Pharmaceutical composition producing antioxidant, antimicrobial, antitoxic human lactoferrin protein, method for preparing it, and method of therapy

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, particularly toxicology and radiology, to drug preparations based on antioxidant proteins and methods of using them. The pharmaceutical composition for treating toxic conditions wherein the therapeutic effect is ensured by the action of antioxidant, antimicrobial, antitoxic human lacroferrin protein on the human body contains non-replicating nanoparticles of human adenovirus serotype 5 genome with inserted human lactoferrin expressing human lactoferrin in the therapeutically effective amount in the body, and an expression buffer with the particle content not less than 2.33×1011 of physical particles per ml of the expressing buffer. The method of therapy involves administering the composition in the therapeutically effective dose of 7×1011 of physical particles to 7×1013 of physical particles per ml of the expressing buffer per an individual; the composition is administered intravenously.

EFFECT: invention provides the stable therapeutic effect after the single administration of the composition.

17 cl, 14 ex, 4 dwg

 

The invention relates to medicine, in particular toxicology and radiology, to medicines on the basis of antioxidant proteins and methods of use thereof.

Renowned pharmaceutical composition comprising lactoferrin person that causes the body's various physiological changes, including immunomodulatory effects, reducing inflammatory responses and inhibiting growth of solid tumors. (U.S. patent No. 6333311).

Renowned pharmaceutical composition comprising recombinant human lactoferrin (U.S. Patent No. 5955316). Known composition intended for the treatment of diabetic ulcers when the application, oral or parenteral application (U.S. Patent No. 7524814).

The known method of prevention and treatment of severe postoperative complications (chronic inflammatory and posthemorrhagic) with the phenomena polyorgano failure and General intoxication by daily intravenous, intracavitary, intratracheal administration of medicinal preparations containing human lactoferrin and ceruloplasmin person (RF Patent No. 2199337).

The obstacles to achieving sustained therapeutic effect when using all of these compositions is the presence in them as active components isolated and purified proteins, which when lubiprostone the rapidly removed from the body of the patient, hence the need for repeated administration. pharmaceutical compositions containing antioxidant protein lactoferrin, to maintain therapeutically effective concentration in the body. In addition to difficulties from a medical point of view, it is economically disadvantageous.

Thus, in this region there is an urgent need to develop pharmaceutical compositions and methods of prevention and treatment based on them, which would be therapeutically highly effective, economically feasible, and require less time and labor costs of medical personnel in the application.

Know rapidly growing trend in the development of gene therapeutic drugs based on recombinant adenoviruses. Among human adenoviruses most characterized is the human adenovirus fifth serotype (Ad) (J.A. Zaia The status of gene vectors for the treatment of diabetes, Cell Biochem, Biophys, 2007, v. 48(2-3), c.183-90 - the Status of genetic vectors for the treatment of diabetes).

One of the advantages of recombinant adenoviruses is to ensure a high level of expression of a target gene in a long time, up to 28 days, some laboratory animals longer (up to 60-90 days) (S.L. Brody, R.G. Crystal, Adenovirus-Mediated in Vivo Gene Transfer, Annals new york acadmy of sciences, 1994, 716, B.C. 90-103 - Provide adenovirus transfer of the ENES in Vivo).

Known successful application of substances on the basis of recombinant adenovirus expressing human lactoferrin in the experiment on mice with breast cancer. After the introduction of substances in the tumor tissue was observed a slowdown due to apoptosis (Inhibition of tumor growth by recombinant adenovirus containing human lactoferrin through inducing tumor cell apoptosis in mice bearing EMT6 breast cancer, Wang J., Li Q., Ou, Y., Han Z., Li, K., Wang, P., Zhou, S., Arch Pharm Res, 2011, Vol 34, No 6, c.987-995 - Inhibition of tumor growth using an adenovirus containing the gene of human lactoferrin by induction of apoptosis of tumor cells in mice with breast cancer EMT).

However, this drug does not provide antioxidant, antimicrobial activity of lactoferrin as described in the method of treatment is provided only its antitumor effect.

It is known that adenovirus can efficiently Express human lactoferrin around tumor cells showing stable growth inhibition of cancer of the cervix, strengthening the immune system and has no toxic side effects (Application for patent No. 200910021705, 19.08.2009, Q. Li, J. Li, Z. Han, G. Wu, J. Hu, Recombinant adenovirus carrying human lactoferrin, preparation and uses thereof, China).

This drug has a narrow antitumor action because the specified method of its use around the tumor cells provides a wide Antioch adantage antimicrobial and antitoxic action of lactoferrin.

The closest pharmaceutical composition of the same purposes of the claimed invention by a combination of traits is antibacterial, antioxidant, detoxifying, immunomodulating and antineoplastic drug as an active substance containing human lactoferrin isolated from breast milk (Patent RF №2165769, adopted for the prototype. Famous drug is made in the form of a lyophilisate containing from 10 to 90% of human lactoferrin. The drug is used for prophylaxis and/or treatment of infectious, inflammatory diseases and toxic conditions of various etiologies.

The obstacles to achieving sustained therapeutic effect when using a specified drug is the need for repeated infusions of the drug because of the rapid excretion of lactoferrin from the body of the patient and, accordingly, the high cost of the drug, medical instruments and time of the medical staff to achieve the desired result of the treatment.

The uniqueness and scarcity of raw - milk seriously limits the scale of production and, consequently, the possibility of its application in the required quantities by medical indications.

The objective of this invention is to provide a pharmaceutical composition, producer is the fact antioxidant, antimicrobial, anti-toxic protein human lactoferrin for achieving sustained therapeutic effect with a single administration of the composition, reducing the cost of the drug, medical instruments and time of the medical staff to achieve the desired result of the treatment, its preparation and method of therapy through the effect of the pharmaceutical composition to the human body.

The problem is solved due to the fact that the pharmaceutical composition in which a therapeutic effect is obtained as a result of exposure antioxidant, antimicrobial, anti-toxic protein human lactoferrin, human body contains rereplacenocase nanoparticles based on the genome of a human adenovirus 5-th serotype with the insertion of a gene coding for human lactoferrin expressing human lactoferrin in a therapeutically effective amount in the body, it contains contains formulating the buffer. Content rereplacenocase nanoparticles is at least of 2.33×1011physical particles per ml formulating buffer. The method of obtaining pharmaceutical compositions containing rereplacenocase nanoparticles on the basis of the genome of adenovirus human serotype 5 with insertion of expressing cassette, comprising a promoter, a gene of human lactoferrin and whitefish is al polyadenylation, is that a given activity is produced by the planting of a permissive cell culture 293 nerealizirane nanoparticles with the gene of human lactoferrin and capacity rereplacenocase nanoparticles in the cells to the desired content, after conducting multistage purification by centrifugation, fourfold peregorazhivaniya-thawing in buffer solution obtained at the previous stage of the solid part, optionally treated with a nuclease with further separation rereplacenocase nanoparticles with the gene of human lactoferrin from destroyed cells by centrifugation and subsequent selection of the obtained supernatant, further cleaning is performed by ultrafiltration, and the resulting supernatant is diluted with buffer and stirred, and then the obtained solution is filtered under pressure, and are cleaned by anion-exchange chromatography and further exlusionary chromatography, the obtained eluate add ethanol and ethylenediaminetetraacetic acid, and then send the normal filtering, then get ready preparation by dilution obtained at the previous stage of the drug makers buffer to a specified content rereplacenocase nanoparticles. While designing rereplacenocase nanoparticles with the insertion of a gene of human lactoferrin done is make the method of homologous recombination in cell culture. A therapeutically effective dose rereplacenocase nanoparticles take 3 ml of the final volume of the composition. Form of release of the drug to 1 ml, 2 ml and 3 ml therapies, is the introduction to the man claimed pharmaceutical compositions, producing antioxidant, antimicrobial, anti-toxic protein human lactoferrin. The pharmaceutical composition is administered to a human intravenously. The pharmaceutical composition can enter the human intravenous drip. Introduction the pharmaceutical composition of conduct for the treatment of toxic conditions due to purulent-inflammatory diseases induced by various microorganisms, physical effects and chemical agents, including means drug and radiation therapy. A therapeutically effective dose rereplacenocase nanoparticles with the insertion of a gene of human lactoferrin is between 7×1011physical particles up to 7×1013physical particles per person. Introduction pharmaceutical compositions performed sequentially in two stages. However, the imposition of a pharmaceutical composition in the second phase, conducted a day after the first injection. The introduction of a pharmaceutical composition for the first stage is carried out in a volume containing 1/3 full therapeutic dose of the pharmaceutical composition, and the second stage input is Yat remaining 2/3 full therapeutic dose of the composition. 1/3 full dose of the pharmaceutical composition is dissolved in 66 ml of a physiologically acceptable solvent, and 2/3 of the total dose is dissolved in 134 ml of a physiologically acceptable solvent. Physiologically acceptable solvent is glucose. The glucose solution is 5%or 10%.

Implementation of the invention.

