CZ308931B6 - Supramolecular complex of platinum metallo-complex oxycellulose matrix with gradual release of platinum metal complex and its use - Google Patents

Abstract

The solution is a supramolecular complex of an oxycellulose matrix with a platinum metal complex with a gradual release of a platinum metal complex, which contains 5 to 30 % by weight platinum metal-complex, which is preferably cisplatin, non-covalently bound to an oxycellulose matrix which is formed by oxidized forms of cellulose with high therapeutic effects in topical intrabody applications to residual tumour tissue after surgical removal.

Description

Supramolecular complex of oxycellulose matrix with platinum metal complex with gradual release of platinum metal complex and its use

Field of technology

The present invention relates to novel supramolecular complexes of oxycellulose matrices with antitumor drugs based on platinum metallo-complexes and their postoperative intrabody application for the elimination of residual tumor cells / tissues after surgery. The presented methodology eliminates the risk of both metastases and re-growth of the tumor at the original site.

Prior art

Complications of surgical removal of solid tumors are both metastases (late diagnosis) and incomplete tumor removal. Both lead to subsequent complications and often end in the patient's death. The usual follow-up therapy includes radiation, photodynamic therapy, cytostatic treatment using other drugs.

Metastases, the spread of diseased cells from the tumor to the whole body, can be estimated to account for up to 90% of all cancer-related deaths. The goal is to find a way to reduce or prevent their spread throughout the body. New tumor deposits in other parts of the body (often the lungs, liver or bones, for example) usually have two unpleasant properties - they tend to be more aggressive (grow faster) and are harder to treat.

Depending on the type and stage of the cancer, we generally treat surgically, with chemotherapy or radiotherapy. One or a combination of the above treatment methods is considered for each patient. Chemotherapy is used as a systemic treatment that affects tumor cells throughout the body and can be applied after surgical removal of tumor tissue to prevent metastasis. This treatment is called adjunctive or adjuvant. We also use chemotherapy to alleviate the symptoms of the disease if the tumor cannot be surgically removed and to control its growth. Chemotherapy is administered in cycles, between individual series followed by different lengths of recovery.

A number of effective anticancer drugs have recently been developed. However, a very serious problem with their clinical application are very strong adverse side effects. One way to solve the problem is to formulate them using suitable excipients. This strategy could potentially improve the therapeutic properties of many of the anticancer drugs used. Systems for the targeted delivery of drugs with controlled release and drug action represent very promising substances for current pharmacotherapy. The goal of their application is to achieve the right amount of drug, at the right time and in the right place. They have the ability to bring the drug unchanged to its site of action with reduced distribution to unwanted parts of the body. Targeting the drug to the site of interaction reduces its administered dose and thus undesirable side effects. This makes it possible to administer highly effective drugs, but with very low solubility, such as taxol derivatives (eg docetaxel) and thus limited applicability, or with high side effects such as anthracyclines (eg doxorubicin) or platinum metal complexes. Docetaxel (L01CD02) and doxorubucin (L01DB01) are among the most intensively studied drugs for these applications. Their appropriate formulation would, inter alia, allow their more gentle administration in the case of docetaxel and cisplatin and a substantial reduction in the cardiotoxicity of doxorubicin.

Currently, polysaccharide polymers, such as oxycellulose, are among the most promising excipients for the formulation of anticancer drugs. The active substance is present in them as a dispersion in a polymeric matrix, or better as a complexed drug in a polymeric matrix. Their application leads to easier control of the level of the drug in the blood and thus its more effective dosing compared to the administration of the free drug. Based on polysaccharide polymers, including oxycellulose, a

- 1 CZ 308931 B6 studied a number of promising excipients, including multifunctional (sometimes called smart excipients). A potential advantage of appropriately applied polymeric excipients, e.g., polysaccharides, could also be easier to control drug entry into the target tumor cell.

Oxidized cellulose (Oxy cellulose, Okcel) is known as a highly promising substance for various pharmaceutical and medical applications. Oxycellulose shows high biocompatibility and biodegradability under physiological conditions leading to non-toxic products. Oxycellulose has long been studied and used in medicine to promote blood clotting, as a prevention for tissue adhesion after surgery, to improve the healing of chronic wounds, to modulate the immune system and for its antitumor effects.

