JP2011256177A - Use of spinosyn for wound healing - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing formulations for treating diseases or illnesses of mammals which need proliferation or regeneration of tissues.SOLUTION: The method includes administration of a spinosyn factor which is a macrolide produced by culture of Saccharopolyspora spinosa, an N-demethyl derivative of each spinosyn factor, a combination thereof, or a physiologically-acceptable salt.

Description

  The present invention relates to the therapeutic use of spinosyns in humans to enhance or promote wound healing in both normal and healing impaired humans. The spinosyns or physiologically acceptable derivatives or salts thereof can be used for therapeutic indications in humans that require soft tissue growth and regeneration. Wound healing disorders are a significant cause of human morbidity and can result in complications such as non-healing wounds. In normal individuals, wound healing is achieved through uncomplicated endogenous processes. In contrast, wound healing disorders are associated with several conditions such as diabetes, infection, immunosuppression, obesity and malnutrition.

  Wound healing is a complex biological process involving mediators such as extracellular matrix, blood cells, parenchymal cells, and cytokines. When the wound is hemostatic (when bleeding stops), the healing process begins. This is divided into three stages: inflammation, tissue formation (proliferation), and tissue regeneration (regeneration). After the damage has occurred, healing begins very quickly. For example, reepithelialization of skin wounds is initiated within a few hours (Singer and Clark, New Eng. J. Med. 341 (10): 738-746 (1999)). The process of wound healing is initiated by myelinated afferent sensory nerves, which in turn are neurogenic inflammation, immune responses (Eglezos et al., Adv. Exp. Biol. Med. 273: 499-503 (1989); Cell Biol.69: 285-294 (1991)) and vascular tone (Khalil & Helme, Brain Res. 500: 256-262 (1989); Brain Res. 527: 292-298 (1990)). These are all essential elements for healing. After initiation of healing by sensory nerves, the healing process is regulated by growth factors and cytokines that affect cell migration, proliferation, and protein production. During hemostasis, proteins such as fibrin and fibronectin interact to coagulate blood and upregulate cytokines and growth factors. After injury, inflammation begins.

  Inflammatory responses occur in three different phases, each mediated by distinctly different mechanisms. (1) an acute transition phase characterized by local vasodilation and increased capillary permeability; (2) a delayed subacute phase most markedly characterized by leukocyte and phagocytic infiltration; and (3) Chronic proliferative phase where tissue degeneration and fibrosis occur. Many different mechanisms are involved in the inflammatory process. The ability to provoke an inflammatory response is essential for survival in the face of environmental pathogens and injuries, but in some situations and diseases there is no obvious benefit and the inflammatory response persists in excess . During inflammation, neutrophils (polymorphonuclear leukocytes (PMNs)), monocytes, and macrophages infiltrate the wound site. These phagocytes release growth factors for the growth phase, enzyme mediators (proteases) that degrade proteins, phagocytose bacteria, dead cells, and dying cells, thus cleaning the wound site.

  In the next phase, growth begins. Collagen deposits and forms scar tissue. Fibroblasts produce proteoglycans that bind collagen fibers. Over time, collagen is broken down by proteases and reformed into a stronger scar structure.

  Spinosyn (also known as factor A83453) is an insecticide for agricultural animals, livestock, and companion animals and is active against: 1) Lepidoptera, 2) members of the order, 3) members of the order Diptera, 4) members of Coleoptera, and 5) members of the lice. Formulations suitable for administration to agricultural animals, livestock, and companion animals include various suspensions, solutions, tablets, capsules, liquids, and treatments.

  Spinosad (a product composed primarily of 85% Spinosyn A and 15% Spinosyn D) is currently approved for the treatment of lice on sheep and the treatment and prevention of flies flocking to sheep in Australia and New Zealand. Yes. In Brazil, spinosad is used for local treatment and protection of certain ticks, flies, and lices, and for the treatment of duck fly flies and skin wounds in cattle, sheep, goats, horses, pigs, birds, and dogs. Is approved as a disinfectant and healing insecticide.

  Spinosyn is known to be useful for preventing the development of lice in humans (US Pat. No. 6,063,771 and European Patent No. 1,252,820). A formulation suitable for use as a lice eradication agent for humans is also described in these patents.

  Despite known anthelmintic activity for spinosyns and commercially approved products, the spinosyn or its physiologically acceptable derivatives or salts have wound healing activity independent of formulations containing disinfectants / disinfectants It was discovered for the first time this time.

  The present invention treats human wounds to enhance wound healing, including incomplete or chronic wounds, skin diseases and allergic diseases, particularly skin related diseases and conditions or symptoms associated therewith, or To facilitate, this relates to a method comprising administering spinosyn or a physiologically acceptable derivative or salt thereof to a person in need thereof. Preferably, these methods are carried out by administering spinosad or a physiologically acceptable derivative or salt thereof.

  The present invention relates to a method for enhancing or promoting human wound healing comprising administering spinosyn or a physiologically acceptable derivative or salt thereof to a person in need thereof. provide. In another aspect, the invention relates to the use of spinosin or a physiologically acceptable derivative or salt thereof, or spinosin or a physiologically acceptable derivative or salt thereof, to enhance or promote human wound healing. The use of a formulation comprising any of the above is provided.

  Spinosyn is a naturally occurring fermentation product. This is a macrolide produced by the culture of Saccharopolyspora spinosa. Fermentation produces a number of factors including spinosyn A and spinosyn D (also called A83543A and A83543D). Spinosyn A and spinosyn D are the two spinosyns most active as insecticides. An agricultural product mainly composed of these two spinosyns (about 85% A and 15% D) is commercially available from Dow AgroSciences under the name Spinosad. Anthelmintic products containing spinosad are commercially available from Eli Lilly and Company. The name “spinosad” comes from a shortened version of spinosyns “A” and “D”.

