AU4438399A

AU4438399A - Wound treatment through inhibition of adenosine diphosphate ribosyl transferase

Info

Publication number: AU4438399A
Application number: AU44383/99A
Authority: AU
Inventors: Samuel J. Leibovich
Original assignee: University of Medicine and Dentistry of New Jersey
Current assignee: Rutgers Biomedical and Health Sciences
Priority date: 1998-06-11
Filing date: 1999-06-11
Publication date: 1999-12-30
Also published as: JP2002517436A; EP1085859A4; WO1999063982A1; EP1085859A1; CA2329160A1

Description

WO 99/63982 PCT/US99/13264 5 10 WOUND TREATMENT THROUGH INHIBITION OF ADENOSINE DIPHOSPHATE RIBOSYL TRANSFERASE 15 BACKGROUND OF THE INVENTION Field of the Invention 20 The present invention pertains to a method for healing a wound in a mammal which comprises the steps of (A) providing a therapeutic wound healing composition comprising a therapeutically effective amount of an inhibitor of mono adenosine diphosphate-ribosyl transferase to inhibit adenosine 25 diphosphate-ribosylation of vascular endothelial growth factor; and (B) contacting the therapeutic wound healing composition with a wound in a mammal. This invention also pertains to wound healing compositions and to methods for preparing and using the wound healing compositions and the pharmaceutical products in which the therapeutic compositions may be used. This invention further pertains to therapeutic 30 dermatological-wound healing compositions useful to minimize and treat diaper WO 99/63982 PCT/US99/13264 2 dermatitis and to methods for preparing and using the therapeutic dermatological wound healing compositions. 5 Description of the Background The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated 10 herein by reference and, for convenience, are referenced in the following text and respectively grouped in the appended bibliography. Wounds are internal or external bodily injuries or lesions caused by physical means, such as mechanical, chemical, viral, bacterial, or thermal means, which 15 disrupt the normal continuity of structures. Such bodily injuries include contusions, wounds in which the skin is unbroken, incisions, wounds in which the skin is broken by a cutting instrument, and lacerations, wounds in which the skin is broken by a dull or blunt instrument. Wounds may be caused by accidents or by surgical procedures. Patients who suffer major or chronic wounds could benefit from an enhancement in 20 the wound healing process. Wound healing consists of a series of processes whereby injured tissue is repaired, specialized tissue is regenerated, and new tissue is reorganized. Wound healing consists of three major phases: a) an inflammation phase (0-3 days), b) a cellular proliferation phase (3-12 days), and (c) a remodeling phase (3 days-6 months). During the inflammation phase, platelet aggregation and clotting 25 form a matrix which traps plasma proteins and blood cells to induce the influx of various types of cells. During the cellular proliferation phase, new connective or granulation tissue and blood vessels are formed. During the remodeling phase, granulation tissue is replaced by a network of collagen and elastin fibers leading to the formation of scar tissue. 30 WO 99/63982 PCT/US99/13264 3 Macrophages play a key role in the induction of angiogenesis in fibroproliferative states, including wound repair, rheumatoid arthritis, and solid tumor development (1-5). Production of angiogenic activity by macrophages depends on the balance of production of positive angiogenic regulators and inhibitors of angiogenesis 5 (6,7,8). Positive angiogenic regulators previously shown to be produced by monocytes and macrophages include the cytokines TNFa and 11-8 (9,10,11); negative regulators include thrombospondin- 1, Ifny -inducible protein- 10 (yTIP- 10) and other as yet uncharacterized protein inhibitors (12,13,14). The mechanisms controlling the balance of positive and negative angiogenesis regulators are not well understood. 10 Non-activated monocytes and macrophages exhibit a non-angiogenic phenotype (1,4). Following activation with agents such as interferon-y and/or endotoxin (LPS), macrophages express angiogenic activity, characterized by the expression of angiogenic cytokines, as well as of inhibitors of angiogenesis (15,16,17,18). Activated cells also produce and release oxygen radicals, nitric oxide (NO) and their 15 derivatives (17,19). These radicals have been shown to play an important role in regulating the angiogenic phenotype of activated macrophages (20,21). Agents such as Ifny and LPS, as well as reduced oxygen tension (hypoxia) and elevated lactate levels, induce macrophages to express angiogenic activity (1-3,9,22). Recently, macrophages in vivo have been shown to express vascular endothelial growth factor 20 (VEGF), an endothelial-specific mitogen that is potently angiogenic (18,23-30). United States patent no. 5,510,391 (Elson) discloses a method of treating blood vessel disorders of the skin and skin disorders caused by photo-aging comprising: a) coformulating a pharmaceutical composition wherein the composition 25 contains from 0.01% to 50% vitamin K; and b) applying the pharmaceutical composition topically to treat blood vessel disorders of the skin and skin disorders caused by photo-aging. The blood vessel disorders of the skin and skin disorders caused by photo-aging includes actinic and iatrogenic purpura, lentigines, telangiectasias of the face, spider angiomas, spider veins of the face and leg. 30 WO 99/63982 PCT/US99/13264 4 SUMMARY OF THE INVENTION 5 The present invention pertains to a method for healing a wound in a mammal which comprises the steps of: (A) providing a therapeutic wound healing composition comprising a therapeutically effective amount of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of 10 vascular endothelial growth factor; and (B) contacting the therapeutic wound healing composition with a wound in a mammal. In a preferred embodiment, the mammal is man. In another preferred 15 embodiment, the inhibitor of mono-adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin KI, Vitamin K2, Vitamin K3, Vitamin K4, Vitamin K5, Vitamin K6, Novobiocin, m-iodo benzyl guanidine, nicotinamide, coumermycin, dicoumarol, and silybin. More preferred inhibitors of mono-adenosine diphosphate-ribosyl transferase are Vitamin K1, Vitamin K3, Novobiocin, and silybin. 20 The inhibitor of mono-adenosine diphosphate-ribosyl transferase is present in the therapeutic wound healing composition in an amount from about 0.1% to about 10%, by weight of the therapeutic wound healing composition. The wound may be selected from the group consisting of pressure ulcers, decubitus ulcers, diabetic ulcers, and burn injuries. The therapeutic wound healing composition may further comprise a 25 pharmaceutically acceptable carrier.

WO 99/63982 PCT/US99/13264 5 The present invention also pertains to a wound healing composition which comprises: (A) a therapeutically effective amount of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of 5 vascular endothelial growth factor; and (B) a pharmaceutically acceptable carrier. The present invention further pertains to a method for treating diaper dermatitis in a human which comprises the steps of 10 (A) providing a therapeutic diaper dermatitis wound healing composition comprising: (a) a therapeutically effective amount of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; 15 (b) a buffering agent to maintain the pH of dermatitis in a range from about 5 to about 8; and (c) an anti-inflammatory agent; and (B) contacting the therapeutic diaper dermatitis wound healing composition with diaper dermatitis in a human. 20 The present invention further pertains to a therapeutic dermatological wound healing composition useful to minimize and treat diaper dermatitis which comprises a therapeutically effective amount of: (1) a therapeutic wound healing composition comprising an inhibitor of mono 25 adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; (2) a buffering agent to maintain the pH of dermatitis in a range from about 5 to about 8; and (3) an anti-inflammatory agent. 30 WO 99/63982 PCT/US99/13264 6 BRIEF DESCRIPTION OF THE FIGURES 5 Figure 1 illustrates the nitrite production by MPMs. Figure 2 illustrates VEGF production by A) RAW264.7 cells, and B) MPMs. 10 Figure 3 illustrates competitive RT-PCR analysis of VEGF mRNA levels in control (non-stimulated) MPMs 24 hours following plating. Figure 4 illustrate RT-PCR analysis of VEGF isoforms produced by Ifny/LPS-activated MPMs, with or without AG treatment. 15 Figure 5 illustrates TNFx production by MPMs. Figure 6 illustrates ADP-Ribosylation of rVEGF 165 by bacterial toxins and by macrophage cytosolic extract. 20 DETAILED DESCRIPTION OF THE INVENTION 25 Production of macrophage-dependent angiogenic activity (MDAA) requires activation by factors such as Interferon-y and/or endotoxin, hypoxia or high concentrations of lactate (Jensen et al. Lab. Invest. 54, 574, 1986). Previous work has demonstrated that the inducible nitric oxide synthase (iNOS) pathway in macrophages regulates MDAA, with inhibition of iNOS down-regulating expression 30 of MDAA (Leibovich et al, PNAS USA 91, 4190, 1994). It has now been found that WO 99/63982 PCT/US99/13264 7 although non-activated macrophages are non-angiogenic, they nevertheless express significant levels of the angiogenic growth factor VEGF. This constitutive expression of VEGF is not hypoxia or lactate dependent. The VEGF produced constitutively by normoxic, non-activated macrophages is found to be in a non-angiogenic form, due to 5 post-translational modification by the process of arginine-specific ADP-ribosylation. In contrast, VEGF produced by LPS-activated, hypoxic, or lactate-treated macrophages is in the non-ADP-ribosylated form, and is angiogenic. Inhibition of the iNOS pathway in LPS-activated macrophages abrogates MDAA expression by a dual mechanism. First, VEGF reverts to the ADP-ribosylated, non-angiogenic state; 10 second, iNOS-inhibited macrophages express an anti-angiogenic factor that blocks the angiogenic activity of several angiogenic factors, including VEGF, TNFc and bFGF. In mice where the iNOS gene has been specifically deleted (iNOS knockout mice, iNOS-/-), wound repair is markedly inhibited (Yamasaki et al., J. Clin. Invest. 101, 967, 1998). This inhibition is manifested in delayed wound closure, and a delay in the 15 formation of granulation tissue. Macrophages from iNOS-/- mice express reduced levels of MDAA in comparison to iNOS+/+ mice, although total VEGF production is not markedly altered. The role of the iNOS pathway and ADP-ribosylation of VEGF in regulating angiogenesis in wound repair and the modalities for pharmacologically modulating macrophage-dependent angiogenic activity and wound repair by targeting 20 iNOS and ADP-ribosylation pathways are under investigation. Murine thioglycolate-induced peritoneal macrophages (MPMs) and the murine RAW264.7 macrophage-like cell line (RAW cells) constitutively produce vascular endothelial growth factor (VEGF). VEGF production is increased under 25 hypoxic conditions or following cell activation with interferon-y (Ifny) and endotoxin (LPS). In contrast, TNFct, is produced only by Ifny/LPS-activated cells. Lactate (20mM) does not increase VEGF production by these cells. However, hypoxia, lactate, and Ifniy/LPS-activated MPMs express angiogenic activity, while normoxic, non-activated MPMs do not. Lack of angiogenic activity is not due to an anti 30 angiogenic factor(s) in the medium of these cells. Angiogenic activity produced by WO 99/63982 PCT/US99/13264 8 hypoxia and lactate-treated MPMs is neutralized by anti-VEGF antibody, which also neutralizes most of the angiogenic activity produced by Ifny/LPS-activated MPMs. The inducible nitric oxide synthase (iNOS) inhibitors Ng nitro-L-arginine-methyl ester (L-NAME) (1.5mM) and aminoguanidine (AG)(lmM) block production of 5 angiogenic activity by MPMs and RAW cells. In RAW cells, L-NAME and AG block Ifny/LPS-activated, but not constitutive VEGF production, while in MPMs, neither constitutive nor Ifny/LPS-activated VEGF synthesis is affected. Synthesis of TNFa is also unaffected. In contrast to normoxic, non-activated MPMs, iNOS-inhibited, Ifny/LPS-activated MPMs produce an anti-angiogenic factor(s). Accordingly, VEGF 10 is a major contributor to macrophage-derived angiogenic activity, and that activation by hypoxia, lactate or Ifny/LPS switches macrophage-derived VEGF from a non angiogenic to an angiogenic state. This switch may involve a post-translational modification of VEGF, possibly by the process of ADP-ribosylation. ADP ribosylation by MPM cytosolic extracts or by cholera toxin switches rVEGF 16 5 from 15 an angiogenic to a non-angiogenic state. In Ifny/LPS-activated MPMs, the iNOS dependent pathway also regulates the expression of an anti-angiogenic factor(s) that antagonizes the bio-activity of VEGF and provides an additional regulatory pathway controlling the angiogenic phenotype of macrophages. 20 In accord with the present invention, the expression of the angiogenic growth factor VEGF by MPMs and RAW cells was examined, and compared with that of TNFc'. The effects of hypoxia, lactate and the L-arginine-dependent inducible NO-synthase (iNOS) pathway on the production of VEGF and TNFu by these cells was also examined. VEGF production was found to be regulated both 25 transcriptionally and translationally by hypoxia and the iNOS pathway, and post translational modification may play an important role in regulating the bio-activity of VEGF as an angiogenic factor. In addition, the iNOS pathway in Ifniy/LPS-activated macrophages regulates the expression of anti-angiogenic factor that antagonizes the angiogenic effects of VEGF, providing an additional regulatory pathway to control 30 the angiogenic phenotype of macrophages.

