U.S. patent application number 17/342694 was filed with the patent office on 2022-03-31 for crystal forms of beta-nicotinamide mononucleotide.
The applicant listed for this patent is Andrew Carr, Philippe Fernandes, David J. Livingston. Invention is credited to Andrew Carr, Philippe Fernandes, David J. Livingston.
Application Number | 20220098229 17/342694 |
Document ID | / |
Family ID | 1000006025974 |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220098229 |
Kind Code |
A1 |
Livingston; David J. ; et
al. |
March 31, 2022 |
CRYSTAL FORMS OF BETA-NICOTINAMIDE MONONUCLEOTIDE
Abstract
The invention relates to crystalline forms of a
.beta.-nicotinamide mononucleotide, methods of their preparation,
and related pharmaceutical preparations thereof. The invention also
relates to preparations suitable for nutraceutical, veterinary, and
agriculturally-relevant uses.
Inventors: |
Livingston; David J.;
(Worcester, MA) ; Carr; Andrew; (Hinxton, GB)
; Fernandes; Philippe; (Turnhout, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Livingston; David J.
Carr; Andrew
Fernandes; Philippe |
Worcester
Hinxton
Turnhout |
MA |
US
GB
BE |
|
|
Family ID: |
1000006025974 |
Appl. No.: |
17/342694 |
Filed: |
June 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16454457 |
Jun 27, 2019 |
11059847 |
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17342694 |
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15765325 |
Apr 2, 2018 |
10392415 |
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PCT/US2016/054776 |
Sep 30, 2016 |
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16454457 |
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62236657 |
Oct 2, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7052 20130101;
C07H 19/048 20130101; C07B 2200/13 20130101 |
International
Class: |
C07H 19/048 20060101
C07H019/048 |
Claims
1-28. (canceled)
29. A solid composition comprising a compound having the structure
of formula (I), ##STR00004## or a salt thereof, and further
comprising one or more solid orally-acceptable excipients or
carriers, wherein said composition is in a unit dosage form for
human administration of 0.01 to 3000 mg, and further wherein said
composition is formulated as a dietary supplement.
30. The solid composition of claim 29, wherein said composition is
pyrogen-free.
31. The solid composition of claim 29, wherein said composition is
non-pyrogenic.
32. The solid composition of claim 29, wherein said unit dosage
form is a pill.
33. The solid composition of claim 29, wherein said unit dosage
form is a tablet.
34. The solid composition of claim 29, wherein said unit dosage
form is a capsule.
35. The solid composition of claim 34, wherein said capsule is a
gelatin capsule.
36. The solid composition of claim 29, wherein said one or more
solid orally-acceptable excipients or carriers comprise
gelatin.
37. The solid composition of claim 29, wherein said one or more
solid orally-acceptable excipients or carriers comprise
hydroxypropylmethyl cellulose.
38. The solid composition of claim 29, wherein said one or more
solid orally-acceptable excipients or carriers comprise sodium
carboxymethyl cellulose.
39. A method of preparing a dietary supplement comprising combining
(i) a compound having the structure of formula (I), ##STR00005## or
a salt thereof, with (ii) one or more solid orally-acceptable
excipients or carriers, to form a solid composition, wherein said
composition is in a unit dosage form for human administration of
0.01 to 3000 mg, and further wherein said composition is formulated
as a dietary supplement.
40. The method of claim 39, wherein said composition is
pyrogen-free.
41. The method of claim 39, wherein said composition is
non-pyrogenic.
42. The method of claim 39, wherein said unit dosage form is a
pill.
43. The method of claim 39, wherein said unit dosage form is a
tablet.
44. The method of claim 39, wherein said unit dosage form is a
capsule.
45. The method of claim 39, wherein said one or more solid
orally-acceptable excipients or carriers comprise gelatin.
46. The method of claim 39, wherein said one or more solid
orally-acceptable excipients or carriers comprise
hydroxypropylmethyl cellulose.
47. The method of claim 39, wherein said one or more solid
orally-acceptable excipients or carriers comprise sodium
carboxymethyl cellulose.
48. A method of supplementing a diet comprising providing a
composition to a human for self-administration, wherein the
composition is a solid composition comprising a compound having the
structure of formula (I), ##STR00006## or a salt thereof, and
further comprising one or more solid orally-acceptable excipients
or carriers, wherein said composition is in a unit dosage form for
human administration of 0.01 to 3000 mg, and further wherein said
composition is formulated as a dietary supplement.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. No. 62/236,657, filed Oct. 2,
2015, which is hereby incorporated by reference herein in its
entirety.
BACKGROUND
[0002] .beta.-Nicotinamide mononucleotide (NMN) has recently gamed
attention for its use in the treatment, amelioration, mitigation,
slowing, arrest, prevention and/or reversal of age-associated
degenerative changes, such as age-related obesity, age-related
increases in blood lipid levels, age-related decreases in insulin
sensitivity, age-related decreases in memory function, and
age-related changes in eye function such as macular
degeneration.
[0003] Given the therapeutic benefits associated with this
compound, there is a need for improved compositions of NMN.
Further, there is a need for improved methods for preparing and
formulating .beta.-nicotinamide mononucleotide.
SUMMARY OF INVENTION
[0004] One aspect of the invention relates to a crystalline
compound having the structure of formula (I),
##STR00001##
[0005] Another aspect of the invention relates to methods for
preparing the crystalline compounds of formula (I).
[0006] In certain embodiments, the present invention provides a
pharmaceutical preparation suitable for use in a human patient,
comprising a crystalline compound of formula (I), and one or more
pharmaceutically acceptable excipients. In certain embodiments, the
pharmaceutical preparations may be for use in treating or
preventing a condition or disease as described herein. In certain
embodiments, the pharmaceutical preparations have a low enough
pyrogen activity to be suitable for intravenous use in a human
patient.
DETAILED DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows the XRPD patterns of .beta.-nicotinamide
mononucleotide (NMN) forms 1 and 2.
[0008] FIG. 2 shows the differential scanning calorimetry
thermogram of Form 1.
[0009] FIG. 3 shows the differential scanning calorimetry
thermogram of Form 2.
[0010] FIG. 4 shows .sup.1H NMR spectra of the amorphous NMN, NMN
Form 1, and NMN Form 2 after drying under vacuum. As shown in the
spectra, Form 1 is substantially anhydrous, and Form 2 has about
1.1-1.2 DMSO molecules per molecule NMN.
[0011] FIG. 5 shows a comparison of XRPD patterns of amorphous NMN,
amorphous NMN post-storage, and NMN Form 1.
[0012] FIG. 6 shows a dynamic vapor sorption isotherm for amorphous
NMN.
[0013] FIG. 7 shows a dynamic vapor sorption change in mass plot
for amorphous NMN. When a sample of amorphous NMN is exposed to
atmospheric humidity, the sample goes through phases. FIG. 7 shows
the mass change over time. The amorphous sample is hygroscopic
until it deliquesces and starts crystallizing. The weight loss
below 1000 min shows a crystallisation event. Once crystallised,
the material remains crystalline, retaining the same XRPD pattern
after a double cycle, but still shows the ability to pick up mass
reversibly (up to 9% w/w change).
[0014] FIG. 8 is an image showing single crystals of NMN Form 1,
observed under a polarized microscope.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In certain embodiments, the invention provides a crystalline
compound having the structure of formula (I),
##STR00002##
[0016] In certain embodiments, a crystalline compound of formula
(I) is not solvated (e.g., the crystal lattice does not comprise
molecules of a solvent). In certain embodiments, the crystalline
compound of formula (I) is anhydrous, or substantially anhydrous.
In certain alternative embodiments, a crystalline compound of
formula (I) is solvated. In certain such embodiments, the
crystalline compound of formula (I) is a dimethylsulfoxide (DMSO)
solvate.
[0017] Any crystalline compound described herein may be used in the
manufacture of a medicament for the treatment of any diseases or
conditions disclosed herein.
[0018] In certain embodiments, the compounds of the present
invention can assemble into more than one crystal formation. In an
exemplary embodiment, the crystalline compound having the structure
of formula (I) exists as "form I" and "form II", as described in
detail below. These different forms are understood as "polymorphs"
herein.
[0019] In certain embodiments, the polymorph of the crystalline
compound is characterized by powder X-ray diffraction (XRD).
.theta. represents the diffraction angle, measured in degrees. In
certain embodiments, the diffractometer used in XRD measures the
diffraction angle as two times the diffraction angle .theta.. Thus,
in certain embodiments, the diffraction patterns described herein
refer to X-ray intensity measured against angle 2.theta..
[0020] In certain embodiments, an anhydrous crystalline compound of
formula (I) has 2.theta. values 20.03; 20.14; 21.83; and 25.73. In
further embodiments, the anhydrous crystalline compound has
2.theta. values 20.03; 20.14; 21.03; 21.83; 23.08; 23.39; 25.73;
and 26.59. In yet further embodiments, the anhydrous crystalline
compound has 2.theta. values 7.70; 11.54; 12.64; 16.03; 18.99;
20.03; 20.14; 20.83; 21.03; 21.83; 23.08; 23.39; 25.48; 25.73;
26.59; and 29.78. In still yet further embodiments, the anhydrous
crystalline compound has 2.theta. values 7.70; 9.95; 11.54; 12.64;
16.03; 18.18; 18.99; 19.16; 19.44; 20.03; 20.14; 20.83; 21.03;
21.83; 22.44; 23.08; 23.39; 23.89; 24.08; 24.53; 24.68; 25.05;
25.48; 25.73; 26.08; 26.59; 27.33; 27.67; 29.78; and 29.92.
[0021] In certain embodiments, an anhydrous crystalline compound of
formula (I) has an XRD pattern substantially as shown in FIG. 1,
labeled Form 1.
[0022] In certain embodiments, a crystalline compound of formula
(I) is not solvated (e.g., the crystal lattice does not comprise
molecules of a solvent). In certain alternative embodiments, a
crystalline compound of formula (I) is solvated.
[0023] In certain embodiments, a crystalline DMSO solvate of the
compound of formula (I) has 2.theta. values 8.29; 17.39; 19.54;
22.78; and 22.98. In further embodiments, the crystalline DMSO
solvate has 2.theta. values 8.29; 17.39; 19.54; 19.74; 20.98;
21.58; 22.03; 22.78; 22.98; and 25.53. In yet further embodiments,
the crystalline DMSO solvate has 2.theta. values 8.29; 16.10;
17.39; 19.24; 19.54; 19.74; 20.33; 20.78; 20.98; 21.18; 21.58;
22.03; 22.78; 22.98; 25.53; 28.48; and 29.48. In further
embodiments, the crystalline DMSO solvate has 2.theta. values 8.29;
13.12; 15.79; 16.10; 16.69; 17.39; 19.03; 19.24; 19.54; 19.74;
20.33; 20.78; 20.98; 21.18; 21.58; 22.03; 22.78; 22.98; 23.95;
24.14; 24.48; 24.64; 25.14; 25.53; 25.87; 26.89; 27.18; 27.67;
28.02; 28.13; 28.48; 28.98; 29.34; 29.48; and 29.92.
[0024] The certain embodiments, a crystalline DMSO solvate of the
compound of formula (I) has an XRD pattern substantially as shown
in FIG. 1, labeled Form 2.
[0025] In certain embodiments, the crystalline DMSO solvate of the
compound of formula (I) contains about 1.0, about 1.1, or about 1.2
molecules of DMSO to one molecule of NMN.
[0026] In certain embodiments, the invention relates to a
pharmaceutical composition comprising a crystalline compound of
formula (I) and one or more pharmaceutically acceptable excipients.
In certain embodiments, the pharmaceutical composition is selected
from tablets, capsules, and suspensions.
[0027] The term "substantially pure" as used herein, refers to a
crystalline polymorph that is greater than 90% pure, meaning that
contains less than 10% of any other compound, including the
corresponding amorphous compound or an alternative polymorph of the
crystalline salt. Preferably, the crystalline polymorph is greater
than 95% pure, or even greater than 98% pure.
Methods of Making the Crystalline Forms of NMN
[0028] In certain embodiments, the invention relates to a method
for the preparation of a crystalline compound having the structure
of formula (I), comprising a) providing a mixture of a compound of
formula (I) in a solvent; and b) crystallizing the compound of
formula (I) from the mixture comprising the compound of formula
(I).
[0029] In certain embodiments, the mixture comprising the compound
of formula (I) is a solution. In certain embodiments, the mixture
is a slurry or a suspension.
[0030] In certain embodiments, the crystalline compound made by the
methods of the invention is anhydrous.
[0031] In certain embodiments, the crystalline compound made by the
methods of the invention is a solvate, e.g., a DMSO solvate.
[0032] In certain embodiments, the mixture comprising the compound
of formula (I) is a solution, and the step of crystallizing the
compound from the mixture comprises bringing the solution to
supersaturation to cause the compound of formula (I) to precipitate
out of solution.
[0033] In certain embodiments, bringing the mixture comprising the
compound of formula (I) to supersaturation comprises the slow
addition of an anti-solvent, such as heptanes, hexanes, ethanol, or
another polar or non-polar liquid miscible with the organic
solvent, allowing the solution to cool (with or without seeding the
solution), reducing the volume of the solution, or any combination
thereof. In certain embodiments, bringing the mixture comprising
the compound of formula (I) to supersaturation comprises adding an
anti-solvent, cooling the solution to ambient temperature or lower,
and reducing the volume of the solution, e.g., by evaporating
solvent from the solution. In certain embodiments, allowing the
solution to cool may be passive (e.g., allowing the solution to
stand at ambient temperature) or active (e.g., cooling the solution
in an ice bath or freezer).
[0034] In certain embodiments, the preparation method further
comprises isolating the crystals, e.g., by filtering the crystals,
by decanting fluid from the crystals, or by any other suitable
separation technique. In further embodiments, the preparation
method further comprises washing the crystals.
