WO2012144535A1 - 急性肝不全抑制剤及びその薬効評価法 - Google Patents
急性肝不全抑制剤及びその薬効評価法 Download PDFInfo
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- WO2012144535A1 WO2012144535A1 PCT/JP2012/060505 JP2012060505W WO2012144535A1 WO 2012144535 A1 WO2012144535 A1 WO 2012144535A1 JP 2012060505 W JP2012060505 W JP 2012060505W WO 2012144535 A1 WO2012144535 A1 WO 2012144535A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a therapeutic agent for acute liver failure and a method for evaluating its efficacy. More specifically, the present invention relates to an acute liver failure inhibitor comprising hepatocyte growth factor (HGF), particularly a prophylactic and / or therapeutic agent for fulminant hepatitis and late liver failure, and treatment with HGF.
- HGF hepatocyte growth factor
- the present invention relates to a method for evaluating the efficacy of human HGF, which comprises measuring the amount of ⁇ -fetoprotein and / or soluble Fas in a given patient with liver failure.
- Acute liver failure is a fatal clinical syndrome characterized by a rapid loss of hepatocyte function, causing coagulopathy, jaundice and brain damage.
- FH fulminant hepatitis
- LOHF late liver failure
- Fulminant hepatitis is a disease with a very poor prognosis, and liver transplantation has been established as the only treatment that improves the survival rate.
- the implementation rate is only over 20%. Since the survival rate from treatment is as low as 20 to 50%, it is urgent to develop a new treatment method with proven effectiveness.
- Hepatocyte growth factor is a growth factor isolated from the plasma of patients with fulminant hepatitis and having a strong effect of promoting liver regeneration (see, for example, Patent Document 1).
- HGF Hepatocyte growth factor
- Patent Documents 1 and 2 it has been found to have various physiological activities such as hepatocyte protective action by anti-apoptosis (for example, see Non-Patent Documents 3 to 5) and anti-fibrotic action.
- Expectation is expected for the treatment of hepatic insufficiency.
- effectiveness has been confirmed in in vitro and animal models, the efficacy and safety of HGF in the human body still remain unknown.
- HGF administration is effective for lifesaving effect in acute liver failure with extremely poor prognosis such as fulminant hepatitis and late liver failure, and in preventing fulminant non-coma acute liver failure with encephalopathy I could't predict it at all.
- HGF administration may cause side effects such as blood pressure reduction and nephrotoxicity, no administration protocol has been established to avoid these side effects in the clinic.
- liver regeneration promoting action has been evaluated from the increase in liver weight and serum albumin level by recombinant human HGF (Non-patent Document 6).
- Non-patent Document 6 Non-patent Document 6
- the serum albumin level reflects the improvement in liver synthesis ability as a result of liver regeneration, and is not suitable as an index of liver regeneration itself.
- ⁇ -fetoprotein is a glycoprotein with a molecular weight of 67 kDa found in the serum of human fetus, and is practically used as a serum immunological diagnostic marker for hepatocellular carcinoma.
- AFP is expressed in immature hepatocytes such as hepatic progenitor cells and hepatoblasts, and clinically, serum AFP levels in fulminant hepatitis and chronic hepatitis slightly increase reflecting liver regeneration. It has been reported (Non-Patent Document 7).
- soluble Fas is one in which Fas present on the cell surface is cleaved and released into the blood, and binding to Fas ligand antagonizes the binding of Fas and Fas ligand on the cell surface to suppress apoptosis.
- the blood HGF level which is important for prognosis prediction, is remarkably increased.
- serum AFP level and soluble Fas level, There was no prediction that AFP or soluble Fas could be used as an index of therapeutic effect (medicine biomarker) by administration of exogenous HGF.
- An object of the present invention is to provide an effective and safe means for treating acute liver failure other than liver transplantation and a means for evaluating the therapeutic effect.
- rh-HGF recombinant human HGF
- rh-HGF is effective in treating acute liver failure associated with hepatic encephalopathy such as fulminant hepatitis and delayed liver failure, and in preventing fulminant non-coma acute liver failure.
- AFP ⁇ -fetoprotein
- soluble Fas were successfully identified as biomarkers that can monitor the regenerative action and the anti-apoptotic (hepatocyte protection) action in real time.
- the administration protocol optimized to avoid side effects such as lowering blood pressure and nephrotoxicity is also effective in the clinic.
- the present inventors have completed the present invention.
- the present invention is as follows.
- An acute liver failure inhibitor comprising a hepatocyte growth factor.
- hepatocyte growth factor is derived from human.
- a method for evaluating the efficacy of hepatocyte growth factor comprising measuring the amount of ⁇ -fetoprotein and / or soluble Fas in a sample collected from a patient having a liver disorder administered with hepatocyte growth factor.
- ⁇ -fetoprotein is an indicator of liver regeneration effect
- soluble Fas is an indicator of anti-apoptotic effect.
- the measured value of ⁇ -fetoprotein and / or soluble Fas is compared with the measured value in a sample collected from the patient before administration of hepatocyte growth factor, and ⁇ -fetoprotein and / or soluble Fas is measured more than before administration.
- the amount of ⁇ -fetoprotein and / or soluble Fas in a sample collected from the patient over time during the administration period of hepatocyte growth factor is monitored, and ⁇ -fetoprotein and / or soluble Fas is monitored during the period.
- Human HGF has a certain lifesaving effect against acute liver failure with extremely poor prognosis, such as fulminant hepatitis and late liver failure, so patients with acute liver failure who cannot receive liver transplantation, especially fulminant It is useful in preventing fulminant in patients with non-coma acute liver failure before hepatitis or late-onset liver failure.
- AFP and soluble Fas reflect the liver regeneration effect and anti-apoptosis (liver protection) effect of human HGF administration, respectively, so that it is determined in real time whether the expected drug effect by human HGF administration is observed.
- the human HGF dose and / or administration period can be optimized (stratified) accordingly.
- FIG. 4 shows that intravenous infusion of rh-HGF reduces blood pressure through capacitive blood vessels in minipigs.
- the effects of intravenously administered rh-HGF on systolic blood pressure (systolic BP), diastolic blood pressure (diastolic BP), heart rate (HR) and cardiac function were investigated in minipigs under general anesthesia.
- systolic BP systolic blood pressure
- diastolic blood pressure diastolic blood pressure
- HR heart rate
- cardiac function were investigated in minipigs under general anesthesia.
- Rh-HGF 0.4 mg / kg was administered over 3 hours with a gradual increase in rate (0.01 mg / kg for the first 60 minutes, 0.03 mg / kg for the next 60 minutes) In the final 60 minutes, 0.36 mg / kg), the blood pressure and heart rate gradually decreased.
- C By injecting 100 mL of physiological saline before rh-HGF administration, a decrease in BP during rh-HGF administration could be prevented.
- FIG. 5 shows that repeated administration of rh-HGF induces increased urinary excretion of albumin and protein in rats.
- Prothrombin time international standardized ratio PT-INR
- total bilirubin T-Bil
- serum albumin before rh-HGF administration day 1 of rh-HGF administration period
- day 7 of rh-HGF administration period day 7 of rh-HGF administration period
- the measurement was made at 1, 7, and 14 days after administration.
- ALT alanine transaminase
- AFP serum alpha-fetoprotein
- Serum HGF and AFP levels were 0.77 and 7.0 ng / mL, respectively, and the liver volume measured by CT was 1055 mL.
- rh-HGF administration (0.6 mg / m 2 / day) was started.
- Administration of diuretics to reduce massive ascites increased serum creatinine levels to 2.0 mg / dL, so protocol treatment was discontinued on day 14 resulting in 13 days of rh-HGF administration.
- the prolongation of PT was stable during rh-HGF administration and the observation period, but T-Bil gradually increased and hepatic encephalopathy did not improve. Liver failure progressed gradually after the observation period. The patient eventually died 68 days after the onset of hepatic encephalopathy.
- PE indicates plasma exchange and CHDF indicates continuous hemofiltration dialysis.
- CHDF indicates continuous hemofiltration dialysis.
- Urinary findings included mild hepatic encephalopathy with flapping tremor, jaundice, and proteinuria and microhematuria with bladder catheter. Platelet count and serum albumin level decreased to 6.9 ⁇ 10 4 / ⁇ L and 3.2 g / dL, respectively, and PT was prolonged to 49% (PT-INR: 1.55).
- serum ALT levels increased to 131 IU / L.
- Serum HGF and AFP levels were 1.94 and 22.9 ng / mL, respectively, and liver volume was 595 mL.
- treatment with rh-HGF was started and the protocol treatment was continued for 14 days without any serious adverse events.
- Consciousness level was not impaired throughout the study period.
- Intravenous rh-HGF administration reduced systolic blood pressure but was conscious and did not complain of any symptoms.
- Prednisolone (PSL) was administered to reduce ALT, but blood biochemistry and patient status remained stable throughout the study. At the end of the study, biochemical findings gradually improved and eventually the patient survived.
