Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
  • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
  • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• NUCLEOTIDE POOLS INCLUDING MITOCHONDRIAL DNA DEPLETION
• the invention relates generally to a pharmacological therapy for a human genetic disease, specifically diseases characterized by unbalanced nucleotide pools, e.g., mitochondrial DNA depletion syndromes, and more specifically, thymidine kinase 2 (TK2) deficiency.
• the pharmacological therapy involves the administration of at least one deoxynucleoside, or mixtures thereof.
• the pharmacological therapy involves the administration of either deoxythymidine (dT) or deoxycytidine (dC), or mixtures thereof.
• This administration of one or more deoxynucleosides is applicable to other disorders of unbalanced nucleoside pools, especially those found in mitochondrial DNA depletion syndrome.
• Mitochondrial diseases are clinically heterogeneous diseases due to defects of the mitochondrial respiratory chain (RC) and oxidative phosphorylation, the biochemical pathways that convert energy in electrons into adenosine triphosphate (ATP).
• the respiratory chain is comprised of four multi-subunit enzymes (complexes I-IV) that transfer electrons to generate a proton gradient across the inner membrane of mitochondria and the flow of protons through complex V drives ATP synthesis (DiMauro and Schon 2003; DiMauro and Hirano 2005).
• Coenzyme Qio (CoQio) is an essential molecule that shuttles electrons from complexes I and II to complex III.
• the respiratory chain is unique in eukaryotic, e.g., mammalian, cells by virtue of being controlled by two genomes, mitochondrial DNA (mtDNA) and nuclear DNA (nDNA).
• mtDNA mitochondrial DNA
• nDNA nuclear DNA
• ROS reactive oxygen species
• TK2 which encodes thymidine kinase (TK2), a mitochondrial enzyme required for the phosphorylation of the pyrirmdine nucleosides (thymidine and deoxycytidine) to generate deoxythyrmdine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) (Saada, et al. 2001). Mutations in TK2 impair the mitochondrial nucleoside/nucleotide salvage pathways required for synthesis of deoxynucleotide triphosphate (dNTP), the building blocks for mDNA replication and repair.
• dNTP deoxynucleotide triphosphate
• TK2 deficiency manifests a wide clinical and molecular genetic spectrum with the majority of patients manifesting in early childhood with a devastating clinical course, while others have slowly progressive weakness over decades.
• the MDS includes disorders of a myopathic form characterized by mutations in TK2, an encephalomyopathic form characterized by mutations in SUCLA2, a neurogastrointestinal encephalopathy form characterized by mutations in TYMP, and a hepatopathic form characterized by mutations in DGUOK, POLG, and MPV17.
• the disorder is a thymidine kinase 2 deficiency, characterized by mutation(s) in the TK2 gene.
• the deoxynucleoside is either deoxythymidine (dT) or deoxycytidine (dC) or mixtures thereof.
• Deoxy adenosine (dA) and deoxyguanosine (dG), alone or together, can also be used in the method of the invention.
• One deoxynucleoside (i.e., dT, dC, dA, or dG) and mixtures of two or more of any of the four deoxynucleosides can be used in the method of the invention.
• Preferred dosages of the deoxynucleoside(s) are between about 100 and about 1,000 mg/kg/day, more preferably between about 300 and about 800 mg/kg/day, and most preferably between about 250 and about 600 mg/kg/day. If the composition comprises a single deoxynucleoside, then the dosages are of the single deoxynucleoside. If the composition comprises more than one deoxynucleoside, the dosages can be of each deoxynucleoside or of the total deoxynucleosides in the composition.
• Administration of the deoxynucleoside(s) can be once daily, twice daily, three times daily, four times daily, five times daily, up to six times daily, preferably at regular intervals.
• Figure 1 depicts a growth curve of wild type (Tk2 +/+ and Tk2 +/" ), and Tk2 _/" mice treated with 260 mg/kg/day or 520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT) from postnatal day 4. Each symbol represents the mean of weight at each time-point. N of each group is indicated in figure.
• Figure 3 are graphs of the relative proportions of dNTPs in isolated mitochondria from brain and liver tissue of wild type (Tk2 +/+ ), and Tk2 _/" , untreated or treated with 200 mg/kg/day dCMP and dAMP, or 260 mg/kg/day or 520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT) at ages postnatal day 13 (top panels) and postnatal day 29 (bottom panels).
