WO2003099282A1 - White matter neuroprotectant pyrroloquinoline quinone compounds and methods of use thereof - Google Patents

Abstract

The invention includes compositions comprising pyrroloquinoline quinone compounds that are useful in methods for the treatment and prevention of white matter injury, e.g., caused by hypoxia or ischemia. The invention also includes methods for the treatment and prevention of white matter injury by contacting a composition of the invention with a human patient.

Description

WHITE MATTER NEUROPROTECTANT PYRROLOQUINOLINE QUINONE COMPOUNDS AND METHODS OF USE THEREOF

FIELD OF THE INVENTION This invention relates to protecting and rehabilitating of neural cells. More particularly, the invention relates to neuroprotective compounds and methods for treatment of white matter lesions such as caused by periventricular leukomalacia or spinal cord injury, and associated conditions like cerebral palsy and multiple sclerosis.

BACKGROUND OF THE INVENTION Different areas of nerve tissue in the central nervous system are either grayish or white.

Gray matter contains nerve cell bodies and forms, e.g., the brain surface layer, and the spinal cord central column. White matter consists largely of nerve fibers covered by myelin, a white, fatty insulating material. White matter forms a layer between the two areas of gray matter in the brain and encloses the column of gray matter in the spinal cord. White matter disease includes demyelinating ("mylelinoclastic") conditions such as multiple sclerosis (MS), and dysmyelinating conditions where myelin is abnormally formed or maintained. Demyelinating conditions involve the destruction of existing myelin. These conditions may be acquired inflammatory diseases, or maybe due to, e.g., radiation damage, tumors, or hypoxic/ischemic conditions. Ischemia leads to rapid changes in myocardial metabolism and cellular injury, the extent of the injury being dependent upon the severity of ischemia. Continued ischemia ultimately leads to total tissue necrosis. Cellular hypoxia and reoxygenation cause ischemia- reperfusion injury in part by generating reactive oxygen species (ROS). Unacceptable concentrations of ROS and free radicals lead to oxidative stress. Differential regional vulnerability to hypoxic /ischemic brain injury may be dependent on the maturational stage of the neuronal and non-neuronal cells in a given region. A common example of such age-dependent regional susceptibility is the exclusively white matter injury seen in infants as a complication of premature birth, referred to as periventricular leukomalacia (PVL). PNL is shown as an (age-dependent) white matter lesion seen in preterm infants and a common antecedent to cerebral palsy. The lesion is defined by focal necrosis of the deep periventricular white matter involving all cellular components, combined with a more diffuse white matter injury that appears selective for developing oligodendrocytes (OLs). Reduced cerebral myelin is the most prominent subsequent cerebral abnormality observed in premature infants with evidence of PNL in the neonatal period. PNL is thought to occur due to too little blood flow to that part of the brain when the baby is a fetus in the womb, at delivery, or after delivery during the first days of life. PNL leads to white matter lesions, which in turn lead to demyelinating conditions such as cerebral palsy (CP), impaired motor functioning and neurocognitive defects.

A propensity to cerebral ischemia caused by impaired cerebrovascular autoregulation, combined with a selective vulnerability of immature OLs to ischemic injury, may contribute to the prevalence of this lesion in the preterm infant. Developing OLs in vitro have been demonstrated to be more vulnerable than are mature, myelin basic protein (MBP)-expressing OLs to oxidative stress and to glutamate receptor (GluR)-mediated ischemic death. The excitatory neurotransmitter glutamate is released from axons and glia under hypoxic/ischemic conditions. OLs appear to be more vulnerable than are other glia when exposed to hypoxia /hypoglycemia in vitro. Furthermore, a number of in vivo studies have demonstrated selective white matter injury after experimental hypoxia /ischemia in the rat brain during early postnatal development.

Both clinical and experimental studies indicate that hypoxia/ischemia is a major underlying cause of PNL. Experimental models of ischemia in immature animals implicate glutamate as a critical factor in the pathogenesis of brain injury. Hypoxic /ischemic conditions result in elevated cerebral glutamate levels in the immature rat brain, measured by in vivo microdialysis. Clinical relevance of the experimental studies is suggested by the demonstration of elevated glutamate in the CSF of term infants after perinatal hypoxia/ischemia. Glutamate has been shown to be toxic to oligodendroglia in vivo and in vitro by receptor-independent and receptor-mediated mechanisms. OLs express functional GluRs in vitro, and these are primarily of the non-ΝMDA subtype.

The cell bodies in the trunk of the spinal cord reside in a gray, butterfly-shaped core that spans the length of the spinal cord. The ascending and descending axonal fibers travel in a surrounding area known as the white matter so called because the axons are wrapped in myelin. Both regions also house glial cells, which help neurons to survive and work properly. The glia include star-shaped astrocytes, microglia (small cells that resemble components of the immune system) and oligodendrocytes, the myelin producers. Each oligodendrocyte myelinates as many as 40 different axons simultaneously.

The majority of clinical deficits in patients with spinal cord injury is due to dysfunction of descending motor tracts and ascending sensory tracts in the spinal cord, i.e., white matter dysfunction. The disease entity multiple sclerosis involves injury to the white matter, i.e. injury to the cellular components of the white matter, including oligodendrocytes, and results in demyelination. Lesions in multiple sclerosis can occur in cerebral, brain stem, or spinal cord white matter where oligodendrocytes are located.

Oligodendrocyte injury and death is an important factor contributing to white matter damage and demyelination in spinal cord injury. Oligodendrocytes are highly vulnerable to, e.g., AMPA/kainate receptor-mediated excitotoxicity. AMPA and kainate-type excitatory amino acid receptors contribute to oligodendrocyte excitotoxicity.