  Each spinosyn consists of a 12-membered macrocyclic ring that is part of a four-membered ring compound to which two different sugars (amino sugar forosamine and neutral sugar 2N, 3N, 4N-tri-O-methylrhamnose) are attached. Have. Spinosyn is distinguished from other macrocycles by this unique structure.

  Spinosyn A (A83543A) was the spinosyn that was first isolated and identified from the fermentation broth of Saccharopolis spora spinosa. Subsequent research on fermentation broth revealed that S. Spinosa parental strains were found to produce a large number of spinosyns (A83543A-J) identified by AJ. Compared to spinosyn A, spinosyns B to J differ in the substitution pattern of the amino group of forosamine at selected sites on the four-membered ring and on 2N, 3N, 4N-tri-O-methylrhamnose. Characterized by. S. currently used. Spinosa strains produce a mixture of spinosyns whose major components are spinosyn A (˜85%) and spinosyn D (˜15%). Additional spinosyns (designated K-W) are described in S. Identified from a spinosa mutant.

  As used herein, the term “spinosine or a physiologically acceptable derivative or salt thereof” refers to the individual spinosyn 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 spinosyn factors, combinations thereof or physiologically acceptable salts Say. For convenience, the term “spinosine” is also used herein to mean individual spinosyns (or physiologically acceptable derivatives or salts thereof), or combinations thereof. Most preferred for human wound healing is spinosad or a physiologically acceptable derivative or salt thereof.

  EP 375316 includes spinosyns A to H and J (which they called A83543 Factors A, B, C, D, E, F, G, H and J), and their salts. It is disclosed. In US Pat. No. 5,202,242, Mynderse, et al. Discloses spinosyn L-N (which they called A83543 Factors L, M, and N), their N-demethyl derivatives and their salts, US Pat. No. 5,591,606 and In US Pat. No. 5,631,155, Turner, et al. Discloses spinosyn Q to T (which they called A83543 Factors Q, R, S, and T), their N-demethyl derivatives and their salts. These patents are incorporated herein by reference. Spinosyns K, O, P, U, V, W, and Y are, for example, Carl V. DeAmicis, James E .; Dripps, Chris J .; Hatton and Laura I.M. 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," is disclosed in pages 146-154 (1997). In US Pat. No. 6,001,981 various synthetic derivatives of spinosyn are described, and in US Pat. No. 6,455,504 various spinosin analogues are disclosed, both of which are It is incorporated herein by reference. Details regarding the fermentation and isolation of spinosyn and procedures for producing synthetic derivatives are described in these references.

  Both US and European patent applications disclose various dosage forms, anthelmintic activity and administration options in animals and agriculture for spinosyn and its physiologically acceptable derivatives or salts.

  U.S. Patent Nos. 6,063,771 and 6,342,482, and European Patent No. 1252820 describe spinosin or a physiologically acceptable derivative or salt thereof for preventing the development of lice in humans. And their use, as well as methods for preparing the formulation.

  As noted above, spinosad formulations are described in Dow AgroSciences, 9330 Zionsville Road, Indianapolis, Indiana, 46268-1054, U.S. Pat. S. A. And Elanco Animal Health, a Division of Eli Lilly and Company, P .; O. Box 708, 2001 W.M. Main Street, Greenfield, Indiana, 46140, U.S. Pat. S. Commercially available from A. In addition, S.M. Spinosa and mutant strains are: Agricultural Research Service Patent Culture Collection (NRRL) National Center for Agricultural Research Research, ARS, USDA, United States, 1815 North United. S. (NRRL 18395, 18537, 18538, 18539, 18719, 18720, 18743, 18823 and 30141 (US Pat. No. 6,455,504)).

  Spinosyn can be reacted to form a salt. Physiologically acceptable salts are also useful in the methods of the invention. Salts are prepared using standard methods for salt preparation. For example, spinosyn A can be neutralized with a suitable acid to form an acid addition salt. Spinosyn acid addition salts are particularly useful. Representative examples of suitable acid addition salts include, for example, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, cholic acid, pamonic acid, mucous acid, glutamic acid, camphor Organic or inorganic acids such as acids, glutaric acid, glycolic acid, phthalic acid, tartaric acid, formic acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid Salts formed by reaction with acids are included.

  All ratios, proportions and parts herein are by weight unless otherwise specified.

  “Wound” means damage to a human caused by a disorder or disease in which tissue has been cut, torn, destroyed, burned, or traumatic, or that causes such damage.

  The “healing” provided by the present invention is an increase or acceleration of the time from when a wound occurs (spinosine is administered) to when the wound closes (the wound fully contracts).

  The term “tissue” refers to a mass of cells in the human body that form a specific function in groups. Tissue includes but is not limited to nerves such as bone, skin, connective tissue, and spinal cord.

  As used herein, the terms “treating”, “treatment”, and “therapy” refer to therapeutic therapy. Those in need of treatment include humans with wounds, diseases, or illnesses.

  Administration “in combination with” one or more further therapeutic agents includes simultaneous administration to humans (in combination) and sequential administration in any order.

  A “therapeutically effective amount” is the minimum amount of active agent (eg, spinosyn, most preferably spinosad) that is necessary to produce a therapeutic effect in humans. For example, a “therapeutically effective amount” for a human having a wound induces, enhances, promotes, or causes a pathological symptom, progression of healing, improvement of the associated physiological condition, or to healing. The amount is to improve tolerance.

  As used herein, a “carrier” is a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to humans exposed to it at the dosage and concentration used. including. Often the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citric acid, and other organic acids, antioxidants including ascorbic acid, hydrophilic polymers such as polyvinylpyrrolidone, glycine, glutamine, asparagine, Monosaccharides, disaccharides and other carbohydrates including amino acids such as arginine or lysine, glucose, mannose or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and Non-ionic surfactants such as polyoxyethylene sorbitan fatty acid ester (TWEEN®), polyethylene glycol (PEG), and polyoxyethylene / polyoxypropylene block copolymer (PLURONIC®) Including.

