Method of treatment of tumors

FIELD: medicine, oncology.

SUBSTANCE: method includes the consecutive stages: (a) administration of at least one dose of anti-angiogenic cyclo-(arginine-glycine-asparagine acid)-containing pentapeptide (pentapeptide cRGD), such as cyclo-(Arg-Gly-Asp-D-Phe-[N-Me]-Val); (b) administration of anti-tumor effective amount of radio immunotherapeutic agent(RIT) not later than in 1 hour following administration of pentapeptide cRGD at stage (a); and (c) administration of at least two additional doses of pentapeptide cRGD, where the first additional dose is administered within 2 days after RIT and each additional dose of pentapeptide cRGD is administered with intervals between doses not more than 2 days.

EFFECT: invention provides the synergic effect in regard to apoptosis of tumor cells and endothelial cells of tumor vessels.

30 cl, 6 dwg, 2 tbl

 

The scope of the invention

The invention relates to methods for treating tumors. More specifically, the invention relates to methods for treating tumors by the combination of radioimmunotherapy and antagonist of integrin receptors.

Cross-reference to related applications

This application is a partial continuation of the simultaneously filed patent application, U.S. serial No. 09/787374, filed September 9, 1999

Statement of government interest

The part described in this work was supported by grant No. RO SA-47829 the National cancer Institute and by grants No. DEFG01-00NE22944 and DEFG03-84ER60233 the U.S. Department of energy. The U.S. government may retain certain rights in the invention.

Background of the invention

New and synergistic therapeutic combination is desirable for the treatment of metastatic breast cancer, prostate cancer, Hodgkin's lymphoma and other cancers, many of which are currently incurable with standard therapy with many therapeutic effects. High frequency of p53 mutations and overexpression of the protein bcl-2 in breast cancer increase resistance to chemotherapy and radiotherapy. The system, aimed at the tumor radioimmunotherapy (RIT) can be specifically targeted to the fabric, and she JV is the capacity to deliver specific for cancer cytotoxic antibodies to widespread metastatic homes. However, studies in xenograft models of cancer of the breast man show that RIT as a stand-alone tool usually does not cure these tumors. Penetration into the tumor antibody labeled with a radioactive label may be uneven and may not be sufficient in all areas of the tumor to ensure cure. Currently uses a combination of RIT with other methods of treatment, but additional chemotherapy or external radiation therapy increases the risk of toxicity to the bone marrow, the primary limiting the dose factor at RIT.

Antiangiogenic funds have been proposed as remedies for the treatment of tumors. These funds genetically targeted to normal endothelial cells, which proliferate at a much higher speed during tumor angiogenesis, compared to very low speeds pack endothelium in normal tissues. It was shown that antiangiogenic funds increase therapeutic efficacy in combination with other chemotherapeutics and when used in combination with external radiation therapy. Receptor integrin αvβ3that binds multiple ligands by the amino acid sequence RGD, expressed in normal vascular system, but who meet high expression in the growing vascular system of the tumor, making it a potential target for antiangiogenic funds. High expression and activation of integrin αvβ3correlated with more metastatic and invasive breast tumors. It was shown that inhibition of activity αvβ3a monoclonal antibody (MAb) and cyclic Pentapeptide RGD caused apoptosis of the endothelium, inhibited angiogenesis and increased permeability of the endothelial monolayer. Inhibition of activity αvβ3was associated with reduced tumor growth in xenografts of breast cancer and melanoma xenografts. Synergy cyclic Pentapeptide RGD with fused protein antibody IL-2 led to increased effectiveness of therapy in mouse models of melanoma, colon cancer and neuroblastoma. Selective capture of the tumor was demonstrated by cyclic pentapeptides RGD labeled with a radioactive label.

The invention

The present invention provides a method of treating a patient having a tumor, such as tumor, prostate tumor, breast cancer, lymphoma and the like. The method is treatment with radioimmunotherapy with many therapeutic effects (CMRIT), including the introduction of the patient antiangiogenic cyclo-(arginine-glycine-aspartic acid is one)Pentapeptide (Pentapeptide cRGD) and radioimmunotherapeutic tool (RIT). The method includes the sequential treatment, including the first stage of the introduction of the patient at least one dose of Pentapeptide cRGD. After this initial treatment Pentapeptide cRGD the patient is given anti-cancer effective amount RIT. After treatment RIT patient is administered at least one additional dose of Pentapeptide cRGD.

The quantity and timing of injection of each dose of Pentapeptide cRGD preferably chosen so that it was equal or close to the maximum tolerated dose for the patient, i.e. at the maximum level at which toxicity Pentapeptide cRGD patient was therapeutically acceptable.

The way CMRIT the present invention provides significantly greater antitumor efficacy compared with treatment one RIT or one Pentapeptide cRGD at the same dosage levels. The way CMRIT also provides greater apoptosis of tumor cells and endothelial cells in the tumor compared with the treatment of one RIT or one Pentapeptide cRGD at the same dosage levels.

In one aspect the present invention also provides a kit comprising a first container comprising at least one standard dose of RIT and one or more additional containers, comprising only at least two standard doses Pentapeptide is cRGD. Each of the containers includes a label describing the contents of the container, optional, sequence of administration and any other relevant information required by the governmental regulations relating to pharmaceutical drugs and radioactive substances. The kit may also include printed instructions for use of the contents of containers for the treatment of tumors, as described in the present description means.

Brief description of drawings

Figure 1 presents a histogram that illustrates the increasing frequency to treat tumors in mice treated way CMRIT of the present invention, in comparison with a method of treating one RIT and one Pentapeptide cRGD, and PR denotes the partial regression, and CR indicates complete regression.

Figure 2 presents a graphical image of the data on toxicity in mice treated way CMRIT of the present invention, in comparison with the methods of treatment for one RIT and one Pentapeptide cRGD.

Figure 3 shows microphotography images of tumor cells that exhibit increased apoptosis in tumors from mice treated way CMRIT of the present invention.

Figure 4 represents a graphical image data showing apoptosis (A) in all cells and (B) in endothelial who lettah (EU) in tumors from mice, treated the way CMRIT of the present invention, according to the TUNEL method.

Figure 5 shows microphotography images of tumor cells that exhibit reduced cell proliferation in tumors of mice treated way CMRIT of the present invention, in comparison with a method of treating one RIT and one Pentapeptide cRGD.

6 shows microphotography image of the tumor cells of the breast NRT 3477, which demonstrates the expression β3and CD31 cells.

A detailed description of the preferred implementation options

The present invention can be implemented in many different forms. Certain embodiments of shown in the drawings and described in detail in the description and the claims. The present description is an illustration of the principles of the invention and is not confined to certain variants of implementation, which are illustrated in the present description.

The method of radioimmunotherapy with many therapeutic effects (CMRIT) for the treatment of tumors in a patient includes successive stages:

(a) introducing the patient at least one dose of antiangiogenic cyclo-(arginine-glycine-aspartic acid)Pentapeptide (Pentapeptide cRGD);

(b) introducing the patient antitumor effective amount radioimmune therapeutic agent (RIT) and

(C) introducing the patient at least one additional dose of Pentapeptide cRGD.

The quantity and timing of injection of each dose of Pentapeptide cRGD preferably chosen so that it was equal or close to the maximum tolerated dose for the patient, i.e. at the maximum level at which toxicity Pentapeptide cRGD patient was therapeutically acceptable. Maximum tolerated dose can be easily determined by methods well known in the pharmaceutical field. For example, the toxicity of Pentapeptide cRGD can be obtained in the clinical studies. The amount of each dosage is preferably in the range of from about 0.05 mg to about 500 mg, preferably from about 0.1 to about 100 mg, most preferably from about 0.2 to about 20 mg total Daily dosage is preferably in the range of from about 0.001 to about 2 mg/kg body weight, preferably, from about 0.002 to about 1 mg/kg, most preferably, from about 0.002 to about 0.2 mg/kg However, the specific dose for each prospective patient depends on many factors, for example, from the activity of a specific used connection Pentapeptide cRGD, age, body weight, General health, sex, diet, time and route of administration and rate you is edenia, pharmaceutical combination and severity of the particular disorder, about which treatment is applied. Preferably parenteral administration, most preferably, intraperitoneal (/b) introduction, however, it is also envisaged orally, in the form of a suppository or local introduction. If desired, additional doses of Pentapeptide cRGD you can keep typing in the period up to several months after the initial RIT.

Pentapeptide cRGD you can make with pharmaceutically acceptable excipients and carriers such as buffers and similar substances, are well known in this field. Suitable excipients are organic or inorganic substances which are suitable for enteral (for example oral or rectal), parenteral (e.g. intravenous injection) or local (e.g., local, dermal, ocular or nasal) administration, or for administration in the form of an inhalation spray, and which do not react with the new compounds, and their examples are water or an aqueous isotonic saline solution, lower alcohols, vegetable oils, benzyl alcohols, polyethylene glycols, glycerol triacetate and other glycerides of fatty acids, gelatin, soy lecithin, carbohydrates such as lactose or starch, magnesium stearate, talc, cellulose and petroleum jelly.

D is I the oral administration of great interest are simple pills, tablets, coated tablets, capsules, syrups, juices or drops; of particular interest are film-coated tablets, and capsules with intersolubility floor. Suppositories are used for rectal administration, solutions, preferably oily or aqueous solutions, and also suspensions, emulsions or implants are used for parenteral administration.

Examples of forms that are suitable for local use, are solutions that can be applied in the form of eye drops, as well as, for example, suspensions, emulsions, creams, ointments or compresses. For administration in the form of an inhalation spray, you can apply the aerosols, which contain the active principle or dissolved or suspended in gas-propellant or mixture of gaseous propellants (e.g., carbon dioxide or ferromagnetically the deputies). In this case, the active principle is properly used in micronized form, with the presence of one or more additional physiologically compatible solvents, such as ethanol. Inhalation solutions can be entered using a conventional inhalers. Pentapeptide cRGD you can liofilizirovanny and received lyophilizate be used, for example, for the manufacture of injectable drugs. Injections can bolus or continuous the CSOs infusion (for example, intravenous, intramuscular, subcutaneous or intrathecal). These preparations can be sterilized and/or may include ancillary ingredients such as preservatives, stabilizers and/or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances, colorants and/or fragrances. If desired, the Pentapeptide cRGD may also contain one or more other active ingredients, including, for example, one or more vitamins, and the like.

