Cancer treatment

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to new drugs and preparations containing effective anticancer agent with anti-Hsp90 antibody.

EFFECT: invention improves clinical effectiveness in treating cancer and leukemia.

48 cl, 25 tbl

 

The present invention relates to new medicines and drugs, including effective pharmaceutical agents with anti-Hsp90 antibody, which together provide increased efficiency in the treatment of various forms of cancer, including cancer of the colon and rectum. Other aspects of the invention relate to the treatment of leukemia.

Cancer treatment and treatment of leukemia

The first aspect of the present invention relates to new medicines and drugs, including effective anti-cancer tool together with anti-Hsp90 antibody, which together provide increased efficiency in the treatment of cancer.

In recent years there have been members of the family of heat shock proteins (Hsp) as playing an important role in oncogenesis and cell death. Indeed, heat shock proteins for many years been identified as a potential target for cancer treatment (Whitesell L, et al., PNAS USA, 1994 Aug 30, 91(18): 8324-8; PMID: 8078881), and members of the family ansamycin (previously called tyrosine kinase inhibitors) have been proposed as tools that can be used to implement the treatment of cancer (Neckers L, et al., Invest New Drugs, 1999, 17(4): 361-73; PMID: 10759403; Schulte TW, et al., Cancer Chemother Pharmacol., 1998, 42(4): 273-9; PMID: 9744771).

It was believed that one heat shock protein, Hsp90, is involved in the development of carcinoma of breast, prostate, melanoma, is Anosov and lymphomas, colon and lung (Banerji U, et al., Curr Cancer Drug Targets, 2003 Oct; 3(5): 385-90; PMID: 14529390), and thyroid carcinomas. The role of Hsp90 is to ensure the correct installation of client proteins, which are involved in a wide variety of cellular processes such as signal transduction. "Client proteins" Hsp90 include transcription factors, such as induced p53 and hypoxia factor 1α, and soluble kinases, including v-Src, Akt, Raf-1 and Bcr-Abl. Hsp90 is constitutively expressed in tumor cells at levels of 2-10 times their level in normal cells, suggesting that it may be important for growth/survival of tumor cells (Schwartz, J., et al. 2003, Semin. Hematol. 40:p87-96). Because the binding of client proteins" Hsp90 can adjust their conformation, stability and fate of Hsp90 may have a significant impact on the way that regulate cellular outcomes, including growth, division, differentiation, movement and cell death (Workman, P., Cancer Lett. 2004 Apr 8; 206(2):149-57; PMID: 15013520). A significant role of Hsp90 in cell processes means that this protein is currently being considered as a possible target for development of therapeutic drugs. Inhibitors of Hsp90 by specific interaction with a single molecular target cause destabilization and ultimate destruction "client protein"s Hsp90.

The second aspect of the present izobreteny which refers to new medicines and drugs, includes effective anti-cancer tool together with anti-Hsp90 antibody, which together provide enhanced efficacy in the treatment of leukemia.

Leukemia is a cancer that affects the bone marrow. In people with leukemia, the bone marrow produces large numbers of abnormal white blood cells. Abnormal leukocytes accumulate in the bone marrow, so the bone marrow is unable to produce sufficient quantities of normal erythrocytes, leukocytes and platelets.

Different types of leukemia can be divided into categories according to their rate of development (acute or chronic) and the type of the affected cells (myeloid or lymphoid cells). Myeloid leukocytes are the first line of defense of the immune system against infection, and they are found mainly in the blood, where they capture and destroy foreign organisms. Lymphoid leukocytes are found in the lymph nodes and blood.

The four most common types of leukemia include chronic lymphatic (lymphocytic) leukemia (CLL), acute myeloid (myeloblastic) leukemia (AML), acute lymphoid (lymphoblastic) leukemia (ALL) and chronic myeloid leukemia (CML).

CLL is a cancer of lymphocytic cells, but it has been slower than ALL. This disease represents the most the more common type of leukemia, striking adults, and is very rarely observed in children.

AML is a cancer that mainly affects myeloid cells known as granulocytes. It creates too many myeloblasts that can block blood vessels and immature myeloid cells. This disease occurs mainly in adults, but can also affect children.

CML (also called chronic granulocytic leukemia) is usually a slowly progressing cancer of neutrophilic cells, which is rare in children and usually more amazing adult men than women. CML is usually easily diagnosed because leukemia cells by more than 95% of patients have a distinct cytogenetic abnormality, the Philadelphia chromosome (Ph1) (Kurzrock, R. et al., 2003, Ann. Intern. Med. 138 (10): p819-30, PMID: 12755554; Goldman, J.M. and Melo, J.V., 2003, N. Engl. J. Med. 349 (15): p1451-64, PMID: 14534339). Ph1 arises from a reciprocal translocation between the long arms of chromosomes 9 and 22, and it can be demonstrated in all hematopoietic precursors (Deininger, M.W. et al., 2000, Blood 96 (10): p3343-56, PMID: 11071626). This translocation leads to the transfer of the Abelson oncogene (abl) on chromosome 9 in the region of chromosome 22, called the cluster area of point break (BCR) (Deininger, M.W. et al., 2000, Blood 96 (10): p3343-56, PMID: 11071626). This, in turn, results in a hybrid gene BCR/ABL, which is the first encodes a chimeric mRNA 8,5 TPN Gene BCR/ABL is an oncogene, which is sufficient to obtain mice diseases such as CML. The transcript of the oncogene BCR/ABL is transmitted to the output protein of 210 kDa 190 kDa. Protein Bcr-Abl is an abnormal protein protein kinase, which causes disturbance of myelopoiesis detected in CML. CML progresses through certain clinical stage, called the chronic phase, accelerated phase and blast crisis. Oncogene BCR/ABL is expressed at all stages, but blast crisis is characterized by multiple additional genetic and molecular changes (Gorre, M.E., et al., 2002, Blood, 100(8): p3041-3044).

Ph1-negative CML is a rare disease characterized by clinical features of CML without cytogenetic or molecular (RT-PCR) evidence of translocation t(9;22)(q34;q11), resulting in condensed mRNA Bcr-Abl. Ph1-negative CML is a low differentiated nosological form, which is less clearly differs from other myeloproliferative syndromes. Previously it was thought that Ph1-negative CML accounts for 5-10% of all clinical forms of CML, but now, under normal availability analysis of RT-PCR for detection of transcript Bcr-Abl, this amount is substantially below 5%. Interestingly, in some patients, this nosologic form may occur in rez is ltate alternative merge with Abl. Merge TEL(ETV6)-ABL as a result of t(9;12) was demonstrated in two cases Ph - CML. Patients with Ph1-negative CML generally have a weaker response to treatment and lower life expectancy than patients with Ph1-positive CML (Onida, F. et al., 2002: Cancer 95 (8): p1673-84, PMID: 12365015).

ALL is a cancer of immature lymphocytic cells, known as lymphoblasts. This disease represents the most common type of leukemia in young children, usually from age 1 to 7 years, and it is quite rare in adults. ALL causes production of many abnormal lymphocytes, which displace the normal red blood cells and platelets. It was thought that protein Bcr-Abl 185 kDa directly associated with the development of ALL.

Two drugs, geldanamycin (GA) and 17 allylamino, 17-demethoxygeldanamycin (17-AAG), which act as inhibitors of Hsp90, have shown promising biological and clinical activity in clinical trials. Indeed, a hybrid protein Bcr-Abl 210 kDa (p210Bcr-Abl) depends on its interaction with Hsp90 for stability, and cell treatment with GA or 17-AAG leads to a rapid destruction of p210Bcr-Abl.

The Hsp90 inhibitor, such as 17-AAG in combination with conventional cytotoxic drugs or other new facilities would also be therapeutically valuable in effect on multistage carcinogenesis (Workman P., Cancer Lett. 2004 Apr 8; 206(2):149-57; PMID: 15013520). In RA the new cells, which are characterized by genetic instability, it is possible that 17AAG by blocking the activity of Hsp90 releases a variety of mutations, which together are "synthetically lethal to tumor. Normal cells that lack the genetic instability of tumor cells that are relatively not affected (Garber, K., 2002, Journal of the National Cancer Institute, vol. 94, No. 22, p1666-1668). A significant problem with 17AAG is that the drug is too toxic for long-term therapy and, therefore, there is a need for non-toxic substitution (Banerji et al., see above).

Imatinib mesylate (Gleevec (RTM)is melkokalibernyh inhibitor of tyrosine kinase, which had a major effect on neoplastic disease as the only means. Originally designed as an inhibitor of tyrosine kinase Bcr-Abl, which is characteristic for malignant processes carrying pathogenic translocation 9;22, imatinib was moderately specific and had a substantial impact on the treatment of chronic myelogenous leukemia (CML) and positive for the Philadelphia chromosome (Ph1+) ALL (Krystal, G.W., 2004, the Leukemia Research 28S1:pS53-S59). One of the problems associated with the treatment of CML with imatinib, is a resistance to the drug as the result of mutations in tyrosinekinase Bcr-Abl. It is important that the cells of CML, which became the persistent to imatinib in vivo, retain their dependence on Hsp90 and, thus, remain sensitive to 17AAG.

In recent publications we are talking about the fact that Hsp90 is present fully in multicomponent complexes that promote malignant progression, and that they are attractive targets for anticancer therapeutics. In particular, we are talking about the fact that Hsp90 in multicomponent complexes derived from tumor cells have affinity binding with 17AAG, which is 100 times higher than Hsp90 from normal cells (i.e. Hsp90 in its latent, not associated in complexes), indicating that in a multicomponent complex it may be epitopes (in particular, Quaternary epitopes), did not show a latent, not bound in complexes of Hsp90.

Antibody mikegrb (RTM) can bind to Hsp90 in its latent, not associated in complexes condition, as well as in multi-component complexes without any side effects on the kinetics of binding.

In WO 01/76627 it comes to compositions for the treatment of fungal infections, and compositions include a combination of (i) polyene or antifungal agents in the form of the inhibitor synthase beta-glucan; and (ii) antibodies that are specific against fungal Hsp90, and the composition is effective against the fungus that causes the infection, despite his resistance to one protivogribkoe the WMD facility.

In accordance with the first aspect of the present invention is the use of

(i) antibodies or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least one anticancer means selected from the group consisting of: doxorubicin, daunorubicin, epirubicin, Herceptin, docetaxel and cisplatin

in the method of manufacturing a medicinal product for the treatment of cancer.

Doxorubicin is an anthracycline antibiotic agent, previously recognized as antitumor agents.

Epirubicin is a less toxic synthetic anthracycline antibiotic, also previously recognized as antitumor agents.

Daunorubicin is antineoplastics a tool used in several therapeutic areas, including use as anti-cancer tool.

Herceptin (trastuzumab) is a monoclonal antibody used to treat metastatic breast cancer, over expressing HER2 protein.

Docetaxel is a recognized anti-cancer agent, and he is an inhibitor of mitosis.

