WO2023114656A1 - Méthodes de traitement et de prévention de maladies et de troubles par modulation de nad - Google Patents

Méthodes de traitement et de prévention de maladies et de troubles par modulation de nad Download PDF

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WO2023114656A1
WO2023114656A1 PCT/US2022/080912 US2022080912W WO2023114656A1 WO 2023114656 A1 WO2023114656 A1 WO 2023114656A1 US 2022080912 W US2022080912 W US 2022080912W WO 2023114656 A1 WO2023114656 A1 WO 2023114656A1
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nad
nicotinamide
adp
subject
ribose
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PCT/US2022/080912
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Paul Mark
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Paul Mark
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives

Definitions

  • compositions that moderate NAD, components of the NAD pathway and moderators of the NAD pathway are provided.
  • Genetic disease can occur when there are whole chromosomes that are missing or added (chromosomal aberrations including aneuploidy such as triploidy, trisomy, monosomy), occuring through mitotic or meiotic nondisjunction. Partial aneuploidy can occur in unbalanced translocations, including Robertsonian and reciprocal translocations. Partial chromosome deletions or duplications can occur (copy number variants- CNVs), and single nucleotide variants (SNVs) also cause genetic disease. Finally, epigenetic changes due to abnormal methylation and acetylation can cause genetic disease.
  • the central dogma of DNA is that DNA is replicated into DNA, transcribed into RNA, and RNA is translated into protein, with subsequent post-translational modification.
  • the assumption has always been that if the DNA is abnormal, the final product, the protein, will also be abnormal.
  • the present disclosure demonstrates application and advantage in comprehensive treatment and prevention methods for diseases and disorder where there are no genetic causes, and as the cause of genetic disease itself. It also demonstrates application and advantage in treating some forms of genetic disease including but not limited to trisomy.
  • the present disclosure provides a method of treating or preventing a disease or disorder.
  • the disease or disorder does not have a genetic basis.
  • the disease or disorder does not have a genetic basis for one or more genes.
  • the disease or disorder does not have a genetic basis for one or more genetic pathways.
  • the present disclosure provides a method of treating or preventing miscarriage in a subject comprising administering to the subject an effective amount of vitamin B6, trimethylglycine, folic acid, apigenin, and optionally, an intermediate of NAD synthesis and/or metformin, and/or NAD precursor.
  • the miscarriage or potential miscarriage is caused by at least one chromosomal aberration of an unborn offspring, wherein the chromosomal aberrations is trisomy, triploidy, monosomy and meiotic nondisjunction in the unborn offspring, or a CNV or SNV.
  • the present disclosure provides a method of preventing diabetic embryopathy in an unborn offspring of a subject comprising administering to the subject an effective amount of vitamin B6, trimethylglycine, folic acid, apigenin, and optionally an intermediate of NAD synthesis and/or NAD precursor, and/or metformin, wherein the diabetic embryopathy is caused by decreased level of NAD due to inflammation, diabetes mellitus, obesity, or reproductive aging of the subject.
  • the diabetic embryopathy is caused by one or more genetic mutations.
  • the present disclosure provides a composition comprising an effective amount of vitamin B6, trimethylglycine, folic acid, apigenin, and optionally an intermediate of NAD synthesis and/or an NAD precursor, and/or metformin.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising 0.1 -5mg of vitamin B6, 500mg-9,000mg of trimethylglycine, 200mg-6000mg folic acid, lmg/kg-40mg/kg apigenin; and a pharmaceutically acceptable carrier.
  • the present disclosure provides a composition further comprising NAD or an intermediate of NAD synthesis.
  • the present disclosure provides a method of treating or preventing biological disorders in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of any preceding claims.
  • the biological disorders are: (i) miscarriage; (ii) diabetic embryopathy; (iii) recurrent congenital malformations (“RCM”); (iv) recurrent constellations of embryonic malformations (“RCEM”); (v) pleiotropism; or a combination thereof.
  • the miscarriage is caused by at least one chromosomal aberration of the subject or the unborn offspring, wherein the chromosomal aberrations is trisomy, triploidy, monosomy or meiotic nondisjunction in the unborn offspring.
  • the diabetic embryopathy is caused by decreased level of NAD due to inflammation, diabetes mellitus, obesity, or reproductive aging of the subject.
  • the method further comprises administration of NAD or an intermediate of NAD synthesis.
  • the method further comprises increasing the NAD level in the unborn offspring or the subject, wherein the unborn offspring or the subject does not have a genetic basis or does not have a genetic influence through DNA variants.
  • the method further comprises increasing the NAD level in the unborn offspring or the subject, wherein the unborn offspring or the subject has a genetic basis.
  • the composition is administered during umbilical cord and embryonic wall development, cranial to caudal ventral body wall closure, neural tube closure, vertebral column development, gut development; limb cartilages and digital ray development, frontal facial development, conotruncal development and ventricular septa development; abdominal wall development; or uterus and upper vagina development.
  • the method further comprises increasing NAD or an intermediate of NAD synthesis prior to or during gestational week 2-3, 3-6, 6-7, 7-8, 9 or a combination thereof.
  • a method to regulate one or more biological processes comprising administration of the pharmaceutical composition of any preceding claims wherein the biological process is DNA replication, DNA repair, aneuploidy, methylation, cancer or a combination thereof.
  • a method of detecting a risk for a biological disorder in a subject or a congenital malformation in an unborn offspring of a subject comprising detecting at least one of: i) a dietary deficiency of one or more of vitamin B6,
  • SUBSTITUTE SHEET (RULE 26) trimethylglycine and folic acid; ii) inadequate absorption of one or more of vitamin B6, trimethylglycine and folic acid; and iii) inadequate level of NAD, wherein the presence of any one of i), ii) or iii), is associated with an increased risk.
  • the method further detects optionally: (i) a dietary deficiency of one or more of tryptophan, nicotinamide, and nicotinamide riboside or (ii) inadequate absorption of one or more of tryptophan, nicotinamide, and nicotinamide riboside.
  • the present disclosure provides a method of preventing recurrent congenital malformations (RCM), which can occur in isolation or in combination, in an unborn offspring of a subject, said method comprises increasing the NAD level by administration of vitamin B6, trimethylglycine, folic acid, or a combination thereof, in the unborn offspring or the subject, wherein the unborn offspring or the subject does not have a genetic basis of the RCM.
  • RCM recurrent congenital malformations
  • the composition further comprises intermediate of NAD synthesis and/or NAD precursor.
  • the recurrent constellations of embryonic malformations includes limb-body wall complex (“LBWC”), pentalogy of Cantrell (“POC”), omphaloceleexstrophy-imperforate anus-spinal defects (“OEIS”) complex, sirenomelia, vertebral-anal- cardiac-tracheoesophageal fistula-renal-limb (“VACTERL”) association, oculoauriculovertebral spectrum (“OAVS”), Mullerian duct aplasia-renal anomalies cervicothoracic somite dysplasia (“MURCS”), urorectal septum malformation (URSM) or a combination thereof.
  • LBWC limb-body wall complex
  • POC pentalogy of Cantrell
  • OEIS omphaloceleexstrophy-imperforate anus-spinal defects
  • sirenomelia sirenomelia
  • VACTERL vertebral-anal- cardiac-tracheoes
  • the genetic basis is HAAO, KYNU, KMO, NADSYN1 mutation or a combination thereof. In one embodiment, the genetic basis involves other genes in the NAD synthetic pathway.
  • a method of preventing recurrent constellations of embryonic malformations (RCEM) in an unborn offspring of a subject comprises administering a composition of the disclosure vitamin B6, trimethylglycine, folic acid, apigenin, metformin or a combination thereof, in the unborn offspring or the subject, wherein the unborn offspring or the subject does not have a genetic basis of the RCEM.
  • RCEM embryonic malformations
  • composition further comprises intermediate of NAD synthesis and/or NAD precursor.
  • the RCEM is POC, LBWC, OEIS, VACTERI, OAVS, MURCS, URSM, sirenomelia or a combination thereof.
  • the disease and disorder have a genetic basis.
  • the genetic basis is HAAO, KYNU, KMO, NADSYN1 mutation or a combination thereof.
  • a method of preventing pleiotropism comprising increasing NAD or administration of the composition of the present disclosure prior to specific times of embryonic development.
  • the timing of treatment or prevention is in relation to umbilical cord and embryonic wall development, such as wk 2-3: ventral body wall closure, cranial to caudal; wk 3-6 neural tube closure, beginning development of vertebral column, gut development; wk 6-7 limb cartilages and digital rays, frontal facial development, conotruncal defects and ventricular septa; wk 7-8 omphalocele; wk 9 formation of uterus and upper vagina.
  • the timing of treatment or prevention occurs prior to or during all trimesters of pregnancy of mother and until the embryo can take over the process of the production of certain compounds.
  • pleiotropism is affected by environmental factors, diet restriction of vitamin B6, trimethylglycine, folic acid or a combination thereof, and hypoxia (such as iron and nitrates).
  • the composition further comprises intermediate of NAD synthesis and/or an NAD precursor.
  • the administration of vitamin B6, trimethylglycine, folic acid and apigenin, metformin occurs prior to weeks 2-3, 3-6, 6-7, 7-8, 8-9 of gestation.
  • the administration of the composition of the present disclosure comprising, for example, vitamin B6, trimethylglycine, and folic acid is provided prior to umbilical cord and embryonic wall development, ventral body wall closure, cranial to caudal, neural tube closure, vertebral column starts developing, gut development, limb cartilages and digital rays, frontal facial development, conotruncal defects and ventricular septa, omphalocele, formation of uterus and upper vagina.