- Thrombocytopenia (7.0 ⁇ 10 4 / ⁇ L), PT: 43% (PT-INR 1.62), T-Bil level: 27.6 mg / dL, ALT level: 253 IU / L, and serum albumin level: 2.9 g / DL but no hepatic encephalopathy (HE) (observed temporarily before enrollment).
- Serum HGF and AFP levels were 1.88 and 39.7 ng / mL, respectively, and liver volume was 1110 mL.
- the rh-HGF administration continued for 14 days and PSL was administered to reduce ALT throughout the study period.
- the increase in T-Bil and prolongation of PT improved moderately during rh-HGF administration and further improved after the observation period. Eventually the patient survived.
- Human hepatocyte growth factor (HGF) which is an active ingredient of the acute liver failure inhibitor of the present invention, is the same as or substantially the same as the amino acid sequence represented by amino acid numbers 30 to 728 or 32-728 in the amino acid sequence represented by SEQ ID NO: 2.
- the origin thereof is not particularly limited, and human or other warm-blooded animal rabbits (e.g., cattle, pigs, mice, rats, hamsters, monkeys, horses, sheep, goats, Rabbits, guinea pigs, chickens, etc.)
- Cellular cells eg, hepatocytes, spleen cells, renal tubular cells, keratinocytes, vascular endothelial cells, bone marrow cells, mesangial cells, neurons, glial cells, pancreatic ⁇ cells, Langerhans cells, epidermal cells , Epithelial cells, goblet cells, smooth muscle cells, skeletal muscle cells, fibroblasts, fibrocytes, adipocytes, immune cells, megakaryocytes, synovial cells Chondrocytes, bone cells, osteoblasts, osteoclasts, breast cells or stromal cells, or precursor cells of these cells, stem cells, cell lines or cancer cells, etc.] s
- amino acid sequence substantially the same as the amino acid sequence shown by amino acid numbers 30 to 728 or 32-728 in the amino acid sequence shown by SEQ ID NO: 2 is the amino acid sequence shown by SEQ ID NO: 2 amino acid numbers 30 to 728 or 32 to An amino acid sequence having about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more of the amino acid sequence represented by 728, comprising the amino acid sequence A sequence in which the protein has substantially the same activity as the protein comprising the amino acid sequence shown in SEQ ID NO: 2.
- Examples of substantially the same activity include liver protecting (apoptosis suppression) action, liver regeneration promoting action and the like. “Substantially homogeneous” indicates that their activities are qualitatively (eg, physiologically or pharmacologically) identical. Therefore, it is preferable that the activities such as apoptosis inhibitory action and liver regeneration promoting action are equivalent (for example, about 0.5 to about 2 times), but quantitative factors such as the degree of these activities and the molecular weight of the protein are different. Also good.
- the HGF used in the present invention is preferably human HGF (pro-HGF) having an amino acid sequence represented by amino acid numbers 30 to 728 or 32-728 in the amino acid sequence represented by SEQ ID NO: 2, or an ortholog thereof in other mammals
- pro-HGF human HGF
- HGF is biosynthesized as a single-chain protein (pro-HGF) having an intramolecular disulfide bond, and then is cleaved by an HGF activator to become a mature double-stranded HGF having biological activity.
- the HGF used in the present invention may be either pro-form or mature protein, but preferably single-chain pro-HGF is used.
- HGF may be a free form or a salt form.
- salts of HGF include physiologically acceptable salts with acids (eg, inorganic acids, organic acids) and bases (eg, alkali metals), and physiologically acceptable acid addition salts are particularly preferable.
- salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
- inorganic acids for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
- organic acids for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid.
- HGF is a protein purification technique known per se, for example, J. from a cell or tissue that naturally produces it or its primary culture or cell line, such as patient plasma obtained by plasma exchange as a treatment for fulminant hepatitis. It can be isolated and purified by a method combining several types of column chromatography, such as the method described in Clin. Invest., 81: 414 (1998).
- HGF can also be produced according to a known peptide synthesis method.
- the peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.
- the target protein can be produced by removing the protecting group.
- the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) or (2). (1) M.
- the protein thus obtained can be purified and isolated by a known purification method.
- the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
- the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, when the protein is obtained as a salt
- the salt can be converted into a free form or other salt by a known method or a method analogous thereto.
- HGF can also be produced by culturing a transformant containing a nucleic acid encoding it and separating and purifying recombinant HGF from the resulting culture.
- the nucleic acid encoding HGF may be DNA or RNA, or may be a DNA / RNA chimera.
- DNA is used.
- the nucleic acid may be double-stranded or single-stranded. In the case of double-stranded DNA, double-stranded DNA, double-stranded RNA or DNA: RNA hybrid may be used, but double-stranded DNA is preferred.
- DNA encoding HGF examples include genomic DNA, human or other warm-blooded animal cells producing HGF, cDNA (cRNA) derived from any tissue or organ in which those cells exist, synthetic DNA (RNA), and the like. Can be mentioned.
- genomic DNA and cDNA encoding HGF the genomic DNA fraction and total RNA or mRNA fraction prepared from the cells and tissues described above were used as templates, respectively, Polymerase Chain Reaction (PCR) method and ReverseReTranscriptase-PCR (RT- PCR can also be directly amplified.
- PCR Polymerase Chain Reaction
- RT- PCR ReverseReTranscriptase-PCR
- genomic DNA and cDNA encoding HGF are genomic DNA library and cDNA library prepared by inserting genomic DNA prepared from the above cells / tissues and total RNA or mRNA fragment into an appropriate vector ( It can also be cloned from a liver, spleen or placenta-derived cDNA library by colony or plaque hybridization method or PCR method, respectively.
- the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
- nucleic acid encoding HGF for example, a nucleic acid containing a nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence capable of hybridizing under stringent conditions with a complementary strand sequence of the nucleotide sequence represented by SEQ ID NO: 1. And a nucleic acid encoding a protein containing and having substantially the same quality of activity as the above-described HGF (eg, apoptosis-inhibiting action, liver regeneration promoting action, etc.).
- a nucleic acid encoding a protein containing and having substantially the same quality of activity as the above-described HGF eg, apoptosis-inhibiting action, liver regeneration promoting action, etc.
- nucleic acid capable of hybridizing with the complementary strand sequence of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions include, for example, about 85% or more, preferably about 90% or more, with the nucleotide sequence represented by SEQ ID NO: 1. More preferably, a nucleic acid containing a base sequence having an identity of about 95% or more, particularly preferably about 97% or more is used.
- Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to highly stringent conditions.
- Highly stringent conditions include: (1) low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C, and (2) formamide 50% (v) with various denaturing agents, for example, 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride, 50 mM sodium phosphate buffer (pH 6.5) containing 75 mM sodium citrate. / v) Reaction conditions characterized by using formamide at 42 ° C. are exemplified.
- stringent conditions include 50% formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated sperm DNA (50 ⁇ g / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, washed with 0.2xSSC and 50% formaldehyde at 55 ° C, followed by 0.1xSSC containing EDTA at 55 ° C High stringency washing may be performed.
- a person skilled in the art can easily achieve a desired stringency by appropriately adjusting the temperature during the hybridization reaction and / or washing, the ionic strength of the buffer, and the like according to factors such as the probe length.
- the nucleic acid encoding HGF used in the present invention is preferably human prepro HGF having the nucleotide sequence shown in SEQ ID NO: 1, its ortholog in other mammals, natural allelic variant or polymorphic variant in human prepro HGF, or These are splice variants.
- the DNA base sequence can be determined using a known kit such as Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), etc., using the ODA-LA PCR method, the Gapped duplex method, Conversion can be performed according to a method known per se such as the Kunkel method or a method analogous thereto.
- the cloned DNA can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. If necessary, the DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
- An expression vector containing DNA encoding HGF can be produced, for example, by excising a target DNA fragment from DNA encoding HGF and ligating the DNA fragment downstream of a promoter in an appropriate expression vector.
- Expression vectors include plasmids derived from E. coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), yeast-derived plasmids (e.g., pSH19, pSH15), insect cell expression Plasmids (e.g. pFast-Bac), animal cell expression plasmids (e.g.
- bacteriophages such as lambda phage, insect virus vectors such as baculovirus ( Examples: BmNPV, AcNPV), animal virus vectors such as retrovirus, vaccinia virus, adenovirus, adeno-associated virus, etc. are used.
- the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
- a cytomegalovirus (CMV) -derived promoter eg, CMV immediate early promoter
- HSV human immunodeficiency virus
- HIV LTR HIV LTR
- RSV rous sarcoma virus
- RSV LTR mouse mammary tumor virus
- MoMLV Moloney murine leukemia virus
- HSV herpes simplex virus
- HSV herpes simplex virus
- SV SV
- SV herpes simplex virus
- SV SV thymidine kinase
- SV40-derived promoter eg, SV40 early promoter
- AAV adeno-associated virus
- a polyhedrin promoter, a P10 promoter and the like are preferable.
