(12) United States Patent (10) Patent No.: US 7,604,797 B2 Hicks (45) Date of Patent: Oct

USOO7604797B2

(12) United States Patent (10) Patent No.: US 7,604,797 B2 Hicks (45) Date of Patent: Oct. 20, 2009

(54) COMPOSITIONS AND METHODS FOR 4490,529 A 12/1984 Rosowsky TREATING BURNS 4,632,920 A * 12/1986 Spillert et al...... 514,158 5,674,912 A * 10/1997 Martin ...... 514,724 (75) Inventor: Terry Lee Hicks, Rego Park, NY (US) 5,863,938 A 1, 1999 Martin

6,060,501 A * 5/2000 Wachtel et al...... 514,423 (73) Assignee: BioMechanisms Inc., Rego Park, NY 6,093,743 A * 7/2000 Lai et al...... 514,599 (US) 2004/0014782 A1 1/2004 Krause ... 514/313 *) NotOt1Ce: Subjubject to anyy d1Sclaimer,disclai theh term off thithis patent is extended or adjusted under 35 U.S.C. 154(b) by 4 days. OTHER PUBLICATIONS (21) Appl. No.: 11/012,210 Bronaugh etal (Journal of Investigative Dermatology, 1978, vol. 71, No. 4, pp. 263-265).* (22) Filed: Dec. 16, 2004 Werner etal (Regional Anesthesia and Pain Medicine, 2002, vol. 27. (65) Prior Publication Data No. 3, pp. 254-260).* International Search Report for Application No. PCT/US04/31917; US 2005/0281820 A1 Dec. 22, 2005 Dated: Apr. 25, 2005; International Searching Authority/USPTO. Written Opinion of the international Searching Authority for Appli Related U.S. Application Data cation N. PCT/US04/31917: Dated: Apr. 25, 2005; International (63) Continuation of application No. PCT/US2004/ Searching Authority/USPTO. 031917, filed on Sep. 30, 2004. * cited by examiner (60) Provisional application No. 60/506,745, filed on Sep. Primary Examiner D L. Jones 30, 2003. (74) Attorney, Agent, or Firm. The Law Offices of Andrew (51) Int. Cl. D. Fortney: Andrew D. Fortney: William K. Nelson A6 IK3I/74 (2006.01) (57) ABSTRACT (52) U.S. Cl...... 424/78.06; 424/78.03; 424/78.05: 424/78.07: 514/886; 514/887 (58) Field of Classification Search ...... 424/1.11, The present invention provides compositions and methods for 424/1.65, 9.19.2, 78.03, 78.05, 78.06, 78.07; treating burns comprising administering to a burn area of a 514/886, 887, 889,903 subject in need thereof of a therapeutically effective amount See application file for complete search history. of a composition comprising an anti-cytokine or anti-inflam matory agent or a functional derivative thereof, and a phar (56) References Cited maceutically acceptable excipient. U.S. PATENT DOCUMENTS 4,132,572 A * 1/1979 Parant et al...... 148.253 43 Claims, 10 Drawing Sheets U.S. Patent Oct. 20, 2009 Sheet 1 of 10 US 7,604,797 B2

Figure 1A

First-degree burn

Second-degree burn

Subcutaneous tissue Third-degree burn

Bood vesses Nerve hair folice U.S. Patent Oct. 20, 2009 Sheet 2 of 10 US 7,604,797 B2

Figure 1B

Skin Cells

BOOd Wesse White Blood Cels

Blood Flow

intact skin and underlying blood vessels are shown, White blood cells are circulating in the blood a concentration of about 4 million cells per milliliter of blood. U.S. Patent Oct. 20, 2009 Sheet 3 of 10 US 7,604,797 B2

Figure 1C

The acute burn injury causes immediate mechanica destruction of skin cells, generating an ulceration. U.S. Patent Oct. 20, 2009 Sheet 4 of 10 US 7,604,797 B2

Figure 1D

BURN UCERAON

TNFO, IL-1, IL-6, -8

The acute burn injury generates immediate inflammation which results in production of the inflammatory cytokines tumor necrosis factor alpha (TNFo), Interleukin(-)-1, IL-6, and L-8. These cytokines originate from cells in the skin and cells in the deeper tissues. U.S. Patent Oct. 20, 2009 Sheet 5 of 10 US 7,604,797 B2

Figure 1E

White BOOd Cels

Under the influence of IL-8 (a white blood cell attractant), white blood cells in the blood Vesses are Caled into the tissues. The White Ces dissolve tiny parts of the blood vessel walls in order to leave the circulation and infiltrate the tissues to arrive at the burn site. The white cells attempt to repair the damaged tissues and fight infection. U.S. Patent Oct. 20, 2009 Sheet 6 of 10 US 7,604,797 B2

Figure 1 F

Edema Fluid escapes through blood vessels that are made porous through the actions of the cytokines

Under the influence of TNFO and IL-1, the Cells that line the blood vessels lose their integrity, and this results in pore formation along the blood vessels. This results in leakage of plasma from the blood vessels that causes edema fluid to form in the surrounding tissues. U.S. Patent Oct. 20, 2009 Sheet 7 of 10 US 7,604,797 B2

Figure 1G

Blood vesses diate

TNFo, IL-1, IL-6, and IL-8 are joined by other inflammatory substances, such as nitric oxide (NO) and free radicals (also called reactive oxygen intermediates or RO). One important effect of these substances is dilation of the blood vessels that causes low blood pressure, and this can result in reduced blood pressure and even shock. U.S. Patent Oct. 20, 2009 Sheet 8 of 10 US 7,604,797 B2

Figure 1H

TNFo, IL-1, IL-6, and IL-8, and the other inflammatory substances, nitric oxide (NO) and free radicals or reactive oxygen intermediates (ROI) can gain access to the bloodstream and cause systemic inflammation, U.S. Patent Oct. 20, 2009 Sheet 9 of 10 US 7,604,797 B2

Figure 1

Blood vessels (vessel dilation and shock), brain, heart, kidneys, lungs, others.

Once TNFO, IL-1, IL-6, IL-8, nitric oxide (NO) and free radicais (ROI) enter the bloodstream, they can cause systemic inflammation. This can damage any organ system in the body, including the organs listed above. This also results in fever, U.S. Patent Oct. 20, 2009 Sheet 10 of 10 US 7,604,797 B2

