阅读说明：本技术 用于防止辐射损伤和促进组织再生的组合物和方法 (For preventing radiation injury and promoting the composition and method of regeneration ) 是由 高塔姆·加特纳卡 于 2018-02-16 设计创作，主要内容包括：本文提供用于面临辐射损伤风险的受试者中治疗或防止这类损伤的组合物和方法。(Provided herein is the compositions and method for the treatment of or prevent this kind of damage in the subject for facing radiation injury risk.)

1. a method of treat or prevent this kind of damage in the subject for facing radiation injury risk, including to described tested Person's application includes the composition of isolated polypeptide, and the polypeptide includes the continuous amino of carboxyl least significant end 4 to 30 of α connection albumen Acid.

2. according to the method described in claim 1, wherein the subject be exposed to radiation before applying said compositions.

3. according to the method described in claim 1, wherein after being exposed to radiation, Xiang Suoshu subject's applying said compositions, Wherein the method inhibits the progress of the radiation injury.

4. according to the method described in claim 1, wherein by the composition local application to the subject.

5. according to the method described in claim 1, wherein the composition also includes hydroxyethylcellulose gel.

6. according to the method described in claim 5, wherein the hydroxyethylcellulose gel is deposited with the concentration of about 1.25% (w/w) In.

7. according to the method described in claim 1, wherein by the composition parenteral administration to the subject.

8. according to the method described in claim 7, wherein the composition is passed through in intravenous, subcutaneous, peritonaeum, intramuscular route It is applied to the subject.

9. according to the method described in claim 1, the composition is wherein applied to the subject by being atomized delivering method.

10. according to the method described in claim 1, wherein the composition is applied to by about 10 μM to about 2000 μM of dosage The subject.

11. according to the method described in claim 1, wherein the composition is applied to by about 100 μM to about 200 μM of dosage The subject.

12. according to the method described in claim 1, wherein the composition is applied by the dosage of about 1mg/kg to about 50mg/kg With the extremely subject.

13. according to the method described in claim 1, wherein to subject's local application include the polypeptide composition simultaneously And the composition to its systemic administration comprising the polypeptide.

14. according to the method described in claim 1, wherein the radiation injury is selected from radiation injury of skin (CRI), acute radiation Syndrome (ARS), recombination radiation damage, radiation burn, radiation dermantitis, the radiation injury of nervous system or brain, lung radiation injury Enteritis caused by scorching, radiation or combinations thereof.

15. according to the method for claim 14, wherein the radiation injury includes CRI and ARS.

16. according to the method for claim 14, wherein the radiation dermantitis is the result of medical intervention.

17. according to the method for claim 16, wherein the medical intervention is treatment of cancer.

18. according to the method for claim 17, wherein the treatment of cancer is radiotherapy.

19. according to the method described in claim 1, wherein the radiation injury is because being exposed to selected from weapons of mass desturction (WMD), caused by the radiation source of nuclear explosion and dirty bomb.

20. according to the method for claim 19, wherein caused by the radiation injury is because being exposed to dirty bomb.

21. according to the method described in claim 1, wherein the radiation injury is because being exposed to selected from diagnostic instrments, the sun or solarization Caused by the radiation source of black bed.

22. according to the method described in claim 1, wherein the radiation injury is appeared in selected from skin, heart, bone, brain, ridge Marrow, cornea, retina and peripheral nerve tissue in.

23. according to the method for claim 22, wherein the radiation injury occurs on the skin.

24. according to the method for claim 19, wherein compared with the control, in the presence of the polypeptide, mean skin scores Reduce at least about 30%.

25. according to the method described in claim 1, wherein the polypeptide prevents intake radionuclide.

26. according to the method described in claim 1, wherein the polypeptide by the α connection albumen carboxyl least significant end 5 to 19 Continuous amino acid composition.

27. according to the method described in claim 1, wherein the α connection albumen is connection protein 37, connection albumen 40, connection Protein 43 or connection albumen 45.

28. according to the method described in claim 1, wherein the polypeptide include selected from SEQ ID NO:1, SEQ ID NO:2, The amino acid sequence of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

29. according to the method for claim 28, wherein the polypeptide includes the amino acid sequence of SEQ ID NO:2.

30. according to the method described in claim 1, wherein the polypeptide also includes cellular internalization sequence.

31. according to the method for claim 30, wherein the cellular internalization sequence includes the amino selected from following protein Acid sequence: rqikiwfqnrrmkwkk, TAT, HIV-Tat, penetrating peptide, Antp-3A (Antp mutant), Buforin II, transit peptides, MAP (mode amphiphilic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB 1, the (double-guanidine-Asia Pep-7, HN-1, BGSC Spermine-cholesterol) and BGTC (double-guanidine-Tren- cholesterol).

32. according to the method for claim 31, wherein the cellular internalization sequence is rqikiwfqnrrmkwkk, and it is wherein described Sequence includes the amino acid sequence of SEQ ID NO:7.

33. according to the method described in claim 1, wherein the polypeptide include selected from SEQ ID NO:8, SEQ ID NO:9, The amino acid sequence of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

34. according to the method for claim 33, wherein the polypeptide includes the amino acid sequence of SEQ ID NO:9.

35. according to the method described in claim 1, wherein the composition includes SEQ ID NO:91.

36. the method that this kind of damage is prevented in the subject for facing radiation injury risk, including apply and wrap to the subject Composition containing isolated polypeptide, the polypeptide include 4 to 30 continuous amino acids of carboxyl least significant end of α connection albumen.

Background of invention

Current event, which has highlighted, may finally be intended to cause nuclear explosion or spread radiation using radiological dispersal device The various acts of terrorism of property substance (for example, dirty bomb).In this case, in addition to traumatic damage, group is probably sudden and violent It is exposed to large dosage of externally and/or internally ionising radiation.In the case where nuclear explosion, skin and subcutaneous tissue injury's (referred to as skin are removed Skin radiation injury (CRI)) except or have no truck with, be exposed to huge external dosage acute ionising radiation be also possible to cause it is acute The symptom of radiation syndrome (ARS).

CRI is defined as the dermal lesions generated because being directly or indirectly exposed to low penetration radiation.In pendant worldly affair part, heat Particle deposits on the skin can lead to the acute partial radiation exposure of large dosage.Being exposed to radiation causes the substrate of damage skin thin Born of the same parents' layer and causing expose the inflammation of tissue, erythema, decortication, acutely it is rubescent, get blister, weakening property fibrosis, infection, ulcer and bad Extremely.

ARS describes one and is exposed to the clinical syndrome or combinations thereof generated after ionising radiation.ARS syndrome includes hematopoiesis Syndrome, gastro-intestinal tract syndrome, neuro-vascular syndrome, heart syndrome and other, and these syndromes respectively can be single Solely or appearance is combined with other syndromes.Under nuclear explosion scene, radioactive exposure continually with other traumatic damages as burn, Blunt force trauma and open wound combination occur, these traumatic damages may concurrent microorganism infection.Recombination radiation damage is retouched The wherein radiation injury symptom associated with other injuries such as burn, wound, infection or blunt force trauma is stated.

Radiation dermantitis is the radiotherapy such as radiotherapy because being applied to subject as a kind for the treatment of (such as cancer treatment) Or the radiation injury that chemotherapy radiotherapy method generates.Radiation dermantitis is the common side effect or toxicity of this kind of therapy.Radiation dermantitis It can be acute, rise in about 90 days and occur away from radiotherapy, and/or be chronic.The performance of radiation dermantitis include erythema, Decortication, necrosis, ulcer, atrophy, telangiectasis, fibrosis and/or other change of skin.Severe radiation dermantitis may need It reduces, interrupting or stopping radiation treatment plan, this may negatively affect the curative effect for the treatment of cancer or other diseases.

Although because the tissue that mechanical injuries, lysis and other reasons are damaged can heal, complicated group Structure and function is knitted also seldom to restore completely if there is crossing.On the contrary, almost all tissue in the mankind and other higher vertebrates Restore from damage to be formed based on cicatricial tissue.Such case most familiar with example be skin incision or scratch healing after wave it The discoloration that do not go and fibrotic scar.It is less popular, after brain or spinal cord injury the formation of glial scar tissue be It damages after central nervous system and restores one of major obstacle (Silver and Miller JH, 2004) of nervous function.It does not deposit at present It treats or prevents after injury this kind of scarring and promote the regenerated means of complex organization's structure and function.

This field needs to can treat, prevent and/or alleviate radiation injury (including radiation dermantitis, CRI, ARS and its group Close) progress suitable medicine.The disclosure meets this demand and other demands.

Invention summary

A kind of isolated polypeptide or its examples of conservative variations are provided, the isolated polypeptide includes the c-terminus of α connection albumen Amino acid sequence (also referred to herein as α connection protein carboxyl terminal (ACT) polypeptide).Provided herein is one kind to face radiation injury risk Subject in prevent, alleviate and treat the method for this kind of damage, the method includes to subject's application provided herein one Kind or numerous compositions (for example, polypeptide, nucleic acid or carrier).For example, provided herein is for preventing, alleviating its progress and treatment skin The method of skin radiation injury (CRI), acute radiation syndrome (ARS), recombination radiation damage or any combination thereof.In some implementations In scheme, present disclose provides prevent, alleviate its progress and treatment be exposed to (such as because of radiological dispersal device, piece together type core What device, nuclear weapon or nuclear power station explosion generated) CRI, ARS, and/or the method for recombination radiation damage after ionising radiation.One In a little embodiments, there is provided herein the methods for preventing, alleviating its progress and treatment radiation dermantitis.

In some embodiments, there is provided herein prevent, alleviate its progress and treat because the sun, X-ray and other examine The method for the radiation injury that disconnected instrument, tanning bed and other ionized radiation sources generate.For example, in some embodiments, herein The composition of offer, which is used in, to be exposed to before ionized radiation source or after exposure but before the paresthesia epilepsy of damage or symptom hair This kind of damage is prevented after work in early days or is alleviated in the method that it is in progress.

In some embodiments, disclosure offer prevents in the subject for facing radiation injury risk, alleviates it sternly Weight degree and the method for treating this kind of damage, the composition including to subject's application including isolated polypeptide, the polypeptide packet Include α connection polypeptide.In some embodiments, composition is applied before subject is exposed to radiation.In other realities It applies in scheme, composition is applied to subject after being exposed to radiation.For example, in some embodiments, composition is existed Subject is applied at least one day after being exposed to radiation.In some embodiments, this method prevents radiation injury to be in progress.One In a little embodiments, by composition local application to subject.In some embodiments, composition is pressed about 10 μM to about 2000 μM of dosage local application is to subject.In other embodiments, composition is pressed into about 50 μM, about 100 μM, about 150 μ M, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM or about 1,000 μM Dosage be applied to subject.In some embodiments, composition is applied by the scheme that is administered daily.In some embodiments In, by composition parenteral administration to subject.In other embodiments, composition is passed through in intravenous, subcutaneous, peritonaeum Or intramuscular route is applied to subject.For example, in some embodiments, by composition by intravenous injection, subcutaneous injection, Intraperitoneal injection or intramuscular injection are applied to subject.In some embodiments, composition is pressed into about 1mg/kg to about 50mg/ The dose parenteral of kg is applied to subject.In other embodiments, by composition by about 1mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg Or the dosage of about 50mg/kg is applied to subject.In some embodiments, composition is applied by the scheme that is administered daily.One In a little embodiments, the parenteral administration treatment of composition, the progress for preventing, and/or inhibiting internal radiation damage.

In some embodiments, radiation injury is selected from CRI, ARS, radiation burn, radiation dermantitis, nervous system or brain Radiation injury, radiation pneumonitis, enteritis and internal radiation caused by radiation.In certain embodiments, radiation injury is CRI. In certain embodiments, radiation injury is ARS.In certain embodiments, radiation injury is recombination radiation damage.Therefore, In some embodiments, radiation injury includes and the radiation injury burnt, wound, infection or blunt force trauma combine.Some In embodiment, caused by radiation injury is because being exposed to the radiation source selected from weapons of mass desturction (WMD), nuclear explosion and dirty bomb. In some embodiments, radiation injury includes radiation dermantitis, and the radiation dermantitis is because of radiotherapy scheme or other Jie The certain types of radiation injury that entering property medical procedure generates.In some embodiments, radiation injury is because being exposed to the sun, X Ray and other diagnostic instrments or ionizing radiation emission source are as caused by tanning bed.In some embodiments, in opacifier, sun-proof Peptide provided herein is provided in white or tanned dew composition.

In some embodiments, radiation injury appear in selected from skin, heart, bone, brain, spinal cord, cornea, retina, In the tissue of hemopoietic system, gastronintestinal system and peripheral nerve.In specific embodiments, radiation injury occurs on the skin. In some embodiments, compared with the control, in the presence of polypeptide, mean skin scoring reduces at least about 30%.In some realities It applies in scheme, polypeptide prevents cicatrization.In some embodiments, polypeptide promotes regeneration.In some embodiments, Polypeptide prevents tissue damage.

In some embodiments, α connection polypeptide includes 4 to 30 Continuance ammines of carboxyl least significant end of α connection albumen Base acid.In some embodiments, polypeptide is made of 5 to 19 continuous amino acids of carboxyl least significant end of α connection albumen.Some In embodiment, α connection albumen is connection protein 37, connection albumen 40, connection protein 43 or connection albumen 45.In other implementations In scheme, polypeptide includes to be selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID The amino acid sequence of NO:5.In some embodiments, polypeptide also includes cellular internalization sequence.In other embodiments, carefully Born of the same parents' internalization sequence includes the amino acid sequence selected from following protein: rqikiwfqnrrmkwkk, TAT, HIV-Tat, penetrating peptide, Antp- 3A (Antp mutant), Buforin II, transit peptides, MAP (mode amphiphilic peptide), K-FGF, Ku70, prion, pVEC, Pep- 1, SynB 1, Pep-7, HN-1, BGSC (double-guanidine-spermidine-cholesterol) and BGTC (double-guanidine-Tren- cholesterol).Some In embodiment, cellular internalization sequence is rqikiwfqnrrmkwkk, and wherein sequence includes the amino acid sequence of SEQ ID NO:7. In some embodiments, polypeptide includes to be selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: The amino acid sequence of 11 and SEQ ID NO:12.In some embodiments, α connection polypeptide polypeptide N-terminal and/or C-terminal includes biotin.In some embodiments, composition includes SEQ ID NO:91.

In some embodiments, topical composition.In some embodiments, composition also includes ethoxy fibre Tie up plain gel.In other embodiments, hydroxyethylcellulose gel is with about 0.25% (w/w), about 0.5% (w/w), about The concentration presence of 0.75% (w/w), about 1.00% (w/w), about 1.25% (w/w), about 1.5% (w/w) or about 2.0% (w/w). In some embodiments, systemic administration composition.For example, in some embodiments, parenteral administration composition.One In a little embodiments, the composition for systemic administration also includes one or more medicines suitable for systemic administration to subject Acceptable excipient on.In some embodiments, can by composition locally and systemically property be applied to demand by Examination person.For example, in some embodiments, to the subject by radiation injury, locally and systemically property applies provided herein group Close object, it is intended to treat, prevent and/or alleviate the progress of both CRI and ARS.For example, in some embodiments, to subject Local application includes the composition of polypeptide provided herein, and includes the composition of herein polypeptides to its systemic administration.In In some embodiments, the composition for the application of locally and systemically property includes identical α connection polypeptide.In some implementations In scheme, to the subject by radiation injury, locally and systemically property applies composition provided herein, it is intended to treat, prevent And/or alleviate be related to burn, the recombination radiation lesion progress for the CRI and/or ARS that wound, infection or blunt force trauma combine.

The additional advantage of disclosed method and composition will be partially elaborated in subsequent specification, and will be from this Specification partly understands it, or can by implement disclosed method and composition acquistion it.Disclosed method and group The advantages of closing object will be realized and be reached by the element and combination specifically noted in the appended claims.It should be appreciated that above General description and being described in detail below be merely illustrative with it is explanatory, and do not limit such as claimed invention.

Brief description

The attached drawing for being incorporated to and constituting the part of this specification shows several embodiments of disclosed method and composition, And together with specification, effect is to explain the principle of disclosed method and composition.

Figure 1A, Figure 1B and Fig. 1 C show that local application ACT peptide effectively prevent, alleviates and treat radiation injury of skin (CRI). Yorkshire is anaesthetized to and is exposed to the low energy electrons (6MeV) of single segmentation amount.Start on day 1 and hereafter every 3 days, Using Kumar scale, the erythema and decortication of skin part are evaluated.Start to use when observing erythema (Kumar scoring > 1.0)(100 μM or 200 μM) or solvent Local treatment and it is applied to exposure portion once a day.Figure 1A is shown(200 μM) of gel processing and the mean skin scoring of vehicle treated are compared.Figure 1B display applies ACT peptide to damage The influence of performance.At the 1st, the 22nd and the 31st day,(200 μM) processing (footline) of gel and vehicle treated (first trip) Compare.Fig. 1 C is shown in the skin from irradiated site and non-irradiated control position for being derived from same animals for the 43rd day (research terminates) The H&E of skin biopsy samples is dyed.

Fig. 2 shows ACT peptide to the prevention effect for preventing and alleviating CRI after exposure.It, will according to identical daily therapeutic scheme The position solvent of radiation or1,000 μM of gel preventative (before CRI symptom is presented) processing (are opened on the 1st day Begin；The same day i.e. after radioactive exposure).Photograph in 21st day compares display compared with Vehicle controls, 1,000 μMGel Preventative process prevents lesion progress and reduces injury severity score.

Fig. 3 shows the production technological process of ACT peptide of the present invention.

Fig. 4 shows that ACT1 polypeptide reduces Deaths in radiation GI toxic model.It is small with those of saline treatment is compareed Mouse is compared, and the C57BL/6 mouse display survival handled daily with systemic delivery peptide (10mg/kg) increases.

Specific embodiments of the present invention

Can by reference to the detailed description of following specific embodiment and embodiment included in it and with reference to attached drawing and its Before and after explanation disclosed method and composition is more readily understood.

A kind of isolated polypeptide or its examples of conservative variations are provided, the isolated polypeptide includes the c-terminus of α connection albumen Amino acid sequence (also referred to herein as α connection protein carboxyl terminal (ACT) polypeptide).In some embodiments, provided herein group It closes object and method is related to the progress for preventing, treating and/or alleviating radiation injury.In some embodiments, provided herein Composition and method are related to the progress for preventing, treating and/or alleviating radiation injury of skin (CRI).In some embodiments, The composition and method of this paper is related to the progress for preventing, treating and/or alleviating acute radiation syndrome (ARS).In some realities It applies in scheme, the composition and method of this paper is related to the progress for preventing, treating and/or alleviating recombination radiation damage.

Surprisingly, to find that composition provided herein and method can play in radiation injury preventative by inventor Effect.For example, composition provided herein and method prevent CRI, ARS or compound after radioactive exposure but before Symptoms Radiation injury and/or alleviation lesion progress.Therefore it provides composition and method can be used to face be exposed to radioactivity dissipate Cloth apparatus, piece together type nuclear device, nuclear weapon, nuclear power station explosion etc. high risk subject group and/or met with recently Meeting prevents, treats or alleviates in the subject of this exposure by this kind of device, weapon and caused specific type radiation damage of exploding The progress of wound.

ARS, CRI and recombination radiation damage are the specific type radiation damages caused by for example exploding because of dirty bomb attack or nuclear power station Wound.In some embodiments, the composition of this paper and method with prevent, treat and/or alleviate the interior of skin and subcutaneous tissue The progress of portion's radiation injury and radiation injury is related.In some embodiments, the composition of this paper and method are especially available The CRI in the subject group for preventing and/or alleviating the high risk for facing experience CRI and/or ARS and/or recombination radiation damage And/or the progress that ARS and/or recombination radiation damage.

In some embodiments, composition provided herein is applied to subject locally to treat, prevent and/or delay Solution is as the skin for the result for being exposed to radiation and the progress of subcutaneous tissue injury.It in some embodiments, will be provided herein is Composition systemic administration treat, prevent and/or alleviate to subject because being exposed to radiogenic systemic injury (such as Visceral injury) progress.In some embodiments, to same subject local application one or more combinations provided herein Object is to treat, prevent and/or alleviate the progress of skin and subcutaneous tissue injury and to its systemic administration to treat, prevent And/or alleviate the progress of internal injury (for example, systemic organ damages).

In addition, the surprising prevention effect of institute's providing method and composition can be used for preventing, treat and alleviating because too The progress of radiation injury caused by sun, diagnostic instrments and tanning bed.Using the diagnostic instrments of ionising radiation for example including x-ray machine With computer tomography (CT) scanner.In some embodiments, these method and compositions can be used to facing cruelly It is exposed to the high risk of a variety of horizontal ionising radiations from the sun, diagnostic instrments or tanning bed or has been subjected to this kind of exposure recently Subject group in prevent, treat or alleviate because of the sun, diagnostic instrments or tanning bed caused by radiation injury progress.Cause This, in some embodiments, by the composition of this paper and method subject be exposed to the sun, diagnostic instrments or tanning bed it Before be applied to subject, and/or be applied to subject after subject is exposed to the sun, diagnostic instrments or tanning bed.

In some embodiments, composition provided herein is in opacifier or suncream or in offer tanning products And/or product form obtained by the mechanism of tanned service (for example, tanned dew, tanned accelerator or suntan oil).In some realities It applies in scheme, the disclosure provides before subject is using tanning bed or is exposed to the forward direction subject application of the sun in subject Method and composition, with prevent, treat or alleviate may by it is this use or exposure caused by radiation injury progress.One In a little embodiments, the disclosure provides after subject is using tanning bed or is exposed to the rear to subject of the sun in subject The method and composition of application, to prevent, treat or alleviate the progress of the radiation injury caused by this use or exposure.

In some embodiments, in the diagnostic instrments or process for being exposed to transmitting ionising radiation, (such as X is penetrated for disclosure offer Line or CT scan) forward direction subject application method and composition, wherein these method and compositions prevent, treat or alleviate The progress of radiation injury caused by this exposure.In some embodiments, disclosure offer is being exposed to transmitting ionising radiation Diagnostic instrments or process backward subject application method and composition, wherein these method and compositions are prevented, are treated Or alleviate the progress of radiation injury caused by this exposure.

In addition, the surprising prevention effect of institute's providing method and composition can be used for preventing, treat and alleviating radiation The progress of property dermatitis.Caused by radiation dermantitis such as treats disease (such as cancer) because of radiotherapy.Therefore, in some embodiments In, the disclosure provide application radiotherapy it is rear to subject apply method and composition, with prevent, treat or alleviate by The progress of radiation injury caused by radiotherapy.In some embodiments, the disclosure is provided after being exposed to radiotherapy The method and composition applied to subject, wherein these method and compositions prevent, treat or alleviate caused by radiotherapy The progress of radiation injury.In some embodiments, by method and composition provided herein in the entire radiation therapy process phase Between be applied to subject, wherein these method and compositions prevent, treat or alleviate radiation injury caused by radiotherapy into Exhibition.In some embodiments, by method and composition provided herein before, during and/or after radiation therapy process It is applied to subject, wherein these method and compositions prevent, treat or alleviate the progress of radiation injury caused by radiotherapy.

