KR100855417B1 - A Pharmaceutical Composition for Wound Healing Comprising Spinosyns - Google Patents

Abstract

The present invention relates to spinosine, or a physiologically acceptable derivative or salt thereof, for promoting or accelerating wound healing in humans.

Wound Healing, Spinosine, Healing Dysfunction, Spinosad, Tissue Proliferation, Regeneration

Description

A pharmaceutical composition for wound healing comprising spinocin {A Pharmaceutical Composition for Wound Healing Comprising Spinosyns}

The present invention relates to the therapeutic use of spinosine in humans for promoting or accelerating wound healing in normal and poorly treated humans. Spinosine, or a physiologically acceptable derivative or salt thereof, can be used for therapeutic conditions in humans in need of soft tissue growth and regeneration. Wound healing insufficiency is a significant source of morbidity in humans and can cause complications such as non-healing wounds. In normal individuals, wound healing occurs through an uncomplicated endogenous process. In contrast, wound healing failure is associated with several conditions, such as diabetes, infection, immunosuppression, obesity and malnutrition.

Wound healing is a complex biological process involving extracellular matrix, blood cells, parenchymal cells, and mediators such as cytokines. After the wound is hemostatic, the bleeding stops and the healing process begins. This occurs in three stages: inflammation, tissue formation (proliferation), and tissue regeneration (remodeling). Healing begins very quickly after injury occurs; For example, re-epithelialization of skin wounds begins within hours [Singer and Clark, New Eng. J. Med . 341 (10): 738-746 (1999). The process of wound healing is characterized by neurological inflammation, immunological reactions [Eglezos et al., Adv . Exp . Biol . Med . 273: 499-503 (1989); Immunol . Cell Biol . 69: 285-294 (1991)] and vascular tone [Khalil & Helme, Brain Res . 500: 256-262 (1989); Brain Res . 527: 292-298 (1990), which are all essential components of healing, are initiated by myelin-formed afferent sensory nerves. After initiation of healing by the sensory nerves, the process of healing is regulated by growth factors and cytokines that affect cell migration, proliferation, and protein production. In hemostasis, proteins such as fibrin and fibrinogen interact to coagulate blood and cytokines and growth factors are upregulated. After the injury, inflammation begins.

The inflammatory response occurs in three distinct stages, each of which is apparently mediated by different mechanisms: (1) an acute transient stage characterized by local vasodilation and increased capillary permeability; (2) a delayed subacute stage, most markedly characterized by infiltration of leukocytes and phagocytes; And (3) chronic proliferative phase in which tissue degeneration and fibrosis are induced. Many different mechanisms are involved in the inflammatory process. The impetus of the inflammatory response is essential for survival despite environmental pathogens and damage, but in some conditions and diseases the inflammatory response is exacerbated and persists for reasons that are not clearly beneficial. During inflammation, neutrophils (polymorphonuclear leukocytes, PMN), monocytes and phagocytes infiltrate the wound. These phagocytes release enzymatic mediators (proteases) that break down protein, a growth factor during the proliferative phase, and prey bacteria, killed and killed cells to remove wounds.

In the next step, propagation begins. Collagen deposits and forms scar tissue. Fibroblasts produce proteoglycans that bind together with collagen fibers. Over time, collagen is broken down by proteases and reformed into stronger scar structures.

Spinosine (also known as factor A83453) is used for the following purposes: 1) insects in Repidoptera neck, 2) members in Homoftera neck, 3) members of Diphthera neck insect, 4) members of Koleopterra neck, and 5) It is an agricultural animal, livestock and pet insecticide that shows activity against members of the Anoflura neck. Formulations suitable for administration of agricultural animals, livestock and pets include various suspensions, solutions, tablets, capsules, solutions and treatments.

 Spinosad (product containing Spinocin A, 85%, and Spinosine D, 15% as the main ingredient) can be used to treat sheep lice, and to treat and prevent sheep fly strikes. Is currently approved in Australia and New Zealand. In Brazil, spinosad is a preservative and wound formation for the local treatment and control of certain mites, flies, and their flies, and for the treatment of horsefly maggots and skin wounds in cattle, sheep, goats, horses, pigs, birds and dogs. It is approved as a repellent.

Spinosine is known to be useful for controlling this infection in humans (US 6,063,771 and EP 1 252 820). Formulations suitable for such insecticidal use in humans are also described in these patents.

Despite what is known in connection with ectoparasitic insecticidal activity and commercial approval of spinosine, spinosine, or a physiologically acceptable derivative or salt thereof, has wound healing activity independently of a formulation containing an antiseptic / antiseptic. Finally found.

Summary of the Invention

The present invention relates to spinosine, or a physiologically acceptable thereof, for patients in need of facilitating or accelerating wound healing, including poor healing or chronic wounds, skin diseases and allergic diseases, in particular these skin related disorders, and conditions or symptoms associated therewith. By administering a derivative or salt to a method for treating wounds in humans for promoting or accelerating wound healing, including poor healing or chronic wounds, skin diseases and allergic diseases, especially these skin related disorders, and conditions or symptoms associated therewith will be. Preferably, these methods are carried out by administering spinosad, or a physiologically acceptable derivative or salt thereof.

The present invention provides a method for promoting or accelerating wound healing in humans, comprising administering spinosine, or a physiologically acceptable derivative or salt thereof, to a human being in need thereof. In another aspect, the present invention provides the use of a preparation containing spinosine, or a physiologically acceptable derivative or salt thereof, or spinosine, or a derivative or salt thereof, for the manufacture of a medicament for promoting or accelerating wound healing in humans. To provide.

Spinosine is a naturally induced fermentation product. These are macrolides produced by the culture of Saccharopolyspora spinosa . The fermentation produces several factors, including Spinosine A and Spinosine D (also referred to as A83543A and A83543D). Spinosine A and Spinosine D are the two types of spinosine that are most active as insecticides. Agricultural products mainly comprising these two spinosins (approximately 85% A and 15% D) are commercially available under the trade name Spinosad from Dow AgroSciences. External parasitic products, including spinosad, are commercially available from Eli Lilly and Company. The trade name "Spinosad" derives from the abbreviation of Spinosine "A" and "D".

Each spinosine has a 12 membered macrocyclic ring that is part of a tetracyclic ring system with two different sugars attached, the amino-sugar porosamine and the natural sugar 2N, 3N, 4N-tri-O-methylramnose. This unique structure forms spinosine independent of other macrocyclic compounds.