  The spinosyns of the present invention, in particular spinosad, stimulate the neurogenic activation of healing and the inflammatory activity involved in subsequent cell growth and proliferation. Thus, the compositions of the present invention can be used in stimulating epithelial cell proliferation and basal keratinocytes for wound healing, particularly skin wounds. These wounds may be superficial or deep, including skin dermal and epidermal damage.

  Spinosyns are useful for treating a variety of wounds and conditions. For example, spinosyn is active in vivo in various wound healing models.

  The human to whom spinosin is administered may be a person whose wound is healed at a normal rate or a person with a healing disorder. When administered to an individual who is not a healing disorder, spinosin is administered to promote a normal healing process. When administered to an individual with a healing disorder, spinosin is administered to facilitate wound healing that would otherwise be slow or not heal at all. Many diseases and conditions can cause healing disorders. Such diseases and conditions include diabetes (eg, type II diabetes), treatment with steroidal and non-steroidal drugs, and ischemic occlusion or injury.

  Many growth factors have been shown to enhance wound healing in healing impaired individuals. These growth factors include growth hormone releasing factor, platelet derived growth factor, and basic fibroblast growth factor. Thus, the present invention also includes administering at least one spinosyn in combination with one or more growth factors or other agents that enhance wound healing.

  The spinosyns of the present invention promote healing of anastomoses and other wounds caused by surgical procedures in normal healing wounds or in people with impaired healing.

  The spinosyns of the present invention, especially spinosad, are found in normal individuals and in conditions that lead to wound healing disorders (uremia, malnutrition, vitamin deficiency, obesity, infections, and steroid drugs, radiation therapy, and anti-cancer / antimetabolites) In individuals with immunosuppression and complications related to systemic treatment), surgical wounds, excision wounds, deep wounds including dermis and epidermis damage, soft tissue injuries such as muscle tears, ocular tissue wounds, dental tissue wounds, Includes oral wounds, gastrointestinal mucosal wounds and ulcers, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, and thermal burns, exposure to extreme high or low temperatures, or exposure to chemicals It is clinically useful in promoting wound healing. The composition also provides skin reconstruction after skin loss to enhance healing of ischemia and wounds associated with ischemic injury, such as chronic venous leg ulcers caused by deficiencies and / or failure of venous circulatory system return. Useful to enhance, increase epidermal tensile strength and epidermal thickness, and enhance skin graft adhesion to the wound bed to promote re-epithelialization from the wound bed. is there.

  As used herein, an “individual” is assumed to be a human.

  For spinosyn formulations, suitable pharmaceutical diluents known to be useful in pharmaceutical compositions may be used. Such diluents include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. Preferably, the pharmaceutical composition is formulated for administration.

  Spinosyn may be administered as a pharmaceutical composition in combination with one or more pharmaceutically acceptable excipients. It will be appreciated that when administered to a human patient, the daily dose of the pharmaceutical composition of the invention will be determined by the attending physician according to a suitable medical diagnosis. The specific therapeutically effective dosage for any particular patient is the specific composition, including the type and extent of the targeted response, and whether other drugs are used, if any Age, weight, overall health, sex, and diet, time of administration of composition, route of administration, and excretion rate, duration of treatment, drugs used in combination with or simultaneously with a particular composition (such as chemotherapeutic drugs) ) As well as various factors including similar factors well known in the medical field. Suitable formulations known in the art can be found in Remington's Pharmaceutical Sciences (latest edition), Mach Publishing Company, Easton, PA. Accordingly, an “effective amount” of spinosyn for purposes herein (including an effective amount of spinosyn) is determined in view of these.

  The pharmaceutical composition of the present invention can be administered in any desired form, such as oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarticular, subcutaneous, intranasal, inhalation, intraocular, or intradermal route. it can. Parenteral administration and topical administration are preferred as administration routes.

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

The pharmaceutical composition is administered in an amount effective to treat the particular condition. In most cases, the dosage of spinosin is about 0.5 μg / kg body weight to about 50 mg / kg body weight per day in consideration of the administration route, symptoms and the like. However, the dose can be reduced to about 0.05 μg / kg body weight. For example, in certain cases for topical administration, the dosage is preferably about 0.2 μg to ² μg per cm ² . For intranasal and intraocular administration, the dosage is preferably from about 0.05 μg / kg to about 50 μg / kg body weight, more preferably from about 0.5 μg / kg to about 5 μg / kg body weight.

  As a general suggestion, the total pharmacologically effective amount of spinosyn administered parenterally ranges from about 0.5 g / kg / day to 5 mg / kg / day of patient weight, but is described above. As such, this is within the discretion of treatment. For continuous administration, spinosyn is typically administered at a dosage rate of about 10 μg / kg / hour to about 100 μg / kg / hour by injection 1 to 4 times per day or by continuous subcutaneous infusion, eg, using a minipump. Be administered. Intravenous bag solutions or bottle solutions may be used.

  The course of spinosin treatment appears to be optimal when it lasts longer than a certain minimum number of days (1-5 days for humans). Depending on the length of treatment, it is necessary to observe changes, and the interval after treatment to produce an effect varies depending on the desired effect.

  For parenteral administration, in one embodiment, the spinosyn is a pharmacologically acceptable carrier, ie, a carrier that is not toxic to the recipient in the amounts and concentrations used and is compatible with the other ingredients of the formulation. In addition, it is generally prepared by mixing in a form (solution, suspension or emulsion) in which a unit dose can be injected in a desired degree of purity.

