New thermally-sensitive liposomes containing therapeutic agents

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a thermally-sensitive liposome for treating cancer in a subject, containing at least one phosphatidylcholine, at least one phosphatidylglycerol, monostearoyl phosphatidylcholine, pegylated phospholipid and an active substance specified in a group consisting of taxotere, docetaxel and carboplatin wherein the liposome has a gel to liquid transition point from approximately 39°C to approximately 45°C.

EFFECT: invention provides storage-stability for a long period of time of one month or more at low temperatures.

9 cl, 8 ex, 8 dwg

 

The LEVEL of TECHNOLOGY

Liposomes are used to deliver a wide variety of drugs. For example, antineoplastic agents, such as actinomycin (U.S. patent 3993754), anthracyclines (U.S. patent 4863739) and Vinca alkaloids (U.S. patent 4952408), enclosed in a capsule of liposomes. Were later obtained thermosensitive liposomes containing the active funds and used to deliver active agents to specific target organs of the subject (U.S. patent No. 6200598 and 6726925 and Yatvin et al., Science 204:188 (1979). When using temperature-sensitive liposomes are delivered to the entity and the target entity is heated. When thermosensitive liposomes reach the heated zone, the phase transition occurs gel-liquid and the release of the active funds. The success of this method requires liposomes with the temperature of the phase transition of the gel-liquid within the temperature range characteristic of the entity.

In the art there remains a need to encapsulate the obtained liposome drug, such as an antitumor agent, which is able to undergo phase transition from the gel-liquid with the temperature characteristic of the subject. This and other conditions are met in the present invention.

DISCLOSURE of INVENTIONS

In one embodiment, the present invention provides the AET thermosensitive liposomes. Thermosensitive liposomes according to the invention usually contain at least one phosphatidylcholine, at least one phosphatidylglycerol and at least one soliped. Thermosensitive liposomes according to the invention will typically have a temperature of phase transition of the gel-liquid from about 39°C to about 45°C. temperature-sensitive liposomes according to the invention optionally can contain one or more additional lipid components, for example, may contain Paglierani phospholipids. Thermosensitive liposome according to the invention may contain one or more active agents, for example, medicines, tools for creating images, diagnostic tools, and combinations thereof.

In some cases, the implementation of phosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol is distearoylphosphatidylglycerol (DSPG) and lysoled is monasteriopiedra.com (MSPH) and thermosensitive liposomes contain Paglierani phospholipids, such as PEG-2000, modified distearoylphosphatidylcholine (DSPE-PEG) or PEG-5000-modified distearoylphosphatidylcholine (DSPE-PEG). Thermosensitive liposomes according to the invention can contain phosphatidylcholine, phosphatidylglycerol, lysoled and Targeted phospholipid in either the proportion allowing the temperature of the phase transition of the gel-liquid be in the range of from about 39°C to about 45°C. Typically, the liposomes according to the invention can contain the following ratios of ingredients by weight in the following ranges, phosphatidylcholine 60-80: phosphatidylglycerol 6-12: lysoled 6-12: Targeted phospholipid 4-15: an active agent 1-30. For example, thermosensitive liposome according to the invention may contain a ratio by weight of DPPC 60-80: DSFG 6-12: MSFH 6-12: DSPE-PEG 4-15: an active agent 1-30.

Thermosensitive liposomes according to the invention can contain one or more active agents. Any active agent, known to experts in the art, can be used in combination with thermosensitive liposomes according to the invention for delivery of active funds in the selected place of the subject. Using here the subject can be any mammal, in particular humans, cats or dogs. In one embodiment, thermosensitive liposomes according to the invention contain one or more anti-cancer remedies. Examples of suitable anti-cancer tools include, but are not limited to, alkylating tools, antimetabolites, cytotoxic vegetable alkaloids, cytotoxic, antitumor antibiotics, anthracycline antibiotics, plant alkaloids, producing the s Taxol, the topoisomerase inhibitors, monoclonal antibodies or fragments thereof, photosensitizers, inhibitors of kinases, antitumor enzymes or enzyme inhibitors, inducers of apoptosis, biological response modifiers, antihormones, retinoids and components containing platinum. In some cases, the implementation of thermosensitive liposomes according to the invention can contain Texan, such as docetaxel. In another separate embodiment, thermosensitive liposomes of the invention may include a compound containing platinum component, such as carboplatin or cisplatin.

The present invention also provides pharmaceutical compositions containing thermosensitive liposomes according to the invention containing the active agent. In such pharmaceutical compositions thermosensitive liposomes according to the invention usually contain at least one phosphatidylcholine, at least one phosphatidylglycerol at least one lysoled or have the temperature of the phase transition of the gel-liquid from about 39°C to about 45°C. temperature-sensitive liposomes for use in the pharmaceutical compositions according to the invention can additionally contain Targeted phospholipid.

In one example of a suitable temperature-sensitive liposomes for use in the pharmaceutical companies who stand according to the invention phosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol is distearoylphosphatidylglycerol (DSPG) and lysoled is monasteriopiedra.com (MSPH) and thermosensitive liposome containing Targeted phospholipid, for example, PEG-2000, modified distearoylphosphatidylcholine (DSPE-PEG). Such thermosensitive liposomes according to the invention can contain phosphatidylcholine, phosphatidylglycerol, lysoled and Targeted phospholipid in any proportion, so that the temperature of the phase transition of the gel-liquid was the range of from about 39°C to about 45°C. Typically, the liposomes for use in the pharmaceutical compositions according to the invention can contain the following ratios of ingredients by weight in the following ranges, phosphatidylcholine 60-80: phosphatidylglycerol 6-12: lysoled 6-12: Targeted phospholipid 4-15: an active agent 1-30. For example, thermosensitive liposome according to the invention may contain a ratio by weight of DPPC 60-80: DSFG 6-12: MSFH 6-12: DSPE-PEG 4-15: an active agent 1-30.

