(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`18 July 2002 (18.07.2002)
`
`
`
`PCT
`
`(10) International Publication Number
`W0 02/055018 A2
`
`(51) International Patent Classification7:
`
`A61K (74) Agents: SPECTOR, Eric, S. et al.; Jones, Tullar &
`Cooper, PC, PO. Box 2266 Eads Station, Arlington, VA
`
`(21) International Application Number:
`
`PCT/USOI/44709
`
`22202 (US)-
`
`(22) International Filing Date:
`14 December 2001 (14.12.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`09/757,610
`
`11 January 2001 (11.01.2001)
`
`US
`
`(71) Applicant: DUKE UNIVERSITY [US/US]; Office of
`Science and Technology, PO. Box 90083, Durham, NC
`(US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
`SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, FT, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR,
`NE, SN, TD, TG).
`
`Published:
`
`STAMLER, Jonathan, S.; 3416 Juniper
`(72) Inventors:
`Place, Chapel Hill, NC 27514 (US). LIU, Limin; 720
`South LaSalle Street, Apt. M, Durham, NC 27705 (US).
`HAUSLADEN, Alfred;
`3600 Tremont Drive, #D5,
`Durham, NC 27705 (US). NUDELMAN, Raphael; 4407
`Beechnut Lane, Durham, NC 27707 (US).
`
`without international search report and to be republished
`upon receipt of that report
`
`For two-letter codes and other abbreviations, refer to the ”Guid-
`ance Notes on Codes andAbbreviations ” appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`O02/055018A2
`
`(54) Title: INHIBITTNG GS—FDH TO MODULATE NO BIOACTIVITY
`
`(57) Abstract: Patients needing NO donor therapy or inhibition of pathologically proliferating cells or increased NO bioactivity are
`treated with a therapeutically effective amount of an inhibitor of glutathione—dependent formaldehyde dehydrogenase.
`
`THORNE - EXHIBIT 1006
`
`THORNE - EXHIBIT 1006
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`JNHEITJNG GS—FDH TO MODULATE NO BIOACTIVITY
`
`I
`
`Technical Field
`
`The invention relates to modulating NO (nitric oxide) bioactivity to obtain
`
`therapeutic efi‘ect.
`
`Background ofthe Invention
`
`Stamler et al. US. Patent No. 6,057,367 is directed to treating mammals for
`
`infections or for conditions associated with pathologically proliferating mammalian cell
`growth (for example, certain cancers, restenosis, benign prostatic hypertrophy) by
`
`administration of a manipulator ofnitrosative stress (an impetus for NO or NO2 group
`
`attachment to proteins, nucleic acids or other biological molecules) to selectively kill or
`
`reduce the growth ofthe microbes or hehninths causing the infection or ofhost cells
`infected with the microbes or ofthe pathologically proliferating mammalian cells.
`
`Stamler et al. US. Application No. 09/695,934 discloses use ofNO donors to
`
`prevent the occurrence ofrestenosis following angioplasty, to inhibit platelets to prevent
`
`coagulation and thrombus formation, to treat angina in patients at risk for coagulation and
`
`thrombus formation, to inhibit microbes, to treat impotence, asthma, cystic fibrosis,
`
`hypoxia and ischemic disorders, heart failure, stroke, arthritis, ARDS, hypertension,
`
`neurodegeneration, painful crisis of sickle cell disease, cancer and any pathological
`
`proliferation of cells and any NMDA related injury. The invention is directed to C-nitroso
`
`compounds and use thereof as NO donors.
`
`Gaston, Stamler and Griflith US. Application No. 08/403,775 is directed to use of
`
`inhibitors of S—nitrosothiol breakdown to treat asthma.
`
`Numerous enzymes have been shown to break down S-nitrosothiols in vitro. These
`
`include (a) thioredoxin system, (b) glutathione peroxidase, (G) gamma glutamyl
`
`transpeptidase, (d) xanthine oxidase, (e) alcohol dehydrogenase Class III, and (f) other
`classes of alcohol dehydrogenase.
