# Patent

## Title
Fibrosis inhibitor for implanted organ

## Abstract

A pharmaceutical preparation comprising a hepatocyte growth factor or a DNA molecule encoding the same and the like according to the present invention can suppress the fibrosis of a transplanted organ after organ transplantation. The present invention is useful in the fields of organ transplantation and regeneration therapy.

## Background
This application is a Divisional of U.S. application Ser. No. 11/795,314, filed Oct. 4, 2007 now U.S. Pat. No. 7,696,170, which is a national stage application of International application No. PCT/JP2005/016653, filed Sep. 9, 2005.


TECHNICAL FIELD
The present invention relates to a medicinal drug comprising a hepatocyte growth factor (hereinafter referred to briefly as “HGF”), which acts to suppress the fibrosis of a transplanted organ as caused by the long-term administration of an immunosuppressive drug. And the present invention is also concerned with an immunologic-tolerance drug comprising, among HGF, HGF having 5 amino acids deleted, which drug can permit the recipient patient to acquire immunologic tolerance.
BACKGROUND ART
Organ transplantation has come to be established as a sort of medical practice where no therapy is available other than organ replacement. In recent years, however, a currently increased number of organ transplant survival and a follow-up survey of transplant recipients, or the patients each having had an organ transplanted, reveal that there is arising the problem of greater concern in that the transplanted organs have been undergoing gradual fibrosis with an elapse of a prolonged period of time after the organ transplantation. In the organ transplantation, immunosuppressive drugs are used, for example, to suppress rejection of the transplanted grafts or the transplanted tissues, and must continue to be taken by the patients throughout their lifetime, except in the case of autografts or isografts. The fibrosis of a transplanted organ is found to develop in the graft-recipient patient who is kept placed on indefinite, continued immunosuppressant regimens. Although the causal relationship between the use of immunosuppressive drugs and the onset of the transplanted organ fibrosis is yet to be clarified, there has not been known so far any means to suppress the fibrosis of transplanted organs in the continued immunosuppressant regimens. It has heretofore been considered necessary for a patient to continue to take the immunosuppressive drugs throughout its lifetime, since such immunosuppressants are essential for the establishment and survival of grafts. Referring particularly to such immunosuppressive drugs, Cyclosporine and FK 506 (Taclolimus), with their potent immunosuppressive activity, have attained excellent performance in suppressing the graft rejection after transplantation of the organs, such as kidney, liver, heart, pancreas, etc., and are attracting increased attention. Namely, Cyclosporine and other immunosuppressive drugs, which can reduce outstandingly the incidence of acute rejection after organ transplantation and find frequent application in organ transplantation, lower the incidence of bone marrow depression, and can offer the advantage that severe infections brought about by a reduced number of leucocytes can be prevented from developing to thereby facilitate the rejection management after organ transplantation to be conducted, thus enabling the survival performance of organ transplantation to be enhanced remarkably. However, such immunosuppressive drugs, Cyclosproin, etc., are also observed to produce side effects or adverse reactions, which include those occurring at sites other than the site of transplantation or systemic ones, such as nephrotoxicity, hepatotoxicity, neuropathy, hypertension, necrosis of caput femoris, cataract, diabetes mellitus, acute pancreatitis, cytomegalovirus infections, etc. It is known that HGH is able to alleviate such systemic side effects caused by the immunosuppressive drugs (refer to the Official Gazette of JP Hei 08-89869 A). HGF is the protein which was discovered by the present inventors, Nakamura et al., to be present in the sera of the regenerated liver of rats with regenerating liver and found to act as a factor capable of proliferating mature hepatocytes in vitro (refer to Biochemical and Biophysical Research Communications, 1984, vol. 122, p. 1450-1459). Furthermore, Nakamura et al. succeeded in isolating HGF from platelets of a rat (refer to Proc. Nat. Acad. Sci., 1986, vol. 83, p. 6489, and FEBS Letters, 1987, vol. 22, p. 311), and identified partly its amino acid sequence. Subsequently, Nakamura et al. conducted cDNA cloning of human and rat HGF on the basis of the amino acid sequence elucidated, and succeeded in producing HGF in the form of a protein through introduction of the resultant cDNA into animal cells by recombinant DNA technology (for example, refer to Nature, 1989, vol. 342, p. 440-443).
DISCLOSURE OF THE INVENTION
The Problem that the Invention is Intended to Solve
The present invention relates to a medicinal drug which acts to suppress the gradually ongoing fibrosis of a transplanted organ and the induction of organ insufficiencies in a recipient patient kept on the continued immunosuppressant regimen. Also, the present invention is concerned with an immunotolerance acquiring agent which can allow a recipient patient to acquire immunologic tolerance against the transplant rejection, when an organ is transplanted from a donor to the recipient.
Means for Solving the Problem
The present inventors found that administration of HGF can result in successful suppression of the fibrosis of a transplanted organ in a recipient animal and that administration of HGF having 5 amino acids deleted allows such recipient animal to acquire immunologic tolerance against the transplanted organ. These findings were followed by further continued research work, leading the present inventors to completion of the present invention.
