JPWO2009125858A1 - 2-Lipid dispersion preparation containing indolinone derivative - Google Patents

Abstract

Anti-malignant tumor agent excellent in safety, convenience, and effect during parenteral administration by lipid dispersion preparation containing Z-3- [2,4- (dimethylpyrrol-5-yl) methylidenyl] -2-indolinone I will provide a.

Description

  The present invention relates to a lipid dispersion preparation containing a 2-indolinone derivative and an antitumor agent containing the lipid dispersion preparation.

ＳＵ５４１６は経口投与した場合は十分な有効性を発揮しないことから、静脈内投与を代表とした非経口投与で治療に用いられている。しかしながら、ＳＵ５４１６は水に極めて難溶であるため、水を溶媒として用いて注射剤とすることは困難である。このため従来は、ＳＵ５４１６を、溶解度を高めるためのエタノール、ポリエチレングリコールなどの有機溶媒と水とを混合したときに析出する結晶を抑制するためにクレモホールやソルトールなどの非イオン性界面活性剤からなる混合溶媒に溶解して製剤とし、投与直前に生理食塩水で希釈して投与液とし、静脈内点滴投与が行われてきた。しかしクレモホールなどの静脈内投与は過敏症の副作用を頻繁に引き起こすため、現状では、抗ヒスタミン剤やステロイド剤の事前投与が必須となっており、患者及び医療従事者の大きな時間的、経済的、精神的負担となっている。また、生理食塩水で希釈された後の投与液は長時間経過すると結晶が析出するおそれがあり、希釈後は数時間以内に投与を開始する必要があり、医療施設での負担がより大きくなっていた。このため、クレモホールや有機溶媒を含有しない、静脈内投与に適した投与液の開発が強く望まれていた。To date, many drugs have been developed and used in medicine for cancer treatment. However, until now, no anticancer agent has been developed that exhibits a selective cell killing effect only on tumor tissues. When an anticancer agent is administered directly into a blood vessel, it may be distributed to organs other than the target that disappear quickly from the blood and does not always accumulate effectively in cancer tissue. For this reason, many anticancer drugs cannot fully exert their antitumor effects (main effects) on cancer tissues, and often have undesirable effects on normal tissues (side effects), causing serious toxicity. Yes. Enhancement of the main effects of anticancer drugs and reduction of side effects are important issues in current cancer chemotherapy, and drug delivery systems (DDS, Drug) that efficiently accumulate drugs in cancer tissues and cells. There is a strong demand for the development of a Delivery System.
Lipid dispersions typified by liposomes and O / W emulsions are closed vesicles that use phospholipids derived from biological components as membrane lipids, and are characterized by low toxicity and low antigenicity when administered to the living body. It has also been shown that encapsulating a drug in a lipid dispersion can improve the reach to the target tissue by changing the blood stability and biodistribution of the drug (Non-patent Document 1, Patent Document 1). ). In addition, the blood vessel walls of new blood vessels that are abundant in cancer tissue are more permeable than those of existing blood vessels, and lipid dispersion vesicles with an average particle size of 50 to 200 nm are likely to accumulate in cancer tissue Is known (Non-Patent Document 2). Therefore, a preparation in which an antitumor active drug is encapsulated in a lipid dispersion is one of DDS that is highly expected to enhance main effects and reduce side effects.
Z-3- [2,4- (dimethylpyrrol-5-yl) methylidenyl] -2-indolinone (Pfizer, Taiho Pharmaceutical Co., Ltd., development code: SU5416) used in the present invention is vascular endothelial growth factor (VEGF). It is known that it exhibits an angiogenesis-inhibiting action in tumor tissues by inhibiting phosphorylation of), and has a strong growth-inhibiting effect on a broad spectrum of human tumors. It is a compound (Patent Literature 2, Non-Patent Literature 3).

Since SU5416 does not exhibit sufficient efficacy when administered orally, it is used for treatment by parenteral administration represented by intravenous administration. However, since SU5416 is extremely hardly soluble in water, it is difficult to prepare an injection using water as a solvent. For this reason, conventionally, SU5416 is made of a nonionic surfactant such as Cremophor or Saltol to suppress crystals that precipitate when an organic solvent such as ethanol or polyethylene glycol for increasing solubility and water are mixed. Intravenous infusion has been carried out by dissolving in a mixed solvent to prepare a preparation and diluting with physiological saline just before administration to prepare a liquid for administration. However, intravenous administration of Cremophor, etc. frequently causes side effects of hypersensitivity, so pre-administration of antihistamines and steroids is essential at the present time, and the patient, medical professionals have a large time, economic and mental It is a burden. In addition, there is a risk that crystals will precipitate after a long time after being diluted with physiological saline, and it is necessary to start administration within several hours after dilution, which increases the burden on medical facilities. It was. For this reason, there has been a strong demand for the development of a dosing solution suitable for intravenous administration that does not contain cremophor or an organic solvent.

