CN1788790A - Extrasin beta-16 for promoting vascularization, tissue regeneration and wound healing - Google Patents
Extrasin beta-16 for promoting vascularization, tissue regeneration and wound healing Download PDFInfo
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Abstract
The present invention relates to one active small molecular polypeptide, thymosin-beta 16, with the functions of promoting vascularization, tissue regeneration and wound healing, and its medicine composition may be used in promoting the tissue repair and healing of damaged skin and mucous membrane, and improving the blood supply of ischemic tissue. Therefore, medicine composition containing thymosin-beta 16 or its bioactive variant or salt in the effective dosage may be sued in treating various kinds of wound to promote its healing, as well as cardiac vascular diseases caused by insufficient blood supply and other ischemic diseases. Furthermore, the present invention may be also used in improving skin health, making skin humidified, delicate, soft and white, and restoring hair.
Description
Technical field
The present invention relates to one group and have the active small molecular polypeptide that promotes neovascularization, tissue regeneration and wound healing effect: thymosin beta 16, its pharmaceutical composition can be used for promoting tissue regeneration, reparation and the healing of skin, mucosa injury, improves the blood supply of ischemic tissue.The present invention also can be used for promoting skin health, makes skin wet, delicacy, softness, pure white and hair regeneration.
Background
Skin is to be wrapped in one deck of whole body surface by membranaceous soft tissue, be one of human body maximum and most important organ, have important physiological function such as sensation, regulate body temperature, absorption, secretion and drainage, it is most important that its integrity is kept normal activities to body.Skin serves as the first line of defence of resisting outside infringement, has important barrier action, therefore also subjects to most various damages.Common impairment factor has mechanicalness: as break, cut, rub; Physics and chemical: as burn and scald, cold injury and strong acid, strong alkali damage; Scalpel edge; And the skin lesion that causes of various chronic diseases: as decubital ulcer, skin ulcer etc.Body has certain Regeneration and Repair ability.Under the normal condition, general wound can natural healing.But often because of being subjected to patient body quality, wound size and the order of severity and having or not the influence of factors such as accompanying infection, wound healing in most cases needs medical intervention.Intractable cutaneous ulcer due to the diabetes is exactly one of example.Diabetes are a kind of common diseases, mainly are because the absolute or relative deficiency of insulin in the blood, and or target cell insulin sensitivity is reduced, cause blood glucose too high, glucose in urine occurs, and then cause fat and protein metabolism disturbance.The sickness rate of present diabetes, up to 2%, wherein the patient of 30%-50% cureless skin ulcer occurs with skin lesion in China, and happening part is many at the relatively poor lower limb of blood supply.Many diabeticss cause amputation at last because slight wound can not heal, even because of infection death.The vascular lesion that dysbolismus causes is the main cause that causes this skin histopathology's property damage, and the key of treatment is that enhancing body is repaired function, improves the metabolism of tissue, and microcirculation improvement promotes the skin tissue regeneration healing.
Except skin injury, gastrointestinal mucosa also is vulnerable to damage.Under normal circumstances; there is the excretory mucus of epithelial cell to cover on the gastric mucosa; and complete epithelial cell membrane is arranged with the cell and the mucous epithelium of mutual close-coupled; common mucus-the mucosal barrier that forms; the protection gastric mucosa; make it not by roughage mechanical wear, in the duodenal epithelium emiocytosis alkalescence intestinal juice and gastric acid.Gastro-duodenal mucosa has good blood circulation, thereby guarantees to supply with the sufficient nutrition material, removes metabolic waste, guarantees epithelial continual renovation, makes mucosa have integrity.Continue or over-drastic psychentonia, excited, can make nervous system and endocrine dysfunction; Drinking and eating irregularly, as roughage, bony spur etc. are to the physical property infringement of mucosa; Edible irritable food is as peracid, maror, ethanol etc.; Take some drugs, as aspirin, indometacin, reserpine etc.; Irregular eating time or the like all can cause the not normal of gastric mucosa injury and gastric juice secretory function.Smoking can cause vasoconstriction, suppresses pancreatic juice and biliary secretion, reduced in duodenum in and the ability of gastric acid, cause bile reflux simultaneously and destroy gastric mucosal barrier, weakened the protection factor, cause the gastrointestinal mucosa damage.
