Liposomal pharmaceutical preparation and method for preparing it

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, and concerns a liposomal drug preparation containing a polyvalent ionic preparation as an active substance with one or more dissociating groups at dissociation constant 4.5-9.5, wherein a liposome has a size of about 30-80 mm and a phospholipid bilayer has a phospholipid with a phase transition temperature above the body temperature so that the phase transition temperature of the liposome is above the body; a method of treating a tumour in a patient involving administering the above liposomal drug preparation.

EFFECT: group of inventions provides the higher therapeutic efficacy index of the liposomal preparation, especially more effective control of liposomal drug preparation recovery after targeted on the involved area, reduced loss of the drug preparation during the blood circulation of the liposomal drug preparation and more effective drug release into the target tissues.

17 cl, 21 ex, 3 dwg, 9 tbl

 

Scope

This invention relates to liposomal preparations containing narcotic drugs liposomal pharmaceutical preparations, in particular liposomal pharmaceutical drug mitoxantrone. Moreover, the invention relates to methods for producing liposomes, liposomal pharmaceutical preparation and their use.

The basis of the invention.

Liposomes can be used as carriers of many drugs, particularly anticancer agents (in particular chemotherapeutic drugs). Liposomes can reduce the distribution of the drug in normal tissues, however, to increase the accumulation of the drug in the tumor tissues, increasing therapeutic index drugs so. The reason why the liposome can passively target tumor is related to the physiological properties of the tumor tissue. The pore size of the tumor blood vessels can reach up to 100-780 nm due to its rapid growth, while the standard size of normal endothelial cells of the vascular walls is about 2 nm. As a result, liposomes can passively accumulate in the tumour region, if they can circulate in the blood for a relative who Thelen long period of time and have a size less than 200 nm, because, after liposomes are small in size are inserted through intravenous injection, they can't get in normal tissue, but can penetrate into the blood vessel affected by the tumor region and to reach the treatment area.

However, it is not easy to achieve therapeutic benefit liposomes must be satisfied the following four requirements: (1) the drug can be encapsulated in the liposome with high encapsulation efficiency and sufficient loading of the medicinal product; (2) the drug should not stand out from the liposomes during storage outside of the body; (3) there should not be a significant loss of drug during the circulation of liposomal drugs in the blood and (4) the drug can be effectively displayed and, thus, to show their therapeutic effect, when liposomes accumulate in the tumour region. With regard to methods of making liposomal preparations currently, three of the above-mentioned tasks have been successfully resolved, therefore, proper removal of liposomal drugs in the body attracts more attention. One very important technical problem that must be addressed for the development of some of liposomal drugs is effective in the positive control proper excretion of liposomal drugs after targeting tumour region. This is especially important for some drugs, such as mitoxantrone.

Group for the study of liposomes in Canada it was found that liposomal form size of about 100 nm, which was prepared using hydrogenated soy phosphatidylcholine (HSPC) and cholesterol as a phospholipid bilayer and download in medicine 300 mmol gradient citric acid, yielded the mitoxantrone not associated with plasma proteins. To improve therapeutic effect of liposomes, the research group, in the end, changed the composition of the phospholipid bilayer on dimyristoyl phosphatidylcholine (DMPC) and cholesterol, and has received a drug with a high therapeutic index. However, leakage medicines may increase during the storage period, as the temperature of the phase transition of DMPC - around 21°C, so that the drug may not be stable (Dipsomania form of mitoxantrone, US 5,858,397).

American Corporation Neopharm used a different way to develop liposomal forms of mitoxantrone in which cardiolipin, negatively charged, was added to the phospholipid bilayer. Through intensive interaction between cardiolipin and mitoxantrone, mitoxantrone could be entered in the phospholipid bilayer in the passive mode boot. This way the liability of the second load differs from the way the active load. By way of actively loading the drug was postponed in vnutrineironalnah aqueous phase in the form of sludge. Phase I clinical studies of the product company Neopharm showed that liposomal drugs can increase the chance of infection compared with the drug, not related to plasma proteins. The development of this product was discontinued due to safety (Liposomal drug mitoxantrone, CN 01817424.8).

Pacific Institute of Pharmacology (Changchun, China) has also filed a patent application for liposomal drug mitoxantrone (Dipsomania introduction of or mitoxantrone hydrochloride m mitoxantrone and the process of its manufacture, CN 200410041612.1). In this application was used by the traditional method pH-gradient to download drugs. This application is going to protect the special form factor, and not to disclose the impact of factors such as the composition of the phospholipids, types of buffer salt in the internal aqueous phase, the size of the liposomes, the ratio of the drug to the liposome, etc. on therapeutic efficacy and toxicity of liposomes.

Girond Jean with co-authors from the Pharmaceutical school of West China, Sichuan University, also studied liposomal drug mitoxantrone. They used soy fo fatiguehair with the temperature of the phase shift (0°C which is sold under the trademark EPIKURON 200) for preparation of liposomes with a size of about 60 nm. In this article, has been studied only pharmacokinetics, without touching the toxicity and therapeutic efficacy obtained liposomally of the drug. Information pertaining to this question can be seen in the Manufacture of liposomes of mitoxantrone circulating for a long time, and their pharmacokinetics", Girond Jean, Botao Yu and Eason Duan, Act of Pharmaceutica Sinica (Chinese journal of pharmaceuticals), 2002, Volume 37, Number 6; study of the preparation of liposomes of mitoxantrone circulating for a long time, with the transmembrane gradients of ammonium sulfate, Girond Jean, Botao Yu, Eason Duan yuan Huang, Chines pharmaceutical Journal (Chinese journal of pharmaceuticals), 2002 Volume 37, Number 12; and ”the Study of methods of preparation of liposomes of mitoxantrone, Eason Duan, West China journal of pharmaceutical Senses (Journal of pharmaceutics Western China), 2001 Volume 16, issue 02.

In the above studies, the size of liposomes is normally regulated at the level of 80~150 nm, as there is a General consensus in research liposomes, the liposome size of about 100 nm is most effective targeting (Pharmacol. Rev. (pharmacological review) 1999 51: 691-744.). However, as already noted, the liposome must have not only an excellent efficiency of targeting, but also a sufficient removal of the drug from limes is soma in order to show their effect.

As noted above, according to the previous segment loss drugs during circulation should, in fact, be prevented, so that the drug could be effectively delivered to tumors, but this requirement also leads to the difficulty of removing drugs from liposomes, when it is aimed at the tumour region. In conventional processes for the manufacture of liposomes, the drug is usually encapsulated by way of the active load, wherein the drug encapsulated in the liposome in the form of a colloidal precipitate that does not have biocatalogue, so that only when the drug is effectively removed from the liposomes, it can turn into a drug with bioactivity. If the rate of introduction of the drug is too slow, this drug can hardly exert its therapeutic effect, even if it was now aimed at the tumour region, and its therapeutic effect may be even worse than the unencapsulated public medicines.

Thus, there is an urgent need in the industry in liposomal drug capable of delivering a drug with good targeting ability and effective is to bring the drug into the targeted tissue, and in the corresponding liposomal pharmaceutical preparation.

Brief description of the invention

The inventors have unexpectedly accidentally discovered that some medicines have many dissociate groups and the tendency to form dense precipitation with polyvalent counterion can be processed to create a small single layer of liposomal drug with significantly higher therapeutic index, so that the above-mentioned technical problem could be solved.

Thus, on the one hand, this invention provides a liposomal preparation of about 30-80 nm with a phospholipid with a melting point higher than the body temperature in the phospholipid bilayer, so that the temperature of the phase shift liposomes above body temperature. Examples of the above-mentioned phospholipid include, but are not limited to, phosphatidylcholine, hydrogenated soy phosphatidylcholine (HSPC), hydrogenated egg yolk phosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC) or distearoyl phosphatidylcholine (DSPC), or combinations thereof.

In one of the embodiments of the present invention, the phospholipid with a melting point higher than the body temperature in the phospholipid bilayer, the equivalent of 50-100 mol/mol%, preferably 55-95 mol/mol %, and still more preferred is elinee 55-95 mol/mol % of the total content of phospholipids.

Additionally, the phospholipid bilayer liposomal preparation of the present invention contains, moreover, an additional phospholipids, for example, a phospholipid with a melting point not higher than the temperature of the body, such as dimyristoyl phosphatidylcholine (DMPC) and the like. The amount of phospholipid in liposomal preparations of this invention can be conventionally determined by those who are versed in this area, provided that the melting temperature of the liposomal drug is clearly not reduced to values lower than the body temperature.

Liposomal preparation of the present invention may also optionally contain cholesterol to regulate the fluidity of the liposomal membrane.

Liposomal preparation of the present invention may also contain additional excipients, in particular excipients for further changes in surface properties of liposomes, to give a liposome great functionality in the body. Such excipients include, for example, lipids and the like substances, modified hydrophilic polymers.

On the other hand, this invention is Liposomal pharmaceutical preparation which contains an investigational drug, in particular medicinal product with n is ivalentine ions, in the liposomal preparation of the present invention. Thus, this invention relates to liposomal pharmaceutical preparation is the size of 30-80 nm, where: (1) Liposomal pharmaceutical preparation contains drug with polyvalent ions as an active ingredient; (2) the phospholipid bilayer contains a phospholipid with a melting point higher than the body temperature so that the temperature of the phase shift liposomes higher than body temperature; and additionally (3) liposomal pharmaceutical preparation contains additional medication and/or additional excipients that are valid in a liposomal pharmaceutical preparation. Preferably, the main peaks of the size of the liposomal pharmaceutical preparation were concentrated in 35-75 nm, in particular about 40-60 nm.