In the pharmaceutical compositions according to the invention include any active agent, such as drugs and/or radioactive means. In one embodiment, the active agent may be an anti-cancer agent. Examples of suitable anti-cancer tools include, but are not limited to, ALK is stimulating agents, antimetabolites, anticancer antibiotics, anthracycline antibiotics, plant alkaloids, Taxol derivatives, topoisomerase inhibitors, monoclonal antibodies, photosensitizers, inhibitors of kinases and components containing platinum. In a separate embodiment, thermosensitive liposomes according to the invention can contain an anthracycline antibiotic, such as docetaxel. In a separate embodiment, thermosensitive liposomes of the invention may include a compound containing platinum, such as carboplatin or cisplatin.

The present invention also provides for methods of treating diseases in a subject using thermosensitive liposomes according to the invention. Such thermosensitive liposomes will usually contain one or more active agents that can be used to treat diseases. A method of treating disease in a subject in need thereof, according to the invention, may provide an introduction to a subject a therapeutically effective amount of a pharmaceutical composition containing a heat-sensitive liposome containing an active agent, where the liposome comprises at least one phosphatidylcholine, at least one phosphatidylglycerol at least one lysoled and the temperature of the phase transition of the gel-fluid status is made from about 39°C to about 45°C. Part of the subject containing some or all of the affected tissue, and then heated to a temperature sufficient to move liposomes from a gel to a liquid, which leads to the release of the active funds directly near the affected tissue. Thermosensitive liposomes for use in the methods according to the invention may also contain Targeted phospholipid, for example DSPE-PEG or DSPE-PEG.

In one example, thermosensitive liposomes for use in the methods according to the invention phosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol is distearoylphosphatidylglycerol (DSPG) and lysoled is monasteriopiedra.com (MSFH). Such thermosensitive liposomes according to the invention can contain phosphatidylcholine, phosphatidylglycerol, lysoled and Targeted phospholipid in any ratio so that the temperature of the phase transition of the gel-liquid was in the range of from about 39°C to about 45°C. Typically, the liposomes for use in the pharmaceutical compositions according to the invention can contain the following ratios of ingredients by weight in the following ranges: phosphatidylcholine 60-80: phosphatidylglycerol 6-12: lysoled 6-12: Targeted phospholipid 4-15: an active agent 1-30. For example, thermosensitive liposome according to the invention can contain is the odds ratio by weight of DPPC 60-80: DSFG 6-12: MSFH 6-12: DSPE-PEG 4-15: an active agent 1-30.

In one embodiment, the present invention includes a method of treating cancer in a subject in need thereof, introducing to the subject a therapeutically effective amount of a pharmaceutical composition containing a heat-sensitive liposome containing an anti-cancer agent, where the liposome comprises at least one phosphatidylcholine, at least one phosphatidylglycerol at least one lysoled, and the temperature of the phase transition of the gel-liquid ranges from about 39°C to about 45°C. Examples of suitable anti-cancer tools include, but are not limited to the above, alkylating tools, antimetabolites, antitumor antibiotics, anthracycline antibiotics, plant alkaloids, Taxol derivatives, topoisomerase inhibitors, monoclonal antibodies, photosensitizers, inhibitors of kinases and compounds containing platinum. In a separate embodiment, thermosensitive liposomes according to the invention can contain an anthracycline antibiotic, such as docetaxel. In a separate embodiment, thermosensitive liposomes of the invention may include a compound containing platinum, such as carboplatin or cisplatin.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 is a curve of differential scanning calorimetry (Dr Who) showing the phase transition of the gel-liquid typical thermosensitive liposomes according to the invention.

Figure 2 is a graph of particle size as a function of the number of cryoprotectant in dried preparation of liposomes.

Figure 3 is a graph of particle size during rehydration of lyophilized liposomes according to the invention as a function of water content in the liposomes at different speeds temperature changes during freezing.

Figa is a schematic of the Protocol used for testing effects on exact observance of particle size rehydration liposomes according to the invention.

FIGU is a graph showing the particle size distributions rehydration liposomes according to the invention after one hour.

Figure 5 is a line graph of the size distribution of the particles of the heat-sensitive carboplatin liposomes.

6 is a histogram of the size distribution of the particles of the heat-sensitive carboplatin liposomes.

7 is a line graph of drug release at 37°C (white diamonds) and at 42°C (black diamonds) as a function of time.

Fig is a histogram showing the release of conteplating at different temperatures for 5 minutes (blue) and 10 minutes (purple).

DETAILED description of the INVENTION

Thermosensitive the liposomes according to the invention typically contain one or more phosphatidylcholine. Suitable examples of phosphatidylcholine that can be used in the practice of the invention include, but are not limited to the mentioned above, 1,2-Dilauroyl-sn-glycerol-3-phosphocholine (DLPH), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSFH), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPH) and 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (PATH).

