JP2010512232A - Near-infrared electromagnetic modification of cell steady-state membrane potential. - Google Patents

Abstract

  A system for applying near-infrared light energy and dosimetry to alter the bioenergetic steady-state transmembrane and mitochondrial potential (ΔΨ-steady) of all irradiated cells by light depolarization effects; and A method is disclosed herein. This depolarization causes a concomitant decrease in the absolute value of the transmembrane potential ΔΨ of irradiated mitochondria and plasma membrane. Many cellular anabolic reactions and drug resistance mechanisms are made less functional and / or weakened by a decrease in membrane potential ΔΨ, a closely related weakening of proton activation force Δp, and an associated reduced phosphorylation potential ΔGp. Can be. Within the irradiated area, a decrease in membrane potential ΔΨ occurs simultaneously in bacterial, fungal and mammalian cells. This depolarization of the membrane provides the ability to enhance antibacterial, antifungal and / or anti-neoplastic agents only to the targeted and unwanted cells.

Description

  The present invention generally relates to a method and system for the generation and dosimetry of infrared radiation at a selected energy, which depolarizes the bioenergetic steady state transmembrane and mitochondrial potential of irradiated cells. More particularly, the method and system for membrane depolarization to enhance antibacterial and antifungal compounds in target bacteria and / or fungi and / or cancer cells It is about.

  The worldwide increase in bacterial, fungal, and other microbial contamination that is resistant to antibacterial agents poses a major threat to human society. Since the advent of sulfa drugs (sulfanilamide, first used in 1936) and pencillin (1942, Pfizer Pharmaceuticals), the development of all kinds of significant amounts of antibacterial drugs across this planet has led to resistant pollutants and pathogens Built a powerful environment for the appearance and expansion of. Certain resistant contaminants are exceptionally epidemiologic because they dominate in hospitals and the general environment. The widespread use of antibiotics not only promotes the production of resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), but also in the infection of fungal microorganisms such as Candida (mycosis). It also has favorable conditions.

  Even in the presence of potent antifungal agents that are microbicides (eg, amphotericin B (AmB)), mortality from candidemia remains at about 38%. In some cases, high doses of AmB must be administered to treat drug-resistant fungi, often resulting in nephrotoxicity and other deleterious effects. Moreover, overuse of anti-mycobacterial agents and antibiotics can cause bioaccumulation in living organisms, which can be cytotoxic to mammalian cells as well. Given the growing global population and the spread of drug-resistant bacteria and fungi, it is expected that the increase in the incidence of bacterial or fungal infections has not diminished for the foreseeable future.

  Recently, available treatments for bacterial and fungal infections include the administration of antibacterial and antifungal agents, and in some cases, the application of surgical debridement of the infected area. In particular, new strategies to treat or prevent microbial infections because antibacterial and antifungal treatments alone are rarely effective due to the extreme prevalence of newly emerging drug resistant pathogens and highly deformed surgical treatments. Developing is inevitable.

  Therefore, the risk of bacterial and fungal infections is reduced at a given target site by biological parts other than the target bacteria and fungi (biological contaminants) (e.g. mammalian tissues, cells or certain biochemicals). There is a need for methods and systems that reduce without undue risk and / or adverse effects on preparations (eg, protein preparations).

  The present invention provides a minimum inhibitory concentration of antimicrobial molecules (antimicrobial agents) and / or anti-neoplastic molecules (anti-neoplastic agents) required to reduce or eliminate microbial and / or neoplastic related pathologies ( Methods and systems for reducing MIC) are directed to methods and systems that, without them, drugs that are no longer functional at safe human dosages will be useful again as an adjunct therapy. In accordance with the method and system of the present invention, irradiation and dosimetry at a selected energy in the near infrared (here known as NIMELS, which stands for “microbe elimination system by near infrared”), all within the irradiated area. Which will change the absolute value of the membrane potential ΔΨ of the irradiated cells.

Other features and advantages of the present invention are set forth in the detailed description of the specific examples which follow, and are in part apparent from the description, or may be learned by practice of the invention. Such features and advantages of the present invention will be understood and confirmed by the systems, methods and apparatus particularly pointed out in the written description and appended claims.
Aspects of the invention can be more fully understood by reading the following description in conjunction with the accompanying drawings, which are to be regarded as illustrative and not limiting in any way. These drawings are not necessarily to scale, and the emphasis is on the principles of the present invention. In these drawings:

FIG. 2 shows a typical phospholipid bilayer. It is the figure which showed the chemical structure of the phospholipid. It is the figure which showed the dipole effect (Ψd) in the phospholipid bilayer membrane. FIG. 2 shows a phospholipid bilayer in a bacterial plasma membrane, a mammalian mitochondrial membrane, or a fungal mitochondrial membrane with a steady-state transmembrane potential prior to NIMELS irradiation. FIG. 5 shows the potential of the transient plasma membrane in bacterial plasma membrane, mammalian mitochondrial membrane, or fungal mitochondrial membrane after NIMELS irradiation. It is the figure which showed the phospholipid bilayer which has a transmembrane protein embedded inside. FIG. 2 shows a general depiction of electron transfer and proton pump. FIG. 2 shows a general overview of mitochondrial membranes of fungal and mammalian cells corresponding to ΔΨ-mito-fungi or ΔΨ-mito-mam. FIG. 5 shows the effect of NIMELS irradiation (in single dose measurement) on MRSA transmembrane potential measured by green fluorescence emission intensity in a control and lasered sample as a function of time (minutes) after laser irradiation. FIG. 6 shows the effect of NIMELS irradiation (at various dose measurements) on the transmembrane potential of C. albicans measured by percent reduction compared to a control of green fluorescence emission intensity in a lasered sample. Effect of NIMELS irradiation (on single dosimetry) on the mitochondrial membrane potential of C. albicans measured by red fluorescence emission intensity in control and lasered samples; and red vs. green fluorescence in control and lasered samples FIG. 5 shows the effect of NIMELS irradiation (in single dosimetry) on the mitochondrial membrane potential of C. albicans measured as a ratio. Effect of NIMELS irradiation on mitochondrial membrane potential of human fetal kidney cells measured by red fluorescence emission intensity in control and lasered samples (in single dosimetry); and red vs. green fluorescence in control and lasered samples It is the figure which showed the effect (by single dosimetry) of NIMELS irradiation with respect to the mitochondrial membrane potential of the human embryonic kidney cell measured as ratio. FIG. 6 shows the reduction in total glutathione concentration in MRSA as it correlates with reactive oxygen species (ROS) production in these cells as a result of NIMELS irradiation (at some dosimetry) (laser The decrease in glutathione concentration in the applied sample is shown as a percentage of the control). C. The decrease in total glutathione concentration in C. albicans is shown as it correlates with reactive oxygen species (ROS) production in these cells as a result of NIMELS irradiation (at some dosimetry) ( The decrease in glutathione concentration in the lasered sample is shown as a percentage of the control). FIG. 6 shows a decrease in total glutathione concentration in human fetal kidney cells as it correlates with reactive oxygen species (ROS) production in these cells as a result of NIMELS irradiation (at some dosimetry). (The decrease in glutathione concentration in the lasered sample is shown as a percentage of the control). FIG. 5 shows the synergistic effect of NIMELS and methicillin in inhibiting growth of MRSA colonies (data shows that methicillin is enhanced by quasi-lethal NIMELS dosimetry). FIG. 2 shows the synergistic effect of NIMELS and bacitracin in inhibiting growth of MRSA colonies (arrows indicate growth of MRSA colonies in the two samples shown or lack thereof) (image shows that bacitracin is quasi-lethal It is enhanced by NIMELS dosimetry). FIG. 5 is a bar graph depicting the synergistic effect of NIMELS with methicillin, penicillin and erythromycin in inhibiting growth of MRSA colonies, as shown by experimental data. 2 is a composite photograph showing the improvement in nail appearance over time of a typical onychomycosis patient treated by the method of the present invention. Panel A shows infected toenails before treatment as a baseline; Panel B shows toenails 60 days after treatment; Panel C shows 80 days after treatment Panel D shows the toenails 100 days after treatment. FIG. 5 illustrates the detection of decreased membrane potential in E. coli by quasi-lethal NIMELS irradiation. FIG. 6 illustrates the detection of increased glutathione in E. coli by quasi-lethal NIMELS irradiation.

  While specific embodiments have been illustrated and described in the drawings, those skilled in the art will envision that the illustrated embodiments are for illustration purposes and variations of those shown, as well as others described herein. It will be appreciated that it can be implemented and is within the scope of the present invention.

  As used in this specification, the singular also includes the plural of that term unless the context clearly indicates otherwise. For example, “NIMELS wavelengths” can encompass a plurality of NIMELS wavelengths as well as combinations of such wavelengths.

  As used herein, unless otherwise indicated, the word “or” is used in the “non-exclusive” sense of “and / or” and in the “exclusive” sense of “any / or”. Absent.

  The term “about” is used herein to mean approximately the approximate range or neighborhood. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the numerical value indicated. In general, the term “about” is used herein to modify the stated numerical value by a variation of 20% up and down.

  The present invention relates to a method for reducing the minimum inhibitory concentration (MIC) of antibacterial molecules (drugs) and / or anti-neoplastic molecules (drugs) required to reduce or eliminate microorganisms and / or neoplastic-related pathogens. And systems, which otherwise would be directed to such methods and systems that are no longer functional at safe human dosages and that are again useful as adjunctive treatment by the methods and systems. In accordance with the method and system of the present invention, near-infrared irradiation and dosimetry at a selected energy (herein known as NIMELS, which stands for “Near-Infrared Microbial Exclusion System”), depolarization of the film in the irradiated region. Used to cause, it will change the absolute value of the membrane potential ΔΨ of irradiated cells.

  This altered ΔΨ will cause a closely related weakening of the proton motive force Δp and the bioenergetics of all affected membranes. Therefore, the effect of NIMELS irradiation (NIMELS effect) can reinforce existing antibacterial molecules against microorganisms that infect human hosts and cause harm. These effects will reduce the function of many cellular anabolic reactions (eg, cell wall formation) and drug resistance mechanisms (requiring chemo-osmotic electrochemical energy to function). Therefore, any membrane-bound cell resistance mechanism or anabolic reaction (using membrane potential ΔΨ, proton activation force Δp, or phosphorylation potential ΔGp for their functional energy requirements) is a method of the invention. And will be affected by the system.

  The methods and systems of the present invention utilize light irradiation to enhance antibacterial and / or antifungal drugs only against undesired target cells (e.g., MRSA or Candida infection of the skin) In general, it has the selectivity enabled by the fact that it is not affected by treatment (with molecules or drugs) intended to damage bacterial or fungal cells.

  In an exemplary embodiment, the applied light irradiation used in accordance with the methods and systems of the present invention includes one or more wavelengths ranging from about 850 to 900 nm in NIMELS dosimetry, as described above. In one aspect, a wavelength of about 865 to 875 nm is used. In another aspect, such applied irradiation has a wavelength of about 905-945 nm in NIMELS dosimetry. In one aspect, such applied light radiation has a wavelength of about 925-935 nm. In a special aspect, a wavelength of 930 nm (or a narrow wavelength range including the wavelength) can be used. In some aspects of the invention, the multiple wavelength ranges include 870 nm and 930 nm, respectively.

  Microbial pathogens whose bioenergy systems can be affected by NIMELS according to the present invention include microorganisms such as bacteria, fungi, filamentous fungi, mycoplasma, protozoa, and parasites.

  In one embodiment, the methods and systems of the present invention are used in treating, reducing and / or eliminating infectious entities known to cause skin infections or wound infections, such as staphylococci and enterococci. . Staphylococcal and enterococcal infections can involve almost any skin surface of the body and are known to cause skin diseases such as erosion, hemorrhoids, bullous impetigo, scaly skin. Staphylococcus aureus is also responsible for infections from staphylococcal food poisoning, enteritis, osteomyelitis, toxic shock syndrome, endocarditis, meningitis, pneumonia, cystitis, sepsis and surgical wounds. Staphylococcal infection can be received even when the patient is in a hospital or long-term care facility. The confined population and widespread use of antibiotics has led to the development of antibiotic-resistant S. aureus strains. These strains are called methicillin resistant Staphylococcus aureus (MRSA). Infections caused by MRSA are often resistant to a wide range of antibiotics (especially β-lactam antibiotics) and have significantly higher rates of morbidity and mortality than infections caused by non-MRSA microorganisms. Associated with higher costs and longer hospital stays. Risk factors for MRSA infection in hospitals include nasal colonization, surgery, previous antimicrobial treatment, entry to intensive care, exposure to MRSA colonized patients or health care, etiology for longer than 48 hours And having an indwelling catheter or other medical device that passes through the skin.

  In other embodiments, the methods and systems of the present invention are used in treating, reducing and / or eliminating an infectious entity known as cutaneous candidiasis. These Candida infections include the skin and can occupy almost any skin surface of the body. However, the most common are warm, moist or wrinkled areas (eg, axilla and groin). Cutaneous candidiasis is very common. Candida is the most common cause of diaper rash, and the warm and moist conditions inside the diaper favor Candida. The most common fungus that causes these infections is Candida albicans. Candida infection is also very common in individuals with diabetes and obesity. Candida can also cause nail infection called onychomycosis, infection of the skin around the nail (peritonitis), and infection around the corner of the mouth (called stomatitis).

The term “NIMELS dosimetry” refers to the values of power density (W / cm ² ) and energy density (J / cm ² ) (where 1 watt = 1 joule / second), which is the subject of the present invention. Can generate reactive oxygen species ("ROS"), thereby reducing the level of biological contaminants at the target site. The term also increases the sensitivity of biological contaminants by reducing ΔΨ with concomitant production of ROS, an antibacterial and anti-neoplastic agent (irradiating cells). If not, the contaminants are also resistant to drugs). This method may be effective without risks and / or side effects that are unacceptable to host patient tissues other than the biological contaminants.

  The “enhancing action” of an antifungal or antibacterial agent or an anti-neoplastic agent refers to the resistance mechanism in fungi, bacteria, or cancer, the growth and / or proliferation of the fungus, bacteria or cancer of the present invention. It means the method and system of the present invention that counteracts enough to block at even lower concentrations than without the method and system. If the resistance is essentially complete, i.e. if the drug has no effect on the cells, the potentiation action prevents the growth and / or proliferation of the pathogenic cells, thereby treating the disease state. It means to be treated with an upper acceptable dosage.

  As used herein, the term “microorganism” refers to an organism that is microscopic and by definition too small to be seen by the human eye. For the purposes of this invention, the microorganism may be a bacterium, fungus, archaea, protist, or the like. The term microbiology is defined as belonging to or related to a microorganism.

  As used herein, the term “cell membrane (or plasma membrane or mitochondrial membrane)” refers to a semi-permeable lipid bilayer, which has a common structure in all living cells. It mainly contains proteins and lipids that are involved in a myriad of important cellular processes. The cell membrane that is the target of the present invention has a protein / lipid ratio greater than 1. Stated another way, none of the target membrane of the contaminant (or part, ie host tissue) contains more than 49.99% lipid by dry weight.

  As used herein, the term “mitochondrion” refers to an organelle surrounded by a membrane found in most eukaryotic cells (mammalian cells and fungi). Mitochondria produce ATP (used as a chemical energy source for cells) required by most eukaryotic cells, and are therefore “intracellular power plants”. Mitochondria include an inner and outer membrane composed of a phospholipid bilayer and a protein. However, these two membranes have different characteristics. The outer mitochondrial membrane surrounds the entire organelle and has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane, and the inner mitochondrial membrane forms an internal fraction known as the crystal, It has a protein to phospholipid ratio similar to that of the organism's plasma membrane. This gives more space to function directly and efficiently for proteins such as cytochromes. The electron transport system (“ETS”) is located on this inner mitochondrial membrane. There are also highly controlled transport proteins within the mitochondrial inner membrane that transport metabolites across this membrane.

  As used herein, the term “flow mosaic model” refers to biology as a structurally and functionally asymmetric lipid bilayer with a variety of embedded proteins that facilitate transport across the membrane. Refers to the conceptualization of the target film. This flow mosaic model allows the phospholipid to change position almost easily in the membrane (flow) and because all phospholipid, protein and glycoprotein combinations present in the membrane give the cell a mosaic image from the outside. , So called. This model is based on a careful balance of thermodynamics and functional considerations. Changes in the thermodynamics of the film affect the function of the film.

  As used herein, the term “membrane dipole potential Ψd” (relative to the transmembrane potential ΔΨ) is between the highly hydrated lipid head on the membrane surface and the less polar interior (hydrophobic) of the bilayer. Refers to the potential formed by Lipid bilayers have a substantial membrane dipole potential Ψd that arises essentially from dipolar groups and molecular structural mechanisms (primarily ester linkages between phospholipids and water).

Ψd is independent of ions at the membrane surface and is used here to describe the following five different dipole potentials:
1) Dipolar potential of mammalian plasma membrane Ψd-plasm-mam;
2) The dipole potential of mammalian mitochondrial membranes Ψd-mito-mam;
3) The dipole potential of the fungal plasma membrane Ψd-plus-fungi;
4) The dipole potential of fungal mitochondrial membranes Ψd-mito-fungi; and 5) The dipolar potential of bacterial plasma membranes Ψd-plus-bact.

As used herein, the term “transmembrane potential” refers to the difference in electrical potential (mV scale) between the aqueous phases separated by the membrane and is given by the symbol (ΔΨ). ΔΨ is independent of ions at the membrane surface and is used here to describe three different plasma transmembrane potentials:
1) Mammalian plasma transmembrane potential ΔΨ-plasm-mam
2) Fungal plasma transmembrane potential ΔΨ-plus-fungi
3) Bacterial plasma transmembrane potential [Delta] [Psi] -plasm-bact.

As used herein, the term “mitochondrial transmembrane potential” refers to the difference in electrical potential (in mV scale) between the fractions separated by the inner mitochondrial membrane, where two different mitochondrial transmembrane potentials: Used to describe
1) Mammalian mitochondrial transmembrane potential ΔΨ-mito-mam
2) Fungal mitochondrial transmembrane potential ΔΨ-mito-fungi.

In mitochondria, potential energy from nutrients (eg, glucose) is converted into active energy available for cellular metabolic processes. The energy released during the continuous redox reaction allows protons (H ⁺ ions) to be pumped from the mitochondrial matrix into the intermembrane space. As a result, the mitochondrial membrane has a difference in chemical osmotic electrical potential (Δψ-mito-mam or Δψ-mito-fungi) as the membrane is polarized. Δψ-mito-mam and Δψ-mito-fungi are important parameters of mitochondrial functionality and give a direct quantitative value for the cellular energy state (redox state).

As used herein, the term “mammalian plasma transmembrane potential (ΔΨ-plasm-mam)” refers to the difference in electrical potential between aqueous phases in the mammalian cell plasma membrane. Mammalian plasma membrane potential differs from bacterial and fungal ΔΨ, mainly caused by H ⁺ ions (protons). In the mammalian plasma membrane, the main driver of ΔΨ is the electrogenic Na ⁺ / K ⁺ -ATPase pump. [Delta] [Psi] -plasm-mam is generated by additional properties of transmembrane K ^<+> diffusion (intracellular to extracellular) and an electrogenic Na ^<+ > / K ^<+> -ATPase pump. Mammalian ATP is produced in the mitochondria by a proton pump.

As used herein, the term “fungal plasma transmembrane potential (ΔΨ-plasm-fungi)” refers to the difference in electrical potential in the fungal cell plasma membrane. The fungal plasma membrane potential is generated by a membrane-bound H ⁺ -ATPase (a high-performance proton pump that requires ATP to function). This H ⁺ -ATPase pump is required for both fungal growth and stable cell metabolism and maintenance. Fungal ATP is produced in mitochondria.

As used herein, the term “bacterial plasma transmembrane potential (ΔΨ-plus-bact)” refers to the difference in electrical potential at the plasma membrane of bacterial cells. The bacterial plasma membrane potential is a steady-state flow (transfer) of electrons and protons (H ⁺ ) across the bacterial plasma membrane, with normal electron transfer and oxidative phosphorylation within the bacterial plasma membrane. Generated by the flow that occurs in A general feature of all electron transport chains is the presence of a proton pump to create a proton gradient through the membrane. Even though bacteria lack mitochondria, aerobic bacteria undergo oxidative phosphorylation (ATP production) by essentially the same process that occurs in eukaryotic mitochondria. As used herein, the term “P-class ion pump” refers to a transmembrane active transport protein assembly that includes an ATP binding site (ie, requires ATP to function). In the transport process, one of the protein subunits is phosphorylated and the transported ions are thought to migrate through the phosphorylated subunit. This class of ion pumps includes Na ⁺ and K ⁺ electrochemical potentials (ΔNa ⁺ / K ⁺ ) and pH gradients (common in animal cells) that maintain Na ⁺ / K ⁺ in the mammalian plasma membrane. -ATPase pump is included. Other important P-class ion pump members transport protons (H ⁺ ions) out of the cell and K ⁺ ions into the cell.

As used herein, the term “Na ⁺ / K ⁺ ATPase” is present in the plasma membrane of all animal cells and exports three Na ⁺ ions in conjunction with the hydrolysis of one ATP molecule. Refers to a P-class ion pump that imports two K ⁺ ions (which maintain Na ⁺ and K ⁺ electrochemical potentials and a pH gradient typical of animal cells). The inner negative membrane potential in fungal cells (and eukaryotic cells) is generated by the transport of H ⁺ ions out of the cell by a proton pump operated by a different ATP.

  As used herein, the terms “ion exchanger and ion channel” refer to transmembrane proteins that are ATP-independent systems that help establish plasma membrane potential in mammalian cells.

As used herein, the term “redox” describes a complex process of sugar oxidation in a cell by a very complex series of processes including, for example, electron transfer. The redox reaction is a chemical reaction in which electrons move from a donor molecule to an acceptor molecule. The term redox comes from two concepts, reduction and oxidation, and can be explained in the following simple terms:
Oxidation describes the loss of electrons by molecules, atoms or ions.
Reduction describes the acquisition of electrons by molecules, atoms or ions.

  As used herein, the term “redox state” describes the redox environment (or level of oxidative stress) of the cell being described.

  As used herein, the term “steady state transmembrane potential (ΔΨ-steady)” is the quantitative plasma membrane potential of a mammalian, fungal or bacterial cell prior to irradiation by the methods and systems of the invention. , Refers to the potential that will remain without such irradiation in the future.

For example, the steady-state flow of electrons and protons across the bacterial cell membrane that occurs in normal electron transfer and oxidative phosphorylation is in a steady state because of the constant flow rate of conventional redox reactions that occur across the membrane. Conversely, any modification of this redox state will cause a transient state membrane potential. ΔΨ-steady is used here to describe the three different steady-state plasma transmembrane potentials based on the species.
1) Steady-state mammalian plasma transmembrane potential ΔΨ-steady-mam
2) Steady-state fungal plasma transmembrane potential ΔΨ-steady-fungi
3) Steady state bacterial transmembrane potential ΔΨ-steady-bact

As used herein, the term “transient plasma membrane potential (ΔΨ-tran)” refers to the plasma membrane potential of mammalian, fungal or bacterial cells after irradiation by the methods and systems of the invention, Irradiation altered the bioenergetics of the plasma membrane. In bacteria, [Delta] [Psi] -tran will also change the redox state of cells because of the presence of ETS and cytochrome in the plasma membrane. ΔΨ-tran is a condition that does not occur without irradiation using the method of the present invention. ΔΨ-tran is used here to describe the plasma transmembrane potential of three different transient states based on the species.
1) Transmembrane potential of a mammal in a transient state ΔΨ-tran-mam
2) Transient fungal plasma transmembrane potential ΔΨ-tran-fungi
3) Transmembrane potential of the bacteria in a transient state ΔΨ-trans-bact

  As used herein, the term “steady state mitochondrial membrane potential (ΔΨ-steady-mito)” is the quantitative mitochondrial membrane potential of mammalian or fungal mitochondria prior to irradiation by the methods and systems of the present invention, It refers to the potential that will remain without such irradiation in the future.

