WO2008073979A2 - Modification électromagnétique proche infrarouge de potentiels de membrane stationnaires cellulaires - Google Patents

Modification électromagnétique proche infrarouge de potentiels de membrane stationnaires cellulaires Download PDF

Info

Publication number
WO2008073979A2
WO2008073979A2 PCT/US2007/087264 US2007087264W WO2008073979A2 WO 2008073979 A2 WO2008073979 A2 WO 2008073979A2 US 2007087264 W US2007087264 W US 2007087264W WO 2008073979 A2 WO2008073979 A2 WO 2008073979A2
Authority
WO
WIPO (PCT)
Prior art keywords
targeted
membrane
nimels
bacterial
agent
Prior art date
Application number
PCT/US2007/087264
Other languages
English (en)
Other versions
WO2008073979A3 (fr
Inventor
Eric Bornstein
Original Assignee
Nomir Medical Technologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/981,431 external-priority patent/US8506979B2/en
Priority claimed from US11/981,340 external-priority patent/US20080131968A1/en
Priority claimed from US11/981,486 external-priority patent/US20090299263A1/en
Application filed by Nomir Medical Technologies, Inc. filed Critical Nomir Medical Technologies, Inc.
Priority to JP2009541559A priority Critical patent/JP2010512232A/ja
Priority to CA002670711A priority patent/CA2670711A1/fr
Priority to AU2007333073A priority patent/AU2007333073A1/en
Priority to EP07865581A priority patent/EP2089107A2/fr
Publication of WO2008073979A2 publication Critical patent/WO2008073979A2/fr
Publication of WO2008073979A3 publication Critical patent/WO2008073979A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/0624—Apparatus adapted for a specific treatment for eliminating microbes, germs, bacteria on or in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/067—Radiation therapy using light using laser light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0601—Apparatus for use inside the body

