CN110494146A - With the method for Microrna treatment diabetic ulcer - Google Patents
With the method for Microrna treatment diabetic ulcer Download PDFInfo
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- CN110494146A CN110494146A CN201780089103.1A CN201780089103A CN110494146A CN 110494146 A CN110494146 A CN 110494146A CN 201780089103 A CN201780089103 A CN 201780089103A CN 110494146 A CN110494146 A CN 110494146A
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- microrna
- ulcer
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- diabetes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
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Abstract
The present invention provides application Microrna -210 or the methods of related compound treatment diabetic's ulcer.
Description
Technical field
The present invention relates to treat the new of diabetic's ulcer by giving Microrna (microRNA) or its carrier
The healing of diabetic ulcer is treated especially with respect to application Microrna -210 and its precursor and/or promoted to method.
Background technique
What is listed or discuss in the present specification is clearly that the file previously published is not considered as the one of the prior art
Part or common knowledge.
Diabetes (diabetes) refer to one group of disease that long-term blood glucose increases caused by internal glucose homeostasis is unbalance.Sugar
The Main Subtype of urine disease has type 1 diabetes (or insulin-dependent diabetes mellitus) and diabetes B (non-insulin-depending type glycosuria
Disease).
Diabetic is the people at highest risk that skin ulcer occurs.The region that the greatest risk of skin ulcer occurs is leg
Portion, especially foot.Chronic hyperglycemia often results in peripheral neuropathy, and then causes the harmony of foot and leg muscle group
It loses, which increase the mechanical stresses during walking, to increase shank or the upper ulcerated possibility of foot.And by week
Neuropathic influence is enclosed, the feeling of pain of four limbs can also reduce, to make patient lack the understanding for implementing preventive measure, further
A possibility that increasing ulcer formation.In other cases, the vascular diseases of advanced diabetes development may also lead to canker
The formation of change.
In addition to increase ulcer formation a possibility that other than, biological factor relevant to diabetes also to open wound just
Often healing damages.Diabetic ulcer, especially diabetic foot ulcers are one of the complication of diabetes most serious.Really
Real, compared with the complication of other diabetes, diabetic foot ulcers more easily lead to patient's hospitalization.In the U.S., diabetes
The main reason for being atraumatic lower limb amputation, is every year foot ulcers there are about 5% diabetic development, there is 1% sugar
Urine patient needs amputation.
Currently, the method that there is no effective treatment diabetic foot ulcer.Therefore, have to such treatment and do not meet significantly
Demand.
Summary of the invention
Inventor is it has now surprisingly been found that the application of mature Microrna -210 or its precursor can promote glycosuria
The ulcer healing of sick ulcers.
Treatment method
In the first aspect of the present invention, method that the present invention provides a kind of in diabetic treats ulcer.The party
Method includes one group of nucleic acid the precursor of the Microrna -210 containing mature Microrna -210 and maturation of effective therapeutic dose
Give the patient for needing this treatment.
It may also be said that the first aspect of the present invention is using the Microrna-including mature Microrna -210 and maturation
The ulcer of one group of exonuclease treatment diabetic of 210 precursor.
Or it may also be said that the first aspect of the present invention is using including the small of mature Microrna -210 and maturation
One group of nucleic acid of the precursor of RNA-210 manufactures the drug for treating the ulcer of diabetic.
Unless otherwise stated, the meaning of all technical and scientific terms used herein with it is of the art
The meaning that those of ordinary skill is generally understood is identical.
It unless the context indicates otherwise, otherwise should be by the preference of specified criteria of the invention, embodiment, feature or parameter
It is considered as with option and any and all priority and option of the every other aspect of the present invention, feature and parameter has been combined to carry out public affairs
It opens.
In order to avoid doubt, the precursor of the Microrna -210 of the mature Microrna -210 and maturation that define here, In
" nucleic acid of the invention " can be referred to as herein.
By reference to resource obtained by those skilled in the art, technical staff will identify conjunction as herein defined
Suitable mature Microrna and its precursor.For example, those skilled in the art can refer to ncbi database and miRBase data
The databases such as library (http://www.mirbase.org) include content intact therein herein.
For example, the skilled person will understand that, mature Microrna -210 (such as multiple including mouse and people
Species) comprising nucleotide sequence CUGGUGGUGUGACAGCGGCUGA (SEQ ID NO:1) or being made of the above nucleotide sequence.
In order to avoid feeling uncertain, Microrna -210 can refer to mature Microrna -210, pre-microRNA-210 and/
Or primary Microrna -210 (pri-microRNA-210), these terms will be understood by those skilled in the art that.