SUMMARY OF THE INVENTION

The invention relates to the discovery that white matter cells may be treated with neuroprotective agents which allow for the regeneration and/or proliferation of the white matter. White matter cells, such as nascent oligodendrocytes, may be protected from cell death or cytoxicity and/or allowed to proliferate by administration of certain agents including those which modulate oxidative stress. As such, conditions prominent in preterm infants like PNL, which often lead to CP, may be treated after diagnosis and administration of the compounds of the invention. In adults, excitotoxic injury, short of death, is believed to be an important mechanism of demyelination, and since oligodendrocyte injury and/or death is an important mechanism of MS, spinal cord injury, white matter insult in spinal cord injury may be attenuated by treatment in accordance with the invention, thus allowing for an important means of treating spinal cord injury. In an aspect, the present invention relates to treating or preventing oxidative stress to oligodendrocytes, e.g., nascent or developing oligodendrocytes (i.e., which attempt to develop after an insult to the white matter) in a subject by administering a compound useful for the prevention or reduction of hypoxic/ischemic white matter cell injury, e.g., one which modulates oxidative stress, such that the cells are protected from cell death and/or allowed to proliferate.

In another aspect of the invention, pyrroloquinoline quinone (PQQ) compounds have been found to modulate free radical damage to white matter cells caused by oxidative stress. Free radicals generated by ischemic or hypoxic conditions have been found to be a significant cause of white matter damage, e.g., as in PNL, leading to white matter cell death. As such, administration of a pyrroloquinoline quinone compound, administered in vivo in non-toxic dosages, is an effective treatment for inhibiting or preventing oxidative stress free radical damage to white matter.

In one aspect, the invention relates to treating or preventing white matter cell death injury caused by hypoxia or ischemia in a subject by administering a pyrroloquinoline quinone compound, e.g., in an amount effective to treat or prevent neural injury. White matter cells may be rescued by administering to white matter cells at risk of cell death an effective amount of a pyrroloquinoline quinone compound, such that the cells, e.g., developing oligodendrocytes, are rescued.

In another embodiment, periventricular leukomalacia may be treated or prevented by administering to a subject in need thereof, e.g., one in utero, an effective amount of a pyrroloquinoline quinone compound, such that periventricular leukomalacia is modulated. Advantageously, the administration is effective if done prior to or at three days postnatal (P3), or from P3 to P7.

White matter lesions or injuries may be treated in another embodiment, wherein a subject having nascent white matter cells at risk of injury is administered an effective amount of a pyrroloquinoline quinone compound, such that the nascent white matter cells, e.g., oligodendrocytes, are protected from toxicity existing in or near the white matter injury. The toxicity existing in or near the white matter injury may be due to free radical toxicity; or ΝMDA-, kainate-, AMP A- or glutamate-mediated toxicity.

The invention also relates to methods for treating subjects at risk of developing multiple sclerosis, e.g., comprising administering to a subject in need thereof an effective amount of a pyrroloquinoline quinone compound, such that the risk of developing multiple sclerosis is reduced or eliminated.

A further embodiment includes methods for rescuing oligodendrocytes, comprising administering to oligodendrocytes at risk of cell death an effective amount of a pyrroloquinoline quinone compound, such that the oligodendrocytes, e.g., developing oligodendrocytes, are rescued or protected. The invention also includes methods for treating a subject at risk of developing or having cerebral palsy, comprising administering to a subject in need thereof an effective amount of a pyrroloquinoline quinone compound, such that the risk of developing cerebral palsy is reduced or eliminated. The invention further includes neuroprotective agents containing a pyrroloquinoline quinone compound, e.g., in an amount effective to protect white matter cells, e.g., oligodendrocytes, from cell death and/or promote their proliferation, and a pharmaceutically acceptable carrier. Also included are kits for treating patients at risk of or suffering from white matter injury, containing in one or more containers, an effective amount of pyrroloquinoline quinone, a pharmaceutically acceptable carrier, and instructions for use.

In another embodiment, white matter cells subjected to oxidative stress and/or free radical injury may be rescued by administering to white matter cells subjected to oxidative stress and/or free radical toxicity an effective amount of a pyrroloquinoline quinone compound, such that said cells are rescued from cell death. The oxidative stress may be caused by hypoxia, ischemia, oxygen-glucose deprivation (OGD), and kainate and/or amino- 3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMP A) (and analogs thereof) toxicity.

A further embodiment relates to methods for modulating OGD-induced oligodendrocyte cell death, wherein oligodendrocytes at risk of cell death are administered an effective amount of a pyrroloquinoline quinone compound, such that oligodendrocytes are rescued from cell death. Advantageously, the oligodendrocytes, e.g., developing oligodendrocytes may be in a subject in utero. These methods are useful for treating in utero subjects at risk of demyelinating conditions such as PNL.

The methods of the invention also include treating a patient at risk of suffering white matter damage, comprising diagnosing a patient as risk of suffering white matter damage, and administering to the patient an effective amount of a pyrroloquinoline quinone compound.

In a further aspect, the invention relates to methods for preventing PNL, white matter cell damage, MS, or CP, by administering a pyrroloquinoline quinone compound in an amount effective to obtain the desired protective effect.

These and other objects of the present invention will be apparent from the detailed description of the invention provided below. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a line graph showing the effect of PQQ and 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX) on OGD-induced death of oligodendrocyte precursor cells, as detailed further in Example 3. Figure 2 is a collection of photographic images of brain sections showing the selective loss of myelin at 96 hours following hypoxic-ischemic white matter injury in p7 rat pups, as detailed further in Example 4.

Figure 3 is a collection of photographic images showing the attenuation of myelin loss following hypoxic-ischemic white matter injury by NBQX, as detailed further in Example 4. Figure 4 is a set of bar graphs showing the age-dependent vulnerability of white matter to AMPA excitotoxicity, as detailed further in Example 4.

