Taxanes covalently bonded with hyaluronic acid or hyaluronic acid derivatives

FIELD: chemistry.

SUBSTANCE: invention relates to taxane, especially to paclitaxel and docetaxel covalently bonded with hyaluronic acid or a hyaluronic acid derivative, used as active substance in pharmaceutical compositions which are used in oncology, for treating tumours, autoimmune diseases and restenosis, as well as a coating for stents and medical devices. The covalent bond is formed between hydroxyl groups of taxane and carboxyl groups or hydroxyl groups of hyaluronic acid or hyaluronic acid derivatives, or amino groups of deacetylated hyaluronic acid. Bonding can take place using a linking compound (spacer compound) which bonds taxane with hyaluronic acid or hyaluronic acid derivative under the condition that, the linking compound is different from hydrazide.

EFFECT: proposed taxane has high therapeutic effectiveness when treating oncological diseases, autoimmune diseases and restenosis, dissolves in water without reduction of its pharmacological activity and is not toxic, which leads to overcoming hypersensitivity and anaphylaxis.

46 cl, 20 ex, 4 dwg

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention relates to similarly to taxanes, especially paclitaxel and docetaxel, covalently linked hyaluronic acid or a derivative of hyaluronic acid, method for their production and their use in the field of Oncology in the treatment of autoimmune diseases and restenosis.

The LEVEL of TECHNOLOGY

Taxanes and, especially, paclitaxel and docetaxel, currently sold on the market under the trade name Taxol and Taxotere are anticancer agents (Huizing M.T. et al., Cancer Inv., 1995, 13: 381-405), which show its antiproliferative effect by acting on the organization of microtubules in cellular cytoskeletal system. Indeed, by inhibiting the depolarization of these microtubules they prevent their normal dynamic reorganization that takes place during mitotic cell division (J.J. Manfredi et al., J. Cell Biol., 1982, 94: 688-696).

The main therapeutic indications for paclitaxel are:

therapy for a running breast cancer;

therapy for Kaposi's sarcoma;

therapy for sarcoma of the lung (not microciona);

- ovarian carcinoma resistant to treatment with standard chemotherapy.

In addition, the specified chemotherapy used for the treatment of carcinoma of the bladder, rotate and endometrium.

Knowing that paclitaxel is insoluble in water, it is mixed with a mixture of cremophor® EL (castor oil) - ethyl alcohol in a ratio of 1:1 in the pharmaceutical compositions currently used in cancer chemotherapy (Pfeifer R.W. et al., Am. J. Hosp.Pharm., 1993, 50:2520-2521). This drug is usually used for continuous intravenous infusion (within 3 to 24 hours) at a dose of 135-175 mg/m².

The presence of cremophor EL in the above preparation is the major cause of adverse reactions, which usually occur during administration of paclitaxel and vary from simple lesions of urticaria to shortness of breath and bronchospasm and even anaphylactic shock (Weiss R.B. et al., J. Clin. Oncol., 1990, 8: 1263-1268).

For this reason, any patient who is going to get the treatment the pharmaceutical composition of paclitaxel-cremophor EL must first follow the Protocol then with the introduction of dexamethasone, possibly associated with antihistamine.

Despite these precautions, up to 40% of patients who receive intravenous infusion of paclitaxel, still feel more or less serious adverse reactions. Therefore, we can say that the clinical use of the drug Taxol in the present, and the methods used for its introduction, are of limited effectiveness. This is the reason why the study is from now directed to the synthesis of new pharmaceuticals and/or for new chemicals above anticancer drugs, which are water-soluble.

For example, researchers have tried to capsulerebel paclitaxel in liposomes, nanocapsules and microspheres composed of polymer walls formed biorazlagaemykh copolymers, such as polylactic acid, biokerosene copolymers such as a copolymer of ethylene and vinyl acetate.

In addition, were obtained microspheres that are loaded with paclitaxel and which is formed biorazlagaemykh polymer, such as polyphosphates, to create a system for sustained release of drugs at a place therapy for the treatment of carcinoma of the lung (Nuijen C. et al., Investigation of New Drugs, 2001, 19: 143-153).

Were taken also attempt to obtain micelles specified anticancer drugs deposition of paclitaxel in an organic solvent system phosphatidylcholine/bile salts (Nuijen C. et al., Investigation of New Drugs, 2001, 19:143-153).

However, these new systems for encapsulation of paclitaxel can be difficult in terms of stability, access, and the ability of reproduction.

In addition, have been made various attempts dissolution of drug-cyclodextrin, but the new drugs have not yielded the desired results (Nuijen C. et al., Investigation of New Drugs, 2001, 19: 143-153).

Chemical research on new drugs tow the Axel, which makes the drug more water soluble while maintaining its effectiveness as an anticancer agent, led to the synthesis of new analogues modified at positions C2' m C7 (application for U.S. patent No. 2001/0018531), as well as to obtain new prodrugs.

Prodrugs are therapeutically inert derived medicines, which are activated when introduced into the body.

Here the active ingredient is released after spontaneous processes of hydrolysis and/or enzymatic hydrolysis.

From this point of view and for the reasons mentioned above have attempted the synthesis of novel prodrugs that led, for example, to obtain medicines, such as acetylacetone (W. Mellado et al., Biochem. Biophys. Res. Commun., 1984, 124(2): 329-336) or the synthesis of new esters of the specified medicines with succinic, glutaric and sulfonic acids at the carbon atom in position C2'. However, it is proved that these esters are unstable in the aquatic environment.

In addition, some derivatives were synthesized with a group phosphonocrotonate ether in position C2 or C7, such as paclitaxel-2'-carbonate, and a number of new esters of paclitaxel with amino acids and their derivatives with patriliny group at position C2'.

It is shown that glucarpidase the-asparagine and glucarpidase-glutamine are the two most vysokochastotnymi products received the type described above synthesis, but they are less effective than paclitaxel per se (Nuijen C. et al., Investigation of New Drugs, 2001, 19: 143-153).

It is also known that paclitaxel was atrificial poly-L-glutamic acid with the formation of new water-soluble derivative of the specified chemotherapeutic drugs with significantly higher half-period of existence than unconjugated paclitaxel (Li, S. et al., Cancer Research, 1998, 58(11): 2404-2409).

Paclitaxel was also made into a derivative with PEG (polyethylene glycol) esterification of medicines chemotherapy in position C2'; however, it is shown that the new molecule is very soluble, but not very stable.

Finally, recently developed a new delivery system drug conjugation of paclitaxel with albumin human serum (HSA). It is proved that the conjugate of paclitaxel-HSA is very soluble in water and is able to "carry" up to 30 molecules of chemotherapeutic drugs. However, the experiments conducted in vitro showed that it is less effective against cancer than paclitaxel per se (Nuijen C. et al., Investigation of New Drugs, 2001, 19: 143-153).

Recently, researchers have synthesized a new delivery system for paclitaxel, esterified pre modificirowan the th hyaluronic acid (hereinafter referred to here denoted by""), which is subjected to interaction with molecules hydrazide associated with a carboxyl group ON the amide bond (Y. Luo et al., Biomacromolecules 2000, 1 (2): 208-218; U.S. patent No. 5874417). This new delivery system for paclitaxel allows the drug to reach directly to the surface of the membrane which is the target of a cancer cell characterized by overexpression of the receptor for ON, CD44. Therefore, it is proved that paclitaxel is associated with, functionalized with a hydrazide, the ability to communicate in a specific way with CD44 cancer cells and, thus, is able through the process of endocytosis) enter into the cytoplasm of a cell where it can be enzymatic way released and activated, launching its mechanism of inhibition of depolarization tubulin and therefore cell division. This mechanism of selective transfer of medicines called "cell alignment".

In addition, it is known that can be used as filler for anti-cancer drugs in pharmaceutical compositions where TO associated (but not covalently associated) with chemotherapeutic drugs such as paclitaxel, to enhance their therapeutic efficacy due to the phenomenon of "aiming"described above (international patent is the first patent application # WO 00/41730), and the ability to reduce the dose normally provided in conventional chemotherapy protocols (international patent application number WO 99/02151).