For example, the steady state flow of electrons and protons across the inner mitochondrial membrane that occurs in normal electron transfer and oxidative phosphorylation is in a steady state due to the constant flow of conventional redox reactions that occur across the membrane. Any modification of this redox state will cause a transient mitochondrial membrane potential. ΔΨ-steady-mito is used to describe two different steady state mitochondrial membrane potentials based on species.
1) Steady-state mammalian mitochondrial potential ΔΨ-steady-mito-mam
2) Steady-state fungal mitochondrial potential ΔΨ-steady-mito-fungi

As used herein, the term “transient mitochondrial membrane potential (ΔΨ-tran-mito-mam or ΔΨ-tran-mito-fungi)” refers to the mammalian or fungal cell after irradiation by the methods and systems of the invention. Membrane potential, thereby referring to the membrane potential at which the irradiation altered the bioenergetics of the inner mitochondrial membrane. In mammalian and fungal cells, ΔΨ-tran-mito will also change the redox state of the cell because of the presence of the electron transport system (ETS) and cytochrome in the inner mitochondrial membrane. ΔΨ-tran-mito can also dramatically affect (proton activation force) Δp-mito-mam and Δp-mito-fungi (because these mitochondrial (H ⁺ ) gradients are innumerable Because it is produced within the mitochondria to produce enough ATP for the cellular function of ΔΨ-tran-mito is a condition that does not occur without irradiation by the method and system of the present invention. ΔΨ-tran-mito will be used to describe the mitochondrial membrane potential of two different transient states based on species.
1) Transient mammalian mitochondrial potential ΔΨ-tran-mito-mam
2) Fungal mitochondrial potential in a transient state ΔΨ-tran-mito-fungi

  As used herein, the term “cytochrome” refers to a hemoprotein bound to a membrane that has a heme atomic group and conducts electrons. As used herein, the term “electron transport system (ETS)” describes an electron carrier (cytochrome) bound to a series of membranes that mediate biochemical reactions that produce (ATP), the energy currency of the cell. In prokaryotic cells (bacteria) this occurs at the plasma membrane. In eukaryotic cells (fungi and mammalian cells) this occurs in mitochondria.

  As used herein, the term “pH gradient (ΔpH)” refers to the difference in pH between the bulk phases on either side of the membrane.

As used herein, the term “proton electrochemical gradient (ΔμH ⁺ ) (dimension kJ mol −1)” refers to the electrical and chemical properties across the membrane, in particular the proton gradient, and a cell that can work in a cell. Represents a kind of potential energy. This difference in proton electrochemical potential between the sides of the membrane engaged in active transport, including the proton pump, is sometimes referred to as the chemical osmotic potential or proton activation force. If ΔμH ⁺ is somewhat reduced, it is hypothesized that the cellular anabolic pathway and resistance mechanisms in the affected cells are inhibited. This can be accomplished by combining λn and Tn to irradiate only the target site, or by simultaneously or sequentially administering drugs arranged and arranged for delivery to the target site. Further enhancement can be achieved (ie, simultaneous targeting of (An and Tn) + (pharmacological molecules) in the anabolic pathway).

As used herein, the term “ionic electrochemical gradient (Δμx +)” refers to the electrical and chemical properties across the membrane caused by a concentration gradient of ions (other than H ⁺ ) and can work in cells. Represents a kind of cell potential energy. In mammalian cells, an electrochemical gradient of Na ⁺ ions is maintained across the plasma membrane by active transport of Na ^{+ out} of the cell. This is a gradient different from the proton electrochemical potential, and is generated from a pump coupled to ATP, and the ATP generated in oxidative phosphorylation is expressed in mammalian mitochondrial proton activation force (Δp-mito-mam). ). If Δμx ⁺ is somewhat reduced, it is hypothesized that the cellular anabolic pathway and resistance mechanisms in the affected cells are inhibited. This can be achieved by combining An and Tn to irradiate only the target site, or by simultaneously or sequentially administering drugs arranged and arranged for delivery to the target site. It can be further enhanced (ie, simultaneous targeting of (λn and Tn) + (pharmacological molecule) in the anabolic pathway).

As used herein, the term "simultaneous targeting of bacterial anabolic pathway", (the cells at influencing target site anabolic pathway (ΔμH ⁺⁾ and / or (lambda] n and Tn lowering Δμx ^{+)) +} (the same Can refer to any of the following bacterial anabolic pathways that can be blocked by pharmacological molecules:
The targeted anabolic pathway is peptidoglycan biosynthesis, which is simultaneously targeted by drugs that bind to the active site of the bacterial transpeptidase enzyme (penicillin-binding protein) that crosslinks the peptidoglycan in the bacterial cell wall. Enzyme inhibition ultimately causes cell lysis and cell death;
The targeted bacterial anabolic pathway is peptidoglycan biosynthesis, which is simultaneously targeted by drugs that bind to acyl-D-alanyl-D-alanyl groups in cell wall intermediates, and thus N-acetylmura. Blocks incorporation of minic acid (NAM)-and N-acetylglucosamine (NAG) -peptide subunits into peptidoglycan matrix (effectively affects peptidoglycan biosynthesis by acting on transglycosylation and / or transpeptideation Block), thereby inhibiting the formation of suitable peptidoglycans in Gram positive bacteria.
Anabolic pathways of bacteria to be targeted is a peptidoglycan biosynthesis, which is C ₅₅ - be targeted simultaneously by isoprenyl pyrophosphate and binding to the drug, pyrophosphatase C ₅₅ - interact with the isoprenyl pyrophosphate Reducing the amount of C ₅₅ -isoprenyl pyrophosphate available to carry building block peptidoglycans out of the inner membrane;
The targeted anabolic pathway is the biosynthesis of bacterial proteins, which are simultaneously targeted by drugs that bind to 23S rRNA molecules in the 50S subunit of the bacterial ribosome, which is responsible for the accumulation of peptidyl tRNA in cells. Resulting in the depletion of the free tRNA required for α-amino acid activation, thus preventing peptide translocation by causing immature dissociation of the peptidyl tRNA from the ribosome;
Simultaneously targeted drugs can simultaneously bind to two domains of the 23S RNA of the 50S bacterial ribosomal subunit, thereby preventing the formation of bacterial ribosomal subunits 50S and 30S (ribosomal subunit assembly) ( Here, the simultaneously targeted drug is chlorinated to increase its lipophilicity to penetrate bacterial cells), and binds to the 23S portion of the 50S subunit of the bacterial ribosome to bind peptidyl-tRNA. Blocks migration from the aminoacyl site (A site) to the peptidyl site (P site), thereby blocking the transpeptidase reaction, resulting in an incomplete peptide being released from the ribosome;
The targeted anabolic pathway is bacterial protein biosynthesis, which is simultaneously targeted by drugs that bind to the bacterial ribosomal subunit of 30S, allowing the binding of the amino-acyl tRNA to the acceptor site (A site) of the ribosome. Block, thereby preventing codon-anticodon interactions and the elongation phase of protein synthesis;
Simultaneously targeted drugs bind more enthusiastically to bacterial ribosomes in a different orientation than the classic subclass of polyketide antibacterials with octahydrotetracene-2-carboxamide skeletons, so they are tet (M) Active against strains of S. aureus having ribosomes and tet (K) outward flux genetic determinants;
The targeted anabolic pathway is the biosynthesis of bacterial proteins, which are concomitant with drugs that bind to specific aminoacyl-tRNA synthetases and prevent esterification of precursors to specific amino acids or one of their compatible tRNAs. Targeted, thus preventing the formation of aminoacyl-tRNA, thus stopping the incorporation of the necessary amino acids into the bacterial protein;
The targeted anabolic pathway is the biosynthesis of bacterial proteins, which bind to 50S rRNA via domain V of 23S rRNA and at the same time the 30S ribosomal subunit by drugs that inhibit bacterial protein synthesis prior to the initiation phase. By simultaneously interacting with the 16S rRNA of the target, thus blocking the binding of the initiator of protein synthesis formyl-methionine (f-Met-tRNA) and the 30S ribosomal subunit. Is the biosynthesis of a bacterial protein, which is simultaneously targeted by a drug that interacts near the peptidyl transferase center at the protein L2 in the region of the 50S subunit of the bacterial ribosome and the P-site of 23S rRNA, and is therefore peptidyl Transformer blowjob It prevents peptidase activity and peptidyl transfer, blocking P-site interactions, interfere with the normal formation of active 50S ribosomal subunit;
Targeted anabolic pathways are DNA replication and transcription, which are simultaneously targeted by drugs that inhibit topoisomerase II (DNA gyrase) and / or topoisomerase IV;
The targeted anabolic pathway is DNA replication and translation, which is a drug that inhibits DNA polymerase IIIC (an enzyme required for chromosomal DNA replication in Gram-positive bacteria but not present in Gram-negative bacteria). Simultaneously targeted by
The targeted anabolic pathway is DNA replication and transcription, which are simultaneously targeted by pharmacological hybrid compounds that inhibit topoisomerase II (DNA gyrase) and / or topoisomerase IV and / or DNA polymerase IIIC;
The targeted anabolic pathway is the biosynthesis of bacterial phospholipids, which are simultaneously targeted by topical drugs that act on the cytoplasmic membrane rich in phosphatidylethanolamine and work well in combination with other topical synergistic drugs Be considered;
The targeted anabolic pathway is the biosynthesis of bacterial fatty acids, which converts the biosynthesis of bacterial fatty acids into β-ketoacyl- (acyl-carrier-protein (ACP)) synthase I / II (FabF / B) Simultaneously targeted by drugs that inhibit by selective targeting of (enzymes essential for type II fatty acid synthesis);
The targeted anabolic pathway is the maintenance of the bacterial plasma transmembrane potential ΔΨ-plasm-bact, and co-targeting drugs disrupt many aspects of bacterial cell membrane function primarily by binding to the Gram-positive cytoplasmic membrane. And does not penetrate the cell, causing depolarization and loss of membrane potential, which leads to inhibition of protein, DNA and RNA synthesis;
Co-targeting drugs increase the permeability of the bacterial cell wall, thus allowing inorganic cations to move through the wall in an unrestricted manner, thereby allowing ions between the cytoplasm and the extracellular environment. Destroy the gradient;
The targeted anabolic pathway is the maintenance of selective permeability of the bacterial membrane and the bacterial plasma transmembrane potential ΔΨ-plus-bact, the co-targeting drug is a cationic bacterial peptide, which is eukaryotic Selective to the negatively charged surface of the bacterial membrane compared to the neutral membrane surface of the cell, leading to permeability of the prokaryotic membrane and final perforation and / or disruption of the bacterial cell membrane, Thereby facilitating leakage of bacterial cell contents and disruption of the transmembrane potential;
Co-targeting drugs inhibit the bacterial protease peptide deformylase, which catalyzes the removal of the formyl group from the N-terminus of newly synthesized bacterial polypeptides; and co-targeting Drugs inhibit two-component regulatory systems in bacteria, such as their ability to respond to the environment through signal transduction across the bacterial plasma membrane, but these signal transduction processes are not present in mammalian membranes Is.

As used herein, the term "simultaneous targeting of anabolic pathways true fungi" (the cells of the target site to affect the anabolic pathway (ΔμH ⁺⁾ and / or (lambda] n and Tn lowering the Δμx ^{+)) +} (Drugs that affect the anabolic pathway of the same fungus) and can refer to any of the following fungal anabolic pathways, which can be inhibited by multiple drugs:
The targeted anabolic pathway is phospholipid biosynthesis, which destroys the structure of existing phospholipids in fungal cell membranes and is simultaneously performed by topical drugs that work well in combination with other topical synergists. Be targeted;
The targeted anabolic pathway is ergosterol biosynthesis, which is a three-step catalyzed ergosterol biosynthesis by the C-14 demethylation step, cytochrome P-450 enzyme 14-a-sterol demethylase. That are simultaneously targeted by drugs that inhibit the oxidative reaction of ergosterol, resulting in depletion of ergosterol and accumulation of lanosterol and other 14-methylated sterols, which has a “bulk” function as a membrane component of ergosterol. Disturbed by disruption of the structure of the plasma membrane;
The targeted anabolic pathway is ergosterol biosynthesis, which is simultaneously targeted by drugs that inhibit the enzyme squalene epoxidase, which in turn inhibits ergosterol biosynthesis in fungal cells, This gives the fungal cell membrane increased permeability;
The targeted anabolic pathway is ergosterol biosynthesis, which is simultaneously targeted by drugs that inhibit two enzymes, d14-reductase and d7, d8-isomerase, at separate points in the ergosterol biosynthesis pathway. Be considered;
The targeted anabolic pathway is biosynthesis of the fungal cell wall, which is a drug that inhibits the enzyme (1,3) β-D-glucan synthase (which in turn directs β-D-glucan synthesis in the fungal cell wall. Simultaneously)
The targeted anabolic pathway is the biosynthesis of fungal sterols, which are simultaneously targeted by drugs that bind to sterols in the fungal cell wall, and the main sterol to which the co-targeting drugs bind is ergosterol, This effectively changes the transition temperature of the cell membrane, causing pores to form in the membrane, resulting in the formation of ion channels that are detrimental to the fungal cell membrane;
Simultaneously targeted drugs are formulated into lipids, liposomes, lipid complexes and / or colloidal dispersions for delivery to prevent toxicity from the drug;
The targeted anabolic pathway is protein synthesis, where 5-FC is taken up by fungal cells by cytosine permease, deaminated to 5-fluorouracil (5-FU), and converted to nucleoside triphosphates. And simultaneously targeted by drugs that are incorporated into RNA and cause miscoding;
The targeted anabolic pathway is fungal protein synthesis, which is the fungal elongation factor EF-2 (which is functionally different from its mammalian analogues) and / or mammalian cells. Simultaneously targeted by drugs that inhibit the absence of fungal elongation factor 3 (EF-3);
The targeted anabolic pathway is fungal chitin biosynthesis (a homopolymer of β- (1,4) -linked N-acetyl-D-glucosamine), which involves the biosynthesis of fungal chitin, the enzyme chitin Synthase 2, simultaneously targeted by drugs that inhibit by inhibiting the action of at least one enzyme required for fungal primary septum formation and cell division;
Co-targeting drugs inhibit the action of the enzyme chitin synthase 3, the enzyme required for chitin synthesis during bud emergence and growth, mating and sporulation;
Co-targeting drugs chelate multivalent cations (Fe ⁺³ or Al ⁺³ ), resulting in inhibition of metal-dependent enzymes responsible for mitochondrial electron transfer and cellular energy production, which is also a fungal cell It also leads to inhibition of the normal degradation of intracellular peroxides; and co-targeting drugs inhibit the ability to respond to the environment by signal transduction across the fungal plasma membrane, such as two-component regulatory systems in fungi.

As used herein, the term "co-targeting of cancer anabolic pathway", (the cell in influencing target site anabolic pathway (ΔμH ⁺⁾ and / or (lambda] n and Tn lowering Δμx ^{+)) +} (the same cancer An anabolic pathway can refer to any of the following cancer anabolic pathways that can be blocked by drugs):
The targeted anabolic pathway is DNA replication, which makes it impossible for DNA replication to crosslink guanine nucleobases in the DNA double helix strand to unwind and separate the strand (required for DNA replication). Simultaneously targeted by drugs that block by
The targeted anabolic pathway is DNA replication, which is simultaneously targeted by drugs that can react with two different 7-N-guanine residues in the same or different strands of DNA;
The targeted anabolic pathway is DNA replication, which is simultaneously targeted by drugs that inhibit DNA replication and cell division by acting as antimetabolites;
The targeted anabolic pathway is cell division, which is simultaneously targeted by drugs that inhibit cell division by interfering with microtubule function;
The targeted anabolic pathway is DNA replication, which inhibits DNA replication and cell division by preventing cells from entering G1 phase (onset of DNA replication) and preventing DNA replication (S phase). Simultaneously targeted by
The targeted anabolic pathway is cell division, which is simultaneously targeted by drugs that promote microtubule stability that prevents chromosomal segregation late; and the targeted anabolic pathway is DNA replication Yes, it is simultaneously targeted by drugs that inhibit DNA replication and cell division by inhibiting type I and type II topoisomerases (this is transcription and replication of DNA by disrupting appropriate DNA supercoiling) Will disturb you).

As used herein, the term “proton activation force (Δp)” refers to storing energy (acting like a type of accumulator) as a combination of protons across the membrane and a voltage gradient. The two components Δp and [Delta] [Psi] (transmembrane potential) and delta pH (chemical gradient of H ^+). In another standard method, Δp consists of H + transmembrane potential Δψ (externally negative (acidic)) and transmembrane pH gradient ΔpH (internally alkaline). The potential energy stored in the form of this electrochemical gradient is by pumping hydrogen ions across the biological membrane (mitochondrial inner membrane or bacterial and fungal plasma membrane) in chemical osmotic pressure. Generated. This Δp can be used for chemical, osmotic or mechanical work in the cell. Proton gradients are commonly used for oxidative phosphorylation to drive ATP synthesis and can be used to drive efflux pumps in bacterial, fungal or mammalian cells, including cancer cells. Δp is used here to describe the proton activation forces in four different membranes based on the species and is mathematically defined as (ΔP = ΔΨ + ΔpH).
1) Mammalian mitochondrial proton activation force (Δp-mito-mam)
2) Proton activation force of fungal mitochondria (Δp-mito-Fungi)
3) Proton activation force of fungal plasma membrane (Δp-plus-Fungi)
4) Proton activation force of bacterial plasma membrane (Δp-plus-Bact)

As used herein, the term “mammalian mitochondrial proton activation force (Δp-mito-mam)” refers to the potential stored in the form of an electrochemical gradient of (H ⁺ ) across the inner mitochondrial membrane of mammals. It refers to energy. Δp-mito-mam is utilized to drive ATP synthesis in mammalian mitochondria in oxidative phosphorylation.

As used herein, the term “fungal mitochondrial proton activation force (Δp-mito-Fungi)” refers to the potential energy stored in the form of an electrochemical gradient across the fungal mitochondrial inner membrane (H ⁺ ). Δp-mito-Fungi is utilized to drive ATP synthesis in fungal mitochondria in oxidative phosphorylation.

As used herein, the term “fungal plasma membrane proton motive force (Δp-plus-Fungi)” is stored in the form of an electrochemical gradient across the fungal plasma membrane (H ⁺ ), and hydrogen ions Refers to the potential energy generated by pumping by membrane-bound H ⁺ -ATPase across the plasma membrane. The H ⁺ -ATPase bound to this plasma membrane is a high-performance proton pump and requires ATP to function. ATP for this H ⁺ -ATPase is generated from Δp-mito-Fungi. Δp-plus-Fungi can be used to drive an efflux pump in fungal cells.

As used herein, the term “bacterial plasma membrane proton motive force (Δp-plus-Bact)” refers to the potential energy stored in the form of an electrochemical gradient (H ⁺ ) across the bacterial plasma membrane. It is generated by pumping hydrogen ions across the plasma membrane under chemical osmotic pressure. Δp-plus-Bact is used in oxidative phosphorylation to drive ATP synthesis at the bacterial plasma membrane and can be utilized to drive efflux pumps in bacterial cells.

  As used herein, the term “anabolic pathway” refers to a cellular metabolic pathway that assembles structural molecules from smaller units. These reactions require energy. Many anabolic pathways and processes are facilitated by adenosine triphosphate (ATP). These processes involve simple and complex molecules such as amino acids such as peptidoglycan, protein, enzyme, ribosome, cellular organelle, nucleic acid, DNA, RNA, glucan, chitin, simple fatty acids, complex fatty acids, cholesterol, steroids and ergosterol. Synthesis can be included.

  As used herein, the term “energy conversion” refers to the pumping of protons across a membrane through a respiratory complex embedded in the membrane to create an electrochemical potential (also known as a proton electrochemical gradient). It refers to the movement of electrons using an electron transfer reaction.

  As used herein, the term “energy transformation” in a cell refers to a chemical bond that is always broken or created to allow energy exchange and conversion. In general, the type conversion of energy from a higher concentration form to a lower concentration form is said to be the driving force for all biological or chemical processes (responsible for cellular respiration).

As used herein, the term “uncoupler” refers to a molecule or device that causes a separation of the energy stored in the electrochemical proton gradient (ΔμH ⁺ ) of the membrane proton from the synthesis of ATP.

As used herein, the term “uncoupled” refers to the use of an uncoupler (molecule or device) to cause separation of the energy stored in the proton electrochemical gradient (ΔμH ⁺ ) of the membrane from the synthesis of ATP. .

  As used herein, the term “adenosine 5′-triphosphate (ATP)” refers to a multifunctional nucleotide that acts as a “molecular currency” for intracellular energy transfer. ATP transports chemical energy within the cell for metabolism and is produced as a source of energy in the process of cellular respiration. ATP is consumed by many enzymes and a wide range of cellular processes, including biosynthetic reactions, efflux pump functions and cell growth and division by assimilation.

  As used herein, the term “adenosine diphosphate (ADP)” is the product of the dephosphorylation of ATP by the ATPase. ADP is returned to ATP by ATP synthesis. In aerobic cells, it is understood that under physiological conditions, ATP synthase produces ATP using the proton activation force Δp created by TES as an energy source. The process of creating all this form of energy is called oxidative phosphorylation. The overall reaction sequence for oxidative phosphorylation is ADP + Pi → ATP. The potential to drive biological reactions is the Gibbs free energy of reactants and products. Gibbs free energy is the energy that can be used to do work ("free"), and the term Gibbs free energy change (ΔG) is the change in free energy that can be used to work in the membrane. Point to. This free energy is a function of enthalpy (ΔH), entropy (ΔS) and temperature. (Enthalpy and entropy are discussed below.)

As used herein, the term “phosphorylation potential (ΔGp)” refers to ΔG for ATP synthesis at any given set of ATP, ADP, and Pi concentrations (dimension: kJ mol ⁻¹ ).

As used herein, the term “CCCP” refers to carbonyl cyanide m-chlorophenylhydrazone, a highly toxic ionophore and uncoupler of the respiratory chain. CCCP acts as a classic uncoupler by increasing proton conductivity through the membrane, uncoupling ATP synthesis from ΔμH ⁺ and extinguishing ΔΨ and ΔpH.

  As used herein, the term “depolarization” (de-energization) refers to a decrease in the absolute value of a cell's plasma or mitochondrial membrane potential ΔΨ. It is hypothesized that any bacterial plasma membrane depolarization leads to loss of ATP and increased free radical formation. It is also hypothesized that any eukaryotic mitochondrial depolarization leads to loss of ATP and increased free radical formation.

  As used herein, the term “enthalpy change (ΔH)” refers to a change in the enthalpy or heat content of a membrane system and is an index or description of the thermodynamic potential of the membrane system.

  As used herein, the term “entropy change (ΔS)” refers to a change in the entropy of a membrane system to a more perturbed state of entropy at the molecular level. The term “redox stress” refers to a cellular state that is different from the normal oxidation / reduction potential (“redox”) state of a cell. Redox stress includes increased levels of ROS, decreased levels of glutathione, and any other situation that alters the redox potential of a cell.

As used herein, the term “reactive oxygen species” refers to one of the following categories:
a) Superoxide ion radical (O ₂ ⁻ )
b) Hydrogen peroxide (not radical) (H ₂ O ₂₎
c) Hydroxyl radical ( ^* OH)
d) Hydroxy ion (OH ⁻ )
These ROS are generally produced by the following reaction chains:
O ₂ → O ₂ ⁻ + 2H ⁺ → H ₂ O ₂ → OH ⁻ + ^* OH → OH ^{−
(e -) (e -)} (e -) (e -)

As used herein, the term “singlet oxygen” refers to (“1O ₂ ”) and is formed by interaction with a triplet excited molecule. Singlet oxygen is a non-radical species with antiparallel spin electrons. Since singlet oxygen 1O ₂ does not have its electron spin restriction, it has very high oxidative power and easily forms membranes (eg, with polyunsaturated fatty acids, ie PUFA), amino acid residues, proteins and It can attack DNA.

  As used herein, the term “energy stress” refers to a condition that alters the ATP level of a cell. This may be exposure to changes in electron transport and uncoupling agents or irradiation that alters ΔΨ in the mitochondria and / or plasma membrane.

  As used herein, the term “NIMELS effect” refers to the modification of the bioenergetic “state” according to the present invention from ΔΨ-steady to ΔΨ-trans at the plasma membrane and mitochondrial level of irradiated cells. Point to. In particular, the NIMELS effect can weaken cellular anabolic pathways or antibacterial and / or cancer resistance mechanisms (utilizing proton motive force or chemo-osmotic potential for energy requirements).

  As used herein, the term “periplasmic space or periplasm” refers to the space between the plasma membrane and the outer membrane of Gram negative bacteria and between the plasma membrane and the cell wall of Gram positive bacteria and fungi such as Candida and Ringworm. Refers to space. This periplasmic space is involved in a variety of biochemical pathways including nutrient acquisition, peptidoglycan synthesis, electron transfer, and modification of substances that are toxic to cells. In Gram positive bacteria such as MRSA, the periplasmic space is of significant clinical importance when the β-lactamase enzyme inactivates penicillin-based antibiotics.

  As used herein, the term “efflux pump” refers to the cytoplasm or periplasm of cells for the exclusion of molecules (such as antibiotics, antifungals or poisons) in an energy-dependent manner from their cells to the external environment. Refers to an active transport protein assembly exported from

  As used herein, the term “efflux pump inhibitor” refers to compounds or electromagnetic radiation delivery systems and methods that interfere with the ability of the efflux pump to export molecules from the cell. In particular, the efflux pump inhibitor of this invention is in the form of electromagnetic radiation, which indicates the ability of the pump to release therapeutic antibiotics, antifungals, anti-neoplastics and toxicants from cells, ΔΨ-steady-mam. , Δψ-steady-fungi or Δψ-steady-bact.

  A cell “uses an efflux pump resistance mechanism” means that a bacterial or fungal or cancer cell transfers antibacterial and / or antifungal and / or antineoplastic agents from the cytoplasm or periplasm to the external environment of the cell. Means exporting, thereby lowering the intracellular concentration of these agents to a concentration lower than that required to inhibit cell growth and / or proliferation.

