JP2009535410A - Pyrroloquinoline quinone and use thereof - Google Patents

Abstract

The present invention comprises a composition containing substantially purified pyrroloquinoline quinone, which is useful in a method for the treatment and prevention of heart damage caused by hypoxia or ischemia. The present invention also includes a method for the treatment and prevention of cardiac injury comprising contacting the composition of the present invention with a human patient. In certain embodiments, the present invention provides a method for preventing, treating or reducing ischemia-reperfusion injury, wherein a therapeutically effective amount of pyrroloquinoline quinone is provided to a subject in need thereof. And thereby protecting the subject's organs and tissues from reperfusion injury caused by ischemic injury.

Description

(Background of the Invention)
The heart is critically dependent on constant blood flow for oxygen and nutrient delivery and removal of harmful metabolites. Ischemia leads to rapid changes in myocardial metabolism and cell damage, the extent of which depends on the severity of the ischemia. Continuous ischemia leads to whole tissue necrosis in a few hours.

  Reperfusion is generally considered beneficial but causes tissue damage by several mechanisms. Clinically, in open heart surgery, heart transplantation, and reversal of heart disease, protection of the myocardium against ischemia-reperfusion injury is a major clinical concern. In addition, exacerbation of hypoxic injury after reperfusion restoration of oxygenation (reoxygenation) is associated with cell damage in other types of organ transplants and cells in liver, intestine, brain, kidney, and other ischemic syndromes. It is an important mechanism of damage. Cell hypoxia and reoxygenation cause ischemia-reperfusion injury in part by generating reactive oxygen species (ROS).

  Since the first isolation of free PQQ from bacteria in the late 1970s, further studies have shown that PQQ is an essential nutrient for vertebrates and probably belongs to the vitamin B group (1). Free PQQ has been confirmed in red blood cells, neutrophils, cerebrospinal fluid, synovial fluid, bile (Non-Patent Document 2), and human milk (Non-Patent Document 3). Trace amounts of free PQQ have also been detected in human spleen, pancreas, lung, brain, heart, intestine, liver and testis, plasma and urine, and rat small intestine, liver and testis (Non-Patent Document 4). . PQQ-dependent dehydrogenase enzymes are crucial for the amino acid lysine degradation pathway in mice. In this reaction, PQQ acts as a mammalian redox cofactor (Non-Patent Document 5). Since PQQ levels in human tissues and body fluids are 5 to 10 times lower than those found in food, PQQ in human tissues can be derived at least in part from dietary sources including vegetables and meat (Non-patent document 6). When mice are fed a diet lacking PQQ, the mice are slow growing, have fragile skin and a reduced immune response and do not reproduce well. Supplementation with PQQ can improve fertility, growth, and neonatal extracellular matrix production and maturation in chemically defined but otherwise fed a nutritionally complete diet. It has been shown that the index of can be adjusted (Non-patent Document 7). Excessive activation of the glutamate receptor N-methyl-D-aspartate (NMDA) subtype is critical in the neuronal damage process in hypoxia / ischemia, and NMDA antagonists are in vitro models of glutamate-mediated neurotoxicity And can improve neuronal damage in both in vivo models. Results from previous studies have demonstrated that PQQ has a protective effect against brain injury in a rodent model of cerebral hypoxia / ischemia, suggesting that PQQ may have potential use in treating seizures (Non-patent document 8). Although PQQ has been shown to be effective in animal models of focal cerebral ischemia and epilepsy, the protective mechanism is not well understood (Non-Patent Document 9).

Only one report investigated the potential cardioprotective effects of PQQ. This study showed that PQQ protects isolated rabbit hearts from reoxygenation damage as measured by LDH activity released into cardiac effluent (Non-Patent Document 10). However, based on this information, PQQ is an effective drug to reduce infarct size when given either prophylactically (pre-treatment) or during reperfusion after ischemia (treatment). It was not possible to decide whether or not there was.
Paz MA et al., The biomedical significance of PQQ. Davidson VL: Principles and Applications of Quinoproteins, 1992, by Marcel Dekker, Inc. P381-393. , Kasahara T, and Kato T. , Nature 2003; 422: 832. Gallop PM et al., Connect Tissue Res 1993; 29: 153-161. Mitchell AE et al., Analytical Biochemistry 1999; 269: 317-325 Kumagawa T et al., Biochim Biophys Acta 1992; 1156: 62-66 Kasahara, T .; And Kato, T .; , Nature 200; 422: 832. Kumazawa T et al., Biochem J 1995; 307: 331-333. Steinberg F et al., Exp Biol Med (Maywood) 2003; 228: 160-166, Steinberg FM et al., J Nutr 1994; 124: 744-753. Jensen FE et al., Neuroscience 1994; 62 (2): 399-406 Zhang Y and Rosenberg PA. European J Neuroscience 2002; 16: 1015-1024. , Jensen FE et al. Xu F et al., Biochemical Biophysical Research Communications 1993, 193: 434-439.

  Here we demonstrate for the first time that pre-treatment or treatment with PQQ can significantly reduce myocardial infarct size in an intact rat model of ischemia or ischemia-reperfusion injury.

(Summary of the Invention)
The present invention relates to the finding that myocardial oxidative stress can be prevented or minimized by the administration of certain cardioprotective factors, and thus has advantages in treating cardiovascular and other diseases. In particular, non-toxic doses of pyrroloquinoline quinone (“PQQ”) drugs are useful as cardioprotective agents, and thus, for example, due to coronary artery blockage, such as ischemia-reperfusion injury, congestive heart failure, cardiac arrest and It has been found to be beneficial in the treatment of a variety of various heart related diseases such as myocardial infarction as well as for cardioprotection. In particular, PQQ has been found to regulate myocardial oxidative stress so that cardiomyocytes (subject to oxidative stress) are protected from cell death.

  The compositions and methods of the present invention provide cardiovascular disease in mammals such as humans in need of reducing or eliminating hypoxia / ischemic heart damage in vivo and ex vivo, and prevention and / or treatment of cardiovascular disease. Surprisingly useful for the prevention and / or treatment of

In another aspect of the invention, PQQ modulates, eg, enhances or maintains, the effects of cardioprotective signaling pathways, such as the mitochondrial channel mitoK _ATP , the nitric oxide-protein kinase C pathway, and the angiotensin converting enzyme pathway. It has been found.

  In another aspect of the invention, the present invention reduces myocardial oxidative stress in a subject's cardiomyocytes by administering an agent that modulates myocardial oxidative stress such that the cardiomyocytes are protected from cell death. Related to treatment or prevention.

  In another aspect of the invention, the invention provides a method for administering myocardial hypoxia in a subject by administering an agent that modulates myocardial hypoxic or ischemic damage such that the cardiomyocytes are protected from cell death. Related to treating or preventing sexual or ischemic damage.

  In another aspect of the invention, PQQ has been found to modulate free radical damage caused by myocardial oxidative stress. Free radicals generated by ischemic or hypoxic conditions have been found to be a significant cause of myocardial damage leading to myocardial death. Thus, administration of PQQ administered at a non-toxic dose in vivo is an effective treatment for inhibiting or preventing free radical damage of myocardial oxidative stress.

  The present invention further relates to a method of improving coronary blood flow by administering a non-toxic amount of PQQ to a subject so that coronary blood flow is improved.

  In one embodiment, the present invention treats cardiac damage caused by hypoxia or ischemia in a subject by administering pyrroloquinoline quinone, for example, in an amount effective to treat or prevent cardiac damage. Relating to preventing. PQQ is generally administered at a non-toxic concentration, eg, less than about 1 nM to less than 10 μM, including less than 900 μM, less than 700 μM, less than 500 μM, less than 300 μM, less than 100 μM, or less than 50 μM. In other embodiments, PQQ may be administered at a concentration of about 1-10 μM. In other embodiments, PQQ is administered according to the weight of the subject. PQQ is generally less than 500 mg / kg, less than 250 mg / kg, less than 100 mg / kg, less than 10 mg / kg, less than 5 mg / kg, less than 3 mg / kg, less than 2 mg / kg, 1 mg / kg per kilogram of subject body weight. at a concentration of about 1 μg / kg to 1 g / kg, including less than kg, less than 500 μg / kg, less than 250 μg / kg, less than 100 μg / kg, less than 10 μg / kg, less than 5 μg / kg, less than 2 μg / kg or less than 1 μg / kg. Can be administered.

  The invention further includes a cardioprotective agent containing, for example, an amount of PQQ effective to provide cardioprotection, and a pharmaceutically acceptable carrier. A kit for treating a patient at risk of heart injury, stroke, or migraine, in one or more containers, an effective amount of pyrroloquinoline quinone, a pharmaceutically acceptable carrier, and Also included is a kit containing instructions for use.

  In another aspect, the present invention relates to the treatment or prevention of cardiac damage caused by hypoxia or ischemia in vivo by administration of a NADPH-dependent methemoglobin reductase substrate; and an effective amount of a NADPH-dependent methemoglobin reductase substrate, pharmacology The invention relates to a kit for use in the treatment or prevention of heart damage, including an acceptable carrier and instructions for use. In some embodiments of the invention, the NADPH-dependent methemoglobin reductase substrate is purified from erythrocytes, such as mammalian erythrocytes (eg, human, bovine, or mouse) or non-mammalian erythrocytes (eg, Rana catsbeiana). The

  In yet another aspect, the present invention provides a method for preventing organ damage during organ or tissue transplantation, wherein PQQ is administered to an organ donor prior to and / or concurrently with removal of the organ or tissue. And a kit used to prevent organ damage during organ or tissue transplantation comprising an effective amount of pyrroloquinoline quinone, a pharmaceutically acceptable carrier, and instructions for use.

  In a further aspect, the invention relates to a method for preventing seizures by administering an amount of PQQ effective to obtain a desired protective effect, eg, in a subject suffering from heart failure. PQQ may desirably be administered at a concentration of, for example, about 1-10 μM. In one embodiment, PQQ can be co-administered with a therapeutically effective amount of tamoxifen to prevent seizures in subjects at risk of suffering from seizures.

  The present invention includes a method for treating heart failure in a subject by administering PQQ and one or more additional therapeutic compounds. In some embodiments, the additional therapeutic compound can be an antiplatelet agent, an anticoagulant agent and / or an antithrombotic agent, or a combination thereof.

  In another aspect, the invention relates to a method of treating myocardial infarction in a subject by administering PQQ at a level such that myocardial infarction is reduced or stabilized.

  In yet another aspect, the invention relates to a method of preventing migraine in a subject by treating the subject with PQQ. PQQ can desirably be administered at a concentration of about 1 to about 10 μM, for example.

  In yet another aspect, the invention relates to a method of preventing reperfusion injury in a subject suffering from or at risk of hypothermia by treating the subject with PQQ. PQQ can desirably be administered at a concentration of about 1 to about 10 μM, for example.

  The invention further relates to a method for preventing vascular occlusion after balloon angioplasty in a subject by pre-treating the subject with PQQ. Subjects also have PQQ and one or more additional therapeutic compounds (such as coumadin, captopril, benazepril, enalapril, fosinopril, lisinopril, quinapril, ramipril, imidapril, peridopril erbumine and trandolapril. It may be pre-treated with angiotensin converting enzyme (ACE) inhibitors and ACE receptor blockers such as losartan, irbesartan, candesartan cilexetil and valsartan.In some embodiments, the additional therapeutic compound is: It can be an antiplatelet agent, an anticoagulant agent and / or an antithrombotic agent, or a combination thereof.

  In another aspect, the invention includes a method for preventing or reducing reperfusion injury in a subject suffering from hypothermic injury by administering PQQ to the subject.

  The present invention further relates to a pharmaceutical composition for treating myocardial infarction in a subject in need of treatment of myocardial infarction comprising a therapeutically effective dose of pyrroloquinoline quinone and a therapeutically effective dose of metoprolol. . In one embodiment of the pharmaceutical composition for treating myocardial infarction, the therapeutically effective dose of pyrroloquinoline quinone is 3 mg / kg. In another embodiment of the pharmaceutical composition for treating myocardial infarction, the therapeutically effective dose of metoprolol is 1 mg / kg.

  The present invention further relates to a kit for treating or preventing cardiac damage associated with hypoxia or ischemia, the kit comprising pyrroloquinoline quinine, metoprolol, a pharmaceutically acceptable carrier in one or more containers. And instructions for using this kit.

  The present invention further includes administering to a subject in need of treatment or prevention of myocardial oxidative stress a therapeutically effective dose of pyrroloquinoline quinone and a therapeutically effective dose of metoprolol. The present invention relates to a method for treating or preventing myocardial oxidative stress.

  The present invention further provides for treating myocardial infarction in a subject comprising administering to a subject in need of treatment or prevention of myocardial infarction a therapeutically effective dose of pyrroloquinoline quinone and a therapeutically effective dose of metoprolol. It relates to a method of treating or preventing.

  The present invention further comprises administering to a subject in need of treatment or prevention of cardiac damage caused by hypoxia or ischemia a therapeutically effective dose of pyrroloquinoline quinone and a therapeutically effective dose of metoprolol. To a method of treating or preventing cardiac damage caused by hypoxia or ischemia in a subject.

  The present invention further provides for the administration of a therapeutically effective dose of pyrroloquinoline quinone alone or in combination with urate to a subject in need of treatment for vascular injury or disease caused by protein nitration. Methods are provided for treating vascular injury or disease resulting from nitration.

  The present invention also provides a subject with a therapeutically effective dose of pyrroloquinoline quinone in combination with probeneso, cilastatin, or other ductal flow blockers for a subject in need of reduction of nephrotoxicity associated with PQQ administration. Also provided are methods of reducing nephrotoxicity associated with PQQ administration by administering.

  These and other objects of the invention will be apparent from the detailed description of the invention provided below.

(Detailed description of the invention)
The features and other details of the invention are described in more detail with reference to the accompanying drawings and set forth in the appended claims. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be used in various embodiments without departing from the scope of the invention. All parts and percentages are by weight unless otherwise specified.

(Definition)
For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, if these definitions change from the meaning prevalent in the field, the following definitions shall prevail.

  “Ischemia” includes a reduction or cessation of blood flow to any organ or tissue of the body. As used herein, the term “ischemia” includes, for example, vascular ischemia (eg, heart and lung), liver ischemia, intestinal ischemia, cerebral ischemia, kidney ischemia, and limb ischemia. It relates to any ischemic syndrome including.

  “Hypoxia” includes a deficiency in the amount of oxygen reaching the body tissue.

  “Hypoxia or ischemia-related injury” includes, but is not limited to, cardiac injury.

  “Reperfusion” includes restoration of blood flow to an organ or tissue whose blood supply has been interrupted, such as after a heart attack or stroke.

  “Oxidative stress” includes conditions that occur in the presence of excess free radicals, decreased levels of antioxidants, or both.

  “Necrosis” specifically includes the death of a cell or tissue due to damage or disease in a local area of the body, such as the heart muscle.

  “Apoptosis” refers to programmed cell death.

  “Beta blockers” include agents that act as competitive antagonists at adrenergic β receptors such as atenolol, metoprolol and propranolol. Such agents also include agents that are more selective with respect to cardiac (β-1) receptors that tolerate reduced systemic side effects. Beta blockers reduce symptoms associated with hypertension, cardiac arrhythmias, migraine, and other disorders associated with the sympathetic nervous system. Beta blockers are also sometimes given to stabilize the heartbeat after a heart attack. Within the sympathetic nervous system, β-adrenergic receptors are mainly located in the heart, lungs, kidneys, and blood vessels. Beta blockers compete with the neurostimulatory hormone epinephrine for these receptor sites, thus preventing the action of epinephrine, lowering blood pressure and heart rate, stopping arrhythmias, and preventing migraine.

  "Heart injury" ischemia-reperfusion injury; congestive heart failure; cardiac arrest; myocardial infarction; cardiotoxicity caused by compounds such as drugs (eg, doxorubicin, herceptin, thioridazine and cisapride); parasite infection (bacteria , Fungi, rickettsia, and viruses such as syphilis, chronic Trypanosoma cruzi infection); fulminant heart amyloidosis; heart surgery; heart transplantation; and traumatic heart injury (eg penetrating or closed heart injury, Any chronic or acute pathological event involving the heart and / or related tissues (eg, pericardium, aorta and other related blood vessels), including aortic valve rupture).

  A “subject” is a living organism such as a human, monkey, cow, sheep, horse, pig, cattle, goat, dog, cat, mouse, rat, cultured cells therefrom, and transgenic species thereof. Including the body. In preferred embodiments, the subject is a human. Administration of the compositions of the invention to the subject to be treated can be carried out using known procedures, at a dosage effective for treating the condition in the subject, and for an effective period of time. The effective amount of therapeutic compound necessary to achieve a therapeutic effect can vary according to factors such as the age, sex, and weight of the subject, and the ability of the therapeutic compound to treat foreign factors in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the urgency of the treatment condition.

  “Substantially pure” includes a compound, eg, a drug, protein or polypeptide, separated from the components that naturally accompany it. Typically, at least 10% of the total material in the sample (by volume, wet or dry weight, or mole percent or mole fraction), more preferably at least 20%, more preferably at least 50%, more preferably A compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% is the compound of interest. Purity can be measured by any appropriate method, for example, in the case of polypeptides, column chromatography, gel electrophoresis or HPLC analysis. A compound, such as a protein, is also substantially purified if it is substantially free of naturally associated components or has been separated from natural contaminants that naturally accompany it. . Included within the meaning of the term “substantially pure” is at least 95% of the total protein in the sample (by volume, wet or dry weight, or mole percent or mole fraction) of the protein of interest Or a polypeptide that is homogeneously pure, such as a protein or polypeptide.

  “Administering” includes a route of administration in which a composition of the invention may exert its intended function, eg, treatment or prevention of cardiac damage caused by hypoxia or ischemia. Depending on the disease or condition to be treated, parenteral administration (eg, intravenous, intraarterial, intramuscular, subcutaneous injection), oral administration (eg, diet), topical, nasal, rectal, or sustained release Various administration routes are possible, including but not necessarily limited to microcarriers. Oral administration, parenteral administration and intravenous administration are preferred dosage forms. The formulation of the compound to be administered will vary according to the route of administration selected (eg, solution, emulsion, gel, aerosol, capsule). Appropriate compositions containing the compounds to be administered can be prepared in physiologically acceptable vehicles or carriers and optionally adjuvants and preservatives. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, including saline and buffered media, sterile water, creams, ointments, lotions, oils, pastes and solid carriers. A suspension is included. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles may include various additives, preservatives or fluids, nutrients or electrolyte replenishers (see generally Remington's Pharmaceutical Sciences, 16th edition, Mack, Ed. (1980)). .

  An “effective amount” performs its intended function, eg, treating or preventing heart damage caused by hypoxia or ischemia, as described herein, partially or completely. Contains the amount of pyrroloquinoline quinone that enables it. The effective amount depends on many factors, including biological activity, age, weight, sex, general health, severity of the condition to be treated, and appropriate pharmacokinetic properties. For example, the dosage of active substance is about 0.0 1 mg / kg / day to about 500 mg / kg / day, advantageously 1 mg / kg / day to about 100 mg / kg / day. A therapeutically effective amount of the active agent can be administered by a suitable route in a single dose or multiple doses. Furthermore, the dosage of the active substance can be increased or decreased proportionally, as indicated by the urgency of the treatment or prevention situation.