As described herein, nucleic acid of the invention includes mature Microrna -210 and its precursor.Suitable precursor includes life
Object precursor, such as mature Microrna -210 can be processed by one or more cell procedure of processings and/or added
Enter the nucleic acid that mature Microrna -210 is formed after cell.Such precursor includes pri-microRNA-210, pre-
The carrier of microRNA-210, the double-strand analogies of mature Microrna -210 and coding Microrna -210.In certain implementations
In scheme, nucleic acid is also possible to the double-strand analogies of Microrna -210.
It should be understood to the one skilled in the art that pri-microRNA-210 and pre-microRNA-210 are mature Micrornas -210
Upstream precursor.It is not wishing to be bound by theory, pri-microRNA-210 can be understood as referring to pass through rna plymerase ii
To the primary transcription sheet that the gene of coding Microrna -210 generates, and pre-microRNA-210 can be considered to mature core
The hairpin precursor of the mature Microrna -210 generated after ribonuclease T. II type enzyme Drosha processing pri-microRNA-210.
In its longer nucleotide sequence, pri-microRNA-210 and pre-microRNA-210 include mature Microrna-
210 nucleotide sequence (CUGUGCGUGUGACAGCGGCUGA (SEQ ID:1)).
The double-strand analogies of mature Microrna -210 include for simulating endogenous -210 function of sexually matured Microrna
Artificial double chain oligonucleotide.This dummy may be with Dicer ribonucleic acid enzymatic lysis pre-microRNA- in structure
The double-stranded RNA generated after 210 hair clips is similar, therefore can be loaded onto RNA induction silencing complex (RISC), is processed into
The Microrna -210 of single-stranded maturation.For example, miRIDIAN miR-210 analogies (miRIDIAN mmu-miR-210-3p；It produces
Article Number: C-310570-05, Dharmacon company) be exactly a suitable Microrna -210 double-strand analogies.
The skilled person will understand that carrier is used as expressing the recombinant nucleotide construct of polynucleotides in target cell.
Carrier generally include to encode the nucleic acid sequence of polynucleotides to be expressed and promote the polynucleotides the one kind effectively transcribed or
It is a variety of to adjust (or control) sequence.The embodiment of carrier includes plasmid, virus and non-virus carrier etc..
As described herein, the carrier for encoding Microrna -210 is understood to be comprising the more of coding Microrna -210
Nucleotide sequence ((such as the pri- of Microrna -210 of pri-microRNA-210, pre-microRNA-210 or maturation
)) and the nucleic acid molecules of optional one or more regulating and controlling sequence microRNA-210.In some instances, carrier is to include
Encode Microrna -210 polynucleotide sequence (pri-microRNA-210, pre-microRNA-210 or maturation it is small
RNA-210 (such as pri-microRNA-210)) and optional one or more regulating and controlling sequence DNA molecular.This field
Technical staff can easily prepare by known method this kind of carrier (for example, Green and Sambrook write by
" Molecular clone:A Laboratory published by Coldspring Harbour Laboratory Press (2012)
Manual, Fourth Edition " described in method, content intact comprising herein).
In some embodiments, mature Microrna -210 includes nucleotide sequence
CUGUGCGUGUGACAGCGGCUGA (SEQ ID NO:1) is made from it.In an alternate embodiment, the present invention can be used
Nucleic acid analog, the mature Microrna only generated after the Microrna -210 of maturation to be applied or its precursor use keeps
Adjust the ability of its target.Such analog includes the miRNA sequence that wherein one or more bases are substituted or lack.
In order to maintain the activity to said target mrna, it should preferentially guarantee the ' kind that the sequence complete complementary in mRNA is targeted with Microrna -210
Son ' sequence remains unchanged.In some embodiments, analog and maturation Microrna -210 at least about 75% it is same
One property or complementarity.For example, having at least about 80%, at least about 85%, at least about 90% or extremely with mature Microrna -210
Few about 99% identity or complementarity.In order to avoid feeling uncertain, every kind of possibility represents independent case study on implementation of the invention.
The skilled person will understand that introducing the treatment (or treatment similar to the illness) to particular condition in medical field
Meaning with its standard.Particularly, which, which can refer to, mitigates the tight of one or more clinical symptoms related with the illness
Principal characteristic.Such as merge in ulcer (i.e. diabetic ulcer) in diabetic, this may refer to promotion ulcer healing, for example, with do not control
The ulcer for the treatment of is compared, and the time range of ulcer healing (that is, skin or mucous membrane (such as skin) healing) is improved.This when
Between range can be in the range of three to 12 months (such as six to 12 months).In certain embodiments, such as it is refreshing
Through property diabetic ulcer, time range is generally three months, and for ischemic diabetic ulcer, time range can be six
Month.In addition to this, successfully treatment also be included within the scope of certain time (such as in one month, in such as three months, for example
In six months) realize the reduction of measurable ulcer size (such as width, depth, volume etc.).In some embodiments, it bursts
The size of ulcer can reduce at least 10%；For example, at least 20%, such as at least 50% (for example, at least 75%).