WO95 / 24201 WO95 / 32187

Journal of Liposome Research, Vol. 4,667,1994 Drug Delivery System, Vol. 14, 433, 1999 Japan Journal of Cancer Research, Vol. 90, 578, 1999

The subject of this invention is providing the lipid dispersion formulation excellent in the safety | security and convenience suitable at the time of parenteral administration of SU5416 which shows an antineoplastic action.
Another object of the present invention is to provide an antitumor agent containing SU5416.

As a result of intensive research to solve the above problems, the present inventors have conceived that SU5416 is fat-soluble and has a high affinity for lipid membranes, and SU5416 is a lipid dispersion such as a liposome or an O / W emulsion. As a result of encapsulating in the body, a dosage form not containing cremophor or an organic solvent was established, and the present invention was completed. Furthermore, it was found that by appropriately adjusting the size of the lipid dispersion and the like, the antitumor activity was significantly increased when SU5416 was administered into the blood vessels of mice, and no increase in toxicity was observed.
That is, the present invention relates to the following invention.
1. A lipid dispersion preparation for parenteral administration containing Z-3- [2,4- (dimethylpyrrol-5-yl) methylidenyl] -2-indolinone.
2. A lipid dispersion preparation wherein at least one lipid component constituting the lipid membrane is a phospholipid.
3. The phospholipid is at least one selected from the group consisting of purified egg yolk phosphatidylcholine, purified soybean phosphatidylcholine, hydrogenated purified egg yolk phosphatidylcholine, hydrogenated purified soy phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylcholine. Said lipid dispersion formulation which is a seed.
4). The above lipid dispersion preparation, wherein the average particle size of the lipid dispersed particles is 1 μm or less.
5. The said lipid dispersion formulation whose phospholipid density | concentration in a formulation is 1-100 mM.
6). The above lipid dispersion preparation, wherein the concentration of Z-3- [2,4- (dimethylpyrrol-5-yl) methylidenyl] -2-indolinone in the preparation is 0.2 to 20 mM.
7). The lipid dispersion preparation, wherein the lipid membrane surface is modified with a polyethylene glycol derivative or a peptide containing a PRP sequence.
8). The lipid dispersion preparation described above, wherein the surface of the lipid membrane is modified with a peptide containing a PRP sequence.
9. The above lipid dispersion preparation, wherein the peptide containing the PRP sequence is APRPG.
10. An antitumor agent for parenteral administration containing the lipid dispersion preparation according to any one of the above.

  The lipid dispersion preparation of the present invention can improve the antitumor effect without increasing toxicity while enhancing convenience. Therefore, the lipid dispersion preparation of the present invention exhibits a particularly remarkable effect as an antitumor agent, and further extends the survival period of cancer patients.

FIG. 1 is a graph showing the distribution of PEG-Lip-SU5416 in Example 1 into the liposome fraction. FIG. 2 is a graph showing the distribution of APRPG-Lip-SU5416 in Example 2 into the liposome fraction. FIG. 3 is a graph showing the change over time of the particle diameter of the liposome preparation according to Test Example 1. FIG. 4 is a graph showing the change over time of the particle size of the O / W emulsion preparation according to Test Example 1. FIG. 5 is a graph showing a change with time of the SU5416 encapsulation rate of the liposome preparation according to Test Example 1. (Initial value is 100%) FIG. 6 is a graph showing the change with time of the SU5416 encapsulation rate of the O / W emulsion preparation according to Test Example 1. (Initial value is 100%) FIG. 7 is a graph showing the results of hemolysis test in Test Example 2. FIG. 8 is a graph showing changes in tumor volume in mice of Test Example 3. FIG. 9 is a graph showing changes in body weight of the mice of Test Example 3.