These common technical, medical and economic results by carrying out the invention by the present invention are achieved by the inventive method, as well as a method of prevention and/or treatment of inflammatory and/or infectious diseases, and/or toxic States perform using antioxidant protein lactoferrin. The feature of the proposed method lies in the fact that the antioxidant protein is produced directly in the human body with the introduction of rereplacenocase nanoparticles with the insertion of a gene antioxidant protein, and are not entered many times in the form selected from a natural source of protein. Downregulation of beloki has a therapeutic effect as antimicrobial and/or anti-inflammatory and/or immunomodulating and/or antioxidantes, and/or detoxifying, and/or anticarcinogenic agent.

For the implementation of the treatment process of the pharmaceutical composition on the basis of rereplacenocase nanocat the CI producing antioxidant protein human lactoferrin is administered in the following doses:

- for the treatment of toxic conditions due to purulent-inflammatory diseases induced by various microorganisms, physical effects and chemical agents, including means drug and radiation therapy, from 7×1011FC to 7×1013FC per person;

- for prevention postinjection complications at the beginning of therapy at the first stage 1/3 full therapeutic dose rereplacenocase nanoparticles with the gene of human lactoferrin;

- for prevention postinjection complications at the beginning of treatment in the second stage, enter 2/3 full therapeutic dose rereplacenocase nanoparticles with the gene of human lactoferrin;

The invention consists in the following. The inventive pharmaceutical composition comprises as the main active component rereplacenocase nanoparticles with the insertion of a gene expressing antioxidant protein human lactoferrin. This protein does to the human body is multifaceted biological properties that determine its antioxidant, detoxifying, immunomodulatory, antimicrobial, anti-inflammatory and anticarcinogenic action. The main significant difference between the claimed pharmaceutical compositions the AI from previously known pharmaceutical compositions, that antioxidant protein for a long time is produced in a single nanostructures directly in the body, and do not enter multiple times in the body in the form isolated from any source of protein. The pharmaceutical composition is compatible with any pharmaceutically suitable solvents. The claimed range of the amount of the pharmaceutical composition introduced into the body of the patient, allow to maintain a high therapeutically effective antioxidant concentration of the target protein in the body for a long time.

The pharmaceutical composition is the starting product for the preparation of various dosage forms, the use of which is determined depending on the disease. The inventive pharmaceutical composition based rereplacenocase nanoparticles with the insertion of a gene encoding antioxidant proteins person, passed preclinical and clinical trials to study specific (therapeutic) efficiency and General toxic effect, which demonstrated the safety of this composition and therapeutic activity as antioxidant, antibacterial and detoxifying substances, as illustrated by the following examples.

To create a pharmaceutical composition, containing in its composition rereplacenocase nano is astitsy on the basis of the genome of a human adenovirus 5-th serotype and prolongirovanne expressing human lactoferrin in human organism in therapeutic quantities should:

1) to construct rereplacenocase nanoparticles with the insertion of a gene lactoferrin;

2) to develop a method of obtaining a pharmaceutical composition;

3) to prove compliance expressed nerealizirane nanoparticles of lactoferrin human native human lactoferrin;

4) to determine the route of administration, therapeutic dose and the permissible dose limits;

5) to prove the prolongation of action of the pharmaceutical composition;

6) to develop a safe and therapeutically effective use of pharmaceutical compositions.

Given further examples, tables and figures reveal the essence of the invention and confirm the efficiency of the decision in this invention.

Example 1.

1) Design rereplacenocase nanoparticles on the basis of the genome of adenovirus human serotype 5 with the insertion of a gene coding for human lactoferrin.

2) Obtaining a pharmaceutical composition.

Design rereplacenocase nanoparticles on the basis of the genome of adenovirus human serotype 5 (the size of 70-80 nm) with the insertion of a gene of human lactoferrin was carried out by the method of homologous recombination in cell culture and was performed using a well-known laboratory techniques (for example, Sambrook J., Fritsch E., Maniatis T. and other Methods of genetic engineering. Molecular cloning. M, Mi Is, 1984, p.205-224, 387-420). The basis was taken recombinant plasmid pJM17 (W.J. McGrory, D.S. Bautista, F.L. Graham. A simple technique for the rescue of early regionl mutations into infectious human adenovirus type 5, Virology, V, 163, No. 2, 1988, s - a Simple technique for removal of early region 1 into infectious human adenovirus type 5), with deletions in the field E1 adenoviral genome. Artificially synthesized cDNA of the gene coding for human lactoferrin ligated in the well-known Shuttle plasmid pRcCMV (Invitrogen, San Diego, CA, No. V75020). Next, for the mutual transformation of the obtained plasmid pRcCMV - Lf and the plasmid pJM17 they were transferrable 293 cells (for example, No. 300192, CLS, Germany) using the method of calcium phosphate precipitation (Graham FL, van der Eb AJ, 1973-A new technique for the assay of infectivity of human adenovirus 5 DNA". Virology, 52 (2) 456-67 - New technique of the method of infection the DNA of human adenovirus 5 serotype). The result was rereplacenocase nanoparticles containing expressing cassette with CMV-promoter, gene of human lactoferrin and a polyadenylation signal. Plaques of recombinant particles were formed on the cell culture in a few days after transfection, they were robbed of a Pasteur pipette, the material obtained was multiplied by the cell line 293 to obtain a titer of 3×1010PC (physical particles/ml (108IU/ml).

Obtaining pharmaceutical compositions.

The required contents of rereplacenocase nanoparticles in the composition would be what about the defined in examples 4 and 5 on the antitoxic effect and should be at least of 2.33×10 11FC/ml (which corresponds to the activity of not less than 6.7×108IU/ml). The amount of drug should be 3 ml, which is associated with ease of packaging and application. Obtaining this composition takes place in several stages.

The result of the above cell suspension, containing rereplacenocase nanoparticles in the titer of 3×1010FC/ml, was used for the further expansion of titles rereplacenocase nanoparticles and preparation of finished pharmaceutical compositions with the specified content is not less of 2.33×1011FC/ml (which corresponds to the activity of not less than 6.7×108IU/ml).

To gain the necessary credits rereplacenocase nanoparticles wave bioreactor with 4500 ml suspension of permissive 293 cell culture were seeded at a cell suspension volume of 500 ml containing rereplacenocase nanoparticles with a titer of 3×1010FC/ml

Cultivated for increasing rereplacenocase nanoparticles inside the cells and achieve their contents 6×1010FC/ml (activity 2×108IU/ml)within approximately 48 hours. To achieve the required content of the nanoparticle-cell mass was submitted for clearing, which consisted of several stages:

1) Carried out the deposition cell mass by centrifugation. Arriving on clearing the suspension had not less than 1014FC 5 l (estimated using the mass spectrum of the tra, 1 TH=1012FC). Centrifugation was carried out at mode 6000 g for 15 min, with liquid adosados was decanted and the remaining solid part containing cells and rereplacenocase nanoparticles were applied for further purification stages.

2) Removing rereplacenocase nanoparticles from the cell culture was performed by cell disruption fourfold primorazhivaniem-thawing. Prepared buffer solution with a pH of 8.0: 5 mM HCl, 0.075 MNaCl, 1 mM MgCl2) 5% sucrose, 1% Polysorbate 80. Obtained in the previous stage sediment resuspendable in 70 ml of buffer (grade ×71)Objem solution was 80 ml.

Freezing was carried out for 2 hours in liquid nitrogen, were thawed in a water bath (at +37°C), without overheating.

3) To facilitate further removal of cellular genomic DNA was performed additional processing by the nuclease. To this was added benzonase to the concentration in solution of 150 U/ml and placed on a soft stirring with a magnetic stirrer for 3 hours at room temperature (21-23°C).

4) Department rereplacenocase nanoparticles from destroyed cells was carried out by centrifugation at 9000 g for 10 min was Collected supernatant containing rereplacenocase nanoparticles.

5) Further purification was performed by ultrafiltration. To this end, the supernatant was diluted with buffer (50 mM TrisHCl p 7.5, 1M NaCl, 2 mM MgCl2, 5% sucrose, pH 7.5) to a volume of not less than 200 ml, stir with a magnetic stirrer. In the filtering process, the volume of the circulating solution (retentate) constantly brought up to the original (200 ml).

6) Further purification was produced by anion-exchange chromatography.

Retentate was applied on a column (AxiChrom 70/300 400 ml)containing aminoalkenes sorbent Q Sepharose virus licenced. Rereplacenocase nanoparticles are sorbed on the column, while the impurities are sorbed not, and washed with buffer A. After removal of impurities rereplacenocase nanoparticles were desirerable washing buffer B. chromatograph Conditions were as follows: flow 193 ml/min, buffer A (40 mM TrisHCl, 0.27 M NaCl, 2 mM MgCl2, 5% Sucrose, 0.1% Polysorbate 80, pH 7.5), conductivity ~28-30 mS/cm; buffer B (40 mM TrisHCl, 0.5M NaCl, 2 mM MgCl2, 5% sucrose, 0.1% Polysorbate 80, pH 7.5) conductivity of ~50 mS/cm. The eluate in a volume of 200 ml was sent to the next stage.