  "Specific binding" or "specifically binds" is a protein such as an antibody that recognizes and binds pyrroloquinoline quinone or its ligand, but does not substantially recognize or bind other molecules in the sample. Is included.

  “Pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents that are compatible with the activity of the compound and are physiologically acceptable to the subject. , Isotonic and absorption delaying agents and the like. An example of a pharmaceutically acceptable carrier is buffered normal saline (0.15M NaCl). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the therapeutic compound, its use in a composition suitable for pharmaceutical administration is contemplated. Supplementary active compounds can also be incorporated into the compositions.

  “Pharmaceutically acceptable esters” include the relatively non-toxic ester-type products of the therapeutic compounds of the present invention. These esters are prepared in-situ during the final isolation and purification of the therapeutic compound or by reacting the purified therapeutic compound in its free acid form or hydroxyl separately with a suitable esterifying agent. Both are methods known to those skilled in the art. The acid can be converted to an ester according to methods well known to those skilled in the art, for example by treatment with an alcohol in the presence of a catalyst.

  “Additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrating agents; Lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; Demulsifiers; Buffers; Salts; Thickeners; Fillers; Emulsifiers; Antioxidants; Antibiotics; Antifungals; Stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences.

  A “unit dose” includes discrete amounts of a pharmaceutical composition containing a predetermined amount of the active ingredient.

  Pyrroloquinoline quinone (PQQ) is a water-soluble anionic quinone that can catalytically transfer electrons between various reducing and oxidizing agents and can be part of a soluble electron transport system in eukaryotic cells. . The original PQQ is of the following general structure:

本明細書で用いられるように、「ピロロキノリンキノン」すなわち「ＰＱＱ」は、ＰＱＱの密接に関連した異性体類似物および立体異性体類似物を含む、化学的類似性を有するピロロキノリンキノンファミリーの任意のメンバーを含み（例えば、Ｚｈａｎｇら，１９９５，Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．２１２：４１−４７，１９９５参照）、さらに任意のＰＱＱ連結ポリマー（例えば、ＰＱＱ連結ポリビニルポリマー）を含む。ＰＱＱは、メトキサチンとしても知られている。ＰＱＱは、動物の組織および体液中に見出される。理論により束縛されることを望まないが、ＰＱＱは、特に反応性酸素種（ＲＯＳ）のフリーラジカルスカベンジャーとして部分的に作用し得る。従って、ＰＱＱは、ＮＡＤＰＨ依存性メトヘモグロビンレダクターゼ基質として機能し得る（例えば、Ｘｕら，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，１９９２，８９（６）：２１３０−４参照）。他のＮＡＤＰＨ依存性メトヘモグロビンレダクターゼ基質は、低酸素または虚血に関連した心臓損傷を減少させるかまたは除去するように機能し得る。

As used herein, “pyrroloquinoline quinone” or “PQQ” refers to a family of pyrroloquinoline quinones having chemical similarities, including closely related isomers and stereoisomers of PQQ. Includes any member (see, eg, Zhang et al., 1995, Biochem. Biophys. Res. Commun. 212: 41-47, 1995), and further includes any PQQ linked polymer (eg, PQQ linked polyvinyl polymer). PQQ is also known as methoxatin. PQQ is found in animal tissues and fluids. While not wishing to be bound by theory, PQQ can act in part as a free radical scavenger, particularly for reactive oxygen species (ROS). Thus, PQQ can function as a NADPH-dependent methemoglobin reductase substrate (see, eg, Xu et al., Proc. Natl. Acad. Sci. USA, 1992, 89 (6): 2130-4). Other NADPH-dependent methemoglobin reductase substrates may function to reduce or eliminate cardiac damage associated with hypoxia or ischemia.

  A composition comprising a substantially purified pyrroloquinoline quinone comprises pyrroloquinoline quinone alone or other components such as beta blockers, and phenylephrine, sphingosine-1-phosphate or ganglioside GM-1. It may be included in combination with a compound that is effective to conveniently modulate the cardioprotective signaling pathway. The pyrroloquinoline quinone can be substantially purified by any of the methods well known to those skilled in the art (eg, EJ Corey and Alfon Tramontano, J. Am. Chem. Soc., 103, 5599-5600 (1981)). J. A. Duine, Review Ann. Rev. Biochem. 58, 403 (1989)).

In one embodiment, the present invention provides PQQ that is linked to one or more polymers, thereby improving the pharmacokinetics, pharmacodynamics, efficacy, and safety of PQQ for tissue protection. The polymer PQQ can be ligated, polyvinyl alcohol, either (by reference in its entirety incorporated herein) PEG-NH 2 or the following Examples 7 and reference documents _A, the polymers disclosed in the Including but not limited to.

  The pyrroloquinoline quinone of the present invention, in one embodiment, is a component of a pharmaceutical composition, which can also be accepted as a pharmaceutical composition, buffers, salts, other proteins, And other ingredients may be included. The present invention also includes modified forms of pyrroloquinoline quinone, which can prevent or reduce hypoxia / ischemic heart damage as described herein.

The structure of the therapeutic compound of the present invention may contain an asymmetric carbon atom. It is to be understood accordingly that the isomers arising from such asymmetry (eg, enantiomers and diastereomers) are included within the scope of the present invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by configurationally controlled synthesis. For the purposes of this application, unless expressly noted otherwise, therapeutic compounds shall be construed to include both R or S stereoisomers at each chiral center. In certain embodiments, the therapeutic compounds of the present invention include cations. If the cationic group is hydrogen H ⁺ , the therapeutic compound is considered an acid. If hydrogen is replaced by a metal ion or its equivalent, the therapeutic compound is an acid salt. Pharmaceutically acceptable salts of the therapeutic compounds are within the scope of the invention, such as pharmaceutically acceptable alkali metal (eg, Li ⁺ , Na ⁺ , or K ⁺ ) salts, ammonium cation salts, Alkaline earth cation salts (eg, Ca ²⁺ , Ba ²⁺ , Mg ²⁺ ), higher valent cation salts, or polycationic counterion salts (eg, polyammonium cations) (eg, Berge et al. (1977)). “Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19). It is understood that the stoichiometry of the anionic compound relative to the salt-forming counterion (if any) varies depending on the charge of the anionic portion of the compound (if any) and the charge of this counterion. Preferred pharmaceutically acceptable salts include sodium, potassium or calcium salts, although other salts are also contemplated within their pharmaceutically acceptable ranges.

  The invention also relates to a method of treating or preventing myocardial oxidative stress, such as caused by hypoxia or ischemia, in a subject. This may be achieved by providing the subject in need with an agent that modulates myocardial oxidative stress, preferably a non-toxic amount of PQQ, so that the cardiomyocytes that are the target of oxidative stress are protected from cell death. By administration. Cell death can be due to, for example, necrosis or apoptosis.

  Cardioprotective signaling pathways are known in the art. These pathways can be targeted for potentiation in patients in need of cardioprotection by administering an amount of pyrroloquinoline quinone effective to enhance or maintain the effects of the cardioprotective signaling pathway.

  Free radicals generated by ischemic or hypoxic conditions have been found to be a significant cause of myocardial damage leading to myocardial death. Thus, for example, administration of PQQ administered in vivo at a non-toxic dose inhibits free radical damage of myocardial oxidative stress, either by PQQ-mediated free radical scavenging, or by free radical production inhibition. It is an effective measure to prevent.

  Where clinically necessary or desirable, for example, administration of a compound of the invention can be made at the beginning of reperfusion or prior to reperfusion.

  It can also be seen that by administering a non-toxic amount of pyrroloquinoline quinone to a subject in need of improvement of coronary flow, coronary flow can be beneficially improved in a subject, for example, a subject suffering from a low blood flow condition. Surprisingly found. This is illustrated in the examples. Coronary flow can be measured by several indicators, such as left ventricular diastolic pressure (“LVDP”) or left ventricular end diastolic pressure (“LVEDP”). Measurement of coronary flow, such as by determining LVDP or LVEDP, is within the skill of the art.

  Thus, cardiac damage caused by hypoxia or ischemia, such as myocardial infarction, can be treated or prevented by administration of a preferably non-toxic dose, eg, pyrroloquinoline quinone at a concentration of less than about 10 μM.

Rats were subjected to PQQ treatment according to two different models. In model 1 (shown schematically in FIG. 10A), male Sprague-Dawley rats were subjected to 2 hours of left anterior descending (LAD) coronary artery ligation without reperfusion. In model 2 (ischemia-reperfusion, shown in FIG. 10B), the rats were subjected to 17 or 30 minutes of LAD occlusion and 2 hours of reperfusion while monitoring left ventricular (LV) hemodynamics. PQQ (15-20 mg / kg) was administered i.v. 30 minutes prior to LAD occlusion to mimic clinical conditions in humans. p. I. By injection (pre-treatment) or at the start of reperfusion. v. Given either by treatment (treatment). The control received vehicle (2% NaHCO ₃ ).

  In model 1, the infarct size (infarct volume / LV volume) after PQQ treatment was smaller than the control (19.1 ± 2.1% of the control, PQQ treatment was 10.0 ± 1.5 Resulting in infarct size; n = 9, P <0.01). In model 2, either PQQ pretreatment or treatment resulted in a reduction in infarct size (infarct volume / risk area) (PQQ pretreatment infarct size was 18.4 ± 2.3, treatment infarct size. 25.6 ± 3.5%, whereas the control is 38.1 ± 2.6%, P <0.01). PQQ prevented ischemia-induced cardiac dysfunction at higher LV generation pressure, LV (+) dP / dt and lower LV (−) dP / dt after 1-2.

  In summary, PQQ had cardioprotective effects in two separate intact rat infarction models consisting of ischemia or ischemia-reperfusion. PQQ reduced infarct size either when given prior to ischemia or ischemia-reperfusion, or when given at the beginning of reperfusion. Furthermore, PQQ had beneficial hemodynamic effects as evidenced by increased left ventricular (LV) pressure and LV (+) dP / dt in 1-2 hours of reperfusion. Pretreatment with PQQ reduced the mean episode of ventricular fibrillation (VF) per rat and the percentage of rats with VF, whereas treatment with PQQ reduced VF during ischemia and reperfusion. The percentage of rats with was reduced. The dose of PQQ was inversely related to infarct size. PQQ reduced the level of malondialdehyde (MDA), an indicator of lipid peroxidation in ischemic myocardium.

  Mortality during the ischemia-reperfusion period of the three groups in model 2 tended to decrease after PQQ (control: 28.6%, pretreatment: 12.9%, treatment: 21.7%) . However, these results did not reach statistical significance. It should be noted that the study has focused on measuring infarct size and hemodynamics and was not designed as a mortality test requiring a larger number of animals.

  PQQ is also effective when given at the beginning of reperfusion. Completed myocardial tissue level studies of malondialdehyde (MDA), a lipid peroxidation product that reacts with thiobarbituric acid. Ischemia / reperfusion increased MDA levels and PQQ prevented this increase. Values comparing PQQ after ischemia / reperfusion (I / R) with sham controls differed 3-fold. Similar effects were seen in distant “normal” myocardium.

  PQQ, given either as a pretreatment or as a treatment at the beginning of reperfusion, reduces myocardial infarct size and improves cardiac function in a dose-related manner in a rat model of ischemia and ischemia-reperfusion It is particularly effective. The results of malondialdehyde (MDA), which indicates that this index of lipid peroxidation was reduced by PQQ, suggests that PQQ acts as a free radical scavenger in ischemic myocardium.

  Without wishing to be bound by theory, one possible PQQ mechanism of action is that PQQ acts as a free radical scavenger. Recent studies have shown that PQQ functions as a free radical scavenger in addition to acting as a cofactor for quinone protein enzymes (Urakami T et al., J Nutr Sci Vitamol (Tokyo) 1997; 43: 19-33, He K et al., Biochemical Pharmacology 2003; 65: 67-74). PQQ can act as a neuroprotective agent by inhibiting peroxynitrate formation (Zhang Y and Rosenberg PA). PQQ was an effective antioxidant that protects mitochondria from oxidative stress-induced lipid peroxidation, protein carbonyl formation and inactivation of the mitochondrial respiratory chain (He K et al., Miyauchi K et al., Antioxido Redox Signal 1999; 1 : 547-554). Phagocytic cells such as monocytes and neutrophils produce superoxide in response to stimuli. Several inhibitors of PQQ redox cycling have proven to be blocking agents for superoxide release by both stimulated neutrophils and monocytes. This suggests that PQQ is involved in respiratory bursts of both macrophages and neutrophils (Bishhop A et al., Free Radic Bio Med 1995; 18: 617-620, Bishop A et al., Free Radic Bio Med 1994; 17: 311-320).

  Our results are consistent with the above study. We have found that pre-treatment with PQQ significantly reduces myocardial MDA levels in the infarct zone and in remote “normal” myocardium. Our data show that MDA was increased by I / R in this putative normal region, indicating the presence of LV dysfunction in remote myocardium during acute ischemia in humans and animals. Consistent with reports by other investigators shown (Yang Z et al., Circulation 2004; 109: 1161-1167, Kramer CM et al., Circulation 1996; 94: 660-666). Our observations are consistent with the hypothesis that PQQ reduces lipid peroxidation and inactivates both ischemic and non-ischemic myocardial superoxide. Thus, the protective effect of PQQ against ischemia-reperfusion injury can be attributed to its action as a free radical scavenger. In our study, PQQ given either as a pretreatment or treatment also reduced the incidence of ventricular fibrillation. Furthermore, PQQ can have a direct antiarrhythmic effect and can cause a decrease in VF due to its anti-ischemic properties.

  The data shown in Example 5 below suggests that PQQ reduces lipid peroxidation and scavenges superoxide. Further evidence that the protective effect of PQQ against I / R damage is due to its action as a free radical scavenger. The observation that MDA increased after I / R in the putative normal region is the observation reported by other investigators showing that myocardium away from the infarct zone exhibits suppressed function in both human and animal models. Match.

  The minimum dose of PQQ that was effective in reducing infarct size upon reperfusion was also determined as shown in Example 5. In addition, we determined the effect of this dose of PQQ on mitochondrial function. For these studies, previously described tissue preparation methods were used. The heart was visually divided longitudinally into an anterior portion containing the region perfused by the left anterior descending coronary artery and a non-infarcted posterior portion. Rats were or were not treated with PQQ (sham control) after 30 minutes of ischemia (I / R) just before the start of 2 hours of reperfusion.

  Treatment at the beginning of reperfusion with 1 mg / kg PQQ did not protect the heart or isolated intact mitochondria from I / R injury. However, treatment with 3 mg / kg PQQ alone was very effective in reducing infarct size by 49% and restoring mitochondrial respiration. In these experiments, hemodynamic results were not different from those described in previous reports.

  Our data also indicate that either prophylactic administration of PQQ or treatment during an active ischemic episode in high-risk patients is beneficial by reducing infarct size and ventricular arrhythmias. Our results also show that when these procedures are used as an early treatment of acute myocardial infarction, treatment with PQQ occurs during reperfusion, which occurs concurrently with chemical thrombolysis or balloon angioplasty / stent surgery. Is also effective. It is also promising that this study has no inhibitory effect on systemic hemodynamics. Further investigation in other models of ischemia-reperfusion injury where free radical production is the greatest cause of injury may be desired. Acute toxicity, particularly adverse side effects on renal function described in rats (Watanabe A et al., Hiroshima J Med Sci 1989; 38: 49-51), and the potential benefits of PQQ in humans, if any, are undecided It remains.

  Therefore, PQQ given either as a pretreatment before ischemia or as a treatment at the start of reperfusion after ischemia reduces myocardial infarct size and increases cardiac function in a dose-related manner in intact rats. It is very effective in improving. PQQ appears to act as a free radical scavenger in ischemic myocardium.

Metoprolol is a β ₁ selective (cardioselective) adrenergic receptor blocker. Metoprolol reduces cardiac oxygen demand, reduces heart rate, and reduces cardiac output at rest and exercise; among other things, reduces systolic blood pressure. This drug is available in the United States as tartrate (LOPRESSOR (R), Geigy Pharmaceuticals) as 50 mg and 100 mg tablets. Effective daily doses are from 100 mg to 450 mg, and LOPRESSOR® is usually administered as 100 mg given in two daily doses. Metoprolol is available in the US as 50 mg, 100 mg and 200 mg extended release tablets as succinate (TOPROL XL®, Astra Pharmaceutical Products, Inc.), which is administered once a day obtain)
PQQ can be co-administered with metoprolol. The results shown in Example 6 below show that the combined use of PQQ and metoprolol tended to reduce infarct size more than using PQQ or metoprolol alone. In one embodiment, metoprolol is administered with a dose of PQQ in a ratio of 1: 3. For example, a 3 mg / kg dose of PQQ is accompanied by a 1 mg / kg dose of metoprolol. In another embodiment, metoprolol is administered at a daily dose of about 50 mg to about 450 mg in combination with a daily dose of PQQ of about 50 mg to about 500 mg / day per day. Myocardial oxidative stress can be prevented or minimized by administration of a combination of PQQ and metoprolol, and therefore has advantages with respect to treating cardiovascular and other diseases. In particular, the combination of PQQ and metoprolol is effective as a cardioprotectant, and thus a variety of different hearts such as ischemia-reperfusion injury, congestive heart failure, cardiac arrest and myocardial infarction, such as due to coronary artery blockage. Useful in the treatment of related diseases and for cardioprotection. This combination is particularly useful for modulating myocardial oxidative stress to protect cardiomyocytes (which are subject to oxidative stress) from cell death.

  The present invention encompasses a method of treating or preventing heart damage caused by hypoxia or ischemia in a subject, wherein PQQ is such that damage associated with hypoxia or ischemia is prevented or reduced. Administered to a subject in need thereof. In certain embodiments, PQQ is administered at a concentration of less than about 10 μM. In other embodiments, PQQ is administered at a concentration ranging from about 10 nM to about 10 μM, from about 10 nM to about 1 μM, from 100 nM to about 10 μM, and from 100 nM to about 500 nM. In yet another embodiment of the invention, PQQ is administered at a concentration such that the concentration of PQQ at the heart tissue site is in the range of 10 nM to about 10 μM. PQQ can also be administered as a function of the subject's body weight. In some embodiments of the invention, the PQQ is less than 500 mg / kg, less than 250 mg / kg, less than 100 mg / kg, less than 10 mg / kg, less than 5 mg / kg, less than 3 mg / kg, less than 2 mg / kg, less than 1 mg / kg about 1 μg and 1 g per kg body weight of the subject, including less than kg, less than 500 μg / kg, less than 250 μg / kg, less than 100 μg / kg, less than 10 μg / kg, less than 5 μg / kg, less than 2 μg / kg or less than 1 μg / kg Administered at a concentration between. In a further embodiment of the invention, the PQQ is administered at a non-toxic concentration, the non-toxic concentration being a cell growth inhibitory but non-cytotoxic PQQ concentration, and one or more intended cell types (eg, Concentrations that are cytotoxic to cell types other than cardiomyocytes). Determination of cytotoxicity of known concentrations of PQQ for one or more cell types is within the ability of one skilled in the art. As a non-limiting example, toxicity to cultured adult mouse cardiomyocytes is observed at a concentration of 100 μM PQQ. In some embodiments, PQQ is administered with other compounds such as antiplatelet agents, anticoagulants and antithrombotic agents.

