Method and composition for treating cancer

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of invention refers to medicine, namely to oncology, and can be used in treating cancer in a patient. The method involves administering at least one encapsulated chemotherapeutic agent, and at least one amphiphilic block copolymer in this patient. What is also presented is a composition, a kit for treating cancer in the patient and using the amphiphilic block copolymer.

EFFECT: group of inventions provides potentiating the encapsulated chemotherapeutic agent by stimulating the active chemotherapeutic agent release from liposomes by the use of the amphiphilic block copolymer, which is poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer, in the composition.

10 cl, 11 dwg, 3 tbl, 4 ex

 

This invention was made with support from the state grant No. 2R01 CA issued by the National Institute of Health. The government has certain rights to this invention.

The technical field to which the invention relates.

This invention generally relates to the treatment of cancer. More specifically, this invention describes compositions and methods for improved delivery of therapeutic substances for the treatment of cancer.

The level of technology

Doxil (DOXIL®) (liposomal doxorubicin HCl for injection) significantly reduces side effects caused by doxorubicin. Need to enter Doxil only once in 4 weeks intravenously also makes it convenient for patients. However, Doxil characterized by a lack of efficiency in comparison with doxorubicin. Therefore, the necessary methods to increase therapeutic activity of Doxil, while preserving its security.

Summary of the invention

In accordance with this invention, methods of inhibiting cancer. In a specific embodiment of the present invention the method includes administration to a patient, at least one encapsulated chemotherapeutic drug and at least one amphiphilic block copolymer. Encapsulated chemotherapeutic the cue drug and an amphiphilic block copolymer can be administered simultaneously and/or sequentially. In the private embodiment, the encapsulated chemotherapeutic drug may be injected first. In a specific embodiment, the present invention is encapsulated chemotherapeutic drug is doxorubicin. In a specific embodiment, the present invention amphiphilic block copolymer contains at least one block of ethylene oxide and at least one block of propylene oxide.

In another embodiment, the invention proposed a composition comprising at least one encapsulated chemotherapeutic drug, at least one amphiphilic block copolymer, and at least one pharmaceutical carrier. In addition, the claimed kits containing: a) a composition containing at least one encapsulated chemotherapeutic drug and at least one pharmaceutical composition; and b) a second composition comprising at least one amphiphilic block copolymer and at least one pharmaceutical composition.

Brief description of drawings

Fig.1A and 1B show the cell capture medicinal drug Doxil cells of ovarian cancer and breast cancer respectively. The average value of the fluorescence of doxorubicin was determined by flow cytometry. Cells were treated with:1) Maxilom within 2 hours, 2) Maxilom together with 0.1% R within 2 hours; 3) Maxilom within 24 hours; 4) first treated with 0.1% RH for 2 hours, washed three times with phosphate-saline buffer (FSB) and then incubated with Maxilom for 24 hours; 5) first processed by Maxilom for 24 hours, washed 3 times the FSB and then treated with 0.1% R within 2 hours. Data are presented as mean ± COC (standard error of the mean) (n=6), *p<0,05, N. C. means insignificant deviation. Values of p (the lowest value level of significance) were obtained using student test after logarithmic transformation of the data.

In Fig.2 shows nuclear accumulation of Doxil in ovarian cancer cells. Cells were treated with: 1) Docelem; 2) Maxilom together with 0.1% R within 2 hours; 3) first treated with 0.1% RH for 2 hours, washed three times FSB and then incubated with Maxilom within 24 hours; 4) first processed by Maxilom for 24 hours, washed 3 times the FSB and then treated with 0.1% R within 2 hours. After treatment, cells were washed 3 times the FSB, lysed using M-PER buffer and the fluorescence of doxorubicin was measured using Specramax M5 tablet reader and normalized to protein content. Data are presented as mean ± COC (n=6), *p<0,05, N. C. means insignificant deviation. The p values were obtained using the criterion t-test after logarithmic transformation of the data.

In Fig.3 shows a confocal micrograph localization of doxorubicin in ovarian cancer cells and breast cancer in the presence and in the absence of pluronic R. Fig.3A shows the capture Doxil cell lines A and A2780/DOX after 2 or 24 hours of incubation. pluronic R was tagged with a fluorescent label Atto 647. In Fig.3B shows the time series of microphotographs of sequential processing cells A 0.1% pluronic after 24-hour incubation with 200 μg/ml Doxil. In Fig.3B presents the capture Doxil/DOX cells A and A2780/DOX in various methods of treatment with 0.1% R. Extreme micrograph represent digital superposition of the two previous images and show co-localization (image represented 63-fold magnification).

In Fig.4 shows fluorescence spectra of free doxorubicin and liposomes of Doxil in the absence (Fig.4A) and the presence (Fig.4B) of 0.1% R.

In Fig.5 shows curves of the release of doxorubicin from liposomes of Doxil mediated HR at 37°C (•) pH 5.5; ( ○ ), pH 5.5 in the presence of 0.1% R; (▲) pH 7,4; (Δ) pH 7.4 in the presence of 0.1% R. Data are presented as mean ± COC (n=4).

Fig.6 illustrates the effect of pluronic R volume A xenografted tumors. Only Doxil (■); Doxil with the subsequent introduction of 0.02% pluronic R after 1 hour (○); Doxil with the subsequent introduction of 0.02% pluronic R h the rez 48 hours (▲); Doxil with the subsequent introduction of 0.02% pluronic R after 96 hours (▼). Treatment consisted of a single intravenous administration of 12 mg/kg Doxil 2 weeks after implantation of the tumor. Data are presented as mean ± COC (n=8), *p<0,05. The p values were obtained using two-factor analysis of variance (two-way ANOVA) by comparing the volumes of the tumors in the group Doxil with other groups.

In Fig.7 presents stained with hematoxilin and eosin tissue of the heart, kidneys, spleen and kidneys of animals of the 4 groups: control, Doxil, Doxil + R after 48 hours, Doxil + R through 96 hours.

In Fig.8 shows the effect R on the distribution of the drug in xenografted tumors A. One week after implantation of the tumor mice intravenously was administered 12 mg/kg Doxil. R (0,02%) was administered intravenously either 48 h or 96 h after medication. Animals killed and identified tumor in 1 or 6 hours later. In Fig.8A presents a fluorescent micrograph (×10) distribution of drugs (red) on sections of the tumors stained for CD31 (green) and cell nuclei (blue). In Fig.8B presents the analysis by high performance liquid chromatography (HPLC) concentrations of doxorubicin in tissue homogenates tumors presented on (A). Data are presented as mean ± SOD (n=5), *p<0.05, N. C - minor (student test for one sample). On Phi is.8B presents confocal micrograph (×10) distribution of drugs on sections of tumors.

In Fig.9 presents the analysis of concentration of drug in the blood of BALB/c mice by HPLC. Mice made a single intravenous injection of 12 mg/kg Doxil. R (0,02%, 100 μl/mouse) was injected intravenously 48 hours after medication, blood samples were collected every 12 hours after injection and analyzed by HPLC. Data are presented as mean ± SOD (n=5), *p<0.05 (student test for one sample).

In Fig.10 shows the effect pluronic copolymers on the release of doxorubicin from liposomes of Doxil. In Fig.10A and 10B - Doxil was dispersively or in acetate buffer (pH 5.5, Fig.10A) or the FSB (pH of 7.4, Fig.10B) in the presence or in the absence of R. The concentration of Doxil in the dispersion was 0.2 mg/ml Concentration pluronic in dispersion were 0.0001%, 0.001%, 0.02%, 0.1%, 0.5%. The release of doxorubicin from the dialysis bag (dashed line) shown for comparison. In Fig.10B and 10G Doxil was dispersively or in acetate buffer (pH 5.5, Fig.10B), or the FSB (pH of 7.4, Fig.10G) in the presence or in the absence 0.02% pluronic copolymers. Data are presented as mean ± SOD (n=6).

In Fig.11 shows the spectra of the emitted fluorescence tetramethylrhodamine In isothiocyanate (TRITC)-R in the absence (curve 1) or presence (curve 2) empty liposomes. The final concentration R the FSB (pH of 7.4) was 0.1%. Liposomes have the same lipid composition as liposomes of Doxil (3,19 mg/ml sodium with the N?-carbonitesetuplite glycol 2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG-2000), 9,58 mg/ml fully hydrogenated soy phosphatidylcholine 3,19 mg/ml cholesterol and approximately 2 mg/ml of ammonium sulfate, Encapsula NanoSciences Corp.Nashville, TN). The final concentration of lipids in the dispersion was 0,232 mg/ml Spectra emitted fluorescence was measured on a FLuorolog (Horiba Jobin Yvon Inc., NJ) with λvasb=550 nm with a spectral width of strip 2 nm. I0and I1correspond to the maxima of the fluorescence intensity of TRITC-R in the presence and in the absence of liposomes, respectively.

A detailed description of the invention

Liposomal drugs played an important role in the development of nano medicines first generation. Drugs such as Doxil, liposomal doxorubicin for the treatment of cancers such as ovarian cancer (Song et al., (2012) J. Liposome Res., 22:77-92; Schwendener, R. A. (2007) Adv. Exp. Med. Biol., 620:17-128; Maurer et al., (2001) Expert Opin. Biol. Ther., 1:923-947; Torchilin, V. P. (2005) Nat. Rev. Drug Discov., 4:145-160), have already reached the clinical stage and showed efficacy in the treatment of patients. After systemic injections of liposomes of Doxil pass through the leaky tumor vessels and linger there, which leads to increased accumulation of liposomal drugs in the tumor compared with low-molecular doxorubicin. This General phenomenon is often called the effect "Increased permeability and detention" (Maedaetal., (2001) JcontrolRelease, 74:47-61). However, the further the permeability of the liposomal drug is tion from vessels in remote areas of the tumour is limited(Kostarelos et al., (2004) Int. J. Cancer, 112:713-721; Chauhan et al., (2011) Annu. Rev. Chem. Biomol. Eng., 2:281-298)), in particular, in the case of fibroid tumors (Torchilin, V. P. (2005) Nat. Rev. Drug Discov., 4:145-160; Yuan et al., (1994) Cancer Res., 54:3352-3356). They form a viscoelastic gel-like extracellular matrix rich in fibronectin and collagen, which can be explained with poor survival (Kalluri et al., (2006) Nat. Rev. Cancer, 6:392-401; Bhowmick et al., (2004) Nature, 432:332-337). Therefore, to improve the distribution and efficacy of liposomal drugs, there are ways of increasing the permeability of the matrix, for example, using inhibitors of the synthesis of collagen, collagenase and hyaluronidase (Diop-Frimpongetal., (2011) Proc. Natl. Acad. Sci., 108:2909-2914; Eikenes et al., (2010) Anticancer Res., 30:359-368; Sugahara et al., (2010) Science 328:1031-1035; Jain et al., (2010) Nat. Rev. Clin. Oncol., 7:653-664). However, these methods lead to increased toxicity of the drug in normal tissues and/or increase the risk of tumor progression and metastasis (Diop-Frimpongetal., (2011) Proc. Natl. Acad. Sci., 108:2909-2914). Other studies have investigated the influence of physical fields, such as ultrasonic treatment, temperature (hyperthermia) or radiation exposure to high energy on the permeability of microvessels and/or microenvironment of tumors (Schroederetal., (2009) J. Control Release, 137:63-68; Kong et al., (2000) Cancer Res., 60:6950-6957; Hagtvet et al., (2011) Radiat. Oncol., 6:135; Harrington et al., (2000) Clin. CancerRes., 6:4939-4949; Khaibullinaetal., (2008) J. Nucl.Med., 49:295 to 302). But these methods are technically complex and of limited application on remote and diffuse is pwhash. Moreover, several studies indicate that cell-mediated capture of intact liposomes in the tumor is limited and that the drug is enclosed in the inner space of the liposomes remains inactive as long as the free in free form (Barenholzetal., (2012) J. Control Release, 160:117-34; Zamboni, W. C. (2008) Oncologist, 13:248-260; Zamboni, W. C. (2005) Clin. CancerRes., 11:8230-8234). Thus, it would be ideal to release low molecular weight drug from the liposomes at the right time and the right place when liposomal drug will accumulate in the tumor vessels. This will allow low molecular weight drug to diffuse from blood vessels in remote areas of the tumor and stimulate cytotoxic effect in cancer cells. Therefore there is an urgent need to develop ways to enhance the delivery of drugs into the tumor with subsequent efficient drug release directly into the tumor with minimal side effects (Kwonetal., (2012) J. Control Release, 164:p.108-114; Bae et al., (2011) J. Control Release, 153:198-205; Florence, A. T. (2012) J. Control Release, 161:399-402).

