Deoxyribonucleoside monophospate bypass therapy for mitochondrial DNA depletion syndrome
11633418 · 2023-04-25
Assignee
Inventors
Cpc classification
A61K31/7068
HUMAN NECESSITIES
A61K31/708
HUMAN NECESSITIES
A61K31/7076
HUMAN NECESSITIES
International classification
A61K31/7068
HUMAN NECESSITIES
A61K31/7072
HUMAN NECESSITIES
A61K31/7076
HUMAN NECESSITIES
Abstract
The invention relates generally to a pharmacological therapy for a human genetic diseases, specifically mitochondrial DNA depletion syndromes, and more specifically, thymidine kinase 2 (TK2) deficiency. The pharmacological therapy involves the administration of at least one deoxyribonucleoside monophosphate, or mixtures thereof. For the treatment of TK2 deficiency, the pharmacological therapy involves the administration of either deoxythymidine monophosphate (dTMP) or deoxycytidine monophosphate (dCMP), or mixtures thereof. This molecular bypass approach is applicable to other disorders of unbalanced nucleotide pools, especially those found in mitochondrial DNA depletion syndrome.
Claims
1. A method of treating thymidine kinase 2 (TK2) deficiency in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP), wherein the therapeutically effective amount is about 100 mg/kg/day of total deoxyribonucleoside monophosphaste in the composition and the composition is administered to the subject parenterally.
2. The method of claim 1, wherein the parenteral administration is intravenous administration.
3. The method of claim 1, wherein the composition is administered once a day, twice a day, three times a day, four times a day, five times daily or six times daily.
4. The method of claim 3, wherein the therapeutically effective amount of the composition per administration is between about 15 mg/kg to about 20 mg/kg.
5. A method of treating mitochondrial DNA depletion syndrome in a human subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising at least one deoxyribonucleoside monophosphate wherein the mitochondrial DNA depletion syndrome is characterized by at least one mutation in a gene chosen from the group consisting of: DGUOK; TYMP; and RRM2B, and wherein the therapeutically effective amount is about 100 mg/kg/day of total deoxyribonucleoside monophosphate in the composition and the composition is administered to the subject parenterally.
6. The method of claim 5, wherein the parenteral administration is intravenous administration.
7. The method of claim 5, wherein the composition is administered, once a day, twice a day, three times a day, four times a day five times daily or six times daily.
8. The method of claim 7, wherein the therapeutically effective amount of the composition per administration is between about 15 mg/kg to about 20 mg/kg.
9. A method of treating mitochondrial DNA depletion syndrome in a human subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising at least one deoxyribonucleoside monophosphate wherein the mitochondrial DNA depletion syndrome is characterized by at least one mutation in a gene chosen from the group consisting of: TK2, DGUOK; TYMP; and RRM2B, and wherein the therapeutically effective amount is about 100 mg/kg/day of total deoxyribonucleoside monophosphate in the composition and the composition is administered to the subject intrathecally.
10. The method of claim 9, wherein the composition is administered, once a day, twice a day, three times a day, four times a day five times daily or six times daily.
11. The method of claim 10, wherein the therapeutically effective amount of the composition per administration is between about 15 mg/kg to about 20 mg/kg.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) For the purpose of illustrating the invention, there are depicted in drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.
(2)
(3)
(4) Abbreviations: CS=citrate synthase; I=NADH-dehydrogenase; II=succinate dehydrogenase; III=cytochrome c reductase; IV=cytochrome c oxidase; V=ATP synthase; I+III=NADH—cytochrome c reductase; II+III=succinate cytochrome c reductase.
(5)
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(10) Statistical analyses were performed using Tk2.sup.−/− versus Tk2.sup.−/−200dCMP/dTMP with P13 cerebral samples (
(11) Abbreviations: CS=citrate synthase; IV=cytochrome c oxidase (COX); I+III=NADH-cytochrome c reductase; I=NADH-dehydrogenase; II=succinate dehydrogenase; III=cytochrome c reductase; V=ATP synthase; P=postnatal day.
(12)
DETAILED DESCRIPTION OF THE INVENTION
(13) The current invention is based upon the surprising discovery that mitochondrial DNA depletion syndromes, including TK2 deficiency, can be treated with a molecular bypass approach. As shown by the results herein, the administration of deoxyribonucleoside monophosphates greatly improved the condition in both a mouse model of TK2 deficiency and human patients with TK2 deficiency.
Definitions
(14) The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.
(15) The term “subject” as used in this application means mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Thus, the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications
(16) The term “patient” as used in this application means a human subject. In some embodiments of the present invention, the “patient” is known or suspected of having mitochondrial disease, mitochondrial DNA depletion syndrome, or TK2 deficiency.
(17) The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease or disorder, or results in a desired beneficial change of physiology in the subject.
(18) The terms “treat”, “treatment”, and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease or disorder, or reverse the disease or disorder after its onset.
(19) The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or minimize the extent of the disease or disorder, or slow its course of development.
(20) The term “in need thereof” would be a subject known or suspected of having or being at risk of having mitochondrial disease, mitochondrial DNA depletion syndrome, or TK2 deficiency.
(21) The term “agent” as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologics, small organic molecules, antibodies, nucleic acids, peptides, and proteins.
(22) The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
(23) Deoxypyrimidine Monophosphate Bypass Therapy Mouse Model of TK2 Deficiency
(24) Attempts to study the pathogenesis and test therapies for TK2 deficiency using cultured fibroblasts from patients had proved unsuccessful because the replicating cells failed to manifest mtDNA depletion. Thus, to elucidate the molecular pathogenesis of TK2 deficiency, a homozygous Tk2 H126N knock-in mutant (Tk2.sup.−/−) mouse that manifests a phenotype strikingly similar to the human infantile encephalomyopathy was generated and previously reported by the inventors (Akman, et al. 2008) (Example 1).
(25) Based upon the understanding of the pathogenesis of Tk2 deficiency, a rationale therapeutic strategy to bypass the enzymatic defect with oral dCMP and dTMP supplementation was designed and implemented (Example 1). Surprisingly, the results herein demonstrate that the molecular bypass therapy with orally administered dCMP and dTMP delayed disease onset, ameliorated the abnormal phenotype and extended the lifespan of Tk2.sup.−/− mice by 2-3 fold. Additionally, no adverse side effects, including malignancies, were observed (Example 2;
(26) Oral dTMP/dCMP crossed biological barriers including the blood-brain barrier (BBB) because treatment increased dTTP in brain and liver in 13-day-old Tk21 mice and augmented levels of mtDNA, restoring the mitochondrial RC activities and protein defects in brain, heart, muscle, liver, and kidney of 13- and 29-day-old mutant mice (Examples 4 and 5;
(27) Previous studies have found that wild-type Tk2 (mitochondrial) activity is constant in the second week of life while cytosolic Tk1 activity decreases significantly between postnatal day 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2.sup.−/− mice and correlates with the onset of mtDNA depletion in brain and heart (Dorado, et al. 2011). The results herein demonstrated that oral dCMP and dTMP delayed the reduction in Tk1 activity (Example 6;
(28) The data in mice shown herein proves that deoxypyrimidine supplementation for Tk2 deficiency is the first effective and safe in vivo treatment option for patients affected by Tk2 mutations. See Garone, et al. 2014, herein incorporated by reference in its entirety. Based upon this data, the administration of deoxyribonucleoside monophosphates was hypothesized to improve the conditions of human patients with TK2 deficiency, even though mouse models have revealed important differences in dNTP homeostasis between mice and humans.