Thus, the present invention relates to:

    (1) An agent for suppressing the fibrosis of a transplanted organ caused by an immunosuppressive drug as administered to a recipient, characterized in that said agent comprises HGF;
    (2) The agent for suppressing the fibrosis of a transplanted organ as described above under (1), characterized in that said HGF is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 or comprising substantially the same amino acid sequence as the one shown in SEQ ID NO: 1 or 2, which peptide acts as HGF, or a partial polypeptide thereof which can also function as HGF;
    (3) An agent for suppressing the fibrosis of a transplanted organ caused by an immunosuppressive drug as administered to the recipient, characterized in that said agent comprises the DNA molecule encoding HGF;
    (4) The agent for suppressing the fibrosis of a transplanted organ as described above under (3), characterized in that the DNA molecule encoding HGF is a DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 3 or 4, or a DNA molecule which hybridizes under stringent conditions with a DNA molecule comprising a nucleotide sequence complementary to the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 3 or 4 and which encodes a peptide acting as HGF;
    (5) The agent for suppressing the fibrosis of a transplanted organ as described above under any one of (1) to (4), characterized in that the immunosuppressive drug is Tacrolimus;
    (6) An immunologic tolerance acquiring agent, characterized in that said agent comprises a peptide consisting of the amino acid sequence as shown in SEQ ID NO: 2, a peptide comprising an amino acid sequence being substantially the same as the one as shown in SEQ ID NO: 2 and which peptide acts as HGF, or a partial polypeptide thereof which can also functions as HGF; and
    (7) An immunologic tolerance acquiring agent, characterized in that said agent comprises the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 4, or a DNA molecule which hybridizes under stringent conditions with a DNA molecule comprising a nucleotide sequence complementary to the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 4 and which encodes a peptide acting as HGF.


The present invention also relates to:

    (8) A method for suppressing the fibrosis of a transplanted organ, characterized in that said method comprises administering HGF to a mammal having had an organ transplanted which is placed on the immunosuppressive-drug regimen;
    (9) The method as described above under (8), characterized in that said HGF is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 or comprising substantially the same amino acid sequence as the one shown in SEQ ID NO: 1 or 2, which peptide acts as HGF, or a partial polypeptide thereof which can also function as HGF;
    (10) A method for suppressing the fibrosis of a transplanted organ, characterized in that said method comprises administering a DNA molecule encoding HGF to a mammal having had an organ transplanted which is placed on the immunosuppressive-drug regimen;
    (11) The method as described above under (10), characterized in that the DNA molecule encoding HGF is a DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 3 or 4, or a DNA molecule which hybridizes under stringent conditions with a DNA molecule comprising a nucleotide sequence complementary to the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 3 or 4 and which codes for a peptide acting as HGF;
    (12) The method as described above under any one of (8) to (11), characterized in that the immunosuppressive drug is Tacrolimus;
    (13) A method for allowing a recipient mammal to acquire immunologic tolerance, characterized in that said method comprises administering to the mammal going to have an organ transplanted a peptide consisting of the amino acid sequence as shown in SEQ ID NO: 2, a peptide comprising an amino acid sequence being substantially the same as the one as shown in SEQ ID NO: 2 and which peptide acts as HGF, or a partial polypeptide thereof which can also functions as HGF; and
    (14) A method for allowing a recipient mammal to acquire immunologic tolerance, characterized in that said method comprises administering to a mammal going to have an organ transplanted the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 4, or a DNA molecule which hybridizes under stringent conditions with a DNA molecule comprising a nucleotide sequence complementary to the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 4 and which encodes a peptide acting as HGF.


Also, the present invention relates to:

    (15) Use of HGF in the manufacture of a medicinal drug for suppressing the fibrosis of a transplanted organ as brought about by administration of an immunosuppressive drug;
    (16) The use as described above under (15), characterized in that said HGF is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 or comprising substantially the same amino acid sequence as the one shown in SEQ ID NO: 1 or 2, which peptide acts as HGF, or a partial polypeptide thereof which can also function as HGF;
    (17) Use of the DNA molecule encoding HGF in the manufacture of a medicinal drug for suppressing the fibrosis of a transplanted organ as brought about by the administration of an immunosuppressive drug;
    (18) The use as described above under (17), characterized in that the DNA molecule encoding HGF is a DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 3 or 4, or a DNA molecule which hybridizes under stringent conditions with a DNA molecule comprising a nucleotide sequence complementary to the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 3 or 4 and which encodes a peptide acting as HGF;
    (19) The use as described above under any one of (15) to (18), characterized in that the immunosuppressive drug is Tacrolimus;
    (20) Use of HGF in the manufacture of a medicinal drug for allowing a recipient patient to acquire immunologic tolerance to a transplanted organ;
    (21) The use as described above under (20), characterized in that said HGF is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or comprising substantially the same amino acid sequence as the one shown in SEQ ID NO: 1 or 2, which peptide acts as HGF, or a partial polypeptide thereof which can also function as HGF;
    (22) Use of DNA encoding HGF in the manufacture of a medicinal drug for allowing a recipient patient to acquire immunologic tolerance to a transplanted organ; and
    (23) The use as described above under (22), characterized in that said DNA is a DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 4, or a DNA molecule which hybridizes under stringent conditions with a DNA molecule comprising a nucleotide sequence complementary to the DNA molecule consisting of the nucleotide sequence as shown in SEQ ID NO: 4 and which encodes a peptide acting as HGF.


As used herein, the term “fibrosis” refers to excessive accumulation in a transplanted organ of extracellular matrix substances, such as collagen fibers, resulting in the morbid conditions of hardening of the transplanted organ, while the term “immunologic tolerance” refers to suppression of destructive actions by the recipient's immune system so that the donor's cells or tissues may not be attacked by the recipient's immune system during organ transplantation.
The Effects of the Invention
The agent for suppressing the fibrosis of a transplanted organ according to the present invention, which acts to suppress the fibrosis of a transplanted organ in an animal having had an organ transplanted which is placed on the long-term immunosuppressant regimen, can prevent the transplanted organ from undergoing gradual fibrosis and also eventually from falling into organ insufficiency.