In the inventive solution presents a new simple and effective strategy to enhance the release of drugs from liposomal carriers in the tumor. This strategy includes the use of amphiphilic block copolymers (e.g. poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), PEO-PPO-PEO) to enhance drug delivery. E is and the strategy can be used with already approved liposomal drugs, such as Doxil.

As noted above, Doxil it Paglinawan liposomal dosage form low molecular weight drugs doxorubicin. In a specific embodiment, the present invention Doxil contains 2 mg/ml doxorubicin, 3,19 mg/ml of sodium salt of N-carbonitesetuplite glycol 2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG-2000), 9,58 mg/ml fully hydrogenated soy phosphatidylcholine and 3.19 mg/ml cholesterol. Doxil widely used in the clinic to treat various types of cancer. However, the effectiveness of Doxil limited.

In the invention it is shown that the introduction amphiphilic block copolymers containing blocks of polyoxyethylene and polyoxypropylene, together with liposomal doxorubicin causes a significant increase in the cytotoxicity of doxorubicin compared with when amphiphilic block copolymer is missing. The presented data show that the treatment of breast cancer cell lines MCF7, MCF7/MX and MCF7/ADR amphiphilic block copolymer and liposomal doxorubicin causes cytotoxicity. At the same time, 2-hour treatment only liposomal doxorubicin did not cause cytotoxicity in this concentration range. Omitting theoretical details, the increase in toxicity can be explained by a significantly large jaw liposomal doctor is Bizina cancer cells in the presence of, or after pre-treatment amphiphilic block copolymer.

It also shows that treatment of cell cultures amphiphilic block copolymer stimulates the release of doxorubicin from liposomes, for example, in the case of pre-treatment amphiphilic block copolymer, or when the amphiphilic block copolymer was added together with or after liposomal doxorubicin.

The effect of the amphiphilic block copolymer on the cell capture liposomal doxorubicin in breast cancer cells was also investigated. Treatment of breast cancer cells is one of liposomal doxorubicin led to the capture and containment of the drug in the intracellular vesicles. When cells were treated with liposomal doxorubicin in the presence of 0.1% of the amphiphilic block copolymer, nuclear localization of the drug was observed after 15 minutes and after 60 minutes, a large part of the drug was localized in the nucleus.

The present invention includes methods of increasing the efficiency of the encapsulated agent (for example a therapeutic drug or diagnostic drug) through a joint injection (e.g., before, together and/or after) at least one amphiphilic block copolymer. The present invention also includes methods of improving the delivery of the encapsulated agent (e.g., a therapeutic drug or diagnostic drug) in the nucleus of cells by compatible with the local administration (for example, before, together and/or after) at least one amphiphilic block copolymer. Therapeutic drug can be encapsulated in micelles or liposomes, particularly liposomes. In a specific embodiment of the present invention, therapeutic drug is active in the nucleus (i.e., therapeutic drug must be delivered into the nucleus to show therapeutic effect). In a specific embodiment of the present invention, therapeutic drug is a chemotherapeutic drug. In particular, the chemotherapeutic drug is a DNA-damaging drug (see below), in particular DNA intercalators, such as anthracyclin. In a specific embodiment of this invention, the chemotherapeutic drug is doxorubicin.

The encapsulated substance can be any biologically active drugs, both therapeutic and diagnostic. This substance can also be an experimental drug being tested as a potential therapeutic drug. Encapsulated substances include, without limitation, polypeptides, peptides, glycoproteins, nucleic acids, synthetic and natural drugs, peptide, polyene, macrocycles, glycosides, terpenes, terpenoids, aliphatic and aromatic substances of low molecular weight, washes the VA and their derivatives and salts. In a specific embodiment of the present invention the encapsulated substance is a low molecular weight substance.

Liposomal dosage forms have been developed for many anticancer drugs in recent years and are currently at different stages of clinical trials. Examples include, but are not limited to: liposomal doxorubicin (DOXIL, Caelyx, Myocet), liposomal daunorubicin (Daunoxome), Targeted liposomal cisplatin (SPI-077, Lobaplatin, LiPlaCis), liposomal encapsulated paclitaxel (LEP-ETU, PNU-93914), liposomal vincristine (OncoTCS), liposomal mitoxantrone (MEM) and others. Liposomal dosage forms of anticancer drugs often have improved toxicity profiles and the best antitumor activity, but it requires further research and development for more effective compositions and preparations. Combination therapy using liposomal dosage forms in combination with other anticancer drugs have shown promising results in various clinical trials (Hofheinz, etal., (2005) Anti-CancerDrugs, 16(7):691-707). In the present invention it is shown that amphiphilic block copolymers, in particular, triblock copolymers, such as poloxamer or pluronic consisting of polyoxyethylene-is polyoxypropylene-polyoxyethylene, can be used in different modes of combination therapy to enhance drug release in tumor: 1) preliminary introduction pluronic block copolymers followed by the introduction of liposomal dosage forms of anti-cancer drug, 2) the simultaneous introduction pluronic block copolymers and liposomal dosage forms of anti-cancer drug, and 3) treatment of liposomal dosage form anticancer drug with sequential introduction of pluronic. It is preferable mode, when the patient is first injected with liposomal dosage form anticancer drug with sequential introduction of the copolymer. The type of cancer to treat such a method can be selected on the basis of to treat what type of cancer primarily used liposomal dosage form of anti-cancer drug.

Due to the fact that the substance of any type can be delivered into the cell, or can be used in the compositions and methods of the present invention, further description of the invention, for simplicity, represented by the example of a therapeutic compound, in particular doxorubicin.

In a specific embodiment of this invention, the amphiphilic block copolymer is a copolymer containing at least one block of poly(oxyethylene) and less the th least one block of poly(oxypropylene). An example of amphiphilic block copolymers are block copolymers with the following formula:

,

where x, y, z, i, and j have values from about 2 to about 800, preferably from about 5 to about 200, more preferably from about 5 to about 80, and where for each R1and R2as shown in formulas IV and V, one is hydrogen and the other is methyl group. Any person with an average level of knowledge in this area will determine that the values x, y and z are typically statistical average and that the values of x and z often, but not necessarily, the same. Formula (I) to (III) are simplified due to the fact that the orientation isopropylene of Radislav in the block B is random. This random orientation is represented in formulas (IV) and (V). Such compounds poly(oksietilenom)-poly(oxypropylene) were described Santon (Am. Perfumer Cosmet. (1958) 72(4):54-58), Schmolka (Loc. Cit. (1967) 82(7):25-30), Schick, ed. Non-ionic Surfactants, Dekker, N. Y. 1967 pp 300-371). A number of such compounds are commercially available under such generic names as "lipolaser" ("lipoloxamers"), "Pluronic" (Pluronics®), "poloxamer"("poloxamers" and "synperonic" ("synperonics"). Polymers of the type pluronic part of the formula-a-b is often referred to as "converted" pluronic, "pluronic R" or "mericarol". In General, the block copolymers can be described from the point of view of the presence of hydrophilic "And" and hydrophobic "B" blocks. For example, the copolymer having a-b-But the formula is a triblock copolymer consisting of a hydrophilic unit connected to a hydrophobic unit connected with other hydrophilic block. "Polioksidony" polymer of formula (IV) available from BASF (BASF, Wyandotte, MI) under the trade name Tetronic® (Tetronic®). Order polyoxyethylenated and polyoxypropylene blocks specified in the formula (IV) can be reversed, giving Tetronic RT® (Tetronic RT®), which is also available from the company BASF (Cm. Schmolka, J. Am. Oil Soc., 59:110 (1979)).

Polyoxypropylene-polyoxyethylene block copolymers can also be obtained with the hydrophilic block comprising a random mixture of repeating units of ethylene oxide and propylene oxide. To maintain the hydrophilicity of the block number of ethylene oxide do dominant. Similarly, the hydrophobic block may be a mixture ethylenoxide and propylenoxide repeating units. Such block copolymers are available from BASF under the trade mark of Pluralit™ (Pluradot™). Unit block Poly(oksietilenom)-poly(oxypropylene comprising the first segment must not consist solely of ethylene oxide. Also not an is) to all segments of type b consisted solely of units of propylene oxide. Alternatively, in the simplest case, for example, at least one of the monomers of the segment And can be replaced by the side chain.

Created many pluronics, which correspond to the following formula:

Examples of poloxamers include, without limitation, pluronic L31, L35, F38, L42, L43, L44, L61, L62, L63, L64, P65, F68, L72, R75, F77, L81, R, R, F87, F88, L92, F98, L101, R, R, R, F108, L121, L122, L123, F127, 10R5, 10R8, 12R3, !7R1, 17R2, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25R5, 25R8, 31R1, 31R2 and 31R4. The name of pluronic includes alphabetic prefix and two - or three-digit number. Letter prefixes (L, R or F) indicate the physical state of each polymer (from the English. Liquid (liquid), paste (paste) or flakeable solid (flocculent solid)). Numeric code identifies the structural parameters of the block copolymer. The last digit of the code indicates the approximate weight of the contents of a block of ethylene oxide in the tens of percent by weight (for example, 80% by weight, if there is a figure 8, or 10% by weight, if figure 1). In the remainder of the first one or two digits encrypted molecular weight of the Central block of propylene oxide. To decipher the code, multiply the corresponding number 300 to get an approximate molecular weight in daltons (Da). Thus, the nomenclature of pluronic provided which allows a convenient method for evaluating characteristics of the block copolymer in the absence of relevant literature. For example, the code F127 will determine the block copolymer in the solid state with a block of propylene oxide 3600 Yes (12×300), and in which 70% of the weight falls on the ethylene oxide. The exact molecular characteristics of each block copolymer of pluronic can be obtained from the manufacturer.

