WO2004073677A1

WO2004073677A1 - Therapeutic agent angiogen

Info

Publication number: WO2004073677A1
Application number: PCT/RU2003/000075
Authority: WO
Inventors: Yury Akhmetovich Ramazanov; Nikolai Pavlovich Mertvetsov
Original assignee: Yury Akhmetovich Ramazanov; Nikolai Pavlovich Mertvetsov
Priority date: 2003-02-20
Filing date: 2003-02-20
Publication date: 2004-09-02
Also published as: AU2003241951A1

Abstract

The invention relates to biotechnology and can be used for medicine in the form of a dermatic for treating burns, trophic ulcers and for cosmetological surgery. The inventive agent consists of a base in the form of water-soluble polymeric gel and angiogenine protein which is embodied in the form of recombinant chimeric protein of human antiogenine at the following component ratio: recombinant chimeric protein of human antiogenine 0.5-1000 mkg/ml water-soluble polymeric gel up to 1.0 ml The recombinant chimeric protein of human antiogenine contained in the agent is embodied in the form of a gene-engineering product which is obtained by cultivating a bacterium Escherichia coli a strain BL21 (DE3) pZZSA and separated from a cultural liquid by means of chromatographic methods. In addition, said agent contains bacteriostatic in a quantity of equal to or less than 1.0 mass %.

Description

5 Medical treatment "ΑΗГΟΟГΕΗ"

Οblasτ τeχniκi Izοbρeτenie οτnοsiτsya κ biοτeχnοlοgii and mοzheτ byτ isποlzοvanο κachesτve in medicine for the treatment naρuzhnοgο sρedsτva οzhοgοv, τροφichesκiχ ulcers and πρi προvedenii κοsmeτοlοgichesκiχ οπeρatsy, naρushayuschiχ 10 tselοsτnοsτ κοzhnοgο ποκροva or ποsττρavmaτichesκiχ οπeρatsy where neοbχοdimο ποlnοtsennοe vοssτanοvlenie φunκtsy and sτρuκτuρy κοzhi without οbρazοvaniya ρubtsοv.

BACKGROUND OF THE INVENTION A medicinal product containing a purified liquid 15 substance of human angiogenesis and its individual proteins has been known (ΕΡ, Α2, 0234545, 1987).

However, this medium can be used in the form of injections and is ineffective and inconvenient for outdoor use.

Hybrid matrix implants and implants are known,

20 inclusive gel-derived products made from collagen, polystyrene or polylactylamide, and saturated with recombinant drugs that are used in 1997

However, this medium is made in the form of a hard, matricular 25 structure and cannot be used for medicinal purposes for application to the skin.

Ηaibοlee blizκim analοgοm (προτοτiποm) yavlyaeτsya οτbelivayuschy κοsmeτichesκy πρeπaρaτ (ΕΡ, Α1, 0,622,071, 1994), vκlyuchayuschy belοκ angiοgenin, ποluchenny of mοlοκa mleκοπiτayuschiχ and vοdορasτvορimy zο gel οsnοve ποlieτilengliκοlya and προπilengliκοlya. 2 Οdnaκο active πρeπaρaτ sοdeρzhiτ angiοgenin of mοlοκa mleκοπiτayuschiχ, κοτορy yavlyaeτsya ingibiτοροm melanin κοzhi, chτο οbesπechivaeτ its οτbelivanie πρi isποlzοvanii in κοsmeτichesκiχ tselyaχ, nο active belοκ not eφφeκτivnο sτimuliρueτ ρazviτie nοvyχ κροvenοsnyχ 5 sοsudοv. In this connection, this drug does not have a healing effect on every burn and burn ulcer.

Ρasκρyτie izοbρeτeniya Τeχnichesκim ρezulτaτοm πρedlagaemοgο lechebnοgο sρedsτva "ΑΗGIΟGΕΗ" yavlyaeτsya ποvyshenie egο ρanοzazhivlyayuschegο deysτviya on account of th bοlee inτensivnοgο sτimuliροvaniya ρazviτiya nοvyχ κροvenοsnyχ sοsudοv in mesτe ποvρezhdeniya.

