Note: Descriptions are shown in the official language in which they were submitted.
CA 2788669 2017-03-14
1
BISMUTH-THIOLS AS ANTISEPTICS FOR BIOMEDICAL USES, INCLUDING
TREATMENT OF BACTERIAL BIOFILMS AND OTHER USES
BACKGROUND
Technical field
The presently disclosed invention embodiments relate to
compositions and methods for the treatment of microbial infections. In
particular, the present embodiments relate to improved treatments for
managing bacterial infections in epithelial tissues, including in wounds such
as
chronic wounds and acute wounds, and in clinical, personal healthcare, and
other contexts, including treatment of bacterial biofilms and other
conditions.
Description of the Related Art
The complex series of coordinated cellular and molecular
interactions that contribute to skin wound healing and responding to and
resisting microbial infections and/or to healing or maintenance of bodily
tissues
generally, may be adversely impacted by a variety of external factors, such as
opportunistic and nosocomial infections (e.g., clinical regimens that can
increase the risk of infection), local or systemic administration of
antibiotics
(which may influence cell growth, migration or other functions and can also
select for antibiotic-resistant microbes), frequent wound dressing changes,
open-air exposure of wounds to speed healing, the use of temporary artificial
structural support matrix or scaffold materials, the possible need for
debridement and/or repeat surgery to excise infected or necrotic tissue and/or
other factors.
Wound healing thus continues to be a formidable challenge for
clinical practitioners worldwide. The current treatments for recalcitrant
wounds
are impractical and ineffective, often requiring multiple surgeries to close
the
wound. For instance, Regranex (becaplermin, Ortho-McNeil Pharmaceutical,
Inc., available from Ethicon, Inc., recombinant platelet-derived growth
factor)
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
2
exemplifies one of the few available treatments for chronic wounds, but is
expensive to produce and has limited clinical utility.
Chronic and Acute Wounds and Wound Biofilms
Wounds occur when the continuity between cells within a tissue,
or between tissues, is disrupted, for instance, by physical, mechanical,
biological, pathological and/or chemical forces (e.g., burns, dermal
infections,
puncture wounds, gunshot or shrapnel wounds, skin ulcers, radiation poisoning,
malignancies, gangrene, autoimmune disease, immunodeficiency disease,
respiratory insult such as by inhalation or infection, gastrointestinal insult
such
as by deleterious ingestion or infection, circulatory and hematologic
disorders
including clotting defects,) or other traumatic injuries, or the like.
While a limited level of bacterial contamination in a wound, or
"colonization" of the wound, may not necessarily interfere with the processes
of
wound healing, the presence of bacteria in numbers sufficient to overwhelm the
host immune defenses can lead to an acute wound or a chronic wound or a
wound in which a bacterial biofilm is present, such as a wound infection in
which bacterial growth proceeds to the detriment of the host. Bryant and Nix,
Acute and Chronic Wounds: Current Management Concepts, 2006 Mosby
(Elsevier), NY; Baronoski, Wound Care Essentials: Practical Principles (2nd
Ed.), 2007 Lippincott, Williams and Wilkins, Philadelphia, PA). For example,
acute wounds such as may result from injury, trauma, surgical intervention, or
other causes, typically lack underlying health deficits and heal rapidly, but
may
on occasion fail to do so due to the presence of an infection; rapidly forming
bacterial biofilms have been described in acute wounds (e.g.,
WO/2007/061942). Additional factors that may contribute to the development
of chronic wounds include losses in mobility (e.g., that result in continued
pressure being applied to a wound site), deficits of sensation or mental
ability,
inaccessibility of the wound site (e.g., in the respiratory or
gastrointestinal
tracts) and circulatory deficits. Infection at a chronic wound site may be
.. detected by the clinical signs of skin redness, edema, pus formation and/or
unpleasant odor, or other relevant, clinically accepted criteria.
Acute wounds that cannot heal properly may thus be present, and
chronic wounds thus may develop, in higher organisms (including but not
limited to humans and other mammals) when the host's immune system has
been overwhelmed by bacterial infection of a wound site (e.g., an acute
wound), creating permissive conditions for bacteria to invade and further
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
3
destroy tissue. In general, chronic wounds are wounds that do not heal within
three months, and instead of becoming smaller they tend to grow larger as the
bacterial infiltration progresses. Chronic wounds may become very painful and
stressful for the patient when nearby nerves become damaged (neuropathy) as
the wound progresses. These wounds affect four million Americans each year
and cost about $9 billion in treatment expenses. Afflicted individuals are
mostly
over the age of 60.
Chronic wounds may in some cases originate as acute wounds
and thus may include, for example, gunshot or shrapnel wounds, burns,
punctures, venous ulcers, pressure ulcers, diabetic ulcers, radiation
poisoning,
malignancies, dermal infections, gangrene, surgical wounds, diabetic foot
ulcers, decubitis ulcers, venous leg ulcers, infected and/or biofilm-
containing
non healing surgical wounds, pyoderma gangrenosum, traumatic wounds, acute
arterial insufficiency, necrotizing fasciitis, osteomyelitis (bone infection),
and
radiation injuries, such as osteoradionecrosis and soft tissue radionecrosis,
or
other types of wounds. Venous ulcers, for example, occur mostly in the legs,
as a result of poor circulation (e.g., ischemia), malfunctioning valves of
veins, or
repeated physical trauma (e.g., repetitive injury). Pressure ulcers may be
present when local pressure that is exerted at or around a wound site is
greater
than blood pressure, for instance, such that poor circulation, paralysis,
and/or
bed sores may contribute to, or exacerbate, the chronic wound. Diabetic ulcers
may occur in individuals with diabetes mellitus, for example, persons in whom
uncontrolled high blood sugar can contribute to a loss of feeling in the
extremities, leading to repetitive injuries and/or neglect on the part of the
individual to attend to injuries. Factors that can complicate or otherwise
influence clinical onset and outcome of chronic wounds include the subject's
immunological status (e.g., immune suppression, pathologically (e.g., HIV-
AIDS), radiotherapeutically or pharmacologically compromised immune system;
age; stress); skin aging (including photochemical aging), and development and
progression of biofilms within the wound. In the case of epithelial tissues in
the
respiratory and/or gastrointestinal tracts, inaccessibility, occlusion,
difficulty in
generating epithelial surface-clearing fluid forces or development of
localized
microenvironments conducive to microbial survival can engender clinical
complications.
Wound-related injuries may be accompanied by lost or
compromised organ function, shock, bleeding and/or thrombosis, cell death
(e.g., necrosis and/or apoptosis), stress and/or microbial infection. Any or
all of
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
4
these events, and especially infection, can delay or prevent the effective
tissue
repair processes that are involved in wound healing. Hence, it can be
important
as early as possible in an individual who has sustained a wound to remove
nonviable tissue from a wound site, a process referred to as debridement, and
also to remove any foreign matter from the wound site, also referred to as
wound cleansing.
Severe wounds, acute wounds, chronic wounds, burns, and
ulcers can benefit from cellular wound dressings. Several artificial skin
products are available for non healing wounds or burns such as: Apligraft
(Norvartis), Demagraft , Biobrane , Transcyte (Advance Tissue Science),
Integra Dermal Regeneration Template (from Integra Life Sciences
Technology), and OrCel . These products, however, are not designed to
address the problem of bacterial tissue infiltration and wound spreading.
Unfortunately, systemic antibiotics are not effective for the
treatment of chronic wounds, and are generally not used unless an acute
bacterial infection is present. Current approaches include administration or
application of antibiotics, but such remedies may promote the advent of
antibiotic-resistant bacterial strains and/or may be ineffective against
bacterial
biofilms. It therefore may become especially important to use antiseptics when
drug resistant bacteria (e.g., methicillin resistant Staphylococcus aureus, or
MRSA) are detected. There are many antiseptics widely in use, but bacterial
populations or subpopulations that are established may not respond to these
agents, or to any other currently available treatments. Additionally, a number
of
antiseptics may be toxic to host cells at the concentrations that may be
needed
to be effective against an established bacterial infection, and hence such
antiseptics are unsuitable. This problem may be particularly acute in the case
of efforts to clear infections from natural surfaces, including internal
epithelial
surfaces, such as respiratory (e.g., airway, nasopharyngeal and laryngeal
paths, tracheal, pulmonary, bronchi, bronchioles, alveoli, etc.) or
gastrointestinal (e.g., buccal, esophageal, gastric, intestinal, rectal, anal,
etc.)
tracts, or other epithelial surfaces.
Particularly problematic are infections composed of bacterial
biofilms, a relatively recently recognized organization of bacteria by which
free,
single-celled ("planktonic") bacteria assemble by intercellular adhesion into
organized, multi-cellular communities (biofilms) having markedly different
patterns of behavior, gene expression, and susceptibility to environmental
agents including antibiotics. Biofilms may deploy biological defense
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
mechanisms not found in planktonic bacteria, which mechanisms can protect
the biofilm community against antibiotics and host immune responses.
Established biofilms can arrest the tissue-healing process.
Common microbiologic contaminants that underlie persistent and
5 .. potentially deleterious infections include S. aureus, including MRSA
(Methicillin
Resistant Staphylococcus aureus), Enterococci, E. coil, P. aeruginosa,
Streptococci, and Acinetobacter baumannii. Some of these organisms exhibit
an ability to survive on non-nutritive clinical surfaces for months. S.
aureus, has
been shown to be viable for four weeks on dry glass, and for between three and
six months on dried blood and cotton fibers (Domenico et al., 1999 Infect.
Immun. 67:664-669). Both E. coil and P. aeruginosa have been shown to
survive even longer than S. aureus on dried blood and cotton fibers (ibicI).
Microbial biofilms are associated with substantially increased
resistance to both disinfectants and antibiotics. Biofilm morphology results
when bacteria and/or fungi attach to surfaces. This attachment triggers an
altered transcription of genes, resulting in the secretion of a remarkably
resilient
and difficult to penetrate polysaccharide matrix, protecting the microbes.
Biofilms are very resistant to the mammalian immune system, in addition to
their very substantial resistance to antibiotics. Biofilms are very difficult
to
eradicate once they become established, so preventing biofilm formation is a
very important clinical priority. Recent research has shown that open wounds
can quickly become contaminated by biofilms. These microbial biofilms are
thought to delay wound healing, and are very likely related to the
establishment
of serious wound infections.
The current guidelines for the care for military wounds, for
example, specify vigorous and complete irrigation and debridement
(Blankenship CL, Guidelines for care of open combat casualty wounds, Fleet
Operations and Support. U.S. Bureau of Medicine and Surgery). While this
early intervention is important, it is not adequate to prevent the development
of
infection. Additional therapeutic steps need to be taken following debridement
to promote healing, reduce the microbial bio-burden, and thereby reduce the
chances of establishing wound infections and wound biofilms.
Because of the complex nature of military traumatic wounds, the
potential for infection is great, particularly considering the introduction of
foreign
objects and other environmental contaminating agents. Both military and
clinical environments (including people within both of these environments) act
as important sources of potentially pathogenic microbes, particularly to those
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
6
suffering from open and/or complex wounds. Acute and chronic wounds,
including surgical and military wounds, have already compromised the body's
primary defense and barrier against infection; the skin. Wounds thus expose
the interior of the body (a moist and nutritive environment) to opportunistic
and
pathogenic infections. Many of these infections, particularly persistent wound
infections, are likely related to biofilm formation, as has been shown to be
the
case with chronic wounds (James et al., 2008). Infection of wounds in
hospitals
constitutes one of the most common causes of nosocomial infection, and
wounds acquired in military and natural disaster environments are particularly
susceptible to microbial contamination. Military wounds are predisposed to
infection because they are typically associated with tissue damage, tend to be
extensive and deep, may introduce foreign bodies and interfere with local
blood
supply, may be associated with fractures and burns, and may lead to shock and
compromised immune defenses.
Skin Architecture and Wound Healing
Maintenance of intact, functioning skin and other epithelial tissues
(e.g., generally avascular epithelial surfaces that form barriers between an
organism and its external environment, such as those found in skin and also
found in the linings of respiratory and gastrointestinal tracts, glandular
tissues,
etc.) is significant to the health and survival of humans and other animals.
The
skin is the largest body organ in humans and other higher vertebrates (e.g.,
mammals), protecting against environmental insults through its barrier
function,
mechanical strength and imperviousness to water. As a significant
environmental interface, skin provides a protective body covering that permits
maintenance of physiological equilibria.
Skin architecture is well known. Briefly, epidermis, the skin outer
layer, is covered by the stratum corneum, a protective layer of dead epidermal
skin cells (e.g., keratinocytes) and extracellular connective tissue proteins.
The
epidermis undergoes a continual process of being sloughed off as it is
replaced
by new material pushed up from the underlying epidermal granular cell, spinous
cell, and basal cell layers, where continuous cell division and protein
synthesis
produce new skin cells and skin proteins (e.g., keratin, collagen). The dermis
lies underneath the epidermis, and is a site for the elaboration by dermal
fibroblasts of connective tissue proteins (e.g., collagen, elastin, etc.) that
assemble into extracellular matrix and fibrous structures that confer
flexibility,
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
7
strength and elasticity to the skin. Also present in the dermis are nerves,
blood
vessels, smooth muscle cells, hair follicles and sebaceous glands.
As the body's first line of defense, the skin is a major target for
clinical insults such as physical, mechanical, chemical and biological (e.g.,
xenobiotic, autoimmune) attack that can alter its structure and function. The
skin is also regarded as an important component of immunological defense of
the organism. In the skin can be found migrating as well as resident white
blood cells (e.g., lymphocytes, macrophages, mast cells) and epidermal
dendritic (Langerhans) cells having potent antigen-presenting activity, which
contribute to immunological protection. Pigmented melanocytes in the basal
layer absorb potentially harmful ultraviolet (UV) radiation. Disruption of the
skin
presents undesirable risks to a subject, including those associated with
opportunistic infections, incomplete or inappropriate tissue remodeling,
scarring, impaired mobility, pain and/or other complications. Like the skin,
other
epithelial surfaces (e.g., respiratory tract, gastrointestinal tract and
glandular
linings) have defined structural attributes when healthy such that infection
or
other disruptions may present serious health risks.
Damaged or broken skin may result, for example, from wounds
such as cuts, scrapes, abrasions, punctures, burns (including chemical burns),
infections, temperature extremes, incisions (e.g., surgical incisions), trauma
and
other injuries. Efficient skin repair via wound healing is therefore clearly
desirable in these and similar contexts.
Although skin naturally exhibits remarkable ability for self-repair
following many types of damage, there remain a number of contexts in which
skin healing does not occur rapidly enough and/or in which inappropriate
cellular tissue repair mechanisms result in incompletely remodeled skin that
as
a consequence can lack the integrity, barrier properties, mechanical strength,
elasticity, flexibility, or other desirable properties of undamaged skin. Skin
wound healing thus presents such associated challenges, for example, in the
context of chronic wounds.
Wound healing occurs in three dynamic and overlapping phases,
beginning with the formation of a fibrin clot. The clot provides a temporary
shield and a reservoir of growth factors that attracts cells into the wound.
It also
serves as a provisional extracellular matrix (ECM) that the cells invade
during
repair. Intermingled with clot formation is the inflammatory phase, which is
characterized by the infiltration of phagocytes and neutrophils into the
wound,
which clear the wound of debris and bacteria, while releasing growth factors
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
8
that amplify the early healing response. The process of restoring the denuded
area is initiated in the proliferation phase of healing and is driven by
chemokines, cytokines, and proteases that have been secreted from the
immune cells and are concentrated within the clot. Keratinocytes are
stimulated to proliferate and migrate, which forms the new layer of epithelium
that covers the wound while wound angiogenesis delivers oxygen, nutrients,
and inflammatory cells to the wounded area. The remodeling phase is the final
phase of wound repair and it is carried out by the myofibroblasts, which
facilitate connective tissue contraction, increase wound strength, and deposit
the ECM that forms the scar (Martin, P. Wound Healing-Aiming for Perfect Skin
Regeneration. Science 1997;4:75-80).
Bismuth Thiol- (BT) based Antiseptics
A number of natural products (e.g., antibiotics) and synthetic
chemicals having antimicrobial, and in particular antibacterial, properties
are
known in the art and have been at least partially characterized by chemical
structures and by antimicrobial effects, such as ability to kill microbes
("cidal"
effects such as bacteriocidal properties), ability to halt or impair microbial
growth ("static" effects such as bacteriostatic properties), or ability to
interfere
with microbial functions such as colonizing or infecting a site, bacterial
secretion
of exopolysaccharides and/or conversion from planktonic to biofilm populations
or expansion of biofilm formation. Antibiotics, disinfectants, antiseptics and
the
like (including bismuth-thiol or BT compounds) are discussed, for example, in
U.S. 6,582,719, including factors that influence the selection and use of such
compositions, including, e.g., bacteriocidal or bacteriostatic potencies,
effective
concentrations, and risks of toxicity to host tissues.
Bismuth, a group V metal, is an element that (like silver)
possesses antimicrobial properties. Bismuth by itself may not be
therapeutically useful and may exhibit certain inappropriate properties, and
so
may instead be typically administered by means of delivery with a complexing
agent, carrier, and/or other vehicle, the most common example of which is
Pepto Bismole, in which bismuth is combined (chelated) with subsalicylate.
Previous research has determined that the combination of certain thiol- (-SH,
sulfhydryl) containing compounds such as ethane dithiol with bismuth, to
provide an exemplary bismuth thiol (BT) compound, improves the antimicrobial
potency of bismuth, compared to other bismuth preparations currently
available.
There are many thiol compounds that may be used to produce BTs (disclosed,
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
9
for example, in Domenico et al., 2001 Antimicrob. Agent. Chemotherap.
45(5):1417-1421, Domenico et al., 1997 Antimicrob. Agent. Chemother.
41(8):1697-1703, and in U.S. RE37,793, U.S. 6,248,371, U.S. 6,086,921, and
U.S. 6,380,248; see also, e.g., U.S. 6,582,719) and several of these
preparations are able to inhibit biofilm formation.
BT compounds have proven activity against MRSA (methicillin
resistant S. aureus), MRSE (methicillin resistant S. epidermidis),
Mycobacterium tuberculosis, Mycobacterium avium, drug-resistant P.
aeruginosa, enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiella
pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila,
Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus
vulgaris, Yersinia enterocolitica, Vibrio cholerae, and Shigella Flexneri
(Domenico et al., 1997 Antimicrob. Agents Chemother 41:1697-1703). There
is also evidence of activity against cytomegalovirus, herpes simplex virus
type 1
(HSV-1) and HSV-2, and yeasts and fungi, such as Candida albicans. BT roles
have also been demonstrated in reducing bacterial pathogenicity, inhibiting or
killing a broad spectrum of antibiotic-resistant microbes (gram-positive and
gram-negative), preventing biofilm formation, preventing septic shock,
treating
sepsis, and increasing bacterial susceptibility to antibiotics to which they
previously exhibited resistance (see, e.g., Domenico et al., 2001 Agents
Chemother. 45:1417-1421; Domenico et al., 2000 Infect. Med. 17:123-127;
Domenico et al., 2003 Res. Adv. In Antimicrob. Agents & Chemother. 3:79-85;
Domenico et al., 1997 Antimicrob. Agents Chemother. 41(8):1697-1703;
Domenico et al., 1999 Infect. Immun. 67:664-669: Huang et al. 1999 J
Antimicrob. Chemother. 44:601-605; Veloira et al., 2003 J Antimicrob.
Chemother. 52:915-919; Wu et al., 2002 Am J Respir Cell Mol Bid. 26:731-
738).
Despite the availability of BT compounds for well over a decade,
effective selection of appropriate BT compounds for particular infectious
disease indications has remained an elusive goal, where behavior of a
particular BT against a particular microorganism cannot be predicted, where
synergistic activity of a particular BT and a particular antibiotic against a
particular microorganism cannot be predicted, where BT effects in vitro may
not
always predict BT effects in vivo, and where BT effects against planktonic
(single-cell) microbial populations may not be predictive of BT effects
against
microbial communities, such as bacteria organized into a biofilm.
Additionally,
limitations in solubility, tissue permeability, bioavailability,
biodistribution and the
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
like may in the cases of some BT compounds hinder the ability to deliver
clinical
benefit safely and effectively. The presently disclosed invention embodiments
address these needs and offer other related advantages.
BRIEF SUMMARY
5 As disclosed herein for the first time, and without wishing to be
bound by theory, according to certain embodiments described herein bismuth-
thiol (BT) compounds may be used as antiseptic agents for use in the treatment
of a wide variety of clinical infectious diseases and conditions and in
personal
healthcare, while also decreasing the costs incurred for the treatment of such
10 infections, including savings that are realized by prevention or
prophylaxis
mediated at least in part by BTs.
Also, in certain embodiments there are contemplated formulations
for treating tissues and/or surfaces that contain bacterial biofilms or
bacteria
related to biofilm formation (e.g., bacteria that are capable of forming or
otherwise promoting biofilms), which formulations comprise one or more BT
compound and one or more antibiotic compound, as described herein, where
according to non-limiting theory, appropriately selected combinations of BT
compound(s) and antibiotic(s) based on the present disclosure provide
heretofore unpredicted synergy in the antibacterial (including anti-biofilm)
effects of such formulations, and/or unpredicted enhancing effects, for
prevention, prophylaxis and/or therapeutically effective treatment against
microbial infections including infections that contain bacterial biofilms.
Also provided herein for the first time are unprecedented bismuth-
thiol compositions comprising substantially monodisperse microparticulate
suspensions, and methods for their synthesis and use.
According to certain embodiments of the invention described
herein there is thus provided a bismuth-thiol composition, comprising a
plurality
of microparticles that comprise a bismuth-thiol (BT) compound, substantially
all
of said microparticles having a volumetric mean diameter of from about 0.4 um
to about 5 m, wherein the BT compound comprises bismuth or a bismuth salt
and a thiol-containing compound. In another embodiment there is provided a
bismuth-thiol composition, comprising a plurality of microparticles that
comprise
a bismuth-thiol (BT) compound, substantially all of said microparticles having
a
volumetric mean diameter of from about 0.4 tim to about 5 firn and being
formed by a process that comprises (a) admixing, under conditions and for a
time sufficient to obtain a solution that is substantially free of a solid
precipitate,
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
11
(i) an acidic aqueous solution that comprises a bismuth salt comprising
bismuth
at a concentration of at least 50 mM and that lacks a hydrophilic, polar or
organic solubilizer, with (ii) ethanol in an amount sufficient to obtain an
admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or 30%
ethanol by volume; and (b) adding to the admixture of (a) an ethanolic
solution
comprising a thiol-containing compound to obtain a reaction solution, wherein
the thiol-containing compound is present in the reaction solution at a molar
ratio
of from about 1:3 to about 3:1 relative to the bismuth, under conditions and
for a
time sufficient for formation of a precipitate which comprises the
microparticles
comprising the BT compound. In certain embodiments the bismuth salt is
Bi(NO3)3. In certain embodiments the acidic aqueous solution comprises at
least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth by weight. In certain
embodiments the acidic aqueous solution comprises at least 0.5%, 1%, 1.5%,
2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% nitric acid by weight. In certain
embodiments the thiol-containing compound comprises one or more agents
selected from 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,
dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-
propanedithiol, 2-
hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol, alpha-lipoic
acid
and dithiothreitol.
In another embodiment there is provided a method for preparing a
bismuth-thiol composition that comprises a plurality of microparticles that
comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles
having a volumetric mean diameter of from about 0.4 mm to about 51.1m, said
method comprising the steps of (a) admixing, under conditions and for a time
sufficient to obtain a solution that is substantially free of a solid
precipitate, (i)
an acidic aqueous solution that comprises a bismuth salt comprising bismuth at
a concentration of at least 50 mM and that lacks a hydrophilic, polar or
organic
solubilizer, with (ii) ethanol in an amount sufficient to obtain an admixture
that
comprises at least about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume;
and (b) adding to the admixture of (a) an ethanolic solution comprising a
thiol-
containing compound to obtain a reaction solution, wherein the thiol-
containing
compound is present in the reaction solution at a molar ratio of from about
1:3
to about 3:1 relative to the bismuth, under conditions and for a time
sufficient for
formation of a precipitate which comprises the microparticles comprising the
BT
compound. In certain embodiments the method further comprises recovering
the precipitate to remove impurities. In certain embodiments the bismuth salt
is
Bi(NO3)3. In certain embodiments the acidic aqueous solution comprises at
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
12
least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth by weight. In certain
embodiments the acidic aqueous solution comprises at least 0.5%, 1%, 1.5%,
2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% nitric acid by weight. In certain
embodiments the thiol-containing compound comprises one or more agents
selected from the group consisting of 1,2-ethane dithiol, 2,3-
dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-
butanedithiol, 1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-
propanol, dithioerythritol, dithiothreitol, alpha-lipoic acid, methanethiol
(CH3SH
[m-mercaptan]), ethanethiol (C2H5SH [e- mercaptan]), 1-propanethiol (C3H7SH
[n-P mercaptan]), 2-propanethiol (CH3CH(SH)CH3 [2C3 mercaptan]),
butanethiol (C41-19SH ([n-butyl mercaptan]), tert-butyl mercaptan (C(CH3)3SH
[t-
butyl mercaptan]), pentanethiols (C5Fl11SH [pentyl mercaptan]), coenzyme A,
lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothreitol,
dithioerythritol, 2-mercaptoindole, transglutaminase, (11-
mercaptoundecyl)hexa(ethylene glycol), (11-mercaptoundecyl)tetra(ethylene
glycol), (11-mercaptoundecyl)tetra(ethylene glycol) functionalized gold
nanoparticles, 1,1',4',1"-terpheny1-4-th101, 1,11-undecanedithiol, 1,16-
hexadecanedithiol, 1,2-ethanedithiol technical grade, 1,3-propanedithiol, 1,4-
benzenedimethanethiol, 1,4-butanedithiol, 1,4-butanedithiol diacetate, 1,5-
pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol,
adamantanethiol, 1-butanethiol, 1-decanethiol, 1-dodecanethiol, 1-
heptanethiol, 1-heptanethiol purum, 1-hexadecanethiol, 1-hexanethiol, 1-
mercapto-(triethylene glycol), 1-mercapto-(triethylene glycol) methyl ether
functionalized gold nanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-
octadecanethiol, 1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-
pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol purum, 1-
undecanethiol, 11-(1H-pyrrol-1-yl)undecane-1-thiol, 11-amino-1-undecanethiol
hydrochloride, 11-bromo-1-undecanethiol, 11-mercapto-1-undecanol, 11-
mercapto-1-undecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecanoic
acid, 11-mercaptoundecyl trifluoroacetate, 11-mercaptoundecylphosphoric
acid, 12-mercaptododecanoic acid, 12-mercaptododecanoic acid, 15-
mercaptopentadecanoic acid, 16-mercaptohexadecanoic acid, 16-
mercaptohexadecanoic acid, 1H,1H,2H,2H-perfluorodecanethiol, 2,2'-
(ethylenedioxy)diethanethiol, 2,3-butanedithiol, 2-butanethiol, 2-
ethylhexanethiol, 2-methy1-1-propaneth101, 2-methyl-2-propanethiol, 2-
phenylethanethiol, 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol purum, 3-
(dimethoxymethylsily1)-1-propanethiol, 3-chloro-1-propanethiol, 3-mercapto-1-
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
13
propanol, 3-mercapto-2-butanol, 3-mercapto-N-nonylpropionamide, 3-
mercaptopropionic acid, 3-mercaptopropyl-functionalized silica gel, 3-methyl-
1-butanethiol, 4,4'-bis(r,nercaptomethyl)biphenyl, 4,4'-dimercaptostilbene, 4-
(6-mercaptohexyloxy)benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-
butanol, 6-(ferrocenyl)hexanethiol, 6-mercapto-1-hexanol, 6-
mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid, 9-
mercapto-1-nonanol, biphenyl-4,4'-dithiol, butyl 3-mercaptopropionate,
copper(I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol
functionalized silver nanoparticles, dodecanethiol functionalized gold
nanoparticles, dodecanethiol functionalized silver nanoparticles,
hexa(ethylene
glycol)mono-11-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3-
mercaptopropionate, nanoTether BPA-HH, NanoThinke 18, NanoThinksTm 8,
NanoThinksTm ACID11, NanoThinksrm ACID16, NanoThinksrm ALC011,
NanolhinksTM THI08, octanethiol functionalized gold nanoparticles, PEG
dithiol average Mr, 8,000, PEG dithiol average mol wt 1,500, PEG dithiol
average mol wt 3,400, S-(11-bromoundecyl)thioacetate, S-(4-
cyanobutyl)thioacetate, thiophenol, triethylene glycol mono-11-
mercaptoundecyl ether, trimethylolpropane tris(3-mercaptopropionate), [11-
(methylcarbonylthio)undecyl]tetra(ethylene glycol), m-carborane-9-thiol, p-
terpheny1-4,4"-dithiol, tert-dodecylmercaptan, tert-nonyl mercaptan.
In another embodiment there is provided a method for protecting
a natural surface, including a biological tissue surface such as an epithelial
tissue surface, against one or more of a bacterial pathogen, a fungal pathogen
and a viral pathogen, comprising contacting the epithelial tissue surface with
an
effective amount of a BT composition under conditions and for a time
sufficient
for one or more of (i) prevention of infection of the surface by the
bacterial,
fungal or viral pathogen, (ii) inhibition of cell viability or cell growth of
substantially all planktonic cells of the bacterial, fungal or viral pathogen,
(iii)
inhibition of biofilm formation by the bacterial, fungal or viral pathogen,
and (iv)
inhibition of biofilm viability or biofilm growth of substantially all biofilm-
form
cells of the bacterial, fungal or viral pathogen, wherein the BT composition
comprises a plurality of microparticles that comprise a bismuth-thiol (BT)
compound, substantially all of said microparticles having a volumetric mean
diameter of from about 0.4 rn to about 5 pm. In certain embodiments the
bacterial pathogen comprises at least one of (i) one or more gram-negative
bacteria; (ii) one or more gram-positive bacteria; (iii) one or more
antibiotic-
sensitive bacteria; (iv) one or more antibiotic-resistant bacteria; (v) a
bacterial
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
14
pathogen that is selected from Staphylococcus aureus (S. aureus), MRSA
(methicillin-resistant S. aureus), Staphylococcus epidermidis , MRSE
(methicillin-resistant S. epidermidis), Mycobacterium tuberculosis,
Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P. aeruginosa,
Escherichia coli, enterotoxigenic E. coli, enterohemorrhagic E. coil,
Klebsiella
pneumoniae, Clostridiurn difficile, Heliobacter pylori, Legionella
pneumophila,
Enterococcus faecalis, methicillin-susceptible Enterococcus faecalis,
Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia
enterocolitica, Vibrio cholera, Shigella flexneri, vancomycin-resistant
Enterococcus (VRE), Burkholderia cepacia complex, Francisella tularensis,
Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-
resistant enterococci, Streptococcus pneumonia, penicillin-resistant
Streptococcus pneumonia, Escherichia coil, Burkholderia cepacia, Bukholderia
multivorans, Mycobacterium smegmatis and Acinetobacter baumannii. In
certain embodiments the bacterial pathogen exhibits antibiotic resistance. In
certain embodiments the bacterial pathogen exhibits resistance to an
antibiotic
that is selected from methicillin, vancomycin, naficilin, gentamicin,
ampicillin,
chloramphenicol, doxycycline and tobramycin.
In certain embodiments the natural surface comprises an
oral/buccal cavity surface. In further embodiments, the natural surface
comprises a biological surface such as bone, joint, muscle, ligament, or
tendon.
In certain embodiments the surface comprises an epithelial tissue
surface that comprises a tissue that is selected from epidermis, dermis,
respiratory tract, gastrointestinal tract and glandular linings.
In certain embodiments the step of contacting is performed one or
a plurality of times. In certain embodiments at least one step of contacting
comprises one of spraying, irrigating, dipping and painting the natural
surface.
In certain embodiments at least one step of contacting comprises one of
inhaling, ingesting and orally irrigating. In certain embodiments least one
step
of contacting comprises administering by a route that is selected from
topically,
intraperitoneally, orally, parenterally, intravenously, intraarterially,
transdermally, sublingually, subcutaneously, intramuscularly, transbuccally,
intranasally, via inhalation, intraoccularly, intraauricularly,
intraventricularly,
subcutaneously, intraadiposally, intraarticularly and intrathecally. In
certain
embodiments the BT composition comprises one or more BT compounds
selected from the group consisting of BisBAL, BisEDT, Bis-dimercaprol, Bis-
DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyrffol, Bis-
Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-
propanethiol.
In certain embodiments the bacterial pathogen exhibits antibiotic
5 resistance. In certain other embodiments the above described method
further
comprises contacting the natural surface with a synergizing antibiotic and/or
with an enhancing antibiotic, simultaneously or sequentially and in any order
with respect to the step of contacting the surface with the BT composition. In
certain embodiments the synergizing and/or enhancing antibiotic comprises an
10 antibiotic that is selected from an aminoglycoside antibiotic, a
carbapenem
antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a
glycopeptide
antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin
antibiotic,
and an aminopenicillin antibiotic. In certain embodiments the synergizing
and/or enhancing antibiotic is an aminoglycoside antibiotic that is selected
from
15 amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.
In another embodiment of the invention described herein there is
provided a method for overcoming antibiotic resistance (e.g,, for a bacterial
pathogen that is resistant to at least one anti-bacterial effect of at least
one
antibiotic known to have an anti-bacterial effect against bacteria of the same
bacterial species, rendering such a pathogen susceptible to-an antibiotic) on
a
natural surface where an antibiotic-resistant bacterial pathogen is present,
comprising contacting the surface simultaneously or sequentially and in any
order with an effective amount of (1) at least one bismuth-thiol (BT)
composition
and (2) at least one antibiotic that is enhanced by, and/or that is capable of
acting synergistically with the at least one BT composition, under conditions
and for a time sufficient for one or more of: (i) prevention of infection of
the
surface by the bacterial pathogen, (ii) inhibition of cell viability or cell
growth of
substantially all planktonic cells of the bacterial pathogen, (iii) inhibition
of
biofilm formation by the bacterial pathogen, and (iv) inhibition of biofilm
viability
or biofilm growth of substantially all biofilm-form cells of the bacterial
pathogen,
wherein the BT composition comprises a plurality of microparticles that
comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles
having a volumetric mean diameter of from about 0.4 pm to about 5 Ilm; and
thereby overcoming antibiotic resistance on the epithelial tissue surface. In
certain embodiments the bacterial pathogen comprises at least one of: (i) one
or more gram-negative bacteria; (ii) one or more gram-positive bacteria; (iii)
one
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
16
or more antibiotic-sensitive bacteria; (iv) one or more antibiotic-resistant
bacteria; (v) a bacterial pathogen that is selected from Staphylococcus aureus
(S. aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus
epidermidis , MRSE (methicillin-resistant S. epidermidis), Mycobacterium
tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant
P. aeruginosa, Escherichia coil, enterotoxigenic E. coli, enterohemorrhagic E.
coli, Kiebsiella pneumoniae, Clostridium difficile, Heliobacter pylori,
Legionella
pneumophila, Enterococcus faecalis, methicillin-susceptible Enterococcus
faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris,
Yersinia enterocolitica, Vibrio cholera, Shigella flexneri, vancomycin-
resistant
Enterococcus (VRE), Burkholderia cepacia complex, Francisella tularensis,
Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-
resistant enterococci, Streptococcus pneumonia, penicillin-resistant
Streptococcus pneumonia, Escherichia coil, Burkholderia cepacia, Bukholderia
multivorans, Mycobacterium smegmatis and Acinetobacter baumannii.
In certain embodiments the bacterial pathogen exhibits resistance
to an antibiotic that is selected from methicillin, vancomycin, naficilin,
gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin
and gatifloxacin.
In certain embodiments the natural surface comprises an
oral/buccal cavity surface. In further embodiments, the natural surface
comprises a biological surface such as bone, joint, muscle, ligament, or
tendon.
In certain embodiments the surface comprises a tissue that is
selected from the group consisting of epidermis, dermis, respiratory tract,
gastrointestinal tract and glandular linings. In certain embodiments the step
of
contacting is performed one or a plurality of times. In certain embodiments at
least one step of contacting comprises one of spraying, irrigating, dipping
and
painting the surface. In certain other embodiments at least one step of
contacting comprises one of inhaling, ingesting and orally irrigating. In
certain
embodiments at least one step of contacting comprises administering by a
route that is selected from topically, intraperitoneally, orally,
parenterally,
intravenously, intraarterially, transdermally, sublingually, subcutaneously,
intramuscularly, transbuccally, intranasally, via inhalation, intraoccularly,
intraauricularly, intraventricularly, subcutaneously, intraadiposally,
intraarticularly and intrathecally. In certain embodiments the BT composition
comprises one or more BT compounds selected from BisBAL, BisEDT, Bis-
dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
17
Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT,
Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-ED1/2-
hydroxy-1-propanethiol. In certain embodiments the synergizing and/or
enhancing antibiotic comprises an antibiotic that is selected from
clindamicin,
gatifloxacin, an aminoglycoside antibiotic, a carbapenem antibiotic, a
cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide
antibiotic, a
lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, and
an
aminopenicillin antibiotic. In certain embodiments the synergizing and/or
enhancing antibiotic is an aminoglycoside antibiotic that is selected from
amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.
Turning to another embodiment there is provided an antiseptic
composition, comprising (a) at least one BT compound; (b) at least one
antibiotic compound that is enhanced by and/or is capable of acting
synergistically with the BT compound; and (c) a pharmaceutically acceptable
excipient or carrier, including a carrier for topical use. In certain
embodiments
the BT compound is selected from BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,
Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT,
Bis-Pyr/Bal., Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,
bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. In
certain embodiments the BT composition comprises a plurality of microparticles
that comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles having a volumetric mean diameter of from about 0.4 gm to
about 5 gm. In certain embodiments the BT compound is selected from
BisEDT and BisBAL. In certain embodiments the antibiotic compound
comprises an antibiotic that is selected from methicillin, vancomycin,
naficilin,
gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin,
gatifloxacin and an aminoglycoside antibiotic. In certain embodiments the
aminoglycoside antibiotic is selected from amikacin, arbekacin, gentamicin,
kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin,
streptomycin, tobramycin and apramycin. In certain embodiments the
aminoglycoside antibiotic is amikacin.
In certain other embodiments there is provided a method for
treating a natural surface that supports or contains bacterial biofilm,
comprising
(a) identifying a bacterial infection on or in the surface as comprising one
of (i)
gram positive bacteria, (ii) gram negative bacteria, and (iii) both (i) and
(ii); and
(b) administering a formulation that comprises one or more bismuth thiol (BT)
CA 2788669 2017-03-14
18
compositions to the surface, wherein (i) if the bacterial infection comprises
gram
positive bacteria, then the formulation comprises therapeutically effective
amounts of at least one BT compound and at least one antibiotic that is
rifamycin, (ii) if the bacterial infection comprises gram negative bacteria,
then
the formulation comprises therapeutically effective amounts of at least one BT
compound and amikacin, (iii) if the bacterial infection comprises both gram
positive and gram negative bacteria, then the formulation comprises
= therapeutically effective amounts of one or a plurality of BT compounds,
rifamycin and amikacin, and thereby treating the surface.