Thermosensitive liposomes according to the invention typically contain one or more phosphatidylglycerol. Suitable examples of phosphatidylglycerol include, but are not limited to the mentioned above, 1,2-dimyristoyl-sn-glycerol-3-fosfoglitserin (DMPG), 1,2-dipalmitoyl-sn-glycerol-3-fosfoglitserin (DPPG), 1,2-distearoyl-sn-glycerol-3-fosfoglitserin (DCFH) and 1-Palmitoyl-2-oleoyl-sn-glycerol-3-fosfoglitserin (POPG).

Thermosensitive liposomes according to the invention typically contain one or more soliped. When used here "lysoled" refers to any derivative of phosphatidic acid (1,2-diacyl-sn-glycerol-3-phosphate), which contains only one acyl chain, covalently linked with glycerol component. Derivatives fosfatidinozitol acids include, but are not limited to the mentioned above, phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. Any lysoled known to specialists in this about the Asti equipment, can be used in the practice of the invention.

Thermosensitive liposomes according to the invention typically contain one or more Targeted phospholipid. Suitable examples Paglierani phospholipids include, but are not limited to the mentioned above, 1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350] (MPEG 350 PE), 1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (MPEG 550 PE), 1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (MPEG 750 PE), 1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (MPEG 1000 PE), 1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (MPEG 2000 PE), 1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (MPEG 3000 PE), 1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (MPEG 5000 PE), modified PEG-2000 distearoylphosphatidylcholine (DSPE-PEG) and modified PEG-5000 distearoylphosphatidylcholine (DSPE-PEG).

Active funds

Thermosensitive liposomes of the invention can be produced for the compilation of one or more active agents. When used here "active agent" includes any compound which is necessary to deliver a specific place in the body. Any active tool can be used in practitionerthe.

As an active tool in thermosensitive liposomes of the invention can be used anticancer tools.

Suitable examples of anti-cancer tools include:

alkylating tools, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, melphalan, nitrosoanatabine (for example, carmustine, fotemustine, lomustin, streptozocin)containing platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, BBR3464), busulfan, dacarbazine, chlormethine, procarbazine, temozolomide, thiotepa and uramustine;

antimetabolites, aimed, for example, folic acid (e.g., aminopterin, methotrexate, pemetrexed, raltitrexed), purine metabolism (e.g., cladribine, Clofarabine, fludarabine, mercaptopurine, pentostatin, tioguanin), pyrimidine metabolism (capecitabine, cytarabine, fluorouracil, floxuridine, gemcitabine);

cytotoxic vegetable alkaloids, for example, taxanes (e.g., docetaxel, paclitaxel and Vinca alkaloids (e.g. vinblastine, vincristine, vindesine, vinorelbine);

cytotoxic/antitumor antibiotics, for example, anthracycline antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubitsin, mitoxantrone, valrubicin, erinomisen, N-acetylcarnitine, rubiazo, 5-imitational, N30 acetylcarnitine and Epirus is Itin), bleomycin, mitomycin, and actinomycin;

the topoisomerase inhibitors, for example, camptothecin (for example, camptothecin, topotecan, irinotecan), pedofil (e.g., etoposide, teniposide);

monoclonal antibodies or fragments thereof, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab, rituximab, tositumomab and trastuzumab;

photosensitizers, for example, aminolevulinate acid, methylaminoethanol, porfimer sodium and verteporfin;

the kinase inhibitors, such as dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib and vandetanib;

enzymes, for example asparaginase, pegaspargase and enzyme inhibitors such as hydroxyurea;

inducers of apoptosis, for example, arsenic trioxide, walked and genasense;

the biological response modifiers, such as denileukin diftitox;

antihormones, for example goserelin acetate, leuprolide acetate, triptorelin pamoate, megestrol acetate, tamoxifen, toremifene, fulvestrant, testolactone, anastrozole, exemestane and letrozole; and

retinoids, such as 9-CIS-retinoic acid and all-TRANS-retinoic acid.

In additional embodiments, the implementation of thermosensitive liposomes according to the invention can contain more than one antineoplastic or more than one heat-sensitive liposome is possible is to use the methods of the invention, each containing different active agents, such as various anti-cancer tool.

Additional active funds that can be used in the practice of the present invention, include but not limited to the above, antibiotics, antifungal, anti-inflammatory agents, immunosuppressive tools, anti-infective tools, antiviral, antihelminthic and antiparasitic compounds.

Thermosensitive liposomes according to the invention containing the active agent may contain lipids or active tool in any relationship until liposome remains thermally sensitive and can release the active agent at a suitable temperature, for example between 39°C and 45°C. Suitable range of proportions by weight phosphatidylcholine: phosphatidylglycerol: lysoled: Targeted phospholipid: active medium are 60-80:6-12:6-12:4-15:1-30. Suitable ranges of ratios by weight phosphatidylcholine: phosphatidylglycerol: lysoled: Targeted phospholipid: an active agent include, but are not limited to 70:8:8:8:4, 71:8:8:8:4, 72:8:8:8:4, 73:8:8:8:4, 74:8:8:8:4, 75:8:8:8:4, 70:8:8:6:4, 71:8:8:6:4, 72:8:8:6:4, 73:8:8:6:4, 74:8:8:6:4, 75:8:8:6:4, 70:8:8:4:4, 71:8:8:4:4, 72:8:8:4:4, 73:8:8:4:4, 74:8:8:4:4, 75:8:8:4:4, 70:9:9:8:4, 71:9:9:8:4, 72:9:9:8:4, 73:9:9:8:4, 74:9:9:8:4, 75:9:9:8:4, 70:9:9:6:4, 71:9:9:6:4, 72:9:9:6:4, 73:9:9:6:4, 74:9:9:6:4, 75:9:9:6:4, 70:9:9:4:4, 71:9:9:4:4, 72:9:9:4:4, 73:9:9:4:4, 74:9:94:4 and 75:9:9:4:4.