Cisplatin is a recognized anti-cancer agent and includes a complex of platinum.

How in detail the order described below, in the experimental results (Experiment A), doxorubicin and daunorubicin are particularly preferred and are especially good synergistic effects with anti-Hsp90 antibody. Herceptin also shows good synergistic effects with anti-Hsp90 antibody. Synergy is also observed with docetaxel and cisplatin in combination with anti-Hsp90 antibody. Synergy between daunorubicin and antibody particularly evident in cells positive for estrogen receptors, and, thus, medicines and methods of treatment using antibodies and daunorubicin can, in particular, to apply (or type) about the presence of cells that have estrogen receptors.

Experiments (Experiments A) also shows that other anti-cancer tool when used in conjunction with anti-Hsp90 antibody, or are uncertain results (paclitaxel) or antagonism (imatinib). This confirms the surprising/unexpected nature of the synergy achieved the above anticancer means, in combination with anti-Hsp90 antibody.

There is also a combination drug that includes:

(i) the antibody or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least one anticancer agent selected from the group consisting of: doxorubin the ina, of daunorubicin, epirubicin, Herceptin, docetaxel and cisplatin,

for simultaneous, separate or sequential use in the treatment of cancer.

Also provided is a method of treatment of cancer, comprising introducing a therapeutically effective amount

(i) antibodies or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least one anticancer means selected from the group consisting of: doxorubicin, daunorubicin, epirubicin, Herceptin, docetaxel and cisplatin,

a needy patient.

Yet another special instructions, used the term "treatment" has a wide meaning. Thus, the term "treatment" or "therapy" refers to any treatment that is designed to cure, alleviate, eliminate or reduce the symptoms of, or prevent or reduce the likelihood of development disorders or disorders of the human or animal. Thus, the term "treatment" refers to and treatment of pathological conditions and their prevention.

The antibody or antigen-binding fragment may be specific for epitope expressed peptide comprising the sequence of SQ ID NO: 1.

As discussed above, although the Quaternary epitopes shown Hsp90 in a lot of komponentnykh complexes, have been suggested as appropriate targets for treatment experiments (below) shows that, in fact, a linear epitope is a useful and effective target for treatment.

Attela receiving them and methods of application are well known and disclosed, for example, Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999.

Antibodies can be generated using standard methods known in this field. Examples of antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single-chain Fab fragments, fragments produced by the library of Fab expression, and antigen-binding fragments of antibodies.

Antibodies can be obtained from a number of hosts, such as goats, rabbits, rats, mice, humans, and others. They can be immunized by injection fungal stress protein or any fragment or oligopeptides, which has immunogenic properties. Depending on the type of host you can use various adjuvants to increase the immune system. Such adjuvants include, but are not limited to, complete adjuvant's adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomies polyols, polyanion, peptides, oil emulsions, hemocyanin mollusk Megathura cremulata and Dini is rophenol. Among adjuvants used in humans, especially useful BCG (Bacillus of Calmet-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to fungal proteins or their fragments or oligopeptides can be obtained using any technique which provides for the production of antibody molecules by permanent cell lines in culture. They include, but are not limited to, the method of hybridoma, methodology hybridoma In human cells and methods of hybridoma EBV (Epstein-Barr) (Koehler et al., 1975, Nature, 256: 495-497; Kosbor et al., 1983, Immunol. Today 4: 72; Cote et al., 1983, PNAS USA, 80: 2026-2030; Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., New York, pp. 77-96).

In addition, you can use the techniques developed for the production of "chimeric antibodies", the splicing of the gene of mouse antibody genes to human antibody to obtain a molecule with appropriate antigen specificity and biological activity (Morrison et al., 1984, PNAS USA, 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314: 452-454). Alternatively, you can adapt the techniques described for obtaining single-chain antibodies using methods known in this field, to obtain specific for fungal stress protein of single-chain antibodies. Antibodies with related specificity, but with excellent idiotypical composition, can be generated by the shuffling circuit from random combinatorial libraries is immunoglobulin (Burton, D.R., 1991, PNAS USA, 88: 11120-11123).

Antibodies can also be obtained by induction generation in vivo of lymphocyte population or by screening libraries of recombinant immunoglobulin or panels of highly specific reagents binding (Orlandi et al., 1989, PNAS USA, 86: 3833-3837; Winter, G. et al., 1991, Nature, 349: 293-299).

You can also obtain antigen-binding fragments, such as fragments, F(ab')2, which can be obtained by digestion of the molecule pepsin antibodies, and Fab fragments, which can be obtained by restoring the disulfide bridges of fragments F(ab')2. Alternatively, a library of expression of Fab can be constructed to allow rapid and easy identification of fragments of monoclonal Fab with the desired specificity (Huse et al., 1989, Science, 256: 1275-1281).

Various immunological assays can be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or Immunoradiometric analyses using either polyclonal or monoclonal antibodies with established specificity are well known in this field. Such immune assays usually involve the measurement of complex formation between fungal stress protein or any fragment or Oligopeptide and its specific antibody. You can use dvuhsimovyiy immune the analysis on a single cell basis using monoclonal antibodies, specific to the two not interfering epitopes fungal stress protein, but you can also use competitive analysis link (Maddox et al., 1983, J. Exp. Med., 158: 1211-1216).

For example, the antibody used in the composition or combined preparation may include the sequence of SEQ ID NO: 2.

It was found that the forms of cancer that can be treated successfully include fibrosarcoma and carcinoma, selected from the group consisting of breast carcinoma, prostate cancer, melanoma, leukemia, lymphoma, carcinoma of the colon, paradisevalley carcinoma of the testes, carcinoma of the pancreas, ovarian, endometrial, thyroid and lungs.

In accordance with the second aspect of the present invention is the use of

(i) antibodies or antigen-binding fragment specific for at least one epitope of Hsp90;

in the method of manufacturing a medicinal product for the treatment of leukemia.

Also provided is the use of

(i) antibodies or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least one anticancer means selected from the group consisting of: imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyacetone,

in the method of manufacturing a pharmaceutical environments is TBA for the treatment of leukemia.

Imatinib, a derivative of 2-phenylaminopyrimidine is melkokalibernyh antagonist with activity against protein kinases and shows strong and specific inhibition of Bcr-Abl. Imatinib is indicated for the treatment of patients with CML in blast crisis, accelerated phase or in chronic phase after failure of treatment with IFN (interferon).

Paclitaxel is a chemotherapeutic agent that is administered as a treatment for some types of cancer. It is most often used to treat ovarian cancer, breast cancer and not small cell lung cancer.

Docetaxel is an anti-cancer agent and an inhibitor of mitosis.

Daunorubicin is antineoplastics a drug used in several therapeutic areas, including use as anti-cancer tool.

Doxorubicin is an anthracycline antibiotic agent, previously recognized as antitumor agents.

Hydroxyurea is antineoplastic agent that is an inhibitor ribonucleotides.

As described in detail below, in the experimental results ("Experiments"), docetaxel and paclitaxel are particularly preferred and are especially good synergistic effects with anti-Hsp90 what nitela. Synergy is also observed with imatinib, doxorubicin, daunorubicin and hydroxyurea in combination with anti-Hsp90 antibody. An anti-cancer agent cisplatin when used in conjunction with anti-Hsp90 antibody showed uncertain results. This confirms the surprising/unexpected nature of the synergy achieved the above anti-cancer agents in combination with anti-Hsp90 antibody.

Also available combined product, including:

(i) the antibody or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least one anti-cancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyacetone,

for simultaneous, separate or sequential use in the treatment of leukemia.

Examples of combination therapies include pharmaceutical packaging containing the antibody (i) and at least one anti-cancer agent (ii) in a separate volume (i.e., not mixed together in the same drug).

There is also a method of treating leukemia, comprising introducing a therapeutically effective amount

(i) antibodies or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least real what cancer means, selected from the group consisting of: imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyacetone,

for treatment need him as a patient.

Leukemia can be a chronic myeloid leukemia or acute lymphoid leukemia, and at least one anti-cancer agent can be a imatinib.

The antibody or antigen-binding fragment may be specific to an epitope displayed by the peptide comprising the sequence of SEQ ID NO:1.

For example, the antibody used in the composition or combined preparation may include the sequence of SEQ ID NO:2.

An anti-cancer agent can be a imatinib.

It was found that the leukemias that can be treated successfully include leukemia, selected from the group consisting of acute myeloblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia.

Leukemia can be a chronic myeloid leukemia or acute lymphoid leukemia.

Chronic myeloid leukemia can be Ph1-Ph1 positive or negative, i.e. characterized by leukemic cells, which contain the Philadelphia chromosome (Ph1-positive), or do not contain the Philadelphia chromosome (Ph1-negative).

B is lo detected, what is chronic myeloid leukemia, which can be successfully treated with imatinib may or Ph1-positive, or Ph1-negative.

Leukemia can be a chronic myeloid leukemia, which is a Ph1-positive and anti-cancer agent can be a imatinib. In particular, the applicant found the treatment of CML, which is a Ph1-positive, can be realized by combination of imatinib and antibodies comprising the sequence of SEQ ID NO:2. Not wishing to be bound to any theory, applicant proposes that Hsp90 may sequestered antibody comprising the sequence of SEQ ID NO:2, which, in turn, means that the abnormal Bcr-Abl-tyrosinekinase (which causes broken milopotas detected in CML), for example, misfolded, aimed at the destruction of the protein, and/or there are barriers to the delivery of its effects on myelopoiesis path.

This treatment, also effective against resistant to imatinib Ph1-positive CML cells. Without the desired connection with any theory of resistance to imatinib, probably associated with the accumulated mutations in the abnormal Bcr-Abl tyrosinekinase, which may, for example, to prevent product contact with the protein and/or inhibit the activity of the drug. It is possible that in cells resistant to imatinib, sequestration of Hsp90 antic the scrap, including the sequence of SEQ ID NO:2, causes the wrong placement, targeting the degradation of protein and/or obstacle rendering effects on myelopoiesis path mutated abnormal tyrosine kinase. Sequestration of Hsp90, which is usually used for "sautereau" genetic mutations associated with cancer cells, binding of abnormal proteins and block their expression may also cause the release of a variety of mutations, which together are synthetically lethal to tumor cells. Normal cells that lack the genetic instability of tumor cells that are relatively not affected.

In addition, and for a special surprise, the applicant has found that the treatment of CML, which is Ph1-negative, can be realized by combination of imatinib and antibodies comprising the sequence of SEQ ID NO:2.

Leukemia can be a chronic myeloid leukemia, which is Ph1-negative and anti-cancer agent can be a imatinib.

These data are surprising, because cells Ph1-negative CML deprived of abnormal protein tyrosine kinase associated with Ph1-positive cells. However, without the desired connection with any theory, it is possible that there are low or basal levels of this kinase (anomalous or otherwise) in Ph1-negative to edah, and, as described above, that the sequestration of Hsp90 is an antibody comprising the sequence of SEQ ID NO:2, means that the protein tyrosine kinase, for example, misfolded or aimed at the destruction of the protein or somehow has barriers to the delivery of its effects on myelopoiesis way. It may also be the case that by sequestration of Hsp90, tumor cells are released various mutations, which together are synthetically lethal.