And finally, it is known that low molecular mass and/or lipid derivative is used to produce liposomes used for delivery of drugs, including anticancer drugs, such as paclitaxel (international patent application number WO 01,39815). From the point of view of the above, there is still a need for new taxan derivatives that are stable and soluble in water and therapeutically effective, at least, what are unmodified taxanes.

The INVENTION

The author claims are now discovered that covalently associated with or derived ON taxanes, not necessarily by connecting the connection, get a stable and water-soluble products, suitable for the production of pharmaceutical compositions for the treatment of tumors, autoimmune disorders, and restenosis. Thus the subject of the invention is Texan, covalently bound to hyaluronic acid or its derivative, and the specified covalent bond selected from the group consisting of:

i) direct broadcasting communication is formed between carboxyl groups of hyaluronic acid or its about wodnego and hydroxyl groups taxane,

ii) bond formed between the carboxyl groups of hyaluronic acid or its derivative and a hydroxyl function taxane through the separator with the specified separator forms the essential link with hyaluronic acid or its derivative, and

iii) bond formed between the hydroxyl function of hyaluronic acid or its derivative and a hydroxyl function taxane through the separator with the specified separator forms the essential link with hyaluronic acid or its derivatives.

The present invention further relates to methods for taxan, covalently linked TO or derived.

The next subject of the invention are pharmaceutical compositions containing as active substance, at least Texan, covalently linked to ON, or derivatives, and their use in the treatment of tumors, autoimmune diseases and restenosis.

Data taxanes covalently associated with or derived, have many advantages that can be summarized as follows:

1) they are instantly soluble in blood;

2) they do not need to be mixed with cremophor EI to obtain drugs that can overcome the above problems related to hypersensitivity and anaphylaxis;

3) because f is rotatively activity of enzymes such as esterase, normally present in plasma, taxanes immediately released and used as filler ON or derived AT from the present compositions into the blood, where they can freely Express their anticancer activity;

4) they provide an opportunity to get a new drug, which in the case of some types of cancer can have unexpected chemotherapeutic activity, which is much higher than the activity you get, with the introduction of unconjugated texana when considering the same dose of the medication.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

The figure 1 shows the percentage of survival after implantation of tumor cells, as described in example 1 for controls (black histogram) and for mice that received paclitaxel (gray histogram) and paclitaxel covalently associated with a complex ether AT 16% of esterification (white histogram)obtained as in example 7.

The figure 2 shows the drugs ability, expressed as IC50 which are the result of experiments in example 2, paclitaxel, covalently linked with ether derivatives, having 16% esterification (gray histogram), 22% esterification (black histogram) and 6.8% esterification (white histogram) for the four cell lines of breast VC is PS compared with the reference product paclitaxel.

The figure 3 shows the percentage of survival after implantation of tumor cells, as described in example 3, the control mice (dotted line) and the mice that received the ASR gel (continuous line).

The figure 4 shows the percentage of paclitaxel covalently associated with a complex ether, as described in example 7, released in the plasma of human blood as described in test example 13, depending on the time.

DETAILED description of the INVENTION

The present invention describes compounds, belonging to the family taxane, preferably, paclitaxel and docetaxel, are presented below by formulas (I) and (II), respectively, covalently linked TO or derived BY, preferably, by connecting groups, as the boundaries between taxonomy component or derivative, covalently linked to both molecules.

Is heteropolysaccharide, consisting of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine and having the following recurring units:

Is a polymer with a linear chain and molecular weight, which can vary between 50,000 and 13×10⁶Yes, depending on its source and method used to obtain it. is at present in nature in pericellular gels, in the main substance of the connective tissue of vertebrate organisms (in which she is one of the main components), in the synovial fluid of joints, in endolite and in the umbilical cord. ON plays an important role in the biological body as a mechanical carrier for the cells of many tissues, such as skin, tendons, muscles and cartilage. It is the main component of the extracellular matrix, but it has other functions, such as tissue hydration, lubrication, and migration, and differentiation of cells.

ON used in the present invention, can be extracted from any source, such as warts, or it can be obtained by enzymatic or technological way and it can have a molecular weight between 400 and 3×10⁶Yes, especially between 400 and 1×10⁶Yes, and preferably between 400 and 230000 Yes.

Derivatives IN the present invention, preferably, selected from the group consisting of the following derivatives:

- In the form of salts with organic and/or inorganic bases;

- Hyaff: esters with aliphatic alcohols, analiticheskogo, cycloaliphatic, aromatic, cyclic and heterocyclic series, with a degree of esterification, which can vary according to the type and chain length used alcohol, and in any case never exceed 50%of the etherification and preferably 0.1-20%, as the final polymer, which is obtained should always be water-soluble, whereas the remaining neeterificirovannah may be converted into salts with organic and/or inorganic bases, and such compounds are described in U.S. patent No. 4851521 included here as links;

- Hyadd^TM: amides, derived from aliphatic amines, analiticheskogo, cycloaliphatic, aromatic, cyclic and heterocyclic series, with a percentage of amidation between 0.1 and 10%, as the final polymer should always be water-soluble, whereas the remaining, which is not amidinophenoxy, can be converted into salts with organic and/or inorganic bases, and such compounds are described in the application for European patent No. 1095064 included here as links;

- O-sulfated derivatives ON the degree of sulfation 4, described in U.S. patent No. 6027741 included here as links;

- ACP: internal ethers with a percentage of esterification of not more than 15%, because the polymer must always be water-soluble, preferably between 0.05 and 10% of esterification, whereas the remaining neeterificirovannah may be converted into salts with organic and/or inorganic bases, and such compounds are described in the European patent No. 0341745 B1, included here as links;

products of deacetylation ON: get them by deacetylation of level N-acetylglucosamine with the percentage of deacetylation, preferably between 0.1 and 30%, while the carboxyl group can be converted into salts with organic and/or inorganic bases, as illustrated in the following structure:

deacetylate, described in International patent application no WO 02/18450, the authors include here as links;

- Oohh^TM: percarboxylic derivatives, obtained by oxidation of the primary hydroxyl level N-acetylglucosamine with a degree of percarboxylic between 1 and 100%, preferably between 25 and 75%. All carboxyl group can be converted into salts with organic and/or inorganic bases, as illustrated in the following structure:

Percarboxylic derivatives described in application for U.S. patent number US 2003181689.

In addition, these compounds, in which taxon, in particular, paclitaxel covalently associated with a complex ether, can be obtained from molecules chemically unmodified and only after synthesis with chemotherapeutic drug modification BY esterification of her with all of the above to enter the alcohols to obtain products Hyaff® or the formation of internal ethers, as in the case of ACP® (see example 8).

Previously listed derivatives, which are particularly important in the method of synthesis of prodrugs ON-Texan, and, in particular, prodrug ON-paclitaxel, are diacetylbenzene and sulfated derivatives, since the same percentage of paclitaxel associated with pre-unmodified hyaluronic acid, they make the end product more soluble in the blood stream.

It is known that receptor D44-membrane mediates many different processes related to cellular physiology and biology, such as proliferation, differentiation and locomote cancer cells and other cells.

In the scientific literature has recently been shown effective IN cancer when injected as such directly in the growing cancer. It is proved that it is able to cause complete regression of 30% of tumors (Herrera-Gayol A. et al., Experimental and Molecular Pathology, 2002, 72: 179-185).

It is also known that can be associated with any chemotherapeutic drug for many different pharmaceutical compositions, as it is able to act as a second anticancer agent, which synergistically enhances the anti-cancer action of the medicinal product associated with it (international Pat is ntna application number WO 01/47561; in an alternative variant reported as anticancer drug that is administered as such in various clinical protocols to reduce/regression growth of cancer cells (international patent application number WO 97/40841).

