JP2006056807A - Preparation for use in photodynamic therapy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preparation desirable for use in photodynamic therapy containing liposomes containing a photosensitive compound and to provide a method for producing the same. <P>SOLUTION: The preparation for use in photodynamic therapy contains liposomes each containing at least one drug containing a photosensitive compound inside a lipid film or in its internal water phase and is substantially free from an organic solvent. The liposomes are made by mixing a lipid film component constituting the lipid film with a supercritical or subcritical carbon dioxide. The photosensitive compound contained in the liposomes desirably includes porphyrin, hematoporphyrin, and their derivatives. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a photodynamic therapy preparation, and more particularly to a photodynamic therapy preparation containing a liposome encapsulating a photosensitizing compound used in photodynamic therapy and a method for producing the same.

Photochemical therapy, also known as photodynamic therapy (also called PDT), is a variety of abnormalities or diseases in the skin or other epithelial organs, especially cancer or precancerous lesions, as well as non-malignant lesions such as psoriasis It is a technique for treating skin diseases and the like. Photodynamic therapy involves applying a photosensitizer to the affected area of the body and then exposing the photosensitizer to a photoactivated light to activate and convert it to a cytotoxic form. This kills the diseased cells or reduces their proliferative capacity.

  A series of photosensitizers are known, and famous ones include psoralens, porphyrins, chlorins and phthalocyanines. Such drugs become toxic or give rise to toxic species when exposed to light. However, many of the photosensitizers still have some limitations in performing PDT to treat all abnormalities or diseases. Thus, there remains a need for better photodynamic therapeutic agents, including formulation improvements to existing photosensitizers, to treat abnormalities or diseases.

Recently, laser therapy has attracted attention as one of the promising cancer treatment methods, and photodynamic therapy belongs to the category. This technology uses a combination of non-toxic photosensitizing drug administration and non-harmful photoirradiation for photosensitization, and is less invasive than surgical excision procedures commonly used for cancer treatment Treatment. It is also selective but has the potential for lower side effects compared to chemotherapy or radiation therapy, where side effects are likely to be problematic. Since organ preservation is possible, the quality of life of the patient being treated (Quality of Life)
It is expected to be a useful treatment to improve

  Such photodynamic therapy for cancer has been studied since the 1980s, and clinical treatment of photodynamic therapy has been started in many countries such as Japan, the United States, Canada and Germany since the 1990s. Recent statistics show that the number of cases of this therapy is increasing year by year.

However, photodynamic therapy for cancer has the following basic problems. i) In the case of cancers that have numerous lesions that have metastasized in the body, this therapy cannot be used because the light cannot penetrate the entire malignant tumor tissue. ii) The price of the photosensitizer is very high. iii) The photosensitizer is gradually decomposed and metabolized in the human body until toxicity is shown. iv) The concentration of the photosensitizer in the malignant tumor cannot be increased, resulting in a reduction in the therapeutic effect.

Improvement of DDS (drug delivery system) -like preparations for PDT has been promoted by utilizing liposomes. A method of encapsulating a photosensitizer in a liposome that is composed of lipids similar to biological membranes and is considered to be highly safe due to its low antigenicity has also been studied. For example, a liposome (Patent Document 1) encapsulating porphyrin and its derivative and used for treatment of a tumor in combination with light, or a complex of 9-hydroxy pheophorbide-a and a liposome (Japanese Patent Publication No. 2004-513948) Has been proposed. In these methods, despite the use of liposomes that are highly safe as materials and have appropriate degradability in vivo, organic solvents such as chloroform, Use a chlorinated solvent such as dichloromethane. Therefore, it has not yet been put into practical use because of the toxicity of the remaining solvent (see, for example, Patent Document 2).

  On the other hand, fat-soluble drugs are easily encapsulated in liposomes, but the encapsulated amount depends on other factors and is not necessarily large. A drug that is a water-soluble electrolyte can be encapsulated in the aqueous phase inside the liposome through the interaction between the charge of the drug and the charge of the charged lipid, but if the drug is a water-soluble non-electrolyte, such means should be used. It cannot be taken.

In JP-A-2003-119120 (Patent Document 3), a cosmetic containing a liposome and an external preparation for skin are disclosed.
A method of producing using supercritical carbon dioxide is disclosed, and an example of producing an external preparation for skin in which a hydrophilic medicinal component or a lipophilic medicinal component is encapsulated in liposomes is shown. However, although examples of water-soluble electrolytes have been shown as hydrophilic medicinal ingredients, it was unclear whether water-soluble non-electrolytes could be efficiently encapsulated in liposomes by this method. Further, in this method, in order to increase the encapsulation rate, it is desired to use a cosolvent such as ethanol, and liposomes having a high encapsulation rate cannot be produced without using an organic solvent.

Even if the anticancer agent or contrast material is successfully encapsulated inside the liposome, the problem of leakage to the outside over time or the situation in which the liposome itself becomes unstable must be considered. Furthermore, it has been pointed out that even when liposomes are administered in vivo, many of them are trapped in the reticuloendothelial tissues such as the liver and spleen, so the desired effect cannot be obtained (Cancer Res., 43,
5328 (1983)). Accordingly, there is a demand for a method for preparing liposomes that can efficiently encapsulate and stably hold liposome preparations or contrast substances, and that does not pose a safety problem.
Patent No. 3020372 Patent No. 2882607 Japanese Patent Laid-Open No. 2003-119120

  The present invention addresses the above-mentioned demands and proposes a photodynamic therapy formulation in which a photosensitizing compound is encapsulated in a liposome prepared without using an organic solvent. This preparation is a highly safe photodynamic therapy preparation with high selectivity to cancer tissues, easy excretion from the body, and low side effects, and is useful for both systemic and local administration. is there.

The present invention includes a photodynamic therapy characterized in that it comprises liposomes containing at least one drug including a photosensitizing compound in a lipid membrane or an internal aqueous phase thereof and substantially free of an organic solvent. It is a formulation.

The liposome is prepared by mixing a lipid membrane component constituting a lipid membrane and carbon dioxide in a supercritical or subcritical state under the condition that at least one compound having a polyethylene glycol (PEG) group is present. It is characterized by that.

The compound having a polyethylene glycol group is preferably PEG-phospholipid.
It is desirable that the lipid membrane is a liposome substantially consisting of a single membrane or several membranes.

The photosensitizing compound is preferably porfimer sodium.
The formulation of the present invention may be delivered directly to the affected area through a catheter.
Moreover, it is preferably used for photodynamic therapy of cancer.

Hyperthermia may be used in combination.
It is a photodynamic therapy preparation that may further contain a contrast material as the drug.
In the method for producing the photodynamic therapy preparation of the present invention, at least one kind selected from a cationic lipid and a sterol together with a phospholipid as a lipid membrane component under a condition in which at least one compound having a polyethylene glycol group is present. A micelle by introducing a solution or suspension containing at least one or more drugs after dissolving or dispersing a compound, and if necessary, a lipophilic drug in carbon dioxide in a supercritical or subcritical state And then adding water to excrete carbon dioxide to produce liposomes containing these drugs inside.

You may use the said compound which has a polyethyleneglycol group in the ratio of 0.01-1 mass% with respect to the carbon dioxide which makes a supercritical state or a subcritical state.
The cancer treatment method of the present invention comprises:
i) Insert a catheter from the artery leading to the cancer's nutritional blood vessels as close to the cancer tissue as possible,
ii) directly injecting the photodynamic therapy preparation into the cancer tissue through the introduction catheter, and iii) inserting a diode laser fiber into the cancer tissue through the catheter,
iv) A method for treating cancer using a photodynamic therapy preparation, comprising treating the cancer tissue by irradiation with light having a wavelength of 600 to 800 nm.
Detailed Description of the Invention
The photodynamic therapy formulation of the present invention is characterized in that it contains at least one or more drugs including a photosensitizing compound in a lipid membrane or in its internal aqueous phase, and contains liposomes substantially free of an organic solvent. It is said.

  In this specification, “photodynamic therapy” refers to photochemical treatment in which a photosensitizing compound that reacts with light such as ultraviolet light, visible light, and infrared light is taken into the body, and the target site is treated by irradiation with light. It is one method.

