WO2011041452A2

WO2011041452A2 - Mouse model for identifying compounds for the treatment of oxidative stress

Info

Publication number: WO2011041452A2
Application number: PCT/US2010/050775
Authority: WO
Inventors: Viktoria Kheifets
Original assignee: Ampere Life Sciences, Inc.
Priority date: 2009-10-01
Filing date: 2010-09-29
Publication date: 2011-04-07
Also published as: WO2011041452A3

Abstract

The Pdss2^kd/kd mutant mouse as an animal model for testing compounds of potential usefulness for the treatment of oxidative stress, particularly for the treatment of mitochondrial diseases and their associated manifestations, and for the delay of the process of aging, is disclosed. Methods of using the mutant mouse of the invention for assessing therapeutic compounds of potential pharmaceutical use in protecting against oxidative stress damage are also disclosed.

Description

MOUSE MODEL FOR IDENTIFYING COMPOUNDS FOR THE TREATMENT

OF OXIDATIVE STRESS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of United States Provisional Patent

Application No. 61/278,071, filed October 01, 2009. The entire content of that application is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides a mouse model for oxidative stress or mitochondrial disease, comprising a mouse having a mutation in the Pdss2 gene, and methods for the testing of therapeutic compounds useful in protecting a human or an animal against oxidative stress or mitochondrial disease in said mouse model.

BACKGROUND OF THE INVENTION

[0003] The present invention provides a method for testing therapeutic compounds of potential pharmaceutical use in protecting against the oxidative damage associated with free radicals in an animal model system. This animal model is the kd/kd mouse, also named Pdss2^kd/kd mouse, which was described over three decades ago as a distinctive model of spontaneous proliferative disease of renal epithelium in a sub-line of CBA/CaH mice, see Lyon et al, J. Med. Genet. (1971) 8:41-48. Although it was not understood at the time, the mice have a mutation in the Pdss2 gene that encodes the subunits of the polyisoprenyl diphosphate synthases which form the isoprenyl side chain of coenzyme Q (CoQ) in mice and humans. These mice develop tubulointerstitial nephritis, dilated tubules and proteinuria. Peng et al, PLoS Genetics (2008) 4 (4) "Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease", have shown that a presumed autoimmune kidney disease in mice with the missense Pdss2^kd/kd genotype can be attributed solely to a

mitochondrial CoQ biosynthetic defect, wherein these mice fail to make coenzyme Q due to a Pdss2 mutation. No manifestations of disease other than glomerulopathy are evident in these animals. However, analysis of livers from these mice reveals that they have significant depletion of CoQ, impairment of mitochondrial respiratory chain function, and disturbance of many other metabolic processes. Peng et al., Kidney Int. (2004) 66(l):20-28 obtained evidence that the kd/kd mouse has dysmorphic mitochondria in the renal tubular epithelial cells. Mutant homozygotes are apparently healthy for the first 8 weeks of life, develop proteinuria interstitial nephritis but eventually die of progressive end-stage renal failure by 4 to 8 months of age. However, Saiki R. et al., Am. J. Physiol. Renal Physiol. (2008 Nov); 295(5): F1535-44; showed that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age. Renal disease results either from inadequate respiratory function due to low levels of CoQ or from increased oxidative stress.

[0004] Oxidative stress is caused by disturbances to the normal redox state within cells. Oxygen has different important roles in biological systems as a terminal electron acceptor in oxidative phosphorylation and other reactions. An imbalance between routine production and detoxification of reactive oxygen species such as peroxides and free radicals can result in oxidative damage to the cellular structure and process. The most important source of reactive oxygen species under normal conditions in aerobic organisms is probably the leakage of activated oxygen from mitochondria during normal oxidative respiration.

Impairments associated with this process are suspected to contribute to mitochondrial disease, neurodegenerative disease, and diseases of aging, due, in part, to cumulative oxidative damage to cellular systems. Oxygen radical injury has also been implicated in for example, pulmonary oxygen toxicity (Tate et al., Chest (1982) 81: 5, and Strausz et al, Am-Rev-Respir- Dis. (1990) 141(1): 124-8); adult respiratory distress syndrome (Hammond, Can. J. Physiol. Pharmacol. (1985) 63(3): 173-187), bronchopulmonary dysplasia (Saugstad, OD Acta Paediatr. (1997) 86: 1277-82), sepsis syndrome, amyotrophic lateral sclerosis (ALS)