Definitions

  • 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 m target bacterial and/or fungal and/or cancer cells
  • 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 ad j unctive therapy
  • MIC minimum inhibitory concentration
  • antimicrobial agents antimicrobial molecules
  • antineoplastice molecules antimicrobial agents
  • antimicrobial agents antimicrobial agents
  • antineoplastice molecules antimicrobial agents
  • neoplastic agents antineoplastice molecules
  • 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 ( ⁇ d)
  • Figure 4A shows a phospholipid bilayer in bacterial plasma membrane, mammalian mitochondrial membrane, or fugal mitochondrial membrane with a steady-state transmembrane 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 ⁇ -mito-fungi or ⁇ -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-lasmg,
  • 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
  • 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
  • NIMELS irradiation at a single dosimetry
  • 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,
  • ROS reactive oxygen species
  • 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,
  • ROS reactive oxygen species
  • 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,
  • ROS reactive oxygen species
  • 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
  • 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 methicilhn, penicillin and erythromycin in growth inhibition of MRSA colonies
  • Figure 18 is a romposite 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
  • panel D shows the toenail 100 days post treatment
  • Figure 19 illustrates the detection of decreased membrane potential m E coh with sublethal NIMELS irradiation
  • Figure 20 illustrates the detection of increased glutathione in E coh with sub-lethal NIMEl S irradiation
  • a NIMELS wavelength includes any wavelength withm the ranges of the NIMELS wavelengths described, as well as combinations of such wavelengths
  • word “or” is used in the “inclusive” sense of “and/or” and not the “exclusive” sense of “either/or "
  • 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
  • MIC minimum inhibitory concentration
  • NIMELS near infrared optical radiation in selected energies and dosimetries
  • 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 mammalian cells are not generally affected by treatments (with molecules or drugs) that are intended to damage the bacterial or fungal cells
  • 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, respecbvely
  • 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
  • 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
  • MRSA methicillin resistant staphylococcus aureus
  • the methods and systems of the present invention are used in treating, reducing and/or eliminating the infectious entities known as cutaneous Candidiasis
  • Candida infections involve the skin, and can occupy almost any skm surface on the body
  • 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 individuals with diabetes and in the obese Candida can also cause infections of the nail, referred to as onychomycosis, infections of the skm surrounding the nail (paronychia) and infections around the corners of the mouth, called angular cheilitis
  • ROS reactive oxygen species
  • the term also includes irradiating a cell to increase the sensitivity of the biological contaminant through the lowering of ⁇ 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
  • 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
  • potentiation means that the agent will inhibit the growth and/or proliferation of pathogenic cells thereby treating the disease state at a therapeutically acceptable dosage
  • microorganism refers to an organism that is microscopic and by definition, too small to be seen by the human eye
  • microorganisms can be bacteria, fungi, archaea, protists, and the like
  • microbial is defined as pertaining or relating to microorganisms
  • cell membrane or plasma membrane or mitochondrial membrane
  • lipid bilayer that has a common structure in all living cells It contains primarily proteins and lipids that are involved m a myriad of important cellular processes
  • Cell membranes that are the target of the present invention have protein/hpid ratios of >1 Stated another way, none of the target membranes in the containment (or moiety, i e , host tissue) contain greater than 49 99% lipid by dry weight
  • mitochondria refers to membrane-enclosed organelles, found in most eukaryotic cells (mamalhan 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 protem-to-phospholipid ratio similar to the eukaryotic plasma membrane, and the inner mitochondrial membrane forms internal compartments known as cristae and has a protem-to-phosphohpid 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
  • Fluid Mosaic Model refers to a widely held conceptualization of biological membranes as a structurally and functionally asymmetric hpid-bilayer, with a larger variety of embedded proteins that aid in cross- membrane transport
  • the Fluid Mosaic Model is so named, because the phospholipids shift position m 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
  • Membrane Dipole Potential ⁇ d (in contrast to the Transmembrane Potential ⁇ ) refers to the potential formed between the highly hydrated lipid heads (hydrophihc) at the membrane surface and the low polar interior of the bilayer (hydrophobic) Lipid bilayers intrinsically possess a substantial Membrane Dipole Potenbal ⁇ d arising from the structural organization of dipolar groups and molecules, primarily the ester linkages of the phospholipids and water ⁇ d does not depend upon the ions at the membrane surface and will be used herein to describe five different dipole potentials
  • 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 ( ⁇ ) ⁇ does depend upon the ions at the membrane surface and will be used herein to describe three different plasma trans-membrane potentials
  • Mitochondrial Trans-Membrane Potential refers to the electrical potential difference between the compartments separated by the mitochondrial inner membrane (dimensions mV) and will be used herein to describe two different mitochondrial trans-membrane potentials
  • mammalian plasma trans-membrane potential ( ⁇ - 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 ⁇ that are primarily generated with H + ions (protons)
  • the major facilitator of the ⁇ is the electrogenic Na + /K + -ATPase pump
  • ⁇ -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 Na + /K + -ATPase pump Mammalian ATP is generated in the mitochondria via the proton pump
  • fungal plasma trans-membrane potential ( ⁇ -plas- fungi) refers to the electrical potential difference m 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 maintenance Fungal ATP is generated in the mitochondria
  • bacterial plasma trans-membrane potential ( ⁇ -plas- bact) refers to the electrical potential difference in the bacterial cell plasma membrane 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
  • H* electrons and protons
  • a common feature of all electron transport chains is the presence of a proton pump to create a transmembrane proton gradient
  • bacteria lack mitochondria, aerobic bacteria carry out oxidative phosphorylation (ATP production) by essentially the same process that occurs in eukaryotic mitochondria
  • P-class ion pump refers to a trans-membrane active transport protein assembly which contains an ATP-bmding site (i e , it needs ATP to function) During the transport process, one of the protein subumts is phosphorylated, and the transported ions are thought to move through the phosphorylated subunit This class
  • 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 mside-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
  • ion exchangers and ion channels refer to transmembrane proteins that are ATP-independent systems, and aid m establishing a plasma membrane potential in mammalian cells
  • Redox shorthand for reduction/oxidation reaction
  • Redox reactions are chemical reactions in which electrons are transferred from a donor molecule to an acceptor molecule
  • Oxidation describes the loss of electrons by a molecule
  • atom or ion Reduction describes the gain of electrons by a molecule, atom or ion
  • redox state describes the redox environment (or level of oxidative stress) of the cells being described
  • steady-state plasma trans-membrane potential ( ⁇ - steady) refers to the quantitative Plasma Membrane Potential of a mammalian, fungal or bacterial 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
  • Transient-state plasma membrane potential 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, ⁇ -tran will also change the redox state of the cell, as the plasma membrane is where the ETS and cytochromes reside ⁇ -tran is a state that would not occur without irradiation using methods of the present invention ⁇ -tran will be used herein to describe three (3) different Transient-state plasma trans-membrane potentials based on species
  • 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 ⁇ -steady-mito will be used herein to describe two (2) different steady-state mitochondrial membrane potentials based on species
  • transient-state mitochondrial membrane potential ( ⁇ -tran-mito-mam or ⁇ -tran-rmto-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
  • ⁇ -tran-mito will also change the redox state of the cell, as the inner mitochondrial membrane is where the electron transport system (ETS) and cytochromes reside ⁇ -tran-mito could also drastically affect (the Proton-mohve force) ⁇ p-mito-mam and ⁇ p-mito-fungi, as these mitochondrial (H*) gradients are generated in the mitochondria, to produce adequate ATP for a myriad of cellular functions
  • ⁇ -tran-mito is a state that would not occur without irradiation in accordance with methods and systems of the present invention ⁇
  • cytochrome refers to a membrane bound hemoprotein that contains heme groups and carries out electron transport
  • ETS electron transport system
  • cytochromes membrane-associated electron carriers
  • pH Gradient refers to the pH difference between two bulk phases on either side of a membrane
  • proton electrochemical gradient (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
  • ⁇ 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 ⁇ n 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 ( ⁇ n and Tn) + (pharmacological molecule or molecules))
  • the term "Ion Electrochemical Gradient ( ⁇ x+)” 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
  • 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 an ATP coupled pump, said ATP produced during oxidative phosphorylation from the mammalian mitochondrial proton-motive force ( ⁇ p-mito-mam)