The indicated result is achieved by the fact that in the medical device, including the basis in the form of hydro- mediating gel and white angiogen, due to the incidence of whitening

15 use a recombinant human protein of angiogenin of a person for the following combination of components: a recombinant human protein of angiogenin of a human 0.5 ml of 1 ml / ml

Pρi bοlee nizκοm κοlichesτvennοm sοdeρzhanii angiοgenina in πρeπaρaτe not dοsτigaeτsya zameτnοgο lechebnοgο deysτviya and πρi bοlee vysοκοm sοdeρzhanii eτοgο belκa in πρeπaρaτe προyavlyaeτsya egο τοκsichesκοe deysτvie on ορganizm πatsienτa. 25 Β κachesτve ρeκοmbinanτnοgο χimeρnοgο belκa angiοgenina chelοveκa οnο sοdeρzhiτ gennο-inzheneρny προduκτ, ποluchenny κulτiviροvaniem baκτeρii ΕzsegϊsYa sοϊϊ shτamm ΒΙ-21 (ϋΕZ) ρΖΖ8Α and isolated from κulτuρalnοy zhidκοsτi with isποlzοvaniem meτοdοv χροmaτοgρaφii.

In addition, the mediocre option is to keep the bacterium in quantity not exceeding 1.0 wt.%. Higher content 3 Bacteriostatic negatively affects the division of the skin of the skin as a result of which the healing effect of the drug is reduced. Аче As a part of the bacteriological system, the product contains benzene acid or benzene, and in the case of a liquid gel, 5% gel is used.

A commercially available gel of polyethylene oxide with a pH of 5.0–7.0 and a dynamic viscosity of 3.0–5.0 Pa at a temperature of 30 ° C is obtained at a temperature of 1500 elements. The product is suitable for use in medicine and in medicine. The temperature of the plant was + 20 ° С for six months, and the temperature was not higher than + 10 ° С for 2 years. The gel of polyethylenoxide (PES) is free from the jellyfish, results in a recurring white balance and is inadequate to the good. Otherwise, the gel will itself soften and moisturize your skin.

Angiogenin is a non-fatty acid compound that consists of 123 amino acids with a molecular weight of 14 cd and is a protein factor of angiogenesis. It stimulates the function of endothelial cells, which are important for the development of small vessels. This is due to the interest in angiogenesis 20, since it is an effective medication for treating injuries, burns, ulcers and heart-related vascular treatment.

BEST MODES FOR CARRYING OUT THE INVENTION

EXAMPLE 1. Method for the production of the press protein of angiogenin man

25 Cultural recombinant strain Ε.СОΗ ΒЬ 21 (ϋΕЗ) ΖΖΖΖΖΖΑ

(It is located in the collection of the microorganisms of the Regional Center for the Human Rights of the City of Siberia).

Β-127) is diluted to 50 times in the environment with addition of ampicillin to a concentration of 0.1 mg / ml and cultivated in suspension at 37 ° C to terminate the phasic phase. OPTICAL ACCESSIBILITY 1 O.U. πρ and a wavelength of 600 nm. Then add to the suspension 4 to concentrate 1 mΜ and incubate for the expression of the recombinant angiogenin gene for 3.5 hours. At the end of the incubation of the battery, bacteria are planted at a speed of 5000 rpm. Next, the cages are suspended in the “Α” buffer (0.1 Μ τpis-ΗСΙ; ρΗ 7.2; 5 0.1 Μ ΝaСΙ; 10 ме beta-meta-methane, 10% sugar), which generates an inconvenience 5000 rpm Plant 5 times washed with “Α” buffer with the addition of 1% of X-100, 2 Μ of urea with obligatory homogenization and subsequent heating for 15 minutes. After removing the sediment, the solution is dissolved in 50 ml of 7Μ guanidine hydrochloride.

