JPH11509834A - Method for making liposomes containing hydro-monobenzoporphyrin photosensitizer - Google Patents

Abstract

  (57) [Summary] A method of making a pharmaceutical composition containing liposomes, wherein the liposomes comprise a hydro-monobenzoporphyrin photosensitizer and a mixture of egg phosphatidylglycerol ("EPG") and dimyristoylphosphatidylcholine ("DMPC"). Containing a therapeutically acceptable amount of a phospholipid comprising the following steps: a. Combining a photosensitizer and a phospholipid with a phospholipid in a molar ratio of about 1: 7.0 or more in the presence of an organic solvent; b. Removing the organic solvent to form a lipid membrane; c. Hydrating the lipid membrane with an aqueous solution at a temperature below 30 ° C. to form a crude liposome containing a photosensitizer-phospholipid complex; and d. Homogenizing or reducing the particle size of the crude liposomes at a temperature below about 30 ° C. to a particle size range below about 300 nm.

Description

FIELD OF THE INVENTION This invention relates to improved pharmaceutical compositions comprising liposomes incorporating porphyrin photosensitizers. and methods for making these liposomes. In particular, the present invention relates to pharmaceutical liposomal compositions containing hydro-monobenzoporphyrin photosensitizers and mixtures of phospholipids containing egg phosphatidylglycerol (“EPG”) and dimyristoyl phosphatidylcholine (“DMPC”) (photosensitizers). A pharmaceutical liposomal composition containing a sensitizer:phospholipid having a molar ratio of about 1:7.0 or greater is described. The liposomes are made in such a way that the particle size ranges from about 150-300 nm.

Photosensitized liposomal compositions are useful for mediating destruction of unwanted cells or tissues or other unwanted substances by irradiation or detecting their presence through fluorescence. Particularly preferred hydro-monobenzoporphyrin photosensitizers in the practice of this invention include those having one or more light absorption maxima in the range of 670-780 nm. Description of the Related Art The use of porphyrin compounds, and in particular hematoporphyrins and hematoporphyrin derivatives (HPDs), has long been known to be useful systemically for the treatment and diagnosis of malignant cells when combined with light. It's here. Porphyrins have a natural tendency to "localize" to malignant cells, which absorb certain wavelengths of light when illuminated, thus providing a means of detecting tumors by the location of fluorescence. Thus, porphyrin-containing formulations are useful for the diagnosis and detection of tumor tissue (see, eg, "Porphyrin Photosensitization," Plenum Press (Kessel et al., eds. 1983)). In addition, porphyrins have the ability to exhibit cytotoxic effects in cells or other tissues in which they are localized when exposed to light of appropriate wavelengths (see, eg, Diamond et al., Lancet , 2 :1175-77 (1972)). See Dougherty et al., The Science of Photo Medicine, 625-38 (Regan et al. eds. 1982); and Dougherty et al., Cancer: Principles and Practice of Oncology, 1836-44 (DeVita Jr. et al. eds. 1982). do). It is postulated that the cytotoxic effect of porphyrins is due to the formation of singlet oxygen when exposed to light. (Weishaupt et al., Cancer Research , 36 :2326-29 The successful treatment of AIDS-related Kaposi's sarcoma of the mouth with sarcomas was described in Schwietzer et al., Otolaryngology--Head and Neck Surgery , 102 :639-49 (1990).

In addition to systemic use for diagnosis and treatment of tumors, porphyrins have a variety of other therapeutic applications. For example, photosensitizers are disclosed in US Pat. Nos. 4,517,762 and and 4,577,636 for the detection and detection of atherosclerotic plaques. It is useful for the treatment of U.S. Pat. Nos. 4,500,507 and 4,485,806 disclose tumor Describes the use of radiolabeled porphyrin compounds for imaging. be. Porphyrin compounds are also disclosed in Japanese Patent No. 4,753,958, Used topically to treat a variety of skin disorders.

A number of porphyrin photosensitizer formulations have been disclosed for therapeutic use. Formulations of photosensitizers that are widely used in the early stages of photodynamic therapy for both detection and therapy are crude derivatives of hematoporphyrin, also referred to as hematoporphyrin derivatives ("HPD") or Lipson derivatives. ) and is prepared as described by Lipson et al., J. Natl. Cancer Inst. , 26 :1-8 (1961). Purified forms of the active ingredient of HPD are pH adjusted as described by Dougherty and co-inventors in U.S. Pat. Nos. 4,649,151; 4,866,168; Prepared by flocculation followed by recovery of flocculate. child used clinically.

Of particular interest in connection with the present invention are one or more A group of modified porphyrins with optical absorption maxima (“green porphyrins”) (known as Gp). These Gp compounds are cell-targeted Provides toxicity at lower concentrations than required for hematoporphyrin or HPD is shown. Gp compounds are protoporphyrins and diverse under appropriate conditions. can be obtained using the Diels-Alder reaction with various acetylene derivatives. Preferred form of Gp The state is a hydro-monobenzoporphyrin derivative (“BPD”). Gp and BPDylation The preparation and use of compounds are disclosed in US Pat. Nos. 4,920,143 and 4,883,790. are shown and incorporated herein by reference into the disclosure of the present application.

Although porphyrin compounds have the natural ability to localize to neoplastic tissues while being cleared from normal surrounding tissue, the selectivity of porphyrin photosensitizers is still somewhat limited. Because tumor tissue generally contains many different components, such as malignant cells, vasculature, macrophages, and fibroblasts, the distribution of photosensitizers within tumor tissue can be very heterogeneous. be. This is especially true for photosensitizers that are not uniform and contain mixtures of components with varying degrees of water or oil solubility. Zhou et al., Photochemistry and Photobiology , 48 :487-92 (1988). The low selectivity of some of these agents as tumor-localizing agents can lead to side effects such as undesirable systemic hypersensitivity to light. An active area of research is therefore to increase the tumor selectivity of known porphyrin photosensitizers and to identify porphyrin photosensitizers that may exhibit greater tumor selectivity.

In general, the more lipophilic a photosensitizer, the greater its ability to target tumors. Spikes et al., Lasers in Medical Science , 2 :3, 3-15 (1986).

Liposome encapsulation of certain drugs prior to administration has recently been shown to significantly affect the pharmacokinetics, tissue distribution, metabolism and efficacy of therapeutic agents. In an effort to increase the tumor selectivity of porphyrin photosensitizers, porphyrin compounds were incorporated into unilamellar liposomes, resulting in greater accumulation of the photosensitizer in both cultured malignant cells and experimental tumors in vivo. and held longer. Jori et al., Br. J. Cancer , 48 :307-309 (1983); Cozzani et al. , In Porphyrins in Tumor Phototherapy , 177-183, Plenum Press (Andreoni et al., eds., 1984). This additional effect of targeting to tumor tissue by liposome-associated porphyrins is due in part to the specific delivery of phospholipid vesicles to serum lipoproteins. Serum lipoproteins have been shown to preferably interact with hyperproliferative tissues such as tumors through receptor-mediated endocytosis. In this manner, the selectivity of porphyrin uptake by tumors is increased compared to the photosensitizer dissolved in aqueous solution. See Zhou et al., supra.