A “liposome” is a structure usually formed from a lipid membrane, ie, a lipid bilayer membrane. In the present specification, the liposome membrane may also be referred to as “lipid membrane”. “Encapsulated” in a liposome means that it is encapsulated in the liposome and associated with the phospholipid membrane, or is present in an aqueous phase (internal aqueous phase) confined inside the phospholipid membrane. Includes both states. The “drug” contained in the lipid membrane or in its internal aqueous phase includes a photosensitizing compound, an anticancer agent, a contrast agent, a formulation aid, and the like. “Cancer” refers to a malignant tumor, sometimes simply “tumor”. “Substantially” means that the upper limit of the concentration of the residual organic solvent in the contrast agent is 10 μg / L.
Photosensitizing compound As used herein, a “photodynamic therapy formulation (sometimes referred to as a“ liposome formulation ”) is a photosensitizing compound, ie, a form of cytotoxicity upon irradiation with a photoactivating light beam. Refers to a formulation containing a photosensitizing substance that is converted to or produces a cytotoxic species. Many photosensitizers are known as photosensitizing compounds encapsulated in liposomes. The liposome contained in the preparation of the present invention can be used for photodynamic therapy by encapsulating any photosensitizing compound depending on the purpose of use. For photosensitizing compounds, they function as psoralens, chlorins, phthalocyanines or porphyrins, or protoporphyrin precursors that are structural precursors of protoporphyrins (eg, naturally occurring precursors) and photochemical therapeutic agents Their derivatives such as ALA, porphobilinogen or their precursors or their derivatives (eg ALA esters) are known.

The photosensitizing compound exerts its effect directly or indirectly by various mechanisms. For example, certain photosensitizing compounds become directly toxic when activated by light. In contrast, others behave to generate oxidants such as toxic species, specifically singlet oxygen or other oxygen-derived free radicals. These chemical species are extremely harmful to biomolecules such as lipids, proteins and nucleic acids and cellular materials. Psoralens are examples of photosensitizing compounds that act directly. A potential risk of using this photosensitizing compound is that unwanted mutagenic and carcinogenic side effects can occur.

Instead of such a photosensitizing compound, a photosensitizing compound having an indirect mode of action is preferably employed. For example, porphyrins that act indirectly by generating toxic oxygen species do not have mutagenic side effects and can be considered as favorable candidates for photochemical treatment. Porphyrins are naturally occurring precursors in the synthesis of heme. In particular, iron (Fe ³⁺ ) reacts with the enzyme ferrochelatase to produce protoporphyrin IX (Pp)
When taken in, heme is formed. Pp is a very powerful photosensitizing compound, but heme has no photosensitizing effect.

Accordingly, the preferred photosensitizing compound is not particularly limited,
Porphyrin;
Protoporphyrin derivatives;
Photofrin ^TM (Quadra Logic Technologies, Inc. (Vancouver, Canada) and hematoporphyrin IX (HpIX) hematoporphyrin, such as porphyrin IX (hematoporphyrin, HpD);
Photosun III (Seehof Laboratorium GmbH, Seehof, Wessel-burenerkoog, Germany); chlorin such as tetra (m-hydroxyphenyl) chlorin (m-THPC);
Those bacteriochlorins (bacteriochlorin, Scotia Pharmaceuticals, Surrey,
(UK), mono-L-aspartyl chlorin e6 (NPe6) (Nippon Petrochemical, CA, USA), chlorin e6 (Porphyrin Products), benzoporphyrins (Quadra Logic Technologies, Vancouver, Canada) (for example, benzoporphyrin Monovalent acid ring A derivatives, BPD-MA) and purpurines (PDT Pharmaceuticals, CA, USA) (eg tin-ethyl etiopurpurin, SnET2);
Phthalocyanines (e.g. zinc-(Quadra Logic Technologies, Vancouver, Canada), aluminum or silicon phthalocyanines, these may be sulfonated, especially aluminum phthalocyanine disulfonate (AlPcS _2a ), aluminum phthalocyanine tetrasulfonate (AlPcS ₄ ) Sulfonated phthalocyanines such as;
Porphycenes;
Hypocrellins;
Protoporphyrin IX (PpIX);
Hematoporphyrin diethers;
Uroporphyrins;
Coproporphyrins;
Deuteroporphyrin;
Polyhematoporphyrin (PHP);
Lutetium texaphyrin (LU-TEX);
5-aminolevulinic acid (ALA);
Porphobilinogen;
ALA esters;
verteporfin, purlytin,
Derivatives of chlorophyll and bacteriochlorophyll (US Pat. No. 5,650,292);
And precursors and derivatives thereof.

Each of the exemplified photosensitizing compounds has unique characteristics, and it is desired to use the characteristics appropriately depending on the application. Hereinafter, some photosensitizing compounds will be described in detail.
One of the porphyrins, Photofrin ^™ has recently been approved as a photosensitizing compound in the treatment of certain cancers and has shown good therapeutic efficacy and stability. Photofrin consists of large oligomers of porphyrin and does not easily penetrate the skin when applied topically. It must therefore be administered parenterally, generally intravenously. However, after intravenous administration of such a photosensitizing compound, the photosensitizing compound still remains in the body for 5 to 6 weeks, causing the side effect of causing skin photosensitization. Furthermore, when compared with the optical spectrum (650 to 800 nm) optimum for photodynamic therapy, the maximum light absorption wavelength of photofurin, 630 nm, is low, and the amount of light transmitted to malignant tumor tissue is small. There are also some obstacles to the preparation of pure photofrin by chemical synthesis.

Other porphyrin-based photosensitizing compounds such as “hematoporphyrin derivative (Hpd)” have also been reported for use in photochemical treatment of cancer (for example, S. Dougherty. J. Natl. Cancer Ins., 1974). Year, 52; 1333; see Kelly and Snell, J. Urol, 1976, 115: 150). Hpd is a complex mixture obtained by treating hematoporphyrin with acetic acid and sulfuric acid and then dissolving the acetylated product with alkali. The use of such a mixture has the disadvantage of being uncertain as a drug. Furthermore, since Hpd must also be administered by injection, there are the same types of side effects seen with photofrin.

Porphyrin, chlorin, bacteriochlorin and porphycene have been developed as next-generation photosensitizing compounds (J. Org. Chem., 63, 1646-1656). Page, 1998). Pheophytin is prepared after removing metal ions from plant chlorophyll and exhibits better absorption on the longer wavelength side than photofrin. Pheophytin itself can be used as a photosensitizing compound, but it can be developed into a more excellent photosensitizing compound by modifying its molecular structure. However, many of them still have problems in terms of stability, excretion, and side effects.

The Pp precursor 5-aminolevulinic acid (ALA) has been studied as a photochemotherapeutic agent for certain skin cancers (see, eg, WO91 / 01727). Because the skin covering basal cell and squamous cell carcinomas is more permeable to ALA than healthy skin and has lower ferrochelatase concentrations in skin tumors, local application of ALA is selectively enhanced in tumors. Has been found to lead to Pp production. However, photochemical treatment using ALA is not always satisfactory for other lesions. ALA cannot penetrate all tumors and other tissues sufficiently effectively to allow treatment of a wide range of tumors or other conditions. For this reason, improvements using derivatives such as ALA esters are underway.

None of the above-mentioned known photosensitizing compounds satisfy all of the requirements required for the use of photodynamic therapy, and each has advantages and disadvantages. In the preparation of the present invention, when a photosensitizing compound suitable for the purpose of use is used, a compound satisfying the following conditions may be selected.
i) having a high photoreaction yield from triplet oxygen to singlet oxygen;
ii) exhibit high absorption in the long wavelength side, especially in the spectrum of 600 nm or more,
iii) After being released from the liposome, it is rapidly excreted from normal cells,
iv) minimal side effects and toxicity of the photosensitizing compound itself, and
v) Mass production with high purity and low cost In the photodynamic therapy preparation of the present invention, it is desirable that the photosensitizing compound is selected from at least one of a porphyrin photosensitizer and a chlorophyll photosensitizer. In particular, in a PDT preparation for cancer, a porphyrin-based sensitizer is preferably used as a photosensitizing compound. It is known that porphyrin-based sensitizers tend to accumulate in malignant tumor tissues (Patent Document 1). Specifically, porphyrin, hematoporphyrin IX, hematoporphyrin derivative (HpD), etc. having tumorigenicity are preferable. In particular, porfimer sodium (Photofurin ^™ ) is preferred because of its ease of loading on liposomes.
Liposome In the photodynamic therapy preparation of the present invention, a photosensitizing compound or the like is used in the form of being encapsulated in a liposome as a microcarrier for efficient delivery to a target organ or tissue lesion. The preferred liposome used in the present invention is a liposome whose lipid membrane is substantially a single membrane or several membranes, has a large inclusion volume, and has improved stability over time and in blood.