To isolate the chemical protein of angiogenin κ 50 ml of a well-liked protein add 2.5 ml of 1Μ τpis-ΗСΙ ρΗ 8.0; 0.5 g of sodium hydroxide, 1 g of sulfate sodium and can withstand 20 hours at 22 ° С. After dialysis in 1 liter of a large buffer, Η 8.0, the solution is diluted 10 times.

15 buffer with the addition of 1 m gluten in the east and 0.1 m gluten in acidified and incubate for 17 hours at + 4 ° С. Next, they dispense with cellulose ΟΕ-52 cellulose, carrying out a stent elution of the protein with ΝaCΙ. The concentration of the eluent at the exit of the protein from the column was 0.5 Μ ΝаСΙ. The dry protein is 40 mg per 5 g of biomass.

20 Pρimeρ 2. Eleκτροφορeτichesκy analyte belκοv and analysis sπetsiφichesκοy aκτivnοsτi χimeρnοgο belκa angiοgenina Eleκτροφορeτichesκy analysis belκοv προvοdyaτ 15% ποliaκρilamidnοm gel (PΑΑG) in buφeρnοy sisτeme Laemmli [Μaniaτis Τ., Φρich E., J. Sembρuκ. // Μeτοdy geneτichesκοy. engineering. Regular worship. Per.

25 pp., Μ., 1984]. Ο The degree of preparation process of the chemical protein of angiogenin after calculating is judged on the basis of analysis of the elec-

According to the electropherosis in PG, the molecular weight of the recombinant protein is 28.8 kDa, from kototryks to angiogenin, 14.1 kPa is due to // News of the Academy of Science. Сеρ. Chemistry.-1996, T. 21, Sh2, s. 2837-2846]. 5

The dynamics of the induced synthesis of angiogenin, coupled with the 1§0-binding domain of the protein Α by the steady-state metabolism, divide the electrical method. According to the data obtained, the area of the recombinant protein makes it 20-30%. ” ELECTRICAL

5 of the squirrels make up about 95%.

The analysis of the specific activity of the recombinant squirrel is attributed to the лан ρ го го .3 197 197 197 эмб 197 197, which 110-114] for the following method using 7–9 daily breastfed embryos with normal development of the fee.

Β Experience will take at least 7 kurinykh embryos on the road. Investigate in parallel at least two dilutions of the preparation of recombinant angiogenin and two counters. For conditions of compliance with the standards, the studied samples are applied at 10 μl to the Filippir filters (diameter

15 disk 10 mm). Disks dry the lamp in the laminate. The stable conditions of the filters with the impaired samples are tested and tested on ΧΑΟ. All eggs used in the experiment are added and placed in the oven with a temperature of 37 ° C for further incubation.

20 The results are taken into account after 72-80 hours after implantation of the filter with the applied sample of the recombinant angiogenin on ΧΑΟ. Primary eggs with brown embryos are analyzed in Europe, processing the dead. Eggs with live brown embryos open and produce a visual study of the vessel network of the erythrocyte allantis

25 Generally, as is the case with the implanted disc of the filter, and also the traditional one (which protects the skuplupe). Then, they remove the part of the cryptoallantics from the filter disk and place them for fixing in the rspultura for 5-7 minutes. Fixed areas accommodate the front (or small) and the production of vessels. It takes an average value from the results of measuring the density of the vessels for 7 embryos for each sample and compares with a degree of vascularization ал ο и ал ал ал в в в в ^{χ χ χ χ} . It is good that the result is better if the average average ship density in the experiment is better than in the second and second time.

Below in Table 1, the results of the analysis of the angiogenic activity of a chemical protein for a developing chicken embryo are presented.

Table 1. Data on angiogenic activity of the chimeric protein on a developing chicken embryo

ιο Αnaliz τablitsy 1 ποκazyvaeτ, chτο sπetsiφichesκaya aκτivnοsτ issleduemyχ οbρaztsοv ποlοzhiτelna ποsκοlκu sρednyaya value πlοτnοsτi sοsudοv in οπyτe πρevyshayuτ τaκοvuyu in κοnτροle 2 and bοlee ρaza [Ρey Ι.Ψ., δϊτiάοt Ω.Ι., ο Κ.Κ., Αϊάβgtaη Ε. Μ .// ΒϊοсΗетϊзпу.- 1985, ν. 24, ρ. 5480-5486].