Spinosine A (A83543A) was the first spinosin isolated and identified from the fermentation broth of Saccharopolypora spinosa. Further investigation of fermentation broth is S. It was found that the parental strain of Spinosa produced a number of spinosins labeled A-J (A83543A-J). Compared to spinosine A, spinosine B to J are characterized by different substitution patterns on the amino group of porosamine at selected sites on the tetracyclic ring system and on 2N, 3N, 4N-tri-O-methyllamnose. S currently used. Spinosa strains produce a mixture of spinosine with a first component of Spinosine A (˜85%) and Spinosine D (˜15%). Additional spinosins named K through W are S. Spinosa mutant strains were identified.

As used herein, the term “spinosine, or a physiologically acceptable derivative or salt thereof” refers to individual spinosin factors (A, B, C, D, E, F, G, H, J, K, L, M, N, O, P, Q, R, S, T, U, V, W or Y), N-demethyl derivatives of individual spinosin factors, combinations thereof or physiologically acceptable salts. For convenience, the term “spinosine” may be used herein to mean individual spinosine, or physiologically acceptable derivatives or salts thereof, or combinations thereof. Most preferred for wound healing in humans is spinosad, or a physiologically acceptable derivative or salt thereof.

EP 375 316 describes spinosine A to H and J (these are referred to as A83543 factors A, B, C, D, E, F, G, H and J), and salts thereof. US Pat. No. 5,202,242 (Mynderse et al.) Describes spinosine L to N (these are referred to as A83543 factors L, M and N), their N-demethyl derivatives and salts; U.S. Patent 5,591,606 and U.S. Patent 5,631,155 (Turner et al.) Describe spinosine Q to T (these are referred to as A83543 factors Q, R, S and T), their N-demethyl derivatives and salts. These patents are incorporated herein by reference. Spinosine K, O, P, U, V, W and Y are described, for example, in Carl V. DeAmicis, James E. Dripps, Chris J. Hatton and Laura I. Karr in American Chemical Society's Symposium Series: Phytochemicals for Pest Control, Chapter 11. "Physical and Biological Properties of Spinosyns: Novel Macrolide Pest-Control Agents from Fermentation," pages 146-154 (1997). US Pat. No. 6,001,981 describes various synthetic derivatives of spinosin, US 6,455,504 describes various spinocin analogues, all of which are incorporated herein by reference. Details regarding the fermentation and isolation of spinosine and methods of preparing synthetic derivatives are provided in these references.

Each US patent and EP patent application describes various agent types, pesticidal activity and selection of administration in animals and agriculture for spinosine, and physiologically acceptable derivatives or salts thereof.

US Pat. Nos. 6,063,771 and 6,342,482 and EP 1 252 820 describe the preparation and use of spinosine, or physiologically acceptable derivatives or salts thereof, for controlling them in humans, and methods of making these preparations. have.

As noted above, the Spinosad formulation is Dow Agrosciences, Zionsville Road 9330, Indianapolis, Indiana, 46268-1054, USA, and Greenfield W., 46140 Indiana, USA. Main Street 2001 P. Oh. Commercially available from Ellanco Animal Health, a spin off of Eli Lilly and Company, Box 708. Also, S. Spinosad and Mutants are Agricultural Research Service Patent Culture Collection (NRRL) at the National Center for Agricultural Utilization Research (ARS, USDA), Peoria North University Street, 1815, 61604, USA. (NRRL 18395, 18537, 18538, 18539, 18719, 18720, 18743, 18823 and 30141 (US Pat. No. 6,455,504)).

Spinosine can act to form salts. Physiologically acceptable salts are also useful in the methods of the present invention. The salts are prepared using standard salt preparation methods. For example, spinosine A can be neutralized with a suitable acid to form an acid addition salt. Acid addition salts of spinosine are particularly useful. Suitable acid addition salts each include organic or inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, cholic acid, pamoic acid, mucinic acid, glutamic acid, camphoric acid, glutaric acid, glycolic acid Salts formed by reaction with phthalic acid, tartaric acid, formic acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid, and the like.

All ratios, percentages, and parts discussed herein are by weight unless otherwise indicated.

"Wound" means damage to a human arising from a disorder or disease in which the tissue is cut, torn, broken, burned, or traumatic, or causes such damage.

“Healing” provided by the present invention is the acceleration or acceleration of the time from when the wound occurs (spinosine administration) to when the wound is closed (complete wound contraction).

The term "tissue" refers to a mass of cells in the human body that collectively form a specific function. Tissues include but are not limited to bones, skin, connective tissue, and nerves such as the spinal cord.

As used herein, “treating”, “treatment” and “therapy” refer to curative therapies. Those in need of treatment include humans with wounds, disorders or diseases.

Administration “combined” with one or more additional therapeutic agents includes simultaneous (simultaneous) and continuous administration in any order to humans.

A “therapeutically effective amount” is the minimum amount of active agent (eg, spinosine and most preferably spinosad) necessary to confer a therapeutic benefit to a human. For example, a “therapeutically effective amount” for a human suffering from a wound is an amount that induces, promotes, accelerates or ameliorates, or alleviates healing resistance, pathological symptoms, healing progression, physiological conditions associated with healing.

As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to humans exposed to the dosages and concentrations employed. Physiologically acceptable carriers are often aqueous pH buffers. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; Antioxidants including ascorbic acid; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming countions such as sodium; And / or nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (TWEEN®), polyethylene glycol (PEG), and polyoxyethylene / polyoxypropylene block copolymers (PLURONIC) (Registered trademark)).

Spinosine and most particularly spinosad of the present invention stimulate healing neuronal activation and subsequent inflammatory activity associated with cell growth and proliferation. Thus, the compositions of the present invention can be used to stimulate epithelial cell proliferation and bottom layer keratinocytes for wound healing, in particular skin wound healing purposes. These wounds can be shallow or deep and include damage to the dermis and epidermis of the skin.

Spinosine is useful for the treatment of many wounds and conditions. Spinosine, for example, is active in vivo in various wound healing models.

Humans to which spinosine is administered can heal wounds at normal rates or may be healed insufficiency. When administered to an individual that is not healing failure, spinosine is administered to accelerate the normal healing process. When administered to a heal insufficiency subject, spinosine is administered to promote healing of a wound that otherwise heals slowly or does not heal at all. Many pains and conditions can cause healing failure. These pains and conditions include diabetes (eg, type II diabetes), treatment of all pharmacological agents of steroids and non-steroids, and ischemic blockage or damage.

A number of growth factors have been shown to promote wound healing in dysfunctional individuals. These growth factors include growth hormone-free factors, platelet-derived growth factors, and basic fibroblast growth factors. Accordingly, the present invention also includes administering one or more spinosins in combination with one or more growth factors or other agents that promote wound healing.

