A liposome formulation for stably incorporating high content of hydrophobic substance is disclosed. The liposome includes two phospholipids with different phase transition temperatures such as saturated and unsaturated phosphatidyl cholines, hydrophobic substances, cholesterol, cholesterol derivatives, antioxidant and hydrophilic polymer-modified lipids such as MPEG-DSPE.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the use of liposomes in the drug delivery system, and more particularly to the stable liposomes capable of incorporating high content of hydrophobic drugs.

2. Description of the Related Art

Liposome technology has been exploited extensively for the purpose of drug delivery for many years. A typical liposome structure is composed of single or multiple layer membranes with hydrophobic domain between the phospholipid bilayers, and the interior aqueous compartment. Hydrophobic or hydrophilic compounds can be entrapped in the hydrophobic domain or encapsulated in the aqueous compartment, respectively. On the other hand, liposomes can be constructed of natural constituents so that the liposome membrane is in principal identical to the lipid portion of natural cell membranes. It is considered that liposomes are quite compatible with the human body when used as drug delivery system. In addition, liposome-based drug formulation also has been reported to be able to achieve the equivalent therapeutic efficacy to free drug, as well as reduce the systemic toxicity in many applications.

The hydrophobic drug, paclitaxel, was sold in the market in 1992, and used in phase II trials for treating breast and ovarian cancer. In 1998, it was used in combination therapy with cisplatin for the treatment of non-small cell lung and ovarian cancer in phase I trials. However, due to its poor solubility in water, paclitaxel is prepared for clinical administration containing Cremophor EL.RTM. (polyethoxylated castor oil) and absolute ethanol in a 50/50 (vol/vol) ratio (Diluent 12). In clinical trials, the problems of anaphylactoid reaction, neutropenia, peripheral neuropathy, bradyarrhythmia and anemia were encountered. Meanwhile, the amount of cremophor EL necessary to solubilize the clinically required dose of paclitaxel is much higher than that administered with any other marketed drug. Cremophor vehicle thus is found to be responsible for hypersensitivity response. Premedication with corticosteroid, diphenhydramine or H.sub.2 antagonist, and slow infusion of a large volume are needed to avoid the side effect. In contrast, owing to the aforementioned advantages of liposome-based drug delivery system, researches of incorporating paclitaxel in liposomes for clinical paclitaxel administration have become a hot topic and been reported regularly.

Conventional paclitaxel-liposomes were prepared at paclitaxel/lipid molar ratio of approximately 3 mole % regardless of whether the liposomes are made of a mixture of phosphatidyl glycerol (PG) and phosphatidyl choline (PC) (U.S. Pat. No. 5,415,869; Sampedro, F et al., J Micrencapsul 11:309-318 (1993); Sharma, A. et al., Pharm Res 11:889-896 (1994); Shien, M. F. et al., J Ferm Bioeng 83:87-90 (1997)), or of unsaturated (U.S. Pat. No. 6,090,955; Bartoli, M. H. et al., J Micrencapsul 7:191-197 (1990); Riondel, J. et al., In Vivo 6:23-28 (1992); Sharma, D. et al., Melanoma Res 8:240-244 (1998)) or partially unsaturated PC (U.S. Pat. No. 5,683,715). At a drug/lipid ratio of 4 mole %, the paclitaxel-liposome system is stable only for 2 days while needle-like crystal precipitates appear during preparation at a drug/lipid ratio up to 8 mole % (Sathyamangalam, V. et al., Biochemistry 33:8941-8947 (1994); Bernsdorff, C. et al., J Biomed Mater Res 46:141-149 (1999)). On the other hand, the liposomes are prepared by employing hydrophilic polymer-conjugated phospholipid (methoxy polyethylene glycol-phosphatidyl ethanolamine) in order to enhance its circulation time in blood post iv administration (Crosasso, P. et al., J Control Release 63:19-30 (2000)). Liposomes with the prolonged circulation time in bloodstream make it possible increasing the availability of the injected liposomes to reach the target cells before being metabolized. However, this formulation of the polymer-engrafted liposomes with a maximal 3 mole % (paclitaxel/lipid ratio) quickly become unstable in one-week storage at 4.degree. C.