  In the liposome preparation of the present invention, a targeting function can be imparted by appropriately designing the particle size of the liposome encapsulating the photosensitizing compound and the like and the lipid membrane thereof. Particularly in the case of systemic administration, it is desirable to consider both passive targeting and active targeting. The former can control the in vivo behavior through adjustment of the liposome particle size, lipid composition, charge, and the like. Adjustment for aligning the liposome particle size within a narrow range is easily performed based on the method described below. In the design of the liposome membrane surface, desired properties can be imparted by changing the type and composition of phospholipids and coexisting substances. In addition, the adoption of active targeting that allows for a higher degree of delivery selectivity and accumulation with respect to the transport and distribution of administered liposomes should also be considered. As an example, introducing a polyalkylene oxide polymer chain or a polyethylene glycol (PEG) group on the surface of the liposome membrane is extremely beneficial because the induction process to the target site can be controlled.

  Liposomes that have not reached cancer tissue or the like are decomposed relatively quickly and excreted outside the body without accumulating in normal tissues. This can be achieved by appropriately controlling the stability of the liposome in relation to the extravasation time. By controlling such clearance, another effect of a DDS dosage form in which a drug for photodynamic therapy containing a large amount of hydrophobic photosensitizing compound is encapsulated in liposomes can be expected. That is, when a photosensitizing compound is encapsulated in liposomes, the photosensitizing compound is deposited nonspecifically in the liver, spleen, kidney, adipose tissue, etc., and it is difficult to fall into a situation where it takes a long time to decompose and excrete. . Therefore, adverse effects caused by staying in the body and delayed side effects can be prevented.

  In fact, patients who have been systemically administered free forms of photosensitizing compounds, such as porphyrin derivatives, will not be easily excreted in the urine, as described above, so that light shielding is avoided to avoid photosensitivity. The limit period is often long. In the case of a positive photosensitivity reaction test, it is required to limit light shielding until it becomes negative. In order to prevent photosensitivity, it is necessary to avoid direct sunlight with a light-shielding curtain and spend in a room where the illuminance is controlled below a certain level. There are many restrictions on daily life, such as the consumption of specific foods that may cause strong photosensitivity and the need to wear clothing such as hats, gloves, and long sleeves and sunglasses when going out.

  In general, phospholipids and / or glycolipids are preferably used as lipid membrane components of liposomes contained in the liposome preparation of the present invention. Preferred neutral phospholipids include lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybeans, egg yolks and the like.

Other phospholipids may be derived from egg yolk, soybeans or other animals or plants, or semi-synthetic phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, synthetically obtained phosphatidic acid, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristolphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylserine (DSPS), distearoylphosphatidylglycerol (DSPG), Dipalmitoylphosphatidylinositol (DPPI),
Examples include distearoyl phosphatidylinositol (DSPI), dipalmitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA).

  It is desirable that the phospholipid of the lipid membrane constituting the liposome of the present invention contains at least a phospholipid having a transition temperature. The “(phase) transition temperature” of a phospholipid is a temperature that causes a phase transition between both the gel and liquid crystal states that the phospholipid can take. The measurement is by differential thermal analysis using a differential scanning calorimeter (DSC). As phospholipids having a phase transition point, dimyristoyl phosphatidylcholine (transition temperature, the same below, 23-24 ° C), dipalmitoylphosphatidylcholine (41.0-41.5 ° C), hydrogenated soybean lecithin (53 ° C), hydrogenated soybean phosphatidylcholine (54 ° C), distearoylphosphatidylcholine (54.1-58.0 ° C) and the like.

The cationic lipid used in the present invention is 1,2-dioleoyloxy-3- (trimethylammonium) propane (DOTAP), N, N-dioctadecylamide glycylspermine (DOGS), dimethyldioctadecylammonium bromide ( DDAB), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2 (spermine-carboxamide) Ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and N- [1- (2,3-dimyristyloxy) propyl] -N, N-dimethyl-N- (2-hydroxy And ethyl) ammonium bromide (DMRIE).

  Cationic phospholipids include esters of phosphatidic acid and amino alcohol, such as esters of dipalmitoyl phosphatidic acid (DPPA) or distearoyl phosphatidic acid (DSPA) and hydroxyethylenediamine. These cationic lipids are contained in an amount of 0.1 to 5% by mass with respect to the total lipid amount, preferably 0.3 to 3% by mass with respect to the total lipid amount, and more preferably 0.5 to 2% by mass with respect to the total lipid amount. What is necessary is just to add.

  These phospholipids are usually used alone, but may be used in combination of two or more. However, when two or more kinds of charged phospholipids are used, it is desirable to use them between negatively charged phospholipids or between positively charged phospholipids from the viewpoint of preventing liposome aggregation. When neutral phospholipids and charged phospholipids are used in combination, the weight ratio is usually 200: 1 to 3: 1, preferably 100: 1 to 4: 1, and more preferably 40: 1 to 5: 1.

  Examples of glycolipids include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, and sphingoglycolipids such as galactosylceramide, galactosylceramide sulfate, lactosylceramide, ganglioside G7, ganglioside G6, and ganglioside G4.

  As a constituent component of the liposome membrane, other substances can be added as necessary in addition to the lipid. Examples include sterols that act as lipid membrane stabilizers, such as cholesterol, dihydrocholesterol, cholesterol esters, phytosterols, sitosterol, stigmasterol, campesterol, cholestanol, or lanosterol. It has also been shown that sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside are effective in stabilizing liposomes (JP-A-5-245357). Among these, cholesterol is particularly preferable.

The amount of sterols used is desirably 0.05 to 1.5 parts by weight, preferably 0.2 to 1 part by weight, and more preferably 0.3 to 0.8 parts by weight with respect to 1 part by weight of phospholipid. If it is less than 0.05 part by weight, stabilization by sterols that improve the dispersibility of the mixed lipid is not exerted, and if it is more than 2 parts by weight, the formation of liposomes is inhibited, or even if formed, it becomes unstable.

Cholesterol in the liposome membrane can also serve as an anchor for introducing polyalkylene oxide. Japanese Patent Application Laid-Open No. 09-3093 discloses a novel cholesterol derivative that can immobilize various functional substances at the end of a polyoxyalkylene chain by covalent bonds and can be used as a component for liposome formation. It is disclosed.
In addition to the sterols, glycols may be added as a constituent of the liposome membrane. When preparing a liposome, if glycols are added together with phospholipid or the like, the retention efficiency of the photosensitizing compound in the liposome is increased. Examples of glycols include ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, trimethylene glycol, and 1,4-butanediol. The amount of glycols used is 0.01 to 20% by mass, preferably 0.5 to 10% by mass, based on the total mass of lipid.
Is desirable.

Depending on the intended purpose of the X-ray contrast agent of the present invention, which is a liposome preparation, a phospholipid or compound having a polyalkylene oxide (polyoxyalkylene chain) (PAO) group or a similar group, which is a polymer chain, is added to the liposome. It may be used as a component of the membrane. By attaching a polyalkylene oxide group or a polyethylene glycol (PEG) group to the surface of the liposome membrane, instability of the liposome itself such as disintegration and aggregation is solved, and stability over time is also improved. Furthermore, a new function can be imparted to the liposome. For example, PEGylated liposomes can be expected to have an effect of being hardly recognized by the immune system. Furthermore, it has been clarified that liposomes can be maintained in blood concentration over a long period of time by increasing the stability in blood by forming a hydrated layer by introduction of PEG groups and having a hydrophilic tendency (Biochim. Biophys. Acta., 1066, 29-36 (1991)). Therefore - by varying (CH ₂ CH ₂ O) _n of oxyethylene units of the PEG group represented by -H length and rate of introduction can be appropriately modulate its function. As the PEG group, polyethylene glycol having oxyethylene units of 10 to 3500, preferably 100 to 2000 is suitable. The amount of polyethylene glycol used is 1 to 40% by mass, preferably about 5 to 25% by mass, with respect to the lipid constituting the liposome. A known technique can be used for PEGylation of the liposome.

