Alginate capsules for use in the treatment of brain tumors

 

The invention relates to the field of medical genetics. The invention is a cell line that can Express the molecule, inhibiting the growth of tumors of the Central nervous system (CNS), and encapsulated in immunohistology alginate gel. Encapsulated cell line provides a new approach to the treatment of tumors that are localized in the Central nervous system such as brain tumors. The invention provides a new technical result - expanding Arsenal of genetic tools for the treatment of brain tumors. 4 C. and 11 C.p. f-crystals, 2 tab., 7 Il.

This invention is in the treatment of tumors that are localized in the Central nervous system (CNS) or primary and secondary (metastatic) cerebral-spinal malignant tumors, and provides new compositions and delivery systems applicable to such therapy.

Primary brain tumors (gliomas) have several unique biological characteristics compared to other metastatic tumors. They are restricted localization in the Central nervous system, and metastatic spread to other organs is not actually happening. Even though t at the same location, where they were originally found. Tumors are vysokopatogennyj and consist of numerous cell types with different phenotypic properties.

Currently elected by the treatment is surgery followed by radiotherapy and chemotherapy. Patients with the most malignant form of brain tumors (glioblastomas) have a poor prognosis for survival of approximately 10 months after diagnosis. Therefore, there is an urgent need for new treatment strategies for this specific group of tumors.

Because tumors have a tendency to relapse in the place of their primary localization required method for local treatment. In addition, because these tumors are composed of numerous tumor cells with different phenotypic properties, the treatment of choice should be able to hit different types of tumor cells.

Other tumors that are localized in the Central nervous system and which are often difficult to treat successfully, include tumors originating from astroglial and oligodendroglial cells, include, for example:

Astrocytomas

- astrocytomas initial stages (astrocytomas stage 1 and 2);

- including secondary glioblastoma, i.e., tumors that differ from the astrocytomas more initial stage;

primary glioblastoma, i.e., tumors that occur in primary glioblastoma de novo;

giant cell glioblastoma;

gliosarcoma;

gliomas cerebral;

Aerodontalgia

- including oligodendroglioma (stage II according to who);

anaplastic oligodendroglioma (stage III according to who);

Mixed gliomas

- oligoastrocytoma (stage II according to who);

- anaplastic oligoastrocytoma (stage III according to who);

Abandonnee tumor

- ependymoma (stage II according to who);

- anaplastic ependymoma (stage III according to who);

subependymoma (stage I by who);

Embryonal tumors

Central neuroblastoma;

- ependymoblastoma;

medulloblastoma;

- supratentorial PNETs;

Neuroblastoma

- olfactory neuroblastoma;

- neuroplasticity tumors of the adrenal gland and sympathetic nervous system.

For most of these tumors is the first treatment of choice is surgery followed by radiotherapy and/or chemotherapy. However, complete removal of the tumor is often difficult operational methods, at the same time, resultaten and/or difficulties in the delivery of therapeutic doses of cytotoxic drugs.

In recent years, much attention focused on gene therapy, where they tested the reversion of malignant phenotype by down-regulating the expression of oncogenes or insertion of a conventional tumor-suppressor genes. Also introduced the immune stimulatory factors, such as cytokines, which aims to strengthen the recognition and rejection of tumors by the immune system. In addition, the modified cells for direct delivery of gene products to tumor cells, increasing their sensitivity to pharmacological agents. Articles that describe these developments include (i) Curr. Opin. Oncol., 7, (1995), pages 94-100; (ii) Curr. Opin. Biotechnol., 5, (1994), pages 611-616; (iii) Cancer Res., 53, (1993), pages 2330-2337; (iv) Hum. Gene Ther., 4, (1993), pages 451-460; (v) Hum. Gene Ther., 5, (1994), pages 153-164; and (vi) Trends Pharmacol. Sci., 14, (1993), pages 202-208.

Despite extensive research over the last years, there are significant obstacles that impede the transition between the experimental study and clinical treatment of malignant brain tumors. One problem is the warning immunoactivity genetically modelirovaniya cells after intracranial implantation. This can be overcome by encapsulating cells-producers.

However, this prospect is, that brain in the immunological relationship is different from other parts of the body such as the loss of b-lymphocytes, it is especially sensitive to the effects of biologically active compounds, such as endotoxins.

The applicants have now found in accordance with the present invention that immunoisolated alginate matrix is particularly suitable for encapsulating cells-producers intended for intracranial implantation in the treatment of tumors of the Central nervous system. Particularly preferably, immunoliposome alginate matrices were microspheres.

Thus, in a broad aspect the present invention provides encapsulated cell-producer, able to Express the molecule that is an inhibitor of the growth of tumors of the Central nervous system, this cell producing encapsulated in immunoisolated alginate matrix. Preferably, this molecule was a peptide, protein or polysaccharide, and most preferably, this molecule was a monoclonal antibody.

The present invention also provides a method of treatment of tumors of the Central nervous system, which includes the implantation at the site of the tumor is encapsulated cells-producer, which is capable expresser provides a method of obtaining a pharmaceutical product for the treatment of tumors of the Central nervous system, which includes encapsulation within immunohistologic alginate matrix cells-producer, able to Express the molecule that is an inhibitor of the growth of the indicated tumor.

The present invention also provides the use of immunohistologic alginate matrices for encapsulation of cells-producers intended for intracranial implantation in the treatment of tumors of the Central nervous system.

In one embodiment of the invention cells-producers intended for use here include genetically engineered cells that produce molecules, such as proteins, peptides and polysaccharides, which will be or directly interact with tumor cells, or indirectly by communication paths of tumor cells and the host. Other suitable cells-producers, considered here, are specialized cells that produce a monoclonal antibody, such as hybridoma cells or even native cells, which are able to Express the molecule, inhibiting tumor.

It is well known that tumor growth depends on the specific cellular communications with the host by means of specific growth factors, which is x substances, supplied by the owner, coming in through the newly formed blood vessels. In recent years it was found and described in the literature several cellular pathways of interaction of tumor/host.

Therefore, one class of cells-producers, suitable here are those who can Express proteins or peptides, which will interact with the communication paths of the tumor/host. For example, suitable cells-producers include those that produce proteins and peptides that act on the neovascularization of the tumor, as, for example, thrombospondin, endostatin, angiostatin and prolactin, proteins that disrupt the relationship of tumor cells with the extracellular matrix, such as protease inhibitors, such as tissue inhibitors of metalloproteinases, and proteins and peptides that act on the immune system, including all the different classes of interleukins.