  • the composition further comprises intermediate of NAD synthesis.
  • the disease and disorder is a spectrum of malformations.
  • the disease or disorder is a single malformation.
  • the malformation(s) are variable in multiple gestation pregnancies.
  • provided herein is a method of preventing pleiotropism in multiple gestation in a subject comprising administration of the composition provided in the present disclosure.
  • the subject has one or more factors including inflammation, type 2 diabetes, obesity, hypoxia, aging, or combination thereof.
  • a tool for measuring pleiotropism In one aspect, provided herein is a tool for measuring pleiotropism. In certain embodiments, the pleiotropism is RCM and RCEM. In one embodiment, provided herein is a method of using the tool for measuring pleiotropism before treatment. In one embodiment, provided herein is a method of using the tool for measuring pleiotropism after treatment.
  • provided herein is a method to treat or prevent recurrent multiple congenital malformations, miscarriages and congenital malformations without a genetic basis by administering a composition provided in the present disclosure.
  • the method reverses NAD deficiency or disturbance.
  • the composition comprises vitamin B6, trimethylglycine, folic acid, apigenin, metformin or a combination thereof.
  • the composition further comprises intermediate of NAD synthesis.
  • the birth defects are caused by extra chromosomes, deletions, duplications or a combination thereof of genetic material.
  • the genetic condition is an aberration of copy number of genetic materials, point mutations (single nucleotide variants) and epigenetic changes.
  • the method comprises administering a compound as provided in the present disclosure.
  • the method reverses NAD deficiency or NAD disturbance.
  • the composition comprises vitamin B6, trimethylglycine, folic acid, apigenin, metformin or a combination thereof.
  • the composition further comprises intermediate of NAD synthesis.
  • the present disclosure provides a method to regulate one or more of the following biological processes: DNA replication, DNA repair, transcription, translation, post-translational modification, aneuploidy, methylation and cancer by administering a composition as provided in the present disclosure.
  • the method modulates NAD deficiency or disturbance.
  • the composition comprises vitamin B6,
  • composition further comprises intermediate of NAD synthesis.
  • the subject is the mother of an unborn offspring, the unborn offspring or the bom offspring.
  • vitamin B6 or its derivative may be administered at a dose of at least about 0.1-0.3 mg/day, at least about 0.3-1.0 mg/day, at least about 1.0-5.0 mg/day, at least about 5-10 mg/day, at least about 10-50mg/day, or at least about 50-100 mg/day.
  • trimethylgycine may be administered at a dose of at least about 1.0-10mg/day, at least about 10-50 mg/day, at least about 50-100 mg/day, at least about 100- 200 mg/day, at least about 200750 mg/day.
  • folic acid may be administered at a dose of at least about 0.4- 1 mg/day, at least about 1-4 mg/day.
  • apigenin may be administer at a dose of at least about 1- 40mg/kg/day, at least about 30-1200mg/day.
  • the intermediate of NAD synthesis may be selected from the group consisting of tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3 -hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, 2- amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, N-methylnicotinamide (MNA), deamido-NADN-methyl-4-pyridone-3- carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, deamido-NAD,
  • the intermediate of NAD synthesis is selected from the group consisting of nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide, deamido-NAD, nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin, a derivative thereof, a salt thereof, and any combination thereof.
  • the intermediate is at least one of vitamin B3, nicotinamide or niacin (nicotinic acid).
  • the NAD or intermediate of NAD synthesis may be administered at a dose of at least about 0.1 -0.3 mg/kg, at least about 0.3-1 mg/kg, at least about 1-25 mg/kg, at least about 25-50 mg/kg, at least about 50-100 mg/kg, at least about 100-150 mg/kg, at least about 150-200 mg/kg, or at least about 200mg/kg body weight of the subject.
  • the NAD or an intermediate of NAD synthesis may be administered orally or by injection, for example by subcutaneous, intramuscular or intravenous injection.
  • the NAD or an intermediate of NAD synthesis may be administered before conception, around the time of conception or during the term of a pregnancy, or after birth if the NAD deficiency persists.
  • the treatment is for a disease or disorder that does not have a genetic basis.
  • a genetic basis is when a gene or its disruption is responsible for the disorder or disease.
  • the treatment is for a disease or disorder that has a genetic basis.
  • the gene is selected from the group consisting of aminoadipate aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1, ADP-ribosyltransferase 1, ADP-ribosyltransferase 2, ADP-ribosyltransferase 3, ADP-ribosyltransferase 4, ADP- ribosyltransferase 5, bone marrow stromal cell antigen 1, catalase, C038, cytochrome 85 reductase 3, cytochrome p450 family 2 subfamily 0 member 6, cytochrome p450 family 8 subfamily B member 1, ectonucleotide pyrophosphatase/phosphodiesterase 1, ectonucleotide pyrophosphatase/phosphodieste
  • kynurenine aminotransferase 1, kynurenine aminotransferase 3, kynureninase, leukosialin, NAD kinase, NAD kinase 2, NAD synthetase 1, nicotinamide phosphoribosyltransferase, nicotinate phosphoribosyltransferase, NAD(P)HX dehydratase, NAD(P)HX epimerase, nicotinamide nucleotide adenylyltransferase 1, nicotinamide nucleotide adenylyltransferase 2, nicotinamide nucleotide adenylyltransferase 3, nicotinamide riboside kinase 1, nicotinamide riboside kinase
  • nicotinamide N-methyltransferase 2, nicotinamide N-methyltransferase, nicotinamide nucleotide transhydrogenase, 5', 3 'nucleotidase-cytosolic, 5 '-nucleotidase - cytosolic IA, 5 '-nucleotidase, cytosolic IB,
  • SUBSTITUTE SHEET ( RULE 26) NT5C1B-RDH14 readthrough, 5'-nucleotidase - cytosolic II, 5'-nucleotidase - cytosolic IIIA, 5'-nucleotidase - cytosolic IIIB, 5'-nucleotidase ecto, 5',3'-nucleotidase-mitochondrial, nudix hydrolase 12, poly(ADP-ribose) polymerase 1, poly(ADP-ribose) polymerase 2, poly(ADP- ribose) polymerase family member 3, poly(ADP-ribose) polymerase family member 4, poly(ADP-ribose) polymerase family member 6, poly(ADPribose) polymerase family member 8, poly(ADP-ribose) polymerase family member 9, poly(ADP -ribose) polymerase family member 10, poly
  • the gene may be selected from the group consisting tryptophan transporter, TD02 (tryptophan 2,3 -dioxygenase), IDO 1,2 (Indoleamine 2,3 -dioxygenase), AFMID (arylformamidase), KYN8 (Kynurenine formamidase), KMO (kynurenine 3- monooxygenase), KYNU (kynureninase), HAAO (3-hydroxyanthranilate 3,4-dioxygenase), ACSMD (aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate phosphoribosyltransferase), NAPRT, NADSYN1, NMAT1, 2, or 3 (nicotinamide nucleotide adenylyltransferase 1, 2, or 3), NADSYN1 (NAD synthetase 1), nicotinamide phosphoribos
  • NMRK1 ,2 Natural Organotase 1 or 2
  • NAMPT nicotinamide Phosphoribosyltransferase
  • PNP Purine Nucleoside Phosphorylase
  • PARP1-6 Poly(ADP-Ribose) Polymerase 1-6
  • ART 1-4 SIRT1-7 (Sirtuin 1-7, also known as NAD- dependent deacetylase sirtuin 1-7)
  • CD38 cyclic ADP ribose hydrolase
  • 8ST1 ADP-ribosyl cyclase 2
  • TRPT-1 tRNA 2-phosphotransferase 1
  • a method of detecting potential for a congenital malformation in a subject comprising detecting at least one of: a. a dietary deficiency of one or more of vitamin B6, trimethylglycine and folic acid;
  • SUBSTITUTE SHEET (RULE 26) b. inadequate absorption of one or more of vitamin B6, trimethylglycine and folic acid; and optionally, c. inadequate synthesis of NAD, wherein the presence of any one of a, b or optionally c is associated with the congenital malformation.
  • the method further detects optionally: (i) a dietary deficiency of one or more of tryptophan, niacin, nicotinamide mononucleotide, and nicotinamide riboside or (ii) inadequate absorption of one or more of tryptophan, niacin, nicotinamide mononucleotide, and nicotinamide riboside.
  • the presence of any one of a, b or optionally c is indicative of a predisposition to a congenital malformation in the mother's unborn offspring.
  • FIG. 1 The NAD de novo synthesis pathway and NAD salvage pathway.
  • 'treat' and 'treating' refer to one or more of the following: (i) preventing development of a malformation (i.e. prophylaxis); (ii) inhibiting or arresting development of the malformation; (iii) relieving a malformation; and (iv) ameliorating, alleviating, lessening, or removing one or more symptoms of a malformation.
  • 'malformation' and 'congenital malformation' are used interchangeably and refer to one or more of the following: (i) an irregular, anomalous, abnormal, or faulty formation, structure or anatomical feature of an embryo, fetus, infant child or adult; (ii) a neurocognitive deficit; and (iii) a consequence of a malformation including miscarriage or spontaneous abortion.
  • vitamin B6' refers to a mixture or complex comprising pyridoxine, pyridoxal, and pyridoxamine, and their phosphorylated derivatives, pyridoxine 5'-phosphate, pyridoxal 5'- phosphate and pyridoxamine 5'-phosphate.
  • SUBSTITUTE SHEET ( RULE 26)
  • the term 'intermediate of NAD synthesis' refers to any compound that is required or can be used, in the synthesis of NAD, including dietary sources such as nicotinic acid, nicotinamide, nicotinamide riboside, vitamin B3, and tryptophan.
