Method of photo inactivation of bird influenza a virus of subtype h5n1

FIELD: medicine.

SUBSTANCE: invention concerns virology. Method involves use of visible spectral range with application of water-soluble photo sensitivisers of cationic type, added to virus-containing medium in 0.25-2 kg/ml concentration before irradiation. Method is characterised by low photo sensitiviser concentrations and safe white or red light, providing for method effectiveness and safety. Invention can be applied in photo decontamination of bird influenza A virus of H5N1 subtype ('bird flu' virus) for various media.

EFFECT: additionally, shrinkage of ever expanding prevalence of diseases caused by 'bird flu' virus.

2 tbl, 5 ex

 

The present invention relates to Virology and can be used for Photobacterium different environments from influenza a birds subtype H5N1 (virus of "bird flu").

Known inactivating, or damaging, the action of light, photocatalytic some organic compounds, primarily related to dyes. A typical example is the photosensitization of various biological systems psoralens with ultraviolet radiation.

Thus, the known method photosensibilisation the psoralens inactivation of viruses, bacteria and protozoa in the blood [Corash L. Inactivation of viruses, bacteria, protozoa, and leukocytes in platelet and red cell concentrates. Vox Sang. 2000. V. 78. P.P. 205-210].

The main disadvantage of psoralens is their mutagenic potential, including in relation to animal cells, due to photoinduced their crosslinks DNA.

The effect of formation of cytotoxic reactive oxygen species (singlet oxygen, superoxide from molecular oxygen, with the participation of dye called photodynamic and appropriate way photoinactivation of pathogens and treatment of infectious diseases : antimicrobial photodynamic therapy (APDT) [Wainwright M. Photodynamic antimicrobial chemotherapy (PACT). J. Antimicrob. Chemother. 1998. V. 42, P.P. 13-28]. The selectivity of antimicrobial PDT is based on the greater sensitivity of the micro is organizmov compared with animal cells to the active forms of oxygen.

The most widely known method of photodynamic inactivation of bacteria. Due to the wide spread of drug resistance among the causative agents of various infectious diseases this way attach great importance and is considered as an alternative to traditional chemotherapy [M.R. Hamblin, Hasan T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem. Photobiol. Sci. 2004. V.3.P.P.436-450].

However, different bacterial species vary in their sensitivity to photodynamic inactivation. The most stable are gram-negative bacteria. This is due to low permeability of their outer cell membrane dye. The negative charge of the surface of bacterial cells determines the active linking to them and, accordingly, a broad antibacterial activity dyes kotonoha Tala [Minnock, A., D.I. Vernon, J. Schofield, J. Griffiths, J.H. Parish, S.T. Brown Photoinactivation of bacteria. Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both gram-negative and gram-positive bacteria. J. Photochem. Photobiol. B: Biol. 1996. V. 32. P.P. 159-164; Jori G. Photodynamic therapy of microbial infections: state of the art and perspectives. J. Environ. Pathol. Toxicol. Oncol. 2006. V. 25. P.P. 505-519. RF patent №2282647, C09B 47/32, 2006].

Mushrooms have a more simply constructed of the outer cell wall. For photodynamic inactivation of fungal pathogens of nature, such as Candida, can be used anionic [Bertoloni, G., Reddi E., Gatta, M., Burlini S., Jori G. Factors infuencing the haematoporphyrin-sensitized photoinactivation of Candida albicans. J. Gen. Environ. 1989. V. 135. P. 957-966], amphiphilic [RF Patent №2230110, C12N 1/14, 2004] and cationic photosensitizers [RF Patent №2282647, SW 47/32, 2006].

Viruses due to the peculiarities of the structure are fundamentally different from the cellular form of target microorganisms for antimicrobial PDT. The highest value for the efficiency of photodynamic inactivation with one or another type of dyes is the presence, composition and structure of the shell of the virion. So, anionic dyes are effective against viruses, which has a shell, and the mechanism of inactivation is the oxidative degradation of its components, in particular, induction of cross-linking proteins. It is generally accepted that viruses do not have shells, are more sensitive to cationic photosensitizers type, which have an affinity for nucleic acids and induce oxidation of their nitrogenous bases, mainly guanosine [Wainwright M Photoinactivation of viruses. Photochem. Photobiol. Sci. 2004. V. 3. P.P. 406-411]. However, due to the structural features of different types of viruses, their sensitivity to photodynamic inactivation with a particular dye is an individual characteristic.