Amazing effect of imatinib and anti-Hsp90 antibodies in Ph1-negative cells may be caused by the presence of the merge TEL(ETV6)-ABL, which was demonstrated in two cases Ph1-negative CLM (Krystal, G.W., 2004, the Leukemia Research 28S1:pS53-S59), and which is sensitive to imatinib.

Leukemia can be characterized by cells that are resistant to imatinib.

Composition or drug of the present invention may further include a known inhibitor of Hsp90, such as AG or 17-AAG.

The third aspect of the present invention (experiments) refers to new medicines and drugs, including effective anti-cancer tool together with anti-Hsp90 antibody, which together provide increased efficiency in the treatment of cancer of the colon and rectum or adenocarcinomas.

Cancer of the colon and rectum is a malignant about whole of the colon and rectum. Cancer of the colon and rectum is a leading cause of morbidity and mortality in cancer. This is the third most common form of cancer in men and the second most common form of cancer in the UK. 95% of cancers of the colon and rectum are adenocarcinomas, which are cancers of the glandular cells that line the inner part of the colon and rectum.

The standard treatment for cancer of the colon and rectum is usually a combination of 5-fluorouracil and leucovorin (folinovoy acid).

5-fluorouracil (5-FU) is used for treatment of several solid tumors, including gastrointestinal cancer and breast cancer. It is usually applied with folinovoy acid when running cancer of the colon and rectum. 5-FU is converted to FdUMP in the cell, which forms a complex with timidilatsintazei (TS), inhibiting the synthesis of DNA, protein and RNA.

Folinovaya acid (leucovorin) is a vitamin that is injected in combination with 5-FU. Folinovaya acid increases the frequency response to 5-fluorouracil with significant prolongation of survival without signs of disease and overall survival. Folinovaya acid increases intracellular amount of folate and stabilizes the complex FdUMP/TS.

Other means, which was found to have an effect, include irisnotes the n and oxalipatin, who is licensed as a tool for first-line in patients with advanced cancer of the colon and rectum in combination with 5-fluorouracil and folinovoy acid. Irinotecan or raltitrexed licensed for use as monotherapy for the second line, when therapy on the basis of fluorouracil unsuccessful or impractical.

Oxaliplatin is a recognized anti-cancer agent and contains a new connection diaminocyclohexane, which forms cross links in DNA and, thus, inhibits DNA replication.

“FOLFOX” is a commonly used combination chemotherapy of 5-fluorouracil, folinovoy acid and oxaliplatin.

Irinotecan (CPT-11, Campto) inhibits topoisomerase I, unwinding the DNA enzyme essential for cell division that leads to stop of replication, single-stranded DNA breaks. In the UK irinotecan licensed for use in patients with advanced cancer of the colon and rectum, is not subjected to chemotherapy, in combination with 5FU/FA and as the sole means of second-line chemotherapy in patients who have installed schema based on 5FU was unsuccessful.

Raltitrexed (ZD 1694, Tomudex) inhibits the enzyme of the thymidylate synthetase, which is involved in DNA synthesis. This is the same enzyme that is targeted 5FU. Rahl is fixed licensed in the UK for the palliative treatment of the running of cancer of the colon and rectum, when schema-based 5FU/FA or intolerable, or inappropriate (see Tebbutt et al., 2002, European Journal of Cancer, 38: 1000-1015; Cutsem et al., 2002, Best Practice and Research Clinical Gastroenterology, 16: 319-330; Beretta et al., 2004, Surgical Oncology, 13: 63-73; NICE guidelines for Irinotecan, Oxaliplatin and raltitrexed for advanced colorectal cancer, 2002).

In accordance with a third aspect of the present invention is the use of

(i) antibodies or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least one anticancer means selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexed

in the method of manufacturing a medicinal product for the treatment of cancer.

An alternative is a combination drug that includes:

(i) the antibody or antigen-binding fragment specific for at least one epitope of Hsp90; and

(ii) at least one anticancer agent selected from the group consisting of: 5-fluorouracil, oxaliplatin, irinotecan and raltitrexed

for simultaneous, separate or sequential use in the treatment of cancer.

In accordance with another aspect of the present invention provides a method of treating cancer, comprising introducing a therapeutically effective amount

(i) antibodies or antigen-binding fragment specific for me the greater extent, to the same epitope of Hsp90; and

(ii) at least one anticancer means selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexed

a needy patient.

Preferably, the cancer is a cancer or adenocarcinoma of the colon and rectum.

Most preferably, an anti-cancer agent 5-fluorouracil, in addition, includes or is injected with folinovoy acid (leucovorin).

Additionally or alternatively, 5-fluorouracil, folinovaya acid and oxaliplatin are entered together.

The composition or preparation in accordance with any aspect of the present invention may optionally include pharmaceutically acceptable carrier, diluent or excipient. Similarly, any method of manufacture of the present invention or application can likewise also include the use of pharmaceutically acceptable carrier, diluent or excipient. Examples of pharmaceutically acceptable carriers, diluents and excipients are well known in this field, for example, see: Remington''s Pharmaceutical Sciences and US Pharmacopoeia, (1984, Mack Publishing Company, Easton, PA, USA).

Drug or combined preparation can, for example, be administered orally, although this does not mean that you should exclude other ways of introduction.

The antibody or antigen-related is yuushi fragment in accordance with the present invention may be labeled with detectable label, or may be conjugated with an effector molecule, for example, a drug such as an anti-cancer agent, such as doxorubicin, daunorubicin, or cisplatin, or 5-fluorouracil, oxaliplatin, irinotecan or pharmaceutical agent that can be used in the treatment of leukemia, such as imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin, and hydroxyurea. Or a toxin, such as ricin, or enzyme using normal procedures, and the invention extends to such labeled antibodies or conjugates of antibodies.

If desired, the mixture of antibodies can be used for diagnosis or treatment of, for example, a mixture of two or more antibodies, which recognize different epitopes of stress protein according to the invention and/or a mixture of antibodies of another class, for example a mixture of IgG and IgM antibodies that recognize the same or different epitope(s) according to the invention.

As discussed above, although the Quaternary epitopes shown Hsp90 in multicomponent complexes have been suggested as appropriate targets for treatment experiments (below) show that, in fact, a linear epitope is a useful and effective target for treatment.

The content of each of the discussed here links, including the links, fully incorporated here by reference.

When you specify “PMID” and for publication is given a reference number, this identifier is operating rooms PubMed, assigned National Library of medicine that includes full bibliographic information and an abstract for each publication on the website . This website can also provide direct access to electronic copies of the full publications, in particular, in the case of, for example, publications PNAS, JBC and MBC.

The present invention will be more apparent from the following description which shows only as an example, certain embodiments of the compositions and experimentation with it.

Experiments

In the first group of experiments (Experiments A"), described below, provides details of a study of anticancer effect of anti-Hsp90 antibody having the sequence of SEQ ID NO: 2, and is specific for epitope expressed peptide having the sequence of SEQ ID NO: 1, used alone or in combination with anticancer means doxorubicin, daunorubicin, docetaxel, Herceptin, imatinib, cisplatin and paclitaxel.

In the second group of experiments ("Experiments"), described below, provides details of a study of the effect of anti-Hsp90 antibody having the sequence of SEQ ID NO: 2, and is specific for epitope expressed peptide having the sequence of SEQ ID NO: 1, used alone or in combination with anticancer means of imatinib, paclitaxel, d is cetacean, by daunorubicin, doxorubicin, cisplatin and hydroxyurea on cell line C chronic myelogenous leukemia people-Caucasoids, and cell line KU-812 myelogenous leukemia person.

In the third group of experiments ("Experiments"), described below, provides details of a study of the effect of anti-SR antibody having the sequence of SEQ ID NO: 2, and is specific for epitope expressed peptide having the sequence of SEQ ID NO: 1, used alone or in combination with anticancer means of 5-fluorouracil (5-FU) and folinovoy acid (leucovorin, LV) and/or oxaliplatin on cell line NT adenocarcinoma of the colon of a person.

General materials and methods

In the absence of other indications, all procedures were performed using standard protocols and, where applicable, in accordance with the manufacturer's instructions. Standard protocols for various techniques, including PCR (polymerase reaction synthesis circuit), molecular cloning, manipulation and sequencing, antibodies, mapping epitopes and design Melitopol, culturing the cells and phage display is described in such documents as McPherson, MJ et al. (1991, PCR: A practical approach, Oxford University Press, Oxford), Sambrook, J. and Russell, D., "Molecular Cloning: A Laboratory Manual", Third Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 2001, Huynh and Davies (1985, DNA Cloning vol. I - A Practica Approach", IRL Press, Oxford, Ed. DM Glover), Sanger, F. et al. (1977, PNAS USA 74(12): 5463-5467), Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998), Jung, G. and Beck-Sickinger AG (1992, Angew. Chem. Int. Ed. Eng., 31: 367-486), Harris, M.A. and Rae, I.F. ("General Techniques of Cell Culture, 1997, Cambridge University Press, ISBN 0521 573645), "Phage Display of Peptides and Proteins: A Laboratory Manual (Eds. Kay, BK, Winter, J., and McCafferty, J., Academic Press Inc., 1996, ISBN 0-12-402380-0).

Reagents and equipment that you can use, along with others, in the ways described here, are available from similar companies Amersham (www.amersham.co.uk), Boehringer Mannheim (www.boehringer-ingeltheim.com), Clontech (www.clontech.com), Genosys (www.genosys.com), Millipore (www.millipore.com), Novagen (www.novagen.com), Perkin Elmer (www.perkinelmer.com), Pharmacia (www.pharmacia.com), Promega (www.promega.com), Qiagen (www.qiagen.com), Sigma (www.sigma-aldrich.com) and Stratagene (http://www.stratagene.com).

Antibody

The antibody used in Experiments a and b below, is an antibody described in WO 01/76627, and it is called here "Milgram" (RTM), having the sequence of SEQ ID NO: 2 and which is specific for epitope expressed peptide having the sequence of SEQ ID NO: 1. The primary antibody solution was a mother solution 4 mg/ml in water. Further breeding carried out in complete RPMI medium.

Briefly, the DNA sequence of the former antibodies specific to the epitope of Hsp90 Candida albicans disclosed in GB 2240979 and EP 0406029, was genetically modified by codon optimization for expression in Escherichia coli (Operon Technologies Inc., Aameda, CA, USA) and inserted into the expression vector in E. coli. Amino acid sequence of anti-Hsp90 antibody comprises the sequence of SEQ ID NO: 2 (includes heavy, light and spacer elements domains). The antibody recognizes an epitope comprising the sequence of SEQ ID NO: 1.

