Composition comprising a biological agent

 

(57) Abstract:

The invention can be used in medicine to treat tumors. The claimed composition comprising an anti-cancer agent and a polyester block copolymers, which are capable of forming liquid composition when mixed with water. Block copolymers includes many linear polymer segments with a molecular weight of 30-500. At least 80% of the links connecting the repeating unit of each segment are simple ether bond. Block copolymers has the critical micellization concentration (CMC) from 0.000001 to 0.5% (weight/vol. or less 37oIn isotonic aqueous solution. The second composition comprises the above components, while block copolymers has a weight percentage of the hydrophobic part is not less than 50% and has a molecular weight of at least 900, or KCM about 0.5% (weight/vol.) or less under the above conditions. Claimed is a method of treatment of an object resistant to the biological agent, the method of treatment of cancer (animal), a method of treating microbial infection, the method of inhibiting or preventing the growth of cancer metastasis, the method of treatment of cancer and the way to overcome multidrug resistance of cancer cells. All of these methods on the delivering drug to the tumor, and to restore the sensitivity of tumors to drug exposure. 8 C. and 49 C.p. f-crystals, 9 Il., 29 table.

This application is a partial continuation of application U.S. N 08/374406, filed January 17, 1995, entitled "improved chemotherapeutic compositions, which, in turn, is a continuation of application U.S. N 07/957998, filed October 8, 1992

The present invention relates, among other things, to (1) pharmaceutical compositions and methods of producing chemotherapeutic agents, and (2) to pharmaceutical compositions comprising biological agents, particularly those that focus on cells and tissues that are resistant to the action of the biological agent.

A number of chemotherapeutic agents has a low solubility and poor stability in physiological fluids. Often chemotherapeutic agents bad pass through the cell membrane. In addition, many of these agents are associated with plasma proteins, as well as engage in other non-specific interactions in the bloodstream before it reaches the cancer target.

Impediment of effective chemotherapy is resistance to biological agents that develop many of up to 103once in the course of chemotherapy. And when such resistance is developing in relation to one agent, often be treated cells become resistant to many other biological agents to which they were not previously exposed (see Goldstein et al., Crit. Rev. Oncol. Hematol., 12: 243-253, 1992; Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 8th Ed., McGraw-Hill, New York, 1994). It is believed that one of the mechanisms through which developing such resistance includes membrane massively protein gp-170 (P glycoprotein or P-gp protein). (See Goldstein et al., Crit. Rev. Oncol. Hematol., 12: 243-253, 1992).

It was found that these difficulties can be overcome by the introduction of a biological agent in a composition containing micelles of one or more copolymers with the following characteristics. In addition, it was shown that a certain set of such copolymers may be especially effective to deliver the drug and restore sensitivity to the biological agent.

A BRIEF DESCRIPTION OF THE INVENTION

In another aspect the invention relates to pharmaceutical compositions, including:

(a) biological agent; and

(b) a polyester block copolymers, including linear polymermat relatively hydrophilic character, the repeating units of which contribute an average value fragmentary constants of Hansa-Leo (Hansch-Leo fragmental constant) at about -0,4 or less and contribute in molecular weight from 30 to 500, while the segment B-type has a relatively hydrophobic character, the repeating units of which contribute an average value fragmentary constants of Hansa-Leo at about -0,4 or more and contribute in molecular weight from 30 to 500, and at least 80% of the links connecting the repeating unit of each of the polymer segments, includes the ester linkage. In a preferred embodiment of the invention polyester block copolymers are selected from the group consisting of polymers of the following formula:

A-B-A'(I)

A-B (II),

B-A-B' (III)

or

L(R1)(R2)(R3)(R4) (IV)

where A and A' denote a linear polymer segments of type A, B and B' denote a linear polymer segments B and R1, R2, R3and R4represent or block copolymers of formulas (I), (II) or (III) or hydrogen, L is a linking group, provided that not more than two of R1, R2, R3and R4are hydrogen.

In the preferred VA is all block copolymers in the process of introduction of the composition or later. Preferably, at least about 0.1% of the biological agent incorporated into micelles, more preferably at least about 1 % of the biological agent, and even more preferably about 5 % of the biological agent.

In a preferred embodiment of the invention the hydrophobic portion of the copolymer in the present compositions is at least about 50%, more preferably at least about 60%, and even more preferably 70%.

In another preferred embodiment of the invention the weight of the hydrophobic portion is at least about 900, more preferably at least about 1700, and even more preferably at least about 2300.

In yet another preferred embodiment of the invention the weight of the hydrophobic portion is at least about 2000, and the hydrophobic portion is at least about 20%, preferably 35%; or the weight of the hydrophobic portion is at least about 2300, and the hydrophobic portion is at least about 20%, preferably 35%.

In yet another preferred embodiment of the invention the copolymer or copolymers that are included in the present compositions, described the ur of the 37oC in isotonic aqueous solution, preferably not more than approximately 0.05% (weight/volume), more preferably not more than about 0.01 percent (weight/volume), and even more preferably not more than about 0,003 % (weight/volume).

Preferably, the copolymers according to the present compositions correspond to the formula (V) below in the text. Particularly preferred among them are those who have the weight of the hydrophobic parts of from about 1500 to about 2000, preferably from about 1710 to about 1780, and the percentage of the hydrophobic part is from about 85% to about 95%, preferably from about 88% to about 92%. Among the most preferred among these copolymers are also those that have the weight of the hydrophobic parts of from about 3000 to about 3500, preferably from about 3200 to about 3300, and the percentage of the hydrophobic part is from about 15% to about 25%, preferably from about 18% to about 22%. Furthermore, in addition to among the most preferred among these copolymers are also those that have the weight of the hydrophobic parts of from about 3500 to about 4000, preferably from about 3700 to about 3800, and the percentage of the hydrophobic parts of the Rianta composition comprises a chemotherapeutic agent.

In its second aspect the invention relates to a pharmaceutical composition comprising a cytotoxic drug, soluble in polymeric micelles.

In another aspect the invention relates to a method of treating microbial infection or tumor by administration of a pharmaceutical composition according to the first or second variant of the present invention.

In another aspect the invention relates to a method of treating diseased tissue exhibiting resistance to biological agent in the treatment of these or other biological agent, at the specified method includes the introduction of a composition comprising (a) a second biological agent, which may be the same or different biological agent, in respect of which the fabric has been resilient, and (b) a micelle-forming composition with the copolymer described for the first or second embodiments of the invention.

In another embodiment, the invention relates to a method of preventing or limiting the formation of metastases through the introduction of one of the anti-cancer compositions of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 illustrates cytotoxic the Oh form.

Fig. 2 shows the kinetics of accumulation of daunorubicin SK-resistant cells or SK-cells, respectively, treated with daunorubicin in free or micellar form.

Fig. 3A and 3B illustrates the inhibition of MCF7-ADRccells incubated in various concentrations of doxorubicin and Pluronic L61 (Pluronic L61).

Fig. 3B illustrates the cytotoxicity Pluronic L61 against MCF7-ADRccells.

Fig. 4 shows the period of occurrence of the clearance of blood from [3H]-Pluronic R and accumulation in the liver.

In Fig. 5 shows comparative data on concentrations in the blood of [3H]-Pluronic R at its introduction, respectively, and/or oral.

In Fig. 6A shows the concentration of daunorubicin in the liver.

In Fig. 6B shows data on concentrations in the blood daunorubicinol over a period of time after its injection.

In Fig. 6B shows data on blood concentrations of daunorubicin during the period of time after its injection.

Fig. 7 shows changes in the course of the treatment object myeloma tumor multidrug-resistant Sp2/Odnrin mice of BALB/c. Volume ornia (Vo).

Fig. 8 shows changes in the course of treatment in the amount of myeloma tumor multidrug-resistant Sp2/0dnrin mice of BALB/c.

Fig. 9 shows the inhibition of tumor metastasis in mice which were injected doxorubicin in combination with Pluronic R, in comparison with mice that were injected only doxorubicin.

IDENTIFY:

The terms and phrases listed below have the following meanings in the context of the present description:

biological agent - the agent used for the diagnosis or receiving images, which can act on a cell, organ or organism, including, but not limited to, those of the medicinal product (pharmaceutical), which lead to changes in the functioning of the cell, organ or organism. Such agents include, but are not limited to, nucleic acids, polynucleotide, antibacterial agents, antiviral agents, antifungal agents, antiparasitic means, means destroying the tumor or anti-cancer tools, proteins, toxins, enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins, immunomodulators, dyes, radioactive labels, radiopaque substances, fluorescent the VA, protivoglaucomny funds midriatichesky funds and local anesthetics.

chemotherapeutic agent is a biological agent that inhibits the growth or reduces the level of survival of tumor or pathogenic microbial cells or inhibits the propagation (which includes unlimited replication, Assembly, viruses or cell infection) virus.

cytotoxic drug is a chemotherapeutic agent used to treat cancer, which is cytotoxic, in particular for rapidly dividing cells.

hydrophobic percentage - the percentage of the molecular weight of blockcopolymer, which is built of blocks of B-type. This value is referred to as "hydrophobic" weight percent".

hydrophobic weight - contribution to the value of the molecular weight of blockcopolymers containing blocks of type C. This value is referred to as "hydrophobic molecular weight".

IR50- the concentration at which achieved 50% cytotoxicity. The value of cytotoxicity can be measured by the method of Alley et al., Cancer Res. , 48: 589-601, 1988 or Scudiero et al. Cancer Res., 48: 4827, 1988. In particular, it can be determined on the basis of the concentration of the medicine, popilnya Deputy, attached to the target side and which is distributed in the lipophilic part of copolymer micelles to bind the target side of such micelles.

the microbe is a bacterium, Mycoplasma, yeast or fungi, virus or a parasite (such as the malaria parasite).

MDR (MDR) cells possess multiple drug resistance, if they are resistant to biological agents that act on cell lines, which are the parent of the MDR cells.

the target part of the molecular structure, which is recognized by the cells, tissues, viral or substrate components, such as a cell surface receptor or acceptor molecule.

