WO2013170191A1 - Procédés d'utilisation d'antagonistes de biosynthèse de nicotinamide adénine dinucléotide à partir de nicotinamide - Google Patents

Procédés d'utilisation d'antagonistes de biosynthèse de nicotinamide adénine dinucléotide à partir de nicotinamide Download PDF

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WO2013170191A1
WO2013170191A1 PCT/US2013/040613 US2013040613W WO2013170191A1 WO 2013170191 A1 WO2013170191 A1 WO 2013170191A1 US 2013040613 W US2013040613 W US 2013040613W WO 2013170191 A1 WO2013170191 A1 WO 2013170191A1
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alkyl
heteroaryl
heterocycloalkyl
aryl
cycloalkyl
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PCT/US2013/040613
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English (en)
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David SHAMES
Lisa Belmont
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Genentech, Inc.
Forma Tm, Llc
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Publication of WO2013170191A1 publication Critical patent/WO2013170191A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4425—Pyridinium derivatives, e.g. pralidoxime, pyridostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4747—Quinolines; Isoquinolines spiro-condensed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • nicotinamide antagonists e.g., Nampt antagonists and/or NMNAT antagonists.
  • Nicotinamide phosphoribosyltransferase converts nicotinamide into to
  • NNN nicotinamide mononucleotide
  • NAD nicotinomide adenine dinucleotide
  • Cancer cells rely heavily on this salvage pathway, as well as another pathway in which Nicotinic Acid Phosphoribosyltransferase 1 (NAPRT1) converts nicotinic acid (NA, niacin) to NA
  • NAPRT1 Nicotinic Acid Phosphoribosyltransferase 1
  • NAMN mononucleotide
  • NaAD NA adenine dinucleotide
  • Inhibitors of Nampt may be toxic to cancer cells because the cells rely more heavily on these salvage pathways as a result of their high metabolic requirements.
  • kits for treating a disease or disorder in an individual comprising administering to the individual an effective amount of an NAD biosynthesis from nicotinamide antagonist, wherein treatment is based upon the individual having a disease or disorder comprising a positive NAPRTl methylation status.
  • a disease or disorder in an individual provided that the individual has been found to have a disease or disorder comprising a positive NAPRTl methylation status, the method comprising administering to the individual an effective amount of an NAD biosynthesis from nicotinamide antagonist.
  • Also provided herein are methods for treating a disease or disorder in an individual comprising: determining that a sample obtained from the individual comprises a positive NAPRTl methylation status, and administering an effective amount of a therapy comprising an NAD biosynthesis from nicotinamide antagonist to the individual, whereby the disease or disorder is treated.
  • Also provided herein are methods of treating a disease or disorder comprising: (a) selecting an individual having the disease or disorder, wherein the disease or disorder comprises a positive NAPRTl methylation status; and (b) administering to the individual thus selected an effective amount of an NAD biosynthesis from nicotinamide antagonist, whereby the disease or disorder is treated.
  • identifying an individual with a disease or disorder who is more or less likely to exhibit benefit from treatment with a therapy comprising an NAD biosynthesis from nicotinamide antagonist comprising: determining presence or absence of a positive NAPRTl methylation status in a sample obtained from the individual, wherein presence of the positive NAPRTl methylation status in the sample indicates that the individual is more likely to exhibit benefit from treatment with the therapy comprising the NAD biosynthesis from nicotinamide antagonist or absence of the positive NAPRTl methylation status indicates that the individual is less likely to exhibit benefit from treatment with the therapy comprising the NAD biosynthesis from nicotinamide antagonist.
  • a therapy comprising an NAD biosynthesis from nicotinamide antagonist
  • the method comprising determining a positive NAPRTl methylation status, whereby presence of the positive NAPRTl methylation status indicates that the individual is more likely to respond effectively to treatment with the NAD biosynthesis from nicotinamide antagonist and absence of the positive NAPRTl methylation status indicates that the individual is less likely to respond effectively to treatment with the NAD biosynthesis from nicotinamide antagonist.
  • methods of predicting the response or lack of response of an individual with a disease or disorder to an therapy comprising an NAD biosynthesis from nicotinamide antagonist comprising detecting in a sample obtained from the individual presence or absence of a positive NAPRTl methylation status, wherein presence of the positive NAPRTl methylation status is predictive of response of the individual to the therapy comprising the NAD biosynthesis from nicotinamide antagonist and absence of the positive NAPRTl methylation status is predictive of lack of response of the individual to the therapy comprising the NAD biosynthesis from nicotinamide antagonist.
  • the NAD biosynthesis from nicotinamide antagonist is an Nampt antagonist.
  • the NAD biosynthesis from nicotinamide antagonist is an NMNAT antagonist.
  • the NAD biosynthesis from nicotinamide antagonist is an antibody, binding polypeptide, small molecule, or polynucleotide.
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • R is bicyclic heteroaryl comprising 1 , 2, 3 or 4 heteroatom(s) independently selected from N, S or O, wherein said heteroaryl may be substituted by one or more substituents selected from the group consisting of amino, oxo, and halo ; and wherein said heteroaryl can comprise one or more N- oxide(s) formed with a N atom member of said heteroaryl;
  • R 1 is -NHR 4 and R 4 is cycloalkyl, heterocycloalkyl, aryl or heteroaryl;
  • each of said cycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents which can be the same or different and are independently selected from the group consisting of:
  • each of said cycloalkyl, heterocycloalkyl, aryl, or heteroaryl may optionally
  • R 2 and R 3 can be independently selected from the group consisting of H and deuterium;
  • R 5 is H, alkyl or arylalkyl-
  • R a and R b are independently selected from the group consisting of H, alkyl, alkoxy, alkoxyalkyl and haloalkyl;
  • n 0, 1, 2, 3, 4, 5 or 6;
  • z 0, 1 or 2.
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • Ar 1 is aryl or heteroaryl, wherein said aryl or heteroaryl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: deuterium, halo, cyano, alkyl, cyanoalkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, aryloxy, -NR a R b , -C(0)N(R a R b ), -C(0)-alkyl, -C(0)-aryl, -S(0)-aryl, -NH-C(O)- alkyl, -NH-C(0)-aryl, (alkoxyalkyl)oxy-, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; Ar 2 is unsubsituted aryl or heteroaryl;
  • R 1 is cycloalkyl, aryl, heterocycloalkyl, or heteroaryl
  • each of said cycloalkyl, aryl, heterocycloalkyl and heteroaryl is either unsubstituted or optionally independently substituted with 1, 2, 3, 4 or 5 substituents which can be the same or different and are independently selected from the group consisting of: deuterium, halo, cyano, alkyl, hydroxyalkyl, cyanoalkyl, haloalkyl, alkenyl, alkynyl, alkoxy, alkylalkoxy, haloalkoxy, aryloxy, -NR a R b , -CONR a R b , -S(0) 2 -alkyl, -S(0) 2 -aryl, - S(0) 2 N(alkyl) 2 , -S(0) 2 -CF 3 , -C(0)alkyl, -NH-C(0)alkyl, -NH-C(0)aryl, methylenedioxy, - (CH 2 ) q cycloalkyl, -
  • each of said cycloalkyl, aryl, heterocycloalkyl, and heteroaryl may additionally be unsubstituted or subsituted by one or more halo, cyano, alkyl or alkoxy or may be be fused with independently selected aryl, heteroaryl, heterocycloalkyl or cyloalkyl;
  • R a and R b are independently H, alkyl, alkoxy, aryl, alkoxyalkyl, -S(0) 2 alkyl and cycloalkyl or
  • R a and R b can form a 5 or 6 membered heterocycloalkyl group together with the nitrogen atom to which they are attached, wherein said heterocycloalkyl group may contain one or more addional heteroatom(s) selected from N, S or O;
  • R 2 and R 3 are H or deuterium
  • n, p and q are independently 0, 1 or 2;
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • R a is 1 , 2, 3 or 4 and can be selected from the group consisting of hydrogen, amino, oxo, halo, alkoxy, alkyl, haloalkyl, -N(alkyl) 2 , -NH(CO)0-alkyl lH-pyrazol, lH-imidazol, and -C(0)NH 2 ; and wherein said pyridine can comprise a N-oxide formed with its N atom member;
  • R 1 is -NR 3 R 4 wherein R 3 is H, alkyl or -S(0) 2 alkyl and R 4 is alkyl, hydroxyalkyl, - S(0) 2 alkyl, -(CH 2 ) q cycloalkyl, -(CH 2 ) q heterocycloalkyl, aryl, arylalkyl-, -(CH 2 ) q heteroaryl; haloalkyl; cycloalkyl; aryl;
  • heterocycloalkyl or heteroaryl
  • each of said cycloalkyl, aryl, heterocycloalkyl or heteroaryl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents which can be the same or different and are independently selected from the group consisting of: halo, cyano, alkyl, hydroxyl, hydroxyalkyl, hydroxyalkoxy, haloalkyl, alkoxy, alkylalkoxy, haloalkoxy, arylalkenyl-, aryloxy, benzyloxy, oxo, -(CH 2 ) q -NR b R c , - (CH 2 ) q -CONR b R c , -S(0) 2 -alkyl, -S(0) 2 NH-alkyl, -S(0) 2 -heterocycloalkyl, -S(0) 2 -CF 3 , -C(0)alkyl, - C(0)aryl, -C(0)alkyleny
  • each of said cycloalkyl, heterocycloalkyl, aryl or heteroaryl may be substituted by one or more halo, nitro, haloalkyl, haloalkoxy, oxo, cyano, alkyl, haloalkyl, or alkoxy;
  • R b and R c are independently selected from the group consisting of H, alkyl, hydroxyalkyl, alkoxy, aryl, alkoxyalkyl, -S(0) 2 alkyl and cycloalkyl or R b and R c can form a 5 or 6 membered
  • heterocycloalkyl group together with the nitrogen atom to which they are attached, wherein said heterocycloalkyl group may contain one or more addional heteroatom(s) selected from N, S or O; q is 0 or 1 ; and pharmaceutically acceptable salts thereof.
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected
  • Ar is aryl or heteroaryl, each of said aryl and heteroaryl being either unsubstituted or optionally independently substituted with 1 , 2, 3 or 4 substituents which can be the same or different and are independently selected from the group consisting of: deuterium, halo, cyano, amino, aminoalkyl-, (amino)alkoxy-, -CONH 2 , -C(0)NH(alkyl), -C(0)N(alkyl) 2 , -C(0)NH(aryl), - C(0)N(aryl) 2 , -CH Z F 3 . Z , -OCH z F 3 .