  • ⁇ 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 ⁇ n 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-target
  • co-targeting of a bacterial anabolic pathway refers to (the ⁇ n and Tn lowering of ( ⁇ H*) and/or ( ⁇ 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-alanme
  • co-targeting of a fungal anabolic pathway refers to (the ⁇ n and Tn lowering of ( ⁇ H ⁇ ) and/or ( ⁇ x*) 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 fungal anabolic pathways that are capable of being inhibited with pharmacological agents wherein the targeted anabolic pathway is phospholipid Biosynthesis that is co-targeted by a topical pharmacological agent that disrupts the structure of existing phospholipids, m fungal cell membranes and workswell in combination with other topical synergistic agents, wherein targeted anabokr 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- 1 I 1 SO enzyme 14-
  • the term "co-targeting of a cancer anabolic pathway” refers to (the An and Tn lowering of ( ⁇ H*) and/or ( ⁇ 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 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
  • 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 ⁇ p are ⁇ (the transmembrane potential) and ⁇ pH (the chemical gradient of H*)
  • ⁇ p consists of the H* transmembrane potential ⁇ (negative (acidic) outside) and a transmembrane pH gradient ⁇ pH (alkaline inside)
  • This potential energy stored in the form of an electrochemical gradient is generated by the pumping of hydrogen ions across 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 cancer
  • ⁇ p-mito-mam refers to the potential energy stored in the form of an (H + ) electrochemical gradient across a mammalian mitochondrial inner membrane ⁇ p-mito- mam is used in oxidative phosphorylation to drive ATP synthesis m the mammalian mitochondria
  • ⁇ p- mito-Fungi refers to the potential energy stored in the form of an (H*) electrochemical gradient across a fungal mitochondrial mner membrane ⁇ p-mito-Fungi is used in oxidative phosphorylation to drive ATP synthesis in the fungal mitochondria
  • the term “Fungal Plasma Membrane Proton-motive force ( ⁇ p- 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 function
  • the ATP for this H*-ATPase is generated from the ⁇ p-mito-Fungi ⁇ p-plas- Fungi can be used to drive efflux pumps in fungal cells
  • ⁇ p- plas-Bact Bacterial Plasma Membrane Proton-motive force ( ⁇ p- plas-Bact )
  • H 4 electrochemical gradient
  • ⁇ p-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
  • 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, ⁇ bosomes, cellular organelles, nucleic acids, DNA, RNA, glucans, chitin, simple fatty acids, complex fatty acids, cholesterols, sterols, and ergosterol
  • 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
  • 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
  • uncoupler refers to a molecule or device that causes the separation of the energy stored in the proton electrochemical gradient ( ⁇ H ⁇ ) of membranes from the synthesis of ATP
  • 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 ( ⁇ H + ) of membranes from the synthesis of ATP
  • ATP adenosine 5'-t ⁇ phosphate
  • ADP adenosine diphosphate
  • the Gibbs free energy is the energy available ("free") to do work, and the term Gibbs free energy change ( ⁇ G) refers to a change in the free energy available in the membrane to do work
  • This free energy is a function of enthalpy ( ⁇ H), entropy ( ⁇ S), and temperature. (Enthalpy and entropy are discussed below.)
  • phosphorylation potential refers to the ⁇ G for ATP synthesis at any given set of ATP, ADP and Pi concentrations (dimensions: kj mol"
  • CCCP refers to carbonyl cyanide m- chlorophenylhydrazone, a highly toxic ionophore and uncoupler of the respiratory chain. CCCP increases the conductance of protons through membranes and acts as a classical uncoupler by uncoupling ATP synthesis from the ⁇ H + and dissipating both the ⁇ and ⁇ pH.
  • depolarization refers to a decrease in the absolute value of a cell's plasma or mitochondrial membrane potential ⁇ . 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.
  • enthalpy change 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.
  • entropy change refers to a change in the entropy of a membrane system to that of a more disordered state at a molecular level.
  • 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.
  • Reactive Oxygen Species refers to one of the following categories: a) The Superoxide ion radical (O2 ) b) Hydrogen Peroxide (non-radical) (H2O2) c) Hydroxyl radical (OH) d) Hydroxy ion (OH )
  • singlet oxygen refers to (“IO2”) 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 IO2 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.
  • membranes e.g., via polyunsaturated fatty acids, or PUFAs
  • 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 ⁇ altering radiation in mitochondrial and/or plasma membranes.
  • 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 ⁇ -steady to ⁇ -trans with the present invention. Specifically, the NIMELS effect ran weaken cellular anabolic pathways or antimicrobial and/or cancer resistance mechanisms that make use of the proton motive force or the chemiosmotic potential for their energy needs
  • pe ⁇ plasrmc 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 periplasms space is involved in various biochemical pathways including nutrient acquisition, synthesis of peptidoglycan, electron transport, and alteration of substances toxic to the cell In gram- positive bacteria like MRSA, the periplasms space is of significant clinical importance as it is where ⁇ -lartamase enzymes inactivate penicillin based antibiotics
  • 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
  • the term "effflux 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
  • the efflux pump inhibitor of this invention is a form of electromagnetic radiation that will interfere with a pump s ability to excrete therapeutic antibiotics, anti-fungal agents, antineoplastic agents and poisons from cells via a modification of the ⁇ -steady-rnam , ⁇ -steady- fungi or, ⁇ -steady bact
  • a cell that ' utilizes an efflux pump resistance mechanism it is meant that the bacterial or fungal or cancer cell exports anti-bacte ⁇ al 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
  • the term inhibit means that the rate of growth and/or proliferation of population of cells is decreased, and if possible, stopped
  • the primary structure refers to the linear arrangement of ammo acids
  • the secondary structure refers to whether the linear amino and structure forms a helical or ⁇ -pleated sheet structure, tertiary structure 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-subumt complex
  • 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-lmking of proteins, both oxygen-dependent and independent oxidation of inter- and mfra-chain bonds, such as disulfide bonds, oxidation of individual ammo acids, and the like
  • pH stress refers to modification of the intracellular pH, i e , a decrease intracellular pH below about 60 or an increase intracellular pH above about 75 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 ⁇ -steady
  • 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
  • anti-bacterial molecule refers to a chemical or compound that is bacteriacidal or bacte ⁇ astatic Another principal efficacy resides in the present invention'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
  • a ' sub-inhibitory concentration' of an antibacterial or anti-fungal molecule refers to a concentration that is less than that required to inhibit a ma j ority 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 )
  • 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
  • Minimal Inhibitory Concentration or MIC is defined as the lowest effective or therapeutic concentration that results in inhibition of growth of the microorganism
  • a therapeutically effective amount ' of a pharmaceutical agent or molecule 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
  • 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 pabent 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
  • Interaction coefficient is defined as a numerical representation of the magnitude of the bacte ⁇ astatic/bacte ⁇ acidal and/or fungistatic/fungicidal interaction between the NIMELS laser and/or the antimicrobial molecule, with the target cells
  • 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
  • the methods and systems of the present invention optically alter and modify ⁇ d-plas-mam, ⁇ d-mito-mam, ⁇ d-plas-fungi, ⁇ d-mito-fungi and ⁇ d-plas-bact to set in motion further alterations of ⁇ and ⁇ p 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 ⁇ d
  • membranes lipid bilayers, see, Figure 1
  • Figure 2 possess a significant dipole potential ⁇ d arising from the structural association of dipolar groups and molecules, primarily the ester linkages of the phospholipids (Figure 2) and water
  • 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 ⁇
  • the transmembrane 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
  • the potential at the cytoplasm side of cell membranes is negative relative to the extracellular physiological solution (Figure 4A)
  • the dipole potential ⁇ d constitutes a large and functionally important part of the electrostatic potential of all plasma and mitochondrial membranes ⁇ d modifies the electric field inside the membrane, producing a virtual positive charge in the apolar bilayer center
  • 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 ⁇ d also plays an important role in the membrane permeability for lipophilic ions
  • the energy transduction in biological membranes generally involves three interrelated mechanisms
  • trans-membrane ionic electrochemical potential also called the membrane proton electrochemical gradient ⁇ 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 mobve force
  • ⁇ Gp is the ⁇ G for ATP synthesis at any given set of ATP, ADP and Pi concentrations
  • 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 ⁇ , in a temporally dependent manner, to move the bioenergetics of a membrane from a thermodynamic steady-state condition ⁇ -steady to one of energy stress and/or redox stress in a transition state ⁇ -trans
  • 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
  • the vibrations or rotations within the lipid bilayer's molecular bonds that absorb the infrared photons must cause a net change m 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 m long chain fatty acids) then radiation will be absorbed causing a change in ⁇ d This can happen in ⁇ d-plas-mam, ⁇ d-mito- mam, ⁇ d-plas-fungi, ⁇ d-mito-fungi and ⁇ d-plas-bact In other words, there can be a direct and targeted change in the enthalpy and entropy ( ⁇ H and ⁇ S) of all cellular lipid bilayers with the methods and systems described herein