For some viruses, which has a shell, known methods of photodynamic inactivation using cationic dyes. So, with skin the herpes virus more efficiently inactivated in PR is the absence of cationic and amphiphilic derivatives of phthalocyanines compared with anionic Merocyanine 540 [Smetana z, Mendelson E., Manor, J., van Lier, E., Ben-Hur e, Salzberg, S., Malik Z. Photodynamic inactivation of herpes viruses with phthalocyanine derivatives. J. Phorochem. Photobiol. B. 1994. V. 22. P.P. 37-43].

Phthalocyanines with positively charged residues on the Central atom of silicon is used for the inactivation of human immunodeficiency virus (HIV) during the disinfection of blood components [Ben Hur E., Moor C.E.,, Margolis-Nunno H, Gottlieb P., Separation M.M. et al. The photodecontamination of cellular blood components: mechanisms and use of photosensitization in transfusion medicine. Transf. Med Rev. 1996. V.10. P 15-22].

Monocationic the dye methylene blue is effective in the inactivation devoid of membrane adenovirus [Schagen F., Moor A., Cheong, S.C., Cramer S.J., van Ormondt, H., van der Eb A j, Dubbelman T., R.C. Hoeben Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation. Gene Therapy. 1999. V. 6 P.P. 873-881] and having a shell dengue virus [O. Huang, Fu W.L., Chen, C., Huang, J.F., Zhang X., Xue O. Inactivation of dengue virus by methylene blue/narrow bandwidth light system. J Pnotochem Photobiol B. 2004. V. 77. P.P. 39-43].

At the same time, cationic porphyrin, causing the inactivation of viruses with lipid membranes has been found to be ineffective in relation to devoid of membrane parvovirus [Trannoy L.L., Terpstra F.G., de Korte D., Lagerberg J.W.,A.J. Verhoeven, Brand A., van Engelenburg F.A. Differential sensitivities of pathogens in red cell concentrates to Tri-P(4)-photoinactivation. 2006. Vox Sang V 91. P.P. 111-118].

Thus, the antiviral activity of the complex depends on the physico-chemical characteristics of the dye and the nature of the object photoinactivation, which complicates the prediction based on the years of literature data on the effectiveness of new photosensitizers and sensitivity of untested kinds of viruses.

Despite the importance of developing effective ways of dealing with the virus of "bird flu", the possibility of its photodynamic inactivation for disinfection of different environments has not been studied.

The known method photoinactivation of influenza virus H5 serogroup virus and avian influenza, selected by the authors for the prototype, using ultraviolet germicidal (253.7 nm) irradiators (newsletter FSUE SRC VB "Vector" from 2005). The disadvantage of this method is the use of short-wave UV radiation, harmful effects on human and animal organisms.

The task of the invention was to provide such a method photoinactivation of the virus avian influenza, which would provide 100% inactivation of the virus in the short-term (several minutes) the action of harmless radiation in the visible spectral range - white or red light.

The problem is solved by the use of photosensitizers is a water - soluble cationic dyes of the type by introducing them to irradiation in virusological medium at a concentration of 0.25-2 µg/ml.

The application of the photosensitizer at a concentration of less than 0.25 μg/ml did not provide effective photoinactivation, and raising it above 2 µg/ml impractical, since does not improve efficiency.

The way sudestada as follows.

In experiments using the virus of the influenza a virus subtype H5N1 bird (RNA-containing, with the shell of the virus) in extraembryonal fluid source title 109EDS50(embryonic lethal doses). Prepare a series of tenfold dilutions of the virus, for which 0.5 cm3vaccinated liquid add 4.5 cm3saline solution.

In vaccinated liquid with a viral dose of 102-107EDS50/ml injected cationic dye at a concentration of 0.25-2 µg ml and after 10 min irradiated 5 min using a halogen white light source (illumination level sample - 65000 Lux) or led source red light (power density on the sample level 17 mW/cm2).

As photosensitizer is used, for example, monocation dyes, proflavin acetate and methylene blue or actication octakis(N-(2-hydroxyethyl)-N,N-dimethylaminomethyl phthalocyanine zinc octaploid (Holocene).

Contamination spend on a 10-day developing chicken embryos. Processed material with different dilutions of the virus to infect 6 embryos. To infect the embryo punch punch in the shell 2 holes to a depth of 1.2±0.3 mm, in which 1 inoculant 0.1 cm3that hole is filled with molten paraffin. Your embryos are placed in thermostat at which the temperature 37±0.5°C and otoscopic 2 times a day. The account of the death of the infected embryos finish in 72 hours (24 hours after the death of the last of embryos). Dead embryos reveal in the day of death. The result of the specificity of the death of chick embryos was confirmed by haemagglutination reaction (DSA). For this purpose each of the deceased embryo separately collect sterile allantoin liquid that check on hemagglutinin activity and identity in RSA.