  Heart damage that can be treated or prevented by the methods and compositions of the present invention includes all heart damage caused by or affected by hypoxia and / or ischemia. Such injuries include ischemia-reperfusion injury, congestive heart failure, myocardial infarction, cardiotoxicity caused by compounds such as drugs (eg, doxorubicin), parasitic infections, fulminant cardiac amyloidosis, cardiac surgery, heart Includes, but is not limited to, transplantation and heart damage resulting from traumatic heart injury. All or part of the heart can be damaged, including tissues such as associated blood vessels and / or pericardium.

  The present invention also administers an NADPH-dependent methemoglobin reductase substrate to a subject in need of treating or preventing heart damage caused by hypoxia or ischemia, thereby preventing hypoxia or ischemia-related damage Or a method of treating or preventing cardiac damage caused by hypoxia or ischemia in a subject, such that it is reduced. In an embodiment of the invention, the NADPH-dependent methemoglobin reductase substrate is purified from red blood cells of mammals (eg, human, bovine or mouse) or red blood cells of non-mammalian (eg, bullfrog: Rana catsbeiana). Those skilled in the art know how to isolate and purify NADPH-dependent methemoglobin reductase substrates using minimal experimentation.

  The present invention relates to organ transplantation or tissue administration by administering pyrroloquinoline quinone to a donor prior to or simultaneously with removal of an organ or tissue so that damage caused by reperfusion of the organ or tissue is reduced or prevented. Further included is a method of preventing damage to an organ or tissue during transplantation. In a preferred embodiment, the organ or tissue to be transplanted is the heart or heart tissue. The PQQ can be contacted with the organ or tissue after surgical removal of the organ or tissue from the donor. In some embodiments, PQQ is added in addition to known organ preservation or tissue preservation solutions such as University of Wisconsin solution or Celsior solution (eg, Tabut et al., Am J Respir Crit Care Med, 2001, 164 (7): 1204-8; see Faenza et al., Transplantation, 2001, 72 (7): 1274-7).

  The present invention also provides a subject in need thereof with a therapeutically effective amount of PQQ alone or in combination with another biologically active agent so that the organ or tissue is protected during ischemia. To treat or reduce organ failure or tissue damage resulting from ischemic syndromes such as intestinal ischemia, liver ischemia, cerebral ischemia, renal ischemia, vascular ischemia, or limb ischemia A method is also provided. Organs and tissues that can be protected include, but are not limited to, kidney, lung, liver, heart, stomach, pancreas, appendix, brain, eye, genital organs, heart tissue, and skin tissue.

  The present invention also provides for the treatment of acute altitude sickness such as elevated pulmonary blood pressure or severe by administering a therapeutically effective amount of tetraiodothyroacetic acid (Tetrac) and / or PQQ to a subject in need thereof. Also provided are methods for treating or reducing symptoms such as disease or high altitude pulmonary edema. Organs and tissues that can be protected include, but are not limited to, kidney, lung, liver, heart, stomach, pancreas, appendix, brain, eye, genital organs, heart tissue, and skin tissue.

  The present invention provides a method of providing neuroprotection by treating a subject with pyrroloquinoline quinone and a pharmaceutically acceptable carrier, thereby preventing seizures in a subject (eg, human) suffering from heart failure. In addition. In some embodiments, pyrroloquinoline quinone is administered to the subject at a concentration of less than about 10 μM. PQQ can be administered before or simultaneously with a surgical procedure that can increase the likelihood of a seizure in the patient. In one embodiment, the procedure is balloon angioplasty. Other procedures include coronary artery bypass surgery and valve replacement surgery. PQQ can be administered before the antithrombotic agent (eg, coumadin), simultaneously with the antithrombotic agent, or after the antithrombotic agent. In yet another embodiment, neuroprotection can be achieved by administering PQQ before, simultaneously with, or after administration of a therapeutically effective dose of tamoxifen to a subject at risk of stroke. .

  The invention also encompasses a method of reducing or preventing headache in a subject (eg, a human) by treating the subject with pyrroloquinoline quinone and a pharmaceutically acceptable carrier. Such headaches include acute and chronic migraine and sinus headache.

  The invention further encompasses a method of preventing reperfusion injury in a subject suffering from hypothermia (eg, a human) by treating the subject with pyrroloquinoline quinone and a pharmaceutically acceptable carrier. The subject can be treated with PQQ prior to or simultaneously with a standard rewarming procedure for treating a person suffering from hypothermia, as is generally known in the art.

  As mentioned above, combination treatment of PQQ and metoprolol is part of the present invention. The combination therapies of the invention are administered by any suitable manner to obtain the desired treatment of myocardial infarction in the patient. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single infusion with a fixed PQQ to metoprolol ratio, or multiple single injections. The components of the combination therapy can be administered by the same route or by different routes, as described above. For example, PQQ is administered orally while metoprolol is administered intravenously; alternatively, all therapeutic agents can be administered by intravenous injection. The order in which therapeutic agents are administered is not considered critical.

  PQQ can also be co-administered with a nephroprotective agent to reduce or prevent nephrotoxicity. Suitable nephroprotective agents suitable for co-administration with PQQ include any compound that is an impedance blocker for ductal flow, ie, a compound that prevents the ductal flow of a compound that causes nephrotoxicity. Exemplary compounds include probenecid and cilastatin. Probenecid is currently marketed for use in the treatment of chronic gout and gouty arthritis. Probenecid is used to prevent seizures rather than to treat them once a gout-related seizure has occurred. Probenecid acts on the kidney (suppresses tubular secretion) and helps the body remove uric acid. Probenecid is also used to make those antibiotics more effective by preventing the body from passing certain antibiotics into the urine.

  Nephrotoxicity with high doses of PQQ alone has been observed in rats (see Example 5). PQQ can be co-administered with probenecid to reduce or prevent nephrotoxicity. The results of Example 8 below show that the combined use of PQQ and probenecid tended to reduce nephrotoxicity. In one embodiment, PQQ is administered at a ratio of 1: 4 to 1: 100 with the dose of probenecid. For example, a 25 mg / kg dose of PQQ is accompanied by a 100 mg / kg dose of probenecid, or a 1 mg / kg dose of PQQ is accompanied by a 100 mg / kg dose of probenecid, or a 2 mg / kg dose of PQQ is 100 mg / kg. Concomitant with a kg dose of probenecid, or a 3 mg / kg dose of PQQ is accompanied by a 100 mg / kg dose of probenecid. Nephrotoxicity can be prevented or minimized by administration of a combination of PQQ and probenecid. Thus, administration of PQQ in combination with probenecid allows treatment of various symptoms using PQQ (eg, cardioprotection) while preventing or minimizing nephrotoxicity.

  In another embodiment, the present invention provides a combination treatment of PQQ and cilastatin to prevent or reduce nephrotoxicity and / or renal failure. Cilastatin is a renal dehydropeptidase-I and leukotriene dipeptidase inhibitor inhibitor. Cilastatin is generally administered with the antibiotic imipenem to increase its effectiveness by preventing its degradation by the kidneys.

  Sequential or substantially simultaneous administration of each therapeutic agent is performed by any suitable route including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. obtain. The therapeutic agents may be administered by the same route or may be administered by different routes. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, while the other therapeutic agent of the combination can be administered orally. Alternatively, for example, all therapeutic agents may be administered orally, or all therapeutic agents may be administered by intravenous injection. The order in which the therapeutic agents are administered is not strictly critical.

  For the combination of PQQ and a renoprotectant, the renoprotectant can be administered before PQQ, simultaneously with PQQ, or after PQQ. In a preferred embodiment, the nephroprotective agent is administered prior to PQQ administration, so that the nephroprotective agent is present in the bloodstream and blocks any potential toxic effects of PQQ. In another embodiment, such as an acute case where sequential administration is not possible, the nephroprotective agent can be administered simultaneously with PQQ or after PQQ. One particularly preferred embodiment includes administering 200 mg / kg probenecid prior to PQQ administration and administering 100 mg / kg probenecid after PQQ administration.

  “Combination therapy” can also include administration of a therapeutic agent as described above in further combination with other biologically active ingredients and non-drug therapies. If the combination therapy further includes a non-drug treatment, the non-drug therapy can be performed at any suitable time as long as a beneficial effect is achieved by the cooperation of the combination of the therapeutic agent and the non-drug treatment. For example, where appropriate, beneficial effects are still achieved when non-drug treatment is temporarily removed from administration of a therapeutic agent, perhaps for days or weeks.

  Thus, the compounds of the present invention and other pharmacologically active agents can be administered to a patient simultaneously, sequentially or in combination. When administered sequentially, the interval between doses typically varies from 0.1 to about 48 hours. It will be appreciated that when using the combinations of the present invention, the compound of the present invention and the other pharmacologically active agent may be present in the same pharmaceutically acceptable carrier and thus can be administered simultaneously. They can be in separate pharmaceutical carriers such as conventional oral dosage forms taken at the same time. The term “combination” further refers to the case where multiple compounds are provided in separate dosage forms and are administered sequentially.

  The beneficial effects of the combination composition of the present invention include, but are not limited to, pharmacokinetic or pharmacodynamic cooperation resulting from the combination of therapeutic agents. In one embodiment, the therapeutic agent cooperation is additive. In another embodiment, the therapeutic agent cooperation is synergistic. In another embodiment, the cooperation of the therapeutic agents improves the therapeutic regimen of one or both of the agents.

  The present invention further relates to a kit for treating a patient suffering from myocardial infarction, which kit contains a therapeutically effective dose of at least one metoprolol and PQQ in the same or separate packaging and uses thereof Provide instructions. Metoprolol is administered at a dose of about 0.1 mg / kg to about 10 mg / kg. Metoprolol is administered at a ratio of about 2: 1 to about 1: 3 with PQQ. For example, if 1 mg / kg metoprolol is administered, one suitable dose to be co-administered is 1 mg / kg PQQ. Another suitable PQQ dose is 2 mg / kg. Another dose is 3 mg / kg PQQ.

  To assess whether a patient is benefiting from (treatment), the patient's symptoms are examined in a quantitative manner, by decreasing the frequency of relapses, or by increasing time to sustained progression. In successful treatment, the patient's condition improves, the number of relapses or the frequency of relapses decreases, or the time to sustained progression increases.

  For any drug, dosage is an important part of treatment success and patient health. In all cases, within the specified range, the physician must determine the best dose for a given patient according to gender, age, weight, height, pathological condition and other parameters.

  The pharmaceutical compositions of the invention comprise a therapeutically effective amount of the active agent. The amount of compound will depend on the patient being treated. Patient weight, severity of illness, mode of administration, and judgment of the prescribing physician should be considered in determining the appropriate amount. Determination of a therapeutically effective amount of PQQ or metoprolol is well within the skill of the artisan.

  In some cases, it may be necessary to use dosages outside the ranges stated in the package insert of the pharmaceutical package to treat the patient. Those cases are obvious to the prescribing physician. If it is necessary, the physician will also know how and when to interrupt, adjust or terminate the treatment in relation to a particular patient response.

  The present invention encompasses the preparation and use of pharmaceutical compositions comprising as an active ingredient a compound that helps prevent or reduce hypoxia / ischemic heart damage. Such pharmaceutical compositions may consist of the active ingredient alone in a form suitable for administration to a subject, or the pharmaceutical composition comprises the active ingredient and one or more pharmaceutically acceptable carriers, One or more additional ingredients or any combination thereof may be included. As is well known in the art, the active ingredient may be present in the pharmaceutical composition in the form of a pharmaceutically acceptable ester or salt, for example, with a physiologically acceptable cation or anion. In addition, pyrroloquinoline quinone is pharmaceutically acceptable as commonly used in pharmaceutical products, as long as it does not adversely affect the efficacy of the preparation, for example, in reducing or inhibiting ischemia or reperfusion injury. May include ingredients necessary to formulate additives (eg, carriers, vehicles and diluents), stabilizers or preparations.

  Examples of additives and stabilizers include sugars such as monosaccharides (eg glucose and fructose), disaccharides (eg sucrose, lactose and maltose) and sugar alcohols (eg mannitol and sorbitol); citric acid, malein Acids and organic acids such as tartaric acid and salts thereof (eg sodium, potassium and calcium salts)); amino acids such as glycine, aspartic acid and glutamic acid and salts thereof (eg sodium, calcium or potassium salts) Surfactants such as polyethylene glycol, polyoxyethylene-polyoxypropylene copolymers and polyoxyethylene sorbitan fatty acid esters; heparin; and albumin.

  Formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or later developed. In general, such preliminary methods involve combining the active ingredient with a carrier or one or more other auxiliary ingredients, and then molding or packaging the product, if necessary or desired, to produce the desired unit. Including single or multiple dose units.

  The description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for prescription administration to humans, but such compositions are generally suitable for administration to all types of animals. It will be understood by those skilled in the art. Modification of pharmaceutical compositions suitable for administration to humans to make the composition suitable for administration to various animals is well understood, and veterinary pharmacologists in the field, if any, Such modifications can be designed and implemented by mere routine experimentation. Subjects intended for administration of the pharmaceutical compositions of the present invention include, but are not limited to, humans and other primates.

  The pharmaceutical compositions useful in the methods of the present invention are suitable for buccal, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ocular, or another route of administration It can be prepared, packaged or sold in a formulation. The preferred form is intravenous administration.

  A pyrroloquinoline quinone and said component are mixed suitably, and a powder, a condyle, a tablet, a capsule, a syrup, an injection etc. are obtained. Other contemplated formulations include planned nanoparticles, liposomal formulations, resealed erythrocytes containing the active ingredient, and immunologically based formulations.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in bulk, as a single unit dose, or as multiple single unit doses. The amount of active ingredient is generally equal to the dose of active ingredient administered to the subject, or a convenient fraction of such dose, eg, half or one third of such dose.

  The relative amounts of active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the invention will depend on the identity, size and condition of the subject to be treated and It will vary further depending on the route to which the composition is to be administered. For example, the composition may comprise between 0.1% (w / w) and 100% (w / w) active ingredient.

  In addition to the active ingredient, the pharmaceutical composition of the present invention may further comprise one or more additional pharmaceutically active agents.

  Additional agents specifically contemplated include antiemetics and scavengers such as cyanide and cyanate scavengers. Controlled or sustained release formulations of the pharmaceutical compositions of the invention can be manufactured using conventional techniques.

  Formulations of the pharmaceutical composition of the present invention suitable for oral administration include tablets, hard capsules or soft capsules, cachets, troches or confectionery tablets each containing a predetermined amount of the active ingredient. It can be prepared, packaged or sold in the form of separate solid dosage units, not limited to. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, or emulsions. Not.

  A tablet containing the active ingredient may be made, for example, by compressing or molding the active ingredient and optionally one or more additional ingredients. Compressed tablets can be combined in a suitable device with an active ingredient in a free flowing form, such as a powder or granular preparation, optionally one of a binder, lubricant, vehicle, surfactant and dispersant. It can be prepared by mixing with the above and compressing. Molded tablets may be made by molding in a suitable device a mixture of the active ingredient, a pharmaceutically acceptable carrier and at least a liquid sufficient to wet the mixture. Pharmaceutically acceptable vehicles used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersing agents include potato starch and sodium starch glycolate. Known surfactants include sodium lauryl sulfate. Known diluents include calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include corn starch and alginic acid. Known binders include gelatin, acacia, pre-gelatinized corn starch, polyvinyl pyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include magnesium stearate, stearic acid, silicon dioxide and talc.

  The tablets may be uncoated, or the tablets may be coated using known methods to achieve delayed disintegration in the subject's gastrointestinal tract and thereby provide sustained release and absorption of the active ingredient. Also good. For example, materials such as glyceryl monostearate or glyceryl distearate may be used to coat the tablets. Further, for example, tablets may be prepared, for example, in US Pat. Nos. 4,256,108, 4,160,452, and 4,265, to form osmotically controlled release tablets. Can be coated using the method described in 874. The tablets may further comprise sweetening, flavoring, coloring, preservatives or some combination of these in order to provide a pharmaceutically sophisticated and palatable preparation.

  Hard capsules containing the active ingredient can be made using a physiologically degradable composition such as gelatin. Such hard capsules contain the active ingredient and may further contain additional ingredients (eg, inert solid diluents such as calcium carbonate, calcium phosphate or kaolin).

  Soft gelatin capsules containing the active ingredients can be made using a physiologically degradable composition such as gelatin. Such soft capsules contain the active ingredient, which can be mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

  Liquid formulations of the pharmaceutical compositions of the present invention suitable for oral administration are in liquid form or in the form of dry products intended to be reconstituted with water or other suitable vehicle prior to use. Either can be prepared, packaged or sold.

  Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredients in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, tonsils, oily esters, ethyl alcohol, peanut oil, olive oil, sesame oil or coconut oil, vegetable oils such as fractionated vegetable oil, and mineral oils such as liquid paraffin. Liquid suspensions include one or more additional agents including, but not limited to, suspending, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffering agents, salts, flavoring agents, coloring agents and sweetening agents. In addition, various components may be included. The oily suspension may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fat, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. . Known dispersants or wetting agents include naturally occurring phospholipids such as lecithin, condensation products of alkylene oxides and fatty acids, condensation products of alkylene oxides and long chain fatty alcohols, fatty acids and hexitols. Product of a partial ester and an alkylene oxide, or a partial ester and an alkylene oxide derived from a fatty acid and hexitol anhydride (for example, polyoxyethylene stearate, heptadecaethyleneoxysetanol, Polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate). Known emulsifiers include lecithin and acacia. Known preservatives include methyl-para-hydroxybenzoate, ethyl-para-hydroxybenzoate or n-propyl-para-hydroxybenzoate, ascorbic acid, and sorbic acid. Known sweeteners include glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

  Liquid solutions of the active ingredient in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. It is to be done. Liquid solutions of the pharmaceutical composition of the present invention can include each of the ingredients described with respect to the liquid suspension, and the suspension does not necessarily aid in dissolving the active ingredient in the solvent. Understood. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, tonsil oil, oily esters, ethyl alcohol, peanut oil, olive oil, sesame oil or coconut oil, vegetable oils such as fractionated vegetable oil, and mineral oils such as liquid paraffin.

  Powdered and granular formulations of the pharmaceutical preparations of the invention can be prepared using known methods. Such formulations may be administered directly to the subject, or an aqueous or oily suspension, for example, by forming a tablet, filling a capsule, or adding an aqueous or oily vehicle. It may be used to prepare a suspension or solution. Each of these formulations may further comprise one or more of a dispersing or wetting agent, suspending agent and preservative. Additional excipients such as fillers and sweeteners, flavors, or colorants may also be included in these formulations.

