The invention relates to the manipulation of enzymatic pathways, such as poly (ADP-ribose) polymerase pathways, which are dependent upon NAD as a substrate. By administering pro-NAD compounds, cell death caused by necrosis and/or apoptosis can be reduced. Further, by inhibiting one or more of these enzymes, the process of cell death can be accelerated, in conditions such as cancer, where acceleration of cellular necrosis and/or apoptosis is desired. Various inhibitors are described

Jacobson, Elaine L.

Jacobson, Myron

English

University of Kentucky

University of KentuckyUKnowledgeGraduate Center for Nutritional Sciences FacultyPatents Nutritional Sciences6-11-2002Method for Controlling Cell Necrosis or ApoptosisElaine L. JacobsonUniversity of KentuckyMyron JacobsonUniversity of KentuckyRight click to open a feedback form in a new tab to let us know how this document benefits you.Follow this and additional works at: https://uknowledge.uky.edu/nutrisci_patentsPart of the Medical Pharmacology CommonsThis Patent is brought to you for free and open access by the Nutritional Sciences at UKnowledge. It has been accepted for inclusion in Graduate Centerfor Nutritional Sciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contactUKnowledge@lsv.uky.edu.Recommended CitationJacobson, Elaine L. and Jacobson, Myron, "Method for Controlling Cell Necrosis or Apoptosis" (2002). Graduate Center for NutritionalSciences Faculty Patents. 12.https://uknowledge.uky.edu/nutrisci_patents/12US006403619B1 (12> Ulllted States Patent (16) Patent N6.= US 6,403,619 B1 Jacobson et al. (45) Date of Patent: Jun. 11, 2002 (54) METHOD FOR CONTROLLING CELL 5,736,529 A * 4/1998 Adams et al. .............. .. 514/46 NECROSIS ()R APOPTOSIS 5,916,906 A * 6/1999 Shaskan . . . . . . . . . . . . .. 514/356 5,939,082 A * 8/1999 Oblong etal. ............ .. 424/401 (75) Inventors: Elaine L. Jacobson; Myron K. 2 21121316 e: ‘111 ~ , , r1 n e a. Jacobson’ both of Lexmgton’ KY (Us) 6,071,888 A * 6/2000 Rihova et al. .............. .. 514/43 . _ . . 6,149,924 A * 11/2000 Paul ............. .. (73) Asslgnee' géllll‘l'gggg'nofggggg?ylgisfsgh 6,231,852 B1 * 5/2001 Bredesen ................. .. 424/944 ’ ’ FOREIGN PATENT DOCUMENTS ( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 W0 W099 646606 12/1999 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS _ M. Jacobson & E. Jacobson, “Discovering neW ADP—ribose (21) App1'NO"09/549’691 polymer cycles: protecting the genome and more.” (22) Filed: Apt; 14, 2000 TIBS24:415—417 (ll/99). _ _ * cited by examiner Related US. Application Data Primary Examiner—Jose‘ G. Dees (63) Continuation-in-part of application No. 09/452,617, ?led on Assistant Examiner—Marina Larnrn DeC- 1, 1999, HOW Pat- NO- 6,337,065- (74) Attorney, Agent, or Firm—Fulbright & J aWorski LLP (60) Provisional application No. 60/110,482, ?led on Dec. 1, 1998. (57) ABSTRACT (51) Int. Cl.7 ......................... .. A61K 31/44; A61K 7/00 The invention relates to the manipulation of enzymatic (52) US. Cl. ..................... .. 514/356; 424/401; 514/351; pathways, such as poly (ADP—ribose) polymerase pathways, 514/353 Which are dependent upon NAD as a substrate. By admin (58) Field of Search ........................ .. 424/401; 514/356, istering pro-NAD compounds, cell death caused by necrosis 514/357, 353 and/or apoptosis can be reduced. Further, by inhibiting one or more of these enzymes, the process of cell death can be (56) References Cited accelerated, in conditions such as cancer, Where acceleration U'S' PATENT DOCUMENTS of cellular necrosis and/or apoptosis is desired. Various inhibitors are described. 5,519,039 A * 5/1996 Leung ...................... .. 