As used herein, patient refers to treated living body, including mammal (for example, people) patient.Therefore, in the present invention
First aspect specific embodiment in, the treatment is carried out in the mammal (such as people).
In substitution second aspect of the invention, a kind of method of promotion diabetic ulcer healing is provided.This method includes
The Microrna -210 for being selected from maturation of dose therapeutically effective and its nucleic acid of precursor (that is, of the invention as herein defined
Nucleic acid) for needing the patient of this treatment.
In another alternative aspect of the invention, provide with selected from the small of mature Microrna -210 and maturation
The nucleic acid (that is, as herein defined of the invention nucleic acid) of the precursor of RNA-210 is manufactured for promoting diabetic ulcer to be cured
The drug of conjunction.
In the third aspect of the present invention, a kind of method for promoting ulcer healing in the patient with diabetes is provided.
This method includes the core the precursor of the Microrna -210 selected from mature Microrna -210 and maturation of dose therapeutically effective
Acid (that is, nucleic acid of the invention as herein defined) is for needing the patient of this treatment.
In the alternative third aspect of the invention, provide using selected from the micro- of mature Microrna -210 and maturation
The nucleic acid (that is, nucleic acid of the invention as herein defined) of the precursor of tiny RNA -210 promotes the ulcer healing of diabetic.
In another optional third aspect of the invention, provide using selected from mature Microrna -210 and maturation
Microrna -210 precursor nucleic acid (that is, nucleic acid of the invention as defined herein) for manufacture promote diabetic
The drug of ulcer healing.
For the avoidance of doubt, mentioned in this article to promote diabetic ulcer healing and promote the ulcer healing of diabetic all
The ulcer healing rate that can be regarded as diabetic's (i.e. diabetic ulcer) improves.For example, compared with untreated ulcer, one
In fixed time range, ulcer healing (i.e. skin or mucous membrane (such as skin)) healing) patient's number increase or ulcer healing
Time improved.In certain embodiments, such as neurogenous diabetes ulcer, time range can be three
Month；For ischemic diabetic ulcer, time range be can be six months.In addition to this, promote diabetic ulcer healing
The ulcer size of measurement within the scope of certain time (such as in one month, in such as three months, in such as six months) can also be passed through
The reduction of (such as width, depth, volume etc.) is assessed.In certain embodiments, it after using exonuclease treatment of the invention, bursts
At least 50% (even, at least 75%) size of ulcer can reduce by least 10%, or at least 20%, or.
As used herein, term diabetes are understood to include type 1 diabetes, diabetes B and more uncommon Asia
Type, such as gestational diabetes mellitus, the adult onset diabetes (MODY) of young man, adult latency autoimmune diabetes
(LADA) and cystic fibrosis dependent diabetes (CFRD) etc..
In of the invention first to third aspect specific embodiment, to the referring to of diabetes (such as in diabetes
In patient) it will refer to 1 type or diabetes B.
The skilled person will understand that term ulcer can refer to due to skin or mucosal breaks and indolence and/or can not heal
Caused by open lesion on internal or external body surface.Ulcer can be formed on a series of body surfaces, such as
On oral cavity, gastrointestinal tract or skin.In of the invention first to third aspect specific embodiment, controlled with nucleic acid of the invention
The ulcer for the treatment of occurs on the skin, and this ulcer is properly termed as skin ulcer.
The skilled person will understand that skin ulcer can be located at any position on treated patient body.In the present invention
First into the specific embodiment of the third aspect, ulcer can be located on the leg or foot (such as sufficient) of treated patient.This
Class ulcer can be referred to as " ulcer of leg " and " ulcer of foot ".In of the invention first to third aspect specific embodiment,
The ulcer is not ischemic ulcer (that is, the ulcer is non-ischemic ulcer).
The ulcer of patient with diabetes can be referred to as diabetic ulcer.In certain embodiments, ulcer is sugar
Urinate sick ulcer.In of the invention first to third aspect more particular embodiment, the ulcer is diabetes ulcer of leg
Or diabetic foot ulcer (such as diabetic foot ulcer).
As used herein, term (diabetes) ulcer of leg can be regarded as referring to the ulcer formed on the leg of afflicted patient.
