HK1111101A - Drug carrier and drug carrier kit for inhibiting fibrosis - Google Patents

Description

Drug carrier and drug carrier kit for inhibiting fibrosis

Technical Field

The present invention relates to a Drug carrier used in a Drug Delivery System (DDS) for stellate cells, a Drug containing the Drug carrier, and a kit for preparing the Drug, and more particularly, to a Drug containing an active ingredient for controlling the activity or proliferation of stellate cells, particularly a Drug targeting extracellular matrix-constituting molecules secreted from stellate cells or targeting 1 or more molecules that function to the production or secretion of extracellular matrix-constituting molecules, and a kit for preparing the Drug.

Background

Liver fibrosis is not limited, but, for example, viral liver diseases caused by hepatitis B or C virus, nonalcoholic steatohepatitis, diabetes with malnutrition, infection diseases such as parasites, tuberculosis, and syphilis, liver blood deficiency due to heart disease, and liver tissue injury accompanying bile passage failure, etc. are healed by trauma, and as a result, Hepatic Stellate Cells (HSCs) are activated, and extracellular matrices (ECMs) such as various kinds of collagen molecules and Fibronectin (fibrinectin) excessively produced or secreted are deposited in the interstitium, thereby causing liver fibrosis. In the end stage of hepatic fibrosis, liver cirrhosis is caused, and hepatic insufficiency, liver cancer, and the like are caused, and therefore, in order to prevent these diseases and/or to suppress their development, development of a drug carrier or a drug carrier kit which at least suppresses hepatic fibrosis is desired.

In addition, in the pancreas, fibrosis of pancreas is also caused by the same mechanism as that of hepatic fibrosis, resulting in chronic pancreatitis (Madro A et al, Med Sci Unit.2004 Jul; 10 (7): RA 166-70.; Jaster R, Mol cancer.2004 Oct 06; 3 (1): 26). However, no effective method for suppressing the development of pancreatic fibrosis, chronic pancreatitis, or the like has been known.

As an effective method for inhibiting fibrosis of the Liver or pancreas, astrocytes are likely to be one of the important target candidates (Fallowfield JA, Iredale JP, Expert Opin therger targets.2004 Oct; 8 (5): 423-35; Pinzani M, Rombouts K. dig Liver Dis.2004 Apr; 36 (4): 231-42.). During fibrosis, stellate cells are activated by cytokines from Kupffer cells or infiltrating cells, transformed into activated cells, and significantly produce extracellular matrix (ECM). Stellate cells are known to be storage cells for vitamin a and belong to the muscle fibroblast family. In addition, stellate cells produce matrix degrading enzymes (MMPs), cytokines such as inhibitory factor (TIMPs), TGF- β, PDGF, and proliferation factors such as HGF, and have a major effect on hepatic fibrosis. The activated stellate cells are associated with an increase in contractile energy to regulate blood flow, and also increase the expression of various cytokine receptors, showing higher sensitivity to cytokines.

As a method for treating fibrosis, methods attempted so far include control of collagen metabolism, promotion of a collagen degradation system, inhibition of activation of stellate cells, and the like. Which comprises the following steps: a truncated TGF-beta type II receptor (Qi Z et al, Proc Natl Acad Sci USA.1999 Mar 2; 96 (5): 2345-9.) or a soluble TGF-beta type II receptor (George J et al, Proc Natl Acad Sci USA.1999 Oct 26; 96 (22): 12719-24.) or HGF (Japanese patent application No. 5-503076, Ueki K et al, Nat Med.1999 Feb; 5 (2): 226-30.) or the like (known as a factor that activates stellate cells to promote the production of extracellular matrix (ECM)), and a matriptase (MMP) production-promoting vector containing HGF or MMP gene (Iimu Y et al, Gastroentero biology research biology; 124: 445: 458.) or a TIMP inhibiting gene using HGF or MMP gene (WormJ 9J 9-9J 316), and the like (Limuld. 9-9: 9-30.) (, Control of stellate cell activation using PPAR γ ligands (Marra F et al, gastroenterology.2000 Aug; 119 (2): 466-78) or angiotensin-II type I receptor antagonists (Yoshiji H et al, hepatology.2001 Oct; 34(4 pt 1): 745-50.), inhibition of PDGF action via PDGF tyrosine kinase inhibitors and the like (Liu XJ et al, World J gastroenterology.2002 Aug; 8 (4): 739-45.) and sodium channel inhibition using amiloride (Benedetti A et al, gastroenterology.2001 Feb; 120 (2): 545-56) and control of stellate cell proliferation using Stroke L et al, World J gastroenterology 2004 October 1; 10) (10J 25J 2831; 2831 JJ 2835) and induction of apoptosis using Glostrich L et al, hepatology.40, JJ.40 et al. However, in either case, since the action specificity and/or the organ specificity are low, there are problems in terms of effects and side effects.

There are many cases where the metabolic pathway is unknown in collagen protein synthesis, and a therapeutic method using a drug that suppresses this problem has not yet been established as an effective and biologically safe therapeutic method from the viewpoint of side effects and the like. That is, in the method of targeting molecules involved in collagen production, since these molecules have various functions, the target specificity cannot be improved, and the possibility of side effects is high. If collagen as a final product can be directly inhibited, it is reasonable as a general therapeutic method for the fibrosis process, but for this purpose, it is necessary to control all of the types of collagen represented by types I to IV.

One of the effective methods for simultaneously inhibiting the synthesis of various collagen molecules without losing the specificity to collagen is to control the function of HSP 47. HSP47 is a collagen-specific chaperone (chaperone) that is common in the synthesis of various types of collagen and is essential for intracellular delivery and molecular maturation. Therefore, if HSP47 function can be specifically controlled in stellate cells, inhibition of hepatic fibrosis can be considered, but there is no report on attempting such a treatment.

The present inventors have produced ribozymes that specifically control the function of HSP47 in a cell system and have shown that this can transiently inhibit collagen production and secretion (Sasaki H, et al. journal of immunology, 2002, 168: 5178-83; Hagiwara S, et al. J Gene Med.2003, 5: 784-94). To specifically inhibit HSP47 synthesis, sirnas that are more easily optimized than ribozymes can be used. The term "siRNA (small interfering RNA)" as used herein refers to a generic term of double-stranded RNA used in RNAi (RNA interference). RNAi refers to the phenomenon in which double-stranded RNA (double-stranded RNA, dsRNA) composed of sense RNA and antisense RNA, which is identical to a certain gene, destroys the same part of the transcription product (mRNA) of the gene, and has been shown in experiments in which nematodes were used at the beginning (Fire A, et al: Nature (1998) 391: 806-811), and it has been clarified that the same induction mechanism exists in mammalian cells (Ui-Tei K, et al: FEBS Lett (2000) 479: 79-82). In addition, Elbashir et al have proposed that short dsRNA of about 21-23 bp in length can induce RNAi in mammalian cell systems without exhibiting cytotoxicity (Elbashir SM, et al: Nature (2001) 411: 494-498). However, in order to effectively exert the effects of these molecules, a method specific to the target organ is required.