(Michikawa, M. et al., J. Neuroscience Research, (2004), 37-1:62-70) and ischemia- reperfusion syndromes including myocardial infarction, stroke, cardiopulmonary bypass (Reber, A et al., British J. of Anaesthesia, (2000) 84, 5, 565-570), acute renal tubular necrosis ( Shah et al., Ren. Fail. (1992) 14: 363-70), Alzheimer's disease (Mcintosh L. et al., Free Radical Biology and Medicine (1997) 23, (2): 183-190) and Tesco G. et al, Ann. NY Acad. Sci. (1992) 673: 149-53) , and Parkinson's disease (Yoshikawa T., European Neurology, (1993) 33: 60-68.

[0005] Coenzyme Q (CoQ) is a coenzyme formed by a biologically active quinone having a polyisoprenoid side-chain of several isoprene units. There are different types of CoQ which can be distinguished by the number of isoprene units contained in the side-chain, e.g. CoQ6, which contains 6 isoprene units and can be found in Saccharomyces cerevisiae, CoQ8, which contains 8 isoprene units and can be found in Escherichia coli, and CoQ9 which contains 9 isoprene units and can be found in Caenorhabditis elegans. In murine species, the most common form of CoQ is CoQ9, which contains 9 isoprene units in the side- chain. In humans, the most common form of CoQ is CoQ 10, which contains 10 isoprene units in the side-chain, also known as Ubiquinone.

[0006] CoQ is present in the membranes of all animal cells where it performs a number of essential functions in the mitochondrial respiratory chain. As the only lipid- soluble antioxidant synthesized endogenously, CoQ also participates in extra-mitochondrial electron transport, functional modification of mitochondrial uncoupling proteins, regulation of the mitochondrial permeability transition core and modulation of the levels of certain receptors on the surface of blood monocytes (Ernster, L. et al.,. Biochim. Biophys. Acta (1995) 1271(1), 195-204; Bentinger, M. et al, Mitochondrion (2007) 7 Suppl. S41-50).

Furthermore, CoQ influences the expression of a large number of genes whose products are involved in a number of metabolic processes (Groneberg, D. A. et al, Int. J. Biochem. Cell Biol. (2005) 37(6), 1208-1218; Doring, F. et al. IUBMB Life (2007) 59(10), 628-633).

[0007] Co-Enzyme Q10 Deficiency is a respiratory chain disorder that has been associated with autosomal recessive neurological disorders that are responsive to CoQ 10 supplementation. Clinical phenotypes include a myopathic form, a childhood-onset cerebellar ataxia, a multisystem infantile variant, Leigh's syndrome, and a childhood onset myopathy. Clinical manifestations include syndromes such as myopathy with exercise intolerance and recurrent myoglobin in the urine manifested by ataxia, seizures or mental retardation, and leading to renal failure (Di Mauro et al., Neuromusc. Disord., (2005) 15:311- 315), childhood-onset cerebellar ataxia and cerebellar atrophy (Masumeci et al., Neurology (2001) 56:849-855 and Lamperti et al., Neurology (2003) 60, 1206: 1208); infantile encephalomyopathy associated with nephrosis; and optic-nerve atrophy and deafness.

Biochemical measurement of muscle homogenates of patients with CoQ 10 deficiency showed severely decreased activities of respiratory chain complexes I and II + III, while complex IV (COX) was moderately decreased (Gempel et al., Brain, (2007) 130(8):2037- 2044).

[0008] Diseases such as cardiomyopathy, neuropathy, nephropathy, muscle degeneration and cancer are associated with significantly lower tissue levels of CoQ (Littarru, G. P. et al. "Clinical aspects of Coenzyme Q: Improvement of cellular bioenergetics or antioxidant protection?", Handbook of Antioxidants, Marcel Dekker, New York, (1996) 203- 239). In addition, genetic disorders characterized by impaired biosynthesis of CoQ involve serious metabolic disturbances.

[0009] Mitochondrial dysfunction contributes to various disease states. If a threshold proportion of mitochondria in the cell is defective, and if a threshold proportion of such cells within a tissue have defective mitochondria, symptoms of tissue or organ dysfunction can result. Some examples of mitochondrial diseases are Friedreich's ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), mitochondrial myopathy, encephalopathy, lactacidosis, and stroke (MELAS), Myoclonus Epilepsy Associated with Ragged-Red Fibers (MERRF), Leigh's syndrome, and respiratory chain disorders. Most mitochondrial diseases manifest the signs and symptoms of accelerated aging, including neurodegenerative diseases, stroke, blindness, hearing impairment, diabetes, and heart failure. In addition to congenital disorders involving inherited defective mitochondria, acquired mitochondrial dysfunction contributes to diseases, particularly neurodegenerative disorders associated with aging like Parkinson's, Alzheimer's, and Huntington's Diseases.