7) Exclusion chromatogaphy

Obtained in the previous stage of the eluate was applied to a column (AxiChrom 100/300 volume 800 ml)containing sorbent Q Sepharose 4 FastFlow. High-molecular substances, not included in the pores of the sorbent, was suirable the first peak (these include rereplacenocase nanoparticles), impurities were suirable after peak output rereplacenocase nanoparticles. Chromatograph conditions were as follows: the flow of 130 ml/min, buffer (10 mM TrisHCl, 75 mm NaCl, 1m MMgCl2, 5% sucrose, 005% Polysorbate 80, pH 8.0).

To poluchennom eluate (80 ml) was added ethanol to a concentration of 0.5% ethylenediaminetetraacetic acid (EDTA) to a concentration of 100 μm, were sent to the next stage.

8) Normal filtering.

For sterilization of the obtained preparation was carried out filtration through a filter with pore size of 22 μm. The final volume of the drug at this stage was 80 ml and contained rereplacenocase nanoparticles in titer of 1×1012FC/ml was diluted formulating buffer (for example, 10 mM TrisHCl, 75 mM NaCl, 1 mM MgCl2, 5% sucrose, 0.05% Polysorbate 80, 0.5% Ethanol, 100 mm EDTA, pH 8.0) to obtain the given content of 2.33×1011FC/ml and sterilized by normal filtering.

Thus, the above method of obtaining a pharmaceutical composition allows on the basis of constructed rereplacenocase nanoparticles with the insertion of a gene of human lactoferrin to get in the drug content rereplacenocase nanoparticles not less of 2.33×1011FC per ml formulating buffer (which corresponds to the activity of the pharmaceutical composition is not less than 6.7×108U/ml), and the full therapeutic dose for humans (7×1011FC) is contained in 3 ml of pharmaceutical composition that corresponds to the task.

Example 2.

Comparison of native human lactoferrin and lactoferrin person, expressio the constituent nerealizirane nanocasting on physico-chemical properties and biological activity.

Rereplacenocase nanoparticles based on the genome of a human adenovirus 5-th serotype Express lactoferrin, called recombinant, its compliance with the native lactoferrin was determined by the physico-chemical properties and biological (antioxidant) activity. For this purpose, recombinant human lactoferrin was isolated from the permissive culture of 293 cells lines transduced nerealizirane nanoparticles with the insertion of a gene encoding human lactoferrin. Purification of recombinant human lactoferrin from the culture fluid was performed by standard method of affinity chromatography on a matrix activated CNBr sepharose 4B, covalently associated with highly purified antibodies to human lactoferrin. Physico-chemical properties were analyzed by well-known methods electorphoresis, Western blot turns, and biological (antioxidant) activity - known method of inhibition of lipid peroxidation (LPO) in liver homogenate of mice. Native lactoferrin was obtained from donor breast milk (Patent RF №2165769, 13.07.2000).

1) Data electrophoretic analysis showed that recombinant human lactoferrin contains protein, molecular weight which is 76 kDa, which corresponds to the molecular mass of human lactoferrin isolated from donor unscorable.

2) by the Method of Western blot turns revealed that the recombinant protein specifically interacts with a polyclonal antibody against human lactoferrin (Sigma, cat. No. L3262).

3) Antioxidant activity of recombinant human lactoferrin is 1.3×10-6mol/ml comparable to the activity of lactoferrin from human milk is 1.4×10-6mol/ml

Thus, the correspondence is established recombinant lactoferrin expressed nerealizirane nanoparticles on the basis of the genome of adenovirus human serotype 5 and native lactoferrin from a female donor milk physico-chemical properties and antioxidant activity.

Example 3.

Comparison of antimicrobial activity.

According antimicrobial activity of recombinant human lactoferrin in comparison with native lactoferrin isolated from breast milk was evaluated against reference strains and clinical isolates of micromethods in broth using guidelines to determine the sensitivity of microorganisms to antibiotics (HOWTO MUK 4.2.1890-04 "Determination of the sensitivity of microorganisms to antibiotics", decl. Chief state sanitary doctor of the Russian Federation on March 4, 2004).

Presented in table 1 data shows the Ute in most cases, equal or smaller in the recombinant lactoferrin (which means even the best antimicrobial activity he compared with native lactoferrin) minimum concentrations of recombinant lactoferrin of human and native human lactoferrin, inhibit the growth standard test microbes.

Thus, it was found that recombinant human lactoferrin expressed nerealizirane nanoparticles on the basis of the genome of a human adenovirus 5-th serotype with the insertion of a gene coding for human lactoferrin and native lactoferrin isolated from breast milk,have a similar antimicrobial activity.

Example 4.

Assessment detoxifying actions determine the minimum effective dose.

Detoxifying action of the pharmaceutical composition was evaluated in vivo using laboratory animal models of toxicity induced cytotoxic drug cisplatin, a widely used schemes of chemotherapy patients with malignant processes.

Antitumor activity of cisplatin is implemented due to the formation of free radical products that disrupt DNA synthesis by intra - and magnievykh crosslinks DNA and induce lipid peroxidation (LPO) of the cell membranes of the kidneys, liver, lungs and other organs. This, ultimately, leads to dysfunction of various organs and extensive complex of toxic reactions, the intensity of which increases with increasing doses of cisplatin.

To evaluate the impact of pharmaceutical HDMI is in the toxic reactions caused by cisplatin, it once was administered at a dose of 16 mg/kg, which resulted in a 50±6%mortality in the control group from acute toxic reactions (figure 1). The death of animals was started 4 days after exposure and continued for 5 days. Toxic effects were reflected in the increased activity of enzymes - ACT and ALT, increased creatinine on the 7th and urea on the 3rd and 7th day of observation (table 2).

As a detoxifying agent, experimental animals for 72 hours prior to the introduction of cisplatin intravenously injected rereplacenocase nanoparticles with the insertion of a gene of human lactoferrin in doses of 1.4×1011of 4.3×1011of 4.3×1012FC/m2and animal groups compare three Modelisation lactoferrin man from a donor female in a dose of 10 mg/kg (dose rate of 30 mg/kg), first, the introduction of 24 hours after infusion of cisplatin.

Figure 1 presents the data, where:

1 - the death of animals in the control group, cisplatin was administered once intravenously at a dose of 16 mg/kg;

2 - the death of animals from the experimental group, which was introduced farmatsevticheskiy composition once intravenously at a dose of 1.4×1011FC/m272 hours before the administration of cisplatin;

3 - the death of animals from the experimental group, which was introduced farmatsevticheskiy composition once intravenously at a dose of 4.3×1011FC/m2for 72 h prior to the introduction of zysblat is on;

4 - the death of animals from the experimental group, which was introduced farmatsevticheskiy composition once intravenously at a dose of 4.3×1012FC/m272 hours before the administration of cisplatin;

5 - the comparison group, the animals which were introduced native human lactoferrin from donor breast milk after 24 h after injection of cisplatin at a dose of 10 mg/kg over the next 3 days (dose rate of 30 mg/kg).

Thus, in the period of implementation of the toxic effects of cisplatin in experimental groups of animals observed maximum production of recombinant human lactoferrin, which has continued for 10 days, which resulted in a reduction in mortality to 13±6% with the introduction of rereplacenocase nanoparticles with the insertion of a gene of human lactoferrin in doses of 1.4×1011of 4.3×1011and 4.3×1012FC/m2. In the comparison group, where animals received three native human lactoferrin, mortality was 19±6%.

The obtained results indicate the presence of detoxifying effect against the toxic effects of cisplatin in pharmaceutical compositions, producing recombinant lactoferrin and native lactoferrin from a female donor milk.

The results of biochemical studies confirmed the decrease in the intensity of the toxic reactions of cisplatin with the introduction of the pharmaceutical composition in D. the sight of 4.3×10 11FC/m2and 4.3×1012FC/m2.

Table 2
ImpactBiochemical parameters of blood of animals
day 4day 7day 10
ACT, IU/l
Cisplatin201±15289±15266±21
Pharmaceutical composition (4,3×1011) + cisplatin174±10167±17140±10
Pharmaceutical composition (4,3×1012) + cisplatin178±8187±23123±12
cisplatin + native human lactoferrin171±13156±17156±10
Phys. solution123±25
ALT, IU/l
Cisplatin79 the 10 125±861±3
Pharmaceutical composition (4,3×1011) + cisplatin56±1468±1364±9
Pharmaceutical composition (4,3×1012) + cisplatin61±169±960±10
cisplatin + native human lactoferrin45±875±1551±8
Phys. solution48±12
Creatinine, µmol/l
Cisplatin65±1297±10124±21
Pharmaceutical composition (4,3×1011) + cisplatin51±1154±1359±13
Pharmaceutical composition (4,3×1012) + cisplatin46±967±1666±11
cisplatin + native human lactoferrin 46±557±1556±6
Phys. solution35±9
Urea, mmol/l
Cisplatin10,5±0,911,0±1,16,1±0,9
Pharmaceutical composition (4,3×1011) + cisplatin5,4±1,85,9±1,24,8±0,5
Pharmaceutical composition (4,3×1012) + cisplatin6,0±0,85,4±0,96,2±1,5
cisplatin + native human lactoferrin5,4±0,36,0±0,55,2±0,5
Phys. solution4,7±1,0

For example, table 2 presents data that show that virtually all of the biochemical indices of the blood of animals in the groups with the introduction of cisplatin on the impact of pharmaceutical compositions and native protein corresponded to the average normal values in all periods of observation. The performance function of the national state of the liver - ACT and ALT levels were not increased in these groups so much as in the control, and the indicators of the functional state of the kidneys, creatinine and urea were maintained close to normal values in contrast to the significantly higher values in the control groups, i.e. recombinant human lactoferrin as native, isolated from breast milk reduces the nephro - and hepatotoxicity caused by cytostatics.