Pentapeptide cRGD can be used as such or in the form of one or more of its physiologically acceptable salts. Pentapeptide cRGD can turn into an internal salt or an acid additive salt with acid. Suitable acids for this reaction are, in particular, those which give physiologically acceptable salts. It is possible to use inorganic acids, their examples are sulfuric acid, nitric acid, halogen acids such as hydrochloric acid or Hydrobromic acid, phosphoric acid such as orthophosphoric acid, sulfamic acid, and also organic acids, in particular aliphatic, alicyclic, analiticheskie, aromatic or heterocyclic mono - or polybasic carboxylic, sulfonic or chamois is e acid, for example, formic acid, acetic acid, propionic acid, Pavlova acid, diethyloxalate acid, malonic acid, succinic acid, Emelyanova acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, benzoic acid, salicylic acid, 2 - or 3-phenylpropionate acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinamide acid, methane - or econsultancy acid, ethicality acid, 2-hydroxyethanesulfonic acid, benzolsulfonat acid, para-toluensulfonate acid, naphthalene-mono - and-desulfonema acid, laurylsulphate acid.

Alternatively, the acid form of Pentapeptide cRGD can turn into one of its physiologically acceptable metal salts or ammonium reaction with base. Particularly suitable salts in this context are the salts of sodium, potassium, magnesium, calcium and ammonium, and substituted ammonium salts, e.g. salts of dimethyl-, diethyl - or Diisopropylamine, salt monoethanol-, diethanol - or triethanolamine, salts of cyclohexylamine, salts of dicyclohexylamine, salts of dibenzylethylenediamine, as well as, for example, salts with N-methyl-D-glucamine or with arginine or lysine.

Preferably, the Pentapeptide cRGD is a

cyclo-(Arg-Gly-Asp-D-Phe-Val (EMD 66203),

cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (EMD 121974, cilengitide, commercially available from Merck KGaA, Darmstadt, Germany) or

cyclo-(Arg-Gly-Asp-D-Phe-1-aminocyclohexanecarboxylic acid) (EMD 270179),

the receipt of which is described in U.S. patent No. 5866540 and 6001961 issued Jonczyk et al., relevant descriptions of which are incorporated into this description by reference. Most preferably, the Pentapeptide cRGD is a cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val).

Preferably, the first dosage of Pentapeptide cRGD administered to the patient no later than 1 hour before the holding of the RIT. Preferably, at least one additional dose of Pentapeptide cRGD administered within about two days after the RIT. In a preferred embodiment of the invention, at least about 2 additional doses of Pentapeptide cRGD administered to the patient after a dose RIT, preferably at least about 3 additional doses of Pentapeptide cRGD, most preferably at least about 4 additional doses. In a particularly preferred implementation, at least about 5 additional doses of Pentapeptide cRGD sequentially injected dose of RIT. Preferably, each additional dose of Pentapeptide cRGD introduced at intervals of not more than about 2 days between doses. If desired, the introduction of additional doses of RIT can continue for several them of months with a frequency of approximately 2 doses per week.

Dosage RIT preferably based on the level of radioactivity of specific tools RIT. The number of used RIT preferably provides radioactive dose in the range of from about 20 MCI to about 200 MCI per dose when using90Y as the radiation source. However, the specific dose for each prospective patient depends on many factors, for example, the specific radioactivity of the used connection RIT, certain used radionuclide, age, body weight, General health, sex, diet, time and route of administration, and rate of excretion, pharmaceutical combination and size and specific gravity of the tumor, to which is applied therapy. For example, in the treatment of metastatic prostate cancer or breast cancer monoclonal antibody against MUC-1 (M170), conjugated with cyklinowanie90Y is usually injected at specific activity of approximately 5 MCI/mg antibody to deliver a total dose in the range of from about 20 MCI to about 200 MCI, depending on the size of the tumor and other factors described above. Preferably parenteral administration RIT, preferably intravenous (IV) injection.

Radioimmunotherapeutic means for treatment of tumors is well known in this field. Suitable means RTI include the any targeted radionuclide therapy of any therapeutically useful attached radionuclides, who can join the tumor or vascular system of the tumor or to capture them. Such useful tools RIT include targeted to the tumor or targeted to the vascular system of the tumor ligand or molecule. The radionuclide can be directly attached to a targeting molecule or ligand or to join helicobasidium substance, United with ligand or associate with him. Alternatively, the ligand may include hematopathology or exciting radionuclide group, and you can enter the patient is cold to bind to the tumor or vascular system of the tumor in the patient. Subsequent introduction of a radionuclide bound ligand can capture the radionuclide in the tumor site (pre-targeted radionuclide therapy). Targeted molecules or ligands include antibodies, antibody fragments, recombinant combinations of fragments of antibodies, peptides or other ligand, which has a selective affinity to tumors or vascular system tumors.

Preferably, RIT represents labeled with a radionuclide complex hematopathy agent-ligand, in which hematopathy agent chemically related to the targeted tumor molecule. Preferred aimed at the tumor molecules include antibodies, such as monoclonal antibodies or fragments of the Academy of Sciences is ITIL. Preferably, aimed at the tumor molecule is an anti-tumor antibody. You can use any anti-tumor antibody. Preferably, the antitumor antibody targeted to the vascular system of the tumor. Alternatively, the antibody may target tumor antigens, such as p185HER2protein cores milk mucin, TAG-72, Lewis a, cancerreally antigen (CEA), melanoma antigens with high relative molecular mass, recognized by the antibody 9.2.27, or associated with ovarian antigens recognized OV-TL3 or MOV18. Preferred anti-tumor antibodies are monoclonalny antibodies against MUC-1, such as M170mAb, commercially available from the company Biomira Inc., Edmonton, Canada, chimeric antitumor monoclonal antibody L6 (ChL6 Mab) and the like.

Any radionuclides suitable for use in the methods of radiation therapy for cancer, can be used in the way CMRIT of the present invention. Suitable radionuclides include, without limitation131I177Lu,67Cu64Cu196Re and90Y. Preferably, the radionuclide is a90Y.

Suitable means RIT and obtaining them are described in U.S. patent No. 5958374 issued by Meares et al., the relevant disclosure of which is incorporated into this description by reference.

Preferably, chelatable the ith agent is polyazamacrocycles group or polyoxometallates group. Preferably, hematopathology group derived from:

1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acids;

1,4,7,10-tetraazacyclotridecane-N,N′,N″,N′′′-tetraoxane acids;

1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′′′-tetraoxane acid or

1,5,9,13-tetraazacyclotetradecane-N,N′,N″,N′′′-tetraoxane acid.

Preferably labeled with a radionuclide complex hematopathy agent-ligand chemically linked to ChL6 mAb or M170 mAb. Most preferably, RIT is a90Y-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acid-peptide-ChL6 (hereinafter90Y-DOTA-peptide-ChL6) or anti-MUC-1 mAb similar.

Radionuclide preferably represents a90Y.111In can be included in the RIT for tumour imaging. Preferably labeled with a radionuclide complex hematopathy agent-ligand chemically linked to ChL6 mAb or M170 mAb.

In a preferred implementation hematopathy agent is a N-substituted 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acid, where N is the Deputy represents-CH2C(=O)-(Gly)3-L-(para-isothiocyanate)-Phe-amide (hereinafter - DOTA-peptide) and the radionuclide is a90Y.

RIT can be costall is but with a variety of pharmaceutically acceptable excipients, suitable for liquid injectable composition as described above in relation to the composition of pentapeptides cRGD.

The way CMRIT of the present invention can be used to treat various forms of cancer. For example, the way CMRIT of the present invention can be used to treat cancers that appear solid tumors, such as breast cancer, colon cancer, lung cancer, thyroid cancer, ovarian cancer and the like. The way CMRIT can also be used to treat forms of cancer, non-solid tumors such as non-Hodgkin's lymphoma, and the like. The way CMRIT of the present invention preferably is used as treatment for breast cancer.

The way CMRIT the present invention provides a significantly higher antitumor efficacy relative to the total overall antitumor activity of treatment for one RIT or one Pentapeptide cRGD at the same dosage levels (i.e. there is a synergistic effect). The way CMRIT also provides greater apoptosis of tumor cells and tumor endothelial cells compared with treatment one RIT or one Pentapeptide cRGD at the same dosage levels.

The present invention in another aspect provides a kit comprising a first container comprising at least one standard doses of the RIT and one or more additional containers, include only at least two standard doses of Pentapeptide cRGD. Each of the containers includes a label describing the contents of the container, optional, sequence of administration and any other relevant information required by the governmental regulations relating to pharmaceutical drugs and radioactive substances. The kit may also include printed instructions for use of the contents of containers for the treatment of tumors, as described in the present description means.

The containers may include bottles, ampules, bottles and the like. Each container preferably includes one standard dose, however, you can also use containers with multiple doses. Instruction materials are also preferably include information on safety and efficacy.

The following non-limiting examples are provided to further illustrate the invention.

The reagents. Yttrium-90 (90Y) without media (Pacific Northwest National Laboratory, Richland, WA or New England Nuclear, Boston, MA) were purchased in the form of chloride in 0,05M HCl. Chimeric L6 (ChL6), a chimeric human-mouse antibody (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA) interacts with a total membrane glikoproteinom, showing high expression in carcinomas of the breast, colon, yaichnikova the human lung. Cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (EMD 121974) is an antagonist selective for integrins αvβ3and αvβ5with the values of the IC50in the low nanomolar range for melanoma cells M21, expressing the isolated integrins αvβ3and in the low micromolar range for melanoma cells M21, expressing αvβ5. Synthesis and characterization of peptides was performed as described previously Dechantsreiter et al., J. Med. Chem. 42: 3033-40 (1999), the relevant disclosure of which is incorporated into this description by reference.

