Note: Descriptions are shown in the official language in which they were submitted.
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Near-Infrared Electromagnetic Modification of Cellular Steady-State
Membrane Potentials
FIELD OF THE INVENTION
The present invention generally relates to methods and systems for generating
infrared optical radiation in selected energies and dosimetries that will
modify the
bioenergetic steady-state trans-membrane and mitochondrial potentials of
irradiated
cells through a depolarization effect, and more particularly, relates to
methods and
systems for membrane depolarization to potentiate antimicrobial and antifungal
compounds in target bacterial and/or fungal and/or cancer cells.
BACKGROUND OF THE INVENTION
The universal rise of bacteria, fungi and other biological contaminants
resistant
to antimicrobial agents presents humanity with a grievous threat to its very
existence.
Since the advent of sulfa drugs (sulfanilamide, first used in 1936) and
penicillin (1942,
Pfizer Pharmaceuticals), exploitation of significant quantities of
antimicrobial agents of
all kinds across the planet has created a potent environment for the
materialization and
spread of resistant contaminants and pathogens. Certain resistant contaminants
take on
an extraordinary epidemiological significance, because of their predominance
in
hospitals and the general environment. Widespread i.ise of antibiotics not
only prompts
generation of resistant bacteria; such as, for example, methicillin-resistant
staphylococcus
aureus (MRSA) and vancomycin-resistant enterococci (VRE); but also creates
favorable
conditions for infection with the fungal organisms (mycosis), such as,
Candida.
While potent antifungal agents exist that are microbicidal (e.g., amphotericin
B
(AmB)), the attributable mortality of candidemia still remains about 38%. In
some
instances, to treat drug-resistant fungi, high doses of AmB must be
administered which
frequently result in nephrotoxicity and other adverse effects. Moreover,
overuse of
antimicrobial agents or antibiotics can cause bioaccumulation in living
organisms which
may also be cytotoxic to mammalian cells. Given the increasing world's
population and
the prevalence of drug resistant bacteria and fungi, the rise in incidence of
bacterial or
fungal infections is anticipated to continue unabated for the foreseeable
future.
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Currently, available therapies for bacteria] and fungal infections include
administration of antibacterial and antifungal therapeutics or, in some
instances,
application of surgical debridement of the infected area. Because
antibacterial and
antifungal therapies alone are rarely curative, especially in view of newly
emergent
drug resistant pathogens and the extreme morbidity of highly disfiguring
surgical
therapies, it has been imperative to develop new strategies to treat or
prevent microbial
infections.
Therefore, there exist a need for methods and systems that can reduce the risk
of
bacterial or fungal infections, in/at a given target site, without intolerable
risks and/or
intolerable adverse effects to biological moieties (e.g., a mammalian tissue,
cell or
certain biochemical preparations such as a protein preparation) other than the
targeted
bacteria and fungi (biological contaminants).
SUMMARY OF THE INVENTION
The present invention is directed to methods and systems for reducing the
minimum inhibitory concentration (MIC) of antimicrobial molecules
(antimicrobial
agents) and/or antineoplastice molecules (antineoplastic agents) necessary to
attenuate
or eliminate microbial and/or neoplastic-related pathology, so that the agents
that
would otherwise be no longer functional at safe human doses will again be
useful as
adjunctive therapy. According to methods and systems of the present invention,
near
infrared optical radiation in selected energies and dosimetries (herein known
as
NIMELS, standing for "near infrared microbial elimination system") are used to
cause a
depolarization of all membranes within the irradiated field, that will alter
the absohzte
value of the membrane potential AT of the irradiated cells.
Other features and advantages of the present invention will be set forth in
the
detailed description of embodiments that follow, and in part will be apparent
from the
description or may be learned by practice of the invention. Such features and
advantages of the invention will be realized and attained by the systems,
methods, and
apparatus particularly pointed out in the written description and claims
appended
hereto.
-2-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
BRIEF DESCRIPTION OF THE DRAWINGS
Aspects of the invention may more fully be understood from the following
description when read together with the accompanying drawings, which are to be
regarded as illustrative in nature, and not limiting. The drawings are not
necessarily to
scale, emphasis instead being placed on the principles of the invention. In
the
drawings:
Figure 1 shows a typical phospholipid bilayer;
Figure 2 shows the chemical structure of a phospholipid;
Figure 3 shows dipole effects in phospholipid bilayer membranes (Td);
Figure 4A shows a phospholipid bilayer in bacterial plasma membrane, mammalian
mitochondrial membrane, or fugal mitochondrial membrane with a steady-state
trans-
membrane potential prior to NIMELS irradiation. Figure 4B shows a transient-
state
plasma membrane potential in bacterial plasma membrane, mammalian
mitochondrial
membrane, or fugal mitochondrial membrane after NIMELS irradiation;
Figure 5 shows a phospholipid bilayer with trans-membrane proteins embedded
therein;
Figure 6 shows a general depiction of electron transport and proton pump;
Figure 7 shows a general view of mitochondrial membrane in fungi and mammalian
cells the corresponding AT-mito-fungi or AT-mito-mam;
Figure 8 shows the effects of NIMELS irradiation (at a single dosimetry) on
MRSA
trans-membrane potential which is measured by green fluorescence emission
intensities
in control and lased samples as a function of time in minutes post-lasing;
Figure 9 shows the effects of NIMELS irradiation (at various dosimetries) on
C. albicans
trans-membrane potential which is measured by percent drop in green
fluorescence
emission intensities in lased samples relative to the control;
-3-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Figure 10 shows the effects of NIMELS irradiation (at a single dosimetry) on
C. albicans
mitochondrial membrane potential which is measured by red fluorescence
emission
intensities in control and lased samples; and the effects of NIMELS
irradiation (at a
single dosimetry) on C. albicans mitochondrial membrane potential which is
measured
as ratio of red to green fluorescence in control and lased samples;
Figure 11 shows the effects of NIMELS irradiation (at a single dosimetry) on
mitochondrial membrane potential of human embryonic kidney cells, which is
measured by red fluorescence emission intensities in control and lased
samples; and
the effects of NIMELS irradiation (at a single dosimetry) on mitochondrial
membrane
potential of human embryonic kidney cells, which is measured as ratio of red
to green
fluorescence in control and lased samples;
Figure 12 shows the reduction in total glutathione concentration in MRSA as it
correlates with reactive oxygen species (ROS) generation in these cells as the
result of
NIMELS irradiation (at several dosimetries); the decrease in glutathione
concentration
in lased samples is shown as percentage relative to the control;
Figure 13 shows the reduction in total glutathione concentration in C.
albicans as it
correlates with reactive oxygen species (ROS) generation in these cells as the
result of
NIMELS irradiation (at several dosimetries); the decrease in glutathione
concentration
in lased samples is shown as percentage relative to the control;
Figure 14 shows the reduction in total glutathione concentration in human
embryonic
kidney cells as it correlates with reactive oxygen species (ROS) generation in
these cells
as the result of NIMELS irradiation (at two different dosimetries); the
decrease in
glutathione concentration in lased samples is shown as percentage relative to
the
control;
Figure 15 shows the synergistic effects of NIMELS and methicillin in growth
inhibition
of MRSA colonies; data show methicillin is being potentiated by sub-lethal
NIMELS
dosimetry; and
-4-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Figure 16 shows the synergistic effects of NIMELS and bacitracin in growth
inhibition of
MRSA colonies; arrows indicate the growth or a lack thereof of MRSA colonies
in the
two samples shown; images show that bacitracin is being potentiated by sub-
lethal
NIMELS dosimetry.
Figure 17 shows a bar chart depicting the synergistic effects, as indicated by
experimental data, of NIMELS with methicillin, penicillin and erythromycin in
growth
inhibition of MRSA colonies
Figure 18 is a composite showing the improvement over time in the appearance
of the
nail of a typical onychomycosis patient treated according to the methods of
the
invention. Panel A shows the baseline, an infected toenail before treatment;
panel B
shows the toenail 60 days post treatment; panel C shows the toenail 80 days
post
treatment; and panel D shows the toenail 100 days post treatment.
Figure 19 illustrates the detection of decreased membrane potential in E. coli
with sub-
lethal NIMELS irradiation.
Figure 20 illustrates the detection of increased glutathione in E. coli with
sub-lethal
NIMELS irradiation.
While certain embodiments depicted in the drawings and described in relation
to the same, one skilled in the art will appreciate that the embodiments
depicted are
illustrative and that variations of those shown, as well as others described
herein, may
be envisioned and practiced and be within the scope of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
As used in this specification, the singular forms "a", "an" and "the" also
encompass the plural forms of the terms to which they refer, unless the
content clearly
dictates otherwise. For example, reference to "a NIMELS wavelength" includes
any
wavelength within the ranges of the NIMELS wavelengths described, as well as
combinations of such wavelengths.
-5-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
As used herein, unless specifically indicated otherwise, the word "or" is used
in
the "inclusive" sense of "and/or" and not the "exclusive" sense of
"either/or."
The term "about" is used herein to mean approximately, in the region of,
roughly, or around. When the term "about" is used in conjunction with a
numerical,
range, it modifies that range by extending the boundaries above and below the
numerical values set forth. In general, the term "about" is used herein to
modify a
numerical value above and below the stated value by a variance of 20%.
The present invention is directed to methods and systems for reducing the
minimum inhibitory concentration (MIC) of antimicrobial molecules (agents)
and/or
antineoplastic molecules (agents) necessary to attenuate or eliminate
microbial and/or
neoplastic-related pathology, so that the antimicrobial agents that would
otherwise be
no longer functional at safe human doses will again be useful as adjunctive
therapy.
According to methods and systems of the present invention, near infrared
optical
radiation in selected energies and dosimetries (herein known as NIMELS,
standing for
"near infrared microbial elimination system") are used to cause a
depolarization of
membranes within the irradiated field, that will alter the absolute value of
the
membrane potential AT of the irradiated cells.
This altered AT will cause an affiliated weakening of the proton motive force
Ap, and the bioenergetics of all affected membranes. Accordingly, the effects
of
NIMELS irradiation (NIMELS effect) can potentiate existing antimicrobial
molecules
against microbes infecting and causing harm to human hosts. These effects will
render
less functional many cellular anabolic reactions (e.g., cell wall formation)
and drug-
resistance mechanisms (e.g., efflux pumps) that require chemiosmotic
electrochemical
energy to function. Hence, any membrane bound cellular resistance mechanisms
or
anabolic reaction that makes use of the membrane potential AiF, proton motive
force
Ap, or the phosphorylation potential AGp for their functional energy needs,
will be
affected by the methods and systems of the present invention.
The methods and systems of the present invention utilize optical radiation to
potentiate antimicrobial and or antifungal drugs against only targeted
undesirable cells
(e.g., MRSA or Candida infection in skin) with a selectivity made possible by
the fact that
-6-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
mammalian cells are not generally affected by treatments (with molecules or
drugs) that
are intended to damage the bacterial or fungal cells.
In exemplary embodiments, the applied optical radiation used in accordance
with methods and systems of the present invention includes one or more
wavelengths
ranging from about 850 nm to about 900 nm, at a NIMELS dosimetry, as described
herein. In one aspect, wavelengths from about 865 nm to about 875 nm are
utilized. In
another aspect, such applied radiation has a wavelength from about 905 nm to
about
945 nm at a NIMELS dosimetry. In one aspect, such applied optical radiation
has a
wavelength from about 925 nm to about 935 nm. In a particular aspect, a
wavelength of
(or narrow wavelength range including) 930 nm can be employed. In some aspects
of
the present invention, multiple wavelength ranges include 870 and 930 nm,
respectively.
Microbial pathogens whose bioenergetic systems can be affected by the NIMELS
according to the present invention include microorganisms such as, for
example,
bacteria, fungi, molds, mycoplasms, protozoa, and parasites.
In one embodiment, the methods and systems of the present invention are used
in treating, reducing and/or eliminating the infectious entities known to
cause
cutaneous or wound infections such as staphyloccocci and enterococci.
Staphyloccoccal
and enterococcal infections can involve almost any skin surface on the body
known to
cause skin conditions such as boils, carbuncles, bullous impetigo and scalded
skin
syndrome. S. aureus is also the cause of staphylococcal food poisoning,
enteritis,
osteomilitis, toxic shock syndrome, endocarditis, meningitis, pneumonia,
cystitis,
septicemia and post-operative wound infections. Staphyloccoccal infections can
be
acquired while a patient is in a hospital or long-term care facility. The
confined
population and the widespread use of antibiotics have led to the development
of
antibiotic-resistant strains of S. aureus. These strains are called
methicillin resistant
staphylococcus aureus (MRSA). Infections caused by MRSA are frequently
resistant to
a wide variety of antibiotics (especially (3-lactams) and are associated with
significantly
higher rates of morbidity and mortality, higher costs, and longer hospital
stays than
infections caused by non-MRSA microorganisms. Risk factors for MRSA infection
in the
-7-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
hospital include colonization of the nares, surgery, prior antibiotic therapy,
admission
to intensive care, exposure to a MRSA-colonized patient or health care worker,
being in
the hospital more than 48 hours, and having an indwelling catheter or other
medical
device that goes through the skin.
In another embodiment, the methods and systems of the present invention are
used in treating, reducing and/or eliminating the infectious entities known as
cutaneous
Candidiasis. These Candida infections involve the skin, and can occupy almost
any skin
surface on the body. However, the most often occurrences are in warm, moist,
or
creased areas (such as armpits and groins). Cutaneous candidiasis is extremely
common. Candida is the most common cause of diaper rash, where it takes
advantage of
the warm moist conditions inside the diaper. The most common fungus to cause
these
infections is Candida albicans. Candida infection is also very common in
individt.tals with
diabetes and in the obese. Candida can also cause infections of the nail,
referred to as
onychomycosis, infections of the skin surrounding the nail (paronychia) and
infections
around the corners of the mouth, called angul.ar cheilitis.
The term "NIMELS dosimetry" denotes the power density (W/cm2) and the
energy density (J/cmz) (where 1 Watt =1 Joule per second) values at which a
subject
wavelength according to the invention is capable of generating a reactive
oxygen
species ("ROS") and thereby reduce the level of a biological contaminant in a
target site.
The term also includes irradiating a cell to increase the sensitivity of the
biological
contaminant through the lowering of 0T with the concomitant generation of ROS
of an
antimicrobial or antineoplastic agent, wherein the contaminant is resistant to
the agent
otherwise. This method can be effected without intolerable risks and/or
intolerable side
effects on the host subject's tissue other than the biological contaminant.
By "potentiation" of an anti-fungal or antibacterial or antineoplastic agent,
it is
meant that the methods and systems of this invention counteract the resistance
mechanisms in the fungi, bacteria, or cancer sufficiently for the agent to
inhibit the
growth and/or proliferation of said fungi, bacteria, or cancer at a lower
concentration
than in the absence of the present methods and systems. In cases where
resistance is
essentially complete, i.e., the agent has no effect on the cells, potentiation
means that the
-8-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
agent will inhibit the growth and/or proliferation of pathogenic cells thereby
treating
the disease state at a therapeutically acceptable dosage.
As used herein, the term "microorganism" refers to an organism that is
microscopic and by definition, too small to be seen by the human eye. For the
purpose
of this invention, microorganisms can be bacteria, fungi, archaea, protists,
and the like.
The word microbial is defined as pertaining or relating to microorganisms.
As used herein, the term "cell membrane (or plasma membrane or
mitochondrial membrane)" refers to a semi-permeable lipid bilayer that has a
common
structure in all living cells. It contains primarily proteins and lipids that
are involved in
a myriad of important cellular processes. Cell membranes that are the target
of the
present invention have protein/lipid ratios of >1. Stated another way, none of
the target
membranes in the contaminent (or moiety, i.e., host tissue) contain greater
than 49.99%
lipid by dry weight.
As used herein, the term "mitochondria" refers to membrane-enclosed
organelles, found in most eukaryotic cells (mamallian cells and fungi).
Mitochondria
are the "cellular power plants," because they generate most of the eukaryotic
cell's
supply of ATP, used as a source of chemical energy for the cell. The
mitochondria
contain inner and outer membranes composed of phospholipid bilayers and
proteins.
The two membranes, however, have different properties. The outer mitochondrial
membrane, encloses the entire organelle, has a protein-to-phospholipid ratio
similar to
the eukaryotic plasma membrane, and the inner mitochondrial membrane forms
internal compartments known as cristae and has a protein-to-phospholipid ratio
similar
to prokaryote plasma membranes. This allows for a larger space for the
proteins such as
cytochromes to function correctly and efficiently. The electron transport
system ("ETS")
is located on the inner mitochondrial membrane. Within the inner mitochondrial
membrane are also highly controlled transport proteins that transport
metabolites
across this membrane.
As used herein, the term "Fluid Mosaic Model" refers to a widely held
conceptualization of biological membranes as a structurally and functionally
asymmetric lipid-bilayer, with a larger variety of embedded proteins that aid
in cross-
-9-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
membrane transport. The Fluid Mosaic Model is so named, because the
phospholipids
shift position in the membrane almost effortlessly (fluid), and because the
combination
of all the phospholipids, proteins, and glycoproteins present within the
membrane give
the cell a mosaic image from the outside. This model is based on a careful
balance of
thermodynamic and functional considerations. Alteration of the membrane
thermodynamics affects the function of the membrane.
As used herein, the term "Membrane Dipole Potential Td" (in contrast to the
Transmembrane Potential4T) refers to the potential formed between the highly
hydrated lipid heads (hydrophilic) at the membrane surface and the low polar
interior
of the bilayer (hydrophobic). Lipid bilayers intrinsically possess a
substantial Membrane
Dipole Potential Td arising from the structural organization of dipolar groups
and
molecules, primarily the ester linkages of the phospholipids and water.
tYd does not depend upon the ions at the membrane surface and will be used
herein to
describe five different dipole potentials:
1) Mammalian Plasma Membrane Dipole Potential LYd-plas-mam;
2) Mammalian Mitochondrial. Membrane Dipole Potential Td-mito-mam;
3) Fungal Plasma Membrane Dipole Potential Td-plas-fungi;
4) Fungal Mitochondrial Membrane Dipole Potential Td-mito-fungi; and
5) Bacterial Plasma Membrane Dipole Potential lFd-plas-bact.
As used herein, the term "Trans-Membrane Potential" refers to the electrical
potential difference between the aqueous phases separated by a membrane
(dimensions mV)
and will be given by the symbol (0T). 4lY does depend upon the ions at the
membrane
surface and will be used herein to describe three different plasma trans-
membrane
potentials.
1) Mammalian Plasma Trans-Membrane Potential 4T-plas-mam
2) Fungal Plasma Trans-Membrane Potential AtY-plas-fungi
3) Bacterial Plasma Trans-Membrane Potential4T-plas-bact
As used herein, the term "Mitochondrial Trans-Membrane Potential" refers to
the electrical potential difference between the coinpartments separated by the
-10-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
mitochondrial inner membrane (dimensions mV) and will be used herein to
describe
two different mitochondrial trans-membrane potentials.
1) Mammalian Mitochondrial. Trans-Membrane Potential AT-mito-mam
2) Fungal Mitochondrial Trans-Membrane Potential AT-mito-fungi
In mitochondria, the potential energy from nutrients (e.g., glucose) is
converted
into active energy available for cellular metabolic processes. The energy
released during
successive oxidation-reduction reactions allows pumping protons (H* ions) from
the
mitochondrial matrix to the inter-membrane space. As a result, there is a
chemiosmotic
electrical potential difference at the mitochondrial membrane as the membrane
is
polarized (AiY-mito-mam or AT-mito-fungi). 0T-mito-mam and 0T-mito-fungi are
important parameters of mitochondrial functionality and give a direct
quantitative
value to the energy status (redox state) of a cell.
As used herein, the term "mammalian plasma trans-membrane potential (0T-
plas-mam)" refers to the electrical potential difference in the mammalian cell
plasma
membrane between the aqueous phases. The mammalian plasma membrane potential
is different from the bacterial and fungal AT that are primarily generated
with H* ions
(protons). In the mammalian plasma membrane the major facilitator of the AT is
the
electrogenic Na'/K*-ATPase pump. AT-plas-mam is generated by the additive
qualities
of trans-membrane K* diffusion (from the inside to the outside of the cell)
and the
electrogenic Nal/K*-ATPase pump. Mammalian ATP is generated in the
mitochondria
via the proton pump.
As used herein, the term "fungal plasma trans-membrane potential (AT-plas-
fungi)" refers to the electrical potential difference in the fungal cell
plasma membrane.
The fungal plasma membrane potential is generated by a membrane-bound H-
ATPase,
a high-capacity proton pump that requires ATP to function. This H'-ATPase pump
is
needed for both fungal growth and stable cell metabolism and niaintenance.
Fungal
ATP is generated in the mitochondria.
As used herein, the term "bacterial plasina trans-membrane potential (DT-plas-
bact)" refers to the electrical potential difference in the bacterial cell
plasma membrane.
-11-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The bacterial plasma membrane potential is generated by the steady-state flow
(translocation) of electrons and protons (H') across the bacterial plasma
membrane that
occurs with normal electron transport and oxidative phosphorylation, within
the
bacterial plasma membrane. A common feature of all electron transport chains
is the
presence of a proton pump to create a transmembrane proton gradient. Although
bacteria lack mitochondria, aerobic bacteria carry out oxidative
phosphorylation (ATP
production) by essentially the same process that occurs in eukaryotic
mitochondria.
As used herein, the term "P-class ion pump" refers to a trans-membrane active
transport protein assembly which contains an ATP-binding site (i.e., it needs
ATP to
function). During the transport process, one of the protein subunits is
phosphorylated,
and the transported ions are thought to move through the phosphorylated
subunit.
This class of ion pumps includes the Na'/K'-ATPase pump in the mammalian
plasma
membrane, which mahltains the Na* and K' electrochemical potential (ANa'/K`)
and the
pH gradients typical of animal cells. Another important member of the P-class
ion
pumps, transports protons (H' ions) out of and K' ions in to the cell.
As used herein, the term "Na`/K' ATPase" refers to a P-class ion pump that is
present in the plasma membrane of all animal cells, and couples hydrolysis of
one ATP
molecule to the export of three Na' ions and the import of two K' ions that
maintains the
Na' and K* electrochemical potential and the pH gradients typical of animal
cells. The
inside-negative membrane potential in fungal cells (also eukaryotic) is
generated by
transport of H' ions out of the cell by a different ATP powered proton pump.
As used herein, the terms "ion exchangers and ion channels" refer to
transmembrane proteins that are ATP-independent systems, and aid in
establishing a
plasma membrane potential in mammalian cells.
As used herein, the term "Redox (shorthand for reduction/oxidation reaction)"
describes the complex processes of the oxidation of, e.g., sugar in cells
through a series
of very complex processes involving electron transfers. Redox reactions are
chemical
reactions in which electrons are transferred from a donor molecule to an
acceptor
molecule. The term redox comes from the two concepts of reduction and
oxidation, and
can be explained in the simple terms:
-12-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Oxidation describes the loss of electrons by a molecule, atom or ion.
Reduction describes the gain of electrons by a molecule, atom or ion.
As used herein, the term "redox state" describes the redox environment (or
level
of oxidative stress) of the cells being described.
As used herein, the term "steady-state plasma trans-membrane potential (AT-
steady)" refers to the quantitative Plasma Membrane Potential of a mammalian,
fungal
or bacteria] cell before irradiation in accordance with the methods and
systems of the
present invention that would continue into the future in the absence of such
irradiation.
For example, the steady-state flow of electrons and protons across a bacterial
cell
membrane that occurs during normal electron transport and oxidative
phosphorylation
would be in a steady-state due to a constant flow of conventional redox
reactions
occurring across the membrane. Conversely any modification of this redox state
would
cause a transient-state membrane potential. AlY-steady will be used herein to
describe
three (3) different steady-state plasma trans-membrane potentials, based on
species.
1) Steady-state mammalian plasma trans-membrane potential AtY-steady-mam
2) Steady-state fungal plasma trans-membrane potential ALY-steady-fungi
3) Steady-state bacterial plasma trans-membrane potential0T-steady-bact
As used herein, the term "Transient-state plasma membrane potential (ALP-
tran)" refers to the Plasma Membrane Potential of a mammalian, fungal or
bacterial cell
after irradiation in accordance with the methods and systems of the present
invention
whereby the irradiation has changed the bioenergetics of the plasma membrane.
In a
bacteria, DT-tran will also change the redox state of the cell, as the plasma
membrane is
where the ETS and cytochromes reside. ALY-tran is a state that would not occur
without
irradiation using methods of the present invention. AT-tran will be used
herein to
describe three (3) different Transient-state plasma trans-membrane potentials
based on
species.
1) Transient-state mammalian plasma trans-membrane potential AtF-tran-mam
2) Transient-state fungal plasma trans-membrane potential AT-tran-fungi
3) Transient -state bacterial plasma trans-membrane potential AT-tran-bact
-13-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
As used herein, the term "steady-state mitochondrial membrane potential (AT-
steady-mito)" refers to the quantitative Mitochondrial Membrane Potential of
mamrnalian or fungal mitochondria before irradiation in accordance with the
methods
and systems of the present invention that would continue into the future, in
the absence
of such irradiation.
For example, the steady-state flow of electrons and protons across
mitochondrial
inner membrane that occurs during normal electron transport and oxidative
phosphorylation would be in a steady-state because of a constant flow of
conventional
redox reactions occurring across the membrane. Any modification of this redox
state
would cause a transient-state mitochondrial membrane potential. AT-steady-mito
will
be used herein to describe two (2) different steady-state mitochondrial
membrane
potentials based on species.
1) Steady-state mitochondrial mammalian potential AT-steady-mito-mam
2) Steady-state mitochondrial fungal potential AT-steady-mito-fungi
As used herein, the term "transient-state mitochondrial membrane potential
(AT-tran-mito-mam or AT-tran-mito-fungi)" refers to the membrane potential of
a
mammalian or fungal cell after irradiation in accordance with the methods and
systems
of the present invention whereby the irradiation has changed the bioenergetics
of the
mitochondrial inner membrane. In mammalian and fungal cells, 0T-tran-mito will
also
change the redox state of the cell, as the iruter mitochondrial membrane is
where the
electron transport system (ETS) and cytochromes reside. AT-tran-mito could
also
drastically affect (the Proton-motive force) Ap-mito-mam and Ap-mito-fungi, as
these
mitochondrial (H*) gradients are generated in the mitochondria, to produce
adequate
ATP for a myriad of cellular functions. AT-tran-mito is a state that would not
occur
without irradiation in accordance with methods and systems of the present
invention.
AlY-tran-mito will be used herein to describe two (2) different transient-
state
mitochondrial membrane potentials based on species.
-14-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
1) Transient-state mitochondrial mammalian potential AT-tran-mito-mam
2) Transient-state mitochondrial fungal potential AT-tran-mito-fungi
As used herein, the term "cytochrome" refers to a membrane-bound
hemoprotein that contains heme groups and carries out electron transport.
As used herein, the term "electron transport system (ETS)" describes a series
of
membrane-associated electron carriers (cytochromes) mediating biochemical
reactions,
that produce (ATP), which is the energy currency of cells. In the prokaryotic
cell
(bacteria) this occurs in the plasma membrane. In the eukaryotic cell (fungi
and
mammalian cells) this occurs in the mitochondria.
As used herein, the term "pH Gradient (ApH)" refers to the pH difference
between two bulk phases on either side of a membrane.
As used herein, the term "proton electrochemical gradient (A H') (dimensions
kJ mol-1)" refers to the electrical and chemical properties across a membrane,
particularly proton gradients, and represents a type of cellular potential
energy
available for work in a cell. This proton electrochemical potential difference
between
the two sides of a membrane that engage in active transport involving proton
pumps, is
at times also called a chemiosmotic potential or proton motive force. When A
H' is
reduced by any means, it is a given that cellular anabolic pathways and
resistance
mechanisms in the affected cells are inhibited. This can be accomplished by
combining
An and Tn to irradiate a target site alone, or can be further enhanced with
the
simultaneous or sequential administration of a pharmacological agent
configured and
arranged for delivery to the target site (i.e., the co-targeting of an
anabolic pathway with
(An and Tn) + (pharmacological molecule or molecules)).
As used herein, the term "Ion Electrochemical Gradient (Apx+)" refers to the
electrical and chemical properties across a membrane caused by the
concentration
gradient of an ion (other than H') and represents a type of cellular potential
energy
available for work in a cell. In mammalian cells, the Na' ion electrochemical
gradient is
maintained across the plasma membrane by active transport of Na* out of the
cell. This
is a different gradient than the proton electrochemical potential, yet is
generated from
-15-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
an ATP coupled pump, said ATP produced during oxidative phosphorylation from
the
mammalian mitochondrial proton-motive force (Ap-mito-mam). When D x' is
reduced
by any means, it is a given that cellular anabolic pathways and resistance
mechanisms
in the affected cells are inhibited. This can be accomplished by combining An
and Tn to
irradiate a target site alone, or can be further enhanced with the
simultaneous or
sequential administration of a pharmacological agent configured and arranged
for
delivery to the target site (i.e., the co-targeting of an anabolic pathway
with (An and Tn)
+ (pharmacological molecule or molecules)).