Method of use

Thermosensitive liposomes according to the invention it is possible to introduce the subject by any suitable means, for example, intravenous, intraarterial introduction, intramuscular, intra-abdominal introduction, subcutaneously, intradermally, intra-articular, intrathecal, intracerebroventricular, nazalnam spray, pulmonary inhalation, by oral administration, and other appropriate methods known to experts in this field of technology. Tissues that can be affected using the methods of the present invention include, but are not limited to, nasal, pulmonary, hepatic, renal, bone, soft tissues, muscles, adrenal tissue, and breast. Tissues that can be affected include as cancer tissue, and patients, and threatened and healthy tissue, if desired. Any tissue or fluid of the body, which can be heated to a temperature above 39.5°C, can be exposed to liposomes according to the invention.

The number of active funds, introduced the subject by using thermosensitive liposomes according to the invention can easily be determined by experts in the art, and accordingly is administered intravenously over a long period of time, for example, within about one minute up to several hours, for example 2, 3, , 6, 24 or more hours. When used here "about" means the variability within 10% when used to modify a numeric value.

The dose of the active means can be adjusted, as is known in the art, depending on the active tool contained in the carrier.

Given the tissue of the subject can be heated before and/or during and/or after the introduction of temperature-sensitive liposomes according to the invention. In one embodiment, first heat a given tissue (e.g., within 10-30 minutes), and liposomes according to the invention are delivered to the subject after heating as soon as practically possible.

You can use any suitable means for heating a given tissue, such as radiation, application of ultrasound, which can be focused high-intensity ultrasound, the use of microwave radiation, any source that generates infrared radiation, such as warm water bath light, as used outside or inside the radiation, such as is generated by radioactive isotopes, electric or magnetic fields, and/or a combination of the foregoing.

The average person skilled in the art will be obvious that other suitable modifications and adaptations of the methods and applications described is here can be performed without deviating from the purpose of the invention, or any variants of its implementation. Having a detailed description of the present invention, the same will be better understood by the following examples, which are included here solely for the purpose of illustration and are not intended to limit the invention.

EXAMPLES

EXAMPLE 1

Preparation and characterization of thermosensitive liposomes with Taxotere

For preparation of liposomes according to the invention the following materials were used:

dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylglycerol (DSPG) and monasteriopiedra (MSPH), Targeted distearoylphosphatidylcholine (DSPE-MPEG), NaCl, KCl, Na2HPO4·12H2O KH2PO4, lactose, CHCl3, methanol, ethanol and distilled water.

For preparation of liposomes according to the invention was used with the following equipment: water bath, rotary evaporator, homogenizer-extruder, lyophilizator, the scattering of laser radiation measuring particles (Smypatec Nanophox) and a thermometer.

Method for the preparation of 20-ml portions of liposomes containing docetaxel.

Measure the following components in the indicated amounts.

ComponentDPPC DSFGMSFHDSPE-PEGTaxotere
Number669 mg75 mg75 mg75 mg37.5 mg

Dissolve these materials in CHCl3/methanol (3:1) at 55°C. Then remove the organic solvent using a rotary evaporator. This can be done by rotary evaporation at 55°C for 1 hour. After drying the dried material can be blow nitrogen during the relevant time period, such as 5 minutes.

Then the dried material rehydration. The corresponding solution for rehydration is a phosphate buffer (PBS), to which you can add lactose or other stabiliausia materials (e.g. sugar). The appropriate Protocol for rehydration is adding 20 ml of PBS-5% solution of lactose (pH 7.3±0.2) and rotation on a rotary evaporator at atmospheric pressure for 1 hour at 50°C. After rehydration solution can degassing under reduced pressure to remove bubbles.

After rehydration, the particle size of liposomes can be adjusted to a desired size, for example 100±15 nm. The appropriate Protocol intrusion is to IP alsowhat homogenizer-extruder filter 200 nm and to perform the extrusion of three times. Change the filter at 100 nm and follow the extrusion of three times. Finally, change the filter at 80 nm and follow the extrusion of three times. The distribution of particle size of liposomes can be measured using any appropriate technique, such as photon cross-correlation spectroscopy (Photon Crosscorrelation Spectroscopy, PCCS) and sensor Nanophox (Sympatec GmbH). After extrusion liposomal solution can be sterilized by filtering through a membrane filter with a pore size of 0.22 μm (Millipore).

After sterilization of liposomes filled into ampoules and lyophilizers. The program lyophilization program is as follows: -50°C 2H, -45°C 1 h, -35°C, 10 h, -15°C, 5h, 0°C, 2H, 10°C 2 h, 20°C 6 hours

Another suitable method for the preparation of liposomes according to the invention is as follows:

Dissolve the same components as described above in CHCl3/methanol (3:1) at 55°C. to Remove the organic solvent using a rotary evaporator as described above. To registrationall 20 ml of PBS-5% solution of lactose at 50°C as described above. Put registrationentry material in the homogenizer and subjected to processing at 15,000 psi for 5 minutes to reduce the particle size. Take homogenized material and to use the extruder to filter 100 nm and spend the extrusion six times to reduce the particle size to 100±15 nm (100 nm × 6) and then sterilized by filtration to 0.22 MK is. After sterilization, filled into ampoules and lyophilizers.

