US20180320148A1 - Acute Respiratory Distress Syndrome Therapeutic Agent - Google Patents

Acute Respiratory Distress Syndrome Therapeutic Agent Download PDF

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US20180320148A1
US20180320148A1 US15/771,874 US201615771874A US2018320148A1 US 20180320148 A1 US20180320148 A1 US 20180320148A1 US 201615771874 A US201615771874 A US 201615771874A US 2018320148 A1 US2018320148 A1 US 2018320148A1
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sod
agent
respiratory distress
distress syndrome
acute respiratory
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Toru Mizushima
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LTT Bio Pharma Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

Definitions

  • the present invention relates to an agent for treating acute respiratory distress syndrome, and specifically, to an agent for treating acute respiratory distress syndrome that contains lecithinized superoxide dismutase (hereinafter may be referred to as “PC-SOD”) as an active ingredient.
  • PC-SOD lecithinized superoxide dismutase
  • ARDS Acute respiratory distress syndrome
  • ARDS Acute respiratory distress syndrome
  • Kyuusei kokyuu sokuhaku shokogun or “Kyuusei kokyuuu kyuuhaku shokogun” in Japanese.
  • This syndrome is one of the main causes of death among patients during intensive care and is a disease of great significance for public health.
  • the annual number of ARDS patients has reached 200,000 in the United States.
  • a reliable method for treating this disease has not been established yet and currently the mortality rate of ARDS is fairly high (40 to 50%).
  • ARDS is a fatal clinical syndrome defined by edema, acute hypoxic respiratory failure with cardiac filling pressure, and bilateral pulmonary infiltration. ARDS is sometimes associated with sepsis and pneumonia characterized by an injury of an alveolar capillary wall, in which edema fluid containing abundant protein leaks into a pulmonary alveolus, leading to reduced surfactant activity and increased lung elastance.
  • the injury of an epithelium and an endothelium also induces serious inflammatory response (for example, recruitment and activation of a leukocyte and production of an inflammatory cytokine precursor), increases vascular permeability (edema), and activates a coagulation system in all tissues as well as in the lung, resulting in a multifunctional failure.
  • ARDS is a disease that suddenly happens to a patient severely-ill due to sepsis, massive blood transfusion, severe pneumonia, a chest injury, pulmonary embolism, mechanical ventilation, pure oxygen inhalation, acute pancreatitis, or the like. Although the early-stage pathophysiology of ARDS is various, the processes leading to the eventual onset is mostly same.
  • a tumor necrosis factor TNF
  • interleukin-1 IL-6
  • IL-8 interleukin-8
  • the lung is an organ through which blood circulating within a body always passes, the lung is susceptible to influence of the blood.
  • a neutrophil is attracted and releases reactive oxygen species and a protease in the lung tissue to injure, for example, an alveolar capillary epithelium and an alveolar epithelial tissue.
  • the neutrophil that settled in the lung further releases, for example, G-CSF and GM-CSF to amplify local inflammation. This leads to an increase in vascular permeability and bloody exudate fills the interstitium and even the inside of the pulmonary alveolus.
  • Capillaries of the pulmonary alveolus constrict under poor ventilation and act to increase blood flow in a well-ventilated area.
  • ventilation becomes poor in many areas of the lung in ARDS and therefore capillaries in these areas constrict to cause pulmonary hypertension.
  • MV mechanical ventilator
  • VILI ventilator-induced lung injury
  • MV with a low tidal volume is clinically recommended; however, a recent research states that even MV with a low tidal volume causes VILI and alveolar injury.
  • ROS Reactive oxygen species
  • the level of reactive oxygen species for example, a superoxide anion is reported to be elevated in blood plasma, exhaled breath condensate, and broncholveolar lavage fluid (BALF).
  • ROS reactive oxygen species
  • BALF broncholveolar lavage fluid
  • This elevation in the level of reactive oxygen species is also observed in a model animal such as a peritonitis model generated by a cecal ligation and puncture technique (CLP technique) (hereinafter may be referred to as “CLP”), an animal that received lipopolysaccharide (LPS) (hereinafter may be referred to as “LPS”), or an animal having an MV-induced tissue damage (mechanical ventilator-induced tissue damage) in the lung.