  In the context of cell growth, the term “inhibit” means to reduce and possibly stop the rate of growth and / or proliferation of a cell population.

  In protein chemistry, primary structure refers to the linear sequence of amino acids; secondary structure refers to whether the linear amino acid structure forms a helix or a β-sheet structure; a protein or any other macromolecule The tertiary structure of is the three-dimensional structure, otherwise, it is the spatial organization (including conformation) of the entire single-stranded molecule; Of protein molecules in a multi-subunit complex.

  As used herein, the term “protein stress” refers to thermodynamic modification of the tertiary and quaternary structure of a protein, including enzymes and other proteins involved in membrane trafficking. The term includes protein denaturation, protein misfolding, protein cross-linking, both oxygen-dependent and independent oxidation of interchain and intrachain bonds (e.g., disulfide bonds), oxidation of individual amino acids, and the like ( However, there is no limit).

  The term “pH stress” refers to a modification of intracellular pH (ie, a reduced intracellular pH of less than about 6.0 or an elevated intracellular pH of greater than about 7.5). This can be caused, for example, by subjecting the cell to the invention described herein to alter cell membrane components or to cause a change in the steady state membrane potential ΔΨ-steady.

  As used herein, the term “antifungal molecule” refers to a fungicidal or fungistatic chemical or compound. The main efficacy is to inhibit antifungal molecules by inhibiting aerobic reactions and / or efflux pump activity in resistant fungal strains, or other resistance mechanisms that require proton motive force or chemo-osmotic potential for energy. It is the ability of the present invention to strengthen by inhibiting.

  As used herein, the term “antibacterial molecule (or drug)” refers to a bactericidal or bacteriostatic chemical or compound. Other main efficacy is that the antibacterial molecules of the present invention can be used to inhibit efflux pump activity in resistant bacterial strains, or to aerobic reactions requiring proton motive force or chemo-osmotic potential with respect to energy and / or Or in the ability to strengthen by inhibiting the resistance mechanism.

  As used herein, the term “quasi-inhibitory concentration” of an antibacterial or antifungal molecule refers to a concentration below that required to inhibit the majority of the population of target cells. (In one aspect, target cells are cells including but not limited to bacterial, fungal, and cancer cells that are targeted for therapy.) Generally, sub-inhibitory concentrations are the minimum inhibitory concentrations. Refers to a lower concentration than (MIC), which is defined as the concentration required to produce at least a 10% reduction in target cell growth or proliferation, unless otherwise stated.

  As used herein, the term “minimum inhibitory concentration” or MIC is defined as the lowest effective or therapeutically effective concentration that results in the inhibition of microbial growth.

  As used herein, the term “therapeutically effective amount” of a drug or molecule (eg, antibacterial or antifungal agent) refers to at least one symptom caused by a target (pathogenic) cell together with NIMELS. Refers to the concentration of drug that is partially or completely removed. In particular, a therapeutically effective amount is (1) if not eliminated, reduces the target cell population in the body, and (2) inhibits (ie, does not stop) the growth of the target cell population in the body, (3) to prevent the spread of infection (ie slow down if not stopped), (4) to reduce symptoms associated with infection (if not to eliminate), drugs with NIMELS Refers to the amount.

  As used herein, the term “interaction rate” is a numerical representation of the grade of bacteriostatic / bactericidal and / or bacteriostatic / fungicidal interaction for a target cell between a NIMELS laser and / or an antimicrobial molecule. Defined.

Thermodynamics of energy conversion in biological membranes The present invention disrupts the biological thermodynamics (bioenergetics) of cell membranes and results in standard energy conversion and energy type conversion of irradiated cells. Is directed to a reduced tolerance for proper reception.

  The method and system of the present invention optically alters and modifies Ψd-plasm-mam, Ψd-mito-mam, Ψd-plus-fungi, Ψd-mito-fungi and Ψd-plus-bact, Drive further changes in ΔΨ and Δp of the film. This is caused by near-infrared irradiation targeted to C—H covalent bonds in the long-chain fatty acids of the lipid bilayer, causing a change in the dipole potential ψd.

  To help understand this process of bioenergetic modification, the following description of the application of thermodynamics to membrane bioenergetics and energy conversion in biological membranes is given. First, the membrane (lipid bilayer, see FIG. 1) has a significant dipole potential Ψd resulting from structural bonds of dipolar groups and molecules, mainly ester bonds between phospholipids (FIG. 2) and water. ing. These bipolar groups are oriented so that the hydrocarbon phase is positive with respect to the outer membrane region (FIG. 3). The degree of dipole potential is usually large, typically a few hundred millivolts. The second major potential, the separation of charge across the membrane, gives rise to a transmembrane potential ΔΨ. This transmembrane potential is defined as the difference in electrical potential between the bulk aqueous phases on either side of the membrane and arises from the selective transport of charged molecules across the membrane. As a rule, this potential on the cytoplasmic side of the cell membrane is negative for extracellular physiological solutions (FIG. 4A).

  The dipole potential ψd constitutes a large and functionally important part of the electrostatic potential of all plasma and mitochondrial membranes. Ψd modifies the electric field inside the film, creating a virtual positive charge at the center of the non-bipolar bilayer. As a result of this “positive charge”, lipid membranes show a significant (eg, up to 6 orders of magnitude) difference in permeability between positive and negatively charged hydrophobic ions. Ψd also plays an important role in membrane permeability to lipophilic ions.

  Many stages of cellular processes such as protein (enzyme) binding and insertion, protein lateral diffusion, ligand-receptor recognition, and membrane fusion to internal and external molecules are the physical properties of the membrane bilayer. It depends critically on Ψd. Studies in model membrane systems have shown the ability of monovalent and multivalent ions to cause isothermal phase transitions, separation of different phases, and distinct clustering of individual components in a mixture in pure lipids. In membranes, charges such as these can physically affect the conformational dynamics of proteins and cytochromes embedded in the membrane (FIG. 4B), and more particularly, large conformational rearrangements in them. Proteins received at the transmembrane domain during the working cycle can be physically affected (Figure 5). Most importantly, the charge at Ψd is believed to modulate the enzymatic activity of the membrane.

Energy conversion Energy conversion in biological membranes generally involves the following three interrelated mechanisms:
1) Conversion of redox energy to “free energy” stored in a transmembrane ionic electrochemical potential (also called membrane proton electrochemical gradient ΔμH ⁺ ). This difference in proton electrochemical potential between the two sides of a membrane engaged in active transport including a proton pump is sometimes referred to as a chemical osmotic potential or proton activation force.
2) In mammalian cells, the (Na ⁺ ) ion electrochemical gradient Δμx ⁺ is maintained across the plasma membrane by active transport of (Na ⁺ ) out of the cell. This is a gradient different from the proton electrochemical potential, and is generated by (pump) by ATP generated from mammalian mitochondrial proton activation force Δp-mito-mamm in oxidative phosphorylation.
3) The use of this “free energy” to build ATP (energy conversion) to drive active transport across the membrane (with concomitant accumulation of solutes and metabolites required in cells) This is called an oxidation potential ΔGp. In other words, ΔGp is ΔG for ATP synthesis in any given set of ATP, ADP and Pi concentrations.

Steady state transmembrane protein (ΔΨ-steady)
The state of the membrane “system” is equilibrium when its chemical potential gradient ΔμH ⁺ and E (energy) are temporarily independent and there is no flow of energy across the edge of the system. If the membrane system variables ΔμH ⁺ and E are constant but there is a net flow of energy transfer across the system, the membrane system is in a steady state and is temporarily dependent.

The focus is this transiently dependent steady state transmembrane and / or mitochondrial potential (ΔΨ-steady) of cells (breathing, growing, dividing cells). This “steady state” of the flow of electrons and protons or Na ⁺ / K ⁺ ions across the mitochondria or plasma membrane during normal electron transfer and oxidative phosphorylation is due to intrinsic or exogenous events. If not disturbed, it is most likely to continue into the future. Any extrinsic modification of membrane thermodynamics results in a transient state of transmembrane and / or mitochondrial potential Δψ-trans, which change from Δψ-steady to Δψ-trans is the object of the present invention.

  The mathematical relationship between the state variables ΔΨ-steady and ΔΨ-trans is called the state equation. In thermodynamics, a state function (state quantity) is a characteristic or system that depends only on the current state of the system. It does not depend on the path that the system has reached in its own state. The present invention facilitates transmembrane and / or state changes in the mitochondrial potential ΔΨ, and in a temporally dependent manner, changes the bioenergetics of the membrane from the thermodynamic steady state ΔΨ−steady to the transient state ΔΨ−. Move to one of energy stress and / or redox stress in trans.

  This can occur in [Delta] [Psi] -steady-mam, [Delta] [Psi] -steady-Bact- [Delta] [Psi] -steady-mito-mam and [Delta] [Psi] -steady-mito-fungi. While not wishing to be bound by theory, this change is caused by near-infrared (wavelength 930 nm) irradiation (dipole potential Ψd change targeted to C—H covalent bonds in long-chain fatty acids of lipid bilayers. And the near infrared (wavelength 870 nm) irradiation targeted to the cytochrome chain (which simultaneously changes the ΔΨ-steady and redox potential of the film).

Essential aspect of the First Law of First Law and membrane thermodynamics thermodynamic (correct for membrane systems), energy of an isolated system is stored, both heat and work, and equivalent forms of energy It is possible. Therefore, the energy level (Ψd and ΔΨ) of the membrane system can be increased over a given distance or in the volume element by force or pressure action, respectively, by increasing or decreasing the mechanical work done; and / or within the membrane Can be changed by non-destructive heat transferred by the temperature gradient.

  This law (the law of conservation of energy) concludes that the total energy of a system isolated from the environment does not change. Therefore, the addition of any amount of heat and work (energy) to the system must be reflected in the change in energy of the system.

Absorption of infrared radiation The individual photons of infrared radiation do not contain enough energy (eg, measured in electron volts) to induce an electrical change (intramolecular) as seen with ultraviolet photons. For this reason, the absorption of infrared radiation is limited to compounds with small energy changes in the vibration and rotation of possible molecular bonds.

  By definition, for membrane bilayers, in order to absorb infrared radiation, vibrations and rotations in the molecular bonds of lipid bilayers that absorb infrared photons must cause a net change in the dipole potential of the membrane. . If the frequency (wavelength) of infrared radiation matches the frequency of the absorbing molecule (ie, the C—H covalent bond in the long chain fatty acid), the radiation is absorbed and causes a change in ψd. This can occur in Ψd-plasm-mam, Ψd-mito-mam, Ψd-plasm-fungi, Ψd-mito-fungi and Ψd-plus-bact. In other words, using the methods and systems described herein, there can be direct and targeted changes (ΔH and ΔS) in the lipid bilayer enthalpy and entropy of all cells.

  The present invention is based on a combination of insights introduced above and partly derived from empirical data, which includes:

Intrinsic, single wavelengths (870 nm and 930 nm) can cause bacterial cells (prokaryotes) such as E. coli and (eukaryotes) such as Hela Chinese hamster ovary cells (CHO) to generate and interact with ROS and toxic singlet oxygen reactions. As a result, it has been recognized that it can be killed. See, for example, US patent application Ser. No. 10 / 649,910 filed on Aug. 26, 2003 and US Patent Application No. 10 / 77,106 filed on Feb. 11, 2004. ) Instead of avoiding individual 870 nm and 930 nm wavelengths using such a NIMELS system, the laser system and method of the present invention (NIMELS system) is typically used in confocal laser microscopes such as optical traps. It has been established that the use of therapeutic laser systems at such wavelengths can be advantageously used in combination with wavelengths of power density 5 logs lower than those (about 500,000 w / cm ² less power).

  This is the change, manipulation and depolarization of ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact, ΔΨ-steady-mito-mam and ΔΨ-steady-mito-fungi of all cells in the irradiation area. Done for the indicated purpose. This is achieved in the present invention by infrared irradiation (930 nm energy) targeted to C—H covalent bonds in long-chain fatty acids of the lipid bilayer, and the dipole potential Ψd of all biological membranes in the irradiated region. -Mito-mam, Ψd-plus-fungi, Ψd-mito-fungi and Ψd-plus-bact changes. Second, near-infrared irradiation (870 nm) of cytochrome chains further alters the ΔΨ-steady and redox potential of cytochrome-containing membranes (ie, bacterial plasma membranes and fungal and mammalian mitochondria).

  Serving as a direct chromophore (C—H bonds in cytochromes and long chain fatty acids), direct enthalpy and entropy in the molecular dynamics of membrane lipids and cytochromes for all cell lipid bilayers in the irradiation light path of the present invention There are changes. This changes the dipole potential ψd of each membrane in the irradiated cell, and at the same time changes the absolute value of the membrane potential Δψ of all membranes.

  These changes occur due to significantly increased molecular motion (ie, ΔS) of the lipid and cytochrome metalloprotein reaction centers when energy is absorbed in a linear one-photon process from the NIMELS system. Since even small thermodynamic changes in the lipid bilayer and / or cytochrome are sufficient to change the dipole potential Ψd, the molecular shape (and hence the enzyme activity) of the attached electron transfer protein, or transmembrane protein, is It becomes less functional. This directly affects and modifies ΔΨ in all membranes in the irradiated cells.

  The NIMELS effect occurs with the methods and systems described herein without thermally or exfoliating mechanical damage to the cell membrane. This combined targeted low-dose approach is a clear change and improvement from existing methods that cause real mechanical damage to all films in the beam path of energy.

Film entropy and second law of thermodynamics The conversion of heat into other forms of energy is by no means complete and must always be accompanied by an increase in entropy (by the second law of thermodynamics). Entropy (in the membrane) is a state function, and its change in the reaction describes the direction of the reaction due to changes in (energy) heat input and output and related molecular rearrangements.

  Even though heat and mechanical energy are equivalent in their basic properties (as forms of energy), the ability to convert thermal energy into work is limited. That is, excessive heat can permanently damage the film structure and prevent work or beneficial energy changes in both directions.

This NIMELS effect alters the entropy “state” of irradiated cells at the lipid bilayer level in a temporally dependent manner. This increase in entropy changes the Ψd of all irradiated membranes (mitochondria and protoplasms), and thus changes the flow of thermodynamically “steady state” electrons and protons across the cell membrane (FIGS. 6 and 7). ). This in turn changes the steady-state transmembrane potential Δψ-steady to the transient state membrane potential (Δψ-tran). This phenomenon occurs at:
1) mammalian plasma transmembrane potential ΔΨ-plasm-mam;
2) Fungal plasma transmembrane potential [Delta] [Psi] -plasm-fungi;
3) Bacterial plasma transmembrane potential [Delta] [Psi] -plasm-bact;
4) Mammalian mitochondrial transmembrane potential ΔΨ-mito-mam; and 5) Fungal mitochondrial transmembrane potential ΔΨ-mito-fungi.

  This is a direct result of targeted enthalpy changes at the level of C—H bonds of long chain fatty acids in a flow mosaic membrane, a dynamic perturbation (in the membrane) that can alter the material properties of the membrane. Produce. This flow mosaic can increase entropy and destroy the tertiary and quaternary properties of the electron transfer protein, resulting in redox stress, energy stress and subsequent generation of ROS, which further damages the membrane, Furthermore, bioenergetics will change.

Since the primary function of the respiratory cell's electron transport system is to convert energy in a steady state, the technique of the present invention temporarily, mechano-opticly, and many related thermodynamic interactions in the conversion process. Used to uncouple the action. This can be done with the clear intention of changing the proton electrochemical gradient ΔμH ⁺ of the membrane and the absolute quantification of the proton activation force and Δp. This phenomenon can occur among others:
1) mammalian mitochondrial proton activation force (Δp-mito-mam);
2) Fungal mitochondrial proton activation force (Δp-mito-Fungi);
3) Fungal plasma membrane proton activation (Δp-plus-Fungi); and 4) Bacterial plasma membrane proton activation (Δp-plus-Bact).

  Such a phenomenon in turn reduces the available Gibbs free energy value ΔG (ΔGp) for phosphorylation and ATP synthesis. The present invention implements these phenomena to enhance the pharmacotherapeutic agent and / or cell resistance mechanisms (antibacterial, antifungal and anti-neoplastic) to inhibit the required energy-dependent aerobic response. (Many of these resistance mechanisms utilize proton motive force or chemical osmotic pressure for their energy requirements to resist and / or shed these molecules) ).

The effects of chemical uncouplers on free radical-generated oxidative phosphorylation and other bioenergetic work as a result of the modification of ΔΨ-steady depend on the energized state of the membrane (protoplasm and mitochondria) Conceivable. Furthermore, the energized state of bacterial membranes or eukaryotic inner mitochondrial membranes is believed to be an electrochemical proton gradient ΔμH ⁺ established by primary proton transfer events that occur during cell respiration and electron transfer. .

Directly extinguish (depolarize) ΔμH ⁺ (eg, by making the coupling membrane permeable for proton or compensatory ion transfer), reducing bioenergetic work to reduce membrane potential ΔΨ-steady Drugs that inhibit by induction. This occurs while breathing (primary proton transfer) continues rapidly.

  For example, carbonyl cyanide m-chlorophenylhydrazone (CCCP), a classic uncoupler of oxidative phosphorylation, induces a decrease in membrane potential ΔΨ-steady and induces concomitant production of ROS while continuing to breathe To do. These drugs (uncouplers) are generally toxic to all bacteria, fungi and mammalian cells and therefore cannot be used as antibacterial, antifungal or anti-neoplastic agents .

  However, in many target cells resistant to antibacterial, antifungal or anti-neoplastic agents, Δp uncouplers (such as CCCP) disrupt the energy gradient required for efflux pumps and hence It has been shown to induce a potent increase in the intracellular accumulation of these drugs. These results clearly show that some resistance mechanisms (eg, drug efflux pumps) are driven by proton activation forces. This selectivity is clearly important for the general damaging nature of the uncoupler if there is a way to use this effect to achieve unique damage to only the “target cell”. Let's go.

  This particular discovery and experimental data of the present invention shows that when the membrane is optically depolarized, the generation of ROS can better enhance the depolarization of the affected cells, It shows that the bacterial effect can be further enhanced (see Example VIII).

Generation of free radicals and ROS by NIMELS laser irradiation By mechanically and optically modifying the thermodynamic interaction of the energy conversion process of many related membranes and also changing ΔΨ-steady, the present invention provides the following irradiation: Can act as an optical uncoupler by reducing the ΔμH ⁺ and Δp of the deposited film:
1) Mammalian mitochondrial proton activation force (Δp-mito-mam)
2) Fungal mitochondrial proton activation force (Δp-mito-Fungi)
3) Fungal plasma membrane proton activation force (Δp-plus-Fungi)
4) Bacterial plasma membrane proton activation force (Δp-plus-Bact).

This reduced Δp creates a series of free radicals and radical oxygen species due to the altered redox state. The production of this free radical and negative oxygen species has been experimentally demonstrated and is described here with the following change of ΔΨ-steady to ΔΨ-trans (see Example VIII):
1) ΔΨ-steady-mam + (NIMELS treatment) →→ ΔΨ-trans-mam
2) ΔΨ-steady-fungi + (NIMELS treatment) →→ ΔΨ-trans-fungi
3) ΔΨ-steady-bact + (NIMELS treatment) →→ ΔΨ-trans-bact
4) ΔΨ-mito-fungi + (NIMELS processing) →→ ΔΨ-trans-mito-fungi
5) ΔΨ-mito-mam + (NIMELS treatment) →→ ΔΨ-trans-mito-mam

  Due to the ΔΨ-steady + (NIMELS treatment) →→ ΔΨ-trans phenomenon, the altered redox state and the generation of free radicals and ROS can cause significant further damage to the biological membrane, for example lipid peroxidation.

Lipid Peroxidation Lipid peroxidation is a common cause of biological cell injury and cell death in the microbial and mammalian kingdoms. In this process, strong oxidants cause the breakdown of membrane phospholipids containing polyunsaturated fatty acids (PUFA). The severity of membrane damage can cause local degradation of membrane fluidity and complete disruption of bilayer integrity.

  Peroxidation of mitochondrial membranes (mammalian cells and fungi) will have deleterious consequences for the respiratory chain, resulting in poor production of ATP and disruption of the cellular energy cycle. Peroxidation of the plasma membrane (bacteria) can affect membrane permeability, membrane proteins such as porin and efflux pumps, inhibition of signal transduction and inappropriate cellular respiration and ATP production (i.e., prokaryotic respiration) The chains are contained in the plasma membrane because prokaryotes do not have mitochondria).

A free radical is defined as an atom or molecule having an unpaired electron. An example of damage that free radicals can do in a biological environment is one-electron removal (due to existing or generated free radicals) from the bis-allylic C—H bond of polyunsaturated fatty acids (PUFA); This produces carbon centered free radicals.
R ^* + (PUFA) -CH (bis-allylic C—H bond) → (PUFA) -C ^* + RH

This reaction can initiate biological membrane damage due to lipid peroxidation. Free radicals can also generate free radical products in addition to non-radical molecules.
(A ^* + B → A−B ^* ) or non-radical product (A ^* + B → A−B)
An example of this is the hydroxylation of aromatics with ^* OH.

Reactive oxygen species (ROS)
Oxygen gas is actually a free radical species. However, since it contains two unpaired electrons in different π-antibonding orbitals that have parallel spins in the ground state, the (spin restriction) rule generally catalyzes electron pairs in which O ₂ has parallel spins. Preventing you from receiving without. Therefore, O ₂ must receive one electron at a time.

There are many significant donors in cells (prokaryotic and eukaryotic cells) that can stimulate the one electron loss of oxygen, which creates additional radical species.
These are generally classified as follows:
Superoxide ion radical (O ₂ ^-)
Hydrogen peroxide (non-radical) (H ₂ O ₂ )
Hydroxyl radical ( ^* OH)
Hydroxy ion (OH ^-)
The reaction chain is:
O ₂ → O ₂ ⁻ + 2H ⁺ → H ₂ O ₂ → OH ⁻ + ^* OH → OH ^{−
(e -) (e -)} (e -) (e -)

Superoxide The risk of these molecules to cells is well classified in the literature. For example, superoxide can act as an oxidizing or reducing agent.
NADH → NAD ⁺

More important for organism metabolism, superoxide can reduce cytochrome C. In general, the reaction rate of superoxide (O ₂ ⁻ ) with lipid (ie, membrane) protein and DNA is considered too slow to have biological significance. The protonated form of the superoxide hydroperoxyl radical (HOO ^* ) has a lower reduction potential than (O ₂ ⁻ ), but can remove hydrogen atoms from the PUFA. Again it should be noted that, pKa values of (HOO ^*) is 4.8, near the (acid) microenvironment of the biological membranes are suitable for the formation of Hydroperoxyl. Moreover, the reaction of superoxide (O ₂ ⁻ ) with any free Fe ⁺³ produces a “peripheryl” intermediate, which can also react with PUFA, and peroxidize lipids (membranes). Can be induced.

Hydrogen peroxide Hydrogen peroxide (H ₂ O ₂ ) is not a good oxidant and cannot remove hydrogen from PUFA. However, it can cross biological membranes (rather easily) and can exert dangerous and harmful effects on other areas of the cell. For example, (H ₂ O ₂ ) is highly reactive with transition metals (eg, Fe ⁺² and Cu ⁺ ) in a microcellular environment, which in turn creates hydroxyl radicals ( ^* OH). (Known as the Fenton reaction). The hydroxyl radical is one of the most reactive species known in biology.

Hydroxyl radical Hydroxyl radical ( ^* OH) reacts with almost all kinds of biological molecules. It has a very fast reaction rate and is essentially controlled by the hydroxyl radical ( ^* OH) diffusion rate and the presence (or absence) of molecules that react near the ( ^* OH) creation site. In fact, the standard reduction potential (E0 ′) of the hydroxyl radical ( ^* OH) is (+2.31 V), which is 7 times greater than (H ₂ O ₂ ) and is biologically relevant. Classified as the most reactive of the free radicals. In addition to damaging proteins and DNA, hydroxyl radicals initiate lipid peroxidation in biological membranes.

Reactive oxygen species created from the peroxidation of PUFAs Furthermore , the generation of lipid peroxidation (from any source) results in the production of three other reactive oxygen intermediate molecules from PUFA.
(a) alkyl hydroperoxide (ROOH);
Like H ₂ O ₂ , alkyl hydroperoxides are not technically radical species, but are unstable in the presence of transition metals such as Fe ⁺² and Cu ⁺ .
(b) an alkyl peroxyl radical (ROO ^* ); and
(c) alkoxyl radical (RO ^*).

  Alkylperoxyl radicals and alkoxyl radicals are highly reactive oxygen species and contribute to further lipid peroxidation propagation. The altered redox state of irradiated cells and the generation of free radicals and ROS (due to the ΔΨ-steady + (NIMELS treatment) →→ ΔΨ-trans phenomenon) are other objects of the invention. This is an additional effect that further alters the bioenergetics of the cell, inhibits the required energy-dependent aerobic response, strengthens the drug therapeutic agent, and / or the cell's antibacterial, antifungal properties And reduce the resistance mechanism to antineoplastic molecules.