Peptic ulcer is a kind of common disease, is because gastric acid-pepsinia and mucous membrane protection function disequilibrium, and gastric acid and pepsin corrode gastric mucosa and the ulcer (tissue disappearance) that forms, penetrable muscular layer of mucosa, serious threat human beings'health.It is reported that the peptic ulcer patient accounts for 1/10th of whole world population.The whole nation is in 1,200,000,000 populations, and then the number of China's trouble peptic ulcer heals 100,000,000 at present.Because the very high sickness rate of peptic ulcer causes this sick medicine of treatment to have comparatively vast market.Factors such as the generation of peptic ulcer and prostaglandin generation, inherited genetic factors, psychentonia, diet are irregular, smoking, medicine, environment are relevant, and there is helicobacter pylori in sticking the touching of most of peptic ulcer patients' gastric antrum portion.Peptic ulcer mostly occurs and is being exposed to acid and pepsic zone.The most common is pyloric cap, claims duodenal bulbar ulcer: lesser gastric curvature, claim gastric ulcer, and also have pyloric canal ulcer, postbulbar ulcer, diverticulum ulcer, stoma ulcer, jejunal ulcer and infraoesophageal ulcer etc.When immune defense function and reparation underactivity, its wound infection rate increase and the state of an illness are increased the weight of, but also cause the delay of tissue repair even be difficult to healing, repairing quality is also relatively poor, relapse rate is higher, brings great inconvenience and misery to the patient, and serious also can cause other complication, as septic shock, digestive tract perforation, canceration etc., but threat to life.Therefore, enhancing body Regeneration and Repair ability, it is an importance of treatment peptic ulcer that its ulcer is healed early.
Generally, when less or damage is light when the wound scope, can repair by the normal healing process.The process of normal healing comprises: wound closure runs off with limit blood and prevents infections; Remove impaired tissue and blood clot; Fibroblast in the surrounding tissue that continues, fibroblast and vascular endothelial cell are invaded wound location and are formed granulation tissue, and various histiocytes substitute granulation tissue and rebuild organizational structure, and wound is healed fully.The Regeneration and Repair of epidermis and tissue thereof is finished by migration of epithelial cells, cell division and three processes of epithelium differentiation.After epithelium was impaired, the damaged basal layer cell of the epithelium broken ends of fractured bone on every side moved to wound surface, and the epithelial cell division is active, covers wound surface gradually, and epithelium further breaks up, and rebuilds the organizational structure and the function that lose, the keratinization of epithelium etc. can occur as skin.Granulation tissue plays following several respects important function in wound healing process: (1) infection and protection wound surface; (2) fast, the slough of machine blood clotting and other foreign bodies; (3) fill up wound or other are damaged.Approximately since the 3rd day, the wound bottom grew granulation tissue, and gos deep in wound.The main component of granulation tissue is vigorous outgrowth new capillary vessel and outgrowth fibroblast, wherein inflammatory cells such as the neutrophilic leukocyte that more or less in addition, macrophage.Secrete many somatomedin and cytokine by platelet generation aggreation at first, then by macrophage to the reaction of hypoxia and lactic acidosis and secrete, these factors can stimulate angiogenesis.Newborn blood capillary promptly begins to obtain employment, and already present venule sprouts and to the injury growth, and is interconnected to form capillary network.Angiogenesis is the key character that tissue injury repairs.
The new drug that is used at present wound healing clinically mainly is the somatomedin albuminoid, as epithelical cell growth factor, and other albumen of regulating cell proliferation and differentiation, for example albumen transforming factor-B family (TGF-B).Gene recombinaton cattle alkali fibroblast growth factor (Recombinant bovine bFGF) and gene recombinaton human epidermal growth factor (Recombinant human EGF) have got the Green Light and have been used for the clinical treatment wound healing.Carry out the four kinds of gene recombinant proteins that also have of clinical three phases experiment, comprising human fibroblastic growth factor etc.This class treatment albumen costliness of using in the wound healing therapy stores unstable and the use trouble, and proteic unstability (protein,split) may cause unexpectedly to the deleterious inflammatory reaction of host.
We find that through series of studies thymosin beta 16 has tangible promotion neovascularization, promote epithelial cell keratinization, tissue regeneration and wound healing effect.Thymosin is to be present in human body and the intravital one group of micromolecule polypeptide of animal, can be divided into α and β type.The main representative of α type family is a thymosin, has the effect of raise immunity, has been widely used in clinical treatment hepatitis.β type family has found 16 members, and aminoacid sequence has high homology, and what wherein content was maximum, tissue distribution is the widest is extrasin beta 4, secondly is extrasin beta-10.Extrasin beta 4 has 43 aminoacid the earliest by separating in the thymus, and molecular weight is 4.9KDa, has now found that this albumen has multiple function, is included in endothelial cell differentiation and migration, T cell differentiation, actin sequestration and the effect in vascularization.Thymosin beta 16 is found in human neuroblastoma at first, is made up of 45 aminoacid, and its aminoacid sequence is as shown in table 1, and molecular weight is 5KDa, is present in people's normal nervous tissue, parotid gland tissues, testis tissue and the superficial cell.Thymosin beta 16 can be incorporated on the actin monomer, thereby suppresses the polymerization of actin.Animal experiment study shows that these polypeptide small molecules can make the skin migration of epithelial cells to wound, stimulate wound to form newborn blood capillary, stimulate fibroblast and hypodermal cell in wound healing process, to produce structural protein and other conjunctive tissue composition, strengthen the formation of granulation tissue, promote tissue regeneration, and then promote wound healing.