[0010] The diseases above appear to be caused by defects in Complex I of the respiratory chain. Electron transfer from Complex I to the remainder of the respiratory chain is mediated by the compound coenzyme Q (also known as Ubiquinone). Oxidized coenzyme Q (CoQ^ox or Ubiquinone) is reduced by Complex I to reduced coenzyme Q (CoQ^red or Ubiquinol). The reduced coenzyme Q then transfers its electrons to Complex III of the respiratory chain (skipping over complex II), where it is re-oxidized to CoQ^ox (Ubiquinone). CoQ^ox can then participate in further iterations of electron transfer.

[0011] When cells in an organism are temporarily deprived of oxygen, anaerobic respiration is utilized until oxygen again becomes available or the cell dies. The pyruvate generated during glycolysis is converted to lactate during anaerobic respiration. The buildup of lactic acid is believed to be responsible for muscle fatigue during intense periods of activity when oxygen cannot be supplied to the muscle cells. When oxygen again becomes available, the lactate is converted back into pyruvate for use in oxidative phosphorylation.

[0012] The present animal model system and methods fill a need for animal models for the testing of therapeutic compounds for the treatment of oxidative stress and particularly for the treatment of mitochondrial diseases, where no animal model exists.

SUMMARY OF THE INVENTION

[0013] The present invention provides a mouse model for oxidative stress comprising a mouse having a mutation in the Pdss2 gene. The present invention also provides a mouse model for mitochondrial disease and its associated manifestations, comprising a mouse having a mutation in the Pdss2 gene. The present invention provides a mouse model for oxidative damage associated with free endogenous radicals, comprising a mouse having a mutation in the Pdss2 gene. The present invention provides a mouse model for oxidative damage, where the oxidative damage results in manifestations of aging.

[0014] The present invention provides a method for testing therapeutic compounds of potential pharmaceutical use in protecting against the oxidative damage associated with free radicals using the Pdss2^kd/kd mouse. Particularly, the invention provides a method for testing therapeutic compounds of potential use in protecting against oxidative damage in disorders such as oxygen toxicity, nephropathy, ischemia, stroke, myocardial infarction, respiratory chain disorders, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease and Alzheimer's disease. Compounds useful for preventing or delaying certain manifestations of aging can be identified by the present methods, especially those manifestations of the aging process, which are the result of free radical damage.

[0015] The present invention provides a method for testing therapeutic compounds of potential pharmaceutical use in protecting a patient with low levels of CoQ. In some embodiments, the invention provides a method for testing therapeutic compounds that stimulate CoQ synthesis and increase CoQ levels efficiently. More particularly the invention provides a method for testing therapeutic compounds of potential use in treating some or all of the symptoms or manifestations of mitochondrial disorders, using the Pdss2^kd/kd mouse. In some embodiments the mitochondrial disorder is selected from Friedreich's ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), mitochondrial myopathy, encephalopathy, lactacidosis, and stroke (MELAS), Myoclonus Epilepsy Associated with Ragged-Red Fibers (MERRF) and Leigh's syndrome.

[0016] The present invention relates to the use of a homozygous Pdss2^kd/kd mutant mouse as a model in a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring renal failure in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, monitoring the untreated controls and the treated mice for symptoms of renal failure damage, and identifying compounds of potential use in therapy as those which prolong survival and/or which delay or prevent symptoms of renal failure.

[0017] In some embodiments, the invention relates to the use of a homozygous

Pdss2^kd/kd mutant mouse as a model in a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring urine production in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, monitoring the untreated controls and the treated mice for quantity of urine produced, and identifying compounds of potential use in therapy as those which cause lower volumes of urine production in the treated mutant mice than in the untreated Pdss2^kd/kd mutant mice.

[0018] In some embodiments, the invention relates to the use of a homozygous

Pdss2^kd/kd mutant mouse as a model in a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring temperature and body weight and composition in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population and/or a wild-type mouse population of the same genetic background as the mutant mice, monitoring the untreated controls and the treated mice for a change of temperature and body weight and composition, and identifying compounds of potential use in therapy as those which keep temperature and body weight and composition higher in the treated mouse than in the untreated mouse, or closer to the temperature and body weight and composition of the wild-type mouse.