DETAILED DESCRIPTION OF THE INVENTION

It was shown that the effectiveness of the copolymers of the present invention to improve the efficacy of chemotherapeutic agents and reversibility MDR highly depends on (a) the percentage of the hydrophobic part, and (b) by weight of the hydrophobic part. Thus the efficiency is increased either by increasing the percentage (a) or weight gain (b), or with increasing values of both factors. The marked increase in the percentage of the hydrophobic part and the Finance micelles for such copolymers occurs at lower concentrations. (See Hurter et al. Macromolecules 26:5030; Hurter et al. Macromolecules 26: 5592, 1993; Alexandris et al., Maromolecules 27: 2414, 1994). He wanting to be limited to any particular theory, we can only note that the formed micelles act as surrogate suitable for measurement of physical properties that lead to improved properties of the biological agent that is important to its delivery to the body. And again, not limited to a particular theory, it should be noted that the efficiency of the biological agent and the reversal of multidrug resistance are not associated with micelles as such. If using as a model the biological agent doxorubicin, apply to the graph of the ratio (a) under IC50(as a measure of effective cytotoxic concentration) for containing copolymer composition (b) if IR50to free doxorubicin against the concentration of the copolymer will be a two-phase graph, with the rapid reduction of the rate observed with increasing concentrations of the copolymer, but which remains within the CMC values for the copolymer. Higher values of CMC is the rapid decline of the coefficient. Cm. Fig. 6B. The maximum increase in the activity of the biological agent occurs when the values conc Pluronic L61, as of 0.0001 % (weight/volume) and below. The presence of micellar form is considered as an important factor in the use of the copolymers for drug delivery in the body and also for other reasons that will be discussed later in the description.

The diagram at the end of the description, allows to understand the relationship between the percentage of hydrophobic part and a weight of the hydrophobic portion of the copolymer with various aspects of the present invention. In the diagram the weights of the hydrophobic part (poly(oxypropylene)) and copolymer are shown directly below each marked with the copolymer. Next to the hydrophobic weight for each copolymer are given values hydrophobic percent.

It was shown that Pluronic F68 has a moderate activity, improve the action of biological agents. Pluronic L61, which has the same weight of a hydrophobic part that Pluronic F68, but higher hydrophobic percentage, represents the most effective block copolymers of the above scheme. Pluronic F108, which has the same percentage of the hydrophobic part that Pluronic F68, but higher hydrophobic weight, is also an effective copolymer, although less effective than Pluronic L61. Pleroo each of the factors here below, than in the case of Pluronic F108 and L61, respectively. Efficiency Pluronic R improve the action of biological agents is the average value between efficiency Pluronic F108 and Pluronic L61. These differences in efficiency are clearly visible when various copolymers at a concentration above the CMC and doxorubicin incubated in vitro with cells with drug resistance. The coefficient IR50for doxorubicin in the absence of the copolymer to the corresponding coefficient in the presence of a copolymer characterized by the index of reversibility sustainability". In table. 1 shows the index values of the resistance of reversibility for the different copolymers.

The value of micellar forms for drug delivery also detected in vivo. In micellar form of biological agents localized in the hydrophobic core of the micelles, closed thus hydrophilic film (consisting of segments of type a), which surrounds the micelles. This protection reduces interaction with proteins of the liver, plasma, other non-target tissues and other molecules that can bind or inactivate the agent, or to turn it into a toxic metabolite. For example, fast metabol is the modification in position C13 (Cm. Mushlin et al., Br.J. Pharmacol. 110: 975-982, 1993). When used as a model drug doxorubicin was shown that the presence of micellar shape reduces its uptake by the liver, reduces the level of transformation in doxorubicinol and reduces the level at which there is a decrease in the concentration of doxorubicin in the blood. Cm. Fig. 4 and 5.

The effectiveness of the copolymers (a) in the formation of micelles (where most performance is reduced PFC) and (b) in furtherance of the distribution of the biological agents most likely in micellar form than in free form, increases in the same order (i.e., with increasing weight hydrophobic or hydrophobic percent). Thus, the hierarchy efficiencies looks again as follows: L61 > R > F108 > F68. The presence of micelles at low concentrations, it is believed, believing that the biological agent remains in contact with the micelles that the biological agent and the copolymer together reach the target tissue. The distribution coefficients, the corresponding micellar form, really give reason to believe that the biological agent will be associated with the micelles. It is also believed that the micellar form of the biological agent protects b inactive or toxic metabolite, and from nonspecific adsorption of blood components, cellular components, etc.

The same character of the effectiveness of the copolymers is applicable, as is evident from the examples, and the treatment of experimental tumors anticancer agents.

At high concentrations of the copolymers can be toxic to the liver, kidneys or other cells of the body (See. BASF Corp., Pluronic Material Safety Data Sheet and Drug Master Files). It was shown that the toxicity of the copolymers is increased in accordance with the parameters of hydrophobicity of the copolymers with increasing efficiency augmentation of biological agents. Fortunately, the level of increase of efficiency when changing the parameters of hydrophobicity significantly exceeds the level of toxicity of the copolymer. So, for example, as shown in Example 8, the value of LD50for L61 in mice of BALB/c 10 times lower than the value LD50for Pluronic F108. However, the difference in therapeutic dose is 100 times more favorably at Pluronic L61 than Pluronic F108 (see Example 9B). Thus, the concentration range in which the efficiency of potentiating the activity of a biological agent can be maintained without concomitant associated with the polymer to promote the sustainability of many tumors responsible representatives of the family of glycoprotein P membrane proteins, whose resistance can be reversed with the use of the composition according to the present invention (See. Goldstein et al. Cancer Res Treatment., 57: 101-119, 1991). It is assumed that these proteins act as pumps which suck biological agent, in respect of which tumors acquire resistance. It is also expected that members of the same family of proteins deposited on the membranes of endothelial cells lining the blood vessels of the brain, determining the existence of the blood-brain barrier (BBB), which prevents many biological agents in the brain. (See for example, Tatsuta et al., J. Biol. Chem., 267: 20383-20391). The compositions of the present invention can be used to enhance the permeability of drugs in the brain, which is discussed in more detail in the application for U.S. Patent filed simultaneously with this, June 7, 1995, and entitled "Compositions containing biological agents to the target exposure" ("Compositions for Targeting Biological Agents"), dossier N attorney 313257-103A, full details of which are given in this paper as a reference. In addition, representatives of the specified protein family responsible, apparently, for the emergence of drug resistance among certain members Candid is her it should be noted that the compositions of the present invention reversiruyut mechanisms, contribute to the release of the representatives of the family of glycoprotein P, and other mechanisms related to drug resistance.

The invention is described hereinafter with reference to the fragmentary constants developed Hansen and Leo (Hansch and Leo). (See Hansch and Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York, 1979; James, Solubility and Related Properties, Marcel Dekker, New York, 1986, pp. 320 or 325). These constants have been developed for use with the aim of assessing the contribution of the molecule and the ability of all molecules are distributed between the phases formed by mixtures of the octanol-water. Such constants are usually referred to as fragmented distribution constants of Hansa-Leo (in the present description as "fragmented constants of Hansa-Leo").

The compositions of the present invention mainly or comprise already prepared for the formation of micelles with the presence of a significant part of the diluted biological agent, or they represent a copolymer composition, which form micelles with the inclusion of a significant part of the biological agent, which is soluble in them during the introduction of biologicheskikh the spruce, suitable for treatment of the part associated with the micelles, the specified target part can be either in a state prepared for such binding to micelles or can connect with micelles directly during the injection. In a particularly preferred embodiment, as the block copolymers using those that have low CMC value in isotope solutions at physiological temperatures. Such copolymers can perform the role of micellar media for delivery of biological agents even after significant dilution of their physiological fluid such as blood in a patient who is undergoing this treatment. These low values of CMC allow you to apply a reduced number of copolymers in the pharmaceutical compositions of the present invention.

Full disclosure application U.S. N 08/374406, filed January 17, 1995, is incorporated into this description by reference.

The number of repeating units of the General hydrophilic block (A-type) or General hydrophobic block (B-type) in the polyester copolymer is preferably from about 4 to about 400. More preferably, the number of repeating units is from about 4 to about 200,type and blocks B-type are mainly a molecular weight of from about 30 to about 500, preferably from about 30 to about 100 and more preferably from about 30 to about 60. Basically in each block A-type or B-type at least about 80% of the relations existing between the repeating units are ether linkages, preferably at least about 90% will be an essential communication and more preferably at least about 95% will be an essential communication. In the context of the present invention, the term "essential connection" refers to the glycosidic linkages (i.e. diabetes relations). However, in one aspect of the invention, the preferred ether linkages.

Preferably, all the recurring units, which form A block-type, had constant of Hansa-Leo for fragments less than about -0,4, more preferably, less than about minus 0.5, even more preferably less than about of-0.7. Preferably, all the recurring units that comprise the block B-type, had constant of Hansa-Leo for fragments of approximately -0,30 or more, and more preferably, about -0,20 or more.

As examples of polymers according to the first variant implementation of the brew is or

< / BR>
or

< / BR>
in which x, y, z, i, and j have values corresponding to the above parameters polyester copolymers, and in which for each pair R1, R2one of the components must be hydrogen, and the other must be a methyl group. The formulae (V) to (VII) is simplified, since, in practice, the orientation isopropylene radicals in the block will be random. This random orientation and are indicated in the formula (VIII), which is therefore more complete. These poly (oksietilenom)-poly (oxypropylene)new compounds were described by Santon (Santon, Am. Perfumer.Cosmet., 72(4): 54-58, (1958); Schmolka, Loc. cit. 82(7): 25-30 (1967); Nonionic Surfactants, Schick ed. (Dekker, NY, 1967), pp. 300 - 371). Many such compounds are commercially available under such trade generic names as "poloxamer", "pluronic" and "synperonic". Polymers of the type pluronic within formula B-A-B is often referred to as "converted" pluronic, "pluronic R" or "mericarol". "Polioksidony" polymer of the formula (VIII) are available from the company BASF (BASF, Wyandotte, MI) under the trade name TetronicTM(TetronicTM). Order polyoxyethylenated and polyoxypropylene blocks specified in the formula (VIII) can be reversed, giving Tetronic RTTM(Tetronic RTTM), to polimery can also be obtained with the hydrophilic blocks, includes a random mix of recurring 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 copolymers are available from BASF under the trade mark of PluralitTM(PluradotTM).

Created many pluronics, which correspond to the following formula:

< / BR>
Undoubtedly, any person with an average level of knowledge in this area will determine that the parameters m and n in the formula usually represent average values, and that the number of recurring units in the first block of a given molecule will not be exactly match the number of repeating units in the third block. In table. 2 in accordance with the formula (IX), specifications for a number pluronics.

Given the CMC values were calculated according to the method of determining surface tension described by Kabanov et al. (Kabanov et al. Macromolecules 28: 2303-2314, 1995).

Additional specific poly(oksietilenom)- poly(oxypropylene)type copolymers in accordance with the present invention is to accept the family of diamine-linked polyoxyethylene - polyoxypropylene polymers of the formula:

< / BR>
where the dashed line denotes the symmetric copies of polyester, extending to the second nitrogen atom, R*denotes alkylene, including from about 2 to about 6 carbon atoms, cycloalkyl comprising from about 5 to about 8 carbon atoms, or phenylene, R1and R2: either (a) they are both hydrogens, or (b) one denotes hydrogen and the other is methyl, R3and R4: either (a) they are both hydrogens, or (b) one denotes hydrogen and the other is methyl, and, if both of the R3and R4are hydrogens, one of R5and R6denotes hydrogen and the other is methyl, and if both of R3and R4are metelli, both R5and R6denote hydrogen. The group-NH2-CH2CH2-NH2in the formula (VIII) and group N-R*N in the formula (X) are examples of the linking group L in formula (IV).