(29) Furthermore, this approach is applicable to individuals with other mitochondrial disorders due to nucleotide pools unbalance.
(30) Patients Benefitting from the Administration of Deoxyribonucleoside Monophosphates
(31) The present invention includes the administration of at least one deoxyribonucleoside monophosphate to a patient in need thereof. In one embodiment, the present invention includes the administration of at least one deoxypyrimidine monophosphate. In a further embodiment, the dexypyrimidine monophosphate is chosen from dCMP, dTMP and mixtures thereof. In yet another embodiment, the present invention includes the administration of at least one deoxypurine monophosphate. In a further embodiment, the deoxypurine monophosphate is chosen from dAMP, dGMP, and mixtures thereof.
(32) As shown in Examples 7-10, the administration of dTMP and dCMP greatly improved the symptoms of TK2 deficiency in patients, especially with regard to muscular-related symptoms. Thus, patients who would benefit from the deoxypyrimidine monophosphate bypass therapy would be those diagnosed with TK2 deficiency. In these patients, at least one deoxypyrimidine monophosphate, dCMP or dTMP, or mixtures thereof would be administered.
(33) A parallel defect of deoxyguanosine kinase (dGK), due to autosomal recessive mutations in DGUOK with deficiencies in dGMP and dAMP, causes mtDNA depletion typically manifesting as early childhood-onset hepatocerebral disease (Mandel et al., 2001). These patients would benefit from the administration of at least one deoxypurine monophosphates, dGMP or dAMP, or mixtures thereof.
(34) Other forms of MDS as well as other disorders related to unbalanced nucleotide pools can be treated by the administration of specific deoxyribonucleoside monophosphate, i.e., dAMP, dGMP, dCMP, or dTMP, or mixtures thereof. These disorders would include but are not limited to deficiencies related to RRM2B (encoding p53R2, the p53-inducible small subunit of ribonucleotide reductase, RNR) and mutations in TYMP (encoding thymidine phosphorylase, TP) which cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).
(35) Additionally, as the mechanisms of other forms of MDS and other disorders become elucidated, the proper deoxyribonucleoside monophosphate(s) for treatment can be determined by the skilled practitioner.
(36) Patients that exhibit the phenotype discussed above for TK2 deficiency including the most typical presentation of progressive muscle disease characterized by generalized hypotonia, proximal muscle weakness, loss of previously acquired motor skills, poor feeding, and respiratory difficulties, can be tested to definitively diagnose the disease.
(37) If the clinical presentation is highly suspicious for mtDNA depletion syndrome, molecular genetic testing using a panel of genes known to cause mtDNA depletion syndrome should be performed (Chanprasert, et al. 2012). The TK2 gene is the only gene in which mutations are known to cause TK2-related mitochondrial DNA depletion syndrome. This testing can include a sequence analysis of the entire coding and exon/intron junction regions of TK2 for sequence variants and deletion/duplication. If compound heterozygous or homozygous deleterious mutations are identified in the sequence analysis, the diagnosis of TK2 deficiency is confirmed, and thus, the subject would benefit from the deoxypyrimidine monophosphate therapy. If sequence analysis does not identify two compound heterozygous or homozygous deleterious mutations, deletion/duplication analysis should be considered to determine and/or confirm a TK2 deficiency diagnosis.
(38) Further tests to determine and/or confirm a TK2 deficiency diagnosis may include testing serum creatine kinase (CK) concentration, electromyography, histopathology on skeletal muscle, mitochondrial DNA (mtDNA) content (copy number), and electron transport chain (ETC) activity in skeletal muscle. If one or more of the following is found in these tests, the TK2 deficiency is determined and/or confirmed. Elevated CK concentration as compared to healthy controls can indicate TK2 deficiency. A skeletal muscle biopsy can be performed, and then a mtDNA content analysis in skeletal muscle performed. If the skeletal muscle biopsy shows prominent variance in fiber size, variable sarcoplasmic vacuoles, variable increased connective tissue, and ragged red fibers as well as increased succinate dehydrogenase (SDH) activity and low to absent cytochrome c oxidase (COX) activity, and mtDNA copy number is severely reduced (typically less than 20% of age- and tissue-matched healthy controls), a diagnosis of TK2 deficiency can be determined and/or confirmed (Chanprasert, et al. 2012).
(39) Additionally, TK2 deficiency is inherited in an autosomal recessive manner. Thus, a sibling of an affected patient can be tested as early as possible after birth to diagnose the disease.
(40) In all of these examples, deoxypyrimidine monophosphate bypass therapy should be started as soon as possible after a diagnosis of TK2 deficiency.
(41) Pharmaceutical Compositions, Methods of Administration, and Dosing
(42) The present invention encompasses the administration of deoxyribonucleoside monophosphates. Most preferred methods of administration are oral, intrathecal and parental including intravenous. The deoxyribonucleoside monophosphates must be in the appropriate form for administration of choice.
(43) The preferred form of the administration of the deoxyribonucleoside monophosphates is in the form of disodium salts of the deoxyribonucleoside monophosphates. These disodium salts (dTMP Na.sub.2, dCMP Na.sub.2 salts) are easily dissolved in liquid (such as water, formula or milk) whereas the free acid form does not readily dissolve in liquid. Once administered, the deoxyribonucleoside monophosphates salts convert to the free acid form. By way of example, for some of the oral administration to human patients, 2000 mg each of dTMP Na.sub.2 and dCMP Na.sub.2 were dissolved in 25 ml of water.
(44) Such compositions for administration may comprise a therapeutically effective amount of the deoxyribonucleoside monophosphates and a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human, and approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
(45) “Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
(46) Oral administration is a preferred method of administration. The deoxyribonucleoside monophosphates can be added to any form of liquid a patient would consume including but not limited to, milk, both cow's and human breast, infant formula, and water.
(47) Additionally, pharmaceutical compositions adapted for oral administration may be capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions. Tablets or hard gelatin capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols. Solutions and syrups may comprise water, polyols, and sugars. An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract. Thus, the sustained release may be achieved over many hours and if necessary, the active agent can be protected from degradation within the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions.
(48) In order to overcome issue of the deoxynucleoside monophosphates crossing the blood/brain barrier, intrathecal administration is a further preferred form of administration of deoxyribonucleoside monophosphates (Galbiati, et al. 2006; Gotz, et al. 2008). Intrathecal administration involves injection of the drug into the spinal canal, more specifically the subarachnoid space such that it reaches the cerebrospinal fluid. This method is commonly used for spinal anesthesia, chemotherapy, and pain medication. Intrathecal administration can be performed by lumbar puncture (bolus injection) or by a port-catheter system (bolus or infusion). The catheter is most commonly inserted between the laminae of the lumbar vertebrae and the tip is threaded up the thecal space to the desired level (generally L3-L4). Intrathecal formulations most commonly use water, and saline as excipients but EDTA and lipids have been used as well.