Accordingly, hematoporphyrin and hematoporphyrin dimethyl ester were formulated in unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) and liposomes of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC). Zhou et al., supra; Ricchelli, New Directions in Photodynamic Therapy , 847 :101-106 (1987); Milanesi, Int. J. Ra. and tetrabenzoporphyrin were formulated in liposomes composed of egg phosphatidylcholine (EPC). Johnson et al., Proc . Photodynamic Therapy : Mechanisms II , Proc. SPIE-Int. Soc. Opt. Eng., 1203 :266-80 (1990). Additionally, lyophilized pharmaceutical formulations have been made containing a porphyrin photosensitizer, a di- or polysaccharide, and one or more phospholipids (eg, EPG and DMPC).

These formulations, when reconstituted with a suitable aqueous vehicle, yield a porphyrin photosensitizer. Liposomes containing an effective amount are formed and this is described in Desai et al., Feb. 6, 1992. U.S. patent application Ser. No. 07/832,542 (now filed under 37 CFR 1.62). Abandoned by continuing application).

The process of Desai et al. up to liters), efficiency may be reduced due to loss of product during the sterile filtration process. Some reduction in force is observed. This loss is related to the particle size reduction process. do. Therefore, due to the importance of photodynamic therapy in cancer therapy, Not only can these photosensitizers be selectively delivered to target tissues, they are also easy to produce on a large scale. There is also a continuing need to identify new photosensitizer formulations.

Farmer et al., U.S. Pat. No. 4,776,991, disclose extensive encapsulation of hemoglobin into liposomes having a narrow liposome size distribution, which, as phospholipids, include (1) hydrogenated soy phosphatidylcholine (“HSPC”; suitably (85% distearoylphosphatidylcholine and 15% dipalmitoylphosphatidylcholine); (2) cholesterol; (3) negatively charged DMPC; line 69). These lipids are mixed in chloroform to form a solution; the chloroform is removed by evaporation to form a lipid film; and sterile hemoglobin is gently washed at 35°C (30-37°C) for 45 minutes. Add to membrane with stirring to form multilamellar liposomes. Rotational agitation of the liposomes is continued for 10-16 hours at 4°C (2-6°C) and the liposomes are passed through a Microfluidizer ^™ to disrupt multilamellar liposomes and produce large unilamellar liposomes. The interaction chamber of the Microfluidizer ^™ is maintained at 5-7°C. (Column 4, lines 1-14; Col. 7, lines 3-9 and 19-21.) However, the lipids selected to make the liposomes were passed through a standard 0.221 m sterile filter. There should be a temporary shrinkage due to the hyperosmolar shock with saline applied prior to sterilization by pressure filtration of . (Col. 4, lines 18-22; col. 9, lines 55-58.) Kappas et al., U.S. Pat. ”) together with the preparation of liposomes containing metalloporphyrins. EPC is dissolved in chloroform, metalloporphyrin is added, and the solution is evaporated to dryness. Phosphate-buffered saline is used at room temperature to hydrate the lipid film and the mixture is "vortexed vigorously." Solids are collected by centrifugation at 4°C. The weight ratio of EPC to metalloporphyrin can be greater than ten. (Kappas et al., column 6, lines 46-65.) However, Kappas et al. recommend sonicating the resulting metalloporphyrin liposomes prior to injection to prevent the formation of large aggregates. ing. (Col. 6, line 66 to col. 7, line 1.) Schneider et al., U.S. Pat. Disclosed are liposomal formulations. (Schneider et al., column 3, lines 52-54.) However, the presence of certain synthetic lipids is required. (Column 2, lines 51-66.) For example, in Example 21, columns 13 and 14, 50 g of a mixture of two such synthetic lipids was dissolved in tertiary butanol, which was then added to 0.5 g zinc-phthalocyanine solution. Using a dynamic mixer, this solution was mixed into 10 liters of lactose medium chilled to 4° C. to produce a blue, slightly opalescent dispersion. Separation and isolation of large liposomes from small liposomes, if necessary, is performed by conventional separation methods such as gel filtration or sedimentation. (Col. 7, lines 11-17.) Thus, DMPC/EPG liposomes containing photosensitizers can be effectively filtered through a standard 0.22 Tm sterile filter. There continues to be a need for a large-scale method of producing the synthetic lipids of Kappas et al., with particle sizes small enough to be easily sterile filtered through the modalities, and without the need to prepare the synthetic lipids of Kappas et al. SUMMARY OF THE INVENTION The present invention includes methods for making pharmaceutical compositions containing liposomes. The liposomes contain a therapeutically acceptable amount of a hydro-monobenzoporphyrin photosensitizer and a mixture of phospholipids including egg phosphatidylglycerol (“EPG”) and dimyristoylphosphatidylcholine (“DMPC”).

Methods of making such liposomes include the following steps: a. A photosensitizer and a phospholipid are mixed in an organic solvent with a phospholipid molar ratio of about 1:7.0 or more. combining in the presence of a medium; b. removing the organic solvent to form a lipid membrane; c. The lipid membrane was hydrated with an aqueous solution at a temperature below 30°C to form a photosensitizer-phospholipid complex. forming crude liposomes containing the body; and d. Reduce the particle size of the crude liposomes to about 150-300 nm at temperatures below about 30°C. homogenizing or reducing to a particle size range of .

Hydration and homogenization/reduction steps of the present invention, "c." d. ” is preferably higher than the glass transition temperature of the photosensitizer: DMPC/EPG mixture composite performed at low temperatures.

By maintaining the hydration temperature and homogenization/reduction step below 30°C, the production of smaller particle sizes was not expected. In fact, the present invention goes against the common knowledge that small grain size is achieved by raising these temperatures rather than lowering them. See, for example, M. Lee et al., "Flow field - size distribution of liposomes by flow fractionation, " J. Pharm . & Biomed. Analysis , 11:10 , 911-20 (1993), equation (6) shows that particle size 'd' is inversely related to temperature 'T' and Figure 6b is for liposome preparation I (approximately 70°C). (prepared at about 23° C.) has a smaller particle size than Preparation II (prepared at about 23° C.).

The present invention also contemplates the pharmaceutical composition per se, which comprises hydro-monobenzo A porphyrin photosensitizer and a mixture of DMPC and EPG phospholipids were used as the photosensitizer - containing liposomes comprising phospholipids in a phospholipid molar ratio of about 1:7.0 or greater, This liposome then has a particle size of about 150-300 nm.

These liposomal compositions provide near 100% encapsulation of hydro-monobenzoporphyrin photosensitizers. Hydro-monobenzoporphyrin photosensitizers can be expensive and usually require complicated synthetic procedures to produce. Thus, no rework is required and there is little photosensitizer waste. Furthermore, due to their small particle size, the liposomes of the present invention exhibit improved filterability, important in large-scale batch production of 500 ml to liter units or larger, as well as improved light transmission. Shows sensitizer retention efficacy. BRIEF DESCRIPTION OF THE FIGURES Other objects, features and advantages arise from the following description of various embodiments and the accompanying drawings, wherein FIG. Gp) Indicates the structure of the compound.