  • Pleiotropism is defined as multiple effects of a single etiologic factor. It is understood to be the phenomenon of a single gene influencing two or more distinct phenotypic traits. In human development, the assumption has always been the etiologic factor is a single gene, as no other single factor has been identified. NAD deficiency through diet alone can cause multiple or single malformations in mice, and humans with decreased NAD production display similar malformations.
  • NAD disturbance solely can cause recurrent multiple congenital malformations including LBWC, POC, OEIS, sirenomelia, VACTERL, OAVS, and MURCS, along with miscarriages and other forms of congenital malformation.
  • NAD nicotinamide adenine dinucleotide
  • the disease and disorder presented in the present disclosure can be caused by perturbances in NAD + , provided herein is a method of preventing the disease and disorder by administering an effective amount of a composition of the disclosure.
  • the composition comprises vitamin B6, trimethylglycine, folic acid, and optionally an NAD intermediate, including but not limited to, nicotinamide mononucleotide or nicotinamide riboside, or NAD+ precursors, including but not limited to, tryptophan or niacin.
  • the cause of genetic diseases is a disturbance in NAD + levels, either through NAD + deficiency or an abnormal NAD + /NADH ratio.
  • SUBSTITUTE SHEET (RULE 26) polymerases (PARPs) and sirtuins, require NAD as a substrate and if there is inadequate substrate then genetic disease will occur through errors in meiotic nondisjunction in eggs and sperm, copy number variants, and single nucleotide variants.
  • the single nucleotide variants can occur through errors in the following: base excision repair, DNA mismatch repair, nucleotide excision repair, nonhomologous end joining, interstrand cross-link repair, and homologous recombination. When these errors occur after the gametes (eggs and sperm) are created through meiosis, somatic errors occur in cells during mitosis which can cause cancer or cell death.
  • NAD deficiency has two critical mechanisms for aneuploidies.
  • reduced NAD has been reported in aging and is a plausible mechanism for the increased rate of meiotic nondisjunction seen in older women of child-bearing age.
  • a hypothesis could be made regarding the increased need for NAD as a substrate to perform replication, transcription, and translation of the increasing number of additional genes found on the third copy of chromosomes 21, 18, and 13 in viable trisomies.
  • the increasing severity of anomalies and poor growth of these infants could be due to decreased NAD availability to perform the usual cellular and organism functions depending on NAD.
  • NAD + deficiency can occur either due to decreased NAD production by the embryo/ fetus or liveborn human, or increased demands for NAD + due to diseases including diabetes, obesity, autoimmune disease and aging. These diseases can occur in the mother prior to or during pregnancy leading to decreased transmission of NAD+ to the developing embryo through the placenta and umbilical cord. This would explain diabetic embryopathy, which are congenital malformations due to maternal diabetes occurring before or during pregnancy. There could also be decreased NAD + available to a developing embryo or fetus due to competition for the maternal NAD + due to multiple gestation.
  • NAD + neurodeficiency -deficiency - thelial -aminoethylcholine
  • NAD + abnormalities also occur due to environmental exposures during pregnancy including hypoxia, hyperthermia, stress, poor nutrition and teratogens including anticonvulsants, folic acid antagonists, aminopterin, methimazole, antithyroid medications, and thalidomide.
  • NAD + abnormalities also occur due to environmental exposures during pregnancy including hypoxia, hyperthermia, stress, poor nutrition and teratogens including anticonvulsants, folic acid antagonists, aminopterin, methimazole, antithyroid medications, and thalidomide.
  • NAD + abnormalities also occur due to environmental exposures during pregnancy including hypoxia, hyperthermia, stress, poor nutrition and teratogens including anticonvulsants, folic acid antagonists, aminopterin, methimazole, antithyroid medications, and thalidomide.
  • Diabetic embryopathy overlaps with RCEM with the following anomalies: anal, caudal dysgenesis, congenital heart defects, vertebral, limb defects, hemifacial microsomia, cleft lip +/- cleft palate, neural tube defects,
  • SUBSTITUTE SHEET ( RULE 26) renal, sirenomelia, and urorectal septum malformation. Diabetes is a risk factor for VACTERL anomalies, along with multiple pregnancies, assisted reproductive technologies, chronic obstructive pulmonary disease, and epilepsy, all of which cause anomalies through diminished NAD + .
  • Neural tube defects have genetic and environmental causes including teratogens, obesity, and diabetes, RCEM, and other anomalies for which a genetic cause has not been identified, including holoprosencephaly, diaphragmatic hernia, cardiac, renal, limb, genitourinary and gastrointestinal defects, among others.
  • Folic acid supplementation has reduced the incidence of NTDs by about 40%.
  • NAD/NADP in folate metabolism is responsible for neural tube defects with or without folic acid supplementation.
  • Recurrent constellations of embryonic malformations including limb-body wall complex (“LBWC”), pentalogy of Cantrell (“POC”), omphalocele-exstrophy-imperforate anus-spinal defects (“OEIS”) complex, sirenomelia, vertebral-anal-cardiac-tracheoesophageal fistula-renal-limb (“VACTERL”) association, oculoauriculovertebral spectrum (“OAVS”), Mullerian duct aplasia-renal anomalies cervicothoracic somite dysplasia (“MURCS”), and urorectal septum malformation (URSM) are a group of conditions each with their own diagnostic criteria for which a known etiology, genetic or otherwise, has yet to be identified.
  • LBWC limb-body wall complex
  • POC pentalogy of Cantrell
  • OEIS omphalocele-exstrophy-imperforate anus-spinal defects
  • Recurrent Constellations of Embryonic Malformations include pentalogy of Cantrell (POC), limb-body wall complex (LBWC), omphalocele-exstrophy-imperforate anus-spinal defects (OEIS) complex, vertebral-anal- cardiac-tracheoesophageal fistula-renal-limb (VACTERL) association,
  • SUBSTITUTE SHEET (RULE 26) oculoauriculovertebral spectrum (OAVS), and Mullerian duct aplasia-renal anomalies cervicothoracic somite dysplasia (MURCS). Additional conditions with multiple embryonic malformations for which no genetic cause has been elucidated include sirenomelia and urorectal septal malformation (USRM), which itself has been considered as part of both OEIS and LBWC.
  • OAVS oculoauriculovertebral spectrum
  • MURCS Mullerian duct aplasia-renal anomalies cervicothoracic somite dysplasia
  • Additional conditions with multiple embryonic malformations for which no genetic cause has been elucidated include sirenomelia and urorectal septal malformation (USRM), which itself has been considered as part of both OEIS and LBWC.
  • VACTERL association the RCEM with by far the most reported cases, also has the greatest number of individuals who do not meet reported criteria, partly because the criteria themselves are not agreed upon. In a survey of 121 clinical geneticists, 15% stated at least two features were necessary to meet criteria, 79% needed three, and 8% required four criteria to be classified as having VACTERL association [Solomon et al, 2012].
  • the RCEM Study Group states there must be the presence of three or more features including at least one major feature (tracheoesophageal fistula with or without esophageal atresia, anal atresia or radial ray defect or postaxial polydactyly). Minor features include structural cardiac malformations, vertebral segmentation anomalies, and renal hypoplasia/aplasia.
  • the EUROCAT VACTERL Study defined STRICT-VACTERL as cases with >3 major VACTERL features and no major congenital anomalies outside the VACTERL spectrum (with specific restrictions mentioned) [van de Putte et al, 2019].
  • POC was defined by Cantrell as the presence of the following five criteria: midline supraumbilical abdominal wall defect, defect of the anterior diaphragm, defect of the lower sternum, defect of the diaphragmatic pericardium and congenital heart defect [Cantrell, Haller, Ravitch, 1958].
  • Toyama’s criteria in 1972 states all five must be present (substituting the term midline supraumbilical abdominal wall defect for omphalocele) for complete POC, probable (or suspected) POC if four of the five (which must include an intracardiac defect and ventral abdominal wall defect) were present, and incomplete if less than four criteria were met [Toyama 1972],
  • the RCEM Study Group lists omphalocele as the suggested abdominal defect [Adam et al, 2020]
  • yet Toyama’s review of 36 cases lists the following abdominal wall defects- omphalocele, diastasis recti, ventral hernia, absent umbilicus, and eventration- suggesting difficulty in finding uniform criteria for at least one aspect (midline supraumbilical abdominal wall defect) of this condition, demonstrating lack of consensus for the diagnostic criteria of POC.
  • LBWC has generally been described as the presence of at least two of the following: exencephaly or encephalocele with facial clefts, thoracoschisis, abdominoschisis or both, and limb defects, [Van Allen, Curry, Gallagher, 1987].
  • Craven, Carey, and Ward presented three cases with no exencephaly/encephalocele, and the only limb defects were contractures and rocker bottom feet, suggesting Limb Body Wall Defect with umbilical cord agenesis as a subgroup [Craven, Carey, Ward, 1997].
  • Martinez-Frias reported fifteen cases of Body Wall Complex without limb defects, five of which had either hydrocephaly or cranial disruption by bands, but not true exencephaly or encephalocele [Martinez-Frias, 1997]. There have also been cases reported with facial clefts (with or without cranial/CNS defect) with limb defects, but without body wall defects [Halder, 2010] which some clinicians believe do not meet the criteria for LBWC (no body wall defect).
  • OEIS complex was originally defined as midline abdominal and pelvic defects of omphalocele, exstrophy of the bladder, imperforate anus, and spine abnormalities in 1978, yet in the original paper 23 of 29 patients described had either two or three of the features, but not complete OEIS [Carey, Greenbaum, Hall, 1978].