Any person with an average level of knowledge in this area will determine in the light of the views expressed in the description of provisions that, if the practical implementation of the invention is limited to, for example, poly (oksietilenom)-poly (oxypropylene)inhibiting compounds in the above formula, too, wadala-Leo for fragments of the monomers, making the block A-type, is about -0,4 or less. Thus, units that make up the first block, not necessarily consist only of ethylene oxide. Similarly, not all block B-type only consists of propyleneoxide units. Instead, these blocks may include monomers other than those defined for formula (V)-(X), so that the parameters of the first variant are stored. So, in the simplest examples, at least one of the monomers in the unit And can be replaced by a lateral linear group from among the groups described above.

In another aspect, the present invention relates to pharmaceutical compositions consisting of blockcopolymer at least one of formulas (I)-(X), where the blocks A-type and B-type consist essentially of recurring units of the formula-O-R5where R5means:

(1) -(CH2)n-CH(R6)-, where n represents zero or an integer from about 1 to about 5 and R6denotes hydrogen, cycloalkyl comprising from about 3 to about 8 carbon atoms, alkyl comprising from about 1 to about 6 carbon atoms, phenyl, alkylphenyl, in which the alkyl contains from about 1 to about 6 carbon atoms, hydroxy, hydroxyalkyl, in which the alkyl contains from about kiuchumi from about 2 to about 7 carbon atoms, alkoxycarbonyl, in which the alkoxy contains from about 1 to about 6 carbon atoms, alkoxycarbonylmethyl, in which the alkoxy and alkyl each comprise independently from each other from about 1 to about 6 carbon atoms, alkylcarboxylic, in which each alkyl includes, independently from each other, from about 1 to about 6 carbon atoms, aminoalkyl, in which the alkyl contains from about 1 to about 6 carbon atoms, alkylamino or dialkylamino, in which each alkyl includes independently from each other from about 1 to about 6 carbon atoms, mono - or dialkylaminoalkyl in which each alkyl includes independently from each other from about 1 to about 6 carbon atoms, chlorine, chloroalkyl, in which the alkyl contains from about 1 to about 6 carbon atoms, fluorine, foralkyl, in which the alkyl contains from about 1 to about 6 carbon atoms, cyano or cianelli, in which the alkyl contains from about 1 to about 6 carbon atoms, or carboxyl;

(2) carbocyclic group which has the ring from about 3 to about 8 carbon atoms, examples of which can be groups such as cycloalkyl or aromatic group, kerno 1 to about 6 carbon atoms, alkylamino, including from about 1 to about 6 carbon atoms, dialkylamino, where each alkyl, independently, comprise from about 1 to about 6 carbon atoms, amino, sulfonylurea, hydroxy, carboxyl, fluorine or chlorine substituents or

(3) heterocyclic group which has the ring from about 3 to about 8 carbon atoms, which may include heterocytolysine or heteroaromatic group, which include from about 1 to about 4 heteroatoms selected from the group consisting of oxygen, nitrogen, sulfur and mixtures thereof, and which may include alkyl containing from about 1 to about 6 carbon atoms, alkoxy comprising from about 1 to about 6 carbon atoms, alkylamino, including from about 1 to about 6 carbon atoms, dialkylamino, where each alkyl, independently, comprise from about 1 to about 6 carbon atoms, amino, sulfonylurea, hydroxy, carboxyl, fluorine or chlorine substituents.

Preferably, n denotes an integer from about 1 to about 3. Carbocyclic or heterocyclic group, R5preferably contain in the ring of from about 4 to about 7 carbon atoms, more preferably predpochtitelno, heterocycles containing one heteroatom. Preferably, the heterocycle is a carbohydrate or similar carbohydrate.

For any specialist with the average level of knowledge in this area it is obvious that the monomers required for the creation of such polymers can be synthesized (See. Vaughn et al., J. Am. Oil Chem. Soc., 28: 294, 1951). In some cases, as it can see any specialist with the average level of knowledge in this area, to conduct polymerization may require the use of appropriate protecting groups. Basically blocks A-type and B-type are at least about 80% OR5-repeating units, more preferably at least about 90%, and even more preferably at least about 95%.

In another aspect, the present invention relates to pharmaceutical compositions consisting of blockcopolymer one of formulas (I)-(X), which blocks A-type and B-type consist essentially of repeating units of-O-R7- where R7represents C1-C4alkylenes group.

Coefficient (P) Hansa-Leo to assess the distribution of organic molecules in a mixture of octanol-water is calculated using the following formula:

Log P = anfn the UPP in the molecule, the value of andenote the number of groups of any kind in the molecule, the values of Fmare coefficients related to certain molecular characteristics, such as ordinary communication or double bond, and the value of bmdetermine the number of such characteristics. So, for example, the constant of Hansa-Leo for a fragment in duplicate ethylenoxide unit (-CH2CH2O-) is:

2fwith+4fn+fabout+(4-1)Fb= 2(0,20)+4(0,23)+(-1,82)+3(-0,12)=-0,86

Constant, Hansa-Leo for a fragment in duplicate propylenoxide unit (-CH2CH(CH3)O-) is:

< / BR>
Specialist with an average level of knowledge in this area will determine what approach Hansa-Leo to assess the distribution constants, which are constants of Hansa-Leo for fragments do not provide an accurate empirical constant values of the distribution (See. Hansch and Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York, 1979; James, Solubility and Related Properties, Marcel Dekker, New York, 1986, pp. 320 or 325). However, this approach is sufficiently accurate to determine the hydrophobicity used to deliver the polymer carrier.

The copolymers used in the present invention, will be preferable to form micelles in itially are supramolecular complexes, which form in aqueous solutions of some amphiphilic molecules in the microphase separation of non-polar parts amphiphiles. Micelles are formed in the case, if the concentration of amphiphilic molecules reaches at a given temperature values KCM,

specific to the amphiphile. Due to the variations in the sizes of the hydrophilic and hydrophobic segments of the block copolymers can be changed as the ability of the copolymers to form under physiological conditions micelles, and the average size of the formed under physiological conditions micelles. The specified capacity may be adjusted by mixing copolymers with different ratios of hydrophobic and hydrophilic blocks. Micelles contain dense core formed of water-insoluble repeating units B units and lipophilic parts of diluted biological agent, and a hydrophilic shell formed from A block and a hydrophobic parts of the biological agent. Micelles are characterized by freedom of translation and rotation in the aquatic environment and water environment containing micelles, has a low viscosity close to water. Micelle formation occurs in a typical case, when the concentration of the copolymer from about 0,0001% to 5% (weight/volume).

Schitka micelles can penetrate into the small capillaries and be captured cells. These micelles may also include large amounts of the biological agent. So, for example, micelles formed by Pluronic L61, can include at least 1 mg of doxorubicin 2 mg of the copolymer.

Effective retention of the drug within the micelles of the present invention can be quantified with the involvement of the distribution coefficient (P), defined by the formula:

P = [Agent]m/[Agent]aq< / BR>
where [Agent]aqdenotes the concentration of the biological agent in the aqueous environment outside of the micelle, and the [Agent]mthe concentration of the agent in the micelles. In some cases, P is easily and accurately calculated, involving differences in fluorescence properties, which have some agents in the water and a more hydrophobic environment.

In some cases, you may need to include using non-covalent binding of the target molecule. See, for example, the work of Kabanov et al. (Kabanov et al., J. Controlled Release, 22: 141 (1992)) and application for U.S. patent filed simultaneously with this, June 7, 1995 and entitled "Compositions containing biological agents to the target exposure" ("Compositions for Targeting Biological Agents"), the dossier were believed who e l e C which has affinity to a particular cell site, and a lipophilic part. Target molecules spontaneously associate with micelles and "anchor" them through the lipophilic part. Such target molecules are typically about 10% or less of the composition.

The target molecules are lipophilic part can represent, among other structures, a lipid group, such as fatty acyl group. In a preferred embodiment, the lipophilic component is a hydrocarbon comprising from about 3 to about 41 carbon atoms, more preferably from about 5 to about 25 carbon atoms and even more preferably from about 9 to about 17 carbon atoms. Alternatively, it may be block copolymers or other natural or synthetic polymer. Target group the target molecule often includes an antibody that is specific, in the typical case, to a specific antigen on the cell surface. It can also be a hormone with a specific nature of the interaction with the receptor on the cell surface, or it can be a drug that carries a receptor for cell surface. For example, glycolipids can serve for the purposes of Italo to a2-GP in glial cells of the brain (a2- glycoprotein), described Chekhonin et al. (Chekhonin et al., FEBS Lett., 287: 149-152, 1991).

In the case of the polyethylene oxide-polypropyleneoxide copolymer of its hydrophilic/hydrophobic properties and micelle-forming properties associated with the value of the coefficient n. The factor n is determined by the following formula:

n = (|B|/|A|)(b/a) = (|B|/|A|)1,32

where |B| and |A| denote the number of repeating units, respectively, in the hydrophobic and hydrophilic blocks of the copolymer, the values of b and a are the molecular weights of the respective repeating units. The value of n typically ranges from about 0.2 to about 0.9, more preferably from about 0.2 to about 1.5. When using mixtures of copolymers parameter N, which represents the weighted average value of n for each copolymer those in the mixture, and the average value determined by the weight parts of components copolymers. The value of N can be used to assess micelle-forming properties of the mixture of copolymers. When using copolymers other than the polyethylene oxide-polypropyleneoxide copolymers, can be developed similar approaches is representative of the class.

According to the second variant of the present invention polymeric micelles is preferably formed from toxic, pharmaceutically acceptable polymers.