  Overproduction of ROS can damage cellular macromolecules, especially lipids. Lipid oxidation has been shown to modify both the small-scale structural dynamics of cell membranes and their more macroscopic lateral organization and packing density that depends on the redirection of components of the altered dipole moment Ψd. ing. Damage due to oxidation of the acyl chain (in the lipid) results in loss of double bonds, chain shortening, and introduction of hydroperoxy groups. Therefore, these changes are thought to affect the structural features and dynamics of the lipid bilayer and the dipole potential ψd.

Antimicrobial resistance Antimicrobial resistance is defined as the ability of a microorganism to survive despite the effects of antimicrobial drugs or molecules. Antimicrobial resistance can evolve naturally, by natural selection, by random mutation, or by genetic engineering. Again, microorganisms can transmit resistance genes to each other by mechanisms such as plasmid exchange. If a microorganism has several resistance genes, it is called multidrug resistance or informally called a “super bug”.

  Multidrug resistance in pathogenic bacteria and fungi is a significant problem in the treatment of patients infected with such microorganisms. Currently, it is terribly expensive and difficult to make or discover new antimicrobial drugs that are safe for human use. Again, there are also evolved resistant mutant microorganisms that challenge all known antibacterial classes and mechanisms. Therefore, only a few antibacterial agents can maintain long-term efficacy. Most mechanisms of resistance to antibacterial drugs are known.

The four main mechanisms by which microorganisms are resistant to antimicrobials are:
a) inactivating or modifying the drug;
b) changing the target site;
c) altering metabolic pathways; and d) reducing drug accumulation by reducing drug permeability and / or increasing cell surface active efflux pumps.

Examples of resistant microorganisms S. aureus is one of the main resistant pathogenic bacteria currently plaguing mankind. This Gram-positive bacterium is found primarily in mucous membranes and skin in nearly half of the world's adults. S. aureus is extremely adaptable to pressure from all known antibiotic classes. S. aureus is the first bacterium found resistant in 1947 to penicillin. Subsequently, almost complete resistance to methicillin and oxacillin was found. “Superbug” MRSA (methicillin-resistant Staphylococcus aureus) was first detected in 1961 and is now widely distributed worldwide in hospitals and the general public. Today, the majority of S. aureus infections in the United States are resistant to penicillin, methicillin, tetracycline and erythromycin. Recently, significant resistance has been reported in a new class of antibiotics (the last reliable antibacterial) glycopeptides and oxazolidineones (Vancomycin since 1996 and Zyvox 2003).

  A new mutant CA-MRSA (community acquired MRSA) has also recently emerged and prevailed and causes a rapidly progressing group of fatal diseases (including necrotizing pneumonia, severe sepsis and necrotizing fasciitis) It is. Outbreaks of community associated (CA) -MRSA infections are reported daily in correctional facilities, athletic teams, military recruits, newborn nurseries, and homosexual men. CA-MRSA infection now appears to be most prevalent in many urban areas, causing most CA-S. Aureus infections.

  The scientific and medical communities have attempted to find enhancers of existing antimicrobial drugs and inhibitors of drug resistance systems in bacteria and fungi. Such enhancers and / or inhibitors would be of great value for the treatment of patients infected with pathogenic drug-resistant microorganisms if they are not toxic to humans. In the United States, as many as 80% of individuals have S. aureus colonies somewhere. Most only have intermittent colonies; 20-30% have persistent colonies. Healthcare workers, diabetics, and dialysis patients all have a high rate of colonies. The external nasal cavity is the dominant colonization site in adults; other potential colonization sites include the axilla, rectum and perineum.

There is a relatively new class of bactericidal antibiotics called selective pharmacologically modified lipopeptides of bacterial ΔΨ-steady , and daptomycin is the first FDA approved member. This antibiotic has shown that virtually all clinically relevant Gram-positive bacteria (eg MRSA) can be killed rapidly by a mechanism of action different from other antibiotics currently on the market (in vitro and In vivo).

  The mechanism of action of daptomycin involves the calcium-dependent incorporation of lipopeptide compounds into the bacterial cytoplasmic membrane. At the molecular level, it reduces the net negative charge and allows it to work better with negatively charged phospholipids typically found in the cytoplasmic membrane of Gram-positive bacteria. Calcium binding between residues (in daptomycin molecule). In general, there is no interaction with fungal or mammalian cells at the therapeutic level and therefore it is a highly selective molecule.

The effect of daptomycin results from this calcium-dependent action on the bacterial cytoplasmic membrane, which has been proposed to eliminate the transmembrane electrical potential gradient ΔμH ⁺ . This lies in effective selective chemical depolarization of bacterial membranes only. Although depolarization (in this manner) is not a lethal effect on bacteria, it is well known that maintenance of a correctly energized cytoplasmic membrane is essential for bacterial cell survival and growth. For example, the antibiotic valinomycin causes depolarization in the presence of potassium ions and is bacteriostatic, but not as bactericidal as in CCCP.

Conversely, in the absence of proton activation force Δp (the main element of which is the transmembrane electrical potential gradient ΔμH ⁺ ), the cell cannot make ATP or is required for growth and reproduction Unable to take in nutrients. The decay of ΔμH ⁺ explains the different (detrimental) effects produced by daptomycin, such as protein, RNA, DNA, peptidoglycan, lipoteichoic acid, and lipid biosynthesis.

Further research of the prior art on the drug daptomycin suggests that the addition of gentamicin or minocycline results in enhanced bactericidal activity against MRSA. Both gentamicin and minocycline can be effluxed from MRSA cells by energy-dependent pumps and are inhibitors of protein synthesis (anabolic function) at the level of the 30S bacterial ribosome. This indicates that the disappearance of the transmembrane electrical potential gradient ΔμH ⁺ by daptomycin can enhance certain antibacterial drugs. This results from a reduced resistance mechanism due to a decrease in membrane potential Δψ and the fact that ATP is not available for the anabolic function of protein synthesis (ie, a concomitant reduced ΔGp).

  Based on the above, the activity and / or anabolic reaction of the drug efflux pump in the target cell safely reduces the membrane potential ΔΨ of the cell in a given target region (ΔΨ−steady + (NIMELS treatment) →→ ΔΨ-trans It is clearly desirable to be able to optically inhibit. The method of the present invention accomplishes this and other tasks by utilizing selected infrared wavelengths such as 870 nm and 930 nm (independent of any exogenous agent such as daptomycin). This is a clear improvement over existing prior art methods that require systemic drugs to accomplish the same task.

Multidrug resistant efflux pumps Multidrug resistant efflux pumps are now known to exist in gram positive bacteria, gram negative bacteria, fungi, and cancer cells. Effluent pumps generally have the multispecificity of transporters that give a broad spectrum of resistance mechanisms. These enhance the effects of other mechanisms of antimicrobial resistance, such as mutation of the antimicrobial target or enzymatic modification of the antimicrobial molecule. Active efflux of antibacterial agents is clinically associated with β-lactam antibacterials, marcolides, fluoroquinolones, tetracyclines and other important antibiotics (most antifungals including itraconazole and terbinafine) With compound).

In the case of resistance by spill pumps, the microorganism has the ability to capture antimicrobial agents or toxic compounds and discharge them out of the cell (environment), thereby reducing the intracellular accumulation of the drug. In general, overexpression of at least one of these efflux pumps is believed to prevent intracellular accumulation of the drug from reaching the threshold required for its inhibitory activity. In general, in microorganisms, drug efflux is coupled with a proton motive force that creates the energy (ATP) required for the electrochemical potential and / or the requirements of these protein pumps. This includes:
1) Mammalian mitochondrial proton activation force (Δp-mito-mam);
2) Fungal mitochondrial proton activation force (Δp-mito-fungi);
3) Fungal plasma membrane proton proton activation force (Δp-plus-fungi); and 4) Bacterial plasma membrane proton activation force (Δp-plus-bact).

Phylogenetically, bacterial antibiotic pumps belong to the following five super families:
(i) ABC (ATP binding cassette), which is the main active transporter energized by hydrolysis of ATP;
(ii) SMR [DMT (drug / metabolite transporter) small multi-drug resistant subfamily];
(iii) MATE [MOP (multidrug / oligoglycolipid / polysaccharide flippase) superfamily efflux subfamily of multiple antimicrobial agents];
(iv) MFS (major accelerator superfamily); and
(v) RND (resistance / nodulation / fission superfamily), all of which are secondary active transporters driven by an ion gradient.

  The approach for inhibiting the efflux pump of the present invention is a general modification (optical depolarization) of the membrane ΔΨ in the irradiated region, which leads to a lower electrochemical gradient, which Reduce the available energy for the oxidation potential ΔGp and the energy requirements for the pump function. It is also an object of the present invention to have the same photobiological mechanism that inhibits many different assimilation and energy driven mechanisms of target cells, including nutrient absorption for normal growth.

Lowering the energy of efflux pumps: Targeting the driving force of the mechanism Today, no drug belongs to the “energy blocker” family of molecules that have been developed clinically as efflux pump inhibitors. There are a few molecules that have been found to be “generic” inhibitors of efflux pumps. Two such molecules are reserpine and verapamil. These molecules were originally recognized as inhibitors of vascular monoamine transporters and blockers of calcium influx across the membrane (or calcium ion antagonists), respectively. Verapamil is known as an inhibitor of the MDR pump in cancer cells and certain parasites and also improves the activity of tobramycin.

  Reserpine inhibits the activity of Bmr and NorA (two Gram-positive bacterial efflux pumps) by altering the production of membrane proton activation force Δp required for the function of the MDR efflux pump. Even though these molecules can inhibit the ABC transporter involved in antibiotic (ie, tetracycline) excretion, the concentration required to block bacterial efflux is neurotoxic in humans. To date, no similar experiments with daptomycin have been mentioned in the literature. Fungal drug efflux is mediated primarily by two groups of membrane-bound transport protein: ATP-binding cassette (ABC) transporters and major facilitator superfamily (MFS) pumps.

Bacterial plasma transmembrane potential ΔΨ-plasm-bact and cell wall synthesis During normal cell metabolism, proteins are excreted from the cytoplasmic membrane to form ΔΨ-plus-bact. This function also acidifies (lowers the pH) a narrow area near the bacterial plasma membrane. In the Gram-positive bacterium Bacillus subtilis, the activity of peptidoglycan autolysis has been shown to be increased (ie, no longer inhibited) when the electron transport system is blocked by the addition of a proton conductor. This suggests that [Delta] [Psi] -plasm-bact and [Delta] [mu] H + () potentially have a profound and available impact on cell wall anabolic function and physiology.

  In addition, the ΔΨ-plus-bact uncoupler is responsible for the formation of peptidoglycan, accumulation of nucleotide precursors involved in peptidoglycan synthesis, and N-acetylglucosamine (GlcNAc), one of the major biopolymers in peptidoglycan. It has been shown to inhibit with inhibition of transport.

  Again, there is a reference to an antibacterial compound called tachypressin that lowers ΔΨ-plasm-bact in gram positive and gram negative bacteria. (Antimicrobial compositions and pharmaceutical preparations thereof, US Pat. No. 5,610,139, the entire teachings of which are incorporated herein by reference.) This compound is a sublethal concentration of β- It has been shown that ampicillin, a lactam antibiotic, has the ability to enhance in MRSA. A number of effects of optically reduced ΔΨ-plus-bact (i.e., increased cell wall autolysis, inhibited cell wall synthesis, and cell wall antibacterial enhancement), and any other that targets bacterial cell walls. It is desirable to couple to related antibacterial treatments. This is particularly appropriate in Gram positive bacteria, such as MRSA, that do not have an efflux pump as a resistance mechanism for antimicrobial compounds that inhibit the cell wall.

  Cell wall inhibitory compounds do not need to enter through the membrane in Gram positive bacteria to be effective against the cell wall (required in Gram negative bacteria). Experimental evidence demonstrated that the NIMELS laser and its attendant optical ΔΨ-plasm-bact reduction phenomenon are synergistic with cell wall inhibitory antibacterial agents in MRSA (see Example XII). This is because MRSA is conjugated to anabolic (periplasmic) ATP because peptidoglycan-inhibiting antibacterial agents do not need to enter cells because they are effective and therefore do not have efflux pumps that function against them. It must function by inhibiting function.

ΔΨ-plus-fungi and ΔΨ-mito-fungi: what is required for correct cell function and antimicrobial resistance In normal cell metabolism, ΔΨ-mito-fungi is produced by the electron transport system in mitochondria, which is then ATP is produced by the mitochondrial ATP synthase enzyme system. It is then ATP that powers the H ⁺ -ATPase bound to the plasma membrane to produce Δψ-plus-fungi. It has previously been shown that fungal mitochondrial ATP synthase is inhibited in a dose-dependent manner by the chemical polygodial (Lunde and Kubo, Antimicrob Agents Chemother. 2000 Jul; 44 (7): 1943 -1953, the entire teachings of which are incorporated herein by reference). This induced decrease in cytosolic ATP concentration leads to suppression of H ⁺ -ATPase bound to the plasma membrane producing ΔΨ-plasm-fungi, and this depletion further increases other cellular activities. Further weakening was found. In addition, the decrease in ΔΨ-plus-fungi causes bioenergetic and thermodynamic collapse of the plasma membrane, which leads to proton influx, which disrupts proton activation forces and hence Inhibits nutrient uptake.

  More importantly, ATP is required for the biosynthesis of the fungal plasma membrane lipid ergosterol. Ergosterol is a structural lipid targeted by the majority of commercially available related antifungal compounds used today as pharmaceuticals (ie, azoles, terbinafine and itraconazole).

  Studies have shown that two antimicrobial peptides (Pep2 and Hst5) have the ability to efflux ATP from fungal cells (ie deplete intracellular ATP concentrations) and this reduced cytosolic ATP is ATP-dependent It has been shown to cause inactivation of ABC transporters CDR1 and CDR2, which are efflux pumps of antifungal agents.

  Utilizing optical methods has the advantage of depolarizing the membrane to deplete cellular ATP in the fungus as an enhancer of efflux pump inhibition and anabolic reactions. Therefore, it may be desirable to optically alter ΔΨ-plasm-fungi and / or ΔΨ-mito-fungi to inhibit necessary cellular functions, ATP production, and enhance antifungal compounds.

  Therefore, one strategy to prevent drug resistance (by efflux pumps) is to reduce the level of intracellular ATP, which inactivates ATP-dependent efflux pumps. To induce. In fungal pathogens, there were no acceptable chemical agents to accomplish this task. However, the NIMELS effect has the ability to optically achieve this goal, and experimental evidence indicates that phenomena in NIMELS lasers and fungi are synergistic with antifungal compounds. (See Example XIII).

  This NIMELS effect is produced by the methods and systems disclosed herein without causing thermal or exfoliative mechanical damage to the cell membrane. This combined, targeted low-dose approach is a distinct and improved version of all existing methods that actually cause mechanical damage to films in all energy beam paths. .

  In a first aspect, the invention relates to the dipole potentials Ψd (Ψd-plasm-mam, Ψd-mito-mam, Ψd-plus-fungi, Ψd-mito-fungi, and (Ψd-plus-bact) is provided to provide a method to drive a cascade of further changes in ΔΨ and Δp of the same membrane.

  Bioenergetics steady-state membrane potentials of all irradiated membranes ΔΨ-steady (ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact, ΔΨ-steady-mito-mam and ΔΨ-steady -Mito-fungi) is changed to ΔΨ-trans values (ΔΨ-trans-mam, ΔΨ-trans-fungi, ΔΨ-trans-Bact, ΔΨ-trans-mito-mam and ΔΨ-trans-mito-fungi). . This is a depolarization of all irradiated cells and a measurable change in the absolute value of Δp (Δp-mito-mam, Δp-mito-Fungi, Δp-plus-Fungi and Δp-plus-Bact). Arise.

  These phenomena can occur without any unacceptable risk and / or unacceptable adverse effects on biological entities (e.g., mammalian tissues, cells or certain biochemical preparations such as protein preparations). Irradiating the target site with optical irradiation of the desired wavelength, power density level, and / or energy density level at a predetermined target site other than the targeted biological contaminants (bacteria and fungi) Caused by.

  In certain embodiments, such applied optical illumination can have a wavelength of about 850-900 nm in NIMELS dosimetry, as described herein. In a typical embodiment, a wavelength of about 865 to 875 nm is utilized. In a further embodiment, such applied irradiation can have a wavelength of about 905-945 nm in NIMELS dosimetry. In certain embodiments, such applied optical illumination can have a wavelength of about 925-935 nm. In the representative non-limiting example illustrated below, the wavelength used is 930 nm.

  The bioenergetic steady state membrane potential can be modified in the exemplary embodiment, as described below, and can employ a number of wavelength ranges, each including 870 and 930 nm as limits.

NIMELS Strengthening Grade Scale (NPMS)
As discussed in more detail earlier, the NIMELS parameters include the average single or additional output power of the laser dipoles and their dipole wavelengths (870 nm and 930 nm). This information plus the area of the laser beam at the target site (cm ² ), the power output of the laser system and the irradiation time can be used to calculate the effective and safe irradiation protocol of the present invention. Provide a set of information.

  Based on these novel resistance reversals and antibacterial enhancement interactions available with NIMELS lasers, a quantitative value for the “enhancement effect” that applies to each specific antibacterial agent and laser dosimetry is needed.

  A new set of parameters will be defined, which will take into account the implementation of any different dosimetry values for the NIMELS laser and any MIC values for a given antimicrobial agent to be tested. This can be easily adjusted for the NIMELS laser system and method by creating only a set of variables that quantify the CFU of pathogenic microorganisms within any given experimental or therapeutic parameter using the NIMELS system. .

  These parameters create a scale called the NIMELS Enhancement Grade Scale (NPMS), which utilizes the phenomenon of reversal of antimicrobial drug resistance and / or MIC enhancement inherent in the NIMELS laser, while at the same time depending on power and / or processing time. Measure safety against burns and injuries in adjacent tissues. This NPMS scale measures a NIMELS effect number (Ne) of 1-10, but the goal is that in any combination of safe antimicrobial concentrations and NIMELS dosimetry, a reduction in pathogen CFU count of 4 or more To get Ne. Even if CFU counting is used here for quantification of pathogenic microorganisms, other quantification means such as staining detection methods or polymerase chain reaction (PCR) methods can also be used to obtain values for the A, B and Np parameters. Can be used.

  This NIMELS effect number Ne is an interaction coefficient that indicates that the combined inhibitory / bacteriostatic effect of antibacterial drugs is synergistic with NIMELS lasers against pathogenic targets without damaging healthy tissue. is there.

The NIMELS enhancement number (Np) is a value that indicates whether a given concentration of an antimicrobial agent is synergistic or antagonistic to a pathogenic target without damaging healthy tissue. is there. Therefore, in any standard given set, the experimental or therapeutic parameters are as follows:
A = CFU count of pathogen when using NIMELS alone;
B = CFU count of pathogens when using antimicrobial agent alone;
Cp counts of pathogens with Np = (NIMELS + antibacterial agent); and Ne = (A + B) / 2Np;
Interpretation of the NIMELS effect number Ne:
If 2Np <A + B, the (predetermined) antimicrobial agent was successfully enhanced by the NIMELS laser at the concentration and dosimetry used.
next:
If Ne = 1, there is no reinforcing effect. If Ne> 1, there is a strengthening effect.
If Ne ≧ 2, there is at least a 50% enhancement effect on the antimicrobial agent. If Ne ≧ 4, there is at least a 75% reinforcing effect on the antimicrobial agent. If Ne ≧ 10, there is at least a 90% reinforcing effect on the antimicrobial agent.
Sample calculation 1:
A = 110 CFU
・ B = 120 CFU
・ Np = 75 CFU
Ne = (110 CFU + 120 CFU) / 2 (75) = 1.5
Sample calculation 2:
A = 150 CFU
・ B = 90 CFU
・ Np = 30CFU
Ne = (150 CFU + 90 CFU) / 2 (30) = 4

  In general, antimicrobial agents are expensive when treating microbial infections, and lower doses of antimicrobial agents should be used because they generally involve undesirable side effects (which can include systemic kidney and / or liver damage) Can be advantageous. It is therefore desirable to devise methods to reduce and / or enhance the MIC of antimicrobial agents. The present invention provides a system and method for reducing the MIC of antibacterial molecules when the area to be treated is simultaneously treated by the NIMELS laser system.

  If the antimicrobial MIC is a topical and resistant focal infection (eg skin, diabetic foot, floor rub), many older, cheaper and safer antimicrobials to treat these infections The therapeutic efficacy of the agent will be restored. Therefore, the MIC of the antibacterial agent is reduced by adding a NIMELS laser (eg, producing a Ne value greater than 1 on one side, 4 or more on the other side, and 10 or more on the other side) This represents an aggressive step towards restoring the once lost therapeutic efficacy of antibiotics.

  Therefore, in one aspect, the present invention reduces the MIC of antibacterial molecules necessary to eradicate or at least attenuate microbial pathogens by depolarizing the membrane within the irradiated zone, which reduces the membrane potential ΔΨ of irradiated cells. Methods and systems for reducing are provided. This weakened ΔΨ causes a closely related weakening of the proton activation force Δp and the associated bioenergetics of all affected membranes. It is a further object of the present invention that this “NIMELS effect” enhances existing antimicrobial molecules against microorganisms that infect human hosts and cause injury.

  In certain embodiments, such applied light irradiation has a wavelength of about 850-900 nm in NIMELS dosimetry, as described herein. In an exemplary embodiment, a wavelength of about 865 to 875 nm is utilized. In a further embodiment, such applied irradiation has a wavelength of about 905-945 nm in NIMELS dosimetry. In certain embodiments, such applied light radiation has a wavelength of about 925-935 nm. In one aspect, the wavelength used is 930 nm.

  Microbial pathogens having a bioenergetic system affected by the NIMELS laser system according to the present invention include, for example, microorganisms such as bacteria, fungi, filamentous fungi, mycoplasma, protozoa, and parasites. As described below, exemplary embodiments can utilize multiple wavelength ranges, including ranges that include 870 nm and 930 nm, respectively, as limits.

  In a method according to one aspect of the invention, irradiation with these contemplated wavelength ranges can be performed independently, sequentially, in a blended ratio, or essentially simultaneously (all of which are pulsed And / or continuous wave, CW, operation can be used).

  Irradiation of contaminants with NIMELS dose irradiation using NIMELS energy is performed before, after, or simultaneously with the administration of the antibacterial agent. However, the NIMELS energy in the NIMELS dosimetry can be applied after reaching the “peak protoplasmic level” in an individual or other mammal infected with the antimicrobial agent. It should be noted that the co-administered antibacterial agent should have antibacterial activity against any naturally sensitive variant of the target resistant contaminant.

  The wavelength irradiated by the method and system of the present invention can cause the susceptibility of contaminants to be less than about 0.01M, or about 0.001M, or similar non-resistant contaminant strains at antibacterial concentrations of about 0.0005M or less. Increase to the level of

  The method of the present invention slows or eliminates the progression of microbial contaminants at the target site to ameliorate symptoms or asymptomatic conditions associated with at least some of the contaminants and / or Increases sensitivity to antibacterial agents. For example, the method of the present invention can reduce the level of microbial contaminants at the target site and / or the activity of antimicrobial compounds, and the sensitivity of biological contaminants to the antimicrobial agents with which they have evolved or acquired resistance. , Resulting in an increase due to an increase without adverse effects on biological patients. The reduction in the level of microbial contaminants may be, for example, at least 10%, 20%, 30%, 50%, 70% or more compared to the pretreatment level. With respect to the sensitivity of biological contaminants to antimicrobial agents, the sensitivity is enhanced by at least 10%.

  In another aspect, the invention provides a system for performing a method according to another aspect of the invention. Such a system includes a laser oscillator for generating radiation, a controller for calculating and controlling the radiation dose, and a delivery assembly (system) for delivering the radiation through the application area to the treatment site. Suitable delivery assemblies / systems include hollow waveguides, fiber optics, and / or free space / beam light transmitting elements. Suitable free space / beam light transmission baskets include collimating lenses and / or aperture stops.

  In one form, the system utilizes one or more solid state dipole lasers as the dual wavelength near infrared light source. Two or more dipole lasers are placed in a single housing for uniform control. The two wavelengths can include emission in two ranges of about 850-900 nm and about 905-945 nm. The oscillator of the present invention is utilized to emit a single wavelength (or peak value, eg, center wavelength) in one of the other ranges disclosed herein. In certain embodiments, such lasers are used to irradiate substantially in the range of about 865-875 nm and about 925-935 nm.

  The system according to the invention can include a suitable light source for each wavelength range that it is desired to produce. For example, suitable solid state laser dipoles, various ultrashort pulse laser dipoles, or ion doped (eg, suitable rare earth) optical fibers or fiber lasers are used. In one form, a suitable near infrared laser includes titanium doped sapphire. Other suitable laser sources can be used within the scope of the present invention, including those by other types of solid state, liquid, or gas enhanced (active) media.