Spinosine of the present invention either heals wounds at normal rates or promotes healing of anastomosis and other wounds caused by surgery in humans with poor healing.

Spinosine, particularly spinosad, of the present invention induce normal wounds and abnormal wound healing, such as uremia, malnutrition, vitamin deficiency, obesity, infection, immunosuppression and systemic treatment with steroids, radiation therapy, and anti-neoplastic Surgical wounds, resection wounds, severe wounds associated with damage to the dermis and epidermis, soft tissue injuries, such as muscle rupture, eye tissue wounds, dental tissue wounds, oral wounds, gastrointestinal mucosa, in individuals with complications related to physical drugs and anti-metabolites Stimulating wound healing of wounds, including wounds and ulcers, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous congestion ulcers, and heat-induced burns, exposure to extreme or cryogenic temperatures, or exposure to chemicals Clinically useful for The compositions may also promote the healing of wounds associated with ischemic and ischemic damage, such as chronic venous lower limb ulcers, caused by impairment of venous circulation and / or dysfunction; Promotion of skin recovery following skin loss; Increase in epidermal tension and epidermal thickness; And increased adhesion of skin grafts to the wound layer and promotion of re-epithelialization from the wound layer.

As used herein, "individual" means a human.

Spinosine preparations may use suitable pharmaceutical diluents known to be useful in pharmaceutical compositions. Such diluents include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should be suitable for the mode of administration. Preferably the pharmaceutical composition will be formulated for administration.

Spinosine may be administered in a pharmaceutical composition in combination with one or more pharmaceutically acceptable excipients. It will be appreciated that when administered to a human patient, the total daily usage of the pharmaceutical compositions of the invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular patient may be the type and extent of response to be achieved; Specific compositions including other agents as needed, if desired; The age, body weight, general health, sex and diet of the patient; The time of administration, route of administration, and rate of secretion of the composition; Duration of treatment; Drugs (eg, chemotherapeutic agents) used in combination or coincidental with the specific composition; And similar factors well known in the medical arts. Suitable formulations known in the art are described in Remington's Pharmaceutical. Sciences (latest edition), Mach Publishing Company, Easton, PA. Thus, the "effective amount" (including effective amount of spinosin) of spinosine for purposes herein is determined by this consideration.

The pharmaceutical compositions of the invention may be administered by conventional means such as oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarticular, subcutaneous, intranasal, inhalation, intraocular, intradermal routes. Parenteral and topical delivery are the preferred routes of administration.

As used herein, the term “parenteral” refers to a mode of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Pharmaceutical compositions are administered in an amount effective to treat a particular condition. In most cases, the spinocin dose is about 0.5 μg / kg to about 50 mg / kg body weight per day, taking into account the route of administration, symptoms, and the like. However, the dosage may be as low as 0.05 μg / kg body weight. For example, in the particular case of topical administration, the dosage is preferably administered at about 0.2 μg to 2 μg / cm 2. For intranasal and intraocular administration, the dosage is preferably administered at about 0.05 μg / kg to about 50 μg / kg body weight, more preferably at about 0.5 μg / kg to about 5 μg / kg body weight.

As a general suggestion, the total pharmaceutically effective amount of spinocin administered parenterally will range from about 0.5 μg to 5 mg / kg patient body weight / day, but as discussed above this will be considered therapeutically. When administered continuously, the spinosine is administered at a rate of about 10 μg / kg / hour to about 100 μg / kg / hour, for example using a mini-pump, by one to four injections per day or continuous subcutaneous infusion. Typically administered. Intravenous bag solutions or bottle solutions may be used.

The course of spinosine treatment appears to be optimal for consecutively longer than a certain minimum day (1-5 days in humans). The duration of treatment required to observe the change and the interval after treatment for the response to be generated will depend on the desired effect.

In the case of parenteral administration, in one embodiment, the spinosine is administered in a unit dose injectable form (solution, suspension, or emulsion) in the desired degree of purity, i.e., the donor at the dosages and concentrations employed. It is non-toxic to and generally formulated by mixing with those compatible with the other ingredients of the formulation.

In general, formulations are prepared by uniformly and intimate contact of spinosine with a liquid carrier or a finely divided solid carrier, or both. Thus, if desired, the product is shaped into the desired formulation. In the case of parenteral carriers, solutions which are isotonic with the blood of the donor are preferably used. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-water soluble vehicles such as fixed oils and ethyl oleate are useful here as well as liposomes. Suitable formulations known in the art are described in Remington's Pharmaceutical Sciences (latest edition), Mach Publishing Company, Easton, PA.

In the case of topical administration, agents such as ointments, creams, and gels can be used in the dosages described above for the composition. Suitable formulations known in the art are described in Remington's Pharmaceutical. Sciences (latest edition), Mach Publishing Company, Easton, PA.

Ointments are generally (1) oily bases, i.e., consisting of fixed oils or hydrocarbons, such as white petrolatum or mineral oil, or (2) absorbent bases, i.e. any anhydrous material or material capable of absorbing water, for example anhydrous Prepared using what consists of lanolin. Typically, after preparation of the substrate, whether oily or absorbent, the active ingredient (spinosine) is added in an amount to provide the desired concentration.

The cream is an oil / water emulsion. These typically consist of fixed oils, hydrocarbons and the like, oil phase (inner phase) including waxes, petrolatum, mineral oil and the like, and water phase (continuous phase) including water and any water soluble substances such as additional salts. Biphasic phases include emulsions, for example surface active agents such as sodium lauryl sulfate; Stabilized by the use of hydrophilic colloids such as acacia colloidal clay, daggers and the like. Upon formation of the emulsion, the active ingredient (spinosine) is usually added in an amount to achieve the desired concentration.

Gels include oily substrates, water, or emulsion-suspension substrates, such as substrates selected from those described above. To the substrate is added a gelling agent which forms a matrix in the substrate and increases its viscosity. Examples of gelling agents include hydroxypropyl cellulose, acrylic acid polymers and the like. Typically, the active ingredient (spinosine) is added to the formulation at the desired concentration before the gelling agent is added.

Oral pharmaceutical formulations are prepared by known methods using well known and readily available ingredients. In preparing the compositions, the active ingredient will generally be mixed with, diluted with, or incorporated into a pharmaceutically acceptable carrier, which carrier may be in the form of a capsule, sachet, paper or other container. When the carrier is provided as a diluent, the carrier can be a solid, semi-solid or liquid substance which acts as a vehicle, excipient or medium with respect to the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, lozenges, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, and the like.