(49) A further preferred form of administration is parenteral including intravenous administration. Pharmaceutical compositions adapted for parenteral administration, including intravenous administration, include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the compositions substantially isotonic with the blood of the subject. Other components which may be present in such compositions include water, alcohols, polyols, glycerine, and vegetable oils. Compositions adapted for parental administration may be presented in unit-dose or multi-dose containers, such as sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile carrier, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
(50) Additionally, since some patients may be receiving enteral nutrition by the time the deoxyribonucleoside monophosphate treatment begins, the dNMPs can be administered through a gastronomy feeding tube or other enteral nutrition means.
(51) Further methods of administration include mucosal, such as nasal, sublingual, vaginal, buccal, or rectal; or transdermal administration to a subject.
(52) Pharmaceutical compositions adapted for nasal and pulmonary administration may comprise solid carriers such as powders, which can be administered by rapid inhalation through the nose. Compositions for nasal administration may comprise liquid carriers, such as sprays or drops. Alternatively, inhalation directly through into the lungs may be accomplished by inhalation deeply or installation through a mouthpiece. These compositions may comprise aqueous or oil solutions of the active ingredient. Compositions for inhalation may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed so as to provide predetermined dosages of the active ingredient.
(53) Pharmaceutical compositions adapted for rectal administration may be provided as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
(54) Pharmaceutical compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient over a prolonged period of time.
(55) Selection of a therapeutically effective dose will be determined by the skilled artisan considering several factors, which will be known to one of ordinary skill in the art. Such factors include the particular form of the deoxyribonucleoside monophosphate, and its pharmacokinetic parameters such as bioavailability, metabolism, and half-life, which will have been established during the usual development procedures typically employed in obtaining regulatory approval for a pharmaceutical compound. Further factors in considering the dose include the condition or disease to be treated or the benefit to be achieved in a normal individual, the body mass of the patient, the route of administration, whether the administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus, the precise dose should be decided according to the judgment of the person of skill in the art, and each patient's circumstances, and according to standard clinical techniques.
(56) A preferred dose ranges from about 100 mg/kg/day to about 1,000 mg/kg/day. A further preferred dose ranges from about 200 mg/kg/day to about 800 mg/kg/day. A further preferred dose ranges from about 250 mg/kg/day to about 400 mg/kg/day. These dosage amounts are of individual deoxyribonucleoside monophosphates or of a composition with a mixture of more than one deoxyribonucleoside monophosphates, e.g., dTMP and dCMP. For example, a dose can comprise 400 mg/kg/day of dTMP alone. In a further example, a dose can comprise a mixture of 200 mg/kg/day of dTMP and 200 mg/kg/day of dCMP. In a further example, a dose can comprise 400 mg/kg/day of a mixture of dTMP and dCMP.
(57) Administration of the deoxyribonucleoside monophosphates can be once a day, twice a day, three times a day, four times a day, five times a day, up to six times a day, preferably at regular intervals. For example, when the deoxyribonucleoside monophosphates are administered four times daily, doses would be at 8:00 AM, 12:00 PM, 4:00 PM, and 8:00 PM.
(58) As shown in the Examples, doses can be adjusted to optimize the effects in the subject. For example, the deoxyribonucleoside monophosphates can be administered at 100 mg/kg/day to start, and then increased over time to 200 mg/kg/day, to 400 mg/kg/day, to 800 mg/kg/day, up to 1000 mg/kg/day, depending upon the subject's response.
(59) A subject can be monitored for improvement of their condition prior to increasing the dosage. Also as shown in the Examples, a subject's response to the therapeutic administration of the deoxyribonucleoside monophosphates can be monitored by observing a subject's muscle strength and control, and mobility as well as changes in height and weight. If one or more of these parameters increase after the administration, the treatment can be continued. If one or more of these parameters stays the same or decreases, the dosage of the deoxyribonucleoside monophosphates can be increased.
(60) The deoxyribonucleoside monophosphates can also be co-administered with other agents. Such agents would include therapeutic agents for treating the symptoms of the particular form of MDS. In particular, for TK2 deficiency, the dTMP and dCMP can be co-administered with an inhibitor of thymidine phosphorylase (e.g. tipiracil) or an inhibitor of cytidine deaminase (e.g. tetrahydrouridine [THU]) (see Example 6). Such inhibitors are known and used in the treatment of some cancers.
EXAMPLES
(61) The present invention may be better understood by reference to the following non-limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention.
Example 1—Materials and Methods for Examples 2-6
(62) Mouse Model of TK2 Deficiency
(63) A homozygous Tk2 H126N knock-in mutant (Tk2) mouse that manifests a phenotype strikingly similar to the human infantile encephalomyopathy has been previously reported (Akman, et al. 2008). Between postnatal day 10 and 13, Tk2.sup.−/− mice rapidly develop fatal encephalomyopathy characterized by decreased ambulation, unstable gait, coarse tremor, growth retardation, and rapid progression to early death at age 14 to 16 days. Molecular and biochemical analyses of the mouse model demonstrated that the pathogenesis of the disease is due to loss of enzyme activity and ensuing dNTP pool imbalances with decreased dTTP levels in brain and both dTTP and dCTP levels in liver, which, in turn, produces mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits, most prominently in the brain and spinal cord (Dorado, et al. 2011).
(64) All experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of the Columbia University Medical Center, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were housed and bred according to international standard conditions, with a 12-hour light, 12-hour dark cycle, and sacrificed at 4, 13, and 29 days of age.
(65) Organs (brain, spinal cord, liver, heart, kidney, quadriceps muscle, lung, and gastrointestinal tract) were removed and either frozen in the liquid phase of isopentane, pre-cooled near its freezing point (−160° C.) with dry ice or fixed in 10% neutral buffered formalin and embedded in paraffin using standard procedures. Paraffin embedded tissue were then stained with hematoxylin and eosin (H&E) for morphological study or processed for immunostaining studies with GFAP, COX I, or complex I subunit as detailed below. All the experiments were performed in at least 3 mice per group. Both heterozygous and homozygous wild type mice were considered as control group (Tk2+) since no clinical and biochemical difference were previously described (Akman et al, 2008; Dorado et al, 2011).
(66) Treatment Administration and Experimental Plan
(67) Deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP) (Hongene Biotech, Inc.) were administered in 50 μl of Esbilac milk formula for small pets (Pet-Ag) by daily oral gavage to Tk2 H126N knock-in mice (Tk2) and aged-matched control wild-type (Tk2+) using 2 doses, 200 mg/kg/day and 400 mg/kg/day, from post-natal day 4 to 29 days. At age 29 days, mice were separated from the mother and the treatment was continued by administration of dCMP and dTMP in drinking water using equimolar doses, respectively of 1.6 mM and 3.2 mM. A negative control group of untreated Tk2 mutant and control wild-type mice were weighted and observed closely for comparison.