FIG. 2 shows hydro-monobenzoporphyrin derivatives of formulas 3 and 4 in FIG. Figure 4 shows the structures of four preferred forms of (BPD).

FIG. 3 is a graphical representation of the relationship of filtration capacity to lipid concentration.

FIG. 4 shows a proposed mechanism for the effect of temperature on the aggregation of hydro-monobenzoporphyrin photosensitizers. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to pharmaceutical liposomal formulations of hydro-monobenzoporphyrin photosensitizers for use in photodynamic therapy or diagnosis of tumors, or for various other therapeutic applications. . Liposomes are completely closed lipid bilayers containing an entrapped volume of water. Liposomes are typically formed upon addition of an aqueous solution to a dry lipid film.

The liposomes of the present invention are unilamellar vesicles with a single bilayer membrane, or multiple can be multilamellar vesicles with bilayer membranes, each bilayer membrane adjacent to the aqueous Separated by a membrane. The liposome bilayer membrane consists of a hydrophobic "tail" region and a hydrophilic It consists of two lipid monolayers with a 'head' region of . The structure of the bilayer membrane is The hydrophobic (non-polar) “tail” of the thin monolayer orients itself toward the middle of the bilayer membrane. while the hydrophilic “head” becomes oriented itself toward the aqueous phase. there is Either unilamellar or multilamellar or other types of liposomes are suitable for the practice of the invention. can be used.

In liposome-drug delivery systems, hydrophilic therapeutic agents are entrapped in the aqueous phase of the liposomes. can be loaded and then administered to the patient. Alternatively, if the therapeutic agent is lipophilic When combined, it can associate with lipid bilayer membranes. Liposomes “target” drugs to active sites. or to solubilize hydrophobic drugs for parenteral administration. can be used for Typically, the hydro-monobenzoporphyrin photoenhancement of the invention Sensitizers are relatively hydrophobic and form stable photosensitizer-lipid complexes .

Liposomes of the Invention Deliver Hydro-Monobenzoporphyrin Photosensitizers have certain properties that make them well suited to formed by the present invention Although the photosensitizer-liposome complex is stable in vivo, liposomes with When administered in vivo, photosensitizers rapidly enter the bloodstream associated with serum lipoproteins. It is "fast breaking" in that it is released quickly. this is the light It is believed that the sensitizer is prevented from accumulating in non-target tissues such as the liver.

Photosensitizer Photosensitizers useful in the practice of this invention are disclosed in U.S. Pat. Nos. 4,920,143 and 4. The hydro-monobenzoporphyrins (so-called "green porphyrin” or “Gp” compounds). Typically these compounds are have one or more optical absorption maxima between about 670 and 780 nm and are practically insoluble in water either soluble (less than 1 mg/ml) or insoluble in water. Preferably, Gp is shown in FIG. One of Formulas 1-6, mixtures thereof, and metallated and labeled forms thereof selected from the group consisting of these compounds having

In FIG. 1, R ¹ and R ² are carboalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and -CONR ⁵ CO- (wherein ^R5 is aryl (6-10C) or alkyl (1-6C)). However, preferably each of R ¹ and R ² is carboalkoxy (2-6C).

In Figure 1, ^R3 can independently be carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone thereof, or is alkyl (1-6C). Preferably ^R3 is _-CH2CH2COOH or _a salt, amide, ester or acylhydrazone thereof.

^R4 is _-CHCH2 ; ^-CHOR4' (wherein R4 ^' is H or alkyl (1-6C), optionally substituted with a hydrophilic group); -CHO; -COOR4 ^' ; -CH(OR4 ^' ) _CH3 ; -CH( _OR4 ^' ) ^CH2OR4' ; -CH(SR4 ^' ) _CH3 ; -CH( ^NR4'2 _{) CH3} ; -CH(CN)CH ₃ ; -CH(COOR4 ^' ) _CH3 ; -CH(OOCR4 ^' ) _CH3 ; -CH(halo) _CH3 ; -CH(halo) _CH2 (halo); direct or indirect derivatives of vinyl groups or R ⁴ consists of a 1-3 tetrapyrrole-type nucleus of formula -LP, where -L- is and P is a second Gp or other porphyrin group, which is one of formulas 1-6 but lacks ^R4 and shown to be occupied by ^R4 Connected to L through a position. When P is another porphyrin group, preferably P has the formula: where: each R is independently H or lower alkyl (1-4C); two of the four bonds not occupied by adjacent rings are attached to ^R3 ; the remaining bonds not occupied by adjacent rings is attached to ^R4 ; and the other bond is attached to L; provided that when ^R4 is _-CHCH2 , both ^R3 groups cannot be carboalkoxyethyl. The preparation and use of such compounds are disclosed in US Pat. Nos. 4,920,143 and 4,883,790, incorporated herein by reference.

Even more preferred are hydro-monobenzoporphyrin compounds, designated as benzoporphyrin derivatives (“BPD”). BPDs are hydrolyzed or partially hydrolyzed forms of the rearrangement products of Formulas 1-3 or Formulas 1-4, wherein one or both of the protected carboxyl groups of ^R3 are hydrolyzed. decomposed. Especially preferred is the compound referred to as BPD-MA in Figure 2, which has two equally active regioisomers.

Many desirable hydro-monobenzoporphyrin photosensitizers (e.g. BPD-MA) are , is not only insoluble in water at physiological pH, but also (1) a pharmaceutically acceptable aqueous - organic co-solvents, (2) aqueous polymer solutions, and (3) surfactant/micelle solutions , is insoluble. However, such photosensitizers use liposomal compositions. can be "solubilized" in a form suitable for parenteral administration. For example, BPD- MA was prepared in an aqueous solution at a concentration of about 2.0 mg/ml using an appropriate mixture of phospholipids. "Solubilized" to form encapsulated liposomes.

Lipid The liposomes of the present invention contain the commonly encountered lipid dimyristoyl phosphatidyl A mixture of choline (“DMPC”) and egg phosphatidylglycerol (“EPG”) including. The presence of DMPC suggests that DMPC is an insoluble hydro-monobenzoporphyrin photosensitizer. to solubilize agents in lipid bilayer membranes and form liposomes capable of encapsulation, It is important because it is the major component in the composition. The presence of EPG indicates that this lipid This is important because a negatively charged, polar head group can prevent liposome aggregation.

In addition to DMPC and EPG, other phospholipids may also be present. Appropriate further Examples of phospholipids, which can also be incorporated into the liposomes of the invention, are dipalmi Toylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine containing phosphatidylcholine (PC), including mixtures of phosphatidylcholines (DSPC). suitable phosph Examples of atidylglycerol (PG) are dimyristoyl phosphatidylglycerol (DMPG), DLPG, etc.