Thymosin beta 16 is different from the medicine of treatment wound healing that use on the market or just under development.Thymosin beta 16 is not a somatomedin, and comparing with various somatomedin on the treatment wound healing has following several respects advantage: 1) stimulate epithelial cell to move specifically, promote the epithelial cell keratinization; 2) stimulating endothelial cell moves specifically, promotes vascularization; 3) molecular weight is very little, has only 45 aminoacid (somatomedin belongs to high molecular weight protein), is easily organizing internal diffusion; 4) have no side effect, can use repeatedly the part; 5) be difficult for being degraded by proteolytic enzyme, acting duration is long, can reduce the medication number of times; 6) thymosin beta 16 can directly act on cytoskeleton system, has the effect of sustenticular cell structural stability.Therefore, thymosin beta 16 has very special effect in the cell regeneration reparation in the wound healing, have important clinical application value.
Description of drawings
The experiment of Fig. 1 mouse skin wound healing.Mice is divided into thymosin beta 16 treatment group and normal saline matched group, every group of each 8 mice.Measured wound area on the 7th day.The area of thymosin beta 16 treatment group is significantly less than matched group, and statistics has extremely significant difference (P＜0.001).
Description of the invention
The present invention originates in the influence of pair cell differentiation of research thymosin beta 16 and on cell migration.Isolated test proof thymosin beta 16 promotes that as cell migration the factor can stimulate the orientation of human umbilical vein's endotheliocyte and epidermis cell to move.Tested the cell of number of different types with Boyden Chomber method, proved that thymosin beta 16 can stimulate the motion of human umbilical vein's endotheliocyte and epidermis cell specifically.Cell movement plays an important role in wound healing.Newborn blood capillary promptly begins to obtain employment, and already present venule sprouts and to the wound growth, need cell movement; The basal layer cell of the epithelium broken ends of fractured bone moves the covering wound surface to wound surface around damaged, needs cell movement.Thymosin beta 16 can promote cell migration, therefore has the effect of accelerating wound healing.
The present invention has determined that further thymosin beta 16 can promote effectively that in vivo vascular endothelial cell moves, and promotes vascularization, promotes tissue regeneration and wound healing.Chick chorioallantoic membrane (CAM) vascularization result of the test shows, thymosin beta 16 promote angiopoietic effect with as the effect of the epithelical cell growth factor of positive control quite or better.The test of animal wound shows that thymosin beta 16 has the effect of accelerating wound healing.After handled with thymosin beta 16 the wound part, wound area dwindled one times than matched group, and there is complete promoting epidermization the wound part, neovascularization widely, and collagen is synthetic also obviously to be increased, and wound healing time is than the obvious shortening of matched group.Actin is a cell intensive amount rich in protein, is cytoskeletal protein, and it can participate in the various kinds of cell functional activity by the conversion of monomer and polymer form, as cell movement, cellular contraction, absorption, secretion, dna replication dna, cell division or the like.Thymosin beta 16 can be incorporated on the actin monomer, comes the functional status of modulate actin by the polymerization that suppresses actin, quickens cell function activities such as cell movement, cell division, promotes neovascularization, tissue regeneration and wound healing.
Therefore, the invention provides a kind of thymosin beta 16 or its acceptable salts or its physiological function derivative that contains effective dose, and the formulated pharmaceutical composition with promotion neovascularization, tissue regeneration and wound healing effect of applicable carrier.
The present invention can local be used for the treatment of the surface damage of scratch, wound, burn, scald, cold injury and other form of skin surface tissue, the skin injury that various dermatosiss cause, the infective ulcer of deep skin tissue, malnutrition ulcer, obstruction ulcer, cornea damage, cornea ulcer also can be used for the gastrointestinal mucosa damage that some disease causes.In addition, the wound that also available compositions-treated surgical operation of the present invention causes for example is coated with active agents etc. on the operation skin wound, to accelerate operation wound heal.
The present invention can be used for treating peptic ulcer.Patient's coat of the stomach secreting mucus amount of suffering from peptic ulcer reduces, the gastric acid secretion amount is corresponding to be increased, too much gastric acid secretion will inevitably cause digesting and the anti-dysequilibrium that digests to gastric mucosa and to the more weak duodenum of sour resistance, and defensive enginery weakens, and causes the self-digestion of tissue.Gastroduodenal ulcer begins and invades and tela submucosa from mucosa, and Chang Shen reaches the flesh layer.Thymosin beta 16 has the damage of promotion local vascular and forms, and improves blood supply, and coat of the stomach secreting mucus amount is increased, and improves anti-digestion power, promotes the local granulation tissue growth of damage, fills up damaged, promotes tissue regeneration, accelerating wound healing.
The present invention can be used for treating the skin ulcer that is caused by diabetes.Minute blood vessel blocks and circulatory disturbance is to suffer from common pathological variation in the diabetics for a long time.Diabetics raises owing to blood glucose continues the higher blood fat that causes, and atherosclerosis and angiemphraxis appear in small artery, make the blood flow volume that flow to lower limb reduce.The function of blood is each tissue that nutrient substance is transported to health, the refuse that produces in also each being organized simultaneously transports out external, and after the blood circulation function reduced, at first affected was foot, because foot apart from heart farthest, blood arrives least easily.Therefore, the easy ischemic necrosis of the skin of lower limb and soft tissue forms the skin ulcer that is difficult to heal.Thymosin beta 16 can be used for skin of lower extremity ulcer place, promotes the damage local vascular to form, and improves blood supply, promotes wound healing.