(Jan Gajdziok, David Vetchý Oxidized Cellulose and its Pharmatetical and Medical Application Cellulose and Cellulose Derivarives Syntheses, Modification and Applications Nova Science Publishers, IncNew York 2015; Schmidt, R, Bogan, D., Moore, J. (2001). Use of oxidized cellulose as free radical scavenger.European Patent EP 1153618; Watt, PW, Harvey, W., Lorimer, E., Wiseman, D. (1998) .Use of oxidized cellulose and complexes thereoffor chronic wound healing.World Patent WO 9800180. Jelínková, M., Briestenský, J., Santar, 1., Říhová, B. (2002) In vitro and in vivo immunomodulatory effects of microdispersed oxidized cellulose Int. Immunopharmacol. 2 (10), 1429-1441; , 1., Kiss, F., Briestenský, J. (1998) Cellulose derivatives, World Patent WO 9833822, Dimitrijevich, SD, Tatarko, M., Gracy, RW (1990) Biodegradation of oxidized regenerated, cellulose, Carbohydr. Res. 195 (2), 247-256; Dimitrijevich, SD, Tatarko, M., Gracy, RW, Wise, GE, Oakford, LX (1990) . In vivo degradation of oxidized, regenerated cellulose. Carbohydr. Res. 198 (2), 331-341; Banker, S. G., Kumar, V. (1995). Microfibrillated oxycellulose. United States Patent US 5405953; Otterlei, M., Espvik, T., Skjak-Braek, G., Smidsrod, O. (1992) Diequatorially bound beta 1-4polyuronates and use of same for cytokine stimulation. United States Patent US5169840; Doyle P. J., Saferstein L., Lorimer E., WattP. W. (1998). Oxidized Oligosaccharides. World Patent WO 9800446; Bajerová, M., Krejčová, K, Rabišková, M., Gajdziok, J., Masteiková, R. (2009). Oxycellulose - significant characteristics in relation to its pharmaceutical and medical applications. Adv. Polym. Tech. 28 (3), 199-208; Banker, G. S., Kumar, V. (1998) Oxidized cellulose. United States Patent US 5780618), (Leidgens V, Seliger C, JachnikB, Welz T, LeukelP, Vollmann-Zwerenz 1, Bogdahn U Kreutz M, Grauer OM, Hau P Ibuprofen and Diclofenac Restrict Migration and Proliferation of Human Glioma Cells by Distinct Molecular Mechanisms. PLoS One. 2015 Oct 20; 10 (10): e0140613; Chai AC, Robinson AL, Chai KX, Chen IM. Ibuprofen regulates the expression and function of membrane-associated serine proteases prostasin and matriptase BMC Cancer. 2015 Dec 29; 15: 1025 doi: 10.1186 / sl2885-015-2039-6; da Silveira EE 1, Chassot JM, Teixeira FC, Azambuja JH, Debom G, Beira FT, Del Pino FA, Lourenco A, Horn AP, Cruz L, Spanevello RM, Braganhol E. Ketoprofen-loaded polymeric nanocapsules selectively inhibit cancer cell growth in vitro and in preclinical model of glioblastoma multiforme Invest New Drugs. 2013 Dec; 31 (6): 1424-35. Doi: 10.1007 / sl0637-013-0016- y. Epub 2013 Sep 27; Damnjanovic 1, Najman S, Stojanovic S, Stojanovic D, Veljkovic A, Kocic H, Langerholc T, Damnjanovic Z, Pesic S. Crosstalk between possible cytostatic and antiinflammatory potential of ketoprofen in the treatment of culture of colon and cervix cancer cell lines Bratisl Lek Listy. 2015; 116 (4): 227-32), Kněžková, Barbora. Modern anticancer drugs. 2008. PhD Thesis. Masaryk University, Faculty of Science.

Applications of oxycellulose derivatives for targeted drug delivery have been described in their combination with magnetic nanoparticles (Sivakumar, Balasubramanian; Aswathy, Ravindran Girija; Nagaoka, Yutaka; Suzuki, Masashi; Fukuda, Takahiro; Yoshida, Yasuhiko; Maekawa, Toru; Sakthikumarif, Das Carboxymethyl Cellulose-Based Magnetic Nanovector as a Theragnostic System for Folate Receptor Targeted Chemotherapy, Imaging, and Hyperthermia against Cancer Langmuir (2013), 29 (10), 3453-3466), or a combination thereof with chitosan and magnetic nanoparticles (Synthesis, characterization and in vitro assessment of the magnetic chitosan-carboxymethylcellulose biocomposite interactions with the prokaryotic and eukaryotic cells Grumezescu, Alexandru Mihai, Andronescu, Ecaterina, Ficai, Anton, Bleotu,

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Coralia; Mihaiescu, Dan Eduard; Chifiriuc, Mariana Carmen International Journal of Pharmaceutics (Amsterdam, Netherlands) (2012), 436 (1-2), 771-777), or a combination of oxycellulose with nanoparticles, or its conjugates with drugs (Soo, B. Chia; Ting, Kang ; Wu, Ben; Zhang, Kevin Transcutaneous multimodal delivery systems comprising dissolvable microneedles PCTInt. Appl. (2012), WO 2012103257 A2 20120802), or a combination thereof with chitosan (Viieira, DB; Kim, V. CFM; Carmona-Ribeiro, AMEdited By.Laudon, Matthew; Romanowicz, Bart Polymer-based delivery vehicle for cisplatin Nanotech Conference & Expo 2011: An Interdisciplinary Integrative Forum on Nanotechnology, Biotechnology and Microtechnology, Boston, MA, United States, June 13 -16, 2011 (2011), 3, 382-385), or the application of an oxycellulose hydrogel for drug release for the treatment of the urinary tract (Konorty, Marina; Hakim, Gil Mucoadhesive thermoreversible topical hydrogels and method for treating internal cavities U.S. Pat. Appl. Publ. . (2014), US20 140105884 Al 20140417)