`
`In respect to alcohol dehydrogenases including alcohol dehydrogenase Class [H
`
`(also known as glutathione-dependent formaldehyde dehydrogenase), see the following
`
`publications which present in vitro data: Kuwada, M., et al. J. Biochem 88, 85 9-869
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`
`(1980); Jensen, D. E., et al., Biochemical Pharmacology 53, 1297-1306 (1997);
`
`Jensen, D. E., et al., Biochem J. 331, 659-668 (1998).
`
`Despite the in vitra results referred to above, the current perspective is that thiols,
`
`ascorbate and copper ions break down S-nitrosothiols in viva. However, there has been no
`
`work heretofore demonstrating how S-nitrosothiols are broken down in viva.
`
`1 It has not heretofore been known that inhibition of glutathione— dep endent
`
`formaldehyde dehydrogenase mediates NO donor therapy, nitrosative stress and NO
`
`bioact'rvity in viva.
`
`Smmag; of the Invention
`
`It has been concluded in the course of making the invention herein that enzyme,
`
`namely glutathione— dependent formaldehyde dehydrogenase known heretofore to oxidize
`
`the formaldehyde glutathione adduct, S-hydroxymethylglutathione, previously thought to
`I be the major enzyme substrate, fimctions in viva to metabolize S-nitrosoglutathione and
`
`protein S-nitro sothiols to modulate NO bioactivity, by controlling the intracellular levels of
`low mass NO donor compounds and preventing protein nitrosylation from reaching toxic
`
`levels.
`
`Based on this, it follows that inhibition of the enzyme potentiates bioactivity in all
`
`diseases in which NO donor therapy is indicated, inhibits the proliferation ofpathologically
`
`proliferating cells, and increases NO bioactivity in diseases where this is beneficial.
`
`One embodiment herein is directed to a method of treating a patient afliicted with a
`
`disorder ameliorated by NO donor therapy, said method comprising administeling to said
`
`patient a therapeutically efi‘ective amount of an inhibitor of glutathione—dependent
`
`formaldehyde dehydrogenase.
`
`Another embodiment herein is directed to a method for treating a patient afilicted
`
`with pathologically proliferating cells, said method comprising administering to said patient .
`
`a therapeutically effective amount of an inhibitor of glutathione-dependent formaldehyde
`
`dehydrogenase. The term “pathologically proliferating cells” is used herein to include
`
`pathologic microbes, pathologic hehninths, and pathologically proliferating mammalian
`
`cells.
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`
`Still another embodiment herein is directed to increasing NO bioactivity for
`
`pharmacological efi‘ect or in the case of diseases associated with a deficiency ofNO. This
`
`embodiment is directed to a method oftreating a patient in need ofincreased nitric oxide
`
`bioactivity, said method comprising administering to said patient a therapeutically efl‘ective
`
`amount of glutathione-dependent formaldehyde dehydrogenase.
`
`Detailed Description
`
`Glutathione-dep endent formaldehyde dehydrogenase (GS-FDH) is a known enzyme
`
`which is conserved from microbes including bacteria and fungi to mammals. It is also
`
`known as alcohol dehydrogenase Class [[I. It has been identified in a variety ofbacteria,
`
`yeasts, plants and animals. The proteins from E. coli, S. cerevisiae and mouse
`
`macrophages share over 60% amino acid sequence identity. In methylotropic
`
`microorganisms, GS-FDH is induced by methanol to prevent formaldehyde accumulation.
`
`The physiological significance of formaldehyde oxidation by GS-FDH is less clear in other
`
`microorganisms and animals. In the course ofmaking the invention herein GS—FDH
`
`associated NADH-dependent S-nitrosoglutathione reductase activity has been detected in
`
`E. coli, in mouse macrophages, in mouse endothelial cells, in mouse smooth muscle cells, in
`
`yeasts, and in human HeLa, epithelial and monocyte cells. As used herein, the term
`
`“glutathione—dependent formaldehyde dehydrogenase” means enzyme that oxidizes S-
`
`hydroxymethylglutathione and also provides NADH-dependent S-nitrosoglutathione
`
`reductase activity (i.e., decomposes S—nitrosoglutathione when NADH is present as a
`
`required cofactor) and shares at least 60% amino acid sequence identity with enzymes
`
`having the same function from E. coli, S. cerevisiae and mouse macrophages. The
`
`glutathione-dependent formaldehyde dehydrogenase may also be referred to as
`
`S-nitrosoglutathione reductase.
