Method for treating oncological patients
FIELD: medicine, veterinary science.
SUBSTANCE: the present innovation deals with treating malignant tumors. For this purpose, its is necessary to provide a blood supply of an agent that destroys extra-cellular blood DNA. This agent should be introduced in dosages providing alteration of electrophoretic profile of extra-cellular blood DNA. Agent, also, should be introduced at the dosages and modes that provide the level of DNA-hydrolytic activity of blood plasma measured in blood plasma being above 150 Kunz units/l plasma during totally above 12 h daily. Therapy may last without intervals for 2 d, not less. As an agent destroying extra-cellular blood DNA one may apply DNAse, in peculiar case, bovine pancreatic DNAse or recombinant human DNAse. The innovation suggests, also, to apply and agent that binds extra-cellular blood DNA, for example, anti-DNA antibodies. The method provides low-toxic and efficient treatment of tumors, particularly at prolonged, even one's life-long therapy with preparations mentioned.
EFFECT: higher efficiency of therapy.
10 cl, 7 ex, 6 tbl
The invention relates to medicine and veterinary medicine and can be used for the treatment of predominantly solid tumors.
Populations of tumor cells developing in the body of the patient, have an extremely high degree of genetic variability is much greater than that in healthy cells. The genetic variability of populations of cancer cells allows them in the disease process to generate phenotypes that are insensitive to the immune and morphogenetic control, capable of invasion and metastasis and are not sensitive to anticancer therapy. It is considered that the selection and clonal expansion of cancer cells underlie biological and clinical "progression" of tumors. In accordance with these views the strategy of modern anticancer therapy based on the principle of destruction clones of tumor cells in the patient using methods of chemotherapy, radiotherapy, immunotherapy, biotherapy, surgical removal, and various combinations thereof.
From non-surgical methods of treatment of oncological diseases, the most widespread methods of chemotherapy, radiotherapy, biotherapy, and more recently and immunotherapy aimed at the destruction, damage or inactivation of intracellular DNA tumor cells.
Known is by chemotherapeutic treatment methods using platinum drugs, see "Molecular mechanisms involved in cisplatin cytotoxicity". Jordan P, Carmo-Fonseca M, Cell Mol Life Sci 2000 Aug. v 57: pp.1229-35, anthracycline antibiotics, see "Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review, Aubel-Sadron G, Londos-Gagliardi D, Biochimie May 1984 v.66: pp.333-52, alkylating agents, see "An overview of cyclophosphamide and ifosfamide pharmacology". Fleming RA, Pharmacotherapy 1997 Sep-Oct v.17: pp.146S-154S, and derived podophyllotoxin, see "Podophyllotoxins: current status and recent developments", Damayanthi Y, Lown JW, Curr Med Chem 1998 Jun 5: v 3 205-52.
Spread radioimmunotherapeutic methods to irradiate the nucleus of tumor cells containing intracellular DNA, alpha-particles of alpha-emitters, specially delivered inside the cancer cells to increase the efficiency effects on intracellular DNA, see "Targeted alpha therapy: evidence for the potential efficacy of the alpha-immunoconjugates in the management of micrometastatic cancer" Alien BJ, Australas Radiol 1999 Nov v.43: pp 480-6.
Known biotherapeutic and immunotherapy methods aimed at the induction of apoptosis of tumor cells, a process of destruction of the cancer cells, triggered by activation of intracellular nucleases and the subsequent destruction of intracellular DNA of tumor cells, for example, by the introduction of patient gene therapy constructs containing genes that initiate the process of apoptosis, see "Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results" Lang FF, Bruner JM, Fuller ON, Aldape K, Prados MD, Chang S, Berger MS, McDermott MW, Kunwar SM, Junck LR, Chandler W, wiebel JA, Kaplan RS, Yung WK, J Clin Oncol 2003 Jul 1 21:13 2508-18, or genes encoding factors that activate intracellular inducing apoptosis nucleases, see "Adenovirus-mediated transfer of caspase-8 augments cell death in gliomas: Memorandum for gene therapy." Shinoura N, Koike engineering Germany H Furitu T, Hashimoto M, Asai A, Kirino T, Hamada H, Hum Gene Ther 2000 May 20 11:8 1123-37, or by introduction of anti-cancer vaccines, see "Vaccine-induced apoptosis: a novel clinical trial end point?" Amin S, Robins RA, Maxwell-Armstrong CA, Scholefield JH, Durrant LG, Cancer Res 2000 Jun 60: 3132-6.
In the patent US 6455250 described using enzyme Endo-SR for the treatment of cancer through its intracellular delivery into the target cell.
Among these methods, the method chemotherapeutic treatment of tumors using Etoposide-4'-Demethylepipodophyllotoxin 9-[4,6-O-R)-ethylidene]-b-D-glucopyranoside elected us as the prototype. Topoisomerase II is an essential cellular enzyme that regulates many aspects of cellular DNA. The enzyme carries out the inter-conversion of different topological forms of intracellular DNA, generating thus a transient double-stranded DNA breaks. Etoposide as a Topoisomerase II inhibitor, increases the intracellular concentration of complexes of Topoisomerase II - cut DNA. As a result of exposure of the drug, the accumulation of a large number of double-strand breaks in cellular DNA, which causes cell death, see "opoisomerase II as a target for anticancer drugs: when enzymes stop being nice". Fortune JM, Osheroff N., Prog Nucleic Acid Res Mol Biol 2000, v.64: pp.221-53.
The disadvantage of the prototype method and other known methods is the low efficiency. This is explained in the following. Prototype method and other known methods, is the target of therapeutic effects of a tumor cell, first and foremost, its intracellular DNA. The experience of such therapy shows that:
- due to the high genetic variability of tumor cells, as a rule, become insensitive to the applied therapy before used the technique allows them to the extent necessary to destroy;
intracellular DNA is relatively inaccessible target that entails the use of high doses of anticancer drugs and/or complex delivery systems (Drug Delivery Systems).
