Proteins from the liver of goats with antitumor activity, a pharmaceutical composition

 

(57) Abstract:

The present invention relates to proteins having a molecular weight of from about 10 to about 14 kDa and partial amino acid sequence. Sequence identification No. 1, as described in the description. Obtaining these sequences is possible by extraction of goat liver with perchloric acid. The present invention describes obtaining a pharmaceutical composition having antitumor activity for parenteral use comprising as active principle proteins in p. 1 in a mixture with a suitable carrier. 2 S. and 2 C.p., table 1.

The present invention relates to proteins extracted from animal tissue, in particular from the liver of mammals, and their application in Oncology.

In WO 92/10197 stated extracts from organs of mammals, in particular liver goats, containing at least three different proteins and different unusual pharmacological and immunological properties. However, it is not given any information about the role they play, or the sequence of amino acid residues of the individual components is m perchloric acid from the liver and kidney of the rat, described in the publication Eur. J. Biochem. 272, 665, 1993. The corresponding complementary DNA sequence deposited in the EMBL data Bank and received when you registered number X70825.

It has been suggested that this protein, which, as indicated, is extracted together with a group of proteins high mobility, plays a role in the process of laying the chain proteins, so it can be considered a member of the class of so-called chaperones.

In WO 93/18146 claimed protein extracted from rabbit liver and having a molecular weight of 59 kilodaltons, which is able to form complexes with chaperones and heat shock proteins with a mass of 90 kilodaltons.

It was shown that the new protein isolated by purification of the extract described in WO 92/10197 has a partial sequence of the amino acid residues are shown as sequence ID number N 1.

Thanks to the following properties of this protein is used in Oncology:

serum of animals after vaccination with protein exhibits cytotoxic activity against cultures of tumor cells of a person;

protein has a pronounced antitumor activity at doses of 0.015 mg/kg Uli Yoshida;

- when the appointment of an animal, including a human, the protein leads to an increase in the number of antibodies that can recognize human cancer cells.

These features allow to explain the activity observed in the clinical trials, which include the appointment of the extracts described in WO 92/10197, patients affected developed cancer of lung, breast, stomach, colon and liver.

Proteins according to the present invention substantially homologous proteins extracted from rat liver and are shown in Eur. J. Biochem. 272, 665, 1993.

Proteins with a high degree of homology with the sequence number identification N 1 were also detected in the liver of different species of animals, in particular, in the liver of the ox and the horse. The invention relates also to these homologous sequences, except for the known sequence of the rat liver.

Thus, discovered a new family of proteins: members of a previously unknown family have a high degree of conservatism and homology among different species of mammals, and their molecular weight is from about 10 kilodalton up rough is Donatella is about 80% or more, mostly 90% or more.

The invention further relates to the use of the previously mentioned proteins, is able to be extracted from the liver of mammals using perchloric acid in Oncology as a therapeutic and/or diagnostic tools.

Thus, in the invention claimed pharmaceutical composition containing a protein having a partial sequence of the amino acid residues corresponding to the sequence number identification N 1, or proteins that are at least 80%, preferably 90% homologous to the sequence identification number N 1.

The pharmaceutical compositions of the present invention assign dropped mainly subcutaneously or intramuscularly, and usually contain from 0.1 to 50 mg of total protein in one single dose. The active part of the protein, purified by conventional means, can be lyophilisate in a suitable non-toxic carrier and placed in vials or flasks.

Suitable solvents include water and physiological salt solutions.

In accordance with another embodiment of the present invention, the proteins of the present invention or their fragments is lokalnych antibodies. Of particular interest are antibodies recognize tumor antigens, and therefore useful for diagnostic, therapeutic and research purposes. Two of these antibodies were deposited on July 27, 1993, at the European collection of cell cultures, animals (ECACC), Porton Down, Salisbury, UK and received when you registered non 930806103 and 930806104.

These antibodies are used in immunological tests, received several biopsy samples of human cancer and allow for their recognition.

Proteins of the present invention when assigning patients affected caused by tumor diseases, in addition to beneficial effects, such as suppression or regression of tumor mass, reduction of pain and improvement in General condition, lead to the induction of antibodies with cytotoxic effects on the culture of tumor cells. For the specified cytotoxic effects of essential whole serum, are not exempt from complementor cascade.

When used for therapeutic purposes or as a vaccine for immunization against neoplastic transformation of the proteins of the present invention may be administered subcutaneously, intramuscularly or votivo period of time until while the concentration of induced antibodies will not reach the normal level.

The concentration of the induced antibodies can be determined by conventional means, using, for example, immunoassay methods. To this end, in the invention claimed diagnostic kits comprising reagents with appropriate labels, in particular, as antigen protein of the present invention or its fragments, optionally immobilized on a suitable carrier, antiimmunoglobulin antibodies and related reagents capable of detecting, for example, using a colorimetric reaction, the complex of antigen-antibody.