  • Urorectal septum malformation includes absence of perineal and anal openings in association with ambiguous genitalia and urogenital, colonic and lumbosacral anomalies [Escobar et al, 1987].
  • Wheeler and Weaver described 25 cases of partial urorectal septum malformation sequence, of which there were many additional findings including renal, gastrointestinal, tracheoesophageal fistulas, neurologic (myelomeningocele), vertebral, limb, cardiac and single umbilical artery [Wheeler and Weaver, 2001].
  • MURCS criteria listed by the RCEM Study Group includes Mullerian duct hypoplasia/aplasia with one or more of the following: short, blind ending vagina (this is the criteria for Mayer-Rokitansky-Kiister-Hauser (MRKH) syndrome), renal agenesis, hypoplasia, malposition, including pelvic kidney, or vertebral segmentation anomalies [Adam et al, 2020].
  • sirenomelia was suggested to be on a spectrum with hemifacial microsomia (also called OAVS) and VATER (now called VACTERL) [Duncan and Shapiro, 1993, Duncan and Shapiro, 1988].
  • Non-traditional findings in sirenomelia included tracheoesophageal fistula with or without esophageal atresia (TEF +/- EA) CNS, ear, face, cleft lip and/or palate, and eye anomalies. These findings suggest a different mechanism besides vitelline artery steal to cause these specific anomalies.
  • Non-traditional findings in hemifacial microsomia patients included anal dysgenesis, TEF +/- EA, cardiovascular, pelvis, CNS, abdominal wall, genital, and respiratory anomalies. These findings distal to the head also suggest a mechanism that is not localized to one region of the body.
  • LBWC patients have been described to have non- traditional anomalies including renal, cardiac, vertebral, rib, intestinal, bladder, genitalia and cleft lip and palate [Hunter, Seaver and Stevenson, 2011, Colpaert et al, 2000].
  • organ systems have been reported to have defects in cases described to have LBWC, POC, OEIS, sirenomelia, OAVS, and VACTERL: cardiovascular, vertebral/ribs, urogenital, sensory, limbs/digits, palate, CNS, respiratory, and gastrointestinal.
  • VACTERL the only two groups in which VACTERL is not noted are those with body wall defects or those with extreme genitourinary anomalies such as cloacal exstrophy and caudal dysgenesis seen in OEIS and sirenomelia, or milder genitourinary anomalies such as MURCS, where genital anomalies are not yet considered part of the VACTERL acronym.
  • VACTERL is actually a default label when more striking features such as body wall defects or distal defects such as caudal dysgenesis or cloacal exstrophy are not present. This makes sense because vertebral, cardiac, renal and limb anomalies are the most common anomalies associated with the RCEM group.
  • NAD disturbance is a cause of recurrent malformations without a known genetic etiology, for twins with discordant anomalies, and for miscarriages. NAD disturbance is an etiologic factor for pleiotropy.
  • Nicotinamide adenine dinucleotide is found in all parts of cells, including the nucleolus, nucleus, mitochondria, cytoplasm, and organelles including ribosomes, endoplasmic reticulum, and Golgi apparatus. It has two primary functions. The first is its indispensable role as an essential cofactor in cellular oxidation/reduction reactions for production of the primary energy source in human cells, adenosine triphosphate (ATP), through glycolysis in the cytoplasm and oxidative phosphorylation in the mitochondria. It is 18
  • SUBSTITUTE SHEET (RULE 26) not possible to produce ATP without NAD.
  • the second is as essential substrate for multiple NAD+ dependent enzyme families including sirtuins, ADP-ribose transferases including Poly (ADP-ribose) polymerases (PARPs), and cyclic ADP (cADP)-ribose synthases.
  • sirtuins are critical for DNA repair, metabolic regulation, and age-related diseases including diabetes and atherosclerosis.
  • PARPs modulate chromatin structure, transcription, replication, recombination, and DNA repair.
  • cADP-ribose synthases are associated with neurologic disorders including autism spectrum disorder, Parkinson’s disease, and schizophrenia CD38, CD157 and RAGE).
  • NAD is produced by two pathways: one requires the essential amino acid tryptophan, and the other requires dietary niacin (FIG. 1).
  • the NAD de novo synthesis pathway catabolizes tryptophan through the kynurenine pathway and the NAD salvage pathway converts niacin and other precursors into NAD [Shi et al, 2017].
  • a failure to produce an adequate amount of NAD due to inhibition of either pathway can have dramatic consequence, and disturbances in the NAD + /NADH ratio in any cellular process can also create serious health ramifications as noted above.
  • NAD is a cause of disease and disorder during embryonic and fetal development.
  • Congenital malformations could be produced solely by NAD deficiency due to environmental factors (dietary restriction of tryptophan and niacin, with or without hypoxia) in wild type mice (all genetically identical).
  • wild-type mice with only diet restriction reduced tryptophan and niacin caused multiple malformations and embryo loss.
  • hypoxia increased the likelihood of embryo malformation in wild-type mice with diet restrictions.
  • maternal Haao LoF mutation exacerbated the effect of dietary restriction on embryonic development.
  • Maternal and embryonic NAD levels are lowered under maternal treatment conditions that cause embryo loss and congenital malformations of mouse embryos.
  • mice that are NAD deficient had malformations in the following locations: skull (exencephaly, a neural tube defect), eyes, palate, vertebrae and ribs, heart, abdominal wall, kidneys, limbs, digits, and tail in the form of caudal agenesis.
  • the mice have caudal agenesis with gastrointestinal and reproductive tract anomalies. Also, the mice have microcephaly or short stature.
  • Congenital malformations are identified in patients with biallelic variants such as, HAAO, KYNU, or NADSYN1 in NAD+ Synthesis Pathway Genes. Initially these patients were labeled as having Vertebral, Cardiac, Renal and Limb Defects syndrome, because of the phenotypic frequency of these four malformations.
  • SUBSTITUTE SHEET (RULE 26) were genetic changes in genes creating enzymes in the NAD Synthesis pathway, the end result was low NAD production which caused the congenital malformations.
  • NAD is synthesized through tryptophan catabolism in the liver, the maternal contribution of NAD in early embryonic development is crucial until the embryo/fetus can take over this process.
  • the risk of developing NAD deficiency is elevated during pregnancy, the time when sufficient NAD supply is required to ensure normal embryonic development.
  • Intra-litter variability (some unaffected, some with one malformation, some with two or more malformations, some embryonic losses). This is due to maternal underproduction of NAD for a multiple gestation, or asymmetric NAD distribution to the developing embryos through the placenta.
  • NAD neurotrophic factor
  • SUBSTITUTE SHEET ( RULE 26) pathophysiologic factors such as inflammation, type 2 diabetes and obesity could influence NAD synthesis. Exact recurrence of an RCEM, or subsequent pregnancies showing different or partial features of RCEM.
  • Body wall, amnion, and umbilical cord abnormalities are observed.
  • the timing of the NAD deficiency has an impact on the specific anomalies generated by this deficiency.
  • One of the greatest impacts is on umbilical cord and body wall development.
  • recurring anomalies include body wall closure, amniotic band development and consequences, and umbilical artery development and anomalies.
  • the amniotic cavity develops above the bilaminar embryonic disc (stage 5, seven to eight days of development) and is lined by amniocytes. The embryo continues to grow in size in relation to the connecting stalk. The amniotic cavity enlarges and encloses the embryo. In the ninth week, the amniotic membrane attaches itself to the chorion and the extraembryonic cavity disappears.
  • amniotic membrane can be damaged at any time during embryogenesis. Although damage to the amnion resulting in amniotic bands is commonly associated with LBWC, there are cases of LBWC without amniotic bands and there are cases with amniotic bands with or without exencephaly with no body wall defects. In addition, there are reports of POC with amniotic bands.
  • NAD abnormalities can cause miscarriage either through genetic disease disclosed herein, through gene/environment interactions, or through NAD + perturbances alone. NAD abnormalities can cause isolated or combined malformations that do not meet the criteria provided for the recurrent constellations of embryonic malformations provided herein.
  • anomalies include caudal regression, body wall defects including gastroschisis and omphalocele, amniotic bands, bowel atresias, cleft lip, cleft palate, club foot, diaphragmatic hernia, cryptorchidism, gastrointestinal defects, heart defects, hip dysplasia, holoprosencephaly, hypospadias, limb malformations, lung agenesis/dysgenesis, microphthalmia/anophthalmia, microtia, neural tube defects, renal agenesis/dysgenesis, skeletal dysplasia, vertebral anomalies, discordant twin anomalies, Fanconi anemia, Potter sequence, arthrogryposis, Tru anomaly, and Prune belly syndrome.
  • the gestational age at which the NAD + perturbance occurs helps determine which embryologic malformations will occur.
  • the vertebral column and gut development starts development, during weeks 6 and 7 development of the renal and collecting systems, limb and digital rays, facial development, cardiac development and external genitalia development occur, during weeks 7-8 mandible and auricular hillocks form, ad during week 9 formation of internal genital organs including the uterus and upper vagina occur.
  • omphalocele can occur during weeks 6-10.
  • the endoderm lining the primary yolk sac is surrounded by mesenchyme, a portion of which differentiates into the vitelline arteries and veins.
  • the umbilical cord is thus formed by the fusion of yolk sac derivatives and connecting stalk mesenchyme.
  • the first organ to form from the primary yolk sac in humans is the allantois.
  • the allantoic arteries do not develop with the connecting stalk. The insult most likely occurs prior to 23 days gestation.
  • Single umbilical artery either due to primary agenesis or atrophy of an existing vessel.