In addition, it should be noted that the prototype method, providing the impact on intracellular DNA of tumor cells, leads to the inevitable destruction of the DNA of healthy cells, which leads to its high toxicity.
The present invention is supposed solution to the problem of creating high-performance and low-toxic way to treat cancer.
According to the invention this problem is solved due to the fact that injected into the blood agent that destroys extracellular DNA blood; this is what the agent can be administered in doses providing the change in electrophoretic profile of extracellular DNA blood detected by the pulse-gelelectrophoresis; this agent may be administered in doses and modes, providing the level of DNA-hydrolytic activity of blood plasma, measured in plasma and in excess of 150 units Kunz per liter of plasma, for a total of more than 12 hours per day; treatment may be carried out continuously for at least 2 days; as an agent that destroys extracellular DNA blood may be used in enzyme Gnkazy, in particular, bovine pancreatic Tnkase, which is administered in parenteral doses of 50,000 units Kunz to 250000000 units Kunz in the day for 5-360 days, or recombinant human Tnkase, this can be used recombinant human Tnkase I (dornase-alpha), which is administered in parenteral doses of 0.15-500 mg/kg of body weight per day during 5-360 days; treatment may be for life; advanced in the blood may be an agent that binds extracellular DNA blood; as this agent can be used anti-DNA antibodies.
The fact of circulating extracellular DNA in the blood of cancer patients is described in several works (P.Anker et al., Clinica Chimica Acta, v.313, 2001, pp.143-146; Fedorov N.A. et.al., Bull.Exp.Biol.Med., v 102, 1986, pp.281-283). In the patent US 5952170 described determination of extracellular D Is in the blood for diagnosing and predicting the course of cancer. In patents US 6465177 and US 6156504 describe the use of extracellular DNA blood for determination of mutations in oncogenes and microsatellite regions of genes, the study of genomic instability in tumors and use of the results of observations for the diagnosis, monitoring and prognosis of the disease.
However, to date no systematic analysis of the spectrum of extracellular DNA blood and its biological role. Studies of extracellular DNA blood without polymerase chain reaction (PCR) in print is not detected. PCR can greatly distort the composition of extracellular DNA blood due to the specificity of the primers used for amplification. Until recently it was carried out mainly genetic analysis of plasma DNA produced by PCR or blot-hybridization, and aimed at studying changes in certain parts of the genome (for example, in microsatellite and individual genes) in the tumor process (Sanchez-Cespedes M., et al., Ann Oncol, 1998, v 9(1), pp.113-116; Sozzi G., et al., Clin Can Res, 1999, 5 v(10), pp.2689-2692; Chen X.Q., et al., Nat Med, 1996, v 2(9), pp.1033-1035).
Thus, known to the applicant authorities lack knowledge about the genetic repertoire of extracellular DNA in the blood of patients with oncopathology, the biological role of extracellular DNA blood in oncopathology and possible therapeutic effect destroyed the I or inactivation for the treatment of these diseases.
The applicant has established that extracellular DNA in the blood of cancer patients contains a unique qualitative and quantitative composition of the repertoire of genes and genetic regulatory elements, dramatically different from the repertoire of DNA, described in the human genome. In contrast to intracellular DNA, extracellular DNA in the blood of cancer patients contains mostly unique human genes, including genes involved in the maintenance and formation of malignant behavior of cancer cells.
It is shown that extracellular DNA blood cancer contributes to malignant growth.
Destruction, including together with modification and binding, extracellular DNA blood in oncopathology prevents malignant growth. Such interference has as an independent therapeutic value, and increases the effectiveness of conventional therapies.
The above new features of the claimed invention based on a fundamentally new ideas about the mechanism of cancer, allow to draw a conclusion on the compliance of the claimed method the criterion of "inventive step".
The claimed method is implemented as follows.
Materials and methods.
Used the following agents that Deplete extracellular DNA blood: biquadrates Tenkasu (Sigma and Samson-Med), recombinant human Tnkase I (Dornase alpha; Genetech), extracellular nuclease of Serratia Mercenses. The solution Gnkazy for injection was prepared by dissolving the matrix solution Gnkazy in sterile phosphate buffer just before the introduction.
DNA in blood plasma was allocated as follows: fresh (not more than 3-4 hours after collection) blood plasma with added anticoagulant (sodium citrate) was centrifuged on a cushion of Ficoll-PlaquePlus (Amersham-Phannacia) at 1500g for 20 minutes at room temperature. Plasma (1/2 of the total number) were carefully selected, not touching the rest of the cells on the pillow ficoll, and Unscrew at 10000g for 30 minutes, to get rid of debris cells and debris. The supernatant was collected without disturbing the precipitate, was added to 1% sarkosyl, 50 mm Tris-HCl, pH to 7.6, 20 mm EDTA, 400 mm NaCl and an equal volume mixture of phenol-chloroform 1:1. The obtained emulsion was incubated at 65°2 hours, then separated phenol-chloroform by centrifugation at 5000g for 20 minutes at room temperature. The procedure of deproteinization phenol-chloroform was repeated the same way three times, after which the aqueous phase was treated with chloroform, and then diethyl ether. Separation from organic solvents produced by centrifugation at 5000g for 15 minutes. To the obtained aqueous phase was added an equal volume of isopropanol and incubated for h is Chi at 0° C. After precipitation of nucleic acids was separated by centrifugation at 0°, 10000g for 30 minutes. The precipitated nucleic acids were dissolved in buffer containing 10 mm Tris-HCl, pH 7,6, 5 mm EDTA, and put on a pillow of speed cesium chloride (1 M, 2.5 M, 5.7 M) in a centrifuge tube rotor SW60Ti. The amount of DNA was 2 ml, the volume of each step CsCL and 1 ml of the Ultracentrifugation was performed in the device L80-80 (Beckman) 3 hours at 250000g. DNA was collected from the surface of the stairs 5.7 M on fractions. Faction deliberately 12 hours at 4°C. the Presence of DNA in the fractions was determined by agarose electrophoresis, visualization of DNA methyl-ethidium. The amount of DNA was determined spectrophotometrically (Beckman DU70) in a cell with a volume of 100 μl, shooting range from 220 to 320 nm.