On the other hand, this invention is a method of preparing the above-mentioned liposomal pharmaceutical product, the method comprising the following steps: (1) preparation of liposomes using a phospholipid with a melting point higher than the body temperature, and, optionally, one or more phospholipids and/or cholesterol; and (2) encapsulating the studied drugs, particularly drugs with polyvalent what areas, in the liposome.

This invention also presents a method of treatment of the disease, including the introduction of liposomal pharmaceutical preparation of the present invention to a patient in need of treatment. It is desirable that such a patient were mammals, in particular man. A brief description of the drawings.

Figure 1 shows the pharmacokinetics of PLM60 in the body of the mouse from manymen (China) and its comparison with the pharmacokinetics of mitoxantrone not associated with plasma proteins in the body, where PLM means pegylated liposomal mitoxantrone, FM means mitoxantrone, not associated with plasma proteins, on the x-axis shows the time (hours) and on the ordinate axis shows the level of mitoxantrone in plasma (µg mitoxantrone/ml plasma).

Figure 2 presents a view in section of PLM60 and FM in the tumor of a mouse, where PLM60 means pegylated liposomal mitoxantrone, FM means mitoxantrone, not associated with plasma proteins, on the x-axis shows the time (hours) and the axis of ordinate shows the concentration of mitoxantrone in tumor tissues (µg mitoxantrone/g tumor tissue).

Figure 3 presents a comparison of the pharmacokinetics of various forms in the body of the mouse, where the x-axis shows the time (hours) and on the ordinate axis shows the level of mitoxantrone in plasma (µg mitoxantrone/ml plasma), and dosages, the various who's form is 4 mg/kg

Description of the preferred variants of the invention

Typically, liposomes made of phospholipids and cholesterol, which is the membrane materials. These two ingredients are not only basic materials create a liposomal bilayer, but also have very important physiological functions.

The physical properties of the liposomal membrane is closely associated with temperature. When the temperature is raised, acyl side chains of the lipid bilayer change form an ordered structure in the form of a disordered structure. These kinds of changes can lead to many changes in the physical properties of lipid membranes. For example, the "gel" state can turn into a "liquid" state, a cross section of the membrane can be increased, the thickness of the bilayer may decrease membrane fluidity can be increased. The temperature at which such changes occur is called the temperature of the phase shift. The temperature of the phase shift of the lipid membrane can be determined by Differential Scanning Calorimetry, Electron Paramagnetic Resonance (EPR) and the like. The temperature of the phase shift liposomal membrane depends on the types of phospholipids. In General, the longer acyl side chain, the higher the temperature of the phase shift; and Vice versa. For example, temperatures of the phase shift of dimyristoyl phosphatidylcholine is 24°C, while dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine - 41°C and 58°C, respectively. Membrane fluidity is an important property of liposomes. When the temperature of the phase shift membrane fluidity will increase, and the drug encapsulated in the liposome, will have a maximum speed of excretion. Thus, the fluidity of the membrane has a direct impact on the stability of liposomes.

In one of the embodiments of the present invention, the invention is a liposomal preparation of about 30-80 nm and a phospholipid with a melting point higher than the body temperature in the phospholipid bilayer, so that the temperature of the phase shift liposomes higher than the body temperature.

It is desirable that liposomal pharmaceutical preparation of this invention was manufactured using phospholipids with a relatively high temperature phase shift of the melting temperature, such as phosphatidylcholine. If the melting point of phosphatidylcholine higher than body temperature, it is desirable that the length of the hydrocarbon chain was not less than 16 carbon atoms. Preferably, the phospholipids of the present invention included, but were not limited to hydrogenated soy phosphatidylcholine, hydrogenated egg-yolk phosphatidyl the Lin dipalmitoyl phosphatidylcholine (DPPC) or distearoyl phosphatidylcholine (DSPC), or their combination.

In the liposomal preparation of the present invention phospholipids with a melting point higher than the body temperature in the phospholipid bilayer is equivalent to approximately 50-100 mol/mol%, preferably about 55-95 mol/mol%, even more preferably about 60-90 mol/mol% relative to the total content of phospholipids. Additionally, the phospholipid bilayer can contain additional phospholipids, for example, phospholipids with a melting point not higher than body temperature, as for example dimyristoyl phosphatidylcholine (DMPC) and the like. These phospholipids may be present in the liposome in any quantity, to meet the requirements, provided that it does not reduces the temperature of the phase shift of liposomal drug is below body temperature. The number who meet the requirements may be determined according to the usual techniques of those who are versed in this field.

Preferably, the liposomal preparation of the present invention could further contain cholesterol. Cholesterol has a function of regulating the fluidity of the membrane. When the liposomal membrane contains 50% (mol/mol) cholesterol, a phase shift of liposomal membrane can disappear. Papahadjopoulos with co-workers called the hall of Teren "buffer fluidity", since the addition of cholesterol to phospholipids is lower than the temperature of the phase shift can reduce the order of the membrane structure and to increase the fluidity of the membrane, whereas the addition of cholesterol to phospholipids above the temperature of the phase shift can increase the orderliness of the membrane structure and reduce membrane fluidity. In the liposomal preparation of the present invention cholesterol can be 2-60 mol/mol %, 5-55 mol/mol % or 10-50 mol/mol % relative to the total number of ingredients liposomes. More specifically, the cholesterol can be 15-45 mol/mol %, for example 20 to 40 mol/mol % relative to the total number of ingredients liposomes. The content of cholesterol in the liposome of the present invention can be easily determined according to the standard techniques of those who are versed in this field.

Note that the phospholipid bilayer in the liposome of the present invention may also contain additional excipients, in particular excipients for further changes in surface properties of liposomes, to give a liposome great functionality in the body. Such excipients include, for example, lipid substances, modified hydrophilic polymers, and their examples include PEG-m is deficieny distearoyl the phosphatidyl ethanolamine (cells of the dspe-PEG), PEG-modified distearoyl the phosphatidyl glycerol (DSPG-PEG), PEG-modified cholesterol (chol-PEG), polyvidone modified distearoyl the phosphatidyl ethanolamine (cells of the dspe-PVP), PEG-modified distearoyl the phosphatidyl glycerol (DSPG-PVP), or PEG-modified cholesterol (chol-PVP). The above-mentioned auxiliary substances may also be membrane materials, modified specific antibody or ligand. The number of such auxiliary substances in the liposome of this invention can be determined according to standard techniques of those who are versed in this area, for example, may be 0.1-20 mol/mol %, preferably 0.3-18 mol/mol %, even more preferably 0.5-15 mol/mol %, in particular 0.8-12 mol/mol %, such as 1-10 mol/mol %, or 2-8 mol/mol %, 2.5-7 mol/mol %, 3-6 mol/mol %, etc. as to the number of moles of phospholipids. When using PEG-modified lipids as auxiliary substances, molecular weight fraction of PEG may be, for example, 400-20000 daltons, preferably 600-15000 daltons, even more preferably 800-10000 daltons, in particular 1000-8000 daltons, for example 1200-5000 daltons. The use of PEG in this invention can also be easily determined on the basis of standard tests those who are versed in this field.

Liposomal is the drug of the present invention is a small single layer of liposomal drug and must have a suitable size. It is desirable that the size of the drug was 30-80 nm, even more preferably 35-70 nm, particularly preferably 40 to 60 nm. The size of the liposomes can be determined using the analyzer, particle size or electron microscope or other means. It should be understood that liposomal particles in this invention may not have exactly the same size, they cover a range of values, due to the nature of the liposomes and properties of the method of manufacture. Thus, in a liposomal preparation of the present invention, is not precluded by the presence of liposomal particles outside the specified range of values, provided that they do not have a clear impact on the properties of liposomal drug or pharmaceutical preparation of the present invention.

Liposome in this invention can be prepared using a variety of appropriate methods, including, for example, methods of film dispersion, method of introduction, methods, ultrasonic dispersion, methods, freeze-drying, methods of freezing-thawing and the like. According to the systems launch, used to prepare liposomes, these techniques can be divided into: (1) methods based on the use of dried lipid membranes, the lipid extract; (2) methods based the data on the use of emulsifying agents; (3) methods of preparation of liposomal preparations based on the use of mixed micelles; and (4) methods of preparation of liposomal preparations based on the use of three-phase mixture of ethanol, phospholipids and water. Encapsulating drugs may be carried out using passive mode loading and active loading. These techniques can be found in many review articles about the liposomes.

During or after preparation of liposomal drug a many appropriate techniques may be used to encapsulate the drug in the liposome and create a liposomal pharmaceutical preparation. Suitable techniques include, for example, methods of active downloads and techniques passive load. The technique of active downloads is usually executed by using gradient methods, for example, the method of gradient of ammonium sulfate, i.e., the use of ammonium sulfate solution as water phase in order, firstly, to prepare a liposome containing ammonium sulfate and vnutripuzarnoe, and valpolini phase, then create a density gradient of ammonium sulfate between vnutripuzarnoe and valpolini phases, removing validatory ammonium sulfate. Nutritionary NH4/sub> +dissociated on NH3and H+that leads to a difference in the concentrations of H+(i.e. pH gradient) between vnutripuzarnoe and valpolini phases, so after valpolini drug in the molecular state is included in vnoutriniouiou water phase, it goes in the ionic state, thus, the drug may not return to validatenow water phase, and the liposome is less loss medicines, and it is more stable. The technique is passive load can be enforced using the method of introduction of the organic solvent, the technique of film dispersion, methods of freezing-thawing and the like.