[0035] In certain embodiments, the preparation method further
comprises inducing crystallization. The method can also comprise
drying the crystals, for example under reduced pressure. In certain
embodiments, inducing precipitation or crystallization comprises
secondary nucleation, wherein nucleation occurs in the presence of
seed crystals or interactions with the environment (crystallizer
walls, stirring impellers, sonication, etc.).
[0036] In certain embodiments, the solvent comprises acetonitrile,
N,N-dimethylacetamide (DMA), dimethylformamide (DMF),
dimethylsulfoxide (DMSO), ethanol, ethyl acetate, heptanes,
hexanes, isopropyl acetate, methanol, methylethyl ketone,
N-methyl-2-pyrrolidone (NMP), tetrahydrofuran, toluene, 2-propanol,
1-butanol, water, or any combination thereof. In certain preferred
embodiments, for example to achieve Form 1, the solvent is methanol
or water. In other preferred embodiments, for example to achieve
Form 2, the solvent is dimethylsulfoxide.
[0037] In certain embodiments, washing the crystals comprises
washing with a liquid selected from anti-solvent, acetonitrile,
ethanol, heptanes, hexanes, methanol, tetrahydrofuran, toluene,
water, or a combination thereof. As used herein, "anti-solvent"
means a solvent in which the salt crystals are insoluble, minimally
soluble, or partially soluble. In practice, the addition of an
anti-solvent to a solution in which the salt crystals are dissolved
reduces the solubility of the salt crystals in solution, thereby
stimulating precipitation of the salt. In certain embodiments, the
crystals are washed with a combination of anti-solvent and the
organic solvent. In certain embodiments, the anti-solvent is water,
while in other embodiments it is an alkane solvent, such as hexane
or pentane, or an aromatic hydrocarbon solvent, such as benzene,
toluene, or xylene. In certain embodiments, the anti-solvent is
methanol.
[0038] In certain embodiments, washing the crystals comprises
washing the crystalline compound of formula (I) with a solvent or a
mixture of one or more solvents, which are described above. In
certain embodiments, the solvent or mixture of solvents is cooled
prior to washing.
[0039] In certain embodiments, the methods of making the
crystalline forms of NMN are used to remove one or more impurities
from NMN. In certain embodiments, the crystallization methods
described herein are used for purifying NMN, e.g., as a final
purification step in the manufacture of the compound.
Uses of Crystal Forms of NMN
[0040] NMN is produced from nicotinamide in the NAD biosynthesis
pathway, a reaction that is catalyzed by Nampt. NMN is further
converted to NAD in the NAD biosynthesis pathway, a reaction that
is catalyzed by Nmnat. "Nicotinamide Adenine Dinucleotide" (NAD),
which corresponds to the following structure,
##STR00003##
is produced from the conversion of nicotinamide to NMN, which is
catalyzed by Nampt, and the subsequent conversion of NMN to NAD,
which is catalyzed by Nmnat. In mammals, the functional homolog of
yeast PNC1 is NAMPT, which also catalyzes the first step in NAD
salvage. NAMPT catalyzes the formation of nicotinamide
mononucleotide (NMN) from NAM, which is then converted to NAD by
NMNAT1, NMNAT2, and NMNAT3. Nicotinamide riboside, a precursor to
NAD, enters the salvage pathway after being converted to NMN by
nicotinamide riboside kinase (NRK) enzymes.
[0041] Thus, diseases, disorders and conditions that are affected
by increasing NAD levels are likewise affected by the amount of NMN
precursor available for NAD biosynthesis, and thus can be treated
by administering the NMN compounds and compositions disclosed
herein.
[0042] In certain embodiments, NMN works through the nicotinamide
mononucleotide adenylyltransferase (Nmnat1) pathway or other
pathways of NAD+ biosynthesis which have nutritional and/or
therapeutic value in improving plasma lipid profiles, prevention of
stroke, and/or prolonging life and well-being. Other embodiments
relate to a method for preventing or treating a disease or
condition associated with the nicotinamide mononucleotide
adenylyltransferase (Nmnat1) pathway or other pathways of NAD+
biosynthesis by administering a composition comprising NMN.
Diseases or conditions which typically have altered levels of NAD+
or its precursors which can be prevented or treated by
supplementing a diet or therapeutic treatment regime with NMN
and/or NAD+ include, but are not limited to, lipid disorders,
(e.g., dyslipidemia, hypercholesterolaemia or hyperlipidemia),
stroke, type I and II diabetes, cardiovascular disease, and other
physical problems associated with obesity.
[0043] Neurodegenerative Diseases
[0044] Axon degeneration occurs frequently in neurodegenerative
diseases and peripheral neuropathies. The degeneration of
transected axons is delayed in Wallerian degeneration slow (Wlds)
mice with the overexpression of a fusion protein with the
nicotinamide adenine dinucleotide (NAD+) synthetic enzyme,
nicotinamide mononucleotide adenylyltransferase (Nmnat1). Both
Wld(s) and Nmnat1 themselves are functional in preventing axon
degeneration in neuronal cultures.
[0045] NAD+ levels decrease in injured, diseased, or degenerating
neural cells and preventing this NAD+ decline efficiently protects
neural cells from cell death. See, Araki & Milbrandt "Increased
nuclear NAD+ biosynthesis and SIRT1 activation prevent axonal
degeneration" Science. 2004 Aug. 13; 305(5686):1010-3 and Wang et
al., "A local mechanism mediates NAD-dependent protection of axon
degeneration" J Cell Biol. 170(3):349-55 (2005) hereby incorporated
by reference in their entirety. As NMN is capable of increasing
intracellular levels of NAD+, NMN is useful as a therapeutic or
nutritional supplement in managing injuries, diseases, and
disorders affecting the central nervous system and the peripheral
nervous system, including, but not limited to, trauma or injury to
neural cells, diseases or conditions that harm neural cells, and
neurodegenerative diseases or syndromes. The correlation of
increased NAD+ synthesis with beneficial outcomes in neural
injuries and diseases or conditions has been discussed in, e.g.,
Stein et al., "Expression of Nampt in Hippocampal and Cortical
Excitatory Neurons Is Critical for Cognitive Function" The Journal
of Neuroscience 2014 34(17):5800-5815; and Stein et al., "Specific
ablation of Nampt in adult neural stem cells recapitulates their
functional defects during aging" EMBO J. 2014 33:1321-1340.
[0046] Some neurodegenerative diseases, neurodegenerative
syndromes, diseases and conditions that harm neural cells, or
otherwise cause injury to neural cells are described below.
[0047] Essential tremor (ET) is the most common movement disorder.
It is a syndrome characterized by a slowly progressive postural
and/or kinetic tremor, usually affecting both upper
extremities.
[0048] Parkinson's disease (PD) is a progressive neurodegenerative
disorder associated with a loss of dopaminergic nigrostriatal
neurons.
[0049] Alzheimer's disease (AD) is the most common form of
dementia. It is a progressive degenerative disease of the brain,
strongly associated with advanced age. Over time, people with the
disease lose their ability to think and reason clearly, judge
situations, solve problems, concentrate, remember useful
information, take care of themselves, and even speak. A number of
neurodegenerative diseases such as Alzheimer's disease execute
their biological impact in the brain. It is preferred that
nicotinamide mononucleotide based compounds disclosed herein are
capable of passing the blood-brain-barrier (BBB).
[0050] Huntington's disease (HD) is an incurable, adult-onset,
autosomal dominant inherited disorder associated with cell loss
within a specific subset of neurons in the basal ganglia and
cortex.
[0051] Ataxia is defined as an inability to maintain normal posture
and smoothness of movement. Neurologic symptoms and signs such as
seizures and movement disorders (e.g., dystonia, chorea) may
accompany ataxia.
[0052] Catatonia is a state of apparent unresponsiveness to
external stimuli in a person who is apparently awake.
[0053] Epilepsy is defined as a chronic condition characterized by
spontaneous, recurrent seizures; seizure is defined as a clinical
event associated with a transient, hypersynchronous neuronal
discharge.
[0054] Neuroleptic malignant syndrome (NMS) refers to the
combination of hyperthermia, rigidity, and autonomic dysregulation
that can occur as a serious complication of the use of
antipsychotic drugs.
[0055] Chorea is an involuntary abnormal movement, characterized by
abrupt, brief, nonrhythmic, nonrepetitive movement of any limb,
often associated with nonpatterned facial grimaces. Chorea
gravidarum (CG) is the term given to chorea occurring during
pregnancy.
[0056] Cortical basal ganglionic degeneration (CBGD) clinical
characteristics include progressive dementia, parkinsonism, and
limb apraxia. Dysfunction of the central or peripheral nervous
system pathways may cause autonomic dysfunction.
[0057] Dystonia is a syndrome of sustained muscle contractions,
usually producing twisting and repetitive movements or abnormal
postures. Writer's cramp is a form of task-specific focal
dystonia.
[0058] Mental retardation (MR) is a condition in which intellectual
capacity is limited significantly. Developmental disability
describes a condition that limits an individual's ability to
perform activities and roles as expected in a certain social
environment. Frequently, MR and developmental disabilities are
present simultaneously as a consequence of brain damage.
[0059] Neuroacanthocytosis is a progressive neurologic disease
characterized by movement disorders, personality changes, cognitive
deterioration, axonal neuropathy, and seizures. Most patients have
acanthocytosis on peripheral blood smear at some point during the
course of the disease.
[0060] Pelizaeus-Merzbacher disease (PMD) and X-linked spastic
paraplegia type 2 (SPG2) are at opposite ends of a clinical
spectrum of X-linked diseases caused by mutations of the same gene,
the proteolipid protein 1 (PLP1) gene, and resulting in defective
central nervous system (CNS) myelination. Clinical signs usually
include some combination of nystagmus, stridor, spastic
quadriparesis, hypotonia, cognitive impairment, ataxia, tremor, and
diffuse leukoencephalopathy on MRI scans.
[0061] Progressive supranuclear palsy (PSP), also known as
Steele-Richardson-Olszewski syndrome, is a neurodegenerative
disease that affects cognition, eye movements, and posture.
[0062] Striatonigral degeneration (SND) is a neurodegenerative
disease that represents a manifestation of multiple system atrophy
(MSA). The other manifestations are Shy-Drager syndrome (e.g.,
autonomic failure predominates) and sporadic olivopontocerebellar
degeneration (sOPCA, cerebellum predominates).
[0063] Ischemic stroke occurs due to a loss of blood supply to part
of the brain, initiating the ischemic cascade. Brain tissue ceases
to function if deprived of oxygen for more than 60 to 90 seconds
and after a few hours will suffer irreversible injury possibly
leading to death of the tissue, i.e., infarction. Atherosclerosis
may disrupt the blood supply by narrowing the lumen of blood
vessels leading to a reduction of blood flow, by causing the
formation of blood clots within the vessel, or by releasing showers
of small emboli through the disintegration of atherosclerotic
plaques. Embolic infarction occurs when emboli formed elsewhere in
the circulatory system, typically in the heart as a consequence of
atria fibriliation, or in the carotid arteries. These break off,
enter the cerebral circulation, then lodge in and occlude brain
blood vessels.
[0064] Due to collateral circulation within the region of brain
tissue affected by ischemia, there is a spectrum of severity. Thus,
part of the tissue may immediately die while other parts may only
be injured and could potentially recover. The ischemia area where
tissue might recover is referred to as the ischemic penumbra.
[0065] As oxygen or glucose becomes depleted in ischemic brain
tissue, the production of high energy phosphate compounds such as
adenine triphosphate (ATP) fails, leading to failure of energy
dependent processes necessary for tissue cell survival. This sets
off a series of interrelated events that result in cellular injury
and death. These include the failure of mitochondria, which can
lead further toward energy depletion and may trigger cell death due
to apoptosis. Other processes include the loss of membrane ion pump
function leading to electrolyte imbalances in brain cells. There is
also the release of excitatory neurotransmitters, which have toxic
effects in excessive concentrations.
[0066] Spinal cord injury, or myelopathy, is a disturbance of the
spinal cord that results in loss of sensation and mobility. The two
common types of spinal cord injury are: trauma: automobile
accidents, falls, gunshots, diving accidents, etc. and disease:
polio, spina bifida, tumors, Friedreich's ataxia, etc. It is
important to note that the spinal cord does not have to be
completely severed for there to be a loss of function. In fact, the
spinal cord remains intact in most cases of spinal cord injury.
[0067] Traumatic brain injury (TBI), also called intracranial
injury, or simply head injury, occurs when a sudden trauma causes
brain damage. TBI can result from a closed head injury or a
penetrating head injury and is one of two subsets of acquired brain
injury (ABI). The other subset is non-traumatic brain injury (e.g.,
stroke, meningitis, anoxia). Parts of the brain that can be damaged
include the cerebral hemispheres, cerebellum, and brain stem.
Symptoms of a TBI can be mild, moderate, or severe, depending on
the extent of the damage to the brain. Outcome can be anything from
complete recovery to permanent disability or death. A coma can also
affect a child's brain. The damage from TBI can be focal, confined
to one area of the brain, or diffuse, involving more than one area
of the brain. Diffuse trauma to the brain is frequently associated
with concussion (a shaking of the brain in response to sudden
motion of the head), diffuse axonal injury, or coma. Localized
injuries may be associated with neurobehavioral manifestations,
hemiparesis or other focal neurologic deficits.
[0068] Another insult to the brain that can cause injury is anoxia.
Anoxia is a condition in which there is an absence of oxygen supply
to an organ's tissues, even if there is adequate blood flow to the
tissue. Hypoxia refers to a decrease in oxygen supply rather than a
complete absence of oxygen, and ischemia is inadequate blood
supply, as is seen in cases in which the brain swells. In any of
these cases, without adequate oxygen, a biochemical cascade called
the ischemic cascade is unleashed, and the cells of the brain can
die within several minutes. This type of injury is often seen in
near-drowning victims, in heart attack patients (particularly those
who have suffered a cardiac arrest), or in people who suffer
significant blood loss from other injuries that then causes a
decrease in blood flow to the brain due to circulatory
(hypovolemic) shock.