The pharmaceutical compositions of the present invention can be administered using various methods, including, but not limited to, oral, local, rectal, vaginal, pulmonary, using, for example, aerosol, or parenterally, which includes, but is not limited to, intramuscular, subcutaneous, intraperitoneal or intravenous methods. Compositions can be entered separately or you can combine them with a pharmaceutically acceptable carrier or excipient commonly used in the pharmaceutical practice. For purposes of oral administration, the composition may be in the form of tablets, capsules, candies, lozenges, powders, syrups, elixirs, aqueous solutions and suspensions, etc. In the case of tablets as carriers can be used lactose, sodium citrate and salts of phosphoric acid. In pills usually include various dezintegriruetsja substances such as starch, and oil, such as magnesium stearate, sodium lauryl sulfate and talc. In the capsules for oral administration include as bodily aqueous suspension, the composition can be combined with emulsifiers and suspendresume agents. If desired, can also be added sweeteners and/or flavouring substances. For parenteral administration is usually prepared sterile solutions of the conjugate, down accordingly pH solutions /and sautereau them. At intravenous introduction it is necessary to control the total concentration of dissolved substances to the drug was isotonic. For eye introduction materials or landfill of liquid can be introduced by known experts systems, such as applicators or eye dropper. These compositions may include mucokinetic, such as hyaluronic acid, chondroitin sulfate, hypromellose or poly (vinyl alcohol), preservatives such as sorbic acid, EDTA or chlorinesilanes, as well as the normal amount of diluents and/or carriers. To implement insertion through pulmonary path you choose such diluents and/or carriers, which are suitable for receiving the aerosol.

The compositions of the present invention may take the form of suppositories for vaginal, urethral and rectal administration. Such suppositories are usually prepared from a mixture of substances, it is used to obtain such carriers substances include theobromine oil, glycerin gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters and polyethylene glycol. For more information about suppozitornyj standard forms cm edition of Remington (Remington''s Pharmaceutical Sciences, 16th Ed., Mack Publishing, Easton, PA, 1980, pp. 1530-1533). Similar gels can also be used for the introduction of vaginal, urethral and rectal methods.

Chemotherapeutic agents suitable for use in the present invention include, but are not limited to, alkaloids Winky, such as vincristine and vinblastine, antibiotics such as mitomycin, such as mitomycin C and N-methyl mitomycin C, antibiotics such as bleomycin, such as bleomycin A2antifolates, such as methotrexate, aminopterin and dideaza-tetrahydrofolate acid, colchicine, dimebolin, etoposide, taxanes, such as paclitaxel (TaxolR), anthracycline antibiotics and other Anthracycline antibiotics are medicines that have problems with delivery in the body related to their low stability, development in the target tissue resistance to these antibiotics or with their fast metabolism. Specified in 7th position with daunosamine. They include, for example, compounds having the following formula:

< / BR>
where R1denotes a hydroxy or methoxy group; R2denotes hydrogen or hydroxy and R3denotes ethyl, acetyl, hydroxyacetic or ether hydroxyacetone. These tetracycline, antibiotics, like many other antitumor agents, are likely by intercalating embedding between the planes of the aromatic ring structures of DNA and thus inhibit DNA replication (CM. Neidle and Waring, Molecular Aspects of Anti-Cancer Drug Action, Pitman Press, 1983). Tumor cells in General are particularly sensitive because they are actively replicated and thus synthesize copious replica of its DNA. These antibiotics include, but are not limited to, doxorubicin, daunorubicin, carminomycin, epirubicin, idarubitsin, mitoxantrone, 4-dimethoxy-daunomycin, 11-dezoksiguanosin, 13-dezoksiguanosin, adriamycin-14-benzoate, adriamycin-14 - octanoate or adriamycin-14-naphthaleneacetic.

Preferred classes of biological agents (including chemotherapeutic agents) include antineoplastic agents, antibacterial agents, antiparasitic funds protivogribkovi the mini-agents in respect of which cells have the propensity to develop resistance mechanisms. Particularly preferred biological agents include anthracyclines, such as doxorubicin, daunorubicin, epirubicin, idarubitsin, mitoxantrone or karminomitsin, alkaloids Winky, antibiotics such as mitomycin, antibiotics such as bleomycin, azole antifungal agents such as fluconazole, polyene antifungal agents such as amphotericin B, taxonomie antineoplastic agents, such as paclitaxel, and immunomodulators, such as tumor necrosis factor alpha cells (TNE ), interferons and cytokines.

Preferred biological agents (including chemotherapeutic agents) include, but are not limited to, antifungal agents such as amphotericin B, flucytosine, ketoconazole, miconazole, Itraconazole, griseofulvin, clotrimazole, econazole, terconazole, butoconazole, ciclopirox alamin, haloprogin, tolnaftate, naftifine, nystatin, natamycin, undecylenoyl acid, benzoic acid, salicylic acid, propionic acid, and Caprylic acid. In addition, such agents include, but are not limited to, antiviral agents such as zidovudine, acyclovir, ganciclovir, is t, not limited to, antibacterial agents, such as compounds similar to penicillin, including-lactam antibiotics, penicillins a wide spectrum of action and penicillins resistant to penicillinase (such as methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, amoxicillin, ampicillin, ampicillin-sulbactam, attilly, bacampicillin, carbenicillin of indanyl, ciclacillin, methacillin, penicillin G, penicillin V, piperacillin, tikarcillin, imipenem, and aztreonam), cephalosporins (cephalosporins include first generation cephalosporins, such as cephapirin, caracoli, cephalexin, cefradine and cephalo-Smoking; second generation cephalosporins, such as cefamandole, cefoxitin, cefaclor, cefuroxime, cefuroxime aksetil, cefonicid, cefalotin and ceforanide; third generation cephalosporins, such as Cefotaxime, ceftizoxime, Ceftriaxone, cefoperazone and ceftazidime), tetracyclines (such as demeclocycline, doxycycline, metatsiklina, minocycline and oxytetracycline), inhibitors of beta-lactamase (such as clavulanic acid), aminoglycosides such as amikacin, gentamicin C, kanamycin A, neomycin B, netilmicin, streptomycin, and tobramycin), chloramphenicol, erythromycin, clindamycin is d, ethionamide, cycloserine, Dapsone, sodium sulfation, clofazimine, sulfonamides (such as sulfanilamide, sulfamethoxazole, sulfacetamide, sulfadiazine and sulfisoxazole), trimethoprimsulfamethoxazole, quinolones such as nalidixic acid, cinoxacin, norfloxacin and ciprofloxacin), methenamine, nitrofurantoin, phenazopyridine. In addition, such agents include funds that are active against protozoal infections, such as chloroquine, diloxanide furoate, emetine or dehydroemetine, 8 - hydroxyquinoline, metronidazole, hengren, melarsoprol, nifurtimox, pentamidine, sodium stibogluconate and suramin.

The dosage of a biological agent in micellar composition is often approximately the same magnitude as in the case of a single biological agent; dosage is determined and prescribed to a patient by a healthcare professional that takes into account many factors, including the age, weight and health status of the patient, as well as the pharmacokinetics of the agent used. Often the amount of the biological agent in micellar form, is necessary for effective treatment, is less than the number applied as a free biological agent. For daunorubicin used in the treatment of R is from about 0.1 mg to 0.2 mg per kg of body weight. In General, biological agents used in the present invention, are administered to animals in an effective amount. When determining the actual amount should take into account the impact on the efficiency of the copolymer used in the composition. In General, the effective amount is that amount which is effective either (1) in reducing the symptoms of the disease to be treated, or (2) induce a pharmacological change relevant to treating the disease. In the case of cancer, an effective amount means an amount effective for: reduction in tumor size; slowing of tumor growth; preventing and inhibiting the formation of metastases; increase the life expectancy of the affected animals.

In many cases, the metabolites of various biological agents create or increase the level of adverse reactions arising from the introduction of drugs. This applies certainly to anthracycline drugs, metabolites which, apparently, are present. (See Mushlin et al. , Br.J. Pharmacol., 110: 975-982, 1993). Copolymer compositions of the present invention can reduce the speed of metasta.

Various antifungal agents successfully treat fungal infections in humans. However, therapeutic dose should often be a compromise between achieving effective levels of medication and avoiding toxic side effects. Recently, the emergence of drug resistance in hereditary sensitive species, such as Candida albicans, and an increase in the frequency of hereditary drug resistance in some species, such as Candida kruset, determined the need for search of new antifungal agents.

Although fluconazole characterized by low frequency of adverse reactions, the frequency of resistance is a growing problem. Therefore, for such agents, as for other antimicrobial agents, it is desirable to have delivering media that would be effective in enhancing the activity of the chemotherapeutic agents, and in the reversal of resistance to these agents.

Below the invention is illustrated by the next non-limiting examples.

Example 1. Ireally conjugates

A solution of 50 μl with 2 mg/ml antibody in2GP; specific to2- the glycoprotein glial the s bis(2-ethylhexyl)sulfosuccinate, available from Serva chemicals Germany (Serva Chemicals)] octane. The reaction is started by adding to a mixture of the two fold molar excess (relative to the polypeptide chloride salt of stearic acid in 0.2 ml of 0.1 M AOTin octane. Chloride stearic acid derived from stearic acid (available from Reakhim, Russia) according to the method described Kabanova et al. (Kabanov et al., Molek. Biologiya (Russian), 22: 473-484 (Engl. Edn.: 382-391), 1988). The reaction is carried out overnight at a temperature of 25oC. the Product precipitated three times with cold acetone, dissolved in medium RPMI 1640 and sterile filtered through a filter with a pore diameter of 0.22 μm. (Polyclonal antibody used in this experiment, interact with acidic glial fibrillar protein).

Example 2. Itinerancy target part of the molecule

Antibody to2GP mark125I using reagent Bolton-hunter (Bolton-Hunter) in the system of reversed micelles of AOTin octane according to the method described Slepnev et al. (Slepnev et al. Bioconjugate Chem., 3, 273-274 (1992)). Specific radioactivity125I-labeled proteins varies from 19 to 21 Ci/mol.

Wistar rats (body weight 80 g, 8 animals per group) injected/in (0.1 ml/10 g body weight) composition, including125I-) copolymer Pluronic L64, dissolved in the medium RPMI 1640. These copolymers, and all others mentioned in the examples, the copolymers obtained from Serva chemicals (Germany).125I-labeled polypeptide dissolved in medium RPMI 1640, administered in the same concentration. Three days later slaughtered and examined for radioactivity selected tissue samples to determine its distribution in tissue, as described in the work Chekhonin et al. (Chekhonin et al. , FEBS Lett., 287: 149-152 (1991)). The distribution of radioactivity was quantitatively assessed using a liquid-scintillation counting. The experiment is repeated at least twice and the obtained results are reproduced with less than 10% variation.

In table. presents the results, expressed as the ratios of radioactivity in the brain radioactivity analyzed in this tissue (C. O.).

Example 3A. Cytotoxicity for resistant cancer cells

Pluronic R dissolved in medium RPMI 1640 [ICN Biochemicals, Inc., (ICN Biomedicals Inc. , Costa Mesa, CA)] to a final concentration of 1% and then the resulting solution is sterilized by filtration for removal of bacterial or fungal contamination. This solution Pluronic R used for obtaining the appropriate dilutions erased the>/P>In studies of cytotoxicity using a line of transformed cells SKOV3 (in the present description - SK-cells and derived cell line SKVLB (in the present description - SK-resistant cells). Both of these cell line was provided by Dr. Ling (V. Ling) from the University of Toronto. SK-resistant cell line is a cell line with multidrug resistance, which is obtained from SK-cell line with long-term cultivation of it in the presence of vinblastine.