In certain embodiments the biofilm comprises one or a plurality of
antibiotic-resistant bacteria. In certain embodiments treating the surface
comprises at least one of: (i) eradicating the bacterial biofilm, (ii)
reducing the
bacterial biofilm, and (iii) impairing growth of the bacterial biohlm. In
certain
embodiments the BT composition comprises a plurality of microparticles that
comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles
having a volumetric mean diameter of from about 0.4 4m to about 5 p.m.
In an embodiment, there is provided a bismuth-thiol (BT)
composition for protecting a natural surface against one or more of a
bacterial
pathogen, a fungal pathogen and a viral pathogen, wherein the bismuth-thiol
composition is for contacting the surface with an effective amount of a BT
composition under conditions and for a time sufficient for one or more of:
(a) prevention of infection of the surface by the bacterial, fungal
or viral pathogen,
(b) inhibition of cell viability or cell growth of substantially all
planktonic cells of the bacterial, fungal or viral pathogen,
(c) inhibition of biofilm formation by the bacterial, fungal or viral
pathogen, and
(d) inhibition of biofilm
viability or biofilm growth of substantially
all biofilm-form cells of the bacterial, fungal or viral pathogen,
wherein the BT composition comprises a plurality of microparticles that
comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles having a volumetric mean diameter of from about 0.4 um to
about 5 m.
CA 2788669 2017-03-14
18a
In another embodiment, there is provided a bismuth-thiol (BT)
composition for overcoming antibiotic resistance on a natural surface where an
antibiotic-resistant bacterial pathogen is present, wherein the bismuth-thiol
.. composition is for contacting the epithelial tissue surface contacting
simultaneously or sequentially and in any order with an effective amount of
(a) the BT composition and
(b) at least one antibiotic that is capable of enhancing or acting
synergistically with the at least one BT composition, under conditions
and for a time sufficient for one or more of:
(i) prevention of infection of the epithelial tissue surface
by the bacterial pathogen,
(ii) inhibition of cell viability or cell growth of substantially
all planktonic cells of the bacterial pathogen,
(iii) inhibition of biofilm formation by the bacterial
pathogen, and
(iv) inhibition of biofilm viability or biofilm growth
of
substantially all biofilm-form cells of the bacterial pathogen,
wherein the BT composition comprises a plurality of microparticles
that comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles having a volumetric mean diameter of from about 0.4 ptm to
about 5 p.m; and thereby overcoming antibiotic resistance on the epithelial
tissue surface, wherein if the BT compound is BisEDT, then the
synergizing or cooperative antimicrobial efficacy enhancing antibiotic
cannot be any of tobramycin, nafcillin, gentamycin, clindamycin,
gatifloxacin, minocycline, vancomycin, and cefazolin.
In still another embodiment, there is provided an antiseptic
composition for treating a natural surface that contains bacterial biofilm,
comprising
(a) at least one bismuth-thiol (BT) composition that comprises a
plurality of microparticles that comprise a bismuth-thiol (BT)
compound, substantially all of said microparticles having a volumetric
mean diameter of from about 0.4 1.1m to about 5 gm; and
(b) at least one antibiotic compound that is capable of
acting
synergistically with, or enhancing, the BT compound, wherein if the BT
CA2788669
18b
Compound is Bis EDT, then the synergizing or cooperative antimicrobial
efficiency
enhancing antibiotic caanot be any tonramycin, nafcillin, gentamycin,
clindamycin,
gatifloxacin, minocycline, vancomycin, and cefazolin.
Various aspects of the disclosure relate to a bismuth-thiol (BT) composition
for use as
an antimicrobial agent, comprising a plurality of solid microparticles, said
microparticles
having a volumetric mean diameter of from 0.4 pm to 5 pm, said microparticles
comprising a
BT compound, wherein the BT compound comprises bismuth and/or a bismuth salt
associated covalently and/or in a coordination complex with one or more thiol-
containing
compounds selected from the group consisting of 1,2-ethanedithiol, 2,3-
dimercaptopropanol,
dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol,
1,3-propanedithiol, and 2-
hydroxypropane thiol.
Various aspects of the disclosure relate to a bismuth-thiol (BT) composition
for
protecting a natural surface against one or more of a bacterial pathogen, a
fungal pathogen
and a viral pathogen, wherein the bismuth-thiol composition is for contacting
the natural
surface with an effective amount of a BT composition under conditions and for
a time
sufficient for one or more of: (a) prevention of infection of the natural
surface by the bacterial,
fungal or viral pathogen, (b)inhibition of cell viability or cell growth of
substantially all
planktonic cells of the bacterial, fungal or viral pathogen, (c) inhibition of
biofilm formation by
the bacterial, fungal or viral pathogen, and (d) inhibition of biofilm
viability or biofilm growth
of substantially all biofilm-form cells of the bacterial, fungal or viral
pathogen, wherein the BT
composition comprises a plurality of microparticles that comprise a bismuth-
thiol (BT)
compound, substantially all of said microparticles having a volumetric mean
diameter of
from about 0.4 pm to about 5 pm.
Various aspects of the disclosure relate to use of a bismuth-thiol (BT)
composition for
protecting a natural surface against one or more of a bacterial pathogen, a
fungal pathogen
and a viral pathogen, wherein the bismuth-thiol composition is for contacting
the natural
surface with an effective amount of a BT composition under conditions and for
a time
sufficient for one or more of: (a) prevention of infection of the natural
surface by the bacterial,
fungal or viral pathogen, (b) inhibition of cell viability or cell growth of
substantially all
planktonic cells of the bacterial, fungal or viral pathogen, (c) inhibition of
biofilm formation by
the bacterial, fungal or viral pathogen, and (d) inhibition of biofilm
viability or biofilm growth
of substantially all biofilm-form cells of the bacterial, fungal or viral
pathogen, wherein the BT
Date recu/Date Received 2020-07-07
CA2788669
18c
composition comprises a plurality of microparticles that comprise a bismuth-
thiol (BT)
compound, substantially all of said microparticles having a volumetric mean
diameter of
from about 0.4 pm to about 5 pm.
Various aspects of the disclosure relate to use of a bismuth-thiol (BT)
composition for
the manufacture of a medicament for protecting a natural surface against one
or more of a
bacterial pathogen, a fungal pathogen and a viral pathogen, wherein the
bismuth-thiol
composition is for contacting the natural surface with an effective amount of
a BT
composition under conditions and for a time sufficient for one or more of: (a)
prevention of
infection of the natural surface by the bacterial, fungal or viral pathogen,
(b) inhibition of cell
viability or cell growth of substantially all planktonic cells of the
bacterial, fungal or viral
pathogen, (c) inhibition of biofilm formation by the bacterial, fungal or
viral pathogen, and (d)
inhibition of biofilm viability or biofilm growth of substantially all biofilm-
form cells of the
bacterial, fungal or viral pathogen, wherein the BT composition comprises a
plurality of
microparticles that comprise a bismuth-thiol (BT) compound, substantially all
of said
microparticles having a volumetric mean diameter of from about 0.4 pm to about
5 pm.
Various aspects of the disclosure relate to a bismuth-thiol (BT) composition
for
overcoming antibiotic resistance on a natural surface where an antibiotic-
resistant bacterial
pathogen is present, wherein the bismuth-thiol composition is for contacting
the natural
surface simultaneously or sequentially and in any order with an effective
amount of: (a) the
BT composition and (b) at least one antibiotic that is capable of enhancing or
acting
synergistically with the at least one BT composition, under conditions and for
a time sufficient
for one or more of: (i) prevention of infection of the natural surface by the
bacterial pathogen,
(ii) inhibition of cell viability or cell growth of substantially all
planktonic cells of the bacterial
pathogen, (iii)inhibition of biofilm formation by the bacterial pathogen, and
(iv) inhibition of
biofilm viability or biofilm growth of substantially all biofilm-form cells of
the bacterial
pathogen, wherein the BT composition comprises a plurality of microparticles
that comprise
a bismuth-thiol (BT) compound, substantially all of said microparticles having
a volumetric
mean diameter of from about 0.4 pm to about 5 pm; and thereby overcoming
antibiotic
resistance on the natural surface, wherein if the BT compound is BisEDT, then
the
synergizing or cooperative antimicrobial efficacy enhancing antibiotic cannot
be any of
tobramycin, nafcillin, gentamycin, clindamycin, gatifloxacin, minocycline,
vancomycin, and
cefazolin.
Date recu/Date Received 2020-07-07
CA2788669
18d
Various aspects of the disclosure relate to use of a bismuth-thiol (BT)
composition for
overcoming antibiotic resistance on a natural surface where an antibiotic-
resistant bacterial
pathogen is present, wherein the bismuth-thiol composition is for contacting
the natural
surface simultaneously or sequentially and in any order with an effective
amount of: (a) the
BT composition and (b) at least one antibiotic that is capable of enhancing or
acting
synergistically with the at least one BT composition, under conditions and for
a time sufficient
for one or more of: (i) prevention of infection of the natural surface by the
bacterial pathogen,
(ii) inhibition of cell viability or cell growth of substantially all
planktonic cells of the bacterial
pathogen, (iii)inhibition of biofilm formation by the bacterial pathogen, and
(iv) inhibition of
biofilm viability or biofilm growth of substantially all biofilm-form cells of
the bacterial
pathogen, wherein the BT composition comprises a plurality of microparticles
that comprise
a bismuth-thiol (BT) compound, substantially all of said microparticles having
a volumetric
mean diameter of from about 0.4 pm to about 5 pm; and thereby overcoming
antibiotic
resistance on the natural surface, wherein if the BT compound is BisEDT, then
the
synergizing or cooperative antimicrobial efficacy enhancing antibiotic cannot
be any of
tobramycin, nafcillin, gentamycin, clindamycin, gatifloxacin, minocycline,
vancomycin, and
cefazolin.
Various aspects of the disclosure relate to use of a bismuth-thiol (BT)
composition for
the manufacture of a medicament for overcoming antibiotic resistance on a
natural surface
.. where an antibiotic- resistant bacterial pathogen is present, wherein the
bismuth-thiol
composition is for contacting the natural surface simultaneously or
sequentially and in any
order with an effective amount of: (a) the BT composition and (b) at least one
antibiotic that
is capable of enhancing or acting synergistically with the at least one BT
composition, under
conditions and for a time sufficient for one or more of: (i) prevention of
infection of the natural
surface by the bacterial pathogen, (ii) inhibition of cell viability or cell
growth of substantially
all planktonic cells of the bacterial pathogen, (iii) inhibition of biofilm
formation by the
bacterial pathogen, and (iv) inhibition of biofilm viability or biofilm growth
of substantially all
biofilm-form cells of the bacterial pathogen, wherein the BT composition
comprises a plurality
of microparticles that comprise a bismuth-thiol (BT) compound, substantially
all of said
microparticles having a volumetric mean diameter of from about 0.4 pm to about
5 pm; and
thereby overcoming antibiotic resistance on the natural surface, wherein if
the BT compound
is BisEDT, then the synergizing or cooperative antimicrobial efficacy
enhancing antibiotic
Date recu/Date Received 2020-07-07
CA2788669
18e
cannot be any of tobramycin, nafcillin, gentamycin, clindamycin, gatifloxacin,
minocycline,
vancomycin, and cefazolin.
Various aspects of the disclosure relate to an antiseptic composition for
treating a
natural surface that contains bacterial biofilm, the antiseptic composition
comprising: (a) at
least one bismuth-thiol (BT) composition that comprises a plurality of
microparticles that
comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles having a
volumetric mean diameter of from about 0.4 pm to about 5 pm; and (b) at least
one antibiotic
compound that is capable of acting synergistically with, or enhancing, the BT
compound,
wherein if the BT compound is BisEDT, then the synergizing or cooperative
antimicrobial
.. efficacy enhancing antibiotic cannot be any of tobramycin, nafcillin,
gentamycin,
clindamycin, gatifloxacin, minocycline, vancomycin, and cefazolin.
Various aspects of the disclosure relate to use of an antiseptic composition
for
treating a natural surface that contains bacterial biofilm, the antiseptic
composition
comprising: (a) at least one bismuth-thiol (BT) composition that comprises a
plurality of
microparticles that comprise a bismuth-thiol (BT) compound, substantially all
of said
microparticles having a volumetric mean diameter of from about 0.4 pm to about
5 pm; and
(b) at least one antibiotic compound that is capable of acting synergistically
with, or
enhancing, the BT compound, wherein if the BT compound is BisEDT, then the
synergizing
or cooperative antimicrobial efficacy enhancing antibiotic cannot be any of
tobramycin,
nafcillin, gentamycin, clindamycin, gatifloxacin, minocycline, vancomycin, and
cefazolin.
Various aspects of the disclosure relate to use of an antiseptic composition
for the
manufacture of a medicament for treating a natural surface that contains
bacterial biofilm,
the antiseptic composition comprising: (a) at least one bismuth-thiol (BT)
composition that
comprises a plurality of microparticles that comprise a bismuth-thiol (BT)
compound,
substantially all of said microparticles having a volumetric mean diameter of
from about 0.4
pm to about 5 pm; and (b) at least one antibiotic compound that is capable of
acting
synergistically with, or enhancing, the BT compound, wherein if the BT
compound is BisEDT,
then the synergizing or cooperative antimicrobial efficacy enhancing
antibiotic cannot be
any of tobramycin, nafcillin, gentamycin, clindamycin, gatifloxacin,
minocycline,
.. vancomycin, and cefazolin.
Date recu/Date Received 2020-07-07
CA2788669
18f
Various aspects of the disclosure relate to a bismuth-thiol composition for
treating a
natural surface that contains bacterial biofilm, wherein the bacterial biofilm
has been
identified to include a bacterial infection by (a) gram positive bacteria, (b)
gram negative
bacteria, and (c) both and (b), and wherein: (i) if the bacterial infection
comprises gram
positive bacteria, then the formulation comprises effective amounts of at
least one BT
compound and at least one antibiotic that is rifamycin, (ii) if the bacterial
infection comprises
gram negative bacteria, then the formulation comprises effective amounts of at
least one BT
compound and amikacin, (iii) if the bacterial infection comprises both gram
positive and gram
negative bacteria, then the formulation comprises effective amounts of one or
a plurality of
BT compounds, rifamycin and amikacin, wherein the BT composition comprises a
plurality
of microparticles that comprise a bismuth-thiol (BT) compound, substantially
all of said
microparticles having a volumetric mean diameter of from about 0.4 pm to about
5 pm, and
wherein if the BT compound is BisEDT, then the synergizing or cooperative
antimicrobial
efficacy enhancing antibiotic cannot be any of tobramycin, nafcillin,
gentamycin,
is clindamycin, gatifloxacin, minocycline, vancomycin, and cefazolin.
Various aspects of the disclosure relate to use of a bismuth-thiol composition
for
treating a natural surface that contains bacterial biofilm, wherein the
bacterial biofilm has
been identified to include a bacterial infection by (a) gram positive
bacteria, (b) gram
negative bacteria, and (c) both (a) and (b), and wherein: (i) if the bacterial
infection comprises
gram positive bacteria, then the formulation comprises effective amounts of at
least one BT
compound and at least one antibiotic that is rifamycin, (ii) if the bacterial
infection comprises
gram negative bacteria, then the formulation comprises effective amounts of at
least one BT
compound and amikacin, (iii) if the bacterial infection comprises both gram
positive and gram
negative bacteria, then the formulation comprises effective amounts of one or
a plurality of
.. BT compounds, rifamycin and amikacin, wherein the BT composition comprises
a plurality
of microparticles that comprise a bismuth-thiol (BT) compound, substantially
all of said
microparticles having a volumetric mean diameter of from about 0.4 pm to about
5 pm, and
wherein if the BT compound is BisEDT, then the synergizing or cooperative
antimicrobial
efficacy enhancing antibiotic cannot be any of tobramycin, nafcillin,
gentamycin,
clindamycin, gatifloxacin, minocycline, vancomycin, and cefazolin.
Various aspects of the disclosure relate to use of a bismuth-thiol composition
for the
manufacture of a medicament for treating a natural surface that contains
bacterial biofilm,
Date recu/Date Received 2020-07-07
CA2788669
18g
wherein the bacterial biofilm has been identified to include a bacterial
infection by (a) gram
positive bacteria, (b) gram negative bacteria, and (c) both (a) and (b), and
wherein: (i) if the
bacterial infection comprises gram positive bacteria, then the formulation
comprises
effective amounts of at least one BT compound and at least one antibiotic that
is rifamycin,
(ii) if the bacterial infection comprises gram negative bacteria, then the
formulation
comprises effective amounts of at least one BT compound and amikacin, (iii) if
the bacterial
infection comprises both gram positive and gram negative bacteria, then the
formulation
comprises effective amounts of one or a plurality of BT compounds, rifamycin
and amikacin,
wherein the BT composition comprises a plurality of microparticles that
comprise a bismuth-
thiol (BT) compound, substantially all of said microparticles having a
volumetric mean
diameter of from about 0.4 pm to about 5 pm, and wherein if the BT compound is
BisEDT,
then the synergizing or cooperative antimicrobial efficacy enhancing
antibiotic cannot be any
of tobramycin, nafcillin, gentamycin, clindamycin, gatifloxacin, minocycline,
vancomycin,
and cefazolin.
These and other aspects of the herein described invention embodiments will be
evident upon reference to the following detailed description and attached
drawings. Aspects
and embodiments of the invention can be modified, if necessary, to employ
concepts of the
various patents, applications and publications to provide yet further
embodiments.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Figure 1 shows surviving numbers (log CFU, colony forming units) from
Pseudomonas aeruginosa colony biofils grown for 24 hours on 10% tryptic soy
agar (TSA)
at 37 C, followed with indicated treatment for 18 hours. Indicated antibiotic
treatments are
TOB, tobramycin 10X KIC, AMK, amikacin 100X MIC, IPM, imipenem 10X MIC, CEF,
cefemine 10X M IC, GIP; ciprofloxaxin 100X MIC, Cpd 2B, compound 2B (Bis-BAL,
1 :1.5).
(MIC,
Date recu/Date Received 2020-07-07
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
19
minimum inhibitory concentration, e.g., lowest concentration that prevents
bacterial growth).
Figure 2 shows surviving numbers (log CFU) from
Staphylococcus aureus colony biofilms grown for 24 hours on 10% tryptic soy
agar, followed by the indicated treatment. Indicated antibiotic treatments are
Rifampicin, RIF 100X MIC; daptomycin, DAP 320X MIC; minocycline, MIN
100X MIC; ampicillin, AMC 10X MIC; vancomycin, VAN 10X MIC; Cpd 2B,
compound 2B (Bis-BAL, 1:1.5), Cpd 8-2, compound 8-2 (Bis-Pyr/BDT (1:1/0.5).
Figure 3 shows scratch closure over time of keratinocytes
exposed to biofilms. (*) Significantly different from control (P<0.001).
Figure 4A and 4B show the subinhibitory BisEDT reversing
antibiotic-resistance to several antibiotics. Effects of antibiotics with and
without BisEDT (0.05 pg/ml) on a lawn of MRSA (Methicillin-resistant S.
aureus)
is shown. Panel A shows standard antibiotic-soaked discs alone, and Panel B
shows discs combined with a BisEDT (BE). [GM= gentamicin, CZ= cefazolin,
FEP= cefepime, IPM= imipenim,SAM= ampicillin/ sulbactam, LVX=
levofloxacin.
Figure 5 shows the effect of BisEDT and antibiotics on biofilm
formation. S. epidermidis grown in TSB + 2% glucose in polystyrene plates for
48h at 37 C. Gatifloxacin (OF), clindamycin (CM), minocycline (MC),
gentamicin (GM), vancomycin (VM), cefazolin (CZ), nafcillin (NC), and
rifampicin (RP). Results were expressed as the mean change in the BPC (in
serial 2-fold dilution steps) at 0.25 M BisEDT (n=3).
Figure 6 shows the effect of BisEDT and antibiotics on growth of
S. epidermidis grown in TSB plus 2% glucose for 48h at 37 C. Results are
expressed as the mean change in MIC (dilution steps) with increasing BisEDT
(n=3). See legend in Figure 5 for antibiotic definitions.
Figure 7 is a bar graph showing the mean S. aureus bacteria
levels detected on the bone and hardware samples from open fractures in an in
vivo rat model following treatment with three BT formulations, Bis-EDT , MB-11
and MB-8-2 with or without Cefazolin antibiotic treatment. Standard errors of
the mean are shown as error bars. Animals euthanized early are not excluded
from the analysis, however samples from one animal in group 2 have been
excluded due to gross contamination.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
DETAILED DESCRIPTION
Particular embodiments of the invention disclosed herein are
based on the surprising discovery that certain bismuth-thiol (BT) compounds as
provided herein (in certain embodiments including BT microparticles having a
5 volumetric mean diameter of from about 0.4 [tm to about 5 m), but not
certain
other BT compounds (even if provided as microparticles), exhibited potent
antiseptic, antibacterial and/or anti-biofilm activity against particular
bacteria,
including bacteria associated with a number of clinically significant
infections
including infections that can comprise bacterial biofilms.
10 Unexpectedly, not all BT compounds were uniformly effective
against such bacteria in a predictable fashion, but instead exhibited
different
potencies depending on the target bacterial species. In particular and as
described herein, certain BT compounds (preferably including BT microparticles
having a volumetric mean diameter of from about 0.4 m to about 5 pm) were
15 found to exhibit higher potency against gram-negative bacteria, while
certain
other BT compounds (preferably including BT microparticles having a
volumetric mean diameter of from about 0.4 gm to about 5 pm) were found to
exhibit greater potency against gram-positive bacteria, in a manner that,
according to non-limiting theory, may for the first time afford clinically
relevant
20 strategies for the management of bacterial infections, including
bacterial biofilm
infections.
Additionally, and as described in greater detail below, certain
embodiments of the invention described herein relate to surprising advantages
that are provided by novel bismuth-thiol (BT) compositions that, as disclosed
herein, can be made in preparations that comprise a plurality of BT
microparticles that are substantially monodisperse with respect to particle
size
(e.g., having volumetric mean diameter from about 0.4 pm to about 5 pm). In
certain of these and related embodiments, the microparticulate BT is not
provided as a component of a lipid vesicle or liposome such as a multilamellar
=
phosphocholine-cholesterol liposome or other multilamellar or unilamellar
liposomal vesicle.
As also disclosed herein, with respect to certain embodiments, it
has been discovered that antibacterial and anti-biofilm efficacies of certain
antibiotics, which antibiotics have previously been found to lack potent
therapeutic effect against such bacterial infections, may be significantly
enhanced (e.g., increased in a statistically significant manner) by treating
the
infection (e.g., by direct application on or in an infected site such as a
natural
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
21
surface) with one or more of these antibiotics in concert, simultaneously or
sequentially and in either order, with a selected BT compound. In a manner
that could not be predicted prior to the present disclosure, certain BT
compounds can be combined with certain antibiotics to provide a synergizing or
enhancing combination as provided herein with respect to antibacterial and/or
anti-biofilm activity against certain bacterial species or bacterial strains.
The
unpredicted nature of such combinations, as described in greater detail below,
is evidenced by the observations that while certain BT/antibiotic combinations
acted synergistically or exhibited enhancement against certain bacteria,
certain
other BT/antibiotic combinations failed to exhibit such synergistic or
enhanced
antibacterial and/or anti-biofilm activity.
According to these and related embodiments, the antibiotic and
the BT compound may be administered simultaneously or sequentially and in
either order, and it is noteworthy that the specific synergizing or enhancing
combinations of one or more antibiotic and one or more BT compound as
disclosed herein for treatment of a particular infection (e.g., a biofilm
formed by
gram-negative or gram-positive bacteria) did not exhibit predictable (e.g.,
merely additive) activities but instead aCted in an unexpectedly synergistic
or
enhancing (e.g., supra-additive) fashion, as a function of the selected
antibiotic,
the selected BT compound and the specifically identified target bacteria.
For example, by way of illustration and not limitation, disclosed
herein in the context of a wide variety of actually or potentially microbially
infected natural surfaces, and further in the context of improved
substantially
monodisperse microparticulate BT formulations, either or both of a particular
antibiotic compound and a particular BT compound may exert limited
antibacterial effects when used alone against a particular bacterial strain or
species, but the combination of both the antibiotic compound and the BT
compound exerts a potent antibacterial effect against the same bacterial
strain
or species, which effect is greater in magnitude (with statistical
significance)
than the simple sum of the effects of each compound when used alone, and is
therefore believed according to non-limiting theory to reflect antibiotic-BT
synergy (e.g., FICI < 0.5) or an enhancing effect (e.g., 0.5 < FICI < 1.0) of
the
BT on the antibiotic potency and/or of the antibiotic on the BT potency.
Accordingly, not every BT compound may synergize with, or be enhancing for,
every antibiotic, and not every antibiotic may synergize with, or be enhancing
for, every BT compound, such that antibiotic-BT synergy and BT-antibiotic
enhancement generally are not predictable. Instead, and according to certain
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
22
embodiments as disclosed herein, specific combinations of synergizing or
enhancing antibiotic and BT compounds surprisingly confer potent antibacterial
effects against particular bacteria, including in particular environments such
as
natural surfaces as described herein, and further including in certain
situations
antibacterial effects against biofilms formed by the particular bacteria.
That is, certain BT-synergizing antibiotics are described herein,
which includes an antibiotic that is capable of acting synergistically (FICI <
0.5)
with at least one BT composition that comprises at least one BT compound as
provided herein, where such synergy manifests as a detectable effect that is
greater (i.e., in a statistically significant manner relative to an
appropriate
control condition) in magnitude than the effect that can be detected when the
antibiotic is present but the BT compound is absent, and/or when the BT
compound is present but the antibiotic is absent. Similarly, certain BT-
antibiotic
combinations exhibit enhancement (0.5 < FICI < 1.0), where such enhancement
manifests as a detectable effect that is greater (i.e., in a statistically
significant
manner relative to an appropriate control condition) in magnitude than the
effect
that can be detected when the antibiotic is present but the BT compound is
absent, and/or when the BT compound is present but the antibiotic is absent.
Examples of such a detectable effect may in certain embodiments
include (i) prevention of infection by a bacterial pathogen, (ii) inhibition
of cell
viability or cell growth of substantially all planktonic cells of a bacterial
pathogen, (iii) inhibition of biofilm formation by a bacterial pathogen, and
(iv)
inhibition of biofilm viability or biofilm growth of substantially all biofilm-
form
cells of a bacterial pathogen, but the invention is not intended to be so
limited,
such that in other contemplated embodiments antibiotic-BT synergy may
manifest as one or more detectable effects that may include alteration (e.g.,
a
statistically significant increase or decrease) of one or more other
clinically
significant parameters, for example, the degree of resistance or sensitivity
of a
bacterial pathogen to one or more antibiotics or other drugs or chemical
agents,
the degree of resistance or sensitivity of a bacterial pathogen to one or more
chemical, physical or mechanical conditions (e.g., pH, ionic strength,
temperature, pressure), and/or the degree of resistance or sensitivity of a
bacterial pathogen to one or more biological agents (e.g., a virus, another
bacterium, a biologically active polynucleotide, an immunocyte or an
immunocyte product such as an antibody, cytokine, chemokine, enzyme
including degradative enzymes, membrane-disrupting protein, a free radical
such as a reactive oxygen species, or the like).
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
23
Persons familiar with the art will appreciate these and a variety of
other criteria by which the effects of particular agents on the structure,
function
and/or activity of a bacterial population may be determined (e.g., Coico et
al.
(Eds.), Current Protocols in Microbiology, 2008, John Wiley & Sons, Hoboken,
NJ; Schwalbe et al, Antimicrobial Susceptibility Testing Protocols, 2007, CRC
Press, Boca Raton, FL), for purposes of ascertaining antibiotic-BT synergy or
enhancement which, as provided herein, is present when the effects of the
synergizing or enhancing antibiotic-BT combination exceed the mere sum of the
effects observed when one component of the combination is not present.
For example, in certain embodiments synergy may be determined
by determining an antibacterial effect such as those described herein using
various concentrations of candidate agents (e.g., a BT and an antibiotic
individually and in combination) to calculate a fractional inhibitory
concentration
index (FICI) and a fractional bactericidal concentration index (FBCI),
according
to Eliopoulos et al. (Eliopoulos and Moellering, (1996) Antimicrobial
combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 330-
96, Williams and Wilkins, Baltimore, MD, USA). Synergy may be defined as an
FICI or FBCI index of 50.5, and antagonism at >4. (e.g., Odds, FC (2003)
Synergy, antagonism, and what the chequerboard puts between them. Journal
of Antimicrobial Chemotherapy 52:1). Synergy may also be defined
conventionally as NI-fold decrease in antibiotic concentration, or
alternatively,
using fractional inhibitory concentration (FIC) as described, e.g., by
Hollander et
al. (1998 Antimicrob. Agents Chemother. 42:744). In certain embodiments,
synergy may be defined as an effect that results from a combination of two
drugs (e.g., an antibiotic and a BT composition) wherein the effect of the
combination is greater (e.g., in a statistically significant manner) than it
would
be if the concentration of the second drug is replaced by the first drug.
Accordingly as described herein and in certain preferred
embodiments, a combination of BT and antibiotic will be understood to
synergize when a FICI value that is less than or equal to 0.5 is observed.
(Odds, 2003). As also described herein, in certain other preferred
embodiments and according to non-limiting theory, it is disclosed that certain
BT-antibiotic combinations may exhibit a FICI value between 0.5 and 1.0 that
signifies a high potential for such synergy, and which may be observed using
non-optimal concentrations of at least one BT and at least one antibiotic that
exhibit unilateral or mutually enhanced cooperative antimicrobial efficacy.
Such
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
24
an effect may also be referred to herein as "enhanced" antibiotic activity or
"enhanced" BT activity.
Enhanced antibiotic and/or BT activity may be detected according
to certain embodiments when the presence both (i) of at least one BT at a
concentration that is less (in a statistically significant manner) than the
characteristic minimum inhibitory concentration (MIC) for that BT for a given
target microbe (e.g., a given bacterial species or strain), and (ii) of at
least one
antibiotic at a concentration that is less (in a statistically significant
manner)
than the characteristic IC50 (concentration that inhibits the growth of 50% of
a
microbial population; e.g., Soothill et al., 1992 J Antimicrob Chemother
29(2):137) and/or that is less than the biofilm-prevention concentration (BPC)
of
that antibiotic for the given target microbe, results in enhanced (in a
statistically
significant manner) antimicrobial efficacy of the BT-antibiotic combination
relative to the antimicrobial effect that would be observed if either
antimicrobial
agent (e.g., the BT or the antibiotic) were used at the same concentration in
the
absence of the other antimicrobial agent (e.g., the antibiotic or the BT). In
preferred embodiments, "enhanced" antibiotic and/or BT activity is present
when a FICI value that is less than or equal to 1.0, and greater than 0.5, is
determined.
As will be appreciated by the skilled person based on the present
disclosure, in certain embodiments synergistic or enhanced antibiotic and/or
BT
activity may be determined according to methods known in the art, such as
using Loewe additivity-based models (e.g., FIC index, Greco model), or Bliss
independence based models (e.g., non-parametric and semi-parametric
models) or other methods described herein and known in the art (e.g.,
Meletiadis et al., 2005 Medical Mycology 43:133-152). Illustrative methods for
determining synergy or enhanced antibiotic and/or BT activity are thus
described, for instance, in Meletiadis et al., 2005 Medical Mycology 43:133-
152
and references cited therein (see also, Meletiadis et al., 2002 Rev Med
Microbiol 13:101-117; White etal., 1996 Antimicrob Agents Chemother
40:1914-1918; Mouton et al., 1999 Antimicrob Agents Chemother 43:2473-
2478).
Certain other embodiments contemplate specific combinations of
one or more antibiotic and one or more BT compound as disclosed herein that
may exhibit synergizing or enhancing effects in vivo for treatment of a
particular
infection (e.g., a biofilm formed by gram-negative or gram-positive bacteria),
even where the BT compound(s) and antibiotic(s) did not exhibit predictable
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
(e.g., merely additive) activities in vivo but instead acted in an
unexpectedly
synergistic or enhancing (e.g., supra-additive; or conferring an effect when
two
or more such agents are present in combination that is greater (e.g., in a
statistically significant manner) than the effect that is obtained if the
5 concentration of the second agent is replaced by the first agent)
fashion, as a
function of the selected antibiotic, the selected BT compound and one or more
of the specifically identified target bacterial species of which the infection
is
comprised. It will therefore be appreciated, according to these and related
embodiments, that in certain in vivo situations FICI or FBCI values (which are
10 determined in vitro) may not be readily available, but that instead BT-
antibiotic
synergizing or enhancing effects may be determined in a manner afforded by
the quantifiable metrics of the infection.
For example, in one embodiment, such as in the in vivo open
fracture Rattus norvegicus femur critical defect model as described in Example
15 11, a statistically significant reduction in bacterial counts observed
post-
treatment for the BT-antibiotic combination as compared to the antibiotic
treatment or BT compound alone, is an indication of synergizing or enhancing
effects. Statistical significance can be determined using methods well-known
to
the skilled person. In certain other embodiments, a reduction observed in this
20 or other in vivo models by at least 5%, 10%, 20%, 30%, 40%, or 50% of
bacterial counts observed in the injury post-treatment for the BT-antibiotic
combination as compared to the antibiotic treatment or BT compound alone is
considered an indication of synergizing or enhancing effects.
Other exemplary indicia of in vivo infections may be determined
25 according to established methodologies that have been developed for
quantification of the severity of the infection, such as a variety of wound
scoring
systems known to the skilled person (see e.g., scoring systems reviewed in
European Wound Management Association (EWMA), Position Document:
Identifying criteria for wound infection. London: MEP Ltd, 2005). Illustrative
wound scoring systems that may be used in assessing synergistic or
enhancement activity of BT-antibiotic combinations as described herein include
ASEPSIS (Wilson AP, J Hosp Infect 1995; 29(2): 81-86; Wilson et al., Lancet
1986; 1: 311-13), the Southampton Wound Assessment Scale (Bailey IS,
Karran SE, Toyn K, et al. BMJ 1992; 304: 469-71). See also, Horan TC,
Gaynes P, Martone WJ, et al. ,1992 Infect Control Hosp Epidemiol 1992; 13:
606-08. Additionally, recognized clinical indicia of wound healing known to
the
skilled clinician may also be measured in the presence or absence of BT
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
26
compounds and/or antibiotics, such as wound size, depth, granulation tissue
condition, infection, etc. Accordingly, and based on the present disclosure,
the
skilled person will readily appreciate a variety of methods for determining
whether a BT composition ¨antibiotic combination alters (e.g., increases or
decreases in a statistically significant manner relative to appropriate
controls) in
vivo wound healing.
In view of these and related embodiments, there are provided
herein a wide variety of methods for treating microbially infected natural
surfaces such as surfaces that support or contain bacterial biofilms, with an
effective amount (e.g., in certain embodiments a therapeutically effective
amount) of a composition or formulation that comprises one or more BT
compounds and, optionally, one or more antibiotic compounds, such as one or
more synergizing antibiotics, or one or more enhancing antibiotics, as
provided
herein. It will be appreciated that based on the present disclosure, certain
antibiotics are now contemplated for use in the treatment of given types of
infections, where such antibiotics had previously been viewed by persons
familiar with the art as ineffective against infections of the same type.
Certain embodiments thus contemplate compositions that
comprise one or more BT compounds for use as antiseptics. An antiseptic is a
substance that kills or prevents the growth of microorganisms, and may be
typically applied to living tissue, distinguishing the class from
disinfectants,
which are usually applied to inanimate objects (Goodman and Gilman's "The
Pharmacological Basis of Therapeutics", Seventh Edition, Gilman et al.,
editors, 1985, Macmillan Publishing Co., (hereafter, Goodman and Gilman") pp.
959-960). Common examples of antiseptics are ethyl alcohol and tincture of
iodine. Germicides include antiseptics that kill microbes such as microbial
pathogens.
Certain embodiments described herein may contemplate
compositions that comprise one or more BT compounds and one or more
antibiotic compound (e.g., a synergizing antibiotic and/or an enhancing
antibiotic as provided herein). Antibiotics are known in the art and typically
comprise a drug made from a compound produced by one species of
microorganism to kill another species of microorganism, or a synthetic product
having an identical or similar chemical structure and mechanism of action,
e.g.,
a drug that destroys microorganisms within or on the body of a living
organism,
including such drug when applied topically. Among embodiments disclosed
herein are those in which an antibiotic may belong to one of the following
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
27
classes: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones,
glycopeptide antibiotics, lincosamides (e.g., clindamycin), penicillinase-
resistant
penicillins, and aminopenicillins. Antibiotics thus may include, but need not
be
limited to, oxacillin, piperacillin, cefuroxime, cefotaxime, cefepime,
imipenem,
aztreonam, streptomycin, tobramycin, tetracycline, minocycline, ciprofloxacin,
levofloxacin, erythromycin, linezolid, phosphomycin, capreomycin, isoniazid,
ansamycin, carbacephem, monobactam, nitrofuran, penicillin, quinolone,
sulfonamide, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol,
Ethionamide, lsoniazid, Pyrazinamide, Rifampicin, Rifampin, Rifabutin,
Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin,
Fusidic acid, Linezolid, Metronidazole, Mupirocin, Platensimycin,
Quinupristin,
Dalfopristin, Rifaximin, Thiamphenicol, Tinidazole, aminoglycoside, beta-
lactam, penicillin, cephalosporin, carbapenem, fluroquinolone, ketolide,
lincosamide, macrolide, oxazolidinone, stretogramin, sulphonamide,
tetracycline, glycylcycline, methicillin, vancomycin, naficilin, gentamicin,
ampicillin , chloramphenicol, doxycycline, tobramycin, amikacin, arbekacin,
gentamicin, kanamycin, neomycin, netilmicin, paromomycin,
rhodostreptomycin, streptomycin, tobramycin, apramycin, clindamicin,
gatifloxacin, aminopenicillin, and others known to the art. Compendia of these
and other clinically useful antibiotics are available and known to those
familiar
with the art (e.g., Washington University School of Medicine, The Washington
Manual of Medical Therapeutics (32"d Ed.), 2007 Lippincott, Williams and
Wilkins, Philadelphia, PA; Hauser, AL, Antibiotic Basics for Clinicians, 2007
Lippincott, Williams and Wilkins, Philadelphia, PA).
An exemplary class of antibiotics for use with one or more BT
compounds in certain herein disclosed embodiments is the aminoglycoside
class of antibiotics, which are reviewed in Edson RS, Terrell CL. The
aminoglycosides. Mayo Clin Proc. 1999 May; 74(5):519-28. This class of
antibiotics inhibits bacterial growth by impairing bacterial protein
synthesis,
through binding and inactivation of bacterial ribosomal subunits. In addition
to
such bacteriostatic properties, aminoglycosides also exhibit bacteriocidal
effects through disruption of cell walls in gram-negative bacteria.