`
`We turn now to the inhibitors of glutathione— dep endent formaldehyde
`
`‘ dehydrogenase for use in the three embodiments herein. These compounds inhibit the
`
`S-nitrosoglutathione reductase activity ofthe glutathione—dependent formaldehyde
`dehydrogenase.
`V
`One class of compounds for use herein as the inhibitors of glutathione-dependent
`
`formaldehyde dehydrogenase is constituted of competitors for NAD+ binding. These
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`
`inhibitors work by binding to the NAD+ cofactor binding site ofthe enzyme and thereby
`
`block the NADH cofactor from binding to the enzyme.
`
`One compound ofthis class is nicotinamide riboside (NR) which has the structure:
`
`1
`
`0
`
`NHZ
`
`HO
`
`I \
`+
`
`O\
`
`OH OH
`
`Other compounds of this class include the following ribonucleoside analogs:
`
`The compound 6-aminonicotinamide (6AN) which has the structure:
`
`0
`
`N H2
`
`H
`
`2
`
`+
`
`HO
`
`OH OH
`
`(1)
`
`(2)
`
`This compound requires additionally metabolization to 6-amino-NAD(P+) by the pentose
`
`phosphate pathway (PPP) enzyme, 6-phosphog1uconate dehydro'genase, for inhibitory
`
`activity.
`
`The compound S—fi-D-iibofilranosylnicotinamide which has the structure:
`
`0
`
`NH2
`
`\
`
`/
`
`HO
`
`OH OH
`
`(3)
`
`This compound requires conversion to the con‘esp onding NAD analog, C—NAD, which is
`
`described later as Compound (13), for inhibitory activity.
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`' The compound 6-fi-D-ribofi1ranosylpicolinamide which has the structure:
`
`-5-
`
`0
`
`OH OH
`
`This compound requires conversion to the corresponding NAD analog, C-PAD, which is
`
`described later, as Compound (14), for inhibitory activity.
`
`The compound 2- B-D-Iibofiiranosylisonicotinamide which has the structure:
`
`HO
`
`\
`
`OH OH
`
`(5)
`
`Other inhibitors of glutathione- dep endent formaldehyde dehydlrogenase which are
`
`ribonucleoside analogs and are competitors for NAD+ binding and thereby inhibit the
`
`S-nitrosoglutathione reductase activity of GS-FDH have the formula:
`
`We
`*
`_
`
`OH OH
`
`(6)
`
`These include thiophenfurin (5-B—D—ribofiiranosylthiophene-S—carboxamide), Compound
`
`(6a), which has the formula (6) where X = S and Y = CH; furanfun'n (S-B—D-
`
`ribofuranosylfirran—3-carboxamide), Compound (6b), which has the formula (6) where X =
`
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`
`O and Y = CH; tiazofurin (2- [3-D-ribofilranosylthiazole—4—carboxamide), Compound (60),
`
`which has the formula (6) where X = S and Y = N; selenazofurin (2- [5-D-
`
`ribofiiranosylselenazole-4- carboxamide), Compound (6d), which has the formula (6) where
`
`X = Se and Y = N; and selenophenfurin (5-[i-D-ribofuranosylselenophene-3-carboxamide),
`
`Compound (6e), which has the formula (6) where X = Se and Y = CH. These compounds
`
`are metabolized to their isosteric NAD analogs for activity. For example, tiazofurin is
`
`phosphorylated by adenosine kinase to the 5'-monophosphate and converted by NAD-
`
`pyrophosphorylate to TAD, described later, for competitive binding.
`
`Still another ribonucleoside analog which is a competitor for NAD+ binding and
`
`thereby inhibits the S-nitro soglutathione reductase activity of GS-FDH is benzamide
`
`ribosome (BR) which has the formula:
`
`(7)
`
`(BR) is metabolized to (BAD), described later, for activity.