Thus, recombinant human lactoferrin has a detoxifying effect against the toxicity of cisplatin, suggesting its functional activity similar to the native protein.

The estimation of the detoxifying action of the pharmaceutical composition in models of chemically induced toxicity has allowed to determine the minimum therapeutically effective dose, equal to 4.3×1011FC/m2(toxic dose (TD) is expressed in the number rereplacenocase nanoparticles on m2the body surface of the animal, the dose of medicines, calculated on the surface area of the body for various animal species and humans are equivalent. (Gabriel RU, Manual on experimental (preclinical) study of new pharmacological substances, 2000, 98 pages).

Example 5

Limits tolerated doses.

Values outside lane is wearable doses of the pharmaceutical composition were determined in experiments on mice after a single intravenous injection (acute toxicity) of laboratory animals at doses: 4,3×10 11FC/m2, 43,0×1011FC/m2, 215,0×1011FC/m2, 430,0×1011FC/m2and 860,0×1011FC/m2.

In the study of acute toxicity, the minimum therapeutic dose, calculated on m2was increased in 10, 50, 100 and 200 times. Excess DT 10-200 times sufficient to obtain reliable information on Toxicological safety of the investigational pharmacological tools. Minimally effective dose, equal to 4.3×1011FC/m2and route of administration were selected on the basis of research results obtained during the study of its pharmacological activity in animals with severe exogenous intoxication caused by the introduction into the organism of animals toxic and lethal doses of highly toxic anticancer drug (example 4).

In experimental animals after a single intravenous pharmaceutical composition in the above doses were recorded clinical signs of possible intoxication, death from toxicity, time loss, change in body mass. Weighing of animals was performed before drug administration (background), and at 3, 7, 10, 14, 21 and 30 days after injection. At 30 days, all surviving animals were euthanized with subsequent autopsy. Conducted post-mortem examination of all dead animals, vkluchaya the macroscopic assessment of the cavities of the body, internal organs and tissues. Irritant effect of the drug was assessed by visual inspection of the injection site. Control animals were injected with a stabilizing buffer solution (control substance No. 1) and isotonic (0.9%) sodium chloride solution (control substance No. 2).

The received data describing the toxicity of pharmaceuticals in a single intravenous administration to mice, are presented in tables 3, 4, 5.

Table 3
no groupThe investigational or control substanceDose, FC/m2The total number of animalsThe death of animals from toxicity
Just died%Time of death, the day
Males
1The pharmaceutical composition4,3×1011600-
2Farmace the political track 43,0×1011600-
3The pharmaceutical composition215,0×1011600-
4The pharmaceutical composition430,0×1011600-
5The pharmaceutical composition860,0×101162332 and 9
6Stabilizing buffer (control substance No. 1)25 ml/kg600-
7Stabilizing buffer (control substance No. 1)50 ml/kg6 00-
80.9% NaCl solution (control substance No. 2)50 ml/kg600-
Female
1The pharmaceutical composition4,3×1011600-
2The pharmaceutical composition43,0×1011600-
3The pharmaceutical composition215,0×1011600-
4The pharmaceutical composition430,0×1011600 -
5The pharmaceutical composition860,0×1011611713
6Stabilizing buffer (control substance No. 1)25 ml/kg600-
7Stabilizing buffer (control substance No. 1)50 ml/kg600-
80.9% NaCl solution (control substance No. 2)50 ml/kg600-

Presented in table 3 the results of the experiment show that a single intravenous administration of a pharmaceutical composition mice - males and mice-females at doses ranging from 4.3×1011FC/m2430×1011FC/m2was satisfactorily transferred animals. Death from toxicity of no is tawala. With the introduction of the pharmaceutical composition at a dose equal to 860×1011FC/m2watched the death of the mice from the toxic effects. Death in the group of male mice was 33%and in the group of mice-females - 17%. The average mortality of mice (males and females) when using this dose was 25%. The dead animals symptoms of intoxication were missing.

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Table 4
no groupThe investigational or control substanceDose, FC/m2Symptoms of intoxication
Males
1The pharmaceutical composition4,3×1011Short, within 3 hours physical inactivity
2The pharmaceutical composition43,0×1011Short, within 3 hours physical inactivity
3The pharmaceutical composition215,0×1011A short course is E. 3 hours physical inactivity
4The pharmaceutical composition430,0×1011Short, within 3 hours physical inactivity
5The pharmaceutical composition860,0×1011Short, within 3 hours physical inactivity
6Stabilizing buffer (control substance No. 1)25 ml/kgsymptoms of intoxication were absent
7Stabilizing buffer (control substance No. 1)50 ml/kgsymptoms of intoxication were absent
80.9% NaCl solution (control substance No. 2)50 ml/kgsymptoms of intoxication were absent
Female
1The pharmaceutical composition4,3×1011Short, within 3 hours physical inactivity
2The pharmaceutical composition43,0×1011Short, within 3 hours physical inactivity
3The pharmaceutical composition215,0×1011Short, within 3 hours physical inactivity
4The pharmaceutical composition430,0×1011Short, within 3 hours physical inactivity
5The pharmaceutical composition860,0×1011Short, within 3 hours physical inactivity
6Stabilizing buffer (control substance No. 1)25 ml/kgsymptoms of intoxication were absent
7Stabilizing buffer (control substance No. 1)50 ml/kgsymptoms of intoxication were absent
80,9% of the NaCl solution (control substance No. 2) 50 ml/kgsymptoms of intoxication were absent

Table 4 presents about the presence of intoxication after the introduction of the pharmaceutical composition in all the tested doses in mice (males and females) observed symptoms of intoxication in the form of short (within 1 - 3 hours) reduction of motor activity (inactivity). 24 hours after injection and then, during the whole period of observation of the animals (30 days) symptoms of intoxication were absent. Animals were active, reaction to human, tactile and painful stimuli was expressed.

As can be seen from the data presented in table 5, in mice treated with the pharmaceutical composition once intravenously at doses ranging from 4.3×1011FC/m2to 860,0×1011FC/m2and in the animals of the control groups treated with a stabilizing buffer solution and isotonic (0.9%) sodium chloride solution, the weight (both males and females) were uniformly increased throughout the period of observation of animals (30 days).

The autopsy of the dead mice revealed venous plethora of internal organs, namely, the spleen and liver. In other organs and tissues of these mice macroscopically pathological is izmenenii, associated with the toxic effect of the composition is not revealed.

As a result of the research has determined the maximum tolerated (MTD) and lethal dose of a pharmaceutical composition for mice when his single intravenous:

- dose 430,0×1011FC/m2described as MTD;

- dose 860,0×1011FC/m2characterized as partially lethal, leading to the death of 25% of the animals.

Differences in sensitivity of males and females taken in the study of animals to the toxic effects of the pharmaceutical composition when a single intravenous injection was not detected.

Symptoms of intoxication in mice using portable and maximum tolerated dose was expressed in inactivity or adynamia, the severity and duration of which increased with increasing dose of the pharmaceutical composition.

Using a lethal dose of the pharmaceutical composition in mice (860,0×1011FC/m2) led to the deaths of animals on 2-13 days after administration without obvious clinical manifestations of intoxication. Autopsy of dead animals and a thorough post-mortem examination was not possible to determine the cause of death of mice.

Thus, the results of the experiment it was concluded that a single intravenous administration of farmacevticheskoi composition mice in doses ranging from 4.3×10 11FC/m2430×1011FC/m2satisfactorily tolerated and can be recommended within the specified limits for intravenous administration to man.

Example 6

Definition of prolongirovannomu actions.

Pharmacokinetics study is one of the main aspects of preclinical drug discovery research, because it allows feasible to develop modes of use of drugs in the clinic.