Another preferred class of cells-producers consists of those that Express proteins or peptides, directly interacting with the actual tumor cells. For example, suitable cell producers in this category include: hybridoma cell lines that produce monoclonal ant the notches, which affect tumor cells, such as the receptor for epidermal growth factor (EGFr), a receptor platelet-derived growth factor AA and BB, acidic or basic receptor fibroblast growth factor, alpha - and beta-receptors of the transforming growth factor, different classes of receptors of vascular endothelial growth factor (VEGFR-1 and VEGFR-2) receptor tyrosine kinase with immunoglobulin and EGF-like domains, as, for example, TIE-1 and TIE-2/tek, a growth factor for hepatocytes (scattering factor) or mono-clonal antibodies, against various classes of integrin receptors; monoclonal antibodies directed against CD-44; monoclonal antibodies directed against complexes of CDK/cyclina; monoclonal antibodies directed against FAS; monoclonal antibodies directed against glycolipids on the cell surface; monoclonal antibodies directed against glycoproteins; monoclonal antibodies directed against proteins derived the expression of specific oncogenes.

Of particular interest in certain circumstances represent cells-producers, whose products inhibit the growth of tumors substances can be started and turned off by pharmacological red tetracycline expression.

According to the present invention can use any transperineal cell line. Cell lines should be constant, i.e., the ability to undergo unlimited cell division and preferably be non-human and neophilaenus.

Examples of such cell lines available from the American Type Culture Collection 10350 Linden Lake Plaza, Manassas, Virginia 20109, USA, are (see Table A):

The present invention cells-producers encapsulate in immunoisolated alginate matrix, which is able to provide stable in situ delivery system expressed protein or other molecules that can disrupt the growth and progression of tumors without immunoactivity cells-producers.

Encapsulation of cells in alginate granules is a well-known method of immobilization of cells and other substances and earlier it was used in the treatment of diabetes, for the production of monoclonal antibodies, as well as in other areas of medicine, as described in the literature.

From PCT/WO 97/44065 this method of drug delivery has been proposed for gene therapy in vivo using encapsulated cells, viewclose two parts: a) the core, including live Packed cells, and b) an outer shell surrounding the specified kernel.

The present invention provides a much more simple way to encapsulate and product, where cells-producers directly encapsulate the one-stage method using alginate with immunoisolation property.

Alginate is a polysaccharide, which is mainly found in brown seaweed. It consists of two monosaccharides types: L-guluronic acid (G) and D-mannurone acid (M). These polysaccharide units are in blocks with alternating sequence of G and M (MG-blocks) and blocks, consisting mainly of either G-or M-unit (G-blocks/M-blocks).

Gelling property is achieved by stapling G-blocks multivalent cations, especially CA2+.

In order alginate was not immunological trigger, the content of G should be above 15%. However, more preferably the present invention to use alginate with a high content of G, ie with G equal to 50% or higher to achieve that alginate was immunoisolation. As is well known in this area, the ratio G/M-blocks and the distribution of various blokovi crosslinking polyvalent cation.

Another crucial aspect is the purity of the alginate used. Thus, one advantage of alginate matrices of the present invention, is that you can get them with the quality of high purity, having a well defined structure and a very low content of impurities, such as endotoxins.

The second advantage of alginate matrices of the present invention, is that the alginate microspheres obtained by the addition dropwise of a solution of alginate containing living cells, sodium, calcium, have an increased concentration of alginate from the center of the microgranules to the outer rim. Through this, you create the optimal space in the center of microgranules for cells to survive, proliferate and to produce, at the same time for cells available a sufficient amount of nutrients and oxygen. External bezel with its higher concentration of alginate gives the possibility of creating a barrier so that no-producing cells do not go out of the internal space or immunological cells do not come into pellets.

Basically, the use of alginate as a matrix for immobilization of cells includes a mixture of su the Lenten cations (usually CA2+). Drops form gel beads, instantly captivating cells in treprostinil lattice of alginate crosslinked by ions. This immobilization procedure can be performed under very mild conditions, and therefore it is compatible with living cells. For a detailed description of how theory and practice of the method the reader is directed to an article, "Alginate as Immobilization Matrix for Cells" by Smidsrod and Skjak-Braek in Trends in Biotechnology, March 1990, Vol.8, No. 3, pages 71-78.

Preferred common way for the formation of calcium-alginate pellets with encapsulated cells producing of the present invention is the following. The sodium alginate is dissolved at a concentration of 1-2% in water or isotonic saline solution. The alginate solution is sterilized by passing through a membrane and then add cells-producers and sum isotonicity. Calcium-alginate granules are formed at prikatyvanie solution of sodium alginate-cell-producers in the bath with calcium chloride (0.05 to 0.25 M), either manually or, preferably, using an electrostatic generator granules, which creates an electrostatic potential 5-7 kV between the needle feed alginate, a gelling bath. By adjusting the diameter of the needle (for example, from 0.1 mm delno the same diameter from 100 to 400 μm. The uniformity of the granules is regulated by summing the salt concentration in the gelling bath from 0 to 200 mm NaCl, at higher salt concentration, giving a higher uniformity. The granules allow to harden in the gelling bath.

It is assumed that the encapsulated cells-producers of this invention will be placed in the cavity tumors after conventional volumetric removal of the tumor by surgery. Soon after surgery, the tumor load is minimal, and many patients have an asymptomatic period before relapse occurs. Because surgery is a traumatic event, the remaining tumor cells will try to establish new ways of biochemical interaction with the host. This includes the formation of new blood vessels and new sources of supply of peptide growth factors to the remaining tumor cells. At this time, when the tumor load is minimal, the treatment by the present invention, will be most effective.

Indeed, a particular advantage of the present invention in accordance with one embodiment is that it allows you to easily slide the simultaneous implantation of cells producing various the exercise on the progression of brain tumors and other tumors. For this purpose it is possible to create a Bank of cells-producers containing encapsulated cells-producers were stored frozen in liquid nitrogen. Then from the Bank can extract cells-producers, the corresponding genotypic expression of the tumor host, which can be treated.

In order to determine which cell producers needed to treat tumors, can be used as an example the following procedure. First biopsy material are characteristic of the tumor, including the identification of receptor status and phenotype. Accordingly, the selected cells-producers, which produce substances, such as monoclonal antibodies directed against the receptor status of the tumor host, then implanted stereotactic up to 60 days after surgical removal of the primary tumor.

Alternatively, it is possible to implant cells-producers, producing angiogenic substances, immediately after surgical removal of the primary tumor.