  • 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
  • the unique, single wavelengths (870 nm and 930 nm) are capable of killing bacterial cells (prokaryotes) such as £ colt and (eukaryotes) such as Chinese HeIa Ovary hampster cells (CHO), as a result of the generation and interaction of ROS and toxic singlet oxygen reaction
  • prokaryotes such as £ colt
  • eukaryotes such as Chinese HeIa Ovary hampster cells (CHO)
  • CHO Chinese HeIa Ovary hampster cells
  • Entropy in a membrane is a state function whose change m a reaction describes the direction of a reaction due to changes in (energy) heat input or output and the associated molecular rearrangements
  • 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 ⁇ d of all irradiated membranes (mitochondrial and plasma) and hence, thermodynamically alter the "steady-state” flow of electrons and protons across a cell membrane ( Figures 6 and 7)
  • This will m turn change the steady-state trans-membrane potential ⁇ -steady to a transient-state membrane potential ( ⁇ -tran) This phenomenon will occur in
  • Such phenomena can in turn decrease the Gibbs free energy value ZlG available for the phosphorylation and synthesis of ATP ( ⁇ Gp)
  • the present invention carries out 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
  • the present invention can act as an optical uncoupler by lowering the ⁇ H + and Ap of the following irradiated membranes
  • Lipid peroxidation is a prevalent cause of biological cell in j ury 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 (PUF A' s) The severity of the membrane damage can cause local reductions in membrane fluidity and full disruption of bilayer integrity
  • Peroxidation of mitochondrial membranes will have detrimental consequences on the respiratory chains resulting m inadequate production of ATP and collapse of the cellular energy cycle
  • Peroxidation of the plasma membrane can affect membrane permeability, disfunction of membrane proteins such as po ⁇ ns 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
  • 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
  • PUFAs polyunsaturated fatty acids
  • 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
  • Reactive Oxygen Species (ROS) Oxygen gas is actually a free radical species.
  • ROS Reactive Oxygen Species
  • the (spin restriction) rule generally prevents Ch from receiving a pair of electrons with parallel spins without a catalyst. Consequently Oz must receive one electron at a time.
  • Superoxide for example, can either act as an oxidizing or a reducing agent.
  • Hydrogen peroxide is not a good oxidizing agent (by itself) and cannot remove hydrogen from PUF A's It can, however, cross biological membranes (rather easily) to exert dangerous and harmful effects in other areas of cells
  • H2O2 is highly reactive with transition metals inside microcellular environments, (such as Fe +2 and Cu*) that can then create hydroxyl radicals (*OH) (known as the Fenton Reaction)
  • *OH hydroxyl radicals
  • An hydroxyl radical is one of the most reactive species known in biology
  • 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
  • the standard reduction potential (EO') for hydroxyl radical (*OH) is (+231 V) a value that is 7x greater than (H2O2), 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
  • alkyl hydroperoxides are not technically radical species but are unstable in the presence of transition metals such as such as Fe +2 and Cu*
  • alkoxyl radicles (RO*) 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 ⁇ -steady + (NIMELS Treatment) ->-> ⁇ - trans phenomenon is another ob j ect 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
  • 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"
  • the four mam mechanisms by which micro-organisms exhibit resistance to antimicrobials are a) Drug lnactivation 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
  • Staphylococcus aureus is one of the ma j or resistant bacterial pathogens currently plaguing civilization This gram positive bacterium is primarily found on the mucous membranes and skm 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 methicillm and oxacillin The "superbug" MRSA (methicillm resistant Staphylococcus aureus) was first detected m 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 oxazohdinones, there have been reports of significant resistance (Vancomycin since 1996 and Zyvo
  • CA-MRSA community-associated MRSA
  • Daptomycm'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
  • ⁇ H + the mam component of which is the transmembrane electrical potential gradient ⁇ H +
  • daptomycm e g , inhibition of protein, RNA, DNA, peptidoglycan, lipoteichoic acid, and lipid biosynthesis
  • Multidrug resistance efflux pumps are now known to be present in gram- posihve 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 ⁇ -lactam antimicrobials, marcolides, fluoroquinolones, tetracyclines and other important antibiotics, along with most antifungal compounds including itraconazole and terbinafine
  • a microbe 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 m 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
  • bacterial antibiotic efflux pumps belong to five superfamilies (i) ABC (ATP-brndmg cassette), which are primary active transporters energized by ATP hydrolysis,
  • MATE multi-antimicrobial extrusion subfamily of the MOP (multidrug/oligosaccharidyl-lipid/polysaccharide flippases) superfamily
  • MFS major facilitator superfamily
  • RND resistance/nodulation/division superfamily
  • the approach of the current invention to inhibit efflux pumps is a general modification (optical depolarization) of the membranes ⁇ withm the irradiated area, leading to lower electrochemical gradients that will lower the phosphorylation potential ⁇ Gp 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
  • Reserpine inhibits the activity of Bmr and NorA, two gram-positive efflux pumps, by altering the generation of the membrane proton-motive force ⁇ p required for the function of MDR efflux pumps Although these molecules are able to inhibit the ABC transporters involved in the extrusion of antibiotics ⁇ 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 daptomycm Fungal drug efflux is mediated primarily by two groups of membrane-bound transport proteins the ATP-bmding cassette (ABC) transporters and the major facilitator superfamily (MFS) pumps Bacterial Plasma Trans-membrane Potential ⁇ -plas-bact and cell wall synthesis
  • ⁇ -plas-bact uncouplers inhibit peptidoglycan formation with the accumulation of the nucleotide precursors involved in peptidoglycan synthesis, and the inhibition of transport of N-acetylglucosamme (GIcNAc), one of the major biopolymers in peptidoglycan
  • tachyplesm that decreases ⁇ -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 ⁇ -lactam antibiotic ampicillm in MRSA
  • ⁇ -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 ⁇ -plas-fungi
  • 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) i943-1953, the entire teaching of which is incorporated herein by reference )
  • this induced reduction of the cytosolic ATP concentration leads to a suppression of the plasma membrane-bound H ⁇ -ATPase that generates ⁇ -plas- fungi, and that this impairment further weakens other cellular activities
  • the lowering of the ⁇ -plas-fungi will cause plasma membrane bioenergetic and thermodynamic disruption leading to an influx of protons that collapses the proton motive force and
  • Ergosterol is the structural lipid that is targeted by the ma j ority of relevant commercial antifungal compounds used in medicine today (i e , azoles, terbmafme and itraconazole)
  • the invention provides a method of modifying the dipole potential ⁇ d of all membranes within the path of a NIMELS beam ( ⁇ d-plas-mam, ⁇ d- mito-marn, ⁇ d-plas-fungi, ⁇ d-mito-fungi, and ⁇ d-plas-bact) to set in motion the cascade of further alterations of ⁇ and ⁇ p in the same membranes
  • the bioenergetic steady-state membrane potentials ⁇ -steady of all irradiated cells are altered to ⁇ -trans values ( ⁇ -trans-mam, ⁇ -trans-fungi, ⁇ -trans-Bact, ⁇ -trans-mito-mam and ⁇ -trans-mito-fungi)
  • ⁇ -trans values ⁇ -trans-mam, ⁇ -trans-fungi, ⁇ -trans-Bact, ⁇ -trans-mito-mam and ⁇ -trans-mito-fungi
  • such applied optical radiation may have a wavelength from about 850 nm to about 9OQ nm, at a NIMELS dosimetry, as described herein
  • wavelengths from about 865 nm to about 875 nm are utilized
  • such applied radiation may have a wavelength from about 905 nm to about 945 nm at a NIMELS dosimetry
  • suoh applied optical radiation may have a wavelength from about 925 nm to about 935 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
  • 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 (cm 2 ) 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
  • NIMELS Potentiation Magnitude Scale measures the NIMELS effect number (Ne) between 1 to 10, where the goal is to gam a Ne of > 4 in reduction of CFU count of a pathogen, at any safe combination of antimicrobial concentration and NIMELS dosimetry
  • CFU count is used here for quantifying pathogenic organism, other means of quantification such as, for example
  • 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
  • NIMELS potentiation number 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
  • Np CFU Count of pathogen with (NIMELS + Antimicrobial)
  • Ne 1 then there is no potentiation effect If Ne > 1 then there is a potentiation effect
  • Ne > 2 then there is at least a 50% potentiation effect on the antimicrobial If Ne ⁇ 4 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
  • 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
  • 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 ⁇ of the irradiated cells This weakened ⁇ will cause an affiliated weakening of the proton motive force ⁇ p, 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
  • such applied optical radiation has a wavelength from about 850 nm to about 9QO urn, 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
  • 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)
  • NIMELS energy at NIMELS dosimetry to the biological contaminant is applied prior to, subsequent to, or concomitant with the administration of an antimicrobial agent
  • 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
  • the co-admimstered 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 contaminant strain at a concentration of the antimicrobial agent of about 001 M or less, or about Q 001 M or less, or about 00005 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