WO 2007147052 discloses a pharmaceutical composition for topical use formed by a film of cellulose or derivatives thereof, comprising a biologically active agent in a supersaturated state, which is, e.g. , volatile solvent; The composition is for antifugal treatment, for the treatment or prevention of varicella zoster infection, HSV-I infection or HSV-2 infection, or for the prevention of onychomycosis.

The invention CN 1843332 A describes prolonged-release antitumor injections comprising angiogenesis inhibitors and cytotoxic agents, which consist of microspheres containing antitumor active substances of 0.5 to 60%, sustained-release excipients of 40 to 99%, such as polylactic acid; a copolymer of polyglycolic acid and glycolic acid; polifeprosan; ethylene vinyl acetate; di-fatty acid and sebacic acid copolymer; poly (erucic acid dimer - sebacic acid) copolymer; poles (fumaric acid - copolymer of sebacic acid). Suspending agents 0.0 to 30%: sodium salts of carboxymethylcellulose, iodine glycerin, dimethicone, propylene glycol, carbomer, mannitol, sorbitol, surfactants, one of Tween 20, Tween 40 and Tween 80, or combinations thereof, the inhibitor is selected from gefitinib , erlotinib, lapatinib, carboxy-amino-triazole, thalidomide, angiostatin, endostatin, endostatin, imatinib mesylate, kanamycin, erlotinib, sorafenib, imatinib. The cytotoxic agent was selected from Nida platinum, melphalan, cyclophosphamide, 4H vinorelbine peroxide, tamoxifen, methotrexate, doxorubicin, epirubicin, actinomycin D, estramustine, semustine or ranimustine.

EP 1491188A1 discloses a pharmaceutical composition for the prevention or treatment of skin disorders containing at least 0.1% of an active ingredient consisting of VPA and / or a salt thereof, which also contains a chemotherapeutic drug which is 5-fluorouracil and further comprises carriers in the form of creams, ointments, pastes and gels. The product is to be used for the treatment of skin tumors, pre-neoplastic skin diseases, inflammation of the skin and / or mucous membranes and photoaging (premature aging of the skin due to sunlight).

The protrusions of the tumor into the surrounding tissue and its complex topology in practice make it almost impossible to surgically remove the entire tumor. Subsequent chemotherapy or systemic drug delivery is often ineffective because the drug concentration at the site of the original tumor is too low.

The essence of the invention

Due to the ineffective postoperative treatment of residual tumor tissue, new methods of applying a cytotoxic drug, based on platinum metallo-complexes, non-covalently bound to an oxycellulose matrix, have been developed topically directly into the wound after tumor removal.

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The invention relates to a new method for the topical intrabody application of stable supramolecular complexes of oxycellulose matrices with platinum metallo-complexes - cytostatics, such as preferably Cisplatin, Carboplatin or Oxaliplatin.

A composition has been developed for postoperative intrabody topical applications consisting of modified oxycellulose and platinum metallo-complexes to eliminate tumor tissue / cells after surgery. The composition allows a controlled gradual release of the drug in the tumor or its residues and thus leads to the elimination of residual tumor tissue and prevents its rapid growth after conventional and laparoscopic surgery.

The composition consists of supra-molecular complexes of oxycellulose matrices, preferably in the form of HD, HL and CaL, with platinum metal complexes, preferably with cisplatin (cis-Pt).

Oxycellulose matrix CaL is a powdered form of calcium salt of oxidized cellulose, is well tolerated and rapidly absorbed by the body, HD is a knitted version of absorbable hemostatic material of oxidized cellulose in textile form and HL is a linteric acid form of oxidized cellulose in powder form which is insoluble in water. In the human body, oxycellulose matrices are absorbed without any residues, are biodegradable, form non-toxic products during degradation and are fully biocompatible. Due to the low pH, it has an antibacterial effect. They help with tissue regeneration, thus also with an increase in therapeutic effects.

Platinum complexes are one of the most effective and most commonly used cytostatics in the treatment of many cancers, especially testicular, ovarian, head, neck, bladder and small cell lung cancers. It is a simple inorganic compound - [czsPtfNHshCh], ie cisdiamino-dichloro platinum complex, which belongs to the so-called alkylating cytostatics. Chloride ligands can be replaced by carboxylates (carboplatin, oxaliplatin), the nature of the ligand affects lipophilicity and drug distribution in the body (iproplatin). The main mechanism of the antitumor effect of cisplatin lies in its interaction with DNA and in the formation of covalent bonds between the cytostatic and purine bases, most often with guanine. The resulting covalent bonds between the chains prevent their separation during replication and at the same time inhibit transcription. Thus, cisplatin induces apoptosis. Although cisplatin is one of the most commonly used cytostatics, its use is limited due to its high toxicity.