Anti-Hsp90 antibody was expressed in host Escherichia coli and then purified by affinity chromatography and imidazole exchange column to a purity of 95%. Used standard protocols in molecular biology (see, for example, Harlow &Lane, supra; Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Sambrook, J. &Russell, D., 2001, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

Drug

Cisplatin was obtained from Bristol-Myers Squibb, Mayne, supplied in the form of a solution of 1 mg/ml

Docetaxel was obtained from Sigma. 5 mg was dissolved initially up to 16 mg/ml dimethylsulfoxide (DMSO).

Doxorubicin was obtained from Pharmacia; 5 ml was supplied in the form of doxorubicin hydrochloride 2 mg/ml

Imatinib (Glivec (RTM)), obtained from Njvartis were supplied in the form of capsules 100 mg Imatinib was initially diluted in water to obtain a stock solution of 10 mg/ml

Paclitaxel was obtained from Sigma, was dissolved in 250 μl of methanol, brought to a volume of 2.5 ml of water to obtain a 2 mg/ml

Daunorubicin was obtained from Sigma; 5 mg diluted in 2.5 ml of water to obtain a 2 mg/m is.

Herceptin (RTM) (trastuzumab) was obtained from Roche and was dissolved in 7.2 ml of water to obtain 21 mg/ml

Hydroxyurea was obtained from Sigma, diluted at 1 g in 4 ml of water to obtain 25 mg/ml

5-fluorouracil (5-FU) was obtained from Sigma; 96 mg was dissolved in 1 ml DMSO, diluted 1/10 in complete RPMI medium, to obtain 9.6 mg/ml

Polynovo acid (LV) were obtained from Sigma; 100 mg was dissolved in 25 ml of water to obtain a stock solution of 4 mg/ml

Oxaliplatin was obtained from Sigma; 12.5 mg was dissolved in 2.5 ml of water to obtain a 5 mg/ml

Each of the above preparations was further diluted in complete RPMI medium.

Determining the concentration and viability of cells

Cells were counted and the percentage viability was determined using the standard hemocytometer, following the staining of an equal volume of 0.4% solution of tripan blue (Sigma).

Analysis of the viability of the cells

Cell viability was assessed after each experiment using analysis of cell titer blue reagent (Promega). The medium was removed from cells and were added 100 μl of fresh complete medium and then adding 20 ál blue reagent cell titer. This solution was incubated at 37°C, 5% CO2within 4 h, and the spectral absorption ability was read at 570 nm using 600 nm as a reference. In this analysis, we used indicator is hydrated dye resazurin (blue) to measure the metabolic capacity of cells. Viable cells restore resazurin in resorufin (pink).

Data interpretation

Cell growth was assessed as described above. Concentration IC50(the dose needed to induce cytotoxicity in 50% of cells) was determined once for each drug during the incubation period of 48 hours

Analysis of the median effect, synergism, additive effect and antagonism, based on the Hill equation was determined by the method of Chou and Talaly using the product calcusyn (BioSoft, Cambridge, UK ). CI (combination index)that reflects the synergy when it is less than 1, an additive effect when it is equal to 1, and antagonism when it is greater than 1 were calculated for varying levels of effect of the drug. Fixed ratio drugs above and below the IC50(the concentration of drug required to provide 50% cytotoxic effect) in the range 0,0156N-8N, where N is a value of about IC50individual drug, was investigated by incubation of drug combinations with the cells for 48 h and then determining the degree of cytotoxicity. Fa50 is determined at the point where the affected 50% of the cells. The values CI are shown for Fa50.

Experiments And

The results show that the antibody testified about the antagonism with imatinib and indifference to paclitaxel. Had the ü some synergy with cisplatin and docetaxel, but the latter is likely to occur in concentrations that cannot be achieved clinically. Doxorubicin demonstrated synergy at clinically achievable levels of the drug with both cell lines, and regardless of whether the estrogen receptor. The results achieved with doxorubicin, interpreted as highly significant synergy. The results when using daunorubicin were equally impressive with the cell line with estrogen receptor, but less than with a cell line that is negative for estrogen receptor, in synergy limited to 6 and 12.5 mg/L. Results for Herceptin has shown a lack of synergy against the line of cells positive for estrogen receptor, but synergy was observed when the cell line is negative for estrogen receptor.

Materials and methods

Information about the cell line and culture

Cell line mammary adenocarcinoma (human Caucasian MCF7 expressing the estrogen receptor and wild-type and variant estrogen receptors, and progesterone receptor, was obtained from ESAS (number ESAS - 86012803).

Other used cell line the following:

HS578T - ECACC number 86082104, carcinoma of the human breast, epithelial. Carcinogenic in mice with depressed immunity and form colonies in semi-solid medium. Negative for the estrogen receptor.

SK-BR-3 - (ATCC) Adanac rcinoma breast cancer. Positive for estrogen receptor. Excessively expresses the gene HER2/C-erb-2.

UACC-812 - (ATCC) - Carcinoma of the duct before the operation the patient was in the intensive chemotherapy. Negative for estrogen receptor, and negative for the progesterone receptor, and negative for P-glycoprotein. Amplification of the sequence of the oncogene HER-2/ neu.

NST - ATS) - Carcinoma of the colon and rectum. Positive expression of TGF beta 1 and beta 2.

Cells were digested using 0.25% trypsin/EDTA (Sigma)and maintained in RPMI medium without phenol red containing 10% fetal calf serum, 1% non-essential amino acids, 2 mm glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Sigma) at 37°C, 5% CO2.

Experiments And

The impact of mikegrb on MCF7 cells

Cell lines were digested and the cells were counted. Cells were added to 12 - or 96-well tablets, flat-bottom tissue culture. In the case of a 12-well plate was added 1 ml of 4×104cells/ml plus 1 ml of medium. In the case of a 96-well plate in the plate was added 100 μl of 4×104cells/ml, followed by adding 100 ál of complete medium. The plates were incubated over night at 37°C, 5% CO2. The next day the cells were observed under phase-contrast microscope to ensure their adhesion to the tablet, and the supernatant medium was removed what spiracies. Then the wells were added with fresh medium containing twice the increasing concentration of mikegrb (1.5 to 200 μg/ml) or composite buffer, or a single environment. The tablets were returned to the incubator for 48 hours After that conducted the analysis of cell titer blue reagent or counting viable cells using hemocytometer.

The impact of cancer means of doxorubicin, daunorubicin, Herceptin, docetaxel, imatinib, paclitaxel and cisplatin in MCF7 cells

Cell lines were digested and the cells were counted. 100 μl of 4×104cells/ml were added to 96-well tablets, flat-bottom tissue culture, and the plate was added 100 μl of complete medium. Then the plates were incubated over night at 37°C, 5% CO2. The next day the cells were observed under phase-contrast microscope to ensure their adhesion to the tablet, and the supernatant medium was removed by aspiration. To the wells were added with fresh medium containing increasing concentrations of the investigational drug (doxorubicin - 0,55-600 µg/ml of daunorubicin - 0,45-1000 µg/ml Herceptin is 0.2 - 200 ág/ml docetaxel - 0.75 to 800 μg/ml imatinib - 4,5-5000 µg/ml cisplatin - 0.04 to 50 μg/ml paclitaxel - 1,8-1000 µg/ml) or a single environment. The tablets were returned to the incubator for 48 h, after which carried out the analysis of cell titer blue reagent.

The impact of mikegrb in combination the with anticancer means doxorubicin, by daunorubicin, Herceptin, docetaxel, imatinib, paclitaxel and cisplatin in MCF7 cells

Cell lines were digested and the cells were counted. 100 μl of 4×104cells/ml were added to 96-well tablets, flat-bottom tissue culture, and the plate was added 100 μl of complete medium. Then the plates were incubated over night at 37°C, 5% CO2. The next day the cells were observed under phase-contrast microscope to ensure their adhesion to the tablet, and the supernatant medium was removed by aspiration. In the tablets was added 100 μl of fresh medium and was "chess Board" of mikegrb in comparison with another drug, as presented below in table 1 (using doxorubicin as an example) to obtain a total volume of 200 µl per well.

The tablets were returned to the incubator for 48 h, after which carried out the analysis of cell titer blue reagent.

Experiments were also performed with HS578T cells using the above methodology, and the results are presented below.

The results of the experiments And

Cell line MCF7

Cisplatin

IC50was 6 mg/l, and no effect after adding mikegrb, except synergies at higher concentrations, see table 2.

Imatinib

IC50amounted to 37.5 mg/l, and no evidence of antagonism with Csp in the range of 3.3-10 picogram the doses of imatinib below 37.5 mg/L. Above this dose imatinib was destroyed cell line.

Docetaxel

IC50amounted to 225 mg/l, and there was no evidence of any synergy with Milgram high doses of docetaxel - see table 3.

Paclitaxel

IC50amounted to 225 mg/l, and no differences with low concentrations of the drug and little synergy at high levels, such as 500 mg/l of paclitaxel. These levels are beyond levels that are clinically relevant.

Doxorubicin

IC50was 1.75 mg/L. there was a clear synergy with picogram the concentration range of the drug - see table 4.

Daunorubicin

IC50was 1 mg/L. Were evidence of synergy with picogram the concentration range of the drug - see table 5.

Herceptin

No detectable activity due to Herceptin and evidence of synergy.

Cell line HS578T

This cell line was insensitive to mikegrb with increasing concentrations up to 400 mg/L. It was not surprising that these tumors are not sensitive to steroids and, thus, there is no endogenous probability of their reactions to the Hsp90 inhibitor, such as mikegrb. However, in combination with anthracyclines doxorubicin and daunorubicin and Herceptin was unexpected synergy.

Doxorubicin

IC50was 1 mg/L. data were Available for the synergy with picogram the concentration range of the drug - see table 6.

Daunorubicin

IC50was 1 mg/L. Were some evidence of synergy, but mostly indifference to mikegrb - see table 7.

Herceptin

With HS578T, one Herceptin could not destroy 50% of the cells at concentrations up to 200 mg/l, but in the presence of mikegrb was observed synergy - see table 8.

Docetaxel

He gave IC5050 mg/l cell line HS578T and did not show data for synergy with Milgram.

Cisplatin

Cisplatin had IC5012.5 mg/l for cell line HS578T and did not show data for synergy with Milgram.

Conclusion

Were evidence of antagonism with imatinib and indifference to paclitaxel. There was a certain synergy with cisplatin and docetaxel, but the latter is likely to occur in concentrations that cannot be achieved clinically. Doxorubicin demonstrated synergy at clinically achievable levels of the drug with both cell lines, and regardless of the presence of the estrogen receptor. Results doxorubicin, interpreted as highly significant synergy. The results of the use of daunorubicin were equally impressive with the cell line with estrogen receptor, but less with the cell line, negative receptor e is trogen, when synergy limited to 6 and 12.5 mg/l

This indicated above, a synergistic effect is observed between the antibody and certain anticancer drugs, but not with other anticancer means, such as imatinib.