Figure 5 is a collection of photographic images of brain sections showing selective hypoxic-ischemic white matter injury in p7 rat pups, as detailed further in Example 4.

Figure 6 is a collection of photographic images of brain sections showing selective myelin loss in hypoxic-ischemic white matter injury in p7 rat pups, as detailed further in Example 4.

Figure 7 is a collection of photographic images of brain sections showing the inability of PQQ to attenuate selective myelin loss in hypoxic-ischemic white matter injury in p7 rat pups, as detailed further in Example 4. Figure 8 is a set of bar graphs showing the inability of PQQ at several doses to prevent hypoxic-ischemic white matter injury in p7 rat pups, as detailed further in Example 4.

Figure 9 is a collection of photographic images of brain sections showing the ability of PQQ to attenuate hypoxic-ischemic cortical injury in ρ3 rat pups, as detailed further in Example 4. Figure 10 is a bar graph demonstrating that PQQ at 5mg/kg reduces hypoxic-ischemic injury in p3 rat pups, as detailed further in Example 4.

Figure 11 is a collection of photographic images of brain sections showing the ipsilateral loss of myelin at p9 in p3 rat pups subjected to hypoxic-ischemic cortical injury, as detailed further in Example 4. Figure 12 is a collection of photographic images of brain sections demonstrating that PQQ attenuates the ipsilateral loss of myelin at p9 in p3 rat pups subjected to hypoxic- ischemic cortical injury, as detailed further in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that particular embodiments described herein are illustrative and limiting of the invention. The principal features of the invention can be employed in various embodiments without departing from the scope of the invention. All parts and percentages are by weight unless otherwise specified.

Definitions

For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined, 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 pertains. However, to the extent that these definitions vary from meanings circulating within the art, the definitions below are to control.

"Ischemia" includes the decrease or cessation of blood flow.

"Hypoxia" includes the deficiency in the amount of oxygen reaching body tissues. "Hypoxia or ischemic-related injury" includes white matter injury such as lesions, and injuries due to toxicity caused by, e.g., glutamate, NMD A, AMP A, OGD, and free radicals.

"Oxidative stress" includes conditions that occur when there is an excess of free radicals, a decrease in antioxidant levels, or both.

"Necrosis" includes the death of cells or tissues through injury or disease, particularly in a localized area of the body such as the white matter.

"Apoptosis" refers to programmed cell death.

"White matter injury" includes any chronic or acute pathological event involving the white matter and/or associated tissue (e.g., glial cells, including astrocytes, microglia and oligodendrocytes), including spinal cord injury or trauma, MS, CP, ischemia-reperfusion injury; congestive heart failure; cardiac arrest; myocardial infarction; cardiotoxicity caused by compounds such as drugs (e.g., doxorubicin, herceptin, thioridazine and cisapride); cardiac damage due to parasitic infection (bacteria, fungi, rickettsiae, and viruses, e.g., syphilis, chronic Trypanosoma cruzi infection); fulminant cardiac amyloidosis; heart surgery; heart transplantation; and traumatic cardiac injury (e.g., penetrating or blunt cardiac injury, aortic valve rupture).

"Subject" includes living organisms such as humans, monkeys, cows, sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, cultured cells therefrom, and transgenic species thereof. In a preferred embodiment, the subject is a human. Administering the compositions of the invention to a subject to be treated can be carried out using known procedures, at dosages and for periods of time effective to treat the condition in the subject. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject, and the ability of the therapeutic compound to treat the foreign agents in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

"Substantially pure" includes compounds, e.g., drugs, proteins or polypeptides that have been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state. Included within the meaning of the term "substantially pure" are compounds which are homogeneously pure, for example, where at least 95% of the total (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. "Administering" includes routes of administration which allow the compositions of the invention to perform their intended function, e.g., treating or preventing white matter trauma. A variety of routes are possible including, but not necessarily limited to, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), oral (e.g., dietary), topical, nasal, rectal, or via slow releasing microcarriers depending on the disease or condition to be treated. Oral, parenteral and intravenous administration are preferred modes of administration. Formulation of the compound to be administered will vary according to the route selected (e.g., solution, emulsion, gels, aerosols, capsule). An appropriate composition comprising the compound to be administered can be prepared in a physiologically acceptable vehicle or carrier and optional adjuvants and preservatives. For solutions or emulsions, suitable carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, sterile water, creams, ointments, lotions, oils, pastes and solid carriers. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. (1980)).

"Effective amount" includes those amounts of pyrroloquinoline quinone which allow it to perform its intended function, e.g., treating or preventing, partially or totally, white matter cell injury or associated conditions, e.g., MS and/or CP, caused by hypoxia or ischemia as described herein. The effective amount will depend upon factors including biological activity, age, body weight, sex, general health, severity of the condition to be treated, as well as appropriate pharrnacokinetic properties. A therapeutically effective amount of the active substance can be administered by an appropriate route in a single dose or multiple doses. Further, the dosages of the active substance can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

"Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are compatible with the activity of the compound and are physiologically acceptable to the subject. 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 media or agent is incompatible with the therapeutic compound, use thereof in the compositions suitable for pharmaceutical administration is contemplated. Supplementary active compoimds can also be incorporated into the compositions. "Pharmaceutically acceptable esters" includes relatively non-toxic, esterified products of therapeutic compounds of the invention. These esters can be prepared in situ during the final isolation and purification of the therapeutic compounds or by separately reacting the purified therapeutic compound in its free acid form or hydroxyl with a suitable esterifying agent; either of which are methods known to those skilled in the art. Acids can be converted into esters according to methods well known to one of ordinary skill in the art, e.g., via 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; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; 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; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, e.g., in Remington 's Pharmaceutical Sciences.