The pharmacokinetics of the pharmaceutical composition was assessed by expression of recombinant lactoferrin enzyme-linked immunosorbent assay with a standard pharmacokinetic parameters: maximum concentration of the target protein in serum (Cmax), time to reach the maximum concentration in serum (tmax), the area under the pharmacokinetic curve of concentration-time (AUC) and elimination half-life (t1/2). The pharmacokinetics were studied following a single intravenous pharmaceutical composition in doses of 4.3×1011of 4.3×1012and 4.3×1013FC/m2. Doses were chosen based on the maximum tolerated doses (MTD) of the pharmaceutical composition defined in experiments to study its acute toxicity in mice, where the MTD for mice is 4.3×1013FC/m2when the introduction of which was observed maximum PR is the production of recombinant human lactoferrin in blood serum of experimental animals.

As the comparison drug ispolzovayoniem man, isolated from breast milk, which was administered once intravenously at a dose of 10 mg/kg

Figure 2 presents:

pharmacokinetic curve 1characterizes the concentration in the blood of native human lactoferrin from donor breast milk;

pharmacokinetic curve 2- concentration in the blood of recombinant human lactoferrin obtained after administration of the pharmaceutical composition to mice at a dose of 4.3×1011FC/m2;

Cmaxmaximum concentration of lactoferrin in whey of blood;

tmaxthe time to reach the maximum concentration of lactoferrin in whey of blood.

Thus, it is shown that the concentration of native human lactoferrin (curve 1) has reached the maximum level of 140±32 µg/ml after 17 min (0,011 days) after administration, the elimination half-life was t1/2hours. Single administration of the pharmaceutical composition at a dose of 4.3×1011FC/m2Cmaxlactoferrin person (curve 2) was 3.6±0.5 μg/ml and reached a maximum level on the 6th day. This concentration of the protein was maintained in serum for 3 days, which indicates a dynamic balance between the production of the target protein and its excretion is. It is seen that the two curves have different shapes, different sizes of high and varies the time to reach maximum concentration, but the area under these curves are close in magnitude, and consequently, both medicines supply blood the same amount of drug. Half-time (t1/2) human lactoferrin with the introduction of the pharmaceutical composition is 8.4 days, which is more than 105 times when such drug native lactoferrin (t1/2=0.08 day) and, respectively, confirms the increase in time of the presence of human lactoferrin in blood flow.

Figure 3 provides:

pharmacokinetic curve 1characterizes the concentration in the blood of native human lactoferrin from donor breast milk;

pharmacokinetic curve 2- concentration in the blood of recombinant human lactoferrin obtained after administration of the pharmaceutical composition to mice at a dose of 4.3×1012FC/m2;

Cmaxmaximum concentration of lactoferrin in whey of blood;

tmaxthe time to reach the maximum concentration of lactoferrin in whey of blood.

Intravenous pharmaceutical composition at a dose of 4.3×1012FC/m2Cmaxmanufacture is by about 4 times on the introduction of the pharmaceutical composition at a dose of 4.3×10 11FC/m2and although it remains lower than when the introduction of native lactoferrin, area under the curve 2 (pharmaceutical composition) 5 times larger than the area under the curve 1, due to the continuous production of recombinant protein in a long time.

Introduction the pharmaceutical composition at a dose of 4.3×1013FC/m2allows you to reliably estimate the flow rereplacenocase nanoparticles (product of the target protein in the organs and deducing from them. So farmacocinetica was assessed with the introduction of the pharmaceutical composition at a dose of 4.3×1013FC/m2.

Figure 4 shows:

pharmacokinetic curve 1characterizes the concentration in the blood of native human lactoferrin from donor breast milk;

pharmacokinetic curve 2- concentration in the blood of recombinant human lactoferrin obtained after administration of the pharmaceutical composition to mice at a dose of 4.3×1012FC/m2;

Cmaxmaximum concentration of lactoferrin in whey of blood;

tmaxthe time to reach the maximum concentration of lactoferrin in whey of blood.

After intravenous administration of the pharmaceutical composition at a dose of 4.3×1013FC/m2through 6.8 days in serum was achieved m ximala concentration of recombinant human lactoferrin, equal 364 µg/ml, followed by two-phase reduction of the concentration produced lactoferrin human serum. The figure shows that the first phase of bearsdley continued to 9.4 days, at which the half-life period (t1/2) amounted to 5.4 days, the second phase - 13.8 days with t1/2=4.8 days. The area under the pharmacokinetic curve 2 (pharmaceutical composition) in 73 times more than under the curve 1.

Thus, compared to the total concentration of recombinant human lactoferrin present in animals with the introduction of various doses of the pharmaceutical compositions allows us to conclude that the dose of 4.3×1011FC/m2producing recombinant human lactoferrin about the amount corresponding single intravenous native lactoferrin in the dose of 10 mg/kg, the dose of the composition in 10 times (4.3×1012FC/m2) leads to increased production of lactoferrin 3.6 times, and the introduction of the pharmaceutical composition at a dose of 4.3×1013FC/m2101 times. Pharmacokinetic curves reflect the prolonged action of the pharmaceutical composition from 12 hours to 30 days after injection.

Example 7

Determination of the route of administration of the pharmaceutical composition.

Evaluation of the irritant action of the pharmaceutical composition when it is once NR is trevenna the introduction was carried out macroscopically and using microscopic (histological) methods.

The study was performed in 12 rabbits breed "Chinchilla", male. The pharmaceutical composition was administered to rabbits intravenously once (in the marginal vein of the ear) in a volume of 1.0 ml at a dose of 4.3×1011FC/m2. Control animals received intravenously was administered isotonic (0.9%) sodium chloride solution in the same mode.

Material (fragment rabbit ear with marginal vein) to the microscopic (histological) studies were selected on the 3rd and 14th day after administration of the pharmaceutical composition.

Fragments ear Vienna were fixed in 10% neutral formalin, after which each sample was taken 2 cross cut section of the ear with Vienna and subjected to further conventional histological processing, including washing in running water, dehydrated in alcohols, soaking in chloroform and paraffin, fill in paraffin, cutting paraffin blocks on the microtome. Slices with a thickness of 5 μm after dewaxing were stained with hematoxylin and eosin, concluded in canadian balsam. Histological specimens were examined in a light microscope series MS 300 firm Micros (Austria) at magnifications of 100, 400, 1000.

Evaluation criteria irritant actions were:

- macroscopic changes: external changes from the side of the vessel (the seal of the vessel, redness of the surrounding skin and inflammatory reaction in the area of the passage is osuda);

microscopic criteria: pathological changes in the walls of the veins, thrombophlebitis, thrombosis.

Visual (macroscopic) analysis of the introduction of pharmaceutical compositions showed that in rabbits at the injection site (marginal vein of the ear) during the entire period of observation was missing: the seal of the vessel, redness of the surrounding skin and inflammatory reaction in the area of passage of the vessel. Thus, macroscopically, evidence of the irritant action of the pharmaceutical composition when her single intravenous injection was not detected.

Histological examination of the field of pharmaceutical compositions observed the following picture:

1) the control rabbits at 3 and 14 days after intravenous injection of 0.9% sodium chloride solution at all rabbits the lumen of the vein was slightly extended, free, with small amount of blood. Pathological changes in the wall of the vein and the surrounding subcutaneous tissue is not found.

2) in rabbits that received the pharmaceutical composition for 3 days after a single intravenous pharmaceutical composition vein in all rabbits were expanded with a small content of blood. In all animals there was a slight bulging of the skin over the vessel. Histological examination in 2 Krol the Cove pathological changes in the wall of the vein and surrounding tissue is not detected. One rabbit slight swelling - out of the plasma into the wall of the vein and the surrounding subcutaneous tissue.

At 14 days after intravenous administration of pharmaceutical compositions in all rabbits the lumen of the vein was not enlarged and contained a small amount of blood. Signs of edema or other pathological changes in the wall of the vein and the surrounding tissue was not detected.

Thus, a single intravenous administration of a pharmaceutical composition had a low-grade and fully reversible irritant effect. Contraindications for intravenous administration of the pharmaceutical composition is not revealed.

Example 8.

Determine whether breeding for implementing the method of administration.

As in previous studies (example 4) was recommended intravenous route of administration of the pharmaceutical composition, carried out an experimental evaluation of its compatibility with blood. This was assessed hemolytic potential pharmaceutical compositions and formulating buffer.

The pharmaceutical composition defines the buffer was diluted with 0.9% sodium chloride solution. Samples were incubated in a thermostat at 37°C.