It should be understood that unless otherwise stated, otherwise disclosed composition and method are not limited to specific synthetic method, tool The analytical technology of body or specific reagent, and itself can change.It should also be understood that its purpose of term used herein only exists In description specific embodiment, and it is not intended to be restrictive.Disclosing can be used, can be applied in combination, can be used for It prepares or as the material of disclosed method and the product of composition, composition and component.There is disclosed herein these materials and Other materials, and it is to be understood that when disclosing combination, subset, interaction, the group etc. of these materials, although can not Each Different Individual of these compounds and the specific meaning of entire combination and arrangement are clearly disclosed, however it is specific herein Conceive and describe each.For example, if open and discuss a kind of carrier and discuss numerous loads comprising promoter Body component, then counter-example unless otherwise indicated, otherwise specifically contemplates promoter and every kind and each combination of other carrier modules With arrangement and possible modification.Therefore, if disclose molecule A, B and C and disclose molecule D, E and F and Combination molecule example A-D also contemplates individually and generally every kind point then even without every kind of molecule and combination is individually described Son and combination.Therefore, in this example, every kind of group of A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are specifically contemplated Merge and it should be considered as disclosure from A, B and C；D, E and F；With the disclosure of example combination A-D.Equally, also specific to conceive simultaneously Disclose the former any subset or combination.Thus, for example, specifically contemplating the subgroup of A-E, B-F and C-E and it should It is considered as open from A, B and C；D, E and F；With the disclosure of example combination A-D.This design is suitable for the whole side of the application Face includes, but are not limited to the step in the method for generating and using disclosed composition.Thus, if there is can be implemented Multiple additional steps, it will be understood that each of these additional steps can use disclosed method any specific embodiment party Case or combination of embodiment are implemented, and specifically conceive each this group of merging and should be considered as disclosure.

A variety of sequences provided herein and these and other sequences can be found in the Genbank at www.pubmed.gov Column.Sequence is inconsistent and difference skilled artisan understands how solving, and how to adjust and be related to a kind of particular sequence Composition and method are to adapt to other correlated series.In view of disclosed herein and information known in the art, it can design to be directed to and appoint The primer and/or probe of what sequence.

Polypeptide presented herein can be any polypeptide of the carboxyl most end terminal amino acid comprising α connection albumen, wherein more Peptide does not include overall length α connection protein matter.Therefore, in one aspect, the polypeptide provided does not include the cytoplasm N of α connection albumen Terminal domains.On the other hand, the polypeptide provided does not include two extracellular domains of α connection albumen.On the other hand, The polypeptide of offer does not include four transmembrane domains of α connection albumen.On the other hand, the polypeptide provided does not include α connection The cytoplasm loop domain of albumen.On the other hand, the polypeptide provided does not include α connection protein cytoplasmic carboxy-terminal domains That part for being close in the 4th transmembrane domain of sequence.Have in α connection albumen and is always positioned at away from carboxyl least significant end ammonia The conservative proline residue or glycine residue (table 2) of about 17 to 30 amino acid of base acid.For example, for people Cx43, amino Proline residue at acid 363 is located at away from carboxyl least significant end isoleucine 19 amino acid backward.In another example, for Chicken Cx43, the proline residue at amino acid 362 are located at away from carboxyl least significant end isoleucine 18 amino acid backward.At another In example, for people Cx45, the glycine residue at amino acid 377 is located at away from carboxyl least significant end isoleucine 19 amino backward Acid.In another example, for rat Cx33, the proline residue at amino acid 258 is located at away from carboxyl least significant end first sulphur ammonia Sour 28 amino acid backward.Therefore, on the other hand, the polypeptide provided does not include the conservative dried meat that albumen is connect with α The amino acid that histidine residue or glycine residue close on.Therefore it provides polypeptide may include the carboxyl least significant end of α connection albumen 4 to 30 amino acid, carboxyl least significant end 4 including α connection albumen, 5,6,7,8,9,10,11,12,13,14,15,16,17, 18,19,20,21,22,23,24,25,26,27,28,29,30 amino acid.

Non-alpha connection albumen or non-ACT can be distributed in α connection protein carboxyl groups most end terminal amino acid in the peptide of offer with side Peptide connects Argine Monohydrochloride.The non-alpha connection albumen of side distribution provided herein connects the example of Argine Monohydrochloride with non-ACT. The example of non-ACT connection Argine Monohydrochloride is 20 to 120 amino acid of c-terminus of people Cx43 (SEQ ID NO:72).Another Example is 20 to 120 amino acid of c-terminus of chicken Cx43 (SEQ ID NO:73).Another example is people Cx45 (SEQ ID NO:74 20 to 120 amino acid of c-terminus).Another example is the c-terminus 20 to 120 of chicken Cx45 (SEQ ID NO:75) A amino acid.Another example is 20 to 120 amino acid of c-terminus of people Cx37 (SEQ ID NO:76).Another example is 20 to 120 amino acid of c-terminus of rat Cx33 (SEQ ID NO:77).

The example of non-alpha connection albumen is the sequence of 239 amino acid of enhanced green fluorescence protein.In another side Face, it is functional when being merged in view of display ACT1 with the c-terminus of the sequence of 239 amino acid of GFP, it is contemplated that when side is distributed with When the disconnected polypeptide of up at least 239 amino acid, ACT peptide reservation function.As long as being given in fact, ACT sequence is used as The free carboxy end for determining polypeptide maintains, and ACT peptide can arrive at its target.Therefore, there is more than 239 amino in addition to ACT peptide The polypeptide of acid can reduce inflammation after injury, promote healing, increases tensile strength, reduces scarring and promote regeneration side Face plays a role.

Connection albumen be responsible for Intercellular communication Gap junctions subelement albumen (Goodenough and Paul, 2003).The gene of conservative pattern based on nucleotide sequence, coding connection albumen is divided into Liang Ge family, referred to as α connection albumen base Cause and β Connexin gene.The carboxyl most end terminal amino acid sequence of α connection albumen is characterized in that multiple unique and conservative spies It levies (referring to table 2).This conservative constitute with ACT peptide formed unique 3D structure, with the interaction of a variety of matching protein, It mediates and interacts, transports and/or block membrane channels with lipid and membrane interaction, with nucleic acid (including DNA) and provide shared Motif is consistent with the ability of phosphorylation for proteolytic cleavage, protein cross, ADP- ribosylation, glycosylation.Therefore it provides Polypeptide and the structural domain of protein interact, the structural domain mediates the protein to connect albumen with α under normal circumstances C-terminus combine.For example, the protein (NOV) of nephroblastoma overexpression and Cx43 carboxy-terminal domain interact (Fu et al., J Biol.Chem.2004 279 (35): 36943-50).Think that this protein and other protein are connect with α The c-terminus of albumen interacts and further interacts with other protein to form macromolecule complex.Therefore, it mentions The polypeptide of confession can inhibit molecule machine (e.g., for example, being related to adjusting a kind of molecule machine of Cx43 Gap junctions polymerization) Operation.

As used herein, " inhibiting ", " inhibition " and " inhibiting effect " mean reduce activity, reaction, symptom, disease or its Allogene parameter.This can include but is not limited to activity, reaction, symptom or disease and completely loses.This can also for example including with Itself or control level are compared, and activity, reaction, symptom or disease reduce 10%.Therefore, such as compared with itself or control level, Reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any amount between it It reduces.

The ACT sequence of provided polypeptide can come from any α connection albumen.Therefore, the α connection protein groups of provided polypeptide Point can come from people, mouse, ox, monotreme (monotrene), marsupial, primate, rodent, cetacean, mammal, Birds, reptile, amphibian animal, fish, notochord class, former rope class or other α connection albumen.

Therefore it provides polypeptide may include selected from it is following connection albumen ACT: mouse connection protein 47, people connection egg White 47, people connects protein 46 .6, ox connection protein 46 .6, mouse connection albumen 30.2, rat connexin 30.2, people's connection egg White 31.9, dog connection protein 31 .9, sheep connection albumen 44, ox connection albumen 44, rat connexin 33, mouse connect albumen 33, people's connexin36, mouse connexin36, rat connexin 36, dog connexin36, chicken connexin36, zebra Fish connexin36, wolf perch connection albumen 35, wolf perch connection albumen 35, newt connection albumen 35, Puffer connexin36, people's connection Protein 37, chimpanzee connection protein 37, dog connection protein 37, hamster connection protein 37, mouse connection protein 37, suslik connect egg White 37, rat connexin 37, mouse connection protein 39, rat connexin 39, people connect albumen 40.1, Africa xenopus connection Albumen 38, zebra fish connection protein 39 .9, people connect albumen 40, chimpanzee connection albumen 40, dog connection albumen 40, ox and connect egg White 40, mouse connection albumen 40, rat connexin 40, hamster connect albumen 40, chicken connects albumen 40, people connects protein 43, Guenon connects protein 43, rabbit connection protein 43, spermophile connection protein 43, hamster connection protein 43, the connection of hair foot mouse Protein 43, rat connexin 43, pig connection protein 43, suslik connection protein 43, mouse connection protein 43, cavy connect albumen 43, ox connection protein 43, hedgehog connection protein 43, chicken connection protein 43, Africa xenopus connection protein 43, rabbit connect albumen 43, carp connection protein 43, zebra fish connection protein 43, wave fish pellet (Danio aequipinnatus) connect protein 43, zebra Fish connects protein 43 .4, zebra fish connection albumen 44.2, zebra fish connection albumen 44.1, people's connection albumen 45, chimpanzee connection Albumen 45, dog connection albumen 45, mouse connection albumen 45, ox connection albumen 45, rat connexin 45, chicken connection albumen 45, Puffer connects albumen 45, chicken connection albumen 45, people's connection protein 46, chimpanzee connection protein 46, mouse connection protein 46, dog connection Protein 46, rat connexin 46, suslik connection protein 46, hamster connection protein 46, chicken connection albumen 56, zebra fish connect egg White 39.9, ox connection albumen 49, people connect albumen 50, chimpanzee connection albumen 50, rat connexin 50, mouse and connect albumen 50, dog connection albumen 50, sheep connection albumen 49, suslik connection albumen 50, hamster connection albumen 50, chicken connection albumen 50, people connect Connect albumen 59 or other α connection albumen.The amino acid sequence of α connection albumen is known in the art and including passing through in table 1 Those of registration number identification.

Table 1: α connects albumen

Therefore it provides polypeptide may include amino acid sequence SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO: 30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:43, SEQ ID NO:89, SEQ ID NO:90 or SEQ ID NO:91 or its examples of conservative variations or segment.

20-30 carboxyl most end terminal amino acid sequence of α connection albumen is characterized in that unique and conservative composition.It is this Unique and conservative composition includes II type PDZ binding motif (Φ-x- Φ；Wherein any amino acid of x=and Φ=hydrophobicity ammonia Base acid；For example, table 2, runic) and proline (P) hinge residues and/or glycine (G) hinge residues that are closed on this motif； High-frequency phosphinylidyne-serine (S) residue and/or phosphinylidyne-threonine (T) residue；With the arginine (R) of high frequency band positive charge, Lysine (K) and negatively charged aspartic acid (D) or glutamic acid (E) amino acid.For many α connection albumen, P residue and G Residue appears in the gathering motif (for example, table 2, italic) for being close in c-terminus II type PDZ binding motif.Most of α connection egg White S and T phosphinylidyne-amino acid generally also constituted with gathering, repetitive sequence sample motif (for example, table 2,It underlines).This structure At being especially in this way, wherein the 90% of 20 carboxyl most end terminal amino acids is made of rear seven amino acid to Cx43.In sequence height In another conservative example of degree, the ACT peptide of Cx43 constitutes highly conserved (for example, people and spot in comparison sheet 2 from the mankind to fish The Cx43 ACT sequence of horse fish).In another example, Cx45 ACT peptide constitute from the mankind to birds it is highly conserved (for example, The Cx45 ACT sequence of people and chicken in comparison sheet 2).In another example, the ACT peptide of Cx36 is constituted from primate to fish height Degree is conservative the Cx36 ACT sequence of chimpanzee and zebra fish (for example, in comparison sheet 2).

Table 2. α connection protein carboxyl terminal (ACT) amino acid sequence

Therefore, in one aspect, the polypeptide provided includes one, two, three or whole amino acidic groups selected from the following Sequence: 1) II type PDZ binding motif, 2) proline (P) hinge residues and/or glycine (G) hinge residues；3) phosphinylidyne-of gathering Serine (S) residue and/or phosphinylidyne-threonine (T) residue；With the arginine (R) and lysine (K) of 4) high frequency band positive charge With negatively charged aspartic acid (D) and/or glutamic acid (E) amino acid).On the other hand, the polypeptide provided includes carboxyl II type PDZ binding motif at end, proline (P) hinge residues and/or glycine (G) hinge for being close in PDZ binding motif Residue and the positively charged residue (K, R, D, E) for being close in hinge residues.

PDZ structural domain is initially identified as postsynaptic density albumen PSD95/SAP90, drosophila (Drosophila) tumor suppression Conserved sequence elements inside albumen dlg-A and tight junction protein ZO-1.Although being initially referred to as GLGF motif or DHR motif, But they are taken as representing these initials for containing first three in the protein of PDZ (PSD95/DLG/ZO-1) now And it is known.Now identified in more than 75 kinds protein these 80-90 amino acid sequences and they single Active site of protein is with the expression of multiple copies characteristic.Therefore, in one aspect, the polypeptide provided can inhibit α connection albumen and packet The protein of the structural domain containing PDZ combines.PDZ structural domain is that specific type has the interaction ' pocket ' sufficiently defined in structure Protein interact module (referred herein as " PDZ motif "), the pocket can be filled by PDZ- binding motif.PDZ Motif is to be located at the consensus sequence that at carboxyl least significant end intracellular but not such was the case under normal circumstances.By four classes The PDZ motif of type is classified: I type (S/T-x- Φ), II type (Φ-x- Φ), type III (Ψ-x- Φ) and IV type (D-x-V), wherein x It is any amino acid, Φ is hydrophobic residue (V, I, L, A, G, W, C, M, F) and Ψ is alkalinity, hydrophilic residue (H, R, K). (Songyang, Z. et al. 1997.Science 275,73-77).Therefore, in one aspect, the polypeptide provided includes II type PDZ Binding motif.

It should be understood that 18 amino acid sequence of carboxyl least significant end of α Cx37 represents the exception variation of ACT peptide theme.Cx37 ACT sample sequence is GQKPPSRPSSSASKKQ*YV (SEQ ID NO:43).Therefore, 4 amino acid of the c-terminus of Cx37 only part Ground meets II type PDZ binding structural domain.Classical II type PDZ binding structural domain is substituted, Cx37 has neutrality Q*, In in position 2 Position expection has hydrophobic amino acid.To which Cx37 includes the sequence that may be referred to as II type PDZ binding structural domain sample.So And Cx37 strictly maintains all other aspects that ACT peptide is constituted, serine residue, frequent R residue and K including gathering Residue and the P for being close in PDZ binding structural domain sample sequence are rich in sequence.In view of ACT sample constitute conservative this aggregate level with Listed above other > 70 kinds of α connection albumen are identical, it is possible to understand that Cx37 ACT sample c-terminus plays work in the ability of offer With.

In order to compare, β connection PROTEIN C x26 is shown in table 2.Cx26 does not have c-terminus II type PDZ binding motif；It is less than 30% carboxyl most end terminal amino acid includes S, T, R, D or E residue；Do not cut with scissors evidence suggests it contains with the P containing gathering and G The motif that the II type PDZ binding motif or PDZ combination sample motif of chain residue close on；And evidence suggests it to contain gathering , repetitive sequence sample serine and threonine phosphinylidyne-amino acid motif.Cx26 has really there are three lysine (K) residue, one by one Gathering is near the c-terminus of sequence.It has however been found that the α connection albumen studied in listed above > 70 kinds of α connection albumen Do not show that (Cx26 is a kind of β connection albumen, therefore according to fixed for this feature in three repetition K residues Structures domains at c-terminus Justice does not have ACT structural domain).

As provided herein, the unique function feature of this relatively short amino acid section covers to a variety of different tissues (such as skins Skin and brain) damage after complex organization's structure and Function reduction answer inflammation, promote healing, reduce scarring, increase drawing It stretches intensity and promotes the unexpected effect of regeneration aspect.Therefore, in one aspect, the polypeptide provided includes II type PDZ combination base Sequence (Φ-x- Φ；Wherein any amino acid of x=and Φ=hydrophobic amino acid).On the other hand, provided ACT polypeptide It is more than that 50%, 60%, 70%, 80%, 90% amino acid is made of following one or more amino acid residues: proline (P), glycine (G), phosphinylidyne-serine (S), phosphinylidyne-threonine (T), arginine (R), lysine (K), aspartic acid (D) or Glutamic acid (E) amino acid residue.

Amino proline (P), glycine (G), arginine (R), lysine (K), aspartic acid (D) and glutamic acid (E) It is the necessary determinant of protein structure and function.Proline residue and glycine residue provide in the 3D structure of protein Tight turn is possibly realized so that generating polypeptide folded conformation required for function.Electrically charged amino acid sequence is frequent Positioned at the protein of folding surface and be polypeptide mediate chemical interaction (including protein-protein interaction, Protein-lipid interaction, enzyme-substrate interaction and protein-nucleic acid interaction) it is required.Therefore, at another Aspect is close in the proline (P) and glycine (G) lysine (K), aspartic acid (D) and paddy ammonia of II type PDZ binding motif It is required characteristic that sour (E), which provides institute's offer effect to ACT peptide rich in region,.On the other hand, the polypeptide provided includes It is close in the proline (P) and glycine (G) lysine (K), aspartic acid (D) and/or glutamic acid of II type PDZ binding motif (E) it is rich in region.

Phosphorylation be the most common protein post-translational modification and to adjust or modification protein structure and function to pass It is important.It include that protein conformation, protein-protein are mutual by the protein structure of phosphorylation modification and the aspects of function Effect, protein-lipid interaction, protein-nucleic acid interaction, the effect of channel gate, protein import and protein Turnover.Therefore, in one aspect, phosphinylidyne-serine (S) and/or phosphinylidyne-threonine (T) are rich in sequence to adjusting ACT peptide function It is required for capable of, increasing or decreasing polypeptide active validity provided by it.On the other hand, the polypeptide provided includes Serine (S) and/or phosphinylidyne-threonine (T) are rich in sequence or motif.

In another example, for the definition of ACT peptide, according to the tissue of high level in lower animal (such as fish)/ Neomorph potentiality, highly advantageously, methionine appears in (table near the aminoterminal of zebra fish Cx43 ACT sequence 2).In addition to encoding methionine, methionine base-pair triplet is alternative translation initiation site.If from this first sulphur Propylhomoserin starting translation, then will generate sequence SSRARPDDLDV (SEQ ID NO:89).This translation product maintains typical case ACT The whole of peptide is guarded and unique feature.Specifically, this peptide include c-terminus II type PDZ binding structural domain and have face It is bordering on the structural domain rich in P, R and D residue of PDZ binding structural domain.In addition, the sequence is in the S base that its aminoterminal includes gathering Sequence has the potentiality for adjusting ACT peptide function.This present significant prospects: dynamic with height tissue/organ regeneration potential Object such as fish can directly translate ACT peptide sequence.

Therefore it provides polypeptide may include the C-terminal sequence of people Cx43.Therefore it provides polypeptide may include amino acid Sequence SEQ ID NO:1 or SEQ ID NO:2.Polypeptide may include 9 amino acid of people's Cx40 c-terminus.Therefore, polypeptide can To include amino acid sequence SEQ ID NO:5.

When specific protein is mentioned above, variant, derivative and segment are contemplated.Protein variant and derivative are this Field technical staff fully understands and can be related to amino acid sequence modifications.For example, amino acid sequence modifications fall generally into One or more in lower classification: displacement, insertion or deletion mutants.Insertion is including aminoterminal and/or c-terminus fusion and individually Or insertion in the sequence of more amino acid.Insertion is usually to merge smaller insertion, example than those aminoterminals or c-terminus Such as, on the order of magnitude of one to four residue.Missing is spy to remove one or more amino acid residues from protein sequence Sign.Generally prepare these variants in the following manner: the locus specificity that nucleotide is carried out in the DNA of coding protein lures Become, thus generates the DNA of coding variant, and hereafter express DNA in recombinant cell culture thing.With known array It is well known that the technology of replacement mutation is generated at predetermined site in DNA, and for example including M13 primer mutagenesis and PCR mutagenesis. Amino acid replacement is usually the displacement of single residue, but can disposably appear in numerous different locations；Insertion usually exists About 1 rank to 10 amino acid residues.Missing or insertion are generated preferably in the pairs of amino acid adjoined, that is, missing 2 2 residues of a residue or insertion.Displacement, missing, insertion or any combination thereof can be combined to realize final construct.Mutation Sequence must not be made to be placed in except open read frame and preferably do not create the complementarity region there may be secondary mRNA structure, unless Need this variation of mRNA secondary structure.Displacement variant be wherein at least one removed residue and its position be inserted into Those of different residues variant.This kind of displacement is made generally according to the following table 3 and is referred to as preservative replacement.

Table 3: amino acid replacement

For example, known to those skilled in the art replace with an amino acid residue in biology and/or be chemically similar Another residue as preservative replacement.For example, one hydrophobic residue to be replaced with another hydrophobic for preservative replacement Property residue, or a polar residues are replaced with another polar residues.Displacement includes the combination shown in table 3.It is each clear The mutation of the preservative replacement of disclosed sequence is contained within the scope of polypeptide provided herein.

Generally, preservative replacement minimal effect or do not influence gained polypeptide biological activity.In a specific example, Preservative replacement is the amino acid replacement that the biological function of the peptide is had no substantial effect in peptide.Peptide may include one or more Amino acid replacement, such as 2-10 preservative replacement, 2-5 preservative replacement, 4-9 preservative replacement, such as 2,5 or 10 guarantors The displacement of keeping property.

The nucleotide sequence of coding polypeptide can be manipulated by using standardization program such as Site-directed mutagenesis or PCR, Generate the polypeptide containing one or more preservative replacements.It is alternatively possible to generate using standard peptide synthesis methods and contain one Or the polypeptide of multiple preservative replacements.Mutagenesis of Alanine-scanning method can be used to identify the ammonia that can allow amino acid replacement in protein Base acid residue.In one example, alanine or other conservative amino acids (those of set forth below) are replaced into one Or when multiple natural amino acids, the biological activity of protein is not reduced more than 25%, such as not more than 20%, such as be not more than 10%.