Specific examples of the lipid dispersion of the present invention include liposomes and O / W emulsions.
The liposome used in the present invention is a closed vesicle having an inner aqueous phase portion surrounded by a lipid bilayer formed by dispersing phospholipids constituting a cell membrane in water as described above, Depending on its size and the number of lipid bimolecules, it is classified into three types: multi-phase liposome (MLV), large unilamellar liposome (LUV), and small unilamellar liposome (SUV). The Any kind of liposome can be used in the present invention. The liposome of the present invention must form a stable liposome structure before in vivo administration.
Examples of the phospholipid constituting the liposome include purified egg yolk phosphatidylcholine (hereinafter referred to as EPC), purified soybean phosphatidylcholine (hereinafter referred to as SPC), hydrogenated purified egg yolk phosphatidylcholine (phase transition temperature 50 to 60 ° C., HEPC), hydrogenated purified soybean phosphatidylcholine (phase transition temperature about 55 ° C., hereinafter HSPC), dipalmitoyl phosphatidylcholine (phase transition temperature about 41 ° C., hereinafter DPPC), distearoyl phosphatidylcholine (phase transition temperature about 58 ° C., hereinafter DSPC) And 1-palmitoyl-2-oleoylphosphatidylcholine (phase transition temperature—3 ° C., hereinafter referred to as POPC), more preferably DPPC and DSPC. These can be used singly or in combination of two or more. When using a phospholipid having a low phase transition temperature, such as POPC, two or more of them are mixed and prepared.
In addition to these phospholipids, the liposomes used in the present invention are preferably used in admixture with stabilizers such as cholesterol derivatives that have been reported to improve liposome stability. The molar ratio of cholesterol derivative and phospholipid is preferably 1: 1 to 10, and more preferably 1: 2.5 to 5.
Further, as an isotonic agent, for example, glycerin, glucose, sodium chloride and the like can be added. Furthermore, preservatives such as parabens, chlorobutanol, benzyl alcohol, propylene glycol and the like may be added.
The molar ratio of the phospholipid used in the present invention to SU5416 is preferably 1: 0.01 to 0.1, more preferably 1: 0.025 to 0.075, and 1: 0.04 to 0.06. Particularly preferred.
The liposome preparation of the present invention is prepared by a known method. As a method for preparing a known liposome preparation, for example, the reverse phase evaporation method [Proc. Natl. Acad. Sci. USA, 75, 4194 (1978), WO 97/48398], freeze-thaw method [Arch. Biochem. Biophys, 212, 186 (1981)], pH gradient method [Biochem. Biophys. Acta, 816, 294 (1985), JP-A-7-165560] and the like.
Among these, the reverse phase evaporation method is useful when encapsulating a poorly water-soluble drug in liposomes at a high inclusion rate, and is also one of the most suitable methods for SU5416. As a method for preparing the liposome preparation of the present invention by the reverse phase evaporation method, for example, SU5416 and a lipid component are dissolved in a solvent such as chloroform, ether, ethanol, etc., and then placed in an eggplant type flask. Form. Next, a buffer solution is added to the thin film, freeze-thawed, and large multiphase liposomes (MLV) whose inner aqueous phase is a buffer solution are prepared. Furthermore, small unilamellar liposomes (SUV) are obtained by an extrusion method or the like. By the above operation, the drug can be encapsulated in the lipid membrane of the liposome at a high rate and quantitatively.
The O / W emulsion used in the present invention is one in which a phospholipid that is a membrane phase lipid contains lipid particles surrounding the inner phase lipid, and the drug is dissolved in the inner phase lipid and in the membrane phase lipid. Exists in a state.
The membrane phase lipid constituting the O / W emulsion needs to have surface activity, and various phospholipids can be used. For example, it is selected from EPC, SPC, HEPC, HSPC, DPPC, DSPC and POPC. These can be used alone or in combination.
On the other hand, as the internal phase lipid constituting the O / W emulsion, a lipid capable of dissolving a drug is desirable. Refined soybean oil, cholesterol, cholesterol ester, triolein, tricaprylin, tri (caprylic / capric acid) glycerin, oleic acid, olein Examples thereof include ethyl acid and tocopherol.
Since the O / W emulsion tends to aggregate between particles, a method of suppressing aggregation by dissolving a polymer such as polyvinylpyrrolidone or serum albumin in the outer aqueous phase can be used. Further, as an isotonic agent, for example, glycerin, glucose, sodium chloride and the like can be added. Furthermore, preservatives such as parabens, chlorobutanol, benzyl alcohol, propylene glycol and the like may be added.
The molar ratio of the lipid used in the O / W emulsion of the present invention to SU5416 is preferably 1: 0.01 to 0.5, more preferably 1: 0.02 to 0.2, and 1: 0.04 to 0.1 is particularly preferred.