Table 6
The time of incubation of the tested substances is STV with blood Hemolysis of erythrocytes in %
The pharmaceutical compositionformulating the buffersodium chloride
without cultivation, of 2.33×1011FC/mldiluted 80 times of 2.9×108FC/mldiluted 800 times of 2.9×107FC/mlwithout cultivationdiluted 80-folddiluted 800 times0.9% solution0.1% solution
30 min2,8±0,71,3±0,11,1±0,21,7±0,61,5±0,11,9±0,32,1±0,4
1 hour3,4±0,91,4±0,11,5±0,13,9±0,42,2±0,72,0±0,52,2±0,2
2 hours4,5±0,61,7±0,65,8±0,72,3±0,41,9±0,11,6±0,4100
3 hours25,9±4,92,1±0,31,9±0,336,0±0,72,8±0,12,8±0,11,8±0,4
4 hours94,5±2,52,6±0,12,4±0,184,5±6,52,3±0,12,6±0,22,8±0,4
24 hours1001,9±0,11,5±0,21002,8±0,22,5±0,32,5±0,4

Presented in table 6 the data show that within the 1st hour hemolysis of erythrocytes was absent (the percentage of hemolysis of erythrocytes in experimental and control (incubation of blood with 0.9% sodium chloride solution) samples was 3.4±0.9% and 2,2±0,2%, respectively. Upon further incubation from 2 to 24 hours observed a gradual increase of hemolysis of erythrocytes in experimental samples from 4.5% to 100%. At the same time, the hemolysis of erythrocytes in the control samples did not exceed the value of 2.8%. so with the introduction of undiluted formulating buffer starting 30 minutes after exposure, the time of complete hemolysis occurred in both cases within 24 hours. Hemolysis of red blood cells in the control medium (0.9% sodium chloride solution). When assessing the hemolytic potential stabilizing buffer solution similar results were obtained. So, during the incubation buffer solution with the blood within 1 hour hemolysis of erythrocytes in experimental samples was absent, while increasing the incubation time from 2 to 24 hours noted increase in hemolysis of red blood cells in samples from the 5.8±0.7% to 100%.

Thus, the data obtained in a model system, indicate that the pharmaceutical composition hemolytic activity. This hemolytic activity of the pharmaceutical composition due to the hemolytic activity of formulating buffer.

It was concluded that the dilution of the solution at 80 and 800-fold with 0.9% sodium chloride solution helped to reduce the hemolytic activity of the drug to control values (0,9% sodium chloride solution).

Example 9.

The definition of acceptable solvent

Intravenous pharmaceutical composition in breeding 80 times (example 8) the person examined th the derivative for intravenous solutions - water for injection, 5% glucose 10% glucose, 0.9% sodium chloride solution. Evaluated the stability contained in the pharmaceutical composition rereplacenocase nanoparticles at a dilution of each of these solutions.

For the production experience 3 ml of the composition with the content of 2.33×1011FC/ml was diluted in 200 ml of solvent were breeding in 80 times that corresponded to 2.9×106FC/ml of the mixture after a certain exposure time at room temperature, was introduced permissive culture cells were incubated at 37°C and 5% CO27 days. Stability is contained in the composition rereplacenocase nanoparticles with the insertion of a gene of human lactoferrin was evaluated by changing their titles (evaluated in terms of activity in the presence of belascoaran).

Table 7
Name of solvent orThe exposure time, min
control substance03060
with 0.9% sodium chloride1×1001×1001×100
Water for injection1×1001×1001×100
5% glucose1×1061×1061×106
10% glucose1×1067,3×1056,5×105
Culture medium (control substance)1×l061×1061×106

Evaluation of the results, presented in table 7, showed stability rereplacenocase nanoparticles without significant loss of titles in 5% and 10% solutions of glucose, as well as the control substance. However, in 0.9% solution of sodium chloride and water for injection titles rereplacenocase nanoparticles dropped to zero, which means no stability rereplacenocase nanoparticles in them.

Thus, the obtained results allow to recommend as a suitable solvent for dilution and intravenous pharmaceutical composition 5% and 10% glucose solutions.

Example 10.

The security proof way of introduction developed the Noah song

Data describing the influence of the pharmaceutical composition, dissolved in 5% and 10% solutions of glucose on blood coagulation in rats ex vivo, are presented in table 8. For the production experience 3 ml of the composition with the content of 2.33×1011FC/ml was diluted in 200 ml of solvent were breeding in 80 times that corresponded to 2.9×108FC/ml In the study used unstabilized blood of rats. Samples were incubated in a thermostat at 37°C. the Samples were evaluated visually on close observation of the samples within 3 minutes.

Table 8
Unstabilized venous bloodThe test substance
The pharmaceutical composition + 5% glucoseThe pharmaceutical composition + 10% glucoseSodium chloride 0,9% (control substance)
The coagulation time, sec368,00±15,31427,33±to 11.56392,67±27,09361,67±15,31

The results presented in table 8 show that the pharmaceutical composition is I, divorced 5% or 10% glucose solution is well mixed with the blood and do not lead to the formation of clots. The clotting time of the blood of experimental samples, as well as 0.9% sodium chloride solution (control substance) was comparable and was 427,33±to 11.56; 392,67±27,09 and 361,67±9.28 are seconds, respectively. The clotting time native blood was 368,00±15,31 seconds.

Thus, when making a pharmaceutical composition in the indicated concentrations and formulating buffer in whole unstabilized venous blood of rats changes in the samples, which would testify to the incompatibility of this pharmaceutical compositions and buffer solution with blood not detected.

Example 11

Determination of method of administration of the pharmaceutical composition to the human.

Previous experimental studies

pharmaceutical compositions were performed on mice that have no preexisting immune response to adenovirus person. However, it is known that many people may have pre-existing immune response to the adenovirus person due to natural infection. A powerful immune response neutralizes the input design that can significantly reduce therapeutic effect of the drug. (Harvey B.-G., Hackett NR, El-Sawy T. et al. Variability of human systemic humoral responses to adenovirus gene transfer vectors administrated to different organs. J. Virol, 1999, v.73, pp.6729-674 - The diversity of the antibody response in humans to the introduction of adenoviral vectors in various organs - str).

The efficiency of the alleged scheme to introduce the pharmaceutical compositions regarding the removal of pre-existing immune response was established in experiments on mice with artificially induced pre-existing immune response to the adenovirus human serotype 5 (Ad5).

For a model of preexisting immune response of mouse strain Balb/c mice weighing 7-9 grams three times intramuscularly were immunized with human adenovirus 5-th serotype, which don't have the insert lactoferrin gene in a dose of 3×1011FC on the mouse. Three weeks after immunization in the blood serum of mice in the neutralization was determined by the level of virus-neutralizing antibodies to adenovirus person fifth serotype. The titer of antibodies to adenovirus human for intramuscular administration to mice was 1:64, and was different from the control group (1:8 - non-specific titles).

The declared scheme is the introduction of pharmaceutical compositions based on the features of the immune system, allowing after the initial introduction of a specific antigen and associating it with a "preexisting" antibodies for some time not to form new antibodies in response to repeated stimulation with a specific antigen. The effectiveness of the claimed scheme is s introduction pharmaceutical compositions experimentally confirmed. The first group of mice with induced pre-existing immune response to the adenovirus human 5-th serotype was administered once intravenously recommended in previous studies on the specific activity of the full dose of the pharmaceutical composition is 4.3×1011FC/m2to achieve the maximum concentration of lactoferrin in blood (Cmax=3.6 µg/ml) for 6 days after injection. The second group of mice with preexisting immune response to the adenovirus 5-th serotype full dose of the composition (4.3×1011FC/m2) was introduced fractional intravenously on the first day was administered one-third (1,43×1011FC/m2through the day introduced a full dose of the pharmaceutical composition (4.3×1011FC/m2). For control formed two groups of mice that do not have predshestvuyuschie immune response to adenovirus human serotype 5, one group was injected farmatsevticheskiy composition comprising a dose of 4.3×1011FC/m2, another equivalent volume of physiological NaCl solution. All experimental and control groups consisted of 5 animals.

For registration of the resulting effect on the removal of pre-existing immune response in all mice took serum for 6 days and determined the concentration of lactoferrin person in them (table 9).

Table 9
no groupGroup nameThe investigational or control substanceDose, FC/m2Scheme introductionThe concentration of lactoferrin, ág/kg
1Having a preexisting immune response to Ad5The pharmaceutical composition4.3×1011once the0,8±0,12
2Having a preexisting immune response to Ad5The pharmaceutical compositionof 1.43×1011+4.3×1011Twice fractional next dayto 3.58±0,51
3No preexisting immune responses to Ad5The pharmaceutical composition4.3×1011once the3,6±0,4
4No preexisting immune responses to Ad50.9% solution
NaCl (who completed substance)
3 mlonce the0

The results of the experiment are presented in table 9 showed that intravenous administration of one third of the full dose of the pharmaceutical composition (1,43×1011FC/m2effectively removes predshestvuyuschei immune response to the adenovirus human 5-th serotype. This allowed intravenously full dose of the composition through the day, part of 4.3×1011FC/m2to achieve expression of human lactoferrin in therapeutic concentrations (to 3.58±0,51 mg/kg) for 6 days after administration of the pharmaceutical composition, which is comparable with the concentration of recombinant lactoferrin obtained after a single injection of the composition to the full dose mice that do not have pre-existing immune response. In mice with preexisting immune responses to the introduction of the composition once the full dose was not possible to obtain a high concentration of recombinant human lactoferrin in blood serum (0,8±0.12 µg/kg).

Thus, the introduction of pharmaceutical compositions based on preliminary intravenously one third primary dose (1,43×1011FC/m2and subsequent intravenous injection of the full dose of the composition through the day (4,3×1011FC/m2)allows to get t repitions concentration of recombinant human lactoferrin in the body, having a pre-existing immune response to the adenovirus person fifth serotype.