WO 99/63982 PCT/US99/13264 9 Vascular endothelial growth factor (VEGF), a polypeptide growth factor that is potently angiogenic (induces the growth of new blood vessels), is chemically modified by macrophages (a cell that plays a key role in regulating 5 angiogenesis) in wound repair. This chemical modification involves the ADP-ribosylation of VEGF by enzymes (ADP-ribosyl transferases) in macrophages. In particular, the cytoplasmic mono-ADP-ribosyl transferases are involved in ADP-ribosylation of VEGF, and this modification results in a change in the properties of the VEGF from being angiogenic to being non-angiogenic. Macrophages make 10 VEGF constitutively, and it seems that macrophages regulate the angiogenic activity of VEGF by this ADP-ribosylation reaction. Inhibitors of mono-ADP-ribosylation such as: Vitamin KI, Vitamin K2, Vitamin K3, Novobiocin, m-iodo benzyl guanidine, and nicotinamide change the phenotype of macrophages from a non-angiogenic to an angiogenic phenotype by inhibiting the ADP-ribosylation of VEGF. Since 15 angiogenesis induction is a key event in normal wound repair, VEGF production by macrophages, the key cells that control angiogenesis by producing angiogenic factors, must require a switch from the non-angiogenic to the angiogenic phenotype, that is, from the ADP-ribosylated to the non-modified form. 20 Since the inhibitors of mono-ADP-ribosylation can block the ADP-ribosylation of VEGF, these inhibitors, and their derivatives and analogs are valuable in the treatment of chronic, non-healing wounds, where angiogenesis is deficient. In many chronic wounds, including but not limited to, pressure ulcers, decubitus ulcers, diabetic ulcers, and certain burn injuries, wounds fail to heal, at least 25 in part due to failures in angiogenesis. The macrophage phenotype in these wounds may be non-angiogenic, with VEGF being produced in the non-angiogenic, ADP-ribosylated form. In this case, treatment of these wounds with inhibitors of ADP-ribosylation would block the ADP-ribosylation of VEGF, and thus result in the production of non-modified, angiogenic VEGF. This VEGF should then participate in 30 stimulating angiogenesis in the wounds, and help promote repair. Accordingly, the WO 99/63982 PCT/US99/13264 10 present invention is directed to a formulation of ADP-ribosyl transferase inhibitors in an appropriate vehicle suitable for local application to wounds. Present technology for the treatment of chronic wounds generally 5 involves intensive wound care, debridement, use of antiseptics, antibiotics, and the use of occlusive dressings. Technologies in development include the use of growth factors, usually prepared by genetic engineering using recombinant DNA technology. Growth factor therapy is currently in clinical trials. Growth factors are extremely expensive, and their efficacy is still in doubt. The advantages of the use of ADP 10 ribosylation inhibitors for the treatment of chronic wounds are: a) the compounds are low molecular weight, well characterized, available, and relatively cheap; b) the compounds modulate the bio-activity of the wound's own biological mediators, shifting them from being non-angiogenic to being angiogenic, rather than attempting to introduce an exogenous growth factor activity; c) formulation of low molecular 15 weight inhibitors for delivery to wounds should be a relatively simple exercise, certainly compared to the formulation of growth factors; and d) vitamin-K compounds, which constitute one of the major groups of mono-ADP ribosylation inhibitors, have been available for other purposes for many years, and have FDA approval. 20 As set out above, the present invention is directed to a method for healing a wound in a mammal which comprises the steps of (A) providing a therapeutic wound healing composition comprising a therapeutically effective amount of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine 25 diphosphate-ribosylation of vascular endothelial growth factor; and (B) contacting the therapeutic wound healing composition with a wound in a mammal. The inhibitor of mono-adenosine diphosphate-ribosyl transferase may be any inhibitor, including active derivatives and analogs, which inhibits 30 ADP-ribosylation of vascular endothelial growth factor, thereby switching vascular WO 99/63982 PCT/US99/13264 11 endothelial growth factor from the non-angiogenic form to the angiogenic phenotype, that is, from the ADP-ribosylated form to the non-ADP-ribosylated form. Preferably, the inhibitor of mono-adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin Kl, Vitamin K2, Vitamin K3, Vitamin K4, Vitamin K5, 5 Vitamin K6, Novobiocin, m-iodo benzyl guanidine, nicotinamide, coumermycin, dicoumarol, and silybin. More preferably, the inhibitor of mono-adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin K1, Vitamin K3, Novobiocin, and silybin. 10 The amount of inhibitor of mono-adenosine diphosphate-ribosyl transferase present in the therapeutic wound healing compositions of the present invention is a therapeutically effective amount. A therapeutically effective amount of inhibitor of mono-adenosine diphosphate-ribosyl transferase is that amount of inhibitor of mono-adenosine diphosphate-ribosyl transferase necessary for the inventive 15 composition to switch the vascular endothelial growth factor from the non-angiogenic form to the angiogenic phenotype, that is, from the ADP-ribosylated form to the non-ADP-ribosylated form, and thereby promote wound healing. The exact amount of inhibitor of mono-adenosine diphosphate-ribosyl transferase is a matter of preference subject to such factors as the type of condition being treated as well as the 20 other ingredients in the composition. In a preferred embodiment, inhibitor of mono adenosine diphosphate-ribosyl transferase is present in the therapeutic wound healing composition in an amount from about 0.1% to about 10%, preferably from about 0.2% to about 8%, and more preferably from about 0.3% to about 5%, by weight of the therapeutic wound healing composition. 25 The types of wounds which may be healed using the wound healing compositions of the present invention are those which result from an injury which causes epidermal damage such as incisions, wounds in which the skin is broken by a cutting instrument, and lacerations, wounds in which the skin is broken by a dull or 30 blunt instrument. The therapeutic compositions may be used to treat pressure ulcers, WO 99/63982 PCT/US99/13264 12 decubitus ulcers, diabetic ulcers, and burn injuries. The therapeutic compositions may also be used to treat various dermatological disorders such as hyperkeratosis, burns, donor site wounds from skin transplants, ulcers (cutaneous, decubitis, venous stasis, and diabetic), psoriasis, skin rashes, and sunburn photoreactive processes. The wound 5 healing compositions can be used for the following indications: a) Healing of cuts and scrapes; b) Burns (heals burns with less scaring and scabbing); c) Decubitus ulcers; d) Bed sores, pressure ulcers; e) Fissures, Hemorrhoids; f) Use in combination with immunostimulators (simulated healing in healing deficient people); g) Post surgical wounds; h) Bandages; i) Diabetic ulcers; j) Venous ulceration; and k) Use in 10 combination with wound cleansing agents. Preferably, the therapeutic compositions may be used to treat pressure ulcers, decubitus ulcers, diabetic ulcers, and burn injuries. In another embodiment, the present invention is directed to a wound 15 healing composition which comprises (A) a therapeutically effective amount of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; and (B) a pharmaceutically acceptable carrier, wherein the amount and type of inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine 20 diphosphate-ribosylation of vascular endothelial growth factor are set out above. In a specific embodiment, the present invention is directed to diaper dermatitis. Diaper dermatitis, or diaper rash, is an irritant contact dermatitis localized to the skin area in contact with the diaper in infants. Diaper dermatitis occurs in about 25 65% of infants ranging from one to 20 months of age. The manifestations of diaper dermatitis vary from diffuse erythema to nodular lesions. Prolonged contact of the skin with urine-soaked diapers results in maceration of the epidermis. Occlusive rubber or plastic pants further aggravates the injury. Diaper dermatitis is caused by ammonia from the urine raising the pH of the skin and combining with constituents of 30 skin oil to form irritants. Bacterial or yeast infections may further complicate diaper WO 99/63982 PCT/US99/13264 13 dermatitis by causing persistent and severe inflammation. Diaper dermatitis is generally treated by keeping the skin dry by changing diapers frequently and applying talcum powder to the irritated area. In severe cases, rubber pants and plastic disposable diaper coverings should be avoided. 5 In accord with the present invention, a method for treating diaper dermatitis in a human is provided which comprises the steps of (A) providing a therapeutic diaper dermatitis wound healing composition comprising: (a) a therapeutically effective amount of an inhibitor of mono-adenosine 10 diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; (b) a buffering agent to maintain the pH of dermatitis in a range from about 5 to about 8; and (c) an anti-inflammatory agent; and (B) contacting the therapeutic diaper dermatitis wound healing composition with diaper dermatitis in a human. Buffering agents can help prevent diaper dermatitis by 15 neutralizing ammonia but do not heal injured mammalian cells. Anti-inflammatory agents can reduce inflammation (erythema) in a patient but do not promote the wound healing process. Wound healing compositions can increase the resuscitation rate of injured mammalian cells and the proliferation rate of new mammalian cells to replace dead cells. Applicants have found that the combination of a buffering agent, an anti 20 inflammatory agent, and a wound healing composition results in a therapeutic dermatological-wound healing compositions useful for minmizing and treating diaper dermatitis. The dermatological-wound healing compositions may optionally contain a therapeutically effective amount of a topical antiseptic to further reduce the duration and severity of diaper dermatitis. 25 Buffering agents are solute compounds which will form a solution to which moderate amounts of either a strong acid or base may be added without causing a large change in the pH value of the solution. In Bronsted's terminology, a buffering agent contains both a weak acid and its conjugate weak base. Buffering solutions 30 usually contain (a) a weak acid and a salt of the weak acid, (b) a mixture of an acid WO 99/63982 PCT/US99/13264 14 salt with the normal salt, or (c) a mixture of two acid salts, for example NaH 2