Cell dose-producers that are implanted, will, of course, depend upon the precise circumstances of each patient, but usually the total number of implanted cells will be within arouse matrix will be of course, to depend on the size of the granules or other encapsulating form. Encapsulated cells-producers will usually be surgically placed in the wound after removal of the primary tumor.

As showed in detail the following experiments, the encapsulated cells-producers can survive, proliferate and maintain their specific expression in vitro and in vivo. This discovery is the possibility of new therapeutic treatment for patients with tumours of the brain, whereby it is possible to encapsulate different cells-producers selected for exposure to selected characteristics of the growth and development of brain tumors. In the experiments described herein, applicants have shown that specific MAT released from the alginate granules, can inhibit the migration of tumor cells, as shown by the interaction with the receptor for epidermal growth factor. Applicants have also shown that specific products released from the encapsulated cells-producers inside the brain, penetrate into the parenchyma of the brain and can spread through the cerebral spinal fluid.

These experiments will help in the understanding of the invention and its advantages. Below is roscopy cells NIH T, encapsulated in alginate. All bars represent 250 microns.

Figure 1A: day encapsulate.

Figure 1B: encapsulated cells after 3 weeks in culture.

Figure 1C: encapsulated cells after 9 weeks in culture.

Figures 1D-1F: micrograph obtained using confocal scanning laser microscopy of cells NIH T encapsulated in alginate. Living cells emit green fluorescence (here shown as lighter areas), while dead cells emit red fluorescence (not visible here). All bars represent 250 microns.

Figure D: the day of encapsulation.

Figure 1E: encapsulated cells after 3 weeks in culture.

Figure 1F: encapsulated cells after 9 weeks in culture.

Figure 1G: activity-galactosidase cells BT4CnVlacZ encapsulated in alginate, after 9 weeks in culture. All bars represent 500 μm.

Figures 2A-2D: histogram of flow cytometry cells NIH T encapsulated in alginate pellets. The horizontal axis expresses the number of the channel in a flow cytometer (relative fluorescence DNA), while the vertical axis expresses the relative number of nuclei of cells in each of the Delhi.

Figure 2C: the cells are encapsulated within 3 weeks.

Figure 2D: the cells are encapsulated within 9 weeks.

Figure 3: the secretion of antibodies hybridoma cells N encapsulated in alginate (average ± average error). The horizontal axis represents the number of days in culture, while the vertical axis shows the release of antibodies in the culture medium. The curve was constructed by regression analysis of the 3-th order.

Figure 4: migration of cells from spheroids GaMg after 4 days, the untreated (control), stimulated by 10 ng/ml EGF (EGF) or stimulated by 10 ng/ml EGF in the presence of encapsulated hybridoma cells (EGF/H528).

Figures 5A-5H: encapsulated hybridoma cells In implanted in the rat brain.

Figure 5A: a longitudinal section of rat brain. Staining with hematoxylin-eosin, bar represents 5 mm.

Figure 5B: the same slice that in figure 5A, showing the encapsulated cells N inside the sites of implantation. Staining with hematoxylin-eosin, bar represents 500 μm.

Figures 5C-5H: micrograph obtained using confocal laser scanning microscopy, release and distribution of monoclonal antibodies inside the brain. Figures 5C, E and F parenchyma brain with encapsulated cells In from the far left side. Bar represents 150 μm. On the left side you can see the intense fluorescence in the parenchyma of the brain, followed by a gradual decrease in the intensity of at least 1000 microns inside the brain.

A gradual change in fluorescence intensity on a horizontal line is additionally shown in figure 5D, where the vertical axis represents the relative fluorescence intensity (0-255). Intense fluorescence is visible on the left side with a gradual decrease in the parenchyma of the brain.

Figure 5E: MAT were found in the subarachnoid space and the underlying brain. Bar represents 75 microns.

Figure 5F: weak fluorescence present in the control could be due to nonspecific binding. Bar represents 75 microns.

Figure 5G: MAT advanced distributed in the perivascular space. Bar represents 50 ám.

Figure 5H: compared to controls show weak binding of immunoglobulins in the perivascular space. Bar represents 50 ám.

Figure 6: radioimmunoassay, which shows the successful survival of cells producing endostatin. The figure shows the radioimmunoassay release of endostatin from the air-conditioned environment in the cell fra is ur 7: effect of alginate therapy with endostatin on tumor growth. Picture a shows an example of a control animal, which were implanted imitating transfetsirovannyh cells encapsulated in alginate pellets. The darker area of the brain shows the tumor site.

Picture shows an example of an animal treated with encapsulated producing endostatin cells. The darker area shows the tumor, and there is a large necrotic area in the center of the tumor.

Experiments

Materials and methods

1. Cell line

In the experiments of the applicants have used four cell lines:

Cell line Items store

1. NIH T ATSC CRL/1658

2. BT4CnVlacZ not stored

3. NTS HB 8509

4. GaMg is not stored

Mouse fibroblasts NIH T represent a potential cell line-producers, in the sense that it is able to be genetically modified in order to ekspressirovali substances which show activity against the growth, progression and tumor development. Cells NIH T were encapsulated in alginate, as described below, was used to study the morphology, viability and cell cycle kinetics in vitro. To study the viability of encapsulated cells in vivo alginate pellets containing cells NIH T them what atomoxetinee rat glioma and its stable was transfusional bacterial gene lacZ, cloned into a plasmid containing the long terminal repeat sequence with the gene of resistance to neomycin virus murine leukemia, Malone, expressed from an internal promoter sarcoma virus Rus. See J. Natl.Cancer Inst., 55 (1975), pages 1177-1187 and Int.J.Cancer, 71 (1997), pages 874-880. The cells were encapsulated in alginate and investigated the in vitro synthesis bacterial-galactosidase.

Cell hybridoma line N received from American standard culture collection (ATSS Rockville, MA). The cell line was obtained by cell fusion of myeloma NS-1-Ag4-1 with spleen cells from BALB/c mice, and it produces murine monoclonal antibodies (MABS) (IgG2a) that bind and block the EGF-binding domain of the human receptor for epidermal growth factor (EGFR). In vitro and in vivo was studied release MAT from encapsulated in alginate cells when using this cell line.