In this invention, can be any suitable ingredients of a medicinal product. Preferably, the active pharmaceutical ingredient in a liposomal pharmaceutical preparation of this invention was the drug with polyvalent ions. The term "medicinal product with polyvalent ions" refers to a drug having two or more dissociate group with dissociation constant KD 4.5~9.5, so that the medicinal product has more positive charges or more negative charges in the range of KD. It is desirable that the s above the dissociation constant was in the range of 5.0-9.5. Even more preferably, the above-mentioned dissociation constant was in the range 5.5-9.5. Particularly preferably, the dissociation constant was in the range of 6.0-9.0 m, in particular from 6.5 to 9.0. The KD value of each dissociate group drugs with ions can be easily determined according to the standard techniques of those who are versed in this field.

In this invention, drugs with polyvalent ions may include, but are not limited to antitumor drugs, such as drugs that can be applied to prevent or treat the following types of cancer: lung cancer (such as non-small cell lung cancer), pancreatic cancer, breast cancer, colorectal cancer or multiple myeloma, liver cancer, cervical cancer, gastric cancer, carcinoma of the prostate, kidney cancer and/or carcinoma of the bladder. Thus, in one of the embodiments of the present invention, the medicinal product with polyvalent ions is an antitumor drug with polyvalent ions. Preferably, as a drug with polyvalent ions were mitoxantrone, vincristine, vinorelbine or vinblastine. Even more preferably, as mentioned above medicines polivalente ions were mitoxantrone and could be further combined, at least one of the auxiliary medicines, which may be, for example, anticancer drug, such as vincristine, vinorelbine or vinblastine, and the like.

Should be particularly noted that in the prevent invention as a drug with polyvalent ions may also be a combination of any one or two or more of the above drugs, for example, a combination of two anticancer drugs, the combination of one or more anticancer drugs with ancillary drugs such as immune, and a combination of two or more other types of drugs.

It is also worth noting that liposomal drug of this invention can also optionally contain one or more auxiliary medicines polivalente ions in addition to the above drugs with polyvalent ions, which can be introduced in combination with drugs with polyvalent ions, as described above. The combined introduction consists of an introduction of all components in a single drug, also consists of a combined introduction in single dosage form.

Should be evaluated for dignity is nstwo the fact, what this drug as the active ingredient, as described herein, is performed not only in its original form, but also in derived forms, such as a solvate (such as hydrates and foods with added alcohol), prodrugs and other physiologically acceptable derivatives, as well as active metabolites, and the like. Derivatives, prodrugs and other physiologically acceptable derivatives, as well as active metabolites of drugs are well known to those versed in this field.

Liposomal pharmaceutical preparation of this invention can, moreover, contain two or more polyvalent counterion with charges opposite to those that have the active ingredient. Examples of polyvalent counterions include, but are not limited to, anions of organic acids, such as the anions of the following acids saturated or unsaturated organic acids: citric acid, tartaric acid, fumaric acid, oxalic acid, malonic acid, succinic acid, malic acid and maleic acid, and the like;

anions of inorganic acids such as sulfate anion, phosphate anion, and the like. Among them, preferred is the citrate anion, the anion sulphate or phosphate anion. Furthermore, as the above-mentioned polyvalent counterions can also be amino acids such as cystine and the like. Without being bound to any particular theory, there are assumptions that the polyvalent counterion capable of creating an insoluble precipitate with the study drug (e.g., a drug with polyvalent ions)encapsulated in a liposome, thus ensure the availability of medicines with polyvalent ions in the liposome.

Liposomal pharmaceutical preparation of this invention includes, furthermore, a further auxiliary substances and carriers are well known in the pharmaceutical industry, such as sucrose, histidine, antioxidants, stabilizers, dispersing agents, preservatives, blood-thinning agents, solvents, salts for influencing osmotic pressure, and the like.

In one of the embodiments of the present invention, the invention provides a method of preparing liposomal pharmaceutical preparation of the present invention includes: first, the preparation of the liposomal preparation of the present invention, as described above, and then incubation of the investigational drug with liposomal drug in suitable condition is Yah. More specifically, the method of preparing liposomal pharmaceutical preparation of the present invention includes the following steps: (1) dissolution of lipid excipients suitable for the preparation of liposomes in an appropriate organic solvent, such as tributylamine alcohol or cyclohexane, and then freeze-drying to obtain a dried powder; (2) hydrating the lyophilized powder with a solution containing a counterion of the active ingredient of the investigational drug for the creation of "empty" liposomes; (3) removing maliboomer of the counterion by appropriate means, such as dialysis or column chromatography, and the like, with the aim of creating a gradient between counterion vnutripuzarnoe phase and valpolini phase; and (4) incubation of the medicinal product with the liposome to receive liposomal drugs. Characterization of phospholipids, cholesterol, excipients and the like, see above for liposomal drug.

Preferably, the lipid was a phospholipid, in particular a lipid with a relatively high temperature phase shift, for example, phosphatidylcholine, hydrogenated soybean phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, dipalmitoyl phosphatidyl the Lin (DPPC) or distearoyl phosphatidylcholine (DSPC), or a combination of them. Additionally, the above-mentioned lipid may also contain cholesterol in number, for example, 2-60 mol/mol%, 5-55 mol/mol % or 10-50 mol/mol %. More specifically, the amount of cholesterol can be 15-45 mol/mol %, for example, 20-40 mol/mol % relative to the total number of moles of all components in the liposome. Those who are versed in this area, can determine the amount of cholesterol depending on specific requirements in relation to the temperature of the phase shift of the liposomes, which is supposed to get and set properties.

As soon as liposomal pharmaceutical preparation is ready, the efficiency of encapsulation of the drug in the liposome can be determined using conventional techniques. Methods of determining the effectiveness of encapsulating liposomes include ultrafiltration, dialysis, column chromatography, centrifugation, and the like. Ultrafiltration is not used because of the high requirements of the experimental device; column chromatography is not used due to the fact that the dilution requires a large amount of eluent, and the content of the medicinal product is very low, so it is difficult to determine the contents, in addition, dilution of a large number of eluent may also lead to loss of lekarstvennogo in the liposome, from the research data you find that the efficiency of encapsulation when dialysis is lower (probably due to rupture of the liposomes after dilution), and time for dialysis requires a lot, so the technique is not suitable. Determination of encapsulation efficiency using centrifugation has the following advantages: small amount of elapsed time, a small degree of dilution of the solution of liposomes, and no need for expensive tools.

Liposomal pharmaceutical preparation of the present invention provides not only a sufficient efficiency of encapsulation and sufficient loading medicines, but also does not allow the allocation of drugs from liposomes during storage outside the body, a significant diversion of drugs from liposomes during circulation to increase toxicity. Important useful effect of liposomal drugs of this invention is that the level of excretion of drugs effectively accelerated, therapeutic index of the liposomes increased the half-life significantly prolonged toxicity is markedly reduced compared to existing products in the field, and, thus, achieved significant therapeutic effect of medicinal cf is DSTV. For example, liposomal pharmaceutical preparation prepared using hydrogenated soy phosphatidylcholine (HSPC) and dipalmitoyl phosphatidylcholine (DPPC), their toxicity is clearly reduced and their therapeutic index is significantly increased. On the contrary, if the phospholipid bilayer consists of dimyristoyl phosphatidylcholine (DMPC), excretion of drugs will be too fast and will cause significant toxicity, even harmlessness to health will give way to a medicinal product that is not associated with plasma proteins. Without being bound to any particular theory, there are suggestions that a small single-layer liposomal preparation of the present invention can accelerate the excretion of drugs, as a small single layer liposomal preparation may contain more liposomal particles, which contain the residue of a medicinal product with a small particle size, compared with the large single layer of liposomal drug, if the ratio of drug/lipid consistently. Sediment medicines with a small particle size would have a relatively large specific surface area, and thus had a greater rate of dissolution under the same conditions.

In addition, liposomal pharmaceutical pre the Arat this invention should be prepared using the appropriate phospholipids, in order to achieve effective introduction of the drug in the target tissue, in particular in tumors. Preferably, the phospholipid bilayer liposomal pharmaceutical preparation of the present invention consisted of phospholipids with a relatively high temperature phase shift. During the experiments it was found that the toxicity of the drug significantly decreased, and therapeutic index was significantly increased if hydrogenated soy phosphatidylcholine (HSPC) and dipalmitoyl phosphatidylcholine (DPPC) or similar substances were used in the preparation. If the phospholipid bilayer consists of dimyristoyl phosphatidylcholine (DMPC), excretion of drugs would be too fast and would cause significant toxicity, even the harmless would match the unencapsulated public drug.

Liposomal pharmaceutical preparation of this invention can be administered to the patient who needs it, by the way, is widely used in the industry. In one of the embodiments of the present invention liposomal drug is a drug for parenteral administration. In one of the preferred embodiments of the present invention liposomal drug is injected through injection the AI.