[0069] Regulating Blood Glucose Concentration
[0070] Provided herein is a process for regulating the
concentration of blood glucose in a mammal. As utilized herein,
regulating the concentration of blood glucose refers to any
increase, decrease, and/or maintenance in or of the concentration
of blood glucose as compared to a previously determined level.
[0071] NMN may be administered to a mammal in need of such
treatment. For example, the mammal may require an increase in blood
glucose concentration. Alternatively, the mammal may require a
decrease in blood glucose concentration. Or, the mammal may require
maintenance of blood glucose concentration above, at, or below a
particular level or within a particular range (e.g., through a
series of increases and/or decreases, or through no increases or
decreases). The blood glucose concentration-regulating NMN may also
be administered to a mammal as a prophylactic measure; that is, the
mammal is in need of treatment to prevent or delay the occurrence
or onset of a medical condition such as, for example, type 1 or
type 2 diabetes.
[0072] The ability to regulate the concentration of blood glucose
in a mammal according to the processes described herein (e.g., by
administering to a mammal a blood glucose regulating amount of a
compound of the present invention may be advantageous in the
treatment and/or prevention of a variety of complications,
diseases, and/or illnesses. The role of increased NAD+ levels on
metabolic diseases and conditions has been described in, for
example, Yoshino et al., "Nicotinamide mononucleotide, a key NAD+
intermediate, treats the pathophysiology of diet- and age-induced
diabetes" Cell Metab. 2011 14:528-536; and Garten, et al., "Nampt:
Linking NAD biology, metabolism, and cancer" Trends Endocrinol
Metab. 2009 20(3):130-138. In general, the present invention may be
utilized to treat a variety of acute, intermediate stage, and
chronic conditions that may be affected by systemic NAD
biosynthesis either directly or indirectly.
[0073] For example, the regulation of blood glucose concentration
may be effective in the treatment and/or prophylaxis of such
medical conditions as brain ischemia-induced hypoglycemia,
hypoglycemic brain injury caused by, e.g., congenital
hyperinsulinism in children, and/or other conditions that severely
reduce blood glucose levels. Alternatively, the regulation of blood
glucose concentration may be effective in counteracting the effects
of the injection of an excessive amount of insulin, or an
insufficient dietary or vitamin intake (e.g., deficiencies in
vitamin B3 (niacin, which is derived from nicotinic acid and
nicotinamide) can result in pellagra, the classic niacin deficiency
disease, characterized by bilateral dermatitis, diarrhea, and
dementia).
[0074] Further, regulation of blood glucose concentration may be
effective in the treatment and/or prophylaxis of hypoglycemia,
hyperglycemia, impaired glucose tolerance, impaired fasting
glucose, and type 1 and type 2 diabetes.
[0075] The regulation of blood glucose concentration according to
the methods described herein may also be advantageous in
counteracting the effects of blood glucose concentration-decreasing
drugs such as acetaminophen, alcohol, anabolic steroids,
clofibrate, disopyramide, gemfibrozil, monoamine oxidase inhibitors
(MAOIs), pentamidine, or sulfonylurea medications (such as
glipizide, glyburide, and glimepiride).
[0076] Other conditions having a plausible connection to NAD
biosynthesis, such as dementia, may also be beneficially treated
and/or prevented by blood glucose regulation. See, e.g., Guest, et
al., "Changes in Oxidative Damage, Inflammation and [NAD(H)] with
Age in Cerebrospinal Fluid" PLOS One. January 2014 9(1):
e85335.
[0077] The increase, decrease, and/or maintenance of blood glucose
concentration can be quantified, for example, by percentage above,
below, or in between one or more previously determined levels, or
can be quantified by a particular blood glucose concentration or a
range thereof.
[0078] For example, the blood glucose concentration may be
increased to at least about 5% above a previously determined level;
to at least about 10% above a previously determined level; to at
least about 25% above a previously determined level; to at least
about 50% above a previously determined level; to at least about
75% above a previously determined level; to at least about 100%
above a previously determined level; to at least about 150% above a
previously determined level; or to at least about 200% above a
previously determined level. By way of another example, the blood
glucose concentration may be decreased to at least about 5% below a
previously determined level; to at least about 10% below a
previously determined level; to at least about 25% below a
previously determined level; to at least about 50% below a
previously determined level; to at least about 75% below a
previously determined level; to at least about 100% below a
previously determined level; to at least about 150% below a
previously determined level; or to at least about 200% below a
previously determined level. By way of yet another example, the
blood glucose concentration may be maintained (e.g., by a series of
increases and/or decreases, or by no increases and/or decreases) at
a concentration that is no more than about 50% greater or about 50%
less than a previously determined level; e.g., no more than about
40% greater or about 40% less; no more than about 30% greater or
about 30% less; no more than about 20% greater or about 20% less;
no more than about 10% greater or about 10% less; or no more than
about 5% greater or about 5% less.
[0079] Alternatively, the blood glucose concentration may be
maintained (e.g., by a series of increases and/or decreases, or by
no increases and/or decreases) at, above, or below a particular
blood glucose concentration or within a desired range of blood
glucose concentrations. For example, the blood glucose
concentration may be maintained at a concentration of greater than
about 60 mg/dL; greater than about 70 mg/dL; greater than about 100
mg/dL; greater than about 110 mg/dL; or greater than about 125
mg/dL. Alternatively, the blood glucose concentration may be
maintained at a concentration of less than about 200 mg/dL; less
than about 175 mg/dL; less than about 150 mg/dL; less than about
125 mg/dL; less than about 110 mg/dL; or less than about 100 mg/dL.
By way of another example, the blood glucose concentration may be
maintained at a concentration of from about 60 mg/dL to about 140
mg/dL; from about 90 mg/dL to about 130 mg/dL; from about 100 mg/dL
to about 125 mg/dL; or from about 110 mg/dL to about 125 mg/dL.
[0080] Drug Toxicity
[0081] In some embodiments, the invention relates to the use of NMN
to prevent adverse effects and protect cells from toxicity.
Toxicity may be an adverse effect of radiation or external
chemicals on the cells of the body. Examples of toxins are
pharmaceuticals, drugs of abuse, and radiation, such as UV or X-ray
light. Both radiative and chemical toxins have the potential to
damage biological molecules such as DNA. This damage typically
occurs by chemical reaction of the exogenous agent or its
metabolites with biological molecules, or indirectly through
stimulated production of reactive oxygen species (e.g., superoxide,
peroxides, hydroxyl radicals). Repair systems in the cell excise
and repair damage caused by toxins.
[0082] Enzymes that use NAD+ play a part in the DNA repair process.
For example, DNA repair syndromes include, but are not limited to,
Cockayne syndrome. Specifically, the poly(ADP-ribose) polymerases
(PARPs), particularly PARP-1, are activated by DNA strand breaks
and affect DNA repair. The PARPs consume NAD+ as an adenosine
diphosphate ribose (ADPR) donor and synthesize poly(ADP-ribose)
onto nuclear proteins such as histones and PARP itself. Although
PARP activities facilitate DNA repair, overactivation of PARP can
cause significant depletion of cellular NAD+, leading to cellular
necrosis. The apparent sensitivity of NAD+ metabolism to
genotoxicity has led to pharmacological investigations into the
inhibition of PARP as a means to improve cell survival. Numerous
reports have shown that PARP inhibition increases NAD+
concentrations in cells subject to genotoxicity, with a resulting
decrease in cellular necrosis. See, e.g., Fang, et al., Defective
Mitophagy in XPA via PARP-1 Hyperactivation and NAD+/SIRT1
Reduction. Cell 2014 157:882-896. Nevertheless, cell death from
toxicity still occurs, presumably because cells are able to
complete apoptotic pathways that are activated by genotoxicity.
Thus, significant cell death is still a consequence of
DNA/macromolecule damage, even with inhibition of PARP. This
consequence suggests that improvement of NAD+ metabolism in
genotoxicity can be partially effective in improving cell survival
but that other proteins that modulate apoptotic sensitivity, such
as sirtuins, may also play important roles in cell responses to
genotoxins.
[0083] Physiological and biochemical mechanisms that determine the
effects of chemical and radiation toxicity in tissues are complex,
and evidence indicates that NAD+ metabolism is an important aspect
of cell stress response pathways. For example, upregulation of NAD+
metabolism, via nicotinamide/nicotinic acid mononucleotide (NMNAT)
overexpression, has been shown to protect against neuron axonal
degeneration, and nicotinamide used pharmacologically has been
recently shown to provide neuron protection in a model of fetal
alcohol syndrome and fetal ischemia. Such protective effects could
be attributable to upregulated NAD+ biosynthesis, which increases
the available NAD+ pool subject to depletion during genotoxic
stress. This depletion of NAD+ is mediated by PARP enzymes, which
are activated by DNA damage and can deplete cellular NAD+, leading
to necrotic death. Another mechanism of enhanced cell protection
that could act in concert with upregulated NAD+ biosynthesis is the
activation of cell protection transcriptional programs regulated by
sirtuin enzymes.
[0084] Aging/Stress
[0085] In certain embodiments, the invention provides a method
extending the lifespan of a cell, extending the proliferative
capacity of a cell, slowing aging of a cell, promoting the survival
of a cell, delaying cellular senescence in a cell, mimicking the
effects of calorie restriction, increasing the resistance of a cell
to stress, or preventing apoptosis of a cell, by contacting the
cell with NMN and/or NAD+. Recent studies have demonstrated the
role NAD+ plays in the aging process and in age-related diseases
and conditions. See, e.g., Imai, et al., "NAD+ and sirtuins in
aging and disease" Trends in Cell Biol. 2014 24(8): 464-471; and
Gomes, et al. "Declining NAD+ Induces a Pseudohypoxic State
Disrupting Nuclear-Mitochondrial Communication during Aging" Cell
2013 155:1624-1638.
[0086] The methods described herein may be used to increase the
amount of time that cells, particularly primary cells (e.g., cells
obtained from an organism, e.g., a human), may be kept alive in an
ex vivo cell culture. Embryonic stem (ES) cells and pluripotent
cells, and cells differentiated therefrom, may also be treated with
a nicotinamide mononucleotide based or derivative compound to keep
the cells, or progeny thereof, in culture for longer periods of
time. Such cells can also be used for transplantation into a
subject, e.g., after ex vivo modification.
[0087] In certain embodiments, cells that are intended to be
preserved for long periods of time may be treated with NMN and/or
NAD+. The cells may be in suspension (e.g., blood cells, serum,
biological growth media, etc.) or in tissues or organs in a
subject. For example, blood collected from an individual for
purposes of transfusion may be treated with NMN and/or NAD+ to
preserve the blood cells for longer periods of time. Additionally,
blood to be used for forensic purposes may also be preserved using
NMN and/or NAD+. Other cells that may be treated to extend their
lifespan or protect against apoptosis include cells for
consumption, e.g., cells from non-human mammals (such as meat) or
plant cells (such as vegetables).
[0088] NMN and/or NAD+ may also be applied during developmental and
growth phases in mammals, plants, insects or microorganisms, in
order to, e.g., alter, retard or accelerate the developmental
and/or growth process.
[0089] In certain embodiments, NMN and/or NAD+ may be used to treat
cells useful for transplantation or cell therapy, including, for
example, solid tissue grafts, organ transplants, cell suspensions,
stem cells, bone marrow cells, etc. The cells or tissue may be an
autograft, an allograft, a syngraft or a xenograft. The cells or
tissue may be treated with NMN and/or NAD+ prior to
administration/implantation, concurrently with
administration/implantation, and/or post
administration/implantation into a subject. The cells or tissue may
be treated prior to removal of the cells from the donor individual,
ex vivo after removal of the cells or tissue from the donor
individual, or post implantation into the recipient. For example,
the donor or recipient individual may be treated systemically with
NMN and/or NAD+ or may have a subset of cells/tissue treated
locally with NMN and/or NAD+. In certain embodiments, the cells or
tissue (or donor/recipient individuals) may additionally be treated
with another therapeutic agent useful for prolonging graft
survival, such as, for example, an immunosuppressive agent, a
cytokine, an angiogenic factor, etc.
[0090] In certain embodiments, cells may be treated with an amount
of NMN that increases the level of NAD+ in vivo, e.g., to increase
their lifespan or prevent apoptosis. For example, skin can be
protected from aging (e.g., developing wrinkles, loss of
elasticity, etc.) by treating skin or epithelial cells with an
amount of NMN that increases the level of intracellular NAD+. In
exemplary embodiments, skin is contacted with a pharmaceutical or
cosmetic composition comprising an amount of NMN that increases the
level of intracellular NAD+. Exemplary skin afflictions or skin
conditions that may be treated in accordance with the methods
described herein include disorders or diseases associated with or
caused by inflammation, sun damage or natural aging. For example,
the compositions find utility in the prevention or treatment of
contact dermatitis (including irritant contact dermatitis and
allergic contact dermatitis), atopic dermatitis (also known as
allergic eczema), actinic keratosis, keratinization disorders
(including eczema), epidermolysis bullosa diseases (including
penfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas
(including erythema multiforme and erythema nodosum), damage caused
by the sun or other light sources, discoid lupus erythematosus,
dermatomyositis, psoriasis, skin cancer and the effects of natural
aging. In other embodiments, an amount of NMN that increases the
level of intracellular NAD+ may be used for the treatment of wounds
and/or burns to promote healing, including, for example, first-,
second- or third-degree burns and/or thermal, chemical or
electrical burns. The formulations may be administered topically,
to the skin or mucosal tissue, as an ointment, lotion, cream,
microemulsion, gel, solution or the like, as further described
herein, within the context of a dosing regimen effective to bring
about the desired result.