[0019] In some embodiments, the invention relates to the use of a homozygous

Pdss2^kd/kd mutant mouse used as a model in a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring renal failure in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, monitoring the CoQ levels from the untreated control and the treated mice, and identifying compounds of potential use in therapy as those which stabilize or elevate the CoQ levels of the treated mutant mouse.

[0020] The invention relates to a method for identifying in vivo the activity of a compound for the treatment of oxidative stress, said method comprising the steps of: (a) providing a first homozygous Pdss2^kd/kd- mutant mouse; (b) providing a second homozygous Pdss2^kd/kd-mutant mouse of the same litter as the first homozygous mouse; (c) optionally providing a third mouse of the same genetic background as the first and second mice, (d) administering the compound only to said first mouse; (e) determining renal failure damage of the first and second mice; and (f) identifying an in vivo pharmaceutical activity of the compound if the profile of the first mouse to which the compound has been administered differs from the profile of the second mouse to which the compound has not been

administered, and/or exhibits changes versus the profile of the third mouse, wherein the profile includes one or more measurements selected from renal failure, urine production quantity, body temperature, body weight and composition, glucose levels, sensorimotor behavior, glutathione levels, lactate levels, brain scans, kidney scans, redox status and CoQ levels; and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered. [0021] In some embodiments, the method for identifying in vivo the activity of a compound for the treatment of oxidative stress comprises the steps of: (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd-mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining renal failure damage of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits fewer or less severe symptoms of renal failure, if administration of the compound delays or prevents symptoms of renal failure, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0022] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of oxidative stress comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) quantifying urine production of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits less urine production than the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0023] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of oxidative stress comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining body temperature and body weight and composition of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits higher body temperature and body weight and composition than the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0024] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of oxidative stress comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining glucose levels of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits higher glucose levels than the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0025] In other embodiments, the method of identifying in vivo the activity of a compound for oxidative stress comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining CoQ levels of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits an increase of the CoQ levels compared to those of the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0026] The invention also relates to a method of identifying in vivo the activity of a compound for the treatment of a patient with low levels of CoQ comprising the steps of : (a) providing a first homozygous Pdss2^kd/kd- mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) optionally providing a third mouse of the same genetic background as the first and second mice; (d) administering the compound only to said first mouse; (e) determining renal failure damage of the first and second mice; and (f) identifying an in vivo pharmaceutical activity of the compound if the profile of the first mouse to which the compound has been administered differs from the profile of the second mouse to which the compound has not been

administered, and/or exhibits changes versus the profile of the third mouse, wherein the profile includes one or more measurements selected from renal failure, urine production quantity, body temperature, body weight and composition, glucose levels, sensorimotor behavior, glutathione levels, lactate levels, brain scans, kidney scans, redox status and CoQ levels; and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0027] In some embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with low levels of CoQ, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd- mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining renal failure damage of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits fewer or less severe symptoms of renal failure, if administration of the compound delays or prevents symptoms of renal failure, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0028] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with low levels of CoQ, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd- mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) quantifying urine production of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits less urine production than the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0029] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with low levels of CoQ, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd- mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining body temperature and body weight and composition of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits higher body temperature and body weight and composition than the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0030] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with low levels of CoQ, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd- mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining glucose levels of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits higher glucose levels than the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0031] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with low levels of CoQ, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd- mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining CoQ levels of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits an increase of the CoQ levels compared to those of the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0032] The invention also relates to a method of identifying in vivo the activity of a compound for the treatment of a patient with a mitochondrial disease comprising the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) optionally providing a third mouse of the same genetic background as the first and second mice; (d) administering the compound only to said first mouse; (e) determining renal failure damage of the first and second mice; and (f) identifying an in vivo pharmaceutical activity of the compound if the profile of the first mouse to which the compound has been administered differs from the profile of the second mouse to which the compound has not been

[0033] In some embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with a mitochondrial disease, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second

homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining renal failure damage of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits fewer or less severe symptoms of renal failure, if administration of the compound delays or prevents symptoms of renal failure, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0034] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with a mitochondrial disease, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second

homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) quantifying urine production of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits less urine production than the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0035] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with a mitochondrial disease, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second

homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining body temperature and body weight and composition of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits higher body temperature and body weight and composition than those of the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0036] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with a mitochondrial disease, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second

homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining glucose levels of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits higher glucose levels than the levels of the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0037] In other embodiments, the method of identifying in vivo the activity of a compound for the treatment of a patient with a mitochondrial disease, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second

homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining CoQ levels of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits an increase of CoQ levels compared to those in the second mouse to which the compound has not been administered, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

[0038] In some embodiments, the therapeutic compound is for the treatment of a mitochondrial disease. In other embodiments, the therapeutic compound is for the treatment of a respiratory chain disease.