This invention also presents a method of treating diseases, in particular tumor in a patient, the method including the introduction of liposomal pharmaceutical preparation of the present invention to a patient in need of treatment. Preferably, therapy (as for example the technique of radioactive thermotherapy) could also be applied in combination to a patient with a tumor to increase therapeutic effect of liposomal pharmaceutical preparation. In this invention as the patient may be a mammal, preferably human.

This invention also relates to the use of liposomal liposomal drug or pharmaceutical product, as defined above, in the manufacture of medicaments for the treatment of a patient with a tumor.

This invention is further illustrated by the following examples which are merely illustrative, and should not be construed as limitations of this invention.

Part 1: Getting liposomes

Example 1

Common methods of obtaining liposomes

1. General method 1

The phospholipid (for example, gidrogenizirovannye soy phosphatidylcholine (HSPC), dipalmitoyl phosphatidylcholine (DPPC) or dimyristoyl phosphatidylcholine (DMPC)and cholesterol (molar ratio of from 1:1 to 10:1) are dissolved in an organic solvent such as tert-butyl alcohol or cyclohexane, to a clear solution is formed. The solution is processed by conventional freeze-drying to obtain dried powder. Dried powder hydrated at 60-65°C (50-1000 mm) solution of ammonium sulfate, citric acid solution or a solution of sulphate of transition metal (such as Nickel sulfate), and shaken for about one hour to obtain a heterogeneous mnogoelementnykh bubbles. The size of the resulting bubbles decreases microfluidizer or extrusives the high-pressure apparatus for obtaining liposomes. The sample obtained liposomes diluted 200-fold with 0.9% NaCl solution and detected ZS. Extremely buffer solution is removed by ultrafiltration apparatus for dynamic transmembrane gradient. A solution of the hydrochloride mitoxantrone (10 mg/ml) is added to an empty liposomes with a corresponding ratio of liposome/drug and download the medicinal product is held at 60-65°C. After incubation for about an hour, used Jeleva size-exclusion chromatography to determine the effectiveness of inclusion of a substance in a gelatin capsule (EE).

2. General method 2

The phospholipid (for example, hydrogenated soy phosphatidylcholine (HSPC), dipalmitoyl phosphatidylcholine (DPPC) or dimyristoyl phosphatidylcholine (DMPC)and the hall of Teren (molar ratio from 1:1 to 10:1) are mixed, and at the same time added distearoyl phosphatidylethanolamine modified polyethylene glycol (cells of the dspe-PEG) in the 0.1-20% by bringing phopholipid. The resulting mixture is dissolved in an organic solvent such as tert-butyl alcohol or cyclohexane, to form a transparent solution. The solution is processed by conventional freeze-drying to obtain dried powder. Dried powder hydrated at 60-65°C (50-1000 mm) solution of ammonium sulfate, citric acid solution or a solution of sulphate of transition metal (such as Nickel sulfate), and shaken for about one hour to obtain a heterogeneous mnogoelementnykh bubbles. The size of the resulting bubbles decreases microfluidizer or extrusives the high-pressure apparatus for obtaining liposomes. The sample obtained liposomes diluted 200-fold with 0.9% solution of Nad and detected HanoZS. Extremely buffer solution is removed by ultrafiltration apparatus for dynamic transmembrane gradient. A solution of the hydrochloride mitoxantrone (10 mg/ml) is added to an empty liposomes with a corresponding ratio of liposome/drug and download the medicinal product is held at 60-65°C. After incubation for about an hour, used Jeleva size-exclusion chromatography on what I determine the effectiveness of inclusion of a substance in a gelatin capsule (EE).

Example 2

Obtain liposomes of mitoxantrone PLM60

HSPC, cholesterol and cells of the dspe-PEG2000 in a weight ratio of 3:1:1 were dissolved in 95% tert-butyl alcohol to a clear solution is formed. The solution is treated by the method of freeze-drying to obtain dried powder. Dried powder hydrated at 60-65°C (50-1000 mm) solution of ammonium sulfate (300 mM) and shaken for about one hour to obtain a heterogeneous mnogoelementnykh bubbles with a final concentration of phospholipid in the amount of 96 mg/ml. the size of the resulting bubbles was reduced microfluidizer to obtain liposomes. The sample obtained liposomes diluted 200 times with 0.9% NaCl solution and detected HanoZS with an average size of about 60 nm and a main peak between 40 nm and 60 nm. Extraliterary solution of ammonium sulfate was removed by ultrafiltration apparatus and replaced with a solution of 250 mm sucrose and 50 mm glycine for dynamic transmembrane gradient. In the empty liposomes added to a solution of hydrochloride mitoxantrone (10 mg/ml) at a ratio of 16:1 liposome/drug, and the drug loading drug at 60-65°C. After incubation for about an hour, the effectiveness of the inclusion of substances in a gelatin capsule (IT) through gilevoy exclusion chromatography was defined as 100%. the received liposomes named PLM60.

Example 3

Obtain liposomes of mitoxantrone PLM85

HSPC, cholesterol and cells of the dspe-PEG2000 in a weight ratio of 3:1:1 were dissolved in 95% tert-butyl alcohol to a clear solution is formed. The solution was treated by the method of freeze-drying to obtain dried powder. Dried powder hydrated at 60-65°C (300 mm) with a solution of ammonium sulfate (300 mm) and shaken for about one hour to obtain a heterogeneous mnogoelementnykh bubbles with a final concentration of phospholipid in the amount of 96 mg/ml. the size of the resulting bubbles reduced extrusives the high-pressure apparatus for obtaining liposomes. The sample obtained liposomes diluted 200 times with NaCl solution and detected HaHoZS with an average size of about 85 nm. Extraliterary solution of ammonium sulfate was removed by ultrafiltration apparatus and replaced with a solution of 250 mm sucrose and 50 mm glycine for dynamic transmembrane gradient. In the empty liposomes was added to a solution of hydrochloride mitoxantrone (10 mg/ml) at a ratio of 16:1 liposomes/medicine, and was conducted by loading the drug at 60-65°C. After incubation for about an hour, the effectiveness of the inclusion of substances in a gelatin capsule (IT) through gilevoy exclusion chromatography was defined as 100%. Received the s liposomes named PLM85.

Example 4

Obtain liposomes of mitoxantrone PLM100

To obtain liposomes hydrochloride mitoxantrone PLM100 having the same composition as PLM60 and PLM85, but the size of liposomes of 100 nm, was used the same method described in Example 3.

Example 5

Obtain liposomes of mitoxantrone PLM60-dppc

DPPC, cholesterol and cells of the dspe-PEG2000 in a weight ratio of 3:1:1 were mixed, the remaining steps are the same as in Example 2. The resulting liposomes are named PLM60-dppc.

Example 6

Obtain liposomes of mitoxantrone PLM60-dmpc

DMPC, cholesterol and cells of the dspe-PEG2000 in a weight ratio of 3:1:1 were mixed, the remaining steps are the same as in Example 2. The resulting liposomes are named PLM60-dmpc.

Example 7

Obtain liposomes of mitoxantrone PLM60-dmpc-0.1

DMPC, cholesterol and cells of the dspe-PEG2000 in a weight ratio of 3:1:0.1 were mixed, the remaining steps are the same as in Example 2. The resulting liposomes are named PLM60-dmpc-0.1.

Example 8

Obtaining liposomes treated with adriamycin PLD60

Adriamycin has been replaced with mitoxantrone during loading of the drug, the remaining steps are the same as in Example 2. The resulting liposomes are named PLD60.

Part 2: the Release of drug from different formulations of liposomes

Example 9

Differences release of the drug between the liposome treated with adriamycin PLD60 and liposome of mitoxantrone PLM60

In this example, mitoxantrone and adriamycin were loaded the od of the pH gradient. If released, the tool adds a certain concentration of ammonium chloride, available molecules of ammonium diffuse into the internal phase of the liposome using a gradient, so that the pH of the internal phase will be increased and protonated drug in the internal phase is transformed into a neutral form, which can diffuse through the membrane. This process will accelerate at the same time the dissolution of the precipitate in the internal phase of the liposome. The rate of release of drug was regulated by dissolving sludge and membrane permeability of liposomes. Conditions of release of the medicinal product were as follows. Liposomes were diluted 25 times released a tool. Released in the medium was isotonic, from 7.4 pH, and concentrations of 2, 10 and 40 mm ammonium chloride, respectively. Diluted liposomes were placed in a dialysis tube, and 2 ml of the diluted liposomes made dialysis through 400 ml of untied funds at 37°C. After 96 hours for analysis at different time points samples.

The obtained data were subjected to regression analysis. In the release medium with 2, 10 and 40 mm ammonium chloride half-lives of release of a drug PLD60 amounted to 94.3, 31.9 and 11.2 hours, respectively. In respect of PLM60 explicit release in the PEX released funds were not observed. As PLD60 PLM60 and do not have differences in composition and size, the difference in dynamic characteristics of release of the drug may be explained by their different pharmaceutical properties. Adriamycin, as mitoxantrone, is anthracycline antibiotic, and their differences lie in the fact that adriamycin contains one splittable group at normal pH, whereas mitoxantrone contains two splittable group (pKa=8.15) at normal pH. The example illustrates that the drug is with multiracialism groups such as mitoxantrone, during the application loading method can form mixed sediment with counterions, so that the release of drug in the laboratory is much slower. On the other hand, the drug with odnorazovij group such as adriamycin, may be released too fast even released the tool without plasma while applying liposomes of small size.