Aminoglycoside antibiotics include gentamicin, amikacin,
streptomycin, and others, and are generally regarded as useful in the
treatment
of gram-negative bacteria, mycobacteria and other microbial pathogens,
although cases of resistant strains have been reported. The aminoglycosides
are not absorbed through the digestive tract and so are not generally regarded
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
28
as being amenable to oral formulations. Amikacin, for example, although often
effective against gentamicin-resistant bacterial strains, is typically
administered
intravenously or intramuscularly, which can cause pain in the patient.
Additionally, toxicities associated with aminoglycoside antibiotics such as
amikacin can lead to kidney damage and/or irreversible hearing loss.
Despite these properties, certain embodiments disclosed herein
contemplate oral administration of a synergizing BT/antibiotic combination
(e.g.,
where the antibiotic need not be limited to an aminoglycoside) for instance,
for
treatment of an epithelial tissue surface at one or more locations along the
oral
cavity, gastrointestinal tract/ alimentary canal. Also contemplated in certain
other embodiments may be use of compositions and methods described herein
as disinfectants, which refers to preparations that kill, or block the growth
of,
microbes on an external surface of an inanimate object.
As also described elsewhere herein, a BT compound may be a
.. composition that comprises bismuth or a bismuth salt and a thiol- (e.g., -
SH, or
sulfhydryl) containing compound, including those that are described (including
their methods of preparation) in Domenico et al., 1997 Antimicrob. Agent.
Chemother. 41(8):1697-1703, Domenico et al., 2001 Antimicob.Agent.
Chemother. 45(5):1417-1421, and in U.S. RE37,793, U.S. 6,248,371, U.S.
6,086,921, and U.S. 6,380,248; see also, e.g., U.S. 6,582,719. Certain
embodiments are not so limited, however, and may contemplate other BT
compounds that comprise bismuth. or a bismuth salt and a thiol-containing
compound. The thiol-containing compound may contain one, two, three, four,
five, six or more thiol (e.g., -SH) groups. In preferred embodiments the BT
compound comprises bismuth in association with the thiol-containing compound
via ionic bonding and/or as a coordination complex, while in some other
embodiments bismuth may be associated with the thiol-containing compound
via covalent bonding such as may be found in an organometallic compound.
Certain contemplated embodiments, however, expressly exclude a BT
compound that is an organometallic compound such as a compound in which
bismuth is found in covalent linkage to an organic moiety.
Exemplary BT compounds are shown in Table 1:
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
29
TABLE 1
Exemplary BT Compounds*
1) CPD 1B-1 Bis-EDT (1:1) BiC2I-14S2
2) CPD 1B-2 Bis-EDT (1:1.5) B1C3H6S3
3) CPD 1B-3 Bis-EDT (1:1.5) BiC3H6S3
4) CPD 1C Bis-EDT (1:1.5) BiC3H6S3
5) CPD 2A Bis-Bal (1:1) BiC3H6S20
6) CPD 2B Bis-Bal (1:1.5) BiC461-1901.6S3
7) CPD 3A Bis-Pyr (1:1.5) B1C7.51-16N1.501.5S1.5
8) CPD 3B Bis-Pyr (1:3) BiC161-112N303S3
9) CPD 4 Bis-Ery (1:1.5) BiC6F11203S3
10) CPD 5 Bis-Tol (1:1.5) BiC1o6H9S3
11) CPD 6 Bis-BDT (1:1.5) B1C6H12S3
12) CPD 7 Bis-PDT (1:1.5) BiC4.61-19S3
13) CPD 8-1 Bis-Pyr/BDT (1:1/1)
14) CPD 8-2 Bis-Pyr/BDT (1:1/0.5)
15) CPD 9 Bis-2hydroxy, propane thiol (1:3)
16) CPD 10 Bis-Pyr/Bal (1:1/0.5)
17) CPD 11 Bis-Pyr/EDT (1:1/0.5)
18) CPD 12 Bis-Pyr/Tol (1:1/0.5)
19) CPD 13 Bis-Pyr/PDT (1:1/0.5)
20) CPD 14 Bis-Pyr/Ery (1:1/0.5)
21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)
*Shown are atomic ratios relative to a single bismuth atom, for comparison,
based
on the stoichiometric ratios of the reactants used and the known propensity of
bismuth to form trivalent complexes with sulfur containing compounds. Atomic
ratios as shown may not be accurate molecular formulae for all species in a
given
preparation. The numbers in parenthesis are the ratios of bismuth to one (or
more)
thiol agents. (e.g. Bi:thio11/thio12) "CPD", compound.
BT compounds for use in certain of the presently disclosed
embodiments may be prepared according to established procedures (e.g., U.S.
RE37,793, U.S. 6,248,371, U.S. 6,086,921, and U.S. 6,380,248; Domenico et
al., 1997 Antimicrob. Agent. Chemother. 41(8):1697-1703, Domenico et al.,
2001 Antimicob.Agent. Chemother. 45(5):1417-1421) and in certain other
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
embodiments BT compounds may also be prepared according to
methodologies described herein. Certain preferred embodiments thus
contemplate the herein described synthetic methods for preparing BT
compounds, and in particular for obtaining BT compounds in substantially
5 monodisperse microparticulate form, in which an acidic aqueous bismuth
solution that contains dissolved bismuth at a concentration of at least 50 mM,
at
least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300
mM, at least 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at
least 700 mM, at least 800 mM, at least 900 mM or at least 1 M and that lacks
a
10 hydrophilic, polar or organic solubilizer is admixed with ethanol to
obtain a first
ethanolic solution, which is reacted with a second ethanolic solution
comprising
a thiol-containing compound to obtain a reaction solution, wherein the thiol-
containing compound is present in the reaction solution at a molar ratio of
from
about 1.3 to about 3:1 relative to the bismuth, under conditions and for a
time
15 sufficient for formation of a precipitate which comprises the
microparticles
comprising the BT compound (such as the conditions of concentration, solvent
strength, temperature, pH, mixing and/or pressure, and the like, as described
herein and as will be appreciated by the skilled person based on the present
disclosure).
20 Accordingly, exemplary BTs include compound 1B-1, Bis-EDT
(bismuth-1,2-ethane dithiol, reactants at 1:1); compound 1B-2, Bis-EDT
(1:1.5);
compound 1B-3, Bis-EDT (1:1.5); compound 1C, Bis-EDT (soluble Bi
preparation, 1:1.5); compound 2A, Bis-Bal (bismuth-British anti-Lewisite
(bismuth-dimercaprol, bismuth-2,3-dimercaptopropanol), 1:1); compound 2B,
25 Bis-Bal (1:1.5); compound 3A Bis-Pyr (bismuth-pyrithione, 1:1.5); compound
3B
Bis-Pyr (1:3); compound 4, Bis-Ery (bismuth-dithioerythritol, 1:1.5); compound
5, Bis-Tol (bismuth-3,4-dimercaptotoluene, 1:1.5); compound 6, Bis-BDT
(bismuth-2,3-butanedithiol, 1:1.5); compound 7, Bis-PDT (bismuth-1,3-
propanedithiol, 1:1.5); compound 8-1 Bis-Pyr/BDT (1:1/1); compound 8-2, Bis-
30 Pyr/BDT (1:1/0.5); compound 9, Bis-2-hydroxy, propane thiol (bismuth-1-
mercapto-2-propanol, 1:3); compound 10, Bis-Pyr/Bal (1:1/0.5); compound 11,
Bis-Pyr/EDT (1:1/0.5); compound 12 Bis-Pyr/Tol (1:1/0.5); compound 13, Bis-
Pyr/PDT (1:1/0.5); compound 14 Bis-Pyr/Ery (1:1/0.5); compound 15, Bis-
EDT/2-hydroxy, propane thiol (1:1/1) (see, e.g., Table 1).
Without wishing to be bound by theory, it is believed that the
presently disclosed methods of preparing a BT compound, which in certain
preferred embodiments may comprise preparing or obtaining an acidic aqueous
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
31
liquid solution that comprises bismuth such as an aqueous nitric acid solution
comprising bismuth nitrate, may desirably yield compositions comprising BT
compounds where such compositions have one or more desirable properties,
including ease of large-scale production, improved product purity, uniformity
or
consistency (including uniformity in particle size), or other properties
useful in
the preparation and/or administration of the present topical formulations.
In particular embodiments it has been discovered that BT
compositions, prepared according to the methods described herein for the first
time, exhibit an advantageous degree of homogeneity with respect to their
occurrence as a substantially monodisperse suspension of microparticles each
having a volumetric mean diameter (VMD) according to certain presently
preferred embodiments of from about 0.4 pm to about 5 pm. Measures of
particle size can be referred to as volumetric mean diameter (VMD), mass
median diameter (MMD), or mass median aerodynamic diameter (MMAD).
These measurements may be made, for example, by impaction (MMD and
MMAD) or by laser (VMD) characterization. For liquid particles, VMD, MMD
and MMAD may be the same if environmental conditions are maintained, e.g.,
standard humidity. However, if humidity is not maintained, MMD and MMAD
determinations will be smaller than VMD due to dehydration during impactor
measurements. For the purposes of this description, VMD, MMD and MMAD
measurements are considered to be under standard conditions such that
descriptions of VMD, MMD and MMAD will be comparable. Similarly, dry
powder particle size determinations in MMD, and MMAD are also considered
comparable.
As described herein, preferred embodiments relate to a
substantially monodisperse suspension of BT-containing microparticles.
Generation of a defined BT particle size with limited geometric standard
deviation (GSD) may, for instance, optimize BT deposition, accessibility to
desired target sites in or on a natural surface, and/or tolerability by a
subject to
whom the BT microparticles are administered. Narrow GSD limits the number
of particles outside the desired VMD or MMAD size range.
In one embodiment, a liquid or aerosol suspension of
microparticles containing one or more BT compounds disclosed herein is
provided having a VMD from about 0.5 microns to about 5 microns. In another
embodiment, a liquid or aerosol suspension having a VMD or MMAD from
about 0.7 microns to about 4.0 microns is provided. In another embodiment, a
liquid or aerosol suspension having aVMD or MMAD from about 1.0 micron to
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
32
about 3.0 microns is provided. In certain other preferred embodiments there is
provided a liquid suspension comprising one or a plurality of BT compound
particles of from about 0.1 to about 5.0 microns VMD, or of from about 0.1,
about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8 or
.. about 0.9 microns to about 1.0, about 1.5, about 2.0, about 2.5, about 3.0,
about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5,
about 7.0, about 7.5 or about 8.0 microns, the particle comprising a BT
compound prepared as described herein.
Accordingly and in certain preferred embodiments, a BT
preparation described for the first time herein which is "substantially"
monodisperse, for example, a BT composition that comprises a BT compound
in microparticulate form wherein "substantially" all of the microparticles
have a
volumetric mean diameter (VMD) within a specified range (e.g., from about 0.4
i_tm to about 5 includes those compositions in which at least 80%, 85%,
90%, 91%, 92%, 93%, or 94%, more preferably at least 95%, 96%, 97%, 98%,
99% or more of the particles have a VMD that is within the recited size range.
These and related properties of BT compositions prepared
according to the herein described synthetic methods offer unprecedented
advantages over previously described BTs, including lower cost and ease of
production, and uniformity within the composition that may permit its
characterization in a manner that facilitates regulatory compliance according
to
one or more of pharmaceutical, formulary and cosmeceutical standards.
Additionally or alternatively, the herein described substantially
monodisperse BT microparticles may advantageously be produced without the
need for nnicronization, i.e., without the expensive and labor-intensive
milling or
supercritical fluid processing or other equipment and procedures that are
typically used to generate microparticles (e.g., Martin et al. 2008 Adv. Drug
Deliv. Rev. 60(3):339; Moribe et al., 2008 Adv. Drug Deily. Rev. 60(3):328;
Cape et al., 2008 Pharm. Res. 25(9):1967; Rasenack et al. 2004 Pharm. Dev.
Technol. 9(1):1-13). Hence, the present embodiments offer beneficial effects
of
substantially uniform microparticulate preparations, including without
limitation
enhanced and substantially uniform solubilization properties, suitability for
desired administration forms such as oral, inhaled or dermatological/ skin
wound topical forms, increased bioavailability and other beneficial
properties.
The BT compound microparticulate suspension can be
administered as aqueous formulations, as suspensions or solutions in aqueous
as well as organic solvents including halogenated hydrocarbon propellants, as
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
33
dry powders, or in other forms as elaborated below, including preparations
that
contain wetting agents, surfactants, mineral oil or other ingredients or
additives
as may be known to those familiar with formulary, for example, to maintain
individual microparticles in suspension. Aqueous formulations may be
aerosolized by liquid nebulizers employing, for instance, either hydraulic or
ultrasonic atomization. Propellant-based systems may use suitable pressurized
dispensers. Dry powders may use dry powder dispersion devices, which are
capable of dispersing the BT-containing microparticles effectively. A desired
particle size and distribution may be obtained by choosing an appropriate
device.
As also noted above, also provided herein according to certain
embodiments is a method for preparing a b'ismuth-thiol (BT) composition that
comprises a plurality of microparticles that comprise a BT compound,
substantially all of such microparticles having a volumetric mean diameter
(VMD) of from about 0.1 to about 8 microns, and in certain preferred
embodiments from about 0.4 microns to about 5 microns.
In general terms, the method comprises the steps of (a) admixing,
under conditions and for a time sufficient to obtain a solution that is
substantially free of a solid precipitate, (i) an acidic aqueous solution that
comprises a bismuth salt comprising bismuth at a concentration of at least 50
mM and that lacks a hydrophilic, polar or organic solubilizer, with (ii)
ethanol in
an amount sufficient to obtain an admixture that comprises at least about 5%,
10%, 15%, 20%, 25% or 30%, and preferably about 25% ethanol by volume;
and (b) adding to the admixture of (a) an ethanolic solution comprising a
thiol-
containing compound to obtain a reaction solution, wherein the thiol-
containing
compound is present in the reaction solution at a molar ratio of from about
1:3
to about 3:1 relative to the bismuth, under conditions and for a time
sufficient for
formation of a precipitate which comprises the BT compound.
In certain preferred embodiments the bismuth salt may be
Bi(NO3)3, but it will be appreciated according to the present disclosure that
bismuth may also be provided in other forms. In certain embodiments the
bismuth concentration in the acidic aqueous solution may be at least 100 mM,
at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least
350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM,
at least 800 mM, at least 900 mM or at least 1 M. In certain embodiments the
acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5%
bismuth by weight. The acidic aqueous solution may in certain preferred
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
34
embodiments comprise at least 5% or more nitric acid by weight, and in certain
other embodiments the acidic aqueous solution may comprise at least 0.5%, at
least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least
3.5%, at
least 4%, at least 4.5% or at least 5% nitric acid by weight.
The thiol-containing compound may be any thiol-containing
compound as described herein, and in certain embodiments may comprise one
or more of 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,
dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-
propanedithiol, 2-
hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol and
dithiothreitol.
Other exemplary thiol-containing compounds include alpha-lipoic acid,
methanethiol (CH3SH [m-mercaptan]), ethanethiol (C2H5SH [e- mercaptan]), 1-
propanethiol (C3H7SH [n-P mercaptan]), 2-Propanethiol (CH3CH(SH)CH3 [2C3
mercaptan]), butanethiol (C4H9SH ([n-butyl mercaptan]), tert-butyl mercaptan
(C(CH3)3SH [t-butyl mercaptan]), pentanethiols (C5H11SH [pentyl mercaptan]),
coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol,
dithiothreitol, dithioerythritol, 2-mercaptoindole, transglutaminase and any
of the
following thiol compounds available from Sigma-Aldrich (St. Louis, MO): (11-
mercaptoundecyl)hexa(ethylene glycol), (11-mercaptoundecyl)tetra(ethylene
glycol), (11-mercaptoundecyl)tetra(ethylene glycol) functionalized gold
nanoparticles, 1,1',4`,1"-terpheny1-4-thiol, 1,11-undecanedithiol, 1,16-
hexadecanedithiol, 1,2-ethanedithiol technical grade, 1,3-propanedithiol, 1,4-
benzenedimethanethiol, 1,4-butanedithiol, 1,4-butanedithiol diacetate, 1,5-
pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol,
adamantanethiol, 1-butanethiol, 1-decanethiol, 1-dodecanethiol, 1-
heptanethiol, 1-heptanethiol purum, 1-hexadecanethiol, 1-hexanethiol, 1-
mercapto-(triethylene glycol), 1-mercapto-(triethylene glycol) methyl ether
functionalized gold nanoparticles, 1-nnercapto-2-propanol, 1-nonanethiol, 1-
octadecanethiol, 1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-
pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol purum, 1-
undecanethiol, 11-(1H-pyrrol-1-yl)undecane-1-thiol, 11-amino-1-undecanethiol
hydrochloride, 11-bromo-1-undecanethiol, 11-mercapto-1-undecanol, 11-
mercapto-1-undecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecanoic
acid, 11-mercaptoundecyl trifluoroacetate, 11-mercaptoundecylphosphoric
acid, 12-mercaptododecanoic acid, 12-mercaptododecanoic acid, 15-
mercaptopentadecanoic acid, 16-mercaptohexadecanoic acid, 16-
mercaptohexadecanoic acid, 1H,1H,2H,2H-perfluorodecanethiol, 2,2'-
(ethylenedioxy)diethanethiol, 2,3-butanedithiol, 2-butanethiol, 2-
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
ethylhexanethiol, 2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-
phenylethanethiol, 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol purum, 3-
(dimethoxymethylsily1)-1-propanethiol, 3-chloro-1-propanethiol, 3-mercapto-1-
propanol, 3-mercapto-2-butanol, 3-mercapto-N-nonylpropionamide, 3-
5
mercaptopropionic acid, 3-mercaptopropyl-functionalized silica gel, 3-methyl-
1-butanethiol, 4,4'-bis(mercaptomethyl)biphenyl, 4,4'-dimercaptostilbene, 4-
(6-mercaptohexyloxy)benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-
butanol, 6-(ferrocenyl)hexanethiol, 6-mercapto-l-hexanol, 6-
mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid, 9-
10 mercapto-1-nonanol, biphenyl-4,4'-dithiol, butyl 3-mercaptopropionate,
copper(1) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol
functionalized silver nanoparticles, dodecanethiol functionalized gold
nanoparticles, dodecanethiol functionalized silver nanoparticles,
hexa(ethylene
glycol)mono-11-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3-
15 mercaptopropionate, nanoTether BPA-HH, NanoThinksTm 18, NanoThinksTm 8,
NanoThinksTm ACID11, NanoThinksTm ACID16, NanoThinksTm ALC011,
NanoThinksTm THI08, octanethiol functionalized gold nanoparticles, PEG
dithiol average Mn 8,000, PEG dithiol average mol wt 1,500, PEG dithiol
average mol wt 3,400, S-(11-bromoundecyl)thioacetate, S-(4-
20 cyanobutyl)thioacetate, thiophenol, triethylene glycol mono-11-
mercaptoundecyl ether, trimethylolpropane tris(3-mercaptopropionate), [11-
(methylcarbonylthio)undecyl]tetra(ethylene glycol), m-carborane-9-thiol, p-
terpheny1-4,4"-dithiol, tert-dodecylmercaptan, and tert-nonyl mercaptan.
Exemplary reaction conditions, including temperature, pH,
25 reaction time, the use of stirring or agitation to dissolve solutes and
procedures
for collecting and washing precipitates, are described herein and employ
techniques generally known in the art.
Unlike previously described methodologies for producing BT
compounds, according to the present methods for preparing BT, BT products
30 are provided as microparticulate suspensions having substantially all
microparticles with VMD from about 0.4 to about 5 microns in certain preferred
embodiments, and generally from about 0.1 microns to about 8 microns
according to certain other embodiments. Further unlike previous approaches,
according to the instant embodiments bismuth is provided in an acidic aqueous
35 solution that
comprises a bismuth salt at a concentration of from at least about
50 mM to about 1 M, and nitric acid in an amount from at least about 0.5% to
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
36
about 5% (w/w), and preferably less than 5% (weight/weight), and that lacks a
hydrophilic, polar or organic solubilizer.
In this regard the present methods offer surprising and
unexpected advantages in view of generally accepted art teachings that
bismuth is not water soluble at 50 pM (e.g., U.S. RE37793), that bismuth is
unstable in water (e.g., Kuvshinova et al., 2009 Russ. J Inorg. Chem
54(11):1816), and that bismuth is unstable even in nitric acid solutions
unless a
hydrophilic, polar or organic solubilizer is present. For example, in all of
the
definitive descriptions of BT preparation methodologies (e.g., Domenico et
al.,
1997 Antimicrob. Agents. Chemother. 41:1697; U.S. 6,380,248; U.S. RE37793;
U.S. 6,248,371), the hydrophilic solubilizing agent propylene glycol is
required
to dissolve bismuth nitrate, and the bismuth concentration of solutions
prepared
for reaction with thiols is well below 15 mM, thereby limiting the available
production modalities for BT compounds.
By contrast, according to the present disclosure there is no
requirement for a hydrophilic, polar or organic solubilizer in order dissolve
bismuth, yet higher concentrations are surprisingly achieved. Hydrophilic,
polar
or organic solubilizers include propylene glycol (PG) and ethylene glycol (EG)
and may also include any of a large number of known solubility enhancers,
including polar solvents such as dioxane and dimethylsulfoxide (DMSO),
polyols (including, e.g., PG and EG and also including polyethylene glycol
(PEG), polypropyleneglycol (PPG), pentaerythritol and others), polyhydric
alchohols such as glycerol and mannitol, and other agents. Other water-
miscible organic of high polarity include dimethylsulfoxide (DMSO),
dimethylformamide (DMF) and NMP (N-methyl-2-pyrrolidone).
Thus, it will be appreciated by those familiar with the art that
solvents, including those commonly used as hydrophilic, polar or organic
solubilizers as provided herein, may be selected, for instance, based on the
solvent polarity/ polarizability (SPP) scale value using the system of Catalan
et
al. (e.g., 1995 Liebigs Ann. 241; see also Catalan, 2001 In: Handbook of
Solvents, Wypych (Ed.), Andrew Publ., NY, and references cited therein),
according to which, for example, water has a SPP value of 0.962, toluene a
SPP value of 0.655, and 2-propanol a SPP value of 0.848. Methods for
determining the SPP value of a solvent based on ultraviolet measurements of
the 2-N,N-dimethy1-7-nitrofluorene/ 2-fluoro-7-nitrofluorene probe/ homomorph
pair have been described (Catalan etal., 1995).
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
37
Solvents with desired SPP values (whether as pure single-
component solvents or as solvent mixtures of two, three, four or more
solvents;
for solvent miscibility see, e.g., Godfrey 1972 Chem. TechnoL 2:359) based on
the solubility properties of a particular BT composition can be readily
identified
by those having familiarity with the art in view of the instant disclosure,
although
as noted above, according to certain preferred embodiments regarding the
herein described synthetic method steps, no hydrophilic, polar or organic
solubilizer is required in order dissolve bismuth.
Solubility parameters may also include the interaction parameter
C, Hildebrand solubility parameter d, or partial (Hansen) solubility
parameters:
op, 6h and 6d, describing the solvent's polarity, hydrogen bonding potential
and
dispersion force interaction potential, respectively. In certain embodiments,
the
highest value for a solubility parameter that describes a solvent or co-
solvent
system in which the bismuth salt comprising bismuth will dissolve may provide
a limitation for the aqueous solution that comprises the bismuth salt, for
instance, according to the presently described method for preparing a
microparticulate BT composition. For example, higher Oh values will have a
greater hydrogen bonding ability and would therefore have a greater affinity
for
solvent molecules such as water. A higher value of maximum observed 6h for
a solvent may therefore be preferred for situations where a more hydrophilic
environment is desired.
By way of non-limiting example, BisEDT having the structure
shown below in formula I may be prepared according to the following reaction
scheme:
/
sH Et0H s ,S¨Bi
Bi(NO3)3
(I)
Briefly, and as a non-limiting illustrative example, to an excess
(11.4 L) of 5% aqueous HNO3 at room temperature may be slowly added 0.331
L (about 0.575 moles) of an aqueous acidic bismuth solution such as a Bi(NO3)3
solution (e.g., 43% Bi(NO3)3 (w/w), 5% nitric acid (w/w), 52% water (w/w),
available from Shepherd Chemical Co., Cincinnati, OH) with stirring, followed
by slow addition of absolute ethanol (4 L). An ethanolic solution (1.56 L) of
a
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
38
thiol compound such as 1,2-ethanedithiol [-0.55 M] may be separately
prepared by adding, to 1.5 L of absolute ethanol, 72.19 mL (0.863 moles) of
1,2-ethanedithiol using a 60 mL syringe, and then stirring for five minutes.
1,2-
ethanedithiol (CAS 540-63-6) and other thiol compounds are available from,
.. e.g., Sigma-Aldrich, St. Louis, MO. The ethanolic solution of the thiol
compound may then be slowly added to the aqueous Bi(NO3)3 / HNO3 solution
with stirring overnight to form a reaction solution. The thiol-containing
compound may be present in the reaction solution, according to certain
preferred embodiments, at a molar ratio of from about 1:3 to about 3:1
relative
to the bismuth. The formed product is allowed to settle as a precipitate
comprising microparhcles as described herein, which is then collected by
filtration and washed sequentially with ethanol, water and acetone to obtain
Bis EDT as a yellow amorphous powdered solid. The crude product may be
redissolved in absolute ethanol with stirring, then filtered and washed
sequentially with ethanol several times followed by acetone several times. The
washed powder may be triturated in 1M NaOH (500mL), filtered and washed
sequentially with water, ethanol and acetone to afford purified
microparticulate
BisEDT.
According to non-limiting theory, bismuth inhibits the ability of
bacteria to produce extracellular polymeric substances (EPS) such as bacterial
exopolysaccharides, and this inhibition leads to impaired biofilm formation.
Bacteria are believed to employ the glue-like EPS for biofilm cohesion.
Depending on the nature of an infection, biofilm formation and elaboration of
EPS may contribute to bacterial pathogenicity such as interference with wound
healing. However, bismuth alone is not therapeutically useful as an
intervention
agent, and is instead typically administered as part of a complex such as a
BT.
Bismuth-thiols (BTs) are thus a family of compositions that includes compounds
that result from the chelation of bismuth with a thiol compound, and that
exhibit
dramatic improvement in the antimicrobial therapeutic efficacy of bismuth. BTs
exhibit remarkable anti-infective, anti-biofilm, and immunomodulatory effects.
Bismuth thiols are effective against a broad-spectrum of microorganisms, and
are typically not affected by antibiotic-resistance. BTs prevent biofilm
formation
at remarkably low (sub-inhibitory) concentrations, prevent many pathogenic
characteristics of common wound pathogens at those same sub-inhibitory
levels, can prevent septic shock in animal models, and may be synergistic with
many antibiotics.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
39
As described herein, such synergy in the antibacterial effects of
one or more specified BT when combined with one or more specified antibiotic
compound is not readily predictable based on profiles of separate antibiotic
and
BT effects against a particular bacterial type, but surprisingly may result
from
selection of particular BT-antibiotic combinations in view of the specific
bacterial
population, including identification of whether gram-negative or gram-positive
(or both) bacteria are present. For instance, as disclosed herein, antibiotics
that synergize with certain BTs may include one or more of amikacin,
ampicillin,
aztreonam, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin
(or
other lincosamide antibiotics), daptomycin (Cubicine), doxycycline,
gatifloxacin,
gentamicin, imipenim, levofloxacin, linezolid (Zyvox8), minocycline, nafcilin,
paromomycin, rifampin, sulphamethoxazole, tetracycline, tobramycin and
vancomycin. In vitro studies showed, for example, that MRSA, which was
poorly or not at all susceptible to gentamicin, cefazolin, cefepime,
suphamethoxazole, imipenim or levofloxacin individually, exhibited marked
sensitivity to any one of these antibiotics if exposed to the antibiotic in
the
presence of the BT compound BisEDT. Certain embodiments contemplated
herein thus expressly contemplate compositions and/or methods in which may
be included the combination of a BT compound and one or more antibiotics
selected from amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,
ciprofloxacin, clindamycin (or another lincosamide antibiotic), daptomycin
(Cubicine),_doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin,
linezolid (Zyvox0), minocycline, nafcilin, paromomycin, rifampin,
sulphamethoxazole, tobramycin and vancomycin, whilst certain other
.. embodiments contemplated herein contemplate compositions and/or methods
in which may be included the combination of a BT compound and one or more
antibiotics from which expressly excluded may be one or more antibiotic
selected from amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,
ciprofloxacin, clindamycin (or other lincosamides), daptomycin (Cubicine),
doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid
(Zyvox0),
minocycline, nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycin
and vancomycin. It is noted in this context that gentamicin and tobramycin
belong to the aminoglycoside class of antibiotics. Also expressly excluded
from
certain contemplated embodiments are certain compositions and methods
described in Domenico et al., 2001 Agents Chemother. 45:1417-1421;
Domenico et al., 2000 Infect. Med. 17:123-127; Domenico et al., 2003 Res.
Adv. In Antimicrob. Agents & Chemother. 3:79-85; Domenico et al., 1997
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
Antimicrob. Agents Chemother. 41(8):1697-1703; Domenico et al., 1999 Infect.
Immun. 67:664-669: Huang et al. 1999 J Antimicrob. Chemother. 44:601-605;
Veloira et al., 2003 J Antimicrob. Chemother. 52:915-919; Wu et al., 2002 Am J
Respir Cell Mol Biol. 26:731-738; Halwani et al., 2008 Int. J Pharmaceut.
5 358:278; Halwani et al., 2009 Int. J. Pharmaceut. 373:141-146; where it
will be
noted that none of these publications teach or suggest the mondisperse
microparticulate BT compositions that are disclosed herein.
Accordingly and as described herein, in certain preferred
embodiments there are provided compositions and methods for treating a
10 subject with a composition that comprises the herein described
microparticulate
BT and that optionally and in certain other embodiments also comprises a
synergizing and/or an enhancing antibiotic. Persons familiar with the relevant
art will, based on the present disclosure, recognize appropriate clinical
contexts
and situations in which such treatment may be desired, criteria for which are
15 established in the medical arts, including inter alia, e.g., surgical,
military
surgical, dermatological, trauma medicine, gerontological, cardiovascular,
metabolic diseases (e.g., diabetes, obesity, etc.), infection and inflammation
(including in the epithelial linings of the respiratory tract or the
gastrointestinal
tract, or other epithelial tissue surfaces such as in glandular tissues), and
other
20 relevant medical specialties and subspecialities.
It will therefore be appreciated that, in certain embodiments as
disclosed herein and known in the art, promoting skin tissue repair (or other
tissue repair, such as epithelial tissue, bone, joint, muscle tendon, or
ligament
repair) is contemplated. In certain embodiments, promoting skin tissue or
other
25 epithelial tissue repair may comprise stimulating or disinhibiting one
or more
cellular wound repair activities selected from (i) epithelial cell (e.g.,
keratinocyte) or dermal fibroblast migration, (ii) epithelial cell (e.g.,
keratinocyte)
or dermal fibroblast growth, (iii) downregulation of epithelial cell (e.g.,
keratinocyte) or dermal fibroblast collagenase, gelatinase or matrix
30 metalloproteinase activity, (iv) dermal fibroblast extracellular matrix
protein
deposition, and (v) induction or potentiation of dermal angiogenesis.
Methodologies for identifying and characterizing such cellular wound repair
activities have been described such that the effects of the herein disclosed
wound tissue repair-promoting compounds, such as compositions comprising
35 BT agents as described herein, on these and related activities can be
determined readily and without undue experimentation based on the present
disclosure. For example, disclosed herein are compositions and methods that
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
41
relate to art accepted models for wound repair based on keratinocyte wound
closure following a scratch wound.
Preferred compositions for treating a microbial infection on or in a
natural surface for use according to the embodiments described herein, may
include in certain embodiments compositions that comprise bismuth-thiol (BT)
compounds as described herein, and which may in certain distinct but related
embodiments also include other compounds that are known in the art such as
one or more antibiotic compounds as described herein. BT compounds and
methods for making them are disclosed herein and are also disclosed, for
example, in Domenico et al. (1997 Antimicrob. Agent. Chemother. 41(8):1697-
1703; 2001 Antimicrob. Agent. Chemother. 45(5)1417-1421) and in U.S.
RE37,793, U.S. 6,248,371, U.S. 6,086,921, and U.S. 6,380,248. As also noted
above, certain preferred BT compounds are those that contain bismuth or a
bismuth salt ionically bonded to, or in a coordination complex with, a thiol-
containing compound, such as a composition that comprises bismuth chelated
to the thiol-containing compound, and certain other preferred BT compounds
are those that contain bismuth or a bismuth salt in covalent bond linkage to
the
thiol-containing compound. Also preferred are substantially monodisperse
microparticu late BT compositions as described herein. Neither from previous
efforts to treat bacterial infections, nor from previous characterization in
other
contexts of any compounds described herein for the first time as having use in
compositions and methods for promoting the herein described treatment of
natural surfaces, could it be predicted that the present methods of using such
compounds would have the herein described beneficial effects.
According to preferred embodiments there are thus provided
methods for treating a natural surface, comprising administering to the
surface
at least one microparticulate BT compound as described herein. In certain
embodiments the method further comprises administering, simultaneously or
sequentially and in either order, at least one antibiotic compound, which in
certain preferred embodiments may be a synergizing antibiotic as described
herein, and which in certain other preferred embodiments may be an enhancing
antibiotic as described herein. The antibiotic compound may be an
aminoglycoside antibiotic, a carbapenem antibiotic, a cephalosporin
antibiotic, a
fluoroquinolone antibiotic, a glycopeptides antibiotic, a lincosamide
antibiotic, a
penicillinase-resistant penicillin antibiotic, or an aminopenicillin
antibiotic.
Clinically useful antibiotics are discussed elsewhere herein and are also
described in, e.g., Washington University School of Medicine, The Washington
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
42
Manual of Medical Therapeutics (32nd Ed.), 2007 Lippincott, Williams and
Wilkins, Philadelphia, PA; and in Hauser, AL, Antibiotic Basics for
Clinicians,
2007 Lippincott, William and Wilkins, Philadelphia, PA.
As described herein, certain embodiments derive from the
unpredictable discovery that for a bacterial infection that comprises gram
positive bacteria, a preferred therapeutically effective formulation may
comprise
a BT compound (e.g., BisEDT, bismuth:1,2-ethanedithiol; BisPyr,
bismuth:pyrithione; BisEDT/Pyr, bismuth:1,2-ethanedithiol/pyrithione) and
rifamycin, or a BT compound and daptomycin (Cubicin , Cubist
Pharmaceuticals, Lexington, MA), or a BT compound and linezolid (Zyvox ,
Pfizer, Inc., NY, NY), or a BT compound (e.g., BisEDT, bismuth:1,2-
ethanedithiol; BisPyr, bismuth:pyrithione; BisEDT/Pyr, bismuth:1,2-
ethanedithiol/pyrithione) and one or more of ampicillin, cefazolin, cefepime,
chloramphenicol, clindamycin (or another lincosamide antibiotic), daptomycin
(Cubicin0), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin,
linezolid (Zyvox0), nafcilin, paromomycin, rifampin, sulphamethoxazole,
tobramycin and vancomycin.
As also described herein, certain embodiments derive from the
unpredictable discovery that for a bacterial infection that comprises gram
negative bacteria, a preferred therapeutically effective formulation may
comprise a BT compound and amikacin. Certain related embodiments
contemplate treatment of an infection comprising gram negative bacteria with a
BT compound and another antibiotic, such as another anninoglycoside
antibiotic, which in certain embodiments is not gentamicin or tobramycin.
Accordingly and in view of these embodiments, other related embodiments
contemplate identifying one or more bacterial populations or subpopulations in
or on a natural surface by the well known criterion of being gram positive or
gram negative, according to methodologies that are familiar to those skilled
in
the medical microbiology art, as a step for selecting appropriate antibiotic
compound(s) to include in a formulation to be administered according to the
present methods.
The presently described compositions and methods may find use
in the treatment of microbes (e.g., bacteria, viruses, yeast, molds and other
fungi, microbial parasites, etc.) in a wide variety of contexts, typically by
application or administration of the herein described compounds (e.g., one or
more microparticulate BTs alone or in combination with one or more
synergizing and/or enhancing antibiotics as disclosed herein) to a microbial
site
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
43
such as a microbial presence on or in a natural surface. Such natural surfaces
include but are not limited to mammalian tissues (e.g., epithelia including
skin,
scalp, gastrointestinal tract lining, buccal cavity, etc.; endothelia, cell
and tissue
membranes such as peritoneal membrane, pericardial membrane, pleural
membrane, periosteal membrane, meningeal membranes, sarcolemal
membranes, and the like; cornea, sclera, mucous membranes, etc.; and other
mammalian tissues such as teeth, bone, joint, tendon, ligament, muscle, heart,
lung, kidney, liver, spleen, gall bladder, pancreas, bladder, nerve, etc.).
The microparticulate antimicrobial agents described herein may
be used to suppress microbial growth, reduce microbial infestation,reduce
biofilm, prevent conversion of bacteria to biofilm, prevent or inhibit
microbial
infection and any other use described herein. These agents are also useful for
a number of antiviral purposes, including prevention or inhibition of viral
infection by herpes family viruses such as cytomegalovirus, herpes simplex
virus Type 1, and herpes simplex virus Type 2, and/or infection by other
viruses. In this regard, the agents are useful for the prevention or
inhibition of
viral infection by a variety of viruses, such as, single stranded RNA viruses,
single stranded DNA viruses, Rous sarcoma virus (RSV), hepatitis A virus,
hepatitis B virus (HBV), Hepatitis C (HCV), Influenza viruses, west nile virus
(WNV), Epstein-Barr virus (EBV), eastern equine encephalitis virus (EEEV),
severe acute respiratory virus (SARS), human immunodeficiency virus (HIV),
human papilloma virus (HPV), and human T cell lymphoma virus (HTLV).
Other internal and external pharmaceutical uses of the herein
described antimicrobial agents include, but are not limited to, treatment or
prevention of bacterial infection, of tuberculosis, of fungal infections such
as
yeast and mold infections (for example, Candida (e.g., Candida albicans,
Candida glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis) or
Cryptococcus or other fungi), of Helicobacter pylori infection, and of peptic
ulcer
disease. In one embodiment, the agent is used at a dosage not generally lethal
to bacteria but which is nonetheless sufficient to reduce protective
polysaccharide coatings that would otherwise resist natural immune response.
This technique is thus believed to aid immune system-mediated eradication of
bacterial infection without harming human symbiotic microorganisms (e.g.,
normal intestinal flora and the like) to the extent that may be the case with
antibiotics.
By way of illustration and not limitation, certain contemplated
embodiments are now described.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
44
In certain embodiments, a microparticulate BT compound
described herein (or composition comprising the microparticulate BT
compound) may be combined with at least one or more anti-biofilm agents for
controlling biofilm development, disrupting a biofilm, or reducing the amount
of
biofilm. As understood in the art, interspecies quorum sensing is related to
biofilm formation. Certain agents that increase LuxS-dependent pathway or
interspecies quorum sensing signal (see, e.g., U.S. Patent No. 7,427,408)
contribute to controlling development and/or proliferation of a biofilm.