  • 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
  • the sensitivity is potentiated by at least 10%
  • the invention provides a system to implement the methods according to other aspects of the invention
  • 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 colhmating lenses and/or aperture stops
  • the system utilizes two or more solid state diode lasers to function as a dual wavelength near-mfrared 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 ran 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
  • Systems according to the present invention can include a suitable optical source for each individual wavelength range desired to be produced
  • a suitable solid stated laser diode, a variable ultra-short pulse laser oscillator, or an ion-doped (e g , with a suitable rare earth element) optical fiber or fiber laser is used
  • 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.
  • 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
  • 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
  • therapeutic systems especially adapted to generate optical radiation substantially m a first wavelength range from about 865 nm to about 875 nm
  • 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 include 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 radiation substantially in the first wavelength range or substantially in the second wavelength range or any combinations thereof
  • 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
  • the controller of the therapeutic system includes a power lirniter 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
  • 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
  • CW continuous wave
  • e g dual-wavelength
  • 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
  • Exemplary embodiments include top-hat or substantially top-hat (e g , trapezoidal, etc ) intensity distributions
  • Other intensity distributions, such as Gaussian may be used
  • 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 used 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
  • illustrative non-hmitmg examples of biological contaminants include, but are not limited to, any bacteria, such as, for example, Escherichia, Enterobader, Bacillus, Campylobacter, Corynebactenum, Klebsiella, Treponema, Vibrio, Streptococcus and Staphylococcus
  • biological contaminants contemplated include, but are not limited to, any fungus, such as, for example, Trichophyton, Microsporum, Epidermophyton, Candida, Scopulariopsis brevicauhs, Fusanum spp , Aspergillus spp , Alternana, Acremomum, Scytalidinum dimi ⁇ iatutn, and Scytahdinium hyahnum Parasites may also be targeted biological contaminants such as Trypanosoma 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, Ep
  • irradiation may be palliative as well as prophylactic
  • 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
  • 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
  • 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 th Ed , McGraw Hill, New York (1994), the entire teaching of which is incorporated herein by reference)
  • 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
  • antimicrobial composition 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 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
  • the physical properties e g , absorption and scattering coefficients, scattering amsotropy, thermal conductivity, heat capacity, and mechanical strength
  • a biological moiety e g , a mammalian cell, tissue, or organ
  • 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 vanes between about 1 J/cm 2 to about 1Q,OOO J/cm 2 (five orders of magnitude), whereas the power density (irradiance) varies from about 1x10 3 W/cm 2 to over about 10 12 W/cm 2 (15 orders of magnitude)
  • laser exposure duration irradiation time
  • This progression describes a suitable method or basic algorithm that can be used for a NIMELS interaction against a biological contaminant in a tissue
  • this mathematical relation is a reciprocal correlation to achieve a laser-tissue interaction phenomena
  • This ratiomale 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
  • a practitioner is able to adjust the power density and time to obtain the desired energy density
  • NIMELS dosimetries exemplified herein e g , Onychomycosis
  • e g Onychomycosis
  • target microbes m vivo were from about 200 J/cm 2 to about 700 J/cm 2 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 (cm 2 ) and the spatial distribution pattern of the energy
  • the illumination pattern of a 1 5 cm irradiation spot with an incident Gaussian beam pattern of the area 1 77 cm 2 can produce at least six different power density values within the 1 77 cm z 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
  • 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 m Biiw embodiments, Tn is from about 100 to about 1200 seconds
  • 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 ⁇ 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
  • an intolerable adverse risk or effect e g , thermal damage such as poration
  • 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)
  • the power density range contemplated herein is from about 025 to about 40 W/cm 2 In other embodiments, the power density range is from about 05 W/cm 2 to about 25 W/cm 2
  • power density ranges can encompass values from about 05 W/cm 2 to about 10 W/cm 2 Power densities exemplified herein are from about 05 W/cm 2 to about 5 W/cm 2 Power densities in vivo from about 1 5 to about 25 W/cm 2 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)
  • the energy density range contemplated herein is greater than 50 J/cm 2 but less than about 25,000 J/cm 2 In other embodiments, the energy density range is from about 750 J/cm 2 to about 7,000 J/cm 2 In yet other embodiments, the energy density range is from about 1,500 J/cm 2 to about 6,000 J/cm 2 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/cm 2 to about "500 J/cm 2 In yet other in vivo embodiments, the energy density is from about 175 J/cm 2 to about 300 J/cm 2 In yet other embodiments, the energy density is from about 200 J/cm 2 to about 250 J/cm 2 In some embodiments, the energy density is from about 300 J/cm 2
  • Power densities empirically tested for various in vitw treatment of microbial species were from about 1 W/cm 2 to about 10 W/cm 2
  • 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 routinely 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)
  • Practitioners e g , dentists
  • near infrared energies in conjunction with periodontal treatment routinely 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)
  • laser treatment of a periodontal infection m a light-colored tissue e g , a melanme 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
  • antibiotic resistant bacteria may be effectively treated according to the methods of the present invention
  • the methods of this invention may be used to augment traditional approaches, to be used in combination with, m lieu of tradition therapy, or even serially as an effective therapeutic approach Accordingly, the invention may be combined with antibiotic treatment
  • antibiotic includes, but is not limited to, ⁇ - lactams, penicillins, and cephalosporins, vancomycins, bacitracins, macrolides (erythromycins), ketohdes (telithromycm), lmcosamides (chndomycin), chloramphenicols, tetracyclines, aminoglycosides (gentamicms), amphote ⁇ cns, anihnouracils, cefazohns, clindamycins, mupirocins, sulfonamides and trimethoprim, ⁇ fampicms, metronidazoles, qumolone
  • antifungal resistant fungi may be effectively treated according to the methods of the invention
  • 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
  • the invention may be combined with antifungal treatment
  • antifungal includes, but is not limited to, polyenes, azoles, imidazoles, t ⁇ azoles, allylammes, echinocandms, cicopirox, flucytosine, g ⁇ seofulvm, amorolofme, soda ⁇ ns and combinations thereof (including salts thereof)
  • antineoplastic resistant cancer may be effectively treated according to the methods of the present invention
  • 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
  • antiineoplastic includes, but is not limited to, actinomycm, anthracyclmes, bleomycin, plicamycm, mitomycin, taxanes, etoposide, teniposide and combinations thereof (including salts thereof)
  • the experimental data supports a universal alteration of ⁇ and ⁇ p 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)
  • MIC Minimum Inhibitory Concentration
  • C albicans ATCC 14053 liquid cultures were grown in YM medium (21g/L, Difco) medium at ⁇ 7°C
  • a standardized suspension was ahquoted 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 5xlO 6 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 Table 4 Method for ⁇ and ROS Assays
  • C albicans ATCC 14053 liquid cultures were grown in YM medium (21g/L, Difco) medium at 37 0 C
  • YM medium 21g/L, Difco
  • a standardized suspension was ahquoted 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
  • HEK293 cells were seeded into appropriate wells of a 24-well plate at a density of 1 x 10 5 cells/ml (0 7ml total volume) in Freestyle medium (Invitrogen) Cells were incubated in a humidified incubator at 37 0 C in 8% CCh for approximately 48 hours prior to the experiment Cells were approximately 9G% confluent at the time of the experiment equating to roughly 3 x IG 5 total cells Immediately prior to treatment, cells were washed in pre-warmed phosphate buffer saline (PBS) and overlaid with 2 ml of PBS during treatment
  • PBS pre-warmed phosphate buffer saline
  • fluorescent dyes that can be taken up by intact cells and accumulate within the intact cells within 15 to 3D 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 ⁇ - 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 wi-chlorophenylhydrazone (CCCP), indicating that maintenance of dye concentration is dependent on the mside-negahve transmembrane potential maintained by functional ETS and ⁇ p
  • the null hypothesis is ⁇ i - 0 ⁇ i is fluorescence intensity in a control cell culture (no laser) sub j ected to carbocyanme dye ⁇ 2 is fluorescence intensity m 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 0
  • BacLightTM Bacterial Membrane Potential Kit (B3495Q, Invitrogen U S ) The BacLightTM Bacterial Membrane Potential Kit provides of carbocyanme 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 destroy membrane potential by eliminating the proton gradient, hence causing higher green fluorescence
  • Green fluorescence emission was calculated using population mean fluorescence intensities for control and lased samples at sub-lethal dosimetry Table 6
  • 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