Patent document 1: japanese Kohyo publication Hei 5-503076

Non-patent document 1: madro A, et al, Med Sci Monit.2004 Jul; 10(7): RA166-70

Non-patent document 2: jaster R, Mol cancer.2004 Oct 06; 3(1): 26

Non-patent document 3: fallowfield JA, Iredale JP, Expert Opin Ther targets.2004 Oct; 8(5): 423-35

Non-patent document 4: pinzani M, Rombouts k. dig Liver dis.2004 Apr; 36(4): 231-42

Non-patent document 5: qi Z et al, Proc Natl Acad Sci USA.1999 Mar 2; 96(5): 2345-9

Non-patent document 6: george J et al, Proc Natl Acad Sci USA.1999 Oct 26; 96(22): 12719-24

Non-patent document 7: ueki K et al, Nat Med.1999 Feb; 5(2): 226-30

Non-patent document 8: iumuro Y et al, Gastroenterology 2003; 124: 445-458

Non-patent document 9: liu WB et al, World J gastroenterol.2003 Feb; 9(2): 316-9

Non-patent document 10: marra F et al, gastroenterology.2000 Aug; 119(2): 466-78

Non-patent document 11: yoshiji H et al, hepatology.2001 Oct; 34(4 Pt 1): 745-50

Non-patent document 12: liu XJ et al, World J gastroenterol.2002 Aug; 8(4): 739-45

Non-patent document 13: benedetti A et al, gastroenterology.2001 Feb; 120(2): 545-56

Non-patent document 14: wang L et al, World J Gastroenterol 2004 October 1; 10(19): 2831-2835

Non-patent document 15: orr JG et al, hepatology.2004 Jul; 40(1): 232-42

Non-patent document 16: sasaki H et al, Journal of Immunology, 2002, 168: 5178-83

Non-patent document 17: hagiwara S et al, J Gene Med.2003, 5: 784-94

Non-patent document 18: fire A, etc.: nature (1998) 391: 806-811

Non-patent document 19: Ui-Tei K, etc.: FEBS Lett (2000) 479: 79-82

Non-patent document 20: elbashir SM et al: nature (2001) 411: 494-498

Non-patent document 21: the new development of the dtitan hydrochloride, drug delivery system DDS technology and its method of use-the most advanced technology for biomedical research and advanced medical treatment-Medicaldo corporation, ISBN: 4944157932, 2003

Non-patent document 22: bridgefield charging, drug delivery system-new challenges for drug development and treatment, new bioscience books, chemists, ISBN: 4759803858, 1995

Disclosure of Invention

The use of a Drug Delivery System (DDS) is one of the effective approaches for targeting tissues and/or organs (Staudinate hydrochloride, new advances in DDS technology of drug delivery systems and methods of their use-the most advanced technology for biomedical research, advanced medicine-medical Co., Ltd., ISBN: 4944157932, 2003; Tantada George, drug delivery systems-new challenges for drug development and treatment, new bioscience books, Chemicals, ISBN: 4759803858, 1995). Drug carriers used in Drug Delivery Systems (DDS) include carriers using polymeric micelles, liposomes, microemulsions, and the like. In order to improve the specificity of these vectors for target organs, there are known a technique of mixing or binding an antigen specific to an organ and/or a tissue, an antibody and/or a ligand of a receptor, and the like into the vector, a technique utilizing physicochemical properties of the vector, and the like.

The present invention relates to drug carriers and drug carrier kits capable of specifically delivering diagnostic and/or therapeutic drugs to stellate cells. The drug carrier of the present invention may be in the form of any one of polymeric micelles, liposomes, emulsions, microspheres, or nanospheres, and can specifically deliver a therapeutic drug to hepatic stellate cells by incorporating or including therein vitamin a (va) or a retinoid (retinoid) derivative such as tretinoin, adapalene (adapalene), vitamin a palmitate, or the like, or a vitamin a analog such as fenretinide (4-HPR) or the like. Further, a pharmaceutical carrier containing 1 or more molecules selected from a TGF-beta activity inhibitor such as a truncated TGF-beta type II receptor or a soluble TGF-beta type II receptor, a growth factor preparation such as HGF, an MMP production promoter such as an adenovirus vector containing an MMP gene, a PPAR γ -ligand, an angiotensin-II type I receptor antagonist, a PDGF tyrosine kinase inhibitor, a cell activation inhibitor and/or a proliferation inhibitor containing a sodium channel inhibitor such as amiloride, and an apoptosis inducer such as compound 861 and gliotoxin is prepared, and oral administration or non-oral administration, such as intravenous or intraperitoneal administration, is performed to a patient at risk of fibrosis or having a fibrotic condition, or a patient having various diseases caused by fibrosis, such as liver cirrhosis, hepatic insufficiency, liver cancer, or chronic pancreatitis, can inhibit activation of stellate cells, and can prevent, inhibit or ameliorate fibrosis and/or the above-mentioned various disease states associated with fibrosis. Alternatively, in addition to this, when a ribozyme, antisense RNA, or siRNA that specifically inhibits HSP47, which is a collagen-specific chaperone, or TIMP, which is an MMP inhibitory factor, is used by encapsulating it in the drug carrier, it is possible to inhibit the secretion of each of type I to type IV collagen at the same time, and as a result, the fibrogenesis can be effectively inhibited.

The present invention therefore relates to stellate cell-specific pharmaceutical carriers comprising a retinoid derivative and/or a vitamin a analogue as a constituent.

The invention also relates to the above pharmaceutical carrier, characterized in that the retinoid derivative comprises vitamin a.

The invention also relates to the drug carrier, which is characterized by containing 0.2-20 wt% of the retinoid derivative and/or the vitamin A analogue.

The invention also relates to the drug carrier, which can be any one of polymer micelle, liposome, emulsion, microsphere and nanosphere.

In addition, the present invention relates to a pharmaceutical product containing the above-mentioned drug carrier and a drug that controls the activity or proliferation of stellate cells, and used for treating a disease associated with stellate cells.

The present invention relates to the above-mentioned pharmaceutical preparation, wherein the disease is selected from the group consisting of hepatitis, hepatic fibrosis, cirrhosis, liver cancer, pancreatitis, pancreatic fibrosis, pancreatic cancer, vocal cord scarring, vocal cord mucosal fibrosis and laryngeal fibrosis.

The present invention relates to the above drug, wherein the drug that controls activation or proliferation of stellate cells is selected from the group consisting of TGF β activity inhibitor, HGF active agent, MMP production promoter, TIMP production inhibitor, PPAR γ ligand, angiotensin activity inhibitor, PDGF activity inhibitor, sodium channel inhibitor, apoptosis inducer, siRNA, ribozyme, antisense nucleic acid, DNA/RNA chimeric polynucleotide, and a vector expressing the same, which targets an extracellular matrix-constituting molecule produced by stellate cells or one or more of molecules that act on production or secretion of the extracellular matrix-constituting molecule.