Figure 1 J

BURN UCERATON

HR341g is thought to block production of the pro-inflammatory molecules TNFa., IL-1, I-6, and IL-8 that initiate the inflammatory process. This not only reduces the local systemic damage that these molecules can cause, but it also blocks the formation of secondary inflammatory molecules like NO and ROl. US 7,604,797 B2 1. 2 COMPOSITIONS AND METHODS FOR Chemical burns can be caused by various irritants and TREATING BURNS poisons, including strong acids and alkalis, phenols and cresols (organic solvents), mustard gas, and phosphorus. CROSS REFERENCE TO RELATED Chemical burns can cause tissue death that can slowly spread APPLICATIONS for hours after the burn. Radiation burns can becaused by nuclear weapons, nuclear This application is a continuation of PCT/US2004/031917 accidents, laboratory exposure, accidents during X-ray radia application entitled. COMPOSITIONS AND METHODS tion chemotherapy, and over-exposure to Sun. Radiation FORTREATING BURNS, filed Sep. 30, 2004, which claims burns can cause inflammation, edema, ulcerations, damage to priority to provisional U.S. application No. 60/506,745, filed 10 underlying endothelium and other cell types, as well as Sep. 30, 2003, the disclosures of which are hereby incorpo mutagenesis resulting in cancer, especially hematologic rated in their entirety by reference. malignancies. After suffering a burn injury, the affected individual can FIELD OF THE INVENTION have usually has severe protein, muscle, and fat wasting in the 15 area of the burn (1). Indeed, loss of up to 20% of body protein This invention relates generally to beneficial effects may occur in the first two weeks following a third degree or obtained via administration of a pharmaceutical composition deep tissue burn injury (2). Increased oxygen consumption, for the treatment ofburns and skin wounds in warm-blooded metabolic rate, urinary nitrogen excretion, fat breakdown and animals, such as mammals and especially humans. In particu steady erosion of body mass are all directly related to burn lar, the present invention is concerned with inflammation size. A return to normal levels as the burn wound heals gradu associated tissue damage and is particularly directed to pro ally restores chemical balance, temperature and pH. To date phylactic and therapeutic methods for treating localized and no one has produced a treatment capable of preventing the life systemic inflammation associated with burns, as well as the threatening inflammatory response a burn victim can endure. treatment of a variety of diseases associated with the inflam Edema In General mation that ensues from a burn. 25 Edema is the term generally used to describe the accumu lation of excess fluid in the intercellular (interstitial) tissue BACKGROUND OF THE INVENTION spaces or body cavities. Edema may occur as a localized phenomenon Such as the Swelling of a leg when the venous Burns outflow is obstructed; or it may be systemic as in congestive Burns are among the oldest, most complex and painful 30 heart failure or renal failure. When edema is severe and gen injuries known. Dating to antiquity, humans have been bat eralized, there is diffuse Swelling of all tissues and organs in tling the devastating effects of burns. Burns are the second the body and particularly pronounced areas are given their leading cause of accidental death in the United States, with own individual names. For example, collection of edema in postburn care being traumatic, painful, lengthy and emotion the peritoneal cavity is known as “ascites'; accumulations of ally draining for the patient. In fact, it has been estimated that 35 fluid in the pleural cavity are termed plueral effusions; and over five million people are involved in burn accidents in the edema of the pericardial sac is termed “pericardial effusion' United States each year. Approximately 150,000 of these or “hydropericardium'. Non-inflammatory edema fluid such patients are hospitalized and over 6000 of these die each year as accumulates in heart failure and renal disease is protein (1). poor and referred to as a “transudate'. In contrast, inflamma Thermal burns are by far the most common types of burns. 40 tory edema related to increased endothelial permeability is Although the skin is usually the part of the body that is protein rich and is caused by the escape of plasma proteins burned, the tissues under the skin can also be burned, and (principally albumin) and polymorphonuclear leukocytes internal organs can be burned even when the skin is not. For (hereinafter “PMNs) to form an exudate. example, drinking a very hot liquid or caustic Substance Such Edema, whether inflammatory or non-inflammatory in as acid can burn the esophagus and stomach. Inhaling Smoke 45 nature, is thus an abnormality in the fluid balance within the or hot air from a fire in a burning building can burn the lungs. microcirculation which includes the Small arterioles, capil Tissues that are burned may die. When tissues are damaged by laries, and post-capillary Venules of the circulatory system. a burn, fluid may leak from blood vessels (capilliary perme Normal fluid balance and exchange is critically dependent on ability), causing Swelling or edema. In an extensive burn, loss the presence of an intact and metabolically active endothe of a large amount of fluid from abnormally leaky blood ves 50 lium. Normal endothelium is a thin, squamous epithelium sels can cause shock. In shock, blood pressure decreases so adapted to permit selective, rapid exchange of water and much that too little blood flows to the brain and other vital Small molecules between plasma and interstitium; but one Organs. which limits the passage of many plasma proteins. Electrical burns may be caused by a temperature of more A variety of different disturbances can induce a condition than 9,000°F., generated by an electric current when it passes 55 ofedema. These include: an elevated venous hydrostatic pres from the electrical source to the body; this type of burn, sure which may be caused by thrombosis of a vein or any Sometimes called an electrical arc burn, usually completely other venous obstruction, heart failure; hypoproteinemia with destroys and chars the skin at the currents point of entry into reduced plasma oncotic pressure resulting from either inad the body. Because the resistance (the body’s ability to stop or equate synthesis or increased loss of albumin; increased slow the currents flow) is high where the skin touches the 60 osmotic pressure of the interstitial fluid due to abnormal current’s source, much of the electrical energy is converted accumulation of sodium in the body because renal excretion into heat there, burning the surface. Most electrical burns also of sodium cannot keep pace with the intake; failure of the severely damage the tissues under the skin. These burns vary lymphatics to remove fluid and protein adequately from the in size and depth and may affect an area much larger than that interstitial space; an increased capillary permeability to fluids indicated by the area of injured skin. Large electrical shocks 65 and proteins as occurs in the inflammatory response to tissue can paralyze breathing and disturb heart rhythm, causing injury; and an increased mucopolysaccharide content within dangerously irregular heartbeats. the interstitial spaces. US 7,604,797 B2 3 4 Currently accepted therapeutic treatments for edema The present invention provides a method for treating burns include those biogenic and synthetic pharmacological agents comprising administering to a burn area of a subject in need used to treat generalized inflammations, of which edema is thereof a therapeutically effective amount of a composition just one clinical manifestation. Such agents are said to inhibit comprising HR341g or a functional derivative thereof; and a the synthesis of pro-inflammatory molecules; and can include 5 pharmaceutically acceptable excipient. Such agents as aspirin, ibuprofen (salicylates and propionate In one aspect, the present invention relates to methods of derivatives), Steroids, and anti-histamines. These agents have controlling or alleviating pain by reducing the severity of a wide scale of effectiveness and, in general, are most valu inflammation and edema associated with a burn comprising able in the treatment of minor inflammatory problems that administering to a subject in need thereof a therapeutically produce only minor, localized edemas. There are few, if any, 10 effective amount of a pharmaceutical composition compris agents that are therapeutically effective in the treatment of ing an anti-cytokine or anti-inflammatory agent or a func severe, local and systemic edemas. Furthermore, as far as is tional derivative thereof, and a pharmaceutically acceptable known, there is no effective agent in present use as a prophy excipient, wherein said pharmaceutical composition inhibits lactic against these conditions. Also, albumin infusion and one or more components of the inflammatory pathway. congestive heart failure medications are useful in treatment of 15 In one aspect, the present invention relates to methods of edema when used appropriately. controlling or alleviating pain by reducing the severity of Current Treatments for Thermally Induced Burns pulmonary edema associated with a burn comprising admin Current treatments for thermally induced burns include the istering to a subject in need thereofatherapeutically effective use of topical agents and various Surgical procedures. The amount of a pharmaceutical composition comprising an anti topical agents that are used to treat burns are limited. Repre cytokine oranti-inflammatory agent or a functional derivative sentative examples of Such topical agents include, without thereof, and a pharmaceutically acceptable excipient, limitation, Bacitracin, Polymyxin B Sulfate, Neomycin, wherein said pharmaceutical composition inhibits one or Polysporin/Neosporin, Povidone, Silver Sulfadiazine, Nitro more components of the inflammatory pathway. fiura sp., Gentamicin, Manfenide Acetate, Nystatin, Sodium The present invention relates to methods of controlling or Hypochlorite Solution, Silver Nitrate, TAB Solution, and 25 alleviating pain by reducing the severity of inflammation and Chlorhexadine Solution. However, none of these drugs stops edema associated with a burn comprising administering to a edema. subject in need thereofatherapeutically effective amount of a Due to the unacceptable rate and risk of infections from pharmaceutical composition comprising HR341g or a func using only topical treatments (without the removal of the tional derivative thereof, and a pharmaceutically acceptable burned tissues), procedures called escharotomy and debride 30 excipient, wherein said pharmaceutical composition inhibits ment were introduced. Escharotomy literally means cutting a one or more components of the inflammatory pathway. hole in the eschar, the thick, rigid barrier of burntissue. It is an The present invention also relates to a method for promot emergency treatment for any full thickness, and almost invari ing rapid healing and/or regeneration of damaged tissues ably, circumferential burn to the dermis. It is relevant particu resulting from a burn comprising administering to a Subject in larly to the neck, thorax and extremities. Burned skin is called 35 need thereofatherapeutically effective amount of a pharma eschar. Debridement is the removal of eschar tissue. Skin ceutical composition comprising an anti-cytokine or anti grafts are layers of skin, which are taken from a Suitable donor inflammatory agent or a functional derivative thereof, and a area of a patient and transplanted to a recipient area of dam pharmaceutically acceptable excipient, wherein said pharma aged skin. Using debridement alone, the rate of infection is ceutical composition promotes rapid healing and/or regenera still extremely high but with the use of skin grafts the infec 40 tion of damaged tissues while retaining the original compo tion rate is lowered. Pig skin and/or allografts may be used sition of the tissue and minimizing complications and instead of the patients own skin. Debridement and skin grafts scarring associated with a burn. in their present form, however, do not completely restore the The present invention also relates to a method for promot function of healthy skin. The transplanted skin lacks oil ing rapid healing and/or regeneration of damaged tissues glands, Sweat glands, hair follicles, and have no nerve endings 45 resulting from a burn comprising administering to a Subject in at the injury site(s). Furthermore, the grafted skin is prone to need thereofatherapeutically effective amount of a pharma deformities such as hypertrophic scarring. Currently it takes ceutical composition comprising HR341g or a functional many months or even years to complete these extremely derivative thereof, and a pharmaceutically acceptable excipi painful procedures. ent, wherein said pharmaceutical composition promotes In view of the above, there is continuing need in the art to 50 rapid healing and/or regeneration of damaged tissues while develop better compositions and methods for treating the retaining the original composition of the tissue and minimiz inflammation with edema that is associated with all forms of ing complications and Scarring associated with a burn. burns. The methods and compositions of the present inven In another aspect, the present invention also relates to a tion provide for the first time a reproducible means for ame method for preventing or ameliorating the adverse affects liorating and/or treating the negative effects associated with 55 associated with controlled thermal induced skin damage burns by blocking one or more of components of the inflam employed in Scar and tattoo removal, cancer excisions, cau matory pathway. The inventors have satisfied these and other tery excision of polyps, ulcers, treatment of decubitus ulcers long felt needs with the following invention. (bedsores), acne, cutaneous fungal infections comprising administering to a subject in need thereof a therapeutically SUMMARY OF THE INVENTION 60 effective amount of a pharmaceutical composition compris ing an anti-cytokine or anti-inflammatory agent or a func The present invention provides a method for treating burns tional derivative thereof, and a pharmaceutically acceptable comprising administering to a burn area of a Subject in need excipient, wherein said pharmaceutical composition pro thereof a therapeutically effective amount of a composition motes rapid regeneration of damaged tissues while retaining comprising an anti-cytokine or anti-inflammatory agent or a 65 the original composition of the tissue and minimizing com functional derivative thereof, and a pharmaceutically accept plications and Scarring associated with the thermally induced able excipient. burn in one or more of the recited conditions. US 7,604,797 B2 5 6 The present invention relates to methods of preventing or For each of the above-recited methods of the present inven ameliorating blistering or pain associated with overexposure tion, the therapeutically effective amount of one or more an to Sun comprising administering to a subject in need thereof a anti-cytokine or anti-inflammatory agents, one or more therapeutically effective amount of a pharmaceutical compo antagonists to the enzyme dihydrofolate reductase, and/or sition comprising an anti-cytokine or anti-inflammatory one or more antagonists to the enzyme enolase may be admin agent or a functional derivative thereof, and a pharmaceuti istered to a subject in need thereof in conjunction with a cally acceptable excipient. therapeutically effective amount of one or more anti-inflam In yet another aspect, the present invention relates to a matory compounds and/or a therapeutically effective amount method for preventing or ameliorating the deleterious inflam of one or more immunomodulatory agents. matory response and/or the adverse sequellae associated with 10 In certain embodiments of the method of the present inven controlled therapeutic thermal induced skin damage tion, the anti-inflammatory compound or immunomodulatory employed in the use of lasers for the treatment of medical drug comprises interferon; interferon derivatives comprising conditions and the use of induced thermal injury in various betaseron, beta.-interferon; prostane derivatives comprising cosmetic procedures comprising administering to a Subject in iloprost, cicaprost; glucocorticoids comprising cortisol, pred need thereofatherapeutically effective amount of a pharma 15 nisolone, methyl-prednisolone, dexamethasone; immunSup ceutical composition comprising an anti-cytokine or anti pressives comprising cyclosporine A, methoXSalene, Sul inflammatory agent or a functional derivative thereof, and a fasalazine, azathioprine, methotrexate; lipoxygenase pharmaceutically acceptable excipient, wherein said pharma inhibitors comprising zileutone, MK-886, WY-50295, ceutical composition prevents or ameliorates the deleterious SC-45662, SC-41661A, BI-L-357; leukotriene antagonists; inflammatory response and/or the adverse sequellae associ peptide derivatives comprising ACTH and analogs thereof. ated with such controlled therapeutic thermal induced skin soluble TNF-receptors; anti-TNF-antibodies; soluble recep damage. tors of interleukins or other cytokines; antibodies against In another aspect of the present invention, a method is receptors of interleukins or other cytokines, T-cell-proteins; provided for the use of pharmaceutical compositions com and calcipotriols and analogues thereof taken either alone or prising HR341g or a functional derivative thereof to diminish 25 in combination. pain or inflammation comprising blocking one or more com In yet another aspect of the invention, a method is provided ponents of the inflammatory pathway. for Suppressing or modulating the immune system in a mam In another aspect of the present invention, a method is malian patient in need of Such immunosuppression compris provided for the use of a synthetic drug comprising an anti ing administering to said patient an immunosuppressing cytokine or anti-inflammatory agent or functional derivative 30 effective amount of a therapeutically effective amount of a thereof to diminish pain or inflammation associated with a pharmaceutical composition comprising an anti-cytokine or burn comprising blocking one or more components of the anti-inflammatory agent or a functional derivative thereof; inflammatory pathway. and a pharmaceutically acceptable excipient. In certain specific embodiments, each of the above-recited In yet another aspect of the invention, a method is provided methods are accomplished by the administration to a subject 35 for Suppressing or modulating the immune system in a mam in need thereof of a therapeutically effective amount of one or malian patient in need of Such immunosuppression compris more antagonists or inhibitors to one or more enzymes or ing administering to said patient an immunosuppressing components of the inflammatory pathway wherein adminis effective amount of a therapeutically effective amount of a tration of the enzyme antagonist or inhibitor is Sufficient to pharmaceutical composition comprising HR341g or a func block one or more components of the inflammatory pathway. 40 tional derivative thereof, and a pharmaceutically acceptable While not intended to be limited to any particular mechanism excipient. of action, the specific enzymes or components of the inflam In yet another aspect of the invention, a method is provided matory pathway which may be inhibited using each of the for Suppressing the synthesis of potentially harmful inflam aforementioned methods of the present invention include, matory molecules comprising cytokines (for example, IL-1, interalia, dihydrofolate reductase, enolase, Interleukin-1beta 45 IL-2, IL-8, IL-12, IL-18, TNF), nitric oxide, reactive oxygen converting enzyme (ICE), tumor necrosis factor alpha con intermediates (ROI), leukotrenes, and/or prostaglandins, or Verting enzyme (TACE), nitric oxide synthase, thromboxane any one or more of the known biological molecules involved synthase, cyclooxygenase, denylate cyclase, histone deacety in inflammatory signal transduction pathways, etc. in a mam lase, elastase, proteinase 3, thrombin, or any combination malian patient in need of Such immunosuppression compris thereof. 