  • Red/green ratios were calculated using population mean fluorescence intensities for control and lased samples at sub-lethal dosimetry
  • ⁇ i fluorescence intensity in a control cell culture mitochondria subjected to a Mitochondrial Membrane Potential Detection Kit
  • ⁇ z 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 0 Will uphold that the addition sub-lethal NIMEL irradiation on the cell culture mitochondria has no effect on ⁇ -steady-mito 0
  • the loss of mitochondrial membrane potential ( ⁇ ) is a hallmark for apoptosis
  • the APO LOGIX JC-I Assay Kit measures the mitochondrial membrane potential in cells
  • JC-I (5,5',6,6'-tetrachloro-l,r,3,3'-tetraethylbenz lmidazolylcarbocyamne iodide) exists as a monomer in the cytosol (green) and also accumulates as aggregates m the mitochondria which stain red
  • JC-I exists in monomeric form and stains the cytosol green
  • the (APO LOGIX JC-I) kit measures membrane potential by conversion of green fluorescence to red fluorescence
  • Figure 1OA 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 1OB 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.
  • ⁇ i is fluorescence intensity in a mammalian control cell culture mitochondria (no laser) subjected to a Mitochondrial Membrane Potential Detection Kit.
  • ⁇ 2 is fluorescence intensity in the same mammalian cell culture pre-irradiated with sub-lethal dosimetry from the NIMELS laser and subjected to a Mitochondrial
  • 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.
  • APO LOGIX JC-I Mitochondrial Membrane Potential Detection Kit
  • JC-I mitochondrial membrane potential in cells
  • JC-I (5,5',6,6'-tetrachloro-l,l',3,3'-tetraefhylbenz- lmidazolylcarbocyanme iodide) exists as a monomer in the cytosol (green) and also accumulates as aggregates m the mitochondria which stain red
  • JC-I exists in monomeric form and stains the cytosol green
  • the (APO LOGIX JC-I) kit measures membrane potential by conversion of green fluorescence to red fluorescence
  • Figure 1 IA 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 1 IB for both control and lased samples
  • ROS reactive oxygen species
  • Glutathione 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-mtrobenzoic acid)), known as EUman'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-mtro-5-thiobenzoic acid and glutathione disulfide (GSSG) Since 2-mtro-5- thiobenzoic acid is a yellow colored product,
  • 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 5OS of the bacterial ⁇ bosome, thereby blocking the exit of the growing peptide chain thus inhibiting the translocation of peptides Erythromycin resistance (as with other marcohdes) is rampant, wide spread, and is accomplished via two significant resistance systems
  • 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
  • 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
  • a ma j or 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
  • antibiotic resistance that is mediated via efflux pumps, is most relevant in gram positive bacteria for marcohdes, tetracyclines and fluoroquinolones In gram negative bacteria, ⁇ -lartam efflux mediated resistance is also of high clinical relevance
  • the null hypothesis is ⁇ i - 0 and ⁇ i 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) ⁇ a 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
  • 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 bacterial 3OS ⁇ bosome through the binding of the enzyme ammoacyl-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 nbosomes from the action of tetracyclines
  • Rifampin Rifampin is a bacterial RNA polymerase inhibitor, and functions by directly blocking the elongation of RNA Rifampicm is typically used to treat mycobacterial infections, but also plays a role in the treatment of methicillm-resistant Staphylococcus aureus (MRSA) m combination with fusidic acid, a bacteriostatic protein synthesis inhibitor
  • MRSA methicillm-resistant Staphylococcus aureus
  • a) ⁇ i is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control and, b) ⁇ 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) ⁇ 3 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
  • Methicillin is a ⁇ -lactam that was previously used to treat infections caused by gram-positive bacteria, particularly ⁇ -lactamase-producing organisms such as S. aureus that would otherwise be resistant to most penicillins, but is no longer clinically used.
  • MRSA methicillin-resistant S. aureus
  • methicillin acts by inhibiting the synthesis of peptidoglycan (bacterial cell walls)
  • ⁇ -plas-bact uncouplers inhibit peptidoglycan formation with the accumulation of the nucleotide precursors involved m peptidoglycan synthesis, and the inhibition of transport of N-acetylglucosamine (GIcNAc), one of the major biopolymers in peptidoglycan
  • Bacitracin will potentiate the multiple influences of an optically lowered ⁇ - 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 - 0 and ⁇ i - 0 where a) ⁇ i is sub-lethal dosimetry from the NIMEL laser system on MRSA as a control and, b) ⁇ ais 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, 0
  • the NIMELS laser and its concomitant optical ⁇ -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 (pe ⁇ plasmic) ATP coupled functions, as MRSA does not have efflux pumps for methicillm
  • Bacitracin is a mixture of cyclic polypeptides produced by Bacillus subhhs 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 Css-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
  • ⁇ -plas-bact uncouplers inhibit peptidoglyran formation with the accumulation of the nucleotide precursors involved in peptidoglycan synthesis, and the inhibition of transport of N-acetylglucosamme (GIcNAc), one of the major biopolymers in peptidoglycan
  • Bacitracin potentiates the multiple influences of an optically lowered ⁇ -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 - 0 and ⁇ i - 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 bacitracin at resistant MIC just below effectiveness level and, 0
  • 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 NIMELS laser at sub-lethal dosimetry on C albicans potentiates lamisil and sporanox due to of an optically lowered ⁇ -plas-fungi and/or ⁇ -rmto-fungi by depolarizing the membranes and depleting cellular ATP in the fungus
  • the null hypothesis is ⁇ i - 0 and ⁇ i - 0 where a) ⁇ i is sub-lethal dosimetry from the NIMEL laser system on C albicans as a control and, b) ⁇ 2 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) ⁇ 3is 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
  • microorganisms exemplified include E coli K-12, multi-drug resistant E colt, Staphylococcus aureus, methicillm- resistant S aureus, Candida albicans, and Trichophyton rubrum
  • 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 (cm 2 ) 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
  • Beam Diameter (cm 2 ) Beam Diameter (cm 2 ) Beam area can be calculated by either
  • 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
  • Total Energy (Joules) Laser Output Power (Watts) * Time (Sees )
  • 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
  • Total energy distribution may be measured as energy density (Joules/cm 2 ) 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 NIMEL S wavelength may be derived as follows
  • the energy density may be derived as follows
  • Energy Density (Joule/cm2) Power Density (1) (W/cm 2 ) * Time (Sees) + Power Density (2) (W/cm 2 ) * Time (Sees)
  • a practitioner may use either the energy density (J/cm 2 ) or energy (J), as well as the output power (W), and beam area (cm 2 ) using either one of the following equations:
  • 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.
  • 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 £. col ⁇ ) to 100% (all other bacteria and fungi).
  • Liquid cultures of E coh ⁇ .12 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 too 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
  • T rubrum ATCC 52022 liquid cultures were grown in peptone-dextrose (PD) medium at 37 0 C
  • PD peptone-dextrose
  • a standardized suspension was ahquoted into selected wells in a 24 - well tissue culture plate Following laser treatments, ahquots were removed from each well and spread onto separate plates The plates were then incubated at 37 0 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
  • 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
  • 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
  • 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 coh antibacterial efficacy when 870 nm is 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
  • FIG. 17 shows (a) the synergistic effects of NIMELS with methi ⁇ llm, 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
  • ATCC and/or of ATCC is ultimately responsible information regarding location Applications resistant to methi ⁇ llm [514761
  • 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 0 C colonies were counted
  • CFU colony forming units
  • the treated and a control untreated suspension were diluted and plated m triplicate on trypic soy agar with or without 30 ⁇ g/ml methicillin After 24hrs of growth at 37 0 C colonies were counted
  • 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 0 C colonies were counted.
  • the treated and a control untreated suspension were diluted and plated in pentuphcate on trypic soy agar with or without 30 ⁇ g/ml methidllin (Groups A4 and B4), 0.5 ⁇ g/ml penicillin G (Groups C4 and D4) or 4 ⁇ g/ml erythromycin (Groups E4 and F4).
  • 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.
  • Time/Temperature assessments were charted to ensure the thermal safety of these laser energies on human dermal tissues (data not shown).
  • he exposed his great toe to both 870 nm and 930 nm for up to 233 seconds, while measuring toenail surface temperature with a laser infrared thermometer He found that using the above dosimetries, at a surface temperature of 375 0 C, with 870 nm and 930 nm together with a combined Power Density of 1 70 W/cm 2 , pain resulted and the laser was turned off
  • 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
  • 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.
  • Treatment #1 and Treatment #2 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 IS shows a comparison of the pretreatment (A), 60 days post- treatment (B), 80 days post-treatment (C), and 120 days post-treatment (D) toenails.
  • healthy and non-infected nail plate was covering 50% of the nail area and growing from healthy cuticle after 120 days.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Radiology & Medical Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Laser Surgery Devices (AREA)
  • Radiation-Therapy Devices (AREA)

