WO2007047582A2

WO2007047582A2 - Porphyrin compounds for enhancing mitochondrial function

Info

Publication number: WO2007047582A2
Application number: PCT/US2006/040374
Authority: WO
Inventors: Gino Cortopassi; Eleonora Napoli; Robert Schoenfeld; Alice Wong
Original assignee: The Regents Of The University Of California
Priority date: 2005-10-18
Filing date: 2006-10-16
Publication date: 2007-04-26
Also published as: WO2007047582A3

Abstract

Prophyrin compounds are useful for ameliorating diseases involving mitochondrial function deficiencies.

Description

PORPHYRIN COMPOUNDS FOR ENHANCING MITOCHONDRIAL

FUNCTION

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 60/728,073, filed October 18, 2005, which is incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant No. ROl AGl 1967 awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] There are multiple, serious mitochondrial diseases, which are the result of deficiencies of of function of the mitochondria. These diseases include the genetic diseases Kearns Sayre Syndrome/Chronic Progressive External Ophthalmoplegia (KSS/CPEO), which causes a deficiency of the mitochondrial enzyme cytochrome oxidase, Leber's Hereditary Optic Neuropathy (LHON), caused by mutations in mitochondrial Complex I; and Friedreich's ataxia, that results in deficiencies in Complex IV. Moreover, mitochondrial dysfunction has been strongly implicated as a causative agent in multiple late-onset neurodegenerative diseases. There is a significant decrease in cytochrome oxidase activity with the progression of Alzheimer's disease. There is a decrease in mitochondrial Complex I activity in both ALS and Parkinson's disease. Drugs that inhibit mitochondrial Complex I cause Parkinsonism in humans and animals. Multiple mitochondrial functions are inhibited in the dopaminergic cells of Parkinson's patients as they age. Although there are some therapies for these diseases, they generally treat the symptoms of the diseases (e.g. acetylcholine depletion in Alzheimer's disease), rather than the root causes of the neurodegenerative conditions. [0004] Currently there are two drugs in use to stimulate mitochondrial function, i.e. Coenzyme Q and idebenone, a Coenzyme Q analog. Neither is very effective, however they are the main drugs prescribed to patients with mitochondrial disease.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides methods and compositions for treating individuals with deficiencies in mitochondrial function, hi some embodiments, the methods comprise administering an effective amount of a porphyrin.

[0006] hi some embodiments, the porphyrin is an iron porphyrin. In some embodiments, the porphyrin is heme. In some embodiments, the porphyrin is hemin.

[0007] hi some embodiments, the deficiency in mitochondrial function is characterized by a deficiency in iron-sulfur cluster enzyme activity, hi some embodiments, the deficiency in mitochondrial function is characterized by a deficiency in cytochrome activity.

[0008] hi some embodiments, the individual is afflicted with an ailment selected fro the group consisting of Friedreich's ataxia (FRDA), Glutaric Aciduria (GA), Parkinson's Disease (PD), Kearns Sayre Syndrome/Chronic Progressive External Ophthalmoplegia (KSS/CPEO), Leber's Hereditary Optic Neuropathy (LHON), Alzheimer's Disease (AD), Cytochrome Oxidase deficiency (COD), Amyotrophic Lateral Sclerosis (ALS), Myoclonic Epilepsy with Lactic Acidosis and Stroke (MELAS), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), and Neurogenic Ataxia with Retinitis Pigmentosum (NARP).

[0009] Other embodiments of the invention are clear from the disclosure herein.