As used herein, the term "co-targeting of a bacterial anabolic pathway" refers
to
(the An and Tn lowering of (4 H') and/or (A x') of cells at the target site to
affect an
anabolic pathway) + (a pharmacological molecule or molecules to affect the
same
bacterial anabolic pathway) and can refer to any of the following bacterial
anabolic
pathways that are capable of being inhibited with pharmacological molecules:
wherein the targeted anabolic pathway is peptidoglycan biosynthesis that is co-
targeted by a pharmacological agent that binds at the active site of the
bacterial
transpeptidase enzymes (penicillin binding proteins) which cross-links
peptidoglycan in
the bacterial cell wall. Inhibition of these enzymes ultimately cause cell
lysis and death;
wherein the targeted bacterial anabolic pathway is peptidoglycan biosynthesis
that is co-targeted by a pharmacological agent that binds to acyl-D-alanyl-D-
alanine
groups in cell wall intermediates and hence prevents incorporation of N-
acetylmuramic
acid (NAM)- and N-acetylglucosamine (NAG)-peptide subunits into the
peptidoglycan
matrix (effectively inhibiting peptidoglycan biosynthesis by acting on
transglycosylation and/or transpeptidation) thereby preventing the proper
formation of
peptidoglycan, in gram positive bacteria;
wherein the targeted bacterial anabolic pathway is peptidoglycan biosynthesis
that is co-targeted by a pharmacological agent that binds with Css-isoprenyl
pyrophosphate and prevents pyrophosphatase from interacting with Css-isoprenyl
pyrophosphate thus reducing the amount of Cas-isoprenyl pyrophosphate that is
available for carrying the building blocks peptidoglycan outside of the inner
membrane;
-16-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
wherein the targeted anabolic pathway is bacteria] protein biosynthesis that
is
co-targeted by a pharmacological agent that binds to the 23S rRNA molecule in
the
subunit 50S subunit of the bacterial ribosome, causing the accumulation of
peptidyl-
tRNA in the cell, hence depleting the free tRNA necessary for activation of a-
amino
acids, and inhibiting transpeptidation by causing premature dissociation of
peptidyl
tRNA from the ribosome;
wherein the co-targeted pharmacological agent binds simultaneously to two
domains of 23S RNA of the 50 S bacterial ribosomal subunit, and can thereby
inhibit the
formation of the bacterial ribosomal subunits 50S and 30S (ribosomal subunit
assembly)
wherein the co-targeted pharmacological agent is chlorinated to increases its
lipophilicity to penetrate into bacterial cells, and binds to the 23S portion
of the 50S
subunit of bacterial ribosomes and prevents the translocation of the peptidyl-
tRNA
from the Aminoacyl site (A-site) to the Peptidyl site (P-site) thereby
inhibiting the
transpeptidase reaction, which results in an incomplete peptide being released
from the
ribosome;
wherein the targeted anabolic pathway is bacterial protein biosynthesis that
is
co-targeted by pharmacological agent that binds to the 30S bacterial ribosomal
subunit
and blocks the attachment of the amino-acyl tRNA from binding to the acceptor
site (A-
site) of the ribosome, tllereby inhibiting the codon-anticodon interaction and
the
elongation phase of protein synthesis;
wherein the co-targeted pharmacological agent binds more avidly to the
bacterial ribosomes, and in a different orientation from the classical
subclass of
polyketide antimicrobials having an octahydrotetracene-2-carboxamide skeleton,
so
that they are active against strains of S. aureus with a tet(M) ribosome and
tet(K) efflux
genetic determinant;
wherein the targeted anabolic pathway is bacterial protein biosynthesis that
is
co-targeted by a pharmacological agent that binds to a specific aminoacyl-tRNA
synthetase to prevent the esterification of a specific amino acid or its
precursor to one of
its compatible tRNA's, thus preventing formation of an aminoacyl-tRNA and
hence
halting the incorporation of a necessary amino acid into bacterial proteins;
-17-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
wherein the targeted anabolic pathway is bacterial protein biosynthesis that
is
co-targeted by a pharmacological agent that inhibits bacterial protein
synthesis before
the initiation phase, by binding the 50S rRNA through domain V of the 23S
rRNA, along
with interacting with the 16S rRNA of the 30S ribosomal subunit, thus
preventing
binding of the initator of protein synthesis formyl-methionine (f-Met-tRNA),
and the
30S ribosomal subunit;
wherein the targeted anabolic pathway is bacterial protein biosynthesis that
is
co-targeted by a pharmacological agent that interacts with the 50S subunit of
bacterial
ribosomes at protein L3 in the region of the 23S rRNA P site near the peptidyl
transferase center and hence inhibits peptidyl transferase activity and
peptidyl transfer,
blocks P-site interactions, and prevents the normal formation of active 50S
ribosomal
subunits;
wherein the targeted anabolic pathway is DNA replication and transcription
that is co-targeted by a pharmacological agent that inhibits Topoisomerase II
(DNA
gyrase) and/or Topoisomerase IV;
wherein the targeted anabolic pathway is DNA replication and translation that
is co-targeted by a pharmacological agent that inhibits DNA polymerase IIIC,
the
enzyme required for the replication of chromosomal DNA in gram-positive
bacteria, but
not present in gram-negative bacteria;
wherein the targeted anabolic pathway is DNA replication and transcription
that
is co-targeted by a pharmacological hybird compound that inhibits
Topoisomerase II
(DNA gyrase) and/or Topoisomerase IV and/or DNA polymerase IIIC;
wherein the targeted anabolic pathway is bacterial phospholipid biosynthesis
that is co-targeted by a topical pharmacological agent that acts on
phosphatidylethanolamine-rich cytoplasmic membranes and works well in
combination
with other topical synergistic agents;
wherein the targeted anabolic pathway is bacterial fatty acid biosynthesis
that is
co-targeted by a pharmacological agent that inhibits bacterial fatty acid
biosynthesis
through the selective targeting of P-ketoacyl-(acyl-carrier-protein (ACP))
synthase I/II
(FabF/B), an essential enzymes in type II fatty acid synthesis;
-18-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
wherein the targeted anabolic pathway is maintenance of bacterial plasma
trans-membrane potential AT-plas-bact and the co-targeting pharmacological
agent
disrupts multiple aspects of bacterial cell membrane function on its own, by
binding
primarily to gram positive cytoplasmic membranes, not penetrating into the
cells, and
causing depolarization and loss of membrane potential that leads to inhibition
of
protein, DNA and RNA synthesis;
wherein the co-targeting pharmacological agent increases the permeability of
the
bacterial cell wall, and hence allows inorganic cations to travel through the
wall in an
unrestricted manner thereby destroying the ion gradient between the cytoplasm
and
extracellular environment;
wherein the targeted anabolic pathway is maintenance of bacterial membrane
selective permeability and bacterial plasma trans-membrane potential 0T-plas-
bact,
and the co-targeting pharmacological agent is a cationic antibacterial peptide
that is
selective for the negatively charged surface of bacterial membranes relative
to the
neutral membrane surface of eukaryotic cells and leads to prokaryotic membrane
permeablization and ultimate perforation and/or disintegration of bacterial
cell
membranes, thereby promoting leakage of bacterial cell contents and a
breakdown of
the transmembrane potential;
wherein the co-targeting pharmacological agent inhibits bacteria protease
Peptide Deformylase, that catalyzes the removal of formyl groups from the N-
termini
of newly synthesized bacterial polypeptides; and
wherein the co-targeting pharmacological agent inhibits two-component
regulatory systems in bacteria, such as the ability to respond to their
environment
through signal transduction across bacterial plasma membranes, these signal
transduction processes being absent in mammalian membranes.
As used herein, the term "co-targeting of a fungal anabolic pathway" refers to
(the An and Tn lowering of (A H') and/or (A xl) of cells at the target site to
affect an
anabolic pathway) + (a pharmacological agent to affect the same fungal
anabolic
pathway) and can refer to any of the following firngal anabolic pathways that
are
capable of being inhibited with pharmacological agents:
-19-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
wherein the targeted anabolic pathway is phospholipid Biosynthesis that is
co-targeted by a topical pharmacological agent that disrupts the structure of
existing
phospholipids, in fungal cell membranes and workswell in combination with
other
topical synergistic agents;
wherein targeted anabolic pathway is ergosterol biosynthesis that is co-
targeted
by a pharmacological agent that inhibits ergosterol biosynthesis at the C-14
demethylation stage, part of the three-step oxidative reaction catalyzed by
the
cytochrome P-450 enzyme 14-a-sterol demethylase, resulting in ergosterol
depletion and
accumulation of lanosterol and other 14-methylated sterols that interfere with
the 'bulk'
functions of ergosterol as a membrane component, via disruption of the
structure of the
plasma membrane;
wherein targeted anabolic pathway is ergosterol biosynthesis that is co-
targeted
with a pharmacological agent inhibits the enzyme squalene epoxidase, that in
turn
inhibits ergosterol biosynthesis in fungal cells that causes the fungal cell
membranes to
have increased permeability;
wherein targeted anabolic pathway is ergosterol biosynthesis that is co-
targeted
with a pharmacological agent inhibits two enzymes in the ergosterol
biosynthetic
pathway at separate and distinct points, d14-reductase and d7, d8-isomerase;
wherein targeted anabolic pathway is fungal cell wall biosynthesis that is co-
targeted with a pharmacological agent that inhibits the enzyme (1,3)(3-D-
Glucan
synthase, that in turn inhibits (3-D-glucan synthesis in the fungal cell wall;
wherein the wherein targeted anabolic pathway is fungal sterol biosynthesis
that
is co-targeted with a pharmacological agent binds with sterols in fungal cell
membranes, the prnlcipal sterol that the co-targeting pharmacological agent
binds being
ergosterol, that effectively changes the transition temperature of the cell
membrane and
causes pores to form in the membrane resulting in the formation of detrimental
ion
channels in fungal cell membranes;
wherein the co-targeted pharmacological agent is formulated for delivery in
lipids, liposomes, lipid complexes and/or colloidal dispersions to prevent
toxicity from
the agent;
-20-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
wherein the wherein targeted anabolic pathway is protein synthesis is co-
targeted with a pharmacological agent that 5-FC is taken up into fungal cells
by a
cytosine permeasc, deaminated to 5-fluorouracil (5-FU), converted to the
nucleosidc
triphosphate, and incorporated into RNA where it causes miscoding;
wherein the wherein targeted anabolic pathway is fungal protein synthesis that
is co-targeted with a pharmacological agent that inhibits fungal elongation
factor EF-2,
which is functionally distinct from its mammalian counterpart and/or fungal
elongation
factor 3 (EF-3)which is absent from mammalian cells;
wherein the wherein targeted anabolic pathway is fungal Chitin bio-synthesis
(the (3 -(1,4)-linked homopolymer of N-acetyl-D-glucosamine), that is co-
targeted with a
pharmacological agent that inhibits fungal chitin biosynthesis by inhibiting
the action of
one or more of the enzymes chitin synthase 2, an enzyme necessary for primary
septum
formation and cell division in fungi;
wherein the wherein the co-targeted pharmacological agent inhibitis the action
of the enzyme chitin synthase 3, an enzyme necessary for the synthesis of
chitin during
bud emergence and growth, mating, and spore formation;
wherein the co-targeting pharmacological agent chelates polyvalent cations
(Fe+3
or A1+3) resulting in the inhibition of the metal-dependent enzymes that are
responsible
for mitochondrial electron transport and cellular energy production, that also
leads to
inhibition of normal degradation of peroxides within the fungal cell; and
wherein the co-targeting pharmacological agent inhibits two-component
regulatory systems in fungi, such as the ability to respond to their
environment through
signal transduction across fungal plasma membranes.
As used herein, the term "co-targeting of a cancer anabolic pathway" refers to
(the An and Tn lowering of (A H`) and/or (A x+) of cells at the target site to
affect an
anabolic pathway) + (a pharmacological agent to affect the same cancer
anabolic
pathway to a greater extent than the non cancerous cells) and can refer to any
of the
following cancer anabolic pathways that are capable of being inhibited with
pharmacological agents:
-21-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
wherein the targeted anabolic pathway is DNA replication that is co-targeted
by
a pharmacological agent that inhibits DNA replication by cross-linking guanine
nucleobases in DNA double-helix strands making the strands unable to uncoil
and
separate, which is necessary in DNA replication;
wherein the targeted anabolic pathway is DNA replication that is co-targeted
by
a pharmacological agent that can react with two different 7-N-guanine residues
in the
same strand of DNA or different strands of DNA;
wherein the targeted anabolic pathway is DNA replication that is co-targeted
by
a pharmacological agent that inhibits DNA replication and cell division by
acting as an
antimetabolite;
wherein the targeted anabolic pathway is cell division that is co-targeted by
a
pharmacological agent that inhibits cell division by preventing microttxbule
function;
wherein the targeted anabolic pathway is DNA replication that is co-targeted
by
a pharmacological agent that inhibits DNA replication and cell division by
preventing
the cell from entering the Gl phase (the start of DNA replication) and the
replication of
DNA (the S phase);
wherein the targeted anabolic pathway is cell division that is co-targeted by
a
pharmacological agent that enhances the stability of microtubules, preventing
the
separation of chromosomes during anaphase; and
wherein the targeted anabolic pathway is DNA replication that is co-targeted
by
a pharmacological agent that inhibits DNA replication and cell division by
Inhibition
of type I or type II topoisomerases, that will interferes with both
transcription and
replication of DNA by upsetting proper DNA supercoiling.
As used herein, the term "proton-motive force (Ap)" refers to the storing of
energy (acting like a kind of battery), as a combination of a proton and
voltage gradient
across a membrane. The two components of Ap are AT (the transmembrane
potential)
and ApH (the chemical gradient of H'). Stated another way, Ap consists of the
H*
transmembrane potential AtiY (negative (acidic) outside) and a transmembrane
pH
gradient ApH (alkaline inside). This potential energy stored in the form of an
electrochemical gradient, is generated by the pumping of hydrogen ions across
-22-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
biological membranes (mitochondrial inner membranes or bacterial and fungal
plasma
membranes) during chemiosmosis. The Ap can be used for chemical, osmotic, or
mechanical work in the cells. The proton gradient is generally used in
oxidative
phosphorylation to drive ATP synthesis and can be used to drive efflux pumps
in
bacteria, fungi, or mammalian cells including cancerous cells. Ap will be used
herein to
describe four (4) different proton motive forces in membranes, based on
species, and is
mathematically defined as (AP = AIY + ApH).
1) Mammalian Mitochondrial Proton-motive force (Ap-mito-mam)
2) Fungal Mitochondrial Proton-motive force (Ap-mito-Fungi)
3) Fungal Plasma Membrane Proton-motive force (Op-plas-Fungi)
4) Bacterial Plasma Membrane Proton-motive force (Ap-plas-Bact)
As used herein, the term of "Mammalian Mitoc.hondrial Proton-motive force
(Op-mito-mam) refers to the potential energy stored in the form of an (H*)
electrochemical gradient across a mammalian mitochondrial inner membrane. Ap-
mito-
mam is used in oxidative phosphorylation to drive ATP synthesis in the
mammalian
mitochondria.
As used herein, the term of "Fungal Mitochondrial Proton-motive force (Ap-
mito-Fungi)" refers to the potential energy stored in the form of an (H`)
electrochemical
gradient across a fungal mitochondrial inner membrane. Ap-mito-Fungi is used
in
oxidative phosphorylation to drive ATP synthesis in the fungal mitochondria.
As used herein, the term "Fungal Plasma Membrane Proton-motive force (Ap-
plas-Fungi)" refers to the potential energy stored in the form of an (H')
electrochemical
gradient, across a fungal plasma membrane and is generated by the pumping of
hydrogen ions across the plasma membrane by a membrane-bound H'-ATPase. This
plasma membrane-bound H`-ATPase is a high-capacity proton pump, that requires
ATP
to ftznction. The ATP for this H'-ATPase is generated from the Ap-mito-Fungi.
Ap-plas-
Fungi can be used to drive efflux pumps in fungal cells.
As used herein, the term "Bacterial Plasma Membrane Proton-motive force (Ap-
plas-Bact )" refers to the potential energy stored in the form of an
electrochemical
-2.3-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
gradient (H'), across a bacterial plasma membrane, and is generated by the
pumping of
hydrogen ions across the plasma membrane during chemiosmosis. Ap-plas-Bact is
used
in oxidative phosphorylation to drive ATP synthesis in the bacterial plasma
membrane
and can be used to drive efflux pumps in bacterial cells.
As used herein, the term "anabolic pathway" refers to a cellular metabolic
pathway that constructs molecules from smaller units. These reactions require
energy.
Many anabolic pathways and processes are powered by adenosine triphosphate
(ATP).
These processes can involve the synthesis of simple molecules such as single
amino
acids and complex molecules such as peptidoglycan, proteins, enzymes,
ribosomes,
cellular organelles, nt.icleic acids, DNA, RNA, glucans, chitin, simple fatty
acids,
complex fatty acids, cholesterols, sterols, and ergosterol.
As used herein, the term "energy transduction" refers to proton transfer
through
the respiratory complexes embedded in a membrane, utilizing electron transfer
reactions to pump protons across the membrane and create an electrochemical
potential
also known as the proton electrochemical gradient.
As used herein the term "energy transformation" in cells refers to chemical
bonds being constantly broken and created, to make the exchange and conversion
of
energy possible. It is generally stated that that transformation of energy
from a more to
a less concentrated form is the driving force of all biological or chemical
processes that
are responsible for the respiration of a cells.
As used herein the term "uncoupler" refers to a molecule or device that causes
the separation of the energy stored in the proton electrochemical gradient (A
H') of
membranes from the synthesis of ATP.
As used herein the term "uncoupling" refers to the use of an uncoupler (a
molecule or device) to cause the separation of the energy stored in the proton
electrochemical gradient (A H') of membranes from the synthesis of ATP.
As used herein the term "adenosine 5-triphosphate (ATP)" refers to a multi-
functional nucleotide that acts as "molecular currency" of intracellular
energy transfer.
ATP transports chemical energy within cells for metabolism and is produced as
an
energy source during the process of cellular respiration. ATP is consumed by
many
-24-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
enzymes and a broad array of cellular processes including biosynthetic
reactions, efflux
pump function, and anabolic cell growth and division.
As used herein the term adenosine diphosphate (ADP)" is the product of ATP
dephosphorylation by ATPases. ADP is converted back to ATP by ATP synthesis.
It is
understood that in aerobic respiring cells, under physiological conditions,
ATP synthase
creates ATP while using the proton-motive force Ap created by the ETS as a
source of
energy. The overall process of creating energy in this fashion is termed
oxidative
phosphorylation. The overall reaction sequence of oxidative phosphorylation
is: ADP +
Pi -> ATP. The underlying force driving biological reactions is the Gibbs free
energy of
the reactants and products. The Gibbs free energy is the energy available
("free") to do
work, and the term Gibbs free energy change (AG) refers to a change in the
free energy
available in the membrane to do work. This free energy is a function of
enthalpy (AH),
entropy (AS), and temperature. (Enthalpy and entropy are discussed below.)
As used herein, the term "phosphorylation potential (AGp)" refers to the AG
for
ATP synthesis at any given set of ATP, ADP and Pi concentrations (dimensions:
kJ mol-
').
As used herein the term "CCCP" refers to carbonyl cyanide m-
chlorophenylhydrazone, a highly toxic ionophore and uncoupler of fl1e
respiratory
chain. CCCP increases the conductance of protons through membranes and acts as
a
classical uncoupler by uncoupling ATP synthesis from the A H' and dissipating
both
the AT and ApH.
As used lierein the term "depolarization" (de-energization) refers to a
decrease
in the absolute value of a cell's plasma or mitochondrial membrane potential.
AT. It is a
given that depolarization of any bacterial plasma membrane will lead to a loss
of ATP
and increased free radical formation. It is also a given that mitochondrial
depolarization
of any eukaryotic cell will lead to a loss of ATP and increased free radical
formation.
As used herein, the term "enthalpy change (AH)" refers to a change in the
enthalpy or heat content of a membrane system, and is a quotient or
description of the
thermodynamic potential of the membrane system.
-25-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
As used herein, the term'"entropy change (AS)" refers to a change in the
entropy
of a membrane system to that of a more disordered state at a molecular level.
The term "redox stress", refers to cellular conditions which differ from the
standard
reduction/oxidation potential ("redox") state of the cell. Redox stress
includes increased
levels of ROS, decreased levels of glutathione and any other circumstances
that alter the
redox potential of the cell.
As used herein, the term "Reactive Oxygen Species", refers to one of the
following categories:
a) The Superoxide ion radical(Oz-)
b) Hydrogen Peroxide (non-radical) (H202)
c) Hydroxyl radical (*OH)
d) Hydroxy ion (OH-)
These ROS generally occur through the reaction chain:
02 4 02- + 2H+ -) H202 4 OH- + *OH 4 OH-
(e-) (e-) (e-) (e-)
As used herein, the term "singlet oxygen" refers to ("102") and is formed via
an
interaction with triplet-excited molecules. Singlet oxygen is a non-radical
species with
its electrons in anti-parallel spins. Because singlet oxygen 102 does not have
spin
restriction of its electrons, it has a very high oxidizing power and is easily
able to attack
membranes (e.g., via polyunsaturated fatty acids, or PUFAs) amino acid
residues,
protein and DNA.
As used herein, the term "energy stress" refers to conditions which alter ATP
levels in the cell. This could be changes in electron transport and exposure
to
uncoupling agents or AT altering radiation in mitochondrial and/or plasma
membranes.
As used herein, the term "NIMELS effect" refers to the modification of the
bioenergetic "state" of irradiated cells at the level of the cell's plasma and
mitochondrial
membranes from AtY-steady to OLY-trans with the present invention.
Specifically, the
NIMELS effect can weaken cellular anabolic pathways or antimicrobial and/or
cancer
-26-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
resistance mechanisms that make use of the proton motive force or the
chemiosmotic
potential for their energy needs.
As used herein, the term "periplasmic space or periplasm" refers to the space
between the plasma membrane and the outer membrane in gram-negative bacteria
and
the space between the plasma membrane and the cell wall in gram-positive
bacteria and
fungi such as the Candida and Trichophyton species. This periplasmic space is
involved in various biochemical pathways including nutrient acquisition,
synthesis of
peptidoglycan, electron transport, and alteration of stibstances toxic to the
cell. In gram-
positive bacteria like MRSA, the periplasmic space is of significant clinical
importance
as it is where P-lactamase enzymes inactivate penicillin based antibiotics.
As used herein, the term "efflux pump" refers to an active transport protein
assembly which exports molecules from the cytoplasm or periplasm of a cell
(such as
antibiotics, antifungals, or poisons) for their removal from the cells to the
external
environment in an energy dependent fashion.
As used herein, the term "efflux pump inhibitor" refers to a compound or
electromagnetic radiation delivery system and method which interferes with the
ability
of an efflux pump to export molecules from a cell. In particular, the efflux
pump
inhibitor of this invention is a form of electromagnetic radiation that will
interfere with
a pump's ability to excrete therapetrtic antibiotics, anti-fungal agents,
antineoplastic
agents and poisons from cells via a modification of the AtY-steady-mam, ALP-
steady-
fungi or, AW-steady-bact.
By a cell that "utilizes an efflux pump resistance mechanism," it is meant
that the
bacterial or fungal or cancer cell exports anti-bacterial and/or anti-fungal
and/or
antineoplastic agents from their cytoplasm or periplasm to the external
environment of
the cell and thereby reduce the concentration of these agents in the cell to a
concentration below what is necessary to inhibit the growth and/or
proliferation of the
cells.
In the context of cell growth, the term "inhibit" means that the rate of
growth
and/or proliferation of population of cells is decreased, and if possible,
stopped.
-27-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
In protein chemistry the primary structure refers to the linear arrangement of
amino acids; the secondary structure refers to whether the linear amino acid
structure
forms a helical or (3-pleated sheet structure; tertiary strtzcture of a
protein or any other
macromolecule is its three-dimensional structure, or stated another way, its
spatial
organization (including conformation) of the entire single chain molecule; the
quaternary structure is the arrangement of multiple tertiary structured
protein
molecules in a multi-subunit complex.
As used herein, the term "protein stress", refers to thermodynamic
modification
in the tertiary and quaternary structure of proteins, including enzymes and
other
proteins that participate in membrane transport. The term includes, but is not
limited
to, denaturation of proteins, misfolding of proteins, cross-linking of
proteins, both
oxygen-dependent and independent oxidation of inter- and intra-chain bonds,
such as
disulfide bonds, oxidation of individual amino acids, and the like.
The term "pH stress", refers to modification of the intracellular pH, i.e., a
decrease intracellular pH below about 6.0 or an increase intracellular pH
above about
7.5. pH. This may be caused, for example, by exposure of the cell to the
invention
described herein, and altering cell membrane components or causing changes in
the
steady-state membrane potential potential AT-steady.
As used herein, the term "anti-fungal molecule" refers to a chemical or
compound that is fungicidal or fungistatic. Of principle efficacy is the
present
invention's ability to potentiate anti-fungal molecules by inhibiting anabolic
reactions
and/or efflux pump activity in resistant fungal strains, or inhibiting other
resistance
mechanisms that require the proton motive force or chemiosmotic potential for
energy.
As used herein, the term "anti-bacterial molecule (or agent)" refers to a
chemical
or compound that is bacteriacidal or bacteriastatic. Another principal
efficacy resides in
the present inverition's ability to potentiate anti-bacterial molecules by
inhibiting efflux
pump activity in resistant bacterial strains, or inhibiting anabolic reactions
and/or
resistance mechanisms that require the proton motive force or chemiosmotic
potential
for energy.
-28-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
As used herein, a"sub-inbibitory concentration" of an antibacterial or anti-
fungal
molecule refers to a concentration that is less than that required to inhibit
a majority of
the target cells in the population. (In one aspect, target cells are those
cells that are
targeted for treatment including, but not limited to, bacterial, fungi, and
cancer cells.)
Generally, a sub-inhibitory concentration refers to a concentration that is
less than the
Minimum Inhibitory Concentration (MIC), which is defined, unless specifically
stated to
be otherwise, as the concentration required to produce at least 10% reduction
in the
growth or proliferation of target cells.
As used herein, the term "Minimal Inhibitory Concentration" or MIC is defined
as the lowest effective or therapeutic concentration that results in
inhibition of growth
of the microorganism.
As used herein, the term "therapeutically effective amount" of a
pharmaceutical
agent or molecule (e.g., antibacterial or anti-fungal agent) refers to a
concentration of an
agent that, together with NIMELS, will partially or completely relieve one or
more of
the symptoms caused by the target (pathogenic) cells. In particular, a
therapeutically
effective amount refers to that amount of an agent with NIMELS that: (1)
reduces, if not
eliminates, the population of target cells in the patient's body, (2) inhibits
(i.e., slows, if
not stops) proliferation of the target cells in the patients body, (3)
inhibits (i.e., slows, if
not stops) spread of the infection (4) relieves (if not, eliminates) symptoms
associated
with the infection.
As used herein, the term "Interaction coefficient" is defined as a numerical
representation of the magnitude of the bacteriastatic/bacteriacidal and/or
fungistatic/fungicidal interaction between the NIMELS laser and/or the
antimicrobial
molecule, with the target cells.
Thermodynamics of Energy Transduction in Biological Membranes
The present invention is directed to perturbing cell membrane biological
thermodynamics (bioenergetics) and the consequent diminished capacity of the
irradiated cells to adequately undergo normal energy transduction and energy
transformation.
-29-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The methods and systems of the present invention optically alter and modify
Td-plas-mam, Wd-mito-mam, tYd-plas-fungi, Td-mito-fungi and tYd-plas-bact to
set in
motion further alterations of OtY and Ap in the same membranes. This is caused
by the
targeted near infrared irradiation of the C-H covalent bonds in the long chain
fatty acids
of lipid bilayers, causing a variation in the dipole potential Td.
To aid with an understanding of the process of this bioenergetic modification,
the following description of the application of thermodynamics to membrane
bioenergetics and energy transduction in biological membranes is presented. To
begin,
membranes (lipid bilayers, see, Figure 1) possess a significant dipole
potential qfd
arising from the structural association of dipolar groups and molecules,
primarily the
ester linkages of the phospholipids (Figure 2) and water. These dipolar groups
are
oriented such that the hydrocarbon phase is positive with respect to the outer
membrane regions (Figure 3). The degree of the dipole potential is usually
large,
typically several hundreds of millivolts. The second major potential, a
separation of
charge across the membrane, gives rise to the trans-membrane potential AT. The
trans-
membrane potential is defined as the electric potential difference between the
bulk
aqueous phases at the two sides of the membrane and results from the selective
transport of charged molecules across the membrane. As a rule, the potential
at the
cytoplasm side of cell membranes is negative relative to the extracellular
physiological
solution (Figure 4A).
The dipole potential Td constitutes a large and functionally important part of
the electrostatic potential of all plasma and mitochondrial membranes. Td
modifies the
electric field inside the membrane, producing a virtual positive charge in the
apolar
bilayer center. As a result of this "positive charge", lipid membranes exhibit
a
substantial (e.g., up to six orders of magnitude) difference in the
penetration rates
between positively and negatively charged hydrophobic ions. Td also plays an
important role in the membrane permeability for lipophilic ions.
Numerous cellular processes, such as binding and insertion of proteins
(enzymes), lateral diffusion of proteins, ligand-receptor recognition, and
certain steps in
membrane fusion to endogenous and exogenous molecules, critically depend on
the
-30-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
physical properties Yrd of the membrane bilayer. Studies in model membrane
systems
have illustrated the ability of mono- and multivalent ions to cause isothermal
phase
transitions in pure lipids, different phase separations, and a distinct
clustering of
individual components in mixtures. In membranes, changes such as these can
exert
physical influences on the conformational dynamics of membrane-embedded
proteins
and cytochromes (Figure 4B), and more specifically, on proteins that go
through large
conformational rearrangements in their transmembrane domains during their
operating
cycles (Figure 5). Most importantly, changes in Td is believed to modulate
membrane
enzyme activities.
Energy Transduction
The energy transduction in biological membranes generally involves three
interrelated mechanisms:
1) The transduction of redox energy to "free energy" stored in a trans-
membrane ionic
electrochemical potential also called the membrane proton electrochemical
gradient
A H'. This proton electrochemical potential difference between the two sides
of a
membrane that engage in active transport involving proton pumps is at times
also
called a chemiosmotic potential or proton motive force.
2) In mammalian cells, the (Na') ion electrochemical gradient A x* is
maintained across
the plasma membrane by active transport of (Na+) out of the cell. This is a
different
gradient than the proton electrochemical potential, yet is generated from a
(pump) via
the ATP produced during oxidative phosphorylation from the Mammalian
Mitochondrial Proton-motive force Ap-mito-mam.
3) The use of this "free energy" to create ATP (energy transformation) to
impel active
transport across membranes with the concomitant buildup of required sohxtes
and
metabolites in the cell is termed the phosphorylation potential AGp. In other
words,
AGp is the AG for ATP synthesis at any given set of ATP, ADP and Pi
concentrations.
Steady-state trans-membrane potential (AT-steady)
-31-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The state of a membrane "system" is in equilibrium when the values of its
chemical potential gradient A H* and E (energy) are temporally independent and
there
is no flux of energy across the margins of the system. If the membrane system
variables
of A H' and E are constant, but there is a net flux of energy moving across
the system,
then this membrane system is in a steady-state and is temporally dependent.