Further, in clinical studies, doses received during the pre-clinical studies and measured on m2body surface were transferred to man, because they are equivalent (average surface area of the body is equal to 1,62 m2). (Gabriel RU, Manual on experimental (preclinical) study of new pharmacological substances, 2000, 98 pages) (Guidance for Industry. Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers - a Guide for industry. Evaluation of the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER).July 2005, Pharmacology and Toxicology - 7, 19 pages)

Example 12

The definition of therapy human Patient L. thyroid Cancer IV senior class. II gr. (T4N2aM1). Assigned to chemotherapy: cyclophosphamide, vincristine, doxorubicin. At the beginning of treatment appeared serious violations on the part of the blood system - erythropenia, lymphopenia, as well as General weakness, nausea, dyspepsia. After intravenous drip pharmaceutical composition in 67 ml of 10% glucose solution at a dose of 2.33·×1011FC and subsequent intravenous drip a full dose of the pharmaceutical is tion of the composition in 200 ml of 10% glucose solution through the day (7×10 11FC) were improved hematological parameters and eliminating nausea and dyspepsia, General condition of the patient improved. Next conducted prophylactic administration of pharmaceutical compositions according to the same scheme for 24 hours before the next chemotherapy cycle.

Example 13

The definition of therapy man

Patient Century Lymphosarcoma Art. IV (generalized form), the state after surgical treatment. Due to the further chemotherapeutic treatment (cyclophosphamide) there is moderate toxic damage of the liver, pneumonia, Erythro - and lymphopenia. To eliminate these toxic effects were used intravenous drip pharmaceutical composition in 67 ml of 5% glucose solution at a dose of 2.33×1011FC and subsequent intravenous drip full dose of the pharmaceutical composition in 200 ml of 5% glucose solution in a day (7×1011FC). The use of pharmaceutical compositions according to this scheme contributed to the relief of pneumonia, liver failure and improved hemodynamic parameters of blood.

Example 14

The definition of therapy to the person.

The patient P. Metachrony cancer of the right breast T3N0M0. after mastectomy. At the beginning of the course of post-operative radiation therapy (single dose of 2 Gy daily for postoperati the config scar) there is a local radiation reaction in the form of an inflammatory reaction of the skin in the area of exposure as well as the patient experienced a reaction of the General dysfunction of the gastrointestinal tract (loss of appetite, nausea, vomiting, diarrhea). To eliminate these toxic effects were used intravenous drip pharmaceutical composition in 67 ml of 10% glucose solution at a dose of 2.33×1011 FC and subsequent intravenous drip full dose of the pharmaceutical composition in 200 ml of 10% glucose solution through the day (7×1011FC). The use of pharmaceutical compositions according to this scheme has improved the General condition of the patient and to eliminate the inflammatory reaction at the site of irradiation. Indicators ACT, ALT, creatinine and urea in normal.

As a result, the claimed pharmaceutical composition contains rereplacenocase nanoparticles based on the genome of a human adenovirus 5-th serotype with the insertion of a gene coding for human lactoferrin, allowing the introduction of a dose of 7×1011FC to 7×1013FC per person, to achieve prolonged (12 hours to 29 days) expression of human lactoferrin. Expressed the pharmaceutical composition of lactoferrin person corresponds to the native lactoferrin from a female donor milk physico-chemical properties, antioxidant activity and antimicrobial properties. A detoxifying effect pharmaceutical com is osili is achieved at the doses of 7×10 11FC to 7×1012FC per person. For the implementation of the claimed method of treatment proposed pharmaceutical composition, which two forms - 1 ml and 3 ml, content rereplacenocase nanoparticles at a dose of not less than of 2.33×1011FC/ml At stated method of therapy for the first time the pharmaceutical composition is administered to a human intravenously at a dose of 2.33×1011FC contained in 1 ml of the composition, which is pre-diluted with 67 ml of a 5% or 10% glucose solution. The second introduction of the composition passes through a day full dose of 7×1011FC contained in 3 ml of composition, pre-diluted with 200 ml of 5% or 10% glucose solution and injected intravenously. The claimed method of treatment allows safe for humans and therapeutically effective to treat the anaemia of various etiologies for people who have prior immune response to the adenovirus human 5-th serotype, indicating the achievement of the objectives of this invention.

Thus, in comparison with the prototype, the advantages of the claimed pharmaceutical compositions on the basis of rereplacenocase nanoparticles, producing antioxidant protein human lactoferrin are:

- a single injection to obtain therapeutic concentrations in humans;

- prolonged action is s (up to 3 weeks);

- reduced costs of drugs, medical instruments, working time of medical personnel;

- falling away of having to use breast milk to obtain lactoferrin;

- reducing the cost of production;

- the possibility of obtaining a relatively larger amount of lactoferrin.

Thus, the goal of the project aimed at creating a pharmaceutical composition based on rereplacenocase nanoparticles, producing directly in the body antioxidant protein human lactoferrin with antioxidant, antimicrobial, anti-toxic protein, suitable for the treatment of toxic conditions of various etiologies and for achieving sustained therapeutic effect with a single administration of the composition is performed. When using the claimed pharmaceutical composition is a reduced cost of the drug, medical instruments and time of the medical staff to achieve the desired result of the treatment.

Use in the pharmaceutical and clinical practice of the claimed pharmaceutical compositions and methods of its use allows to achieve several technical, medical and economic results:

- the claimed pharmaceutical composition is biocompatible with the human body is and and therapeutically highly effective;

- rereplacenocase nanoparticles, producing antioxidant proteins person is compatible with various media - solvents for intravenous use;

pharmaceutical composition suitable for the treatment of diseases of different localization;

1. Pharmaceutical composition for the treatment of toxic conditions, characterized in that it contains rereplacenocase nanoparticles based on the genome of a human adenovirus 5-th serotype with the insertion of a gene coding for human lactoferrin expressing human lactoferrin, and formulating a buffer when the content rereplacenocase nanoparticles not less 2,33·1011physical particles per ml formulating buffer.

2. The pharmaceutical composition according to claim 1, characterized in that therapeutically effective dose rereplacenocase nanoparticles take 3 ml of the final volume of the composition.

3. The pharmaceutical composition according to claim 2, characterized in that the release form of the drug 1 ml.

4. The pharmaceutical composition according to claim 2, characterized in that the release form of the drug 2 ml.

5. The pharmaceutical composition according to claim 2, characterized in that the release form of the drug 3 ml.

6. Therapies toxic conditions, including the introduction of a composition according to claim 1 in a therapeutically effective dose.

7. The method of treatment according to claim 6, characterized in that the pharmaceutical HDMI is tion is administered to a human intravenously.

8. The method of treatment according to claim 7, characterized in that the pharmaceutical composition is administered to a human intravenously.

9. The method of treatment according to claim 6, characterized in that the introduction of the pharmaceutical composition of conduct for the treatment of toxic conditions due to purulent-inflammatory diseases induced by various microorganisms, physical effects and chemical agents, including means drug and radiation therapy.

10. The method of treatment according to claim 6, characterized in that a therapeutically effective dose rereplacenocase nanoparticles with the insertion of a gene of human lactoferrin is from 7·1011physical particles up to 7·1013physical particles per person.

11. The method of treatment according to claim 6, characterized in that the introduction of pharmaceutical compositions performed sequentially in two stages.

12. The method of treatment according to claim 11, characterized in that the introduction of the pharmaceutical composition in the second phase, conducted a day after the first injection.

13. The method of treatment according to claim 11, characterized in that the introduction of a pharmaceutical composition for the first stage is carried out in a volume containing 1/3 full therapeutic dose, and in the second stage, enter the remaining 2/3 of the full therapeutic dose of the pharmaceutical composition.

14. The method of treatment according to item 13, wherein the 1/3 full dose is farmacevticheskoi composition is dissolved in 66 ml of a physiologically acceptable solvent, and 2/3 of the total dose is dissolved 134 ml of a physiologically acceptable solvent.

15. The method of treatment according to item 15, wherein the physiologically acceptable solvent is a solution of glucose.

16. The method of treatment according to item 15, wherein the physiologically acceptable solvent is 5%glucose solution.

17. The method of treatment according to item 15, wherein the physiologically acceptable solvent is 10%glucose solution.



 

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FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Described is a plasmid which codes esterase Psychrobacter cryohalolentis K5T and contains Ndel/Xhol - a DNA fragment of plasmid pET32a with length of 5.366 thousand base pairs, which includes a promoter of bacteriophage T7, a translation enhancer of gene 10 of the bacteriophage T7, a transcription terminator of the bacteriophage T7, a bla β-lactamase gene which determines resistance of cells transformed by plasmid pPC023 to ampicillin, a replication initiation section ori; and Ndel/Sall - a DNA fragment with size of 0.869 thousand base pairs, which contains a gene which codes the mature form of esterase Psychrobacter cryohalolentis K5T with an amino acid sequence SEQ ID NO: 2, given in the description, having an C-end hexahestidine linker. Provided is an Escherichia coli bacteria strain which is modified by said plasmid, a producer of a polypeptide having esterase Psychrobacter cryohalolentis K5T activity.