It has been reported that the particle size of liposomes and O / W emulsions has a great influence on the biodistribution of encapsulated drugs and the delivery to tumor tissues [Biological and Pharmaceutical Bulletin, 17, 935 (1994)]. . Therefore, in the present invention, it is preferable to perform sizing treatment in order to make the particle size of the lipid dispersion encapsulating the drug appropriate and uniform, for example, using an extruder (manufactured by Lipex Biomembrane, etc.). Prepare by passing several times through a membrane filter of appropriate pore size or by using an ultrasonic homogenizer.
The average particle diameter of the liposome is preferably 1 μm (1000 nm) or less, more preferably 50 to 500 nm, still more preferably 100 to 300 nm, particularly preferably 75 to 150 nm, and most preferably 75 to 125 nm. The average particle size of the O / W emulsion is preferably 1 μm (1000 nm) or less, more preferably 50 to 500 nm, still more preferably 100 to 300 nm, and particularly preferably 150 to 300 nm.
The concentration of phospholipid and SU5416 in the formulation should be adjusted to increase the encapsulation rate of SU5416 in the lipid dispersion, prevent the precipitation of SU5416, and bring the solution viscosity to a range suitable for parenteral administration. The phospholipid concentration is 1 to 100 mM, preferably 5 to 50 mM, and the SU5416 concentration is 0.2 to 20 mM, preferably 0.5 to 10 mM.
Moreover, the stability in the blood of a liposome can be improved by modifying the membrane surface of a liposome and O / W emulsion with a polyethylene glycol (PEG) derivative as needed. In many cases, a PEG derivative having a molecular weight of 500 to 20000 and a phospholipid covalent conjugate is used, and a molecular weight of 2000 to 5000 PEG and distearoylphosphatidylethanolamine (hereinafter DSPE) conjugate (hereinafter DSPE-PEG). Is the most widely used.
In addition, by modifying with ligands such as peptides, lectins, antibodies, carbohydrates, glycoproteins and glycolipids, the stability of liposomes and O / W emulsions in blood, tissue distribution, transferability to tumor tissues, etc. Further improvement can be achieved. In particular, it has been confirmed that a peptide containing a PRP sequence is specifically accumulated in a new blood vessel present in a large amount in cancer tissue, and is one of VEGF receptors, fms-like tyrosine kinase-1 (flt-1). Has been suggested [Oncogene, 21, 2662 (2002), WO 00/23476]. By modifying the membrane surface with a polyethylene glycol derivative and a peptide containing a PRP sequence, it is possible to obtain liposomes and O / W emulsions that are stable in blood and highly transferable to tumor tissues.
Here, the peptide containing the PRP sequence used may be a peptide containing the amino acid sequence of proline-arginine-proline, and can be prepared by a known method in the field of peptide synthesis. For example, APRPG (alanine-proline-arginine-proline-glycine), GPRPL (glycine-proline-arginine-proline-leucine), GPRPR (glycine-proline-arginine-proline-arginine) can be exemplified.
Through the above operations, the lipid dispersion preparation of the present invention is prepared, and if necessary, by performing ultracentrifugation treatment, gel filtration treatment, ultrafiltration treatment and dialysis treatment alone or in combination, liposomes and O / W Drugs not encapsulated in the emulsion can be removed.
The lipid dispersion preparation of the present invention obtained by the above method can be used as it is, but in consideration of the storage period, storage conditions, etc., an excipient such as mannitol, trehalose, lactose, glycine is added and lyophilized. You can also. Further, a cryopreservation agent such as glycerin may be added and stored frozen.
The lipid dispersion preparation of the present invention is generally used as an injection (intravenous, intramuscular, subcutaneous, intraperitoneal administration preparation) after being suspended or diluted with a physiologically acceptable aqueous solution at the time of use. They can also be used as nasal drops, inhalants, eye drops, suppositories, transdermal absorbents, transmucosal absorbents, and the like. The final formulation consists of malignant tumors [lung cancer in mammals such as humans, digestive organ cancer (esophageal cancer, stomach cancer, rectal cancer, colon cancer), breast cancer, head and neck cancer, liver cancer, gallbladder cancer, Pancreatic cancer, gynecological cancer (uterine cancer, cervical cancer, ovarian cancer, etc.), urological cancer (renal cancer, bladder cancer, prostate cancer), brain tumor, leukemia, melanoma, malignant lymphoma, etc. It suffices if it is designed so that an effective amount for treatment and remission is administered. For example, SU5416 should just contain 0.01-10000 mg as an intravenous administration liposome formulation. The dosage of SU5416 contained in the liposome preparation of the present invention is preferably 0.01 to 1000 mg / day.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited thereto.