  In general, the formulations are prepared by uniformly and intimate contact of spinosyn with a liquid carrier or a finely divided solid carrier, or both. Then, if necessary, the preparation is shaped into the desired dosage form. For parenteral carriers, solutions that are isotonic with the blood of the recipient are preferably used. Examples of such carrier media include water, saline, Ringer's solution, and dextrose solution. Similar to liposomes, non-aqueous solvents such as non-volatile oils and ethyl oleate are also useful herein. Suitable formulations known in the art can be found in Remington's Pharmaceutical Sciences (latest edition), Mach Publishing Company, Easton, PA.

  For topical administration, formulations such as ointments, creams, gels, etc. may be used at dosages for the compositions described above. Suitable formulations known in the art can be found in Remington's Pharmaceutical Sciences (latest edition), Mach Publishing Company, Easton, PA.

  Ointments are generally (1) oleaginous bases, i.e. consisting of non-volatile oils or hydrocarbons such as white petrolatum or mineral oil, or (2) absorbent bases, i.e. any capable of absorbing water such as dehydrated lanolin Prepared from any of the anhydrous materials. Conventionally, following formation of the substrate, the active ingredient (spinosine), whether oily or absorbent, is added in an amount that provides the desired concentration.

  Cream is an oil / water emulsion. This typically consists of an oil phase (inner phase) containing solidified oils such as wax, petrolatum, mineral oil, hydrocarbons, etc. (internal phase) and an aqueous phase containing any water soluble material such as water and added salts (continuous phase). ). The two phases are stabilized by using surfactants such as emulsifiers such as sodium lauryl sulfate, hydrocolloids (eg acacia colloidal clay, bagum, etc.). During the formation of the emulsion, the active ingredient (spinosine) is conventionally added in an amount to achieve the desired concentration.

  The gel comprises a base selected from an oleaginous base such as those described above, water or an emulsion suspension base. To the substrate is added a gelling agent that forms a matrix in the substrate and increases its viscosity. Examples of gelling agents include hydroxypropyl cellulose and acrylic acid polymers. Conventionally, the active ingredient (spinosine) is added to the formulation at the desired concentration in the state prior to the addition of the gelling agent.

  Oral pharmaceutical formulations are prepared by known procedures using well-known and readily available ingredients. In preparing the compositions, the active ingredients are usually mixed with a pharmacologically acceptable carrier, or diluted with a carrier, or in a carrier in the form of a capsule, sachet, paper, or other container. Encapsulate. When a carrier is used as a diluent, the carrier can be a solid, semi-solid, or liquid material that acts as a medium, excipient, or medium for the active ingredient. Thus, the composition can be in the form of tablets, pills, powders, lozenges, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules and the like.

  Tablets contain a number of inert materials, known as additives or excipients, in addition to the dose of active or therapeutic ingredient for the composition described above. These materials, including diluents, binders, glidants, and lubricants help to provide sufficient processing and compression properties to the formulation. The additional group of substances added serves to give more desirable physical characteristics to the finished tablet. Substances included in this group include disintegrants, colorants, and flavors and sweeteners in the case of chewable tablets, and polymers or waxes or other materials that delay dissolution in the case of sustained release tablets.

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

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

The composition is also envisaged for use in a wound dressing or bandage comprising a therapeutically effective amount of spinosin or a physiologically acceptable derivative or salt thereof. A wound protective bandage or bandage with an outer knitted fabric support, preferably an elastomeric knitted fabric support;
An inner pad. The inner pad preferably has an outer membrane surface made of a film-forming material and has a therapeutically effective amount of spinosyn or a physiologically acceptable derivative or salt thereof, preferably spinosad or a physiologically acceptable thereof, in the matrix. Derivatives or salts are incorporated at the dosages described above for the compositions. The pad may be integral with or separated from the outer knitted fabric support.

  Spinosyn is ideally incorporated into the matrix of the membrane, but may be incorporated into the material of the inner pad encased by the membrane.

  The therapeutically active agent is retained in the polymer matrix so that migration is prevented and spinosyn is gradually released over time.

  In another aspect, the wound dressing has an absorbent pad with a liquid permeable body side liner, a separate outer cover sheet, optionally an impermeable liquid, and an absorbent body disposed therebetween. The inner and / or absorbent body is made of a material that incorporates a therapeutically effective amount of spinosyn into the matrix or intermediate structural space to ensure that the spinosyn is near the wound site.

  The internal surface or pad of the bandage is preferably manufactured from natural or synthetic membranes, from organic or inorganic, animal or plant derived film forming materials, or from plastic materials. For example, plastic materials that are formed or coated in the usual manner as films or membranes, such as from gelatin or from vegetable gums or from hydrophilic or hydrophobic films, polyvinyl chloride polyacetate or polyamide. Manufactured.

  Suitable polymer materials include silastic or other silicone-based materials, polyethylene terephthalate (PET), Dacron, knitted Dacron, velor dacron, polygrasine, nylon, silk, polyethylene (PE), polyurethane, polyvinyl chloride sila Stick elastomer, silicone, rubber, PMMA (poly- (methyl methacrylate)), latex, polypropylene (PP), polyolefin, cellulose, polyvinyl alcohol (PVA), poly (hydroxyethyl methacrylate) (PHEMA), poly (glycolic acid), Poly (acrylonitrile) (PAN), fluoroethylene-cohexa-fluoropropylene (FEP), teflon (PTFE), copolymers thereof, and mixtures thereof, But it is not limited to these.

  The simplest way to incorporate a therapeutically active compound into a polymeric material is to incorporate the therapeutically active substance directly into the plastic resin prior to molding or the like.

  The film or membrane is ideally made from a hydrophobic polymer that is permeable to both liquids and gases, but not microbial. The hydrophobicity of the film or membrane is a useful feature in that it reduces the tendency of the film or membrane to adhere to the wound site.

  The amount of spinosyn incorporated into the formulation is not critical. The concentration may be in an amount sufficient to provide the desired amount of spinosin and in a range sufficient to allow the formulation to be easily applied to the wound site.