  According to one embodiment of the invention, the therapeutic system is an optical radiation generation system adapted to generate optical radiation essentially in the first wavelength range (approximately 850-900 nm), throughout the application range. A delivery assembly for producing optical radiation to be transmitted, and a controller for controlling the dose of radiation transmitted through the coverage, operatively connected to the optical radiation emitting device, and The time integral of irradiation power density and energy density transmitted per unit area is lower than a predetermined threshold. Again, within this embodiment, the therapeutic system is particularly adapted to generate optical radiation substantially within the first wavelength range (approximately 865-875 nm).

  According to a further embodiment, the therapeutic system includes an optical radiation generator arranged to substantially generate optical radiation in the second wavelength range (about 905-945 nm); The first wavelength range noted is generated by the optical radiation generator simultaneously or simultaneously / sequentially. Again, within the scope of this embodiment, the therapeutic system is particularly adapted to generate optical radiation substantially within the first wavelength range (about 925-935 nm).

  The therapeutic system further includes a delivery assembly (system) for transmission of optical radiation within a second wavelength range (and first wavelength range, if applicable) through the application region, and An optical radiation generator is operatively controlled to selectively generate radiation substantially in the first wavelength range or substantially in the second wavelength range or any combination thereof. A controller for

  According to one embodiment, the delivery assembly includes at least one optical fiber having a distal end configured and arranged for insertion into a patient's tissue located within the light transmission range of the medical device; Here, this radiation is delivered to the tissue surrounding the medical device in NIMELS dosimetry. The delivery device can also include a free beam optical system.

  According to a further embodiment, the treatment system controller includes a power limiter for controlling the radiation dose. The controller may further include a memory for storing patient profiles and a dosimetry calculator for calculating the dose required for a particular target site based on information entered by the operator. it can. In one aspect, this memory can also be used to store information about different types of diseases and their treatment profiles, such as radiation patterns and radiation doses for specific applications. The light irradiation can be delivered from the treatment system to the application site in different patterns. This irradiation can be delivered as a continuous wave (WC) or pulsed or generated as a combination of each. For example, in a single wavelength pattern or in a pattern of multiple wavelengths (eg, dual wavelengths). For example, two wavelengths of radiation can be multiplexed (optically combined) or transmitted simultaneously to the same treatment site. Appropriate optics including (but not limited to) the use of polarizing beam splitters (combiners) and / or superimposition of output collected from a suitable mirror at the focus and / or other suitable multiplexing / combining techniques Can be used. Alternatively, this irradiation can be delivered in another pattern that alternatively delivers the two wavelengths to the same treatment site. Two or more pulse intervals can be selected as desired by the NIMELS technique of the present invention. Each treatment can be combined with any manner of transmission. The intensity distribution of the delivered light irradiation can be selected as desired.

  Typical examples include the intensity distribution of the top layer or substantially the top layer (eg, trapezoid, etc.). Other intensity distributions such as a Gaussian distribution can also be used.

  As used herein, the term “biological contaminant” refers to a target site (eg, an infected patient's tissue or organ) or, in the case of a mammal, the target site by direct or indirect contact with the target site. Closely intended (for example, in the case of cells, tissues or organs transplanted into a recipient, or in the case of a device used in a patient), intended to mean contaminants that may have undesirable and / or harmful effects. ing. The biological contaminants of this invention are generally microorganisms such as bacteria, fungi, filamentous fungi, mycoplasma, protozoa, parasites known to those skilled in the art to be found at the target site.

  One skilled in the art will appreciate that the methods and systems of the present invention can be used with a variety of biological contaminants generally known to those skilled in the art. The following list is merely illustrative of a wide range of microorganisms that can be targeted by the methods and devices of the present invention and is not intended to limit the scope of the invention.

  Thus, non-limiting examples for explanation of biological contaminants (pathogens) include, for example, Escherichia, Enterobacter, Bacillus, Campylobacter, Corynebacterium, Klebsiella, Treponema, Vibrio, Including, but not limited to, the genus Cocci and Staphylococcus.

  To further illustrate, biological contaminants include, but are not limited to, any fungus such as Trichophyton, Microspores, Epidermis, Candida, Skoplaropsis brebicauris, Fusarium, Aspergillus, Arte Lunaria, Acremonium, Citaridinum medium, and Citaridinum hiarinam. Parasites such as trypanosomes and malaria parasites are also biological contaminants that can be targeted (plasmodium species, and filamentous fungi; including mycoplasma and prions). Viruses include, for example, human immunodeficiency virus and other retroviruses, herpes virus, parvovirus, filovirus, silkovirus, paramyxovirus, cytomegalovirus, hepatitis virus (including hepatitis B and C), poxvirus Togaviruses, Epstein-Barr viruses and parvoviruses can also be targeted.

  It will be appreciated that the target site to be irradiated need not already be infected with biological contaminants. Indeed, the methods of the invention can be used “prophylactically” prior to infection. Further embodiments include medical devices such as catheters (eg IV catheters, central venous lines, arterial catheters, peripheral catheters, dialysis catheters, peritoneal dialysis catheters, epidural catheters), artificial junctions, stents, external Includes use for fixation pins, chest tubes, esophageal drug delivery tubes, and the like.

  In certain instances, irradiation may be palliative as well as prophylactic. Therefore, the method of the present invention is used to irradiate tissue over a therapeutically effective time to treat or alleviate symptoms of infection. The expression “treat or alleviate” means the reduction, prevention and / or reversal of the symptoms of an individual treated according to the present invention compared to the symptoms of an individual who has not received such treatment.

One skilled in the art will appreciate that the invention is useful for a variety of diseases caused by or related to any microbial, fungal, and viral infection (Harrison's Principles of Internal Medicine , 13th edition, McGraw Hill, New York (1994), the entire teachings of which are incorporated herein by reference). In certain embodiments, the methods and systems according to the present invention are utilized in conjunction with traditional therapeutic approaches available in the art for treating infections by administration of known antimicrobial compositions (e.g., Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 8th edition, 1990, Pergmon Press, the entire teachings of which are incorporated herein by reference). The terms "antibacterial composition", "antibacterial agent" refer to compounds (e.g., antibacterial, antifungal, and antiviral agents) that are administered to animals, including humans, to inhibit the growth of microbial infections and combinations thereof Point to.

  A wide range of contemplated applications includes, for example, various dermatological, foot treatment and general purpose drugs. The interaction between the site to be treated and the applied energy uses wavelength; chemical and physical properties of the target site; beam power density or radiant flux density; either continuous wave (CW) or pulsed irradiation Laser beam spot size; defined by several parameters including exposure time, energy density, and any change in physical properties of the target site as a result of laser irradiation using any of these parameters. In addition, the physical properties of the target site (eg, absorption and scattering coefficients, scattering anisotropy, thermal conductivity, heat capacity, and mechanical strength) also affect the overall effect and results.

NIMELS dosimetry shows power density (W / cm ² ) and energy density (J / cm ² ; 1 Watt = 1 Joule / second), so that the subject wavelength can produce ROS, Can reduce the level of biological contaminants at the target site and / or irradiate the contaminants to make the biological contaminants susceptible to resistant antimicrobial agents, with concomitant generation of ROS. By increasing ΔΨ, increasing unacceptable risk and / or unacceptable side effects without biological parts other than biological contaminants (eg, mammalian cells, tissues, or periods) Can do.

  As discussed in Boulnois 1986, (Lasers Med. Sci. 1: 47-66 (1986), the entire teachings of which are incorporated herein by reference), low power density (also called radiant flux density) and In energy, laser-tissue interactions can be described purely optically (photochemically), but at higher power densities, photo-thermal interactions are assured. In certain embodiments, as illustrated below, the NIMELS dose parameter is a measure of the photothermal parameter of the region traditionally used with known drugs and photodynamic treatments with foreign drugs, dyes and / or chromophores. In between, it can function without the need for foreign drugs, dyes and / or chromophores within the scope of photodynamic therapy.

The energy density for medical laser applications in the art (which can also be expressed as a flux or as a product (or integral) of a particle or radiant flux and time) is typically about 1 to 10,000 J. Although varying / cm ² (5-digit grades), power density (irradiance) varies from about ^{1 × 10 -3 ~10 12 W /} cm 2 (15 digits grade). In view of the reciprocal relationship between power density and irradiation exposure time, it is recognized that roughly the same energy density is required for any intended specific laser-tissue interaction. As a result, laser exposure duration (irradiation time) is the primary parameter that determines the nature and safety of laser-tissue interactions. For example, if you are mathematically looking for thermal evaporation (not exfoliation) of tissue in vivo (based on Boulnois 1986), if you generate an energy density of 1000 J / cm ² (see Table 1), then It can be seen that any of the dosimetric parameters can be used:

  This process describes a suitable method or basic algorithm that can be used for NIMELS interaction with biological contaminants in tissue. In other words, this mathematical relationship is a reciprocal relationship that achieves the laser-tissue interaction phenomenon. This principle can be used as the basis for dosimetric calculations for the observed antimicrobial phenomena given by NIMELS energy, with the insertion of NIMELS experimental data of energy density and time and power parameters.

  Specific interactions at the target site to be irradiated (eg, chemical and physical properties of the target site; whether to use continuous wave (CW) or pulsed irradiation; laser beam spot size; and physical properties of the target site For example, the practitioner is based on any change in absorption and scattering coefficients, scattering anisotropy, thermal conductivity, heat capacity, and mechanical strength as a result of laser irradiation using any of these parameters) The power density and time can be adjusted to obtain the desired energy density.

The examples given here illustrate such a relationship in the context of both in vitro and in vivo treatment. Thus, for example in the context of the treatment of onychomycosis or infected wounds of 1 to 4 cm in diameter, the power density value is a safe, non-injury / least injured thermal laser-tissue interaction It was changed at about 0.5-5 W / cm ² to stay in effect and well below the level of “degeneration” and “tissue overheating”. Other suitable spot sizes can also be used. Using this reciprocal relationship, the threshold energy density required for NIMELS interaction with these wavelengths can be maintained regardless of the spot size as long as the desired energy is delivered. In an exemplary embodiment, light energy is delivered to the tissue with a uniform geometric distribution (eg, a flat tip or top layer process). Using such a technique, a suitable NIMELS dosimetry (NIMELS effect) sufficient to produce ROS is calculated to reduce the level of biological contaminants and / or the contaminants of biological contaminants. Can reach the threshold energy density required to increase susceptibility to antibacterial agents that are resistant to (but below the level of “degeneration” and “tissue overheating”).

The NIMELS dosimetry (e.g. onychomycosis) targeting microorganisms in vivo as exemplified herein was approximately 200-700 J / cm < ² > and spanned approximately 100-700 seconds. These power values are not close to those associated with photodetachment or photothermal (laser / tissue) interactions.

The intensity distribution of a parallel laser beam is given by the power density of the beam and is defined as the ratio of laser power to the area of the circle (cm ² ) and the spatial distribution pattern of energy. Therefore, the illumination pattern of the irradiation spots of 1.5cm having an incident Gaussian beam pattern area 1.77 cm ² may generate at least six different power density values to the irradiation of 1.77 cm ² area. The intensity (or power concentration) of these varying power densities increases from 1 (at the outer edge) to 6 beyond the surface area of the spot at the center point. In certain embodiments of the present invention, a beam pattern is provided that overcomes this inherent error associated with traditional laser beam radiation. The NIMELS parameter can be calculated as a function of treatment time (Tn) as follows:
Tn = energy density / power density.

  In certain embodiments (see, for example, the in vitro experiments below), Tn is about 50-300 seconds; in other embodiments, Tn is about 75-200 seconds; In the specific example, Tn is about 100 to 150 seconds. In an in vivo embodiment, Tn is about 100-1200 seconds.

  Using the above relationship and the desired light intensity distribution, eg, flat tip illumination profile as described herein, a series of in vivo energy parameters can be validated in vitro for NIMELS microbial decontamination therapy. Proven experimentally. Thus, the key parameter for a given target site has been shown to be the energy density required for NIMELS therapy at a variety of different spot sizes and power densities.

  “NIMELS dosimetry” refers to the power density and / or energy density of the subject wavelength of the invention from the first threshold point at which ΔΨ at the target site can be optically reduced to the second endpoint. To increase the susceptibility of the biological contaminants to the antimicrobial agent to which the contaminants are resistant due to the occurrence of ROS, such as unacceptable harmful risks or effects at the biological site (e.g. , Including the range up to just before the value at which thermal damage (eg protein) is detected. Those skilled in the art will appreciate that in certain circumstances, adverse effects and / or risks at target sites (e.g., mammalian cells, tissues or organs) may be tolerated due to the inherent benefits obtained by the methods of the invention. Will. Thus, the intended stopping point is a point where adverse effects are not negligible and therefore undesirable (eg, cell death, protein denaturation, DNA damage, morbidity, or mortality).

In certain embodiments (eg, in vivo applications), the power density range contemplated here is about 0.25-40 W / cm ² . In other embodiments, the power density range is about 0.5-25 W / cm ² .

In further embodiments, the power density range can include values of about 0.5-10 W / cm ² . The power density exemplified here is about 0.5 to 5 W / cm ² . In vivo, power densities of about 1.5-2.5 W / cm ² have been shown to be effective against a variety of microorganisms.

  Empirical data indicates that higher energy density values are generally used when targeting biological contaminants in in vitro settings (e.g., plates) rather than in vivo (e.g., toenails). It seems to show.

In certain embodiments (see Example in vitro below), the energy density range contemplated herein is greater than 50 J / cm ² is less than about 25,000J / cm ^2. In other embodiments, the energy density range is about 750-7,000 J / cm ² . In yet another embodiment, the energy density range is about 1,500 to 6,000 J / cm ² , which means that biological contaminants are targeted in in vitro settings (eg, plates). Depends on whether it should be targeted in vivo (eg around toenails or medical devices). In certain embodiments (see in vivo examples below), the energy density is about 100-500 J / cm ² . In yet another in vivo embodiment, the energy density is about 175-300 J / cm ² . In yet another embodiment, the energy density is about 200-250 J / cm ² . In some embodiments, the energy density is about 300~700J / cm ^2. In some other embodiments, the energy density is about 300-500 J / cm ² . In yet another case, the energy density is about 300-450 J / cm ² .

The power density empirically investigated for various in vitro treatments of microbial species is about 1-10 W / cm ² .

  Those skilled in the art will appreciate that identification of a particularly suitable NIMELS dosimetry within the power density and energy density ranges contemplated herein for a given environment can be made empirically by routine experimentation. Let's go. Practitioners (e.g., dentists) who use near infrared energy with peri-dental treatment routinely adjust power density and energy density based on the urgency associated with each given patient (e.g., tissue Adjust parameters as a function of color, tissue structure, and depth of pathogen invasion). For example, laser treatment of peridental infections in light-colored tissues (e.g., melanin-deficient patients), darker tissues more efficiently absorb near-infrared energy, and hence more near-infrared energy in the tissue Because it converts quickly to heat, it will have a greater thermal safety parameter than darker tissue. Therefore, a clear need for practitioner capabilities is to identify multiple different NIMELS dosimetry values for various treatment protocols.

  As explained below, it has been found that antibiotic resistant bacteria can be effectively treated by the methods of the present invention. In addition, the method of the present invention can be used to increase traditional approaches and can be used in conjunction with traditional treatment, instead or even as an effective therapeutic approach. Has been found. Thus, this invention can be combined with antibiotic therapeutics. The term `` antibiotics '' includes β-lactam antibiotics, penicillins, and cephalosporin, vancomycin, bacitracin, macrolide antibiotics (erythromycin), ketride antibiotics (telisromycin), lincosamide antibiotics ( Lindamycin), chloramphenicol, tetracycline, aminoglycoside (gentamicin), amphotericin, anilinouracil, cefazolin, clindamycin, mupirocin, sulfonamide and trimethoprim, rifampicin, metronidazole, quinolone, novobiocin, polymyxin, oxazolidine class (e.g. Linezolid), glycylcycline (e.g., tigecycline), cyclic lipopeptide (e.g., daptomycin), pleuromutilin (e.g., retapamulin) and gramicidin Including etc. as well as any salts or variants of these, but not limited to. It is also understood that tetracyclines are within the scope of the present invention including, but not limited to, immunocycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline and minocycline. It is further understood that aminoglycoside antibiotics include but are not limited to gentamicin, amikacin, neomycin and the like.

  As explained below, it has been found that antifungal resistant fungi can be effectively treated by the methods of this invention. In addition, the method of the present invention can be used to augment traditional approaches, and can be used in conjunction with, or instead of, traditional treatments, or even continuously as an effective treatment approach. It has been found that. Thus, this invention can be combined with antifungal therapeutic agents. The term “antifungal agent” includes, but is not limited to, polyenes, azoles, triazoles, allylamines, echinocandin, ciclopirox, flucytosine, griseofulvin, amorolophine, sodarin and combinations thereof.

  As explained below, it is postulated that antineoplastic resistant cancer can be effectively treated by the methods of the present invention. In addition, the method of the present invention can be used to increase traditional approaches, can be used in conjunction with traditional treatments, or even used continuously as an effective therapeutic approach. Has been found to be possible. Thus, this invention can be combined with antineoplastic treatment. The term “anti-neoplastic agent” includes, but is not limited to, actinomycin, anthracycline, bleomycin, priomycin, mitomycin, taxane, etoposide, teniposide and combinations thereof (including salts thereof).

  The common doctrine of the prior art that attempts to find inhibitors of drug resistance systems in bacteria and fungi, or potentiators of antibacterial agents, is always that in order for such agents to have some inherent value, an infected mammal Has had to be non-toxic to tissues. Moreover, there has always been the fact that antibacterial agents affect bacterial or fungal cell processes that are not common to mammalian hosts and, therefore, are generally safe and therapeutically active in nature and design. In the prior art, if antibacterial agents, potentiators, and / or resistance reversal entities affect mammalian cells in the same manner that they damage pathogens, they are safe for treatment. It could not be used.

  In the present invention, experimental data (see, eg, Examples I-X) supports general changes in ΔΨ and Δp between all cell types, and thus not only the electrical mechanism of the whole cell membrane. It leads to the idea that the electrodynamic surface does not have different properties that can be adequately separated. This indicates that not only pathogenic cells (unwanted cells) but all cells in the beam path are affected by depolarization.

  Reconfirming the photobiological and cellular energetics data of this NIMELS system already showed (ie that all membrane energetics are affected in the same way across prokaryotic and eukaryotic species). The technology according to the present invention exploits this general photodepolarization effect independently in unwanted cells by adding antimicrobial molecules to the treatment regimen to (only) enhance such molecules in unwanted cells. To do. The result of such targeted therapy can take advantage of the general depolarization effect of the NIMELS laser, which is better evaluated against bacteria and fungi against mammalian cells in the path of the therapeutic beam. And transient. Therefore, as experimental data suggest, the measurement of transient energetic strength of mammalian cells is greater than that seen in bacterial or fungal cells, despite the occurrence of photodepolarization and ROS. Must be.

  The examples below provide experimental evidence to prove the concept of general photomembrane depolarization that leads to an understanding of our current photobiology and cellular energetics and conservation of thermodynamics when applied to cellular processes.

  The following examples are included to illustrate exemplary embodiments of the invention and are not intended to limit the scope of the invention. Those skilled in the art will appreciate that many changes can be made in a particular embodiment and that they will still yield similar results without departing from the spirit and scope of the invention.

Example I

Example II
Bacterial method: MINELS treatment parameters for MRSA in vitro The following parameters show the general bacterial method according to the invention when applied to MRSA for experiments V and VIII-XII in vitro. ing.

A. Experimental materials and methods for MRSA:

Similar cell culture and kinetic protocols were used for all NIMELS irradiation in E. coli and C.I. It was performed in an in vitro test using albicans. Therefore, for example, C.I. An albicans ATCC 14053 liquid culture was grown at 37 ° C. in YM medium (21 g / L, Difco). An aliquot from the standardized suspension was taken into selected wells in a 24-well tissue culture plate. After laser treatment, 100 μL was taken from each well and serially diluted 1: 1000, resulting in a final dilution of 1: 5 × 10 ⁶ of the initial culture. An aliquot of each final dilution was spread on a separate plate. The plates were then incubated at 37 ° C. for about 16-20 hours. Manual colony counts were made and recorded.

  Again, similar cell culture and kinetic protocols were used for all NIMELS irradiations in E. coli and C.I. An in vitro assay test was performed using albicans. Therefore, for example, C.I. ATCC 14053 liquid culture was grown at 37 ° C. in YM medium (21 g / L, Difco). An aliquot from the standardized suspension was taken into selected wells in a 24-well tissue culture plate. After laser treatment, samples that received each laser and control samples were processed according to individual assay instructions.

Example III
Mammalian Cell Method: NIMELS Treatment Parameters for In Vitro HEK293 (Human Embryonic Kidney Cell) Experiments The following parameters apply the method for general bacteria according to the invention to HEK 293 cells for in vitro experiments. Explains the case.

A. Experimental Materials and Methods for HEK293 Cells HEK293 cells were seeded in freestyle medium (Invitrogen) at a density of 1 × 10 ⁵ cells / ml (total volume 0.7 ml) in appropriate wells of a 24-well plate. Cells were incubated for about 48 hours at 37 ° C. in 8% CO ₂ in a humidified incubator prior to the experiment. The cells were approximately 90% confluent at the time of the experiment, which is comparable to approximately 3 × 10 ⁵ total cells. Just prior to treatment, cells were washed with pre-warmed phosphate buffered saline (PBS) and overlaid with 2 ml PBS during treatment.

  After laser treatment, the cells were mechanically removed from the wells and transferred to a 1.5 ml centrifuge tube. Mitochondrial membrane potential and total glutathione were measured according to kit manufacturer's instructions.

Example IV
NIMELS In Vitro Test for CRT + (Yellow) and CRT- (White) S. aureus Experiments We have engineered the crt gene (yellow carotenoid pigment) to remove S. aureus crt- (white ) Experiments were performed with mutants and these mutants were subjected to a predetermined non-lethal dose of NIMELS laser against wild-type (yellow) Staphylococcus aureus. The purpose of this experiment is to test for the free radical oxygen species (ROS) generation phenomenon and / or singlet oxygen generation by the NIMELS laser. In the scientific literature, Liu et al. Previously used a similar model to test the antioxidant protective activity of staphylococcus aureus carotenoid pigments against neutrophils. (Liu et al., Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity, Vol. 202, No. 2, 2005, July 18, pp. 209-215, the entire teachings of which are incorporated herein by reference. (Incorporated inside.)

Gold in Staphylococcus aureus is given by carotenoid (antioxidant) dyes which can be protected from singlet oxygen organisms, variants that do not produce such carotenoid pigment (crt ^-) when is isolated, It has been previously determined that the mutant colonies are “white” in appearance, more susceptible to the bactericidal action of oxidants and impair neutrophil survival.

  NIMELS laser non-lethal dosimetry consistently (relative to wild-type S. aureus) consistently kills less than 90% of mutant “white” cells and does not kill normal S. aureus. It was issued. The only genetic difference in these two S. aureus strains is the lack of antioxidant pigments in the mutants.

Example V
MRSA, C.I. NIMELS in vitro test for changes in ΔΨ in albicans and E. coli selected by uptake into intact cells, accumulate in those intact cells within 15-30 minutes, and without detectable staining of other cytoplasmic components There is a fluorescent dye. These membrane potential dye indicators have been available for many years and have been used to study cell physiology. The fluorescence intensity of these dyes can be easily monitored because their fluorescence spectral characteristics are responsible for the change in the value of the transmembrane potential ΔΨ-steady.

  These dyes generally work by partitioning between the extracellular medium and the membrane or the cytoplasm of the membrane, depending on the potential. This occurs due to the redistribution of the dye due to the interaction of the voltage potential on the dye with the ionic charge. This fluorescence can be removed in about 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). This shows that the maintenance of dye concentration depends on the negative transmembrane potential inside maintained by functional ETS and Δp.

Hypothesis test:
The null hypothesis is μ ₁ −μ ₂ = 0
μ ₁ is the fluorescence intensity in a control cell culture (without laser) exposed to carbocyanine dye, and μ ₂ is the fluorescence intensity in the same cell culture pre-irradiated with quasi-lethal dosimetry from a NIMELS laser. is there

This data shows that pre-treatment of the NIMELS laser system (cells) extinguishes the fluorescence of the cells (less than the unirradiated control or “no laser” cells), It shows that the NIMELS laser interacted with the cellular respiratory process and oxidative phosphorylation through the plasma membrane.
μ ₁ −μ ₂ = 0
Supports that the additional quasi-lethal NIMEL irradiation on this cell culture has no effect on ΔΨ-steady.
μ ₁ −μ ₂ > 0
Support that additional quasi-lethal NIMEL irradiation on this cell culture has a dissipative or depolarizing effect on ΔΨ-steady.