Cell line. NRT 3477, cell line adenocarcinoma of the breast of man, received from the Scientific and pharmaceutical research Institute Bristol-Myers Squibb (Seattle, WA). More than 70% of the cells NRT 3477 intensively stained L6. In NRT cells 3477 expressed bcl-2 and p53 is a mutant with meaningless mutation in the exon 10 leading to deletions in the region of the protein p53, which functions when tetramerization and in the detection of double-strand breaks of DNA. Cells 3477 NW Express a functional integrin αvβ5but not integrin αvβ3because the connection with vitronectin is blocked specific to αvβ5antibody P1F6, but not locked-specific αvβ3what nitela LM609. Cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) blocks the connection of NRT cells 3477 with vitronectin with IC50approximately 5 microns.

90Y-DOTA-peptide-ChL6. ChL6 conjugatively with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acid (DOTA) and were labeled radioactive label90Y, as described DeNardo, et al. J. Nucl. Med., 36: 829-836, with efficiency greater than 80% or its equivalent to obtain90Y-DOTA-peptide-ChL6.90Y-DOTA-peptide-ChL6 investigated the structural and functional integrity of high-performance liquid chromatography (HPLC) with sifting through the molecular sieve, the electrophoresis film of cellulose acetate (SAE) and radioimmunodetection analysis (RIA) binding to cells NRT 3477. HPLC and SAE indicated that more than 90%90Y-DOTA-peptide-ChL6 was in Monomeric form at less than 4% of the species with high molecular weight, according to the definition of SAE. Immunoreactive binding to living cells is indicated on the reactivity of more than 92% with the introduction of a single dose of 200, 230 or 260 µci90Y-DOTA-peptide-ChL6.

Mouse. Female Nude mice Balb/c nu/nu (ages 7-10 weeks old; Harlan Sprague Dawley, Inc., Frederick, MD) were kept in accordance with the recommendations of the University of California for the care of animals. Cells NRT 3477 (3,0×106collected in the logarithmic phase, were subcutaneously injected with one side of the abdomen for research therapy (h the exception noted) and in both directions for studies of immunopathology. RIT were injected with the tail vein, and cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) was delivered venturepoint (b) injection. "0 day" was defined as the time of injection RIT or the first injection of Pentapeptide cRGD for the group with the introduction of only Pentapeptide cRGD. Mice were killed offset neck for studies of immunopathology in the specified time point when the tumor exceeded the permissible limits, or through 84 days for research therapy.

Group control treatment (without RIT). The group consisted of mice not treated (24 mouse, 14 mice with 2 tumors each, and 10 mice with 1 tumor); receiving unlabeled antibody ChL6 (315 g) (8 mice with 2 tumors each) and treated with cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), is administered in 6 doses of 250 mcg at 0, 2, 4, 6, 8 and 10 day (18 mice with 1 tumor each).

Group treatment high-dose RIT. The group consisted of mice treated with RIT as the sole agent (260 µci90Y-DOTA-peptide-ChL6 (39 mice, 15 with 2 tumors each, and 24 with 1 tumor)and RIT (260 µci90Y-DOTA-peptide-ChL6 in combination with cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) in 6 doses of 250 mcg, starting with 0 day, 1 hour before RIT c followed by the introduction of another 5 doses at 2, 4, 6, 8 and day 10 (42 mouse, all have 1 of each tumor).

Group treatment low dose of RIT. The group consisted of mice treated with RIT as the sole agent (200-230 µci90Y-DOTA-peptide is ID-ChL6 (28 mice, including 9 with 2 tumors each of the previous research that has received 230 µci90Y-DOTA-peptide-ChL6, and 19 mice with 1 of each tumor, treated with 200 µci90Y-DOTA-peptide-ChL6)), and the group treated with RIT (200 µci90Y-DOTA-peptide-ChL6) in combination with the 6-dose (250 μg each) cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val). These doses were administered at 0 day, 1 hour before RIT c followed by the introduction of another 5 doses at 2, 4, 6, 8 and 10 day (30 mice, all with 1 of each tumor).

Destroying tumor cells effect. Tumors were measured with calipers in 3 orthogonal diameters of 3 times/week. Tumor volume was calculated using the formula for powelliphanta (DeNardo et al. Clin. Cancer Res., 3: 71-79 (1997)). The initial tumor volume was defined as the amount on the day before treatment. Tumors that underwent complete regression, were considered to have zero volume. The response of tumors classified as follows: recovery (C), the tumor disappeared and not re-grow to the end of the study (84 days); complete regression (CR), the tumor disappeared, at least on the 7th day, but later rose again; partial regression (PR), tumor volume decreased by 50% or more at least, on the 7th day, but later rose again; no response (NR), tumor volume decreased less than 50%. For mice with 2 tumors with different reactions, the reaction of tumor was described in accordance with what eczema both tumors. Mice that died before the 30th day from toxicity were excluded from the results of the evaluation of tumour response.

The toxicity. The magnitude of the mass and the blood was measured 2-3 times/week for 12 weeks after injection to death. Blood samples were taken from the tail vein using a microcapillary pipettes capacity of 2 µl. Samples from mice in the group doses were pooled and diluted 1:200 in saline phosphate buffer (PBS, 0.9% saline solution/10 mm phosphate, pH 7,6)for the number of erythrocytes (RBC); 1:100 in 1% (wt./about.) the ammonium oxalate for the number of platelets; or 1:20 in 3% (wt./about.) acetic acid for determination of leukocyte (WBC).

Group of cell immunopathology. In the absence of other guidance, the group consisted of 2 mice each had 2 tumors, the analysis of all 4 tumors at each time point. The group consisted of mice not treated (4 mice, 7 tumors); 250 μg each) cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), is administered as a single dose followed by killing 2 hours, 6 hours and 1 to 5 days after injection of the peptide; only RIT (260 µci90Y-DOTA-peptide-ChL6) with the subsequent killing of 2 hours, 6 hours and 1-6 days (3 mouse 5 tumors on day 5); and RIT (260 µci90Y-DOTA-peptide-ChL6) in combination with cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (250 µg)administered 1 hour before RIT (the way CMRIT) and repeated every other day for up to 10 days with PEFC is blowing killing 2 hours, 6 hours and 1-6 days after RIT. The tumor was removed, cut in half, froze in an environment with optimal cutting temperature (O.C.T.) and stored at about -70°to obtain slices (slice thickness 10 μm). At all time points evaluated for the detection of apoptosis by analysis of tagging the end of single-stranded gap dUTP-mediated terminal deoxynucleotidyltransferase (TUNEL, Carvieli, et al. J. Cell Bio., 119: 493-501 (1992)), and the selected time point (untreated, 1, 5, and 6 days) were evaluated for differences in the rate of proliferation (Ki67) and density of microvessels (CD31).

Analysis of TUNEL total of apoptosis and endothelial apoptosis. The tumor was cut into slices of 10 μm and placed on glass slides Fisher superplus (Fisher, Pittsbugh, PA), dried with air for 1 hour and was frozen at about -70°to the analysis of TUNEL kit ApopTag Red (rhodamine used as the label, Intergen, Purchase, NY), following the manufacturer's instructions after fixation for about 10 min in 1% paraformaldehyde. After TUNEL slides were washed and incubated over night at 4°with mouse anti-mouse antibody Mab against CD31 at a dilution of 1:100 (Pharmingen, San Diego, CA) to identify endothelial cells. Slides were washed and incubated for 1 hour with anti-mouse antibody linked with FITC (dilution 1:50) (Pharmingen). Slides were washed briefly oguzeli 4.6-diamidino-2-phenylindol (DAPI, 0.2 ág/ml) for background nuclear staining, again washed and set and then stored in the dark at about 4°to quantitative analysis.

Quantitative analysis of total and endothelial apoptosis. For the quantitative analysis of 6 selected by random sampling fields ×600 (150000 μm2/field) in deprived necrosis areas of each slice used the Olympus microscope, equipped with a set of Chroma Pinkle Filter Set (Chroma, Brattleboro, VT) with excitation filters for UV, FITC (fluorescein isocyanate) and rhodamine filters and double/triple the bandwidth to allow simultaneous visualization of multiple wavelengths. Fields were chosen to cover the entire area of inspection using DAPI labels, which usually consisted of about 300-350 cells. Apoptosis of all cells was determined by the average number of positive cores on the field for each tumor, whereas apoptosis of endothelial cells was determined using the same fields filter with double bandwidth for counting cells, labeled and FITC (CD31), and rhodamine (TUNEL). Fields were chosen from clearly not necrotic areas of the tumor slices as xenografts NRT 3477 usually grow quickly in "Nude" mice, doubling its volume in 6 days, leading to Central necrosis untreated tumors. Since TUNEL may mark necroticism the e cells, although less intensively, this strategy was chosen by a completely random process.

Analyses of proliferation and density of microvessels. The slice thickness of 10 μm tumors from treated mice and mice treated with pentapeptides cRGD, RIT and way CMRIT, and destroyed after 1, 5 and 6 days after treatment, were fixed for 10 min in ice-cold acetone, washed in PBS and briefly incubated in methanol with 0.6% of N2About2(5 min). After rinsing in PBS, the sections were blocked for 10 min with 10% goat serum and 1% bovine serum albumin in PBS. Mouse antibody against Ki67 Mab (Pharmingen, clone V) were made in blocking solution (at 6.25 µg/ml), and slides were incubated at room temperature for 2 hours, followed by rinsing in PBS. Put goat antimurine labeled with rhodamine or goat antimurine labeled with Cy-3 antibody (Jackson ImmunoResearch Laboratories Inc, West Grove, PA, 1:100), and slides were incubated for 1 hour at room temperature. After rinsing in PBS, the sections were incubated for 1 hour at room temperature with mouse artemisinin CD31 antibody (Pharmingen, 1:100) followed by rinsing in PBS followed by incubation for 1 hour with goat Anticriminal antibody labeled with FITC (Pharmingen, 1:50). After rinsing in PBS, slides were subjected to protiva is rasiwasia DAPI (0.4 µ g/ml), histological preparations were made in the Biomeda gel (Fisher) under a glass cover. Quantitative analysis of Ki67 was performed using an Olympus microscope at magnification X1000 to assess proliferation. The mean total number of Ki67-positive cells/field in the tumor was determined by calculations with 6 fields per tumor, selected by random sampling DAPI staining. The density of microvessels was determined by counting the number of vessels, painted CD31, on a randomly selected field with increasing H. Any endothelial cell or group of cells positive for CD31, which was separate from the next group, considered as one micronised. 6 randomly selected fields on the slice of the tumor used to establish an average value for each tumor. The average density of microvessels for the treatment group was determined by averaging values for the 4 tumors/group.