Can Ben-Bassat et al. (J.Bacteriol.169:751-7,1987), O'Regan et al. (Gene 77: 237-51,1989), Sahin-Toth et al. (Protein Sci.3:240-7,1994), Hochuli et al. (Bio/ Technology 6:1321-5,1988) it neutralizes in science of heredity and standard molecular biological teaching material, together with elsewhere, finding pass In the further information of preservative replacement.

Displacement or deletion mutagenesis can be used to be inserted into the glycosylation site (Asn-X-Thr/Ser) N- or O- glycosylation (Ser or Thr) site.Missing cysteine or other unstability residues can also be desirable.Such as it is residual by missing alkalinity One of base or with glutamine residue or histidine residues replace an alkaline residue realize potential proteolytic cleavage site (such as Arg missing or displacement).

Derivatization is that recombinant host cell acts on the result for having expressed polypeptide after certain translations.Glutamine residue and day Deamidation is at corresponding glutamyl and aspartyl resi dues after winter amide residues are often translated.Alternatively, these residues are micro- Deamidation under acid condition.Other posttranslational modifications include that the hydroxylation, serine residue or threonine of proline and lysine are residual The phosphorylation of the hydroxyl of base, lysine, arginine and histidine side chains o- amino group methylation (T.E.Creighton, Proteins:Structure and Molecular Properties, W.H.Freeman&Co., San Francisco 79-86 pages [1983]), the acetylation of N-terminal amino and in some cases, the amidation of C-terminal carboxyl.

It is appreciated that in the presence of numerous amino acid and the peptide analogues that can be incorporated in disclosed composition.For example, existing many More D amino acid or the amino acid compared to amino acid shown in table 3 with different functionalities substituent group.Disclose naturally occurring peptide Counter stereoisomer and peptide analogues stereoisomer.It can be by loading tRNA molecule and structure with selected amino acid The gene construct for example using amber codon by amino acid analogue according to site-specific fashion insertion peptide chain is built, by this A little amino acid are incorporated in polypeptide chain (Thorson et al., Methods in Molec.Biol.77:43-73 (1991) easily； Zoller,Current Opinion in Biotechnology,3:348-354(1992)；Ibba,Biotechnology& Genetic Engineering Reviews 13:197-216 (1995), Cahill et al. TIBS, 14 (10): 400-403 (1989)；Benner,TIB Tech,12:158-163(1994)；Ibba and Hennecke, Bio/technology, 12:678- 682 (1994), the document are incorporated herein by reference, at least for the associated materials with amino acid analogue).

It can produce the molecule for looking like polypeptide but not being keyed by native peptides.For example, amino acid or amino acids Key like object may include CH₂NH--、--CH₂S--、--CH₂----, -- CH ═ CH-- (cis and trans), -- COCH₂--、-- CH(OH)CH₂-- and -- CHH₂SO-- (can find these keys and other keys in the following documents: Spatola, A.F. quoted from Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.Weinstein volume It writes, Marcel Dekker, New York, page 267 (1983)；Spatola, A.F., Vega Data (March nineteen eighty-three), the 1st Volume, the 3rd phase, Peptide Backbone Modifications (general summary)；Morley,Trends Pharm Sci (1980) the 463-468 pages；Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (-- CH₂NH--,CH₂CH₂--)；Spatola et al. Life Sci 38:1243-1249 (1986) (-- CH H₂--S)；Hann J.Chem.Soc Perkin Trans.I 307-314 (1982) (-- CH----, cis and trans)；Almquist et al. J.Med.Chem.23:1392-1398(1980)(--COCH₂--)；Jennings-White et al. Tetrahedron Lett 23:2533(1982)(--COCH₂--)；Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982)(--CH(OH)CH₂--)；Holladay et al. Tetrahedron.Lett 24:4401-4404 (1983) (-- C (OH) CH₂--) and Hruby Life Sci 31:189-199 (1982) (-- CH₂--S--)；The document each by reference mode It is incorporated herein.It is appreciated that peptide analogues can have more than one atom between key atom, such as Beta-alanine, gamma-amino Butyric acid etc..

Amino acid analogue and peptide analogues often have enhancing an or desirable attribute, e.g., more economical production, Stronger chemical stability, the pharmacological property of enhancing (half-life period, absorption, potency, validity etc.), change specificity (for example, Broad-spectrum biological activity), reduce it is antigenic, stronger across biological barrier (for example, alimentary canal, blood vessel, blood brain barrier) ability And other.

D- amino acid can be used to generate more stable peptide, because D amino acid is not identified by peptase etc..With same type One or more amino acid of D- amino acid (for example, D-Lys substitution L-lysine) systematicness displacement consensus sequence can be used To generate more stable peptide.Cysteine residues can be used to that two or more peptides are cyclized or are bonded together.May have Benefit is that peptide is made to be constrained to specific conformation.(Rizo and Gierasch Ann.Rev.Biochem.61:387 (1992), by drawing It is incorporated herein with mode).

Therefore it provides polypeptide may include the examples of conservative variations (ACT) at α connection PROTEIN C end.As shown in table 4, sequence The example of single preservative replacement inside sequence SEQ ID NO:2 is provided in SEQ ID NO:3.It is given in sequence SEQ ID NO:4 The example of three preservative replacements in the inside sequence SEQ ID NO:2 out.Therefore it provides polypeptide may include amino acid SEQ ID NO:3 or SEQ ID NO:4.

Table 4.ACT polypeptide variants sequence

It is appreciated that the side of a kind of any variant for limiting gene and protein disclosed herein, modifier or derivative Formula is to limit variant, modifier and derivative according to specific known array sequence identity (also referred to herein as homology).It is special Do not disclose the variant of nucleic acid and polypeptide disclosed herein, the variant and described or known sequence have at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.Those skilled in the art are readily understood upon how to determine two kinds of protein or core The sequence identity of acid.For example, can after being aligned two sequences sequence of calculation identity so that sequence identity is in Its highest level.

It can be in such a way that disclosed algorithm implements another sequence of calculation identity.It can implement in the following way Optimal sequence for comparing compares: the local sequence of Smith and Waterman Adv.Appl.Math.2:482 (1981) is same One property algorithm, Needleman and Wunsch, J.Mol.Biol.48:443 (1970) sequence identity alignment algorithm, The similarity retrieval method of Pearson and Lipman, Proc.Natl.Acad.Sci.U.S.A.85:2444 (1988), these calculations The computerization of method executes (Wisconsin Genetics software package, Genetics Computer Group, 575Science Dr., GAP, BESTFIT, FASTA and TFASTA in Madison, Wis.) or inspection.The side that these bibliography pass through reference Formula is completely incorporated herein the method for sequence of calculation identity.

Zuker, M.Science 244:48-52,1989 can for example be passed through；Jaeger et al. Proc.Natl.Acad.Sci.USA 86:7706-7710,1989；Jaeger et al. Methods Enzymol.183:281- Algorithm disclosed in 306,1989 obtains the sequence identity of same type to nucleic acid, and the document is incorporated to by reference Herein at least for comparing relevant material to nucleic acid.

Therefore it provides polypeptide may include connect with α albumen C-terminal (ACT) have at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the amino acid sequence of 99% sequence identity.Therefore, in one aspect, the polypeptide provided include with SEQ ID NO:1, SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO: 34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:89 or SEQ ID NO:90 have at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, The amino acid sequence of 99% sequence identity.As an example, one kind is provided and appears in people Cx43's (SEQ ID NO:2) Identical nine amino acid segment on c-terminus has the polypeptide (SEQ ID NO:4) of 66% sequence identity.

It can be by provided herein is the tissue damages that polypeptide is added directly to subject.But by suitable with the polypeptide of offer Formula or the trans- cell internalizing transport protein being connected chemically enhance the cytoplasm location efficiency of the polypeptide.By light or cell to Tat- HA peptide cotransduction further enhances the efficiency of cell internalizing transport protein.

Therefore it provides polypeptide may include cell internalizing transport protein or sequence.Cellular internalization sequence can be ability Domain is known or newfound any internalization sequence or its examples of conservative variations.The non-limiting example of cell internalizing transport protein and sequence Attached bag include rqikiwfqnrrmkwkk (Antennapedia) sequence, TAT, HIV-Tat, penetrating peptide, Antp-3A (Antp mutant), Buforin II, transit peptides, MAP (mode amphiphilic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (double-guanidine-spermidine-cholesterol and BGTC (double-guanidine-Tren- cholesterol) (referring to table 5).

Table 5: cell internalizing transport protein

Therefore it provides polypeptide can also include amino acid sequence below: SEQ ID NO:7, SEQ ID NO:14 (Bucci, M. et al. 2000.Nat.Med.6,1362-1367), SEQ ID NO:15 (Derossi, D. et al. 1994.Biol.Chem.269,10444-10450), SEQ ID NO:16 (Fischer, P.M. et al. 2000.J.Pept.Res.55,163-172), SEQ ID NO:17 (Frankel, A.D. and Pabo, C.O.1988.Cell 55, 1189-1193；Green, M. and Loewenstein, P.M.1988.Cell 55,1179-1188), SEQ ID NO:18 (Park, C.B. et al. 2000.Proc.Natl.Acad.Sci.USA 97,8245-8250), SEQ ID NO:19 (Pooga, M. Et al. 1998.FASEB J.12,67-77), SEQ ID NO:20 (Oehlke, J. et al. 1998.Biochim.Biophys.Acta.1414,127-139), SEQ ID NO:21 (Lin, Y.Z. et al. 1995.J.Biol.Chem.270,14255-14258), SEQ ID NO:22 (Sawada, M. et al. 2003.Nature Cell Biol.5,352-357), SEQ ID NO:23 (Lundberg, P. et al. 2002.Biochem.Biophys.Res.Commun. 299,85-90), SEQ ID NO:24 (Elmquist, A. et al. 2001.Exp.Cell Res.269,237-244), SEQ ID NO:25 (Morris, M.C. et al. 2001.Nature Biotechnol.19,1173-1176), SEQ ID NO:26 (Rousselle, C. et al. 2000.Mol.Pharmacol.57,679-686), SEQ ID NO:27 (Gao, C. et al. 2002.Bioorg.Med.Chem.10,4057-4065) or SEQ ID NO:28 (Hong, F.D. and Clayman, G.L.2000.Cancer Res.60,6551-6556).The polypeptide of offer can also (double-guanidine-spermidine-gallbladder be solid comprising BGSC Alcohol) or BGTC (biguanides-Tren- cholesterol) (Vigneron, J.P. et al. 1998.Proc.Natl.Acad.Sci.USA.93, 9682-9686).Aforementioned reference is thus completely incorporated herein by reference for instructing cell internalizing carrier and sequence Column.Any other internalization sequence currently known or identify later can be combined with peptide of the invention.

The polypeptide of offer may include any ACT sequence combined with any one of cellular internalization sequence provided herein (for example, any one of ACT peptide disclosed herein).The combined example is provided in table 6.Therefore it provides polypeptide can wrap Containing the rqikiwfqnrrmkwkk sequence comprising amino acid sequence SEQ ID NO:7.Therefore it provides polypeptide may include amino acid sequence SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

Table 6: the ACT polypeptide with cellular internalization sequence (CIS)

The isolated nucleic acid for encoding polypeptide provided herein is also provided.Disclosed nucleic acid is for example by nucleotide, ucleotides It is formed like object or nucleotide surrogate.The non-limitative example of these molecules and other molecules described herein.It is understood that example Such as, in cell when expression vector, the mRNA of expression is generally made of A, C, G and U.

" isolated nucleic acid " or " nucleic acid of purifying " means the DNA without gene, in the biology for deriving DNA of the present invention Gene side is distributed in naturally occurring property genome.Therefore, which includes for example, being incorporated to carrier (such as autonomous science Plasmid or virus)；Or it is incorporated in prokaryotes or Eukaryotic genomic DNA (for example, transgenosis)；Or as independent molecule In the presence of (for example, the cDNA, the genome that pass through PCR, restriction endonuclease digestion or chemical synthesis or be synthetically produced in vitro Segment or cDNA segment) recombinant DNA.It further includes the recombination as a part of the heterozygous genes of encoding additional polypeptide sequence DNA.Term " isolated nucleic acid " also refers to RNA, for example, mRNA molecule, by isolated DNA molecular coding or through chemical synthesis, Or through separation or substantially free of at least some of cellular component, for example, other kinds of RNA molecule or peptide molecule.

Therefore it provides a kind of isolated nucleic acid for encoding polypeptide, the polypeptide includes amino acid sequence SEQ ID NO:1, SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

Therefore it provides nucleic acid may include nucleic acid sequence SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO: 81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87SEQ ID NO:88 or SEQ ID NO:89.

Nucleic acid provided herein can be connect with expression control sequence operability.Carrier is also provided, it includes mention herein The one or more nucleic acid supplied, amplifying nucleic acid are connect with expression control sequence operability.In the presence of can be used to deliver nucleic acid extremely The numerous compositions and method of cell in vitro or in vivo.These method and compositions can mainly be divided into two classes: based on virus Delivery system and be not based on virus delivery system.For example, can be by numerous direct delivery systems such as, electroporation, rouge Plasmids, calcium phosphate precipitation, plasmid, viral vectors, viral nucleic acid, bacteriophage nucleic acid, bacteriophage, clay, or by Inhereditary material is shifted in cell or carrier (such as cationic-liposome), to deliver nucleic acid.Suitable transfection means, including virus carry Body, chemical transfectants or physical-mechanical method (such as DNA electroporation and direct diffusion method), such as by Wolff, J.A. et al., Science,247,1465-1468,(1990)；And Wolff, J.A.Nature, 352,815-818, (1991) description.This kind of side Method is well known in the art and can be transformed match with composition as described herein and method easily.In certain situations Under, it will these methods are modified specially to cooperate huge DNA molecular to play a role.In addition, these methods can be used to pass through Using the targeting feature of carrier, certain diseases and cell colony are targeted.

Transfer vector, which can be, enters cell (for example, plasmid) for delivery of gene or as the overall plan of delivery of gene Any constructs (Ram et al. of a part (for example, as recombinant retrovirus or a part of adenovirus) slightly Cancer Res.53:83-88,(1993))。

As used herein, plasmid or viral vectors are by disclosed nucleic acid (such as SEQ ID NO:6) in non-degradable situation It is lower transport into cell and include promoter substance, the promoter generates gene in the cell of delivery of gene thereto Expression.In some embodiments, promoter is derived from virus or retrovirus.Viral vectors is, for example, adenovirus, gland connection Virus, herpesviral, vaccinia virus, poliovirus virus, AIDS virus, thermophilic neuron virus, sindbis virus With other RNA virus, including with HIV main chain these virus.Also disclose share these virus make it suitable for be used as carry Any Viraceae of the attribute of body.Retrovirus includes mouse Maloney leukemia virus, MMLV and expression MMLV as carrier Required attribute retrovirus.Retroviral vector can carry bigger hereditary payload than other viral vectors, that is, Transgenosis or marker gene, and for this reason, it is common carrier.But they are not useable in nonproliferating cell. Adenovirus vector is relatively stable and easily operated, has high titre, and can deliver in aerosol preparation, and can turn Contaminate overstepping one's bounds fragility cell.Poxvirus vector is huge and has several positions for being inserted into gene, they have thermal stability and Room temperature can be stored in.A kind of viral vectors is also disclosed, engineering is had been subjected to, thus the place for inhibiting viral antigen to excite Main biological immune response.Such carrier can carry the code area of interleukin 8 or 10.

Viral vectors can have the transfer ability for introducing gene to cell more higher than chemically or physically method and (introduce base The ability of cause).Generally, viral vectors contains unstructuredness early gene, structural late gene, rna plymerase iii transcription Inverted terminal repeat necessary to object, duplication and capsidation and the promoter for controlling viral genome transcription and replication.Work as work When Cheng Huawei carrier, virus generally removes one or more early genes and has inserted gene or gene/promoter box Enter to substitute the viral DNA of removal in viral genome.Such construct can carry the external hereditary object of up to about 8kb Matter.The required function of removed early gene is generally by having been subjected to engineering with the gene product of trans- expression early gene Cell line supply.

Retrovirus is the animal virus for belonging to Retroviridae (Retroviridae), including any type, Asia Section, category or preferendum.Retroviral vector is generally by Verma, I.M., Retroviral vectors for gene Transfer. quoted from Microbiology-1985, American Society for Microbiology, the 229-232 pages, Washington, (1985) description, the document are incorporated herein by reference.U.S. Patent number 4,868,116 and 4, 980,286；PCT application WO 90/02806 and WO 89/07136；And Mulligan, (Science 260:926-932 (1993)) example that the method for gene therapy is carried out using retroviral vector is described in；Professor's content of the document Incorporated herein by reference.

Retrovirus is substantially that nucleic acid loading is made to be packaged into packing material therein.Nucleic acid loading is believed with its carrier pack Number, efficient packet is loaded on inside packaging capsid by the progeny molecule that the packaging signal ensures to replicate.In addition to packaging signal, also deposit Virus is being replicated and numerous molecules of cis- needs for duplication and packaging.Generally, reverse transcription virus gene group contains and relates to produce Gag, pol and env gene of raw egg white matter capsid.General exactly gag, pol and env gene is by be transferred external to target cell DNA replacement.Retroviral vector, which typically contains, to be incorporated to the packaging signal of packaging capsid, sends the letter that gag transcript unit starts Number sequence, element necessary to reverse transcription, the primer binding site of the tRNA primer including combining process of reverse-transcription, DNA synthesis The initiation position of the second chain of synthetic DNA synthesis process is served as at the terminal repeat of period guide RNA chain switching, the 5' of 3'LTR The abundant sequence of the purine of point and the retrovirus that can be realized DNA state near the end LTR are inserted into host genome Specific sequence.Remove gag, pol and env gene allow about 8kb foreign sequence insertion viral genome in, be reverse transcribed and It is packaged into new retroviral particles once duplication.Depending on the size of every kind of transcript, this nucleic acid capacity is enough The extremely many a genes of delivering one.

Due to removed reproducing unit and packaging protein (gag, pol and env) in most of retroviral vector, one As by by carrier be placed in package cell line in, generate these carriers.Package cell line is to have used containing duplication and packed dress Set but lack the Retroviral Transfer of any packaging signal or the cell line of conversion.The carrier for carrying selected DNA is transfected into When these cell lines, by the device of the cis- offer of auxiliary cell, the carrier containing target gene is replicated and is packaged into new inverse Retroviral particle.The unpackaged genome corresponding to the device, because they lack required signal.

Building (Berkner et al., the J.Virology 61:1213-1220 of replication-defective adenoviral has been described (1987)；Massie et al., Mol.Cell.Biol.6:2872-2883 (1986)；Haj-Ahmad et al., J.Virology 57:267-274(1986)；Davidson et al., J.Virology61:1226-1239 (1987)；Zhang"Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis"BioTechniques 15:868-872(1993)).Made using these viruses It is had an advantage that for carrier, they are limited in terms of the degree that they can diffuse to other cell types, the reason is that they can With in initial infection cell internal reproduction, but new infectious virus particle cannot be formed.Have shown that recombined adhenovirus It is directly delivered to airway epithelia, liver cell, blood vessel endothelium, CNS parenchymal tissue [plant] and other numerous tissue sites in vivo Realize that High-efficiency gene shifts (Morsy, J.Clin.Invest.92:1580-1586 (1993) afterwards；Kirshenbaum, J.Clin.Invest.92:381-387(1993)；Roessler,J.Clin.Invest.92:1085-1092(1993)； Moullier,Nature Genetics 4:154-159(1993)；La Salle,Science 259:988-990(1993)； Gomez-Foix,J.Biol.Chem.267:25129-25134(1992)；Rich,Human Gene Therapy 4:461- 476(1993)；Zabner,Nature Genetics6:75-83(1994)；Guzman,Circulation Research 73: 1201-1207(1993)；Bout,Human Gene Therapy 5:3-10(1994)；Zabner,Cell 75:207-216 (1993)；Caillaud,Eur.J.Neuroscience 5:1287-1291(1993)；And Ragot, J.Gen.Virology 74:501-507(1993)).Recombined adhenovirus by being implemented in combination with gene transfer with cell surface special receptor, hereafter according to Wild type or the identical mode of replication-defective adenoviral, virus are internalized by (Chardonnet by receptor mediated endocytosis And Dales, Virology 40:462-477 (1970)；Brown and Burlingham, J.Virology 12:386-396 (1973)；Svensson and Persson, J.Virology 55:442-449 (1985)；Seth et al., J.Virol.51:650- 655(1984)；Seth et al., Mol.Cell.Biol.4:1528-1533 (1984)；Varga et al., J.Virology 65: 6061-6070(1991)；Wickham et al., Cell 73:309-319 (1993)).

Viral vectors can be a kind of carrier based on the adenovirus of removed E1 gene, and these virions are thin Born of the same parents system in 293 cell line of people as generated.In one aspect, E1 gene and E3 gene are removed from adenoviral gene group.

The viral vectors of another type is based on adeno-associated virus (AAV).This deficiency parvovirus can infect many Cell type is simultaneously not pathogenic to the mankind.AAV type carrier can transport about 4 to 5kb, and known wild type AAV stablizes insertion the In 19 chromosomes.As an example, this carrier can be Avigen, the P4.1C of San Francisco, Calif. production Carrier can contain herpes simplex virus thymidine kinase gene, HSV-tk and/or marker gene, such as encoding green fluorescent egg The gene of white GFP.

In the AAV virus of another type, AAV contain it is a pair of contain at least one instruct cell specific expression The inverted terminal repeat (ITR) of the box side distribution of promoter, the promoter are connect with heterologous gene operability.In In this case, heterologous finger is relative to AAV or B19 parvovirus and non-natural any nucleotide sequence or gene.

Generally, the code area AAV and the code area B19 have been lacked, to generate safe no cytotoxicity carrier.AAV ITR or its modifier assign infectious and site-specific integration, but do not assign cytotoxicity, and promoter instructs cell special Opposite sex expression.It is incorporated herein U.S. Patent number 6,261,834 by reference for material relevant to AAV carrier.

The disclosed carrier DNA that therefore offer can be integrated into mammalian chromosome in the case where no overt toxicity divides Son.

The gene being inserted into virus and retrovirus usually contains the promoter of auxiliary control target gene product expression And/or enhancer.Promoter is usually that one or more plays when being in relatively fixed position for transcription initiation site The DNA sequence dna of effect.Promoter contains RNA polymerase and core element necessary to basic interaction occurs for transcription factor, And upstream element and response element can be contained.