50 ing administering to said patient an immunosuppressing In one specific embodiment, each of the above-recited effective amount of a pharmaceutical composition compris methods are accomplished by the administration to a subject ing an anti-cytokine or anti-inflammatory agent or a func in need thereof of a therapeutically effective amount of one or tional derivative thereof, and a pharmaceutically acceptable more antagonists to the enzyme dihydrofolate reductase Suf excipient, wherein said pharmaceutical composition Sup ficient to block one or more components of the inflammatory 55 presses the synthesis of cytokines, or any one or more of the pathway. In one embodiment, the therapeutically effective known biological molecules involved in the activation of amount of one or more antagonists to the enzyme dihydro inflammatory signal transduction pathways leading to a folate reductase is sufficient to block one or more components blockade of inflammation or reduced immune response, or a of the glycolytic pathway. combination thereof. In another specific embodiment, each of the above-recited 60 In yet another aspect of the invention, a method is provided methods are accomplished by the administration to a subject for Suppressing the synthesis of potentially harmful inflam in need thereof of a therapeutically effective amount of one or matory molecules comprising cytokines (for example, IL-1, more antagonists to the enzyme enolase Sufficient to block IL-2, IL-6, IL-8, IL-12, IL-18, TNF), nitric oxide, reactive one or more components of the inflammatory pathway. In one oxygen intermediates (ROD, leukotrenes, and/or prostaglan embodiment, the therapeutically effective amount of one or 65 dins, or any one or more of the known biological molecules more antagonists to the enzyme enolase is sufficient to block involved in inflammatory signal transduction pathways, etc. one or more components of the glycolytic pathway. in a mammalian patient in need of Such immunosuppression US 7,604,797 B2 7 8 comprising administering to said patient an immunosup disarray because of thermal injuries: cachexia, diarrhea, pressing effective amount of a pharmaceutical composition encephalopathy, myglobulinuria, and neurities. comprising HR341g or a functional derivative thereof; and a In yet another aspect of the invention, a method is provided pharmaceutically acceptable excipient, wherein said pharma for modulating expression of major histocompatibility com ceutical composition Suppresses the synthesis of cytokines, or 5 plex molecules in a mammalian patient in need of Such any one or more of the known biological molecules involved inflammatory-suppression comprising administering to said in the activation of inflammatory signal transduction path patient an inflammatory-suppression effective amount of a ways leading to a blockade of inflammation or reduced therapeutically effective composition comprising HR341g or immune response, or a combination thereof. a functional derivative thereof, and a pharmaceutically 10 acceptable excipient, wherein said pharmaceutical composi In yet another aspect of the invention, a method is provided tion modulates expression of major histocompatibility com for ameliorating the diseases associated with inflammatory plex molecules. mediators and the systemic response to a burn injury. The In another embodiment, the pharmaceutical compositions initial burn or inflammation and edema involves oxidant and of the present invention are thus useful to treat the pain asso arachidonic acid metabolites, which trigger neutrophils and 15 ciated with and/or prevent a disease or disorder often accom macrophages to release cytokines, including, but not limited panying a burn wherein said disease or disorder is selected to, tumor necrosis factor, IL-1, IL-2, IL-8, IL-12, IL-18, as from the group consisting of myocardialischemia, tissue and well as nitric oxide. Endotoxins from pathogens in the wound muscle-associated ischemia, extremity-associated ischemia, and/or the gastrointestinal tract initiate and enhance inflam stroke, sepsis, amyotrophic lateral Sclerosis (ALS), seizures, mation and can result in the translocation of microorganisms extension of strokes after initial tissue damage, functional across the gut and generate pathology at distant sites which brain damage secondary to primary and secondary brain would otherwise be unaffected by the trauma. The exagger tumors, local brain damage secondary to meningitis or brain ated response is called the “two hit hypothesis, but “after abscess, viral meningitis, viral encephalitis, and/or local burn' is more descriptive. The post-burn septic response is brain damage secondary to trauma, transplantation of organs caused by excessive inflammatory mediators derived from the 25 or tissue, graft-Versus-host diseases brought about by trans host, especially IL-1, IL-2, TNF, IL-8, NO, reactive oxygen plantation, autoimmune syndromes including rheumatoid intermediates (ROI), and its complications. These complica arthritis, Systemic lupus erythematosus, Hashimoto's thy tions or “associated disease responses” (ADRs) are caused by roiditis, multiple Sclerosis, myasthenia gravis, type I diabetes edema, inflammation, and the translocation of microbial mellitis,juvenile-onset or recent-onset diabetes mellitus, pos flora. Since an anti-cytokine oranti-inflammatory agents such 30 terior uveitis, allergic encephalomyelitis, glomerulonephri as, but not limited to HR341g and functional derivatives tis, post-infectious autoimmune diseases including rheumatic thereof inhibit the edema and inflammatory response, anti fever and post-infectious glomerulonephritis, inflammatory cytokine oranti-inflammatory agents such as, but not limited and hyperproliferative skin diseases, psoriasis, atopic derma to HR341g and functional derivatives thereofhave the ability titis, contact dermatitis, eczematous dermatitis, Seborrhoeic to treat diseases where inflammation contributes to the dis 35 dermatitis, Lichen planus, Pemphigus, bullous pemphigoid, ease process. Epidermolysis bullosa, urticaria, angioedemas, vasculitis, A list of the typical ADRs includes, but is not limited to, erythema, cutaneous eosinophilia, Lupus erythematosus, those that are post-burn complications such as compartment acne. Alopecia areata, keratoconjunctivitis, Vernal conjunc syndrome, acidosis, acute renal failure, acute tubular necro tivitis, uveitis associated with Behcet’s disease, keratitis, her sis, cellulitis, secondary seizures, contractures, reduced end 40 petic keratitis, dystrophia epithelialis corneae, ocular pem organ perfusion, endotoxemia, eXotoxemia, gangrene, noso phigus, Mooren’s ulcer, Scleritis, Graves opthalmopathy, comial pneumonia (50% of patients with burn/smoke Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen aller inhalation injury develop this type), ARDS (acute respiratory gies, reversible obstructive airway disease, bronchial asthma, distress syndrome), ventilator associated pneumonia, sepsis, allergic asthma, intrinsic asthma, extrinsic asthma, dust septic shock, cachexia, diarrhea, encephalopathy, myglobu 45 asthma, chronic or inveterate asthma, late asthma and airway linuria, Smoke inhalation-induced lung injury, thromboem hyper-responsiveness, bronchitis, gastric ulcers, vascular bolic complications, and those other non-burn associated dis damage caused by ischemic diseases and thrombosis, eases with an inflammatory component Such as, but not ischemic bowel diseases, inflammatory bowel diseases, limited to, anemia, cancer, congestive heart failure, coagu necrotizing enterocolitis, intestinal lesions associated with lated blood vessels (thrombosis), dermatomyositis (DM), 50 thermal burns, Celrac diseases, proctitis, eosinophilic gastro dermatitis, alveolar proteinosis pneumonia, bronchcolotis enteritis, mastocytosis, Crohn's disease, ulcerative colitis, obliterans organizing pneumonia (BOOP), chronic aspiration rhinitis, eczema, interstitial nephritis, Goodpasture's Syn lipoid pneumonia, community acquired pneumonia (CAP), drome, hemolytic-uremic syndrome, diabetic nephropathy, coronavirus pneumonia, cryptoccal pneumonia, chlamydia myositis, Guillain-Barre syndrome, polyneuritis, mononeu pneumonia, descquamative interstitial pneumonia, eosino 55 ritis, radiculopathy, pure red cell aplasia, aplastic anemia, philic pneumonia, haemophilus influenza pneumonia, hae hypoplastic anemia, idiopathic thrombocytopenic purpura, mophilus parainfluenzae pneumonia, idiopathic pneumonia, autoimmune hemolytic anemia, pernicious anemia, megalo influenza associated pneumonia, idiopathic interstitial pneu blastic anemia, osteoporosis, sarcoidosis, fibroid lung, idio monia, kliebsiella pneumonia, mycoplasma pneumonia, non pathic interstitial pneumonia, dermatomyositis, photoallergic specific interstitial pneumonia (associated with dermatomyo 60 sensitivity, cutaneous T cell lymphoma, atherosclerosis, aor sitis-DM), pasteurella multocida pneumonia, pneumocystis titis syndrome, polyarteritis nodosa, myocardosis, Sclero carinnii-(PCP) pneumonia, pseudomonas aeruginosa pneu derma, Wegener's granulomatosis, Sjogren's syndrome, monia, respiratory synctial virus infection, staphylococcal eosinophilic fasciitis, lesions of gingiva, male patternalopecia necrotising pneumonia, tuberculosis pneumonia, usual inter or alopecia senilis by preventing epilation or providing hair Stitial pneumonitis (UIP), Varicella Zoster virus pneumonia, 65 germination and/or promoting hair generation and hair toxic shock syndrome, and toxic epidermal necrosis (TEN). growth, Sezary's syndrome, Addison's disease, ischemia The following list of diseases are associated with metabolic reperfusion injury of organs, endotoxin-shock, pseudomem US 7,604,797 B2 9 10 branous colitis, colitis caused by drug or radiation, ischemic pseudomembranous colitis, colitis caused by drug or radia acute renal insufficiency, chronic renal insufficiency, lung tion, ischemic acute renal insufficiency, chronic renal insuf cancer, pulmonary emphysema, dermatitis erythema multi ficiency, lung cancer, pulmonary emphysema, dermatitis forme, linear IgA ballous dermatitis, carcinogenesis, erythema multiforme, linear IgA ballous dermatitis, carcino metastasis of carcinoma, Behcet’s disease, autoimmune genesis, metastasis of carcinoma, Behcet’s disease, autoim hepatitis, primary biliary cirrhosis, Sclerosing cholangitis, mune hepatitis, primary biliary cirrhosis, Sclerosing cholan partial liver resection, acute liver necrosis, necrosis caused by gitis, partial liver resection, acute liver necrosis, necrosis toxin, viral hepatitis, shock, or anoxia, cirrhosis, alcoholic caused by toxin, viral hepatitis, shock, or anoxia, cirrhosis, cirrhosis, hepatic failure, fulminant hepatic failure, late-onset alcoholic cirrhosis, hepatic failure, fulminant hepatic failure, hepatic failure. “acute-on-chronic' liver failure, augmenta 10 late-onset hepatic failure, “acute-on-chronic' liver failure, tion of chemotherapeutic effect, cytomegalovirus infection, augmentation of chemotherapeutic effect, cytomegalovirus cancer, trauma, and chronic bacterial infection. infection, cancer, trauma, and chronic bacterial infection. In one aspect of the invention, the therapeutically effective In one embodiment, the reduction or inhibition of pathol amount of the one or more anti-cytokine oranti-inflammatory ogy and/or symptoms associated with one or more of each of agents administered to a subject in need thereof is that amount 15 the above-recited indications is on the order of about 10-20% Sufficient to reduce or inhibit, interalia, the pathology asso reduction or inhibition. In another embodiment, the reduction ciated with one or more of the following diseases: myocardial or inhibition of pathology and/or symptoms is on the order of ischemia, tissue and muscle-associated ischemia, extremity 30-40%. In another embodiment, the reduction or inhibition associated ischemia, stroke, sepsis, amyotrophic lateral scle of pathology and/or symptoms is on the order of 50-60%. In rosis (ALS), seizures, extension of strokes after initial tissue yet another embodiment, the reduction or inhibition of the damage, functional brain damage secondary to primary and pathology and/or symptoms associated with each of the secondary brain tumors, local brain damage secondary to recited indications is on the order of 75-100%. It is intended meningitis or brain abscess, viral meningitis, viral encepha herein that the ranges recited also include all those specific litis, and/or local brain damage secondary to trauma, trans percentage amounts between the recited range. For example, plantation of organs or tissue, graft-Versus-host diseases 25 the range of about 75 to 100% also encompasses 76 to 99%, brought about by transplantation, autoimmune syndromes 77 to 98%, etc., without actually reciting each specific range including rheumatoid arthritis, systemic lupus erythemato therewith. Sus, Hashimoto's thyroiditis, multiple Sclerosis, myasthenia In yet another aspect, the present invention is directed to a gravis, type I diabetes mellitis,juvenile-onset or recent-onset method of relieving or ameliorating the pathology or symp diabetes mellitus, posterior uveitis, allergic encephalomyeli 30 toms associated with any one or more of the above-identified tis, glomerulonephritis, post-infectious autoimmune diseases diseases or indications in a mammal Suffering from any one or including rheumatic fever and post-infectious glomerulone more of the above-identified diseases or indications which phritis, inflammatory and hyperproliferative skin diseases, comprises administering to the mammal in need thereof a psoriasis, atopic dermatitis, contact dermatitis, eczematous therapeutically effective pathology or symptom-reducing dermatitis, Seborrhoeic dermatitis, Lichen planus, Pemphi 35 amount of a pharmaceutical composition comprising one or gus, bullous pemphigoid, Epidermolysis bullosa, urticaria, more anti-cytokine or anti-inflammatory agents, either alone angioedemas, vasculitis, erythema, cutaneous eosinophilia, or in combination with one or more anti-inflammatory com Lupus erythematosus, acne, Alopecia areata, keratoconjunc pounds or immunomodulatory agents; and a pharmaceuti tivitis, vernal conjunctivitis, uveitis associated with Behcet’s cally acceptable carrier or excipient, wherein said anti-cytok disease, keratitis, herpetic keratitis, dystrophia epithelialis 40 ine or anti-inflammatory agent is Sufficient to inhibit one or corneae, ocular pemphigus, Mooren’s ulcer, Scleritis, more components of the inflammatory pathway. Graves’ opthalmopathy, Vogt-Koyanagi-Harada syndrome, The present invention also relates to the use of the HR341g sarcoidosis, pollen allergies, reversible obstructive airway pharmaceutical composition in combination with one or more disease, bronchial asthma, allergic asthma, intrinsic asthma, antibacterial or antiviral compositions or any combination extrinsic asthma, dust asthma, chronic or inveterate asthma, 45 thereof for treating any one of the aforementioned diseases, or late asthma and airway hyper-responsiveness, bronchitis, any combination thereof. gastric ulcers, vascular damage caused by ischemic diseases The present invention provides methods for therapeutically and thrombosis, ischemic bowel diseases, inflammatory or prophylactically treating edema in a Subject. bowel diseases, necrotizing enterocolitis, intestinal lesions The method for therapeutically treating edema comprises associated with thermal burns, Celrac diseases, proctitis, 50 the step of administering an effective amount of an anti eosinophilic gastroenteritis, mastocytosis, Crohn's disease, cytokine or anti-inflammatory agent or derivative thereof to ulcerative colitis, rhinitis, eczema, interstitial nephritis, the subject after occurrence of the edema. Goodpasture's syndrome, hemolytic-uremic syndrome, dia The method for prophylactically treating edema comprises betic nephropathy, myositis, Guillain-Barre Syndrome, poly the step of administering an effective amount of an anti neuritis, mononeuritis, radiculopathy, pure red cell aplasia, 55 cytokine or anti-inflammatory agent or derivative thereof to aplastic anemia, hypoplastic anemia, idiopathic thrombocy the Subject prior to the occurrence of edema. topenic purpura, autoimmune hemolytic anemia, pernicious Either methodology inhibits the permeability of the anemia, megaloblastic anemia, osteoporosis, Sarcoidosis, microvasculature fluid, macromolecules, and blood cells fibroid lung, idiopathic interstitial pneumonia, dermatomyo thereby acting directly on the clinical edema and reducing the sitis, photoallergic sensitivity, cutaneous T cell lymphoma, 60 activation of detrimental metabolic cascades and pathways atherosclerosis, aortitis syndrome, polyarteritis nodosa, myo that require activation of the inflammatory pathway. cardosis, Scleroderma, Wegener's granulomatosis, Sjogren's In one aspect of the invention, the pharmaceutical compo syndrome, eosinophilic fasciitis, lesions of gingiva, male pat sitions of the present invention are administered orally, sys tern alopecia or alopecia senilis by preventing epilation or temically, via an implant, intravenously, topically, intrathe providing hair germination and/or promoting hair generation 65 cally, or nasally. and hair growth, Sezary's syndrome. Addison's disease, In one aspect of the invention, the pharmaceutical compo ischemia-reperfusion injury of organs, endotoxin-shock, sitions of the present invention are administered to the burn US 7,604,797 B2 11 12 area within 5, 10, 20, 30, 40, 50, and 60 minutes of the event disease responses that accompany various types of burns. causing the burn. Preferably, the pharmaceutical composi What follows is a brief description of the types of burns and tions of the present invention are administered to the burn area associated edemas and other diseases that may be treated with within 10 to 20 minutes of the event causing the burn. Most the compositions and methods of the present invention. preferably, the pharmaceutical compositions of the present Types of Burns invention are administered to the burn area as soon as possible Superficial or 1st degree burns. The body will always following the event causing the burn. The pharmaceutical develop edema after a burn, with Sunburns and 1 st degree compositions of the present invention should be administered burns not having visible blisters because a minimal amount of to the burn area as soon as possible but may also be admin plasma is lost. Clinical signs include painful erythema. His istered up to twelve hours following the burn event. 10 tologically, the epidermis is partially destroyed and the basal In one embodiment of the invention, the burn being treated membrane remains intact. Usually the prognosis for a first is a thermally induced burn, a thermally induced controlled degree burn is that it heals in a few days. burn, a chemical burn, a radiation burn, an electrical burn, an Partial Thickness or 2nd degree burns. These burns will ice burn, or a burn caused by exposure to lightening. almost always blister as will some 3rd degree burn injuries. In each of the above described methods, the burns are either 15 Clinical signs include erythema, blisters, underlying tissue first, second, third or fourth degree burns or any combination blanches with pressure. Histologically, the basal membrane is thereof. partially destroyed. Usually the prognosis for a partial thick In certain embodiments of the methods of the present ness or 2nd degree burn is that it heals in ten to fifteen days. invention, the Subject or mammal is a human. Deep 2nd degree burns. Clinical signs include erythyema, In other embodiments of the methods of the present inven blisters, and that the underlying tissue does not blanche with tion, the Subject or mammal is a veterinary and/or a domes pressure. Histologically, the basal membrane is entirely ticated mammal. destroyed; the dermis is partially destroyed, epidermal cells In yet another aspect, the present invention provides a kit are still present around hair follicles. Usually the prognosis for use in emergency burn accidents or injuries for application for a deep 2nd degree burn is that it heals in three to four of the pharmaceutical composition for immediate application 25 weeks, or does not heal and therefore may require grafting. to the skin as soon after the accidental burn or burn injury as Full Thickness or 3rd degree burns. These are not as likely possible. to blister because the skin is usually destroyed. Clinical signs In another aspect, the present invention provides a topical include brown, black or white; no blister, no sensitivity. His burn treatment formulation suitable for use in fire extinguish tologically, the epidermis and dermis is totally destroyed; ers that may be used to cover individuals whose bodies and/or 30 Subcutaneous tissues are more or less injured. Usually the clothes are engulfed inflames, wherein said formulation com prognosis for a full thickness or 3rd degree burn is that it does prises an anti-cytokine, an anti-inflammatory agent, or not heal except from the edges and therefore requires graft HR341g or a functional derivative thereof. 1ng. There has been thus outlined, rather broadly, the important Fourth degree burns. These burns can involve the destruc features of the invention in order that a detailed description 35 tion of underlying muscle and tendons. Clinical signs include thereofthat follows can be better understood, and in order that blackened appearance, dryness, severe pain. Histologically, the present contribution can be better appreciated. There are the skin, underlying tissue, muscles, tendons, and bones, are additional features of the invention that will be described destroyed. Usually the prognosis for a fourth degree burn is hereinafter. that it does not heal and therefore requires debridement and In this respect, before explaining at least one embodiment 40 grafting. of the invention in detail, it is to be understood that the Edema invention is not limited in its application to the details as set Edema is found in all types of burns, including, for forth in the following description and figures. The present example, those caused by heat, extreme cold, radiation, invention is capable of other embodiments and of being prac chemicals and electricity. It related to pain, infection, debri ticed and carried out in various ways. Additionally, it is to be 45 dement, skin grafts, amputation, Scarring, shock and death. If understood that the terminology and phraseology employed true Success at treating a burn is to be achieved, it is going to herein are for the purpose of description and should not be be during the pre-edema window of opportunity. Edema usu regarded as limiting. ally begins to form 30 minutes to two (2) hours after receiving As such, those skilled in the art will appreciate that the the burn with peak formation occurring at 48 hrs. A thirty conception, upon which this disclosure is based, can readily 50 minute to 2 hour window gives ample time to treat the patient be used as a basis for designing other methods for carrying out with the compositions and methods of the present invention. the several features and advantages of the present invention. It Thus, by preventing and/or treating inflammation in all is important, therefore, that the claims be regarded as includ types of burns, it is possible to reduce edema formation. The ing Such equivalent constructions insofar as they do not depart compositions and methods of the present invention trigger the from the spirit and scope of the present invention. 