Carboplatin is less toxic than cisplatin. The mechanism of action is the same as for cisplatin, but the activation of the molecule is slower. Compared to cisplatin, it has milder side effects, but contributes to bone marrow damage and is highly haematotoxic.

Oxaliplatin has low nephrotoxicity, but causes frequent nausea, diarrhea and vomiting, and causes peripheral neuropathy of sensitive nerves in treated patients.

In the case of platinum metal complexes, the process for preparing the supramolecular complex is based on activating the absorbency of the oxycellulose matrix by immersion in water and subsequent addition of a solvent solution containing the drug. The solvent is selected from the group consisting of methanol, ethanol, isopropanol, 2-methoxyethanol, N, N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran, acetonitrile, acetone or a mixture thereof, and as additional water. The mixing is carried out in a controlled manner so that the drug contained in the solvent remains dissolved in the resulting mixture of water or with the organic solvent. Subsequently, the aqueous organic solvent systems containing the oxycellulose matrix and the drug are shaken, preferably for 24 hours. The modified matrix, i.e. with a non-covalently bound drug, is then isolated by filtration, then washed with the solvent system used, and finally vacuum dried. The entire preparation process is controlled so that the temperature of the oxycellulose matrices or their solutions does not exceed 20 ° C.

The prepared compositions consist of supramolecular structures of oxycellulose matrices and platinum metal complexes, wherein the platinum metal complexes are present in the matrix in an amount of 5 to

-4 CZ 308931 B6% by weight. Structural analyzes have shown that platinum metallo-complexes are indeed an integral part of the oxycellulose matrix and are therefore not a mere mechanical mixture but a supramolecular structure. High stability of the formed structures was also verified, which proves that the release of cytotoxic platinum metallo-complexes does not occur prematurely into the surrounding healthy tissue. Furthermore, the increased permeability through simulated biological membranes compared to the permeability of platinum metallo-complexes alone was verified, which is attributed to the supra-molecular structure and increases the efficiency of drug transport directly into the tumor tissue.

Two basic drug release mechanisms are known for cellulose matrices. In an aqueous environment, the matrix is first moistened with water, releasing an initial concentration of dissolved drug from the surface of the matrix. Thanks to this gel layer, water can release the drug from the matrix without breaking it (preferably with hydrophilic drugs). In the subsequent phase, the surface of the matrix is hydrated, the mobility of the polymer chains increases and a swollen gel is formed. In the next phase, the polymer chains are loosened and released into the environment. Another gel layer is formed on the new surface of the matrix and the whole process is repeated until it disappears (preferred for hydrophobic drugs). In the case of sparingly soluble drugs, both mechanisms are involved in their release from the matrix. The stability of the used matrix has a great influence on the given process. In the case of more stable matrices, drug elution will be preferred. In contrast, in the case of less stable matrices, most of the bound drug is released already during the disintegration of the matrix itself. Thus, there is a gradual release of the drug from the modified oxycellulose only at the site after the operation of the tumor, which rapidly reduces the side effects of the cytostatics used.

It has been shown that complex tests of modified cellulose matrices stable for at least 6 months.

The advantage of intrabody topical application was realized only on the basis of a number of experiments, where preclinical tests were performed on mouse nuu nu models after removal of breast and pancreatic cancer tumor tissue and subsequent application of the composition both orally, in daily doses and once topically directly to the site. after the operated tumor in the same dose. Comparison of application in the form of oral and intrabody topical postoperative application showed the advantage of topical application, because oral administration was associated with mortality 9 to 33% higher, and especially the observed increase in tumor size was 34 to 70% higher.

It has been found that the supramolecular complex of oxycelluloses with cisplatin when administered intracellularly has the ability to inhibit the growth of breast and pancreatic cancer by up to 29% compared to surgery alone and 56% compared to surgery followed by cisplatin alone. Also, the mortality of experimental mice when the body was applied topically to the body was 18 to 37% lower compared to cisplatin alone.

Only on the basis of these results were it concluded that the use of only intrabody topical application of platinum metal complexes and only in the form of supramolecular oxycellulose structures, which have proven to be the most gentle and effective.

Explanation of drawings

Giant. 1 Table of overview of cisplatin loading relative to oxycellulose loading, percentage of drug in oxycellulose and loading of drug into oxycellulose, when EtOH / water mixture (1: 1, V / V) was used as solvent

Giant. 2 Table of overview of cisplatin loading relative to oxycellulose loading, percentage of drug in oxycellulose and loading of drug to oxycellulose, where water was used as solvent.

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Giant. 3 Table of overview of cisplatin loading relative to oxycellulose loading, percentage of drug in oxycellulose and loading of drug into oxycellulose, where acetone / water (1: 1, V / V) was used as solvent.