(68) Phenotype Assessment
(69) To define the degree of safety and efficacy of dTMP/dCMP, survival time, age-at-onset of disease, type and severity of symptoms, occurrence of side effects, and proportion of treatment termination due to adverse events, in treated and untreated Tk2 mice were compared. General behavior, survival time, and body weights of the mice were assessed daily beginning at postnatal day 4. Videotaping and open-field test with an Opto-Varimetrix-3 sensor system (Columbus Instruments) were performed at 13 and 29 days by counting horizontal and vertical movements, recording ambulatory and resting time, and measuring the total distance traveled in 10 minutes.
(70) Brain Histology
(71) Brain and spinal cord samples from 13- to 29-day-old mice were fixed with 10% neutral-buffered formalin and embedded in paraffin using standard procedures. Cerebellum, brainstem, hippocampus, cerebral cortex, and cervical, thoracic and lumbar tracts of the spinal cord were analyzed.
(72) Sections (5 μm thick) were stained with H&E and luxol fast blue to analyze the overall structure of the tissue. Immunostaining with antibodies against GFAP, complex I (NDUFB6), or COX subunit 2 was also performed. Briefly, paraffin-embedded brain and spinal cord slides were deparaffinized, rehydrated, and rinsed in phosphate-buffered saline solution (PBS). To block endogenous peroxidase activity, sections were incubated with 3% hydrogen peroxide in methanol. Slides were then placed in 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven for 15 minutes, for antigen retrieval. Slides were incubated with mouse anti-GFAP antibody (1:100) (Novocastra. NCL-GFAP-GA5) or mouse monoclonal antibody anti-complex I 17 kDa (NDUFB6) subunit (1:100) (A21359; Molecular Probes) or mouse monoclonal antibody anti-COX subunit 2 (1:100) (clone COX 229, A6404; Molecular Probes) overnight at 4° C. Sections were subsequently rinsed in PBS and incubated with anti-mouse M.O.M. Peroxidase kit, 1:200 dilution for 60 minutes at room temperature. Immunoreactivity was detected by avidin-biotin complex (ABC) with DAB substrate (Vector Laboratories, Burlingame, Calif., USA). Slides were examined by light microscopy using an Olympus BX51 microscope, and images were captured with a QImaging Retiga EXi digital camera, using QCapture software version 2.68.6.
(73) dNTP Pool by Polymerase Extension Assay
(74) Tissues were homogenized on ice in 10 volumes (w/v) of cold MTSE buffer (210 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl pH 7.5, 0.2 mM EGTA, 0.5% BSA) and centrifuged at 1,000 g for 5 minutes at 4° C., followed by three centrifugations at 13,000 g for 2 minutes at 4° C. Supernatant was precipitated with 60% methanol for the mitochondrial fraction and 100% methanol for the cytosolic fraction, kept 2 hours at −80° C., boiled 3 minutes, stored at −80° C. (from 1 hour to overnight), and centrifuged at 20,800 g for 10 minutes at 4° C. Supernatants were evaporated until dry, and pellet was resuspended in 65 μl of water and stored at −80° C. until analyzed.
(75) To minimize ribonucleotide interference, total dNTP pools were determined as previously reported (Ferraro, et al. 2010; Marti, et al. 2012). Briefly, 20 μl volume reactions was generated by mixing 5 μl of sample or standard with 15 μl of reaction buffer (0.025 U/ml ThermoSequenase DNA polymerase (GE Healthcare, Piscataway, N.J., USA) or Taq polymerase (Life Technologies, NY, USA), 0.75 μM .sup.3H-dTTP or .sup.3H-dATP (Moravek Biochemicals), 0.25 μM specific oligonucleotide, 40 mM Tris-HCl, pH 7.5, 10 mM MgCl.sub.2, 5 mM DTT). After 60 minutes at 48° C., 18 ml of reaction were spotted on Whatman DE81 filters, air dried, and washed three times for 10 minutes with 5% Na.sub.2HPO.sub.4, once in distilled water and once in absolute ethanol. The retained radioactivity was determined by scintillation counting.
(76) Nucleosides Measurement by HPLC
(77) Deoxythymidine (dT), deoxyuridine (dU), uracil (U), and thymine (T) levels were assessed by a gradient-elution HPLC method as described previously (Lopez, et al. 2009; Marti, et al. 2012), with minor modifications. Briefly, deproteinized samples were injected into an Alliance HPLC system (Waters Corporation) with an Alltima C18NUC reversed-phase column (Alltech) at a constant flow rate of 1.5 ml/min (except where indicated) using three buffers: eluent A (20 mM potassium phosphate, pH 5.6), eluent B (water), and eluent C (methanol). Samples were eluted over 60 minutes with a gradient as follows: 0-5 min, 100% eluent A; 5-25 min, 100-71% eluent A, 29% eluent B; 25-26 min, 0-100% eluent C; 26-30 min, 100% eluent C; 30-31 min, 0-100% eluent B; 31-35 min, 100% eluent B (1.5-2 ml/min); 35-45 min, 100% eluent B (2 ml/min); 45-46 min, 100% eluent B (2-1.5 ml/min); 46-47 min, 0-100% eluent C; 47-50 min, 100% eluent C; 50-51 min, 0-100% eluent A; and 51-60 min, 100% eluent A.
(78) Absorbance of the elutes was monitored at 267 nm, and dThd and dUrd peaks were quantified by comparing their peak areas with a calibration curve obtained with aqueous standards. For definitive identification of dT, dU, U, and, T peaks for each sample, a second aliquot treated with excess of purified E. coli TP (Sigma) was used to specifically eliminate dT and dU. The detection limit of this method is 0.05 mmol/1 for all nucleosides.
(79) RT-qPCR: Mitochondrial DNA Quantification
(80) Real-time PCR was performed with the primers and probes for murine COX I gene (mtDNA) and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH, nDNA) (Applied Biosystems, Invitrogen, Foster City, Calif., USA) as described using standard curve quantification, in an ABI PRISM 7,000 Sequence Detection System (Applied Biosystems) (Dorado, et al. 2011). MtDNA values were normalized to nDNA values and expressed as percent relative to wild type (100%).
(81) Mitochondrial Respiratory Chain Protein Levels
(82) Thirty micrograms of whole brain cerebrum or cerebellum extracts was electrophoresed in an SDS-12% PAGE gel, transferred to Immun-Blot™ PVDF membranes (Bio-Rad, Hercules, Calif., USA) and probed with MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail of antibodies (MitoSciences, Eugene, Oreg., USA). Protein-antibody interaction was detected with peroxidase-conjugated mouse anti-mouse IgG antibody (Sigma-Aldrich, St Louis, Mo., USA), using Amersham™ ECL Plus western blotting detection system (GE Healthcare Life Sciences, UK). Quantification of proteins was carried out using NIH ImageJ 1.37V software. Average gray value was calculated within selected areas as the sum of the gray values of all the pixels in the selection divided by the number of pixels.
(83) Mitochondrial Respiratory Chain Enzyme Activities by Spectrophotometer Analysis
(84) Mitochondrial RC enzymes analysis was performed in cerebrum and cerebellum tissues as previously described (DiMauro, et al. 1987).