  • vitelline artery disruption blood flow within the umbilical cord segment of the right vitelline artery may be interrupted. This can result in a hole in the abdominal wall and herniation of intestine (gastroschisis).
  • clinicians should suspect a possible NAD + deficiency disorder when identifying a newborn with vertebral, cardiac, renal, limb, neural tube, central nervous system including holoprosencephaly, ocular or auricle anomalies, gastrointestinal or pulmonary malformations.
  • NAD + perturbance can cause other disease not known to have a genetic etiology including cancer, immune disease including infectious disease sequelae and autoimmune disease, diseases of metabolic/endocrine origin including diabetes and mitochondrial disorders, nutritional diseases including atherosclerosis, hypertension, and obesity, neuropsychiatric disorders including neurodegenerative conditions and autism, and diseases of aging and death.
  • Disease and disorder that may be treated by the methods of the present disclosure includes:
  • SUBSTITUTE SHEET ( RULE 26) • vertebral defects, in particular vertebral defects predominantly affecting the thoracolumbar spine;
  • cardiac defects such as patent ductus arteriosus (POA), atrial septal defect (ASO) and hypoplastic left heart (HLH);
  • POA patent ductus arteriosus
  • ASO atrial septal defect
  • HHL hypoplastic left heart
  • limb abnormalities such as talipes (club foot);
  • laryngeal web for example with persistent laryngeal tracheomalacia
  • vocal cord palsy such as iatrogenic vocal cord palsy
  • restrictive lung disease for example restrictive lung disease due to spondylocostal defects
  • malformations caused by or associated with defects in NAD synthesis include the following:
  • Sacral agenesis including sacral agenesis with spinal lipoma
  • Sacral tethered cord including sacral tethered cord with a terminal lipoma and/or spinal dysraphism
  • the malformation may be a neurocognitive deficit.
  • the neurocognitive deficit may be any deficit in a subject’s perception, memory, association and recall in the thought process and behavior.
  • the neurocognitive deficit may be a developmental delay, an intellectual disability, a delay in speech development or a delay in behavioral development.
  • the malformations may occur in isolation or in any combination. In some embodiments the malformations occur in combination. In one or more embodiments the malformation may be a cardiac defect, vertebral defect, limb defect, or any combination of one or more thereof. In some embodiments the same defect in AD synthesis will cause different malformations or combination of malformations in different subjects.
  • malformation is used to refer to the consequence of a physical or morphological malformation or neurocognitive deficit such as those set out above.
  • a malformation may be miscarriage for example a pregnant female may be gestating an embryo with a malformation.
  • the epigenetic modification refers to stably-heritable phenotype(s) resulting from chromosomal alterations without alterations to the DNA sequence.
  • Epigenetic modifications can, in some instances lead to a disease or disorder such as cancer. Berger, S L, et al., Genes Dev. 23:781-783 (2009).
  • Epigenetic modifications typically involve changes in the chromatin structure that can result in overexpression and/or repression of genes that control cellular processes such as differentiation, proliferation, and/or apoptosis.
  • Such modifications can involve, e.g., DNA methylation and histone acetylation. See, e.g., Gnyska, A., et al., Anticancer Res. 33:2989- 2996 (2013).
  • epigenetic modifications lead to treatment or prevention of a disease or disorder such as birth defects.
  • the composition is an epigenetic modulating agent, e.g., a therapeutic agent, which can affect, e.g., block, reduce, reverse, or alleviate, a diseasecausing epigenetic modification, thereby treating a disease or disorder, e.g., malformation.
  • the subject that are being treated does not have a genetic basis for a disease or disorder. In certain embodiments, the subject that are being treated have a genetic basis for the disease or disorder. Whether the subject has a genetic basis or not, the genetic basis may be in genes encoding any one of the enzymes involved in NAD synthesis, for example via the de novo NAD synthesis pathway, Preiss-Handler pathway or the NAD salvage pathway may lead to defects in the pathway.
  • the gene may be selected from the group consisting of any gene including but not limited to aminoadipate, aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1, ADP-ribosyltransferase 1, ADP- ribosyltransferase 2, ADP-ribosyltransferase 3, ADPribosyltransferase 4, ADP- ribosyltransferase 5, bone marrow stromal cell antigen 1, catalase, CD38, cytochrome B5 reductase 3, cytochrome p450 family 2 subfamily D member 6, cytochrome p450 family 8 subfamily 8 member 1, ectonucleotide pyrophosphatase/phosphodiesterase 1, ectonucleotide pyrophosphatas
  • SUBSTITUTE SHEET ( RULE 26) family 3 member 2, solute carrier family 5 member 8, solute carrier family 6 member 19, solute carrier family 7 member 5, solute carrier family 7 member 8, solute carrier family 16 member 10, solute carrier family 22 member 13, solute carrier family 36 member 4, solute carrier family 52 member 1, solute carrier family 52 member 2, solute carrier family 52 member 3, tryptophan 2, 3 -dioxygenase, tankyrase, tankyrase 2, and xanthine dehydrogenase.
  • a gene encoding a tryptophan transporter TDO2 (tryptophan 2,3-dioxygenase), IDO 1,2 (Indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB (Kynurenine formamidase), KMO (kynurenine 3 -monooxygenase), KYNU (kynureninase), HAAO (3- hydroxyanthranilate 3,4-dioxygenase), ACSMD (aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate phosphoribosyltransferase), NAPRT, NADSYN1, NMAT1, 2, or3 (nicotinamide nucleotide adenylyltransferase 1, 2, or 3), NADSYN1 (NAD synthetase 1), nicotinamide phosphoribosyltrans
  • NMRK1 ,2 Natural Organotase 1 or 2
  • NAMPT nicotinamide Phosphoribosyltransferase
  • PNP Purine Nucleoside Phosphorylase
  • PARP1-6 Poly (ADP-Ribose) Polymerase 1-6
  • ART 1-4 SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7)
  • CD38 cyclic ADP ribose hydrolase
  • BST1 ADP -ribosyl cyclase 2
  • TRPT-1 tRNA 2-phosphotransferase 1
  • the gene is HAAO, KYNU, KMO or NADSYN 1.
  • the type of mutations that lead to defects in a NAD synthesis pathway include a point mutation such as a missense mutation, nonsense mutation or a synonymous mutation, a duplication, a deletion, an insertion or a splice mutation.
  • the disease or disorder may have a genetic basis where the genetic basis is a mutation.
  • the mutation may be homozygous or heterozygous.
  • the malformation may not be as severe as the corresponding homozygous mutation.
  • a subject with a heterozygous mutation may exhibit fewer malformations or a different combination of malformations than a subject with the corresponding heterozygous malformation.
  • a subject with a heterozygous mutation may exhibit only a single malformation. Subjects with a heterozygous mutation may exhibit reduced severity of a malformation compared to a subject with a corresponding homozygous mutation.
  • a genetic basis whether they are present or absent in a subject that may be treated by the method of the present disclosure can be detected by any means known in the art.
  • fluorescence in situ hybridization FISH
  • comparative genomic hybridization CGH
  • PCR amplification of a gene or portion thereof FISH
  • SUBSTITUTE SHEET (RULE 26) sequencing of the amplification product; sequencing such as whole-genome sequencing (WGS) or whole exome sequencing (WES), use of microarrays, multiplex ligation-dependent probe amplification (MLP A), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis or restriction fragment length polymorphism (RELP).
  • WGS whole-genome sequencing
  • WES whole exome sequencing
  • MLP A multiplex ligation-dependent probe amplification
  • SSCP single strand conformational polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • RELP restriction fragment length polymorphism
  • mutations are detected by exome or genome sequencing.
  • Nucleic acid for mutation detection can be obtained from samples of blood, skin, amniotic fluid, chorionic villi.
  • concentrations of metabolites produced by the enzymes of a pathway may be determined by measured either quantitatively or qualitatively.
  • the metabolites may be selected from the group comprising tryptophan, N-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3- hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA), xanthurenic acid, quinolinic acid (also known as quinolinate), 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide (NaAD)(or deamido-NAD), nicotinamide adenine dinucleotide (NAD), nicotinamide, N-methylnicotinamide (MNA), NNl-methyl-4-pyridone-3 -carboxamide (4-Py), N-methyl-2-pyridone-5-carboxamide (2-Py), nicotinamide monon
  • the metabolite may be anthranilate, kynurenate, xanthurenate or any combination thereof.
  • NAD and metabolites can be detected, quantified or both using mass spectrometry, typically in conjunction with liquid chromatography.
  • mass spectrometry typically in conjunction with liquid chromatography.
  • 3 -hydroxykynurenine and 3-hydroxyanthranilic acid can be detected and quantified by liquid chromatography-tandem mass spectrometry (LCMS/MS).
  • LCMS/MS liquid chromatography-tandem mass spectrometry
  • a serum sample is deproteinated with trichloroacetic acid before neutralization. The sample is then applied to a Cl 8 column and metabolites eluted using as gradient of 0-5 % mobile phase (e.g. acetic acid in acetonitrile).
  • metabolite's are then applied to a mass spectrometer for example a triple quadrupole mass spectrometer to detect 3HK and 3HAA by multiple reaction monitoring (MRM) in positive ion mode.
  • MRM multiple reaction monitoring
  • the metabolite's can be quantified against commercial standards for example those available from Sigma Aldrich (USA).
  • the measured concentration of NAD or a metabolite of the pathway can be compared to the concentration of NAD or a metabolite in a normal individual.
  • NAD or metabolites can be measured in samples of blood, serum, plasma, saliva, urine, tear, lymph fluid, cerebrospinal 30
  • SUBSTITUTE SHEET (RULE 26) fluid, mucosal secretion, peritoneal fluid, ascitic fluid, fecal matter, amniotic fluid, chorionic villus and body exudate.