Giant. 4 Comparison of Raman spectra for modified oxycellulose HL with cisplatin ("CisPt-water", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HL").

Giant. 5 Comparison of Raman spectra for modified oxycellulose HL with cisplatin ("CisPt-water / EtOH", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HL").

Giant. 6 Comparison of Raman spectra for modified oxycellulose HL with cisplatin ("CisPt-water / Acet.", Content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HL").

Giant. 7 Comparison of Raman spectra for modified oxycellulose HL with cisplatin ("CisPt-water / THF", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HL").

Giant. 8 Comparison of Raman spectra for modified oxycellulose HD with cisplatin ("CisPt-water", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HD").

Giant. 9 Comparison of Raman spectra for modified oxycellulose HD with cisplatin ("CisPt-water / EtOH", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HD").

Giant. 10 Comparison of Raman spectra for modified oxycellulose HD with cisplatin ("CisPt-water / Acet.", Content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HD").

Giant. 11 Comparison of Raman spectra for modified oxycellulose HD with cisplatin ("CisPt-water / THF", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("HD").

Giant. 12 Comparison of Raman spectra for modified CaL oxycellulose with cisplatin ("CisPt-water", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("CaL").

Giant. 13 Comparison of Raman spectra for modified CaL oxycellulose with cisplatin ("CisPt-water / EtOH", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("CaL").

Giant. 14 Comparison of Raman spectra for modified CaL oxycellulose with cisplatin ("CisPt-water / Acet.", Content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("CaL").

Giant. 15 Comparison of Raman spectra for modified CaL oxycellulose with cisplatin ("CisPt-water / THF", content 5 wt.% Cisplatin), pure drug ("cisplatin") and unmodified oxycellulose ("CaL").

Giant. 16 Intracellular topical application of supramolecular complexes of oxycellulose matrices with cisplatin after surgical treatment of nu-nu mice with PATU human pancreatic carcinoma.

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Giant. 17 Oral administration of supramolecular complexes of oxycellulose matrices with cisplatin after surgical treatment of nu-nu mice with PATU human pancreatic cancer.

Giant. 18 Intracellular topical application of supramolecular complexes of oxycellulose matrices with cisplatin after surgical treatment of nu-nu mice with BT-474 human breast cancer.

Giant. 19 Oral administration of supramolecular complexes of oxycellulose matrices with cisplatin after surgical treatment of nu-nu mice with BT-474 human breast cancer and its comparison with cisplatin alone and oxycellulose matrices.

Giant. 20 In vitro study of tissue cell tumor cell mortality by cisplatin alone compared to their oxycellulose complex.

Giant. 21 Results of elemental analysis of modified cellulose (CaL, HL and HD) with cisplatin (5 wt.%) For different solvent systems.

Examples of embodiments of the invention

Example 1

Preparation and characterization of supra-molecular complexes of oxycellulose matrices (HD, HL and CaL) with cisplatin

Example 1A

Using solvent ethanol / water (1: 1, V / V) mg of cisplatin was dissolved in ethanol / water (1: 1, V / V; 5 ml) and added to oxycellulose (HL, HD or CaL) (100 mg) . After shaking for 24 hours at 20 ° C, the modified oxycellulose was filtered off, washed with ethanol / water (1: 1, V / V; 3 ml), water (3 ml) and then dried under high vacuum at 20 ° C. Elemental analysis determined that approximately 5% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 1.

mg of cisplatin was dissolved in ethanol / water (1: 1, V / V; 5 ml) and added to oxycellulose (HL, HD or CaL) (100 mg). After shaking for 24 hours at 20 ° C, the modified oxycellulose was filtered off, washed with ethanol / water (1: 1, V / V; 3 ml), water (3 ml) and then dried under high vacuum at 20 ° C. Elemental analysis determined that approximately 9.5% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 1.

mg of cisplatin was dissolved in ethanol / water (1: 1, V / V; 5 ml) and added to oxycellulose (HL, HD or CaL) (100 mg). After shaking at 19 ° C for 36 hours, the modified oxycellulose was filtered off, washed with ethanol / water (1: 1, V / V; 3 ml), water (3 ml) and then dried under high vacuum at 19 ° C. Elemental analysis determined that approximately 28% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 1.

The Raman spectra of the modified oxycelluloses thus prepared were measured and compared with those of pure cisplatin and unmodified oxycellulose. By comparing the position of the peaks and their ratio of the intensities of the modified oxycelluloses, cisplatin itself and the unmodified oxycelluloses, it can be concluded that the active substance cisplatin is not bound by purely physical absorption but forms an inclusion complex with the matrix.

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During the disintegration of the cellulose matrices, the individual fibers are released into the solution. The phenomenon is manifested by a change in the absorption spectrum compared to the spectrum measured at time to. If no significant changes in contrast are observed at a given time, the matrix can be considered stable at that time. The stability of the tested matrices is large enough to apply both mechanisms of drug release. Complete release of the drug in the body can be expected within one month after application of oxycellulose to the body.