Thymosin beta 16 among the present invention can be used for the skin treating or prevent to be caused by epidermolysis bullosa and all hypotypes thereof and degenerative, immune inflammation and other disorderly symptom of surrounding tissue.Epidermolysis bullosa is a kind of rare genetic disorder disease, shows as skin blister, and then causes open skin ulcer wound, ulcer and scab.The degree of this disease can be from slightly to seriousness deformity, even crisis life.Thymosin beta 16 has the ability that promotes reparation, healing and prevention, has the level of inducing terminal deoxy-nucleotide transferase, reducing inflammatory cytokine or chemokines, as a kind of endotheliocyte chemotactic factor and angiogenesis factor, thus healing and prevention patient's dermatosis.
The present invention can be used for the regeneration and the reparation of stimulatory organs tissue.For example behind excision part colon or other tissue, coat pharmaceutical composition of the present invention at the organ-tissue position of operation and form and tissue regeneration, accelerate the agglutination of organ with the new vessels that promotes organ-tissue.
On the other hand, the present invention can reduce the formation of scar tissue and the reconstruction and the absorption of the scar tissue that promotes to be caused by a variety of causes.Cause the various factors of scar tissue to comprise operation process, radiation, broken wound, chemical toxicant, virus and bacterial infection or wound." scar tissue " used herein is meant that the recovery process neutralizes and forms fiber and collagen tissue in other pathological process.For example, thymosin beta 16 is made intermixture also throw near damaged tissue, and then promote tissue regeneration, blood vessel hyperplasia and microcirculqtory system form, and the reconstruction that acceleration is organized reduces the formation of scar tissue.
The present invention can be used for improving skin and function of anti-skin aging, promotes metabolic turnover, the enhancement skin health of skin histology, makes skin wet, delicacy, softness, pure white.Keratinization is one of most important functions of epidermis cell.The basal cell horny layer of dividing a word with a hyphen at the end of a line gradually develops into seedless pancake horn cell by cone shape cell, and this develops required time is growth cycle, needs for 3～4 weeks approximately, and each confluent monolayer cells difference conversion time is called epidermis and renews rate.Epidermis renews rate and reduces gradually during skin aging.Thymosin beta 16 has tangible promotion migration of epithelial cells function, can quicken by the horny layer transition to the surface of the basal cell of deep layer, thereby promote the epithelial cell keratinization, keeps the continuous metabolic turnover of epidermis, therefore has function of anti-skin aging.Thymosin beta 16 may be the special molecular that skin homergy function and anti-aging effects are kept in have relevant with skin regeneration.
The present invention can stimulating endothelial cell migration and growth, vasostimulant propagation increases the blood flow of tissue, thus the treatment cardiovascular disease.The feature of cardiovascular disease is that the blood supply of heart or other target organ suffers damage." cardiovascular disease " used herein is meant the disease that heart and blood vessel cause owing to blood supply insufficiency, including, but not limited to, coronary artery disease, congestive heart failure and periphery angiopathy.Heart disease is meant the heart change that causes owing to stenosis or occlusion coronarius, and characteristics are the required nutritional labeling of heart and hypoxgia and cause myocardial infarction.The thymosin beta 16 of effective dose can be by promoting blood vessel hyperplasia, induce extra blood capillary, set up collateral circulation, thereby increase the blood flow of cardiac muscular tissue, provide heart required nutritional labeling, improve hypoxgia, the oxygen of cardiac muscular tissue and effect are increased, play the tissue injury that myocardial ischemia that prevention and treatment cause owing to partially or completely thromboembolism coronarius causes.
The present invention also can be used for the cranial nerve tissue injury that prevents and treat the blood supply deficiency to cause, spinal cord injury and peripheral nervous tissue injury, and various diseases and old and feeble cerebral tissue and the neural tissue injury that causes.This neural prevention, treatment and protective effect are not limited only to thymosin beta 16; a plurality of allotype bodies that also should comprise thymosin beta 16 are as the oxidized form of extrasin beta 4, extrasin beta 9, extrasin beta-10, extrasin beta 11, thymosin, extrasin beta 13, extrasin beta 14, extrasin beta 15 and above-mentioned allotype body.
The present invention provides a kind of promotion neovascularity outgrowth method for insufficient some tissue of blood supply.For example, the alopecia that the hair follicle blood supply minimizing that causes because of a variety of causes, hair follicle malnutrition (not destroying but there is hair follicle structure) cause.The thymosin beta 16 of effective dose can induce extra blood capillary by promoting the blood vessel hyperplasia of hair follicle tissue, thereby increases the blood flow of hair follicle tissue, improves blood supply, for the hair follicle tissue of ischemia provides nutrition.In addition, thymosin beta 16 also has and promotes hair follicle substrate stem cell to divide a word with a hyphen at the end of a line and the effect of newborn basal cell, improves the nutrition of hair follicle tissue, promotes hair regeneration.