It is this temporally dependent steady-state trans-membrane and/or
mitochondrial potential (ALY-steady) of a cell (a respiring, growing and
dividing cell)
that is of focus. This "steady-state" of the flow of electrons and protons, or
Na'/K' ions
across a mitochondrial or plasma membrane dtiring normal electron transport
and
oxidative phosphorylation, would most likely continue into the future, if
unimpeded by
an endogenous or exogenous event. Any exogenous modification of the membrane
thermodynamics, would bring about a transient-state trans-membrane and/or
mitochondrial potential OLP-trans, and this change from AT-steady to Atp-trans
is an
object of the present invention.
Mathematical relationships between the state variables AT-steady and AT-trans
are called equations of state. In thermodynamics, a state function (state
quantity), is a
property or a system that depends only on the current state of the system. It
does not
depend on the way in which the system attained its particular state. The
present
invention facilitates a transition of state in a trans-membrane and/or
mitochondrial
potential AT, in a temporally dependent manner, to move the bioenergetics of a
membrane from a thermodynamic steady-state condition AT-steady to one of
energy
stress and/or redox stress in a transition state DT-trans.
This can occur in AW-steady-mam, 0T-steady-fungi, AT-steady-Bact-AlY-
steady-mita=mam and 0T-steady-mito-fungi. Not wishing to be bound by theory,
it is
believed that this transition is caused by the targeted near infrared
irradiation of the C-
H covalent bonds in the long chain fatty acids of lipid bilayers (with 930 nm
wavelength), causing a variation in the dipole potential tYd, and the targeted
near
infrared irradiation of cytochrome chains (with X of 870 nm), that will
concurrently alter
AT-steady and the redox potential of the membranes.
-32-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The First Law of Thermodynamics and Mecnbranes
An elemental aspect of the First Law of Thermodynamics (which holds true for
membrane systems) is that the energy of an insulated system is conserved and
that heat
and work are both considered as equivalent forms of energy. Hence, the energy
level of
a membrane system (lYd and ALP ) can be altered by an increase or decrease of
mechanical work exerted by a force or pressure acting, respectively, over a
given
distance or within an element of volume; and/or non-destructive heat
transmitted
through a temperature gradient in the membrane.
This law (the law of conservation of energy), posits that the total energy of
a
system insulated from its surroundings does not change. Thus, addition of any
amounts
of (energy) heat and work to a system must be reflected in a change of the
energy of the
system.
Absorption of infrared radiation
The individual photons of infrared radiation do not contain sufficient energy
(e.g., as measured in electron-volts) to induce electronic transitions (in
molecules) as is
seen with photons of ultraviolet radiation. Because of this, absorption of
infrared
radiation is limited to compounds with small energy differences in the
possible
vibrational and rotational states of the molecular bonds.
By definition, for a membrane bilayer to absorb infrared radiation, the
vibrations
or rotations within the lipid bilayer's molecular bonds that absorb the
infrared photons,
must cause a net change in the dipole potential of the membrane. If the
frequency
(wavelength) of the infrared radiation matches the vibrational frequency of
the
absorbing molecule (i.e., C-H covalent bonds in long chain fatty acids) then
radiation
will be absorbed causing a change in YJd. This can happen in Td-plas-mam, tiYd-
mito-
mam, Td-plas-fungi, Td-mito-fungi and kFd-plas-bact. In other words, there can
be a
direct and targeted change in the enthalpy and entropy (AH and AS) of all
cellular lipid
bilayers with the methods and systems described herein.
-33-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The present invention is based upon a combination of insights that have been
introduced above and are derived in part from empirical data, which include
the
following:
It has been appreciated that the unique, single wavelengths (870 nm and 930
nm)
are capable of killing bacterial cells (prokaryotes) such as E.co1i and
(eukaryotes) such as
Chinese Hela Ovary hampster cells (CHO), as a result of the generation and
interaction
of ROS and toxic singlet oxygen reaction. See, e.g., U.S. Application Serial
No.
10/649910 filed 26 August 2003 and U.S. Application Serial No. 10/776106 filed
11
February 2004, the entire teachings of which are incorporated herein by
reference.
With such NIMEL systems, it has been established that instead of avoiding the
individual 870 nm and 930 nm wavelengths, the laser system and process of the
present
invention (NIMEL system) combine the wavelengths at 51og less power density
than is
typically found in a confocal laser microscope such as that used in optical
traps (- to
500,000 w/cmz less power) to advantageously exploit the use of such
wavelengths for
therapeutic laser systems.
This is done for the expressed purpose of alteration, manipulation and
depolarization of the AT-steady-mam, AtY-steady-fungi, ALY-steady-Bact, AT-
steady-
mito-mam and AIV-steady-mito-fungi of all cells within the irradiation field.
This is
accomplished in the present invention by the targeted near infrared
irradiation of the C-
H covalent bonds in the long chain fatty acids of lipid bilayers (with 930 nm
energy),
resulting in a variation in the dipole potentials Td-plas-mam, Td-rnito-mam,
Td-
plas-fungi, Td-mito-fungi and LPd-plas-bact of all biological membranes within
the
irradiation field. Secondly, the near infrared irradiation of cytochrome
chains (with 870
nm), will additionally alter ALY-steady and the redox potential of the
membranes that
have cytochromes (i.e., bacterial plasma membranes, and fungal and mammalian
mitochondria).
Serving as direct chromophores (cytochromes and C-H bonds in long chain fatty
acids), there will be a direct enthalpy and entropy change in the molecular
dynamics of
membrane lipids and cytochromes for all cellular lipid bilayers in the
irradiation path of
the present invention. This will alter each membrane dipole potential Td, and
-34-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
concurrently alter the absolute value of the membrane potential AT, of all
membranes
in the irradiated cells.
These changes occur through significantly increased molecular motions (viz.
AS)
of the lipids and metallo-protein reaction centers of the cytochromes, as they
absorb
energy from the NIMEL system in a linear one-photon process. As even a small
thermodynamic shift in either the lipid bilayer and/or the cytochromes would
be
enough to change the dipole potential Td, the molecular shape (and hence the
enzymatic reactivity) of an attached electron transport protein, or trans-
membrane
protein would be rendered less functional. This will directly affect and
modify the AT
in all membranes in the irradiated cells.
The NIMELS effect occurs in accordance with methods and systems described
herein, importantly, without thermal or ablative mechanical damage to the cell
membranes. This combined and targeted low dose approach is a distinct
variation and
improvement from existing methods that would otherwise cause actual mechanical
damage to all membranes within the path of a beam of energy.
Membrane Entropy and the Second Law of Thermodynamics
The conversion of heat into other forms of energy is never perfect, and
(according to the Second Law of Thermodynamics) must always be accompanied by
an
increase in entropy. Entropy (in a membrane) is a state function whose change
in a
reaction describes the direction of a reaction due to changes in (energy) heat
input or
output and the associated molecular rearrangements.
Even though heat and mechanical energy are equivalent in their fundamental
nature (as forms of energy), there are limitations on the ability to convert
heat energy
into work. i.e., too much heat can permanently damage the membrane
architecture and
prevent work or beneficial energy changes in either direction.
The NIMELS effect will modify the entropy "state" of irradiated cells at the
level
of the lipid bilayer in a temporally dependent manner. This increase in
entropy will
alter the Td of all irradiated membranes (mitochondrial and plasma) and hence,
thermodynamically alter the "steady-state" flow of electrons and protons
across a cell
-35-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
membrane (Figures 6 and 7). This will in turn change the steady-state trans-
membrane
potential AT-steady to a transient-state membrane potential (AT-tran). This
phenomenon will occur in:
1) Mammalian Plasma Trans-membrane Potential 0T-plas-mam;
2) Fungal Plasma Trans-membrane Potential AT-plas-fungi;
3) Bacterial Plasma Trans-membrane Potential AT-plas-bact;
4) Mammalian Mitochondrial Trans-membrane Potential AtY-mito-mam; and
5) Fungal Mitochondrial Trans-membrane PotentialOiP-mito-fungi.
This is a direct result of the targeted enthalpy change at the level of the C-
H
bonds of the long chain fatty acids in the fluid mosaic membrane, causing a
measure of
dynamic disorder (in the membrane) which can alter the membranes corporeal
properties. This fluid mosaic increases in entropy and can disrupt the
tertiary and
quaternary properties of electron transport proteins, cause redox stress,
energy stress
and subsequent generation of ROS, that will further damage membranes and
additionally alter the bioenergetics.
Since a prime function of the electron transport system of respiring cells is
to
transduce energy under steady-state conditions, techniques according to the
present
invention are utilized to temporarily, mechano-optically uncouple many of the
relevant
thermodynamic interactions on that transduction process. This can be done with
the
express intent of altering the absolute quantitative value of the proton
electrochemical
gradient A H' and proton-motive force and Ap of the membranes. This phenomenon
can occur, inter alia, in:
1) Mammalian Mitochondrial Proton-motive force (Ap-mito-mam);
2) Fungal Mitochondrial Proton-motive force (Ap-mito-Fungi);
3) Fungal Plasma Membrane Proton-motive force (Ap-plas-Fungi); and
4) Bacterial Plasma Membrane Proton-motive force (Ap-plas-Bact).
Such phenomena can in turn decrease the Gibbs free energy value 4G available
for the phosphorylation and synthesis of ATP (AGp). The present invention
carries out
-36-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
these phenomena in order to inhibit the necessary energy dependent anabolic
reactions,
potentiating pharmacological therapies, and/or lowering cellular resistance
mechanisms
(to antimicrobial, antifungal and antineoplastic molecules) as many of these
resistance
mechanisms make use of the proton motive force or the chemiosmotic potential
for their
energy needs, to resist and/or efflux these molecules.
Free Radical Generation in consequence of modifications of 0T-steady
The action of chemical uncouplers for oxidative phosphorylation and other
bioenergetic work is believed to depend on the energized state of the membrane
(plasma or mitochondrial). Further, it is believed that the energized state of
the
bacterial membrane or eukaryotic mitochondrial inner membrane, is an
electrochemical
proton gradient A H* that is established by primary proton translocation
events
occurring during cellular respiration and electron transport.
Agents that directly dissipate (depolarize) the A H*, (e.g., by permeabilizing
the
coupling membrane to the movement of protons or compensatory ions) short-
circuits
energy coupling, and inhibit bioenergetic work, by inducing a reduction in the
membrane potential DT-steady. This will occur while respiration (primary
proton
translocation) continues apace.
For example, the classic uncoupler of oxidative phosphorylation, carbonyl
cyanide m-chlorophenylhydrazone (CCCP), induces a reduction in membrane
potential
ALP-steady and induces a concomitant generation of ROS, as respiration
continues.
These agents (uncouplers) generally cannot be used as antimicrobials,
antifungals, or
antineoplastics, because their effects are correspondingly toxic to all
bacterial, fungal
and mammalian cells.
However, it has been shown that in many target cells that are resistant to
antimicrobials, antifungals, or antineoplastics, a Ap uncoupler (like CCCP)
will collapse
the energy gradient required for an efflux pump and hence induce a strong
increase in
the intracellular accumulation of these drtzgs. These results clearly indicate
that some
resistance mechanisms (such as drug efflux pumps) are driven by the proton
motive
force. If there were a way to harness this effect to uniquely achieve only
"target cell"
-37-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
damage, this selectivity would be a clear improvement upon the universal
damaging
nature of uncouplers.
The scientific findings and experimental data of the present invention show
that
as a membrane is depolarized optically, the generation of ROS may well
fi.trther
potentiate the depolarization of affected cells, and further potentiate the
antibacterial
effects of the present invention. (See, Example VIII).
Free radical and ROS generation by irradiation with the NIMELS laser
By mechano-optically modifying many of the relevant thermodynamic
interactions of the membrane energy transduction process, along with altering
AT-
steady, the present invention can act as an optical uncoupler by lowering the
A H' and
Ap of the following irradiated membranes:
1) Mammalian Mitochondrial Proton-motive force (Ap-mito-mam)
2) Fungal Mitochondrial Proton-motive force (Ap-mito-Fungi)
3) Fungal Plasma Membrane Proton-motive force (Ap-plas-Fungi)
4) Bacterial Plasma Membrane Proton-motive force (Ap-plas-Bact)
This lowered 4p will cause a series of free radicals and radical oxygen
species to
be generated because of the altered redox state. The generation of free
radicals and
reactive oxygen species has been proven experimentally and described herein
with the
alteration of AT-steady to A\11-trans in the following (see, Example VIII):
1) AT-steady-mam + (NIMELS Treatment) AT-trans-mam
2) 4tiY-steady-fungi + (NIMELS Treatment) AIF-trans-fungi
3) 41IY-steady-bact + (NIMELS Treatment) ->-> OLY-trans-bact
4) AT-mito-fungi + (NIMELS Treatment) ~~ AT-trans-mito-fungi
5) 4LY-mito-mam + (NIMELS Treatnient) 4 4 ALY-trans-mito-mam
The altered redox state and generation of free radicals and ROS because of the
4T-steady + (NIMELS Treatment) 44 4T- trans phenomenon, can cause serious
further damage to biological membranes such as lipid peroxidation.
-38-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Lioid peroxidation
Lipid peroxidation is a prevalent cause of biological cell injury and death in
both
the microbial and mammalian world. In this process, strong oxidents cause the
breakdown of membrane phospholipids that contain polyunsaturated fatty acids
(PUFA's). The severity of the membrane damage can cause local reductions in
membrane fluidity and full disruption of bilayer integrity.
Peroxidation of mitochondrial membranes (mamallian cells and fungi) will have
detrimental consequences on the respiratory chains resulting in inadequate
production
of ATP and collapse of the cellular energy cycle. Peroxidation of the plasma
membrane
(bacteria) can affect membrane permeability, disfunction of membrane proteins
such as
porins and efflux pumps, inhibition of signal transduction and improper
cellular
respiration and ATP formation (i.e., the respiratory chains in prokaryotes are
housed in
the plasma membranes as prokaryotes do not have mitochondria).
Free radical
A free radical is defined as an atom or molecule that contains an unpaired
electron. An example of the damage that a free radical can do in a biological
environment is the one-electron (via an existing or generated free radical)
removal from
bis-allylic C-H bonds of polyunsaturated fatty acids (PUFAs) that will yield a
carbon
centered free radical.
R* + (PUFA)-CH(bis-allylic C-H bond) ~ (PUFA)-C* + RH
This reaction can initiate lipid peroxidation damage of biological membranes.
A free radical can also add to a nonradical molecule, producing a free radical
product.
(A* + B-> A-B*) or a nonradical product (A*+B 4 A-B)
An example of this would be the hydroxylation of an aromatic compound by *OH.
Reactive Oxygen Species (ROS)
-39-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Oxygen gas is actually a free radical species. However, because it contains
two
unpaired electrons in different rT-anti-bonding orbitals that have parallel
spin in the
ground state, the (spin restriction) rule generally prevents 02 from receiving
a pair of
electrons with parallel spins without a catalyst. Consequently 02 must receive
one
electron at a time.
There are many significant donors in a cell (prokaryotic and eukaryotic) that
are
able to stimulate the one-electron reduction of oxygen, that will create an
additional
radical species.
These are generally categorized as:
The Superoxide ion radical (Oz )
Hydrogen Peroxide (non-radical) (H202)
Hydroxyl radical ('OH)
Hydroxy ion (OH-)
The Reaction Chain is:
02 --> 02- + 2H* --> HzOz -> OH- + *OH 4 OH-
(e-) (e-) (e-) (e-)
Superoxide
The danger of these molecules to cells is well categorized in the literature.
Superoxide, for example, can either act as an oxidizing or a reducing agent.
NADH 4 NAD'
Of higher importance to an organism's metabolism, superoxide can reduce
cytochrome C. It is generally believed that the reaction rates of superoxide
(Oz=) with
lipids (i.e., membranes) proteins, and DNA are too slow to have biological
significance.
The protonated form of superoxide hydroperoxyl radical (HOO*) has a lower
reduction
potential than (Oz ), yet is able to remove hydrogen atoms from PUFA's. Also
of note,
the pKa value of (HOO*) is 4.8 and the (acid) microenvironment near
biologiocal
membranes will favor the formation of hydroperoxyl radicals. Furthermore, the
-40-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
reaction of superoxide (02-) with any free Fe+3 will produce a "perferryl"
intermediate
which can also react with PUFA's and induce lipid (membrane) peroxidation.
Hydrogen Peroxide
Hydrogen peroxide (HzOz) is not a good oxidizing agent (by itself) and cannot
remove hydrogen from PUFA's. It can, however, cross biological membranes
(rather
easily) to exert dangerous and harmful effects in other areas of cells. For
example,
(HxOz) is highly reactive with transition metals inside microcellular
environments, (such
as Fe+z and Cu*) that can then create hydroxyl radicals (*OH) (known as the
Fenton
Reaction). An hydroxyl radical is one of the most reactive species known in
biology.
Hydroxyl Radical
Hydroxyl radicals (*OH) will react with almost all kinds of biological
molecules.
It has a very fast reaction rate that is essentially controlled by the
hydroxyl radical
(*OH) diffusion rate and the presence (or absence) of a molecule to react near
the site of
(*OH) creation. In fact, the standard reduction potential (E0') for hydroxyl
radical (*OH)
is (+2.31 V) a value that is 7x greater than (HzOz), and is categorized as the
most reactive
among the biologically relevant free radicals. Hydroxyl radicals will initiate
lipid
peroxidation in biological membranes, in addition to damaging proteins and
DNA.
Reactive Oxygen Species Created from the Peroxidation of PUFAs
Furthermore, the development of lipid peroxidation (from any source) will
result in the genesis of three other reactive oxygen intermediate molecules
from
PUFA's.
(a) alkyl hydroperoxides (ROOH);
Like H202, alkyl hydroperoxides are not technically radical species but are
unstable in
the presence of transition metals such as such as Fe+z and Cu".
(b) alkyl peroxyl radicles (ROO*); and
(c) alkoxyl radicles (RO*).
-41-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Alkyl peroxyl radicles and alkoxyl radicles are extremely reactive oxygen
species and also contribute to the process of propagation of further lipid
peroxidation.
The altered redox state of irradiated cells and generation of free radicals
and ROS
because of the AW-steady + (NIMELS Treatment) 4 -) ALY- trans phenomenon is
another
object of the present invention. This is an additive effect to further alter
cellular
bioenergetics and inhibit necessary energy dependent anabolic reactions,
potentiate
pharmacological therapies, and/or lower cellular resistance mechanisms to
antimicrobial, antifungal and antineoplastic molecules.
ROS overproduction can damage cellular macromolecules, above all lipids.
Lipid oxidation has been shown to modify both the small-scale structural
dynamics of
biological membranes as well as their more macroscopic lateral organization
and altered
a packnlg density dependent reorientation of the component of the dipole
moment tYd.
Oxidative damage of the acyl chains (in lipids) causes loss of double bonds,
chain
shortening, and the introduction of hydroperoxy groups. Hence, these changes
are
believed to affect the structural characteristics and dynamics of lipid
bilayers and the
dipole potential LYd.
Antimicrobial Resistance
Antimicrobial resistance is defined as the ability of a microorganism to
survive
the effects of an antimicrobial drug or molecule. Antimicrobial resistance can
evolve
naturally via natural selection, through a random mutation, or through genetic
engineering. Also, microbes can transfer resistance genes between one another
via
mechanisms such as plasmid exchange. If a microorganism carries several
resistance
genes, it is called multi-resistant or, informally, a "superbug."
Multi-drug resistance in pathogenic bacteria and fiingi are a serious problem
in
the treatment of patients infected with such organisms. At present, it is
tremendously
expensive and difficult to create or discover new antimicrobial drugs that are
safe for
human use. Also, there have been resistant mutant organisms that have evolved
challenging all known antimicrobial classes and mechanisms. Hence, few
antimicrobials
-42-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
have been able to maintain their long-term effectiveness. Most of the
mechanisms of
antimicrobial drug resistance are known.
The four main mechanisms by which micro-organisms exhibit resistance to
antimicrobials are:
a) Drug inactivation or modification;
b) Alteration of target site;
c) Alteration of metabolic pathway; and
d) Reduced drug accumulation: by decreasing drug permeability and/or
increasing
active efflux on the cell surface.
Resistant Microbe Example
Staphylococcus aureus (S. aureus) is one of the major resistant bacterial
pathogens currently plaguing humanity. This gram positive bacterium is
primarily
found on the mucous membranes and skin of close to half of the adult world-
wide
population. S. aureus is extremely adaptable to pressure from all known
classes of
antibiotics. S. aureus was the first bacterium in which resistance to
penicillin was found
in 1947. Since then, almost complete resistance has been found to methicillin
and
oxacillin. The "superbug" MRSA (methicillin resistant Staphylococcus aureus)
was first
detected in 1961, and is now ubiquitous in hospitals and communities
worldwide.
Today, more than half of all S. aureus infections in the United States are
resistant to
penicillin, methicillin, tetracycline and erythromycin. Recently, in what were
the new
classes of antibiotics (antimicrobials of last resort) glycopeptides and
oxazolidinones,
there have been reports of significant resistance (Vancomycin since 1996 and
Zyvox
since 2003).
A new variant CA-MRSA, (community acquired MRSA) has also recently
emerged as an epidemic, and is responsible for a group of rapidly progressive,
fatal
diseases including necrotizing pneumonia, severe sepsis and necrotizing
fasciitis.
Outbreaks of community-associated (CA)-MRSA infections are reported daily in
correctional facilities, athletic teams, military recruits, in newborn
nurseries, and among
-43-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
active homosexual men. CA-MRSA infections now appear to be almost endemic in
many urban regions and cause most CA-S. aureus infections.
The scieniific and medical community has been attempting to find potentiators
of existing antimicrobial drugs and inhibitors of drug resistance systems in
bacteria and
fungi. Such potentiators and/or inhibitors, if not toxic to humans, would be
very
valuable for the treatment of patients infected with pathogenic and drug-
resistant
microbes. In the United States, as many as 80% of individuals are colonized
with S.
aureus at some point. Most are colonized only intermittently; 20-30% are
persistently
colonized. Healthcare workers, persons with diabetes, and patients on dialysis
all have
higher rates of colonization. The anterior nares are the predominant site of
colonization
in adults; other potential sites of colonization include the axilla, rectum,
and perineum.
Selective Pharmacological Alteration of AT-steady in bacteria
There is a relatively new class of bactericidal antibiotics called the
lipopeptides
of which daptomycin is the first FDA approved member. This antibiotic has
demonstrated (in vitro and in vivo) that it can rapidly kill virtizally all
clinically relevant
gram-positive bacteria (such as MRSA) via a mechanism of action distinct from
those of
other antibiotics on the market at present.
Daptomycin's mechanism of action involves a calcium-dependent incorporation
of the lipopeptide compound into the cytoplasmic membrane of bacteria. On a
molecular level, it is calcium binding between two aspartate residues (in the
daptomycin molecule) that decreases its net negative charge and permits it to
better act
with the negatively charged phospholipids that are typically found in the
cytoplasmic
membrane of gram-positive bacteria. There is generally no interaction with
fungi or
mammalian cells at therapeutic levels, so it is a very selective molecule.
The effects of daptomycin have been proposed to result from this calcium-
dependent action on the bacterial cytoplasmic membrane that dissipates the
transmembrane membrane electrical potential gradient A H'. This is in effect
selective
chemical depolarization of only bacterial membranes. It is well known that the
maintenance of a correctly energized cytoplasmic membrane is essential to the
survival
-44-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
and growth of bacterial cells, nevertheless depolarization (in this manner) is
not in and
of itself a bacterially lethal action. For example, the antibiotic
valinomycin, which causes
depolarization in the presence of potassium ions, is bacteriostatic but not
bactericidal as
would be the case with CCCP.
Conversely, in the absence of a proton motive force Ap, the main component of
which is the transmembrane electrical potential gradient A H', cells cannot
make ATP
or take up necessary nutrients needed for growth and reproduction. The
collapse of
A H' explains the dissimilar (detrimental) effects produced by daptomycin
(e.g.,
inhibition of protein, RNA, DNA, peptidoglycan, lipoteichoic acid, and lipid
biosynthesis).
Further research into the prior-art concerning the drug daptomycin, suggests
that the addition of gentamicin or minocycline (to daptomycnl) results in the
enhancement of its bactericidal activity against MRSA. As both gentamicin and
minocycline can be effluxed out of MRSA cells through energy dependent pumps,
and
are inhibitors of protein synthesis (an anabolic function) at the level of the
30S bacterial
ribosome. This indicates that dissipation of the transmembrane electrical
potential
gradient A H' by daptomycin can potentiate certain antimicrobial drugs. This
should
occur as a result of resistance mechanisms that are rendered less useful by a
reduction
in the membrane potential AT and the fact that ATP is not available (i.e., the
concomitant lowered d Gp) for the anabolic function of protein synthesis.
Based on the above, it would clearly be desirable to be able to optically
inhibit
the activity of drug efflux pumps and/or anabolic reactions in target cells by
safely
reducing the membrane potential AT (0T-steady+ (NIMELS Treatment) 3--> AT-
trans) of the cells in a given target area. Methods according to the present
invention
accomplish this and other tasks with the use of selected infrared wavelengths,
e.g., 870
nm and 930 nm, independent of any exogenous chemical agents such as
daptomycin.
This is a clear improvement over the existing prior art methods that require a
systemic
drug to accomplish the same task.
Multidrug resistance efflux pumps
-45-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Multidrug resistance efflux pumps are now known to be present in gram-
positive bacteria, gram-negative bacteria, fungi, and cancer cells. Efflux
pumps
generally have a poly-specificity of transporters that confers a broad-
spectrum of
resistance mechanisms. These can strengthen the effects of other mechanisms of
antimicrobial resistance such as mutations of the antimicrobial targets or
enzymatic
modification of the antimicrobial molecules. Active efflux for antimicrobials
can be
clinically relevant for (3-lactam antimicrobials, marcolides,
fluoroquinolones,
tetracyclines and other important antibiotics, along with most antifungal
compounds
including itraconazole and terbinafine.
With efflux pump resistance, a microbe has the capacity to seize an
antimicrobial
agent or toxic compound and expel it to the exterior (environment) of the
cell, thereby
reducing the intracellular accumulation of the agent. It is generally
considered that the
over-expression of one or more of these efflux pumps prevents the
intracellular
accumulation of the agent to thresholds necessary for its inhibitory activity.
Universally
in microbes, the efflux of drugs is coupled to the proton motive force that
creates
electrochemical potentials and/or the energy necessary (ATP) for the needs of
these
protein pumps. This includes:
1) Mammalian mitochondrial proton-motive force (Ap-mito-mam);
2) Fungal mitochondrial proton -motive force (Op-mito-fungi);
3) Ftmgal plasma membrane proton-motive force (Ap-plas-fungi); and
4) Bacterial plasma membrane proton-motive force (Ap-plas-bact).
Phylogenetically, bacterial antibiotic efflux pumps belong to five
superfamilies:
(i) ABC (ATP-binding cassette), which are primary active transporters
energized by
ATP hydrolysis;
(ii) SMR [small multidrug resistance subfamily of the DMT (drug/metabolite
transporters) superfamily];
(iii) MATE [multi-antimicrobial extrusion subfamily of the MOP
(multidrug/oligosaccharidyl-lipid/polysaccharide flippases) superfamily];
(iv) MFS (major facilitator superfamily); and
-46-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
(v) RND (resistance/nodulation/division superfamily), which are all secondary
active transporters driven by ion gradients.
The approach of the current invention to inhibit efflux pumps is a general
modification (optical depolarization) of the membranes ALY within the
irradiated area,
leading to lower electrochemical gradients that will lower the phosphorylation
potential
OGp and energy available for the pumps functional energy needs. It is also the
object of
the present invention to have the same photobiological mechanism inhibit the
many
different anabolic and energy driven mechanisms of the target cells, including
absorption of nutrients for normal growth.
Reduction of efflux pump energ-y: Targeting the driving force of the mechanism
Today, there are no drugs that belong to the "energy-blocker" family of
molecules that have been developed for clinical use as efflux pump inhibitors.
There are a couple of molecules that have been found to be "general"
inhibitors of efflux
pumps. Two such molecules are reserpine and verapamil. These molecules were
originally recognized as inhibitors of vesicular monoamine transporters and
blockers of
transmembrane calcium entry (or calcium ion antagonists), respectively.
Verapamil is
known as an inhibitor of MDR pumps in cancer cells and certain parasites and
also
improves the activity of tobramycin.
Reserpine inhibits the activity of Bmr and NorA, two gram-positive efflux
pumps, by altering the generation of the membrane proton-motive force Ap
required for
the function of MDR efflux pumps. Although these molecules are able to inhibit
the
ABC transporters involved in the extrusion of antibiotics (i.e.,
tetracycline), the
concentrations necessary to block bacterial efflux are neurotoxic in humans.
To date,
there has been no mention in the literature of similar experiments with
daptomycin.
Fungal drug efflux is mediated primarily by two groups of membrane-bound
transport
proteins: the ATP-binding cassette (ABC) transporters and the major
facilitator
superfamily (MFS) pumps.
-47-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Bacterial Plasma Trans-membrane Potential OlP-plas-bact and cell wall
synthesis
During normal cellular metabolism, protons are extruded through the
cytoplasmic membrane to form AT-plas-bact. This function also acidifies (lower
pH) the
narrow region near the bacterial plasma membrane. It has been shown in the
gram
positive bacterium Bacillus subtilis, that the activities of peptidoglycan
autolysins are
increased (i.e., no longer inhibited) when the electron transport system was
blocked by
adding proton conductors. This suggests that 0IP-plas-bact and A H'
(independent of
storing energy for cellular enzymatic functions) potentially has a profound
and
exploitable influence on cell wall anabolic functions and physiology.
In addition, it has been shown that 0T-plas-bact uncouplers inhibit
peptidoglycan formation with the accumulation of the nucleotide precursors
involved
in peptidoglycan synthesis, and the inhibition of transport of N-
acetylglucosamine
(GlcNAc), one of the major biopolymers in peptidoglycan.
Also, there is reference to an antimicrobial compound called tachyplesin that
decreases AT-plas-bact in gram positive and gram negative pathogens.