55 proper healing sequence required in all forms of burns and therefore prevents the destructive biochemical reactions typi BRIEF DESCRIPTION OF THE DRAWINGS cally brought on by a burn. FIG. 1A-J illustrates the formation of edema following a Moreover, by preventing and/or treating edema, it is also burn injury and the effect of administration of HR341g on possible to modulate the “after-burn’ sequence of events so 60 that the burn is prevented from becoming deeper and wider. inflammatory cytokines such as cytokines tumor necrosis The compositions and methods of the present invention are factor alpha (TNFa), IL-1, IL-6, and IL-8, and other inflam able to effectively reduce after-burn by blocking ongoing matory molecules such as NO and ROI. inflammation. After the composition of the present invention DETAILED DESCRIPTION OF THE INVENTION has been applied to a burn, it prevents tissue damage that 65 would otherwise occur. The composition of the present inven The present invention in its simplest form provides a com tion adheres to the walls of the epitheal lining and the lining position and method for treating burns, edema and associated of the hair follicles, thereby protecting each in the after-burn US 7,604,797 B2 13 14 period. The compositions of the present invention will also with burn injuries. Patients will develop reduced edema and prevent microorganisms from invading the burn site. Patients burns will heal naturally, reducing the necessity for the more will therefore also suffer less because they remain free from invasive treatment of debridement and skin grafting. Skin various infections commonly associated with typical burns. grafting can be complicated by infection and can leave The composition thus prevents burninjuries from progressing 5 unsightly and disfiguring Scars. In serious burns that require to greater severity. debridement, the opportunity to grow an individual’s own By preventing and/or treating inflammation, it is possible skin may be lost making long and painful skin graft proce to inhibit the complex chemical changes, which often become dures necessary. In some cases, amputation is the only solu the determining factors in a patients outcome. The compo tion. Many full thickness burns can now be healed using the sition of the present invention curtails these chemical changes 10 composition of the present invention with reduced need for making the body react the way it would after lesser traumas debridement and/or skin grafting. Such as a mild wound or cut instead of a burn. After a mild cut By preventing inflammation, it is also possible to prevent or wound, the body begins to clog the wound with platelets so hypertrophic scarring that typically occurs with more serious the healing stage can begin. This is a normal response that burns. Burns from partial superficial to full thickness can be promotes repair of the injured area. healed without infection or hypertrophic scarring if inflam With the compositions and methods of the present inven 15 mation and tissue damage is reduced. With full thickness or tion it is also possible to prevent and/or treat infections caused 3rd degree burns, there is no dermis, so collagen fibers are not by bacteria, as well as being able to prevent and/or treat aligned vertically and horizontally, but are present in random, numerous Associated Disease Responses (ADRs). The use disordered masses. The proliferation phase begins, yet of compositions and methods of the present invention pre progress is difficult. During remodeling, the collagen fibers vents the tissue damage that is the breeding ground for micro are Supposed to be tightly aligned but because of infections, organisms in most burns. This reduced rate of infection trans debridement, skin grafts and other disorders, the process is lates to reduced disease, disorders and deformities. The not organized. This dysfunction can lead to hypertrophic ability to interfere with the cycle of infection in sequellae can scarring. If there is reduced inflammation/infection present, halt the disease process. Gram-positive and gram-negative there is a reduced requirement to remove the patient’s remain organism infections usually develop after edema. The 25 ing skin, and so the body can repair itself with fewer compli destructive consequences of these pathophysiological phases cations and with little to no scarring. The compositions and are related to MOD (Multiple Organ Dysfunction) at an early methods of the present invention allow the body to elicit the stage. The translocation of microorganisms can be prevented proper repair sequence. if the area of plasma leakage can be blocked. The composi HR341g Composition and Derivatives Thereof tions of the present invention prevent the accumulation of 30 Representative examples of naturally occurring and non neutrophils, and their release of oxygen free radicals and naturally generated anti-cytokine or anti-inflammatory various proteases by limiting inflammation, thereby prohib agents or functional derivatives thereof that may be used in iting further tissue damage. the prophylactic and therapeutic methods for treating local A list of the typical ADRs includes, but is not limited to, ized and systemic inflammation associated with burns those which are burn-associated Such as compartment syn 35 include, for example, but not limited to, pharmaceutical com drome, acidosis, acute renal failure, acute tubular necrosis, positions comprising HR341g, aminopterin, methotrexate, cellulitis, secondary seizures, contractures, reduced end-or pyramethamine, and trimethoprim or any combination gan perfusion, endotoxemia, exotoxemia, gangrene, nosoco thereof. mial pneumonia (50% of patients with burn/smoke inhalation Aminopterin (AMT, 4-amino-4-deoxy-pteroylglutamic injury develop this type), ARDS (acute respiratory distress acid) is a dihydofolate reductase inhibitor. Dihydrofolate syndrome), ventilator associated pneumonia, sepsis, septic 40 reductase (DHFR) catalyzes the reaction of 7,8-dihydrofolate shock, thromboembolic complications, and those other non and NADPH to form 5,6,7,8-tetrahydofolate and NADP". burn associated diseases with an inflammatory component Tetrahydofolate is essential for the biosynthesis of purines, Such as, but not limited to, anemia, cancer, congestive heart thymidylate and several amino acids (Rajagopalan et al. failure, reduced end-organ perfusion, dermatomyositis PNAS vol. 99 (21), 13481-13486 (2002), incorporated by (DM), dermatitis, alveolar proteinosis pneumonia, bronchco 45 reference herein). Aminopterinacts as an antineoplastic agent lotis obliterans organizing pneumonia (BOOP), chronic aspi by interfering with one or more biosynthetic steps involving ration lipoid pneumonia, community acquired pneumonia folate coenzymes of the cell. The structural formula of ami (CAP), coronavirus pneumonia, cryptoccal pneumonia, nopterin is as follows: chlamydia pneumonia, descquamative interstitial pneumonia, eosinophilic pneumonia, haemophilus influenza pneumonia, 50 haemophilus influenza pneumonia, haemophilus parainflu Structure of Aminopterin enzae pneumonia, idiopathic pneumonia, influenza associ ated pneumonia, idiopathic interstitial pneumonia, kliebsiella pneumonia, mycoplasma pneumonia, non-specific interstitial pneumonia (associated with dermatomyositis-DM), pas teurella multocida pneumonia, pneumocystis carinnii-(PCP) 55 pneumonia, pseudomonas aeruginosa pneumonia, respira tory synctial virus infection, staphylococcal necrotising s' N O pneumonia, tuberculosis pneumonia, usual interstitial pneu N N ND C-N J monitis (UIP), Varicella Zoster virus pneumonia, toxic shock H. H syndrome, and toxic epidermal necrosis (TEN). The follow 60 NH2 NH O ing list of diseases are associated with metabolic disarray because of thermal injuries: cachexia, diarrhea, encephalopa OH-C-C-C-C-C-OH thy, myglobulinuria, and neurities. | H. H. H It is also possible to prevent the usual debridement and O requirement for skin grafting often necessary for third degree 65 or higher burns. By using the composition of the present Methotrexate (MTX: 4-amino-4-deoxy-N. Sup. 10-methyl invention, patients will Suffer less pain and trauma associated pteroylglutamic acid) and Aminopterin (AMT, 4-amino-4- US 7,604,797 B2 15 16 deoxy-pteroylglutamic acid) are dihydrofolate reductase sized as described by U.S. Pat. Nos. 5,965,106, 5,140,104, inhibitors () and act as antineoplastic agents by interfering and 4,956,461—the texts of each of which are being with one or more biosynthetic steps involving folate coen expressly incorporated by reference herein. These issued pat Zymes of the cells. The structure of MTX differs from AMT in ents not only provide representative embodiments of natu that the former contains a methyl group in the N.sup.10 posi- 5 rally occurring and non-naturally generated dihydrofolate tion while the latter does not, having hydrogen instead. The reductase inhibitors or dihydrofolate reductase inhibitor structural formula of MTX is as follows: functional derivatives thereof; but also provide complete and detailed procedures and techniques for synthesizing and puri Structure of MTX fying such dihydrofolate reductase inhibitor functional ana 10 logues for use in the methodologies of the present invention. Additionally, pyramethamine, and trimethoprim are, by chemical formulation and structure, intended to be part of the II members forming the class of compounds of dihydrofolate NH2 N N reductase inhibitors or dihydrofolate reductase inhibitor 21 n 15 functional derivatives thereof. Thus pyramethamine, and tri methoprim and all substituted and derivatized forms of N 2 N N C-N C pyramethamine, and trimethoprim are also intended to be H. encompassed within the scope of methodologies of the NH2 CH N present invention. The dihydrofolate reductase inhibitor compounds or func tional derivatives thereof for use in the methods of the present invention may be prepared by the reaction of 4-amino-4- deoxy-pteroic acid or 4-amino-4-deoxy-N. Sup. 10-meth ylpteroic acid with cysteic or homocysteic acid. Thus, repre The following references describe the preparation of meth 25 sentative examples of compounds for use in the methods of otrexate see Seeger et al., J. Am. Chem. Soc., 1949, the present invention comprise, without limitation, those 71:1753: the metabolism of methotrexate see Freeman, J. MTX derivative compounds such as: 4-amino-4-deoxy Pharmacol. Exp. Ther. 1958, 122:154; and Henderson et al., N.sup. 10-methylpteroyl-D.L-homocysteic acid (mAPA-D, Cancer Res. 1965,25:1008, 1018: the toxicity of methotrex L-HCySA), 4-amino-4-deoxy-N. Sup. 10-methylpteroyl-L- ate Condit et al., Cancer 1960, 13:222-249; the pharmaco 30 cysteic acid (mAPA-L-CySA), 4-amino-4-deoxy-N.Sup.10 kinetic models of methotrexate Bischoff, et al., J. Pharm. Sci methylpteroyl-L-homocysteic acid (mAPA-L-HCySA), 1970, 59:149; the metabolism and pharmacokinetics of 4-amino-4-deoxypteroyl-D.L-homocysteic acid (APA-D.L- methotrexate Evans, Appl. Pharmacokinet. 1980. 518-548: HCySA), 4-amino-4-deoxypteroyl-L-cysteic acid (APA-L- the clinical pharmacology of methotrexate Bertino, Cancer CySA), and 4-amino-4-deoxypteroyl-L-homocysteic acid Chemother, 1981, 3:359-375: Jolivet et al., N. Engl.J. Med., 35 (APA-L-HCySA). 1983, 309: 1094-1 104 the texts of each of which references For example, and not by way of limitation, the compounds are expressly incorporated by reference herein. for use in the methods of the present invention comprise MTX MTX and AMT have been found to be effective clinically and AMT analogues in which the glutamic acid moiety of against certain malignant tumors: for example, good to excel MTX or AMT is replaced by cysteic acidor homocysteic acid. lent tumor response has been seen in patients with acute 40 Additional non-limiting examples of aminopterin deriva lymphocytic leukemia, Burkitt's lymphoma, carcinoma of tives that may be used in the methods of the present invention the breast, mycosis fungoides, epidermoid cancer of the head are provided as follows: alpha-carboxyl Substituted aminop and neck area, and osteogenic sarcoma. In addition, MTX is terin derivatives—for example, in one embodiment of ami the drug of choice in the treatment of choriocarcinoma and is nopterin derivatives, alpha-carboxyl-substituted aminopterin also used for certain non-neoplastic conditions such as gen- 4s derivatives including alpha-carboxylester derivatives, alpha eralized psoriasis and certain autoimmune diseases Such as carboxylamide derivatives, alpha-carboxylpeptide deriva rheumatoid arthritis and lupus erythematosus. tives, and alpha-carboxylhydrazide derivatives may be used; However, it should be noted that chemotherapy with MTX alpha-carboxylamide derivatives—non-limiting examples of or AMT is accompanied by a variety of toxicities, partly which include alpha-carboxylester derivatives of aminopterin related to their ability to form polyglutamates, which limit the 50 include the alpha-methylester, alpha-ethylester, alpha-propy effectiveness of the compounds and their long-term use. lester, alpha-butylester, alpha-pentylester, alpha-hexylester, It will be recognized and appreciated that Formulas I and II alpha-heptylester and alpha-octylester of aminopterin, in are presented herein using conventional chemical structure, which the esters may beformed from the n- or iso-form of the format, and notations for amino acids and peptide organiza corresponding alcohols—Further examples include other tion as those found in Albert L. Lehninger's text, Biochem- 55 ester derivatives such as the alpha-benzylester of aminop istry, The Molecular Basis Of Cell Structure And Function, terin; alpha-carboxylamide derivatives—non-limiting 2nd edition, Worth Publishers, Inc., 1977 the text of which examples of which include the alpha-amide, alpha-butyla is expressly incorporated by reference herein. Moreover, For mide, alpha-benzylamide, and the alpha-amidoethane Sul mulas I and II by their definitions intend that all presently fonic acid derivative of aminopterin; alpha-carboxylpeptide known and future embodiments of naturally occurring and 60 derivatives—non-limiting examples of which include the non-naturally generated Substances—which are by chemical alpha-glycyl derivative, alpha-aspartyl derivative, alpha formulation and structure members forming the class of com glutamyl derivative and the alpha-polyglutamyl 1-5 deriva pounds of dihydrofolate reductase inhibitors or dihydrofolate tive of aminopterin; alpha-carboxylhydrazide derivatives— reductase inhibitor functional derivatives thereof (including non-limiting examples of which include the alpha all substituted and derivatized forms)—lie within the scope of 65 carboxylhydrazide derivative of aminopterin; gamma the present invention. However, representative, non-limiting, carboxyl Substituted aminopterin derivatives—In one examples of embodiments are those formulated and synthe embodiment of aminopterin derivatives, gamma-carboxyl US 7,604,797 B2 17 18 Substituted aminopterin derivatives including gamma-car non-limiting examples of include the alpha, gamma-diamide boxylester derivatives, gamma-carboxylamide derivatives, derivatives; alpha, gamma-dipeptide derivatives; alpha, gamma-carboxylpeptide derivatives, and gamma-carboxyl gamma-dihydrazide derivatives; alpha-ester, gamma-amide hydrazide derivatives may be used; gamma-carboxylester derivatives; alpha-ester, gamma-peptide derivatives; alpha derivatives—non-limiting examples of which include amide, gamma-ester derivatives; alpha-amide, gamma-pep gamma-carboxylester derivatives include the gamma-methy tide derivatives; alpha-amide, gamma-hydrazide derivatives; lester, gamma-ethylester, gamma-propylester, gamma-buty alpha-peptide, gamma-ester derivatives; alpha-peptide, lester, gamma-pentylester, gamma-hexylester, gamma-hep gamma-ester derivatives; alpha-peptide, gamma-amide tylester, and the gamma-octylester of aminopterin, of which derivatives; alpha-peptide, gamma-hydrazide derivatives; the esters may be synthesized from the n- or iso-form of the 10 alpha-hydrazide, gamma-ester derivatives; alpha-hydrazide, corresponding alcohols—Further examples include other gamma-amide derivatives; and the alpha-hydrazide, gamma ester derivatives such as the gamma-benzylester derivative of peptide derivatives of aminopterin. aminopterin; gamma-carboxylamide derivatives—non-limit Additional non-limiting examples of MTX derivatives that ing examples of which include the gamma-amide, gamma may be used in the methods of the present invention are butylamide, gamma-benzylamide, and the gamma-amidoet 15 provided as follows: alpha-carboxyl substituted MTX deriva hane Sulfonic acid derivative of aminopterin; gamma tives—for example, in one embodiment of MTX derivatives, carboxylpeptide derivatives—non-limiting examples of alpha-carboxyl-substituted MTX derivatives including which include the gamma-glycyl derivative, gamma-asparty1 alpha-carboxylester derivatives, alpha-carboxylamide derivative, gamma-glutamyl derivative, and the gamma-poly derivatives, alpha-carboxylpeptide derivatives, and alpha glutamyl 1-5 derivative of aminopterin; gamma-carboxyl carboxylhydrazide derivatives may be used; alpha-carboxy hydrazide derivatives—non-limiting examples of which lamide derivatives—non-limiting examples of which include include the gamma-carboxylhydrazide derivative of aminop alpha-carboxylester derivatives of MTX include the alpha terin; alpha, gamma-homobisubstituted aminopterin deriva methyl ester, alpha-ethylester, alpha-propylester, alpha-buty tives—One embodiment of aminopterin derivatives comprise lester, alpha-pentylester, alpha-hexylester, alpha-heptylester alpha, gamma-homobisubstituted aminopterin derivatives 25 and alpha-octylester of MTX, in which the esters may be including alpha, gamma-dicarboxylester derivatives, alpha, formed from the n- or iso-form of the corresponding alco gamma-dicarboxylamidederivatives, alpha, gamma-dicar hols—Further examples include other ester derivatives such boxylpeptide derivatives, and alpha, gamma-dicarboxylhy as the alpha-benzylester of MTX; alpha-carboxylamide drazide derivatives may be used; alpha, gamma-dicarboxy derivatives—non-limiting examples of which include the lester derivatives—non-limiting examples of which include 30 alpha-amide, alpha-butylamide, alpha-benzylamide, and the the alpha, gamma-dimethylester, alpha, gamma-diethylester, alpha-amidoethane sulfonic acid derivative of MTX; alpha alpha, gamma-dipropylester, alpha, gamma-dibutyl ester, carboxylpeptide derivatives—non-limiting examples of alpha, gamma-dipentyl ester alpha, gamma-dihexylester, which include the alpha-glycyl derivative, alpha-asparty1 alpha, gamma-diheptylester, and the alpha, gamma-diocty derivative, alpha-glutamyl derivative and the alpha-poly lester of aminopterin, the esters of which may be synthesized 35 glutamyl 1-5 derivative of MTX; alpha-carboxylhydrazide from the n- or iso-form of the corresponding alcohols. Further derivatives—non-limiting examples of which include the examples include other diester derivatives Such as the alpha, alpha-carboxylhydrazide derivative of MTX; gamma-car gamma-dibenzylester derivative of aminopterin; alpha, boxyl substituted MTX derivatives—In one embodiment of gamma-dicarboxylamide derivatives—non-limiting MTX derivatives, gamma-carboxyl-substituted MTX deriva examples of which include the alpha, gamma-diamide, alpha, 40 tives including gamma-carboxylester derivatives, gamma gamma-dibenzylamide, and the alpha, gamma-diami carboxylamide derivatives, gamma-carboxylpeptide deriva domethane Sulfonic acid derivative of aminopterin...; alpha, tives, and gamma-carboxylhydrazide derivatives may be gamma-dicarboxylpeptide derivatives—non-limiting used; gamma-carboxylester derivatives—non-limiting examples of which include the alpha, gamma-diglycyl, alpha, examples of which include gamma-carboxylester derivatives gamma-diaspartyl, alpha, gamma-diglutamyl, and the alpha, 45 include the gamma-methylester, gamma-ethylester, gamma gamma-dipolyglutamyl 1-5 derivative of aminopterin. propylester, gamma-butylester, gamma-pentylester, gamma alpha, gamma-dicarboxylhydrazide derivatives—non-limit hexylester, gamma-heptylester; and the gamma-octylester of ing examples of which include the alpha, gamma-dicarboxy MTX, of which the esters may be synthesized from the n- or lhydrazide derivatives of aminopterin; alpha, gamma-hetero iso-form of the corresponding alcohols—Further examples bisubstituted aminopterin derivatives—One embodiment of 50 include otherester derivatives such as the gamma-benzylester aminopterin derivatives comprise alpha, gammaheterobisub derivative of MTX; gamma-carboxylamide derivatives— stituted aminopterin derivatives including alpha, gamma-di non-limiting examples of which include the gamma-amide, carboxylester derivatives, alpha-ester, gamma-amide deriva gamma-butylamide, gamma-benzylamide, and the gamma tives, and alpha-ester, gamma-hydrazide derivatives; alpha, amidoethane sulfonic acid derivative of MTX; gamma-car gamma-dicarboxylester derivatives—non-limiting examples 55 boxylpeptide derivatives—non-limiting examples of which of which include the alpha-methylester, gamma-butylester of include the gamma-glycyl derivative, gamma-asparty1 aminopterin and the alpha-methylester, gamma-benzylester derivative, gamma-glutamyl derivative, and the gamma-poly of aminopterin; alpha-ester, gamma-amide derivatives—non glutamyl 1-5 derivative of MTX; gamma-carboxylhy limiting examples of which include the alpha-benzylester, drazide derivatives—non-limiting examples of which include gamma-butylamide derivative; alpha-benzylester, gamma 60 the gamma-carboxylhydrazide derivative of MTX; alpha, benzylamide derivative; alpha-benzylester, gamma-butyla gamma-homobisubstituted MTX derivatives—One embodi mide-p-toluene Sulfonic acid derivative; and the alpha-ben ment of MTX derivatives comprise alpha, gamma-homo Zylester, gamma-benzylamide-p-toluene Sulfonic acid bisubstituted MTX derivatives including alpha, gamma-di derivative of aminopterin; alpha-Ester, gamma-hydrazide carboxylester derivatives, alpha, gamma-dicarboxylamide derivatives—non-limiting examples of which include the 65 derivatives, alpha, gamma-dicarboxylpeptide derivatives, alpha-t-butylester, gamma-hydrazide derivative of aminop and alpha, gamma-dicarboxylhydrazide derivatives may be terin; other alpha, gamma-heterobisubstituted derivatives— used; alpha, gamma-dicarboxylester derivatives—non-limit US 7,604,797 B2 19 20 ing examples of which include the alpha, gamma-dimethyl gammahydrazide, methotrexate-alpha-hydrazide, 3'5-dichlo ester, alpha, gamma-diethylester, alpha, gamma-dipropy romethotrexate-gammahydrazide, 3',5-dichloromethotrex lester, alpha, gamma-dibutylester, alpha, gamma ate-alpha-hydrazide, methotrexate-alpha-alpha-lysyl-glycyl dipentylester alpha, gamma-dihexylester, alpha, gamma glycyl-tyrosyl hydrazide (SEQ ID NO: 1), methotrexate diheptylester, and the alpha, gamma-dioctylester of MTX, the 5 gamma-tyrosyl hydrazide, methotrexate-alpha-alpha-lysyl esters of which may be synthesized from the n- or iso-form of hydrazide, methotrexate-alpha-alpha-lysine, methotrexate the