Experiments In

In the second group of experiments (described below) described in detail the study of the effect of anti-Hsp90 antibody having the sequence of SEQ ID NO: 2, and is specific for epitope expressed peptide having the sequence of SEQ ID NO: 1, used separately or in combination with anticancer means of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin, cisplatin and hydroxyurea on cell line C chronic myelogenous leukemia people-Caucasoids, and cell line KU-812 myelogenous leukemia person.

The results show that antibody gave evidence of the synergy with imatinib, paclitaxel, docetaxel, daunorubicin. Antibody gave a testimony about some synergy with doxorubicin and hydroxyurea. The results show that antibody gave a testimony of indifference to this drug called cisplatin.

The results achieved with docetaxel and paclitaxel viewed as highly significant synergy.

Materials and methods

Information about the cell line and culture

Line KU-812 myelogenous leukemia person received from ESAS (number ESAS - 90071807). In this cell line was identified, the Philadelphia chromosome (Ph1). Cells morphologically characteristic of basophils.

Cell line C chronic myelogenous leukemia people-Caucasoids received from ESAS (number ESAS - 89121407). C was established from the pleural effusion of a 53-year-old woman with chronic myelogenous leukemia in terminal blast crisis. Karyological studies of various Pollini K-562 were divided into 3 groups (A, B, C) (Dimery, I. W. et al., 1983, Exp. Hematol.; 11(7):p601-10). The line used in these experiments was CV. Experiments have demonstrated that these lines are generally the same, from the point of view of morphology, growth kinetics in liquid suspension culture, the efficiency of cloning in soft agar culture, the binding of anti-C monoclonal antibodies and surface proteins. KW compared with CA and CS in relation to the kinetics of growth, markers of cell surface proteins, surface antigens, cytogenetics and production of hemoglobin. Observed differences between cell lines, with the important difference is that while more than 90% of the cells CA or was Ph1-positive, less than 15% of the cells KV contained Ph1 (Dimery, IW, et al., 1983, Exp. Hematol.; 11(7):p601-10). Although cytogenetic tests do not reveal the true presence of the Ph1 chromosome, it seems that C contain cha is th chromosome Ph1, which, at least, amplified four times. This part of the Ph1 chromosome encodes a chimeric transcript bcr/c-abl, which when broadcast network composite protein bcr/c-abl (Grosveld, G., et al., 1986, Mol. Cell. Biol. 6, No. 2: p607-616). Composite protein bcr/c-abl has increased tyrosine kinase activity, which is responsible for the pathogenesis of CML.

Cells were maintained at a concentration of 2×106up to 9×106cells/ml in RPMI medium without phenol red containing 10% fetal calf serum, 2 mm glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Sigma) at 37°C, 5% CO2.

Experiments

The impact of mikegrb cells C

The cell line was calculated. Cells were added to 12 - or 96-well tablets, flat-bottom tissue culture using aliquot amount of 100 μl containing 4×105cells/ml tablet was added to fresh medium containing or twice increasing concentration of mikegrb (RTM) (1.5 to 200 μg/ml), or a single environment. The tablets were returned to the incubator for 48 h, after which carried out the analysis of cell titer blue reagent or determined the number of viable cells using hemocytometer.

The impact of cancer means of doxorubicin, daunorubicin, docetaxel, paclitaxel, imatinib, cisplatin and hydroxyacetone cells C

The cell line was calculated. 100 μl of 2×105and the and 4×10 5cells/ml were added to 96-well tablets, flat-bottom tissue culture. Then the plates were incubated over night at 37°C, 5% CO2. In the tablet was added to fresh medium containing increasing concentrations of the investigational drug (doxorubicin - 0,55-600 µg/ml of daunorubicin - 0.07 to 100 µg/ml docetaxel - 0.75 to 800 μg/ml paclitaxel - 0.5 to 500 μg/ml imatinib - 4,5-5000 µg/ml cisplatin - 0,04-50 µg/ml) or a single environment. The tablets were returned to the incubator for 48 h, after which carried out the analysis of cell titer blue reagent.

The impact of mikegrb in combination with anticancer means doxorubicin, daunorubicin, docetaxel, paclitaxel, imatinib, cisplatin and hydroxyurea on cell C

The cell line was calculated. 100 μl of 2×105or 4×105cells/ml were added to 96-well tablets, flat-bottom tissue culture. Then the plates were incubated over night at 37°C, 5% CO2. In the tablets was added 100 μl of fresh medium and was "chess Board" of mikegrb, in comparison with another drug, as presented below in table (using doxorubicin as an example) to obtain a total volume of 200 µl per well.

Experiments were also performed with cells KU-812, using the above methodology, and the results are presented below.

Results

The impact of mi is agraba (RTM) on cells C

In relation to cell line K one mikegrb at a concentration of 12.5 µg/ml has been shown to reduce cell viability by 40%.

The impact of mikegrb (RTM) and funds for cancer cells C

Imatinib

IC50amounted to 16 mcg/ml have Been some evidence of synergy between imatinib and Milgrom (RTM) in the concentration range of the drug (see table 10).

Doxorubicin

IC50was 1 µg/ml Were some evidence of synergy between doxorubicin and Milgrom (RTM) at certain concentrations of the drug, but mostly indifference to mikegrb (RTM).

Daunorubicin

IC50amounted to 0.75 µg/ml Were some evidence of synergy between daunorubicin and Milgrom (RTM) at low concentrations of the drug (see tab. 11).

Docetaxel

IC50was 70 µg/ml Was clear evidence of synergy between docetaxel and Milgrom (RTM) in the concentration range of the drug (see tab. 12).

Paclitaxel

IC50was 32 µg/ml Was clear evidence of synergy between paclitaxel and Milgrom (RTM) in the concentration range of the drug (see table 13).

Cisplatin

IC50was 12.5 µg/ml there was No evidence of synergy between cisplatin and Milgrom (RTM).

Hydroxyurea

IC50Niko is Yes not achieved hydroxyurea as the only means. However, there was some evidence of synergy between hydroxyurea and Milgrom (RTM) at low concentrations of the drug (see table 14).

Cell line KU-812

The impact of mikegrb (RTM) on cells of the KU-812

In relation to cell line KU-812 one mikegrb at a concentration of 50 mcg/ml has been shown to reduce cell viability by 40%.

The impact of mikegrb (RTM) and funds for cancer cells KU-812

Imatinib

IC500.12 µg/ml Were some evidence of synergy between imatinib and Milgrom (RTM) at low concentrations of the drug (see table).

Summary

When using a cell line K was evidence of synergy with imatinib, paclitaxel and docetaxel. There was evidence of some synergy with daunorubicin, doxorubicin and hydroxyurea. Indifference was observed for this drug called cisplatin.

When using a cell line KU-812 was evidence of some synergy with imatinib.

Conclusions

The data presented here clearly demonstrate that the antibody mikegrb (RTM) separately can reduce the viability and Ph1-positive, and Ph1-negative CML cell lines. In addition, there is an amazing synergy between anticancer means, including imatinib, and anti-Hsp90 antibody in Ph1-positive CML cell lines. The data also de will Instituut, there is a synergy between paclitaxel, docetaxel, daunorubicin, doxorubicin, hydroxyurea, and anti-Hsp90 antibody in Ph1-positive cell lines of leukemia. These results allow us to apply the composition, including anticancer agents such as imatinib, together with anti-Hsp90 antibody (Milgrom RTM) for the treatment of CML. The synergism shown by a combination of anti-cancer tools and antibodies mikegrb (RTM), potentially enables the use of a lower therapeutic dosages that would be very important with regard to problematic toxicity of many anticancer funds and, in particular, imatinib, or more effective and longer courses of treatment at the same dosage, reducing, through this, unwanted side effects.

Possible aspects of clinical use of the present invention include: (i) obtaining a synergistic combination of anti-cancer agents, e.g. of imatinib, and anti-Hsp90 antibodies in the treatment of CML should be the treatment of choice. It probably would have led to a reduction in mortality from CML; (ii) imatinib-toxic, and synergy provided by the present invention, means that you can use a lower dose of imatinib while maintaining efficacy while reducing toxicity; and (iii) reduce the toxicity effect of anti-Hsp90 antibodies would provide moznosti use clinical efficacy of higher doses of imatinib and additionally would improve clinical outcome.

Experiments With

In the third group of experiments (described below) described in detail the study of the effect of anti-Hsp90 antibody having the sequence of SEQ ID NO: 2, and is specific for epitope expressed peptide having the sequence of SEQ ID NO: 1, used separately or in combination with anticancer means of 5-FU and folinovoy acid and/or oxaliplatin on cell line adenocarcinoma of the colon NT.

The results show that antibody gave a testimony of synergy with 5-FU and folinovoy acid and oxaliplatin. There was also evidence of synergy with the combination of four drugs of mikegrb/anti-Hsp90 antibody, 5-FU, folinovoy acid and oxaliplatin. It was found that especially useful concentration of 5-FU more than 75 µg/ml and oxaliplatin more than 10,5 mg/ml

Materials and methods

Information about the cell line and culture

Cell line NT stage II adenocarcinoma of the colon of a person of Caucasian received from ESAS (number ESAS - 91072201).

Cells were digested using 0.25% trypsin/EDTA (Sigma), and maintained in an environment McCoy 5a containing 10% fetal calf serum, 2 mm glutamine, 1000 IU of penicillin, 0.1 mg of streptomycin (Sigma) at 37°C, 5% CO2.

Other cell lines include NST.

Experiments

The impact of mikegrb cells NC

Cell lines were digested and the cells were counted. Cells were added to 12 - or 96-well tablets, flat-bottom tissue culture. In the case of a 12-well plate, each well was added 1 ml of 4×104cells/ml or 4×105cells/ml plus 1 ml of complete medium McCoy 5a. In the case of a 96-well plate, each well was added 100 μl of 4×104or 4×105cells/ml, followed by addition of 100 μl of complete medium McCoy 5a. The tablets are then incubated over night at 37°C, 5% CO2. The next day the cells were observed under phase-contrast microscope to ensure their adhesion to the tablet, and the supernatant medium was removed by aspiration. Then the wells were added with fresh RPMI medium containing twice the increasing concentration of mikegrb (RTM) (1.5 to 200 μg/ml) or a single environment. The tablets were returned to the incubator for 48 h, after which carried out the analysis of cell titer blue reagent or counting of viable cells.

The impact of anti-cancer tools 5FU and folinovoy acid and oxaliplatin cells NC

Cell lines were digested and the cells were counted. 100 μl of 4×104cells/ml or 4×105cells/ml were added to 96-well tablets, flat-bottom tissue culture and in tablets was added 100 μl of complete medium McCoy 5a. Then the plates were incubated over night at 37°C, 5% CO2. The next day etki observed under phase-contrast microscope for to ensure their adhesion to the tablet, and the supernatant medium was removed by aspiration. Then the wells were added with 100 μl of fresh complete RPMI medium containing twice the increasing concentration of the study drug (5-FU 4,5-2400 ág/ml plus 1 mg/ml folinovoy acid or oxaliplatin 1-500 μg/ml) or one environment (environment + 2,5% DMSO for control 5-FU). The tablets were returned to the incubator for 48 h, after which carried out the analysis of cell titer blue reagent.