The controls are vaccinated liquid without processing light (Control 1); vaccinated liquid drug without processing light (Control 2); vaccinated liquid without the drug, the processed light (Control 3); intact chicken embryos (Control 4).

About inactivation of virus in the inoculum used to infect chicken embryos judged by total fall infectivity, i.e. the loss of the ability to cause the death of embryos. Determine the dose of the virus, which can be inactivated using photodynamic treatment at a certain concentration of the cationic dye.

The invention is illustrated by the following examples.

Example 1

In vaccinated liquids with different doses of virus injected dye, proflavin acetate at a concentration of 1.0 or 2.0 µg/ml) and after 10 min irradiated with white light (dose of 15 j/cm2). The results are presented in t the blitz 1. See the complete fall of infectivity (100% survival of chick embryos) at the dose of virus in the treated liquid is not more than 104EDS50/ml. the Results are presented in table 1.

Example 2

Similarly, when using 1.0 microgram/ml of the dye methylene blue see full drop infectivity dose of virus is not more than 106EDS50/ml. the Results are presented in table 1.

Example 3

Similarly, when you use 2.0 µg/ml methylene blue and see the full drop in infectivity at all tested doses of the virus for up to 107EDS50/ml. the Results are presented in table 1.

Example 4

Similarly, when using 1.0 or 2.0 µg/ml of Halosense see the full drop in infectivity at all tested doses of the virus for up to 107EDS50/ml. the Results are presented in table 1.

Example 5

In vaccinated liquids with different doses of virus injected Halogens at a concentration of 0.25, 0.5 or 1.0 μg/ml and 10 min irradiated with red light (dose of 5.1 j/cm2). The results are presented in table 2. See the full drop in infectivity at doses of virus in the treated fluid 104, 105or 107EDS50/ml. the Results are presented in table 2.

Thus, the introduction of vaccinated environment available to monocation the dyes of proflavine acetate or methylene blue and the subsequent irradiation for 5 min using a source of white light, creating a light similar to natural sunlight, providing 100% inactivation of the virus avian influenza in certain titles. Proflavin acetate at a concentration of 1.0-2.0 µg/ml can be used for photoinactivation of the virus avian influenza in the credits, no more than 104EDS50/ml. In the case of higher viral contamination, it is advisable to use methylene blue in a concentration of up to 2.0 mg/ml

Using the proposed method with the use of new actication dye of Holocene helps to disinfect liquids with high titers of the virus avian influenza. Processing Halosense concentrations of 1.0-2.0 µg/ml and irradiated for 5 min either red or white light provides 100% inactivation of the virus avian influenza in all tested titles, including 107EDS50/ml.

Antiviral, and antibacterial and antifungal activity [RF Patent №2282647, SW 47/32, 2006] Holocene occur under similar regimes photodynamic treatment, and therefore Halogens it is also advisable to use for Photobacterium in the case of mixed microbial contamination.

In contrast, when photoinactivation with the use of germicidal UV radiation revealed a large (3,4-fold) resistance compared to vegetative bacteria [Chag J., Ossoff S.F., Lobe, D.C., M.H. Dorfman, Dumais C.M., Qualls R.G., Johnson J.D. UV inactivation of pathogenic and indicator microorganisms. Applied and Enviromental Microbiology. 1985. P.P. 1361-1365], resulting for water disinfection with mixed contamination it is necessary to apply higher doses of harmful UV radiation.

Implementing a new way photoinactivation of the virus of the influenza a virus subtype H5N1 bird can reduce the increasing prevalence of diseases caused them.

The proposed method involves the use of low concentrations of photosensitizers and harmless white or red light, which ensures its safety and economic benefits.

Table 1. Virucidal activity of cationic dyes of the type in respect of the virus of the influenza a virus subtype H5N1 bird when irradiated for 5 minutes with white light
SubstanceConcentrationDose of EDS virus50/ml% survival ECThe titer in RSA
102100,00
103100,0 0
1.0 microgram/ml104100,00
Example 110533,3>1:16
1060>1:16
Proflavin1070>1:16
acetate102100,00
103100,00
2.0 µg/ml104100,00
Example 110550,0>1:16
1060>1:16
1070>1:16
102100,00
103100,00
1.0 microgram/ml104100,00
Example 2105100,00
106100,00
Methylene1070>1:16
blue102 100,00
103100,00
2.0 µg/ml104100,00
Example 3105100,00
106100,00
107100,00
102100,00
103100,00
1.0 microgram/ml104100,00
Example 4105100,00
106100,00
Holocene107100,00
102100,00
103100,00
2.0 µg/ml104100,00
Example 4105100,00
106100,00
107 100,00
Control 1-1070>1:16
Control 22.0 µg/ml
Proflavin
acetate
1070>1:16
Control 22.0 µg/ml
Methylene blue
1070>1:16
Control 22.0 µg/ml Holocene1070>1:16
Control 3-1070>1:16
Control 4--100,00