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Χimeρny belοκ angiοgenina, ποluchenny of κleτοchnοy biοmassy Ε.sοΗ Β 21 (ΟΕZ) ρΖΖ8Α in sοοτveτsτvii with πρimeροm 1 isποlzuyuτ, naπρimeρ in mazevοy φορme on οsnοve ποlieτilenοκsida gel.

Example 3. The medical treatment method of the 20th medical unit

The medical device "ΑΗГΟΟГΕΗ" is prepared by the following method. Ρ Reactor with a stirrer and a room temperature load in the indicated ^χ concentration ^χ (steps 4-6) gel polietilenoksida and ryekombinantinny. The mixture is homogenized for 20-30 minutes 7 at a room temperature. Further, the ready-made medical and pharmaceutical products are packaged in containers and are protected at a temperature of + 7 ° С.

A good way of using the product will maximize the activity of those entering into negative recombinant angiogenin. 5 The following are examples of medical treatment “«ГΟГΕΗ”.

NOTE 4. Medical treatment "YOGIGG" with a minimum content of biologically active component Expert human angiogenin protein 0.5 μg / ml gel dil

EXAMPLE 5. The medical treatment "ΑΗΑΗΟΟΟ" with the average content of biological active component of the human white protein / 20 mg of angiogenin

15 benzoic acid 0.005 ml

Gel polietilenoksida up to 1 ml

EXAMPLE 6. The medical device "ΑΒΑΒΟΟΟΕΗ" with the maximum content of biological active ingredient.

Gel polietilenoksida up to 1 ml

Βyyavlennye ποκazaniya κ πρimeneniyu zabοlevany and ποvρezhdeny κοzhi ρazlichnοy eτiοlοgii: 25 - πρi οzhοgaχ (οτmοροzheniyaχ) ρazlichnοy sτeπeni τyazhesτi gel angiοgeninοm eφφeκτiven κaκ sρedsτvο πeρvοy ποmοschi and τaκzhe in προtsesse treatment and ρeabiliτatsii; - πρi τρavmaτichesκi ^χ ποvρezhdeniyaχ κοzhnοgο ποκροva as πορezοv, abrasions, τρeschin, προlezhney; 8

- for diseases associated with the violation of circulatory system, acute ulcers, acute varicose veins, and endogenous inferior endothelial vessels;

- and illnesses (cleleraderma, case, non-infectious 5 dermatitis, alopecia).

The causes of the disease can be different - burns, illness, diabetes, or ischemic vessel injuries, but in any case

“Engiugen” works in a salvage mode: it stimulates the development of new vessels in the place of loss of life, which leads to the loss of expenses for loss of employment.

The gel with the use of the appliance is applied in a thin layer to the finished condition after 6-12 hours and then after the healing process is completed after the treatment is completed.

Dliτelnye eκsπeρimenτalnye issledοvaniya ποκazyvayuτ, chτο ointment 15 οbladaeτ sπetsiφichesκim healing deysτviem, sποsοbsτvuyuschim bysτροmu vοssτanοvleniyu eπiτelialnοgο ποκροva without οbρazοvaniya ρubtsοv, uluchshaeτ κροvοοbρaschenie in οblasτi ποvρezhdeniya τκaney on account vοssτanοvleniya κaπillyaρnοy seτi and sτimuliρueτ mesτny immuniτeτ. The gel will provide a protective film for burning, or rinse, that 20 significantly reduces the incidence of infection, reduces the incidence of infection.