DETAILED DESCRIPTION OF THE INVENTION

[0010] We have demonstrated that heme stimulates expression of mitochondrial proteins that are deficient in mitochondrial disease, restores mitochondrial iron-sulfur functions, and counteracts the effects of the disease, returning mitochondrial protein levels to their original level. Accordingly, the present invention is based on the surprising observation that administration of metalloporphyrins, such as heme, to cells that are deficient in mitochondrial functions result in an increase in those functions. The present invention provides for therapeutic and prophylactic administration of metalloporphyrins to treat diseases, preferably human diseases, associated with, and/or involving, reduced mitochondrial functions. [0011] Energy production by the mitochondrial electron transport proteins is made possible by several heme-containing (cytochrome) proteins containing 5 hemes/cytochromes, including: Cytochrome c reductase (cytochromes b and cϊ), cytochrome c, and cytochrome c oxidase (cytochromes a and «₃). Hemes and cytochromes are made by the biosynthetic pathway shown in the Figure 1, and its efficiency is dependent on metabolic status. Without intending to limit the scope of the invention to a particular mechanism, it is believed that porphyrin supplementation may drive cytochrome synthesis, and thus increase the synthesis of mitochondrial proteins that contain those cytochromes, and thereby increase mitochondrial activity.

[0012] Any number of porphyrins can be used according to the methods of the present invention. In some embodiments, the porphyrins will be metalloporphyrins. An exemplary metal in metalloporphyrins is iron. Exemplary porphyrins that can be used in the invention include, e.g., heme and hemin (discussed further below). Additional porphyrins that can be used in the invention include, e.g., delta-Aminolevulinic acid hydrochloride, Al(III) Phthalocyanine Chloride Tetrasulfonic Acid, 3,6-Bis(decyl)phthalonitrile, Bilirubin (alpha), Biliverdin dimethyl ester, Bilirubin dimethyl ester, Biliverdin hydrochloride, Bilirubin conjugate, Coproporphyrin I dihydrochloride, Coproporphyrin III dihydrochloride, Coproporphyrin I tetramethyl ester, Coproporphyrin III tetramethyl ester, Cu(II) meso- Tetra(4-carboxyphenyl)porphine, Cu(II) meso-Tetra(4-sulfonatophenyl) porphine (acid form), Chlorin e6, Co(III) Protoporphyrin IX chloride, Cr(III) Mesoporphyrin IX chloride, Deuteroporphyrin IX dihydrochloride, Deuteroporphyrin IX dimethyl ester, 2,4-Dimethyl Deuteroporphyrin IX dimethyl ester, 2,4-Diacetyl deuteroporphyrin IX dimethyl ester, Deuteroporphyrin IX 2,4 bis ethylene glycoln Deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride, Deuteroporphyrin DC 2,4-disulfonic acid dimethyl ester disodium salt, Etioporphyrin I, Fe(III) Mesoporphyrin IX chloride, Fe(III) meso-Tetra(4- sulfonatophenyl)porphine chloride (acid form), Fe(III) meso-Tetra(4-carboxyphenyl)porphine chloride, Hematoporphyrin IX dihydrochloride, Hematoporphyrin DC dimethyl ester, Hematoporphyrin IX base, Hexacarboxylporphyrin I dihydrochloride, Hexacarboxylporphyrin I hexamethyl ester, Heptacarboxylporphyrin I dihydrochloride, Heptacarboxylporphyrin I heptamethyl ester, Deuteroporphyrin DC 2,4 (4,2) hydroxyethyl vinyl, Isohematoporphyrin DC, Mesoporphyrin DC dihydrochloride, Mesobilirubin, Mesoporphyrin DC dimethyl ester, Mn(III) Protoporphyrin DC chloride, Mn(III) meso- Tetra(4-sulfonatophenyl)porphine chloride (acid form), Pyropheophorbide-a methyl ester, Ni(II) Octaethylporphine, Ni(II) meso-Tetraphenylporphine chlorin free, N-Methyl Mesoporphyrin IX, N-Methyl Protoporphyrin IX, meso-Tetraphenylporphine , Octaethylporphine, Porphobilinogen, Porphine, Phthalocyariine, Protopoφhyrin IX, Protoporphyrin IX dimethyl ester, Protoporphyrin IX disodium salt, Pentacarboxylporphyrin I dihydrochloride, Pentacarboxylporphyrin I pentamethyl ester , Pd(II) meso-Tetra(4- carboxyphenyl)porphine, Pd(II) meso-Tetra(pentafluorophenyl)poφhine, Pt(II) Octaethylporphine , Pt(II) meso-Tetra(pentafluorophenyl)porphine, Sn(IV) Protoporphyrin IX dichloride, Sn(IV) Mesoporphyrin IX dichloride, meso-Tetra(4-sulfonatophenyl)porphine dihydrochloride, Tetrabenzoporphine, meso-Tetraphenylporphine chlorin free, meso-Tetra(4- pyridyl)porphine, meso-Tetra(m-hydroxyphenyl)poφhine, meso-Tetra(p- hydroxyphenyl)porphine, meso-Tetra(4-carboxyphenyl)porphine, meso-Tetra(4- carboxyphenyl)porphine tetramethyl ester, meso-Tetra(o-dichlorophenyl) poφhine, meso- Tetra(2,4,6-trimethylphenyl)poφhine, meso-Tetra(4-N,N,N-trimethylanilinium) poφhine tetrachloride, meso-Tetra(pentafluorophenyl)poφhine chlorin free, meso-Tetra (N-methyl-4- pyridyl) poφhine tefra tosylate, Uropoφhyrin I dihydrochloride, Uropoφhyrin III dihydrochloride, Deuteropoφhyrin IX 2-vinyl, 4-hydroxymethyl, Vanadyl octaethylpoφhine, Vanadyl meso-tetraphenylpoφhine chlorin free, Zn(II) Protopoφhyrin IX, Zn(II) Deuteropoφhyrin IX 2,4 bis ethylene glycol, Zn(II) Phthalocyanine tetrasulfonic acid.