Table 2. Virucidal activity of Halosense the virus of the influenza a virus subtype H5N1 bird when irradiated for 5 min with red light (5,1 j/cm 2)
The viral dose% survival ECThe titer in RSA
102100,00
10366,6>1:16
0.25 microgram/ml1040>1:16
Example 51050>1:16
1060>1:16
1070>1:16
102100,00
103100,00
Holocene0.5 μg/ml10466,6>1:16
Example 510533,3>1:16
1060>1:16
1070>1:16
102100,00
103100,00
1.0 microgram/ml104100,00
Example 5105100,0/td> 0
106100,00
107100,00
Control 1-1070>1:16
Control 21.0 microgram/ml of Holocene1070>1:16
Control 3-1070>1:16
Control 4--100,00

The way photoinactivation of the virus of the influenza a virus subtype H5N1 bird, characterized in that vaccinated Wednesday introduced a water-soluble cationic photosensitizers type at a concentration of 0.25-2 µg/ml, and then irradiated with white or red light at a dose of 5-15 j/cm2not less than 5 minutes



 

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EFFECT: enhanced effectiveness of treatment; localized treatment; reduced risk of complications and stress action upon child.

FIELD: medicine.

SUBSTANCE: the present innovation deals with treating vascular cutaneous neoplasms, such as nevus flammeus and gemangiomas. Light-thermal impact at energy ranged 39-47 J/sq. cm should be performed in two stages, and between them, 2-3 wk after the onset of vascular resistance at the first stage one should perform beta-therapy daily for 2-3 d at single dosage being 20 g. Then, 3 wk later it is necessary to conduct the second stage of light-thermal impact by starting at energy value being 42 J/sq. cm, not less. The method enables to shorten therapy terms due to applying combined method to affect vascular cutaneous neoplasms.

EFFECT: higher therapeutic and cosmetic effect.

1 ex

FIELD: medicine.

SUBSTANCE: method involves intravitreously introducing two electrodes into intraocular neoplasm after carrying out vitrectomy and retinotomy to expose the intraocular neoplasm. The electrodes are manufactured from platinum group metal. Electrochemical destruction is carried out with current intensity of 100 mA during 1-10 min or 10 mA during 10 min in changing electrodes polarity and their position in the intraocular neoplasm space, and the electrodes are removed. 0.1-1% aqueous solution of khlorin as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, is intravenously introduced at a dose of 0.8-1.1 mg/kg. Visual control of intraocular neoplasm cells fluorescence is carried out by applying fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, intravitreous laser radiation is carried out in parallel light beam of wavelength equal to 661-666 nm is applied at a dose of 30-120 J/cm2.The transformed retina and tumor destruction products are intravitreally removed. Boundary-making endolasercoagulation of retinotomy area is carried out after having smoothed and compressed retina with perfluororganic compound. The operation is finished with placing sutures on sclerotomy and conjunctiva. Platinum, iridium or rhodium are used as the platinum group metals. Another embodiment of the invention involves adjusting position and size of the intraocular neoplasm in trans-scleral diaphanoscopic way. Rectangular scleral pocket is built above the intraocular neoplasm to 2/3 of sclera thickness with its base turned away from limb. Several electrodes are introduced into intraocular neoplasm structure via the built bed. The electrodes are manufactured from platinum group metal. Electrochemical destruction is carried out with the same current intensity in changing electrodes polarity and their position in the intraocular neoplasm space, and the electrodes are removed. Superficial scleral flat is returned to its place and fixed with interrupted sutures. 0.1-1% aqueous solution of khlorin as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, is intravenously introduced at a dose of 0.8-1.1 mg/kg after having carried out vitrectomy and retinotomy. Visual control of intraocular neoplasm cells fluorescence is carried out by applying fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, intravitreous laser radiation is carried out in parallel light beam of wavelength equal to 661-666 nm is applied at a dose of 30-120 J/cm2. The transformed retina and tumor destruction products are intravitreally removed using vitreotome. Boundary-making endolasercoagulation of retinotomy area is carried out after having smoothed and compressed retina with perfluororganic compound. The operation is finished with placing sutures on sclerotomy and conjunctiva. Platinum, iridium or rhodium are used as the platinum group metals. The number of electrodes is equal to 4-8.