  • CLP technique cecal ligation and puncture technique
  • LPS lipopolysaccharide
  • MV-induced tissue damage mechanical ventilator-induced tissue damage
  • Sivelestat is a neutrophil elastase inhibitor and is a drug used for ARDS patients in Japan.
  • Neutrophil elastase is a protease produced by a neutrophil and the neutrophil elastase inhibitor prevents acute lung injury in an animal model.
  • the clinical effect of sivelestat is not necessarily satisfactory. For example, administration of sivelestat did not decrease the mortality rate of ARDS patients.
  • sivelestat is not directly effective in suppressing ROS production and therefore indicates that treatment of a tissue injury mediated by ROS in ARDS cannot be achieved clinically by sivelestat.
  • an antioxidative function is operative in a living body.
  • catalase which exists in a peroxisome in a cell and performs oxidation and detoxification by using hydrogen peroxide
  • SOD superoxide dismutase
  • glutathione peroxidase have an antioxidative function and this function counters an oxidizing system effectively.
  • SOD is an enzyme that eliminates a reactive oxygen anion, and so far an isozymic form of SOD such as Cu/Zn SOD, magnesium-SOD, or an extracellular (EC)-SOD has been found to exist. Reduction of the activity and production level of this kind of antioxidant enzyme is observed in the ARDS patient and the VILI patient, as well as the animal model.
  • antioxidant enzymes may be an effective therapeutic drug for ARDS and there have been many clinical and nonclinical reports based on this idea.
  • the lung function of the ARDS patient is improved in response to antioxidant therapy using N-acetylcysteine (NAC), and the occurrence of organ failure is reduced by taking an antioxidant supplement such as ⁇ -tocopherol or ascorbic acid.
  • NAC N-acetylcysteine
  • the treatment period of the ARDS patient in the ICU treatment room (central treatment room) is shortened.
  • efficacy of this kind of antioxidant enzyme there have been many reports on efficacy of this kind of antioxidant enzyme.
  • Cu/Zn SOD isozymic (iso) form of SOD
  • PC-SOD lecithinized SOD
  • Patent Literatures 1 and 2 The PC-SOD is a lecithinized SOD obtained by preparing a human Cu/Zn-superoxide dismutase (SOD) by gene recombination technology, and then chemically binding an average of four molecules of lecithin derivative (phosphatidylcholine derivative: PC) relative to one molecule of SOD (dimer).
  • SOD human Cu/Zn-superoxide dismutase
  • PC-SOD idiopathic pulmonary fibrosis
  • the present inventors have developed an inhalation (inhalant) of the PC-SOD and have confirmed, albeit confirmation at the animal level, that the inhalation was effective against bleomycin-induced pulmonary fibrosis, elastase-induced, and smoking-induced pneumonia, as well as pulmonary emphysema (chronic obstructive pulmonary disease: COPD) (Patent Literature 3).
  • COPD chronic obstructive pulmonary disease
  • the present inventors evaluated an effect of PC-SOD on ARDS and VILI. Consequently, the present inventors have confirmed that PC-SOD was effective against edema, tissue injury, and inflammation in the lung and other organs that neither the steroid nor sivelestat was effective against, and have found that PC-SOD could be a therapeutic drug for ARDS and VILI.
  • an object of the present invention to provide an agent for treating ARDS (acute respiratory distress syndrome), and in particular, an agent for treating acute respiratory distress syndrome containing lecithinized superoxide dismutase (PC-SOD) as an active ingredient.
  • ARDS acute respiratory distress syndrome
  • PC-SOD lecithinized superoxide dismutase
  • an aspect of the present invention is:
  • an agent for treating acute respiratory distress syndrome including, as an active ingredient, a lecithinized superoxide dismutase represented by the following general formula (I):
  • SOD′ represents a residue of a superoxide dismutase
  • Q represents a chemical crosslinking
  • B represents a residue of lysolecithin, in which a hydrogen atom of a hydroxyl group is removed from the lysolecithin having the hydroxyl group at the 2-position of glycerol
  • m is an average number of bonds of the lysolecithin relative to one molecule of the superoxide dismutase and represents an integer of 1 or more
  • the present invention includes the following aspects:
  • the present invention provides an agent for treating acute respiratory distress syndrome that contains PC-SOD as an active ingredient.