"Unit dose" includes a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. Pyrroloquinoline quinone (PQQ) is a water soluble anionic quinone that can transfer electrons catalytically between a variety of reductants and oxidants, and may be part of a soluble electron transport system in eukaryotic cells. PQQ proper is of the general structure

"Pyrroloquinoline quinone compounds" includes any member of the pyrroloquinoline quinone family having chemical similarity, including closely related isomeric and stereoisomeric analogs of PQQ (See e.g., Zhang et al., 1995, Biochem. Biophys. Res. Commun. 212: 41-47, 1995). PQQ is also known as methoxatin. Without wishing to be bound by theory, PQQ may act in part as a free-radical scavenger, particularly of reactive oxygen species (ROS). As such, PQQ may function as an NADPH-dependent methemoglobin reductase substrate (See e.g., Xu et al., Proc. Natl. Acad. Sci. USA, 1992, 89(6):2130-4). Other NADPH-dependent methemoglobin reductase substrates may function to decrease or eliminate hypoxia or ischemia-related white matter cell injury and associated conditions; "pyrroloquinoline quinone" refers to these compounds.

Compositions comprising a pyrroloquinoline quinone compound, e.g., substantially purified pyrroloquinoline quinone, may include pyrroloquinoline quinone alone, or in combination with other components which are effective to treat white matter cell injury or associated conditions, such as 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX), the structure of which is shown below:

Pyrroloquinoline quinone may be substantially purified by any of the methods well known to those skilled in the art. (See, e.g., E. J. Corey and Alfonso Tramontano, J. Am. Chem. Soc. 103, 5599-5600 (1981); J.A. Duine, Review Ann. Rev. Biochem. 58, 403 (1989)).

The pyrroloquinoline quinone compounds of the invention are, in one embodiment, a component of a pharmaceutical composition, which may also comprise buffers, salts, other proteins, and other ingredients acceptable as a pharmaceutical composition. The invention also includes a modified form of pyrroloquinoline quinone, which is capable of preventing or reducing hypoxic/ischemic white matter cell injury as described herein.

The structure of the therapeutic compounds of this invention may include asymmetric carbon atoms. It is to be understood accordingly that the isomers (e.g., enantiomers and diastereomers) arising from such asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis. For the purposes of this application, unless expressly noted to the contrary, a therapeutic compound shall be construed to include both the R or S stereoisomers at each chiral center. In certain embodiments, an therapeutic compound of the invention comprises a cation. If the cationic group is hydrogen, H ^", then the therapeutic compound is considered an acid. If hydrogen is replaced by a metal ion or its equivalent, the therapeutic compound is a salt of the acid. Pharmaceutically acceptable salts of the therapeutic compound are within the scope of the invention, e.g., pharmaceutically acceptable alkali metal (e.g., Li⁺, Na⁺, or K⁺) salts, ammonium cation salts, alkaline earth cation salts (e.g., Ca²⁺, Ba²⁺, Mg²⁺), higher valency cation salts, or polycationic counter ion salts (e.g., a polyammonium cation). (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J Phαrm. Sci. 66:1-19). It will be appreciated that the stoichiometry of an anionic compound to a salt- forming counter ion (if any) will vary depending on the charge of the anionic portion of the compound (if any) and the charge of the counter ion. Preferred pharmaceutically acceptable salts include a sodium, potassium or calcium salt, but other salts are also contemplated within their pharmaceutically acceptable range. The invention relates to methods of treating or preventing the deleterious effects of oxidative stress, such as is caused by hypoxia or ischemia, on white matter in a subject. This is done by administering to a subject in need thereof a preferably non-toxic amount of an agent such as PQQ which modulates oxidative stress such that the white matter cells which are the target of the oxidative stress are protected from cell death. The cell death may be due, e.g., to necrosis or apoptosis.

Free radicals generated by ischemic or hypoxic conditions have been found to be a significant cause of white matter cell damage leading to cell death. As such, administration of a pyrroloquinoline quinone compound, administered in vivo, e.g., in non-toxic dosages, is an effective treatment for inhibiting or preventing oxidative stress free radical damage, either by pyrroloquinoline quinone compound-mediated free radical scavenging, or by inhibition of free radical generation. White matter cell injury caused by hypoxia or ischemia, may therefore be treated or prevented by administration of pyrroloquinoline quinone, preferably in a non-toxic dosage.

The invention encompasses methods of treating or preventing white matter cell injury caused by hypoxia or ischemia in a subject, wherein a pyrroloquinoline quinone compound is administered to a subject in need thereof, such that hypoxia or ischemic-related injury is prevented or decreased. In further embodiments of the invention, the pyrroloquinoline quinone compound is administered at a non-toxic concentration, which includes concentrations of pyrroloquinoline quinone compound which are cytostatic but not cytotoxic, and concentrations which are cytotoxic to cell types other than the intended one or more cell types (e.g., oligodendrocytes). The determination of the cytotoxicity of a known concentration of a pyrroloquinoline quinone compound to one or more cell types is within the abilities of one of ordinary skill in the art. By way of non-limiting example, toxicity to cultured oligodendrocytes has been observed at a concentration of 30μM. In some embodiments, a pyrroloquinoline quinone compound is administered in combination with other compounds, such as anti-platelet drugs, anti-coagulant drugs, and anti-fhrombotic drugs. The invention encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for the prevention or reduction of hypoxic/ischemic white matter cell injury as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a pharmaceutically acceptable ester or salt, such as in combination with a physiologically- acceptable cation or anion, as is well known in the art. Further, the pyrroloquinoline quinone may contain pharmacologically acceptable additives (e.g., carrier, excipient and diluent), stabilizers or components necessary for formulating preparations, which are generally used for pharmaceutical products, as long as it does not adversely affect the efficacy of the preparation, e.g., in decreasing or inhibiting white matter cell injury.

