with color drawings will be provided by the US
Patent and Trademark Office upon request and payment of the necessary fee.
[0036] Figure l a shows Trichmichosis axillaris in the armpit of a human subject. Figure lb
shows complete resolution of Trichmichosis axillaris in the armpit of the subject following
treatment in about 48 hours.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The terms used in this specification generally have their ordinary meanings in the art,
within the context of the invention, and in the specific context where each term is used.
Certain terms that are used to describe the invention are discussed below, or elsewhere in the
specification, to provide additional guidance to the practitioner regarding the description of
the invention. For convenience, certain terms may be highlighted, for example using italics
and/or quotation marks. The use of highlighting has no influence on the scope and meaning
of a term; the scope and meaning of a term is the same, in the same context, whether or not it
is highlighted. It will be appreciated that same thing can be said in more than one way.
Consequently, alternative language and synonyms may be used for any one or more of the
terms discussed herein, nor is any special significance to be placed upon whether or not a
term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of
one or more synonyms does not exclude the use of other synonyms. The use of examples
anywhere in this specification including examples of any terms discussed herein is illustrative
only, and in no way limits the scope and meaning of the invention or of any exemplified
term. Likewise, the invention is not limited to various embodiments given in this
specification.
[0038] Where a range of values is provided, it is understood that each intervening value, to
the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between
the upper and lower limit of that range and any other stated or intervening value in that stated
range, is encompassed within the invention. The upper and lower limits of these smaller
ranges may independently be included in the smaller ranges, and are also encompassed within
the invention, subject to any specifically excluded limit in the stated range. Where the stated
range includes one or both of the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0039] Throughout this application, various publications, patents and published patent
applications are cited. The inventions of these publications, patents and published patent
applications referenced in this application are hereby incorporated by reference in their
entireties into the present invention. Citation herein of a publication, patent, or published
patent application is not an admission the publication, patent, or published patent application
is prior art.
[0040] As used herein and in the appended claims, the singular forms "a," "and," and "the"
include plural referents unless the context clearly dictates otherwise. Thus, for example, "a
transport enhancer" encompasses a plurality of transport enhancers as well as a single
transport enhancer. Reference to "a chelating agent" includes reference to two or more
chelating agents as well as a single chelating agent, and so forth. In this specification and in
the claims that follow, reference will be made to a number of terms, which shall be defined to
have the following meanings:
[0041] When referring to a formulation component, it is intended that the term used, e.g.,
"agent," encompass not only the specified molecular entity but also its pharmaceutically
acceptable analogs, including, but not limited to, salts, esters, amides, prodrugs, conjugates,
active metabolites, and other such derivatives, analogs, and related compounds.
[0042] The terms "treating" and "treatment" as used herein refer to the administration of an
agent or formulation to a clinically symptomatic individual afflicted with an adverse
condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of
symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate
improvement or remediation of damage. The terms "preventing" and "prevention" refer to the
administration of an agent or composition to a clinically asymptomatic individual who is
susceptible to a particular adverse condition, disorder, or disease, and thus relates to the
prevention of the occurrence of symptoms and/or their underlying cause. Unless otherwise
indicated herein, either explicitly or by implication, if the term "treatment" (or "treating") is
used without reference to possible prevention, it is intended that prevention be encompassed
as well, such that "a method for the treatment of gingivitis" would be interpreted as
encompassing "a method for the prevention of gingivitis."
[0043] "Optional" or "optionally present" - as in an "optional substituent" or an "optionally
present additive" means that the subsequently described component (e.g., substituent or
additive) may or may not be present, so that the description includes instances where the
component is present and instances where it is not.
[0044] By "pharmaceutically acceptable" is meant a material that is not biologically or
otherwise undesirable, e.g., the material may be incorporated into a formulation of the
invention without causing any undesirable biological effects or interacting in a deleterious
manner with any of the other components of the dosage form formulation. However, when
the term "pharmaceutically acceptable" is used to refer to a pharmaceutical excipient, it is
implied that the excipient has met the required standards of toxicological and manufacturing
testing and/or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food
and Drug Administration. As explained in further detail infra, "pharmacologically active" (or
simply "active") as in a "pharmacologically active" derivative or analog refers to derivative or
analog having the same type of pharmacological activity as the parent agent. The terms
"treating" and "treatment" as used herein refer to reduction in severity and/or frequency of
symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of
symptoms and/or their underlying cause, and improvement or remediation of an undesirable
condition or damage. Thus, for example, "treating" a subject involves prevention of an
adverse condition in a susceptible individual as well as treatment of a clinically symptomatic
individual by inhibiting or causing regression of the condition. The term "chelating agent" (or
"active agent") refers to any chemical compound, complex or composition that exhibits a
desirable effect in the biological context, i.e., when administered to a subject or introduced
into cells or tissues in vitro. The term includes pharmaceutically acceptable derivatives of
those active agents specifically mentioned herein, including, but not limited to, salts, esters,
amides, prodrugs, active metabolites, isomers, analogs, crystalline forms, hydrates, and the
like. When the term "chelating agent" is used, or when a particular chelating agent is
specifically identified, it is to be understood that pharmaceutically acceptable salts, esters,
amides, prodrugs, active metabolites, isomers, analogs, etc. of the agent are intended as well
as the agent per se.
[0045] By an "effective" amount or a "therapeutically effective" amount of an active agent is
meant a nontoxic but sufficient amount of the agent to provide a beneficial effect. The
amount of active agent that is "effective" will vary from subject to subject, depending on the
age and general condition of the individual, the particular active agent or agents, and the like.
Unless otherwise indicated, the term "therapeutically effective" amount as used herein is
intended to encompass an amount effective for the prevention of an adverse condition and/or
the amelioration of an adverse condition, i.e., in addition to an amount effective for the
treatment of an adverse condition.
[0046] As will be apparent to those of skill in the art upon reading this invention, each of the
individual embodiments described and illustrated herein has discrete components and features
which may be readily separated from or combined with the features of any of the other
several embodiments without departing from the scope or spirit of the present invention. Any
recited method can be carried out in the order of events recited or in any other order that is
logically possible.
[0047] Unless otherwise indicated, the invention is not limited to specific formulation
components, modes of administration, chelating agents, manufacturing processes, or the like,
as such may vary.
[0048] Unless otherwise defined, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
pertains. In the case of conflict, the present document, including definitions will control.
Definitions
[0049] The term "biofilm" refers to matrix-enclosed microbial accretions to biological or
non-biological surfaces. Biofilm formation represents a protected mode of growth that allows
cells to survive in hostile environments.
[0050] The term "biofilm formation" is intended to include the formation, growth, and
modification of the bacterial colonies contained with biofilm structures, as well as the
synthesis and maintenance of the polysaccharide matrix of the biofilm structures.
[0051] The term "gram positive bacteria" refers to bacteria having cell walls with high
amounts of peptidoglycan. Gram positive bacteria are identified by their tendency to retain
crystal violet and stain dark blue or violet in the Gram staining protocol.
[0052] The term "gram negative bacteria" refers to bacteria having thinner peptidoglycan
layers which do not retain the crystal violet stain in the Gram staining protocol and instead
retain the counterstain, typically safranin. Gram negative bacteria stain red or pink in the
Gram staining protocol.
[0053] The term "antimicrobial agent" refers to any substance that kills or prevents the
growth of bacteria or other microbes.
[0054] A non-limiting list of bacteria that may be susceptible to the antimicrobial
compositions of the invention include: Acidothermus cellulyticus, Actinomyces odontolyticus,
Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus
amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus licheniformis, Bacillus
pumilus, Bacillus subtilis, Bifidobacterium adolescentis, Bifidiobacterium longum,
Caldicellulosiruptor saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium
acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium cellulolyticum,
Clostridium difficile, Clostridium kluyveri, Clostridium leptum, Clostridium novyi,
Clostridium perfringens, Clostridium tetani, Clostridium thermocellum, Corynebacterium
diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium
jeikeium, Corynebacterium urealyticum, Desulfitobacterium hafniense, Desulfotomaculum
reducens, Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna,
Geobacillus kaustophilus, Geobacillus thermodenitrificans, Janibacter sp., Kineococcus
radiotolerans, Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua, Listeria
welshimeri, Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium paratuberculosis,
Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium ulcerans,
Mycobacterium vanbaalenii, Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora erythraea,
coagulase-negative Staphylococcus species, Staphylococcus aureus, methicillin resistant
Staphylococcus aureus (MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus agalactiae, Streptococcus gordonii,
Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus
sanguinis, Streptococcus suis, Streptomyces avermitilis, Streptomyces coelicolor,
Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis, and combinations
thereof.