  • This agent is effective against edema, tissue injury, and inflammation in the lung and other organs that neither the steroid nor sivelestat was effective against, and thus brings new hope to treatment of ARDS and VILI.
  • FIG. 1 includes graphs showing the results of Test Example 1 (A and B: survival rate).
  • FIG. 2 includes graphs showing the results (A to D) of Test Example 2.
  • FIG. 3 includes graphs showing the results (A to C) of Test Example 3.
  • FIG. 4.1 includes diagrams showing the results (A, B) of Test Example 4.
  • FIG. 4.2 includes graphs showing the results (C to E) of Test Example 4.
  • FIG. 5.1 includes diagrams showing the results (A to D) of Test Example 5.
  • FIG. 5.2 is a graph showing the results (E) of Test Example 5.
  • FIG. 6 includes graphs showing the results (A to D) of Test Example 6.
  • FIG. 7.1 includes diagrams showing the results (A and B) of Test Example 7.
  • FIG. 7.2 includes diagrams showing the results (C and D) of Test Example 7.
  • FIG. 7.3 includes diagrams showing the results (E and F) of Test Example 7.
  • FIG. 8 includes diagrams showing the results (A to D) of Test Example 8.
  • FIG. 9 includes graphs showing the results (A and B) of Test Example 9.
  • FIG. 10 includes graphs showing the results (A and B) of Test Example 10.
  • lecithin refers to normal lecithin, which means phosphatidylcholine
  • lysolecithin refers to a compound in which one molecule of fatty acid bound at the 2-position of glycerol in lecithin is removed and a hydroxyl group is bound to the carbon atom at the 2-position.
  • the PC-SOD used in the present invention can be usually obtained by binding one or more lecithin derivatives, in which a chemical crosslinking agent is bound to the hydroxyl group at the 2-position of lysolecithin, to the SOD.
  • the PC-SOD can be represented by the following formula (I):
  • SOD′ represents a residue of the superoxide dismutase
  • Q represents a chemical crosslinking
  • B represents a residue of lysolecithin, in which a hydrogen atom of a hydroxyl group is removed from the lysolecithin having the hydroxyl group at the 2-position of glycerol
  • m is the average number of bonds of the lysolecithin
  • m is the average number of bonds of lysolecithin relative to one molecule of the superoxide dismutase and represents an integer of 1 or more
  • SOD′ used herein is not particularly limited as long as the SOD′ can exert an essential function of decomposing reactive oxygen species (O 2 ⁇ ) in a living organism.
  • SOD residues derived from various plants, animals, or microorganisms can be widely used.
  • antigenicity thereof in the living organism it is preferable that antigenicity thereof in the living organism be reduced as much as possible. Accordingly, it is preferable to suitably select an appropriate SOD residue as an SOD′ to be used, depending on a subject, to which the agent for treating acute respiratory distress syndrome of the present invention is administered.
  • the SOD′ is intended to be administered to an actual patient with acute respiratory distress syndrome as the subject. Therefore, in order to reduce antigenicity in the living organism due to the administration as much as possible, a human-derived SOD residue is preferably used. Accordingly, in view of antigenicity, the human-derived SOD may be used advantageously as the agent for treating acute respiratory distress syndrome of the present invention.
  • a human-derived Cu/Zn SOD (human-derived SOD containing copper and zinc in the active center; hereinafter may be abbreviated as human Cu/Zn SOD) is particularly preferably used as the human-derived SOD. This is because the human Cu/Zn SOD is expressed in a large amount in cells and the production technology therefor based on a genetic engineering method has been already established, whereby the human Cu/Zn SOD can be prepared in a large amount.