In our experiments we used strains of mouse lung carcinoma Lewis and Ehrlich carcinoma. Cells were grown in medium RPMI-1640 with the addition of 10% fetal calf serum, 1% penicillin-streptomycin in an environment with 5% carbon dioxide.
For the induction of tumors in mice cells were grown to monolayer, were separated using a solution of trypsin-EDTA. Cells are washed three times by centrifugation in phosphate buffer and resuspendable 0.5·107in milliliter. Viability was determined by the inclusion of methylene blue in hemocytometer. For injection, the animals were used suspense is not less than 95% viable cells.
Used mouse strain C57B1 and white outbred mice, obtained from the kennel "Rapolano". The weight of the animals 24-26 grams. The animals were kept 6-7 pieces in a cage on a standard diet without water restrictions. LLC cells in a dose of 5·105100 μl of phosphate buffer was introduced into the soft tissues of the thigh. The Ehrlich tumor was perepevalas under the skin of the right side of the introduction of 0.2 ml of 10%suspension of tumor cells in isotonic sodium chloride
In some experiments, we studied the content of extracellular DNA in the blood plasma. DNA was extracted according to a previously described Protocol. The DNA content was measured using sets Pico Green. Electrophoresis of extracellular DNA blood was performed in 1% agarose gel. DNA was stained with ethidium-bromide. A comparative content of high molecular weight (>300 base pairs) fraction of DNA in the panels were evaluated by densitometric. As a token used the phage lambda treated restrictase EcoR and Hind III.
Example 1. Inhibition of tumor development Ehrlich.
Used recombinant human Tnkase I (Genetech).
group 1 - 10 mice inoculated with Ehrlich carcinoma - control. Mice received twice a day 3 to day 7 after inoculation of the tumor intraperitoneal injection of 200 μl of phosphate buffer.
group 2 - 10 mice inoculated with Ehrlich carcinoma treated with Tnkase four times a day is Geneve 3 to day 7 after inoculation of the tumor at a dose of 1 mg/kg intraperitoneally in 200 ál of phosphate buffer.
group 3 - 10 mice inoculated with Ehrlich carcinoma treated with Tnkase four times per day 3 to day 7 after inoculation of the tumor at a dose of 0.5 mg/kg intraperitoneally in 200 ál of phosphate buffer.
group 4 - 10 mice inoculated with Ehrlich carcinoma treated with Tnkase four times per day 3 to day 7 after inoculation of the tumor at a dose of 0.1 mg/kg intraperitoneally in 200 ál of phosphate buffer.
group 5 - 10 mice inoculated with Ehrlich carcinoma treated with Tnkase four times per day 3 to day 7 after inoculation of the tumor at a dose of 0.05 mg/kg intraperitoneally in 200 ál of phosphate buffer.
The results of experiments was assessed by the inhibition of tumor growth (SRW, expressed as a percentage) on the last day of the introduction of Gnkazy. TRO was determined by the standard method. Determined the content of extracellular DNA blood and its electrophoretic fractionation.
The results are shown in table 1: the size of the tumor, the content of extracellular DNA and electrophoretic profile through 7 days after inoculation of the tumor.
|Group||Tumor volume||Inhibition in %||The content of extracellular DNA (ng/ml)||The presence of high molecular weight fractions in the extracellular DNA|
|1 mgkg||23+/-6||76%||to 38.3||0|
|0.1 mg / kg||58+/-12||37%||78,0||70%|
|0.05 mg / kg||87+/-11||10%||98,7||100%|
These data indicate that to achieve the best therapeutic effect it is necessary to use a high enough dose of the drug Gnkazy I.
Example 2. Inhibition of tumor development Ehrlich.
Used recombinant human Tnkase I (Genetech).
Participated in experiment 5 groups of mice inoculated LLC.
group 1 - 7 mice control.
group 2 - 6 mice treated intraperitoneally therapy Dnazol dose of 1 mg/kg 2 times per day 3 to day 5 after inoculation.
group 3 - 6 mice treated intraperitoneally therapy Dnazol dose of 1 mg/kg 2 times per day 3 to day 10 after inoculation.
group 4 - 6 mice received therapy Dnazol dose of 1 mg/kg 2 times per day 3 to day 15 after inoculation.
group 5 - 6 mice received therapy Dnazol dose of 1 mg/kg 2 times per day 3 to day 18 after inoculation.
group 6 - 6 mice received therapy Dnazol dose of 1 mg/kg 2 times in the TCI 3, 5, 7, 9, 11, 13, 15 and 17 days after inoculation.
group 7 - 6 mice treated intraperitoneally therapy Dnazol at a dose of 0.5 mg/kg 4 times per day 3 to day 10 after inoculation.
The results of the experiment were assessed by survival of the animals at 30 and 50 days after inoculation of tumor - table 2
|Group||30 day(the number of live/dead animals in the group)||50 day(the number of live/dead animals in the group)|
These data indicate that the effectiveness of the treatment increases with the duration of treatment. The effectiveness of treatment decreases if it is not continuous. Repeated introduction during the day is preferred.