Another type of suitable lipid that can be included is phosphatidylethanolamine (PE ), phosphatidylic acid (PA), phosphatidylserine, and phosphatidyl Nositol.

of hydro-monobenzoporphyrin photosensitizers to DMPC/EPG phospholipid mixtures. The molar ratio can be as low as 1:7.0 or higher proportions of phospholipids (e.g. 1:7.5). Preferably, this molar ratio is 1:8 or more. less phospholipid (eg 1:10, 1:15, or 1:20). This molar ratio is Depending on the exact photosensitizer used, most hydro-monobenzoporphyrins A sufficient number of DMPC and EPG lipid molecules to form stable complexes with photosensitizer molecules guarantee the existence of When the number of lipid molecules is insufficient to form a stable complex , the lipophilic phase of the lipid bilayer membrane is saturated with photosensitizer molecules. Then the process conditions Any small change in the Forced leakage to the outside of the parcel, onto the surface of the lipid bilayer membrane, or even into the aqueous phase can be made

This is practical if the concentration of hydro-monobenzoporphyrin photosensitizer is high enough. Sometimes it precipitates from the aqueous layer and can promote aggregation of the liposomes. not encapsulated The more photosensitizer present, the higher the degree of aggregation. about increased aggregation As a result, the average particle size increases and aseptic or sterile filtration becomes more difficult. world Thus, as described in Example 1 below, even minor changes in molar ratios can be applied to the present invention. It can be important in achieving the improved filtration capabilities described.

Thus, small increases in lipid content can significantly increase the filterability of liposomal compositions by increasing the ability to form and maintain small particles. This is particularly advantageous when working with large volumes of 500 ml, 1 liter, 5 liters, 40 liters or more, as opposed to smaller batches of about 100-500 ml or less. It is believed that this volume effect occurs because larger homogenizing devices tend to provide less effective agglomeration than can be easily achieved on a small scale. For example, a larger size Microfluidizer ^™ has a less effective interaction chamber than a smaller size.

1.05:3:5 BPD-MA:EPG:DMPC molar ratio (i.e. photosensitizer:phospholipid 1 : slightly less than 8.0 phospholipids) is acceptable in small batches up to 500 ml It can provide maximum filtering capability. However, making a larger volume of the composition In the case of higher phospholipid molar ratios, assurance of reliable sterile filtration capability is assured. provide more. In addition, large-scale A substantial loss of capacity common to mock batches can thus be avoided.

Cryoprotectants and tonicity agents Any known to be useful in the art of preparing lyophilized formulations cryoprotectants (e.g. di- or polysaccharides, or other bulky agents such as lysine) ing agents) may be used in the present invention. In addition, to maintain equimolar concentrations with body fluids, An isotonicity agent typically added for this purpose can be used. In a preferred embodiment, two Sugars or polysaccharides are used and function as both cryoprotectants and isotonic agents. can

In a particularly preferred embodiment, phospholipids, DMPC and EPG, and disaccharides or certain formulations of polysaccharides have particularly narrow particle size distribution of liposomes A liposomal composition is formed. If the process of hydrating the lipid membrane is prolonged, more Large liposomes tend to form or the photosensitizer may begin to precipitate. be. Addition of disaccharides or polysaccharides results in instantaneous hydration and drug-phospholipid complex provides the most extensive surface area for placing thin films of This thin film allows faster water and thus the shape when the liposomes are first formed by the addition of an aqueous phase. The resulting liposomes have smaller and more uniform particle sizes. this offers significant advantages in terms of ease of manufacture.

However, when saccharides are present in the compositions of the invention, the saccharides are present in the dry lipid film After formation, it may also be added as part of the aqueous solution used in hydration. It is possible. In a particularly preferred embodiment, the saccharide is a dry product of the invention during hydration. Added to the dry lipid film.

Di- or polysaccharides are preferred over monosaccharides for this purpose. of the present invention To keep the osmotic pressure of the liposomal composition similar to that of blood, No more than 4-5% monosaccharides could be added. In contrast, about 9-10% of Sugars can be used without creating unacceptable osmotic pressure. higher amount of disaccharide The genus provides a higher surface area, resulting in a smaller size during hydration of the lipid film. grains are formed.

Accordingly, preferred liposomal compositions of the present invention include a photosensitizer and DMPC and It contains a mixture of EPG phospholipids plus di- or polysaccharides. disaccharide or Polysaccharides, if present, are preferably lactose, trehalose, maltose , the group consisting of maltotriose, palatinose, lactulose, or sucrose is selected from Lactose or trehalose are preferred. even more preferred Alternatively, the liposomes contain lactose or trehalose.

Also, disaccharides or polysaccharides, if present, should be mixed with 0.5-6.0 DMPC/EPG phospholipids. with a preferred ratio of about 10-20 saccharides (even more preferably 1.5-4 at a ratio of about 10 to .0) respectively. In one embodiment, the preferred A new formulation is lactose or trehalose and a mixture of DMPC and EPG. with a concentration ratio of about 10:0.94-1.88 to about 0.65-1.30, respectively, but limited to not specified.

The presence of disaccharides or polysaccharides in the composition is extremely small and narrow particles Not only does it tend to produce liposomes with a range of sizes, it also The mode in which nobenzoporphyrin photosensitizers are effective (i.e., exposure reaching 80-100% The present invention provides a liposome composition that can be incorporated particularly stably with high encapsulation efficiency. moreover, Liposomes made with sugars typically have improved physical and chemical stability , and therefore these are the photoenhancers of the incorporated hydro-monobenzoporphyrins. Sensitizer compounds can be stored in reconstituted liposomal suspensions without leakage during long-term storage. It can be held either as a suspension or as a cold dry powder.

Excipient Other additional ingredients may include small amounts of harmless, ancillary substances in the liposomal composition. . For example, antioxidants (e.g. butylated hydroxytoluene, α-tocopherol and ascorbyl palmitate) and pH buffers (e.g. phosphates and and glycine).

Preparation Selected hydro-monobenzoporphyrins as described herein Photosensitizer-containing liposomes contain photosensitizers and DMPC and EPG phospholipids ( and any other additional excipients such as phospholipids or antioxidants) in an organic solvent can be prepared by combining in the presence of Suitable organic solvents are any volatile organic solvents such as diethyl ether, acetone, methylene chloride, loform, piperidine, piperidine-water mixture, methanol, tert-butanol , dimethylsulfoxide, N-methyl-2-pyrrolidone, and mixtures thereof.

Preferably, the organic solvent is immiscible with water, such as methylene chloride, but Immiscibility with water is not required. In any case, the selected solvent should be It should not only be capable of dissolving lipid membrane constituents, but also to a significant degree Should not react with (or otherwise adversely affect) ingredients.

The organic solvent is then removed from the resulting solution to form a dry lipid film by any known laboratory technique that is not harmful to the dry lipid film and photosensitizer. Preferably, the solvent is removed by placing the solution under vacuum until the organic solvent evaporates. The solid residue is the dried lipid film of the invention. The thickness of the lipid film is not critical, but usually varies from about 30 to about 45 mg/ ^cm2 , depending on the amount of solid residue and the total area of the glass wall of the flask. Once formed, the membrane can be kept for an extended period of time (preferably no more than 4-21 days) before hydration. On the other hand, the temperature during retention of the lipid film is also not a critical factor, although the temperature is preferably below room temperature, most preferably in the range of about -20 to about 4°C.