(Antimicrobial
compositions and pharmaceutical preparations thereof United States Patent
5,610,139,
the entire teaching of which is incorporated herein by reference.) This
compound was
shown at sub-lethal concentrations to have the ability to potentiate the cell
wall
synthesis inhibitor (3-lactam antibiotic ampicillin in MRSA. It is desirable
to couple the
multiple influences of an optically lowered AtY-plas-bact (i.e., increased
cell wall
autolysis, inhibited cell wall synthesis, and cell wall antimicrobial
potentiation) to any
other relevant antimicrobial therapy that targets bacterial cell walls. This
is especially
relevant in gram positive bacteria such as MRSA that do not have efflux pumps
as
resistance mechanisms for cell wall inhibitory antimicrobial compounds.
Cell wall inhibitory compounds do not need to gain entry through a membrane
in gram positive bacteria, as is necessary with gram negative bacteria, to
exhibit effects
against the cell wall. Experimental evidence has proven (see, Example XII)
that the
NIMELS laser and its concomitant optical AlY-plas-bact lowering phenomenon is
synergistic with cell wall inhibitory antimicrobials in MRSA. This must
function via the
inhibition of anabolic (periplasmic) ATP coupled functions, as MRSA does not
have
-48-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
efflux pumps that function on peptidoglycan inhibitory antimicrobials, as they
do not
need to enter the cell to be effective.
4W-plas-fungi and 4W-mito-fungr: Necessities for correct cellular function and
antifungal
resistance
During normal cellular metabolism AT-mito-fungi is generated in the
mitochondria via the electron transport system that then generates ATP via the
mitochondrial ATP synthase enzyme system. It is the ATP that then powers the
plasma
membrane-bound H'-ATPase to generate 0T-plas-fungi. It has previously been
found
that fungal mitochondrial ATP synthase is inhibited by the chemical,
polygodial, in a
dose-dependent manner (Lunde and Kubo, Antimicrob Agents Chemother. 2000 July;
44(7): 1943-1953, the entire teaching of which is incorporated herein by
reference.) It
was further found that this induced reduction of the cytosolic ATP
concentration leads
to a suppression of the plasma membrane-bound H*-ATPase that generates OlY-
plas-
fungi, and that this impairment further weakens other cellular activities.
Additionally,
the lowering of the AW-plas-fungi will cause plasma membrane bioenergetic and
thermodynamic disruption leading to an influx of protons that collapses the
proton
motive force and hence inhibits nutrient uptake.
Of further importance, ATP is necessary for the biosynthesis of the fungal
plasma membrane lipid ergosterol. Ergosterol is the structural lipid that is
targeted by
the majority of relevant commercial antifungal compounds used in medicine
today (i.e.,
azoles, terbinafine and itraconazole).
Studies have shown that two antimicrobial peptides (Pep2 and Hst5) have the
ability to cause ATP to be effluxed out of fungal cells (i.e., depleting
intracellular ATP
concentrations) and that this lowered cytosolic ATP causes the inactivation of
ABC
transporters CDRI and CDR2 which are ATP-dependent efflux pumps of antifungal
agents.
There is an advantage to using an optical method to depolarize membranes and
deplete cellular ATP in fungus, as a potentiator to efflux pump inhibition and
anabolic
reactions. Hence, it would be desirable to optically alter either the AT-plas-
fungi and/or
-49-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
AT-mito-fungi to inhibit necessary cellular functions, ATP generation, and
potentiate
antifungal compounds.
Therefore, one of the strategies for preventing drug resistance (via efflux
pumps)
is to decrease the level of intracellular ATP which induces inactivation of
the ATP-
dependent efflux pumps. In fungal pathogens, there have been no acceptable
chemical
agents to accomplish this task. The NIMELS effect however has the ability to
accomplish
this goal optically, and experimental evidence has demonstrated that the
NIMELS laser
and phenomenon in fungi, is synergistic with antifungal compounds. (See,
Example
XIII).
This NIMELS effect will occur in accordance with methods and systems
disclosed herein, without causing thermal or ablative mechanical damage to the
cell
membranes. This combined and targeted low dose approach is a distinct
variation and
improvement from all existing methods that would otherwise cause actual
mechanical
damage to all membranes within the path of a beam of energy.
In a first aspect, the invention provides a method of modifying the dipole
potential Td of all membranes within the path of a NIMELS beam (Td-plas-mam,
Td-
mito-mam, Td-plas-fungi, LYd-mito-fungi, and Td-plas-bact) to set in motion
the
cascade of further alterations of AT and Ap in the same membranes.
The bioenergetic steady-state membrane potentials AT-steady of all irradiated
cells (AT-steady-mam, AT-steady-fungi, AT-steady-Bact, AT-steady-mito-mam and
AT-steady-mito-fungi) are altered to AT-trans values (AtY-trans-mam, AT-trans-
fungi,
0T-trans-Bact, AT-trans-mito-mam and AT-trans-mito-fungi). This results in a
concomitant depolarization and quantifiable alteration in the absolute value
of the Ap
for all irradiated cells (Ap-mito-mam, Ap-mito-Fungi, Ap-plas-Fungi and Ap-
plas-Bact).
These phenomena occur without intolerable risks and/or intolerable adverse
effects to biological subjects (e.g., a mammalian tissue, cell or certain
biochemical
preparations such as a protein preparation) in/at the given target site other
than the
targeted biological contaminants (bacteria and fungi), by irradiating the
target site with
optical radiation of desired wavelength(s), power density level(s), and/or
energy
density level(s).
-50-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
In certain embodiments, such applied optical radiation may have a wavelength
from about 850 nm to about 900 nm, at a NIMELS dosimetry, as described herein.
In
exemplary embodiments, wavelengths from about 865 nm to about 875 nm are
utilized.
In further embodiments, such applied radiation may have a wavelength from
about 905
nm to about 945 nm at a NIMELS dosimetry. In certain embodiments, such applied
optical radiation may have a wavelength from about 925 nm to about 935 nm. In
representative non-limiting embodiments exemplified hereinafter, the
wavelength
employed is 930 nm.
Bioenergetic steady-state membrane potentials may be modified, in exemplary
embodiments, as noted below, and may employ multiple wavelength ranges
including
ranges bracketing 870 and 930 nm, respectively.
The NIMELS Potentiation Magnitude Scale (NPMS)
As discussed in more detail supra, NIMELS parameters include the average
single or additive output power of the laser diodes and the wavelengths (870
nm and
930 nm) of the diodes. This information, combined with the area of the laser
beam or
beams (cmz) at the target site, the power output of the laser system and the
time of
irradiation, provide the set of information which may be used to calculate
effective and
safe irradiation protocols according to the invention.
Based on these novel resistance reversal and antimicrobial potentiation
interactions available with the NIMELS laser, there needs to be a quantitative
value for
the "potentiation effect" that will hold true for each unique antimicrobial
and laser
dosimetry.
A new set of parameters are defined that will take into account the
implementation of any different dosimetric value for the NIMELS laser and any
MIC
value for a given antimicrobial being examined. This can be simply tailored to
the
NIMELS laser system and methods by creating only a set of variables that
quantify
CFU's of pathogenic organisms within any given experimental or treatment
parameter
with the NIMELS system.
-51-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
These parameters create a scale called the NIMELS Potentiation Magnitude Scale
(NPMS) and exploits the NIMELS lasers inherent phenomenon of reversing
resistance
and/or potentiating the MIC of antimicrobial drugs, while also producing a
measure of
safety against burning and injuring adjacent tissues, with power, and/or
treatment time.
The NPMS scale measures the NIMELS effect number (Ne) between I to 10, where
the
goal is to gain a Ne of _ 4 in reduction of CFU count of a pathogen, at any
safe
combination of antimicrobial concentration and NIMELS dosimetry. Although CFU
count is used here for quantifying pathogenic organism, other means of
quantification
such as, for example, dye detection methods or polymerase chain reaction (PCR)
methods can also be i.rsed to obtain values for A, B, and Np parameters.
The NIMELS effect number Ne is an interaction coefficient indicating to what
extent the combined inhibitory/bacteriostatic effect of an antimicrobial drug
is
synergistic with the NIMELS laser against a pathogen target without harm to
healthy
tissue.
The NIMELS potentiation number (Np) is a value indicating whether the
antimicrobial at a given concentration is synergistic, or antagonistic, to the
pathogen
target without harm to healthy tissue. Hence, within any given set of standard
experimental or treatment parameters:
= A CFU Count of pathogen with NIMELS alone;
= B CFU Count of pathogen with antimicrobial alone;
= Np = CFU Count of pathogen with (NIMELS + Antimicrobial); and
= Ne = (A+B) / 2Np;
Interpretation of NIMELS effect number Ne:
where:
If 2Np < A + B then the (given) antimicrobial has been successfully
potentiated with the
NIMELS laser at the employed concentrations and dosimetries.
then:
If Ne =1 then there is no potentiation effect. If Ne > 1 then there is a
potentiation effect.
If Ne ? 2 then there is at least a 50% potentiation effect on the
antimicrobial. If Ne >_ 4
-52-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
then there is at least a 75% potentiation effect on the antimicrobial. If Ne
10 then there
is at least a 90% potentiation effect on the antimicrobial.
Sample calculation 1:
= A=110CFU
= B = 120 CFU
= Np = 75 CFU
= Ne = (110 CFU + 120 CFU) / 2(75) = 1.5
Sample calculation 2:
=A=150CFU
= B = 90 CFU
= Np = 30 CFU
= Ne = (150 CFU + 90 CFU) / 2(30) = 4
In general, it can be advantageoi.is to use a lower dose of antimicrobials
when
treating microbial infections, as the antimicrobials are expensive and by and
large
associated with undesirable side effects that can include systemic kidney
and/or liver
damage. Therefore, it is desirable to devise methods to lower and or
potentiate the MIC
of antimicrobials. The present invention provides systems and methods to
reduce the
MIC of antimicrobial molecules when the area being treated is concomitantly
treated
with the NIMELS laser system.
If the MIC of an antimicrobial is reduced for a localized and resistant focal
infection (e.g., skin, diabetic foot, bedsore), the therapeutic efficacy of
many of the older,
cheaper and safer antimicrobials to treat these infections will be restored.
Therefore,
decreasing the MIC of an antimicrobial, by the addition of the NIMELS laser
(e.g.,
generating a value of Ne that is in one aspect > 1 and in another aspect _ 4
and yet in
another aspect _ 10), represents a positive step forward in restoring the once
lost
therapeutic efficacy of antibiotics.
Therefore, in one aspect, this invention provides methods and systems that
will
reduced the MIC of antimicrobial molecules necessary to eradicate or at least
attenuate
microbial pathogens via a depolarization of membranes within the irradiated
field
which will decrease the membrane potential AT of the irradiated cells. This
weakened
-53-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
4T will cause an affiliated weakening of the proton motive force Ap, and the
associated
bioenergetics of all affected membranes. It is a further object of the present
invention
that this "NIMELS effect" potentiate existing antimicrobial molecules against
microbes
infecting and causing harm to human hosts.
In certain embodiments, such applied optical radiation has a wavelength from
about 850 nm to about 900 nm, at a NIMELS dosimetry, as described herein. In
exemplary embodiments, wavelengths from about 865 nm to about 875 nm are
utilized.
In further embodiments, such applied radiation has a wavelength from about 905
nm to
about 945 nm at a NIMELS dosimetry. In certain embodiments, such applied
optical
radiation has a wavelength from about 925 nm to about 935 nm. In one aspect,
the
wavelength employed is 930 nm.
Microbial pathogens that have their bioenergetic systems affected by the
NIMELS laser system according to the present invention include microorganisms
such
as, for example, bacteria, fungi, molds, mycoplasmas, protozoa, and parasites.
Exemplary embodiments, as noted below may employ multiple wavelength ranges
including ranges bracketing 870 and 930 nm, respectively.
In the methods according to one aspect of the invention, irradiation by the
wavelength ranges contemplated are performed independently, in sequence, in a
blended ratio, or essentially concurrently (all of which can utilize pulsed
and/or
continuous-wave, CW, operation).
Irradiation with NIMELS energy at NIMELS dosimetry to the biological
contaminant is applied prior to, subsequent to, or concomitant with the
administration
of an antimicrobial agent. However, said NIMELS energy at NIMELS dosimetry can
be
administered after antimicrobial agent has reached a "peak plasma level" in
the infected
individual or other mammal. It should be noted that the co-administered
antimicrobial
agent ought to have antimicrobial activity against any naturally sensitive
variants of the
resistant target contaminant.
The wavelengths irradiated according to the present methods and systems
increase the sensitivity of a contaminant to the level of a similar non-
resistant
-54-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
contaminant strain at a concentration of the antimicrobial agent of about 0.01
M or less,
or about 0.001 M or less, or about 0.0005 M or less.
The methods of the invention slow or eliminate the progression of microbial
contaminants in a target site, improve at least some symptoms or asymptomatic
pathologic conditions associated with the contaminants, and/or increase the
sensitivity
of the contaminants to an antimicrobial agent. For example, the methods of the
invention result in a reduction in the levels of microbial contaminants in a
target site
and/or potentiate the activity of antimicrobial compounds by increasing the
sensitivity
of a biological contaminant to an antimicrobial agent to which the biological
contaminant has evolved or acquired resistance, without an adverse effect on a
biological subject. The reduction in the levels of microbial contaminants can
be, for
example, at least 10%, 20%, 30%, 50%, 70% or more as compared to pretreatment
levels.
With regard to sensitivity of a biological contaminant to an antimicrobial
agent, the
sensitivity is potentiated by at least 10%.
In another aspect, the invention provides a system to implement the methods
according to other aspects of the invention. Such a system includes a laser
oscillator for
generating the radiation, a controller for calculating and controlling the
dosage of the
radiation, and a delivery assembly (system) for transmitting the radiation to
the
treatment site through an application region. Suitable delivery
assemblies/systems
include hollow waveguides, fiber optics, and/or free space/beam optical
transmission
components. Suitable free space/beam optical transmission components include
collimating lenses and/or aperture stops.
In one form, the system utilizes two or more solid state diode lasers to
function
as a dual wavelength near-infrared optical source. The two or more diode
lasers may be
located in a single housing with a unified control. The two wavelengths can
include
emission in two ranges from about 850 nm to about 900 nm and from about 905 nm
to
about 945 nm. The laser oscillator of the present invention is used to emit a
single
wavelength (or a peak value, e.g., central wavelength) in one of the ranges
disclosed
herein. In certain embodiments, such a laser is used to emit radiation
substantially
within the about 865-875 nm and the about 925-935 nm ranges.
-55-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Systems according to the present invention can include a suitable optical
source
for each individual wavelength range desired to be produced. For example, a
suitable
solid stated laser diode, a variable ultra-short pxlse laser oscillator, or an
ion-doped
(e.g., with a suitable rare earth element) optical fiber or fiber laser is
used. In one form,
a suitable near infrared laser includes titanium-doped sapphire. Other
suitable laser
sources including those with other types of solid state, liquid, or gas gain
(active) media
may be used within the scope of the present invention.
According to one embodiment of the present invention, a therapeutic system
includes an optical radiation generation system adapted to generate optical
radiation
substantially in a first wavelength range from about 850 nm to about 900 nm, a
delivery
assembly for causing the optical radiation to be transmitted through an
application
region, and a controller operatively connected to the optical radiation
generation device
for controlling the dosage of the radiation transmitted through the
application region,
such that the time integral of the power density and energy density of the
transmitted
radiation per unit area is below a predetermined threshold. Also within this
embodiment, are therapeutic systems especially adapted to generate optical
radiation
substantially in a first wavelength range from about 865 nm to about 875 nm.
According to further embodiments, a therapeutic system includes an optical
radiation generation device that is configured to generate optical radiation
substantially
in a second wavelength range from about 905 nm to about 945 nm; in certain
embodiments the noted first wavelength range is simultaneously or
concurrently/sequentially produced by the optical radiation generation device.
Also
within the scope of this embodiment, are therapeutic systems especially
adapted to
generate optical radiation substantially in a first wavelength range from
about 925 nm
to about 935 nm.
The therapeutic system can further nzclude a delivery assembly (system) for
transmitting the optical radiation in the second wavelength range (and where
applicable, the first wavelength range) through an application region, and a
controller
operatively for controlling the optical radiation generation device to
selectively generate
-56-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
radiation substantially in the first wavelength range or substantially in the
second
wavelength range or any combinations thereof.
According to one embodiment, the delivery assembly comprises one or more
optical fibers having an end configured and arranged for insertion in patient
tissue at a
location within an optical transmission range of the medical device, wherein
the
radiation is delivered at a NIMELS dosimetry to the tissue surrounding the
medical
device. The delivery assembly may further comprise a free beam optical system.
According to a further embodiment, the controller of the therapeutic system
includes a power limiter to control the dosage of the radiation. The
controller may
further include memory for storing a patient's profile and dosimetry
calculator for
calculating the dosage needed for a particular target site based on the
information input
by an operator. In one aspect, the memory may also be used to store
information about
different types of diseases and the treatment profile, for example, the
pattern of the
radiation and the dosage of the radiation, associated with a particular
application.
The optical radiation can be delivered from the therapeutic system to the
application
site in different patterns. The radiation can be produced and delivered as a
continuous
wave (CW), or pulsed, or a combination of each. For example, in a single
wavelength
pattern or in a multi-wavelength (e.g., dual-wavelength) pattern. For example,
two
wavelengths of radiation can be multiplexed (optically combined) or
transmitted
simultaneously to the same treatment site. Suitable optical combination
techniques can
be used, including, but not limited to, the use of polarizing beam splitters
(combiners),
and/or overlapping of focused outputs from suitable mirrors and/or lenses, or
other
suitable multiplexing/combining techniques. Alternatively, the radiation can
be
delivered in an alternating pattern, in which the radiation in two wavelengths
are
alternatively delivered to the same treatment site. An interval between two or
more
pulses may be selected as desired according to NIMELS techniques of the
present
invention. Each treatment may combine any of these modes of transmission. The
intensity distributions of the delivered optical radiation can be selected as
desired.
Exemplary embodiments include top-hat or substantially top-hat (e.g.,
-57-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
trapezoidal, etc.) intensity distributions. Other intensity distributions,
such as Gaussian
may be used.
As used herein, the term "biological contaminant" is intended to mean a
contaminant that, upon direct or indirect contact with the target site, is
capable of
undesired and/or deleterious effect(s) on the target site (e.g., an infected
tissue or organ
of a patient) or upon a mammal in proximity of the target site (e.g., such as,
for example,
in the case of a cell, tissue, or organ transplanted in a recipient, or in the
case of a device
tzsed on a patient). Biological contaminants according to the invention are
microorganisms such as, for example, bacteria, fungi, molds, mycoplasmas,
protozoa,
parasites, known to those of skill in the art to generally be found in the
target sites.
One of skill in the art will appreciate that the methods and systems of the
invention may be used in conjunction with a variety of biological contaminants
generally known to those skilled in the art. The following lists are provided
solely for
the purpose of illustrating the broad scope of microorganisms which may be
targeted
according to the methods and devices of the present invention and are not
intended to
limit the scope of the ulvention.
Accordingly, illustrative non-limiting examples of biological contaminants
(pathogens) include, but are not limited to, any bacteria, such as, for
example,
Escherichia, Enterobacter, Bacillus, Campylobacter, Corynebacterium,
Klebsiella, Treponema,
Vibrio, Streptococcus and Staphylococcus.
To further illustrate, biological contaminants contemplated include, btxt are
not
limited to, any fungus, such as, for example, Trichophyton, Microsporum,
Epidermophyton,
Candida, Scopulariopsis brevicaulis, Fusarium spp., Aspergillus spp,,
Alternaria, Acremonium,
Scytalidinum dimidiatum, and Scijtalidinium hyalinum. Parasites may also be
targeted
biological contaminants such as Tn,/panosoma and malarial parasites, including
Plasmodium species, as well as molds; mycoplasms and prions. Viruses include,
for
example, human immuno-deficiency viruses and other retroviruses, herpes
viruses,
parvoviruses, filoviruses, circoviruses, paramyxoviruses, cytomegaloviruses,
hepatitis
viruses (including hepatitis B and hepatitis C), pox viruses, toga viruses,
Epstein-Barr
virus and parvoviruses may also be targeted.
-58-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
It will be understood that the target site to be irradiated need not be
already
infected with a biological contaminant. Indeed, the methods of the present
invention
may be used "prophylactically," prior to infection. Further embodiments
include use
on medical devices such as catheters, (e.g., IV catheter, central venous line,
arterial
catheter, peripheral catheter, dialysis catheter, peritoneal dialysis
catheter, epidural
catheter), artificial joints, stents, external fixator pins, chest tubes,
gastronomy feeding
tubes, etc.
In certain instances, irradiation may be palliative as well as prophylactic.
Hence,
the methods of the invention are used to irradiate a tissue or tissues for a
therapeutically
effective amount of time for treating or alleviating the symptoms of an
infection. The
expression "treating or alleviating" means reducing, preventing, and/or
reversing the
symptoms of the individual treated according to the invention, as compared to
the
symptoms of an individual receiving no such treatment.
One of skill in the art will appreciate that the invention is useful in
conjunction
with a variety of diseases caused by or otherwise associated with any
microbial, fungal,
and viral infection (see, Harrison's, Principles of Internal Medicine, 13'h
Ed., McGraw
Hill, New York (1994), the entire teaching of which is incorporated herein by
reference).
In certain embodiments, the methods and the systems according to the invention
are
used in concomitance with traditional therapeutic approaches available in the
art (see,
e.g., Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 8th ed,
1990,
Pergmon Press, the entire teaching of which is incorporated herein by
reference.) to treat
an infection by the administration of known antimicrobial agent compositions.
The
terms "antimicrobial composition", "antimicrobial agent" refer to compounds
and
combinations thereof that are administered to an animal, including human, and
which
inhibit the proliferation of a microbial infection (e.g., antibacterial,
antifungal, and
antiviral).
The wide breath of applications contemplated include, for example, a variety
of
dermatological, podiatric, pediatric, and general medicine to mention but a
few.
The interaction between a target site being treated and the energy imparted is
defined
by a number of parameters including: the wavelength(s); the chemical and
physical
-59-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
properties of the target site; the power density or irradiance of beam;
whether a
continuous wave (CW) or pulsed irradiation is being used; the laser beam spot
size; the
exposure time, energy density, and any change in the physical properties of
the target
site as a result of laser irradiation with any of these parameters. In
addition, the
physical properties (e.g., absorption and scattering coefficients, scattering
anisotropy,
thermal conductivity, heat capacity, and mechanical strength) of the target
site may also
affect the overall effects and outcomes.
The NIMELS dosimetry denotes the power density (W/cm2) and the energy
density (J/cmz; where 1 Watt = 1 Joule/second) values at which a subject
wavelength is
capable of generating ROS and thereby reducing the level of a biological
contaminant in
a target site, and/or irradiating the contaminant to increase the sensitivity
of the
biological contaminant through the lowering of AIP with concomitant generation
of
ROS to an antimicrobial agent that said contaminant is resistant to without
intolerable
risks and/or intolerable side effects on a biological moiety (e.g., a
mammalian cell,
tissue, or organ) other than the biological contaminant.
As discussed in Boulnois 1986, (Lasers Med. Sci. 1:47-66 (1986), the entire
teaching of which is incorporated herein by reference), at low power densities
(also
referred to as irradiances) and/or energies, the laser-tissue interactions can
be described
as purely optical (photochemical), whereas at higher power densities photo-
thermal
interactions ensue. In certain embodiments, exemplified hereinafter, NIMELS
dosimetry parameters lie between known photochemical and photo-thermal
parameters
in an area traditionally used for photodynamic therapy in conjunction with
exogenous
drugs, dyes, and/or chromophores, yet can function in the realm of
photodynamic
therapy without the need of exogenous drugs, dyes, and/or chromophores.
The energy density - also expressible as fluence, or the product (or integral)
of
particle or radiation flux and time - for medical laser applications in the
art typically
varies between about 1 J/cmz to about 10,000 J/cmz (five orders of magnitude),
whereas
the power density (irradiance) varies from about 1x10-3 W/cmz to over about
1012 W/cm2
(15 orders of magnitude). Upon taking the reciprocal correlation between the
power
density and the irradiation exposure time, it can be observed that
approximately the
-60-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
same energy density is required for any intended specific laser-tissue
interaction. As a
result, laser exposure duration (irradiation time) is the primary parameter
that
determines the nature and safety of laser-tissue interactions. For example, if
one were
mathematically looking for thermal vaporization of tissue in vivo (non-
ablative) (based
on Boulnois 1986), it can be seen that to produce an energy density of 1000
J/cm2 (see,
Table 1) one could use any of the following dosimetry parameters:
Table 1: Example of Vahxes Derived on the Basis of the Boulnois Table
POWERDENSITY TIME ENERGYDENSIIY
1x105 W/cmz 0.01 sec. 1000 J/cm2
1x104 W/cmz 0.10 sec. 1000 J/cm2
1x10' W/cm2 1.00 sec. 1000,J/cm2
This progression describes a suitable method or basic algorithm that can be
used
for a NIMELS interaction against a biological contaminant in a tissue. In
other words,
this mathematical relation is a reciprocal correlation to achieve a laser-
tissue interaction
phenomena. This rationlale can be used as a basis for dosimetry calculations
for the
observed antimicrobial phenomenon imparted by NIMELS energies with insertion
of
NIMELS experimental data in the energy density and time and power parameters.
On the basis of the particular interactions at the target site being
irradiated (such
as the chemical and physical properties of the target site; whether continuous
wave
(CW) or pulsed irradiation is being used; the laser beam spot size; and any
change in the
physical properties of the target site, e.g., absorption and scattering
coefficients,
scattering anisotropy, thermal conductivity, heat capacity, and mechanical
strength, as a
result of laser irradiation with any of these parameters), a practitioner is
able to adjust
the power density and time to obtain the desired energy density.
The examples provided herein show such relationships in the context of both in
vitro and in vivo treatments. Hence, in the context of treating, e.g.,
onychomycosis or
infected wounds for spot sizes havnlg a diameter of 1-4 cm, power density
values were
varied from about 0.5 W/cm2 to about 5 W/cmz to stay within safe and non-
damaging/minimally damaging thermal laser-tissue interactions well below the
level of
"denaturization" and "tissue overheating". Other suitable spot sizes may be
used.
-61-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
With this reciprocal correlation, the threshold energy density needed for a
NIMELS
interaction with these wavelengths can be maintained independent of the spot-
size so
long as the desired energies are delivered. In exemplary embodiments, the
optical
energy is delivered through a uniform geometric distribution to the tissues
(e.g., a flat-
top, or top-hat progression). With such a technique, a suitable NIMELS
dosimetry
sufficient to generate ROS (a NIMELS effect) can be calculated to reach the
threshold
energy densities required to reduce the level of a biological contaminant
and/or to
increase the sensitivity of the biological contaminant to an antimicrobial
agent that said
contaminant is resistant to, but below the level of "denaturization" and
"tissue
overheating .
NIMELS dosimetries exemplified herein (e.g., Onychomycosis) to target
microbes in vivo, were from about 200 J/cm2 to about 700 J/cm2 for
approximately 100 to
700 seconds. These power values do not approach power values associated with
photoablative or photothermal (laser/tissue) interactions.
The intensity distribution of a collimated laser beam is given by the power
density of the beam, and is defined as the ratio of laser output power to the
area of the
circle in (cmz) and the spatial distribution pattern of the energy. Hence, the
illumination
pattern of a 1.5 cm irradiation spot with an incident Gaussian beam pattern of
the area
1.77 cmz can produce at least six different power density values within the
1.77 c.mz
irradiation area. These varying power densities increase in intensity (or
concentration
of power) over the surface area of the spot from 1 (on the outer periphery) to
6 at the
center point. In certain embodiments of the invention, a beam pattern is
provided
which overcomes this inherent error associated with traditional laser beam
emissions.
NIMELS parameters may be calculated as a function of treatment time (Tn) as
follows:
Tn = Energy Density/Power Density.
In certain embodiments (see, e.g., the in vitro experiments hereinbelow), Tn
is
from about 50 to about 300 seconds; in other embodiments, Tn is from about 75
to about
200 seconds; in yet other embodiments, Tn is from about 100 to about 150
seconds. In in
vivo embodiments, Tn is from about 100 to about 1200 seconds.
-62-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Utilizing the above relationships and desired optical intensity distributions,
e.g.,
flat-top illumination geometries as described herein, a series of in vivo
energy
parameters have been experimentally proven as effective for NIMELS microbial
decontamination therapy in vitro. A key parameter for a given target site has
thus been
shown to be the energy density required for NIMELS therapy at a variety of
different
spot sizes and power densities.
"NIMELS dosimetry" encompasses ranges of power density and/or energy
density from a first threshold point at which a subject wavelength according
to the
invention is capable of optically reducing DT in a target site to a second end-
point
and/or to increase the sensitivity of the biological contaminant to an
antimicrobial agent
that said contaminant is resistant to via generation of ROS, immediately
before those
values at which an intolerable adverse risk or effect is detected (e.g.,
thermal damage
such as poration) on a biological moiety. One of skill in the art will
appreciate that
under certain circumstances adverse effects and/or risks at a target site
(e.g., a
mammalian cell, tissues, or organ) may be tolerated in view of the inherent
benefits
accruing from the methods of the invention. Accordingly, the stopping point
contemplated are those at which the adverse effects are considerable and,
thus,
undesired (e.g., cell death, protein denaturation, DNA damage, morbidity, or
mortality).
In certain embodiments, e.g., for in vivo applications, the power density
range
contemplated herein is from about 0.25 to about 40 W/cmz. In other
embodiments, the
power density range is from about 0.5 W/cmz to about 25 W/cm2.
In further embodiments, power density ranges can encompass values from
about 0.5 W/cmz to about 10 W/cmz. Power densities exemplified herein are from
about
0.5 W/cmzto about 5 W/cm2. Power densities in vivo from about 1.5 to about 2.5
W/cmz
have been shown to be effective for various microbes.
Empirical data appears to indicate that higher power density values are
generally used when targeting a biological contaminant in an in vitro setting
(e.g.,
plates) rather than in vivo (e.g., toe nail).
In certain embodiments (see, in vitro examples below), the energy density
range
contemplated herein is greater than 50 J/cmz but less than about 25,000 J/cmz.
In other
-63-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
embodiments, the energy density range is from about 750 J/cm2 to about 7,000
J/cmz. In
yet other embodiments, the energy density range is from about 1,500 J/cmz to
about
6,000 J/cm2 depending on whether the biological contaminant is to be targeted
in an in
vitro setting (e.g., plates) or in vivo (e.g., toe nail or surrounding a
medical device).