EFFECT: reduced risk of metastasizing.

4 cl, 13 dwg

FIELD: medicine.

SUBSTANCE: method involves building tunnel to posterior eyeball pole in inferoexterior and superexterior quadrants. The tunnel is used for implanting flexible polymer magnetolaser implant to the place, the subretinal neovascular membrane is localized. The implant has a permanent magnet shaped as a cut ring and is provided with drug delivery system and a short focus scattering lens of laser radiator connected to light guide. The permanent implant magnet is axially magnetized and produces permanent magnetic field of 5-7 mTesla units intensity. It is arranged with its north pole turned towards sclera at the place of the subretinal neovascular membrane projection with extrascleral arrangement of laser radiator lens membrane being provided in the subretinal neovascular membrane projection area. The other implant end is sutured to sclera 5-6 mm far from the limb via holes made in advance. The implant is covered with conjunctiva and retention sutures are placed thereon. Light guide and drug supply system lead is attached to temple with any known method applied. Drugs are supplied via the implant drug supply system in retrobulbary way in any order. Triombrast is given in the amount of 0,4-0,6 ml and dexamethasone or dexone in the amount of 0,4-0,6 ml during 3-4 days every 12 h. 0.1-1% aqueous solution of khlorin is intravenously introduced at the third-fourth day after setting the implant as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, at a bolus dose of 0.8-1.1 mg/kg. Visual control of subretinal neovascular membrane cells fluorescence is carried out by applying fluorescent diagnosis methods. After saturating the subretinal neovascular membrane with the photosensitizer to maximum saturation level, intravitreous, transretinal laser radiation of 661-666 nm large wavelength is applied at general dose of 30-120 J/cm2. The flexible polymer magnetolaser implant is removed and sutures are placed on conjunctiva. Permanent magnet of the flexible polymer magnetolaser implant is manufactured from samarium-cobalt, samarium-iron-nitrogen or neodymium-iron-boron system material. The photosensitizer is repeatedly intravenously introduced at the same dose in 2-3 days after the first laser radiation treatment. Visual intraocular neoplasm cells fluorescence control is carried out using fluorescent diagnosis techniques. Maximum level of saturation with the photosensitizer being achieved in the subretinal neovascular membrane via laser light guide and implant lens, repeated laser irradiation of the subretinal neovascular membrane is carried out with radiation dose of 30-60 J/cm2.

EFFECT: accelerated subretinal edema and hemorrhages resorption; regression and obliteration of the subretinal neovascular membrane; prolonged vision function stabilization.

6 cl

FIELD: medicine.

SUBSTANCE: method involves filling vitreous cavity with perfluororganic compound. Two electrodes manufactured from platinum group metal are intravitreally, transretinally introduced into intraocular neoplasm. Electrochemical destruction is carried out with current intensity of 10-100 mA during 1-10 min in changing electrodes polarity and their position in the intraocular neoplasm space, and the electrodes are removed. 0.1-1% aqueous solution of khlorin as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, is intravenously introduced at a dose of 0.8-1.1 mg/kg. Visual control of intraocular neoplasm cells fluorescence is carried out by applying fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, intravitreous, transretinal laser radiation of 661-666 nm large wavelength is applied at a dose of 30-120 J/cm2 in perfluororganic compound medium. The transformed retina and tumor destruction products are intravitreally removed with perfluororganic compound volume being compensated with its additional introduction. Boundary-making endolasercoagulation of retinotomy area is carried out. The perfluororganic compound is substituted with silicon oil. The operation is ended in placing sutures over sclerotmy areas and over conjunctiva. Perfluormetylcyclohexylperidin, perfluortributylamine or perfluorpolyester or like are used as the perfluororganic compound for filling vitreous cavity. Platinum, iridium or rhodium are used as the platinum group metals.

EFFECT: complete destruction of neoplasm; reduced dissemination risk.

6 cl, 12 dwg

FIELD: medicine, applicable for stopping of pains of various nature.

SUBSTANCE: the device has a quantum-mechanical oscillator located in a casing, magnet, vessel for medicinal agent and a hollow cylinder. The magnet is installed between the oscillator and the vessel. Positioned in the vessel is a hollow cylinder having through holes on its surface.

EFFECT: quick and absolute anestesia.

2 ex, 1 dwg

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