Exemplary agents include, by way of example, N-(3-oxododecanoyI)-L-
homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine
lactone (BHL) analogs, either in combination or separately (see, e.g., U.S.
Patent No. 6,455,031). An oral hygiene composition comprising a
microparticulate BT compound and at least one anti-biofilm agent can be
delivered locally for disruption and inhibition of bacterial biofilm and for
treatment of periodontal disease (see, e.g., U.S. Patent No.6,726,898).
Compositions Comprising Microparticulate Bismuth-Thiols and
Uses for Oral Hygiene and for Treating Inflammation and Infection of the
Mouth.
In another embodiment, compositions comprising microparticulate BT
compounds are formulated for oral use and may be used in methods for
preventing or reducing microbial growth in the mouth and for preventing and/or
treating microbial infections and inflammation of the oral cavity. These
compositions are therefore useful for preventing or treating (i.e., reducing
or
inhibiting development of, reducing the likelihood of occurrence or recurrence
of) dental plaque, halitosis, periodontal disease, gingivitis, and other
infections
of the mouth. The oral compositions comprising microparticulate BT compound
may also be useful for preventing and/or controlling (i.e., slowing,
retarding,
inhibiting) biofilm development, disrupting a biofilm, or reducing the amount
of
biofilm present on an oral surface, particularly a tooth or gums.
Trapped food particles, poor oral hygiene and poor oral health,
and improper cleaning of dentures can promote microbial growth between
teeth, around the gums, and on the tongue. Continued microbial growth and
the presence of dental caries may result in halitosis, dental plaque (i.e., a
biofilm formed by colonization of microorganisms), gingivitis, and
inflammation.
In the absence of proper oral care (e.g., tooth brushing, flossing), more
serious
infections, such as periodontal disease and infections of the jaw, may ensue.
Good oral hygiene is important not only for oral health, but for
prevention of several chronic conditions. Controlling bacterial growth in the
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
mouth may help lower risk of heart disease, preserve memory, and reduce the
risk of infection and inflammation in other areas of the body. People with
diabetes are at greater risk for developing severe gum problems, and reducing
the risk of gingivitis by maintaining good oral health may help control blood
5 sugar. Pregnant women may be more likely to experience gingivitis, and
some
research suggests a relationship between gum disease in pregnant women and
delivery of preterm, low-birth-weight infants.
Bacteria are the primary etiologic agents in periodontal disease.
More than 500 bacterial strains may be found in dental plaque (Kroes et al.,
10 Proc. Natl. Acad. Sci. USA 96:14547-52 (1999)). Bacteria have evolved to
survive in the environment of the tooth surface, gingival epithelium, and oral
cavity as biofilms, which contributes to the difficulty in treating
periodontitis.
Bactericidal agents as well as antibiotics that are currently used to treat
such
infections often do not kill all of offending organisms. Use of an agent that
is
15 ineffective against certain bacteria species may result in proliferation
of
resistant bacterial species. Moreover, these agents may cause unpleasant side
effects, such allergic reactions, inflammation, and tooth discoloration.
Dental bacterial plaque is a biofilm that adheres tenaciously to
tooth surfaces, restorations, and prosthetic appliances. The primary means to
20 control biofilms in the mouth is through mechanical cleaning (i.e.,
tootbrushing,
flossing, etc.). Within the first two days after which no such cleaning has
been
undertaken, the tooth's surface is colonized predominantly by gram-positive
facultative cocci, which are primarily streptococci species. The bacteria
excrete
an extracellular slime layer that helps anchor the bacteria to the surface and
25 provides protection for the attached bacteria. Microcolony formation
begins
once the surface of the tooth has been covered with attached bacteria. The
biofilm grows primarily through cell division of adherent bacteria, rather
than
through the attachment of new bacteria. Doubling times of bacteria forming
plaque are rapid in early development and slower in more mature biofilms.
30 Coaggregation occurs when bacterial colonizers subsequently
adhere to bacteria already attached to the pellicle. The result of
coaggregation
is the formation of a complex array of different bacteria linked to one
another.
After a few days of undisturbed plaque formation, the gingival margin becomes
inflamed and swollen. Inflammation may result in creation of a deepened
35 gingival sulcus. The biofilm extends into this subgingival region and
flourishes
in this protected environment, resulting in the formation of a mature
subgingival
plaque biofilm. Gingival inflammation does not appear until the biofilm
changes
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
46
from one composed largely of gram-positive bacteria to one containing gram-
negative anaerobes. A subgingival bacterial microcolony, composed
predominantly of gram-negative anaerobic bacteria, becomes established in the
gingival sulcus between 3 and 12 weeks after the beginning of supragingival
plaque formation. Most bacterial species currently suspected of being
periodontal pathogens are anaerobic, gram-negative bacteria.
Bacterial microcolonies protected within the biofilm are typically
resistant to antibiotics (administered systemically), antiseptics or
disinfectants
(administered locally), and immune defenses. Antibiotic doses that kill free-
floating bacteria, for example, need to be increased as much as 1,500 times to
kill biofilm bacteria. At this high concentration, these antimicrobials tend
to be
toxic to the patient as well (see, e.g., Coghlan 1996, New Scientist 2045:32-
6;
Elder et al., 1995, Eye 9:102-9).
Diligent and frequent physical removal of bacterial plaque biofilms
is the most effective means of eliminating and controlling plaque. However,
subgingival plaque within pockets cannot be reached by brushes, floss, or oral
rinses, Therefore, frequent periodontal debridement of subgingival root
surfaces by a dental hygienist or dentist is an essential component in
prevention and treatment of periodontitis.
In certain embodiments, a microparticulate BT compound may be
incorporated into oral hygiene compositions , such as but not limited to,
toothpaste, mouthwash (i.e., mouth rinse), oral gels, dentifrice powders, oral
sprays (including a spray dispersed by an oral inhaler), edible film, chewing
gum, oral slurry, denture liquid cleaners, denture storage liquids, and dental
floss, which may be routinely used by any subject. A microparticulate BT
compound may be incorporated into oral hygiene compositions that are used
primarily by dental care professions, including for example, fluoride liquid
treatments, cleaning compositions, buffing compositions, oral rinses, and
dental
floss. The present embodiments contemplate replacement of antimicrobials
formulated with oral hygiene compositions, which are described in the art,
with
the presently described microparticulate BT compounds to provide the
advantages disclosed herein, including the range of antimicrobial activities,
solubility and bioavailability, anti-biofilm effects, non-toxicity,
enhancement of
antibiotic efficacies, and other properties as described herein.
A microparticulate BT compound may also be used for preventing
or treating caries and/or inflammation (i.e., reducing the likelihood of
occurrence
or recurrence of caries and/or inflammation, respectively) by administering
the
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
47
microparticulate BT compound to the surface of the teeth. A composition
comprising a microparticulate BT compound may be a mucoadhesive
composition that is applied to the surface of a tooth and/or gum or oral
mucous
membrane may be in any form that adheres to some extent to a surface or that
delivers a pharmaceutically effective amount of the active ingredient(s) to
the
desired surface. A microparticulate BT compound can also be formulated to
release slowly from the composition applied to the tooth. For example, the
composition may be a gel (e.g., a hydrogel, thiomer, aerogel, or organogel) or
liquid. An organogel may comprise an organic solvent, lipoic acid, vegetable
oil, or mineral oil. Such gel or liquid coating formulations may be applied
interior or exterior to an amalgam or composite or other restorative
composition.
A slow-release composition may deliver a pharmaceutically effective amount of
microparticulate BT compound for 1, 2, 3, 4, 5, 6, or 7 (a week) days or for
2, 3,
4, 5, 6, 7 weeks, or 1, 2, 3, 4, 5, or 6 months. Such compositions can be
prepared by a person skilled in the art using any number of methods known in
the art.
In certain other embodiments, and as described herein,
= antimicrobial compositions are provided for oral use that comprise
microparticulate BT compound and one or more additional antimicrobial
compounds or agents. Particularly useful are the compositions comprising s
and a second antimicrobial agent that when administered in combination have
enhanced or synergistic antimicrobial effects, as described herein. By way of
example, an enhanced antimicrobial effect may be observed when a
microparticulate BT compound is administered together with an antimicrobial
agent that chelates iron. In other particular embodiments, a microparticulate
BT
compound is formulated with an anti-inflammatory agent, compound, small
molecule, or macromolecule (such as a peptide or polypeptide).
Any of the microparticulate BT compounds described herein may
be formulated for oral use. In certain embodiments, microparticulate BT
compounds that are prepared with hydrophobic thiols (e.g., thiochlorophenol)
may be used and which may exhibit greater capability than less hydrophobic BT
compounds to adhere to teeth and tissues of the mouth. BT compounds that
have a net negative charge, such as those having a 1:2 molar ratio (bismuth to
thiol) may also have favorable adhesive properties.
The oral hygiene compositions comprising a microparticulate BT
compound may further comprise one or more active ingredients and/or one or
more orally suitable excipients or carriers. In one embodiment, the oral
hygiene
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
48
compositions may further comprise baking soda or another alkaline compound
or substance. Because of the chemical and physical properties of baking soda,
it has wide range of applications, including cleaning, deodorizing, and
buffering.
Baking soda neutralizes odors chemically, rather than masking or absorbing
them. Baking soda can be combined with a microparticulate BT compound
either as a mixture of powders, or dissolved or suspended in any one of the
dentifrice powders, gels, pastes, and liquids described herein. In other
embodiments, a microparticulate BT compound can be combined with other
alkali metal bicarbonate or carbonate substances (e.g., potassium bicarbonate
or calcium carbonate) that help maintain a desired alkaline pH and that also
possess cleansing and deodorizing properties.
Oral hygiene compositions comprising a microparticulate BT
compound may further comprise one or more of the following ingredients.
Antimicrobial agents: for example, chlorhexidine; sang uinarine extract;
metronidazole; quaternary ammonium compounds (such as cetylpyridinium
chloride); bis-guanides (e.g., chlorhexidine digluconate, hexetidine,
octenidine,
alexidine); halogenated bisphenolic compounds (e.g., 2,2' methylenebis-(4-
chloro-6-bromophenol) or other phenolic antibacterial compounds;
alkylhydroxybenzoate; cationic antimicrobial peptides; aminoglycosides;
quinolones; lincosamides; penicillins; cephalosporins, macrolides;
tetracyclines;
other antibiotics known in the art; Coleus forskohlii essential oil; silver or
colloidal silver antimicrobials; tin- or copper-based antimicrobials; Manuka
oil;
oregano; thyme; rosemary; or other herbal extracts; and grapefruit seed
extract.
Anti-inflammatory or antioxidant agents: for example, ibuprofen, flurbiprofen,
aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, cloves,
panthenol,
retinal, omega-3 fatty acids, gamma-linolenic acid (GLA), green tea, ginger,
grape seed, etc. Anti-caries agents: for example, sodium- and stannous
fluoride, aminefluorides, sodium monofluorophosphate, sodium
trimetaphosphate, zinc citrate or other zinc agents, and casein. Plaque
buffers:
for example, urea, calcium lactate, calcium glycerophosphate, and strontium
polyacrylates. Vitamins: for example, Vitamins A, C and E. Plant extracts.
Desensitizing agents: for example, potassium citrate, potassium chloride,
potassium tartrate, potassium bicarbonate, potassium oxalate, potassium
nitrate, and strontium salts. Anti-calculus agents: for example, alkali-metal
pyrophosphates, hypophosphite-containing polymers, organic phosphonates
and phosphocitrates etc. Biomolecules: for example, bacteriocins,
bacteriophages, antibodies, enzymes, etc. Flavors: for example, peppermint
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
49
and spearmint oils, fennel, cinnamon, etc. Proteinaceous materials: for
example, collagen. Preservatives. Opacifying agents. Coloring agents. pH-
adjusting agents. Sweetening agents. Pharmaceutically acceptable carriers:
for example, starch, sucrose, water or water/alcohol systems etc. Surfactants:
for example, anionic, nonionic, cationic and zwitterionic or amphoteric
surfactants, saponins from plant materials (see, e.g., U.S. Patent No.
6,485,711). Particulate abrasive materials: for example, silicas, aluminas,
calcium carbonates, dicalcium phosphates, calcium pyrophosphates,
hydroxyapatites, trimetaphosphates, insoluble hexametaphosphates,
agglomerated particulate abrasive materials, chalk, fine ground natural chalk
and the like. Humectants: for example, glycerol, sorbitol, propyleneglycol,
xylitol, lactitol etc. Binders and thickeners: for example, sodium carboxy
methyl
cellulose, hydroxyethyl cellulose (Natrosole), xanthan gum, gum arabic,
synthetic polymers (e.g., polyacrylates and carboxyvinyl polymers such as
Carbopole). Polymeric compounds that enhance the delivery of active
ingredients such as antimicrobial agents. Buffers and salts to buffer the pH
and
ionic strength of the oral care composition. Bleaching agents: for example,
peroxy compounds (e.g., potassium peroxydiphosphate). Effervescing systems:
for example, sodium bicarbonate/citric acid systems. Color change systems. In
particular embodiments, an abrasive is silica or fine ground natural chalk.
The oral hygiene compositions comprising a microparticulate BT
compound that are formulated for use as a toothpaste may further comprise a
humectant (for example, glycerol or sorbitol), a surface-active agent, binding
agent, and/or a flavoring agent. The toothpastes may also include a
sweetening agent, whitening agent, preservative, and antimicrobial agent. The
pH of a toothpaste and other compositions for oral use is typically between pH
5.5 and 8.5. In certain embodiments, oral hygiene compositions, including
toothpaste, have a pH between 7 and 7.5, between 7.5 and 8, between 8 and
8.5, or between 8.5 and 9, which may enhance the antimicrobial activity of the
microparticulate BT compound. The toothpaste compositions described herein
may include one or more of chalk, dicalcium phosphate dihydrate, sorbitol,
water, hydrated aluminum oxide, precipitated silica, sodium lauryl sulfate,
sodium carboxymethyl cellulose, flavoring, sorbitan monooleate, sodium
saccharin, tetrasodium pyrophosphate, methyl paraben, propyl paraben. One
or more coloring agents, for example, FD&C Blue, can be employed if desired.
Other suitable ingredients that may be including in a toothpaste formulation
are
described in the art, for example, in U.S. Pat. No. 5,560,517.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
In one particular embodiment, the oral hygiene composition is a
nnouthspray and comprises a microparticulate BT compound, an alkaline buffer
(e.g., potassium bicarbonate), an alcohol, a sweetener component, and a flavor
system. The flavor system may also have or more of the following: a flavorant,
5 a humectant, a surfactant, a sweetener, and a colorant agent (see, e.g.,
U.S.
Patent No. 6,579,513). Surfactants described herein and known in the art for
use in oral hygiene compositions may be anionic, nonionic, or amphoteric.
In another embodiment, the microparticulate BT-containing oral
hygiene composition may be combined with additional active ingredients such
10 as taurolidine and taurultam, which have been described in the art as
useful for
including in toothpastes, tooth gels, and mouthwashes for treating treat
serious
infections (see, e.g., United Kingdom Patent Application No., GB 1557163, U.S.
Patent No. 6,488,912). As described herein, microparticulate BT can also be
combined with one or more additional antimicrobial agents that when combined
15 with microparticulate BT, the combination has additive or synergistic
effects.
In yet another particular embodiment, an oral hygiene composition
described herein may further comprise at least one or more anti-biofilm agents
for controlling biofilm development, disrupting a biofilm, or reducing the
amount
of biofilm. As understood in the art, interspecies quorum sensing is related
to
20 biofilm formation. Certain agents that increase LuxS-dependent pathway
or
interspecies quorum sensing signal (see, e.g., U.S. Patent No. 7,427,408)
contribute to controlling development and/or proliferation of a biofilm.
Exemplary agents include, by way of example, N-(3-oxododecanoyI)-L-
homoserine lactone (OdDHL) blocking compounds and N-butyryl-L-homoserine
25 lactone (BHL) analogs, either in combination or separately (see, e.g.,
U.S.
Patent No. 64550311. An oral hygiene composition comprising a
microparticulate BT compound and at least one anti-biofilm agent can be
delivered locally for disruption and inhibition of bacterial biofilm and for
treatment of periodontal disease (see, e.g., U.S. Patent No.6,726,898).
30 An oral hygiene composition described herein may contain a
sufficient amount of a microparticulate BT compound that effects substantial
antimicrobial action during the time required for a normal tooth brushing,
mouth
rinsing, or flossing. As described herein a microparticulate BT compound may
be retained on oral surfaces (such as tooth, amalgam, composite, mucous
35 membrane, gums). A microparticulate BT compound retained on the teeth
and
gums after completion of brushing, rinsing, flossing, for example, may
continue
to provide extended anti-biofilm and anti-inflammatory action.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
51
In other embodiments, microparticulate BT compounds are slowly
released from muco-adhesive polymers or other agents that contribute to
retention of microparticulate BT compound on mucosal and tooth surfaces.
Microparticulate BTcompounds may be added to stable, viscous,
mucoadhesive aqueous compositions, which may also be used for the
prevention and treatment of ulcerative, inflammatory, and/or erosive disorders
of mucous membranes and/or the delivery of pharmaceutically active
compounds to mucosal surfaces for topical treatment or transfer to the
systemic
circulation (see, e.g., U.S. Patent No. 7,547,433).
In another embodiment, oral hygiene compositions comprising a
microparticulate BT compound further comprise olive oil, which may enhance
plaque removal. The use of olive oil in a product intended for oral hygiene,
such as a toothpaste, a mouthwash, a spray, oral inhaler, or chewing gum, may
contribute to elimination or reduction (a decrease) of bacterial plaque and/or
to
elimination or reduction (decrease of) in the numbers of bacteria present in
the
buccal cavity, thereby achieving a reduction in the occurrence of dental
diseases (e.g., tooth decay, periodontal disease) and halitosis (see, e.g.,
U.S.
Patent No. 7,074,391).
In other embodiments, an oral hygiene composition comprising a
microparticulate BT compound may further comprise a mucosal disinfectant
preparation for topical application in the mouth. An oral hygiene composition
may further comprise an aqueous slurry useful for cleaning the tongue and
throat (see, e.g., U.S. Patent No. 6,861,049). In still another embodiment, an
oral hygiene composition comprising a microparticulate BT compound may
further comprise at least one mint that is used for preventing (i.e., reducing
the
likelihood of occurrence) formation of a cavity (dental caries) or reducing
the
number of cavities. One such mint, called CaviStat (Ortek Therapeutics, Inc.,
Roslyn Heights, NY), contains arginine and calcium, which helps neutralize
acid
pH and promotes adherence of calcium to enamel surfaces. The inclusion of
mint in an oral hygiene composition comprising a microparticulate BT
compound may thus increase pH and enhance adherence of a microparticulate
BT compound to oral surfaces.
Compositions Comprising Microparticulate Bismuth-Thiols
Formulated for Orthopedic Use. In a particular embodiment, methods are
provided for using compositions comprising a microparticulate BT compound for
preventing and/or treating microbial infections and inflammation resulting
from
an orthopedic procedure (e.g., orthopedic surgery, orthopedic therapy,
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
52
arthroplasty (including two-step arthoplasty), orthodontic therapy). The
cornpositions comprising microparticulate BT compounds as described herein
are therefore useful for preventing and/or treating (i.e., reducing or
inhibiting
development of, reducing the likelihood of occurrence or recurrence of)
microbial infections of the skeleton and supporting structure (i.e., bones,
joints,
muscles, ligaments, tendons) such as osteomyelitis. The compositions
described herein comprising a microparticulate BT compound may also be
useful for preventing and/or controlling (i.e., slowing, retarding,
inhibiting)
biofilm development, disrupting a biofilm, or reducing the amount of biofilm
present in a joint or on the surface of a bone, ligament, tendon, or tooth.
The compositions described herein for orthopedic use that
comprise a microparticulate BT compound may further comprise one or more
additional antimicrobial compounds or agents. Particularly useful are the
compositions comprising a microparticulate BT compound and a second
antimicrobial agent that when administered in combination have enhanced or
synergistic antimicrobial effects, as described herein. By way of an
additional
example, an enhanced antimicrobial effect may be observed when a
microparticulate BT compound is administered together with an antimicrobial
agent that chelates iron. In other particular embodiments, a microparticulate
BT
.. compound is formulated with an anti-inflammatory agent, compound, small
molecule, or macromolecule (such as a peptide or polypeptide).
Compositions comprising a microparticulate BT compound may
be combined with at least one other antimicrobial agent (i.e., a second,
third,
fourth, etc. antimicrobial agent) that when administered in combination have
enhanced or synergistic antimicrobial effects (i.e., greater than an additive
effect). By way of example, an enhanced antimicrobial effect may be observed
when a microparticulate BT compound is administered together with an
antimicrobial agent that chelates iron. In particular embodiments,
compositions
comprising a microparticulate BT compound may be combined with at least one
other antimicrobial agent and/or anti-inflammatory agent selected from the
following: Antimicrobial agents: for example, chlorhexidine; sanguinarine
extract; metronidazole; quaternary ammonium compounds (such as
cetylpyridinium chloride); bis-guanides (e.g., chlorhexidine digluconate,
hexetidine, octenidine, alexidine); halogenated bisphenolic compounds (e.g.,
2,2' methylenebis-(4-chloro-6-bromophenol) or other phenolic antibacterial
compounds; alkylhydroxybenzoate; cationic antimicrobial peptides;
aminoglycosides; quinolones; lincosamides; penicillins; cephalosporins,
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
53
macrolides; tetracyclines; other antibiotics known in the art; Coleus
forskohlii
essential oil; silver or colloidal silver antimicrobials; tin- or copper-based
antimicrobials; Manuka oil; oregano; thyme; rosemary; or other herbal
extracts;
and grapefruit seed extract. Anti-inflammatory or antioxidant agents: for
example, ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric,
olive leaf extract, cloves, panthenol, retinol, omega-3 fatty acids, gamma-
linolenic acid (GLA), green tea, ginger, grape seed, etc. In particular
embodiments, the compositions comprising microparticulate BT compound may
further comprise an antibiotic selected from clindamycin, vancomycin,
daptomycin, cefazolin, gentamicin, tobramycin, metronidazole, cefaclor,
ciprofloxacin, or other antimicrobial such as a quaternary ammonium compound
(e.g., benzalkonium chloride, cetyl pyridinium chloride), an anti-microbial
zeolite, alkali metal hydroxide, or an alkaline earth metal oxide. The
compositions may optionally comprise one or more pharmaceutically suitable
carriers (i.e., excipients), surfactants, buffers, diluents, and salts, and
bleaching
agents, which are described herein. Accordingly, these and certain of the
related herein disclosed embodiments contemplate inclusion in such products
and processes of the presently disclosed microparticulate BT compositions,
which may include one or more microparticulate BT, and which may also
optionally further include an antibiotic such as a synergizing or an enhancing
antibiotic as described herein.
Biological, Biomedical and Other Uses for Microparticulate BTs.
Certain other embodiments contemplate use of the herein described
microparticulate BTs, whether as individual BTs or BTs in which the bismuth
moiety is replaced with a different Group V metal such as antimony (Sb) or
arsenic (As), and/or as such BTs in combination with one or more antibiotic
with
which, as described herein, the BT exhibits synergizing or enhanced
antimicrobial activity, in orally ingested nutritional formulations.
According to non-limiting theory, the inclusion of microparticulate
BTs in such formulations along with other components such as vitamins,
minerals, amino acids, hydrocarbons including carbohydrates, fatty acids,
oils,
phytonutrients, teas, herbs or herbal extracts, and/or other nutritional or
food
products, may in certain embodiments result in the blockage or retardation of
nutrient uptake by microbial populations in the gastrointestinal tract, in a
manner that promotes increased (e.g., in a statistically significant manner
relative to an appropriate control) bioavailability of the BT and optionally
the
antibiotic and/or of the additional nutritional component(s) to the host
digestive
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
54
tract. In certain other embodiments, by varying the particular vitamins,
minerals, amino acids, hydrocarbons including carbohydrates, fatty acids,
oils,
phytonutrients, teas, herbs or herbal extracts, and/or other nutritional or
food
products that are included in the oral microparticulate BT (or AsT or SbT)
formulation, bioavailability of the BT and optionally of the antibiotic and/or
of the
additional nutritional component(s) to the host digestive tract may be
decreased
(e.g., in a statistically significant manner relative to an appropriate
control).
For instance, it may be desirable when a pathologic
gastrointestinal (GI) tract infection is present to administer a
microparticulate BT
formulation that discourages intestinal absorption of the BT compounds so that
they remain bioavailable within the GI tract in order to exert antimicrobial
effects
against the infectious pathogens. Those familiar with the art will be aware of
a
number of vitamins, minerals, amino acids, hydrocarbons including
carbohydrates, fatty acids, oils, phytonutrients, teas, herbs and/or herbal
extracts that promote or discourage GI tract absorption of nutrients, such
that
formulations for increasing or decreasing the GI tract presence of one or more
components (e.g., the microparticulate BT compound, the antibiotic, or one or
more particular nutrients) may be prepared using the presently disclosed
microparticulate BTs (or AsT or SbT).
Certain other embodiments provided herein contemplate inclusion
of the presently disclosed microparticulate BT compounds in compositions for
oral delivery to reduce fecal or digestive gas odors, for instance in patients
who
have undergone colostomy, and in other compositions for topical delivery to
reduce underarm, foot or other body odors associated with topical microbial
presence. A number of skin and GI tract microbial populations, including
planktonic and biofilm bacteria, are susceptible to low concentrations of the
herein described microparticulate BT compounds, including such BT
compounds when present with enhancing or synergizing antibiotics as
described herein.
Accordingly, certain embodiments contemplate orally delivered
and topically delivered microparticulate BT formulations to decrease (e.g., in
a
statistically significant manner relative to an appropriate control)
populations of
GI-resident or skin-resident bacteria in a manner that reduces or alleviates
the
problem of unwanted odor. Oral and topical pharmaceutical formulations are
described below, such that these and related embodiments offer advantages
associated with the present microparticulate formulation of BT, such as
compatible bioavailability and solubility properties and low toxicity; other
factors
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
that may influence the selection of antimicrobial compositions are described
elsewhere herein and may also be found, e.g., in U.S. 6,582,719.
An exemplary BT compound, BisEDT, has been applied (50 uL of
a 1 mg/mL solution in DMSO) to the axillary area in human test subjects and
5 shown to neutralize body odors for two to three days. A mixture of BisEDT
in
talcum powder applied to the feet of a human test subject substantially
reduced
foot odor. Laboratory mice fed 1 mg/kg BisEDT orally twice daily for five days
exhibited 90% reductions in the number of fecal flora. Related embodiments
also contemplate a generally useful deodorant for any thiol-containing
solution
10 that emits odors (e.g., fish oils such as salmon oil), comprising a
microparticulate BT preparation as described herein that is made with an
excess of bismuth, and that can be added to the thiol-containing solution as
an
odor quenching agent. The resulting mixture retains the antimicrobial
properties of the microparticulate BT. Other contemplated applications include
15 solvents such as other biological source oils or butters, for instance,
hemp oil,
tea tree oil, shea butter, flax seed oil, fish oils, and in certain
embodiments such
oils as may have an independent or synergistic anti-inflammatory and/or pain-
reducing and/or other beneficial physiologic effect.
Pharmaceutical Compositions and Administration
20 Certain embodiments also relate to a pharmaceutical composition
containing the microparticulate BT compounds disclosed herein; in certain such
embodiments the pharmaceutical composition may further comprise one or
more antibiotics such as an antibiotic with which the BT compound exhibits a
synergizing or enhancing effect as described herein. In one embodiment, there
25 is provided a composition comprising one or more such microparticulate
BT
compounds in a pharmaceutically acceptable carrier, excipient or diluent and
in
a therapeutic amount, as disclosed herein, when administered to an animal,
preferably a mammal, most preferably a human patient.
Administration of the microparticulate BT compounds, or their
30 pharmaceutically acceptable salts, in pure form or in an appropriate
pharmaceutical composition, can be carried out via any of the accepted modes
of administration of agents for serving similar utilities. The pharmaceutical
compositions can be prepared by combining a microparticulate BT compound
with an appropriate pharmaceutically acceptable carrier, diluent or excipient,
35 and may be formulated into preparations in solid, semi-solid, liquid or
gaseous
forms, such as tablets, capsules, powders, granules, ointments, solutions,
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
56
suppositories, injections, inhalants, gels, microspheres, and aerosols.
Typical
routes of administering such pharmaceutical compositions include, without
limitation, oral, topical, transdermal, inhalation, parenteral, sublingual,
rectal,
vaginal, and intranasal. The term parenteral as used herein includes
subcutaneous injections, intravenous, intramuscular, intrasternal injection or
infusion techniques. Pharmaceutical compositions are formulated so as to
allow the active ingredients contained therein to be bioavailable upon
administration of the composition to a patient. Compositions that will be
administered to a subject or patient take the form of one or more dosage
units,
where for example, a tablet may be a single dosage unit, and a container of a
compound in aerosol form may hold a plurality of dosage units. Actual methods
of preparing such dosage forms are known, or will be apparent, to those
skilled
in this art; for example, see The Science and Practice of Pharmacy, 20th
Edition (Philadelphia College of Pharmacy and Science, 2000). The
composition to be administered will, in any event, contain a therapeutically
effective amount of a compound, or a pharmaceutically acceptable salt thereof,
for treatment of a disease or condition of interest in accordance with the
teachings herein.
The pharmaceutical compositions useful herein also contain a
pharmaceutically acceptable carrier, including any suitable diluent or
excipient,
which includes any pharmaceutical agent that does not itself induce the
production of antibodies harmful to the individual receiving the composition,
and
which may be administered without undue toxicity. Pharmaceutically
acceptable carriers include, but are not limited to, liquids, such as water,
saline,
glycerol and ethanol, and the like. A thorough discussion of pharmaceutically
acceptable carriers, diluents, and other excipients is presented in
REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current
edition).
A pharmaceutical composition may be in the form of a solid or
liquid. In one aspect, the carrier(s) are particulate, so that the
compositions
are, for example, in tablet or powder form. The carrier(s) may be liquid, with
the
compositions being, for example, an oral syrup, injectable liquid or an
aerosol,
which is useful in, for example, inhalatory administration.
When intended for oral administration, the pharmaceutical
composition is preferably in either solid or liquid form, where semi-solid,
semi-liquid, suspension and gel forms are included within the forms considered
herein as either solid or liquid.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
57
As a solid composition for oral administration, the pharmaceutical
composition may be formulated into a powder, granule, compressed tablet, pill,
capsule, chewing gum, wafer or the like form. Such a solid composition will
typically contain one or more inert diluents or edible carriers. In addition,
one or
more of the following may be present: binders such as carboxymethylcellulose,
ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin;
excipients
such as starch, lactose or dextrins, disintegrating agents such as alginic
acid,
sodium alginate, Primogel, corn starch and the like; lubricants such as
magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide;
sweetening agents such as honey, sucrose or saccharin; a flavoring agent such
as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
When the pharmaceutical composition is in the form of a capsule,
for example, a gelatin capsule, it may contain, in addition to materials of
the
above type, a liquid carrier such as polyethylene glycol or oil.
The pharmaceutical composition may be in the form of a liquid, for
example, an elixir, syrup, solution, emulsion or suspension. The liquid may be
for oral administration or for delivery by injection, as two examples. When
intended for oral administration, preferred composition contain, in addition
to
the present compounds, one or more of a sweetening agent, preservatives,
dye/colorant and flavor enhancer. In a composition intended to be administered
by injection, one or more of a surfactant, preservative, wetting agent,
dispersing
agent, suspending agent, buffer, stabilizer and isotonic agent may be
included.
The liquid pharmaceutical compositions, whether they be
solutions, suspensions or other like form, may include one or more of the
following adjuvants: sterile diluents such as water for injection, saline
solution,
preferably physiological saline, Ringer's solution, isotonic sodium chloride,
fixed
oils such as synthetic mono or diglycerides which may serve as the solvent or
suspending medium, polyethylene glycols, glycerin, propylene glycol or other
solvents; antibacterial agents such as benzyl alcohol or methyl paraben;
antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such
as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates and agents for the adjustment of tonicity such as sodium chloride
or dextrose. The parenteral preparation can be enclosed in ampoules,
disposable syringes or multiple dose vials made of glass or plastic.
Physiological saline is a preferred adjuvant. An injectable pharmaceutical
composition is preferably sterile.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
58
A liquid pharmaceutical composition intended for either parenteral
or oral administration should contain an amount of a microparticulate BT
compound such that a suitable dosage will be obtained. Typically, this amount
is at least 0.01% of a microparticulate BT compound in the composition. When
.. intended for oral administration, this amount may be varied to be between
0.1
and about 70% of the weight of the composition. Preferred oral pharmaceutical
compositions contain between about 4% and about 50% of the BT compound.
Preferred pharmaceutical compositions and preparations according to the
present invention are prepared so that a parenteral dosage unit contains
between 0.01 to 10% by weight of the microparticulate BT compound prior to
dilution.
The pharmaceutical composition may be intended for topical
administration, in which case the carrier may suitably comprise a solution,
emulsion, ointment or gel base. The base, for example, may comprise one or
more of the following: petrolatum, lanolin, polyethylene glycols, bee wax,
mineral oil, shea butter, tea tree oil, flax seed oil, hemp oil or other plant
or
vegetable oils including those known to have anti-inflammatory and/or anti-
pain
or other beneficial effects, salmon oil or other fish oils including those
known to
have anti-inflammatory and/or anti-pain or other beneficial effects, diluents
such
as water and alcohol, and emulsifiers and stabilizers. Thickening agents may
be present in a pharmaceutical composition for topical administration. If
intended for transdermal administration, the composition may include a
transdermal patch or iontophoresis device. Topical formulations may contain a
concentration of the microparticulate BT compound from about 0.1 to about
10% w/v (weight per unit volume).
The pharmaceutical composition may be intended for rectal
administration, in the form, for example, of a suppository, which will melt in
the
rectum and release the drug. The composition for rectal administration may
contain an oleaginous base as a suitable nonirritating excipient. Such bases
include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The pharmaceutical composition may include various materials,
which modify the physical form of a solid or liquid dosage unit. For example,
the composition may include materials that form a coating shell around the
active ingredients. The materials that form the coating shell are typically
inert,
and may be selected from, for example, sugar, shellac, and other enteric
coating agents. Alternatively, the active ingredients may be encased in a
gelatin capsule.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
59
The pharmaceutical composition in solid or liquid form may
include an agent that binds to the microparticulate BT compound and thereby
assists in the delivery of the compound. Suitable agents that may act in this
capacity include a monoclonal or polyclonal antibody, a protein or a liposome.
Certain contemplated embodiments, however, expressly exclude the inclusion
of a liposome in the pharmaceutical composition.
The pharmaceutical composition may consist of dosage units that
can be administered as an aerosol. The term aerosol is used to denote a
variety of systems ranging from those of colloidal nature to systems
consisting
of pressurized packages. Delivery may be by a liquefied or compressed gas or
by a suitable pump system that dispenses the active ingredients. Aerosols of
the microparticulate BT compounds may be delivered in single phase,
bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s).
Delivery of the aerosol includes the necessary container, activators, valves,
subcontainers, and the like, which together may form a kit. One skilled in the
art, without undue experimentation may determine preferred aerosols.
The pharmaceutical compositions may be prepared by
methodology well known in the pharmaceutical art. For example, a
pharmaceutical composition intended to be administered by injection can be
prepared by combining a compound of the invention with sterile, distilled
water
so as to form a solution. A surfactant may be added to facilitate the
formation
of a homogeneous solution or suspension. Surfactants are compounds that
non-covalently interact with the compound of the invention so as to facilitate
dissolution or homogeneous suspension of the compound in the aqueous
delivery system.
The herein described microparticulate BT compounds, or their
pharmaceutically acceptable salts, are administered in a therapeutically
effective amount, which will vary depending upon a variety of factors
including
the activity of the specific compound employed; the metabolic stability and
length of action of the compound; the age, body weight, general health, sex,
and diet of the patient; the mode and time of administration; the rate of
excretion; the drug combination; the severity of the particular disorder or
condition; and the subject undergoing therapy. Generally, a therapeutically
effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e.,
0.07
mg) to about 100 mg/kg (i.e., 7.0 g); preferaby a therapeutically effective
dose
is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 7 mg) to about 50 mg/kg
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
(i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70
kg
mammal) from about 1 mg/kg (Le., 70 mg) to about 25 mg/kg (i.e., 1.75 g).
The ranges of effective doses provided herein are not intended to
be limiting and represent preferred dose ranges. However, the most preferred
5 dosage will be tailored to the individual subject, as is understood and
determinable by one skilled in the relevant arts. (see, e.g., Berkowet al.,
eds.,
The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992;
Goodmanetna., eds.,Goodman and Cilman's The Pharmacological Basis of
Therapeutics, 10th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001);
10 Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology
and
Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore,
MD. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985);
Osolci al., eds.,Remington's Pharmaceutical Sciences, 18th edition, Mack
Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology,
15 Appleton and Lange, Norwalk, CT (1992)).
The total dose required for each treatment can be administered by
multiple doses or in a single dose over the course of the day, if desired.
Generally, treatment is initiated with smaller dosages, which are less than
the
optimum dose of the compound. Thereafter, the dosage is increased by small
20 increments until the optimum effect under the circumstances is reached.
The
diagnostic pharmaceutical compound or composition can be administered alone
or in conjunction with other diagnostics and/or pharmaceuticals directed to
the
pathology, or directed to other symptoms of the pathology. The recipients of
administration of microparticulate BT compounds and/or compositions can be
25 any vertebrate animal, such as mammals. Among mammals, the preferred
recipients are mammals of the Orders Primate (including humans, apes and
monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta
(including mice, rats, rabbits, and hamsters), and Carnivora (including cats,
and
dogs). Among birds, the preferred recipients are turkeys, chickens and other
30 members of the same order. The most preferred recipients are humans.
For topical applications, it is preferred to administer an effective
amount of a microparticu late BT-containing pharmaceutical composition to a
target area, e.g., skin surfaces, mucous membranes, and the like. This amount
will generally range from about 0.0001 mg to about 1 g of a BT compound per
35 application, depending upon the area to be treated, whether the use is
diagnostic, prophylactic or therapeutic, the severity of the symptoms, and the
nature of the topical vehicle employed. A preferred topical preparation is an
CA 2788669 2017-03-14
61
ointment, wherein about 0.001 to about 50 mg of active ingredient is used per
cc of ointment base. The pharmaceutical composition can be formulated as
transdermal compositions or transdermal delivery devices ("patches"). Such
compositions include, for example, a backing, active compound reservoir, a
control membrane, liner and contact adhesive. Such transdermal patches may
be used to provide continuous pulsatile, or on demand delivery of the
compounds of the present invention as desired.
The microparticulate BT compositions can be formulated so as to
provide quick, sustained or delayed release of the active ingredient after
administration to the patient by employing procedures known in the art.
Controlled release drug delivery systems include osmotic pump systems and
dissolutional systems containing polymer-coated reservoirs or drug-polymer
matrix formulations. Examples of controlled release systems are given in U.S.