In certain embodiments (see, in vivo examples below), the energy density is
from about
100 J/cm2 to about 500 ,J/cmz. In yet other in vivo embodiments, the energy
density is
from about 175 J/cmz to about 300 J/cm2. In yet other embodiments, the energy
density
is from about 200 J/cm2 to about 250 J/cmz. In some embodiments, the energy
density is
from about 300 J/cmz to about 700 J/cmz. In some other embodiments, the energy
density is from about 300 J/cm2 to about 500 J/cmz. In yet others, the energy
density is
from about 300 J/cmz to about 450 J/cm2.
Power densities empirically tested for various in vitro treatment of microbial
species were from about 1 W/cm2 to about 10 W/cmz.
One of skill in the art will appreciate that the identification of
particularly
suitable NIMELS dosimetry values within the power density and energy density
ranges
contemplated herein for a given circumstance may be empirically done via
routine
experimentation. Practitioners (e.g., dentists) using near infrared energies
in
conjunction with periodontal treatment routinnely adjust power density and
energy
density based on the exigencies associated with each given patient (e.g.,
adjust the
parameters as a function of tissue color, tissue architecture, and depth of
pathogen
invasion). As an example, laser treatment of a periodontal infection in a
light-colored
tissue (e.g., a melanine deficient patient) will have greater thermal safety
parameters
than darker tissue, because the darker tissue will absorb near-infrared energy
more
efficiently, and hence transform these near-infrared energies to heat in the
tissues faster.
Hence, the obvious need for the ability of a practitioner to identify multiple
different
NIMELS dosimetry values for different therapy protocols.
As illustrated infra, it has been found that antibiotic resistant bacteria may
be
effectively treated according to the methods of the present invention. In
addition, it has
been found that the methods of this invention may be used to augment
traditional
approaches, to be used in combination with, in lieu of tradition therapy, or
even serially
- 64 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
as an effective therapeutic approach. Accordingly, the invention may be
combined
with antibiotic treatment. The term "antibiotic" includes, but is not limited
to, (3-
lactams, penicillins, and cephalosporins, vancomycins, bacitracins, macrolides
(erythromycins), ketolides (telithromycin), lincosamides (clindomycin),
chloramphenicols, tetracyclines, aminoglycosides (gentamicins), amphotericns,
anilinouracils, cefazolins, clindamycins, mupirocins, sulfonamides and
trimethoprim,
rifampicins, metronidazoles, quinolones, novobiocins, polymixins,
oxazolidinone class
(e.g., linezolid), glycylcyclines (e.g., tigecycline), cyclic lipopeptides
(e.g., daptomycin),
pleuromutilins (e.g., retapamulin) and gramicidins and the like and any salts
or variants
thereof. It also understood that it is within the scope of the present
invention that the
tetracyclines include, but are not limited to, immunocycline,
chlortetracycline,
oxytetracycline, demeclocycline, methacycline, doxycycline and minocycline and
the
like. It is also further understood that it is within the scope of the present
invention that
aminoglycoside antibiotics include, but are not limited to, gentamicin,
amikacin and
neomycin, and the like.
As illustrated below, it has been found that antifungal resistant fungi may be
effectively treated according to the methods of the invention. In addition, it
has been
found that the methods of the present invention may be used to augment
traditional
approaches, to be used in combination with, in lieu of, traditional therapy,
or even
serially as an effective therapeutic approach. Accordingly, the invention may
be
combined with anHfungal treatment. The term "antifungal" includes, but is not
limited
to, polyenes, azoles, imidazoles, triazoles, allylamines, echinocandins,
cicopirox,
flucytosine, griseofulvin, amorolofine, sodarins and combinations thereof
(including
salts thereof).
As illustrated below, it has been postulated that antineoplastic resistant
cancer
may be effectively treated according to the methods of the present invention.
In
addition, it has been found that the methods of the invention may be used to
augment
traditional approaches, to be used in combination with, in lieu of tradition
therapy, or
even serially as an effective therapeutic approach. Accordingly, the invention
may be
combined with antineoplastic treatment. Ther term "antineoplastic" includes,
but is not
-65-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
limited to, actinomycin, anthracyclines, bleomycin, plicamycin, mitomycin,
taxanes,
etoposide, teniposide and combinations thereof (including salts thereof).
A common tenet in the prior art of trying to find an inhibitor of drug
resistance
systems in bacteria and fungi, or a potentiator of antimicrobial agents has
always been
that such agents must be non-toxic to the mammalian tissues that are infected
to have
any intrinsic value. Furthermore, it has always been a fact that
antimicrobials affect
bacterial or fungal cellular processes that are not common to the mammalian
host, and,
hence, are generally safe and therapeutic in nature and design. In the prior
art, if
antimicrobials, potentiators, and/or resistance reversal entities were to also
affect the
mammalian cells in the same manner as they damage the pathogens, they could
not be
used safely as a therapy.
In the current invention, the experimental data (see, e.g., Examples I-X)
supports
a universal alteration of ALY and Ap among all cell types, and hence leads to
the notion
that not only the electro-mechanical, but also the electro-dynamical aspects
of all cell
membranes, have no differing properties that can adequately be separated. This
indicates that all cells in the path of the beam are affected with
depolarization, not only
the pathogenic cells (non-desired cells).
By reaffirming what the photobiology and cellular energetics data of the
NIMELS system has already illuminated (i.e., that all of membrane energetics
are
affected in the same way across prokaryotic and eukaryotic species),
techniques
according to the present invention utilize this universal optical depolarizing
effect to be
independently exploited in non-desired cells, by adding antimicrobial
molecules to a
therapeutic regimen, and potentiating such molecules in (only) non-desired
cells.
Such a targeted therapeutic outcome can exploit the NIMELS laser's effect of
universal
depolarization, which can be more graded and transient to the mammalian cells
in the
path of the therapeutic beam, than to the bacteria and fungi. Hence, as the
experimental
data suggests, the measures of temporal and energetic robushless of the
mammalian
cells must be greater in the face of optical depolarization and ROS
generation, than is
seen in the bacterial or fungal cells.
-66-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The examples below provide experimental evidence proving the concept of
universal optical membrane depolarization coupled to our current understanding
of
photobiology and cellular energetics and the conservation of thermodynamics as
applied to cellular processes.
EXAMPLES
The following examples are utcluded to demonstrate exemplary embodiments of
the present invention and are not intended to limit the scope of the
invention. Those of
skill in the art, will appreciate that many changes can be made in the
specific
embodiments and still obtain a like or similar result without departing from
the spirit
and scope of the present invention.
EXAMPLE I
Table 2: MIC values for Susceptible, Intermediate and Resistant S. aureus
Minimum Inhibitory Concentration (MIC) Interpretive Standards ( /m1) for Stn
hi lococcus sp.
Antimicrobial Agent Susce tible Intermediate Resistant
Penicillin <0.12 >0.25
Methicillin <8 >16
Amino 1 cosides
Gentamicin <_4 8 >_16
Kanamycin <_16 32 >_64
Macrolides
Er throm cin <_0.5 1-4 28
Tetracycline
Tetracycline <_4 8 >16
Fluoro uinolone
Ciprofloxacin :51 2 >4
Folate Pathway Inliibitors
Trimethoprim <8 - >_16
Ansamycins
Rifam in <1 2 >4
EXAMPLE rI
-67-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
BACTERIAL METHODS: NIMELS TREATMENT PARAMETERS FOR IN VITRO MRSA
EXPERIMENTS
The following parameters illustrate the general bacterial methods according to
the invention as applied to MRSA for the in vitro Experiments V and VIII-XII.
A, Experiment Materials and Methods for MRSA:
Table 3. Method: for CFU counts
Time Task FTE
(hrs) (hrs)
Inoculate overnight culture
T-18 1
50 ml directly from glycerol stock
Set up starter cultures
T 4 1
Three dilutions 1:50, 1:125,1:250 LB Media
Monitor ODeoo of starter cultures 4
Preparation of plating culture
At 10:00am, the culture which is at ODeoo =1,0 is diluted 1:300 in PBS (50 mis
T 0 1
final volume) and stored at RT for 1 hour.
(Rooin temp should be -25 C)
Seeding of 24-well plates
T+1 2 ml aliquots are dispensed into pre-designated wells in 24-well plates
and 1
transferred to NOMIR
T +2 to Dilution of treated samples
+8 After laser treatment, 100 l from each well is diluted serially to a final
4
dilution of 1:1000 in PBS.
Plating of treated samples
100 l of final dilution is plated in quintuplicate (5X) on TSB agar with and
2
without antibiotics. (10 TSB plates per well)
Plates are incubated at 37 C 18-24hrs.
T +24 Colonies are counted on each plate 6
Similar cell culture and kinetic protocols were performed for all NIMELS
irradiation with E. coli and C. albicans in vitro tests. Hence, for example,
C. albicans
ATCC 14053 liquid cultures were grown in YM medium (21g/L, Difco) medium at 37
C.
A standardized suspension was aliquoted into selected wells in a 24-well
tissue culture
plate. Following laser treatments, 100 L was removed from each well and
serially
diluted to 1:1000 resulting in a final dilution of 1:5x106 of initial culture.
An aliquot of
each final dilution were spread onto separate plates. The plates were then
incubated at
37 C for approximately 16-20 hours. Manual colony counts were performed and
recorded.
-68-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Table 4. Method: for AT and ROS Assays
Time Task FTE
(hrs) (hrs)
T 18 Inoculate overnight culture 1
50 ml directly from glycerol stock
Set up starter cultures
T 4 1
Three dilutions 1:50, 1:125, 1:250 LB Media
Monitor ODeoo of starter cultures 4
Preparation of plating culture
At 10:00am, the culture which is at OD6oo = 1.0 is diluted 1:300 in PBS (50
mis
T 0 1
final volume) and stored at RT for 1 hour.
(Rooin temp should be -25 C)
Seeding of 24-well plates for Assays
T+1 2 ml aliquots are dispensed 'uito pre-designated wells in 24-well plates
and 1
transferred to NOMIR
T +2 to Dilution of treated samples
+8 After laser treatment each control and Lased sample were treated as per 4
directions of individual assay.
Again, similar cell culture and kinetic protocols were performed for all
NIMELS
irradiation with E. coli and C. albicans in vitro assay tests. Hence, for
example, C. albicans
ATCC 14053 liquid cultures were grown in YM medium (21g/L, Difco) medium at 37
C.
A standardized suspension was aliquoted into selected wells in a 24 -well
tissue culture
plate. Following laser treatments each lased and control sample were treated
as per
directions of individual assay.
EXAMPLE III
MAMMALIAN CELL METHODS: NIMELS TREATMENT PARAMETERS FOR IN VITRO HEK293
(HUMAN EMBRYONIC KIDNEY CELL)EXPERIMENTS
The following parameters illustrate the general bacterial methods according to
the invention as applied to HEK293 cells for the in vitro experiments.
A. Experiment Materials and Methods for HEK293 cells.
HEK293 cells were seeded into appropriate wells of a 24-well plate at a
density
of 1 x 105 cells/ml (0.7m1 total volume) in Freestyle medium (Invitrogen).
Cells were
incubated in a humidified incubator at 37 C in 8% C02 for approximately 48
hours
-69-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
prior to the experiment. Cells were approximately 90% confluent at the time of
the
experiment equating to roughly 3 x 105 total cells. Immediately prior to
treatment, cells
were washed in pre-warmed phosphate buffer saline (PBS) and overlaid with 2 ml
of
PBS dtxring treatment.
After laser treatment, cells were mechanically dislodged from the wells and
transferred to 1.5 ml centrifuge tubes. Mitochondrial membrane potential and
total
glutathione was determined according to the kit manufacturer's instructions.
EXAMPLE IV
NIMELS IN VITRO TESTS FOR CRT+ (YELLOW)AND CRT - (WHITE) S. AUREUS EXPERIMENTS
We conducted experiments with crt- (white) mutants of S. aureus that were
genetically engineered with the crt gene (yellow carotenoid pigment) removed,
and
these mutants were subjected to previously determined non-lethal doses of
NIMELS
laser against wild type (yellow) S. aureus. The purpose of this experiment was
to test for
the phenomenon of Radical Oxygen Species (ROS) generation and/or singlet
oxygen
generation with the NIMELS laser. In the scientific literature, Liu et al. had
previously
used a similar model, to test the antioxidant protection activity of the
yellow S. aureus
*caratenoid) pigment against neutrophils. (Liu et al., Staphylococcus aureus
golden
pigment impairs neutrophil killing and promotes virulence through its
antioxidant
activity, Vol. 202, No. 2, July 18, 2005 209-215, the entire teaching of which
is
incorporated herein by reference.)
It has previously been determined that the golden color in S. aureus is
imparted
by carotenoid (antioxidant) pigments capable of protecting the organism from
singlet
oxygen, andwhen a mutant is isolated (crt-) that does not produce such
carotenoid
pigments, the mutant colonies are "white" in appearance and more susceptible
to
oxidant killing, and have impaired neutrophil survival.
It was found that non-lethal dosimetries of the NIMELS laser (to wild type S.
aureus) consistently killed up to 90% of the mutant "white" cells and did not
kill the
normal S. aureus. The only genetic difference in the two strains of S. aureus
is the lack of
-70-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
an antioxidant pigment in the mutant. This experimental data strongly suggests
that it is
the endogenous generation of radical oxygen species and/or singlet oxygen that
are
killing the "white" S. aureus.
Table 5. Data:
Dl - D4 Yellow Wild Type S. aureus.
D5 - D6 White "crt" Mutant S. Aureus.
Total
Output Beam Time Energy Energy Density Power Density
Plate No Power (W) Spot (cm) (sec) Joules Q/cm~) (W/cm~)
Dl 11 1.5 720 7920 4481.793 6.224712
D2 11.5 1.5 720 8280 4685.511 6.507654
D3 12 1.5 720 8640 4889.228 6.790595
D4 12.5 1.5 720 9000 5092.946 7.073536
Total
Output Beam Time Energy Energy Density Power Density
Plate No Power (W) S ot (cm) (sec) Joules (J/cm') (W/cmz)
D5 11 1.5 720 7920 4481.793 6.224712
D6 115 1.5 720 8280 4685.511 6.507654
D7 12 1.5 720 8640 4889.228 6.790595
D8 12.5 1.5 720 9000 5092.946 7.073536
Samples Dl - D4 Yellow Wild Type S. aureus.
Samples D5 - D6 White "crt-" Mutant S. aureus.
Table 6.
-=---...._ ..............
S. Aureus stud (ATCC 12600 WT & CRTM-)
Laser-
Control treated
Sam le CFU's CFU' Percent of Control
203 44
D1 274 55 18.48
291 35
241 46
268 56
270 155
303 133
D2 266 110 46.76
245 111
321 148
D3 315 87 25.32
344 101
310 100
-71-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
350 71
395 75
405 23
472 31
D4 401 30 7.21
403 32
359 31
530 163
534 194
D5 520 192 35.05
552 194
520 188
252 54
262 46
D6 248 50 20.00
273 70
270 41
276 40
169 30
D7 260 38 14,68
259 35
296 42
323 6
348 3
D8 423 9 1.68
408 6
340 7
EXAMPLE V
NIMELS IN VITRO TESTS FOR AT ALTERATION IN MRSA, C. ALBICANS AND E. COLI
There are selected fluorescent dyes that can be taken up by nltact cells and
accumulate withul the intact cells within 15 to 30 minutes without appreciable
staining
of other protoplasmic constituents. These dye indicators of membrane potential
have
been available for many years and have been employed to study cell physiology,
The
fluorescence intensity of these dyes can be easily monitored, as their
spectral fluorescent
properties are responsive to changes in the value of the trans-membrane
potentials AT-
steady.
These dyes generally operate by a potential-dependent partitioning between the
extracellular medium and either the membrane or the cytoplasm of membranes.
This
occurs by redistribution of the dye via interaction of the voltage potential
with an ionic
charge on the dye. This fluorescence can be eliminated in about 5 minutes by
the
protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that
-72-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
maintenance of dye concentration is dependent on the inside-negative
transmembrane
potential maintained by functional ETS and Ap.
Hypothesis Testing:
The null hypothesis is l - z= 0:
i is fluorescence intensity in a control cell culture (no laser) subjected to
carbocyanine
dye
z is fluorescence intensity in the same cell culture pre-irradiated with sub-
lethal
dosimetry from the NIMELS laser
The data indicates that the fluorescence of cells is dissipated (less than
control of
unirradiated or "unlased" cells) by pre-treatment (of the cells) with the
NIMELS laser
system, indicating that the NIMELS laser interacted with respiratory processes
and
oxidative phosphorylation of the cells via the plasma membranes.
- x=0
Will uphold that the addition sub-lethal NIMEL irradiation on the cell culture
has no
effect on AtY-steady.
pu- z>0
Will uphold that the addition sub-lethal NIMEL irradiation on the cell culture
has a
dissipation or depolarization effect on AT-steady.
Materials and Methods:
BacLightT"' Bacterial Membrane Potential Kit (B34950, Invitrogen U.S.).
The BacLightT"' Bacterial Membrane Potential Kit provides of carbocyanine dye
DiOC2(3) (3,3'-diethyloxacarbocyanine iodide, Component A) and CCCP (carbonyl
cyanide 3-chlorophenylhydrazone, Component B), both in DMSO, and a 1 x PBS
solution (Component C).
DiOC2(3) exhibits green fluorescence in all bacterial cells, but the
fluorescence shifts
toward red emission as the dye molecules self associate at the higher
cytosolic
concentrations caused by larger membrane potentials. Proton ionophores such as
CCCP
-73-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
destroy membrane potential by eliminating the proton gradient, hence causing
higher
green fluorescence.
Detection of membrane potential AiY in MRSA
Green fluorescence emission was calculated using population mean fluorescence
intensities for control and lased samples at sub-lethal dosimetry:
Table 6.
MRSA Dosimetry Progression
First lasing Procedure : Both 870 and 930
Second lasing procedure 930 alone
Parameters Otttptit Beam Spot Area of Spot(cm2) Time
Power (W) (cm) (sec)
870 at 4.25 W and 930 at 4.25 W for 16 min 8.5 1.5 1.77 960
followed by
930 at 8.5W for 7 min 8.5 1.5 1.77 420
The data shows that t - 2> 0 as the lased cells had less "Green
fluorescence" as
seen in Figure S. These MRSA samples showed clear alteration and lowering of
ALP-
steady-bact to one of AW-trans-bact with sub-lethal NIMELS dosimetry.
Detection of membrane potential AT in C. albicans
Green fluorescence emission was calculated using population mean fluorescence
intensities for control and lased samples at sub-lethal dosimetry listed in
the table
below:
Table 7.
First lasin rocedure : Both 870 and 930
Second lasing procedure 930 alone
Output Beam Spot Area of Time
Parameters Power (W) (cm) Spot(cm2) (sec)
Laser #1
Test (H-1) 870 at 4 W and 930 at 4 W for 18 min 8.0 1.5 1.77 1080
followed by
Test (H-1) 930 at 8W for 8 min 8.0 1.5 1.77 480
Laser #2
Test (H-2) 870 at 4.25 W and 930 at 4.25 W for 18 8.5 1.5 1.77 1080
min followed by
Test (H-2) 930 at 8.5 W for 8 min 8.5 1.5 1.77 480
Laser #3
Test (H-3) 870 at 4 W and 930 at 4 W for 20 min 8.0 11.5 11.77 1200
-74-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
followed b
Test (H-3) 930 at 8 W for 10 min 8.0 1.5 1.77 600
The data shows that ~a - z> 0 as the lased C. albicans cells had less "Green
fluorescence" as seen in Figure 9. These C. albicans samples showed clear
alteration and
lowering of AtY-steady-fungi to one of OtY-trans-fungi with sub-lethal NIMELS
dosimetry with increasing (sub-lethal) NIMELS laser dosimetry.
Detection of membrane potential OT in E. coli
Red/green ratios were calculated using population mean fluorescence
intensities
for control and lased samples at sub-lethal dosimetry:
The data shows that i - z > 0 as the lased cells had less "Green
fluorescence" as
seen in Figure 19. These E. coli samples showed clear alteration and lowering
of 0T -
steady-bact to one of AT -trans-bact with sublethal NIMELS dosimetry.
EXAMPLE VI
NIMELS IN VITRO TESTS FOR AT-mito IN C. ALBICANS WITH SUB-LETHAL LASER
DOSIMETRY
Hypothesis Testing:
The null hypothesis is l - z= 0:
a) 1 is fluorescence intensity in a control cell culture mitochondria
subjected to a
Mitochondrial Membrane Potential Detection Kit.
b) z is fluorescence intensity in the same cell culture pre-irradiated with
sub-lethal
dosimetry from the NIMELS laser and subjected to a Mitochondrial Membrane
Potential Detection Kit.
The data shows that the fluorescence of mitochondria is dissipated (less than
control unlased cells) by pre-treatment (of the cells) with the NIMELS laser
system, the
results indicate that the NIMELS laser interacted with respiratory processes
and
oxidative phosphorylation of the cells in mitochondria of fungal and mammalian
cells.
~ - z= 0
-75-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Will uphold that the addition sub-lethal NIMEL irradiation on the cell culture
mitochondria has no effect on 0T-steady-mito.
- 2>0
Will uphold that the addition sub-lethal NIMEL irradiation on the cell
culture has a dissipation or depolarization effect on AiY-steady-mito.
Materials and Methods:
Mitochondrial Membrane Potential Detection Kit (APO LOGIX JC-1) (Cell
Technology Inc., 950 Rengstorff Ave, Suite D; Mountain View CA 94043).
The loss of mitochondrial membrane potential (AT) is a hallmark for apoptosis.
The APO LOGIX JC-1 Assay Kit measures the mitochondrial membrane potential in
cells.
In non-apoptotic cells, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenz==
imidazolylcarbocyanine iodide) exists as a monomer in the cytosol (green) and
also
accumulates as aggregates in the mitochondria which stain red. Whereas, in
apoptotic
and necrotic cells, JC-1 exists in monomeric form and stains the cytosol
green.
Table 8. Candida Albicans Dosimetry Table
First lasing procedure : Both 870 and 930
Second lasing procedure 930 alone
Test Parameters Output Beam Area of Time
Power (W) Spot (cm) Spot(cm2) (sec)
Cand Test (H-3) 870 at 4.25 W and 930 at 8.5 1.5 1.77 960
Mito 1 4.25 W for 16 min followed by
Test (H-3) 930 at 8.5 W for 10 min 8.5 1.5 1.77 600
The (APO LOGIX JC-1) kit measures membrane potential by conversion of green
fluorescence to red fluorescence. In Figure 10A, the appearance of red color
has been
measured and plotted, which should only occur in cells with intact membranes,
and the
ratio of green to red is shown in Figure 10B for both control and lased
samples.
-76-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Clearly in this test, the red fluorescence is reduced in the lased sample
while the ratio of
green to red increases, indicating depolarization. These results are the same
as the
trans-membrane AT tests (i.e., both data show depolarization).
These results also show that , - z> 0 and that sub-lethal NIMEL irradiation
on
the cell mitochondria has a dissipation or depolarization effect on ALY-steady-
mito,
indicating a clear reduction of Candida Albicans ALF-steady-mito-fungi to AT-
trans-mito-
fungi.
EXAMPLE VII
NIMELS IN VITRO TESTS FOR AT-mito HUMAN EMBRYONIC KIDNEY CELLS WITH SUB-
LETHAL LASER DOSIMETRY
Hypothesis Testing:
The null hypothesis is y - z= 0:
a) pi is fluorescence intensity in a mammalian control cell culture
mitochondria (no
laser) subjected to a Mitochondrial Membrane Potential Detection Kit.
b) z is fluorescence intensity in the same mammalian cell culture pre-
irradiated with
sub-lethal dosimetry from the NIMELS laser and subjected to a Mitochondrial
Membrane Potential Detection Kit.
The data shows that the fluorescence of mitochondria is dissipated (less than
control unlased cells) by pre-treatment (of the cells) with the NIMELS laser
system, the
results indicate that the NIMELS laser interacted with respiratory processes
and
oxidative phosphorylation of the cells in mitochondria of mammalian cells.
- z= 0
Will uphold that the addition sub-lethal NIMEL irradiation on the mammalian
cell
culture mitochondria has no effect on OLY-steady-mito-mam.
F,ti- z> 0
Will uphold that the addition sub-lethal NIMEL irradiation on the mammalian
cell
culture has a dissipation or depolarization effect on 0T-steady-mito-mam.
-77-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Materials and Methods:
Mitochondrial Membrane Potential Detection Kit (APO LOGIX JC-1) (Cell
Technology Inc., 950 Rengstorff Ave, Suite D; Mountain View CA 94043).
The loss of mitochondrial membrane potential (AT) is a hallmark for apoptosis.
The
APO LOGIX JC-1 Assay Kit measures the mitochondrial membrane potential in
cells.
In non-apoptotic cells, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenz-
imidazolylcarbocyanine iodide) exists as a monomer in the cytosol (green) and
also
accumulates as aggregates in the mitochondria which stain red. Whereas, in
apoptotic
and necrotic cells, JC-1 exists in monomeric form and stains the cytosol
green.
Table 9. Mamallian Cell Dosimetries
First lasing rocedure : Both 870 and 930
Second lasing procedure 930 alone
Output Beam Area of Time
Parameters Power Spot Spot(cm2) (sec)
(W) (cm)
Test (H-2) 870 at 4.25 W and 930 at 4.25 W 8.5 1.5 1.77 1080
for 18 min followed by
Test (H-2) 930 at 8.5 W for 10 min 8.5 1.5 1.77 600
HEK-293 (Human Embryonic Kidney Cells) AT-mito tests:
The (APO LOGIX JC-1) kit measures membrane potential by conversion of green
fluorescence to red fluorescence. In Figure 11A, the appearance of red color
has been
measured and plotted, which should only occur in cells with intact membranes,
and the
ratio of green to red is shown in Figure 11B for both control and lased
samples.
Clearly in this test, the red fluorescence is reduced in the lased sample
while the
ratio of green to red increases, indicating depolarization. These results show
that i - 2
> 0 and that sub-lethal NIMELS irradiation on the mammalian cell mitochondria
has a
dissipation or depolarization effect on OtY-steady-mito-mam, indicating a
clear
reduction in mammalian OlY-steady-mito-mam to AT-trans-mito-xnam.
EXAMPLE VIII
-78-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
NIMELS IN VITRO TESTS FOR REACTIVE OXYGEN SPECIES (ROS)
These in vitro tests for generation of reactive oxygen species (ROS) were
carried
on after laser alteration of bacterial trans-membrane AT-steady-bact to AT-
trans-bact,
AT-steady-mito-fungi to AtY-trans-mito-fungi, and ALY-steady-mito-mam to ALF-
trans-
mito-mam with sub-lethal laser dosimetry comparable to those used in 0T tests
above
in previous examples.
Materials and Methods:
Total Glutathione Quantification Kit (Dojindo Laboratories; Kumamoto Techno
Research Park, 2025-5 Tabaru, Mashiki-machi, Kamimashiki-gun; Kumamoto 861-
2202,
JAPAN)
Glutathione (GSH) is the most abundant thiol (SH) compound in animal tissues,
plant tissues, bacteria and yeast. GSH plays many different roles such as
protection
against reactive oxygen species and maintenance of protein SH groups. During
these
reactions, GSH is converted into glutathione disulfide (GSSG: oxidized form of
GSH).
Since GSSG is enzymatically reduced by glutathione reductase, GSH is the
dominant
form in organisms. DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)), known as
Eliman's
Reagent, was developed for the detection of thiol compounds. In 1985, it was
suggested
that the glutathione recycling system by DTNB and glutathione reductase
created a
highly sensitive glutathione detection method. DTNB and glutathione (GSH)
react to
generate 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG). Since 2-
nitro-5-
thiobenzoic acid is a yellow colored product, GSH concentration in a sample
solution
can be determnled by the measurement at 412 nm absorbance. GSH is generated
from
GSSG by glutathione reductase, and reacts with DTNB again to produce 2-nitro-5-
thiobenzoic acid. Therefore, this recycling reaction improves the sensitivity
of total
glutathione detection.
At significant concentrations ROS will react rapidly and specifically with the
target at a rate exceeding the rate of its reduction by the components of the
glutathione
antioxidant system (catalases, peroxidases, GSH).
-79-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Detection of Glutathione in MRSA at sub-lethal NIMELS dosimetry that alters
AtY-
steady-bact to one of 0T-trans-bact
The results as shown in Figure 12 clearly show a reduction in total
glutathione in
MRSA at sub-lethal NIMELS dosimetry that alters that alters AT-steady-bact to
one of
AT-trans-bact, which is a proof of generation of ROS with sub-lethal
alteration of Trans-
membrane AT-steady-bact to one of AIY-trans-bact.
Detection of Glutathione in E.coli at sub-lethal NIMELS dosimetry that alters
Trans-
membrane AlY-steady to one of AT-trans
The results as shown in Figure 20 clearly shows a reduction in total
glutathione
in E. coli at sub-lethal NIMELS dosimetry that alters AT-steady-bact to one of
AT -
trans-bact, which is evidence of generation of ROS with sub-lethal alteration
of Trans-
membrane ALY-steady-bact to one of AT -trans-bact.
Detection of glutathione in C. albicans at sub-lethal NIMELS that alters AtY-
steady-mito-fungi to Agf-trans-mito-fungi and subsequently 0T-steady-fungi to
one of
AW-trans-fungi.
Detection of Glutathione in C. albicans at sub-lethal NIMELS dosimetry that
alters 0T-
steady-mito-fungi to AW-trans-mito-fungi and subsequently OlF-steady-fungi to
one of
OT-trans-fun¾i
The results as shown in Figure 13 clearly show a reduction in total
glutathione in
C. albicans at sub-lethal NIMELS dosimetry that alters DT-steady-mito-hmgi to
AT-
trans-mito-fungi and subsequently OLY-steady-fungi to one of AT-trans-fungi,
which is
a proof of generation of ROS with sub-lethal alteration of Trans-membrane AW-
steady-
mito-fungi to AT-trans-mito-fungi and subsequently AT-steady-fungi to one of
AT-
trans-fungi.