Abstract

L'invention concerne des systèmes et procédés d'application d'énergies et de dosimétries optiques proche infrarouge pour modifier les potentiels trans-membrane et mitochondriaux stationnaires bioénergétiques (stable Dy) de toutes les cellules irradiées par un effet de dépolarisation optique. Cette dépolarisation entraîne une diminution concomitante de la valeur absolue des potentiels trans-membrane Dy des membranes mitochondriales et plasmiques irradiées. Un grand nombre de réactions anaboliques cellulaires et de mécanismes de résistance aux médicaments peut être rendu moins fonctionnel et/ou atténué par une diminution d'un potentiel de membrane Dy, l'affaiblissement affilié de la force motrice de proton Dp et le potentiel de phosphorylation réduit associé DGp. Dans la zone d'exposition d'irradiation, la diminution des potentiels de membrane Dy se produira dans les cellules de bactérie, de champignon et de mammifère à l'unisson. Cette dépolarisation de membrane donne la capacité à potentialiser les médicaments antimicrobiens, antifongiques et/ou anti-néoplasiques contre uniquement les cellules non souhaitables ciblées.
PCT/US2007/087264 2006-12-12 2007-12-12 Modification électromagnétique proche infrarouge de potentiels de membrane stationnaires cellulaires WO2008073979A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2009541559A JP2010512232A (ja) 2006-12-12 2007-12-12 細胞の定常状態の膜ポテンシャルの近赤外線による電磁的改変
CA002670711A CA2670711A1 (fr) 2006-12-12 2007-12-12 Modification electromagnetique proche infrarouge de potentiels de membrane stationnaires cellulaires
AU2007333073A AU2007333073A1 (en) 2006-12-12 2007-12-12 Near-infrared electromagnetic modification of cellular steady- state membrane potentials
EP07865581A EP2089107A2 (fr) 2006-12-12 2007-12-12 Modification électromagnétique proche infrarouge de potentiels de membrane stationnaires cellulaires

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US87442406P 2006-12-12 2006-12-12
US60/874,424 2006-12-12
US11/981,431 US8506979B2 (en) 2002-08-28 2007-10-31 Near-infrared electromagnetic modification of cellular steady-state membrane potentials
US11/981,340 US20080131968A1 (en) 2002-08-28 2007-10-31 Near-infrared electromagnetic modification of cellular steady-state membrane potentials
US11/981,486 US20090299263A1 (en) 2002-08-28 2007-10-31 Near-Infrared electromagnetic modification of cellular steady-state membrane potentials
US11/981,486 2007-10-31
US11/981,340 2007-10-31
US11/981,431 2007-10-31