Pat. Nos. 3,845,770 and 4,326,525 and in P. J. Kuzma et al, Regional
Anesthesia 22 (6): 543-551 (1997).
The microparticulate BT compositions can also be delivered
through intra-nasal drug delivery systems for local, systemic, and nose-to-
brain
medical therapies. Controlled Particle Dispersion (CPD)TM technology,
.. traditional nasal spray bottles, inhalers or nebulizers are known by those
skilled
in the art to provide effective local and systemic delivery of drugs by
targeting
the olfactory region and paranasal sinuses.
The invention also relates in certain embodiments to an
intravaginal shell or core drug delivery device suitable for administration to
the
human or animal female. The device may be comprised of the active
pharmaceutical ingredient in a polymer matrix, surrounded by a sheath, and
capable of releasing the compound in a substantially zero order pattern on a
daily basis similar to devices used to apply testosterone as described in WO
98/50016.
Current methods for ocular delivery include topical administration
(eye drops), subconjunctival injections, periocular injections, intravitreal
injections, surgical implants and iontophoresis (uses a small electrical
current to
transport ionized drugs into and through body tissues). Those skilled in the
art
would combine the best suited excipients with the compound for safe and
effective intra-occular administration.
The most suitable route will depend on the nature and severity of
the condition being treated. Those skilled in the art are also familiar with
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
62
determining administration methods (oral, intravenous, inhalation, sub-
cutaneous, rectal etc.), dosage forms, suitable pharmaceutical excipients and
other matters relevant to the delivery of the compounds to a subject in need
thereof.
The presently described compositions and methods may also find
use in the treatment of acute and chronic wounds and wound biofilms,
including, for example, as burn creams, as topicals for the treatment of
existing
wounds including those described herein, for prevention of chronic wounds, for
treatment of MRSA skin infections, and for other related indications as
disclosed herein and as will be apparent to the skilled person in view of the
present disclosure.
Non-limiting examples of bacteria against which the herein
described compositions and methods may find beneficial use, according to
certain embodiments as described herein, include Staphylococcus aureus (S.
aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus epidermidis,
MRSE (methicillin-resistant S. epidermidis), Mycobacterium tuberculosis,
Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P. aeruginosa,
Escherichia coil, enterotoxigenic E. coil, enterohemorrhagic E. coil,
Klebsiella
pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophilaõ
Enterococcus faecalis, methicillin-susceptible Enterococcus faecalis,
Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia
enterocolitica, Vibrio cholera, Shigella flexneri, vancomycin-resistant
Enterococcus (VRE), Burkholderia cepacia complex, Francisella tularensis,
Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-
sensitive and vancomycin-resistant enterococci (e.g., E. faecalis, E.
faecium),
methicillin-sensitive and methicillin-resistant staphylococci (e.g., S.
aureus, S.
epidermidis) and Acinetobacter baumannii, Staphylococcus haemolyticus,
Staphylococcus hominis, Enterococcus faecium, Streptococcus pyo genes,
Streptococcus agalactiae, Bacillus anthracis, Klebsiella pneumonia, Proteus
mirabilis, Proteus vulgaris, Yersinia enterocolytica, Stenotrophomonas
maltophilia, Streptococcus pneumonia, penicillin-resistant Streptococcus
pneumonia, Burkholderia cepacia, Bukholderia multivorans, Mycobacterium
smegmatis and E. cloacae.
The practice of certain embodiments of the present invention will
employ, unless indicated specifically to the contrary, conventional methods of
microbiology, molecular biology, biochemistry, cell biology, virology and
immunology techniques that are within the skill of the art, and reference to
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
63
several of which is made below for the purpose of illustration. Such
techniques
are explained fully in the literature. See, e.g., Sambrook, et al. Molecular
Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular
Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I
& II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic
Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and
Translation (B. Flames & S. Higgins, eds., 1984); Animal Cell Culture (R.
Fresh ney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).
Unless the context requires otherwise, throughout the present
specification and claims, the word "comprise" and variations thereof, such as,
"comprises" and "comprising" are to be construed in an open, inclusive sense,
that is as "including, but not limited to".
Reference throughout this specification to "one embodiment" or
"an embodiment" or "an aspect" means that a particular feature, structure or
characteristic described in connection with the embodiment is included in at
least one embodiment of the present invention. Thus, the appearances of the
phrases "in one embodiment" or "in an embodiment" in various places
throughout this specification are not necessarily all referring to the same
embodiment. Furthermore, the particular features, structures, or
characteristics
may be combined in any suitable manner in one or more embodiments.
Certain embodiments relate to methods, compositions and kits for
treating an acute or chronic wound or a wound biofilm in a subject, which may
comprise promoting skin tissue repair in the subject, or for altering one or
more
cellular wound repair activity in a cell or plurality of cells. A cell
generally
indicates a single cell, whereas a plurality of cells indicates more than one
cell.
The cells may comprise a tissue, organ or entire organism. Furthermore, the
cell or cells may be located in vivo, in vitro, or ex vivo. Maintaining cell,
tissue
and organ cultures are routine procedures for one of skill in the art, the
conditions and media for which can be easily ascertained. (See, for example,
Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss 5th
Ed. (2005); Davis, Basic Cell Culture, Oxford University Press 2'd Ed.
(2002)).
As disclosed herein, certain embodiments relate to methods for
treating an acute or chronic wound or a wound biofilm in a subject that
comprises administering to the subject a therapeutically effective amount of a
composition comprising a BT compound as described herein for use in such
method (e.g., as provided in the form of a plurality of substantially
monodisperse microparticles), and optionally in certain further embodiments
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
64
also comprising an antibiotic compound as described herein for use in such
method, for example, a BT compound such as BisEDT or BisBAL or other
compounds presented in Table 1 herein, or any other BT agent such as those
described in Domenico et al. (1997 Antimicrob. Agent. Chemother. 41:1697;
2001 Antimicrob. Agent. Chemother. 45:1421) and/or in U.S. RE37,793, U.S.
6,248,371, U.S. 6,086,921, and U.S. 6,380,248 and/or as prepared according to
the methods disclosed herein. Certain other embodiments relate to methods
that comprise contacting any natural surface with a composition comprising
one or more of the herein described microparticulate BT compounds, where
such step of contacting may comprise one or more of directly applying,
coating,
dipping, irrigating, spraying, painting or otherwise bringing the BT
composition
into contact with the natural surface.
The step of administering to a subject such as a human or other
mammalian subject may be performed by any means known to the art, for
example, topically (including via direct administration to skin or to any
epithelial
tissue surface, including such surfaces as may be present in glandular tissues
or in the respiratory and/or gastrointestinal tracts), vaginally,
intraperitoneally,
orally, parenterally, intravenously, intraarterially, transdermally,
sublingually,
subcutaneously, intramuscularly, transbuccally, intranasally, via inhalation,
intraoccularly, subcutaneously, intraadiposally, intraarticularly or
intrathecally.
In preferred embodiments administering may be performed
topically, where pharmaceutical excipients or carriers for topical use are
described herein and known in the art.
As noted above, certain invention embodiments described herein
relate to topical formulations of the described BT compounds (e.g., BisEDT
and/or BisBAL), which formulations may in certain further embodiments
comprise one or more antibiotic compounds as described herein, for instance,
amikacin, ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin,
clindamycin (or another lincosamide antibiotic), daptomycin (Cubicin ),
doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid
(Zyvoxe),
minocycline, nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycin
and vancomycin; or a carbapenem antibiotic, a cephalosporin antibiotic, a
fluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamide
antibiotic, a
pen icillinase-resistant penicillin antibiotic, and/or an aminopenicillin
antibiotic,
and/or an aminoglycoside antibiotic such as amikacin, arbekacin, gentamicin,
kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin,
streptomycin, tobramycin or apramycin, and/or a lipopeptide antibiotic such as
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
daptomycin (Cubicine), or an oxazolidinone antibiotic such as linezolid
(Zyvox ). These and related formulations may comprise the BT compound(s)
(and optionally one or more antibiotics) in a pharmaceutically acceptable
carrier, excipient or diluent and in a therapeutic amount, as disclosed
herein,
5 when administered topically to an animal, preferably a mammal, and most
preferably a human, and in particularly preferred embodiments, a human having
an acute or chronic wound or a wound that contains a bacterial infection which
may be biofilm-related (e.g., in which bacteria capable of promoting biofilm
formation may be present but a biofilm is not yet detectable) or that contains
a
10 bacterial infection such as a biofilm or other bacterial presence.
Topical administration of the BT compounds described herein, or
their pharmaceutically acceptable salts, in pure form or in an appropriate
pharmaceutical composition, can be carried out via any of the accepted modes
of topical administration of agents for serving similar utilities. Topical
15 application or administration of a composition includes, in preferred
embodiments, directly contacting the composition (e.g., a topical formulation)
with skin and/or another epithelial tissue surface (e.g., respiratory tract,
gastrointestinal tract and/or glandular epithelial linings) of the subject
undergoing treatment, which may be at one or more localized or widely
20 distributed skin and/or other epithelial tissue surface sites and which
may
generally refer to contacting the topical formulation with an acute or chronic
wound site that is surrounded by intact stratum corneum or epidermis but need
not be so limited; for instance, certain embodiments contemplate as a topical
application the administration of a topical formulation described herein to
25 injured, abraded or damaged skin, or skin of a subject undergoing
surgery,
such that contact of the topical formulation may take place not only with
stratum
corneum or epidermis but also with skin granular cell, spinous cell, and/or
basal
cell layers, and/or with dermal or underlying tissues, for example, as may
accompany certain types of wound repair or wound healing or other skin tissue
30 remodeling.
Such skin tissue repair may therefore comprise, in certain
preferred embodiments, dermal wound healing, as may be desirable, for
example, in preventing or ameliorating an acute chronic wound or a wound
biofilm or, as another example, in preventing or ameliorating skin wound
35 dehiscence, or in improving, accelerating or otherwise enhancing dermal
wound
healing when an acute or chronic wound and/or skin wound dehiscence may be
present. Certain other embodiments that contemplate topical administration to
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
66
an epithelial tissue surface present in respiratory tract, gastrointestinal
tract
and/or glandular linings similarly may comprise administration of the topical
formulation by an appropriate route as will be known in the art for delivering
a
topical preparation as provided herein, to one or more epithelial tissue
surfaces
present in respiratory (e.g., airway, nasopharyngeal and laryngeal paths,
tracheal, pulmonary, bronchi, bronchioles, alveoli, etc.) and/or
gastrointestinal
(e.g., buccal, esophageal, gastric, intestinal, rectal, anal, etc.) tracts,
and/or
other epithelial surfaces.
According to certain contemplated embodiments topical
administration may comprise direct application into an open wound. For
instance, an open fracture or other open wound may include a break in the skin
that may expose additional underlying tissues to the external environment in a
manner that renders them susceptible to microbial infection. Such a situation
is
not uncommon in certain types of acute traumatic military wounds, including,
for
example, Type III (severe) open fractures. In accord with these and related
embodiments, topical administration may be by direct contact of the herein
described BT composition with such damaged skin and/or another epithelial
surface and/or with other tissues, such as, for instance, connective tissues
including muscle, ligaments, tendons, bones, circulatory tissues such as blood
vessels, associated nerve tissues, and any other organs that may be exposed
in such open wounds. Examples of other tissues that may be exposed, and
hence for which such direct contact is contemplated, include kidney, bladder,
liver, pancreas, and any other tissue or organ that may be so detrimentally
exposed to opportunistic infection in relation to an open wound.
The topical formulations (e.g., pharmaceutical compositions) may
be prepared by combining the described BT compound (e.g., comprising a
compound described in U.S. RE37,793, U.S. 6,248,371, U.S. 6,086,921, and/or
U.S. 6,380,248 and/or prepared according to the present disclosure such as the
herein described microparticulate BT suspensions), and in certain related
embodiments by combining one or more desired antibiotics (e.g., an
aminoglycoside antibiotic such as amikacin) separately or together with the BT
compound, with an appropriate pharmaceutically acceptable carrier, diluent or
excipient for use in a topical formulation preparation, and may be formulated
into preparations in solid, semi-solid, gel, cream, colloid, suspension or
liquid or
other topically applied forms, such as powders, granules, ointments,
solutions,
washes, gels, pastes, plasters, paints, bioadhesives, microsphere suspensions,
and aerosol sprays.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
67
Pharmaceutical compositions of these and related embodiments
are formulated so as to allow the active ingredients contained therein, and in
particularly preferred embodiments the herein described BT compound(s) alone
or in combination with one or more desired antibiotics (e.g., a carbapenem
antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a
glycopeptide
antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin
antibiotic,
and an aminopenicillin antibiotic, or an aminoglycoside antibiotic such as
amikacin, or rifamycin) which may be applied simultaneously or sequentially
and in either order, to be bioavailable upon topical administration of the
formulation containing the BT compound(s) and/or antibiotic composition(s) to
an acute or chronic wound and optionally to surrounding skin of a subject,
such
as a mammal, including a human, and in certain preferred embodiments a
human patient having an acute or chronic wound, or being at increased risk for
having, an acute or chronic wound or a wound biofilm or wound dehiscence
(e.g., an obese and/or diabetic individual). Certain embodiments disclosed
herein contemplate topical administration of a BT compound and of an
antibiotic, including administration that may be simultaneous or sequential
and
in either order, but the invention is not intended to be so limited and in
other
embodiments expressly contemplates a distinct route of administration for the
BT compound relative to the route of administration of the antibiotic. Thus,
the
antibiotic may be administered orally, intravenously, or by any other route of
administration as described herein, while the BT compound may be
administered by a route that is independent of the route used for the
antibiotic.
As a non-limiting, illustrative example, the BT compound may be administered
topically as provided herein, while the antibiotic may be simultaneously or
sequentially (and in any order) administered by a distinct route, such as
orally,
intravenously, transdermally, subcutaneously, intramuscularly and/or by any
other route of administration.
The topical formulations described herein deliver a therapeutically
3D effective amount of the antiseptic or wound-healing agent(s) (and
optionally the
antibiotic(s)) to the wound site, for instance, to skin cells such as dermal
fibroblasts. Preferred formulations may be contacted with a desired site such
as a topical wound site, a chronic wound, an epithelial tissue surface or
other
intended site of administration by spraying, irrigating, dipping and/or
painting;
such formulations therefore may exhibit ready permeability into the skin, as
can
be determined according to any of a number of established methodologies
known to the art for testing the skin permeability of a drug composition (see,
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
68
e.g., Wagner et al., 2002 J. Invest. DermatoL 118:540, and references cited
therein; Bronaugh et al., 1985 J. Pharm. Sci. 74:64; Bosman et al., 1998 J.
Pharm. Biomed. Anal. 17:493-499; Bosman et al., 1996 J. Pharm Biomed Anal.
1996 14:1015-23; Bonferoni et al., 1999 Pharm. Dev. TechnoL 4:45-53; Frantz,
Instrumentation and methodology for in vitro skin diffusion cells in
methodology
for skin absorption. In: Methods for Skin Absorption (Kemppainen & Reifenrath,
Eds), CRC Press, Florida, 1990, pp. 35-59; Tojo, Design and calibration of in
vitro permeation apparatus. In: Transdermal Controlled Systemic Medications
(Chien YW, Ed), Marcel Dekker, New York, 1987, 127-158; Barry, Methods for
studying percutaneous absorption. In: Dermatological Formulations:
Percutaneous absorption, Marcel Dekker, New York, 1983, 234-295).
Compositions, and formulations comprising such compositions,
that will be administered to the skin of a subject or patient may in certain
embodiments take the form of one or more dosage units, where for example, a
liquid-filled capsule or ampule may contain a single dosage unit, and a
container of a topical formulation as described herein in aerosol form may
hold
a plurality of dosage units. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in this art; for example, see The
Science and Practice of Pharmacy, 20th Edition (Philadelphia College of
Pharmacy and Science, 2000). The composition or formulation to be
administered will, in any event, contain a therapeutically effective amount of
an
antiseptic and/or wound healing-promoting compound as provided herein (e.g.,
a BT compound), or a pharmaceutically acceptable salt thereof, in accordance
with the present teachings.
As noted above, the present topical formulations may take any of
a wide variety of forms, and include, for example, creams, lotions, solutions,
sprays, gels, ointments, pastes or the like, and/or may be prepared so as to
contain liposomes, micelles, and/or microspheres. See, e.g., U.S. Patent No.
7,205,003. For instance, creams, as is well known in the arts of
pharmaceutical
and cosmeceutical formulation, are viscous liquids or semisolid emulsions,
either oil-in-water or water-in-oil. Cream bases are water-washable, and
contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also
called the "internal" phase, is generally comprised of petrolatum and a fatty
alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although
not necessarily, exceeds the oil phase in volume, and generally contains a
humectant. The emulsifier in a cream formulation is generally a nonionic,
anionic, cationic or amphoteric surfactant.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
69
Lotions, which are preferred for delivery of cosmetic agents, are
preparations to be applied to the skin surface without friction, and are
typically
liquid or semi-liquid preparations in which solid particles, including the
active
agent, are present in a water or alcohol base. Lotions are usually suspensions
of solids, and preferably comprise a liquid oily emulsion of the oil-in-water
type.
Lotions are preferred formulations herein for treating large body areas,
because
of the ease of applying a more fluid composition. It is generally preferred
that
the insoluble matter in a lotion be finely divided. Lotions will typically
contain
suspending agents to produce better dispersions as well as compounds useful
for localizing and holding the active agent in contact with the skin, e.g.,
methylcellulose, sodium carboxymethyl-cellulose, or the like.
Solutions are homogeneous mixtures prepared by dissolving one
or more chemical substances (solutes) in a liquid such that the molecules of
the
dissolved substance are dispersed among those of the solvent. The solution
may contain other pharmaceutically acceptable and/or cosmeceutically
acceptable chemicals to buffer, stabilize or preserve the solute. Common
examples of solvents used in preparing solutions are ethanol, water, propylene
glycol or any other pharmaceutically acceptable and/or cosmeceutically
acceptable vehicles.
Gels are semisolid, suspension-type systems. Single-phase gels
contain organic macromolecules distributed substantially uniformly throughout
the carrier liquid, which is typically aqueous, but also, preferably, contain
an
alcohol, and, optionally, an oil. Preferred "organic macromolecules," i.e.,
gelling
agents, may be chemically crosslinked polymers such as crosslinked acrylic
acid polymers, for instance, the "carbomer" family of polymers, e.g.,
carboxypolyalkylenes, that may be obtained commercially under the Carbopol
trademark. Also preferred in certain embodiments may be hydrophilic polymers
such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers
and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose,
hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl
methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and
xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel,
dispersing agents such as alcohol or glycerin can be added, or the gelling
agent
can be dispersed by trituration, mechanical mixing or stirring, or
combinations
thereof.
Ointments, as also well known in the art, are semisolid
preparations that are typically based on petrolatum or other petroleum
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
derivatives. The specific ointment base to be used, as will be appreciated by
those skilled in the art, is one that will provide for a number of desirable
characteristics, e.g., emolliency or the like. As with other carriers or
vehicles,
an ointment base should be inert, stable, nonirritating, and nonsensitizing.
As
5 explained in Remington: The Science and Practice of Pharmacy, 19th Ed.
(Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases
may be grouped in four classes: oleaginous bases; emulsifiable bases;
emulsion bases; and water-soluble bases. Oleaginous ointment bases include,
for example, vegetable oils, fats obtained from animals, and semisolid
10 hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also
known as absorbent ointment bases, contain little or no water and include, for
example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic
petrolatum.
Emulsion ointment bases are either water-in-oil (W/0) emulsions or oil-in-
water
(0/W) emulsions, and include, for example, cetyl alcohol, glyceryl
15 monostearate, lanolin, and stearic acid. Preferred water-soluble ointment
bases are prepared from polyethylene glycols of varying molecular weight (see,
e.g., Remington,. Id.).
Pastes are semisolid dosage forms in which the active agent is
suspended in a suitable base. Depending on the nature of the base, pastes are
20 divided between fatty pastes or those made from single-phase aqueous
gels.
The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or
the like. The pastes made from single-phase aqueous gels generally
incorporate carbmmethylcellulose or the like as a base.
Formulations may also be prepared with liposomes, micelles, and
25 microspheres. Liposomes are microscopic vesicles having one
(unilamellar) or
a plurality (multilamellar) of lipid walls comprising a lipid bilayer, and, in
the
present context, may encapsulate and/or have adsorbed to their lipid
membranous surfaces one or more components of the topical formulations
herein described, such as the antiseptic, wound healing/skin tissue/
epithelial
30 tissue repair-promoting compounds (e.g., microparticulate BT compounds,
optionally along with one or more antibiotics) or certain carriers or
excipients.
Liposomal preparations herein include cationic (positively charged), anionic
(negatively charged), and neutral preparations. Cationic liposomes are readily
available. For example, N[1-2,3-dioleyloxy)propyI]-N,N,N-triethylammonium
35 (DOTMA) liposomes are available under the tradename Lipofectin0 (GI BCO
BRL, Grand Island, N.Y.). Similarly, anionic and neutral liposomes are readily
available as well, e.g., from Avanti Polar Lipids (Birmingham, AL), or can be
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
71
easily prepared using readily available materials. Such materials include
phosphatidyl choline, cholesterol, phosphatidyl ethanolamine,
dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG),
and dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials
can also be mixed with DOTMA in appropriate ratios. Methods for making
liposomes using these materials are well known in the art.
Micelles are known in the art as comprised of surfactant
molecules arranged so that their polar headgroups form an outer spherical
shell, while the hydrophobic, hydrocarbon chains are oriented towards the
center of the sphere, forming a core. Micelles form in an aqueous solution
containing surfactant at a high enough concentration so that micelles
naturally
result. Surfactants useful for forming micelles include, but are not limited
to,
potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium
dodecane sulfonate, sodium lauryl sulfate, docusate sodium,
decyltrimethylammonium bromide, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium chloride,
dodecylammonium chloride, polyoxy1-8 dodecyl ether, polyoxyl-12 dodecyl
ether, nonoxynol 10, and nonoxynol 30.
Microspheres, similarly, may be incorporated into the presently
described topical formulations. Like liposomes and micelles, microspheres
essentially encapsulate one or more components of the present formulations.
They are generally, but not necessarily, formed from lipids, preferably
charged
lipids such as phospholipids. Preparation of lipidic microspheres is well
known
in the art.
Various additives, as known to those skilled in the art, may also
be included in the topical formulations. For example, solvents, including
relatively small amounts of alcohol, may be used to solubilize certain
formulation components. It may be desirable, for certain topical formulations
or
in cases of particularly severe skin injury such as a post-surgical acute or
chronic wound or post-surgical dermal wound dehiscence, to include in the
topical formulation an added skin permeation enhancer in the formulation.
Examples of suitable enhancers include, but are not limited to, ethers such as
diethylene glycol monoethyl ether (available commercially as Transcutol ) and
diethylene glycol monomethyl ether; surfactants such as sodium laurate,
sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride,
Poloxamer (231, 182, 184), Tween (20, 40, 60, 80), and lecithin (U.S. Pat.
No. 4,783,450); alcohols such as ethanol, propanol, octanol, benzyl alcohol,
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
72
and the like; polyethylene glycol and esters thereof such as polyethylene
glycol
monolaurate (PEGML; see, e.g., U.S. Pat. No. 4,568,343); amides and other
nitrogenous compounds such as urea, dimethylacetamide (DMA),
dimethylformamide (DMF), 2-pyrrolidone, 1 -methyl-2-pyrrolidone,
ethanolamine, diethanolamine, and triethanolamine; terpenes; alkanones; and
organic acids, particularly citric acid and succinic acid. Azone and
sulfoxides
such as DMSO and C10MS0 may also be used, but are less preferred.
Most preferred skin permeation enhancers are those lipophilic co-
enhancers typically referred to as "plasticizing" enhancers, i.e., enhancers
that
have a molecular weight in the range of about 150 to 1000 daltons, an aqueous
solubility of less than about 1 wt %, preferably less than about 0.5 wt %, and
most preferably less than about 0.2 wt %. The Hildebrand solubility parameter
of plasticizing enhancers is in the range of about 2.5 to about 10, preferably
in
the range of about 5 to about 10. Preferred lipophilic enhancers are fatty
esters, fatty alcohols, and fatty ethers. Examples of specific and most
preferred
fatty acid esters include methyl laurate, ethyl oleate, propylene glycol
monolaurate, propylene glycerol dilaurate, glycerol monolaurate, glycerol
monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Fatty alcohols
include, for example, stearyl alcohol and oleyl alcohol, while fatty ethers
include
compounds wherein a diol or triol, preferably a 02-C4 alkane diol or triol,
are
substituted with one or two fatty ether substituents. Additional skin
permeation
enhancers will be known to those of ordinary skill in the art of topical drug
delivery, and/or are described in the relevant literature. See, e.g.,
Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, Boca
Raton, FL, 1995).
Various other additives may be included in the topical
formulations according to certain embodiments of the present invention, in
addition to those identified above. These include, but are not limited to,
antioxidants, astringents, perfumes, preservatives, emollients, pigments,
dyes,
humectants, propellants, and sunscreen agents, as well as other classes of
materials whose presence may be cosmetically, medicinally or otherwise
desirable. Typical examples of optional additives for inclusion in the
formulations of certain embodiments of the invention are as follows:
preservatives such as sorbate; solvents such as isopropanol and propylene
glycol; astringents such as menthol and ethanol; emollients such as
polyalkylene methyl glucosides; humectants such as glycerine; emulsifiers such
as glycerol stearate, PEG-100 stearate, polyglycery1-3 hydroxylauryl ether,
and
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
73
polysorbate 60; sorbitol and other polyhydroxyalcohols such as polyethylene
glycol; sunscreen agents such as octyl methoxyl cinnamate (available
commercially as Parsol MCX) and butyl methoxy benzoylmethane (available
under the tradename Parsol 1789); antioxidants such as ascorbic acid (vitamin
C), a-tocopherol (Vitamin E), 8-tocopherol , 7-tocopherol, 6-tocopherol, s-
tocopherol , 2-
tocopherol, fi-tocopherol , and retinol (vitamin A);
essential oils, ceramides, essential fatty acids, mineral oils, wetting agents
and
other surfactants such as the PLURONICO series of hydrophilic polymers
available from BASF (Mt. Olive, NJ), vegetable oils (e.g., soy bean oil, palm
oil,
liquid fraction of shea butter, sunflower oil), animal oils (e.g.,
perhydrosqualene), mineral oils, synthetic oils, silicone oils or waxes (e.g.,
cyclomethicone and dimethicone), fluorinated oils (generally
perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes (e.g.,
beeswax, carnauba wax, and paraffin wax); skin-feel modifiers; and thickeners
and structurants such as swelling clays and cross-linked carboxypolyalkylenes
that may be obtained commercially under the Carbopol trademark.
Other additives include beneficial agents such as those materials
that condition the skin (particularly, the upper layers of the skin in the
stratum
corneum) and keep it soft by retarding the decrease of its water content
and/or
protect the skin. Such conditioners and moisturizing agents include, by way of
example, pyrrolidine carboxylic acid and amino acids; organic antimicrobial
agents such as 2,4,4'-trichloro-2-hydroxy diphenyl ether (triclosan) and
benzoic
acid; anti-inflammatory agents such as acetylsalicylic acid and glycyrrhetinic
acid; anti-seborrhoeic agents such as, retinoic acid; vasodilators such as
nicotinic acid; inhibitors of melanogenesis such as kojic acid; and mixtures
thereof. Other advantageously included cosmeceutically active agents may be
present, for example, a-hydroxyacids, a-ketoacids, polymeric hydroxyacids,
moisturizers, collagen, marine extracts, and antioxidants such as ascorbic
acid
(vitamin C), a-tocopherol (Vitamin E) or other tocopherols such as those
described above, and retinol (vitamin A), and/or cosmetically acceptable
salts,
esters, amides, or other derivatives thereof. Additional cosmetic agents
include
those that are capable of improving oxygen supply in skin tissue, as
described,
for example, in WO 94/00098 and WO 94/00109. Sunscreens may also be
included.
Other embodiments may include a variety of non-carcinogenic,
non-irritating healing materials that facilitate treatment with the
formulations of
certain embodiments of the invention. Such healing materials may include
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
74
nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts,
honey, glandular or animal extracts, or safe therapeutic agents that may be
added to the formulation to facilitate dermal healing. The amounts of these
various additives are those conventionally used in the cosmetics field, and
.. range, for example, from about 0.01% to about 20% of the total weight of
the
topical formulation.
The formulations of certain embodiments of the invention may
also include conventional additives such as opacifiers, 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 methyl and propyl
esters of p-hydroxybenzoic acid (e.g., methyl and propyl paraben), sodium
benzoate, sorbic acid, imidurea, and combinations thereof. The formulations
may also contain irritation-mitigating additives to minimize or eliminate the
possibility of skin irritation or skin damage resulting from the anti-
infective acute
or chronic wound healing and skin tissue repair-promoting compound to be
administered, or from other components of the composition. Suitable irritation-
mitigating additives include, for example: a-tocopherol ; monoamine oxidase
inhibitors, particularly phenyl alcohols such as 2-phenyl-1-ethanol; glycerin;
salicylates; ascorbates; ionophores such as monensin; amphiphilic amines;
ammonium chloride; N-acetylcysteine; capsaicin; and chioroquine. The
irritation-mitigating additive, if present, may be incorporated into the
topical
formulation at a concentration effective to mitigate irritation or skin
damage,
typically representing not more than about 20 wt %, more typically not more
than about 5 wt %, of the formulation.
The topical formulations may also contain, in addition to the
antiseptic/ wound healing/ anti-biofilm/ skin tissue repair-promoting compound
(e.g., a BT compound, preferably as substantially homogeneous microparticles
as provided herein, and optionally in combination with one or more synergizing
antibiotics as described herein), a therapeutically effective amount of one or
more additional pharmacologically active agents suitable for topical
administration. Such agents may include an asymmetrical lamellar aggregate
consisting of phospholipids and oxygen-loaded fluorocarbon or a fluorocarbon
compound mixture, which are capable of improving oxygen supply in skin
tissue, as described, for example, in International Patent Publication Nos. WO
94/00098 and WO 94/00109.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
Suitable pharmacologically active agents that may be
incorporated into the present topical formulations and thus topically applied,
may include but are not limited to, the following: agents that improve or
eradicate pigmented or non-pigmented age spots, keratoses, and wrinkles;
5 antimicrobial agents; antibacterial agents; antipruritic and antixerotic
agents;
antiinflammatory agents; local anesthetics and analgesics; corticosteroids;
retinoids (e.g., retinoic acid; vitamins; hormones; and antimetabolites. Some
examples of topical pharmacologically active agents include acyclovir,
amphotericins, chlorhexidine, clotrimazole, ketoconazole, econazole,
10 miconazole, metronidazole, minocycline, nystatin, neomycin, kanamycin,
phenytoin, para-amino benzoic acid esters, octyl methoxycinnamate, octyl
salicylate, oxybenzone, dioxybenzone, tocopherol, tocopheryl acetate, selenium
sulfide, zinc pyrithione, diphenhydramine, pramoxine, lidocaine, procaine,
erythromycin, tetracycline, clindamycin, crotamiton, hydroquinone and its
15 monomethyl and benzyl ethers, naproxen, ibuprofen, cromolyn, retinoic
acid,
retinol, retinyl palmitate, retinyl acetate, coal tar, griseofulvin,
estradiol,
hydrocortisone, hydrocortisone 21-acetate, hydrocortisone 17-valerate,
hydrocortisone 17-butyrate, progesterone, betamethasone valerate,
betamethasone dipropionate, triamcinolone acetonide, fluocinonide, clobetasol
20 propionate, minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide,
and
5-fluorouracil. As also noted above, certain embodiments contemplate
inclusion in the formulation of an antibiotic such as a carbapenem antibiotic,
a
cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide
antibiotic, a
lincosamide antibiotic, a pen icillinase-resistant penicillin antibiotic, an
25 aminopenicillin antibiotic, or an aminoglycoside antibiotic such as
amikacin.
A pharmacologically acceptable carrier may also be incorporated
in the topical formulation of certain present embodiments and may be any
carrier conventionally used in the art. Examples include water, lower
alcohols,
higher alcohols, honey, polyhydric alcohols, monosaccharides, disaccharides,
30 polysaccharides, sugar alcohols such as, for example, glycols (2-
carbon),
glycerols (3-carbon), erythritols and threitols (4-carbon), arabitols,
xylitols and
ribitols (5-carbon), rnannitols, sorbitols, dulcitols and iditols (6-carbon),
isomaltols, maltitols, lactitols and polyglycitols, hydrocarbon oils, fats and
oils,
waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants,
silicone
35 surfactants, and water-based mixtures and emulsion-based mixtures of
such
carriers.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
76
Topical formulation embodiments of the present invention may be
applied regularly to whatever acute or chronic wound site (e.g., the wound
itself
and surrounding tissue, including surrounding tissue that appears unaffected
by
infection or otherwise normal or healthy) or skin area or other epithelial
tissue
surface (e.g., gastrointestinal tract, respiratory tract, glandular tissue)
requires
treatment with the frequency and in the amount necessary to achieve the
desired results. The frequency of treatment depends on the nature of the skin
(or other epithelial tissue) condition (e.g., an acute or chronic wound or
other
skin wound such as may be found in dehiscence that results from a surgical
incision, or other types of skin wounds), the degree of damage or
deterioration
of the skin (or other tissue), the responsiveness of the user's skin (or other
tissue), the strength of the active ingredients (e.g., the herein described
wound-
healing! antiseptic/ anti-biofilm/ skin tissue repair-promoting compounds such
as a BT compound and optionally one or more additional pharmaceutically
active ingredients, such as an antibiotic, e.g., amikacin or other antibiotic)
in the
particular embodiment, the effectiveness of the vehicle used to deliver the
active ingredients into the appropriate layer of the skin (or other epithelial
surface-containing tissue), the ease with which the formula is removed by
physical contact with bandages or other dressings or clothing, or its removal
by
.. sweat or other intrinsic or extrinsic fluids, and the convenience to the
subject's
or patient's activity level or lifestyle.
Typical concentrations of active substances such as the BT
compound antiseptic/ anti-biofilm/ wound-healing/ skin tissue repair-promoting
compositions described herein can range, for example, from about 0.001-30%
by weight based on the total weight of the composition, to about 0.01-5.0%,
and
more preferably to about 0.1-2.0%. As one representative example,
compositions of these embodiments of the present invention may be applied to
an acute or chronic wound and/or to the skin at a rate equal to from about 1.0
mg/cm2 of skin to about 20.0 ring/cm2 of skin. Representative examples of
topical formulations include, but are not limited to, aerosols, alcohols,
anhydrous bases (such as lipsticks and powders), aqeuous solutions, creams,
emulsions (including either water-in-oil or oil-in-water emulsions), fats,
foams,
gels, hydro-alcoholic solutions, liposomes, lotions, microemulsions,
ointments,
oils, organic solvents, polyols, polymers, powders, salts, silicone
derivatives,
and waxes. Topical formulations may include, for example, chelating agents,
conditioning agents, emollients, excipients, humectants, protective agents,
thickening agents, or UV absorbing agents. One skilled in the art will
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
77
appreciate that formulations other than those listed may be used in
embodiments of the present invention.
Chelating agents may be optionally included in topical
formulations, and may be selected from any agent that is suitable for use in a
cosmetic composition, and may include any natural or synthetic chemical which
has the ability to bind divalent cationic metals such as Ca2+, Mn2+, or Mg2+.
Examples of chelating agents include, but are not limited to EDTA, disodium
EDTA, EGTA, citric acid, and dicarboxylic acids.