Such ulcer can on thigh (such as in groin and/or on thigh), on knee/around, and/or more specifically
On shank (such as on shin bone, shank and/or ankle/ankle around).Term (diabetes) ulcer of foot can be understood as referring to
The ulcer formed on the foot of afflicted patient.This ulcer may on ankle/around, on foot, on heel/around, on toe and/
Or it is more particularly formed on the bottom of foot (i.e. sole).In order to avoid feel uncertain, on the ankle of afflicted patient/around formation
Ulcer may belong to the definition of (diabetes) leg and ulcer of foot.
Diabetic ulcer (such as diabetic foot ulcer) can be divided into different hypotypes according to the potential cause of disease for causing it to fall ill.
The ulcer that patient in suffering limb with the peripheral nerve disease as caused by diabetes (preferably there is no vascular diseases) occurs
It is referred to alternatively as neurogenous diabetes ulcer, and in the patient's suffering limb for suffering from vascular diseases (such as vascular diseases caused by diabetes)
The ulcer of middle generation may be known as ischemic diabetic ulcer.Diabetic ulcer may also have the cause of disease of mixing, this ulcer
It is properly termed as neural ischemia ulcer.
It is not intending to be bound by theory, it is believed that since chronic hyperglycemia causes the hypoxemia in all diabetic ulcers anti-
It should be damaged, it is therefore not necessary to consider the cause of disease, the application of nucleic acid of the invention can be diabetic ulcer (such as diabetes skin ulcer)
Effective treatment is provided.
Therefore, in of the invention first to the third aspect certain embodiments, (diabetes) ulcer is (for example, glycosuria
Sick leg or ulcer of foot (for example, diabetic foot ulcer)) it is nervous glycosuria characteristic of disease skin ulcer (for example, neurogenous diabetes leg
Or ulcer of foot (for example, neurogenous diabetes ulcer of foot)).In certain embodiments, neurogenous diabetes ulcer (for example,
Neurogenous diabetes leg or ulcer of foot (such as neurogenous diabetes ulcer of foot)) it is not ischemic ulcer (that is, neurogenous diabetes
Property ulcer be Ischemic ulcer).
In the alternate embodiment in terms of of the invention first to third, diabetes skin ulcer is (for example, sugar
Urinate sick ulcer of leg or ulcer of foot (for example, diabetic foot ulcer)) it is ischemic Diabetic Skin Ulcer (for example, ischemic
Diabetes leg or ulcer of foot (for example, ischemic diabetic foot ulcer)).
In other alternate embodiments, diabetes skin ulcer is (for example, diabetes ulcer of leg or ulcer of foot are (for example, sugar
Urine foot disease ulcer)) be neural ischemia diabetes skin ulcer (for example, neural ischemia diabetes leg or ulcer of foot (for example,
Neural ischemia diabetic foot ulcer)).
In order to avoid feeling uncertain, above the of the invention first to the third aspect all preferred embodiments and case study on implementation described
It is equally suitable for the invention the 4th to the 7th aspect, as described below.
Other treatment method
It is not wishing to be bound by theory, it is believed that Microrna -210 can enhance in diabetic's body due to long-term chronic height
Anoxic response weakens caused by sugar, and then promotes the healing of diabetic ulcer.Fibroblast is responsible for generating fibr tissue
Such as the cell of collagen and extracellular matrix, under the conditions of high sugar, it is scarce that fibroblastic proliferation is affected with migration
A part that oxygen response is damaged.
According to the fourth aspect of the invention, a kind of method for enhancing the hypoxic effect in diabetic ulcer is provided.This
Method will include the nucleic acid of the precursor selected from mature Microrna -210 and mature Microrna -210 (that is, defined herein
Nucleic acid of the invention) for needing its patient.
The skilled person will understand that enhancing hypoxic effect, which can refer under low oxygen conditions, to be improved by hypoxic inducing factor-1 (HIF-
1) the one or more processes mediated.Such as angiogenesis, cell Proliferation, cell survival, cell migration, RBC acceptor garland rate and thin
Born of the same parents break up (such as fibroblast proliferation, migration and differentiation), endothelial precursor cell recruitment and antibacterial ability.
According to the fifth aspect of the invention, provide one kind in diabetic ulcer restore Fibroblast Function (such as
The proliferation and migration and/or synthesis of extracellular matrix and collagen) method.This method will include selected from the small of maturation
The nucleic acid (that is, nucleic acid of the invention as defined herein) of the precursor of RNA-210 and the Microrna -210 of maturation is for needing it
Patient.
According to the sixth aspect of the invention, it provides one kind and promotes extracellular base in dermal tissue in diabetic ulcer
The method that matter and/or collagen are formed.This method will include the Microrna-selected from mature Microrna -210 and maturation
The nucleic acid (that is, nucleic acid of the invention as defined herein) of 210 precursor is used to need its patient.
The skilled person will understand that the critical process of the final stage (such as diabetic ulcer healing) of ulcer healing is wound
Re-epithelialization.