Example 3. Treatment of carcinoma of the lung.
Sick man, 54 years old was admitted to the hospital with a diagnosis of carcinoma of the lung.
With the consent of the patient, taking into account the exhaustion of all possible is erodov treatment, he was appointed as a daily subcutaneous injection of dornase Alfa. Treatment began with the introduction of a daily dose of 50 mcg/kg Every other day was carried out by measurement of extracellular DNA blood and its electrophoretic fractionation. Once a week was carried out by NMR and x-ray control of the primary tumor and metastases. After seven days in the absence of changes in the content of extracellular DNA in the blood, its electrophoretic pattern and the lack of response from the primary tumor and metastases daily dose was increased to 100 μg/kg in the absence of changes after 7 days produced an increase in the daily dose to 150 mg/kg 2 days after the first injection of the drug at a dose of 150 mcg/kg in the preparation of extracellular DNA blood on the background of insignificant decrease the total amount of extracellular DNA in the blood (less than 20%) had significant (>50%) decrease in the fraction content of extracellular DNA blood larger than 300 pairs grounds. In the next 4 days, the patient improved significantly, and the follow-NMR studies at the end of the seven-day cycle was a decrease of the size of the primary tumor by 25% and there were radiographic signs of regression of the two bone metastatic nodes.
Samples of extracellular DNA of this patient, atie before treatment and 21 days after start of therapy, were cloned using the method that allows you to design reamplification plasmid library extracellular DNA blood representativeness of up to a million clones with an average size of 300-500 base pairs.
Allocated according to a previously described Protocol DNA was subjected to more thorough deproteinization using proteinase K (Sigma) at 65°With removal strongly related proteins. After deproteinization and one treatment of phenol-chloroform at 65°C, DNA was besieged by 2.5 volumes of ethanol overnight. Then DNA was either treated with restriction enzyme EcoRI for 3 hours or Pfu polymerase (Stratagene) in the presence of 300 µm all deoxynucleotidase to remove the sticky ends. Completed DNA was fosforilirovanii polynucleotides T4 (30U, 2 h). Received drugs ligated into the plasmid pBluescript (Stratagene), digested EcoRI or PvuII, respectively, and dephosphorylating alkaline phosphatase CIP (Fermentas) for 1 hour. For ligating usually used 1 μg of vector and 0.1-0.5 ág of serum DNA. Ligation was performed using the Rapid Ligation Kit (Roche) for 10 hours at 16°C. the Amount of ligase mixture was 50 μl. Legirovannoi library transformed into DH12S cells (Life Technologies) using tame the electroporator porator E. coli (BioRad). To transform one library used to 12-20 electroporation the data ditch. For control of the Cup with 1.5% agar and LB medium containing 100 μg/ml ampicillin, were sown cultivation library 10-4, 10-5and 10-6. In both cases, the representativeness of the library was approximately 2-3·106the clones.
Analysis of randomly selected 96 clones with a length of from 300 to 1000 base pairs from a library derived from extracellular DICK blood of the patient before treatment, showed that 55 out of 96 clones represent a unique sequence human DNA. Of the 55 unique fragments of DNA function or product of the corresponding gene using Human Gene Bank were identified for 15 sequences:
|Gene or corresponding protein product||Reported role in cancerogenesis and cancer progression|
|G-protein coupled receptor protein||Key role in neoplastic transformation,apoptosis inhibition, hormone independence and metastasis|
|Snf2 coupled activator CBP (SCARP)||Reduced activator, reported in synovial sarcoma and leukemia.|
|SRY-box containing gene||Reduced modulator expressed in embryogenesis. Reported in medulloblastoma, gonadal tumors, highly metastatic melanoma.|
|Tyrosine kinase||Key role in cancer cell regulation network. Some class homologues are the products of cellular oncogenes.|
|Fibroblast activation protein, a cell surface protease||Involved into cancer invasion and metastasis.|
|Brain testican||Reorted in embryonic rhabdomyosarcoma.|
|KRAB domain Zn-finger protein.||Reported in early embryogenesis, neuroblastoma, Ewing sarcoma, T-cell lymphoma, linked with acquisition of drug resistance in lung cancer.|
|Melanoma associated antigen||Antigen expressed in melanoma cells.|
|N-cadherin||Involved into cancer invasion and metastasis.|
|Interleukin 7||Proposed essential autocrine-paracrine growth factor for many cancers|
|DEAD Box RNA helicaselike protein||Expressed in highly proliferating and cancer cells.|
|Lipin-1||Involved into cancer cell response to cytotoxic drugs.|
|Dynein||Participate in p53 intracellular traffic, reported in prostate cancer and hepatocellular carcinoma.|
|Ramp protein||Reported in human embryonic carcinoma|
Analysis of randomly selected 100 clones from a library derived from extracellular DNA of the patient's blood through 21 days after the start of treatment, showed that more than 90% of the identified sequences of the clones are short fragments of repetitive DNA in the human genome, mainly alpha satellite DNA.
Thus, the use of doses Gnkazy, sufficient for destruction of extracellular DNA blood larger than 300 base pairs, leading to the disappearance of extracellular DNA blood of unique fragments of the human genome, including those involved in the formation and maintenance of the malignant behavior of cancer cells. This results in regression of the tumor according to the Declaration, the act method.
Example 4. Treatment of malignant poorly differentiated lymphoma with diffuse lesions of the spleen and the hilus of the liver with metastatic sites in the liver.