514/356 5,693,671 A * 12/1997 Niihara et al. ............ .. 514/563 15 Claims, 18 Drawing Sheets U.S. Patent Jun. 11,2002 Sheet 1 0f 18 US 6,403,619 B1 2052326 :Ewcmm 92:0: $25.5 “.8; 252m vcmcwwmzou m526 2 EE 055:22E_:__wu-coZ Ucm268 £530 28 @525 52552mm EU& 2% 838%EswFSU 355F A W \~ \\ 120325: a? $559523 a+ 0 m n s 0?Un  % HU[0V H M/W P .07U.S. Patent Jun. 11,2002 Sheet 4 0f 18 US 6,403,619 B1 FIG. 4A [Nam] Control - Sub-optimal — Niacin Fibroblasts I I I I 0 500 1000 I 500 2000 2500 pmol NAD/mg protein FIG. 4B [Nam] Control Sub-optimal Niacin Kerati nocytes I I I I 0 5000 10000 I 5000 20000 25000 pmol NAD/mg protein U.S. Patent Jun. 11,2002 Sheet 5 0f 18 US 6,403,619 B1 FIG. 5 l l l l l 15 - _ Malmb Subjects Controls 10 Mean=l75i24_ _ N = 705 Mean : 160 i 38 Frequency < 126 = 15% 5 _ _ 0 50 10C 150 200 250 300 Niacin Number FIG. 6 G 4.0- Q _ :2: (3]) ® 3.0- 9 V _ NAD/ protein lpmol/Mg) 2.0- - EU .68) 1.0- - l l l Non-diseased Skin from Individuals with Actinic Squamous Keratosis Cell Carcinoma U.S. Patent Jun. 11,2002 Sheet 8 0f 18 US 6,403,619 B1 FIG. 9A P53 std Low NAD Normal NAD Z. FIG. 9B Q33 00300330030~0003000Hu00000003 17.5 A N D033000033| A 030003030000 3 Nm 00 N l._ 300033003303330 mm a 3 _ q0. 5O55 2O701| p53 relative to actm Hours after DNA damage U.S. Patent Jun. 11,2002 Sheet 11 0f 18 US 6,403,619 B1 100 _ High NAM 100 — Normal NAM 75- UTYPQ3 75- l:lType3 Comets of Ii-i; Type 4 Comets of .;f_-ii Type 4 Increasing 50 — Increasing 5O - Tail Length Tail Length (0/0 Of _ (0/0 Of _ Population) Population) I $—r I | | | | '_|' O“ - = _' ‘ O 2448 96 O 2448 96 O 2448 96 O 2448 96 Untreated MNNG-Teated Untreated MNNG-Treated Hours Hours 30 —— - Normal NAD High Nam 20 — p53 relative to actin l5 — 1O — 5 __ O 2 Hours after DNA damage U.S. Patent Jun. 11,2002 Sheet 12 0f 18 US 6,403,619 B1 FIG. 13 O \ HO \ Nicotinic acid I / O N Methylnicotinate (C1) I / N O /\O \ Ethylnicotiniate (C2) I / N O /\/\O I \ Butylnlcotmate (C4) N/ O /\/\/\O \ Hexylnicotinate (C6) I O N/ /\/\/\/\O \ Octylnicotinate (C8) I / O N /\/\/\/\/\/\/\Q \ Tetradecylnicotinate (C1 4) I O N/ O \ Octadecylnicotinate (C18) I / N / U.S. Patent Jun. 11,2002 Sheet 13 0f 18 US 6,403,619 B1 15 § ’. § 1O — ‘x.’ _ Log PO/W "x' R2:O.9767 ' 5 _ . _ §Predicted values 0 | l I O 5 1O 15 2O Nicotinate Ester Chain Length U.S. Patent Jun. 11,2002 Sheet 14 0f 18 US 6,403,619 B1 882;. _obcoU IIllllllllllll‘llll'lllllllll'llll mm; .UElllllllllllllllllllllllllllllllll mm om mm 00? mg om? mm? UQHQQC. _obcoU IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII xumm <mP .UElllllllllllllllllllllllllllllllll mm om mm 00? mNP om? U.S. Patent Jun. 11,2002 Sheet 15 0f 18 US 6,403,619 B1 FIG. 16 130 - I Back, Treated wlth Pro-NAD 125 v Abdomen, No treatment 120 NAD/protein, 115 % of Day 0 110 105 100: Days U.S. Patent Jun. 11,2002 Sheet 16 0f 18 US 6,403,619 B1 FIG. 17A 100 FIG. 17D - N0 Nam Added - - No Nam Added 75 — — 75 — — — . 5O — — % Necrosis _ % Apoptosis _ _ 25 - 25 — — 0 %I O | I l__' l 0 24 48 0 24 48 Time (hr) Time (hr) FIG. 173 FIG. 17E 100 100 50 MM Nam - 50 “M Nam 75 — — 75 — — % Necrosis 5O 0/0 Apoptosis 5O 25 — — 25 — — O %l F O l I |—- —|-—: 0 24 0 24 48 Time (hr) Time (hr) FIG. 17C FIG. 17F 100 100 500 MM Nam - 500 MM Nam 75 — — 75 5O — — . % Necrosis _ _ °/0 APOPtOSIS 50 _ 25 25 O O U.S. Patent Jun. 11,2002 Sheet 17 0f 18 US 6,403,619 B1 FIG. 18A FIG. 18C 50 5O - U ntreated - - Treated 40 — — 40 — - 3O - — 3O - J % Apoptosis 201 __ % Apoptosis 20L _— 101 ‘I i 101 0 0 Time (hr) Time (hr) FIG. 183 FIG. 18D 50 5O - U ntreated ' - Treated - 40 — — 40 — — 3O — — 30 _ % Necrosis 20L .