  The pharmaceutical compositions of the invention can also be prepared, packaged or sold in the form of an oil-in-water emulsion or a water-in-oil emulsion. The oily phase can be a vegetable oil such as olive or peanut oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions include naturally occurring gums such as gum arabic or tragacanth, naturally occurring phospholipids such as soy phospholipids or lecithin phospholipids, fatty acids such as sorbitan monooleate and hexitol anhydride. One or more emulsifiers may also be included such as esters or partial esters derived from the combination of and a condensation product of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

  The pharmaceutical compositions of the invention may be prepared, packaged or sold in a formulation suitable for rectal administration. Such compositions can be in the form of, for example, suppositories, retention enemas preparations, and solutions for rectal or colonic irrigation.

  Suppository formulations are non-irritating pharmaceutically acceptable that are solid at normal room temperature (ie, about 20 ° C.) and liquid at the rectal temperature of the subject (ie, about 37 ° C. in healthy humans). Can be made by combining excipients and active ingredients. Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycol, and various glycerides. Suppository formulations may further comprise various additional ingredients including but not limited to antioxidants and preservatives.

  A retention enema preparation or a solution for rectal or colonic irrigation can be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations can be administered using a delivery device adapted to the rectal anatomy of the subject and can be packaged within such a delivery device. . Enema preparations can further include various additional ingredients including, but not limited to, antioxidants and preservatives.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in a formulation suitable for intravaginal administration. Such compositions can be in the form of, for example, suppositories, impregnated or coated vaginal inserts such as tampons, irrigation preparations, gels or creams or vaginal cleansing solutions.

  Methods for impregnating or coating a material with a chemical composition are known in the art, and methods for depositing or bonding a chemical composition onto a surface, methods for incorporating a chemical composition into a material structure during material synthesis (I.e., for example, when using physiologically degradable materials) and methods of absorbing aqueous or oily solutions or suspensions into absorbent materials (with or without subsequent drying) ), But is not limited thereto.

  Irrigation preparations or vaginal irrigation solutions can be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is known in the art, irrigation preparations can be administered and packaged in a delivery device adapted to the subject's vaginal anatomy.

  The irrigation preparation may further comprise various additional ingredients including but not limited to antioxidants, antibiotics, antifungals and preservatives.

  Additional delivery methods for administration of the compound include drug delivery devices such as those described in US Pat. No. 5,928,195.

  As used herein, “parenteral administration” of a pharmaceutical composition is any of the administration characterized by physical disruption of a subject's tissue and administration of the pharmaceutical composition through a tissue tear. Includes route. Thus, parenteral administration includes administration of the pharmaceutical composition by injection of the composition, administration of the pharmaceutical composition by application of the composition through a surgical incision, composition through a non-surgical wound that penetrates the tissue. Including, but not limited to, administration of a pharmaceutical composition by application of the product. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and renal dialysis infusion techniques.

  Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dose forms such as ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained or biodegradable formulations in oily or aqueous vehicles. Such formulations may further comprise one or more additional ingredients including but not limited to suspending, stabilizing or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is used in a suitable vehicle (eg, sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Provided in a dry (ie, powder or granular) form for reconstitution.

  The pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oleaginous suspension or solution. This suspension or solution can be formulated according to techniques in the art and, in addition to the active ingredient, additional ingredients such as dispersing, wetting or suspending agents described herein. May be included. Such sterile injectable formulations can be prepared using nontoxic parenterally acceptable diluents or solvents such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation are pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts. Can be included.

  Formulations suitable for topical administration include liquid or semi-liquid preparations (eg, pastes, lotions, oil-in-water emulsions or water-in-oil emulsions (eg, creams, ointments or pastes) and solutions or Formulations that can be administered topically can contain, for example, from about 1% to about 10% (w / w) of the active ingredient, although the concentration of the active ingredient can be The solubility limit of the active ingredient therein may be as high as the formulation for topical administration may further comprise one or more of the additional ingredients described herein.

  The pharmaceutical compositions of the invention can be prepared, packaged, or sold in a formulation that is suitable for pulmonary administration via the oral vestibule. Such formulations can comprise dry particles, the dry particles comprising the active ingredient and having a diameter in the range of about 0.5 to about 7 nanometers, preferably about 1 to about 6 nanometers. Have. Such compositions conveniently use a device with a dry powder reservoir to which a propellant stream can be directed to disperse the powder, or a self-propelled solvent / powder dispersion container (eg, in a sealed container). A device comprising an active ingredient dissolved or suspended in a low-boiling propellant in the form of a dry powder for administration. Preferably, such powder comprises particles, at least 98% by weight of the particles have a diameter greater than 0.5 nanometers, and at least 95% of the number of particles have a diameter less than 7 nanometers. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% of the number of particles have a diameter less than 6 nanometers. The dry powder composition preferably comprises a solid fine powder diluent such as sugar and is conveniently provided in a unit dose form.

  Low boiling propellants generally include liquid propellants having a boiling point of less than 65 ° F. (18.3 ° C.) at atmospheric pressure. Generally, the propellant can make up 50 to 99.9% (w / w) of the composition, and the active ingredient can make up 0.1 to 20% (w / w) of the composition. The propellant contains additional components such as liquid nonionic surfactants (preferably having a particle size in the same order as the particles containing the active ingredient) or solid anionic surfactants or solid diluents. Further may be included.

  The pharmaceutical compositions of the invention formulated for pulmonary delivery can also provide the active ingredients in the form of solutions or suspension droplets. Such formulations may be prepared, packaged or sold as sterile, aqueous or dilute alcoholic solutions or suspensions containing the active ingredients as required and using any nebulizing or atomizing device And can be conveniently administered. Such formulations contain one or more additional ingredients including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, or preservatives such as methyl hydroxybenzoate. Further may be included. The droplets provided by this route of administration preferably have an average diameter in the range of about 0.1 to about 200 nanometers.

  The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of the invention.

  Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such formulations are administered in the manner in which snuff is taken, ie by rapid inhalation through the nasal passage from a powder container held near the nose.

  Formulations suitable for nasal administration may contain, for example, as little as 0.1% (w / w) and as much as 100% (w / w) of the active ingredient and may be added as described herein One or more of the various ingredients may further be included.

  The pharmaceutical compositions of the invention can be prepared, packaged or sold in a formulation suitable for buccal administration. Such formulations can be, for example, in the form of tablets or confectionery tablets made using conventional methods and can contain, for example, 0.1-20% (w / w) active ingredient, the remainder Orally soluble or degradable compositions, and may optionally include one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension containing the active ingredient. Such powdered, aerosolized or atomized formulations, when dispersed, preferably have an average particle size or droplet size in the range of about 0.1 to about 200 nanometers and It may further comprise one or more of the additional ingredients described in the document.

  The pharmaceutical compositions of the invention may be prepared, packaged or sold in a formulation suitable for ocular administration. Such formulations are, for example, in the form of eye drops comprising, for example, a solution or suspension of, for example, 0.1-1.0% (w / w) active ingredient in an aqueous or oily liquid carrier. obtain. Such eye drops may further comprise a buffer, salt, or one or more other of the additional ingredients described herein. Other useful formulations that can be administered to the eye include formulations that are in microcrystalline form or that contain the active ingredient in a liposomal preparation.

  A mixture of pyrroloquinoline quinone and a pharmacologically acceptable additive is preferably prepared as a lyophilized product and dissolved upon use. Such a preparation can be prepared in a solution containing about 0.01-100.0 mg / ml pyrroloquinoline quinone by dissolving pyrroloquinoline quinone in distilled water for injection or sterile purified water. More preferably, the preparation is adjusted to have a physiologically isotonic salt concentration and a physiologically desirable pH value (pH 6-8).

  The dose is appropriately determined according to symptoms, body weight, sex, animal species, etc., but it is generally assumed that a treatment option that maintains a blood concentration of about 1 μM is preferred. This plasma concentration can be achieved by administration of one to several doses per day. When pyrroloquinoline quinone is to be administered to a subject, 0.1 ng to 10 mg / kg body weight (eg, 1 ng to 1 mg / kg body weight) of pyrroloquinoline quinone can be administered intravenously.

  The compound may be given to the animal several times a day, or the compound may be once a day, once a week, once every two weeks, once a month, once a month, or It may be administered once a year or less frequently. The frequency of the dose will be readily apparent to those skilled in the art and depends on many factors such as, but not limited to, the type and severity of the disease being treated, the type of animal and the age.

  These examples are provided for purposes of illustration only, and the present invention should in no way be construed as limited to these examples, but rather as a result of the teaching provided herein. It should be construed to encompass all and all variations that become apparent.

Example 1. In vitro study of PQQ storage of cardiomyocyte viability An in vitro model of cultured adult mouse cardiomyocytes was developed to study cardioprotection by PQQ. These cells can survive in culture at physiological pH for up to 48 hours, with> 90% consisting of rod-shaped cells. These cells can be readily used for determination of cell viability by trypan blue exclusion and for biochemical, immunochemical and molecular studies. In this model, approximately 35% of the cells die when exposed to 0% oxygen in a hypoxic chamber for 2-3 hours. As shown in FIG. 1, 1 μM PQQ added 1 hour prior to subjecting the cells to high hypoxia (2-3 hours at 0% oxygen) is viable cells as shown by trypan blue exclusion. A significant increase in the ratio of Higher concentrations of PQQ (100 μM) are very toxic under normoxic conditions, as evidenced by 100% cell death. FIG. 2 demonstrates that 1 μM PQQ protection against hypoxia-induced cell death is not inhibited by 10 μM 5-hydroxydecanoic acid (mitochondrial _KATP channel inhibitor). Without wishing to be bound by theory, these data suggest that PQQ does not exert cardioprotection by opening mitochondrial _KATP channels.

Example 2 Ex vivo study of PQQ preservation of cardiac function Ex vivo studies were performed using mouse heart preparations isolated using Langendorff technology. In this approach, the heart can be removed and placed on a perfusion device, where drug can be delivered via the aortic cannula. The heart is paced at a constant rate, and left ventricular pressure [LVDP; (left ventricular systolic pressure) − (left ventricular end diastolic pressure)], left ventricular end diastolic pressure [LVEDP], and positive and Record the negative maximum first derivative [+ dP / dtmax and -dP / dtmax]. Equilibrate the heart for 20 minutes. Following drug or vehicle injection, the heart is subjected to 20 minutes of ischemia [coronary flow complete arrest] followed by 30 minutes of reperfusion. Coronary sinus blood flow as a reflection of coronary blood flow is also measured. This protocol results in severe myocardial damage as measured by hemodynamic parameters.

  As seen in FIG. 3, 100 nM PQQ infused for only 2 minutes prior to complete arrest of coronary blood flow results in significant preservation of LVDP baseline 1. VDP averages 60 mmHg. Similar results are obtained using LVEDP [Figure 4]. The LVEDP baseline is an average of 8 mmHg. (Note that increasing LVEDP represents an adverse response). As expected, the data for + dP / dtmax and -dP / dtmax follow the LVDP results [FIGS. 5 and 6]. Similarly, coronary flow is significantly improved by PQQ pretreatment compared to controls [Figure 7].

  In FIG. 8, it is shown that pretreatment for 2 minutes with PQQ at the indicated concentrations has a progressively better response between 10 nM and 1 μM, but toxicity occurs at 10 μM. Of primary interest is that 100 nM PQQ given at the beginning of reperfusion (PQQ treatment) is equivalent to pretreatment. Thus, PQQ is useful both in pre-treatments, such as cardiac procedures or other surgical procedures, and after a symptom has occurred, for example, in an acute critical event.

  FIG. 9 shows the measurement experiment results of infarct size after PQQ pretreatment. As shown, the infarct size gradually decreases between 10 nM and 1 μM, corresponding to hemodynamic data. Consistent with the latter, infarct size does not decrease with 10 μM PQQ.

Example 3 FIG. PQQ preservation of cells subjected to oxidative stress Cultured cardiomyocytes are subjected to oxidative stress by in vitro administration of H ₂ O ₂ . Two studies are performed, in which one heart muscle cell is added with a concentration of PQQ between 10 nM and less than 10 μM followed by H ₂ O ₂ . In the other study, cardiomyocytes are subjected to a 2 hour in vitro administration of H ₂ O ₂ followed by addition of PQQ at a concentration between 10 nM and less than 10 μM. In both studies, PQQ is found to protect.

Example 4 Use of PQQ to prevent / reduce oxidative stress in vivo Male Sprague-Dawley rats were randomly treated with pyrroloquinoline quinone (PQQ) prior to ischemia or ischemia-reperfusion. PQQ (15-20 mg / kg) was given 30 minutes prior to left anterior descending coronary artery (LAD) occlusion by intraperitoneal administration (pretreatment) or at the beginning of reperfusion by intravenous injection (treatment). While monitoring left ventricular (LV) hemodynamics, rats were subjected to 17 or 30 minutes of LAD occlusion and 2 hours of reperfusion. PQQ given as either pretreatment or treatment reduced infarct size in these rat models. PQQ protects against cardiac dysfunction induced by ischemia, and after 1-2 hours of reperfusion, the LV generation pressure, LV (+) dP / dt is higher and LV (-) dP / dt is lower. It was. There were fewer ventricular fibrillation (VF) episodes in PQQ-treated rats. Myocardial malondialdehyde (MDA) (an index of lipid peroxidation) was decreased by PQQ. Thus, PQQ given either as a pretreatment or as a treatment at the beginning of reperfusion reduces myocardial infarct size and improves cardiac function in a dose-related manner in a rat model of ischemia and ischemia-reperfusion It is particularly effective. MDA results suggest that PQQ acts as a free radical scavenger in ischemic myocardium.

Statistical analysis All results are presented as mean ± SEM. The two treatment groups (pretreatment and treatment) were compared to the normal control group using one-way analysis of variance (ANOVA) with regression equations for multiple group comparisons. Differences in mortality during the period of occlusion and reperfusion among the three groups were assessed by chi-square test. The percentage of rats with VF was assessed by Fisher's exact test. All calculations were performed using the general linear model procedure of Minitab version 7.2 (Minitab Statistical Software) or Primer of Biostatistics: The program version 3.03 (McGraw-Hill). Statistical significance was set at p <0.05.

Was dissolved reperfusion model PQQ in the vehicle (2% NaHCO ₃₎ - ischemia and ischemia. The volume given either intraperitoneally (ip) or intravenously (iv) was 1 ml. All controls were treated with 1 ml vehicle. In model 1, 20 mg / kg of PQQ was administered 30 minutes before 2 hours of ischemia induced by LAD ligation, i. p. Gave in. In model 2, 15 mg / kg PQQ was administered i.p. 30 minutes prior to 17 or 30 minutes ischemia. p. Followed by 2 hours of reperfusion (pretreatment). In another model 2 experiment, 15 mg / kg PQQ was administered i.v. via the femoral vein at the start of reperfusion. v. Given by bolus injection (treatment). These protocols are summarized in FIG.

  After induction of anesthesia (ketamine 80 mg / kg, xylazine 4 mg / kg body weight, intraperitoneal), a tracheotomy was performed and the animals were aerated in a Harvard Respirator (model 683, Harvard Apparatus). Model 1 rats were subjected to 2-hour proximal left anterior descending (LAD) coronary artery ligation without reperfusion. Model 2 used ischemia-reperfusion as previously described (Sievers RE et al., Magn Reson Med 1989; 10: 172-81). In this model, a reversible coronary snare occluder was placed around the proximal LAD coronary artery by a midline sternotomy. Rats were then subjected to 17 or 30 minutes of LAD occlusion and 120 minutes of reflow. In addition, model 2 rats were recorded hemodynamic measurements. A 4F Millar catheter was inserted through the right carotid artery into the left ventricle (LV). After 20 minutes of equilibration, the heart rate (HR), systolic pressure (LVSP), end diastolic pressure (LVEDP), LV (+) dP / dtmax, LV (−) dP / dtmax were expressed as MacLab / 4S (Milford , MA). LV generation pressure (LVDP) was calculated by subtracting LVEDP from LVSP.

  Body weights in the three groups of rats in both Model 1 and Model 2, Set 1 and Set 2 were not different (values for the control, pretreatment and treatment groups were as follows: 320 ± 16 321 ± 22 and 306 ± 18 g; by analysis of variance (ANOVA), p = 0.799).

  At baseline, there was no significant difference in heart rate, LVSP, LVEDP, LV (+) dP / dt, and LV (−) dP / dt between model 2, control, pretreatment and treatment groups. Whether given by pretreatment or as treatment, PQQ prevents ischemia-induced cardiac dysfunction, and higher LVSP, LVDP, LV (+) after 1-2 hours of reperfusion dP / dt and lower LV (−) dP / dt (FIGS. 11A-B; 12A-B).

Infarct size Infarct size was measured as previously described (Sievers RE et al., Magn Reson Med 1989; 10: 172-81, Zhu B-Q et al., Jam Coll Cardiol 1997; 30: 1878-85). In model 1, the heart was excised at the end of the 2 hour ischemic period. The sections were then incubated for 10-15 minutes in a 1% solution of triphenyltetrazolium chloride (TTC) until viable myocardium was colored red brick.

  In model 2, after 2 hours of reperfusion, LAD was reoccluded and phthalocyanine dye (Engelhard Cooperation, Louisville, KY) was injected into the LV cavity and the normally perfused myocardium stained blue. The heart was then excised, rinsed with excess dye, and sliced transversely from the top to the base into 2 mm thick sections. Sections were incubated in TTC as described above. Infarcted myocardium cannot be stained with TTC. Tissue sections were then fixed in 10% formalin solution and weighed. Color digital images of both sides of each transverse slice were obtained using a video camera (Leica DC 300F) connected to a microscope (Stereo Zoom 6 Photo, Leica). Areas showing tissue stained blue (non-ischemic), tissue stained red (ischemic but non-infarcted) and unstained tissue (infarcted) are outlined on each color image. Draw and measure in a blinded fashion using NIH Image 1.59 (National Institutes of Health, Bethesda, MD). On each side, the absolute weight of the infarct-related tissue was determined by multiplying the proportion of LV area representing the infarct-related tissue (average of two images) by the weight of the section. The infarct size for each heart was expressed as follows:

次いで、危険領域のパーセンテージとしての梗塞サイズを、以下の通り計算した：

The infarct size as a percentage of the risk area was then calculated as follows:

虚血モデル（モデル１）では、ＰＱＱ後の梗塞サイズ（梗塞量／ＬＶ量、フタロシアニン青色色素は注射されていない）は、対照よりも小さかった（図１３）。モデル２における実験の第１セットにおいて、虚血は１７分間であり、その後、２時間の再灌流が行われ、梗塞サイズ（梗塞量／危険領域、梗塞量／ＬＶ量）は、２０ｍｇ／ｋｇのＰＱＱでの前処置によって減少した（図１４）。モデル２の実験の第２セットにおいて、虚血は３０分間であり、その後、２時間の再灌流が行われ、１５ｍｇ／ｋｇのＰＱＱでの前処置または処置のいずれかの後での梗塞サイズは、対照よりも小さかった（図１５）。

In the ischemia model (Model 1), the infarct size after PQQ (infarct volume / LV volume, phthalocyanine blue dye was not injected) was smaller than the control (FIG. 13). In the first set of experiments in Model 2, ischemia is 17 minutes, followed by 2 hours of reperfusion, and the infarct size (infarct volume / risk area, infarct volume / LV volume) is 20 mg / kg. Decreased by pretreatment with PQQ (FIG. 14). In the second set of Model 2 experiments, ischemia is 30 minutes, followed by 2 hours of reperfusion, and the infarct size after either pretreatment or treatment with 15 mg / kg PQQ is , Smaller than the control (Figure 15).