[0039] In some of the foregoing embodiments, the progression of the renal failure is quantified by the amount of urine produced.

[0040] The present invention also relates to a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring evaluation of redox status in various organs by imaging with tracer techniques such as HMPAO, Tc99m-HMPAO or other imaging agents, in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, and identifying compounds of potential use in oxidative stress therapy as those which keep redox status at or near normal levels.

[0041] The present invention also relates to a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring evaluation of redox status in various organs by imaging with tracer techniques such as HMPAO, Tc99m-HMPAO, or other imaging agents, in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, and identifying compounds of potential use in the treatment of mitochondrial disease as those which keep redox status at or near normal levels. [0042] The present invention also relates to a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring evaluation of glutathione localization in various organs by imaging with tracer techniques such as HMPAO, Tc99m-HMPAO, or other imaging agents, in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, and identifying compounds of potential use in oxidative stress therapy as those which keep glutathione at or near normal levels.

[0043] The present invention also relates to a method comprising the steps of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring evaluation of glutathione localization in various organs by imaging with tracer techniques such as HMPAO, Tc99m-HMPAO, or other imaging agents, in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, and identifying compounds of potential use in the treatment of mitochondrial disease as those which keep glutathione at or near normal levels.

[0044] The present invention also relates to a method comprising the step of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring glucose uptake by imaging with tracer techniques such as FDG or other imaging agents or other imaging methods such as positron emission tomography (PET), in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, and identifying compounds of potential use in the treatment of oxidative stress as those which keep glucose at higher levels than those of the untreated control.

[0045] The present invention also relates to a method comprising the step of administering a potentially therapeutic compound to a homozygous Pdss2^kd/kd mutant mouse population, monitoring glucose uptake by imaging with tracer techniques such as FDG or other imaging agents or other imaging methods such as positron emission tomography (PET), in comparison to an untreated homozygous Pdss2^kd/kd mutant mouse population, and identifying compounds of potential use in the treatment of mitochondrial disease as those which keep glucose at higher levels than those of the untreated control.

[0046] The present invention relates to the use of the homozygous Pdss2^kd/kd mutant mouse as a mouse model for the oxidative stress or mitochondrial disease and their associated manifestations. Potential compounds for the treatment of oxidative stress or mitochondrial disease and their associated manifestations would prolong survival and/or delay or prevent symptoms of renal failure in the homozygous Pdss2^kd/kd mutant mice used as models. DETAILED DESCRIPTION OF THE INVENTION

[0047] Oxygen radical-mediated tissue damage has been implicated in a variety of pathological conditions including, without limitation, ischemia reperfusion injury to brain and heart, Parkinson's disease, certain other neurodegenerative diseases, neonatal hyperoxic lung injury, atherosclerosis, mitochondrial disease, as well as normal aging.

[0048] In the normal, healthy individual, most oxygen free radicals are produced by the mitochondria as byproducts of the electron transport processes of oxidative

phosphorylation for physiological energy generation. Superoxide dismutase enzymes, glutathione peroxidase, and reduced glutathione function to protect the cells against the oxidative stress associated with normal cellular metabolism.

[0049] The Pdss2^kd/kd mutant mouse provides a model system in which to test potentially therapeutic compositions, such as, but not limited to, redox therapeutics, antioxidants or free radical scavengers for the ability to prevent fatal damage from

endogenous oxygen free radicals, especially those generated in the mitochondria. The Pdss2^kd/kd mutant mouse provides a model system in which to test potentially therapeutic compositions useful for stabilizing or increasing levels of CoQ in tissues. The Pdss2^kd/kd mutant mouse provides a model system in which to test potentially therapeutic compositions useful for the impairment of the respiratory chain system. The Pdss2^kd/kd mutant mouse provides a model system in which to test potentially therapeutic compositions useful for treating mitochondrial diseases.