According to the seventh aspect of the invention, the method for promoting diabetic ulcer re-epithelialization is provided.This method will include
The nucleic acid of the precursor of Microrna -210 selected from mature Microrna -210 and maturation is (that is, as defined herein of the invention
Nucleic acid) it is used to need its patient.
Drug composition
As described herein, the nucleic acid in the present invention can be used as drug.This nucleic acid, which both can be used alone, to be added
It is used into known pharmaceutical composition/preparation.
It will be understood by those skilled in the art that the nucleic acid being defined herein can be administered in the form of pharmaceutical composition, controlling
It can promote to heal in treatment, enhance hypoxia response, restore Fibroblast Function, promote extracellular matrix and/or collagen
Formation and promote re-epithelialization as defined herein (for example, first to the 7th aspect of the invention).
Therefore, in the eighth aspect of the present invention, a kind of pharmaceutical composition is provided.It includes selected from mature Microrna-
210 and mature Microrna -210 precursor nucleic acid (that is, nucleic acid of the invention as defined herein) and any one or it is more
The pharmaceutically acceptable adjuvant of kind, diluent and/or carrier, for treatment as defined herein and method (for example, being used for this hair
The first to the 7th bright aspect).
In substitution eighth aspect of the invention, (therapeutically effective amount) pharmaceutical composition method as herein defined is provided
(for example, for first to the 7th aspect of the invention).This pharmaceutical composition include selected from mature Microrna -210 and at
The nucleic acid (that is, nucleic acid of the invention as defined herein) of the precursor of ripe Microrna -210 and any one or more pharmacy
Upper acceptable excipient, for needing its patient.
Pharmaceutically acceptable excipient includes medium, adjuvant, carrier, diluent, pH adjusting agent and buffer, tonicity tune
Save agent, stabilizer, wetting agent etc..
The skilled person will understand that nucleic acid of the invention can be with topical application (i.e. in ulcer spot).Therefore, technical staff will
Understand, nucleic acid described in of the invention first to eighth aspect and composition can locally be made with pharmaceutically acceptable dosage form
With.Suitable administration route may include being directly administered in skin (skin) or mucomembranous surface, subcutaneous, intradermal or cutaneous penetration (example
Such as pass through microneedle injection or transdermal patch).
Suitable dosage form includes such as liposome system (such as suspension) and emulsion, and can be liquid, lotion, cream
The form of cream, ointment and/or aerosol.Liquid preparation can be water base or oil base, can also prepare, make in dry powder form
It is redissolved with preceding suitable solvent.
According to some embodiments, nucleic acid of the invention can be shifted/is introduced into cell using a variety of delivery systems, such as
It is encapsulated in liposome, target liposomes, dendron shape polyglycereol amine nano-carrier, nano particle, particle, microcapsules, electroporation, core
Transfection, based on ultrasound, recombinant cell based on laser, that Microrna -210 can be expressed, receptor mediated endocytosis,
Building Microrna -210 as a part of viral vectors or other carriers, can not be killed in course of infection cell progress
The viral vectors of the viral vectors of duplication, not reproducible, injection can generate the cell of the viral vectors of nucleic acid of the present invention, injection
Transfection of polynucleotides, electroporation, calcium phosphate mediation etc. or any other method well known by persons skilled in the art.
The skilled person will understand that nucleic acid of the invention and composition comprising it can be with different dosage (for example, as above
The preparation) it gives, those skilled in the art can readily determine that suitable dosage.It can be according to the nucleic acid to be delivered
The dosage and frequency of the pharmacological property selection administration of (i.e. naked RNA, carrier, used delivery of particles etc.).In some implementations
In scheme, nucleic acid of the invention (combining individually or with other reagents) can be with each use/treatment about 0.01mg daily to about
Dosage between 10mg is applied.
For example, can be in each administration/treatment about 0.01mg between about 8mg, it can also administration/treatment about 0.01mg every time
To between about 2mg, can also every time administration/treatment about 0.05mg between about 4mg.Sometimes can also every time administration/treatment about
0.05mg to about 2mg, more particularly can also every time administration/treatment about 0.08mg to about 2mg (for example, every time administration/treatment about
0.08mg to about 1mg).Or the amount of use/treatment can be in about 0.5mg between about 9mg every time.In some exemplary implementations
In scheme, nucleic acid of the invention can be prepared in salting liquid (such as PBS).In some embodiments, disclosed herein dose
Where amount in office can be applied under case, such as daily 1 to 5 time, weekly 1 to 10 time or monthly 1 to 15 time, wherein such application can
In identical or different time progress.This application can have different time intervals and can be in identical or different in one day
Between apply.