The conventional amount of formulation applied to the wound depends on the size of the wound and the concentration of spinosyn in the formulation. Generally, the formulation is applied in an amount that provides about 0.1 μg to about 5 g of spinosyn per cm ^{2 of} wound. Preferably, the amount of spinosin applied ranges from about 0.2 μg to about 2 g / cm ² , most preferably from about 0.25 μg to about 0.5 g / cm ² .

  Spinosyns may also be administered to the eye in the form of liquids, droplets or concentrates or gels to treat human lacrimal gland injuries, disorders and conditions.

  Spinosyns can also be administered intranasally to the nasal mucosa in the form of droplets or sprays to treat human nasal mucosa and sinus epithelial disorders, injuries, and conditions.

  Spinosyns typically have a concentration in the medium of about 0.01 μg / ml to 50 mg / ml, preferably 0.01 μg / ml to 10 mg / ml, pH of about 5 to about 8, preferably about 6 to about. 7, most preferably at pH 6.2. It will be appreciated that by using certain of the above-described excipients, carriers, or stabilizers, a salt of spinosine is formed.

  Spinosyns are usually stored in single or multiple dose containers, such as sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 3 ml vial is filled with 1 ml of sterile filtered 1% (w / v) aqueous spinosin solution and the mixture is lyophilized. Infusions are prepared by redissolving lyophilized spinosin using distilled water for injection, which may optionally contain one or more antioxidants.

  The dose may be adjusted in a manner specific to the patient so that the blood spinosyn activity becomes a predetermined concentration determined by, for example, the RIA technique. Therefore, the dose of the patient may be adjusted so as to continue the regular bottom level of blood concentration measured by RIA as 50 to 1000 ng / ml, preferably about 150 to 500 ng / ml.

<Example 1> Topical application of spinosad improves wound healing by applying mulesing to sheep (study number T9CAL0205)

  Castration and mulling the cleaved merino lambs (Mulsing involves the surgical removal of woolen skin from the buttock, and when healed, the sensitivity of the sheep to the development of wrinkles is reduced. ). Fifty sheep were left untreated and a group of 381 were treated with 7.1 g of 4 mg / g spinosad aerosol (see Table 1 for formulations). Spinosad aerosol is applied topically until the wound site is moistened with the formulation, paying attention to the area.

  Mulesing wounds of 18 lambs per group are observed in detail on treatment days 7 and 17 to assess signs of wound healing (Table 1). Scap formation and wound contraction are recorded from 0 (complete or normal healing) to 3 (equivalent to no healing). Hemorrhagic / serous exudates and healing disorders are recorded visually from 0 (no bleeding / exudate, normal healing) to 3 (severe bleeding or healing disorders).

^＃幾何平均。低スコアほど治癒している。
^＠１８匹のうちの影響されたヒツジの数。

^# Geometric mean. The lower the score, the better.
^@ Number of affected sheep out of 18.

  Seven days after mulesing, sheep treated with spinosad show contraction of the mulesing wounds compared to untreated controls, which can be confirmed by a lower average score for wound contraction. By day 17 after mulesing, sheep treated with spinosad have improved scab formation, wound contraction, reduced bleeding and serous exudates, and improved wound healing compared to untreated control sheep did.

  It is believed that topical application of spinosad to the wound by sheep mulesing improves wound healing after 17 days of treatment compared to untreated animals. This improvement in wound healing occurs without any confounding factors such as the development of the moth fly. However, the formulation used in this example also contained an antimicrobial compound (chlorhexidine). The ability of chlorhexidine to prevent bacterial infection is also believed to have had a beneficial effect on wound healing in this example.

<Example 2> Topical application of spinosad improves wound healing by applying mules to sheep (study number T9CAL0206)

  Mulling a female Merino lamb that has been trimmed and trimmed. Approximately 46 ml of diluted (125 ppm) excitinosad (see Table 3 for formulations) using a small (2 or 5 L) pressurized horticultural hand spray applicator available at a garden retail store Apply to mule wounds of 150 lambs. The container or reservoir is formed in a cone and is pressurized by manual pumping. The spray is ejected from the cone nozzle. Spinosad aerosol 4 mg / g (see Table 1 for formulation) is applied to the wound using an aerosol spray until the wound and surrounding wool are moistened with the formulation. 50 sheep are left untreated after mulesing (control).

  After 14 days of treatment, the lamb is collected and placed in a cage. The lamb is observed over a 15 minute period to identify any signs of ulcers such as tail cramps, biting of the area, or foot trampling. Twenty-five lambs from each group are randomly selected and placed on the rails to evaluate these mulesing wounds for signs of ulcer development and wound healing (Table 4). Scap formation and wound contraction are recorded from 0 (complete or normal healing) to 3 (equivalent to no healing). Hemorrhagic / serous exudates and healing disorders are recorded visually from 0 (bleeding / no exudate normal healing) to 3 (severe bleeding or healing disorders).

^＃幾何平均。低スコアほど治癒している。
^＠２５匹のうちの影響されたヒツジの数。

^# Geometric mean. The lower the score, the better.
^@ Number of affected sheep out of 25.

  Sheep treated with spinosad either as an aerosol formulation (4 mg / L) or as a manual spray (125 mg / L) have reduced scores for scab formation, wound contraction and wound site bleeding and the presence of serous exudate Wound healing improved as evidenced by Furthermore, of the 25 sheep examined in each group, none of the animals are healing disorders compared to the 4 healing impaired animals highlighted in the untreated control group. There are sheep fly larvae in 4 of the 25 untreated control sheep compared to the sheep from the group treated with excitinosad and the group treated with spinosad aerosol. Infection at the wound site is present in 6 of the 25 sheep in the untreated group, but not in the group treated with spinosad.