[0055] The term "antibiotic" refers to a substance that is antagonistic to the growth of
microorganisms. Suitable antibiotics may be naturally-occurring, chemically-modified, or
synthetically-produced.
[0056] The term "surgical rinse" refers to a solution used during surgery to irrigate the site of
an implanted medical device, with the intent to prevent initial formation of biofilms in the
vicinity of the medical device.
[0057] The term "dental rinse" refers to a solution containing one or more zinc chelators used
as a mouthwash or rinse to prevent the establishment of oral biofilms that lead to dental
caries.
[0058] The term "personal cleansing composition" refers to a composition that is used for
personal hygiene. Personal cleansing compositions include, but are not limited to: gels,
creams, suspensions, colloids, soaps, deodorants, body washes, shampoos, and the like. In
one embodiment, the personal cleansing compositions of the present invention inhibit
biofilm-related infections including, but not limited to, community-acquired methicillinresistant
Staphylococcus aureus (CA-MRSA) infection.
[0059] The term "hard surface cleaning composition" refers to a composition that is used to
clean and/or sanitize a hard or solid surface. In one embodiment, the invention provides a
composition that prevents bacterial biofilm growth on hard surfaces including, but not limited
to, surgical instruments, storage tanks, pipelines, trays, containers, walls, floors, countertops,
locker room floors, benches, lockers, showers, bathrooms, toilets, water filtration units, and
the like.
[0060] Chelating agent: Chelation is a chemical combination with a metal in complexes in
which the metal is part of a ring. An organic ligand is called a chelator or chelating agent, the
chelate is a metal complex. The larger number of ring closures to a metal atom the more
stable is the compound. The stability of a chelate is also related to the number of atoms in the
chelate ring. Monodentate ligands which have one coordinating atom like H20 or N¾ are
easily broken apart by other chemical processes, whereas polydentate chelators, donating
multiple binds to metal ion, provide more stable complexes. Chlorophyll, a green plant
pigment, is a chelate that consists of a central magnesium atom joined with four complex
chelating agent (pyrrole ring). Heme is an iron chelate which contains iron (II) ion in the
center of the porphyrin. Chelating agents offers a wide range of sequestrants to control metal
ions in aqueous systems. By forming stable water soluble complexes with multivalent metal
ions, chelating agents prevent undesired interaction by blocking normal reactivity of metal
ions. EDTA (ethylenediamine tetraacetate) is a good example of common chelating agents
which have nitrogen atoms and short chain carboxylic groups.
[0061] Examples of chelators of iron and calcium include, but are not limited to, Diethylene
triamine pentaacetic acid (DTPA), ethylene diamine tetraacetic acid (EDTA), nitrilotriacetic
acid (NTA), 1,3-propylene diamine tetraacetic acid (PDTA), Ethylene diamine disuccinic
acid (EDDS), and ethylene glycol tetraacetic acid (EGTA). Any suitable chelating agent
known in the art, which is biologically safe and able to chelate iron, calcium or other metals,
is suitable for the invention.
[0062] Compounds useful as chelating agents herein include any compounds that coordinate
to or form complexes with a divalent or polyvalent metal cation, thus serving as a sequestrant
of such cations. Accordingly, the term "chelating agent" herein includes not only divalent and
polyvalent ligands (which are typically referred to as "chelators") but also monovalent
ligands capable of coordinating to or forming complexes with the metal cation.
[0063] Suitable biocompatible chelating agents useful in conjunction with the present
invention include, without limitation, monomeric polyacids such as EDTA,
cyclohexanediamine tetraacetic acid (CDTA), hydroxyethylethylenediamine triacetic acid
(HEDTA), diethylenetriamine pentaacetic acid (DTPA), dimercaptopropane sulfonic acid
(DMPS), dimercaptosuccinic acid (DMSA), aminotrimethylene phosphonic acid (ATPA),
citric acid, pharmaceutically acceptable salts thereof, and combinations of any of the
foregoing. Other exemplary chelating agents include: phosphates, e.g., pyrophosphates,
tripolyphosphates, and hexametaphosphates.
[0064] EDTA and ophthalmologically acceptable EDTA salts are particularly preferred,
wherein representative ophthalmologically acceptable EDTA salts are typically selected from
diammonium EDTA, disodium EDTA, dipotassium EDTA, triammonium EDTA, trisodium
EDTA, tripotassium EDTA, and calcium disodium EDTA.
[0065] EDTA has been widely used as an agent for chelating metals in biological tissue and
blood, and has been suggested for inclusion in various formulations. For example, U.S. Pat.
No. 6,348,508 to Denick Jr. et al. describes EDTA as a sequestering agent to bind metal ions.
In addition to its use as a chelating agent, EDTA has also been widely used as a preservative
in place of benzalkonium chloride, as described, for example, in U.S. Pat. No. 6,21 1,238 to
Castillo et al. U.S. Pat. No. 6,265,444 to Bowman et al. discloses use of EDTA as a
preservative and stabilizer. However, EDTA has generally not been applied topically in any
significant concentration formulations because of its poor penetration across biological
membranes and biofilms including skin, cell membranes and even biofilms like dental
plaque.
[0066] Among the chelating/sequetering materials which may be included in the
compositions there may be mentioned biocompatible chelating agents include, without
limitation, monomeric polyacids such as EDTA, cyclohexanediamine tetraacetic acid
(CDTA), hydroxyethylethylenediamine triacetic acid (HEDTA), diethylenetriamine
pentaacetic acid (DTPA), dimercaptopropane sulfonic acid (DMPS), dimercaptosuccinic acid
(DMSA), aminotrimethylene phosphonic acid (ATPA), citric acid, pharmaceutically
acceptable salts thereof, and combinations of any of the foregoing.
[0067] Other exemplary chelating agents include: phosphates, e.g., pyrophosphates,
tripolyphosphates, and hexametaphosphates. Other exemplary chelating agents include:
phosphates, e.g., pyrophosphates, tripolyphosphates, and hexametaphosphates; chelating
antibiotics such as chloroquine and tetracycline; nitrogen-containing chelating agents
containing two or more chelating nitrogen atoms within an imino group or in an aromatic ring
(e.g., diimines, 2,2'-bipyridines, etc.); and polyamines such as cyclam (1,4,7,1 1-
tetraazacyclotetradecane), N-(Ci-C 3o alkyl)-substituted cyclams (e.g., hexadecyclam,
tetramethylhexadecylcyclam), diethylenetriamine (DETA), spermine, diethylnorspermine
(DENSPM), diethylhomo-spermine (DEHOP), deferoxamine ( '-{5-
[Acetyl(hydroxy)amino]pentyl} -N-[5-( {4- [(5-aminopentyl)(hydroxy)amino]-4-
oxobutanoyl} amino)pentyl]-N-hydroxysuccinamide, or N'-[5-(Acetyl-hydroxyamino)
pentyl]-N- [5-[3-(5-aminopentyl-hy droxy-carbamoyl) propanoylamino]pentyl] -Nhydroxy-
butane diamide); also known as desferoxamine B, desferoxamine B, DFO-B,
DFOA, DFB or desferal), deferiprone, pyridoxal isonicotinoyl hydrazone (PIH),
salicylaldehyde isonicotinoyl hydrazone (SIH), ethane- l,2-bis(N-l-amino-3-ethylbutyl-3-
thiol).
[0068] Additional, suitable biocompatible chelating agents which may be useful for the
practice of the current disclosure include EDTA-4-aminoquinoline conjugates such as ([2-
(Bis-ethoxycarbonylmethyl-amino)-ethyl]-{[2-(7-chloro-quinolin-4-ylamino)-
ethylcarbamoyl]-methyl}-amino)-acetic acid ethyl ester, ([2-(Bis-ethoxycarbonylmethylamino)-
propyl]-{[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic
acid ethyl ester, ([3-(Bis-ethoxycarbonylmethyl-amino)-propyl]-{[2-(7-chloro-quinolin-4-
ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic acid ethyl ester, ([4-(Bisethoxycarbonylmethyl-
amino)-butyl]-{[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-
methyl}-amino)-acetic acid ethyl ester, ([2-(Bis-ethoxymethyl-amino)-ethyl]-{[2-(7-chloroquinolin-
4-ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic acid ethyl ester, ([2-(Bisethoxymethyl-
amino)-propyl]-{[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-methyl}-
amino)-acetic acid ethyl ester, ([3-(Bis-ethoxymethyl-amino)-propyl]-{[2-(7-chloro-quinolin-
4-ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic acid ethyl ester, ([4-(Bis-ethoxymethylamino)-
butyl]-{[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic
acid ethyl ester as described in Solomon et al, Med. Chem. 2 : 133-138, 2006.