The dried lipid film is then treated with an aqueous solution (preferably containing disaccharides or polysaccharides) and homogenized to form particles of desired size. hydration Examples of useful aqueous solutions used during the process are sterile water; Phosphate-buffered (pH 7.2-7.4) sodium chloride solution without nesium; 9.75 % w/v lactose solution; lactose-saline solution; 5% glucose solution; including any other physiologically acceptable aqueous solution of one or more electrolytes. but However, preferably the aqueous solution is sterile. body of aqueous solution used during hydration The product can vary widely, but should be no more than about 98% and no more than about 30-40%. Shouldn't be. A typical useful volume range is about 75% to about 95%, preferably is about 85% to about 90%.

Upon hydration, crude liposomes are formed, which contain a therapeutically effective amount of A lo-monobenzoporphyrin photosensitizer-phospholipid complex is incorporated. "Treatment effective The "amount" depends on the tissue to be treated and the target-specific ligand (eg, antibody or or immunologically active fragment). can change to The various parameters used in selective photodynamic therapy are interrelated. It should be noted that Therefore, a therapeutically effective amount may also include other parameters. parameters (e.g., light flux, dose, duration, and and time interval between administration of photosensitizer and treatment irradiation).

In general, all these parameters are adjusted to significantly damage the surrounding tissue. tissue that is considered undesirable (e.g., neovascular tissue or tumor). without significant damage to surrounding tissue) , allowing observation of such undesirable tissue.

Typically, a therapeutically effective amount is about 0.1 to about 20 mg/kg, preferably about 0.15 to 2.0 mg/kg , and even more preferably within the range of about 0.25 to about 0.75 mg/kg. The amount is such that it produces a dose of porphyrin photosensitizer. the mixture by conventional means If the gel becomes so thick that it cannot be handled or administered to a subject, Preferably, the w/v concentration of photosensitizer in the composition ranges from about 0.1 to about (8.0-10.0) g/L. It is surrounded. Most preferably, the concentration is about 2.0-2.5 g/L.

The hydration step is carried out at a temperature not exceeding about 30°C (preferably the photosensitizer-phosphorus at a temperature below the glass transition temperature of the lipid complex, even more preferably at room temperature or It should be carried out at a lower temperature (eg 15-20°C). Photosensitizer - Phosphorus The glass transition temperature of lipid complexes can be measured using a differential scanning calorimeter.

that the aqueous solubility of a substance should increase as the temperature at which it is used increases At temperatures around the transition temperature of the complex, lipid membranes are “solid ’ from the gel state to the pre-transition state and finally to a further ‘fluid’ liquid crystalline state and tends to undergo a phase transition. However, at higher temperatures than these, the fluidity Not only does it increase, but the degree of phase separation and the rate of membrane defects also increase. This tie As a result, the extent to which the photosensitizer leaks from the interior of the membrane to the interface and further into the aqueous phase is increased. do. Minor changes in conditions (e.g., temperature even a slight drop in ) will shift the equilibrium from the aqueous "solubility" due to precipitation of the photosensitizer. can be lifted. In addition, once the typically water-insoluble photosensitizer begins to precipitate, this Reencapsulation inside the lipid bilayer membrane is not possible. This precipitate is It is thought that it will contribute significantly to the task.

Furthermore, the normal thickness of lipid bilayer membranes in the "solid" gel state (~47/) is It decreases to about 37/ in the transition to the 'liquid crystal' state. Thus, hydro-monobene The available uptake volume for the soporphyrin photosensitizer to occupy is reduced. less than The small "chamber" cannot contain large amounts of photosensitizers, and then photosensitizers are saturated lipid bilayers. It can be squeezed out from the interstices of the layer film. In the photosensitizer leaching process, any two These vesicles can aggregate with each other for reduced particle size and ease of sterile filtration. and introduce further difficulties. This surprise in aggregation of photosensitizer liposomes See FIG. 4 which illustrates one proposed mechanism to explain the temperature effect. . In addition, the use of higher hydration temperatures (e.g., about 35-45°C) is also beneficial during sterile filtration. loss of ability of the photosensitizer to either precipitate or agglomerate in the can occur.

The particle size of the crude liposomes first formed on hydration then preferably also does not exceed about 30° C. (preferably the photosensitizer-phospholipid complex glass formed in the hydration step). homogenized to a more uniform size of about 150-300 nm or reduced to a smaller size range of about 150-300 nm at a temperature below the transition temperature, more preferably below room temperature of about 25°C. (or both). A variety of high speed stirring devices can be used in the homogenization process. For example, a Microfluidizer ^™ (eg, Microfluidics ^™ Model 110F); a sonicator; a high shear mixer; a homogenizer; or a standard laboratory shaker.

The homogenization temperature should be room temperature or lower (e.g. 15-20°C). What should be done is found. higher homogenization temperature (e.g. 32-42°C) ), the relative filtration capacity of the liposomal composition was initially could be improved due to increased fluidity, but then unpredictably, particle size Since the noise increases, it tends to decrease as the stirring continues.

Preferably a Microfluidizer^TMsA high pressure device such as is used for agitation. microphone During rofluidization the fluid passes through the high pressure interaction chamber A large amount of heat is generated in a short period of time. Two streams of fluid in the interaction chamber but at high speed, they collide with each other at 900 degrees. of microfluidization temperature As it increases, the fluidity of the membrane also increases, so initially, as expected, , particle size reduction becomes easier. For example, the filtration capacity^TMs The first few passes through the device can increase as much as four-fold. Fluidity of bilayer membrane The increase facilitates a decrease in particle size, which makes the final composition easier to filter. Become. In the first few passes, this mechanism of increased fluidity makes the process more efficient. dominate profit.

However, as both the number of passes and temperature increase, more Hydrophobic hydro-monobenzoporphyrin photosensitizer molecules squeeze out of liposomes , increasing the tendency of liposomes to aggregate into larger particles. Aggregation of vesicles The size cannot be reduced any further, at the point when the starts to dominate the process. surprised Surprisingly, particle size then actually tends to increase through agglomeration.

For this reason, in the method of the present invention, the homogenization temperature is cooled and maintained at a temperature not exceeding room temperature after the composition has passed through a zone of maximum agitation (e.g., the interaction chamber of a Microfluidizer ^™ device). . Suitable cooling systems include cooling water in any standard agitation device (e.g., Microfluidizer ^™ ) in which homogenization is performed, e.g., in a suitable cooling jacket surrounding a mixing chamber or other zone of maximum turbulence. can be readily provided by circulating the The pressure used in such high pressure devices is not critical, and pressures of about 10,000 to about 16,000 psi are not uncommon.