`
`Still another rib onucleoside analog which is a competitor for NAD+ binding and
`
`thereby inhibits the S-nitrosoglutathione reductase activity of GS-FDH is ribavirin which
`
`has the formula:
`
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`' Still another ribonucleoside analog which is a competitor for NAD+ binding and
`
`thereby inhibits the S-nitrosoglutathione reductase activity of GS-FDH is mizoribine
`
`(MZR) which has the formula:
`
`Still another ribonucleoside analog for use herein as inhibitor of GS-FDH is 5-
`
`ethynyl—1-B-D-ribofiiranosylimidazole-4-carboxamide (EICAR) which has the formula:
`
`NHZ '
`
`HO
`
`I
`
`N Q
`
`OH OH
`
`This compound metabolizes to (EAD), Compound (17) described later, for inhibitory
`
`activity.
`
`_
`
`Another compound which is an inhibitor of GS—FDH by Viitue ofbeing a
`
`competitor for NAD” binding is NAD+ which has the formula:
`
`OH OH
`
`(10)
`
`(11)
`
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`
`' Still other compounds which are inhibitors of GS-FDH by virtue ofbeing
`
`competitors for NAD+ binding and thereby inhibit the S-nitrosoglutathione reductase
`
`activity of GS-FDH are NAD+ derivatives.
`One such NAD+ derivative is 6-amino-NAB which has the formuia:
`
`o
`
`‘\
`l +
`
`Nth
`
`“2
`
`S?
`*fLi}~E~O
`OH OH
`
`om
`
`H
`
`N\\’
`N I, A,
`
`”2
`
`Another such NAD+ derivative is S-fi-D-ribofilranosylnicotinamide adenine
`
`dinucleotide (C-NAD) which has the formula:
`
`(12)
`
`(13)
`
`This compound is a metabolite of Compound (3) described above.
`
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`
`Another such NAD+ derivative is 6-B—D-ribofi1ranosy1picolinamide adenine
`
`dinucleotide (C—PAD) which has the formula:
`
`1J2N0€|00fi
`
`”Hz
`
`O—E—O
`
`OH
`
`H
`
`OH OH
`
`(14)
`
`This compound is a metabolite of Compound (4) desc1ibed above.
`
`Other such NAD+ derivatives have the structural formula:
`
`EEO/”y“;
`x/v
`OHOH
`
`“
`
`(15)
`
`H2
`
`These include TFAD, Compound (1521), which has the formula (15) where X is S and Y is
`
`CH and is a metabolite of Compound (6a); FFAD, Compound (15b), which has the formula
`
`(15) where X = O and Y = CH and is a metabolite of Compound (6b); TAD (thiazole—4-
`
`carboxamide adenine dinucleotide), Compound (150), which has the formula (15) where X
`
`= S and Y = N and is a metabolite of Compound (60); SFAD, Compound (15d), which has
`
`the formula (15) where X = Se and Y = N, and is a metabolite of Compound (6d); and
`
`SAD, Compound (156), which has the fonnula ( 15) where X = Se and Y = CH, and is a
`
`metabolite of Compound (6e).
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`Yet another such NAD+ derivative is benzamide adenine dinucleotide (BAD) which
`
`-10-
`
`has the formula:
`
`.
`
`O
`
`NFfi
`
`Dior
`6»: OH
`
`OH OH
`
`H
`
`H
`
`<
`
`[NW
`
`/N
`
`NH2
`
`(16)
`
`Compound (16) is a metabolite of (BR) which is Compound (7) described above.
`
`Yet another such NAD+ derivative is (EAD) which has the formula:
`
`(17)
`
`Compound (17) is a metabolite of EICAR, Compound (10) described above.
`
`Still other such NAD+ derivatives have the formula:
`
`0 can i
`{11:? OH
`OH
`
`(18)
`
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`These include [3—CH2—TAD, Compound (18a), which has the formula (18) where X and Y =
`
`OH, W = H, and Z = CH2; B-CFZ-TAD, Compound (18b), which has the formula (18)
`
`where X and Y = OH, W = H and Z = CFz; 3'F—TAD, Compound (18c), which has the
`
`formula (18) where X = OH, Y =. F, W = H and Z = O; Z'Fara-TAD, Compound (18d),
`
`which has the fo1mula (18) where X and Y = OH, W = F and Z = O; Z'Fara-B—CHZ—TAD,
`
`Compound (18e), which has the formula (18) where X and Y = OH, W = F and Z = CH2;
`
`and Z'Fara- B-CFz—TAD, Compound (18f), which has the formula (18) where X and Y =
`
`OH,W=FandZ= CF2.