Herpesvirus infection can allowed whereby by being had been provided using the molecular genetic experiment of National People's Congress type herpesviral Cell in clone, propagate and establish huge heterologous DNA fragment means (Sun et al., Nature genetics 8:33-41, 1994；Cotter and Robertson, Curr Opin Mol Ther 5:633-644,1999).These large-scale DNA virus are (single Pure herpesviral (HSV) and Epstein-Barr viral (EBV) have people's heterologous DNA fragment to specific cells delivering > 150kb Potentiality.EBV recombinant can maintain huge DNA small pieces in the B cell of infection as episomal DNA.Carrying is up to The independent cloning of 330kb human genome insert seems genetically stable.These episomes are maintained to need to infect the phase in EBV Between constitutive expression special EBV nucleoprotein, EBNA1.In addition, these carriers can be used for transfecting, wherein a large amount of protein can With of short duration generation in vitro.Simplex viral amplicon system is also being used to pack > the DNA small pieces of 220kb, and the dye felt is thin Born of the same parents, which can stablize DNA as episome, to be maintained.

The non-replicating vaccinia virus vector that other useful systems are limited for example including science and host.

Disclosed composition can be delivered to target cell in many ways.For example, by electroporation or can pass through Lipofection passes through calcium phosphate precipitation delivering compositions.The delivery mechanism of selection will partly depend on targeted cell Type and delivering whether for example occur in vivo or in vitro.

Therefore, in addition to disclosed polypeptide, nucleic acid or carrier, composition can be for example comprising lipid, such as liposome, such as sun Cationic liposomal (for example, DOTMA, DOPE, DC-cholesterol) or anionic liposome.If desired, liposome can also include Promote the protein of targeting specific cells.Composition comprising compound and cationic-liposome can be applied to and flow into target device The blood of official or the target cell for sucking respiratory tract to respiratory tract.About liposome, for example, see Brigham et al. Am.J.Resp.Cell.Mol.Biol.1:95-100(1989)；Felgner et al. Proc.Natl.Acad.Sci.USA84: 7413-7417(1987)；U.S. Patent number 4,897,355.In addition, can be used as can be targeted to particular cell types for compound The component of the microcapsules of (such as macrophage) is applied, or in which spreads compound from microcapsules for special speed or dose design Or it is applied in the case where delivering compound.

It is including applying and absorbing the above-mentioned side in the cell that exogenous DNA enters subject (that is, gene transfer or transfection) In method, number of mechanisms delivering compositions to cell can be passed through.As an example, commercially available liposome product can be used such as LIPOFECTIN、LIPOFECTAMINE(GIBCO-BRL,Inc.,Gaithersburg,Md.)、SUPERFECT(Qiagen, Inc. Xi Erdeng, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.) and according to this field Other liposomes of standardization program exploitation, by liposome delivery.Furthermore, it is possible to pass through its technology from Genetronics, Inc. Electroporation obtained by (San Diego, California) and by SONOPORATION instrument (ImaRx Pharmaceutical Corp., Tucson, Ariz.), disclosed nucleic acid or carrier are delivered in vivo.

The nucleic acid for being delivered to cell should be integrated into host cell gene group, typically contain integration sequence.These sequences warp It is often the relevant sequence of virus, when particularly using the system based on virus.Also the system delivering for being not based on nucleic acid can be used, Such as liposome, these viral integrase systems are incorporated in nucleic acid to be delivered, so that nucleic acid contained in delivery system can be whole It is incorporated into host genome.

Other are integrated into the general technology of host genome for example including being designed to promote and host genome homologous recombination System.These systems commonly rely on the sequence that side is distributed nucleic acid to be expressed, in the sequence and host cell gene group The target sequence in portion has enough homologys, to recombinate between vector nucleic acid and target nucleic acid, causes the nucleic acid integration of delivering Enter host genome.These system and method necessary to homologous recombination are promoted to be known to the skilled in the art.

It can be by number of mechanisms well known in the art (for example, intake naked DNA, liposome merge, by particle gun DNA intramuscular injection, endocytosis etc.), by composition in vivo and/or ex vivo delivered to subject cell.

If using ex vivo approach cell or tissue can be taken out according to standard scheme well known in the art and in body Outside maintains.It can be by any gene transfer mechanism, e.g., for example, the gene delivery of calcium phosphate mediation, electroporation, micro note It penetrates or proteoliposome, composition is introduced into cell.It can will then be turned according to the standard method for cell or tissue type The cell infusion (for example, pharmaceutically in acceptable carrier) led homotopy is implanted into subject again.It is known by various cells Transplant or be infused into the standard method of subject.

The nucleic acid for being delivered to cell typically contains expression control system.For example, in virus system and retroviral systems The gene of insertion usually contains the promoter and/or enhancer of auxiliary control target gene product expression.Promoter is usually one A or multiple DNA sequence dnas for playing a role when being in relatively fixed position for transcription initiation site.Promoter contains RNA Core element necessary to basic interaction occurs for polymerase and transcription factor, and can contain upstream element and reaction member Part.

The promoter transcribed from carrier is controlled in mammalian host cell can obtain from various sources, for example, viral The genome of (such as polyomavirus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis type B virus, cytomegalovirus) Or come from heterologous mammal promoter, such as β actin promoter.The early promoter and late promoter of SV40 virus Advantageously (Fiers et al., Nature, 273:113 are obtained as the SV40 restriction fragment also containing SV40 virus origin of replication (1978)).The immediate early promoter of human cytomegalovirus is advantageously used as Hind III E restriction fragment to obtain (Greenway, P.J. et al., Gene 18:355-360 (1982)).Certainly, it is also used herein from host cell or correlation The promoter of species.

Enhancer is often referred to apart from the unfixed distance of transcription initiation site and can also be away from transcriptional units 5' (Laimins, L. et al., Proc.Natl.Acad.Sci.78:993 (1981)) or 3'(Lusky, M.L. et al., Mol.Cell.Bio.3:1108 (1983)) sequence of DNA that plays a role.In addition, enhancer may be at introne (Banerji, J.L. et al., Cell 33:729 (1983)) it is internal and in Encoding sequence itself (Osborne, T.F., Et al., Mol.Cell.Bio.4:1293 (1984)) it is internal.Their length is usually between 10 and 300bp, and they are suitable Formula plays a role.Enhancer plays the effect for increasing the transcription from neighbouring promoter.Enhancer also often contains mediate transcription The response element of adjusting.The response element that promoter can also be adjusted containing mediate transcription.Enhancer often determines gene expression Adjusting.Although currently known many enhancer sequences come from mammalian genes (globin, elastoser, albumin, α- Fetoprotein and insulin), but for General Expression, it will usually use the enhancer from eukaryotic cell virus.Example is multiple The SV40 enhancer of starting point advanced stage processed side (bp 100-270), cytomegalovirus early promoter enhancer, in replication orgin Polyoma enhancer and adenovirus cancers on advanced stage side.

Promoter and/or enhancer can be activated by the light or specified chemical event-specific for triggering its function.It can be with All systems are adjusted with reagent such as tetracycline and dexamethasone.There is also by being exposed to irradiation such as gamma-radiation or alkylation chemotherapeutic Object carrys out the mode of enhanced virus vector gene expression.

In certain embodiments, promoter and/or Enhancer district can serve as constitutive promoter and/or enhancer, So that the expression to transcript regions of transcript unit maximizes.In certain constructs, promoter and/or Enhancer district are complete It is active in portion's eukaryocyte type, even if it is only also such in specific time expression in specific cell type.It is this The promoter of type is CMV promoter (650 base).Other such promoters are SV40 promoter, cytomegalovirus (overall length Promoter) and retroviral vector LTR.

It has been shown that whole specific regulatory elements can clone and be used to construct in particular cell types (such as melanocyte Oncocyte) in selective expression expression vector.Glial fibrillary acidic protein (GFAP) promoter has been used in mind Selective expression's gene in cell through colloid origin.

The expression vector used in eukaryotic host cell (yeast, fungi, insect, plant, animal, people or karyocyte) Can also contain be possible to influence mRNA expression to sequence necessary to tanscription termination.These regional transcriptions are encoding tissue factor Polyadenylation segment in the untranslated part of the mRNA of albumen.3' non-translational region also includes translational termination site.Transcription is single Position can also contain poly-adenosine area.One benefit in this region is that it increases, and transcript unit will be as mRNA A possibility that processing and transhipment.Sufficiently establish the identification and purposes of poly-adenosine signal in expression construct.Homology is more Poly- adenylation signal can be used in transgenic constructs.In certain transcript units, poly-adenosine area is early derived from SV40 Phase poly-adenosine signal and by about 400 base compositions.The unit of transcription contain individually or with above-mentioned combined sequence its His standard sequence, to improve the stability of expression or construct from construct.

Viral vectors may include the nucleic acid sequence of coded markings product.Whether this marked product be used to determine gene If being delivered to cell and having been delivered, if expression.Example markup gene is coding-galactosidase Escherichia coli (E.Coli) lacZ gene and green fluorescent protein.

In some embodiments, label can be selected marker.Suitable selected marker for mammalian cell Example is dihyrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin and puromycin. When this kind of selected marker is successfully transferred to mammalian host cell, if being placed under selection pressure, the mammal of conversion Host cell can survive.There are two widely used different classes of selection schemes.The first kind is based on cell metabolism With use the mutational cell line of ability for lacking the culture medium for not depending on supplement and growing.Two examples are: Chinese hamster ovary (CHO)DHFR^-Cell and mouse LTK^-Cell.These cells, which lack, is not adding this kind of nutrient such as thymidine or hypoxanthic feelings The ability grown under condition.Because these cells, which lack nucleotide, synthesizes certain genes necessary to complete approach, therefore except non-supplemental The nucleotide of loss is supplemented in culture medium, otherwise they cannot survive.Supply these culture mediums be alternatively will be complete DHFR gene or TK gene are introduced into the cell for lacking respective gene, therefore change their growth requirement.Without DHFR or TK The individual cells of genetic transformation will not be able to survive in unsupplemented culture medium.

Second classification be dominant selection, be related to the selection scheme used in any cell type and do not need using Mutational cell line.These schemes are generally using the drug for stagnating host cell growth.Those cells with new gene will express It assigns the protein of drug resistance and will continue to survive under selection.The example of this kind of dominant selection uses following drug: new mould Element (Southern P. and Berg, P., J.Molec.Appl.Genet.1:327 (1982)), Mycophenolic Acid (Mulligan, R.C. and Berg, P.Science 209:1422 (1980)) or hygromycin (Sugden, B. et al., Mol.Cell.Biol.5: 410-413(1985)).Three examples utilize the bacterial gene under eukaryon control to be assigned respectively for suitable drugs G418 or new The resistance of mycin (Geneticin (Geneticin)), xgpt (Mycophenolic Acid) or hygromycin.Other include neomycin analog G418 and puromycin.

Cell comprising one or more carriers provided herein is also provided.As used herein, " cell ", " cell line " and " cell culture " may be used interchangeably and all this class name includes offspring.Disclosed cell can be for cloning or increasing Grow any cell of carrier provided herein.Therefore, cell can come from the cell line of any primary cell culture or foundation. Method can be adapted for any cell, including prokaryotic cell or eukaryocyte, such as bacterium, plant, zooblast.Cell type It can be selected based on the selection of carrier and required purposes by those skilled in the art.

It discloses by produced by the method with cell inside any nucleic acid molecules disclosed herein or carrier transfected animal Animal.It discloses by caused by the method with cell inside any nucleic acid molecules disclosed herein or carrier transfected animal Animal, wherein animal is mammal.It also discloses by in any nucleic acid molecules disclosed herein or carrier transfected animal Animal caused by the method for portion's cell, wherein mammal is mouse, rat, rabbit, ox, sheep, pig or primate.

A kind of composition is provided, it includes provided herein one or more more in pharmaceutically acceptable carrier Peptide, nucleic acid or carrier.Therefore it provides a kind of composition, it includes provided herein in pharmaceutically acceptable carrier Two or more combination in what ACT polypeptide.For example, providing a kind of composition, it includes pharmaceutically acceptable carriers In SEQ ID NO:1 and SEQ ID NO:5.

As used herein, " pharmaceutically acceptable " means on biology or is not in addition unfavorable material, that is, the material can To be applied to subject with nucleic acid or carrier, any unfavorable biological impact is not caused or not with harmful way and wherein Any other component of pharmaceutical composition containing the material interacts.Carrier will natively be selected to reduce to greatest extent Any degradation of effective component simultaneously reduces any adverse side effect in subject to greatest extent, such as this general of those skilled in the art It is known.

Composition provided herein can also be comprising any known or newfound be applied to wound, tissue damage inflammation The substance of position or cancer.For example, the composition provided can also include one of following classification or more persons: antibiotic (for example, Aminoglycoside, cephalosporins, chloramphenicol, clindamycin, erythromycin, fluoroquinolones, macrolides, Azolide, first Nitre azoles, penicillins, Tetracyclines, trimethoprim-sulfamethoxazole, vancomycin), steroids is (for example, androstane class (Andranes) (for example, testosterone), cholestane (for example, cholesterol), cholic acid (for example, cholic acid), corticosteroid (for example, Dexamethasone), female alkenes (Estraenes) (for example, estradiol), pregnane (for example, progestational hormone), narcotic analgeiscs and non-fiber crops Liquor-saturated property antalgesic (for example, morphine, codeine, heroin, Hydromorphone, levorphanol, pethidine, methadone (Methadone), Oxydone, dextropropoxyphene, fentanyl (Fentanyl), methadone (Methadone), naloxone, buprenorphine, butorphanol, Nalbuphine, pentazocine), chemotherapeutic is (for example, anticancer drug such as, but not limited to, hemel (Altretamine), aspartoyl Amine enzyme, bleomycin, busulfan, carboplatin, Carmustine (Carmustine), Chlorambucil, cis-platinum, carat Li Bin, ring phosphorus Amide, cytarabine, Dacarbazine, diethyl diethylstilbestrol, ethinyloestradiol, Etoposide, floxuridine, fludarabine, fluorine urine are phonetic Pyridine, Flutamide, Goserelin, hydroxycarbamide, idarubicin, ifosfamide, Leuprorelin, levamisol, Luo Mositing, dichloro First diethylamine, Medroxyprogesterone, megestrol acetate, melphalan, purinethol, methotrexate (MTX), mitomycin, mitotane, mitoxantrone, Taxol, Pentostatin (pentastatin), pipobroman, Primycin, prednisone, procarbazine, streptozotocin, his Moses Sweet smell, Teniposide, vincaleukoblastinum, vincristine), anti-inflammatory agent is (for example, alclofenac (Alclofenac)；Double propionic acid Ah cyanogen rice pines； AtgestoneAcetonide (Algestone Acetonide)；Alpha amylase；Amcinafal (Amcinafal)；Amicinafide (Amcinafide)；Amfenac sodium；Amiprilose hydrochloride；Anakinra；Anirolac (Anirolac)；Anitrazafen (Anitrazafen)；Apazone (Apazone)；Balsalazide disodium；Bendazac (Bendazac)；Benoxaprofen (Benoxaprofen)；Benzydamine hydrochloride；Bromelain；Broperamole (Broperamole)；Budesonide；Carprofen (Carprofen)；Cicloprofen (Cicloprofen)；Cinnopentazone (Cintazone)；Cliprofen (Cliprofen)；Propionic acid Clobetasol；Clobetasone butyrate；Clopirac (Clopirac)；Propionic acid cloticasone (Cloticasone Propionate)； Cormetasone Acetate (Cormethasone Acetate)；Cortodoxone (Cortodoxone)；Decylate；Deflazacort； Delatestryl；Depo- testosterone；Desonide；Desoximetasone；Dexamethasone dipropionate；Diclofenac Potassium；C14H10Cl2NNaO2；It is double Vinegar diflorasone；Diflumidone sodium；Diflunisal；Difluprednate (Difluprednate)；Diftalone (Diftalone)； Dimethyl sulfoxide；Drocinonide (Drocinonide)；Endrisone (Endrysone)；Enlimomab (Enlimomab)；According to promise Sharp health sodium (Enolicam Sodium)；Epirizole (Epirizole)；Etodolac；Etofenamate (Etofenamate)；Connection Phenylacetic acid (Felbinac)；Fenamole (Fenamole)；Fenbufen；Fenclofenac (Fenclofenac)；Fenclorac (Fenclorac)；Fendosal (Fendosal)；Benzene pyrrole dislikes diketone (Fenpipalone)；Fentiazac (Fentiazac)；Fu Lazha Ketone (Flazalone)；Fluazacort (Fluazacort)；Flufenamic acid；Flumizole (Flumizole)；Flunisolide acetate；Fluorine Ni Xin (Flunixin)；Flunixin meglumine；Fluocortin butyl (Fluocortin Butyl)；Fluorometholone acetate；Fluquazone (Fluquazone)；Flurbiprofen (Flurbiprofen)；Fluretofen (Fluretofen)；Fluticasone propionate；Furans Lip river Fragrant (Furaprofen)；Furobufen (Furobufen)；Halcinonide；Halobetasol propionate；Halopredone (Halopredone Acetate)；Ibufenac (Ibufenac)；Brufen (Ibuprofen)；Ibuprofen aluminum；Ibuprofen Piconol (Ibuprofen Piconol)；Ilonidap (Ilonidap)；Indomethacin；Indometacin sodium；Indoprofen (Indoprofen)；Indoxole (Indoxole)；Intrazole (Intrazole)；Isoflupredone acetate (Isoflupredone Acetate)；Isoxepac (Isoxepac)；According to rope former times health (isoxicam)；Ketoprofen (Ketoprofen)；Lofemizole hydrochloride (Lofemizole Hydrochloride)；Lornoxicam (Lomoxicam)；Lotepredenol etabonate (Loteprednol Etabonate)；First chlorine That fragrant sour sodium；Meclofenamic Acid；Meclorisone dibutyrate (Meclorisone Dibutyrate)；Mefenamic acid；Mesalazine (Mesalamine)；Meseclazone (Meseclazone)；Mesterolone；Protobolin (Methandrostenolone)；U.S.A is replaced Nandrolone；Metenolone acetate；Methylprednisolone Suleptanate (Methylprednisolone Suleptanate)；Momiflumate；Naphthalene Fourth U.S. ketone；Nandrolone Phenylpropionate；Naproxen；Naproxen sodium；Naproxol (Naproxol)；Nimazone (Nimazone)；Olsalazine Sodium (Olsalazine Sodium)；Orgotein (Orgotein)；Orpanoxin (Orpanoxin)；Anavar (Oxandrolane)；Olsapozine (Oxaprozin)；Oxyphenbutazone (Oxyphenbutazone)；Oxymetholone；Hydrochloric acid Renyi support Woods (Paranyline Hydrochloride)；Pentosan Polysulfate Sodium；Phenbutazone sodium glycerate (Phenbutazone Sodium Glycerate)；Pirfenidone；Piroxicam；Piroxicam cinnamate；Piroxicam olamine (Piroxicam Olamine)；Pirprofen (Pirprofen)；Prednazate (Prednazate)；Prifelone (Prifelone)；Prodolic acid (Prodolic Acid)；Proquazone (Proquazone)；Proxazole (Proxazole)；Proxazole citrate；Li Mei Suo Long；Romazarit (Romazarit)；Salcolex (Salcolex)；Sha Naxiding；Salsalate；Sanguinarium Chloride (Sanguinarium Chloride)；Seclazone (Seclazone)；Sermetacin (Sermetacin)；Stanozolol；Shu Duo Former times health (Sudoxicam)；Sulindac；Suprofen；Talmetacin (Talmetacin)；Talniflumate (Talniflumate)；His Lip river Willow ester (Talosalate)；Tebufelone (Tebufelone)；Tenidap；Tenidap sodium；Tenoxicam；Tesicam (Tesicam)；Tesimide；Testosterone；It mixes testosterone (Testosterone Blends)；Tetridamine (Tetrydamine)； Tiopinac (Tiopinac)；Pivalic acid Tixocortol；Tolmetin；Tolmetin sodium；Triclonide (Triclonide)；Triflumidate (Triflumidate)；Zidometacin (Zidometacin)；Zomepirac sodium) or antihistamine (for example, ethanol amine (e.g., benzene sea Lamine, carbinoxamine), ethylenediamine (e.g., bent, pyrilamine quicker than that), alkylamine (e.g., chlorphenamine, dexchlorpheniramine, bromobenzene That quick, triprolidine), other antihistamines such as astemizole, Loratadine, fexofenadine, Brompheniramine (Bropheniramine), clemastine (Clemastine), paracetamol, pseudoephedrine, triprolidine).

Composition can locally, oral or extra-parenteral administration.For example, composition can in external, encephalic, intravaginal, anus, Subcutaneously, intradermal, intracardiac, stomach is interior, it is intravenous, intramuscular, pass through intraperitoneal injection, percutaneous, intranasal or pass through inhalant application.Such as this Used in text, " encephalic application " mean direct delivered substance to brain, for example including by conduit or the intrathecal of needle, brain pond, the ventricles of the brain It is interior or delivered through sphenoid sinus.

If used, the parenteral administration of composition is usually characterized by injection.Injection can routinely form conduct Liquid solution agent or suspension, the solid-state form suitable for forming solution or suspension before injecting are prepared as emulsion.More It is related to for modified parenteral administration scheme recently using slow release or slow-released system, to maintain constant dosage.Referring to, For example, U.S. Patent number 3,610,795 incorporated herein by reference.

As used herein, " local intranasal administration " means composition being delivered to nose and nasal meatus by one or two nostril It and may include being delivered by sprinkling mechanism or dropping liquid mechanism or by the atomization of nucleic acid or carrier.It is applied by inhalant Composition can be by being carried out with sprinkling or the delivering of dropping liquid mechanism intranasal or mouth.Breathing can also be directly delivered to by intubation Any region of system (for example, lung).

It in some embodiments, can once a day, twice daily, three times a day, four times a day, five times a day or more Repeatedly part applies composition provided herein.It in some embodiments, can be with primary, every three days every two days primary, every four At primary, every six days primary, every five days primary, every four days day is primary or part applies composition provided herein once a week.In In some embodiments, composition provided herein can be applied based on required part.For example, in some embodiments, it can To apply composition provided herein during or after the Symptoms of radiation injury or its severity increase.In some realities Apply in scheme, can locally apply composition provided herein once a day, for about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100 days or More consecutive days.In some embodiments, composition provided herein includes ACT peptide provided herein and gel, and Can locally it apply.In other embodiments, composition provided herein includes SEQ ID NO:91.In some embodiments In, composition provided herein includes SEQ ID NO:91 and hydroxyethylcellulose gel, wherein part applies composition.

In some embodiments, composition provided herein includes being referred to asTopical gels agent. In some embodiments,Include 1.25% hydroxyethylcellulose gel and ACT1 peptide.In The chemical structure of ACT1 peptide is: biotin-Ahx-Arg-Gln-Pro-Lys-Ile-Trp-Phe-Pro-Asn-Arg-Arg- Lys-Pro-Trp-Lys-Lys-Arg-Pro-Arg-Pro-Asp-Asp-Leu-Glu-Ile- OH (SEQ ID NO:91), wherein Ahx is L-2- aminocaproic acid (6-aminocaprolc acid).In some embodiments,Also comprising one or more anti- Rotten agent, solvent, buffer, stabilizer, complexing agent and/or any additional pharmaceutically acceptable auxiliary material or carrier.