Cell line human glycome GaMg described in Anticancer Res., 8 (1988), pages 874-880, and has previously been shown that it expresses EGFR (Acta Neuropathol. Berl., 84 (1992), pages 190-197). Studied specific inhibition of cell migration in GaMg coculturing system between multicellular spheroids GaMg and encapsulated cells is 80 cm2(Nunc, Roskilde, Denmark) with a complete nutrient medium consisting of eagle medium, modified by way of Dulbecco (DMEM) with the addition of 10% integrirovannoi warmth of a newborn calf serum, 4 times increased compared to the words in the concentration of essential amino acids, 2% L-glutamine, penicillin (100 IU/ml) and streptomycin (100 μg/ml) (all reagents from BioWhittaker, Verviers, Belgium). Cell lines hybridoma N and GaMg grew up in mattresses for the cultivation area of 80 cm2(Nunc) in culture medium RPMI 1640 with the addition of 10% horse serum (BioWhittaker). Upon reaching confluence, the monolayer GaMg was treated with 3 ml of 0.025% trypsin (BioWhittaker) and spheroids stimulated by seeding 5106cells in 20 ml of complete RPMI medium in mattresses for the cultivation area of 80 cm2(Nunc) coated with 0.5 percent inert agar (Difco, Detroit, MI). All cell lines were kept in a conventional incubator for tissue cultures at 37C, 100% relative humidity, 95% air and 5% CO2.

3. Structure and properties of alginate

In these experiments to microencapsulate cells-producers used sodium alginate from brown algae Laminaria hyperborea (LF 10/60) (Protanal, Drammen, Norway). It consisted of two of monochorionic connected together in blocks of three different types: GG MM and MG, and the proportion and distribution of these blocks determine chemical and physical properties of the alginate molecules. Some divalent cations, like CA2+firmly contact between the individual and G-blocks, which begins the formation of a wide alginate network, in which G-blocks form a rigid connection. Alginate, which is used by the applicants, has a high content of above 60%, G-block, resulting in high mechanical strength and porosity, making it suitable for encapsulating cells in order to obtain secondary metabolites (see Trends in Biotechnology, 8 (1990), pages 71-78). Scanning electron microscopy it was shown that the pore size in alginate granules is in the range between 5 and 200 nm (33, 34). Mechanical strength, volume stability and porosity of the granules is correlated with the content of guluronic acid.

4. Encapsulating cells

Used method of encapsulation is described in detail in "Alginate as Immobilization Matrix for Cells" by Smidsrod and Skjak-Braek in Trends in Biotechnology, March 1990, Vol.8, No. 3, pages 71-78.

Briefly drops cells, diluted in 1.5% sodium alginate, poured into 0.1 M CA2+-solution. After polymerization of the alginate pellets three times washed in phosphate buffered saline Blew the La cultivation area of 175 cm2(Nunc) containing 50 ml of culture medium. Culture medium was changed every three days, and the vials were replaced once a week. All encapsulated in alginate cells were kept in a conventional incubator for tissue cultures at 37C, 100% humidity, 95% air and 5% CO2. For all experiments with cell lines NIH T and BT4CnVlacZ used the density of cells 6106cells/ml of alginate and granules of a size between 0.8 and 1.2 mm For in vitro experiments with cell line N used the density of cell 3105cells/ml of alginate and the diameter of the granules is between 2.3 and 2.5 mm For in vivo studies with cell lines N used the density of cell 3105cells/ml of alginate and the diameters of the granules is between 0.8 and 1.2 mm

In vitro experiments

1. The morphology and viability of encapsulated in alginate cell

The morphology of the cells NIH T encapsulated in alginate, researched on the day of encapsulation and after 3 and 9 weeks 6 granules, transferred to 6-well plate (Nunc) with a top layer of 1.0 ml DPBS. Pellets were investigated by light microscope (Nikon Diaphot and photographed by digital camera Nikon F-301. Experiments on the morphology conducted in two Parallels.

Jiznesposobnoe fluorescence viability (test live/deadviability/cytotoxicity, Molecular Probes, Eugene, OR). Solution label was prepared from 2 μm calcein AM and 4 μm ethidium of glycosilated in complete culture medium. Alginate pellets were placed individually in 16-mm multilangue plates (Nunc) with a top layer of 0.5 ml of solution labels for 30 minutes at room temperature. After that they were transferred in DPBS and immediately investigated. Fluorescence was measured in the optical blocks through alginate, using confocal laser scanning microscope with an argon-krypton laser (Biorad MRC-1000, Hemel Hempstead, UK) using optics with filters on Theheavy red and FITZ. Fluorescence was recorded in the plane of 120 μm inside alginate pellets. Experiments on the viability was performed in three Parallels.

Studied education-galactosidase in cells BT4CnVlacZ encapsulated in alginate for 1, 3 and 9 weeks. Pellets were washed for 1 min in DPBS (pH 8,4) and fixed for 10 min in 0.2% glutaraldehyde and 2% formaldehyde in DPBS. They were then washed 35 min in DPBS and stained for activity-galactosidase 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (x-Gal, Sigma). The substrate solution consisted stictocardia potassium and 2 mm MgCl2dissolved in DPBS (all reagents from E. Merck, Darmstadt, Germany). They were incubated at 4With at least 24 hours and determined-galactosidase activity, represented as colored blue cytoplasm.

2. The kinetics of the cell cycle in cells encapsulated in alginate

The distribution of cell cycle encapsulated cells NIH T was determined by flow cytometrical DNA analysis. Encapsulated cells were released from the alginate by dissolving the pellet in complete nutrient medium containing 1.5% trinacria citrate dehydrate (E. Merck) for 15 min, followed by centrifugation at 140 g for 4 min and remove supernatant. Cells are again double-suspended in complete culture medium, centrifuged at 140 g for 4 min, fixed in chilled on ice in 96% ethanol and kept at 4C. Before analysis flow cytometry cells were incubated for 15 min with 0.5% pepsin (Sigma) in 0.9% saline solution (pH of 1.5) at 37With before the selected cores were washed in 0.9% saline solution and treated for 1 min with ribonuclease (Sigma) (1 mg/ml in 0.9% physiological age is) to the nuclei. The DNA content in the cells was determined using flow cytometer Becton Dickinson FACSort (Becton Dickinson, Palo Alto, CA). DNA histograms obtained by setting the two-parameter window a direct and adverse cytogeography scattering to single-parameter DNA histogram. Each histogram was obtained when counting 5000 excited nuclei. Experiments cytometry was repeated three times and the distribution of cell cycle was determined as described in Radiat Environ Biophys., 12 (1975), pages 31-39.

3. The release of antibodies from encapsulated hybridoma cells

Prepared alginate pellets with a diameter ranging between 2.3 and 2.5 mm, containing 1.5103cells In pellets on the day of encapsulation, as described above. Respectively through 0, 1, 5, 12, 19, 23, 30 and 33 days 10 pellets were removed from the mother culture and investigated the release of MAT in RPMI medium. The pellets were transferred to 24-well plates (Nunc) in 0.5 ml of complete RPMI medium (37C). After 6 hours incubation collected four samples of 100 μl each, were placed in centrifuge tubes with a capacity of 1.5 ml (Treff AG, Degersheim, Switzerland) and frozen at -20C.