Examples of additives and stabilizers include saccharides such as monosaccharides (e.g., glucose and fructose), disaccharides (e.g., sucrose, lactose and maltose) and sugar alcohols (e.g., mannitol and sorbitol); organic acids such as citric acid, maleic acid and tartaric acid and salts thereof (e.g., sodium salt, potassium salt and calcium salt); amino acids such as glycine, aspartic acid and glutamic acid and salts thereof (e.g., sodium, calcium or potassium salt); surfactants such as polyethylene glycol, polyoxyethylene-polyoxypropylene copolymer and polyoxyethylenesorbitan fatty acid ester; heparin; and albumin. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. The preferred mode is intravenous administration.

The pyrroloquinoline quinone and the above-mentioned ingredients are admixed as appropriate to give powder, granule, tablet, capsule, syrup, injection and the like. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. The amount of the active ingredient is generally equal to the dosage of the active ingredient, which would be administered to a subject, or a convenient fraction of such a dosage such as, for example, one- half or one-third of such a dosage. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1 % and 100% (w/w) active ingredient. In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers. Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include potato starch and sodium starch glycollate. Known surface active agents 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 binding agents include gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets maybe coated using methods described in, e.g., U.S. Patent Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

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

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstirution with water or another suitable vehicle prior to use. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersing or wetting agents include naturally- occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include lecithin and acacia. Known preservatives include methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents 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 may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products 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. A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.

Suppository formulations may be made by combining the active ingredient with a non- irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20°C) and which is liquid at the rectal temperature of the subject (i.e., about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives. Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, a gel or cream or solution for vaginal irrigation. Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives. Additional delivery methods for administration of compounds include a drug delivery device, such as that described in U.S. Patent No. 5,928,195.

"Parenteral administration" includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection, by application through a surgical incision, by application through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. 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 provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3 -butane diol, for example. 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 parentally-administrable formulations that are useful include those, which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nm, and preferably from about 1 to about 6 nm. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non- ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient). Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nm.

The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition 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 a formulation is admimstered in the manner in which snuff is taken i. e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nm, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically-administrable formulations that are useful include those, which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

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

While the dose is appropriately determined depending on symptom, body weight, sex, animal species and the like, it is generally assumed that treatment options holding the blood concentration at about 1 μM will be preferred. This plasma concentration may be achieved through administration of one to several doses a day. When pyrroloquinoline quinone is to be administered to a subject, 0. Ing to lOmg/kg body weight (e.g., Ing to lmg/kg body weight) of pyrroloquinoline quinone can be given intravenously.

The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

EXAMPLES These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXAMPLE 1 Hypoxic/ischemic (H/I) white matter (WM) injury occurs in periventricular leukomalacia in preterm infants. Since H/I involves GluR-mediated excitotoxicity and oxidative stress, we examined the effects of pyrroloquinoline quinone on H/I WM injury in immature rats. We examined the protective efficacy of PQQ post treatment following H/I WM injury at three days postnatal (P3) or P7. Unilateral carotid ligation (UCL) and hypoxia (6% O₂, lh) at P7 results in selective, subcortical WM injury and ipsilateral decrease in MBP expression at PI 1. PQQ post treatment (lmg/kg, n=4, 5mg/kg, n=8, 10 mg/kg, n=4, 20mg/kg, n=4) or vehicle control (equal number rats/group) was administered i.p. every 12h for 48h post hypoxia. After H/I at P7, rats were sacrificed at PI 1. WM injury was not attenuated by PQQ at any dose vs. vehicle. At P3, UCL and hypoxia (6% O₂, 2h) resulted in WM and cortical injury at P9. At P3, PQQ (5mg/kg x 4, q 12h) significantly attenuated WM injury (injury scale 1.67 + .44, n=8) compared to vehicle treated controls (0.5 + 0.22, n=8, ρ<0.03). At P3, when there is little or no GluR expression, PQQ is protective.

EXAMPLE 2

Protective effect of pyrroloquinoline quinone on oligodendroglial excitotoxicity in vitro. To further examine mechanisms of PQQ protection of oligodendroglia (OL), we tested the effect of PQQ on excitotoxicity induced by kainate or oxygen-glucose deprivation (OGD) in cultured OL precursor cells. Primary OL precursors were isolated from newborn rat brains. Cultures were exposed to 0, 2.5, 5, 10, or 20 μM PQQ 10 min before exposure to 50 μM kainate continuously or OGD for 2 hr, and cell survival was assessed 24h later. Dose- response studies showed that PQQ significantly reduced kainate-induced (at >10 μM, p<0.001) or OGD-induced OL death (at >5 μM, p<0.001). Co treatment of PQQ and the non- NMDA glutamate receptor antagonist NBQX (lOOμM) did not differ from NBQX alone. PQQ significantly attenuated kainate- or OGD-evoked calcium accumulation measured by ⁴⁵Ca²⁺ uptake (p<0.01). Whole-cell patch clamp recordings of OL precursors showed that PQQ (lOOμM) attenuated kainate-induced currents (kainate 500μM). While PQQ may be in part acting to scavenge free radicals in the OGD model, these results show that PQQ may also modulate AMPA/kainate receptor-mediated excitotoxicity. PQQ compounds (and related agents like NBQX) therefore are valuable in treatment of hypoxic/ischemic white matter injury in the developing brain.

EXAMPLE 3

Effect of PQQ on perinatal white matter injury and excitotoxicity to immature oligodendrocytes.

I. The effect of PQQ on oxygen glucose deprivation in developing oligodendrocytes in vitro. Since both glutamate receptor mediated injury and oxidative stress are believed to be responsible for oligodendrocyte (OL) injury in periventricular leukomalacia, we tested the protective effects of PQQ in an in vitro model of hypoxia/ischemia: oxygen glucose deprivation. The data below show that PQQ is highly effective in preventing OGD-induced injury of developing OLs. The mechanism of this protection, without limitation to a particular theory, may involve blockade of glutamate receptors; antioxidant actions; or a combination of both.