The immunologic-tolerance acquiring agent of the present invention can be given to a recipient animal (patient) immediately after having had a donor's organ transplanted to thereby allow the recipient animal (patient) to acquire the immunologic tolerance to the transplanted organ, and consequently can suppress rejection to the transplanted organ. The immunologic-tolerance acquiring agent of the present invention can be administered to a transplant-recipient animal to allow the animal to acquire the immunologic tolerance to the donor, thereby enabling such animal to induce the conditions of being thereafter rendered unreactive to any immunologic response to the donor or donor's organ, and can reduce the dose of the immunosuppressive drug or suspend the administration of such drug. In advance of transplantation of an organ, furthermore, the immunologic-tolerance acquiring agent of the present invention can also be administered to a recipient, simultaneously with implantation of a donor's tissue (e.g., a tissue involved in the immunity to be described below) other than the organ to be transplanted, to thereby allow the recipient to acquire the immunologic tolerance to the donor's organ. On the other hand, the immunologic-tolerance acquiring agent of the present invention can also be administered to a donor, simultaneously with implantation of a donor's tissue (e.g., a tissue involved in the immunity to be described below) other than the organ to be transplanted, to thereby allow the donor to acquire the immunologic tolerance to the recipient. By allowing the immunologic tolerance to be acquired by either one of the above-described procedures, it may become possible to perform organ transplantation between the donor and recipient for both of which the organ transplantation has heretofore been infeasible owing to absence or lack of the complex histocompatibility antigen matching. Also, the immunologic-tolerance acquiring agent of the present invention can suppress or reduce rejection after organ transplantation in a recipient having acquired the immunologic tolerance to a donor who has had an organ transplanted from the donor or having had an organ transplanted from the donor who has acquired the immunologic tolerance to the recipient. The above-described suppressed or reduced rejection can permit administration of an immunosuppressive drug or a dose thereof to be suspended or reduced. As used herein, the term “animal” is understood to comprehend mammals inclusive of humans.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a view illustrating the pathological picture of the heart transplanted in the mouse which survived 60 days after transplantation as well as a graph (B) showing a proportion of the cross-section area of the fibrosis portion to the one of the total cross-section area of the heart wall.
FIG. 2 is a view illustrating the survived graft of a white-haired skin section of a BALB/c mouse implanted onto a black-haired skin section of a C3H/He mouse.



THE BEST MODE FOR CARRYING OUT THE INVENTION
HGF that is usable in the present invention is the known substance, and use can be made of any one of HGFs as prepared by various methods, only if it is purified to such an extent as may be usable as a medicinal drug. Referring to the method for preparing HGF, for example, cells producing HGF in primary culture or as cell line can be cultivated, followed by separation and purification from the supernatant to yield HGF. By following the genetic engineering procedure, alternatively, a gene encoding HGF is inserted into a suitable vector, followed by introduction into a suitable host cell to effect transformation, and the transformant can be cultivated, followed by isolation from the supernatant liquid to give the objective recombinant HGF (for example, refer to the official Gazette of JP No. Hei 5-111382A, Biochem. Biophys. Res. Commun. 1989, vol. 163, p. 967, etc.). The above-described host cell is not particularly limited, and there can be used a variety of host cells as conventionally utilized in the genetic engineering procedure, such as E. coli, yeasts or animal cells, etc. HGF variants as obtained by these manners may allow one to a plural number (e.g., several (e.g., 1 to 8); the same is understood to hold true hereinafter)) of amino acids in the amino acid sequence to undergo substitution, deletion or addition, and may allow the sugar chain similarly to undergo substitution, deletion or addition, only if these variants have substantially the same action as the naturally occurring HGF. Such HGF variant may be exemplified by the 5 amino-acids deleted type of HGF to be described below. In this context, the expression “to allow one or a plural number of amino acids to undergo substitution, deletion or addition” as referred to in the amino acid sequence is understood to denote that such a number (one to several) as may be caused through the well known technical procedures, such as the genetic engineering procedure, site-specific mutagenesis procedure, etc., or may occur naturally is allowed to undergo deletion, substitution or addition and others. The expression “HGF having allowed the sugar chain to undergo substitution, deletion or addition” refers to, for example, naturally occurring HGF having had the sugar chain deleted through treatment with enzymes, etc., naturally HGF having allowed the amino acid sequence at the sugar-chain addition site to undergo mutation in such a manner as may not add sugar-chains, or naturally occurring HGF having allowed the amino acid sequence to undergo mutation in such a manner as may add sugar chains to sites different from the site of sugar-chain addition.
Such variant HGF also includes proteins which show amino acid sequences of at least about 80% or more homology with the amino acid sequence of HGF, preferably not less than about 90% homology, more preferably not less than about 95% homology, and can act as HGF. The term “homology” in the above-described amino acid sequence means the degree of agreement or coincidence among the amino acid sequences in terms of amino acid residues constituting individual sequences as found when the primary structures of proteins are compared.
The above-mentioned HGF includes, for example, the amino acid sequences shown by SEQ ID NO: 1 or 2. The variant HGF as represented by the SEQ ID NO: 2 is a 5 amino-acids deleted type of HGF which has 5 amino acid residues deleted at the positions 161 to 165 in the amino acid sequence shown by SEQ ID NO: 1. Both of the proteins possessing the amino acid sequences shown by SEQ ID NOS: 1 and 2, respectively, are naturally occurring HGF of human origin, which elicit mitogen and motogen activities, etc. as HGF.
Peptides comprising substantially the same amino acid sequences as the one shown by SEQ ID NO: 1 or 2 preferably include peptides comprising amino acid sequences of at least about 80% or more homology with the amino acid sequence shown by SEQ ID NO: 1 or 2, preferably not less than about 90% homology, more preferably not less than about 95% homology, such as peptides which are generated by subjecting one to several amino acid residues out of the amino acid sequence shown by SEQ ID NO:1 or 2 to insertion, deletion or substitution with different amino acid residues and which can act as HGF. The amino acids to be inserted or substituted may be non-natural amino acids other than 20 kinds of amino acids encoded by genes. Such non-natural amino acids may be any amino acids, only if they possess amino and carboxyl groups, and may be exemplified by γ-aminobutyric acid, etc.
Such peptides each may be used as a single constituent or as mixtures thereof.