  Topical treatment of mulesing wounds with spinosad improves wound healing compared to untreated control animals, as shown in Example 1 above. As wound healing improves, the infection rate decreases and the prevalence of sheep fly larvae also decreases. Improvement in wound healing is seen regardless of the dose of spinosad applied to the formulation or mulesing wound. Furthermore, spinosad can enhance wound healing even in the absence of chlorhexidine (exitinosad). This data suggests that the enhanced wound healing seen in Examples 1 and 2 may be an effect of spinosad itself rather than the property of inhibiting chlorhexidine infection.

Example 3 Spinosad Improves Wound Healing in Rats Using a Thermal Injury Model a) Effect of Spinosad on Laser-Induced Wound Healing in Normal, Young Rats Young outbred male SD rats (3 About 300 g), hair was removed from the interscapular region 24 hours before wound induction. Rats are anesthetized and a CO ₂ laser is used to induce burns (beam spot diameter set to 10 mm, each stimulated 4 times with 25 watts power, 0.5 second pulse), A 2 cm ² annular wound area is created. Groups of 12 rats are treated with saline, citric acid (5%), or spinosad (0.5% 5% citric acid solution). Treatment is performed by two intradermal injections of 100 μl on the opposite side of the wound twice a day for 5 days after wound induction.

  Scab formation temporarily reduces the rate of wound contraction (Snowden et al., Aust. J. exp. Biol. Med. Sci. 60: 73-82 (1982)). Therefore, the scab and lightly adhering scab are gently removed as soon as they are found to retain all corresponding wounds so that the wound area can be accurately tracked.

  Wounds are measured daily for 6 consecutive days after wound induction and thereafter every 48 hours until complete wound closure / re-epithelialization occurs. For accuracy, the area of burn (maximum wound diameter) was tracked under a stereo microscope and then measured with a digital planimeter. The final point of healing is when complete wound contraction occurs (Table 5).

  The effect of spinosad on wound healing was noticeable on the second day after wound induction, with rats treated with spinosad having 30% smaller wounds than controls treated with saline or citric acid. Rats treated with spinosad have full wound healing on day 12, which is 4 days earlier than those treated with saline or citric acid.

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

b) Effect of spinosad on laser-induced wound healing in diabetic (healing disorder) rats Diabetes induction using streptozotocin Diabetes is induced in 3 month old outbred male SD rats using streptozotocin (STZ) To do. Streptozotocin (75 mg / kg) is dissolved in 0.1 M cold sodium citrate buffer (pH 4) and kept on ice to avoid degradation. After 24 hours of fasting, rats are injected intraperitoneally with a single dose of freshly prepared (cold) STZ (Rakienten et al., Cancer Chemotherapy Report 29: 73-82 (1963)). Diabetes symptoms appear in these rats within 2-3 days and their diabetic status is confirmed by a urine glucose test. Insulin therapy is used to provide regular interruptions from catabolic dominance of the condition and to regular and severe hyperglycemia (Willers et al., J. Neurol. Sci. 91: 153-164 (1989)). . Depending on the severity of these physiological conditions (for example, eating and drinking almost, weight loss to about 115 g, extremely inactive), one or two injections of insulin (protophan 2IU / 100 g) are given to diabetic rats And administer freshly subcutaneously. Rats that could not improve or lost more than 15% of their body weight are sacrificed.

Approximately 200 g of diabetic rats had their hair removed from the interscapular region 24 hours prior to wound induction. Rats are anesthetized and a CO ₂ laser is used to induce burns (beam spot diameter set to 10 mm, each stimulated 4 times with 25 watts power, 0.5 second pulse), A 2 cm ² annular wound area is created. Groups of 12 rats are treated with saline, citric acid (5%), or spinosad (0.5% 5% citric acid solution). Treatment is performed by two intradermal injections of 100 μl on the opposite side of the wound twice a day for 5 days after wound induction. The scab and lightly glued scab are gently removed as soon as they are found to retain all corresponding wounds so that accurate tracking of the wound area is possible. Wounds are measured as described above (Example 3a) until wound closure occurs (Table 6).

  Diabetic rats treated topically with spinosad improved wound healing compared to saline controls on day 3 after wound induction, as can be seen from the reduced wound area. The size of the wound contracts more rapidly in spinosad-treated rats, and the wound heals 13-4 days earlier than the saline-treated control.

c) Effect of spinosad on laser-induced wound healing in aged (healing disorder) rats Hair was removed from the interscapular region for 24 hours prior to wound induction in crossbred male SD rats (24 months old, approximately 600 g) . Rats are anesthetized and burns are induced using a CO ₂ laser (beam spot diameter set to 10 mm, each stimulated 4 times with 25 watt power, 0.5 second pulse, 2 cm ^Two circular wound areas are generated, and groups of 12 rats are treated with saline, citric acid (5%), or spinosad (0.5% 5% citric acid solution). Over a period of 5 days, twice a day by two intradermal injections of 100 μl on the opposite side of the wound, removing the scab and the lightly adhering scab as soon as it is found, The wound is measured as described above (Example 3a) until wound closure occurs (Table 7).

  Older rats treated topically with spinosad improved wound healing compared to saline controls on day 3 after wound induction, as can be seen from the reduced wound area. Wound size contracts more rapidly in spinosad-treated rats and increases in wound size in saline-treated rats for up to 6 days. In rats treated with spinosad, the wound size continues to decrease compared to the saline control and the wound heales 13 and 4 days earlier than the saline treated control.

  Spinosad improves the rate of wound healing, whether topically or intradermally applied.

  Spinosad improves wound healing in elderly rats with chronic sensory nerve deficits. Such data suggests that spinosad can enhance wound healing through sensory nerve dependent and independent mechanisms. Thus, spinosad can heal chronic ulceration associated with aging.

  Spinosad improves wound healing in rats with induced diabetes, another form of sensory nerve deficiency. This data also suggests that the wound healing properties of spinosad can be adjusted via sensory nerve-dependent and independent pathways. Thus, spinosad can also heal other wounds associated with chronic ulceration and inadequate healing in diabetic patients.