Flow cytometry was used to determine the concentration of MAT samples. Monolayer cell cultures is Alili and the cells were fixed in 2% solution of paraformaldehyde in DPBS for 1 minutes Then the cells were centrifuged at 140 g for 4 min, the supernatant was removed. The cells are then re-diluted in DPBS containing 2 mm EDTA, 1% bovine serum albumin and 1 g/l glucose and distributed in a cone-shaped 96-well tablet (Nunc) at the rate of 1.7105cells/well. The cells were centrifuged at 340 g for 4 min and the supernatant removed. After that, cells were treated on a vortex and incubated for 2 hours at 4With the collected MAT in RPMI medium (undiluted and at a dilution of 1:5, 1:20 and 1:100 in DPBS). As the standard used antibodies MAT (528) EGFR (Santa Cruz Biotechnology, Santa Cruz, CA) with a known concentration of MAT (concentration 20, 5, 1, and 0.2, 0.1 and 0.05 mg/ml). Cells were washed twice in 2 mm EDTA, 1% BSA, 1 g/l glucose in DPBS and then incubated with goat labeled FITZ artemisinine immunoglobulins (Dako A/S, Glostrup, Denmark) (dilution 1:20) for 30 min at 4°C. Flow cytometry was done on a flow cytometer Becton Dickinson FACSort. Were detected and made visible the individual cells of the two-parameter direct and adverse they dissipation and set the box on the one-parameter FITZ-column chart, which was determined by the fluorescence intensity. Received standard and GaMg. When comparing results obtained with the medium collected from hybridoma containing alginate granules, got a curve of concentration of the MAT.

4. Cell migration

Spheroids GaMg separately transferred in 16-mm multilangue plates (Nunc) in 1.0 ml of complete RPMI medium containing 10 ng/ml EGF (Sigma). After that the tumor cells were subjected to alginate pellets containing cells N (three alginate pellets in each well). As a control spheroids were subjected to a complete RPMI medium with and without 10 ng/ml EGF. Daily for four days was measured orthogonal to the diameter of each colony, using a light microscope with a measuring grid in the eyepiece. Then defined circular area covered by the cells migrated out of the spheroids, and used as an indicator of migration. Experiments conducted in two Parallels with six spheroids in each experience.

5. The survival rate of cells producing endostatin and proof of release of endostatin from granules

5A. The survival rate of cells producing endostatin

Methods:

Cell line and culturing conditions

As a cell line-producers used human embryonic kidney 293 cells (293-EBNA), expressyou rser-Ri, containing the gene encoding human endostatin, liposomes and selected with 0.5 μg/ml puromycin.

Transfetsirovannyh cells (293-endo) were grown to confluence in mattresses for the cultivation area of 175 cm (Nunc, Roskilde, Denmark) containing nutrient medium consisting of modified from the method of Dulbecco environment Needles (DMEM) supplemented with 10% heat inactivated fetal bovine serum, 4.5 g/l D-glucose, penicillin (100 IU/ml) and streptomycin (100 μg/ml), 205 µg/ml geneticin (G-418) and 0.5 μg/ml puromycin. Simulating the transfectants were obtained by transferowania of 293 cells with the vector rser-Ri without endostatin gene and grown in the same conditions, except puromycin (all reagents from Biowhitaker, Verviers, Belgium).

Tumor cell line (VTS) chosen for these experiments was obtained from induced ethylnitrosourea rat gliosarcoma (passage 26), and it is syngeneic in BD-IX. Cells were grown to confluence in mattresses for the cultivation area of 80 cm2with a complete nutrient medium consisting of modified according to the method of Dulbecco eagle medium (DMEM) supplemented with 10% integrirovannoi warmth of a newborn calf serum, 4 times increased compared to the concentration in words interchangeable aminoxy is Tina from granules

Immunoblot

Collected air-conditioned environment of the encapsulated cells endo-29 and 293-EBNA and used for the production of conventional Western blotting SDS/PAGE to determine is released if endostatin from granules.

Briefly, samples were separated in 12% SDS-gel and hybridisable on the nitrocellulose PVDP. The blots were washed in 100% methanol for 5 min, distilled water - 1 min blocking solution (0.05 M Tris/HCl, 0.45 M NaCl, 2% Tween, pH 10.2) - 4 min, and finally a buffer for washing (0.05 M Tris/HCl, 0.15 M NaCl, 0.05% Tween-20, pH 10.2) for 15 minutes and Then the blots were incubated overnight with rabbit anti-human anticorodal (1:1000 in buffer for washing). After incubation overnight, the blots were washed in DPBS and incubated with swine anti-rabbit conjugated with alkaline phosphatase IgG (DAKO, Denmark).

The band made visible by incubation of the substrate with a dye solution (2-4 min).

Experiments in vivo

1. Intracranial implantation

Inbred rats male BD IX (36) with body mass 160-250 g were kept on a standard pellet diet was given unlimited access to tap water and were kept in the cells individually at a constant temperature and humidity under the regime of 12 hours light and dark. Cu is the first skin incision was performed trepanation of the skull by means of a drill 3.5 mm 4.2 mm rear bregma and 2.5 mm to the right of the sagittal suture. Removed the cortical and white matter tissue by suction to a depth of 2.0 mm and cavity tissue was placed from 8 to 14 alginate pellets (granules day) containing either cells NIH T or cells N. Hole after trephination was closed with bone wax, the skin sewed nylon thread. Within 1 hour the animals were allowed to depart from the operation under a warming lamp. Care of animals were in accordance with the established rules. Rats were observed once daily and every other day weighed. All animals recovered soon after implantation and no noticeable signs of disease or neurological disorders during the follow-up period.