[0013] Heme is an iron-protopoφhyrin complex consisting of four substituted pyrrole rings linked by -CH group. When the iron atom is in the ferrous state, the complex is called ferroprotopoφhyrin or heme, and the molecule is electrically neutral. When the iron atom is in the ferric state, the complex is called ferriprotopoφhyrin or hemin, and the molecule carries a unit positive charge.

[0014] Heme is an essential molecule to living aerobic organisms and plays a role in various biological reactions. It is an essential component of mitochondrial energy-generating subsystems cytochrome oxidase, cytochrome c, and cytochrome reductase (See Figure 1). Several types of heme, which differ in the composition of the side chains of the pyrrole rings, are present in nature. The final steps of the heme pathway are in the mitochondria, and make heme b which is the most ubiquitous and constitutes the prosthetic moiety of almost all hemeproteins. Heme c, which is made from heme b, is incoφorated into cytochrome c and cytochrome cl of cytochrome reductase. Heme a also arise from heme b, and is then incoφorated into cytochrome oxidase, as heme a and a.3. Any such type of heme can be used according to the methods of the invention. Heme biosynthesis makes use of iron-sulfur proteins ferrochelatase and adrenodoxin, which may be consumed by this activity.

[0015] Hemin is a non-toxic FDA orphan drug which is normally derived from processed red blood cells. It is approved for treatment of porphyria and myelodysplastic syndrome in humans and the pharmacokinetics toxicology of hemin are well understood (Tenheunen, et al. (1987) J Pharm. Pharmocol. 39:780-786; and Volin, et al. (1988) Blood 71:625-628). Chemically, hemin is chloro[7,12-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18- dipropan oato(2-)-N₂i, N₂₂,N₂₃,N₂₄]iron. Hemin for administration by injection is commercially available from Ovation Pharmaceuticals, Inc. under the trade name Panhematin®. and has been known as hematin. Hematin, which is suitable for use according to this invention, is the chemical reaction product of hemin with aqueous sodium carbonate solution. Heme arginate, can also be used in the methods of the present invention and is available commercially from Orphan Europe (UK) Ltd. under the tradename Normosang®.

[0016] Examples of deficiencies in mitochondrial function that can be treated in the methods of the present invention include, but are not limited to, down regulation of expression or activity of electron transport components, ATP synthesis proteins, TCA cycle components, or coproporphyrinogen oxidase (CPOX). These defects also include defects in mitochondrial membrane potential, and defects in ATP synthesis. These defects result in muscle weakness, deafness, diabetes, dementia, ataxia, blindness (optic neuropathy), and cardiomyopathy, and myoclonus.