(85) Nucleosides and Nucleotides Metabolic Enzymes
(86) Thymidine phosphorylase and thymidine kinase 1 and 2 activities were measured as previously described (Martf, et al. 2003; Lopez, et al. 2009; Dorado, et al. 2011).
(87) Statistical Methods
(88) Data are expressed as the mean±SD of at least three experiments per group. Gehan-Breslow-Wilcoxon test was used to compare the survival proportion of each group of mice. Unpaired t-test with Welch's correction and Mann-Whitney U-test were used to compare 13-day-old Tk2.sup.+ versus untreated Tk2.sup.−/−, 13-day-old untreated Tk2.sup.−/− versus Tk2.sup.−/−200dCMP/dTMP, 29-day-old wild-type versus Tk2.sup.−/−200dCMP/dTMP and Tk2.sup.−/−400dCMP/dTMP, for molecular and biochemical studies. Response to treatment was evaluated comparing Tk2.sup.−/− versus Tk2.sup.−/−200dCMP/dTMP at 13 days and Tk2.sup.−/−200dCMP/dTMP versus Tk2.sup.−/−400dCMP/dTMP. A P-value of <0.05 was considered to be statistically significant.
Example 2—dCMP/dTMP Delays Disease Onset, Prevents Neuromuscular Manifestations, and Prolongs Lifespan of Tk2-Deficient Mice
(89) Oral treatment with dCMP+dTMP 200 mg/kg/day each in milk (Tk2.sup.−/−200dCMP/dTMP) beginning at postnatal day 4 delayed disease onset to 20-25 days when the mutant mice developed a mild tremor and stopped gaining weight (data not shown). In the fourth week, they manifested weakness and reduced movements. In contrast, Tk2.sup.−/− mice treated from day 4 with dCMP+dTMP 400 mg/kg/day each in milk (Tk2.sup.−/−400dCMP/dTMP) appeared normal until day 21, when weight gain decelerated and mild head tremor developed (
(90) Untreated Tk2.sup.−/− mice had a mean lifespan of 13.2±2.5 days (mean±SD), whereas Tk2.sup.−/−200dCMP/dTMP survived to 34.6±3.2 days (P=0.0028; n=7; Gehan-Breslow-Wilcoxon test) while Tk2.sup.−/−400dCMP/dTMP lived to 44.3±9.1 days (P=0.0071; n=7; Gehan-Breslow-Wilcoxon test) (
(91) Open-field assessment of motor function in 29-day-old Tk2.sup.−/−200dCMP/dTMP Tk2.sup.−/−400dCMP/dTMP, and wild-type Tk2 mice showed no differences in the distance traveled, horizontal and vertical movements, or resting time (
(92) Relative to 29-day-old Tk2.sup.+ mice, age-matched Tk2.sup.−/−200dCMP/dTMP and Tk2.sup.−/−400dCMP/dTMP animals showed decreases in gross muscle mass and muscle fiber diameter that were independent of the treatment dose but paralleled to body weight (
Example 3—Histological and Histochemical CNS Studies Confirmed dCMP/dTMP Efficacy
(93) Efficacy of treatment in central nervous system (CNS) was demonstrated in histological studies that showed dramatic reductions in the numbers of vacuoles in neurons of the spinal cord and cerebellar and brain stem nuclei of 13-day-old Tk2.sup.−/−200dCMP/dTMP mice relative to untreated 13-day-old Tk2.sup.−/− mice (
Example 4—Treatment Crosses Biological Barriers
(94) To confirm that the treatment crosses biological barriers, dNTP levels in isolated mitochondria were assessed by polymerase extension assay described in Example 1. In 13-day-old untreated Tk2.sup.−/− mice relative to Tk2.sup.+ littermates, isolated brain mitochondria showed decreased levels of dTTP (0.67±0.1 pmol/mg-protein versus 2.52±1.0), while isolated liver mitochondria revealed reduced dCTP levels (1.07±0.8 versus 2.9±1.0) (Table 1). The treatment crossed the blood-brain barrier increasing the level of dTTP in isolated brain mitochondria of 13-day-old Tk2.sup.+200dCMP/dTMP (3.55±1) and Tk2.sup.−/−200dCMP/dTMP (1.5±0.7) and as a consequence, restored the proportion of dTTP relative to total dNTP in treated mutants. In contrast, levels of dCTP in isolated mitochondria were stable in brain of 13-day-old Tk2.sup.+200dCMP/dTMP (3.07±2), decreased in brain of Tk2.sup.−/−200dCMP/dTMP (1.13±0.5), and decreased in liver of 13-day-old Tk2.sup.+200dCMP/dTMP (0.13±0.4) and Tk2.sup.−/−200dCMP/dTMP (0.56±0.5) (Table 1).
(95) In 29-day-old Tk2.sup.−/−200dCMP/dTMP relative to Tk2.sup.+ mice, absolute levels of dTTP and dCTP were markedly reduced in isolated mitochondria from brain (dTTP 0.11±0.05 and dCTP 0.6±0.2) and from liver (dTTP 0.15±0.04 and dCTP 0.04±0.03) (Table 1); when these data were expressed as percentage of total dNTPs, there were striking decreases in dTTP/dNTP in brain (P=0.0322; n=7; Mann-Whitney U-test) and dCTP/dNTP in liver (P=0.0338; n=3; Mann-Whitney U-test) (
(96) TABLE-US-00001 TABLE 1 dNTP pools level in tissues Untreated Untreated Untreated Brain Tk2.sup.+ Tk2.sup.−/− Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP Tk2.sup.+ Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP Mitochondria (P13; n = 5) (P13; n = 4) (P13; n = 5) (P13; n = 5) (P29; n = 4) (P29; n = 5) (P29; n = 8) dATP 0.91 ± 0.2 1.51 ± 1 0.85 ± 0.5 1.03 ± 0.8 0.44 ± 0.2 0.86 ± 0.4 0.5 ± 0.3 dTTP 2.52 ± 1 0.67 ± 0.1* 3.55 ± 1 1.52 ± 0.7 1.87 ± 0.9 3.5 ± 1 0.11 ± 0.05** dGTP 1.06 ± 0.4 1.17 ± 0.7 1.9 ± 1 0.68 ± 0.3 1.3 ± 0.5 1.9 ± 1 0.87 ± 0.6 dCTP 1.99 ± 0.9 3.9 ± 3 3.07 ± 2 1.13 ± 0.5 0.32 ± 0.2 1.2 ± 0.6 0.6 ± 0.2 Untreated Untreated Untreated Liver Tk2.sup.+ Tk2.sup.−/− Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP Tk2.sup.+ Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP Mitochondria (P13; n = 3) (P13; n = 3) (P13; n = 3) (P13; n = 3) (P29; n = 3) (P29; n = 5) (P29; n = 4) dATP 1.47 ± 1 1.34 ± 0.8 1.42 ± 0.8 2.6 ± 1 0.32 ± 0.2 0.5 ± 0.3 0.31 ± 0.3 dTTP 0.61 ± 0.08 0.54 ± 0.2 0.67 ± 0.3 0.36 ± 0.3 0.46 ± 0.3 1.2 ± 0.8 0.15 ± 0.04* dGTP 2.2 ± 2 2.64 ± 1 0.93 ± 0.9 1.2 ± 1 0.58 ± 0.25 0.75 ± 0.4 0.8 ± 0.6 dCTP 2.9 ± 1 1.07 ± 0.8 1.13 ± 0.4 0.56 ± 0.5 0.36 ± 0.3 0.67 ± 0.2 0.04 ± 0.03* Data expressed in pmol normalized to mg-protein (mean ± SD). Statistical analyses were performed with untreated Tk2.sup.−/− vs untreated Tk2.sup.+ (P13) and Tk2.sup.−/−2004CMP/dTMP vs Tk2.sup.+200dCMP/dTMP (P13 and P29). * = p < 0.05; ** = p < 0.005. P = postnatal day