The HGFs which are usable in the present invention may have either one of a carboxyl (—COOH), carboxylate (—COO—), amide (—CONH2) or ester (—COOR) group at the C-terminus. In this context, R as used in the ester group may be exemplified by C1-6alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc., C3-8cycloalkyl groups, such as cyclopentyl, cyclohexyl, etc., C6-12aryl groups, such as phenyl, α-naphthyl, etc., C7-14aralkyl groups, such as phenyl-C1-2alkyl groups, e.g., benzyl, phenetyl, etc., or α-naphthyl-C1-2alkyl groups, e.g., α-naphthylmethyl, etc., as well as pivaloyloxymethyl group frequently used as an ester for oral preparations. In cases where the HGF as used in this present invention has carboxyl groups (or carboxylates) at sites other than the C-terminus, the HGF which has its carboxyl group amidated or esterified is also included in the HGF of the present invention, whereby such ester includes the above-mentioned C-terminus ester. Furthermore, the HGF as used in the present invention includes the above-described peptides whose N-terminal methionine residue has its amino group protected with a protective group (e.g., C1-6acyl groups, such as C2-6alkanoyl groups inclusive of formyl and acetyl groups, etc.), whose glutamyl group as generated by in vivo truncation of its N-terminal site is converted to pyroglutamic acid, or whose intramolecular amino acid has its side-chain substituent (e.g., —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) protected with an appropriate protective group (e.g., acyl groups, such as C2-6 alkanoyl groups, inclusive of formyl and acetyl groups, etc.), or conjugated proteins, such as glycoproteins derived through addition of sugar chains to the above-mentioned peptides, and the like.
The partial polypeptides (hereinafter referred to in some instances briefly as “partial polypeptide”) of HGF as used in the present invention may be any peptides, only if they are partial polypeptides of the above-described HGFs. Referring to a number of the amino acids of such partial polypeptides in the present invention, preferred are peptides comprising not less than at least about 20 amino acids out of the constituent amino acid sequence of the above-mentioned HGF, preferably not less than about 50 amino acids and more preferably not less than 100 amino acids. The partial polypeptides of the present invention may have any one out of carboxyl (—COOH), carboxylate (—COO—), amide (—CONH2) and ester (—COOR) groups at their C-termini. Furthermore, the partial polypeptides of the present invention, as is the case with the above-mentioned HGF include the peptides having the amino group of their N-terminus methionine residue protected with a protective group, the peptides having Gln as generated by in vivo truncation of their N-terminal side converted to pyroglutamic acid, the peptides whose intramolecular amino acid has its side-chain substitute protected with an appropriate protective group, and conjugated peptides, such as the so-called glycopeptides generated through addition of sugar chains.
Salts of the HGFs and partial polypeptides thereof as used in the present invention may be exemplified by physiologically allowable acid-addition salts with acids or bases, with the physiologically allowable salts, among others, being preferable. Such salts include, for example, salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acids, etc.), or salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
The HGFs and partial polypeptides thereof which are usable in the present invention can be produced in accordance with the known peptide synthesis methods or through truncation of HGF with a suitable peptidase. The peptide synthesis method may be either of the solid-phase and liquid-phase synthesis methods. Thus, a partial polypeptide or amino acids which constitutes HGF can be allowed to condensate with the remaining portion, followed by elimination of any protective groups from the condensate, if such groups exist, to thereby yield the objective peptide. The known condensation and protective-group elimination procedures include, for examples, the procedures and methods as described M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966), Schroeder and Luebke, The peptide, Academic Press, new York (1965) (1965), etc. After completion of the reaction, the ordinarily employed purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc., can be combined to isolate and purify the partial polypeptide of the HGFs. When the partial polypeptide as obtained by the above-described methods is in the free form, such peptide can be converted to a suitable salt by the known procedure, or conversely when the partial polypeptide is in the form of a salt, the known procedures can be used to convert the same to a free form.
In the present invention, the HGF encoding DNA molecules can be contained as an active ingredient.
The DNA molecule encoding HGF includes, for example, DNA molecules comprising the nucleotide sequence as shown by SEQ ID NO: 3 or 4, or DNA molecules which hybridize under stringent conditions with a DNA molecule comprising a nucleotide sequence complementary to the DNA molecule consisting of the nucleotide sequence as shown by SEQ ID NO: 3 or 4 and which encode the protein showing activities substantially equivalent to those of HGF, such as mitogen and motogen activities. In this context, the DNA molecule, which hybridizes with the DNA molecule consisting of the nucleotide sequence as shown by SEQ ID NO: 3 or 4, denotes DNA molecules obtained by colony hybridization, plaque hybridization or Southern blot hybridization and the like with use of the above-described DNA molecule as a probe. Specifically, there can be mentioned, for example, the DNA molecule identified by carrying out hybridization at a temperature of about 65° C. in the presence of sodium chloride of a concentration in the range of about 0.7 to 1.0 M with use of a filter having a colony- or plaque-derived DNA molecule immobilized thereto, and subsequently washing the filter with an SSC solution (SSC solution having one-fold or standard concentration is composed of 150 mM of sodium chloride and 15 mM of sodium citrate) having about 0.1- to 2-fold concentration.
Specific examples of the DNA molecule, which hybridizes with DNA consisting of the nucleotide sequence shown by SEQ ID NO: 3 or 4 as described above, include DNA molecules showing not less than about 80% of homology with the nucleotide sequence shown by SEQ ID NO: 3 or 4, preferably not less than about 90% homology and more preferably not less than about 95% homology. Hybridization can be conducted in accordance with the known procedures, such as the procedures described in Molecular Cloning, A Laboratory Manual, Third Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001: hereinafter referred to briefly as “Molecular Cloning 3rd Edition”). When a commercially available library is used, hybridization can be carried out in accordance with the procedure described in Instruction Manual as attached thereto.