corresponding alcohols. Further examples include other alpha-alpha-lysyl-epsilon-arginine-glycine-glycine-tyrosine diester derivatives such as the alpha, gamma-dibenzylester (SEQ ID NO: 2), aminopterin-gamma-hydrazide, aminop derivative of MTX; alpha, gamma-dicarboxylamide deriva terin-alpha-hydrazide, 3'5'dichloraminopterin-gamma-hy tives—non-limiting examples of which include the alpha, 10 drazide, 3',5'-dichloroaminopterin-alpha-hydrazide, aminop gamma-diamide, alpha, gamma-dibenzylamide, and the terin-gamma-tyrosyl hydrazide, aminopterin-alpha-alpha alpha, gamma-diamidomethane Sulfonic acid derivative of lysyl-glycyl-tyrosyl hydrazide, aminopterin-alpha-alpha MTX.; alpha, gamma-dicarboxylpeptide derivatives—non lysyl hydrazide, aminopterin-alpha-alpha-lysine, and limiting examples of which include the alpha, gamma-digly aminopterin-alpha-alpha-lysyl-epsilon-arginine-glycine cyl, alpha, gamma-diaspartyl, alpha, gamma-diglutamyl, and 15 glycine-tyrosine (SEQID NO:3). Reactive amine-containing the alpha, gamma-dipolyglutamyl 1-5 derivative of MTX. derivatives of folic acid analogs such as 5.8dideazamethotr alpha, gamma-dicarboxylhydrazide derivatives—non-limit exate, 5,8-dideaza 5,6,7,8-tetrahydromethotrexate, 5.8,- ing examples of which include the alpha, gammadicarboxy dideaza 5,6,7,8-tetrahydroaminopterin, 5,8,10-trideazatet lhydrazide derivatives of MTX; alpha, gamma-heterobisub rahydrofolic acid, and 8,10-dideazaminopterinare also useful Stituted MTX derivatives. One embodiment of MTX according to the methods of the present invention. derivatives comprise alpha, gammaheterobisubstituted MTX It is also specifically contemplated within the scope of the derivatives including alpha, gamma-dicarboxylester deriva invention that the amine derivatives of the afore-mentioned tives, alpha-ester, gamma-amide derivatives, and alpha-ester, folic acid analogs or derivatives thereof are particularly well gamma-hydrazide derivatives; alpha, gamma-dicarboxy Suited for use in the preparation of therapeutic antibody con lester derivatives—non-limiting examples of which include 25 jugates, which therapeutic antibody conjugates may be used the alpha-methylester, gamma-butylester of MTX and the in the methods of the present invention to prevent edema alpha-methylester, gamma-benzylester of MTX; alpha-ester, associated with all types of burns. Thus, these derivatives gamma-amide derivatives—non-limiting examples of which represent intermediates in the preparation of therapeutic anti include the alpha-benzylester, gamma-butylamide derivative; body-folic acid analog conjugates. Selective attachment of alpha-benzylester, gamma-benzylamide derivative; alpha 30 the folic acid analogs via a reactive amine to an oxidized benzylester, gamma-butylamide-p-toluene Sulfonic acid carbohydrate moiety of an antibody or antibody fragment derivative; and the alpha-benzylester, gamma-benzylamide results in a conjugate that retains the antibody specificity and p-toluene sulfonic acid derivative of MTX; alpha-Ester, immunoreactivity. gamma-hydrazide derivatives—non-limiting examples of It is also specifically contemplated within the scope of the which include the alpha-t-butylester, gamma-hydrazide 35 invention that the anti-cytokine or anti-inflammatory agent derivative of MTX; other alpha, gamma-heterobisubstituted pharmaceutical compositions comprising HR341g, aminop derivatives—non-limiting examples of include the alpha, terin, methotrexate or a functional derivative thereof may be gamma-diamide derivatives; alpha, gamma-dipeptide deriva “concurrently administered to a patient. Concurrently tives; alpha, gamma-dihydrazide derivatives; alpha-ester, administering means the anti-cytokine or anti-inflammatory gamma-amide derivatives; alpha-ester, gamma-peptide 40 agents are administered to the Subject either (a) simulta derivatives; alpha-amide, gamma-ester derivatives; alpha neously in time (optionally by formulating the two togetherin amide, gamma-peptide derivatives; alpha-amide, gamma-hy a common carrier), or (b) at different times during the course drazide derivatives; alpha-peptide, gamma-ester derivatives; of a common treatment schedule. In the latter case, the anti alpha-peptide, gamma-ester derivatives; alpha-peptide, cytokine or anti-inflammatory agent compounds are admin gamma-amide derivatives; alpha-peptide, gamma-hydrazide 45 istered sufficiently close in time to achieve the intended derivatives; alpha-hydrazide, gamma-ester derivatives; effect. The active agents may be administered together in a alpha-hydrazide, gamma-amide derivatives; and the alpha single pharmaceutical composition or separately. The active hydrazide, gamma-peptide derivatives of MTX. agents of HR341 g (i.e., the anti-cytokine or anti-inflamma Other possible examples of folic acid analogues that may tory agents comprising sodium monofluorophosphate and/or be used in the methods of this invention include: 3',5' Dichlo 50 aminopterin, methotrexate or a functional derivative thereof, romethotrexate, 3',5' Dichloroaminopterin, 5.8-Didea as well as the other components of HR341 g) should be Zamethotrexate, 5.8 Dideaza 5,6,7,8-tetrahydromethotrexate, present in the patient at sufficient combined levels to be 5.8-Dideaza 5,6,7,8-tetrahydroaminopterin, 5.8, 10-Tridea therapeutically effective. The routes of administration of the Zaminopterin, 5,10-Dideazatetrahydrofolic acid, 8, 10-Didea anti-cytokine or anti-inflammatory agents comprising Zaminopterin. 55 HR341g.(e.g., sodium monofluorophosphate, and/or ami Also specifically contemplated for use within the methods nopterin, methotrexate or a functional derivative thereof) may of the invention are amine derivatives of the aforementioned be the same or different. For any route of administration, and other folic acid analogs. Such amine derivatives encom single or divided doses may be used. pass any folic acid analog containing or modified to contain a In one embodiment, the pharmaceutical composition used reactive amine moiety. The term “reactive amine' is intended 60 for the methods of the present invention comprises an to encompass any nitrogen-containing functional group that HR341g-based composition comprised of the following can be covalently attached or bonded through a nitrogenatom ingredients in the recited percentages: Dicalcium phosphate to an aldehyde functional group either by a single chemical dihydrate (DCP) 21.4% (w/v), insoluble sodium metaphos condensation reaction or by a chemical condensation reaction phate 13% (w/v); sorbitol syrup (70% solution) 23.3% (w/v) followed by reduction to stabilize the covalent bond formed. 65 guar gum 4.2% (w/v), Xanthan gum 1.7% (w/v); monosodium Thus amine derivatives offolic acid analogs useful according phosphate 0.28% (w/v); sodium monofluorophosphate 8.9% to the invention include but are not limited to: methotrexate (w/v); aminopterin 0.0015% (w/v); titanium dioxide 0.56% US 7,604,797 B2 21 22 (w/v); sodium dodecylbenzene sulphate 0.46% (w/v); water also blocks the formation of secondary inflammatory mol 22.4% (w/v); trimagnesium phosphate 0.74% (w/v); and ecules Such as nitric oxide or reactive oxygen intermediates. hydroxethyl cellulose ester 2.9% (w/v). Example 1 outlines The anti-inflammatory effects of HR341g will serve one or the procedure for preparing one of the pharmaceutical com more of the following functions, either alone or any combi positions of the invention. nation thereof: prevent the destruction of the epidermis and In another embodiment, the pharmaceutical composition dermis by debridement and skin grafts; prevent hypertrophic used for the methods of the present invention comprises an scarring and other deformities-including loss of hair growth; HR341g-based composition comprised of the following stop the depletion of various metabolic fluids; act as a ingredients in the recited percentages: Dicalcium phosphate molecular antibiotic; act as a protease inhibitor; acts as a dihydrate (DCP) 21.4% (w/v), insoluble sodium metaphos 10 signal transduction inhibitor-blocks cell signaling channels phate 13% (w/v); sorbitol syrup (70% solution) 23.3% (w/v) between activated receptors on cells and intracellular compo guar gum 4.2% (w/v), Xanthan gum 1.7% (w/v); monosodium nents; prevent infections and return the dermis and epidermis phosphate 0.28% (w/v); sodium monofluorophosphate 8.9% back to the original form, texture, elasticity and strength; (w/v); titanium dioxide 0.56% (w/v); sodium dodecylben promote hair growth and hair restoration at the area of treat Zene sulphate 0.46% (w/v); water 22.4% (w/v); trimagnesium 15 ment; inhibits overexpression of enolase in a number of bio phosphate 0.74% (w/v); and hydroxethylcellulose ester 2.9% chemical recognition processes. The chemoenzymatic (w/v). approach appears Vulnerable to exploitation by fluoride While not intended to be limited by any particular mecha reagents (such as, but not limited to, sodium monofluoro nism of action, the brief description provided herein below phosphate) as a substrate. (Harper's Biochemistry, 25th Edi provides one possible mechanism of action for the composi tion, Eds. Murray et al. Chapter 19 (2000), incorporated by tions of the present invention. Thus, by way of illustration reference herein). only, and not by way of limitation, FIGS. 1A-1I illustrates in In particular, after triggering an inflammatory response as diagramatic form the formation of edema following a burn a result of a burn, burn patients have been found to have an injury, and the effect of administration of HR341g on inflam increased Susceptibility to Subsequent inflammatory stimuli matory cytokines such as tumor necrosis factoralpha (TNFa), 25 and infections. For example, if levels of lymphocyte and IL-1, IL-6, and IL-8, and other inflammatory molecules such macrophage derived cytokines are examined, evidence shows as NO and ROI. that increased vascular permeability and inflammatory cytok In particular, with respect to FIG. 1, FIG. 1A illustrates ine activation (interleukin-1, interleukin-6 and tumor necro intact skin and the underlying blood vessels. White blood sis factor-alpha) were induced in patients with burns. It was cells are circulating in the blood at a concentration of about 4 30 further found that patients were at increased risk for immu million cells per milliliter of blood. FIG.1B illustrates that the nosuppression after a burn, which in turn increases the risk of acute burn injury causes immediate mechanical destruction infection. of skin cells, generating an ulceration. FIG.1C illustrates that The compositions and methods of the present invention the acute burn injury generates immediate inflammation prevent edema (in part) by blocking cytokine production by which results in production of the inflammatory cytokines 35 human peripheral mononuclear cells found in the blood and tumor necrosis factor alpha (TNFa), IL-1, IL-6, and IL-8. those produced by fibroblasts in the skin. In fact, burns induce These cytokines originate from cells in the skin and cells in tumor necrosis factor and interleukin-1, which in turn causes the deepertissues. FIG.1D illustrates that under the influence an increase in interferon-gamma (IFN-gamma) and lower of IL-8 (a white blood cell attractant), white blood cells in the levels of interleukin-12 (IL-12) expression. These pro-in blood vessels are called into the tissues. The white blood cells 40 flammatory cytokines cause capilliary leaks (increased per dissolve small portions of the blood vessel walls in order to meability) which results in edema formation. This can be leave the blood circulation and infiltrate the tissues to arrive at counteracted with the compositions of the present invention. the burn site. The white cells attempt to repair the damaged For example, anti-cytokine oranti-inflammatory agents com tissues and fight infection. FIG. 1E illustrates that under the prising aminopterin, methotrexate or a functional derivative influence of TNFa and IL-1, the cells that line the blood 45 thereof, including HR341g or a functional derivative thereof, vessels lose their integrity, and this results in pore formation will act as a cytokine inhibitor. For example, HR341g and along the blood vessels. This results in leakage of plasma derivatives thereof may reduce capillary membrane perme from the blood vessels that causes edema fluid to form in the ability by inhibiting cytokines and nitric oxide. Thus, the surrounding tissues. FIG.1F illustrates that TNFa. IL-1, IL-6, dehydration of the intra-vascular system is prevented and and IL-8 arejoined by other inflammatory Substances, such as 50 there is no overflow of plasma in the intercellular space, nor nitric oxide (NO) and free radicals (also called reactive oxy between the epidermal, dermal junction. Edema does not gen intermediates or ROI). One important effect of these develop because the plasma remains in the intravascular sys substances is dilation of the blood vessels that causes low tem. blood pressure, and this results in reduced blood pressure and Methods of Use even shock. FIG. 1G illustrates that TNFa. IL-1, IL-6, and 55 Thus, in its simplest aspect, the present invention provides IL-8 and the other inflammatory substances, such as NO or a method for treating all forms of burns comprising adminis ROI gain access to the blood stream and cause systemic tering to a burn area of a subject in need thereof of a thera inflammation. FIG. 1H illustrates that once TNFa. IL-1, IL-6, peutically effective amount of a composition comprising an and IL-8 and the other inflammatory substances, such as NO anti-cytokine or anti-inflammatory agent or both, or a func or ROI enter the blood stream, they cause systemic inflam 60 tional derivative thereof, and a pharmaceutically acceptable mation. This can damage any organ system in the body, excipient. including organs Such as heart kidneys, lungs and the brain. In another aspect, the present invention relates to methods The systemic inflammation also causes fever. FIG. 1I illus of controlling or alleviating pain by reducing the severity of trates that HR341g is believed to block production of TNFa. edema associated with a burn comprising administering to a IL-1, IL-6, and IL-8 that initiate the inflammatory process. 65 subject in need thereofatherapeutically effective amount of a This not only reduces the local and systemic damage that pharmaceutical composition comprising an anti-cytokine or these molecules can cause, but administration of HR341 g anti-inflammatory agent or both, or a functional derivative US 7,604,797 B2 23 24 thereof, and a pharmaceutically acceptable excipient, from recombinant cell culture and biologically active equiva wherein said pharmaceutical composition inhibits one or lents of the native sequence cytokines. more components of the inflammatory pathway. In yet another aspect of the invention, a method is provided In another aspect, the present invention also relates to a for modulating expression of major histocompatibility com method for promoting rapid regeneration of damaged tissues plex (MHC) molecules in a mammalian patient in need of resulting from a burn comprising administering to a subject in Such immunosuppression comprising administering to said need thereofatherapeutically effective amount of a pharma patient an immunosuppressing effective amount of a thera ceutical composition comprising an anti-cytokine or anti peutically effective amount of a pharmaceutical composition inflammatory agent or both, or a functional derivative thereof; comprising HR341 g or a functional derivative thereof; and a and a pharmaceutically acceptable excipient, wherein said 10 pharmaceutically acceptable excipient, wherein said pharma pharmaceutical composition promotes rapid regeneration of ceutical composition modulates expression of major histo damaged tissues while retaining the original composition of the tissue and minimizing the complications and Scarring compatibility complex molecules. associated with a burn. In yet another aspect of the invention, a method is provided for limiting intramolecular nucleophilic reactions that occur In certain embodiments of the invention, the burn being 15 in most pathways that affects the reactivity of intramolecular treated is a chemical, radiation, electrical, Sunburn, heat, and intermolecular groups comprising administering to a extreme cold- or thermally-induced burn, or any combination patient in need thereofatherapeutically effective amount of a thereof. pharmaceutical composition comprising an anti-cytokine or Thus, in yet another aspect, the present invention also anti-inflammatory agent or a functional derivative thereof. relates to a method for preventing orameliorating the adverse and a pharmaceutically acceptable excipient. A number of affects associated with controlled thermal induced skin dam oxygen groups or ROI are unstable in burn injuries and treat age employed in Scar and tattoo removal, cancer excisions, ment with an anti-cytokine or anti-inflammatory agent Such cautery excision of polyps, ulcers, treatment of decubitus as HR341g will inhibit these oxygen free radicals or oxidants. ulcers (bedsores), and/or acne comprising administering to a subject in need thereofatherapeutically effective amount of a In another embodiment, the pharmaceutical compositions pharmaceutical composition comprising an anti-cytokine or 25 of the present invention are thus useful to treat the pain and anti-inflammatory agent or both or a functional derivative tissue dysfunction associated with and/or prevent a diseases thereof, and a pharmaceutically acceptable excipient, or disorders often accompanying a burn. Since HR341 g wherein said pharmaceutical composition promotes rapid reduces inflammation, it may be used to treat diseases where regeneration of damaged tissues while retaining the original inflammation is thought to cause pathology or tissue damage. 30 A list of the typical ADRs includes, but is not limited to, those composition of the tissue and minimizing the complications which are burn-associated Such as compartment syndrome, and scarring associated with the thermally induced burn in acidosis, acute renal failure, acute tubular necrosis, cellulitis, one or more of the recited conditions. secondary seizures, contractures, reduced end-organ perfu In yet another aspect of the invention, a method is provided Sion, endotoxemia, exotoxemia, gangrene, nosocomial pneu for Suppressing or modulating the immune system in a mam monia (50% of patients with burn/smoke inhalation injury malian patient in need of such immunosuppression compris 35 develop this type), ARDS (acute respiratory distress syn ing administering to said patient an immunosuppressing drome), ventilator associated pneumonia, sepsis, septic effective amount of a therapeutically effective amount of a shock, thromboembolic complications, and those other non pharmaceutical composition comprising an anti-cytokine or burn associated diseases with an inflammatory component anti-inflammatory agent or both or a functional derivative Such as, but not limited to, anemia, cancer, congestive heart thereof, and a pharmaceutically acceptable excipient. 40 failure, reduced end-organ perfusion, dermatomyositis In yet another aspect of the invention, a method is provided (DM), dermatitis, alveolar proteinosis pneumonia, bronchco for Suppressing the synthesis of potentially harmful inflam lotis obliterans organizing pneumonia (BOOP), chronic aspi matory molecules comprising cytokines, interleukins (for ration lipoid pneumonia, community acquired pneumonia example, IL-1, IL-8, IL-12, IL-18, TNF), nitric oxide, reac (CAP), coronavirus pneumonia, cryptoccal pneumonia, tive oxygen intermediates (ROI), prostaglandins, or any one 45 chlamydia pneumonia, descquamative interstitial pneumonia, or more of the known biological molecules involved in eosinophilic pneumonia, haemophilus influenza pneumonia, inflammatory signal transduction pathways, in a mammalian haemophilus influenza pneumonia, haemophilus parainflu patient in need of such anti-inflammation comprising admin enzae pneumonia, idiopathic pneumonia, influenza associ istering to said patient an antiinflammatory effective amount ated pneumonia, idiopathic interstitial pneumonia, kliebsiella of a therapeutically effective amount of a pharmaceutical pneumonia, mycoplasma pneumonia, non-specific interstitial composition comprising HR341 g or a functional derivative 50 pneumonia (associated with dermatomyositis-DM), pas thereof, and a pharmaceutically acceptable excipient, teurella multocida pneumonia, pneumocystis carinnii-(PCP) wherein said pharmaceutical composition Suppresses the Syn pneumonia, pseudomonas aeruginosa pneumonia, respira thesis of interleukins (for example, IL-1, IL-2, IL-8, IL-12, tory synctial virus infection, staphylococcal necrotising IL-18, TNF), nitric oxide, reactive oxygen intermediates pneumonia, tuberculosis pneumonia, usual interstitial pneu (ROI), prostaglandins, or any one or more of the known 55 monitis (UIP), varicella Zoster virus pneumonia, toxic shock biological molecules involved in inflammatory signal trans syndrome, and toxic epidermal necrosis (TEN). The follow duction pathways. ing list of diseases are associated with metabolic disarray As used herein, the term “cytokine' is a generic term for because of thermal injuries: cachexia, diarrhea, encephalopa proteins released by one cell population which act on another thy, myglobulinuria, and neurities. cell as intercellular mediators. Examples of Such cytokines 60 The drugs and/or topical agents that are conventionally are tumor necrosis factor-alpha and -beta; colony stimulating used to treat burns are limited in their use and scope. Table 1 factors (CSFs) such as macrophage-CSF (M-CSF); granulo indicates the drugs, listed with their benefits and drawbacks. cyte-macrophage-CSF (GM-CSF); and granulocyte-CSF None of these drugs, however, prevent or stop edema associ (G-CSF); interleukins (ILS) such as IL-1, IL-2, IL-8, IL-12, or ated with burns, since they have not been demonstrated to IL-18; and other polypeptide factors including leukemia 65 block the inflammatory response. The present invention spe inhibitory factor (LIF) and kit ligand (KL). As used herein, cifically provides for inclusion of one or more of the conven the term cytokine includes proteins from natural Sources or tional drugs in combination with HR341g. US 7,604,797 B2 25 26