PO

4 and Na 2

HPO

4 . A weak acid becomes a buffering agent when alkali is added and a weak base becomes a buffering agent when acid is added. The buffering agents in the dermatological-wound healing compositions of the present invention may be selected 5 from a wide range of therapeutic agents and mixtures of therapeutic agents. Buffering agents which occur in nature include phosphates, carbonates, ammonium salts, proteins of plant and animal tissues, and the carbonic-acid-bicarbonate system in blood. Nonlimiting illustrative specific examples of buffering agents include citric acid-sodium citrate solution, phosphoric acid-sodium phosphate solution, and acetic 10 acid-sodium acetate solution. Preferably, the buffering agent is phosphoric acid sodium phosphate. The amount of buffering agent used in the present invention is an effective amount and may vary depending upon the dosage recommended or permitted 15 for the particular buffering agent. In general, the amount of buffering agent present is the ordinary dosage required to obtain the desired result. Such dosages are known to the skilled practitioner in the medical arts and are not a part of the present invention. In a preferred embodiment, the buffering agent in the dermatological-wound healing composition is present in an amount to maintain the pH of the dermatitis in a range 20 from about 5 to about 8, preferably from about 5.5 to about 7.5, and more preferably from about 6 to about 7. Anti-inflammatory agents are compounds that counteract or suppress the inflammatory process. The anti-inflammatory agents in the dermatological-wound 25 healing compositions of the present invention may be selected from a wide variety of steroidal, non-steroidal, and salicylate water-soluble and water-insoluble drugs and their acid addition or metallic salts. Both organic and inorganic salts may be used provided the anti-inflammatory agent maintains its medicament value. The anti inflammatory agents may be selected from a wide range of therapeutic agents and 30 mixtures of therapeutic agents which may be administered in sustained release or WO 99/63982 PCT/US99/13264 15 prolonged action form. Nonlimiting illustrative specific examples of non-steroidal anti-inflammatory agents include the following medicaments: ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac 5 tromethamine, diclofenac, and evening primrose oil (containing about 72% linoleic acid and about 9% gamma-linolenic acid). Nonlimiting illustrative specific examples of salicylate anti-inflammatory agents include the following medicaments: acetylsalicylic acid, mesalamine, salsalate, diflunisal, salicylsalicylic acid, and choline magnesium trisalicylate. Nonlimiting illustrative specific examples of steroidal anti 10 inflammatory agents include the following medicaments: flunisolide, triamcinoline, triamcinoline acetonide, beclomethasone diproprionate, betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone, predinisone, methyl prednisolone, and prednisolone. 15 Preferred anti-inflammatory agents to be employed may be selected from the group consisting of ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine, salsalate, diflunisal, salicylsalicylic acid, choline 20 magnesium trisalicylate, flunisolide, triamcinoline, triamcinoline acetonide, beclomethasone diproprionate, betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone, predinisone, methyl prednisolone, and prednisolone. In a preferred embodiment, the anti-inflammatory agent is selected from the group consisting of ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, 25 etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, and evening primrose oil. In a more preferred embodiment, the anti-inflammatory agent is evening primrose oil. The anti-inflammatory agent of the present invention may be used in 30 many distinct physical forms well known in the pharmaceutical art to provide an initial WO 99/63982 PCT/US99/13264 16 dosage of the anti-inflammatory agent and/or a further time-release form of the anti inflammatory agent. Without being limited thereto, such physical forms include free forms and encapsulated forms, and mixtures thereof 5 The amount of anti-inflammatory agent used in the present invention is a therapeutically effective amount and may vary depending upon the therapeutic dosage recommended or permitted for the particular anti-inflammatory agent. In general, the amount of anti-inflammatory agent present is the ordinary dosage required to obtain the desired result. Such dosages are known to the skilled 10 practitioner in the medical arts and are not a part of the present invention. In a preferred embodiment, the anti-inflammatory agent in the dermatological-wound healing composition is present in an amount from about 0.01% to about 10%, preferably from about 0.1% to about 5%, and more preferably from about 1% to about 3%, by weight. 15 In another specific embodiment, the present invention is directed to a therapeutic dermatological-wound healing composition useful to minimize and treat diaper dermatitis which comprises a therapeutically effective amount of: (1) a therapeutic wound healing composition comprising an inhibitor of mono 20 adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; (2) a buffering agent to maintain the pH of dermatitis in a range from about 5 to about 8; and (3) an anti-inflammatory agent. 25 Once prepared, the inventive therapeutic wound healing compositions may be stored for future use or may be formulated in effective amounts with pharmaceutically acceptable carriers to prepare a wide variety of pharmaceutical compositions. Examples of pharmaceutically acceptable carriers are pharmaceutical 30 appliances and topical vehicles. Examples of pharmaceutical appliances are sutures, WO 99/63982 1 7 PCT/US99/13264 staples, gauze, bandages, burn dressings, artificial skins, liposome or micell formulations, microcapsules, aqueous vehicles for soaking gauze dressings, and the like, and mixtures thereof Topical compositions employ topical vehicles, such as creams, gels formulations, foams, ointments and sprays, salves, and films, which are 5 intended to be applied to the skin or body cavity and are not intended to be taken by mouth. Oral topical compositions employ oral vehicles, such as mouthwashes, rinses, oral sprays, suspensions, and dental gels, which are intended to be taken by mouth but are not intended to be ingested. The preferred topical vehicles are water and pharmaceutically acceptable water-miscible organic solvents such as ethyl alcohol, 10 isopropyl alcohol, propylene glycol, glycerin, and the like, and mixtures of these solvents. Water-alcohol mixtures are particularly preferred and are generally employed in a weight ratio from about 1:1 to about 20:1, preferably from about 3:1 to about 20:1, and most preferably from about 3:1 to about 10:1, respectively. 15 A variety of traditional ingredients may optionally be included in the pharmaceutical composition in effective amounts such as buffers, preservatives, tonicity adjusting agents, antioxidants, polymers for adjusting viscosity or for use as extenders, and excipients, and the like. Other conventional additives include humectants, emollients, lubricants, stabilizers, dyes, and perfumes, providing the 20 additives do not interfere with the therapeutic properties of the therapeutic wound healing composition. Specific illustrative examples of such traditional ingredients include acetate and borate buffers; thimerosol, sorbic acid, methyl and propyl paraben and chlorobutanol preservatives; sodium chloride and sugars to adjust the tonicity; and excipients such as mannitol, lactose and sucrose. Other conventional 25 pharmaceutical additives known to those having ordinary skill in the pharmaceutical arts may also be used in the pharmaceutical composition. The ultimate pharmaceutical compositions are readily prepared using methods generally known in the pharmaceutical arts.

WO 99/63982 PCT/US99/13264 18 In accordance with this invention, therapeutically effective amounts of the therapeutic wound healing compositions of the present invention may be employed in the pharmaceutical appliance. These amounts are readily determined by those skilled in the art without the need for undue experimentation. The exact amount of 5 the therapeutic wound healing composition employed is subject to such factors as the type and concentration of the therapeutic wound healing composition and the type of pharmaceutical appliance employed. Thus, the amount of therapeutic wound healing composition may be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the 10 need for undue experimentation. In a preferred embodiment, the pharmaceutical composition will comprise the therapeutic wound healing composition in an amount from about 0.1% to about 10%, by weight of the pharmaceutical composition. In a more preferred embodiment, the pharmaceutical composition will comprise the therapeutic wound healing composition in an amount from about 0.2% to about 8%, 15 by weight of the pharmaceutical composition. In a most preferred embodiment, the pharmaceutical composition will comprise the therapeutic wound healing composition in an amount from about 0.3% to about 5%, by weight of the pharmaceutical composition. 20 The present invention extends to methods for making the pharmaceutical compositions. In general, a pharmaceutical composition is made by contacting a therapeutically effective amount of a therapeutic wound healing composition with a pharmaceutically acceptable carrier and the other ingredients of the final desired pharmaceutical composition. The therapeutic wound healing 25 composition may be in a solvent and may be absorbed onto a pharmaceutical appliance.

WO 99/63982 PCT/US99/13264 19 RESULTS Production of Nitrite by RAW264.7 Cells and MPMs 5 Figure 1 shows the production of nitrite by MPMs. Nitrite was not produced by non-activated cells, either with or without lactate. Following challenge with Ifny/LPS, nitrite production was strongly induced, with nitrite accumulating over the 48 hr. incubation period. L-NAME (1.5mM) blocked nitrite production by about 70-80%; AG (1mM) blocked nitrite production by >95%. RAW264.7 cells produced 10 nitrite in a similar manner, and L-NAME and AG blocked nitrite synthesis by RAW264.7 cells to a similar extent (data not shown). Production of VEGF by RAW264.7 Cells and Murine Peritoneal Macrophages 15 The production of VEGF by RAW cells is shown in Figure 2A. Non stimulated RAW cells produced VEGF in an apparently constitutive manner over the 48 hour incubation period. This spontaneous production of VEGF was similar in regular culture plates and in gas-permeable Permanox plates. Stimulation of cells with Ifny and LPS increased the production of VEGF by RAW cells over the constitutive 20 level produced by non-stimulated cells by about 3-4 fold by 18 hours. By 48 hours, the stimulated VEGF levels were only 2 fold increased over the constitutive level. The iNOS inhibitors AG (1.0mM) and L-NAME (1.5mM) did not block the constitutive production of VEGF by non-stimulated RAW cells, but reduced the production of VEGF by Ifny/LPS-activated RAW cells, to a level markedly below that 25 of the non-stimulated cells. Sodium lactate (25mM) did not alter the production of VEGF by these cells, either with or without Ifny/LPS activation. RAW cells cultured under hypoxic conditions produced increased amounts of VEGF. After 18 hours, VEGF levels in the media of cells cultured under hypoxic conditions were about 3 fold greater than those in the media of control, normoxic cells. This differential was 30 less marked by 48 hours. Analyses of the dissolved oxygen levels in the conditioned WO 99/63982 20 PCT/US99/13264 media directly following harvesting indicated clearly that under normoxic conditions, oxygen levels were consistently high (pO2 > 145). After 24 and 48 hours incubation under hypoxic conditions (95% N 2 /5%CO 2 ), the p 0 2 was 71mm and 46 mm respectively. 5 The production of VEGF by MPMs was similar to that of RAW cells, with constitutive production occurring over 48 hours (Figure 2B). Increased production was induced by Ifny/LPS. However, in contrast to RAW cells, iNOS inhibitors did not significantly reduce the production of VEGF by Ifny/LPS-activated 10 MPMs. As was observed for RAW cells, sodium lactate did not modulate the production of VEGF by these cells. Culture of MPMs under hypoxic conditions resulted in an increase in VEGF production in the first 18 hours; after 48 hours, however, constitutive production of VEGF was only slightly higher than that of hypoxic cells. Oxygen levels determined in the conditioned media of MPMs were 15 similar to those found in RAW cell media. Quantitative RT-PCR Analysis of VEGF mRNA Levels A typical example of a quantitative RT-PCR dilution series using the 20 VEGF RNA minigene as internal standard is shown in Figure 3. The PCR amplification product of the minigene is 293 bp in size. The native mRNA PCR amplification band is 362 bp in size. The point of equivalence for the amplified minigene and the amplified native mRNA is readily determined from the dilution series. The values determined from these analyses were normalized to the levels of 25 G3PDH mnRNA determined in parallel samples, although little variation in the G3PDH mRNA levels were in fact observed between samples. On this basis, the relative amounts of VEGF mRNA in the various macrophage preparations are shown in Table 1. Both hypoxia and Ifny/LPS activation upregulated VEGF steady state mRNA levels in MPMs at 4 and 10 hours. By 24 hours, however, the levels of VEGF 30 mRNA were similar in all the groups. In RAW cells, VEGF mRNA levels remained WO 99/63982 PCT/US99/13264 21 elevated at 24 hours. Aminoguanidine treatment of Ifny/LPS-treated MPMs did not significantly reduce their steady-state VEGF mRNA levels at any time point; in RAW cells, however, the VEGF mRNA levels were reduced by 70-80% at 4, 10 and 24 hours. 5 RT-PCR Analysis of VEGF mRNA Isoforms Three isoforms of VEGF were found to be produced by both non activated and Ifny/LPS-activated MPMs. These isoforms corresponded to VEGF-1 10 (652bp), VEGF-2 (580bp) and VEGF-3 (448 bp)(45). The relative proportions of the VEGF isoforms expressed by MPMs at each time point following Ifniy/LPS activation were only slightly modulated by Ifny/LPS-activation and by inhibition of iNOS with AG (Figure 4). In RAW cells, VEGF mRNA isoforms were similarly unaffected by Ifny/LPS activation and by AG treatment. 15 Production of TNFa by MPMs and RAW264.7 Cells TNFx was not produced by either non-stimulated MPMs or by RAW264.7 cells over the 48 hour test period. Production of TNFc by MPMs is 20 shown in Figure 5. Following stimulation with Ifny/LPS, TNFo expression was strongly induced, with increased TNFcc in the conditioned media being apparent by 8 hours following challenge. There was no significant difference in TNFcX production in cells treated with or without sodium lactate. Similarly, culture of cells in Permanox dishes, under either normoxic or hypoxic conditions, did not modulate TNFu 25 production. The iNOS inhibitors L-NAME and AG had no significant effect on the production of TNFc by MPMs. Production of TNFou by RAW cells was similar to that observed in MPMs (data not shown).