  • defects in NAD synthesis of a subject in utero can be detected by measuring the concentrations of metabolites in a maternal sample.
  • a sample of blood, serum, plasma, saliva, or urine may be obtained from a pregnant female for analysis.
  • NAD or metabolites of NAD synthesis or nicotinamide degradation are measured in a blood or urine sample.
  • a defect in NAD synthesis is detected, for example by measuring metabolite levels or detecting a gene mutation, in a subject that is considering becoming pregnant. In this way the methods described herein can be used as pre-pregnancy testing for a defect in NAD synthesis.
  • the risk of a subject's offspring having a congenital malformation caused by a defect in NAD synthesis can be determined by assessing whether one or more of the offspring's biological parents have a defect in NAD synthesis.
  • a sample from one of the biological parents can be tested for a mutation in a gene encoding an enzyme involved in NAD synthesis. If a mutation is found, then this is indicative of a predisposition to a congenital malformation in the subject's offspring.
  • a sample from one of the biological parents can be tested the concentration of NAD, a metabolite of NAD synthesis or degradation, or a stable product from a metabolite in the sample. That concentration can be compared to the concentration of NAD, the metabolite or the stable product to a predetermined concentration of NAD, the metabolite or the stable product in a normal subject (i.e. one without a defect in NAD synthesis). If the concentration of NAD, the metabolite or the stable product is lower than that of a normal subject then that is indicative of a predisposition to a congenital malformation in the subject's offspring.
  • the risk of a subject's offspring having a congenital malformation caused by a defect in NAD synthesis can be determined before conception of the offspring, for example by detecting defects in NAD synthesis using any of the methods disclosed herein.
  • SUBSTITUTE SHEET (RULE 26) malformation caused by a defects in NAD synthesis is indicated to reduce the risk that the offspring will develop a malformation or to reduce the severity of a symptom associated with a malformation in the subject whether that malformation is apparent or not.
  • compounds useful for the treatment of disease or disorder are compounds, or salts or derivatives thereof, of vitamin B6, trimethylglycine, folic acid, apigenin or a combination thereof.
  • additional compounds useful for the treatment of disease or disorder are compounds, or salts or derivatives thereof, that are intermediates in the pathway downstream of the defect or the end product of the pathway, NAD or substrates and metabolites of the salvage pathway of NAD synthesis.
  • Additional compounds useful for the treatment of a malformation include the following: tryptophan, N-formylkynurenine, kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, 2-amino-3-carboxymuconate-6-semialdehyde, quinolinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide (or deamido-NAD), nicotinamide, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, niacin (nicotinic acid), vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
  • Useful compounds for the treatment disclosed herein include tryptophan, N- formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA), xanthurenic acid, quinolinic acid (also known as quinolinate), 2-aminomuconate, 2-oxoadipate, glutaryl-CoA, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide (NaAD)(or deamido-NAD), nicotinamide adenine dinucleotide (NAD), nicotinamide, N-methylnicotinamide (MNA), -NNl-methyl-4-pyridone- 3-carboxamide (4-Py), N-methyl-2-pyridone-5 -carboxamide (2-Py), and nicotinamide mononucleot
  • compounds useful for the treatment disclosed herein include 3- hydroxyanthranilic acid, 2-amino-3-carboxymuconate-6-semialdehyde; quinolinic acid, niacin, nicotinamide adenine dinucleotide, nicotinamide and nicotinamide mononucleotide, 2-amino-3- carboxymuconic semialdehyde; quinolinic acid, niacin, nicotinamide adenine dinucleotide, nicotinamide and nicotinamide mononucleotide.
  • the malformation may be treated by tryptophan, 3- hydroxyanthranilic acid, 2-amino-3-carboxymuconic semialdehyde; quinolinic acid, niacin, nicotinamide adenine dinucleotide, nicotinamide, nicotinamide mononucleotide, derivatives thereof, salts thereof, and any combination thereof.
  • compositions comprising the compounds disclosed herein for administration to a patient, preferably a human patient.
  • the pharmaceutical composition comprises suitable formulations of carriers, stabilizers and/or excipients.
  • the pharmaceutical composition comprises a composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or by direct injection into tissue.
  • the composition is administered to a patient via infusion or injection.
  • Administration of the suitable compositions may be through different routes, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present disclosure provides for an uninterrupted administration of the suitable composition.
  • uninterrupted, i.e. continuous administration may be realized by a small pump system worn by the patient for metering the influx of therapeutic agent into the body of the patient.
  • the continuous administration may be transdermal by way of a patch worn on the skin and replaced at intervals.
  • a patch worn on the skin worn on the skin and replaced at intervals.
  • patch systems for drug delivery suitable for this purpose. It is of note that transdermal administration is especially amenable to uninterrupted administration, as exchange of a first exhausted patch can advantageously be accomplished simultaneously with the placement of a new, second patch, for example on the surface of the skin immediately adjacent to the first exhausted patch and immediately prior to removal of the first exhausted patch.
  • compositions may further comprise a pharmaceutically acceptable carrier.
  • suitable pharmaceutical carriers are well known in the art and include solutions, e.g. phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, liposomes, etc.
  • Compositions comprising such carriers can be formulated by well-known conventional methods.
  • Formulations can comprise carbohydrates, buffer solutions, amino acids and/or surfactants.
  • Carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol.
  • pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption 33
  • SUBSTITUTE SHEET (RULE 26) delaying agents, compatible with pharmaceutical administration.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counter-ions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, asparagine, 2-phenylalanine, and threonine; sugars or sugar alcohols, such as trehalose, sucrose, octasulfate, sorbitol or xylitol stachyose, mannose, sorbose, xylose, ribos
  • Such formulations may be used for continuous administrations which may be intravenuous or subcutaneous with and/or without pump systems.
  • Amino acids may be charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and/or histidine.
  • Surfactants may be detergents, preferably with a molecular weight of >1.2 KD and/or a poly ether, in one embodiment, with a molecular weight of >3 KD.
  • Non-limiting examples for detergents are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85.
  • Non-limiting examples for polyethers are PEG 3000, PEG 3350, PEG 4000 or PEG 5000.
  • Buffer systems used in the present disclosure can have a preferred pH of 5-9 and may comprise citrate, succinate, phosphate, histidine and acetate.
  • compositions of the present disclosure can be administered to the subject at a suitable dose which can be determined e.g., by dose escalating studies by administration of increasing doses of the composition described herein exhibiting cross-species specificity described herein to non-chimpanzee primates, for instance macaques.
  • the composition described herein can be advantageously used in identical form in preclinical testing in non-chimpanzee primates and as drug in humans.
  • the composition can also be administered in combination with additional other proteinaceous and non-proteinaceous drugs. These drugs may be administered simultaneously with the composition described herein as defined herein or separately before or after administration of said composition in timely defined intervals and doses.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one 34
  • SUBSTITUTE SHEET (RULE 26) patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • the composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.
  • the composition further biologically active agents, depending on the intended use of the composition.
  • Such agents might be drugs acting on the gastrointestinal system, drugs acting as cytostatica, drugs preventing hyperuricemia, drugs inhibiting immunoreactions (e.g., corticosteroids), drugs modulating the inflammatory response, drugs acting on the circulatory system and/or agents such as cytokines known in the art.
  • the composition disclosed herein is applied in an additional cotherapy, i.e., in combination with another medicament.
  • a method to regulate gene expression in mammalian cells via an epigenetic control mechanism a compound described herein can be prepared as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to a derivative of the disclosed compounds where the parent compound is modified by making an acid or base salt.
  • Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, 35
  • SUBSTITUTE SHEET (RULE 26) fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
  • conventional nontoxic salts include those derived from bases, such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like.
  • salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
  • a compound described herein can be formulated in combination with one or more other compounds including another compound described herein.
  • a compound described herein can be formulated as a pharmaceutical composition or as a dietary supplement and administered to a mammalian host, such as a human patient or non-human animal, in a variety of forms adapted to the chosen route of administration.
  • a composition or supplement sometimes includes a diluent, buffer, preservative and the like.
  • Various sustained release systems for drugs have also been devised and can be applied to the compounds described herein.
  • compositions and preparations sometimes contain at least 0.1 % of active compound.
  • the percentage of the compositions and preparations may be varied and sometimes are about 2% to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically usefill compositions is such that an effective dosage level will be obtained.
  • SUBSTITUTE SHEET (RULE 26) Tablets, troches, pills and capsules, may also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • binders such as gum tragacanth, acacia, com starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as com starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, fructos
  • the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • a compound may be administered by infusion or injection (for example subcutaneous, intramuscular or intravenous injection).
  • Solutions of a compound or a pharmaceutically acceptable salt thereof can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof. These preparations sometimes contain a preservative to prevent the growth of microorganisms.
  • a dosage form can include a sterile aqueous solution or dispersion or sterile powder comprising an active ingredient, which are adapted for the preparation of sterile solutions or dispersions.
  • the ultimate dosage form can be a sterile fluid which is stable under the conditions of manufacture and storage.
  • a liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), nontoxic glyceryl esters, and suitable mixtures thereof.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • An isotonic agent for example, a sugar, buffer or sodium chloride is included in some embodiments.
  • Prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile solutions often are
  • SUBSTITUTE SHEET (RULE 26) prepared by incorporating an active compound in a required amount in an appropriate solvent, sometimes with one or more of the other ingredients enumerated above, followed by filter sterilization, bi the case of sterile powders for the preparation of sterile injectable solutions, preparation methods sometimes utilized are vacuum drying and the freeze-drying techniques, which yield a powder of an active ingredient in addition to any additional desired ingredient present in the previously sterile-filtered solutions.