Example 10

The nature of the release of liposomes of mitoxantrone in various sizes

To compare the nature of the release of liposomes of mitoxantrone different sizes were taken two conditions of release.

Condition of release 1: liposome was diluted 25 times released a tool. The released medium contained 50% of people who aceskay plasma, using glucose was isotonic and had a pH of 7.4. Other conditions are identical to those described in Example 9. The obtained data are subjected to regression analysis. The result showed that the half-life of release of PLM60 amounted to 56.4 hours, while PLM85 was not significantly released under the same conditions.

Condition of release 2: used the released product containing 50% human plasma and 20 mm ammonium chloride, other conditions are identical to those described in Example 9. The obtained data are subjected to regression analysis. The result showed that the half-life of release of PLM60 amounted to 26.2 hours, while the half-life release PLM85 36.7 hours.

This example is sufficiently showed that the release of drug can be significantly increased by reducing the size of the liposomes.

Example 11

The nature of the release of liposomes of mitoxantrone with different formulations membrane

Were used to the same conditions of release as described in Example 9. The result showed that the half-life of release of PLM60-DPPC was 116 hours, the half-life release PLM60-DMPC was 26 hours, and the half-life of release of PLM60-DMPC-0.1 amounted to 15 hours. This example showed that the use of phospholipid at a lower temperature phase of the BoD of the yoke Tm can accelerate the release of the drug. However, if the release of the drug was excessively accelerated, also may increase toxicity, and this is further confirmed by the following examples.

Part 3: Pharmacokinetics in vivo

Example 12

The pharmacokinetic character of PLM60 in mice Kunimine and comparison of PLM60 and free mitoxantrone

This example was carried out on male mice Kunimine with body weight of about 20 GV vein tails of mice were injected with different levels of doses of mitoxantrone. Dosing PLM60 were 1, 2 and 4 mg/kg, and the dosage of free mitoxantrone (FM) was 2 mg/kg At different time points samples were collected plasma. Methods of treatment and diagnostics of plasma samples described in the document: Methods in Enzymology, Vol: 391, str-185. The results are shown in Table 1 and figure 1, which clearly shows that the half-life of mitoxantrone increased significantly by encapsulating liposomes. At the same dosage, the time delay PLM60 in circulation amounted to 32 times the time delay FM, and AUC 3700 times the time delay FM. The dependence of the AUC dose showed that PLM60 has linear pharmacokinetics in vivo.

Table 1
Pharmacokinetics PLM60 and FM in mice Kunimine
PLM604 mg/kgPLM602 mg/kgPLM60 1 mg/kgFM mg/kg
OptionsValueValueValueValue
AUC 0-48(mg/l*h)1451.666728.398452.7090.198
AUC 0-∞(mg/l*h)1654.543892.437503.0780.199
AUMC 0-4821838.03412050.6817049.2590.103
AUMC 0-∞36234.61124686.91710488.8110.135
MRT 0-48(h)15.04316.54415.5710.517
MRT 0-∞ (h)21.90027.66220.8490.675
Tmax(h) 0.0830.0830.2500.083
Cmax(mg/l)86.32947.51325.9700.699

Example 13

The distribution of PLM60 and FM in the tissues of mice-carriers of tumour

There were clear differences between the tissue distribution PLM60 and FM in mice-the carriers of tumour. In this example, we used mouse males Kunimine with body weight of about 20 g Mice were inoculated in the right armpit cell sarcoma S-180 in a dose of 5×105. Drugs were injected into the vein of the mice, the tumor increased to 0.4 to 0.7 after the introduction of the drug, the mice were euthanized at different time points and tissues were removed to determine the concentration of mitoxantrone. Cloth included heart, liver, spleen, lungs, kidneys, intestine, bone marrow and tumors. The results showed that PLM60 clearly aimed at the tumor tissue. Detailed data are shown in Table 2 and figure 2.

Table 2: distribution Data PLM60 and FM in the tissues of normal mice
ClothPLM-60 4 mg/kg FM 4 mg/kg
t (h)With-µg/gSDt (h)With-µg/gSD
Heart14.010.3815.3850.679
43.390.3843.5170.952
83.480.6483.1970.357
162.830.57242.9430.549
242.060.48
Liver16.780.7814.770 0.997
45.990.6743.5560.543
86.310.3882.6590.439
166.220.95241.9370.346
244.520.65
Spleen14.661.3714.0440.414
44.360.6744.4600.494
84.781.7083.7742.676
16 7.562.13247.7522.469
245.911.00
Light18.441.08110.2051.732
44.582.3648.0241.859
86.331.4387.0180.728
165.121.24248.0820.844
242.890.23
Kidney17.09 0.84118.2431.238
47.121.17417.1925.010
87.040.96813.4091.251
166.751.16247.4631.209
245.820.84
Ulcer11.660.6611.5320.309
42.330.6642.1400.655
82.340.648 2.5511.204
162.420.51243.9361.625
242.250.32
Bone marrow11.090.5410.1270.041
40.640.14
80.730.16
160.540.24
240.120.02
Tumor191.287.4110.06140.0078
463.908.5640.01330.0027
854.018.04
1638.619.19
2410.412.67

Example 14

Pharmacokinetic comparison of different formulations of liposomes

Used in this example, the animals are similar to those used in Example 12. PLM60-DPPC, PLM60-DMPC-0.1 and PLM60-DMPC at a dose of 4 mg/kg were injected into the vein in the tail of the mice. The data displayed in Table 3 and Figure 3. It is shown that the pharmacokinetics of liposomal drugs has changed significantly with the change in the composition of the membrane of liposomes. MRT C is achene on PLM60-DPPC, PLM60-DMPC-0.1 and PLM60-DMPC in vivo was 14.22, 7.914 and 10.123 hours, respectively. The difference between PLM60-DPPC and PLM60-DMPC was the length of the hydrocarbon chains of the phospholipids, which accounted for 16 and 14 carbons, respectively. Length Alloway chain could significantly affect the permeability of the membrane phospholipid bilayer. The temperature of the phase shift DPPC was 41°C and the temperature of the phase shift DMPC - 23°C. the Difference between PLM60-DMPC-0.1 and PLM60-DMPC was to level PEG. The release of liposomal drug in the plasma depends on two factors: the first is the release of liposomal drug through the phospholipid bilayer, and the other is the ground clearance by lipoprotein and reticuloendothelial system (RES). As PEG drug PLM60-DMPC-0.1 was not full, release, due to components of plasma, had a greater influence.

Value
Table 3
Comparison of the in vivo pharmacokinetics of different formulations of liposomes in mice
PLM60-DPPC4 mg/kgPLM60-DMPC-0.14 mg/kgPLM60-DMPC4 mg/kg
ValueValue
AUC 0-48(mg/l*h)1506.710174.849337.641
AUC 0-∞ (mg/l*h)1581.242175.705344.134
AUMC 0-4821425.2741383.7573417.981
AUMC 0-∞26235.6131478.2673818.856
MKT 0-48(h)14.2207.91410.123
MRT 0-∞ (h)16.5928.41311.097
Tmax(h)1.0001.0001.000
Cmax(mg/l)81.97619.85339.115

Part 4: Comparison of the toxicity of various compounds

Example 15 Comparison of short-term toxic effects between PLM60 and FM

100 mice Kunimine (half males and half females) with a body weight of 18-22 g were divided into two groups, each g is the SCP 10 mice half males and half females. The mice 1-5 groups were applied different levels of dosage FM, while the 6-10 mice groups was applied equivalent dose level of liposomal drug. Observed changes in body weight, and recorded the time of death of each animal. Dead animals were dissected. The results of all groups are shown in Table 4, which showed that short-term toxic effect PLM60 was much less than the toxicity FM.

Table 4: Comparison of short-term toxic effect of PLM60 FM in mice Kunimine

td align="left"> 8
Liposome and doseThe survival time of mice males (days)The survival time of mice females (days)
mg/kgNo. 1No. 2No. 3No. 4No. 5No. 1No. 2No. 3No. 4No. 5
FM 2078896889ND
FM 12181313ND712121314ND
FM 7.2NDNDNDNDNDNDNDNDNDND
FM 4.32NDNDNDNDNDNDNDNDNDND
FM 2.59NDNDNDNDNDNDNDND NDND
PLM60 2017ND12NDNDNDNDNDNDND
PLM60 12NDNDNDNDNDNDNDNDNDND
PLM60 7.2NDNDNDNDNDNDNDNDNDND
PLM60 4.32NDNDNDNDNDNDNDNDNDIs d
PLM60 2.59NDNDNDNDNDNDNDNDNDND
ND: No data, then there is no animal died at the end of experimental research.