- the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter and the like are preferable.
- the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
- yeast PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
- dhfr dihydrofolate reductase
- MTX metalhotrexate
- Amicillin resistance Ampicillin resistance
- Neo r neomycin resistance
- G418 resistance G418 resistance
- the target gene can also be selected using a medium not containing thymidine.
- a base sequence (signal codon) encoding a signal sequence suitable for the host may be added to the 5 'end of the DNA encoding HGF.
- the host is an animal cell
- the host is Escherichia
- an ⁇ -amylase signal sequence, a subtilisin signal sequence, and the like are used.
- yeast an MF ⁇ signal sequence, a SUC2 signal sequence, and the like are used.
- animal cells for example, animal cells, insect cells, insects, Escherichia bacteria, Bacillus bacteria, yeasts and the like are used.
- animal cells include monkey cells (eg: COS-1, COS-7 , CV-1, Vero), hamster-derived cells (eg: BHK, CHO, CHO-K1 , CHO-dhfr -), mice Cells (e.g. NIH3T3, L, L929, CTLL-2, AtT-20), rat-derived cells (e.g. H4IIE, PC-12, 3Y1, NBT-II), human-derived cells (e.g. HEK293, A549, HeLa, HepG2, HL-60, Jurkat, U937) are used.
- monkey cells eg: COS-1, COS-7 , CV-1, Vero
- hamster-derived cells eg: BHK, CHO, CHO-K1 , CHO-dhfr -
- mice Cells e.g. NIH
- insect cells for example, when the virus is AcNPV, larvae-derived cell lines derived from night stealing (Spodoptera frugiperda cell; Sf cells), Trichoplusia ni midgut-derived MG1 cells, Trichoplusia ni egg-derived High Five TM cells , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used.
- insect-derived cell lines Bacthelial cells
- Sf cells examples include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13: 213-217 (1977)) and the like.
- insects include silkworm larvae.
- genus Escherichia examples include Escherichia coli K12, DH1, JM103, JA221, HB101, C600, and the like.
- Bacillus bacteria examples include Bacillus subtilis MI114 and 207-21.
- yeast examples include Saccharomyces cerevisiae AH22, AH22R ⁇ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71, and the like.
- Transformation can be performed according to a known method depending on the type of host.
- Animal cells can be transformed, for example, according to the method described in Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52: 456 (1973).
- Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6: 47-55 (1988).
- Recombinant HGF can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
- a method of separating the culture supernatant from the culture by centrifugation or filtration is used. Isolation and purification of HGF contained in the obtained culture supernatant can be performed according to a method known per se.
- Such methods include the use of solubilities such as salting out and solvent precipitation; mainly using differences in molecular weight such as hemodialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
- the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein is obtained as a salt, a method known per se Alternatively, the salt can be converted into a free form or other salt by a method analogous thereto.
- HGF may be used as it is, but it can also be used after mixing with a pharmacologically acceptable carrier as necessary to obtain a pharmaceutical composition.
- a pharmacologically acceptable carrier various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Solvents, solubilizers, suspending agents, isotonic agents, buffering agents in liquid formulations It is formulated as a soothing agent. Moreover, formulation additives, such as antiseptic
- the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
- solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
- the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, such as polyvinyl alcohol, polyvinyl Examples include pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and other hydrophilic polymers, polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
- the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
- Preferable examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate.
- Preferable examples of the soothing agent include benzyl alcohol.
- Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
- Preferable examples of the antioxidant include sulfite and ascorbate.
- Suitable examples of the colorant include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellows Nos. 4 and 5, and edible blue Nos. 1 and 2), water-insoluble lake dyes (Example: Aluminum salt of water-soluble edible tar pigment, etc.), natural pigment (eg, ⁇ -carotene, chlorophyll, Bengala, etc.).
- Examples of the dosage form of the pharmaceutical composition include injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and parenterals such as drops.
- the pharmaceutical composition can be produced by a method commonly used in the field of pharmaceutical technology, such as the method described in the Japanese Pharmacopoeia.
- the content of the active ingredient in the pharmaceutical composition varies depending on the dosage form, the dose of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight.
- Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions. May contain antioxidants, buffers, antibacterial agents, tonicity agents and the like. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
- the protein preparation can be enclosed in a container in unit doses or multiple doses such as ampoules and vials.
- HGF and a pharmacologically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.
- HGF human HGF
- HGF is converted to an immunoconjugate in which the antibody is cross-linked.
- Medium stability and delivery efficiency to hepatocyte surface can be improved.
- hepatocyte surface molecules include, but are not limited to, EGFR (HER1), HER2, HER3, HER4, and the like.
- EGFR HER1
- HER2, HER3, HER4, and the like examples include, but are not limited to, EGFR (HER1), HER2, HER3, HER4, and the like.
- HER1 HER1
- HER2, HER3, HER4, and the like When using an anti-EGFR antibody, it is desirable to use a non-neutralizing antibody as a targeting antibody so as not to inhibit signal transduction from EGFR.
- the antibody against the hepatocyte surface molecule may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.
- the antibody can be prepared by a well-known immunological technique.
- the antibody may be a complete antibody molecule or a fragment.
- the fragment may be any fragment as long as it has an antigen binding site (CDR) for hepatocyte surface molecules, and examples thereof include Fab, F (ab ′) 2 , ScFv, and minibody.
- CDR antigen binding site
- the HGF preparation finally prepared as a liquid preparation as described above can be administered to a patient by injection or instillation intravenously, intraarterially, subcutaneously, intramuscularly, intraperitoneally or the like.
- the dosage is appropriately adjusted depending on the patient's symptoms, age, weight, etc.
- 0.1 mg / kg / day which has been confirmed to be effective in rats, is converted to 0.6 mg in humans.
- / m 2 / day, once or several amounts between safety (reversible side effects) is 24 mg / m 2 / day for a 4.0 mg / kg / day, which has been confirmed in terms of human rat It can be administered in divided doses, but is not limited thereto.
- a single administration over a long period of time by instillation.
- a daily dose is administered once, it is administered for 1 to 12 hours, preferably 2 to 6 hours.
- Specific administration protocols include, for example, about 10% of a single dose in the first 1/3 period of a single administration time, about 30% of a single dose in the next 1/3 period, and Examples include, but are not limited to, intravenous administration of about 60% of a single dose during the last 1/3 period.
- a method in which a physiological saline is infused prior to administration of HGF can be mentioned.
- the administration period of the HGF preparation is not particularly limited, and can be appropriately adjusted within a range in which sufficient drug efficacy is obtained and no serious side effects occur according to the patient's symptoms and the like. Is about 10-20 days.
- the dose and / or administration period of the HGF preparation can be appropriately increased or decreased based on the results of the evaluation of the efficacy of HGF described later.
- a subject of treatment with the HGF preparation of the present invention is an acute liver failure patient.
- Acute liver failure is defined as a case of acute liver injury due to various causes that has a prothrombin time (PT) of 50% or less or a prothrombin inhibition-international unit (PI-INR) of 1.5 or more.
- PT prothrombin time
- PI-INR prothrombin inhibition-international unit
- Fulminant hepatitis is identified as a hepatitis that develops hepatic encephalopathy within 8 weeks after the appearance of symptoms, with PT less than 40% of the standard value. Fulminant hepatitis is further classified into two subtypes, acute (FHA) and subacute (FHSA) (encephalopathy develops within 10 days and after 11 days, respectively). On the other hand, patients with PT less than 40% who develop encephalopathy 8-24 weeks after the appearance of symptoms are diagnosed with late liver failure. As shown in the examples below, the lifesaving rate based on a nationwide survey on patients with fulminant hepatitis and late liver failure in Japan was about 18%, whereas the treatment with the HGF preparation of the present invention was 4 people.
- the administration protocol of the HGF preparation used in this example is a dose that has been confirmed to have a liver regeneration effect in an experiment using animals that do not have severe liver damage, such as normal rats and partially hepatectomized rats, and fulminant. Based on the treatment period predicted to be necessary and sufficient based on the treatment results for hepatitis and late liver failure, the results of the non-clinical safety study show that the minimum dose should not occur without causing irreversible side effects.
- the survival rate in patients with fulminant hepatitis and / or late liver failure can be further improved by optimizing the dose and the administration period.
- the treatment using the HGF preparation of the present invention is performed on a patient with non-coma acute liver failure before progressing to fulminant hepatitis or delayed liver failure, thereby causing fulminant hepatitis and / or delayed onset. Progression to fulminant liver failure (fulminant) can be prevented, and the mortality of patients with acute liver failure can be significantly reduced as a result of the progress-inhibiting effect.
- the HGF preparation of the present invention can be used in combination with other means for treating acute liver failure as necessary.
- Other therapies that can be combined with HGF include, for example, corticosteroid treatment [Tygstrup, N. & Juhl, E., Gut, 1979; 20: 620-623], lamivudine administration for acute hepatitis B [Kumar, M. et al., Hepatology, 2007; 45: 97-101], plasma exchange therapy [Clemmesen, JO et al., Am. J. Gastroenterol., 2001; 96: 1217-1223].