  • niacin niacin, nicotinic acid and nicotinamide are approved for therapeutic use in humans for treating niacin deficiency.
  • the recommend daily allowance (RD A) for niacin is 16 milligrams per day for men and 14 milligrams per day for women.
  • Conventional medical advice indicates that excess niacin or nicotinic acid can be harmful and may adversely affect liver function.
  • the compound may be administered as a controlled release tablet.
  • a controlled release tablet may comprise about 5-30% high viscosity hydroxypropyl methyl cellulose, about 2-15% of a water-soluble pharmaceutical binder, about 2-20% of a hydrophobic component such as a waxy material, e.g., a fatty acid, and about 30-90% niacin.
  • a suitable controlled release tablet comprises: (a) about 5-20 percent by weight hydroxypropyl methylcellulose having a viscosity of about 10,000 CPS or greater, a substitution rate for the methoxyl group of about 7-30% and a substitution rate for the hydroxypropoxyl group of about 7-20%; (b) about 2-8 percent hydroxypropyl methylcellulose having a viscosity of less than about 100, CPS methyl cellulose, or polyvinyl pyrollidone; (c) about 5-15 percent by weight hydrogenated vegetable oil or stearic acid; and (d) about 30-90% of active ingredient.
  • the controlled released tablets can also be coated so as to further prolong the release of the active ingredient into the gastrointestinal tract, or to prevent its release into the stomach, in order to prevent or attenuate the gastrointestinal side effects which can accompany an active ingredient administration.
  • coatings predominantly comprising a polymeric material having a high degree of swelling on contact with water or other aqueous liquids can be used to further prolong the release of the compound from the tablet.
  • suitable polymers include cross-linked sodium carboxymethylcellulose, cross-linked hydroxypropylcellulose, hydroxymethylpropylcellulose, e.g., Methocel®, carboxymethylamide, potassium methylacrylate divinylbenzene copolymer, polymethyl methacrylate, cross-linked polyvinylpyrrolidine, high molecular weight polyvinylalcohol, and the like.
  • SUBSTITUTE SHEET (RULE 26) Hydroxypropylmethyl cellulose is available in a variety of molecular weights and viscosity grades for example from Dow Chemical Co. under the Methocel® brand name. These polymers may be dissolved in suitable volatile solvents, along with dyes, lubricants, and flavorings, and coated onto the controlled release tablets, e.g., in amounts equal to 0.1- 5% of the total tablet weight, by methods well known to the art. For example, see Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Publishing Co., Easton, Pa. (16th ed. 1980).
  • the controlled release tablets can be formulated to contain about 5mg, lOmg, 15mg, 20, mg, 30mg, 40mg, 50mg, 75mg, lOOmg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg or about 1,000 mg, l,250mg, l,500mg, l,750mg, 2,000mg, 2,250mg, 2,500mg
  • the controlled release tablets will release about 10-35 wt-% of the total niacin within about 2 hours in an in vitro dissolution test, and about 40-70 wt-% of the total niacin in eight hours.
  • the concentration of a compound described herein in a liquid composition is about 0.1 wt%, 0.25wt %, 0.5wt %, 0.75wt %, Iwt %, 2wt %, 3wt %, 4wt %, 5wt %, 6wt %, ?wt%, 8wt %, 9wt %, lOwt %, 1 Iwt %, 12wt %, 13wt %, 14wt %, 15wt %, 16wt %, 17wt %, 18wt %, 19wt %, 20wt %, 2 Iwt %, 22wt %, 23wt %, 24wt %, 25wt %, 26wt %, 27wt %, 28wt %, 29wt %, 30wt %, 3 Iwt %, 32wt %, 33wt %, 34wt %, 3
  • Useful dosages, whether by controlled release or not, of compounds can be determined by their in vivo activity in animal models, such as the mouse models described herein. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
  • compositions and formulation disclosed herein are useful for the treatment or prevention of disease or disorder in a subject that may or may not have a genetic basis.
  • a subject that may or may not have a genetic basis.
  • the disclosed composition can be useful as an epigenetic modulating agent.
  • Administration of the epigenetic modulating agent can be useful for the treatment or prevention of a disease or disorder.
  • the present disclosure teaches a method of treatment for a disorder or disease.
  • the method comprises administration of a therapeutically effective amount of the disclosed composition to a subject.
  • Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt or slow the progression of an existing diagnosed pathologic condition or disorder.
  • Terms such as “prevent,” “prevention,” “avoid,” “deterrence” and the like refer to prophylactic or preventative measures that prevent the development of an undiagnosed targeted pathologic condition or disorder.
  • “those in need of treatment” can include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • the term "therapeutically effective amount” refers to an amount of a drug effective to "treat" a disease or disorder in a subject or mammal.
  • the therapeutically effective amount of the drug can reduce the number of cancer cells; retard or stop cancer cell division, reduce or retard an increase in tumor size; inhibit, e.g., suppress, retard, prevent, stop, delay, or reverse cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit, e.g., suppress, retard, prevent, shrink, stop, delay, or reverse tumor metastasis; inhibit, e.g., suppress, retard, prevent, stop, delay, or reverse tumor growth; relieve to some extent one or more of the symptoms associated with the cancer, reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • the drug prevents growth and/or kills existing cancer cells, it can be referred to as cytostatic and/or cytotoxic.
  • subject is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears etc.
  • the genetic basis may be based on genes encoding enzymes active in NAD synthesis such as the genes which include genes encoding a tryptophan transporter, TDO2 (tryptophan 2,3 dioxygenase), IDO 1,2 (Indoleamine 2,3-dioxygenase), AFMID (arylformamidase), KYNB
  • SUBSTITUTE SHEET (RULE 26) semialdehyde decarboxylase), QPRT (quinolinate phosphoribosyltransferase), NAPRT, NADSYN1, NMAT1, 2, or3 (nicotinamide nucleotide adenylyltransferase 1, 2, or 3), NADSYN1 (NAD synthetase 1), nicotinamide phosphoribosyltransferase.
  • NMRK1 ,2 Natural Organotase 1 or 2
  • NAMPT nicotinamide Phosphoribosyltransferase
  • PNP Purine Nucleoside Phosphorylase
  • PARP1-6 Poly (ADP-Ribose) Polymerase 1-6
  • ART 1-4 SIRT1-7 (Sirtuin 1-7, also known as NAD-dependent deacetylase sirtuin 1-7)
  • CD38 cyclic ADP ribose hydrolase
  • BST1 ADP-ribosyl cyclase 2
  • TRPT-1 tRNA 2- phosphotransferase 1
  • Malformation includes for example cardiac malformations, vertebral, renal dysplasia, limb anomalies, and tracheoesophageal fistula, for example tracheoesophageal fistula with esophageal atresia (TE).
  • TE esophageal atresia
  • the malformations occur in the absence of a known genetic cause.
  • therapeutically effective amount refers to an amount of a compound provided herein, or an amount of a combination of compounds provided herein, to treat or prevent a malformation, or to treat a symptom of the malformation, in a subject.
  • the terms 'subject' and 'patient' are used interchangeably to refers to an individual who will receive or who has received treatment (e.g., administration of a compound described herein) according to a method described herein, or who has a malformation caused by a defect in NAD synthesis, or who is at risk of having a malformation caused by a defect in NAD synthesis.
  • a compound described herein may be in a therapeutically effective amount in a formulation or medicament, which is an amount that can lead to a physiological effect (e.g., inhibiting the malformation), or lead to ameliorating, alleviating, lessening, relieving, diminishing or removing symptoms of the malformation, for example.
  • Examples of a subject include mammal, human, ape, monkey, ungulate (e.g., equine, bovine, caprine, ovine, porcine, buffalo, camel), canine, feline, rodent (e.g. mouse, rat).
  • a subject may be male or female.
  • the compound can be administered to a subject in any age group, including, for example, neonate, infant, juvenile, pediatric, adolescent and adult. In some embodiments, for example where the subject is in utero the compound can be administered to the pregnant mother of the subject. In certain embodiments, the subject does not have any genetic basis for the malformation. In certain embodiments, the subject has one or more genetic basis for the malformation.
  • a compound described herein can be administered to any subject to reduce the risk of a malformation caused by a defect in NAD synthesis.
  • a compound described herein can be administered to any subject to increase the amount of NAD in the subject to reduce the risk of a malformation caused by a defect in a NAD synthesis pathway, or to prevent such a malformation becoming apparent.
  • a compound described herein can be administered to a subject who is, or is contemplating becoming pregnant in order to reduce the risk of a malformation.
  • a compound described herein can be administered to a subject who is, or is contemplating becoming pregnant in order to reduce the risk of miscarriage, for example miscarriage caused by a malformation.
  • the methods involve administering a therapeutically effective amount of a compound to the subject with the malformation.
  • the compound may be administered by any route.
  • the compound may be administered by oral, parenteral, intravenous, intramuscular, topical, subcutaneous or intradermal routes.
  • the route of administration will vary depending on the nature of the compound and the way it is formulated. A skilled person will be able to determine the appropriate route of administration.
  • the subject is in utero and administration of the compound to the subject can be achieved by administering the compound to the mother of the subject.
  • the compound is administered by injection, for example a subcutaneous, intramuscular or intravenous injection. In other embodiments, the compound is administered orally.
  • the amount of the compound, or a salt or derivative thereof, required for treatment varies not only with a particular salt selected but also with the route of administration, the nature of the malformation being treated and the age and condition of the subject and will be ultimately at the discretion of the physician or clinician.