  • R 1 is -NR a R b , wherein R a is H, alkyl or -S(0) 2 alkyl and R b is alkyl, hydroxyalkyl, -
  • cycloalkyl heterocycloalkyl; aryl; heteroaryl;
  • each of said cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents which can be the same or different and are independently selected from the group consisting of: deuterium, halo, cyano, alkyl, hydroxyl, hydroxyalkyl, hydroxyalkoxy, cyanoalkyl, haloalkyl, alkenyl, alkynyl, alkoxy, alkylalkoxy, haloalkoxy, arylalkenyl-, aryloxy, benzyloxy, oxo, -(CH 2 ) q -NR c R d , -(CH 2 ) q -CONR c R d , -S(0) 2 -alkyl, -S(0) 2 -aryl, S(0) 2 NH 2 , -S(0) 2 NH- alkyl, -S(0) 2 N(alkyl)
  • each of said cycloalkyl, heterocycloalkyl, aryl or heteroaryl may be substituted by one or more halo, nitro, haloalkyl, haloalkoxy, oxo, cyano, alkyl, haloalkyl, or alkoxy and;
  • R c and R d are independently selected from the group consisting of H, alkyl, hydroxyalkyl, alkoxy, aryl, alkoxyalkyl, -S(0) 2 alkyl and cycloalkyl or R c and R d can form a 5 or 6 membered heterocycloalkyl group together with the nitrogen atom to which they are attached, wherein said heterocycloalkyl group may contain one or more addional heteroatom(s) selected from N, S or O; z is 0, 1 or 2;
  • q 0, 1, 2, 3 or 4;
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group
  • W is -C(O)-, -S(O)- or -S(0) 2 -;
  • R is an aryl or bicyclic heteroaryl wherein the heteroatoms of each of said heteroaryl numbers 1, 2 or 3, and are independently selected from N, S or O, wherein each of said aryl, heteroaryl is optionally substituted with one or more substituents which can be the same or different and are independently selected from the group consisting of deuterium, halo, cyano, amino, aminoalkyl, (amino)alkoxy, -CONH 2 , -C(0)NH(alkyl), -C(0)N(alkyl) 2 , -C(0)NH(aryl), - C(0)N(aryl) 2 , -CF 3 , -CHF 2 , -CH 2 F, -alkyl, alkoxy, hydroxyl, hydroxyalkyl, (alkoxyalkyl) amino, - N(R 3 )-C(0)-alkyl, -N(R 3 )-aryl, cycloalkyl, heterocycloalkyl, hetero
  • G is aryl, heteroaryl, cycloalkyl, heterocycloalkyl or -NR R 2 , with each of said aryl, heteroaryl, heterocycloalkyl and cycloalkyl being either unsubstituted or independently substituted with 1 , 2, 3 or 4 substituents which can be the same or different and are independently selected from the group consisting of deuterium, halo, cyano, amino, aminoalkyl, (amino)alkoxy-, -CONH 2 , - C(0)NH(alkyl), -C(0)N(alkyl) 2 , -C(0)NH(aryl), -C(0)N(aryl) 2 , -CF 3 , -CHF 2 , -CH 2 F, alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxyalkyl, aryloxy, (alkoxyalkyl)amino, -N(R 3 )-C(0)-al
  • R 1 and R 2 are the same or they are different, and are independently selected from H, Ci to C7 alkyl, Ci to C 7 alkoxy, Ci to C 4 hydroxyalkyl, aryl, heteroaryl, heterocycloalkyl and cycloalkyl, and wherein heteroatoms of said heteroaryl and heterocycloalkyl are independently selected from one or more N, O and S, with the proviso that no two adjacent ring heteroatoms are both S or both O, further wherein R 1 and R 2 can be either unsubstituted or optionally independently substituted with one or more substituents which can be the same or different and are independently selected from the group consisting of deuterium, halo, cyano, amino, aminoalkyl, (amino)alkoxy, -CONH 2 , -C(0)NH(alkyl), - C(0)N(alkyl) 2 , -C(0)NH(aryl), -C(0)N(aryl) 2 , -CF 3 , -
  • R 3 is H, alkyl or arylalkyl
  • n 4, 5 or 6;
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • A is CH or N
  • E is O or is absent
  • R is (a) a bicyclic heteroaryl comprising one or more heteroatom ring members independently selected from N, S or O, wherein said bicyclic heteroaryl is unsubstituted or is substituted with one or more substituents selected from the group consisting of deuterium, amino, alkylamino, dialkylamino, alkyl, halo, cyano, haloalkyl, hydroxy, hydroxyalkyl, and alkoxy; and wherein one or more N ring members of said bicyclic heteroaryl is optionally an N-oxide; or
  • R 1 is (1) R m or -alkylenyl-R m , where R m is cycloalkyl, heterocycloalkyl, phenyl, or monocyclic heteroaryl; wherein each of said cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl is unsubstituted or is substituted with one or more substituents R x ; wherein each R x substituent is independently selected from the group consisting of: deuterium, halo, hydroxy, hydroxy alkyl, cyano, -NR a R b , -alkylenyl-NR a R b , oxo, alkyl, cyanoalkyl, haloalkyl, alkoxy, -S-alkyl, haloalkoxy, alkoxyalkyl-, alkenyl, alkynyl, -C(0)Calkyl, -C0 2 alkyl, -C0 2
  • each of said cycloalkyl, heterocycloalkyl, aryl, and heteroaryl within R x is unsubstituted or is substituted with one or more substituents independently selected from the group consisting of deuterium, alkyl, halo, hydroxy, cyano, alkoxy, amino, -C(0)alkyl, and -C0 2 alkyl; wherein R a and R b are each independently H, alkyl, alkoxy, alkoxyalkyl, cyanoalkyl, or haloalkyl; and R c is H, alkyl or arylalkyl-;
  • R s and R l are each independently H, alkyl, alkoxyalkyl, haloalkyl, - C(0)alkyl, or -C0 2 alkyl; or
  • R n is H, R m , -alkylenyl-R m , hydroxyalkyl, cyanoalkyl, alkoxyalkyl, haloalkyl, -CONR , or -C(0)R j ;
  • R m is as defined in (1) above;
  • R h and R 1 are each independently H or alkyl, or R h and R 1 taken together with the nitrogen to which they are attached form a monocyclic heterocycloalkyl;
  • R J is an alkyl unsubstituted or substituted with one or more substituents selected from the group consisting of: deuterium, halo, amino, hydroxy, alkoxy, cycloalkyl, heteroaryl, phenyl, and heterocycloalkyl; or a cycloalkyl, heterocycloalkyl, phenyl, or heteroaryl, each unsubstituted or substituted with one or more substituent
  • R 2 and R 3 are each independently selected from the group consisting of H and deuterium; and pharmaceutically acceptable salts of compounds of Formula I.
  • R x is independently selected from the group consisting of: -C(0)alkyl or -C(0)alkyl-0-alkyl.
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • E is O or is absent
  • R is (a) an 8-, 9-, or 10-membered bicyclic heteroaryl comprising one heteroatom selected from N, S, and O, and one, two, or three additional N atoms, wherein said bicyclic heteroaryl is unsubstituted or is substituted with one or more substituents selected from the group consisting of deuterium, amino, alkylamino, dialkylamino, alkyl, halo, cyano, haloalkyl, hydroxy, hydroxyalkyl, and alkoxy, and wherein one or more N atoms of said bicyclic heteroaryl is optionally an N-oxide; or
  • R 1 is (1) a saturated, monocyclic heterocycloalkyl, which is unsubstituted or substituted with one or more substituents R x ; wherein each R x substituent is independently selected from the group consisting of: deuterium, halo, hydroxy, cyano, -NR a R b , -alkylenyl-NR a R b , oxo, alkyl, hydroxyalkyl, cyanoalkyl, haloalkyl, alkoxy, haloalkoxy, alkoxyalkyl-, -S-alkyl, alkenyl, alkynyl, aryl, arylalkyl-, aryloxy-, arylalkoxy-, cycloalkyl, cycloalkoxy, (cycloalkyl) alkyl-, heterocycloalkyl,
  • heterocycloalkyl alkyl-, (heterocycloalkyl) alkoxy-, -C(0)alkyl, -C0 2 alkyl, -C0 2 H, -C(0)cycloalkyl, -C(0)heterocycloalkyl, -S(0)-alkyl, -S0 2 -alkyl, -S0 2 -aryl, -S0 2 -(haloalkyl), -CONH 2 ,
  • R x is independently unsubstituted or substituted with one or more substituents selected from the group consisting of alkyl, halo, hydroxy, cyano, alkoxy, amino, -C(0)alkyl, and -C0 2 alkyl; wherein R a and R b are each independently H, alkyl, alkoxy, alkoxyalkyl, cyanoalkyl, or haloalkyl; and R c is H or alkyl; or
  • heterocycloalkyl comprises a fused, bridged, or spiro bicyclic system
  • said heterocycloalkyl is unsubstituted or substituted with one or more substituents independently selected from the group consisting of: alkyl, halo, haloalkyl, hydroxy, hydroxyalkyl, alkoxy, cyano, cyanoalkyl, oxo, -alkylenyl-NR d R e , -C(0)alkyl, -C0 2 alkyl, and -S0 2 alkyl; wherein R d is H or alkyl and R e is H, alkyl, haloalkyl, -C(0)alkyl, -C0 2 alkyl, or -S0 2 alkyl; and
  • R 2 and R 3 are each independently H or deuterium
  • R x is selected from -C(0)aryl.
  • each of said cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and phenyl substituents within R x is substituted with one or more -NHC0 2 alkyl.
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group
  • R is (a) an 8-, 9-, or 10-membered bicyclic heteroaryl comprising one heteroatom selected from N, S, and O, and one, two, or three additional N atoms, wherein said bicyclic heteroaryl is unsubstituted or is substituted with one or more substituents selected from the group consisting of deuterium, amino, alkylamino, dialkylamino, alkyl, halo, cyano, haloalkyl, hydroxy, hydroxyalkyl, and alkoxy, and wherein one or more N atoms of said bicyclic heteroaryl is optionally an N-oxide; or
  • R 1 is H, -(Ci_ 4 alkylene) 0 _iC(O)R a , -(Ci_ 4 alkylene) 0 _iCO 2 R a , -(Ci_ 4 alkylene) 0 _iS(O)R a , -(Ci. 4 alkylene) 0 .iSO 2 R a , -C(0)NH(R a ), or -C(0)N(R a ) 2 ; wherein each R a is independently
  • each R m is independently selected from the group consisting of hydroxy, -NR b R c , alkoxy, cyano, halo, -C(0)alkyl, -C0 2 alkyl, -CONR b R c , -S(0)alkyl, -S0 2 alkyl, -S0 2 NR b R c , aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein R b is H or alkyl; R c is H, alkyl, alkoxyalkyl, haloalkyl, -C(0)alkyl, -C0 2 alkyl, or -S0 2 alkyl; and each aryl, heteroaryl, cycloalkyl, and heterocycloalkyl group within R m is unsubstituted or substituted with one or more substituent
  • R x is H or alkyl
  • R y is H, alkyl, alkoxyalkyl, haloalkyl, - C(0)alkyl, -C0 2 alkyl, or -S0 2 alkyl;
  • R 2 and R 3 are each independently H or deuterium
  • n 1 or 2;
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • R is (a) an 8-, 9-, or 10-membered bicyclic heteroaryl comprising one heteroatom selected from N,
  • bicyclic heteroaryl is unsubstituted or is substituted with one or more substituents selected from the group consisting of deuterium, amino, alkylamino, dialkylamino, alkyl, halo, cyano, haloalkyl, hydroxy, hydroxyalkyl, and alkoxy, and wherein one or more N atoms of said bicyclic heteroaryl is optionally an N-oxide; or
  • R 1 is H, -(Ci_ 4 alkylene) 0 _iC(O)R a , -(Ci_ 4 alkylene) 0 _iCO 2 R a , -(Ci_ 4 alkylene) 0 _iS(O)R a ,
  • each R m is independently selected from the group consisting of hydroxy, -NR b R c , alkoxy, cyano, halo, -C(0)alkyl, -C0 2 alkyl,
  • R b is H or alkyl
  • R c is H, alkyl, alkoxyalkyl, haloalkyl, -C(0)alkyl, -C0 2 alkyl,
  • each aryl, heteroaryl, cycloalkyl, and heterocycloalkyl group within R m is
  • each alkyl or alkoxy is unsubstituted or substituted with -NR b R c ,
  • heterocycloalkyl heteroaryl, or -C(0)alkyl
  • each aryl, heteroaryl, cycloalkyl, and heterocycloalkyl is unsubstituted or
  • phenyl, cycloalkyl, heteroaryl, or heterocycloalkyl each unsubstituted or substituted with one or more substituents selected from the group consisting of alkyl, haloalkyl, hydroxy, -NR b R c , alkoxy, haloalkoxy, cyano, halo, oxo, -C(0)alkyl, -C0 2 alkyl, -C(O)- heterocycloalkyl, -CONR b R c , -S(0)alkyl, -S0 2 alkyl, -S0 2 -haloalkyl, -S0 2 NR b R c , aryl, heteroaryl, cycloalkyl, and heterocycloalkyl;
  • each alkyl or alkoxy is unsubstituted or substituted with -NR b R c ,
  • heterocycloalkyl heteroaryl, or -C(0)alkyl
  • each aryl, heteroaryl, cycloalkyl, and heterocycloalkyl is unsubstituted or substituted with alkyl, halo, or -C(0)alkyl; or
  • R x is H or alkyl
  • R y is H, alkyl, alkoxyalkyl, haloalkyl, -C(0)alkyl, -C0 2 alkyl, or
  • R 2 and R 3 are each independently H or deuterium
  • n 1 or 2;
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • R is (a) a bicyclic heteroaryl comprising 1 , 2, 3, or 4 heteroatom ring members independently selected from N, S or O, wherein said bicyclic heteroaryl is unsubstituted or is substituted with one or more substituents selected from the group consisting of deuterium, amino, alkylamino, dialkylamino, alkyl, halo, cyano, haloalkyl, hydroxy, hydroxyalkyl, and alkoxy; and wherein one or more N ring members of said heteroaryl is optionally an N-oxide; or
  • R 1 is alkyl, R m , or -alkylenyl-R m ,
  • alkyl is unsubstituted or substituted with one or more substituents selected from the group consisting of deuterium, halo, hydroxy, cyano, alkoxy, -NR y R z , hydroxyalkyl, cyanoalkyl, haloalkyl, haloalkoxy, alkoxyalkyl, -S-alkyl, -C(0)alkyl, -C0 2 alkyl, -C0 2 H, -C(0)NH 2 ,
  • R y and R z are each independently H, alkyl, alkoxyalkyl, haloalkyl,
  • R m is cycloalkyl, phenyl, monocyclic heteroaryl, or heterocycloalkyl, each unsubstituted or substituted with one or more substituents R x ;
  • each R x substituent is independently selected from the group consisting of:
  • halo hydroxy, hydroxyalkyl, cyano, -NR a R b , -alkylenyl-NR a R b , oxo, alkyl, cyanoalkyl, haloalkyl, alkoxy, -S-alkyl, haloalkoxy, alkoxyalkyl-, alkenyl, alkynyl, -C(0)alkyl, -CONH 2 , C(0)NH(alkyl), -C(0)NH(haloalkyl), -C(0)N(alkyl) 2 , -C(0)NH(cycloalkyl), arylalkyl-, arylalkoxy-, aryloxy-, cycloalkyl, cycloalkyloxy, (cycloalkyl) alkyl-, heterocycloalkyl, aryl,
  • heterocycloalkyl alkyl-, (heterocycloalkyl) alkoxy-, -C(0)heterocycloalkyl, heteroaryl,
  • each of said cycloalkyl, heterocycloalkyl, aryl, and heteroaryl within R x is unsubstituted or is substituted with 1 , 2, or 3 substituents independently selected from the group consisting of alkyl, halo, hydroxy, cyano, alkoxy, amino, -C(0)alkyl, and -C0 2 alkyl;
  • R a and R b are each independently H, alkyl, alkoxy, alkoxyalkyl, cyanoalkyl, or haloalkyl;
  • R c is H, alkyl, or arylalkyl-
  • R 2 and R 3 are each independently selected from the group consisting of H and deuterium; or a pharmaceutically acceptable salt thereof.