Example 5—dCMP/dTMP Treatment Ameliorates Biochemical and Molecular Genetic Abnormalities
(97) Treatment with dCMP and dTMP enhanced mtDNA levels in the mutant mice. At pre-treatment baseline, 4-day-old Tk2.sup.−/− mice did not manifest clinical abnormalities, but showed reductions of mtDNA copy numbers in brain cerebrum (38±13% mtDNA relative to wild-type brain, P=0.0002; n=5; Mann-Whitney U-test), cerebellum (54±1%, P=0.0228; n=4; Mann-Whitney U-test), muscle (28±12%), and kidney (62±11%) with normal mtDNA levels in heart and liver (
(98) At age 29 days, relative to Tk2.sup.+ mice, Tk2.sup.−/−200dCMP/dTMP mice showed mtDNA depletion that was severe in cerebellum (23±8%) and brain cerebrum (11±1%) and moderate in muscle (48±23%), liver (70±13%), and kidney (55±6%) (
(99) To assess the impact of treatment on mitochondrial RC enzymes, their activities and steady-state protein levels in brain cerebrum and cerebellum were measured. In 13-day-old untreated Tk2.sup.−/− mice, relative to untreated wild-type, brain cerebrum showed reduced COX activity (57±19%, P=0.0159; n=5; Mann-Whitney U-test) and significantly increased citrate synthase (CS) activity (148±17%; P=0.0317; n=5; Mann-Whitney U-test) (Table 2;
(100) The RC defects were more severe in cerebellum with significant reductions in all of the complexes when normalized either to CS (
(101) Western blot analysis of mitochondrial RC complex subunits in 13-day-old untreated Tk2.sup.−/− animals compared with untreated control mice revealed reductions in steady-state levels of complex I (55±39% brain cerebrum; 38±13% cerebellum, P=0.025; n=6; Mann-Whitney U-test) and complex IV (74±32% brain cerebrum; 44±16% cerebellum, P=0.0017; n=6; Mann-Whitney U-test), while RC protein levels were normal in 13-day-old Tk2.sup.−/−200dCMP/dTMP mice (
(102) TABLE-US-00002 TABLE 2 Mitochondrial respiratory chain enzyme activities in brain hemisphere Normalized Untreated Untreated to Tk2.sup.+ Tk2.sup.−/− Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP mg-proteins (P13; n = 5) (P13; n = 5) (P13; n = 4) (P13; n = 5) (P29; n = 4) (P29; n = 6) Complex I 0.05 ± 0.01 0.05 ± 0.03 0.04 ± 0.0.03 0.07 ± 0.03 0.24 ± 0.13 0.29 ± 0.06 Complex 0.79 ± 0.1 0.88 ± 0.1 0.60 ± 0.1 0.89 ± 0.4 0.62 ± 0.38 0.71 ± 0.16 I + III Complex 0.36 ± 0.04 0.38 ± 0.03 0.33 ± 0.03 0.30 ± 0.06 0.47 ± 0.05 0.44 ± 0.01 II + III Complex IV 0.3 ± 0.1* 0.17 ± 0.05* 0.17 ± 0.1 0.22 ± 0.13 1.98 ± 0.29 1.55 ± 0.17 Complex II 0.4 ± 0.1 0.51 ± 0.1 0.27 ± 0.1 0.43 ± 0.06 0.98 ± 0.18 0.79 ± 0.04 CS 5.3 ± 1** 7.5 ± 1** 6.05 ± 0.8 7.2 ± 1 7.2 ± 1.2 7.09 ± 0.9 Normalized Untreated Untreated to Tk2.sup.+ Tk2.sup.−/− Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP CS (P13; n = 5) (P13; n =5 ) (P13; n = 4) (P13; n = 5) (P29; n = 4) (P29; n = 6) Complex I 0.009 ± 0.003 0.007 ± 0.003 0.007 ± 0.004 0.01 ± 0.005 0.03 ± 0.022 0.042 ± 0.009 Complex 0.15 ± 0.05 0.11 ± 0.01 0.1 ± 0.03 0.12 ± 0.056 0.08 ± 0.04 0.1 ± 0.02 I + III Complex 0.07 ± 0.014 0.05 ± 0.006* 0.05 ± 0.01 0.04 ± 0.01 0.066 ± 0.011 0.062 ± 0.007 II + III Complex IV 0.057 ± 0.03 0.02 ± 0.008* 0.028 ± 0.01 0.03 ± 0.017 0.28 ± 0.06 0.22 ± 0.04 Complex II 0.07 ± 0.02 0.069 ± 0.01 0.045 ± 0.019 0.06 ± 0.017 0.14 ± 0.03 0.11 ± 0.01 Normalized Untreated Untreated to Tk2.sup.+ Tk2.sup.−/− Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP Tk2.sup.+200dCMP/dTMP Tk2.sup.−/−200dCMP/dTMP II (P13; n = 5) (P13; n = 5) (P13; n = 4) (P13; n = 5) (P29; n = 4) (P29; n = 6) Complex I 0.13 ± 0.05 0.11 ± 0.07 0.11 ± 0.04 0.18 ± 0.1 0.25 ± 0.12 0.37 ± 0.08 Complex 2.07 ± 0.6 1.75 ± 0.3 2.34 ± 0.5 2.06 ± 0.9 0.74 ± 0.7 0.89 ± 0.15 I + III Complex 0.97 ± 0.25 0.76 ± 0.15 1.37 ± 0.4 0.7 ± 0.05 0.50 ± 0.15 0.55 ± 0.02 II + III Complex IV 0.79 ± 0.3 0.36 ± 0.1 0.66 ± 0.3 0.55 ± 0.3 2.06 ± 0.4 1.96 ± 0.13 CS 14.2 ± 4.2 15.01 ± 3.2 19.4 ± 4.6 17.05 ± 4.1 7.78 ± 3 8.9 ± 1.2 Data expressed in micromole/min/mg tissue and normalized to mg-proteins (mean ± SD) or normalized to citrate synthase (CS) activity or to complex II activity (mean ± SD). Statistical analyses were performed with untreated Tk2.sup.−/− vs untreated Tk2.sup.+ (P13) and Tk2.sup.−/−200dCMP/dTMP vs TK2.sup.+200dCMP/dTMP (P13 and P29). * = p < 0.05; ** = p < 0.005). P = postnatal day