WO 99/63982 PCT/US99/13264 22 ADP-Ribosylation of VEGF by Bacterial Toxins and Macrophage Extracts Labeling of rVEGF with 3 2 P-NAD was observed using cholera toxin and macrophage cytosolic extracts (Figure 6). Labeling with cholera toxin resulted in 5 a single 3 2 P-labeled band corresponding to the size of rVEGF 16 5 standard (Figure 6C). Labeling with macrophage cytosolic extracts resulted in the 32 P-labeling of a large number of bands, due to the endogenous labeling of macrophage cytosolic proteins (Figure 6A). To clearly demonstrate labeling of rVEGF 16 5 in this mixture, immunoprecipitation of the macrophage cytosolic labeling mixture with anti-VEGF 10 antibody was necessary. Following immunoprecipitation, a prominent labeled band corresponding to rVEGF 16 5 was clearly visible (Figure 6B). This band was not present in control reactions carried out in the absence of rVEGF 16 5 . Labeling of VEGF using Pertussis toxin was not observed (Figure 6E). 15 Angiogenic and Anti-Angiogenic Responses in Rat Corneas The angiogenic responses induced in rat corneas by the concentrated conditioned media from the MPMs cultured under various conditions are shown in Table 2. Medium from non-activated MPMs cultured under normoxic conditions did 20 not induce angiogenesis. This medium did not contain anti-angiogenic activity, as the angiogenic effects of VEGF (25ng) were unaffected by this medium. Medium from Ifniy/LPS-activated MPMs was potently angiogenic, while medium from iNOS inhibited Ifny/LPS-activated MPMs showed markedly reduced angiogenic activity. In contrast to medium from normoxic, non-activated MPM, this medium was found to 25 contain anti-angiogenic activity, as we have reported previously (36). Medium from normoxic, lactate-treated non-activated MPMs showed significant angiogenic activity. Similarly, medium from non-activated MPMs cultured under hypoxic conditions showed significant angiogenic activity. In both these cases, a polyclonal antibody to VEGF neutralized the angiogenic activity in the conditioned media. Angiogenic 30 responses induced by rVEGF 16 5 were neutralized by anti-VEGF antibody in control WO 99/63982 PCT/US99/13264 23 experiments, while those induced by bFGF (20ng/implant) and TNFa (20ng/implant) were unaffected. The angiogenic responses induced by rVEGF 16 5 that was ADP 5 ribosylated using cholera toxin or MPM cytosolic extract are shown in Table 3. While control VEGF (taken through a sham labeling procedure in the absence of cholera toxin and MPM cytoplasmic extracts) strongly induced angiogenesis, both cholera toxin-mediated and MPM cytoplasmic extract-mediated ADP-ribosylated VEGF showed greatly reduced angiogenic responses, indicating that the ADP-ribosylation 10 abrogated the angiogenic activity of the VEGF. Since the VEGF was purified from the reaction mixtures using heparin-Sepharose binding and elution, we also tested eluates from control VEGF-free reactions prepared with cholera toxin or macrophage cytosolic extract, to determine first if these extracts contained angiogenic activity in their own right, and second, if any anti-angiogenic activity might be enriched in the 15 eluates through this procedure, and interfere with the angiogenic activity of the VEGF. The eluates were therefore tested alone, and then with the post-reaction addition of rVEGF 16 5 . The sham eluates did not exhibit direct angiogenic activity, nor did they exhibit anti-angiogenic activity when combined with VEGF. 20 DISCUSSION In this study, it has been shown that murine macrophages (MPMs) produce VEGF, a potent, endothelial cell specific, angiogenic growth factor (23,24). VEGF production by MPMs does not require activation, with significant VEGF levels 25 being released into the conditioned media over 18-48 hours without the addition of external stimulants. This constitutive level of VEGF production was, however, markedly increased by stimulation of the cells with Iffiy/LPS (Figure 2). In contrast, the production of TNFo was strictly dependent on macrophage activation with Ifny and LPS, as has been shown in many previous studies (Figure 4)(37-39). 30 WO 99/63982 PCT/US99/13264 24 VEGF expression has been shown to be regulated by oxygen tension both in vivo and in vitro (40-44), with low levels of oxygen (hypoxia) resulting in the upregulation of VEGF expression. This increased expression has been shown to be regulated both at the transcriptional level and at the level of mRNA stability, 5 depending upon the cell type. In our studies, oxygen concentrations were measured in the conditioned media of macrophages cultured in both normal and Permanox culture dishes. These measurements indicated that under these conditions, the media on the MPMs and RAW cells were normoxic, suggesting that the constitutive VEGF production observed was not due to induction of VEGF gene expression by low 10 oxygen tension. However, when cells were specifically incubated under hypoxic conditions, significant upregulation of VEGF, but not of TNFu or nitrite production, was observed in both cell types. This upregulation of VEGF expression was apparent both at the mRNA and the protein level. These observations suggest that the expression of the VEGF gene is regulated by oxygen tension in macrophages, as 15 observed in other cell types. It is not yet clear, however, whether this regulation occurs at the level of transcription or at the level of mRNA stability. Knighton and coworkers have shown previously that the expression of angiogenic activity by rabbit bone-marrow-derived macrophages is regulated by 20 hypoxia, and that the high levels of lactate that accumulate in the conditioned media of hypoxic macrophages are important in regulating the expression of macrophage derived angiogenic activity (5). In MPMs and RAW cells, culture in the presence of high lactate concentrations (25mM), under normoxic conditions, did not modulate the level of expression of VEGF mRNA or protein. However, it is important to note that 25 while non-stimulated MPMs express significant levels of VEGF, the conditioned media from these cells is non-angiogenic (4,8,22). Following lactate or hypoxia treatments, the media exhibit angiogenic activity (Table 1). This raises the important question of how the angiogenic activity of VEGF is regulated. First, VEGF may operate in synergy with TNFa to stimulate the microvasculature in the conditioned 30 media from IfnTy/LPS-activated macrophages. However, the fact that medium from WO 99/63982 PCT/US99/13264 25 lactate-treated or hypoxia-treated non-activated macrophages, which do not contain TNFu, express potent angiogenic activity suggests that under the appropriate conditions, VEGF can be angiogenic in the absence of TNFa. This is supported by the fact that the angiogenic activity in these media is neutralized by anti-VEGF 5 antibodies (Table 1). A second possibility tested was that medium from normoxic, non-activated MPMs might contain an anti-angiogenic factor(s) that blocks the angiogenic effects of VEGF. This hypothesis was tested using the rat corneal bio assay, by combining concentrated conditioned medium from these cells with rVEGF 16 5 , to determine if the angiogenic effects of the VEGF were inhibited. No 10 inhibition of the effects of VEGF were in fact observed in this system, clearly indicating that anti-angiogenic factor(s) were not present in this conditioned medium. This is in contrast to the conditioned medium from iNOS-inhibited, Ifny/LPS-activated macrophage medium, as discussed further below. 15 It was then hypothesized that the VEGF produced by non-stimulated MPMs may differ structurally from the VEGF produced by stimulated MPMs. This structural difference could relate to alternatively spliced isoforms of VEGF with differing angiogenic activities, or to post-translational modification of VEGF by, for example, ADP ribosylation-dependent mechanisms (32,45,46). Our results using RT 20 PCR indicate that the isoforms of VEGF are not markedly changed during macrophage activation, by lactate, or by inhibition of iNOS. VEGF1, 2 and 3 mRNA isoforms are produced in similar proportions under all conditions tested. It thus seems that the most likely mechanism for regulation of VEGF angiogenic activity might involve post-translational mechanisms, as has been suggested recently by 25 Hussain et al (32,47). In support of this hypothesis, rVEGF was shown to be a substrate for ADP-ribosylation, and ADP-ribosylation was shown to abrogate the angiogenic activity of rVEGF. Since macrophages are impermeable to NAD + , metabolic labeling of endogenously synthesized VEGF by macrophages using 3 2

P

labeled NAD

+

, is not possible (32,33). However, we demonstrated labeling of 30 rVEGF165 in vitro, using cytosolic extracts of macrophages, as well as by the WO 99/63982 PCT/US99/13264 26 bacterial arginine-specific ADP-ribosyl transferase, cholera toxin subunit A (Figure 6)(34,35). Pertussis toxin, on the other hand, which is a cysteine-specific ADP-ribosyl transferase, did not modify rVEGF 16 5 (48). In addition, we showed that ADP ribosylation of VEGF abrogates its angiogenic activity. In contrast to unmodified 5 rVEGF 16 5 , rVEGF 1 65 derivatized using either cholera toxin or macrophage cytosolic extract was found to be non-angiogenic (Table 3). It has been shown that the production of angiogenic activity by human monocytes and by murine macrophages is induced by activation of the cells with 10 Ifny/LPS (1-3,20,21,49). In addition, the L-arginine-dependent inducible nitric oxide synthase (iNOS)-dependent pathway plays an important role in regulating the expression of angiogenic activity by Ifny/LPS-activated macrophages (21). Inhibitors of iNOS, such as L-NAME, Ng-monomethyl-L-arginine (L-NMMA), diphenyleneiodonium (DPI) and AG block the production of angiogenic activity by 15 activated macrophages, without inhibiting the production of the angiogenic cytokines TNFc. and 11-8 (21,49,50). In this study, we show that the iNOS inhibitors L-NAME and AG markedly inhibit the production of VEGF by Ifny/LPS-activated RAW cells (>70% inhibition), but have little effect on the constitutive (non-stimulated) production of VEGF by these cells. Interestingly, in Ifny/LPS-activated RAW cells, 20 L-NAME and AG inhibit VEGF production to a level significantly below that of non stimulated cells. This suggests that the pathways involved in the regulation of VEGF production in non-activated and activated RAW cells are different, with only the activated pathway being sensitive to iNOS products. This might relate to the nature of the transcriptional promotors involved in the expression of the VEGF gene under 25 constitutive and activated conditions. In MPMs, on the other hand, the iNOS inhibitors had no significant effect on the production of either the constitutive or Infy/LPS-stimulated VEGF. However, it is again important to note that the angiogenic activity of the MPM conditioned media was markedly down-regulated by the iNOS inhibitors. Our results suggest that two mechanisms are involved in the 30 regulation of expression of angiogenic activity by the iNOS-inhibited, Ifny/LPS- WO 99/63982 PCT/US99/13264 27 activated MPMs. The first is analogous to that observed in the activation of macrophages by hypoxia and lactate; namely the regulation of the ADP-ribosylation of VEGF, and hence of its angiogenic activity. Infy/LPS activation switches the production of VEGF from the ADP-ribosylated, non-angiogenic form to the 5 unmodified, angiogenic form. Second, the iNOS-dependent pathway regulates the expression of an inhibitor of angiogenesis. When the iNOS pathway is active and NO is produced, the inhibitor is inactive or absent; when the iNOS pathway is blocked with AG or L-NAME, the inhibitor is active. We have previously reported that this anti-angiogenic activity is present in the conditioned medium of iNOS-inhibited 10 Ifny/LPS-activated MPMs (36). The nature of this inhibitor is not yet clear; however it is not neutralized by specific antibodies to thrombospondin-1 or yIP-10, both of which are potent anti-angiogenic agents that may be produced by macrophages (51,52). Specific antibodies to TNFc and TGFP also do not neutralize the anti angiogenic activity. The inhibitor binds weakly to heparin-Sepharose and has an 15 apparent molecular weight >100kDa (36). Hussain and coworkers (32,47) have suggested that ADP-ribosylation dependent mechanisms may be involved in the post-translational modification of angiogenic factors, resulting in non-angiogenic forms. Our results suggest that this 20 may indeed be one of the mechanisms regulating the production of angiogenic activity by macrophages. We suggest that VEGF produced by the constitutive pathway is normally in the ADP-ribosylated, non-angiogenic form, while VEGF produced by Ifny/LPS-activated MPMs is in the unribosylated, angiogenic form. Activation may thus regulate the post-transcriptional modification of VEGF from the ADP-ribosylated 25 non-angiogenic form to the unmodified angiogenic form. In addition, the iNOS pathway in activated MPMs appears to regulate the production (or bio-activity) of an anti-angiogenic factor, that is apparent only in Ifny/LPS-activated, iNOS-inhibited MPM medium.