The method that the present invention sets forth also can be applied to inductivity tissue regeneration engineering.At present, the medical skill personnel adopt inductivity tissue regeneration engineering to promote and accelerating wound when carrying out invasive surgery.Method is to promote the bioactive substance of histiocyte migration, differentiation to grow into the wound with having, and the cell of formative tissue structure is assembled in a large number in the wound.The present invention can be used to help the regeneration of human or animal's granulation tissue.Method is to contain the polymer of Bioabsorbable, and the solvent of solubility and the complex of thymosin beta 16 are positioned over regeneration place of human or animal's meat dental tissue.This complex promotes people or other mammiferous granulation tissue regeneration by the thymosin beta 16 that discharges effective dose.
In addition, the growth of solid tumor and transfer rely on angiogenesis.New vessels provides sufficient oxygen and nutrition for tumor cell, and the endotheliocyte of new vessels is expressed multiple somatomedin, stimulates tumor cell proliferation.Tumor vessel lacks pericyte, and basement membrane is imperfect, and therefore, tumor cell is easy to enter blood circulation.Because neovascularization plays an important role in tumor growth, be one of preferred oncotherapy embodiment so suppress the angiogenesis of tumor tissues.If there is not neovascularization in the tumor tissues, tumor tissues just can not obtain required nutrition, and tumor growth is slow or stop growing the also final necrosis of degenerating.Thymosin beta 16 has the promotion angiogenesis function among the present invention.Therefore, the inhibitor of thymosin beta 16 can be used for suppressing the angiogenesis of tumor tissues, thereby reaches the purpose of treatment tumor.
The thymosin beta 16 that the present invention describes is an inherent polypeptide composition in the normal body.Therefore, use highly purified thymosin beta 16 safety, reliable.In embodiment of the present invention, thymosin beta 16 can be a separation and Extraction from histiocyte, can be that direct chemical is synthetic, also can be artificial expression.In addition, any have the bioactive substance that can bring out or stimulate body tissue, cell to produce thymosin beta 16 and also can add in the skin care compositions and methods of the present invention.Below all belong to category of the present invention.
Thymosin beta 16 has a plurality of allotype bodies, and amino acid sequence homology can reach 80% or higher.Thymosin beta 16 has aminoacid sequence LKKTNT, and this sequence is with the sequestration of mediation actin or in conjunction with relevant.Although be not bound by any particular theory, the activity of thymosin beta 16 allotype body may partly be owing to have the ability of modulate actin polymerization.For example, the polymerization that thymosin beta 16 can modulate actin (for example, β-thymosin as if can by making the F-actin depolymerizing) in conjunction with free G-actin.The ability of thymosin beta 16 modulate actin polymerization is whole or in part because it can be by combination of LKKTNT sequence or sequestration actin.Therefore, other has sequestration or in conjunction with the protein of actin or modulate actin polymerization function, the allotype body that comprises the thymosin beta 16 that contains aminoacid sequence LKKTNT, also can promote vascularization, improve the function of tissue metabolism, accelerating wound healing, so be also included among the present invention.
Thymosin beta 16 is distributed in many tissues and the cell, and therefore the reagent that can stimulate thymosin beta 16 to produce can add to and promote tissue and/or cell generation thymosin beta 16 in the skin care compositions and methods.This class reagent includes but not limited to the member of growth factor family, as somatomedin (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF-β), basic fibroblast growth factor (BFGF), thymosin and the VEGF (VEGF) in insulin like growth factor (IGF-1), platelet source.Especially preferred reagent is other member of transforming growth factor (TGF-β) or TGF-beta superfamily.On the other hand, the preparation that can regulate thymosin beta 16 gene expression also can add in the skin care compositions and methods of the present invention.
Can also go into into to have the various antiinflammatories of anti-infectious function in the pharmaceutical composition of the present invention, have the Chinese medicine preparation of anti-infectious function, and other natural materials with anti-infectious function.Antiinflammatory is meant various steroids, non-steroidal anti-inflammatory agents and various antibiotics.
In this article, " effective dose " is meant the requirement that contains the thymosin beta 16 composition.Will promote the tissue regeneration effect when this composition uses therapeutic dose in process of tissue reparation.Thymosin beta 16 can promote and nurse one's health process of tissue reparation.Process of tissue reparation usually occurs in after the injury disease.Process of tissue reparation obstacle occurs often owing to causing the reflection of growth is not enough at the various cells of wound such as fibroblast, stem cell, vascular endothelial cell, histiocyte.Therefore, the reparation that adding can be induced or the activating agent of stimulating cellular growth can help organizing." promotion " used herein speech is meant that cell growth mechanism or process have activation, stimulation, potentiation, initiation and keep effect.These cell growth mechanisms or generating process are that tissue forms, and repair or grow necessary.
In this article, " carrier " is meant substrate or the adjuvant that contains active thymosin beta 16, is used for serving as delivery system.In the present invention, " delivery system " refers to active thymosin beta 16 to be the body of passing the human or animal when pharmaceutical composition (medicament preparation or dosage form) when being used.Therefore term " delivery system " comprises the substrate or the adjuvant of the form of ownership that common pharmaceutical composition can use, as solid, semisolid and fluid composition.Special relevant compositions is, for example, ointment, hydrophilic ointment, emulsifiable paste, paste, frost, gel, hydrogel, solution, emulsion, suspension, washing liquid, liniment, shampoo, soap agent, paste, spray, powder, thin film, bedding and padding, application, binder, plaster etc.