Instead of the polyethylene glycol, various known polyalkylene oxide groups,-(AO) _n -Y, may be introduced on the surface of the liposome membrane. Here, AO represents an oxyalkylene group having 2 to 4 carbon atoms, n is an average addition mole number of the oxyalkylene group, and is a positive integer of 1 to 2000, preferably 10 to 500, more preferably 20 to 200. is there. Y represents a hydrogen atom, an alkyl group (for example, an aliphatic hydrocarbon group having 1 to 5 carbon atoms which may be branched) or a functional functional group.

  Examples of the oxyalkylene group having 2 to 4 carbon atoms (represented by AO) include oxyethylene group, oxypropylene group, oxytrimethylene group, oxytetramethylene group, oxy-1-ethylethylene group, oxy-1,2 -A dimethylethylene group etc. are mentioned.

When n is 2 or more, the types of oxyalkylene groups may be the same or different. In the latter case, it may be added in a random manner or a block shape. In the case of imparting hydrophilicity to the polyalkylene oxide chain, the oxyalkylene group is preferably an ethylene oxide added alone, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the polyalkylene oxide chain, the number of added moles of oxyalkylene groups other than ethylene oxide is increased. For example, a liposome containing a block copolymer of polyethylene oxide and polypropylene oxide is a preferred embodiment of the present invention.

The functional functional group of Y is for attaching a “functional substance” such as sugar, glycoprotein, antibody, lectin, and cell adhesion factor to the end of the polyalkylene oxide chain. For example, amino group, oxycarbonylimidazole group And a reactive functional group such as an N-hydroxysuccinimide group.

  Liposomes with a polyalkylene oxide chain immobilized with a “functional substance” at the tip are identified as functions of the “functional substance”, eg, “recognition element”, in addition to the effect of introducing the polyalkylene oxide chain. Effects such as organ directivity and tumor tissue directivity are sufficiently exerted. In order to impart tumor tissue directivity, anti-tumor monoclonal antibodies corresponding to tumor-specific antigens present only in tumor cells are bound to liposome membranes as functional substances, resulting in liposomes with higher target selectivity (for example, JP-A-11-28087).

A known technique can be used for introducing the polyalkylene oxide chain into the liposome membrane. A phospholipid or compound having a polyalkylene oxide group can be used alone or in combination of two or more. The content thereof is 0.001 to 50 mol%, preferably 0.01 to 25%, based on the total amount of the liposome membrane constituents.
It is mol%, More preferably, it is 0.1-10 mol%.

  Other examples of compounds that can be added include dialkyl phosphates such as dicetyl phosphate, which is a negatively charged substance, and aliphatic amines such as stearylamine, as compounds that give a positive charge.

  In the liposome preparation of the present invention, the adjustment of the size and distribution of liposomes as fine particles is closely related to high targeting properties and delivery efficiency. The particle size (particle size) is measured by freezing the dispersion containing the liposome encapsulating the photosensitizing compound, then depositing carbon on the crushed interface, and observing the carbon with an electron microscope (freeze crushing TEM method). be able to. Here, “average particle diameter” refers to a simple average of a certain number of observed contrast agent particles, for example, 20 diameters. This usually coincides with or is approximately similar to the “center particle size”, which refers to the particle size having the highest frequency in the particle size distribution.

In order to give the liposome the passive targeting ability as described above, it is necessary to prepare the liposome by appropriately aligning the particle size when producing the liposome. Japanese Patent No. 2619037 describes that by excluding liposomes having a particle size of 3 μm or more, inadvertent retention of liposomes in lung capillaries is avoided. However, liposomes with a particle size range of 0.5-3 μm are not necessarily tumorigenic in nature.

In order to make the liposome encapsulating the photosensitizing compound pro-tumorous, the aim of using the “EPR effect (Enhanced permeability and retention)”
The average particle size is preferably 0.1 to 0.2 μm, more preferably 0.11 to 0.13 μm. By aligning the average particle size of the liposome within the range of 0.11 to 0.13 μm, the liposome preparation can be selectively concentrated on the cancer tissue. The pores of the neovascular wall in solid cancer tissue are abnormally large compared to the pore size of the capillary wall window (fenestra) of normal tissue, 0.03 to 0.08 μm, and even molecules with a size of approximately 0.1 μm to 0.2 μm Leaks from the vessel wall. Thus, the EPR effect is due to the higher permeability of the neovascular wall in cancer tissue than the microvascular wall of normal tissue.

Liposomes leaking from the pores of the blood vessel wall do not return to the blood vessels again because the lymphatic vessels are not sufficiently developed around the cancer cells, and remain in the place for a long time. Since the EPR effect is based on passive transport using the bloodstream, the retention in the blood must be improved in order to effectively develop the EPR effect. That is, it is necessary that the liposome particles encapsulating the photosensitizing compound stay in the blood for a long time and pass through the blood vessels near the cancer cells many times. When the liposome encapsulating the photosensitizing compound is not particularly large as described above, it is difficult to be a target of capture phagocytosis by reticuloendothelial cells. Such an EPR effect would rather be considered in the case of systemic chemotherapy or systemic administration of liposome formulations. Furthermore, it is also useful in the case of angiography and when encapsulating a contrast agent used for imaging of cancer tissue together with liposomes.

The preferred average particle size of the liposomes differs when the photodynamic therapy preparation is locally administered in vivo. In intraarterial injection chemotherapy, which is a typical topical chemotherapy, when the liposome encapsulating a photosensitizing compound is directly applied through a catheter to a blood vessel near the cancer lesion, the average particle size of the liposome is usually 0.7 to 0. 0.9 μm, more preferably 0.75 to 0.85 μm,
Particularly preferably, it is desirable that the thickness is about 0.8 μm. Liposomes with such an average particle size concentrate in the target cancer tissue without leaking from the vascular wall pores of the trophozoites that lead to cancer tissue but increased permeability, and accumulate in the tumor at a high concentration. Achieved.
Liposome Production Methods Various methods have been proposed for producing liposomes. When the production method is different, the shape and characteristics of the final liposome are often significantly different (JP-A-6-80560). Therefore, the production method is appropriately selected according to the desired form and characteristics of the liposome. In general, liposomes are prepared by mixing lipid membrane components such as phospholipids and sterols in a container together with organic solvents such as chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, THF, etc.
Start by dissolving. In particular, chlorinated solvents are often used. Such preparations of liposomes always contain an organic solvent. In order to remove these remaining organic solvents, a plurality of steps and a long time treatment are required at present. Such residual organic solvents, particularly chlorinated organic solvents, are concerned about adverse effects on living bodies, such as side effects.

The method for producing a liposome of the present invention comprises a compound selected from at least one of a cationic lipid and a sterol together with a phospholipid as a lipid membrane component under a condition in which at least one compound having a polyethylene glycol group is present, and Depending on the solution, a lipophilic drug is dissolved or dispersed in carbon dioxide in a supercritical or subcritical state, and then a micelle is formed by introducing a solution or suspension of at least one drug, followed by water. And discharging carbon dioxide to produce a liposome containing a drug containing a photosensitizing compound. By such a liposome production method, a liposome having a high encapsulation rate of a photosensitizing compound can be produced without using any organic solvent. Another feature is that the photosensitizing compound, which is a water-soluble non-electrolyte, is also efficiently encapsulated in liposomes.

The liposome according to the present invention is produced using supercritical carbon dioxide or subcritical carbon dioxide without using an organic solvent. Carbon dioxide has a critical temperature of 31.1 ° C and a critical pressure of 75.3 kg / cm ^{2, which} is relatively easy to handle.
High purity fluid is preferable because it is inexpensive and easily available. Liposomes prepared by this method have various favorable properties and advantages for encapsulating photosensitizing compounds as described later. The temperature of carbon dioxide in the supercritical state (including subcritical state) used in the production method of the present invention is usually 25 to 200 ° C, preferably 31 to 100 ° C, and more preferably 35 to 80 ° C. Suitable pressure is usually 50 to 500 kg / cm ^2, preferably 100 to 400 kg / cm ^2, particularly preferably in the range of 90~150 kg / cm ^2.

