This invention relates to a liposome having active targeting property and ensured stability in blood, which can be used in the diagnosis and/or treatment of diseases, particularly renal diseases, that accompany production of various types of proteoglycan in injured portions of tissues and/or organs.
In recent years, there have been increased studies on a drug delivery system (DDS) based on a so-called targeting technique in which a drug is efficiently distributed in an organ of interest. Liposomes are one of the most extensively studied means as such a DDS because of their ability to include, or trap, drugs therein.
However, there are various problems to be resolved in putting these liposomes into practical use, particularly, the difficulties in escaping from the mechanism of living body for recognizing foreign bodies and in controlling intracorporeal kinetics. That is, liposomes cause aggregation in blood by their mutual reaction with various blood plasma proteins, including opsonin, and are captured by a reticuloendothelial system (RES) such as in the liver or spleen, so that it has been difficult to deliver liposomes selectively to the target tissues or cells.
In recent years, it became possible to prevent aggregation of liposomes in blood and avoid being captured by RES, by coating the surface of liposome with a hydrophilic polymer such as polyethylene glycol (PEG) and thereby preventing adsorption of various blood plasma proteins, including opsonin, to the liposome surface (U.S. Pat. No. 5,013,556, U.S. Pat. No. 5,676,971).
There are a number of studies based on these techniques with regard to the targeting at tissues of interest, such as tumor tissues, regions of enhanced vascular permeability, inflamed tissues, the liver, the brain and lymph tissues, but all of these cases are based on the effect of so-called passive targeting property, which is induced as a result of the improvement of the stability in blood by avoiding the capture by RES (Advanced Drug Delivery Reviews, 24 (1997), 337-334), so that concern has been directed toward the development of a liposome having the ability to bind to the tissues of interest, in which the targeting function is reinforced by so-called active targeting techniques.
Regarding the active targeting techniques, methods for the modification of the liposome surface with target factors such as antibodies, antibody fragments, amino acids, peptides or saccharides have been studied, as well as techniques for making the liposome surface into cationic nature. Particularly, the cation formation can be cited as one of the desirable modification techniques, because it has the ability to deliver genes and the like into cells, as a gene introducing technique, and it improves accumulation of liposome on, for example, an injured portion of vascular endothelium (JP-A-7-89874; the term xe2x80x9cJP-Axe2x80x9d as used herein means an xe2x80x9cunexamined published Japanese patent applicationxe2x80x9d). However, strong aggregation and the like phenomena mediated by the binding of blood protein to liposome were observed by the cationic liposome when compared with a neutral or anionic liposome (Biochimica et Biophysica Acta, 1280 (1996), 149-154), so that its sufficient active targeting property in the living body was not obtained as such.
On the other hand, proteoglycans which keep the cell surface anionic are known as the components which their interactions to cationic liposome are expected. It has been reported that overproduction of various types of proteoglycans occurs in tissues of fibrosis in a large number of organs (the liver, the lungs, the heart, the pancreas, the bone marrow and arteries) of tumors such as large bowel cancer, of cell proliferative nephritis and of other inflammatory diseases (Acta Pathol. Jpn., 36 (6): 827 (1986), FEBS Lett., 244: 315 (1988), J. Rheumatol., 18 (10): 1466, (1991), J. Dent. Res., 71 (9): 1587, (1992). This reaction in the living body is considered to be a result of excess repairing reaction which occurs during the step of wound healing or of excess cell proliferation in a tumor or the like tissue, so that it is used as a pathological marker of the foci of the aforementioned diseases.
However, very little is known about reports which aim at achieving active targeting for tissues and/or organs in which such proteoglycan is overproduced. For example, JP-A-8-27030 describes that a drug carrier whose surface is modified with a basic compound which takes positive charge within a physiological pH range is accumulated on a cortical portion of the kidney, but there are no reports which disclose a targeting technique for the specific accumulation of a carrier on renal glomerulus, particularly renal glomerulus which caused inflammation by an injury, and proteoglycan producing tissues and/or organs typified thereby.
Glomerulonephritis is one of the diseases so far reported in which production of various types of proteoglycan is accelerated in injured portions (Clin. Exp. Immunol., 108: 69, (1997), Kidney International, 49: supple. 54, s 55 (1996), J. Am. Soc. Nephrol., 2: s 88 (1992)). Importance of the targeting of a drug for a tissue and/or organ overproducing proteoglycan can easily be understood by merely citing this case of glomerulonephritis.