[0069] Additionally, natural chelators including, but not limited to citric acid, phytic acid,
lactic acid, acetic acid and their salts. Other natural chelators and weak chelators include but
are not limited to curcumin (turmeric), ascorbic acid, succinic acid, and the like .
[0070] In some embodiments, the chelating agents are selected from the tetrasodium salt of
iminodisuccinic acid (Baypure® CXI 00; LANXESS GMBH (previously Bayer Chemicals)
Leverkusen, DE) or salts of poly-asparatic acid (Baypure® DS100; LANXESS GMBH,
Leverkusen, DE). In some embodiments, the chelating agents are tetra sodium salts of Lglutamic
acid N,N-diacetic acid (GLDA - Dissolvine®, AkzoNobel, Netherlands).
[0071] In some embodiments, the chelating agent incorporated in the formulation is a
prochelator. A prochelator is any molecule that is converted to a chelator when exposed to the
appropriate chemical or physical conditions. For example, BSIH (isonicotinic acid [2-
(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide) prochelators are
converted by hydrogen peroxide into SIH (salicylaldehyde isonicotinoyl hydrazone) ironchelating
agents that inhibit iron-catalyzed hydroxyl radical generation.
[0072] The inactivated metal ion sequestering agent is sometimes referred to herein as a
"prochelator," although sequestration of metal ions can involve sequestration and
complexation processes beyond the scope of chelation per se. The term "prochelator" is
analogous to the term "prodrug" insofar as a prodrug is a therapeutically inactive agent until
activated in vivo, and the prochelator, as well, is incapable of sequestering metal ions until
activated in vivo.
[0073] Transport Enhancer: The transport enhancer is selected to facilitate the transport of a
chelating agent through the tissues, extra-cellular matrices, and/or cell membranes of a body.
An "effective amount" of the transport enhancer represents an amount and concentration
within a formulation of the invention that is sufficient to provide a measurable increase in the
penetration of a chelating agent through one or more of the sites of oral cavity or teeth in a
subject than would otherwise be the case without the inclusion of the transport enhancer
within the formulation.
[0074] In certain instances, the transport enhancer may be present in a formulation of the
invention in an amount that ranges from about 0.01 wt.% or less to about 30 wt.% or more,
typically in the range of about 0.1 wt.% to about 20 wt.%, more typically in the range of
about 1 wt.% to about 11 wt.%, and most typically in the range of about 2 wt.% to about 8
wt .%, for instance, 5 wt.%>.
[0075] The transport enhancer is generally of the formula (I)
(I)
o
Q 2
O
[0076] wherein R1 and R2 are independently selected from C2-C6 alkyl, Ci-C 6 heteroalkyl,
C -Ci 4 aralkyl, and C2-Ci 2 heteroaralkyl, any of which may be substituted, and Q is S or P.
Compounds wherein Q is S and R1 and R2 are C1-C3 alkyl are preferred, with
methylsulfonylmethane (MSM) being the optimal transport enhancer.
[0077] The phrase "having the formula" or "having the structure" is not intended to be
limiting and is used in the same way that the term "comprising" is commonly used. With
respect to the above structure, the term "alkyl" refers to a linear, branched, or cyclic saturated
hydrocarbon group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, t-butyl, cyclopentyl, cyclohexyl and the like. If not otherwise
indicated, the term "alkyl" includes unsubstituted and substituted alkyl, wherein the
substituents may be, for example, halo, hydroxyl, sulfhydryl, alkoxy, acyl, etc. The term
"alkoxy" intends an alkyl group bound through a single, terminal ether linkage; that is, an
"alkoxy" group may be represented as -O-alkyl where alkyl is as defined above. The term
"aryl" refers to an aromatic substituent containing a single aromatic ring or multiple aromatic
rings that are fused together, directly linked, or indirectly linked (such that the different
aromatic rings are bound to a common group such as a methylene or ethylene moiety).
Preferred aryl groups contain 5 to 14 carbon atoms. Exemplary aryl groups are contain one
aromatic ring or two fused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl,
diphenylether, diphenylamine, benzophenone, and the like. "Aryl" includes unsubstituted and
substituted aryl, wherein the substituents may be as set forth above with respect to optionally
substituted "alkyl" groups. The term "aralkyl" refers to an alkyl group with an aryl
substituent, wherein "aryl" and "alkyl" are as defined above. Preferred aralkyl groups contain
6 to 14 carbon atoms, and particularly preferred aralkyl groups contain 6 to 8 carbon atoms.
Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3 -phenylpropyl,
4-phenyl-butyl, 5 -phenyl -pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-
phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like. The term "acyl" refers to
substituents having the formula -(CO)-alkyl, -(CO)-aryl, or -(CO)-aralkyl, wherein "alkyl,"
"aryl, and "aralkyl" are as defined above. The terms "heteroalkyl" and "heteroaralkyl" are
used to refer to heteroatom-containing alkyl and aralkyl groups, respectively, i.e., alkyl and
aralkyl groups in which one or more carbon atoms is replaced with an atom other than
carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or
sulfur.
[0078] The term "implantable medical device" refers to any medical device implanted or
inserted in the human body. Such devices can be temporarily or permanently implanted or
inserted. An implantable medical device can be, for example, catheters, orthopedic devices,
prosthetic devices, vascular stents, urinary stents, pacemakers, implants, or the like.
[0079] The term "bathing a device" refers to submerging a device in a solution in order to
pre-treat the device, for example, prior to surgical implantation. Bathing a device can also
occur after the device has been surgically implanted, for example, by irrigating the surgical
site with a sterile solution.
[0080] The term "coating a device" refers to pre-treating a device with a composition prior to
surgical implantation. Suitable compositions for pre-treating the device may include, for
example, solutions, gels, polymer coatings, and the like. A variety of means may be
employed to coat a device, such as spraying or submerging the device. The coated device
comprises a surface layer having desirable properties conferred by the coating composition.
In one embodiment, the coating composition comprises at least one zinc chelator. In another
embodiment, the coating composition comprises one or more soluble G5 domains or zinc
adhesion modules.
[0081] The term "topical pharmaceutical composition" refers to pharmaceutical compositions
suitable for dermal administration to a mammal. Suitable topical pharmaceutical
compositions include, but are not limited to, gels, creams, lotions, ointments, tinctures,
sprays, and solids. In one embodiment, a topical pharmaceutical composition of the present
invention is applied on the outer surface of the skin or in the vicinity of cuts, abrasions, turf
burn injuries, lacerations, burns, or puncture wounds in order to treat, prevent, or inhibit the
formation of bacterial biofilms.
Biofilms
[0082] Biofilms are bacterial communities that adhere to biological or abiotic substrata,
differentiate into micro- and macrocolonies, and produce an extracellular matrix typically
comprised of polysaccharides and proteins. Bacteria in biofilms are resistant to antibiotics
and host immune responses and are extremely difficult to eradicate. For example, devicerelated
infections due to staphylococcal biofilms often require surgical removal of the
implanted device, debridement of the surrounding tissue, and prolonged antibiotic treatment.
[0083] The formation of biofilms includes a series of steps that begins with the initial
colonization of the surface and ends with the complex formation of a mature biofilm.
Biofilms exist on a variety of surfaces including tissues, smooth surfaces and biological
crevices, however they are most likely to be seen in their mature state in the more stagnant
sites, like fissures and crevices, as these places provide protection from the forces of removal,
like fluid flow and mechanical action. Additionally, through the growth process of the
biofilm, the microbial composition changes from one that is primarily gram-positive and
streptococcus-rich to a structure filled with gram-negative anaerobes in its more mature state.
[0084] The first step in biofilm development is the adsorption of host and bacterial molecules
to the surface. Within minutes of a cleaning, biofilm formation begins, which can be defined
as a thin coat of microbes. This layer acts like an adhesive by sticking to the surface and
encouraging a conditioning film of bacteria to attach to the surface. This conditioning film
directly influences the initial microbial colonization, and continues to adsorb bacteria to the
tooth surface.