• dC deoxycytidine
• dT deoxythymidine
• Figure 4 are graphs showing the ratio of mtDNA/nDNA in brain, liver, intestine, and muscle in wild type Tk2 mice (Tk2 +/+ ) (left hand bar) as compared to Tk2 _/" mice, untreated or treated with 260 mg/kg/day or 520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT), at ages postnatal days 13 and 29. Data are represented as mean ⁇ standard deviation (SD) of the percent of mtDNA copies relative to Tk2 + . p-values were assessed by Mann- Whitney tests. (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001).
• Figure 5 are graphs depicting the results of HPLC measuring dT and uracil in plasma of untreated wild type (Tk2 +/+ ) mice, wild type (Tk2 +/+ ) mice treated with 260 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT), Tk2 _/" mice treated with 260 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT), and Tk2 _/" mice treated with 200 mg/kg/day of dCMP and dTMP, 30 minutes after treatment. Data are expressed as mean ⁇ SD.
• Figure 6 are graphs of levels of respiratory chain enzyme activities in Tk2 _/" mice treated with 400 mg/kg/day of dCMP and dTMP and THU at 13 days postnatal, 260 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT) at 13 and 29 days postnatal, or 520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT) 29 days postnatal.
• Data are represented as the percent of the RCE activities in Tk2 _/" mouse tissues normalized to protein levels and relative to Tk2 + for each treatment, p-values determined by Mann-Whitney tests. *p ⁇ 0.05.
• Figure 7A is an immunoblot of respiratory chain proteins in wild type mice treated with 260 mg/kg/day or 520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT), and Tk2 ⁇ / ⁇ mice treated with 260 mg/kg/day or 520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT) at 29 days postnatal.
• Figure 7B are graphs showing the RCE levels normalized to complex II, represented as percent of the RCE levels in TK2 +/+ mice, p-values were assessed by Mann-Whitney tests.
• CS citrate synthase
• CI NADH-dehydrogenase
• CII succinate dehydrogenase
• CIII cytochrome c reductase
• CIV cytochrome c oxidase (COX)
• CI+III NADH-cytochrome c reductase
• CII + 111 succinate dehydrogenase-cytochrome c reductase.
• the current invention is based upon the surprising discovery that mitochondrial DNA depletion syndromes, including TK2 deficiency, can be treated with deoxynucleosides. As shown by the results herein, the administration of deoxynucleosides greatly improved the condition in both a mouse model of TK2 deficiency and human patients with TK2 deficiency. Definitions
• 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.
• the term "subject" as used in this application means mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates.
• 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
• patient as used in this application means a human subject.
• the "patient” is known or suspected of having a disease or disorder characterized by unbalanced nucleotide pools, mitochondrial disease, mitochondrial DNA depletion syndrome, or TK2 deficiency.
• terapéuticaally 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.
• treat refers 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.
• prevent refers 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.
• the term "in need thereof would be a subject known or suspected of having or being at risk of having a disease or disorder characterized by unbalanced nucleotide pools, mitochondrial disease, mitochondrial DNA depletion syndrome, or TK2 deficiency.
• agent means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.
• deoxynucleoside as used herein means deoxythymidine or dT, deoxycytidine or dC, deoxyadenosine or dA, and deoxyguanosine or dG.
• deoxynucleosides also include physiologically functional derivatives of the deoxynucleosides.
• physiologically functional derivative refers to a compound (e.g, a drug precursor) that is transformed in vivo to yield a deoxynucleoside.
• the transformation may occur by various mechanisms (e.g., by metabolic or chemical processes), such as, for example, through hydrolysis in blood.
• Prodrugs are such derivatives, and a discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
• an adverse effect is an unwanted reaction caused by the administration of a drug. In most cases, the administration of the deoxynucleosides caused no adverse effects. The most expected adverse effect would be a minor gastrointestinal intolerance.
• 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.
• “about” can mean within 1 or more than 1 standard deviations, per the practice in the art.