The impact of mikegrb (RTM) in combination with anticancer means 5FU and folinovoy acid and oxaliplatin on cell NT

Cell lines were digested and the cells were counted. 100 μl of 4×104cells/ml or 4×105cells/ml were added to 96-well tablets, flat-bottom tissue culture, and the tablets were added 100 μl of complete medium McCoy 5a. Then the plates were incubated over night at 37°C, 5% CO2. The next day the cells were observed under phase-contrast microscope to ensure their adhesion to the tablet, and the supernatant medium was removed by aspiration. To the wells were added with 100 μl of fresh complete RPMI medium and were "chess Board" of mikegrb (RTM) in comparison with the investigational drug ((5-FU 4,5-2400 ág/ml plus 1 mg/ml folinovoy acid or oxaliplatin 1-500 μg/ml) or one environment (environment + 2,5% DMSO for control 5-FU)), as presented in table 16, for Poluchenie volume of 200 µl per well.

The impact of mikegrb (RTM) in combination with anticancer means 5FU and folinovoy acid and oxaliplatin on cell NT

Cell lines were digested and the cells were counted. 100 μl of 4×104cells/ml or 4×105cells/ml were added to 96-well tablets, flat-bottom tissue culture, and the tablets were added 100 μl of complete medium McCy 5a. Then the plates were incubated over night at 37°C, 5% CO2. The next day the cells were observed under phase-contrast microscope to ensure their adhesion to the tablet, and the supernatant medium was removed by aspiration. To the wells were added with 100 μl of fresh complete RPMI medium and were "chess Board" of mikegrb (RTM) in comparison with the investigational drug ((5-FU:folinovaya acid:oxaliplatin in the ratio 3:1:0,42) or one environment (environment +2,5% DMSO for control 5-FU)), as presented in table 16 for 5-FU, to obtain a total volume of 200 µl per well.

Results

The impact of mikegrb (RTM) on cells NC

In relation to cell line NT one picogram (RTM) at a concentration of 125 µg/ml has been shown to reduce cell viability by 50%.

The impact of anti-cancer tools 5FU or oxaliplatin separately and mikegrb (RTM) in combination with anticancer means 5FU or oxaliplatin cells NC

5FU

IC505-fluorouracil was 150 μg/ml was the clear evidence of synergy between 5-FU and Milgrom (RTM) in the concentration range of the drug (see table 17-20).

Oxaliplatin

IC50for oxaliplatin was 16 μg/ml Were some evidence of synergy between oxaliplatin and Milgrom (RTM) in the concentration range of the drug (see table 21).

The impact of mikegrb (RTM) in combination with anticancer means 5FU and oxaliplatin on cell NT

IC50LV (leucovorin/5FU/ox was 25/75/10,5 µg/ml Were some evidence of synergy between 5-FU and oxiplatin and Milgrom (RTM) in the concentration range of the drug (see table 22-24).

Summary

The results show that antibody gave a testimony about synergy with 5FU and folinovoy acid or oxaliplatin and some evidence of synergy with the combination of four drugs with 5-FU and folinovoy acid and oxaliplatin at concentrations of more than 75 µg/ml of 5-FU and more than 10,5 mg/ml oxaliplatin. There was no evidence of synergy with 5-FU and oxaliplatin, in table 25 shows the values of CI at ED50, ED75and ED90.

Conclusions

The data presented here clearly demonstrate that the antibody mikegrb (RTM) separately can reduce the viability of the cell line adenocarcinoma of the colon. Was synergy between anticancer means, including 5-fluorouracil, oxiplatin, and anti-Hsp90 antibody on the cell line adenocarcinoma of the colon. The data also show that there is a synergy between 5-fluorouracil and oxaliplatin with anti-Hsp90 antibody on the cell line adenocarcinoma of the colon.

Table 1
Chess Board mikegrb (MG) (μg/ml), in comparison with doxorubicin (DR) (µg/ml)

3
Table 2
CisplatinMikegrbCI
12,5250,012
25500,024
Table 3
MikegrbDocetaxelCI
12,51000,400
252000,098
50400,002
Table 4
MikegrbDoxorubicinCI
0,752,250,249
1,54,50,261
390,248
6180,476
12,537,50,186
25750,155
50150strength of 0.159
Table 5
MikegrbDaunorubicinCI
0,750,750,189
1,51,50,257
330,512
660,676
12,512,50,469
25 250,082
50500,176
Table 6
DoxorubicinMikegrbCI
0,550,750,573
1,121,50,541
2,2530,824
4,560,254
9120,507
18,5250,538
37501,033
Table 7
MikegrbDaunorubicinCI
0,750,751,153
1,51,51,300
31,557
660,763
12,512,50,367
25251,014 registered
Table 8
MikegrbHerceptinCI
0,750,750,007
1,51,50,008
330,005
660,034
12,512,50,025
25250,170

Table 9
Chess Board mikegrb (MG) (mcg/ml)
in comparison with doxorubicin (DR) (µg/ml)

0,05
Table 10
Imatinib (µg/ml)Milgram, ug/mlCI
10,750,033
21,50,069
430,114
860,218
1612,50,366
32250,558
64500,799
Table 11
Daunorubicin (µg/ml)Mikegrb (µg/ml)CI
0,751,50,696
1,530,872
Table 12
Docetaxel (µg/ml)Mikegrb (µg/ml)CI
1,51,50,001
330,008
660,707
12,512,50,051
25250,013
50500,003
1001000,004
Table 13
Paclitaxel (µg/ml)Mikegrb (µg/ml)CI
11,50,064
230,145
460,011
812,5
16250,04
32500,06
641000,077
Table 14
Hydroxyurea (µg/ml)Mikegrb (µg/ml)CI
0,30,750,798
0,60,750,694
Table 15
Imatinib (µg/ml)Mikegrb (µg/ml)CI
10,3750,131
21,50,093
430,016
860,001
1612,50,002
32250,327

Table 16
Chess Board mikegrb (RTM) (MG ág/ml), in comparison with 5-fluorouracil (5-FU in µg/ml)
123456789101112
AEnvironment
a 2.5% DMSO
5FU
4,5
5FU 95FU 18,55FU 375FU 755FU 1505FU 3005FU 6005FU 12005FU 2400Only medium without cells
Ina 2.5% DMSO 3 MG5FU
4,5 MG 3
5FU 9 MG 35FU
18,5 MG 3
5FU 37 MG 35FU 75 MG 3 5FU 150 MG 35FU 300 MG 35FU 600 MG 35FU 1200
MG 3
5FU 2400 MG 3Only medium without cells
a 2.5% DMSO MG 65FU 4,5 MG 65FU 9 MG 65FU
18,5 MG 6
5FU 37 MG 65FU 75 MG 65FU 150 MG 65FU 300 MG 65FU 600 MG 65FU 1200
MG 6
5FU 2400 MG 6Only medium without cells
Da 2.5% DMSO MG 12,55FU
4,5
MG
12,5
5FU 9 MG 12,55FU
18,5
MG
12,5
5FU 37 MG 12,55FU 75 MG 12,55FU 150 MG 12,55FU 300 MG 12,55FU 600 MG 12,55FU 1200
MG 12,5
5FU 2400 MG 12,5Only medium without cells
Ea 2.5% DMSO 25 MG5FU
the 4.5 MG 25
5FU 9 MG 25 5FU
18,5 MG 25
5FU 37 MG 255FU 75 MG 255FU 150 MG 255FU 300 MG 255FU 600 MG 255FU 1200
MG 25
5FU 2400 MG 25Only medium without cells
Fa 2.5% DMSO 50 MG5FU 4,5 50 MG5FU 9 MG 505FU
18,5 50 MG
5FU 37 50 MG5FU 75 MG 505FU 150 MG 505FU 300 MG 505FU 600 MG 505FU 1200
50 MG
5FU 2400 MG 50Only medium without cells
Ga 2.5% DMSO 100 MG5FU 4,5 100 MG5FU 9 100 MG5FU 18,5 100 MG5FU 37 MG 1005FU 75 MG 1005FU 150 MG 1005FU 300 MG 1005FU 600 MG 1005FU 1200
MG 100
5FU 2400 MG 100Only medium without cells
H2.5% of the DSO 200 MG 5FU 4,5 200 MG5FU 9
200 MG
5FU 18,5 200 MG5FU 37 MG 2005FU 75 MG 2005FU 150
200 MG
5FU 300 MG 2005FU 600 MG 2005FU 1200
200 MG
5FU 2400 MG 200Only medium without cells

0,153 9
Table 17
5-fluorouracil and mikegrb ratio of 0.37:1
5-FU (mg/ml)Mikegrb (µg/ml)CI
4,512,50,001
9250,004
18,5500,008
371000,049
Table 18
5-fluorouracil and mikegrb in the ratio of 0.75:1
5-FU (mg/ml)Mikegrb (µg/ml)CI
4,560,184
912,50,295
Table 19
5-fluorouracil and mikegrb in the ratio of 12:1
5-FU (mg/ml)Mikegrb (µg/ml)CI
3730,557
7560,228
15012,50,198
300250,540
600500,250
120010024001000,212
Table 20
5-fluorouracil and mikegrb in the ratio of 50:1
5-FU (mg/ml)Mikegrb (µg/ml)CI
751,50,004
15030,342
30060,482
Table 21
Oxaliplatin and mikegrb in the ratio of 1.25:1
Oxaliplatin (µg/ml)Mikegrb (µg/ml)CI
7,560,378
15,512,5of 0.182
3125
62,5500,046
1251000,616
2502000,185
Table 22
5-fluorouracil, oxaliplatin and mikegrb in the ratio 3:1:0,42
5-FU (mg/ml)Mikegrb (µg/ml)Oxaliplatin (µg/ml)CI
931,30,053
18,562,60,109
3712,5the 5.250,239
752510,5 3,120
Table 23
5-fluorouracil, oxaliplatin and mikegrb in the ratio 3:2:0,42
5-FU (mg/ml)Mikegrb (µg/ml)Oxaliplatin (µg/ml)CI
961,30,074
18,512,52,60,152
3725the 5.250,405
755010,51,519
Table 24
5-fluorouracil, oxaliplatin and mikegrb in the ratio 3:0,5:0,42
5-FU (mg/ml)Mikegrb (µg/ml)Oxaliplatin (µg/ml)CI
1,51,30,043
18,532,60,089
376the 5.250,184
7512,510,52,202
Table 25
Drug (ratio)The magnitude of the combined rate at
ED50ED75ED90Dmr
5-FUN/AN/AN/A297,14930,95571
MikegrbN/AN/AN/A96,465430,87307
OxaliplatinN/AN/AN/A27,771330,94124
5-FU/mikegrb/
oxaliplatin
0,77008 0,114190,0225664,920050,87996
(3:1:0,42)
5-FU/mikegrb/
oxaliplatin
0,850850,164150,0392455,547980,97163
(1,5:1:0,21)
5-FU/mikegrb/
oxaliplatin
0,622670,099160,0222461,080420,9142
(6:1:0,85)

1. Use
(i) antibodies or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anticancer means selected from the group consisting of doxorubicin, daunorubicin, epirubicin, Herceptin, docetaxel and cisplatin
in the method of manufacturing a medicinal product for the treatment of cancer.