  Spinosad improves healing in normal young healthy rats with normal sensory nerves. Such findings suggest that spinosad can operate through sensory nerve-independent mechanisms or enhance sensory nerve function. Thus, spinosad can also enhance wounds in healthy human patients such as those resulting from injury or surgery.

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

  Diabetic and aged rats generally have a lag phase (Tables 6 and 7) before any significant response to injury compared to healthy adolescent rats (Table 5). This lag phase is diabetic (Gibran et al., J. Surg. Res. 108: 122: 128 (2002)) and elderly (Khalil & Helme, J. Gerontol. Biol. Sci. 51A (5): B354-B361. (1996)) due to a general decline in the activity of afferent sensory nerves in rats and becomes apparent during the inflammatory phase of wound healing. Spinosad may produce a wound healing effect in diabetic rats, either locally by stimulating afferent sensory nerves or by utilizing a mechanism that is independent of afferent sensory nerve activation.

  The effect of spinosad is more pronounced in control animals with normal sensory nerves (Table 5). This effect is particularly evident in the early elements of the early inflammatory phase (2-5 days), proliferative phase (2-6 days), the proliferative phase (6-8 days) and the remodeling phase (9-12 days). There is a further advantageous effect in the later elements.

  Spinosad also appears to have an effect during the remodeling phase of wound repair, including cell infiltration and proliferation. The skin responds to injury by producing collagen and matrix proteins during this phase, which causes the wound to contract and heal. Thus, spinosad can have effects on mediators that affect cell invasion and activation, such as cytokines, chemokines, and other cell signaling substances, and can directly produce matrix production and skin remodeling at the wound site. Can be enhanced.

  Example 3 shows that spinosad improves wound healing throughout the three phases of healing. The data of Example 3 confirms the results of Examples 1 and 2 that spinosad helps wound healing induced by surgery or burns. Spinosad enhanced wound healing in two species.

Example 4 Study of the mode of action of spinosad in enhancing wound healing in rats using a herpes model of neurogenic inflammation a) Effect of spinosad dose on blood flow The neurogenic inflammatory response is well established (Khalil & Helme, Brain Res. 500: 256-262 (1989)). Anesthesia is induced in outbred male SD rats with pentobarbitone sodium (60 mg / kg ip). General anesthesia is maintained by supplemental injection of 15 mg / kg. This method of anesthesia has been shown not to affect the basal vasodilator response in the peripheral microvasculature (Khalil & Helme, Brain Res. 500: 256-262 (1989)). The left jugular vein is cannulated with a polyethylene tube for intravenous administration of heparin / saline or drug. Body temperature is maintained at 37 ° C. At the end of the experiment, animals are sacrificed by overdose of barbiturate.

  Herpes is induced in the middle region of the hind paw of anesthetized rats using a -40 kPa vacuum press applied through a metal suction cap heated to 40 ° C. for 30 minutes. Once herpes is established, remove the epidermis (surface epithelium) and perfuse the Ringer's solution over the base of the herpes in a perspex chamber with an air inlet and outlet to establish a baseline measurement of 4 ml / ml. Maintain with a peristal pump at time. Relative blood flow is monitored over time by a laser Doppler flow meter via a probe placed directly above the herpes base, and relative blood flow (volts) is continuously monitored with a chart recorder.

  Spinosad is diluted in Ringer's solution of 5% citric acid and perfused through the shingles base for 30 minutes.

  Spinosad perfused throughout the herpes wound has the greatest effect on blood flow at a concentration of 0.5% with respect to response magnitude and duration of response. A solution of 0.05% spinosad reduces the magnitude of the reaction compared to 0.5% spinosad but has a beneficial effect on blood flow. A 5% concentration of spinosad results in an intermediate reaction and reduces the duration and peak size compared to the reaction obtained with 0.5% spinosad. Maintaining the magnitude of the response seen with 0.5% spinosad suggests an interaction with the calcitonin gene-related peptide (CGRP) neural pathway, while seen with higher concentrations of spinosad Desensitization is often associated with a substance P-mediated pathway. These data indicate that spinosad has a beneficial effect on the blood flow essential for wound healing. This effect may be due to direct action on blood vessels or through the release of peptides such as CGRP and substance P from sensory nerves that in turn mediate vascular responses.

b) Effect of Sensory Neuropeptide Antagonists and Nitric Oxide Synthase Inhibitors on Rat Vascular Response to 0.5% Spinosad The neurogenic inflammatory response is assessed using well-established methods (Khalil & Helme, Brain Res., 527: 292-298 (1990)). Anesthesia is induced in outbred male SD rats with pentobarbitone sodium (60 mg / kg ip). General anesthesia is maintained by a 15 mg / kg supplemental injection. This method of anesthesia has been shown not to affect the basal vasodilator response in the peripheral microvasculature (Khalil & Helme, Brain Res. 500: 256-262 (1989)). The left jugular vein is cannulated with a polyethylene tube for intravenous administration of heparin / saline or drug. Body temperature is maintained at 37 ° C. At the end of the experiment, animals are sacrificed by overdose of barbiturate.

  Herpes is induced in the middle region of the hind paw of anesthetized rats using a -40 kPa vacuum press applied through a metal suction cap heated to 40 ° C. for 30 minutes. Once herpes is established, remove the epidermis (surface epithelium) and perfuse the Ringer's solution over the base of the herpes in a perspex chamber with an air inlet and outlet to establish a baseline measurement of 4 ml / ml. Maintain with a peristal pump at time. Relative blood flow is monitored over time by a laser Doppler flow meter via a probe placed directly above the herpes base, and relative blood flow (volts) is continuously monitored with a chart recorder.