[0017] Diseases associated with a deficiency in mitochondrial function that can be treated or ameliorated according to methods of the invention include, e.g., FRDA, PD, KSS/CPEO, LHON, PD, AD, COD, MM, MERRF, MELAS, NARP. Deficiencies in mitochondrial function can include deficiencies (compared to levels in healthy individuals) in iron sulfur cluster enzyme activity (including, but not limited to, deficiencies in the activity of one or more of the enzymes aconitase, ferrochelatase, adrenodoxin, NADH Dehydrogenase, Succinate Dehydrogenase, Cytochrome c reductase, electron-transfer flavoprotein) and/or cytochrome c activity.

[0018] Pharmaceutical compositions containing metalloporphyrin compounds can be formulated for human and animal prophylactic and therapeutic applications by those having ordinary skill in the art. Pharmaceutical formulations for administering hemin for the treatment of porphyrias are well understood. Consequently, pharmaceuticals for administering metalloporphyrins, including iron porphyrin compounds, to treat diseases associated with deficiency in mitochondrial function can be formulated based on known hemin formulations.

[0019] According to the invention, individuals suffering from diseases associated with deficiency in mitochondrial function are identified and treated with one or more iron porphyrin compounds. The range of dose amounts and frequency of delivery of iron porphyrin compound to be administered to mammals, and particularly to humans, to be effective in treating, preventing or ameliorating the symptoms of diseases associated with a deficiency in mitochondrial function can be determined by those having ordinary skill in the art. A methodology for determining appropriate dosage includes determining the existing state of disease of a patient; administering at a preselected frequency, a preselected amount of pharmaceutical formulation containing iron porphyrin compound; determining the state of disease exhibited by the patient at a later time when the disorder, if untreated, would have increased; and applying an adjustment to the dosage amount and/or delivery rate to reduce, maintain or increase the effect of preventing or reducing the disease or disease symptoms.

[0020] Doses of iron porphyrin compound can be delivered, for example, in a single dosage, divided dosages or in a sustained release during a period, e.g., less than 24 hours. Some iron porphyrin compounds, such as hemin, are susceptible to rapid conversion to billirubin and thus are excreted in substantial fraction via the liver. Dosage amount and frequency of administration may need to be increased to compensate for the therapeutic agent that is so purged prior to reaching the target. This need to boost dosage and frequency is primarily an issue for systemic methods of delivery.

[0021] Pharmaceutical compositions that include a iron porphyrin compound may be administered by any method that can deliver iron porphyrin to the site in the body of a mammal where activity is to occur. These methods include but are not limited to oral, subcutaneous, transdermal, intravenous, intramuscular, liposomal and parenteral methods of administration. The iron porphyrin compound can be combined with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed. The preparation of such compositions can entail combining the iron porphyrin compound with buffers, antioxidants such as ascorbic acid, low molecular weight (e.g., less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with serum albumin can be used. A composition of the invention can be formulated as a lyophilized product using appropriate excipient solutions (e.g., sucrose) as diluents.

[0022] Hemin, hematin and hemin arginate can be, e.g., administered intravenously in sterile liquid dosage forms. Hemin may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. For example, see Remington's Pharmaceutical Sciences, 17th ed., A. R. Gennaro, Mack Publishing Company, Easton, Pa. (1985), p. 842. Panhematin® hemin is sold in single doses as a sterile, lyophilized black powder suitable for intravenous infusion after reconstitution with USP grade sterile water as described in the Physicians Desk Reference. Medical Economics Data Production Co., Montvale, NJ., (1995) pp. 447-448.

EXAMPLES 1. Exposure to hemin rescues heme c deficiency in Friedreich's ataxia.

[0023] Blood lymphocytes from patients with the mitochondrial disease Friedreich's ataxia have defects in cytochrome c (Schoenfeld et al, 2005; Napoli et al, 2006).

[0024] The levels of heme inserted in cytochrome c (heme c) and the levels of cytochrome c were analyzed in mitochondrial preparations obtained from 3 controls and 5 FRDA mutant lymphoblast lines, untreated or treated with 5 μM hemin for 18 hours. See Figure 2. Both the levels of heme c and cytochrome c in Friedreich's ataxia mutants are significantly lower than the controls (p<0.05); the analysis in the same cell lines after treatment with hemin shows a significant and complete rescue of the heme c deficiency (p<0.01).