  • Examples of the human Cu/Zn SOD include: a natural human Cu/Zn SOD produced from human tissues or cultured cells; a human Cu/Zn SOD produced by the genetic engineering method; a recombinant human Cu/Zn SOD having substantially the same amino acid sequence as in the natural human Cu/Zn SOD; an SOD where some amino acids in the amino acid sequences of these human Cu/Zn SODs are deleted, added, substituted, or chemically modified or changed. Any human Cu/Zn SOD may be used.
  • a human Cu/Zn SOD in which an amino acid (cysteine: Cys) at the 111-position of the amino acid sequence of the natural human Cu/Zn SOD has been converted into S-(2-hydroxyethylthio)cysteine is preferable.
  • Such a human Cu/Zn SOD is described in detail in, for example, Japanese Patent Application Laid-Open No. Hei. 9-117279, and can be obtained by the method described therein.
  • R is a fatty acid residue (acyl group)
  • the fatty acid residue (acyl group) represented by R is preferably a saturated or unsaturated fatty acid residue of 10 to 28 carbon atoms, more preferably a myristoyl group, a palmitoyl group, a stearoyl group, an icosanoyl group, a docosanoyl group, or other saturated fatty acid residues of 14 to 22 carbon atoms, and particularly preferably a palmitoyl group, which is a saturated fatty acid residue of 16 carbon atoms.
  • the chemical crosslinking represented by Q in the general formula (I) is not particularly limited as long as an SOD and lecithin can be crosslinked to be chemically (covalently) bound with each other.
  • Such a chemical crosslinking is particularly preferably a residue:
  • This residue is a residue without hydroxyl groups at both the ends of a linear dicarboxylic acid represented by a formula: HO—C(O)—(CH 2 ) n —C(O)—OH, an anhydride, ester, or halide thereof, or the like (provided that in the case of the anhydride, ester, and halide, moieties corresponding to the hydroxyl groups at both the ends are removed).
  • n is an integer of 2 or more, and preferably an integer of 2 to 10.
  • m represents the average number of bonds of lysolecithin relative to one molecule of SOD. Accordingly, m is an integer of 1 or more, preferably 1 to 12, and particularly preferably 4.
  • a method for producing PC-SOD used in the present invention that is, a method for binding a lecithin derivative with an SOD, and preferably with a human Cu/Zn SOD can be performed, for example, by using the method described in Japanese Patent Application Laid-Open No. Hei. 9-117279.
  • PC-SOD The chemical structure of the preferable PC-SOD is schematically shown below and the following PC-SOD is particularly preferable.
  • the PC-SOD is obtained by covalently binding an average of four molecules of lecithin derivative to a free amino group of a human Cu/Zn SOD produced by genetic recombination using E. coli as a host cell.
  • the PC-SOD used in the agent for treating acute respiratory distress syndrome of the present invention be purified to such an extent that it is usable as a medicine and does not substantially contain substances that are not permitted to be mixed as a medicine.
  • a purified PC-SOD having a specific SOD activity of 2,500 U/mg or more, and more preferably a purified PC-SOD having a specific SOD activity of 3,000 U/mg or more may be used as the PC-SOD.
  • 1U (unit) represents an enzyme amount of PC-SOD that inhibits the NBT (nitro blue tetrazolium) reduction rate by 50% as measured using NBT under a condition of pH 7.8 and 30° C., in accordance with a method described in J. Biol. Chem., vol. 244, No. 22 6049-6055 (1969).
  • the agent for treating acute respiratory distress syndrome provided by the present invention is an agent for treating acute respiratory distress syndrome containing the PC-SOD thus prepared as an active ingredient, and may preferably be an agent for treating acute respiratory distress syndrome containing the PC-SOD and a stabilizing agent.
  • the stabilizing agent may include a sugar component.
  • the sugar component is not particularly limited as long as it can be used pharmaceutically; however sucrose is particularly preferable. Therefore, the most preferable agent for treating acute respiratory distress syndrome provided by the present invention is a composition containing PC-SOD and sucrose.
  • sucrose sucrose purified to such an extent that it may be used as a medicine is preferably used, and sucrose treated with activated carbon is particularly preferably used.