As used herein, term " about " is used to refer to series of values, be understood to be described value ±
10% variation, especially ± 5% can also refer to the variation of ± 2% (such as ± 1%).
Nucleic acid of the invention, which can permit, to be delivered at least one copy to each treated cell (i.e. mature is small
RNA-210 any dosage).Higher dosage can produce improved effect, and for example, at least 5 copies or at least ten are copied
Shellfish, for example, at least 100 copies or at least 500 copies (for example, at least 1000 copies).
In order to avoid feeling uncertain, technical staff (for example, doctor) should be able to determine practical dose of the individuation of most suitable patient
Amount.The dosage may with administration route, patient to be treated state of an illness type and severity and treated patient
Ethnic group, the age, weight, gender, renal function, liver function and patient treatment after special reaction and change.Above-mentioned dosage is
Illustratively.Certainly, on rare occasion, higher or lower dosage range should be set, and this is also in the scope of the present invention
Within.
Nucleic acid of the present invention and method have the advantage, that compared with existing treatment method, either according to above-mentioned
The method or other modes use, they may it is more effective, toxicity is lower, action time is longer, potency is higher, generates
Side effect is less, and/or with other useful pharmacological properties.Especially such method has the further advantage that they are aobvious
The ulcer healing for improving diabetic ulcer patient is write, so as to improve its prognosis.
Detailed description of the invention
Fig. 1 illustrates influence of the miR-210 analogies to diabetic mice wound healing.Compared with negative control, miR-
210 analogies are obviously improved wound healing rate.
Fig. 2 illustrates influence of the miR-210 analogies compared with the control to human fibroblasts migration.It is often sugared in normal oxygen
(N5), its effect is explored under hypoxemia normal sugared (H5) and high sugared (H30) state of hypoxemia.Under the high sugared state of hypoxemia, and compare
Group is compared, and miR-210 analogies can significantly improve the migration of fibrocyte.
Fig. 3 illustrates influence of the miR-210 analogies compared with the control to human fibroblasts proliferation.It is often sugared in normal oxygen
(N5), its effect is explored under normal oxygen high sugared (N30), hypoxemia normal sugared (H5) and high sugared (H30) state of hypoxemia.In hypoxemia height
Under sugared state, compared with the control group, miR-210 analogies can significantly improve the migration of fibrocyte.
Example
The present invention is explained by following embodiment, but is never limited to this.
Embodiment 1: influence of -210 analogies of locally injecting Microrna to diabetic mice wound healing
(miR-210) analogies of intracutaneous injection Microrna -210 and negative control
Use the 12 week old male diabetes of C57BI/6J background (Charles Rivers, Jackson Labs, USA)
(db/db) mouse.All experimental groups are age, weight, blood glucose matching.3% isoflurane (Abbott) is by mouse anesthesia.With scraping
The hair of knife and depilatory cream removal back of mice.After alcohol washes skin, punched in dorsal midline two sides using 6mm biopsy
Device manufactures wound.Before mouse is alleviated by (0 03 mg kg of body weights) of buprenorphine injections twice daily after wound
Two days any potential pains, and single cage raising is placed in after wound.
After wound, (miRIDIAN mmu-miR-210-3p (is based on 0.125nmol miRIDIAN miR-210 analogies
Sequence MIMAT0000658 (CUGUGCGUGUGACAGCGGCUGA (SEQ ID NO:1)；Catalog number (Cat.No.): C-310570-05；
Dharmacon)) or negative control (miRIDIAN cel-miR-67 (and be based on sequence MIMAT0000039
(UCACAACCUCCUAGAAAGAGUAGA (SEQ ID NO:2)；Cat#:CN-001000-01；Dharmacon) along edge of wound
Intracutaneous injection.Duplicate injection in 6th day after wound.Wound and complete skin are taken out from injection site after 12 days after wound.
Wound healing evaluation
It takes pictures every other day to wound on the wound same day and before putting to death.The circular reference of known area is placed on
It takes pictures simultaneously on the side of injured mouse.Wound area and known circle are calculated using ImageJ software (National Institutes of Health)
The ratio of shape area of reference, and it is expressed as the percentage of initial wound area.It will be seen from figure 1 that compared with negative control,
MiR-210 analogies improve the rate of wound healing.
Embodiment 2: in normal sugar under height sugar, hypoxemia and normal oxygen environment, -210 analogies of Microrna move fibroblast
The influence of shifting
Transfection of (miR-210) analogies of Microrna -210 in HDF cell:
At first day, fibroblasts of adult human dermis (HDF) cell is inoculated with the density of 80-90%.Second day, with 1,10
With 20nM miR-210 analogies (miRIDIAN mmu-miR-210-3p；Catalog number (Cat.No.): C-310570-05；Dharmacon is public
Department) or negative control (miRIDIAN cel-miR-67；Catalog number (Cat.No.): CN-001000) use RNAiMAX (Thermo Fisher
Scientific company, catalog number (Cat.No.): 13778075) transfection agents are transfected.Cell migration assay is carried out after 48 hours.