Materials and methods:
BacLight ™ bacterial membrane potential kit (B34950, Invitrogen US). This BacLight ™ bacterial membrane potential kit contains carbocyanine dyes DiOC2 (3) (3,3′-diethyloxacarbocyanine iodide, component A) and CCCP (carbonyl cyanide 3-chlorophenylhydrazone, component B) (both Both in DMSO) and 1 × PBS solution (component C).
DiOC2 (3) exhibits green fluorescence in all bacterial cells, but this fluorescence changes towards red emission as the dye molecules self-associate at higher cytosolic concentrations caused by the greater membrane potential. Proton ionophores, such as CCCP, disrupt the membrane potential by eliminating the proton gradient, causing higher green fluorescence.

The detection green fluorescence emission of the membrane potential ΔΨ in MRSA was calculated using the population mean fluorescence intensity for the samples lasered with control and quasi-lethal dosimetry:

This data shows that μ ₁ −μ ₂ > 0 (laser irradiated cells) had less “green fluorescence” as shown in FIG. These MRSA samples clearly showed a change in ΔΨ-steady-bact to one of ΔΨ-trans-bact by quasi-lethal NIMELS dosimetry.

C. The detected green fluorescence emission of the membrane potential ΔΨ in albicans was calculated using the population mean fluorescence intensity for the control and the samples irradiated with quasi-lethal dosimetry listed in the table below:

This data shows that μ ₁ −μ ₂ > 0 (laser-irradiated C. albicans cells) had less “green fluorescence” as shown in FIG. These C.I. The albicans sample clearly showed a change and decline of ΔΨ-steady-fungi to one of ΔΨ-trans-fungi by quasi-lethal NIMELS dosimetry and increasing (quasi-lethal) NIMELS dosimetry.

Detecting red / green ratio of the membrane potential ΔΨ in E. coli, were calculated using the population mean fluorescence intensity for samples laser irradiation in control and sublethal dosimetry:

This data shows that μ ₁ −μ ₂ > 0 (laser irradiated cells) had less “green fluorescence” as shown in FIG. These E. coli samples clearly showed a change and drop in ΔΨ-steady-bact to one of ΔΨ-trans-bact by quasi-lethal NIMELS dosimetry.

Example VI
C. NIMELS in vitro test for Δψ-mito by quasi-lethal laser dosimetry in Albicans Hypothesis test:
The null hypothesis is μ ₁ −μ ₂ = 0
a) μ ₁ is the fluorescence intensity in mitochondria of control cell cultures exposed to the mitochondrial membrane potential detection kit.
b) μ ₂ is the fluorescence intensity in the same cell culture pre-irradiated from the NIMELS laser with quasi-lethal dosimetry and exposed to the mitochondrial membrane potential detection kit.

This data indicates that mitochondrial fluorescence is extinguished (less than control non-irradiated cells) by (cell) pre-treatment with the NIMELS laser system, and these results indicate that the NIMELS laser It has been shown to interact with cellular respiratory processes and oxidative phosphorylation in the mitochondria of fungal and mammalian cells.
μ ₁ −μ ₂ = 0
Supports that the additional quasi-lethal NIMEL irradiation on the mitochondria of this cell culture has no effect on ΔΨ-steady-mito.
μ ₁ −μ ₂ > 0
Support that additional quasi-lethal NIMEL irradiation on this cell culture has a dissipative or depolarizing effect on ΔΨ-steady-mito.

Materials and methods:
Mitochondrial membrane potential detection kit (APO LOGIX JC-1) (Cell Technology Inc., 950 Rengstorff Ave, Sute D; Mountain View CA 94043).

  Loss of mitochondrial membrane potential (ΔΨ) is a hallmark of apoptosis. This APO LOGIX JC-1 assay kit measures the mitochondrial membrane potential in cells.

  In non-apoptotic cells, JC-1 (5,5 ′, 6,6′-tetrachloro-1,1 ′, 3,3′-tetraethylbenz-imidazolylcarbocyanine iodide) is the cytosol as a monomer. It exists in (green) and accumulates in mitochondria as aggregates (red). However, in cells that have undergone apoptosis and necrosis, JC-1 exists in monomeric form and stains the cytosol green.

  This (APO LOGIX JC-1) kit measures membrane potential by conversation of green fluorescence to red fluorescence. In FIG. 10A, the appearance of red is measured and plotted, which only occurs in cells with intact membranes, and the ratio of green to red is shown in FIG. 10B (control and laser irradiated). For both samples). Obviously, in this test, the red fluorescence is decreased in the sample that received the laser, but the ratio of green to red is increased, indicating depolarization. These results are the same as the transmembrane ΔΨ test (ie, both data indicate depolarization).

These results also, μ ₁ -μ _2> 0 and sublethal NIMEL irradiation on cell mitochondria also shown to have dissipated or depolarizing effect on ΔΨ-steady-mito, Candida albicans [Delta] [Psi]-steady -Shows a clear decrease of -mito-fungi to [Delta] [Psi] -trans-mito-fungi.

Example VII
NIMELS in vitro test for Δψ-mito of human fetal kidney cells by quasi-lethal laser dosimetry Hypothesis test:
The null hypothesis is μ ₁ −μ ₂ = 0
a) μ ₁ is the fluorescence intensity in mitochondria (no laser irradiation) of mammalian control cell cultures exposed to the mitochondrial membrane potential detection kit.
b) μ ₂ is the fluorescence intensity in the same mammalian cell culture pre-irradiated from the NIMELS laser with quasi-lethal dosimetry and exposed to the mitochondrial membrane potential detection kit.

Membrane Potential Detection Kit This data shows that mitochondrial fluorescence is extinguished (less than control non-irradiated cells) by (cell) pretreatment with the NIMELS laser system, and these results , Indicating that the NIMELS laser interacted with the cellular respiratory process and oxidative phosphorylation in the mitochondria of mammalian cells.
μ ₁ −μ ₂ = 0
Supports that the additional quasi-lethal NIMEL irradiation on mitochondria of this mammalian cell culture has no effect on ΔΨ-steady-mito-mam.
μ ₁ −μ ₂ > 0
Support that additional quasi-lethal NIMEL irradiation for this mammalian cell culture has a dissipative or depolarizing effect on ΔΨ-steady-mito-mam.

Materials and methods:
Mitochondrial membrane potential detection kit (APO LOGIX JC-1) (Cell Technology Inc., 950 Rengstorff Ave, Sute D; Mountain View CA 94043).
Loss of mitochondrial membrane potential (ΔΨ) is a hallmark of apoptosis. This APO LOGIX JC-1 assay kit measures the mitochondrial membrane potential in cells. In non-apoptotic cells, JC-1 (5,5 ′, 6,6′-tetrachloro-1,1 ′, 3,3′-tetraethylbenz-imidazolylcarbocyanine iodide) is the cytosol as a monomer. It exists in (green) and accumulates in mitochondria as aggregates (red). However, in cells that have undergone apoptosis and necrosis, JC-1 exists in monomeric form and stains the cytosol green.

ΔΨ-mito test of HEK-293 (human embryonic kidney cells):
This (APO LOGIX JC-1) kit measures membrane potential by conversation of green fluorescence to red fluorescence. In FIG. 11A, the appearance of red is measured and plotted, which only occurs in cells with intact membranes, and the ratio of green to red is shown in FIG. 11B (control and laser irradiated). For both samples).

Apparently, in this test, the red fluorescence is decreased in the sample that received the laser, but the ratio of green to red is increased, indicating depolarization. These results show that μ ₁ −μ ₂ > 0 and quasi-lethal NIMEL irradiation on mammalian cell mitochondria have a dissipative or depolarizing effect on ΔΨ-steady-mito-mam, in mammals It shows a clear drop from ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam.

Example VIII
NIMELS In Vitro Test for Reactive Oxygen Species (ROS) An in vitro test for the generation of these reactive oxygen species (ROS) was performed using bacterial transmembrane ΔΨ-steady-bact to ΔΨ-trans-bact. , .DELTA..PSI.-steady-mito-fungi to .DELTA..PSI.-trans-mito-fungi, and .DELTA..PSI.-steady-mito-mam to .DELTA..PSI.-trans-mit-mamma. After the laser change, with comparable quasi-lethal laser dosimetry.

Materials and methods:
Kit for determination of total glutathione (Dojindo Laboratories; Kumamoto Techno Research Park, 2025-5 Tabaru, Mashiki-machi, Kamimashiki-gun; Kumamoto 861-2202, Japan)

  Glutathione (GSH) is the most abundant thiol (SH) compound in animal tissues, plant tissues, bacteria and yeast. GSH plays many different roles, such as protecting against reactive oxygen species and maintaining the SH groups of proteins. In these reactions, GSH is converted to glutathione disulfide (GSSG: oxidized form of GSH). Since GSSG is enzymatically reduced by glutathione reductase, GSH is the predominant form in organisms. DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), known as the Elman reagent, was developed for the detection of thiol compounds. In 1985, it was suggested that the glutathione recycling system with DTNB and glutathione reductase creates a highly sensitive glutathione detection method. DTNB and glutathione (GSH) react to produce 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG). Since 2-nitro-5-thiobenzoic acid is a yellow product, the GSH concentration in the sample solution can be measured by measuring the absorbance at 412 nm. GSH is produced from GSSG by glutathione reductase and reacts with DTNB to again produce 2-nitro-5-thiobenzoic acid. This recycling reaction therefore improves the sensitivity of total glutathione detection.

  At significant concentrations, ROS reacts rapidly and specifically with the target at a rate that exceeds its reduction rate by components of the glutathione antioxidant system (catalase, peroxidase, GSH).

Detection of glutathione in MRSA in quasi-lethal NIMELS dosimetry changing ΔΨ-steady-bact to one of ΔΨ-trans-bact As shown in FIG. 12, these results indicate that ΔΨ-steady-bact is ΔΨ− It clearly shows a decrease in total glutathione in MRSA in quasi-lethal NIMELS dosimetry that converts to one of the trans-bacts, which translates from transmembrane ΔΨ-steady-bact to one of ΔΨ-trans-bact. Evidence for the occurrence of ROS due to quasi-lethal changes.

Detection of glutathione in E. coli in quasi-lethal NIMELS dosimetry changing ΔΨ-steady to one of ΔΨ-trans As shown in FIG. 20, these results indicate that ΔΨ-steady-bact is changed to ΔΨ-trans-bact. It clearly shows the reduction of total glutathione in E. coli in quasi-lethal NIMELS dosimetry changing to one, which is a quasi-lethal change of transmembrane ΔΨ-steady-bact to one of ΔΨ-trans-bact This is evidence of the occurrence of ROS.

  C. in quasi-lethal NIMELS changing ΔΨ-steady-mito-fungi to ΔΨ-trans-mito-fungi and then changing ΔΨ-steady-fungi to one of ΔΨ-trans-fungi. Detection of glutathione in albicans

C. in quasi-lethal NIMELS dosimetry changing [Delta] [Psi] -steady-mito-fungi to [Delta] [Psi] -trans-mito-fungi and then changing [Delta] [Psi] -steady-fungi to one of [Delta] [Psi] -trans-fungi. Detection of glutathione in albicans As shown in FIG. 13, these results indicate that ΔΨ-steady-mito-fungi is changed to ΔΨ-trans-mito-fungi and then ΔΨ-steady-fungi is changed to ΔΨ-trans-fungi. C. in quasi-lethal NIMELS dosimetry It clearly shows a decrease in total glutathione in the albicans, which is transmembrane ΔΨ-steady-mito-fungi to ΔΨ-trans-mito-fungi, then ΔΨ-steady-fungi ΔΨ-trans- Evidence for the occurrence of ROS due to a quasi-lethal change to one of the fungi.

Detection of glutathione in HEK-293 (human embryonic kidney cells) in quasi-lethal NIMELS dosimetry changing ΔΨ-steady-mito-mam to ΔΨ-trans-mam As shown in FIG. It clearly shows a decrease in total glutathione in HEK-293 (human embryonic kidney cells) in quasi-lethal NIMELS dosimetry that changes ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam, Evidence for the occurrence of ROS by NIMELS-mediated transmembrane Δψ-steady-mito-mam to Δψ-trans-mito-mam.

Example IX
Evaluation of the effect of quasi-lethal dose of NIMELS laser on MRSA with erythromycin and trimethoprim In this example, the quasi-lethal dose of NIMELS laser enhances the effect of antibiotic erythromycin more than the antibiotic trimethoprim in MRSA Measured whether or not. In erythromycin resistance, the efflux pump plays a major factor. The resistance mechanism of the trimethoprim efflux pump has not been reported in Gram-positive S. aureus.

Background: Erythromycin is a marcolide antibiotic with an antibacterial spectrum very similar to β-lactam penicillin. In the past, it has been considered one of the safest and most effective antibiotics for the treatment of a wide range of Gram-positive bacterial skin and respiratory tract infections. In the past, erythromycin has been used for people with allergies to penicillin. The mechanism of action of erythromycin is to prevent bacterial growth and replication by interfering with bacterial protein synthesis. This is achieved because erythromycin binds to the 23S rRNA molecule in the 50S bacterial ribosome, thereby blocking the growth of the growing peptide chain and thus inhibiting peptide migration. Erythromycin resistance (and other marcolides) is uncontrollable and extensive and is achieved by two significant resistance systems:
A) Modification that makes 23S rRNA in the 50S ribosomal subunit unreactive B) Drug outflow

  Trimethoprim is an antibiotic that has historically been used to treat urinary tract infections. It is a member of a class of antibacterial agents known as inhibitors of dihydrofolate reductase. The mechanism of action of trimethoprim is to interfere with the bacterial dihydrofolate reductase (DHFR) system because it is an analog of dihydrofolate. This causes competitive inhibition of DHFR due to an affinity 1000 times higher than the natural substrate for this enzyme.

  Therefore, trimethoprim inhibits the synthesis of tetrahydrofolate molecules. Tetrahydrofolic acid is an essential precursor in the de novo synthesis of DNA nucleotide thymidylate. Bacteria cannot take up folic acid from the environment (ie, the infected host) and are therefore dependent on the de novo synthesis of their own tetrahydrofolic acid. Inhibition of this enzyme ultimately prevents DNA replication.

  Trimethoprim resistance generally arises from overproduction of normal chromosome DHFR, or drug-resistant DHFR enzymes. Reports of trimethoprim resistant Staphylococcus aureus have shown that the resistance is either chromosomally mediated or encoded in a large plasmid. Several strains have been reported to show both chromosomal and plasmid-mediated trimethoprim resistance. In Staphylococcus aureus, a Gram-positive pathogen, resistance to trimethoprim is due to genetic mutation, and it has not been reported that trimethoprim was actively effluxed extracellularly.

Pumps for efflux in bacteria The main pathway of drug resistance in bacteria and fungi is the active transport (efflux) of antibiotics out of the cell, preventing therapeutic concentrations from being obtained in the cytoplasm of the cells.

  Active efflux of antibiotics (and other harmful molecules) is mediated by a series of transmembrane proteins in the cytoplasmic membrane of gram positive bacteria and the outer membrane of gram negative bacteria.

  Clinically, antibiotic resistance mediated by efflux pumps is most relevant to marcolide antibiotics, tetracyclines and fluoroquinolones in Gram-positive bacteria. In gram negative bacteria, β-lactam efflux mediated resistance is also clinically relevant.

The hypothesis testing null hypothesis is μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ = 0, where:
a) μ ₁ is a quasi-lethal dosimetry from the NIMELS laser system to MRSA as a control;
b) μ ₂ is a quasi-lethal dosimetry with addition of a resistant MIC just below the effective level of trimethoprim to MRSA from the same NIMELS laser system;
c) μ ₃ is a quasi-lethal dosimetry with the addition of a resistant MIC just below the effective level of erythromycin to MRSA from the same NIMELS laser system.

This data shows that the addition of antibiotics trimethoprim or erythromycin alters quasi-lethal irradiation, resulting in a decrease in the growth of these MRSA colonies as follows:
μ ₁ −μ ₂ = 0
It supports that the addition of trimethoprim does not produce deleterious effects after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.
μ ₁ −μ ₂ > 0
Support that the addition of trimethoprim produces a deleterious effect after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.
μ ₁ −μ ₃ = 0
Support that the addition of erythromycin does not produce a deleterious effect after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.
μ ₁ -μ _3> 0
Support that the addition of erythromycin produces a deleterious effect after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.

result:
This experiment clearly showed that μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ > = 0 under quasi-lethal laser parameters with the NIMELS system. This indicates that the efflux pump is inhibited, and that erythromycin resistance is reversed by the NIMELS effect on ΔSA-steady-bact of MRSA.

Example X
Evaluating the effects of NIMELS laser quasi-lethal dose on MRSA with tetracycline and rifampin The purpose of this experiment is to increase the effect of antibiotic tetracycline over antibiotic rifampin in MRSA. Was to see if. Outflow pumps are well-studied and play a major factor in tetracycline resistance. However, the resistance mechanism by rifampin efflux pumps has not been reported in Gram-positive S. aureus.

  This experiment was previously performed with erythromycin and trimethoprim, and the data showed that the NIMELS effect can damage the resistance mechanism by the efflux pump in erythromycin.

tetracycline:
Tetracycline is a bacteriostatic antibiotic, which means that it prevents bacterial growth by inhibiting protein synthesis. Tetracycline accomplishes this by inhibiting the action of the bacterial 30S ribosome by binding of the enzyme aminoacyl-tRNA. Tetracycline resistance is often due to the acquisition of a new gene that encodes an energy-dependent tetracycline efflux or a protein that encodes a protein that protects the bacterial ribosome from the action of tetracycline.

Rifampin:
Rifampin is an inhibitor of bacterial RNA polymerase and functions by directly blocking RNA elongation. Rifampicin is typically used to treat mycobacterial infections, but also plays a role with fusidic acid (a bacteriostatic protein synthesis inhibitor) in the treatment of methicillin-resistant Staphylococcus aureus (MRSA). There is no report that rifampin resistance is due to spill pumps in MRSA.

hypothesis:
The null hypothesis is μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ = 0, where:
a) μ ₁ is a quasi-lethal dosimetry from the NIMELS laser system to MRSA as a control;
b) μ ₂ is a quasi-lethal dosimetry with the addition of a resistant MIC just below the effective level of tetracycline to MRSA from the same NIMELS laser system;
c) μ ₃ is a quasi-lethal dosimetry with the addition of a resistant MIC just below the effective level of rifampin to MRSA from the same NIMELS laser system.

This data shows that the addition of antibiotic tetracycline or rifampin results in a decrease in the growth of these MRSA colonies after sublethal irradiation, as follows:
μ ₁ −μ ₂ = 0
Support that the addition of tetracycline does not produce a deleterious effect after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.
μ ₁ −μ ₂ > 0
Support that the addition of tetracycline produces a detrimental effect on normally grown MRSA colonies after quasi-lethal NIMEL irradiation.
μ ₁ −μ ₃ = 0
Support that the addition of rifampin does not produce a deleterious effect after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.
μ ₁ -μ _3> 0
Support that the addition of rifampin produces deleterious effects after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.

result:
This experiment clearly showed that μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ > = 0 under quasi-lethal laser parameters with the NIMELS system. This indicates that the efflux pump is inhibited and that tetracycline resistance is reversed by the NIMELS effect on MRSA ΔΨ-steady-bact.

Example XI
Evaluation of the effect of NIMELS laser quasi-lethal dose on MRSA with methicillin and cell wall synthesis with ΔΨ-plus-bact inhibition
Methicillin:
Methicillin was previously used to treat infections caused by gram-positive bacteria, particularly β-lactamase producing microorganisms such as S. aureus (resistant to most penicillins), but is no longer used clinically It is a β-lactam antibiotic. The term methicillin-resistant Staphylococcus aureus (MRSA) continues to be used to describe S. aureus that is resistant to all penicillins.

Mechanism of Action Like other β-lactam antibiotics, methicillin acts by inhibiting the synthesis of peptidoglycan (bacterial cell wall).

The Gram-positive bacterium Bacillus subtilis has been shown to increase (ie, no longer inhibit) peptidoglycan autolytic activity when ETS is blocked by the addition of a proton conductor. This suggests that ΔΨ-plus-bact and ΔμH ⁺ (regardless of energy storage for cellular enzyme function) potentially have an abyssable effect on cell wall anabolic function and physiology. is doing.

  In addition, the ΔΨ-plasm-bact uncoupler is responsible for peptidoglycan formation, accumulation of nucleotide precursors involved in peptidoglycan synthesis, and transport of N-acetylglucosamine (GlcNAc), one of the major biopolymers in peptidoglycan. It has been reported that inhibition is caused by inhibition of.

Hypothesis test:
Bacitracin will enhance the multiple effects of optically reduced ΔΨ-plasm-bact on the growing cell wall (ie, increased cell wall autolysis, inhibited cell wall synthesis). This is particularly relevant in Gram positive bacteria such as MRSA that do not have an efflux pump as a resistance mechanism for cell wall inhibitory antibacterial compounds.
The null hypothesis is μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ = 0, where:
a) μ ₁ is a quasi-lethal dosimetry from the NIMELS laser system to MRSA as a control;
b) μ ₂ is a quasi-lethal dosimetry with the addition of a resistant MIC just below the effective level of methicillin to MRSA from the same NIMELS laser system;
μ ₁ −μ ₂ = 0
Support that the addition of methicillin does not produce a deleterious effect after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.
μ ₁ −μ ₂ > 0
Support that the addition of methicillin produces deleterious effects after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.

result:
As shown in FIG. 15, this experiment clearly shows that μ ₁ −μ ₂ > = 0 under quasi-lethal laser parameters with the NIMELS system, which indicates that the addition of methicillin is a CFU count. As shown by the above, it means that the MRSA colonies that have grown normally have a harmful effect after quasi-lethal NIMEL irradiation. This suggests that methicillin (regardless of efflux pump) was enhanced by the NIMELS effect on MRSA ΔΨ-steady-bact.

  Therefore, the NIMELS laser and its associated optical ΔΨ-plus-bact reduction phenomenon are synergistic with cell wall inhibitory antibacterial agents in MRSA. Without wishing to be bound by theory, this must function by inhibition of anabolic (periplasmic) ATP-coupled functions, since MRSA does not have an efflux pump for methicillin.

Example XII
Assessment of the effect of a sublethal dose of NIMELS laser on MRSA with bacitracin and ΔΨ-plasm-bact inhibition of cell wall synthesis Bacitracin is a mixture of cyclic polypeptides produced by Bacillus subtilis. Because it is a toxic and difficult-to-use antibiotic, bacitracin is generally not used orally but is used topically.

Mechanism of action:
Bacitracin, C ₅₅ - to disturb the dephosphorylation of isoprenyl pyrophosphate, the pyrophosphate, a molecule that carries the building blocks of the outer peptidoglycan bacterial cell walls of the plasma membrane of the inner layer and gram-positive microorganisms Gram-negative microorganisms is there.

In the Gram-positive bacterium Bacillus subtilis, it has been shown that when ETS is blocked by the addition of a proton conductor, the autolytic activity of peptidoglycan is increased (ie, no longer inhibited). This indicates that ΔΨ-plus-bact and ΔμH ⁺ (regardless of energy storage for cellular enzyme function) potentially have an abyssable effect on cell wall anabolic function and physiology. ing.

  In addition, the ΔΨ-plasm-bact uncoupler is responsible for peptidoglycan formation, accumulation of nucleotide precursors involved in peptidoglycan synthesis, and transport of N-acetylglucosamine (GlcNAc), one of the major biopolymers in peptidoglycan. It has been reported that inhibition is caused by inhibition of.

Hypothesis test:
Bacitracin enhances the multiple effects of optically reduced ΔΨ-plasm-bact on the growing cell wall (ie, increased cell wall autolysis, inhibited cell wall synthesis). This is particularly relevant in Gram positive bacteria such as MRSA that do not have an efflux pump as a resistance mechanism for cell wall inhibitory antibacterial compounds.
The null hypothesis is μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ = 0, where:
a) μ ₁ is a quasi-lethal dosimetry from the NIMELS laser system to MRSA as a control;
b) μ ₂ is a quasi-lethal dosimetry with the addition of a resistant MIC just below the effective level of bacitracin to MRSA from the same NIMELS laser system;
μ ₁ −μ ₂ = 0
Support that the addition of bacitracin does not produce a deleterious effect after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.
μ ₁ −μ ₂ > 0
Support that the addition of bacitracin produces deleterious effects after quasi-lethal NIMEL irradiation on normally grown MRSA colonies.

result:
As shown in FIG. 16, this experiment clearly shows that μ ₁ −μ ₂ > = 0 under quasi-lethal laser parameters with the NIMELS system, indicating that the addition of bacitracin is normal It means that the MRSA colonies that have grown have a deleterious effect after quasi-lethal NIMEL irradiation. In FIG. 16, the arrows indicate MRSA growth or lack thereof in the two samples shown. This indicates that bacitracin (regardless of efflux pump) was enhanced by the NIMELS effect on MRSA ΔΨ-steady-bact. Therefore, the NIMELS laser and its associated optical ΔΨ-plus-bact reduction phenomenon are synergistic with cell wall inhibitory antibacterial agents in MRSA. Without wishing to be bound by theory, this must function by inhibition of anabolic (periplasmic) ATP-coupled functions, since MRSA does not have an efflux pump for bacitracin.