Example 16

Comparison of short-term toxic effect of different formulations of liposomes

90 mice male Balb/c mice with a body weight of 18-22 g were divided into 9 groups, each of 10 mice. Mice of group 1 were assigned to 6 mg/kg FM, while the remaining 8 mice groups were assigned to the 6 and 12 mg/kg PLM60, PLM60-DPPC and PLM60-DMPC-0.1 and PLM60-DMPC, respectively. Observed changes in body weight, and recorded the time of death of each animal. Dead animals were dissected. The results of the death of the mice group treated FM, and groups who liposomal drug, displayed in Table 5. This experiment showed the following order of short-term toxic effects: PLM60<PLM60-DPPC<PLM60-DMPC-0.1 FM<PLM60-DMPC. This experiment also confirmed that the release of drug can be further accelerated by using a small unilaminar pusy Kow and phospholipid with a lower Tm as the composition of the bilayer, such as PLM60-DMPC, thus causing greater toxicity in the body. It should be noted that the toxicity of liposomes with incomplete PEG was lower than the toxicity of liposomes with a more complete PEG. This can be attributed to the fact that under the influence of lipoprotein and destructive actions of the immune system during circulation, PLM60-DMPC-0.1 bulk PEG will release the drug earlier than PLM60-DMPC and will not release suddenly in important tissues, thus showing less toxicity, but toxicity PLM60-DMPC-0.1 was still approximately equal to the toxicity of free mitoxantrone.

td align="left"> ND
Table 5
Comparison of short-term toxic effect of the different liposomes
The form and dose (mg/kg)The survival time of mice (days)
12345678910
FM-6NDNDNDNDNDNDNDNDND
PLM60-6NDNDNDNDNDNDNDNDNDND
PLM60-12NDNDNDNDNDNDNDNDND11
Plm60DPPC-6NDNDNDNDNDNDNDNDNDND
Plm60DPPC-1210101211/td> NDNDND13ND14
Plm60-DMPC-64NDND36776NDND
Plm60DMPC-123353334333
Plm60DMPC-0.1-6NDNDNDNDNDNDNDNDNDND
Plm60DMPC-0.1-12101210121010111010
ND: no animal died at the end of experimental studies

Example 17

Comparison of the toxicity of the formulations of liposomes of various sizes

In comparison of the toxicity of PLM60, PLM85 and PLM100 used mouse males S with body weight 18-22 g Dose was 9 mg/kg the Results showed that the difference in body weight caused by the three compositions of liposomes were equivalent, which confirms the absence of significant differences in the toxicity of three compositions of liposomes in experimental conditions. The mice groups that received FM, body weight decreased by 30% and about 20% of the mice died.

Part 5: Antitumor action in the body

Example 18

Comparison of therapeutic action PLM60 and FM on sarcoma S-180

Mouse media ascitic tumor, which 7 days ago were inoculated with S180 tumor cells were euthanized by decapitation, and was extracted and diluted with RPMI 1640 medium dairy viscous ascitic fluid. After dilution number of tumor cells was set to 2.5×106cells/ml 0.2 ml suspension of tumor cells, containing about 5×105tumor cells were grafted into the armpit of the anterior limb of mice males KM from the weight of those who and 18-22, After inoculation of the cells in the cell suspension residual tumor were counted under an optical microscope; live tumor cells were more than 95%. The number of vaccinated mice was 80.

Seven days after inoculation were selected and divided into 5 groups according to the tumor size and body weight 39 mice with distinct tumors with a diameter of about 5 mm; that is, 7 mice in full control group, 8 mice in each group receiving 4 mg/kg PLM60, or 6 mg/kg PLM60, or 4 mg/kg FM, or 6 mg/kg FM. Mice did intravenous.

After the introduction of mice bred normally. The diameter of the tumors were measured 3 times a week using calipers at the same time was measured body weight. The size of the tumor (TV) was calculated by the following formula: V=1/2×a×b2where a and b denote the length and width respectively. The sizes of the tumors were calculated using the measurement results. Mice killed by decapitation on the 21st day after inoculation, tumors were removed and weighed. The level of tumor suppression (%) calculated according to the following formula: level of tumor suppression = (1 - average tumor weight in the treated group, drug / average tumor weight in the control group)×100%. The results of the experiment were tested by t-test.

The results presented in table 6 showed that the growth of solid tumor S180 who was b significantly suppressed in the group, took 4 mg/kg LM60, and the treated group, 6 mg/kg PLM60.

Table 6
Action PLM60 on the weight of solid tumor S180
GroupThe tumor weight (mg)The ratio of tumor suppression (%)
Control2813.8±884.2
PLM60 4 mg/kg421.9±215.4a85.00
PLM60 6 mg/kg332.4±162.5aAt 88.19
Free mitoxantrone 4 mg/kg2828.5±1067.8-
Free mitoxantrone 6 mg/kg2293.3±1720.018.50
a: in comparison with the control group, p<0.01

Example 19

therapeutic effect of PLM60 and FM on L1210 sample of ascites

Mouse media ascitic tumor BDF1, grafted cells ascitic tumor L1210 seven days ago, were euthanized by decapitation, and was extracted under aseptic condition and diluted with RPMI 1640 media is the your dairy viscous ascitic fluid. After dilution number of tumor cells was set to 2.5×106cells/ml 0.2 ml suspension of tumor cells, containing about 5×105tumor cells were inoculated into the abdominal cavity 7-8 weeks mice female BDF1. After inoculation of the cells in the cell suspension residual tumor were counted under an optical microscope; live tumor cells were more than 95%.

After 24 hours, mice were divided into 8 groups by body weight and assigned FM at doses of 2, 4 and 8 mg/kg and PLM60 at doses of 2, 4, 6 and 8 mg/kg by injection in a volume of 20 ml/kg in a vein in the tail of the mice, respectively. After the introduction of mice bred normally. Body weight was measured 3 times per week, were observed and recorded the time of death of each animal was calculated survival time. To assess survival time of each group were used, the average survival time (MST) and the median time of survival. An experimental study was conducted within 60 days after inoculation.

Data were analyzed using the statistical program SPSS 11.5. The results showed that all in all administrative groups showed a significant increase in survival time compared with the control group, and the group who PLM60 (8 mg/kg)demonstrated significant improvement in treatment compared to the treated group, FM at the same dose (P<0.05). Raza is taty displayed in Table 7.

Example 20

Therapeutic action of PLM60 and FM on a sample of liver metastasis L1210

Mouse media ascitic tumor BDF1, grafted cells ascitic tumor L1210 seven days ago, were euthanized by decapitation, and was extracted under aseptic condition and diluted with RPMI 1640 medium dairy viscous ascitic fluid. After dilution number of tumor cells was set to 2.5×105cells/ml 0.2 ml suspension of tumor cells, containing about 5×104tumor cells were inoculated intravenously 7-8 week old mice male BDF1. After inoculation of the cells in the cell suspension residual tumor were counted under an optical microscope; live tumor cells were more than 95%. Just was inoculated 62 mouse.

After 24 hours, mice were divided into groups and assigned medication. After the introduction of mice bred normally. The body weight of mice was measured three times per week, and were observed and recorded the time of death of each mouse was also calculated survival time. An experimental study was performed within 60 days after inoculation.

The result showed that all mice in the control group died between the 11th and 14th day after inoculation, all mice in groups, taking three degrees dosage FM, died between the 11th and 17th day after inoculation, all mice the group, took 2 mg/kg PLM60, died on the 39th day after inoculation, and none of the mice in the treated group, 8 mg/kg PLM60 not died during the study.

Data were analyzed using the statistical program SPSS 11.5. The results showed that the group who took 6 mg/kg FM, and all groups who liposomal drug, showed a significant increase in survival time of mice in comparison with the free mitoxantrone. The results are shown in Table 8.

Example 21

Therapeutic effect of liposomal mitoxantrone different size ascitic tumor L1210

The experimental design and method of data processing are the same as in Example 19. It was found 5 groups, including the control group, the treated group, FM group who PLM60, the group took PLM85, and the treated group, PLM100. Dosage injection to mice of each group was 4 mg/kg the Results are shown in Table 9. The results showed that liposome smaller had the best therapeutic effect.

Some preferred examples of the present invention described above, but these examples are in no way intended to limit the scope of the invention. Besides what has been described and illustrated in this text, the ordinary skilled technicians the East in this area will clearly understand other modifications, variations and modifications of the present invention after reading the disclosure of this invention, and all of them should fall under the scope of protection of this invention. All patents, published patent applications and publications cited herein are incorporated by reference as their full texts are included in the document.

1. Liposomal drug, in which (1) liposomal drug contains multivalent ionic drug as the active substance with two or more dissotsiiruut groups with the dissociation constant of 4.5 to 9.5; (2) liposome liposomal drug has a size of 30-80 nm; (3) two-layer structure of the liposome contains a phospholipid with a transition temperature (Tm) higher than body temperature, cholesterol and hydrophilic polymer-modified lipid; and (4) intrahippocampal phase of the liposome contains a multivalent counterion; and (5) transition temperature (Tm) liposomes above the temperature of the body where the content of the phospholipid with the transition temperature (Tm) higher than the body temperature, is about 50-100 mol./mol.%, preferably 55-95 mol./mol.%, more preferably 60-90 mol./mol.% relative to the total content of the phospholipids in the phospholipid bilayer structure.

2. Liposomal drug according to claim 1, characterized in that the size of the liposomes is 35-75 nm, preferably 40-70 nm, especially 40-60 nm.

3. Liposomal drug according to any one of claim 1 or 2, characterized in that the dissociation constant pKa of the active substance is 5.0 to 9.5, preferably of 5.5 to 9.5, more preferably 6,0-9,0, especially 6,5-9,0.

4. Liposomal drug according to any one of claims 1 or 2, wherein the multivalent ionic drug is an anticancer drug selected from mitoxantrone, vincristine, vinorelbine, vinblastine, or any combination thereof, preferably of mitoxantrone.