As a final step, the compositions of the present invention are preferably sterile filtered through filters with extremely small pore size (ie 0.22 Tm). The filter pressure used during sterile filtration can vary widely depending on the composition volume, density, temperature, filter type, filter pore size, and liposome particle size. However, as an indication, a typical set of filtration conditions is: filtration pressure 15-25 psi; filtration load 0.8-1.5 ml/ ^cm2 ; and filtration temperature about 25°C.

A typical, general procedure is described below, along with further illustrative details: (1) Sterile filtration of organic solvent through a hydrophobic 0.22 Tm filter.

(2) EPG, DMPC, hydro-monobenzoporphyrin photosensitizers, and excipients , addition to filtered organic solvent, dissolution of both excipient and photosensitizer.

(3) Filtration of the product solution through a 0.22 Tm hydrophobic filter.

(4) Filtrate rotary evaporator device (for example, Rotoevaporator name (commercially available under the name).

(5) removing the organic solvent to form a dry lipid film;

(6) Analyze the lipid membrane to determine the level of organic solvent concentration.

(7) Preparation of 10% lactose solution.

(8) Filtration of the lactose solution through a 0.22 Tm hydrophilic filter.

(9) Hydrate the lipid membrane with a 10% lactose solution to form crude liposomes.

(10) Reduction of particle size of crude liposomes by passing them through Microfluidizer ^™ three times.

(11) Measurement of particle size distribution of reduced liposomes.

(12) Sterile filtration of the liposomal composition through a 0.22 Tm hydrophilic filter (requires Optionally, the solution can be prefiltered with a 5.01m prefilter first).

(13) Analysis of photosensitizer efficacy.

(14) Filling vials with the liposomal composition.

(15) Lyophilization.

freeze drying Once formulated, the liposomal compositions of the present invention can be stored for extended periods of time if desired. can be lyophilized for For example, the preferred hydro-monobenzoporphyrin BPD-MA, a photosensitizer, was prepared in a cryodesiccate liposomal composition. maintains its efficacy for at least 9 months at room temperature and for at least 2 years is designed for a shelf life of If the composition is lyophilized, subsequent suitable aqueous solution (e.g. sterile water, or sterile containing sugars and/or other excipients) water) for reconstitution prior to administration (e.g. injection) can be called.

Preferably, the liposomes to be lyophilized contain disaccharides or polysaccharides during hydration. Formed upon addition of an aqueous vehicle containing sugars. The composition is then collected , placed in vials, lyophilized and stored (ideally refrigerated) .

The lyophilized composition can then be prepared by simply adding water for injection immediately prior to administration. can thus be reconstructed.

particle size The liposomal compositions of the present invention can be used without significant clogging of filters. Efficient and large volumes from 500ml to over 1 liter pass through 0.22Tm hydrophilic filters A liposome of sufficiently small and fine particle size that aseptic filtration of the composition can be achieved system. A particularly preferred particle size range is less than about 300 nm, more preferably or less than about 250 nm. Most preferably, the particle size is less than about 220 nm.

As noted above, the present invention provides an unexpected degree of particle size reduction. Controls three major parameters that can affect ease. As a result, the filtering capacity The filterability (especially standard sterile filtration) of the liposomal compositions of the present invention significantly improved in These parameters are: (1) DMPC-EPG lipid mixture (2) water temperature during the summation step; and (3) during the homogenization or size reduction step temperature. The photosensitizer/lipid molar ratio was the highest for these three parameters. is also considered to have a large effect.

Filtration Ability Microfluidizers Liposome Compositions^TMsthree times, and then It can be tested by withdrawing the sample in a vacuum. The syringe is a 0.22Tm hydrophilic connected to a filter and then placed in a syringe pump. part of piston motion A constant speed was set at 10 ml/min, and the liposomes flocculated heavily by flocculation. Collect the filtrate until the filter is set. The volume of the filtrate was then measured and ml/cm² or g/cm²Record in units (square centimeters is the effective filtration area). servant Thus, filtration capacity for the purposes of this invention can be filtered through a 0.22 Tm filter. defined as the maximum volume or weight of the liposomal composition.

Dosing and use Hydro-monobenzoporphyrin Photosensitizers Incorporated into Liposomes of the Invention The use of the agent is typically for cancer diagnosis or treatment. Liposome composition to irradiate neoplastic cells or other abnormal tissue (including infectious agents) with light (preferably visible). useful for making them susceptible to destruction by exposure to light). upon photoactivation , to promote the formation of singlet oxygen (which is responsible for the cytotoxic effects) Photosensitizers are considered. By incorporating a photosensitizer into the liposomes of the present invention, More efficient sensitivity of tumor tissue can be obtained.

Furthermore, when the photosensitizers of the present invention are photoactivated by a suitable excitation wavelength, this They are typically visibly fluorescent. This fluorescence can then be detected by tumors or can be used to localize other target tissues.

Generally speaking, hydro-monobenzoporphyrin photosensitizers in liposomes The concentration depends on the nature of the photosensitizer used. For example, if BPD-MA is used When used, the photosensitizer is generally incorporated into the liposome at a concentration of about 0.10%-0.5% w/v. be When lyophilized and reconstituted, this typically results in about 5.0 mg/ Obtain a reconstituted solution of photosensitizer up to ml.

The liposomal compositions of the invention are typically administered parenterally. the injection can be intravenous, subcutaneous, intramuscular, intrathecal, or intracardiac Even intramembranous injection is possible. However, liposomes can also be delivered to the nose by aerosols. It can be administered intracavitary or it can be administered intrapulmonary.

of hydro-monobenzoporphyrin photosensitizer liposomal formulations to be administered The amount depends on the choice of active ingredient, the condition to be treated, the mode of administration, the respective subject, and depends on the judgment of the doctor. However, generally speaking, in the range of 0.05-10 mg/kg doses may be required. Due to the large number of variables for individual treatment regimens, the above Ranges are of course only indicative. Therefore, considerable range of motion is required for these expected from recommended values.

Diagnosis in localized tumor tissue or localized atherosclerotic plaque The pharmaceutical composition of the present invention is known for photodynamic therapy, for use as administered systemically in the same general manner as clear the drug from the tissue ) (the drug does not accumulate there) The latency is about the same, e.g. 10 hours. After localizing the composition of the invention, the presence of the photosensitizer can be detected. determines the location of the target tissue.

Hydro-monobenzoporphyrin light incorporated into liposomes for diagnostic purposes A sensitizer compound may be used with a radioisotope or other means of detection, or It can be labeled with it. In that case the detection means will depend on the nature of the label. technet Scintigraphic labels such as chromium or indium can be used with ex vivo scanners. can be detected using Certain luminescent labels may also be used, but the photosensitizer itself These labels may require prior illumination, such as the fluorescence-based detection of .

For the activity of the photosensitizer hydro-monobenzoporphyrins of the present invention, any suitable Appropriate absorption wavelengths are used. It may mediate cytotoxicity or fluorescence emission can be supplied using various methods known in the art for (e.g., visible light (includes incandescent or fluorescent sources or light diodes such as light emitting diodes) including)). Laser light also delivers light in situ to localized photosensitizers. can be used for For example, in a typical protocol, several hours before irradiation about 0.5–1.5 mg/kg green porphyrin photosensitizer intravenously Inject and then excite at the appropriate wavelength.