The present invention also relates to the above pharmaceutical product, wherein the molecule that acts on the production or secretion of extracellular matrix-constituting molecules is HSP 47.

The present invention also relates to the above-mentioned pharmaceutical product, which is formed by mixing a drug and a drug carrier at or near a medical site.

The present invention also relates to a kit for preparing the above drug, comprising: a drug that controls the activity or proliferation of the stellate cells, a drug carrier-forming substance, and 1 or more containers containing one or more of a retinoid derivative and/or a vitamin a analog.

The present invention relates to a method for treating a stellate cell-related disease, which comprises administering an effective amount of the above drug to a subject in need thereof.

The present invention relates to the above method wherein the disease is selected from the group consisting of hepatitis, liver fibrosis, cirrhosis, liver cancer, pancreatitis, pancreatic fibrosis, pancreatic carcinoma, vocal cord scarring, vocal cord mucosal fibrosis and laryngeal fibrosis.

The present invention relates to the above method, wherein the pharmaceutical product is administered non-orally.

The invention relates to the use of the aforementioned pharmaceutical carrier for the manufacture of a medicament for the treatment of a disease associated with astrocytes.

The invention also relates to a method for delivering the drug to the stellate cells by using the drug carrier.

The invention also relates to the following pharmaceutical carriers: characterized in that a drug carrier for inhibiting fibrosis, which comprises a retinoid derivative and/or a vitamin A analog as a component and specifically delivers a drug for controlling the activity or proliferation of stellate cells to the stellate cells; the above-mentioned drug carrier for inhibiting fibrosis characterized in that a retinoid derivative contains vitamin A; the drug carrier for inhibiting fibrosis is characterized by containing 0.2-20% of a retinoid derivative and/or a vitamin A analogue; the drug carrier for inhibiting fibrosis is any form of polymer micelle, liposome, emulsion, microsphere and nano-microsphere; characterized in that the drug for controlling the activity or proliferation of stellate cells comprises the above-mentioned drug carrier for inhibiting fibrosis of 1 or more drugs selected from the group consisting of a TGF β activity inhibitor, an HGF active agent, an MMP production promoter, a TIMP production inhibitor, a PPAR γ ligand, an angiotensin activity inhibitor, a PDGF activity inhibitor, a sodium channel inhibitor and an apoptosis inducer; a drug carrier for inhibiting fibrosis, characterized in that the drug for controlling the activity or proliferation of stellate cells comprises an siRNA, a ribozyme, an antisense RNA, or a vector expressing the siRNA, the ribozyme, the antisense RNA, or the vector, which targets an extracellular matrix-constituting molecule produced by stellate cells, or one or more of molecules that act on the production or secretion of the extracellular matrix-constituting molecule; the above-mentioned drug carrier for inhibiting fibrosis, which molecule exerts an effect on the production or secretion of extracellular matrix-constituting molecules, is HSP 47.

The invention also relates to the following pharmaceutical carrier kit: a drug carrier kit for inhibiting fibrosis, which comprises a drug for controlling the activity or proliferation of stellate cells, a drug carrier-constituting substance, and 1 or more containers containing 1 or more of a retinoid derivative and/or a vitamin a analog; the above pharmaceutical carrier kit for inhibiting fibrosis characterized in that a retinoid derivative contains vitamin a; the drug carrier kit for inhibiting fibrosis is characterized by containing 0.2-20% of a retinoid derivative and/or a vitamin A analogue; the drug carrier kit for inhibiting fibrosis is in any form of polymer micelle, liposome, emulsion, microsphere and nanosphere; a pharmaceutical carrier kit for inhibiting fibrosis characterized in that the drug for controlling the activity or proliferation of stellate cells comprises 1 or more drugs selected from the group consisting of a TGF β activity inhibitor, an HGF active agent, an MMP production promoter, a TIMP production inhibitor, a PPAR γ ligand, an angiotensin activity inhibitor, a PDGF activity inhibitor, a sodium channel inhibitor and an apoptosis inducer; a pharmaceutical carrier kit for inhibiting fibrosis, characterized in that the drug for controlling the activity or proliferation of stellate cells comprises an siRNA, a ribozyme, an antisense RNA, or a vector expressing the siRNA, the ribozyme, the antisense RNA, or the vector targeting an extracellular matrix-constituting molecule secreted by stellate cells or one or more of molecules that act on the production or secretion of the extracellular matrix-constituting molecule; the above-mentioned pharmaceutical carrier kit for inhibiting fibrosis, in which the molecule that acts on the production or secretion of extracellular matrix-constituting molecules is HSP 47.

As an effective method for preventing, inhibiting or ameliorating fibrosis and/or various diseases caused by accompanying fibrosis, the intended therapeutic effect as seen in the examples can be provided by using the drug carrier and the drug carrier kit of the present invention capable of specifically delivering a diagnostic and/or therapeutic drug to stellate cells. That is, the drug carrier and the drug carrier kit of the present invention use astrocytes as specific targets, and therefore can suppress a pathological state such as fibrosis or the like, which is expressed by astrocytes as a center, with high efficiency, and minimal side effects.

Drawings

FIG. 1 is a diagram showing the protocol for in vitro determination of the effect of gp46-siRNA using NRK cells and determination of the optimal sequence, period and concentration.

FIG. 2 is a photograph showing the results of immunoblotting of gp46 and actin (study of optimal sequence after 24-hour culture).

FIG. 3 is a photograph showing the results of immunoblotting of gp46 and actin (study of optimum concentration in 24-hour culture).

FIG. 4 is a photograph showing the results of immunoblotting of gp46 and actin (at a concentration of 50nM, for optimum culture time).

FIG. 5 is a graph showing the protocol for evaluating the inhibition of collagen expression by gp46-siRNA in NRK cells.

FIG. 6 is a graph showing the inhibition of collagen synthesis by siRNA.

FIG. 7 is a photograph showing introduction of siRNA specific to HSC.

FIG. 8 is a photograph for evaluating the introduction rate of siRNA specific to HSC.

FIG. 9 is a photograph for evaluating inhibition of gp46 expression by siRNA.

FIG. 10 is a photograph showing Azan staining of the liver of a rat to which DMN was administered.

Fig. 11 is a diagram showing an LC rat treatment protocol.

FIG. 12 is a photograph showing Azan staining of the liver of LC rats administered with VA-Lip-gp46 siRNA.

FIG. 13 is a diagram showing a method of extracting a stained portion using an NIH image. 6 positions were randomly photographed from the Azan stain images.

Fig. 14 is a graph showing the area ratio (collagen area ratio,%) of the fibrosis portion in the liver tissue image.

Fig. 15 is a graph showing the amount of hydroxyproline in liver tissue.

FIG. 16 is a graph showing survival curves of cirrhosis rats administered with VA-Lip-gp46siRNA intraportally.