In addition to the active or therapeutic ingredients, the tablets in the dosages described above for the composition contain a number of inert materials known as additives or excipients. These materials help to impart sufficient processing and compression properties to formulations including diluents, binders, lubricants and lubricants. An additional group of additional materials help to provide additional desired physical properties to the finished tablet. Included in this group include disintegrants, colorants, flavors and sweeteners in the case of chewable tablets, and polymers or waxes or other dissolution-delaying materials in the case of controlled-release tablets.

“Pharmaceutically acceptable” means that the carrier, diluent or excipient is compatible with the other ingredients of the formulation and should not be harmful to its recipient.

Further details, including representative formulations, can be found in Remington's Pharmaceutical Sciences (latest edition), Mach Publishing Company, Easton, PA.

The compositions of the present invention also contemplate wound care dressings or bandages comprising a therapeutically effective amount of spinosine, or a physiologically acceptable derivative or salt thereof. Bandages or dressings for wound care may comprise an outer fabric support, preferably an elastomeric fabric support; And an inner pad, the inner pad preferably comprising an outer membrane surface made from a film-forming material and having a therapeutically effective amount of spinosine, or a physiologically acceptable derivative or salt thereof, preferably spinosad, Or a physiologically acceptable derivative or salt thereof is introduced into the matrix at the dosages described above for the composition. The pad may be integral with or separate from the outer fabric support.

Spinosine is ideally introduced into the membrane matrix, but may be introduced into the material of the inner pad contained by the membrane.

The therapeutically active agent is contained in the polymeric matrix to inhibit migration and to allow gradual release of spinosine over time.

In another aspect, the wound dressing includes a liquid permeable body-side liner, an individual outer cover sheet, optionally a liquid impermeable sheet, and an absorber disposed therebetween. The internal and / or absorbent is made from a substance that has introduced a therapeutically effective amount of spinosine into the matrix and the gap so that the spinosine is invariably consistently very close to the wound.

The inner surface or pad of the bandage is preferably made from natural or synthetic members or film-forming materials or plastic materials of organic or inorganic, animal or vegetable origin. For example, it is prepared from gelatin or vegetable gums, such as polyvinylchloride polyacetate or polyamide, coated or cast as a film or film in a general manner, or from hydrophilic or hydrophobic film forming plastic materials.

Suitable polymeric materials include, but are not limited to, silicone or other silicone-based materials, polyethylene terephthalate (PET), darkon, braided darkron, velour darkron, polyglycine, nylon, silk, polyethylene (PE), poly Urethanes, polyvinyl chloride silicone elastomers, silicones, rubbers, PMMA (poly- (methylmethacrylate)), latex, polypropylene (PP), polyolefins, cellulose, polyvinyl alcohol (PVA), poly (hydroxyethylmet Acrylates (PHEMA), poly (glycolic acid), poly (acrylonitrile) (PAN), fluoroethylene-cohexa-fluorophosphylene (FEP), teflon (PTFE), copolymers and mixtures thereof, although It is not included.

The simplest way to introduce a therapeutically active compound into a polymeric material is by blending the therapeutically active material directly into a plastic resin prior to casting or the like.

Films or membranes are ideally made from hydrophobic polymers that are liquid and gas permeable but impermeable to the passage of microorganisms. Hydrophobicity of the film or membrane is a useful property that reduces the tendency for the film or membrane to adhere to the wound site.

The amount of spinosine introduced into the formulation is not critical; The concentration should only be in a range sufficient to allow rapid application of the agent to the wound area in an amount that will deliver the desired amount of spinosine.

The usual amount of formulation applied to the wound will depend on the wound size and the concentration of spinosine in the formulation. In general, the formulation will be applied to the wound in an amount that provides about 0.1 μg to about 5 μg spinosin / cm 2 wound. Preferably, the dosage of spinosine will range from about 0.2 μg to about 2 μg / cm 2, most preferably from about 0.25 μg to about 0.5 μg / cm 2.

Spinosine may be administered as a liquid, drop, or thickening, gel to the eye to treat lacrimal injury, disorder and pathology in humans.

Spinosine may also be administered intranasally in the form of liquids, drops, or sprays to the nasal mucosa to treat disorders, injuries and pathologies of human nasal mucosa and sinus epithelium.

Spinosine is about 0.01 μg / ml to 50 mg / ml, preferably 0.01 μg / ml to 10 mg / ml at a pH of about 5 to about 8, preferably about 6 to about 7, most preferably about pH 6.2. It is typically formulated into a vehicle at a concentration of. It will be appreciated that such excipients, carriers, or stabilizers may form spinosine salts.

Spinosine will usually be stored as an aqueous solution or lyophilized formulation for reconstitution in unit or multi-dose containers, eg sealed ampoules or vials. As an example of a lyophilized formulation, a 3-ml vial is filled with 1 ml of a sterile-filtration 1% (w / v) spinosin aqueous solution and the resulting mixture is lyophilized. Infusion solutions are prepared by reconstitution of lyophilized spinosine using water for injection, which may optionally include one or more antioxidants.

Dosages may also be adjusted in a patient specific manner to provide a predetermined concentration of spinocin activity in the blood, as measured by, for example, RIA techniques. Patient dosage can be adjusted to achieve controlled progression to a state of 50-1000 ng / ml, preferably 150-500 ng / ml via blood levels measured by RIA.

Example  One

Spinosad  Topical application is in sheep Mulzing ( mulesing Improve wound healing from work. (Study number T9CAL0205 )

Castration and trimmed Merino lambs were emulated (the emulsification involves surgical removal of fleece-containing skin from the area of the limbs and degrades sheep's susceptibility to fly blow upon regeneration). Fifty sheep were left untreated and 381 groups of 1 were treated with 7.1 g of 4 mg / g spinosad aerosol (see Table 1 below for formulation). The spinosad aerosol was applied topically until the wool was carefully cleaved and moistened with the product.

Spinosad Aerosol Formulations ingredient Quantitative% w / w Spinosad @ 89.9% 0.445 Alcohol 100AGF4 20 Chlorhexidine Digluconate 20% 0.08 Brilliant Blue FCF 0.02 Profile gallate 0.01 Lactic acid 85% 0.06 Propylene glycol 10 Deionized water 64.43 Twin 20 2 PVP K30 3

Mulling wounds of 18 lambs per group were examined closely and evaluated for evidence of wound healing 7 and 17 days after treatment (Table 1). Scab formation and wound shrinkage were scored from 0 (complete or normal healing) to 3 (corresponding to no healing). Bleeding / serum effusion and damaged healing were visually scored from 0 (no bleeding / exudation and normal healing) to 3 (severe bleeding or damaged healing).