WO 99/63982 PCT/US99/13264 28 These results clearly indicate that VEGF is a substrate for ADP ribosylation, and that ADP-ribosylation of VEGF abrogates its angiogenic activity. Preliminary results (manuscript in preparation) also indicate that vitamin-K3 and novobiocin, both inhibitors of mono-ADP-ribosylation reactions (34,53), result in the 5 production of angiogenically active VEGF by non-activated normoxic macrophages, without affecting the level of VEGF production or the production of TNFxt, suggesting the involvement of mono-ADP-ribosylation in the regulation of angiogenic activity in macrophages. Ultimate proof, however, of the role of mono-ADP ribosylation in the regulation of VEGF bio-activity by macrophages, will require the 10 direct demonstration that VEGF is differentially ADP-ribosylated in macrophages under conditions that modify oxygen tension or Ifniy/LPS-induced macrophage activation and the iNOS-dependent pathway. In summary, on the basis of these observations, it appears that VEGF 15 is an important contributor to macrophage-dependent angiogenic activity. VEGF production in macrophages is regulated at several levels. Constitutively expressed VEGF is normally angiogenically inactive. Hypoxia and Ifny/LPS activation increase the absolute amount of VEGF produced, but also result in the expression of angiogenic VEGF. High lactate does not increase the amount of VEGF produced, but 20 also results in the production of angiogenic VEGF. The change in the angiogenic phenotype of VEGF may be due to post-translational modification, perhaps by the process of ADP-ribosylation, that modulates VEGF bio-activity. rVEGF 16 5 is a substrate for ADP-ribosylation by cholera toxin and by MPM cytoplasmic extracts, and ADP-ribosylation of rVEGF 16 5 was shown to abrogate its angiogenic activity. 25 In hypoxic and Ifny/LPS-activated MPMs, activation upregulated VEGF mRNA expression, and also shifted the balance of post-translational modification of VEGF from the non-angiogenic to the angiogenic form. In RAW264.7 cells, the Ifhy/LPS activation-dependent modulation of VEGF mRNA levels is regulated in part by the iNOS pathway, but the constitutive production of VEGF in non-activated cells is not. 30 In MPMs on the other hand, the regulation of VEGF mRNA level by Ifny/LPS WO 99/63982 PCT/US99/13264 29 activation is not significantly dependent on the iNOS pathway. VEGF angiogenic activity in these cells appears to be regulated at the level of post-translational modification. Finally, when the iNOS pathway is inhibited in Ifniy/LPS-activated MPMs, an anti-angiogenic factor is expressed that blocks the angiogenic activity of 5 VEGF. Together, regulation of VEGF bio-activity by post-translational modification, and iNOS-dependent regulation of the expression an anti-angiogenic factor, provide novel mechanisms for controlling the angiogenic phenotype of macrophages, and may play a key role in the regulation of macrophage-dependent angiogenic activity in vivo, in wound repair, fibroproliferation, and possibly in solid tumor development. 10 The present invention is further illustrated by the following examples which are not intended to limit the effective scope of the claims. All parts and percentages in the examples and throughout the specification and claims are by weight of the final composition unless otherwise specified. 15 EXAMPLES Materials & Methods 20 Murine Peritoneal Macrophages (MPMs) and RAW264.7 cells Balb-c mice (male, 6-8 weeks, Taconic, Germantown, NY) were injected intraperitoneally with 2.5 ml sterile Brewer's thioglycollate broth (3% w/v) (Difco Labs., Detroit, MI). Five days later, the mice were sacrified and MPMs were 25 harvested using PBS containing 100 U/ml of heparin. Cells were centrifuged at 300g for 5 mins. at 40C., washed twice with serum-free DMEM, and resuspended in DMEM containing 10% FCS and 50tg/ml gentamycin (DMEM-10%FCS). Cells were seeded into 60 mm tissue culture dishes (Costar, Cambridge, MA)(4x10 6 cells/dish) and incubated at 370C in a humidified incubator in 95% air/5% CO 2 for 4 30 hrs to allow the cells to adhere. In some experiments, cells were seeded in Contur WO 99/63982 PCT/US99/13264 30 Permanox gas-permeable dishes (Miles, Naperville, IL) rather than regular tissue culture dishes, to increase the availability of ambient gases to the cells on the base of the dishes. Non-adherent cells were removed by washing with serum free DMEM, and the cells were refed with DMEM/1% FCS. MPMs were activated using 100U/ml 5 murine Ifny (Sigma Chemical Co., St. Louis, MO) and 100ng/ml of LPS (E.coli serotype 055:B5, Sigma) either in the presence or absence of the iNOS inhibitors L NAME (1.5mM) or AG (1mM). To test the effects of lactate on MPMs, sodium lactate (25 mM) was added to the cultures at the start of the incubation period. To test the effects of hypoxia, MPMs were incubated in Permanox dishes, either under 10 normoxic conditions (95% air, 5%CO 2 ) or under hypoxic conditions (95%N 2 , 5%CO 2 ). Media and cells were harvested at the indicated time points following addition of Ifn-y/LPS and/or lactate. Aliquots of media were sampled immediately following incubation, and analyzed in a Blood Gas Analyzer (Instrumentation Lab., Lexington, MA). The remaining media were centrifuged at 4 0 C for 5 mins at 15,000 15 g to remove cellular debris, and stored at -80 0 C. prior to analysis. RAW264.7 cells were obtained from ATTC, and routinely maintained in DMEM-10%FCS. Cells were passaged by scraping, and plated in either regular or Permanox dishes, with or without Ifny/LPS, with or without sodium lactate, and 20 under hypoxic conditions, as described above. The effects of L-NAME and AG on the production of VEGF and TNFat by these cells were also tested. Media and cells were harvested and treated as described above. Isolation of Total Cellular RNA 25 Total cellular RNA was isolated from macrophage cell cultures using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH). Medium was removed from the cells, TRI REAGENT added directly to the culture dishes, and the cell lysate passed several times through a 21 gauge syringe needle. Samples were 30 stored at RTo for 5 mins., 0.2ml chloroform was then added per milliliter lysis WO 99/63982 PCT/US99/13264 31 reagent, the mixture vortexed for 15 secs. and then incubated at RT 0 for 10 mins. The resultant mixture was centrifuged at 12,000g for 15 mins. at 4 0 C. The aqueous (upper) phase was transferred to a fresh microfuge tube, and RNA precipitated by adding 0.5ml isopropanol per Iml TRI REAGENT used for the original extraction. 5 Samples were incubated at RTo for 5 mins. and then centrifuged at 12,000g for 10 mins. at 4 0 C. The RNA pellets were washed with 75% ethanol, air dried for 5 mins. and dissolved in RNAase-free water. Quantitative RT-PCR Analysis of VEGF mRNA levels 10 VEGF mRNA levels were determined by RT-PCR using an internal minigene RNA standard that is present through both the RT and the PCR reaction stages. The 293 bp VEGF minigene RNA standard, containing a 69 bp gene deletion, was prepared as follows: Total RNA from MPMs was subjected to RT and PCR 15 through 35 cycles, using the following primers: Sense minigene primer: (18-mer) in exon 1 (positions 41-58): 5' GGACCCTGGCTTTACTGC.3' Anti-sense minigene primer (39mer), starting in exon 5, spanning an intron, and continuing into exon 4 to position 387, deleting 69 bp of the gene to position 318, and 20 continuing to position 300. The primer thus spans an intron, and contains a 69 bp deletion. 5' TTGGTCTGCATTCACATCGGC-GTGATGTTGCTCTCTGAC 3'. The PCR band was purified from primers by ethanol precipitation, and blunt end ligated into the pCR-Script AmpSK(+) vector (Stratagene, La Jolla, CA). The 25 orientation of the minigene fragment in the vector was determined by dideoxy sequence analysis. A clone containing the minigene insert in an antisense orientation was used for subsequent in vitro transcription for the preparation of the RNA minigene. The vector was linearized with Notl, treated with proteinase-K (4p.g/mi) for 1 hr. at 37 0 C., and purified by phenol extraction and ethanol precipitation. The 30 linearized plasmid was then transcribed in vitro using a VTRAN-7 transcription kit WO 99/63982 PCT/US99/13264 32 (Sigma), using T7 RNA polymerase, yielding sense RNA. The reaction product was treated with RNAase-free DNAase-1 (10u/mg DNA in the transcription reaction) (Promega, Madison, WI) for 2 hours at 37 0 C. The reaction mixture was then heated to 90 0 C. for 5 mins., cooled, and 10x transcription stop solution (5M. ammonium 5 acetate, 0.1M. EDTA) were added, followed by phenol extraction and isopropanol precipitation. The RNA concentration was determined spectrophotometrically. VEGF RNA minigene (2.5pg per reaction) was then incorporated into the RT-PCR reactions. Total RNA from macrophages treated under various conditions was added to the RT-PCR reactions in amounts ranging from 1-200ng/reaction. The 10 oligonucleotide primers used for the competitive RT-PCR reaction were 18-mers nested into the initial primers used to prepare the minigene: Sense primer in exon 1: 5' ACCCTGGCTTTACTGCTG 3' Antisense primer (intron spanning): 5' GGTCTGCATTCACATCGG.3' The antisense primer was used for the initial RT reaction; the reverse transcriptase 15 was inactivated at 990C. for 5 mins., and added to a PCR mix containing an equivalent amount of sense primer. PCR was then carried out for 25 cycles. The reactions were analyzed by electrophoresis on 1.5% agarose gels in TAE buffer, stained with ethidium bromide, and scanned using the Molecular Dynamics Fluorlmage Analyzer. The concentrations of input RNA that gave bands of equal 20 intensity to that of the internal VEGF RNA minigene were then determined. Although intron-spanning primers were used throughout, controls for genomic DNA contamination of total RNA preparations were routinely carried out. These controls involved the performance of parallel reactions in the absence of reverse transcriptase. 25 As a control for a housekeeping gene that is not markedly modulated by the various culture conditions used, an RT-PCR procedure for the enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was also developed (details not shown). Parallel reactions for G3PDH mRNA levels were performed on the various macrophage RNA samples, and the VEGF mRNA levels determined by RT-PCR were 30 normalized to the G3PDH levels.

WO 99/63982 PCT/US99/13264 33 RT-PCR Analysis of VEGF mRNA Isoforms For reverse transcription, 1.0jg of total RNA was reverse transcribed 5 using 100 ng of the reverse VEGF specific primer indicated below, using 50 U MuLv reverse transcriptase with an RNA PCR Kit (Perkin Elmer, Foster City, CA), following the manufacturer's protocol. Following the initial RT reaction step, the 20 tl reaction volumes were boiled for 5 mins. to inactivate the reverse transcriptase. 100ng forward primer (see below) were added, together with 80[1 of a PCR master 10 mix, to give a final concentration of 1 mM MgCl 2 , IX PCR buffer II, and 2.5 U Taq polymerase (Perkin-Elmer) per reaction. PCR primers were selected to enable the amplification of the three differentially spliced murine isoforms of VEGF mRNA formed from the VEGF gene. These VEGF mRNA isoforms are derived from a gene containing 8 exons (45). The largest, VEGF-1, is formed using all 8 exons. VEGF-2 15 lacks exon 7, and VEGF-3 lacks exons 6 and 7. By using PCR primers in exons 3 and 8, the three different isoforms of VEGF generate PCR amplification products of different sizes, and since they amplify from the same primers, the ratio of intensities of the three bands gives an estimate of the relative abundance of the three differentially spliced mRNA isoforms.The primers selected for the PCR amplifications were: 20 Forward primer, located in exon 3: 5'GATGAAGCCCTGGAGTGC3' Reverse Primer, located in exon 8: 5'TCCCAGAAACAACCCTAA3' The following cycling program for PCR was used: Denaturation at 94 0 C for 1 min., annealing at 54 0 C for 1min., and extension for 2 mins. at 72 0 C, for 25 cycles, with a final extension at 72 0 C for 15 mins. PCR reactions were then analyzed by 25 electrophoresis on 1.5% agarose gels using TAE buffer, and stained with ethidium bromide. Gels were scanned using a Molecular Dynamics Fluorlmage analyzer, and the staining intensities of the PCR-amplified VEGF isoform bands were analyzed using the ImageQuant image analysis software package (Molecular Dynamics).