FIG. 17 is a photograph showing Azan staining of liver tissue of a liver cirrhosis rat administered with VA-Lip-gp46siRNA in the portal vein.

FIG. 18 is a graph showing survival curves of cirrhosis rats administered with VA-Lip-gp46siRNA intraportally.

FIG. 19 is a photograph showing Azan staining of liver tissue of a liver cirrhosis rat administered with VA-Lip-gp46siRNA intraportally.

FIG. 20 is a graph showing survival curves of cirrhosis rats intravenously administered VA-Lip-gp46 siRNA.

FIG. 21 is a graph showing survival curves of cirrhosis rats intravenously administered VA-Lip-gp46 siRNA.

FIG. 22 is a photograph showing Azan staining of liver tissue of a liver cirrhosis rat to which VA-Lip-gp46siRNA was intravenously administered.

FIG. 23 is a graph showing that the introduction efficiency of VA-Lip-gp46siRNA is improved by RBP.

FIG. 24 is a view showing inhibition of introduction of VA-Lip-gp46siRNA by an anti-RBP antibody.

Detailed Description

The retinoid derivative and/or vitamin a analog of the present invention contains vitamin a, and further includes a retinoid derivative and/or a vitamin a analog dissolved or mixed in a solvent capable of dissolving or retaining the vitamin a.

The retinoid derivative and/or vitamin a analog of the present invention may be any type as long as they can be positively accumulated in stellate cells, and examples of the retinoid derivative include, but are not limited to, tretinoin, adapalene, and retinol, and particularly tretinoin, and examples of the vitamin a analog include, but are not limited to, fenretinide (4-HPR). The present invention utilizes the property of actively taking in retinoid derivatives and/or vitamin a analogs by stellate cells, and can deliver desired substances or objects specifically to stellate cells by using these retinoid derivatives and/or vitamin a analogs as a drug carrier or by incorporating or including them in other components constituting the drug carrier.

Therefore, the pharmaceutical carrier of the present invention may further contain a pharmaceutical carrier constituting component other than the retinoid derivative and/or the vitamin a analog. The ingredient is not particularly limited, and any ingredient known in the fields of medicine and pharmacy may be used, and preferably, an ingredient that may contain a retinoid derivative and/or a vitamin a analog, or may be combined therewith may be contained. Examples of such components include, but are not limited to, lipids, phospholipids such as glycerophosphate, sphingolipids such as sphingomyelin, sterols such as cholesterol, vegetable oils such as soybean oil and poppy oil, mineral oils, and lecithins such as egg yolk lecithin. Among them, preferred are those which can constitute liposomes, for example, natural phospholipids such as lecithin, semisynthetic phospholipids such as Dimyristoylphosphatidylcholine (DMPC), Dipalmitoylphosphatidylcholine (DPPC) and Distearoylphosphatidylcholine (DSPC), cholesterol, and the like.

In addition, the pharmaceutical carrier of the present invention may also contain a substance that improves uptake into stellate cells, such as Retinol Binding Protein (RBP).

The incorporation of the retinoid derivative and/or vitamin a analog on the pharmaceutical carrier of the invention may alternatively comprise chemically and/or physically incorporating the retinoid derivative and/or vitamin a analog in or with other components of the pharmaceutical carrier. Alternatively, the binding or inclusion of a retinoid derivative and/or a vitamin a analog on a pharmaceutical carrier of the invention can also be accomplished by incorporating a retinoid derivative and/or a vitamin a analog having an affinity for the basic pharmaceutical carrier components at the time of manufacture of the pharmaceutical carrier. The amount of retinoid derivative and/or vitamin a analogue incorporated or contained in the pharmaceutical carrier of the invention is 0.01 to 100%, preferably 0.2 to 20%, more preferably 1 to 5% by weight of the ingredients constituting the pharmaceutical carrier.

The form of the drug carrier of the present invention is not limited as long as it can deliver a desired substance or object to target stellate cells, and may be any form, for example, polymer micelles, liposomes, emulsions, microspheres, nanospheres, or the like. The drug carrier of the present invention may contain a mediator inside, may exist as attached to the outside of the mediator, or may be mixed with the mediator, as long as the retinoid and/or vitamin a analog contained therein is at least partially exposed to the outside of the preparation at the latest before reaching the stellate cells.

The drug carrier of the present invention is capable of achieving a desired effect, such as inhibition or prevention of fibrosis, with a maximum effect and minimal side effects, by efficiently delivering a desired substance or object, such as a drug that controls the activity or proliferation of stellate cells, to stellate cells with stellate cells as a specific target. The substance or substance to be delivered by the drug carrier is not particularly limited, but is preferably of a size that can physically move in vivo from the administration site to the liver, pancreas, or the like where stellate cells are present. Therefore, the drug carrier of the present invention can deliver not only substances such as atoms, molecules, compounds, proteins, nucleic acids, etc., but also substances such as drug delivery systems and micromachines composed of 1 or more elements in carriers, virus particles, and cells. The substance or object preferably has properties that confer an effect on the stellate cells, such as labeling the stellate cells, or controlling the activity or proliferation of the stellate cells.

Therefore, in one embodiment of the present invention, the substance to be delivered by the drug carrier is "a drug for controlling the activity or proliferation of stellate cells", and may be any drug which directly or indirectly inhibits the physicochemical action of stellate cells involved in the promotion of fibrosis, and examples thereof include, but are not limited to, TGF-beta activity inhibitors such as truncated TGF-beta type II receptor and soluble TGF-beta type II receptor, proliferation factor preparations such as HGF and expression vectors thereof, MMP production promoters such as adenovirus vectors containing MMP genes, TIMP production inhibitors such as antisense TIMP nucleic acids, PPAR γ ligands, angiotensin activity inhibitors, PDGF activity inhibitors, cell activation inhibitors and/or cell proliferation inhibitors containing sodium channel inhibitors, and apoptosis inducers such as compound 861 and gliotoxin, adiponectin (see Japanese patent laid-open publication No. 2002-363094), Rho kinase inhibitory activity, such as (+) -trans-4- (1-aminoethyl) -1- (4-pyridylcarbamoyl) cyclohexane (see WO 00/64478). The "drug for controlling the activity or proliferation of stellate cells" of the present invention may be any drug which directly or indirectly promotes the physicochemical action of stellate cells directly or indirectly involved in the inhibition of fibrosis, and includes, but is not limited to, for example, drugs which promote a collagen decomposition system, MMP production promoters such as MMP expression vectors, and HGF, HGF analogs, or their expression vectors, and drugs having HGF-like activity.