In a specific embodiment of this invention, the amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer. In a specific embodiment, the present invention PEO-PPO-PEO triblock copolymer contains from about 15 to about 35, in particular from about 20 to about 30 monomers EO (ethylene oxide) at each end and from about 30 to about 50, in particular from about 35 to about 45 monomers ON (propylene oxide) in the Central unit. In one embodiment of the present invention the ratio of units of monomers PEO/PPO/PEO is 26/40/26. In a specific embodiment of this invention, the amphiphilic block copolymer is poloxamer 235.

Amphiphilic block copolymers can contain at 30% by weight, 40% by weight or at least 50% by weight of the CEA. The molecular weight of the PPO block of the amphiphilic block copolymer may be at least from about 120 or 1700 to about 3000, 3600 or 4200. In a specific embodiment of this invention, the amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with sod is the neigh of the CEA, at least 30% by weight and a molecular weight of PPO block from about 1200 to about 4200; the content of the CEA, at least 40% by weight and a molecular weight of PPO block from about 1700 to about 3600; or, more specifically, the content of the CEA, at least 50% by weight and a molecular weight of PPO block from about 1700 to about 3000.

It follows from the above that may be used more than one block copolymer. In other words, this may be a mixture or combination of block copolymers. For example, the mixture may contain at least one amphiphilic block copolymer and at least one second amphiphilic block copolymer. In a specific embodiment of the present invention (1) the first amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 30% by weight, and the second amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 70% by weight or less; (2) the first amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 40% by weight, and the second amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 60% by weight or less; (3) the first amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, what about the least 50% by weight, and the second amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 50% by weight or less;(4) the first amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 60% by weight, and the second amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 40% by weight or less; or (5) the first amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 70% by weight, and the second amphiphilic block copolymer is a PEO-PPO-PEO triblock copolymer with the content of the CEA, at least 30% by weight or less. In the case of mixtures of PEO-PPO-PEO block copolymers, independently from each other, can have the CEA unit with a molecular weight of from about 900 to about 4200, from about 1700 to about 3600 or, more specifically, from about 1700 to about 3000.

Medication

The invention includes compositions containing at least one amphiphilic block copolymer, at least one encapsulated therapeutic drug (e.g., liposomal doxorubicin) and at least one pharmaceutically acceptable carrier. The invention also includes a composition containing at least one amphiphilic block copolymer and less than the least one pharmaceutically acceptable carrier, and a second composition comprising at least one encapsulated therapeutic agent and at least one therapeutic drug. The compositions of this invention may also contain other therapeutic agents (e.g., other chemotherapy drugs).

The present invention also encompasses methods of preventing, inhibiting and/or treatment of diseases or disorders (e.g. cancer/neoplasm) patients. Methods include the introduction of the patient, at least one amphiphilic block copolymer and at least one encapsulated therapeutic drug (related to the disease or disorder). Pharmaceutical(s) composition(I) of this invention can be administered to animals, in particular mammals, more specifically the people, for the treatment or inhibition of cancer. Amphiphilic block copolymer may be administered simultaneously and/or sequentially with encapsulated therapeutic drug. For example, the amphiphilic block copolymer may be administered to the patient prior to the introduction of encapsulated therapeutic drug may be injected simultaneously with the encapsulated therapeutic drug and/or may be introduced after the introduction of the encapsulated therapeutic drug.

In particular Varian is the first implementation of the present invention, the amphiphilic block copolymer is introduced after the encapsulated therapeutic drug (liposomal doxorubicin). Amphiphilic block copolymer may be injected through a sufficient amount of time to ensure the accumulation (the largest number) of the encapsulated drug in the region of interest (e.g., tumors). The time of maximum accumulation (tmax[h]) Doxil in model tumors after a single injection was determined previously (PenateMedinaetal. (2011) J. Drug Deliv., 2011:160515; Laginha et al., (2005) Clin. Cancer Res., 11:6944-6949; Charrois et al., (2003) J. Pharmacol. Exp. Ther., 306:1058-1067; Vaage et al., (1997) Br. J. Cancer, 75:482-486). In a specific embodiment of this invention, the amphiphilic block copolymer is introduced within 96 hours, within 72 hours, within 48 hours, within 12 hours, within 6 hours 4 hours 2 hours 1 hour 0.5 hours or less after the introduction of encapsulated therapeutic drug. The optimal time of introduction amphiphilic block copolymer is determined by the time of maximum accumulation of liposomes of Doxil in tumors that depend on the pharmacokinetic characteristics of Doxil. In a specific embodiment of this invention, the amphiphilic block copolymer is introduced through from about 1 hour to 96 hours after administration of the encapsulated therapeutic drug. Studies of the pharmacokinetics and biological distribution of liposomal doxorubicin in patients in whom Asali, that 1/3 entered paglinawan liposomal doxorubicin distributed fabric in the first 4-5 hours after administration, why should a prolonged phase of clearance with an average half-life in 45-55 hours (Gabizon et al., (1994) Cancer Res, 54:987-992). It was also shown that the greatest accumulation paglinawan liposomal doxorubicin observed a few days after injection (from 3 to 5 days) (Stewart et al., (1997) Oncology 11(Suppl 11:)33-37). In a specific embodiment of the present invention to patients amphiphilic block copolymer may be preferably from 10 hours up to 168 hours after administration of the encapsulated therapeutic compound, more preferably from 24 to 144 hours after administration of the encapsulated therapeutic compound, more preferably from 48 to 120 hours after administration of the encapsulated therapeutic drug.

Amphiphilic block copolymer may be present in the composition in any concentration. In a specific embodiment of this invention, the amphiphilic block copolymer was present at concentrations of from about 0,001% to about 5%. In a specific embodiment of this invention, the amphiphilic block copolymer was used in a concentration of from about 0.05% to about 2%.

In a specific embodiment of the present invention, therapeutic is Reparata is a chemotherapy drug. The term "chemotherapeutic drug" means cancer or any other product for hyperproliferative diseases. Chemotherapeutic drugs are substances that have anticancer activity and/or damaging the cell. Chemotherapeutic agents include, without limitation: (1) DNA-damaging drugs (for example, drugs that inhibit DNA synthesis): anthracyclines (e.g. doxorubicin, daunorubicin, epirubicin), alkylating agents (e.g., nitrogen mustards, bendamustine, altretamine, esters of methanesulfonate, busulfan, carboplatin, nitrosamine, carmustine, cisplatin, chlorambucil, cyclophosphamide, dacarbazine, hexamethylmelamine, sofabed, ifosfamide, lomustin, mechlorethamine, alkyl sulphonates, epirubicin, idarubitsin, triazine, ethylenimine, melphalan, mitotane, mitomycin, pipobroman, procarbazine, streptozocin, aziridine, thiotepa, uramustine and triethylenemelamine), platinum derivatives (for example cisplatin, carboplatin, tetraploid, ormaplatin, triplatin, satraplatin, nedaplatin, oxiplatin, heptaplatin, iproplatin, transplatin, lobaplatin and cis-diaminedichloroplatinum), telomerase inhibitors and topoisomerase (e.g., topotecan, irinotecan, etoposide, teniposide, amsacrine, menogaril, amonafide, dactinomycin, daunorubicin, N,N-dibenzyl daunorubicin, ellipticine, daunomycin pyrazoloacridine, idarubitsin, mitoxantrone, m-ANSA, doxorubicin, doxirubicin, oxytrol, rubiazo, epirubicin, and bleomycin), drugs that bind to the minor groove of DNA (for example, plicamycin); (2) drugs that interact with tubulin (e.g., vincristine, vinblastine, paclitaxel, taxanes, docetaxel and BAY 59-8862); (3) antimetabolites, such as capecitabine, chlorodeoxyadenosine (cladribine, cytarabine, cytosine arabinoside, asparaginase, azacytidine, dakabin, floxuridine, fludarabine, 5-fluorouracil, doxifluridine, gemcitabine, hydroxyurea, 6-mercaptopurine, methotrexate, pentostatin, pemetrexed, trimetrexate and 6-tioguanin; (4) antiangiogenic drugs (e.g., Avastin, thalidomide, sunitinib, lenalidomide) and drugs affecting the blood vessels (e.g. flavonoids/flavones, vadiesen, derivatives combretastatin such as CA4DP, ZD6126, AVE8062A); (5) antibodies or fragments of antibodies (e.g., trastuzumab, bevacizumab, rituximab, ibritumomab, gemtuzumab, alemtuzumab, cetuximab, ranibizumab); and (6) hormones/endocrine therapy: aromatase inhibitors (for example, 4-hydroinjection, exemestane, aminoglutetimid, anastrozole, letrozole), antiestrogens (for example Tamoxifen, Toremifene, Raloxifen, Faslodex), antiandrogenna drugs (for example, flutamide), antigranulocyte drugs (for example, mitotane and aminoglutetimid) and with eroei (for example, adrenal corticosteroids, prednisone, dexamethasone, methylprednisolone and prednisolone); and (7) antimitoticescoe drugs such as navelbine, epothilone, taxanes (e.g. paclitaxel, Taxotere, docetaxel), Vinca alkaloids, estramustin, vinblastine, vincristine, vindesine and vinorelbine.

Cancers that can be treated by this method include, but are not limited to, the following: prostate cancer, colon and rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelioma of the pleura, white blood cells (including lymphoma and leukemia), esophagus, breast, muscle, connective tissue, lung (including small cell carcinoma and non-small cell lung carcinoma), adrenal, thyroid, kidneys, or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma, horiokartsinoma, bocletoccna carcinoma of skin, squamous cell carcinoma of the skin and carcinoma of the ovary. In a specific embodiment, the present invention cancerous disease that can be treated this way, may be ovarian cancer, lung cancer, including small cell carcinoma and non-small cell lung carcinoma, stomach cancer, breast cancer, Kaposi's sarcoma, uterine cancer,blood cancer, including multiple myeloma, lymphoma, Hodgkin's lymphoma and nehodgkinski lymphoma.

The compositions described in the invention will be administered to a patient as a pharmaceutical preparation. The term "patient" in the present description applies to people or animals. The compositions can be used therapeutically under a doctor's supervision.

The compositions described in this invention can be prepared in a standard way for the introduction of any pharmaceutically acceptable carrier(s). For example, drugs can be prepared in any appropriate medium, such as water, buffered saline, ethanol, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol, etc.,), dimethyl sulfoxide (DMSO), oils, detergents, suspendisse tool or in a suitable mixture of environments. The concentration of drugs in the selected environment may vary, and the selection may be based on the desired method of introducing pharmaceutical product. Except when any commonly used environment or the drug is incompatible with the input preparation, their use in the pharmaceutical preparation is provided by the invention. It should be noted that the applied composition and doses of liposomal doxorubicin (DOXIL) is well-known in their field (see DOXIL® Product information (2010) Centocor Ortho Biotech Products).

ACC is accordance with the invention, the dose and regimen compositions suitable for administration to a particular patient can be determined by a physician taking into account age, gender, specific patient's condition and severity of disease, for treatment using the compositions. The physician may also take into account the method of introducing pharmaceutical carrier and biological activity of the composition.