  • the agent for treating acute respiratory distress syndrome can be prepared as a composition, in which use of such sucrose with PC-SOD can prevent reduction in the activity of the PC-SOD due to long term storage, the stability is high, and the property is particularly favorable even if it is lyophilized.
  • a mixing ratio of the PC-SOD to sucrose in the agent for treating acute respiratory distress syndrome of the present invention can be suitably determined depending on an administration amount, a form of the formulation, or the like, and is not particularly limited.
  • a weight ratio of the PC-SOD to sucrose is preferably within a range of about 0.1:100 to 80:100, and more preferably about 0.4:100 to 60:100.
  • another medical active component and a commonly-used formulation component such as an excipient, a binder, a lubricant, a colorant, a disintegrator, a buffer, a tonicity adjusting agent, a preservative, and a soothing agent can be added as long as they do not affect the activity of PC-SOD and the effect of the formulation.
  • the agent for treating acute respiratory distress syndrome provided by the present invention can be prepared using PC-SOD and sucrose by a commonly-used method that is pharmaceutically known.
  • the PC-SOD used for a formulation composition of the present invention is preferably in a solution form, a frozen form, or a lyophilized form.
  • the agent for treating acute respiratory distress syndrome provided by the present invention may preferably be administered in the form of an injection.
  • the injection is preferably in the form of solution, suspension, emulsion, or a solid formulation that is dissolved before use. These formulations can be prepared in accordance with a method described in General Rules for Preparations of The Japanese Pharmacopoeia.
  • the agent for treating acute respiratory distress syndrome provided by the present invention may preferably be administered in the form of an inhalant.
  • Such an inhalant means a pharmaceutical composition for delivery to the trachea, bronchus, lung, and the like, and is suitably a composition suitable for a nasal drop or administration through the nose or lung, and particularly a composition suitable for administration through the lung.
  • the inhalant can be produced in the form of powder, solution, or suspension using the above-described PC-SOD as an active ingredient.
  • the above-described PC-SOD as an active ingredient may be pulverized as it is or with additives such as an excipient, a lubricant, a binder, a disintegrator, a stabilizing agent, or a corrective, to produce the inhalant.
  • PC-SOD When the inhalant is produced as a solution or suspension, for example, PC-SOD may be dissolved or suspended in water or a mixture of water and an auxiliary solvent, for example, an alcohol auxiliary solvent such as ethanol, propylene glycol, or polyethylene glycol, to produce the inhalant.
  • an auxiliary solvent for example, an alcohol auxiliary solvent such as ethanol, propylene glycol, or polyethylene glycol
  • Such a solution or suspension can additionally contain an antiseptic, a solubilizer, a buffer, a tonicity adjusting agent, an absorption promoter, a thickener, and the like.
  • the inhalant produced as described above is directly administered inside the nasal or mouth cavity or to the trachea, bronchus, lung, or the like by using common means in the field of inhalants, for example, a dropper, a pipette, a cannula, or a sprayer such as an atomizer or a nebulizer, the sprayer turning the inhalant into a nebulized form.
  • a dropper a pipette, a cannula
  • a sprayer such as an atomizer or a nebulizer
  • the inhalant can be administered by spraying it as an aerosol packaged in a pressurized container with an appropriate propellant (for example, gases of chlorofluorocarbons, such as dichlorofluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or the like), or by using a nebulizer.
  • an appropriate propellant for example, gases of chlorofluorocarbons, such as dichlorofluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or the like
  • the amount of PC-SOD that is an active ingredient in the agent for treating acute respiratory distress syndrome of the present invention and the administration amount of the formulation vary depending on a method for preparing the formulation, a dosage form, a target disease degree, or age or body weight of a patient and are not particularly limited.
  • an exemplary clinical amount can be 5 to 500 mg (15,000 to 1,500,000 U) per day per adult, and preferably, 40 to 200 mg (120,000 to 600,000 U) per day per adult.
  • the number of doses is not particularly limited, but the administration can be performed once or more daily.