  FIG. 16 shows that the dose of PQQ given as a pretreatment inversely correlated with infarct size. In these experiments, 17 hours of ischemia was followed by 2 hours of reperfusion.

Ventricular fibrillation (VF)
An electrocardiogram (ECG, lead II) was obtained by inserting a hypodermic needle electrode into the limb. ECG was monitored during ischemia and reperfusion and episodes of paroxysmal VF were recorded. The number of VF episodes per rat and the percentage of rats with VF in each group were calculated. None of the rats received antiarrhythmic drugs before or during occlusion and reperfusion. The VF episode was successfully treated by quickly striking the exposed myocardium with the thumb and index finger of one hand.

  Pretreatment with 15 or 20 mg / kg PQQ reduced the mean episode of VF per rat (FIG. 17A). Pretreatment or treatment with 15 or 20 mg / kg PQQ reduced the percentage of rats with VF (FIG. 17B).

  In another additional experiment in which malondialdehyde was measured (see below), there were no episodes of VF in either the sham group or the PQQ group (n = 5 each). However, the VF in the I / R group (30 minutes ischemia followed by 2 hours of reperfusion) averages 1.8 ± 0.4 episodes / rat, and is 0. 0 by pretreatment with 15 mg / kg PQQ. Reduced to 2 ± 0.2 episodes / rat (n = 5 in each group, P <0.001 by binary ANOVA, with P = 0.002 significant interaction between PQQ and I / R) .

Myocardial malondialdehyde (MDA) measurement Myocardial tissue MDA (lipid peroxidation product that reacts with thiobarbituric acid) was determined spectrophotometrically at an absorbance of 532 nm. The concentration of the sample was calculated using an extinction coefficient of 1.56 × 10 5 / Mcm and the results were expressed as nmol / g wet weight heart (Ohkawa H et al., Analytical Biochemistry 1979; 95: 351-358, Moritz F Et al., Cardiovascular Research 2003; 59: 834-843).

  Various tissue preparation methods were used to measure myocardial tissue MDA (an index of lipid peroxidation). The heart was visually divided from the top to the base into an anterior portion containing the area perfused by LAD and the remaining non-ischemic portion. Rats were pre-treated or untreated with 15 mg / kg PQQ (sham control) and subjected to 30 minutes of ischemia and 2 hours of reperfusion. In these additional experiments, the hemodynamic results were not different from the above results. As seen in FIG. 18A, I / R increased MDA levels and PQQ prevented this increase. PQQ values and control values after I / R differed by a factor of 3 (FIG. 18A). A similar effect was seen in distant “normal” myocardium (FIG. 18B). When given at the beginning of reperfusion, 15 mg / kg PQQ also reduced MDA levels in ischemic myocardium from 176 ± 16 to 123 ± 17 nmol / g (n = 8, P <0.05).

Embodiment 5 FIG. PQQ restores mitochondrial respiration at low concentrations and is cardioprotective in an in vivo model of ischemia / reperfusion injury Adult male rats undergo 30 minutes of left anterior descending (LAD) coronary artery occlusion and 2 hours of reperfusion. It was. To assess the potential benefits of reperfusion therapy in humans, PQQ was administered i.e. at a dose of 1 and 3 mg / kg body weight at the start of reperfusion. v. Given by injection. After removal, the heart was divided from the top to the base into an anterior and posterior segment containing the LAD perfused area, and mitochondria were isolated. Mitochondrial respiration of each myocardial segment was measured and compared to mitochondrial respiration isolated from pre-conditioned and sham-operated hearts (without PQQ).

  Treatment with 1 mg / kg PQQ did not protect mitochondria from I / R injury. However, treatment with 3 mg / kg PQQ was very effective in restoring mitochondrial respiration. Respiratory regulation and ADP-oxygen consumption rate in the ischemic region (RCR: 8.0 ± 0.5, ADP / O; 4.5), and state 3 respiratory rate (RR: 41 nmol O atom / min / mg protein, n = 5) is consistent with false ones (RCR: 8.2 ± 0.3, ADP / O: 3.7, RR = 43 nmol O atom / min / mg protein), while pre-acclimated without PQQ The heart RCR value was surprisingly 20% lower. The respiratory response from ischemic naïve heart mitochondria was reduced by 50-100%. Electron micrographs of tissues and mitochondria treated with PQQ did not show the morphology typical of myocardial damage. PQQ also reduced infarct size and myocardial malondialdehyde (MDA) tissue concentration by 49% and 61%, respectively.

  The low dose of 3 mg / kg PQQ given during reperfusion very effectively restores mitochondrial respiration inhibited by ischemia and reduces infarct size in oxidative and I / R damage to mitochondria. PQQ may exert its cardioprotective function as a lipid peroxidation inhibitor or radical scavenger. Therefore, PQQ treatment may emerge as a powerful therapeutic agent for acute ischemic syndrome.

  Respiratory regulation and ADP-oxygen consumption rate in ischemic region (RCR: 8.0 0.5, ADP / O, 4.5), and state 3 respiratory rate (RR: 41 nmol O atom / min / mg protein, n = 5) was consistent with the sham one, while the RCR value of the heart previously conditioned without PQQ was surprisingly 20% lower. The respiratory response from ischemic naïve heart mitochondria was reduced by 50-100%. RCR data is visually shown in FIG. Electron micrographs of PQQ treated tissues and mitochondria did not show the morphology typical of myocardial injury. Therefore, the low dose of 3 mg / kg PQQ given during reperfusion very effectively restores mitochondrial respiration that is inhibited by ischemia and reduces infarct size in oxidative and I / R damage to mitochondria. Decreased.

  Toxicity studies were also performed. We found no nephrotoxicity or hepatotoxicity at doses of 3 mg / kg or 10 mg / kg (see Table 1). As noted above, 3 mg / kg PQQ given during reperfusion appeared to be an effective cardioprotective dose. At 15 mg / kg, 7/8 animals showed no nephrotoxicity or liver toxicity, but one rat excluded from the data shown in Table 1 developed uremic disease on day 4 and 10 Died on the day. Rats that received 20 mg / kg had previously received 10 mg / kg at a cumulative dose of 30 mg / kg for 2 weeks. As shown in Table 1, all of these animals developed uremia and died on the 10th day. All laboratory studies were performed in a blinded fashion by a clinical laboratory at San Francisco VA Medical Center. Note that baseline levels of some scales, such as albumin, are lower in rats than in humans, while others such as creatine kinase (CK) are higher.

実施例６．心機能に対するＰＱＱおよびメトプロロールの効果の研究
１５ｍｇ／ｋｇのＰＱＱでの前処置または処置は、梗塞サイズを低減させ、虚血／再灌流（Ｉ／Ｒ）のラットモデルにおける心機能を改善した。β遮断剤メトプロロールは、急性心筋梗塞症患者における標準処置として使用される。従って、メトプロロールおよび低用量ＰＱＱを用いた心筋梗塞の併用処置を各薬物単独と比較して研究するために実験を行った。心臓保護機構を決定するために、変化を、ミトコンドリア機能および脂質過酸化において測定した。

Example 6 Study of effects of PQQ and metoprolol on cardiac function Pretreatment or treatment with 15 mg / kg PQQ reduced infarct size and improved cardiac function in a rat model of ischemia / reperfusion (I / R). The beta blocker metoprolol is used as a standard treatment in patients with acute myocardial infarction. Therefore, an experiment was conducted to study the combined treatment of myocardial infarction with metoprolol and low dose PQQ compared to each drug alone. Changes were measured in mitochondrial function and lipid peroxidation to determine cardioprotective mechanisms.

  Intact male rats were subjected to 30 minutes of left anterior descending coronary artery occlusion and 2 hours of reperfusion while monitoring left ventricular hemodynamics. In preliminary experiments, a dose of 1 mg / kg of metoprolol was found to be optimal in this system to reduce infarct size. Therefore, to mimic clinical treatment, metoprolol (1 mg / kg) and / or PQQ (3 mg / kg) were given by femoral vein injection at the beginning of reperfusion. In separate experiments, after ischemia / reperfusion, mitochondrial respiratory regulation and ADP-oxygen consumption rate (RCR) of ischemic and non-ischemic myocardium and levels of malondialdehyde (MDA) (an index of lipid peroxidation) Was measured.

  The results show that treatment with either metoprolol or PQQ reduced myocardial infarct size (infarct weight / risk area). Using these drugs in combination tended to further reduce infarct size. Metoprolol and / or PQQ also protected against left ventricular (LV) dysfunction induced by ischemia after 1-2 hours of reperfusion. Therefore, LV generation pressure increased and LV end-diastolic pressure decreased. Metoprolol and / or PQQ also decreased CK release. Mitochondrial RCR in ischemic and non-ischemic myocardium was enhanced primarily by PQQ and not so much by metoprolol. PQQ reduced MDA in ischemic and non-ischemic myocardium. These results are summarized in Table 2 below.

  These experiments suggest that PQQ and metoprolol are effective in the treatment of myocardial infarction, but the combination of PQQ and metoprolol may be more effective than either drug alone. For mitochondrial protection, PQQ is superior. It should be noted that in a recent large study of 45,852 acute myocardial infarction patients randomized to metoprolol or placebo, the incidence of cardiac shock was higher than about 30% in the metoprolol group ( Collins R, et al., COMMIT / CCS-2; Placebo-controlled trial of early metoprolol in 46,000 acute myocardial infarction patients.Late-breaking trials presented at the American College of Cardiology Annual Scientific Session 2005.March 6-9,2005.Orlando , Fla ). This may be due at least in part to metoprolol being unable to restore mitochondrial function.

実施例７：ＰＱＱ連結ポリビニルアルコールおよびＰＱＱ連結ポリマーの合成
ＰＱＱを最初に活性化させ、次いで、ポリマーと反応させてＰＱＱ連結ポリマーを得た。９ｋ〜１００の異なる分子量のポリ（ビニルアルコール）（ＰＶＡ）を、本出願において試験した。ＰＶＡは、その多くの望ましい特徴のため、特に様々な薬学的お生物医学的用途にとって大いに関心のあるポリマーである。

Example 7 Synthesis of PQQ-Linked Polyvinyl Alcohol and PQQ-Linked Polymer PQQ was first activated and then reacted with the polymer to obtain a PQQ-linked polymer. Poly (vinyl alcohol) (PVA) of 9k-100 different molecular weight was tested in this application. PVA is a polymer of great interest especially for various pharmaceutical and biomedical applications because of its many desirable characteristics.

  The synthesis procedure is shown in FIG. 20A. This is a two step reaction. In the first step, PQQ reacted with a dehydrating agent (eg, DCC or CDI) to give an active immediate. In the second step, active PQQ reacted with PVA to form an ester bond and PQQ was chemically bonded to the PVA backbone.

  The experimental design is shown in Table 3. The PQQ loading level was 1-10% and the reaction temperature was controlled at 0, 25 and 50 ° C.

＊実験は、ＤＭＦ溶液中で実施した。３つの分子量のＰＶＡを試験した（Ｍ．Ｗ．〜１０Ｋ、４０Ｋ、および１０Ｋ）。

* Experiment was performed in DMF solution. Three molecular weights of PVA were tested (MW-10K, 40K, and 10K).

Synthesis and purification procedures:
1. PQQ was dissolved in N, N-dimethylformamide (DMF) solution at controlled temperatures (0 ° C., 25 ° C., and 50 ° C.).

  2. A dehydrating agent (DCC or CDI) was added to the solution to form activated PQQimmediate.

  3. PVA was added to the solution and the reaction was held for 20 hours.

  4). The solution was transferred to a dialysis tube (COMW 4,000) and dialyzed in deionized water for 2 days. The water was changed 3 times a day.

  5). After dialysis, the solution was concentrated under vacuum and dried at 50 ° C. in a vacuum oven.

H-NMR analysis Proton NMR spectra of the received PQQ and PQQ / PVA conjugates are shown in FIG. 45 and FIG. Two peaks were shown at 8.58 ppm and 7.19 ppm, and these peaks were assigned to the two aromatic protons shown in PQQ. The d ₆ -DMSO peak appeared at 2.50 ppm and the peak from the water residue was shown at 3.31 ppm. Purity was calculated by comparing proton integration areas and was> 95 ± 5% for the two resources. The NMR spectrum of the PQQ / PVA conjugate clearly showed the formation of a linkage bond. In the low field, the two peaks at 8.58 ppm and 7.19 ppm were from PQQ, and the 4 to 1 ppm multiple peaks in the high field were from PVA.

FT-IR spectra of PQQ and PVA ATR mode FT-IR spectra of PVA, PQQ, and concatenated PQQ / PVA are shown in FIG. 47, FIG. 48, and FIG. The characteristic peak near 3,000 cm ⁻¹ is from the hydroxyl group in PVA. In the PQQ / PVA conjugate, this peak is clearly reduced by the ligation reaction. In addition, a strong peak at 1720 cm ⁻¹ was formed as a result of the new ester bond.

XRD spectrum of PVA powder X-ray diffraction was performed on a Shimadzu XRD-6000. Crystallinity has a significant effect on PQQ release kinetics. Crystallinity is determined by many factors (eg, molecular weight and molecular weight polydispersity, thermal history, etc.). The received PVA (MW: 10K) shows similar crystallinity, and this crystal excess is about 32-34%, as shown in FIG. 50, with a main peak at 19-20 (2θ). Have. When PQQ reacted with PVA to form a PQQ / PVA linkage, the product showed only an amorphous peak. This ligation reaction completely changed the microscopic structure.

PQQ Quantitative Assay by HPCL A standard calibration curve is shown in FIG. 51, which can detect PQQ amounts up to 0.1 ng / 20 μl. The volume limit and detection limit are 0.2 ng / 20 μl and 0.1 ng / 20 μl, respectively, which means that PQQ can be effectively detected when the amount of PQQ is> 0.1 ng / 20 μl (FIGS. 52 and 53). If the amount of PQQ is> 0.2 ng / 20 μl, this amount can be known using the calibration curve.

Confirmation of PQQ coupled PVA system by GPC During dialysis, the solution was checked for effluent PQQ by UV lamp. After 2 days of dialysis, there was no detectable amount of PQQ. The PQQ linked PVA solution was concentrated by rotary evaporator and the final composite was oven dried.

  GPC analysis was performed to confirm the binding of PQQ to the polymer. FIG. 21 shows a GPC spectrum of PQQ using a fluorescence detector. A sharp peak appears at 14.90 minutes, which is the retention of pure PQQ at this operating condition. The peak structure was confirmed by the UV absorption spectrum as shown in FIG.

  When PVA was detected by a fluorescence detector, the intensity was very weak compared to the intensity of PQQ. This is because PVA showed almost no fluorescence.

  The GPC spectrum of PQQ-linked PVA is shown in FIG. 23, with peaks at 10.19 minutes, 13.67 minutes, and overlapping peaks at 14.90 minutes. From the calculations, the molecular weight was about 40K, 10K and low molecular weight molecules. All molecules contained PQQ because PQQ can be detected by a fluorescence detector. Further confirmation was made by examining the UV absorption spectrum of each peak. In general, the peaks are the same, with slight differences due to new ester bond formation in the molecule (FIG. 24).

  Various analytical methods can be used for this PQQ-linked PVA product. Proton NMR studies were performed to investigate PQQ binding to PVA in more detail, with the exception of GPC.

  The PQQ-PVA conjugate may also include 20 wt% PQQ and 80 wt% PVA. In other words, all 30 repeating PVA units contain one PQQ molecule, and all PVA molecules contain about 6 to about 16 PQQ molecules, preferably about 7 to about 8 PQQ molecules. See FIGS. 20B and 20C. PQQ / PVA conjugates were synthesized by reacting PQQ with PVA in DMF solution using CDI as a dehydrating agent. A typical load level is about 20 wt%. The pure product was obtained after hydrolysis and lyophilization. Purity was confirmed by HPLS. The ligated product was characterized by various methods.

PQQ-Linked Polymer Additional PQQ-linked polymers can be synthesized as disclosed below and in FIGS. 54A-JJ. These compounds provide a variety of candidates for controlling PQQ release. PEG-PNH ₂ is one of the candidates. Various molecular weights (thousands to 20K) and 1 to 2 amine groups at the end of the main chain are commercially available. Amide bonds are expected to have longer release times compared to ester bonds in PQQ linked PVA systems.

PQQ linked polymer: mooring masking and non-tethering masking 1. Linkage with polymer having neutral charge and minimum atomic volume 2. Linkage with minimum / minimum lipophilic polymer. 3. Linkage with intermediate level lipophilic polymer 4. Linkage with highly lipophilic polymer 5. Linkage with highly hydrophilic polymer 6. Linkage with amphoteric polymer Linking with a polymer with basic properties 8. 8. Link with borderline acidic polymer 9. Linkage with short polymer-for increased biosorption 10. Linkage with a polymer having a long backbone length to suppress bioadsorption 11. Linkage with short chain fatty alcohol moiety (<C6) 12. Linkage with long chain aliphatic moieties (> C6-C14) 13. Linkage with very long chain aliphatic moiety (> C14) 15. Linking with telechelic and non-telechelic polymers 15. Linkage with aromatic non-bioactive alcohol Ligation with small amines (non-hydrolyzable products)
17. Ligation with medium amines 18. Ligation with large amines (long half-life) 19. Swellability / Linking with hydrogel polymer 20. Connection with thermosensitive polymer 21. Linkage with electroactive polymer 22. Linkage with time-resolved linker-mediated polymer 23. pH-reactive polymer linkage 24. Photoreactive polymer linkage 25. Surface & Matrix Assisted Adsorbable Polymer Linkage 26. Kinetic polymer linkage 27. Linkage with biopermeable polymer 28. Cell-absorbing polymer coupling 29. Thermodynamic equilibrium (reciprocating) polymer linkage 30. Ligation with homo and heteropolymers of isotactic, syndiotactic and atactic nature In vitro PQQ release kinetics PQQ release kinetics can be tested in vitro using human plasma or pure esterase. Provides results for future clinical studies. Established HPLC methods can also be used for this study.

Example 8: PQQ in combination with probenecid reduces nephrotoxicity Method:
PQQ / provenesid / PQQ analog-rat Run Date: January 11-January 13, 2005 Dose PQQ: 25 mg / kg
PQQ analog: Based on PQQ equivalents (25 mg / kg), not by total weight.

Volume probenecid: 100 mg / kg IP (5 ml / kg of 20 mg / ml solution)
Formulation:
PQQ:
Made from 2% NaHCO ₃ just before use:
1) Add DD H ₂ O to 2 grams of NaHCO ₃ to make 100 ml = 2% NaHCO ₃
2) 75 mg PQQ + 15 ml 2% NaHCO ₃ = 5 mg PQQ / ml
Probenecid:
1) Weigh 600 mg probenecid 2) Add 27 ml DD H ₂ O 3) 4-5 drops 19.1N NaOHOH
4) Stirring 5) Bring pH to 7.4 with 1.0N KH ₂ PO 4 (add DDH ₂ O to 1.36 grams potassium dihydrogen phosphate to 10 ml)
(Requires 0.5ml to 1.2ml)
6) Add DD H ₂ O to 30 ml ft just before use
PQQ analog: 10 mg / ml solution in saline. Note that analog 81 did not become 100% solution.