Taxan this application, covalently linked to ON, or derived from, as mentioned above, differs from all drugs taxan, in particular, the covalent bond of paclitaxel with or derivatives, optionally using a connecting group, makes paclitaxel soluble in water without reducing its pharmacological activity.

Indeed, the in vivo experiment described in example 1 clearly demonstrates the same anti-cancer effectiveness of this conjugated and unconjugated paclitaxel paclitaxel when administered at equal doses.

In addition, paclitaxel may be unexpected pharmacological properties that differ from the pharmacological properties of unconjugated paclitaxel, especially in the case of some types of tumors.

Indeed, in example 2 clearly shows that this ether derivative ON associated with paclitaxel, has a new anticancer pharmacological activity: on the model of cytotoxicity in vitro, described below, ON-paclitaxel of the present invention neojidannogo the shows anticancer activity, which is much higher than the activity shown individually unconjugated paclitaxel.

New antitumor properties means that the data taxanes, especially paclitaxel conjugated with or derivatives, can be used to produce pharmaceutical compositions applicable as chemotherapeutic drugs not only for treatment of all forms of tumors, for which treatment is administered Taxol®, but also for other forms of tumors, which are usually not treated with Taxol®, such as stomach cancer and liver cancer, colon cancer, melanoma and leukemia. In addition, it can be used in systemic autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus, autoimmune glomerulonephritis and, finally, Hashimoto's thyroiditis.

The use of these products in a new pharmacological treatment for the above mentioned pathologies is possible because a new connection ON-paclitaxel reduces the systemic toxicity of Taxol®, thus increasing therapeutic efficacy of the medicinal product, as it:

- is water-soluble;

- not associated with cremophor® EL and, therefore, do not show any toxic effect, which manifests cremophor;

- is equally effective at doses definitely b is more low, than the doses commonly used in clinical protocols or equal to these doses.

It is also known the use of paclitaxel as a drug, which is used for inhibition of restenosis, which usually follows after plastic operations on vessels (preferably, blood pressure), coronary bypass surgery and organ transplants.

These taxanes, in particular, paclitaxel covalently associated with or derived, can also be used to prevent restenosis, or they can be used for the formation of internal coatings for stents and devices, implantable after the above vascular surgeries, it was shown that they may be contacted by a chemical image of the surface of these stands or easily adsorbed on it.

In any case, the time data products on the surface of the stent and, consequently, their gradual release into the bloodstream more than unconjugated paclitaxel, due to the fact that the chemical-physical characteristics contribute TO a progressive, slow but continuous release of Taxol® with the surface of the device.

Pharmaceutical compositions comprising these taxanes covalently associated with or derived, can be wedenesday way (intravenous or arterial, intramuscular, intraperitoneal, subcutaneous or oral routes), they can be used for local applications (percutaneous absorption) or they can be entered directly in place of the cancer by injection.

Or its derivative, covalently associated with paclitaxel, may also act as anti-cancer drugs per se.

In the following example 1 the author of the proposal shows how the treatment of experimentally induced tumor growth in "Nude" mice stitched derived AT, ACP®, detects significant tumor regression compared with untreated controls.

The author of the application therefore describes the first time the new role and its derivatives, which are part of these products Texan-ON or taxon-derived, as anticancer agents and their comparative use in the field of Oncology. In addition, these taxanes covalently associated with or derived, can be associated with a variety of biologically and pharmacologically active molecules, such as steroids, hormones, proteins, trophic factors, vitamins, nonsteroidal anti-inflammatory drugs, chemotherapeutic drugs, calcium antagonists, antibiotics, antiviral agents, interlay the ins and cytokines, such as interferon. Thus, it is possible to obtain many different associations of the above medicines and on the various pharmaceutical compositions containing taxanes of the invention.

The present invention relates also to a method for real taxan, especially paclitaxel covalently linked TO or derived; these products can be obtained in the following ways:

1) indirect synthesis, which includes the introduction of a linking group (spacer, spacer) between taxonom and ON or derived ON, or

2) direct synthesis between taxonom and ON or derived.

Functional groups or derivatives, which can react with taxonom directly or not directly via a connecting group (spacer), are the following groups:

1) a hydroxyl group;

2) carboxyl group.

Connecting the connection can be, for example, selected from the group consisting of aliphatic or analiticheskoi chain, unbranched or branched, substituted by one or more groups selected from hydroxyl, carboxyl or carbonyl groups, epoxides, acylchlorides, mercaptans, NITRILES, halides, anhydrides, isocyanates and isothioscyanates and amino groups.

Among the possible connections connecting predpochtite lname are bromine derivatives of carboxylic acids, having from 2 to 18 carbon atoms, and especially acids having from 3 to 10 carbon atoms, more preferred are 3-bromopropionic acid and 4-bromacleanse acid.

The fusion reaction between the hydroxyl functional groups ON (or its derivatives) and taxonomy component, such as paclitaxel, can be carried out by way of indirect or direct synthesis.

Indirect synthesis can lead to the formation of the following types of covalent bond between the linking group and ON or derived ON:

ester groups:

- comprising carboxyl functional group appropriately selected linking compounds that activate the activating agent such as, for example, carbodiimide (scheme 1, below);

- includes a hydroxyl group or a derivative ON that bromilow or replace toiley group followed by nucleophilic substitution by carboxyla appropriately selected linking compounds (scheme 2 below); or includes anhydrite function appropriately selected linking compounds (scheme 3 below);

urethane or tourmanova connection:

- includes the amino group suitably selected linking groups (scheme 4, below), or

simple essential connection:

- includes functional apachegroup (appropriately chosen) connecting group (scheme 6, below);

- includes a hydroxy-group ON or derived ON that bromilow or replace toiley group with subsequent nucleophilic substitution of the hydroxyl group is appropriately selected linking groups (scheme 7, below).

acetaldol or Catalinas connection:

- includes aldehyde and/or ketone group appropriately selected linking groups (scheme 8 below);

- includes a hydroxyl group appropriately selected linking groups and require the presence of simple carbonyl compounds, such as formaldehyde (scheme 9 below).

The above methods can be carried out using agents, activating a hydroxyl group or derivative, FOR example, selected from the group consisting of carbonyldiimidazole and di-(N-Succinimidyl)carbonate.

Direct reaction synthesis IU the DN hydroxyl groups or derivatives and taxonom, such as paclitaxel, can lead to the formation of the following types of covalent bonds:

acetaldol connection:

- includes a hydroxyl group taxane and hydroxyl groups or derivatives ON which covalently bind the accession of simple carbonyl compounds, such as formaldehyde (scheme 10).

The reaction between carboxyl groups or derivatives and taxonom, such as paclitaxel, can be carried out by way of direct or indirect synthesis. Indirect synthesis can lead to the formation of the following types of covalent bond between the linking connection and ON or derived ON:

ester link:

- carboxyl group appropriately selected linking compounds such as 4-bromacleanse acid, activate the activating agent such as carbodiimide, and thus make it suitable for synthesis with a hydroxyl group taxane (preferably, a group on the carbon atom at the C2'), such as paclitaxel. Then direct contacts in anhydrous solvent with a Quaternary ammonium salt, in particular with tetrabutylammonium (TBA) salt or derivative TO get nucleophilic substitution of carboxyl ON or derived ON bromoadenosine connection. In this way receive ester bond m is waiting ON or derived ON and connecting the group in turn is associated with paclitaxel. In the alternative case, the nucleophilic substitution of carboxyl group or a derivative ON bromoadenosine connection can be performed before the formation of communication between itself connecting the connection and taxonom (scheme 11 below).

through the use of activating agents carboxyl group or a derivative thereof, such as carbodiimide, you can form an ester bond between the specified functional group and a hydroxyl group (appropriately chosen) connecting the connection, previously or subsequently associated with paclitaxel (scheme 12 below).

amide connectivity:

- activation of carboxyl groups or derivatives ON activating agent capable of contacting amino group suitably selected connecting connections, except hydrazides, previously or subsequently associated with taxonom, such as paclitaxel (scheme 13 below).