WO 99/63982 PCT/US99/13264 34 Assay of VEGF Protein Levels by ELISA VEGF in conditioned media was assayed using a sandwich ELISA kit (Quantikine M, R & D Systems, Minneapolis, MN.), following the manufacturer's 5 protocol. This assay detects murine VEGF with sensitivity in the range of 3-500 pg/ml. Samples with VEGF concentrations above this range were diluted with RPMI and re-assayed. All samples were assayed in triplicate. Results are presented as means +/- standard deviations of the mean (S.D.). 10 Assay of TNFa by ELISA Murine TNFot was assayed using a sandwich ELISA kit (TNF-A Minikit, Endogen, Woburn, MA), following the procedure of the manufacturer. All samples were assayed in triplicate. Results are presented as means +/- S.D. 15 Assay of Nitrite To determine the production of nitric oxide (NO) by the cells under the various conditions tested, the media were analyzed for nitrite using the Griess 20 reaction, as described previously. Briefly, 50l culture medium were placed in a 96 well plate, followed by 50pL of cold 350mM ammonium chloride, pH 9.6. 100 p.l of a mixture of 1 part 5mM sulfanilic acid, 1 part 5mM N-(1-Naphthyl) ethylenediamine and 3 parts glacial acetic acid was added. After 10 minutes of incubation in the dark at room temperature, absorbance at 570nm was determined using a microplate 25 scanner (BioTek Instruments, Burlington. VT). The system was calibrated using freshly-prepared standard nitrite solutions. A linear regression line was determined from the standards, and the experimental nitrite concentrations calculated. Results are means + S.D.

WO 99/63982 PCT/US99/13264 35 Assay of Angiogenic and AntiAngiogenic Activity Conditioned media from MPM cultures were concentrated 20 fold and diafiltered using Amicon centrifugal spin filters (3KDa cut-off)(Beverly, MA). Five ptl 5 concentrated media were incorporated into equal volumes of slow-release Hydron (12% w/v in 95% ethanol) (Interferon Sciences, New Brunswick, NJ) and allowed to dry. Hydron pellets were implanted aseptically into pockets within rat corneal stromas, 2mm from the limbal vasculature, as described previously (1,2,4,9). Corneas were examined daily for seven days using a stereomicroscope and perfused with 10 colloidal carbon at the end of the observation period to provide a permanent record of the angiogenic responses. Corneas were examined histologically for any evidence of non-specific inflammation. Angiogenic responses were assessed on a graded scale as follows: No response, or slight budding of the limbal vasculature that regresses rapidly = 0; Formation of a few capillary buds and sprouts that progress less the 0.2mm from 15 the limbus, and start to regress = 1; Persistent growth of a network of capillary buds and sprouts that grow at least 1mm towards the implant, but do not reach and invade the implant = 2; strong growth of a dense network of capillary buds and sprouts that reaches and surrounds the implant = 3. Four corneal implants were prepared per test sample, and the responses summed. A maximal response thus has a score of 12, while 20 a minimal response has a score of 0. For the assay of anti-angiogenic activity, test conditioned media (20x concentrated) were combined with 20ng recombinant human

VEGF

16 5 (gift of Dr. Napoleone Ferrara, Genentech Inc., S. San Francisco, CA). The effects of the test media on the angiogenic activity of the rVEGF were then determined using the corneal bio-assay. 25 Effects of Anti-VEGF Antibodies on Macrophage Angiogenic Activity To determine the contribution of VEGF to the angiogenic activity of the MPM conditioned media, an affinity purified neutralizing polyclonal antibody to 30 VEGF (gift of Dr. Napoleone Ferrara) was used. Concentrated conditioned media WO 99/63982 PCT/US99/13264 36 prepared as described above were incubated with anti-VEGF antibody at a final concentration of 10 tg/ml at 37 0 C. for 2 hours. Controls were incubated with pre immune IgG at the same concentration. These treated media were then assayed for angiogenic activity in the rat corneal bio-assay. 5 ADP-Ribosylation of rVEGF Initial attempts to metabolically label VEGF endogenously synthesized in MPMs, using 3 2 p-NAD + were unsuccesful, as macrophages are impermeable to 10 NAD+, which cannot enter the cells and provide a substrate for the cytoplasmic ADP ribosyl transferases (32,33). We therefore used either permeabilized MPMs (data not shown) or macrophage cytoplasmic extracts to determine whether exogenous rVEGF is a substrate for macrophage ADP-ribosyl transferases. Similarly, rVEGF was tested as a substrate for cholera toxin (an arginine-specific ADP-ribosyl transferase) and for 15 pertussis toxin (a cysteine-specific ADP-ribosyl transferase) (34,35). i) Cytosolic extracts of MPMs were prepared as follows: MPMs were plated in 100mm culture dishes (10 x 106 cells per dish in 10ml medium) in RPMI1640 medium containing 10% fetal calf serum, and incubated at 370C. 20 overnight. The medium was then removed, and the cells were washed (x2) with cold PBS. The cells were then harvested by scraping into cold PBS (lml/dish). The cells were spun down at 300g and resuspended on ice in 20mM Tris-HCl pH7.5, ImM EDTA, 5mM MgCl 2 , 1mM DTT, 2mM mercaptoethanol, 1mM PMSF, 1tg/ml leupeptin, 1 lg/m aprotinin, and 0.25M sucrose (1ml/50x10 6 cells) and sonicated 25 briefly. The extract was centrifuged in the cold for 15 mins. at 1100g to remove nuclei and insoluble debris. The protein content of the extracts was determined using the Bradford method (BioRad, Richmond, CA), and the extracts were stored at 800C. until use. To determine whether these extracts were able to ADP-ribosylate rVEGF 16 5 , labeling reactions were set up containing: 500ng VEGF 16 5 , 0lpg 30 macrophage protein extract, 20mM Tris-HCl pH7.8, 20mM isoniazid, 120mM WO 99/63982 PCT/US99/13264 37 MgCl 2 , 10mM NaF, 0.02% leupeptin, 0.54mM NADP, 0.4mM isobutyl methylxanthine, 0.1% lubrol, 2mM DTT, 10mM thymidine, and 7.tCi 3 2 P-labelled

NAD

+ (800Ci/mmol)(DuPont-NEN, Wilmington, DE). After 2 hours incubation at 300C., the reaction mixture was placed on ice, and pre-cleared for 30 mins. with 10lt 5 of Protein-A/G-agarose (Santa Cruz Biotech., Santa Cruz, CA). 10ptg of a murine anti-VEGF monoclonal antibody (gift of Texas Biotechnology, Inc., Dallas, TX), was added to the supernatant, and the mixture was incubated on ice for 2 hours. 10l Protein-A/G-agarose beads were then added, and the mixture was further incubated for 2 hours. at 4 0 C. with gentle rocking. The beads were harvested by centrifugation, 10 and washed (x3) with cell lysis buffer. The beads were then incubated in an equal volume of 2x electrophoresis sample buffer (final concentration of 100mM DTT), and heated at 95 0 C. for 10 ins. to elute bound VEGF from the beads. The samples were then separated using 0.1% SDS-15% PAGE, and the fractionated proteins were transferred to a nitrocellulose membrane by semi-dry electrophoretic transfer. The 15 filters were then immunostained using anti-VEGF antibody, and the VEGF bands were detected using enhanced fluorecence detection reagents (Amersham Vistra reagents) and a Fluorimage Analyzer (Molecular Dynamics). The nitrocellulose blots were then analyzed using a PhosphorImage analyzer (Molecular Dynamics, Sunnyvale, CA), to determine the localization of 3 2 P-labeled bands. 20 ii) rVEGF 16 5 was incubated for up to 2 hours at 300C. with cholera toxin as follows: 500ng rVEGF, 250pg cholera toxin (A-subunit, Sigma Chemical Co., St. Louis, MO), in the reaction buffer described above. The reaction was terminated by the addition of an equal volume of cold 10% TCA. The precipitated 25 protein was washed (x3) with water-saturated chloroform, and finally resuspended in an equal volume of 2x PAGE sample buffer, as above. The samples were separated by SDS-PAGE, and transferred to a nitrocellulose membrane as described above. iii) 500ng rVEGF 16 5 was incubated for up to 2 hours at 30 0 C. with 30 25pg pertussis toxin (Sigma, cat. no. P-0317) in the reaction mixture described above.

WO 99/63982 PCT/US99/13264 38 Pertussis toxin was pre-activated by incubation for 30 mins. with 10mM ATP and 20mM DTT prior to addition to the VEGF reaction mixture. The reaction was terminated and analyzed as described above. 5 Effects of ADP-Ribosylation on the Angiogenic Activity of VEGF To determine whether ADP-ribosylation of VEGF modulates its bio activity as an angiogenic factor, rVEGF 16 5 was treated as described above with either cholera toxin, or macrophage cytosolic extract, but in the presence of unlabeled 10 NAD+. To facilitate the recovery of rVEGF from the reaction mixture, rather than using immunoprecipitation for the recovery of VEGF, which requires the use of harsh, denaturing conditions for the recovery of VEGF from the Protein-A/G-agarose beads, heparin-Sepharose binding was used to recover the VEGF. Following the labeling reaction, 10pl washed heparin-Sepharose beads were added, and the mixture was 15 incubated at 40C. for 4 hours with gentle agitation. The beads were then washed (x3) with 100pl 20mM Tris-HCl pH7.8 containing 0.4M. NaCl. VEGF was eluted from the beads by incubation with 20pl Tris-HCl containing 1.5M. NaCl. Recovery of VEGF was determined by specific ELISA. Control reactions were carried out in the absence of bacterial toxins and macrophage extract. To ensure that anti-angiogenic 20 activity was not present in the macrophage extracts or the cholera toxin preparations, similar labeling reactions were carried out in the absence of VEGF, and the heparin Sepharose eluates from these reactions were tested in the anti-angiogenesis assay. Figure 1 illustrates the nitrite production by MPMs. Cells were 25 incubated in DMEM/1%FCS, with or without sodium lactate (25mM), Ifny (100u/ml) and LPS (100ng/ml), L-NAME (1.5mM), or AG (1mM), as indicated. Media were harvested 8, 24 and 48 hours after challenge with IfnTy/LPS. Results are means +/ S.D. of triplicate determinations in a typical experiment. Similar results were found in at least three separate experiments. 30 WO 99/63982 PCT/US99/13264 39 Figure 2 illustrates VEGF production by A) RAW264.7 cells, and B) MPMs. Cells were incubated in DMEM/1%FCS, with or without sodium lactate (25mM), Ifny (100u/ml) and LPS (100ng/ml), L-NAME (1.5mM), or AG (1mM), as indicated. Media were harvested 18 and 48 hours after challenge with Ifny/LPS. 5 Results are means +/- S.D. of triplicate determinations in a typical experiment. Similar results were found in at least three separate experiments. Figure 3 illustrates competitive RT-PCR analysis of VEGF mRNA levels in control (non-stimulated) MPMs 24 hours following plating. Varying 10 amounts of total RNA (1-200ng) isolated from MPMs were reverse transcribed and amplified by PCR through 25 cycles in the presence of a VEGF RNA minigene (2.5pg) that amplifies using the same primers as the native VEGF mRNA, as described in Methods. The RNA minigene yields an amplified PCR product of 293bp, the native VEGF mRNA yields a 362bp fragment. The amount of total RNA that 15 yields an amplification band of the same intensity as the minigene is determined from these analyses. Figure 4 illustrate RT-PCR analysis of VEGF isoforms produced by Ifny/LPS-activated MPMs, with or without AG treatment. Total RNA isolated from 20 MPMs was reverse transcribed and amplified by PCR, as described in Methods. PCR primers were located in exons 3 and 8, resulting in the amplification of 3 PCR products corresponding to 652, 580 and 448bp. Figure 5 illustrates TNFat production by MPMs. Cells were incubated 25 in DMEM/I1%FCS, with or without sodium lactate (25mM), Ifny (100u/ml) and LPS (100ng/ml), L-NAME (1.5mM), or AG (1mM), as indicated. Media were harvested 8, 24 and 48 hours after challenge with Ifny/LPS. Results are means +/- S.D. of triplicate determinations in a typical experiment. Similar results were found in at least three separate experiments. 30 WO 99/63982 PCT/US99/13264 40 Figure 6 illustrates ADP-Ribosylation of rVEGF 16 5 by bacterial toxins and by macrophage cytosolic extract. A. rVEGF (500ng) was incubated with macrophage cytosolic extract (see Methods) in the presence of 3 2 p-NAD