• “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.
• the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
• the term "about” meaning within an acceptable error range for the particular value should be assumed.
• Mitochondrial DNA (mtDNA) depletion syndrome comprises several severe autosomal diseases characterized by a reduction in mtDNA copy number in affected tissues. Most of the MDS causative nuclear genes encode proteins that belong to the mtDNA replication machinery or are involved in deoxyribonucleoside triphosphate (dNTP) metabolism.
• dNTP deoxyribonucleoside triphosphate
• TK2 thymidine kinase deficiency
• TK2 encoded by the nuclear gene, TK2 is a mitochondrial matrix protein that phosphorylates thymidine and deoxycytidine nucleosides to generate deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP), which in turn, are converted to deoxynucleotide triphosphates (dNTPs) required for mitochondrial DNA synthesis.
• dTMP deoxythymidine monophosphate
• dCMP deoxycytidine monophosphate
• dNTPs deoxynucleotide triphosphates
• TK2 mutations cause devastating neuromuscular weakness with severe depletion of mitochondrial DNA (mtDNA) in infants and children, as well as progressive external ophthalmoplegia with mtDNA multiple deletions in adults.
• mtDNA mitochondrial DNA
• Many patients cannot walk and require some type of mechanical ventilation and feeding tube.
• the central nervous system is variably involved in these disorders, with symptoms that include seizures, encephalopathy, cognitive impairment, and hearing loss. Less than 7% of patients live more than 42 years.
• the experiments set forth herein using the mouse model of Tk2 deficiency show the administration of deoxynucleosides to be effective and safe for the treatment of the disease. Additionally, as shown in Example 5, the administration of dT and dC greatly improved the symptoms of TK2 deficiency in patients.
• the present invention includes the administration of at least one deoxynucleoside to a patient in need thereof.
• the present invention includes the administration of at least one deoxpyrimidine.
• the deoxypyrimidine is chosen from dC, dT and mixtures thereof.
• the present invention includes the administration of at least one deoxypurine.
• the deoxypurine is chosen from dA, dG, and mixtures thereof.
• deoxynucleosides Patients who would benefit from the administration of deoxynucleosides would be those diagnosed with TK2 deficiency. In these patients, at least one deoxypyrimidine, dC or dT, or mixtures thereof would be administered.
• dGK deoxyguanosine kinase
• MDS as well as other disorders related to unbalanced nucleotide pools can be treated by the administration of specific deoxynucleosides, i.e., dA, dG, dC, or dT, or mixtures thereof.
• deoxynucleosides i.e., dA, dG, dC, or dT, 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).
• Additional nuclear genes that disrupt mitochondrial dNTP pools include but are not limited to SUCLA2, SUCLG1 and MPV17. Disorders related to these genes can also be treated by the administration of one or more deoxynucleosides
• TK2 deficiency 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.
• 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 deoxynucleoside 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.
• 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.
• CK serum creatine kinase
• electromyography histopathology on skeletal muscle
• mtDNA mitochondrial DNA
• ETC electron transport chain
• TK2 deficiency can be determined and/or confirmed (Chanprasert, et al. 2012).
• TK2 deficiency is inherited in an autosomal recessive manner.
• a sibling of an affected patient can be tested as early as possible after birth to diagnose the disease.
• deoxynucleoside therapy should be started as soon as possible after a diagnosis of TK2 deficiency.
• the present invention encompasses the administration of deoxynucleosides, more specifically one or more deoxynucleosides.
• Most preferred methods of administration are oral, intrathecal and parental including intravenous.
• the deoxynucleosides must be in the appropriate form for administration of choice.
• Deoxynucleosides are easily dissolved in liquid are easily dissolved in liquid (such as water, formula or milk) whereas the free acid form does not readily dissolve in liquid.
• compositions comprising one of more deoxynucleosides for administration may comprise a therapeutically effective amount of the deoxynucleosides and a pharmaceutically acceptable carrier.
• 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.