Alternatively, a formulation of paclitaxel-liposomes comprising a special phospholipid, cardiolipid, and phosphatidyl choline (PC) was disclosed in U.S. Pat. No. 5,424,073 and Int J Oncol 12:1035-1040 (Cabanes, A. et al., 1998). The molecular structure of cardiolipid is composed of one huge hydrophilic head and four aliphatic chains. The liposomes prepared in accordance with this formulation increase the paclitaxel/lipid molar ratio to 9 mole %, however, it is stable only for 1 month when stored in liquid form at 4.degree. C.

Generally, paclitaxel incorporated within the bilayer membrane of liposomes is thermodynamically prone to self-aggregation, then precipitating from liposomes. Previous researches have reported that the optimal paclitaxel/lipid molar ratio in a typical liposome formulation is ranged from 3 to 4 mole %, and paclitaxel-liposomes are more stable when the drug/lipid ratio is kept at approximately 3 mole %. When the molar ratio is increased, needle-like crystal precipitates appear during the preparation process. Besides, it is known by person skilled in the art that drugs with a low drug/lipid ratio are commonly unsuitable for clinical administration. A high dose of liposomes still may result in certain extent of toxicity due to the injection of excessive amounts of lipids in the body. Furthermore, increasing liposome concentration also raises the cost of production. Therefore, it is important to elevate the hydrophobic drug/lipid ratio in liposome-based drug delivery system by which the above drawbacks may be avoided.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a liposome-based drug delivery system that is able to incorporate large amounts of hydrophobic compounds. Accordingly, the formulated liposomes capable of incorporating high content of hydrophobic compounds can maintain considerably stable for months, as well as reduce the possible side effects in the versatile applications.

The invention achieves the above-identified objects by providing the formulated liposomes for incorporating large amount of hydrophobic compounds. The composition of the liposomes at least comprises a first and second phospholipids, hydrophobic drugs and other additives such as lipids modified by hydrophilic polymer (such as MPEG-DSPE), cholesterol, cholesterol derivatives and antioxidants. According to the invention, the phase transition temperature of the first phospholipids, T.sub.g1, is in the range from 40 to 70.degree. C., and preferably from 50 to 65.degree. C. The phase transition temperature of the second phospholipids, T.sub.g2, is in the range from -30 to 20.degree. C., and preferably from -20 to 4.degree. C. Also, the phase transition temperature of the first phospholipids, T.sub.g1, is higher than that of the second phospholipids, T.sub.g2 while the drug delivery temperature T.sub.1 and storage temperature T.sub.2 are chosen at specified ranges subject to the order of T.sub.g1>T.sub.1>T.sub.2>T.sub.g2. The first phospholipids having higher phase transition temperature forms the gel state phase, and the second phospholipids having lower phase transition temperature forms the liquid-crystal phase. Each membrane lipid bilayer consists of several regions of gel state phases and liquid-crystal phases, and the hydrophobic drugs can be held within the lipid bilayer. The phase boundary barrier between the regions of gel state phase and liquid-crystal phase is able to reduce lateral movement and aggregation of the hydrophobic drugs, thereby stabilizing the liposomes. Thus, this liposome composition results in coexistence of multiple discontinuous immiscible phases (gel state phase and liquid-crystal phase) occurring on each bilayer membrane of liposomes regardless of unilamellar or mulitlamellar structure when the drug is delivered or stored. The drug delivery temperature T.sub.1 is optionally from 30 to 38.degree. C., while the storage temperature T.sub.2 is optionally from 4 to 25.degree. C.