[0085] There are many distinct habitats most of which are bathed in ionic solutions. In order
to survive bacteria must attach to one of its surfaces or risk being at the risk of air and fluid
flows. Bacteria attaching to exposed smooth surfaces must be quite firmly attached to resist
the flow of air and water. Any build-up of cells due to multiplication is more easily dislodged
because the mass of bacteria experiences a greater shear force. This does not mean that the
exposed, smooth, surfaces of teeth are devoid of attached bacteria because some species have
evolved efficient adhesion mechanisms. It does mean, however, that any significant build-up
is inhibited and that plaque accumulation is limted to sheltered sites such as interproximal
areas, the gingival margin and fissures. Bacteria will also accumulate in defects.
[0086] Before plaque can accumulate, the surface has to be colonised by bacteria which then
multiply and attract further colonisers. These "first colonisers" are known as pioneer species.
[0087] The surfaces of these cells and, in fact the surfaces of nearly all cells, are negatively
charged because of the presence of proteins and other wall and cell membrane components
which contain phosphate, carboxyl and other acidic groups. Furthermore, nearly all nonbiological
surfaces are also negatively charged. Sometimes this is due to the accumulation of
organic material which adsorbs to the surface from the environment and sometimes because
the surface is inherently negatively charged because of its chemistry. However, the presence
of high amounts of positively charged ionic multivalent metals in the surrounding fluid, and
in the biofilm fluid, causes the bacteria to be attracted to the negatively charged surface
(DLVO Theory.)
[0088] As the concentration of multivalent metals continues to build in the biofilm, it reaches
levels, where small changes in pH can cause the precipitation of metal salts onto the surface.
These precipitates build up over time. This deposits will then cause erosion and damage to
the surface.
[0089] Multivalent metals like calcium being common are often involved in biofilm
production and its detrimental effects, hence a reduction in metal levels will play a key role in
treating the adverse conditions on surfaces. Current treatment modalites do not take this
approach, but rather depend upon mechanical removal of biofilms on teeth and other wet
surfaces.
[0090] Removal of multivalent metals like calcium could be accomplished by means of
calcium chelators. However, chelators are also negatively charged molecules, and are
therefore repelled from the biofilm surface. Therefore to accomplish the task of getting these
chelators into the plaque and close to the metals, a charge-masking, permeation-enhancing
carrier allows the chelators to get to the target metal ion, e.g. calcium. The sequestration
inactivating moiety may also facilitate transport of the metal ion sequestering agent through
biological membranes.
[0091] Without wishing to be bound by theory, it appears that a significant role played by the
biocompatible chelating agent in the present formulations is in the removal of the metals
(such as copper, iron, and calcium) from the biofilm which allow easier mechanical removal
of the biofilm and slows down the rebuilding of the unhealthy biofilms.
[0092] Accordingly, the chelating agent is multifunctional in the context of the present
invention, insofar as the agent serves to decrease unwanted proteinase (e.g., collagenase)
activity, prevent formation of mineral deposits, and/or reduce mineral deposits that have
already formed, and reduce calcification, in addition to acting as a preservative and
stabilizing agent. The formulation also includes an effective amount of a transport enhancer
that facilitates penetration of the formulation components through cell membranes, tissues,
and extracellular matrices, including the gums and other oral tissue. The "effective amount"
of the transport enhancer represents a concentration that is sufficient to provide a measurable
increase in penetration of one or more of the formulation components through membranes,
tissues, and extracellular matrices as just described. Suitable transport enhancers include, by
way of example, methylsulfonylmethane (MSM; also referred to as methyl sulfone),
combinations of MSM with dimethylsulfoxide (DMSO), or a combination of MSM and, in a
less preferred embodiment, DMSO, with MSM particularly preferred.
[0093] MSM is an odorless, highly water-soluble (34% w/v @ 79° F.) white crystalline
compound with a melting point of 108-1 10° C. and a molecular weight of 94.1 g/mol. MSM
serves as a multifunctional agent herein, insofar as the agent not only increases cell
membrane permeability, but also acts as a "transport facilitating agent" (TFA) that aids in the
transport of one or more formulation components to oral tissues. Furthermore, MSM per se
provides medicative effects, and can serve as an anti-inflammatory agent as well as an
analgesic. MSM also acts to improve oxidative metabolism in biological tissues, and is a
source of organic sulfur, which assists in the reduction of scarring. MSM additionally
possesses unique and beneficial solubilization properties, in that it is soluble in water, as
noted above, but exhibits both hydrophilic and hydrophobic properties because of the
presence of polar S=0 groups and nonpolar methyl groups. The molecular structure of MSM
also allows for hydrogen bonding with other molecules, i.e., between the oxygen atom of
each S=0 group and hydrogen atoms of other molecules, and for formation of van der Waal
associations, i.e., between the methyl groups and nonpolar (e.g., hydrocarbyl) segments of
other molecules. Ideally, the concentration of MSM in the present formulations is in the range
of about 0.1 wt. % to 40 wt. %, or from about 1 wt.% to about 4, 5, 6, 7, 8, 10, 15 wt.%, and
preferably between about 1.5 wt. % to 8.0 wt. %.
[0094] Other optional additives in the present formulations include secondary enhancers, i.e.,
one or more additional transport enhancers. For example, formulation of the invention can
contain added DMSO. Since MSM is a metabolite of DMSO (i.e., DMSO is enzymatically
converted to MSM), incorporating DMSO into an MSM-containing formulation of the
invention will tend to gradually increase the fraction of MSM in the formulation. DMSO also
serves as a free radical scavenger, thereby reducing the potential for oxidative damage. If
DMSO is added as a secondary enhancer, the amount is preferably in the range of about 1.0
wt. % to 2.0 wt. % of the formulation, and the weight ratio of MSM to DMSO is typically in
the range of about 1:50 to about 50:1.
Formulations
[0095] A variety of means can be used to formulate the compositions of the invention.
Techniques for formulation and administration may be found in "Remington: The Science
and Practice of Pharmacy," Twentieth Edition, Lippincott Williams & Wilkins, Philadelphia,
PA (1995). For human or animal administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards comparable to those required by the FDA.
Administration of the pharmaceutical formulation can be performed in a variety of ways, as
described herein.
[0096] Other possible additives for incorporation into the formulations that are at least
partially aqueous include, without limitation, thickeners, isotonic agents, buffering agents,
and preservatives, providing that any such excipients do not interact in an adverse manner
with any of the formulation's other components. It should also be noted that preservatives are
not generally necessarily in light of the fact that the selected chelating agent itself serves as a
preservative. Suitable thickeners will be known to those of ordinary skill in the art of
formulation, and include, by way of example, cellulosic polymers such as methylcellulose
(MC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose
(HPMC), and sodium carboxymethylcellulose (NaCMC), and other swellable
hydrophilic polymers such as polyvinyl alcohol (PVA), hyaluronic acid or a salt thereof (e.g.,
sodium hyaluronate), and crosslinked acrylic acid polymers commonly referred to as
"carbomers" (and available from B.F. Goodrich as Carbopol® polymers). Various organic
gums such as but not limited to Xanthan gum and Konjac gum. The preferred amount of any
thickener is such that a viscosity above 10,000 cps is provided, as a gel having a viscosity
above this figure generally considered optimal for both comfort and retention of the
formulation on the oral tissues. Any suitable isotonic agents and buffering agents commonly
used in oral formulations may be used, providing the pH of the formulation is maintained in
the range of about 4.5 to about 9.0, preferably in the range of about 6.8 to about 7.8, and
optimally at a pH of about 7.4.
[0097] The formulations of the invention also include a pharmaceutically acceptable carrier,
which will depend on the particular type of formulation. For example, the formulations of the
invention can be provided as a solution, suspension, paste or gel, in which case the carrier is
at least partially aqueous. The formulations may also be ointments, in which case the
pharmaceutically acceptable carrier is composed of an ointment base. Preferred ointment
bases herein have a melting or softening point close to body temperature, and any ointment
bases commonly used in oral preparations may be advantageously employed. Common
ointment bases include petrolatum and mixtures of petrolatum and mineral oil.