[0091] Topical formulations comprising an amount of NMN that
increases the level of intracellular NAD+ may also be used as
preventive, e.g., chemopreventive, compositions. When used in a
chemopreventive method, susceptible skin is treated prior to any
visible condition in a particular individual.
[0092] In certain embodiments, an amount of NMN that increases the
level of intracellular NAD+ may be used for treating or preventing
a disease or condition induced or exacerbated by cellular
senescence in a subject; methods for decreasing the rate of
senescence of a subject, e.g., after onset of senescence; methods
for extending the lifespan of a subject; methods for treating or
preventing a disease or condition relating to lifespan; methods for
treating or preventing a disease or condition relating to the
proliferative capacity of cells; and methods for treating or
preventing a disease or condition resulting from cell damage or
death. In certain embodiments, the method does not act by
decreasing the rate of occurrence of diseases that shorten the
lifespan of a subject. In certain embodiments, a method does not
act by reducing the lethality caused by a disease, such as
cancer.
[0093] In certain embodiments, an amount of NMN that increases the
level of intracellular NAD+ may be administered to a subject in
order to generally increase the lifespan of its cells and to
protect its cells against stress and/or against apoptosis. Treating
a subject with NMN may be similar to subjecting the subject to
hormesis, i.e., mild stress that is beneficial to organisms and may
extend their lifespan.
[0094] An amount of NMN that increases the level of intracellular
NAD+ can also be administered to subjects for treatment of
diseases, e.g., chronic diseases, associated with cell death, in
order to protect the cells from cell death. Exemplary diseases
include those associated with neural cell death, neuronal
dysfunction, or muscular cell death or dysfunction, such as
Parkinson's disease, Alzheimer's disease, multiple sclerosis,
amyotropic lateral sclerosis, and muscular dystrophy; AIDS;
fulminant hepatitis; diseases linked to degeneration of the brain,
such as Creutzfeld-Jakob disease, retinitis pigmentosa and
cerebellar degeneration; myelodysplasis such as aplastic anemia;
ischemic diseases such as myocardial infarction and stroke; hepatic
diseases such as alcoholic hepatitis, hepatitis B and hepatitis C;
joint-diseases such as osteoarthritis; atherosclerosis; alopecia;
damage to the skin due to UV light; lichen planus; atrophy of the
skin; cataract; and graft rejections. Cell death can also be caused
by surgery, drug therapy, chemical exposure or radiation
exposure.
[0095] An amount of NMN that increases the level of intracellular
NAD+ can also be administered to a subject suffering from an acute
disease, e.g., damage to an organ or tissue, e.g., a subject
suffering from stroke or myocardial infarction or a subject
suffering from a spinal cord injury. An amount of NMN that
increases the level of intracellular NAD+ may also be used to
repair an alcoholic's liver.
[0096] Cardiovascular Disease
[0097] In certain embodiments, the invention provides methods for
treating and/or preventing a cardiovascular disease by
administering to a subject in need thereof an amount of NMN that
increases the level of intracellular NAD+. The benefits of NAD+ in
treating cardivasular diseases has been described in several
studies, such as Borradaile, et al., "NAD+, Sirtuins, and
Cardiovascular Disease" Current Pharmaceutical Design 2016
15(1):110-117.
[0098] Cardiovascular diseases that can be treated or prevented by
an amount of NMN that increases the level of intracellular NAD+
include cardiomyopathy or myocarditis; such as idiopathic
cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy,
drug-induced cardiomyopathy, ischemic cardiomyopathy, and
hypertensive cardiomyopathy. Also treatable or preventable using
compounds and methods described herein are atheromatous disorders
of the major blood vessels (macrovascular disease) such as the
aorta, the coronary arteries, the carotid arteries, the
cerebrovascular arteries, the renal arteries, the iliac arteries,
the femoral arteries, and the popliteal arteries. Other vascular
diseases that can be treated or prevented include those related to
platelet aggregation, the retinal arterioles, the glomerular
arterioles, the vasa nervorum, cardiac arterioles, and associated
capillary beds of the eye, the kidney, the heart, and the central
and peripheral nervous systems.
[0099] Yet other disorders that may be treated with an amount of
NMN that increases the level of intracellular NAD+ include
restenosis, e.g., following coronary intervention, and disorders
relating to an abnormal level of high density and low density
cholesterol. Another disease that can benefit from NAD+ treatment
is nonalcoholic steatohepatitis (NASH) which a fatty liver
disease.
[0100] Circadian Rhythm
[0101] The circadian clock is encoded by a
transcription-translation feedback loop that synchronizes behavior
and metabolism with the light-dark cycle. It has been unexpectedly
discovered that both the rate-limiting enzyme in mammalian NAD+
biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT), and
levels of NAD+, display circadian oscillations which are regulated
by the core clock machinery in mice. Inhibition of NAMPT promotes
oscillation of the clock gene Per2 by releasing CLOCK:BMAL1 from
suppression by SIRT1. In turn, the circadian transcription factor
CLOCK binds to and up-regulates Nampt, thus completing a feedback
loop involving NAMPT/NAD+ and SIRT1/CLOCK:BMAL1. See, e.g., Ramsey
et al., "Circadian clock feedback cycle through NAMPT-mediated NAD+
biosynthesis" Science 2009 324:651-654.
[0102] Thus, the periodic variation in NAMPT-mediated NAD+
biosynthesis suggests that it impacts physiologic cycles and
possibly the sleep-wake and fasting-feeding cycle. Without being
bound by a single theory, it is believed that NAD+ serves as a
critical "metabolic oscillator" for the rhythmic regulation of
response to environmental cues through control of SIRT1 activity.
Compounds disclosed herein may be used to affect a circadian
feedback loop through NAMPT-mediated NAD+ biosynthesis and/or a
pathway underlying the temporal coupling of metabolic, physiologic,
and circadian cycles in mammals.
[0103] The recognition of a regulatory pathway involving
NAMPT/NAD+-SIRT1/CLOCK:BMAL1 has broad implications for
understanding how physiologic and behavioral cycles are coordinated
with the environmental light-dark cycle. For instance, during
sleep, when animals are normally quiescent and fasting, the levels
of NAMPT steadily increase, peaking at the beginning of the
wakefulness period and coinciding with feeding. As a result of the
increase in NAMPT, NAD+ rises to stimulate SIRT1, which
orchestrates an appropriate metabolic response in liver involving a
switch from catabolic to anabolic pathways.
[0104] In certain embodiments, the present invention provides
methods for regulation of the core clock machinery (sometimes also
referred to as the circadian clock) of a mammal, thereby affecting
behaviors, activities, and/or biological functions that occur in or
are affected by a diurnal or circadian cycle and that are
regulated, at least in part, by the circadian clock. Generally, the
methods involve the administration of a therapeutic or prophylactic
amount of a circadian clock-regulating compound to a patient or
mammal in need of regulation of the circadian clock.
[0105] The methods of treatment disclosed herein are generally
directed to methods of regulating the circadian clock, thereby
regulating or affecting biological functions that are regulated by
(sometimes also said to be affected by, affiliated with, or
mediated by) the activity of the circadian clock. Typically, these
biological functions display a pattern of activity and inactivity
that is generally repeated approximately every 24 hours,
oscillating between "active" and "inactive" states during the 24
hour period.
[0106] Thus, the present invention provides methods of regulating
the activity of the circadian clock by administering to a mammal in
need thereof a circadian-clock regulating compound. Generally, the
regulation of the activity of the circadian clock is the result of
the regulation of CLOCK:BMAL1, which is achieved according to the
present methods by regulating the activity of SIRT1. The activity
of SIRT1 is generally regulated according to the present methods by
administration of a circadian clock-regulating compound, and in
certain embodiments, by administration of an amount of NMN that
affects the NAD+ pathway. The regulation of the circadian clock
thereby permits regulation of activities mediated by the circadian
clock.
[0107] According to the present invention, the activity of the
circadian clock may be increased, decreased, or maintained by the
administration of a circadian clock-regulating compound.
Accordingly, biological functions (sometimes also referred to as
biological activities) that are regulated by the activity of the
circadian clock may also be increased, decreased, or maintained. In
addition, these biological functions may also be time shifted; that
is to say, an activity that typically occurs during a particular
period, such as for example, during daytime or daylight hours
(sometimes also referred to as the light cycle) or during the night
or nighttime hours (sometimes also referred to as the dark cycle)
may be shifted such that the activity occurs during the dark or
light cycle, respectively, instead.
[0108] Any of a number of biological functions that are typically
affected by the activity of the circadian clock may be regulated by
the methods of the present invention. Thus, the present methods may
be used to treat disorders or disease states that are the result
of, for example, the irregular, inadequate, or pathological
function of the circadian clock. Similarly, the present methods may
be used to treat disorders or symptomatology caused by exogenous
factors that affect the proper function or activity of the
circadian clock or that require a "resetting" of the clock. For
example, administration of circadian clock-regulating compound to a
patient experiencing a metabolic disorder provides therapeutic
benefit not only when the patient's serum NMN or NAD level is
increased, but also when an improvement is observed in the patient
with respect to other disorders that accompany the metabolic
disorder, like weight loss or gain. In some treatment regimens, the
circadian clock-regulating compound of the invention may be
administered to a patient at risk of developing a disorder as
described herein or to a patient reporting one or more of the
physiological symptoms of such a disorder, even though a diagnosis
of a metabolic disorder may not have been made.
[0109] Examples of disorders, disease states, or symptomatology
that may be treated according to the methods of the present
invention include, but are not limited to, travel to or across one
or more time zones, a change in work shifts, night shift work, or a
change in the physical status of a mammal caused by, for example,
pregnancy or administration of medications of any kind.
Accordingly, the methods of the present invention may be used to
treat or prevent disorders, symptoms of disorders, or symptoms
caused by exogenous factors. Such disorders and symptoms may
include, for example, metabolic disorders, such as improper cycling
or timing of feeding and fasting cycles, hyperglycemia,
hypoglycemia, or diabetes; sleep disorders, such as insomnia,
advanced sleep phase syndrome, delayed sleep phase syndrome,
inconsistent sleep/wake cycles, or narcolepsy or to improve
wakefulness in individuals suffering from excessive sleepiness; and
symptoms caused by exogenous factors, such as, travel to or across
one or more time zones (jet lag), shifting into or out of daylight
savings time, a change in work shifts or night shift work,
pregnancy, or medications being taken for unrelated diseases or
disorders.
[0110] Accordingly, in certain embodiments, the present invention
is directed to a method of regulating a biological function in a
mammal, the function being affected by the circadian clock. The
method comprises administering a therapeutic or prophylactic
(sometimes also referred to as a circadian clock-regulating) amount
of a circadian clock-regulating compound to the mammal. The
biological function can be, for example, any one of the biological
functions described herein. In certain embodiments, the invention
comprises a method of treating a metabolic disorder in a mammal and
comprises administering a therapeutic or prophylactic amount of a
circadian clock-regulating compound to the mammal. In other
embodiments, the invention comprises a method of treating a
disorder in a mammal mediated by the function of the circadian
clock and comprises administering a therapeutic or prophylactic
amount of a circadian clock-regulating compound to the mammal.
According to any one of these embodiments, the circadian
clock-regulating compound may be, for example, nicotinamide,
nicotinamide mononucleotide (NMN), nicotinamide adenine
dinucleotide (NAD); salts and prodrugs thereof; nicotinamide
phosphoribosyltransferase (NAMPT); and combinations thereof, as
described in greater detail below. In other embodiments, the
circadian clock-regulating compound may be an antagonist of any one
of the compounds listed above, thereby exacting an effect opposite
that of nicotinamide, nicotinamide mononucleotide (NMN),
nicotinamide adenine dinucleotide (NAD); salts and prodrugs
thereof; nicotinamide phosphoribosyltransferase (NAMPT); and
combinations thereof.
[0111] In certain embodiments, the present invention is directed to
a method of regulating metabolic activity of a mammal comprising
administering to the mammal a therapeutic amount of a circadian
clock-regulating compound. In certain embodiments, the metabolic
activity of the mammal is increased. In other embodiments, the
metabolic activity is decreased. In yet other embodiments, the
metabolic activity of the mammal is maintained at a desired level,
thereby preventing fluctuations in activity/inactivity. In still
other embodiments, the metabolic activity is caused to occur in the
light cycle (as opposed to its typical occurrence in the dark
cycle). In other embodiments, the metabolic activity is caused to
occur in the dark cycle (as opposed to its typical occurrence in
the light cycle). In certain embodiments, the circadian
clock-regulating compound is administered to the mammal in order to
increase the anabolic activity of the liver (e.g., increase the
activity of the metabolic pathways of the liver or shift or switch
liver activity from catabolism to anabolism). In other embodiments,
the circadian clock-regulating compound is administered to the
mammal in order to increase the catabolic activity of the liver
(e.g., decrease the activity of the metabolic process).
[0112] Mitochondrial Diseases and Metabolic Effects
[0113] In addition to regulating circadian rhythms and protect
neural cells from cell death, sirtuins such as SIRT3, SIRT4, and
SIRT5 are found in mitochondria. SIRT3 is expressed at high levels
in metabolically active tissue. Modulation of SIRT3 has a variety
of physiological applications for muscle cells including mimicking
calorie restriction or exercise, increasing mitochodrial biogenesis
or metabolism, sensitizing a cell to glucose uptake, increasing
fatty acid oxidation, and decreasing reactive oxygen species. In
addition, SIRT3 is demonstrated herein to be involved in promoting
cell survival during genotoxic stress. Thus modulation of SIRT3
levels also has applications in mediating cell survival.