- % Necrosis 2O _ 10L l 10 _ O 24 48 O 24 48 Time (hr) Time (hr) III UnT E] iOpig/mL @20 ug/mL low NAD control NAD US 6,403,619 B1 1 METHOD FOR CONTROLLING CELL NECROSIS OR APOPTOSIS RELATED APPLICATIONS This application is a continuation in part of application Ser. No. 09/452,617, ?led Dec. 1, 1999, now US. Pat. No. 6,337,065, Which claims priority provisional patent appli cation serial No. 60/110,482, ?led Dec. 1, 1998. Both applications are incorporated by reference in their entirety. BACKGROUND 1. Field of the Invention The invention is directed to methods and composition of using organic molecules termed pro-NAD agents, capable of enhancing dermal and epidermal skin cell NAD content With a resulting enhancement of DNA repair and other protective responses to genotoxic stress in skin. Also a part of the invention are methods for manipulating cell regulation path Ways involving NAD via, e.g., enhancement or inhibition of enZymatic pathWays. 2. Description of the Background The present application relates to methods and composi tions capable of modulating and upregulating the cellular nicotinamide-adenine-dinucleotide (NAD) content by the topical application of chemical agents for the purpose of enhancing natural protective responses of skin cells to DNA damage. Also a part of the invention is the manipulation of enZymatic pathWays associated thereWith. The methods and compositions are effective, e.g., for the prevention and treatment of skin deterioration that results from DNA dam age to cells of the skin, as Well as in the treatment of other conditions Which involve enZymatic pathWays Where NAD is a substrate. The symptoms of such skin deterioration are many and typically include the loss of moisture, ?ne lines, deep lines, Wrinkles, and loss of elasticity as Well as atrophic sclerosis and other blemishes of skin. Skin deteriorates With age as a natural consequence of prolonged exposure to internal and external factors. Internal deterioration factors include natural metabolic byproducts such as free radicals Which cause the aging of all tissues. External deterioration factors include ioniZing radiation such as sunlight and chemical insults such as pollution and cigarette smoke. In theory, skin care methods and compositions should inhibit, or sloW the process of skin deterioration by counteracting these internal and external factors. Unfortunately, current methods and compositions for skin care are generally reac tive rather than proactive. That is, current methods and compositions reduce or obscure the signs of aging but have minimal or no effect on the underlying progressive and cumulative biochemical processes that cause skin deterio ration. It is therefore desirable to have a skin care method and composition Which not only reduce the symptoms of deterioration but also treat the underlying causes of skin deterioration in such a Way that deterioration can actually be retarded. To understand the limitations of current methods and compositions, it is necessary to understand the function and structure of the skin and the mechanisms of skin deterioration. At ten pounds, the skin is the largest organ in the body. FIG. 1 shoWs a diagram of skin marking the location of the tWo major cell types present in skin, namely ?broblasts, Which are located in the dermal layer of the skin and keratinocytes, Which are located in the epidermal layer of the skin. The skin provides the ?rst line of defense betWeen the body’s interior and harmful environmental insults by Well established physical and biochemical mechanisms. Physical 10 15 20 25 30 35 40 45 55 60 65 2 protection mechanisms include the relatively impermeable barrier the skin provides. The skin can, to some extent, repel and absorb insults such as chemicals and ultraviolet light so that While the skin may be damaged, the underlying tissue