Example 1B

Using a solvent water mg cisplatin was dissolved in water 5 ml and added to oxycellulose (HL, HD or CaL) (100 mg). After shaking for 24 hours at 20 ° C, the modified oxycellulose was filtered off, washed with water (6 ml) and then dried under high vacuum at 20 ° C. Elemental analysis determined that approximately 4% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 2.

mg of cisplatin was dissolved in 5 ml of water and added to oxycellulose (HL, HD or CaL) (100 mg). After shaking for 24 hours at 20 ° C, the modified oxycellulose was filtered off, washed with water (6 ml) and then dried under high vacuum at 20 ° C. Elemental analysis determined that approximately 8.5% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 2.

mg of cisplatin was dissolved in 5 ml of water and added to oxycellulose (HL, HD or CaL) (100 mg). After shaking at 19 ° C for 36 hours, the modified oxycellulose was filtered off, washed with water (6 ml) and then dried under high vacuum at 19 ° C. Elemental analysis determined that approximately 26% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 2.

The Raman spectra of the modified oxycelluloses thus prepared were measured and compared with those of pure cisplatin and unmodified oxycellulose. By comparing the position of the peaks and their ratio of the intensities of the modified oxycelluloses, cisplatin itself and unmodified oxycelluloses, it can be concluded that the active substance cisplatin is not bound by purely physical absorption but forms an inclusion complex with the matrix.

Example 1C

Using the solvent acetone: water (1: 1, V / V) mg of cisplatin was dissolved in acetone / water (1: 1, V / V; 5 ml) and added to oxycellulose (HL, HD or CaL) (100 mg) . After shaking for 24 hours at 20 ° C, the modified oxycellulose was filtered off, washed with acetone / water (1: 1, V / V; 3 ml), water (3 ml) and then dried under high vacuum at 20 ° C. Elemental analysis determined that approximately 5% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 3.

mg of cisplatin was dissolved in acetone / water (1: 1, V / V; 5 ml) and added to oxycellulose (HL, HD or CaL) (100 mg). After shaking for 24 hours at 20 ° C, the modified oxycellulose was filtered off, washed with acetone / water (1: 1, V / V; 3 ml), water (3 ml) and then dried under high vacuum at 20 ° C. Elemental analysis determined that approximately 10% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 3.

mg of cisplatin was dissolved in acetone / water (1: 1, V / V; 5 ml) and added to oxycellulose (HL, HD or CaL) (100 mg). After shaking at 19 ° C for 36 hours, it was modified

The oxycellulose is filtered off, washed with acetone / water (1: 1, V / V; 3 ml), water (3 ml) and then dried under high vacuum at 19 ° C. Elemental analysis determined that approximately 30% of cisplatin was bound to oxycellulose with the exact weight concentrations of Figure 3.

The Raman spectra of the modified oxycelluloses thus prepared were measured and compared with those of pure cisplatin and unmodified oxycellulose. By comparing the position of the peaks and their ratio of the intensities of modified oxycelluloses, cisplatin alone and unmodified oxycelluloses in different solvents, it can be concluded that the active substance cisplatin is not bound by purely physical absorption but forms an inclusion complex with the matrix.

Example 2

Preparation of Nu-nu mice with human cancer and subsequent surgical treatment.

Naked immunodeficient mice lack T-lymphocytes due to the absence of thymus, so they cannot generate an immune response to foreign tissues. Therefore, xenotransplantation of human cancer cells is possible.

Cancer cell lines (human breast tumor - BT-474, pancreas - PATU) were created by transfection of cells. Breast and pancreatic cancer cells were maintained in T-medium supplemented with 5% fetal bovine serum. All cells were maintained at 5% CO 2 (V / V) at 37 ° C in a humidified atmosphere. All experiments were performed on cells in exponential growth phase.

Subcutaneous implantation of human cancer cells into the flanks of immunodeficient nude nu-nu mice (females, athymic, age 6 weeks, weight 18 to 20 g). Tumor cells (1x10 ⁷ ) were resuspended in 0.1 ml PBS and 0.1 ml Matrigel (BD Biosciences) and administered subcutaneously to the flank of mice using an insulin syringe and a size 27 needle.

Once the tumor mass reached a size of about 200 mm ³ (about 10 days after transplantation, corresponding to 0.1 to 0.2 g of tumor tissue), 95 to 97% surgical removal of the tumor and measurement of tumor size was started.