[0114] Increasing the protein or activity level of SIRT3 in a
muscle cell can mimic the benefits of calorie restriction or
exercise. In some embodiments, the invention relates to methods for
increasing mitochondrial biogenesis or metabolism or for boosting
mitochondrial activity/endurance in a muscle cell by contacting a
muscle cell with an agent IS that increases the protein or activity
level of SIRT3 in the cell. In some embodiments, the invention
relates to methods for sensitizing a muscle cell to glucose uptake
by contacting a muscle cell with an agent that increases the
protein or activity level of SIRT3 in the cell. Further embodiments
of the invention relate to methods for increasing fatty acid
oxidation in a muscle cell by contacting a muscle cell with an
agent that increases the protein or activity level of SIRT3 in the
cell. Some embodiments of the invention relate to methods for
decreasing reactive oxygen species (ROS) in a muscle cell by
contacting the muscle cell with an agent that increases the protein
or activity level of SIRT3 in the cell.
[0115] Increasing levels of SIRT3 benefits many diseases and
disorders affected by metabolism within mitochondria. Increasing
SIRT3 may be useful in any subjects in need of metabolic activation
of one or more of their muscles, e.g., smooth muscles or cardiac
muscles or muscle cells thereof. A subject may be a subject having
cachexia or muscle wasting.
[0116] Increasing SIRT3 may also be used to increase or maintain
body temperature, e.g., in hypothermic subjects. Alternatively,
inhibiting SIRT3 may be used to reduce body temperature, e.g., in
subjects having fever or hyperthermia.
[0117] Generally, activation of SIRT3 may be used to stimulate the
metabolism of any type of muscle, e.g., muscles of the gut or
digestive system, or the urinary tract, and thereby may be used to
control gut motility, e.g., constipation, and incontinence.
[0118] Other embodiments in which it would be useful to increase
SIRT3 include repair of muscle, such as after a surgery or an
accident, increase of muscle mass; and increase of athletic
performance.
[0119] Thus the invention provides methods in which beneficial
effects are produced by contacting one or more muscle cells with an
amount of NMN that increases the protein or activity level of SIRT3
in the cell. These methods effectively facilitate, increase or
stimulate one or more of the following: mimic the benefits of
calorie restriction or exercise in the muscle cell, increase
mitochondrial biogenesis or metabolism, increase mitochondrial
activity and/or endurance in the muscle cell, sensitize the muscle
cell to glucose uptake, increase fatty acid oxidation in the muscle
cell, decrease reactive oxygen species (ROS) in the muscle cell,
increase PGC-1a and/or ucp3 and/or GLUT4 expression in the muscle
cell, and activate AMP activated protein kinase (AMPK) in the
muscle cell.
[0120] Various types of muscle cells can be contacted in accordance
with the invention. In some embodiments, the muscle cell is a
skeletal muscle cell. In certain embodiments, the muscle cell is a
cell of a slow-twitch muscle, such as a soleus muscle cell. The
methods of the invention include, in some embodiments,
administering, to a subject in need of such treatment, an amount of
NMN that increases the protein or activity level of SIRT3 in cells
of the subject.
[0121] The cell that is contacted or the subject that is treated in
the aforementioned methods preferably is a cell in need of SIRT3
increase in protein or activity level. In certain embodiments, the
cell is a diseased cell of a subject.
[0122] Also provided are methods for regulating skeletal muscle
metabolism or skeletal muscle energy homeostasis in a subject. In
such methods, an agent that modulates the protein or activity level
of SIRT3 in the subject, i.e., the SIRT3 modulators described
herein, is administered to a subject in need thereof.
[0123] Also provided are methods for increasing the protein level
of SIRT3 in a muscle cell or in muscles of a subject. Such methods
include subjecting a cell or a subject to caloric restriction or
fasting, or administering to a subject in need thereof an amount of
NMN that increases the protein or activity level of SIRT3 in a
muscle cell. Diseases, disorders and conditions in which such
methods are useful include mitochondrial diseases, metabolic
disorders, neurologic disorders, muscular disorders, cardiovascular
diseases, and excessive weight or obesity. Specific metabolic
disorders, diseases or conditions include insulin resistance,
diabetes, diabetes related conditions or disorders, or metabolic
syndrome. Other metabolic disorders will be known to the skilled
person.
[0124] Mitochondrial diseases that can be treated include diseases
that show a variety of symptoms caused by dysfunction of
mitochondria in cells. The mitochondrial diseases may be classified
in various ways by biochemical abnormalities, clinical symptoms or
types of DNA abnormalities. Types named as KSS (chronic progressive
external ophthalmoplegia), MERRF (myoclonus epilepsy associated
with ragged-red fibers; Fukuhara syndrome), MELAS, Leber's disease,
Leigh encephalopathia and Pearson's disease are widely known. Among
them, MELAS is a type mainly showing stroke-like episodes, occupies
30% or more of the whole and is believed to be the most frequent
type in the mitochondrial disease.
[0125] In certain embodiments, NMN is useful in treating diseases
or disorders associated with DNA repair defects or mitochondrial
dysfunction (e.g., resulting from the deregulation of mitochondrial
homeostasis). In some embodiments, "mitochondrial dysfunction" or
"deregulation of mitochondrial homeostasis" means that one or more
mitochondrial component (e.g., ETC component) is depleted, for
example by a decrease in mitochondrial gene expression or
mitochondrial DNA content, resulting in compromised mitochondrial
function (e.g., loss of or decreased oxidative phosphorylation
(OXPHOS) capacity). Examples of DNA repair diseases include
Cockayne syndrome and TTD.
[0126] Retinal Diseases and Disorders
[0127] Photoreceptor neuronal cell death and vision can be rescued
by NMN administration. In certain embodiments, nicotinamide
phosphoribosyl transferase (NAMPT)-mediated NAD biosynthesis can
play a role in for rod and/or cone PR neuron survival. In certain
embodiments, decreased NAD levels can cause impaired mitochondrial
function in PR neurons, alterations in TCA cycle metabolites, and
can lead to cell death and blindness.
[0128] Deleting NAMPT can lead to photoreceptor death, loss of
normal retinal structure and function, and vision loss. In some
cases, damage to photoreceptor neurons and their function can be
reversed with supplementation of NMN, an NAMPT enzymatic reaction
product. Disclosed herein are methods of administering NMN to
restore NAD levels in the retina. In some embodiments, NMN
supplementation can be an effective therapeutic intervention for
many retinal degenerative diseases.
[0129] Provided herein are methods of treating, preventing, and
reducing risk of diseases associated with photoreceptor
dysfunction, including, without limitation, age-related macular
degeneration (AMD), inherited and acquired retinal diseases such
as, without limitation, retinitis pigmentosa (RP), rod and cone
dystrophism, and Leber's congenital amaurosis (LCA) by
administration of NMN to a subject. In certain embodiments, NMN
administration can be an effective intervention for the prevention
and/or treatment of orphan retinal degenerative diseases including
but not limited to rod dystrophy, cone dystrophy, retinitis
pigmentosa, other inherited retinal degenerations, Leber's
congenital amaurosis (LCA) and acquired retinal degenerations such
as, but not limited to, age-related macular degeneration,
photoreceptor degeneration following retinal detachment.
[0130] In some embodiments, these methods can comprise
administering to a subject a pharmaceutically effective amount of
nicotinamide mononucleotide (NMN). In some embodiments, a
pharmaceutically effective amount of nicotinamide mononucleotide
(NMN) can be an amount effective for increasing retinal NAD
levels.
[0131] Disclosed herein are methods of treating macular
degeneration in a subject. In some embodiments, the methods include
treating aberrant retinal NAD levels in a subject, including
aberrantly low retinal NAD levels. These methods comprise
administering NMN to a subject. In some embodiments, the methods
include treating retinal degeneration in a subject. In some
embodiments, the methods include treating photoreceptor damage in a
subject. In some embodiments, the methods include treating
photoreceptor degeneration in a subject.
[0132] In some embodiments, the methods include treating vision
loss associated with retinal degeneration in a subject. In some
embodiments, the methods include treating aberrant retinal
structure in a subject. In some embodiments, the methods include
increasing retinal NAD levels in a subject.
[0133] In some embodiments, the methods include reducing the risk
of developing macular degeneration in a subject. In some
embodiments, the methods include reducing risk of developing
aberrant retinal NAD levels in a subject. In some embodiments, the
methods include reducing the risk of developing retinal
degeneration in a subject. In some embodiments, the methods include
reducing the risk of developing photoreceptor damage/degeneration
in a subject. In some embodiments, the methods include reducing the
risk of developing vision loss associated with retinal degeneration
in a subject. In some embodiments, the methods include reducing the
risk of developing aberrant retinal structure in a subject.
[0134] In some embodiments, the methods include treating a retina
disease in a subject. In some embodiments, a retinal disease that
can be treated by administration of NMN can be retinitis pigmentosa
(RP), Leber's congenital amaurosis (LCA), rod dystrophy, cone
dystrophy, rod-cone dystrophy, cone-rod dystrophy, age-related
macular degeneration, photoreceptor degeneration following retinal
detachments, or a combination thereof.
[0135] In certain embodiments, the crystal forms of
.beta.-nicotinamide mononucleotide (NMN) (and formulations prepared
using such crystal forms) may be used for treating, ameliorating,
mitigating, slowing, arresting, preventing or reversing
age-associated obesity in a subject. In some embodiments, the
invention relates to methods of treating, ameliorating, mitigating,
slowing, arresting, preventing or reversing age-associated
increases in blood lipid levels in a subject. In some embodiments,
the invention relates to methods of treating, ameliorating,
mitigating, slowing, arresting, preventing or reversing
age-associated loss of insulin sensitivity in a subject. In some
embodiments, the invention relates to methods of treating,
ameliorating, mitigating, slowing, arresting, preventing or
reversing age-associated impairment of memory function in a
subject. In some embodiments, the invention relates to methods of
treating, ameliorating, mitigating, slowing, arresting, preventing
or reversing age-associated decline in eye function in a subject.
In some embodiments, the invention relates to methods of treating,
ameliorating, mitigating, slowing, arresting, preventing or
reversing age-associated retinal degeneration in a subject. In some
embodiments, the invention relates to methods of treating,
ameliorating, mitigating, slowing, arresting, preventing or
reversing dry eye. In some embodiments, the invention relates to
methods of treating, ameliorating, mitigating, slowing, arresting,
preventing or reversing age-associated dry eye. In some
embodiments, the invention relates to methods of treating,
ameliorating, mitigating, slowing, arresting, preventing or
reversing infertility.
[0136] In some embodiments, the invention provides methods of
treating age-associated defects in neural stem/progenitor cell
(NSPC) functionality in a subject through administration of a
crystal form of NMN or a formulation prepared using such a crystal
form. In some embodiments, the invention provides methods of
reducing age-associated decrease in a NSPC population in a subject
through administration of a crystal form of NMN or a formulation
prepared using such a crystal form. In some embodiments, the
invention provides methods of maintaining at least one NSPC in a
subject through administration of a crystal form of NMN or a
formulation prepared using such a crystal form. In some
embodiments, the invention provides methods of enhancing NAD
biosynthesis in a subject through administration of a crystal form
of NMN or a formulation prepared using such a crystal form. In some
embodiments, the invention provides methods of promoting NSPC
proliferation in a subject, in which the methods comprise
administration of a crystal form of NMN, or a formulation prepared
using such a crystal form, to the subject. The methods of each of
these embodiments can comprise, consist essentially of, or consist
of administration of a therapeutically effective amount of a
crystal form of NMN or a formulation prepared using such a crystal
form.
[0137] In some embodiments, the invention provides methods of
increasing bone density levels in a subject. In some embodiments,
the invention provides methods of treating aberrantly low bone
density levels in a subject. In some embodiments, the invention
provides methods of treating an age-associated bone density
decrease in a subject. In some embodiments, the invention provides
methods of treating osteoporosis in a subject. In some embodiments,
the invention provides methods of preventing an age-associated bone
density decrease in a subject. The methods of each of these
embodiments can comprise, consist essentially of, or consist of
administration of a therapeutically effective amount of a crystal
form of NMN or a formulation prepared using such a crystal
form.
[0138] In certain embodiments, the invention relates to methods of
preventing, methods of reducing risk of, and methods of treating
various diseases associated with photoreceptor dysfunction,
including, without limitation, age-related macular degeneration
(AMD), inherited and acquired retinal diseases such as, without
limitation, retinitis pigmentosa (RP), rod and cone dystrophism,
and Leber's congenital amaurosis (LCA) by administration of NMN. In
various embodiments, administration of an NMN crystal form or a
formulation prepared using such a crystal form can be an effective
intervention for the prevention and/or treatment of orphan retinal
degenerative diseases including but not limited to rod dystrophy,
cone dystrophy, retinitis pigmentosa, other inherited retinal
degenerations, Leber's congenital amaurosis (LCA) and acquired
retinal degenerations such as, but not limited to, age-related
macular dengeration photoreceptor degeneration following retinal
detachment. In various embodiments, a crystal form of NMN or a
formulation prepared using such a crystal form can be administered
by any administration route known to skilled artisans, such as,
without limitation, oral, parenteral, intraocular, intraperitoneal,
intravenous or intramuscular routes. In various embodiments, NMN
can be administered with or without an excipient.
[0139] In some embodiments, NMN treats an age-related disease. In
certain embodiments, the age-related disease is Alzheimer's
disease, amniotropic lateral sclerosis, arthritis, atherosclerosis,
cachexia, cancer, cardiac hypertrophy, cardiac failure, cardiac
hypertrophy, cardiovascular disease, cataracts, colitis, chronic
obstructive pulmonary disease, dementia, diabetes mellitus,
frailty, heart disease, hepatic steatosis, high blood cholesterol,
high blood pressure, Huntington's disease, hyperglycemia,
hypertension, infertility, inflammatory bowel disease, insulin
resistance disorder, lethargy, metabolic syndrome, muscular
dystrophy, multiple sclerosis, neuropathy, nephropathy, obesity,
osteoporosis, Parkinson's disease, psoriasis, sarcopenia, sleep
disorders, sepsis and/or stroke. In some embodiments, the
mitochondrial disease is mitochondrial myopathy, diabetes mellitus
and deafness (DAD), Leber's hereditary optic neuropathy (LHON),
Leigh syndrome, neuropathy, ataxia, retinitis pigmentosa and
petosis (NARP), myoclonic epilepsy with ragged red fibers (MERRF),
myoneurogenic gastrointestinal encephalopathy (MNGIE),
mitochondrial myopathy, encephalomyopathy, lactic acidosis,
stroke-like symptoms (MELAS), Kearns-Sayre syndrome (KSS), chronic
progressive external opthalmoplegia (CPEO) and/or mtDNA
depletion.