Influence of topical spinosad application on wound healing in sheep after the emulsifying operation. group ^# Scab formation ^# Wound shrinkage ^# Hemorrhage / serous effusion Heal Damage ^@ Days 7 17 7 17 7 17 7 17 Unprocessed 0.04 0.57 0.12 0.88 0.12 0.08 0 0 Spinosad Aerosol 0.04 0 0.08 0.36 0.12 0 0 0

^# Geometric mean. Low scores indicate better healing.

^@ 18 can be a crazy amount of influence.

After 7 days of emulsification, the amount treated with spinosad was greater in shrinkage of the emulsifying wound than the untreated control as measured by the lower mean score for wound contraction. By 17 days after mulching, the amount treated with spinosad improved scab formation, wound contraction, and decreased bleeding and serous exudation, indicating improved wound healing compared to untreated control amounts.

Topical application of spinosad to sheep mulching wounds will improve wound healing 17 days after treatment compared to untreated animals. This improvement in wound healing occurred in the absence of confounding factors such as black fly blows. However, the formulations used in this example also included an antibacterial compound, chlorhexidine. Chlorhexidine's ability to inhibit bacterial infection may have a positive effect on wound healing in this example.

Example  2

Spinosad  Topical application is in sheep Mulzing  Improve wound healing from work. (Study number T9CAL0206 )

The sweet and lean dependent merino female lambs were emulated. Approximately 46 ml of diluted (125 ppm) xtinosad (see Table 3 below for formulations) was used in 150 litters using a small amount (2 or 5 L) of pressurized garden manual spray available at a retail store for garden articles. The amount was applied to the emulsifying wounds. The container or reservoir is conical shaped and pressurized by manual pumping. The spray is delivered from conical nozzles. Spinosad aerosol 4 mg / g (see Table 1 above for formulation) was applied to the wound using an aerosol spray until the wound and surrounding wool were wetted by the product. Fifty sheep were left untreated after emulation (control).

Extinosad (25 g / L Spinosad) Formulation ingredient Quantitative% w / w Spinosad @ 92% 2.7 Dagger One Proxel GXL 0.2 Propylene glycol 10 Xantum sword 0.2 Pluronic P123 or Pluronic P105 1.0 Antifoam C (30%) 0.2 Loma PWA (44.5% solution) 4.5 Deionized water 80.2

After 14 days of treatment, the lambs were collected and caged in cages. Over 15 minutes, the lambs were observed and identified with any signal of fly blow, such as tail cramps, biting in the area or trampling of the feet. Twenty-five lambs from each group were randomly selected, placed on a rail and their mulching wounds evaluated for evidence of flies blow and wound healing (Table 4). Scab formation and wound shrinkage were scored from 0 (complete or normal healing) to 3 (corresponding to no healing). Bleeding / serum effusion and damaged healing were visually scored from 0 (no bleeding / exudation and normal healing) to 3 (severe bleeding or damaged healing).

Influence of topical spinosad application delivered to two different formulations on wound healing, and the presence and infection of fly strikes in sheep 14 days after the emulsifying operation and treatment. group ^# Scab formation ^# Wound shrinkage ^# Hemorrhage / serous effusion Heal Damage ^{@ @} Larva present ^@ Infection present Unprocessed 0.15 0.15 0.15 4 4 6 Extinosad (Spinosad 125mg / L) 0.06 0.03 0 0 One 0 Spinosad Aerosol (4mg / L) 0.03 0.06 0 0 0 0

^# Geometric mean. Low scores indicate better healing.

The number of sheep affected 25 ^@ .

The amount treated with spinosad as an aerosol formulation (4 mg / L) or manual spray (125 mg / L) is evidenced by reduced scores for scab formation, wound contraction, and the presence of bleeding and serous exudation at the wound site. It also showed improved wound healing. Moreover, none of the 25 sheep irradiated with each treatment group compared to the 4 heal failure animals recorded with the untreated control group was non-heal failure. Larvae from Lucilia cuprina were 4 out of 25 untreated control sheep compared to 1 sheep from the group treated with Extinosad and 0 from the group treated with Spinosad aerosol. It was in Marie. Infection at the wound site was present in 6 of 25 sheep in the untreated group, but absent in the group treated with spinosad.

Topical treatment of the mulling wounds using spinosad improved wound healing compared to untreated control animals as shown in Example 1 above. This improvement in wound healing was accompanied by a lowered infection rate and a lower presence of Lucilia cuprina larvae. Improvements in wound healing occurred regardless of the formulation or dosage of the spinosad applied to the wound in mulching. Moreover, spinosad can enhance wound healing in the absence of chlorhexidine (extinosad). This data indicates that the increased wound healing shown in Examples 1 and 2 may be due to the effects of spinosad itself rather than the infection-inhibition of chlorhexidine.

Example  3

Spinosad  Using thermal damage model In the rat  Improve wound healing

a) normal immature In the rat  For laser-induced wound healing Spinosad  effect

Hair was removed from the inter-shoulder site 24 hours before wound induction in 3 month old and approximately 300 g immature randomized male Sprague Dawley rats. Rats were anesthetized and thermal burns were induced with a CO ₂ laser (beam spot diameter setting of 10 mm and 25 watts power, 4 consecutive stimulation periods at 0.5 sec pulses), resulting in a circular wound area of 2 cm 2. Groups of 12 rats were treated with saline, citric acid (5%) or spinosad (0.5% in 5% citric acid). Treatments were delivered twice daily for 5 days post wound induction via 100 μl of two intradermal injections on the opposite side of the wound.

Scab formation caused a transient decrease at the rate of wound contraction [Snowden et al., Aust . J. Exp . Biol . Med . Sci . 60: 73-82 (1982). Thus, the scab and lightly attached skin were gently removed upon detection to keep all wounds comparable and allow accurate tracking of the wound area.

Wounds were examined daily for 6 consecutive days after wound induction and every 48 hours thereafter until complete wound closure / re-epithelialization was performed. The image area (maximum diameter of the wound) was tracked under stereomicroscope for accuracy and then measured with a digital planetimeter. The healing endpoint is when complete wound contraction occurs (Table 5).