The selection of a suitable pharmaceutical mixture will also depend on the selected method of administration. For example, the compositions of this invention can be administered by direct injection into the desired area (e.g., the tumor). In this case, the pharmaceutical preparation will contain the substances dispersed in the environment that is compatible with the site of injection. Compositions of the present invention can be administered by any method. For example, compositions of the present invention can be, without limitation, parenteral, subcutaneous, oral, topical, pulmonary, rectal, vaginal, intravenous, intraperitoneal, intrathecal, vnutriarterialno, epidurally, intramuscularly, intradermally or intracarotid. In a specific embodiment, the present invention compositions are introduced by injection, such as intravenous or intraperitoneal. Pharmaceutical mixture for injection known in the field. If the injection is selected as a method of introduction of the composition, it is necessary to take measures to both is that the delivery of a sufficient number of molecules to cells purpose to demonstrate a biological effect.

Pharmaceutical compositions containing the drug of the present invention as the active component in the direct mixture with a pharmaceutically acceptable carrier, can be prepared according to conventional pharmaceutical methods. The media can have many different forms depending on the form of preparation desired for administration, e.g. intravenous or direct injection.

In a specific embodiment, the present invention is encapsulated therapeutic drug (such as liposomal doxorubicin) is administered by injection, including intravenous injection. In a specific embodiment of this invention, the amphiphilic block copolymer is administered by injection, e.g. intravenously, subcutaneously or intramuscularly.

The pharmaceutical preparation of this invention can be prepared in the form of dosage units for ease of administration and uniformity of treatment. Each dosage unit should contain a certain amount of active ingredient calculated to produce the desired effect, in connection with the selected pharmaceutical carrier. Methods for the determination of the appropriate dosage units are well known experienced in this field people.

Dosiro the data units can be proportionally increased or decreased depending on the weight of the patient. Suitable concentrations to facilitate a specific pathological condition can be determined based on a calculation of the curve of concentration, as is customary in this field.

In accordance with this invention suitable dosage units for administration of the compositions of this invention can be determined by examining the toxicity of molecules or cells in animal models. Various concentrations of drugs in the pharmaceutical mixture can be injected into mice, and the minimum and maximum dose can be determined on the basis of the positive results and side effects observed as a result of treatment. Suitable dosage units may also be calculated by examining the effectiveness of drug treatment in combination with other standard drugs. Dosage unit compositions may be determined individually or in combination for each treatment in accordance with the fixed effect.

Pharmaceutical mixture containing preparations of the present invention, may be injected through a suitable period of time, for example, at least twice a day or more, up until the pathological symptoms are reduced or partially removed, after which the dosage may be reduced to the maintenance level. The appropriate period of time in specific with what you learn will typically depend on the condition of the patient.

The methods proposed in this invention can be used in combination with other cancer therapies. For example, other chemotherapeutic agents may be used (e.g., simultaneously and/or sequentially). Cancer treatments such as radiotherapy and/or surgery (for example, cutting the tumor), can also be used with the compositions described in this invention.

Definition

"Pharmaceutically acceptable" means approved by the governing body of the Federal or local government or listed in the U.S. Pharmacopoeia or other generally recognized Pharmacopoeia for use in animals and humans.

"Carrier" refers to, for example, the solvent adjuvant, preservative (for example, thimersol, benzyl alcohol), antioxidants (e.g. ascorbic acid, metabisulfite sodium), solubilizer (e.g., tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris-HCl, acetate or phosphate), antimicrobial solution, substances that increase the volume (for example, lactose, mannitol), fillers, excipients, or media that is introduced is the active agent of the present invention. Suitable pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, R is stiteler or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol are preferred as carriers, particularly for solution for injection. Suitable pharmaceutical carriers are described in the book by E. C. Martin “Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, 20th Edition (Lippincott, Williams and Wilkins), 2000; Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

The term "cure" in this context means any kind of treatment, which provides benefit to a patient suffering from a disease, including improvement in the condition of the patient (for example, one or more symptoms), delay in the development of state and so on

"Therapeutically effective amount" of a substance or pharmaceutical composition refers to the amount, effectively preventing, inhibiting, healing or reducing the symptoms of certain disorders or diseases. Cancer treatment in this context can mean a cure, relief, and/or inhibition of cancer, its symptoms, or the predisposition towards it.

The term "therapeutic drug" in this context means a chemical or biological molecule, including, without limitation, nucleic acids, peptides, proteins and antibodies used for the treatment of a pathological condition is, diseases or disorders or to reduce the symptoms of pathological conditions, diseases or disorders.

The term "low-molecular substance (a small molecule)" in this context means a substance or a substance having a relatively low molecular weight (e.g. less than 4,000 less than 2000, in particular, less than 1 kDa or 800 Da). Typically, small molecules are organic, but not proteins, polypeptides or nucleic acids, although they may be amino acids or dipeptides.

The term "amphiphilic" in this context means the ability to dissolve in both water and lipids/nonpolar environments. Usually amphiphilic substance contains a hydrophilic part and a hydrophobic part. "Hydrophobic" means the preference to non-polar environments (for example, a hydrophobic substance or group dissolves better or wetted non-polar solvents, such as hydrocarbons than water). In this context, the term "hydrophilic" means the ability to dissolve in water.

In this context, the term "polymer" determines molecules formed by the chemical bonding of two or more repeating units or monomers. In the simplest view, the term "block copolymer" is defined by the conjugates of at least two different polymer segments, where each polymer segment contains two and more consecutive units of the same type.

The following examples describe exemplary methods of application of the present invention and in no way limit the scope of the invention.

EXAMPLE 1

Mahilyowskaya liposomal dosage form low molecular weight anticancer drugs doxorubicin, Doxil widely used in clinical practice to treat various types of cancer (e.g. ovarian cancer, AIDS-associated Kaposi's sarcoma and multiple myeloma) (Amanteaetal., (1997) ., 61:301-311; Sharpe et al., (2002) Drugs 62:2098-2126; Gabizon et al., (2001) Cancerlnvest., 19:424-436). However, the efficiency of Doxil as monotherapy is limited, and it was shown that the combination of Doxil with other chemotherapeutic drugs (Taxol, Hycamtin) is well tolerated by patients and have a higher efficiency (Camposetal., (2003) Gynecol.Oncol., 90:610-618; Dunton, C. J. (1997) Semin. Oncol., 24:S5-2 S5-11). Thus, trials of new combinations of Doxil with other drugs are of great interest for the development of new cancer treatments. Amphiphilic block copolymers pluronic comprising blocks of polyoxyethylene-polyoxypropylene, was used in this study in several modes of combination therapy: pre-processing the block copolymer, followed by the introduction of Doxil, administered together Doxil and pluronic and the introduction of Doxil with the subsequent introduction of a PLU is Onika. Studies of cytotoxicity on cell lines of ovarian cancer, sensitive A and resistant to medication A2780/DOX as well as sensitive and resistant cell lines breast cancer MCF7 and MCF7/ADRinvitro showed that co-administration with Maxilom, pre-or post-treatment of the cells with pluronics at non-toxic concentrations leads to the accumulation of much larger amounts of doxorubicin in the cell nucleus, compared with one Maxilom. Curves release of doxorubicin from liposomes Doxil in vitro and analysis of fluorescence quenching showed that pluronic causes an increase in the mobility and permeability of liposomal membranes that may cause drug release from liposomes. Introduction pluronic 1 and 48 hours after Doxil showed significantly better antitumor activity in comparison with the group, where he entered one Doxil.

The combination of well-characterized drugs have been used successfully in clinical practice for the treatment of cancer patients with new materials/activators, as well as the application of new treatment regimens may provide a solution and to provide significant benefits compared to therapeutic drugs used as monotherapy. Mahilyowskaya liposomal formulation of doxorubicin, Doxil actively used the camping in phase I and phase II combination studies for the treatment of various types of cancer. The frequency of clinical efficacy of these combinations, particularly in ovarian cancer resistant to platinum therapy was higher compared with monotherapy Paglierani liposomal doxorubicin (Roseetal., (2008) Am. J. Clin. Oncol., 31:476-480; Markman et al., (2004) Semin. Oncol., 31:91-105; Gabizon et al., (1994) Acta Oncol., 33:779-776; Eltabakh et al., (2001) Expert Opin. Pharmacother., 2:109-124). However, combination therapy is not without side effects, and the introduction of more than one cytotoxic agent can lead to more serious overall toxicity caused by dosage form, compared with monotherapy. The ideal agent for combination therapy should have low toxicity and have a synergistic effect with other medication the medicines. Described the combination of liposomal doxorubicin with sensitizers of ATP (adenosine triphosphate) (DiNicolantonioetal., (2002) Anticancer Drugs, 13:625-630; Cree I. A. (2003)Cancer Res., 161:119-125; Knight et al., (2009) BMC Cancer, 9:38). Pluronic block copolymers are very strong chemosensitization cancers multidrug-resistant (MDR) (Batrakova et al., (2010) J. Control Release, 143:290-301; Batrakova et al., (2003) Pharm Res., 20:1581-1590). Pluronic block copolymers inhibit the ATPase activity of Pgp, the main protein associated with multidrug resistance in some cancers, responsible for the release of cytotoxic drugs from the cell (Kabanov et al., (203) J. Control Release 91:75-83). Moreover, pluronic causes a decrease in the level of ATP, inhibition of oxygen uptake and inhibition of complexes I and IV of the respiratory chain of mitochondria in MDR cells. Taken together, these effects significantly increase the cytotoxicity of doxorubicin in drug-resistant cells (Alakhova et al., (2010) J. Control Release, 142:89-100). Especially important, SP1049C, dosage form containing pluronic L61 and F127 and doxorubicin, has successfully completed phase II clinical trials in advanced cancer of the esophagus (Batrakova et al., J Control Release 130:98-106).

Here we describe a new therapeutic approach for treating cancer using a combination of liposomal doxorubicin Doxil and pluronic block copolymer and drug scheme that greatly enhances anticancer activity of Doxil.

Materials and methods

Chemical reagents and materials

Doxil (liposomal doxorubicin (DOX) HCL for incci) was acquired ALZA Sof (Mountain View, CA). The assay kit for measuring the concentration of ATP (#FLAA-1KT), thiazolyl blue tetrazolium bromide (MTT, #M5655-1G), phosphate saline buffer, Dulbecco (FSB) were purchased at Sigma-Aldrich (St.Louis, MO), pluronic R (lot #WPYE357B) was provided by BASF Corporation (North Mount Olive, NJ). The ratio of monomers PEO-PPO-PEO in R is 26/40/26. The working solution had the following composition: 122 mm sodium chloride, 10 mm HEPES, 3 mm potassium chloride, 1,2 the M magnesium sulfate, 1.4 mm calcium chloride and 0.4 mm doonally potassium phosphate, pH increased to 7.4.

Cell lines and culturing conditions

Cell lines A, Msrbl acquired in ATSS. Cells A and A2780/DOX were cultured in medium RPMI 1640, cells MCF7 and MCF7/ADR were maintained in DMEM with addition of 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 u/ml penicillin and 100 mg/ml streptomycin. Cells A2780/DOX and MCF7/ADR6bmH cultivated in the presence of 1 μg/ml of doxorubicin. Cells were grown at 37°C in a humid air atmosphere with 5%content of CO2(by volume). All experiments the cell was held in a stage of exponential growth.