Conditioning agents may also be optionally included in topical
formulations. Examples of skin conditioning agents include, but are not
limited
to, acetyl cysteine, N-acetyl dihydrosphingosine, acrylates/behenyl
acrylate/dimethicone acrylate copolymer, adenosine, adenosine cyclic
phosphate, adensosine phosphate, adenosine triphosphate, alanine, albumen,
algae extract, allantoin and deriviatives, aloe barbadensis extracts, aluminum
PCA, amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk
powder, butylene glycol, caffeine, calcium gluconate, capsaicin,
carbocysteine,
carnosine, beta-carotene, casein, catalase, cephalins, ceramides, chamomilla
recutita (matricaria) flower extract, cholecalciferol, cholesteryl esters,
coco-
betaine, coenzyme A, corn starch modified, crystallins, cycloethoxymethicone,
cysteine DNA, cytochrome C, darutoside, dextran sulfate, dimethicone
copolyols, dimethylsilanol hyaluronate, DNA, elastin, elastin amino acids,
epidermal growth factor, ergocalciferol, ergosterol, ethylhexyl PCA,
fibronectin,
folic acid, gelatin, gliadin, beta-glucan, glucose, glycine, glycogen,
glycolipids,
glycoproteins, glycosaminoglycans, glycosphingolipids, horseradish peroxidase,
hydrogenated proteins, hydrolyzed proteins, jojoba oil, keratin, keratin amino
acids, and kinetin, lactoferrin, lanosterol, lauryl PCA, lecithin, linoleic
acid,
linolenic acid, lipase, lysine, lysozyme, malt extract, maltodextrin, melanin,
methionine, mineral salts, niacin, niacinamide, oat amino acids, oryzanol,
palmitoyl hydrolyzed proteins, pancreatin, papain, PEG, pepsin, phospholipids,
phytosterols, placental enzymes, placental lipids, pyridoxal 5-phosphate,
quercetin, resorcinol acetate, riboflavin, RNA, saccharomyces lysate extract,
silk amino acids, sphingolipids, stearamidopropyl betaine, stearyl palmitate,
tocopherol, tocopheryl acetate, tocopheryl linoleate, ubiquinone, vitis
vinifera
(grape) seed oil, wheat amino acids, xanthan gum, and zinc gluconate. Skin
36 conditioning agents other than those listed above may be combined with a
disclosed composition or preparation provided thereby, as can be readily
appreciated by one skilled in the art.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
78
Topical formulations may also optionally include one or more
emollients, examples of which include, but are not limited to, acetylated
lanolin,
acetylated lanolin alcohol, acrylates/C10-30 alkyl acrylate crosspolymer,
acrylates
copolymer, alanine, algae extract, aloe barbadensis extract or gel, althea
officinalis extract, aluminum starch octenylsuccinate, aluminum stearate,
apricot
(prunus armeniaca) kernel oil, arginine, arginine aspartate, arnica montana
extract, ascorbic acid, ascorbyl palmitate, aspartic acid, avocado (persea
gratissima) oil, barium sulfate, barrier sphingolipids, butyl alcohol,
beeswax,
behenyl alcohol, beta-sitosterol, BHT, birch (betula alba) bark extract,
borage
(borago officinalis) extract, 2-bromo-2-nitropropane-1,3-diol, butcherbroom
(ruscus aculeatus) extract, butylene glycol, calendula officinalis extract,
calendula officinalis oil, candelilla (euphorbia cerifera) wax, canola oil,
caprylic/capric triglyceride, cardamon (elettaria cardamomum) oil, carnauba
(copernicia cerifera) wax, carrageenan (chondrus crispus), carrot (daucus
carota sativa) oil, castor (ricinus communis) oil, ceramides, ceresin,
ceteareth-
5, ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20, ceteth-24, cetyl
acetate, cetyl octanoate, cetyl palmitate, chamomile (anthemis nobilis) oil,
cholesterol, cholesterol esters, cholesteryl hydroxystearate, citric acid,
clary
(salvia sclarea) oil, cocoa (theobroma cacao) butter, coco-caprylate/caprate,
coconut (cocos nucifera) oil, collagen, collagen amino acids, corn (zea mays)
oil, fatty acids, decyl oleate, dextrin, diazolidinyl urea, dimethicone
copolyol,
dimethiconol, dioctyl adipate, dioctyl succinate, dipentaerythrityl
hexacaprylate/hexacaprate, DMDM hydantoin, DNA, erythritol, ethoxydiglycol,
ethyl linoleate, eucalyptus globulus oil, evening primrose (oenothera biennis)
oil, fatty acids, tructose, gelatin, geranium maculatum oil, glucosamine,
glucose
glutamate, glutamic acid, glycereth-26, glycerin, glycerol, glyceryl
distearate,
glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glyceryl
myristate,
glyceryl oleate, glyceryl stearate, glyceryl stearate SE, glycine, glycol
stearate,
glycol stearate SE, glycosaminoglycans, grape (vitis vinifera) seed oil, hazel
(corylus americana) nut oil, hazel (corylus avellana) nut oil, hexylene
glycol,
honey, hyaluronic acid, hybrid safflower (carthamus tinctorius) oil,
hydrogenated castor oil, hydrogenated coco-glycerides, hydrogenated coconut
oil, hydrogenated lanolin, hydrogenated lecithin, hydrogenated palm glyceride,
hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenated tallow
glyceride, hydrogenated vegetable oil, hydrolyzed collagen, hydrolyzed
elastin,
hydrolyzed glycosaminoglycans, hydrolyzed keratin, hydrolyzed soy protein,
hydroxylated lanolin, hydroxyproline, imidazolidinyl urea, iodopropynyl
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
79
butylcarbamate, isocetyl stearate, isocetyl stearoyl stearate, isodecyl
oleate,
isopropyl isostearate, isopropyl lanolate, isopropyl myristate, isopropyl
palmitate, isopropyl stearate, isostearamide DEA, isostearic acid, isostearyl
lactate, isostearyl neopentanoate, jasmine (jasminum officinale) oil, jojoba
(buxus chinensis) oil, kelp, kukui (aleurites moluccana) nut oil, lactamide
MEA,
laneth-16, laneth-10 acetate, lanolin, lanolin acid, lanolin alcohol, lanolin
oil,
lanolin wax, lavender (lavandula angustifolia) oil, lecithin, lemon (citrus
medica
limonum) oil, linoleic acid, linolenic acid, macadamia ternifolia nut oil,
magnesium stearate, magnesium sulfate, maltitol, matricaria (chamomilla
recutita) oil, methyl glucose sesquistearate, methylsilanol PCA,
microcrystalline
wax, mineral oil, mink oil, mortierella oil, myristyl lactate, myristyl
myristate,
myristyl propionate, neopentyl glycol dicaprylate/dicaprate, octyldodecanol,
octyldodecyl myristate, octyldodecyl stearoyl stearate, octyl hydroxystearate,
octyl palmitate, octyl salicylate, octyl stearate, oleic acid, olive (olea
europaea)
oil, orange (citrus aurantium dulcis) oil, palm (elaeis guineensis) oil,
palmitic
acid, pantethine, panthenol, panthenyl ethyl ether, paraffin, PCA, peach
(prunus
persica) kernel oil, peanut (arachis hypogaea) oil, PEG-8 C12 18 ester, PEG-15
cocamine, PEG-150 distearate, PEG-60 glyceryl isostearate, PEG-5 glyceryl
stearate, PEG-30 glyceryl stearate, PEG-7 hydrogenated castor oil, PEG-40
hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-20 methyl
glucose sesquistearate, PEG-40 sorbitan peroleate, PEG-5 soy sterol, PEG-10
soy sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, PEG-32
stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate, PEG-150
stearate, pentadecalactone, peppermint (mentha piperita) oil, petrolatum,
phospholipids, polyamino sugar condensate, polyglycery1-3 diisostearate,
polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60,
polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate,
potassium sorbate, potassium stearate, propylene glycol, propylene glycol
dicaprylate/dicaprate, propylene glycol dioctanoate, propylene glycol
dipelargonate, propylene glycol laurate, propylene glycol stearate, propylene
glycol stearate SE, PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18
hectorite, quaternium-22, retinal, retinyl palmitate, rice (oryza sativa) bran
oil,
RNA, rosemary (rosmarinus officinalis) oil, rose oil, safflower (carthamus
tinctorius) oil, sage (salvia officinalis) oil, salicylic acid, sandalwood
(santalum
album) oil, serine, serum protein, sesame (sesamum indicum) oil, shea butter
(butyrospermum parkii), silk powder, sodium chondroitin sulfate, sodium DNA,
sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
polyglutamate, sodium stearate, soluble collagen, sorbic acid, sorbitan
laurate,
sorbitan oleate, sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate,
sorbitol, soybean (glycine soja) oil, sphingolipids, squalane, squalene,
stearamide MEA-stearate, stearic acid, stearoxy dimethicone,
5 stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate, stearyl
heptanoate, stearyl stearate, sunflower (helianthus annuus) seed oil, sweet
almond (prunus amygdalus dulcis) oil, synthetic beeswax, tocopherol,
tocopheryl acetate, tocopheryl linoleate, tribehenin, tridecyl neopentanoate,
tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil, water,
waxes,
10 wheat (triticum vulgare) germ oil, and ylang ylang (cananga odorata) oil.
In some embodiments a topical formulation may contain a suitable
excipient, which typically should have a high affinity for the skin, be well
tolerated, stable, and yield a consistency that allows for easy utilization.
Suitable topical excipients and vehicles can be routinely selected for a
15 particular use by those skilled in the art, and especially with
reference to one of
many standard texts in the art, such as Remington's Pharmaceutical Sciences,
Vol. 18, Mack Publishing Co., Easton, Pa. (1990), in particular Chapter 87.
Optionally one or more humectants are also included in the topical
formulation.
Examples of humectants include, but are not limited to, amino acids,
20 chondroitin sulfate, diglycerin, erythritol, fructose, glucose, glycerin,
glycerol,
glycol, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey,
hydrogenated starch hydrolysate, inositol, lactitol, maltitol, maltose,
mannitol,
natural moisturization factor, PEG-15 butanediol, polyglyceryl sorbitol, salts
of
pyrollidone carboxylic acid, potassium PCA, propylene glycol, sodium
25 glucuronate, sodium PCA, sorbitol, sucrose, trehalose, urea, and xylitol.
Certain embodiments contemplate topical formulations containing
one or more additional skin protective agent. Examples of skin protective
agents may include, but are not limited to, algae extract, allantoin, aluminum
hydroxide, aluminum sulfate, betaine, camellia sinensis leaf extract,
30 cerebrosides, dimethicone, glucuronolactone, glycerin, kaolin, lanolin,
malt
extract, mineral oil, petrolatum, potassium gluconate, and talc. One skilled
in
the art will readily appreciate that skin protectants other than those listed
above
may also be combined with a disclosed composition of the present invention or
preparation provided thereby.
36 Surfactants may also desirably be included in certain topical
formulations contemplated herein, and can be selected from any natural or
synthetic surfactants suitable for use in cosmetic compositions, such as
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
81
cationic, anionic, zwitterionic, or non-ionic surfactants, or mixtures
thereof.
(See Rosen, M., "Surfactants and Interfacial Phenomena," Second Edition,
John Wiley & Sons, New York, 1988, Chapter 1, pages 431). Examples of
cationic surfactants may include, but are not limited to, DMDAO or other amine
oxides, long-chain primary amines, diamines and polyamines and their salts,
quaternary ammonium salts, polyoxyethylenated long-chain amines, and
quaternized polyoxyethylenated long-chain amines. Examples of anionic
surfactants may include, but are not limited to, SDS; salts of carboxylic
acids
(e.g., soaps); salts of sulfonic acids, salts of sulfuric acid, phosphoric and
polyphosphoric acid esters; alkylphosphates; monoalkyl phosphate (MAP); and
salts of perfluorocarboxylic acids. Examples of zwitterionic surfactants may
include, but are not limited to, cocoamidopropyl hydroxysultaine (CAPHS) and
others which are pH-sensitive and require special care in designing the
appropriate pH of the formula (i.e., alkylaminopropionic acids, imidazoline
carboxylates, and betaines) or those which are not pH-sensitive (e.g.,
sulfobetaines, sultaines). Examples of non-ionic surfactants may include, but
are not limited to, alkylphenol ethoxylates, alcohol ethoxylates,
polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans,
long-chain carboxylic acid esters, alkonolamides, tertiary acetylenic glycols,
polyoxyethylenated silicones, N-alkylpyrrolidones, and alkylpolyglycosidases.
Wetting agents, mineral oil or other surfactants such as non-ionic detergents
or
agents such as one or more members of the PLURONICS series (BASF, Mt.
Olive, NJ) may also be included, for example and according to non-limiting
theory, to discourage aggregation of BT microparticles within the
microparticulate suspension. Any combination of surfactants is acceptable.
Certain embodiments may include at least one anionic and one cationic
surfactant, or at least one cationic and one zwitterionic surfactant which are
compatible, i.e., do not form complexes which precipitate appreciably when
mixed.
Examples of thickening agents that may also be present in certain
topical formulations include, but are not limited to, acrylamides copolymer,
agarose, amylopectin, bentonite, calcium alginate, calcium carboxymethyl
cellulose, carbomer, carboxymethyl chitin, cellulose gum, dextrin, gelatin,
hydrogenated tallow, hydroxytheylcellulose, hydroxypropylcellulose,
hydroxpropyl starch, magnesium alginate, methylcellulose, microcrystalline
cellulose, pectin, various PEG's, polyacrylic acid, polymethacrylic acid,
polyvinyl
alcohol, various PPG's, sodium acrylates copolymer, sodium carrageenan,
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
82
xanthan gum, and yeast beta-glucan. Thickening agents other than those listed
above may also be used in embodiments of this invention.
According to certain embodiments contemplated herein, a topical
formulation may comprise one or more sunscreening or UV absorbing agents.
Where ultraviolet light- (UVA and UVB) absorbing properties are desired, such
agents may include, for example, benzophenone, benzophenone-1,
benzophenone-2, benzophenone-3, benzophenone-4, IDenzophenone-5,
benzophenone-6, benzophenone-7, benzophenone-8, benzophenone-9,
benzophenone-10, benzophenone-11, benzophenone-12, benzyl salicylate,
butyl PABA, cinnamate esters, cinoxate, DEA-methoxycinnamate, diisopropyl
methyl cinnamate, ethyl dihydroxypropyl PABA, ethyl diisopropylcinnamate,
ethyl methoxycinnamate, ethyl PABA, ethyl urocanate, glyceryl octanoate
dimethoxycinnamate, glyceryl PABA, glycol salicylate, homosalate, isoamyl p-
methoxycinnamate, oxides of titanium, zinc, zirconium, silicon, manganese, and
cerium, PABA, PABA esters, Parsol 1789, and isopropylbenzyl salicylate, and
mixtures thereof. One skilled in the art will appreciate that sunscreening and
UV absorbing or protective agents other than those listed may be used in
certain embodiments of the present invention.
Topical formulations disclosed herein are typically effective at pH
values between about 2.5 and about 10Ø Preferably, the pH of the
composition is at or about the following pH ranges: about pH 5.5 to about pH
8.5, about pH 5 to about pH 10, about pH 5 to about pH 9, about pH 5 to about
pH 8, about pH 3 to about pH 10, about pH 3 to about pH 9, about pH 3 to
about pH 8, and about pH 3 to about pH 8.5. Most preferably, the pH is about
pH 7 to about pH 8. One of ordinary skill in the art may add appropriate pH
adjusting ingredients to the compositions of the present invention to adjust
the
pH to an acceptable range. "About" a specified pH is understood by those
familiar with the art to include formulations in which at any given time the
actual
measured pH may be less or more than the specified value by no more than
0.7, 0.6, 0.5, 0.4., 0.3, 0.2 or 0.1 pH units, where it is recognized that
formulation composition and storage conditions may result in drifting of pH
from
an original value.
A cream, lotion, gel, ointment, paste or the like may be spread on
the affected surface and gently rubbed in. A solution may be applied in the
same way, but more typically will be applied with a dropper, swab, or the
like,
and carefully applied to the affected areas. The application regimen will
depend on a number of factors that may readily be determined, such as the
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
83
severity of the wound and its responsiveness to initial treatment, but will
normally involve one or more applications per day on an ongoing basis. One of
ordinary skill may readily determine the optimum amount of the formulation to
be administered, administration methodologies and repetition rates. In
general,
it is contemplated that the formulations of these and related embodiments of
the
invention will be applied in the range of once or twice or more weekly up to
once, twice, thrice, four times or more daily.
As also discussed above, the topical formulations useful herein
thus also contain a pharmaceutically acceptable carrier, including any
suitable
diluent or excipient, which includes any pharmaceutical agent that does not
itself harm the subject receiving the composition, and which may be
administered without undue toxicity. Pharmaceutically acceptable carriers
include, but are not limited to, liquids, such as water, saline, glycerol and
ethanol, and the like, and may also include viscosity enhancers (e.g., balsam
fir
resin) or film-formers such as colloidion or nitrocellulose solutions. A
thorough
discussion of pharmaceutically acceptable carriers, diluents, and other
excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES
(Mack Pub. Co., N.J. current edition).
When the topical formulation is in the form of a gel- or liquid-filled
capsule, for example, a gelatin capsule, it may contain, in addition to
materials
of the above type, a liquid carrier such as polyethylene glycol or oil. The
liquid
pharmaceutical compositions of certain embodiments of the invention, whether
they be solutions, suspensions or other like form, may include one or more of
the following: sterile diluents such as water for injection, saline solution,
preferably physiological saline, Ringer's solution, isotonic sodium chloride,
fixed
oils such as synthetic mono or diglycerides which may serve as the solvent or
suspending medium, polyethylene glycols, glycerin, propylene glycol or other
solvents; antibacterial agents such as benzyl alcohol or methyl paraben;
additional antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as
acetates, citrates or phosphates and agents for the adjustment of tonicity
such
as sodium chloride or dextrose.
For topical administration the carrier may suitably comprise a
solution, emulsion, ointment or gel base. The base, for example, may comprise
one or more of the following: petrolatum, lanolin, polyethylene glycols, bee
wax, mineral oil, diluents such as water and alcohol, and emulsifiers and
stabilizers. Thickening agents may be present in a pharmaceutical or
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
84
cosmeceutical composition for topical administration. If intended for
transdermal administration, the composition may include a transdermal patch or
iontophoresis device. Topical formulations may contain a concentration of the
compound of certain embodiments of the invention from about 0.1 to about
10% w/v (weight per unit volume). A topical formulation may be provided in the
form of a cream, lotion, solution, spray, gel, ointment, paste or the like,
and/or
may contain liposomes, micelles, microspheres and/or other microparticle or
nanoparticle delivery elements. A topical formulation may also be provided in
the form of time-release or sustained release particles or pellets, for
example,
slow-release ethylene vinyl acetate polymer (e.g., Elvax040, Aldrich,
Milwaukee, WI) pellets, that can be directly administered to a wound site.
The topical formulation may include an agent that binds to the
skin tissue repair-promoting compound and thereby assists in its delivery to
skin epithelial cells (e.g., keratinocytes) and/or fibroblasts. Suitable
agents that
may act in this capacity include clathrating agents such as cyclodextrins;
other
agents may include a protein or a liposome.
The topical formulation of certain embodiments of the invention
may also be provided in the form of dosage units that can be administered as
an aerosol. The term aerosol is used to denote a variety of systems ranging
from those of colloidal nature to systems consisting of pressurized packages.
Delivery may be by a liquefied or compressed gas or by a suitable pump
system that dispenses the active ingredients. Aerosols of compounds of certain
embodiments of the invention may be delivered in single phase, bi-phasic, or
tri-phasic systems in order to deliver the active ingredient(s). Delivery of
the
aerosol includes the necessary container, activators, valves, subcontainers,
and the like, which together may form a kit. One skilled in the art, without
undue experimentation may determine preferred aerosols for delivering topical
formulations to the skin or to a wound site.
The topical formulations may be prepared by methodology well
known in the pharmaceutical art. For example, a pharmaceutical composition
intended to be administered to a wound site or to the skin as a spray, wash or
rinse can be prepared by combining a BT antiseptic/ wound-healing/ anti-
biofilm/ skin tissue repair-promoting compound as described herein with
sterile,
distilled water so as to form a solution. A surfactant may be added to
facilitate
the formation of a homogeneous solution or suspension. Surfactants are
compounds that non-covalently interact with the antioxidant active compound
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
so as to facilitate dissolution or homogeneous suspension of the compound in
the aqueous delivery system.
The BT antiseptic/ wound-healing/ anti-biofilm/ skin tissue repair-
promoting compounds for use in topical formulations, or their pharmaceutically
5 acceptable
salts, are administered in a therapeutically effective amount, which
will vary depending upon a variety of factors including the nature of the
wound
site (where relevant), the activity of the specific BT compound employed
(including the inclusion or absence from the formulation of an antibiotic,
such as
an aminoglycoside antibiotic, e.g., amikacin); the metabolic stability and
length
10 of action of
the compound; the age, body weight, general health, sex, skin type,
immune status and diet of the subject; the mode and time of administration;
the
rate of excretion; the drug combination; the severity of the particular skin
wound
for which skin tissue repair is desired; and the subject undergoing therapy.
Generally, a therapeutically effective daily dose is (for a 70 kg mammal) from
15 about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g);
preferably a
therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg
(i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a
therapeutically
effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to
about 25 mg/kg (i.e., 1.75 g).
20 The ranges of
effective doses provided herein are not intended to
be limiting and represent preferred dose ranges. However, the most preferred
dosage will be tailored to the individual subject, as is understood and
determinable by one skilled in the relevant arts. (see, e.g., Berkow et al.,
eds.,
The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodman
25 et al., eds., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 101h edition, Pergamon Press, Inc., Elmsford, N.Y., (2001);
Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and
Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore,
MD. (1987); Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985);
30 Osolci al., eds., Remington's Pharmaceutical Sciences, 18th edition, Mack
Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology,
Appleton and Lange, Norwalk, CT (1992)).
The total dose required for each treatment can be administered by
multiple doses or in a single dose over the course of the day, if desired.
Certain
35 preferred embodiments contemplate a single application of the topical
formulation per day. Generally, and in distinct embodiments, treatment may be
initiated with smaller dosages, which are less than the optimum dose of the
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
86
compound. Thereafter, the dosage is increased by small increments until the
optimum effect under the circumstances is reached.
The topical formulation can be administered alone or in
conjunction with other treatments and/or pharmaceuticals directed to the skin
wound, or directed to other associated symptoms or etiologic factors. For
example, and as also noted above, the topical formulation may further comprise
retinoic acid. As another example, the topical formulation may comprise one or
more skin tissue repair-promoting compounds described herein, or may
comprise two or more such compounds having different cellular wound repair
activities.
The recipients of the topical formulations described herein can be
any vertebrate animal, such as mammals. Among mammals, the preferred
recipients are mammals of the Orders Primate (including humans, apes and
monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta
(including mice, rats, rabbits, and hamsters), and Carnivora (including cats,
and
dogs). Among birds, the preferred recipients are turkeys, chickens and other
members of the same order. The most preferred recipients are humans, and
particularly preferred are humans having one or more acute or chronic wounds
or wounds that contain biofilms.
For topical applications, it is preferred to administer an effective
amount of a pharmaceutical composition comprising a BT compound antiseptic/
wound-healing/ anti-biofilm/ skin tissue repair-promoting compound according
to the herein described embodiments, to a target area, e.g., a skin wound such
as an acute or chronic wound, and/or an at-risk area (e.g., for wound
dehiscence) of the skin, and the like. This amount will generally range from
about 0.0001 mg to about 1 g of a compound of certain embodiments of the
invention per application, depending upon the area to be treated, the severity
of
the wound (or of a past or contemplated surgical incision), and the nature of
the
topical vehicle employed. A preferred topical preparation is an ointment or
slow-release pellets, wherein about 0.001 to about 50 mg of active ingredient
is
used per cc of ointment base or pellet suspension. The pharmaceutical
composition can be formulated as transdermal compositions or transdermal
delivery devices ("patches"). Such compositions include, for example, a
backing, active compound reservoir, a control membrane, liner and contact
adhesive. Such transdermal patches may be used to provide continuous
pulsatile, or on demand delivery of the compounds of the present invention as
desired.
CA 2788669 2017-03-14
87
The compositions of certain embodiments can be formulated so
as to provide quick, sustained or delayed release of the active ingredient
after
administration to the patient by employing procedures known in the art.
Controlled release drug delivery systems include osmotic pump systems and
dissolutional systems containing polymer-coated reservoirs or drug-polymer
matrix formulations. Examples of controlled release systems are given in U.S.
Pat. Nos. 3,845,770 and 4,326,525 and in P. J. Kuzma et al, Regional
Anesthesia 22 (6): 543-551 (1997).
The most suitable route will depend on the nature and severity of
the condition being treated. Those skilled in the art are also familiar with
determining topical administration methods (sprays, creams, open application,
occlusive dressing, soaks, washes, etc.), dosage forms, suitable
pharmaceutical excipients and other matters relevant to the delivery of the
compounds to a subject in need thereof.
Throughout this specification, unless the context requires
otherwise, the words "comprise'', "comprises" and "comprising" will be
understood to imply the inclusion of a stated step or element or group of
steps
or elements but not the exclusion of any other step or element or group of
steps
or elements. By "consisting of' is meant including, and limited to, whatever
follows the phrase "consisting of." Thus, the phrase "consisting of' indicates
that the listed elements are required or mandatory, and that no other elements
may be present. By "consisting essentially of' is meant including any elements
listed after the phrase, and limited to other elements that do not interfere
with or
contribute to the activity or action specified in the disclosure for the
listed
elements. Thus, the phrase "consisting essentially of' indicates that the
listed
elements are required or mandatory, but that no other elements are required
and may or may not be present depending upon whether or not they affect the
activity or action of the listed elements.
In this specification and the appended claims, the singular forms
"a," "an" and "the" include plural references unless the content clearly
dictates
otherwise. As used herein, in particular embodiments, the terms "about" or
"approximately" when preceding a numerical value indicates the value plus or
minus a range of 5%, 6%, 7%, 8% or 9%. In other embodiments, the terms
"about" or "approximately" when preceding a numerical value indicates the
value plus or minus a range of 10%, 11%, 12%, 13% 01 14%. In yet other
embodiments, the terms "about" or "approximately' when preceding a numerical
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
88
value indicates the value plus or minus a range of 15%, 16%, 17%, 18%, 19%
or 20%.
The following Examples are presented by way of illustration and
not limitation.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
89
EXAMPLES
EXAMPLE 1
PREPARATION OF BT COMPOUNDS
The following BT compounds were prepared either according to
the methods of Domenico et al. (U.S. RE37,793, U.S. 6,248,371, U.S.
6,086,921, U.S. 6,380,248) or as microparticles according to the synthetic
protocol described below for BisEDT. Shown are atomic ratios relative to a
single bismuth atom, for comparison, based on the stoichiometric ratios of the
reactants used and the known propensity of bismuth to form trivalent
complexes with sulfur containing compounds. The numbers in parenthesis are
the ratios of bismuth to one (or more) thiol agents (e.g. Bi:thio11/thio12;
see also
Table 1).
1) CPD 1B-1 Bis-EDT (1:1) BiC2H4S2
2) CPD 1B-2 Bis-EDT (1:1.5) BiC3H6S3
3) CPD 1B-3 Bis-EDT (1:1.5) BiC3H6S3
4) CPD 1C Bis-EDT (soluble Bi prep.) (1:1.5) BiC3H6S3
5) CPD 2A Bis-Bal (1:1) BiC3H6520
6) CPD 2B Bis-Bal (1:1.5) BiC4.6H901 6S3
7) CPD 3A Bis-Pyr (1:1.5) BiC761-16N1.5015S1.5
8) CPD 3B Bis-Pyr (1:3) BiC161-112N303S3
9) CPD 4 Bis-Ery (1:1.5) BiC6H1203S3
10) CPD 5 Bis-Tol (1:1.5) B1C10.6H9S3
11) CPD 6 Bis-BDT (1:1.5) BiC6H12S3
12) CPD 7 Bis-PDT (1:1.5) BiC461-19S3
13) CPD 8-1 Bis-Pyr/BDT (1:1/1)
14) CPD 8-2 Bis-Pyr/BDT (1:1/0.5)
15) CPD 9 Bis-2hydrm, propane thiol (1:3)
16) CPD 10 Bis-Pyr/Bal (1:1/0.5)
17) CPD 11 Bis-Pyr/EDT (1:1/0.5)
18) CPD 12 Bis-Pyr/Tol (1:1/0.5)
19) CPD 13 Bis-Pyr/PDT (1:1/0.5)
20) CPD 14 Bis-Pyr/Ery (1:1/0.5)
21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
Microparticulate bismuth-1,2-ethanedithiol (Bis-EDT, soluble
bismuth preparation) was prepared as follows:
To an excess (11.4 L) of 5% aqueous HNO3 at room temperature
in a 15 L polypropylene carboy was slowly added by dropwise addition 0.331 L
5 (-0.575 moles) of an aqueous Bi(NO3)3 solution (43% Bi(NO3)3 (w/w), 5%
nitric
acid (w/w), 52% water (w/w), Shepherd Chemical Co., Cincinnati, OH, product
no. 2362; 6 -1.6 g/mL) with stirring, followed by slow addition of absolute
ethanol (4 L). Some white precipitate formed but was dissolved by continued
stirring. An ethanolic solution (-1.56 L, -0.55 M) of 1,2-ethanedithiol (CAS
540-
10 63-6) was separately prepared by adding, to 1.5 L of absolute ethanol,
72.19
mL (0.863 moles) of 1,2-ethanedithiol using a 60 mL syringe, and then stirring
for five minutes. The 1,2-ethanedithiol/ Et0H reagent was then slowly added
by dropwise addition over the course of five hours to the aqueous Bi(NO3)3/
HNO3 solution, with continued stirring overnight. The formed product was
15 allowed to settle as a precipitate for approximately 15 minutes, after
which the
filtrate was removed at 300 mL/min using a peristaltic pump. The product was
then collected by filtration on fine filter paper in a 15-cm diameter Buchner
funnel, and washed sequentially with three, 500-mL volumes each of ethanol,
USP water, and acetone to obtain BisEDT (694.51 gm/ mole) as a yellow
20 .. amorphous powdered solid. The product was placed in a 500 mL amber glass
bottle and dried over CaCl2 under high vacuum for 48 hours. Recovered
material (yield -200 g) gave off a thiol-characteristic odor. The crude
product
was redissolved in 750 mL of absolute ethanol, stirred for 30 min, then
filtered
and washed sequentially with 3 x 50 mL ethanol, 2 x 50 mL acetone, and
25 washed again with 500 mL of acetone. The rewashed powder was triturated
in
1M NaOH (500 mL), filtered and washed with 3 x 220 mL water, 2 x 50 mL
ethanol, and 1 x 400 mL acetone to afford 156.74 gm of purified BisEDT.
Subsequent batches prepared in essentially the same manner resulted in yields
of about 78-91%.
30 The product was characterized as having the structure shown
above in formula I by analysis of data from 1H and 13C nuclear magnetic
resonance (NMR), infrared spectroscopy (IR), ultraviolet spectroscopy (UV),
mass spectrometry (MS) and elemental analysis. An HPLC method was
developed to determine chemical purity of BisEDT whereby the sample was
35 prepared in DMSO (0.5mg/mL), The Amax was determined by scanning a
solution of BisEDT in DMSO between 190 and 600nm. lsocratic HPLC elution
at 1 mL/min was performed at ambient temperature in a mobile phase of 0.1%
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
91
formic acid in acetonitrile:water (9:1) on a Waters (Millipore Corp., Milford,
MA)
model 2695 chromatograph with UV detector monitoring at 265 nm (Xrnax), 2 pL
injection volume, equipped with a YMC Pack PVC Sil NP, 5pm, 250X4.6 mm
inner diameter analytical column (Waters) and a single peak was detected,
reflecting chemical purity of 100 0.1%. Elemental analysis was consistent with
the structure of formula (I).
The dried particulate matter was characterized to assess the
particle size properties. Briefly, microparticles were resuspended in 2%
Pluronic F-68 (BASF, Mt. Olive, NJ) and the suspension was sonicated for 10
minutes in a water bath sonicator at standard setting prior to analysis using
a
Nanosizer/Zetasizer Nano-S particle analyzer (model ZEN1600 (without zeta-
potential measuring capacity), Malvern Instruments, Worcestershire, UK)
according to the manufacturer's recommendations. From compiled data of two
measurements, microparticles exhibited a unimodal distribution with all
detectable events between about 0.6 microns and 4 microns in volumetric
mean diameter (VMD) and having a peak VMD at about 1.3 microns. By
contrast, when BisEDT was prepared by prior methods (Domenico et al., 1997
Antimicrob. Agents Chemother. 41(8):1697-1703) the majority of particles were
heterodisperse and of significantly larger size, precluding their
characterization
on the basis of VMD.
EXAMPLE 2
COLONY BIOFILM MODEL OF CHRONIC WOUND INFECTION:
INHIBITION BY BT COMPOUNDS
Because bacteria that exist in chronic wounds adopt a biofilm
lifestyle, BTs were tested against biofilms for effects on bacterial cell
survival
using biofilms prepared essentially according to described methods (Anderl et
al., 2003 Antimicrob Agents Chemother 47:1251-56; Walters et al., 2003
Antimicrob Agents Chemother 47:317; Wentland et al., 1996 Biotchnol. Prog.
12:316; Zheng et al., 2002 Antimicrob Agents Chemother 46:900).
Briefly, colony biofilms were grown on 10% tryptic soy agar for 24
hours, and transferred to Mueller Hinton plates containing treatments. After
treatment the biofilms were dispersed into peptone water containing 2% w/v
glutathione (neutralizes the BT), and serially diluted into peptone water
before
being spotted onto plates for counting. Two bacteria isolated from chronic
wounds were used separately in the production of colony biofilms for testing.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
92
These were Pseudomonas aeruginosa, a gram negative bacterial strain, and
Methicillin Resistant Staphylococcus aureus (MRSA), which is gram positive.
Bacterial biofilm colonies were grown on top of micro porous
membranes resting on an agar plate essentially as described (Anderl et al.,
2003 Antimicrob Agents Chemother 47:1251-56; Walters et al., 2003
Antimicrob Agents Chemother 47:317; Wentland et al., 1996 Biotchnol. Prog.
12:316; Zheng et al., 2002 Antimicrob Agents Chemother 46:900) The colony
biofilms exhibited many of the familiar features of other biofilm models,
e.g.,
they consisted of cells densely aggregated in a highly hydrated matrix. As
also
reported by others (Brown et al., J Surg Res 56:562; Millward et al, 1989
Microbios 58:155; Sutch et al., 1995 J Pharm Pharmacol 47:1094; Thrower et
al., 1997 J Med Microbio146:425) it was observed that bacteria in colony
biofilms exhibited the same profoundly reduced anti-microbial susceptibility
that
has been quantified in more sophisticated in vitro biofilm reactors. Colony
biofilms were readily and reproducibly generated in large numbers. According
to non-limiting theory, this colony biofilm model shared some of the features
of
an infected wound: bacteria grew at an air interface with nutrients supplied
from beneath the biofilm and minimal fluid flow. A variety of nutrients
sources
was used to cultivate colony biofilms, including blood agar, which is believed
to
mimic in vivo nutrient conditions.
Colony biofilms were prepared by inoculating 5 pl spots of
planktonic bacterial liquid cultures onto a 25 mm diameter polycarbonate
filter
membrane. The membranes were sterilized prior to inoculation, by exposure to
ultraviolet light for 10 min per side. The inocula were grown overnight in
bacterial medium at 37 C and diluted in fresh medium to an optical density of
0.1 at 600 nm prior to deposition on the membrane. The membranes were then
placed on the agar plate containing growth medium. The plates were then
covered and placed, inverted, in an incubator at 37 C. Every 24 h, the
membrane and colony biofilm were transferred, using sterile forceps, to a
fresh
plate. Colony biofilms were typically used for experimentation after 48 hours
of
growth, at which time there were approximately 109 bacteria per membrane.
The colony biofilm method was successfully employed to culture a wide variety
of single species and mixed species biofilms.
To measure susceptibility to antimicrobial agents (e.g., BT
compounds including combinations of BT compounds; antibiotics; and BT
compound-antibiotic combinations), colony biofilms were transferred to agar
plates supplemented with the candidate antimicrobial treatment agent(s).
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
93
Where the duration of exposure to antimicrobial treatment exceeded 24 hours,
the colony biofilms were moved to fresh treatment plates daily. At the end of
the treatment period, the colony biofilms were placed in tubes containing 10
ml
of buffer and vortexed for 1-2 min to disperse the biofilm. In some cases, it
was
.. necessary to briefly process the sample with a tissue homogenizer to break
up
cell aggregates. The resulting cell suspensions were then serially diluted and
plated to enumerate surviving bacteria, which were reported as colony forming
units (CFU) per unit area. Survival data were analyzed using logio
transformation.
For each type of bacterial biofilm colony cultures (Pseudomonas
aeruginosa, PA; methicilin resistant Staphylococcus aureus, MRSA or SA) five
antibiotics and thirteen BT compounds were tested. Antimicrobial agents tested
against PA included the BTs referred to herein as BisEDT and Compounds 2B,
4, 5, 6, 8-2, 9, 10, 11 and 15 (see Table 1), and the antibiotics tobramycin,
amikacin, imipenim, cefazolin, and ciprofloxacin. Antimicrobial agents tested
against SA included the BTs referred to herein as BisEDT and Compounds 2B,
4, 5, 6, 8-2, 9, 10 and 11 (see Table 1), and the antibiotics rifampicin,
daptomycin, minocycline, ampicillin, and vanconnycin. As described above
under "brief descriptions of the drawings", antibiotics were tested at
concentrations of approximately 10-400 times the minimum inhibitory
concentrations (MIC) according to established microbiological methodologies.
Seven BT compounds exhibited pronounced effects-on PA
bacterial survival at the concentrations tested, and two BT compounds
demonstrated pronounced effects on MRSA survival at the concentrations
tested; representative results showing BT effects on bacterial survival are
presented in Figure 1 for BisEDT and BT compound 2B (tested against PA) and
in Figure 2 for BT compounds 2B and 8-2 (tested against SA), in both cases,
relative to the effects of the indicated antibiotics. As also shown in Figures
1
and 2, inclusion of the indicated BT compounds in combination with the
indicated antibiotics resulted in a synergistic effect whereby the potency of
reducing bacterial survival was enhanced relative to the anti-bacterial
effects of
either the antibiotic alone or the BT compound alone. In the PA survival
assay,
compound 15 (Bis-EDT/2hydroxy, propane thiol (1:1/1)) at a concentration of 80
pg/mL exhibited an effect (not shown) that was comparable to the effect
obtained using the combination of 1600 pg/mL AMK plus 80 pg/mL BisEDT
(Fig. 1).
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
94
EXAMPLE 3
DRIP FLOW BIOFILM MODEL OF CHRONIC WOUND INFECTION:
INHIBITION BY BT COMPOUNDS
Drip flow biofilms represent an art accepted authentic model for
forming, and testing the effect of candidate anti-bacterial compounds against,
bacterial biofilms. Drip flow biofilms are produced on coupons (substrates)
placed in the channels of a drip flow reactor. Many different types of
materials
can be used as the substrate for bacterial biofilm formation, including
frosted
glass microscope slides. Nutritive liquid media enters the drip flow
bioreactor
cell chamber by dripping into the chamber near the top, and then flows the
length of a coupon down a 10 degree slope.
Biofilms are grown in drip flow bioreactors and exposed to BT
compounds individually or in combinations and/or to antibiotic compounds
individually or in combinations with other antibacterial agents, including BT
compounds, or to other conventional or candidate treatments for chronic
wounds. BT compounds are thus characterized for their effects on bacterial
biofilms in the drip-flow reactor. Biofilms in the drip-flow reactor are
prepared
according to established methodologies (e.g., Stewart et al., 2001 J Appl
Microbiol. 91:525; Xu et al., 1998 App!. Environ. Microbiol. 64:4035). This
design involves cultivating biofilms on inclined polystyrene coupons in a
covered chamber. An exemplary culture medium contains 1 g/I glucose, 0.5 g/I
NH4NO3, 0.25g/I KCI, 0.25 g/I KH2PO4, 0.25 g/I MgSO4-7H20, supplemented
with 5% v/v adult donor bovine serum (ph 6.8) that mimics serum protein-rich,
iron limited conditions that are similar to biofilm growth conditions in vivo,
such
as in chronic wounds. This medium flows drop-wise (50m1/h) over four coupons
contained in four separate parallel chambers, each of which measures 10cm x
1.9cm by 1.9cm deep. The chambered reactor is fabricated from polysulfone
plastic. Each of the chambers is fitted with an individual removable plastic
lid
that can be tightly sealed. The biofilm reactor is contained in an incubator
at
37 C, and bacterial cell culture medium is warmed by passing it through an
aluminum heat sink kept in the incubator. This method reproduces the
antibiotic tolerant phenotype observed in certain biofilms, mimics the low
fluid
shear environment and proximity to an air interface characteristic of a
chronic
wound while providing continual replenishment of nutrients, and is compatible
with a number of analytical methods for characterizing and monitoring the
effects of introduced candidate antibacterial regimens. The drip-flow reactor
has been successfully employed to culture a wide variety of pure and mixed-
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
species biofilms. Biofilms are typically grown for two to five days prior to
application of antimicrobial agents.
To measure the effects of anti-biofilm agents on biofilms grown in
drip-flow reactors, the fluid stream passing over the biofilm is amended or
5 supplemented with the desired treatment formulation (e.g., one or more BT
compounds and/or one or more antibiotics, or controls, and/or other candidate
agents). Flow is continued for the specified treatment period. The treated
biofilm coupon is then briefly removed from the reactor and the biofilm is
scraped into a beaker containing 10 ml of buffer. This sample is briefly
10 processed (typically 30s to 1 min) with a tissue homogenizer to disperse
, bacterial aggregates. The suspension is serially diluted and plated to
enumerate surviving microorganisms according to standard microbiological
methodologies.
EXAMPLE 4
15 WOUND BIOFILM INHIBITION OF KERATINOCYTE SCRATCH REPAIR:
BIOFILM SUPPRESSION BY BT COMPOUNDS
This Example describes a modification of established in vitro
keratinocyte scratch models of wound healing, to arrive at a model having
relevance to biofilm-associated wound pathology and wound healing, and in
20 .. particular to acute or chronic wounds or wounds containing biofilms as
described herein. According to the keratinocyte scratch model of the effects
of
chronic wound biofilms, cultivation of mammalian (e.g., human) keratinocytes
and bacterial biofilm populations proceeds in separate chambers that are in
fluid contact with one another, to permit assessment of the effects of
conditions
25 that influence the effects, of soluble components elaborated by
biofilms, on
keratinocyte wound healing events.