- the present invention also provides a method for evaluating the efficacy of HGF, comprising measuring the amount of ⁇ -fetoprotein and / or soluble Fas in a sample collected from a patient having a liver disorder who has been administered HGF.
- the increase in serum AFP levels in acute liver failure was thought to be due to liver regeneration induced following liver injury (Taketa, K., Hepatology, 12: 1420-1432 (1990)).
- serum HGF levels are important for predicting fulminant acute hepatitis and predicting prognosis of fulminant hepatitis.
- serum AFP levels do not increase due to an increase in blood HGF concentration due to endogenous HGF induced by liver injury, and therefore, exogenously HGF (rh- Even if HGF) was administered to raise the blood HGF concentration, it could not be predicted that the serum AFP level would rise.
- the present inventors have repeatedly administered HGF to patients with fulminant hepatitis and late liver failure.
- the serum AFP level rises during the administration period and gradually decreases after the end of the administration.
- AFP treats patients with chronic liver failure such as fulminant hepatitis and other acute liver failure, severe acute hepatitis, and decompensated cirrhosis with HGF, an index of the effect of promoting liver regeneration (Biomarker) was clarified.
- Biomarker an index of the effect of promoting liver regeneration
- Soluble Fas is one in which Fas present on the cell surface is cleaved and released into the blood, and binding to Fas ligand antagonizes the binding between Fas on the cell surface and Fas ligand and suppresses apoptosis.
- Soluble Fas treated with HGF in patients with chronic liver failure such as acute liver failure including fulminant hepatitis, severe acute hepatitis, decompensated cirrhosis, etc. Marker).
- the patient to be evaluated by the method for evaluating the efficacy of the present invention is not particularly limited as long as it has liver damage and is administered HGF as a treatment thereof, and various diseases associated with liver damage / hepatocyte death,
- acute liver failure including fulminant hepatitis, delayed liver failure, non-coma acute liver failure
- acute hepatitis chronic hepatitis
- autoimmune liver disease autoimmune hepatitis, primary biliary cirrhosis
- Viral hepatitis AE (type AE) liver fibrosis, cirrhosis
- liver cancer alcoholic liver injury, drug-induced liver injury ⁇ (addictive drug-induced liver disease, allergic drug-induced liver injury), liver abscess, liver parasitic disease ⁇ (Japan)
- Examples include schistosomiasis, hepatosomiasis), liver amyloidosis, lupoid hepatitis), and the like, preferably acute liver failure patients.
- a sample derived from a patient as a test sample is not particularly limited as long as an increase in AFP associated with the liver regeneration effect and / or an increase in soluble Fas associated with the anti-apoptotic effect of hepatocytes can be detected.
- the invasion to the patient is small, for example, blood, plasma, serum, saliva, urine, tears that can be easily collected from a living body, cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid Among them, those collected relatively easily such as aqueous humor and vitreous humor are mentioned, and serum or plasma is more preferable.
- serum or plasma it can be prepared by collecting blood from a patient according to a conventional method and separating the liquid component.
- Detection of AFP and / or soluble Fas in a test sample can be performed by, for example, subjecting the sample to various molecular weight measurement methods such as gel electrophoresis and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography).
- various molecular weight measurement methods such as gel electrophoresis and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography).
- ionization methods eg, electron impact ionization, field desorption, secondary ionization, fast atom bombardment, matrix-assisted laser desorption ionization (MALDI), Electrospray ionization, etc.
- mass spectrometers eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, etc.
- a band or spot that matches the molecular weight of the marker protein There could be done by detecting a peak, but not limited to.
- a method of producing an antibody that recognizes the amino acid sequence and detecting the protein by Western blotting or various immunoassays is more preferably used. Furthermore, the hybrid detection method of the above method is also effective.
- AFP and soluble Fas can be measured by using an antibody against them and constructing an optimized immunoassay system and making it a kit without using a special device such as a mass spectrometer. This is particularly useful in that the protein can be detected with high accuracy.
- An antibody against AFP or soluble Fas can be prepared, for example, by isolating and purifying the protein from a human sample expressing the protein and immunizing an animal using the marker protein or a partial peptide thereof as an antigen.
- the antibody against AFP or soluble Fas may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique.
- the antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2 , ScFv, and minibody.
- a polyclonal antibody is commercially available using AFP or soluble Fas or a partial peptide thereof (if necessary, a complex cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin)) as an antigen. 2 to 4 times subcutaneously or intraperitoneally in animals every 2-3 weeks (by measuring the antibody titer of partially collected serum by a known antigen-antibody reaction and confirming its increase) It can be obtained by collecting whole blood about 3 to about 10 days after the final immunization and purifying the antiserum.
- animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.
- Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, principles of cell fusion methods and preparation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal antibodies and cancer-basics and clinics”, 2-14. Page, Science Forum Publishing, 1985).
- AFP or soluble Fas or a partial peptide thereof is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and spleen or lymph nodes are collected about 3 days after the final administration, and lymphocytes are collected.
- the lymphocytes and myeloma cells are cell-fused to obtain a hybridoma that produces a monoclonal antibody against AFP or soluble Fas.
- the cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)].
- a hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method.
- the hybridoma producing the monoclonal antibody can be cultured in vitro, or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.
- the method for evaluating the efficacy of the present invention using anti-AFP or anti-soluble Fas antibody is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is chemically determined.
- any measurement method may be used as long as it is a measurement method that is detected by physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
- nephrometry, competition method, immunometric method and sandwich method are preferably used.
- a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
- the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
- the enzyme is preferably stable and has a large specific activity.
- ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
- the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
- luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
- a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.
- the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
- a test sample is reacted with an insolubilized anti-AFP or anti-soluble Fas antibody (primary reaction), and another labeled anti-AFP or anti-soluble Fas antibody is reacted (secondary reaction).
- the amount of AFP or soluble Fas in the test sample can be quantified by measuring the amount (activity) of the labeling agent on the insolubilized carrier.
- the primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.
- Monoclonal antibodies against AFP or soluble Fas can also be used in measurement systems other than the sandwich method, for example, competitive methods, immunometric methods, nephrometry, and the like.
- competition method the antigen in the test sample and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeled B or F is measured, and the amount of antigen in the test sample is quantified.
- a soluble antibody is used as an antibody
- B / F separation is performed using polyethylene glycol
- a liquid phase method using a second antibody against the antibody and a solid-phased antibody is used as the first antibody
- a soluble method is used, and a solid phase method using a solid phase antibody as the second antibody is used.
- the immunometric method the antigen of the test sample and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test sample is separated from the antigen.
- the solid phase and the liquid phase are separated.
- the amount of label in any phase is measured to quantify the amount of antigen in the test sample.
- the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured.
- Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test sample is small and only a small amount of precipitate is obtained.
- the level of AFP in a sample from a patient administered with HGF is significantly increased compared to the level of AFP in a sample collected from the patient prior to administration of HGF. If so, it can be determined that liver regeneration has been induced in the patient and the efficacy of HGF administration is observed.
- the level of AFP in the sample from the patient receiving HGF is not significantly changed or rather lower than the level of AFP in the sample collected from the patient before the administration of HGF It can be determined that liver regeneration is not induced in the patient, and the effect of promoting liver regeneration by HGF administration is not observed.
- the planned administration protocol may be carried out as it is, but if the effect is not sufficiently obtained, the dosage of HGF Increase and / or prolongation of the administration period can be considered.
- the level of soluble Fas in a sample from a patient receiving HGF is significantly increased compared to the level of soluble Fas in a sample taken from the patient prior to administration of HGF If it is, it can be determined that the apoptosis of hepatocytes is suppressed in the patient, and the efficacy of HGF administration is observed. On the other hand, the level of soluble Fas in the sample from the patient who received HGF was not significantly changed or rather decreased compared to the level of soluble Fas in the sample collected from the patient before administration of HGF.
- the planned administration protocol may be carried out as it is, but if the effect is not sufficiently obtained, the dosage of HGF may be Increase and / or prolongation of the administration period can be considered.
- the method for evaluating the efficacy of the present invention is preferably carried out by collecting samples from patients in time series, measuring the amount of AFP and / or soluble Fas in each sample, and examining their changes over time.
- the sample collection interval is not particularly limited, but it is desirable to sample as frequently as possible within a range that does not impair the patient's QOL.
- plasma or serum is used as a sample, for example, 1 to 10 days, preferably 3 to 7 days It is preferable to collect blood at intervals of.
- the level of AFP increases with time, it can be determined that liver regeneration is induced in the patient, and that the efficacy of HGF administration is observed.
- liver regeneration is not induced in the patient and that the effect of promoting liver regeneration by HGF administration is not observed. Can do. In the latter case, if the anti-apoptotic effect using soluble Fas as an index is sufficient, the planned administration protocol may be carried out as it is, but if the effect cannot be obtained sufficiently, the dosage of HGF Increase and / or prolongation of the administration period can be considered.