Direct synthesis can lead to the formation of the following types of covalent bonds:

ester link:

- activation of carboxyl groups ON or derived BY an activating agent, giving the possibility to associate it with a hydroxyl group taxane (scheme 14 below).

activation guide is auxillou group tecknologi component activating agent, giving the possibility of linking it with functional carboxyl group or a derivative (scheme 14 below);

the next type of communication requires bromides or tosylate derived taxane. The indicated relationship is obtained by reaction of nucleophilic substitution bromides or toiley group, a carboxyl group or a derivative (scheme 15 below).

The reaction between the amine groups deacetylating ON and taxonomy component, such as paclitaxel, can be carried out by way of indirect or direct synthesis.

Indirect synthesis can lead to the formation of the following types of covalent bond between the linking compound, and AT:

amide linkages:

- includes carboxyl group appropriately selected binding compounds (scheme 16);

urethane or tourmanova connection:

- includes hydroxyl or Tilney group appropriately selected linking compounds (scheme 17).

Direct synthesis can lead to the formation of the following types of covalent bonds:

urethane links:

- includes a hydroxyl group taxane and functional amino group deacetylating ON (and therefore is and 18).

According to the same method of communication, comprising connecting the connection and Texan, such as paclitaxel, may be the type of ester (scheme 19), urethane or thiourethane (scheme 20), acetal or Catala (scheme 21), for her education may require the presence of an activating agent, especially for ester and urethane linkages.

Connecting the connection can be associated with taxonom, such as paclitaxel, before or after bonding with the functional groups ON or derived, depending on the type of functional groups appropriately selected connecting the connection.

The percentage of direct or indirect binding taxane, such as paclitaxel, or a derivative may vary between 0.1% and 100%, preferably between 0.1% and 35%.

The following examples are given to provide non-limiting illustrations of the present invention.

Example 1

The impact of the new ether derivatives ON with paclitaxel in "Nude" mice after implantation of tumor cells

For this experiment, the authors were inoculable cells of human ovarian adenocarcinoma, OVCAR-3, immunocompromised Nude mice belonging to the species Athymic CD-1. Each mouse was inoculable in wirebrushing by 5×10 ⁶cancerous cells.

Pilot scheme:

The subjects of the medicinal product:

- Taxol, 5 animals were treated;

- HYTAD1p20: ether derivative, covalently associated with paclitaxel, with 16% of esterification of carboxyl (wt./wt.). Molecular weight is used for the synthesis of this new drug was 200000 Yes (see example 7 for details of its receipt). For this medication also used the five animals.

Treated animals: 10 animals were first inoculable cells OVCAR-3.

Five animals were used for the experiment with Taxol® and the other five were used for experiments with HYTAD1p20:

all ten animals then received an intraperitoneal injection of 3 doses of pharmacological tools (on the 6th, 13th and 20th days after inoculation of cancer cells), equal to 20 mg/kg of body weight Taxol® or 125 mg/kg of body weight HYTAD (corresponds to 20 mg/mouse of paclitaxel).

Control animals: 5 animals were first inoculable inducing cancer suspension cells OVCAR-3, after which they had not received any treatment.

Determination of the survival curve

The curve of survival was calculated from the data of therapeutic intervention at the 92nd day after inoculation of cancer cells into the abdominal cavity.

Results: the obtained results are illustrated in figure 1.

Three control what lnyh animals had developed adenocarcinoma of the ovary and died between 70th and 75th day after inoculation of cancer cells. At the 92nd day after treatment, the last day of the experiment, none of the animals that had received pharmacological treatment with paclitaxel or HYTAD, not killed.

Example 2

Experiment in vitro

The purpose of the experiment in vitro was mainly the definition of the activity profile of new ether derivative ON associated with paclitaxel, and evaluation/comparison of antitumor activity of derivatives HYTAD with paclitaxel, in order thus to determine their pharmacological potential in comparison with the anticancer drug.

Pilot scheme:

Tested products:

- Taxol: a reference product

- HYTAD1p20 - HYTAD2p20 - HYTAD2p10: ether derivative, covalently associated with paclitaxel with 16% esterification of carboxyl (wt./wt.) (in case HYTAD1p20 molecular weight is used in the synthesis of this new drug, is 200,000 Da) (see example 7 for details of its receipt) or 22% (in the case of HYTAD2p20 molecular weight is used ON 39000 Yes), or 6.8% (in the case of HYTAD2p20, molecular weight is used ON 39000 Yes).

Cell line

Cell lines of human origin

Used four cell lines breast cancer person. All four of the strains tested cells are usually susceptible to paclitaxel and clearly expressyou the receptor CD44 with the same amplification.

MCF-7

- MDA-MB-231

- MDA-MB-468

- SKBR-3

Experimental Protocol:

1) the Test cell line is seeded at a concentration of 3000 cells per well in 96-well-plate with a flat bottom;

2) after 24 hours in the cell type tested solution, appropriately diluted in culture medium;

3) even after 72 hours the cells experience by colorimetry using bromide 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole (MTT) assessment of their viability, this test also detects different susceptibility of the cells to the subject drug. This is possible because the mitochondrial dehydrogenase is able to restore tetrazolium salt (yellow) in the crystal blue formazan. More or less color intensity assessed by spectrophotometry (Dezinot F. et al., J. Immunol. Methods, 1986, 22 (89): 271-277).

Results

Below the authors in table and graphic form in figure 2 the results obtained in terms of the IC₅₀(the concentration of drug required to inhibit cell growth by 50% in respect of the test product and used different cell lines).

In figure 2 the x-axis represents the pharmacological activity, expressed as IC₅₀and calculated as the ratio between the molar concentrations compared to reference the product (paclitaxel), which usually take in such a way that it had the value zero. Therefore, the shaded columns indicate the pharmacological activity, which is higher than the reference product.

IC₅₀(expressed as nm or μm paclitaxel or its HYTAD-derivatives in the culture medium)

Conclusion

As indicated in the literature, all used cell lines are sensitive to Taxol, a drug mainly used for the treatment of metastatic carcinoma of the breast and ovaries. As for the cell lines of breast cancer, it is shown that different HYTAD are much more powerful tools than paclitaxel, with factor +150 relative to HYTAD1p20 on the cell line MCF-7 cell.

Example 3

The impact of ASR gel on naked mice after implantation of tumor cells

For this experiment, the authors used a cell NT carcinoma of the colon human body immunodepression naked mice, referring to the form of Athymic Nude-nu (nu-nu). Each animal was anestesiology and 0.3 ml of cell suspension NT were injected with in his abdominal cavity at a concentration of 166000 cells/ml So that each mouse received 50000 cancer cells.

Pilot scheme:

Treated animals: 113 animals were first inoculable NT and immediately after that they had received one dose of treatment, 0.2 ml of ASR gel 40 mg/ml

Control animals: 117 animals were inoculable suspensions of cancer cells NT, but they did not receive the treatment.

Curve of survival: survival curve was calculated from the day of inoculation until the day of death. Death or established, libvisual killing animals weight was reduced by more than 20% of their initial mass, and in the case of hemopericardium indicating diffuse metastases. The percentage survival in the two groups was determined daily and expressed in the form of a graph to obtain the curve shown in figure 3.

The experiment lasted 120 days, after which all surviving animals were killed and examined mikroskopicheskim way to control the presence of tumors in the abdominal cavity.

Results: 32 of animals from 230 showed no development no significant neoplasia. 22 of these animals belonged to the group of mice treated with ASR gel®, 10 - K the control group.

Gel ACP®: 19.5% of treated animals not found neoplasia;

control: 8.5% of control animals not found neoplasia.