MATERIALS AND METHODS

Culture and treatment of oligodendrocyte precursor cells. Primary oligodendrocyte precursor cells were isolated from mixed glial cultures of the forebrains of newborn Sprague— Dawley rats using a selective detachment procedure as described previously. Precursor cultures were routinely characterized by immunochemical detection of cell-specific markers, and usually consisted of approximately 90% A2B5 oligodendrocyte precursors and less than 5% glial fibrillary acidic protein (GFAP)-positive astrocytes. Cells were plated on poly-DL- ornithine-coated glass cover slips in 24-well plates and maintained in a serum-free, chemically defined medium.

Oxygen-glucose Deprivation (OGD). Cultures were switched to the same medium that was deoxygenated and glucose-free (Gibco). PQQ (Sigma) and NBQX (Sigma-RBI) were directly dissolved in the culture medium and applied 10 min prior to the onset of deoxygenation when these agents were present in glucose-free medium. Cultures were then transferred to an anaerobic chamber (Billups-Rothenberg, Inc., Del Mar, CA) at 37°C. Following OGD for 2 hr, D-glucose as a concentrated stock solution made in the glucose-free medium was added back to the cultures to a final concentration of 25 mM, and cultures were returned to a normoxic 5% CO incubator at 37°C. Cell death was assessed 24 hr later. Cell viability assay. Cell death was quantitatively assessed using the trypan blue exclusion method. Cells in 5-7 adjacent fields (x200) per well were counted to determine cell survival. At least 1000 cells per condition were counted in control cultures. The mean ± SEM values for each experimental group were obtained from at least three separate experiments, and each was performed in triplicate. Statistical analysis was performed using one-way analysis of variance (ANON A) with

hoc test. Statistical significance was accepted for/? <0.05. RESULTS

PQQ protects oligodendrocyte precursor cells from OGD-induced cell death. In this study, PQQ was found to be toxic to cultured oligodendrocyte precursor cells at concentrations of >30 μM (data not shown). Cultures were thus given 0, 2.5, 5, 10, or 20 μM PQQ before exposure to OGD for 2 hr. Additionally, 100 μM NBQX was co treated with 0, 5, and 20 μM PQQ. Cell survival was assessed 24 hr later. The dose-response curve is shown in Fig. 1. PQQ at >5 μM significantly OGD-induced oligodendroglial death with approximately 70% of cell survival at 20 μM. NBQX (100 μM) achieved approximately 78% of cell survival. The combination of NBQX and PQQ, however, did not differ from NBQX alone. Figure 1 shows the effect of PQQ and NBQX on OGD-induced death of oligodendrocyte precursor cells. Cultures were given 0, 2.5, 5, 10, or 20 μM PQQ before exposure to OGD for 2 hr. In addition, 100 μM NBQX was co treated with 0, 5, and 20 μM PQQ. Cell survival was assessed 24 hr later. *Lowest concentration showing significant difference when compared to the controls (p < 0.05 by ANOVA-Tukey analysis). II. The effect of PQQ post treatment in a rodent model of periventricular leukomalacia (PVL).

The effects of PQQ was tested in two models of PNL. The first is a well-characterized model at seven days postnatal (P7). Below are the results from the P7 study, which shows some repeat-dose toxicity, and also results from a study done at lower doses in P3 animals, which show a positive effect and no toxicity.

PQQ adversely effects weight gain in a dose dependent manner. Treatment with PQQ at p7 for four doses, once every 12 hours following hypoxia showed that PQQ caused weight loss at higher doses. Analysis of the effect of 20mg/kg PQQ (n=5) versus littermate controls (n=4), and lOmg/kg PQQ (n=5) versus littermate controls (n=5) showed an adverse effect on weight gain at 20 (p<0.02) and 10 mg/kg (p <0.01) despite small numbers. There is no significant difference in weight loss between lOmg/kg and 20mg/kg of PQQ. However, at lower doses lmg/kg of PQQ (n=4) versus littermate controls (n=4) and 5mg/kg PQQ (n=5) versus littermate controls (n=5), there is no significant change in body weight at lmg/kg (p<0.5) and 5mg kg (ρ<0.26). Effect of PQQ on hypoxic/ischemic white matter injury. At high doses, any potential effect of PQQ is masked by toxicity as described above. At lower doses there is a suggestion of protection, but numbers of animals evaluated to date are insufficient to determine significance. At 1 mg/kg PQQ (n=4) versus littermate controls (n=4), a slight decrease in white matter injury is seen, which may be significant following additional repetition. The experiments at 5 mg/kg (n=5) versus littermate controls (n=5) were potentially skewed by technical difficulties (the amount of injury in that litter was much less than usual) and is currently being repeated. Evaluation of MBP ice at pi 1 following 4 doses of 20mg/kg PQQ (n=5) versus littermate controls (n=4) did not demonstrate a clear neuroprotective effect between the two groups. Similarly, lOmg/kg PQQ (n=5) versus littermate controls (n=5) also failed to demonstrate a neuroprotective effect. MATERIALS AND METHODS In vivo unilateral carotid ligation in combination with hypoxia: Long Evans rat pups postnatal day 7 (P7) underwent ligation of the right common carotid artery via a unilateral midline incision under ether anesthesia. The vagus nerve and sympathetic chain were carefully reflected away from the carotid artery. The carotid artery was first cauterized then bisected. The incision was closed using 6.0 nylon suture. Animals were allowed to recover for one hour prior to being placed in an airtight chamber containing 6% O₂/ balance N₂. Temperature was controlled at 33°C in the chamber during hypoxia. The pups were exposed to hypoxia for one hour and then returned to room air.