When producing liposomes using supercritical or subcritical carbon dioxide, it is necessary to dissolve, disperse or mix the lipid membrane components in carbon dioxide in the supercritical state (including subcritical state). It becomes. At that time, among the drugs to be encapsulated, poorly water-soluble photodynamic therapy drugs, specifically photosensitizing compounds, anticancer compounds, lipophilic contrast agents, etc. are mixed with supercritical carbon dioxide together with lipid membrane components. It is good. Furthermore, it is preferable to dissolve, disperse or mix in the presence of at least one compound having a hydroxyl group as a dissolution aid (or cosolvent).

  Examples of the compound having a hydroxyl group (that is, a hydroxyl group-containing compound) include a compound having a hydroxyl group, a polyol group, a polyalkylene glycol ether group, or a combination of a polyol / polyglycol ether group as a hydrophilic group. included. As a hydroxyl group-containing compound that can actually be used as a solubilizing agent, a compound that exhibits affinity with a lipid membrane component such as phospholipid or cholesterol and is mixed with these components is desirable. Furthermore, in order to disperse and dissolve the lipid membrane component well in the polar liquid carbon dioxide, an amphiphilic material having both appropriate hydrophilicity and hydrophobicity is preferable.

  In the case of the above-mentioned compound having a hydroxyl group, it is desirable not to use a lower alcohol or the like from the viewpoint of safety when there is a concern about the toxicity of the remaining dissolution aid. Therefore, in view of efficacy and safety, a more preferable solubilizer is a compound having a polyethylene glycol (PEG) group, preferably a lipid having a polyethylene glycol group, more preferably PEG-phospholipid. The oxyethylene unit is 10-3500, preferably 100-2000.

Use of one or more such compounds having a hydroxyl group is desirable in order to improve the encapsulation rate. The compound having a hydroxyl group may be used as a solubilizing agent at a ratio of 0.01 to 1% by mass, preferably 0.1 to 0.8% by mass, of carbon dioxide to be in a supercritical state or a subcritical state.

The production method of the liposome used for the photodynamic therapy preparation of the present invention is specifically performed as follows. Liquid carbon dioxide is added to the pressure vessel to bring it to the supercritical or subcritical state under the preferred pressure and temperature described above. In the presence of at least one compound having a hydroxyl group, a phospholipid and a lipid membrane stabilizing substance as a membrane lipid component of a liposome, or a poorly water-soluble drug are dissolved in carbon dioxide in a supercritical (or subcritical) state. Disperse. A compound selected from cationic phospholipids and sterols as membrane lipid components is mixed and dissolved and dispersed together with the phospholipid. Alternatively, liquid carbon dioxide may be added to a pressure vessel to which these compounds have been added in advance, and then the temperature and pressure are adjusted to obtain a supercritical state and mixed. In the supercritical carbon dioxide containing the phospholipids and lipid membrane stabilizing substances that were subsequently produced, water-soluble photosensitizing compounds, anticancer agents, water-soluble iodine-based contrast agents among the drugs to be encapsulated, as needed Accordingly, micelles are formed by introducing an aqueous solution containing the formulation aid. The side to be added and the side to be added may be reversed. After sufficiently mixing, when water is added to the system and the pressure is reduced to discharge carbon dioxide, an aqueous dispersion in which liposomes encapsulating a photosensitizing compound and the like are dispersed is generated. In this case, a photosensitizing compound may be contained in the aqueous phase inside and outside the liposome membrane. Since the aqueous solution is also encapsulated inside the liposome, the photosensitizing compound exists mainly in the aqueous phase inside the liposome in addition to the external aqueous phase (aqueous medium) of the liposome, and is in a so-called “encapsulation” state. Further, the liposome is 0.1 to 1.0 μm.
It is preferably passed through a filtration membrane having a pore size of 0.1 to 0.45 μm. Liposomes can be separated by conventional techniques such as ultrafiltration, centrifugation, gel chromatography, and dialysis. The preparation may then be lyophilized for storage. Such a dry preparation is resuspended in an aqueous medium immediately before use to form a dispersion. The osmolarity of the liposome after reconstitution is typically 250 to 500 mosmol / L, preferably 290 to 350 mosmol / L.

The liposome preparation method using supercritical carbon dioxide or subcritical carbon dioxide has been shown to have higher liposome production rate, encapsulation efficiency of encapsulated drug, and retention rate of encapsulated drug in the liposome than conventional methods. (See Patent Document 3 above). Furthermore, application on an industrial scale is also possible. This method capable of efficiently encapsulating non-electrolyte-containing drugs in liposomes substantially without using chlorinated solvents and other organic solvents is useful for the production of the photodynamic therapy formulation and the like of the present invention. Is the method.
Production of Photodynamic Therapy Formulation The photodynamic therapy formulation of the present invention can be produced by a technique known in the art using the above-mentioned liposome and further using one or more types of physiologically acceptable formulation aids. . The formulations of the present invention are preferably marketed in sterilized form. As for the sterilization, a sterile preparation is obtained by γ-ray irradiation, autoclave sterilization, or heat sterilization. The preparation of a preferred embodiment of the present invention contains a formulation aid in the aqueous phase inside the lipid membrane of the liposome and the aqueous medium in which the liposome is suspended. This formulation aid is a substance that is added together with a photosensitizing compound or the like when formulating liposomes, and various substances are used as necessary based on conventional contrast agent production techniques. Specifically, various physiologically acceptable buffers, chelating agents, pharmacologically active substances (for example, vasodilators, coagulation inhibitors, etc.), osmotic pressure regulators, stabilizers, antioxidants ( For example, α-tocopherol, ascorbic acid), a viscosity modifier, a preservative and the like can be mentioned. Preferably, both an amine buffer and a chelating agent are included. As a pH buffer, a water-soluble amine buffer and a carbonate buffer are preferably used. Particularly preferred is an amine buffer, with trometamol being preferred.

  The “aqueous medium” is a water-based solvent that dissolves or suspends a photosensitizing compound, a formulation aid, and the like. As the water, use sterilized pyrogen-free water. When a formulation aid is contained in an aqueous solution (that is, an aqueous medium in which the liposome is suspended) other than the aqueous phase (an aqueous solution encapsulated by the lipid membrane) inside the lipid membrane of the liposome, There is no osmotic pressure difference, which maintains the structural stability of the liposomes. Even during storage, it is possible to prevent destabilization due to the osmotic pressure effect of the liposome encapsulating the photosensitizing compound and the like, and the retention stability of the drug in the liposome is improved.

It is desirable that the liposome contained in the liposome preparation of the present invention is a liposome substantially consisting of a single membrane or several membranes. Single-membrane liposomes are liposomes composed of a membrane (unilamellar vesicle) having a single phospholipid bilayer. In observation with a transmission electron microscope (TEM) by the Freeze fracture replica method,
A liposome composed of a phospholipid bilayer in which a replica is generally recognized as one layer is called a single membrane liposome. That is, the observed particle traces left on the carbon film are determined to be a single film, and those having two or more steps are determined to be “multilayer films”. Liposomes composed of two or three membranes are stronger than single membrane liposomes. “Substantially” means that in the liposome preparation of the present invention, such a single membrane liposome or a liposome composed of several membranes is at least 80% of the total liposomes contained in the contrast agent, Preferably it means containing 90% or more.

  The single membrane liposome or the liposome composed of several membranes can be efficiently prepared by the phase separation method using water using the supercritical carbon dioxide or the subcritical carbon dioxide as a solvent of the lipid membrane component. On the other hand, according to the conventional method for preparing liposomes, liposomes composed of multilamellar vesicles (MLV) of various sizes and forms are often present in a considerable proportion. Therefore, in order to increase the ratio of single membrane or several membrane liposomes, it is necessary to further irradiate ultrasonic waves or pass through a filter having a fixed pore size many times. Single-membrane or several-membrane liposomes also have the advantage that the dose of liposomes, in other words, the amount of lipid administered does not increase, compared to MLV.