Western blotting

To determine the cellular phenotype associated with Pgp, we used the method of Western blot turns. Monoclonal antibodies to Pgp, S (Dako Corp. Carpinteria, CA) were used at a dilution of 1:100. Monoclonal antibodies to β-actin (Sigma Inc.) were used at a dilution of 1:15000. Antimachine secondary antibodies with horseradish peroxidase (dilution 1:20000) were purchased at Sigma Inc.

Determination of cytotoxicity

Cells were seeded in 96-well tablets when the initial density of 8×103cells/well 24 hours before the experiment. The next day cells were treated with doxorubicin or Maxilom in the presence or absence of 0.1% (weight/volume) R for 2 or 24 hours at 37°C in a humid in the stuffy atmosphere with 5% CO 2. After treatment, the medium was removed, cells were washed three times with PBS and cultured for three days in fresh medium. Cytotoxicity was determined using the standard MTT method, the absorption was measured at 562 nm using Spectramax MX M5. Each value of the concentration was determined from samples of eight separate wells. Values IR50were calculated from the percentage of treated cells and untreated controls using the software GraphPad Prizm 5 (GraphPad Software, San Diego, CA).

Studies of cell capture

Cells were seeded in 24-hole tablets when the initial density of 8×103cells/well on the day of the experiment cells were formed 70-80% of a continuous layer. After 2 or 24 hours incubation in the presence or absence of 0.1% R, cells were washed 3 times the FSB, was trypsinization, added 1 ml of medium and besieged by centrifugation at 1500 rpm for 3 minutes. Cellular precipitate resuspendable in 1 ml FSB with 1% bovine serum albumin (BSA). Fluorescence of doxorubicin was analyzed by flow cytometry at the center for cell analysis of the Medical Center of the University of Nebraska.

Confocal microscopy

Cells were seeded in 8-hole slides for 48 hours prior to the experiment with an initial density of 1×104cells/well. A continuous layer of cells treated with Maxilom (200 or 400 MK is/ml) for 24 or 48 hours, to determine the intracellular localization of doxorubicin. Cell nuclei were additionally painted with Hoechst 33258 (Sigma, St.Louis, MO). Atto 647 fluorescently labeled pluronic R was synthesized as described previously (Yi et al., (2010) Free It. Biol. Med., 49:548-558). 0,007% Atto 647-tagged P85, mixed with 0.1% unlabeled R, was used to study colocalization pluronic. Cells were visualized by confocal system visualization of living cells (Carl Zeiss LSM 510 Meta, Peabody, MA).

Study of the release of doxorubicin

The effect R on the release of doxorubicin from liposomes of Doxil was studied by the method of equilibrium dialysis. 1 ml of Doxil with the concentration of doxorubicin 0.2 mg/ml in the FSB was placed in a dialysis bag and dialserver against 25 ml FSB with constant stirring at 37°C in the dark. Samples of 1 ml detalizirovannoi solution were taken at specific time points (1, 2, 4, 8 and 24 hours) and replace with an equal amount of fresh medium. The concentration of doxorubicin in the sample dialysate was determined by measuring absorption at 485 nm using a Lambda 25 UV/VIS spectrophotometer. The amount of doxorubicin released from the liposomes of Doxil, was expressed as a percentage of the total number of doxorubicin and plotted as a function of time.

Fluorescent analysis

Fluorescence spectra of free doxorubicin and Li is Osom Doxil in the presence of 0.1% R were recorded, using Fluorolog spectrofluorimeter® HORIBA Jobin Yvon Inc., NJ at an exciting wavelength of 480 nm with a bandwidth of excitation and emission of 5 nm. Used solutions of free doxorubicin and doxorubicin contained 50 µm doxorubicin. Measuring the fluorescence of TRITC-labeled R was carried out at λvasb=550 nm with a bandwidth of excitation and emission of 2 nm.

Measurement of size and ζ-potential.

Effective hydrodynamic diameter (Deff) and Zeta-potential of the particles was measured using Zetasizer (Marven Instruments Limited. U. K.) at 25°C. the particle Size, the indices of polydispersity and Zeta-potential of liposomes were analyzed using software provided by the manufacturer. Average values were calculated from at least three measurements.

Animals

All experiments were carried out with the permission of the Committee on institutional animal care and use Medical Center University of Nebraska and, in accordance with the guidelines of the National Institute of Health for use of laboratory animals. To create a tumor model in this study was used Nude mice (6 - and 8-week-old females, the national Institute of Health, Frederick, MD). Animals were kept in groups of 5 with unlimited access to food.

Animal tumor model and protivoopujoleve the th activity

Xenotransplantation model of human ovarian carcinoma were used as described previously (Rakunlu et al., (2006) Clin. Cancer Res., 14:3607-3616). Cell line A2780 human ovarian carcinoma (4×106) were introduced subcutaneously into the right side of the waist females Nude nu/nu mice. When the tumors had reached a size of 0.5 cm3(10-15 days after transplantation), the mice did intravenous injection Doxil (12 mg doxorubicin/kg per injection). In the group of sequential processing through 1, 48, or 96 hours the mice were injected with 0.02% R in the same amount as Doxil. Mice weight and volume of tumors were measured every second day.

Statistical analysis

Differences between different groups were analyzed by t-test t-test for paired comparisons and univariate variance analysis for multiple comparisons. A p value less than 0.05 was accepted statistically significant. All statistical analysis was performed using the software Prism Software (Version 5.0 GraphPad Software, San Diego, CA, USA).

Results

The effect of Pluronic on the cytotoxicity of Doxil in cancer cells in vitro

In most experiments we used sensitive and resistant to medication ovarian cancer cells A and A2780/DOX. In some experiments were used sensitive and resistant to medication breast cancer cells MCF7 and MCF7/ADR. First, the cell is treated by increasing concentrations of Doxil for 2 hours in the presence or in the absence of R (0.1% weight/volume). As shown in table 1, a two-hour processing one Maxilom at concentrations up to 200 mg/ml (based on the concentration of doxorubicin in the liposomal preparation) did not cause cytotoxicity in either cell line. However, the combined treatment with R increased toxicity Doxil as sensitive and MDR cells. Two-hour processing one of 0.1% R did not cause toxicity in these cells. Similar results were obtained for breast cancer cells (table 2).

Table 1
ProcessingDoxil® IR50µg/mla
A2780A2780/Dox
Doxil® (2 h)H. O.§H. O.
Doxil® + 0.1% R (2 h, co)b7.55±0.728.32±1.12
Doxil® (24 h)48.02±9.70H. O.
Doxil® (24 h)+0.1% R (2 h, pre)c17.48±2.28(*)H. O.
Doxil® (24 h)+0.1% R (2 h, sledovatelno) d14.76±6.48(*)H. O.
Table 1. Values IR50(ág/ml) Doxil in ovarian cancer cells under different treatments.andExperiments were performed in four repetitions, data are presented as mean ± SOS at least four independent experiments. H. O. - not determined up to 200 ug/ml statistical comparison was performed by t-test t-test between group Doxil (24 h) and groups pre-and co-processing: *p<0,05.bCells were incubated together with Maxilom and 0.1% RH for 2 hours, then washed three times FSB and cultured in fresh medium for 72 hours before measurement of cytotoxicity.cCells are first treated with 0.1% RH for 2 hours, then washed 3 times the FSB and then incubated with Maxilom within 24 hours, washed 3 times the FSB and cultured in fresh medium for 72 hours before measurement of cytotoxicity.dCells are first processed by Maxilom within 24 hours, washed 3 times the FSB, and then for 2 hours and incubated with 0.1% R.

Table 2
ProcessingDoxil® IR50µg/mla
MCF7MCF7/ADR
Doxil® (2 h)H. O.aH. O.b
Doxil® + 0.1% R (2 h, co)b43,24±0,38852,49±0,73
Doxil® (24 h)6,24±0,26H. O.
Doxil® (24 h)+0.1% R (2 h, pre)cH. O.
Doxil® (24 h)+0.1% R (2 h, sequentially)dH. O.
Table 2. Values IR50(ág/ml) Doxil in breast cancer cells with different treatments. Cm. Table 1 for explanations.

The effect R on the cytotoxicity of Doxil were studied with 24-hour processing. To avoid toxicity, caused R, the copolymer was added to the cells for 2 hours, or immediately before or after treatment with Maxilom. Thus, also prevented direct interaction R and Doxil in the environment. One Doxil for 24-hour processing caused toxicity in sensitive but not in MDR cells (Tables 1 and 2). Prior and successive processing R also increase the La toxicity Doxil in cell line A compared only with Maxilom.

The effect of Pluronic R for seizure medications in cancer cells.

Intracellular accumulation of Doxil was investigated by flow cytometry. This method does not allow to distinguish between doxorubicin, in liposomes, and freed from particles Doxil, but allows you to measure the total fluorescence of doxorubicin in cells. R added exactly the same as in the measurement of cytotoxicity described above: either simultaneously with Maxilom in the case of a 2-hour treatment, either 2 hours before or after Doxil in the case of a 24-hour processing. In each case, except for pre-treatment in MDR cells with 0.1% R caused a significant increase in intracellular accumulation of the drug (Fig.1A and 1B). In a similar experiment, cells were lysed and the fluorescence was normalized to the protein content. The result of this experiment was similar to data flow cytometry (Fig.2), suggesting that changes in the accumulation of drugs are the result of common seizure medications in cells, rather than redistribution within the cell between free and encapsulated form, which have different fluorescence.

Caused R increase seizure medications was expressed most strongly in the case of joint processing, when the copolymer and Doxil was present in the cellular environment at the same time. This indicates n is then, joint processing R with Maxilom stimulated drug release from liposomes. This trend was observed both in sensitive and resistant cells. It should be noted that in MDR cells, the level of seizure medications was significantly lower than in the sensitive (Fig.1A and 1B). This may be due to either a lower rate of internalization of particles Doxil or rapid release of free doxorubicin released from captured liposomes inside the cell. As R is a well-known inhibitor of Pgp, it is likely that in this case it also inhibited the release of doxorubicin from MDR cells. Like that, most likely, could occur in the case of joint processing Maxilom with R when the conditions for the release of doxorubicin from liposomes induced by copolymer, the most favorable. As a result, when such conditions, differences in seizure medications between sensitive and MDR cells is strongly reduced. In the case of pre-treatment R becomes less likely that the copolymer will affect the drug release from liposomes, which may explain the less pronounced effect R for seizure medications in MDR cells.

Cellular transport Doxil in cancer cells in the presence and in the absence of Pluronic

Intracellular localization of drugs plays in inachou role for their activity and toxicity. Doxorubicin incorporated into DNA and inhibits topoisomerase II, which razvenchivaet DNA for transcription and thus stops the replication process (Gewirtz, D. A. (1999) Biochem. Pharmacol., 57:727-741). It is therefore important that doxorubicin has reached the nucleus. Capture liposomal doxorubicin, Doxil, a slow process and after 24-hour incubation with 200 μg/ml Doxil, the capture was low and the fluorescence of doxorubicin was observed in intracellular vesicles, but not in the nucleus (Fig.3A). However, in the presence of 0.1% R cell capture doxorubicin and nuclear localization significantly increased in sensitive and MDR cells (Fig.3A). In the presence of 0.1% R cell seizure medications was observed already after 5 minutes and doxorubicin was rapidly accumulated in the nucleus (Fig.3B). Intracellular localization R observed using Atto 647 fluorescently labeled pluronic. Cells were treated with 0,007% labeled pluronic with the addition of 0.1% of unlabeled pluronic (Fig.3A).