+

. The total labeling reaction was analyzed on the 0.1% SDS-15% PAGE gel. B. The rVEGF 16 5 5 -macrophage cytosolic extract labeling mixture was immuno-precipitated with anti VEGF antibody, and the immunoprecipitated VEGF was analyzed by SDS-PAGE. A dominant 3 2 P-labeled band migrating in the same position as rVEGF 16 5 (determined by Western analysis of the same blot) is indicated. C. rVEGF 16 5 was incubated with cholera toxin subunit A and 32

P-NAD

+ as decribed in Methods. D. Cholera toxin 10 was incubated with 3 2

P-NAD

+ in the absence of rVEGF 16 5 . E. rVEGF 16 5 was incubated with pertussis toxin and 32

P-NAD

+

, as described in Methods.

WO 99/63982 PCT/US99/13264 41 Table 1 Relative VEGF mRNA Levels* in Macrophages Determined by Competitive RT-PCR 5 Time (hours) 4 10 24 Control (unstimulated) MPMs 1 1 1 Hypoxic MPMs 2.8 5 1.4 10 Ifny/LPS-activated MPMs 2.2 4.8 1 Iffiy/LPS-activated MPMs 2 4.7 1 + AG (1mM) Control (unstimulated) RAW cells 1 1.3 1.2 Hypoxic RAW cells 3 5.8 2.5 15 Ifny/LPS-activated RAW cells 2.4 5.4 2.2 Ifny/LPS-activated RAW cells 0.9 1.4 1.2 + AG (1mM) 20 * VEGF mRNA levels for each group are compared with the G3PDH mRNA level in the same RNA samples..

WO 99/63982 PCT/US99/13264 42 Table 2 Angiogenic and Anti-Angiogenic Responses Induced in Rat Corneas by Conditioned Media from Mouse Peritoneal Macrophages (MPMs) Cultured Under Various 5 Conditions In Vitro Macrophage Culture Conditions 1 Angiogenic Score 2 1. Normoxia 1 2. Hypoxia 9 10 3. Normoxia + Lactate (25mM) 8 4. Ifny (100U/ml))/LPS (100ng/ml) 11 5. Ifny/LPS + Aminoguanidine (1mM) 2 6. Group 2 + anti-VEGF Ab (10tg/ml) 1 7. Group 3 + anti-VEGF Ab (10g/ml) 2 15 8. Group 4 + anti-VEGF Ab (10g/ml) 4 9. rVEGF1 6 5 (20ng) 11 10. bFGF (20ng) 12 11. TNFoc (20ng) 10 12. Group 9 + anti-VEGF Ab (10ptg/ml) 2 20 13. Group 10 + anti-VEGF Ab (10g/ml) 11 14. Group 11 + anti-VEGF Ab (10pg/ml) 11 15. Group 1 + rVEGF 16 5 (20ng) 11 16. Group 5 + rVEGF 16 5 (20ng) 2 25 1 Macrophages were incubated for 48 hours under the indicated conditions, concentrated (x20) and diafiltered using Centricon 3 (3000 M.Wt. cut-off) filters (Amicon). Samples were then combined with equal volumes of Hydron (Interferon Sciences, Inc.)(12% w/v in 95% ethanol). 10il droplets were then allowed to dry on 30 the cut ends of 2mm diameter Teflon rods. These pellets were then implanted aseptically in the corneas of rats. 2 Angiogenic responses were assessed 7 days following implantation. The angiogenic score represents the sum of the graded angiogenic responses from 4 individual corneas for each test sample. A maximal response would score 12; a minimal response 0 (see 35 Methods).

WO 99/63982 PCT/US99/13264 43 Table 3 Effects of ADP-Ribosylation# on the Angiogenic Activity of rVEGF 16 5 5 Test Material Angiogenic score 1 Sham-reacted rVEGF 16 5 (20ng) 11 2. Cholera toxin-treated rVEGF 16 5 (20ng) 2 10 (Heparin-Sepharose eluate) 3. Cholera toxin control 1 (Heparin-Sepharose eluate) 4. rVEGF 16 5 (20ng) + Cholera toxin control 10 5. Macrophage cytosolic extract-treated rVEGF 16 5 (20ng) 3 15 (Heparin-Sepharose eluate) 6. Macrophage cytosolic extract control 2 7. rVEGF 1 65 (20ng) + Macrophage cytosolic extract control 11 20 # rVEGF165 was treated in a reaction mixture with either cholera toxin or macrophage cytosolic extracts, as described in the Methods section. Controls of VEGF treated in the absence of cholera toxin or macrophage cytosolic extract, were performed to determine the effects of the buffers on VEGF. Controls of the cholera toxin and macrophage cytosolic.extract incubated without VEGF were also 25 performed, to determine whether extraneous angiogenic or anti-angiogenic factors were present in these reagents. All reactions were treated with heparin-Sepharose as described in Methods, to recover the VEGF from the reaction mixtures.