2. Hemin stimulates the expression of several mitochondrial proteins.

[0025] The levels of the 39Kda subunit of Complex I, subunit II of Complex IV (COXII) _. and cytochrome c were analyzed by Western blot followed by densitometry of the bands. The levels of such proteins are increased in lymphoblasts treated for 18 hours with different concentrations (1-20 μM) of hemin, Complex I subunit being the one responding the most and at the lowest dose. Figure 3 displays the result of a representative experiment and the graph shows the relative expression of the proteins considered as % of the expression of the same proteins in the untreated cells. Expression values are normalized to those of the 51Kda subunit of complex V, which isn't affected by the treatment with hemin.

3. Hemin stimulates gene expression in multiple cell types.

[0026] To understand if the response to hemin was general we also evaluated the expression of cytochrome c protein, Complex I (subunit 39 Kda) and Complex IV (subunit II) proteins , and cytochrome c heme, i.e. heme c in human 143B osteosarcoma tumor cells. Figure 4. The same proteins whose expression was stimulated by the hemin treatment in lymphoblasts were induced also in this cell model. These mcreases were observed with similar levels of Complex V (subunit 51 Kda), a non-heme containing protein, suggesting that the increases were not the result of an increase in mitochondrial number, but rather, an increase in mitochondrial proteins per mitochondrion.

4. Hemin rescues functional defects caused by a specific mitochondrial deficiency.

[0027] The frataxin gene encodes a mitochondrial protein whose deficiency causes Friedreich's ataxia, heme deficiency, and iron-sulfur cluster deficiency. The frataxin gene was knocked down by siRNA technology in human cells, and heme deficiency and iron- sulfur cluster deficiency was observed.

[0028] Hemin supplementation restored the activity of the iron-sulfur cluster enzyme adrenodoxin to normal levels, and the mean activity of cytochrome oxidase increased. Thus hemin not only stimulates mitochondrial proteins but also restores iron-sulfur functions.

Conclusions

[0029] There are several human mitochondrial diseases that are caused by deficiencies in respiratory chain enzymes and there are no effective therapies. Coenzyme Q therapy, and idebenone, a more bioavailable form of Coenzyme Q, are given for mitochondrial disease, but there is little beneficial effect.

[0030] We showed that the treatment with hemin mcreases the levels of at least one subunit of Complex IV, Complex I and of cytochrome c, and of heme c, while no effect was observed on Complex V. The cytochrome c molecule in fact possesses a heme molecule (heme c), as does cytochrome c oxidase (hemes a and αj). Complex I is the first and largest protein of the mitochondrial respiratory chain and between its 43 subunits there are 3 iron-sulfur centers. Hence, by providing iron to mitochondria in a bioavailable form hemin stimulates the expression of those proteins which need iron for their activity.

Methods

Cell culture

[0031] All cells were maintained at 37° in a humidified atmosphere containing 5% CO₂.

[0032] Lymphocytes were grown in RPMI 1640 supplemented with 500 mg/L glutamate, 1 mM sodium pyruvate, 50 μg/ml uridine, 100 μM nonessential amino acids (Invitrogen, Carlsbad, CA), 20% fetal calf serum and penicillin/streptomycin.

[0033] Human 143B osteosarcoma cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g/L glucose, 110 mg/L pyruvate, supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin.

[0034] The day before the mitochondrial isolation, fresh media was added to control and FRDA lymphoblasts and to 143B osteosarcoma. For the studies of rescue with hemin one fraction of the cells was added of 5 μM hemin and stored in incubator for 18 hours. For the dose-response study, controls, FRDA mutants and 143B osteosarcoma were treated with increasing doses of hemin (0, 1, 5 10, 20 and 50 μM) for 18 hours.