Liposomes with a small number of lipid membranes, in particular LUV (Large unilamellar veislcles), which are monolayer liposomes with a large particle size, have the advantage of providing a large encapsulation capacity compared to multilamellar liposomes. Liposomes preferably used in the contrast agent of the present invention have a retention volume larger than that of SUV, and are excellent in trapping efficiency of water-soluble anticancer compounds, in other words, inclusion efficiency. On the other hand, even in the case of a single-film or several-film liposome with good encapsulation efficiency of the photosensitizing compound, the stability of the liposome is lowered if the weight of the encapsulating photosensitizing compound is relatively large. In particular, a tendency to be vulnerable to rapid changes in ionic strength was observed. The contrast medium liposome of the present invention is adjusted to a relatively small average particle size. Furthermore, the lipid membrane is stabilized by containing at least one compound selected from a compound having a polyalkylene oxide group (for example, phospholipid), sterols, and glycol in the liposome membrane. As a result, such liposomes were found to be stable against salt shock.

The particle size can be adjusted by changing the formulation or process conditions. For example, when the pressure in the supercritical state is increased, the formed liposome particle size is decreased. In order to make the particle size distribution of the liposome to be produced in a narrower range, filtration may be performed with a polycarbonate membrane, a cellulose membrane, or the like. For example, by passing through an extruder equipped with a filter having a pore size of 0.6 to 1 μm as a filtration membrane, liposomes having an average particle size of 0.8 μm can be efficiently prepared. To make the average particle size even smaller, filter with a filter such as 0.45 μm. This extrusion filtration method is described, for example, in Biochim. Biophys. Acta 557, 9 (1979). By incorporating such an “extrusion” operation, in addition to the above sizing, it is possible to adjust the concentration of the photosensitizing compound existing outside the liposome, exchange the liposome dispersion, and remove undesirable substances. There are also advantages.

  When a drug such as a photosensitizing compound is encapsulated in a microcarrier such as a liposome as in the liposome preparation of the present invention, in addition to the encapsulation efficiency and encapsulation stability of the photosensitizing compound, the weight of the liposomal membrane lipid must be considered. Don't be. As the membrane lipid weight of the liposome increases, the viscosity of the preparation increases. As the amount of drug encapsulated in the liposome, the total amount of drug in the aqueous solution encapsulated in the liposome is 1 to 8, preferably 3 to 8, more preferably 5 to 8 with respect to the liposome membrane lipid. It is desirable to contain.

When the weight ratio of all drugs encapsulated in the liposome is less than 1, it is necessary to inject a relatively large amount of lipid, and the viscosity of the preparation increases, resulting in poor drug delivery efficiency. Single-membrane or several-membrane liposomes are advantageous because they have excellent encapsulation volume and encapsulation efficiency. On the other hand, when the encapsulated weight ratio of all drugs to liposome membrane lipid exceeds 8, liposomes are also structurally unstable, and drug diffusion and leakage outside the liposome membrane were injected during storage or in vivo. Inevitable later. Immediately after the liposome suspension drug is produced and separated, it is 100
It is described that even if% encapsulation is achieved, the encapsulated components decrease as quickly as possible based on destabilization due to the osmotic pressure effect (Japanese Patent Publication No. 9-505821).
Photodynamic therapy preparation The photodynamic therapy preparation of the present invention can be used for ordinary photodynamic therapy. Specifically, it can be used for either systemic administration or local administration (eg, delivery to the intestinal tract, cheek, sublingual, gingiva, palate, nose, lung, vagina, rectum, or eyeball). The photodynamic therapy preparation of the present invention can also be provided in a dosage form suitable for oral or parenteral administration. Among these, photodynamic therapy preparations for topical administration include gels, creams, ointments, sprays, lotions, waxes, sticks, soaps, powders, tablets, films, vaginal suppositories, aerosols Formulated in any of drops, solutions, and conventional pharmaceutical forms in the art. In the case of topical administration or oral administration, it further contains an absorption enhancer such as a lubricant, a humectant, an emulsifier, a suspending agent, a preservative, a sweetener, a flavoring agent, and the following surface penetration aid as necessary. Also good.

  It may be administered with other active ingredients that enhance the photochemotherapeutic effect. As mentioned above, a chelating agent can also be included. These improve the stability of the photochemotherapeutic agent or enhance the accumulation of Pp. That is, the chelation of iron by this chelating agent prevents the reaction in which iron is taken into Pp and forms heme by the action of the enzyme ferrochelatase, and as a result, Pp is accumulated. Thus, the photosensitization effect is enhanced.

As for the chelating agent in this case, it is particularly preferred to use an aminopolycarboxylic acid chelating agent, for example described in the literature on detoxification of metals or chelation of paramagnetic metal ions in magnetic resonance contrast agents. All chelating agents of In particular, mention may be made of EDTA, CDTA (cyclohexanediaminetetraacetic acid), DTPA, DOTA and their known derivatives and analogues. EDTA is particularly preferable. To obtain an iron chelating effect, desferrioxamine and other iron carriers can also be used in combination with an aminopolycarboxylic acid chelating agent such as EDTA. The chelating agent can be appropriately used at a concentration of 1 to 20%, for example 2 to 10% (w / w).

  Furthermore, it is known that surface penetration aids and in particular dialkyl sulfoxides such as dimethyl sulfoxide (DMSO) have beneficial effects in enhancing photochemical therapeutic effects. Details are described in WO95 / 07077.

The photodynamic therapy formulation of the present invention can be formulated and / or administered with other drugs to further enhance the effectiveness of photodynamic therapy. Examples of other drugs include pharmacologically active substances (for example, vasodilators, coagulation inhibitors). Specifically, for the treatment of tumors, angiogenesis inhibitors (anti-angiogenic agents) known to be effective for tumor treatment (O'Reilly et al., Nature Medicine, 2, p689-692, 1996; Yamamoto et al. Anticancer Research, 14, p1-4, 1994; and Brooks et al., J. Clin. Invest., 96, p1815-1822, 1995) together with the photodynamic therapy formulation of the present invention for photodynamic therapy, The tumor vasculature can be further destroyed. Angiogenesis inhibitors used include TNP-470 (AGM-1470, a synthetic analog of a fungal secretion called fumagillin; Takeda Pharmaceutical Co., Ltd. (Osaka)), Angiostatin (Harvard Medical School Children's Hospital, Surgical Research Lab. ) And integrin α _V β ₃ antagonists (eg, monoclonal antibodies to integrin α _V β ₃ , The Scripps Research Institute, LaJolla, CA).

In addition, an immunotherapeutic agent (for example, an effector such as an antibody or macrophage activating factor) or a chemotherapeutic agent can be used separately or additionally to enhance the effect of the photodynamic therapy according to the present invention. Such supplemental agents are administered by known methods for use of these supplemental agents with respect to route of administration, concentration, formulation. These supplements may be administered before, after or during treatment with photodynamic therapy depending on their function. For example, an angiogenesis inhibitor can be administered 5-10 days after photodynamic therapy to prevent tumor regrowth.
Similarly, an anti-cancer agent can be used in combination with the photodynamic therapy formulation of the present invention, either contained as part of the drug, or separately or sequentially with the photodynamic therapy formulation as a separate therapeutic agent. Can be administered.

Also, local or systemic administration of glucose has been found useful in photodynamic therapy. It appears that administration of glucose lowers the pH, thereby increasing the hydrophobicity of protoporphyrins such as ALA, thus facilitating their penetration into cells. When considering topical administration, the drug, such as a cream, preferably contains 0.01 to 10% (w / w) glucose.

Depending on the condition of the site to be treated and the nature of the photodynamic therapy formulation, the photodynamic therapy formulation used in the present invention can be administered alone or in combination with any agent as described above. For example, the optimal dose and light irradiation conditions of a photodynamic therapy preparation for cancer treatment are usually individually set in consideration of the type of lesion, site, symptom, patient-side conditions, and the like. You may make it the quantity of the photosensitizing compound in a liposome become comparable as the conventional dosage. The concentration of the photochemotherapeutic agent in the photodynamic therapy formulation of the present invention depends on the nature of the compound in the photodynamic therapy formulation, the mode of administration and symptoms, and the condition of the patient, and can be adjusted as necessary. If the solution has an excessively high concentration, the disadvantageous situation of aggregation of liposomes and increase in viscosity must be taken into consideration. Usually, the concentration of the photochemotherapeutic agent (W / W relative to the final drug to be administered) is preferably 0.01 to 50%, for example 0.05 to 20%, 1 to 10% (w / w). For therapeutic applications, the concentration of the photochemotherapeutic agent is suitably 0.1-50%, for example 0.2-30% (w / w).