Methods of preparing the liposomal compositions of the invention, the compositions themselves, and photodynamic treatments The use of this composition in is described in further detail in the examples below. child These examples illustrate the simple substitution of suitable hydro-monobenzoporphyrin photosensitizers. , by simple substitution of additional phospholipids, or by another method, similarly described lipids. It is easily adapted for the manufacture and use of posomes. The following examples illustrate preferred embodiments of the present invention. Although preferred embodiments, preferred uses and preferred properties are presented, these do not limit the invention. not meant to limit For example, for liposome formation, hydro-monoben Although BPD-MA is used as a soporphyrin photosensitizer, the present invention is directed to this particular photosensitizer. It is not intended to be limited to sensitizers.

Example 1 Effect of Molar Ratio on Filtration Capacity Three BPD-MA liposomes (100 ml batches) were prepared at room temperature (approximately 20°C) using the following general procedure. BPD-MA, butylated hydroxytoluene (“BHT”), ascorbyl palmitate, and phospholipids DMPC and EPG were dissolved in methylene chloride and the resulting solution filtered through a 0.22 Tm filter. The solution was then dried under vacuum using a rotary evaporator until the amount of methylene chloride in the solid residue was no longer detectable by gas chromatography.

A 10% lactose/water solution for injection was then prepared and passed through a 0.22Tm filter. filtered. This lactose/water solution contains a photosensitizer/phospholipid solid residue. Added to the flask. The solid residue is dispersed in a 10% lactose/water solution, and Stir for about 1 hour, cool, and pass through a homogenizer. This solution is then Filtered through 0.22 Tm Durapore (hydrophilic filter).

Using the above procedure, three different preparations of the liposomal composition of BPD-MA, each with a different photosensitizer:EPG:DMPC molar ratio, were prepared as follows: The filtration ability of these three batches was tested according to the following method: After passing the liposomal composition through the Microfluidizer ^™ three times, samples were collected with a syringe. The volume of sample collected was dependent on a visual estimate of the filtration capacity of the composition. The syringe was connected to a 0.22Tm hydrophilic filter and then placed in the syringe pump. The speed of piston movement was set to 10 ml/min and the filtrate was collected until the filter was clogged by flocculation of large liposomes. The volume of filtrate was measured and recorded in ml/cm ² . The results are summarized in Table 2 below.

Table 1 shows that the filtration capacity of the first formulation (slightly less phospholipid than the photosensitizer/DMPC-EPG lipid mixture (1:8.0)) compared to the second formulation (having a molar ratio of 1:8.0). ) and about 8 times less than the third formulation (molar ratio 1:10.0).

FIG. 3 shows a plot of g/cm ² versus total lipid concentration (%, w/v) on filtration capacity. The relationship was surprisingly linear with an r ^- squared of 0.9985. The plot yielded the following empirical formula: Filtration Capacity = [23.36 Total Lipid Concentration (%, w/v)] - [35.51] According to the slope of this formula, 1 gram increase in DMPC or EPG lipids present. Each time there is a 23-fold improvement in the filtration capacity of the liposomal formulation. This plot further demonstrates that when the total lipid concentration falls below 1.52% (BPD-MA photosensitizer concentration of 2.1 mg/ml), the liposomal composition is completely incapable of filtration through a 0.22 Tm filter.

Example 2 Filtration Capacity and Effectiveness of Scaled-Up Batch A larger batch of BPD-MA liposomes (1.2 liters, i.e., 12 times the 100 ml batch (Example 1)) was prepared at a molar ratio of 1:3:5 ( Photosensitizer: EPG:DMPC) was used and prepared at room temperature (approximately 20°C). The ingredients of the composition are listed in Table 3 below: Twice the thickness of the lipid film was used in this larger batch to provide a more intense study for a molar ratio of 1:8.0. The results, shown in Table 4 below, showed significantly improved filtration capacity compared to batches 1/12 times the size with only slightly less DMDC and EPG lipids.

A slight increase in lipid content significantly increased the filtration capacity of the liposomal composition. Furthermore, the yield is nearly 100% and 99.5% (by HPLC analysis) of photosensitizer efficacy is maintained after sterile filtration.

Example 3 Four batches of the liposomal composition of hydration temperature BPD-MA, each having the same photosensitizer:DMPC-EPG lipid molar ratio (1.05:3:5), were mixed with different membrane water. Prepared at sum temperature.

The data for these four batches (shown in Table 5 below) were compared to determine efficacy. showed the relationship between the loss of water and the membrane hydration temperature.

These results indicated that increasing the hydration temperature above room temperature was associated with an unwanted and significant loss of photosensitizer efficacy.

EXAMPLE 4 Effect of Increasing Homogenization Temperature The relationship between filtration capacity and homogenization temperature was studied using a pair of 0.53 liter batches (photosensitizer:EPG:DMPC=1.05:3:5). The composition was prepared as in Example 1 above using a hydration temperature of 45°C and a Microfluidizer ^™ (exit temperature set at 35°C). The data showed that: As shown below, at high hydration temperatures and high homogenization temperatures, the relative filtration capacity of the BPD-MA liposomal compositions decreased, as expected, to Microfluidizer ^™ . It increases after the first pass (#1-#2) but decreases for further passes (#2-#4).

A second 0.53 liter batch (photosensitizer:EPG:DMPC=1.05:3:5) was used to study the relationship between number of passes through the Microfluidizer ^™ , filtration capacity, and particle size distribution. In this batch, a high hydration temperature (40°C) and a high homogenization temperature (Microfluidizer ^™ outlet temperature set at 42°C) were used. The data are listed in Table 7 below.

The data show that the filtration capacity increases from pass #3 to pass #5 as expected, but decreases back to the original value in passes #6-#9. In contrast, the average particle size of the liposomes in the composition decreases slightly from 710 nm in pass #1 to 247 nm in pass #6. However, on further passes the particle size surprisingly increased to about 300 nm.

Example 5 Preparation of Liposomes of the Invention BPD-MA liposomes (100 ml batches) are prepared at room temperature (approximately 20° C.) using the following general procedure. BPD-MA, butylated hydroxytoluene (“BHT”), ascorbyl palmitate, and phospholipids DMPC and EPG are dissolved in methylene chloride. The photosensitizer:EPG:DMPC molar ratio is 1.0:3:7 and has the following composition: Using the above formulation, the total lipid concentration (% w/v) is approximately 2.06. The resulting solution is filtered through a 0.22 Tm filter and then dried under vacuum using a rotary evaporator. Drying is continued until the amount of methylene chloride in the solid residue is no longer detectable by gas chromatography.

A 10% lactose/water solution for injection is then prepared and filtered through a 0.22 Tm filter. Instead of warming the temperature to about 35°C, the lactose/water solution is maintained at room temperature (about 25°C) for addition to the flask containing the photosensitizer/phospholipid solid residue. The solid residue is dispersed in a 10% lactose/water solution at room temperature, stirred for about 1 hour, and passed through a Microfluidizer ^™ homogenizer (exit temperature controlled at about 20-25°C) 3-4 times. This solution is then filtered through a 0.22 Tm Durapore (hydrophilic filter).