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule selected from the group consisting of:
  • E is O or is absent
  • R is (a) an 8-, 9-, or 10-membered bicyclic heteroaryl comprising one heteroatom selected from N, S, and O, and one, two, or three additional N atoms, wherein said bicyclic heteroaryl is unsubstituted or is substituted with one or more substituents selected from the group consisting of the group consisting of deuterium, amino, alkylamino, dialkylamino, alkyl, halo, cyano, haloalkyl, hydroxy, hydroxyalkyl, and alkoxy, and wherein one or more N atoms of said bicyclic heteroaryl is optionally an N-oxide; or
  • R 1 is (1) R x , where R x is a phenyl, cycloalkyl, heterocycloalkyl, or monocyclic heteroaryl, unsubstituted or substituted with one or more R m substituents;
  • each R m substituent is independently selected from the group consisting of: halo, hydroxy, cyano, -NR a R b , -alkylenyl-NR a R b , oxo, alkyl, hydroxyalkyl, cyanoalkyl, haloalkyl, alkoxy, haloalkoxy, alkoxyalkyl-, -S-alkyl, alkenyl, alkynyl, aryl, arylalkyl-, aryloxy-, arylalkoxy-, cycloalkyl, cycloalkyloxy, (cycloalkyl)alkyl-, heterocycloalkyl, (heterocycloalkyl)alkyl-,
  • R m substituents taken together form a phenyl ring, wherein each of said cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and phenyl substituents within R m is independently unsubstituted or substituted with one or more substituents selected from the group consisting of alkyl, halo, hydroxy, cyano, alkoxy, amino, -C(0)alkyl, and - C0 2 alkyl;
  • R a and R b are each independently H, alkyl, alkoxy, alkoxyalkyl, cyanoalkyl, or haloalkyl; and R c is H or alkyl;
  • each R z is independently -CONR h R ⁇ hydroxy, cyano, alkoxy, halo, or -C(0)R j ;
  • R h and R 1 are each independently H or alkyl, or R h and R 1 taken together with the nitrogen to which they are attached form a monocyclic heterocycloalkyl;
  • R J is alkyl, cycloalkyl, heterocycloalkyl, phenyl, or benzyl, each unsubstituted or substituted with one or more substituents selected from the group consisting of: alkyl, halo, amino, hydroxy, and alkoxy;
  • R 2 and R 3 are each independently H or deuterium
  • R 4 is H; an alkyl unsubstituted or substituted with one or more substituents selected from the group consisting of: deuterium, halo, amino, hydroxy, alkoxy, cycloalkyl, heteroaryl, phenyl, and heterocycloalkyl, wherein each cycloalkyl, heteroaryl, phenyl, and heterocycloalkyl is unsubstituted or substituted with one or more substituents selected from the group consisting of: deuterium, alkyl, halo, amino, hydroxy, and alkoxy; or a cycloalkyl, heterocycloalkyl, phenyl, or heteroaryl, each unsubstituted or substituted with one or more substituents selected from the group consisting of: deuterium, alkyl, halo, amino, hydroxy, and alkoxy; with the proviso that R 4 is not H when R 1 is as defined in (1) above;
  • the method further comprises administering niacin.
  • niacin is Niaspan®.
  • the niacin reduces toxicity of the NAD biosynthesis from nicotinamide antagonist and/or increases the therapeutic index of the NAD biosynthesis from nicotinamide antagonist.
  • the positive NAPRTl methylation status is methylation of at least one cytosine within a NAPRTl DNA region. In some embodiments, the positive NAPRTl methylation status is methylation of at least one cytosine within a CpG island of an NAPRTl gene. In some embodiments, the positive NAPRTl methylation status is methylation of at least one cytosine between about chromosome coordinates 144659500 and 144661000 of human chromosome.
  • the positive NAPRTl methylation status is methylation of at least one cytosine in the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: l. In some embodiments, the positive NAPRTl methylation status is methylation of at least one cytosine in the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l.
  • the positive NAPRTl methylation status is methylation of at least 25% of the cytosines within a portion of an NAPRTl gene. In some embodiments, the positive NAPRTl methylation status is methylation of at least 25% of the cytosines within a portion of an NAPRTl gene, wherein the portion of the NAPRTl gene corresponds to a portion of the human NAPRTl sequence represented by SEQ ID NO: l. In some embodiments, the positive NAPRTl methylation status is methylation of at least 25% of the cytosines within a promoter in an NAPRTl gene.
  • the positive NAPRTl methylation status is methylation of at least 25% of the cytosines within a CpG island of an NAPRTl DNA region. In some embodiments, the positive NAPRTl methylation status is methylation of at least 25% of the cytosines in a sequence corresponding to the sequence between about chromosome coordinates 144659500 and 144661000 of human chromosome 8. In some embodiments, the positive NAPRTl methylation status is methylation of at least 25% of the cytosines in the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: l. In some embodiments, the positive NAPRTl methylation status is methylation of at least 25% of the cytosines in the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l.
  • the disease or disorder or disease or disorder cell is cancer.
  • the cancer or cancer cell is breast cancer, colorectal cancer, endometrium cancer, kidney cancer, lung cancer, lymphoid cancer, ovarian cancer, pancreatic cancer, or stomach cancer.
  • identifying a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of an NAPRT1 gene wherein greater than 25% methylation of the NAPRT1 gene indicates that the individual is more likely to benefit from treatment.
  • identifying a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of an NAPRT1 promoter wherein greater than 25% methylation of the NAPRT1 promoter indicates that the individual is more likely to benefit from treatment.
  • identifying a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of a CpG island in an NAPRT1 gene wherein greater than 25% methylation of the CpG island indicates that the individual is more likely to benefit from treatment
  • identifying a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: l wherein greater than 25% methylation of the sequence that the individual is more likely to benefit from treatment.
  • identifying a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l wherein greater than 25% methylation of the sequence that the individual is more likely to benefit from treatment .
  • identifying an individual who is more likely to exhibit benefit from a therapy comprising an NAD biosynthesis from nicotinamide antagonist comprising a) isolating DNA from a tumor sample the individual; b) incubating the DNA sample with sodium bisulfite sufficient to convert unmethylated cytosine in the DNA to uracil; c) sequencing the NAPRT1 promoter region of the DNA; d) determining the methylation level of the NAPRT1 promoter region by determining the number of cytosine residues that were not converted to uracil in step b); wherein greater than 25% methylation of the sequence that the individual is more likely to benefit from treatment.
  • identifying an individual who is more likely to exhibit benefit from a therapy comprising an NAD biosynthesis from nicotinamide antagonist comprising a) isolating DNA from a tumor sample the individual; b) incubating the DNA sample with sodium bisulfite sufficient to convert unmethylated cytosine in the DNA to uracil; c) sequencing a fragment of the DNA comprising a portion NAPRT1 CpG island; d) determining the methylation level of the NAPRT1 CpG island by determining the number cytosine residues that were not converted to uracil in step b); wherein greater than 25% methylation of the sequence that the individual is more likely to benefit from treatment.
  • the portion of the NAPRTl CpG island comprises the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: l.
  • identifying an individual who is more likely to exhibit benefit from a therapy comprising an NAD biosynthesis from nicotinamide antagonist comprising a) isolating DNA from a tumor sample the individual, b) incubating the DNA sample with sodium bisulfite sufficient to convert unmethylated cytosine in the DNA to uracil, c) amplifying a portion of the CpG island of the NAPRTl gene of the DNA sample using quantitative methylation specific PCR, d) determining the methylation level of the NAPRTl region by determining the -dCt value of the tumor sample with the -dCt value obtained from quantitative methylation specific PCR of non-methylated DNA, wherein greater than 25% methylation of the NAPRTl sequence that the individual is more likely to benefit from treatment.
  • the portion of the CpG island of the NAPRTl gene comprises the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l
  • the DNA from the tumor sample is isolated from a formalin-fixed paraffin embedded tumor sample.
  • step c) further comprises a pre-amplification of the portion of the CpG island of the NAPRTl gene of the DNA sample prior to quantitative methylation specific PCR.
  • kits for treating a human cancer patient with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of an NAPRTl gene in a tumor sample from the patient wherein greater than 25% methylation of the NAPRTl gene indicates that the patient is more likely to benefit from treatment; and administering an effective amount of an NAD biosynthesis from nicotinamide antagonist to the patient more likely to benefit from treatment.
  • kits for treating a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of an NAPRTl promoter in a tumor sample from the patient wherein greater than 25% methylation of the NAPRTl promoter indicates that the patient is more likely to benefit from treatment, administering an effective amount of an NAD biosynthesis from nicotinamide antagonist to the patient more likely to benefit from treatment.
  • kits for treating a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of a CpG island in an NAPRTl gene in a tumor sample from the patient wherein greater than 25% methylation of the CpG island indicates that the patient is more likely to benefit from treatment; and administering an effective amount of an NAD biosynthesis from nicotinamide antagonist to the patient more likely to benefit from treatment
  • kits for treating a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: l in a tumor sample from the patient wherein greater than 25% methylation of the sequence that the individual is more likely to benefit from treatment, and administering an effective amount of an NAD biosynthesis from nicotinamide antagonist to the patient more likely to benefit from treatment.
  • kits for treating a human cancer patient suitable for treatment with an NAD biosynthesis from nicotinamide antagonist comprising determining the level of methylation of cytosines of the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l in a tumor sample from the patient wherein greater than 25% methylation of the sequence that the patient is more likely to benefit from treatment, and administering an effective amount of an NAD biosynthesis from nicotinamide antagonist to the patient more likely to benefit from treatment.
  • kits for treating human cancer patient with an NAD biosynthesis from nicotinamide antagonist comprising a) isolating DNA from a tumor sample the patient; b) incubating the DNA sample with sodium bisulfite sufficient to convert unmethylated cytosine in the DNA to uracil; c) sequencing the NAPRT1 promoter region of the DNA; d) determining the methylation level of the NAPRT1 promoter region by determining the number of cytosine residues that were not converted to uracil in step b) wherein greater than 25% methylation of the sequence that the patient is more likely to benefit from treatment; and e) administering an effective amount of an NAD biosynthesis from nicotinamide antagonist to the patient more likely to benefit from treatment.
  • the portion of the NAPRT1 CpG island comprises the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: 1.
  • the portion of the CpG island of the NAPRTl gene comprises the
  • the DNA from the tumor sample is isolated from a formalin-fixed paraffin embedded tumor sample.
  • step c) further comprises a pre- amplification of the portion of the CpG island of the NAPRTl gene of the DNA sample prior to quantitative methylation specific PCR.
  • the method further comprises administering niacin.
  • the niacin reduces toxicity of the NAD biosynthesis from nicotinamide antagonist and/or increases the therapeutic index of the NAD biosynthesis from nicotinamide antagonist.
  • Figure 1 is a representative series of graphs depicting nicotinic acid (NA) Rescue (A and B) or Non-Rescue (C and D) of CALU-6 or NCI-460 cells, respectively, after undergoing treatment with nicotinamide phosphoribosyltransferase (Nampt) inhibitors Compound B as measured by quantification of ATP (CellTiterGlo, Promega) or DNA (CyQuant, Promega).
  • NA nicotinic acid
  • Ammonit nicotinamide phosphoribosyltransferase
  • Figure 2 is a series of graphs demonstrating that a low or intermediate Nicotinic Acid Phosphoribosyltransferase 1 (NAPRTl) methylation status correlates with high NAPRTl gene expression and NA Rescue in a number of cancer cell lines.
  • NAPRTl Nicotinic Acid Phosphoribosyltransferase 1
  • A Correlation between NAPRTl mRNA and NAPRTl protein levels.
  • B Correlation between NAPRTl mRNA levels and NAPRTl methylation status.
  • C Correlation between high NAPRTl methylation status (solid bar) or low/intermediate NAPRTl methylation status (striped bar) and no NA Rescue or NA Rescue in cancer cell lines, respectively.
  • Figure 3 is a graph depicting the relationship between average methylation across seven CpG island sites in the NAPRTl promoter and NA Rescue in a number of cancer lines. Hatched bars indicate no rescue cells.