[0050] Thus, the compounds that protect the Pdss2^kd/kd mutant mouse from death due to renal failure at an average of 4-8 months of age, as seen in the untreated control homozygous Pdss2^kd/kd mutant mouse, are identified as being able to possibly relieve mitochondrial disease symptoms. Comparable assessment of drugs which might be effective in the treatment of mitochondrial diseases due to a CoQ deficiency is also possible using the Pdss2^kd/kd mutant mouse and the present methods as those which prolong survival

significantly past about four to eight months and which prevent or postpone the onset of renal failure.

[0051] The term "patient" includes a human or an animal.

[0052] "Composition" in the term "body weight and composition" refers to body composition parameters such as total body fat, lean mass, body fluids, and total body water, particularly body fat. Such parameters can be measured, for example, by the Echo Magnetic Resonance Imaging (EchoMRI) system from Echo Medical System, Houston, TX, or by other methods known to one of skill in the art. [0053] "Glucose level" refers to blood serum glucose level.

[0054] "CoQ level" can be measured in any tissues, fluids or organs that contain

CoQ, such as blood, spinal fluid, muscle, brain, kidney or milk.

[0055] "Genetic background" is defined as described in "Genetic Background:

Understanding its importance in mouse-based biomedical research" (A Jackson Laboratory Resource Manual), Bar Harbor, Maine, USA: The Jackson Laboratory, September 2009, accessible at World Wide Web URL

j axmice . j ax . org/manual/genetic_background_manual .pdf .

[0056] All references and patent publications cited in the present application are incorporated herein by reference.

[0057] The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention.

EXAMPLES

Example 1

Animal Model

[0058] Although the first mutation in the Pdss2 as described by Lyon et al, /. Med.

Genet. (1971) 8:41-48, arose spontaneously in the CBA/CaH colony, the mouse line used in the present invention is a congenic line derived by transferring the kd allele, along with closely linked micro satellite markers to the B6 background (Hancock, WW et al, J. Immunol (2003); 171: 2778-2781). Homozygous mice obtained after several generations of back crossing are used.

[0059] The genotypes of one-day-old pups are determined by genetic analysis of tissue surgically excised from the toe or tail tip (2-3 mm). In these mice the third exon of the Pdss2 gene is deleted, thus destroying the ability to synthesize an active mitochondrial manganese superoxide dismutase.

Example 2

Animal Husbandry

[0060] Mice are housed under standard animal housing conditions with a normal day/night cycle and fed normal mouse chow (not high-fat) (Labdiet 5001, PMI Feeds Inc., St. Louis, Mo.). Nursing mothers are housed in individual cages with their offspring. They are provided free access to food and water ad libitum. Fresh bedding and a change of cage litter are provided twice per week.

[0061] Test mice are weighed daily to allow calculation of appropriate dosage, but otherwise, handling is kept to a minimum to reduce stress on the nursing mother.

Example 3

Administration of Test Compounds

[0062] Mice are given chow comprising 20 or 100 mg/kg of test compounds per day.

Chow is given ad libitum. The intake of 20 mg/kg or 100 mg/kg of drug is accomplished by determining the average food consumption of the mice before treatment, and formulating the drug into the chow so that the mouse ingests the desired amount of drug when eating the average food consumption.

Example 4

Statistical Analysis

[0063] Survival analysis is carried out by examining the animals daily to determine renal failure and mortality, and entering the results into a life-table in the program Statistica (Statsoft, Tulsa, Okla.). Comparison between groups is carried out using the Kaplan and Meier survival function, non-parametric t- tests, and the Gehans Wilcoxon statistic from within the program to compare groups.

Example 5

Behavioral Analysis

[0064] Treated Pdss2 animals are observed daily from 3 days of age through to 19 weeks for behavioral abnormalities compared to litter mate controls. Daily video recordings of up to 10 minutes can also be made to longitudinally track the development of behavioral changes.

Example 6

Evaluation of Test Compounds

[0065] In general, experiments are carried out with Pdss2 mice, starting at 3 days of age, and the mice are weighed and divided into test and control groups. Test mice receive alpha tocotrienol quinone (20 mg/kg or 100 mg/kg), CoQIO (100 mg/kg), or compounds for which evaluation as pharmaceuticals is desired, at a dosage which is below the level of toxicity. Unless otherwise indicated, the test compounds are administered in a

pharmaceutically acceptable carrier in the chow by the oral route. Unless otherwise determined to be advantageous, the test compounds are administered at the same time each day (+/- 0.2 hours).