5. Liposomal drug according to any one of claims 1 or 2, wherein the multivalent counterion carries two or more charges, opposite to the current substance of the medicinal product, where the multivalent counterion is chosen from an anion of a saturated or unsaturated organic acid, the anion of an inorganic acid or the ionic form amino acids and where the organic acid is chosen from citric acid, tartaric acid, fumaric acid, oxalic acid, malonic acid, succinic acid, malic acid and maleic acid; an inorganic acid selected from sulfate and phosphate anions; amino acid selected from cystine; preferred by multivalent counterion is a citrate anion, sulfate or phosphate anion.

6. Liposomal drug according to claim 5, characterized in that Thu is multivalency the counterion is a sulfate anion.

7. Liposomal drug according to any one of claims 1 or 2, characterized in that the phospholipid with the transition temperature (Tm) higher than the body temperature in the phospholipid bilayer structure selected from phosphatidylcholine, hydrogenated soybean phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine or any combination thereof.

8. Liposomal drug according to claim 7, characterized in that the two-layer structure of a phospholipid, in addition, contains another phospholipid with the transition temperature (Tm) higher than the body temperature.

9. Liposomal drug according to any one of claims 1 or 2, characterized in that the cholesterol is about 2-60 mol./mol.%, preferably 5-55 mol./mol.%, especially 10-50 mol./mol.%, in particular 15-45 mol./mol.%, more specifically 20-40 mol./mol.% relative to the total content of the ingredients in the phospholipid bilayer structure.

10. Liposomal drug according to any one of claims 1 or 2, characterized in that the lipids modified with hydrophilic polymers are selected from distearyldimethyl ethanolamine modified PEG (cells of the dspe-PEG), distearoylphosphatidylglycerol modified PEG (DSPG-PEG), cholesterol-modified PEG (cholo-PEG), distearoylphosphatidylcholine modified by polyvidone (cells of the dspe-PVP), distearoylphosphatidylcholine is acerola, modified by polyvidone (DSPG-PVP), or cholesterol-modified polyvidone (chol-PVP), or any combination thereof, in an amount of 0.1-20 mol./mol.%, preferably 0.3 to 18 mol./mol.%, more preferably 0.5 to 15 mol./mol.%, even more preferably 0.8 to 12 mol./mol.%, most preferably 3-6 mol./mol.% relative to the total content of the phospholipids in the phospholipid bilayer structure.

11. Liposomal preparation of claim 10, characterized in that it contains hydrogenated soy phosphatidylcholine, cholesterol and distearoyl a phosphatidylethanolamine, a modified PEG in a weight ratio of 3:1:1, in which distearoylphosphatidylcholine modified PEG is preferably distearoylphosphatidylcholine modified PEG.

12. A method of obtaining a liposomal pharmaceutical preparation according to any one of claims 1 to 11, characterized in that it comprises the following stages: (1) preparation of liposomes by size of 30-80 nm using a phospholipid with a transition temperature (Tm) higher than body temperature, cholesterol and hydrophilic modified polymer-lipid; and (2) encapsulating multivalent ionic drug in the liposome, where the multivalent ionic drug has two or more dissociating groups with a dissociation constant pKa of 4.5 to 9.5, and anthroposophy phase liposomes contain multi is alenty the counterion.

13. Liposomal pharmaceutical preparation containing liposomal pharmaceutical preparation according to any one of claims 1 to 11, and optionally a pharmaceutically acceptable carrier and/or excipient.

14. Liposomal pharmaceutical preparation according to item 13, characterized in that it contains salt for changing the osmotic pressure, buffer agent and/or antioxidant.

15. A method of treating tumors in a patient, comprising the introduction of a liposomal pharmaceutical preparation according to any one of claims 1 to 11, or liposomal pharmaceutical preparation according to any one of p-14 to a patient in need of such treatment.

16. The method according to item 15, wherein the additional treatment is applied to the patient thermotherapy, preferably radioactive thermotherapy.

17. The use of liposomal pharmaceutical preparation according to any one of claims 1 to 11 or liposomal pharmaceutical preparation according to any one of p and 14 for the manufacture of a medicinal product for the treatment of a tumor in a patient.



 

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12 cl, 3 tbl, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention describes a prostate-specific membrane antigen (PSMA) compounds having a structure wherein Z represents tetrazolium or CO2Q; each Q is independently specified in hydrogen or a protective group; and wherein (A) m has a value of 0, 1, 2, 3, 4, 5 or 6; R represents a pyridine ring specified in a group including and wherein X represents fluorine, iodine, fluorine radioisotope, iodine radioisotope, chlorine, bromine, bromine radioisotope, astatine radioisotope, NO2, NH2, N+(R2)3, Sn(R2)3, Si(R2)3, Hg(R2), B(OH)2, -NHNH2, -NHN=CHR3, -NHNH-CH2R3; n has a value of 1, 2, 3, 4 or 5; Y represents O, S, N(R), C(O), NR'C(O), C(O)N(R'), OC(O), C(O)O, NR'C(O)NR, NR'C(S)NR', NR'S(O)2, S(CH2)P, NR(CH2)p, O(CH2)p, OC(O)CHR8NHC(O), NHC(O)CHR8NHC(O) or a covalent bond; wherein p has a value of 1, 2 or 3, R' represents H or C1-C6 alkyl, and R8 represents hydrogen, alkyl, aryl or heteroaryl, each of which may be substituted; R2 represents C1-C6 alkyl; and R3 represents alkyl, alkenyl, alkinyl, aryl or heteroaryl each of which is substituted by fluorine, iodine, fluorine radioisotope, iodine radioisotope, chlorine, bromine, bromine radioisotope or astatine radioisotope NO2, NH2, N+(R2)3, Sn(R2)3, Si(R2)3, Hg(R2) or B(OH)2; or (B) m has a value of 0, 1, 2, 3, 4, 5 or 6; Y represents O, S, N(R'), C(O), NR'C(O), C(O)N(R'), OC(O), C(O)O, NR'C(O)NR', NR'C(S)NR', NR'S(O)2, S(CH2)P, NR'(CH2)P, O(CH2)p, OC(O)CHR8NHC(O), NHC(O)CHR8NHC(O) or a covalent bond; wherein p has a value of 1, 2 or 3, R' represents H or C1-C6 alkyl, and R8 represents hydrogen, alkyl, aryl or heteroaryl, each of which may be substituted; R represents wherein X' is specified in a group including NHNH2, -NHN=CHR3 and -NHNH-CH2R3; wherein R3 represents alkyl, alkenyl, alkinyl, aryl or heteroaryl each of which is substituted by fluorine, iodine, fluorine radioisotope, iodine radioisotope, bromine, bromine radioisotope and astatine radioisotope; NO2, NH2, N+(R2)3, Sn(R2)3, Si(R2)3, Hg(R) or B(OH)2; R2 represents C1-C6 alkyl; n has a value of 1, 2, 3, 4 or 5; or (C) m has a value of 4, Y represents NR', and R represents wherein G represents O, NR' or a covalent bond; R' represents H or C1-C6 alkyl; p has a value of 1, 2, 3 or 4, and R7 is specified in a group including NH2, N=CHR3, NH-CH2R3, wherein R3 represents alkyl, alkenyl, alkinyl, aryl or heteroaryl, each of which is substituted by fluorine, iodine, fluorine radioisotope, iodine radioisotope, chlorine, bromine, bromine radioisotope or astatine radioisotope, NO2, NH2, N+(R2)3, Sn(R2)3, Si(R2)3, Hg(R2), B(OH)2; and R2 represents C1-C6 alkyl; also described are a method for visualising cells, organs or tissues involving the cell effect or introduction of the above compound into the body, as well as a method of treating a tumour and a kit comprising the above compound.

EFFECT: there are prepared and described the new compounds that enables introducing radionuclides for SPECT-images and positron-emission tomography (PET) easily for the purpose of visualising, eg prostate cancer cells and angiogenesis.

24 cl, 5 tbl, 24 ex, 5 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: device refers to new styrene derivatives with the structure formula A in the form of geometrical isomers or tautomers and their pharmaceutical acceptable salts. In structural formula (A) R1 represents hydrogen; R2 represents hydrogen or C1-C6alkyl; R3, R4, R5 and R6 are identical or different and independently represent hydrogen, halogen, C1-C6alkyl or -OR12; R7 represents hydrogen or C1-C6alkyl; R8 represents hydrogen; R9 represents hydrogen, C1-C6alkyl, or -C(=O)R13; R10 represents hydrogen or C1-C6alkyl; Z represents W-Y, wherein W represents -C(R14)(R15)-; Y represents -C(R16)(R17)-; each R12 independently represents hydrogen or C1-C6alkyl; each R13 independently represents C1-C6alkyl; R14 and R15 are identical or different, and independently specified in hydrogen, fluoro, methyl, ethyl, trifluoromethyl, -OH, -OCH3 or -NH2; or R14 and R15 together form oxo; R16 and R17 are identical or different and independently represent hydrogen, halogen, C1-C6alkyl or -OR12. The other radical values are specified in the patent claim.

EFFECT: compounds may be used for treating an ophthalmic disease or disorder in an individual which can represent age-related macular degeneration or Stargardt macular degeneration.

17 cl, 14 tbl, 143 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry and represents a biodegradable polymer carrier for the delivery of an anti-cancer drug with its macromolecule having a linear chain configuration and consisting of links of general formula wherein m=1 or 2; the molecular mass of the polymer carrier is 10000 to 200000 Da.

EFFECT: invention provides creating the new biodegradable polymer carrier able for chemical or physical binding to the anti-cancer drug to form easily degradable bonds in the human physiological environments.