  • FIG. 4 shows 5-aza-2-deoxycytidine (5-azadC) treatment induces NAPRTl expression in cell lines with NAPRTl CpG island methylation. These data show that most cell lines that cannot be rescued by nicotinic acid express very low levels of NAPRTl transcript (DMSO) as determined by quantitative PCR. NAPRTl is inducible by the DNA demethylating agent 5-azadC. Conversely, cells lines that could be rescued already express NAPRTl transcript, which was not further induced by 5- azadC treatment.
  • Figure 5 is a diagram depicting nucleotide regions of the NAPRT1 CpG island aligned with regions amplified by BSP primers 1 through 8. From 5' to 3' of NAPRT1, alignment of DNA region amplified by BSP primers 6, 7, 8, 1, 5, 2, 4, and 3.
  • Figure 6 is a diagram depicting nucleotide regions of the NAPRT1 CpG island aligned with DNA regions amplified by BSP primers 1 through 8 along with DNA regions amplified by quantitative methylation-specific PCR primers and probes 1 through 7 used for mapping of methylation sites with a quantitative methylation specific PCR assay (QMSP) after initial sodium bisulfite modification. From 5' to 3' of NAPRT1, alignment of regions amplified by QMSP primers and probe sets 1, 2, 4, 5, and 3.
  • QMSP quantitative methylation specific PCR assay
  • Figure 7A and 7B present the sequence of the NAPRT1 CpG island region following modification by sodium bisulfite treatment (SEQ ID NO: 2) aligned with regions amplified by BSP primers 1 through 8 along with regions amplified by methylation-specific PCR primers and probes 1 through 7 used for mapping of methylation sites with a quantitative methylation specific PCR assay (QMSP).
  • QMSP forward and reverse primers as well as QMSP probes are depicted along with the amplicon produced by the quantitative methylation specific PCR assay.
  • Figure 8 is the NAPRT1 CpG island DNA sequence with 600 base pairs added upstream and downstream (SEQ ID NO: 1).
  • Figure 9 is the NAPRT1 CpG island DNA sequence with 600 base pairs added upstream and downstream, and after initial modification with sodium bisulfite assuming the sequence was fully methylated (SEQ ID NO: 2).
  • Figure 10A is the NAPRT1 genomic CpG island DNA sequence (SEQ ID NO: 3).
  • Figure 10B is the NAPRT1 CpG island DNA sequence following modification with sodium bisulfite assuming the sequence is fully methylated (SEQ ID NO: 4).
  • Figure IOC is the reverse complement strand of the NAPRT1 CpG island DNA sequence following modification with sodium bisulfite assuming the sequence was fully methylated (SEQ ID NO:5).
  • FIG 11 is a depiction of NAPRT1 CpG island overlaid with representative sodium bisulfite sequencing data from a pool of peripheral blood monocyte DNA from 20 healthy donors.
  • For the bisuflite sequencing data black and white grids.
  • Each row represents a single TA clone from the PCR product and each column represents an individual CpG site within the PCR amplified amplicon.
  • Open boxes represent unmethylated CpG sites; filled boxes represent methylated sites; shaded boxes are undetermined.
  • the black and white box on the left, labeled BSP7 shows the methylation pattern at the 3' end of the CpG island. A commercially available methylation assay is targeted to this region as shown by the bar.
  • FIG. 12 is a diagram depicting the DNA methylation profile of CpG sites in NAPRTl expressed by breast cancer cell lines.
  • A A diagram of NAPRTl genomic DNA and CpG island site recognized by BSP primers for sodium bisulfite sequencing.
  • B Methylation profile of CpG sites in the HCC70 breast cancer cell line that demonstrated no rescue by NA when treated with Nampt inhibitors.
  • C Methylation profile of CpG sites in MDA-MB-231 and CAL120 breast cancer cell lines that demonstrated rescue by NA when treated with Nampt inhibitors.
  • Each row represents a cloned PCR product (alleles) of BSP1/2; each column represents a single CpG site within the cloned region. Open boxes represent unmethylated CpG sites; filled boxes represent methylated sites.
  • Figure 13 is a methylation profile of various non-small cell lung cancer (NSCLC) cell lines.
  • NSCLC non-small cell lung cancer
  • A Methylation profile of CpG sites in HI 155, HI 650, HI 703, and LXFL529 NSCLC cell lines that demonstrated no rescue by NA when treated with Nampt inhibitors.
  • B Methylation profile of CpG sites in HI 838, H2030, H2122, and H226 NSCLC cell lines that demonstrated rescue by NA when treated with Nampt inhibitors.
  • Each row represents a cloned PCR product (alleles) of BSP1/2; each column represents a single CpG site within the cloned region. Open boxes represent unmethylated CpG sites; filled boxes represent methylated sites.
  • Figure 14 shows analysis of methylation (QMSP3) and gene expression of NAPRTl in tumors (NSCLC) and matched normal (benign adjacent lung) tissue.
  • Panel A shows methylation in matched tumor/normal;
  • Panel B shows expression in the same tissues.
  • FIG. 15 Analysis of methylation of NAPRTl in DNA derived from IHC slides. Inserts show immunohistochemistry showing expression NAPRTl in tumor samples.
  • Tumor samples HP- 7770 a small cell lung cancer tumor, has an IHC score of zero indicating no detectable expression of NAPRTl (blue area). The brown area is non-malignant tissue.
  • Tumor sample HP-7489 a non-small cell lung cancer tumor, has an IHC score of 3+ indicating expression of NAPRTl.
  • the graph represents QMSP analysis of methylation of DNA isolated directly from the IHC slides.
  • Figure 16 is a diagram demonstrating the RNA level of NAPRTl in tissues.
  • Low levels of NAPRTl RNA in malignant tumors originating from breast, colon, endometrium, head and neck, kidney, lung, lymphoid, neuroendocrine, ovary, pancreas, prostate, skin, soft tissue, thyroid, urinary tract, and stomach indicate potential benefits of Nampt inhibitor treatment in patients with these cancer types.
  • Figure 17A is a graph showing siRNA knockdown of NAPRTl prevents NA rescue of A549 tumor cells following treatment with an Nampt inhibitor.
  • Control siRNA indicates samples that received siRNAs not directed toward NAPRTl.
  • NAPRTl siRNA indicates cells treated with siRNAs specific for NAPRTl.
  • +NA indicates cells further treated with nicotinic acid. Error bars represent the standard error of each data point (SEM).
  • Figure 17B is shows results of a western blot analysis showing NAPRTl expression is specifically inhibited by siRNAs directed toward NAPRTl in A549 cells. Bottom panel shows an actin loading control. DETAILED DESCRIPTION
  • NAPRTl refer herein to a native NAPRTl from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the terms encompass the genomic location (e.g. , 8q24.3 cytogenetic band, chromosome 8- 142,656,955 - 146,364,022 bp, and/or GC08M144656), "full-length,” unprocessed NAPRTl as well as any form of NAPRTl that result from processing in the cell.
  • NAPRTl also encompasses naturally occurring variants of NAPRTl , e.g., splice variants or allelic variants.
  • sequence of an exemplary human NAPRTl nucleic acid sequence is NM_145201 or an exemplary human NAPRTl is amino acid sequence NP_660202.3.
  • Nampt refer herein to a native Nampt from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the terms encompasses "full-length,” unprocessed Nampt as well as any form of Nampt that result from processing in the cell.
  • Nampt e.g., splice variants or allelic variants
  • Nampt variant means an Nampt polypeptide or polynucleotide, generally being or encoding an active Nampt polypeptide, as defined herein having at least about 80% amino acid sequence identity with any of the Nampt as disclosed herein.
  • Such Nampt variants include, for instance, Nampt wherein one or more nucleic acid or amino acid residues are added or deleted.
  • an Nampt variant will have at least about 80% sequence identity, alternatively at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, to Nampt as disclosed herein.
  • Nampt variant are at least about 10 residues in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 in length, or more.
  • Nampt variant will have or encode a sequence having no more than one conservative amino acid substitution as compared to Nampt, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to Nampt.
  • NMNAT nicotinamide phosphoribosyltransferase
  • mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the terms encompasses "full-length,” unprocessed NMNAT as well as any form of NMNAT that result from processing in the cell.
  • the term also encompasses naturally occurring variants of NMNAT, e.g., splice variants or allelic variants. Examples of sequences for NMNAT include NMNAT 1 : NM_022787; NMNAT2:
  • NMNAT variant means an NMNAT polypeptide or polynucleotide, generally being or encoding an active NMNAT polypeptide, as defined herein having at least about 80% amino acid sequence identity with any of the NMNAT as disclosed herein.
  • NMNAT variants include, for instance, NMNAT wherein one or more nucleic acid or amino acid residues are added or deleted.
  • an NMNAT variant will have at least about 80% sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, to NMNAT as disclosed herein.
  • NMNAT variant are at least about 10 residues in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 in length, or more.
  • NMNAT variant will have or encode a sequence having no more than one conservative amino acid substitution as compared to NMNAT, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to NMNAT.
  • NAD biosynthesis from nicotinamide antagonist is any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity mediated by, for example, Nampt and/or NMNAT.
  • the antagonist is an antagonist binding polypeptide.
  • the antagonist is an antagonist antibody.
  • the antagonist is a small molecule antagonist.
  • the antagonist is a polynucleotide antagonist.
  • Non-limiting examples of small molecule Nampt antagonists which can be used in methods according to the invention are given herein, for example Nampt antagonists as described in published PCT patent applications Nos. WO 2011006988, WO 2011109441, WO2012031196, WO2012031197, and WO2012031199.
  • DNA methylation refers to the presence of a methyl group at the C5 position of cytosine (i.e. 5-methylcytosine).
  • Methylation of cytosine bases in DNA provides a layer of epigenetic control of gene expression in eukaryotes.
  • the DNA contains 5-hydroxymethylcytosine.
  • CpG island refers to a short stretch of DNA in which the frequency of the CpG sequence is higher than other regions.
  • CpG islands are typically located around the promoters of housekeeping genes or other genes frequently expressed in a cell. In some cases, CpG islands play a role in the epigenetic regulation of gene expression. For example, genes are often expressed when the CpG sequences are hypomethylated, and are inactive when the CpG island is methylated or hypermethylated.
  • the CpG island of human NAPRT1 is located between coordinates 144659746 and 144660635 of human chromosome 8.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction.
  • polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after synthesis, such as by conjugation with a label.
  • Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g.
  • methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages e.g. , phosphorothioates, phosphorodithioates, etc.
  • pendant moieties such as, for example, proteins (e.g. , nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g. , acridine, psoralen, etc.), those containing chelators (e.g. , metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g.
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0- allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S("thioate”), P(S)S ("dithioate”), "(0)NR 2 ("amidate”), P(0)R, P(0)OR', CO or CH 2 ("formacetal”), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • Oligonucleotide generally refers to short, single stranded
  • polynucleotides that are, but not necessarily, less than about 250 nucleotides in length.
  • Oligonucleotides may be synthetic.
  • the terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • primer refers to a single stranded polynucleotide that is capable of hybridizing to a nucleic acid and following polymerization of a complementary nucleic acid, generally by providing a free 3' -OH group.
  • small molecule refers to any molecule with a molecular weight of about 2000 daltons or less, preferably of about 500 daltons or less.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self -replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors.”
  • an "isolated" antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g. , SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g. , ion exchange or reverse phase HPLC).
  • electrophoretic e.g. , SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g. , ion exchange or reverse phase HPLC
  • An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • the term "antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. , bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • anti-Nampt antibody and "an antibody that binds to Nampt” refer to an antibody that is capable of binding Nampt with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting Nampt.
  • the extent of binding of an anti-Nampt antibody to an unrelated, non-Nampt protein is less than about 10% of the binding of the antibody to Nampt as measured, e.g. , by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an anti-Nampt antibody binds to an epitope of Nampt that is conserved among Nampt from different species.
  • anti-NMNAT antibody and "an antibody that binds to NMNAT” refer to an antibody that is capable of binding NMNAT with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting NMNAT.
  • the extent of binding of an anti-NMNAT antibody to an unrelated, non-NMNAT protein is less than about 10% of the binding of the antibody to NMNAT as measured, e.g. , by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an anti-NMNAT antibody binds to an epitope of NMNAT that is conserved among NMNAT from different species.
  • a “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds.
  • Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
  • Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. , an antibody) and its binding partner (e.g. , an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. , antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • An "affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • HVRs hypervariable regions
  • an "antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • an "antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
  • An exemplary competition assay is provided herein.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • full length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g. , containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage -display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g. , CDRs) correspond to those of a non- human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, e.g. , a non-human antibody refers to an antibody that has undergone humanization.
  • an “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • Percent ( ) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No.
  • TXU510087 The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • detection includes any means of detecting, including direct and indirect detection.
  • biomarker refers to an indicator, e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample.
  • the biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and/or clinical features.
  • a biomarker is a polypeptide and polynucleotide modifications (e.g., methylation).