In some embodiments, about 1 before being exposed to ionising radiation, about 2, about 5, about 10, about 15, about 20, about 24, About 48 hours or more, part and/or systemic administration composition provided herein.In some embodiments, In It is exposed to ionising radiation about 1, about 2, about 5, about 10, about 15, about 20, about 24, about 48 hour or more later, part And/or systemic administration composition provided herein.In some embodiments, about 1 to about 48 after being exposed to ionising radiation Hour or exposure after about 10 to about 36 hours window times, part and/or systemic administration after about 4 to about 24 hours or exposure Composition provided herein.In specific embodiments, about 24 hours after being exposed to ionising radiation, part applies to be mentioned herein The composition of confession.In some embodiments, by composition provided herein (for example, including ACT peptide provided herein and gel Composition) thin layer be applied on the region for being exposed to ionising radiation.Therefore, in some embodiments, the disclosure provides For treating the method for being exposed to ionising radiation, applying the method includes part includes SEQ ID NO:91 and hydroxy ethyl fiber The composition of plain gel is to exposed region.

It in some embodiments, can once a day, twice daily, three times a day, four times a day, five times a day or more Multiple systemic administration composition provided herein.It in some embodiments, can be primary, every with primary, every three days every two days Primary or systemic administration combination provided herein once a week in primary, every six days primary, every five days primary, every four days on the four Object.In some embodiments, it can be mentioned herein before being exposed to ionising radiation or later based on required systemic administration The composition of confession.For example, in some embodiments, it can be during the Symptoms of radiation injury or its severity increase Or composition provided herein is applied later.In some embodiments, can once a day systemic administration it is provided herein Composition, for about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100 days or more.In some embodiments, provided herein Composition for systemic delivery to the subject for having demand includes ACT peptide provided herein.In certain embodiments, originally The composition for systemic delivery to the subject for having demand that text provides includes ACT1 polypeptide (SEQ ID NO:2).At it In his embodiment, the composition provided herein for systemic delivery to the subject for having demand includes one or more medicines Acceptable carrier on.

In some embodiments, about 1 after being exposed to ionising radiation, about 2, about 5, about 10, about 15, about 20, about 24, About 48 hours or more, systemic administration composition provided herein.In specific embodiments, it is being exposed to electricity From about 24 hours after radiation, systemic administration composition provided herein.In some embodiments, it is being exposed to ionising radiation Afterwards and before the Symptoms of ARS, systemic administration composition provided herein.In some embodiments, it is being exposed to electricity From after radiation and after the Symptoms of ARS, systemic administration composition provided herein, wherein composition treatment, inhibit or Alleviate the progress of ARS.

The exact amount of required composition will be different between subjects, this depends on species, age, the body of subject Weight and overall state, the severity, specific nucleic acid used or the carrier that are receiving the anaphylactia treated, its application mould Formula etc..Therefore, it is not possible to provide the exact amount of every kind of composition.But in view of teaching content herein, suitable amount can be with Routine experiment is used only by those of ordinary skill in the art to determine.

Material may be in solution or suspension (for example, in the particulate of incorporation, liposome or cell).It can be by anti- Body, receptor or receptors ligand are by these materials targeted to specific cell type.Being below with reference to document will using this technology Specific protein targeted to tumor tissues example (Senter et al., Bioconjugate Chem., 2:447-451, (1991)；Bagshawe,K.D.,Br.J.Cancer,60:275-281,(1989)；Bagshawe et al., Br.J.Cancer, 58:700-703,(1988)；Senter et al., Bioconjugate Chem., 4:3-9, (1993)；Battelli et al., Cancer Immunol.Immunother.,35:421-425,(1992)；Pietersz and McKenzie, Immunolog.Reviews,129:57-80,(1992)；And Roffler et al., Biochem.Pharmacol, 42:2062- 2065,(1991)).The liposome of solvent such as " steal into another country viral (stealth) " and other antibody conjugates (including targets colon The drug that the lipid of cancer mediates), by the receptor-mediated DNA target of cell specific ligand to, lymphocyte guiding performance tumour Targeting and in vivo high degree of specificity treatment retrovirus are to rat glicoma cells targeting.It is to use this below with reference to document Technology is by specific protein targeted to example (Hughes et al., the Cancer Research, 49:6214- of tumor tissues 6220,(1989)；With Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).In general, receptor participates in endocytic pathway caused by composing type or ligand.These receptor gatherings are small in clathrin envelope In nest, enter cell by clathrin coated vesicl, pass through the acidification inner body of sorting receptor, and is then recycled to Cell surface, it is intracellular to store or degrade in lysosome.Internalization pathway plays multiple functions, and such as nutrient intake removes activating Protein, opportunistic entrance, dissociation of ligand and the degradation and acceptor levels adjustment effect for removing macromolecular, virus and toxin.Perhaps Polyceptor follows more than one approach intracellular, this depends on cell type, acceptor density, ligand classes, ligand valence state and ligand Concentration.The molecular mechanism and cell mechanism (Brown and Greene, DNA and Cell of receptoe mediated endocytosis are reviewed Biology 10:6,399-409(1991))。

Remington:The Science and Practice of Pharmacy (19th ed.) A.R.Gennaro is compiled It writes, describes suitable carrier and their preparation in Mack Publishing Company, Easton, Pa.1995.Generally, Pharmaceutically acceptable salt in preparation using appropriate amount keeps preparation isotonic.The example of pharmaceutically acceptable carrier includes but not It is limited to salt water, Ringer's solution and dextrose solution.The pH of solution can be about 5 to about 8, about 7 to about 7.5.The example of other carriers Including sustained release formulation, such as solid hydrophobic polymers semipermeable matrices antibody-containing, the matrix is in molding system Product (for example, film, liposome or particulate) form.Those skilled in the art will be evident, such as depending on The administration method and concentration of the composition of application, certain carriers can be more preferable.

Pharmaceutical carrier known to those skilled in the art.These major parts are generally used for application drug to the standard of people and carry Body, including solution such as sterile water, salt water and in the buffer solution of physiological pH.Composition can intramuscular or subcutaneous administration.By root Other compounds are applied according to the standardization program that those skilled in the art use.

In addition to the molecule of selection, pharmaceutical composition may include carrier, thickener, diluent, buffer, preservative, Surfactant etc..Pharmaceutical composition can also include one or more effective components such as antimicrobial, anti-inflammatory agent, arcotic Deng.

Pharmaceutical composition can be applied in numerous ways, this depends on whether topically or systemically to treat and depend on In region to be treated.Can local (including eye, vagina, rectum, intranasal), it is oral, by sucking or parenteral administration, such as By in intravenous drip, subcutaneous, peritonaeum or intramuscular injection application.In certain embodiments, it is administered by local application. In certain embodiments, it is administered by systemic administration.Systemic administration is for example including enteral or parenteral administration.In In some embodiments, systemic administration is carried out by intravenous injection, subcutaneous injection, intracutaneous injection, intramuscular injection or sucking. In some embodiments, row systemic administration is delivered by atomization.

Product for parenteral administration includes sterile aqueous pharmaceutical or non-aqueous liquor, suspension and emulsion.It is non-aqueous The example of solvent is propylene glycol, polyethylene glycol, vegetable oil such as olive oil and injectable organic ester such as ethyl oleate.Aqueous carrier Including water, alcohol/aqueous solution, emulsion or suspension, the medium including salt water and buffering.Parenteral vehicles include sodium chloride solution, Ringer dextrorotation liquid glucose, dextrose and sodium chloride, Lactated Ringer liquid or fixed oil.Intravenous vehicles may include stream Body and nutritional supplement, electrolyte supplements (such as based on those of Ringer dextrose).Preservative and other additives It can be such as there are antimicrobial, antioxidant, complexing agent and inert gas.

Formulations for topical administration may include ointment, lotion, cream, gelling agent (for example, poloxamer gel Agent), drops, suppository, spray, liquid agent and pulvis.Conventional pharmaceutical carrier, aqueous, powdery or oleaginous bases, thickener etc. It can be required or want.Can for example microfibre, polymer (for example, collagen), nanosphere, sprayer, lotion, Cream, fabric, plastics, engineered bracket, host material, tablet, implantation container, powder, oil, resin, wound dressing, Pearl, microballon, slow release pearl, capsule, injection, intravenous drip, pump installation, siloxanes implants or any Bioengineered material Disclosed composition is applied in material.In some embodiments, formulations for topical administration is opacifier, suncream or similar Preparation.This kind of composition can be absorbed, filters, reflects or block to be radiated from the UV of the sun or other UV radiation sources.Therefore, In In some embodiments, the disclosure, which provides, plays the following preparation acted on: reducing UV radioactive exposure and prevents, treats or alleviate Radiation injury progress from UV radiation.In other embodiments, formulations for topical administration be tanned lotion, it is tanned plus Fast agent, suntan oil or similar formulations.In some embodiments, opacifier, suncream, tanned dew, tanned accelerator or tanned Oil includes the polypeptide provided herein measured in this way, the amount can prevent, treat or alleviate by ultraviolet light (for example, come from the sun, Tanning bed or other sources) caused by radiation injury progress.For example, in some embodiments, opacifier includes certain concentration The polypeptide provided herein of (for example, about 0.001% (w/w) to about 5.0% (w/w) or about 10 μM to about 2000 μM).

In one aspect, the pharmaceutically acceptable carrier provided is poloxamer.Pass through trade markIt refers to Poloxamer be in water formed heat-convertible gel nonionic surfactant.Poloxamer is polyethylene glycol oxide-polycyclic Ethylene Oxide-polyethylene glycol oxide (PEO-PPO-PEO) triblock copolymer.Two polyethylene oxide chains are hydrophilic, but polypropylene chains are dredged Water.When being placed in aqueous solution, these hydrophobic characters and hydrophilic profile electrification.PEO-PPO-PEO chain is taken in wherein hydrophobicity The heart will collect in the form of the chainlet for forming micella.Then, micella tends to gelation characteristics, because they collect in groups with shape The solid content (gel) being only slightly present near hydrophilic end at wherein water.When cooled, it becomes liquid, but when heating, It is hardened.This characteristic makes it can be used for middle drug compounding, because when it is ice-cold, it can be by its inhalation syringe for accurate Dosage measurement.When it is warming up to body temperature (when being applied to skin), it thicken to perfect consistency (especially with soybean lecithin When the combination of rouge/isopropyl palmitate), with embrocating of promoting to be suitable for and stick.It is widely usedF127 (F127), because It is easy to get for it and therefore it is used in this kind of medicinal application.F127 has the EO:PO:EO ratio of 100:65:100, institute Stating ratio by weight has the PEO:PPO ratio of 2:1.Pluronic gel is aqueous solution and typically contains 20-30% F-127.Therefore, the composition of offer can be provided in F127.

It is it is well known that and can be in excipient substance handbook (Rowe, R.C. etc. for the auxiliary material in topical gels People, APhA Publications；5th edition, 2005) example is found in.Exemplary auxiliary material may include wax, it is various sugar and it is a variety of Starch, polymer, gel, softening agent, thickener, rheology modifier, moisturizer, glycerol, organic basic compound, the fibre of type Tie up plain derivative, gelatin, vegetable oil, polyethylene glycol and solvent.The example of rheology modifier includes carbopol, hydroxy propyl cellulose Element, C_26-28Alkyl dimethicone, C_26-28Alkyl methicone, polyphenylene silicon silsesquioxane, trimethyl silane The friendship of oxygroup esters of silicon acis, ring five dimethyl silicone polymer and dimethyl silicone polymer/vinyl trimethylsilane oxygroup esters of silicon acis Linked polymer, fumed silica (for example, Cab-O-Sil M5P) and its mixture.The example of softening agent includes glycerol, penta 2 Alcohol, pyrrolidone sodium carboxylate, lanolin, carbohydrate isomer, stearic oxygroup dimethione, the poly- diformazan silicon oxygen of stearyl Alkane and its mixture.Softening agent can be used for the cuticle dehydration for preventing from occurring because using anhydrous solvent in preparation.It is organic The example of alkali includes 2- amino-2-methyl propyl alcohol, niacinamide, carbinolamine, triethanolamine, Trisamino, AMP-95, AmP- Ultra PC 2000, triisopropanolamine, diisopropanolamine (DIPA), Neutrol TE, Ethomeen and its mixture.Organic base can be with Make the pH alkalinity or neutral of drug.

Other exemplary auxiliary materials include water soluble pore formers.Water soluble pore formers are can to promote water intake and diffuse into solidifying The additive of glue.Any suitable pore-foaming agent can be used, but in some embodiments, pore-foaming agent may include chlorination Sodium, potassium chloride, sucrose, glucose, lactose, sorbierite, xylitol, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol or its Mixture.

Polymer can function as the auxiliary material in topical gels agent.Illustrative polymers include hydrophilic polyurethane, parent Water polyacrylic acid, the copolymer of carboxymethyl cellulose and acrylic acid, N- vinylpyrrolidone, poly- (hydroxy acid), polyanhydride, poly- original Acid esters, polycarbonate, polyalkylene (for example, polyethylene and polypropylene), polyglycols (for example, poly(ethylene glycol)), gathers polyamide Alkylene oxide (for example, polyethylene oxide), polyakylene terephthalate (for example, polyethylene terephthalate), Polyvinyl alcohol, polyvinylether, polyvinyl ester, polyvinylhalide (for example, poly- (vinyl chloride)), polyvinylpyrrolidone, polysiloxanes, Poly- (vinylacetate), polystyrene, polyurethane copolymer, cellulose, cellulose derivatization are (for example, hydroxyethyl cellulose, hydroxyl Propyl cellulose, hypromellose, methylcellulose, ethyl cellulose, carboxymethyl cellulose or cellulose acetate), algae Hydrochlorate, poly- (acrylic acid), poly- (acrylic acid) derivative, acrylic copolymer, methacrylic acid, methacrylic acid derivative, first Base acrylic copolymer, poly- (butyric acid), poly- (valeric acid), poly- (lactide-co-caprolactone), its copolymer and its admixture.

In some embodiments of the present invention, polymer can be super absorbent polymer (SAP).Such as according to IUPAC Definition, if certain polymer can be absorbed and retain extremely a large amount of water relative to its own quality, the polymer be considered as have it is super Absorbability.SAP can be absorbed the water of up to 500 times its own weight and can be swollen up to 1000 times of its initial volumes.Have The specific SAP of meaning includes Sodium Polyacrylate, polyurethane Tecophilic TG-2000 and by using polyacrylamide amine copolymer Object, ethylidene maleic anhydride copolymers, cross-linking type carboxy-methyl-cellulose, polyvinyl alcohol copolymer, polyvinylpyrrolidone and The polymer of cross-linking type polyethylene glycol oxide preparation.

In some embodiments of the present invention, the polymer of relative hydrophobic can be used.It can be used any suitable Hydrophobic polymer.But the illustrative polymers of relative hydrophobic include aromatic polyurethane, silicon rubber, polysiloxanes, gather in oneself Ester, polyvinyl chloride, polyethylene, poly-L-lactide, poly- DL- glycolide, polyether-ether-ketone (PEEK), polyamide, gathers polycarbonate Amide and polyvinyl acetate.In addition it is possible to use hydrophobic gel matrix and/or rheology modifier.

In some embodiments of the present invention, polymer can serve as the thickener in drug.Specifically, gel Polymer moieties can serve as visco-elastic material and can retain gel in site of administration, together with the α connection being dispersed therein Polypeptide.

In some other embodiments, the gel comprising polymer can have spreadability, thus on a skin surface When application, it forms film.This film can to apply contained α connection polypeptide in become in broad regions can Can, and can play the role of maintaining α connection polypeptide on skin involvement region.

Other auxiliary materials may include the different kinds of ions or nonionic compound for maintaining preparation stability, thus taking precautions against may The formulation components demulsification of its therapeutic value or aesthetic values is reduced, sedimentation, reunites or degrades.

The example of ionic compound may include salt such as sodium chloride, potassium chloride；Cationic, anionic or amphoteric ion Type surfactant such as lauryl sodium sulfate (SDS), per-fluoro octanoate (PFOA), perfluoro octane sulfonate (PFOS), laurel Alcohol sulfuric ester ammonium (ALS), sodium laureth sulfate (SLES), alkylbenzene sulfonate, cetyl trimethylammonium bromide (CTAB), Hexadecylpyridinium chloride drone (CPC), polyethoxylated tallow base amine (POEA), benzalkonium chloride (BAC), benzethonium chloride, 12 Alkyl betaine, Cocoamidopropyl betaine and cocounut oil acyl both sexes base Glycinates.

Can serve as the nonionic compound of auxiliary material example include nonionic surface active agent such as Pluronic, Tween, AMP and Brij surfactant families；With the surfactant of derivative biological origin, for example, natural or semi-synthetic Property surfactant, such as oleic acid, sorbitan trioleate, sorbitan mono-oleic acid ester, lecithin, coconut oleoyl amine MEA, coconut palm Oleamide DEA and Cocoamidopropyl betaine.Surfactant (ionic and non-ionic) can reduce interface can and Topical preparation can be promoted to spread on wider region.

In some embodiments of the present invention, solvent auxiliary material can be used as α connection polypeptide and other auxiliary materials Carrier vehicle.Polymer chain, which can interact with solvent and swelling occurs, can assign viscoelastic to topical preparation to be formed The network of performance.In some embodiments of topical preparation, solvent can be evaporated when applying, and leave the remnants of polymer α connection polypeptide of the film together with embedding.

The exemplary solvent auxiliary material that can be used in hydrophilic formulation may include isobide acid anhydride dimethyl ether, propylene glycol, Or mixtures thereof glycerol, isopropanol, ethyl alcohol, benzyl alcohol, ethylene glycol, polyethylene glycol, ethoxydiglycol.It can be used for hydrophobicity Exemplary solvent auxiliary material in preparation may include capric acid/Trivent OCG, isopropyl myristate, mineral oil, different 12 Alkane, Dermol 105, butanediol, pentanediol, hexylene glycol, methoxy poly (ethylene glycol), ring five dimethyl silicone polymer, ring four are poly- Or mixtures thereof dimethyl siloxane, dimethyl silicone polymer, octyl polymethyl siloxane.

In addition to α connection polypeptide and auxiliary material, topical preparation can also be comprising at least one additional medicine such as Antimicrobial, anti-acne drug, anti-inflammatory agent, antalgesic, arcotic, antihistamine, preservative, immunosuppressor, hemostatic, blood Pipe vasodilator, wound healing medicine, antibiont envelope medicine and its mixture.

The example of antimicrobial includes penicillin and related drugs, carbapenem, cephalosporins and correlation Drug, erythromycin, aminoglycoside, bacitracin, gramicidins, mupirocin, chloramphenicol, Thiamphenicol, sodium fusidate, woods Can mycin, clindamycin, macrolides, ovobiocin, polymyxins, rifamycin,Spectinomycin (spectinomysin), Tetracyclines,Vancomycin (vanomycin), teicoplanin, link-typed circuit, antifolic, packet Include sulfamido, trimethoprim and combinations thereof and pyrimethamine, synthetic antibacterium medicine, including itrofurans, mandelic acid crow Lip river It is tropine and methenamine hippu, nitro glyoxaline, quinolone, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, right Aminosalicylic acid (PAS), seromycin, capreomycin, 2-ethylisonicotinthionamide, protionamide (prothionamide), thiacetazone (thiacetazone), viomycin, everninocin (eveminomycin), glycopeptide, glycylcycline (glyclyclycline), ketolide, oxazolidone；Imipenem (imipenen), amikacin, Netilmicin, phosphorus are mould Element, gentamicin, ceftriaxone, Ziracin (Ziracin), Linezolid, Xin Neiji (Synercid), aztreonam and first nitre Azoles, Epiroprim (Epiroprim), sanfetrinem sodium (Sanfetrinem sodium), Biapenem (Biapenem), up to interior Mycin, cefluprenam (Cefluprenam), Cefoselis (Cefoselis), sanfetrinem cilexitil (Sanfetrinem Celexetil), Cefpirome, mersacidin (Mersacidin), rifalazil, Kosan, lenapenem (Lenapenem), Veneprim, Sulopenem (Sulopenem), Ritipenem acetoxymethyl (ritipenem acoxyl), Cyclothialidine, Micacocidin A, carumonan (carumonam), Cefozopran (Cefozopran) and cephalo he U.S. ester.

The example of topical anti-acne medicine includes Adapalene (adapalene), azelaic acid, benzoyl peroxide, crin Mycin and clindamycin phosphate, Doxycycline, erythromycin, keratolytic such as salicylic acid and retinoic acid (" Retin-A "), promise Pregnant ester, organic peroxide, vitamin A acid such as Accutane and vitamin A acid, albucid soluble and tazarotene.Specific anti-acne Medicine include Adapalene, azelaic acid, benzoyl peroxide, clindamycin (for example, clindamycin phosphate), Doxycycline (for example, Doxycycline monohydrate), erythromycin, Accutane, norgestimate, albucid soluble, tazarotene, etretinate and acetretin。

The example of antihistamine include bagodryl hydrochloride, diphenhydramine salicylate, diphenhydramine, hydrochloric acid chlorphenamine, Chlorphenamine maleate, Dimethoxanate Hydrochloride, salt love song be quicker than that, promethazine hydrochloride, methdilazine hydrochloride etc..Local anesthetic Example includes cinchocaine hydrochloride, cinchocaine, lidocaine hydrochloride, lidocaine, benzocainum, the p- butylamino of hydrochloric acid Benzoic acid -2- (two-ethylaminos) ethyl ester, procaine hydrochloride, totokaine, tetracaine hydrochloride, chloroprocaine hydrochloride, hydrochloric acid Hydroxyprocaine (oxyprocaine hydrochlorid), mepivacaine, ***ehydrochloride, piperocaine hydrochloride, Dacroment Peace reaches hydrochloric acid Cronin.

The example of preservative includes alcohols, quaternary ammonium compound, boric acid, Chlorhexidine and Chlorhexidine derivative, iodine, phenol, terpene Class, bactericide, disinfectant, including thimerosal, phenol, thymol, benzalkonium chloride, benzethonium chloride, Chlorhexidine, povidone Iodine, Cetylpyridinium Chloride, eugenol and trimethylammonium bromide.