The expression β3and CD31 tumors NRT 3477 "Nude" mice. The tumor was cut in half was frozen in O.C.T. medium (Tissue Tek, Miles, Inc., Ekhart, IN) and stored at about -70°to obtain slices. Then the sections were fixed in ice-cold acetone for 10 min, washed in PBS and blocked in 10% goat serum in PBS for 30 min hamster Antibody against mouse CD61 (β3) Mab (Pharmingen) was applied at a concentration of 10 μg/ml and slides were incubated for 3 hours at the room for the Noah temperature. After rinsing in PBS was applied antihuman associated with rhodamine antibody (Jackson ImmunoResearch Laboratories, Inc.) (1:50) followed by incubation for 1 hour at room temperature. After rinsing in PBS was applied antibody against mouse CD31 (1:100, Pharmingen), and slides were incubated for 1 hour at room temperature. After rinsing in PBS was applied anticrimine associated with FITC antibody (1:50, Pharmingen), and slides were incubated for 1 hour at room temperature and washed in PBS. Slides were immersed in DAPI (0.2 ág/ml), and histological preparations were made in the Biomeda gel (Fisher) under a glass cover. Joint expression β3and CD31 was observed by Olympus microscope, equipped with a set of Chroma Pinkle Filter Set (Chroma, Brattleboro, VT) with excitation filters for UV, FITC and rhodamine filters and double/triple the bandwidth to allow simultaneous visualization of multiple wavelengths.

The statistical analysis. Statistical analysis mortality of mice treated RIT and way CMRIT, was performed using Fisher's exact test software StatExact to determine whether mortality is different. Statistical analysis data treatment was performed using a criterion Cochran Mantel Haenszel to evaluate the effect of doses of RIT on the outcome for groups RIT and techniques is as CMRIT. Comparison of frequency of treatment with all other reactions for groups treated with RIT, in comparison with the method CMRIT, was performed using Fisher's exact test when used for statistical purposes the best tumour response in animals with two tumors. Statistical differences between groups of immunopathology at different time points were evaluated by variance analysis (ANOVA) (Fisher PLSD) using appropriate software STATview at p<0.05 is considered as significant difference.

Destroying tumor cells effect. The majority of tumors in mice not treated, mice treated with one cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), and mice treated not labeled antibody ChL6, grew without interruption. The visible effect of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (total 6 doses × 250 μg, administered every other day) on tumor growth was not observed, which led to the absence of a cure in 18 of the tested mice. 2 mice treated not labeled antibody ChL6 in addition to the 2 mice not treated, occurred spontaneous regression of their tumors, resulting in cure rates of 8% (4/50) for mice not treated with RIT (table 1, figure 1).

In table 1 the numbers in parentheses represent the total number of produced using the best reaction from mice that had 2 tumors (mice affected 2 tumors). For example, if the mouse had 2 tumors, one tumor was treated and the other tumor was subjected to partial regression this is considered a partial regression (PR) in the table in the analysis of the worst reactions (figures without parentheses), but would be considered a cure (C) when using the analysis of the best response (numbers in parentheses). Mice treated with RIT, treated90Y-DOTA-peptide-ChL6 at a dose of 260 µci (high dose) or 200-230 µci (low dose) in isolation or in combination with 6 doses of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (method CMRIT). Reaction of tumors in mice that died from toxicity before the expiry of the period of 30 days after RIT, excluded from assessment of effectiveness. One RIT in the high dose resulted in 4 cases cure the 26 mice (15%), while RIT in the low dose resulted in 5 cases of cure in 20 mice (25%). The way CMRIT with low-dose RIT resulted in 8 cases of cure from 22 mice (36%, p=0,514), then as a way CMRIT with high-dose RIT led to 10 instances of healing in 19 mice (53%, p=0.011). Statistical analysis showed that there was no difference in the outcome of the treatment on the basis of dose RIT corrected at RIT and the way CMRIT. Because the outcome did not change with dose (p>0,8), outcomes for RIT were compared with outcomes for a method CMRIT. The results show that the method CMRIT resulted in a significantly higher number of cases of cure (cure rates 44%)than RIT (cure rates 20%) (p=0,020), which is consistent with the increased efficiency of the method CMRIT, compared with one RIT.

Figure 1 shows the increased effectiveness of the treatment when xenotransplant is the ATA breast cancer in mice obtained by way CMRIT. Shows of tumour response90Y-DOTA-peptide-ChL6 and cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), used as the sole means, and with the combined treatment (method CMRIT). The results illustrate the combined results of high dose and low dose for RIT (200-260 µci90Y-DOTA-peptide-ChL6). The group consisted of mice not receiving treatment, mice treated 315 µg not labeled ChL6, mice treated with 6 doses of 250 mcg cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), mice treated one RIT, and mice treated way CMRIT (6 doses of 250 mcg cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) and 1 dose of RIT (200 and 260 µci)). Reactions were assessed at the end of the 84-day period. Significantly increased the effectiveness of the treatment was observed in mice treated by way CMRIT, compared with mice treated RIT (using the worst outcome, as shown, or the best outcome for mice with two tumors with p=0.02 for the last of these outcomes).

1 (3)
Table 1
The treatmentPentapeptide cRGDRIT (µci)MouseCureCRPRNR
Without treatment--2420 (1)21 (18)
ChL6, 315 mcg--81 (2)007 (6)
Pentapeptide cRGD250 mcg×6-1800018
RIT low dose-200-230204 (5)49 (11)3 (0)
RIT high dose-2602642 (6)20 (16)0
CMRIT low dose250 mcg×62002283101
CMRIT high dose250 mcg×62601910351
RIT (combined)-200-260468 (9)6 (10)29 (27)3 (0)
CMRIT (combined)250 mcg×6200-26041186152

In table 1 Pentapeptide cRGD is a cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val); CR pre who is a full regression; and NR represents the absence of regression; figures in parentheses are based on the best response in mice with two tumors.

The toxicity of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val). One cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (250 mg, 6 doses for 10 days) did not cause increased mortality or toxicity compared to mice who did not receive treatment (mice from all groups not treated with RIT (not treated, not labeled ChL6, cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)), not died from toxicity. Mice treated cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), and mouse not treated, showed the same changes in body weight and the levels of RBC, WBC and platelets. Mice treated or high or low dose of RIT, and mice treated with the combination of RIT and cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), showed reduced body weight and the number of RBC, WBC and platelets, compared with mice not treated with RIT, but the combination of RIT with cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) inhibited these figures are lower than the values observed when one RIT (figa-D). High-dose RIT mortality was higher than that of non-treated mice and in the RIT group (13/39 mice (33%)and in group method CMRIT (23/42 mice (55%), but mortality was not statistically increased under the influence of way CMRIT (Fisher exact test p=0,0736). Similarly, mortality was increased in the groups of low-dose RIT and how CMRIT, compared to mice not treated, but with Artest mice, treated the way CMRIT (8/30 (27%)), not increased, in comparison with RIT (8/28 mice (29%)) (Fisher exact test p=1,000). When combining results mortality at low and high dose RIT way CMRIT did not lead to increased mortality, compared with RIT (Fisher exact test p=0,1652). These results indicate that the Pentapeptide cRGD, such as cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), introduced separately or in combination with RIT, does not cause a significant increase in toxicity.

Figure 2 shows that neither the treatment of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), no treatment method CMRIT does not increase toxicity. A: RBC, B: platelet: WBC and D: the body weight of mice in groups of mice not treated (5), mice treated one cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (6 doses of 250 μg), mice treated one RIT (260 µci90Y-DOTA-peptide-ChL6) (5), and mice treated way CMRIT (260 µci90Y-DOTA-peptide-ChL6 and 6 doses of 250 mcg cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)) (13) one test. Shows the results represent pooled blood samples for each group and mean values of body weight ± the standard error for each group is shown in days. The Pentapeptide cRGD or in combination with RIT did not increase toxicity.

Apoptosis in tumor and endothelial cells (all cells). Apoptosis was assessed by TUNEL method in combination with staining CD31 to identify endotel the sexual cells (figure 3). The number of TUNEL-positive tumor and endothelial cells (all cells) were averaged for 1 tumor in 6 random fields (H) not necrotic tissue is selected so that the cells covered the entire area of the field, with an average of approximately 350 cells/field. Tumors without treatment had an average of 9±1,0 positive cells/field (2.6 per cent). A single dose of the Pentapeptide cRGD cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)) was significantly increased apoptosis after 1 day after treatment (16,2±1,89, 4,6%), but this level of apoptosis in subsequent decline. Compared to non-treated tumors, one RIT resulted in significantly increased apoptosis (as indicated in figure 4), with the largest number of apoptotic cells observed after 6 days (21,4±or 2.9, 6.1 per cent). Apoptosis after method was higher than in all treatment groups at all time points, except for 6 days (marked significantly increased apoptosis, figure 4). Apoptosis after application of the method CMRIT peaked 5 days after RIT (30,7±2.0 cells/field, 8,8%), with lower peak 1 day after the application of the method CMRIT (21,9±2,6, 6,3%). These 2 peak consistent with apoptosis occurred two "waves" of apoptosis. However, the difference in the apoptosis of all cells that have occurred in the tumors of the treated way CMRIT, compared with tumors subjected to RIT, is additive and, thus, indicates t the m what other mechanisms can also affect the efficiency.