In medium RPMI or in solution Pluronic R described above, make different breeding of many anticancer agents. For this experiment, prepare the cells, placing equal volumes of cell suspension (2000-3000 cells) cell 96-alopecia microtiter tablet [Costar Cambridge, MA (Costar, Cambridge, MA)] and cultivating them within 2 days. All processes of the cultivation is carried out at a temperature of 37oC and when the content in an atmosphere of 5% CO2. Then add 100 ál to each well of fresh medium (RPMI medium 1630 with the addition of 10% serum amniotic calf). Then in cells contribute 100-ál volumes of free anti-cancer agent or copolymer plus breeding anti-cancer agent. Klechkovychi cells washed three times with fresh medium. Then the washed cells were cultured in fresh medium for another four days.

The number of viable cells in each culture is evaluated by the method of standard XTT assay, which allows to determine the activity of mitochondrial enzymes. (See Scudiero et al. Cancer Res., 48: 4827 (1988)). To the cells was added 50 μl per each well of sterile XTT concentration of 1 mg/ml (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl] - 2H-tetrazole-5-carboxanilide inner salt. Sigma (Sigma, St. Louis, MO) in RPMI-1640 containing 5 μl/ml phenazine of methosulfate (Sigma) in the FBI in the concentration of 1.54 mg/ml. Cells incubated for 16 hours, after which in each cell determine the level of absorption at 450 nm. For each value determines SEM (mean value of three determinations), which always remains within 10% of the value. The value MK50(i.e. the concentration at which achieved 50% inhibition) is determined by extrapolation on the graph, where the number of viable cells (i.e., the activity of mitochondrial enzymes) applied in accordance with the concentration introduced into the cells of the medicinal product. For SK-resistant cells were obtained the results presented in table. 5.

The data in the first the practical form in Fig. 1, where the concentration of drugs plotted on a graph against the activity of mitochondrial enzymes in SK-resistant cells, and as for the free drugs (line 1) and its micellar form (line 2). In Fig.1 shows the corresponding data for SK-cells and also for the free form of daunorubicin and its micellar form (line 3 and 4, respectively).

Example 3B - Processing SK-resistant cells with different agents.

In the experiment with SK-resistant cells apply the procedure described in example 3A. Cells were provided by Dr. Ling (Dr. V. Ling) from the University of Toronto. Results have been presented in table. 6.

Example 3B - Kinetics of accumulation of daunorubicin

The kinetics of accumulation of daunorubicin in SK-cells and SK-resistant cells when processing their daunorubicin at a concentration of 10 ng/ml determined by measuring the fluorescence of accumulated cells daunorubicin (ex= 471 nmeat= 556 nm). In Fig. 2 presents the data on accumulation of specified drugs in SK-resistant cells (line 1: free medication; line 2: micellar form); Fig. 2 presents data for SK cells (line 3: free lekarstvennoj

In these experiments, use CHrC5 cell line carcinoma of the ovary of the Chinese hamster (presented by Dr. Ling of the University of Toronto) and the method of example 3A. When using Pluronic L61 concentration introduced into the cells of the copolymer is 0.01% (weight/volume); when using Pluronic R this concentration is 0.01% (weight/volume); when using Pluronic F108 this concentration is 0.01% (weight/volume); and when using Pluronic F68 this concentration is 5.0 % (weight/volume). Were the values obtained IR50presented in table. 7.

Example 3D - Titration copolymer

Was used the method of example 3A except for two details. The first difference was that instead of S cells using doxorubicin-resistant MCF7 cells (MCF7 - ADRccells, which will be described further in example 21). The second difference was that in addition to variations in concentrations of doxorubicin in this experiment were also used different concentrations of the copolymer. Figure 3A shows the percentage of inhibition in cultures that have different concentrations Pluronic L61 in response to changes in the concentration of doxorubicin. Line 1 refers to doxorubicin; the line is outstay 0.3 to 10-5M Pluronic L61; line 4 refers to doxorubicin in the presence of 0.16 10-4M Pluronic L61; line 5 refers to doxorubicin in the presence of 0.8 to 10-4Pluronic L61; line 6 refers to doxorubicin in the presence of 0.4 to 10-3M Pluronic L61; and line 7 refers to doxorubicin in the presence of 0.4 to 10-1Pluronic L61. In Fig. 3B, these data are combined so that the figure illustrates the value MK50for doxorubicin introduced into cells in the presence of these varying concentrations Pluronic L61.

Example 4 - Cytotoxicity copolymer

Cells MCF7 - ADRwith(cells resistant to doxorubicin, which will be described later in example 21) incubated with Pluronic L61 at different concentrations and by the method of example 3A determine cytotoxicity. The results obtained are shown in Fig. 3V.

Example 5A - Distribution of the polymer in the body

From the Institute of atomic energy. Kurchatov (Moscow, Russia) receive radioactive tritium-labeled derivatives Pluronic R. Introducing/in 100 μl per 20 g of body weight of 1% (weight/volume) isotonic radioactive copolymer (2107imp./min/20 g body weight) (a) mice of BALB/c (obtained from the Veterinary Department R is eriment were injected with 3106mouse myeloma cells SP2/0dnr(described in example 9A). Distribution of the polymer in the body in different periods of time after injection of radioactive polymer was measured after killing at different time points treated animals, tissue excision, listed in the table below, and quantitative determination of the distribution of radioactivity using liquid scintillation counting. For preparation of samples for liquid scintillation account such samples are placed in 1 ml of tissue solubilizer (available from Serva chemicals, Germany) and homogenized in the cold. The resulting homogenates incubated for 14 hours at room temperature, discolor by adding 50 μl of 30% hydrogen peroxide and incubated over night at room temperature. For BALB/c mice that were not injected tumor, were obtained the results presented in table. 8.

For mice BALB/C injected with tumor were obtained the results presented in table. 9.

From this series of experiments were obtained additional observations related to the fact that (1) the degradation products of the polymers are not detected until 24-hour time point after administration of polar 6A - The concentration of copolymer in the blood

6-week old mice of BALB/c injected/100 μl/20 g body weight [3H]- Pluronic R of example 4. In Fig. 4 shows the levels of radioactivity detected in the blood of mice at different time points after injection (dark line) and in the liver (dotted line).

Example 6B - Concentration of copolymer in the blood

6-week old mice of BALB/c enter/injection or oral 100 μl/20 g body weight [3H]-Pluronic R of example 4. In Fig. 5 shows the levels of radioactivity detected in the blood of mice at different time points after injection, where the first arrow in each pair points to the/in the injected polymer, and the second arrow on oral introduced polymer.

Example 7 Pharmacokinetics of daunorubicin and daunorubicinol

Metabolism of daunorubicin followed by means of HPLC method. Daunorubicin (Sigma, St Louis, MO) introducing/7 week old mice C57B1/6 of 10 mg/kg body weight using as a carrier of saline or saline containing 1% (weight/volume) Pluronic 85. The injection volume is 100 μl/20 g body weight. At various times after injection, animals kill and spend extracts triperoxonane acid (TFU). The solubilized extract contribute to the column for HPLC with treatment phases 4,h mm C18 [15 microns of Ultrastar, Beckman, USA (Ultrasphere, Beckman)]. Through the column flow gradient of acetonitrile from 0 to 40 in 0.1% TFU, identify and quantify peaks of daunorubicin and its metabolite - daunorubicinol. Figure 6A shows the concentration of daunorubicin in the liver at 150 minutes after injection, while the arrow a indicates the level of free daunorubicin (μg to 10 mg liver tissue), and the arrow In indicates the level in the composition with the copolymer. Fig. 6B illustrates the timing of the accumulation in the blood of mice introduced free daunorubicinol (line 1) and its copolymer of the form (line 2). Fig. 6B illustrates the timing of the accumulation in the blood of mice entered the free daunorubicin (line 1) or a copolymer of the form (line 2).

Example 8 - Acute toxicity

Acute toxicity Pluronic F108, R and L61 study on 5-week-old male mice of BALB/c. Each experimental group consists of 6 mice. Intraperitoneally administered various doses of isotonic solutions Pluronic. Daily for 14 days tracking the mortality of animals. The value of LD50and maximiliam) is calculated using proitalia. (CM. Chan and Hayes in Principles and Methods of Toxicology, Hayes, A. W., ed. Raven Press, New York, 1989, pp. 169-189). Results have been presented in table. 10.

Example 9A - Treatment tumor

Line of tumor cells with multidrug resistance receive a series of passages of the cell line murine myeloma SP2/0 in the presence of 100 ng/ml of daunorubicin. It was shown that the stable cell line SP2/0dnr10 times more resistant to epirubicin (IR50= 0,7 µg/ml for the parent cell line; IR50= to 8.0 μg/ml for line SP2/0dnr). And when such cells are used for the formation of tumors in mice and 50 days after the development of solid tumors secrete cells from tumors, such highlighted cells show the same resistance.

6-week old mice BALB/c mice injected subcutaneously with cells of the SP2/0drr(3 of 105cells). When processing on day 0, which begin 14 days after injection of tumor cells, mice injected/injection of 20 μl/20 g body weight (1) saline, (2) isotonic solution of epirubicin (5 mg/kg body weight) or (3) isotonic solution of epirubicin, dissolved in 1% Pluronic R (1 mg/kg body weight). In Fig. 7 shows the results, expressed as changes from the cylinder the 60-day course of treatment. Similar results were obtained with the use of daunorubicin and Pluronic L61 and F108.

Example 9B - Optimal therapeutic dose

Was used the same procedure as in example 9A, except that it was taken parental cell line (SP2/0, not to be resistant cell line), and the copolymer and its concentration are presented in table. 11.

If Pluronic F108 optimal antitumor efficacy was achieved using 10% of the copolymer. For Pluronic R optimal antitumor efficacy was achieved using 1% of the copolymer. For Pluronic L61 optimal antitumor efficacy was achieved using 0.1% of the copolymer. These values were taken as the optimal therapeutic doses (CTD) for the corresponding copolymers.

Example 9B - treatment with the use of the copolymers in amounts corresponding CTD

The procedure of example 9B quantity of each: Pluronic F108, Pluronic R and Pluronic L61 - were taken equal to the CTD as carriers for epirubicin. The obtained results are shown in figure 8 (the ordinate shows the ratio V/Vo).

Example 10

Blue 30 minutes at 37oC and then sterilized by filtration through a 0.22 μm filter. To this mixture is added an equal volume of a solution of 200 mg of daunorubicin in medium RPMI 1640 and the resulting mixture is incubated for 30 minutes at 37oC.