2. The release and distribution of immunoglobulins inside the brain of rats

After 3 and 9 weeks rats were scored by inhalation of CO2. The brain was removed, placed in a solution of Tissue Tec (Miles Laboratories Inc., Naperville, IL) and frozen in 2-methylbutane (E. Merck), cooled in liquid nitrogen. Longitudinal sections (14 µm) were made on cryotome Reichert-Jung cryocut 1800 (Leica, Wetzlar, Germany) and stored at ~20C. Crisisi obtained from rats that were implanted encapsulated cells N and scored after 3 weeks, fixed in acetone for 5 min at room temperature and then washed twice in DPBS for 5 Uchenie 1 hour at room temperature and then washed in DPBS for 5 min. Sections were treated for 30 with ribonuclease (Sigma) (0.5 mg/ml in 0.9% saline solution) and staining of nuclei obtained by adding the slices of propecia iodide (Sigma) (50 mg/ml in 0.9% saline solution). In addition, the slices were washed in DPBS for 5 min and then placed with Vectashield (Vector Laboratories Inc., Burlingame, CA). Fluorescence measurements were done using a confocal laser scanning microscope Leica TCS NT with argon-krypton laser (Leica), using optics with TRITC filters and FITZ. Investigated sections taken at the same depth inside the brain of experimental animals, and studied plots the maximum fluorescence in both groups. Crisisi obtained from rats that were implanted cells NIH T and scored in 9 weeks, were stained with hematoxylin and eosin for histological examination.

3. Immune responses to cell-producers were encapsulated in alginate

Methods

After 1, 3 and 9 weeks after implantation was estimated percentage immunologically cells in the border zone between the brain and alginate granules in rats BD-IX. The brain was placed on the substrate, poured into a solution of tissue-tec and frozen in liquid nitrogen. Serial longitudinal sections of 5-20 μm made on the cryostat Reichert Jung (Leica, Wetzlar, Germany) were placed for 5 min, incubated for 30 min at room temperature with 10% normal rabbit serum diluted in PBS, and then incubated overnight at 4With in a humid chamber with mouse monoclonal antibodies (MABS), diluted in 10% rabbit serum.

Used the following MAT: H, ED1 and ED2 anti-rabbit macrophage MAT, H against D5-positive T cells and OF reactive D45-positive b-cells. MAT was obtained from Serotec, Oxford, UK.

Within 30 min was used biotinylated rabbit antimurine immunoglobulins, diluted 1:300. Avidin-Biotin-peroxidase complex (ABC complex/HRP, Dakopatts, Glostrup, Denmark) was prepared as recommended by the manufacturer, and gave it to react with the sections for 30 minutes Finally, the sections were treated with buffer containing 3-amino-9-ethylcarbazole, for the development of the colored reaction product. After all incubations were followed by washing in PBS. All drugs contrasting stained with hematoxylin, put in glyceryl (Dakopatts) and examined by light microscopy.

4. The effect of alginate therapy with endostatin on tumor growth

Young adult rats BD-IX of both sexes (8 rats in General plus 20 controls) were analizirovali stance (David Kopf Instruments, Tujunga, USA), made incisions in the skin and had a craniotomy with a hole of 2 mm by 1 mm at the rear and 3.0 mm to the right from bregma and at a depth of 2.5 mm was placed alginate pellets. After that introduced cells gliosarcoma 1104 VTS 1 mm lies lateral in relation to alginate granules to a depth of 2 mm Alginate granules contained or 293 cells producing endostatin or imitating 293 transfectants as control. Eight animals received implants of each form cell lines. In addition, as control of the normal progression of a tumor 8 animals introduced only some cells VTS.

Finally, as a control cell viability inside the granules in vivo remaining 4 control animals received alginate pellets containing some cells 293-endo. The syringe was slowly pulled within 3 min (for all users) and the hole was closed with bone wax and I was stitched up. Animals were allowed to recover after surgery under supervision. During the experimental period, animals were placed in pairs at a constant temperature and humidity, fed conventional granular feed and ad libitum were provided tap water.

Results

In vitro experiments

1. Morphology and IMEMO 6,5102cells NIH T on the day of encapsulation (Fig.1A). Cells evenly inside alginate granules, with the outer, free from cells rim 25-50 microns. During cultivation was observed cell proliferation inside the alginate, which led after 3 weeks to increased cell density (Fig.1B). After 9 weeks in culture, within alginate granules observed the presence of multicellular spheroids (Fig.1C). After 9 weeks, more than 90% of the granules remained intact in culture, which is assessed by light microscopy. After about a week a few individual cells in culture migrated from alginate granules in a nutrient medium, and this limited movement of individual cells continued during the subsequent 8 weeks of culture.

A study using confocal laser scanning microscopy showed that about 90% of the encapsulated cells remained alive on the day of encapsulation (Fig.1D). After 3 weeks, about 50% in the culture of the initially encapsulated cells were alive (Fig.1E). Some surviving cells were adapted to the alginate and formed multicellular spheroids, which can be clearly observed after 9 weeks (Fig.1F). At this point in time the total number provided in Fig.1F, most cells were in spheroids was alive.

Encapsulated cells BT4CnVlacZ expressed constant and uniformly distributed-galactosidase activity during the whole observation period for 9 weeks (Fig.1G).

2. The kinetics of the cell cycle in cells encapsulated in alginate

Histograms of flow cytometry NIH T cells showed a change in the ploidy of cells within alginate granules after 1 week after encapsulation (Fig.2B). Perhaps it is the polyploidization in comparison with diploid set in the control (Fig.2A). However, after 3 and 9 weeks, respectively Fig.2C, 2D, observed the normalization with the same ploidy diploid distribution, as in the control. The fraction of proliferating cells in S and G2M phases in vitro was 50% for the control, compared with 55% and 60% after 3 and 9 weeks, respectively.

3. The release of antibodies from encapsulated hybridoma cells

At the end of the first day of encapsulation was the release of 13 ng/(mlh) MAT in a nutrient medium (Fig.3). Diffusion of immunoglobulins of the granules and in the environment has increased steadily over the subsequent days of cultivation and cherine about 400 ng/(mlh) during the last 3 weeks of the observation period.

4. Cell migration

Cell migration from spheroids GaMg stimulated EGF, was extensive, and the average value of gains doubled in comparison with the control (Fig.4). However, when alginate pellets containing cells N added in the presence of EGF, cell migration is strongly inhibited, indicating that the encapsulated cells producing N effectively Express antibodies directed against the EGF receptor.

5. Establishing release of endostatin from granules

As can be seen from the Western blots of conditioned medium collected from granules, released considerable amount of endostatin from granules (Fig.6). The radioimmunoassay showed that 10 alginate granules, producing endostatin (400 μm) with 25000 encapsulated cells, secretively 2.5 µg/ml/24 h

Experiments in vivo

1. Intracranial implantation

Longitudinal sections of rat brain showed little or no change in the parenchyma of the brain, adjacent to the site of implantation encapsulated in alginate cells NIH T (Fig.5A). After 9 weeks observed a slight intracranial edema or swelling. In alginate granules were absent any gains cledet in the center and on the periphery of the granules, free from cell sites of the alginate between cells. Watched the minimum aggregation of cells around the border area between the hole for implantation and parenchyma of the brain.