[Example 1]
(SU5416 liposome preparation modified with polyethylene glycol derivative, hereinafter referred to as PEG-Lip-SU5416)
30 μmol of DPPC, 30 μmol of POPC, 15 μmol of cholesterol, 6 μmol of DSPE-PEG, 3 μmol of SU5416 (Chloroform solution) are placed in an eggplant type flask (lipid composition: DPPC / POPC / cholesterol / DSPE-PEG / SU5416 = 10/10/5/2/1), 5 mL of chloroform was added. Chloroform was distilled off under reduced pressure with an evaporator to form a lipid thin film on the inner wall surface of the eggplant type flask. Furthermore, it was vacuum-dried in a desiccator for 1 hour. Next, it was hydrated with 1.5 mL of 20 mM HEPES [4- (2-hydroxyethyl) -1-piperazine etheric acid] buffer (pH 7.4), and freeze-thawed three times. After ultrasonic treatment for 10 minutes using a bath sonicator, a liposome preparation was prepared by passing 5 times through a polycarbonate membrane filter (Nuclepore) with a pore size of 100 nm using an extruder (manufactured by Lipex Biomembrane). The average particle size of the liposome was about 120 nm.
[Example 2]
(SU5416 liposome preparation modified with polyethylene glycol derivative and APRPG peptide, hereinafter referred to as APRPG-Lip-SU5416)
DPPC 30 μmol, POPC 30 μmol, cholesterol 15 μmol, DSPE-PEGylated APRPG sequence-containing peptide (DSPE-PEG-APRPG) 6 μmol, SU5416 (Chloroform solution) 3 μmol were placed in an eggplant type flask (lipid composition: DPPC / POPC / cholesterol / DSPE- PEG-APRPG / SU5416 = 10/10/5/2/1), 5 mL of chloroform was added. Hereinafter, a liposome preparation was prepared according to the method of Example 1. The average particle diameter of the liposome was about 130 nm.
[Example 3]
(SU5416 emulsion formulation consisting of EPC and triolein, hereinafter referred to as O / W-EPC-SU5416)
A chloroform solution (100 mM) of EPC and triolein (oil) were fractionated into a 50 mL eggplant type flask using a microsyringe so that the molar ratio of EPC: triolein was 2: 8. Next, the required amount of SU5416 in chloroform (50 mM) was added to the eggplant type flask so that the molar ratio of EPC: triolein: SU5416 was 2: 8: 1. Further, a small amount of chloroform was added and lightly mixed so that the lipid solution was uniform. Chloroform was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film on the bottom of the eggplant-shaped flask. While maintaining at 80 ° C., 0.3 M glucose / 1% polyvinylpyrrolidone solution was added and hydrated by vigorous stirring with a vortex mixer. In order to make the particle diameter uniform, ultrasonic treatment was performed for 90 minutes with an ultrasonic homogenizer (SONIFER Model 250, BRANSON). The final lipid concentration is 20 mM as EPC. The average particle size of the emulsion was about 240 nm.
[Example 4]
(SU5416 O / W emulsion preparation modified with PEG derivative and consisting of DPPC, cholesterol, cholesterol oleate, hereinafter referred to as O / W-DPPC-PEG-SU5416)
DPPC, cholesterol, cholesterol oleate in chloroform (100 mM each) and DSPE-PEG in chloroform / methanol (1/3) (100 mM) in a molar ratio of DPPC: cholesterol: cholesterol oleate: DSPE-PEG of 10 : 10: 20: 2 was dispensed into a 50 mL eggplant-shaped flask using a microsyringe. Next, the required amount of SU5416 in chloroform (50 mM, DPPC: cholesterol: cholesterol oleate: DSPE-PEG: SU5416 molar ratio was added to the eggplant type flask was 10: 10: 20: 2: 1). A small amount of chloroform was added, and the mixture was lightly mixed so that the lipid solution was uniform, and the chloroform was distilled off under reduced pressure using a rotary evaporator to form a thin lipid film on the bottom of the eggplant-shaped flask. Then, 0.3M HEPES / 1% polyvinylpyrrolidone solution was added and hydrated by vigorous stirring with a vortex mixer.To homogenize the particle size, sonication was performed with an ultrasonic homogenizer (SONIFER Model 250, BRANSON). 90 minutes, final lipid concentration is D A 10mM as PC. The average particle size of the emulsion was about 160 nm.
[Comparative Example 1]
(SU5416 solution not lipid dispersion, hereinafter referred to as Free SU5416 formulation)
Polyethylene glycol 400, Cremophor EL (Polyoxyl 35 castor oil), benzyl alcohol, and absolute ethanol were mixed at a weight ratio of 45: 31.5: 2: 21.5 to obtain a solvent for Free SU5416. SU5416 was dissolved in this so that it might become 1.2 mg / mL. Immediately before administration to animals, it was diluted 4-fold with 0.45% sodium chloride solution.
[Test Example 1]
(SU5416 lipid dispersion encapsulation rate and stability over time)
The liposome solution prepared in Example 1 and Example 2 was fractionated into 6 fractions by gel filtration using a PD-10 column filled with Sephadex G-25. To each fraction, 50 μL of 10% reduced Triton X-100 solution and 20 mM HEPES buffer were added and mixed, and sonication was performed to solubilize the liposome membrane. The absorbance of the solution was measured using HPLC at an absorption wavelength of 440 nm, and the SU5416 concentration of each fraction was determined from the calibration curve. On the other hand, turbidity (absorption wavelength 750 nm) was measured for each fraction, and the liposome concentration was determined (FIGS. 1 and 2, fractions 1-3).