The batch's filtration ability is tested by the following procedure: After passing the liposomal composition through the Microfluidizer ^™ three times, a sample is collected with a syringe. The collected sample volume is approximately 20 ml and is dependent on visual estimation of the filtration capacity of the composition and assessment of liposome mass. The syringe is connected to a 0.22Tm hydrophilic filter and then placed in the syringe pump. The speed of piston movement is set to 10 ml/min and the filtrate is collected until the filter clots by flocculation of large liposomes. Measure the volume of the filtrate and record in ml/cm ² or g/cm ² .

The filtration capacity of the composition in g/cm ² is typically greater than about 10. Furthermore, the yield is about 100% (by HPLC analysis) and the efficacy of the photosensitizer is typically maintained even after sterile filtration. Average particle size varies from about 150 nm to about 300 nm (±50 nm).

Modifications and/or variations of the disclosed subject matter may fall within the scope of the invention as claimed below. It is clear to the person skilled in the art that it can be done without departing from the scope.

Claims (1)

[Claims] 1. A method for preparing a pharmaceutical composition containing liposomes, comprising the liposome Is a hydro-monobenzoporphyrin photosensitizer, as well as egg phosphatidylglycol. Serol ("EPG") and dimyristoyl phosphatidylcholine ("DMPC") Comprising a therapeutically acceptable amount of a phospholipid comprising a mixture of including:     a. Photosensitizers and phospholipids can be combined with phospholipids in a molar ratio of Combining in the presence of a solvent;     b. Removing the organic solvent to form a lipid membrane;     c. Hydrating the lipid membrane with an aqueous solution at a temperature lower than 30 ° C. to form a photosensitizer-phospholipid Forming a crude liposome containing the complex; and     d. The particle size of the crude liposome is reduced to about 300 nm at a temperature below about 30 ° C. Homogenizing or reducing to a lower particle size range. 2. Said temperature is the glass transition temperature of said photosensitizer-phospholipid complex; and / Or   The molar ratio of hydro-monobenzoporphyrin photosensitizer to phospholipid is about 1: a phospholipid of 8.0 or more; and / or   The organic solvent is methylene chloride; and / or   The organic solvent is removed by evaporation under reduced pressure in step "b." And / or   The aqueous solution comprises a disaccharide or polysaccharide; and / or   The hydro-monobenzoporphyrin photosensitizer comprises one of the formulas 1 to 6 shown in FIG. Has one and has one or more light absorption maxima between 670 and 780 nm, or Its metallized or labeled form,   Where each R¹And R^TwoIs carboalkoxy (2-6C), alkyl (1-6C) sulf Honyl, aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and -C ONR^FiveCO- (where R^FiveIs a group consisting of aryl (6-10C) or alkyl (1-6C) Independently selected from:   Each R^ThreeIs independently carboxyalkyl (2-6C) or a salt, amide, A stele, or an acylhydrazone, or an alkyl (1-6C); do it   Each R^FourBut -CHCH_Two; -CHOR^Four'(Where R^Four'Is H or alkyl (1-6C) -CHO; -COOR^Four'; -CH (OR^Four') CH_Three; -CH (OR^Four') CH_Two OR^Four'; -CH (SR^Four') CH_Three; -CH (NR^Four' _Two) CH_Three; -CH (CN) CH_Three; -CH (COOR^Four') CH_Three; -CH (OOCR^Four' ) CH_Three; -CH (halo) CH_Three; -CH (halo) CH_Two(Halo); direct or indirect induction of vinyl group An organic group of less than 12C generated from the conductorization; or R^FourIs 1-3 tetrapi of formula -L-P It is a roll-type core, where -L- Selected from the group consisting of   And P is the second Gp, which is one of equations 1-6 but R^FourMissing And then R^FourJoined to L through the position shown to be occupied by, or other A porphyrin group; and / or   Said hydration step "c." Is accomplished at or below room temperature; and / or Is   Wherein said homogenizing or reducing step "d." Is performed at room temperature or below. And / or   The particle size is reduced to a range less than about 250 nm; The method of claim 1. 3. Each R^ThreeBut -CH_TwoCH_TwoCOOH or its salts, amides, esters or acyl Do Razon, and each R¹And R^TwoIs a carboalkoxy (2-6C). 3. The method according to 2. 4. 4. The method of claim 3, wherein said photosensitizer is BPD-MA having the formula of FIG. 5. A pharmaceutical composition containing liposomes in the particle size range of about 150-300 nm. So, the liposome a. A therapeutically acceptable amount of a photosensitizer and b. Phospholipid mixture containing (1) egg phosphatidylglycerol ("EPG") and (2) Dimyristoyl phosphatidylcholine ("DMPC") Containing A phospholipid having a molar ratio of the photosensitizer and the phospholipid mixture of about 1: 7.0 or more; Pharmaceutical composition. 6. A molar ratio of the photosensitizer to the phospholipid mixture of about 1: 8.0 or more. And / or   The hydro-monobenzoporphyrin (Gp) is represented by the formulas 1 to 6 shown in FIG. Has one and has one or more light absorption maxima between 670 and 780 nm, or Its metallized or labeled form,   Each R¹And R^TwoIs carboalkoxy (2-6C), alkyl (1-6C) sulfonyl, Aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and -CONR^FiveCO- (Where R^FiveIs independent of the group consisting of aryl (6-10C) or alkyl (1-6C) Selected   Each R^ThreeIs, independently, carboxyalkyl (2-6C) or a salt, amide, An ester or an acylhydrazone or an alkyl (1-6C);   R^FourBut -CHCH_Two; -CHOR^Four'(Where R^Four'Is H or alkyl (1-6C); optional Is substituted with a hydrophilic substituent); -CHO; -COOR^Four'; -CH (OR^Four') CH_Three; -CH (OR^Four') CH_TwoO R^Four'; -CH (SR^Four') CH_Three; -CH (NR^Four' _Two) CH_Three; -CH (CN) CH_Three; -CH (COOR^Four') CH_Three; -CH (OOCR^Four' ) CH_Three; -CH (Halo) CH_Three; -CH (halo) CH_Two(Halo); generated from direct or indirect derivatization of vinyl group An organic group of less than 12C formed; or R^FourIs a 1-3 tetrapyrrole type nucleus of the formula -L-P Yes, where -L- Selected from the group consisting of   And P is the second Gp, which is one of equations 1-6 but R^FourMissing And then R^FourConnected to L through the position indicated to be occupied by Is another porphyrin group; and / or The particle size is below about 220 nm; A composition according to claim 5. 7. Each R^ThreeBut -CH_TwoCH_TwoCOOH or its salt, amide, ester or acyl hydride Razon; and   Each R¹And R^TwoIs a carboalkoxy (2-6C). . 8. The hydro-monobenzoporphyrin photosensitizer is BPD-MA having the formula of FIG. The composition of claim 7, wherein