• 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.
• Oral administration is a preferred method of administration.
• the deoxynucleosides 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.
• 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
• intrathecal administration is a further preferred form of administration (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.
• compositions adapted for parenteral 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.
• the dNs can be administered through a gastronomy feeding tube or other enteral nutrition means.
• Further methods of administration include mucosal, such as nasal, sublingual, vaginal, buccal, or rectal; or transdermal administration to a subject.
• 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.
• 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.
• 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.
• 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.
• the deoxynucleoside therapy comprises the administration of one or more deoxy nucleosides chosen from the group consisting of deoxy thymidine (dT), deoxy cytidine (dC), deoxyadenosine (dA) and deoxyguanosine (dG).
• deoxy nucleosides chosen from the group consisting of deoxy thymidine (dT), deoxy cytidine (dC), deoxyadenosine (dA) and deoxyguanosine (dG).
• deoxynucleosides are beneficial based upon the deficiency. It is also within the skill of the art for the practitioner to determine if mixtures of the deoxynucleosides should be administered and in what ratio. If two deoxynucleosides are to be administered, they can be in a ratio of 50/50 of each deoxynucleoside, e.g., dC and dT, or in ratios of about 5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/10, and 95/5.
• dC and dT e.g., dC and dT
• dT and dC are administered in mixture of equal amounts for TK2 deficiency.
• 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 deoxynucleoside, 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.
• 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 indi vidual deoxynucleosides or of a composition with a mixture of more than one deoxynucleosides, e.g., dT and dC.
• a dose can comprise 400 mg/kg/day of dT alone.
• a dose can comprise a mixture of 200 mg/kg/day of dT and 200 mg/kg/day of dC.
• a dose can comprise 400 mg/kg/day of a mixture of dT and dC.
• Administration of the deoxynucleosides 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 deoxynucleosides are administered four times daily, doses would be at 8:00 AM, 12:00 PM, 4:00 PM, and 8:00 PM.
• Doses can also be lowered if being administered intravenously or intrathecally.
• Preferred dose ranges for such administration are from about 50 mg/kg/day to about 500 mg/kg/day.
• doses can be adjusted to optimize the effects in the subject.
• the deoxynucleosides 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 and tolerability.
• a subject can be monitored for improvement of their condition prior to increasing the dosage.
• a subject's response to the therapeutic administration of the deoxynucleosides 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 deoxynucleosides can be increased.
• the deoxynucleosides are well tolerated. Any observed adverse effects were minor and were mostly diarrhea, abdominal bloating and other gastrointestinal manifestations. A subject can also be monitored for any adverse effects, such as gastrointestinal intolerance, e.g., diarrhea. If one or more adverse effects are observed after administration, then the dosage can be decreased. If no such adverse effects are observed, then the dosage can be increased. Additionally, once a dosage is decreased due to the observation of an adverse effect, and the adverse effect is no longer observed, the dosage can be increased.
• the deoxynucleosides can also be co-administered with other agents.
• agents would include therapeutic agents for treating the symptoms of the particular form of MDS.
• the dT and dC can be co- administered with an inhibitor of ubiquitous nucleoside catabolic enzymes, including but not limited to enzyme inhibitors such as tetrahydrouridine (inhibitor of cytidine deaminase) and immucillin H (inhibitor of purine nucleoside phosphorylase) and tipiracil (inhibitor of thymidine phosphorylase).
• enzyme inhibitors such as tetrahydrouridine (inhibitor of cytidine deaminase) and immucillin H (inhibitor of purine nucleoside phosphorylase) and tipiracil (inhibitor of thymidine phosphorylase).
• tetrahydrouridine inhibitor of cytidine deaminase
• Tk2 H126N knock-in mutant (712 ⁇ A ) 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 ⁇ / ⁇ 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.