- the planned administration protocol may be carried out as it is, but if the effect is not sufficiently obtained, the dosage of HGF may be Increase and / or prolongation of the administration period can be considered.
- AFP expression is known to be affected by the glucocorticoid response element (GRE) present in the 5 ′ flanking region of the AFP gene [Ido, A. et al., Cancer Res., 1995; 55: 3105-3109], because it may not accurately reflect the liver regeneration effect of HGF administration. Therefore, in patients who are also using steroidal anti-inflammatory agents, the efficacy of HGF can be evaluated with emphasis on the determination of the anti-apoptotic effect of hepatocytes using soluble Fas as an index.
- GRE glucocorticoid response element
- the present invention also provides a kit for evaluating the efficacy of HGF, comprising an anti-AFP antibody and / or an anti-soluble Fas antibody.
- the kit further includes other components preferable for carrying out the above-described method for evaluating the efficacy of the present invention, such as a reaction buffer solution, a washing solution, an insolubilizing carrier, a labeling agent, an AFP and / or a soluble Fas preparation. May be included.
- the HGF preparation of the present invention is preferably used in combination with the drug efficacy evaluation kit of the present invention.
- renal impairment ⁇ 1 mg / mL protein urinary excretion, red blood cell deformation or RBC casts in sediment urine, serum creatinine level greater than 2.0 mg / dL, or urine volume less than 400 mL / day ) Were also excluded.
- rh-HGF was prepared as a GMP grade material.
- the initial dose of rh-HGF was fixed at 0.6 mg / m 2 / day (determined by multiple preclinical animal studies as a dose ensuring safety and clinical efficacy). In dose escalation studies, the dose of rh-HGF can be increased from the initial dose (0.6 mg / m 2 ) to 1.2, 1.8 or 2.4 mg / m 2 .
- rh-HGF was intravenously administered in increments of 3 hours up to 14 days and then observed for 14 days. All patients were followed and treatment outcomes were determined after the study period (up to 28 days).
- the first evaluation item was the safety of repeated intravenous administration of rh-HGF, and was evaluated based on the occurrence, frequency, and severity of adverse events. All patients were treated in the intensive care unit. During the study period, patients were regularly observed for safety from the start of rh-HGF administration until after completion of study drug administration. Safety assessment was performed by physical examination, clinical examination and observation of adverse events. Adverse events were observed throughout the study and graded according to a common toxicity criteria grading system. The causal relationship between adverse events and rh-HGF was determined by the physician's best judgment. All adverse events were treated appropriately regardless of their cause; patients were removed from the study when necessary.
- the incidence of adverse events was calculated from the number of patients who received at least one rh-HGF and experienced at least one adverse event.
- the second endpoint was the pharmacokinetics of intravenously injected rh-HGF, and clinical effects including survival and treatment outcome.
- ELISA enzyme-linked immunosorbent assay
- PT-INR PT International Normalized Ratio
- T-Bil serum albumin
- ALT alanine aminotransferase
- AFP ⁇ -fetoprotein
- echocardiography showed a decrease in left ventricular end-diastolic volume (LVEDV) and ejection fraction (EF), but no abnormality in left ventricular movement was observed (Fig. 1A).
- LVEDV left ventricular end-diastolic volume
- EF ejection fraction
- Reference example 2 Assessment of nephrotoxicity induced by repeated administration of rh-HGF in rats
- Urinary excretion of albumin increased in a dose-dependent manner from day 4 in rats treated with rh-HGF (FIG. 2).
- Example Phase I / II study of rh-HGF administration for patients with subacute fulminant hepatitis (FHSA) and late liver failure (LOHF) (1) Patient characteristics Between September 2005 and June 2008, 20 patients with FHSA or LOHF were evaluated for participation in a rh-HGF clinical trial. As a result, 16 patients were excluded because they met one or more exclusion criteria, and finally only 4 patients were enrolled. Fulminant hepatitis is a relatively rare syndrome in Japan (698 patients between 1998 and 2003), and serious complications to more accurately assess the safety and efficacy of treatment. Because patients who had s were excluded, it was difficult to adopt the study subjects.
- Patients 1, 2 and 4 were diagnosed with FHSA and patient 3 was diagnosed with LOHF. Because patients 1, 3 and 4 did not have an appropriate donor, and patient 2 was over 70 years of age, none were able to receive a liver transplant. FHSA in patients 1 and 4 is due to supplements containing HEV and coenzyme Q-10, respectively, and the cause of liver failure in patients 2 and 3 is unknown. Two patients with FHSA (patients 1 and 2) and one patient with LOHF (patient 3) showed hepatic encephalopathy with coma II and V, respectively, and the consciousness level of patient 4 with FHSA was not impaired at the time of enrollment .
- PT prothrombin time
- T-Bil total bilirubin
- serum HGF serum HGF
- Rh-HGF pharmacokinetics of slow-injected gradual infusions
- rh-HGF was administered after plasma exchange. Serum levels of HGF increased in parallel with a gradual increase in rh-HGF dose and reached the highest drug concentration (C max ) at the end of the 3 hour rh-HGF infusion (FIG. 3). C max gradually increased from 18.8 ⁇ 6.0 ng / mL on day 1 to 22.3 ⁇ 9.6 ng / mL on day 11 during the HGF administration period (Table 2).
- T 1/2 serum HGF half-life
- Patient 2 (awake from hepatic encephalopathy on day 3 of the HGF administration period) did not suffer from any symptoms, although the heart rate increased by up to about 30% during HGF administration (FIG. 4). All patients showed a slight to mild increase in urinary excretion of albumin at enrollment and decreased urine output during the study period. However, repeated administration of rh-HGF did not increase urinary excretion of albumin. Urine volume was also affected by several factors other than rh-HGF administration, such as infusion volume, circulating plasma volume, and diuretic administration.
- the doses chosen for this study are based on the scaling of doses used in preclinical animal studies and have been shown to be safe in some repeated dose toxicity studies, but animal studies have at least the results It shows that a 4-fold dose increase is possible.
- This dose (corresponding to 0.1 mg / kg in rodents) has been reported to promote liver regeneration in normal and partially hepatectomized rats [Ishii, T. et al., J. Biochem., 1995; 117: 1105-1112], does not guarantee efficacy in cases involving severe liver damage.
- the treatment period is based on a nationwide survey of FH and LOHF in Japan from 1998 to 2002.
- liver volume was measured by abdominal computed tomography (CT) examination before rh-human HGF administration, immediately after completion of administration, and 14 days after administration.
- CT computed tomography
- the serum AFP values before administration, on the 8th day after administration, the next day after administration, on the 7th day after administration and on the 14th day after administration are plotted in FIG.
- the serum AFP value was 7.0 ng / mL before rh-HGF administration, which was within the standard value (10.0 ng / mL or less), but increased to 15.0 ng / mL on day 3 of rh-HGF administration. On day 11 of administration, it reached 38.9 ng / mL.
- the serum AFP level rapidly increased during rh-HGF administration and decreased after the end of administration, and thus it was considered to be a biomarker for observing the efficacy of HGF, that is, the effect of HGF on promoting liver regeneration.
- the serum AFP level was within the reference value throughout the test period from before rh-HGF administration, and it is considered that induction of liver regeneration did not occur even after rh-HGF administration.
- patients 1 and 3 are both fatal, the clinical course is very different.
- the rh-HGF administration period (13 days) and the subsequent observation period (14 days) are stable and the general condition is stable.
- the period from onset to death was 79 days (68 days from the onset of encephalopathy to death), while patient 3 was an extremely severe case, with an 11-day period. Died on day 13 of the observation period after rh-HGF administration. That is, it is considered that patient 1 induced liver regeneration by rh-HGF administration, and had a life-prolonging effect, but patient 3 did not induce liver regeneration.
- liver volume (patient 1), which has been conventionally said to be an indicator for induction of liver regeneration, is 1055 mL before rh-HGF administration, 1076 mL the day after the end of administration, and 984 mL on the 14th day after the end of administration. No significant change was observed, and it decreased slightly after the end of administration (Table 3). Serum albumin levels did not change significantly during rh-HGF administration in any of patients 1 to 4 (FIG. 5). Therefore, it was shown that AFP can be an index (biomarker) of liver regeneration effect superior to liver volume and serum albumin.
- ALT which is considered to be a serum marker for hepatocellular injury
- rh-HGF serum marker for hepatocellular injury
- soluble Fas before rh-HGF administration was at the same level, and it was compensated for hepatocellular injury (as a biological response to protect hepatocytes).
- Patient 2 with a decreased serum ALT level did not require anti-apoptotic action (increased soluble Fas) by rh-HGF, whereas patient 4 had hepatocyte damage (increased serum ALT). Because it persisted, in addition to compensatory increase in soluble Fas, soluble Fas by rh-HGF was induced to suppress apoptosis, suggesting that this could eventually lead to survival.