[0140] Examples of diseases, disorders, or conditions associated
with mitochondrial dysfunction include, but are not limited to,
aging, aging-related diseases, mitochondrial diseases (e.g.,
Alper's disease, Barth syndrome, beta-oxidation defects,
carnitine-acyl-carnitine deficiency, carnitine deficiency, creatine
deficiency syndromes, co-enzyme Q10 deficiency, complex I
deficiency, complex II deficiency, complex III deficiency, complex
IV deficiency/COX deficiency, complex V deficiency, chronic
progressive external ophthalmoplegia syndrome, CPT I deficiency,
CPT II deficiency, Kearns-Sayre syndrome, lactic acidosis,
long-chain acyl-CoA dehydrongenase deficiency, Leigh syndrome, Luft
disease, glutaric aciduria type II, mitochondrial cytopathy,
mitochondrial DNA depletion, mitochondrial encephalopathy,
mitochondrial myopathy, and Pearson syndrome), metabolic diseases
and disorders (e.g., amino acid deficiency), diseases resulting
from mitochondrial and energy deficiency, lethargy, heart
disorders, cardiovascular disease, stroke, infarction, pulmonary
hypertension, ischemia, cachexia, sarcopenia, neurodegenerative
diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease), dementia, lipodystrophy, liver steatosis,
hepatitis, cirrhosis, kidney failure, preeclampsia, male
infertility, obesity, diabetes (e.g., diabetes type I), muscle
disorders, and muscle wasting.
[0141] In some embodiments, NMN is useful for promoting cell
viability (in various species), vascular remodeling, wound healing
and healing in general (e.g., treating wounds resulting from cuts,
scrapes, surgery, bodily insults, trauma, burns, abrasions,
sunburns, etc.). In some embodiments, the methods and compositions
are useful for promoting iron homeostasis and/or erythropoiesis. In
some embodiments, methods and compositions provided herein are
useful to promote successful organ and tissue transplantation, or
to promote recovery from organ and tissue transplantation. In some
embodiments, provided methods and compositions are useful for
preserving cells and organs. In some embodiments, methods and
compositions provided herein have cosmetic applications, for
example for treating conditions associated with mitochondrial
dysfunction which relate to the skin or scalp/hair, such as skin
aging (e.g., loss in volume and elasticity, discoloration, liver
spots (lentigo senislis)), wrinkles, baldness, and loss of hair
pigmentation. In some embodiments, agents or compositions described
herein are useful for products or methods relating to cosmetics,
energy drinks, and/or animal and plant industries (e.g. livestock,
pets and agricultural products).
[0142] Subjects that may be treated as described herein include
eukaryotes, such as mammals, e.g., humans, ovines, bovines,
equines, porcines, canines, felines, non-human primate, mice, and
rats. Cells that may be treated include eukaryotic cells, e.g.,
from a subject described above, or plant cells, yeast cells and
prokaryotic cells, e.g., bacterial cells. For example, NMN may be
administered to farm animals to improve their ability to withstand
farming conditions longer.
[0143] The compound may also be used to increase lifespan, stress
resistance, and resistance to apoptosis in plants. In certain
embodiments, NMN is applied to plants, e.g., on a periodic basis,
or to fungi. In other embodiments, plants are genetically modified
to produce NMN. In other embodiments, plants and fruits are treated
with NMN prior to picking and shipping to increase resistance to
damage during shipping. Plant seeds may also be contacted with NMN,
e.g., to preverse them.
[0144] NMN may also be used to increase lifespan, stress resistance
and resistance to apoptosis in insects. In certain embodiments, NMN
would be applied to useful insects, e.g., bees and other insects
that are involved in pollination of plants. In preferred
embodiments, NMN would be applied to bees involved in the
production of honey. Generally, the methods described herein may be
applied to any organism, e.g., eukaryote, that may have commercial
importance. For example, they can be applied to fish (aquaculture),
shrimp, pigs, and birds (e.g., chicken and fowl).
[0145] Other uses of NMN include favorably modulating the
microbiome or serving as a form of vitamin B3. As the precursor of
NAD, NMN could also serve in assays for diseases and conditions
affected by NAD biosynthesis, such as use as a standard.
[0146] As used herein, a therapeutic that "prevents" a disorder or
condition refers to a compound that, in a statistical sample,
reduces the occurrence or frequency of the disorder or condition in
the treated sample relative to an untreated control sample, or
delays the onset or reduces the severity of one or more symptoms of
the disorder or condition relative to the untreated control sample.
Thus, prevention of cancer includes, for example, reducing the
number of detectable cancerous growths in a population of patients
receiving a prophylactic treatment relative to an untreated control
population, and/or delaying the appearance of detectable cancerous
growths in a treated population versus an untreated control
population, e.g., by a statistically and/or clinically significant
amount. Prevention of an infection includes, for example, reducing
the number of diagnoses of the infection in a treated population
versus an untreated control population, and/or delaying the onset
of symptoms of the infection in a treated population versus an
untreated control population. Prevention of pain includes, for
example, reducing the magnitude of, or alternatively delaying, pain
sensations experienced by subjects in a treated population versus
an untreated control population.
[0147] The term "treating" includes prophylactic and/or therapeutic
treatments. The term "prophylactic or therapeutic" treatment is
art-recognized and includes administration to the host of one or
more of the subject compositions. If it is administered prior to
clinical manifestation of the unwanted condition (e.g., disease or
other unwanted state of the host animal) then the treatment is
prophylactic (i.e., it protects the host against developing the
unwanted condition), whereas if it is administered after
manifestation of the unwanted condition, the treatment is
therapeutic (i.e., it is intended to diminish, ameliorate, or
stabilize the existing unwanted condition or side effects
thereof).
Pharmaceutical Compositions
[0148] In certain embodiments, the present invention relates to
pharmaceutical compositions comprising a crystalline compound of
formula (I) and one or more pharmaceutically acceptable excipients,
as well as formulations prepared using such a crystalline compound
and one or more pharmaceutically acceptable excipients. In certain
embodiments, the pharmaceutical preparations may be for use in
treating or preventing a condition or disease as described herein.
In certain embodiments, the pharmaceutical preparations have a low
enough pyrogen activity to be suitable for intravenous use in a
human patient. In certain embodiments, the invention also relates
to preparations suitable for nutraceutical, veterinary, and
agriculturally-relevant uses.
[0149] Exemplary pharmaceutically acceptable excipients are
presented herein, and include, for example binders, disintegrating
agents, lubricants, corrigents, solubilizing agents, suspension
aids, emulsifying agents, coating agents, cyclodextrins, and/or
buffers. Although the dosage will vary depending on the symptoms,
age and body weight of the patient, the nature and severity of the
disorder to be treated or prevented, the route of administration
and the form of the drug, in general, a daily dosage of from 0.01
to 3000 mg of the compound is recommended for an adult human
patient, and this may be administered in a single dose or in
divided doses. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect.
[0150] The precise time of administration and/or amount of the
composition that will yield the most effective results in terms of
efficacy of treatment in a given patient will depend upon the
activity, pharmacokinetics, and bioavailability of a particular
compound, physiological condition of the patient (including age,
sex, disease type and stage, general physical condition,
responsiveness to a given dosage, and type of medication), route of
administration, etc. However, the above guidelines can be used as
the basis for fine-tuning the treatment, e.g., determining the
optimum time and/or amount of administration, which will require no
more than routine experimentation consisting of monitoring the
subject and adjusting the dosage and/or timing.
[0151] In certain embodiments, the individual to which the
composition is administered is a mammal such as a human, or a
non-human mammal. When administered to an animal, such as a human,
the composition or the compound is preferably administered as a
pharmaceutical composition comprising, for example, a compound of
the invention and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known in the art and
include, for example, aqueous solutions such as water or
physiologically buffered saline or other solvents or vehicles such
as glycols, glycerol, oils such as olive oil, or injectable organic
esters. In a preferred embodiment, when such pharmaceutical
compositions are for human administration, particularly for
invasive routes of administration (i.e., routes, such as injection
or implantation, that circumvent transport or diffusion through an
epithelial barrier), the aqueous solution is pyrogen-free, or
substantially pyrogen-free. The excipients can be chosen, for
example, to effect delayed release of an agent or to selectively
target one or more cells, tissues or organs. The pharmaceutical
composition can be in dosage unit form such as tablet, capsule
(including sprinkle capsule and gelatin capsule), granule, lyophile
for reconstitution, powder, solution, syrup, suppository, injection
or the like. The composition can also be present in a transdermal
delivery system, e.g., a skin patch. The composition can also be
present in a solution suitable for topical administration, such as
an eye drop, through ophthalmic mucous membrane administration.
[0152] A pharmaceutically acceptable carrier can contain
physiologically acceptable agents that act, for example, to
stabilize, increase solubility or to increase the absorption of a
compound such as a compound of the invention. Such physiologically
acceptable agents include, for example, carbohydrates, such as
glucose, sucrose or dextrans, antioxidants, such as ascorbic acid
or glutathione, chelating agents, low molecular weight proteins or
other stabilizers or excipients. The choice of a pharmaceutically
acceptable carrier, including a physiologically acceptable agent,
depends, for example, on the route of administration of the
composition. The preparation or pharmaceutical composition can be a
self-emulsifying drug delivery system or a self-microemulsifying
drug delivery system. The pharmaceutical composition (preparation)
also can be a liposome or other polymer matrix, which can have
incorporated therein, for example, a compound of the invention.
Liposomes, for example, which comprise phospholipids or other
lipids, are nontoxic, physiologically acceptable and metabolizable
carriers that are relatively simple to make and administer.
[0153] The phrase "pharmaceutically acceptable" is employed herein
to refer to those 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.
[0154] The phrase "pharmaceutically acceptable carrier" as used
herein means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material. Each carrier must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as pharmaceutically
acceptable carriers include: (1) sugars, such as lactose, glucose
and sucrose; (2) starches, such as corn starch and potato starch;
(3) cellulose, and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such
as cocoa butter and suppository waxes; (9) oils, such as peanut
oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil
and soybean oil; (10) glycols, such as propylene glycol; (11)
polyols, such as glycerin, sorbitol, mannitol and polyethylene
glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13)
agar; (14) buffering agents, such as magnesium hydroxide and
aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;
(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) phosphate buffer solutions; and (21) other non-toxic
compatible substances employed in pharmaceutical formulations. In
certain embodiments, pharmaceutical compositions of the present
invention are non-pyrogenic, i.e., do not induce significant
temperature elevations when administered to a patient.
[0155] The term "pharmaceutically acceptable salt" refers to the
relatively non-toxic, inorganic and organic acid addition salts of
the compounds. These salts can be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting a purified compound in its free base form with a suitable
organic or inorganic acid, and isolating the salt thus formed.
Representative salts include the hydrobromide, hydrochloride,
sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,
palmitate, stearate, laurate, benzoate, lactate, phosphate,
tosylate, citrate, maleate, fumarate, succinate, tartrate,
naphthylate, mesylate, glucoheptonate, lactobionate,
laurylsulphonate salts, and amino acid salts, and the like.
Preparation of the crystalline salts is detailed in the Examples,
below (See, for example, Berge et al. (1977) "Pharmaceutical
Salts", J. Pharm. Sci. 66: 1-19.).
[0156] In other cases, the compounds useful in the methods of the
present invention may contain one or more acidic functional groups
and, thus, are capable of forming pharmaceutically acceptable salts
with pharmaceutically acceptable bases. The term "pharmaceutically
acceptable salts" in these instances refers to the relatively
non-toxic inorganic and organic base addition salts of a compound.
These salts can likewise be prepared in situ during the final
isolation and purification of the compound, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate, or bicarbonate of
a pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary, or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts,
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine, and the
like (see, for example, Berge et al., supra).
[0157] A pharmaceutical composition (preparation) can be
administered to a subject by any of a number of routes of
administration including, for example, orally (for example,
drenches as in aqueous or non-aqueous solutions or suspensions,
tablets, capsules (including sprinkle capsules and gelatin
capsules), boluses, powders, granules, pastes for application to
the tongue); absorption through the oral mucosa (e.g.,
sublingually); anally, rectally or vaginally (for example, as a
pessary, cream or foam); parenterally (including intramuscularly,
intravenously, subcutaneously or intrathecally as, for example, a
sterile solution or suspension); nasally; intraperitoneally;
subcutaneously; transdermally (for example as a patch applied to
the skin); and topically (for example, as a cream, ointment or
spray applied to the skin, or as an eye drop). The compound may
also be formulated for inhalation. In certain embodiments, a
compound may be simply dissolved or suspended in sterile water.
Details of appropriate routes of administration and compositions
suitable for same can be found in, for example, U.S. Pat. Nos.
6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970
and 4,172,896, as well as in patents cited therein.
[0158] The formulations may conveniently be presented in unit
dosage form and may be prepared by any methods well known in the
art of pharmacy. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will vary depending upon the host being treated, the particular
mode of administration. The amount of active ingredient that can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 1 percent to about ninety-nine percent
of active ingredient, preferably from about 5 percent to about 70
percent, most preferably from about 10 percent to about 30
percent.