[0098] The pharmaceutical formulation may be a solid, semi-solid or liquid, such as, for
example, a liquid, a cream, a suspension, an emulsion, beads, a powder, or the like,
preferably in unit dosage form suitable for single administration of a precise dosage. Suitable
pharmaceutical formulations and dosage forms may be prepared using conventional methods
known to those in the field of pharmaceutical formulation and described in the pertinent texts
and literature, e.g., in Remington: The Science and Practice of Pharmacy, cited previously
herein.
[0099] The formulations of the invention may also be prepared as a hydrogel, dispersion, or
colloidal suspension. Hydrogels are formed by incorporation of a swellable, gel-forming
polymer such as those set forth above as suitable thickening agents (i.e., MC, HEC, HPC,
HPMC, NaCMC, PVA, or hyaluronic acid or a salt thereof, e.g., sodium hyaluronate), except
that a formulation referred to in the art as a "hydrogel" typically has a higher viscosity than a
formulation referred to as a "thickened" solution or suspension. In contrast to such preformed
hydrogels, a formulation may also be prepared so as to form a hydrogel in situ following
application into the oral cavity. Such gels are liquid at room temperature but gel at higher
temperatures (and thus termed "thermoreversible" hydrogels), such as when placed in contact
with body fluids. Biocompatible polymers that impart this property include acrylic acid
polymers and copolymers, N-isopropylacrylamide derivatives, and ABA block copolymers of
ethylene oxide and propylene oxide (conventionally referred to as "poloxamers" and available
under the Pluronic® trade name from BASF-Wyandotte). The formulations can also be
prepared in the form of a dispersion or colloidal suspension. Preferred dispersions are
liposomal, in which case the formulation is enclosed within "liposomes," microscopic
vesicles composed of alternating aqueous compartments and lipid bilayers. Colloidal
suspensions are generally formed from microparticles, i.e., from microspheres, nanospheres,
microcapsules, or nanocapsules, wherein microspheres and nanospheres are generally
monolithic particles of a polymer matrix in which the formulation is trapped, adsorbed, or
otherwise contained, while with microcapsules and nanocapsules, the formulation is actually
encapsulated. The upper limit for the size for these microparticles is about 5m to about 10m.
[0100] The formulations may also be incorporated into a sterile insert that provides for
controlled release of the formulation over an extended time period, generally in the range of
about 1 hours to 60 days, and possibly up to 12 months or more, following implantation of
the insert into any tissue. One type of insert is an implant in the form of a monolithic polymer
matrix that gradually releases the formulation to the oral tissues through diffusion and/or
matrix degradation. With such an insert, it is preferred that the polymer be completely soluble
and or biodegradable (i.e., physically or enzymatically eroded in the tissues) so that removal
of the insert is unnecessary. These types of inserts are well known in the art, and are typically
composed of a water-swellable, gel-forming polymer such as collagen, polyvinyl alcohol, or
a cellulosic polymer. Another type of insert that can be used to deliver the present
formulation is a diffusional implant in which the formulation is contained in a central
reservoir enclosed within a permeable polymer membrane that allows for gradual diffusion of
the formulation out of the implant. Osmotic inserts may also be used, i.e., implants in which
the formulation is released as a result of an increase in osmotic pressure within the implant
following application to the oral tissue and subsequent absorption.
[0101] The chelating agent may be administered, if desired, in the form of a salt, ester,
crystalline form, hydrate, or the like, provided it is pharmaceutically acceptable. Salts, esters,
etc. may be prepared using standard procedures known to those skilled in the art of synthetic
organic chemistry and described, for example, by J . March, Advanced Organic Chemistry:
Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley- Interscience, 1992).
[0102] The amount of chelating agent administered will depend on a number of factors and
will vary from subject to subject and depend on the particular chelating agent, the particular
disorder or condition being treated, the severity of the symptoms, the subject's age, weight
and general condition, and the judgment of the prescribing physician. The term "dosage
form" denotes any form of a pharmaceutical composition that contains an amount of
chelating agent and transport enhancer sufficient to achieve a therapeutic effect with a single
administration or multiple administrations. The frequency of administration that will provide
the most effective results in an efficient manner without overdosing will vary with the
characteristics of the particular active agent, including both its pharmacological
characteristics and its physical characteristics, such as hydrophilicity.
[0103] The formulations may also include conventional additives such as opacifiers,
flavoring agents, antioxidants, fragrance, colorant, gelling agents, thickening agents,
stabilizers, surfactants, and the like. Other agents may also be added, such as antimicrobial
agents, to prevent spoilage upon storage, i.e., to inhibit growth of microbes such as yeasts and
molds. Suitable antimicrobial agents are typically selected from the methyl and propyl esters
of p-hydroxybenzoic acid (i.e., methyl and propyl paraben), sodium benzoate, sorbic acid,
imidurea, and combinations thereof.
[0104] The active formulation of the invention can be formulated in combination with one
or more pharmaceutically-acceptable anti-microbial agents. In this regard, combinations of
different antimicrobial agents may be tailored to target different (or the same)
microorganisms that contribute towards morbidity and mortality. The pharmaceutically
acceptable anti-microbial agents of the present invention are suitable for internal
administration to an animal, for example, a human. However, if the formulation is to be used
in industrial sterilizing, sterilizing chemicals such as detergents, disinfectants, and
ammonium-based chemicals (e.g. quaternary ammonium compounds such as QUATAL) can
be used in combination with, or prior to or after the treatment with the formulation. Such
sterilizing chemicals are typically used in the art for sterilizing industrial work surfaces (e.g.
in food processing, or hospital environments), and are not suitable for administration to an
animal.
[0105] The invention further contemplates preparations, formulations, coatings, films, oils,
and composite materials that contain the formulation of the invention. Such materials are
useful in many varied industrial and medical applications. Industrial applications include
marine applications such as fouling-release treatments for surfaces of ships and boats such as
the hull, offshore marine structures such as oil rigs, sea water conduit systems for seaside
plants, buoys, heat exchangers, cooling towers, desalination equipment, filtration membranes,
docks, aquatic zoo and aquarium and other structures which may all experience some degree
of fouling when continually exposed to fresh or salt water. Medical applications include use
as treatments for devices, including implantable devices, such as tubing, catheters, stents,
vascular implants, cardiac regulation devices, and other devices that come into contact with
body fluids.
[0106] The formulation can be incorporated into any cleaning agent. If EDTA or some
other chelator is already present in the cleaning agent then only the transport enhancer (e.g.,
MSM) need to be included. If not, a chelator and a transport enhancer (MSM/EDTA) are
added.
[0107] Methods of using the formulation of the invention are also included. The
formulation can be used as a cleaning agent for specific medical devices such as a contact
lens. Devices such as tubes (e.g., intra-venous tubing) and catheters may be treated with the
formulation by rinsing the interior surface for a period of time and a number of applications
that are found suitable for removal of a desired level of biofilm. Tubes intended for industrial
use can also be rinsed or otherwise treated with the formulation for removal of biofilm, or
prevention of formation of biofilm.
[0108] The formulation can be used as a cleaning agent generally as a wipe. A wipe
according to the invention may comprise a fabric suitable for wiping a surface wherein the
wipe is pre-soaked with a lotion containing the formulation. Alternately, the formulation can
be applied directly (e.g., by spraying, pouring, etc.) followed by wiping.
[0109] In some embodiments, the formulation may be applied to a surface susceptible to
biofilm formation. The formulations can be applied to coat or form surfaces of articles used
in industrial, marine, and medical applications.
[0110] The treatment regimen will depend on a number of factors that may readily be
determined, such as severity of the condition and responsiveness of the microbial infection to
be treated, but will normally be one or more treatments per day, with a course of treatment
lasting from a day or several days to several months, or until a significant reduction of
biofilm is achieved.
[0111] The compositions of the invention may further include additional drugs or
excipients as appropriate for the indication. In one aspect of the embodiment, the
pharmaceutical composition further comprises a therapeutically effective amount of at least
one antimicrobial agent. In a more specific aspect, the antimicrobial agent is an antibiotic.
[0112] In another embodiment, the present invention provides a method for inhibiting
formation of a biofilm on a device, the method comprising contacting the bacteria with an
effective amount of a formulation comprising a transport enhancer (such as MSM) and a
chelating agent (such as EDTA), whereby formation of a biofilm on the device is inhibited.