2. Combined product, including:
(i) the antibody or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, Herceptin, docetaxel and cisplatin,
for simultaneous, separate or sequential use in the treatment of cancer.

3. The use according to claim 1, where the specified cancer selected from the group consisting of fibrosarcoma, breast carcinoma, prostate cancer, melanoma, leukemia, lymphoma, carcinoma of the colon, paradisevalley carcinoma of the testes, carcinoma of the pancreas, ovarian, endometrial, thyroid and lungs.

4. Combined preparation according to claim 2, where the specified cancer selected from the group consisting of fibrosarcoma, breast carcinoma, predstaviteley, melanoma, leukemia, lymphoma, carcinoma of the colon, paradisevalley carcinoma of the testes, carcinoma of the pancreas, ovarian, endometrial, thyroid and lungs.

5. A method of treating cancer, comprising introducing a therapeutically effective amount of:
(i) antibodies or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anticancer means selected from the group consisting of doxorubicin, daunorubicin, epirubicin, Herceptin, docetaxel and cisplatin,
a needy patient.

6. Use
(i) antibodies or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1;
in the method of manufacturing a medicinal product for the treatment of leukemia.

7. Use
(i) antibodies or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anticancer means selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyacetone,
in the method of manufacturing a medicinal product for the treatment of leukemia.

8. Combined preparation containing
(i) the antibody or antigen-binding fragment that recognizes the epitope is within the sequence of SEQ ID NO:1; and
(ii) at least one anti-cancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyacetone,
for simultaneous, separate or sequential injection in the treatment of leukemia.

9. The use according to any one of p or 7, where the specified leukemia selected from the group consisting of acute myeloblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia.

10. Combined preparation of claim 8, where the specified leukemia selected from the group consisting of acute myeloblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia.

11. The use according to any one of p or 7, where the aforementioned at least one anti-cancer agent is imatinib.

12. Combined preparation of claim 8, where the aforementioned at least one anti-cancer agent is imatinib.

13. The use according to any one of p or 7, where the specified leukemia is a chronic myeloid leukemia or acute lymphoid leukemia.

14. Combined preparation of claim 8, where the specified leukemia is a chronic myeloid leukemia or acute lymphoid leukemia.

15. Use item 13 where specified leukemia is characterized by cells, which are positive for the Philadelphia chromosome, or cells that are negative for the Philadelphia chromosome.

16. Combined preparation according to 14, where the specified leukemia is characterized by cells that are positive for the Philadelphia chromosome, or cells that are negative for the Philadelphia chromosome.

17. Use item 13, where the specified anti-cancer agent is imatinib, and the specified leukemia is characterized by cells that are positive for the Philadelphia chromosome.

18. Combined preparation according to 14, where the specified anti-cancer agent is imatinib, and the specified leukemia is characterized by cells that are positive for the Philadelphia chromosome.

19. Use item 13, where the specified anti-cancer agent is imatinib, and the specified leukemia is characterized by cells that are negative for the Philadelphia chromosome.

20. Combined preparation according to 14, where the specified anti-cancer agent is imatinib, and the specified leukemia is characterized by cells that are negative for the Philadelphia chromosome.

21. The use according to any one of p or 7, where the specified leukemia is characterized by cells that JW is Auda resistant to imatinib.

22. Combined preparation according to any one of p,16,18 or 20, where the specified leukemia is characterized by cells that are resistant to imatinib.

23. A method of treating leukemia, comprising introducing a therapeutically effective amount of:
(i) antibodies or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anticancer means selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyacetone,
needs it for the patient.

24. The method according to item 23, where the specified leukemia is a chronic myeloid leukemia, and said at least one anti-cancer agent is imatinib.

25. Use
(i) antibodies or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anticancer means selected from the group consisting of: 5-fluorouracil, oxaliplatin, irinotecan and raltitrexed
in the method of manufacturing a medicinal product for the treatment of cancer.

26. Combined product, including:
(i) the antibody or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anti-cancer the tool, selected from the group consisting of: 5-fluorouracil, oxaliplatin, irinotecan and raltitrexed
for simultaneous, separate or sequential use in the treatment of cancer.

27. The use according to any one of claims 1, 6, 7 or 25, where the aforementioned antibody or antigen-binding fragment specific for the epitope represented by the peptide having the sequence of SEQ ID NO:1.

28. Combined preparation according to any one of claim 2,8 or 26, where the aforementioned antibody or antigen-binding fragment specific for the epitope represented by the peptide having the sequence of SEQ ID NO:1.

29. The use according to any one of claims 1, 6, 7 or 25, where the specified antibody comprises the sequence of SEQ ID NO:2.

30. Combined preparation according to any one of claim 2, 8 or 26, where the specified antibody comprises the sequence of SEQ ID NO:2.

31. Use A.25 where specified cancer selected from the group consisting of fibrosarcoma, adenocarcinoma, breast cancer, prostate cancer, melanoma, leukemia, lymphoma, carcinoma of the colon and rectum, paradisevalley carcinoma of the testes, carcinoma of the pancreas, ovarian, endometrial, thyroid and lungs.

32. Combined preparation for p where specified cancer selected from the group consisting of fibrosarcoma, adenocarcinoma, breast cancer, prostate cancer, melanoma, leukemia,lymphoma, carcinoma of the colon and rectum, paradisevalley carcinoma of the testes, carcinoma of the pancreas, ovarian, endometrial, thyroid and lungs.

33. Use p where specified cancer is a cancer of the colon, rectum or adenocarcinoma.

34. Combined preparation for p where specified cancer is a cancer of the colon, rectum or adenocarcinoma.

35. A method of treating cancer, comprising introducing a therapeutically effective amount of
(i) antibodies or antigen-binding fragment that recognizes the epitope having the sequence of SEQ ID NO:1; and
(ii) at least one anticancer means selected from the group consisting of: 5-fluorouracil, oxaliplatin, irinotecan and raltitrexed,
a needy patient.

36. Use A.25, where an anti-cancer agent is 5-fluorouracil and, in addition, includes polynovo acid (leucovorin).

37. Combined preparation for p, where an anti-cancer agent is 5-fluorouracil and, in addition, includes polynovo acid (leucovorin).

38. The method according to p, where an anti-cancer agent is 5-fluorouracil and, in addition, includes polynovo acid (leucovorin).

39. Use p, where an anti-cancer agent comprises 5-fluorouracil, polynovo KIS the GTC (leucovorin and oxaliplatin.

40. Combined preparation according to clause 37, where an anti-cancer agent comprises 5-fluorouracil, polynovo acid (leucovorin and oxaliplatin.

41. The method according to § 38, where an anti-cancer agent comprises 5-fluorouracil, polynovo acid (leucovorin and oxaliplatin.

42. The method according to any of pp.5, 23, 24, 35, 38, or 41, where the specified composition or combined preparation is administered orally.

43. The use according to any one of claims 1, 6, 7 or 25, where the aforementioned antibody or antigen-binding fragment Machen detectable label.

44. Combined preparation according to any one of claim 2, 8 or 26, where the aforementioned antibody or antigen-binding fragment Machen detectable label.

Cab on any of pp.5, 23, 24, 35, 38, or 41, where the aforementioned antibody or antigen-binding fragment Machen detectable label.

46. Use item 43, where the aforementioned antibody or antigen-binding fragment anywhereman with effector molecule.

47. Combined preparation according to item 44, where the aforementioned antibody or antigen-binding fragment anywhereman with effector molecule.

48. The method according to item 45, where the aforementioned antibody or antigen-binding fragment anywhereman with effector molecule.



 

Same patents:
Therapy of mycoses // 2380116

FIELD: medicine.

SUBSTANCE: invention covers a composition applied in therapy of infections caused by fungi Aspergillus in human and animals. The composition contains an antibody or its antigen binding fragment specific at least to one epitope hsp90 from an organism Aspergillus, and at least one antimycotic means chosen from the group containing itraconazole, posaconazole and voriconazole. Epitope is presented by peptides with certain amino acid sequences presented in the description. There is a combined preparation based on the composition and intended for simultaneous, separate or consecutive application in treating said infection. The invention shows possibility to use the composition in implementing the method for preparing a drug of said appointment. There is also described therapy of aspergillosis with using the composition and also a set comprising the drug described above, used in treating the infections caused by fungi Aspergillus.

EFFECT: invention allows administering lower doses of drugs without reducing clinical effectiveness, and improving clinical effectiveness in standard doses with reducing herewith undesired by-effects.

29 cl, 4 tbl

FIELD: biotechnology.

SUBSTANCE: DNA is constructed encoding protein, advancing fungus resistance to certain group of compounds. Alternatively DNA is constructed having defect in function and encoding protein, which advances lowering of GPI-anchored protein amount in fungus cell wall. Encoded protein is useful in production of antibody thereto which may by applied as active ingredient of antifungal agent.

EFFECT: new method for production of antifungal agent.

11 cl, 8 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to tetrahydropyridoindole derivatives of general formula , where R1, R2, R3 and R4 independently represent hydrogen; C1-C5alkyl, which can be optionally substituted and represents trifluoromethyl if C1-C5alkyl is substituted; C1-C3alkoxy or halogen, and R5 is C1-C6alkylcarbonyl, C1-C5alkylcarbamoyl, C1-C5alkoxycarbonyl, C2-C5alkenylcarbonyl, C3-C6cycloalkylcarbonyl, C3-C6cycloalkyl(C1-C3)alkylcarbonyl, C3-C6cycloalkylcarbamoyl, C3-C6cycloalkylthiocarbamoyl, phenylcarbonyl or phenyl(C1-C3)alkylcarbonyl, where the phenyl residue in these two groups contains one, two, three or four substitutes, independently selected from a group comprising C1-C4alkyl, C1-C3alkoxy, halogen, trifluoromethyl and trifluoromethoxy, monosubstituted with a C3-C6cycloalkyl group, or monosubstituted with a phenyl group which in turn is substituted with a C1-C3alkyl group; phenyl(C1-C3)alkoxycarbonyl, phenylcarbamoyl or phenylthiocarbamoyl (where these two groups are optionally independently monosubstituted with a C1-C5alkyl group or halogen atoms); phenyl(C1-C3)alkylcarbamoyl, phenyl(C1-C3)alkylthiocarbamoyl, biphenylcarbamoyl, naphthylcarbonyl, naphthyl(C1-C3)alkylcarbonyl or naphthylcarbamoyl (where the naphthyl residues in these three groups are optionally monosubstituted with substitutes independently selected from a group comprising C1-C3alkyl, C1-C3alkoxy and halogen); fluorenylcarbonyl, optionally substituted with an oxo group, fluorenyl(C1-C3)alkoxycarbonyl; or 5-9-member heteroarylcarbonyl groups containing one or two heteroatoms, independently selected from a group comprising oxygen, nitrogen and sulphur, where the said groups can be substituted with one or two groups independently selected from C1-C3alkyl and halogen, provided that if R1, R2, R3, R4 are hydrogen, R5 is not ethoxycarbonyl or tert-butoxycarbonyl, or salt thereof. The invention also relates to a pharmaceutical composition based on the compound of formula I and to use of the compound in preparing a medicinal agent.