Example 4

Getting ON with a molecular mass of between 5000 and 10000 daltons (for possible synthesis-paclitaxel with low molecular weight)

2,40 g sodium salt with a molecular mass 990000 Yes dissolved in 240 ml of 0.15 M NaCl. The solution is then complement of 7.9 ml of a 14% solution of NaOCl. At a constant temperature of +4°C, the solution is treated with ultrasound for 120 minutes at a frequency of 20 Hz and 150 watts. After completion of the reaction the pH adjust to 6.5 0.1 G. of HCl and the solution is then treated to precipitate in 1000 ml of a mixture of 2:1 methanol:acetone. The product was collected by filtration and sushi is whether in vacuum for 48 hours at 45°C. So get of 1.65 g of sodium salt. Analysis of high performance liquid chromatography (HPLC) shows that the faction has an average molecular mass (MM) 5850, srednekamennogo molecular weight (MN) 3640 and the index polydispersity 1,61.

Example 5

Getting ether derivative ON associated with paclitaxel, with esterification of carboxyl about 4 wt./wt.%

51 mg of paclitaxel was dissolved in CH₂Cl₂and the complement solution of 104 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 20 mg of 4-pamakani acid. Then the solution is distributed in the water. After removing residual carbodiimide and bromide the reaction solvent is dried with anhydrous sodium sulfate and removed by rotary evaporator. Thus obtained product (21 mg dissolved in N-organic (NMP) and added to 20 mg/ml solution, turned into tetrabutylammonium (TBA) salt in NMP (200 mg in 10 ml of NMP). After the reaction for seven days at ambient temperature the solution was diluted with 5 ml water and 1 ml of saturated NaCl solution. Thus obtained solution is stirred for 1 hour to replace the sodium ion TION. Then slowly dropwise once added ethanol, and the thus obtained fibrous product is dissolved in water, dialist and finally dried by freezing.

Getting ether derivative ON associated with paclitaxel, with esterification of carboxyl approximately 10 wt./wt.%

As in example 5, 308,7 mg of paclitaxel dissolved in 15 ml of dichloromethane, complement 117,2 mg 4-pamakani acid and 614,1 mg EDC. Then to the solution was added water to remove all bromide and carbodiimide. Thus obtained organic solution supplemented with sodium sulfate for drying it, while the solvent is removed by rotary evaporator. In the end receive 363 mg of the intermediate product.

175 mg of the thus obtained intermediate product is added to 1 g of TBA dissolved in anhydrous NMP. The solution was stirred at ambient temperature for 7 days, then add 20 ml of water and 4 ml of saturated NaCl solution. The mixture is stirred for 1 hour to carry out the exchange of the sodium ion TION. Then slowly dropwise once added ethanol, and the thus obtained fibrous product is dissolved in water, dialist and finally dried by freezing.

Example 7

Receiving a live derivative with paclitaxel with esterification of carboxyl about 16 wt./wt.%

164 mg of the intermediate product obtained by the procedure described in the previous examples 5 and 6, are added to a solution of 680 mg ON-TBA dissolved in 25 ml bezvodno the NMP. After 7 days of reaction at ambient temperature solution supplemented with 20 ml water and 4 ml of saturated NaCl solution. After 1 hour, slowly dropwise once add ethanol. Educated product is collected by filtration and dissolved in water, dialist and when the conductivity of the dialysis solution is reduced below 10 µs, its frozen. The frozen solution is then dried by freezing.

Example 8

Receiving a live derivative with paclitaxel with esterification of carboxyl approximately 10 wt./wt.%

102,6 mg of paclitaxel was dissolved in 5 ml of dichloromethane and the solution complement of 20.4 mg of succinic anhydride. After three hours the solvent is removed by evaporation using a rotary evaporator. Thus obtained product is dissolved in 5 ml of dimethyl sulfoxide (DMSO) with low content of water and add 27.3 mg of dicyclohexylcarbodiimide. After 5 minutes the solution is supplemented with a solution of NA-TION, obtained by dissolution of 327 mg of polymer in 15 ml of DMSO with low water content. The solution was stirred at ambient temperature for 24 hours. Then to the solution add a few ml of water and 3 ml of a saturated solution of NaCl. After 1 hour the precipitate is precipitated by adding ethanol. The fibrous product is collected by filtration, dissolved in water, dialist and finally dried by freezing.

Example 9

Getting the EF is REGO derived ON with paclitaxel with esterification of carboxyl about 4 wt./wt.%

510,1 mg of paclitaxel dissolved in 6 ml dichloromethane, complement of 95.4 mg 3 bromopropionic acid and 525,0 mg EDC. Then to the solution was added water to remove bromide and carbodiimide distribution, while using 10 volumes of water to remove these chemicals. The organic solution supplemented with sodium sulfate for dehydration him and the solvent is removed by rotary evaporator.

The thus obtained intermediate product in the amount of 155,5 mg added to 1,46 g-TBA dissolved in anhydrous NMP, and thus the resulting solution was stirred at ambient temperature for 7 days. Then add 20 ml of water and 4 ml of saturated NaCl solution. The solution is stirred for 1 hour to carry out an exchange of the sodium ion TION. Then slowly dropwise once added ethanol, and the thus obtained fibrous product is dissolved in water, dialist and finally dried by freezing.

Example 10

Getting ether derivative of hyaluronic acid by esterification of carboxyl about 30 wt./wt.%

500 mg of paclitaxel was dissolved in CH₂CL₂and the solution is complemented 397,6 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 300,9 mg 4-pamakani acid. Then the solution is distributed in the water. After removing residual carbodiimide and bromide the reaction solution was dried soo what hatom sodium and the solvent is removed by rotary evaporator. Thus obtained product is dissolved in NMP and added to a solution containing ~20 mg/ml of hyaluronic acid, converted into a salt with TION in NMP (1,95 g in 100 ml of NMP). After 7 days of reaction at ambient temperature the solution was diluted with 20 ml of water and 4.5 ml of a saturated solution of NaCl. The solution is stirred for 1 hour to carry out the exchange of the sodium ion TION. Then slowly dropwise once added ethanol, and the thus obtained fibrous product is dissolved in water, dialist and finally dried by freezing.

Example 11

Getting partially autoschedule ether (approximately 10% substitution) with 8% wt./wt. paclitaxel

3,10 g, converted into a salt with TBA, dissolved in 150 ml of DMSO with low content of water at ambient temperature. The solution is then complemented 541,0 mg of the intermediate of paclitaxel obtained by the method described in examples 5, 6 and 7. After keeping it for a reaction time within 7 days at ambient temperature the reaction solution is added 126,5 g of triethylamine and the whole mixture is stirred for 30 minutes.

The solution 319,5 g iodide, 2-chloro-1-methylpyridine in 30 ml of DMSO is slowly added dropwise over a 45 minute interval, and the mixture was incubated at 30°C for 15 hours.

Add a solution formed by 50 ml of water and 1.7 g of sodium chloride, and formed with the ect slowly poured into 400 ml of acetone with continuous stirring. A precipitate, which is separated by filtration and washed with three times 50 ml of a mixture of 5:1 acetone and water and three times with acetone (50 ml). Thus, the final product is dried in vacuum at 38°C.

Example 12

Test the solubility of ether AT-paclitaxel obtained in example 5, in 5% glucose solution

14,6 mg product ON-paclitaxel obtained by esterification according to example 7 (based ON the molecular mass of 200 kDa) with a degree of substitution of carboxyl of 16.3 wt./wt.%, was dissolved in 1 ml of aqueous 5% glucose solution. The solution is mixed magnetic stirring bar, can be filtered through 20 μm sterile filter, mounted on the syringe. The concentration of paclitaxel in solution is 2.38 mg/ml

The authors tried to find the maximum concentration of product per ml of 5% aqueous glucose solution. When the concentration is 32.8 mg product ON-paclitaxel per ml glucose solution obtain a viscous solution with a concentration of paclitaxel to 5.35 mg/ml

Example 13

Tests for selection of paclitaxel from human plasma

Get the solution, which consists of 101.3 mg product ON-paclitaxel in 10 ml of water. ON-receive paclitaxel as described in example 7. Tests on the allocation of spend by location 40 mg of the above solution in contact with 2 ml of human plasma at 37°C.