TABLE 1.

Drug Type Benefits Drawbacks Bacitracin Ointment Effective against gram ineffective against gram Polypeptide positive cocci and bacilli. negative organisms, and Antibiotic Inhibits cell wall synthesis fungi. Shown to have a of bacteria. Enchances re negative effect on epithelialization. keratinocyte Safe, non-toxic proliferation. Some incidences of resistant strains. Use a petrolatum base that increase maceration. Ineffective against thickness injuries. Must be used up to 3 imes a day. Polymyxin Ointment Effective agains gram ineffective against gram B Sulfate Simple, negative organisms. positive organisms. It is basic Contains Surface bacteria. petrolatum based and peptide anti Affects cell membrane herefore promotes biotic permeability, killing maceration. Must be used microorganisms. Non-toxic up to 3 times a day. unless used for prolonged Caused massive periods reductions in keratinocyte proliferation. Is ineffective against gram negative strains of Paeruginosa. Suppresses PMN ability to destroy microorganisms. Neomycin Ointment Particularly effective Resistant organisms are Broad against gram-negative common. Is ineffective spectrum organisms and the gram against Some gram anti-biotic positive strain of S. aurents. negative and some gram also can be Controls the protein positive organisms. used. Can synthesis of bacteria by Ointment form promotes be used as a binding to a ribosomal maceration. C8 subunit. Inhibits the Hypersensitivity occurs proliferation of bacteria on requently (5% to 8%). wound Surfaces. Ototoxicity and Nephrotoxicity has been reported in cases with TBSA of 20% or more. Polysporin? Ointment Effective against gram Cannot be used on burns Neosporin (Polysporin) positive cocci and bacilli. of 20% or more of is a Effective against Some TBSA. Petrolatum base combination gram-negative organisms. promotes maceration. of Contains Surface bacteria. Causes massive Polymyxin inhibits the proliferation of reductions in B Sulfate bacteria on wound keratinocyte and Surfaces. proliferation. Is Bacitracin. ineffective against most (Neosporin) strains of Paeruginosa. is a Suppresses PMN ability combination o destroy of microorganisms. Neomysin, ineffective when used on Polymyxin full thickness injuries. B Sulfate. Povidone Ointment Effective against most Causes substantial delays Iodine Wide strains of gram-positive in wound healing. Toxic bactericidal and gram-negative o fibroblast cells and spectrum organisms. Able to oxidize keratinocytes. PMN or microbial protoplasm. Also Polymorphonuclear effective against candida eukocytes are inhibited and most fungi. by exposure to this drug. Toxic to children and pregnant women. Silver Cream Particularly effective Retardation of healing Sulfadiazine Topical against a wide range of ime likely to be expected 1%- Sulfonamide flora which include several because drug is toxic to Cream of Silver strains of gram-negative keratinocytes and Nitrate and bacteria and a few gram fibroblasts. Inhibits the Sodium positive oraganisms. effects of PMN in killing Sulfadiazine Superinfection and microorganisms. Also and resistance is rare. Promotes imits local lymphocyte prepared in wound healing because of function. The a 1% water its bactericidal properties. development of miscible Easy to use. Causes no kernicterus puts pregnant US 7,604,797 B2 27 28

TABLE 1-continued

Drug Type Benefits Drawbacks

C8. pain. Used for deep partial women and infants at and full thickness wounds. extreme risks for damage. Nitrofura Cream Effective against several Development of contact Zone 0.2% Broad anti gram-negative and a couple dermatitis, rash, local Compound bacterial of gram-positive edema, and pruitus has spectrum organisms. The mechanism been reported. Not of action appears to be by effective against any inhibition of bacterial fungal organisms, or enzymes. Causes no pain against gram-negative following application. Paeruginosa. Very toxic Formation of resistant to fibroblasts. A bacterial is rare. Can be detrimental effect on the mixed with other drugs growth and migration of keratinocytes. Gentamicin Cream Effective against several Not effective against O.1% Broad anti gram-negative organisms. mostgram-positive and bacterial Inhibits protein synthesis Some gram-negative spectrum and messenger ribonucleic organisms. Resistant acid translation. Not organisms are common excessively toxic to with its use. keratinocytes. Easy to Hypersensitivity is apply. No pain is common also. associated with its Ototoxicity and application. nephrotoxicity Sometimes occur, especially when the drug is used in large volumes over an extended period of time. Manfenide Cream Wide range of antibacterial The risk of toxcity is Acetate Methylated activity against mostgram high with risk to O.S9/o Sulfonamide positive and gram-negative respiratory status and ph Cream Compound pathogens. The formation status. Cases of Super (Sulfamylon) of resistant organisms is infection with candida rare. Controls Superficial can develop infections. Readily occassionally. Rashes absorbed into eschar and occur in 50% of the therefore high effective patients treated. Toxicity against invasive wound increases in correlation to infection. the TBSA burned and treated. Is toxic to both keratinocytes and fibroblasts. Nystatin Cream Effective against the most Several strains of candida Fungicide common candida fungal can develop resistance. It infection. Aid healing by is not effective against containing contagions. either gram-positive or Hypersentivity reactions gram-negative are rare, even with microorganisms. Must be extended use. Not toxic to applied 3 times a day. keratinocytes or Not effective when fibroblasts. Increased cell combined with other wall permeability is the agents. Limited in its use, mechanism for the drugs and is prone to Super lungicidal action. infection outbreaks in burn units. Acetic Solution Effective against many Reduced epithelial cell Acid 0.5% Acid based gram-positive and gram proliferation. Very toxic anti negative microorganisms, o regenerating bacterial especially Paeruginosa. epithelium. Reduces agent Penetrates cell wall and PMN function. Skin disrupts the cell membrane. irritation is common. Acidosis results from protracted use over large Surface area wounds. Toxic to fibroblasts. Must be applied requently to keep the wound moist. Must wash he wound between aplications. Sodium Solution Effective against most Toxic to fibroblast cells, Hypochlorite General gram-positive and some keratinocytes, and inhibits (Dakin's bactericidal, gram-negative he viability of of Solution) fungicidal, microorganisms. Also polymorphonuclear and effective against most eukocytess. The drug virucidal fungal infections. Toxicity dissolves blood clots and agent is rare. Effective against delays clotting. Bleeding is some viral infections. Used common in over 70% of th to irrigate wounds. patients that are treated. US 7,604,797 B2 29 30

TABLE 1-continued Drug Type Benefits Drawbacks Acidosis is common when TBSA is over 20% Silver Solution- Most effective against Extremely hypotonic Nitrate General gram-positive bacteria. Is because electrolytes O.S9/o bactericidal invulnerable to resistant leach into dressing, agent. Wet organisms. No cases of leading to chemical and to moist hypersensitivity reactions electrolyte imbalance. dressing have occurred. The wound Must be used on smaller healing is enhanced by the TBSA burns because of control of local infection. toxicity. The application Mildly effective against calls for frequent soaking some gram-negative every 2 hours. The organisms. Solution is painful to apply. Patient must be monitered for blood methemoglobinemia. TAB Solution- Low level of tissue Gram-positive organisms Solution Triple toxicity. A moderate level Such as Paeruginosa are (Triple antibiotic. of activity against of a not effected by this drug. Anti-biotic Wetto variety of gram-negative Occurences of Solution) moist and gram-positive hyperSensitivity reactions dressing. organisms. No resistant have been recorded. Also in an organisms are known to Inhibits the ability of ointment. exist. Limited level of PMN's to destroy toxicity to keratinocytes. ingested microorganisms. Uses Bacitracin, Skin rashes occur often Polymyxin B and up to 10% of the patients Neomycin studied. Ototoxity and nephrotoxicity have been reported. Chlorhexi- Solution- Effective against Some Ineffective against Dine General gram-positive several varieties of both Solution Antibiotic microorganisms. gram-negative and gram Apparently shows low positive organisms. Has levels of toxicity to cells no effect on fungal and there are no data on infections. Causes skin tissue toxicity. Can be used reactions with prolonged for different depths and use, including contact sizes ofburns. dermatitis and skin rashes. Must be changed constantly.