  • Biomarkers include, but are not limited to, polynucleotides (e.g., DNA, and/or RNA), polypeptides, polypeptide and polynucleotide modifications (e.g. posttranslational modifications), carbohydrates, and/or glycolipid-based molecular markers.
  • the "amount” or “level” of a biomarker associated with an increased clinical benefit to an individual is a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to the treatment.
  • level of expression or “expression level” in general are used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide).
  • Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis.
  • "Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).
  • Elevated expression refers to an increased expression or increased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control (e.g., housekeeping biomarker).
  • a control such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control (e.g., housekeeping biomarker).
  • Reduced expression refers to a decrease expression or decreased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control (e.g., housekeeping biomarker).
  • housekeeping biomarker refers to a biomarker or group of biomarkers (e.g., polynucleotides and/or polypeptides) which are typically similarly present in all cell types.
  • the housekeeping biomarker is a "housekeeping gene.”
  • a “housekeeping gene” refers herein to a gene or group of genes which encode proteins whose activities are essential for the maintenance of cell function and which are typically similarly present in all cell types.
  • Amplification generally refers to the process of producing multiple copies of a desired sequence.
  • Multiple copies mean at least two copies.
  • a “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence.
  • copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and/or sequence errors that occur during amplification.
  • multiplex-PCR refers to a single PCR reaction carried out on nucleic acid obtained from a single source (e.g., an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.
  • Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.
  • stringency of hybridization reactions see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • "Stringent conditions” or “high stringency conditions”, as defined herein, can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) overnight hybridization in a solution that employs 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ug/ml
  • Modely stringent conditions can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and SDS) less stringent that those described above.
  • moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • diagnosis is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer).
  • diagnosis may refer to identification of a particular type of cancer.
  • Diagnosis may also refer to the classification of a particular subtype of cancer, e.g., by histopathological criteria, or by molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).
  • a method of aiding diagnosis of a disease or condition can comprise measuring certain biomarkers in a biological sample from an individual.
  • sample refers to a composition that is obtained or derived from a subject and/or individual of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics.
  • disease sample and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.
  • Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof.
  • a tumor sample includes but is not limited to a sample from a solid tumor, a sample from a liquid tumor such as a leukemia, a sample from a primary tumor, a sample from a metastatic tumor, cultured tumor cells, and tumor lysates.
  • the tumor sample can be cells derived from a tumor and found in a fluid such as plasma, serum, whole blood, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, and mucus.
  • tissue sample or “cell sample” is meant a collection of similar cells obtained from a tissue of a subject or individual.
  • the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject.
  • the tissue sample may also be primary or cultured cells or cell lines.
  • the tissue or cell sample is obtained from a disease tissue/organ.
  • the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • a “reference sample”, “reference cell”, “reference tissue”, “control sample”, “control cell”, or “control tissue”, as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual.
  • healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue e.g., cells or tissue adjacent to a tumor.
  • a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or individual.
  • the sample can be peripheral blood mononuclear cells.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the subject or individual.
  • a "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis, provided that it is understood that the same section of tissue sample may be analyzed at both morphological and molecular levels, or analyzed with respect to both polypeptides and polynucleotides.
  • correlate or “correlating” is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.
  • “Individual response” or “response” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including slowing down and complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down or complete stopping) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e. reduction, slowing down or complete stopping) of metasisis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase in the length of progression free survival; and/or (9) decreased mortality at a given point of time following treatment.
  • disease progression e.g., cancer progression
  • a reduction in tumor size i.e., reduction, slowing down or complete stopping
  • inhibition i.e. reduction, slowing down or complete stopping
  • metasisis i.e. reduction, slowing down or complete stopping
  • the term "substantially the same,” as used herein, denotes a sufficiently high degree of similarity between two numeric values, such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values or expression).
  • the difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the
  • the phrase "substantially different,” as used herein, denotes a sufficiently high degree of difference between two numeric values such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).
  • the difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.
  • label when used herein refers to a detectable compound or composition.
  • the label is typically conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or an antibody, and facilitates detection of the reagent to which it is conjugated or fused.
  • the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which results in a detectable product.
  • an "effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a "therapeutically effective amount" of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • anti-cancer therapy refers to a therapy useful in treating cancer.
  • anti-cancer therapeutic agents include, but are limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer , anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g., GleevecTM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-beta, BlyS, APRIL, BCMA receptor(s), TRAIL/Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also included in the invention.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents
  • a "chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
  • methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); do
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (
  • dromostanolone propionate epitiostanol, mepitiostane, testolactone
  • anti-adrenals such as aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elf ornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet
  • pirarubicin pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2' , 2' -trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxe
  • ABRAXANETM docetaxel
  • TXOTERE® docetaxel
  • chloranbucil 6-thioguanine
  • mercaptopurine methotrexate
  • platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin
  • vincas which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene
  • VAXID® vaccine e.g., LURTOTECAN®
  • rmRH e.g., ABARELIX®
  • BAY439006 sorafenib; Bayer
  • SU-11248 sunitinib, SUTENT®, Pfizer
  • perifosine, COX-2 inhibitor e.g., celecoxib or etoricoxib
  • proteosome inhibitor e.g., PS341
  • VELCADE® CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of
  • cyclophosphamide an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.
  • ELOXATINTM oxaliplatin
  • Chemotherapeutic agents as defined herein include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375- 382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985).
  • the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate -containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D- amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide- containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell (e.g., a cell whose growth is dependent upon NAD biosynthesis from nicotinamide (e.g., Nampt and/or NMNAT) expression either in vitro or in vivo).
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,
  • Docetaxel (TAXOTERE®, Rhone -Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • radiation therapy is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one time administration and typical dosages range from 10 to 200 units (Grays) per day.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non- human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).
  • Reduce or inhibit is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • An "article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein.
  • the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.
  • a "target audience” is a group of people or an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individuals, populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.
  • NAPRT1 biomarkers comprising a NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • kits for treating a disease or disorder in an individual comprising administering to the individual an effective amount of a NAD biosynthesis from nicotinamide antagonist, wherein treatment is based upon the individual having a disease or disorder comprising one or more NAPRT1 biomarkers.
  • methods of treating a disease or disorder in an individual comprising administering to the individual an effective amount of a NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist), wherein treatment is based upon the individual having a disease or disorder comprising a positive NAPRT1 methylation status.
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • methods of treating a disease or disorder cell comprising one or more NAPRT1 biomarkers, the method comprising providing an effective amount of a NAD biosynthesis from nicotinamide antagonist.
  • methods of treating a disease or disorder cell, wherein the disease or disorder cell comprises a positive NAPRT1 methylation status, the method comprising providing an effective amount of a NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • kits for treating a disease or disorder in an individual provided that the individual has been found to have a disease or disorder comprising one or more NAPRT1 biomarkers comprising administering to the individual an effective amount of a NAD biosynthesis from nicotinamide antagonist.
  • a NAD biosynthesis from nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist.
  • method for treating a disease or disorder in an individual comprising: determining that a sample obtained from the individual comprises one or more NAPRT1 biomarkers, and administering an effective amount of a therapy comprising a NAD biosynthesis from nicotinamide antagonist to the individual, whereby the disease or disorder is treated.
  • a sample obtained from the individual comprises one or more NAPRT1 biomarkers
  • administering an effective amount of a therapy comprising a NAD biosynthesis from nicotinamide antagonist to the individual, whereby the disease or disorder is treated.
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • kits for treating a disease or disorder comprising: (a) selecting an individual having the disease or disorder, wherein the disease or disorder comprises one or more NAPRTl biomarkers; and (b) administering to the individual thus selected an effective amount of a NAD biosynthesis from nicotinamide antagonist, whereby the disease or disorder is treated.
  • methods of treating a disease or disorder comprising: (a) selecting an individual having the disease or disorder, wherein the disease or disorder comprises a positive NAPRTl methylation status; and (b) administering to the individual thus selected an effective amount of a NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist), whereby the disease or disorder is treated.
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • identifying an individual with a disease or disorder who is more or less likely to exhibit benefit from treatment with a therapy comprising a NAD biosynthesis from nicotinamide antagonist comprising: determining presence or absence of one or more NAPRTl biomarkers in a sample obtained from the individual, wherein presence of the one or more NAPRTl biomarkers in the sample indicates that the individual is more likely to exhibit benefit from treatment with the therapy comprising the NAD biosynthesis from nicotinamide antagonist or absence of the one or more NAPRTl biomarkers indicates that the individual is less likely to exhibit benefit from treatment with the therapy comprising the NAD biosynthesis from nicotinamide antagonist.
  • identifying an individual with a disease or disorder who is more or less likely to exhibit benefit from treatment with a therapy comprising a NAD biosynthesis from nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • the method comprising: determining presence or absence of a positive
  • NAPRTl methylation status in a sample obtained from the individual wherein presence of the positive NAPRTl methylation status in the sample indicates that the individual is more likely to exhibit benefit from treatment with the therapy comprising the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) or absence of the positive NAPRTl methylation status indicates that the individual is less likely to exhibit benefit from treatment with the therapy comprising the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • a therapy comprising a NAD biosynthesis from nicotinamide antagonist
  • the method comprising determining one or more NAPRTl biomarkers, whereby presence of the one or more NAPRTl biomarkers indicates that the individual is more likely to respond effectively to treatment with the NAD biosynthesis from nicotinamide antagonist and absence of the one or more NAPRTl biomarkers indicates that the individual is less likely to respond effectively to treatment with the NAD biosynthesis from nicotinamide antagonist.
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • the method comprising determining a positive NAPRTl methylation status, whereby presence of the positive NAPRTl methylation status indicates that the individual is more likely to respond effectively to treatment with the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) and absence of the positive NAPRTl methylation status indicates that the individual is less likely to respond effectively to treatment with the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • kits for predicting the response or lack of response of an individual with a disease or disorder to an therapy comprising a NAD biosynthesis from nicotinamide antagonist comprising detecting in a sample obtained from the individual presence or absence of one or more NAPRTl biomarkers, wherein presence of the one or more NAPRTl biomarkers is predictive of response of the individual to the therapy comprising the NAD biosynthesis from nicotinamide antagonist and absence of the one or more NAPRTl biomarkers is predictive of lack of response of the individual to the therapy comprising the NAD biosynthesis from nicotinamide antagonist.
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • methods of predicting the response or lack of response of an individual with a disease or disorder to an therapy comprising a NAD biosynthesis from nicotinamide antagonist comprising detecting in a sample obtained from the individual presence or absence of a positive NAPRTl methylation status, wherein presence of the positive NAPRTl methylation status is predictive of response of the individual to the therapy comprising the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) and absence of the positive NAPRTl methylation status is predictive of lack of response of the individual to the therapy comprising the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • the one or more NAPRTl biomarkers is expression levels of NAPRT.
  • the expression levels of NAPRTl are expression levels of NAPRTl mRNA.
  • the expression levels of NAPRTl are expression levels of NAPRTl polypeptide.
  • the one or more NAPRTl biomarkers is NAPRTl methylation status.
  • the NAPRTl methylation status is a positive NAPRTl methylation status.
  • the positive NAPRTl methylation status is methylation of at least one cytosine within a NAPRTl DNA region; for example, the NAPRTl region corresponding to SEQ ID NO: l.
  • the positive methylation status is methylation of at least one cytosine in the promoter region of the NAPRTl gene.
  • the positive methylation status is methylation of at least one cytosine in the 5' region of the NAPRTl gene.
  • the positive methylation status is methylation of at least one cytosine in the CpG island of the NAPRTl gene. In some embodiments, the positive methylation status is methylation of at least one cytosine between about chromosome coordinates 144659500 and 144661000 of human chromosome 8. In some embodiments, the positive methylation status is methylation of at least one cytosine between about chromosome coordinates 144659746 and 144660635 of human chromosome 8. In some embodiments, the positive methylation status is methylation of at least one cytosine between about chromosome coordinates 144659146 and 144661235 of human chromosome 8.
  • the positive methylation status is methylation of at least one cytosine between about chromosome coordinates 144660163 and 144660106 of human chromosome 8. In some embodiments, the positive methylation status is methylation of at least one cytosine between about chromosome coordinates 144660163 and 144660690 of human chromosome 8. In some embodiments, the positive methylation status is methylation of at least one cytosine between about chromosome coordinates 144660411 and 144660433 of human chromosome 8. In some embodiments, the positive methylation status is methylation of at least one cytosine in the sequence represented by SEQ ID NO: l or SEQ ID NO:3.