`
`Still other such NAD+ derivatives have the fonnula:
`
`(19)
`
`These include B-CHz-BAD, Compound (19a), which has the formula (19) Where X = OH,
`
`W = H and Z = CH2, and 2'Fara-13-CHz-BAD, Compound (19b) which has the formula (19)
`
`WhereX=H,W=F andZ=CHT
`
`The derivatives of formulas (18) and (19) including Compounds (18a), (18b), (18c),
`
`(18d), (18e), (18f), (19a) and (19b) are TAD (Compound (150)) and BAD (Compound
`
`(16)) analogs which are metabolically stable and cell membrane permeable, methylene or
`
`difluoromethylene bz‘s (pho sphonate)s, and analogs substituted with fluorine in the ribose
`
`moiety of adenosine which are more hydrophobic than their hydroxy congeners.
`
`Other compounds which are inhibitors of GS-FDH are mimickers of the
`
`nicotinamide portion ofNAD and a water molecule and include mycophenolic acid (MPA),
`
`Compound (20), and its morpholinoethyl ester prodrug mycophenolate mofetil(M1\/IF),
`
`Compound (21).
`
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`
`Compound (20) has the formula:
`
`(20)
`
`(21)
`
`Still other compounds which are inhibitors of GS—FDH are competitive substrates
`
`for NADH binding. These include 6-thioanologs ofnatural purine bases, e. g., 6-
`
`mercaptopurine (Compound 22) and 6~thioguanine (Compound 23).
`
`Compound (22) has the formula:
`
`'
`
`(22)
`
`Compound (23) has the formula:
`
`0
`
`H
`
`The above Compounds (1) - (23) are considered also to inhibit the activity of other
`
`NADH dependent dehydrogenases such as inosine monophosphate dehydrogenase
`
`(IMPDH). Other inhibitors of lMPDH by virtue of competition for NAD+ cofactor binding
`site, are also effective as inhibitors of GS-FDH herein.
`I
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`
`The compounds specifically described above are available commercially or their
`
`synthesis is described in or obvious from the literature.
`
`Some ofthe above compounds have been utilized for some ofthe utilities herein
`
`without knowledge that at least part oftheir function may have been due to GS-FDI-I
`
`inhibition; these compounds for these uses are excluded from the invention herein, but are
`
`not excluded from the invention herein for other uses.
`For example, tiazofinin has been used previously for antineoplastic activity against
`
`tumors as has thiophenfurin and selenazofurin. Moreover, selenazofurin, ribavirin, MZR,
`
`EICAR and MMF have been used for antiviral or potential antiviral activity. Moreover,
`
`mycophenolic acid has been evaluated as an anticancer, antiviral, antifungal and
`
`antibacterial agent, as well as for its therapeutic use in psoriasis and rheumatoid arthritis.
`
`Moreover, furanfurin and ribavirin have been shown to be inactive as an antitumor agents.
`These cases are excluded from the invention herein. However, the same compounds are
`
`not excluded from the invention herein for other uses.
`
`,
`
`Another class of compounds useful herein to inhibit GS-FDH is constituted of
`
`glutathione derivatives including D-glutathione and S—alkyl glutathione containing from 1
`
`to 6 carbon atoms in the S—alkyl group.
`
`The use of gold-based compounds to treat asthma and cystic fibrosis is excluded
`
`from the invention herein.
`
`We turn now to the embodiment directed to a method oftreating a patient afllicted
`
`with a disorder ameliorated by NO donor therapy where the method comprises
`
`administering to the patient a therapeutically elfective amount of an inhibitor of
`
`glutathione—dep endent formaldehyde dehydrogenase. This embodiment may be referred to
`
`as the first embodiment herein.
`
`The disorders applicable to this embodiment include, for example, breathing
`
`disorders (e.g., asthma, cystic fibrosis, and ARDS), heart disease, hypertension, ischemic
`
`coronary syndromes, atherosclerosis, glaucoma, diseases characterized by angiogenesis
`
`(e.g., coronary artery disease), disorders where there is risk ofthrombosis occurring,
`
`disorders where there is risk of restenosis occurring, chronic inflammatory diseases (e. g,
`
`AID dementia and psoriasis), diseases where there is risk of apoptosis occurring (e. g, heart
`
`failure, atherosclerosis, degenerative neurologic disorders, arthritis and liver injury
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`(ischemic or alcoholic)), impotence, obesity caused by eating in response to craving for
`
`food, stroke, reperfusion injury (e. g., traumatic muscle injury in heart or lung or crush
`
`injury), and disorders where preconditioning of heart or brain for NO protection against
`
`subsequent ischemic events is beneficial.