Wound topical application pharmaceutical composition of the present invention preferably contains the ointment or the frost of active component.When making ointment, active component can use paraffinic base or water miscibility ointment base, perhaps, makes the cream that active component contains water bloom base.The example of external type compositionss such as preparation masking liquid, ointment comprises mixing with additive aequum effective dose.These additives are selected from the substrate commonly used or the adjuvant of external used medicine or cosmetics usually, for example vaseline, liquid paraffin, lanoline, stearic acid list glyceride, Polyethylene Glycol, hexadecanol, glycerol, second alcohol and water etc.
If desired, the water of frost can comprise, for example, the polyhydric alcohol of 30%W/W at least promptly contains the alcohol of two or more bases, as propylene glycol, fourth-1, and 3-glycol, mannitol, sorbitol, glycerol and Polyethylene Glycol and their mixture.Topical preparation can increase or reduce contained active component as required.
Pharmaceutical composition of the present invention can also be an injecta composition, is used in the intravenous, skin film, intramuscular or subcutaneous injection.The example of preparation injecta composition comprises the thymosin beta 16 or derivatives thereof of effective dose and the fluid matrix mixing manufacture of aequum is formed that the example of this substrate comprises water or normal saline, wherein also can add other beneficiating ingredient or adjuvant.
Pharmaceutical composition of the present invention can be solid dosage forms compositionss such as oral capsule, tablet, powder, comprises the thymosin beta 16 or derivatives thereof dry powder of effective dose and the pharmaceutic adjuvant mixing manufacture of aequum are formed.Pharmaceutical composition of the present invention can also be the oral liquid compositions.The example of preparation oral liquid compositions comprises the thymosin beta 16 or derivatives thereof of effective dose and the fluid matrix mixing manufacture of aequum is formed that the example of this substrate comprises water or normal saline, wherein also can add other beneficiating ingredient or adjuvant.
The present invention can also use with the mode that may command discharges intermixture.Herein " may command release intermixture " is meant any complex.This complex can be used for slowly discharging the effective thymosin beta 16 composition that comprises wherein.This complex contains biological effective components can be solid shape, infiltrative, and half shape admittedly is colloidality or aqueous.
Thymosin beta 16 of the present invention also can be used in the compositions of cosmetics or other skin nursing products.These compositionss include but not limited to cleaning agent, eye cream, eye shadow, facial cream, milk shape lotion, skin lotion, perfume, XIANGFEN, oil for cosmetic purpose, pasty state perfume, powder, cold cream, skin-care gel, skin lotion, shave and use cream, shave and use lotion, suntan lotion, the anti-oil that shines, sun-proof lotion, the anti-lotion that shines, nail polish paste, clean lotion for skin care, cleansing cream, cleaning lotion, clean skin milk, the cleaning breast, the cosmetic base material, facial film, contain the zinc astringent, hand cream, XIANGFEN, whitening powder, vanishing cream, cosmetic end powder, the shower cosmetics, kermes, mascara, lip pomade, the lip frost, eyeliner, hair oil, hair caring liquid, suppurative mastitis, hair gel, mayonnaise agent, the hair moisturizing agent, shampoo, hair conditioner, deodorization, cologne etc.
The present invention has determined that thymosin beta 16 can promote moving of vascular endothelial cell effectively external, in vivo can the vascularization of inducing mouse cornea.Thymosin beta 16 promote angiopoietic effect with as the effect of the epidermal growth stimulating factor of positive control quite or better.And the test of animal wound shows that thymosin beta 16 has the function of accelerating wound healing.Compare with matched group after handling with thymosin beta 16 in animal wound part, more neovascularization are widely arranged, and wound collagen is synthetic also to be increased, and illustrates that thymosin beta 16 can accelerated in wounds contracture and promotion wound healing.
Embodiment
Embodiment 1
Cell culture
Human umbilical vein endothelial cells (HUVECS) is used the RPMI1640 culture medium culturing.Culture medium contains: 5% Ox blood serum, and 100 units per ml green grass or young crops/chain enzymes, 50 mcg/ml are celebrated big enzyme element, the plain B of 2-7 mcg/ml both sexes enzyme, supplement such as 5 units per ml heparin and 200 units per ml vascular endothelial cell growth factor.People's keratinization of epidermis cell and forehead fibroblast are cultivated with DMEM and 10% calf serum, and human breast cancer cell's strain MDA-MB435 cell is cultivated with RPMI1640 and 10% calf serum.Neutrophilic granulocyte separates from fresh human peripheral.Human vas smooth muscle cell smooth muscle cell culture medium culturing.