PQQ treatment: Rat pups were administered PQQ via i.p. injection in a NaHCO₃ vehicle (2%) or an equivalent volume of vehicle alone. Two low doses (lmg/kg) and (5mg/kg), and two higher doses lOmg/kg and 20mg/kg of PQQ were used in this trial. P7 pups were given 4 doses, once every 12 hours following hypoxia.

Measurement of severity of white matter injury: Animals were sacrificed at PI 1 with an overdose of sodium pentobarbitol and then perfused with 4% paraformaldehyde in phosphate buffer. The brains were post-fixed for 2-4 hours, then transferred to 30% sucrose and sectioned (coronal) at 15μm. Slides were stained with hematoxylin and eosin (H & E) for light microscopy. Sections were immunostained with MBP to derive the severity of white matter injury. Severity of white matter injury was obtained from immunostained sections averaged from six stereotactically determined planes from each animal in each treatment group, and the means calculated for each group. Serial sections from the anterior, middle and posterior of the dorsal hippocampus in each rat were evaluated for loss of MBP in the periventricular white matter. Staining of the contralateral hemisphere was compared and graded on a scale of 0-3 as follows: 0, no injury; 1, injury limited to the loss of cortical processes without thinning of the capsule; 2, more severe injury and limited to the loss of processes with thinning of capsule; 3, injury resulting in the loss of processes and thinning of the capsule including full thickness loss.

Attenuation of white matter and cortical injury with PQQ at PS: Carotid ligation was performed on rat pups at P3. After 1 hour recovery they were subjected to 2 hours of hypoxia at 6% 02. Littermates were given either vehicle or 5 mg/kg PQQ every 12 hours, x 4 doses, commencing immediately after hypoxia. One hour of hypoxia following carotid ligation at P3 (similar to model at P7) was previously found to result in no discemable injury. 5 mg/kg was found to be the highest dose, at this regimen, to have no toxic effect on P7 animals.

Pups were sacrificed and evaluated at P9. % vehicle treated littermates had ipsilateral injury loss of MBP (by immunocytochemistry staining) involving cortical processes and marked thinning and disruption of the white matter tracks. On H&E untreated pups had necrosis and apoptotic bodies throughout the ipsilateral white matter. Cortical layers N and NI showed laminar necrosis of neurons and oligodendrocytes. Pups treated with PQQ had remarkably little injury as evaluated by either MBP or H&E. No injury was detectable in any animals on the contralateral side.

EXAMPLE 4

Protective Effect of PQQ on perinatal white matter and cortical injury.

As noted above, the age-dependent vulnerability of white matter involves selective injury to pre-myelinating oligodendrocytes (ODs), and is most severe at postnatal day 7 (p7), as shown in Fig. 2. The vulnerability to hypoxic/ischemic white matter injury can be attenuated with the AMP A receptor antagonist NBQX in a rodent model of PNL, as shown in Fig. 3. These results correlate with results of AMP A injections that demonstrate a parallel vulnerability to glutamate receptor (GluR)-mediated excitotoxicity (shown in Fig. 4). White matter injury following intracerebral injections of AMP A is age-dependent as shown in Fig. 4, left panel, and is blocked by ΝBQX (Fig. 4, right panel), supporting a receptor-mediated mechanism of injury. White matter injury following AMPA injection is significantly more severe at p7 compared with younger (p4) and older (pi 1) animals, or with MK-801 alone (P7CTL). The ability of PQQ to block hypoxic/ischemic perinatal white matter and cortical injury was demonstrated in a rat carotid ligation model, and is detailed below. MATERIALS AND METHODS

Carotid ligation with hypoxia: Selective white matter injury was produced in P3 and P7 Long Evans rats by unilateral carotid ligation followed by hypoxia (6% oxygen, balance nitrogen, for 1-2 hours) where the body temperature was kept at 33-35°C. P3 pups received PQQ (5mg/kg) or vehicle every twelve hours for forty-eight hours. P7 pups received PQQ (1, 5, 10 or 20 mg/kg) or vehicle every twelve hours for forty-eight hours.

Assessment of white matter injury: Following UCL/hypoxia P3 animals were sacrificed at P9; P7 animals were sacrificed at P 11. Coronal sections of cryoprotected brains perfused with 4% paraformaldehyde, then stained with hematoxylin and eosin or processed for immunocytochemistry with myelin basic protein (MBP). Three adjacent pairs of coronal sections were assessed for each rat. MBP localization was assessed by comparing hemispheric staining ipsilateral and contralateral to the ligation. Lesion severity was graded as follows: 0- ipsilateral and contralateral regions are similar; 1- injury ipsilateral to the ligation is limited to a loss of staining in the cortical processes; 2- loss of staining includes thinning of the periventricular white matter; 3- injury of the white matter tracts includes a full- thickness loss of staining in the capsule.

Selective white matter injury induced by UCL/hypoxia is not attenuated by PQQ inp7 rats. Selective white matter injury with minimal cortical injury, induced by UCL hypoxia in p7 rats, is apparent after sacrifice at pi 1 (See Fig. 5). The white matter injury includes ipsilateral loss of myelin at pi 1, as shown in Fig. 6. As shown in Fig. 7, PQQ did not significantly attenuate white matter injury including ipsilateral loss of myelin at pi 1. As shown in Fig. 8, UCL/hypoxia (6% oxygen for 1 hour) at p7 results in selective white matter injury with minimal cortical injury when evaluated at p9 or p 11. Post-hypoxia treatment with PQQ (1_? 5, or lOmg/kg, every twelve hours for forty-eight hours) did not significantly attenuate selective white matter injury at any dose. PQQ at 20 mg/kg demonstrated increased toxicity.