WO 99/63982 PCT/US99/13264 44 Throughout this application, various publications have been referenced. The disclosures in these publications are incorporated herein by reference in order to more fully describe the state of the art. 5 References 1. Polverini PJ, Cotran RS, Gimbrone MA Jr., Unanue, ER: Activated macrophages induce vascular proliferation. Nature 1977. 269: 804-806 10 2. Koch, AE; Polverini PJ, Leibovich SJ: Induction of neovascularization by activated human monocytes. J Leukocyte Biol 1985, 37: 279 288 3. Polverini PJ: Macrophage-induced angiogenesis: A review. 15 Cytokines. 1989, 1: 54-73 4. Polverini PJ, Leibovich SJ: Induction of neovascularization and nonlymphoid mesenchymal cell proliferation by macrophage cell lines. Lab Invest 1985, 51: 635-642 20 5. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C: Macrophages and angiogenesis. J Leukocyte Biol 1994, 55: 410-422 6. DiPietro LA, Polverini PJ: Angiogenic macrophages produce the 25 angiogenic inhibitor thrombospondin-1. Am J Pathol 1993, 143: 678-684 7. Polverini PJ: The pathophysiology of angiogenesis. Crit Revs Oral Biol Med 1995, 6: 230-247 WO 99/63982 PCT/US99/13264 45 8. Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1995, 1: 27-31 9. Leibovich SJ, Polverini PJ, Shepard HM, Wiseman DM, Shively V, 5 Nuseir N: Production of angiogenic activity by human monocytes requires an L arginine/nitric oxide synthase-dependent effector mechanism. Nature 1987, 329: 630-632 \ 10. Koch A, Polverini PJ, Kunkel SL, Harlow LA, Dipietro LA, Elner VM, Elner SG, Strieter RM: Interleukin-8 as a Macrophage-Derived Mediator of 10 Angiogenesis. Science 1992, 258: 1798-1801 11. Frater-Schroder M, Risau W, Hallmann P, Gautschi R, Bohlen P: Tumor necrosis factor type-o, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc Natl. Acad Sci USA 1987, 84: 5277-5281 15 12. DiPietro LA, Polverini PJ: Angiogenic Macrophages Produce the Angiogenic Inhibitor Thrombospondin-1. Am J Pathol 1993, 143: 678-684 13. Xiong M, Lanahan M, Elson E. Leibovich SJ. P53 protein levels 20 in murine peritoneal macrophages are modulated by the inducible-nitric oxide synthase (iNOS) pathway. Mol Biol Cell 1996, 7(Suppl): 23a 14. Besner EB, Klagsbrun M: Macrophages Secrete a Heparin Binding Inhibitor of Endothelial Cell Growth Microvasc Res 1991, 42:187-197 25 15. Polverini PJ: Cytokines 1989, 1: 54-73 16. Sunderkotter C, Goebeler M Schulze-Osthoff K, Bhardwaj R, Sorg C: Macrophage-Derived Angiogenesis Factors Pharmacol Therapeut 1991, 51: 30 195-216 WO 99/63982 PCT/US99/13264 46 17. Nathan CF: Secretion of oxygen intermediates: Role in effector functions of activated macrophages. Fed Proc 1982, 41: 2206-2211 5 18. Nagashima M, Yoshiro S, Ishiwata T, Asano G: Role of vascular endothelial growth factor in angiogenesis of rheumatoid arthritis. J Rheumatol 1995, 22: 1624-1630 19. Hibbs JB Jr, Vavrin Z, Taintor RR: L-Arginine is required for the 10 expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J Immunol 1987, 138: 550-565 20. Koch AE, Cho M, Burrows JC, Polverini PJ, Leibovich SJ: Inhibition of production of monocyte/macrophage-derived angiogenic activity by 15 oxygen free-radical scavengers. Cell Biol Int Reps 1992, 16: 415-411 21. Leibovich SJ, Polverini PJ, Fong TW, Harlow LA, Koch AE: Production of angiogenic activity by human monocytes requires an L-arginine/nitric oxide-synthase-dependent effector mechanism. Proc Natl Acad Sci USA 1994, 91: 20 4190-4194 22. Knighton DR, Hunt TK, Scheuenstahl H, Halliday BJ, Werb Z, Banda MJ: Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 1984, 221: 1283-1285 25 23. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N: Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989, 244: 1306-1309 WO 99/63982 PCT/US99/13264 47 24. Connolly DT, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, Haymore BL, Leimgruber R, Feder J: Human vascular permeability factor. J Biol Chem 1989, 264: 20017-20024 5 25. Gospodarowicz D, Abraham JA, Schilling J: Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculostellate cells. Proc Natl Acad Sci. USA 1989, 86: 7311-7315 26. Berse B, Brown LF, Van de Water L, Dvorak HF, Senger DR: 10 Vascular Permeability Factor (Vascular Endothelial Growth Factor) Gene Is Expressed Differentially in Normal Tissues, Macrophages, and Tumors. Mol Biol Cell 1992, 3: 211-220 27. Sharkey AM, Charnock-Jones DS, Boocock CA, Brown, KD, 15 Smith SK: Expression of mrna for vascular endothelial growth factor in human placenta. J Reprod Fertil 1993, 99: 609-615 28. Fava RA, Olsen NJ, Spencer-Green G, Yeo TK, Berse B, Jackman RW, Senger DR, Dvorak HF, Brown LF: Vascular permeability factor/endothelial 20 growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J Exp Med. 1994, 180: 341-346 29. Torry RJ, Labarrere CA, Torry DS, Holt VJ, Faulk WP: Vascular endothelial growth factor expression in transplanted human hearts. Transplantation 25 1995, 60: 1451-1457 30. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK: Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J 30 Clin Invest 1996, 98: 482-489 WO 99/63982 PCT/US99/13264 48 31. Shima DT, Kuroki M, Deutsch U, Ng YS, Adamis AP, D'Amore PA: The mouse gene for vascular endothelial growth factor. J Biol Chem 1996, 271: 3877-3887 5 32. Zabel, DD, Feng JJ, Scheuenstahl H, Hunt TK, Hussain MZ. Lactate stimulation of macrophage-derived angiogenic activity is associated with inhibition of poly(ADP-ribose) synthesis. Lab Invest 1996, 74: 644-649. 10 33. Aktories K, Just I: In vitro ADP-ribosylation of Rho by bacterial ADP-ribosyl transferases. Methods in Enzymol. 1995, 256: 184-195 34. Okazaki IJ, Moss J: Mono-ADP-ribosylation: A reversible post translational modification of proteins. Adv Pharmacol 1996, 35: 247-280 15 35. Moss J, Vaughan M: Mechanism of action of cholaregen. Evidence for ADP-ribosyl transferase activity with arginine as an acceptor. J Biol Chem 1997, 252: 2455-2457 20 36. Leibovich SJ, Xiong M, Elson E, Sharma S, Seo C, Lanahan M: The role of macrophages in the control of angiogenesis in wound repair: Nitric oxide (NO), angiogenesisinhibitors and tumor suppresser genes. In: Bone Formation and Repair. eds. Rabie AM, Urist MR. 1997, 101-111 25 37. Beutler B, Cerami A: Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 1986, 320: 584-588 38. Burchett SK, Weaver WM, Westall JA, Larsen A, Kronheim S, Wilson CB: Regulation of tumor necrosis factor/cachectin and Il-1 secretion in 30 human mononuclear phagocytes. J Immunol 1988, 140: 3473-3481 WO 99/63982 PCT/US99/13264 49 39. Mukaida N, Harada A, Yasumoto K, Matsushima K: Properties of pro-inflammatory cell type-specific leukocyte chemotactic cytokines, interleukin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF). Microbiol Immunol 5 1992, 36: 773-789 40. Shweiki D, Itin A, Soffer D, Keshet E: Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. Nature 1992, 359: 843-845 10 41. Ladoux A, Frelin C: Hypoxia is a strong inducer of vascular endothelial growth factor messenger RNA expression in the heart. Biochem Biophys Res Comms 1993, 195: 1005-1010 15 42. Goldberg MA, Schneider TJ: Similarities between the oxygen sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem 1994, 269: 4355-4359 43. Shima DT, Adamis AP, Ferrara N, Yeo KT, Allende R, 20 Folkman,J, D'Amore P: Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterisation of vascular endothelial growth factor (VEGF) as the mitogen. Mol Med 1995, 1:182-193 44. Levy AP, Levy NS, Wegner S, Goldberg MA: Transcriptional 25 regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem 1995, 270: 13333-13340 45. Brune B, Lapetina EG: Activation of a cytosolic ADP-ribosyl transferase by nitric oxide-generating agents. J Biol Chem 1989, 264: 8455-8458 30 WO 99/63982 PCT/US99/13264 50 46. Molina-Vedia L, McDonald B, Reep B, Brune B, DiSilvio M, Billiar TR, Lapetina EG Nitric oxide induced S-nitrosylation of glyceraldehyde-3 phosphate dehydrogenase inhibits enzymatic activity and increases endogenous ADP ribosylation. J Biol Chem 1992, 267: 24929-24932 5 47. Feng JJ, Hunt TK, Ghani P, Hussain MZ: Macrophage-derived angiogenic activity potential can be reversibly inhibited by ADP-ribosylation. Wound Repair Regeneration 1997, 5: A 111 10 48. West RE, Moss J, Vaughan M, Liu T-Y: Pertussis toxin catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site. J Biol Chem 1985, 260: 14428-14430 49. Leibovich SJ, Golczewski J: Expression of inducible nitric oxide 15 synthase (iNOS) is required for production of angiogenic activity by murine macrophages and IC-21 cells. Mol Biol Cell (Suppl): 373a 50. Upperman JS, Leibovich SJ, Xiong M, Golczewski,J, Deitch EA Diphenyleneiodonium (DPI), an inhibitor of nucleotide-requiring flavoproteins, 20 inhibits the production of macrophage-derived angiogenic activity. Surg Forum 1996, 47: 708-710. 51. Good DJ, Polverini PJ, Rastinejad F, LeBeau MM, Lemons RS, Frazier WA, Bouck NP: A tumor suppresser-dependent inhibitor of angiogenesis is 25 immunologically indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad Sci. USA 1990, 87: 6624-6628. 52. Angiolillo AL, Sgadari C, Taub DD, Liao F, Farber JM, Maheshawari S, Kleinman HK, reaman GH, Tosato G: Human interferon-inducible WO 99/63982 PCT/US99/13264 51 protein 10 is a potent inhibitor of angiogenesis in vivo. J Exp Med 1995, 182: 155 159 53. Banasik M, Ueda K: Inhibitors and activators of ADP 5 ribosylation reactions. Mol. Cell Biochem 1994, 138: 185-197. Throughout this disclosure, applicant will suggest various theories or mechanisms by which applicant believes the components in the therapeutic wound healing compositions function to inhibit adenosine diphosphate-ribosylation of 10 vascular endothelial growth factor. While applicant may offer various mechanisms to explain the present invention, applicant does not wish to be bound by theory. These theories are suggested to better understand the present invention but are not intended to limit the effective scope of the claims. 15 While the invention has been particularly described in terms of specific embodiments, those skilled in the art will understand in view of the present disclosure that numerous variations and modifications upon the invention are now enabled, which variations and modifications are not to be regarded as a departure from the spirit and scope of the invention. Accordingly, the invention is to be broadly 20 construed and limited only by the scope and spirit of the following claims.

Claims

1. A method for healing a wound in a mammal which comprises the steps of 5 (A) providing a therapeutic wound healing composition comprising a therapeutically effective amount of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; and (B) contacting the therapeutic wound healing composition with a wound in a 10 mammal.

2. The method according to claim 1, wherein the mammal is man.

3. The method according to claim 1, wherein the inhibitor of mono 15 adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin KI, Vitamin K2, Vitamin K3, Vitamin K4, Vitamin KS, Vitamin K6, Novobiocin, m-iodo benzyl guanidine, nicotinamide, coumermycin, dicoumarol, and silybin. 20

4. The method according to claim 3, wherein the inhibitor of mono adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin KI, Vitamin K3, Novobiocin, and silybin.

5. The method according to claim 1, wherein the inhibitor of mono 25 adenosine diphosphate-ribosyl transferase is present in the therapeutic wound healing composition in an amount from about 0.1% to about 10%, by weight of the therapeutic wound healing composition. WO 99/63982 53 PCT/US99/13264

6. The method according to claim 1, wherein the wound is selected from the group consisting of pressure ulcers, decubitus ulcers, diabetic ulcers, and burn injuries. 5

7. The method according to claim 1, wherein the therapeutic wound healing composition further comprises a pharmaceutically acceptable carrier.

8. A wound healing composition which comprises: (A) a therapeutically effective amount of an inhibitor of mono-adenosine 10 diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; and (B) a pharmaceutically acceptable carrier.

9. The wound healing composition according to claim 8, wherein the 15 inhibitor of mono-adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin KI, Vitamin K2, Vitamin K3, Vitamin K4, Vitamin K5, Vitamin K6, Novobiocin, m-iodo benzyl guanidine, nicotinamide, coumermycin, dicoumarol, and silybin. 20

10. The method according to claim 9, wherein the inhibitor of mono adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin K1, Vitamin K3, Novobiocin, and silybin.

11. The wound healing composition according to claim 8, wherein the 25 inhibitor of mono-adenosine diphosphate-ribosyl transferase is present in the therapeutic wound healing composition in an amount from about 0.1% to about 10%, by weight of the therapeutic wound healing composition.

12. A method for treating diaper dermatitis in a human which 30 comprises the steps of: WO 99/63982 PCT/US99/13264 54 (A) providing a therapeutic diaper dermatitis wound healing composition comprising: (a) a therapeutically effective amount of an inhibitor of mono-adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of 5 vascular endothelial growth factor; (b) a buffering agent to maintain the pH of dermatitis in a range from about 5 to about 8; and (c) an anti-inflammatory agent; and (B) contacting the therapeutic diaper dermatitis wound healing composition 10 with diaper dermatitis in a human.

13. The method according to claim 12, wherein the inhibitor of mono adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin Ki, Vitamin K2, Vitamin K3, Vitamin K4, Vitamin KS, Vitamin K6, 15 Novobiocin, m-iodo benzyl guanidine, nicotinamide, coumermycin, dicoumarol, and silybin.

14. The method according to claim 13, wherein the inhibitor of mono adenosine diphosphate-ribosyl transferase is selected from the group consisting of 20 Vitamin KI, Vitamin K3, Novobiocin, and silybin.

15. The method according to claim 12, wherein the inhibitor of mono adenosine diphosphate-ribosyl transferase is present in the therapeutic wound healing composition in an amount from about 0.1% to about 10%, by weight of the 25 therapeutic wound healing composition.

16. The method according to claim 12, wherein the buffering agent is selected from the group consisting of citric acid-sodium citrate, phosphoric acid sodium phosphate, and acetic acid-sodium acetate. 30 WO 99/63982 PCT/US99/13264 55

17. The method according to claim 12, wherein the anti-inflammatory agent is selected from the group consisting of ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine, 5 diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine, salsalate, diflunisal, salicylsalicylic acid, choline magnesium trisalicylate, flunisolide, triamcinoline, triamcinoline acetonide, beclomethasone diproprionate, betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone, predinisone, methyl prednisolone, and prednisolone. 10

18. The method according to claim 12, wherein the therapeutic wound healing composition further comprises a pharmaceutically acceptable carrier.

19. A therapeutic dermatological-wound healing composition useful to 15 minimize and treat diaper dermatitis which comprises a therapeutically effective amount of: (1) a therapeutic wound healing composition comprising an inhibitor of mono adenosine diphosphate-ribosyl transferase to inhibit adenosine diphosphate-ribosylation of vascular endothelial growth factor; 20 (2) a buffering agent to maintain the pH of dermatitis in a range from about 5 to about 8; and (3) an anti-inflammatory agent.

20. The composition according to claim 19, wherein the inhibitor of 25 mono-adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin KI, Vitamin K2, Vitamin K3, Vitamin K4, Vitamin K5, Vitamin K6, Novobiocin, m-iodo benzyl guanidine, nicotinamide, coumermycin, dicoumarol, and silybin. WO 99/63982 PCT/US99/13264 56

21. The composition according to claim 20, wherein the inhibitor of mono-adenosine diphosphate-ribosyl transferase is selected from the group consisting of Vitamin KI, Vitamin K3, Novobiocin, and silybin. 5

22. The composition according to claim 19, wherein the inhibitor of mono-adenosine diphosphate-ribosyl transferase is present in the therapeutic wound healing composition in an amount from about 0.1% to about 10%, by weight of the therapeutic wound healing composition. 10

23. The composition according to claim 19, wherein the buffering agent is selected from the group consisting of citric acid-sodium citrate, phosphoric acid-sodium phosphate, and acetic acid-sodium acetate.

24. The composition according to claim 19, wherein the anti 15 inflammatory agent is selected from the group consisting of ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine, salsalate, diflunisal, salicylsalicylic acid, choline magnesium trisalicylate, flunisolide, 20 triamcinoline, triamcinoline acetonide, beclomethasone diproprionate, betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone, predinisone, methyl prednisolone, and prednisolone.

25. The composition according to claim 19, wherein the therapeutic 25 wound healing composition further comprises a pharmaceutically acceptable carrier.