  • LPS lipopolysaccharide
  • Diff-Quik was purchased from Sysmex Corporation (Kobe).
  • a DRI-CHEM slide (used for identification of BUN) was purchased from Fujifilm Corporation.
  • L-012 (luminescent probe), LabAssay creatinine, and Evans blue were purchased from Wako Pure Chemical Industries, Ltd.
  • Novo-Heparin (5000 units) was purchased from Mochida Pharmaceutical Co., Ltd.
  • Pentobarbital was purchased from Tokyo Chemical Industry Co., Ltd.
  • ICR mice (6 to 7-week old) were purchased from Charles River Laboratories, Inc.
  • a mouse was anesthetized with pentobarbital (10 mg/kg, intraperitoneally) and a small abdominal midline incision was made to expose the cecum thereof. Subsequently, the cecum was ligated at approximately 2 cm with a silk thread and punctured twice with an 18G needle (manufactured by Terumo Corporation). Then, the abdomen was closed.
  • Sham mice Sham operation group were subjected to the same operation except the ligation and puncture of the cecum.
  • the survival rate was checked every 12 hours, and CLP-induced multiple organ failure was observed 8 hours after the CLP treatment.
  • a mechanical ventilator for small animals was used to induce ventilator-induced lung injury (VILI).
  • VILI ventilator-induced lung injury
  • a mouse was anesthetized with pentobarbital (10 mg/kg, intraperitoneally), tracheotomy was performed and an 8 mm metal tube was inserted into the trachea thereof.
  • the mouse was mechanically ventilated with a tidal volume of 17.5 mL/kg, a positive end-expiratory pressure of 0 cm H 2 O, and a rate of 150 breaths/min.
  • Total respiratory elastance was measured every 30 minutes for 120 minutes by a snapshot technique.
  • a mouse was anesthetized with isoflurane and 1 mg/kg LPS (in 0.9% NaCl) was administered thereto intratracheally once using a micropipette (P200).
  • PC-SOD or dexamethasone 3 or 15 KU/kg (1 or 5 mg/kg) of PC-SOD (in 0.45% NaCl) or 20 or 200 ⁇ g per mouse of dexamethasone (in 0.9% NaCl) was administered intravenously using a 26G needle under isoflurane anesthesia.
  • sivelestat 10 or 100 mg/kg of sivelestat (in 0.9% NaCl) was administered intraperitoneally using a 21G needle.
  • the timing of the first drug administration after CLP, LPS, or MV procedure was immediately before the procedure for PC-SOD and dexamethasone, and 30 minutes before the procedure for sivelestat.
  • EBD Evans blue
  • the wet weight of a lung was measured to determine a wet-to-dry weight ratio of the lung. Then, the lung was dried at 60° C. overnight and reweighed to obtain the dry weight thereof.
  • the levels of BUN and creatinine in blood were measured by using a DRI-CHEM slide and LabAssay creatinine, respectively, according to the protocols.
  • the level of inflammatory cytokines in plasma was measured by an Elisa kit according to the protocol.
  • BALF was collected by inserting a cannula into a trachea and performing lavage twice with 1 mL of 50 U/mL heparin (in sterile 0.9% NaCl). Usually, about 1.8 mL of BALF was collected from each mouse. The amount of proteins in BALF was determined by a Bradford assay. The level of inflammatory cytokines was determined by the Elisa kit as described above.
  • ROS reactive oxygen species
  • L-012 in saline
  • SHOSHIN EM in vivo imaging system
  • Okazaki Japan
  • An imaging system including a chamber equipped with an electron multiplying CCD camera
  • L-012 in saline
  • the mouse was euthanized 2 minutes after injection of L-012, subjected to abdominal midline incision, and imaged (five-minute intervals).
  • the mouse was euthanized 5 minutes after injection of L-012, and the lung thereof was anatomized promptly and imaged (five-minute intervals).
  • a tissue specimen was fixed in 10% neutral buffered formalin for 24 hours and then embedded in paraffin before being cut into a section 4 ⁇ m thick.
  • the section was stained firstly with Mayer's hematoxylin and secondly with 1% alcoholic eosin solution (H&E staining).