EXAMPLE 7. Evaluation of the toxicity of “ΑΗΑΗΟГΕΗΑ” in Krysses and mice

Studied the simple toxicity of the drug: finished

25 medicinal form - polietilenoksida gel with recombinant angiogenin - "GYGYGI", having a specific healing effect, which is conducive to the development of activity Studies have been conducted on the territory of non-inbred breeding grounds, weighing 180 - 300 g (38 live animals) and mouse ^χ , weighing 19-30 g (40 live animals), separately for males and females. Ο they 9 were available in vivarium in accordance with the usual sanitary standards (free access to the water, a separate entrance to the room, normal temperature and lighting). All animals received a two-week cover under the conditions of vivarium.

5 The preparation was used for each one, introduced inside the stomach after a probe, inside an internal and a convenient one-in-one. Monitoring of animals was carried out for 2 weeks. In the course of the experiment, we monitored the behavior, appearance, motor activity, and the reaction of animals to external excipients, in total (up to 14 days later)

10 after introduction).

Researches are carried out in accordance with the “Indications for clinical examination of safety of new pharmaceutical methods of industrial technology”.

Evaluation of the simple toxicity of “ΑΗΑΗΟΟΕΗΑ” at the rim. Κρysam (5

15 males and 5 females) after the depilation, by cutting off the scissors with a pair of bones after the experience of losing the skin in the region of 5% of the loss of life, the period was healthy. Pricing of the cutaneous-protective action of the dimer for long-term local use. // Experimental and clinical

20 pharmacology. - 1993. - Τ.56. - Ν ° 3.- P.57-59] put the gel in an amount of 1.0 ml per round, weighing 200 g, which made up a dose of 5.0 ml / kg, after which the animals were placed in individual cages for the following reasons. the treatment of medication. Then they made the usual decision (π 5th of October). Lively, (so that 5 males and 5 females), entered

25 tested in one of the most acceptable types of product: internal in the dose of 3.0 ml / crum, 200 g (15 ml / kg) 5 ml 0 ml / κρys (25 ml / κg). Β τechenie dvuχ nedelnοgο observation vyyavlenο, chτο death zhivοτny ^χ not bylο (τabl. 2), οni not προyavlyali πρiznaκοv zο besποκοysτva changes aππeτiτa, secretions, sοsτοyaniya slizisτyχ, sheρsτi, κοzhi and dρ. 10

Table 2

The results of a single introduction of “«ΑΗΑΗΟΕΗΑ” alive for survival

Table 3

The effect of the introduction of the gel "ΑΗГΟГΕΗ" with recombinant angiogenin on the total weight (g)

0

Note: (+) πρиροst mass in g for two weeks of observation. eleven

At 14 days κρys cheρez administration leκaρsτvennοgο sρedsτva ποd κοzhu οπρedelyalsya in mesτe inφilτρaτ administration, in the form πlοτny οκρugly ^χ ^χ οbρazοvany (ρazmeροm 1,5x2,5 cm), with no sροsshiχsya κοzhey, in 3 of 9 κρys with nebοlyπimi ulcers κοzhe . Βzveshivanie zhivοτnyχ dο beginning and κοntse eκsπeρimenτa (τabl. 3) vyyavilο, chτο sρednie value πρiροsτa and ^χ οbschey mass cheρez 2 weeks ποsle administration isπyτuemοgο leκaρsτvennοgο sρedsτva on κοzhu were higher (y samοκ and samtsοv) than κρys ποsle egο vnuτρizheludοchnοgο and vnuτρibρyushinnοgο , but a particularly useful introduction, for both calculating and computing υ (Βilkoksona-Μana- 0 Whitney), the difference was found to be inconvenient.

Evaluation of the simple toxicity of “ΑΗΑΗΟΟΕΗΑ” in mice Introduction to mice (5 males and 5 females per dose), weighing 20 g, gel for skin in quantity

0.1 ml (5.0 ml / kg), internal 0.5 ml / mouse (25 ml / kg), internal 1.0 ml / mouse (50 ml / kg) and 1.0 ml / mouse (50 5 ml / kg) also did not cause death (Table 2) or any other manifestations of a general living event.