Proteins of the present invention can be combined with suitable carriers, playing the role of AIDS. Suitable auxiliary means can be, for example, from non-toxic proteins, mainly xenogenic proteins, in particular proteins of the same species, which carried out the extraction of the immunogenic protein.

Proteins of the present invention receives, subjecting the crude extract obtained by extraction of organs perchloric acid, and then hypertonic saline solutions (e.g., 3 M solution of chloride of Kali is Noah exchange chromatography (liquid Express chromatography of proteins), as described in the examples.

A protein isolated from the liver of goats, block N-end and partially define the sequence of its amino acid residues after cleavage with CNBr, receiving two main fragment with a molecular weight (determined by the method of MALDI-TOF), respectively, 10263, and 4063 daltons, while the molecular weight to the cleavage is 14290 daltons, which is consistent with the value obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

The invention is illustrated by the following examples.

Example 1

Extract from the liver of goats, obtained as described in WO 92/10197, and which is hereinafter referred to as UK 101, concentrate on the membrane Amicon PM 10 and then subjected to dialysis against 0.01 M solution of NaH2PO4/Na2HPO4with a pH of 6.5. The resulting product was then purified by liquid chromatography high resolution on a column of TSK DEAE 5 PW, balanced the specified buffer solution; the source buffer is selected, and the protein absorbed on the resin, elute 1 M solution of sodium chloride. Peak eluruumist in the source buffer, then purified by liquid chromatography high resolution on a column of TSK SW 3000.

olcu it consists mainly of glycogen; second, the most enriched in proteins with low molecular weight, then purified by the method of liquid Express chromatography of proteins on a column of Protein-RAS HIC Phenyl 5 PW.

Clearance at the specified column hydrophobic exchange chromatography is carried out in the following conditions: first elute the source buffer, 20 mm Tris HCl pH 7, containing 1 M solution of ammonium sulfate, and then spend the elution linear gradient to 20 mm solution of Tris-HCl without ammonium sulfate. The source buffer is cast away, and the area eluroomus in the gradient polyarnosti of ammonium sulfate from 0.6 to 0.8 M, harvested and subjected to dialysis against water.

Get the sample, which is then denoted as UK 114 that indicates the protein band of about 14 kilodaltons when conducting electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate and the degree of purity of about 90%.

Example 2

When performing immunocytochemical tests using polyclonal antibodies induced in rabbits, which weekly for two months subcutaneously administered vaccine extract from the liver of the goat (WO 92/10197) in phosphate buffered saline, containing the full beta-blockers.

Monopoles Freund's adjuvant. Fusion with myeloma cells leukocytes isolated from animals vaccinated against UK 101, carried out in the usual ways. Two, from the obtained hybridomas deposited on 27 July 1993 in the European collection of cell cultures, animals (ESAS), Porton Down, Salisbury, UK, and when you register assign them numbers 930806103 and 930806104.

Antibodies secreted by the specified hybridomas, recognize the present invention.

Mono - and polyclonal antibodies examined using immunocytochemical tests conducted at 30 obtained by the method of biopsy samples of malignant tumors isolated from various organs such as mammary gland, lung, bladder, stomach, colon/rectum, uterus, soft tissue, the prostate gland. Tissue fixed in 10% buffered formalin and drugs in paraffin paint, using a set of Mistostatin Kit SP, manufactured by the company "Zymed Lab. Inc."

The slices are incubated at a temperature of 4oC in an incubator with antibodies (0.5 μg/l of immunoglobulin in phosphate buffered saline containing 1% bovine serum albumin) overnight. After washing the sections for 60 minutes to stand in an incubator with antigallican streptavidin-Biotin. The binding of peroxidase was determined by reaction of 3,3-diaminobenzidine/hydrogen peroxide. Positive are considered tissue showing specific response against the antibody in the cytoplasm. The immune activity of normal tissues is assessed as negative, slightly positive, positive (++) and strongly positive (+++). The results are shown in the table. Immunocytochemical activity with different polyclonal antibodies against extracts from the liver of the goat, the calf and the horse is found in the majority of malignant tumors (82.7 per cent for antibodies against extract from the liver of horses and 100% against the extract from the liver of a calf). Monoclonal antibody Sekretareva hybridomas number 930806103, gives positive results for 93.7% of the investigated tumors.

1. Proteins having a molecular weight of from about 10 to about 14 kDa and partial amino acid sequence of Sequence ID number 1 or sequences that are at least 80% homologous to the sequence shown with the identification number 1, extracted from goat liver perchloric acid, and then hypertonic saline solutions with subsequent dialysis and eyes

2. Proteins in p. 1, at least 90% homologous to the sequence shown with the identification number 1.

3. Proteins in p. 1, showing antitumor activity for use in anticancer therapy.

4. Pharmaceutical composition having antitumor activity for parenteral use, characterized in that it contains as active principle proteins in p. 1 in a mixture with a suitable carrier.

 

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