Analytical methods

The morphology of the liposomes can be analyzed on the electron microscope. Liposomes were negatively stained phosphomevalonate acid and placed in a copper grid. Water could evaporate and the samples were investigated using an electron microscope. Liposomes prepared by the methods according to the invention, the analysis under the electron microscope were homogenized.

For liposomes, prepared as described above was measured by the percentage of the encapsulated drug (% encapsulation). % encapsulation = number of encapsulated drug/drug total × 100%. percent encapsulation was determined as follows: 1 ml of liposomes were centrifuged at 6000 rpm for 5 minutes Docetaxel in the supernatant was measured by high performance liquid chromatography (HPLC). The content of docetaxel liposomes was determined by extraction of docetaxel from liposomes and measurement extracted docetaxel using HPLC. For the extraction of 0.1 ml of liposomes were dissolved in water:acetonitrile (45:55) to 0.5 ml was Added to 4 ml of tert-butyl methyl ether and stirred for 30 seconds. The mixture was centrifuged at 300 g for 15 minutes, 3 ml of the organic layer was removed and dried by rotary evaporation. The dried material was again dissolved in 200 ml of water is:acetonitrile (45:55) and took 5-10 ml for analysis by HPLC.

The HPLC analysis was performed under the following conditions: mobile phase water:acetonitrile (45:55) was used column Venusil C 18 (column reversed-phase 18) at 1 ml/min column Temperature was 30°C. UV detection was set at 230 nm. Under these conditions, the detection limit of the drug is in the range 20-800 ng.

Was determined the ability of the above Protocol to restore docetaxel in the sample. To 0.1 ml of liposomes, prepared as described above, was added 0.1 ml of a normal solution of docetaxel. The sample was dissolved in water:acetonitrile (45:55) to 0.5 ml was Added to 4 ml of tert-butyl methyl ether and stirred for 30 seconds. Then the sample was centrifuged at 300 g for 15 minutes, 3 ml of the organic layer was dried by rotary evaporation. To the residue was added 200 ml of water:acetonitrile (45:55) and took 5-10 ml for analysis by HPLC. The following table shows the degree of recovery at different concentrations of docetaxel.

The drug concentrationRecovery, %Recovery, %Recovery, %Average
80 mg/ml100,34of 99.9799.91 per
100 mg/ml99,1596,6398,0897,95
120 mg/ml97,6899,0199,4198,70

Was defined transition temperature of liposomes prepared according to the invention. Measurements were performed differential scanning calorimetry (DSC) using a Q100 (TA Instruments, Inc. New Castle DE) c blank hermetically sealed aluminum crucibles as a control. The lipid concentration was reduced to 20 mg/ml, and 10 ml of liposomal suspension was carefully placed in a sealed sealed aluminum crucibles. The scanning frequency was set at 2°C per minute. Figure 1 shows a graph of the DSC received on the liposomes according to the invention. Range of the DSC shows that the transition temperature for a temperature-sensitive liposomes with Taxotere is approximately 42°C.

Stability of liposomes prepared with the above method were evaluated by periodic measurements of the particle size during storage. The results in the table below show that liposomes prepared as described above, stable at least 3 m the month.

TimeBefore lyophilizationAfter lyophilization1 month2 months3 months
Size97 nm101 nm107 nm106 nm106 nm

Also monitor the content of the drug. The results show that after lyophilization of liposomes are stable at 2-8°C for at least 3 months.

TimeAfter lyophilization1 month2 months3 months
Taxotere (mg/ml)1,1321,1311,1321,130

The rate of encapsulation of the drug is also tracked. The results show that after lyophilization the amount of the drug encapsulated by the liposome, is stable at 2-8°C for at least 3 months.

Encapsulation of the drug

TimeAfter lyophilization1 month2 months3 months
Encapsulation, %99,098,698,198,3

We have tested the effect of different cryoprotectants on the particle size during lyophilization. Were tested lactose, trehalose, sucrose and mannitol. The results show that lactose and sucrose is more effective than mannitol and trehalose. Figure 2 shows a graph of particle size as a function of % by weight cryoprotectant, in dried mud.

The rate at which liposomes freeze for freeze-drying, and the water content in the liposomes affect the particle size. Figure 3 shows a graph of particle size as a function of water content in the liposomes at three different speeds freeze.

Wednesday rehydration is also set on the particle size of liposomes. Tested water, 5% dextrose in water (DW) and 0.9% NaCl. 0.9% NaCl and 5% dextrose in water supports the particle size of liposomes. The following table shows the results of two different formations of liposomes with three independent solvents. The average diameter of liposomes following nanometre (nm). The composition of F4-1 contains the following components DPPC:DSPG:DSPE-PEG:MFH:docetaxel in the following mass. % 71,56:8,15:8,24:8,02:4,00 and F4-2 contains the same components in concentrations 71,78:8,06:8,10:8,07:3,98 wt.%.

Wednesday rehydrationComposition1 (nm)2 (nm)3 (nm)
WaterF4-1136133139
F4-2132128141
DVF4-197101103
F4-2102104105
0.9% NaClF4-1101105102
F4-2106103101

They studied the stability of liposomes after rehydra the promotion. Lyophilized liposomes rehydratable with 0.9% NaCl and tested as shown schematically in Figa. The distribution of particle size was monitored with the help of dynamic light scattering by repetitive scans over a period of 1 hour. The results show that the distribution of particles rehydration liposomes stable for 1 hour (Pigv).