According to the materials produced in 1996 by The Japanese Society of Dialysis Medicine, it is said that about 27,000 patients are subjected to dialysis due to renal insufficiency, and about 50% of the cases is caused by chronic nephropathy, and about 30% thereof by diabetic nephropathy. Since the primary disease in both of the chronic nephropathy and diabetic nephropathy is glomerulonephritis, these patients will be released from the struggling against dialysis when an effective method for the treatment of glomerulonephritis is established. Up to now, however, no effective glomerulonephritis treating method has been found.
For example, a glomerulonephritis, called IgA nephritis, is a glomerulonephritis most frequently found in the Japanese people, and it is said that its patients are estimated to be close to 300,000 in Japan alone and it occupies around 40% of the glomerulonephritis in adults and close to 30% of that in children. As a result of a follow up study carried out in Japan for recent 20 years, it was found that about 40% of IgA nephritis cases, estimated to be 5,000 to 6,000 cases per year (1995), reached terminal renal insufficiency and were subjected to dialysis, so that it is considered that the development of a therapeutic method specific for IgA nephritis is a world-wide pressing need. However, close to 5,000 patients have been subjected to dialysis every year because of the absence of sufficient means for its treatment.
It has been reported that, as a result of 10 years of clinical efficacy evaluation on IgA nephritis, the steroid therapy which is now actively used in Japan for nephrotic syndrome and rapidly progressive glomerulonephritis, and its efficacy is said to be established, also has a possibility of keeping renal functions for a prolonged period of time by its 2 to 3 years of continuous use at an early stage of progressive IgA nephritis.
On the other hand, however, side effects become a serious problem when it is continuously administered for a certain period for such a chronic disease, because problems such as arteriosclerosis, osteoporosis and immunity reduction in adults, slow development in children and the like cannot be avoided. As a result, it becomes a problem that its sufficient efficacy cannot be obtained, because not only QOL (quality of life) of the patient is reduced but also its administration cannot be continued due to side effects and, particularly, its administration for a prolonged period of time is impossible.
In order to resolve these problems, it is necessary to think out a means for expressing its drug effect alone, by a dose which does not express the aforementioned side effects, and it is desirable for this purpose to design a system for efficiently delivering a drug to the glomerulus which caused inflammation by an injury.
The same requirement is also applied to other diseases in which proteoglycan is excessively produced in injured portions of tissues and/or organs, so that it is expected to develop a method which can exert sufficient therapeutic effect without inducing side effects, by actively targeting the organ of interest with a drug which shows a strong drug effect but cannot be used due to its toxicity.
In consequence, this problem is resolved by including a drug into a liposome which is efficiently accumulated on tissues and/or organs that accompany production of proteoglycan, typified by renal glomerulus which caused inflammation by an injury, and by delivering the drug efficiently to such tissues and allowing it to perform continuous action. This purpose is achieved by realizing a liposome having active targeting property and ensured stability in blood, which also has the ability to be accumulated on injured portions of tissues and/or organs that accompany production of various types of proteoglycan.
Accordingly, this invention contemplates providing a liposome having active targeting property and ensured stability in blood, which can be used in the diagnosis and/or treatment of diseases, particularly renal diseases, that accompany production of various types of proteoglycan in injured portions of tissues and/or organs.
In the process of carrying out studies on factors which control intracorporeal kinetics of liposome, the present inventors have found surprisingly that, when a liposome is prepared by incorporating a basic compound which takes positive charge within a physiological pH range, within a certain range of mixing ratio, and combining a hydrophilic polymer to its surface in an amount controlled within a certain range, it shows such properties that not only its stability in blood is ensured, its capture by the liver and the like can be avoided and its retention in blood is improved, but also its ability to recognize proteoglycan in injured portions is sharply improved.
It was found also that, when its major particle size range is from 90 to 200 nm, its accumulation on and selectivity and persistency of the effects are improved for tissues and/or organs in the living body, where proteoglycan is produced in excess amount, particularly for renal glomeruli. In this case, the term xe2x80x9cmajor particle size rangexe2x80x9d means a range of particle size in which 70% or more of particles are included when the particle size distribution of liposomes is defined by scattering intensity distribution in the particle size measurement based on the laser light scattering methods.