Example XIII
NIMELS laser quasi-lethal dose of C. with ramisyl and sporanox. Assessment of the effects on albicans The purpose of this experiment was to determine whether a sublethal dose of NIMEL laser was applied to C. It was to observe whether to enhance the effect in albicans.

Introduction:
A decrease in the ATP concentration in the cytosol of fungal cells leads to suppression of H ⁺ -ATPase bound to the plasma membrane that produces ΔΨp-fungi and this loss is seen to attenuate other cellular activities. Has been issued. In addition, the decrease in ΔΨp-fungi causes bioenergetic and thermodynamic disruption of the plasma membrane, which leads to proton influx, which disrupts proton activation and thus nutrients Inhibits uptake. It is further noted that ATP is required for the biosynthesis of the fungal plasma membrane lipid ergosterol. Ergosterol is targeted by a large number of commercially available related antifungal compounds (i.e., azole, terbinafine and itraconazole) used today in medicine, including ramisyl and sporanox (and unregistered counterparts) It is a structured lipid.

  Also recently, two new antimicrobial peptides (Pep2 and Hst5) have the ability to efflux ATP from fungal cells (ie deplete intracellular ATP concentration) and this reduced cytosolic ATP is It has been shown to result in inactivation of ABC transporters CDR1 and CDR2, which are efflux pumps for dependent antifungal agents.

Ramisil:
Ramisyl (as well as other allylamines) inhibits ergostero synthesis by inhibiting squalene epoxidase, an enzyme that is part of the fungal cell wall synthesis pathway.

Sporanox:
The mechanism of action of itraconazole (Sporanox) is the same as other azole antifungal agents; it inhibits fungal cytochrome P450 oxidase-mediated ergosterool synthesis.

hypothesis:
C. NIMELS laser in semi-lethal dosimetry to albicans can be used to reduce ramisyl and sporanox to optically reduced ΔΨ-plasm-fungi and / or ΔΨ-mito--by fungal membrane depolarization and intracellular ATP depletion. Strengthen for fungi.
The null hypothesis is μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ = 0, where:
a) μ ₁ is the C.I. as a control from the NIMELS laser system. Quasi-lethal dosimetry to albicans; and;
b) μ ₂ is C.I. from the same NIMELS laser system. Quasi-lethal dosimetry with addition to albicans with a resistant MIC just below the effective level of Sporanox;
c) μ ₃ is a quasi-lethal dosimetry with the addition of a resistant MIC just below the effective level of ramisyl to MRSA from the same NIMELS laser system.

This data shows that the addition of the antibiotic ramisyl and / or sporanox, after quasi-lethal irradiation, indicated that these C.I. Shows that it results in reduced growth of albicans colonies:
μ ₁ −μ ₂ = 0
C. was successfully grown with the addition of Sporanox. Supports albicans colonies that do not produce deleterious effects after quasi-lethal NIMEL irradiation.
μ ₁ −μ ₂ > 0
C. was successfully grown with the addition of Sporanox. Supports producing a detrimental effect on albicans colonies after quasi-lethal NIMEL irradiation.
μ ₁ −μ ₃ = 0
C. was successfully grown with the addition of ramisyl. Supports albicans colonies that do not produce deleterious effects after quasi-lethal NIMEL irradiation.
μ ₁ -μ _3> 0
C. was successfully grown with the addition of ramisyl. Supports producing a detrimental effect on albicans colonies after quasi-lethal NIMEL irradiation.

result:
This experiment clearly showed that μ ₁ −μ ₂ = 0 and μ ₁ −μ ₃ > = 0 under quasi-lethal laser parameters with the NIMELS system. This is because C. It means that a harmful effect is produced on albicans colonies after quasi-lethal NIMEL irradiation. This suggests that egosterol biosynthesis inhibitors (ramisyl and sporanox) were enhanced by quasi-lethal dosimetric irradiation of the NIMELS laser system.

Example XIV
NIMELS dosimetry calculations The following examples describe selected experiments depicting the ability of the NIMELS approach to affect the viability of various commonly found microorganisms at the wavelengths described herein. These exemplified microorganisms include E. coli K-12, multi-drug resistant Escherichia coli, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Candida albicans, and S. aureus.

二重波長処理について：
２）

ビーム面積は、下記の何れかにより計算することができる：
３）

特定の出力パワーである期間にわたって作動する一のＮＩＭＥＬＳレーザーダイオードシステムにより組織に送達される全光エネルギーは、ジュールで測定されて、下記のように計算される：
４）

特定の出力パワーである期間にわたって両ＮＩＭＥＬＳレーザーダイオードシステムにより同時に組織に送達される全光エネルギー(両波長)は、ジュールで測定されて、下記のように計算される：
５）

実際には、全エネルギーの、照射治療領域にわたる分布及び分配を知ることは、最大ＮＩＭＥＬＳ利益のある応答のための線量を正確に測定するために有用である(但し、必須ではない)。全エネルギー分布は、エネルギー密度(ジュール／ｃｍ²)として測定することができる。以下に記載のように、所定の波長の光について、エネルギー密度は、組織反応の測定において最も重要な因子である。一のＮＩＭＥＬＳ波長についてのエネルギー密度は、下記のように得られる：
６）

７）

２つのＮＩＭＥＬＳ波長が用いられる場合には、エネルギー密度は、下記のように得られる：
８）

９）

特定の線量についての治療時間を計算するためには、実務家は、エネルギー密度(Ｊ／ｃｍ²)又はエネルギー(Ｊ)、並びに出力パワー(Ｗ)、及びビーム面積(ｃｍ²)を利用することができる(下記の方程式の何れか一つを利用)：
１０）

１１）

As discussed in more detail earlier, the NIMELS parameters include the average single or additive power of the laser diode and the wavelength of the diode (870 nm and 930 nm). This information, along with the area of the laser beam (cm ² ), gives an initial set of information that can be used to calculate an effective and safe irradiation protocol according to the present invention. The power density of a given laser measures the potential effect of NIMELS at the target site. The power density is a function of any given laser power and beam area and can be calculated by the following equation:
For a single wavelength:
1)

About dual wavelength processing:
2)

The beam area can be calculated by either:
3)

The total light energy delivered to the tissue by one NIMELS laser diode system operating over a period of time with a specific output power is measured in joules and calculated as follows:
4)

The total light energy (both wavelengths) delivered to the tissue simultaneously by both NIMELS laser diode systems over a period of a specific output power is measured in joules and calculated as follows:
5)

In fact, knowing the distribution and distribution of total energy across the irradiated treatment area is useful (but not essential) to accurately measure the dose for a maximal NIMELS beneficial response. The total energy distribution can be measured as energy density (joule / cm ² ). As described below, for a given wavelength of light, energy density is the most important factor in measuring tissue response. The energy density for one NIMELS wavelength is obtained as follows:
6)

7)

If two NIMELS wavelengths are used, the energy density is obtained as follows:
8)

9)

To calculate treatment time for a specific dose, practitioners should use energy density (J / cm ² ) or energy (J), and output power (W), and beam area (cm ² ) (Use one of the following equations):
10)

11)

  Since dosimetric calculations as illustrated in this example can be cumbersome, the treatment system can also include a computer database that stores all studied therapeutic possibilities and dosimetry. The computer (dose measurement and parameter calculator) in this controller is pre-programmed using an algorithm based on the above equation so that the operator can easily retrieve data and parameters on the screen. Additional required data (e.g. spot size, desired total energy, time and pulse width for each wavelength, irradiated tissue, irradiated bacteria) can be entered along with any other necessary information, so Any and all algorithms and calculations necessary for a suitable treatment result can be generated by dosimetry and parameter calculations, and therefore a laser can be implemented.

  In summary, in the following examples, when a bacterial culture is exposed to a NIMELS laser, the bactericidal rate (measured by counting colony forming units or CFU in the treated culture plate) is 93.7 (multidrug resistant E. coli). To 100% (all other bacteria and fungi).

Example XV
Bacteria method: NIMELS treatment parameters for E. coli targeting in vitro The following parameters apply to E. coli at a final temperature well below that associated with thermal damage according to the literature. The method according to will be described.

A. Experimental materials and methods for E. coli K-12:
E. coli K-12 liquid cultures were grown in Luria-Bertani (LB) medium (25 g / L). The plate contained 35 mL of LB plate medium (25 g / L LB, 15 g / L bacterial agar). The culture was diluted with PBS. All protocols and manipulations were performed using aseptic techniques.

B. Growth Kinetics Removed from seed cultures, inoculated into many 50 mL LB media and cultured overnight at 37 ° C. The next morning, select a heralthiest culture and use it for 5% inoculation of 50 mL LB at 37 ° C, using OD ₆₀₀ over time, every 30-45 minutes until the culture reaches stationary phase. Monitored by measuring.

C. Masterstock population Starting from a log phase culture (approximately 0.75 OD ₆₀₀ ), place 10 mL at 4 ° C, add 10 mL 50% glycerol, and take aliquots into 20 cryovials. Quick-frozen in liquid nitrogen. These cryovials were then stored at -80 ° C.

D. Liquid culture A liquid culture of E. coli K12 was prepared as described above. A 100 μL aliquot was removed from the subculture and serially diluted 1: 1200 with PBS. This dilution is used to ensure that these cells reach steady state (growth) in PBS suspension without significant division and a relatively constant number of cells can be aliquoted for further testing. The mixture was incubated at room temperature for about 2 hours or until no further increase in OD ₆₀₀ was observed.

  Once it was determined that the K12 dilution had reached a steady state, 2 mL of this suspension was aliquoted into selected wells of a 24-well tissue culture plate with selected dosimetry parameters. Taken for NIMELS experiments. The plates were incubated at room temperature until ready for use (about 2 hours).

After laser treatment, 100 μl was removed from each well and serially diluted 1: 1000, ultimately producing a 1: 12 × 10 ⁵ dilution of the initial K12 culture. A 3 × 200 L aliquot of each final dilution was spread in triplicate onto separate plates. The plates were then incubated for about 16 hours at 37 ° C. Manual colony counts were made and recorded. I also took a digital photo of each plate. Similar cell culture and kinetic protocols were used for S. aureus and C.I. All NIMELS irradiation tests using albicans were performed in vitro. For example, C.I. An albicans ATCC 14053 liquid culture was grown at 37 ° C. in YM medium (21 g / L, Difco). From the standardized suspension, aliquots were taken into selected wells in 24-well tissue culture plates. After laser treatment, 100 μL was removed from each well and serially diluted 1: 1000 to give a final dilution of 1: 5 × 10 ⁵ of the initial culture. 3 × 100 μL of each final dilution was spread on a separate plate. The plates were then incubated at 37 ° C. for about 16-20 hours. Manual colony counts were made and recorded. I also took a digital photo of each plate.

  S. aeruginosa ATCC52022 liquid culture was grown at 37 ° C. in peptone-dextrose (PD) medium. From the standardized suspension, aliquots were taken into selected wells in 24-well tissue culture plates. After laser treatment, aliquots were incubated at 37 ° C. for approximately 91 hours. Manual colony counts were made and recorded after 66 and 91 hours of incubation. All microorganisms grew in the control wells, but there was no growth in 100% of the laser treated wells described here. I also took a digital photo of each plate.

  Thermal testing was performed with PBS solution starting from room temperature. Ten watts of NIMELS laser energy was available for use with a 12 minute laser cycle before the temperature of the system rose to near the critical threshold of 44 ° C.

Example XVI
Dosimetry for NIMELS laser (wavelength 930 nm) for in vitro targeting to E. coli This experiment shows that NIMELS single wavelength λ = 930 nm is quantifiable antibacterial against E. coli within safe thermal parameters for mammalian tissues Show in vitro that it is related to efficacy.

The experimental data is in vitro, and if the threshold of total energy of 5400 J and energy density of 3056 J / cm ² at 930 nm alone into the system is achieved in 25% less time, 100% antibacterial efficacy is still present. It shows what can be achieved.

  In vitro experimental data also show that treatment with a single energy of λ = 930 nm has in vitro antibacterial efficacy against the bacterial species S. aureus within thermal parameters that are safe for mammalian tissues. It also shows.

If a threshold of 5400 J total energy and 3056 J / cm ² energy density into the system is achieved in 25% less time for S. aureus and other bacterial species, 100% antimicrobial efficacy is still achieved. It is also considered that.

  In vitro experimental data also show that the NIMELS single wavelength at λ = 930 nm is in vitro, C.I. It has also been shown to have antifungal efficacy against albicans within the range of safe heat parameters for mammalian tissues.

It is also contemplated that if a threshold of 5400 J total energy and 3056 J / cm ² energy density into the system is achieved in 25% less time, 100% antimicrobial efficacy will still be achieved.

Example XVII
In vitro experimental data for in vitro NIMELS lasers (wavelength 870 nm) also show that for E. coli total energy below 7200 J and energy density below 4074 J / cm ² and 5.660 W / It also shows that significant sterilization is not achieved with a power density below cm ² and a wavelength of 870 nm alone.

Example XVIII
NIMELS-specific, alternating synergistic in vitro experimental data between light energy of 870 nm and 930 nm also shows that they alternate between two NIMELS wavelengths (λ = 870 nm and 930 nm) (870 nm before 930 nm). ) It also shows an additive effect when used. The presence of the 870 nm NIMELS wavelength as the first irradiation has been found to enhance the antimicrobial efficacy effect of the second 930 nm NIMELS wavelength irradiation.

  In vitro experimental data show that this synergistic effect (combining the wavelength of 870 nm with the wavelength of 930 nm) makes it possible to reduce the light energy of 930 nm. As shown here, the light energy is approximately 1/3 of the total energy and energy density required for NIMELS 100% E. coli antibacterial efficacy when the wavelengths (870 nm before 930 nm) are combined in an alternating manner. Decrease.

  In vitro experimental data also show that this synergistic mechanism is that if an equal amount of light energy at 870 nm is added to the system before 930 nm energy (20% increased power density), then light energy at 930 nm It can also be shown that can be reduced to about 1/2 of the total energy density required for NIMELS 100% E. coli antimicrobial efficacy.

  This synergistic ability is important for human tissue safety because the light energy at 930 nm heats the system at a higher rate than the light energy at 870 nm and generates the least amount of heat possible during processing. This is beneficial for mammalian systems.

  It is also conceivable that NIMELS light energy (870 nm and 930 nm) is used alternately in other ways in other bacterial species in the manner described above, and its 100% antibacterial effect is essentially the same.

  The in vitro experimental data also shows that if you use two NIMELS wavelengths (870 nm and 930 nm) alternately (870 nm before 930 nm), there is also an additive effect between them when irradiating the fungus. It also shows. The presence of the 870 nm NIMELS wavelength as the first irradiation mathematically enhances the effect of the antifungal efficacy of the irradiation at the second 930 nm NIMELS wavelength.

  In vitro experimental data (see table below) indicates that this synthesis mechanism is required for the antibacterial efficacy of bacterial species prior to 930 nm energy into this system if an equivalent amount of 870 nm light energy is present in this system. If applied at a power density 20% higher than 930 nm, it may be possible to reduce the light energy (total energy and energy density) of 930 nm to about 1/2 of the total energy density required for NIMELS 100% antifungal efficacy It is shown that.

  This synergistic effect is important for human tissue safety because 930 nm light energy heats the system at a much higher rate than 870 nm light energy, which generates as little heat as possible during processing. Useful for mammalian systems.

  It is also conceivable that if NIMELS light energy (870 nm and 930 nm) is used alternately with other fungal species in the manner described above, 100% of the antifungal effect will be essentially the same.

Example XIX
NIMELS unique, simultaneous synergistic in vitro experimental data between λ = 870 nm and λ = 930 nm light energy also shows that if two NIMELS wavelengths (870 nm combined with 930 nm) are used simultaneously ( It also shows that there is an additive effect between 870 nm and 930 nm. The presence as a simultaneous irradiation of the 870 nm NIMELS wavelength and the 930 nm NIMELS wavelength absolutely enhances the effectiveness of the antibacterial efficacy of the NIMELS system.

  In vitro experimental data (see, eg, Tables IX and X below) shows that combining the energy and density of light at λ = 870 nm and λ = 930 nm utilizes a single process according to the present invention. It has been shown to effectively reduce the energy and density of light at 930 nm by about half of the total energy and energy density required for.

  This simultaneous synergistic ability is important for human tissue safety because 930 nm light energy heats the system much faster than 870 light energy, and for mammalian systems the smallest amount possible during processing. It is beneficial to generate heat.

  If NIMELS light energy (870 nm and 930 nm) is used simultaneously with other bacterial species in the above manner, it is also possible that the 100% antimicrobial effect is essentially the same. (See FIGS. 17, 18 and 19) The in vitro experimental data also shows that if two NIMELS wavelengths (870 nm and 930 nm) are used simultaneously on a fungus, there is an additive effect between them. It also shows. The presence of a 870 nm NIMELS wavelength and a 930 nm NIMELS wavelength as simultaneous irradiation has been found to enhance the antifungal efficacy of the NIMELS system.

  In vitro experimental data show that this synergistic effect (for simultaneous illumination, combining the wavelength of 870 nm with the wavelength of 930 nm) is 930 nm when the wavelengths of (870 nm and 930 nm) are combined in a simultaneous manner. Light energy is measured in NIMELS 100% C.I. It shows that it is possible to reduce to about half of the total energy and energy density required for albicans antifungal efficacy.

  Thus, using NIMELS wavelengths (λ = 870 nm and 930 nm), antibacterial and antifungal efficacy can be achieved in an alternating manner or simultaneously or in any combination of such manners, Reduces the exposure at λ = 930 combined with a minimized temperature increase.

  In vitro experiments also showed that when E. coli was irradiated at the wavelength λ = 830 nm alone (for control) with the following parameters (12 minutes irradiation cycle, 100% antibacterial and antifungal with λ = 930 nm irradiation) This also shows that this control 830 nm laser did not produce any antibacterial efficacy (with the same parameters for minimal NIMELS dosimetry combined with efficacy).

  In vitro experimental data also show that there is little additive effect when using λ = 830 nm and λ = 830 emission when applied in safe temperature dosimetry. The presence of a control wavelength of 830 nm as the first emission is far superior to the enhancement effect of the 870 nm NIMELS wavelength in producing synergistic antibacterial efficacy with the second 930 nm NIMELS wavelength.

  In vitro experimental data also show that when applied in safe thermal dosimetry, the additive effects due to the 830 nm and NIMELS 930 nm wavelengths are less severe when used simultaneously. In fact, in vitro experimental data show that when 870 nm is combined with 930 nm, it achieves 100% E. coli antibacterial efficacy, with 17% less energy and 17% less energy density than the commercially available 830 nm. And 17% less power density is required. This again significantly reduces the heat and harm to in vivo systems handled by NIMELS wavelengths.

Sterilization amount:
In vitro data includes 2,000,000 (2 × 10 ⁶ ) colony forming units (CFU) of E. coli and Staphylococcus aureus, with the NIMELS laser system (within heat tolerance) in vitro It was also shown to be effective against bacterial solutions. This is twice as much as typically seen per gram of infected human ulcer tissue. Brown et al. Found that in 75% of diabetic patients examined, all microbial cells were at least 100,000 CFU / g, and in 37.5% of those patients, the amount of microorganisms was 1,000 , 000 (1 × 10 ⁶ ) CFU (Brown et al., Ostomy Wound Management, 40147, published in October 2001, the entire teachings of which are incorporated herein by reference).

Thermal Parameters In vitro experimental data also show that the NIMELS laser system can achieve 100% antibacterial and antifungal efficacy within a thermal tolerance that is safe for human tissue.

Example XX
Effects of colder temperatures in NIMELS Bacterial species cooling In vitro experimental data also show that optical antibacterial activity can be achieved by lowering the bacterial sample in PBS to a starting temperature of substantially 4C over 2 hours. It has also been shown that sexual efficacy is not achieved by any current reproducible antibacterial energy and NIMELS laser system.

Example XXI
The NIMELS effect on S. gonorrhoeae This example shows the NIMELS wavelength (870 nm and 930 nm) effect when used in an alternating or simultaneous manner.

Example XXII
MRSA / antibacterial enhancement This example demonstrates the use of NIMELS wavelengths targeted at MRSA (λ = 830 nm and 930 nm) in vitro to increase antimicrobial susceptibility to methicillin. Four separate experiments were performed. Data sets for these four experiments are given in the table below. See also FIG. It is (a) the synergistic effect of NIMELS with methicillin, penicillin and erythromycin in the inhibition of growth of MRSA colonies (data show that proton activating power of bacterial plasma membranes by quasi-lethal NIMELS dosimetry ( (Indicating that it has been enhanced by inhibiting the anabolic process of peptidoglycan synthesis, which is also targeted by the drug simultaneously), and (b) ) Erythromycin has been shown to be enhanced to a greater extent (because the Nimels effect is an anabolic process of protein synthesis and a bacterial plasma membrane that supplies energy for the efflux pump for erythromycin resistance) Proton activation force (Δp-plus-Bact) is inhibited).

material:

General method for CFU counting:

Independent report of the MRSA study on 7 November 2006
(MRSA Data Progression 11-07-06 Experiment # 1)
Experiment 1-Design:
Eight different laser doses were used to treat logarithmically growing MRSA salt solution suspensions labeled A1-H1.

Treated and control untreated suspensions were diluted and plated in triplicate on soybean agar with or without 30 μg / ml methicillin.
After growing for 24 hours at 37 ° C., colonies were counted.

  CFU (colony forming units) were compared for both control (untreated) and treated MRSA between plates with and without these methicillins.

Experiment 1-Results:
Conditions D1-H1 showed similar CFU reductions in treated and untreated samples on methicillin plates.

  Conditions A1, B1, and C1 showed 30%, 33%, or 67% less CFU in the laser treated samples, respectively, compared to the untreated controls.

  This indicates that the treatment performed on samples A1, B1 and C1 made MRSA sensitive to the effects of methicillin.

Independent report of the MRSA study on 8 November 2006
(MRSA Data Progression 11-08-06 Experiment # 2)
Experiment 2-Design:
Based on the effective dose established in Experiment 1 and previously, eight different laser doses were used to treat logarithmically growing MRSA salt solution suspensions labeled A2-H2.

Treated and control untreated suspensions were diluted and plated in triplicate on soybean agar with or without 30 μg / ml methicillin.
After growing for 24 hours at 37 ° C., colonies were counted.

Experiment 2-Results:
Comparison of CFU on plates with and without methicillin showed a significant increase in methicillin potency in all laser-treated samples, excluding A2 and B2. This data is summarized in the table below.

Independent report on MRSA study, November 9-10, 2006
(MRSA Data Progression 11-10-06 Experiment # 3)
Experiment 3-Design:
Logarithmically growing MRSA salt solution suspensions labeled A3-H3 were treated using Experiments 1 and 2 and 8 different laser doses based on previously established effective doses.

Treated and untreated control suspensions were diluted and plated in triplicate with or without 30 μg / ml methicillin on agar containing trypsinized soy.
Colonies were counted after growth for 24 hours at 37 ° C.

Experiment 3-Results:
Comparison of CFU on plates with and without methicillin showed a significant increase in the efficacy of methicillin in all laser treated samples. This data is summarized in the table below.

Independent report on MRSA, November 10-11, 2006
(MRSA Data Progression 11-10-06 Experiment # 4)
Experiment 4-Design:
Two different laser doses based on the effective dose established in previous experiments were used to treat logarithmically growing MRSA salt solution suspensions labeled A4-F4.

  Dilute treated suspension and control untreated suspension to 30 μg / ml methicillin (groups A4 and B4), 0.5 μg / ml penicillin G (groups C4 and D4) or 4 μg / ml erythromycin Plated in triplicate on agar containing trypsinized soybeans with or without (Groups E4 and F4).

  Colonies were counted after growth for 24 hours at 37 ° C.

Experiment 4-Results:
Laser treatment increases the sensitivity of MRSA to each antibiotic tested several fold. This data is summarized below.

Example XXIII
In vivo safety studies-After studying fibroblasts in human patients in vitro, the inventor performed a dosimetric titration on himself to determine a safe maximum energy level and exposure time. Confirmed (it does not cause burns to human skin tissue or damage to irradiated tissue).

  The methodology used by the inventor was to illuminate his big toe with varying time and NIMELS laser power settings. The results of this self-irradiation experiment are described below.

Time / temperature assessments were charted to ensure the thermal safety of these laser energies for human skin tissue (data not shown). In one laser procedure, he exposed his toe thumb to both 870 nm and 930 nm for up to 233 seconds while simultaneously measuring the toe surface area with a laser infrared thermometer. He found that using the above dosimetry at a surface temperature of 37.5 ° C. at 870 nm and 930 nm with a combined power density of 1.70 W / cm ² , he found pain and turned off the laser.

In the second laser procedure, he exposed his toe's thumb to 930 nm for up to 142 seconds, while again measuring the toe's surface temperature with a laser infrared thermometer. He found that at 930 nm alone at a surface temperature of 36 ° C. and a power density of 1.70 W / cm ² , he found pain and turned off the laser.