`
`‘
`
`The inhibitors of glutathione—dependent formaldehyde dehydrogenase are described
`
`above.l
`
`The term “therapeutically efli‘ective amount” for this first embodiment means a
`
`glutathione-dependent formaldehyde dehydrogenase inhibiting amount in vivo that causes
`
`amelioration ofthe disorder being treated or protects against a risk associated with the
`
`disorder. For example, for asthma, a therapeutically effective amount is a bronchodilating
`
`effective amount; for cystic fibrosis, a therapeutically efl‘ective amount is an airway
`
`obstruction ameliorating effective amount; for ARDS, a therapeutically efi‘ective amount is
`
`a hypoxemia ameliorating effective amount; for heart disease, a therapeutically effective
`
`amount is an angina relieving or angiogenesis inducing efi‘ective amount; for hypertension,
`
`a therapeutically efi‘ective amount is a blood pressure reducing eifective amount; for
`
`ischemic coronary disorders, a therapeutic amount is a blood flow increasing effective
`
`amount; for atherosclerosis, a therapeutically elfective amount is an endothelial dysfunction
`
`reversing efi‘ective amount; for glaucoma, a therapeutic amount is an intraocular pressure
`
`reducing effective amount; for diseases characterized by angiogenesis, a therapeutically
`
`elfective amount is an angiogenesis inhibiting effective amount; for disorders where there is
`
`risk ofthrombosis occurring, a therapeutically effective amount is a thrombosis preventing
`
`efi'ective amount; for disorders where there is risk of restenosis occurring, a therapeutically
`
`efi‘ective amount is a restenosis inhibiting efi‘ective amount; for chronic inflammatory
`
`diseases, a therapeutically effective amount is an inflammation reducing effective amount;
`
`for disorders where there is risk of apoptosis occurring, a therapeutically effective amount
`
`is an ap optosis preventing effective amount; for impotence, a therapeutically effective is an
`
`erection attaining or sustaining efl‘ective amount; for obesity, a therapeutically effective
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`amount is a satiety‘causing efl‘ective amount; for stroke, a therapeutically effective amount
`is a blood flow increasing or a TIA protecting effective amount; for reperfilsion injury, a
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`therapeutically efi‘ective amount is a function increasing efl‘ective amount; and for
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`preconditioning ofheart and brain, a therapeutically effective amount is a cell protective
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`efiective amount, e.g., as measured by triponin or CPK.
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`In general, the dosage, i. e, the therapeutically effective amount, ranges from 1 ug to
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`10 g/kg and often ranges from 10. pg to 1 g/kg‘ or 10 pg to 100 mg/kg body weight ofthe
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`patient, per day.
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`I The patients include mammals including humans.
`The preferred route of administration is oral administration although other routes of
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`administration including parenteral are useful. Topical administration can be appropriate
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`for localized disorders.
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`I
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`Preferred treating agents for the first embodiment include D-glutathione, ribavirin,
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`D-glutathione together with ribavirin, and mycophenolic acid. D-Glutathione can be given,
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`for example, intravenously at 10-100 mg/kg and/or inhaled in 1-10 mM concentration for
`asthma; inhaled at l-lO mM concentration for cystic fibrosis and ARDS; and intravenously
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`at 10—3 00 mg/kg for heart disease including angina, ischemic coronary syndrome, and
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`disease where there is risk of apoptosis occurring (e. g., acetaminophen induced liver
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`injury), and at 100 to 1,000 mg for hypertension. Ribavirin can be given, for example,
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`injected in an amount of l-lO g at a concentration of 5—25 mg/ml for angina fiom coronary
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`artery disease, inhaled in amount of l to 10 grams to prevent thrombosis from occurring,
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`e.g., where pulmonary embolism is found, and topically at l-5% in a topical composition
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`for psoriasis. Mycophenolic acid can be given, for example, coated on a stent (drug
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`concentration 10-40% per polymer) for treating restenosis or topically at a concentration of
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`l to 10% in a paste to treat impotence.