Lyophilized liposomes were stored at various temperatures for 9 months. Liposomes were tested for % encapsulation and the average particle size after 0, 1, 3, 6 and 9 months. Results in the following table show that the liposomes were stable for up to 9 months at 4°C.

Storage time (months)Storage temperature -20°CStorage temperature 4°CStorage temperature 20°C
0to 92.1% (98 nm)to 92.1% (98 nm)to 92.1% (98 nm)
191,8% (101 nm)91,5% (103 nm)91,7% (107 nm)
2of 91.6% (102 nm)91,4% (105 nm)91.1% of 106 nm)
391,5% (105 nm)91,2% (106 nm)90,8% (109 nm)
691,4% (108 nm)of 91.3% (107 nm)90,9% (111 nm)
990,8 % (112 nm)90,5% (110 nm)90,2% (116 nm)

EXAMPLE 2

Compared the in vivo distribution of the drug from the liposomes according to the invention and distribution of free docetaxel.

Six female mice of BALB/C (20±2 g) randomly divided into three groups. Spent anesthesia of mice and put them on a Styrofoam panel with a hole in it. One leg of the mouse passed through the hole on the other side of the panel. The panel floated in a water bath to heat the leg up to 43.5±0.5°C for 15 minutes Then the mice did an injection in the tail vein or liposomes at a dose of 10 mg/kg or the same dose of Taxotere (control: prepared according to manufacturer specifications). One leg of each mouse was then heated for 30 min after injection, after which it was slaughtered animal. Muscles heated and unheated leg cut. The drug was extracted from a fixed weight of muscle tissue using the extraction method described above. The resulting preparation analysis is Aravali using HPLC. The results are shown in the following table.

Docetaxel liposomesDocetaxel
APRA/Acont, % (hot leg)APRA/Acont,
% (unheated leg)
APRA/Acont, %
(hot leg)
APRA/Acont, %
(unheated leg)
Group 113,3a 4.95,465,48
Group 214,3the ceiling of 5.606,536,29
Group 311,15,695,485,32

Data show that temperature-sensitive liposome delivers in a hot leg more than twice doxetaxel than in the unheated leg.

EXAMPLE 3

Compared the in vivo distribution of the drug, obtained with liposomes according to the invention and obtained with neurocysticercosis containing docetaxel liposomes.

Thermochemical the e liposomes and heterosubstituted liposomes were obtained by the formulas, in the following table:

TrackDocetaxelDPPCDSFGDSPE-PEGMSFH
Thermosensitive25 mg450 mg50 mg50 mg50 mg
Not heat-sensitive25 mg450 mg50 mg50 mg0

Six female mice of BALB/C (20±2 g) randomly divided into three groups. Spent anesthesia of mice and put them on a Styrofoam panel with a hole in it. One leg of the mouse passed through the hole on the other side of the panel. The panel floated in a water bath to heat the leg up to 43.5±0.5°C for 15 minutes Then the mice did an injection in the tail vein or liposomes at a dose of 10 mg/kg or the same dose of Taxotere (control: prepared according to manufacturer specifications). One leg of each mouse was then heated for 30 min after injection, after which it was slaughtered animal. Muscles heated and unheated leg cut. The drug was extracted from a fixed weight of muscle tissue using the extraction method described above. The resulting preparation was analyzed using HPLC. The results are shown in the following table.

GroupThermosensitive liposomeNot thermosensitive liposome
Heat/no heatHeatedNot heatedHeatedNot heated
APRA/Acont %0,7040,4280,4430,444

In the group with thermosensitive liposomes concentration of the drug in heated tissues were more than two times higher than in unheated tissues. In groups with injection of docetaxel (example 2) and heterosubstituted liposomes concentration of the drug was similar in heated and unheated tissues. These results indicate that temperature-sensitive liposome releases the drug into the tissue in these experimental conditions.

EXAMPLE 4

Compared the in vivo delivery efficiency of docetaxel in use is the so called liposomes according to the invention and a free docetaxel in mice suffering from carcinoma of the lung Lewis.

Used twelve female mice of Kunming at the age of 7-9 weeks weighing 20±2 g Cell carcinoma of the lung Lewis (3×106cells in 0.1 ml PBS) were implanted under the skin in the lower part of the right leg of each mouse. Before beginning processing tumors grew to a size of 4-6 mm in diameter.

Twelve mice were divided according to the size of the tumor and randomly divided into 3 experimental groups: salt, free docetaxel and thermosensitive liposomes according to the invention.

GroupAnimals/groupProcessingDose (mg/m2)Heating time (min)
12Sol030
24Injection of docetaxel7530
36Thermosensitive liposome with docetaxel7530

Containing docetaxel termokos is indeed liposomes according to the invention were obtained as described above and stored at 2-8°C until use. Experimental animals were injected with 75 mg/m2docetaxel or in thermosensitive liposome according to the invention or as liposomally Taxotere prepared according to the manufacturer's specifications.

Treatment started on day 8 after tumor implantation and repeated at 12 and 16 days. In all experimental groups spent anesthesia of mice with intra-abdominal injection of pentobarbital (80 mg/kg); the drug was administered in a volume of 0.2 ml by injection into the tail vein. This dose of anesthesia provided sufficient immobilization during the time period of exposure.