For determination of paclitaxel, which wisweb is given in the plasma by separating yourself from, established a three-time probe: 6, 30 and 60 minutes. At the end of each interval contacting paclitaxel was extracted from the solution plasma-ON-paclitaxel by 3 washes, each time with 1.5 ml of tert-butyl methyl ether (TME), the product of the three extraction gathered together, evaporated to dryness natural evaporation at 65°C and again suspended in 400 μl of absolute ethanol to determine the content of the examined drugs HPLC (high performance liquid chromatography). The obtained results are shown in figure 4: after 6 minutes more than 80% of paclitaxel was separated from and this percentage has not increased at the later time of observation.

Example 14

Preparation of ether derivative of hyaluronic acid with the paclitaxel direct esterification carboxyl about 10% w/w (wt./wt.%)

0,5211 g of paclitaxel was dissolved in a vessel with a round bottom 15 ml dichloromethane and added 0.1185 g P-toluene chloride (p-Toluenesulfonil Chloride) and 0.0751 g dimethylaminopyridine (DMAP/DMAP). The reaction of the activated hydroxyl occurred at room temperature with stirring overnight. Further, the reaction was brought to the receipt of the product ("knockout" of the product, worked up by addition of water and extraction via DCM; the organic phase was then supplemented with magnesium sulfate for is gidratatsii and after filtration was dried and concentrated on a rotary evaporator the rotavapor.

320 mg of the intermediate product, thus obtained, were added to 1.987 g NATA dissolved in anhydrous NMP. The solution was mixed at ambient temperature for 7 days, after which 20 ml of water and 8 ml of a saturated solution of NaCl was added. The solution thus obtained was mixed for 1 hour to allow the exchange of sodium ions TION. Then slowly added ethanol drop by drop, and thus obtained fibrous product was dissolved in water, dializiruetsa and, finally, subjected to freeze dry.

Example 15

Preparation of ether derivative of hyaluronic acid with paclitaxel direct esterification carboxyl about 15% w/w (wt./wt.%)

0,4122 g of paclitaxel was dissolved in a vessel with a round bottom 15 mg dichloromethane and added 0.0952 g P-toluene chloride(p-Toluenesulfonil Chloride) and 0.0601 g dimethylaminopyridine (DMAP). Activation reactions of hydroxyl allowed to occur at room temperature with stirring overnight. Further the reaction was brought to the receipt of the product (brought to "knockout" of the product) (worked up by addition of water and extraction via DMC; the organic phase was then supplemented with magnesium sulfate for dehydration and after filtration was dried and concentrated on the rotavapor rotary evaporator).

300 mcpromoter product, thus obtained, were added to 1.2451 g NATA dissolved in anhydrous NMP. The solution was mixed at ambient temperature for 7 days, after which 20 ml of water and 5 ml of saturated NaCl solution was added. The solution thus obtained was mixed for 1 hour to allow the exchange of sodium ions TION. Then slowly added ethanol drop by drop, and thus obtained fibrous product was dissolved in water, dializiruetsa and, finally, subjected to freeze dry.

Example 16

Conversion in vitro data of example 2 as a function of equivalents of Taxol

The correct value of IC50 for Taxol on MDA/MB/231 cells

Correct data for IC50 for Taxol on MDA/MB/231 cells and is specified in the table in example 2 is 0.35 nm, but is 0.35 μm, it is obvious that a careful reading of figure 2, where the ratio of IC50 of Taxol to the components according to the invention are specified in the form of a graph. And, in fact, the correct unit of measurement for IC50 could not be nm, but is microns.

In fact 0.35 μm correspond to 350 nm, so the above ratio would be 350/2 .58=135, which corresponds to the value specified in figure 2.

Analysis of the results from example 2

In the specified example 2 tested the following mixture according to the invention: HYTAD1p20 ether-derived from (hyaluronic acid), covalent is associated with paclitaxel with the degree of esterification, calculated as w/w of 16% (wt./wt.%), with an average molecular weight of hyaluronic acid is 200,000 Da, HYTAD1p20 ether is a derivative of hyaluronic acid covalently associated with paclitaxel with the degree of esterification, calculated as w/w 22%(wt./wt.%), with an average molecular weight of hyaluronic acid is 39000 Da, HYTAD1p20 ether is a derivative of hyaluronic acid covalently associated with paclitaxel with the degree of esterification, calculated as w/w of 6.8%(wt./wt.%), with an average molecular weight of hyaluronic acid is 39000 Da. The determination of the degree of molar derivatization of hyaluronic acid(ON) paclitaxel, and the above-mentioned derivatives with paclitaxel are above the degree of esterification, which is expressed as mass percentages:

1) calculate the number of repeating units, where the carboxyl group derivativea Taxol - spacer (taxol-spacer % R.U. ON-Tah):

where

w/w % is the degree of mass derivatization

Mw_Dermolecular weight units, connecting the Taxol-spacer (Mw_R.U.+MW_spacer+Mw_taxol)

MW_taxmolecular weight of paclitaxel (853.92 g/mol)

2) Calculate the number of duplicate rederivation units, where the carboxyl groups are sodium (% R.U. ON Na):

%R.U. ON Na=100-(%R.U. H-Tax)

3) Calculate the number of moles of recurring units IN derivatizing by paclitaxel (mol HA-tax):

4) Calculate the number of moles rederivation repeating units HA (mol FOR Na):

where

Mw_Namolecular weight rederivation repeating units, where the carboxyl groups are sodium (401.38)

5) calculate the molar percentage of Taxol bound to hyaluronic acid (IN):

Thus, the above-mentioned mixture

HYTAD1p20:

w/w%=16%

- Mw_der=(Na-Na)+CH₂CH₂CH₂CO+Tax=1301.41

- Mw_tax=853.92

% R.U. HA-Tax=16*1301.41/853.92=24.38

% R.U. HA Na=100-24 .38=75.62

mol HA-Tax=24.38/1301.41=0.018

mol HA-Na=75.62/401.38=0.188

The second derivative HYTAD2p20:

w/w%=22%

- Mw_der=(Na-Na)+CH₂CH₂CH₂CO+Tax=1301.41

- Mw_tax=853.92

% R.U. HA-Tax=22*1301.41/853.92=33.52

% R.U. ON Na=100-33 .52=66.48

mol HA-Tax=33.52/1301.41=0.0257

mol HA-Na=66.48/401.38=0.165

3rd HYTAD2p10:

w/w%=6.8%

- Mw_der=(Na-Na)+CH₂CH₂CH₂CO+Tax=1301.41

- Mw_tax=853.92

% R.U. HA-Tax=6.8*1301.41/853.92=10.36

% R. U. AT Na=100-10 .36=89.64

mol HA-Tax=10.36/1301.41=0.0079

mol HA-Na=89.64/401.38=0.223

Now we preconvention the values specified in the table note the ru 2 (fixed in terms of values S to paclitaxel, as noted in paragraph(1)), in order to determine S, expressed in quantities equivalent Taxol (IC50_{taxol equivalents(equivalent Taxol)})contained in the three tested above mixtures from S expressed as nm of the tested product (S_{nM tested conpounds}by application of the following mathematical formulas

IC50_{taxol equivalents (equivalent Taxol)}=IC50_{PM tested products(tested products)}×% degree of substitution_{mol Taxol/mol HA}.

We have the following table:

It follows, then, that the mixture according to the invention is therefore more effective than Taxol on:

- MCF/7 cells, respectively: 3.5/0,11=31.8 times (times)

3.5/0.0022=1590 times 3.5/0.023=152.1 time,

- MDA/MB/231 respectively nm / nm=1100 times (times), PM/8.16=RS

SKBR/3: 0.23/ 0.0047=48.9 times.