Female 49 years old was admitted to the hospital in serious condition, with fever up to 39°With progressive jaundice and signs of hepatic failure with suspected acute hepatitis. In the study revealed malignant poorly differentiated lymphoma with diffuse lesions of the spleen and the hilus of the liver with multiple metastatic sites in the liver. With the consent of the patient, considering the impossibility of application of specific treatment and constantly progressive picture of the disease, she was prescribed intravenous infusion of bovine pancreatic Gnkazy. Twice a day was carried out by measurement of extracellular DNA blood and its electrophoretic fractionation. On the first day it was introduced 500000 IU of enzyme in the form of two six-hour infusion. In the future, the daily dose was increased by 1,000,000 UNITS per day on a daily basis.
When reaching a daily dose of 5500000 ED showed a significant (>50%) decrease in the content of extracellular DNA in the blood and the disappearance of the fraction of DNA larger than 300 base pairs in an electrophoretic fractionation. On the background of the continuous infusion dose 5500000 ED day the patient's condition began to improve, did Radka and jaundice, improved blood biochemical parameters. Control ultrasound examination, held on the 20th day after the start of treatment, revealed a significant (>40%) reduction in the area of the lesion of the spleen and the disappearance of more than half of the metastatic sites in the liver. The patient was transferred to another medical facility for conducting chemotherapeutic treatment.
Thus, the use of doses Gnkazy, sufficient for destruction of extracellular DNA blood larger than 300 base pairs leads to regression of the tumor according to the claimed method.
Example 5. The study of the influence of polyclonal sera containing antibodies against DNA, on the growth of Ehrlich carcinoma in mice receiving treatment with Dnazol.
Antibodies against DNA was extracted from blood of patients with systemic lupus erythematosus according to the method Shuster A.M. (Shuster A.M. et.al., Science, v.256, 1992, pp.665-667). Such anti-DNA antibodies can not only communicate, but also to carry out the hydrolysis of DNA. As Gnkazy used human recombinant Tnkase I (Genetech).
group 1 - 7 mice inoculated with Ehrlich carcinoma - control.
group 2 - 6 mice inoculated with Ehrlich carcinoma, received on the third day after inoculation of the tumor intravenous injection fraction of human anti-DNA antibodies (IgG) at 200 μg per animal. The animals received intraperitoneal therapy DNA is zoé at a dose of 0.5 mg/kg 4 times per day 3 to day 7 after inoculation.
group 3 - 6 mice inoculated with Ehrlich carcinoma, received on the third day after inoculation of the tumor intravenous injection fraction of nonspecific human immunoglobulin (IgG) at 200 μg per animal. The animals received intraperitoneal therapy Dnazol at a dose of 0.5 mg/kg 4 times per day 3 to day 7 after inoculation.
group 4 - 6 mice inoculated with Ehrlich carcinoma received intraperitoneal therapy Dnazol at a dose of 0.5 mg/kg 4 times per day 3 to day 7 after inoculation.
The effect was determined by the inhibition of tumor growth on day 7 after inoculation (SRW, expressed as a percent)
The size of the tumor 7 days after inoculation are shown in table 4
|Group||Tumor volume||T %|
These data indicate that the combined use of Gnkazy agent and the binding of extracellular DNA in the blood, leading to more significant antitumor effect. Below is an electrophoretic profile of extracellular DNA in the blood plasma of mice in Gruppo, 2 and 3 through 7 days after inoculation of the tumor.
Group: 1 2 3
Clearly noticeable that the combined use of anti-DNA antibodies and Gnkazy leads to a more pronounced destruction of extracellular DNA in the blood, which is manifested not only in the reduction of its content, but also the disappearance of the heavier fractions.
Example 6. The study of the kinetics of degradation of fraction of high molecular weight fraction (larger than 300 base pairs) of extracellular DNA in the blood of a patient with breast cancer in the presence of bovine pancreatic Gnkazy, Proteinase K and bovine pancreatic Gnkazy, Lipase and bovine pancreatic Gnkazy and extracellular deoxyribonuclease Serratia Mercenses with ribonuclease activity and which is as destructive and the modifying agent.
In the patient plasma samples were added to the enzyme, and incubated 45 minutes at 37°C. After 45 minutes the reaction was stopped and carried out the isolation and electrophoretic fractionation by densitometry extracellular DNA blood. The results are presented in table 5.
|The way to handle||% degradation of high molecular weight fractions|
|Proteinase K (0.1 mg/ml)||0|
|Pancreatic lipase (0.1 ág/ml)||0|
|Bovine pancreatic Tnkase (1 Kuntz Units/ml)||25|
|Bovine pancreatic Tnkase (1 Kuntz Units/ml) + Proteinase K (0.1 mg/ml)||35|
|Bovine pancreatic Tnkase (1 Kuntz Units/ml) + Pancreatic lipase (0.1 ág/ml)||40|
|Extracellular deoxyribonuclease Serratia Mercenses (1 Kuntz Units/ml)||45|
These data indicate that the combined use of Gnkazy and agent, modifying the relationship of extracellular DNA blood proteins, lipids and ribonucleic acids, leads to more efficient degradation of high molecular weight (>300 base pairs) fraction of extracellular DNA blood.
Example 7. The effect of different methods of destruction of extracellular DNA on its pathogenic properties.
Mouse C57B1 has been vaccinated highly metastatic or nizkopotentsialnogo strain tumors LLC. On day 9 after inoculation, animals were euthanized and collected a total blood plasma of mice. The total fraction of extracellular DNA blood after extraction were stored at -20°in phosphate buffer.