Experiment: 5 female SD / group Group 1: Control-no treatment Group 2: 5 ml PQQ / kg IV
Group 3: 5 ml probenecid / kg IP; 5 ml PQQ IV 30 minutes, probenecid repeat after 6.0 hours Group 4: PVA-PQQ-80, 1.9 mg PQQ / ml, 13.2 ml / kg
Group 5: PVA-PQQ-81, 2.2 mg PQQ / ml, 11.3 ml / kg
After 48 hours, animals are sacrificed, blood is drawn for BUN, creatinine, serum phosphorus, and kidneys are removed for weight and histopathology.

結論：
図２７は、単独投与のＰＱＱと対照とが有意に異なることを示している。しかしながら、プロベネシドと組み合わせたＰＱＱ（または類似物８０および８１）は、対照と比較して有意には異ならなかった。従って、プロベネシドと組み合わせて投与されたＰＱＱは、腎毒性を低減するのに有用である。

Conclusion:
FIG. 27 shows that the single dose PQQ and the control are significantly different. However, PQQ (or analogues 80 and 81) in combination with probenecid was not significantly different compared to the control. Thus, PQQ administered in combination with probenecid is useful for reducing nephrotoxicity.

Example 9: PQQ prevents actin nitration and TNF-induced barrier failure in endothelial cell monolayers The small pulmonary artery is a major determinant of pulmonary artery pressure and vascular resistance. Their endothelium regulates lung resistance, remodeling, and blood fluidity. The effect of PQQ on lung microvascular endothelial cell culture was studied to determine the benefits of using PQQ to treat vascular injury and vascular injury related diseases.

Materials / Reagents:
All reagents were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.

Lung Microvascular Endothelial Cell Culture Rat pulmonary microvascular endothelial cells (RLMVEC) and bovine pulmonary microvascular endothelial cells (BLMVEC) were obtained in the fourth passage (Vec Technologies, Rensselaer, NY). The preparation was confirmed by Vec Technologies to be a pure population by: (i) a characteristic “cobblestone” appearance as assessed by phase contrast microscopy, (ii) Factor VIII related antigens (Iii) uptake of acylated low density lipoprotein and (iv) absence of smooth muscle actin (indirect immunofluorescence). For all studies, both RLMVEC and BLMVEC are both 20% fetal calf serum (Hyclone, Hyclone Laboratories, Logan, Utah), 15 μg / ml Endothelial Cell Growth Supplement (Upstream Biopenement (Upplk). 10% fetal calf serum (VEC Technologies) for RLMVEC in medium containing Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY) supplemented with% non-essential amino acids (Gibco-BRL) In MCDB-131 complete medium containing The sage was cultured. Both cell lines were maintained at 37 ° C. in 5% CO ₂ + humid air. The confluent pulmonary microvascular endothelial cell monolayer (PMEM) was reached in 2 to 3 population doublings that took 3-4 days.

Treatment TNF treatment: Highly purified recombinant human TNFα from Escherichia coli (Calbiochem-Novabiochme, La Jolla, Calif.) In 10 μg / ml stock solution was used. Endotoxin levels were less than 0.1 ng / μg TNFα as determined by standard Limulus assay. We have previously shown that boiling TNFα for 0.75 hours prevents the effect of TNF in our system (14), which shows no endotoxin contamination. PMEM was treated with TNFα at 100 ng / ml. This is because dose response studies show that this dose consistently causes increased permeability.

Anti-ONOO ^- agent: ONOO ^- inhibitors used were urate (5FM) and PQQ (1 μM). The inventors have previously shown that urate scavenges TNF-induced ONOO ⁻ and has no effect on cell viability in the endothelium (30). PQQ is a putative vitamin and superoxide anion radical scavenger. Cells were treated with urate or PQQ alone or co-treated with urate, PQQ and TNF.

  Treatment vehicle: For all studies, incubation of PMEM with TNF, PQQ, urate and all corresponding controls was phenol-free DMEM supplemented with 10% FBS to avoid the potential antioxidant effect of phenol. (Pf-DMEM, Gibco BRL).

Endothelial permeability assay Nucleopore Track-Etch Polycarbonate Membrane (13 mm diameter, pore size 0.8 mm; Corning Costar, Cambridge, Mass.) Coated with gelatin (type B from bovine skin; Sigma) and modified Boyden chemotaxis In a chamber (9 mm inner diameter; Adaps, Dedham, MA) MF cement no. 1 (Millipore, Bedford, Mass.) And sterilized with ultraviolet light for 12.0-24.0 hours as previously described (8, 16). BLMVEC or RLMVCEC (1.5 × 10 5 in 0.50 ml DMEM) were cultured on gelatinized membranes to reach fusion within 3-5 days (37 ° C., 5% CO ₂ ).

  An experimental device for the study of transendothelial transport without hydrostatic and colloid osmotic gradients has been described (16). Briefly, this system consists of two compartments separated by a microporous polycarbonate membrane lined with an endothelial cell monolayer as described above. The luminal (upper) compartment (0.7 ml) was suspended in the abluminal (lower) compartment (25 ml). The lower compartment was continuously stirred for complete mixing. The entire system was kept in a water bath at a constant temperature of 37 ° C. The fluid height in both compartments was the same to eliminate convection.

  Endothelial permeability was characterized by the clearance rate of Evans Blue-labeled albumin using a modification of the authors of the original technique described by Patterson et al. (29) (16). A buffer solution containing Hanks'Blansed Salt Solution (HBSS, Gibco-BRL) containing 0.5% bovine serum albumin and 20 mM (2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES) buffer. Used on both sides of the monolayer. The luminal compartment buffer was labeled with a final concentration of 0.057% Evans Blue dye in a volume of 700 μl. The absorbance of free Evans Blue in the luminal and aluminal compartments was less than 1% of the total absorbance of Evans blue in the buffer. At the beginning of each study, the luminal compartment sample was diluted 1: 100 to determine the initial absorbance of the compartment. Abluminal compartment samples (300 ml) were taken every 5 minutes for 60 minutes. The absorbance of the samples was measured at 620 nm with a SpectraMax Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, California). The clearance rate of Evans Blue labeled albumin was determined by least square linear regression between 10 and 60 minutes for the control and experimental groups.

Immunofluorescence and confocal microscopy Cell preparation and antibody treatment: RLMVCEC or BLMVEC (1 × 104 / 0.20 ml medium) were grown on 18 mm coverslips in 35 mm culture dishes and incubated for 2 hours at 37 ° C. for attachment And then grown to fusion in additional 2 ml of medium (16). PMEM is treated as indicated, washed with Dulbecco's phosphate buffered saline (DPBS, Gibco BRL), fixed with 3.7% formaldehyde solution for 20 minutes at room temperature (RT), then 1 in DPBS. Permeabilized with% Triton X-100 for 5 minutes at RT. Cells were washed with DPBS and then blocked in 10% normal goat serum (Ngs, Gibco BRL) for 5 minutes at RT. PMEM was incubated with mouse monoclonal anti-nitrotyrosine antibody (clone 1A6, Upstate) diluted 1: 1000 in 10% NGS. A secondary antibody, Alexa Fluor 488 labeled goat anti-mouse IgG (Molecular Probes, Eugene, OR) was added at a 1: 1000 dilution in 10% NGS, incubated for 1 hour at RT, and then washed thoroughly. Total β-actin was stained with mouse monoclonal anti-β-actin antibody (clone AC74) followed by Alexa Fluor 568-labeled goat anti-mouse IgG (Molecular Probes).

  The quantification procedure for the fluorescence image is as follows. PMEM is visualized and quantified by confocal microscopy using Leica Confocal System TCS SP2 (Leica Microsystems Inc., Exton, Pa.). There were 4 separate studies with 4 treatment groups and 2 treatment times per study. All fields were selected by random movement of the microscope stage to another area within the intact endothelial monolayer. All six fields per treatment group were analyzed with one image per field. All treatment groups were normalized for fluorescence intensity by first adjusting the settings for noise, brightness and contrast as determined by the slide with maximum fluorescence (16).

  The specificity of the anti-nitrotyrosine antibody was confirmed by antibody-antigen competition. A 10: 1 molar ratio of nitrotyrosine antibody and nitrotyrosine antigen was preincubated at 37 ° C. for 30 minutes in 10% NGS before application to PMEM. Cover slips were mounted on clean glass slides with Permafluor mounting medium (Thermo Shandon, Pittsburgh PA). POT is a Spot RT color camera (Diagnostic Instruments, Inc., SterlingHeighings, Inc.) installed on an Olympus IX70 inverted microscope (Olympus America, Inc., Melville, NY) equipped for phase, light, and fluorescence detection. ) And visualized. An illustrative image was captured at 100 × magnification and an exposure time of 8 seconds, and the image was downloaded to Spot RT imaging software (Diagnostic Instruments, Inc.) (16).

Statistics One-way analysis of variance (ANOVA) was used to compare values between treatments. If significance was confirmed between treatments, a post hoc multiple comparison test was performed with Bonferrone (parametric-equal variance) or Duncan (nonparametric-unequal variance) test to determine significant differences between groups (37 ). When appropriate, a log10 transformation was performed to smooth the data. Each PMEM well and flask represents a single experiment. All data are reported as mean ± SEM. The superiority was p <0.05. In all studies, there are 5-10 samples per group.

CONCLUSION We have found that tumor necrosis factor-α (TNF-α) is a peroxy-subductant in the permeability of pulmonary microvascular endothelial monolayer (PMEM) associated with nitrated β-actin (NO ₂ -β-actin). The hypothesis that it causes an increase in nitrate (ONOO ⁻ ) dependence was tested. The permeability of PMEM was evaluated by the clearance rate of albumin labeled with Evans Blue. Cell compartmentalization of NO ₂ -β-actin was indicated by showing confocal localization of nitrotyrosine-immunofluorescence with β-actin-immunofluorescence. Incubation of PMEM with TNF (100 ng / ml) for 0.5 and 4.0 hours resulted in increased albumin permeability. At 0.5 hours, there was confocal localization of nitrotyrosine-immunofluorescence with β-actin-immunofluorescence. Increased confocal localization and permeability of nitrotyrosine-immunofluorescence with TNF-induced β-actin-immunofluorescence was prevented by the anti-ONOO ^- drug urate (5 μM) and PQQ (1 μM). This _data, ONOO related to the generation of _NO 2-.beta.-actin ^- indicates that caused by the dependence of barrier dysfunction TNF.

Our studies have shown that PQQ is (i) TNF-induced nitrotyrosine increase, (ii) coexistence of nitrotyrosine and β-actin, and (iii) increased permeability of lung microvascular endothelial monolayers. It is further shown to prevent this. Thus, PQQ prevents TNF-induced ONOO ^- dependent endothelial cell failure. Thus, the development of strategies using PQQ and urate provides a new direction for the treatment of vascular injury and vascular injury related diseases.

Example 10: Neuroprotection by PQQ Pyrroloquinoline quinone (PQQ) is a free, water-soluble anionic compound, which is a redox cycling planar orthoquinone with potential free radical scavenger properties. PQQ-dependent enzymes such as methyl alcohol and alcohol dehydrogenase bind to PQQ as a prosthetic group and also contain cytochrome c that accepts electrons and provides them to ubiquinones that function as electron carriers in the mitochondrial respiratory chain. .

  PQQ has been demonstrated to suppress N-methyl-D-aspartate (NMDA) -induced electrical responses and is neuroprotective against NMDA-mediated neurotoxic injury in vitro, Jensen et al. (Neuroscience 62 (1994) 399-406) showed that PQQ administered intraperitoneally 30 minutes before hypoxia was neuronal in an in vivo cerebral hypoxia / ischemia (bilateral carotid artery ligation combined with hypoxia) model in 7-day-old pups. It has been shown to reduce infarct size without causing behavioral side effects. However, it was performed to determine whether PQQ given systemically could improve neurobehavioral outcome and protect the infarcted brain caused by a localized cerebral ischemia model in adult animals There is no previous research. Therefore, the efficacy of PQQ in providing neuroprotection as assessed by 2 hours of reversible middle cerebral artery occlusion (rMCAo) following neurobehavioral and infarct size measurements in adult rats was evaluated. A dose response curve for PQQ versus infarct volume was also characterized.

Materials and Methods Animal Models All animal experimental procedures were approved by the Institutional Animal Care and Use Committee following guidelines for laboratory animal care and use. Male Sprague-Dawley rats (300-350 g, Taconic, Germantown, NY) were anesthetized with isoflurane in a sealed chamber after intramuscular injection of 50 mg / kg atropine sulfate (Sigma, St. Louis, MO). The rats were then tracheally inserted and mechanically ventilated with 2.0% isoflurane in 30% O ₂ / residue N ₂ . Blood gas analysis demonstrated that PaCO ₂ was 30 mm to 45 mm Hg and PaO ₂ was 90 mm Hg or more. Body temperature was monitored with a rectal probe and maintained between 37.0 ° C. and 37.5 ° C. using a heating pad. Temporal muscle temperature was used to reflect brain temperature and was maintained between 36.0 ° C. and 37.0 ° C. using a heating lamp. One femoral artery was cannulated for pressure monitoring and blood gas sampling.

  Reversible middle cerebral artery occlusion was performed as described by Long et al. (Stroke 20 (1989) 84-91), as used previously in our laboratory. A 4-0 nylon intraluminal suture was introduced into the right internal carotid artery (ICA) via the external carotid artery (ECA). The common carotid artery and ICA were temporarily clipped and the suture was placed in the ECA stump, moved away from the ICA and gently advanced ~ 20 mm until resistance was felt. The suture was left in place for 2 hours and then withdrawn. PQQ (10 mg / kg, Sigma, St. Louis, MO) is dissolved in phosphate buffered saline (10 mM solution), and a volume of 1 ml is 10, 3, or The jugular vein was injected to deliver a 1 mg / kg dose. Vehicle treated controls received an equal volume of phosphate buffered saline. The investigator was unaware of whether the animals were treated with vehicle or PQQ injection. Body temperature and brain temperature were maintained throughout the experiment until the animals were fully recovered from anesthesia and returned to their cages. After 72 hours, the animals were killed and the brain was examined.

Neurobehavioral defect scoring Neurobehavioral defect scoring was based on the 18-point scale described by Garcia et al. (Stroke 26 (1995) 627-634). Neurological status was recorded daily in each rat for 3 days starting 24 hours after ischemia. Each subject was tested late in the afternoon to avoid any effects of circadian rhythm. Investigators assessing neurobehavioral deficiencies were not informed whether the vehicle was administered or PQQ was administered. The neurobehavioral scale consisted of the following six trials: 1) spontaneous activity (0-3 points); 2) limb movement symmetry (0-3 points); 3) forelimb extension (0 ~ 3 points); 4) climbing (1-3 points); 5) body self-acceptance (1-3 points), and 6) nasal hair contact response (1-3 points). The score given to each rat upon completion of the evaluation is the sum of a total of 6 individual tests. The minimum neurological score is 3 and the maximum is 18.

Measurement of infarct volume Infarct volume was measured as 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St. Clinton) as used previously in our laboratory (Neuroport 11 (2000) 2675-2679). (Louis, MO) staining. 72 hours after ischemia, rats were injected with 120 mg of pentobarbital. The brain was then removed and cut into 2 mm sections. Slices were placed in a Petri dish containing 2% TTC for 30 minutes, periodically stirred so that none of the slices remained at the bottom, and then placed in 10% formaldehyde. The total volume of unstained tissue (mm ² ) (SigmaScan Pro, SPSS software) was multiplied by the 2 mm slice thickness to calculate the damage volume.

Statistical analysis Statistical evaluation of neurobehavioral scores was performed by repeated measures ANOVA (Statistica, StatSoft Inc.). A comparison was made between the treatment group and the corresponding vehicle group for assessment of infarct volume. A non-parametric Mann-Whitney test was used to evaluate the volume of the non-normal distribution. Differences were considered statistically significant at P <0.05.

Results Neuroprotection with 10 mg / kg PQQ PQQ was initially studied at a dose of 10 mg / kg based on a previous report by Jensen et al. (Neuroscience 62 (1994) 399-406). The infarct volume is 319 mm ³ (SD: 96.2; n = 7) in vehicle-treated animals and 50 mm ³ (SD: 39; n = 7) in animals given 10 mg / kg PQQ immediately before the onset of ischemia. 8) was significantly less (p <0.01; Mann-Whitney test). Infarct volume is 362 mm ³ (SD: 110; n = 5) in vehicle-treated animals and significantly less at 67 mm ³ (SD: 53; n = 8) in animals given PQQ 3 hours after the onset of ischemia (P <0.05; Mann-Whitney test). These data are shown in FIG. 31A and FIG. Behavioral scores were also better in the PQQ treated group when given PQQ just before the onset of ischemia and 3 hours after the start of ischemia, as shown in FIGS. 33A and 33B, compared to the corresponding vehicle treated controls. there were.

Neuroprotection by PQQ at 3 mg / kg and 1 mg / kg 10 mg / kg PQQ given 3 hours after the onset of ischemia appeared to be as effective as PQQ administration at the same time as ischemia. The effect of different capacities after time was tested. This is because 3 hours after the onset of ischemia provides a therapeutic opportunity for treatment (Stroke 30 (1999) 2752-2758). When PQQ was given at 3 mg / kg 3 hours after the start of ischemia, the infarct volume was 406 mm ³ (SD: 114; n = 10) in the vehicle-treated animals and 120 mm ³ (SD: 47; n = 8) (p <0.01; Mann-Whitney test; FIG. 2A, FIG. 3). At this dose, behavioral scores were also better in the PQQ group compared to the vehicle group (FIG. 33C). A dose response curve is shown in FIG. 31B.

When PQQ was given at 1 mg / kg 3 hours after ischemia, the infarct volume was 316 mm ³ (SD: 132; n = 6) in the vehicle treated animals and 328 mm ³ (SD: 112) in the PQQ treated animals. N = 6) and no significant difference was observed (p>0.05; Mann-Whitney test; FIG. 2A). Behavioral scores were also not significantly different in the PQQ treated group compared to the vehicle treated group (FIG. 33D).

  FIG. 32 shows four representative slides of normal sham control animals, vehicle treated animals, PQQ 10 mg / kg treated animals and PQQ 3 mg / kg treated animals.

DISCUSSION This study is the first study to investigate the neuroprotection of PQQ assessed by both infarct volume and neurobehavioral outcome in a widely used model of localized reversible middle cerebral ischemia / reperfusion in adult rats. The data show that PQQ is effective in providing neuroprotection at the behavioral and infarcted volume when given before ischemia or 1 hour after reperfusion, and the neuroprotection provided by PQQ is capacitive It has proved to be relevant.