Embodiment 2
The cell migration experiment
Whether can cause specific cell migration in order to test after thymosin beta 16 stimulates, we use Boyden Chomber method to various kinds of cell strain test.Nitre film (aperture 0.8mm) both sides with Boyden Chamber two interventricular septum all cover Fibronection earlier, and concentration is 15 mcg/ml, place 60 minutes down for 37 ℃.Last chamber adds cell suspension, and following chamber is a complete culture solution, 5%CO
2Left standstill in the incubator 4 hours, and took out filter membrane, will go up chamber face cell and strike off, after fixing dyeing, at the microscopically cell counting.Every group of 3 films.Statistical variable is represented with mean standard deviation.The result shows the specific migration that causes Human umbilical vein endothelial cells and people's keratinization of epidermis cell of thymosin beta 16 energy, the quantity showed increased of wandering cell (the results are shown in Table 2).Experimental results show that thymosin beta 16 all obviously quickens the migration of Human umbilical vein endothelial cells in the concentration range of 10 to 1000 millimicro grams per milliliters, and be amount-result relation.On the contrary, thymosin beta 16 does not cause MDA-MB435 human breast cancer cell strain and the isocellular obvious migration of human vas smooth muscle cell.
Embodiment 3
" galling " wound is closed experiment
With one milliliter of suction pipe tip the monolayer Human umbilical vein endothelial cells of adherent growth is scratched scratch one, clean the cell that comes off with the PBS buffer.Continue to cultivate after adding the thymosin beta 16 of variable concentrations, observe broken trace and close situation.The thymosin beta 16 of 1000 millimicro grams per milliliters was handled after 4 hours, and Human umbilical vein endothelial cells is obviously supported the matched group of base more than only adding embryo to the quantity that the scratch place moves, and the time that scratch is closed also obviously accelerates.
Embodiment 4
Promote the blood vessel hyperplasia experiment
Thymosin beta 16 adopts chick chorioallantoic membrane (CAM) experiment to detect to the facilitation of angiogenesis, opens a 1cm hatching on 7 days the fertilization Embryo Gallus domesticus eggshell
2Left and right sides window on the sterilization filter paper, and is positioned over CAM with the chemosynthesis thymosin beta 16 albumen application of sample of variable concentrations, continues down for 37 ℃ to cultivate 48 hours at the constant temperature and humidity incubator then, observes the angiogenic growth situation.This experiment is the test contrast with the normal saline, phorbol myristate acetate (PMA) 0.5 microgram/positive contrast of 10 microlitres.The measuring result shows that thymosin beta 16 albumen application of sample group chick chorioallantoic membrane angiogenic growth is obviously vigorous, and caliber is thick and branch is many, is lobate distribution.With normal saline group angiogenic growth degree in contrast, the blood vessel hyperplasia degree of thymosin beta 16 5 micrograms/10 microlitres significantly increases than normal saline group, and the blood vessel hyperplasia degree of 10 micrograms/10 microlitres and PMA positive controls blood vessel hyperplasia degree are quite or slightly many.This experimental result shows that thymosin beta 16 has the effect of obvious promotion blood vessel hyperplasia.
Embodiment 5
The wound healing experiment
Behind the mouse anesthesia, remove the skin of mouse back, cause diameter to be about the wound of 4 millimeters sizes, at random animal is divided into thymosin beta 16 treatment group and matched group (every group of 8 animals) subsequently with 4 millimeters sample of tissue device.The medicine liquid droplet of 50 microlitres (containing 5 microgram thymosin beta 16s) is on wound, and matched group uses normal saline.Animal every other day receives seance, observes continuously after 7 days and puts to death mice.Wound area is used to assess the curative effect of thymosin beta 16.Wound skin is fixed in 10% formalin solution.H﹠amp; E and collagen staining method are used to observe the pathology variation of skin.Repeated experiments comes to the same thing.The result shows that the wound area of thymosin beta 16 processed group dwindles one times than matched group, and there is complete promoting epidermization the wound part, neovascularization widely, and collagen is synthetic also obviously to be increased.Wound healing time is than the obvious shortening of matched group, and statistics has extremely significant difference (P＜0.001).These results show that thymosin beta 16 has tangible promotion wound healing effect (seeing accompanying drawing 1).
Table 1: thymosin beta 16 protein sequence
N-terminal M SDKPDLSEVEKFDRSKLKKTNTEEKNTLPSKETIQQEKECVQTS C-end
Table 2: the influence of thymosin alpha-1 6 pair cell migration
| Cell category | Thymosin alpha-1 6 concentration (millimicro grams per milliliter) | |||||
| 1000 | 100 | 10 | 1 | 0 | Positive control | |
| People's keratinization of epidermis cell Human umbilical vein endothelial cells vascular smooth muscle cell human breast cancer cell | 81±2.2 72±2.3 1.1±0.4 0.8±0.4 | 65±1.1 58±1.4 0.5±0.5 1.3±0.7 | 54±1.4 46±1.5 1.0±0.4 1.8±0.4 | 21±1.0 14±1.3 1.0±0.3 2.4±0.8 | 7±0.3 11±0.6 0.9±0.3 2.0±0.5 | 58±1.4 54±1.6 36±3.5 66±3.7 |
Annotate: the data that example goes out in the table are meansigma methodss of cell migration number in three experiments
Sequence table
＜110〉grand duke Wang, Bao Leri
＜120〉thymosin beta 16 promotes vascularization, tissue regeneration and wound healing
<160>1
<210>1
<211>45
＜212〉aminoacid
<400>1
Met Ser Asp Lys Pro Asp Leu Ser Glu Val 10
Glu Lys Glu Asp Arg Ser Lys Leu Lys Lys 20
Thr Asn Thr Glu Glu Lys Asn Thr Leu Pro 30
Ser Lys Glu Thr Ile Gln Gln Glu Lys Glu 40
Cys Val Gln Thr Ser
Claims (10)
1 one kinds of pharmaceutical compositions that promote neovascularization, tissue regeneration and wound healing contain thymosin beta 16, its biological activity variant or salt and its pharmaceutically useful carrier of effective dosage.