Example 3

Application of supramolecular complexes of oxycellulose matrices with cisplatin after surgical treatment of nu-nu mice with PATU human pancreatic cancer

Example 3 A

Intra-body topical application

Immunodeficient nude nu-nu mice, prepared and surgically treated according to Example 2, i.e. with the pancreatic tumor removed, were divided into 10 experiments of 6 mice each. Group A consisted of two experiments, it was used as a control without any further application. The other groups were administered topically, in powder form, to the site of the operated tumor. Group B received cisplatin alone in an amount of 0.5 mg, it also consisted of 2 experiments, group C received oxycellulose HL in experiment 1 and oxycellulose CaL in experiment 2, each in an amount of 4.5 mg. Group D6 was given the supramolecular complex of oxycellulose CaL with cisplatin in two experiments and in group D7 the supramolecular complex of oxycellulose HL with cisplatin was administered in two experiments, in an amount of 5 mg with 10% by weight of cisplatin, corresponding to cisplatin alone.

Subsequently, tumor size was monitored and measured 50 days after surgery.

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Inhibition of tumor regrowth in a drug-dependent manner was monitored. The influence of local intrabody topical application of supramolecular complexes of oxycellulose matrices with cytostatics, oxycellulose alone and cytostatics alone on tumor regrowth, resp. possible elimination of residual tumor tissue. A significant therapeutic effect was observed for topical intrabody application of oxycellulose complexes manifested by markedly limited tumor growth. This approach represents a significant benefit over control experiments in final tumor volume. Changes in tumor size were used to determine antitumor activity. It has been found that the supramolecular complex of oxycelluloses with cisplatin when administered intracellularly has the ability to inhibit cancer growth by up to 46% compared to surgery alone and to 75-80% compared to surgery followed by cisplatin alone. Also, the mortality of experimental mice after intracellular topical application of the complex was 18 to 35% lower compared to administration of cisplatin alone. The detailed results are presented in the table in Fig. 16.

Example 3 B

Oral administration

Immunodeficient nude nu-nu mice, prepared and surgically treated according to Example 2, i.e. with removal of the pancreatic tumor, were divided into five experiments of three mice each. Group A was used as a control without any further application. The other groups received the substances orally directly into the stomach. Group B received cisplatin alone in an amount of 0.5 mg, group C received oxycellulose HL in experiment 1 and oxycellulose CaL in experiment 2, each in an amount of 4.5 mg. Group D6 was administered supramolecular complex of oxycellulose CaL with cisplatin and in group D7 was administered supramolecular complex of oxycellulose HL with cisplatin, in an amount of 5 mg with 10% by weight, containing cisplatin, which corresponds to the amount of cisplatin alone.

The size of the tumors was monitored and measured 50 days after surgery.

Inhibition of regrowth by tumors was monitored mainly as a comparison with intrabody topical application.

The influence of oral and intrabody topical application of supramolecular complexes of oxycellulose matrices with cytostatics, oxycellulose alone and cytostatics alone on the regrowth of tumors, resp. possible elimination of residual tumor tissue. Changes in tumor size were used to determine antitumor activity. It has been found that the supramolecular complex of oxycelluloses with cisplatin when administered orally has a 77% inhibition of cancer growth compared to chemotherapy alone. The mortality of the complex when administered orally was relatively high, at 85 to 88%. Detailed results are presented in the table in Fig. 17.

Comparison of application in the form of oral and intrabody topical postoperative application showed the advantage of topical application, because oral administration was associated with mortality 20 to 33% higher, and especially the observed increase in tumor size was about 74 to 77% compared to control.

Example 4

Application of supramolecular complexes of oxycellulose matrices with cisplatin after chimeric treatment of nu-nu mice with BT-474 human breast cancer

Example 4 A

Intra-body topical application

Immunodeficient nude nu-nu mice, prepared and treated chemically according to Example 8, i.e. with removal of breast cancer, were divided into ten experiments of 6 mice each. Group A consisted of two experiments, it was used as a control without any further application. The other groups were substances

- 10EN 308931 B6 administered topically, in powder form to the site of the operated tumor. Group B received cisplatin alone in an amount of 0.5 mg, it also consisted of two experiments, group C received oxycellulose HL in experiment 1 and oxycellulose CaL in experiment 2, each in an amount of 4.5 mg. Group D6 was given the supramolecular complex of oxycellulose CaL with cisplatin in two experiments and in group D7 in two experiments the supramolecular complex of oxycellulose HL with cisplatin, in an amount of 5 mg with 10% by weight, containing cisplatin, corresponding to cisplatin alone.

Subsequently, tumor size was monitored and measured 50 days after surgery.

Inhibition of tumor regrowth in a drug-dependent manner was monitored. The influence of local intrabody topical application of supramolecular complexes of oxycellulose matrices with cytostatics, oxycellulose alone and cytostatics alone on tumor regrowth, resp. possible elimination of residual tumor tissue. A significant therapeutic effect was observed for topical intrabody application of oxycellulose complexes manifested by markedly limited tumor growth. This approach represents a significant benefit over control experiments in final tumor volume. Changes in tumor size were used to determine antitumor activity. It has been found that the supramolecular complex of oxycelluloses with cisplatin when administered intracellularly has the ability to inhibit cancer growth by up to 29% compared to surgery alone and to 56% compared to surgery followed by cisplatin alone. The mortality of experimental mice during intrabody topical application of the complex is in the range of 55 to 63%. Detailed results are presented in the table in Fig. 18.