Other examples of the "drug for controlling the activity or proliferation of stellate cells" of the present invention include drugs for controlling the metabolism of extracellular matrix, for example, collagen; for example, siRNA, ribozyme, or antisense nucleic acid (including RNA, DNA, PNA, or a complex thereof) that has an effect of inhibiting the expression of a target molecule, which targets an extracellular matrix-constituting molecule produced by a stellate cell, or which targets 1 or more molecules that contribute to the production or secretion of the extracellular matrix-constituting molecule; or a substance having a dominant negative effect, or a vector expressing the substance or the vector.

siRNA is double-stranded RNA having a sequence specific to a target molecule such as mRNA, and promotes degradation of the target molecule to inhibit expression of a substance formed thereby, for example, a protein (RNA interference). Since Fire et al published its principles (Nature, 391: 806-811, 1998), extensive research has been conducted on the optimization of siRNA, and those skilled in the art are well versed in this technology. In addition, RNA interference other than siRNA and other substances causing a response of inhibiting gene expression have been studied intensively, and the number of such substances is also large at present.

For example, Japanese patent laid-open No. 2003-219893 describes a double-stranded polynucleotide composed of DNA and RNA for inhibiting the expression of a target gene. The double strand of the polynucleotide may be a DNA/RNA hybrid in which one of the strands is DNA and the other strand is RNA, or a DNA/RNA chimera in which a portion of the same strand is DNA and the other portion is RNA. The polynucleotide is preferably composed of 19 to 25, more preferably 19 to 23, further preferably 19 to 21 nucleotides, and in the case of a DNA/RNA hybrid, the sense strand is preferably DNA and the antisense strand is preferably RNA; in the case of a DNA/RNA chimera, a portion of the upstream side of the double-stranded polynucleotide is preferably RNA. The polynucleotide can be prepared into a substance having an arbitrary sequence by a chemical synthesis method known per se and according to a conventional method.

The target molecule is preferably a molecule capable of inhibiting all secretion of extracellular matrix-constituting molecules, and examples of such a molecule include, but are not limited to, HSP 47. The gene sequence of HSP47 or its homologues is disclosed, for example, in the form of GenBank accession No. ab010273 (human), X60676 (mouse), M69246 (rat, gp 46).

Therefore, examples of preferable substances to be delivered by the drug carrier of the present invention include siRNA targeting HSP47, DNA/RNA hybrid, chimeric polynucleotide, antisense nucleic acid, and the like.

Examples of the drug carrier delivery material of the present invention include agents for inhibiting fibrosis, such as G-CSF (see WO 2005/082402), thrombomodulin-like protein (see Japanese patent application laid-open No. 2002-371006), and keratan sulfate oligosaccharide (see Japanese patent application laid-open No. 11-269076).

The substance or object delivered by the drug carrier of the present invention may or may not be labeled. By labeling, it is possible to monitor whether or not transmission, increase or decrease of stellate cells, and the like, and it is particularly useful at the level of test and study. The label may be selected from any substance known to those skilled in the art, for example, any radioisotope, a substance (for example, an antibody) bonded to a labeled substance, a fluorescent substance, fluorescein (Fluorophore), a chemiluminescent substance, an enzyme, and the like.

The present invention also relates to a pharmaceutical product for treating a disease associated with stellate cells, which contains the above-mentioned drug carrier and a drug that controls the activity or proliferation of the above-mentioned stellate cells, and to the use of the above-mentioned drug carrier for producing a pharmaceutical product for treating a disease associated with stellate cells. Here, the diseases related to astrocytes are diseases directly or indirectly related to the processes of astrocytes and diseases, that is, the onset, progression, amelioration, remission, cure, etc. of diseases, and include, for example, liver diseases such as hepatitis, particularly chronic hepatitis, hepatic fibrosis, cirrhosis, and liver cancer; pancreatitis, especially chronic pancreatitis, pancreatosis, pancreaticofibrosis, and pancreatic cancer. In addition, recent reports have indicated that vocal cords and throat diseases such as vocal scar formation, vocal cord mucosal fibrosis and laryngeal fibrosis are also included in the above-mentioned diseases because stellate cells are also present in the vocal cords (see, for example, Fuja TJ et al, Cell Tissue Res.2005; 322 (3): 417-24).

In the drug of the present invention, the drug carrier may contain a drug therein, may be present as attached to the outside of the drug-containing body, or may be mixed with a drug, as long as at least a part of the retinoid derivative and/or the vitamin a analog contained in the drug carrier is exposed to the outside of the preparation at the latest before reaching the stellate cells. Therefore, the drug may be coated with an appropriate material, for example, an enteric coating agent or a time-disintegrable material, depending on the administration route, drug release pattern, or the like, and may be incorporated into an appropriate drug release system.

The pharmaceutical product of the present invention can be administered by various routes including both oral and non-oral routes, for example, oral, intravenous, intramuscular, subcutaneous, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, and the like, and can be formulated into dosage forms suitable for the respective routes of administration without limitation. The dosage form and the preparation method may be any known dosage form and method (for example, refer to standard pharmacy, editions such as Bianbian Xie, etc., Nanjiang Tang, 2003, etc.).

For example, the dosage form suitable for oral administration is not limited, and includes, for example, powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, syrups and the like, and the dosage form suitable for non-oral administration includes injections such as solution injections, suspension injections, emulsion injections, and extemporaneous injections. The preparation for parenteral administration may be in the form of an aqueous or non-aqueous isotonic sterile solution or suspension.

The pharmaceutical carrier or drug of the present invention may be supplied in any form, but from the viewpoint of storage stability, it is preferably supplied in a form that can be prepared at the time of use, for example, in a form that can be prepared by a doctor, a pharmacist, a nurse, or other medical support (paramedical) at or near a medical site. In this case, the pharmaceutical carrier or drug of the present invention is provided in the form of 1 or more containers containing therein at least one of the essential components, and is prepared before use, for example, within 24 hours, preferably within 3 hours, and more preferably immediately before use. In the preparation, a reagent, a solvent, a preparation kit, and the like, which are generally available at the preparation site, can be used as appropriate.

Accordingly, the present invention also includes a kit for preparing a pharmaceutical carrier or a pharmaceutical product, wherein the pharmaceutical carrier comprises 1 or more containers containing 1 or more of a pharmaceutical carrier-constituting substance, a retinoid derivative and/or a vitamin a analog, and/or a pharmaceutical product, and the present invention also includes the necessary components of the pharmaceutical carrier or the pharmaceutical product provided in the form of the kit. The kit of the present invention may further comprise, in addition to the above, instructions describing the pharmaceutical carrier of the present invention, a method for preparing a drug, a method for administering a drug, and the like. The kit of the present invention may contain all of the components for completing the pharmaceutical carrier or drug of the present invention, or may not contain all of the components. Thus, the kit of the present invention may further contain reagents, solvents, such as sterile water, physiological saline, glucose solution, and the like, which are generally available in medical fields, laboratory facilities, and the like.

The present invention also relates to a method for treating an astrocyte-related disease comprising administering an effective amount of the above drug to a subject in need thereof. The effective amount here means an amount that reduces the onset of the subject disease, alleviates symptoms, or prevents progression, and is preferably an amount that prevents the onset of the subject disease or cures the subject disease. In addition, it is preferred that the amount does not adversely affect the benefits obtained by administration. The amount can be determined appropriately by in vitro tests using cultured cells or the like, and tests using model animals such as mice, rats, dogs, or pigs, and such test methods are well known to those skilled in the art.