Furthermore, the DNA molecule encoding HGF of the present invention is not limited to the above-described ones, and any DNA molecules can be used as the DNA molecule encoding HGF, insofar as the expressed proteins exhibit substantially the same activities as HGF. For example, the DNA molecules encoding partial polypeptides of HGF also fall into the scope of the present invention, only if they encode the partial polypeptide which can act as HGF. The DNA molecule encoding the partial polypeptide of HGF may be any DNA molecules, only if they comprise the nucleotide sequence encoding the above-described partial polypeptide. As is the case with the above-described DNA molecule encoding HGF, additionally, it may be any one of genomic DNA, genomic DNA library, the above-described cell/tissue derived cDNA, the above-described cell/tissue derived cDNA library, and synthetic DNA molecules. The vector to be used in the library may be any one of bacteriophages, plasmids, cosmids and phagemids. The mRNA fraction as prepared from the above-described cells/tissues can be used to make amplification directly by the RT-PCR. Specific examples of the DNA molecule encoding the partial polypeptide of the present invention include (a) DNA molecules comprising fractional nucleotide sequences of the DNA consisting of the nucleotide sequence as shown by SEQ ID NO: 3 or 4, (b) DNA molecules which hybridize under stringent conditions with a DNA molecule comprising nucleotide sequence complementary to a DNA molecule comprising a fragmental nucleotide sequence of the DNA molecule consisting of the nucleotide sequence as shown by SEQ ID NO: 3 or 4, or (c) DNA molecules comprising the fragmental nucleotide sequence as described above under (a) or (b), and the like.
The DNA can be readily obtained, for example, by the ordinary hybridization and PCR, etc., specifically by consulting with the reference books, such as the previously described Molecular Cloning, etc.
The RNA molecules encoding the HGF or partial polypeptides thereof which are usable in the present invention can also be used in the present invention and fall into the scope of the present invention, if such RNA molecules are capable of expressing such HGF or partial polypeptides thereof by reverse transcriptases. Such RNA molecules can be obtained by the known procedures, as well.
The agent for suppressing the fibrosis of a transplanted organ or immunologic-tolerance acquiring agent according to the present invention can be applied to humans, as well as mammals (e.g., cows, horses, pigs, sheep, dogs, cats, etc.).
The organs to which the agent for suppressing the fibrosis of a transplanted organ can be applied include, for example, the heart, kidney, liver, small intestine, pancreas, skin or corneum, with the heart among others being preferable. Also, the immunologic-tolerance acquiring agent of the present invention is also applicable to the transplantation of hematopoietic cells, in addition to the above-mentioned organ transplants.
The agent for suppressing the fibrosis of a transplanted organ or immunologic-tolerance acquiring agent according to the present invention can assume various dosage forms, such as liquid preparations, solid preparations or capsules, etc., wherein HGF alone or together with conventionally used carrier is generally processed into an injectable solution, inhalant, suppository or oral preparation. The above-mentioned injectable solution may be either aqueous or oily injectable solution. In the case of manufacture of an aqueous injectable solution, such aqueous injectable solution can be prepared in accordance with the known process by appropriately admixing an aqueous solvent (e.g., water for injection uses, purified water, etc.) with pharmaceutically allowable additives, such as isotonic agent (e.g., sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffering agent (e.g., phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, tris-buffer, glutamate buffer, ε-aminocaproate buffer, etc.), preservative (e.g., methyl p-oxybenzoate, ethyl p-oxybenzoate, propyl p-oxybenzoate, butyl p-oxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc.), thickening agent (e.g., hydroxymethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol, etc.), stabilizer (e.g., sodium hydrogensulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutyl hydroxytoluene, etc.) or pH regulating agent (e.g., hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.), and dissolving HGF in the resultant solution, followed by sterilization by filtration through a filter and filling into a sterile container. Also, use is made of a suitable solubilizing agent, such as alcohols (e.g., ethanol, etc.), polyhydric alcohols (e.g., propylene glycol, polyethyleneglycol, etc.) or a nonionic surfactant (e.g., Polysorbate 80, polyoxyethylene hardened castor oil 50, etc.), and the like. In cases where an oily injectable solution is produced, there can be used an oily solvent, such as sesame oil or soybean oil, etc., while use may be made of a solubilizing agent, such as benzyl benzoate or benzyl alcohol, etc. The injectable solution as prepared is ordinarily filled into a suitable ampoule or vial. The content of HGF in the injectable solution is generally adjusted to a level in the range of about 0.0002 to 0.2% in w/v, preferably in the range of about 0.001 to 0.1% in w/v. The liquid preparations, inclusive of injectable solutions, etc., are preferably stored through freezing or lyophilization, etc. to remove the moisture. The lyophilisate is used by adding distilled water for injection, etc. for reconstitution.