  • a suitable dose is in the range of from about 0.1 mg to about 200 mg/kg, e.g., from about 50 to about 125 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, and often is in the range of 6 to 90 mg/kg/day, or about 15 to 60 mg/kg/day.
  • a suitable dose in general, sometimes is in the range of from about 1 to 150 mg/kg body weight of the recipient per day, e.g. from about 10 to about 130 mg/kg, from about 40 to about 120 mg/kg, from about 50 to about 100 mg/kg, from about 60 to 90 mg/kg, from about 65 to 85 mg/kg, or, for example, about 80 mg/kg/day.
  • the compound may be administered at a dose of at least about 50mg/kg body weight of the subject.
  • the compound may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 42
  • SUBSTITUTE SHEET (RULE 26) 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45,46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 5758, 59,60, 61,62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
  • a compound may be conveniently administered in unit dosage form.
  • a desired amount of compound may be administered in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four, five, six, seven, eight, nine, ten or more than ten doses per day.
  • a suitable dose is significantly more than the RD A for that compound.
  • the dose may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 170 or more times the RDA for that compound.
  • a pharmaceutical composition comprising 0.1 -5mg of vitamin B6, 500mg-9,000mg of trimethylglycine, 200mg-6000mg folic acid, lmg/kg-40mg/kg apigenin; and a pharmaceutically acceptable carrier.
  • composition of any preceding item further comprising NAD or an intermediate of NAD synthesis.
  • a method of treating or preventing biological disorders in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of any preceding items.
  • SUBSTITUTE SHEET (RULE 26) The method of any preceding items wherein the miscarriage is caused by at least one chromosomal aberration of the subject or the unborn offspring, wherein the chromosomal aberrations is trisomy, triploidy, monosomy or meiotic nondisjunction in the unborn offspring.
  • the method of any preceding items further comprising administration of NAD or an intermediate of NAD synthesis.
  • any preceding items further comprising increasing the NAD level in the unborn offspring or the subject, wherein the unborn offspring or the subject does not have a genetic basis or does not have a genetic influence through DNA variants.
  • the method of any preceding items further comprising increasing the NAD level in the unborn offspring or the subject, wherein the unborn offspring or the subject has a genetic basis.
  • the method of any preceding items wherein the genetic basis is HAAO, KYNU, KMO, or NADSYN1 mutation.
  • NAD level is increased by further administration of tryptophan, niacin, nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a combination thereof.
  • RCEM is POC, LBWC, OEIS, VACTERL, OAVS, MURCS, URSM or sirenomelia.
  • composition is administered during umbilical cord and embryonic wall development, cranial to caudal ventral body wall closure, neural tube closure, vertebral column development, gut development; limb cartilages and digital ray development, frontal facial development, conotruncal development and ventricular septa development; abdominal wall development; or uterus and upper vagina development.
  • SUBSTITUTE SHEET (RULE 26) The method of any preceding items comprising increasing NAD or an intermediate of NAD synthesis prior to or during gestational week 2-3, 3-6, 6-7, 7-8, 9 or a combination thereof.
  • the method of any preceding items wherein the subject has inflammation, diabetes mellitus, obesity, aging, hypoxia or a combination thereof.
  • the method of any preceding items further comprising administration of a compound that reverses NAD deficiency or disturbance.
  • a method to regulate one or more biological processes comprising administration of the pharmaceutical composition of any preceding items wherein the biological process is DNA replication, DNA repair, aneuploidy, methylation, cancer or a combination thereof.
  • a method of detecting a risk for a biological disorder in a subject or a congenital malformation in an unborn offspring of a subject comprising detecting at least one of: i) a dietary deficiency of one or more of vitamin B6, trimethylglycine and folic acid; ii) inadequate absorption of one or more of vitamin B6, trimethylglycine and folic acid; and iii) inadequate level of NAD, wherein the presence of any one of i), ii) or iii), is associated with an increased risk.
  • the method of any preceding items further detects optionally: (i) a dietary deficiency of one or more of tryptophan, nicotinamide, and nicotinamide riboside or (ii) inadequate absorption of one or more of tryptophan, nicotinamide, and nicotinamide riboside.
  • SUBSTITUTE SHEET (RULE 26) The method of any preceding items wherein the subject is the mother of an unborn offspring, the unborn offspring or a bom offspring.
  • nicotinamide mononucleotide NPN
  • nicotinic acid mononucleotide nicotinic acid adenine dinucleotide
  • deamido-NAD nicotinamide
  • nicotinamide mononucleotide nicotinamide adenine dinucleotide
  • niacin vitamin B3, a derivative thereof, a salt thereof, and any combination thereof.
  • recurrent congenital malformation is selected from the group consisting of a vertebral defect, sacral agenesis, spinal dysraphism, cardiac defect, a hypoplastic kidney, a solitary kidney, a shortened long bone; a limb abnormality, submucous cleft palate, bifid uvulua; laryngeal web, vocal cord palsy, anterior anus, restrictive lung disease, microcephaly, and any combination thereof.
  • the genetic basis is a mutation of a gene selected from the group consisting of aminoadipate aminotransferase, aminocarboxymuconate semialdehyde decarboxylase, acid phosphatase 5, arylformamidase, an alkaline phosphatase, aldehyde oxidase 1, ADP- ribosyltransferase 1, ADP-ribosyltransferase 2, ADP-ribosyltransferase 3, ADP- ribosyltransferase 4,ADP- ribosyltransferase 5, bone marrow stromal cell antigen 1, catalase, CD38, cytochrome 85 reductase 3, cytochrome p450 family 2 subfamily D member 6, cytochrome p450 family 8 subfamily B member 1, ectonucleotide pyrophosphatase/phosphodiesterase 1, ectonucleo
  • SUBSTITUTE SHEET (RULE 26) pyrophosphatase/phosphodiesterase 2, flavin adenine dinucleotide synthetase 1, glutamate-anunonia ligase, 3-hydroxyanthranilate 3,4-dioxygenase, indoleamine 2,3- dioxygenase 1, indoleamine 2,3 -dioxygenase 2, kynurenine 3- monooxygenase, kynurenine aminotransferase 1, kynurenine aminotransferase 3, kynureninase, leukosialin, NAD kinase, NAD kinase 2, NAD synthetase 1, nicotinamide phosphoribosyltransferase, nicotinate phosphoribosyltransferase, NAD(P)HX dehydratase, NAD(P)HX epimerase, nicotin
  • the genetic basis is a mutation of a gene wherein the gene is selected from the group consisting of a tryptophan transporter, TD02 (tryptophan 2, 3 -dioxygenase), IDO 1,2 (indoleamine 2,3 -dioxygenase), AFMID (arylformamidase), KYNB (kynurenine formamidase), KMO (kynurenine 3-
  • SUBSTITUTE SHEET (RULE 26) monooxygenase), KYNU (kynureninase), HAAO (3-hydroxyanthranilate 3,4- dioxygenase), ACSMD (aminocarboxymuconate semialdehyde decarboxylase), QPRT (quinolinate phosphoribosyltransferase), NAPRT, NADSYN 1, NMAT1, 2, or 3 (nicotinamide nucleotide adenylyltransferase 1, 2, or 3), NADSYN 1 (NAD synthetase 1), nicotinamide phosphoribosyltransferase, NMRK1 ,2 (Nicotinamide riboside kinase 1 or 2), NAMPT (nicotinamide phosphoribosyltransferase), PNP (purine nucleoside phosphorylase), PARP1-6 (poly(ADP-ribose) polyme
  • Salinas-Torres VM De La O-Espinoza EA, Salinas-Torres RA. Severe Intrauterine Amputations in One Dichorionic Twin With Pentalogy of Cantrell: Further Evidence and Consideration for Mechanical Teratogenesis. Pediatr Dev Pathol. 2017 Sep-0ct;20(5):440- 443. doi: 10.1177/1093526617689896. Epub 2017 Jan 25. PMID: 28812462.

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Abstract

L'invention concerne des méthodes de traitement et de prévention de troubles par modulation de NAD. L'invention concerne des compositions qui contiennent du NAD, des composants de la voie NAD et des modérateurs de la voie NAD. L'invention concerne également un outil de classification de troubles provoqués par la mauvaise régulation de NAD.
PCT/US2022/080912 2021-12-17 2022-12-05 Méthodes de traitement et de prévention de maladies et de troubles par modulation de nad WO2023114656A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20070116779A1 (en) * 2005-11-23 2007-05-24 Elizabeth Mazzio Comprehensive nutraceutical agent for treatment/ prevention of Parkinson's disease
WO2009074366A1 (fr) * 2007-12-10 2009-06-18 Henkel Ag & Co. Kgaa Produits pour le lavage des cheveux à action combinée détergente/soignante
US8367121B2 (en) * 2005-11-23 2013-02-05 Florida A & M University Nutraceutical agent for attenuating the neurodegenerative process associated with Parkinson's disease
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US20070116779A1 (en) * 2005-11-23 2007-05-24 Elizabeth Mazzio Comprehensive nutraceutical agent for treatment/ prevention of Parkinson's disease
US8367121B2 (en) * 2005-11-23 2013-02-05 Florida A & M University Nutraceutical agent for attenuating the neurodegenerative process associated with Parkinson's disease
US20200381230A1 (en) * 2007-06-14 2020-12-03 Quest Diagnostics Investments Incorporated Mass spectrometry method for measuring vitamin b6 in body fluid
WO2009074366A1 (fr) * 2007-12-10 2009-06-18 Henkel Ag & Co. Kgaa Produits pour le lavage des cheveux à action combinée détergente/soignante

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