With the exception of the group, which was introduced salt, all experimental groups received an equivalent dose of docetaxel at the rate of 75 mg/m2. Immediately after injection, mice were placed in specially designed racks that allow the tumor to the isolated leg to be in a water bath for 30 minutes. The temperature of the water bath was 43°C. This temperature of the water bath was obtained in the initial calibration, the aim of which was the temperature of the tumor 42°C. All mice killed on day 18. The tumor was excised surgically and registered their weight. Inhibition of tumor growth was evaluated as follows:

the reduction ratio of the tumor = (Vs-Vx)/Vs

where: Vs - tumor volume of mice treated with salt, Vx - tumor volume of the experimental group.

The results are shown in the following table.

GroupThe number of mouseWeight of tumorAverageReduction, %
Sol15,8744,5370
23,199
Injection of docetaxel30,5001,00277,91
4amount of 0.118
51,380
62,010
Thermosensitive liposome with docetaxel70,0310,078598,27
80,009
90,078
100,12
110,151
120,050

Delivery of docetaxel in the form of thermosensitive liposomes and local heating of the tumor resulted in greater tumor suppression than one delivery of docetaxel. The two mice who had been used thermosensitive liposome, the swelling had almost disappeared.

EXAMPLE 5

Preparation of thermosensitive liposomes containing carboplatin

Liposomes can be produced by any method known to specialists in this field of technology. One acceptable method is as follows.

Liposomes prepared from lipids that are listed in the following table.

LipidsWeight (mg)
DPPC2000
MSFH150
DSFG250
DSPE-MPEG250

Dissolve the lipids in 3 ml of chloroform. Evaporate the chloroform on a rotary evaporator at 60°C under reduced pressure to form a thin film. Continue heating for 40 minutes to remove the institutions the institutions solvent. Add 25 ml of water for hydration of the dried lipid film at 60°C for 10 minutes. Lower pressure at room temperature to remove air bubbles within 10 minutes. Once again heated for 10 minutes at 60°C. to Carry out the extrusion of the lipid suspension through a membrane with a pore size of 200 nm to 10 times. To carry out the extrusion through a membrane with pore size of 100 nm to 4 times. The liposomes thus prepared can be stored at 4°C.

Further, liposomes can be filled with an active agent, such as carboplatin, using suitable methods known in the art. One of the suitable methods is the following:

Put 800 mg carboplatin and 1000 mg of lactose in 20 ml of empty liposomes with a concentration of 106 mg of liposomes on Jr. to Heat the mixture to 60°C in a water bath and stirred at 300 rpm for 30 minutes. Filled liposomes can be stored at 4°C.

Excess product can be removed from the filled liposomes using any methods known in the art, for example, exclusion chromatography or dialysis. One suitable method looks like this:

The solution-filled liposomes can be placed in a dialysis bag (molecular limit: 8000~14000). The solution allows the dialysis liposomes in 200 ml of a 5%solution of lactose at 4°C for 2 hours. Replace dialysis solution fresh 200 ml of a 5%solution of lactose and spend dialysis and for a further 2 hours at 4°C. Filled liposomes can be removed from the dialysis bag and stored at 4°C. Filled liposomes should be protected from exposure to light.

EXAMPLE 6

Physical characteristics containing carboplatin liposomes.

After splitting filled with preparation of liposomes and a pure preparation of liposomes are the ratio of drug/lipid 0,04, as can be seen from the following table, and the average particle size of 95 nm (Figure 5 and 6). The lipid concentration is 106 mg/ml

The size of the liposomal particlesThe total number of medicationNet medicationEncapsulation, %The drug/lipid
100 nm4,63 mg/ml0,0 mg/ml100%0,04

Because carboplatin liposomes for clinical use must be diluted, was studied stability containing carboplatin liposomes according to the invention in 5% glucose solution and water as solvents. The stability of liposomes in these solvents was studied by dissolving 10 μl of liposomes in 990 ál of solvent at room temperature for 6 hours. The fluidity of the drug after dissolve the texts analyzed by comparing the encapsulation of the drug before and after dissolution. The following table contains the results obtained. Data show that carboplatin liposomes are compatible with water and with 5% glucose.

SolventThe fluidity of medicationAverage
5% glucose0,23%0,19%
0,23%
0,12%
Water-0,35%-0,08%
0,00%
0,12%

EXAMPLE 7

The characteristic profile of release of the drug containing carboplatin liposomes

Profile release carboplatino of the drug was analyzed at 37°C and 42°C. the Detailed method is as follows:

Water baths were heated to 38°C and 43°C, respectively (the temperature of the test specimen was one degree lower than the water bath). a 1.0 ml aliquot of liposomes was diluted with 9.0 ml of 5% glucose solution. 5 ml of the diluted solution was heated at 38°C in a water bath. The remaining 5 ml of the diluted solution was heated at 43°C in a water bath. At various time points after the start of heating took 200 μl of the sample from the sample is both exhibitions temperature groups. Samples were taken after 0, 0,25, 0,5, 1, 2, 4, 8, 16, 32, 64 and 128 min from a sample with a temperature of 42°C after 0, 2, 8, 32 and 128 min from a sample with a temperature of 37°C. the Samples were cooled in ice water immediately after capture.

Analyzed the total content of the drug and the concentration of the pure drug in the samples. The release of drug was calculated by the following equation:

The release of the drug = C net/C total × 100%.

The following table and 7 contain the results.