• 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.
• 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 described in the supplemental procedures. 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).
• H&E hematoxylin and eosin
• Deoxycytidine (dC) and deoxythymidine (dT) were administered in 50 ⁇ of Esbilac milk formula for small pets (Pet-Ag) by daily oral gavage to 712 H126N knockin mice (Tk2 "A ) and aged matched control wild-type (7k2 + ) using 2 doses, 260 mg/kg/day and 520 mg/kg/day, from post-natal day 4 to 29 days. At age 21 days, mice were separated from the mother and the treatment was continued by administration of dC and dT in drinking water using equimolar doses respectively of 1.6mM and 3.2mM. A negative control group of untreated Tk2 mutant and control wild-type mice were weighed and observed closely for comparison.
• Body weight was assessed daily, since it has been previously observed that incapacity of gaining weight is the first sign of disease (Akman, et al. 2008).
• 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 lOOOg for 5 minutes at 4°C, followed by three centrifugations at 13,000g for 2 minutes at 4°C. Supernatant was precipitated with 60% methanol, kept 2 hours at -80°C, boiled 3 minutes, stored at -80°C (from 1 hour to overnight) and centrifuged at 20,800g for 10 minutes at 4°C.
• cold MTSE buffer 210 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl pH 7.5, 0.2 mM EGTA, 0.5% BSA
• reaction buffer [0.025 U/ml ThermoSequenase DNA polymerase (GE Healthcare, Piscataway, NJ, USA) or Taq polymerase (Life Technologies, NY, USA), 0.75 ⁇ 3H-dTTP or 3H-dATP (Moravek Biochemicals), 0.25 ⁇ specific oligonucleotide, 40 mM Tris-HCl, pH 7.5, 10 mM MgC12, 5mM DTT].
• Deoxy thymidine (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. 2012b), 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 four buffers: eluent A (20 mM potassium phosphate, pH 5.6), eluent B (water) and eluent C (methanol).
• eluent A (20 mM potassium phosphate, pH 5.6
• eluent B water
• 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.
• 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, CA, USA) as described as described using ddCt method in a Step One Plus Real Time PCR System (Applied Biosystems) (Dorado, et al. 2011).
• MtDNA values were normalized to nDNA values and expressed as percent relative to wild- type (100%).
• Antibody Cocktail of antibodies (MitoSciences, Eugene, OR, USA). Protein-antibody interaction was detected with peroxidase-conjugated mouse anti-mouse IgG antibody (Sigma-1)
• Mitochondrial RC enzymes analysis was performed in cerebrum tissue as previously described (DiMauro, et al. 1987).
• Example 2- The Administration of dC/dT to Tk2 ⁇ / ⁇ Mice Delayed the Clinical Onset of TK2 Deficiency and Increased Survival
• a dose of 260 and 520 mg/kg/day each of deoxynucleosides were administered to the Tk2 ⁇ / ⁇ mice. These doses of deoxynucleosides were the molar equivalent of 400 and 800 mg/kg/day of dCMP+dTMP respectively.
• Mice treated with oral dC+dT (260 or 520mg/kg/day from age 4 days) appeared normal until postnatal day 21 ( Figure 1). After age 21 days, mutant mice treated with 260 mg/kg/day dose (Tk2 "/_ 260 "w* ⁇ dC/dT ) stopped gaining weight and developed mild head tremor and weakness that led to death at postnatal day 31+4.3 ( Figure 2).
• mice treated with the 520 mg/kg/day dC+dT continued to gain weight for one additional week, but subsequently manifested deterioration similar to ⁇ "7"260 mg/Kg/day dC/dT , and died at postnatal day 43+10.
• Tk2 _/ mice treated with 200 or 400mg/kg/day of oral dCMP/dTMP treatment.
• Tk2 ' 260 mg/Kg/day dC/dT and Tk2 ' 520 mg/Kg/day dC/dT mice at postnatal day 13 the treatment prevented mtDNA depletion in heart, liver, kidney, intestine and muscle (Figure 4).
• mtDNA copy number was only partially ameliorated in brain at postnatal day 13 in a dose-dependent manner with mtDNA/nDNA ratios relative to control brain reaching 39% with 260 mg/kg/day of dC+dT and 52% with 520 mg/kg/day.
• Respiratory chain enzyme (RCE) activities and protein levels were completely rescued in brain of TK2 _/" 260 m s /K s /da y dC/dT at postnatal day 13 ( Figure 6). RCE activities were also restored at postnatal day 29, and only a slight decrease of complex I activity could be observed in TK2 _/ ⁇ 52 0m g /K g /da y dC/dT ( Figure 6).
• Thymidine kinase 2 (H126N) knock in mice show the essential role of balanced deoxy ucleotide pools for mitochondrial DNA maintenance.
• MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nature Genet. 38:570-575
• TK2 thymidine kinase 2 gene