[0066] During the course of an experiment, each mouse is evaluated daily for weight, lethargy, lack of appetite, vital signs and for any indication of renal disorders (observed via monitoring the amount of urine release or measured, or by monitoring for decreased levels of glutathione in the kidneys with Tc99m-HMPAO imaging).

[0067] Additionally video records can be made for later evaluation, for example for side-by-side comparisons or for observing changes over time.

[0068] If desired, cardiac histopathology and central nervous system tissue analysis are carried out after death of the animal or after sacrifice of the animal.

Example 7

Evaluations of Sensorimotor Behavior

A. Fore and Hindlimb Grip Strength Test in Pdss2 -mutant Mice

[0069] Animals are tested for grip strength, a standard model of neuromuscular function and sensorimotor integration, using a Computerized Grip Strength Meter for Mice (Dual Stand Model 1027CDM, Columbus Instruments, Columbus, Ohio).

[0070] Animals are moved into the testing room for 30 minutes before testing. Prior to testing, each gauge is calibrated with a set of known weights and the apparatus is adjusted for the size of animal, according to manufacturer's instructions. The forelimb measurements are carried out with the meter in the tension peak mode to freeze the reading as the subject is pulled away from the grip bar. The hindlimb measurements are carried out with the meter in the compression peak mode to freeze the reading as the subject's hindlimbs are pulled over the bar toward the meter. Each animal is hand-held by the investigator as pulled past the grip bars, using a consistent technique, leaving the fore and hind limbs free to grasp the grip bars.

[0071] Testing is carried out on day 2 and repeated, in a blind-randomized fashion, twice weekly for a defined interval. Typically, three successive readings are taken for each animal with an inter-trial interval long enough to record the data and zero both meters for the next trial. B. Rota-Rod Test in Mice

[0072] Apparatus: Rota-Rod Treadmill for Mice (575M USB Model, from Med

Associates, Georgia (St. Albans) Vermont).

[0073] Procedure: Animals are tested in this study, using a Rota-Rod Treadmill for

Mice (575M USB Model, from Med Associates, Georgia (St. Albans) Vermont). The animals are moved into the testing room 30 minutes before testing. Every mouse receives 2-3 training runs of 1-2 minutes at intervals of 2-3 hours before testing.

[0074] The cylinder on the apparatus is set in motion before placing the mice in position. The motor is set at a constant selected speed in 7700 on RESET mode, and the mice are placed, one by one, in their sections.

[0075] Testing is carried out on day 2 and repeated, in a blind-randomized fashion, twice weekly for a defined interval. Typically, three successive readings are taken for each animal with an inter-trial interval long enough to record the data and zero both meters for the next trail.

Example 8

Positron emission tomography (PET) Procedures

6²Cu-ATSM-PET and ¹⁸FDG-PET

[0076] Regional oxidative stress and glucose metabolism in the brain may be evaluated by imaging as typified by positron emission tomography (PET). Novel functional, double imaging of redox and energy states using PET with [⁶²Cu]-diacetyl-bis(N4- methylthiosemicarbazone ( 62 Cu-ATSM) and [ 18 F]-fluorodeoxyglucose ( 18 FDG) as described in Ikawa M et al., (2009) Mitochondrion 9, 144-148 can be performed in vivo. ⁶²Cu-ATSM-

PET can be applied to evaluate oxidative stress and cerebral blood flow, whereas 18 FDG-PET can be applied to diagnose glucose metabolism.

[0077] ⁶²Cu is eluted from a ⁶²Zn/⁶²Cu positron generator and ⁶²Cu-ATSM is obtained by simple mixing of generator eluate (⁶²Cu-glycine) and ATSM synthesized by a previously reported method (Fujibayashi et al., (1997) J. Nucl. Med. 38 (7) 1155-1160). A 20-min dynamic PET scan is performed with bolus injection of ⁶²Cu-ATSM via the antecubital vein in approximatively 555 MBq with frame durations of 10s x 12, 60s x 8 and 10 min x 1. Early and delayed images are calculated using the first 3 min of PET data and the last frame of the dynamic data. ⁶²Cu-ATSM accumulation in the early phase reflects coronary blood flow; (Fujibayashi et al., (1997) see supra; Lewis et al., (2001) J. Nucl. Med. 42, 655-661; Obata et al., (2001) Ann. Nucl. Med. 15, 499-504; and Dearling et al , .(2002) J. Biol. Inorg. Chem.l ^',

249-259. For 18 FDG-PET, approximately 150 MBq or tracer is administered about 1 h after the ⁶²Cu-ATSM injection. Fifty minutes after the tracer injection, 10 min-PET acquisition is started. The reconstructed images are then converted to semi-quantitative images corrected by the injection dose and subject's body weight for data analysis.