The first phospholipids, with higher phase transition temperature (T.sub.g1) from 40 to 70.degree. C., are preferably hydrogenated naturally-occurring phospholipids and saturated phospholipids with long carbon chain (--(CH2).sub.n--, the value of n is at least 14), such as phosphatidyl choline (PC), phosphatidyl glycerol (PG), phosphatidyl serine (PS), phosphatidyl acid (PA), or phosphatidyl ethanolamine (PE). Examples of hydrogenated phosphatidyl choline (PC) are hydrogenated egg phosphatidyl choline (HEPC) (T.sub.g=50.about.55.degree. C.) and hydrogenated soy phosphatidyl choline (HSPC) (T.sub.g=55.degree. C.), while examples of saturated phsopholipids with long carbon chains (--(CH2).sub.n--, the value of n is at least 14) are dipalmitoyl phosphatidyl choline (DPPC) (T.sub.g=42.degree. C.), distearyloyl phosphatidyl choline (DSPC) (T.sub.g=55.degree. C.), diarachidoyl phosphatidyl choline (Tg=66.degree. C.), dimyristoyl phosphatidyl ethanolamine (DMPE) (Tg=49.5.degree. C.), dipalmitoyl phosphatidyl ethanolamine (DPPE) (Tg=64.degree. C.), distearoyl phosphatidyl ethanolamine (DSPE) (Tg=74.degree. C.), diarachidoyl phosphatidyl ethanolamine (Tg=82.degree. C.), dipalmitoyl phosphatidyl glycerol (DPPG) (Tg=41.5.degree. C.), distearoyl phosphatidyl glycerol (Tg=54.5.degree. C.), dimyristoyl phosphatidyl acid (DMPA) (Tg=50.degree. C.), dipalmitoyl phosphatidyl acid (DPPA) (Tg=66.degree. C.), dipalmitoyl phosphatidyl serine (DPPS) (Tg=54.degree. C.), and distearoyl phosphatidyl serine (DSPS) (Tg=70.degree. C.). The desired phospholipids may also be a combination of two or more phospholipids listed above. The lists of PC above are illustrations of specific phospholipids but are in no way intended to limit the scope thereof.

The second phospholipids, with lower phase transition temperature (T.sub.g2) from -30 to 10.degree. C., are preferably unsaturated phospholipids or saturated phospholipids with short carbon chains (--(CH2).sub.n--, the value of n is at most 14), such as phosphatidyl choline (PC), phosphatidyl glycerol (PG), phosphatidyl serine (PS), phosphatidyl acid (PA), or phosphatidyl ethanolamine (PE). Examples of synthetic or naturally-occurring unsaturated phospholipids are egg phosphatidyl choline (EPC) (T.sub.g=-8.degree. C.) and soy phosphatidyl choline (SPC) (T.sub.g=0.degree. C.), oleoyl palmitoyl phosphatidyl choline (Tg=-10.degree. C.), dioleoyl phosphatidyl choline (Tg=-19.degree. C.), dipetroselinoyl phosphatidyl choline (Tg=1.degree. C.), dipalmitelaidoyl phosphatidyl choline (Tg=-4.degree. C.), dipalmitoleoyl phosphatidyl choline (Tg=-36.degree. C.), dipalmitelaidoyl phosphatidyl ethanolamine (Tg=-33.5.degree. C.), dioleoyl phosphatidyl ethanolamine (Tg=-16.degree. C.),dioleoyl phosphatidyl serine (Tg=-10.degree. C.), while examples of synthetic or naturally-occurring saturated phospholipids with short carbon chains is dilauroyl phosphatidyl choline (DLPC) (T.sub.g=-2.degree. C.). diundecanoyl phosphatidyl choline (Tg=-15.5.degree. C.), didecanoyl phosphatidyl choline (Tg=-34.7.degree. C.), dinonanoyl phosphatidyl choline (Tg=-55.2.degree. C.), didecanoyl phosphatidyl ethanolamine (Tg=3.6.degree. C.), dinonanoyl phosphatidyl ethanolamine (Tg=-14.5.degree. C.). The desired phospholipids may also be a combination of two or more phospholipids listed above. The list of phospholipids above are illustrations of specific phospholipids but are in no way intended to limit the scope thereof.

DETAILED DESCRIPTION OF THE INVENTION

It is disclosed in the invention that at special ranges of two phospholipid combination and temperature, liposome composed of two phospholipids such as an unsaturated phospholipid (the second phospholipid) and a saturated phospholipid (the first phospholipid ) with different phase transition temperatures are able to form two separated phases, a gel state phase and liquid-crystal phase, in the phospholipid bilayer, as shown in FIG. 1 (see Original Patent). The two immiscible phases coexist in the liposomes and create several discontinuous regions. The first phospholipid having higher phase transition temperature forms the gel state phase, and the second phospholipid having lower phase transition temperature forms the liquid-crystal phase. Each membrane bilayer consists of several regions of gel state phases and liquid-crystal phases, and the hydrophobic compounds can be held within the lipid bilayer. The phase boundary barrier between the regions of gel state phase and liquid-crystal phase is able to reduce lateral movement and aggregation of the hydrophobic compounds, thereby stabilizing the liposome.