The example of anti-inflammatory agent includes non-steroidal anti-inflammatory drugs (NSAID)；Propanoic derivatives such as brufen and naproxen；Acetic acid spreads out Biology such as Indomethacin；Enolic acid derivative such as Meloxicam, paracetamol；Gaultherolin；Ethylene glycol list salicylic acid Ester；Aspirin；Mefenamic acid；Flufenamic acid；Indomethacin；Diclofenac；Alclofenac；C14H10Cl2NNaO2；Brufen；Ketone Ibuprofen；Naproxen；Moor rice brufen；Fenoprofen；Sulindac；Fenclofenac；Clidanac；Flurbiprofen；Fentiazac；Butylbenzene hydroxyl Acid；Piroxicam；Bute；Oxyphenbutazone；Clofezone (clofezone)；Pentazocine；Mepiprazole (mepirizole)；Tiaramide hydrochloride (tiaramide hydrochloride)；Steroids such as clobetasol propionate, dipropyl Sour betamethasone, halobetasol propionate (halbetasol proprionate), diflorasone diacetate, Fluocinonide, Ha Xi How are Nai De, Amcinonide, Desoximetasone, Triamcinolone acetonide, momestasone furoate, fluticasone propionate, dipropium dipropionate, Qu An How are moral, fluticasone propionate, desonide, Flucloronide, hydrocortisone valerate, prednicarbate (prednicarbate), Qu An Moral, Flucloronide, hydrocortisone and it is known in the art other, prednisolone (predonisolone), dexamethasone, fluorine Chloronaphthalene moral, hydrocortisone acetate, Econopred (predonisolone acetate), methylprednisolone (methylpredonisolone), dexamethasone acetate, betamethasone, betamethasone valerate, flumethasone (flumetasone), Fluorometholone, beclomethasone dipropionate, Fluocinonide, topical corticosteroids class, and can be following inefficient cortex class One of steroid, as hydrocortisone, hydrocortisone -21- monoesters (for example, hydrocortisone -21- acetic acid esters, hydrogenation can Pine -21- butyrate, hydrocortisone -21- propionic ester, hydrocortisone -21- valerate etc.), hydrocortisone -17,21- two Ester is (for example, hydrocortisone -17,21- diacetate esters, hydrocortisone -17- acetic acid esters -21- butyrate, hydrocortisone - 17,21- dibutyrate etc.), Ah cyanogen's rice pine, dexamethasone, flumethasone (flumethasone), prednisolone (prednisolone) or methylprednisolone, or it can be efficient corticosteroid, such as clobetasol propionate, formic acid times his rice Loose benzene, dipropium dipropionate, diflorasone diacetate, Fluocinonide, momestasone furoate, Triamcinolone acetonide.

The example of antalgesic includes alfentanil, benzocainum, buprenorphine, butorphanol, butamben (butamben), capsaicine, clonidine, codeine, cinchocaine, enkephalins, fentanyl, hydrocodone, Hydromorphone, indoles beauty Pungent, lidocaine, levorphanol, pethidine, methadone, morphine, nicomorphine, opium, oxybuprocaine (oxybuprocaine), Oxycodone, Oxymorphone, pentazocine, pramocaine, keracaine, dextropropoxyphene, proparacaine (proxymetacaine), Sufentanil (sufentanil), totokaine and C16H25NO2.

The example of arcotic includes alcohols such as phenol；Ergol；Calamine；Dichloroxylenol；Dyclonine；Chloramines Ketone；Menthol；Pramocaine；Resorcinol；Triclosan (troclosan)；Procaine drug class such as benzocainum, Bu Bika Cause, chloroprocanine；Cinchocaine (cinchocaine)；Cocaine；Dexivacaine (dexivacaine)；Diamocaine (diamocaine)；Cincaine；Etidocaine；Hexylcaine；Chirocaine (levobupivacaine)；Lidocaine； Mepivacaine；Oxetacaine (oxethazaine)；prilocalne；Procaine；Keracaine；Propoxycaine；Pyrroles's card Cause；Risocaine；Rodocaine；Ropivacaine；Totokaine；And derivative, such as pharmaceutically acceptable salt and ester, including hydrochloric acid Bupivacaine, chloroprocaine hydrochloride, diamocaine cyclamate, quinocaine, dyclonine hydrochloride, hydrochloric acid are according to replacing Cacaine, Levobupivacaine HCL, lidocaine hydrochloride, Mepivacaine HCL, Pramoxine HCL, hydrochloric acid prilocalne, salt Sour procaine, proparacaine hydrochloride, ranocaine, Ropivacaine HCL and tetracaine hydrochloride.

The example of hemostatic includes fibrin ferment, phytondione, protamine sulfate, aminocaproic acid, tranexamic acid, kappa Crow, Carbazochrome Sodium Sulfonate (carbaxochrome sodium sulfanate), rutin sophorin and aurantiamarin.

Exceptionally except biologically active polypeptide group, the present invention can also containing other activating agents, as niacinamide, phytantriol, Farnesol, bisabolol and salicylic acid.It is expected that certain additional activating agents will synergistically play a role with bioactivity peptide composition Or the shelf-life that preparation will be enhanced.

The example for the Wound healing and bone regeneration medicine that can be used together with drug products of the present invention includes molten fibrin enzyme (such as fibre Lyase, deoxyribonuclease, streptokinase and dornase), the cutting tissue agent containing chlorinated lysozyme is big mould containing sulfuric acid celebrating Element, flamazine, bacitracin and sulfuric acid fradiomycin antimicrobial, contain Trafermin (trafermin), Bucladesine (dimension A gives birth to alcohol ester for sodium (bucladesine sodium), vitamin A acid dimension E ester (tretinoin tocoferil) (tocoretinate)), (Lai Ziyou ox laky blood blood mentions for Alprostadil Alfadex, solcoseryl (solcoseryl) Take object) and Crow sand aluminium granulation growth stimulator, the Operand containing white soft sweets, povidone iodine and iodine, and contain Bendazac (bendazac), the preparation of dimethylisopropyl azulenes (Kessazulen) and adrenaline as effective component.

In addition to α connection polypeptide, auxiliary material and other treatment medicine, gel can also include the sense of improvement topical preparation Other compounds of official's performance.

The example of this kind of compound includes essence, dyestuff and colorant；Complexing agent includes but is not limited to natrium adetate (EDTA), EGTA, CP94, citric acid；Preservative includes but is not limited to quaternary ammonium compound, such as benzalkonium chloride, benzethonium chloride, west three Bromo-amine (cetrimide), Dequalinium Chloride and Cetylpyridinium Chloride；Mercurial, such as phenylmercuric nitrate, phenyl mercuric acetate and thimerosal；Alcohol agent, example Such as, methaform, phenethyl alcohol and benzyl alcohol；Antibacterium ester, for example, metagin；With other antimicrobials such as chlorine oneself Fixed, chloreresol, benzoic acid and polymyxins.

In certain embodiments, it provides a kind of comprising at least one α connection polypeptide and hydroxyethylcellulose gel Topical preparation, wherein hydroxyethylcellulose gel make α connection polypeptide stabilize.In certain embodiments, hydroxyl second Base cellulose gel stabilizes α connection polypeptide, to can be detected at least after 5 DEG C store 3 months by analysis method 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% α connection polypeptide.In some embodiments, α connection polypeptide With about 0.0025% (w/w), about 0.005% (w/w), about 0.0075% (w/w), about 0.010% (w/w), about 0.015% (w/ W), about 0.020% (w/w), about 0.025% (w/w), about 0.030% (w/w), about 0.035% (w/w), about 0.040% (w/ W), about 0.045% (w/w), about 0.050% (w/w), about 0.055% (w/w), about 0.060% (w/w), about 0.065% (w/ W), about 0.070% (w/w), about 0.075% (w/w), about 0.080% (w/w), about 0.085% (w/w), about 0.090% (w/ W), about 0.095% (w/w), about 0.100% (w/w), about 0.150% (w/w), about 0.200% (w/w), about 0.250% (w/ W), about 0.500% (w/w), about 0.750% (w/w), about 1.00% (w/w), about 1.50% (w/w), about 2.00% (w/w), about 2.50% (w/w) or about 3.00% (w/w) or about 3.50% (w/w) or about 4.00% (w/w) or about 4.50% (w/w) or about 5.00% (w/w) or bigger concentration are present in preparation.In one embodiment, α connection polypeptide is with about 0.005% (w/w) concentration between about 1.00% (w/w) is present in preparation.

In other embodiments, drug products of the invention be the ACT peptide containing 0.0072% (w/w) (20 μM), The ACT peptide of 0.018% (w/w) (50 μM), the ACT peptide of 0.036% (w/w) (100 μM), 0.072% (w/w) (200 μM) ACT The Clear colourless gel of the ACT peptide of peptide or 0.36% (w/w) (1000 μM).ACT peptide can be dissolved in containing > 0% water, > 10% Water, > 20% water, > 30% water, > 40% water, > 50% water, > 60% water, > 70% water, > 80% water or > 90% water and 0.25% glue Solidifying agent (polymer), 0.55% gelling agent (polymer), 0.75% gelling agent (polymer), 1.00% gelling agent (polymer), 1.25% gelling agent (polymer), 1.50% gelling agent (polymer), 1.75% gelling agent (polymer), 2.00% gelling agent (polymer), 2.25% gelling agent (polymer) or 2.50% gelling agent (polymer) or 3.00% gelling agent (polymer) or 3.50% gelling agent (polymer) or 4.00% gelling agent (polymer) or 4.50% gelling agent (polymer) or 5.00% gelling In the semisolid dosage form of agent (polymer).

In certain embodiments, ACT peptide can be relative to about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5 or about pH 8 is well saved and is sufficiently buffered.In certain embodiments of topical preparation, qualitative and quantitative composition is That listed in table 7.

Table 7: the qualitative and quantitative composition of drug gel

ACT1 peptide sequence is listed in the following table 8, wherein Ahx refers to L-2- aminocaproic acid, also referred to as 6-aminocaprolc acid:

Table 8: peptide 328967 (ACT1) sequence (SEQ ID NO:91)

The general property of peptide 328967 is listed in the following table 9.

Table 9: the general physical characteristic of peptide 328967

Physical appearance	White is to off-white powder
		Molecular weight	3597.33±2.0amu
Counter ion	AcOH
		Solubility	During room temperature is water-soluble

In some aspects, drug products locally the auxiliary material used in product be selected from or it is following one or more:

Methylparaben

Propylben

Glycerine

Sodium dihydrogen phosphate

Disodium hydrogen phosphate

Propylene glycol

Natrium adetate (EDTA)

D-mannital

Hydroxyethyl cellulose, 250HHX

Purified water

In some embodiments, drug products part product includes peptide, D-mannital, hydroxyethyl cellulose and pure Change water.The product can also comprising it is following one or more: methyl hydroxybenzoate, Nipasol, glycerine, sodium dihydrogen phosphate, phosphorus Sour disodium hydrogen, propylene glycol and natrium adetate (EDTA).In other embodiments, hydroxyethyl cellulose is 250HHX.At it In his embodiment, peptide is α connection polypeptide.

In some embodiments, drug products part product includes concentration in about 0.001% (w/w) and about 0.5% (w/w) peptide of (for example, about 0.0072%, 0.018%, 0.036% or 0.072% (w/w)) between；Concentration is about 0.10% (w/w) between about 0.25% (w/w) (for example, about 0.17% (w/w)) methyl hydroxybenzoate；Concentration in about 0.01% (w/w) and The Nipasol of (for example, about 0.02% (w/w)) between about 0.03% (w/w)；Concentration is in about 1% (w/w) and about 10%w/w) Between glycerine (for example, about 5% (w/w))；Concentration is between about 0.1% (w/w) and about 0.5% (w/w) (for example, about 0.263% (w/w)) sodium dihydrogen phosphate；Concentration between about 0.02% and about 0.06% (for example, about 0.044% (w/w)) Disodium hydrogen phosphate；Concentration between about 1% (w/w) and about 5% (w/w) (for example, about 3% (w/w)) propylene glycol；Concentration is about EDTA between 0.01% and about 0.1% (for example, about 0.05% (w/w))；Concentration is in about 0.01% (w/w) and about 0.1% (w/ W) D-mannital of (for example, about 0.05% (w/w)) between；Concentration is between about 0.5% and about 2.5% (for example, about 1.25% (w/w)) hydroxyethyl cellulose and concentration about 0.1% to about 10% (for example, about 1%) purified water.Other In embodiment, peptide is α connection polypeptide.

It is tested and is derived by vitro and in vivo, these stabilizers and auxiliary material is incorporated in drug products, because they are stingless Swash property, dye and non-immunogenicity.Stability study shows that compared with Pluronic gel, ACT1 peptide is in gelling agent ethoxy It is more stable in cellulose 250HHX (1.25%).When storing three months for 5 DEG C, in the drug containing 1.25% hydroxyethyl cellulose The ACT peptide of interiors of products is only reduced to the 98% of labelled amount (that is, initial concentration), and at 25 DEG C of storage identical time limits, drop Down to the 84% of labelled amount.In one aspect of the invention, natrium adetate (EDTA) and mannitol are incorporated to drug products Inside, to provide stability to ACT1 peptide.In some embodiments of the present invention, mannitol presses 0.01% (w/w) extremely 1.6% (w/w), 0.01% (w/w) to 1.5% (w/w), 0.01% (w/w) to 1.4% (w/w), 0.01% (w/w) to 1.3% (w/w), 0.01% (w/w) to 1.2% (w/w), 0.01% (w/w) to 1.1% (w/w), 0.01% (w/w) to 1.0% (w/ W), 0.01% (w/w) to 0.9% (w/w), 0.01% (w/w) to 0.8% (w/w), 0.01% (w/w) to 0.7% (w/w), 0.01% (w/w) to 0.6% (w/w), 0.01% (w/w) to 0.5% (w/w), 0.01% (w/w) to 0.4% (w/w), 0.01% (w/w) to 0.3% (w/w), 0.01% (w/w) to 0.2% (w/w), 0.01% (w/w) to 0.1% (w/w) or 0.01% (w/w) to 0.0.05% (w/w) is present in preparation.In a particular embodiment, mannitol presses about 0.05% (w/ W) it is present in preparation.

It in other respects, include buffer in topical preparation to maintain pH to be within the scope of certain.Suitable buffer can To include but is not limited to acetate buffer, citrate buffer agent, phosphate buffer, lactate buffers, apple acid buffering Agent, succinic acid buffer agent, borate buffer, sodium hydroxide, potassium hydroxide and ammonium hydroxide.Also phosphate such as phosphorus can be used Acid dihydride sodium (NaH₂PO₄；Also referred to as sodium dihydrogen phosphate), disodium hydrogen phosphate (Na₂HPO₄；Also referred to as disodium hydrogen phosphate), di(2-ethylhexyl)phosphate Hydrogen potassium (KH₂PO₄), dipotassium hydrogen phosphate (K₂HPO₄) and its mixture.In certain embodiments, with citrate buffer agent phase Than phosphate buffer provides superior stability.In some aspects, it is found that the buffer capacity of 25mM is enough to drug products With.Buffer is needed to control pH gel systems and maintain the stability of peptide medicine.In certain embodiments, topical preparation PH range can be pH 2 to pH 12, pH 4 to pH 10 or pH 6 to pH 8.In some embodiments, optimum PH range It is about pH 5.0 to about pH 7.0.In other embodiments, the pH of topical preparation of the present invention be 4.0,4.5,5.0,5.5, 6.0,6.5,7.0,7.5 or 8.0.In other respects, it measures and provides better paraben esters in Aquo System for 3% propylene glycol Class solubilization.

In some embodiments, ACT peptide is incorporated to the preparation in hydroxyethyl cellulose and can be realized large-scale production and has The product of the feature of storage and stability requirement needed for making it be practically applicable to clinical treatment and meet.Although solidifying using Pluronic The ACT1 preparation of glue may need the about 2.5 hours relatively long incorporation time and only generate 50 grams of batches, but what is provided contains Having the preparation of hydroxyethyl cellulose allows ACT peptide significantly faster to mix gel and generate much bigger batch.For example, in office When using Pluronic F127 gel in portion's preparation, time-consuming is super to mix polymer after an hour, and needs for preparation to be placed in Incorporation is assisted in water bath.On the contrary, hydroxyethyl cellulose (for example, 250 HHX of HEC) is mixed in room temperature easily and 30 Aquation in minute.Therefore, can promote to be mass produced using hydroxyethyl cellulose.Using the production technology of Pluronic gel Cryostat may be needed, so that its viscosity enters required range and needs more than using the production technology of hydroxyethyl cellulose Energy.In addition, possible 5 DEG C of the condition of storage of finished product preparation in Pluronic gel is very thin.

It has been found that hydroxyethyl cellulose (HEC) is the suitable gelling agent and acceptable carriers of drug products of the present invention.In In some embodiments, gelling agent is hydroxyethyl cellulose (HEC), 250HHX.In certain embodiments, the percentage of HEC (w/w) within the scope of 1-5%.In other embodiments, the percentage (w/w) of HEC is 1.25%.It is pure in HEC manufacture The cellulose of change is reacted with sodium hydroxide to generate the alkali cellulose of swelling.The cellulose of alkali process more has chemical anti-than cellulose Ying Xing.By making alkali cellulose and reacting ethylene oxide, a series of hydroxyethyl ether cellulose is generated.In this reaction, hydroxyl Hydrogen atom in base cellulose is replaced by ethoxy, to assign water solubility to gel.Conceive in the present invention, can be used Single HEC ether, or the mixture of molecular weight and the discrepant HEC ether of structure can be used.The pharmaceutical purpose HEC of suitable grade is ripe It is knowing and described comprehensively in pharmacy literature.Suitable commercially available HEC brand includes but is not limited to Fuji HEC-HP；Fuji HEC-AG 15；NATRO-SOL 250HR；NATROSOL 250MH；NATROSOL 250G；CELLOSIZE QP 30000； TYLOSE H SERIES；NATROSOL 180L；NATROSOL 300H；TYLOSE P-X；NATROSOL 250M； CELLOSIZE WP 4400；CELLOSIZE UT 40；NATROSOL 250H4R；Tylose H 20P；NATROSOL LR； TYLOSE MHB；NATROSOL 250HHP；HERCULES N 100；CELLOSIZE WP 300；TYLOSE P-Z SERIES； NATROSOL 250H；TYLOSE PS-X；Cellobond HEC 400；CELLOSIZE QP；CELLOSIZE QP 1500； NATRO-SOL 250；Hydroxyethyl ether cellulose；HESPAN；TYLOSE MHB-Y；NATROSOL 240JR；Hydroxyethyl starch； CELLOSIZE WP；CELLOSIZE WP 300H；2- hydroxyethyl ether cellulose；BL 15；CELLOSIZE QP 4400； CELLOSIZE QP3；TYLOSE MB；CELLULOSE HYDROXY-ETHYLATE；CELLOSIZE WPO 9H17； CELLOSIZE 4400H16；Natrosol；Hydroxyethyl cellulose；Hydroxyethyl cellulose (HEC)；Hydroxyethyl cellulose 100H(celocell 100h)；TYLOSE MH-XP；NATROSOL 250HX；Natrosol；Daicel EP 500；HEC- Unicel；HEC (hydroxyethyl cellulose)；Cellosize；HEC-Al 5000；Fuji HEC-AL 15；HEC-Unicel QP 09L；Cellulose, ether, 2- hydroxyl ether；Unicel QP 52000H；HEC-QP 4400；SP 250 (cellulose)； Hetastarch；Cellulose, ether, 2- hydroxyl ether；Glutofix 600；FL 52；Fuji HEC-AX 15F；Tylose H 300P；HEC-Unicel QP 300H；Tylose H 300；Daicel SP 550；Daicel SE 600；Unicel QP 15000；HEC-QP 100MH；HEC-QP 9H；OETs；Daicel EP 850；H.E. cellulose；Cellobond 25T； Unicel QP 100MH；Tylose H 4000；SE 850K；Tylomer H 20；Daicel SE 850K；Tylose H 30000YP；Unicel QP 4400；SP 407；Tylose H 100000；Daicel SP 200；Culminal HEC 5000PR；Tylopur H 300；Daicel SP 750；Sanhec；BL 15 (cellulose derivative)；Unicel QP 300H； Tylomer H 200；J 164；Tylose H 10；Tylose H 20；AH 15；Daicel SP 600；Daicel SE 900；HEC-Unicel QP 4400H；AX 15；Daicel SP 800；Fuji HEC-AW 15F；HEC-SE 850；HEC-A 5-25CF；Metolose 90SEW；AW 15 (polysaccharide)；Cellobond HEC 5000；HEC-QP 100M；Cellobond HEC15A；Tylose H 15000YP2；Walocel HT 6.000 PFV；2- hydroxyethyl cellulose (Natrosol Type 250HRCS)；Fuji HEC-BL 20；Fuji HEC-SY 25F；Telhec；HEC-SP 200；HEC-AH 15；HEC- Unicel QP 30000H；Referring to；HEC 10A；Daicel SP 400；Admiral 3089FS；Fuji HEC-A 5000F； HEC-SP 400；Hydroxyethylmethylcellulose (HEMC)；Hydroxyethyl cellulose (HEC)；Hydroxyethyl starch (CAS number: 9004- 62-0)；Hydroxyethyl cellulose；"Natrosol"[Aqualon]；HEC；2- hydroxyethyl cellulose；NATROSOL 150L； TYLOSE MHB-YP；Hydroxyethyl ether cellulose；NATROSOL 250L；CELLOSIZE WP 400H；TYLOSE P；Cellulose, 2- hydroxyethyl ether；TYLOSE MH-K；NATROSOL 250HHR.

In other embodiments, drug products of the invention are packaged in 20mL bottle (USP I type, borosilicate are transparent Scintillation glass bottle, the more sealing taper urea screw lids of band) in.Use the methyl hydroxybenzoate and 0.02% (w/w) hydroxyl of 0.17% (w/w) The mixture of phenylpropyl alcohol ester is as preservative.

In some embodiments, the present invention includes a kind for the treatment of or preventing in the subject for facing radiation injury risk The method of this kind of damage, the method includes including at least one α connection polypeptide and hydroxy ethyl fiber to subject's application The topical preparation of plain gel, wherein hydroxyethylcellulose gel stabilizes α connection polypeptide.In certain embodiments In, drug products of the invention can be used to alleviate excessive cicatrization related to radiation injury.In these embodiments, originally The drug products of invention can when being exposed to radiation, be exposed to radiation after 1 hour, be exposed to radiation after 2 hours, sudden and violent Be exposed to radiation after 3 hours, be exposed to radiation after 4 hours, be exposed to radiation after 5 hours, be exposed to radiation after 6 hours, Be exposed to radiation after 7 hours, be exposed to radiation after 8 hours, be exposed to radiation after 9 hours, be exposed to radiation after 10 Hour, be exposed to radiation after 11 hours, be exposed to radiation after 12 hours, be exposed to radiation after 13 hours, be exposed to 14 hours after radiation, be exposed to radiation after 15 hours, be exposed to radiation after 16 hours, be exposed to radiation after 17 hours, Be exposed to radiation after 18 hours, be exposed to radiation after 19 hours, be exposed to radiation after 20 hours, after being exposed to radiation 21 hours, be exposed to radiation after 22 hours be exposed to radiation after 23 hours, be exposed to radiation after 24 hours, be exposed to 72 hours or hereafter apply 48 hours after radiation, after being exposed to radiation.

In other respects, drug products are manufactured with following steps:

Step 1: in the beaker of suitable size, addition propylene glycol, glycerine, methyl hydroxybenzoate and Nipasol.Use spiral Rotor (propeller) mixes until paraben esters are completely dissolved.