The SU5416 concentration and O / W emulsion concentration of the O / W emulsion solution prepared in Example 3 were determined in the same manner as described above.
As a result of obtaining the SU 5416 encapsulation rate in the lipid dispersion from the SU 5416 concentration and the lipid dispersion concentration of each fraction of Example 1, Example 2 and Example 3, the encapsulation rate of SU 5416 in the lipid dispersion is PEG-Lip-SU5416 obtained in 1 was 86.0%, APRPG-Lip-SU5416 obtained in Example 2 was 85.8%, and O / W-EPC-SU5416 obtained in Example 3 was 86.9%. It was confirmed that it was sealed with high efficiency. Moreover, about any lipid dispersion formulation, the average particle diameter did not change for 14 days after preparation (FIGS. 3 and 4). In addition, for any lipid dispersion preparation, it was revealed that the SU5416 encapsulation rate did not change for 14 days from immediately after preparation and was stable as a preparation (FIGS. 5 and 6).
HPLC condition detector: UV-visible spectrophotometer (measurement wavelength: 440 nm)
Column: TSKGEL ODS-80TS 4.6 mm x 25 cm (TOSOH)
Column temperature: 40 ° C
Mobile phase: Methanol / 35 mM KH ₂ PO ₄ (pH 3.0) mixed solution (75:25)
Flow rate: 1.0 mL / min (Pressure about 16 kgf / cm ² )
Injection volume: 10 or 20 μL
Measurement time: 25 min (a peak is detected at about 15 minutes)
[Test Example 2]
(Hemolytic properties of lipid dispersion SU5416 preparation and Free SU5416 preparation)
The blood collected from the mouse was centrifuged (2000 × g, 5 min) and the supernatant was collected. Phosphate buffered saline (PBS, pH 7.4) was added to the residue and mixed, then centrifuged (2000 × g, 5 min), the supernatant was collected, and this operation was repeated 5 times. It was diluted to 5% (v / v) with 20 mM HEPES buffered saline to obtain an erythrocyte solution. 20 μL each of the lipid dispersion SU5416 liposome solution prepared in Example 1 and Example 2, the Free SU5416 solution prepared in Comparative Example 1 and the solvent for dissolving Free SU5416 was added to 180 μL of the erythrocyte solution, and mixed with a vortex mixer. Thereafter, the cells were incubated at 37 ° C. for 10, 30, 60 min, and centrifuged (10,000 × g, 5 min). 100 μL of the supernatant was taken, the wavelength of 570 nm was measured with a microplate reader, and the hemolysis rate was calculated.
As a result, as shown in FIG. 7, remarkable hemolysis was observed in the Free SU5416 solution and the Free SU5416 solvent, while almost no hemolysis was observed in the PEG-Lip-SU5416 and APRPG-Lip-SU5416 solutions. The reason for this was considered that since the liposome preparation does not contain Cremophor EL and an organic solvent, it does not have an action of destroying the erythrocyte membrane.
[Test Example 3]
(Anti-tumor effect and toxicity evaluation of lipid dispersion SU5416 preparation and Free SU5416 preparation)
Mouse colon cancer cell line Colon26NL17 used for evaluation of antitumor effect was modified in the presence of 5% CO ₂ at 37 ° C. and 10% FBS (Fetal Bovine Serum, fetal bovine serum) -DMEM (Dulbecco's Modified Eagle's Medium, Dulbecco modification) Eagle medium) / Ham F12 = 1/1 and subcultured. Colon 26NL17 prepared to 5 × 10 ⁶ cells / mL was subcutaneously administered (1 × 10 ⁶ cells / mouse) to the left ventral part of 4-week-old male Balb / C mice to give solid cancer-bearing mice. Created. Five times on days 5, 7, 9, 11, and 13 after Colon 26NL17 transplantation, HEPES buffered saline as a control and APRPG-Lip-SU5416 solution prepared in Example 2 or Free SU5416 solution prepared in Comparative Example 1 0.2 mL / body / day was administered into the tail vein of mice (the dose was 3 mg / kg as SU5416). The antitumor effect of the SU5416-encapsulated liposome solution was examined by measuring the tumor volume 4 days after cancer transplantation. Tumor volume was calculated using the following formula.
Tumor volume = 0.4 × a × b2
(A: major axis of the tumor site, b: minor axis of the tumor site)
As a result, as shown in FIG. 8, the APRPG-Lip-SU5416 treatment group showed a significant tumor growth inhibitory effect compared to the Control treatment group and the SU5416 treatment group (Free SU5416) that was not made into liposomes.
As a result of examining the change in body weight as an index of side effects, as shown in FIG. 9, no significant weight loss was observed in all treatment groups.
These results indicate that the SU5416 liposome preparation significantly enhances the antitumor activity as compared to the non-liposomal solution. The cause of the enhancement of the anticancer activity is to improve the pharmacokinetics of SU5416 by using SU5416 as a liposome preparation, prolong the blood half-life, which has been a problem, and improve the accumulation in new blood vessels by targeting And so on.