Example 4 B

Oral administration

Immunodeficient nude nu-nu mice, prepared and surgically treated according to Example 8, i.e. with breast tumor removal, were divided into five experiments of three mice each. Group A was used as a control without any further application. The other groups received the substances orally directly into the stomach. Group B received cisplatin alone in an amount of 0.5 mg, group C received oxycellulose HL in experiment 1 and oxycellulose CaL in experiment 2, each in an amount of 4.5 mg. Group D6 was administered supramolecular complex of oxycellulose CaL with cisplatin and in group D7 was administered supramolecular complex of oxycellulose HL with cisplatin, in an amount of 5 mg with 10% by weight, containing cisplatin, which corresponds to the amount of cisplatin alone.

Tumor size was monitored and measured 50 days after surgery.

Inhibition of tumor regrowth was monitored primarily as a comparison to intrauterine topical application.

The influence of oral and intrabody topical application of supramolecular complexes of oxycellulose matrices with cytostatics, oxycellulose alone and cytostatics alone on tumor regrowth, respectively, was evaluated. possible elimination of residual tumor tissue. A significant therapeutic effect was observed for topical intrabody application of oxycellulose complexes manifested by markedly limited tumor growth. This approach represents a significant benefit over control experiments in final tumor volume. Changes in tumor size were used to determine antitumor activity. It has been found that the supramolecular complex of oxycelluloses with cisplatin when administered orally has the ability to inhibit cancer growth by only 34-45% compared to surgery alone, and oral administration of cisplatin in supramolecular complexes with oxycellulose has the ability to inhibit cancer growth by 46-60%. The mortality of the complex was relatively high, 72 to 75%. Detailed results are presented in the table in Fig. 19

Comparison of application in the form of oral and intrabody topical postoperative application showed the advantage of topical application, because oral application was associated with mortality by 11 to 20% higher, and especially the observed increase in tumor size was about 0 to 10% compared to topical application.

- 11 CZ 308931 B6

Example 5

Study of the effectiveness of therapy of oxycellulose matrix complexes with cytostatics on tissue cultures of tumor cells in vitro

Solid tumor cell lines were obtained from the ETCC. All cells were grown exponentially in RPMI 1640 medium with 10% fetal calf serum in 35 mm petri dishes. For experiments, cells were cultured in pure growth medium for 16 hours to reach the exponential growth phase. Then, 5 x 10 ⁵ cells were seeded in 1.8 to 2 cm wells and incubated for 16 hours in growth medium with supramolecular complex of oxycellulose CaL and cisplatin. After washing, cell death was observed with a DMB Leica IRB microscope with a DFC 480 camera using immersion X63 oil and a Leica cube N2.1 filter.

Cell mortality in tissue cultures was monitored after treatment with cisplatin alone in comparison with CaL oxycellulose complex with 10% by weight, cisplatin at 24 hours after application. The CaL oxycellulose complex with cisplatin was at a concentration of 0.1 pmol / ml of growth medium. Cell mortality was determined by sulforhodamine B (SRB) assay, alternatively by trypan blue exclusion staining, results expressed as% of cells killed from the total number of individual cell lines (as shown in Figure 20).

Claims (7)

PATENT CLAIMS A supramolecular complex of an oxycellulose matrix with a platinum metal complex with a gradual release of a platinum metal complex, characterized in that it contains 5 to 30% by weight of a platinum metal complex non-covalently bound to an oxycellulose matrix, the platinum metal complex being cislatin / carblatin, . The supramolecular complex of an oxycellulose matrix with a platinum metal complex with a sustained release of a platinum metal complex according to claim 1, characterized in that the oxycellulose matrix is oxidized cellulose. The supramolecular complex of an oxycellulose matrix with a platinum metal complex with a gradual release of a platinum metal complex according to claim 2, characterized in that the oxidized cellulose is in the linter acid form. The supramolecular complex of an oxycellulose matrix with a platinum metal complex with a gradual release of a platinum metal complex according to claim 2, characterized in that the oxidized cellulose is in the form of a calcium salt. The supramolecular complex of an oxycellulose matrix with a platinum metal complex with a gradual release of a platinum metal complex according to claim 2, characterized in that the oxidized cellulose is in a textile knitted form. Use of a supramolecular complex of an oxycellulose matrix with a platinum metal complex with a sustained release of a platinum metal complex according to claim 1, for the preparation of a medicament for the topical cytostatic treatment of tumor tissue. Use of a supramolecular complex of an oxycellulose matrix with a platinum metal complex with a gradual release of a platinum metal complex according to claim 6, wherein the topical cytostatic treatment of tumor tissue is postoperative intrabody topical application to residual tumor cells / tissue after surgery.