Human cell carcinoma of the ovary (cells CRL157) will precultured in 1% solution Pluronic F68 in medium RPMI 1640 containing 10% serum amniotic calf. Add a solution of daunorubicin in Pluronic and the mixture is incubated for 60 minutes at 37oC, after which the cells washed three times with RPMI medium 1640 and cultured in medium RPMI 1640 containing 10% amniotic calf serum for 3 days. Determine the cytotoxicity for this drug, and for parallel drug free daunorubicin, using the method of Alley et al. (Alley et al. Cancer Res., 48, 589-601 (1988)). Results have been presented in table. 12.

Using the same procedure determine the cytotoxicity to cells of the human T-lymphoma (Jurkat) (see tab. 13).

Using the same procedure determine the cytotoxicity for small cell lung carcinoma human (H-69) (see table. 14).

Example 11

Block copolymers of poly(oksietilenom)-poly(oxypropylene), characterized coefficien lower concentrations. The mixture is incubated for 40 minutes at 30oC. the Average diameter of the micelles was measured by the method of quasielastic light scattering. (See Kabanov et al. Macromolecules 28: 2303-2314, 1995). Thus were obtained the results presented in table. 15.

Example 12

The mixture Pluronic P85 and Pluronic L64 in the ratio 1:1.5, which have individual coefficients (n) relations (oxypropylene) units to (oxyethylene) in the range from 1.00 to 1.50, respectively, and the combined value of N equal to 1.30, dilute with medium RPMI 1640 to a final concentration of 2.0 % at 4oC. the Mixture is incubated for 30 minutes at 37oC and then sterilized by filtration through a filter with a pore size of 0.22 μm. Add an equal volume of a solution of 200 mg of daunorubicin in medium RPMI 1640 and incubated the mixture for 30 minutes at 37oC.

Determine the cytotoxicity to human cells of ovarian cancer (CRL157) for this drug, and parallel to free daunorubicin, as described in example 3A. Results have been presented in table. 16.

Composition of daunorubicin to assess cytotoxicity for (i) cells of the human T-lymphoma (Jurkat) and (ii) normal managernew human cells. Results were obtained that predse IR50regarding (i) the cells of the human T-lymphoma (Jurkat) and (ii) normal managernew human cells and compare them with the corresponding values for the free daunorubicin. The measurements were carried out at the specified time intervals contact drugs with cells. While getting the results presented in table. 18.

Example 14

The antitumor agent vinblastine include in block-copolymer mixture described in example 12. Value IR50this drug is relatively SK cells was defined as 0,121 µg/ml; value IR50against SK-resistant cells 0.0012 µg/ml Value IR50for free vinblastine relatively SK cells was determined in the amount of 0,095 μg/ml and the amount of 0.615 against SK-resistant cells.

Example 15

Anticancer agent mitomycin C include in block-copolymer mixture described in example 12. Value IR50this drug is relatively SK cells was defined as 0,265 µg/ml; value IR50against SK-resistant cells is 0,005 µg/ml Value IR50for free mitomycin C relative to SK cells was determined in the amount of 0,320 µg/ml; value IR50against SK-resistant glue the polymer mixture, described in example 12. Value IR50this drug is relatively SK cells was defined as 0,880 g/ml; is IR50against SK-resistant cells is 0,0175 µg/ml Value IR50for free

methotrexate relatively SK cells was determined in the amount of kgs 1,090 mg/ml; and against SK-resistant cells is 1,340 mg/ml

Example 17

Antitumor agent include colchicine in block-copolymer mixture described in example 12. Value IR50this drug is relatively SK cells was determined as determined as 0.720 mg/ml; value IR50against SK-resistant cells is 0.045 µg/ml Value IR50for a free colchicine relatively SK cells was determined in the amount of 0,950 μg/ml; and against SK-resistant cells is 7,450 µg/ml.

Example 18

The antitumor agent daunorubicin include in block-copolymer mixture described in example 12. Value IR50this drug is relatively SK cells was defined as 0,600 µg/ml; value IR50against SK-resistant cells is 0,0068 µg/ml Value IR50to free daunorubicin relatively SK cells was determined in the amount of 0,620 μg/ml; and against SK-ostoich phosphate buffer solution with a concentration of 20 mg/ml add 30 μl of a solution of daunorubicin in a mixture of blockcopolymer, described in example 12. Simultaneously prepare the second composition on the basis of free daunorubicin.

Preparations incubated for 10 min at 25oC and then analyzed by HPLC on a column of TSK-3000 SW gel filtration in the FBI, containing 0.3 M sodium chloride and 5% acetonitrile. Detection was performed at 280 nm and 470 nm. Part associated with the BSA, is determined from the formula:

Db = Sb/Sf

where Sb denotes the relative area of the peak at 470 nm (corresponding to daunorubicin), which coincides with the retention time for the peak at 280 nm (corresponding BSA); and

Sf denotes the relative area of the peak (or peaks) corresponding to daunorubicin(s) does not match (s) in retention time with the peak of the BSA.

The results presented in the table. 19.

Example 20

Micellar daunorubicin obtained by the method of example 12, and free daunorubicin incubated in the dark at 37oC and using the example 1 method to determine the cytotoxicity for cells CRL157 (cells from human carcinoma of the ovary).

Results have been presented in table. 20.

Example 21

Daunorubicinol composition according to example 12 and free down the ear cell lines, resistant to drugs: daunorubicin/verapamil-resistant cells (MCF-7AU), not expressing P-170, and daunorubicin-resistant, verapamil-sensitive (MCF7-ADRc) expressing P-170. These cells were obtained from cell Bank of Moscow Research Center of molecular diagnostics, Moscow, Russia. Results have been presented in table. 21.

Free daunorubicin demonstrates higher IR50(i.e., is less toxic) against both resistant lines. And Daunorubicin, are included in blakesophiey composition demonstrates a lower IR50(i.e., is more toxic) against both resistant lines.

Example 22

Animals in groups (6 animals per dose), including 7-week-old female mice of C57 B1/6 inoculant intraperitoneally daunorubicin, free and micellar obtained by the method of example 12. The mice are observed for 14 days. Concentrations of drugs are selected so that each mouse to be injected volume, amounting to a maximum of 0.5 ml.

Determine the MTD as the maximum dose that does not lead to lethality from daunorubicin (any higher dose in autonaut twice. The results are reproduced with less than 10% variation.

It was shown that the MTD for free and micellar daunorubicin, respectively 2.0 and 1.0 microgram/kg body weight.

Example 23

Daunorubicin causes the repression of the bone marrow and leads to the development of reversible leukopenia, i.e., decrease in leukocyte count (low white blood cells), during drug administration. Suppression of the bone marrow, as well as the anticancer activity of daunorubicin linked, as is, with its DNA-binding activity, whereas it is more harmful side effects anthracyclines as cardiotoxicity, associated mainly with the appearance of metabolites (which have low anti-cancer activity and does not exert any appreciable influence on the bone marrow). For this reason, the level of blood when introduced in vivo MTD daunorubicin used to evaluate the ratio between the specific (DNA-binding) activity of the medicinal product and its non-specific toxicity.

Animals in groups (6 animals per group), including 7-week-old female mice of S B1/6 inoculant intraperitoneally daunorubicin, free and micellar obtained by the method of example 12. The pharmacy is ISI to be injected volume, amounting to a maximum of 0.5 ml are Taken blood samples and count the number of viable leukocytes, as described in the work of Maisha et al. (Michisch et al., Proc. Natl. Acad. Sci. USA 88, 547-551 (1991)). The number of cells determined after injection of 0.1 ml of the FBI, is used as the reference value. This value is 15-16 million cells/ml Experiment was repeated twice. The results are reproduced with less than 10% variation.

The results obtained are presented in table. 22.

Example 24

Examine the impact of free and micellar daunorubicin obtained by the method of example 12, the number of cells three days after injection, as described in example 23.

Results have been presented in table. 23.

The data given in examples 22-24 show that solubilization of daunorubicin in block-copolymer micelles are not affected in a noticeable extent on the General toxicity of the drug (which is MTD in 2 mg/kg and 1 mg/kg for free and micellar drug, respectively), whereas this solubilization increases bone marrow suppression, which leads to increased anti-cancer actions.

Example 25

Antitumor activity of the composition (see Valeriola et al., Cancer Chemother. Pharmacol., 29, 133-140, 1991).

Animals in groups (6 animals per group), including 7-week-old female mice of S B1/6 inoculant/(via tail vein) daunorubicin, free and micellar obtained by the method of example 12. Pharmaceutical composition comprising MTD for the corresponding composition adjusted so that each mouse to be injected volume, amounting to a maximum of 0.1 ml. Experiment was repeated twice. The results are reproduced with less than 10% variation.

To obtain samples of blood plasma (10 µl) was taken from the tail artery through hour after administration of the medicinal product, diluted with sterile RPMI medium 1640 at a ratio of 1:10 and then centrifuged at 400g for 15 minutes. The resulting supernatant diluted as indicated in the table using plasma obtained in a similar way from mice that did not inoculable drug (plasma of mice that were not inoculated drug, no significant cytotoxic effect on H-69 cells), and mixed with an equal volume of a suspension of N-69 cells in medium RPMI 1640 containing 10% serum amniotic calf. Cells are incubated for two hours at 37ed RPMI 1640, containing 10% amniotic calf serum, at 37oC and 5% CO2within three days, after which determine cytotoxicity according to the method of example 10.

Get the results, presented in table. 24.

Thus, the serum of mice that were injected micellar composition, characterized by a higher cytotoxicity.

Example 26

Repeat the procedure of example 25 using SK-cells and SK-resistant cells. The results obtained are as follows:

a) When mice injected composition with daunorubicin dose equal to the MTD, and measure the cytotoxicity of the resulting plasma, the results look that way, as in table. 25.

b) When each mouse injected with 10 mg/kg daunorubicin, was found in the resulting plasmas have the values given in the table. 26.

Example 27 - Processing Candida fluconazole Cultural environment

Used to test the sensitivity of the environment represents the environment of high resolution (HR). This environment is optimal for testing, and it is prepared in two parts as follows. Part A consists of 0.2 M phosphate buffer, pH 7.5, prepared using Na2o
C. Before use, mix equal amounts of part a and part B.

Organisms

Used in this study organisms represent a fresh clinical isolates of Candida species or indicator organisms used in routine studies of sensitivity to antifungal agents. Selected isolates range of sensitivity of fluconazole, ranging from sensitivity to fluconazole (minimum inhibitory concentration (MIC) is 0.2 µg/ml) to resistance to fluconazole (MIC = 100 μg/ml). All isolates are subculturing on agar Saburo and incubated for 48 hours prior to determination of sensitivity.