Effect of spinosad (0.5%) on wound healing rats in rats exposed to thermal burn injury compared to saline and citric acid controls. Days after wound start Wound size cm 2 (group mean ± SEM, n = 12) Brine Citric acid Spinosad 0.5% One 2.5 ± 0 2.6 ± 0.1 2.6 ± 0.1 2 2.8 ± 0.1 3.1 ± 0.1 1.9 ± 0.1 3 4.1 ± 0.2 4.5 ± 0.2 1.7 ± 0.1 4 4.3 ± 0.1 4.4 ± 0.2 1.4 ± 0.1 5 4.5 ± 0.1 4.8 ± 0.1 1.3 ± 0.1 7 3.4 ± 0.1 3.6 ± 0.2 1.2 ± 0.1 9 2.3 ± 0.3 2.5 ± 0.1 1.0 ± 0.1 11 1.9 ± 0.1 1.5 ± 0.2 0.4 ± 0.1 12 * * Resolved 13 1.1 ± 0.1 0.6 ± 0.2 15 0.4 ± 0.2 0.1 ± 0 16 Dissipated Dissipated

The effect of spinosad on wound healing was significant until 2 days post wound induction, and spinosad treated rats were 30% smaller in wounds than those of saline and citric acid treated controls. Rats treated with spinosad showed complete dissipation of the wound up to 12 days, which was 4 days faster than their counterparts treated with saline or citric acid.

These data show that a simple aqueous solution of spinosad can promote wound healing in normal immature healthy rats.

b) Diabetes mellitus In the rat  Spinosa for the healing of laser-induced wounds De  effect

Induction of Diabetes Using Streptozotocin

Diabetes was induced in 3-month-old randomized male Sprague Dawley rats using streptozotocin (STZ). Streptozotocin (75 mg / kg) was dissolved in 0.1 M cold sodium citrate buffer (pH 4) and kept on ice to prevent degradation. 24 hours after fasting, rats were injected intraperitoneally with a single dose of freshly prepared (cold) STZ (Rakienten et al., Cancer Chemotherapy Report 29: 73-82 (1963)). Symptoms of diabetes become evident in these rats within 2 to 3 days and their diabetes status was confirmed by urine glucose test. Insulin treatment has been used to allow regular and severe hyperglycemia from the catabolic predominance of the state and to allow regular and severe hyperglycemia [Willars et al. , J. Neurol . Sci . 91: 153-164 (1989). One to two insulin injections (Protopane 2 IU / 100 g) were administered subcutaneously to new diabetic rats according to the severity of their physical state (e.g., little to eat or drink, weight loss to about 115 g, extremely inert) ). Rats that were not improved or who lost at least 15% of body weight were slaughtered.

Approximately 200 g of diabetic rats had their hair removed from the area between the shoulders 24 hours before wound induction. Rats were anesthetized and thermal burns were induced with a CO ₂ laser (beam spot diameter setting of 10 mm and 25 watts power, 4 consecutive stimulation periods at 0.5 sec pulses), resulting in a circular wound area of 2 cm 2. Groups of 12 rats were treated with saline or spinosad (0.5% in 5% citric acid). Treatments were delivered twice daily daily for 5 days after wound induction by dropping 100 μl of solution directly onto the wound site.

The scab and lightly attached skin were gently removed upon detection to keep all wounds comparable and to allow accurate tracking of the wound area. Wounds were measured as described above (Example 3a) until the wound closure was clear (Table 6).

Effect of spinosad (0.5%) on wound healing rats in diabetic rats exposed to thermal burn injury compared to saline control. Days after wound start Wound size, cm 2 (group mean, n = 12) Brine Spinosad 0.5% One 2.2 ± 0 2.1 ± 0 2 2.2 ± 0.1 2.1 ± 0.1 3 2.1 ± 0.1 1.5 ± 0.1 4 2.1 ± 0.1 1.6 ± 0.1 5 2.4 ± 0.2 1.9 ± 0.1 6 3 ± 0 2.5 ± 0.2 8 3 ± 0.1 2.1 ± 0.1 10 2.3 ± 0.2 1.2 ± 0.2 12 1.2 ± 0.1 0.6 ± 0.1 13 * Dissipated 14 0.6 ± 0.1 16 0.2 ± 0.1 17 Dissipated

Diabetic rats treated locally with spinosad showed improved wound healing compared to the saline control 3 days after wound induction as indicated by the lowered wound area. Wound size contracted more rapidly and continuously in spinosad-treated rats, resulting in wound dissipation on day 13 four days earlier than wound dissipation occurred in saline-treated controls.

c) Diabetes mellitus In the rat  Effect of Spinosad on Healing of Laser-Induced Wounds

Twenty-four-month-old and approximately 600 g randomized male Sprague Dawley rats had their hair removed from the area between the shoulders 24 hours before wound induction. Rats were anesthetized and thermal burns were induced with a CO ₂ laser (beam spot diameter setting of 10 mm and 25 watts power, 4 consecutive stimulation periods at 0.5 sec pulses), resulting in a circular wound area of 2 cm 2. Groups of 12 rats were treated with saline or spinosad (0.5% in 5% citric acid). Treatments were delivered twice daily daily for 5 days after wound induction by dropping 100 μl of solution directly onto the wound site.

The scab and lightly attached skin were gently removed upon detection to keep all wounds comparable and to allow accurate tracking of the wound area. Wounds were measured as described above (Example 3a) until the wound closure was clear (Table 7).

Effect of spinosad (0.5%) on wound healing rats in mature rats exposed to thermal burn injury compared to saline control. Days after wound start Wound size, cm2 (group mean, n = 12) Brine Spinosad 0.5% One 2.3 ± 0 2.4 ± 0 2 2.3 ± 0 2.9 ± 0.1 3 3.5 ± 0.2 2.7 ± 0.2 4 4 ± 0.2 2.3 ± 0.2 5 4.9 ± 0.2 2 ± 0.2 6 5.6 ± 0.2 3.3 ± 0.1 8 4.6 ± 0.1 3.5 ± 0.1 10 3.2 ± 0.1 2.5 ± 0.2 12 2.2 ± 0.1 1.2 ± 0.1 14 1.6 ± 0.1 0.8 ± 0.1 15 * Dissipated 16 1.2 ± 0 18 0.9 ± 0.1 20 0.5 ± 0.1 21 Dissipated

Mature rats treated locally with spinosad showed improved wound healing compared to the saline control 3 days after wound induction as indicated by the degraded wound area. The wound size contracted continuously in spinosad-treated rats up to 6 days while the wound size was increased in saline-treated rats. Rats treated with spinosad continuously reduced wound size compared to the saline control group, resulting in wound dissipation on day 15, six days earlier than wound dissipation in the saline-treated control group.

Spinosad improved the rate of wound healing administered topically or intradermally.

Spinosad improved wound healing in mature rats with chronic sensorineural deficiency. These data suggest that spinosad can enhance wound healing through sensory nerve dependence and sensory nerve independent mechanisms. Thus, spinosad can cure chronic ulceration associated with aging.