2 is described according to claim 1, thymosin beta 16 is meant whole aminoacid sequences of thymosin beta 16, also can be partial amino-acid series, main being meant has the function fragment LKKTNT of actin combination, and the allotype body with this function fragment.
3 is described according to claim 2, and its thymosin beta 16 can be nature extraction, chemosynthesis or artificial the expression.
4 is described according to claim 1, and its pharmaceutical composition can comprise that other has various somatomedin, cytokine and other factor that promotes that thymosin beta 16 generates.
5 is described according to claim 1, its pharmaceutical composition can local be used for the treatment of the scratch of skin surface tissue, wound, burn, scald, the surface damage of cold injury and other form, the skin injury that various dermatosiss cause, the infective ulcer of deep skin tissue, the malnutrition ulcer, stop up ulcer, the cornea damage, cornea ulcer, the chronic skin ulcer that various diseases causes, the skin ulcer that diabetes cause, digestive tract ulcer, gastroduodenal ulcer, the disturbance of blood circulation ulcer, surgical incision also can be used for cosmetics and improves skin and anti aging effect.
6 is described according to claim 1, its pharmaceutical composition can be used for the cardiovascular disease for the treatment of and preventing to cause owing to blood supply insufficiency, include but not limited to coronary artery disease, congestive heart failure and periphery vascular lesion, can be used for also treating that the skin follicle blood supply reduces and the alopecia that causes.
7 is described according to claim 1, and its pharmaceutical composition can be used for treatment, prevention and protection cerebral tissue and peripheral nervous tissue injury, and spinal cord injury is used for neurocyte protection and promotes nervous tissue's regeneration.
8 is described according to claim 1, and its pharmaceutical composition can be used for the skin treating or prevent to be caused by epidermolysis bullosa and all hypotypes thereof and degenerative, immune inflammation and other disorderly symptom of surrounding tissue.
9 is described according to claim 1, and pharmaceutically useful carrier comprises the substrate or the adjuvant of the form of ownership that common pharmaceutical composition can use in its pharmaceutical composition, as solid, semisolid and fluid composition.Special relevant compositions is, for example, ointment, hydrophilic ointment, emulsifiable paste, paste, frost, gel, hydrogel, solution, emulsion, suspension, washing liquid, liniment, shampoo, soap agent, paste, spray, powder, thin film, bedding and padding, application, binder, plaster etc.
10 is described according to claim 1, and pharmaceutically useful carrier can be solid dosage forms compositionss such as oral capsule, tablet, powder in its pharmaceutical composition, can also be injecta composition, is used in the intravenous, skin film, intramuscular or subcutaneous injection.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009068688A3 (en) * | 2007-11-30 | 2009-09-24 | Immundiagnostik Ag | Composition for treatment and prevention of hair loss and premature graying of hair |
| CN108531543A (en) * | 2018-03-12 | 2018-09-14 | 黄健聪 | A kind of external combined method of evaluation hair tonic/Anti-hair loss effect |
| CN113198004A (en) * | 2021-05-19 | 2021-08-03 | 天津市宝恒生物科技有限公司 | Bacillus coagulans preparation for repairing intestinal mucosa |
| CN113242737A (en) * | 2018-11-21 | 2021-08-10 | 沃特蒂阿姆集团有限公司 | Healing composition comprising electrolyzed water |
-
2004
- 2004-12-13 CN CN 200410098483 patent/CN1788790A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009068688A3 (en) * | 2007-11-30 | 2009-09-24 | Immundiagnostik Ag | Composition for treatment and prevention of hair loss and premature graying of hair |
| CN108531543A (en) * | 2018-03-12 | 2018-09-14 | 黄健聪 | A kind of external combined method of evaluation hair tonic/Anti-hair loss effect |
| CN113242737A (en) * | 2018-11-21 | 2021-08-10 | 沃特蒂阿姆集团有限公司 | Healing composition comprising electrolyzed water |
| CN113242737B (en) * | 2018-11-21 | 2024-01-16 | 韦奥公司 | Healing composition comprising electrolyzed water |
| CN113198004A (en) * | 2021-05-19 | 2021-08-03 | 天津市宝恒生物科技有限公司 | Bacillus coagulans preparation for repairing intestinal mucosa |
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