PQQ attenuates hypoxic/ischemic white matter and cortical injury inp3 rats. UCL/hypoxia at p3 results in diffuse white matter injury with overlying cortical necrosis by p9 that is attenuated by PQQ treatment (See Figs. 9 and 10). Fig. 9 shows H & E- stained p9 brain sections of rats subjected to UCL/hypoxia at p3, showing white matter injury with overlying cortical necrosis (left panel and left panel inset); treatment with PQQ attenuates this cortical injury (right panel and right panel inset). Fig. 10 shows that UCL/hypoxia (6% oxygen for two hours) at p3 results in diffuse white matter injury with overlying cortical injury when evaluated in H & E-stained brain sections in vehicle-treated rats sacrificed at p9 (n=8). Post-hypoxia treatment with PQQ (every twelve hours for forty-eight hours) significantly attenuated the ipsilateral loss of MBP expression as compared with vehicle-treated control (n=8; p <0.01), showing that the cortical injury as assessed by H & E staining was minimal in PQQ-treated rats. This effect includes an attenuation in the amount of myelin loss at p9 (Compare Fig. 11 (no PQQ) with Fig. 12 (5mg/kg PQQ, administered every twelve hours for forty-eight hours)).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the following claims. Narious substitutions, alterations, 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.

Claims

CLAIMSWhat is claimed is:

1. A method for preventing or inhibiting periventricular leukomalacia, comprising administering to a subject in need thereof an effective amount of a pyrroloquinoline quinone compound, such that periventricular leukomalacia is modulated.

2. The method of claim 1, wherein said subject is in utero.

3. The method of claim 2, wherein said administration is done < P3.

4. The method of claim 2, wherein said administration is done > P3.

5. The method of claim 2, wherein said administration is done from P3 > P7.

6. The method of claim 1 , further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

7. A method for treating a white matter lesion or injury, comprising administering to a subject having nascent white matter cells an effective amount of a pyrroloquinoline quinone compound, such that said nascent white matter cells are protected from toxicity existing in or near said white matter injury.

8. The method of claim 7, wherein said toxicity existing in or near said white matter injury is due to free radical toxicity; or NMDA-, kainate-, AMP A- or glutamate-mediated toxicity.

9. The method of claim 7, wherein said nascent white matter cells are oligodendrocytes.

10. The method of claim 7, further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

11. A method for treating a subject at risk of developing multiple sclerosis, comprising administering to a subject in need thereof an effective amount of a pyrroloquinoline quinone compound, such that said risk of developing multiple sclerosis is reduced or eliminated.

12. The method of claim 11, wherein periventricular leukomalacia is prevented or inhibited.

13. The method of claim 11, wherein said subject is a infant.

14. The method of claim 13, wherein said infant is in utero.

15. The method of claim 13, wherein said administration is done < P3.

16. The method of claim 13, wherein said administration is done > P3.

17. The method of claim 13, wherein said administration is done from P3 > P7.

18. The method of claim 11, further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

19. A method for rescuing white matter cells, comprising administering to white matter cells at risk of cell death an effective amount of a pyrroloquinoline quinone compound, such that said cells are rescued.

20. The method of claim 19, wherein said white matter cells are oligodendrocytes.

21. The method of claim 20, wherein said oligodendrocytes are developing oligodendrocytes.

22. The method of claim 19, further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

23. A method for rescuing oligodendrocytes, comprising administering to oligodendrocytes at risk of cell death an effective amount of a pyrroloquinoline quinone compound, such that said oligodendrocytes are rescued or protected.

24. The method of claim 23, wherein said oligodendrocytes are developing oligodendrocytes.

25. The method of claim 23, further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

26. A method for treating a subject at risk of developing cerebral palsy, comprising administering to a subject in need thereof an effective amount of a pyrroloquinoline quinone compound, such that said risk of developing cerebral palsy is reduced or eliminated.

27. The method of claim 26, wherein periventricular leukomalacia is prevented or inhibited.

28. The method of claim 26, wherein said subject is a infant.

29. The method of claim 28, wherein said infant is in utero.

30. The method of claim 28, wherein said administration is done < P3.

31. The method of claim 28, wherein said administration is done > P3.

32. The method of claim 28, wherein said administration is done from P3 > P7.

33. The method of claim 26, further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

34. A method of treating spinal cord injury, comprising administering to a subject suffering from a spinal cord injury an effective amount of a pyrroloquinoline quinone compound, such that said spinal cord injury is treated.

35. A method for rescuing white matter cells subjected to oxidative stress and/or free radical injury, comprising administering to white matter cells subjected to oxidative stress and/or free radical toxicity an effective amount of a pyrroloquinoline quinone compound, such that said cells are rescued from cell death.

36. The method of claim 35, wherein said oxidative stress is caused by hypoxia, ischemia, oxygen-glucose deprivation (OGD), and kainate and/or amino-3-hydroxy-5-methyl-4- isoxazoleproprionic acid (AMP A) (and analogs thereof) toxicity.

37. The method of claim 35, further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

38. A method for modulating OGD-induced oligodendrocyte cell death, comprising administering to oligodendrocytes at risk of cell death an effective amount of a pyrroloquinoline quinone compound, such that oligodendrocytes are rescued from cell death.

39. The method of claim 38, wherein said oligodendrocytes are in a subject in utero.

40. The method of claim 38, wherein said oligodendrocytes are developing oligodendrocytes.

41. The method of claim 38, further comprising administration of 6-nitro-7- sulfamoylbenzo(f)quinoxaline-2,3-dione.

42. A method of treating a patient at risk of suffering white matter damage, comprising: diagnosing a patient as risk of suffering white matter damage; and administering to a said patient an effective amount of a pyrroloquinoline quinone compound.