  Some properties of PQQ could be involved in neuroprotection. First, PQQ can inhibit peroxynitrite formation. It has been proposed that the neurotoxicity of nitric oxide in ischemic stroke depends on its conversion to peroxynitrite. As a cofactor for free radical scavengers and quinoprotein enzymes, PQQ can inhibit peroxynitrite formation. Second, PQQ can oxidize NMDA receptor redox sites. Pathological activation of NMDA receptors has been implicated in various CNS diseases including ischemia. Third, PQQ has been shown to be able to function as an effective antioxidant in protecting mitochondrial lipids and proteins and to protect mitochondrial function from oxidative damage.

  In summary, we found that PQQ reduces infarct size and improves behavioral scores when given as a single dose 3 hours after the start of 2 hours of rMCAo. Under these conditions, PQQ is effective at 3 mg / kg and 10 mg / kg, but not at 1 mg / kg. Thus, PQQ, acting as an essential nutrient, antioxidant and redox modulator in various systems, is a new class of drugs that provides effective neuroprotection and has potential use in the treatment of adult seizures.

Example 11: Pharmacokinetics of PQQ in rats In order to evaluate the response to PQQ with and without probenecid, we started to measure PQQ concentrations over time in rat plasma. Group A (rats 1-3) received 20 mg PQQ / kg i. v. Administered. Group B (rats 4-6) received 100 mg probenecid / kg i. p. 30 mg later, 20 mg PQQ / kg i. v. Administered. Blood was collected from both Group A and Group B rats at 0 minutes, 5 minutes, and 30 minutes after administration, and 1 hour, 2 hours, 4 hours, and 6 hours after administration.

Sample preparation:
Rat blood: 100 μl rat blood + 60 μl heparinized saline, centrifuged; 60 μl plasma was quantitatively pipetted into test tubes and frozen at −80 ° C. until analysis.

  Calibration sample: Dilute rat plasma with saline (80:60, v / v) and use it to add a set of calibration curve samples with PQQ standards ranging from 31.25 to 2500 ng / ml rat plasma Prepared by. See FIG.

result:
The results of rat plasma PQQ concentrations for each of Group A and Group B rats are shown in FIG. 35 and Table 4 below. Rat plasma samples were prepared by two-step extraction and diluted 2-100 fold prior to HPLC analysis.

図３６は、群Ａおよび群Ｂにおける各時点について平均値の血漿ＰＱＱ濃度−時間曲線の比較を例示している。

FIG. 36 illustrates a comparison of mean plasma PQQ concentration-time curves for each time point in Group A and Group B.

Example 12 Use of PQQ and probenecid to prevent / reduce oxidative stress in vivo Male Sprague-Dawley rats are randomized with pyrroloquinoline quinone (PQQ), probenecid, or both prior to ischemia or ischemia-reperfusion. Processed. PQQ (1-3 mg / kg) and / or probenecid (100 mg / kg) injected intraperitoneally (pretreatment) 30 minutes prior to left anterior descending coronary artery (LAD) occlusion, or intravenously at the beginning of reperfusion (treatment) Given by. Rats were subjected to 15 or 30 minutes of LAD occlusion and 30 minutes, 1 hour or 2 hours of reperfusion with left ventricular (LV) hemodynamic monitoring. PQQ in combination with probenecid reduced infarct size in these rat models. PQQ in combination with probenecid prevents ischemia-induced cardiac dysfunction, and after 30 minutes to 2 hours of reperfusion, higher LV systolic pressure, LV onset pressure, LV (+) dP / dt and lower It had LV (−) dP / dt. Creatine kinase (CK) production was reduced by PQQ in combination with probenecid. Thus, PQQ in combination with probenecid is highly effective in reducing myocardial infarct size and improving cardiac function in a dose-related manner in a rat model of ischemia and ischemia-reperfusion.

Statistical analysis All results are presented as mean ± SEM. Two treatment groups (pretreatment and treatment) were compared to normal control groups using one-way analysis of variance (ANOVA) with regression equations for multiple group comparisons. Differences in mortality during the period of occlusion and reperfusion among the three groups were assessed by chi-square test. The percentage of rats with VF was assessed by Fisher's exact test. All calculations were performed using the general linear model procedure of Minitab version 7.2 (Minitab Statistical Software) or Primer of Biostatistics: The program version 3.03 (McGraw-Hill). Statistical significance was set at p <0.05.

Was dissolved reperfusion model PQQ in the vehicle (2% NaHCO ₃₎ - ischemia and ischemia. The volume given either intraperitoneally (ip) or intravenously (iv) was 1 ml. All controls were treated with 1 ml vehicle. 1-3 mg / kg PQQ i. 30 min before 15 or 30 min ischemia. v. Followed by 30 minutes of 1 or 2 hours of reperfusion.

600 mg probenecid was dissolved in 27 ml ddH ₂ O. 4-5 drops of 19.1N NaOH were added and the pH was adjusted to 7.4 with 1.0N KH ₂ PO ₄ . Probenecid was given 30 minutes prior to 15 or 30 minutes of ischemia followed by 30 minutes, 1 hour or 2 hours of reperfusion.

  After induction of anesthesia (ketamine 80 mg / kg, xylazine 4 mg / kg body weight, intraperitoneal), a tracheotomy was performed and the animals were aerated in a Harvard Respirator (model 683, Harvard Apparatus). Infarct-sized rats were subjected to 2 hours proximal left anterior descending (LAD) coronary artery ligation without reperfusion. This model of ischemia followed by reflow used ischemia-reperfusion as previously described (Sievers RE et al. Magn Reson Med 1989; 10: 172-81). In this model, a reversible coronary snare occluder was placed around the proximal LAD coronary artery by a midline sternotomy. Rats were then subjected to 15 or 30 minutes of LAD occlusion and 30, 60 or 120 minutes of reflow. In addition, these rats were recorded hemodynamic measurements. A 4F Millar catheter was inserted through the right carotid artery into the left ventricle (LV). After 20 minutes of equilibration, the heart rate (HR), systolic pressure (LVSP), end diastolic pressure (LVEDP), LV (+) dP / dtmax, LV (−) dP / dtmax were expressed as MacLab / 4S (Milford , MA). LV generation pressure (LVDP) was calculated by subtracting LVEDP from LVSP.

  At baseline, there were no significant differences in heart rate, LVSP, LVEDP, LV (+) dP / dt, and LV (−) dP / dt between the control, pretreatment and treatment groups. PQQ in combination with probenecid, whether given by pretreatment or given as treatment, prevents ischemia-induced cardiac dysfunction, as shown in FIGS. 5-9, for 30 minutes, 1 hour Or after 2 hours of reperfusion, it had higher LVSP, LVDP, LV (+) dP / dt, and lower LV (−) dP / dt. (* <0.05 vs. previously published I / R (control) data by ANOVA using the Student-Newman-Keuls test. Zhura, Journal of Cardiovascular Pharmaceutical and Therapeutics; 11 (2): 119-128 (200-200). )) And FIGS.

梗塞サイズ
虚血−再灌流ラットモデルを用い、ラットを、１７分間または３０分間の左前下降冠動脈結紮および２時間の再灌流に供し、梗塞サイズを、以前に記載された通りに測定した（Ｓｉｅｖｅｒｓ ＲＥら，Ｍａｇｎ Ｒｅｓｏｎ Ｍｅｄ １９８９；１０：１７２−８１、Ｚｈｕ Ｂ−Ｑら，Ｊ Ａｍ Ｃｏｌｌ Ｃａｒｄｉｏｌ １９９７；３０：１８７８−８５）。心臓を、２時間の虚血期間の終わりに切り出した。次いで、切片を、生存可能な心筋が赤レンガ色に着色されるまで塩化トリフェニルテトラゾリウム（ＴＴＣ）の１％溶液中で１０〜１５分間インキュベートした。

Infarct size Using an ischemia-reperfusion rat model, rats were subjected to 17 min or 30 min left anterior descending coronary artery ligation and 2 hr reperfusion, and infarct size was measured as previously described (Sievers RE Et al., Magn Reson Med 1989; 10: 172-81, Zhu B-Q et al., J Am Coll Cardiol 1997; 30: 1878-85). The heart was excised at the end of the 2 hour ischemic period. The sections were then incubated for 10-15 minutes in a 1% solution of triphenyltetrazolium chloride (TTC) until viable myocardium was colored red brick.

  In model 2, after 2 hours of reperfusion, LAD was reoccluded and phthalocyanine dye (Engelhard Cooperation, Louisville, KY) was injected into the LV cavity and the normally perfused myocardium stained blue. The heart was then excised, rinsed with excess dye, and sliced transversely from the top to the base into 2 mm thick sections. Sections were incubated in TTC as described above. Infarcted myocardium cannot be stained with TTC. Tissue sections were then fixed in 10% formalin solution and weighed. Color digital images of both sides of each transverse slice were obtained using a video camera (Leica DC 300F) connected to a microscope (Stereo Zoom 6 Photo, Leica). Areas showing tissue stained blue (non-ischemic), tissue stained red (ischemic but non-infarcted) and unstained tissue (infarcted) are outlined on each color image. Draw and measure in a blinded fashion using NIH Image 1.59 (National Institutes of Health, Bethesda, MD). On each side, the absolute weight of the infarct-related tissue was determined by multiplying the proportion of LV area representing the infarct-related tissue (average of two images) by the weight of the section. The infarct size for each heart was expressed as follows:

次いで、危険領域のパーセンテージとしての梗塞サイズを、以下の通り計算した：

The infarct size as a percentage of the risk area was then calculated as follows:

虚血モデル（モデル１）では、ＰＱＱ後の梗塞サイズ（梗塞量／ＬＶ量、フタロシアニン青色色素は注射されていない）は、対照よりも小さかった（図１３）。モデル２における実験の第１セットにおいて、虚血は１７分間であり、その後、２時間の再灌流が行われ、梗塞サイズ（梗塞量／危険領域、梗塞量／ＬＶ量）は、２０ｍｇ／ｋｇのＰＱＱでの前処置によって減少した（図１４）。モデル２の実験の第２セットにおいて、虚血は３０分間であり、その後、２時間の再灌流が行われ、１５ｍｇ／ｋｇのＰＱＱでの前処置または処置のいずれかの後での梗塞サイズは、対照よりも小さかった（図１５）。

In the ischemia model (Model 1), the infarct size after PQQ (infarct volume / LV volume, phthalocyanine blue dye was not injected) was smaller than the control (FIG. 13). In the first set of experiments in Model 2, ischemia is 17 minutes, followed by 2 hours of reperfusion, and the infarct size (infarct volume / risk area, infarct volume / LV volume) is 20 mg / kg. Decreased by pretreatment with PQQ (FIG. 14). In the second set of Model 2 experiments, ischemia is 30 minutes, followed by 2 hours of reperfusion, and the infarct size after either pretreatment or treatment with 15 mg / kg PQQ is , Smaller than the control (Figure 15).

  Figures 42 and 43 show that the combination of PQQ and probenecid reduces infarct size as both a percentage of risk area and a percentage of left ventricular volume. FIG. 44 shows that the combination of PQQ and probenecid decreased the increase in creatine kinase. Therefore, the combination of PQQ and creatine kinase is effective in preventing and reducing oxidative stress in vivo, particularly related to cardiac pathology. Tables 10-15 below more fully illustrate the toxicity of PQQ administration.

（表１４ 病理学）
生理食塩水 対照
１−５ 正常な組織学的構造
１００ｍｇプロベネシド
６−１０ 正常な組織学的構造
１０ｍｇＰＱＱ／ｋｇ
１１−１５ 正常な組織学的構造
２０ｍｇＰＱＱ／ｋｇ
１６−２０ 細管が若干拡張
４０ｍｇＰＱＱ／ｋｇ
２１ 非常に重篤な皮質細管上皮外傷
全面的な細管上皮壊死（近位および遠位細管上皮）
糸球体および集合管は存続
２２ ＃２１に同じ
２３ 軽度の外傷、５％壊死細管
２４ ＃２１に同じ
２５ ＃２１に同じ
１００ｍｇプロベネシド／ｋｇ、１０ｍｇ ＰＱＱ／ｋｇ
２６−３０ 正常な組織学的構造
１００ｍｇプロベネシド／ｋｇ、１０ｍｇ ＰＱＱ／ｋｇ
３１−３５ １６−２０と同様に、細管が若干拡張
１００ｍｇプロベネシド／ｋｇ、１０ｍｇ ＰＱＱ／ｋｇ
３６−４０ 本質的に＃２１に同じ
イヌを用いた予備的研究におけるプロベネシドと併用または併用しないＰＱＱ投与の毒性学研究を以下の表１５に示す。

(Table 14 Pathology)
Saline Control 1-5 Normal histological structure
100 mg probenecid 6-10 normal histological structure
10mgPQQ / kg
11-15 Normal histological structure
20mg PQQ / kg
16-20 Capillary tube slightly expanded
40mgPQQ / kg
21 Very severe cortical tubule epithelial trauma Total tubular epithelial necrosis (proximal and distal tubule epithelium)
Glomeruli and collecting ducts continue to be the same as 22 # 21
100 mg probenecid / kg, 10 mg PQQ / kg
26-30 Normal histological structure
100 mg probenecid / kg, 10 mg PQQ / kg
31-35 Similar to 16-20, narrow tube slightly expanded
100 mg probenecid / kg, 10 mg PQQ / kg
36-40 The toxicology study of PQQ administration with or without probenecid in a preliminary study with essentially the same dog as # 21 is shown in Table 15 below.

イヌ１３５７：注入毒性無；２日目および３日目に重篤な外傷；用量がＭＴＤを十分上回ると思われたため３日目に屠殺；臨床所感は、もう１日も持たないであろうと思われた。

Dog 1357: no infusion toxicity; severe trauma on days 2 and 3; sacrificed on day 3 because dose seemed well above MTD; clinical experience would not have another day It was broken.

  Dog 1475: Vomiting and nausea (injection toxicity) 1 hour after treatment; thereafter seemed completely normal.

  The results of treatment with probenecid in rats for ischemia-reperfusion are shown below. Note that the first injection is probenecid and PQQ or NS 30 minutes after ischemia. The second injection is reperfusion 1 hour probenecid. P values are from 2-sample t-test in the case of PQQ group vs. control group.

均等物
当業者は、本明細書に記載される特定の手順に対する多数の均等物を認識するか、または慣例的実験法に過ぎないものを使用してこれを確認することが可能である。このような均等物は、本発明の範囲内にあると考えられ、特許請求の範囲によりカバーされる。様々な置換、変更、および改変は、特許請求の範囲によって規定される本発明の精神および範囲から逸脱することなく、本発明に対して行われ得る。他の態様、利点、および改変は、本発明の範囲内である。本出願全体にわたって引用される全ての参照文献、発行特許、および公開された特許出願の内容は、参照により本明細書に援用される。

Equivalents Those skilled in the art will recognize a number of equivalents to the specific procedures described herein, or may confirm this using only routine experimentation. Such equivalents are considered to be within the scope of this invention and are covered by the claims. Various substitutions, changes and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. Other aspects, advantages, and modifications are within the scope of the invention. The contents of all references, issued patents, and published patent applications cited throughout this application are hereby incorporated by reference.