  Spinosad is diluted in Ringer's solution of 5% citric acid and perfused through the shingles base for 30 minutes. One footpad of each rat is perfused with 0.5% spinosad, while the other footpad of each rat is first perfused with an antagonist or inhibitor for 10 minutes, followed by 0. Coperfuse 5% spinosad for 30 minutes. CGRP8-37 (CGRP antagonist; Auspep, VIC, Australia) was perfused at 1 μM and N-nitro L-arginine methyl ester (L-NAME, endothelial nitric oxide synthase inhibitor; Cayman Chemical Co., MI, USA) Is perfused at 100 μM, and perforated II substance P antagonist II (Austep, VIC, Australia) is perfused at 10 μM.

  In the presence of CGRP and SP peptide antagonists and NOS inhibitors, blood flow is reduced in response to spinosad stimulation (Table 9). These data indicate that the effect of spinosad on vascular blood flow is mediated through the CGRP, substance P, and nitric oxide pathways. The contributions of CGRP, substance P, and eNO to the effect of spinosad are 27%, 34%, and 24%, respectively (Table 9). The vascular response profile in the presence of a CGRP antagonist has a similar magnitude response throughout perfusion, suggesting that the mediation of spinosad effects by CGRP is constant over time. The vascular response profile in the presence of substance P antagonist (spun) or eNOS inhibitor (L-NAME) indicates that the response decreases with time. This suggests that substance P and eNOS are more involved during the late stages of the reaction than mediating the effects of spinosad.

  These data collectively indicate that the effect of spinosad on blood flow mediates sensory neuropeptide substances P and CGRP and mediates endothelial nitric oxide. The involvement of eNO in this process suggests that spinosad may be acting directly on the vascular endothelium as well as via sensory innervation of blood vessels in the wound. Furthermore, the effect of spinosad is mediated through different effectors at different reaction stages over a long period of time. Such findings account for differences in wound healing profiles between spinosad-treated and control rats in the early recovery of wounds in heat injury models (Example 3), particularly healing impaired (old or diabetic) rats. Can do. The ability of neuropeptide inhibitors and antagonists to inhibit the action of spinosad on the bloodstream suggests that the initial wound healing properties of spinosad act via the sensory nerve pathway and include a combination of neuropeptides. Such activity enhances the early inflammatory stage of the wound healing process initiated primarily by sensory nerve activity (Steinhoff et al., Arch. Dermatol. 139: 1479-1488 (2003)). In fact, the data from Example 3 show that the effect of spinosad on wound contraction is evident in the first 5 days of healing in normal young rats and healing impaired rats.

c) Effect of spinosad on vascular response in early and late phases of acute inflammation The neurogenic inflammatory response is assessed using well-established methods (Khalil & Helme, Brain Res. 527: 292-298 ( 1990)). Anesthesia is induced in outbred male SD rats with pentobarbitone sodium (60 mg / kg ip). General anesthesia is maintained by a 15 mg / kg supplemental injection. This method of anesthesia has been shown not to affect the basal vasodilator response in the peripheral microvasculature (Khalil & Helme, Brain Res. 500: 256-262 (1989)). The left jugular vein is cannulated with a polyethylene tube for intravenous administration of heparin / saline or drug. Body temperature is maintained at 37 ° C. At the end of the experiment, animals are sacrificed by overdose of barbiturate.

  Herpes is induced in the middle region of the hind paw of anesthetized rats using a -40 kPa vacuum press applied through a metal suction cap heated to 40 ° C. for 30 minutes. Once herpes is established, remove the epidermis (surface epithelium) and perfuse the Ringer's solution over the base of the herpes in a perspex chamber with an air inlet and outlet to establish a baseline measurement of 4 ml / ml. Maintain with a peristal pump at time. Relative blood flow is monitored over time by a laser Doppler flow meter via a probe placed directly above the herpes base, and relative blood flow (volts) is continuously monitored with a chart recorder.

  Spinosad is diluted in Ringer's solution of 5% citric acid and perfused through the shingles base for 30 minutes. The shingles on one footpad of each rat are immediately perfused with spinosad (early phase), while the shingles on the other footpad of each rat are perfused 5 hours after the induction of herpes ( Delay phase).

  Neurogenic effectors are primarily involved in the early phase of the inflammatory response, while the delayed phase of the inflammatory response includes components of the immune system such as neutrophils and monocytes. Because spinosad has approximately the same effect in the early and late stages of the inflammatory response (Table 10), spinosad is not only acting on neurogenic mediators of inflammation (also shown in Example 4b), This suggests that it also has a significant effect on immunological mediators of inflammatory responses. This result means that immune mediators are involved in cell mobilization and activation during the proliferative to remodeling phase of wound healing (6-12 days). As described above in Example 3, the effect of spinosad on wound healing is seen in the inflammatory, proliferative, and remodeling phases. The data of the current example provides evidence that spinosad stimulates neurogenic and immunological pathways to mediate its wound healing effect.

Claims (11)

  Use of a therapeutically effective amount of spinosyn or a salt thereof for the manufacture of a preparation for the treatment of a disease or disorder in a mammal in need of tissue growth or regeneration.   Use according to claim 1, wherein the spinosyn is spinosad or a physiologically acceptable salt thereof.   Use according to claim 1, wherein the spinosyn is administered topically.   Use according to claim 1, wherein the spinosyn is administered orally.   Use according to claim 1, wherein the spinosyn is administered parenterally.   2. Use according to claim 1, wherein the mammal is a sheep.   2. Use according to claim 1, wherein the mammal is a human.   Use of a therapeutically effective amount of spinosyn or a salt thereof for the manufacture of an oral formulation for the treatment of a mammalian disease or disorder in need of tissue growth or regeneration.   Use according to claim 8, wherein the spinosyn is spinosad or a physiologically acceptable salt thereof.   9. Use according to claim 8, wherein the mammal is a sheep.   9. Use according to claim 8, wherein the mammal is a human.