2. The release and distribution of immunoglobulins within the rat brain

Implanted beads with encapsulated hybridoma cells can be easily seen after 3 weeks of intensive green fluorescence (Fig.5C). Immunoglobulins detected in the brain tissue at a distance of at least 1 mm from alginate granules (Fig.5C, 5D), with a gradual decrease in the intensity of fluorescence from the boundaries of the sites of implantation and inside the brain. Two experimental animals MAT were detected throughout the brain, in which there were implants (data not shown). Advanced MAT were found in leptomeninges both hemispheres of the brain (Fig.5E) with the strongest fluorescence, visible in the subarachnoid region in the right hemisphere. Negative controls showed weak fluorescence in leptomeninges, possibly caused by non-specific binding between antibodies and epitopes cells leptomeninges (Fig.5F).

However, the parenchyma of the brain gave a negative result. Chrome is in fluorescence intensity between the two hemispheres (Fig.5G). Weak fluorescence present in the control was probably caused by nonspecific binding (Fig.5H).

3. Immune responses to cell-producers were encapsulated in alginate

In the brain, connecting with alginate granules observed infiltration of mononuclear cells. The number of cells in the infiltrate decreased from week 1 to week 9. One week after implantation was visible OH-positive microglia with dendritic morphology in the parenchyma and reactive microglia and infiltrating monocytes appeared in edge to alginate granules zone. ED1 and ED2 were stained monocytes, close to the border zone, while a few cells were stained these MAT everywhere in the parenchyma of the brain. Also in the border to the granules zone was a limited number of T - and b-cells (table). The number OH-positive cells in the border zone decreased from 62% in the first week up to 20% on week 9, while the number of ED1-positive cells decreased from 37% in the first week to 7% at week 9. The number of ED2-positive cells (5%), T cells (14%) and b cells (1%) changed utterly during the observation period (table).

4. The effect of alginate therapy with EN is Nate lived for 20±4% longer than processed imitating transfitsirovannykh cells. Detailed histological examination showed large necrotic areas in tumors that received alginate therapy with endostatin (see Fig.7, part b). These necrotic areas were never visible in the control (imitating transfetsirovannyh cells encapsulated in alginate, Fig.7, part a).

Discussion

The results of the above experiments clearly show that microencapsulating cells survive, proliferate and maintain their phenotypic expression over a long period of time. It was also shown that MAT released from the alginate pellets have the ability to inhibit the migration of tumor cells in vitro, interacting with EGFR, and that MAT is released and distributed within the rat brain.

As can be seen from the results of light microscopy, cells NIH T adapted to alginate in vitro and began to proliferate for several days after encapsulation. The CLSM study revealed that cell viability is at about 90% on the day of encapsulation. During the first three weeks in culture approximately 50% of the originally included cells died within capsules. However, after 9 nadamay death of cells within the alginate was also reported by other authors, and it may be the result of reduced diffusion of oxygen, nutrients and waste products, which ultimately can lead to equilibrium between the number of proliferating and dying cells. A more favorable rate of diffusion can be achieved by reducing the grain size, the increase in the content of G-units, which will increase the pore size, or by changing the concentration of alginate. In addition, diffusion depends on the number of initially encapsulated within granule cells. Alginate itself is non-toxic and, therefore, should not expect that he contributes to the observed cell death inside the granules.

Cells BT4CnVlacZ showed a high and evenly distributed activity-galactosidase within 9 weeks of culture.

These results show that also can be formed of specific gene products during prolonged periods within alginate beads.

Research flow cytometry showed that cells NIH T week change from diploid to multiplaying population. This indicates that the cell nuclei are divided, but in the limited space inside the hard alginate network cells initially n is). However, after 3 weeks, the distribution of the cell cycle was similar to the control. This may indicate that cells need a certain period of adaptation within the alginate, and that individual cells with two and three cores or complete their cytokinesis, or perish. Histogram after 9 weeks were similar to those in 3 weeks, but there were indicators indicating the growth of cells in the proliferating phase. Analysis of the distribution of cell cycle showed an increase in the number proliferous cells from 50% in the control to about 60% after 9 weeks. This may be the result of selection of cells within alginate pellets with a higher proliferative capacity during prolonged cell culture NIH T.

The release of antibodies from encapsulated hybridoma cells N was basically constant at about 400 ng/(mlhc) from 12 noon on day 33, which shows that in the culture after 12 days was established stable density MAT-secreting hybridoma cells. This discovered fact is important for the clinical situation, because it shows that achieved stable production of monoclonal antibodies at a high level.

The migration of cells from spheroids GaMg promoting what was inovalis and area gains was similar in control. Based on this we can assume that paracrine mechanisms of cell proliferation is inhibited these MAT, possibly by blocking EGF-binding domain of EGFR.

It has been shown that implantation of encapsulated in alginate cell producers in other organs outside the Central nervous system (CNS) occurs gains fibroblasts in alginate granules, leading to cell death and destruction of the graft (Transplantation, 54 (1992), pages 769-774). Due to the unique localization and the absence of fibroblasts in the Central nervous system, this cell gains were not observed in the present study (Fig. 5A, B). It was shown that, depending on composition, alginates, in some cases, trigger the immune system in the body by stimulating monocytes to produce high levels of cytokines. Cytogenetically part of alginate are the M-unit. Therefore, for the experiments of the applicants was selected alginate with a high level of G-units in order to minimize the immune response in the brain. In subsequent experiments, the applicants have established low immune response against encapsulated in alginate cells inside the brain, only with some education of microglia in brain tissue close to the implanted pellets. These on the d treatment method inside the brain. Also watched the minimum aggregation of cells around the border zone between the site of implantation and the parenchyma of the brain. This may be due to cells NIH T released from the alginate granules, due to the weak immune response to the implant, as discussed above, and/or due to ranozazivluschee process in the tissue. However, it is believed that a small number of cells-producers, which are released from the alginate is the problem, because these cells are provided with normal graft against the mechanisms of rejection by the host. However, if you prefer, you can take steps to prevent the release of cells, for example, covering the granules with a layer of poly-L-lysine or irradiation of cells prior to encapsulation, thereby inhibiting the proliferative ability. Immunoglobulins were released from alginate granules and distributed in the parenchyma of the brain at a distance of at least 1 mm from the border of the sites of implantation. Two experimental animals MAT was also detected in the whole hemisphere of the brain where the implants. This distribution may be due to passive diffusion. MAT was also in the subarachnoid space and within the perivascular space Virchow-Ro is inside the Central nervous system. Interestingly, tumor cells follow the same pathways in the brain that makes them available for components produced encapsulated in alginate cells.