An oral preparation may be exemplified by such dosage forms as a tablet, granule, fine granule, powder, capsule, liquid preparation, emulsion, suspension or syrup, etc. such dosage forms are produced by the known processes. In cases where the granule or tablet is manufactured, such dosage form is produced by using pharmaceutically allowable additives, such as an excipient (e.g., lactose, white sugar, glucose, starch, crystalline cellulose, etc.), lubricant (e.g., magnesium stearate, talc, stearic acid, calcium stearate, etc.), disintegrant (e.g., starch, sodium carmellose, calcium carbonate, etc.) or binder (e.g., starch paste solution, hydroxypropylcellulose solution, carmellose solution, gum-arabic solution, gelatin solution, sodium-alginate solution, etc.), while the granule or tablet may be provided with a coating film by use of a suitable coating agent (e.g., gelatin, white sugar, gum arabic, carnauba wax, etc.) or enteric coating agent (e.g., cellulose acetate phthalate, methacrylic-acid copolymers, hydroxypropylcellulose phthalate, carboxymethyl ethylcellulose, etc.), etc. In the case of manufacture of a capsule, suitable use is made of the known excipients, magnesium stearate, calcium stearate, talc or light anhydrous silicate for the purpose of enhancing the fluidity and lubricating property, crystalline cellulose and lactose for the purpose of increased pressurized fluidity, or the above-mentioned disintegrant, and the like. HGF may be mixed uniformly with the above-mentioned excipient or granulated, or the granule as granulated is provided with a coating film by use of a suitable coating agent, followed by filling into a capsule or enclosure-molding with a capsule base as plasticized by adding glycerol or sorbitol, etc. to a suitable capsule base (e.g., gelatin, etc.). Such capsules can be incorporated with a coloring agent or preservative (e.g., sulfur dioxide, methyl p-oxybenzoate, ethyl p-oxybenzoate, propyl p-oxybenzoate, butyl p-oxybenzoate, etc.), and the like. Such capsules can be provided in the forms of an ordinary capsule, as well as an enteric-coating capsule, gastric-resistant capsule or release-controlled capsule. In the case of the enteric capsule, HGF coated with an enteric coating agent or HGF after being admixed with a suitable excipient is filled into an ordinary capsule. Alternatively, HGF or HGF admixed with the above-mentioned excipient can be filled into a capsule coated with an enteric coating agent or a capsule molded with an enteric polymer used as a base. In cases where a syrup is produced, there can be appropriately selected and used, for example, a stabilizer (e.g., sodium edentate, etc.), suspending agent (e.g., gum arabic, carmellose, etc.), flavoring agent (e.g., simple syrup, glucose, etc.) or fragrance, etc.
A suppository can also be prepared by the ordinary pharmaceutical-preparation process with use of the conventionally used base (e.g., cacao fat, laurin fat, glycerogelatin, macrogol, whittep sol, etc.).
Also, an inhalant can be prepared by the conventional pharmaceutical-preparation means, and in the case of manufacture of such inhalant, the additives to be used may be any additives which are used in the inhalant preparation, and there are used, for example, a propellant as well as the above-described excipient, binder, lubricant, preservative, stabilizer, isotonic agent, pH regulating agent or flavoring agent (e.g., citric acid, menthol, ammonium glycyrrhizate, glycine, fragrance, etc.) and the like. As a propellant, use is made of liquefied gas propellants or compressed gases, etc. The liquefied gas propellant may be exemplified by fluorinated hydrocarbons (replacement Freons, such as HCFC22, HCFC-123, HCFC-134a, HCFC142, etc. and the like), liquefied petroleum gas, dimethyl ether, and the like. The compressed gas includes, for example, soluble gases (e.g., carbon dioxide gas, nitrogen suboxide gas, etc.), or insoluble gases (e.g., nitrogen gas, etc.), and the like.
HGF as usable in the present invention, together with a biodegradable polymer, can be processed into the controlled-release preparation. HGF, especially when processed into a controlled-release preparation, can be expected to produce the beneficial effects, such as sustained maintenance of the blood level, reduction of the number of administration and alleviation of side effects, etc. The said controlled-release preparation can be produced, for example, in accordance with the known process as described in Drug Delivery System, Chapter 3 (published by CMC, 1986).
The in vivo degradable polymer which is used in the controlled release preparation can be appropriately selected among the known in vivo degradable polymers, and may be exemplified by polysaccharides, such as starch, dextran or chitosan, etc., proteins, such as collagen or gelatin, etc., polyamino acids, such as poly(glutamic acid), polylysine, polyleucine, polyalanine or polymethionine, etc., polyesters, such as poly(lactic acid), poly(glycolic acid), polymer or copolymer of lactic acid/glycolic acid, polycaprolactone, poly-(β-hydroxybutyric acid), poly(malic acid), polyacid anhydride or copolymer of fumaric acid/polyethylene glycol/vinyl-pyrrolidone, polyalkylcyanoacrylic acids, such as polyorthoesters or poly(methyl α-cyanoacrylate), polycarbonates, such as polyethylene carbonate or polypropylene carbonate, etc., and the like, with polyesters being preferable and poly(lactic acid) or polymers or copolymers of lactic acid/glycolic acid being more preferable. In the case of use of the polymer or copolymer of lactic acid/glycolic acid, its composition ratio (lactic acid/glycolic acid) (mole %) varies with the length of time of the controlled release, and ranges from about 100/0 to 50/50, in cases where the length of time of controlled release is, for example, about 2 weeks to 3 months, preferably about 2 weeks to one month. The said polymer or copolymer of lactic acid/glycolic acid generally shows a weight-average molecular weight of about 5,000 to 20,000. The copolymer of lactic acid/glycolic acid can be produced, for example, by the known production process, such as the one described in the Official Gazette of JP Sho 61-28521 A. The formulation ratio of the in vivo biodegradable polymer to HGF is not particularly limited, and HGF is for example in the range of about 0.01 to 30 w/w % against the in vivo biodegradable polymer.
The content of HGF in each of the above-described dosage forms can be suitably adjusted depending upon the dosage form, disease to be treated, severity of the disease or patient's age, etc.
DNA encoding HGF can be administered to a patient in accordance with the conventionally employed methods, such as the ones described in “Jikken Igaku” (Experimental Medicine), an extra number entitled “Fundamental Techniques of Gene Therapy”, published by Yohdo-sha in 1996, “Jikken Igaku” (Experimental Medicine), an extra number entitled “Experimental Methods of Gene Transfer & Expression Analysis”, published by Yohdo-sha in 1997, and Handbook of Developmental Research on Gene Therapy, edited by Japanese Society of Gene Therapy, published by N.T.S. in 1999, and the like.