Cell migration assay
HDF cell is inoculated with and transfected in 24 well culture plates.After 48 hours, scrape, by cell in normal oxygen (21%O2) or
Hypoxemia (1%O2) often sugared (5mM) or high sugared (30mM) is exposed under environment.Cell is in serum starvation state, and it is mould that mitogen is added
(catalog number (Cat.No.): 10107409001) 10ug/mL, Roche company inhibit cell Proliferation to element-C.It is cell-free with 0 hour and 24 hours
Region area computation migration.As shown in Fig. 2, -210 analogies of Microrna significantly increase into fibre under the conditions of hypoxemia high sugar
Tie up cell migration.
Embodiment 3: under normal and high sugar and hypoxemia and normoxic condition, -210 analogies of Microrna are at fiber finer
The influence of born of the same parents' proliferation
Transfection Microrna -210 (miR-210) analogies in HDF cell
The first day density with 80%-90% is inoculated with fibroblasts of adult human dermis (HDF) cell.Second day, 1,10 and
20nM miR-210 analogies (miRIDIAN μ-miR-210-3p；Catalog number (Cat.No.): C-310570-05；Dharmacon company) or yin
Property control (miRIDIAN cel-miR-67；Catalog number (Cat.No.): CN-001000-01；Dharmacon company) it is transfected and is tried with RNAiMAX
(catalog number (Cat.No.): 13778075) ThermoFisher Scientific company transfects for agent.Analysis of cell proliferation is carried out after 48 hours.
Analysis of cell proliferation
HDF cell is 24 hours hungry, it is exposed in normal oxygen (21%O2) or hypoxemia (1%O2) and it is often sugared (5mM) or high
Sugared (30mM) 24 hours.Cell Proliferation is measured using BrdU cell proliferation ELISA kit (Abcam company, ab-126556).
From figure 3, it can be seen that -210 analogies of Microrna significantly increase fibroblastic proliferation under the conditions of hypoxemia and high sugar.
Claims (33)
1. a kind of method for treating diabetic's ulcer, which is characterized in that the method includes by the choosing for the treatment of effective dose
The patient for needing this treatment is given from the nucleic acid of the precursor of the Microrna -210 of mature Microrna -210 and maturation.
2. a kind of nucleic acid of the precursor of the Microrna -210 selected from mature Microrna -210 and maturation, for treating diabetes
The skin ulcer of patient.
3. the nucleic acid preparation treatment diabetes of the precursor of Microrna -210 of the application selected from mature Microrna -210 and maturation
The drug of patient skin ulcer.
4. the method described in any one of claims 1 to 3 uses the small of the Microrna -210 and maturation for containing maturation
The compound of the precursor of RNA-210, which is characterized in that the precursor of mature Microrna -210 is selected from pri-microRNA-210,
Pre-microRNA-210, the double-strand analogies of mature Microrna -210 or the carrier for encoding Microrna -210.
5. the use of method described in any one of Claims 1-4 or compound, which is characterized in that the nucleic acid is into
The double-strand analogies of ripe Microrna -210.
6. the use of method described in any one of claims 1 to 5 or compound, which is characterized in that the diabetes are 1
Type or diabetes B.
7. the use of method described in any one of claims 1 to 6 or compound, which is characterized in that the ulcer is skin
Skin ulcer.
8. the use of method described in any one of claims 1 to 7 or compound, which is characterized in that the ulcer is sugar
Urinate characteristic of disease leg or ulcer of foot.
9. the use of method described in any item of the claim 1 to 8 or compound, which is characterized in that the skin ulcer
It is diabetic foot ulcer.
10. a kind of method for promoting diabetic ulcer healing, this method includes effective to needing the patient of this treatment to apply
The nucleic acid of the precursor of the Microrna -210 selected from mature Microrna -210 and maturation of therapeutic dose.
11. a kind of Microrna -210 selected from mature Microrna -210 and maturation that can be used for that diabetic ulcer is promoted to heal
Precursor nucleic acid.
12. the nucleic acid preparation of precursor of the application selected from mature Microrna -210 and mature Microrna -210 promotes diabetes
The drug of ulcer healing.
13. the use of method described in any one of claim 10 to 12 or compound, which is characterized in that the maturation
The precursor of Microrna -210 is selected from pri-microRNA-210, pre-microRNA-210, pair of mature Microrna -210
Chain simulative or the carrier for encoding Microrna -210.