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`Treatment is continued as long as symptoms and/or pathology ameliorate.
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`We turn now to the embodiment directed to a method oftreating a patient afilicted
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`with pathologically proliferating cells where the method comprises administering to said
`
`patient a therapeutically efl‘ective amount of an inhibitor of glutathione-dep endent
`
`formaldehyde dehydrogenase. This embodiment may be referred to as the second
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`embodiment herein‘.
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`We turn now to the case ofthe second embodiment herein where the pathologically
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`proliferating cells are pathologically proliferating microbes.
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`The microbes involved are those where glutathione-dependent formaldehyde
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`dehydrogenase is expressed to protect the microbe from nitrosative stress or where host
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`cell infected with said microbe expresses said enzyme thereby protecting the microbe from
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`nitrosative stress.
`
`The term “pathologically proliferating microbes” is used herein to mean pathologic
`microorganisms including but not limited to pathologic bacteria, pathologic viruses,
`pathologic Chlamydia, pathologic protozoa, pathologic Rickettsia, pathologic fungi, and
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`pathologic mycoplasmata. More detail on the applicable microbes is set forth at columns
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`11 and 12 of U. S. Patent No. 6,057,3 67 which are incorporated here by reference.
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`The term "host cells infected with pathologic microbes" includes not only
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`mammalian cells infected with pathologic viruses but also mammalian cells containing
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`intracellular bacteria or protozoa, e. g, macrophages containing Mycobacrerz'um
`tuberculosis, Mycobacterz‘um leper (leprosy), or Salmonella typhi (typhoid fever).
`
`We turn now to the case ofthe second embodiment herein where the pathologically
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`proliferating cells are pathologic helminths. The term “pathologic hehninths” is used herein
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`to refer to pathologic nematodes, pathologic trematodes and pathologic cestodes. More
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`detail on the applicable helminths is set forth at column 12 of US. Patent No. 6,057,367
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`which is incorporated herein by reference.
`
`We turn now to the case of the second embodiment where the pathologically
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`proliferating cells are pathologically proliferating mammalian cells.
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`The term "pathologically proliferating mammalian cells" as used herein means cells
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`of the mammal that grow in size or number in said mammal so as to cause a deleterious
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`effect in the mammal or its organs. The term includes, for example, pathologically
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`proliferating cancer cells, the pathologically proliferating or enlarging cells causing
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`restenosis, the pathologically proliferating or enlarging cells causing benign prostatic
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`hypertrophy, the pathologically proliferating cells causing myocardial hypertrophy and
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`proliferating cells at inflammatory sites such as synovial cells in arthritis. Pathologically
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`proliferating cancer cells include the cell proliferation in Hodgkin's disease, in small cell
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`lung cancer, in cancer ofthe breast, and in testicular and prostate cancer.
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`The inhibitors of glutathione-dependent formaldehyde dehydrogenase for this
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`second embodiment herein are those described above.
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`The therapeutically effective amount for this second embodiment means a
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`glutathione-dependent formaldehyde dehydrogenase inhibiting amount in viva which is an
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`antiproliferatrve effective amount. Such antiproliferative effective amount as used herein
`
`means an amount causing reduction in rate ofproliferation of at lest 10%.
`
`In general, the dosage, i.e., the therapeutically effective or antiproliferative effective
`
`amount, ranges from 1 pg to 10 g/kg and often ranges from 10 pg to 1 g/kg or 10 pg to
`100 mg/kg body weight ofthe patient being treated, per day.
`
`fimpMEMSmenmmmflsmdmmghmmms
`
`The preferred route of administration in respect to inhibiting growth ofmicrobes is
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`oral administration although other routes of administration including parenteral and topical
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`are useful. Topical administration is especially useful for exposed infections, e. g., filngal
`
`infections such as athlete's foot, viral infections such as herpes and microbe-caused oral or
`skin lesions. For inhibiting the growth ofhelminths, the preferred route of administration is
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`oral administration although other routes of administration including parenteral are useful.