Preparation of yeast suspensions

The yeast suspension is prepared by taking five individual colonies and suspending them in sterile water to obtain a slightly turbid suspension. Then the suspension is diluted in the ratio 1:100 in HR environment with obtaining a suspension which contains 2 to 104organisms/ml.

Complexes of polymer/fluconazole

Polymers derived from the company Serva Who is cut filter with pore size of 0.4 μm (Sartorius) to ensure sterility.

Preparation of dilutions fluconazole/polymer

First appreciate five polyester block copolymers on the efficacy of fluconazole in the presence of the polymer and in his absence. For cultivation solutions fluconazole/polymer use solutions containing the same polymer. By such dilution receive a standard solution containing 1250 µg/ml of fluconazole. From the standard solution further do cultivation in the polymer to obtain concentrations of fluconazole in the range from 1250 to 2.5 µg/ml as a control use of dissolved fluconazole in concentrations of from 1250 to 2.5 µg/ml.

Determination of minimum inhibitory concentration (MIC)

To determine MIC add 8 ál of each dilution of fluconazole in the polymer to the cell in sterile microtiter plateau. Prepare two sets of controls, which contain either 8 ál of water, or 8 µl of fluconazole without polymer. The first serves as a control for the absence of medication, while the second allows you to evaluate any influence of the polymer on the yeast cells. Then for each cell up to a volume of 100 ál add the HR environment. And at the end add 100 ál of each yeast suspension to the appropriate series of dilutions of fluconazole. Each d is to believe the desired size of the inoculum. Each yeast suspension is then examined against fluconazole at final concentrations of 50 - 0.1 ág/ml in five different polymers or water. The final concentration of each polymer in each cell is 4% and the final concentration of yeast cells is 1 to 104organisms/ml Microtiter plateau gently mixed and then incubated for 48 hours at 37oC in a humid atmosphere. After 24 hours, the plateau is assessed visually. After 48 hours, the plateau is placed on a rocking chair for mixing for five minutes to the suspension of yeast cells in the environment and then measure OP490using sterile culture medium as blind samples.

Interpretation MICK

In the case of each organism assess its growth in the cell with the positive control, free from drugs, and the result is compared then with the cell that contains the control on the polymer, and if OP490polimersoderzhashchie cell is < 90% of the OP490for control cells, conclude that the polymer inherent inhibitory ability against the body and MICK cannot be assessed. If OP490polymer control can be carried on the remaining results.

For each polymer and each organism as the value MICK take the lowest concentration of fluconazole, which reduces growth (OP490up to 50% of the OP490positive control. We used the following valuation assumptions for the study isolates: < 6,2 mg/ml - sensitive; 6,2 and 15.2 ág/ml - intermediate sensitivity; and > 12.5 µg/ml is stable. The results obtained are presented in table. 27.

Example 28 - TNF Activity

Cytotoxic activity of TNE relatively stable SK-cells determined using XTT analysis, as described in example 3A. TNE added to the cells for 24 hours in various concentrations: 1) free TNF ; 2) TNF 0.1% Pluronic P85 and 3) TNF 0.01% Pluronic L61. After 24 hours incubation analyze with TNE cells are washed with medium RPM1 and analyzed by XTT method. All experimental points are examined in the triple repetition. Data represent average values of standard error. The results are shown in table. 28.

Example 29 - the Treatment of experimental tumors-gliomas

Antibodies (Ab) for GFAP and d2-glycoprotein modify the remnants of stearic acid as described in example 1. They are also covalently bind with Pluronic R as op is you want to make doxorubicin in the treatment of gliomas. Glioma C6 inoculant vnutriarterialno groups (n=25) male mice of sprag-Share (Sprague-Dawley), weighing 280-300 g), obtained from a nursery in Kryukovo, Russian Academy of Sciences. After 10, 15, 20 and 25 days after inoculation injected intraperitoneally in a volume of 1 ml/300 g body weight) (a) 10 mg/kg of free doxorubicin, (b) doxorubicin in 1% Pluronic R, (C) doxorubicin 10% Pluronic R containing 0.1 mg/ml Ab, modified chloride stearic acid, and (d) doxorubicin 10% Pluronic R containing 0.1 mg/ml Ab, associated with Plutonium. The controls do intraperitoneal injections of equal volume of saline. Daily clinical observations. Animals are weighed weekly for the first two months and monthly thereafter. Check vital signs, to make sure that the animal died, and within 5 minutes after the death of the animal are necropsy. Analyze the data on survival of animals in order to rank the impact of drugs on the frequency and latency of tumors. The data below represent the ratio of the average time of survival in the treatment group (T) and in the control group (C). For necropsy take the t in formalin together with the tissues of the animal. Whole brains were removed and cut in three different positions. Select three different slice of the spinal cord in the cervical, thoracic and lumbar levels. Cut the sample is placed in a cassette for tissue Tissue Tek and processed in the processor to tissues. The tissue sections are cut into sections with a thickness of 4-6 mm by using a microtome and stained them with hematoxylin-eosin. Using histopathological studies of the brain assess: (i) the total number of tumors in animals; (ii) the number of animals with tumors; and (iii) conduct histopathological classification and grading of tumors. The results obtained are presented in table. 29.

Histopathological studies revealed that 1) free doxorubicin does not affect the size and number of tumors compared to controls; 2) all 3 micellar compositions cause a significant reduction in the size and number of tumors; 3) the most pronounced effect is observed in the case of micellar doxorubicin + starolitovsky antibodies, and in this case the tumor is practically not observed.

Example 30 - Inhibition of the formation of metastases

Cells N of Lewis lung carcinoma (2105) injected subcutaneously 6-week old male mice of C57B1/6. Mice, umemulo method described in the literature (Wilmanns, C.; Fan, D; O Brian, C. A. et al., (1992) Int. J. Cancer. Inst., 52, 98-104). Injection of cancer cells leads to the formation of solid tumors at the injection site and metastasized tumors in the lung and liver. Selected samples of tumors from two mice and adapt to cultivation as described in the literature method (Dong, Z.; Radinsky, R.; Fan, D.; Tsan, R; Bucana, C. D.; Wilmans, C. and Fidler, I. J. (1994) Int. J. Cancer Inst., 86, 913 - 920). The sensitivity of in vitro cell samples for free and micellar doxorubicin (0.01% Pluronic L61) is determined using an XTT analysis, as described in example 3A. The results are presented in Fig. 9. The cells of the liver metastases are characterized by their resistance to free doxorubicin. In the case of micellar drug that reverses resistance. The cells of the liver metastasis, sensitive to the free drug, have value IR50approximately equal IR50celebrated in the parent cell line N. However, the cytotoxicity against cells of liver metastases in micellar drug remains significantly higher cytotoxicity than free drug.

Example 31

A composition suitable for parenteral administration, prepare the dissolution of the 37oC and then sterilized by filtration through a filter with a pore size of 0.22 μm. The filtered solution is mixed with a solution of 10 mg of sterile lyophilized powder daunorubicin, dissolved in 50 ml of RPMI medium and incubated for 30 minutes at 37oC.

The composition can be stored in the dark at room temperature for 7 days without loss of activity, or it can be dried and be stored for at least 1 year in the dark at room temperature.

Example 32

Another composition suitable for parenteral administration, is prepared by dissolving 400 mg Pluronic P-85 and 600 mg Pluronic L-64 in 50 ml FBI at 4oC. the Mixture is incubated for 30 minutes at 37oC and then sterilized by filtration through a filter with a pore size of 0.22 μm. The filtered solution is mixed with a solution of 1 mg of sterile lyophilized powder of daunorubicin and 5 mg of glucose dissolved in 50 ml of the FBI, and the mixture is incubated for 30 minutes at 37oC.

The composition can be stored in the dark at room temperature for 7 days without loss of activity, or it can be dried and be stored for at least 1 year in the dark at room tempera is rhenium 100 mg of sodium ascorbate in 100 ml of a 9% aqueous solution of sodium chloride. Half of the solution is added at 4oC 400 mg Pluronic P-85 and 600 mg Pluronic L-64. The mixture is incubated for 30 minutes at 37oC and then sterilized by filtration through a filter with a pore size of 0.22 μm. Separately in the remainder of the ascorbate-sodium-chloride solution dissolve 10 mg of sterile lyophilized powder of daunorubicin and 50 mg of glucose, two solutions are mixed and incubated for 30 minutes at 37oC.

The composition can be stored in the dark at room temperature for 30 days without loss of activity, or it can be dried and be stored for at least 1 year in the dark at room temperature.

Example 34

Another composition suitable for parenteral administration, is prepared by dissolving 100 mg of sodium ascorbate in 100 ml of a 9% aqueous solution of sodium chloride. To this solution was added at 4oC 10 mg Pluronic L-61. The mixture is incubated for 30 minutes at 37oC and then sterilized by filtration through a filter with a pore size of 0.22 μm. The solution is Packed together with a container containing 10 mg of doxorubicin.

1. Composition comprising an anti-cancer chemotherapeutic agent and a polyester block copolymers having the critical end of the thief, capable of forming liquid composition when mixed with water, the specified block copolymers includes many linear polymer segments, repeating units which have a molecular weight between about 30 and 500, in which at least 80% of the links connecting the repeating unit of each of the polymer segments, includes simple radio link.

2. The composition according to p. 1, characterized in that it contains micelles that include polyester block copolymers or forms micelles containing polyester block copolymers, in the process of introduction into the organism of an animal or thereafter.

3. The composition according to p. 2, characterized in that at least about 0.1% of the chemotherapeutic agent is contained in the micelles prior to the introduction or implementation process.

4. The composition according to p. 3, characterized in that at least about 1% of the chemotherapeutic agent is contained in the micelles prior to the introduction or implementation process.

5. The composition according to p. 1, wherein the polyester block copolymers comprises at least one polyester segment specified polyester segment includes a homopolymer oksietilenovomu monomer-och2CH2- or copolymer, or block copolymers specified OK RIT about 5 to 400 Monomeric units.

6. The composition according to p. 1, characterized in that the said repeating units for each of the polymer segments have a molecular weight from about 30 to 100.

7. The composition according to p. 6, characterized in that at least about 90% of the links connecting the repeating unit of each polymer segment include ether linkages.