14. the use of method described in any one of claim 10 to 13 or compound, which is characterized in that the nucleic acid is
The double-strand analogies of mature Microrna -210.
15. the use of method described in any one of claim 10 to 14 or compound, which is characterized in that the diabetes
Ulcers are with 1 type or diabetes B.
16. the use of method described in any one of claim 10 to 15 or compound, which is characterized in that the diabetes
Ulcer is skin ulcer.
17. the use of method described in any one of claim 10 to 16 or compound, which is characterized in that the diabetes are burst
Ulcer is diabetes leg or ulcer of foot.
18. the use of method described in any one of claim 10 to 17 or compound, which is characterized in that the diabetes are burst
Ulcer is diabetic foot ulcer.
19. a kind of method for promoting ulcer healing in the patient with diabetes comprising to the patient for needing this treatment
Apply the nucleic acid of the precursor selected from mature Microrna -210 and mature Microrna -210 of dose therapeutically effective.
20. a kind of nucleic acid is selected from the precursor of mature Microrna -210 and mature Microrna -210, for promoting diabetes
The ulcer healing of patient.
21. the nucleic acid preparation of precursor of the application selected from mature Microrna -210 and mature Microrna -210 promotes with sugar
Urinate the drug of patient's ulcer healing of disease.
22. the use of method described in any one of claim 19 to 21 or compound, which is characterized in that mature is small
The precursor of RNA-210 is selected from pri-microRNA-210, pre-microRNA-210, the double-strand mould of mature Microrna -210
Quasi- object or the carrier for encoding Microrna -210.
23. the use of method described in any one of claim 19 to 22 or compound, which is characterized in that the nucleic acid is
The double-strand analogies of mature Microrna -210.
24. the use of method described in any one of claim 19 to 23 or compound, which is characterized in that the diabetes
It is 1 type or diabetes B.
25. the use of method described in any one of claim 19 to 24 or compound, which is characterized in that the ulcer is
Skin ulcer.
26. the use of method described in any one of claim 19 to 25 or compound, which is characterized in that the skin is burst
Ulcer is diabetes leg or ulcer of foot.
27. the use of method described in any one of claim 19 to 26 or compound, which is characterized in that the skin is burst
Ulcer is diabetic foot ulcer.
28. a kind of method for the anoxic response for enhancing diabetic ulcer comprising apply to patient with this need selected from maturation
Microrna -210 and mature Microrna -210 precursor nucleic acid.
29. a kind of method for restoring Fibroblast Function in diabetic ulcer comprising applied to patient with this need
The nucleic acid of precursor selected from mature Microrna -210 and mature Microrna -210.
30. a kind of promote the method that extracellular matrix and/or collagen are formed in dermal tissue in diabetic ulcer comprising
The nucleic acid of the precursor selected from mature Microrna -210 and mature Microrna -210 is applied to patient with this need.
31. a kind of method for promoting diabetic ulcer re-epithelialization comprising apply to patient with this need selected from maturation
The nucleic acid of the precursor of Microrna -210 and mature Microrna -210.
32. a kind of pharmaceutical composition, it includes Microrna -210 mature defined in any one of preceding claims and at
It the nucleic acid of the precursor of ripe Microrna -210 and chooses any one kind of them or a variety of pharmaceutically acceptable excipient, it is aforementioned to be applied to
Any one claim.
33. the method for any one of preceding claims, which is characterized in that the nucleic acid is applied in the form of pharmaceutical composition,
Described pharmaceutical composition includes the small of Microrna -210 and maturation mature defined in any one of preceding claims
It the nucleic acid of the precursor of RNA-210 and chooses any one kind of them or a variety of pharmaceutically acceptable excipient.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/GB2017/050923 WO2018178608A1 (en) | 2017-03-31 | 2017-03-31 | Methods of treatment of diabetic ulcers with microrna |
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| CN110494146B CN110494146B (en) | 2024-08-27 |
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| WO (1) | WO2018178608A1 (en) |
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| WO2024157253A1 (en) * | 2023-01-24 | 2024-08-02 | Rambam Med-Tech Ltd. | Polynucleotides and use of same for wound healing |
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| WO2012020308A2 (en) * | 2010-08-13 | 2012-02-16 | The University Court Of The University Of Glasgow | Cellular and molecular therapies |
| US10260067B2 (en) * | 2014-10-01 | 2019-04-16 | The Brigham And Women's Hospital, Inc. | Enhancing dermal wound healing by downregulating microRNA-26a |
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2017
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- 2017-03-31 WO PCT/GB2017/050923 patent/WO2018178608A1/en active Application Filing
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| WO2018178608A1 (en) | 2018-10-04 |
| CN110494146B (en) | 2024-08-27 |
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