The dosage form can assume any known forms being suited for each of the above-described mode of administration. In the case of a microcapsule, for example, the core substance of host cells having the HGF-encoding DNA or the expression plasmid containing the HGF-encoding DNA transfected therein is covered with the known coating-film substance in accordance with the known method (e.g., coacervation method, interfacial polymerization method or double-nozzle method, etc.) to produce such microcapsule in the form of a microparticle of about 1 to 500 μm in diameter, preferably about 100 to 400 μm, whereby the coating-film substance includes, film-forming polymers, such as Carboxymethylcellulose, cellulose acetate phthalate, ethyl-cellulose, alginic acid or its salts, gelatin, gelatin/gumarabic, nitrocellulose, polyvinyl alcohol or hydroxypropylcellulose, poly(lactic acid), poly(glycolic acid), chitosan-alginate, cellulose sulfate-poly(dimethyldiallyl)-ammonium chloride, hydroxyethyl methacrylate/methyl methacrylate, chitosan-carboxymethylcellulose, alginate-polylysine-alginate, etc.
The content of DNA in the preparation can be appropriately adjusted depending upon the disease to be treated, the patient's age and weight, etc., and its daily dose ordinarily ranges from about 0.0001 to 100 mg as DNA of the present invention, preferably from about 0.001 to 10 mg.
Also, the HGF-encoding DNA and HGF can be used independently and both can be utilized concomitantly, as well.
The immunosuppressive drug is understood to comprehend all the immunosuppressive drugs which are usable in the organ transplantation. Such immunosuppressive drug includes, for example, Cyclosporin and Tacrolimus (FK506) which act as a inhibitor against calcineurin; Zenapax and Simulect which belong to the IL-2 antibody; Rapamycin, an inhibitor against TOR (target of rapamycin: a factor which constitutes the signal transduction pathway for modifying the physiologic functions involved in cellular growth in response to the conditions of nutrition), etc.; CellCept which is an antimetabolite, and the like. The agent for suppressing the fibrosis of a transplanted organ according to the present invention is useful, among others, for the prevention, amelioration or therapy of the fibrosis of a transplanted organ as caused by the long-term administration of Tacrolimus.
Also, the agent for suppressing the fibrosis of a transplanted organ or immunologic-tolerance acquiring agent according to the present invention may appropriately be incorporated with other medicinal active ingredients, unless contrary to the objects of the present invention. Such medicinal active ingredient may be exemplified by coronary vasodilators (e.g., amyl nitrite, isosorbide nitrate, nitroglycerin, trapidil, etc.), β-blockers (e.g., oxprenolol, carteolol, bucumolol, bufetolol, propranolol, pindolol, etc.), calcium antagonists (e.g., diltiazem, verapamil, nifedipine, nicardipine, etc.), peripheral circulation disorder remedies (e.g., alprostadylalfadex, kalidinogenase, tocopherol, nicomol, etc.), antiarrhythmic drugs (e.g., ajmarine, procainamide, lidocaine, etc.), antihypertensive drugs (e.g., furosemide, trichlormethiazide, hydralazine, sympathomimetics depressant drugs, calcium blockers, etc.), antihyperlipemic drugs (e.g., clofibrate, pravastatin, simvastatin, lovastatin, nicomol, etc.), anticoagulant drugs (e.g., heparin, warfarin, dicumarol, aspirin, etc.), thrombolytic drugs (e.g., urokinase, etc.), antidiabetci drugs (e.g., tolubutamide, chlorpropamide, acetohexamide, glibenclamide, metformin, acarbose, etc.), anti-inflammatory drugs (e.g., diclofenac sodium, ibuprofen, indomethacin, etc.), antimicrobial drugs (e.g., cefixime, cefdinir, ofloxacin, tosufloxacin, etc.) or antifungal drugs (e.g., fluconazole, itraconazole, etc.), and the like. The pharmaceutical preparations containing such medicinal active ingredients can be used concomitantly with the preparations of the present invention. Such medicinal active ingredients are not particularly limited, insofar as the object of the present invention can be attained, and can be used in appropriate formulation proportions or concomitant-use proportions.
The agent for suppressing the fibrosis of a transplanted organ according to the present invention can be administered to a patient through a suitable route of administration according to their dosage forms. For example, the agent can be processed into a dosage form of an injectable solution for the intravenous, intraarterial, subcutaneous or intramuscular injection. The dose may be appropriately adjusted depending upon the symptoms, age or weight of a patient, etc., and ordinarily ranges from about 0.001 to 1000 mg as HGF per adult patient, preferably from about 0.01 to 100 mg, which can be suitably given to a patient once a day or as divided into several times a day. Also, the agent for suppressing the fibrosis of a transplanted organ according to the present invention is desirably administered to a patient simultaneously with, before or immediately after, administration of an immunosuppressive drug after organ transplantation. Furthermore, the agent for suppressing the fibrosis of a transplanted organ according to the present invention may start be administered to a patient in advance of organ transplantation.
The immunologic-tolerance acquiring agent of the present invention is administered to a recipient patient simultaneously with, before or after, organ transplantation, and referring to the term of administration, the said immunologic-tolerance acquiring agent is desirably administered to the patient for at least two weeks after organ transplantation. Prior to organ transplantation, furthermore, it is desirable to transplant a donor animal's tissue different from the organ transplant, for example, the donor animal's lymphocytes in the case of transplantation of the heart, into a recipient animal to thereby administer to the recipient animal the immunologic-tolerance acquiring agent of the present invention together with an immunosuppressive drug for at least about two weeks. Prior to organ transplantation, alternatively, a recipient's tissue, such as lymphocytes, etc., is transplanted into a donor for organ transplantation, and then the immunologic-tolerance acquiring agent of the present invention is given to the donor together with an immunosuppressive drug to thereby enable the donor's organ to acquire the immunologic tolerance to the recipient. By following the latter procedural technique, the donor's organ which has acquired the immunologic tolerance to the recipient can be transplanted into the recipient to thereby permit the recipient to suppress the rejection reaction of the transplanted organ.