2 cl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely oncology and concerns treating locally advanced vaginal cancer. For this purpose, the therapy is staged. The first stage involves the chemotherapeutic course that provides administering cisplatin 100 mg/m and doxorubicine 30 mg/m2 intravenously drop-by-drop; the gamma-ray teletherapy covering a primary centre in a dose of 20 Gy is followed by two fractions 2.5 Gy of the intracavitary Co60 therapy every 5 hours twice a week; in the middle of the 5-hour period, platidiam 5 mg dissolved in saline solution 1.0 ml is introduced into surface layers of the tumour which is exposed to ultrasound of frequency 880 kHz and intensity 0.4 Wt/cm2 through the licensed dimexide gel tissue for 5 minutes; the therapy requires the gamma-ray teletherapy covering the lymphatic nodes alternated with 10-11 sessions of the intracavitary Co60 therapy in total to reach a total dose of the primary centre exposure of 70-75 Gy, of the lymphatic nodes exposure of 40 Gy.

EFFECT: specified integrated therapy provides the effective treatment and prevention of the recurrences also ensured by using the chemotherapy combined with the ultrasonic exposure that promotes the deeper chemopreparation penetration into tumour tissues.

1 ex

FIELD: biotechnologies.

SUBSTANCE: antagonistic antibody is proposed, which specifically binds with a conformational epitope from a fragment 1378-1640 of an amino acid sequence Notch3, given in the description, and inhibits activation of Notch3. The specified antibody is characterised by the fact that it contains variable areas of light and heavy chain, with a set of appropriate CDR1-CDR3, amino acid sequences of which are given in the description. An antibody may additionally contain a mark. The following is proposed: versions of a coding nucleic acid, an expressing vector, a cell for antibody production, containing the specified vector. The method is described to produce antibodies by means of cell cultivation. Versions of antibodies application are disclosed: to detect a disease related to Notch3, for production of a medicinal agent for treatment of an individual with a disorder or a disease related to Notch3, for treatment of Notch3-related disease or disorder.

EFFECT: using the invention may find application in medicine in treatment or prevention of Notch3-related diseases or disorders.

25 cl, 18 dwg, 4 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to 5'-deoxy-5-fluoro-N4-pentyloxycarbonyl)cytidine, having anti-tumour activity with a high level of safety, as well as a pharmaceutical composition based thereon.

EFFECT: improved properties of the composition.

2 cl, 7 tbl

FIELD: medicine.

SUBSTANCE: there are presented: a liposomal preparation for the pulmonary delivery, wherein the liposomes a surface of which is modified by at least one polymer specified in a group consisting of terminally hydrophobisated polyvinyl alcohols and chitosan, an encapsulated gene, a method for preparing and a method of treating a pulmonary tissue disease involving the stage of administering the above liposomal preparation into the patient's lung. It has been shown that the liposome modified by terminally hydrophobisated polyvinyl alcohol may be kept on the pulmonary tissue surface for a long time.

EFFECT: high effectiveness of the pulmonary delivery of the claimed liposomal preparation for a relatively short time.

10 cl, 1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to an anti-cancer agent and an agent improving anti-cancer effect and combined with a liposomal agent prepared by oxaliplatin encapsulation in a liposome, and a combined therapeutic agent containing tegafur, gimeracil and oteracil potassium. The liposome in the declared agents consists of dipalmitoyl phosphatidylcholine, cholesterol and 1,2-distearoyl-8n-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000; or hydrogenised soybean phosphatidylcholine, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)- 2000; or dipalmitoyl phosphatidylcholine, cholesterol and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine polyglycerol. The invention also refers to using the above liposomal agent for preparing the anti-cancer agent and for intensifying anti-cancer activity of the combined therapeutic agent containing tegafur, gimeracil and oteracil potassium. Also, the invention relates to a method of treating cancer by administering the liposomal agent comprising oxaliplatin encapsulated in a liposome, and the combination drug containing tegafur, gimeracil and oteracil potassium.

EFFECT: group of inventions enhances anti-cancer activity of the therapeutic agent without toxicity increase.

12 cl, 10 dwg, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine, namely to pharmaceutical compositions for topical application containing insulin and liposomes. What is described is a pharmaceutical composition for topical application, containing insulin and liposomes in the form of a biofilm having rapid cutaneous penetration, and an ability to reduce blood glucose.

EFFECT: what is presented is the composition having rapid cutaneous penetration.

4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine, namely to pharmaceutical compositions for topical application. What is described is a pharmaceutical composition for topical application, containing insulin and liposomes bound thereto containing hydrated lecithines in a combination with cholesterol in the form of a plaste having rapid cutaneous penetration, and an ability to reduce blood glucose.

EFFECT: usability and ease of dosing.

4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is the use of L-carnosine for making a nanopreparation having antihypoxic and antioxidant activity combined with a combination of substances selected from the group of phospholipids, non-polar lipids in the following ratio, wt %: L-carnosine - 1.1-1.2, non-polar lipids such as triglycerides, cholesterol, free fatty acids, DL-α-Tocopherol - 1.2-2.5, phospholipids such as phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomyelin - 95.3-96.3 for preparing a drug having antihypoxic and antioxidant activity. The drug can be presented in the form of liposomes containing L-carnosine.

EFFECT: invention provides higher stability of L-carnosine and its lifetime up to three days with underlying higher effectiveness in small doses, as well as to improve the cerebral ischemia tolerance, the recovery after acute hypoxia and to increase the antioxidant status of the brain tissue.

3 cl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutical compositions for local application. Claimed compositions include sildenafil, minoxidil, testosterone, or alprostadil, euphyllin, yohimbine, or sildenafil, minoxidil, yohimbine as active agents respectively. Claimed compositions also contain hydrogenated lecithins in combination with cholesterol, emulsifying agent, preservative and demineralised water in specified weight ratios.

EFFECT: group of inventions ensures fast penetration through skin and complex therapy of erectile dysfunction taking into account impact on psychogenic and endocrine factors.

3 cl, 4 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of molecular medicine and pharmacology and describes liposome, which contains, at last, one lipid bi-layer surrounding water compartment, in which said lipid bi-layer contains, at least, one amphiphilic synthetic pyridinium derivative, in which total amount of amphiphilic pyridinium derivative constitutes from 2 to 25 mol % of total content of lipids in liposome. Liposomes are suitable for delivery of medicinal compounds.

EFFECT: liposome is stable and makes it possible to improve intracellular release of medication.

18 cl, 1 ex, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a magnetic system which has a structure containing magnetic nanosized particles of formula MIIMIII2O4, wherein MII=Fe, Co, Ni, Zn, Mn; MIII=Fe, Cr, or maghemite which are functionalized by bifunctional compounds of formula R1-(CH2)n-R2 where n = 2-20, R1 is specified in: CONHOH, CONHOR, PO(OH)2, PO(OH)(OR), COOH, COOR, SH, SR; R2 is an external group, and is specified in: OH, NH2, COOH, COOR; R is an alkyl group or an alkaline metal specified in C1-6-alkyl and K, Na or Li, respectively). The structure also includes a polymer optionally containing a pharmacologically active molecule, and an external pharmacologically active molecule may be specified in anticancer agents, antimicrobial agents, anti-inflammatory agents, immunomodulators, molecules acting on the central nervous system or able to mark cells so as to enable the identification thereof by means of the usual diagnostic detectors. The invention also refers to a method for preparing the nanosized particles of formula MIIMIII2O4 which consists in adding a metal salt to alcohol, heating to complete solubilisation of salts, adding water to facilitate salt hydrolysis and heating to temperature 150-180°C to prepare a suspension to be then functionalised. The invention also refers to a method for preparing the magnetic system, wherein the functionalised nanoparticles and the pharmacologically active molecules are integrated into the matrix of a water-insoluble polymer, and the prepared structure is continuously and one-stage coated with adequate surfactants.

EFFECT: invention aims at preparing the magnetic system suitable for hyperthermia procedures.

16 cl, 4 tbl, 1 dwg, 26 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a pharmaceutical composition for treating and preventing viral and bacterial infections, containing as active ingredients: lysocyme, peroxidase, poviargol, as anti-inflammatory ingredients: escin and glycyrrhizic acid or a salt thereof, as carriers - liposome based on high-active hydrogenated lecithins in a combination with cholesterol and pharmaceutically acceptable carriers and excipients, and the ingredients of the composition are taken in a certain ratio, wt %.

EFFECT: invention ensures maintaining prolonged activity of the enzymes being the ingredients of the composition, fast skin penetration and absorption.

4 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. A dosage form for treating mycotic infections contains nanosomes of cholesterol-containing amphotericin B of the diameter within the range of 20-200 nm in physiologic saline, the ratio of lipid to amphotericin B ranges from 45:1 to 45:15. Amphotericin B is exposed to ultrasound to destruct at the final stage to transform the particles into smaller and less lamellar nanosomes.

EFFECT: invention provides lower nephrotoxicity and effective antimycotic activity.

3 cl, 11 tbl, 3 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: method involves applying a composition comprising liposomes having gene structures encoding growth factors. The composition is administered for making injections into wound and impregnating materials for covering or closing wounds with the materials. Advanced bandage has coverage material and liposomes. Introducing liposome gene structures directly into wounds contributes to better healing results.

EFFECT: wide range of functional applications; enhanced treatment effectiveness and safety.

31 cl, 13 dwg, 4 tbl

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