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Molecular Biology (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Neurology (AREA)
• Physical Education & Sports Medicine (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Biotechnology (AREA)
• Hematology (AREA)
• Immunology (AREA)
• Physiology (AREA)
• Nutrition Science (AREA)
• Biomedical Technology (AREA)
• Genetics & Genomics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Urology & Nephrology (AREA)
• Analytical Chemistry (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biochemistry (AREA)
• Obesity (AREA)
• Child & Adolescent Psychology (AREA)
• Diabetes (AREA)
• General Engineering & Computer Science (AREA)
• Cell Biology (AREA)

Priority Applications (37)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (2)

Family

ID=57546446

Family Applications (1)

Country Status (24)

Cited By (17)

Families Citing this family (6)

Citations (1)

Family Cites Families (6)

• 2016
  • 2016-06-17 US US15/736,092 patent/US10471087B2/en active Active
  • 2016-06-17 LT LTEP19156021.8T patent/LT3505174T/lt unknown
  • 2016-06-17 ES ES16812537T patent/ES2748556T3/es active Active
  • 2016-06-17 CN CN202310757609.2A patent/CN116726035A/zh active Pending
  • 2016-06-17 BR BR122020021913-0A patent/BR122020021913B1/pt active IP Right Grant
  • 2016-06-17 CN CN201680045355.XA patent/CN107847512A/zh active Pending
  • 2016-06-17 HU HUE16812537A patent/HUE046399T2/hu unknown
  • 2016-06-17 RU RU2018101305A patent/RU2721492C2/ru active
  • 2016-06-17 SM SM20200399T patent/SMT202000399T1/it unknown
  • 2016-06-17 PL PL16812537T patent/PL3310362T3/pl unknown
  • 2016-06-17 AU AU2016280293A patent/AU2016280293B2/en active Active
  • 2016-06-17 CA CA2989653A patent/CA2989653A1/en active Pending
  • 2016-06-17 BR BR112017027079-0A patent/BR112017027079B1/pt active IP Right Grant
  • 2016-06-17 RS RS20200779A patent/RS60572B1/sr unknown
  • 2016-06-17 KR KR1020187001496A patent/KR20180039624A/ko not_active Ceased
  • 2016-06-17 KR KR1020257009842A patent/KR20250048602A/ko active Pending
  • 2016-06-17 EP EP19156021.8A patent/EP3505174B1/en active Active
  • 2016-06-17 EP EP16812537.5A patent/EP3310362B1/en active Active
  • 2016-06-17 HR HRP20191794TT patent/HRP20191794T1/hr unknown
  • 2016-06-17 HK HK18111524.2A patent/HK1252133A1/zh unknown
  • 2016-06-17 KR KR1020227002790A patent/KR20220018623A/ko not_active Ceased
  • 2016-06-17 PT PT191560218T patent/PT3505174T/pt unknown
  • 2016-06-17 PL PL19156021T patent/PL3505174T3/pl unknown
  • 2016-06-17 RS RS20191295A patent/RS59724B1/sr unknown
  • 2016-06-17 MX MX2017016425A patent/MX2017016425A/es active IP Right Grant
  • 2016-06-17 LT LT16812537T patent/LT3310362T/lt unknown
  • 2016-06-17 SI SI201630439T patent/SI3310362T1/sl unknown
  • 2016-06-17 IL IL275256A patent/IL275256B2/en unknown
  • 2016-06-17 JP JP2017566000A patent/JP6599484B2/ja active Active
  • 2016-06-17 DK DK16812537.5T patent/DK3310362T3/da active
  • 2016-06-17 SI SI201630830T patent/SI3505174T1/sl unknown
  • 2016-06-17 MX MX2020000269A patent/MX389016B/es unknown
  • 2016-06-17 PT PT168125375T patent/PT3310362T/pt unknown
  • 2016-06-17 SM SM20190571T patent/SMT201900571T1/it unknown
  • 2016-06-17 HU HUE19156021A patent/HUE050678T2/hu unknown
  • 2016-06-17 EP EP19178700.1A patent/EP3569236A1/en active Pending
  • 2016-06-17 WO PCT/US2016/038110 patent/WO2016205671A1/en not_active Ceased
  • 2016-06-17 DK DK19156021.8T patent/DK3505174T3/da active
  • 2016-06-17 ES ES19156021T patent/ES2808148T3/es active Active
• 2017
  • 2017-12-14 IL IL256331A patent/IL256331B/en active IP Right Grant
• 2019
  • 2019-09-26 US US16/583,852 patent/US11110111B2/en active Active
  • 2019-10-02 JP JP2019182191A patent/JP6675037B2/ja active Active
  • 2019-10-02 JP JP2019182192A patent/JP7036782B2/ja active Active
  • 2019-10-30 CY CY20191101127T patent/CY1122605T1/el unknown
• 2020
  • 2020-06-09 IL IL275255A patent/IL275255B/en active IP Right Grant
  • 2020-06-15 HR HRP20200949TT patent/HRP20200949T1/hr unknown
  • 2020-06-17 AU AU2020204042A patent/AU2020204042B2/en active Active
  • 2020-06-25 CY CY20201100578T patent/CY1123107T1/el unknown
• 2021
  • 2021-04-28 US US17/242,822 patent/US11666592B2/en active Active
  • 2021-10-11 AU AU2021250841A patent/AU2021250841B2/en active Active
• 2023
  • 2023-04-24 US US18/138,432 patent/US12251392B2/en active Active
• 2025
  • 2025-02-11 US US19/050,489 patent/US20250360154A1/en active Pending

Patent Citations (1)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events