Newborn human foreskin cells are cultured as monolayers in
treated plastic dishes, in which monolayers a controlled "wound" or scratch is
formed by mechanical means (e.g., through physical disruption of the
30 monolayer such as by scraping an essentially linear cell-free zone
between
regions of the monolayer with a suitable implement such as a sterile scalpel,
razor, cell scraper, forceps or other tool). In vitro keratinocyte monolayer
model
systems are known to undergo cellular structural and functional process in
response to the wounding event, in a manner that simulates wound healing in
35 vivo. According to the herein disclosed embodiments, the influence of
the
presence of bacterial biofilms on such processes, for instance, on the healing
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
96
time of the scratch, is observed, and in these and related embodiments the
effects are also assessed of the presence of selected candidate antimicrobial
(e.g., antibacterial and antibiofilm) treatments.
Wounded keratinocyte monolayers cultured in the presence of
biofilms are examined according to morphological, biochemical, molecular
genetic, cell physiologic and other parameters to determine whether
introduction of BT comopunds alters (e.g., increases or decreases in a
statistically significant manner relative to appropriate controls) the
damaging
effects of the biofilms. Wounds are first exposed to each BT compound alone,
and to contemplated combinations of BT compounds, in order to test the
toxicity
of each BT compound treatment prior to assessing the effects of such
treatments on biofilm influences toward the model wound healing process.
In a representative embodiment, a three-day biofilm is cultured on
a membrane (e.g., a TransWell membrane insert or the like) that is maintained
in a tissue culture well above, and in fluid communication with, a
keratinocyte
monolayer that is scratched to initiate the wound healing process. Biofilms
cultured out of authentic acute or chronic wounds are contemplated for use in
these and related embodiments.
Thus, an in vitro system has been developed for evaluating
soluble biofilm component effects on migration and proliferation of human
keratinocytes. The system separates the biofilm and keratinocytes using a
dialysis membrane. Keratinocytes are cultured from newborn foreskin as
previously described (Fleckman et al., 1997 J Invest. Dermatol. 109:36;
Piepkorn et al., 1987 J Invest. Dermatol. 88:215-219) and grown as confluent
monolayers on glass cover slips. The keratinocyte monolayers can then be
scratched to yield "wounds" with a uniform width, followed by monitoring
cellular
repair processes (e.g., Tao et al., 2007 PLoS ONE 2:e697; Buth et al. 2007
Eur. J Cell Biol. 86:747; Phan et al. 2000 Ann. Acad. Med. Singapore 29:27).
The artificial wounds are then placed in the bottom of a sterile double-sided
chamber and the chamber is assembled using aseptic technique. Both sides of
the chamber are filled with keratinocyte growth medium (EpiLife) with or
without
antibiotics and/or bismuth-thiols. Uninoculated systems are used as controls.
The system is inoculated with wound-isolated bacteria and
incubated in static conditions for two hours to enable bacterial attachment to
surfaces in the upper chambers. Following the attachment period, liquid
medium flow is initiated in the upper chamber to remove unattached cells. Flow
of medium is then continued at a rate that minimizes the growth of planktonic
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
97
cells within the upper chamber, by washout of unattached cells. After
incubation periods ranging from 6 to 48 hours, the systems (keratinocyte
monolayers on coverslips and bacterial biofilm on membrane substrate) are
disassembled and the cover slips removed and analyzed. In related
embodiments, mature biofilms are grown in the upper chamber prior to
assembling the chamber. In other related embodiments, the separate co-
culturing of biofilms and scratch-wounded keratinocyte monolayers is
conducted in the absence and presence of one or more BT compounds,
optionally with the inclusion or exclusion of one or more antibiotics, in
order to
determine effects of candidate agents such as BT compounds, or of potentially
synergizing BT compound-plus-antibiotic combinations (e.g., a BT compound
as provided herein such as a BT that is provided in microparticulate form, and
one or more of amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,
ciprofloxacin, clindamycin (or another lincoasamide antibiotic), daptomycin
(Cubicin ),_doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin,
linezolid (Zyvoxe), minocycline, nafcilin, paromomycin, rifampin,
sulphamethoxazole, tobramycin and vancomycin), on keratinocyte repair of the
scratch wound, e.g., to identify an agent or combination of agents that alters
(e.g., increases or decreases in a statistically significant manner relative
to
appropriate cOntrols) at least one indicator of scratch wound healing, such as
the time elapsing for wound repair to take place or other wound-repair indicia
(e.g., Tao et al., 2007 PLoS ONE 2:e697; Buth et al. 2007 Eur. J Cell Biol.
86:747; Phan et al. 2000 Ann. Acad. Med. Singapore 29:27).
EXAMPLE 5
WOUND BIOFILM INHIBITION OF KERATINOCYTE SCRATCH REPAIR
Isolated human keratinocytes were cultured on glass coverslips
and scratch-wounded according to methodologies described above in Example
4. Wounded cultures were maintained under culture conditions alone or in the
presence of a co-cultured biofilnn on a membrane support in fluid
communication with the keratinocyte culture. The scratch closure time interval
during which keratinocyte cell growth and/or migration reestablishes the
keratinocyte monolayer over the scratch zone was then determined. Figure 3
illustrates the effect that the presence in fluid communication (but without
direct
contact) of biofilms had on the healing time of scratched keratinocyte
monolayers.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
98
Accordingly there are contemplated in certain embodiments a
method of identifying an agent for treating a chronic wound, comprising
culturing a scratch-wounded cell (e.g., keratinocyte or fibroblast) monolayer
in
the presence of a bacterial biofilm with and without a candidate anti-biofilm
agent being present; and assessing an indicator of healing of the scratch-
wounded cell monolayer in the absence and presence of the candidate anti-
biofilm agent, wherein an agent (e.g., a BT compound such as a substantially
monodisperse BT microparticle suspension as described herein, alone or in
synergizing combination with an antibiotic, such as one or more of amikacin,
ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin,
daptomycin (Cubicine),_doxycycline, gatifloxacin, gentamicin, imipenim,
levofloxacin, linezolid (Zyvox0), minocycline, nafcilin, paromomycin,
rifampin,
sulphamethoxazole, tobramycin and vancomycin) that promotes at least one
indicator of healing is identified as a suitable agent for treating an acute
or
chronic wound or a wound that contains a biofilm.
EXAMPLE 6
SYNERGIZING BISMUTH-THIOL (BT)-ANTIBIOTIC COMBINATIONS
This example shows instances of demonstrated synergizing
effects by combinations of one or more bismuth-thiol compounds and one or
more antibiotics against a variety of bacterial species and bacterial strains,
including several antibiotic-resistant bacteria.
Materials & Methods. Susceptibility studies were performed by
broth dilution in 96-well tissue culture plates (Nalge Nunc International,
Denmark) in accordance with NCCLS protocols (National Committee for Clinical
Laboratory Standards. (1997). Methods for Dilution Antimicrobial
Susceptibility
Tests for Bacteria that Grow Aerobically: Approved Standard M7-A2 and
Informational Supplement M100-S10. NCCLS, Wayne, PA, USA).
Briefly, overnight bacterial cultures were used to prepare 0.5
McFarland standard suspensions, which were further diluted 1:50 (-2 x 106
cfu/mL) in cation-adjusted Mueller¨Hinton broth medium (BBL, Cockeysville,
MD, USA). BTs (prepared as described above) and antibiotics were added at
incremental concentrations, keeping the final volume constant at 0.2 mL.
Cultures were incubated for 24 h at 37 C and turbidity was assessed by
absorption at 630 nm using an ELISA plate reader (Biotek Instruments,
Winooski, VT, USA) according to the manufacturer's recommendations. The
Minimum Inhibitory Concentration (MIC) was expressed as the lowest drug
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
99
concentration inhibiting growth for 24 h. Viable bacterial counts (cfu/mL)
were
determined by standard plating on nutrient agar. The Minimal Bactericidal
Concentrations (MBC) was expressed as the concentration of drug that
reduced initial viability by 99.9% at 24 h of incubation.
The checkerboard method was used to assess the activity of
antimicrobial combinations. The fractional inhibitory concentration index
(FICI)
and the fractional bactericidal concentration index (FBCI) were calculated,
according to Eliopoulos et al. (Eliopoulos and Moellering, (1996)
Antimicrobial
combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 330-
96, Williams and Wilkins, Baltimore, MD, USA). Synergy was defined as an
FICI or FBCI index of 5Ø5, no interaction at >0.5-4 and antagonism at >4
(Odds, FC (2003) Synergy, antagonism, and what the chequerboard puts
between them. Journal of Antimicrobial Chemotherapy 52:1). Synergy was also
defined conventionally as M--fold decrease in antibiotic concentration.
Results are presented in Tables 2-17.
TABLE 2
S. aureus Nafcilin resistant
NAF MIC NAF/BEMIC
Strain (pg/ml) (pg/ml) A Synergy
60187-2 10.00 0.6 16.7
52446-3 175.00 40.0 4.4 +
M1978 140.00 50.0 2.8
W54793 130.00 33.3 3.9
S24341 210.00 65.0 3.2
H7544 28.33 15.0 1.9
H72751 145.00 43.3 3.3
W71630 131.67 46.7 2.8
X22831 178.33 75.0 2.4
X23660 123.33 43.3 2.8
036466 191.67 93.3 2.1
BE = 0.2 ug/mI BisEDT; Bacterial strains were obtained from the Clinical
Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY.
Nafcillin
was obtained from Sigma (St. Louis, MO).
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
100
TABLE 3
S. aureus Nafcilin resistant
GM/BE
GM MIC MIC
Strain (pg/ml) (pg/ml) A Synergy
60187-2 0.233 0.004 58.3 +
52446-3 10.667 1.500 7.1
M1978 32.500 4.000 8.1
W54793 0.250 0.080 3.1
S24341 0.250 0.058 4.3 +
H7544 0.383 0.093 4.1
H72751 0.200 0.072 2.8
W71630 17.667 3.800 4.6 +
X22831 0.085
X23660 22.500 4.000 5.6 +
036466 0.267 0.043 6.2 +
BE = 0.2 pg/mIBisEDT; Bacterial strains were obtained from the Clinical
Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY.
Nafcillin was
obtained from Sigma.
TABLE 4
S. aureus
Rifampin/Neomvcin/Paromomycin
MIC MIC + BE
ATCC 25923 (pg/ml) (pg/ml) A Synergy
RIF 0.033 0.003 13.0 +
NEO 0.500 0.200 2.5 -
PARO 1.080 0.188 5.7 +
MRSA S2446-3
RIF 2.500 2.500 1.0 -
NE0 13.400 8.500 1.6 -
PARO 335.000 183.300 1.8 -
BE = 0.2 pg/ml BisEDT; Strain S2446-3 was obtained from the Clinical
Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY.
Antibiotics
were obtained from Sigma.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
101
TABLE 5
S. epidermidis - GM resistant
strain ATCC 35984 strain S2400-1
BisEDT MIC MBC MIC MBC
(pg/m1) (pg/ml GM)
(pg/ml GM) (pg/ml GM) (pg/ml GM)
0 53.3 384.0 85.3 426.7
0.005 20.0 96.0 96.0 512.0
0.01 37.3 117.3 64.0 256.0
0.02 21.3 26.7 28.0 128.0
0.04 2.0 16.0 2.0 128.0
0.08 2.0 10.7 2.0 53.3
0.16 (MIC) 3.0 10.0
0.32 2.0 4.0
GM = gentamicin; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Gentamicin was
obtained
from the Pharmacy Department at Winthrop; synergy in bold
TABLE 6
S. epidermidis - S2400-1
Biofilm Prevention
BisEDT (pg/ml) A
Antibiotic 0 0.05 0.1 (0.05 BE) Synergy
cefazolin 28 10 1 2.8 -
vancomycin 3.2 0.9 0.1 3.6 -
gatifloxacin 1.6 0.1 0.1 16.0 ++
rifampicin 0.03 0.04 0.04 0.7 -
nafcillin 48 64 8 0.8 -
clindamycin 1195 48 12 24.9 ++++
gentamicin 555 144 12 3.9
borderline
minocycline 0.85 0.73 0.08 1.2 -
Data in pg/ml; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were
obtained
from the Pharmacy Department at Winthrop.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
102
TABLE 7
S. epidermidis - S2400-1
MIC
BisEDT (pg/ml) A
Antibiotic 0 0.05 0.1 (0.05 BE) Synergy
cefazolin 32 8 1 4.00 +
vancomycin 3.2 2.3 0.3 1.40 -
gatifloxacin 1.7 0.8 0.3 2.13 -
rifampicin 0.03 0.04 0.04 0.75 -
nafcillin 171 192 68 0.89 -
clindamycin 2048 768 24 2.67 -
gentamicin 2048 320 80 6.40 +
minocycline 1.13 0.43 0.10 2.63 -
Data in pg/ml; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were
obtained
from the Pharmacy Department at Winthrop.
TABLE 8
S. epidermidis - S2400-1
MBC
BisEDT (pg/ml) A
Antibiotic 0.0 0.1 (0.1 BE) Synergy
cefazolin 48 10 4.80 +
vancomycin 5.4 1.4 3.86 borderline
gatifloxacin 2.8 1.4 2.00 -
rifampicin 0.03 0.07 0.43 -
nafcillin 256 128 2.00 -
clindamycin 2048 768 2.67 -
gentamicin 1536 256 6.00 +
minocycline 1.20 1.20 1.00 -
Data in pg/ml; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were
obtained
from the Pharmacy Department at Winthrop.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
103
TABLE 9
S. epidermidis
ATCC 35984
MIC
BisEDT (pg/ml)
Antibiotic 0.0 0.05 A Synergy
Nafcillin 16.00 5.00 3.2
Clindamycin 2048.00 1024.00 2
Gentamicin 213.33 16.00 13.3 ++
Minocycline 0.13 0.04 3.3
Rifampicin 0.021 0.014 1.5
Data in pg/m1; Antibiotics were obtained from the Pharmacy Department at
Winthrop-University Hospital, Mineola, NY.
TABLE 10
E. coli - Ampicillin/Chloramphenicol resistant
MIC
MIC AB AB/BE MIC BE
Strain (pg/ml) (pg/ml AB) A Synergy
(pg/ml)
MC4100/TN9 (CM) 220 12.7 17.4 + 0.6
MC4100/P9 (AM) 285 49 5.8 + 0.5
MC4100 (AM) 141.7 35 4.0 + 0.6
AB = antibiotic; CM = chloramphenicol; AM = ampicillin; BE = BisEDT at 0.3
pg/ml;
Strains were obtained from the laboratory of Dr. MJ Casadaban, Department of
Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL.
Antibiotics were obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
TABLE 11
E. coil - Tetracycline-resistant:
Doxycycline + BisEDT
DOX MIC DOX/BE MIC BE MIC
Strain (pg/ml) (pg/ml DOX) A Synergy
(pg/ml)
TET M 16.50 4.50 4.0 + 0.85
TET D 20.50 0.03 820.0 ++++
0.85
TET A 15.00 10.00 1.5 0.40
TET B 20.13 10.33 2.0 0.60
DOX = doxycycline; BE = BisEDT at 0.3 pg/ml; Strains were obtained from the
laboratory of Dr. I Chopra, Department of Bacteriology, The University of
Bristol,
Bristol, UK. Antibiotics were obtained from the Pharmacy Department at
Winthrop-
University Hospital, Mineola, NY.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
104
,
TABLE 12
P. aeruciinosa - Tobramycin-resistant:
BisEDT Synergy
NN NN+BE BE MIC
Strain (pg/ml) (pg/ml NN) A Synergy (pg/ml)
Xen5 0.32 0.19 1.68 - 0.9
Agr PA E 115 70 1.64 - 0.9
Agr PA I 200 73 2.74 - 1
Agr PA K 4.8 3 1.60 - 0.82
Agr PA 0 130 20.5 6.34 + 0.98
Agr =aminoglycoside resistant; NN = tobramycin; PA = Pseudomonas aeruginosa;
BE = BisEDT, 0.3 pg/ml; Strains were obtained from the laboratory of Dr. K.
Poole,
Department of Microbiology and Immunology, Queens University, Ontario, CN.
Tobramycin was obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
TABLE 13
B. cepacia
Tobramycin+BE Synergy
MIC
NN NN+BE BE MIC
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml)
13945 200 50 4 + 2.4
25416 125 10 12.5 ++ 1.2
HI 2229 64 8 8 + 0.8
AU 0267 128 2 64 ++++ 0.8
AU 0259 1024 256 4 + 1.6
HI 2255 64 8 8 + 1.6
AU 0273 512 32 16 ++ 1.6
HI 2253 64 16 4 + 1.6
HI 2147 512 8 64 ++++ 1.6
NN = Tobramycin; BE = BisEDT, 0.4 pg/ml; Strains were obtained from the
laboratory of Dr. J.J. LiPuma, Department of Pediatrics and Communicable
Diseases, University of Michigan, Ann Arbor, MI; also Veloira et al. 2003.
Tobramycin was obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
105
TABLE 14
B. cepacia
Tobramycin+BE Synergy
MBC
NN NN+BE BE MIC
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml)
HI 2249 256 8 32 ++ 3.2
HI 2229 128 32 4 + 6.4
AU 0267 256 32 8 + 6.4
AU 0259 1024 1024 1 - 12.8
HI 2255 128 32 4 + 12.8
HI 2711 512 8 64 ++++ 6.4
AU 0284 1024 64 16 ++ 0.8
AU 0273 512 32 16 ++ 1.6
HI 2253 128 64 2 - 3.2
HI 2147 512 128 4 + 6.4
NN = Tobramycin; BE = BisEDT, 0.4 pg/ml; Strains were obtained from the
laboratory of Dr. J.J. LiPuma, Department of Pediatrics and Communicable
Diseases, University of Michigan, Ann Arbor, Ml; also Veloira et al. 003.
Tobramycin was obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
TABLE 15
Tobramycin Resistant Strains
MIC
NN NN+BE . Lipo-BE-NN
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml NN)
M13637 512 32 16 ++ 0.25
M13642R 128 64 2 - 0.25
PA-48913 1024 256 4 + 0.25
PA-48912-2 64 8 8 + 0.25
PA-10145 1 4 0.25 - 0.25
SA-29213 2 1 2 - 0.25
NN = Tobramycin; BE = BisEDT, 0.8 pg/ml; Lipo-BE-NN = liposomal BE-NN;
Strains were obtained from the laboratory of Dr. A. Omri, Department of
Chemistry
and Biochemistry, Laurentian University, Ontario, CN; (M strains are mucoid B.
cepacia; PA=P. aeruginosa; SA=S. aureus). Tobramycin was obtained from the
Pharmacy Department at Winthrop-University Hospital, Mineola, NY.
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
106
TABLE 16
Tobramycin Resistant Strains
MBC
Lipo-BE-
NN NN+BE NN
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml NN)
M13637 1024 64 16 ++ 8
M13642R 256 128 2 16
PA-48913 4096 512 8 + 4
PA-48912-2 128 32 4 + 0.5
PA-10145 1 8 0.125 4
SA-29213 2 1 2 0.25
NN = Tobramycin; BE = BisEDT, 0.8 ug/m1; Lipo-BE-NN = liposomal BE-NN;
Strains were obtained from the laboratory of Dr. A. Omri, Department of
Chemistry
and Biochemistry, Laurentian University, Ontario, ON; (M strains are mucoid B.
cepacia; PA=P. aeruginosa; SA=S. aureus). Tobramycin was obtained from the
Pharmacy Department at Winthrop-University Hospital, Mineola, NY.
TABLE 17
BisEDT-Pyrithione Synergy
S. aureus
P. aeruginosa E. coil ATCC
NaPYR ATCC 27853 ATCC 25922 25923
(ug/ml) (pg/ml BE) (pg/ml BE) (pg/ml BE)
0 0.25 0.1 0.25
0.025 0.1 0.125
0.05 0.025 0.063
0.1 0.125 0.0125 0.063
0.2 0.125 0.0125 0.031
0.4 0.00625 0
0.8 0.125 0.00625
1.6
(MIC) 0.063 0.00625
3.2 0.063 0
6.4 0.063
12.8 0
BE = BisEDT; NaPYR = sodium pyrithione; Chemicals were obtained from Sigma-
Aldrich; synergy in bold. Indicated bacterial strains were from American Type
Culture Collection (ATCC, Manassas, VA).
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
107
EXAMPLE 7
COMPARATIVE BISMUTH-THIOL (BT) AND ANTIBIOTIC EFFECTS AGAINST GRAM-
POSITIVE AND GRAM-NEGATIVE BACTERIA INCLUDING ANTIBIOTIC-RESISTANT
BACTERIAL STRAINS
In this example the in vitro activities of BisEDT and comparator
agents were assessed against multiple clinical isolates of Gram-positive and ¨
negative bacteria that are responsible for skin and soft tissue infections.
Materials and Methods. Test compounds and test concentration
ranges were as follows: BisEDT (Domenico et al., 1997; Domenico et al.,
Antimicrob. Agents Chemother. 45(5):1417-1421. and Example 1), 16-0.015
pg/mL; linezolid (ChemPacifica Inc., #35710), 64-0.06 pg/mL; Daptomycin
(Cubist Pharmaceuticals #MCB2007), 32-0.03 pg/mL and 16-0.015 pg/mL;
vancomycin (Sigma-Aldrich, St. Louis, MO, # V2002), 64-0.06 pg/mL;
ceftazidime, (Sigma #C3809), 64-0.06 pg/mL and 32-0.03 pg/mL; imipenem
(United States Pharmacopeia, NJ, #1337809) 16-0.015 pg/mL and 8-0.008
pg/mL; ciprofloxacin (United States Pharmacopeia, # I0C265), 32-0.03 pg/mL
and 4-0.004 pg/mL; gentamicin (Sigma #G3632) 32-0.03 pg/mL and 16-0.015
pg/mL. All test articles, except gentamicin, were dissolved in DMSO;
gentamicin was dissolved in water. Stock solutions were prepared at 40-fold
the highest concentration in the test plate. The final concentration of DMSO
in
the test system was 2.5%.
Organisms. The test organisms were obtained from clinical
laboratories as follows: CHP, Clarian Health Partners, Indianapolis, IN; UCLA,
University of California Los Angeles Medical Center, Los Angeles, CA; GR
Micro, London, UK; PHRI TB Center, Public Health Research Institute
Tuberculosis Center, New York, NY; ATCC, American Type Culture Collection,
Manassas, VA; Mt Sinai Hosp., Mount Sinai Hospital, New York, NY; UCSF,
University of California San Francisco General Hospital, San Francisco, CA;
Bronson Hospital, Bronson Methodist Hospital, Kalamazoo, MI; quality control
isolates were from the American Type Culture Collection (ATCC, Manassas,
VA). Organisms were streaked for isolation on agar medium appropriate to
each organism. Colonies were picked by swab from the isolation plates and put
into suspension in appropriate broth containing a cryoprotectant. The
suspensions were aliquoted into cryogenic vials and maintained at -80 C.
Abbreviations are: BisEDT, bismuth-12-ethanedithiol; LZD, linezolid; DAP,
daptomycin; VA, vancomycin; CAZ, ceftazidime; IPM, imipenem; CIP,
ciprofloxacin; GM, gentamicin; MSSA, meth icillin-susceptible Staphylococcus
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
108
aureus; CLSI QC, Clinical and Laboratory Standards Institute quality control
strain; MRSA, methicillin-resistant Staphylococcus aureus; CA-MRSA,
community-acquired methicillin-resistant Staphylococcus aureus; MSSE,
methicillin-susceptible Staphylococcus epidermidis; MRSE, methicillin-
resistant
Staphylococcus epidermidis; VSE, vancomycin-susceptible Enterococcus.
The isolates were streaked from the frozen vials onto appropriate
medium: Trypticase Soy Agar (Becton-Dickinson, Sparks, MD) for most
organisms or Trypticase Soy Agar plus 5% sheep blood (Cleveland Scientific,
Bath, OH) for streptococci. The plates were incubated overnight at 35 C.
Quality control organisms were included. The medium employed for the MIC
assay was Mueller Hinton ll Broth (MHB II- Becton Dickinson, # 212322) for
most of the organisms. MHB II was supplemented with 2% lysed horse blood
(Cleveland Scientific Lot # H13913) to accommodate the growth of
Streptococcus pyo genes and Streptococcus agalactiae. The media were
prepared at 102.5% normal weight to offset the dilution created by the
addition
of 5 pL drug solution to each well of the microdilution panels. In addition,
for
tests with daptomycin, the medium was supplemented with an additional
25mg/L Ca2+.
The MIC assay method followed the procedure described by the
Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards
Institute. Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria
That Grow Aerobically; Approved Standard¨Seventh Edition. Clinical and
Laboratory Standards Institute document M7-A7 [ISBN 1-56238-587-9].
Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400,
Wayne, Pennsylvania 19087-1898 USA, 2006) and employed automated liquid
handlers to conduct serial dilutions and liquid transfers. Automated liquid
handlers included the Multidrop 384 (Labsystems, Helsinki, Finland), Biomek
2000 and Multimek 96 (Beckman Coulter, Fullerton CA). The wells of Columns
2-12 of standard 96-well microdilution plates (Falcon 3918) were filled with
150pL of DMSO or water for gentamicin on the Multidrop 384. The drugs (300
pL) were dispensed into Column 1 of the appropriate row in these plates.
These would become the mother plates from which the test plates (daughter
plates) were prepared. The Biomek 2000 completed serial transfers through
Column 11 in the mother plates. The wells of Column 12 contained no drug
and were the organism growth control wells in the daughter plates. The
daughter plates were loaded with 185 pL of the appropriate test media
(described above) using the Multidrop 384. The daughter plates were prepared
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
109
on the Multimek 96 instrument which transferred 5 pL of drug solution from
each well of a mother plate to each corresponding well of each daughter plate
in a single step.
Standardized inoculum of each organism was prepared per CLSI
methods (ISBN 1-56238-587-9, cited supra). Suspensions were prepared in
MHB to equal the turbidity of a 0.5 McFarland standard. The suspensions were
diluted 1:9 in broth appropriate to the organism. The inoculum for each
organism was dispensed into sterile reservoirs divided by length (Beckman
Coulter), and the Biomek 2000 was used to inoculate the plates. Daughter
plates were placed on the Biomek 2000 work surface reversed so that
inoculation took place from low to high drug concentration. The Biomek 2000
delivered 10 pL of standardized inoculum into each well. This yielded a final
cell concentration in the daughter plates of approximately 5 x 105 colony-
forming-units/mL. Thus, the wells of the daughter plates ultimately contained
185 pL of broth, 5 pL of drug solution, and 10 pL of bacterial inoculum.
Plates
were stacked 3 high, covered with a lid on the top plate, plaaed in plastic
bags,
and incubated at 35 C for approximately 18 hours for most of the isolates. The
Streptococcus plates were read after 20 hours incubation. The microplates
were viewed from the bottom using a plate viewer. For each of the test media,
an uninoculated solubility control plate was observed for evidence of drug
precipitation. The MIC was read and recorded as the lowest concentration of
drug that inhibited visible growth of the organism.
Results. All marketed drugs were soluble at all of the test
concentrations in both media. BisEDT exhibited a trace precipitate at 32
pg/mL,
but MIC readings were not affected as the inhibitory concentrations for all
organisms tested were well below that concentration. On each assay day, an
appropriate quality control strain(s) was included in the MIC assays. The MIC
values derived for these strains were compared to the published quality
control
ranges (Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Susceptibility Testing; Eighteenth Informational Supplement.
CLSI
document M100-S18 [ISBN 1-56238-653-0]. Clinical and Laboratory Standards
Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898
USA, 2008) for each agent, as appropriate.
On each assay day, an appropriate quality control strain(s) was
included in the MIC assays. The MIC values derived for these strains were
compared to the published quality control ranges (Clinical and Laboratory
Standards Institute. Performance Standards for Antimicrobial Susceptibility
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
110
Testing; Eighteenth Informational Supplement. CLSI document M100-S18
[ISBN 1-56238-653-0]) for each agent, as appropriate. Of 141 values for
quality
control strains where quality control ranges are published, 140(99.3%) were
within the specified ranges. The one exception was imipenem versus S. aureus
29213 which yielded one value on a single run (5. 0.008 pg/mL) that was one
dilution below the published QC range. All other quality control results on
that
run were within the specified quality control ranges.
BisEDT demonstrated potent activity against both methicillin-
susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus
(MRSA), and community-acquired MRSA (CA-MRSA), inhibiting all strains
tested at 1 pg/mL or less with an MIC90 values of 0.5 pg/mL for all three
organism groups. BisEDT exhibited activity greater than that of linezolid and
vancomycin and equivalent to that of daptomycin. lmipenem was more potent
than BisEDT against MSSA (MIC90 = 0.03 pg/mL). However, MRSA and
CAMRSA were resistant to imipenem while BisEDT demonstrated activity
equivalent to that shown for MSSA. BisEDT was highly-active against
methicillin-susceptible and methicillin¨resistant Staphylococcus epidermidis
(MSSE and MRSE), with MIC90 values of 0.12 and 0.25 pg/mL, respectively.
BisEDT was more active against MSSE than any of the other agents tested
except imipenem. BisEDT was the most active agent tested against MRSE.
BisEDT demonstrated activity equivalent to that of daptomycin,
vancomycin, and imipenem against vancomycin-susceptible Enterococcus
faecalis (VSEfc) with an MIC90 value of 2pg/mL. Significantly, BisEDT was the
most active agent tested against vancomycin-resistant Enterococcus faecalis
(VREfc) with an MIC90 value of 1 pg/mL.
BisEDT was very active against vancomycin-susceptible
Enterococcus faecium (VSEfm) with an MIC90 value of 2 pg/mL; its activity was
equivalent to that or similar to that of daptomycin and one-dilution higher
than
that of vancomycin. BisEDT and linezolid were the most active agents tested
against vancomycin-resistant Enterococcus faecium (VREfm), each
demonstrating an MIC90 value of 2 pg/mL. The activity of BisEDT against
Streptococcus pyogenes (MIC90 value of 0.5 pg/mL) was equivalent to that of
vancomycin, greater than that of linezolid, and slightly less than that of
daptomycin and ceftazidime. The compound inhibited all strains tested at 0.5
pg/mL or less. In these studies, the species that was least sensitive to
BisEDT
was Streptococcus agalactiae where the observed MIC90 value was 16 pg/mL.
BisEDT was less active than all of the agents tested except gentamicin.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
1 1 1
The activity of BisEDT and comparators against Gram-negative
bacteria included demonstrated BisEDT potency against Acinetobacter
baumanii (MIC90 value of 2 pg/mL) making BisEDT the most active compound
tested. Elevated MICs for a significant number of test isolates for the
comparator agents resulted in off-scale MIC90 values for these agents.
BisEDT was a potent inhibitor of Escherichia coil, inhibiting all strains at 2
pg/mL or less (MIC90 = 2 pg/mL). The compound was less active than
imipenem, but more active than ceftazidime, ciprofloxacin, and gentamicin.
BisEDT also demonstrated activity against Klebsiella pneumoniae with an
MIC90 value of 8 pg/mL which was equivalent to that of imipenem. The
relatively high MIC90 values exhibited by imipenem, ceftazidime,
ciprofloxacin,
and gentamicin indicated that this was a highly antibiotic-resistant group of
organisms. BisEDT was the most active compound tested against
Pseudomonas aeruginosa with an MIC90 value of 4 pg/mL. There was a high
level of resistance to the comparator agents for this group of test isolates.
In summary, BisEDT demonstrated broad-spectrum potency
against multiple clinical isolates representing multiple species, including
species
commonly involved in acute and chronic skin and skin structure infections in
humans. The activity of BisEDT and key comparator agents was evaluated
against 723 clinical isolates of Gram-positive and Gram¨negative bacteria. The
BT compound demonstrated broad spectrum activity, and for a number of the
test organisms in this study, BisEDT was the most active compound tested in
terms of anti-bacterial activity. BisEDT was most active against MSSA, MRSA,
CA-MRSA, MSSE, MRSE, and S. pyogenes, where the MIC90 value was 0.5
pg/mL or less. Potent activity was also demonstrated for VSEfc, VREfc,VSEfm,
VREfm, A. baumanii, E. coil, and P. aeruginosa where the MIC90 value was in
the range of 1 - 4 pg/mL. MIC90 values observed were, for K. pneumoniae
(MIC90 = 8 pg/mL), and for S. agalactiae (MIC90= 16 pg/mL).
EXAMPLE 8
MICROPARTICULATE BT-ANTIBIOTIC ENHANCING AND SYNERGIZING ACTIVITIES
This example shows that microparticulate bismuth thiols (BTs)
promote antibiotic activity through enhancing and/or synergizing interactions.
A major complicating factor in treating infections is the emerging
resistance of bacteria to antibiotics. Methicillin resistance in S.
epidermidis
(MRSE) and S. aureus (MRSA) actually reflects multiple drug resistance,
making these pathogens very difficult to eradicate. However, no staphylococci
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
112
from hundreds of strains tested showed resistance to BTs. Furthermore, BTs at
subinhibitory (subMIC) concentrations reduced resistance to several important
antibiotics.
Staphylococcus aureus. A graphic demonstration of the
antibiotic-resensitizing effects of subMIC bismuth ethanedithiol (BisEDT)
against MRSA is provided (Figure 4) showing enhanced antibiotic action of
several classes of antibiotics, including gentamicin, cefazolin, cefepime,
imipenim, sulphamethoxazole, and levofloxacin. Thus, BisEDT nonspecifically
enhanced the activity of most antibiotics.
Broth dilution antimicrobial susceptibility studies were performed
against 12 MRSA strains using several antibiotics combined with subMIC levels
of BisEDT (Table 18). Both the biofilm-prevention concentration (BPC) and the
minimum inhibitory concentration (MIC) were determined in a special biofilm
culture medium (BHIG/X). The MIC and BPC for gentannicin and cefazolin were
reduced by subMIC BisEDT (BisEDT MIC, 0.2-0.4 pg/ml), but not below the
breakpoint for sensitivity. subMIC BisEDT enhanced the sensitivity of MRSA to
gatifloxacin and cefepime close to the breakpoint for sensitivity. These
strains
were already sensitive to vancomycin, but were made considerably moreso in
the presence of subMIC BisEDT. Generally, the MIC and BPC were reduced 2-
to 5-fold with subMIC BisEDT.
TABLE 18.
Antimicrobial Activity of BT-Antibiotic Combinations against MRSA
MIC Standards
Antibiotic BisEDT (pg/mL) ( g/m1)
0 0.025 0.05 0.1
Gentamicin
BPC 81 41 63 30 53 31 33 25
MIC 81 40 60 27 58 30 48 31 ..
16
Cefazolin
BPC 109 86 76 86 76 105 34 28
MIC 93 75 99 76 90 60 45 32
Gatifloxacin
BPC 3.6 2.6 2.6 0.9 2.4 1.1 0.9
0.8
MIC 3.6 2.6 4.0 2.8 4.0 2.8 2.4
1.1
Vancomycin
BPC 2.5 1.7 1.5 0.6 1.3 0.5 0.7
0.4
MIC 2.5 1.7 2.5 1.7 1.5 0.6 1.3
0.5
Cefepime
BPC 24 37 27 28 18 16 5.0 7.3
MIC 45 32 32 28 37 24 9.3 6.1
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
113
12 MRSA clinical isolates were grown in BHIG/X and exposed to serial dilutions
of
antibiotics in the presence of 0-0.1 pg/ml BisEDT. The MIC and BPC, calculated
in
pg/ml, are the means standard deviations from at least three trials. The
right hand
column lists the Standard MIC for antibiotic senstivity (S) and resistance (R)
A broth dilution study of cefepime-resistant MRSA isolates is
shown in Table 19. BisEDT at 0.1 pg/ml significantly enhanced the inhibitory
activity of cefepime in 11 of 12 isolates. In this particular study, the data
indicated synergy between BisEDT and cefepime (FIC < 0.5), with many of the
isolates at the breakpoint for sensitivity.
TABLE 19
Cefepime-resistant MRSA Sensitized by BisEDT
MIC for Cefepime (ug/mL) in subMIC BisEDT
BE BE BE
0 pg/mL 0.05 pg/mL 0.1 pg/mL
MRSA MIC MIC MIC
Strain #
4 256 256 16
6 256 256 32
7 128 256 32
10 128 32 16
18 256 128 8
24 256 64 8
28 256 128 8
35 256 256 8
37 128 128 8
41 128 256 8
46 256 256 256
47 32 8 8
Twelve cefepime-resistant MRSA were tested in BHIG/X medium in polystyrene
plates for sensitivity to cefepime combined with subMIC BisEDT at 37 C for
48h.
Results for combination studies with nafcillin or gentamicin are
shown in Table 20. Combined with nafcillin, BisEDT (0.2 g/m1) reduced the
MIC90 for nafcillin by over 4-fold against MRSA (FIC, 0.74). Combined with
gentamicin, BisEDT reduced the MIC90 for gentamicin over 10-fold against
MRSA (FIC, 0.6). BTs reversed the resistance of all four gentamicin-resistant
isolates tested to clinically relevant concentrations [Domenico et al., 2002].
The
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
114
MICs for these antimicrobial agents was reduced substantially, especially for
gentamicin. The broth used in these studies was Trypticase Soy Broth (TSB)
with 2% glucose, which showed results similar to that seen in Mueller-Hinton
II
broth fortified with 1% sheep's blood.
TABLE 20
MRSA: Nafcillin or Gentamicin + BisEDT Synergy
NAF NAF+BE GM GM+BE
Strain MIC MIC A MIC MIC A
60187-2 10.00 0.60 16.67 0.23
0.00 58.33
52446-3 175.00 40.00 4.38
10.67 1.50 7.11
M1978 140.00 50.00 2.80
32.50 4.00 8.13
W54793 130.00 33.33 3.90 0.25 0.08 3.13
S24341 210.00 65.00 3.23 0.25 0.06
4.29
H7544 28.33 15.00 1.89 0.38 0.09
4.11
H72751 145.00 43.33 3.35 0.20 0.07
2.79
W71630 131.67 46.67 2.82 17.67 3.80 4.65
X22831 178.33 75.00 2.38
X23660 123.33 43.33 2.85
22.50 4.00 5.63
036466 191.67 93.33 2.05 0.27 0.04
6.15
AVG A 4.21 AVG A 10.43
NAF or GM in pg/ml; BE at 0.2 pg/ml
Staphylococcus epidermidis. The activities of most classes of
antibiotic were promoted in the presence of BisEDT. With regard to the BPC,
clindamycin and gatifloxacin showed significantly more antibiofilm activity
against S. epidermidis when combined with BisEDT (Figure 5). Stated in
different terms, the BPC for clindamycin, gatifloxacin and gentamicin were
reduced 50-fold, 10-fold and 4-fold, respectively, in the presence of subMIC
BisEDT.