The composition may be, for example, a spray, lotion, solution, gel, cream, ointment, surgical
rinse, or dental rinse. In another embodiment, the composition may be a device-soaking
solution, a personal cleaning composition, or a hard surface cleaning composition. In such
compositions, the proportion of the EDTA to MSM is in the range of about 1:100- 100:1, and
the percentages of EDTA and MSM in the composition are in the ranges of about 0.1% to
15% and about 0.1% to 40%> by weight, respectively.
[0113] In another embodiment, the present invention provides a topical pharmaceutical
composition for inhibiting formation of a biofilm on or within a mammal, comprising an
effective amount of a formulation comprising a transport enhancer (such as MSM) and a
chelating agent (such as EDTA, tetrasodium salt of iminodisuccinic acid, poly-asparatic acid
and/or salts thereof, or tetra sodium salts of L-glutamic acid N,N-diacetic acid (GLDA), and
at least one pharmaceutically acceptable carrier.
[0114] In a further embodiment, the present invention provides a surgical rinse for
inhibiting formation of a biofilm comprising bacteria, wherein the surgical rinse comprises an
effective amount of a formulation comprising a transport enhancer (such as MSM) and a
chelating agent (such as EDTA). In one embodiment, the surgical rinse may be, for example,
a buffered saline solution or a Ringer's solution. A surgical rinse of the present invention may
be applied before, during, or after surgery and may be aspirated from the surgical area or left
on the surgical area to inhibit biofilm formation
[0115] In yet another embodiment, the present invention provides a method for inhibiting
formation of a biofilm comprising bacteria, the method comprising contacting the bacteria
with an effective amount of a formulation comprising a transport enhancer (such as MSM)
and a chelating agent (such as EDTA), wherein the bacteria are selected from the group
consisting of Acidothermus cellulyticus, Actinomyces odontolyticus, Alkaliphilus
metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus
amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus licheniformis, Bacillus
pumilus, Bacillus subtilis, Bifidobacterium adolescentis, Bifidiobacterium longum,
Caldicellulosiruptor saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium
acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium cellulolyticum,
Clostridium difficile, Clostridium kluyveri, Clostridium leptum, Clostridium novyi,
Clostridium perfringens, Clostridium tetani, Clostridium thermocellum, Corynebacterium
diphtheriae, Corynebacterium efificiens, Corynebacterium glutamicum, Corynebacterium
jeikeium, Corynebacterium urealyticum, Desulfitobacterium hafniense, Desulfotomaculum
reducens, Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna,
Geobacillus kaustophilus, Geobacillus thermodenitrificans, Janibacter sp., Kineococcus
radiotolerans, Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua, Listeria
welshimeri, Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium paratuberculosis,
Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium ulcerans,
Mycobacterium vanbaalenii, Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora erythraea,
coagulase-negative Staphylococcus species, Staphylococcus aureus, methicillin resistant
Staphylococcus aureus (MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus agalactiae, Streptococcus gordonii,
Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus
sanguinis, Streptococcus suis, Streptomyces avermitilis, Streptomyces coelicolor,
Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis, and combinations
thereof, whereby formation of the biofilm is inhibited.
[0116] A further embodiment of the present invention provides bandages, sponges or
gauzes impregnated with a safe and effective amount of a formulation comprising a transport
enhancer (such as MSM) and a chelating agent (such as EDTA), wherein the bandage, sponge
or gauze inhibits the formation of a biofilm on the skin. In one embodiment, the bandage,
sponge or gauze is suitable for use in patients with cuts, burns, turf burns, abrasions,
lacerations, puncture wounds, regions of bacterial infection such as boils and pustules, and
the like.
[0117] Yet another embodiment of the present invention provides a personal cleansing
formulation comprising a transport enhancer (such as MSM) and a chelating agent including
but not limited to, EDTA and salts thereof, or tetrasodium salt of iminodisuccinic acid, or
salts of poly-asparatic acid, or tetra sodium salts of L-glutamic acid N,N-diacetic acid
(GLDA).), wherein the personal cleansing composition inhibits formation of a biofilm on the
skin. Suitable personal cleansing compositions include, but are not limited to, surgical scrubs,
shower gels, body washes, soaps, deodorants, and the like. In another embodiment of the
invention, the personal cleansing composition is applied as a part of a personal hygiene
routine. Personal cleansing compositions of the present invention are suitable for use by a
variety of individuals, including, for example, people recovering from Staph infections,
athletes using team locker rooms, and healthcare professionals.
[0118] A further embodiment of the present invention provides a hard surface cleaning
formulation comprising a transport enhancer (such as MSM) and a chelating agent (such as
EDTA), wherein the composition inhibits formation of a biofilm on a hard surface. The
present hard surface cleaning composition has a variety of useful applications, including use
in industrial applications as well as medical, veterinary, or livestock environments. For
example, hard surface cleaners of the present invention are useful in the cleaning and treating
of pipeline systems, cooling water systems in power plants, refineries, chemical plants, air
conditioning systems, storage tanks, trays, containers, walls, floors, countertops, locker room
floors, benches, lockers, showers, bathrooms, toilets, water filtration units, and the like, as
part of a standard cleaning routine.
[0119] A further embodiment of the present invention provides a dental rinse for inhibiting
formation of a biofilm, the dental rinse comprising a formulation comprising a transport
enhancer (such as MSM) and a chelating agent (such as EDTA).
[0120] For topical administration to the epidermis, a formulation comprising a transport
enhancer (such as MSM) and a chelating agent (such as EDTA) of the present invention may
be formulated in an ointment, cream, or lotion, or as a transdermal patch. Ointments and
creams, may, for example, be formulated with an aqueous or oily base with the addition of
suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily
base and will in general also contain one or more emulsifying agents, stabilizing agents,
suspending agents, thickening agents, or coloring agents. Formulations suitable for topical
administration in the mouth include lozenges comprising a formulation comprising a
transport enhancer (such as MSM) and a chelating agent (such as EDTA) in a flavored base,
usually sucrose and acacia or tragacanth; pastilles comprising the active ingredients in an
inert base such as gelatin and glycerin or sucrose and acacia; and mouth washes comprising
the active ingredients in a suitable liquid carrier. For topical administration to the eye, the
formulation comprising a transport enhancer (such as MSM) and a chelating agent (such as
EDTA) can be made up in solution or suspension in a suitable sterile aqueous or non-aqueous
vehicle. Additives such as buffers (e.g. sodium metabisulphite or disodium edeate) and
thickening agents such as hypromellose can also be included.
[0121] For intra-nasal administration, a formulation comprising a transport enhancer (such
as MSM) and a chelating agent (such as EDTA) of the present invention can be provide in a
liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an
aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing
agents, or suspending agents. Liquid sprays are conveniently delivered from pressurized
packs.
[0122] For administration by inhalation, a formulation comprising a transport enhancer
(such as MSM) and a chelating agent (such as EDTA) of the present invention can be
delivered by insufflator, nebulizer or a pressurized pack or other convenient means of
delivering the aerosol spray. Pressurized packs may comprise a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount.
[0123] In another embodiment of the present invention, wound dressings including but not
limited to sponges or gauzes can be impregnated with a formulation comprising a transport
enhancer (such as MSM) and a chelating agent (such as EDTA) to prevent or inhibit
bacterial or fungal attachment and reduce the risk of wound infections. Similarly, catheter
shields as well as other materials used to cover a catheter insertion sites can be coated or
impregnated with a formulation comprising a transport enhancer (such as MSM) and a
chelating agent (such as EDTA) to inhibit bacterial or fungal biofilm attachment thereto.
Adhesive drapes used to prevent wound infection during high risk surgeries can be
impregnated with the isolated protein or active fragment or variant thereof as well. Additional
medical devices which can be coated with a formulation comprising a transport enhancer
(such as MSM) and a chelating agent (such as EDTA) include, but are not limited, central
venous catheters, intravascular catheters, urinary catheters, Hickman catheters, peritoneal
dialysis catheters, endrotracheal catheters, mechanical heart valves, cardiac pacemakers,
arteriovenous shunts, schleral buckles, prosthetic joints, tympanostomy tubes, tracheostomy
tubes, voice prosthetics, penile prosthetics, artificial urinary sphincters, synthetic pubovaginal
slings, surgical sutures, bone anchors, bone screws, intraocular lenses, contact lenses,
intrauterine devices, aortofemoral grafts and vascular grafts. Exemplary solutions for
impregnating gauzes or sponges, catheter shields and adhesive drapes or coating catheter
shields and other medical devices include, but are not limited to, phosphate buffered saline
(pH approximately 7.5) and bicarbonate buffer (pH approximately 9.0).