is preserved. Biochemical mechanisms include the innate and acquired immune systems. Microbiological pathogens are repelled by immune responses at the epidermal level involv ing Langerhans cells, keratinocytes, cytokines, polynuclear cells, endothelial cells, mast cells, and lymphocytes. Structurally, the skin comprises epithelial tissue (the epidermis) in the outer layer and beneath it, connective tissue (the dermis), and beneath that, the fatty tissue (hypodermis). The epidermis is not vasculariZed and regen erates every four to six Weeks. Its primary function is to maintain the body’s skin integrity, acting as a physical barrier to toxic agents, dirt, bacteria, microorganisms, and physical insults. The dermis is beneath the epidermis and functions by providing strength, support, blood, and oxygen to the skin. The principal cell components of the dermis are ?broblasts, although it also contains sWeat glands, sebaceous glands, hair follicles, and small fat cells. Hypodermis, also knoWn as the super?cial fascia, attaches the dermis to the underlaying structures. Its function is to promote an ongoing blood supply to the dermis for regeneration. The mechanisms of skin deterioration involve a gradual and progressive process that begins at birth. Internal factors that contribute to skin aging include toxic metabolic byproducts, autoimmune diseases, and genetic predisposi tion. The consequences of internal deterioration can be observed over the entire body from the skin to the internal organs. While the mechanisms of internal deterioration are not completely understood, somatic mutation has been shoWn to be a contributing factor. Under the somatic muta tion theory, cells gradually lose their youthful characteristics and their capacity to divide by the accumulation of muta tions (errors) in their genetic code. These mutations may be caused by free radicals or alkylating agents generated in metabolism that lead to unrepaired DNA damage. Over time, mutations accumulate in the body until the cell can no longer divide or produce functional proteins. External factors such as chemical and physical agents in the environment can also cause DNA damage that leads to skin deterioration. The external factors include sunlight, pollution, and ingested chemicals from smoking or from food. Deterioration of skin leads to changes in dermal thickness and elasticity due to increased crosslinking of collagen. Epidermal regeneration increases in activity While metabolism, sWeat glands, and vasculariZation, all decrease in activity. The damage from internal and external factors is progressive and cumulative and results in the appearance of deterioration associated With aged skin. Related to the somatic mutation theory, both internal and external factors contribute to oxidative stress, Which in turn results in DNA damage. In humans, oxidative stress and DNA damage is caused by factors such as hyperbaric oxygen, gamma radiation, ultraviolet radiation, oZone, peroxides, free radicals, alkylating agents, and redox cycling drugs. While total oxidative stress and DNA damage may be reduced by living in a loW pollution environment and avoiding sunlight, they cannot be eliminated. Some factors like ioniZing radiation are present in all environments at a loW level and other factors are byproducts of metabolism and cannot be totally eliminated. Further, urban environ ments have high levels of ground level pollution from a variety of sources that are not likely to be reduced in the near 

Method for Controlling Cell Necrosis or Apoptosis

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Method for Controlling Cell Necrosis or Apoptosis

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