(103) TABLE-US-00003 TABLE 3 Cerebellar mitochondrial respiratory chain enzyme activities Normalized to CS COX I II + III II I + III Tk2.sup.+ 0.24 ± 0.07 0.08 ± 0.03 0.11 ± 0.02 0.11 ± 0.03 0.14 ± 0.03 Tk2.sup.−/− 0.13 ± 0.07* 0.015 ± 0.01** 0.06 ± 0.02* 0.06 ± 0.01* 0.067 ± 0.05* Tk2.sup.+13 days 200 0.24 ± 0.07 0.065 ± 0.04 0.11 ± 0.03 0.11 ± 0.003 0.11 ± 0.06 Tk2.sup.−/−days 200 0.19 ± 0.01 0.036 ± 0.01 0.10 ± 0.02 0.087 ± 0.06 0.11 ± 0.01 Tk2.sup.+29 days 0.20 ± 0.02 0.069 ± 0.007 0.072 ± 0.004 0.073 ± 0.008 0.10 ± 0.01 Tk2.sup.+29 days 200 0.17 ± 0.03 0.049 ± 0.004 0.071 ± 0.02 0.065 ± 0.01 0.11 ± 0.03 Tk2.sup.−/− .sup.29 days 200 0.11 ± 0.03* 0.034 ± 0.01 0.057 ± 0.01 0.080 ± 0.02 0.05 ± 0.005* Tk2.sup.+29 days 0.027 ± 0.002 0.002 ± 0.002 0.039 ± 0.009 0.07 ± 0.01 0.028 ± 0.002 Tk2.sup.+29 days 400 0.036 ± 0.022 0.005 ± 0.001 0.017 ± 0.012 0.035 ± 0.02 0.027 ± 0.019 Tk2.sup.−/−29 days 400 0.04 ± 0.024 0.004 ± 0.002 0.02 ± 0.006 0.05 ± 0.01 0.028 ± 0.019 Normalized to II COX I II + III CS I + III Tk2.sup.+ 2.1 ± 0.6 0.74 ± 0.3 1.01 ± 0.1 9.1 ± 2.5 1.31 ± 0.2 Tk2.sup.−/− 2.1 ± 0.7 0.29 ± 0.2 1.06 ± 0.1 18.5 ± 7.4 0.97 ± 0.6 Tk2.sup.+13 days 200 2.09 ± 0.2 0.43 ± 0.3 0.98 ± 0.1 9.1 ± 2.5 0.92 ± 0.3 Tk2.sup.−/−13 days 200 1.7 ± 0.4 0.29 ± 0.1 0.84 ± 0.0.5 8.8 ± 1.8 0.94 ± 0.1 Tk2.sup.+29 days 2.8 ± 0.02 0.95 ± 0.003 0.99 ± 0.1 13.7 ± 1.5 1.48 ± 0.08 Tk2.sup.+29 days 2.62 ± 0.38 0.65 ± 0.4 0.97 ± 0.1 15.9 ± 3.6 1.76 ± 0.2 Tk2.sup.−/− .sup.29 days 200 1.43 ± 0.4 0.43 ± 0.2 0.72 ± 0.1 13.3 ± 3.5 0.68 ± 0.2 Tk2.sup.+29 days 0.38 ± 0.05 0.031 ± 0.04 0.53 ± 0.04 14 ± 2.2 0.4 ± 0.03 Tk2.sup.+29 days 400 0.43 ± 0.3 0.14 ± 0.02 0.35 ± 0.03 16 ± 4.3 0.56 ± 0.3 Tk2.sup.−/−29 days 400 0.47 ± 0.3 0.05 ± 0.06 0.41 ± 0.01 19.4 ± 3.4 f 0.5 ± 0.2 Data expressed in micromole/min/mg tissue and normalized to mg-proteins or normalized to citrate synthase (CS) activity or to complex II activity (mean ± SD). Statistical analyses were performed with untreated Tk2.sup.−/− vs untreated Tk2.sup.+ and Tk2.sup.−/−200dCMP/dTMP vs Tk2.sup.+200dCMP/dTMP. * = p < 0.05; **= p < 0.005) P = postnatal day
Example 6—Deoxynucleotide Metabolism
(104) To understand the metabolism of dCMP/dTMP after oral gavage administration, levels of dCMP/dTMP and their metabolites in muscle and liver tissues and in plasma were measured after 30 minutes of oral gavage. In Tk2.sup.−/−200dCMP/dTMP mice, deoxynucleoside monophosphates were not detectable. Levels of deoxyuridine and deoxythymidine were markedly increased at age 13 days, but subsequently lower in 29-day-old mice (
(105) To understand the cause for differences in deoxyuridine and deoxythymidine plasma levels between 13 and 29 days of age, the activity level of TP in small intestine, brain, and liver was measured. TP activity was higher in the small intestine at P29 (
(106) Tk2 activity was confirmed to be normal in treated and untreated Tk2.sup.+ mice and reduced in Tk2.sup.− mice in muscle and brain. Unexpectedly, Tk1 activity was increased in brain and muscle of 13- and 29-day-old treated mice (
(107) TABLE-US-00004 TABLE 4 Deoxypyrimidine monophosphates metabolism Plasma Levels of nucleoside metabolites Mice Age Treatment Dose Time Uracil (μM) Thymine (μM) dUrd (μM) Thd (μM) Tk2.sup.+ (N = 1) 13 dCMP + dTMP 80 nmol/kg/day 0 19.1 UND 5.5 1.0 Tk2.sup.+ (N = 3) 13 dCMP + dTMP 80 nmol/kg/day 30 59.3 ± 33.9 3.6 ± 3.3 29.0 ± 18.5 71.6 ± 53.3 Tk2.sup.−/− (N = 1) 13 dCMP + dTMP 80 nmol/kg/day 0 21.2 5.0 4.2 6.8 Tk2.sup.−/− (N = 3) 13 dCMP + dTMP 80 nmol/kg/day 30 78.0 ± 56.7 5.4 ± 3.5 28.3 ± 9.1 70.4 ± 19.2 Tk2.sup.+ (N = 3) 29 dCMP + dTMP 80 nmol/kg/day 0 79.7 ± 20.5 0.1 ± 0.1 2.2 ± 0.1 5.0 ± 5.4 Tk2.sup.+ (N = 2) 29 dCMP + dTMP 80 nmol/kg/day 30 109.3 ± 1.3 68.7 ± 53.3 29.3 ± 13.4 73.4 ± 50.2 Tk2.sup.−/− (N = 3) 29 dCMP + dTMP 80 nmol/kg/day 30 61.4 ± 10.9 11.2 ± 3.4 18 ± 1.9 30.8 ± 13.2 Thymidine Phosphorylase activity Mice Age Treatment Dose TISSUE Activity (nmol/h/mg-proteins) Tk2.sup.+ (N = 3) 13 dCMP + dTMP 80 nmol/kg/day BRAIN 6.2 ± 3.3 Tk2.sup.−/− (N = 4) 13 dCMP + dTMP 80 nmol/kg/day BRAIN 11.6 ± 0.8 Tk2.sup.+ (N = 2) 29 dCMP + dTMP 80 nmol/kg/day BRAIN 5.9 ± 0.4 Tk2.sup.−/− (N = 2) 29 dCMP + dTMP 80 nmol/kg/day BRAIN 8.0 ± 0.8 Tk2.sup.+ (N = 4) 13 Untreated — LIVER 4.9 ± 2.4 Tk2.sup.−/− (N = 2) 13 Untreated — LIVER 6.1 ± 2.6 Tk2.sup.+ (N = 3) 13 dCMP + dTMP 80 nmol/kg/day LIVER 9.0 ± 2.9 Tk2.sup.−/− (N = 3) 13 dCMP + dTMP 80 nmol/kg/day LIVER 6.0 ± 3.1 Tk2.sup.+ (N = 6) 29 dCMP + dTMP 80 nmol/kg/day LIVER 4.9 ± 2.45 Tk2.sup.−/− (N = 4) 29 dCMP + dTMP 80 nmol/kg/day LIVER 4.6 ± 2.5 Tk2.sup.+ (N = 6) 3 Untreated — S.I. 26.1 ± 11.0 Tk2.sup.−/− (N = 3) 3 Untreated — S.I. 28 ± 14 Tk2.sup.+ (N = 2) 13 dCMP + dTMP 80 nmol/kg/day S.I. 29.6 ± 3.9 Tk2.sup.−/− (N = 3) 13 dCMP + dTMP 80 nmol/kg/day S.I. 30.9 ± 10.6 Tk2.sup.+ (N = 9) 29 dCMP + dTMP 80 nmol/kg/day S.I. 278.8 ± 75 Tk2.sup.−/− (N = 4) 29 dCMP + dTMP 80 nmol/kg/day S.I. 244.1 ± 80 S.I. = small intestine; dUrd = deoxyuridine; Thd = thymidine
Example 7—Materials and Protocols Used for Examples 8-10