[0124] In yet another embodiment, a formulation comprising a transport enhancer (such as
MSM) and a chelating agent (such as EDTA) can be incorporated in a liquid disinfecting
solution. Such solutions may further comprise antimicrobials or antifungals such as alcohol,
providone-iodine solution and antibiotics as well as preservatives. These solutions can be
used, for example, as disinfectants of the skin or surrounding area prior to insertion or
implantation of a device such as a catheter, as catheter lock and/or flush solutions, and as
antiseptic rinses for any medical device including, but not limited to catheter components
such as needles, Leur-Lok connectors, needleless connectors and hubs as well as other
implantable devices. These solutions can also be used to coat or disinfect surgical instruments
including, but not limited to, clamps, forceps, scissors, skin hooks, tubing, needles, retractors,
scalers, drills, chisels, rasps and saws.
EXAMPLES
[0125] The following examples are put forth so as to provide those skilled in the art with a
complete invention and description of how to make and use embodiments in accordance with
the invention, and are not intended to limit the scope of what the inventors regard as their
discovery. Efforts have been made to ensure accuracy with respect to numbers used (e.g.
amounts, temperature, etc.) but some experimental errors and deviations should be accounted
for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.
Example 1: Reduction of Plaque development after single brushing
[0126] Toothpaste was prepared comprising EDTA (tetrasodium salt) and MSM, which
were purchased from Sigma. Control was a leading "anti-plaque" toothpaste available in the
market.
[0127] The MSM/EDTA toothpaste showed 106% greater reduction in plaque. This
showed a high degree of statistical significance.
Example 2 : Reduction of Plaque development after repeated brushings
[0128] Toothpaste was prepared comprising EDTA (tetrasodium salt) and MSM, which
were purchased from Sigma. Control was a leading "anti-plaque" toothpaste available in the
market. Loe Sillness dental plaque index was measured on subjects after 2 weeks of twice
daily brushing, after an initial prophylaxis.
5.0% MSM / 2.0%sodium citrate gel 0.95
5.0% MSM / 2.0% sodium phytate gel 1.18
[0129] The MSM+chelator results compared to control showed extremely high statistical
significance.
Example 3 : 96-Well Microtiter Plate Biofilm Cell Detachment Assay
[0130] The wells of a 96-well microtiter plate (Falcon no. 353072) were filled with 100 mΐ
of medium containing 102 to 104 CFU of bacteria and incubated at 37 °C in 10% C0 2 for 20
hours. Ten mΐ of enzyme solution [ 1 mg/ml in phosphate buffered saline (PBS)], or 10 mΐ of
PBS in the case of controls, was added to each well and the plates were incubated for an
additional 6 hours. The wells were washed extensively under running tap water and the
bacteria remaining attached to the surface were stained with crystal violet, rewashed, and
destained with ethanol in accordance with procedures described by Kachlany et al. Mol.
Microbiol. 2001 40:542-554). The optical density (O.D.) of the ethanol-dye solution was
measured in a BioRad Benchmark microtiter plate reader set to 590 nm.
Example 4 : Effect of the formulation on underarm polymicrobial biofilms that lead to
underarm body odor.
[0131] A relatively benign polymicrobial infection called Trichomichosis axillaris infests
the underarm skin and hair of a large number of people. This infestation produces unsightly
deposits on axillary hair as shown in the figure below. This infestation also leads to an
intensely unpleasant body odor.
[0132] The standard treatment for this condition is to shave the affected area, and then to
apply antimicrobial creams and lotions, for example benzoyl peroxide or erythromycin cream
— this treatment modality often results in irritation and contact dermatitis. The average time
for resolution of the disease has been characterized as 3 weeks (Kim Comparative Study of
Benzoyl Peroxide Versus Erythromycin in Trichomycosis Axillaris and Pubis, Korean J Med
Mycol. 2005 Jun; 10(2):70-75.). A gel of MSM/EDTA of the formulation was applied once a
day in five patients with Trichomichosis axillaris. The infection in each case disappeared
within three days. At the same time, all underarm malodor in the patients resolved, and no
patient showed any irritation or contact dermatitis. A picture of the before and after of one
patient is shown in Figures la- lb.
Example 4 : Effect of the formulation on cat bite infections.
[0133] 20 to 80% of all cat bite wounds get infected with a polymicrobial infection within
24 hours, with the first signs appearing after 12 hours. Treatment with traditional
antimicrobials is often prolonged, and it takes weeks to months to heal the badly infected
wounds. The infection occurs because remnants of cat dental plaque get left behind in the
wound and cannot be cleaned by traditional wound cleaning methods. (A. Freshwater, Why
Your Housecat's Trite Little Bite Could Cause You Quite a Fright: A Study of Domestic
Felines on the Occurrence and Antibiotic Susceptibility of Pasteurella multocida, Journal
compilation 2008 Blackwell Verlag Zoonoses Public Health. 55 (2008) 507-513; J . SiUery; et
al, Pasteurella multocida Peritonitis: Another Risk of Animal-Assisted Therapy, Source:
Infection Control and Hospital Epidemiology, Vol. 25, No. 1 (January 2004), pp. 5-6,
Published by: The University of Chicago Press on behalf of The Society for Healthcare
Epidemiology of America; Itzhak Brook, et al, Animal bite-associated infections:
microbiology and treatment, Expert Review of Anti-Infective therapy, 9.2 (feb 201 1): p215).
Over a hundred cat bite wounds were treated with one of a lotion or a gel comprising of
MSM and EDTA within an hour of injury. None of the wounds were infected, and all wounds
healed within three days.
[0134] All publications and patent applications cited in this specification are herein
incorporated by reference as if each individual publication or patent application were
specifically and individually indicated to be incorporated by reference.
[0135] Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be readily apparent to
those of ordinary skill in the art in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the spirit or scope of the
appended claim.

CLAIMS
What is claimed is:
1. An antimicrobial formulation, comprising:
a chelating agent or salts thereof;
a transport enhancer; and
an acceptable vehicle or base for such composition;
wherein the chelating agent and the transport enhancer are present in a proportion
effective to bring about a significant reduction in bacterial and/or fungal biofilm on a surface to
which it is applied, and
wherein the percentage of chelator is about 0.1% to 15% and the percentage of transport
in the composition is about 0.1% to 40% by weight, respectively..
2. The formulation of claim 1, wherein the transport enhancer is MSM.
3. The formulation of claim 1, wherein the transport enhancer is DMSO.
4. The formulation of claim 2, wherein the proportion of the chelator to MSM is in the range of
about 10:1-1:20.
5. The formulation of claim 1, wherein the composition comprises a formulation selected from
solid, liquid, inhalant, spray, lotion, solution, gel, cream, ointment, surgical rinse, or dental rinse.
6. The formulation of claim 1, wherein the chelating agent is selected from ethylenediamine t et
raacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), cyclohexanediamine tetraacetic
acid (CDTA), hydroxyethylethylenediamine triacetic acid (HEDTA), diethylenetriamine
pentaacetic acid (DTPA), dimercaptopropane sulfonic acid (DMPS), dimercaptosuccinic acid
(DMSA), aminotrimethylene phosphonic acid (ArPA), citric acid, acetic acid and acceptable
salts thereof, and any combinations thereof.
7. The formulation of claim 7, wherein the EDTA salt is selected from diammonium EDTA,
disodium EDTA, dipotassium EDTA, triammonium EDTA, trisodium EDTA, tripotassium
EDTA, tetrasodium EDTA, tetrapotassium EDTA, calcium disodium EDTA, and combinations
thereof.
8. The formulation of claim 1, wherein the chelating agent is selected from phosphates,
pyrophosphates, tripolyphosphates, and hexametaphosphates.
9. The formulation of claim 1, wherein the chelating agent is a chelating antibiotic, chloroquine
or tetracycline.
10. The formulation of claim 1, wherein the chelating agent is a nitrogen-containing chelating
agents containing two or more chelating nitrogen atoms within an imino group or in an aromatic
ring, diimines, or 2,2'-bipyridines.