Participated in experiment 7 groups of mice inoculated nizkotitanistym strain LLC.
group 1 - 6 mice inoculated with nizkotitanistym strain LLC.
group 2 - 6 mice inoculated with either kopetestatusaction strain LLC + intravenously twice (on the seventh and eighth day after inoculation) introduction the total fraction of extracellular DNA of mice inoculated with highly metastatic strain (0.05 µg DNA before the introduction of was dissolved in 500 μl of fresh heparinized blood).
group 3 - 6 mice inoculated with nizkotitanistym strain LLC + intravenously twice (on the seventh and eighth day after inoculation) introduction the total fraction of the DNA of the mice inoculated with highly metastatic strain (0.05 µg DNA before the introduction was dissolved in 500 μl of fresh plasma). Before the introduction of the DNA sample was subjected to photochemical disinfection (Appendix 1 μm methylene blue followed by irradiation with red light for 10 minutes (˜60000 Lux)).
group 4 - 6 mice inoculated with nizkotitanistym strain LLC + intravenously twice (on the seventh and eighth day after inoculation) introduction the total fraction of the DNA of the mice inoculated with highly metastatic strain (0.05 µg DNA before the introduction was dissolved in 500 μl of fresh plasma). Before the introduction of the DNA sample was mixed with 10 μg of hydrolytic anti-DNA antibodies.
group 5 - 6 mice inoculated with nizkotitanistym strain LLC + intravenously twice (on the seventh and eighth day after inoculation) introduction the total fraction of the DNA of the mice inoculated with highly metastatic strain (0.05 µg DNA before the introduction was dissolved in 500 μl of fresh heparinized blood). Before the introduction of the sample was added to 1 μg of the fragment And the plant toxin Ricin, and incubated 1 hour at 37°C. the Ricin is a representative with whom the family RIP (proteins inactivating ribosomes) toxins, widely used to create immunotoxins. In addition to the ability to inactivate ribosomes these proteins have the ability to degenerate and to hydrolyze DNA. For the implementation of the toxic effect of the catalytic unit And toxins RIP type II must be delivered into the cell subunit Century In the absence of a subunit In a chain And not toxic, but polynucleotide-adenylylation activity chain And can be used for the destruction of DNA circulating in the plasma.
group 6 - 6 mice inoculated with nizkotitanistym strain LLC + intravenously twice (on the seventh and eighth day after inoculation) introduction the total fraction of the DNA of the mice inoculated with highly metastatic strain (0.05 µg DNA before the introduction was dissolved in 500 μl of fresh heparinized blood. The DNA. The DNA sample before the introduction was subjected to enzymatic methylation (I.Muiznieks et al., FEBS Letters, 1994, v.344, pp.251-254).
group 7 - 6 mice mice inoculated with nizkotitanistym strain LLC + intravenously twice (on the seventh and eighth day after inoculation) introduction the total fraction of extracellular DNA mice inoculated nizkotitanistym strain LLC.
8 group 6 mice mice inoculated with nizkotitanistym strain LLC + intravenously twice (on the seventh and eighth day after inoculation) introduction the total fraction of the DNA of the mice inoculated with viscometer the political strain (0.05 µg DNA before the introduction was dissolved in 500 μl of fresh heparinized blood). The DNA sample before the introduction incubated in the presence of 200 ng/ml of dornase - alpha 30 minutes at 37°C.
Estimated number of metastatic nodes in the lungs on day 15 after inoculation.
The results of the experiment are shown in table 6.
Thus, extracellular DNA in the blood of mice with vysokokachestvenny strain enhances tumor metastasis less of a malignant tumor. Destruction, linking and modification of extracellular DNA blood hinder the process according to the claimed method.
1. The method of treatment of malignant tumors, characterized in that provide an introduction to blood agent that destroys extracellular DNA blood.
2. The method according to claim 1, characterized in that the agent that destroys extracellular DNA blood is injected in doses providing change electrophoretic profile of extracellular DNA blood detected by the pulse-gelelectrophoresis.
3. The way p is 1, characterized in that the agent that destroys extracellular DNA blood is injected in doses and modes, providing the level of DNA-hydrolytic activity of blood plasma, measured in plasma and in excess of 150 units Kunz per liter of plasma, for a total of more than 12 hours a day.
4. The method according to claim 3, characterized in that the treatment is carried out continuously for at least 2 days.
5. The method according to claim 1, characterized in that as an agent that destroys extracellular DNA blood using the enzyme Tnkase.
6. The method according to claim 5, characterized in that use bovine pancreatic to Tenkasu, which is administered in parenteral doses of 50,000 units Kunz to 250000000 units Kunz in the day during 5-360 days.
7. The method according to claim 5, characterized in that the use of recombinant human Tnkase.
8. The method according to claim 7, characterized in that the use of recombinant human Tnkase I (dornase-alpha), which is administered in parenteral doses of 0.15-500 mg/kg of body weight per day during 5-360 days.
9. The method according to claim 1, characterized in that the treatment for life.
10. The method according to claim 1, characterized in that it further provides an introduction to blood agent that binds extracellular DNA blood.
11. The method according to claim 10, wherein the agent that binds extracellular DNA blood using anti-DNA anti-Christ. ate.
FIELD: medicine, oncology.
SUBSTANCE: the present innovation deals with treating gastric Helicobacter pylori-associated MALT-lymphomas. For this purpose, one should perorally introduce "Vitaflor" preparation as a ferment at daily dosage being not less than 40 g for 4 wk to an empty stomach or in intervals between meals, and in case of no complete regress of lymphoma at monotherapy with "Vitaflor" it is necessary to conduct additional courses of chemotherapy. The method provides cytostatic impact directly upon the tumor and suppresses proliferation of tumoral cells in case of complete absence of chemotherapy-accompanying side effects.
EFFECT: higher efficiency of therapy.
3 cl, 4 ex
SUBSTANCE: method involves administering local tumor cell radiomodification with following radiation therapy and radical surgical operation being applied. The local radiomodification is carried out by applying endovascular tumor tissue perfusion with 5% Metronidasole via superior rectal artery in superoampular rectal cancer cases and via superior rectal artery and one of internal iliac arteries in medial and inferoampular rectal cancer cases with said blood vessel occlusion done using non-lytic radiopaque emboli. Single remote radiation therapy is carried out at a dose of 10 Gy 1 h later after radiomodification being over. Surgical operation is done not later than in 24 h after the irradiation.