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`For inhibiting the growth ofpathologically proliferating cancer cells, the route of
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`administration can be oral or parenteral and local administration is possible, for example, by
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`infirsion directly into a tumor or into the blood vessels delivering blood to the tumor, or by
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`forming the agent into a slow release pellet or into a polymer matrix and then implanting
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`the pellet or polymer matrix device in or on the tumor. The preferred routes of
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`administration in the case of inhibiting growth ofpathologically proliferating mammalian
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`cells that would cause restenosis is from attachment on a stent implanted in angioplasty but
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`systemic including oral and intravenous administration can be acceptable. The preferred
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`route of administration in the case of inhibiting growth of pathologically proliferating
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`mammalian cells causing benign prostatic hypertrophy is fiom attachment on a prostatic
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`implant or by local injection.
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`Where the pathologically proliferating cells comprise pathologic bacteria or fungus
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`and the patient is afliicted with a bacterial or fungal infection, the administering kills or
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`reduces the growth ofthe pathologic bacteria or fungus. Where the pathologically
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`proliferating cells are pathologically proliferating mammalian cells, the administering kills or
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`reduces the growth ofthe pathologically proliferating mammalian cells.
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`Preferred treating agents for the second embodiment include D-glutathione,
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`ribavirin, D-glutathione together with Iibavirin, and mycophenolic acid. D-Glutathione can
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`be given, for example, inhaled at l~10 mM concentration for viral pneumonia; orally at a
`
`dose of 0.2 to 2 grams daily for pinworm and Hodgkin’s disease, to inhibit restenosis after
`
`angioplasty and to resolve benign prostatic hypertrophy; and intravenously at 100 to 300
`
`mg/kg Ifor squamous cell lung cancer. The combination ofD-glutathione and ribavirin is
`preferably given for bacterial pneumonia (e.g., 100 mg - l g D-glutathione orally and 1-10
`
`g ribavirin inhaled) and for metastatic breast cancer, metastatic testicular cancer and
`
`metastatic prostate cancer (egg, 10.0 - 300 grams/kg D-glutathione intravenously up to four
`
`times or more a day and 1—10 g ribavirin intravenously).
`
`Treatment is continued as long as symptoms and/or pathology ameliorate.
`
`The inhibitor of glutathione-dependent formaldehyde dehydrogenase can be
`administered alone for the second embodiment or in combination with conventional therapy
`
`for the disorder being treated or in combination with newly discovered agents for use for
`
`therapy of the disorder treated and/or in combination with any other agent that imposes
`
`nitrosative or oxidative stress.
`
`Agents for selectively imposing nitrosative stress to inhibit proliferation of
`
`pathologically proliferating cells in combination therapy with GS-FDH inhibitors herein and
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`dosages and routes of administration therefor include those disclosed in U. S. Patent No.
`
`6,057,367, the whole ofwhich is incorporated herein.
`
`Supplemental agents for imposing oxidative stress (i.e., agents that increase GSSG
`
`(oxidized glutathione) over GSH (glutathione) ratio or NAD(P) over NAD(P)H ratio or
`
`increase thiob arbituric acid derivatives) in combination therapy with GS-FDH inhibitors
`
`herein include, for example, Irbuthionine-S-sulfoximine (BSO), glutathione reductase
`
`inhibitors (e. g., BCNU), inhibitors or uncouplers ofmitochondrial respiration and drugs
`
`that increase reactive oxygen species (ROS), e.g., adriamycin, in standard dosages with
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`standard routes of administration. For example, BSO can be given intravenously or orally
`
`at 10-30 grams per‘day.
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`We turn now to the embodiment directed to a method oftreating a patient in need
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`ofincreased nitric oxide bioactivity, said method comprising administering to said patient a
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`therapeutically efi‘ective amount of glutathione-dependent formaldehyde dehydrogenase.
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`This embodiment may be referred to as the third embodiment herein.
`
`In one subset, i.e., the first subset, of the third embodiment, the patient has a
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`disorder associated with a deficiency in nitric oxide. The term “disorder associated with a
`
`deficiency in nitric oxide” is used herein mean disorder where NO deficiency is a feature
`
`and the deficiency constitutes less NO than the norm or less than the normal NO bioactive
`response. Disorders associated with a deficiency in nitric oxide include atherosclerosis,
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`restenosis, and disorders involving deficiency in NO in tissues w