Next, the effect of sequential treatment of the cells with 0.1% R transport Doxil/doxorubicin was investigated in cells A (Fig.3C). Cells were incubated with 200 μg/ml Doxil within 24 hours, washed, and 1 hour was added to 0.1% R. After one hour incubation with pluronic his colocalization with doxorubicin from Doxil was practically absent. After 24 hours it was possible to observe a partial is localizatio doxorubicin and pluronic, however, nuclear seizure medications were not. Further incubation with Maxilom was extended to 48 hours, and pre-and sequential processing R up to 2 hours (Fig.3C).

Analysis of quenching the fluorescence of doxorubicin in Docile in the presence and absence of 0.1% R

In liposomes, Doxil doxorubicin is in crystalline form, resulting in its fluorescence is quenched (approximately 82%). As the release of the doxorubicin fluorescence intensity increases. The intensity of the fluorescence Doxil was measured in the presence and absence of 0.1% R to study the release of doxorubicin from liposomes. The emission fluorescence of one Doxil not changed within the hour. Adding pluronic after 30 minutes there was a significant increase in fluorescence, indicating rapid drug release, which peaked in the first 30 minutes (Fig.4).

The effect of pluronic on the release of doxorubicin from Taxila in vitro was also investigated. As can be seen in Fig.5, doxorubicin quickly released from the liposomes in the presence of pluronic as at pH 5.5 and at pH of 7.4.

The antitumor activity of Doxil in combination with Pluronic

The antitumor activity of Doxil in the presence or in the absence of pluronic was investigated to determine whether pluronic enhance the accumulation of drugs in the tumor in order to cause tumor suppression. Mice with tumors formed by cells of the ovarian cancer line A, received a single intravenous injection of 12 mg/kg Doxil. After 1, 48, or 96 hours mice received the same volume of 0.02% R intravenously. The volume of tumors in the control and experimental groups is shown in Fig.6. Sequential introduction R caused a significant inhibition of tumor growth compared with a one-time introduction of Doxil even in the group where R was administered one hour after Doxil. The strongest effect was observed in the group where R was administered 48 hours after Doxil. The observed difference in antitumor effects Doxil in the presence or in the absence of pluronic may be associated with farmakokineticheskimi characteristics Doxil, which is well known as long-circulating in the blood product doxorubicin. As was shown above (Fig.4), pluronic causes an increase in the mobility and permeability of the liposomal lipid bilayer, resulting in rapid release of doxorubicin from liposomes.

To explore the systemic toxicity of different treatment regimens, heart, liver, spleen and kidneys were isolated from mice with tumors at the end of the experiment. Histological analysis revealed no toxicity in all cases (Fig.7).

EXAMPLE 2

Pluronic R was administered either 48 or 96 hours after Doxil that corresponds to itself is the large increase in antitumor activity and when changes were observed, respectively. Animals killed after 1 or 6 hours after administration of the copolymer for the analysis of fluorescence drugs on sections of the tumors. Blood vessels (CD31) and cell nuclei (DAPI) were stained for comparison. As can be seen from the analysis of fluorescence, with the progressive introduction R after 48 hours there was a significant increase in the total fluorescence of doxorubicin on tumor slices compared with one Maxilom (Fig.8A). This effect was most pronounced after 6 hours after the introduction of the block copolymer. It is worth noting that in the absence of R fluorescence drugs were mainly colocalizes with blood vessels, while after adding R medicine spread throughout the tumor, reaching remote areas. From this it follows that after 48 hours of particles Doxil was mainly accumulated in the blood vessels, whereas R stimulated the release of doxorubicin from liposomes in tumor tissue. Images of the full sections of tumors strongly support the increase in fluorescence of the drug in the tumor after injection of the copolymer (Fig.8B, upper row). However, a different picture was observed when the copolymer is inserted through 96 hours after Doxil. In this case, the drug was released from the liposomes and its fluorescence in the tumor did not change p is after the introduction of the copolymer (Fig.8B, the bottom row).

Analysis of the accumulation of drugs by HPLC shows that R caused a small but significant increase after 6 hours after administration of the copolymer when the copolymer was injected 48 hours after introduction of Doxil (Fig.8B). Maximum accumulation of drugs in solid tumors in mice was observed between 24 and 48 hours after drug administration (Laginhaetal., (2005) Clin.CancerRes., 11:6944-6949). Thus, the introduction of the copolymer after Doxil in that moment, when there is maximum accumulation of drugs in solid tumors, leads to increased release of the drug throughout the tumor and, as shown in Fig.7, increases the antitumor activity. In contrast, the introduction of the copolymer is too early, for example one hour after Doxil, or too late, for example, through 96 hours after Doxil, does not lead to increased drug release and improves antitumor activity.

Materials and methods

Animal tumor model analogously to example 1.

The immunohistochemistry. Immunohistochemical analysis was performed on sections of tumors isolated from Nude mice with developed A tumors who were treated either by Maxilom or in combination with R. Fixed by formalin and paraffin embedded tumor tissue were stained FITZ-labeled anti-CD31 antibody is mi (BD Biosciences., dilution 1:100). At the end of colouring for 5 minutes was added Hoechst 33342 (Sigma-Aldrich, St.Louis, MO) for staining of cell nuclei. Visualization of the samples was performed on a microscope (Zeiss Axioplan 2.

EXAMPLE 3

A significant number of liposomes of Doxil still circulates in the blood at 48 hours after injection. The direct method of measuring the concentrations of free and liposomal drugs does not exist. Method HPLC allows to measure only the total concentration of drugs. Introduction R 48 hours after Doxil practically not changed farmakokineticeski profile drugs, which remained similar to the profile one has Docile (Fig.9). This indicates that the sequential introduction of pluronic did not cause the drug release from circulating liposomes of Doxil as free doxorubicin was quickly removed from the blood. Thus, this example shows that the introduction of the block copolymer after Doxil does not cause drug release from circulating liposomes, and as shown above in example 1, releases the drug from liposomes accumulated in the tumor.

Materials and methods

Animal tumor model analogously to example 1.

Determining the concentration of drugs in plasma.

Six-week female BALB/C mice were purchased from Charles River Breeding Laboratories (Raleigh, NC, USA). Mice made disposable intravenous inject the Yu 12 mg/kg Doxil. R (0,02%, 100 μl/mouse) was injected intravenously within 48 hours after medication. Animals umertvlâl for obtaining blood samples every 12 hours after drug administration. The blood samples were subjected to hematological analysis and analysis by HPLC.

High-performance liquid chromatography (HPLC).

The concentration of doxorubicin in tissue tumors was determined by HPLC. Before analysis of the tumor is first homogenized using a homogenizer TEADOR® (Biospec Products, Inc.). The tissue was added an equal volume of water qualification "HPLC" and homogenized. 40 μl of tissue homogenate 8 μl of daunorubicin (0.2 mg/ml) was used as internal standard (SU). To each sample was added 100 μl of trichloroacetic acid (THU 10%) and mixed on a vortex-mixer for 30 sec. Then the samples were centrifuged at 15000 g for 10 min at 4°C. the Supernatant (40 μl) was placed in a test tube and evaporated for 2 hours under a flow of gas in 40-degree heat mate. The residue was dissolved in 80 l mobile phase and an aliquot, 60 µl was used for HPLC analysis. The analysis was carried out using a C18 column with reversed phase (Agilent Eclipse XDB, 150×4.6 mmi.d., the particle size 5 µm) on Agilent 1200 HPLC (UV detector G1353B, the detector G1321A fluorescence, pump G1311A, G1329A Autosampler, thermostat G1316A column) at a flow rate of 1.2 ml/min Mobile phase: 1% acetic acid is: acetonitrile in the ratio of 48%:52%. Fluorescent detection of doxorubicin was performed at λvasb=480 nm and λCOI=590 nm.

EXAMPLE 4

Investigation of drug release from liposomes of Doxil was conducted by the method of equilibrium dialysis (nominal cutoff molecular weight of 3500 kDa). Stock solutions Doxil (2 mg/ml doxorubicin) were diluted 10-fold in the presence or in the absence of R or FSB, pH 7.4, or in acetate buffer, pH 5.5. The obtained solutions Doxil, whether or not containing pluronic copolymers (1 ml) were placed in dialysis bags and cialisbuy against 25 ml of the corresponding buffer with constant stirring at 37°C in the dark. Samples (1 ml) dialysate were collected at different points in time(1, 2, 4, 8, 24 and/or 48 h) and was replaced with an equal volume of the corresponding buffer. The concentration of doxorubicin in the sample dialysate was determined spectrophotometrically by measuring the absorbance at 485 nm using a Lambda 25 UV/VIS spectrophotometer (PerkinElmer). The amount of doxorubicin released from the liposomes DOXIL, was expressed as a percentage of the total number of doxorubicin and depicted graphically as a function of time.

Investigation of drug release from liposomes of Doxil method of equilibrium dialysis showed that adding pluronic block copolymers (R, F127, L61, F68) to the liposomes at pH of 7.4 or 5.5 greatly enhances / min net is ü release of doxorubicin (Fig.10). In this case, approximately 50% of drug was released during the experiment in the presence of pluronic R(Fig.10A, 10B) or other pluronic block copolymers (Fig.10B, 10D), while only 15% of doxorubicin released from Doxil without adding pluronic. It is worth noting that the ability of pluronic R to stimulate the release of drugs from liposomes depends on the concentration of the copolymer. In particular, increased drug release was quite significant when the concentration of R 0,02%-0,05% much less when 0,001% R and did not manifest when 0,0001% R (Fig.10A, 10B).