[0159] Methods of preparing these formulations or compositions
include the step of bringing into association an active compound,
such as a compound of the invention, with the carrier and,
optionally, one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association a compound of the present invention with liquid
carriers, or finely divided solid carriers, or both, and then, if
necessary, shaping the product.
[0160] Formulations of the invention suitable for oral
administration may be in the form of capsules (including sprinkle
capsules and gelatin capsules), cachets, pills, tablets, lozenges
(using a flavored basis, usually sucrose and acacia or tragacanth),
lyophile, powders, granules, or as a solution or a suspension in an
aqueous or non-aqueous liquid, or as an oil-in-water or
water-in-oil liquid emulsion, or as an elixir or syrup, or as
pastilles (using an inert base, such as gelatin and glycerin, or
sucrose and acacia) and/or as mouthwashes and the like, each
containing a predetermined amount of a compound of the present
invention as an active ingredient. Compositions or compounds may
also be administered as a bolus, electuary or paste.
[0161] To prepare solid dosage forms for oral administration
capsules (including sprinkle capsules and gelatin capsules),
tablets, pills, dragees, powders, granules and the like), the
active ingredient is mixed with one or more pharmaceutically
acceptable carriers, such as sodium citrate or dicalcium phosphate,
and/or any of the following: (1) fillers or extenders, such as
starches, lactose, sucrose, glucose, mannitol, and/or silicic acid;
(2) binders, such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;
(3) humectants, such as glycerol; (4) disintegrating agents, such
as agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain silicates, and sodium carbonate; (5) solution
retarding agents, such as paraffin; (6) absorption accelerators,
such as quaternary ammonium compounds; (7) wetting agents, such as,
for example, cetyl alcohol and glycerol monostearate; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such
a talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate, and mixtures thereof; (10)
complexing agents, such as, modified and unmodified cyclodextrins;
and (11) coloring agents. In the case of capsules (including
sprinkle capsules and gelatin capsules), tablets and pills, the
pharmaceutical compositions may also comprise buffering agents.
Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0162] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0163] The tablets, and other solid dosage forms of the
pharmaceutical compositions, such as dragees, capsules (including
sprinkle capsules and gelatin capsules), pills and granules, may
optionally be scored or prepared with coatings and shells, such as
enteric coatings and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as
to provide slow or controlled release of the active ingredient
therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile, other
polymer matrices, liposomes and/or microspheres. They may be
sterilized by, for example, filtration through a bacteria-retaining
filter, or by incorporating sterilizing agents in the form of
sterile solid compositions that can be dissolved in sterile water,
or some other sterile injectable medium immediately before use.
These compositions may also optionally contain opacifying agents
and may be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples
of embedding compositions that can be used include polymeric
substances and waxes. The active ingredient can also be in
micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0164] Liquid dosage forms useful for oral administration include
pharmaceutically acceptable emulsions, lyophiles for
reconstitution, microemulsions, solutions, suspensions, syrups and
elixirs. In addition to the active ingredient, the liquid dosage
forms may contain inert diluents commonly used in the art, such as,
for example, water or other solvents, cyclodextrins and derivatives
thereof, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0165] Besides inert diluents, the compositions of the present
invention can also include adjuvants such as wetting agents,
lubricants, emulsifying and suspending agents such as sodium lauryl
sulfate and magnesium stearate, or sweetening, flavoring, coloring,
perfuming, preservative, or anti-oxidant agents.
[0166] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0167] Formulations of the pharmaceutical compositions for rectal,
vaginal, or urethral administration may be presented as a
suppository, which may be prepared by mixing one or more active
compounds with one or more suitable nonirritating excipients or
carriers comprising, for example, cocoa butter, polyethylene
glycol, a suppository wax or a salicylate, and which is solid at
room temperature, but liquid at body temperature and, therefore,
will melt in the rectum or vaginal cavity and release the active
compound.
[0168] Formulations of the pharmaceutical compositions for
administration to the mouth may be presented as a mouthwash, or an
oral spray, or an oral ointment.
[0169] Alternatively or additionally, compositions can be
formulated for delivery via a catheter, stent, wire, or other
intraluminal device. Delivery via such devices may be especially
useful for delivery to the bladder, urethra, ureter, rectum, or
intestine.
[0170] Formulations which are suitable for vaginal administration
also include pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing such carriers as are known in the art
to be appropriate.
[0171] Dosage forms for the topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches and inhalants. The active compound may be mixed
under sterile conditions with a pharmaceutically acceptable
carrier, and with any preservatives, buffers, or propellants that
may be required.
[0172] The ointments, pastes, creams and gels may contain, in
addition to an active compound, excipients, such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0173] Powders and sprays can contain, in addition to an active
compound, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of
these substances. Sprays can additionally contain customary
propellants, such as chlorofluorohydrocarbons and volatile
unsubstituted hydrocarbons, such as butane and propane.
[0174] The compounds described herein can be alternatively
administered by aerosol. This is accomplished by preparing an
aqueous aerosol, liposomal preparation, or solid particles
containing the composition. A nonaqueous (e.g., fluorocarbon
propellant) suspension could be used. Sonic nebulizers are
preferred because they minimize exposing the agent to shear, which
can result in degradation of the compound.
[0175] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular composition, but typically include nonionic surfactants
(Tweens, Pluronics, sorbitan esters, lecithin, Cremophors),
pharmaceutically acceptable co-solvents such as polyethylene
glycol, innocuous proteins like serum albumin, oleic acid, amino
acids such as glycine, buffers, salts, sugars, or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0176] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
active compound in the proper medium. Absorption enhancers can also
be used to increase the flux of the compound across the skin. The
rate of such flux can be controlled by either providing a rate
controlling membrane or dispersing the compound in a polymer matrix
or gel.
[0177] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention. Exemplary ophthalmic formulations are described in
U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697 and
2005/004074 and U.S. Pat. No. 6,583,124, the contents of which are
incorporated herein by reference. If desired, liquid ophthalmic
formulations have properties similar to that of lacrimal fluids,
aqueous humor or vitreous humor or are compatable with such fluids.
A preferred route of administration is local administration (e.g.,
topical administration, such as eye drops, or administration via an
implant).
[0178] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and infusion.
Pharmaceutical compositions suitable for parenteral administration
comprise one or more active compounds in combination with one or
more pharmaceutically acceptable sterile isotonic aqueous or
nonaqueous solutions, dispersions, suspensions or emulsions, or
sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0179] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a ligand,
drug, or other material other than directly into the central
nervous system, such that it enters the patient's system and thus,
is subject to metabolism and other like processes, for example,
subcutaneous administration.
[0180] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0181] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0182] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0183] Injectable depot forms are made by forming microencapsulated
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions that are
compatible with body tissue.
[0184] The preparations of agents may be given orally,
parenterally, topically, or rectally. They are, of course, given by
forms suitable for each administration route. For example, they are
administered in tablets or capsule form, by injection, inhalation,
eye lotion, ointment, suppository, infusion; topically by lotion or
ointment; and rectally by suppositories. Oral administration is
preferred.
[0185] For use in the methods of this invention, active compounds
can be given per se or as a pharmaceutical composition containing,
for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active
ingredient in combination with a pharmaceutically acceptable
carrier.
[0186] Methods of introduction may also be provided by rechargeable
or biodegradable devices. Various slow release polymeric devices
have been developed and tested in vivo in recent years for the
controlled delivery of drugs, including proteinacious
biopharmaceuticals. A variety of biocompatible polymers (including
hydrogels), including both biodegradable and non-degradable
polymers, can be used to form an implant for the sustained release
of a compound at a particular target site.
[0187] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally, and topically, as
by powders, ointments or drops, including buccally and
sublingually.
[0188] Regardless of the route of administration selected, the
compounds, which may be used in a suitable hydrated form, and/or
the pharmaceutical compositions of the present invention, are
formulated into pharmaceutically acceptable dosage forms by
conventional methods known to those of skill in the art.
[0189] Actual dosage levels of the active ingredients in the
pharmaceutical compositions may be varied so as to obtain an amount
of the active ingredient that is effective to achieve the desired
therapeutic response for a particular patient, composition, and
mode of administration, without being toxic to the patient.
[0190] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound or
combination of compounds employed, or the ester, salt or amide
thereof, the route of administration, the time of administration,
the rate of excretion of the particular compound(s) being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compound(s)
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts. In general, the compositions of this
invention may be provided in an aqueous solution containing about
0.1-30% w/v of a compound disclosed herein, among other substances,
for parenteral administration. Typical dose ranges are from about
0.01 to about 50 mg/kg of body weight per day, given in 1 single or
2-4 divided doses. Each divided dose may contain the same or
different compounds of the invention.
[0191] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the therapeutically effective
amount of the pharmaceutical composition required. For example, the
physician or veterinarian could start doses of the pharmaceutical
composition or compound at levels lower than that required in order
to achieve the desired therapeutic effect and gradually increase
the dosage until the desired effect is achieved. A "therapeutically
effective amount" of a compound with respect to the subject method
of treatment, refers to an amount of the compound(s) in a
preparation which, when administered as part of a desired dosage
regimen (to a mammal, preferably a human) alleviates a symptom,
ameliorates a condition, or slows the onset of disease conditions
according to clinically acceptable standards for the disorder or
condition to be treated or the cosmetic purpose, e.g., at a
reasonable benefit/risk ratio applicable to any medical treatment.
It is generally understood that the effective amount of the
compound will vary according to the weight, sex, age, and medical
history of the subject. Other factors which influence the effective
amount may include, but are not limited to, the severity of the
patient's condition, the disorder being treated, the stability of
the compound, and, if desired, another type of therapeutic agent
being administered with the compound of the invention. A larger
total dose can be delivered by multiple administrations of the
agent. Methods to determine efficacy and dosage are known to those
skilled in the art (Isselbacher et al. (1996) Harrison's Principles
of Internal Medicine 13 ed., 1814-1882, herein incorporated by
reference).
[0192] In general, a suitable daily dose of an active compound used
in the compositions and methods of the invention will be that
amount of the compound that is the lowest dose effective to produce
a therapeutic effect. Such an effective dose will generally depend
upon the factors described above.
[0193] If desired, the effective daily dose of the active compound
may be administered as one, two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms. In certain
embodiments of the present invention, the active compound may be
administered two or three times daily. In preferred embodiments,
the active compound will be administered once daily.
[0194] The patient receiving this treatment is any animal in need,
including primates, in particular humans, and other mammals such as
equines, cattle, swine and sheep; and poultry and pets in
general.
[0195] In certain embodiments, compounds of the invention may be
used alone or conjointly administered with another type of
therapeutic agent. As used herein, the phrase "conjoint
administration" refers to any form of administration of two or more
different therapeutic compounds such that the second compound is
administered while the previously administered therapeutic compound
is still effective in the body (e.g., the two compounds are
simultaneously effective in the patient, which may include
synergistic effects of the two compounds). For example, the
different therapeutic compounds can be administered either in the
same formulation or in a separate formulation, either concomitantly
or sequentially. In certain embodiments, the different therapeutic
compounds can be administered within one hour, 12 hours, 24 hours,
36 hours, 48 hours, 72 hours, or a week of one another. Thus, an
individual who receives such treatment can benefit from a combined
effect of different therapeutic compounds.
[0196] This invention includes the use of pharmaceutically
acceptable salts of compounds of the invention in the compositions
and methods of the present invention. In certain embodiments,
contemplated salts of the invention include, but are not limited
to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In
certain embodiments, contemplated salts of the invention include,
but are not limited to, L-arginine, benenthamine, benzathine,
betaine, calcium hydroxide, choline, deanol, diethanolamine,
diethylamine, 2-(diethylamino)ethanol, ethanolamine,
ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole,
lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine,
piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium,
triethanolamine, tromethamine, and zinc salts. In certain
embodiments, contemplated salts of the invention include, but are
not limited to, Na, Ca, K, Mg, Zn or other metal salts.
[0197] The pharmaceutically acceptable acid addition salts can also
exist as various solvates, such as with water, methanol, ethanol,
dimethylformamide, dimethylsulfoxide, and the like. Mixtures of
such solvates can also be prepared. The source of such solvate can
be from the solvent of crystallization, inherent in the solvent of
preparation or crystallization, or adventitious to such solvent. In
some embodiments, a solvate of a disclosed compound can be a
dimethylsulfoxide solvate.
[0198] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0199] Examples of pharmaceutically acceptable antioxidants
include: (1) water-soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal-chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0200] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
Examples
Analytical Methods for Examples 1-5
X-Ray Powder Diffraction
[0201] X-Ray Powder Diffraction patterns were collected on a Bruker
D8 diffractometer using Cu K.alpha. radiation (40 kV, 40 mA),
.theta.-2.theta. goniometer, and divergence of V4 and receiving
slits, a Ge monochromator and a Lynxeye detector. The instrument is
performance checked using a certified Corundum standard (NIST
1976). The software used for data collection was Diffrac Plus XRD
Commander v2.6.1 and the data were analysed and presented using
Diffrac Plus EVA v15.0.0.0.
[0202] Samples were run under ambient conditions as flat plate
specimens using powder as received. The sample was gently packed
into a cavity cut into polished, zero-background (510) silicon
wafer. The sample was rotated in its own plane during analysis. The
details of the data collection are: [0203] Angular range: 2 to
42.degree. 2.theta. [0204] Step size: 0.05.degree. 2.theta. [0205]
Collection time: 0.5 s/step
HPLC
[0206] Purity analysis was performed on an Agilent HP1100 series
system equipped with a diode array detector and using ChemStation
software vB.04.03 using the method detailed below in Table 1.
TABLE-US-00001 TABLE 1 HPLC Parameters Parameter Value Type of
method Reversed phase with gradient elution Sample Preparation 1
mg/mL in aqueous 20 mM ammonium acetate, pH 5.0 Column Waters
Atlantis C18, 100 .ANG., 3 .mu.M Column Temperature 25.degree. C.