  • the positive methylation status is methylation of at least one cytosine in the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: l. In some embodiments, the positive methylation status is methylation of at least one cytosine in the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l. In some embodiments, the positive methylation status is methylation of at least one cytosine in any one of the genomic sequences corresponding to the sequences amplified by the primers set forth in Tables 2 and 3.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in the NAPRTl region corresponding to SEQ ID NO: l.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in the promoter region of NAPRTl gene.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in the 5' region of the NAPRTl gene. In some embodiments of any of the methods, the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in the CpG island of the NAPRTl gene.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines between about chromosome coordinates 144659500 and 144661000 of human chromosome 8.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines between about chromosome coordinates 144659746 and 144660635 of human chromosome 8.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines between about chromosome coordinates 144659146 and 144661235 of human chromosome 8.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines between about chromosome coordinates 144660163 and 144660106 of human chromosome 8.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines between about chromosome coordinates 144660163 and 144660690 of human chromosome 8.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines between about chromosome coordinates 144660411 and 144660433 of human chromosome 8.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in the sequence represented by SEQ ID NO: l or SEQ ID NO:3.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in the sequence represented between about position 1018 and about position 1545 of SEQ ID NO: l.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l.
  • the positive methylation status is a methylation of more than about any of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosines in any one of the genomic sequences corresponding to the sequences amplified by the primers set forth in Tables 2 and 3.
  • cancers and cancer cells include, but are not limited to, carcinoma, lymphoma, blastoma (including meduUoblastoma and retinoblastoma), sarcoma (including liposarcoma, synovial cell sarcoma and chondrosarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
  • carcinoma lymphoma
  • blastoma including meduUoblastoma and retinoblastoma
  • sarcoma including liposarcoma, synovial cell sarcoma and chondrosarcoma
  • neuroendocrine tumors including carcinoid tumors, gastrinoma, and islet cell cancer
  • cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, as well as head and neck cancer.
  • the cancer is breast cancer, colorectal cancer, endometrium
  • Presence and/or levels/amount of a biomarker can be determined qualitatively and/or quantitatively based on any suitable criterion known in the art.
  • presence and/or levels/amount of a biomarker in a first sample is increased as compared to presence/absence and/or levels/amount in a second sample.
  • presence/absence and/or levels/amount of a biomarker in a first sample is decreased as compared to presence and/or levels/amount in a second sample.
  • the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosures for determining presence/absence and/or levels/amount of a biomarker are described herein.
  • elevated levels/amount refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein or nucleic acid modification), detected by standard art known methods such as those described herein, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
  • biomarker e.g., protein or nucleic acid modification
  • the elevated levels/amount refers to the increase in level/amount of a biomarker in the sample wherein the increase is at least about any of 1.5X, 1.75X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 25X, 50X, 75X, or 100X the level/amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
  • elevated levels/amount refers to an overall increase of greater than about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold as compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
  • reduced levels/amount refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein or nucleic acid modification), detected by standard art known methods such as those described herein, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
  • biomarker e.g., protein or nucleic acid modification
  • reduced levels/amount refers to the decrease in level/amount of a biomarker in the sample wherein the decrease is at least about any of 0.9X, 0.8X, 0.7X, 0.6X, 0.5X, 0.4X, 0.3X, 0.2X, 0.1X, 0.05X, or 0.01X the level/amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
  • Presence and/or level/amount of various biomarkers in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemical ("IHC"), Western blot analysis,
  • Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.
  • MSD Meso Scale Discovery
  • presence and/or level/amount of a biomarker is determined using a method comprising: (a) performing gene expression profiling, PCR (such as rtPCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on a sample (such as a subject cancer sample); and b) determining presence and/or level/amount of a biomarker in the sample.
  • the microarray method comprises the use of a microarray chip having one or more nucleic acid molecules that can hybridize under stringent conditions to a nucleic acid molecule encoding a gene mentioned above or having one or more polypeptides (such as peptides or antibodies) that can bind to one or more of the proteins encoded by the genes mentioned above.
  • the PCR method is qRT-PCR.
  • the PCR method is multiplex- PCR.
  • gene expression is measured by microarray.
  • gene expression is measured by qRT-PCR.
  • expression is measured by multiplex- PCR.
  • methods for evaluating methylation include randomly shearing or randomly fragmenting the genomic DNA, cutting the DNA with a methylation-dependent or methylation- sensitive restriction enzyme and subsequently selectively identifying and/or analyzing the cut or uncut DNA.
  • Selective identification can include, for example, separating cut and uncut DNA (e.g., by size) and quantifying a sequence of interest that was cut or, alternatively, that was not cut. See, e.g., U.S. Pat. No. 7,186,512.
  • the method can encompass amplifying intact DNA after restriction enzyme digestion, thereby only amplifying DNA that was not cleaved by the restriction enzyme in the area amplified. See, e.g., U.S. Patent Application Nos. 10/971,986;
  • amplification can be performed using primers that are gene specific.
  • adaptors can be added to the ends of the randomly fragmented DNA, the DNA can be digested with a methylation-dependent or methylation-sensitive restriction enzyme, intact DNA can be amplified using primers that hybridize to the adaptor sequences.
  • a second step can be performed to determine the presence, absence or quantity of a particular gene in an amplified pool of DNA.
  • the DNA is amplified using realtime, quantitative PCR.
  • the methods comprise quantifying the average methylation density in a target sequence within a population of genomic DNA.
  • the method comprises contacting genomic DNA with a methylation-dependent restriction enzyme or methylation-sensitive restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved; quantifying intact copies of the locus; and comparing the quantity of amplified product to a control value representing the quantity of methylation of control DNA, thereby quantifying the average methylation density in the locus compared to the methylation density of the control DNA.
  • the quantity of methylation of a locus of DNA can be determined by providing a sample of genomic DNA comprising the locus, cleaving the DNA with a restriction enzyme that is either methylation-sensitive or methylation-dependent, and then quantifying the amount of intact DNA or quantifying the amount of cut DNA at the DNA locus of interest.
  • the amount of intact or cut DNA will depend on the initial amount of genomic DNA containing the locus, the amount of methylation in the locus, and the number (i.e., the fraction) of nucleotides in the locus that are methylated in the genomic DNA.
  • the amount of methylation in a DNA locus can be determined by comparing the quantity of intact DNA or cut DNA to a control value representing the quantity of intact DNA or cut DNA in a similarly-treated DNA sample.
  • the control value can represent a known or predicted number of methylated nucleotides.
  • the control value can represent the quantity of intact or cut DNA from the same locus in another (e.g., normal, non-diseased) cell or a second locus.
  • methylation-sensitive restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be directly proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample.
  • a methylation-dependent restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be inversely proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample.
  • Such assays are disclosed in, e.g., U.S. patent application Ser. No. 10/971,986.
  • quantitative amplification methods e.g., quantitative PCR or quantitative linear amplification
  • methods of quantitative amplification can be used to quantify the amount of intact DNA within a locus flanked by amplification primers following restriction digestion.
  • Additional methods for detecting DNA methylation can involve genomic sequencing before and after treatment of the DNA with bisulfite. See, e.g., Frommer et al., Proc. Natl. Acad. Sci. USA 89: 1827-1831 (1992). When sodium bisulfite is contacted to DNA, unmethylated cytosine is converted to uracil, while methylated cytosine is not modified.
  • restriction enzyme digestion of PCR products amplified from bisulfite -converted DNA is used to detect DNA methylation. See, e.g., Sadri & Hornsby, Nucl. Acids Res. 24:5058-5059 (1996); Xiong & Laird, Nucleic Acids Res. 25:2532-2534 (1997).
  • a MethyLight assay is used alone or in combination with other methods to detect DNA methylation (see, Eads et al., Cancer Res. 59:2302-2306 (1999)). Briefly, in the MethyLight process genomic DNA is converted in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil). Amplification of a DNA sequence of interest is then performed using PCR primers that hybridize to CpG dinucleotides.
  • amplification can indicate methylation status of sequences where the primers hybridize.
  • the amplification product can be detected with a probe that specifically binds to a sequence resulting from bisulfite treatment of an
  • kits for use with MethyLight can include sodium bisulfite as well as primers or detectably-labeled probes (including but not limited to Taqman or molecular beacon probes) that distinguish between methylated and unmethylated DNA that have been treated with bisulfite.
  • kit components can include, e.g., reagents necessary for amplification of DNA including but not limited to, PCR buffers, deoxynucleotides; and a thermostable polymerase.
  • a Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) reaction is used alone or in combination with other methods to detect DNA methylation (see Gonzalgo & Jones Nucleic Acids Res. 25:2529-2531 (1997)).
  • the Ms-SNuPE technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single -nucleotide primer extension. Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite -converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest.
  • a methylation-specific PCR (“MSP”) reaction is used alone or in combination with other methods to detect DNA methylation.
  • An MSP assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. See, Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, (1996); U.S. Pat. No. 5,786,146.
  • DNA methylation is detected by a QIAGEN PyroMark CpG Assay predesigned Pyrosequencing DNA Methylation assays.
  • cell methylation status is determined using high-throughput DNA methylation analysis to determine sensitivity to NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • genomic DNA is isolated from a cell or tissue sample (e.g. a tumor sample or a blood sample) and is converted in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil) using standard assays in the art.
  • the bisulfite converted DNA product is amplified, fragmented and hybridized to an array containing CpG sites from across a genome using standard assays in the art.
  • the array is imaged and processed for analysis of the DNA methylation status using standard assays in the art.
  • the tissue sample is formalin-fixed paraffin embedded (FFPE) tissue.
  • the tissue sample is fresh frozen tissue.
  • the DNA isolated from the tissue sample is preamplified before bisulfite conversion.
  • the DNA isolated from the tissue sample is preamplified before bisulfite conversion by using the Invitrogen Superscript III One-Step RT-PCR System with Platinum Taq.
  • the DNA isolated from the tissue sample is preamplified before bisulfite conversion using a Taqman based assay.
  • the sodium bisulfite reaction is conducted using the Zymo EZ DNA Methylation Kit.
  • the bisulfite converted DNA is amplified and hybridized to an array using the Illumina Infinium HumanMethylation450 Beadchip Kit.
  • the array is imaged on an Illumina iScan Reader.
  • the images are processed with the GenomeStudio software methylation module.
  • the methylation data is analyzed using the Bioconductor lumi software package. See Du et al.,
  • NAPRT1 DNA methylation sites are identified using bisulfite sequencing PCR (BSP) to determine sensitivity to NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • BSP bisulfite sequencing PCR
  • genomic DNA is isolated from a cell or tissue sample (e.g., a tumor sample or a blood sample) and is converted in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil) using standard assays in the art.
  • the bisulfite converted DNA product is amplified using primers designed to be specific to the bisulfite converted DNA (e.g., bisulfite-specific primers) and ligated into vectors for transformation into a host cell using standard assays in the art. After selection of the host cells containing the PCR amplified bisulfite converted DNA product of interest, the DNA product is isolated and sequenced to determine the sites of methylation using standard assays in the art.
  • the tissue sample is formalin-fixed paraffin embedded (FFPE) tissue.
  • the tissue sample is an FFPE tissue that has been processed for IHC analysis; for example, for NAPRTl expression.
  • the tissue sample is an FFPE tissue that showed little or no NAPRTl expression by IHC. In some embodiments, the tissue sample is fresh frozen tissue. In some embodiments, the DNA isolated from the tissue sample is preamplified before bisulfite conversion. In some embodiments, the DNA isolated from the tissue sample is preamplified before bisulfite conversion using the Invitrogen Superscript III One-Step RT-PCR System with Platinum Taq. In some embodiments, the DNA isolated from the tissue sample is preamplified before bisulfite conversion using a Taqman based assay. In some embodiments, the sodium bisulfite reaction is conducted using the Zymo EZ DNA Methylation-Gold Kit.
  • the primers designed to be specific to the bisulfite converted DNA are designed using Applied Biosystems Methyl Primer Express software.
  • the bisulfite converted DNA is amplified using the bisulfite-specific primers in Table 2.
  • the bisulfite-specific primers are BSP1 primers and/or BSP2 primers presented in Table 2.
  • the bisulfite converted DNA product is PCR amplified using the Invitrogen Superscript III One-Step RT-PCR System with Platinum Taq.
  • the PCR amplified bisulfite converted DNA product is ligated into a vector using the Invitrogen TOPO TA Cloning kit.
  • the host cell is bacteria.
  • the isolated PCR amplified bisulfite converted DNA product of interest is sequenced using Applied Biosystems 3730x1 DNA Analyzer.
  • the primers designed to be specific to the bisulfite converted DNA are designed using Qiagen PyroMark Assay Design software.
  • the bisulfite converted DNA product is PCR amplified using the Invitrogen Superscript III One-Step RT-PCR System with Platinum Taq.
  • the PCR amplified bisulfite converted DNA product is sequenced using Qiagen Pyromark Q24 and analyzed Qiagen with PyroMark software.
  • the coding DNA strand of all or a portion of the NAPRTl gene is sequenced.
  • the complementary DNA strand of all or a portion of the NAPRTl gene is sequenced.
  • the sense DNA strand and the complementary DNA strands of all or a portion of the NAPRTl gene are sequenced.
  • a mixture of sense strands and complementary strands of all or a portion of the NAPRTl gene is sequenced.
  • the coding strand of one PCR amplicon from the NAPRTl is sequenced and the complementary strand of another PCR amplicon from NAPRTl in sequenced.