When the concentration of the copolymer of 0.02% by weight similar to pluronic R pluronic F127, and the mixture pluronic F127 and L61 (1:8 by weight) significantly increased the drug release from liposomes for 48 hours was recovered about 50% of the drugs compared with 15% in the case of only Doxil. However, pluronic F68 had less effect on the drug release from liposomes that may be associated with a more hydrophilic nature F68 and less interaction with the membrane of liposomes in comparison with other copolymers used in this example (Fig.10B, 10G). Thus, it was shown that copolymers with a high content of the CEA (about 30% in pluronic F127, or about 50% pluronic R, or about 90% pluronic L61), as well as copolymers with greater forefront of the popular mass unit CEA (about 2250 in pluronic R, approximately 3800 pluronic F127) is most effective release of the drug from the liposomes Doxil. The lowest activity had copolymers with the lowest amount of CEA (about 20% in pluronic F68) and the lowest molecular weight of the block CEA (about 1740). The molecular weight of the blocks PPO and PEO and the mass percentage of the block copolymers represented in the literature and is proportional to the number of units of propylene oxide and ethylene oxide in the respective blocks (Kabanov, A. V., Batrakova, E. V., Alakhov, V. Y. (2002) Pluronic® block copolymers as novel polymer therapeutics for drug and gene delivery, J. Control. Release 82 (2-3), 189-212). Molecular weight units of propylene oxide and ethylene oxide is 58 and 44, respectively. It is also worth noting that the highest activity is exhibited relatively more hydrophobic pluronic with hydrophilic-lipophilic balance (products HLB) 22 or less (22 to pluronic F127, 16 to pluronic R and 3 for pluronic L61), while the lowest activity are hydrophilic pluronic with more products HLB 22 (29 pluronic F68). It is also important to note that the most active pluronic have a low critical concentration of mitselloobrazovaniya (PFC) at 37°C - sample 0.0028 mm at pluronic F127, or about 0,067 mm at pluronic R, or a sample of 0.11 mm at pluronic L61, while the lowest activity was shown hydrophilic pluronic F68 with KCM about 0.48 mm. The products HLB values and KCM, conditions and methods of their measurement and the relationship with the lengths of the blocks of PPO and PEO can be found in the literature (Batrakova, E., Lee, S., Li, S., Venne, A., Alakhov, V., Kabanov, A. (1999) Fundamental relationships between the composition of Pluronic block copolymers and their hypersensitization effect in MDR cancer cells. Pharm. Res. 16 (9), 1373-1379; Kozlov, M. Y., Melik-Nubarov, N. S., Batrakova, E. V., Kabanov, A. V. (2000) Relationship between Pluronic® block copolymer structure, critical micellization concentration and partitioning coefficients of low molecular mass solutes, Macromolecules 33, 3305-3313; Kabanov, A. V., Batrakova, E. V., Alakhov, V. Y. (2002) Pluronic® block copolymers as novel polymer therapeutics for drug and gene delivery, J. Control. Release 82 (2-3), 189-212).

Interestingly, the size and polydispersity of the liposomes in the presence of higher concentrations R has not changed over the same time period (table 3), which means that the increase in drug release in the presence of the block copolymer is not associated with destruction of liposomes. At the same time when mixing TRIS-labeled R with empty liposomes was observed quenching fluorescence TRIS (about 50%), which means that the copolymer is incorporated in the liposomal membrane (Fig.11).

The ability R to increase the speed of diffusion of doxorubicin through planar lipid and liposomal membranes was previously described. The research presented in this paper also showed that the block copolymer significantly increases the rate of release of doxorubicin from liposomes DOXIL. Moreover, it was shown that the copolymer does not affect the size of liposomes. However, it fits into the liposomal membrane, which presumably includes incorpor is of the hydrophobic block of polyoxypropylene in non-polar part of the lipid bilayer. The study also showed that hydrophobic pluronic are embedded in the lipid membrane and disrupt the packing of the lipids, which leads to permeabilization membrane.

Table 3
DeffPDI
Doxil (pH5.5)83.9±2.200.036±0.01
Doxil (pH7.4)82.9±1.210.011±0.01
Doxil + 0.5% R (pH5.5)89.8±0.440.032±0.01
Doxil + 0.5% R (pH7.4)87.1±0.680.067±0.01
Doxil + 0.1% R (pH5.5)88.2±1.070.067±0.01
Doxil + 0.1% R (pH7.4)85.5±0.910.067±0.01
Doxil + 0.02% R (pH5.5)88.1±0.950.049±0.01
Doxil + 0.02% R (pH7.4)85.5±0.730.067±0.01
Doxil + 0.01% R (pH5.5) 88.6±0.060.036±0.01
Doxil + 0.01% R (pH7.4)86.8±0.470.046±0.01
Doxil + 0.001% R (pH5.5)88.0±0.250.058±0.01
Doxil + 0.001% R (PH7.4)84.8±0.940.059±0.01
Doxil + 0.0001% R (pH5.5)87.2±0.360.056±0.01
Doxil + 0.0001% R (pH7.4)85.2±1.070.027±0.02
Table 3. Effective hydrodynamic diameter (Deff) and index of polydispersity (PDI) particles Doxil in dispersion (0.2 mg/ml in the calculation of the concentration of doxorubicin in the presence of different concentrations of pluronic R at pH 5.5 and pH of 7.4 were measured by photon-correlation spectroscopy in a thermostatted cell at an angle of dispersion of 90°.

In the foregoing specification cited some publications and patents, to describe the condition of the area that includes the present invention. A full description of each of these articles are listed in the references.

While the foregoing invention has been thoroughly proillyustriroval what about using examples, for clarity of understanding can be made of numerous changes and modifications, without departing from the essence of the invention which is limited solely by the scope of the attached claims.

1. A method of treating cancer in a patient, comprising the introduction of at least one encapsulated chemotherapeutic agent and at least one amphiphilic block copolymer of this patient.

2. The method according to p. 1, characterized in that the encapsulated chemotherapeutic drug and the amphiphilic block copolymer is contained in a single composition.

3. The method according to p. 1, characterized in that the encapsulated chemotherapeutic drug and the amphiphilic block copolymer is contained in different compositions.

4. The method according to p. 3, characterized in that the encapsulated chemotherapeutic drug and the amphiphilic block copolymer is injected, at least consistently.

5. The method according to p. 4, characterized in that the encapsulated chemotherapeutic drug is administered to the specified amphiphilic block copolymer.

6. The method according to p. 1, characterized in that the encapsulated chemotherapeutic drug and the amphiphilic block copolymer is injected at least at the same time.

7. The method according to p. 1,characterized in that that specified encapsulated chemotherapeutic drug is liposomal doxorubicin.

8. The method according to p. 1, characterized in that the amphiphilic block copolymer contains at least one block of ethylene oxide and at least one block of propylene oxide.

9. The method according to p. 8, characterized in that the amphiphilic block copolymer is poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer.

10. The method according to p. 9, characterized in that the content of poly(propylene oxide)and the amphiphilic block copolymer is at least 30%.

11. The method according to p. 10, characterized in that the molecular weight of the block poly(propylene oxide)and the amphiphilic block copolymer is in the range from about 1200 to about 4200.

12. The method according to p. 1, characterized in that includes the introduction of one amphiphilic block copolymer and the second amphiphilic block copolymer, while the first and second block copolymers different.

13. Composition for treating cancer in a patient, comprising at least one encapsulated chemotherapeutic drug, at least one amphiphilic block copolymer and at least one pharmaceutical carrier.

14. The composition according to p. 13, characterized in that said encapsulated chemotherapeutic preparetoplay liposomal doxorubicin.

15. The composition according to p. 13, characterized in that the amphiphilic block copolymer contains at least one block of ethylene oxide and at least one block of propylene oxide.

16. A kit for treating cancer in a patient, comprising: a) a composition containing at least one encapsulated chemotherapeutic drug and at least one pharmaceutical composition; and b) a second composition comprising at least one amphiphilic block copolymer and at least one pharmaceutical composition.

17. Set under item 16, characterized in that said encapsulated chemotherapeutic drug is liposomal doxorubicin.

18. Set under item 16, characterized in that the amphiphilic block copolymer contains at least one block of ethylene oxide and at least one block of propylene oxide.

19. The use of amphiphilic block copolymer to enhance the action encapsulated chemotherapeutic drug by stimulating the release of active chemotherapeutic agent from the liposomes.

20. Application under item 19, characterized in that the amphiphilic block copolymer is administered after the encapsulated chemotherapeutic drug over a period of time sufficient for the accumulation of encapsulated chemioterapico the definition of the drug in tumors.



 

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FIELD: medicine.

SUBSTANCE: invention represents a method for preparing a substance with anti-tumour activity in hepatocellular carcinoma consisting in the fact that individual's enteric mucosa is homogenised with water in ratio 1:3-5 at +3-+5°C; the homogenate is centrifuged; the supernatant is heated for 7-10 min at temperature 20° to 38°C increased to 100°C and heated to 15-20 min at this temperature, centrifuged again; the cooled supernatant cooled to +4-+6°C is added with ethanol cooled (-15)-(-20)°C to the concentration of 65-70%; 15-17 h later it is centrifuged again; the alcohol is removed from the supernatant; the prepared end product is frozen and stored at (-65)-(-70)°C until used.

EFFECT: extended range of products possessing the anti-tumour activity.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology and immunology, and can be used in treating an individual with a stable pathogenic infection or a tumour. That is ensured by administering a therapeutically effective amount of a programmed death (PD-1) peptide antagonist and a therapeutically effective amount of a pathogen or tumour antigen molecule (vaccine).

EFFECT: invention provides the synergetic action of the antigen and PD-1 antagonist (anti-PD-L1 antibody) combination by intensifying the T-cell immune response to the pathogen or tumour.

14 cl, 29 dwg, 5 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to MUC1 cytoplasmic domain peptides, and can be used in the anticancer therapy. A method for inhibiting MUC1-positive cancer cell in an individual involves administering into an individual the MUC1-peptide of the length of at least 6 sequential MUC1 residues and no more than 20 sequential MUC1residues and containing the sequence CQCRRK, wherein the amino terminal cysteine from CQCRRK is closed at its NH2 terminal by at least one amino acid residue, which shall not conform with the native transmembrane sequence MUC-1. Alternatively, there can be used MUC-1 peptide of the length of at least sequential MUC1 residues and no more than 20 sequential MUC1 residues, which contains the sequence CQCRRK with all amino acid residues of the above peptide being D-amino acids.

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SUBSTANCE: invention relates to field of biochemistry, in particular to single variable domain, aimed against IL-6R, to polypeptide and construction, directed against IL-6R, containing said single variable domain, as well as to methods of obtaining them. Disclosed are nucleic acids, coding said single variable domain, polypeptide and construction, as well as genetic constructions, containing said nucleic acids. Described are host cells and host organisms, containing said nucleic acids. Invention also deals with composition for blocking interaction of IL-6/IL-6R, containing effective quantity of described single variable domain, polypeptide, construction, nucleic acid or genetic construction. Also disclosed is method of prevention and/or treatment of at least one of diseases or disorders, associated with IL-6, IL-6R, complex IL-6/IL-6R and/or signal pathways, in which IL-6, IL-6R or complex IL-6/IL-6R is involved and/or biological functions and reactions, win which IL-6, IL-6R or complex IL-6/IL-6R takes part with application of described single variable domain, polypeptide, construction or composition.

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SUBSTANCE: invention refers to biotechnology, in particular to tumour-specific promoters, and can be used in the anti-cancer therapy. There are constructed the broad-spectrum tumour-specific promoters providing the therapeutic gene expression inside a cancer cell. The invention also involves expression cassettes, expression vectors, pharmaceutical compositions, methods of treating cancer and using the expression cassettes and vectors.

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29 cl, 19 dwg, 4 tbl, 20 ex

FIELD: medicine, pharmaceutics.