It has been reported that hydrophobic compounds such as paclitaxel has a tendency to undergo concentration-dependent aggregation in hydrophobic environment, forming intermolecular hydrogen bonds (Sathyamanglam, V. et al., J Pharm Sci 83: 1470-76(1994)). Similarly, as a large amount of paclitaxel was embedded in the hydrophobic domain within bilayer membrane, it is thermodynamically prone to self-aggregating, destablizing the liposomes. Accordingly, when the formulated liposomes are prepared, two immiscible phases are formed and phase boundaries are speculated to construct a barrier stopping the self-aggregation process of hydrophobic molecules. As a result, stable liposomes capable of incorporating high content of hydrophobic compound become possible. The existence of lateral phase-separated phospholipid-regions is advantageous for incorporating large amount of hydrophobic molecules into the phospholipid bilayer. The formulated liposomes can incorporate higher content of paclitaxel and remain more stable than any other liposome formulations ever reported.

The invention, hence, provides a liposome-based drug delivery system composing of two phospholipids with different phase transition temperatures. The phospholipids with high (T.sub.g1) and low (T.sub.g2) phase transition temperatures can be saturated and unsaturated phospholipids, respectively. The coexistence of several discontinuous immiscible phases (e.g. gel phase and liquid-crystal phase) occurs in phospholpiid bilayer at a specific phospholipid composition and temperature (T), wherein T.sub.g1>T>T.sub.g2. The specific temperature T can be the liposome delivery temperature T.sub.1 (about 30.about.38.degree. C.) or storage temperature T.sub.2 (about 4.about.25.degree. C.). Therefore, T is commonly considered as a range that includes the liposome delivery and storage temperatures. The temperature range provides the requirement to design the particular combination of two phospholipids to achieve phase separation. For example, when the intravenous injection is administrated (intravenous administration temperature is 37.degree. C. (T.sub.1) and liposomes are stored at is 4.degree. C. (T.sub.2)), a phospholipid with phase transition temperature larger than 40.degree. C. (T.sub.g1>40.degree. C.) could be carefully chosen as the first phospholipid, and preferably with phase transition temperature ranged from 40 to 70.degree. C. Also, a phospholipid with phase transition temperature lower than 4.degree. C. (T.sub.g2<4.degree. C.) could be carefully chosen as the second phospholipid, and preferably in a range from -30 to 4.degree. C. When the subcutaneous injection is administrated (administration temperature is 32.degree. C. (T.sub.1) and liposomes are stored at is 25.degree. C. (T.sub.2)), a phospholipid with phase transition temperature larger than 35.degree. C. (T.sub.g1>35.degree. C.) could be carefully chosen as the first phospholipid, and preferably with phase transition temperature ranged from 35 to 60.degree. C. Also, a phospholipid with phase transition temperature lower than 25.degree. C. (T.sub.g2<25.degree. C.) could be carefully chosen as the second phospholipid, and preferably in a range from -20 to 10.degree. C.

The first phospholipids,with higher phase transition temperature (T.sub.g1) from 40 to 74 0.degree.C, are preferably hydrogenated naturally-occurring phospholipids and saturated phospholipids with long carbon chain (-(CH2).sub.n-, the value of n is at least 14), such as phosphatidyl choline (PC), phosphatidyl glycerol (PG), phosphatidyl serine (PS), phosphatidyl acid (PA), or phosphatidyl ethanolamine (PB). Examples of hydrogenated phosphatidyl choline (PC) are hydrogenated egg phosphatidyl choline (HEPC) (T.sub.g=50.about.55 .degree.C) and hydrogenated soy phosphatidyl choline (HSPC) (T.sub.g=55 0C), while examples of saturated phsopholipids with long carbon chains (-(CH.sub.2).sub.n-, the value of n is at least 14) are dipalmitoyl phosphatidyl choline (DPPC) (T.sub.g=42 0C), distearyloyl phosphatidyl choline (DSPC) (T.sub.g=55 .degree. C.), diarachidoyl phosphatidyl choline (Tg=66.degree. C.), dimyristoyl phosphatidyl ethanolamine (DMPE) (Tg =49.5 .degree. C.), dipalmitoyl phosphatidyl ethanolamine (DPPE) (Tg=64.degree. C.), distearoyl phosphatidyl ethanolamine (DSPE) (Tg =74.degree. C.), diarachidoyl phosphatidyl ethanolamine (Tg=82.degree. C.), dipalmitoyl phosphatidyl glycerol (DPPG) (Tg=41.5.degree. C.), distearoyl phosphatidyl glycerol (Tg=54.5.degree. C.), dimyristoyl phosphatidyl acid (DMPA) (Tg=50.degree. C.), dipalmitoyl phosphatidyl acid (DPPA) (Tg=66.degree. C.), dipalmitoyl phosphatidyl serine (DPPS) (Tg=54.degree. C.), and distearoyl phosphatidyl serine (DSPS) (Tg=70.degree. C.). The desired phospholipids may also be a combination of two or more phospholipids listed above.