Figure 3 shows microphotography images that show the way CMRIT leads to increased apoptosis. Apoptosis (TUNEL with ApopTagRed) and CD31 (FITC), illustrated in sections of tumors by 10 μm (a) mice not treated (C) mice treated with pentapeptides cRGD (1 dose of 250 micrograms cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)), (C) mice treated RIT (260 µci90Y-DOTA-peptide-ChL6), and (D) mice treated way CMRIT (260 µci90Y-DOTA-peptide-ChL6 and 6 doses of 250 mcg cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)), was evaluated 5 days after the start of treatment. Apoptosis (rhodamine) was observed in tumor cells and in endothelial cells (FITC)where co-localized dyes rhodamine and FITC (green cells marked by the points of the arrows). Background nuclear staining was obtained DAPI. Sections were photographed with increasing H an Olympus microscope equipped with filters Pinkle. Increased apoptosis of tumor and endothelial cells was observed in mice treated by way CMRIT, compared with mice treated one RIT (ANOVA, p<0,05).

Endothelial apoptosis. Because earlier it was reported that the Pentapeptide cRGD causes apoptosis of endothelial cells of blood vessels, apoptosis in tumors treated with pentapeptides cRGD, compared with unexposed Le the structure of the tumors. Significantly increased apoptosis was observed after 1 day (2,5±0.5) and 5 days (2,4±0,7) after treatment with pentapeptides cRGD. Compared with tumors not treated with 0.6±0,1). However, these differences in apoptosis between tumors treated with pentapeptides cRGD, and not treated tumors was not affected by differences in growth or tumor volume even when multiple (6) doses of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val). One RIT has also led to a significant increase in endothelial apoptosis, compared with non-treated tumors at an early time point (figure 4). CMRIT was associated with 2 peaks of apoptosis of endothelial cells, after 1 day and 5 days after RIT, at the highest levels after 1 day (3,9±1.2 cells/field) (figure 4). The average level of endothelial apoptosis in the tumors of the treated way CMRIT, was higher. Than in all other groups at all time points, except for 3 days and 6 days. However, it appeared that increased endothelial apoptosis was not preceded by apoptosis of all cells in the tumors of the treated way CMRIT, with peaks occurring on the 1st and 5th days.

Figure 4 gives a graphical representation of data showing that cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) leads to increased apoptosis of tumor and endothelial cells after 1 and 5 days after treatment for one Pentapeptide cRGD or lehenespena CMRIT. A. Apoptosis of all cells (filled symbols) and Century endothelial cells (EC) (not filled symbols) were assessed by analysis of TUNEL and CD31 immunohistochemical analysis of the tumors NRT 3477 from non-treated mice (▪), mice treated with pentapeptides cRGD (1 dose of 250 micrograms cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (•), mice treated RTI (260 µci90Y-DOTA-peptide-ChL6 (▴), and mice treated way CMRIT (260 µci90Y-DOTA-peptide-ChL6 and 6 doses of 250 mcg cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)) (◆). Apoptosis was quantitatively evaluated with increasing H on 6 randomly selected fields per tumor at 4 tumors used to determine treatment. Apoptosis of all cells specified colored markers, and endothelial apoptosis is not colored markers. The "whiskers" show the standard error of the mean. Values at the way CMRIT significantly greater (ANOVA, p<0.05)than the value of RIT, is shown by an icon *; size when using Pentapeptide cRGD much greater than in the absence of treatment (ANOVA, p<0,05), shown by an icon &; RIT values significantly greater than in the absence of treatment, is shown by an icon $. After 2 hours, 1 day and 5 days after RIT apoptosis of all cells to tumors, treated by the method CMRIT, significantly different from the tumors of the treated RIT. After 6 hours, 2 days and 5 days after RIT endothelial apoptosis would be the greatly increased in mice treated the way CMRIT, compared with mice treated RIT. The X-axis is not drawn to scale to illustrate changes in the early time point. Interrupt Y-axis shows the change in scale between the apoptosis of all cells and apoptosis of endothelial cells.

Total cellular proliferation. The average number of Ki67-positive cells was determined for tumors, untreated, through 1, 5, and 6 days (6 days did not define for tumors treated with pentapeptides cRGD). The Ki67 antibody recognizes a protein present in the nuclei of proliferating cells in all active phases of the cell cycle, but it does not recognize a protein found in cells at the stage of G0. The results show that RIT or RGD Pentapeptide separately significantly reduced the rate of cell proliferation, active in the cell cycle, after 5 days, compared with non-treated mice (table 2). The way CMRIT similarly reduced proliferation compared with non-treated mice, and resulted in significantly reduced cell proliferation after 6 days, compared with one RIT (figure 5).

Figure 5 shows microphotography images of tumor cells showing reduced proliferation of tumor cells after treatment method CMRIT, in comparison with the treatment of RIT. Proliferate the s cells in tumors, treated (A) RIT and the way CMRIT, identified by 10-μm frozen sections of tumors after 6 days after RIT Ki67 mouse Mab, followed by the introduction antimelanoma antibodies associated with rhodamine. Groups of endothelial cells, were used to calculate the density of microvessels were identified mouse antibody Mab against CD-31 followed by the application of Anticriminal antibodies associated with FITC. Nuclear dye DAPI was. Proliferation was quantitatively determined at magnification X1000 6 randomly selected fields per tumor, at 4 tumors used to determine treatment. Fewer Ki67-positive cells were observed in the tumors of the treated way CMRIT, compared with tumors treated RIT, 6 days after RIT, although the number of microvessels was not different at a given point of time. Sections were photographed with an increase H an Olympus microscope equipped with filters Pinkle.

The density of microvessels. The density of microvessels in each tumor was determined by averaging the total number of non-adjacent painted CD-31 areas in 6 randomly selected fields from one section of each tumor from mice not treated, from mice treated cRGD, RIT and the way in CMRIT key time points after treatment (table 2). Significantly reduced densely shall be the microvessels, compared to non-treated mice was observed after 6 days after treatment RIT of mice treated with one or RIT, RIT in combination with pentapeptides cRGD cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)). It was expected that increased endothelial apoptosis (above RIT), observed during the 1-day and 5-day, not preceded apoptosis in all cells. These data indicate that increased epithelial apoptosis, associated with the way CMRIT, may participate in the mechanism influencing the outcome of treatment, but is hardly the only one. In addition, the data to 6-day does not indicate that reduced the number of microvessels explain the difference in treatment outcome between RIT and the way CMRIT.

Table 2
DayNot treatedcRGD aRIT bCMRIT c
The density of microvessels d
016±3
112±215±115±2
520±413±112±1
6Ȋ 9±1 e9±2 e
Proliferating cells f
017±1
114±2 f18±119±1
513±1 f13±1 f13±1 f
67±13±1 g
and one in b/W dose of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) 250 mcg
b - one dose 315 mcg90Y-DOTA-peptide-ChL6 (260 µci).
with one in/dose 315 mcg90Y-DOTA-peptide-ChL6 (260 µci) and 4/b of each dose of 250 µg cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) 250 mcg every other day, starting 1 hour before RIT.
d - average number of ± standard error of CD31-positive areas in 6 fields for each tumor, counted with increasing X.
e - significantly different from non-treated (ANOVA, p<0,05).
f - the average number of proliferating cells, determined by taking the average number of Ki67-positive cells is to 6 fields for each tumor, counted at magnification 1000X (approximately 112 ± 10 cells/field).
g - way CMRIT significantly less than RIT (ANOVA, p<0,05).

The expression αvβ3the tumors NRT 3477. Immunohistochemistry with an antibody against β3interacting with murine integrin (CD61), demonstrated the limited tagging of selected areas of tumors NRT 3477 "Nude" mice (6). The field labeled β3included region, manifested in the form of annular vascular structures that were co-labeled antibody that recognizes CD31, which is consistent with the expression αvβ3in the blood vessels that supply blood to these tumors. Observed significantly lower expression αvβ3than CD31, which could be expected if only a small fraction of endothelial cells, labeled CD31, was neovascular.

6 is a microphotography image slice of the tumor, showing, β3and CD31 expressed on the tumor cells of the breast NRT 3477. The tumor is removed from a not-treated mice were analyzed for the expression of integrin β3and endothelial CD31 protein using immunohistochemical techniques. Used hamster antibody Mab to mouse β3then use stigmatophora antibodies associated with rhodamine. Then applied the rat antibody Mab against murine CD31 then use Anticriminal antibodies associated with FITC. Joint localization (orange color specified by arrows) rhodamine (β3) and FITC (CD31) is consistent with the expression of integrin αvβ3in the blood vessels supplying blood to the tumor. Slice tumors were photographed with increasing H an Olympus microscope equipped with filters Pinkle.

The results show that the method CMRIT of the present invention increased the effectiveness of treatment of a tumor without increased toxicity relative to conventional monotherapies RIT. The way CMRIT with the use of high-dose RIT resulted in 53% of cases cured, compared with 15% of cases, a cure for one RIT and 0% of cases, a cure for one cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), whereas the levels of the combined doses resulted in 44% of cases cured, compared with 20% of cases, a cure for one RIT. Although it increased the number of deaths associated with the way CMRIT at the higher dose level, the mortality rates for the method CMRIT and RIT were not statistically different. These results indicate that significantly better treatment outcome related to the way in CMRIT, compared with treatment by the same means without an accompanying statistical increase toxicity.

DeNardo et a. reported that 250 mcg Pentapeptide cRGD (EMD 270179)given 1 hour before RIT, increased capture RIT tumors NRT 3477 up to 50% (Cancer Biother. Radiopharm. 15: 71-79 (2000)). However, in a previous study, this model NRT 3477 using90Y-DOTA-peptide-ChL6 in doses in the range from 110 µci up to 330 µci increased number of frequencies of cases, cure is not followed increased injected doses above 260 µci90Y-DOTA-peptide-ChL6. These results are strong evidence that increased capture RIT-related peptides Pentapeptide cRGD, such as cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), is not the main factor responsible for the increase in the number of cases of cure.