- AFP and soluble Fas can be used as biomarkers for promoting liver regeneration and anti-apoptosis of HGF, respectively, and measuring changes in levels of both markers before and after administration of HGF in patients with liver damage.
- administration of HGF in the patient may induce liver regeneration and / or suppress apoptosis. It has been shown that it can be determined whether one effect is induced.
- hepatic encephalopathy such as fulminant hepatitis or delayed liver failure has progressed while ensuring reversibility of side effects.
- HGF preparations at the stage of non-coma acute liver failure, even patients who are not indicated for liver transplantation can prevent progression to fulminant hepatitis or late liver failure, and dramatically increase the survival rate Can be improved.
- HGF biomarkers of HGF of the present invention it becomes possible to stratify patients with hepatic failure, and to determine the optimal dose and / or administration period of HGF according to each patient group Therefore, it is extremely useful in that it can provide a safer and more effective HGF treatment.
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Abstract
Description
一方、可溶性Fasは細胞表面に存在するFasが切断されて血中に放出されるものであり、Fasリガンドと結合することで、細胞表面のFasとFasリガンドの結合に拮抗してアポトーシスを抑制する。
しかしながら、劇症肝炎では、予後予測に重要な血中HGF値は顕著に上昇するが、血中HGF値と血清AFP値及び可溶性Fas値との間には相関関係が認められていないことから、外因性HGFの投与による治療効果の指標(薬効バイオマーカー)としてAFPや可溶性Fasを利用できるとの予測はなかった。
[1]肝細胞増殖因子を含有してなる、急性肝不全抑制剤。
[2]劇症肝炎もしくは遅発性肝不全の治療又は非昏睡型急性肝不全から劇症肝炎もしくは遅発性肝不全への進展抑制のための、上記[1]記載の剤。
[3]1回の投与において投与速度を段階的もしくは連続的に増加させながら全身投与することを特徴とする、上記[1]又は[2]記載の剤。
[4]1回の投与時間の最初の1/3の期間に1回投与量の約10%、次の1/3の期間に1回投与量の約30%、並びに最後の1/3の期間に1回投与量の約60%を静脈内投与することを特徴とする、上記[3]記載の剤。
[5]肝細胞増殖因子がヒト由来である、上記[1]~[4]のいずれかに記載の剤。
[6]肝細胞増殖因子を投与された肝臓障害を有する患者から採取した試料中のα-フェトプロテイン及び/又は可溶性Fasの量を測定することを含む、肝細胞増殖因子の薬効評価方法。
[7]α-フェトプロテインが肝再生効果の指標であり、可溶性Fasが抗アポトーシス効果の指標である、上記[6]記載の方法。
[8]α-フェトプロテイン及び/又は可溶性Fasの測定値を、肝細胞増殖因子の投与前の該患者から採取した試料中の測定値と比較し、投与前よりもα-フェトプロテイン及び/又は可溶性Fasの量が上昇していた場合に、肝細胞増殖因子による治療効果が認められると判定することを特徴とする、上記[6]又は[7]記載の方法。
[9]肝細胞増殖因子の投与期間中に該患者から経時的に採取した試料中のα-フェトプロテイン及び/又は可溶性Fasの量をモニタリングし、当該期間中にα-フェトプロテイン及び/又は可溶性Fasの量が上昇していた場合に、肝細胞増殖因子による治療効果が認められると判定することを特徴とする、上記[6]~[8]のいずれかに記載の方法。
[10]前記試料が血清又は血漿である、上記[6]~[9]のいずれかに記載の方法。
[11]前記患者が急性肝不全患者である、上記[6]~[10]のいずれかに記載の方法。
[12]前記患者が劇症肝炎もしくは遅発性肝不全患者又は非昏睡型急性肝不全患者である、上記[11]記載の方法。
[13]α-フェトプロテインを指標とする場合に、患者がステロイド系抗炎症剤の投与を受けていないことを特徴とする、上記[6]~[12]のいずれかに記載の方法。
[14]上記[6]~[13]のいずれかに記載の方法によりヒト肝細胞増殖因子の薬効が認められなかった場合に、投与量を増大させる及び/又は投与期間を延長することを特徴とする、上記[1]~[5]のいずれかに記載の剤。
[15]抗α-フェトプロテイン抗体及び/又は抗可溶性Fas抗体を含んでなる、ヒト肝細胞増殖因子の薬効評価用キット。
[16]上記[15]記載のキットと組み合わせることを特徴とする、上記[1]~[5]のいずれかに記載の剤。
本明細書におけるアミノ酸配列の同一性は、相同性計算アルゴリズムNCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) を用い、以下の条件 (期待値=10; ギャップを許す; マトリクス=BLOSUM62; フィルタリング=OFF) にて計算することができる。
実質的に同質の活性としては、例えば、肝保護 (アポトーシス抑制) 作用、肝再生促進作用などが挙げられる。「実質的に同質」とは、それらの活性が定性的に(例: 生理学的に、または薬理学的に) 同一であることを示す。したがって、アポトーシス抑制作用、肝再生促進作用などの活性は同等 (例えば、約0.5~約2倍) であることが好ましいが、これらの活性の程度やタンパク質の分子量などの量的要素は異なっていてもよい。
HGFは遊離体であってもよいし、塩の形態であってもよい。HGFの塩としては、酸 (例: 無機酸、有機酸) や塩基 (例: アルカリ金属) との生理学的に許容される塩が挙げられ、とりわけ生理学的に許容される酸付加塩が好ましい。この様な塩としては、例えば、無機酸 (例えば、塩酸、リン酸、臭化水素酸、硫酸) との塩、あるいは有機酸 (例えば、酢酸、ギ酸、プロピオン酸、フマル酸、マレイン酸、コハク酸、酒石酸、クエン酸、リンゴ酸、蓚酸、安息香酸、メタンスルホン酸、ベンゼンスルホン酸) との塩などが用いられる。
(1) M. Bodanszky and M.A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York(1965)
このようにして得られたタンパク質は、公知の精製法により精製単離することができる。ここで精製法としては、例えば、溶媒抽出、蒸留、カラムクロマトグラフィー、液体クロマトグラフィー、再結晶、これらの組み合わせなどが挙げられる。当該方法で得られるタンパク質が遊離体である場合には、該遊離体を公知の方法あるいはそれに準じる方法によって適当な塩に変換することができるし、逆にタンパク質が塩として得られた場合には、該塩を公知の方法あるいはそれに準じる方法によって遊離体または他の塩に変換することができる。
配列番号1で示されるヌクレオチド配列の相補鎖配列とストリンジェントな条件下でハイブリダイズし得る核酸としては、例えば、配列番号1で示されるヌクレオチド配列と約85%以上、好ましくは約90%以上、さらに好ましくは約95%以上、特に好ましくは約97%以上の同一性を有する塩基配列を含有する核酸などが用いられる。
本明細書における塩基配列の同一性は、相同性計算アルゴリズムNCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) を用い、以下の条件 (期待値=10; ギャップを許す; フィルタリング=ON; マッチスコア=1; ミスマッチスコア=-3) にて計算することができる。