Detection of Glutathione in HEK-293 (Human Embryonic Kidney Cells) at sub-
lethal
NIMELS dosimetry that alters ALY-steady-mito-mam to AT-trans-mito-mam
-80-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The results as shown in Figure 14 clearly show a reduction in total
Glutathione
in HEK-293 (Human Embryonic Kidney Cells) with sub-lethal NIMELS dosimetry
that
alters AT-steady-mito-mam to OlY-trans-mito-mam, which is proof of generation
of
ROS with NIMELS-mediated sub-lethal alteration of Trans-membrane AT-steady-
mito-
mam to AT-trans-mito-mam.
EXAMPLE IX
Assessment of the impact of Sub-lethal doses of NIMELS Laser on MRSA with
Erythromycin and Trimethoprim
In this example, it was determined whether a sub-lethal dose of the NIMEL
laser
will potentiate the effect of the antibiotic erythromycin more than the
antibiotic
trimethoprim in MRSA. Efflux pumps play a major factor in erythromycin
resistance.
There are no reported trimethoprim efflux pump resistance mechanisms in the
gram
positive S. aureus.
Background: Erythromycin is a marcolide antibiotic that has an antibacterial
spectrum
of action very similar to that of the (3-lactam penicillin. In the past, it
has been effective
in the treatment of a wide range of gram-positive bacterial infections
effecting the skin
and respiratory tract, and has been considered one of the safest antibiotics
to use. In the
past, erythromycin has been used for people with allergies to penicillins.
Erythromycin's mechanism of action is to prevent growth and replication of
bacteria by
obstructing bacterial protein synthesis. This is accomplished because
erythromycin
binds to the 23S rRNA molecule in the 50S of the bacterial ribosome, thereby
blocking
the exit of the growing peptide chain thus inhibiting the translocation of
peptides.
Erythromycin resistance (as with other marcolides) is rampant, wide spread,
and is
accomplished via two significant resistance systems:
A) modification of the 23S rRNA in the 50S ribosomal subunit to insensitivity
B) efflux of the drug out of cells
-81-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Trimethoprim is an antibiotic that has historically been used in the treatment
of
urinary tract infections. It is a member of the class of antimicrobials known
as
dihydrofolate reductase inhibitors. Trimethoprim's mechanism of action is to
interfere
with the system of bacterial dihydrofolate reductase (DHFR), because it is an
analog of
dihydrofolic acid. This causes competitive inhibition of DHFR due to a 1000
fold higher
affinity for the enzyme than the natural substrate.
Thus, trimethoprim inhibits synthesis of the molecule tetrahydrofolic acid.
Tetrahydrofolic acid is an essential precursor in the de novo synthesis of the
DNA
nucleotide thymidylate. Bacteria are incapable of taking up folic acid from
the
environment (i.e., the infection host) and are thus dependent on their own de
novo
synthesis of tetrahydrofolic acid. Inhibition of the enzyme ultimately
prevents DNA
replication.
Trimethoprim resistance generally results from the overproduction of the
normal chromosomal DHFR, or drug resistant DHFR enzymes. Reports of
trimethoprim resistance S. aureus have indicated that the resistance is
chromosomally of
the mediated type or is encoded on large plasmids. Some strains have been
reported to
exhibit both chromosomal and plasmid-mediated trimethoprim resistance.
In the gram positive pathogen S. aureus, resistance to trimethoprim is due to
genetic
mutation, and there have been no reports that trimethoprim is actively
effluxed out of
cells.
Efflux Pumps in Bacteria
A major route of drug resistance in bacteria and fungi is the active export
(efflux)
of antibiotics out of the cells such that a therapeutic concentration in not
obtained in the
cytoplasm of the cell.
Active efflux of antibiotics (and other deleterious molecules) is mediated by
a
series of transmembrane proteins in the cytoplasmic membrane of gram positive
bacteria and the outer membranes of gram negative bacteria.
Clinically, antibiotic resistance that is mediated via efflux pumps, is most
relevant in gram positive bacteria for marcolides, tetracyclines and
fluoroquinolones. In
-82-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
gram negative bacteria, (3-lactam efflux mediated resistance is also of high
clinical
relevance.
Hypothesis Testing
The null hypothesis is i - 42= 0 and 41 - 3= 0 where:
a) i is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control
and;
b) 2 is the same sub-lethal dosimetry from the NIMEL laser system on MRSA
with the
addition of trimethoprim at resistant MIC just below effectiveness level and;
c) 3 is the same sub-lethal dosimetry from the NIMEL laser system on MRSA
with the
addition of erythromycin at resistant MIC just below effectiveness level.
The data shows that the addition of the antibiotic trimethoprim or
erythromycin,
after sub-lethal irradiation, results in the reduction in growth of these MRSA
colonies,
as follows:
1 - 2=0
Will uphold that the addition of trimethoprim produces no deleterious effect
after sub-
lethal NIMEL irradiation, on normal growth of MRSA colonies.
i- 2>0
Will uphold that the addition of trimethoprim produces a deleterious effect
after sub-
lethal NIMEL irradiation, on normal growth of MRSA colonies.
i - s= 0
Will uphold that the addition of erythromycin produces no deleterious effect
after sub-
lethal NIMEL irradiation, on normal growth of MRSA colonies.
i - 3> 0
Will uphold that the addition of erythromycin produces a deleterious effect
after sub-
lethal NIMEL, irradiation, on normal growth of MRSA colonies.
Table 10.
EXPERIMENTAL CONTROL (no laser)
trimeth erythro i trimeth er thro
AGAR 2 u/ml 4 ug/ml AGAR 2 u/ml 4 u/ml
B-4 1 84 110 39 B-4 1 180 213 196
B-4 2 88 125 35 B-4 2 230 198 168
-83-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
B-4 3 120 138 39 B-4 3 241 240 175
B-4 4 114 115 28 B-4 4 220 220 177
B-4 5 117 100 27 i I B-4 5 smeared 145 195
Results:
This experiment clearly showed that under sub-lethal laser parameters with the
NIMELS system, - z= 0 and I - a>= 0. This indicates that an efflux pump is
being
inhibited, and resistance to erythromycin being reversed by the NIMELS effect
on AtY-
steady-bact of the MRSA.
EXAMPLE X
Assessment of the impact of Sub-lethal doses of NIMELS Laser on MRSA with
Tetracycline and Rifampin
The purpose of this experiment was to observe if a stzb-lethal dose of the
NIMEL
laser will potentiate the effect of the antibiotic tetracycline more than the
antibiotic
rifampin in MRSA. Efflux pumps are well researched, and play a major factor in
tetracycline resistance. However, there are no reported rifampin efflux pump
resistance
mechanisms in the gram positive S.aureus.
This experiment was also previously run with erythromycin and trimethoprim,
with data indicating that the NIMELS effect is able to damage efflux pump
resistance
mechanisms in erythromycin.
Tetracycline:
Tetracycline is considered a bacteriostatic antibiotic, meaning that it
hampers the
growth of bacteria by inhibiting protein synthesis. Tetracycline accomplishes
this by
inhibiting action of the bacteria130S ribosome through the binding of the
enzyme
aminoacyl-tRNA. Tetracycline resistance is often due to the acquisition of new
genes,
which code for energy-dependent efflux of tetracyclines, or for a protein that
protects
bacterial ribosomes from the action of tetracyclines.
Rifampin:
-84-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Rifampin is a bacterial RNA polymerase inhibitor, and functions by directly
blocking the elongation of RNA. Rifampicin is typically used to treat
mycobacterial
infections, but also plays a role in the treatment of methicillin-resistant
Staphylococcus
auretts (MRSA) in combination with fusidic acid, a bacteriostatic protein
synthesis
inhibitor. There are no reports of rifampin resistance via efflux pumps in
MRSA.
Hypothesis:
The null hypothesis is I - z= 0 and pi a= 0 where:
a) i is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control
and;
b) z is the same sub-lethal dosimetry from the NIMEL laser system on MRSA
with the
addition of tetracycline at resistant MIC just below effectiveness level and;
c) s is the same sub-lethal dosimetry from the NIMEL laser system on MRSA
with the
addition of rifampin at resistant MIC just below effectiveness level.
The data shows that the addition of the antibiotic tetracycline or rifampin,
after
sub-lethal irradiation, results in the reduction in growth of these MRSA
colonies, as
follows:
i- z=0
Will uphold that the addition of tetracycline produces no deleterious effect
after sub-
lethal NIMEL irradiation, on normal growth of MRSA colonies.
i - z> 0
Will uphold that the addition of tetracycline produces a deleterious effect
after sub-
lethal NIMEL irradiation, on normal growth of MRSA colonies.
, - a= 0
Will uphold that the addition of rifampin produces no deleterious effect after
sub-lethal
NIMEL irradiation, on normal growth of MRSA colonies.
'- 3>0
Will uphold that the addition of rifampin produces a deleterious effect after
sub-lethal
NIMEL irradiation, on normal growth of MRSA colonies.
- 85 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Table 11.
EXPERIMENTAL CONTROL
rifampin tetrac c. rifam in tetracyc.
AGAR 90u /ml 4u /ml AGAR 90 u/ml 4ug/ml
E1-1 307 210 42 E1-1 270 183 240
E1-2 300 200 56 El-2 210 210 256
E1-3 300 280 46 El-3 224 166 268
E'1-4 310 378 48 E1-4 semared 228 310
E1-5 250 280 42 E1-5 215 188 255
E2-1 246 272 18 E2-1 240 274 280
E2-2 254 320 28 E2-2 310 210 283
E2-3 174 330 27 E2-3 190 180 263
E2-4 170 semared 16 E2-4 257 240 260
E2-5 240 284 18 E2-5 275 310
E3-1 310 270 72 E3-1 280 288 368
E3-2 280 225 67 E3-2 320 280 380
E3-3 260 284 45 E3-3 310 210 375
E3-4 210 200 47 E3-4 320 290 390
E3-5 220 smeared 74 E3-5 320 300 smeared
Results:
This experiment clearly showed that under sub-lethal laser parameters with the
NIMELS system, - z= 0 and - 3>= 0. This indicates that an efflux pump is
being
inhibited, and resistance to tetracycline is being reversed by the NIMELS
effect on AtY-
steady-bact of the MRSA.
EXAMPLE XI
Assessment of the impact of Sub-lethal doses of NIMELS Laser on MRSA with
Methicillin and ALY-plas-bact inhibition of cell wall synthesis
Methicillin:
Methicillin is a(3-lactam that was previously used to treat infections caused
by
gram-positive bacteria, particularly (3-lactamase-producing organisms such as
S. aureus
that would otherwise be resistant to most penicillins, but is no longer
clinically used.
The term methicillin-resistant S. aureus (MRSA) continues to be used to
describe S.
attreus strains resistant to all penicillins.
-86-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Mechanism of action
Like other (3-lactam antibiotics, methicillin acts by inhibiting the synthesis
of
peptidoglycan (bacterial cell walls).
It has been shown in the gram positive bacterium Bacillus subtilis, that the
activities of peptidoglycan autolysins are increased (i.e., no longer
inhibited) when the
ETS was blocked by adding proton conductors. This suggests that AT-plas-bact
and
A H' (independent of storing energy for cellular enzymatic functions)
potentially has a
profound and exploitable influence on cell wall anabolic functions and
physiology.
In addition, it has been reported that AT-plas-bact uncouplers inhibit
peptidoglycan formation with the accumulation of the nucleotide precursors
involved
in peptidoglycan synthesis, and the inhibition of transport of N-
acetylglucosamine
(GIcNAc), one of the major biopolymers in peptidoglycan,
Hypothesis Testing:
Bacitracin will potentiate the multiple influences of an optically lowered Aw-
plas-bact on a growing cell wall (i.e., increased cell wall autolysis,
inhibited cell wall
synthesis). This is especially relevant in gram positive bacteria such as
MRSA, that do
not have efflux pumps as resistance mechanisms for cell wall inhibitory
antimicrobial
compounds.
The null hypothesis is i - z = 0 and , - a= 0 where:
a) i is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control
and;
b) z is the same sub-lethal dosimetry from the NIMEL laser system on MRSA
with the
addition of methicillin at resistant MIC just below effectiveness level and;
,- z=0
Will uphold that the addition of methicillin produces no deleterious effect
after sub-
lethal NIMEL irradiation, on normal growth of MRSA colonies.
,- z>0
Will uphold that the addition of methicillin produces a deleterious effect
after sub-lethal
NIMEL irradiation, on normal growth of MRSA colonies.
-87-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Results:
As shown in Figure 15, this experiment clearly showed that under sub-lethal
laser parameters with the NIMELS system, , - z>= 0, meaning that the
addition of
methicillin produces a deleterious effect after sub-lethal NIMEL irradiation
on normal
growth of MRSA colonies as shown by CFU count. This suggest that methicillin
(independent of an efflux pump) is being potentiated by the NIMELS effect on
AYf-
steady-bact of the MRSA.
Hence, the NIMELS laser and its concomitant optical AT-plas-bact lowering
phenomenon is synergistic with cell wall inhibitory antimicrobials in MRSA.
Without
wishing to be bound by theory, this must function via the inhibition of
anabolic
(periplasmic) ATP coupled functions, as MRSA does not have efflux pumps for
methicillin.
EXAMPLE XII
Assessment of the impact of Sub-lethal doses of NIMELS Laser on MRSA with
Bacitracin and 0T-12las-bact inhibition of cell wall synthesis
Bacitracin is a mixture of cyclic polypeptides produced by Bacillus subtilis.
As a
toxic and difficult-to-use antibiotic, bacitracin cannot generally be used
orally, but used
topically.
Mechanism of action:
Bacitracin interferes with the dephosphorylation of the Cas-isoprenyl
pyrophosphate, a molecule which carries the building blocks of the
peptidoglycan
bacterial cell wall outside of the inner membrane in gram negative organisms
and the
plasma membrane in gram positive organism.
It has been shown in the gram positive bacterium Bacillus subtilis, that the
activities of peptidoglycan autolysins are increased (i.e., no longer
inhibited) when the
ETS was blocked by adding proton conductors. This indicates that 0T-plas-bact
and
-88-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
A H` (independent of storing energy for cellular enzymatic functions)
potentially has a
profound and exploitable influence on cell wall anabolic functions and
physiology.
In addition, it has been reported that AT-plas-bact uncouplers inhibit
peptidoglycan formation with the accumulation of the nucleotide precursors
involved
in peptidoglycan synthesis, and the inhibition of transport of N-
acetylglucosamine
(GIcNAc), one of the major biopolymers in peptidoglycan.
Hypothesis Testing:
Bacitracin potentiates the multiple influences of an optically lowered 0T-plas-
bact on a growing cell wall (i.e., increased cell wall autolysis, inhibited
cell wall
synthesis). This is especially relevant in gram positive bacteria such as
MRSA, that do
not have efflux pumps as resistance mechanisms for cell wall inhibitory
antimicrobial
compounds.
The null hypothesis is i - z= 0 and - a= 0 where:
a) is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control
and;
b) z is the same sub-lethal dosimetry from the NIMEL laser system on MRSA
with the
addition of bacitracin at resistant MIC just below effectiveness level and;
,- z=0
Will uphold that the addition of bacitracin produces no deleterious effect
after sub-
lethal NIMEL irradiation, on normal growth of MRSA colonies.
- z>0
Will uphold that the addition of bacitracin produces a deleterious effect
after sub-lethal
NIMEL irradiation, on normal growth of MRSA colonies.
Results:
As shown in Figure 16, this experiment clearly showed that under sub-lethal
laser parameters with the NIMELS system, u - z>= 0, meaning that the
addition of
bacitracin produces a deleterious effect after sub-lethal NIMEL irradiation,
on normal
-89-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
growth of MRSA colonies. In Figure 16, arrows point to MRSA growth or a lack
thereof
in the two samples shown. This indicates that bacitracin (independent of an
efflux
pump) is being potentiated by the NIMELS effect on AW-steady-bact of the MRSA.
Hence, the NIMELS laser and its concomitant optical AT-plas-bact lowering
phenomenon is synergistic with cell wall inhibitory antimicrobials in MRSA.
Without
wishing to be bound by theory, this most likely functions via the inhibition
of anabolic
(periplasmic) ATP coupled functions as MRSA does not have efflux pumps for
bacitracin.
EXAMPLE XIII
Assessment of the impact of Sub-lethal doses of NIMELS Laser on C. albicans
with
Lamisil and Sporanox
The purpose of this experiment was to observe if a sub-lethal dose of the
NIMEL
laser will potentiate the effect of the antifungal compounds Lamisil and/or
sporanox in
C. aibicans.
Introduction:
It has been found that a reduction of the cytosolic ATP concentration in
fungal
cells leads to a suppression of the plasma membrane-bound HI-ATPase that
generates
AtYp-fungi, and that this impairment weakens other cellular activities.
Additionally, the
lowering of the ALYp-fungi causes plasma membrane bioenergetic and
thermodynamic
disruption, leading to an influx of protons that collapse the proton motive
force and,
hence, inhibits nutrient uptake. Of further note, ATP is necessary for the
biosynthesis of
the fungal plasma membrane lipid ergosterol. Ergosterol is the structural
lipid that is
targeted by the majority of relevant commercial antifungal compounds used in
medicine today (i.e., azoles, terbinafine and itraconazole) including lamisil
and
sporanox (and generic counterparts thereof).
-90-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Also, recently, it has bee shown that two novel antimicrobial peptides (Pep2
and
Hst5) have the ability to cause ATP to be effluxed out of fungal cells (i.e.,
depleting
intracellular ATP concentrations) and that this lowered cytosolic ATP causes
the
inactivation of ABC transporters CDRl and CDR2 which are ATP-dependent efflux
pumps of antifungal agents.
Lamisil:
Lamisil (like other allylamines) inhibits ergosterol synthesis by inhibiting
squalene expoxidase, an enzyme that is part of the fungal cell wall synthesis
pathway.
Sporanox:
The mechanism of action of itraconazole (Sporanox) is the same as the other
azole antifungals: it inhibits the fungal cytochrome P450 oxidase-mediated
synthesis of
ergosterol.
Hypothesis:
The NIMELS laser at sub-lethal dosimetry on C. albicans potentiates lamisil
and
sporanox due to of an optically lowered AW-plas-fungi and/or OtY-mito-fungi by
depolarizing the membranes and depleting cellular ATP in the fungus.
The null hypothesis is pi z= 0 and - a= 0 where:
a) Vi is sub-lethal dosimetry from the NIMEL, laser system on C. albicans as a
control
and;
b) z is the same sub-lethal dosimetry from the NIMEL laser system on C.
albicans with
the addition of Sporanos at resistant MIC just below effectiveness level and;
c) sie the same sub-lethal dosimetry from the NIMEL laser system on C.
albicans with
the addition of Lamisil at resistant MIC just below effectiveness level.
The data indicates that the addition of the antifungal lamisil and/or sporanox
after sub-lethal irradiation, results in the redtxction in growth of these C.
albicans
colonies, as follows:
l - 2= 0
Will uphold that the addition of Sporanox produces no deleterious effect after
sub-lethal
NIMEL irradiation, on normal growth of C. albicans colonies.
-91-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
,- z>0
Will uphold that the addition of Sporanox produces a deleterious effect after
sub-lethal
NIMEL irradiation, on normal growth of C. albicans colonies.
,- 3= 0
Will uphold that the addition of Lamisil produces no deleterious effect after
sub-lethal
NIMEL irradiation, on normal growth of C. albicans colonies.
,- 3>0
Will uphold that the addition of Lamisil produces a deleterious effect after
sub-lethal
NIMEL irradiation, on normal growth of C. albicans colonies.
Table 12. Candida Albicans NIMELS Dosimetry Charts
First lasing procedure : Both 870 and 930
Second lasing procedure 930 alone
Output Beam Area of Time
Test Parameters Power Spot
Spot(cm2) (sec)
(W) (cm)
AF-8 Test (H-1) 870 at 4.25 W and 930 at 8.0 1.5 1.77
4.25 W for 18 min followed by
AF-8 Test (H-1) 930 at 8.5W for 12 min 8.0 1.5 1.77
Table 13. Colony Counts:
Control Experimental
Lamisil Lamisil
Sporanox Sporanox
Group Replicate AGAR 0.5ml 0.5 ug/ml AGAR 0.5 ml 0.5 ug/ml u /
1 220 280 311 n.d. 78 80
2 320 n.d. 295 249 74 107
AF8 3 266 290 360 330 101 110
4 248 335 332 209 70 86
190 334 320 244 90 91
Results:
This experiment clearly showed that under sub-lethal laser parameters using
the
NIMELS system, i - 2> 0 and , - 3> 0, meaning that the addition of lamisil
produces
a deleterious effect after sub-lethal NIMEL, irradiation, on normal growth of
C. albicans
-92-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
colonies. This suggest that egosterol biosynthesis inhibitors (lamisil and
sporanox) are
potentiated by a sub-lethal dosimetry irradiation of the NIMELS Laser system.
EXAMPLE XIV
NIMELS Dosimetry Calculations
The examples that follow describe selected experiments depicting the ability
of
the NIMELS approach to impact upon the viability of various commonly found
microorganisms at the wavelengths described herein. The microorganisms
exemplified
include E. coli K-12, multi-drug resistant E. coli, Staphylococcus aureus,
methicillin-
resistant S. aureus, Candida albicans, and Tricliophyton rubrum.
As discussed in more details supra, NIMELS parameters include the average
single or additive output power of the laser diodes, and the wavelengths (870
nm and
930 nm) of the diodes. This information, combined with the area of the laser
beam or
beams (cmz) at the target site, provide the initial set of information which
may be used
to calculate effective and safe irradiation protocols according to the
invention.
The power density of a given laser measures the potential effect of NIMELS at
the target
site. Power density is a function of any given laser output power and beam
area, and
may be calculated with the following equations:
For a single wavelength:
1) Power Density (W/cm2) = Laser Output Power
Beam Diameter (cmz)
For dual wavelength treatments:
2) Power Density (W/cm2) = Laser(1) Output Power + Laser (2) Outvut
Power
Beam Diameter (cm2) Beam Diameter (cm2)
Beam area can be calculated by either:
3) Beam Area (cmz) = Diameter (Em)z * 0.7854 or Beam Area (cmz) = Pi *
Radius (cm)z
-9.3-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The total photonic energy delivered into the tissue by one NIMELS laser diode
system operating at a particular output power over a certain period is
measured in
Joules, and is calculated as follows:
4) Total Energy (Joules) = Laser Output Power (Watts) * Time (Secs.)
The total photonic energy delivered into the tissue by both NIMELS laser diode
systems (both wavelengths) at the same time, at particular output powers over
a certain
period, is measured in Joules, and is calculated as follows:
5) Total Energy (Joules) =[Laser(1) Output Power (Watts) * Time (Secs)] +
[Laser (2) Output Power (Watts) * Time(Secs)]
In practice, it is useful (but not necessary) to know the distribution and
allocation of the total energy over the irradiation treatment area, in order
to correctly
measure dosage for maximal NIMELS beneficial response. Total energy
distribution
may be measured as energy density (Joules/cm2). As discussed infra, for a
given
wavelength of light, energy density is the most important factor in
determining the
tissue reaction. Energy density for one NIMELS wavelength may be derived as
follows:
6) Energy Density (Joules/ cmz) = Laser Output power (Watts) * Time (secs)
Beam Area (cmz)
7) Energy Density (Joule/cmz) = Power Density (W/cm2) * Time (secs)
When two NIMELS wavelengths are being used, the energy density may be derived
as
follows:
8) Energy Density (Joules/ cmz) = Laser (I)Output power (Watts) * Time
secs
Beam Area (cm2)
+ Laser (2) OutRut vower (Watts) * Time (secs)
Beam Area (cmz)
or,
9) Energy Density (Joule/cm2) = Power Density (1) (W/cmz) * Time (Secs)
+ Power Density (2) (W/cmz) * Time (Secs)
-94-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
To calculate the treatment time for a particular dosage, a practitioner may
use
either the energy density (J/cmz) or energy (J), as well as the output power
(W), and
beam area (cm2) using either one of the following equations:
10) Treatment Time (seconds) = Energy Density
(Joules/cmz)
Output power Density (W/cm2)
11) Treatment Time (seconds) = Energy
(Joules)
Laser Output Power (Watts)
Because dosimetry calculations such as those exemplified in this Example can
become burdensome, the therapeutic system may also include a computer database
storing all researched treatment possibilities and dosimetries. The computer
(a
dosimetry and parameter calculator) in the controller is preprogrammed with
algorithms based on the above-described formulas, so that any operator can
easily
retrieve the data and parameters on the screen, and input additional necessary
data
(such as: spot size, total energy desired, time and pulse width of each
wavelength, tissue
being irradiated, bacteria being irradiated) along with any other necessary
information,
so that any and all algorithms and calculations necessary for favorable
treatment
outcomes can be generated by the dosimetry and parameter calculator and hence
run
the laser.
In the examples that follow, in summary, when the bacterial cultures were
exposed to the NIMELS laser, the bacterial kill rate (as measured by counting
Colony
Forming Units or CFU on post-treatment culture plates) ranged from 93.7%
(multi-drug
resistant E. coli) to 100% (all other bacteria and fungi).
EXAMPLE XV
BACTERIAL METHODS: NIMELS TREATMENT PARAMETERS FOR IN VITRO E. COLI
TARGETING
-95-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
The following parameters illustrate the methods according to the invention as
applied to E. coli, at final temperatures well below those associated in the
literature with
thermal damage.
A. Experiment Materials and Methods for E. coli K-12:
E. coli K12 liquid cultures were grown in Luria Bertani (LB) medium (25 g/L).
Plates contained 35 mL of LB plate medium (25 g/L LB, 15 g/L bacteriological
agar).
Culture dilutions were performed using PBS. All protocols and manipulations
were
performed using sterile techniques.
B. Growth Kinetics
Drawing from a seed culture, multiple 50 mL LB cultures were inoculated and
grown at 37 C overnight. The next morning, the healthiest culture was chosen
and
used to inoculate 5% into 50 mL LB at 37 C and the O.D.600 was monitored over
time
taking measurements every 30 to 45 minutes until the culture was in stationary
phase.
C. Master Stock Production
Starting with a culture in log phase (O.D.6oo approximately 0.75), 10 mL were
placed at 4 C 10 mL of 50% glycerol were added and was aliquoted into 20
cryovials
and snap frozen in liquid nitrogen. The cryovials were then stored at -80 C.
D. Liquid Cultures
Liquid cultures of E. coli K12 were set up as described previously. An aliquot
of
100 L was removed from the subculture and serially diluted to 1:1200 in PBS.
This
dilution was allowed to incubate at room temperature approximately 2 hours or
until
no further increase in O.D.6oo was observed in order to ensure that the cells
in the PBS
suspension would reach a static state (growth) with no significant doubling
and a
relatively consistent number of cells could be aliquoted further for testing.
Once it was determined that the K12 dilution was in a static state, 2 mL of
this
suspension were aliquoted into selected wells of 24-well tissue culture plates
for
selected NIMELS experiments at given dosimetry parameters. The plates were
incubated at room temperature until ready for use (approximately 2 hrs).
Following laser treatments, 100 l was removed from each well and serially
diluted to 1:1000 resulting in a final dilution of 1:12x105 of initial K12
culture. Aliquots
-96-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
of 3 x 200 L of each final dilution were spread onto separate plates in
triplicate. The
plates were then incubated at 37 C for approximately 16 hours. Manual colony
counts
were performed and recorded. A digital photograph of each plate was also
taken.
Similar cell culture and kinetic protocols were performed for all NIMELS
irradiation
tests with S. aureus and C. albicans in vitro tests. For example, C. albicans
ATCC 14053
liquid cultures were grown in YM medium (21g/L, Difco) medium at 37 C. A
standardized suspension was aliquoted into selected wells in a 24-well tissue
culture
plate. Following laser treatments, l00 L was removed from each well and
serially
diluted to 1:1000 resulting in a final dilution of 1:5x105 of initial culture.
3x100 f.tL of
each final dilution were spread onto separate plates. The plates were then
incubated at
37 C for approximately 16-20 hours. Manual colony counts were performed and
recorded. A digital photograph of each plate was also taken.
T. rubrum ATCC 520221iquid cultures were grown in peptone-dextrose (PD)
medium at 37 C. A standardized suspension was aliquoted into selected wells
in a 24 -
well tissue culture plate. Following laser treatments, aliquots were removed
from each
well and spread onto separate plates. The plates were then incubated at 37 C
for
approximately 91 hours. Manual colony counts were performed and recorded after
66
hours and 91 hours of incubation. While control wells all grew the organism,
100% of
laser-treated wells as described herein had no growth. A digital photograph of
each
plate was also taken.
Thermal tests performed on PBS solution, starting from room temperature. Ten
(10) Watts of NIMELS laser energy were available for use in a 12 minute lasing
cycle,
before the temperature of the system is raised close to the critical threshold
of 44 C.
Table 14. Time & Tem erature measurements for In Vitro NIMELS Dosimetries
NIMEL SEAMSPOT TREATMENT TOTAL ENERGY POWER TEMPERATURE TEMP
OUTPtIT 1.5 CM TIME (SEC) ENERGY DENSITY DENSITY START FINISH
POWER (W) DIAMEI'ER QOULES) (RADIANT (IRRADIANCE) OVERLAP EXPOSURE) (W/CW)
AREA (CMZ) QJCMZ)
Plate 1-N
3.0+3.0=6.0
W 1.76 720 4320 2448 3.40 20.5 'C 34.0 C
Plate 2-N -- 1.76 720 5040 2858 3.97 20.7 C 36,5 C3.5 +3.5 = 7.0
-97-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
NIMEL BEAMSPOT TREATMENT TOTAL ENERGY POWER TEMPERATURE TEMP
OUTPUT 1.5 CM TIME (SEC) ENERGY DENSITY DENSITY START FINISH
POWER (W) DIAMETER ()OULES) (RADIANT (IRRADIANCE)
OVERLAP EXPOSURE) (W/CMZ)
AREA (CM~) Q/CMZ)
W
Plate 3-N - 4.D
+ 4.0 = 8.0 W 1.76 720 5760 3268 4.54 21.0 C 38.5 C
Plate 4-N - 4.5
+4.5=9.0W 1.76 720 6480 3679 5.11 2.0 C 41.0 C
Plate 5-N - 5.0
+5.0=10.W 1.76 720 7200 4089 5.68 21.0 C 40.5 'C
Plate 6-N - 5.5
+5.5 =11 W 1.76 720 7920 4500 6.25 21.0 C 46.0 C
Plate 7-N - 7.0
+7,0 =14.0 W 116 360 5040 2863 7.95 21.0 C 47.0 C
Plate 8-N - 7.5
+ 7.5 = 15 W 1.76 360 5400 3068 8.52 21.7 C 47.2 C
EXAMPLE XVI
DOSIMETRY VALUES FOR NIMELS LASER WAVELENGTH 930 NM FOR E. COLI IN VITRO
TARGETING
The instant experiment demonstrates that the NIMELS single wavelength A
930 nm is associated with quantitatable antibacterial efficacy against E. coli
in vitro
within safe thermal parameters for mammalian tissues.