EFFECT: increased radiation therapy destruction effectiveness.
FIELD: organic chemistry, medicine, oncology, biochemistry, pharmacology.
SUBSTANCE: invention relates to the development of a method for inhibition in interaction of metalloproteinase 2 with integrin αvβ3 in host cells. Method involves contact of integrin with the amount of compound inhibiting this interaction and represented by the formula (I): wherein each of G1 and G2 represents independently -NH-C(O)-O-R1, -NH-C(O)-O-(CH2)v-(C6H4)-X3, -NH-C(O)-NH-(CH2)v-(C6H4)-X3, -O-C(O)-NH-(CH2)v-(C6H4)-X3, -O-C(O)-O-(CH2)v-(C6H4)-X3 or -NH-C(O)-CH2-(C6H4)-X3; each of Y1 and Y2 represents independently -OH, (C1-C4)-alkyl, (C1-C4)-hydroxyalkyl, (C1-C4)-alkoxy-group, phenyl, benzyl or -NH2; R1 represents (C1-C4)-alkyl; each of X1 and X2 represents independently halogen atom or (C1-C4)-alkoxy-group; X3 represents halogen atom, nitro-group, (C1-C4)-alkyl, (C1-C4)-alkoxy-group or (C1-C4)-perfluoroalkyl; Z represents -C≡C-, -C6H4-, cis-CH=CH-, trans-CH=CH-, cis-CH2-CH=CH-CH2-, trans-CH2-CH=CH-CH2-, 1,4-naphthyl, cis-1,3-cyclohexyl, trans-1,3-cyclohexyl, cis-1,4-cyclohexyl or trans-1,4-cyclohexyl; A represents hydrogen atom (H) or a covalent bond; each of m and n represents independently a whole number o or 1; t represents a whole number o or 1; each of p, r and v represents a whole number 1 or 2 and under condition that when A means hydrogen atom (H) then t is 0; when A means a covalent bond then t = 1, and when m = 0 then Y1 represents (C1-C4)-hydroxyalkyl, and when n = o then Y2 represents (C1-C4)-hydroxyalkyl. Also, invention describes a method for apoptosis induction involving administration of abovementioned substance in the therapeutically effective dose.
EFFECT: improved method for tumor inhibition, expanded assortment of antitumor agents.
37 cl, 9 dwg, 7 ex
FIELD: organic chemistry, amino acids, medicine, pharmacy.
SUBSTANCE: invention relates to using derivatives of cysteine for preparing a medicinal agent. The proposed agent is designated for treatment of diseases arising as a result of formation of heterotrimeric protein G, and to new derivatives of cysteine, and pharmaceutical composition based on thereof. Derivatives of cysteine, in particular, involve the following compounds: bis-1,1'-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo-[2,2a]-pyrazine]-disulfide and bis-1,1'-[7-(2-amino-1-oxo-3-thiopropyl)-2-91-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo-[1,2a]-pyrazine-7-yl]-disulfide. Invention provides high effectiveness of treatment.
EFFECT: valuable medicinal properties of compounds.
6 cl, 7 dwg, 2 tbl, 7 ex
SUBSTANCE: method involves carrying out radiation treatment combined with textile material application on the tumor. Tumor-transformed vaginal uterus neck portion volume is determined with ultrasonic examination techniques. Koletex napkin impregnated with therapeutic cytostatic preparation dose is used as the textile material. The napkin pattern is produced on the basis of ultrasonic examination data. The napkin is quilted with ligature along the perimeter and fixed at the level of vagina fornix by drawing tightly in purse-string mode. Napkin is changed every 24 h within 10-20 days long treatment course.
EFFECT: improved life quality.
FIELD: medicine, biology.
SUBSTANCE: invention relates to application of tissue factor agonist, namely FVII or FVIIa to induce or increase cell journey, as well as to application tissue factor antagonist modified with FVII to reduce or avoid cell journey in treatment of pathological conditions associated with specific control of cell journey or chemotaxis.
EFFECT: method for treatment of improved effectiveness.
14 cl, 14 ex, 10 dwg
SUBSTANCE: disclosed are combine product and kits useful in treatment for solid tumor by application of ZD6126 compound having formula in combination with platinum antitumor drug (e.g. cysplatine) and/or taxane. Treatment methods may include ionizing radiation. In was discovered that certain doses of abovementioned combined substances according to method of present invention have synergic action on solid tumors (as well as on angiogenesis neoplasm).
EFFECT: new method for solid tumor treatment.
12 cl, 5 tbl, 9 dwg
FIELD: medicine, oncology.
SUBSTANCE: one should carry out chemoradiation therapy at applying a cytostatic preparation followed by distance and intracavitary irradiation. Depending upon development of tumor lesion during the first 3 or 6 d it is necessary to conduct monochemotherapy only due to introducing proxiphen together with dimethyl sulfoxide at weight ratio of 4.5-5.0 : 0.5-1.5, correspondingly by applications in "Coletex" napkins. Moreover, a napkin should be pre-impregnated in 20%-dimethylsulfoxide solution and fixed with a tough vaginal tamponade by changing napkins every 24 h. Then since the 4th d or the 7th d simultaneously with application it is necessary to carry out contact irradiation and distance impact onto minor pelvis every 4-6 h at single focal dosage (SFD) being 2 Gy at 10 seances 5 times/weekly with high-activity sources of SFD 2 Gy. The innovation provides tumor regress under conditions of no therapeutic complications, thus, improving patients' quality of life.
EFFECT: higher therapy.