Example 17

Preparation of ether derivative ON (hyaluronic acid) with paclitaxel by direct esterification (firesale) of the carboxylic acid with the use of an activating agent other than tosilata or bromide

0,6035 g of di(N-Succinimidyl) carbonate was dissolved in 20 ml of acetonitrile in a vessel with a round bottom under a stream of nitrogen. After complete dissolution, 0.5010 g of paclitaxel, pre-dissolved in 20 ml of acetonitrile, and 140 ml of pyridine was added. The reaction was allowed to flow while stirring at room temperature under a stream of nitrogen. After 24 hours the solvent was removed under vacuum in the rotavapor and the raw product was placed in dichloromethane. White precipitate was filtered, and the organic phase was washed five times with water weak acidity (pH about 4). Polyextremophile organic phase was supplemented with sodium sulfate to remove water, filtered and dried in a rotary evaporator.

The second step 0.534 mg activated paclitaxel, thus obtained, was dissolved in 50 ml of NMP and added to 2.1787 g NATA, pre-dissolved in anhydrous NMP. The mixture is stirred at 35°C for 24 hours, then 12 ml of a saturated solution of NaBr was added, and stirring is continued for one hour, in order to allow the replacement of ions TION of sodium. Then the conjugate was injected into the sediment by adding ethanol drop by drop, and purified by washing with a mixture of ethanol/water 90:10, to remove salts and unbound paclitaxel.

Example 18

Preparation of ether derivative ON (hyaluronic acid) with paclitaxel by indirect epirizole in hydroxyl groups, pre-activated by tosylation

In the solution paclitaxel in dry pyridine (0.4 g/ml) was added 10 equivalents of succinic anhydride in order to obtain 2'-O-succinyl-paclitaxel derivative (Speiser-paclitaxel). The reaction mixture was kept at room temperature for 3 hours while bubbling nitrogen. Then the solvent was evaporated under vacuum and the solid residue was purified free from succinic anhydride by suspension in subacid the distillated water, p and stirring for 30 minutes, and a five-fold centrifugation. Then the final purified product was dried.

Meanwhile 2.1241 g HA-TBA was dissolved in 500 ml of DMSO and added 0.0961 g R-toluensulfonyl chloride and 0.0641 g dimethylaminopyridine (DMAP) in order to activate the hydroxyl group of hyaluronic acid. Activation reactions of hydroxyl allowed to occur at room temperature with stirring over night, then the solution was diluted with 300 ml of DMSO.

Then 1.8535 g 2'-O-succinyl-paclitaxel derivative, dissolved in 6 ml of pyridine, was added drop by drop in the activated hyaluronic acid and the reaction of epirizole was carried out at 35°C for 48 hours, then 12 ml of a saturated solution of NaBr was added. Thus the resulting mixture was stirred for another one hour, in order to allow the replacement of ions TION of sodium. Then the conjugate was injected into the sediment by adding ethanol and purified by washing with a mixture of ethanol/water 90:10 to remove salts and unbound 2'-O-succinyl-paclitaxel.

Example 19

Preparation of ether derivative of hyaluronic acid with paclitaxel by indirect esterification (firesale) hydroxyl groups through a separator containing functional group of the anhydride

0.6157 g of Di(N-succinimidyl) carbonate was dissolved in 20 ml acetone the sludge in the vessel with a round bottom under a stream of nitrogen. After complete dissolution, 0.5105 g of paclitaxel, pre-dissolved in 60 ml of acetonitrile, and 140 ml of pyridine was added. The reaction was allowed to flow while stirring at room temperature under a stream of nitrogen. After 24 hours the solvent was removed under vacuum in the rotavapor and the raw product was placed in dichloromethane. White precipitate was filtered, and the organic phase was washed five times with water weak acidity (pH about 4). After extraction the organic phase was supplemented with sodium sulfate to remove water, filtered and dried in a rotary evaporator.

The second step 2.1125 g HA-TBA was dissolved in 500 ml of anhydrous DMF, supplemented 0.0532 g L-aspartic anhydride-hydrochloride. The reaction was allowed to proceed for 24 hours at room temperature to achieve esterification of hydroxyl groups of hyaluronic acid.

Then 0.4120 g activated paclitaxel, prepared in advance, was dissolved in 50 ml of anhydrous DMF was slowly added to a derivative of hyaluronic acid. The reaction for 24 hours was carried out at room temperature in the presence of triethylamine. At the end of 12 ml of a saturated solution of NaBr was added. Thus obtained mixture was mixed for one hour in order to allow the replacement of ions TBA intothree the m Then the conjugate was injected into the sediment by adding ethanol, and purified by washing with a mixture of ethanol/water 90:10, to remove salts and unbound paclitaxel.

Example 20

Preparation of ether derivative of hyaluronic acid with paclitaxel by indirect esterification in the carboxylic acid via a separator containing a hydroxyl group HA+CDI att cooh

First derivative of paclitaxel-separator was synthesized as follows.

To a solution of 1.0254 g of paclitaxel in 50 ml of dichloromethane was added 0.2521 g R-toluensulfonyl chloride and 0.1583 g dimethylaminopyridine. The reaction of the activated hydroxyl took place over night at room temperature; then it was purified by several washings with water and extraction of the organic phase, after which it is concentrated in the rotavapor" for subsequent reaction with the separator. Thus it was dissolved in 100 ml of anhydrous DMF and added 0.1854 g of 4-hydroxybutyrate sodium. Reaction epirizole allowed to proceed at 35°C for 4 hours, then the product was injected into the sediment in water, washed several times and dried under vacuum.

After dehydration of the sodium sulfate solution was filtered and dried under vacuum.

In the second place 1.2589 hyaluronic acid TION b is lo dissolved in 200 ml of anhydrous DMSO and to the activation of hydroxyl groups of hyaluronic acid was added 0.1156 p 1,1'-carbonyldiimidazole (CDI); the reaction was carried out for 1 hour at 42°C, and then the solution was diluted with 600 ml of DMSO. At the end was added a pre-prepared p-toluensulfonyl-paclitaxel in excess to allow hydroxyl separator to react the formation of the ether.

The reaction was allowed to occur at 42°C overnight with stirring, and then 12 ml of a saturated solution of NaBr was added, and stirring is continued for one hour, in order to allow the replacement of ions TION of sodium. Then the conjugate was injected into the sediment by adding ethanol drop by drop and purified by washing with a mixture of ethanol/water 90:10 to remove salts and unbound paclitaxel.

As described, therefore, of the invention it is clear that these methods can be modified in various ways. Such modifications should not be construed as a deviation from the essence and purpose of the invention, and all such modifications that may seem obvious to the expert in this area, are within the scope of the following claims.

1. Texan, covalently linked hyaluronic acid or a derivative of hyaluronic acid, where the covalent bond is selected from the group consisting of:
(1) direct ester bonds between carboxyl groups of hyaluronic acid or its produced in the water and hydroxyl groups taxane,
(2) bond formed between the carboxyl groups of hyaluronic acid or its derivative and hydroxyl groups taxane by connecting the connection (spacer elements compounds), where it connects the connection forms an ester bond with hyaluronic acid or a derivative of hyaluronic acid,
(3) bond formed between the hydroxyl groups of hyaluronic acid or its derivative and hydroxyl groups taxane by connecting the connection where it connects the connection forms an ester bond with hyaluronic acid or a derivative of hyaluronic acid.

2. Texan according to claim 1, where taxon selected from paclitaxel and docetaxel.

3. Texan according to claim 1, where the specified taxonom is paclitaxel.

4. Texan according to claim 1, where the hyaluronic acid has a molecular weight between 400 and 3·10⁶Yes.

5. Texan according to claim 4, where the hyaluronic acid has a molecular weight of between 400 and 1·10⁶Yes.

6. Texan according to claim 4, where the hyaluronic acid has a molecular weight between 400 and 230000 Yes.

7. Texan according to claim 1, where hyaluronic acid is converted into a salt with an organic and/or inorganic bases.

8. Texan according to claim 1, where the derivative of hyaluronic acid selected from the group consisting of esters of hyaluronic acid with aliphatic alcohols, analiticheskogo cycloaliphatic, aromatic, cyclic and heterocyclic series, and these esters have a degree of esterification of 50% or less than 50%.