Example XXIV
In Vivo Safety Study-Limited Clinical Preliminary Study After the above experiments, additional patients with onychomycosis of the foot were treated. All of these patients were free, volunteers and signed informed consent. The essential goal of this limited preliminary study is to achieve in vivo the same level of fungal decontamination as obtained in vitro using the NIMELS laser apparatus. We have also determined the maximum exposure time and temperature limits allowed by the inventors in self-exposure experiments. In a highly controlled and monitored environment, 3-5 laser exposure procedures were performed for each patient. Four patients were recruited and received treatment. Patients who signed informed consent were not rewarded and were informed that they could be stopped at any time, even during the procedure.

  The dosimetry used for treatment of the first patient was the same as that used during the inventor's own exposure (above). The surface temperature of the nail was also comparable to the temperature found by the inventors in their exposure.

  Treated toes showed significantly reduced tinea pedis and peri-nail bed scaly, which indicated removal of nail plate decontamination that had acted as a fungal reservoir. Control nails were shaved with a tissue bar across and the shavings were stored for plating on mycological media. The treated nails were shaved and plated in the exact same manner.

To cultivate nail shavings, Sabourodetran agar (2% dextrose) medium was prepared with the following additives: chloramphenicol (0.04 mg / ml) (for general fungal testing) Chloramphenicol (0.04 mg / ml) and cycloheximide (0.4 g / ml) (selective for dermatophytes); chloramphenicol (0.04 mg / ml) and griseofulvin (20 μg / ml) (fungi Serve as a negative control for growth).
The bacteriological results on Day 9 of Treatment # 1 and Treatment # 2 (3 days after Treatment # 1) were the same (the control toenails had dermatophyte growth and the treated foot It was not on the fingernails). Treated nail plate did not show any growth, while untreated control culture plates showed significant growth. The first patient was followed for 120 days and received 4 treatments under the same protocol. FIG. 18 shows a comparison of toenails before treatment (A), 60 days after treatment (B), 80 days after treatment (C), and 120 days after treatment (D). In particular, the healthy and uninfected nail plate covered 50% of the nail area and grew from a healthy epidermis after 120 days.

While certain embodiments have been described herein, those skilled in the art will appreciate that the methods, systems, and apparatus of the present invention may be embodied in other specific forms without departing from the spirit thereof. Will. Therefore, the specific examples of the present application should be considered as explanations of the present invention in all points, and do not limit the present invention. It is understood that the human nail acts as an intractable lens and disperses and / or reflects a portion of NIMELS infrared energy. Therefore, dose / tolerance studies on pig skin were conducted to titrate the maximum NIMELS dosimetry without burns / damage. Pig skin was used as a model for human skin. These studies were conducted according to the Animal Protection Act by the NIH Guide for the Care and Use of Laboratory Animals. These tests are shown below.
Study of pig skin dose / tolerance

Example XXV
Non-thermal NIMELS interaction Evidence for non-thermal NIMELS interaction:
Experiments (in vitro water bath studies) showed that the temperature achieved in in vitro NIMELS experiments is not high enough to neutralize pathogens.

  In the chart below, if E. coli alone was challenged at 47.5 ° C continuously for 8 minutes in a test tube in a water bath, they achieved 91% colony growth. Understand clearly. Therefore, it has been shown essentially that the NIMELS reaction is of entirely photochemical nature and occurs in the absence of exogenous drugs and / or dyes.

Claims (75)

An effective amount of a suitable drug; and a NIMELS light generating laser configured and arranged to generate the output of NIMELS irradiation λn at NIMELS dosimetry Tn to irradiate the target site;
A therapeutic system comprising:
The output can change the membrane dipole potential ψd of the irradiated cell membrane, thereby producing a NIMELS effect, which enhances the medicinal efficacy of the drug.   The therapeutic system of claim 1, wherein the system is configured and arranged to avoid undesirable damage to a biological portion of the target site.   The therapeutic system of claim 1, wherein the NIMELS light generating laser is configured and arranged to generate an output of the NIMELS radiation at a wavelength of 870 nm or 930 nm or both.   The therapeutic system according to claim 1, wherein the NIMELS light-generating laser is configured and arranged to irradiate a target site for a time (Tn) of 50 to 1200 seconds. The therapeutic system according to claim 1, wherein the NIMELS dosimetry provides an energy density of 100 to 2000 J / cm ² at the target site.   The therapeutic system of claim 1, further comprising a light dispersal tip configured and arranged to scatter the output at the target site.   The therapeutic system of claim 1, further comprising a light delivery subsystem configured and arranged to generate an output having a geometrically flat tip intensity distribution.   The therapeutic system of claim 7, wherein the light delivery subsystem comprises a flat tip lens.   The therapeutic system of claim 1, further comprising a light delivery system comprising an optical fiber.   The energy output can be adjusted based on the calculation of total energy at the target site or the power or time of the output to change the steady state transmembrane potential ΔΨ-steady to the transient state transmembrane potential ΔΨ-trans. The therapeutic system of claim 1, further comprising a dosimetry controller configured and arranged to create a Nimels effect.   The therapeutic system of claim 1, wherein the drug is selected from the group consisting of an antibacterial agent, an antifungal agent, an antineoplastic agent, and combinations thereof.   The antibacterial agent is β-lactam antibiotic, glycopeptide, cyclic polypeptide, macrolide antibiotic, ketolide antibiotic, anilinouracil, lincosamide, chloramphenicol, tetracycline, aminoglycoside, bacitracin, cefazolin, Cephalosporin, mupirocin, nitroimidazole, quinolone and fluoroquinolone, novobiocin, polymyxin, cationic detergent antibiotic, oxazolidineone or other heterocyclic organic compounds, glycylcycline, lipopeptide, cyclic lipopeptide, pleuromutilin, And gramicidin, daptomycin, linezolid, ansamycin, carbacephem, carbapenem, monobactam, platencimycin, streptogramin, tinidazole, and these It is selected from the group consisting of combinations including any salts, therapeutic system of claim 11.   The antifungal agent is selected from the group consisting of polyenes, azoles, imidazoles, triazoles, allylamines, echinocandin, ciclopirox, flucytosine, griseofulvin, amorolophine, sodarin, and any combination thereof. 11. The therapeutic system according to 11.   The therapeutic system of claim 11, wherein the anti-neoplastic agent is selected from the group consisting of actinomycin, anthracycline, bleomycin, plicamycin, mitomycin, taxane, etoposide, teniposide, and combinations thereof. A therapeutically effective amount of the drug;
NIMELS light arranged and configured to generate NIMELS irradiation λn with NIMELS dosimetry Tn to irradiate the target site to selectively irradiate CH covalent bonds in long chain fatty acids of lipid bilayers A generating laser, wherein the targeted irradiation of the covalent bond is effective to alter the membrane dipole potential Ψd of the irradiated cell membrane, and the combination of λn and Tn is: (i) irradiation of cytochrome chains, And (ii) a light-generating laser suitable for changing the bioenergy state of the membrane from one thermodynamic steady-state condition to one transient energy stress and / or one of redox stresses; and the NIMELS A light delivery system suitable for delivering illumination λn to a target site with NIMELS dosimetry Tn, wherein the light delivery system converts the NIMELS illumination λn Light delivery system is configured by being arranged to deliver the desired host cells to the target site in NIMELS dosimetry Tn without damaging;
Including therapeutic system.   16. The therapeutic system of claim 15, wherein the NIMELS light generating laser is configured and arranged to irradiate a target site for a time (Tn) of 50 to 1200 seconds. The therapeutic system of claim 15, wherein the NIMELS dosimetry provides an energy density of 100-2000 J / cm ^{2 at} the target site.   16. The therapeutic system of claim 15, wherein the drug is selected from the group consisting of antibacterial agents, antifungal agents, anti-neoplastic agents, and combinations thereof.   19. The therapeutic system of claim 18, wherein the anti-neoplastic agent is selected from the group consisting of actinomycin, anthracycline, bleomycin, pricamycin, mitomycin, and combinations thereof.   The antibacterial agent is β-lactam antibiotic, glycopeptide, cyclic polypeptide, macrolide antibiotic, ketolide antibiotic, anilinouracil, lincosamide, chloramphenicol, tetracycline, aminoglycoside, bacitracin, cefazolin, Cephalosporin, mupirocin, nitroimidazole, quinolone and fluoroquinolone, novobiocin, polymyxin, cationic detergent antibiotic, oxazolidineone or other heterocyclic organic compounds, glycylcycline, lipopeptide, cyclic lipopeptide, pleuromutilin, And gramicidin, daptomycin, linezolid, ansamycin, carbacephem, carbapenem, monobactam, platencimycin, streptogramin, tinidazole, and these Including meaning of salt is selected from the group consisting of combinations, the therapeutic system according to claim 18.   The antifungal agent is selected from the group consisting of polyenes, azoles, imidazoles, triazoles, allylamines, echinocandin, ciclopirox, flucytosine, griseofulvin, amorolophine, sodarin, and any combination thereof. 18. The therapeutic system according to 18.   The system is arranged and arranged to change the proton activation force Δp, so that cells or biological contaminants in the biological part generate the appropriate ATP, or the biological part Or prevented from taking essential nutrients required for proper growth and reproduction, including the synthesis of proteins, RNA, DNA, peptidoglycan, lipoteichoic acid, and lipids in biological contaminants. The therapeutic system as described.   The system seems to enhance the drug by reducing the ATP available by the altered Gibbs free energy value, ΔGp, so as to impair the energy-dependent efflux of the drug conjugated with the proton activation force Δp. 24. The therapeutic system of claim 22, wherein the therapeutic system is configured and arranged.   16. The therapeutic system of claim 15, wherein the therapeutic system is configured to generate a NIMELS effect, Ne having a value of 2-10, wherein Ne is a microorganism in any tissue, portion, or solution. The therapeutic system representing the level of antibacterial enhancement against pathogens.   The therapeutic system of claim 15, wherein the light delivery system comprises a flat tip lens, an optical fiber, or a dispersing tip. To inhibit the cell anabolic pathways weaken cellular resistance mechanisms to antimicrobial molecules, a method of reducing DerutamyuH ⁺ or Derutamyux ⁺ of target sites in the cell:
irradiating the target site with a combination of λn and Tn;
Simultaneously reducing Δp-mito-mam and / or Δp-plus-Bact of the target site; and simultaneously or sequentially administering one antimicrobial agent to the target site, The method provides for inhibition of at least one cell anabolic pathway.   27. The method of claim 26, wherein the targeted anabolic pathway is peptidoglycan biosynthesis, which is simultaneously targeted by the antimicrobial agent binding to the active site of a bacterial transpeptidase enzyme.   The targeted bacterial anabolic pathway is peptidoglycan biosynthesis, which is simultaneously targeted by the antimicrobial agent binding to the acyl-D-alanyl-D-alanine group in the cell wall intermediate, thereby Incorporation of N-acetylmuramic acid (NAM)-and N-acetylglucosamine (NAG) -peptide subunits into peptidoglycan matrix, thereby preventing proper formation of peptidoglycan in gram positive bacteria. 26. The method according to 26. Bacterial anabolic pathway to the target is a peptidoglycan biosynthesis, it is targeted at the same time by the antimicrobial agent, the antimicrobial agent is C ₅₅ - combined with isoprenyl pyrophosphate, thereby pyrophosphatase There C ₅₅ - to prevent interacting with isoprenyl pyrophosphate, whereby, C ₅₅ can be utilized to carry building blocks peptidoglycan outside of the inner membrane - reducing the amount of isoprenyl pyrophosphate, 27. The method of claim 26.   The targeted anabolic pathway is the biosynthesis of bacterial proteins, which bind to 23S rRNA molecules within the 50S subunit of the bacterial ribosome, thereby accumulating peptidyl-tRNA in the cell, thereby Claims simultaneously targeted by said antibacterial agent that depletes free tRNA required for α-amino acid activation and inhibits transpeptideation by causing premature dissociation of peptidyl tRNA from the ribosome. 26. The method according to 26.   31. The method of claim 30, wherein the antimicrobial agent binds simultaneously to two domains of the 23S RNA of 50S bacterial ribosomal subunit, thereby inhibiting the formation of bacterial ribosomal subunits 50S and 30S.   The antibacterial agent is chlorinated to increase its lipophilicity and binds to the 23S portion of the 50S subunit of the bacterial ribosome, resulting in a peptidyl-tRNA active site (A site) to a peptidyl site (P site). 32. The method of claim 30, wherein the transfer to) is prevented, thereby inhibiting the transpeptidase reaction.   The targeted anabolic pathway is bacterial protein biosynthesis, which binds to the 30S bacterial ribosomal subunit, thereby blocking the binding of aminoacyl-tRNA to the acceptor site (A site) of the ribosome; 32. The method of claim 30, wherein the method is simultaneously targeted by the antimicrobial agent thereby inhibiting codon-anticodon interactions and the elongation phase of protein synthesis.   34. The method of claim 33, wherein the antimicrobial agent binds more strongly to the bacterial ribosome in a different orientation than a typical subclass polyketide antimicrobial agent having an octahydrotetracene-2-carboxamide backbone.   Said targeted anabolic pathway is the biosynthesis of bacterial proteins, which bind to a specific aminoacyl-tRNA synthetase and thereby to one of its compatible tRNAs of a specific amino acid or its precursor. 27. The method of claim 26, wherein the method is simultaneously targeted by the antibacterial agent that prevents esterification of and thus prevents the formation of aminoacyl-tRNA.   The targeted anabolic pathway is bacterial protein biosynthesis, which binds to 50S rRNA via domain S of 23S rRNA and interacts with 16S rRNA of the 30S ribosomal subunit to initiate protein synthesis. 27. Simultaneously targeted by the antibacterial agent that inhibits bacterial protein synthesis prior to initiation by preventing binding of formylmethionine (f-Met-tRNA) to the 30S ribosomal subunit. the method of.   The targeted anabolic pathway is the biosynthesis of bacterial proteins, which interact with the 50S subunit of the bacterial ribosome at protein L3 in the region of the 23S rRNAP site near the peptidyl transferase center, thereby 27. The method of claim 26, wherein the method is simultaneously targeted by the antibacterial agent that inhibits peptidyl transferase activity and peptidyl transfer, blocks P-site interaction, and prevents normal formation of active 50S ribosomal subunits. .   27. The targeted anabolic pathway is DNA replication and transcription, which is simultaneously targeted by the antimicrobial agent that inhibits topoisomerase II (DNA gyrase) and / or topoisomerase IV. The method described.   The targeted anabolic pathway is DNA replication and translation, which inhibits DNA polymerase IIIC (an enzyme required for chromosomal DNA replication in Gram-positive bacteria but not present in Gram-negative bacteria) 27. The method of claim 26, wherein the method is simultaneously targeted by the antimicrobial agent.   The targeted anabolic pathway is DNA replication and transcription, which are simultaneously targeted by the antibacterial agents that inhibit topoisomerase II (DNA gyrase) and / or topoisomerase IV and / or DNA polymerase IIIC. 27. The method of claim 26.   27. The method of claim 26, wherein the targeted anabolic pathway is bacterial phospholipid biosynthesis, which is simultaneously targeted by the antimicrobial agent acting on a phosphatidylethanolamine-rich cytoplasmic membrane.   The targeted anabolic pathway is the biosynthesis of bacterial fatty acids, which converts the biosynthesis of bacterial fatty acids into β-ketoacyl- (acyl-carrier protein (ACP)) synthase I / II (FabF / B) 27. The method of claim 26, simultaneously targeted by said antimicrobial agent that inhibits by selective targeting of (an enzyme essential for biosynthesis of type II fatty acids).   The targeted anabolic pathway is the maintenance of the bacterial plasma transmembrane potential ΔΨ-plasm-bact, where the antimicrobial agent disrupts bacterial cell membrane function by binding to the Gram positive cytoplasmic membrane, thereby 27. The method of claim 26, causing depolarization and loss of membrane potential, which leads to inhibition of protein, DNA and RNA synthesis.   The antimicrobial agent increases the permeability of the bacterial cell wall, thereby allowing inorganic cations to freely traverse the wall, thereby eliminating the ionic gradient between the cytoplasm and the extracellular environment, 27. The method of claim 26.   The targeted anabolic pathway is the selective permeability of the bacterial membrane and the maintenance of the bacterial transmembrane potential ΔΨ-plus-bact, and the antimicrobial agent is a cationic antimicrobial peptide; It is more selective for the negatively charged surface of the bacterial membrane than the neutral membrane surface of eukaryotic cells, so that membrane permeabilization of the bacterial cell membrane and final perforation and / or 27. The method of claim 26, wherein disruption occurs, thereby facilitating leakage of bacterial cell contents and disruption of the transmembrane potential.   27. The method of claim 26, wherein the antimicrobial agent inhibits bacterial protease peptide deformylase, and the deformylase catalyzes the removal of the formyl group from the N-terminus of a newly synthesized bacterial polypeptide.   The antibacterial agent inhibits a two-component regulatory system in bacteria that includes the ability to respond to their environment via signal transduction across the bacterial plasma membrane, these signal transduction processes comprising: 27. The method of claim 26, wherein the method is absent from the mammalian membrane.   Any protein or enzyme that the antibacterial agent binds to or is delivered by a second molecule, which is produced as a resistance mechanism by which the targeted bacterium weakens or inactivates the antibacterial agent 27. The method of claim 26, wherein the method acts as a competitive inhibitor and / or acts as an inhibitor of an efflux pump, thereby assisting in restoring the efficacy of the antimicrobial agent. To inhibit the anabolic pathways of the cell weaken the cell resistance mechanisms to antimicrobial molecules, a method of reducing DerutamyuH ⁺ or Derutamyux ⁺ in cells of the target site:
irradiating the target site with a combination of λn and Tn;
Simultaneously reducing Δp-mito-mam and / or Δp-plus-Bact of the target site; and simultaneously or sequentially administering a plurality of antimicrobial agents to the target site, wherein at least one at the target site A method of achieving inhibition of one cell anabolic pathway.   Any protein or enzyme that combines at least one of the antimicrobial agents with a second molecule, which is produced as a resistance mechanism for the targeted bacteria to weaken or inactivate one of the antimicrobial agents 50. The method of claim 49, wherein the method acts as a competitive inhibitor against and / or acts as an inhibitor of an efflux pump, thereby assisting in restoring the efficacy of the antimicrobial agent. To weaken the cell resistance mechanisms to inhibit anabolic pathways of the cells to the antifungal molecule, a method of reducing DerutamyuH ⁺ or Derutamyux ⁺ in cells of the target site:
irradiating the target site with a combination of λn and Tn;
Simultaneously reducing Δp-mito-mam, Δp-mito-Fungi, Δp-plus-Fungi of the target site; and simultaneously or sequentially administering one antifungal agent to the target site, The method provides for inhibition of at least one cellular anabolic pathway at a target site.   52. The targeted anabolic pathway is phospholipid biosynthesis, which is simultaneously targeted by the antifungal agent that disrupts the structure of existing phospholipids in fungal cell membranes. Method.   The targeted anabolic pathway is ergosterol biosynthesis, which is simultaneously targeted by the antifungal agent that inhibits ergosterol biosynthesis at the C-14 demethylation stage, the inhibition being 52. The method of claim 51, wherein depletion of ergosterol results in the accumulation of lanosterol and other 14 methylated sterols that interfere with the function of ergosterol as a membrane component by disrupting the structure of the plasma membrane.   The targeted anabolic pathway is the biosynthesis of ergosterol, which is simultaneously targeted by the antifungal agent that inhibits squalene epoxidase, which inhibition is then followed by ergosterol biosynthesis in fungal cells. 52. The method of claim 51, wherein the method inhibits synthesis, which causes the fungal cell membrane to have increased permeability.   52. The anabolic pathway targeted is ergosterol biosynthesis, which is simultaneously targeted by the antifungal agent that inhibits d14-reductase and d7, d8-isomerase. Method.   The targeted anabolic pathway is fungal cell wall biosynthesis, which is simultaneously targeted by the antifungal agent that inhibits (1,3) β-D-glucan synthesis, which inhibition is then 52. The method of claim 51, wherein the method inhibits β-D-glucan synthesis in fungal cell walls.   The targeted anabolic pathway is the biosynthesis of fungal sterols, which are simultaneously targeted by the antifungal agent binding to sterols in the fungal cell membrane, the main sterol being ergosterol, 52. The method of claim 51, effectively changing the transition temperature of the cell membrane to form pores in the membrane, resulting in the formation of ion channels that are detrimental to the fungal cell membrane.   58. The method of claim 57, wherein the antifungal agent is formulated in a lipid, liposome, lipid complex and / or colloidal dispersion for delivery to prevent toxicity of the agent.   The targeted anabolic pathway is protein synthesis, and the antifungal agent is taken up by fungal cells by cytosine permease, deaminated to 5-fluorouracil (5-FU), and nucleoside triphosphate 52. The method of claim 51, wherein the method is 5-FC that is converted to and incorporated into RNA, resulting in miscoding.   52. The method of claim 51, wherein the targeted anabolic pathway is fungal protein synthesis, which is simultaneously targeted by the antifungal agent that inhibits the fungal elongation factor EF-2.   The targeted anabolic pathway is biosynthesis of fungal chitin, which is caused by the antifungal agent that inhibits the biosynthesis of fungal chitin by inhibiting the action of at least one enzyme chitin synthase 2. 52. The method of claim 51, which is targeted simultaneously.   62. The method of claim 61, wherein the antifungal agent inhibits the action of the enzyme chitin synthase 3, an enzyme required for chitin synthesis during budding and growth, mating and sporulation. Said antifungal agent chelates a polyvalent cation (Fe ⁺³ or Al ⁺³ ) resulting in inhibition of metal-dependent enzymes responsible for mitochondrial electron transfer and cellular energy production, which also 52. The method of claim 51, wherein the method also leads to inhibition of normal degradation of the peroxide in the.   52. The method of claim 51, wherein the antifungal agent inhibits a two-component regulatory system in a fungus that responds to the environment by signal transduction across the fungal plasma membrane.   Combining the antifungal agent with a second molecule, the molecule is a competitive inhibitor against any protein or enzyme produced as a resistance mechanism for the targeted fungus to weaken or inactivate the antifungal agent. 52. The method of claim 51, wherein the method is in combination with a second molecule that acts as an inhibitor of the efflux pump and thus assists in restoring the efficacy of the antifungal agent. In order to inhibit cellular anabolic pathways and weaken cell resistance mechanisms against antifungal molecules,
A method of reducing DerutamyuH ⁺ or Derutamyux ⁺ in cells of the target site:
irradiating the target site with a combination of λn and Tn;
Simultaneously reducing intracellular Δp-mito-mam and / or Δp-mito-fungi and / or Δp-plus-fungi at the target site; and simultaneously or sequentially, adding a plurality of antifungal agents to the target site The method of achieving inhibition of at least one cellular anabolic pathway at a target site.   Any protein that combines at least one of the above antifungal agents with a second molecule, which is produced as a resistance mechanism for the targeted fungus to weaken or inactivate one of the antifungal agents 68. The method of claim 66, which acts as a competitive inhibitor for an enzyme and as an inhibitor of an efflux pump, thereby assisting in restoring the efficacy of the antifungal agent. In order to inhibit the cell's anabolic pathway and weaken the cell resistance mechanism against antineoplastic agents,
A method of reducing DerutamyuH ⁺ or Derutamyux ⁺ in cells of the target site:
irradiating the target site with a combination of λn and Tn;
Reducing the Δp-mito-mam and / or the mammalian transmembrane potential Δψ-plasm-mam; and simultaneously or sequentially, administering an anti-neoplastic agent to the target site, The method provides for inhibition of at least one cell anabolic pathway.   The targeted anabolic pathway is DNA replication, which cross-links guanine nucleobases in DNA to generate DNA strands that are necessary for DNA replication and cannot be separated by helix separation. 69. The method of claim 68, simultaneously targeted by the antineoplastic agent that inhibits replication.   Said targeted anabolic pathway is DNA replication, which is said anti-neoplasm that reacts with two different 7-N-guanine residues in the same strand of DNA or in different strands of DNA 69. The method of claim 68, wherein the method is simultaneously targeted by agents.   The targeted anabolic pathway is DNA replication, which is simultaneously targeted by the antineoplastic agent that inhibits DNA replication and cell division by acting as an antimetabolite. Item 69. The method according to Item 68.   69. The method of claim 68, wherein the targeted anabolic pathway is cell division, which is simultaneously targeted by the anti-neoplastic agent that inhibits cell division by interfering with microtubule function. .   The targeted anabolic pathway is DNA replication, which is simultaneously performed by the anti-neoplastic agent that inhibits DNA replication and cell division by preventing cells from entering the G1 phase and DNA replication. 69. The method of claim 68, targeted.   69. The targeted anabolic pathway is cell division, which is simultaneously targeted by the antineoplastic agent that enhances microtubule stability and prevents late chromosome segregation. The method described in 1.   The targeted anabolic pathway is DNA replication, which is simultaneously targeted by the anti-neoplastic agent that inhibits DNA replication and cell division by inhibiting type I or type II topoisomerase, and the topoisomerase 69. The method of claim 68, wherein inhibition of hinders DNA transcription and replication by disrupting proper DNA supercoiling.

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