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel compounds of formula Ia, their stereoisomers or pharmaceutically acceptable salts, inhibiting JAK kinase activity. Compounds can be applied in treatment of inflammatory diseases, such as rheumatoid arthritis, psoriasis, contact dermatitis, in treatment of autoimmune diseases, such as lupus, multiple sclerosis, neurodegenerative diseases, such as Alzheimer's disease, etc. In formula Ia R1 represents H; R2 represents -OR4, -NR3R4- or -NR3S(O)2R4; R3 represents H or C1-C6alkyl, where said alkyl is optionally substituted with ORa; R4 represents H, C1-C6alkyl, -(C0-C5alkyl)(C3-C6cycloalkyl), -(C0-C5alkyl)(C4-C5heteroaryl), where heteroaryl contains 1-2 nitrogen atoms as heteroatoms, or -(C0-C5alkyl)(C6aryl), where said alkyl is optionally substituted with group R8 and said aryl, cycloalkyl and heteroaryl are optionally substituted with group R9; or R3 and R4, taken together with nitrogen atom, which they are bound to, form C3heterocyclyl, containing 1 nitrogen atom as heteroatom, optionally substituted with group R13; Z represents -NR5R6; R5 represents H; R6 represents H, C1-C10alkyl, -(C0-C5alkyl)(C4-C5heterocyclyl), where heterocyclyl contains oxygen atom as heteroatom, -(C0-C5alkyl)(C3-C8cycloalkyl), -(C0-C5alkyl)(C3-C5heteroaryl), where heteroaryl contains 1 nitrogen atom or 1 oxygen atom or contains 2 atoms, selected fromoxygen, nitrogen and sulphur, as heteroatoms, -(C0-C5alkyl)(C6aryl), where said alkyl is optionally substituted with group R10, and said aryl, cycloalkyl, heteroaryl and heterocyclyl are optionally substituted with group R11; R7 represents H; R8 and R10 each independently represents halogen or ORa; R9 independently represents -CN, -CF3, halogen, -C(O)ORa, -C(O)NRaRb, -(C0-C5alkyl)NRaRb, -(C0-C5alkyl)ORa, -(C0-C5alkyl)SRa, -O[C(Ra)2]1-3O-, C1-C3alkyl, optionally substituted with F, -(C0-C5alkyl)(C3-C6cycloalkyl), optionally substituted with group oxo or F, -(C0-C5alkyl)C3-C6heterocyclyl, where heterocyclyl contains 1-2 heteroatoms, selected from atoms of oxygen and nitrogen, and where heterocyclyl is optionally substituted with halogen or C1-C3alkyl, -(C0-C5alkyl)C6aryl, optionally substituted with halogen, or -(C0-C5alkyl)C4-C5heteroaryl, where heteroaryl contains 1 nitrogen atom or 1 oxygen atom or contains 2 atoms, selected from atom of oxygen, nitrogen and sulphur as heteroatoms, and where heteroaryl is optionally substituted with or C1-C3alkyl; R10 independently represents halogen or ORa. Other values of radicals are given in the invention formula.

EFFECT: obtaining pharmaceutically acceptable salts, inhibiting JAK kinase activity.

15 cl, 4 tbl, 452 ex

FIELD: medicine, pharmaceutics.

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27 cl, 1 tbl, 5 dwg, 25 ex

FIELD: medicine.

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EFFECT: method enables avoiding the compulsory gaps of the radiation therapy by reducing a rate of severe local radiation reactions, and provides the target delivery and accurate dosage of metronidazole administered into the patient's body leading to the partial death of well-oxygenated cells and re-oxygenation of hypoxic tumour cells.

2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula or its therapeutically acceptable salts, wherein A1 represents furyl, imidazolyl, isothiazolyl, isoxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, thienyl, triazolyl, piperidinyl, morpholinyl, dihydro-1,3,4-thiadiazol-2-yl, benzothien-2-yl, banzothiazol-2-yl, tetrahydrothien-3-yl, [1,2,4]triazolo[1,5-a]pyrimidin-2-yl or imidazo[2,1-b][1,3]-thiazol-5-yl; wherein A1 is unsubstituted or substituted by one, or two, or three, or four, or five substitutes independently specified in R1, OR1, C(O)OR1, NHR1, N(R1)2, C(N)C(O)R1, C(O)NHR1, NHC(O)R1, NR1C(O)R1, (O), NO2, F, Cl, Br and CF3; R1 represents R2, R3, R4 or R5; R2 represents phenyl; R3 represents pyrazolyl or isoxazolyl; R4 represents piperidinyl; R5 represents C1-C10alkyl or C2-C10alkenyl each of which is not specified or specified by substitutes specified in R7, SR7, N(R7)2, NHC(O)R7, F and Cl; R7 represents R8, R9, R10 or R11; R8 represents phenyl; R9 represents oxadiazolyl; R10 represents morpholinyl, pyrrolidinyl or tetrahydropyranyl; R11 represents C1-C10alkyl; Z1 represents phenylene; Z2 represents piperidine unsubstituted or substituted by OCH3, or piperazine; both Z1A and Z2A are absent; L1 represents C1-C10alkyl or C2-C10alkenyl each of which is unsubstituted or substituted by R37B; R37B represents phenyl; Z3 represents R38 or R40; R38 represents phenyl; R40 represents cyclohexyl or cyclohexenyl; wherein phenylene presented by Z1 is unsubstituted or substituted by the group OR41; R41 represents R42 or R43; R42 represents phenyl, which is uncondensed or condensed with pyrrolyl, imidazolyl or pyrazole; R43 represents pyridinyl, which is uncondensed or condensed with pyrrolyl; wherein each cyclic fragment presented by R2, R3, R4, R8, R9, R10, R38, R40, R42 and R43 is independently unsubstituted or substituted by one or more substitutes independently specified in R57, OR57, C(O)OR57, F, Cl CF3 and Br; R57 represents R58 or R61; R58 represents phenyl; R61 represents C1-C10alkyl; and wherein phenyl presented by the group R58 is unsubstituted or substituted by one or more substitutes independently specified in F and Cl.

EFFECT: invention refers to a pharmaceutical composition containing the above compounds, and to a method of treating diseases involving the expression of anti-apoptotic Bcl-2 proteins.

7 cl, 2 tbl, 48 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry, namely represents a method for ensuring a uniform dissolution profile of a pharmaceutical composition of cyclobenzaprine containing inert coated particles with the cyclobenzaprine containing composition of a drug layering for forming IR granules to be coated with a prolonged-release coating for forming ER granules.

EFFECT: developing the method for ensuring the uniform dissolution profile of the pharmaceutical composition.

53 cl, 6 ex, 5 dwg, 4 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of microcapsulation of heterocyclic compounds of triazine series, applied in pharmaceutical industry and agriculture as pesticides. Method of obtaining microcapsules includes physical-chemical method of precipitation with non-solvent with application of polyvinyl alcohol as microcapsule envelope. Carbinol and acetone are used as non-solvent. Preparation E 472c is used as emulsifier.

EFFECT: application of invention simplifies the process of obtaining microcapsules, increase of preparation output by weight.

3 ex

FIELD: chemistry.

SUBSTANCE: invention provides a method of encapsulating antiseptic-stimulator Dorogova (ASD) fraction 2. The method is a physical-chemical non-solvent deposition method. When carrying out the method, the cladding of the microcapsules used is sodium alginate and the precipitation agent is carbon tetrachloride.

EFFECT: simple and faster process of producing microcapsules and higher mass output.

2 ex

FIELD: chemistry.

SUBSTANCE: method of encapsulating creatine is a physical-chemical non-solvent deposition method. The envelope used is sodium alginate, which is deposited from a solution in butanol in the presence of a glycerol ester with one or two molecules of edible fatty acids and one or two molecules of citric acid by adding chloroform as a precipitant.

EFFECT: simple and faster process of producing microcapsules and higher mass output.

3 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: distinctive feature of the present method is the use of betaine as an antioxidant and sodium alginate as a microcapsule envelope, as well as the use of tetrachloromethane as a precipitant when producing microcapsules using a physical-chemical non-solvent deposition method.

EFFECT: simple and faster process of producing microcapsules and higher mass output.

2 ex

FIELD: chemistry.

SUBSTANCE: described is method of obtaining particles of encapsulated with fat-soluble polymer envelope flavour enhancer, which possess supramolecular properties, with fat-soluble polymer being used as microcapsule envelope and flavour enhancer "gooseberry" being used as core in production of encapsulated particles by method of precipitation with non-solvent with application of acetone and butanol as precipitators.

EFFECT: simplification and acceleration of the process of producing microcapsules, reduction of loss in production of microcapsules.

1 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: method represents physico-chemical of precipitation with non-solvent, in which as microcapsule envelope xanthan gum, as core - ADS 2 fraction, and as precipitating agent - benzene are used.

EFFECT: simplification and acceleration of the process of obtaining microcapsules and increase of output by their weight.

3 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and represents an oral pharmaceutical composition for treating intestinal disorders, containing butyric acid or its salt in a combination with water-soluble or water-dispersed dietary fibre specified in inulin, maltodextrin or a mixture thereof, at least one flavouring agent specified in vanillin, vanilla essence or a mixture thereof and one or more pharmacologically acceptable excipients differing by the fact that it contains: a) a matrix containing lipophilic compounds with a melting point of less than 90°C, and an amphiphilic matrix wherein an active ingredient is at least ball-shaped; b) an amphiphilic matrix; c) an outer hydrophilic matrix wherein the lipophilic matrix and the amphiphilic matrix are dispersed; e) a coating; and a portion of the above at least one flavouring agent is dispersed in one or more of the above matrixes, and a portion of the flavouring agent is dispersed in the coating.

EFFECT: preparing the pharmaceutical composition for treating intestinal disorders.

22 cl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is a group of inventions involving a pharmaceutical composition, a method of preparing it, a method of treating Parkinson's disease and a method of reducing a 'wear' effect in the given patients by administering the same. The pharmaceutical composition in the form of a single oral dose for treating Parkinson's disease consists of a mixture of a) Levodopa or its salt in an amount of 50 mg to 300 mg in the form of prolonged release, b) Carbidopa or its salt in an amount of 10 mg to 100 mg in the form of prolonged release, wherein the prolonged release is ensured by coating or mixing Levodopa and Carbidopa with one or more rate control polymers, and c) Entacapone or its salt in an amount of 100 mg to 1000 mg in the form of prolonged release, optionally with other pharmaceutically acceptable excipients.

EFFECT: group of inventions promotes patient's treatment compliance; using it leads to a stable blood content of active antigens and to reducing administration rate that provides reducing the 'wear' effect in the patients with Parkinson's disease; besides, the additional technical effect ensured by the composition consists in its stability at high temperature and humidity.

9 cl, 15 tbl

FIELD: medicine.

SUBSTANCE: invention refers to using a polyurethane polymer as a drug delivery system to provide the biologically active risperidone delivery at a constant speed for a relatively long period of time, as well as to methods for using it.

EFFECT: system provides high biological compatibility and biological stability and is applicable as an implant in patients (humans and animals) for the risperidone delivery to tissues and organs.

25 cl, 13 dwg, 3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: inventions refers to medicine, namely to pulmonology, and can be used for treating a pulmonary disorder in a patient. That is ensured by administering an effective dose of the nebulised liposomal amikacin formulation of 100 to 2,500 mg daily within the cycle of treatment, which involves the period of administration from 15 to 75 days and the following withdrawal period from 15 to 75 days. The cycle of treatment repeats at least twice.

EFFECT: invention provides improving the pulmonary function, which is supported for at least 15 days after the termination of treatment, and increasing the one-second forced expiratory volume (FEV1).

28 cl, 16 tbl, 11 dwg, 3 ex

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