  The lipid dispersion preparation of the present invention can enhance the convenience and enhance the antitumor effect without increasing toxicity. Therefore, the lipid dispersion preparation of the present invention is expected to exhibit a particularly remarkable effect as an antitumor agent, and is expected to be applied to a therapeutic method having a high therapeutic effect that further extends the survival period of cancer patients.

Claims (10)

  A lipid dispersion preparation for parenteral administration containing Z-3- [2,4- (dimethylpyrrol-5-yl) methylidenyl] -2-indolinone.   The lipid dispersion preparation according to claim 1, wherein at least one lipid component constituting the lipid membrane is a phospholipid.   The phospholipid is at least one selected from the group consisting of purified egg yolk phosphatidylcholine, purified soybean phosphatidylcholine, hydrogenated purified egg yolk phosphatidylcholine, hydrogenated purified soy phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylcholine. The lipid dispersion preparation according to claim 2, which is a seed.   The lipid dispersion preparation according to any one of claims 1 to 3, wherein the lipid dispersed particles have an average particle size of 1 µm or less.   The lipid dispersion preparation according to claim 1, wherein the concentration of phospholipid in the preparation is 1 to 100 mM.   The lipid dispersion preparation according to claim 1, wherein the concentration of Z-3- [2,4- (dimethylpyrrol-5-yl) methylidenyl] -2-indolinone in the preparation is 0.2 to 20 mM.   The lipid dispersion preparation according to claim 1, wherein the surface of the lipid membrane is modified with a polyethylene glycol derivative or a peptide containing a PRP sequence.   The lipid dispersion preparation according to claim 7, wherein the surface of the lipid membrane is modified with a peptide containing a PRP sequence.   The lipid dispersion preparation according to claim 8, wherein the peptide containing a PRP sequence is APRPG.   The antitumor agent for parenteral administration containing the lipid dispersion formulation of any one of Claims 1-9.

Images