Only modest decreases in the biofilm prevention concentration
(BPC) were noted for minocycline, vancomycin, and cefazolin, while rifampicn
and nafcillin remained unaffected at 0.05 pg/m1 BisEDT. At 0.1 lig/m1BisEDT
no biofilm was detected, regardless of antibiotic employed, signifying that no
antagonism occurred. This BisEDT concentration was close to the MIC for S.
epidermidis [Domenico et al., 2003] (See Figure 5).
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
115
With regard to growth inhibition, seven of eight antibiotics tested
were significantly enhanced in the presence of 0.1 pg/ml (0.5 pM) BisEDT
against S. epidermidis (Figure 6). The MIC change was most pronounced for
clindamycin and gentamicin, followed by vancomycin, cefazolin, minocycline,
gatifloxacin and nafcillin, with rifampicin unaffected. Of the antibiotics
this strain
was resistant to (NC, CZ, GM, CM), only cefazolin resistance was reversed to
clinically relevant levels by BisEDT.
Minimum bactericidal concentration (MBC) for most antibiotics
tested against S. epidermidis decreased slightly with subMIC BisEDT.
Gentamicin showed the greatest reduction in MBC (4- to 16-fold), followed by
cefazolin (4- to 5-fold), vancomycin and nafcillin (3- to 4-fold), minocycline
and
gatifloxacin (2- to 3-fold), while clindamycin and rifampicin MBC remained
largely unaffected. Clindamycin is a bacteriostatic agent, which explains its
lack of bactericidal activity. Cefazolin resistance was reversed with respect
to
the MBC [Domenico et al., 2003]. These effects were additive.
The potentiation of antimicrobial agents was also demonstrated in
vivo in a graft infection rat model (Table 21). BisEDT levels as low as 0.1
pg/ml
were able to promote the prevention of resistant S. epidermidis biofilm for 7
days.
As summarized in Table 21, implants impregnated with 0.1 tig/ml
BisEDT, 10 tig/ml RIP and 10 ilg/mIrifampin, alone or combined were
implanted s.c. into rats. Physiological solution (1 ml) containing the MS and
MR
strains at 2x107 cfu/ml was inoculated onto the graft surface using a
tuberculin
syringe. All grafts were explanted at 7 days following implantation and
sonicated for 5 minutes in sterile saline solution to remove the adherent
bacteria. Quantitation of viable bacteria was obtained by culturing dilutions
on
blood agar plates. The limit of detection was approximately 10 cfu/cm2.
TABLE 21
RIP, BTs, and rifampin against S. epidermidis in a graft infection model
Quantitative graft culture
Group' Graft-bonded drugb (cfu/cm2)
No MSSE <10
Untreated MSSE 5.0 x 107 7.7
x 106
MS1' RIP 4.3 x
102+ 1.2 x 102
MS2' BTs 5.8 x
102 0.9 x 102
MS3c Rifampin 5.9 x 103 1.8
x 103
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
116
Quantitative graft culture
Groupa Graft-bonded drug' .. (cfu/cm2)
mscd RIP plus BTs <10
MS50d RIP plus rifampin 2.0 x 101 0.6 x 101
MS6bd BTs plus rifampin 1.9 x 101 0.4 x 101
No MRSE <10
Untreated MRSE 7.8 x 107 2.0 x 107
MR1c RIP 6.7 x 102 2.1 x102
MR2c BTs 6.2 x 102 2.3 x 102
MR3c Rifampin 7.6 x 104 2.1 x 104
MR4ba RIP plus BTs <10
MR5c RIP plus rifampin 4.3 x 101 1.1 x 101
MR6c BTs plus rifampin 3.0 x 101 1.1 x 101
a Each group had 15 animals; MS, methicillin-susceptible S. epidermidis; MR,
methicillin-resistant S. epidermidis
Dacron graft segments impregnated with 0.1 mg/I of BTs, 10 mg/I of RIP, 10
mg/I of
rifampin
C Statistically significant when compared with control groups MS and MR
d Statistically significant when compared with MS3 group
e Statistically significant when compared with MR1, MR2, and MR3 groups
Gram-negative Bacteria. Tobramycin activity against resistant
Pseudomonas aeruginosa was enhanced several-fold with subMIC BisEDT
(Table 22). In these trials, the MIC was defined more precisely as the IC24.
TABLE 22
Tobramycin-resistant P. aeruginosa: BisEDT Effect
NN MIC BE MIC NN-FBE MIC
Strain (pg/ml) (pg/ml) (pg/ml) A
PA Xen5 0.3 0.9 0.2 1.7
Agr PA E 115.0 0.9 70.0 1.6
Agr PA I 200.0 1.0 73.0 2.7
Agr PA K 4.8 0.86 3.0 1.6
Agr PA 0 130.0 0.98 20.5 6.3
Resistant strains of P. aeruginosa were cultured in Mueller-Hinton II broth at
37 C
in the presence of tobramycin (NN) and BisEDT (BE; 0.33 pg/ml). The MIC was
determined as the antibiotic concentration that inhibited growth for 24 1 h.
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
117
Against tobramycin-resistant Burkholderia cepacia, 0.4 g/ml
BisEDT rendered seven of 10 isolates tobramycin sensitive (mean FIC; 0.48),
and reduced the MIC90 by 10-fold (Table 23). Both the MIC and MBC of
tobramycin were reduced significantly to achievable levels against 50 clinical
Burkhoideria cepacia isolates with subMIC BisEDT [Veloira et al., 2003].
BisEDT and tobramycin in liposomal form have proven highly synergistic
against P. aeruginosa. (Halwani et al., 2008; Halwani et al., 2009).
TABLE 23
Tobramycin and BisEDT versus B. cepacia
MIC for
Strain Tobramycin BisEDT Tobramycin
FIC
(pg/ml) (pg/ml) (BisEDT at
0.4 pg/ml) Index
B. multivorans
HI 2249 256 0.4 a a
HI 2229 64 0.8 8 0.63
AU 0267 128 0.8 2 0.52
AU 0259 1024 1.6 256 0.50
HI 2255 64 1.6 8 0.38
B. cenocepacia
H12711 256 0.4 a a
AU 0284 512 0.4 a a
AU 0273 512 1.6 32 0.31
HI2253 64 1.6 16 0.50
H12147 512 1.6 8 0.27
a The three strains inhibited by BisEDT at 0.4 pg/m1 were excluded from
further
study.
FIC Index 0.5 indicates synergy: FICI >0.5 and <1.0 indicates enhancement.
Chloramphenicol and ampicillin resistant Escherichia coli were
made sensitive to these drugs by the addition of subMIC BisEDT (Table 24).
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
118
TABLE 24
Chloramphenicol/Ampicillin Resistant E. coli: BisEDT Effect
Drug + BE
Drug MIC BE MIC MIC
Strain Drug (pg/ml) (Pgin11) (pg/ml) A
MC4100/TN9 CM 220.0 0.6 12.7 17.4
MC4100/P9 AMP 285.0 0.5 49.0 5.8
MC4100 AMP 141.7 0.6 35.0 4.0
Resistant strains of Ecoli were cultured in Mueller-Hinton II broth at 37 C in
the
presence of chloramphenicol (CM) or ampicillin (AMP) and BisEDT alone or in
combination (BE; 0.33 pg/ml). The MIC was determined as the antibiotic
concentration that inhibited growth for 24 1 h.
Tetracycline resistant Escherichia coli were made sensitive to
doxycycline by the addition of subMIC BisEDT (Table 25). The combination
exhibited synergy against the TET M and TET D strains (FIG 5. 0.5), with
additive effects against the TET A and TET B strains.
TABLE 25
Tetracycline Resistant E. Coll: BisEDT Effect
DOX MIC BE MIC DOX+BE MIC
Strain (pg/ml) (pg/ml) (pg/ml) A
TET M 16.5 1.3 0.85 4.5 2.7 4.0
TET D 20.5 1.1 0.85 0.03 0.0 820.0
TET A 15.0 1.8 0.40 10.0 1.0 1.5
TET B 20.1 2.4 0.60 10.3 3.2 2.0
Resistant strains of E.coli were cultured in Mueller-Hinton II broth at 37 C
in the
presence of doxycycline (DOX) and BisEDT alone or in combination (BE; 0.33
pg/ml). The MIC was determined as the antibiotic concentration that inhibited
growth for 24 1 h.
References
Domenico P, R O'Leary, BA Cunha. 1992. Differential effect of
bismuth and salicylate compounds on antibiotic sensitivity of Pseudomonas
aeruginosa. Eur J Clin Microbiol lnfec Dis 11:170-175; Domenico P, D Parikh,
BA Cunha. 1994. Bismuth modulation of antibiotic activity against
gastrointestinal bacterial pathogens. Med Microbiol Lett 3:114-119; Domenico
P, Kazzaz JA, Davis JM, Niederman MS. 2002. Subinhibitory bismuth
ethanedithiol (BisEDT) sensitizes resistant Staphylococcus aureus to nafcillin
or
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
119
gentannicin. Annual Meeting, ASM, Salt Lake City, UT; Domenico P, Kazzaz JA,
Davis JM. 2003. Combating antibiotic resistance with bismuth-thiols. Research
Advances in Antimicrob Agents Chemother 3:79-85; Domenico P, E Gurzenda,
A Giaconnetti, 0 Cirioni, R Ghiselli, F Orlando, M Korem, V Saba, G Scalise, N
Balaban. 2004. BisEDT and RIP act in synergy to prevent graft infections by
resistant staphylococci. Peptides 25:2047-2053; Halwani M, Blomme S,
Suntres ZE, Alipour M, Azghani AO, Kumar A, Omri A. 2008. Liposomal
bismuth-ethanedithiol formulation enhances antimicrobial activity of
tobramycin.
Intl J Pharmaceut 358:278-84; Halwani M, Hebert S, Suntres ZE, Lafrenie RM,
Azghani AO, Omri A 2009. Bismuth-thiol incorporation enhances biological
activities of liposonnal tobramycin against bacterial biofilm and quorum
sensing
molecules production by Pseudomonas aeruginosa. Int J Pharmaceut 373:141-
6; Veloira WG, Gurzenda EM, Domenico P, Davis JM, Kazzaz JA. 2003.
Synergy of tobramycin and bismuth thiols against Burkholderia cepacia. J
Antimicrob Chemother 52:915-919.
EXAMPLE 9
MICROPARTICULATE BT-ANTIBIOTIC ENHANCING AND SYNERGIZING ACTIVITIES
This example shows that the microparticulate bismuth thiol
BisEDT promotes antibiotic activity through enhancing and/or synergizing
interactions with specific antibiotics against specific microbial target
organisms.
Single-point data for each indicated combination in Table 26 were generated
essentially according to the methods used in Example 8.
TABLE 26
FICI Values for single-point BisEDT-antibiotic combinations
Anti SA MRSA E Fc SP PRSP EC EC KP PA Bcep Bmult Abau Msmeg
biotic
100 773 3121 1195 5348 102 2232 1231 1380 1756 5665 2594 817
Oxacillin 1.28 2.28 0.92 1.03
Piperacillin 0.57 1.28 1.11
1.11 0.87 1.29 2.23 0.67 1.12 1.12 1.12
Cefuroxime 1.11 4.23 1.11 1.03
Cefotaxime
1.11 2.23 0.73 1.11 1.11 1.37 1.29 0.61 0.64 1.29 1.11 1.29
Cefepime
0.87 0.96 1.11 0.62 1.34 0.96 0.71 0
I mipenem 0.67 1.48 0.73 0.92 0.43 1.11 1.29
1.23 1.12 0.73 1.23 0.81 CD
CD
Aztreonam
0.74 1.29 0.73 0.55 0.67 0.96 0.87
1\)
Streptomycin 0.95 0.61 0.66
1.29 1.04 1.98 1.37 1.12 2.62 1.13 o o
Tobramycin 0.73 0.78 0.47
0.57 0.96 0.87 1.29 0.91 0.67 1.12
CD
Tetracycline
0.89 1.23 0.92 1.23 0.34 0.62 0.79 1.29 1.29 1.96 1.12 1.12 0
Minocycline 1.09 1.23 1.11
0.46 1.37 1.04 1.29 0.99 2.23 1.12 1.29
Ciprofloxacin
1.14 1.29 1.29 2.75 2.23 2.29 1.04
Levofloxacin 1.23 1.11 1.08 0.95 0.70
Erythromycin 1.28 0.67 0.92 0.78 1.03
Linezolid 1.23 1.23 1.23 1.01 1.11
Phosphomycin 0.61 1.23 1.45
1.96 1.02 1.86 1.29 1.23 1.12
Capreomycin
0.75
Isoniazid
0.88
fli
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
121
SA, Staphylococcus aureus; MRSA, methicillin-resistant
Staphylococcus aureus; E Fc, Enterococcus faecalis; SP, Streptococcus
pneumoniae; PRSP, penicillin-resistant Streptococcus pneumoniae; EC,
Escherichia coil; KP, Klebsiella pneumoniae; PA, Pseudomonas aeruginosa;
Bcep, Burkholderia cepacia; Bmult, Bukholderia multivorans; Abau,
Acinetobacter baumanii; Msmeg, Mycobacterium smegmatis.
EXAMPLE 10
MICROPARTICULATE BT-ANTIBIOTIC ENHANCING AND SYNERGIZING ACTIVITIES
The effects of combinations of microparticulate Bis-EDT and four
Bis-EDT analogs prepared as described above, and other agents against
representative strains of several Gram-negative pathogenic bacteria were
tested. A modification of a common laboratory method was used to determine
synergism (FICI <0.5), enhancement (0.5 < FICI < 1.0), antagonism (FICI >
4.0) and indifference (1.0 < FICI <4.0) used fractional inhibitory
concentrations
(FICs) and FIC indices (FICI) (Eliopoulos G and R Moellering. 1991.
Antimicrobial combinations. In Antibiotics in Laboratory Medicine, Third
Edition,
edited by V Lorian. Williams and Wilkins, Baltimore, MD, pp. 432-492; Odds,
2003 J. Antimicrob. Chemother. 52(1):1). The checkerboard technique was
used to determine FIC indices and was employed in this study.
TABLE 27
Test Components
FIC Highest Stock Conc. Range
Test Cpd Lot No. Solvent Concentration Tested in FIC
(pg/mL) (pg/mL)
Bis-EDT MB-1B-3 DMSO 320 0.12-8
Bis-EDT (analog) MB-2B DMSO 320 0.12-8
Bis-EDT (analog) MB-8-2 DMSO 320 0.12-8
Bis-EDT (analog) MB-11 DMSO 320 0.12-8
Bis-EDT (analog) MB-15 DMSO 320 0.12-8
095K1324 10% 0.06-64
Aztreonam 2,560
(Sigma) DMSO
GOD116 0.06-64
Cefepime HCI dH20 2,560
(USP)
084K0674 0.015-16
Cefotaxime dH20 640
(Sigma)
014K1362 0.06-64
Piperacillin (Sigma) dH20 2,560
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
122
Stock solutions of all test articles were prepared at 40X the final
target concentration in the appropriate solvent. All test articles were in
solution
under these conditions. The final drug concentrations in the FIC assay plates
were set to bracket the MIC value of each agent for each test organism, unless
the strain was totally resistant to the test agent. The concentration ranges
tested are displayed in Table 27. The test organisms were originally received
from clinical sources, or from the American Type Culture Collection. Upon
receipt, the isolates were streaked onto Tryptic Soy Agar II (TSA). Colonies
were harvested from these plates and a cell suspension was prepared in an
appropriate broth growth medium containing cryoprotectant. Aliquots were then
frozen at -80 C. The frozen seeds of the organisms to be tested in a given
assay were thawed, streaked for isolation onto TSA plates, and incubated at
35 C. All organisms were tested in Mueller Hinton II Broth (Becton Dickinson,
Lot No.9044411). The broth was prepared at 1.05X normal weight/volume to
offset the 5 % volume of the drugs in the final test plates.
Minimal Inhibitory Concentration (MIC) values were previously
determined using the broth microdilution method for aerobic bacteria (Clinical
and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard¨
Eighth Edition. CLSI document M07-A8 [ISBN 1-56238-689-1]. Clinical and
Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898 USA, 2009.).
FIC values were determined using a broth microdilution method
.. previously described (Sweeney et al., 2003 Antimicrob. Agents Chemother.
47(6):1902-1906). To prepare the test plates, automated liquid handlers
(Multidrop 384, Labsystems, Helsinki, Finland; Biomek 2000 and Multimek 96,
Beckman Coulter, Fullerton CA) were used to conduct serial dilutions and
liquid
transfers.
The appropriate wells of standard 96-well microdilution plates
(Falcon 3918) were filled with 150 pL of the appropriate solvent in columns 2-
12
using the Multidrop 384. Three hundred microliters of each secondary test drug
was added to each well in Column 1 of the plates. These plates were used to
prepare the drug "mother plates" which provided the serial drug dilutions for
the
drug combination plates. The Biomek 2000 was used to transfer 150 pL of
each secondary drug solution (40X) from the wells in Column 1 of the mother
plate and to make eleven 2-fold serial dilutions. Mother plates of Bis-EDT
(and
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
123
analogs) were serial diluted top to bottom by hand, using a multichannel
pipette. Two mother plates, one for each secondary drug and one for Bis-EDT
(or analogs), were combined to form a "checkerboard" pattern by transfer of
equal volumes (using a multi-channel pipette) to the drug combination plate.
Row H and Column 12 each contained serial dilutions of one of the agents
alone for determination of the MIC.
The "daughter plates" were loaded with 180 pL of test medium
using the Multidrop 384. Then, the Multimek 96 was used to transfer 10 L of
drug solution from each well of the drug combination mother plate to each
corresponding well of the daughter plate in a single step. Finally, the
daughter
plates were inoculated with test organism. Standardized inoculum of each
organism was prepared per published guidelines (CLSI, 2009). For all isolates,
the inoculum for each organism was dispensed into sterile reservoirs divided
by
length (Beckman Coulter), and the Biomek 2000 was used to inoculate the
plates. The instrument delivered 10 p,L of standardized inoculum into each
well
to yield a final cell concentration in the daughter plates of approximately 5
x 105
colony-forming-units/mL.
The test format resulted in the creation of an 8 x 12 checkerboard
where each compound was tested alone (Column 12 and Row H) and in
combination at varying ratios of drug concentration. All organism plates were
stacked three high, covered with a lid on the top plate, placed in plastic
bags,
and incubated at 35 C for approximately 20 hours. Following incubation, the
microplates were removed from the incubators and viewed from the bottom
using a ScienceWare plate viewer. Prepared reading sheets were marked for
the MIC of drug 1 (row H), the MIC of drug 2 (column 12) and the wells of the
growth-no growth interface.
An Excel program was used to determine the FIG according to the
formula: (MIC of Compound 1 in combination/MIC of Compound 1 alone) +
(MIC of Compound 2 in combination/MIC of Compound 2 alone). The FICI for
the checkerboard was calculated from the individual FICs by the formula: (FICi
+ FIC2 + FICn)/n, where n = number of individual wells per plate for which
FICs were calculated. In instances where an agent alone yielded an off-scale
MIC result, the next highest concentration was used as the MIC value in the
FIG calculation.
Microparticulate Bis-EDT, the four microparticulate BT analogs,
and all of the other agents (and combinations of agents) were soluble at all
final
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
124
test concentrations. The MIC and FICI values that were determined are
presented in the Tables below.
TABLE 28
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1B-3 and Piperacillin
Compound 1 Compound 2
FICI2
Organism.' MIC' MIC
Name (PgifilL) Name (PgimL)
Alone Alone
P. aeruginosa 1381 1 >64 0.83
P. aeruginosa 1384 1 8 0.96
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-1B-3 0.5 Piperacillin 8 1.12
P. aeruginosa 2566 0.5 32 1.37
P. aeruginosa 2568 1 8 0.71
P. aeruginosa 103 1 8 0.79
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 29
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1B-3 and Aztreonam
Compound 1 Compound 2
MIC
Organism.' Name (PgimL) Name
(ligim I-) FICI2
Alone Alone
P. aeruginosa 1381 1 32 1.04
P. aeruginosa 1384 1 8 0.71
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-1B-3 0.5 Aztreonam 8 0.87
P. aeruginosa 2566 0.5 16 1.37
P. aeruginosa 2568 1 8 0.71
P. aeruginosa 103 1 4 1.29
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
125
TABLE 30
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Piperacillin
Compound 1 Compound 2
MIC1 MIC
Organism.' Name (pg/mL)
Name (Pg/m1-) FICI2
Alone Alone
P. aeruginosa 1381 1 >64 1.29
P. aeruginosa 1384 1 16 0.71
P. aeruginosa 1474 1 8 1.12
P. aeruginosa 1479 MB-15 1 Piperacillin 8 1.29
P. aeruginosa 2566 1 32 1.04
P. aeruginosa 2568 1 8 1.12
P. aeruginosa 103 2 8 0.73
1MIC, Minimum Inhibitory Concentration
FICI, Fractional Inhibitory Concentration Index
TABLE 31
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Aztreonam
Compound 1 Compound 2
MIC' MIC
Organism.' Name (Pgin/L) Name
(Pgin1L) FIC12
Alone Alone
P. aeruginosa 1381 2 32 1.11
P. aeruginosa 1384 1 8 0.79
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-15 2 Aztreonam 8 0.67
P. aeruginosa 2566 0.5 16 1.12
P. aeruginosa 2568 1 8 0.79
P. aeruginosa 103 2 4 1.23
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
126
TABLE 32
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Piperacillin
Compound 1 Compound 2
MIC1 MIC
Organism.' Name (pg/mL)
Name (Pgiml-) FICI2
Alone Alone
P. aeruginosa 1381 2 >64 1.23
P. aeruginosa 1384 2 16 0.73
P. aeruginosa 1474 2 8 1.23
P. aeruginosa 1479 MB-8-2 2 Piperacillin 8 1.23
P. aeruginosa 2566 2 32 1.23
aeruginosa 2568 2 8 0.98
P. aeruginosa 103 4 8 1.19
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE33
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Aztreonam
Compound I Compound 2
MIC1 MIC
Organism.' Name (PgimL) Name (pg/mL) F1c12
Alone Alone
P_ aeruginosa 1381 2 32 1.11
P. aeruginosa 1384 2 8 1.11
P. aeruginosa 1474 2 8 0.73
P. aeruginosa 1479 MB-8-2 2 Aztreonam 8 0.98
P. aeruginosa 2566 2 16 1.23
P. aeruginosa 2568 2 8 0.98
P. aeruginosa 103 4 8 1.19
lmic, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
127
TABLE 34
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Piperacillin
Compound 1 Compound 2
MIC1 MIC
Organism" Name (pg/mL) Name (ligiml-) FICI2
Alone Alone
P. aeruginosa 1381 1 >64 1.12
P. aeruginosa 1384 1 16 0.71
P. aeruginosa 1474 1 8 1.12
P. aeruginosa 1479 MB-11 1 Piperacillin 8 1.29
P. aeruginosa 2566 0.5 32 1.12
P. aeruginosa 2568 1 8 1.12
P. aeruginosa 103 2 8 1.11
1MIC, Minimum Inhibitory Concentration
FICI, Fractional Inhibitory Concentration Index
TABLE 35
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Aztreonam
Compound 1 Compound 2
MIC1 MIC
Organism" Name (pg/mL)
Name (pg/mL) Flo
Alone Alone
P. aeruginosa 1381 2 32 0.92
P. aeruginosa 1384 1 8 0.96
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-11 1 Aztreonam 8 0.79
P. aeruginosa 2566 0.5 16 1.12
P. aeruginosa 2568 1 8 0.96
P. aeruginosa 103 2 8 1.11
IMIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
=
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
128
TABLE 36
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Piperacillin
Compound 1 Compound 2
MIC' MIC
Organism.' Name (PgirnL) Name
(Pgin11-) FICI2
Alone Alone
P. aeruginosa 1381 2 >64 1.02
P. aeruginosa 1384 8 16 0.79
P. aeruginosa 1474 8 8 0.91
P. aeruginosa 1479 MB-2B 8 Piperacillin 8 1.08
P. aeruginosa 2566 8 32 1.04
P. aeruginosa 2568 8 8 0.97
P. aeruginosa 103 8 8 1.16
1MIC, Minimum Inhibitory Concentration
FICI, Fractional Inhibitory Concentration Index
TABLE 37
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Aztreonam
Compound 1 Compound 2
MICI MIC
Organism.' Name
(PgirriL) Name (ligiml-) FICI2
Alone Alone
P. aeruginosa 1381 8 64 0.89
P. aeruginosa 1384 8 8 0.91
P. aeruginosa 1474 8 8 0.54
P. aeruginosa 1479 MB-2B 8 Aztreonam 8 0.87
P. aeruginosa 2566 8 16 0.91
P. aeruginosa 2568 8 8 0.87
P. aeruginosa 103 8 8 1.08
imic, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
129
TABLE 38
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1B-3 and Cefotaxime
Compound 1 Compound 2
MIC1 MIC
Organism.' Name (pg/mL)
Name (pg/mL)
F1c12
Alone Alone
K. pneumoniae 1346 2 0.06 1.23
K. pneumoniae 1355 1 0.06 2.29
K. pneumoniae 2238 1 16 1.29
K. pneumoniae 2541 MB-1B-3 2 Cefotaxime 0.12 1.23
K. pneumoniae 2546 1 0.25 1.12
K. pneumoniae 2549 1 0.12 0.79
P. aeruginosa 103 1 16 0.96
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 39
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1B-3 and Cefepime
Compound 1 Compound 2
MIC1 MIC
Organisml Name (PgimL) Name Alone
FICI2
Alone
P. aeruginosa 1381 1 32 1.29
P. aeruginosa 1384 1 2 0.79
P. aeruginosa 1474 1 2 0.79
P. aeruginosa 1479 MB-1B-3 1 Cefepime 4 1.12
P. aeruginosa 2566 0.5 " 8 1.37
P. aeruginosa 2568 1 2 0.79
P. aeruginosa 103 1 2 0.71
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
130
TABLE 40
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Cefotaxime
Compound 1 Compound 2
MIC
Organism.' Name (pg/mL)
Name (Pg111114
FICI2
Alone Alone
K. pneumoniae 1346 2 0.06 1.23
K. pneumoniae 1355 1 0.12 2.37
K. pneumoniae 2238 2 16 1.23
K. pneumoniae 2541 .MB-15 2 Cefotaxime 0.12 1.23
K. pneumoniae 2546 2 0.25 0.97
K. pneumoniae 2549 2 0.06 1.23
P. aeruginosa 103 1 16 0.96
WIC, Minimum Inhibitory Concentration
FICI, Fractional Inhibitory Concentration Index
TABLE 41
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Cefepime
Compound 1 Compound 2
MIC
Organism.' Name (pg/mL)
Name (pg/mL) Fici2
Alone Alone
P. aeruginosa 1381 1 32 1.29
P. aeruginosa 1384 1 2 0.79
P. aeruginosa 1474 1 2 1.12
P. aeruginosa 1479 MB-15 1 Cefepime 4 1.12
P. aeruginosa 2566 0.5 8 1.37
P. aeruginosa 2568 1 2 1.12
P. aeruginosa 103 1 1 1.12
1Mic, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
131
TABLE 42
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Cefotaxime
Compound 1 Compound 2
MIC
Organism.' Name (PginiL) Name (Pgin11-)
FICI2
Alone Alone
K. pneumoniae 1346 0.5 0.06 1.37
K. pneumoniae 1355 0.5 0.06 1.37
K. pneumoniae 2238 0.5 16 1.37
K. pneumoniae 2541 MB-8-2 1 Cefotaxime 0.12
1.12
K. pneumoniae 2546 1 0.25 1.29
K. pneumoniae 2549 1 0.06 1.12
P. aeruginosa 103 2 16 1.11
1MIC, Minimum Inhibitory Concentration
FICI, Fractional Inhibitory Concentration Index
TABLE 43
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Cefepime
Compound 1 Compound 2
MIC1 MIC
Organisml Name (PgImL) Name (PgimL) FICI2
Alone Alone
P. aeruginosa 1381 2 32 1.23
P. aeruginosa 1384 2 2 0.80
P. aeruginosa 1474 2 2 1.11
P. aeruginosa 1479 MB-8-2 2 Cefepime 4 1.23
P. aeruginosa 2566 2 8 1.23
P. aeruginosa 2568 2 2 0.98
P. aeruginosa 103 2 1 1.11
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
132
TABLE 44
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Cefotaxime
Compound 1 Compound 2
MIC' MIC
Organism" Name (pg/mL)
Name (pg/mL) m12
Alone Alone
K. pneumoniae 1346 0.5 0.06 1.37
K. pneumoniae 1355 0.5 0.06 1.87
K. pneumoniae 2238 0.5 8 1.37
K. pneumoniae 2541 MB-11 0.5 Cefotaxime 0.25
0.73
K. pneumoniae 2546 0.5 0.25 1.37
K. pneumoniae 2549 0.5 0.06 1.37
P. aeruginosa 103 1 16 1.12
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 45
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Cefepime
Compound 1 Compound 2
MIC' MIC
Organism.' Name (PgimL) Name FICI2
Alone Alone
P. aeruginosa 1381 1 32 1.12
P. aeruginosa 1384 1 2 1.12
P. aeruginosa 1474 0.5 2 1.12
P. aeruginosa 1479 MB-11 0.5 Cefepime 8 0.87
P. aeruginosa 2566 0.5 16 0.93
P. aeruginosa 2568 0.5 2 0.87
P. aeruginosa 103 1 1 0.12
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
133
TABLE 46
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Cefotaxime
Compound 1 Compound 2
MICI MIC
Organism' Name (pg/mL)
Name (Pgin11-) FIC 12
Alone Alone
K. pneumoniae 1346 4 0.06 1.19
K. pneumoniae 1355 4 0.06 1.19
K. pneumoniae 2238 4 8 1.64
K. pneumoniae 2541 MB-2B 8 Cefotaxime 0.25 0.64
K. pneumoniae 2546 8 0.25 1.16
K. pneumoniae 2549 8 0_12 0.83
P. aeruginosa 103 2 16 1.11
1MIC, Minimum Inhibitory Concentration
FICI, Fractional Inhibitory Concentration Index
TABLE 47
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Cefepime
Compound 1 Compound 2
MIC' MIC
Organism' Name (PginiL) Name (Pgin11-) FICI2
Alone Alone
P. aeruginosa 1381 4 32 1.09
P. aeruginosa 1384 4 2 0.94
P. aeruginosa 1474 2 2 0.98
P. aeruginosa 1479 MB-2B 2 Cefepime 4 1.11
P. aeruginosa 2566 2 8 1.23
P. aeruginosa 2568 2 2 1.11
P. aeruginosa 103 2 2 0.61
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
CA 02788669 2012-08-01
WO 2011/097347
PCT/US2011/023549
134
EXAMPLE 11
THE EFFECT OF BISMUTH THIOLS ON INFECTION IN A RATTUS NORVEGICUS
FEMUR CRITICAL DEFECT
The current standard of care for open fractures is irrigation,
debridement and antibiotics; this is intended to reduce the bacterial load in
the
wound to the point that infection does not occur. Despite these treatments,
infections still complicate up to 75% of severe combat open tibia fractures.
Interestingly, even though early infections are often caused by gram negative
bacteria, late infections that are implicated in healing problems and
amputation
are due to gram positive infections, frequently Staphylococci species (Johnson
2007).
One of the reasons that S. aureus are resistant to standard
treatment is their ability to form a biofilm. Bacteria in biofilms are able to
resist
concentrations of antimicrobial compounds which would kill similar organisms
in
a culture medium (Costerton 1987).
The aim of this study was to determine whether BTs will reduce
infection in a contaminated open fracture model either on their own or with
antibiotics. The contaminated rat femur critical defect model is a well-
accepted
model and was used for the experiments described in this Example. This
model offers a standardized model for comparing various possible treatments
and their effects on reducing infection and/or improving healing.
Compounds (CPD) CPD-8-2 (bismuth pyrithione/ butanedithiol;
Table 1) and CPD-11 (bismuth pyrithione/ ethanedithiol; Table 1) are two
analogues of BIS-Bis that have shown potential against Biofilm secreting
bacteria in vitro, though with a different spectrum of activity than Bis-EDT.
The three BT formulations, Bis-EDT, CPD-11 and CPD-8-2 (see
Table 1) demonstrated inhibitory effects on S. aureus strains in vitro when
used
with and without Tobramicin and Vancomycin in a Poly Methyl Methacrylate
(PMMA) cement bead vehicle. Three formulations of microparticulate BTs were
produced in a clinically useful hydrogel gel form as described herein. These
BTs were tested suspended in a gel at a concentration of 5mg/m1-1 as has been
found to be an appropriate concentration for gel delivery. The gel
formulations
conformed to the wound contours, and did not require removal following
application.
Two treatment arms were used: in the first arm, BT was used
singularly; in the second arm BT was used in conjunction with a systemic
antibiotic (ABx).
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
135
(a) BT Singularly.
Six hours after inoculation with S. aureus, the wound was
debrided, irrigated with saline and lml of BT gel inserted within the defect.
(b) BT with Systemic Antibiotics (ABx).
Six hours after inoculation with S. aureus, the wound was
debrided, irrigated with saline and 1m1 of BT gel added inserted within the
defect. The antibiotic used was Cefazolin at a dose equivalent to 5mgKg-1
delivered via sub-cutaneous injection twice daily for a total of 3-days
following
the injury. The first dose was administered immediately prior to debridement.
Previous data suggested that this dose would result in a reduction in bacteria
levels from ..-z106 to ==--104 and therefore still allow the relative effect
of different
BTs to be measured.
(c) Control
Six hours after inoculation with S. aureus, the wound was
debrided and irrigated with saline. The control animals were also treated with
Cefazolin as per the regime described above.
PROCEDURE:
The procedure for the in vivo rat injury model was performed as
described by Chen etal. (2002 J. Orthop. Res. 20:142; 2005 J. Orthop. Res.
23:816; 2006 J. Bone Joint Surg. Am. 88:1510; 2007 J. Orthop. Trauma
21:693). The rats were anesthetized and prepped for surgery. The
anterolateral aspect of the femoral shaft was exposed through a 3-cm incision.
The periosteum and attached muscle was stripped from the bone. A polyacetyl
plate (27 x 4 x 4 mm) was placed on the anterolateral surface of the femur.
The
plates were predrilled to accept 0.9-mm diameter threaded Kirschner wires.
The bases of these plates were formed to fit the contour of the femoral shaft.
Pilot holes were drilled through both cortices of the femur using the plate as
a
template and threaded Kirschner wire was inserted through the plate and
femur. The notches that were 6 mm apart on the plate served as a guide for
.. bone removal. A small oscillating saw was used to create the defect while
the
tissue was cooled by continuous irrigation in an effort to prevent thermal
damage.
Several groups of 10 animals each were inoculated with 1x105
CFU of S. aureus and treated with BT alone or in combination with antibiotics
6
hours post-inoculation as described above, The groups were as follows: Bis-
EDT gel; MB-11 gel; MB-8-2 gel; Bis-EDT gel & Abx; MB-11 gel & Abx; MB-8-2
gel & Abx; Control (Abx alone).
CA 02788669 2012-08-01
WO 2011/097347 PCT/US2011/023549
136
Animals were euthanized 14 days after surgery and bone and
hardware sent for microbiological analysis, the results of which are shown in
Figure 7.
Based on the power analysis, 10 animals per group will give a
power of 80% to detect a 25% difference between the treatment and control
groups. This is with an expected standard deviation of 35% and alpha of 0.05.
As shown in Figure 7, in combination with Bis-EDT, MB-11 and
MB-8-2, Cefazolin antibiotic activity was enhanced as compared to Cefazolin or
any of the Bis compounds alone to reduce S. aureus infection of injured bone.
Cefazolin in combination with MB-11 and MB-8-2 showed enhanced antibiotic
activity as compared to Cefazolin alone to reduce S. aureus infection detected
on hardware. Bis-EDT did not appear to affect Cefazolin activity in this
capacity.
References:
Costerton JW, Cheng KJ, Geesey GG, et al. Bacterial Biofilms in
Nature and Disease. Ann Rev Microbiol. 1987; 41:435-64
Domenico P, Baldassarri L, Schoch PE, Kaehler K, Sasatsu M,
Cunha BA. Activities of Bismuth Thiols against Staphylococci and
Staphyloccocal Biofilms. Antimicrob Agents and Chemother. 2001; 45(5):1417-
21
Halwani M, Blomme S, Suntres ZE, etal. Liposomal bismuth-
ethanedithol formulation enhances antimicrobial activity of tobramycin. Int J
Pharm. 2008; 358:278-84
Johnson EN, Burns TC, Hayda RA, Hospenthal DR, Murray CK.
Infection complications of open type III tibial fractures among combat
casualties. Cfin Infect Dis. 2007; 45(4):409-415
Other Cited Documents and Related Documents
Domenico et al., Canadian J. MicrobioL 31:472-78 (1985);
Domenico et al., Reduction of capsular polysaccharides and potentiation of
aminoglycoside inhibition in gram-negative bacteria with bismuth
subsalicylate.
J Antimicrob Chemo 1991;28:801-810; Domenico et al., Infection 20:66-72
(1992); Domenico et al., Infect. Immun. 62:4495-99 (1994); Domenico et al., J.
AntimicroL Chemother. 38:1031-40 (1996); Domenico et al., Enhancement of
bismuth antibacterial activity with lipophilic thiol chelators. Antimicrob
Agents
Chemother 1997;41:1697-703; Domenico et al., Surface antigen exposure by
bismuth-dimercaprol suppression of Klebsiefia pneumoniae capsular
CA 2788669 2017-03-14
137
polysaccharide. Infect Immun 67:664-669 (1999); Domenico et al., 2000. The
potential of bismuth-thiols for treatment and prevention of infection. Infect
Med
17:123-127; Domenico et at., Activities of bismuth thiols against
staphylococci
and staphylococcal biofilms. Anfimicrob Agents Chemother 200145:1417-21;
Domenico et al., Combating antibiotic resistance with bismuth-thiols. Research
Advances in Antimicrob Agents Chemother 2003;3:79-85; Domenico et at.,
Reduction of capsular polysaccharides and potentiation of aminoglycoside
inhibition in gram-negative bacteria with bismuth subsalicylate. J Antimicrob
Chemo 1991;28:801-810; Domenico et al., BisEDT and RIP act in synergy to
prevent graft infections by resistant staphylococci. Peptides 2004.;25:2047-
53;
Domenico et al., 2005. Pyrithione enhanced antimicrobial activity of bismuth.
Antibiotics for Clinicians 9:291-.297; U.S. Patent No. 6,582,719; U.S.
RE37,793
; U.S. Patent No. 6,248,371; U.S. Patent No. 6,086,921; U.S. Patent No.
6,380,248; U.S. Patent No. 6,582,719; U.S. Patent No. 6,380,248; U.S. Patent
No. 6,875,453.
The various embodiments described above can be combined to
provide further embodiments. Aspects of the embodiments can be modified, if
necessary to employ concepts of the various patents, applications and
publications to provide yet further embodiments.
These and other changes can be made to the embodiments in
light of the above-detailed description. In general,,in the following claims,
the
terms used should not be construed to limit the claims to the specific
embodiments disclosed in the specification and the claims, but should be
construed to include all possible embodiments along with the full scope of
equivalents to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