  • the specimen was mounted with malinol (mounting medium) and scanned using a NanoZoomer-XR digital slide scanner (Hamamatsu Photonics K.K.).
  • mice Male ICR mice were subjected to a CLP technique and PC-SOD (kU/kg) or vehicle was administered thereto.
  • (A) in the diagram shows the results when administration was performed immediately before the CLP operation and 12, 24, and 48 hours after the CLP operation.
  • (B) in the diagram shows the results when administration was performed 1, 12, 24, and 48 hours after the CLP operation.
  • a CLP operation or a sham operation was performed on male ICR mice.
  • Drugs were administered once before the CLP operation or the sham operation.
  • PC-SOD kU/kg
  • dexamethasone ⁇ g/mouse
  • Sivelestat mg/kg
  • EBD vans blue dye
  • plasma samples were prepared 8 hours after the CLP operation and the levels of BUN and creatinine were measured.
  • (A) in the diagram shows the results of intravenous administration of PC-SOD and demonstrates that PC-SOD administration suppressed an increase in vascular permeability caused by CLP (kidney and liver).
  • (B) in the diagram shows the results of intravenous administration of PC-SOD and demonstrates that PC-SOD administration suppressed an increase of BUN (blood urea nitrogen) and creatinine (indicator of kidney function) caused by CLP.
  • BUN blood urea nitrogen
  • creatinine indicator of kidney function
  • a CLP operation or a sham operation was performed on male ICR mice.
  • Drugs were administered once before the CLP operation or the sham operation.
  • PC-SOD kU/kg
  • dexamethasone ⁇ g/mouse
  • Sivelestat mg/kg
  • EBD vans blue dye
  • (A) in the diagram shows the results of intravenous administration of PC-SOD and demonstrates that PC-SOD administration suppressed an increase of TNF- ⁇ and IL-6, which are inflammatory cytokines, caused by CLP.
  • (B) and (C) in the diagram show the results of administration of dexamethasone (Dex) and sivelestat (Siv) and demonstrate that administration thereof failed in suppressing an increase of TNF- ⁇ and IL-6.
  • LPS (1 mg/kg) or vehicle (control) was administered once into a trachea of a male ICR mouse.
  • PC-SOD lipopolysaccharide
  • LPS lipopolysaccharide
  • a section of the lung tissue was prepared 24 hours after the LPS administration and was subjected to a histopathological examination (H&E staining) (Test B).
  • the lesion area was determined by ImageJ software (Test C).
  • BALF was prepared 48 hours after the LPS administration and the levels of protein (Test D) and inflammatory cytokines (Test E) were determined.
  • PC-SOD administration suppressed an increase of protein in the BALF (bronchoalveolar lavage fluid) (this increase indicates lung injury and edema) caused by lipopolysaccharide (LPS).
  • results shown in (E) in the diagram demonstrated that PC-SOD suppressed an increase of TNF- ⁇ , IL-1 ⁇ , and IL-6 (inflammatory cytokines) in the BALF (bronchoalveolar lavage fluid) caused by lipopolysaccharide (LPS).
  • mice Male ICR mice were subjected to MV (mechanical ventilator) treatment or were not subjected to MV treatment (control).
  • MV mechanical ventilator
  • PC-SOD 15 kU/kg or vehicle was administered intravenously once immediately before the MV treatment.
  • EBD Evans blue dye
  • the wet-to-dry weight ratio of the lung was determined 2 hours after the MV treatment (Test B).
  • a section of the lung tissue was prepared 2 hours after the MV treatment and was subjected to a histopathological examination (H&E staining) (Test C).
  • the lesion area was determined by ImageJ software (Test D). Total respiratory elastance was measured every 30 minutes for 120 minutes (Test E).
  • mice Male ICR mice were subjected to MV treatment or were not subjected to MV treatment (control), as with the above-mentioned Test 5.
  • Dexamethasone (Dex) 200 ⁇ g/mouse or vehicle was administered intravenously once immediately before the MV treatment.