Time
(min)
00,250,51248163264128
42ºC (%)0,918,569,383,2for 95.3for 95.3for 95.395,897,099,599,0
37 degrees C (%)0,9the 0,80,91,01,9

These data show that liposomes according to the invention release the drug immediately after heating.

For further characteristics of the release profile of drug from the liposomes according to the invention were studied containing carboplatin liposomes at different temperatures from 37°C to 43°C at 5 and 10 minutes of heating. The following table and Fig shows the fraction of released drug. At 37°C releases almost never happens, but starting from 40°C the drug is released quickly.

Temperature (°C)37383940414243
5 min0,9%2,6%5,6%17,4%78,5%of 98.2%95,4%
10 min0,9%4,6%21,8%48,7%85,5%96,6%95,8%

EXAMPLE 8

Characteristic stability containing carboplatin liposomes

Estimated turnover of the drug during storage at different temperatures.

Liposomes were stored at -20°C, 4°C and 25°C for 5 or 10 days. Analyzed the % encapsulation of the drug. The results presented in the following table, show that the liposomes according to the invention is stable at 4°C for 10 days.

Temperature4°C25°C
Time (days)510510
Fluidity0,01%0,01%1,55%2,52%

To determine whether you can sterilize liposomes according to the invention by filtering, evaluating their stability when filtering. 2 ml aliquot of liposomes was filtered through a 0.22 μm filter. Interest is Olu encapsulated drug was evaluated before and after filtering. The data in the following table show that liposomes can be filtered in a small volume.

Before filteringAfter filtering
Turnover (%)0%0,62%

Long-term stability of liposomes was evaluated as a result of storage of liposomes at 4°C. the Fluidity of the drug was evaluated at 0, 1 and 2 months. As shown in the following table, the liposomes according to the invention is stable at 4°C for at least 6 months.

Time (months)Content preparationFluidityParticle size
04,63 mg/ml0,00%99,79 nm
14,60 mg/ml0,03%106,01 nm
2to 4.62 mg/ml0,09%99,24 nm
34.59 mg/ml 0,14%101,78 nm
64,60 mg/ml0,24%100,09 nm

All publications, patents and patent applications mentioned in this description correspond to the level of a specialist who is experienced in this area of technology, which is addressed to this invention, and shown here as a reference to the same extent as if each individual publication, patent or patent application were specifically and individually listed as incorporated by reference.

1. Temperature-sensitive liposome for the treatment of cancer in a subject, containing at least one phosphatidylcholine, at least one phosphatidylglycerol, monasteriopiedra (MSPH), Targeted phospholipid and an active agent selected from the group consisting of Taxotere, docetaxel and carboplatin, where the liposome has a temperature of phase transition of the gel-liquid from about 39°C to about 45°C.

2. Thermosensitive liposome according to claim 1, in which the phosphatidylcholine is dipalmitoylphosphatidylcholine (DPPC), and phosphatidylglycerol is distearoylphosphatidylglycerol (DSPG).

3. Thermosensitive liposome according to claim 1, in which Targeted the phospholipid is PEG-2000-modified distearoylphosphatidylcholine (DSPE-PEG).

p> 4. Thermosensitive liposome according to claim 3, containing DPPC:DSPG:MFH:DSPE-PEG: an active agent in the ratio of 60-80:6-12:6-12:4-15:1-30 mass.

5. Pharmaceutical composition for treating cancer in a subject, containing heat-sensitive liposome according to any one of claims 1 to 4.

6. The composition according to claim 5, where the active agent is docetaxel, and the particle size of liposomes and the number of doxetaxel contained within liposomes remain essentially stable during storage of the composition at 2-8°C for 1-9 months.

7. The composition according to claim 5, where the active agent is carboplatinum, the number carboplatin contained within liposomes remains essentially stable during storage of the composition at 4°C for 1-6 months.

8. The composition according to claim 5, in which the active medium is carboplatinum, and at least 83,2% carboplatin released from liposomes during heating of liposomes within 1-128 min to 42°C.

9. A method of treating cancer in a subject in need thereof, including:
introduction to a subject a therapeutically effective amount of the pharmaceutical composition according to claim 5; and
the heated region of the subject containing all or part of the disease.

10. Method of manufacture of temperature-sensitive liposomes containing anti-cancer agent, including:
the dissolution of DPPC, DSFG, MSPH, DS the e-PEG and cancer funds selected from the group consisting of Taxotere, docetaxel and carboplatin, in an organic solvent for the formation of a lipid solution;
removing the organic solvent from the above-mentioned lipid solution is to form a dried material;
hydration of the dried material in an aqueous solution of PBS and lactose;
obtaining a dispersion containing liposomes, and the regulation of the particle size mentioned liposomes in the dispersion; and
sterilization mentioned dispersion of liposomes.

11. Method of manufacture of temperature-sensitive liposomes containing anti-cancer agent, including:
the dissolution of DPPC, DSFG, MSPH, DSFA-PEG in an organic solvent;
the removal of organic solvent for the formation of a dried material;
hydration of the dried material in an aqueous solution for the formation of hydrated lipid material;
extrusion mentioned hydrated lipid material to form a dispersion containing liposomes;
adding anticancer means selected from the group consisting of Taxotere, docetaxel and carboplatin, in the above-mentioned dispersion during the heating and mixing to form a dispersion of liposomes loaded with an anticancer agent; and
remove excess anti-cancer agent that is not part of providing Utah loaded with anti-cancer liposomes.

12. The product obtained using the method according to claim 10 or 11.



 

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