Mitochondria isolation

[0035] Mitochondria were isolated from 3 controls and 5 FRDA mutant lines of human immortalized lymphoblasts following the method of Trounce et al (Trounce,et al. Methods Enzymol 264: 484-509 (1996)). Approximately IxIO⁸ cells were harvested by centrifugation and the pellet was re-suspended with 4 ml of isolation buffer (210 mM mannitol, 70 mM sucrose, ImM EGTA and 5mM HEPES, pH 7.2) for each gram of packed cells, treated with a final concentration of 0.3 mg/ml digitonin for 1 min 30 sec and centrifuged at 3500 rpm for 5 minutes. The pellet was then resuspended with 5 ml for each gram of initial packed cells and homogenized with a chilled class homogenator (20 passes). After several centrifugation steps at 1510 rpm, the final supernatant was centrifuged at lOOOOg and the final pellet was suspended with 0.1 ml of isolation buffer per gram of starting cells, giving a protein concentration of approximately 8-12 mg/ml. For determination of mitochondrial protein concentration, 5 μl of the mitochondrial suspension was diluted 1:20 in double distilled water and the protein concentration was estimated using the Bradford assay (Bio-Rad). The lysates were prepared resuspending the mitochondrial pellet in about 100 μl per initial gram of packed cell of the following lysis buffer: 5OmM Tris, pH 7.8, 100 mM NaCl, 1 mM PMSF and 1% detergent (IGEP AL-C A630) for 30 minutes at 0°C and insoluble material removed by centrifugation at 1600Og. The supernatant was collected, the protein amount evaluated using the Bradford assay (Bio-Rad) and the mitochondrial fraction stored at -80°C.

Western blot analysis

[0036] Equal amounts of lysates (40μg) untreated or treated with hemin were resolved on a 15% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Millipore, Bedford, MA) by electroblotting. After blocking with 4% non-fat dry milk, the blot was incubated with anti-cytoclirome c (1:1500), or anti-COXII (1:2000), or anti-Complex I- 39Kda subunit (1:1000) and was developed with AP-conjugated secondary antibodies using a chemiluminescent substrate.

Heme staining of cytochrome c

[0037] Untreated and treated mitochondrial extracts were separated on a 15% SDS/PAGE gel. Heme staining was performed as previously described (Schulz, et al, MoI Microbiol 37(6): 1379-88 (2000)). Forty micrograms of mitochondrial protein from 3 different controls and 5 different FRDA lymphoblast lines were loaded onto a 15% polyacrylamide gel and the electrophoresis was performed at 13OmV for 60'. After the run the gel was fixed in 10% TCA fori 10', washed 4 times for 5' in double distilled water. The heme staining is based on the oxidation of σ-dianisidine, a probe which, in presence of hydrogen peroxide (H₂O₂), can be oxidized by the peroxidase activity of some heme-proteins (such as cytochrome c) changing color. The gel was soaked in a solution of 5OmM trisodium citrate, 0.7% H₂O₂ and 1 mg/ml o-dianisidine for 40-60' at 45°C. 0.5μg of horse heart purified cytochrome c was used as positive controls. The band in correspondence of cytochrome c became evident after 20 minutes. Statistical analysis of the data.

[0038] ANOVA followed by Bonferroni's post test was carried out to calculate the level of significance in the rescue experiments.

[0039] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0040] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating an individual with a deficiency in mitochondrial function, the method comprising administering to the individual an effective amount of a porphyrin.

2. The method of claim 1, wherein the porphyrin is heme.

3. The method of claim 1 , wherein the porphyrin is hemin.

4. The method of claim 1 , wherein the porphyrin is an iron porphyrin.

5. The method of claim 1 , wherein the deficiency in mitochondrial function is characterized by a deficiency in iron-sulfur cluster enzyme activity.

6. The method of claim 1 , wherein the deficiency in mitochondrial function is characterized by a deficiency in cytochrome activity.

7. The method of claim 1, wherein the individual is afflicted with an ailment selected from the group consisting of Friedreich's ataxia (FRDA), Glutaric Aciduria (GA), Parkinson's Disease (PD), Kearns Sayre Syndrome/Chronic Progressive External Ophthalmoplegia (KSS/CPEO), Leber's Hereditary Optic Neuropathy (LHON), Alzheimer's Disease (AD), Cytochrome Oxidase deficiency (COD), Amyotrophic Lateral Sclerosis (ALS), Myoclonic Epilepsy with Lactic Acidosis and Stroke (MELAS), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), and Neurogenic Ataxia with Retinitis Pigmentosum (NARP).