FIG. 1 is a bar graph showing the increase in viable adult mouse cardiomyocytes after hypoxia by pretreatment with PQQ. FIG. 2 is a bar graph showing that PQQ protection is not inhibited by 10 μM 5-hydroxydecanoic acid (mitochondrial _KATP channel inhibitor). FIG. 3 is a line graph showing that pre-ischemic PQQ treatment maintains left ventricular pressure (LVDP). FIG. 4 is a line graph showing that pre-ischemic PQQ treatment maintains left ventricular end diastolic pressure (LVEDP) (left ventricular systolic pressure-left ventricular end diastolic pressure). FIG. 5 is a line graph showing the effect of PQQ treatment prior to ischemia as measured by the positive maximum first derivative of left ventricular pressure (LVDP). FIG. 6 is a line graph showing the effect of pre-ischemic PQQ treatment as measured by the negative maximum first derivative of left ventricular pressure (LVDP). FIG. 7 is a line graph showing that coronary blood flow is significantly improved by PQQ treatment compared to controls. FIG. 8 is a bar graph showing that a 2 minute pre-treatment with PQQ at several indicated concentrations has an increasingly favorable response between 10 nM and 1 μM, but toxicity occurs at 10 μM. FIG. 9 is a bar graph showing changes in myocardial infarct size after PQQ pretreatment. Infarct formation size gradually decreases between 10 nM and 1 μM, but infarct formation size is not reduced with 10 μM PQQ. FIG. 10 is a schematic diagram showing experimental protocol model 1 (2 hours ischemia) and model 2 (ischemia / reperfusion). Model 2 included two separate experimental sets, set 1: (17 minutes ischemia / 2 hours reperfusion) and set 2: (30 minutes ischemia / 2 hours reperfusion). Pretreated rats are i.p. prior to 30 minutes of ischemia. p. PQQ was received by injection. In the treatment group, PQQ is i. v. Given by injection. Control rats received an equal volume of vehicle at the times indicated. Arrows indicate the timing of PQQ administration. i. p. = Intraperitoneal; i. v. = Intravenous; I = ischemia; LAD = left anterior descending coronary artery. FIG. 11A is a line graph showing left ventricular systolic pressure (LVSP) in model 2 rats during ischemia / reperfusion. Pretreatment with PQQ (PQQ by ip injection prior to 30 minutes of ischemia) or treatment with PQQ (PQQ by iv injection at the start of reperfusion) is LVSP at 2 hours of reperfusion Brought about an increase. B = baseline; I = ischemia; R = reperfusion. FIG. 11B is a line graph showing left ventricular pressure (LVDP) in model 2 rats during ischemia / reperfusion. Treatment with PQQ increased LVDP both after 30 minutes of reperfusion and after 2 hours of reperfusion. Pretreatment with PQQ increased LVDP at 2 hours of reperfusion. B = baseline; I = ischemia; R = reperfusion. FIG. 12A is a line graph showing left ventricle (LV) (+) dP / dt in model 2 rats during ischemia / reperfusion. Pretreatment with PQQ or treatment with PQQ significantly increased LV (+) dP / dt at 2 hours of reperfusion. B = baseline; I = ischemia; R = reperfusion. FIG. 12B is a line graph showing left ventricle (LV) (−) dP / dt in model 2 rats during ischemia / reperfusion. Pre-treatment with PQQ or treatment with PQQ significantly reduced LV (−) dP / dt at 2 hours of reperfusion. B = baseline; I = ischemia; R = reperfusion. FIG. 13 is a bar graph showing myocardial infarct size in Model 1 (only ischemia). Pretreatment with 20 mg / kg PQQ significantly reduced infarct size (infarct volume / LV volume%). Ischemia was induced by 2 hour LAD ligation without reperfusion. FIG. 14 is a bar graph showing myocardial infarct size in model 2 (ischemia / reperfusion). In these experiments, ischemia was induced by 17 minutes of LAD occlusion followed by 2 hours of reflux (reperfusion). Pretreatment with 20 mg / kg PQQ significantly reduced infarct size (when measured in either infarct volume / risk area% or infarct volume / LV volume%). FIG. 15 is a bar graph showing myocardial infarct size for additional experiments in model 2 (ischemia / reperfusion). In these experiments, 30 hours of ischemia was followed by 2 hours of reperfusion. Pretreatment with 15 mg / kg PQQ treated with 15 mg / kg PQQ significantly reduced infarct size (as measured by either infarct volume / risk area% or infarct volume / LV volume%) . P values refer to the respective I / R infarct size measurements. FIG. p. FIG. 5 is a line graph showing the effect of pretreatment with different doses of PQQ on the infarct size of 5 groups of rats pretreated with the indicated range of PQQ doses by injection. There was a strong negative correlation between infarct size and PQQ dose. FIG. 17A is a bar graph showing the average episode of ventricular fibrillation (VF) per rat when data from both Model 1 and Model 2 are combined. Pretreatment with 15-20 mg / kg PQQ significantly reduced the average episode of VF per rat. Analysis was performed by one-way analysis of variance (ANOVA). FIG. 17B is a bar graph showing the percentage of rats with VF using Kumi combined data from Model 1 and Model 2. Pre-treatment with 15-20 mg / kg PQQ or treatment with 15-20 mg / kg PQQ significantly reduced the percentage of rats with VF. Analysis was performed by Fisher's exact test. FIG. 18A is a line graph showing myocardial MDA levels from the anterior segment of LV subjected to 30 minutes of LAD occlusion followed by 2 hours of reperfusion. Pretreatment with 15 mg / kg PQQ significantly reduced MDA in ischemic myocardium. Differences between I / R and treated and non-treated rats (control) were significant by two-way analysis of variance. Sham = Rats subjected to unoccluded LAD coronary artery isolation for the entire study period. FIG. 18B is a line graph showing myocardial MDA levels from the posterior segment of LV (non-ischemic). Pretreatment with 15 mg / kg PQQ also reduced MDA in this non-ischemic isolated myocardium. FIG. 19 is a bar graph showing the respiratory control ratio of mitochondria isolated from rat heart under the following conditions: (i) Control: 3 hours pentobarbital anesthesia, n = 4, (ii) PQQ treatment: 3 mg / Kg, 20 minutes equilibration period, 30 minutes ischemia, PQQ injection, 2 hours reperfusion, n = 5; and (iii) ischemia / reperfusion: 20 minutes equilibration period, 30 minutes imaginary Blood followed by 2 hours of reperfusion, n = 3. FIG. 20A is a schematic diagram showing the synthesis mechanism of PQQ-linked PVA. FIG. 20B is a schematic diagram showing a PVA unit with one PQQ molecule. FIG. 20C is a schematic diagram showing a PVA molecule with multiple PQQ molecules. FIG. 21 shows the retention time of PQQ using a fluorescence detector. FIG. 22 shows the absorption spectrum of PQQ in water. FIG. 23 shows the GPC spectrum of PQQ linked PVA. FIG. 24 shows the GPC spectrum of PQQ using a fluorescence detector. FIG. 25A shows the following UV absorption spectra: (A) PQQ linked PVA (40K molecular weight) with a retention time of 10.19 minutes. FIG. 25B shows the following UV absorption spectra: (B) PQQ linked PVA (10K molecular weight) with a retention time of 13.67 minutes. FIG. 25C shows the following UV absorption spectra: (C) PQQ residue. FIG. 26 is based on the integrated areas of the aromatic peaks at 8.45 ppm and 7.25 ppm and the aliphatic peaks at 3.84 ppm, 1.93 ppm and 1.50 ppm, with loading levels of approximately 1-1.5 (± 0 4) (PQQ units per PVA molecular chain). The load dump level is about 4 (± 2) wt% PQQ in the linked product. FIG. 27 is a photograph of macroscopic pathology of kidney in control mice, mice treated with PQQ alone, mice treated with PQQ combined with probenecid, and mice treated with PQQ combined with PVA. FIG. 28 is a representative microscope of nitrotyrosine fluorescence of PMEM immunostained with monoclonal anti-nitrotyrosine showing the effect of urate and PQQ on anti-nitrotyrosine immunocytological specificity and TNF-induced nitrotyrosine augmentation. It is a photograph (bovine cell). Photomicrograph of PMEM immunostained with the same antibody after pre-incubation of the antibody for 30 minutes at a 10: 1 antigen: antibody ratio with 3-nitrotyrosine. Confocal histogram analysis of nitrotyrosine fluorescence obtained from both rat and bovine controls, urate, PQQ and TNF treated PMEM after 0.5 to 4 hours (N = 4, 6 samplings per treatment) . Statistical differences are determined by one-way analysis of variance with Kruskal-Wallis rank followed by multiple comparisons using Dunn's method. * = Different from control group # = different from each TNF group 29A-B. Urate and PQQ prevent TNF-induced coexistence with nitrotyrosine and β-actin in PMEM. FIG. 2 shows confocal micrographs (bovine cells) of control, PQQ, urate and TNF treated PMEM after 0.5 hours (A) and 4.0 hours (B). Nitrotyrosine is immunostained with anti-nitrotyrosine and appears as green fluorescence. β-actin is immunostained with anti-β-actin and appears as red fluorescence. The color change that results from the combined red and green micrographs appears as yellow when the coexistence is demagnetized (inset: arrow). A total of 4 preparations were generated from both rat and bovine PMEM for each treatment and time point. 29A-B. Urate and PQQ prevent TNF-induced coexistence with nitrotyrosine and β-actin in PMEM. FIG. 2 shows confocal micrographs (bovine cells) of control, PQQ, urate and TNF-treated PMEM after 0.5 hours (A) and 4.0 hours (B). Nitrotyrosine is immunostained with anti-nitrotyrosine and appears as green fluorescence. β-actin is immunostained with anti-β-actin and appears as red fluorescence. The color change that results from the combined red and green micrographs appears as yellow when the coexistence is demagnetized (inset: arrow). A total of 4 preparations were generated from both rat and bovine PMEM for each treatment and time point. FIG. Urate and PQQ prevent the increase in albumin clearance rate in TNF-induced PMEM. The combined data albumin clearance response was obtained from rat and bovine PMEM. Treatment is control, urate, PQQ and TNF over 4.0 hours. Statistical differences are determined by one-way analysis of variance with Kruskal-Wallis rank followed by multiple comparisons using Dunn's method. * = Different from control group # = different from TNF group FIG. Effect on treatment of cerebral infarct size with PQQ 10 mg / kg, 3 mg / kg and 1 mg / kg (iv) (31A). PQQ administered immediately before ischemia (0 hours, vehicle and PQQ 10 mg group) and 3 hours after (3 hours, vehicle and PQQ 10 mg group) significantly reduced infarct volume (p <0.05; Mann− Whitney test). When administered 3 hours after ischemia, PQQ at 3 mg / kg (3 hours, vehicle PQQ 3 mg group) but not 1 mg / kg (3 hours, vehicle PQQ 1 mg group) reduced infarct volume. There is a significant treatment effect in the 3 mg / kg group (p <0.05; Mann-Whitney test), but no significant effect in the 1 mg / kg group (p> 0.05; Mann-Whitney test). FIG. Dose response curve of treatment with PQQ 10 mg / kg, 3 mg / kg and 1 mg / kg (iv) (31B). PQQ administered immediately before ischemia (0 hours, vehicle and PQQ 10 mg group) and 3 hours after (3 hours, vehicle and PQQ 10 mg group) significantly reduced infarct volume (p <0.05; Mann− Whitney test). When administered 3 hours after ischemia, PQQ at 3 mg / kg (3 hours, vehicle PQQ 3 mg group) but not 1 mg / kg (3 hours, vehicle PQQ 1 mg group) reduced infarct volume. There is a significant treatment effect in the 3 mg / kg group (p <0.05; Mann-Whitney test), but no significant effect in the 1 mg / kg group (p> 0.05; Mann-Whitney test). FIG. Normal animal slice (A); Vehicle treated animal slice (B); PQQ 10 mg / kg treated (3 hours after ischemia) animal slice (C); PQQ 3 mg / kg treated animal slice (D) is represented. FIG. Effect on neurobehavioral score of treatment with PQQ 10 mg / kg, 3 mg / kg and 1 mg / kg (iv). Treatment with PQQ 10 mg / kg immediately before ischemia (33A) and 3 hours after (33B) results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect in both groups 32A and 32B (p <0.05; repeated measures ANOVA). Treatment with PQQ 3 mg / kg 3 hours after ischemia results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect when given 3 hours after ischemia in the 3 mg / kg group (4C, p <0.05; repeated measures ANOVA), but 3 hours after ischemia in the 1 mg / kg group There is no significant treatment effect when given (33D, p> 0.05, repeated measures ANOVA). FIG. Effect on neurobehavioral score of treatment with PQQ 10 mg / kg, 3 mg / kg and 1 mg / kg (iv). Treatment with PQQ 10 mg / kg immediately before ischemia (33A) and 3 hours after (33B) results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect in both groups 32A and 32B (p <0.05; repeated measures ANOVA). Treatment with PQQ 3 mg / kg 3 hours after ischemia results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect when given 3 hours after ischemia in the 3 mg / kg group (4C, p <0.05; repeated measures ANOVA), but 3 hours after ischemia in the 1 mg / kg group There is no significant treatment effect when given (33D, p> 0.05, repeated measures ANOVA). FIG. Effect on neurobehavioral score of treatment with PQQ 10 mg / kg, 3 mg / kg and 1 mg / kg (iv). Treatment with PQQ 10 mg / kg immediately before ischemia (33A) and 3 hours after (33B) results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect in both groups 32A and 32B (p <0.05; repeated measures ANOVA). Treatment with PQQ 3 mg / kg 3 hours after ischemia results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect when given 3 hours after ischemia in the 3 mg / kg group (4C, p <0.05; repeated measures ANOVA), but 3 hours after ischemia in the 1 mg / kg group There is no significant treatment effect when given (33D, p> 0.05, repeated measures ANOVA). FIG. Effect on neurobehavioral score of treatment with PQQ 10 mg / kg, 3 mg / kg and 1 mg / kg (iv). Treatment with PQQ 10 mg / kg immediately before ischemia (33A) and 3 hours after (33B) results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect in both groups 32A and 32B (p <0.05; repeated measures ANOVA). Treatment with PQQ 3 mg / kg 3 hours after ischemia results in improved neurobehavioral scores at 24, 48 and 72 hours. There is a significant treatment effect when given 3 hours after ischemia in the 3 mg / kg group (4C, p <0.05; repeated measures ANOVA), but 3 hours after ischemia in the 1 mg / kg group There is no significant treatment effect when given (33D, p> 0.05, repeated measures ANOVA). FIG. 34 shows a calibration curve for PQQ (31.25 to 2500 ng / ml) in rat plasma processed with two-step extraction and determined with an HPLC fluorescence detector (360/460 nm). FIG. 35 shows rat plasma PQQ concentrations for Group A (PQQ alone) and Group B (PQQ + provenesid) rats. FIG. 36 shows rats (n = 20 mg PQQ / kg, i.v.) and B (100 mg probenecid / kg, i.p. followed by 20 mg PQQ / kg, i.v.) rats. The plasma QQ concentration-time curve in 3) is shown. Figure 37 shows baseline, 15 min occlusion, 30 min occlusion, 30 min reperfusion, 60 min reperfusion with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. 2 is a graph showing rat left ventricular systolic pressure (LVSP) during perfusion and 120 minutes of reperfusion. Figure 38 shows baseline, 15 min occlusion, 30 min occlusion, 30 min reperfusion, 60 min reperfusion with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. FIG. 6 is a graph showing rat left ventricular end diastolic pressure (LVEDP) during perfusion and 120 minutes of reperfusion. Figure 39 shows baseline, 15 min occlusion, 30 min occlusion, 30 min reperfusion, 60 min reperfusion with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. FIG. 6 is a graph showing rat left ventricular pressure (LVDP) during perfusion and 120 minutes of reperfusion. FIG. 40 shows baseline, 15 min occlusion, 30 min occlusion, 30 min reperfusion, 60 min reperfusion with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. FIG. 6 is a graph showing the positive maximum first derivative (LV + dp / dt) of the left ventricle of a rat during perfusion and 120 minutes of reperfusion. Figure 41 shows baseline, 15 min occlusion, 30 min occlusion, 30 min reperfusion, 60 min reperfusion with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. FIG. 5 is a graph showing the negative maximum first derivative (LV-dp / dt) of the rat left ventricle during perfusion and 120 minutes of reperfusion. FIG. 42 is a bar graph showing percent infarct size in rats with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. FIG. 43 is a bar graph showing infarct size / risk area percentage and infarct size / left ventricular volume in rats with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. FIG. 44 is a bar graph showing the increase in creatine kinase in rats with or without treatment with a combination of 100 mg / kg probenecid and 2 or 3 mg / kg PQQ. FIG. 45 is a proton NMR spectrum of received PQQ and PQQ / PVA conjugates using d ₆ -DMSO as the solvent labeled at 2.50 ppm. FIG. 46 is a proton H-NMR spectrum of the PQQ / PVA conjugate (using D ₂ O as a solvent labeled as internal reference at 4.79 ppm). FIG. 47 is an ATR mode FT-IR spectrum of PVA powder. FIG. 48 is an ATR mode FT-IR spectrum of PVA powder. FIG. 49 is an ATR mode FT-IR spectrum of the PVA / PQQ conjugate powder. FIG. 50 is an XRD spectrum of two PVA powders. (A) PQQ / PVA conjugate. (B) PVA powder. (D) PQQ powder. (D) Steel substrate. FIG. 50 is an XRD spectrum of two PVA powders. (A) PQQ / PVA conjugate. (B) PVA powder. (D) PQQ powder. (D) Steel substrate. FIG. 50 is an XRD spectrum of two PVA powders. (A) PQQ / PVA conjugate. (B) PVA powder. (D) PQQ powder. (D) Steel substrate. FIG. 50 is an XRD spectrum of two PVA powders. (A) PQQ / PVA conjugate. (B) PVA powder. (D) PQQ powder. (D) Steel substrate. FIG. 51 is a standard calibration curve for a PQQ (10-1,000 ng / ml) assay using HPLC-FLD. FIG. 52 is the quantitative limit (0.2 ng / 20 μl) of the HPLC-FLD method for the PQQ assay. FIG. 53 is the detection limit (0.1 ng / 20 μl) of the HPLC-FLD method for the PQQ assay. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates. Figures 54A-JJ show small molecular weight (SMW) PQQ conjugates.

Claims (39)

  A method for preventing, treating or reducing ischemia-reperfusion injury, wherein a subject in need thereof is administered a therapeutically effective amount of pyrroloquinoline quinone, whereby the subject's Protecting organs and tissues from reperfusion injury caused by ischemic injury.   The method of claim 1, wherein the ischemia-reperfusion injury is heart injury.   The method of claim 1, wherein the pyrroloquinoline quinone is administered in an amount between 1 mg and 10 mg per kg body weight of the subject.   4. The method of claim 3, wherein the pyrroloquinoline quinone is administered in an amount of about 3 mg / kg body weight of the subject.   The method of claim 1, wherein the pyrroloquinoline quinone is administered to the subject prior to the ischemic injury.   The method of claim 1, wherein the pyrroloquinoline quinone is administered to the subject after the ischemic injury.   2. The method of claim 1, wherein the pyrroloquinoline quinone is administered to the subject before, at the start of, or after the start of reperfusion.   The method of claim 1, wherein the ischemia-reperfusion injury is a seizure.   9. The method of claim 8, wherein the stroke is a result of a heart attack.   9. The method of claim 8, further comprising administering a therapeutically effective amount of tamoxifen to a subject in need thereof.   8. The method of claim 7, wherein the pyrroloquinoline quinone is administered in an amount between 1 mg and 10 mg per kg body weight of the subject.   10. The method of claim 9, wherein the pyrroloquinoline quinone is administered to the subject prior to the ischemic injury.   10. The method of claim 9, wherein the pyrroloquinoline quinone is administered to the subject after the ischemic injury.   13. The method of claim 12, wherein the pyrroloquinoline quinone is administered to the subject before, at the start of, or after the start of reperfusion.   The method of claim 1, wherein the ischemia-reperfusion injury is kidney injury.   16. The method of claim 15, wherein the kidney damage is the result of intestinal ischemia or gastrointestinal tract injury.   16. The method of claim 15, wherein the pyrroloquinoline quinone is administered in an amount between 1 mg and 10 mg per kg body weight of the subject.   16. The method of claim 15, wherein the pyrroloquinoline quinone is administered to the subject prior to the ischemic injury.   16. The method of claim 15, wherein the pyrroloquinoline quinone is administered to the subject after the ischemic injury.   16. The method of claim 15, wherein the pyrroloquinoline quinone is administered to the subject before, at, or after the start of reperfusion.   A pharmaceutical composition for treating ischemia-reperfusion injury in a subject in need of treating ischemia-reperfusion injury, comprising the treatment of pyrroloquinoline quinone linked to one or more polymers. A pharmaceutical composition comprising a pharmaceutically effective amount.   The pharmaceutical composition according to claim 21, wherein the polymer is polyvinyl alcohol. Wherein the polymer is a PEG-NH _2, the pharmaceutical composition according to claim 21.   A pharmaceutical composition for treating ischemia-reperfusion injury in a subject in need of treating ischemia-reperfusion injury, comprising a therapeutically effective amount of pyrroloquinoline quinone and a renal protective agent A pharmaceutical composition comprising:   25. A pharmaceutical composition according to claim 24, wherein the nephroprotective agent is probenecid.   The therapeutically effective dose of pyrroloquinoline quinone is between 1 mg / kg and 10 mg / kg, and the therapeutically effective dose of probenecid is between 100 mg / kg and 200 mg / kg. 25. A pharmaceutical composition according to 24.   25. A pharmaceutical composition according to claim 24, wherein the nephroprotective agent is cilastatin.   25. The pharmaceutical composition of claim 24, wherein the pyrroloquinoline quinone is linked to one or more polymers.   The method of claim 1, wherein the ischemia-reperfusion injury is a seizure injury.   The method of claim 1, wherein the ischemia-reperfusion injury occurs in a limb.   32. The method of claim 30, wherein the limb injury is a result of diabetes or a diabetes related disorder.   The method of claim 1, wherein the ischemia-reperfusion injury occurs in the lung.   A pharmaceutical composition for treating ischemia-reperfusion injury in a subject in need of treating ischemia-reperfusion injury, comprising a therapeutically effective dose of pyrroloquinoline quinone and metoprolol , Pharmaceutical composition.   34. The pharmaceutical composition of claim 33, wherein the therapeutically effective dose of pyrroloquinoline quinone is between 1 mg / kg and 10 mg / kg.   35. The pharmaceutical composition of claim 34, wherein the therapeutically effective dose of pyrroloquinoline quinone is 3 mg / kg.   36. The pharmaceutical composition of claim 35, wherein the therapeutically effective dose of metoprolol is 1 mg / kg.   34. The pharmaceutical composition of claim 33, wherein the ischemia-reperfusion injury is myocardial infarction.   A kit for treating or preventing damage resulting from ischemic injury, comprising in one or more containers a therapeutically effective dose of pyrroloquinoline quinone, a pharmaceutically acceptable carrier, and the kit And, if necessary, one or more compounds selected from the group consisting of metoprolol, probenecid, and cilastatin.   40. The kit of claim 38, wherein the pyrroloquinoline quinone is linked to one or more polymers.

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