ハイブリダイゼーションは、自体公知の方法あるいはそれに準じる方法、例えば、Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、ハイブリダイゼーションは、添付の使用説明書に記載の方法に従って行なうことができる。ハイブリダイゼーションは、好ましくは、ハイストリンジェントな条件に従って行なうことができる。ハイストリンジェントな条件としては、(1) 洗浄に低イオン強度及び高温、例えば、50℃で0.015 M 塩化ナトリウム/0.0015 M クエン酸ナトリウム/0.1% 硫酸ドデシルナトリウムを使用し、(2) ホルムアミドのような変性剤、例えば、0.1% ウシ血清アルブミン/0.1% フィコール/0.1% ポリビニルピロリドン/750 mM 塩化ナトリウム、75 mM クエン酸ナトリウムを含む50 mM リン酸ナトリウム緩衝液 (pH 6.5) とともに、50% (v/v) ホルムアミドを42℃で使用することを特徴とする反応条件が例示される。あるいは、ストリンジェントな条件は、50% ホルムアミド、5xSSC (0.75 M NaCl、0.075 M クエン酸ナトリウム)、50 mM リン酸ナトリウム (pH 6.8)、0.1% ピロ燐酸ナトリウム、5xデンハート溶液、超音波処理鮭精子DNA (50 μg/ml)、0.1% SDS、及び10% 硫酸デキストランを42℃で使用し、0.2xSSC及び50% ホルムアルデヒドで55℃で洗浄し、続いて55℃でEDTAを含有する0.1xSSCからなる高ストリンジェント洗浄を行うものであってもよい。当業者は、プローブ長等のファクターに応じて、ハイブリダイゼーション反応および/または洗浄時の温度、緩衝液のイオン強度等を適宜調節することにより、容易に所望のストリンジェンシーを実現することができる。
クローン化されたDNAは、目的によりそのまま、または所望により制限酵素で消化するか、リンカーを付加した後に、使用することができる。該DNAは、必要に応じてその5’末端側に翻訳開始コドンとしてのATGを有し、また3’末端側には翻訳終止コドンとしてのTAA、TGAまたはTAGを有していてもよい。これらの翻訳開始コドンや翻訳終止コドンは、適当な合成DNAアダプターを用いて付加することができる。
発現ベクターとしては、大腸菌由来のプラスミド(例: pBR322、pBR325、pUC12、pUC13)、枯草菌由来のプラスミド (例: pUB110、pTP5、pC194)、酵母由来プラスミド (例: pSH19、pSH15)、昆虫細胞発現プラスミド (例: pFast-Bac)、動物細胞発現プラスミド (例: pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo)、λファージなどのバクテリオファージ、バキュロウイルスなどの昆虫ウイルスベクター (例: BmNPV、AcNPV)、レトロウイルス, ワクシニアウイルス, アデノウイルス, アデノ随伴ウイルスなどの動物ウイルスベクターなどが用いられる。
例えば、宿主が動物細胞である場合、サイトメガロウイルス (CMV) 由来プロモーター (例: CMV前初期プロモーター)、ヒト免疫不全ウイルス (HIV) 由来プロモーター (例: HIV LTR)、ラウス肉腫ウイルス (RSV) 由来プロモーター (例: RSV LTR)、マウス乳癌ウイルス (MMTV) 由来プロモーター (例: MMTV LTR)、モロニーマウス白血病ウイルス (MoMLV) 由来プロモーター (例: MoMLV LTR)、単純ヘルペスウイルス (HSV) 由来プロモーター (例: HSVチミジンキナーゼ(TK) プロモーター)、SV40由来プロモーター (例: SV40初期プロモーター)、エプスタインバーウイルス (EBV) 由来プロモーター、アデノ随伴ウイルス (AAV) 由来プロモーター (例: AAV p5プロモーター)、アデノウイルス (AdV) 由来プロモーター (Ad2またはAd5主要後期プロモーター) などが用いられる。
宿主が昆虫細胞である場合、ポリヘドリンプロモーター、P10プロモーターなどが好ましい。
宿主がエシェリヒア属菌である場合、trpプロモーター、lacプロモーター、recAプロモーター、λPLプロモーター、lppプロモーター、T7プロモーターなどが好ましい。
宿主がバチルス属菌である場合、SPO1プロモーター、SPO2プロモーター、penPプロモーターなどが好ましい。
宿主が酵母である場合、PHO5プロモーター、PGKプロモーター、GAPプロモーター、ADHプロモーターなどが好ましい。
動物細胞としては、例えば、サル由来細胞 (例: COS-1、COS-7、CV-1、Vero)、ハムスター由来細胞 (例: BHK、CHO、CHO-K1、CHO-dhfr-)、マウス由来細胞 (例: NIH3T3、L、L929、CTLL-2、AtT-20)、ラット由来細胞 (例: H4IIE、PC-12、3Y1、NBT-II)、ヒト由来細胞 (例: HEK293、A549、HeLa、HepG2、HL-60、Jurkat、U937) などが用いられる。
昆虫細胞としては、例えば、ウイルスがAcNPVの場合、夜盗蛾の幼虫由来株化細胞 (Spodoptera frugiperda cell; Sf細胞)、Trichoplusia niの中腸由来のMG1細胞、Trichoplusia niの卵由来のHigh FiveTM細胞、Mamestra brassicae由来の細胞、Estigmena acrea由来の細胞などが用いられる。ウイルスがBmNPVの場合、昆虫細胞としては、蚕由来株化細胞 (Bombyx mori N 細胞; BmN細胞) などが用いられる。該Sf細胞としては、例えば、Sf9細胞 (ATCC CRL1711)、Sf21細胞 (以上、Vaughn, J.L. et al., In Vivo, 13: 213-217 (1977))などが用いられる。
昆虫としては、例えば、カイコの幼虫などが用いられる。
エシェリヒア属菌としては、例えば、Escherichia coli K12、DH1、JM103、JA221、HB101、C600などが用いられる。
バチルス属菌としては、例えば、Bacillus subtilis MI114、207-21などが用いられる。
酵母としては、例えば、Saccharomyces cerevisiae AH22、AH22R-、NA87-11A、DKD-5D、20B-12、Schizosaccharomyces pombe NCYC1913、NCYC2036、Pichia pastoris KM71などが用いられる。
動物細胞は、例えば、細胞工学別冊8 新細胞工学実験プロトコール, 263-267 (1995) (秀潤社発行)、Virology, 52: 456 (1973) に記載の方法に従って形質転換することができる。
昆虫細胞および昆虫は、例えば、Bio/Technology, 6: 47-55 (1988) などに記載の方法に従って形質転換することができる。
ここで、薬理学的に許容される担体としては、製剤素材として慣用の各種有機あるいは無機担体物質が用いられ、液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤などとして配合される。また必要に応じて、防腐剤、抗酸化剤、着色剤などの製剤添加物を用いることもできる。
溶剤の好適な例としては、注射用水、生理的食塩水、リンゲル液、アルコール、プロピレングリコール、ポリエチレングリコール、ゴマ油、トウモロコシ油、オリーブ油、綿実油などが挙げられる。
溶解補助剤の好適な例としては、ポリエチレングリコール、プロピレングリコール、D-マンニトール、トレハロース、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウム、サリチル酸ナトリウム、酢酸ナトリウムなどが挙げられる。
懸濁化剤の好適な例としては、ステアリルトリエタノールアミン, ラウリル硫酸ナトリウム, ラウリルアミノプロピオン酸, レシチン, 塩化ベンザルコニウム, 塩化ベンゼトニウム, モノステアリン酸グリセリンなどの界面活性剤、例えばポリビニルアルコール, ポリビニルピロリドン, カルボキシメチルセルロースナトリウム, メチルセルロース, ヒドロキシメチルセルロース, ヒドロキシエチルセルロース, ヒドロキシプロピルセルロースなどの親水性高分子、ポリソルベート類、ポリオキシエチレン硬化ヒマシ油などが挙げられる。
等張化剤の好適な例としては、塩化ナトリウム、グリセリン、D-マンニトール、D-ソルビトール、ブドウ糖などが挙げられる。
緩衝剤の好適な例としては、リン酸塩、酢酸塩、炭酸塩、クエン酸塩などの緩衝液などが挙げられる。
無痛化剤の好適な例としては、ベンジルアルコールなどが挙げられる。
防腐剤の好適な例としては、パラオキシ安息香酸エステル類、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸などが挙げられる。
抗酸化剤の好適な例としては、亜硫酸塩、アスコルビン酸塩などが挙げられる。
着色剤の好適な例としては、水溶性食用タール色素(例: 食用赤色2号および3号、食用黄色4号および5号、食用青色1号および2号などの食用色素)、水不溶性レーキ色素 (例: 前記水溶性食用タール色素のアルミニウム塩など)、天然色素 (例: β-カロチン、クロロフィル、ベンガラなど) などが挙げられる。
医薬組成物は、製剤技術分野において慣用の方法、例えば日本薬局方に記載の方法等により製造することができる。医薬組成物中の有効成分の含量は、剤形、有効成分の投与量などにより異なるが、例えば約0.1ないし100重量%である。
後述の実施例に示されるとおり、わが国における劇症肝炎及び遅発性肝不全患者に関する全国的調査に基づく救命率は約18%であったのに対し、本発明のHGF製剤による治療は4名中2名の患者を救命することができ、死亡例のうち1例についても肝性脳症発症から68日という長期生存が得られたことから、極めて予後不良で、従来肝移植しか有効な治療法がなかった劇症肝炎及び/又は遅発性肝不全に対しても、治療効果を示すことが強く示唆された。当該実施例で用いられたHGF製剤の投与プロトコルは、正常ラットや部分肝切除ラット等の重篤な肝障害を有しない動物を用いた実験において、肝再生作用が確認された用量と、劇症肝炎及び遅発性肝不全の治療実績から必要十分であると予測された投与期間を採用したものであり、非臨床安全性試験の結果から、不可逆性の副作用を生じることなく、最低でも用量を4倍に増量できると予測されることから、投与量および投与期間を最適化することにより、劇症肝炎及び/又は遅発性肝不全患者における救命率をさらに向上させ得ると考えられる。さらに、本発明のHGF製剤を用いた治療は、劇症肝炎や遅発性肝不全に進行する前の非昏睡型急性肝不全患者に対して実施することにより、劇症肝炎及び/又は遅発性肝不全への進行(劇症化)を阻止することができ、当該進展抑制効果によって、結果的に急性肝不全患者の死亡率を著しく低減することができる。