  • NAPRT1 DNA methylation sites are identified using quantitative methylation specific PCR (QMSP) to determine sensitivity to NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • QMSP quantitative methylation specific PCR
  • genomic DNA is isolated from a cell or tissue sample and is converted in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil) using standard assays in the art.
  • the tissue sample is formalin-fixed paraffin embedded (FFPE) tissue.
  • the tissue sample is an FFPE tissue that has been processed for IHC analysis; for example, for NAPRT1 expression.
  • the tissue sample is an FFPE tissue that showed little or no NAPRT1 expression by IHC.
  • the tissue sample is fresh frozen tissue.
  • the bisulfite converted DNA product is amplified using primers designed to be specific to the bisulfite converted DNA (e.g., quantitative methylation specific PCR primers).
  • the bisulfite converted DNA product is amplified with quantitative methylation specific PCR primers and analyzed for methylation using standard assays in the art.
  • the tissue sample is formalin-fixed paraffin embedded (FFPE) tissue.
  • the tissue sample is fresh frozen tissue.
  • the DNA isolated from the tissue sample is preamplified before bisulfite conversion using the Invitrogen Superscript III One-Step RT-PCR System with Platinum Taq. In some embodiments, the DNA isolated from the tissue sample is preamplified before bisulfite conversion. In some embodiments, the DNA isolated from the tissue sample is preamplified before bisulfite conversion using a Taqman based assay. In some embodiments, the sodium bisulfite reaction is conducted using a commercially available kit. In some embodiments, the sodium bisulfite reaction is conducted using the Zymo EZ DNA Methylation-Gold Kit.
  • the primers designed to be specific to the bisulfite converted DNA are designed using Applied Biosystems Methyl Primer Express software.
  • the bisulfite converted DNA is amplified using a Taqman based assay.
  • the bisulfite converted DNA is amplified using the quantitative methylation specific PCR primers and probes in Tables 3 and 4, respectively.
  • the bisulfite converted DNA is amplified using the QMSP3 primers and probes presented in Tables 3 and 4.
  • the bisulfite converted DNA is amplified on an Applied Biosystems 7900HT and analyzed using Applied Biosystems SDS software.
  • the invention provides methods to determine NAPRT1 methylation by 1) IHC analysis of tumor samples, followed by 2) quantitative methylation specific PCR of DNA extracted from the tumor tissue used in the IHC ananlysis of step 1.
  • coverslips from IHC slides are removed by one of two methods: the slide are placed in a freezer for at least 15 minutes, then the coverslip is pried off of the microscope slide using a razor blade. Slides are then incubated in xylene at room temp to dissolve the mounting media. Alternatively, slides are soaked in xylene until the coverslip fell off. This can take up to several days. All slides are taken through a deparaffinization procedure of 5 min xylene (x3), and 5 min 100% ethanol (x2).
  • Tissues are scraped off slides with razor blades and placed in a tissue lysis buffer containing proteinase K and incubated overnight at 56°C. In cases where tissue is still present after incubation, an extra 10 ⁇ Proteinase K may be added and the tissue is incubated for another 30 min. DNA extraction was continued; for example, by using a QIAamp DNA FFPE Tissue kit. DNA extracted directly from IHC slides was subject to QMSP analysis using the QMSP3 primers and probes as described above.
  • the bisulfite -converted DNA is sequenced by a deep sequencing.
  • Deep sequencing is a process, such as direct pyrosequencing, where a sequence is read multiple times. Deep sequencing can be used to detect rare events such as rare mutations. Ultra-deep sequencing of a limited number of loci may been achieved by direct pyrosequencing of PCR products and by sequencing of more than 100 PCR products in a single run. A challenge in sequencing bisulphite-converted DNA arises from its low sequence complexity following bisulfite conversion of cytosine residues to thymine (uracil) residues.
  • RRBS Reduced representation bisulphite sequencing
  • RRBS Reduced representation bisulphite sequencing
  • RRBS Reduced representation bisulphite sequencing
  • Targeting may be accomplished by array capture or padlock capture before sequencing.
  • targeted capture on fixed arrays or by solution hybrid selection can enrich for sequences targeted by a library of DNA or RNA oligonucleotides and can be performed before or after bisulphite conversion.
  • padlock capture provides improved enrichment efficiency by combining the increased annealing specificity of two tethered probes, and subsequent amplification with universal primers allows for a more uniform representation than amplification with locus-specific primers.
  • Additional methylation detection methods include, but are not limited to, methylated CpG island amplification (see Toyota et al., Cancer Res. 59:2307-12 (1999)) and those described in, e.g., U.S. Patent Publication 2005/0069879; Rein et al., Nucleic Acids Res. 26 (10): 2255-64 (1998); Olek et al., Nat Genet. 17(3): 275-6 (1997); Laird, PW Nature Reviews 11 : 195-203 (2010); and PCT Publication No. WO 00/70090).
  • the expression of NAPRT1 in a cell is determined by evaluating NAPRT1 mRNA in a cell.
  • Methods for the evaluation of mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
  • the expression of NAPRT1 in a test sample is compared to a reference sample.
  • the test sample may be a tumor tissue sample and the reference sample may be from normal tissue or cells such as PBMCs.
  • Samples from mammals can be conveniently assayed for mRNAs using Northern, dot blot or PCR analysis.
  • such methods can include one or more steps that allow one to determine the levels of target mRNA in a biological sample (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member).
  • the sequence of the amplified target cDNA can be determined.
  • Optional methods of the invention include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies.
  • mRNAs such as target mRNAs
  • test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes.
  • the probes are then hybridized to an array of nucleic acids immobilized on a solid support.
  • the array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes whose expression correlates with increased or reduced clinical benefit of anti-angiogenic therapy may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
  • presence and/or level/amount is measured by observing protein expression levels of an aforementioned gene.
  • the method comprises contacting the biological sample with antibodies to a biomarker described herein under conditions permissive for binding of the biomarker, and detecting whether a complex is formed between the antibodies and biomarker.
  • a biomarker described herein Such method may be an in vitro or in vivo method.
  • an antibody is used to select subjects eligible for therapy with NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist), e.g., a NAPRTl biomarker for selection of individuals.
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • NAPRTl expression in a test sample is determined by IHC analysis using an NAPRTl detection agent such as an anti-NAPRTl antibody.
  • the presence and/or level/amount of biomarker proteins in a sample are examined using IHC and staining protocols.
  • IHC staining of tissue sections has been shown to be a reliable method of determining or detecting presence of proteins in a sample.
  • level of biomarker is determined using a method comprising: (a) performing IHC analysis of a sample (such as a subject cancer sample) with an antibody; and b) determining level of a biomarker in the sample.
  • IHC staining intensity is determined relative to a reference value.
  • RNA is isolated from a tumor sample, incubated with sodium bisulfite sufficient to convert unmethylated cytosine in the DNA to uracil, and sequencing all or part of the NAPRTl promoter region of the DNA.
  • the methylation level of the NAPRTl gene is measured by determining the number of CpG and CpHpG sites where the cytosine residue was not converted to uracil.
  • the part of the NAPRTl gene is the NAPRTl promoter. In some embodiments, the part of the NAPRTl gene is a CpG island. In some embodiments, the part of the NAPRTl gene is between about chromosome coordinates 144659500 and 144661000 of human chromosome 8.
  • the part of the NAPRTl gene is between about chromosome coordinates 144659746 and 144660635 of human chromosome 8. In some embodiments, the part of the NAPRTl gene is a between about chromosome coordinates 144659146 and 144661235 of human chromosome 8. In some embodiments, the part of the NAPRTl gene is a between about chromosome coordinates 144660163 and 144660106 of human chromosome 8. In some embodiments, the part of the NAPRTl gene is between about chromosome coordinates 144660163 and 144660690 of human chromosome 8.
  • the part of the NAPRTl gene is a between about chromosome coordinates 144660411 and 144660433 of human chromosome 8. In some embodiments, the part of the NAPRTl gene is the sequence represented by SEQ ID NO: l or SEQ ID NO: 3. In some embodiments, the part of the NAPRTl gene comprises the sequence represented between about position 1221 and about position 1545 of SEQ ID NO: l.
  • a therapy comprising an NAD biosynthesis from nicotinamide antagonist
  • DNA is isolated from a tumor sample, incubated with sodium bisulfite sufficient to convert unmethylated cytosine in the DNA to uracil.
  • the methylation level of the NAPRTl gene is measured by amplifying a portion of the NAPRTl gene of the DNA sample using quantitative methylation specific PCR and determining the methylation level of the NAPRTl region by determining the -dCt value of the tumor sample with the -dCt value obtained from quantitative methylation specific PCR of non- methylated DNA.
  • the non-methylated DNA is recombinant DNA.
  • the non-methylated DNA is from normal tissue, for example, PBMCs or non-cancerous tissue. In some embodiments, greater than about any one of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% methylation of the NAPRTl sequence indicates that the individual is more likely to benefit from treatment.
  • the portion of the NAPRTl gene amplified is from the NAPRTl promoter. In some embodiments, the portion of the NAPRTl gene amplified is from the a CpG island. In some embodiments, the portion of the NAPRTl gene amplified is between about chromosome coordinates 144659500 and
  • the portion of the NAPRTl gene amplified is between about chromosome coordinates 144659746 and 144660635 of human chromosome 8. In some embodiments, the portion of the NAPRTl gene amplified is between about chromosome coordinates 144659146 and 144661235 of human chromosome 8. In some
  • the portion of the NAPRTl gene amplified is between about chromosome coordinates 144660163 and 144660106 of human chromosome 8. In some embodiments, the portion of the NAPRTl gene amplified is between about chromosome coordinates 144660163 and 144660690 of human chromosome 8. In some embodiments, portion of the NAPRTl gene amplified is from the between about chromosome coordinates 144660411 and 144660433 of human chromosome 8. In some embodiments, the portion of the NAPRTl gene amplified is from the sequence represented by SEQ ID NO: 1 or SEQ ID NO:3.
  • the portion of the NAPRT1 gene amplified comprises the sequence represented between about position 1221 and about position 1288 of SEQ ID NO: l. In some embodiments, the portion of the NAPRT1 gene amplified comprises a genomic sequence corresponding to the sequence amplified by any of the primers set forth in Table 3. In some embodiments, the DNA from the tumor sample is isolated from a formalin-fixed paraffin embedded tumor sample. In some embodiments, the method portion of the NAPRT1 gene is pre-amplified prior to quantitative methylation specific PCR.
  • IHC may be performed in combination with additional techniques such as morphological staining and/or fluorescence in-situ hybridization.
  • Two general methods of IHC are available; direct and indirect assays.
  • binding of antibody to the target antigen is determined directly.
  • This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme- labeled primary antibody, which can be visualized without further antibody interaction.
  • a labeled primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody.
  • a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used for IHC typically will be labeled with a detectable moiety.
  • Numerous labels are available which can be generally grouped into the following categories: (a) Radioisotopes, such as 35S, 14C, 1251, 3H, and 1311; (b) colloidal gold particles; (c) fluorescent labels including, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially available fluorophores such SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of the above; (d) various enzyme-substrate labels are available and U.S.
  • Patent No. 4,275,149 provides a review of some of these.
  • Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate
  • HRPO horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • saccharide oxidases e.g
  • dehydrogenase dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase
  • lactoperoxidase lactoperoxidase
  • microperoxidase and the like.
  • enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate; alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl- -D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl- -D- galactosidase).
  • HRPO horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -D-galactosidase ⁇ -D-Gal
  • a chromogenic substrate e.g., p-nitrophenyl- -D-galactosidase
  • fluorogenic substrate e.g., 4-methylumbelliferyl- -D- gal
  • Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, e.g., using a microscope, and staining intensity criteria, routinely used in the art, may be employed.
  • a staining pattern score of about 1+ or higher is diagnostic and/or prognostic.
  • a staining pattern score of about 2+ or higher in an IHC assay is diagnostic and/or prognostic.
  • a staining pattern score of about 3 or higher is diagnostic and/or prognostic.
  • staining is generally determined or assessed in tumor cell and/or tissue (as opposed to stromal or surrounding tissue that may be present in the sample).
  • the sample may be contacted with an antibody specific for said biomarker under conditions sufficient for an antibody-biomarker complex to form, and then detecting said complex.
  • the presence of the biomarker may be detected in a number of ways, such as by Western blotting and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum.
  • a wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two- site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target biomarker.
  • Presence and/or level/amount of a selected biomarker in a tissue or cell sample may also be examined by way of functional or activity-based assays. For instance, if the biomarker is an enzyme, one may conduct assays known in the art to determine or detect the presence of the given enzymatic activity in the tissue or cell sample.
  • the samples are normalized for both differences in the amount of the biomarker assayed and variability in the quality of the samples used, and variability between assay runs.
  • normalization may be accomplished by detecting and incorporating the level of certain normalizing biomarkers, including well known housekeeping genes, such as ACTB.
  • normalization can be based on the mean or median signal of all of the assayed genes or a large subset thereof (global normalization approach).