FIELD: medicine, in particular angiogenesis prophylaxis and treatment.
SUBSTANCE: invention relates to 2-cyclooxygenase inhibitors selected from group containing 4-[5-(4-chlorophenyl)-3-phenyl-1H-pyrazole-1-yl] benzenesulfonamide; 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl] benzenesulfonamide; 4-[5-methyl-3-phenyloxazole-4-yl] benzenesulfonamide or pharmaceutically acceptable salts thereof and pharmaceutical composition containing the same in therapeutically effective amount. Said composition are useful in treatment and/or prophylaxis of angiogenesis disorders such as metastasis, eye angiogenesis, diabetic retinopathy, etc. in subjects are needed in such treatment and/or prophylaxis.
EFFECT: new pharmaceuticals for angiogenesis treatment and/or prophylaxis.
SUBSTANCE: method involves introducing antitumor chemo preparations with blood components. To do it, 300 ml of patient autoblood is subjected to centrifuging during 20 min at 2200 rpm. The produced 150-200 ml of autoplasma and 100 ml of packed red blood cells are placed into separate reservoirs. Cys-platinum as single dose of 100 mg is incubated with the autoplasma and cyclophosphane as single dose of 1 g is incubated with the packed red blood cells. Single doxorubicin dose of 30-50 mg is concurrently introduced with one of the preparations. When combined with cis-platinum, doxorubicin is incubated with the packed red blood cells. When combined with cyclophosphane, doxorubicin is incubated with the autoplasma. Reinfusion is carried out to bring total dose of the preparations to 150 mg of doxorubicin, 3-5 g of cyclophosphane and 200 mg of cis-platinum. Pause between the procedures is 3-4 days long.
EFFECT: avoided risk of adverse side effects; increased preparation activity; accelerated treatment course.
SUBSTANCE: method involves administering pancreatine and/or a digestive enzyme mixture acting like pancreatine, in addition to insulin therapy. The remedy is introduced at a dose of 40000 lipase units when eating and 20000 lipase units between food intakes. Enzyme treatment is carried out on the background of patient fecal elastase-1 level not exceeding 100 mcg/g of patient stool.
EFFECT: enhanced effectiveness of glycemia control.
4 cl, 5 tbl
FIELD: medicine, cosmetology.
SUBSTANCE: one should introduce enzymes-containing liposomes of DNAase activity into skin and/or subcutaneous fiber, moreover, one should apply multi-layer MLV-liposomes at their size ranged 500-2000 nm at temperature of phase transition of liposomal membrane being 30-60 C, moreover, one should deliver and deposit an enzyme into intercellular space, and as enzymes with DNAase activity one should apply endo- and/or exonucleases that destroy single and/or double stranded DNA. The innovation prevents the damage of intracellular DNA of alive skin cells and/or its adnexa and/or subcutaneous fiber.
EFFECT: higher efficiency of prophylaxis and correction.
4 ex, 4 tbl
FIELD: medicine, biotechnology, pharmaceutical industry, veterinary science.
SUBSTANCE: method involves addition of tris-HCl buffer (pH 7.5) containing 0.1 M of lysine and 5 mM of EDTA to the Cohn's fraction followed by incubation and centrifugation. Prepared suspension is incubated with 10% PEG-3000 and after centrifugation the solution is applied onto column with lysine-Sepharose at pH 7.5 and plasminogen is eluted with buffer solution at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine and 5 mM of caprylic acid. Then plasminogen is incubated with streptokinase in tris-buffer (pH 7.5) containing 15 mM of lysine, 10 mM of ε-aminocaproic acid and 50% of glycerol. Then method involves chromatography on column with benzamidine-Sepharose at pH 8.5 and elution with buffer at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine, and the end product is lyophilized that comprises about 30 IU/ml of plasmin with purity above 98%, at least 10 mM of lysine and 10 mM of glycine in an aqueous solution at pH about 3.5. The prepared product shows apyrogenic property, absence of toxicity in experiments in laboratory animals and it doesn't cause allergic or other adverse response reactions in intracutaneous or intravenous administrations, and the preparation doesn't cause defects in the vision field after its an intraorbital route of administration.
EFFECT: improved preparing method, valuable medicinal properties of plasmin.
7 cl, 1 ex
FIELD: medicine, neurology.
SUBSTANCE: method involves intravenous administration of autolymphocytes treated with an immunomodulating agent by extracorporal method using cycloferon (250 mg) as an immunomodulating agent. Simultaneously, the following medicinal mixture comprising lidocaine, 100 mg; lidazum, 32 U; dexamethasone, 4 mg; leukinferon, 10 000 U; 40% glucose solution, 4 ml is administrated into interspinal ligaments of spinal column at levels corresponding to thoracal and lumbar enlargements of the spinal cord. The procedure is repeated three times with interval for 48-72 h. Method provides enhancing the effectiveness of lymphostimulation and immunomodulation in cerebrospinal sclerosis. Invention can be used for lymphostimulation and immunomodulation in cerebrospinal sclerosis.
EFFECT: improved method for treatment.
1 tbl, 1 ex
FIELD: biochemistry, pharmaceutical chemistry.
SUBSTANCE: invention relates to preparing conjugate of naturally occurring or recombinant urate oxidase (uricase) bound covalently with poly-(ethylene glycol) or poly-(ethylene oxide) (both are designated as PEG) wherein in average from 4 to 10 PEG strands are conjugated with each subunit of uricase and molecular mass of PEG is about between 20 and 40 kDa. Prepared PEG-uricase conjugates are nonimmunogenic practically and retain at least 75% of uricolytic activity of nonmodified enzyme.
EFFECT: improved preparing method, valuable properties of conjugates.
22 cl, 17 dwg, 12 ex