The second phospholipids, with lower phase transition temperature (T.sub.g2) from -30 to 10.degree. C., are preferably unsaturated phospholipids or saturated phospholipids with short carbon chains (--(CH2).sub.n--, the value of n is at most 14), such as phosphatidyl choline (PC), phosphatidyl glycerol (PG), phosphatidyl serine (PS), phosphatidyl acid (PA), or phosphatidyl ethanolamine (PE). Examples of synthetic or naturally-occurring unsaturated phospholipids are egg phosphatidyl choline (EPC) (T.sub.g=-8.degree. C.) and soy phosphatidyl choline (SPC) (T.sub.g=0.degree. C.), oleoyl palmitoyl phosphatidyl choline (Tg=-10.degree. C.), dioleoyl phosphatidyl choline (Tg=-19.degree. C.), dipetroselinoyl phosphatidyl choline (Tg=1.degree. C.), dipalmitelaidoyl phosphatidyl choline (Tg=-4.degree. C.), dipalmitoleoyl phosphatidyl choline (Tg=-36.degree. C.), dipalmitelaidoyl phosphatidyl ethanolamine (Tg=-33.5.degree. C.), dioleoyl phosphatidyl ethanolamine (Tg=-16.degree. C.),dioleoyl phosphatidyl serine (Tg=-10.degree. C.), while examples of synthetic or naturally-occurring saturated phospholipids with short carbon chains is dilauroyl phosphatidyl choline (DLPC) (T.sub.g=-2.degree. C.). diundecanoyl phosphatidyl choline (Tg=-15.5.degree. C.), didecanoyl phosphatidyl choline (Tg=-34.7.degree. C.), dinonanoyl phosphatidyl choline (Tg=-55.2.degree. C.), didecanoyl phosphatidyl ethanolamine (Tg=3.6.degree. C.), dinonanoyl phosphatidyl ethanolamine (Tg=-14.5.degree. C.). The desired phospholipids may also be a combination of two or more phospholipids listed above.
 

Claim 1 of 44 Claims

1. A formulated liposome for incorporating a high content of hydrophobic substances therein, comprising: a first phospholipid which is selected from the group consisting of a hydrogenated naturally-occurring phospholipid and a saturated phospholipid having long carbon chains (--(CH2).sub.n--, in which n is at least 14), and which has a phase transition temperature T.sub.g1 ranging between 40 and 74.degree. C.; a second phospholipid which is selected from the group consisting of an unsaturated phospholipid and a saturated phospholipid having short carbon chains (--(CH2).sub.n--, in which n is at most 14), and which has a phase transition temperature T.sub.g2 ranging between -30 and 10.degree. C.; liposome-forming materials effective to form a liposome in which the first phospholipid and the second phospholipid coexist in two immiscible phases and create several discontinuous regions, and in which a molar ratio of the first phospholipid to the second phospholipid is at least 3:16; and one or more hydrophobic substances incorporated in the liposome in an amount of at least 20 mole % to form the formulated liposome, wherein a drug delivery temperature T.sub.1 and a drug storage temperature T.sub.2 are chosen at specified ranges subject to an order of T.sub.g1>T.sub.1>T.sub.2>T.sub.g2, and wherein the formulated liposome has an incorporation efficiency which remains at least about 70% of incorporation efficiency for six months or more.
 

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