Vascular contribution to the way CMRIT was investigated by assessment of endothelial apoptosis and its trends in dynamics compared to the apoptosis of tumor cells. If Pentapeptide cRGD caused endothelial apoptosis, can be expected to experience increased endothelial apoptosis that occurs before the increased apoptosis in all cells. Pentapeptide cRGD (cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val)) in combination with RIT (for example, the way CMRIT) have increased levels of apoptosis and endothelial, and all cells above the level that was observed at RIT in almost all points of time at significantly increased levels in the 1st and 5th days. In addition, one dose (cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) significantly increased the endothelial apoptosis in the same point of time, compared with no treatment. Although there was a clear type of endothelial apoptosis, preceding the apoptosis of all cells, was persistent increase endothelial apoptosis in the way CMRIT, compared to RIT. However, the effect of this distinction is not reflected directly by differences in the density of microvessels in the evaluated time point. It is possible that (cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) influenced the quality of the microvascular organization, which would not reflect the density of microvessels. This is consistent with a decrease in proliferation observed in tumors treated the way CMRIT, compared with tumors treated RIT, on the 6th day, the latest estimated time point, when the number of microvessels decreased in the tumors of the treated and RIT, and the way CMRIT.

Other possible differences between the way CMRIT and RIT could be associated with any abscopal indirect effects pentapeptides cRGD, such as cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val). It was shown that irradiation causes the accumulation and activation of integrin β3 blood vessels of the tumor within 1-4 hours of exposure. This increase is due to the accumulation of platelets in the lumen of the vessels irradiated tumors. Adhesion of platelets to endothelial cells is inhibited by blockade of antibody to the integrin αvβ3and the blood clot is city potentially contribute to tumor angiogenesis by growth factors, contained in α-granules. It was reported that cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) has the IC50420 nm for the isolated receptor α2bβ3compared to 5 nm for αvβ3. It is possible that cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) in the dose of this mouse model inhibited the accumulation of platelets in the circulatory system tumors in response to irradiation and, thus, reduced the paracrine interaction with tumor cells. In addition, it was reported that the receptor integrin αvβ3participates in the full activation of growth factor receptor-2 in vascular endothelium (VEGFR-2). In addition, inhibition of Pentapeptide cRGD could interfere provided below in chain the effects of receptor VEGFR-2 on endothelial cells, such as reduced release of growth factor to neighboring tumor cells, leading to an overall inhibition of proliferation of tumor cells.

The lack of reactivity of the growth of subcutaneous tumors NRT 3477 on the inhibition of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) (one therapeutic effect), probably indicates that the growth of this model uses existing tumor blood vessels, or that sufficient growth of blood vessels occurs even in the presence of Pentapeptide cRGD. After RIT, at a time when the density of blood vessels was significantly reduced, ing the repression of Pentapeptide cRGD can lead to reduced recovery of tumor cells and endothelial cells from radiation damage, leading to increased tumor cell death when the waves of apoptosis associated with increased therapeutic efficacy.

The way CMRIT the present invention provides increased efficiency RIT for the treatment of tumors in the combined treatment of angiogenic cyclic Pentapeptide RGD. Therapeutic synergism observed for the method CMRIT of the present invention, probably due to the combined effects of several mechanisms leading to increased apoptosis and reduced cell proliferation. However, a higher level of endothelial apoptosis observed way CMRIT, would be consistent with the loss of endothelium acting on the loss of tumor cells and by contributing to the observed increase in the number of cases of cure.

1. A method of treating tumors in a patient, comprising successive stages:

(a) introduction to the patient at least one dose of antiangiogenic cyclo-(arginine-glycine-aspartic acid)-containing Pentapeptide (Pentapeptide cRGD);

(b) introduction to patient effective antitumor amount radioimmunotherapeutic tool (RIT) not later than approximately 1 h after injection of Pentapeptide cRGD in stage (a); and

(C) the introduction of the patient, at least two additional doses of PE is tapatia cRGD, where the first extra dose of Pentapeptide cRGD administered within about two days after the introduction of RIT and each additional dose of Pentapeptide cRGD introduced at intervals of not more than about two days between doses.

2. The method according to claim 1, where Pentapeptide cRGD is a cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val).

3. The method according to claim 1, where at stage (C) sequentially injected at least 5 additional doses of Pentapeptide cRGD.

4. The method according to claim 1, where RIT represents the complex is labeled with a radionuclide hematopathy agent-ligand, in which hematopathy agent chemically linked to a molecule that is aimed at the tumor.

5. The method according to claim 4, where aimed at the tumor molecule is a monoclonal antibody.

6. The method according to claim 5, where the monoclonal antibody is an anticancer monoclonal antibody.

7. The method according to claim 6, where the antitumor monoclonal antibody is a monoclonal antibody against MUC-1.

8. The method according to claim 6, where the antitumor monoclonal antibody is a chimeric monoclonal antibody L-6.

9. The method according to claim 6, where the antitumor monoclonal antibody is a monoclonal antibody M against MUC-1.

10. The method according to claim 4, where hematopathy agent is polyazamacrocycles group or proximally is a mini-group.

11. The method according to claim 10, where the specified hepatoblastoma group derived from a member of the group consisting of

1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acids;

1,4,7,10-tetraazacyclotridecane-N,N′,N″,N′′′-tetraoxane acids;

1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′′′-tetraoxane acids;

1,5,9,13-tetraazacyclotetradecane-N,N′,N″,N′′′-tetraoxane acid.

12. The method according to claim 4, where the radionuclide is a90Y.

13. The method according to claim 4, where hematopathy agent is a N-substituted 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acid, where N is the Deputy represents-CH2C(=O)-(Gly)3-L-(parasiticidal)-Phe-amide, and the radionuclide is a90Y.

14. The method according to claim 1, where the tumor is a tumor of the breast.

15. A method of treating tumors in a patient, comprising successive stages:

(a) introduction to the patient at least one dose of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val);

(b) introduction to patient effective antitumor amount radioimmunotherapeutic tool (RIT) not later than approximately 1 h after administration of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) from stage (a); and

(c) the introduction of the patient, at least two updat the additional doses of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val), where the first additional dose of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) is administered within about 2 days after administration of RIT, and where each additional dose doses of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) is administered at intervals of not more than about 2 days between each dose.

16. The method according to clause 15, where at the stage (C)at least 5 additional doses of cyclo-(Arg-Gly-Asp-D-Phe-N-Me]-Val) is administered sequentially.

17. The method according to clause 15, where RIT represents labeled with a radionuclide complex hematopathy agent-ligand, in which hematopathy agent chemically linked to a molecule that is aimed at the tumor.

18. The method according to 17, where aimed at the tumor molecule is a monoclonal antibody.

19. The method according to p, where a monoclonal antibody is an anticancer monoclonal antibody.

20. The method according to claim 19, where the antitumor monoclonal antibody is a monoclonal antibody against MUC-1.

21. The method according to claim 19, where the antitumor monoclonal antibody is a chimeric monoclonal antibody L6.

22. The method according to claim 19, where the antitumor monoclonal antibody is a monoclonal antibody M170 against MUC-1.

23. The method according to claim 19, where hematopathy agent is polyazamacrocycles group or polyoxometallates group.

24. The method according to claim 19, where pointed to by the I hepatoblastoma group derived from a group member, consisting of

1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acids;

1,4,7,10-tetraazacyclotridecane-N,N′,N″,N′′′-tetraoxane acids;

1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′′′-tetraoxane acids;

1,5,9,13-tetraazacyclotetradecane-N,N′,N″,N′′′-tetraoxane acid.

25. The method according to claim 19, where the radionuclide is a90Y.

26. The method according to claim 19, where hematopathy agent is a N-substituted 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraoxane acid, where N is the Deputy represents-CH2C(=O)-(Gly)3-L-(parasiticidal)-Phe-amide, and the radionuclide is a90Y.

27. The method according to clause 15, where the tumor is a tumor of the breast.

28. The kit includes a first container comprising at least one standard dose of RIT and one or more additional containers, comprising only at least two standard doses of Pentapeptide cRGD.

29. Set p, in which each container includes a label describing the contents of the container, and any other pertinent information required by the governmental regulations relating to pharmaceutical drugs and radioactive substances.

30. Set p in addition, includes printed instructions for use of the contents of containers for the treatment of tumors.



 

Same patents:

FIELD: medicine, oncology.

SUBSTANCE: invention is related to pharmaceutical composition for photodynamic treatment of malignant tumors, which contains therapeutically efficient amount of at least one of fullerene С60 derivatives from group of compounds, which have fullerene С60 molecule that is bound with one molecule of amino acid or dipeptide, or its pharmaceutically acceptable derivative, or complex of association with biocompatible synthetic polymers or biolpoimers and tetra pyrroles, or conjugate of association with amino compounds. Patented composition which includes the above mentioned compounds, allows using emission spectrum up to 1 mcm for photodynamic treatment, which in turn, enables broadening the application area of photodynamic treatment due to penetration of light with such wave length into deep tissues. The invention also relates to the method of photodynamic treatment of malignant tumors, when the patented pharmaceutical composition is applied.

EFFECT: composition has low toxicity and high accumulation selectivity in tumor tissues.

7 cl, 7 ex, 12 dwg

FIELD: organic chemistry, medicine, biochemistry, pharmacy.