Injection Volume 5 .mu.L Detector Wavelength, 254 nm, 4 nm
Bandwidth Flow Rate 1.0 mL/min Mobile Phase A 20 mM aqueous
ammonium acetate, pH 5.0 Mobile Phase B methanol Gradient Timetable
Time (min) % Mobile Phase A 0 100 5 5 8 5 8.1 100 16.1 100
Polarised Light Microscopy (PLM)
[0207] Samples were studied on a Leica LM/DM polarised light
microscope with a digital video camera for image capture. A small
amount of each sample was placed on a glass slide, 15 mounted in
immersion oil and covered with a glass slip, the individual
particles being separated as well as possible. The sample was
viewed with appropriate magnification and partially polarised
light, coupled to a .lamda. false-color filter.
Example 1: Synthesis of Form 1 by Crystallisation from Methanol
[0208] Amorphous nicotinamide mononucleotide (565 mg) was weighed
into a glass vial and methanol (10.0 mL) added. The resulting
slurry was stirred at room temperature for 4 h and then a further
portion of MeOH (10.0 mL) added. The white solid present was
isolated by filtration and then dried under vacuum at room
temperature for ca. 16 h to give crystalline beta nicotinamide
mononucleotide Form 1, as shown by XRPD analysis. (459 mg, 81%
recovery).
Example 2: Synthesis of Form 1 by Crystallisation from Water
[0209] Amorphous beta nicotinamide mononucleotide (605 mg) was
weighed into a glass vial and deionised water (600 .mu.L) added.
After brief vortexing, a clear solution was formed. A portion of
this solution (ca. 200 .mu.L) was dispensed into a separate vial
and cooled to 5.degree. C. for ca. 16 h. An evolved white solid was
isolated by filtration and shown by XRPD analysis to be crystalline
beta nicotinamide mononucleotide Form 1 (yield not determined).
Example 3: Synthesis of Form 1 by Vapor Diffusion for SCXRD
[0210] Amorphous beta nicotinamide mononucleotide (605 mg) was
weighed into a glass vial and deionised water (600 .mu.L) added.
After brief vortexing, a clear solution was formed. A portion of
this solution (100 .mu.L) was dispensed into a separate vial, which
itself was placed into a larger vial containing methanol (500
.mu.L) such that vapor can diffuse freely between both vials. The
larger vial was sealed and stored at RT for ca. 16 h, after which
time a white solid had evolved. This solid was sampled and shown by
SCXRD to be crystalline beta nicotinamide mononucleotide Form
1.
Example 4: Synthesis of Form 2
[0211] Amorphous beta nicotinamide mononucleotide (568 mg) was
weighed into a glass vial and DMSO (10.0 mL) added. The resulting
slurry was stirred at room temperature for ca. 20 h. The white
solid present was then isolated by filtration, washed with acetone
(3.times.1 mL) and dried under vacuum at room temperature for ca.
16 h to give crystalline beta nicotinamide mononucleotide Form 2
(501 mg, 72% recovery*). * based on assumption that material is a
DMSO mono-solvate
Example 5: Three Month Stability and Forced Degradation Study for
Amorphous and Crystalline NMN
[0212] Samples of amorphous NMN and NMN crystalline Form 1 were
stored as solids at 25.degree. C./0% Relative Humidity (RH), and
40.degree. C./75% RH for three months. The samples were prepared in
duplicate with an offset of 2 weeks. Each replicate was stored in a
different container.
[0213] Replicate 1 was charged in an open HPLC vial that was placed
in a sealed scintillation vial containing a saturated solution of
the relevant inorganic salt (Table 2). These samples were analysed
by HPLC, 1H NMR, XRPD and Polarized Light Microscopy (PLM) at 4, 8,
and 12 week time points.
[0214] Replicate 2 was stored in sealed box with a recipient
containing a saturated solution of the relevant inorganic salt
(Table 2). These samples were analysed by HPLC at 2, 6, 10 and 12
week time points. Samples at 25.degree. C./0% RH were stored in a
sealed box containing a desiccant agent (P.sub.2O.sub.5).
TABLE-US-00002 TABLE 2 Inorganic Salt/ Conditions Dessicant Agent
25.degree. C./0% RH P.sub.2O.sub.5 25.degree. C./60% RH
NH.sub.4NO.sub.3 25.degree. C./97% RH K.sub.2SO.sub.4 40.degree.
C./75% RH NaCl
[0215] Amorphous material remained unchanged in terms of solid form
and particle morphology after storage for 12 weeks at 25.degree.
C., 0% RH. A drop in purity was observed from 98.2% (time 0) to
95.5% (time 12 weeks). The main growing impurity observed by HPLC
eluted at RRT 1.73 and corresponded to nicotinamide (2.8% at the 12
weeks' time point). (See Tables 3 and 4.)
TABLE-US-00003 TABLE 3 HPLC Purity Profiles of Amorphous NMN and
Crystal Form 1 NMN at t = 0 J07086 RME-1304-063-01 (Amorphous)
(Crystalline) RRT Area % RRT Area % 0.70 0.47 0.69 0.15 0.76 0.10
0.75 0.09 1.00 98.56 1.00 99.52 -- -- 1.31 0.06 -- -- 1.46 0.01 --
-- 1.48 0.03 -- -- 1.53 0.01 1.76 0.87 1.74 0.12
[0216] The amorphous material crystallized when stored under 25% or
greater relative humidity, giving Form 1 with acicular morphology.
Chemical purity was shown to decrease upon storage at all the
tested conditions, especially at 40.degree. C./75% RH. The main
growing impurity observed was also nicotinamide (RRT=1.73). Purity
of the material after 12 weeks at 40.degree. C./75% RH was
determined as 67.5% (replicate 2) and the nicotinamide abundance
was 20.6%.
[0217] Crystalline Form 1 remained unchanged in terms of solid form
and particle morphology at all conditions tested. The material
remained chemically pure (.about.99.3%) after 4 weeks of storage at
all the tested conditions. A slight drop in purity was observed
beyond that time point. Storage at 25.degree. C./0% RH proved to be
the most favorable conditions for sample stability as the analysis
after 12 weeks showed the sample to be 98.9% pure and 0.38%
nicotinamide. The largest change in purity was observed for the
samples stored at high temperature (40.degree. C.) or high humidity
(75% RH, 97% RH). Purity dropped from 99.5% to 95.8% after 12 weeks
at 25.degree./97% RH and to 90.7% after 12 weeks at 40.degree.
C./75% RH. The most abundant impurity at all conditions was
nicotinamide (RRT=1.73 min). This impurity increased from 0.12%
(time 0) to 3.46% after 12 weeks at both, 40.degree. C./75% RH and
25.degree. C./97% RH.
[0218] The HPLC results were consistent among replicates, however,
faster degradation was observed with Replicate 1. A potential
influence may have been the smaller size of the containers which
required less time to equilibrate to the corresponding storage
conditions. Tables 4-7 provide results obtained for Replicates 1
and 2.
TABLE-US-00004 TABLE 4 Stability and Forced Degradation Study:
Results for Amorphous Material Replicate 1 Known Time Purity
Impurity* Sample ID Conditions Point Observations XRPD (%) (%) PLM
J07087_25_0_4w 25.degree. C., 4 weeks white solid Unchanged 96.9
1.81 Unchanged J07087_25_0_8w 0% RH 8 weeks white solid Unchanged
95.8 2.44 Unchanged J07087_25_0_12w 12 weeks white solid Unchanged
95.0 2.81 Unchanged J07087_25_60_4w 25.degree. C., 4 weeks brown
solid Form 1 96.9 2.13 acicular 60% RH particles up to ~75-100
.mu.m J07087_25_60_8w 8 weeks brown solid, Form 1 95.2 3.46 --
compacted J07087_25_60_12w 12 weeks brown solid, Form 1 95.7 3.46
acicular compacted particles up to ~75-100 .mu.m J07087_25_97_4w
25.degree. C., 4 weeks white solid, Form 1 97.1 2.44 acicular 97%
RH very wet particles up to ~75-100 .mu.m J07087_25_97_8w 8 weeks
partially n/a -- -- -- deliquesced. yellow liquid J07087_25_97_12w
12 weeks deliquesced. n/a -- -- -- yellow liquid J07087_40_75_4w
40.degree. C., 4 weeks brown Form 1 91.3 6.70 acicular 75% RH
compacted particles up to solid ~75-100 .mu.m J07087_40_75_8w 8
weeks brown Form 1 76.9 14.23 -- compacted solid J07087_40_75_12w
12 weeks black solid, Form 1 -- -- -- very wet.sup.$ *nicotinamide
(RRT = 1.73 min) .sup.$HPLC analysis not performed as weight could
not be stabilised for sample preparation. .sup.1H NMR spectra were
consistent with the stmcture of the materials of all samples.
TABLE-US-00005 TABLE 5 Stability and Forced Degradation Study:
Results for Crystalline Material Replicate 1 Known Time Purity
Impurity Sample ID Conditions Point Observations XRPD (%) * (%) PLM
RME-1304-63- 25.degree. C., 4 weeks white solid Unchanged 99.4 0.25
acicular 01_25_0_4w 0% RH particles up to~75 .mu.m RME-1304-63- 8
weeks white solid Unchanged 99.2 0.33 acicular 01_25_0_8w particles
up to~75 .mu.m RME-1304-63- 12 weeks white solid Unchanged 98.9
0.38 acicular 01_25_0_12w particles up to~75 .mu.m RME-1304-63-
25.degree. C., 4 weeks light brown Unchanged 99.3 0.34 acicular
01_25_60_4w 60% RH solid particles up to~75 .mu.m RME-1304-63- 8
weeks light brown Unchanged 98.9 0.63 acicular 01_25_60_8w solid,
particles up loose particles to~75 .mu.m RME-1304-63- 12 weeks
light brown Unchanged 98.2 0.98 acicular 01_25_60_12w solid,
particles up loose particles to~75 .mu.m RME-1304-63- 25.degree.
C., 4 weeks white solid Unchanged 99.3 0.49 acicular 01_25_97_4w
97% RH particles < 75 .mu.m RME-1304-63- 8 weeks white solid
Unchanged 98.3 1.38 acicular 01_25_97_8w particles up to 75-100
.mu.m RME-1304-63- 12 weeks white solid, Unchanged 95.8.sup.$ 3.46
acicular 01_25_97_12w wet particles < 75 .mu.m RME-1304-63-
40.degree. C., 4 weeks white solid Unchanged 99.2 0.46 acicular
01_25_75_4w 75% RH particles up to~75 .mu.m RME-1304-63- 8 weeks
white solid Unchanged 97.7 1.38 acicular 01_25_75_8w with orange
particles up spots to~75 .mu.m RME-1304-63- 12 weeks brown solid,
Unchanged 90.7 3.46 acicular 01_25_75_12w very wet particles <
75 .mu.m *nicotinamide (RRT = 1.73 min) .sup.$Weight could not be
stabilised. Approximate value was taken. .sup.1HNMR spectra were
consistent with the structure of the materials of all samples.
TABLE-US-00006 TABLE 6 Forced Degradation Study Results for
Amorphous Material Replicate 2 Known Time Purity Impurity * Sample
ID Conditions Point Observations (%) (%) J07087_25_60_2w 25.degree.
C., 2 weeks light yellow solid, 97.4 2.14 60% RH very compacted
J07087_25_60_6w 6 weeks light brown, 97.4 2.06 compacted
J07087_25_60_10w 10 weeks light brown, 96.0 2.92 compacted
J07087_R2_25_60_12w 12 weeks light brown, 96.2 2.32 compacted
J07087_25_97_2w 25.degree. C., 2 weeks white compacted 97.8 1.79
97% RH solid J07087_25_97_6w 6 weeks white solid, 96.5 3.01 very
wet J07087_25_97_10w 10 weeks white solid, 94.9 4.39 very wet
J07087_25_97_12w 12 weeks white solid, 91.5 7.02 very wet
J07087_40_75_2w 40.degree. C., 2 weeks brown compacted 94.6 4.61
75% RH solid J07087_40_75_6w 6 weeks dark brown solid 88.0 8.22
J07087_40_75_10w 10 weeks black solid 75.9 16.18
J07087_R2_40_75_12w 12 weeks black solid, 67.5.sup.$ 20.60 very wet
* nicotinamide (RRT = 1.73 min) .sup.$Weight could not be
stabilised. Approximate value was taken.
TABLE-US-00007 TABLE 7 Forced Degradation Study Results for
Amorphous Material Replicate 2 Known Sample ID Time Purity
Impurity* RME-1304-63-01-.sub.-- Conditions Point Observations (%)
(%) _25_60_2w 25.degree. C., 2 weeks white solid 99.6 0.19
_25_60_6w 60% RH 6 weeks light brown 99.5 0.21 compacted solid
_25_60_10w 10 weeks light brown 99.1 0.35 compacted solid
R2_25_60_12w 12 weeks light brown 98.8 0.23 compacted solid
_25_97_2w 25.degree. C., 2 weeks white solid 99.5 0.22 _25_97_6w
97% RH 6 weeks white solid, 99.4 0.38 very wet _25_97_10w 10 weeks
white solid, wet 98.6 0.77 R2_25_97_12w 12 weeks white solid, wet
98.4 0.55 _40_75_2w 40.degree. C., 2 weeks white solid 99.5 0.22
_40_75_6w 75% RH 6 weeks white solid 99.4 0.27 with orange spots
_40_75_10w 10 weeks light brown 98.8 0.49 loose particles
R2_40_75_12w 12 weeks light brown 97.4 0.77 loose particles
*nicotinamide (RRT = 1.73 min)
INCORPORATION BY REFERENCE
[0219] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
EQUIVALENTS
[0220] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification and
the claims below. The full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such
variations.
* * * * *