11. The formulation of claim 1, wherein the chelating agent is a polyamine selected from cyclam
(1,4,7,1 1-tetraazacyclotetradecane), N-(Ci-C o alkyl)-substituted cyclams (e.g., hexadecyclam,
tetramethylhexadecylcyclam), diethylenetriamine (DETA), spermine, diethylnorspermine
(DENSPM), diethylhomo-spermine (DEHOP), deferoxamine (N'-{5-
[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-
oxobutanoyl} amino)pentyl]-N-hydroxysuccinamide, or N'-[5-(Acetyl-hydroxy-amino)pentyl]-N-
[5-[3-(5-aminopentyl-hydroxy-carbamoyl) propanoylamino]pentyl]-N-hydroxy-butane diamide),
desferrioxamine B, desferoxamine B, DFO-B, DFOA, DFB, desferal, deferiprone, pyridoxal
isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH), ethane- l,2-bis(N-
1-amino-3-ethylbutyl-3-thiol).
12. The formulation of claim 1, wherein the chelating agent is a EDTA-4-aminoquinoline
conjugate selected from ([2-(Bis-ethoxycarbonylmethyl-amino)-ethyl]-{[2-(7-chloro-quinolin-4-
ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic acid ethyl ester, ([2-(Bisethoxycarbonylmethyl-
amino)-propyl]-{[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-
methyl}-amino)-acetic acid ethyl ester, ([3-(Bis-ethoxycarbonylmethyl-amino)-propyl]-{[2-(7-
chloro-quinolin-4-ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic acid ethyl ester, ([4-(Bisethoxycarbonylmethyl-
amino)-butyl]-{[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-
methyl}-amino)-acetic acid ethyl ester, ([2-(Bis-ethoxymethyl-amino)-ethyl]-{[2-(7-chloroquinolin-
4-ylamino)-ethylcarbamoyl]-methyl}-amino)-acetic acid ethyl ester, ([2-(Bisethoxymethyl-
amino)-propyl]-{[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-methyl}-
amino)-acetic acid ethyl ester, ([3-(Bis-ethoxymethyl-amino)-propyl]-{[2-(7-chloro-quinolin-4-
ylamino)-ethylcarbamoyl] -methyl} -amino)-acetic acid ethyl ester, ([4-(Bis-ethoxymethylamino)-
butyl]- {[2-(7-chloro-quinolin-4-ylamino)-ethylcarbamoyl]-methyl} -amino)-acetic acid
ethyl ester.
13. The formulation of claim 1, wherein the chelating agent is a tetrasodium salt of
iminodisuccinic acid.
14. The formulation of claim 1, wherein the chelating agent is poly-asparatic acid or a salt
thereof.
15. The formulation of claim 1, wherein the chelating agent is a tetra sodium salt of L-glutamic
acid N,N-diacetic acid.
16. The formulation of claim 1, wherein the chelating agent is a natural chelator selected from
citric acid, phytic acid, lactic acid, acetic acid and their salts and curcumin.
17. The formulation of claim 1, further comprising an antibiotic agent.
18. The formulation of claim 17, wherein the antibiotic is antibacterial or antifungal.
19. The formulation of claim 1, wherein the formulation is for oral administration.
20. The formulation of claim 19, wherein the formulation is a lozenge or a mouth wash.
2 1. The formulation of claim 1, wherein the formulation is for parenteral administration.
22. The formulation of claim 1, wherein the formulation is for topical administration.
23. The formulation of claim 1, wherein the formulation is an ointment, cream, or lotion.
24. The formulation of claim 1, wherein the formulation is for intra-nasal administration.
25. The formulation of claim 1, wherein the formulation is for timed release.
26. A method for inhibiting a biofilm associated bacterial infection comprising administering the
formulation of claim 1 in combination with or prior to administration of an antibiotic.
27. A medical device coated with the formulation of claim 1.
28. The medical device of claim 27, wherein the medical device is an implantable device.
29. The medical device of claim 28, wherein the medical device is selected from the group
consisting of a central venous catheter, an intravascular catheter, an urinary catheter, a Hickman
catheter, a peritoneal dialysis catheter, an endrotracheal catheter, a mechanical heart valve, a
cardiac pacemaker, an arteriovenous shunt, a schleral buckle, a prosthetic joint, a tympanostomy
tube, a tracheostomy tube, a voice prosthetic, a penile prosthetic, an artificial urinary sphincter, a
synthetic pubovaginal sling, a surgical suture, a bone anchor, a bone screw, an intraocular lens, a
contact lens, an intrauterine device, an aortofemoral graft, and a vascular graft.
30. The medical device of claim 30, wherein the medical device is a surgical instrument.
3 1. The medical device of claim 30, wherein the surgical instrument is a clamp, forceps, scissor,
skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, or saw.
32. A wound dressing impregnated with the formulation of claim 1.
33. The wound dressing of claim 32, wherein the wound dressing is a sponge, gauze, or catheter
shield.
34. A transdermal patch comprising the formulation of claim 1.
35. A method for promoting detachment of bacterial or fungal cells from a biofilm comprising
contacting bacterial cells with the formulation of claim 1.
36. A method of inhibiting infection on a medical device or surgical instrument by bacteria or
fungi comprising contacting the medical device or surgical instrument with the formulation of
claim 1.
37. A method of inhibiting infection on a medical device or surgical instrument by bacteria or
fungi comprising coating the medical device or surgical instrument with the formulation of claim
1.
38. A method of inhibiting infection on a medical device or surgical instrument by bacteria or
fungi comprising bathing the medical device or surgical instrument in a solution comprising the
formulation of claim 1.
39. A method of inhibiting or treating bacterial or fungal infections comprising administering a
pharmaceutically acceptable composition comprising the formulation of claim 1.
40. The method of claim 39 wherein the bacteria is selected from the group consisting of
Acidothermus cellulyticus, Actinomyces odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus
oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus
halodurans, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bifidobacterium
adolescentis, Bifidiobacterium longum, Caldicellulosiruptor saccharolyticus, Carboxydothermus
hydrogenoformans, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium
botulinum, Clostridium cellulolyticum, Clostridium difficile, Clostridium kluyveri, Clostridium
leptum, Clostridium novyi, Clostridium perfringens, Clostridium tetani, Clostridium
thermocellum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium
glutamicum, Corynebacterium jeikeium, Corynebacterium urealyticum, Desulfitobacterium
hafniense, Desulfotomaculum reducens, Eubacterium ventriosum, Exiguobacterium sibiricum,
Fingoldia magna, Geobacillus kaustophilus, Geobacillus thermodenitrificans, Janibacter sp.,
Kineococcus radiotolerans, Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua,
Listeria welshimeri, Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium paratuberculosis,
Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium ulcerans,
Mycobacterium vanbaalenii, Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora erythraea, coagulasenegative
Staphylococcus species, Staphylococcus aureus, methicillin resistant Staphylococcus
aureus (MRSA), Staphylococcus epidermidis, methicillin resistant Staphylococcus epidermidis
(MRSE), Streptococcus agalactiae, Streptococcus gordonii, Streptococcus mitis, Streptococcus
oralis, Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus suis, Streptomyces
avermitilis, Streptomyces coelicolor, Thermoanaerobacter ethanolicus, Thermoanaerobacter
tengcongensis, and combinations thereof.
4 1. The method of claim 39 wherein the bacteria is a gram-positive bacteria.
42. A bandage impregnated with a safe and effective amount of the formulation of claim 1,
wherein the bandage inhibits the formation of a biofilm on the skin.
43.A personal cleansing composition comprising an effective amount of the formulation of
claim 1, wherein the personal cleansing composition inhibits formation of a biofilm on the skin.
44. The personal cleansing composition of claim 43, wherein the composition is a surgical scrub,
shower gel, body wash, or soap.
45. A hard surface cleaning composition comprising an effective amount of the formulation of
claim 1, wherein the composition inhibits formation of a biofilm on the hard surface.
46. A dental rinse for inhibiting formation of a biofilm, the dental rinse comprising an effective
amount of the formulation of claim 1.
47. The formulation of claim 1, wherein the formulation is suitable for application to a surface,
the formulation comprising:
about 0.4-15% of chelator;
about 0.5-30% of MSM;
one or more thickening and gelling agents;
10-99% water; and
optionally contains surfactants, detergents and/or soaps.
48. The formulation of claim 47, comprising:
about1-5% of chelator;
about 1-10% of MSM;
0.1-6% of one or more thickening agents; and
80-97% water.
49. The formulation of claim 1, wherein the formulation is suitable for oral application, the
formulation comprising:
about 0.4-8% of a chelator; and
about 0.5-16% of MSM.