EFFECT: obtaining novel tetrahydropyridoindole derivatives which have CRTH2 receptor antagonistic activity.

14 cl, 14 tbl, 171 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to novel substituted derivatives of N-(3-benzoylaminophenyl)-4-pyridyl-2-pyrimidine amine of general formula (I), with inhibitory activity towards protein kinase, method of producing said derivatives and pharmaceutical compositions based on the derivatives. In the compound of formula 1 R1 is hydrogen and R2 is NR5R6, or R1 is NR5R6 and R2 is hydrogen; R3 is trifluoromethyl; R4 is lower alkyl; and R5 and R6 are independently hydrogen, lower alkyl, di(lower alkyl)amino-lower alkyl, N-lower alkylpiperidinyl, N-lower alkylpyrrolidinyl, or lower alkyl, or NR5R6 together represent pyrrolidino, piperidino, morpholino, N-lower alkylpiperazino, 1N-imidazolyl, 1H-2-lower alkylimidazolyl, 1H-4-lower alkylimidazolyl or 1H-2,4-di-lower alkylimidazolyl, or a pharmaceutically acceptable salt of such a compound.

EFFECT: compounds can be used in treating diseases related to inhibition of protein kinase activity, such as neoplastic diseases or leukaemia.

13 cl, 21 ex

FIELD: medicine.

SUBSTANCE: group of inventions is related to medicine, namely to oncology and may be used for treatment of malignant tumors. Versions of method, according to invention, include introduction of complex - heat shock protein - antigenic peptide of tumor and inhibitor of tyrosine kinase or retinoid. Set of invention includes container with complex - heat shock protein - antigenic peptide of tumor and container with inhibitor of tyrosine kinase or retinoid.

EFFECT: invention makes it possible to amplify immune response to tumor, to reduce its mass by synergetic effect of complexes - heat shock protein - antigenic peptide of tumor and inhibitor of tyrosine kinase or retinoid.

54 cl, 2 tbl, 1 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to molecular salt of 4-[(4-methyl-1-piperazinyl) methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamide chosen from the group consisting of (D)-tartrate, (L)-tartrate, succinate and malonate, and to a based pharmaceutical composition.

EFFECT: there are produced and described new salts which can find application in medicine, namely in therapy of tumours.

3 cl, 23 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to new derivatives of imidazo[1,2-c]pyrimidinyl acetic acid of formula (I) or to its salts: , where R1 is ,, in which n is an integer ranging from 0 to 6; Y is aryl, where the said aryl is optionally substituted at a substitutable position with one or more substitutes selected from a group which consists of halogen or C1-6alkyl, optionally substituted with mono-, di- or trihalogen; R2 is hydrogen; R3 is hydrogen or halogen; and R4 is hydrogen. The invention also relates to derivatives of imidazo[1,2-c]pyrimidinyl acetic acid of formula (I-i) or to its salts, to a drug, to use of compounds in paragraph 1, as well as to a drug in form of a standard single dosage.

EFFECT: obtaining new biologically active compounds, which are active towards CRTH2.

23 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medical products and covers applications of O- and S-glycosides 5-hydroxy-1,4-naphthoquinone (judlone) derivatives of formula 1 as an agent that stimulates human leukaemia cell apoptosis. Disclosed compounds selectively stimulate human leukemia cell apoptosis as comparrf with a prototype juglone without affecting normal cells of human immune system (neutrophils).

EFFECT: invention allows extending range of products selectively stimulating leukaemia cell apoptosis.

4 cl, 13 dwg, 5 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: present invention relates to pharmaceutics and a tablet with high drug content, which contains an active component in form of a formula (I) compound or its pharmaceutically acceptable salt in amount of approximately 30-80 wt % of active component, in total mass of the tablet and at least one binding substance, which contains microcrystalline cellulose or hydroxypropylmethicellulose.

EFFECT: design of a tablet with high drug content.

16 cl, 1 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: triterpene glycosides from holothurians are applied to prepare the pharmaceutical composition stimulating apoptosis of human leukaemia cells.

EFFECT: agent described above stimulates apoptosis of human leukaemia cells effectively and does not affect normal cells of human immune system.

3 cl, 14 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to oncology, and concerns development of a medicinal agent for angiogenesis control. It is ensured by application of sphingosine-1-phosphate receptor agonist representing 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3 diol, or its hydrochloride, or phosphate. The present invention also can include these substances combined with chemotherapeutic agents.

EFFECT: invention provides angiogenesis regulation including inhibition of uncontrolled neoangiogenesis, particularly in solid tumour therapy.

9 cl, 7 dwg

FIELD: chemistry.

SUBSTANCE: in compound of formula I , R1 is hydrogen; R2 is phenyl substituted by trifluoromethyl and optionally by other substitute selected out of a group including lower hydroxyl alkyl, lower alkylamino, lower hydroxyl alkylamino, dilower alkylamino, 1H-imidazolyl, lower alkyl-1H-imidazolyl, carbamoyl, lower alkylcarbamoyl, pyrrolidino, piperazino, lower alkylpiperazino, morpholino, lower alkoxy, trilfuoro-lower alkoxy, phenyl, pyridyl and halogenyl; R4 is methyl; where 'lower' prefix denotes radical with up to 7 carbon atoms. Also invention concerns pharmaceutical composition and method of treatment, as well as application of the claimed compounds in obtaining pharmaceutical composition.

EFFECT: improved proteinkinase inhibition properties.

9 cl, 98 ex

FIELD: medicine.

SUBSTANCE: group of the inventions refers to medicine, namely to oncology and immunology and can be used for treating renal cell carcinoma or thymoma. The method is implemented as follows: Polypeptide containing amino acid precipitations 1-182 of a sequence SEQ ID NO:29 or precipitations 1-176 of a sequence SEQ ID NO: 159, is used for preparing a drug for treating renal cell carcinoma. Another aspect of the invention concerns application of polypeptide containing amino acid precipitations 1-176 of the sequence SEQ ID NO: 159 for preparing a drug for treating thymoma.

EFFECT: application of the inventions allows improving clinical effectiveness in said diseases due to tumour induction polypeptide reaction with reducing cytotoxic by-effects.

5 cl, 35 tbl, 6 dwg, 46 ex

FIELD: medicine.

SUBSTANCE: invention concerns biochemistry and medicine. There is offered therapeutic application of yessotoxins as inhibitors of human tumour cell growth. The mechanism of action of yessotoxin (YTX) is associated with activation of cell phosphodiesterases and respectively with decreasing the content of cyclic adenosine monophosphate in cytosol. This cytotoxic effect of YTX with respect to tumour cells can be applied as a strategy to develop preparation effective in treating tumour processes.

EFFECT: inhibition of human hepatocellular carcinoma cell growth following introduction of YTX.

3 cl, 1 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to application of transcript modifying agents to prevent the genetic grading of malignant cells required to resist cell damage and their survival in chemotherapy or radiotherapy. There is applied a pharmaceutical composition containing hydralazine and valproic acid or its salt, e.g. magnesium valproate, in a pharmaceutically acceptable carrier to assist in cancer treatment along with chemotherapy or radiotherapy.

EFFECT: invention provides synergetic effect in cancer treatment by conventional therapeutic agents.

5 cl, 5 ex, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to drugs and concerns a pharmaceutical composition for percutaneous delivery of 4-hydroxy-tamoxsifen which contains 4-hydroxy-tamoxifen in an alcohol water solution where amount of spirit is 35-99.9 wt % of the composition weight, and about 45%-55%, about 46%-54%, about 47%-53%, about 48%-52%, about 49%-51% or about 50% of said 4-hydroxy-tamoxifen is found as an isomer Z and remained amount of said 4-hydroxy-tamoxifen is in the form of isomer E. There is also disclosed method for preparing a pharmaceutical composition for percutaneous delivery of 4-hydroxy-tamoxifen.

EFFECT: preparation of the pharmaceutical composition which contains an equilibrium ratio of isomers E and Z of 4-hydroxy-tamoxifen.

13 cl, 9 dwg, 4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to four defined crystalline polymorphous forms of the monohydrate of a compound A having the following chemical composition:

.

Compound A. These four polymorphous forms, denoted here as forms I, II, III and IV, exhibit CXC chemokine receptor ligand activity.

EFFECT: invention relates to a pharmaceutical composition based on said compounds, to use thereof for treating different diseases and to methods for synthesising the said polymorphous forms.

58 cl, 2 tbl, 4 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to new crystalline modifications of N-α-(2,4,6-triisopropylphenylsulphonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonyl piperazide and/or its salts. Such crystalline modifications have high stability particularly at low hygroscopicity compared to known amorphous forms of the compound.

EFFECT: invention relates to a method of obtaining such new crystalline modifications, to pharmaceutical compositions containing these new crystalline modifications and their use as an anti-tumour agent.

26 cl, 7 tbl, 13 ex, 20 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology and can be used for treatment of pulmonary cancer. Essence of invention lies in the fact that before initiation of treatment an extracorporeal ray treatment of 300 ml of patient's blood in red diapason range with λ=0.67 mcm in continuous-wave mode during 3 minutes at maximum radiant power 17 mlW, radiation level w=3.06 J/cm2 is conducted. Then irradiated blood is incubated with cytostatic in therapeutic dose during 40 minutes at T=37°C, whereafter the irradiated blood with cytostatic is inserted to patient intravenously by drop infusion. During course of treatment three or four such procedures are conducted at three days interval, in total one or two courses of treatment at three weeks interval are conducted.

EFFECT: use of this invention enables to transfer patients with tumors of stage three or four into resectable condition by means of stimulating involution and regress of neoplastic deposits from lymphonodus at neoadjuvant therapy delivery of photo- modified autoblood.

2 ex

FIELD: medicine.

SUBSTANCE: group of invention concerns medicine, namely to oncology and can be used in prostate cancer treatment. The methods of the invention imply administration of a composition containing therapeutically effective quantities of supercritical extracts of rosemary, curcuma, marjoram and ginger, as well as therapeutically effective quantities of water-alcoholic extracts of holy basil, ginger, curcuma, Scutellaria baicalensis, rosemary, green tea, Polygonum cuspidatum, Coptis chinensis Franchet and barberries.

EFFECT: inhibition of prostate cancer cell growth due to the effect of COX-2 composition without any significant side-effects.

36 cl, 1 tbl, 5 dwg, 4 ex

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