The amount of the drug to be administered in the method of the present invention varies depending on the kind of the drug, retinoid derivative and/or vitamin A analog used, and when, for example, siRNA to HSP47 is used as the drug, the weight of the drug is, for example, 0.01 to 45 mg/kg/day, preferably 0.1 to 30 mg/kg/day, more preferably 1 to 20 mg/kg/day, most preferably 4 to 6 mg/kg/day. When vitamin A is used as a retinoid derivative and/or vitamin A analogue, vitamin A is typically administered in an amount of 10-20 mg/kg/day. The amount of retinoid derivative and/or vitamin a analog contained in the pharmaceutical carrier and the drug used in the method of the present invention is known to those skilled in the art, or can be determined appropriately by the above-mentioned tests and the like.

The specific amount of the drug to be administered in the method of the present invention may be determined in consideration of various conditions related to the subject to be treated, such as the severity of symptoms, general health status of the subject, age, body weight, sex, diet, administration period and frequency of the subject, drugs used in combination, responsiveness to treatment, compliance with treatment, and the like, and thus may be different from the typical amounts described above, and even in such a case, these methods are still included in the scope of the present invention.

As the administration route, various routes including both oral and non-oral routes, for example, routes including oral, intravenous, intramuscular, subcutaneous, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, and intrauterine, and the like.

The frequency of administration varies depending on the nature of the drug to be used or the conditions of the subject, and may be, for example, 1 day (i.e., 2, 3, 4 or5 or more times per day), 1 time per day (i.e., 1 time per 2, 3, 4, 5, 6, 7 days, etc.), 1 time per week, and 1 time per week (i.e., 1 time per 2, 3, 4 weeks, etc.).

In the method of the present invention, the term "subject" refers to any biological subject, preferably an animal, more preferably a mammal, and even more preferably a human. In the present invention, a subject may be healthy or may be suffering from a disease, and when an attempt is made to treat a disease, it typically refers to a subject suffering from the same disease or at risk of suffering from the same disease.

In addition, the term "treatment" includes all types of medically allowable preventive and/or therapeutic interventions for the purpose of cure, temporary relief, prevention, or the like of a disease. For example, when the disease is hepatic fibrosis, the term "treatment" includes medically acceptable interventions for various purposes such as delaying or stopping the progression of fibrosis, regression or disappearance of lesions, prevention of the onset or recurrence of fibrosis, and the like.

The invention also relates to a drug delivery method for stellate cells by using the drug carrier. The method includes, but is not limited to, a step of supporting the drug carrier with the transfer substance, and a step of administering or adding the drug carrier with the transfer substance to an organism, a medium, such as a culture medium, or the like containing stellate cells. These steps can be suitably realized by any known method, a method described in the present specification, or the like. The above-described delivery method may be combined with other delivery methods, for example, with other delivery methods targeting an organ in which stellate cells exist, and the like.

Examples

The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the specific numerical values and sequences shown in the examples.

EXAMPLE 1 preparation of siRNA to gp64

Among the optimal sequences for siRNA recognition targeting the base sequence of HSP47 which is a common chaperone for collagen (types I to IV), sequences A and B were prepared according to the siRNA oligomerization program of IGENE Corp. The sequence C was prepared by searching 19-base sequences selected as targets for rat gp64 (human HSP47 analog, GenBank Accession No. M69246) on the Internet using siRNA Target Finder (http:// www.ambion.com/techlib/misc/siRNA _ Finder. html) manufactured by Ambion. In the design, attention is paid to 1) the position of the AA dimer at which the initial AA dimer is labeled, 2) the position of the AA dimer at which the AA dimer is labeled, and 3) the GC content of 30 to 70%. In this example, siRNA having the following sequence was prepared.

A: GUUCCACCAUAAGAUGGUAGACAAC (forward strand siRNA of 25 nucleotides from 757 th on the sequence, SEQ ID NO: 1)

B: CCACAAGUUUUAUAUCCAAUCUAGC (forward strand siRNA of 25 nucleotides from 1626 th on the sequence, SEQ ID NO. 2)

C: GAAACCUGUAGAGGCCGCA (19-base forward strand siRNA from the 64 th on the sequence, SEQ ID NO. 3)

Example 2 inhibition of gp46 expression by the siRNA prepared

Gp46 was obtained from rat, and each siRNA was transfected at 0.1nM to 50nM into normal rat kidney cells (NRK cells) of collagen-producing fibroblasts, and cultured for 12 to 48 hours (FIG. 1). The expression level of gp46 was confirmed by immunoblotting (see FIGS. 2 to 4, the upper band is gp46, and the lower band is actin as a control). Either siRNA significantly inhibited expression of gp46 protein compared to vehicle (figure 2). In the following experiments, siRNA having sequence a among them most effective was used. Using concentration-dependent inhibition by siRNA (FIG. 3), protein expression of gp46 was inhibited by 50nM siRNA by about 90% after 48 hours (FIG. 4).

Example 3 inhibition of collagen Synthesis by the produced siRNA

Rat fibroblast (NRK cells) culture supernatants for studies of collagen synthesis amount were added under the conditions as described above (siRNA concentration 50nM, treatment time 48 hours)³H-proline in secreted proteins after transfection³Amount of H (FIG. 5). The amount of collagen synthesis isAccording to the report of Peterkofsky et al (Peterkofsky et al, biochemistry.1971Mar 16; 10 (6): 988-94), in³Gp46siRNA was cultured in the presence of H-proline and introduced into fibroblasts, and the ratio of the amount of protein secreted into the supernatant to the amount of protein degraded by collagenase was calculated.

Mathematical formula 1

Collagen synthesis rate collagenase sensitive part x 100/(5.4 x collagenase non sensitive part + collagenase sensitive part)

The collagen synthesis rate of rat fibroblasts was reduced by about 40% compared to the control group (fig. 6).

EXAMPLE 4 Hepatic Stellate Cell (HSC) specific introduction of nucleic acids

An emulsion (VA-Lip-GFP) in which a liposome obtained by coating VA with 10% VA mixed with a liposome and GFP-expressing plasmid was prepared, and after administering the mixture into the portal vein of rats, liver tissues were collected and fixed. The emulsion was prepared in a manner assuming that the plasma volume of 200g rat was about 10ml and the concentration of VA and GFP in portal blood reached 10. mu.M. Specifically, 25mg of all-trans retinol (VA) was dissolved in 87. mu.l DMSO to prepare a 100mM stock solution. Mu.l of lipofectin and 179. mu.l of PBS were added to 1. mu.l of this VA stock solution, and 10. mu.g of GFP expression plasmid was further added thereto to make the total amount 200. mu.l, and vortexed for 3 minutes to prepare VA-Lip-GFP. SD rats were laparotomized and VA-Lip-GFP was slowly injected into the peripheral portal vein. Liver tissue was collected 48 hours after injection. Since the intramuscular line protein of the intermediate fiber (intermediate membrane) specifically expresses Hepatic Stellate Cells (HSC) compared to other liver cells, fixed liver tissues were stained with an Alexa Fluor 568-labeled intramuscular line protein antibody, and a fluorescent double image with GFP was observed, confirming that GFP was expressed in the Hepatic Stellate Cells (HSC) (fig. 7). In the group without treatment control or with GFP-expressing plasmid medium alone, no expression was observed in rat astrocytes, but in the group with VA-Lip-GFP, it was found that the astrocytes specifically expressed GFP.