SUBSTANCE: invention relates to derivatives of quinazoline of the general formula (I): and their pharmaceutically acceptable salts and in vivo hydrolyzed esters as aurorakinase inhibitors and their using, to a method for inhibition and pharmaceutical composition based on thereof, and to a method for their synthesis. In compound of the general formula (I) X represents -NR6 wherein R6 represents hydrogen atom or (C1-C6)-alkyl; R5 represents group of the formula (a): or (b): wherein * means a point for adding to group X in compound of the formula (I); R1, R2, R3 and R4 are chosen independently from hydrogen atom or -X1R9 wherein X1 represents -O-, and R9 is chosen from one of the following groups: (1) hydrogen atom or (C1-C5)-alkyl; (3) (C1-C5)-alkyl-X3R20 wherein X3 represents -O- or -NR25 wherein R25 represents hydrogen atom, (C1-C3)-alkyl or (C1-C3)-alkoxy-(C2-C3)-alkyl, and R20 represents hydrogen atom, (C1-C3)-alkyl, cyclopentyl, cyclohexyl or 5- or 6-membered saturated heterocyclic group with 1 or 2 heteroatoms that are chosen independently from nitrogen atom (N) wherein (C1-C3)-alkyl group can carry 1 or 2 substitutes that are chosen from oxo, hydroxy group, halogen atom and (C1-C4)-alkoxy group, and wherein cyclic group can carry 1 or 2 substitutes that are chosen from (C1-C4)-alkyl; (4) (C1-C5)-alkyl-X4-(C1-C5)-alkyl-X5R26 wherein X4 and X5 can be similar or different, and each means -O- or -NR31- wherein R31 represents hydrogen atom, (C1-C3)-alkyl or (C1-C3)-alkoxy-(C2-C3)-alkyl, and R26 represents hydrogen atom or (C1-C3)-alkyl; (5) R32 wherein R32 represents 5- or 6-membered saturated heterocyclic group added through carbon atom or nitrogen atom with 1 or 2 heteroatoms that are chosen independently from oxygen (O), sulfur (S) and N atoms wherein heterocyclic group can carry 1 or 2 substitutes that are chosen from hydroxy, (C1-C4)-alkyl, (C1-C4)-hydroxyalkyl, (C1-C4)-alkoxy, (C1-C4)-alkoxy-(C1-C4)-alkyl; (6) (C1-C5)-alkyl-R32 wherein R32 is given above; (18) (C1-C5)-alkyl optionally substituted with 1, 2 or 3 halogen atoms; (19) (C1-C5)-alkyl-X10-(C1-C5)-alkyl-X11R90 wherein X10 and X11 that can be similar or different each means -O- or -NR95- wherein R95 represents (C1-C5)-alkyl, (C1-C3)-alkyl substituted with 1, 2 or 3 halogen atoms, (C1-C4)-alkyl or (C1-C4)-alkoxy groups, (and wherein 2 (C1-C4)-alkoxy groups by (C1-C4)-alkyl groups alkoxy can form in common 5- or 6-membered saturated heterocyclic group that comprises 2 oxygen atoms), (C2-C5)-alkenyl, (C2-C5)-alkynyl, (C3-C6)-cycloalkyl, (C3-C6)-cycloalkyl-(C1-C3)-alkyl or (C1-C3)-alkoxy-(C2-C3)-alkyl; R90 represents hydrogen atom or (C1-C3)-alkyl; (22) (C1-C5)-alkyl-R96 wherein R96 represents 5- or 6-membered heterocyclic group that can be saturated or unsaturated (added through carbon or nitrogen atom) with 1 or 2 heteroatoms that are chosen independently from N wherein heterocyclic group can carry 1 or 2 substitutes that are chosen from (C1-C4)-hydroxyalkyl, (C1-C4)-alkyl, hydroxy and (C1-C4)-alkoxy-(C1-C4)-alkyl, and wherein R60 represents hydrogen atom; R61 represents group of the subformula (k): wherein p represents 0 or 1; q represents 1; R'1 and R''1 represent independently hydrogen atom or (C1-C10)-alkyl; T represents C=O; V represents -N(R63)R64 wherein R63 represents -(CH2)q'R70 or phenyl optionally substituted with 1 or 2 groups chosen independently from halogen atom, (C1-C4)-alkyl, (C1-C4)-alkoxy, trifluoromethyl, trifluoromethoxy, nitro, difluoromethyl, difluoromethoxy and cyano group; R64 represents hydrogen atom or (C1-C3)-alkyl; q' = 0; R70 represents -K-J wherein K represents a bond, and J represents phenyl optionally substituted with 1, 2 or 3 groups that are chosen from halogen atom, (C1-C3)-alkyl, cyano, (C1-C3)-alkoxy, and R62 represents hydrogen atom. Proposed compounds can be used in treatment and prophylaxis of diseases mediated by aurorakinase activity, for example, proliferative diseases, such as cancer.

EFFECT: valuable medicinal properties of compounds.

32 cl, 7 tbl, 2 sch, 147 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to therapeutic agents showing effectiveness in treatment of pain, cancer, cerebrospinal sclerosis, Parkinson's disease, Huntington's chorea and/or Alzheimer's disease. Invention describes compound of the formula (I): or its pharmaceutically acceptable salts wherein RF1 and RF2 represent independently electron-acceptor groups; Z is chosen from O=; R1 is chosen from (C1-C10)-alkyl, heterocyclyl-(C1-C6)-alkyl, substituted heterocyclyl-(C1-C6)-alkyl; R2 is chosen from (C1-C6)-alkyl; X represents bivalent (C1-C10)-group that separates groups added to it by one or two atoms; Ar represents bivalent (C4-C12)-aromatic group, and Y is chosen from =CH=. Also, invention describes fields wherein compounds of the formula (I) are used, a pharmaceutical composition based on thereof, and methods for their synthesis. Invention provides synthesis of novel compounds possessing useful biological properties.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

17 cl, 2 tbl, 35 ex

FIELD: medicine, pharmacology, organic chemistry.

SUBSTANCE: invention relates to a composition used for photoaffinity labeling of proteins. Composition comprises analogues of tamandarine of the formula (1): or analogue of didemnine of the formula (2): , or fragment of didemnine of the formula (3): . Also, invention relates to a method for synthesis of analogues of didemnine, fragment of didemnine, a backing containing analogue of tamandarine or didemnine, and a method for inhibition of growth or proliferation of cells, and a method for inhibition of oncogenesis and apoptosis in cell. Invention provides decreasing toxicity and enhancing the therapeutic index.

EFFECT: valuable biological and medicinal properties of analogues.

67 cl, 3 tbl, 51 dwg, 7 ex

FIELD: medicine, oncology, organic chemistry, antibiotics.

SUBSTANCE: invention proposes a combination and corresponding method for treatment of tumors that are positive with respect hormone receptors. Combination comprises rapamycin or 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin, or ABT578, or compound of the formula (1): or an aromatase inhibitor: atamestan, exemestan, formestan, aminoglutetinide, rogletimide, pyridoglutemife, trilostan, testolactone, ketocona-zol, vorozol, fandrozol, anastrozol or letrozol. In the formula (1) R1 means -CH3 or (C3-C6)-alkynyl; R2 means hydrogen atom (H) or -CH3-CH3-OH; X means oxygen atom (O), (H, H) or (H, -OH) under condition that R2 doesn't mean H if X means O, and R1 means -CH3. Proposed composition shows synergism in suppression of breast cancer proliferation.

EFFECT: valuable medicinal properties of combination.

14 cl

FIELD: medicine, oncology, organic chemistry, antibiotics.

SUBSTANCE: invention proposes a combination and corresponding method for treatment of tumors that are positive with respect hormone receptors. Combination comprises rapamycin or 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin, or ABT578, or compound of the formula (1): or an aromatase inhibitor: atamestan, exemestan, formestan, aminoglutetinide, rogletimide, pyridoglutemife, trilostan, testolactone, ketocona-zol, vorozol, fandrozol, anastrozol or letrozol. In the formula (1) R1 means -CH3 or (C3-C6)-alkynyl; R2 means hydrogen atom (H) or -CH3-CH3-OH; X means oxygen atom (O), (H, H) or (H, -OH) under condition that R2 doesn't mean H if X means O, and R1 means -CH3. Proposed composition shows synergism in suppression of breast cancer proliferation.

EFFECT: valuable medicinal properties of combination.

14 cl

FIELD: medicine, pharmacy.

SUBSTANCE: invention proposes a system for controlled release of temosolomide that comprises 10 wt.-% of temosolomide and biodegradable polymeric material representing polyanhydride. This polyanhydride is synthesized by condensation of 3,4-bis-(p-carboxyphenoxy)propane with sebacic acid in their ratio = 20:80, respectively. The system for controlled release of temosolomide represents implanted tablets. Implants are able to release anticancer preparation temosolomide by the controlled manner in vivo for prolonged time from 1 h to 4 weeks.

EFFECT: improved and valuable medicinal and pharmaceutical properties of system.

13 cl, 1 tbl, 2 dwg, 8 ex

FIELD: medicine, oncology, pharmacy.

SUBSTANCE: invention proposes using malachite green in form of tetramethyldiaminotriphenylcarbinol anhydrooxalate [(C23H25N2) x (C2HO4)]2 x C2H2O4 of the structural formula (I): as an agent for treatment of malignant neoplasms in the dosing from 1 mg to 2 g. Proposed agent shows the broad spectrum of its curative effect with respect to different oncological diseases: carcinoma, adenocarcinoma, sarcoma, melanoma, glyoblastoma, hepatoma, lymphoma. This treatment provides both disappearance of tumor in kidney, bladder, brain, liver and elimination of metastasis.

EFFECT: valuable medicinal properties and enhanced effectiveness of agent.

7 cl, 18 ex

FIELD: medicine, oncology, pharmacy.

SUBSTANCE: invention proposes using malachite green in form of tetramethyldiaminotriphenylcarbinol anhydrooxalate [(C23H25N2) x (C2HO4)]2 x C2H2O4 of the structural formula (I): as an agent for treatment of malignant neoplasms in the dosing from 1 mg to 2 g. Proposed agent shows the broad spectrum of its curative effect with respect to different oncological diseases: carcinoma, adenocarcinoma, sarcoma, melanoma, glyoblastoma, hepatoma, lymphoma. This treatment provides both disappearance of tumor in kidney, bladder, brain, liver and elimination of metastasis.

EFFECT: valuable medicinal properties and enhanced effectiveness of agent.

7 cl, 18 ex

FIELD: organic chemistry, medicine, pharmacology, biochemistry.