Step 2: in production containers, addition purified water (part i), EDTA, sodium dihydrogen phosphate, disodium hydrogen phosphate and D- Mannitol.With helical rotor mixing until obtaining clear solution.

Step 3: solution of the addition from step 1 to production containers.With purified water (part ii, be divided into about 3 it is equal Part) elution beaker and leacheate is returned container is added.Continue to be mixed until solution visual homogeneity with helical rotor.

Step 4: under homogenizing mixing, hydroxyethyl cellulose being added into the production containers from step 3.Mixing is until poly- It is fully dispersed to close object.

Step 5: in independent beaker, addition purified water (Section III part) connects polypeptide with α (for example, peptide 328967, ACT1 peptides).With stirring rod or helical rotor mixer mixing until peptide be completely dissolved and gel-forming until.

Step 6: in the case where helical rotor continuously mixes, drug solution of the addition from step 5 to production containers.Use purified water (Section IV part is divided into about 3 moieties), which elutes beaker and leacheate is returned, is added container.Mixing is until gel is equal Until even.

Production technological process is provided in Fig. 3.

The α connection protein polypeptide preparation that can be used in the present invention is detailed in U.S. Patent number 8,846,605, it is described Document is incorporated herein by reference.U.S. Patent number 7,786,074.7,888,319；8,357,668；8,809,257； 8,859,733；8,916,515；With 394,351；9,408,381；9,844,214；Complete content with 9,9,855,313 also leads to The mode for crossing reference is incorporated herein.

The composition of oral administration include pulvis or granule, aqueous medium or non-aqueous media suspension or solution, Capsule, small pouch or tablet.It may need thickener, corrigent, diluent, emulsifier, dispersing aid or binder.

Some compositions can potentially serve as it is pharmaceutically acceptable acid or base addition salts application, wherein by with it is inorganic Acid such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid and organic acid such as formic acid, acetic acid, propionic acid, ethyl alcohol Acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid reaction or by with inorganic base such as hydrogen-oxygen Change the reaction shape of sodium, ammonium hydroxide, potassium hydroxide and organic base such as mono-, di-, trialkylamine and arylamine and substituted ethanol amine At the addition salts.

It can empirically determine the effective dose and scheme of application composition, and make this kind of determination and be in this field skill Within the scope of art.Dosage range for applying composition is those of effect needed for being large enough to generate range, wherein influencing disease The symptom of disease.Dosage should not be so big, so that causing adverse side effect, such as undesired cross reaction, allergy. In general, whether dosage will include other in severity extent, administration method or therapeutic scheme in age, situation, gender and patient Drug and change, and can be determined by those skilled in the art.It, can be by personal doctor's tune in the case where any contraindication Save dosage.Dosage can change and can continue one day or a couple of days with daily administration one or more administration dosage.For given Classification drug products can find the guide of optimal dose in the literature.Dosage range depends primarily on applying for confectionery composition Add, the severity of symptom and its administration method.

For example, ACT peptide combinations can be by the agent down to 0.01%w/v in the application of such as research experimental tool Amount uses.Dosage can be in topical dermatological Wound healing and bone regeneration medicine down to 0.0002%w/v and may up to 20%w/v.It is aobvious It writes the composition of more concentration itself or can use or make in the application of such as cancer/tumor therapy with other compound combinations It is used immediately after acute tissue injury (such as CRI) for injecting for early stage concentration.Depending on the severity of damage, it to be used for intestines The outer administration method of stomach is for example intramuscular, intracerebral, intracardiac and intraspinal tube the recommended dose upper limit can be up to 1%w/v or v/v.This Upper dosage limit can be changed because of preparation, this depends on how such as polypeptide plays a role with its effect of promotion or with polypeptide cooperation Other pharmaceutical agent combinations.

For continuously delivering the polypeptide of offer, for example, process over time can be used when combining with intravenous drip, Improve the upper limit of the 0.01g/kg weight determined by doctor based on situation.In another example, local delivery is (for example, in skin In skin wound) the upper concentration of provided nucleic acid will be 5-10 μ g/cm²Wound, this depend on for example nucleic acid how with promotion It acts on or cooperates other pharmaceutical agent combinations to play a role with nucleic acid.This by with doctor based on improve situation determine frequency repeatedly It carries out.In another example, the upper limit of the provided nucleic acid of internal (for example, intramuscular, intracerebral, intracardiac and intraspinal tube) delivering is dense Degree will be 50-100 μ g/ml solution.Again, frequency will be determined by doctor based on situation is improved.

It also discloses the preoperative polypeptide with offer and pre-processes certain region.The concentration of polypeptide can be and 10-30% Pluronic gel or appoint can realize in the preoperative peptide infiltration in purpose part interior continue at least 3-6 hour time how it is this kind of 10-200 μM of carrier mixing.The adjusting of this preoperative state can improve the subsequent healing reaction of operation, including reduce inflammatory reaction.

Also disclose the subject that radioactive exposure risk is faced with the polypeptide therapeutic provided.In some aspects, this treatment Prevent subsequent irradiation from damaging, such as radiation injury of skin.In certain embodiments, the polypeptide provided is with about 10 μM to about 1000 μM Concentration application.In some embodiments, the polypeptide provided is with about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 110 μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160 μM, about 170 μM, about 180 μM, about 190 μM, about 200 μM, about 225 μM, about 250 μM, about 275 μM or about 300 μM or about 400 μM Or about 500 μM or about 600 μM or about 700 μM or about 800 μM or about 900 μM or about 1000 μM or about 1200 μM or about 1500 μM or About 2000 μM of concentration application.In some embodiments, the polypeptide provided is applied at least about 100 μm of concentration.At other In embodiment, the polypeptide provided is at least about 200 μm.Concentration application in a further embodiment, the polypeptide provided with At least about 1000 μm of concentration application.

In some embodiments, by composition systemic administration to subject.For example, in some embodiments, it will Composition passes through sucking application.In other embodiments, by composition parenteral administration to subject.For example, in some realities It applies in scheme, composition is applied to subject by intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection.In In some embodiments, by composition by about 0.01mg/kg to about 100mg/kg or about 0.1mg/kg to about 100mg/kg or About 0.5mg/kg to about 50mg/kg or about 1mg/kg to about 50mg/kg or about 2mg/kg to about 25mg/kg or about 5mg/kg Dose parenteral to about 25mg/kg is applied to subject.In some embodiments, by composition by about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/ Kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about The dosage of 50mg/kg, about 60mg/kg, about 70mg/kg, about 80mg/kg, about 90mg/kg or about 100mg/kg are applied to tested Person.

In certain embodiments, it will thus provide polypeptide be applied to subject by dosage regimen once a day.In its other party Face, it will thus provide polypeptide be applied to subject by dosage regimen once a week.In other respects, it will thus provide polypeptide by monthly one Secondary dosage regimen is applied to subject.

In certain embodiments, it will thus provide polypeptide be exposed to radiation before at least about 2 years, at least about 1 year, at least About 6 months, at least about 60 days, at least about 30 days, at least about 20 days, at least about 10 days, at least about 7 days, at least about 3 days, at least About 1 day, at least about 23 hours, at least about 22 hours, at least about 21 hours, at least about 20 hours, at least about 19 hours, at least about 18 hours, at least about 17 hours, at least about 16 hours, at least about 15 hours, at least about 14 hours, at least about 13 hours, at least About 12 hours, at least about 11 hours, at least about 10 hours, at least about 9 hours, at least about 8 hours, at least about 7 hours, at least about It is applied within 6 hours, at least about 5 hours, at least about 4 hours, at least about 3 hours, at least about 2 hours or at least about 1 hour tested Person.In other embodiments, it will thus provide polypeptide be exposed to radiation after at least about 1 hour, at least about 2 hours, at least about 3 Hour, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, At least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, it is at least about 15 small When, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 Hour, at least about 22 hours, at least about 23 hours, at least about 1 day, at least about 3 days, at least about 7 days, at least about 10 days, at least It is applied to subject within about 20 days, at least about 30 days, at least about 60 days, at least about 6 months, at least about 1 year or at least about 2 years.In Some aspects, it will thus provide polypeptide from be exposed to radiation after at least about 1 day at least about 30 days apply.In specific embodiment In, it will thus provide polypeptide be exposed to radiation after at least about 1 day apply.

In some embodiments, the tissue for being exposed to radiation includes but not limited to skin, heart, bone, brain, spinal cord, angle Film, retina and peripheral nerve.In some aspects, the tissue for being exposed to radiation is skin.In some aspects, average by measuring Skin scores, which are assessed, prevents radiation injury to the subject's offer polypeptide for facing the risk for being exposed to this kind of damage.Certain In embodiment, such as compared with the control, in the presence of the polypeptide of offer, mean skin scoring reduce at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least About 90%.In a particular embodiment, as compared with the control, in the presence of the polypeptide of offer, mean skin scoring is reduced at least About 30%.

As used herein, term " skin scores " refers to the means of any assessment skin and subcutaneous tissue injury's degree.For example, In some embodiments, skin scores are obtained using the scale of referred to as Kumar scale, provides the scale in table 11 herein Example.Therefore, as used herein, term " mean skin scoring " is using any assessment and evaluation skin and subcutaneous tissue injury Method scoring obtained average.

It in certain embodiments, include but not limited to radiation injury of skin because being exposed to radiogenic radiation injury (CRI；That is, because acute exposed to radiogenic skin and subcutaneous tissue injury), acute radiation syndrome (ARS；Acute big The severe disease occurred after dosage penetrability full-body exposure), radiation burn, radiation dermantitis, nervous system or brain radiation injury, Enteritis caused by radiation pneumonitis and radiation.In some aspects, radiation injury is radiation injury of skin.In other respects, radiation damage Wound is radiation burn.In addition in terms of other, radiation injury is radiation dermantitis.

In some embodiments, ARS includes any one following or more person: hematopoiesis syndrome or marrow syndrome；Stomach and intestine Trace integration sign；Neuro-vascular syndrome；Cardiovascular syndrome or central nervous system syndrome.These terms with it is known in the art Other terms such as ARS of hemopoietic system " targeting ", " ARS of targeting marrow ", " ARS of targeting gastrointestinal tract (GI) ", " the targeting heart Dirty ARS " etc. is used interchangeably.In some embodiments, the subject for being exposed to ionising radiation can have these syndromes Any of or any combination thereof.In some embodiments, syndrome is referred to as, for example,

In some embodiments, composition provided herein and method are used to treat with the tested of recombination radiation damage Person.In some embodiments, there is skin trauma, radiation burn, CRI, ARS, radiation with the tested of recombination radiation damage Property dermatitis, and/or it is relevant to ionising radiation is exposed to any other damage or syndrome.Therefore, in some embodiments, Present disclose provides the method and compositions that treatment is exposed to the various complication of ionising radiation, and may include locality with And the systemic composition that offer is provided.

In some embodiments, caused by radiation injury is because of dirty bomb attack.Dirty bomb is the easy of spreading harmful radioactive materials Quick-fried device.Being exposed to the high levels of radiation from dirty bomb may cause the symptom of acute radiation syndrome or radiation burn.Exposure In radiation causes the basal cell layer of damage skin and cause the inflammation at exposure position, erythema, decortication, acutely it is rubescent, water Bubble and ulcer.

In certain embodiments, the wound of radionuclide contamination and burn promote the systemic of radionuclide to take the photograph It takes, this makes local detoxify and identify to classify to complicate significantly.In other embodiments, radionuclide contamination also interference wound Mouth healing.Therefore, in some aspects, the polypeptide provided is prevented from the systemic intake radionuclide in radiation injury position.At it In terms of him, is formed, provided at radiation injury position by reducing bleeding, the severity of thermal burn damage and cicatricial tissue Polypeptide prevents or treats radiation injury.In addition in terms of other, the polypeptide provided promotes the regeneration at radiation injury position.

Material comprising composition presented herein (for example, polypeptide, nucleic acid or carrier) is also provided.It is used to for example, providing It treats wound and/or the material of radiation injury, wherein material is coated with ACT polypeptide.Non- limit for the material treated wound Property example processed includes bandage, exempts from seam adhesive tape (steri-strip), suture, nail (staple) or graft (for example, dermatoplasty Object).

For example, can in the provided polypeptide of 10-200 μM of concentration range soak-out material (for example, bandage, exempt from stitch glue Band (steri-strip), suture, nail or graft).It by material drying and can then be sealed in sterile chamber.Material It can be immersed at 4 DEG C in the liquid 10-30%pluronic gel for containing polypeptide by 10-200 μM of concentration.Material can then be made Material reaches approximate room temperature, so that gel polymerisation, leaves the gel coating for surrounding the polypeptide dipping of the material, the coating can be close It is enclosed in sterile chamber.Polypeptide can also be mixed crosslinkable aquogel system for example poly- (lactic-co-glycolic acid) (PLGA) or In polyurethane, material that the aquogel system then can be shaped as treating wound (for example, bandage, exempt to stitch adhesive tape, suture, Nail or graft).Therefore it provides composite hydrogel-fret peptide.

It is also disclosed in the Medical implant coated before implantation with provided polypeptide in subject.For example, this kind of implantation hand In art to be formed around implant because of cicatricial tissue formation common problem encountered is that shrinking pouch, person causes purpose excessive tissue hard Change, contraction and final shape are bad.It is implanting or this shape can be reduced or prevented using polypeptide of the invention thereon It is bad.The non-limitative example of Medical implant includes: limbs prosthese, breast prosthesis, penis implant, testis implant, people Work eye, facial implant, joint prosthesis, heart valve prosthesis, blood vessel prosthesis, dental prosthesis, facial prostheses, oblique dish valve (tilted disc valve), caged ball valve (caged ball valve), ear prosthese, ose implant, pacemaker, cochlear implantation prosthesis With skin substitute (for example, porcine xenograft valve/porcine skin, BIOBRANE, culture keratinocyte).

Provided herein is a kind of to promote the method to heal after tissue damage in subject, and the method includes applying to subject One or more compositions (for example, polypeptide, nucleic acid or carrier) provided herein in pharmaceutically acceptable carrier.In In some embodiments, wound is the result of radiation injury of skin.Also providing a kind of treat has tissue damage (for example, CRI) Subject method, one kind provided herein that the method includes being applied in pharmaceutically acceptable carrier to subject Or numerous compositions (for example, polypeptide, nucleic acid or carrier).

" promotion ", " promoting " and " promotion " refers to that activity, reaction, symptom, disease or other biological parameter increase.This can be with Including but not limited to activity, reaction, the starting of symptom and disease.This can also for example including such as with natural or control level phase Than activity, reaction, symptom and disease increase by 10%.Therefore, such as compared with natural or control level, increase can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or between it any amount increase.

A kind of method that " treatment " or " treatment " means influence for reducing disease or symptom.Treatment can also refer to a kind of reduction The basic cause of disease itself of disease or symptom rather than only reduce symptom method.Treatment can be any reduction away from natural horizontal And the symptom that can be but not limited to disease, symptom or disease or symptom melts completely.For example, if with same subject or When natural horizontal in control subject is compared, one or more symptoms of disease reduce 10% in the subject with disease, Then assert that promoting the disclosed method of wound healing is treatment.Therefore, such as compared with natural or control level, reduction be can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or between it any amount reduction.

In some respects, disclosure offer prevents ARS, CRI, recombination radiation damage, and/or spoke in specific group of patients Penetrating property dermatitis；Or composition and the side of lesion progress caused by alleviating because of ARS, CRI, recombination radiation damage, and/or radiation dermantitis Method, wherein PATIENT POPULATION is comprising being the subject for facing the risk of this damage.

As used herein, " subject " include but is not limited to the animal of nucleic acid, plant, bacterium, virus, parasitic body and Any other biology or entity.Subject can be vertebrate, more specifically mammal (for example, people, horse, pig, rabbit, Dog, sheep, goat, non-human primates, ox, cat, cavy or rodent), fish, bird or reptiles or amphibian animal.Subject can be Invertebrate, more specifically arthropod (for example, insect and shell-fish).The term does not indicate specific age or property Not.It is therefore intended that cover adult and newborn subject and fetus, it is no matter male or female.In some embodiments, patient Refer to the subject of disease or illness.In some embodiments, PATIENT POPULATION refers to be formed with disease or illness or face Specific, restriction the set of the subject of the risk of specified disease or illness.For example, in some embodiments, the disclosure mentions For by war and/or terrorism region occur PATIENT POPULATION (including soldier and/or common people) in treatment, prevent or Alleviate the method for the severity of radiation injury.In some embodiments, the disclosure is provided comprising receiving radiotherapy The method that the severity of radiation injury is treated, prevented or alleviated in the PATIENT POPULATION of oncological patients.Term " patient " includes Human experimenter and veterinary subject.

The method of offer can reduce cicatricial tissue after tissue damage and be formed in subject." cicatricial tissue " means in body Threadiness (fibrosis) connective tissue formed at damage or disease location in any tissue of body, by unordered collagen and The excess generation of other connective tissue proteins causes, and plays effect damaged in patchery tissue.Cicatricial tissue can substitute impaired Skin and lower section muscle, impaired cardiac muscle or internal organ (such as liver) affected areas.Fine and close and thick and solid, it is usually than surrounding Organize it is paler, the reason is that its blood supply is bad, although and it the tissue of damage is substituted in structure, it cannot execute forfeiture group The function of knitting.It is made of collagenous fibres, and person will often limit the normal elasticity in involved tissue.Therefore cicatricial tissue can limit The mobile range of muscle processed or when influencing lymphatic system or the circulatory system, prevents appropriate body fluid circulatory.Brain or spinal cord injury The formation of glial scar tissue is to restore one of the major obstacle of nervous function after damaging central nervous system afterwards.It can be according to It is reduced according to the population evaluation cicatricial tissue of damaged part intra cell type.For example, can be according to neuronal cell/astrocyte Ratio increases estimation glial scar tissue and reduces.It can be by measuring scar width or cicatricial tissue area measurement scar merely The reduction (Wilgus et al., 2003) that trace organizes the formation of.Furthermore, it is possible to make compared with normal tissue about callus The Histological assessment of internal structure complexity recovery situation

Other than fibrosed tissue is formed in subject after reduce tissue damage, provided composition and method can also Be used to treat relevant to fibrosed tissue forming process morbid state increase in subject illness (for example, psoriasis, it is Cutaneous and It is systemic mastocytosis, asthma, eczema, nasosinusitis, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, more The hardening of hair property, pulmonary fibrosis and cystic fibrosis).The reduction that fibrosed tissue is formed in subject can be sentenced by the clinic of doctor Disconnected measurement gives tissue in subject after doctor's assessment treatment and/or whether the normal configuration and function of organ is recaptured.Make For an example, for psoriasis, doctor is by the skin for assessing subject to determine the red covered by sheet white deposit Whether reduced swell skin patch.Certain form of psoriasis is with papule sample (pustular psoriasis) or burns (red skin) Appearance is characterized.In these cases, doctor will determine whether treatment already leads to reducing these symptoms.Biopsy is judged in doctor It, will in the case where clinically feasible and/or necessary subject tissue or organ or in the animal model of human diseases The histological structure for preparing the fragment of tissue of biopsy samples, and organizing will be by CIin Path and/trained histopathology Scholar's assessment, to determine that fibrosis is reduced with whether normal organization and functional rehabilitation have already appeared.It is also possible to type group Knit the area for learning prepared product qualitative assessment fibrosis relative to normal tissue.

The method of offer can restore normal organization mechanics performance such as tensile strength in subject after tissue damage. " tensile strength " refers to stress or the amount of strain required for destroy tissue or wound.

The tensile strength treated wound can with 60% of intact tissue in 3 months after the treatment, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.Therefore it provides organization mechanics performance in a kind of recovery subject (including increase Healing impainnent is to be close to or up to the tensile strength of normal intact tissue) method, the method includes applying to subject One or more compositions (for example, polypeptide, nucleic acid or carrier) provided herein in pharmaceutically acceptable carrier.

Critically important wound type includes to musculoskeletal structure/tissue and covering this in terms of tensile strength/elongation The damage of the skin of a little structures.For example, the stretching that the method provided can improve occlusion joint, bone, cartilage, tendon or ligament is strong Degree.The stretching that the method for offer can also improve skin under higher degree stress/strain (such as the skin of covering elbow, knee or foot) is strong Degree.It is that excessive epulosis in these regions causes to shrink and the hinge area that healed to the joint injury relevant most common problem that heals Domain does not extend.This is with serious beauty and psychological consequence.Help is adjusted and is mitigated the shape of this kind of cicatricial tissue by the attribute of peptide At leading to bigger joint mobility.

The method of offer can improve regeneration after tissue damage in subject." regeneration " means after injury or makees For normal body processes, update, regrowth or the recovery of body or body part, tissue or substance.With scarring on the contrary, tissue Regeneration is related to organized renewing to its prototype structure, function and physiological status.This is also referred to as tissue " complexity " herein.Recovery can To be part or complete, it is intended that such as compared with natural or control level, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% recovery or the recovery of any amount in-between.As an example, the skin injury the case where Under, regeneration can be related to the recovery of hair follicle, glandular structure, blood vessel, muscle or fat.In the case where cerebral injury, tissue is again Life can be related to neuron and maintain or restore.It as an example, in the case of skin, can be according to fibrous scar group The magnitude of the amount of normal skin is knitted/regenerated as ratio, assesses the regenerated improvement of tissue.As another example, Ke Yiji Number is just dispersed in structure regenerated, such as normalized to wound area total amount just in regenerated skin body of gland.

In one aspect, regeneration includes convening and breaking up to replace damaged cell for stem cell.As used herein, " stem cell " be exist between cell differentiated in tissue or organ or from external source introduce neoblast, for example, Embryonic stem cell, Adult bone marrow stem cell, the neoblast can be with self-renewing and differentiation is to generate tissue or device The main specialized cell of official.The main function of stem cell is to maintain and repair wherein there are these stem cells in living organism Tissue.Stem cell differentiation mean non-specialized cell (for example, stem cell) obtains whereby specialized cell (such as Skin Cell, nerve carefully Born of the same parents, heart cell, liver cell or muscle cell) feature process.As an example, in the case where skin injury, group The stem cell that knitting regeneration can be related to being present in epithelium is divided into hair follicle (Alonso and Fuchs, 2003).In the feelings of cerebral injury Under condition, regeneration can be related to stem cell and be divided into neuron.The method of offer can increase in subject after tissue damage Capable and experienced cell differentiation.Can by provide clinically-acceptable label endogenous cell or the gene means of transplanted cells or its His means and determine that marked stem cell is divided into and mix the frequency in normal organization, the stem cell for measuring enhancing is divided Change.As another example, it is known that certain structures such as hair follicle regeneration is from endogenous retinal stem cells after tissue damage.Thus, counting pair The hair follicle of tissue damage area normalization breaks up the stem cell for playing the role of being quantitatively evaluated enhancing.