  The preparation of the present invention is preferably administered parenterally to a patient as an injection or infusion, specifically by intravascular administration or intravenous administration. When it is necessary to administer a relatively high concentration of liposome preparation in a large amount in a short time, the requirements for enabling such bolus injection are the fluidity and low viscosity of the photodynamic therapy preparation. The viscosity of the liposome dispersion of the present invention (when measured by the Ostwald method) is 20 mPa · s or less at 37 ° C. in order to reduce infusion resistance and reduce patient pain and avoid the risk of extravasation. , Preferably 18 mPa · s or less, more preferably 15 mPa · s or less.

When the osmolality of the liposome preparation to be administered is high, the burden on the heart and circulatory system is large. To obtain a solution or suspension that is isotonic with blood, the photosensitizing compound is dissolved or suspended in a medium at a concentration that provides the isotonic solution. For example, if the photosensitizing compound alone cannot provide an isotonic solution because the solubility of the photosensitizing compound is low, other non-toxic water-soluble substances are formed so that an isotonic solution or suspension is formed. For example, salts such as sodium chloride, saccharides such as mannitol, glucose, sucrose, and sorbitol may be added to the medium.
Photodynamic therapy (PDT)
The photodynamic therapy preparation of the present invention can be applied to ordinary photodynamic treatment methods. Abnormalities and diseases that can be treated according to the present invention include malignant, pre-malignant, and non-malignant abnormalities or diseases that are responsive to photodynamic therapy. Examples include tumors or other neoplasms, particularly basal cell carcinomas, dysplasias or other proliferative diseases, and other diseases or bacterial, viral or fungal infections such as herpes virus infections. The photodynamic therapy preparation of the present invention is particularly suitable for the treatment of diseases, diseases or abnormalities in which lesions are formed discretely and the preparation can be directly administered (the lesion here refers to a broad meaning including tumors and the like). Used).

  The photodynamic therapy preparation of the present invention can be administered either systemically or locally, but is preferably administered locally. However, like oral administration for gastric disorders, administration is performed remotely at the upper end of the duct including the administration site. Local administration to sites where direct administration is not possible can be accomplished by techniques known in the art using, for example, a catheter or other suitable drug delivery system.

Photodynamic therapy can be repeated at the same site without causing resistance. Depending on the mechanism of drug redistribution or resynthesis, partial irradiation may rather enhance the effect. A side effect is photosensitivity. Normal tissue is also damaged but is repaired relatively well.

After administration of the preparation of the present invention, the administration site is irradiated with light in order to obtain a photochemical therapeutic effect. Lamps or lasers are used to illuminate various body parts (eg Van den Be
rgh, Chemistry in Britain, May 1986, pp 430-439). Irradiation can be performed using an optical fiber to a portion that cannot be directly irradiated. The length of time for which the light irradiation is performed depends on the symptoms, the nature of the photodynamic therapy formulation of the present invention and the dosage form. Generally, the exposure time is on the order of about 0.5 to 48 hours, for example about 1 to 10 hours. Irradiation is usually performed at a dose of 40 to 200 Joules / cm ² , for example 100 Joules / cm ² .

The wavelength of light used for irradiation can be selected so as to obtain a photochemotherapeutic effect more effectively. Usually, when porphyrins are used for photochemical treatment, irradiation is performed with light near the absorption maximum of porphyrins. For example, in the case of the prior art using ALA for photochemical treatment of skin cancer, a wavelength region of 350 to 640 nm, preferably 610 to 635 nm has been adopted. However, the photosensitization effect can be enhanced by selecting and irradiating a wider range of wavelengths that exceed the absorption maximum of porphyrin. This is thought to be due to the fact that when Pp and other porphyrins are exposed to light having a wavelength within its absorption spectrum, they are broken down into various photoproducts, particularly protoporphyrin (PPp). . PPp is a kind of chlorin and has a considerable photosensitizing effect. Further, the absorption spectrum further extends to the long wavelength side beyond the wavelength that Pp absorbs, and specifically extends to almost 700 nm (Pp hardly absorbs light exceeding 650 nm). Therefore, PPp is not excited at the wavelengths used in conventional photochemical treatment, and therefore a higher photosensitizing effect benefit cannot be obtained. It is known that irradiation is particularly effective when irradiation is carried out using light in the wavelength range of 500 to 850 nm, preferably 600 to 800 nm, more preferably 650 to 700 nm. It is particularly important to include wavelengths of 630 and 690 nm.
For clinical application of PDT for cancer treatment by photodynamic therapy , so far, it is suitable for photochemical treatment method or diagnostic method for disease or abnormality on epithelial coating surface, preferably mucosal coating surface of human or animal body, suitable for affected surface The focus was on photodynamic therapy formulations that could be applied. Such diseases or abnormalities include surface cancers. Cancer treatment by photodynamic therapy is a chemical reaction between oxygen caused by in-situ photosensitization in the body when light (photon) is supplied from the outside together with a photosensitizer sensitive to photon. Is based on the mechanism that singlet oxygen or free radicals produced by selenium destroy cancer cells or malignant tumor tissue. It has been pointed out that the photosensitizer porphyrin compound inherently has a tendency to localize in neoplastic tissue, but such tumor selectivity is limited (Patent Document 1). The uneven distribution of porphyrin compounds in the tumor tissue results in hypersensitivity that later appears as a side effect. The widespread use of photodynamic therapy has so far been limited due to the large size of the laser device and the long light shielding period to avoid photosensitivity.

  However, a second-generation drug with a short light-shielding period was developed, and small semiconductor lasers were approved for manufacturing for early lung cancer. Is expected to be enforced. Encapsulating porphyrin compounds in liposomes has also been shown to promote non-specific uptake into tumor tissue (Zhou C. et al., Photochem. Photobiol (1988), 48: 487-492). Therefore, the liposome preparation of the present invention can change the targeting ability for a tumor as needed, and is suitable for such use.

  Photodynamic therapy has no interaction with other therapies, i.e. surgery, radiation therapy, chemotherapy, etc. and can be performed in combination with these therapies. There is also a close interaction with hyperthermia.

  The formulations of the present invention are used as systemic chemotherapy. Suitable subjects are effective against several types of cancer including melanoma tumors, malignant brain tumors, ovarian cancer, breast cancer, skin cancer, lung cancer, esophageal cancer and bladder cancer.

The actual cancer photodynamic therapy is performed as follows. The liposome preparation of the present invention is intravenously administered to a patient in advance. Wait for the drug to accumulate in the tumor. Laser light having a wavelength that matches the excitation wavelength of the photosensitizing compound is irradiated 24 to 72 hours after the drug concentration difference between the cancer tissue and the normal tissue becomes maximum. The laser used in photodynamic therapy is almost 1 of the laser knife.
In addition to low output of / 100, liposomes encapsulating photosensitizing compounds can accumulate in cancer tissues, minimizing damage to normal tissues and selectively killing only cancer lesions. . Irradiate light with a wavelength that acts on the photoreactive substance and can reach the tissue sufficiently. For example, infrared rays having a wavelength of 630 nm are often used. Fiber may be used for irradiation for deep lesions. In the light irradiation, a laser beam such as an excimer excitation die laser is used as a light source. Laser light has problems such as a small treatment area and a large and expensive apparatus body. A metal halide lamp for photodynamic therapy, which has a large treatment area and is inexpensive and compact, has been developed and may be used.

  The photodynamic therapy preparation of the present invention is also preferably used in intraarterial infusion chemotherapy which is local chemotherapy. In arterial chemotherapy, angiography identifies the arteries leading to the tumor lesion. Since the artery is often branched in the middle, the catheter is made to reach as close to the tumor lesion as possible under angiography. If there are multiple vegetative vessels, select the best vessel possible (selective intra-arterial chemotherapy). A microcatheter is inserted into the vegetative blood vessel of the tumor, and a high-concentration liposome preparation is introduced directly through this catheter in a short time. One-shot arterial injection is used in combination with surgery and radiation therapy.