Spinosad improved wound healing in rats suffering from induced diabetes, another form of sensory neurodeficiency. This data also suggests that the wound-healing properties of spinosad can be modulated through both sensory nerve dependent and independent pathways. Thus, spinosad can heal other wounds associated with chronic ulceration and poor healing in diabetes.

Spinosad improved healing in normal immature healthy rats with intact sensory neurons. These findings suggest that spinosad can work through sensory nerve independent mechanisms, or enhance sensory nerve function. Thus, spinosad may improve wound healing in healthy human patients, such as patients with injuries or surgery.

The results of Example 3 show that spinosad can stimulate wound healing through a number of mechanisms and will be effective in both healing failure and normal humans.

Diabetic and mature rats generally showed a delay before showing any significant response to injury (Tables 6 and 7) compared to healthy immature rats (Table 5). This delay is apparent during the inflammatory phase of wound healing [Gibran et al., J. Surg . Res . 108: 122: 128 (2002) and maturation [Khalil & Helme, J. Gerontol . Biol . Sci . 51A (5): B354-B361 (1996)] due to a general decline in the activity of afferent sensory neurons in rats. This spinosad may possibly produce a wound healing effect in diabetic rats due to the use of a local afferent sensory nerve stimulation or an independent mechanism of afferent sensory nerve activation independent mechanisms.

The effects of spinosad were more pronounced in control animals with intact sensory neurons (Table 5). This effect, together with additional positive effects during the late component of the proliferative phase (6-8 days) and during the remodeling phase (9-12 days), is the initial inflammatory phase (2-5 days), the initial component of the multiplication phase (2-6 days). Is especially obvious.

Spinosad has also been shown to have an effect during the remodeling phase of wound repair, including cell infiltration and proliferation. The dermis responds to damage during this period by producing collagen and matrix proteins that cause wound shrinkage and dissipation. Thus, spinosad may have an effect on mediators affecting cell infiltration and activation, such as cytokines, chemokines and other cellular signaling agents, or directly enhance skin production and skin remodeling at the wound site. Can be.

Example 3 shows that wound healing is improved over three periods of healing. The data of Example 3 confirmed the results of Examples 1 and 2 that the spinosad aids in the healing of wounds induced by surgery or burns. Spinosad enhanced wound healing in two species.

Example  4

Using a bullous model of neurological inflammation In the rat  To improve wound healing Spinosad  Investigate how it works

a) blood flow Spinosad  Dose Effect

Neurological inflammatory responses are well established methods [Khalil & Helme, Brain Res . 527: 292-298 (1990). Randomized male Sprague Dawley rats were anesthetized with pentobarbitone sodium (60 mg / kg intraperitoneal). General anesthesia was maintained by supplemental injections of 15 mg / kg. Anesthesia of this method has not been shown to modulate basal vasodilated neuronal response in peripheral microvascular [Khalil & Helme, Brain] Res . 500: 256-262 (1989). The jugular jugular vein was intubated with a polyethylene tube for intravenous administration of heparin / saline solution or drug. Body temperature was maintained at 37 ° C. At the completion of the experiment, animals were slaughtered by barbiturate excess.

The blisters were induced in the middle region of the hind paw of the anesthetized rat with a vacuum pressure of -40 kPa applied for 30 minutes through a metal suction cap heated to 40 ° C. Once blisters developed, the epidermis (surface epithelium) was removed and a perspex chamber with inlet and outlet outer ports was fixed on the blister base. Ringer's solution was perfused on the blister surface and maintained with a peristaltic pump at 4 ml / hr to determine baseline. Relative blood flow was monitored over time by a laser Doppler rheometer through a probe placed directly on the bleb base and relative blood flow (volts) continuously monitored on a chart recorder.

Spinosad was diluted in 5% citric acid in Ringer's solution and perfused on the blister base for up to 30 minutes.

Influence of spinosad at different concentrations on blood flow on the blister base compared to Ringer's solution. Spinosad concentration 0.05% 0.5% 5% Area under the curve cm ² (mean ± SEM, n = 6) 32 (± 5) 113.6 (± 9.4) 62.4 (± 4.8)

Spinosads perfused across blister wounds at 0.5% concentration had the highest impact on blood flow in terms of height and duration of response. The 0.05% spinosad solution had a positive effect on blood flow, but the magnitude of the response was reduced compared to 0.5% spinosad. Spinosad at 5% concentration showed an intermediate reaction with peaks of reduced duration and magnitude compared to the reaction obtained with 0.5% spinosad. The sustained magnitude of the response, represented by 0.5% spinosad, suggests interaction with the calcitonin gene related peptide (CGRP) neural pathways, but desensitization with higher concentrations of spinosad is often associated with the Substance P mediated pathway. Related. These data indicate that they had a positive effect on blood flow, an essential requirement for wound healing. This action can be performed either due to direct action on blood vessels or through the release of peptides such as CGRP and substance P from sensory nerves that mediate the vascular response again.

b) 0.5% To spinosad  About Rat  Effects of Sensory Neuropeptide Antagonists and Nitric Oxide Synthase Inhibitors on Vascular Responses

Neurological inflammatory responses are well established methods [Khalil & Helme, Brain Res. 527: 292-298 (1990). Randomized male Sprague Dawley rats were anesthetized with pentobarbitone sodium (60 mg / kg intraperitoneal). General anesthesia was maintained by supplemental injections of 15 mg / kg. Anesthesia of this method has not been shown to modulate basal vasodilated neuronal response in peripheral microvascular [Khalil & Helme, Brain Res. 500: 256-262 (1989). The jugular jugular vein was intubated with a polyethylene tube for intravenous administration of heparin / saline solution or drug. Body temperature was maintained at 37 ° C. At the completion of the experiment, animals were slaughtered by barbiturate excess.

The blisters were induced in the middle region of the hind paw of the anesthetized rat with a vacuum pressure of -40 kPa applied for 30 minutes through a metal suction cap heated to 40 ° C. Once blisters developed, the epidermis (surface epithelium) was removed and a perspective chamber with inlet and outlet outer ports was fixed on the blister base. Ringer's solution was perfused on the blister surface and maintained with a peristaltic pump at 4 ml / hr to determine baseline. Relative blood flow was monitored over time by relative blood flow (volts) continuously monitored on a chart recorder and by a laser Doppler rheometer through a probe placed directly on the bleb base.