The method of offer can reduce inflammation in subject." inflammation ", " inflammatory reaction " or " immune response " means to live Organize the reaction acted on injury, infection or irritation, it is characterised in that rubescent, warm, swelling, pain and function are lost, because of blood Flow increases with flowing in immunocyte and secretion and generates.Inflammation is reaction of the body to the infective micro-organisms invaded And causes the blood flow of affected area to increase, discharges the chemicals for attracting leucocyte, plasmaflux increases and removes residue Monocyte (or astrocyte in the case where brain) arrives.The anything for stimulating inflammatory reaction, which is referred to as, inflammatory.Therefore, In addition in reduction subject in response to tissue damage inflammation, it is thin with inflammatory that the composition and method provided can also be used to treatment The horizontal pathologic of born of the same parents increases relevant illness, and the illness is for example including asthma, eczema, nasosinusitis, atherosclerosis, class wind Wet arthritis, inflammatory bowel disease, Cutaneous and systemic mastocytosis, psoriasis and multiple sclerosis.With offer Polypeptide processing can also reduce itch, such as the wound itch to heal.In general, the histamine institute that itch is discharged by mast cell It causes.The polypeptide of offer can reduce mast cell degranulation and histamine release.Therefore it provides polypeptide can be used to treat and be related to The symptom of histamine release, the symptom includes but is not limited to itch, tickle, nasal sinus stimulation, allergic cough, blood-shot eye illness, asthma and Eczema.

Inflammation can be measured according to the reduction of inflammatory cell type (for example, monocyte or astrocyte) density to reduce. It can be according to the drop of inflammatory cell type (for example, neutrophil leucocyte, mast cell, basophilic granulocyte and monocyte) density Low measurement inflammation is reduced.Inflammation can be calculated by in-vivo measurement neutrophil activity reduce (Jones et al., 1994). In addition, the frequency or histamine levels of factor such as mast cell degranulation or the magnitude of active oxygen category level may be used as inflammation and subtract Few magnitude.Can also by with qRT-PCR check certain genes (such as interferon-' alpha ' ,-β and-γ, tumor necrosis factor-alpha, Interleukin-11 β, -2, -4, -5, -6, -8, -12, -18, -23, -27, CD4, CD28, CD80, CD86, MHCII and iNOS gene) Transcriptional level measures level of inflammation indirectly.Pro-inflammatory cytokine levels in tissue and/or body fluid (including blood plasma) to subject Measurement can measure inflammation reduction.It is worth noting that, ACT peptide mechanism of action can by inhibit Inflammatory cell emigration and/ Or inhibit proinflammatory chemicals (histamine, active oxygen classification) and pro-inflammatory cytokine such as interleukin (IL) -1, IL-6, IL-8 and Tumor necrosis factor (TNF).

The method of offer can inhibit in subject the proliferation of transformed cells.The cell of conversion means to grow out-of-control condition Tumour, cancer or the tumour cell of lower abnormal division and duplication.Therefore, inhibiting the cell Proliferation (that is, hyperplasia) of the conversion causes Growth is reduced and therefore the grade of malignancy of cancer reduces.The representative but non-limit of the cancer of disclosed composition and method treatment can be used Property inventory processed is as follows: glioma, lymthoma, B cell lymphoma, t cell lymphoma, mycosis fungoides, lymphogranulomatosis (Hodgkin's disease), myeloid leukemia, bladder cancer, the cancer of the brain, nervous system cancer, head and neck cancer, incidence squamous cell Cancer, kidney, lung cancer such as Small Cell Lung Cancer and non-small cell lung cancer, neuroblastoma, glioblastoma, oophoroma, pancreas Cancer, prostate cancer, cutaneum carcinoma, liver cancer, melanoma, mouth, larynx, larynx and squamous cell lung carcinoma, colon cancer, cervical carcinoma, cervical carcinoma, cream Gland cancer and epithelioma, kidney, genitourinary system carcinoma, lung cancer, the cancer of the esophagus, head and neck cancer, colorectal cancer, hemopoietic system cancer, carcinoma of testis, Colon cancer and the carcinoma of the rectum, carcinoma of prostate or cancer of pancreas.Therefore it provides method can be used to treat cancer in subject. For example, the method provided can be used to treat the glioma in subject.

Various kinds of cell proliferation marker and kit such as Ki67/MIB-1 immunostaining, titrtated thymidine or bromine can be passed through BrdU label index, DNA S- phase ratio, Proliferative type cells Expression of PCNA, potential doubling time and nucleosome composition area phase Close albumen (AgNORs) analysis, the measurement inhibition that transformed cells are proliferated.It is fixed to follow since the proliferation activity of tumour depends on The speed of ratio (the growth ratio) and cell cycle of the cell of ring can pass through equation [PA=Ki67 or MIB-1 scoring completely X AgNORs] measurement tumour practical proliferation activity (Pich et al., 2004).In another example, there is silk using simple It divides qualitative and quantitative target and measures proliferation in the cell colony of conversion, histopathologist skillfully assesses biopsy and cuts Piece.

The various mouse models for cancer research are developed.In the presence of the dedicated mouse mould for being directed to specific types of cancer Type.For example, bladder cancer, cervical carcinoma, carcinoma of endometrium, human primary gastrointestinal cancers, genitourinary system carcinoma, head and neck cancer, hemopoietic system cancer, kidney Cancer, lung cancer, breast cancers, melanoma, myeloma, nervous system cancer, carcinoma of mouth, oophoroma, cancer of pancreas, prostate cancer, meat Tumor, cutaneum carcinoma.It fully describes and uses these models.Can be studied in any one of these models polypeptide provided herein, The advantageous effect of nucleic acid or carrier.For example, cutaneum carcinoma mouse model can be readily used for showing.It can be using developing Human cancer tissue's heteroplastic transplantation model is cultivated cancer (Yoo, 2004) using no-special pathogen, homology Inbred Mouse (nude mice). Polypeptide, nucleic acid or carrier provided herein can be locally applied to for example Bioengineered material such as hollow-fibre membrane (Orlandini and Margaria.1983；Ming Chu et al., 1998) it and microfibre, slow release pearl, hypodermic needle, stays Set conduit, the Bioengineered material can be partially inserted into cancerous growths object or systemic administration (such as intravenous infusion, Intramuscular, intraperitoneal injection) to arrive at its target.This treatment can with self application or with other therapeutic compound such as chemotherapeutic Combination.

The method of offer can inhibit the transfer of transformed cells in subject." transfer " means cancer cell from initial site One or more positions elsewhere in body are traveled to, are usually propagated by blood vessel or lymphatic vessel.Transfer can resolve into Sequence of events.Firstly, cancer cell migration starts from tumour cell so as to leaving the process of primary growth site, substrate is often penetrated Film is simultaneously mobile to local vessel system.Interior infiltration describes cancer cell and enters vascular system and the process for being distributed to distal site.Outside Seep the process for referring to that cancer cell is gone out from vascular system.It is profoundly affected finally, cancer cell is proliferated in distal site by following aspect: office The availability of limitization growth factor, the influence of interstitial cell and surrounding extracellular matrix environment (so-called " soil ") and by thus The availability of the tumor vascularization the being growing nutrient provided and the factor that generate.Therefore it provides composition and method can To inhibit the transfer of cell described in subject by the migration (that is, metastatic migration) for inhibiting transformed cells.Tumour occurs It is cell cycle entanglement as a result, leading to uncontrolled cellular proliferation.Specific cells process-control cell cycle progress and checkpoint warp The mechanism imbalance that each interkinesis passes through.Under normal circumstances, these events are because there are conservative mechanism and molecule such as cell cycle Gene and its product and it is highly conserved.It can be according to this kind of cell cycle gene and product (such as cyclin, cell week Phase protein dependent kinase (Cdks), Cdk inhibit albumen (CKI) and extracellular factor (i.e. growth factor)) level, measure pair The inhibition of metastatic migration.It is counted using Laser Cell and the subversiveness technology of business software can be used for quantitative and evaluate cell week Phase process and cell growth.Use histogram measurement S- phase ratio (including ploidy value) and each index such as mitotic index of estimation It is invasive to evaluate tumour that enough information is provided with tumor doubling time index, for clinical worker.

As used herein, tissue damage can be scraped, be cut, lacerated wound, crush injury, compression trauma for example because of following generation (compression wound), stretch injury, bite, abrade, bullet wound, explosion damage, perforated body, stab, burn, Wind toast, day burn, chemical burn, operation wound, operation intervention, medicine intervention, cell, host rejection, medicine after tissue or organ transplant Object influence, drug side-effect, bedsore, radiation injury, beautifying skin wound, visceral injury, lysis (for example, asthma, cancer), Infection, infectious agent, growth course, maturation (for example, acne), hereditarian malformation, developmental anomaly, environmental toxin, change Ying Yuan, scalp injury, facial lesion, jaw portion damage Foot -injuries, toe damage, finger injury, bone injury, sexual organ damage, joint damage Wound, excretory organs damage, eye injury, corneal injury, muscle damage, adipose tissue damage, injury of lungs, Airway damage, hernia, anus Damage, hemorrhoid, injury of ear, retinal damage, skin injury, abdominal injury, arm damage, Leg-wheeled robot, injury gained in sports, back injury, Parturition injury, premature labor damage, toxicity is bitten, tendon injury, ligament injury, heart injury, heart valve damages, vascular system damages Wound, cartilage damage, lymphatic system damage, craniocerebral trauma, dislocation, esophageal perforation, fistula, nail damage, foreign matter, fracture, frostbite, hand Slap damage, Heat stress disorders, lacerated wound, neck injury, autotomy, shock, traumatic soft tissue injury, spinal cord injury, spinal cord Damage, ligament injury, cartilage damage, thorax damage, tooth damage, wound, nervous system injury, declines at strain, bacterial strain, tendon injury Always, aneurysm, stroke, injury to alimentary tract, infarct or ischemic injury.

In certain embodiments, caused by tissue damage is because of radiation injury.In certain embodiments, radiation injury includes And it is comprehensive to be not limited to radiation injury of skin (that is, because acute exposed to radiogenic skin and subcutaneous tissue injury), acute radiation Simulator sickness (that is, the severe disease occurred after acute high dose penetrability full-body exposure), radiation burn, radiation dermantitis, nerve Enteritis caused by system or brain radiation injury, radiation pneumonitis and radiation.In some aspects, radiation injury is radiation injury of skin. In other respects, radiation injury is radiation burn.In addition in terms of other, radiation injury is radiation dermantitis.In some implementations In scheme, caused by radiation injury of skin is attacked because of dirty bomb.

In some embodiments, radiation injury appear in include but not limited to, skin, heart, bone, brain, spinal cord, angle In the tissue of film, retina and peripheral nerve.In some aspects, radiation injury occurs on the skin.In certain embodiments, Tissue damage caused by radiating causes the basal cell layer of skin to damage.In some respects, the damage of skin base cell layer caused by radiating It is bad to lead to a variety of symptoms, including but not limited to the inflammation, erythema at exposure position, decortication, acutely it is rubescent, get blister and ulcer.

In certain embodiments, the wound of radionuclide contamination and burn promote the systemic of radionuclide to take the photograph It takes, this makes local detoxify and identify to classify to complicate significantly.In other embodiments, radionuclide contamination also interference wound Mouth healing.Therefore, in some aspects, by preventing at radiation injury position from wound or the systemic intake radioactive nucleus of burn site Element, the polypeptide provided prevent or treat radiation injury.In other respects, the severity by reducing bleeding, thermal burn damages It is formed with cicatricial tissue at radiation injury position, the polypeptide provided prevents or treat radiation injury.In addition it in terms of other, is providing Polypeptide promote radiation injury position at regeneration.

Specific manifestation peptide of the invention and/or other preparations will adjust cell migration and proliferation, thus in all tissues Reduce inflammation, accelerating wound healing, reparation, regeneration and the recovery for reducing scarring and finally promoting structure and function.Apply The healing of wound will be related to substantially reducing fibrosis after peptide, therefore reduce inside scarring and tissue damage in skin wound Fibrous patch promotes the structure and function of regeneration and recovery organization and organ.

The peptide and/or other preparations can be used to treat internal injury caused by external wounds.In certain embodiments In, external wounds are caused by radiation injury.In some embodiments, internal injury can by through being infected with burn and wound make Radioactive nucleotides at internal systemic contamination cause.Therefore, in some embodiments, method provided herein and combination Object provides to the treatment of Internal radiation injury, prevents and/or inhibit its progress.In some embodiments, Internal radiation injury is because of spoke Caused by the systemic contamination penetrated.Radiation systemic contamination can because directly apply radioactive nucleotides or radioactive nucleotides from Caused by the burn of contamination and wound enter.In some aspects, radiation injury includes but not limited to radiation injury of skin (that is, because of urgency Property is exposed to radiogenic skin and subcutaneous tissue injury), acute radiation syndrome is (that is, complete in acute high dose penetrability The severe disease that occurs after body irradiation), radiation burn, radiation dermantitis, nervous system or brain radiation injury, radiation pneumonitis and Enteritis caused by radiating.In some aspects, radiation injury is radiation injury of skin.In other respects, radiation injury is radiation burn. In addition in terms of other, radiation injury is radiation dermantitis.In some embodiments, radiation injury of skin is drawn by dirty bomb attack It rises.

Damage internal organ cause fiberization, lead to the forfeiture of structure and function in tract.In central nervous system (CNS) in, this injury response is mediated by astrocyte (fibroblast-like cell in CNS) and therefore then be will be called Astrocyte reaction.Embodiment of the present invention will alleviate this fibrosis/astrocyte reaction, therefore assist damaged tissues Repair and regenerate and organize the recovery with organ structure and function.Other embodiments of the invention include using the peptide and/ Or other preparations improve angiogenesis and improve blood vessel group by stimulation angiogenic factors (e.g., but being not limited to VEGF) Differentiation is knitted, thus improves the blood flow for arriving at site of tissue damage.Increase wound location blood supply stimulation by our medicine It will lead to scarring in external wounds and internal wounds to reduce and the reparation of tissue and organ and regeneration is promoted to improve.The present invention Additional embodiment include with stem cell and/or promote stem cell mobilization and/or regeneration cell and/or drug and/ Or other endogenous and/or clinical protocol combine application, the peptide and/or other preparations are used for the use of tissue and neomorph On the way.Stem cell will help regeneration and our medicine will be by including but is not limited to reduce fibrosis/astrocyte The process of cicatrization directly and/or indirectly promotes differentiation, thus restores normal institutional framework and function.

Composition and method promote the internal admissibility environment for the structure and function of tissue and organ regeneration and restoring It generates.The regenerative process assisted by peptide of the present invention include but is not limited to inside and outside damage after the following terms, tissue, The regeneration of organ or other body parts, the recovery from illness of function and recovery: vascular occlusion and ischaemic, cerebral apoplexy, myocardial infarction, Spinal cord injury, peripheral nerve injury, retinal damage, bone injury, is exposed to radiation and damages, damages or no cerebral lesion Then because of damage, operation, cancer, congenital and developmental anomaly but other histologic lesions caused by being not limited to the former and cause group Knit disease (the including but not limited to diabetes, bacillary, viral and prion correlation disease of structure and function progressive loss Disease, Alzheimer disease, Parkinson's disease, AID and other heredity for causing tissue/organ/body part structure and function to lose Decision type, environment decision type or idiopathic disease process).Further it is provided that peptide can with promote tissue and cytothesis medicine Object or other compounds are applied together, and the drug or other compounds include but is not limited to the trophic factors in various procedures, The process include but is not limited to brain, retina, spinal cord and peripheral neverous system regeneration (for example, NGF, FGF, neurotrophy because Son, neuregulin, Endothelin, GDNF, BDNF, BMP, TGF, Wnt).

Additional embodiment of the invention includes delivering the peptide and/or other preparations, the technology using following technology Such as, but not limited to whole rqikiwfqnrrmkwkk (Antennapedia) sequences and relevant cell internalizing carrier are (for example, Tat albumen Transduction structural domain, whole tat peptide, whole TAT fusion protein), virus gene delivery vehicle, DNA expression vector and may help In make peptide of the present invention itself or in conjunction with other medicaments in the case of reach any other delivering at tissue and/or cytosis position Method, other described medicaments include but is not limited to the co-factor (e.g., including but be limited to TAT-HA2) for assisting this delivering And/or stem cell, drug and the other preparations of accessory organ and institutional framework and function reparation, regeneration and recovery.

Unless otherwise specifically indicated, it otherwise can be used well known by persons skilled in the art for this specific reagent or change Close object any method, generate compositions disclosed herein and to implement disclosed method be required composition.

For example, provide nucleic acid can be used standard chemical synthetic method generate or can be used enzymatic method or it is any its He generates known method.Such methods can be from standard enzymic digestion, then nucleotide fragments separation (see, for example, Sambrook et al., Molecular Cloning:A Laboratory Manual, second edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) the 5th, the 6th chapter) to pure synthetic method, for example, making With Milligen or Beckman System 1Plus DNA synthesizer (for example, Milligen-Biosearch, Burlington, Mass.8700 type Fully automated synthesis instrument or ABI 380B type) cyano ethyl phosphorimide method.It can be used for producing The synthetic method of raw oligonucleotides also by Ikuta et al., Ann.Rev.Biochem.53:323-356 (1984) (phosphotriester and Phosphite ester-triester method) and Narang et al., Methods Enzymol., 65:610-620 (1980) (phosphotriester method) are retouched It states.Known method can be used, such as Nielsen et al., those of Bioconjug.Chem.5:3-7 (1994) description method is produced Raw egg white matter nucleic acid molecules.

A kind of method generating disclosed polypeptide (such as SEQ ID NO:2) is that two or more peptides or polypeptide are passed through egg White matter chemical technology links together.It is, for example, possible to use laboratory equipment is currently available that, Fmoc (9- fluorenes methoxy carbonyl is utilized Base) or Boc (tertbutyloxycarbonyl) chemistry, chemical synthesising peptide or polypeptide.(Applied Biosystems,Inc.,Foster City,Calif.).Those skilled in the art can be readily understood upon, for example, can by standard chemical react synthesis with openly The corresponding peptide of protein or polypeptide.For example, can be cut with synthetic peptide or polypeptide and not from its synthetic resin, and can close At peptide or protein matter other segments and then cut these segments from resin, thus make in other segments by functional blockade End group exposure.By peptide condensation reaction, both segments can be total in its carboxyl terminal and amino terminal respectively by peptide bond Valence engagement, to form protein or its segment.(Grant G A(1992)Synthetic Peptides:A User Guide.W.H.Freeman and Co.,N.Y.(1992)；Bodansky M and Trost B.,Ed.(1993) (the side that the document passes through reference Principles of Peptide Synthesis.Springer-Verlag Inc., NY Formula, which is incorporated herein, at least possesses material relevant to peptide synthesis).Alternatively, as described herein, independently in vivo synthetic peptide or Polypeptide.Once separation, can connect these independent peptides or polypeptide, to form peptide or its piece by similar peptide condensation reaction Section.

For example, by clone's or the connection of synthesis peptide fragment enzymatic allows to connect relatively short peptide fragment, it is biggish to generate Peptide fragment, polypeptide or whole protein structural domain (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).It is standby The native chemical connection of selection of land, synthetic peptide can be used to from synthetically constructing huge peptide or polypeptide compared with short peptide stretch.This side Method forms (Dawson et al. Synthesis of Proteins by Native Chemical by a two step chemicals reaction Ligation.Science,266:776-779(1994)).First step is unshielded synthetic peptide-thioesters and contains aminoterminal Chemo-selective reaction occurs for another unprotect peptide fragment of Cys residue, to generate the intermediate of thioesters connection as initially Covalent product.In the unconverted situation of reaction condition, this intermediate undergoes spontaneous, quick inner molecular reaction, even It connects site and forms natural peptide bond (Baggiolini M et al. (1992) FEBS Lett.307:97-101；Clark-Lewis I etc. People, J.Biol.Chem., 269:16075 (1994)；Clark-Lewis I et al., Biochemistry, 30:3128 (1991)； Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

Alternatively, it is coupled chemically unshielded peptide fragment, wherein as being connected chemically as a result, the key formed between peptide fragment It is non-natural (non-peptide) key (Schnolzer, M et al. Science, 256:221 (1992)).This technology has been used to synthesize egg The analog of white matter structural domain and a large amount of relatively pure protein (deLisle Milton R with complete bio activity C et al., Techniques in Protein Chemistry IV.Academic Press, New York, the 257-267 pages (1992))。

It discloses and prepares composition and the method for the intermediate that generates these compositions.It can be used for generating there are a variety of The method of these compositions such as synthesizes chemical method and standard molecular biology method.This is generated it is appreciated that particularly disclosing A little and composition disclosed in other method.The nucleic acid molecules generated by this method are disclosed, the method includes can grasp The nucleic acid for encoding polypeptide disclosed herein is connected as mode and controls the sequence of the expression of nucleic acid.Disclose by be disclosed herein Any nuclear transformation cell method caused by cell.Disclose the method institute by expressing any nucleic acid disclosed herein Any disclosed polypeptide generated.It discloses through the side with cell inside any nucleic acid molecules transfected animal disclosed herein Animal caused by method.It discloses by produced by the method with cell inside any nucleic acid molecules transfected animal disclosed herein Animal, wherein animal is mammal.It also discloses by thin with any nucleic acid molecules transfected animal inside disclosed herein Animal caused by the method for born of the same parents, wherein mammal is mouse, rat, rabbit, ox, sheep, pig or primate.It also discloses and passes through Animal caused by the method for any cell disclosed herein is added to animal.

Above-described material and other materials can be packaged in any suitable combination, as can be used for Implement or assist implementing the kit of disclosed method.If designing and the reagent constituents be transformed in given kit being so as to one It rises in disclosed method, this to be useful.For example, disclosing the kit for promoting wound healing, the kit packet One or more polypeptides, nucleic acid or the carrier provided herein being contained in pharmaceutically acceptable carrier.This kind of kit may be used also With include gel, bandage, Millipore adhesive tape, dosing Q- suction nozzle, spray, supporter, syrup, liquid, disposable pipe or Bag.Kit can also illustrate the security information with product or preparation containing Correct.Kit can be true containing such as doctor The fixed dosage information based on application and medication.

Disclosed method and composition is suitable for various fields, including but not limited to laboratory research tool.These systems Agent plays adjustment effect in several cell processes (such as cell Proliferation, cell migration).These preparations can be in the lab For in vitro and in vivo model system, with study various cell processes, Cycle Regulation, cell behavior, cell, organ or Organize the reaction etc. to test compound.Preparation itself can be supplied or with supply when other compound combinations or as kit The part of (as being used for Cell Proliferation Assay Kit).Kit can be changed containing preparation referred to herein itself or with other Close the preparation of object combination.This kit will include the explanation for being intended to promote experiment.Disclose from the disclosure it is apparent and/or Those skilled in the art will be understood that other purposes.

Embodiment