The immunologic-tolerance acquiring agent of the present invention can be administered to a patient through a suitable route of administration by an appropriate method of administration at doses, as is the same with the agent for suppressing the fibrosis of a transplanted organ according to the present invention.
The term “tissue” as mentioned above in the donor's tissues or recipient's tissue refers to the tissues involved in the immunity, inclusive of the organs and tissues, such as the bone marrow, thymus, spleen, lymph nodes, tonsils, blood vessels, skin, intestinal tracts, etc., or the immunocytes, such as leucocytes, macrophage, lymphocytes (NK (natural killer) cell, helper T-cell, killer T-cell, B cell), dendritic cells, etc., cytokines, antibodies (immunoglobulins (Ig), IgG, IgA, IgM, IgD, IgE, etc.), granulocytes (neutrophils, eosinophils and basophils), and the like.
To be described below are the present invention by way of the examples, but the present invention is not understood to be limited thereto.
In the examples, the term “%” denotes “% by mass”, unless particularly specified otherwise, and HGF used was the variant HGF (whose nucleotide sequence is shown by SEQ ID NO: 2) having had 5 amino acids deleted.
Example 1
Suppressory Effect on the Fibrosis of the Cardiac Muscle in a Mouse Having the Heart Transplanted
(1) Generation of a Heart Transplanted Model:
Used were 8-weeks aged, male mice weighing ca. 20 g of two species (BALB/c and C3H/He mice). A BALB/c mouse used as a donor animal were kept under anesthesia with the mixed drug of ketamine (100 μg/kg) and xylazine (10 μg/kg), and the heart was subjected to perfusion with 1 mL of physiologic saline supplemented with 7.5% by mass of heparin and removed through excision to use as the heart for transplantation. A C3H/He mouse was maintained under anesthesia with the mixed drug consisting of ketamine (100 μg/kg) and xylazine (10 μg/kg), then subjected to laparotomy and allowed to undergo heterotopic transplantation of the heart for transplantation excised from the BALB/c mouse into the abdomen between the abdominal aorta and abdominal inferior venae cava under microscope.
(2) Administration of Immunosuppressive Drug and HGF
As an immunosuppressive drug, use was made of Prograf-Injectable Solution 5 mg (containing 5 mg/ml of Tacrolimus; manufactured by Fujisawa Pharmaceutical Co.).
Immediately after the transplantation as described above under (1), Tacrolimus was given subcutaneously to the mouse at a dose of 0.1 mg/kg/day once a day for 60 consecutive days with use of a Gauge 27 needle for injection. Immediately after the transplantation, additionally, a solution (0.2 mL; 250 μg of HGF/kg) as prepared by dissolving 1 mg of HGF in 20 mL of physiologic saline was given to animals of the group treated through administration of HGF at the regular interval of 12 hours (500 μg/kg/day) for 14 consecutive days, while the control animals were treated similarly through subcutaneous administration of physiologic saline (0.2 mL) for 14 consecutive days using a Gauge 27 needle for injection.
(3) Histopathological Analysis
While taking as Day 0 the day when the transplantation surgery was performed, the transplanted heart was incised on Day 14, and was fixed in formalin and embedded in paraffin in accordance with the conventional procedure to thereby prepare the heart tissue slices. The tissue slices were Masson-trichrome stained. The stained tissue images were loaded into a computer with a microscopic CCD camera (manufactured by Olympus Co.), and the NIH software package for analysis of images (free software) was used to calculate a surface-area ratio of the fibrosed green-stained section to the total area of the coronary cross-section image of the transplanted heart.
(4) Results
In the control group of animals, the fibrosis-formation ratio of the transplanted heart was shown to be 22.3±7.7% in, whereas it was found to be suppressed at a level as low as 15.6±1.3% in the group treated through administration of HGF (refer to FIG. 1).
This demonstrated that the recombinant human HGF can suppress the fibrosis of the transplanted heart.
Example 2
Acquisition of Immunologic Tolerance
Method:
In the same manner as described under (1) of Example 1, using a BALB/c mouse as a donor animal, the heart was transplanted from the BALB/c mouse to a C3H/He mouse in the abdomen between the abdominal aorta and abdominal inferior venae cava. Immediately after the transplantation, a solution (0.2 mL; 250 μg of HGF/kg) as prepared by dissolving 1 mg of HGF in 20 mL of physiologic saline started, and continued, being administered subcutaneously to the animals at the regular interval of 12 hours (500 μg/kg/day) for 14 consecutive days with use of a Gauge 27 needle for injection. The survived mouse 60 days after the transplantation was subjected to skin excision from the back, followed by transplantation of a 1-cm2 skin piece removed from a BALB/c or C57BL/10 mouse into the skin-excised portion.
Results:
In the C3H/He mouse, which had survived for 60 days after transplantation of the heart, followed by transplantation of a piece of the back skin from the donor animal of the BALB/c mouse, it was found that the transplanted white-haired skin piece of BALB/c mouse had been able to be implanted successfully on the black-haired skin of the BALB/c mouse. This finding demonstrates that the C3H/He mouse having the heart transplanted from the BALB/c mouse acquired the immunologic tolerance to the BALB/c mouse.
INDUSTRIAL APPLICABILITY
The agent for suppressing the fibrosis of a transplanted organ and immunologic-tolerance acquiring agent according to the present invention are useful in the fields of organ transplantation and regeneration therapy.

## Claims

1. A method for allowing a recipient patient having a donor organ or tissue transplant to acquire immunologic tolerance to the donor organ or tissue, which comprises administering to the patient an effective amount of hepatocyte growth factor simultaneously with or immediately after the transplantation of the donor organ or tissue,
wherein the hepatocyte growth factor is a peptide consisting of an amino acid sequence having not less than about 95% homology with SEQ ID NO: 2 and which promotes immunological tolerance in the patient.