Therefore, the cancer treatment method according to the present invention preferably comprises the following steps.
i) Insert a catheter from the artery leading to the cancer's nutritional blood vessels as close to the cancer tissue as possible,
ii) The photodynamic therapy preparation is directly injected into the cancer tissue through the introduction catheter.
iii) Inserting a diode laser fiber into the cancer tissue through the introduction catheter,
iv) treating said cancer tissue by irradiation with light having a wavelength of 600-800 nm, preferably 650-700 nm. Arterial chemotherapy is suitable for localized cancer tissue with few metastases, for example It is preferably implemented for solid cancers such as brain tumors, head and neck cancer, lung cancer, breast cancer, liver cancer, cervical cancer, and bladder cancer. According to such intra-arterial chemotherapy, liposome preparations containing liposomes are released through the catheter into blood vessels that nourish the cancer tissue in the vicinity of the cancer lesions. It reaches the cancer lesion directly without being leaked from the wall, affected by dilution, or captured by reticuloendothelial cells. In a preferred embodiment, the lipid membrane of the liposome may be PEGylated as described above, but it is desirable that the average particle size is around 0.8 μm. Liposomes consisting essentially of a single membrane or several membranes can be administered with a small number of liposomes because the encapsulating capacity is large and a large amount of photosensitizing compound can be encapsulated. In this case, the photosensitizing compound encapsulated in the liposome may be a photosensitizing substance having low tumorigenicity and tissue permeability.

The arterial local administration method includes a technique for identifying a nutrient blood vessel to a cancer lesion by angiography and guiding a catheter. Therefore, it is convenient for the intraarterial injection chemotherapy that the liposome preparation of the present invention contains a contrast agent used for angiography and cancer tissue imaging in the liposome. Specifically, based on a dosage form that embodies the DDS concept, a preparation combining an X-ray contrast agent specialized for cancer diagnosis and a photosensitizing compound is conceivable. Since this preparation also contains a contrast agent, tumors can be diagnosed by contrast enhancement. Liposomes with an average particle size of 0.8 μm
Disperses within the tumor tissue and stays for a long time without causing leakage from the tumor-feeding blood vessel wall with increased permeability.

Contrast agents suitable for the liposome preparation of the present invention include water-soluble nonionic iodine compounds and oil-based contrast agents for X-ray examination. These contrast agents may be encapsulated in the liposome separately or together with the photosensitizing compound. In addition to water-soluble iodine compounds, lipiodol having tumor affinity can be further improved in delivery to cancer lesions by encapsulating in liposomes having an average particle size of 0.8 μm. Preferred iodo compounds are iomeprol, iopamidol, iohexol, iopromide, ioxirane, iotasulfur, iotrolane or iodixanol. Examples of oil-based contrast agents include iodinated poppy oil fatty acid ethyl ester. Particularly for hepatocellular carcinoma and the like, lipiodol with high hepatic tumor accumulation is suitable. By administering a liposome preparation encapsulating a photosensitizing compound and a contrast agent through a catheter placed in a blood vessel, tumor imaging and cancer treatment can be performed.

Moreover, a combination of a photodynamic therapy formulation of liposomes containing phospholipids having a transition temperature and cancer hyperthermia is possible, and it is expected that the therapeutic effect will be enhanced. When a photodynamic therapy preparation containing liposomes encapsulating a photosensitizing compound is administered to a patient and then heated from outside the body after a certain period of time, local hyperthermia using microwaves or electromagnetic waves is mainly performed. Is called. Alternatively, a method of heating by putting an instrument in a lumen such as the esophagus, rectum, uterus, bile duct, or a method of heating by inserting several electrode needles into cancer tissue is also performed. In any case, liposomes that contain a phospholipid having a transition temperature and are composed of one or several membranes change the structure of the liposome membrane when heat is applied, and the encapsulated drug is released outside the liposome. It becomes easy to be done. Thereafter, treatment is performed by irradiating with laser light as described above.
[The invention's effect]
The photodynamic therapy preparation of the present invention has i) high selectivity to malignant tumor tissue, ii) high absorption of spectrum at a wavelength of 600 nm or more, iii) high excretion from the human body after administration, iv) minimal side effects and toxicity It has the characteristic. In addition, the preparation of the present invention enables efficient delivery of the photosensitizing compound to the affected area, and does not substantially contain a chlorinated solvent and other organic solvents unlike the conventional liposome preparation. This further reduces the burden on the subject.
[Example]
The present invention will be more specifically described by the following examples. However, the examples are intended to be described by way of examples, and are not intended to limit the scope of the present invention in any way.

A mixture of 86 mg of dipalmitoylphosphatidylcholine (DPPC), 38.4 mg of cholesterol, and 19.2 mg of PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and the inside of the autoclave was heated to 60 ° C. Then, 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 50 kg / cm ² , is increased to 120 kg / cm ² by reducing the volume in the autoclave, carbon dioxide is brought into a supercritical state, and lipids are stirred while stirring. Dispersed and dissolved. Subsequent production was performed in a dark room equipped with a red coral. While stirring the above system, a solution obtained by adding 15 mL of Japanese Pharmacopoeia 5% glucose injection solution per 75 mg of porfimer sodium was continuously injected with a metering pump. After completion of the injection, the system was depressurized to discharge carbon dioxide to obtain a liposome dispersion containing a porfimer sodium solution. The obtained dispersion was heated to 60 ° C., passed through a cellulosic filter manufactured by Advantech and a 1.0 μm filter, and further filtered under pressure using 0.45 μm, 0.3 μm and 0.2 μm filters. The obtained liposome had an average particle size of 0.12 μm and an encapsulation rate of 15%.

The obtained sample was sealed in a Spectrapro Biotech RC dialysis tube, and immersed in a large amount of Japanese Pharmacopoeia 5% glucose injection solution to perform dialysis to remove porfimer sodium that could not be taken into the liposomes. A liposomal preparation of the present invention of porfimer sodium 0.75 mg / mL was obtained. Such a photodynamic therapy preparation can be used in a usual photodynamic treatment method.

Claims (12)

A photodynamic therapy preparation comprising liposomes containing at least one or more drugs including a photosensitizing compound and substantially free of an organic solvent in a lipid membrane or an internal aqueous phase thereof. The liposome is prepared by mixing a lipid membrane component constituting a lipid membrane and carbon dioxide in a supercritical or subcritical state under the condition that at least one compound having a polyethylene glycol (PEG) group is present. The photodynamic therapy formulation according to claim 1.   The photodynamic therapy preparation according to claim 1 or 2, wherein the compound having a polyethylene glycol group is PEG-phospholipid.   The photodynamic therapy preparation according to any one of claims 1 to 3, wherein the lipid membrane is a liposome substantially consisting of a single membrane or several membranes.   The photodynamic therapy preparation according to any one of claims 1 to 4, wherein the photosensitizing compound is porfimer sodium.   The photodynamic therapy preparation according to any one of claims 1 to 5, wherein the preparation is directly delivered to an affected area through a catheter.   The photodynamic therapy preparation according to any one of claims 1 to 6, which is used for photodynamic therapy of cancer.   8. The photodynamic therapy preparation according to claim 7, wherein thermotherapy is used in combination.   8. The photodynamic therapy preparation according to claim 1, further comprising a contrast substance as the drug. Under conditions where at least one compound having a polyethylene glycol group is present, a compound selected from at least one of a cationic lipid and a sterol together with a phospholipid as a lipid membrane component, and further, if necessary, a lipophilic drug After dissolving or dispersing in supercritical or subcritical carbon dioxide, a solution or suspension containing at least one drug is introduced to form micelles, and then water is added to discharge carbon dioxide. And the manufacturing method of the photodynamic therapy formulation in any one of Claims 1-9 including producing the liposome which contains those drugs inside. 11. The photodynamic therapy preparation according to claim 10, wherein the compound having a polyethylene glycol group is used in a ratio of 0.01 to 1% by mass with respect to carbon dioxide to be in a supercritical state or a subcritical state. Method. i) Insert a catheter from the artery leading to the cancer's nutritional blood vessels as close to the cancer tissue as possible,
ii) directly injecting the photodynamic therapy preparation into the cancer tissue through the introduction catheter, and iii) inserting a diode laser fiber into the cancer tissue through the catheter,
iv) A method for treating cancer using a photodynamic therapy preparation, comprising treating the cancer tissue by irradiation with light having a wavelength of 600 to 800 nm.