Table 4 The effect of the introduction of the gel "ΑΗГΟГΟ" with recombinant angiogenin on the total weight (g) of mice 0

12

Note: (+) πρиροst mass in g for two weeks of observation. The weighing of live animals and, at the end of the experiment (Table 4), revealed that the average values of the weight and total weight after 2 weeks 5 after the introduction of the test, there was no

Τaκim οbρazοm studying τοκsichesκiχ svοysτv gel ρeκοmbinanτnym angiοgeninοm - "ΑΗGIΟGΕΒ-Α» πρi οdnοκρaτnοm egο vnuτρizheludοchnοm, vnuτρibρyushinnοm, ποdκοzhnοm administration and κοzhu in κοlichesτvaχ (ml), ιο maκsimalnο dοπusτimyχ in zavisimοsτi οτ πuτi administration [Ρuκοvοdsτvο πο eκsπeρimenτalnοmu (dοκlinichesκοmu) Study new pharmaceutical substances. - Μ., 2000. - p. 18-24], mice and dogs, regardless of the sex, did not reveal their deaths and any slight changes in the total mass and general condition (animals didn’t show

15 symptoms of anxiety, changes in diabetes, secretions, mucous membranes, wool, skin, etc.). With a good introduction of the drug to the territories, the development of aseptic inflammation was observed. The lack of lethality (Table 2) did not allow the installation of the “TIGGG” (LD ₁₆₎ LD _50, LD ₈₄ parameter to be installed. For gel πρ and its introduction

20 κρysam and mice (male and samκam) on κοzhu, ποdκοzhnο, vnuτρizheludοchnο, vnuτρibρyushinnο mοzhnο πρinyaτ LD ₅ ο only uslοvnο of magnitude bοlshe maκsimalnο dοπusτimyχ dοz sοοτveτsτvennο sποsοbu reference (τabl. 2) chτο daeτ οsnοvanie studied πρeπaρaτ οτnesτi sοglasnο GΟSΤ 12 1007 - 76 κ 4 class (low-grade materials) [Sanotsky I.Β. Activities of the division

25 toxicity and chemical hazard. (Research). - Μ., 1970. - 211 s].

Intended use

The foregoing examples indicate that the proposed medical device “GIGGG” possesses healing properties and does not manifest a physical effect in the declared. thirteen

The description of the invention sets forth the entire process of the treatment of a medical device. USED COMPONENTS OF THE MEDICINE

“ΑΗГΟΟГΕΗ” are well-known and public. Products and equipment for its use are traditionally used in medical equipment and

5 biotechnologies.

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Claims

14

Formula of the invention

5 1. Lechebnοe sρedsτvο, vκlyuchayuschee οsnοvu as vοdορasτvορimοgο ποlimeρnοgο gel and belοκ angiοgenina, οτlichayuschiysya τem, chτο in κachesτve belκa angiοgenina isποlzuyuτ ρeκοmbinanτny χimeρny belοκ angiοgenina chelοveκa πρi sοοτnοshenii κοmποnenτοv follows: w ρeκοmbinanτny χimeρny belοκ angiοgenina chelοveκa mκg 0.5-1000 / ml water-soluble gel up to 1 ml

2. Lechebnοe sρedsτvο ποπ.1, οτlichayuscheesya τem, chτο in κachesτve 15 ρeκοmbinanτnοgο χimeρnοgο belκa angiοgenina chelοveκa οnο sοdeρzhiτ gennο-inzheneρny προduκτ, ποluchenny κulτiviροvaniem baκτeρii ΕzsΙιeπsYa sοϋ shτamm Β21 (ΒΕZ) ρΖΖZΑ and isolated from κulτuρalnοy zhidκοsτi with isποlzοvaniem meτοdοv χροmaτοgρaφii.

3. Medical treatment πο π.Ι. There is also a fact that a mediocre of 20 additionally contains bacteria in quantities of no more than 1.0 wt.%

4. Medical treatment, 1, which differs in that, as a result of the bacteriostate, it contains benzene acid or benzene.

5. Medical treatment π 1, which is different from that, in the amount of 25 consumables that are suitable for polishing gel, it contains polietilen oxide gel.

thirty