The present inventors have accomplished this invention by conducting intensive studies based on the aforementioned new knowledge.
Accordingly, the present invention provides a liposome in which a drug is included, which comprises (1) a basic compound which takes positive charge within a physiological pH range, (2) a lipid derivative of a hydrophilic polymer and (3) a lipid which constitutes the liposome, as its membrane constituting components, wherein their constituting ratios are from 1 to 20 mol % of (1) based on (3) and from 0.2 to 5 mol % of (2) based on the total of (1) and (3).
More preferred is a liposome which comprises (1) a basic compound which takes positive charge within a physiological pH range, (2) a lipid derivative of a hydrophilic polymer and (3) a lipid which constitutes the liposome, as its membrane constituting components, wherein their constituting ratios are from 5 to 15 mol % of (1) based on (3) and from 0.2 to 5 mol % of (2) based on the total of (1) and (3).
The aforementioned basic compound which takes positive charge within a physiological pH range is preferably a basic compound which has an amidino group, a basic compound which has two or more amino groups, a basic compound which has a piperidine ring or a basic compound which has a quaternary amine, more preferably any one of those represented by the following formulae 1 to 4. 
In this connection, the compound of formula 1 is described in WO 97/42166, the compounds of formulae 2 and 3 are described in JP-A-9-263579 and the compound of formula 4 is described in Chem. Papers, 39 (1), 125-134 (1985), but these documents describe nothing about a liposome having active targeting property and ensured safety in blood, which can be used in the diagnosis and/or treatment of diseases, particularly renal diseases, that accompany production of various types of proteoglycan in injured portions of tissues and/or organs. 
(In the formula 1, A represents an aromatic ring, each of R1 and R2 represents an alkyl or alkenyl group having from 10 to 25 carbon atoms, wherein R1 and R2 may be the same or different from each other, each of X1 and X2 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94OCOxe2x80x94, xe2x80x94CONHxe2x80x94 or xe2x80x94NHCOxe2x80x94, wherein X1 and x2 may be the same or different from each other, m is 0 or 1 and n is 0 or an integer of from 1 to 6.) 
(In the formula 2, R3 represents hydrogen or an alkyl or alkenyl group having from 1 to 8 carbon atoms, R4 represents hydrogen or an alkyl or alkenyl group having from 1 to 8 carbon atoms, each of R5 and R6 represents hydrogen or an alkyl or alkenyl group having from 1 to 25 carbon atoms (excluding a case in which R5 and R6 are both hydrogen), wherein R5 and R6 may be the same or different from each other, X3 represents xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, p is 0 or 1 and q is 0 or an integer of from 1 to 10.) 
(In the formula 3, each of R7 and R8 represents an alkyl or alkenyl group having from 1 to 8 carbon atoms, wherein R7 and R8 may be the same or different from each other, R9 represents hydrogen or an alkyl or alkenyl group having from 1 to 8 carbon atoms, each of R10 and R11 represents hydrogen or an alkyl or alkenyl group having from 1 to 25 carbon atoms (excluding a case in which R10 and R11 are both hydrogen), wherein R10 and R11 may be the same or different from each other, X4 represents xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, r is 0 or 1 and s is 0 or an integer of from 1 to 10.) 
The aforementioned lipid derivative of a hydrophilic polymer is preferably a lipid derivative of polyethylene glycol, more preferably, said lipid derivative of polyethylene glycol is a compound which contains a polyethylene glycol chain and a diacyl glycerol in one molecule.
It is desirable that the aforementioned hydrophilic polymer has a molecular weight of from 1,000 to 7,000.
Regarding the aforementioned liposome, it is desirable that its major particle size range is from 90 to 200 nm.
It is desirable that the aforementioned drug to be included in the liposome is a drug for use in the diagnosis and/or treatment of renal diseases or a drug for use in the diagnosis and/or treatment of diseases which accompany overproduction of proteoglycan in injured portions of tissues and/or organs.
It is desirable that the aforementioned drug for use in the diagnosis and/or treatment of renal diseases is an adrenocortical steroid and/or a derivative thereof.
It is desirable to use the aforementioned liposome as a drug for the treatment of inflamed kidney, and it is desirable to produce a medicament for the treatment of inflamed kidney making use of such liposomes.
The invention also provides a method for the treatment of inflamed kidney, which comprises administering these liposomes to mammals including human.