  • Sivelestat (Siv) 100 mg/kg or vehicle was administered intraperitoneally once immediately before the MV treatment.
  • the wet-to-dry weight ratio of the lung was determined 2 hours after the MV treatment (Tests A and C). Total respiratory elastance was measured every 30 minutes for 120 minutes (Tests B and D).
  • lung edema caused by MV was not suppressed by administration of a steroid (dexamethasone: Dex) or sivelestat (Siv).
  • CLP operation (Tests A and B), LPS administration (Tests C and D), and MV treatment (Tests E and F) were performed as described above.
  • PC-SOD (3 or 15 kU/kg) was administered intravenously once before the CLP operation, LPS administration, or MV treatment.
  • a luminescent probe (L-012) (75 mg/kg) was administered 4 hours after the CLP operation, 6 hours after the LPS administration, and 2 hours after the MV treatment.
  • An abdominal cavity (Test A) or a lung (Tests C and E) of the mouse was imaged using a Lumazone in vivo imaging system.
  • the sum intensity of reactive oxygen species (ROS) was determined by Slide Book6 software (Tests B, D, and F).
  • PC-SOD administration also suppressed an increase of reactive oxygen species in the lung caused by lipopolysaccharide (LPS) as well as an increase of reactive oxygen species in the lung caused by the mechanical ventilator (MV).
  • LPS lipopolysaccharide
  • MV mechanical ventilator
  • PC-SOD solution was heated at 100° C. for 60 minutes for inactivation.
  • a CLP operation or a sham operation was performed on a male ICR mouse.
  • the heat-inactivated PC-SOD (kU/kg; kU before heat-inactivation) was administered intravenously once before the CLP operation.
  • a plasma sample was prepared 8 hours after the CLP investigation and the levels of BUN and creatinine (Test A) and the level of inflammatory cytokines (Test B) were measured.
  • a CLP operation or a sham operation was performed on a male ICR mouse.
  • NAC (mg/kg) was administered intraperitoneally or unmodified SOD (kU/kg) was administered intravenously, once before the CLP operation.
  • a plasma sample was prepared 8 hours after the CLP operation and the levels of BUN and creatinine (Test A) and the level of inflammatory cytokines (Test B) were measured.
  • PC-SOD of the present invention is specific for CLP-induced renal dysfunction and systemic inflammation.
  • PC-SOD of the present invention is beneficial to acute respiratory distress syndrome (ARDS) patients.
  • MV mechanical ventilation
  • VILI ventilator-induced lung injury
  • ROS Reactive oxygen species
  • ARDS ARDS
  • VILI Reactive oxygen species
  • PC-SOD provided by the present invention is found to be very effective in mice against the cecal ligation and puncture technique (CLP) (a peritonitis model) or administration of lipopolysaccharide (LPS), or MV-induced tissue damage (mechanical ventilator-induced tissue damage), edema, or inflammation in the lung.
  • CLP cecal ligation and puncture technique
  • LPS lipopolysaccharide
  • MV-induced tissue damage mechanical ventilator-induced tissue damage
  • PC-SOD intravenous administration of PC-SOD increased the survival rate and reduced the vascular permeability in the mice treated by the CLP technique, and suppressed CLP-induced inflammation of the whole body as well as the kidney in contrast to dexamethasone or sivelestat.
  • LPS lipopolysaccharide
  • ROS reactive oxygen species
  • an agent for ameliorating acute respiratory distress syndrome contains a specific PC-SOD as an active ingredient, and has a higher affinity for, for example, cell membranes than a conventional SOD and a greater ability to eliminate superoxide anions in a lesion.
  • the present invention provides an agent for treating acute respiratory distress syndrome that contains PC-SOD as an active ingredient.
  • This agent is effective against edema, a tissue injury, and inflammation in a lung and other organs that neither a steroid nor sivelestat was effective against, and thus brings new hope to treatment of ARDS and VILI and has a great value in medical care.

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US20110262420A1 (en) * 2008-12-03 2011-10-27 Ltt Bio-Pharma Co., Ltd. Inhalant comprising modified superoxide dismutase
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