Experimental data in vitro demonstrates that if the threshold of total energy
into
the system with 930 nm alone of 5400 J and an energy density of 3056 J/cmz is
met in
25% less time, 100% antibacterial efficacy is still achievable.
Table 15. Sub-thermal NIMELS(A = 930) Dosimetry for In ViEro E. coli Targeting
OUTPUT TOTAL ENERGY POWER
POWER TIME ENERGY DENSITY DENSr1Y E-COLIKILL
(W) BEAMSPOT(CM) (SEC.) JOULES Q/CM~) (W/CM~) PERCENTAGE
7.0 1.5 720 5040 2852 3.96 40.2%
8.0 1.5 720 5760 3259 4.53 100.0%
10.0 1.5 540 5400 3056 5.66 100.0%
-98-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Experimental data in vitro also demonstrates that treatments using a single
energy with l= 930 nm has antibacterial in vitro efficacy against the
bacterial species S.
aureus within safe thermal parameters for mammalian tissues.
It is also believed that if the threshold of total energy into the system of
5400
and an energy density of 3056 J/cmz is met in 25% less time with S. aureus and
other
bacterial species, that 100% antibacterial efficacy will still be achieved.
Table 16. Sub-thermal NIMELS (A = 930) Dosimetry for In Vitro S. aureus
Targeting
OUTPUT TOTAL ENERGY POWER
POWER BEAM ENERGY DENSITY DENSITY 5AUREUSKILL
(W) SPOT(CM) TIME(SEC) JOULES (J/CMZ) (W/CM~) PERCENTAGE
7.0 1.5 720 5040 2852 3.96 24.1%
8.0 1.5 720 5760 3259 4.53 100.0%
Experimental in vitro data also showed that the NIMELS single wavelength of A
= 930 nm has anti-fungal efficacy against in vitro C. albicans at ranges
within safe thermal
parameters for mammalian tissues.
It is also believed that if the threshold of total energy into the system of
5400 J
and an energy density of 3056 J/cmz is met in 25% less time, that 100%
antifungal
efficacy will still be achieved.
Table 17. Sub-thermal NIMELS(l= 930) Dosimetry for In Vitro C. albicans
Targeting
CANDIDA
OUTPUT TOTAL ENERGY POWER ALDICANS
POWER BEAM TIME ENERGY DENSITY DENSITY KILL
(W) SPOT(CM) (SEC.) JOULES Q/CMZ) (W/CM2) PERCENTAGE
8.0 1.5 720 5760 3259 4.53 100.0%
9.0 1.5 720 6840 3681 5.11 100.0%
EXAMPLE XVII
DOSIMETRY VALUES FOR NIMELS LASER WAVELENGTH 870 NM IN VITRO
Experimental in vitro data also demonstrates that no significant kill is
achieved
up to a total energy of 7200 J, and energy density of 4074 J/cmz and a power
density of
5.66 0 W/cmz with the wavelength of 870 nm alone against E. coli.
-99-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Table 18. E. coli Studies- Single wavelength A = 870 nm
OUTPUT BEAM TOTAL ENERGY POWER DIFFERENCE POWER SPOT TIME ENERGY DENSITY
DENSITY CONIROL NIMELS CONTROL- E.COLIKILL
(W) (CM) (SEC.) JOULES Q/CM1) (W/CMZ) CFUS CFUs NIMEL PERCENTAGE
6.0 1.5 720 4320 2445 3.40 90 95 (5) -5.6%
7.0 1.5 720 5040 2852 3.96 94 94 0 0.0%
8.0 1.5 720 5760 3259 4.53 93 118 (25) -26.9%
9.0 1.5 720 6480 3667 5.09 113 112 1 0.9%
10.0 1.5 720 7200 4074 j5.66 103 111 (8) -7.8%
10.0 1.5 540 5400 3056 5.66 120 101 19 15.8%
Comparable results using radiation having A = 870 nm alone were also observed
with S.
aureus.
EXAMPLE XVIII
NIMELS UNIQUE ALTERNATING SYNERGISTIC EFFECT BETWEEN 870 NM AND 930 NM
OPTICAL ENERGIES
Experimental in vitro data also demonstrates that there is an additive effect
between the two NIMELS wavelengths (A = 870 nm and 930 nm) when they are
alternated (870 nm before 930 nm). The presence of the 870 nm NIMELS
wavelength as
a first irradiance has been found to enhance the effect of the antibacterial
efficacy of the
second 930 nm NIMELS wavelength irradiance.
Experimental in vitro data demonstrates that this synergistic effect
(combining
the 870 nm wavelength to the 930 nm wavelength) allows for the 930 nm optical
energy
to be reduced. As shown herein, the optical energy was reduced to
approximately 1/3
of the total energy and energy density required for NIMELS 100 % E. coli
antibacterial
efficacy, when the (870 nm before 930 nm) wavelengths are combined in an
alternating
manner.
Experimental in vitro data also demonstrates that this synergistic mechanism
can
allow for the 930 nm optical energy (total energy and energy density) to be
reduced to
approximately 1/2 of the total energy density necessary for NIMELS 100% E.
coli
antibacterial efficacy if equal amounts of 870 nm optical energy are added to
the system
before the 930 nm energy at 20% higher power densities.
Table 19. E coli data from Alternating NIMELS Wavelengths
- 100-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
OUTPUT
POWER POWER
(W) SPOT TOTALENERGY ENERGYDENSITY DENSITY E.COL(KILL
(CM) TIME (SEC.) JOULES (I/CMZI (W/CMZ) PERCENTAGE
8 W/ 540 / 180 4320 / 1440 2445 / 815
8W 1.5 12 min. = 5760 = 3529 4.53 / 4.53 100.0%
lOW / 240 / 240 2400 / 2400 1358 / 1358
10W 1.5 8 min. = 4800 = 2716 5.66 / 5.66 100.0%
This synergistic ability is significant to human tissue safety, as the 930 nm
optical
energy, heats up a system at a greater rate than the 870 nm optical energy,
and it is
beneficial to a mammalian system to produce the least amount of heat possible
during
treatment.
It is also believed that if the NIMELS optical energies (870 nm and 930 nm)
are
alternated in the above manner with other bacterial species, that the 100%
antibacterial
effect will be essentially the same.
Experimental in vitro data also demonstrates that there is also an additive
effect
between the two NIMELS wavelengths (870 nm and 930 nm) when they are
alternated
(870 nm before 930 nm) while irradiating fungi. The presence of the 870 nm
NIMELS
wavelength as a first irradiance mathematically enhances the effect of the
anti-fungal
efficacy of the second 930 nm NIMELS wavelength irradiance.
Experimental in vitro data (see, table infra) demonstrates that this
synergistic
mechanism can allow for the 930 nm optical energy (total energy and energy
density) to
be reduced to approximately 1/2 of the total energy density necessary for
NIMELS 100%
antifungal efficacy if equal amounts of 870 nm optical energy is added to the
system
before the 930 nm energy at 20% higher power densities than is required for
bacterial
species antibacterial efficacy.
Table 20. C. albicans Data from Alternatin NIMEL Wavelen ths
OUTPUT CANDIDA
POWER POWER ALBICANS
(W) SPOT TOTALENERGY ENERGYDENSITY DENSITY KILL
(CM) TIME (SEC) JOULES (J/CM1) (W/CM2) PERCENTAGE
lOW / 240 / 240 2400 / 2400 1358 / 1358
lOW 1.5 8 min = 4800 = 2716 5.66 / 5.66 100.0%"
-101-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
This synergistic effect is significant to human tissue safety, as the 930 nm
optical
energy, heats up a system at a greater rate than the 870 nm optical energy,
and it is
beneficial to a mammalian system to produce the least amount of heat possible
during
treatment.
It is also believed that if the NIMELS optical energies (870 nm and 930 nm)
are
alternated in the above manner with other fungi species, that the 100% anti-
fungal effect
will be essentially the sazne.
EXAMPLE XIX
NIMELS UNIOUE SIMULTANEOUS SYNERGISTIC EFFECT BETWEEN 11= 870 NM AND A = 930
NM OPTICAL ENERGIES
Experimental in vitro data also demonstrates that there is an additive effect
between the two NIMELS wavelengths (870 nm and 930 nm) when they are used
simultaneously (870 nm combined with 930 nm). The presence of the 870 nm
NIMELS
wavelength and the 930 nm NIMELS wavelength as a simultaneous irradiance
absolutely enhances the effect of the antibacterial efficacy of the NIMELS
system.
In vitro experimental data (see, for example, Tables IX and X below)
demonstrated that by combining A = 870 nm and A = 930 nm (in this example used
simultaneously) effectively reduces the 930 nm optical energy and density by
about half
of the total energy and energy density required when using a single treatment
according to the invention.
Table 21. E. coli data from Combined NIMEL Wavelen ths
OUTPUT
POWER
(W) BEAM TOTAL ENERGY
870NM/ SPOT TIME ENERGY DENSI7Y POWERDENSITY E-COLIKILL
930NM (CM) (SEC) ]OULES Q/CM1) (W/CMI) PERCENTAGE
5W+5W= 720 3600 (x2) 2037 (x2)
1.5 = 7200 = 4074 5.66 100 %
Table 22. S. aureus data from Combined NIMELS Wavelen ths
OUTPUT
POWER
(W) BEAM TOTAL ENERGY S.AUREUS
870NM/ SPOT TIME ENERGY DENSITY POWER DENSITY KILL
930NM (CM) (SEC) )OULES (]/CM2) (W/CM=) PERCENTAGE
-102-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
5W +5W= 720 3600 (x2) 2037 (x2)
W 1.5 - 7200 = 4074 5.66 98.5 %
5.5W +5.5 720 3960 (x2) 2241 (x2)
=11 W 1.5 = 7920 = 4482 6.22 100%
This simultaneous synergistic ability is significant to human tissue safety,
as the
930 nm optical energy, heats up a system at a greater rate than the 870 nm
optical
energy, and it is beneficial to a mammalian system to produce the least amount
of heat
possible during treatment.
It is also believed that if the NIMELS optical energies (870 nm and 930 nm)
are
used simultaneously in the above manner with other bacterial species, that the
100%
antibacterial effect will be essentially the same. (See, Figures 17, 18, and
19.)
Experimental in vitro data also demonstrates that there is an additive effect
between the
two NIMELS wavelengths (870 nm and 930 nm) when they are used simultaneously
on
fungi. The presence of the 870 nm NIMELS wavelength and the 930 nm NIMELS
wavelength as a simultaneous irradiance have been found to enhance the effect
of the
anti-flxngal efficacy of the NIMELS system.
Experimental in vitro data demonstrates that this synergistic effect
(connecting
the 870 nm wavelength to the 930 nm wavelength for simultaneous irradiation)
allows
for the 930 nm optical energy to be reduced to approximately 1/2 of the total
energy and
energy density required for NIMELS 100 % C. albicans anti-fungal efficacy,
when the
(870 nm before 930 nm) wavelengths are combined in a simultaneous manner.
Table 23. Candida albicans from Combined NIMELS Wavelengths
ouTPCrr
POWER
(W) BEAM T07AL ENERGY POWER C.ALBICANS
870NM/ SPOT TIME ENERGY DENSITY DENSrrY KILL
930NM (CM) (SEC) JOULES (J/CMl) (W/CM') PERCENTAGE
5W + 720 3600 (x2) 2037 (x2) =
5 W=10 1.5 = 7200 4074 5.66 100 %
Thus, NIMELS wavelengths (A = 870 nm and 930 nm) may be used to achieve
antibacterial and anti-fungal efficacy in an alternating mode or
simultaneously or in any
combination of such modes thereby reducing the exposure at the l= 930
associated with
temperature increases which are minimized.
-103-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Experimental in vitro data also demonstrates that when E. coli is irradiated
alone
with a (control) wavelength of l= 830 nm, at the following parameters, the
control 830
nm laser produced zero antibacterial efficacy for 12 minutes irradiation
cycles, at
identical parameters to the minimum NIMELS dosimetry associated with 100%
antibacterial and anti-fungal efficacy with radiation of A = 930 nm.
Table 24. E coli Single Wavelength l= 830 nm
OUTPUT BEAM TOTAL ENERGY POWER
POWER SPOT TIME ENERGY DENSITY DENSITY
(W) (CM) (SEC.) JOULES Q/CM2) (W/CMZ)
8.0 1.5 720 5760 3259 4.53
9.0 1.5 720 6480 3667 5.09
Experimental in vitro data also demonstrates that when applied at safe thermal
dosimetries, there is little additive effect when using radiance of A = 830 nm
in
combination with A = 930 nm. The presence of the 830 nm control wavelength as
a first
irradiance is far inferior to the enhancement effect of the 870 nm NIMELS
wavelength in
producing synergistic antibacterial efficacy with the second 930 nm NIMELS
wavelength.
Table 25. E. coli data from Substituted alternating 830 nm control Wavelength
OUTPUT
POWER
(W) BEAM TOTAL
830NM/ SPOT TIME ENERGY ENERGY DENSITY POWER DENSITY E. COLI KILL
930NM (CM) (SEC) JOULES (J/CM1) (W/C0) PERCENTAGE
540 /
180 4320 / 1440 =
8W/8W 1.5 12min 5760 2445/815=3529 4.53/4.53 0%
240 /
W/ 240 2400 / 2400 =
10W 1.5 8 min 4800 1358 / 1358 = 2716 5.66 / 5.66 65%
Experimental in vitro data also demonstrates that when applied at safe thermal
dosimetries, there is less additive effect with the 830 nm wavelength, and the
NIMELS
930 nm wavelength when they are used simultaneously. In fact, experimental in
vitro
data demonstrates that 17% less total energy, 17% less energy density, and 17%
less
power density is required to achieve 100 % E. coii antibacterial efficacy when
870 nm is
-104-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
combined simultaneously with 930 nm vs. the commercially available 830 nm.
This,
again, substantially reduces heat and harm to an in vivo system being treated
with the
NIMELS wavelengths.
Table 26. E. coli data from Substituted Simultaneous 830 nm control Wavelen th
OUTPUT
POWER (W) BEAM TOTAL
830NM/ SPOT TIME ENERGY ENERGY DENSITY POWER DENSITY E-COLI KILL
930NM (CM) (SEC) JOULES Q/CM2) (w/CM1) PERCENTAGE
720 3600(x2)
5W + 5 W=10 1.5 =7200 2037(x2) =4074 5.66 91 %
5.5W+5.5 720 3960(x2)
=11 W 1.5 =7920 2250(x2) =4500 6.25 90 %
3960 (x2)
6 W+6 W 720 f 2454(x2)
=12 W 1.5 =864D' =4909" 6.81" 100 %
Amount of Bacteria Killed:
In vitro data also showed that the NIMELS laser system in vitro is effective
(within thermal tolerances) against solutions of bacteria containing 2,000,000
(2 x 106)
Colony Forming Units (CFU's) of E. coli and S. aureus. This is a 2x increase
over what is
typically seen in a 1 gm sample of infected human ulcer tissue. Brown et al.
reported
that microbial cells in 75% of the diabetic patients tested were all at least
100,000
CFU/gm, and in 37.5% of the patients, quantities of microbial cells were
greater than
1,000,000 (1x106)CFU (see Brown et aC., Ostomy Wound Management, 401:47, issue
10,
(2001), the entire teaching of which is incorporated herein by reference).
Thermal Parameters:
Experimental in vitro data also demonstrates that the NIMELS laser system can
accomplish 100% antibacterial and anti-fungal efficacy within safe thermal
tolerances
for human tisstzes.
EXAMPLE XX
THE EFFECTS OF LOWER TEMPERATURES ON NIMELS
Cooling of Bacterial species:
- 105 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Experimental in vitro data also demonstrated that by substantially lowering
the
starting temperature of bacterial samples to 4 C for two hours in PBS before
lasing
cycle, that optical antibacterial efficacy was not achieved at any currently
reproducible
antibacterial energies with the NIMELS laser system.
EXAMPLE XXI
NIMELS EFFECTS ON TRYCHOPHYTON RUBRUM
This example demonstrates the effects NIMELS wavelengths (870 nm and 930
nm) when used in alternating or simultaneous modes.
Table 27. NIMELS T. rubrutn Tests Alternating Wavelengths
OUTPUT
POWER (W)
870 NM/ BEAM TOTAL ENERGY ENERGY DENSITY POWER DENSI7Y
Exp. NO. 930 NM SPOT (CM) T1ME (SEC.) JOULES (J/CM1) (W/CM=)
540 / 180
4320 / 1440 2445 / 815
1 8W/8W 1.5 12min. =5760 =3529 4.53/4.53
240 / 240
2400 / 2400 1358 / 1358
2 lOW/IOW 1.5 8min. =4800 =2716 5.66/5.66
Experiment No.1 = Minimal Effect
Experiment No.2 = 100% Kill in all plates
Table 28. NIMELS T. rubrum -- Simultaneous Wavelengths
OUTPUT
POWER(W)
Ex 870 NM & BEAM TOTAL ENERGY ENERGY DENSITY POWER DENSTTY
NO. 930 NM SPOT (CM) TSME (SEC.) JOULES Q/CMI) (W/CM2)
720 2037 (x2)
3 5+5= 10 1.5 12 min. 3600 (x2) =7200 =4074 5.66
5.5W+5.5W 720
4 =11 W 1.5 3960 (x2) =7920 2250(x2) =4500 6.25
6 W+6 W 720
=12 W 1.5 3960 (x2) =8640 2454(x2) =4909 6.81
Experiments Nos. 3, 4, and 5= 100% Kill in all plates
- 106 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Table 29. NIMELS T. rubrum - Single Wavelength
EXP No BEAM TOTAL
OUTPUT SPOT ENERGY ENERGY DENSTPY POWER DENSTTY
/,=930 POWER(W) (CM) TIME(SEC.) JOULES Q/CMZ) (W/CMZ)
6 8.0 1.5 720 5760 3259 4.53
720
7 9.0 1.5 6840 3681 5.11
Experiments Nos. 6 and 7 = 100% Kill in all plates
Table 30. Control T. rubrurn -- 830 nm / 930nm Alternating
EXPERIMENT
No.
J1830 & OUTPUT
POWER BEAM TOTAI.ENERGY ENERGYDENBITY POWERDENSITY
A=930 (W) SPOT (CM) TIME (MIN.) JOULES Q/CMl) (W/CMl)
540 / 180
8 8W/SW 1.5 12min 4320 / 1440 = 5760 2445 / 815 = 3529 4.53/4.53
240 / 240
9 10W/lOW 1.5 8min 2400/2400=4800 1358 / 1358 = 2716 5.66/5.66
Experiment No. 8 = No Effect
Experiment No. 9 =100% Kill
Treatments as described in the above Table XVIII resulted in 100% kill.
Table 31 In Vitro Targeting of T ratbrum using A = 830 nm and 930nm
BEAM TOTAL
OIJTPUTPOWER SPOT TIME ENERGY ENERGYDENSI7Y POWER DENSITY
(W) (CM) (SEC.) IOULES (J/CMZ) (W/CMZ)
720 3600(x2)
+ 5=10 1.5 =7200 2037(x2) =4074 5.66
EXAMPLE XXII
MRSA/ ANTIMICROBIAL POTENTIATION
-107-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
This example shows the use of NIMELS wavelengths (A = 830 nm and 930nm)
in in vitro targeting of MRSA to increase antimicrobial sensitivity to
methicillin. Four
separate experiments have been performed. The data sets for these four
experiments
are presented in the tables that follow. See, also, Figure 17, which shows:
(a) the
synergistic effects of NIMELS with methicillin, penicillin and erythromycin in
growth
inhibition of MRSA colonies; data show that penicillin and methicillin is
being
potentiated by sub-lethal NIMELS dosimetry by inhibiting the Bacterial Plasma
Membrane Proton-motive force (Ap-plas-Bact) thereby inhibiting peptidoglycan
synthesis anabolic processes that are co-targeted with the drug; and (b) that
erythromycin is potentiated to a greater extent, because the Nimels effect is
inhibiting
the Bacterial Plasma Membrane Proton-motive force (Ap-plas-Bact) that supplies
the
energy for protein synthesis anabolic processes and erythromycin resistance
efflux
pumps.
Materials:
Table 32.
Bacteria
ATCC Number:
Top of Form BAA-43T"' Price:
Bottom of Form
Organism: Staphylococcusaureu Staphylococcus aureus subsp. aureus Rosenbach;
deposited as
s Rosenbach
3hospital, Lisbon, Portugal, 1998
Designations: HSJ216 Isolation: 1476
Depositor: H De Lencastre F__
Biosafety Level: 2 Shipped: freeze-dried
Growth ATCC medium 260: Trypticase soy agar with defibrinated sheep blood
Growth conditions: aerobic
Conditions: Temperature: 37.OC
In addition to the MTA mentioned above, other ATCC and/or
regulatorv permits may be required for the transfer of this ATCC
Permits/Forms: material. Anyone purchasing ATCC material is ultimately
responsible
for obtaining the permits. Please click here for information regarding
the specific reqtiirements for shipment to your location.
Related Products
Comments: Brazilian clone of MRSA 12386
-108-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Applications: liresistant to methicillin [51476]
51476: de Sousa MA, et al. Intercontinental spread of a multidrug-
resistant methicillin-resistant Staphylococcus aureus clone. J. Clin.
References: Microbiol. 36: 2590-2596, 1998. PubMed: 9705398
12386: Herminia De Lencastre, personal communication, the entire
teaching isincorporated herein by reference.
General Methods for CFU counts:
Table 33.
ime
Task TE
(hrs)
hrs)
Inoculate ovemight ctilture
-18 50 ml directly from glycerol stock
Set up starter cultures
-4 Three dilutions 1:50, 1:125, 1:250
Monitor OD6oo of starter cultures
Preparation of plating culture
At 10:00am, the culture which is at ODsoo =1.0 is diluted 1:300 in
0 PBS (50 mis final volume) and stored at RT for 1 hour.
(Room temp should be -25 C)
Seeding of 24-well plates
+1 2 ml aliquots are dispensed into pre-designated wells in 24-well
plates and transferred to NOMIR (8 24-well plates total)
Dilution of treated samples
+2 to After laser treatment, 100 l from each well is diluted serially to
+8
a final dilution of 1:1000 in PBS.
Plating of treated samples
100 l of final dilution is plated in triplicate on TSB agar with
and without 30 g/ml methicillin. (6 TSB plates per well)
- 109 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Plates are incubated at 37 C 18-24hrs.
+24 Colonies are counted on each plate (96 plates total)
Table 34. MRSA Dosimetry Progression 11-06-06 Experiment #1
First lasing procedure :
Both 870 and 930
Second lasing procedure
930 alone
Output Beam Total Energy Power
Power Spot Area of Time Energy Density Density Temp Temp
Parameters (W) (cm) S ot(cm2) (sec) Joules Ulcm') (Wlcm') Initial C Final C
Test (1) 870 at 5W and 930 at SW
for 12 min followed by 10.0 1.5 1.77 720 7200 4074 5.66 24.4 44
Test (1) 930 at 8W for 6 min 8.0 1.5 1.77 360 2880 1630 4.53 44 46.8
Teat (2) 870 at 55W and 930 at
5.5W for 12 min followed by 11.0 1.5 1.77 720 7920 4482 6,22 26.5 48.1
Test (2) 930 at SW for 6 min 8.0 1.5 1.77 360 2880 1630 4.53 48.1 47.4
Test (3) 870 at 5.5W and 930 at
5.5W for 10 min follawed by 10.0 1.5 1.77 600 6000 3395 5.66 25.7 43.1
Test (3) 930 al8W for 4 min 8.0 1.5 1.77 240 1920 1086 453 43.1
Test 44.8
(4) 870 at 5.5W and 930 at
S.5W for 10 rnin followed 6 11.0 1.5 1.77 600 6600 3735 6.22 22.9 45.2
Test (4) 930 at BW for 4 min 8.0 1.5 1.77 240 1920 1086 4.53 45.2 45.3
Test (5) 870 at 5W and 930 at 5W
for 8 min followed 6 10.0 1.5 1.77 480 4800 2716 5.66 24.2 43.2
Test (5) 930 at 7W for 4 min 7.0 1.5 1.77 240 1680 951 3.96 43.2 43.8
Test (6) 870 at 5.5W and 930 at
5.5W for 8 min followed by 1110 1.5 1.77 480 5280 2968 6.22 25.3 42.7
Test (6) 930 at 7W for 4 min 7.0 1.5 1.77 240 1680 951 3.96 42.7 44.9
Test (7) 870 at 5W and 930 at 5W
for 6 min followed b 10.0 1.5 1.77 360 3600 2037 5.66 26.2 40.6
Test (7) 930 at 7W for 4 min 7.0 1.5 1.77 240 1680 951 3.96 40.6 44
Test (8) 870 at 5.5W and 930 at
5.5W for 6 min followed 6 11.0 1.5 1.77 360 3960 2241 6.22 26 42
Test (8) 930 at 7W for 4 min 7.0 1.5 1.77 240 1660 951 3.96 42 ' 44.2
Independent Report for MRSA studies, 07 NOV 2006
-110-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
(MRSA Data Progression 11-07-06 Experiment #1)
Experiment 1 - design:
Eight different laser dosages were used to treat a saline-suspension of
logarithmically growing MRSA, labeled Al to Hl.
The treated and a control untreated suspension were diluted and plated in
triplicate on trypic soy agar with or without 30 g/ml methicillin.
After 24hrs of growth at 37 C colonies were counted.
CFU (colony forming units) were compared between the plates with and
without methicillin for both control (untreated) and treated MRSA.
Experiment 1 - results:
Conditions Dl through Hl showed a similar reduction in CFU on the methicillin
plates in treated and untreated samples.
Conditions Al, Bl and Cl showed 30%, 33%, or 67% fewer CFU in the laser
treated samples compared to the untreated controls, respectively.
This indicates that the treatments performed on sample Al, Bl and Cl
sensitized
the MRSA to the effects of methicillin.
Table 35 MRSA Data Progression 11-07-06 Experiment #1
Methicillin CPU AVG CFU/ml Meth Laser
(Meth) Effect Effect
(+Meth)
1 224 203.7 6.11E+08
no 2 266
3 121
Cont 1 207 141.7 4.25E+08 0.695581
yes 2 137
Al 3 81
1 132 134.3 4.03E+08
no 2 143
Exp 3 128
1 99 99.7 2.99E+08 0.741935 0.7035
yes 2 96
3 104
Bl Cont no 1 235 188.3 5.65E+08
- 111 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
2 1 166 169.3 5.08E+08 0.899115
yes 2 192
3 150
1 213 200.3 6.01E+08
no 2 199
3 189
Exp
1 102 113.3 3.40E+08 0.565724 0.6693
yes 2 107
3 131
1 280 320.3 9.61 E+08
no 2 242
Cont 3 439
1 240 406 1.22E+09 1.26743
yes 2 466
C1 3 512
1 187 184 5.52E+08
no 2 189
Exp 3 176
1 95 132.3 3.97E+08 0.719203 0.3259
yes 2 176
3 126
1 251 184 5.52E+08
no 2 125
3 176
Cont 1 171 154 4.62E+08 0.836957
yes 2 141
D 1 3 150
1 221 203.7 6.11E+08
no 2 180
3 210
Exp 1 164 155.3 4.66E+08 0.762684 1.0087
yes 2 153
3 149
1 142 225.3 6.76E+08
no 2 268
Cont 3 266
1 147 131.3 3.94E+08 0.58284
yes 2 121
3 126
El 1 226 258.3 7.75E+08
no 2 217
Exp 3 332
1 181 214.3 6.43E+08 0.829677 1.632
yes 2 232
3 230
Fl Cont no 1 223 226.7 6.80E+08
- 112 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
2 260
3 197
1 197 198 5.94E+08 0.873529
yes 2 188
3 209
1 223 237.7 7.13E+08
no 2 256
Exp 3 234
1 206 197 5.91E+08 0.828892 0.9949
yes 2 179
3 206
1 214 224 6.72E+08
no 2 217
3 241
Cont 1 246 219.3 6.58E+08 0,979167
yes 2 222
G1 3 190
1 243 242.7 7.28E+08
no 2 261
Exp 3 224
1 193 210.7 6.32E+08 0.868132 0.9605
yes 2 237
3 202
1 252 255.3 7.66E+08
no 2 267
Cont 3 247
1 188 192.3 5.77E+08 0.753264
yes 2 206
Hl 3 183
1 232 245 7.35E+08
no 2 232
Exp 3 271
1 211 199.7 5.99E+08 0.814966 1.0381
yes 2 21
3 176
Table 36. MRSA Dosimetry Progression 11-07-06 Experiment #2
MRSA Dosimetry Progression 11-07-06
First lasing procedure : Both 870 and 930
Second lasing rocedure 930 alone
geam Energy Power
Output Spot Area of Total Energy Density Density Temp Temp
Parameters Power (W) (cm) 5 ot(cm2) Time (sec) Joules Q/cm') (W/em') Initial C
Final C
Test (1) 870 at
5W and 930 at 10.0 1.5 1.77 720 7200 4074 5.66 23.4 45.3
-113-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
5W for 12 min
followed by
Test (1) 930 at
8W for 6 min 8.0 1.5 1.77 360 2880 1630 4.53 45.3 46.8
Test (2) 870 at
5W and 930 at
5W for 12 min
followed 6 10.0 1.5 1.77 720 7200 4074 5.66 21.2 45.5
Test (2) 930 at
8W for 6 min 8.0 1.5 1.77 360 2880 1630 4.53 45.5 47.7
Test (3) 870 at
SW and 930 at
5W for 12 min
followed 6 10.0 1.5 1.77 720 7200 4074 5.66 21.6 47.0
Test (3) 930 at
SW for 6 min 8,0 1.5 1.77 360 2880 1630 4.53 47.0 49.0
Test (4) 870 at
5.5W and 930
at 55W for 12
min followed
by 11.0 1.5 1,77 720 7920 4482 6.22 20.4 50,3
Test (4) 930 at
8W for 6min 8.0 1.5 1.77 360 2880 1630 4.53 50.3 50,1
Test (5) 870 at
5.SW and 930
at55W for
12min
followed by 11.0 1.5 1.77 720 7920 4482 6.22 24.0 50.9
Test (5) 930 a t
SW fur 6 min 8.0 1.5 1.77 360 2880 1630 4.53 50.9 502
Test (6) 870 at
5.5W and 930
at 5.5W for 12
min followed
by 11.0 1.5 1.77 720 7920 4482 6.22 23.0 48.2
Test (6) 930 at
8W for 6 min 80 1.5 1.77 360 2860 1630 4.53 48.2 48.3
E Test (7) 870 at
5W and 930 at
5W for 14 min
followed 6 10.0 1.5 1.77 840 8400 4753 5.66 22.0 48.3
Test (7) 930 at
7W for 8 min 7.0 1.5 1.77 480 3360 1901 3.96 48.3 44.2
Test (8) 870 at
5W and 930 at
5W for 14 min
followed by 11.0 1.5 1.77 840 9240 5229 6.22 22.0 47.6
Test (8) 930 at
7W for 8 min 7.0 1.5 1.77 480 3360 1901 3.96 47.6 46.2
Independent Report for MRSA studies, 08 NOV 2006
(MRSA Data Progression 11-08-06 Experiment #2)
- 114 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Experiment 2 - design:
Eight different laser dosages based on an effective dose established in
experiment 1 and previously were used to treat a saline-suspension of
logarithmically
growing MRSA, labeled A2 to H2.