SUBSTANCE: invention relates a pharmaceutical composition, pharmaceutical combination and a set for treatment of cancer forms mediated by histone deacetylase. Proposed composition and combination comprises the effective amount of benzamide derivatives of the formula (5): and other anticancer active substance chosen from group consisting of cisplatin, etoposide, camptothecin, 5-fluorouracil, gemcitabine, paclitaxel, docetaxel, carboplatin, oxaplatin, doxorubicin and vinblastine. Invention provides high effectiveness of treatment and decreasing toxicity.

EFFECT: valuable medicinal properties of pharmaceutical composition, enhanced effectiveness of treatment.

106 cl, 13 tbl, 1 dwg

FIELD: medicine, radionuclide diagnostics.

SUBSTANCE: 199Т1 chloride intravenous injection is followed by early planar scintigraphy in 20 min, visual determination of increased marker accumulation area, quantitative estimation of early accumulation in lesion (ER), additionally, delayed imaging is taken in 180 min following 199Т1 chloride injection, at that, area of increased marker accumulation is determined repeatedly, accumulation intensity is determined quantitatively (DR), retention index (RI) is calculated as DR/ER and nonspecific inflammatory process is diagnosed if at least 2 of 3 following signs are found: RI<-0.047ER+0.979 in early and delayed imaging, borders of early marker hyperfixation area in early imaging are more distinct than in delayed phase of test, homogeneity of hyperfixation area in delayed imaging, while malignant tumour process is diagnosed if at least 2 of 3 following signs are found: RI>-0.047ER+0.979 in early and delayed imaging, less distinct borders of marker hyperfixation area in early imaging as compared to delayed imaging or without significant changes depending on imaging phase, heterogeneity of hyperfixation area in delayed imaging.

EFFECT: precision and informative value of differential diagnosis of nonspecific inflammatory and malignant processes of musculoskeletal system is increased as a result of enhanced qualitative and quantitative evaluation of scintigraphy image.

4 dwg, 1 tbl, 2 ex

FIELD: medicine, pulmonology.

SUBSTANCE: it is necessary to study initial values of functional reserve ability (FRA) of pulmonary-capillary circulation in %, average pressure value in pulmonary artery (AvPPA) in mm mercury column and daily variability of peak volumetric expiration rate (ΔPVRexp.) in % to calculate the following equation: D=+1.376·FRA-2.087·AvPPA-1.023·ΔPVRexp. At D value being above -25.71 one should predict instable flow of bronchial asthma. The innovation enables to carry out integral evaluation of functional state of pulmonary microcirculation, pressure in pulmonary artery and reactivity of respiratory tract.

EFFECT: higher efficiency and accuracy of prediction.

2 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to an improved solid-phase method for synthesis of radioisotope indicators, in particular, for synthesis of compounds labeled with 18F that can be used as radioactive indicators for positron- emission tomography (PET). In particular, invention relates to a method for synthesis of indicator labeled with 18F that involves treatment of a precursor fixed on resin if the formula (I): SOLID CARRIER-LINKER-X-INDICATOR wherein X means a group promoting to nucleophilic substitution by a definite center of a fixed INDICATOR with 18F- ion for preparing a labeled indicator of the formula (II): 18F-INDICATOR; to compound of the formula (Ib):

and compound of the formula (Ih): ;

to radiopharmaceutical set of reagents for preparing indicator labeled with 18F for using in PET; to a cartridge for radiopharmaceutical set of reagents for preparing indicator labeled with 18F for using in positron-emission tomography.

EFFECT: improved method of synthesis.

13 cl, 1 sch, 3 ex

FIELD: pharmaceutical chemistry, radioactive nuclide diagnosis and therapy.

SUBSTANCE: claimed method includes sequential mixing under cooling solutions of sodium perrhenate, gelatin, sodium thiosulfate, and hydrochloric acid at sodium thiosulfate and sodium perrhenate molar ratio of 1.5-3.4. Obtained mixture is heated on boiling water bath under continuous stirring for 10 min, cooled, conditioned for 2-3 h, passed through chromatography column filled with anion exchange resin and containing sterilizing filter. Filtrate is collected up to acidic fraction breakthrough, pre-packed at aseptic conditions into sterile bottles for drugs and stored in refrigerator. Said nanocolloid has 80 % or more particles with size of 2-100 nm; 5 % or less particles with size of <20 nm; nanocolloid solution has storage stability of 6 months or more; radioactive preparation obtained on the base of such nanocolloid has radiochemical purity of 90 % or more wherein said radiochemical purity is kept for not less than 4 hours.

EFFECT: new radiopharmaceutical agents useful in lymphoscintigraphy.

3 ex

FIELD: medicine.

SUBSTANCE: method involves introducing 2 ml of Pyrphotech into cubital vein. 370 MBq of Tc-99m-per technetate is intravenously introduced 20 min later. Small pelvis veins emission-type computer tomographic examination is carried out with gamma-chamber. Radiopharmacological preparation activity is recorded in the region of interest. Pelvic venous plethora degree is determined from the number of pulsations per second in the region of interest.

EFFECT: high accuracy of the method.

1 tbl

FIELD: medicine, instrumentation engineering, and biology.

SUBSTANCE: proposed radioactive source in the form of radioactive yttrium or strontium oxide that can be used, for instance, in medicine for preparation of drugs incorporating radioactive materials, for treatment of oncological diseases, as well as for producing β-sources used in instrumentation engineering and biologic investigations is solidly capsulated by molten bio-inert glass. Powdered yttrium and strontium oxides are mixed up with glass frit, mixture is applied to metal holder, dried out, and fused; rough surface is produced using definite combination of differently dispersed powders.

EFFECT: simplified design, facilitated production of radioactive source; inactivity of radioactive source with respect to organism tissue.

2 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves applying cuffs in upper shank half and lower thigh half regions under pressure of 100 mm of mercury column. Radiopharmaceutical preparation is introduced along posterolateral surface of the upper shank half above the cuff under the proper fascia. Examination is carried out in three stages in rest state with cuffs applied, in rest state after having removed the cuffs and after physical effort. Resorption function of deep lower extremity lymph collectors, available retrograde lymph circulation and deep lymph collector valves state of shank is studied at the first stage. The cuffs are removed at the second stage and lower extremity lymph circulation, retrograde lymph circulation availability and valve apparatus state of superficial shank lymph collectors is studied. After having applied 1 h long physical effort at the third stage, lymph circulation is estimated in deep and superficial lymph collectors of the lower extremity, retrograde lymph circulation availability and valve apparatus state of the deep and superficial lymph collectors of the lower extremity.

EFFECT: wide range of functional applications.

1 dwg

FIELD: oncology.

SUBSTANCE: invention characterizes compositions, their employment, and embodiments of a method of treatment of B-cellular lymphomas, leucosis, and other malignant tumors CD40+. Principal active therapeutical agent is anti-CD40L antibody or another CD40L antagonist inhibiting CD40-CD40L intermediate. In combination or composition with indicated CD40L antagonist any one or several of the following components can be additionally used: anti-CD20 antibody, a chemotherapeutical agent or a combination thereof, and radiotherapy.

EFFECT: enhanced mechanisms of apoptosis of tumor CD40+ cells due to sensitization of these earlier destruction-resistant cells.

88 cl, 4 dwg, 6 tbl, 8 ex

FIELD: medicine, functional diagnostics.

SUBSTANCE: one should introduce a radionuclide indicator to register the dynamics of its distribution in limbs' tissues due to scintigraphic method in the state of the rest and after loading testing. Moreover, loading test should be carried out due to placing limbs into thermostated membranes to heat for about 20-45 min at temperature that should not achieve the threshold of tissue lesion. Then it is necessary to detect the values of volumetric circulation and blood volume in affected limb to compare these values with those of contralateral limb. In case of decreased both values after loading test in affected limb against with the parameters of these values in contralateral one the state of circulation reserve should be evaluated as unfavourable. The innovation suggested widens the number of preparations for predicting the value of reserve possibilities of limb's circulation.

EFFECT: higher accuracy of evaluation.

3 dwg, 3 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention provides novel compounds of general formula (I) and pharmaceutically acceptable salts thereof, wherein G represents glycin; D aspartic acid; R1 group -(CH2)n- or -(CH2)n-C6H4-; n is integer from 1 to 10; h is 1 or 2; X1 represents amino acid residue having one functional deviation such as amine; X2 and X4 independently represent amino acid residue capable of forming disulfide bond; X3 represents hydrophobic amino acid such as phenylalanine; X6 represents group -NH-[CH-]-C(O)-; X7 is missing or represents biomodifying grouping consisting of monodisperse poly(ethylene glycol): (II), wherein is integer from 1 to 10; C-end unit represents amide group or 1-10 amino acid residues; Z1 chelating group or reporter group; and W1 is missing or represents spacing moiety derived from glutaric or succinic acid. Compounds of invention are designed as diagnostic agents for visualization or as therapeutic agents, the two agent types being directional vectors capable of binding to integrin receptors.

EFFECT: expanded diagnostic and therapeutic possibilities.

19 cl, 3 tbl, 5 ex

FIELD: medicine, molecular biology, antibodies.

SUBSTANCE: invention relates to an antibody raised against CCR5 and comprising: (i) two light chains wherein each light chain comprises product of plasmid expression and designated as pVK:HuPRO140-VK (ATCC - PTA-4097), and (ii) two heavy chains wherein each heavy chain comprises product of plasmid expression and designated as pVg4:HuPRO140 HG2-VH (ATCC - PTA-4098), or plasmid designated as pVg4:HuPRO140 (mut B+D+I)-VH (ATCC - PTA-4099), or fragment of such antibody binding with CCR5 on a human cell surface. Invention relates to nucleic acid encoding light and heavy chains of antibody, expression vector, cell-host transformed with at least one vector, and a method for preparing antibody. Antibody is used as an active component in composition used for inhibition of infection of cells CD4 + HIV-1, and to a pharmaceutical composition used in treatment of a patient with HIV-1 infection. Also, invention relates to antibody conjugate against CCR5 and its using. Use of antibodies provides enhancing effectiveness of prophylaxis and treatment of HIV-1 infection.

EFFECT: valuable medicinal properties of antibody.

31 cl, 23 dwg, 3 ex

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