Example 5 quantification of nucleic acid introduction Rate

An emulsion containing VA-coated liposomes and FITC-labeled gp46siRNA (VA-Lip-gp46siRNA (FITC)) was prepared and administered intraportally to SD rats (amount of siRNA 10 μ g/200 μ l) in the same manner as in example 4, except that FITC-labeled gp46siRNA was used instead of GFP-expressing plasmid. Liver tissues were collected 48 hours after the administration, and compared with other liver cells, α SMA (smooth muscle actin) specifically expressed in HSC was stained with Alexa Fluor 568-labeled anti- α SMA antibody, cell nuclei were stained with DAPI, respectively, and fluorescence images were observed with a confocal Laser Scanning Microscope (LSM). As shown on the left side of fig. 8, in the VA-Lip-gp46siRNA (FITC) administration group, cells emitting both green fluorescence by FITC and red fluorescence by Alexa Fluor568 were observed in many cases, and when quantification was performed by NIHimage (arbitrary 10 fields of x1000 fluorescence micrographs were selected and the number of cells was counted), the introduction efficiency was 77.6% (average value of 10 fields). On the other hand, in the group administered with Lip-gp46siRNA (FITC) containing no VA, the introduction efficiency was as low as 14.0%, and the introduction into cells other than astrocytes was 3.0% (see right side of fig. 8). From the above results, it was found that the introduction efficiency into stellate cells was greatly improved by the inclusion of VA.

Example 6 inhibition of gp46 expression Using VA-Lip-gp46siRNA

In other sections of the tissue collected by example 5, gp46 was stained with an Alexa Fluor 568-labeled anti-HSP 47 antibody, cell nuclei were stained with DAPI, respectively, and fluorescence images were observed with a confocal laser scanning microscope. As shown in fig. 9, in the VA-Lip-gp46 siRNA-administered group, the expression of gp46, which was visible as red fluorescence (right side of fig. 9), was significantly reduced compared to the control group (left side of fig. 9) to which VA-Lip-random siRNA containing random siRNA non-specific to gp46 was administered. The expression inhibition rate of the control group was 75% as high as that of example 7 in the 6-field average value, and the number of gp 46-negative cells was calculated by NIH image selection x1000 fluorescence micrographs at 10 fields.

EXAMPLE 7 treatment of LC rats (intraportal administration 1)

LC model rats were prepared using Dimethylnitrosamine (DMN) according to the report of Jezeque et al (Jezeque AM et al, J hepatol.1987 Oct; 5 (2): 174-81) (FIG. 10). Specifically, 1% Dimethylnitrosamine (DMN) was administered to 5-week-old SD rats (male) at a dose of 1ml/kg (intraperitoneal administration) and administered 3 consecutive days per week. As reported above, an increase in fibers was observed from week 2, and significant fibrosis, destruction of hepatic lobular structure, and formation of regenerative nodules were observed at week 4 (fig. 11). Therefore, gp46siRNA was lipidated by the same method as example 4, and mixed with 10% VA to form an emulsion (VA-Lip-gp46siRNA), and the emulsion was administered. VA-Lip-gp46siRNA was administered starting at week 3 where fibrosis was well visible and evaluated at weeks 4 and 5. Since the effect was confirmed up to 48 hours using the in vitro test of example 2, the administration was 2 times per week (fig. 11). The amount of siRNA to be administered was adjusted to 40. mu.g in total according to the conventional report on direct injection of siRNA (McCaffery et al, Nature.2002 Jul 4; 418 (6893): 38-9). In the azan staining of liver after siRNA administration, no significant difference was observed between the general diet group, siRNA (random) administration group and siRNA (gp46) administration group at week 4, but a decrease in the amount of fibers occurred in the gp46siRNA administration group at week 5 (FIG. 12). To quantify the amount of fiber, the stained portion was extracted using NIH image, the area was calculated (fig. 13), and a significant reduction in collagen area was seen in the gp46 siRNA-administered group (fig. 14). In order to evaluate the degree of fibrosis by another standard, hydroxyproline, which is an index of fibrosis, was quantified by a conventional method. Specifically, 20mg of freeze-dried liver tissue was hydrolyzed with HCl for 24 hours, and then the reaction solution was centrifuged, and the supernatant was treated with a reagent such as Euclidean solution, and centrifuged. The supernatant was recovered and the absorbance at 560nm was measured to determine the amount of hydroxyproline in liver tissue (Hepatology1998 Nov; vol.28: 1247-. As shown in FIG. 15, the amount of hydroxyproline was extremely small in the gp46 siRNA-administered group.

EXAMPLE 8 treatment of LC rats (intraportal administration 2)

To investigate the change in survival rate resulting from administration of the drug product of the invention, LC model rats were prepared using Dimethylnitrosamine (DMN) in 20% increments over the usual ones according to the method of Qi Z et al (Proc Natl Acad Sci USA.1999 Mar 2; 96 (5): 2345-9.). In this model, 4 intraportal administrations were performed on weeks 1 and 2. The administration contents were PBS, Lip-gp46siRNA, VA-Lip-random siRNA and VA-Lip-gp46siRNA (each group was n ═ 7). After 3 weeks, all of the control groups (PBS-administered group, VA-Lip-random siRNA-administered group, and Lip-gp46 siRNA-administered group) died, whereas 6 of 7 survived in the VA-Lip-gp46 siRNA-administered group (FIG. 16). In addition, a significant reduction in fiber amount was seen in gp46 siRNA-administered group in liver azan staining at day 21 (fig. 17).

EXAMPLE 9 treatment of LC rats (intraportal administration 3)

In another experiment, the LC model rats (1% DMN administered intraperitoneally 3 times per week at 1 mg/kg) prepared according to the methods of Qi Z et al and Ueki T et al (Nat med.1999 Feb; 5 (2): 226-30) described above were administered intraportally starting from week 3 as shown in the following table (n ═ 6 for each group). Each drug was administered in a total volume of 200 μ l after adding PBS, and the number of administrations was 1 time per week.