The treated and a control untreated suspension were diluted and plated in
triplicate on trypic soy agar with or without 30 g/ml methicillin.
After 24hrs of growth at 37 C colonies were counted.
Experiment 2 - results:
Comparison of CFU on plates with and without methicillin showed a significant
increase in the effectiveness of methicillin in all laser treated samples with
the exception
of A2 and B2. This data is summarized in tabular form below.
Table 37.
Grouping Fold increase in methicillin sensitivity
A2 0.84
B2 0.91
C2 3.20
D2 2.44
E2 4.33
F2 2.13
G2 3.47
H2 1.62
Table 38. MRSA Data Progression 11-08-06 Experiment #2
NOMIR MRSA Study 07-08 NOV 2006
Methicillin CFU AVG CFU/ml Meth Effect Laser
(Meth) Effect
(+Meth)
A2 1 51 49.3 1.48E+08
no 2 43
3 54
Cont 1 35 35.7 1.07E+08 0.72
yes 2 47
3 25
Exp no 1 49 47 1.41E+08
2 45
- 115 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
3 47
1 39 41 1.23E+08 10.87 1.15
yes 2 48
3 36
1 97 71.3 2.14E+08
no 2 47
3 70
Cont 1 47 49.7 1.49E+08 0.7
yes 2 56
B2 3 46
1 32 34.7 1.04E+08
no 2 34
3 38
Exp 1 27 26.7 8.OOE+07 0.77 0.54
yes 2 28
3 25
1 60 55.7 1.67E+08
no 2 65
3 42
Cont 1 42 55.3 1.66E+08 0.99
yes 2 71
C2 3 53
1 35 40.3 1.21E+08
no 2 38
3 48
Exp 1 16 12.7 3.80E+07 0.31 0.23
yes 2 12
3 10
1 108 85.3 2.56E+08
no 2 85
3 63
Cont 1 20 52 1.56E+08 0.61
yes 2 65
D2 3 71
1 9 9.3 2.80E+07
no 2 9
3 10
Exp
1 5 2.3 7.00E+06 0.25 0.04
yes 2 1
3 1
E2 1 52 59.7 1.79E+08
no 2 60
3 67
Cont 1 68 62.3 1.87E+08 1.04
yes 2 66
3 53
Exp no 1 8 11 3.30E+07
2 12
-116-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
3 13
1 2 2.7 8.00E+06 0.24 0.04
yes 2 2
3 4
1 125 87.7 2.63E+08
no 2 73
3 65
Cont 1 62 71 2.13E+08 0.81
yes 2 64
F2 3 87
1 37 41 1.23E+08
no 2 43
Exp 3 43
1 13 15.7 4.70E+07 0.38 0.22
yes 2 15
3 19
1 77 80 2.40E+08
no 2 110
3 53
Cont 1 75 83.3 2.50E+08 1.04
yes 2 92
G2 3 83
1 26 28 8.40E+07
no 2 28
3 30
Exp 1 10 8.3 2.50E+07 0.3 0.1
yes 2 7
3 8
1 77 105.7 3.17E+08
no 2 156
3 84
Cont 1 76 76.7 2.30E+08 0.73
yes 2 72
H2 3 82
1 28 28.3 8.50E+07
no 2 36
3 21
Exp
1 13 12.7 3.80E+07 0.45 0.17
yes 2 12
3 13
Table 39. Outlined Protocol for NOMIR MRSA study - November 09 2006
(11-09-06 Experiment #3)
Method:
- 117 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Time
Task FTE
(~s) (hrs)
Inoculate overnight culture
T-18 1
50 ml directly from glycerol stock
Set up starter cultures
T-4 1
Three dilutions 1:50, 1:125, 1:250
Monitor ODeoo of starter cultures 4
Preparation of plating culture
At 10:00am, the culture which is at OD600 = 1.0 is diluted
T 0 1
1:300 in PBS (50 mls final volume) and stored at RT for 1 hour.
(Room temp should be -25 C)
Seeding of 24-well plates (S plates in total)
T+1 2 ml aliquots are dispensed into pre-designated wells in 1
24-well plates and transferred to NOMIR (8 24-well plates total)
Dilution of treated samples
T +2 to +8 After laser treatment, 100 pl from each well is diluted 4
serially to a final dilution of 1:1000 in PBS.
Plating of treated samples
100 41 of final dilution is plated in quintu~licate (5X) on 2
TSB agar with and without 30 g/mI methicillin. (10 TSB plates per
well)
Plates are incubated at 37 C 18-24hrs.
T +24 Colonies are counted on each plate (160 plates total) 6
Table 40. MRSA Dosimetry Progression 11-09-06 Experiment #3
MRSA Dosimetry Progression 11-09-06
First lasing procedure : Both 870 and 930
Second lasing procedure 930 alone
- 118 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Beam Area of Total Energy Power
Output Spot Spot Time Energy Density Density Temp Temp
Parameters Power (W) (rm) (cm2) (sec) Joules (J/,,m2) (W/cmz) Initial C Final
C
Test (1) 870 at
5.5W and 930 at
5.5W for 12 min
followed by 11.0 1.5 1.77 720 7920 4482 6.22 22.0 48.1 Test (1) 930 at
8W for 6 min 8.0 1.5 1.77 360 2880 1630 4.53 48.1 47.7
Test (2) 870 at
5.5W and 930 at
5.5W for 12 min
followed by 11.0 1.5 1.77 720 7920 4482 6.22 22.9 48.8
Test (2) 930 at
8W for 6 min 8.0 1.5 1.77 360 2880 1630 4.53 48.8 48.7
Test (3) 870 at
5.5W and 930 at
5.5W for 12 min
followed by 11,0 1.5 1.77 720 7920 4482 6.22 22.8 48.9
Test (3) 930 at
8W for 6 min 8.0 1.5 1.77 360 2880 1630 4.53 48.9 48.9
Test (4) 870 at
5.5W and 930 at
5.5W for 12 min
followed 6 11.0 1.5 1.77 720 7920 4482 6.22 24.0 50.3
Test (4) 930 a t
SW for 6min 8.0 1.5 1.77 360 2880 1630 4.53 50.3 50.5
Test (5) 870 at
5W and 930 at
5W for14min
followed b 10.0 1.5 1.77 840 8400 4753 5.66 23.7 48.4
Test (5) 930 at
6W for 9 min 6.0 1.5 1.77 540 3240 1833 140 48.4 45.0
Test (6) 870 at
5W and 9.30 at
5W for 14 min followed by 10.0 1.5 1.77 840 8400 4753 5.66 23.5 49.2
Test (6) 930 at
6W for 9 min 6.0 1.5 1.77 540 3240 1833 3.40 42.9 46.3
Test (7) 870 at
5W and 930 at
5W for 14 min
followed 6 10.0 1.5 1.77 840 8400 4753 5.66 25.6 49.9
Test (7) 930 at
6W for 9 min 6.0 1.5 1.77 540 3240 1833 3.40 49.9 46.3
Test (8) 870 at
5W and 930 at
5W for 14 min
followed by 10.0 1.5 1.77 840 8400 4753 5.66 22.1 48.0
Test (8) 930 at
6W for 9 min 6.0 1.5 1.77 540 3240 1833 3.40 48.0 46.0
- 119 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Independent Report for MRSA studies, 09-10 NOV 2006
MRSA Data Progression 11-10-06 Experiment #3
Experiment 3 - design:
Eight different laser dosages based on an effective dose established in
experiments 1 and 2 and previously were used to treat a saline-suspension of
logarithmically growing MRSA, labeled A3 to H3.
The treated and a control untreated suspension were diluted and plated in
pentuplicate on trypic soy agar with or without 30 g/ml methicillin.
After 24hrs of growth at 37 C colonies were counted.
Experiment 3 - results:
Comparison of CFU on plates with and without methicillin showed a significant
increase in the effectiveness of inethicillin in all laser treated samples.
This data is
summarized in tabular form below.
Table 41.
Grouping Fold increase in methicillin sensitivity
A3 1.98
B3 1.62
C3 1.91
D3 2.59
E3 2.09
F3 2.08
G3 3.16
H3 2.97
Table 42. MRSA Data Pro ression 11-10-06 Ex eriment #3
NOMIR MRSA Study 09-10 NOV 2006
Methicillin CFU AVG CFU/ml Meth Effect Laser Effect (+M)
(Meth)
A3 Cont no 1 41 47 1.41E+08
- 120 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
2 63
3 46
4 49
36
1 35 48.4 1.45E+08 1.03
2 45
yes 3 52
4 66
5 44
1 24 31.4 9.42E+07
2 34
no 3 26
4 33
Exp 5 40
1 23 16.2 4.86E+07 0.52 0.33
2 15
yes 3 14
4 16
5 13
1 109 72 2.16E+08
2 61
no 3 59
4 61
Cont 5 70
1 61 71.4 2.14E+08 0.99
2 79
yes 3 51
4 68
B3 5 98
1 27 31.2 9.36E+07
2 25
no 3 39
4 24
Exp 5 41
1 9 19 5.70E+07 0.61 0.27
2 22
yes 3 23
4 25
5 16
C3 Cont 1 46 57.6 1.73E+08
2 60
no 3 60
4 66
5 56
yes 1 70 58.4 1.75E+08 1.01
2 54
3 52
4 51
- 121 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
65
1 52 38.2 1.15E+08
2 34
no 3 38
4 34
Exp 5 33
1 12 20.2 6.06E+07 0.53 0.35
2 26
yes 3 22
4 24
5 17
1 50 50.6 1.52E+08
2 45
no 3 55
4 54
Cont 5 49
1 58 51.2 1.54E+08 1.01
2 46
yes 3 43
4 59
D3 5 50
1 7 9.2 2.76E+07
2 10
no 3 8
4 9
Exp 5 12
1 6 3.6 1.08E+07 0.39 0.07
2 3
yes 3 1
4 5
5 3
E3 1 47 54.8 1.64E+08
2 55
no 3 71
4 45
Cont 5 56
1 56 50.6 1.52E+08 0.92
2 48
yes 3 48
4 52
5 49
Exp 1 50 53.2 1.60E+08
2 65
no 3 49
4 46
5 56
yes 1 15 23.6 7.08E+07 0.44 0.47
2 24
-122-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
3 26
4 27
26
1 57 72.4 2.17E+08
2 142
no 3 62
4 52
Cont 5 49
1 65 53.2 1.60E+08 0.73
2 50
yes 3 54
4 40
F3 5 57
1 29 28.4 8.52E+07
2 39
no 3 25
4 23
Exp 5 26
1 13 9.8 2.94E+07 0.35 0.18
2 10
yes 3 14
4 5
5 7
1 60 57.8 1.73E+08
2 53
no 3 54
4 66
Cont 5 56
1 56 67.6 2.03E+08 1.17
2 56
yes 3 70
4 63
C,3 5 93
1 23 218 6.84E+07
2 24
no 3 21
4 21
Exp 5 25
1 9 8.4 2.52E+07 0.37 0.12
2 11
yes 3 5
4 8
5 9
H3 Cont 1 64 72.4 2.17E+08
2 86
no 3 72
4 45
5 95
-123-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
1 72 75.2 2.26E+08 1.04
2 75
yes 3 71
4 79
79
1 20 23.8 7.14E+07
2 17
no 3 23
4 28
Exp 5 31
1 6 8.4 2.52E+07 0.35 0.11
2 12
yes 3 4
4 9
5 11
- 124 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Table 43. Outlined Protocol for NOMIR MRSA study - November 10 2006
Method:
Time Task FTE
(hrs) (hrs)
Inoculate overnight culture
T-18 1
50 ml directly from glycerol stock
T-4 Set up starter cultures 1
Three dilutions 1:50,1:125, 1:250
Monitor ODeoo of starter cultures 4
Preparation of plating culture
At 10:00am, the culture which is at ODsoo =1.0 is diluted 1:300 in
T 0 1
PBS (50 mis final volume) and stored at RT for 1 hour.
(Room temp sJnould be -25 C)
Seeding of 24-well plates (6 plates in total)
T+1 2 ml aliquots are dispensed into pre-designated wells in 24-well 1
plates and transferred to NOMIR (6 24-well plates total)
Dilution of treated samples
T +2 to +8 After laser treatment, 100 l from each well is diluted serially to
4
a final dilution of 1:1000 in PBS.
Plating of treated samples
100 l of final dilution is plated in Quintuplicate (5X) on TSB
agar in the following manner:
24 well Plate # 1 and 2 with and without 30 g/ml methicillin. 2
24 well Plate # 3 and 4 with and without g/ml Penicillin
24 well Plate # 5 and 6 with and without g/ml Erythromycin
(10 TSB plates per well)
Plates are incubated at 37 C 18-2411rs.
T +24 Colonies are counted on each late (120 plates total) 6
Table 44. MRSA Dosimetr,~~gression 11-10-06 Experiment #4
MRSA Dosimetry Progression 11-10-06
First lasing procedure : Both 870 and 930
Second lasing procedure 930 alone
Output Beam Area of Total Energy Power
Power Spot Spot Time Energy Density Density Temp Temp
Parameters (W) (cm) (cm2) (sec) Joules Q/cm2) (W/cm') InitialC Fina1C
Test (1) 870 at 5.5W and
930 at 5.5W for 12 min
followed by 11.0 1.5 1.77 720 7920 4482 6.22 22.3 46.3
Test (1) 930 at 8W for 6
min (METHICILLIN
PLATES) 8.0 1.5 1.77 360 2880 1630 4.53 46.3 47.6
Test (2) 870 at 5W an1930
at 5W for 14 min followed 10.0 1.5 1.77 840 8400 4753 5.66 23.1 47.1
-125-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
by
Test (2) 930 at 6W for 9
min (METHICILLIN
PLATES) 6.0 1.5 1.77 540 3240 1833 3.40 47.1 44.3
Test (3) 870 at 5.5W and
930 at 5.5W for 12 min
followed by 11.0 1.5 1.77 720 7920 4482 6.22 24.4 48.4
Test (3) 930 at 8W for 6
min (PENICILLIN G
PLATES) 8.0 1.5 1.77 360 2880 1630 4.53 48.4 47.1
Test (4) 870 at 5W and 930
at 5W for 14 min followed
by 10.0 1.5 1.77 840 8400 4753 5.66 23.3 47.9
Test (4) 930 at 6W for 9min
(PENICILLIN G PLATES) 6.0 1.5 1.77 540 3240 1833 3.40 47.9 45.0
Test (5) 870 at 5.5W and
930 at 5.5W for 12min
followed by 11.0 1.5 1.77 720 7920 4482 6.22 22.9 50.2
Test (5) 930 at 8W for 6
min (ERYTHROMYCIN
PLATES) 8.0 1.5 1.77 360 2880 1630 4.53 50.2 51.6
Test (6) 870 at 5W and 930
at 5W for 14 min followed
b 10.0 1.5 1.77 840 8400 4753 5.66 24.2 50.3
Test (6) 930 at 6W for 9
min (ERYTHROMYCIN
PLATES) 6.0 1.5 1.77 540 3240 1833 3.40 50.3 43.6
Independent Report for MRSA studies, 10-11 NOV 2006
(MRSA Data Progression 11-10-06 Experiment #4)
Experiment 4 - design:
Two different laser dosages based on an effective dose established in previous
experiments were used to treat a saline-suspension of logarithmically growing
MRSA,
labeled A4 to F4.
The treated and a control untreated suspension were diluted and plated in
pentuplicate on trypic soy agar with or without 30 g/ml methicillin (Groups
A4 and
134), 0.5 g/ml penicillin G (Groups C4 and D4) or 4 fag/ml erythromycin
(Groups E4
and F4).
After 24hrs of growth at 37 C colonies were counted.
-126-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Experiment 4 - results:
Laser treatment increases sensitivity of MRSA to each antibiotic tested by
several
fold. This data is summarized below.
Series Drug
A4 Methicillin
B4 Methicillin
C4 Penicillin
D4 Penicillin
E4 Erythromycin
F4 Er throm cin
Table 45.
Grouping Fold increase in antibiotic sensitivity
A4 2.19
B4 2.63
C4 2.21
D4 3.45
E4 50.50
F4 9.67
Table 46. MRSA Data Progression 11-10-06 Experiment #4
NOMIR MRSA Study 10-11 NOV 2006
I I Drug? CFU AVG CFU/ml Drug Laser Effect
Effect (+Dru ) A4 1 84 92 2.76E+08
2 95
no 3 69
4 106
106
Cont 1 97 86.2 2.59E+08 0.94
2 104
yes 3 82
4 58
5 90
Exp 1 82 84.4 2.53E+08
2 80
no 3 85
4 90
5 85
yes 1 37 36.2 1.09E+08 0.43 0.42
2 33
-127-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
3 36
4 39
36
1 86 105 3.15E+08
2 142
no 3 105
4 97
5 95
Cont 1 149 132.6 3.98E+08 1.26
2 101
yes 3 119
4 153
5 141
B4 1 73 88.8 2.66E+08
2 84
no 3 109
4 89
5 89
Exp
I 46 42.4 1.27E+08 0.48 0,32
2 34
yes 3 42
4 44
5 46
1 211 143.8 4.31E+48
2 138
no 3 114
4 145
5 111
Cont 1 106 108.4 3.25E+08 0.75
2 99
yes 3 102
4 113
5 122
C4 1 84 90.2 2.71E+98
2 84
no 3 87
4 107
5 89
Exp 1 25 30.4 9.12E+07 0.34 0,28
2 33
yes 3 19
4 33
5 42
D4 Cant 1 111 123.6 3.71E+08
2 110
no 3 135
4 107
5 155
-128-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
1 101 132.8 3.98E+08 1.07
2 111
yes 3 138
4 132
182
1 73 75.6 2.27E+08 2 86
no 3 93
4 74
5 52
Exp 1 14 233.8 7.14E+07 0.31 0.18
2 23
yes 3 22
4 29
5 31
1 122 125.6 377E+08
2 154
no 3 127
4 116
5 109
Cont 1 199 127 3.SiEt08 1,01
2 125
yes 3 103
4 101
5 107
E4 1 17 17.6 5.28E+07
2 20
no 3 18
4 21
5 12
Exp 1 0 0.4 1.20E+06 0.02 0
2 1
yes 3 0
4 0
5 1
F4 1 117 177.8 5.33Ei08
2 126
no 3 318
4 166
5 162
Cont 1 186 155.4 4.66E+08 0.87
2 170
yes 3 121
4 132
5 168
Exp no 1 60 66.4 1.99E+08
2 54
3 60
-129-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
4 102
56
1 2 5.8 1.74E+07 009 0.04
2 7
yes 3 6
4 6
5 8
EXAMPLE XXIII
IN VIVO SAFETY TESTING - HUMAN PATIENT
Following the in vitro fibroblast studies, the inventor performed a dosimetry
titration on himself to ascertain the safe, maximum level of energy and time
of exposure
that could be delivered to human dermal tissue without burning or otherwise
damaging
the irradiated tissues.
The methodology he used was to irradiate his great toe for varying lengths of
time and power settings with the NIMELS laser. The results of this self-
exposure
experiment are described below.
Table 47. Combined Wavelength Dosimetries
O11'IPU7 BEAM AREA OF TIME TOTAL ENERGY POWER
PARAMETERS POWER SPOT SPOT(CM(SEC) ENERGY DENSITY DENSITY
(W) (CM) JOULES Q/CM~) (W/CMZ)
870 nm 1.5 1.5 1.77 250 375 212 0.85
930 nm 1.5 1.5 1.77 250 375 212 0.85
Combined 3.0 1.5 1.77 250 750 424 1.70
Table 48: Dosimetry at A = 930 nm
OUiTUT BEAM AREAOF TIME TOTAL ENERGY POWER
PARAMETERS POWER SPOT SPOT(CM2) (SEC) ENERGY DENSffY DENSIFY
(W) (CM) JOULES U/CMZ) (W/CMZ)
930nm 3.0 1.5 1.77 120 360 204 1.70
Time/Temperature assessments were charted to ensure the thermal safety of
these laser energies on human dermal tissues (data not shown). In one laser
procedure,
he exposed his great toe to both 870 nm and 930 nm for up to 233 seconds,
while
-130-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
measuring toenail surface temperature with a laser infrared thermometer. He
found
that using the above dosimetries, at a surface temperature of 37.5 C, with 870
nm and
930 nm together with a combined Power Density of 1.70 W/cm2, pain resulted and
the
laser was turned off.
In a second laser procedure, he exposed his great toe to 930 nm for up to 142
seconds, while again measuring toenail surface temperature with a laser
infrared
thermometer. He found that, at a surface temperature of 36 C, with 930 nm
alone at a
Power Density of 1.70 W/cm2, pain resulted and the laser was turned off.
EXAMPLE XXIV
IN VIVO SAFETY TESTING - LIMITED CLINICAL PILOT STUDY
Following the experiment above, additional patients with onychomycosis of the
feet were treated. These patients were all unpaid volunteers, who provided
signed
informed consent. The principle goal of this limited pilot study was to
achieve the same
level of fungal decontamination in vivo, as was obtained in vitro with the
NIMELS laser
device. We also decided to apply the maximum time exposure and temperature
limit
that was tolerated by the inventor during his self- exposure experiment.
In a highly controlled and monitored environment, three to five laser exposure
procedures were performed on each subject. Four subjects were recruited and
underwent the treatment. Subjects provided signed informed consent, were not
compensated, and were informed they could withdraw at any time, even during a
procedure.
The dosimetry that was used for the treatment of the first subject was the
same
as that used during the inventor's self-exposure (shown above). The
temperature
parameters on the surface of the nail also were equivalent to the temperatures
found by
the inventor on self-exposure.
The treated toes showed significantly reduced Tinea pedis and scaling
surrounding the nail beds, which indicated a decontamination of the nail plate
that was
acting as a reservoir for the fungus. The control nails were scraped with a
cross-cut
- 131 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
tissue bur, and the shavings were saved to be plated on mycological media. The
treated
nails were scraped and plated in the exact same manner.
For culturing the nail scrapings, Sabouraud dextrose agar (2% dextrose) medium
was prepared with the following additions: chloramphenicol (0.04 mg/ml), for
general
fungal testing; chloramphenicol (0.04 mg/ml) and cycloheximide (0.4g/ml),
which is
selective for dermatophytes; chloramphenicol (0.04 mg/ml) and griseofulvin (20
g/ml),
which served as a negative control for fungal growth.
Nine-day mycological results for Treatment #1 and Treatment #2 (performed
three days
after Treatment #1) were the same, with a dermatophyte growing on the control
toenail
plates, and no growth on the treated toenail plates. Treated plates did not
show any
growth whereas untreated control culture plates showed significant growth.
The first subject was followed for 120 days, and received four treatments
under the
same protocol. Figure 18 shows a comparison of the pretreatment (A), 60 days
post-
treatment (B), 80 days post-treatment (C), and 120 days post-treatment (D)
toenails.
Notably, healthy and non-infected nail plate was covering 50% of the nail area
and
growing from healthy cuticle after 120 days.
While certain embodiments have been described herein, it will be understood by
one skilled in the art that the methods, systems, and apparatus of the present
invention
may be embodied in other specific forms without departing from the spirit
thereof. The
present embodiments are therefore to be considered in all respects as
illustrative and
not restrictive of the present invention. It is understood that the Human nail
acts as a
refractory lens, and disperses and/or reflects portions of the NIMELS infrared
energy.
Hence, Porcine skin dose/tolerance studies were performed to titrate maximum
NIMELS dosimetry without burn/damage to tissues. Porcine skin was used as a
model
for human skin. These studies were carried out in compliance with the Animal
Protection Act and according to the NIH Guide for the Care and Use of
Laboratory
Animals. These tests are shown below.
Porcine Skin Dose/Tolerance studies
Table 49.
- 132 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Output Beam Area of Total Energy Power
Dose Parameters Power Spot Spot Time Energy Density Density
ID (nm) (W) (cm) (cm2) (sec) Ooules) (J/cm2) (W/cm2)
870 1.3 1.5 1.77 120 156 88 0.74
1 930 1.3 1.5 1.77 120 156 88 0.74
Combined 2.6 1.5 1.77 120 312 177 1.47
930 Alone 2.6 1.5 1.77 50 130 74 1.47
870 1.3 1.5 1.77 140 182 103 0.74
2 930 1.3 1.5 1.77 140 182 103 0.74
Combined 2.6 1.5 1.77 140 364 206 1.47
930 Alone 2.6 1.5 1.77 60 156 88 1.47
870 1.3 1.5 1.77 160 208 118 0.74
3 930 1.3 1.5 1.77 160 208 118 0.74
Combined 2.6 1.5 1.77 160 416 235 1.47
930 Alone 2.6 1.5 1.77 70 182 103 1.47
870 1.3 1.5 1.77 180 234 132 0.74
4 930 1.3 1.5 1.77 180 234 132 0.74
Combined 2.6 1.5 1.77 180 468 265 1.47
930 Alone 2.6 1.5 1.77 80 208 118 1.47
870 1.5 1.5 1.77 100 150 85 0.85
930 1.5 1.5 1.77 100 150 85 0.85
Combined 3 1.5 1.77 100 300 170 1.7
930 Alone 3 1.5 1.77 40 120 68 1.7
870 1.5 1.5 1.77 120 180 102 0.85
6 930 1.5 1.5 1.77 120 180 102 0.85
Combined 3 1.5 1.77 120 360 204 1.7
930 Alone 3 1.5 1.77 50 150 85 1.7
870 1.5 1.5 1.77 140 210 119 f 0.85
-133-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
7 930 1.5 1.5 1.77 140 210 119 0.85
Combined 3 1.5 1.77 140 420 238 1.7
930 Alone 3 1.5 1.77 60 180 102 1.7
870 Control Control Control Control Control Control Control
8 930 Control Control Control Control Control Control Control
Combined Control Control Control Control Control Control Control
930 Alone Control Control Control Control Control Control Control
870 1.15 2 3.14 100 115 37 0.37
9 930 1.15 2 3.14 100 115 37 0.37
Combined 2.3 2 3.14 100 230 73 0.73
930 Alone 2.3 2 3.14 40 92 29 0.73
870 1.15 2 3.14 120 138 44 0.37
930 1.15 2 3.14 120 138 44 0.37
Combined 2.3 2 3.14 120 276 88 0.73
930 Alone 2.3 2 3.14 50 115 37 0.73
870 1.15 2 3.14 140 161 51 0.37
11 930 1.15 2 3.14 140 161 51 0.37
Combined 2.3 2 3.14 140 322 102 0.73
930 Alone 2.3 2 3.14 60 138 44 0.73
870 1.15 2 3.14 160 184 59 0.37
12 930 1.15 2 3.14 160 184 59 0.37
Combined 2,3 2 3.14 160 368 117 0.73
930 Alone 2.3 2 3.14 70 161 51 0.73
870 1.15 2 3.14 180 207 66 0.37
13 930 1.15 2 3.14 180 207 66 0.37
Combined 2.3 2 3.14 180 414 132 0.73
930 Alone 2.3 2 3.14 80 184 59 0.73
- 134 -
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
870 1.15 2 3.14 200 230 73 0.37
14 930 1.15 2 314 200 230 73 0.37
Combined 2.3 2 3.14 200 460 146 0.73
930 Alone 2.3 2 3.14 90 207 66 0.73
870 1.15 2 3.14 240 276 88 0.37
15 930 1.15 2 3.14 240 276 88 0.37
Combined 2.3 2 3.14 240 552 176 0.73
930 Alone 2.3 2 3.14 120 276 88 0.73
870 Control Control Control Control Control Control Control
20 930 Control Control Control Control Control Control Control
Combined Control Control Control Control Control Control Control
930 Alone Control Control Control Control Control Control Control
EXAMPLE XXV
Non-thermal NIMELS Interaction
Evidence for non-thermal NIMELS interaction:
It was demonstrated through experimentation (in vitro water bath studies),
that
the temperatures reached in the in vitro NIMEL,S experimentation, were not
high
enough in and of themselves to neutralize the pathogens.
In the chart that follows, it can clearly be seen that when simple E. coli
Bacteria
were challenged at 47.5 C continuously for 8 minutes in a test tube in a water
bath, they
achieved 91% growth of colonies. Therefore, it was demonstrated essentially
that the
NIMELS reaction is indeed photo-chemical in nattire, and occurs in the absence
of
exogenous drugs and/or dyes.
-135-
CA 02670711 2009-05-26
WO 2008/073979 PCT/US2007/087264
Table 50.
Water Bath Test
Bacteria placed in PBS on bench at room
temperature for 3 hours; then in water bath
at 47.5C for 8 min and lated.
Control Final
8/26/2005 8/26/2005
A 73 D 64
B 82 E 73
75 F 72
Average % 90.9 %
Growth Lived
after 47.5C for 8 min.
- 136 -