In each of the aforementioned aspects and embodiments of agent or procured from a commercial Source (e.g., pharma the invention, combination therapies other than those listed 40 ceutical company, such as Eli Lilly, Indianapolis, Ind., Sigma, above in Table 1 are also specifically contemplated herein. In St. Louis, Mo.). particular, the compositions of the present invention may be Anti-bacterial antibiotic agents include, but are not limited administered with one or more macrollide or non-macrollide to, penicillins, cephalosporins, carbacephems, cephamycins, antibiotics, anti-bacterial agents, anti-fungal agents, anti-vi carbapenems, monobactams, aminoglycosides, glycopep ral agents, anti-parasitic agents, and/or anti-inflammatory or 45 tides, quinolones, tetracyclines, macrollides, oxazalidiinones, immunomodulatory drugs or agents. streptogramins, and fluoroquinolones. Examples of antibiotic Examples of macrollide antibiotics that may be used in agents include, but are not limited to, lineZolid (Zyvax), dal combination with the composition of the present invention fopristine, quinupristine, Penicillin G (CAS Registry No.: include, interalia, the following synthetic, semi-synthetic or 61-33-6); Methicillin (CAS Registry No.: 61-32-5); Nafcillin naturally occurring microlidic antibiotic compounds: methy 50 (CAS Registry No.: 147-52-4): Oxacillin (CAS Registry No.: mycin, neomethymycin, YC-17, litorin, erythromycin A to F. 66-79-5); Cloxacillin (CAS Registry No.: 61-72-3); Diclox oleandomycin, roXithromycin, dirithromycin, flurithromy acillin (CAS Registry No.: 3116-76-5); Ampicillin (CAS cin, clarithromycin, davercin, azithromycin, josamycin, Registry No.: 69-53-4); Amoxicillin (CAS Registry No.: kitasamycin, spiramycin, midecamycin, rokitamycin, mioka 26787-78-0): Ticarcillin (CAS Registry No.: 34787-01-4); mycin, lankacidin, and the derivatives of these compounds. 55 Carbenicillin (CAS Registry No.: 4697-36-3); Mezlocillin Thus, erythromycin and compounds derived from erythromy (CAS Registry No.: 51481-65-3): Azlocillin (CAS Registry cin belong to the general class of antibiotics known as “mac No.: 37091-66-0); Piperacillin (CAS Registry No. 61477 rollides.” Examples of preferred erythromycin and erythro 96-1): Imipenem (CAS Registry No.: 74431-23-5): Aztre mycin-like compounds include: erythromycin, onam (CAS Registry No. 78110-38-0); Cephalothin (CAS clarithromycin, azithromycin, and troleandomycin. 60 Registry No.: 153-61-7); Cefazolin (CAS Registry No.: Additional antibiotics, other than the macrolidic antibiot 25953-19-9); Cefaclor (CAS Registry No.: 70356-03-5); ics described above, which are suitable for use in the methods Cefamandole formate sodium (CAS Registry No.: 42540-40 of the present invention include, for example, any molecule 9); Cefoxitin (CAS Registry No.: 35607-66-0); Cefuroxime that tends to prevent, inhibit or destroy life and as such, and as (CAS Registry No. 55268-75-2); Cefonicid (CAS Registry used herein, includes anti-bacterial agents, anti-fungal 65 No. 61270-58-4); Cefinetazole (CAS Registry No.: 56796 agents, anti-viral agents, and anti-parasitic agents. These 20-4); Cefotetan (CAS Registry No.: 697 12-56-7); Cefprozil agents may be isolated from an organism that produces the (CAS Registry No. 92665-29-7); Loracarbef (CAS Registry US 7,604,797 B2 31 32 No. 121961-22-6); Cefetamet (CAS Registry No.: 65052 e.g., by stimulating or Suppressing a cellular activity of a cell 63-3); Cefoperazone (CAS Registry No. 62893-19-0); Cefo in the immune system, e.g., T-cells, B-cells, macrophages, or taxime (CAS Registry No.: 63527-52-6); Ceftizoxime (CAS other antigen presenting cells (APC), or by acting upon com Registry No.: 68401-81-0); Ceftriaxone (CAS Registry No.: ponents outside the immune system which, in turn, stimulate, 73384-59-5); Ceftazidime (CAS Registry No.: 72558-82-8); Suppress, or modulate the immune system, e.g., hormones, Cefepime (CAS Registry No. 88040-23-7); Cefixime (CAS receptor agonists or antagonists, and neurotransmitters; Registry No. 79350-37-1); Cefpodoxime (CAS Registry immunomodulators can be, e.g., immunosuppressants or No. 80210-62-4); Cefsulodin (CAS Registry No. 62587-73 immunostimulants. By “anti-inflammatory drugs, it is 9); Fleroxacin (CAS Registry No. 79660-72-3): Nalidixic meant, e.g., agents which treat inflammatory responses, i.e., a acid (CAS Registry No. 389-08-2); Norfloxacin (CAS Reg 10 tissue reaction to injury, e.g., agents which treat the immune, istry No. 70458-96-7); Ciprofloxacin (CAS Registry No.: vascular, or lymphatic systems. 85721-33-1): Ofloxacin (CAS Registry No. 82419-36-1): Anti-inflammatory or immunomodulatory drugs or agents Enoxacin (CAS Registry No.: 7401 1-58-8); Lomefloxacin suitable for use in this invention include, but are not limited (CAS Registry No.: 98.079-51-7); Cinoxacin (CAS Registry to, interferon derivatives, e.g., betaseron, beta.-interferon; No. 28657-80-9); Doxycycline (CAS Registry No. 564-25 15 prostane derivatives, e.g., compounds disclosed in PCT/ 0); Minocycline (CAS Registry No.: 10118-90-8); Tetracy DE9370013, e.g., iloprost, cicaprost; glucocorticoid, e.g., cor cline (CAS Registry No.: 60-54-8); Amikacin (CAS Registry tisol, prednisolone, methylprednisolone, dexamethasone; No. 37517-28-5); Gentamicin (CAS Registry No.: 1403-66 immunsuppressives, e.g., cyclosporine A, FK-506, methox 3); Kanamycin (CAS Registry No. 8063-07-8); Netilmicin salene, thalidomide, SulfaSalazine, azathioprine, methotrex (CAS Registry No. 56391-56-1); Tobramycin (CAS Regis ate; lipoxygenase inhibitors, e.g., Zileutone, MK-886, try No. 32986-56-4); Streptomycin (CAS Registry No.: WY-50295, SC-45662, SC-41661A, BI-L-357; leukotriene 57-92-1): Azithromycin (CAS Registry No. 83905-01-5); antagonists, e.g., compounds disclosed in DE 40091171 Ger Clarithromycin (CAS Registry No. 81 103-11-9); Erythro man patent application P 42 42 390.2: WO 9201675: mycin (CAS Registry No.: 114-07-8); Erythromycinestolate SC-41930; SC-50605; SC-51146; LY 255283 (D. K. Herron (CAS Registry No.: 3521-62-8); Erythromycin ethyl succi 25 et al., FASEB J. 2: Abstr. 4729, 1988); LY 223982 (D. M. nate (CAS Registry No.: 41342-53-4); Erythromycin gluco Gapinski et al. J. Med. Chem. 33: 2798-2813, 1990); heptonate (CAS Registry No.: 23067-13-2); Erythromycin U-753.02 and analogs, e.g., described by J. Morris et al., lactobionate (CAS Registry No. 3847-29-8); Erythromycin Tetrahedron Lett. 29: 143-146, 1988, C. E. Burgos et al., stearate (CAS Registry No.: 643-22-1); Vancomycin (CAS Tetrahedron Lett. 30: 5081-5084, 1989; B. M. Taylor et al., Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.: 30 Prostaglandins 42: 211-224, 1991; compounds disclosed in 61036-64-4); Chloramphenicol (CAS Registry No. 56-75 U.S. Pat. No. 5,019,573; ONO-LB-457 and analogs, e.g., 7); Clindamycin (CAS Registry No.: 18323-44-9); Trimetho described by K. Kishikawa et al., Adv. Prostagl. Thombox. prim (CAS Registry No.:738-70-5); Sulfamethoxazole (CAS Leukotriene Res. 21: 407-410, 1990; M. Konno et al., Adv. Registry No.: 723-46-6); Nitrofurantoin (CAS Registry No.: Prostagl. Thrombox. Leukotriene Res. 21: 411-414, 1990; 67-20-9); Rifampin (CAS Registry No.: 13292-46-1); Mupi 35 WF-11605 and analogs, e.g., disclosed in U.S. Pat. No. 4,963, rocin (CAS Registry No. 12650-69-0); Metronidazole (CAS 583; compounds disclosed in WO 9118601, WO 9118879; Registry No.: 443-48-1); Cephalexin (CAS Registry No.: WO 9118880, WO 9118883, antiinflammatory substances, 15686-71-2); Roxithromycin (CAS Registry No. 80214-83 e.g., NPC 16570, NPC 17923 described by L. Noronha-Blab. 1); Co-amoxiclavuanate; combinations of Piperacillin and et al., Gastroenterology 102 (Suppl.): A 672, 1992: NPC TaZobactam, and their various salts, acids, bases, and other 40 15669 and analogs described by R. M. Burchet al., Proc. Nat. derivatives. Acad. Sci. USA 88: 355-359, 1991; S. Pou et al., Biochem. Anti-fungal agents include, but are not limited to, terbin Pharmacol. 45: 2123-2127, 1993; peptide derivatives, e.g., afine hydrochloride, nystatin, amphotericin B, griseofulvin, ACTH and analogs; IL-1 receptor antagonists, IL-18 binding ketoconazole, miconazole nitrate, flucytosine, fluconazole, protein, activated protein C (Xigris), soluble TNF-receptors: itraconazole, clotrimazole, benzoic acid, Salicylic acid, Vori 45 TNF-antibodies; soluble receptors of interleukins, other conazole, caspofungin, and selenium sulfide. cytokines, T-cell-proteins; antibodies against receptors of Anti-viral agents include, but are not limited to, amanta interleukins, other cytokines, and T-cell-proteins (the text of dine hydrochloride, rimantadin, acyclovir, famciclovir, fos each of the afore-mentioned references is expressly incorpo carnet, ganciclovir Sodium, idoxuridine, ribavirin, Sorivu rated by reference herein). dine, trifluridine, Valacyclovir, Vangancyclovir, pencyclovir, 50 Additional Uses Vidarabin, didanosine, stavudine, Zalcitabine, Zidovudine, The present invention also has applications in emergency interferon alpha, and edoxudine. kits outfitted to contain a pharmaceutical composition com Anti-parasitic agents include, but are not limited to, pire prising an anti-cytokine oranti-inflammatory agent or a func thrins/piperonyl butoxide, permethrin, iodoquinol, metron tional derivative thereof, including HR341g or a functional idazole, diethylcarbamazine citrate, piperazine, pyrantel 55 derivative thereof so that pharmaceutical formulations com pamoate, mebendazole, thiabendazole, praziquantel, prising an anti-cytokine oranti-inflammatory agent or a func albendazole, proguanil, quinidine gluconate injection, qui tional derivative thereof such as HR341g can be made avail nine Sulfate, chloroquine phosphate, mefloquine hydrochlo able for use in every emergency first aid kit. Such topical ride, primaquine phosphate, atovaquone, co-trimoxazole formulations can be applied to the skin immediately or (sulfamethoxazole/trimethoprim), and pentamidineisethion 60 shortly after an accident or injury. For example, such emer ate. gency kits would be invaluable in each household for use in In another aspect, in the method of the present invention, emergency household accidents, in the car, including residen one may, for example, Supplement the composition by admin tial vehicles, commercial vehicles, and most emergency istration of a therapeutically effective amount of one or more response and police vehicles. an anti-inflammatory or immunomodulatory drugs or agents. 65 The present invention also has applications in all types of By “immunomodulatory drugs or agents', it is meant, e.g., Sunburn and would be employed in post-Sun exposure care to agents which act on the immune system, directly or indirectly, prevent skin cancer, prevent blistering, sooth, cool and US 7,604,797 B2 33 34 reduce/eliminate the pain of sunburns. The present invention tissues of humans and lower animals without undue toxicity, also has applications in artificial Suntanning salons. irritation, allergic response and the like, and are commensu The present invention also has applications in all fields of rate with a reasonable benefit/risk ratio. Pharmaceutically professional uses including for example, hospitals, emer acceptable salts are well known in the art. For example, S. M gency and burn treatment, doctor office, general practitio Berge, et al. describe pharmaceutically acceptable salts in ners office, ambulances and emergency vehicles, high risk detail in J. Pharmaceutical Sciences, 1977, 66:1-19. The salts industries, fire fighting, military, navy, law enforcement, can be prepared in situ during the final isolation and purifi mechanical workshops, auto repair, welding etc., and restau cation of the compounds of the invention, or separately by rantS. reacting the free base function with a suitable organic acid. The present invention also has applications in the field of 10 Representative acid addition salts include acetate, adipate, fire extinguishers and fire retardant materials in general, as alginate, ascorbate, aspartate, benzene-Sulfonate, benzoate, well as possible uses in mandatory safety equipment that are bisulfate, borate, butyrate, camphorate, campherSulfonate, modified to contain HR341g. citrate, cyclopentanepropionate, digluconate, dodecylsulfate, The present invention additionally has applications in the ethanesulfonate, fumarate, glucoheptonate, glycerophos field of cosmetics, including for example, Sunburn care, burn 15 phate, hemisulfate, heptonate, hexanoate, hydrobromide, treatment, treatment of certain cancers, scar removal, post hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lac laser treatment care, including, for example, lasers used in tobionate, lactate, laurate, lauryl Sulfate, malate, maleate, hair removal and other cosmetic procedures, as well as malonate, methanesulfonate, 2-naphthalenesulfonate, nicoti wrinkle removal. nate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, Modes of Administration and Pharmaceutical Composi persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, tions propionate, Stearate. Succinate, Sulfate, tartrate, thiocyanate, In general, the composition of the present invention is toluenesulfonate, undecanoate, Valerate salts, and the like. intended to be applied topically and directly to the burns or Representative alkali or alkaline earth metal salts include wound as described above. When the wound is deep, or the Sodium, lithium, potassium, calcium, magnesium, and the burn severe, it is preferred that the composition is in the form 25 like, as well as nontoxic ammonium, quaternary as ammo of an ointment, Salve or cream which is spread directly onto nium, and mine cations, including, but not limited to ammo the wound and then covered with a standard sterile dressing nium, tetramethylammonium, tetraethylammonium, methy pad or other appropriate dressing material. Alternatively, the lamine, dimethylamine, trimethylamine, triethylamine, ointment, cream or salve of the present composition is applied ethylamine, and the like. directly onto the dressing pad or other appropriate dressing 30 The present invention also provides pharmaceutical com material. The pad or dressing material is then placed over the positions which comprise one or more of the anti-cytokine or wound or burn with the medicine-side down. This latter anti-inflammatory agent compounds described above formu approach works better when applying dressing to severe lated together with one or more non-toxic pharmaceutically burns and shallow wounds. For first degree burns and slight acceptable carriers. The pharmaceutical compositions may abrasions, in addition, the composition may be applied in 35 be specially formulated for oral administration in solid or aerosolized form. liquid form, for parenteral injection, or for rectal administra Thus, the pharmaceutical composition of the present tion. invention is applied to a wound so as to cover the injured The pharmaceutical compositions of this invention can be Surface completely, e.g., with, for example, and not by way of administered to humans and other animals orally, rectally, limitation, one-quarter inch thickness of the pharmaceutical 40 parenterally, intracistemally, intravaginally, intraperito composition. The only limitation on the application is that the neally, topically (as by powders, ointments, or drops), pharmaceutical composition should be applied within the first bucally, or as an oral or nasal spray. The term "parenteral twenty minutes following the burn or injury but may also be administration as used herein refers to modes of administra applied as soon as possible but preferably before 12 hours. tion which include intravenous, intramuscular, intraperito Dressing-change schedules are of course dictated by the con 45 neal, intrathecally, intrasternal. Subcutaneous and intraarticu dition of the wound. In highly-contaminated (wounds exhib lar injection and infusion. iting significant amounts of pus) or weeping wounds or severe Pharmaceutical compositions of this invention for burns, dressing changes may be performed every four to six parenteral injection comprise pharmaceutically acceptable hours; in other wounds or burns, changes are performed less sterile aqueous or nonaqueous solutions, dispersions, Suspen frequently, sometimes only one or two times per day. 50 sions, or emulsions as well as sterile powders for reconstitu Dressings are advantageously changed three to four times tion into sterile injectable solutions or dispersions just prior to a day. Repeated daily dressing changes are continued until the use. Examples of Suitable aqueous and nonaqueous carders, wound or burn is healed. Healing time varies, depending upon diluents, solvents, or vehicles include water, ethanol, polyols the type and depth of the wound or the severity of the burn. (such as glycerol, propylene glycol, polyethylene glycol, and The present pharmaceutical composition is effective in the 55 the like), and suitable mixtures thereof, vegetable oils (such treatment of a large variety of wounds and burns to a mammal, as olive oil), and injectable organic esters such as ethyl oleate. subject or patient in need thereof where bacterial and fungal Proper fluidity can be maintained, for example, by the use of contamination would ordinarily occur in the absence of treat coating materials such as lecithin, by the maintenance of the ment. The present medicinal composition can of course also required particle size in the case of dispersions, and by the use be used to treat burns and wounds in other mammals, such as 60 of Surfactants. Veterinary animals including, without limitation, dogs, cats, These compositions may also contain adjuvants such as other household pets, horses, farm animals, and the like. preservative, wetting agents, emulsifying agents, and dispers The compounds of the present invention include pharma ing agents. Prevention of the action of microorganisms may ceutically acceptable salts that can be prepared by those of be ensured by the inclusion of various antibacterial and anti skill in the art. As used herein, by “pharmaceutically accept 65 fungal agents, for example, paraben, chlorobutanol, phenol able salt it is meant those salts which are, within the scope of sorbic acid, and the like. It may also be desirable to include Sound medical judgment, Suitable for use in contact with the isotonic agents such as Sugars, Sodium chloride, and the like. US 7,604,797 B2 35 36 Prolonged absorption of the injectable pharmaceutical form used in the art Such as, for example, water or other solvents, may be brought about by the inclusion of agents which delay solubilizing agents and emulsifiers such as ethyl alcohol, absorption Such as aluminum monostearate and gelatin. isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alco In some cases, in order to prolong the effect of the drug, it hol, benzyl benzoate, propylene glycol. 1,3-butylene glycol, is desirable to slow the absorption of the drug from subcuta dimethyl formamide, oils (in particular, cottonseed, ground neous or intramuscular injection. This may be accomplished nut, corn, germ, olive, castor, and sesame oils), glycerol, by the use of a liquid Suspension of crystalline or amorphous tetrahydrofurfuryl alcohol, polyethylene glycols and fatty material with poor water solubility. The rate of absorption of acid esters of sorbitan, and mixtures thereof. the drug then depends upon its rate of dissolution which, in Besides inert diluents, the oral compositions can also turn, may depend upon crystal size and crystalline form. 10 include adjuvants such as wetting agents, emulsifying and Alternatively, delayed absorption of a parenterally adminis Suspending agents, Sweetening, flavoring, and perfuming tered drug form is accomplished by dissolving or Suspending agents. the drug in an oil vehicle. Suspensions, in addition to the active compounds, may Injectable depot forms are made by forming microencap contain Suspending agents as, for example, ethoxylated isos Sule matrices of the drug in biodegradable polymers such as 15 tearyl alcohols, polyoxyethylene Sorbitol and Sorbitan esters, polylactide-polyglycolide. Depending upon the ratio of drug microcrystalline cellulose, aluminum metahydroxide, bento to polymer and the nature of the particular polymer nite, agar-agar and tragacanth, and mixtures thereof. employed, the rate of drug release can be controlled. Compositions for rectal or vaginal administration are pref Examples of other biodegradable polymers include poly erably suppositories which can be prepared by mixing the (orthoesters) and poly(anhydrides). Depot injectable formu compounds of this invention with Suitable non-irritating lations are also prepared by entrapping the drag in liposomes excipients or carriers such as cocoa butter, polyethylene gly or microemulsions which are compatible with body tissues. col, or a Suppository wax which are solid at room temperature The injectable formulations can be sterilized, for example, but liquid at body temperature and therefore melt in the rec by filtration through a bacteria-retaining filter or by incorpo tum or vaginal cavity and release the active compound. rating sterilizing agents in the form of sterile solid composi 25 The pharmaceutical compositions of the present invention tions which can be dissolved or dispersed in sterile water or can also be administered in the form of liposomes. As is other sterile injectable medium just prior to use. known in the art, liposomes are generally derived from phos Solid dosage forms for oral administration include cap pholipids or other lipid Substances. Liposomes are formed by Sules, tablets, pills, powders, and granules. In Such solid dos mono- or multi-lamellar hydrated liquid crystals that are dis age forms, the active compound is mixed with at least one 30 persed in an aqueous medium. Any non-toxic, physiologi inert, pharmaceutically acceptable excipient or carrier Such as cally acceptable and metabolizable lipid capable of forming sodium citrate or dicalcium phosphate and/or (a) fillers or liposomes can be used. The present compositions in liposome extenders such as starches, lactose, Sucrose, glucose, manni form can contain, in addition to a pharmaceutical composi tol, and silicic acid, (b) binders such as, for example, car tion of the present invention, stabilizers, preservatives, boxymethylcellulose, alginates, gelatin, polyvinylpyrroli 35 excipients, and the like. The preferred lipids are the phospho done. Sucrose, and acacia, (c) humectants such as glycerol, lipids and the phosphatidylcholines (lecithins), both natural (d) disintegrating agents such as agar-agar, calcium carbon and synthetic. ate, potato or tapioca starch, alginic acid, certain silicates, and Methods to form liposomes are known in the art. See, for Sodium carbonate, (e) solution retarding agents such as par example, Prescott, Ed., Methods in Cell Biology, Volume affin, (f) absorption accelerators such as quaternary ammo 40 XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq. nium compounds, (g) wetting agents such as, for example, Dosage forms for topical administration of a pharmaceu cetyl alcohol and glycerol monostearate, (h) absorbents such tical compositions of this invention include powders, sprays, as kaolin and bentonite clay, and (i) lubricants such as talc, ointments, and inhalants. The active compound is mixed calcium Stearate, magnesium Stearate, Solid polyethylene gly under Sterile conditions with a pharmaceutically acceptable cols, Sodium lauryl Sulfate, and mixtures thereof. In the case 45 carrier and any needed preservatives, buffers, or propellants of capsules, tablets and pills, the dosage form may also com which may be required. Opthalmic formulations, eye oint prise buffering agents. ments, powders and solutions are also contemplated as being Solid compositions of a similar type may also be employed within the scope of this invention. as fillers in Soft and hard-filled gelatin capsules using Such Actual dosage levels of active ingredients in the pharma excipients as lactose or milk Sugar as well as high molecular 50 ceutical compositions of this invention may be varied so as to weight polyethylene glycols and the like. obtainanamount of the active compound(s) that is effective to The solid dosage forms of tablets, dragees, capsules, pills, achieve the desired therapeutic response for a particular and granules can be prepared with coatings and shells such as patient, compositions, and mode of administration. The enteric coatings and other coatings well known in the phar selected dosage level will depend as upon the activity of the maceutical formulating art. They may optionally contain 55 particular pharmaceutical compound or analogue thereof of opacifying agents and can also be of a composition that they the present invention, the route of administration, the severity release the active ingredient(s) only, or preferentially, in a of the condition being treated, and the condition and prior certain part of the intestinal tract, optionally, in a delayed medical history of the patient being treated. However, it is manner. Examples of embedding compositions which can be within the skill of the art to start doses of the pharmaceutical used include polymeric Substances and waxes. 60 compound at levels lower than required to achieve the desired The active compounds can also be in micro-encapsulated therapeutic effect and to gradually increase the dosage until form, if appropriate, with one or more of the above-men the desired effect is achieved. tioned excipients. The pharmaceutical compositions of the present invention Liquid dosage forms for oral administration include phar can be used in both veterinary medicine and human therapy. maceutically acceptable emulsions, Solutions, Suspensions, 65 The magnitude of a prophylactic or therapeutic dose of the syrups, and elixirs. In addition to the active compounds, the pharmaceutical composition of the invention in the acute or liquid dosage forms may contain inert diluents commonly chronic management of pathology and pain associated with US 7,604,797 B2 37 38 above-mentioned diseases or indications will vary with the tion initially receive low doses, and that they then be titrated severity of the condition to be treated and the route of admin based on individual response(s) or blood level(s). It may be istration. The dose, and perhaps the dose frequency, will also necessary to use dosages outside these ranges in Some cases, vary according to the age, body weight, and response of the as will be apparent to those of ordinary skill in the art. Further, individual patient. In general, the total dose range of the it is noted that the clinician or treating physician will know, pharmaceutical composition of this invention is generally with no more than routine experimentation, how and when to between about 0.001 to about 100 mg, preferably about 0.01 interrupt, adjust, or terminate therapy in conjunction with to about 20 mg, and more preferably about 16 mg of active individual patient response. compound per kilogram of body weight are administered The term “unit dose' is meant to describe a single dose, topically to a mammalian patient. If desired, the effective 10 although a unit dose may be divided, if desired. Although any dose may be divided into multiple doses for purposes of suitable route of administration may be employed for provid administration, e.g. two to four separate doses. ing the patient with an effective dosage of the composition Alternatively, the total dose range of the active ingredients according to the methods of the present invention, topical of this invention is generally between about 1 and 500 mg per administration is preferred. Suitable routes include, for 70 kg of body weight per day, or about 10 and 500 mg per 70 15 example, topical, transdermal, Subcutaneous, intramuscular, kg of body weight per day, between about 50 and 250 mg per by inhalation, and like forms of administration may be 70 kg of body weight per day, and more preferably between employed. Suitable dosage forms include nasal sprays, tro about 100 and 150 mg per 70 kg of body weight per day. ches, dispersions, Suspensions, solutions, patches, and the It is intended herein that by recitation of such specified like, although topical and/or nasal dosage forms are pre ranges, the ranges cited also include all those dose range ferred. amounts between the recited range. For example, in the range Useful dosages of the compounds of the present invention about 1 and 500, it is intended to encompass 2 to 499, 3-498, can be determined by comparing their in vitro activity, and in etc., without actually reciting each specific range. The actual vivo activity in animal models. Methods for the extrapolation preferred amounts of the active ingredient will vary with each ofeffective dosages in mice, and other animals, to humans are case, according to the species of mammal, the nature and 25 known to the art; for example, see U.S. Pat. No. 4.938,949. severity of the particular affliction being treated, and the method of administration. The present invention is illustrated by the Examples that It is also understood that doses within those ranges, but not follow, it being understood, however, that the invention is not explicitly stated, such as 30 mg, 50 mg, 75 mg, etc. are limited to the specific details of these Examples. encompassed by the stated ranges, as are amounts slightly 30 outside the stated range limits. EXAMPLE ONE Alternatively, the total dose range of the pharmaceutical compositions of this invention is generally between about Introduction 10 and 10 molar range per 70kg of body weight, or about 107 and 10 molar range per 70 kg of body weight, prefer 35 This example outlines the procedure for preparing the ably between about 10 and 10 molar range per 70 kg of pharmaceutical composition of the present invention. body weight, and more preferably between about 10" molar Materials and Methods range per 70 kg of body weight (in cream form, aminopterin Preparation of HR341g may be included up to 100 micromolar). It is intended herein HR341g is made according to the following procedure for that by recitation of Such specified ranges, the ranges cited 40 the topical formulation. In brief, Dicalcium phosphate dihy also include all those concentration amounts between the drate (DCP). Insoluble sodium metaphosphate, Sorbitol recited range. For example, in the range about 10 and 10 syrup (70% solution), Guar gum, Xanthan gum or Pluronic molar range, it is intended to encompass 1.1x10 to F87, Monosodium phosphate, Sodium monofluorophos 9.9x10, 1.2x10 to 9.8x10", etc., without actually reciting phate, Aminopterin, Titanium dioxide, Sodium dodecylben each specific range. The actual preferred amounts of the 45 Zene Sulphate, Water, Trimagnesium phosphate, and active ingredients will vary with each case, according to the Hydroxethyl cellulose ester are added to a high sheer mixer in species of mammal, the nature and severity of the particular the amounts shown in Table 2 (w/v), and filtered through a affliction being treated, and the method of administration. In 0.007 inch screen. any event, the concentration of the active ingredients in the topical cream formulation should include aminopterin or a 50 TABLE 2 functional derivative thereof in a concentration of 1-100 uM and/or sodium monoflourophosphate of 0.1 to 1.0M. Particu Ingredients Weight (w/v) larly preferred concentrations foraminopterin or a functional Dicalcium phosphate dihydrate (DCP) 1150 grams derivative thereof are at a concentration of 33.13 LM Insoluble sodium metaphosphate 700 grams (0.0015%) and/or sodium monoflourophosphate at a concen 55 Sorbitol syrup (70% solution) 1250 grams Guar gum 225 grams tration of 0.663 M (8.9%). Xanthan gum 90 grams In general, the pharmaceutical compositions of the present Monosodium phosphate 15 grams invention are periodically administered to an individual Sodium monofluorophosphate 477 grams patient as necessary to improve symptoms of the particular Aminopterin 80 milligrams Titanium dioxide 30 grams disease being treated. The length of time during which the 60 Sodium dodecylbenzene sulphate 25 grams compositions are administered and the total dosage will nec Water 1200 grams essarily vary with each case, according to the nature and Trimagnesium phosphate 40 grams severity of the particular affliction being treated and the Hydroxethyl cellulose ester 157.5 grams physical condition of the Subject or patient receiving Such treatment. 65 It is further recommended that children, patients of age This formulation can be made in an approximately 5 kilogram over 65 years, and those with impaired renal or hepatic func quantity in a Molteni TM5 mixer or any other high shear US 7,604,797 B2 39 40 mixer known to those of skill in the art, in the following Material and Methods stages. Mixing should be carried out under vacuum. Patient A In stage 1, the ingredients are added in the following order Patient A arrives at the hospital 15 minutes after he was and amounts: 90 degree Centigrade Water (1105 g) and Sor burned. The patientis suffering from 2" and 3" degree burns, bitol Syrup (417 g) are put in the mixer. Monosodium Phos and fluid resuscitation based upon the Parkland formula is phate (15g), Sodium Monofluorophosphate (477 g) and Ami administered to the patient. Within 2 hours inflammation and nopterin (80 mg) are then added and mixed for 12 minutes at edema develop. The burn results in the release of local media 6,000 rpm. In stage 2, dry mix Guar Gum (225 g), Xanthan tors. These mediators cause even more inflammation and Gum (90 g), Titanium Dioxide (30 g), Dicalcium Phosphate edema to develop, and complement activation is triggered Dihydrate (DCP) (1150 g). Insoluble Sodium Metaphosphate 10 that causes further systemic mediator production. (700 g), and Hydroxethyl cellulose ester (157.5g) in a con Among these mediators, arachidonic acid, cytokine pro tainer and then slowly add dry mix combination into the stage duction (including IL-1 and TNF), NO, reactive oxygen inter I mix. Mix for 10 minutes at 7,000 rpm. In stage 3, sorbitol mediates (ROI), are produced. This results in neutrophil Syrup (417 g) is added into the mixer and mixed for 5 minutes sequestration and priming of both neutrophils and macroph at 7,000 rpm. In stage 4, Sodium Dodecylbenzene Sulphate 15 ages locally and systematically. (25 g) and Sorbitol Syrup (417 g) are added to the remainder The progression of burn wound inflammation increases, of the room temperature mix for 5 minutes at 7,000 rpm. including the generation of circulating immunosuppressive Separately mix dodecylbenzene sulphate (25g) and sorbitol compounds. IL-6 is released, which initiates liver acute phase syrup (416 g) and 95 groom temperature water. This wet mix protein production. Hyper metabolism develops resulting in is then added into the high sheer mixer and mixed for 15 muscle catabolism. The patient loses weight if inflammation minutes at 7,000 rpm. Pumping the mixture through a 0.007 is prolonged. Since both the neutrophils and macrophages are inch screen enhances the Smoothness of the cream. The mix primed, massive amounts of oxidants, arachidonic acid should be smooth with no grainy texture. Viscosity should be metabolites, cytokines and proteases can be produced. This consistent throughout. When complete, the mixture is then action causes further local and systemic inflammation that packed into tubes. 25 induces tissue damage. The higher levels of mediators, par Results ticularly cytokines, increase the damage, leading to even more inflammation. The finish product should be in the proportions shown in Patient B Table 3. Patient Barrives at the hospital 15 minutes after he was 30 injured; the patient is suffering from 2" and 3" degree burns TABLE 3 and fluids based upon the Parkland formula are administered. Ingredients Weight (w/v) Percentage (w/v) HR341g is applied to the burn areas. Edema is substantially reduced at the burn site. There is some inflammation, which is Dicalcium phosphate dihydrate 1150 grams 21.4 (DCP) necessary for proper healing, but there are no excessive reac Insoluble sodium metaphosphate 700 grams 13.0 35 tions as in Patient A. Even though mediators such as cytok Sorbitol syrup (70% solution) 1250 grams 23.3 ines, oxidants and arachidonic acid are released, the absolute Guar gum 225 grams 4.2 amounts are Smaller than in Patient A, and edema remains at Xanthan gum 90 grams 1.7 Monosodium phosphate 15 grams O.28 manageable levels. Cytokine production and growth factors Sodium monofluorophosphate 477 grams 8.9 affect “target cells' through receptors found on the target Aminopterin 80 milligrams O.OO15 40 cells. Most individual receptors are highly specific and can Titanium dioxide 30 grams O.S6 only recognize one molecule. So in the case of a burn wound, Sodium dodecylbenzene sulphate 25 grams O46 Water 1200 grams 22.4 several cells may react to a single growth factor, yet each cell Trimagnesium phosphate 40 grams O.74 may respond differently. The specific receptors for each Hydroxethyl cellulose ester 157.5 grams 2.9 growth factor ensure that the cellular response will also be 45 unique. Interleukin I (IL-1) is produced by macrophages, monocytes, skin cells, and its release can cause fever. IL-2 further stimulates T-lymphocytes and activates natural killer EXAMPLE TWO (NK) cells. Other interleukins stimulate the proliferation of Introduction bone marrow cells, either broadly or very selectively. Patient 50 B has an increased recruitment of cells into the wound, increased collagen formation and organization and wound This example outlines the testing of the pharmaceutical strength. Furthermore, Patient B suffered minimum associ composition in the methods of the present invention. The ated disease responses (ADRs), because microorganisms study of three hypothetical burn patients is presented. These need the environment of a burn wound to proliferate, that studies are designed to represent typical patients. Patients A, 55 environment has been altered with HR431 g. B and C were admitted to the hospital at the same time, with Patient C total burn surface area (TBSA) burns of 30%. The patients Patient C was intubated immediately because of facial burns were in the upper chest area, and on their upper backs. burns. The risk is two fold because if the patient has pulmo Patient C, in addition had small burns on the side of his face. nary injury, then over 50% develop nosocomial pneumonia. Post-burn injury in these patients is due to inflammation 60 There is also a 35% chance that if the patient is placed on a with associated edema that peaks several days post burn. ventilator, he will also develop pneumonia. If pulmonary Also, without Surgery, 48 hours after a burn, bacterial micro edema develops before the patient is placed on a ventilator, organisms may invade the burn wound. In some patients, there is a near 100% chance that the patient will die. Patient C there is an extreme systematic inflammatory response to the didn't suffer detectable pulmonary injury, but to be safe burn. In what is described as “after burn', the systematic 65 Patient C was placed on a ventilator. inflammatory response progresses until an “associated dis Patient C develops thermal edema 2 hours after being ease response' is evident. admitted to the hospital. The results of inflammatory changes US 7,604,797 B2 41 42 caused by the release of cytokines such as TNF, IL-1, IL-2, Patient B recovers, because edema was controlled. The IL-8, and IL-6. These increases cause additional priming of patient needs no Surgery and is released in 3 months with a neutrophils released from the bone marrow. IL-1 action high quality of life. causes T-cell proliferation by inducing more IL-2 receptors. Patient C never recovered from his injuries. The patient Immediately, Patient C begins to show the effects of post-burn suffered several ADRs, debridments, escharotomies and skin hemodynamic instability. The patient’s blood pressure is grafts. The patient also suffered several deformities including altered, cardiac output falls, and signs of hypovolemia are contractures, hypertophic scarring, and several operations. revealed. However, intravascular Volume is maintained and Patient C will spend over 5 years in and out of hospitals, and cardiac output returns to normal over the next 24 hours. A require a doctor's care for the rest of his life. generalized capillary leak occurs in unburned areas. How 10 No burn patients suffer the same inflammatory reactions. ever, this capillary permeability is only transiently changed in Inflammation that supercedes certain thresholds interfere the unburned areas. Vasoactive amine release is the cause of with the healing process. These changes can induce capillary the increased microvascular permeability. The edema may be leaks that alter chemical balances. The key component of the exacerbated due to burn induced hypoproteinemia. equation is the elimination of inflammation an associated In the next 48 hours the patientis infected by gram-positive 15 edema and irreversible ischemia. Once inflammatory edema microorganism and 72 hours after that, by gram-negative is reduced, patients will have a decreased requirement for not pathogens. Since the capillary leak that occurred immediately need skin grafts, debridment, escharotomies or any other type after the burn was never addressed. Patient C’s burn was of Surgery. HR341g blocks inflammation and edema forma incompletely addressed. Patient C’s circulating immunosup tion by preventing further damage from after burn. In hospi pressive compounds, such as adrenal corticosteroids, anti tals today inflammation and edema management is a primary inflammatory cytokines (for example, IL-10), Vasodilator focus. HR341g in conjunction with other treatments such as prostaglandins PGE, PGE PG1, are increased drastically. nutritional Support and oxygen therapy can help patients Increased IL-1 and TNF, above certain levels, increases recover faster and more completely hypermetabolism and organ dysfunction. Patient C suffers Various publications have been referred to throughout this from circulating endotoxin, even in the absence of a clinically 25 application. The disclosures of these publications in their salient septic focus. The mechanism of the endotoxemia is entireties are hereby incorporated by reference into this appli absorption from the bacteria-colonized burn wound, or from cation to more fully describe the state of the art to which this leaks in the gastrointestinal tract due to increased gut perme invention pertains. ability. Endotoxin initiates the release of several mediators including arachidonic acid, metabolites, oxygen free radicals 30 EQUIVALENTS and cytokines. The increased permeability can amplify inflammation and induce a form of ischemia-reperfusion The above examples have been depicted solely for the injury. Patient C also suffers from blood flow maldistribution purpose of exemplification and are not intended to restrict the and increased skeletal muscle catabolism. Anemia, and scope or embodiments of the invention. Other embodiments increasing liver acute phase protein production occurs. 35 not specifically described should be apparent to those of Results ordinary skill in the art. Such other embodiments are consid Patient A's condition deteriorates and hospital staff decide ered to fall, nevertheless, within the scope and spirit of the to perform surgery. It is estimated that it will take several present invention. Thus, the invention is properly limited operations to restore the patient’s former quality of life. solely by the claims that follow.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 3