9. Texan according to claim 1, where the derivative of hyaluronic acid selected from the group consisting of amides of hyaluronic acid with aliphatic amines, analiticheskogo, cycloaliphatic, aromatic, cyclic and heterocyclic series, with these amides have a degree of amidation between 0.1 and 10%.

10. Texan according to claim 1, where the derivative of hyaluronic acid selected from the group consisting of O-sulfated derivatives of hyaluronic acid with a degree of sulfation to 4.

11. Texan according to claim 1, where the derivative of hyaluronic acid selected from the group consisting of inner esters of hyaluronic acid having a degree of esterification that is equal to or less than 15%.

12. Texan according to claim 1, where the derivative of hyaluronic acid selected from the group consisting of deacetylated hyaluronic acid, formed by deacetylation of link N-acetylglucosamine and having a degree of deacetylation between 0.1 and 30%.

13. Texan according to claim 1, where the derivative of hyaluronic acid selected from the group consisting of percarboxylic derivatives of hyaluronic acid obtained by oxidation of the primary hydroxyl level N-acetylglucosamine and having a degree of percarboxylic between 1 is 100%.

14. Texan according to claim 1, where the connecting connecting connecting Texan with hyaluronic acid or a derivative of hyaluronic acid selected from the group consisting of aliphatic or alifaticheskih chains, unbranched or branched, substituted by one or more groups selected from hydroxyl, carboxyl, acylchlorides, halogen, anhydrite.

15. Texan on 14, where the linking compound selected from the group consisting of carboxylic acids having from 2 to 18 carbon atoms in the aliphatic or analiticheskoi chain, substituted by bromine.

16. Texan on 14, where the linking compound selected from the group consisting of carboxylic acids having from 3 to 10 carbon atoms in the aliphatic or analiticheskoi chain, substituted by bromine.

17. Texan on clause 16, where the linking compound selected from 3-bromopropionic acid and 4-pamakani acid.

18. Taxon of claim 8, where hyaluronic acid etherification after the formation of covalent bonds with taxonom.

19. Texan according to claim 11, where hyaluronic acid etherification after the formation of covalent bonds with taxonom.

20. Texan according to claim 1, where the covalent bond is an ester bond between taxonom and connecting the connection.

21. Texan according to claim 1, where the percentage of binding between hyaluronic acid and taxonom is between 0.1 and 100%.

23. Texan according to claim 1, where hyaluronic acid or a derivative of hyaluronic acid enhances the antineoplastic effect of taxane.

24. Texan according to claim 11, where the inner ester of hyaluronic acid enhances the antineoplastic effect of taxane.

25. Texan according to claim 1, where hyaluronic acid enhances the antineoplastic effect of taxane.

26. Pharmaceutical composition for use in Oncology for the treatment of tumors, autoimmune diseases and restenosis, containing as active substance, at least Texan, covalently linked hyaluronic acid or a derivative of hyaluronic acid, as described in claims 1 to 25, in combination with pharmaceutically acceptable excipients or diluents.

27. The pharmaceutical composition according p for the introduction of oral, intravenous, arterial, intramuscular, subcutaneous, intraperitoneal or percutaneous or by direct injection into the site of the tumor.

28. The pharmaceutical composition according to item 27 for insertion through the mouth.

29. The pharmaceutical composition according to item 27, where hyaluronic acid or a derivative of hyaluronic acid is capable of releasing Texan in the introduction.

30. The pharmaceutical composition according to any one of p-29, optionally containing one who does several biologically or pharmacologically active substances.

31. The pharmaceutical composition according to item 30, where the specified biologically or pharmacologically active substance is selected from the group consisting of steroids, hormones, trophic factors, proteins, vitamins, nonsteroidal anti-inflammatory drugs, chemotherapeutic drugs, blockers of calcium, antibiotics, antiviral agents, interleukins and cytokines.

32. The pharmaceutical composition according to item 30, where the specified biologically or pharmacologically active substance is interferon.

33. Use taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid according to claims 1 to 25 for obtaining pharmaceutical compositions suitable for treatment of tumors.

34. Use p, where the treatment of tumors, includes chemotherapy for breast cancer, ovarian cancer and/or endometrial, melanoma, lung cancer, liver cancer, prostate and/or bladder, cancer of the stomach and/or intestines, leukemia, and Kaposi's sarcoma.

35. Use taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid according to claims 1 to 25 for obtaining pharmaceutical compositions applicable for the treatment of autoimmune pathologies.

36. Use p where these autoimmune diseases selected from the group consisting of rawmat odnogo arthritis, the Hashimoto Hashimoto's disease, systemic lupus erythematosus and autoimmune glomerulonephritis.

37. Use taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid according to claims 1 to 25 for obtaining pharmaceutical compositions applicable for the treatment of restenosis.

38. Use taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid according to claims 1 to 25 for coating stents and medical devices.

39. Stents and medical devices covered by taxonom, covalently linked hyaluronic acid or a derivative of hyaluronic acid according to claims 1 to 25.

40. The method of obtaining taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid class (1) according to claim 1, comprising the following stages:
A) activation of the hydroxyl group taxane or, respectively, the carboxyl group of hyaluronic acid or a derivative of hyaluronic acid with the aid of an activating agent;
B) the addition of hyaluronic acid or a derivative of hyaluronic acid or, respectively, taxane dissolved in a suitable solvent;
(C) optional purification of the thus obtained product.

41. The method of obtaining taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid class (1) according to claim 1, VK is uchumi the following stages:
A') obtaining bromide or tosilata taxane;
In') performing a nucleophilic substitution of the bromide or tosilata taxane obtained in stage A'), a carboxyl group of hyaluronic acid or a derivative of hyaluronic acid;
With') optional purification of the obtained product.

42. The method of obtaining taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid class (3) according to claim 1, comprising the following stages:
L) activating the carboxyl group connecting the connection may previously associated with taxonom;
M) adding hyaluronic acid or a derivative of hyaluronic acid;
N) optional purification of the thus obtained product and the reaction taxonom, if it was not previously associated with connecting the connection.

43. The method of obtaining taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid class (3) according to claim 1, where connecting the connection has at least a carboxyl group and is associated with a hydroxyl group of hyaluronic acid or a derivative of hyaluronic acid ester bond, which includes the following stages:
L') substitution of the hydroxyl group of hyaluronic acid or a derivative of hyaluronic acid by group Totila or bromide;
M') adding connecting connect the tion, possible pre-associated with taxonom;
N') optional purification of the thus obtained product and carrying out the reaction with taxonom, if he previously had not been associated with connecting the connection.

44. The method of obtaining taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid class (3) according to claim 1 through a linking compound having at least one anhydrous group and linking the hydroxyl group of hyaluronic acid or a derivative of hyaluronic acid ester bond, which includes the following stages:
L) add a linking compound to a solution containing hyaluronic acid or a derivative of hyaluronic acid;
M) optional purification of the thus obtained product;
N") conducting the reaction of the product obtained at stage L) or M) with taxonom.

45. The method of obtaining taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid class (3) according to claim 1, by connecting the connection having at least a hydroxyl group and linking the carboxyl group of hyaluronic acid or a derivative of hyaluronic acid through ester bonds, which includes the following stages:
a) adding an activating agent to a solution containing Gia the uronic acid or a derivative of hyaluronic acid;
b) adding connecting the connection may previously associated with taxonom, in the solution obtained in stage a);
c) optional purification of the thus obtained product and carrying out the reaction with taxonom, if he previously had not been associated with connecting the connection.

46. The method of obtaining taxane, covalently linked hyaluronic acid or a derivative of hyaluronic acid class (2) according to claim 1 through a linking compound having at least halogen, such as bromine, and linking the carboxyl group of hyaluronic acid or a derivative of hyaluronic acid through ester bonds, which includes the following stages:
a') adding connecting the connection may previously associated with taxonom, in a solution of hyaluronic acid or a derivative of hyaluronic acid;
b') an optional purification of the thus obtained product, and carrying out reaction with taxonom, if he preferred was not associated with connecting the connection.