(108) The compounds used in Examples 8-10 were dTMP.Na.sub.2 and dCMP.Na.sub.2 were obtained through Hongene Biotechnology USA, Inc. (Morrisville. N.C.). The compounds were verified as greater than 99% pure by HLPC and by mass spectrometry. Each bottle of dTMP.Na.sub.2, and dCMP.Na.sub.2 was labeled with the following but not limited to: the product name, product lot number, protocol number, recommended storage conditions, expiration date, “Caution: New Drug-Limited by Federal Law to Investigational Use.” Hongene Biotech' company name and address, and the Investigator's name or investigation site. Labeling complied with the requirements of 21 CFR 312.6.
(109) The Investigator ensured that all study drugs were stored and dispensed in accordance with the Food and Drug Administration (FDA) regulations concerning the storage and administration of investigational drugs.
Example 8—dTMP and dCMP Treatment on Patients—Case 1
(110) The first patient was a 29 year-old Spanish man (JJM) with TK2 deficiency that initially manifested at age 3 years with leg weakness causing difficulty running and frequent falls (Vilà, et al. 2003). The weakness progressed at age 12, began using BiPAP ventilatory support and one year later, he became wheelchair-bound. Because of arm weakness, he stopped playing the violin and due to respiratory muscle weakness, he has suffered recurrent bouts of pneumonia and uses BiPAP 23 hours daily. Despite the severe myopathy, this young man completed medical school.
(111) In August, 2011, he began dTMP and dCMP (each 100 mg/kg/day supplied by Hongene Biotechnology as described in Example 7), and the dose was increased to 200 mg/kg/day. The compounds were mixed in water and administered orally as a single daily dose. After initiating this therapy, the patient increased weight from a baseline of 35 kg to 44 kg, muscle strength improved slightly at the biceps, triceps, and quadriceps and the reduced time on BiPAP from 24 to 22 hours daily. During this period, the developed hearing loss, which was likely due to disease progression as this clinical manifestation has been reported in another patient with TK2 deficiency (Marti et al., 2010). No other potential side-effects were noted by this patient.
Example 9—dTMP and dCMP Treatment on Patients—Case 2
(112) The second patient was a 10 year-old Italian boy (LS DOB Jun. 3, 2004) who presented at age 2 years with hypotonia, severe limb weakness, and delayed motor development. He was relatively stable and was able to sit and walk unassisted until September, 2011, when he became very weak and lost the ability to walk. He was barely able to stand without support. In March, 2012, he started dTMP+dCMP therapy (dTMP and dCMP (each 100 mg/kg/day supplied by Hongene Biotechnology as described in Example 7). The compounds were mixed in water and administered orally as a single daily dose, which was prescribed by an Italian physician. In August, 2012, significant improvement of strength was noted. He was able to walk 10 steps independently. In addition to the improved limb strength, his voice and head control improved. He grew 5 centimeters in height and gained 3.5 kilograms in weight. In November, 2012, the doses were increased to 200 mg/kg/day. In late 2014, his 6-minute walk test showed a 3-fold improvement over pre-treatment baseline. As of March, 2016, he is able to walk unassisted, climb stairs using arm rails for support, ride a bicycle, and swim.
Example 10—dTMP and dCMP Treatment on Patients—Case 3
(113) A third patient was a 3 year-old child (AE, DOB Feb. 11, 2011) with infantile-onset myopathy. He had documented mtDNA depletion (11% of normal in skeletal muscle), and compound heterozygous mutations in the TK2 gene: c.144_145DelGA mutation which causes a frameshift, and c.323C>T missense mutation (p.T108M) (identified in the Medical Genetics Laboratory at the Baylor College of Medicine). The child developed normally until age 11 months, when he was able to pull to a standing position and walk a few steps using his arms to hold onto furniture. He lost the ability to walk and has become unable to sit, stand, roll over in bed, or lift his limbs or head off of a bed. Baseline serum creatine kinase levels were 1290-2098 U/L and venous lactate was elevated at 3.9 mmol/L (normal 0.5-2.2). The diagnosis of mitochondrial disease was made at Johns Hopkins Medical Center in May, 2012, when a muscle biopsy revealed severe cytochrome c oxidase (complex IV) deficiency. He started a cocktail of coenzyme Q10, L-carnitine, and creatine, which did not lead to significant improvements. The diagnosis of TK2 deficiency was achieved by genetic test results in July, 2012. After developing an upper respiratory viral infection on September 2012, he was put on non-invasive ventilation and subsequently received a tracheostomy and placed on mechanical ventilation.
(114) An emergency IND was requested on Oct. 24, 2012 to initiate dCMP+dTMP therapy in AE. At that time, he weighed 10.4 kg and was unable to grip objects or lift his legs from the bed. An eIND was obtained and he started the therapy in November, 2012 at 100 mg/kg/day. One month later, the doses were increased to 200 mg/kg/day. He showed improvement in his, weight, arm strength and leg movements. At age 4 years 9 months, he weighed 19.5 kg and was able to grip and hold small objects, lift his arm using triceps muscle, and lift his legs from the bed.
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