[0078] The animal models of the present invention may be evaluated by these methods with and/or without treatment with therapeutic compounds of interest.

Tc99m-HMPAO SPECT

[0079] Serum levels of glutathione are assessed in vivo by HMPAO SPECT imaging using Tc99m-HMPAO. The onset of mitochondrial nephropathy is associated with decreases in serum glutathione and renal HMPAO uptake. Treatment of kd/kd mice with 20 mg/kg or 100 mg/kg of alpha- tocotrienol quinone at the age of 4 weeks prevents or significantly delays the onset of renal disease at 16 weeks and its severity in a dose -dependent fashion up to 19 weeks, at which time they are imaged and sacrificed.

[0080] The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety.

[0081] Although the foregoing invention has been described in some detail by way of example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

CLAIMS What is claimed is:

1. A method for identifying in vivo the activity of a compound for the treatment of oxidative stress, said method comprising the steps of: (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) optionally providing a third mouse of the same genetic background as the first and second mice; (d) administering the compound only to said first mouse; (e) determining renal failure damage of the first and second mice; and (f) identifying an in vivo pharmaceutical activity of the compound if the profile of the first mouse to which the compound has been administered differs from the profile of the second mouse to which the compound has not been administered, and/or exhibits changes versus the profile of the third mouse, wherein the profile includes one or more measurements selected from renal failure, urine production quantity, body temperature, body weight and

composition, glucose levels, sensorimotor behavior, glutathione levels, lactate levels, brain scans, kidney scans, redox status and CoQ levels; and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

2. The method of claim 1, for identifying in vivo the activity of a compound for the treatment of oxidative stress, said method comprising the steps of: (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining renal failure damage of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits fewer or less severe symptoms of renal failure, if administration of the compound delays or prevents symptoms of renal failure, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

3. The method according to claim 2, wherein the renal failure is quantified by the amount of urine produced by the mice.

4. The method according to claim 1, wherein said compound is for the treatment of a mitochondrial disease.

5. The method according to claim 4, wherein the compound is for the treatment of a respiratory chain disease.

6. A method of identifying in vivo the activity of a compound for the treatment of a patient with low levels of CoQ, comprises the steps of : (a) providing a first homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) optionally providing a third mouse of the same genetic background as the first and second mice (d) administering the compound only to said first mouse; (e) determining renal failure damage of the first and second mice; and (f) identifying an in vivo pharmaceutical activity of the compound if the profile of the first mouse to which the compound has been administered differs from the profile of the second mouse to which the compound has not been administered, and/or exhibits changes versus the profile of the third mouse, wherein the profile includes one or more measurements selected from renal failure, urine production quantity, body temperature, body weight and

7. The method of claim 6, for identifying in vivo the activity of a compound for patient with low levels of CoQ, said method comprising the steps of: (a) providing a first

homozygous Pdss2^kd/kd-mutant mouse; (b) providing a second homozygous Pdss2^kd/kd mutant mouse of the same litter as the first homozygous mouse; (c) administering the compound only to said first mouse; (d) determining renal failure damage of the first and second mice; and (e) identifying an in vivo pharmaceutical activity of the compound if the first mouse to which the compound has been administered exhibits fewer or less severe symptoms of renal failure, if administration of the compound delays or prevents symptoms of renal failure, and/or if the first mouse to which the compound has been administered survives longer than the second mouse to which the compound has not been administered.

8. The method according to claim 7, wherein the renal failure is quantified by the amount of urine produced by the mice.

9. The method according to claim 7, wherein said compound is for the treatment of a mitochondrial disease.

10. The method according to claim 9, wherein the compound is for the treatment of a respiratory chain disease.

11. A mouse model for oxidative stress comprising a mouse having a mutation in the Pdss2 gene.

12. The mouse model according to claim 11, where the oxidative stress symptom is a mitochondrial disease.

13. The mouse model according to claim 11, where the oxidative stress is a result of oxidative damage caused by free endogenous radicals.

14. The mouse according to claim 13, where the oxidative damage results in manifestations of aging.