8. The composition according to p. 1, wherein each polymer segment consists essentially of recurring units of the formula-O-R5where R5represents -(CH2)n-CH(R6)-, where n represents zero or an integer from about 1 to 5 and R6denotes hydrogen, cycloalkyl containing from about 3 to 8 carbon atoms, alkyl containing from about 1 to 6 carbon atoms, phenyl, alkylphenyl, in which the alkyl contains from about 1 to 6 carbon atoms, hydroxy, hydroxyalkyl, in which the alkyl contains from about 1 to 6 carbon atoms, alkoxy containing from about 1 to 6 carbon atoms, alkylaryl containing from about 2 to 7 carbon atoms, alkoxycarbonyl, in which the alkoxy contains from about 1 to 6 carbon atoms, alkoxycarbonylmethyl, in which the alkoxy and alkyl each independently from each contains from about 1 to 6 carbon atoms is of Pereda, aminoalkyl, in which the alkyl contains example from 1 to 6 carbon atoms, alkylamino or dialkylamino in which each alkyl contains independently from each other from about 1 to 6 carbon atoms, mono - or dialkylaminoalkyl, in which each alkyl contains independently from each other from about 1 to 6 carbon atoms, chlorine, chloroalkyl, in which the alkyl contains from about 1 to 6 carbon atoms, fluorine, foralkyl, in which the alkyl contains from about 1 to 6 carbon atoms, cyano or cianelli, in which alkyl contains from about 1 to 6 carbon atoms, or carboxyl; carbocyclic group which has the ring from about 3 to 8 carbon atoms, examples of which can be groups such as cycloalkyl or aromatic group, and which may include alkyl containing from about 1 to 6 carbon atoms, alkoxy containing from about 1 to 6 carbon atoms, alkylamino containing from about 1 to 6 carbon atoms, dialkylamino, in which each alkyl independently has about 1 to 6 carbon atoms, amino, sulfonylurea, hydroxy, carboxyl fluorine or chlorine substituents or a heterocyclic group which has the ring from about 3 to 8 carbon atoms, which may include hetero the s group, consisting of oxygen, nitrogen, sulfur and mixtures thereof, and which may include alkyl containing from about 1 to 6 carbon atoms, alkoxy containing from about 1 to 6 carbon atoms, alkylamino containing from about 1 to 6 carbon atoms, dialkylamino, in which each alkyl independently has about 1 to 6 carbon atoms, amino, sulfonylurea, hydroxy, carbocyanine, fluorine or horsenettle.

9. The composition according to p. 1, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 50%.

10. The composition according to p. 9, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 60%.

11. The composition according to p. 1, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 900.

12. The composition according to p. 11, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 1700.

13. The composition according to p. 12, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 2000, and the weight percent hydro is lar weight of the hydrophobic parts of the polyester blockcopolymer is at least about 2300 and the weight percentage of the hydrophobic portion is at least about 20%.

15. The composition according to p. 1, characterized in that the polyester block copolymers has the formula

< / BR>
where n and m denote integers from about 4 to 400.

16. The composition according to p. 15, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 50%.

17. The composition according to p. 16, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 60%.

18. The composition according to p. 15, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 900.

19. The composition according to p. 18, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 1700.

20. The composition according to p. 15, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 2000, and the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 20%.

21. The composition according to p. 20, characterized in that the molecular weight of hydrophobin and polyester blockcopolymer is at least about 20%.

22. The composition according to p. 15, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer approximately from 1500 to 2000, and the weight percentage of the hydrophobic part is about 85 to 95%.

23. The composition according to p. 15, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is from about 3000 to 3500, and the weight percentage of the hydrophobic portion is approximately from 15 to 95%.

24. The composition according to p. 15, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is about 3500 to 4000, and the weight percentage of the hydrophobic part is about 25 to 35%.

25. The composition according to p. 1, wherein the anti-cancer chemotherapeutic agent is an alkaloid Winky, antibiotic mitomycin type, type antibiotic bleomycin, antifolate, colchicine, dimebolin, etoposide, Texan or anthracycline antibiotic.

26. The composition according to p. 25, wherein the anti-cancer chemotherapeutic agent is doxorubicin, daunorubicin, karminomitsin, epirubicin, idarubitsin, mitoxantrone, 4-dimethoxy-daunomycin, 11-dezoksiguanosin, 13-dezoksiguanosin, adriamycin-14-b is, however, what anti-cancer chemotherapeutic agent comprises an effective amount of at least one cytotoxic drug selected from the group consisting of anthracycline and means causing multidrug resistance.

28. A composition comprising a biological agent and a polyester block copolymers that can form a liquid composition when mixed with water, and the specified block copolymers includes many linear polymer segments, repeating units which have a molecular weight between about 30 and 500, and at least 80% of the links connecting the repeating unit of each of the polymer segments, includes simple radio communication, and polyester block copolymers has a weight percentage of the hydrophobic parts of at least about 50%, and polyester block copolymers has a molecular weight of the hydrophobic parts of at least about 900, or composition includes one or more specified polyester block copolymers, which have a CMC of about 0.5% (weight. /about.) or less at 37°C in isotonic aqueous solution.

29. The composition according to p. 28, characterized in that the polyester block copolymers comprises at least one polyester segment, indicated the or block copolymers specified oksietilenovomu monomer and monomer-och(CH3)CH2and each of these polyester segments contains from about 5 to 400 Monomeric units.

30. The composition according to p. 29, wherein the repeating unit of each polymer segments have a molecular weight from about 30 to 100.

31. The composition according to p. 30, characterized in that at least about 90% of the links connecting the repeating unit of each polymer segment includes a simple radio link.

32. The composition according to p. 4, wherein each polymer segment consists essentially of recurring units of the formula-O-R5where R5means -(CH2)n-CH(R6)-, where n represents zero or an integer from about 1 to 5 and R6denotes hydrogen, cycloalkyl comprising from about 3 to 8 carbon atoms, alkyl containing from about 1 to 6 carbon atoms, phenyl, alkylphenyl, in which the alkyl contains from about 1 to 6 carbon atoms, hydroxy, hydroxyalkyl, in which the alkyl contains from about 1 to 6 carbon atoms, alkoxy comprising from about 1 to 6 carbon atoms, alkylsulphonyl, including from about 2 to 7 carbon atoms, alkoxycarbonyl, in which the alkoxy contains from about 1 to 6 carbon atoms, alkoxy is erode, alkylcarboxylic, in which each alkyl includes independently from each other from about 1 to 6 carbon atoms, aminoalkyl, in which the alkyl contains from about 1 to 6 carbon atoms, alkylamino or dialkylamino, in which each alkyl contains independently from each other from about 1 to 6 carbon atoms, mono - or dialkylaminoalkyl, in which each alkyl contains independently from each other from about 1 to 6 carbon atoms, chlorine, chloroalkyl, in which the alkyl contains from about 1 to 6 carbon atoms, fluorine, foralkyl, in which alkyl contains from about 1 to 6 carbon atoms, cyano or cianelli, in which the alkyl contains from about 1 to 6 carbon atoms, or carboxyl; carbocyclic group which has the ring from about 3 to 8 carbon atoms, examples of which can be groups such as cycloalkyl or aromatic group, and which may include alkyl containing from about 1 to 6 carbon atoms, alkoxy containing from about 1 to 6 carbon atoms, alkylamino containing from about 1 to 6 carbon atoms, dialkylamino, in which each alkyl, independently, contains from about 1 to 6 carbon atoms, amino, sulfonylurea, hydroxy, carboxyl, fluorine or horsenettle or heterocytolysine or heteroaromatic group, which may include from about 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, sulfur and mixtures thereof, and which may include alkyl containing from about 1 to 6 carbon atoms, alkoxy containing from about 1 to 6 carbon atoms, alkylamino containing from about 1 to 6 carbon atoms, dialkylamino, in which each alkyl independently has about 1 to 6 carbon atoms, amino, sulfonylurea, hydroxy, carboxyl, fluorine or horsenettle.

33. The composition according to p. 28, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 60%.

34. The composition according to p. 28, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 1700.

35. The composition according to p. 28, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 2000, and the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 20%.

36. The composition according to p. 35, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 2300x2">

37. The composition according to p. 28, characterized in that the polyester copolymers included in the composition have a CMC of about 0.05% (wt./about.) or less at 37°C in isotonic aqueous solution.

38. The composition according to p. 28. characterized in that the polyester block copolymers has the formula

< / BR>
where n and m denote integers from about 4 to 400.

39. The composition according to p. 38, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 60%.

40. The composition according to p. 38, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 1700.

41. The composition according to p. 39, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 2000, and the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 20%.

42. The composition according to p. 41, wherein the molecular weight of the hydrophobic parts of the polyester blockcopolymer is at least about 2300, and the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 20%.

43. Compositedata, -lactam antibiotics, cephalosporins, tetracycline, inhibitors of beta-lactamases, aminoglycosides, chloramphenicol, erythromycin, clindamycin, spectinomycin, vancomycin, bacitracin, isoniazid, rifampin, ethambutol, aminosalicylic acid, pyrazinamide, ethionamide, cycloserine, Dapsone, sodium sulfation, clofazimine, sulfonamides, trimetho-Prim-sulfamethoxazole, quinolones, methenamine, nitrofurantoin or phenazopyridine.

44. The method of treatment of the object on which the object is resistant to biological agent and includes the introduction of the composition under item 1.

45. The method according to p. 44, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer entered the composition is at least about 50%.

46. The method according to p. 45, characterized in that the weight percentage of the hydrophobic parts of the polyester blockcopolymer entered the composition is at least about 60%.

47. The method according to p. 44, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer entered the composition is at least about 900.

48. The method according to p. 47, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer input composition is hydrophobes parts of polyester blockcopolymer entered the composition is at least about 2000, and the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 20%.

50. The method according to p. 49, characterized in that the molecular weight of the hydrophobic parts of the polyester blockcopolymer entered the composition is at least about 2300, and the weight percentage of the hydrophobic parts of the polyester blockcopolymer is at least about 20%.

51. A method of treating cancer, comprising introducing into the animal organism in need of treatment an effective amount of an anti-cancer composition under item 1.

52. A method of treating a microbial infection, comprising introducing into the animal organism in need of such treatment, an effective amount against microbial infection of the composition under item 1.

53. Method of inhibiting or preventing the growth of cancer metastasis, including the introduction of inhibiting or preventing the growth of metastases number of songs under item 1.

54. A method of treating cancer, comprising introducing an effective amount of an anti-cancer composition under item 1.

55. A method of treating cancer by p. 54, wherein the cancer is leukemia, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, myeloma, melanoma, glioma and the Oh Hodgkin's lymphoma, lymphoma non-Hodgkin's lymphoma, acute lymphocytic lymphoma, acute miliitary lymphoma, acute't lymphatic leukemia, Kaposi's sarcoma, small cell lung cancer, not small cell lung cancer or glial astrocytoma.

57. The way to overcome multidrug resistance of cancer cells to the cytotoxic substances arising in the treatment of cancer cytotoxic drugs, which is administered to the patient during chemotherapy effective amount of a composition according to p. 27.

 

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