Spinosad was diluted in 5% citric acid in Ringer's solution and perfused on the blister base for up to 30 minutes. One paw of each rat was perfused with 0.5% spinosad, the other paw of each rat was first perfused for 10 minutes with an antagonist or inhibitor and then 0.5% spinosad was co-perfused for 30 minutes. _{8 -37} CGRP (CGRP antagonists; ahwooseu peptidase (Auspep) of Victoria, Australia, main material) and perfused at 1μM, N- nitro-L- arginine methyl ester (L-NAME, endothelial nitric oxide synthase inhibitors; of Michigan material Saiman Chemical Co.) was perfused at 100 μM and Spandide II (Substant P antagonist II; Auspep, Victoria, Australia) was perfused at 10 μM.

The effect of sensory neuropeptide antagonists and eNOS inhibitors on the vascular response of rat plantar blebs to perfusion of 0.5% spinosad, and the relative contribution of effectors to the activity of spinosad. Perfused substance Effector Blood flow (cm ² ) as area under the curve Mean ± SEM, n = 6 Effector's contribution to the vascular response of spinosad (%) Control Nil 113.6 (± 9.4) 100 L-NAME eNOS 87.2 (± 5.2) 24 CGRP _8-37 CGRP 83.2 (± 6.5) 27 Spanded Substance P 74.9 (± 3.6) 34

Blood flow in response to spinosad stimulation decreases in the presence of CGRP and SP peptide antagonists and NOS inhibitors (Table 9). These data indicate that the influence of spinosad on vascular blood flow is mediated through the CGRP, substant P and nitric oxide pathways. Contributions of CGRP, Substance P and eNO to the effect of Spinosad are 27%, 34% and 24%, respectively (Table 9).

Vascular response profiles in the presence of CGRP antagonists have a similar magnitude of response throughout perfusion, suggesting that the mediation of the spinosad effect by CGRP is constant over time. Vascular response profiles in the presence of substance P antagonists (spantides) or eNOS inhibitors (L-NAMEs) indicate that the response decreases with time. This suggests that Substance P and eNOS have a greater relevance to mediating the effects of Spinosad during the later stages of the response.

These data collectively indicate that the influence of spinosad on blood flow is mediated through sensory neuropeptide substance P and CGRP, and endothelial nitric oxide. The relevance of eNO in this process suggests that spinosad can act not only directly on the vascular endothelium but also through the sensory nerve distribution of blood vessels in the wound. Moreover, the influence of spinosad is long and is mediated through different effectors at different stages of the reaction. This finding may explain the difference in wound healing profiles (especially the initial dissipation of wounds in healing-deficient (mature or diabetic) rats) in spinosad treated rats and control rats in the thermal injury model (Example 3). . The ability of neuropeptide inhibitors and antagonists to inhibit the action of spinosad on the bloodstream suggests that the early wound healing properties of spinosad act through the sensory nerve pathways and are related to the combination of neuropeptides. This activity will enhance the early inflammatory phase of the wound-healing process, which is mostly initiated by sensory neuronal activity. Steinhoff et al., Arch . Dermatol . 139: 1479-1488 (2003). Furthermore, the data of Example 3 show that the spinosad effect on wound contraction in normal immature rats and heal-deficiency rats was significant during the first 5 days of healing.

c) response to vascular responses during the early and late stages of acute inflammation Spinosad  effect

Neurological inflammatory responses are well established methods [Khalil & Helme, Brain Res. 527: 292-298 (1990). Randomized male Sprague Dawley rats were anesthetized with pentobarbitone sodium (60 mg / kg intraperitoneal). General anesthesia was maintained by supplemental injections of 15 mg / kg. Anesthesia of this method has not been shown to modulate basal vasodilated neuronal response in peripheral microvascular [Khalil & Helme, Brain Res. 500: 256-262 (1989). The jugular jugular vein was intubated with a polyethylene tube for intravenous administration of heparin / saline solution or drug. Body temperature was maintained at 37 ° C. At the completion of the experiment, animals were slaughtered by barbiturate excess.

The blisters were induced in the middle region of the hind paw of the anesthetized rat with a vacuum pressure of -40 kPa applied for 30 minutes through a metal suction cap heated to 40 ° C. Once blisters developed, the epidermis (surface epithelium) was removed and a perspective chamber with inlet and outlet outer ports was fixed on the blister base. Ringer's solution was perfused on the blister surface and maintained with a peristaltic pump at 4 ml / hr to determine baseline. Relative blood flow was monitored over time by relative blood flow (volts) continuously monitored on a chart recorder and by a laser Doppler rheometer through a probe placed directly on the bleb base.

Spinosad was diluted in 5% citric acid in Ringer's solution and perfused on the blister base for up to 30 minutes. Spinosad was immediately perfused to the blisters on one sole of each rat (initial) and perfused 5 hours after blister induction to the blisters on the other plantar (end).

Effect of spinosad 0.5% on the vascular response of plantar blisters during the early (immediately after injury) and late (5 hours after injury) of acute inflammation. Inflammation stage Early last period Area under the curve (cm 2) Mean ± SEM, n = 6 113.6 (± 9.4) 95.5 (± 9.3)

Neural effectors are predominantly associated with the early stages of the inflammatory response, but the late stages of the inflammatory response are associated with components of the immune system, such as neutrophils and monocytes. The fact that spinosad has almost the same effect at the beginning and end of the inflammatory response (Table 10) shows that spinosad acts on the neuronal mediator of inflammation (as shown in Example 4b) as well as a significant effect on the immunological mediator of the inflammatory response. Suggest that we have This result is significant because of the involvement of immune mediators in recruitment and activation during the proliferative and remodeling phases of wound healing (6-12 days). As described above in Example 3, the effect of spinosad on wound healing is seen in the inflammatory, steam and remodeling phases. The data from this example provide evidence that spinosad stimulates neurological and immunological pathways to mediate its wound healing effect.

Claims (11)

A pharmaceutical composition for promoting or accelerating wound healing in a mammal, comprising spinosine or a physiologically acceptable salt thereof. The pharmaceutical composition of claim 1, wherein the spinosine is spinosad or a physiologically acceptable salt thereof. The pharmaceutical composition of claim 1, wherein the mammal is not curative of dysfunction. The pharmaceutical composition of claim 1, wherein the mammal is curative insufficiency. The pharmaceutical composition of claim 1, wherein the spinosine is applied topically to the wound. The pharmaceutical composition of claim 1, wherein the spinosine is administered orally. The pharmaceutical composition of claim 1, wherein the spinosine is administered parenterally. The pharmaceutical composition of claim 1, wherein the mammal is a ruminant. The pharmaceutical composition of claim 8, wherein the ruminant is a sheep. The pharmaceutical composition of claim 1, wherein the mammal is a human. The pharmaceutical composition of claim 1, wherein the mammal is a cat, dog or horse.