TABLE 1

Treatment group	Administration of drugs	Dosage of drug
			9-1	VA	VA 200nmol
9-2	Lip-gp46siRNA	Liposome 100nmol, gp46siRNA 20 ug
			9-3	VA-Lip-random siRNA	VA 200nmol, liposome 100nmol, random-siRNA 20 μ g
9-4	VA-Lip-gp46siRNA	VA 200nmol, liposome 100nmol, gp46siRNA 20 μ g

The results showed that 6 subjects died after 45 days from the start of DMN administration, except for the group to which the drug of the present invention was administered (treatment group 9-4), but all subjects died after 36 days from the start of DMN administration, and survived for more than 70 days from the start of DMN administration (fig. 18). In addition, it was found that, in the dead individuals, the amount of liver fibers was quantified from the collagen area in the same manner as in example 7, and that the administration of VA-Lip-gp46siRNA significantly suppressed the increase in the amount of liver fibers (fig. 19).

EXAMPLE 10 treatment of LC rats (intravenous administration)

LC model rats (1 μ g/bw (g)) prepared in the same manner as in example 9 were intraperitoneally administered 3 times a week with 1% DMN, and intravenous administration was performed from week 3 as shown in the following table (n ═ 6 in each group). Each drug was administered in such a manner that the total volume reached 200 μ l after adding PBS. The administration time was 10-4 groups to 7 weeks, 10-10 groups to 6 weeks, and all the other groups were administered until death.

TABLE 2

Treatment group	Administration of drugs	Dosage of drug	Number of administrations
				10-1	VA	VA 200nmol	2 times per week
10-2	Lip-gp46siRNA	Liposome 100nmol, gp46siRNA 100. mu.g
			10-3	VA-Lip-randomsiRNA	VA 200nmol, liposome 100nmol, random-siRNA 100 μ g
10-4	VA-Lip-gp46siRNA	VA 200nmol, liposome 100nmol, gp46siRNA 100 μ g
			10-5	PBS	200μl	3 times per week
10-6	VA	VA 200nmol
			10-7	VA-Lip	VA 200nmol, liposome 100nmol
10-8	Lip-gp46siRNA	Liposome 100nmol, gp46siRNA 150. mu.g
			10-9	VA-Lip-randomsiRNA	VA 200nmol, liposome 100nmol, random-siRNA 150 μ g
10-10	VA-Lip-gp46siRNA	VA 200nmol, liposome 100nmol, gp46siRNA 150 μ g

The results showed that 6 subjects died after 45 days from the start of DMN administration except for the group to which the drug of the present invention was administered (treatment groups 10-4 and 10-10), but all subjects survived for more than 70 days from the start of DMN administration except for two subjects died at 45 days in treatment group 4 (fig. 20 and 21). In addition, it was found that, in the dead individuals, the amount of liver fibers was quantified in the same manner as in example 7, and that the administration of VA-Lip-gp46siRNA significantly suppressed the increase in the amount of liver fibers (fig. 22).

The above results indicate that the drug of the present invention is extremely effective for the prevention and treatment of stellate cell-related fibrosis.

Example 11 improvement of Effect by RBP (retinol binding protein)

The effect of RBP on the efficiency of introduction of VA-Lip-gp46siRNA was investigated using LI90, a human hepatic stellate cell-derived cell line. First, 100nM of the VA-Lip-gp46siRNA (FITC) prepared in example 5 was added to LI90 in culture together with FBS (fetal bovine serum) at various concentrations (i.e., 0, 0.1, 0.5, 1, 2, 4, or 10%), and after 48 hours of culture, fluorescence images were observed with LSM, and the amount of siRNA entering each cell was quantified using FACS. In addition, the RBP content in FBS is about 0.7 mg/dl. As shown in FIG. 23, FBS (RBP) concentration-dependently increased the amount of siRNA introduced. Next, 100nM VA-Lip-gp46siRNA (FITC), 4% FBS and 10. mu.g (21,476nmol) of anti-RBP antibody were added together to LI90 in culture, and the siRNA introduction efficiency was evaluated in the same manner. As shown in fig. 24, the amount of introduction increased by RBP is significantly reduced by the addition of the anti-RBP antibody. The above results indicate that RBP is effective for further improving the introduction of the drug of the present invention.

Sequence listing

<110> Sadhi Hodok medical university of Hokkaido university

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<151>2004-12-22

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Claims (15)

1. A stellate cell specific drug carrier comprises a retinoid derivative and/or a vitamin A analogue as a constituent.

2. The pharmaceutical carrier of claim 1, wherein the retinoid derivative comprises vitamin a.

3. The pharmaceutical carrier according to claim 1, wherein the carrier comprises 0.2-20 wt% of a retinoid derivative and/or a vitamin A analog.

4. The drug carrier according to any one of claims 1 to 3, which is in the form of any one of polymeric micelles, liposomes, emulsions, microspheres and nanospheres.

5. A pharmaceutical preparation comprising the pharmaceutical carrier according to any one of claims 1 to 4 and a drug for controlling the activity or proliferation of stellate cells, which is used for treating a disease caused by stellate cells.

6. The pharmaceutical product of claim 5, wherein the disease is selected from the group consisting of hepatitis, liver fibrosis, cirrhosis, liver cancer, pancreatitis, pancreatic fibrosis, pancreatic carcinoma, vocal cord scarring, vocal cord mucosal fibrosis, and laryngeal fibrosis.

7. The pharmaceutical product of claim 5 or 6, wherein the drug that controls the activity or proliferation of stellate cells is selected from the group consisting of a TGF β activity inhibitor, an HGF active agent, an MMP production promoter, a TIMP production inhibitor, a PPAR γ ligand, an angiotensin activity inhibitor, a PDGF activity inhibitor, a sodium channel inhibitor, an apoptosis inducer, and siRNA, ribozyme, antisense nucleic acid, DNA/RNA chimeric polynucleotide, and a vector that expresses them, targeting extracellular matrix constituent molecules produced by stellate cells or targeting one or more of molecules that act on the production or secretion of the extracellular matrix constituent molecules.

8. The pharmaceutical product according to claim 7, wherein the molecule that acts on the production or secretion of extracellular matrix-constituting molecules is HSP 47.

9. A pharmaceutical product according to any one of claims 5 to 8, which is formed by mixing the drug and the drug carrier at or near a medical site.

10. A kit for preparing a pharmaceutical product according to any one of claims 5 to 9, comprising: a drug that controls the activity or proliferation of the stellate cells, a drug carrier-forming substance, and 1 or more containers containing 1 or more of a retinoid derivative and/or a vitamin a analog.

11. A method for treating a disease caused by stellate cells, which comprises administering an effective amount of the pharmaceutical product according to any one of claims 5 to 9 to a subject in need thereof.

12. The method of claim 11, wherein the disease is selected from the group consisting of hepatitis, liver fibrosis, cirrhosis, liver cancer, pancreatitis, pancreatic fibrosis, pancreatic cancer, vocal cord scarring, vocal cord mucosal fibrosis, and laryngeal fibrosis.

13. The method of claim 11 or 12, wherein the pharmaceutical product is administered non-orally.

14. Use of a pharmaceutical carrier according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a disease caused by stellate cells.

15. A method for delivering a drug to stellate cells using the drug carrier according to any one of claims 1 to 4.