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2 cl, 2 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: method involves transformation of a Clostridium acetobutylicum cell by a vector containing: a replicon origin enabling its replication in C. acetobutylicum; a replacement cartridge containing a first marker gene surrounded by two sequences homologous to selected sites around the DNA target sequence, enabling recombination of said cartridge; a second marker gene representing upp counter-selection marker. The cells expressing the first marker gene are selected with the cartridge integrated in their genome. The cells not expressing the second marker gene with the eliminated said vector are selected.

EFFECT: invention enables producing the transformed Clostridium acetobutylicum cell which is genetically stable and marker-free.

31 cl, 6 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: there are presented a polynucleotide coding an antibody, an expression vector, a host cell, compositions containing the antibody, and also a method for preparing the antibody and methods for using the antibody.

EFFECT: invention may be used for preparing therapeutic and diagnostic agents to be used for the purpose of detecting the pathological conditions associated with expression or activity of ephrine B2 ligand pathways.

27 cl, 10 dwg, 3 tbl, 6 ex

Modified phytases // 2329301

FIELD: biotechnologies.

SUBSTANCE: polypeptide with phytase activity is proposed. The sequences are shown in the description. A polynucleotide coding the said polypeptide is described. The application of the said polypeptide for the use in a feed mix or a feed supplement is described.

EFFECT: allows to increase modified phytase resistance against temperature and humidity.

6 cl, 6 ex, 12 tbl, 10 dwg

FIELD: biotechnology.

SUBSTANCE: disclosed is procariotic recombinant host cell containing heterologous protein of replication activation, which activated condition-dependent replicon origin, and extrachromosomal DNA molecule, containing heterologous therapeutic gene and condition-dependent replicon origin. Also disclosed are method for producing of plasmid containing heterologous therapeutic gene and condition-dependent replicon origin, plasmid representing suicide vector for gene transferring, containing heterologous therapeutic gene and condition-dependent replicon origin.

EFFECT: method for avoiding of non-controlled overexpression of therapeutic gene and resistance genes.

44 cl, 41 dwg, 10 tbl, 14 ex

FIELD: biotechnology, genetic engineering, pharmaceutical industry.

SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.

EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.

2 cl, 3 dwg, 4 ex

The invention relates to biotechnology, in particular genetic engineering

The invention relates to immunology

The invention relates to biotechnology, genetic engineering, Microbiology and medical industry and is a engineered in vitro recombinant plasmid, which provides a synthesis of HBsAg

FIELD: metallurgy.

SUBSTANCE: ingot manufacturing method involves tempering of an ingot, multiple forging with series change of orientation axis through 90° at the temperature interval of 773-923 K with total true deformation degree of not less than 3 and further annealing at the temperature above isothermic forging temperature by 50 K during 1-5 hours.

EFFECT: obtaining an austenitic steel ingot with nanocrystalline structure and improved strength properties.

2 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to cold curing epoxide compositions and can be used in making structures, including large-sized structures, from polymer composite materials by vacuum infusion in engineering fields. The epoxide composition includes an epoxide base containing epoxy-diane resin, an active diluent and a curing system based on an amine curing agent and a surfactant, characterised by that the epoxy-diane resin used is a resin or a mixture of resins with molecular weight 340-430, the active diluent used has viscosity of up to 0.1 Pa·s, the amine curing agent is a mixture of a curing agent basedd on an aromatic amine and a cold curing catalyst, and the curing system further includes a heterocyclic imidazole-type compound and a nanomodifier. The technical result is preparation of a high-technology epoxy composition, curable without the need for additional heat and without a large exothermic effect, and characterised by improved physical and mechanical properties.

EFFECT: composition is characterised by high modulus of elasticity of 3,8-4,2 GPa, which allows its use in making deformation-resistant articles from polymer composite materials with higher structural strength.

7 cl, 3 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: invention can be used in chemical industry. Cerium oxide CeO2 nanoparticles are obtained by mixing 0.2 M Ce(NO3)3 · 6H2O solution with supercritical water. The reaction is carried out at temperature of 370-390°C and pressure of 240-260 atm. The ratio of the volume of the cerium salt solution to the volume of supercritical water is preferably equal to 2:10.

EFFECT: synthesis of metal oxide nanoparticles and creating an ecologically clean, wasteless technology.

2 cl, 1 ex, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention is meant for electronics and microelectronics and can be used in making coatings performing carrier transfer or storage functions, in transistors, electrodes, light sources, solar cells, field-emission cathodes, displays and sensors. The coating contains molecules of carbon nanobuds linked to each other by at least one fullerene group 2. Functional groups can be bonded to molecules of carbon nanobuds.

EFFECT: coating has on-off ratio greater than 1, which increases its stability, reduces the size of electric elements, increases their rate of operation and efficiency.

11 cl, 12 dwg

FIELD: chemistry.

SUBSTANCE: invention is meant for electronics and microelectronics and can be used in making coatings performing carrier transfer or storage functions, in transistors, electrodes, light sources, solar cells, field-emission cathodes, displays and sensors. The coating contains molecules of carbon nanobuds linked to each other by at least one fullerene group 2. Functional groups can be bonded to molecules of carbon nanobuds.

EFFECT: coating has on-off ratio greater than 1, which increases its stability, reduces the size of electric elements, increases their rate of operation and efficiency.

11 cl, 12 dwg

FIELD: chemistry.

SUBSTANCE: invention can be used in chemical industry. Fullerene-containing soot is obtained first. The soot is then mixed with a fullerene solvent in order to extract fullerenes from the soot and fullerene solution is filtered in order to separate from spent soot. The solvent used is α-bromonaphthalene. In order to separate C70 fullerene, first extraction is carried out using such aromatic solvents as o-dichlorobenzene or o-xylene, and second extraction of spent soot is then carried out by mixing with α-bromonaphthalene (α-C10H7Br). When mixing with α-bromonaphthalene, the soot is heated to temperature not below 40°C.

EFFECT: high degree of extraction of fullerenes from soot and providing extraction of the selected fullerene first.

3 cl

FIELD: process engineering.

SUBSTANCE: invention relates to powder metallurgy, particularly, to production of silicon dioxide nanopowders. To simplify production of silicon dioxide nanopowders and to clean obtained powder from unsublimated impurities minced silicon dioxide is loaded into reaction crucible top section while carbonic reducing agent is fed into crucible bottom section separated form said top section by baffle. Silicon dioxide is reduced by blowing with water vapors at feeding them into reaction crucible bottom section at 1150-1250°C. Reaction products are discharged via reaction crucible top section with deposition of reduced amorphous silicon dioxide nanopowder.

EFFECT: simplified process.

1 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, and concerns a pharmaceutical composition for the therapy of acute toxic conditions. The composition for the therapy of acute toxic conditions contains a protein - human lactoferrin, and additionally contains non-replicating nanoparticles with an inserted gene of human lactoferrin, and an expressing buffer in the following proportions per a dose: human lactoferrin 50 to 100 mg; non-replicating nanoparticles - 7×1011 of physical particles; the expressing buffer - the rest, ml. Human lactoferrin is either donor breast milk lactoferrin, or any human lactoferrin.

EFFECT: invention provides fast onset and prolonged antitoxic action.

4 cl, 8 ex, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention can be used in chemical industry. Gallium oxide Ga2O3 nanoparticles are obtained by mixing 0.1 M aqueous solution of Ga(NO3)3·8H2O with supercritical water. The reaction is carried out at temperature of 365-384°C and at pressure of 220-240 atm. The ratio of the volume of the gallium salt solution to the volume of supercritical water is preferably equal to 2:10.

EFFECT: synthesis of metal oxide nanoparticles and ecologically clean, wasteless technology.

2 cl, 1 ex, 2 dwg

FIELD: chemistry.

SUBSTANCE: method involves preparation of a suspension of nanodiamond powder and a liquid phase, adding the suspension to an electrolyte and conducting electrolysis to deposit a composite coating. The liquid phase used to prepare the suspension is ethyl alcohol or acetone, wherein the nanodiamond powder is added to the liquid phase in amount of 60-80 vol. %, and the powder in the suspension is dispersed by crushing and attrition grinding with a lap, after which the suspension is added to the electrolyte.

EFFECT: dispersing nanodiamond powder and endowing the powder with resistance to sedimentation and coagulation in the electrolyte.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to a cardioprotective and antioxidant drug. The cardioprotective and antioxidant drug prepared by the three-staged extraction of ground tick trefoil (Hedysarum alpinum) herb in aqueous-alcoholic solutions to be randomly agitated at room temperature in a blacked-out place under certain circumstances; then the fractions are poured out and dried in a thermostat.

EFFECT: drug prepared by the above method possesses no side action and shows manifested cardioprotective and antioxidant action.

2 tbl

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