Publications (2)

Publication Number Publication Date
WO2008073979A2 true WO2008073979A2 (fr) 2008-06-19
WO2008073979A3 WO2008073979A3 (fr) 2008-10-23

Family

ID=39512464

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/087264 WO2008073979A2 (fr) 2006-12-12 2007-12-12 Modification électromagnétique proche infrarouge de potentiels de membrane stationnaires cellulaires

Country Status (6)

Country Link
EP (1) EP2089107A2 (fr)
JP (1) JP2010512232A (fr)
KR (1) KR20090097925A (fr)
AU (1) AU2007333073A1 (fr)
CA (1) CA2670711A1 (fr)
WO (1) WO2008073979A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102725027A (zh) * 2010-01-21 2012-10-10 皇家飞利浦电子股份有限公司 控制装置、可穿戴装置和用于光治疗目的的照明系统
EP2995346A1 (fr) * 2008-10-29 2016-03-16 Nomir Medical Technologies, Inc Modification électromagnétique dans l'infrarouge proche de potentiels de membrane cellulaire à l'état stationnaire
CN111175103A (zh) * 2020-01-16 2020-05-19 江西业力医疗器械有限公司 一种真菌性阴道炎荧光检测的白带样本前处理液及其制备方法
US10702706B2 (en) 2013-07-16 2020-07-07 Nomir Medical Technologies, Inc. Apparatus, system, and method for generating photo-biologic minimum biofilm inhibitory concentration of infrared light
CN111781175A (zh) * 2020-06-18 2020-10-16 中国人民解放军军事科学院国防科技创新研究院 一种用于提高细胞中线粒体活性的方法及装置和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6042603A (en) * 1996-10-30 2000-03-28 Photogen, Inc. Method for improved selectivity in photo-activation of molecular agents
US20030130709A1 (en) * 2001-06-26 2003-07-10 D.C. Constance Haber Therapeutic methods using electromagnetic radiation
US20040034341A1 (en) * 1998-03-27 2004-02-19 Palomar Medical Technologies, Inc. Method and apparatus for the selective targeting of lipid-rich tissues
US20040111132A1 (en) * 2002-12-10 2004-06-10 Olga Shenderova Phototherapeutic treatment methods and apparatus
US20040156743A1 (en) * 2002-08-28 2004-08-12 Eric Bornstein Near infrared microbial elimination laser system
US20050075703A1 (en) * 2001-01-22 2005-04-07 Eric Larsen Photodynamic stimulation device and methods

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6042603A (en) * 1996-10-30 2000-03-28 Photogen, Inc. Method for improved selectivity in photo-activation of molecular agents
US20040034341A1 (en) * 1998-03-27 2004-02-19 Palomar Medical Technologies, Inc. Method and apparatus for the selective targeting of lipid-rich tissues
US20050075703A1 (en) * 2001-01-22 2005-04-07 Eric Larsen Photodynamic stimulation device and methods
US20030130709A1 (en) * 2001-06-26 2003-07-10 D.C. Constance Haber Therapeutic methods using electromagnetic radiation
US20040156743A1 (en) * 2002-08-28 2004-08-12 Eric Bornstein Near infrared microbial elimination laser system
US20040111132A1 (en) * 2002-12-10 2004-06-10 Olga Shenderova Phototherapeutic treatment methods and apparatus

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2995346A1 (fr) * 2008-10-29 2016-03-16 Nomir Medical Technologies, Inc Modification électromagnétique dans l'infrarouge proche de potentiels de membrane cellulaire à l'état stationnaire
CN102725027A (zh) * 2010-01-21 2012-10-10 皇家飞利浦电子股份有限公司 控制装置、可穿戴装置和用于光治疗目的的照明系统
US9579521B2 (en) 2010-01-21 2017-02-28 Koninklijke Philips N.V. Control device, wearable device and lighting system for light therapy purposes
US10702706B2 (en) 2013-07-16 2020-07-07 Nomir Medical Technologies, Inc. Apparatus, system, and method for generating photo-biologic minimum biofilm inhibitory concentration of infrared light
CN111175103A (zh) * 2020-01-16 2020-05-19 江西业力医疗器械有限公司 一种真菌性阴道炎荧光检测的白带样本前处理液及其制备方法
CN111175103B (zh) * 2020-01-16 2023-07-04 江西业力医疗器械有限公司 一种真菌性阴道炎荧光检测的白带样本前处理液及其制备方法
CN111781175A (zh) * 2020-06-18 2020-10-16 中国人民解放军军事科学院国防科技创新研究院 一种用于提高细胞中线粒体活性的方法及装置和应用

Also Published As

Publication number Publication date
CA2670711A1 (fr) 2008-06-19
JP2010512232A (ja) 2010-04-22
KR20090097925A (ko) 2009-09-16
EP2089107A2 (fr) 2009-08-19
AU2007333073A1 (en) 2008-06-19
WO2008073979A3 (fr) 2008-10-23

Similar Documents

Publication Publication Date Title
EP2995346B1 (fr) Modification électromagnétique dans l'infrarouge proche de potentiels de membrane cellulaire à l'état stationnaire
US8506979B2 (en) Near-infrared electromagnetic modification of cellular steady-state membrane potentials
WO2009117672A2 (fr) Modification de ptoentiels de membranes stables cellulaires dans des bactéries résistantes aux médicaments
WO2009059233A2 (fr) Modification électromagnétique proche infrarouge de potentiels de membrane cellulaires stables
US8983257B2 (en) Therapeutic light delivery apparatus, method, and system
AU2006278464A1 (en) Near infrared microbial elimination laser systems (NIMELS) for use with medical devices
WO2008073979A2 (fr) Modification électromagnétique proche infrarouge de potentiels de membrane stationnaires cellulaires
WO2010019800A1 (fr) Appareil de délivrance de lumière thérapeutique, procédé et système
US20090299263A1 (en) Near-Infrared electromagnetic modification of cellular steady-state membrane potentials
US20230256261A1 (en) Bactericidal methods and compositions
Feng et al. Dramatic destruction of methicillin-resistant Staphylococcus aureus infections with a simple combination of amoxicillin and light-activated methylene blue
WO2022087213A1 (fr) Procédés et composés pour le traitement de microbes positifs à la catalase
CN101663066A (zh) 近红外电磁改变稳态细胞膜电位
Aysan et al. Biostimulative effect of 809-nm diode laser and indocyanine green on p. aeruginosa instead of photodynamic therapy
Özhan et al. Potassium Chloride Potentiates Chlorin e6-Mediated Antimicrobial Photodynamic Inactivation
Kurachi Antimicrobial PDT

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780051272.2

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07865581

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2670711

Country of ref document: CA

Ref document number: 2007865581

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2007333073

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2009541559

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2007333073

Country of ref document: AU

Date of ref document: 20071212

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1020097014482

Country of ref document: KR