<21 Oc SEO ID NO 1 <211 LENGTH: 4 <212 TYPE PRT <213> ORGANISM: Artificial Sequence <22 Oc FEATURE; <223> OTHER INFORMATION: The sequence was artificially synthesized. The alpha carbon of the lysine residue is bound to the alpha carbon of methotrexate. FEATURE; NAME/KEY: Binding LOCATION: (1) . . (1)

<4 OO SEQUENCE: 1 Lys Gly Gly Tyr 1.

SEO ID NO 2 LENGTH: 4 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: The sequence was artificially synthesized. The US 7,604,797 B2 43 44

- Continued arginine residue is bound to the epsilon carbon of methotrexate. &220s FEATURE: <221 NAMEAKEY: Binding <222> LOCATION: (1) ... (1)

<4 OO SEQUENCE: 2 Arg Gly Gly Tyr 1.

<210 SEQ ID NO 3 <211 LENGTH: 4 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: OTHER INFORMATION: The sequence was artificially synthesized. The arginine residue is bound to the epsilon carbon of aminopterin. &220s FEATURE: <221 NAMEAKEY: Binding <222> LOCATION: (1) ... (1)

<4 OO SEQUENCE: 3 Arg Gly Gly Tyr 1.

What is claimed is: carboxylpeptides of aminopterin; gamma-carboxylhy 1. A method for reducing the severity of inflammation and drazides of aminopterin; alpha-gamma-dimethylester, edema associated with a burn injury, comprising administer alpha-gamma-diethylester, alpha-gamma-dipropy ing a pharmaceutical composition to a mammal having a burn 30 lester, alpha-gamma-dibutyl ester, alpha-gamma-dipen injury in need thereof, the pharmaceutical composition com tyl ester, alpha-gamma-dihexylester, alpha-gamma prising: diheptylester, alpha-gamma-dioctylester, and alpha a) a therapeutically effective amount of sodium monofluo gamma-dibenzylester dicarboxylesters of aminopterin; rophosphate: alpha-gamma-diamide, alpha-gamma-dibenzylamide, b) a therapeutically effective amount of a dihydrofolate 35 and alpha-gamma-diamidomethane Sulfonic acid dicar reductase inhibitor selected from the group consisting of boxylamides of aminopterin; alpha-gamma-diglycyl, aminopterin, methotrexate, pyramethamine, trimethop alpha-gamma-diaspartyl, alpha-gamma-diglutamyl. rim, a functional derivative of aminopterin, and a func and alpha-gamma-dipolyglutamyl dicarboxylpeptides tional derivative of methotrexate, wherein the functional of aminopterin; alpha-gamma-dicarboxylhydrazides of derivatives of aminopterin are selected from the group 40 aminopterin; alpha-methylester-gamma-butylester, and consisting of 3',5'-dichloroaminopterin; 5,8-dideaza alpha-methylester-gamma-benzylesterdicarboxylesters 5,6,7,8-tetrahydroaminopterin; 5,8,10-trideazaminop of aminopterin; alpha-benzylester-gamma-butylamide, terin; 5,10-dideazatetrahydrofolic acid; 8,10-didea alpha-benzylester-gamma-benzylamide, alpha-benzy Zaminopterin; aminopterin-gamma-hydrazide; aminop lester-gamma-butylamide-p-toluene Sulfonic acid, and terin-alpha-hydrazide; 3',5'-dichloraminopterin 45 alpha-benzylester-gamma-benzylamide-p-toluene Sul gamma-hydrazide; 3',5'-dichloroaminopterin-alpha fonic acid alpha-ester-gamma-amides of aminopterin; hydrazide; aminopterin-gamma-tyrosyl-hydrazide; alpha-t-butylester-gamma-hydrazides of aminopterin; aminopterin alpha-alpha-lysyl-glycyl-tyrosyl-hy alpha-ester-gamma-peptides of aminopterin; alpha drazide; aminopterin-alpha-alpha-lysyl hydrazide; ami amide-gamma-esters of aminopterin; alpha-amide nopterin-alpha-alpha-lysine; aminopterin-alpha-alpha 50 gamma-peptides of aminopterin; alpha-amide-gamma lysyl-epsilon-arginine-glycine-glycine-tyrosine (SEQ hydrazides of aminopterin; alpha-peptide-gamma ID NO: 3); alpha-methyl, alpha-ethyl, alpha-propyl. esters of aminopterin; alpha-peptide-gamma-amides of alpha-butyl, alpha-pentyl, alpha-hexyl, alpha-heptyl, aminopterin; alpha-peptide-gamma-hydrazides of ami alpha-octyl, and alpha-benzyl carboxylesters of ami nopterin; alpha-hydrazide-gamma-amides of aminop nopterin; alpha-amide, alpha-butylamide, alpha-benzy 55 terin; and alpha-hydrazide-gamma-peptides of aminop lamide, and alpha-amidoethane Sulfonic acid carboxy terin; and the functional derivatives of methotrexate are lamides of aminopterin; alpha-glycyl, alpha-aspartyl, Selected from the group consisting of 4-amino-4-deoxy alpha-glutamyl, and alpha-polyglutamyl carboxylpep No-methylpteroyl-D.L-homocysteic acid (mAPA-D.L- tides of aminoptering gamma-methylester, gamma-ethy HCySA), 4-amino-4-deoxy-N-methylpteroyl-L-cys lester, gamma-propylester, gamma-butylester, gamma 60 teic acid (mAPA-L-CySA), 4-amino-4-deoxy-No pentylester, gamma-hexylester, gamma-heptylester, methylpteroyl-L-homocysteic acid (mAPA-L-HCySA); gamma-octylester, and gamma-benzylester alpha-car 4-amino-4-deoxypteroyl-D.L-homocysteic acid (APA boxylhydrazide-gamma-carboxylesters of aminopterin; D.L-HCySA), 4-amino-4-deoxypteroyl-L-cysteic acid gamma-amide, gamma-butylamide, gamma-benzyla (APA-L-CySA), 4-amino-4-deoxypteroyl-L-homocys mide, and gamma-amidoethane Sulfonic acid carboxy 65 teic acid (APA-L-HCySA); 3',5'-dichloromethotrexate; lamides of aminopterin; gamma-glycyl, gamma-aspar 5,8-dideaza-5,6,7,8-tetrahydromethotrexate; methotr tyl, gamma-glutamyl, and gamma-polyglutamyl exate-gamma hydrazide; methotrexate-alpha-hy US 7,604,797 B2 45 46 drazide: 3',5'-dichloromethotrexate-gamma-hydrazide: lipoxygenase inhibitor, a leukotriene antagonist; ACTH, a 3',5'-dichloromethotrexate-alpha-hydrazide; methotr soluble TNF-receptor; an anti-TNF-antibody; a soluble exate-alpha-alpha-lysyl-glycyl-glycyl-tyrosyl receptor of interleukin, a cytokine, an II-1 receptor antago hydrazide (SEQ ID NO: 1); methotrexate-gamma-ty nist, or a T-cell-protein; an antibody against a receptor of rosyl hydrazide; methotrexate-alpha-alpha-lysyl interleukin, a cytokine, or a T-cell-protein; or a calcipotriol. hydrazide; methotrexate-alpha-alpha-lysine; methotr 5. The method of claim 2, wherein the pharmaceutical exate-alpha-alpha-lysyl-epsilon-arginine-glycine-gly composition further comprises a therapeutically effective cine-tyrosine (SEQ ID NO: 2); 5,8-dideazamethotrex amount of one or more additional anti-inflammatory com ate; alpha-methyl, alpha-ethyl, alpha-propyl, alpha pounds and/or a therapeutically effective amount of one or butyl, alpha-pentyl, alpha-hexyl, alpha-heptyl, alpha 10 more additional immunomodulatory agents. octyl, and alpha-benzyl carboxylesters of methotrexate; 6. The method of claim 5, wherein the additional anti alpha-amide, alpha-butylamide, alpha-benzylamide, inflammatory compound or additional immunomodulatory and alpha-amidoethane Sulfonic acid carboxylamides of drug comprises at least one of interferon; betaseron or n-in methotrexate; alpha-glycyl, alpha-aspartyl, alpha terferon; a prostane; iloprost or cicaprost; a glucocorticoid; an glutamyl, and alpha-polyglutamyl carboxylpeptides of 15 immunosuppressive; a lipoxygenase inhibitor, a leukotriene methotrexate; gamma-methylester, gamma-ethylester, antagonist; ACTH; a soluble TNF-receptor; an anti-TNF gamma-propylester, gamma-butylester, gamma-penty antibody; a soluble receptor of interleukin, an II-1 receptor lester, gamma-hexylester, gamma-heptylester, gamma inhibitor, a cytokine, or a T-cell-protein; an antibody against octylester, and gamma-benzylester alpha-carboxylhy a receptor of interleukin, or a T-cell-protein; or a calcipotriol. drazide-gamma-carboxylesters of methotrexate; gamma-amide, gamma-butylamide, gamma-benzyla 7. The method according to claim 1, wherein the burn is mide, and gamma-amidoethane Sulfonic acid carboxy either a first, second or third degree thermal burn, or a com lamides of methotrexate; gamma-glycyl, gamma-aspar bination thereof. tyl, gamma-glutamyl, and gamma-polyglutamyl 8. The method according to claim 1, wherein the mammal carboxylpeptides of methotrexate, gamma-carboxylhy 25 is a human being. drazides of methotrexate; alpha-gamma-dimethylester, 9. The method according to claim 4, wherein the glucocor alpha-gamma-diethylester, alpha-gamma-dipropy ticoid is selected from the group consisting of cortisol, pred lester, alpha-gamma-dibutyl ester, alpha-gamma-dipen nisolone, methyl-prednisolone, and dexamethasone. tyl ester, alpha-gamma-dihexylester, alpha-gamma 10. The method according to claim 4, wherein the immu diheptylester, alpha-gamma-dioctylester, and alpha 30 nosuppressive is selected from the group consisting of gamma-dibenzylester dicarboxylesters of methotrexate; cyclosporine A, methoXSalene, thalidomide, Sulfasalazine, alpha-gamma-diamide, alpha-gamma-dibenzylamide, and azathioprine. and alpha-gamma-diamidomethane Sulfonic acid dicar 11. The method according to claim 4, wherein the lipoxy boxylamides of methotrexate, alpha-gamma-diglycyl, genase inhibitor comprises Zileutone. alpha-gamma-diaspartyl, alpha-gamma-diglutamyl, 35 12. The method according to claim 6, wherein the gluco and alpha-gamma-dipolyglutamyl dicarboxylpeptides corticoid is selected from the group consisting of cortisol, of methotrexate; alpha-gamma-dicarboxylhydrazides of prednisolone, methyl-prednisolone, or dexamethasone. methotrexate; alpha-methylester-gamma-butylester, 13. The method according to claim 6, wherein the immu and alpha-methylester-gamma-benzylester dicarboxy nosuppressive is selected from the group consisting of lesters of methotrexate; alpha-benzylester-gamma-bu 40 cyclosporine A, methoXSalene, thalidomide, Sulfasalazine, tylamide, alpha-benzylester-gamma-benzylamide, and azathioprine. alpha-benzylester-gamma-butylamide-p-toluene Sul fonic acid, and alpha-benzylester-gamma-benzylamide 14. The method according to claim 6, wherein the lipoxy p-toluene Sulfonic acid alpha-ester-gamma-amides of genase inhibitor comprises Zileutone. methotrexate; alpha-t-butylester-gamma-hydrazides of 45 15. The method according to claim 6, wherein the II-1 methotrexate; alpha-ester-gamma-peptides of methotr receptor inhibitor comprises an IL-1 receptor antagonist. exate; alpha-amide-gamma-esters of methotrexate; 16. The method according to claim 6, wherein the burn is alpha-amide-gamma-peptides of methotrexate; alpha either a first, second or third degree thermal burn, or a com amide-gamma-hydrazides of methotrexate; alpha-pep bination thereof. tide-gamma-esters of methotrexate; alpha-peptide 50 17. The method according to claim 1, comprising admin gamma-amides of methotrexate; alpha-peptide-gamma istering the pharmaceutical composition topically. hydrazides of methotrexate; alpha-hydrazide-gamma 18. The method according to claim 17, wherein the phar amides of methotrexate; and alpha-hydrazide-gamma maceutical composition comprises an ointment, salve or peptides of methotrexate; and CCa: c) a pharmaceutically acceptable carrier. 55 19. The method according to claim 17, comprising admin 2. The method of claim 1, wherein the dihydrofolate reduc istering the pharmaceutical composition from 2 to 4 times. tase inhibitor is aminopterin or methotrexate; and said phar maceutical composition inhibits dihydrofolate reductase. 20. The method according to claim 1, wherein the sodium 3. The method of claim 1, wherein the pharmaceutical monofluorophosphate is presentinanamount of from 1.5% to composition further comprises a therapeutically effective 60 15% by weight per unit volume of the pharmaceutical com amount of one or more anti-inflammatory compounds and/or position. a therapeutically effective amount of one or more immuno 21. The method according to claim 1, comprising admin modulatory agents. istering the pharmaceutical composition within 12 hours of 4. The method of claim 3, wherein the anti-inflammatory the burn injury. compound or immunomodulatory drug comprises at least one 65 22. The method according to claim 21, comprising admin of interferon; betaseron or B-interferon; a prostane; iloprost istering the pharmaceutical composition within 20 minutes of or cicaprost; a glucocorticoid; an immunosuppressive; a the burn injury. US 7,604,797 B2 47 48 23. The method according to claim 1, wherein the thera 35. The method according to claim 1, wherein the pharma peutically effective amount of the dihydrofolate reductase ceutical composition further comprises one or more non inhibitor is a dose of from 0.001 to 100 mg per 70 kg of body macrollide antibacterial agents. weight of said mammal. 36. The method according to claim 1, wherein the pharma 24. The method according to claim 20, wherein the thera ceutical composition further comprises one or more anti peutically effective amount of the dihydrofolate reductase fungal agents. inhibitor is a dose of from 0.01 to 20 mg per 70 kg of body 37. The method according to claim 1, wherein the pharma weight of said mammal. ceutical composition further comprises one or more anti-viral 25. The method according to claim 1, comprising admin agents. istering the pharmaceutical composition parenterally. 10 38. The method according to claim 1, wherein the pharma 26. The method according to claim 25, wherein the phar ceutical composition further comprises one or more anti maceutical composition comprises a pharmaceutically parasitic agents. acceptable solution, dispersion, Suspension or emulsion. 39. The method according to claim 34, wherein the one or 27. The method according to claim 1, further comprising more macrollide antibiotics comprises at least one of methy administering an antibacterial agent to said mammal. 15 mycin, neomethymycin, litorin, erythromycin A to F, olean 28. The method according to claim 27, wherein the phar domycin, roXithromycin, dirithromycin, flurithromycin, maceutical composition comprises the antibacterial agent. clarithromycin, davercin, azithromycin, josamycin, kitasa 29. The method according to claim 1, wherein the admin mycin, spiramycin, midecamycin, rokitamycin, miokamy istering the pharmaceutical composition comprises oral, sys cin, and lankacidin. temic, implant, intravenous, topical, intrathecal, or nasal 40. The method according to claim 35, wherein the one or administration. more non-macrollide antibacterial agents comprises at least 30. The method according to claim 1, wherein administer one of penicillins, cephalosporins, carbacephems, cephamy ing the pharmaceutical composition comprises spraying a cins, carbapenems, monobactams, aminoglycosides, glyco burn victim with a fire extinguisher containing the pharma peptides, quinolones, tetracyclines, macrollides, oxazalidii ceutical composition. 25 nones, streptogramins, and fluoroquinolones. 31. The method according to claim 1, wherein the pharma 41. The method according to claim 36, wherein the one or ceutically acceptable carrier comprises dicalcium phosphate more anti-fungal agents comprises at least one ofterbinafine dihydrate (DCP), insoluble sodium metaphosphate, sorbitol hydrochloride, nystatin, amphotericin B, griseofulvin, keto syrup Solution, guar gum, Xanthan gum, monosodium phos conazole, miconazole nitrate, flucytosine, fluconazole, itra phate, titanium dioxide, Sodium dodecylbenzene Sulphate, 30 conazole, clotrimazole, benzoic acid, Salicylic acid, Vori water, trimagnesium phosphate, and hydroxethyl cellulose conazole, caspofungin, and selenium sulfide. ester. 42. The method according to claim 37, wherein the one or 32. The method according to claim 1, wherein the pharma more anti-viral agents comprises at least one of amantadine ceutical composition comprises about 21.4% dicalcium phos hydrochloride, rimantadin, acyclovir, famciclovir, foScamet, phate dihydrate (DCP) by weight, about 13% insoluble 35 ganciclovir Sodium, idoxuridine, ribavirin, Sorivudine, triflu sodium metaphosphate by weight, about 23.3% sorbitol ridine, Valacyclovir, Vangancyclovir, pencyclovir, Vidarabin, syrup Solution by weight, about 4.2% guar gum by weight, didanosine, stavudine, Zalcitabine, Zidovudine, interferon about 1.7% xanthan gum by weight, about 0.28% monoso alpha, and edoxudine. dium phosphate by weight, about 0.56% titanium dioxide by 43. The method according to claim 38, wherein the one or weight, about 0.46% sodium dodecylbenzene sulphate by 40 more anti-parasitic agents comprises at least one of pire weight, about 22.4% water by weight, about 0.74% trimag thrins/piperonyl butoxide, permethrin, iodoquinol, metron nesium phosphate by weight, and about 2.9% hydroxethyl idazole, diethylcarbamazine citrate, piperazine, pyrantel cellulose ester by weight. pamoate, mebendazole, thiabendazole, praziquantel, 33. The method according to claim 31, wherein the phar albendazole, proguanil, quinidine gluconate injection, qui maceutical composition comprises about 8.9% of sodium 45 nine Sulfate, chloroquine phosphate, mefloquine hydrochlo monofluorophosphate by weight, and about 0.0015% of the ride, primaquine phosphate, atovaquone, co-trimoxazole dihydrofolate reductase inhibitor by weight. (sulfamethoxazole/trimethoprim), and pentamidineisethion 34. The method according to claim 1, wherein the pharma ate. ceutical composition further comprises one or more mac rollide antibiotics. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. 7,604,797 B2 Page 1 of 1 APPLICATION NO. : 1 1/O12210 DATED : October 20, 2009 INVENTOR(S) : Terry Lee Hicks It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the Title Page:

The first or sole Notice should read --

Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 148 days.

Signed and Sealed this Fifth Day of October, 2010

David J. Kappos Director of the United States Patent and Trademark Office