In conclusion, the experiments described above show that the encapsulated cells-producers survive and proliferate inside the alginate over an extended period of time both in vitro and in vivo. Inside the cytoplasm of the encapsulated cells BT4CnVlacZ produced gene products, such as-galactosidase, within a few weeks of cultivation. Encapsulated hybridoma cells also produce and release large quantities of MAT in vitro and in vivo. Migration of tumor cells GaMg inhibited in the presence of encapsulated cells N. The implants of encapsulated cells In produce and release MAT inside the brain of rats, and MAT spread within the brain parenchyma and within the subarachnoid and perivascular space. Therefore, the present invention represents a promising tool for the treatment of tumors of the Central nervous system.

Claims

1. Cell line that can Express the molecule, which is inhibitory (G), where this molecule represents a) a molecule capable of interacting with components of the paths connecting the tumor and the host; (b) a molecule that can have an impact on the neovascularization of the tumor; (c) a molecule that can disrupt communications tumor cells with the extracellular matrix, or (d) a monoclonal antibody that is able to interact directly with tumor receptor, where the specified monoclonal antibody binds or interacts with the receptor AA and BB platelet-derived growth factor, receptors acidic or basic fibroblast growth factor, receptors alpha and beta transforming growth factor, different classes of receptors of vascular endothelial growth factor (VEGFR-1 and VEGFR-2), tyrosinekinase receptors with immunoglobulinemia and EGF-like domains, such as, for example, TIE-1 and TIE-2/tek, growth factor hepatocyte (scattering factor); CD 44; complexes CDR/cyclin; glycolipids on the cell surface; glycoproteins and proteins resulting from the expression of specific oncogenes.

2. Encapsulated cell line under item 1, where alginate contains more than 50% guluronic acid.

3. Encapsulated cell line under item 1 or 2, where oderit 80-100% guluronic acid.

5. Encapsulated cell line according to any one of the preceding paragraphs, where the specified expression of the indicated molecules can be switched on and off external pharmacological agent.

6. Encapsulated cell line according to any one of the preceding paragraphs, where the specified cell line is in pellet or microgranule.

7. Encapsulated cell line according to any one of the preceding paragraphs, where the CNS tumor is a brain tumor.

8. Encapsulated cell line according to any one of the preceding paragraphs, where alginate is highly purified, contains a very small amount of endotoxin or does not contain endotoxins.

9. Encapsulated cell line according to any one of the preceding paragraphs, immobilized in granule or microgranule, in which the concentration of alginate increases toward the outer surface of the granules or microgranules.

10. Encapsulated cell line according to any one of paragraphs.1-9, where this molecule is a molecule (e.g., protein, peptide, or polysaccharide) that can have an impact on the neovascularization of the tumor.

11. Encapsulated cell line according to any one of paragraphs.1-9, where pointed to by alewyn receptor, where the specified monoclonal antibody binds or interacts with the receptor AA and BB platelet-derived growth factor, receptors acidic or basic fibroblast growth factor, receptors alpha and beta transforming growth factor, different classes of receptors of vascular endothelial growth factor (VEGFR-1 and VEGFR-2), tyrosinekinase receptors with immunoglobulinemia and EGF-like domains, such as, for example, TIE-1 and TIE-2/tek, growth factor hepatocyte (scattering factor); CD 44; complexes CDR/cyclin; glycolipids on the cell surface; glycoproteins and proteins, the resulting expression of specific oncogenes.

12. Encapsulated cell line according to any one of paragraphs.1-11 intended for use in the drug.

13. The method of obtaining the encapsulated cell line according to any one of paragraphs.1-12, providing one-step encapsulation by drip adding a solution of alginate containing viable cells in a solution containing polyvalent cations.

14. Drug for the treatment of Central nervous system tumors, representing encapsulated cell line according to any one of paragraphs.1-12.

15. A method of treating Central nervous system tumors, produce.14.

 

Same patents:

The invention relates to microencapsulating secretory cells and the method of treatment of diseases caused by impaired functioning of the secretory cells

The invention relates to the field of biotechnology

The invention relates to microencapsulating secretory cells in the hydrophilic gel, therapeutic methods that are used macroencapsulation secretory cells, and to the preservation of secretory cells by macroencapsulation
The invention relates to the food industry

The invention relates to medicine, namely to immunology, and can be used to determine the in vitro activity of complement by the classical pathway activation in a variety of diseases that can occur with the involvement of the complement system, including
The invention relates to biotechnology and can be used to obtain a porous wound healing materials
Antitumor agent // 2228757
The invention relates to the field of medicine and for the creation of anti-cancer drugs
The invention relates to chemistry and medicine

The invention relates to medicine, more specifically to Oncology, and can be used in the treatment of patients with malignant prostate tumors with a significant prevalence of process

The invention relates to a LHRH antagonists - compounds of General formula Iin which a represents acetyl or 3-(4-forfinal)propionyloxy group Xxx1mean D-Nal(1) or D-Nal(2), Xxx2-Xxx3mean D-Cpa-D-Pal(3) or a simple link, Xxx4means Ser, Xxx5means N-Me-Tyr, Xxx6mean D-Hci or a residue of D-amino acids of General formula (II)

where n means the number 3 or 4, a R1means a group of the General formula IIIwhere R denotes an integer from 1 to 4, R2means hydrogen or alkyl group, and R3means unsubstituted or substituted aryl group or heteroaryl group, or R1mean 3-amino-1,2,4-triazole-5-carbonyl group,Xxx7means Leu or Nle, Xxx8means Arg or Lys(iPr), Xxx9means Pro and Xxx10means A1A or Sar, and their salts with pharmaceutically acceptable acids: process for the preparation of these compounds, pharmaceutical compositions having the properties of an LHRH antagonist, comprising as an active narushenie compounds according to the invention has a high solubility in water
The invention relates to the field of pharmaceutical industry and relates to a composition for the prevention and treatment of fibrocystic mastopathy

The invention relates to compounds, which are Aza-derivatives, such as Aza-indiani, Aza-tinalley or Aza-valley, to pharmaceutical compositions and to a method of treatment of pathological conditions such as cancer, bacterial and viral infection

The invention relates to new compounds to metabolites ecteinascidin, namely ETM-305, ETM-204 and ETM-775, having the following structural formula:

These compounds are strong antitumor agents

The invention relates to the production of endostatin mouse and man
Up!