  • measured normalized amount of a subject tumor mRNA or protein is compared to the amount found in a reference set. Normalized expression levels for each mRNA or protein per tested tumor per subject can be expressed as a percentage of the expression level measured in the reference set. The presence and/or expression level/amount measured in a particular subject sample to be analyzed will fall at some percentile within this range, which can be determined by methods well known in the art.
  • relative expression level of a gene is determined as follows:
  • Relative expression genel sample 1 2 exp (Ct housekeeping gene - Ct genel) with Ct determined in a sample.
  • Relative expression genel reference RNA 2 exp (Ct housekeeping gene - Ct genel) with Ct determined in the reference sample.
  • Normalized relative expression genel sample 1 (relative expression genel sample 1 / relative expression genel reference RNA) x 100
  • Ct is the threshold cycle.
  • the Ct is the cycle number at which the fluorescence generated within a reaction crosses the threshold line.
  • RNA is a comprehensive mix of RNA from various tissue sources (e.g., reference RNA #636538 from Clontech, Mountain View, CA). Identical reference RNA is included in each qRT-PCR run, allowing comparison of results between different experimental runs.
  • the sample is a clinical sample.
  • the sample is used in a diagnostic assay.
  • the sample is obtained from a primary or metastatic tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue.
  • tumor cells can be obtained indirectly in the form of tissues or fluids that are known or thought to contain the tumor cells of interest. For instance, samples of lung cancer lesions may be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushings, or from sputum, pleural fluid or blood.
  • the sample includes circulating tumor cells; for example, circulating cancer cells in blood, urine or sputum.
  • Genes or gene products can be detected from cancer or tumor tissue or from other body samples such as urine, sputum, serum or plasma.
  • body samples such as urine, sputum, serum or plasma.
  • the same techniques discussed above for detection of target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells may be sloughed off from cancer lesions and appear in such body samples. By screening such body samples, a simple early diagnosis can be achieved for these cancers. In addition, the progress of therapy can be monitored more easily by testing such body samples for target genes or gene products.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or combined multiple samples from the same subject or individual that are obtained at one or more different time points than when the test sample is obtained.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained at an earlier time point from the same subject or individual than when the test sample is obtained.
  • Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more healthy individuals who are not the subject or individual.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject or individual.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual.
  • a disease or disorder e.g., cancer
  • the NAD biosynthesis from nicotinamide antagonist is an antibody, binding polypeptide, small molecule, or polynucleotide.
  • the NAD biosynthesis from nicotinamide antagonist is an antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a human, humanized, or chimeric antibody.
  • the antibody is an antibody fragment and the antibody fragment binds NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • the NAD biosynthesis from nicotinamide antagonist is a small molecule.
  • the NAD biosynthesis from nicotinamide antagonist is a Nampt antagonist.
  • the NAD biosynthesis from nicotinamide antagonist is a NMNAT antagonist. Further description of antagonists useful in these methods are provided below.
  • the individual according to any of the above embodiments may be a human.
  • the method comprises administering to an individual having such cancer an effective amount of a NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.
  • the individual may be a human.
  • the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) described herein can be used either alone or in combination with other agents in a therapy.
  • a NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) described herein may be co-administered with at least one additional therapeutic agent including another NAD biosynthesis from nicotinamide antagonist.
  • the NAD biosynthesis from nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • the niacin reduces toxicity of the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist). In some embodiments, the niacin increases the therapeutic index of the NAD biosynthesis from nicotinamide antagonist. In certain embodiments, an additional therapeutic agent is a
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • NAD biosynthesis from nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • a NAD biosynthesis from nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • an antibody, binding polypeptide, and/or small molecule e.g., an antibody, binding polypeptide, and/or small molecule described herein (and any additional therapeutic agent)
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time- points, bolus administration, and pulse infusion are contemplated herein.
  • NAD biosynthesis from nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • an antibody, binding polypeptide, and/or small molecule e.g., an antibody, binding polypeptide, and/or small molecule
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the NAD biosynthesis from nicotinamide antagonist need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question.
  • the effective amount of such other agents depends on the amount of the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • a NAD biosynthesis from nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • the appropriate dosage of a NAD biosynthesis from nicotinamide antagonist will depend on the type of disease to be treated, the severity and course of the disease, whether the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist) is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist), and the discretion of the attending physician.
  • NAD biosynthesis from nicotinamide antagonist is suitably administered to the individual at one time or over a series of treatments.
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist
  • One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the individual receives from about two to about twenty, or e.g., about six doses of the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist)).
  • An initial higher loading dose, followed by one or more lower doses may be
  • An exemplary dosing regimen comprises administering. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the invention in place of or in addition to the NAD biosynthesis from nicotinamide antagonist (e.g., Nampt antagonist and/or NMNAT antagonist).
  • nicotinamide antagonist e.g., Nampt antagonist and/or NMNAT antagonist.
  • nicotinamide antagonists e.g., Nampt antagonists and/or NMNAT antagonists
  • the NAD biosynthesis from nicotinamide antagonists are an antibody, binding polypeptide, small molecule, and/or polynucleotide.
  • an antibody that binds to Nampt and/or NMNAT.
  • an antibody is humanized.
  • an anti- NAD biosynthesis from nicotinamide antibody e.g., anti-Nampt antibody and/or anti-NMNAT antibody
  • nicotinamide antibody e.g., anti-Nampt antibody and/or anti-NMNAT antibody
  • a monoclonal antibody including a chimeric, humanized or human antibody.
  • an anti-NAD biosynthesis from nicotinamide antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.
  • the antibody is a full length antibody, e.g., an intact IgGl" antibody or other antibody class or isotype as defined herein.
  • an anti-NAD biosynthesis from nicotinamide antibody may incorporate any of the features, singly or in combination, as described in Sections below:
  • an antibody provided herein has a dissociation constant (Kd) of ⁇ 1 ⁇ .
  • Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay.
  • Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).
  • MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 °C).
  • a non-adsorbent plate (Nunc #269620)
  • 100 pM or 26 pM [1251] -antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)).
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached.
  • Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE ®-3000 (BIAcore, Inc., Piscataway, NJ) at 25 °C with immobilized antigen CM5 chips at -10 response units (RU).
  • CM5 carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N'- (3-dimethylaminopropyl)- carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml (-0.2 ⁇ ) before injection at a flow rate of 5 ⁇ /minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25°C at a flow rate of
  • association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).
  • an antibody provided herein is an antibody fragment.
  • Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below.
  • Fab fragment antigen
  • Fab' fragment antigen binding domain
  • Patent Nos. 5,571,894 and 5,587,458 For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161 ; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single -domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B l).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.
  • recombinant host cells e.g., E. coli or phage
  • an antibody provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non- human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non- human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non- human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); US Patent Nos.
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit” method (see, e.g., Sims et al., J. Immunol.
  • an antibody provided herein is a human antibody.
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated.
  • Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
  • Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
  • Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., in Methods in Mol. Biol. 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.
  • phage display methods repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • PCR polymerase chain reaction
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
  • Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
  • an antibody provided herein is a multispecific antibody, e.g., a bispecific antibody.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for Nampt and/or NMNAT and the other is for any other antigen.
  • bispecific antibodies may bind to two different epitopes of Nampt and/or NMNAT. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Nampt and/or NMNAT. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
  • Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No.
  • Engineered antibodies with three or more functional antigen binding sites are also included herein (see, e.g., US 2006/0025576).
  • the antibody or fragment herein also includes a "Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to Nampt and/or NMNAT as well as another, different antigen (see, US 2008/0069820, for example).
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to "defucosylated” or “fucose- deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621 ; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech.
  • Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108, Presta, L; and
  • WO 2004/056312 Adams et al., especially at Example 11
  • knockout cell lines such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells
  • Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)
  • Kanda Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)
  • WO 2004/056312 Adams et al., especially at Example 11
  • knockout cell lines such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells
  • Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No.
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). [0261] Fc region variants
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
  • non-radioactive assays methods may be employed (see, for example, ACTITM nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652- 656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No.
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821 ; and WO 94/29351 concerning other examples of Fc region variants.
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541.
  • immunoconjugates comprising an anti-NAD biosynthesis from nicotinamide antibody (e.g., anti-Nampt antibody and/or anti-NMNAT antibody) herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos.
  • ADC antibody-drug conjugate
  • drugs including but not limited to a maytansinoid
  • a maytansinoid see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl
  • an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos.
  • an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from
  • Pseudomonas aeruginosa Pseudomonas aeruginosa
  • ricin A chain abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.
  • a radioactive atom to form a radioconjugate.
  • radioactive isotopes are available for the production of radioconjugates. Examples include At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu.
  • the radioconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc99 or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-I l l, fluorine-19, carbon-13, nitrogen- 15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl- 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutar aldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide -containing linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used.
  • the immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo- GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
  • cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo- GMBS, sulfo
  • Binding polypeptides are polypeptides that bind, preferably specifically, to Nampt and/or NMNAT as described herein.
  • the binding polypeptides are NAD biosynthesis from nicotinamide antagonists (e.g., Nampt antagonist and/or NMNAT antagonist).
  • Binding polypeptides may be chemically synthesized using known polypeptide synthesis methodology or may be prepared and purified using recombinant technology.
  • Binding polypeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such binding polypeptides that are capable of binding, preferably specifically, to a target, Nampt and/
  • Binding polypeptides may be identified without undue experimentation using well known techniques.
  • techniques for screening polypeptide libraries for binding polypeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and W 084/03564; Geysen et al., Proc. Natl. Acad. Sci.
  • bacteriophage (phage) display is one well known technique which allows one to screen large polypeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a target polypeptide, Nampt and/or NMNAT.
  • Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J.K. and Smith, G. P. (1990) Science, 249: 386).
  • the utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity.
  • Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments.
  • WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands.
  • WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech., 9: 187).
  • WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO
  • Small molecules for use as a NAD biosynthesis from nicotinamide small molecule antagonists (e.g., Nampt small molecule antagonist and/or NMNAT small molecule antagonist).
  • nicotinamide small molecule antagonists e.g., Nampt small molecule antagonist and/or NMNAT small molecule antagonist.
  • Small molecules are preferably organic molecules other than binding polypeptides or antibodies as defined herein that bind, preferably specifically, to Nampt and/or NMNAT as described herein. Binding organic small molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). Binding organic small molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic small molecules that are capable of binding, preferably specifically, to a polypeptide as described herein may be identified without undue experimentation using well known techniques.
  • Binding organic small molecules may be, for example, aldehydes, ketones, oximes, hydrazones,
  • the NAD biosynthesis from nicotinamide small molecule antagonist is a Nampt antagonist. In some embodiments, the NAD biosynthesis from nicotinamide antagonist is a NMNAT small molecule antagonist.
  • the Nampt small molecule antagonist can be chosen from Nampt antagonists described in PCT Publication No. WO 2011006988, and in particular the following compounds:
  • the Nampt small molecule antagonist can also be chosen from Nampt antagonists described in PCT Publication No. WO 2011109441, and in particular, compounds of Tables 1A, IB, 2, 3A, 3B and 4.
  • the Nampt small molecule antagonist can further be chosen from Nampt antagonists described in PCT patent application Nos. WO2012031196, WO2012031197, WO2012031199 and in particular the following compounds or pharmaceutically acceptable salts thereof described in unpublished PCT patent application Nos. WO2012031197:
  • R is bicyclic heteroaryl comprising 1 , 2, 3 or 4 heteroatom(s) independently selected from N, S or O, wherein said heteroaryl may be substituted by one or more substituents selected from the group consisting of amino, oxo, and halo ; and wherein said heteroaryl can comprise one or more N-oxide(s) formed with a N atom member of said heteroaryl;
  • R 1 is -NHR 4 and R 4 is cycloalkyl, heterocycloalkyl, aryl or heteroaryl;
  • each of said cycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with 1, 2, 3, 4 or 5 substituents which can be the same or different and are independently selected from the group consisting of:
  • each of said cycloalkyl, heterocycloalkyl, aryl, or heteroaryl may optionally
  • R 2 and R 3 can be independently selected from the group consisting of H and deuterium
  • R 5 is H, alkyl or arylalkyl-
  • R a and R b are independently selected from the group consisting of H, alkyl, alkoxy, alkoxyalkyl and haloalkyl;
  • n 0, 1, 2, 3, 4, 5 or 6;
  • z 0, 1 or 2.
  • the Nampt small molecule antagonist can further be chosen from Nampt antagonists selected from the group consisting of:

Abstract

L'invention concerne la biosynthèse de nicotinamide adénine dinucléotide à partir d'antagonistes de nicotinamide (par exemple, antagonistes de Nampt et/ou antagonistes de NMNAT), ainsi que des procédés d'utilisation associés.
PCT/US2013/040613 2012-05-11 2013-05-10 Procédés d'utilisation d'antagonistes de biosynthèse de nicotinamide adénine dinucléotide à partir de nicotinamide WO2013170191A1 (fr)

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