急性肝不全における血清AFP値の上昇は、肝障害に引き続いて誘導される肝再生によると考えられていた(Taketa, K., Hepatology, 12: 1420-1432 (1990))。一方、血清HGF値は急性肝炎の劇症化予知および劇症肝炎の予後予測に重要であり、劇症肝炎では血清HGF値1.0 ng/ml以上(基準値0.40 ng/ml以下)に上昇するが、劇症肝炎および遅発性肝不全の全国調査データ(1998年~2009年)の439例において、血中HGF値と血清AFP値の両者に相関関係は見出せなかった。すなわち、劇症肝炎および遅発性肝不全患者において、肝障害で誘導された内因性HGFによる血中HGF濃度の上昇によって血清AFP値が上昇することはなく、そのため、外因性にHGF(rh-HGF)を投与して血中HGF濃度を上げたとしても、血清AFPレベルが上昇するとは予測できなかった。かかる状況の下、本発明者らは、劇症肝炎及び遅発性肝不全患者にHGFを反復投与したところ、意外にもその投与期間中に血清AFP値が上昇し、投与終了後に漸減することを見出し、AFPが、劇症肝炎をはじめとした急性肝不全、重症の急性肝炎、非代償性肝硬変などの慢性肝不全患者に対してHGFによる治療を行った際、その肝再生促進効果の指標(バイオマーカー)となることを明らかにした。投与期間中、肝重量(肝容量)や血清アルブミン値に大きな変化は認められず、AFPが従来公知の肝再生促進マーカーよりも肝再生効果を鋭敏かつリアルタイムで反映することが示された。
血清や血漿を用いる場合、常法に従って患者から採血し、液性成分を分離することにより調製することができる。
競合法では、被検試料中の抗原と標識抗原とを抗体に対して競合的に反応させた後、未反応の標識抗原(F)と、抗体と結合した標識抗原(B)とを分離し(B/F分離)、B、Fいずれかの標識量を測定し、被検試料中の抗原量を定量する。本反応法には、抗体として可溶性抗体を用い、B/F分離をポリエチレングリコール、前記抗体に対する第2抗体などを用いる液相法、および、第1抗体として固相化抗体を用いるか、あるいは、第1抗体は可溶性のものを用い第2抗体として固相化抗体を用いる固相化法とが用いられる。
イムノメトリック法では、被検試料の抗原と固相化抗原とを一定量の標識化抗体に対して競合反応させた後固相と液相を分離するか、あるいは、被検試料中の抗原と過剰量の標識化抗体とを反応させ、次に固相化抗原を加え未反応の標識化抗体を固相に結合させた後、固相と液相を分離する。次に、いずれかの相の標識量を測定し被検試料中の抗原量を定量する。
また、ネフロメトリーでは、ゲル内あるいは溶液中で抗原抗体反応の結果生じた不溶性の沈降物の量を測定する。被検試料中の抗原量が僅かであり、少量の沈降物しか得られない場合にもレーザーの散乱を利用するレーザーネフロメトリーなどが好適に用いられる。
例えば、入江 寛編「ラジオイムノアッセイ」(講談社、昭和49年発行)、入江 寛編「続ラジオイムノアッセイ」(講談社、昭和54年発行)、石川栄治ら編「酵素免疫測定法」(医学書院、昭和53年発行)、石川栄治ら編「酵素免疫測定法」(第2版)(医学書院、昭和57年発行)、石川栄治ら編「酵素免疫測定法」(第3版)(医学書院、昭和62年発行)、「Methods in ENZYMOLOGY」 Vol. 70 (Immunochemical Techniques (Part A))、同書 Vol. 73 (Immunochemical Techniques (Part B))、同書 Vol. 74 (Immunochemical Techniques (Part C))、同書 Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays))、同書 Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods))、同書 Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (以上、アカデミックプレス社発行)などを参照することができる。
rh-HGF投与の安全性確保のための動物実験
動物
クラウンミニブタ(雌、6~7月齢)及びウィスターラット(雄、7週齢)を、それぞれジャパンファーム(鹿児島、日本)及び日本チャールス・リバー株式会社(横浜、日本)から購入した。動物は、研究期間を通して一定の室温(25℃)、自由飲水及び表示された食餌で維持した。動物試験のプロトコルは、京都大学大学院医学研究科(京都、日本)の倫理委員会により承認された。全ての動物実験は、1~3週間の標準食餌での順化の後行なった。
クラウンミニブタ(雌)をセボフルラン、二酸化窒素及び酸素の吸引により麻酔した後、カテーテルを内頸静脈(rh-HGFの注入のため)及び総頸動脈(BPを測定するため)に挿入した。1mg/kgのrh-HGFを20分間にわたり内頸静脈から注入した。HRを心電図モニタリングにより記録し、心機能を心エコーにより測定した。rh-HGFの段階的注入のBPへの影響を評価するために、投与速度を段階的に増加させながら(最初の60分間で総用量の10%、次の60分間で30%、そして最後の60分間で60%)、内頸静脈に挿入したカテーテルから、0.4mg/kgのrh-HGFを3時間にわたり注入した。
rh-HGF(0.4、1.0及び4.0mg/kg)を14日間ラットに静脈内ボーラス投与した後、2週間観察した。アルブミン及びタンパク質の尿中排泄を、rh-HGFの投与中及び投与後に経時的に測定した。動物を、rh-HGF投与の終了時(14日目)及び観察期間の終了時(28日目)に屠殺し、血清クレアチニン及び組織学的所見を含めて、腎障害を評価した。
概要
この単群/非盲検/用量漸増試験は、京都大学病院(京都、日本)において実施した。試験プロトコルは、患者登録の開始前に、京都大学病院の治験審査委員会及び倫理委員会の審査及び承認を受けた。試験は、GCPの原則に従い、ヘルシンキ宣言の倫理指針を遵守して行なった。参加した全ての患者、又は法定代理人(参加者が肝性脳症のため署名できない場合)から、本試験への登録前に、書面によるインフォームド・コンセントを得た。
同意した患者を2005年9月から2008年6月にあらかじめ選抜した。肝移植を受けられなかったFHSA又はLOHF患者であって、以下の4パラメーターの少なくとも1つを満たす患者を適格とした:(1)45歳以上、(2)PTが標準値の10%以下、(3)総ビリルビン(T-Bil)レベルが18.0mg/dL以上、又は(4)直接/総ビリルビン比が0.67未満。以下の患者は不適格とした:16歳未満;登録前48時間にグルカゴン及びインスリン、又はプロスタグランジンE1で治療された者;悪性腫瘍を有するか又はその病歴を有する者;心不全を有する者;肺炎、敗血症、播種性血管内凝固症候群又は消化管出血を含む重篤な合併症を有する者; 及びrh-HGFに対するアレルギー反応を有する者。女性における生殖発生へのrh-HGFの毒性は調べられていないため、妊娠可能な年齢の女性も不適格とした。また、腎障害(≧1mg/mLタンパク質の尿中排泄、沈渣尿中の赤血球変形又は赤血球円柱(RBC casts)、2.0mg/dL以上の血清クレアチニンレベル、或いは400mL/日未満の尿容量を含む)を有する患者も除外した。
rh-HGFをGMPグレードの材料として調製した。rh-HGFの初回用量は、0.6mg/m2/日に固定した(安全性及び臨床効果を確保する用量として、複数の前臨床動物試験により決定した)。用量漸増試験においては、rh-HGFの用量を、初回用量(0.6mg/m2)から1.2、1.8又は2.4mg/m2に増加させることができる。rh-HGFを、14日間まで、3時間段階的に増加させて静脈内投与した後、14日間観察した。全ての患者を追跡し、試験期間(28日まで)後の治療成績を決定した。
第一の評価項目は、rh-HGFの静脈内反復投与の安全性であり、有害事象の発生、頻度、及び深刻度に基づき評価した。全ての患者を集中治療室で治療した。試験期間中、rh-HGF投与の開始から治験薬投与の完了後まで、安全性について定期的に患者を観察した。安全性評価は、身体検査、臨床検査及び有害事象の観察により行なった。有害事象は、試験期間を通して観察し、共通毒性基準グレーディングシステムに従って等級付けした。有害事象とrh-HGFとの因果関係は、医師の最善の判断により決定した。全ての有害事象は、その原因に関わらず適切に治療した;必要な場合、患者を試験から外した。有害事象の発生率は、少なくとも1回のrh-HGFの投与を受けた患者のうち、少なくとも1の有害事象を経験した患者の数から計算した。
第二の評価項目は、静脈内注射されたrh-HGFの薬物動態、並びに生存期間及び治療成績を含む臨床効果であった。rh-HGFの薬物動態を調べるために、血液試料を1、3、5、8、及び11日目の複数の時点で採取した。HGFの血清中濃度は、酵素結合免疫吸着測定法(ELISA)(Otsuka Co.,Ltd.、徳島、日本)(Tsubouchi et al., Hepatology 1991, 13:1-5)により決定した。検査データ(PT国際標準化比(PT-INR)、T-Bil、血清アルブミン、アラニンアミノトランスフェラーゼ(ALT)、α-フェトプロテイン(AFP)を含む)を、血漿交換又はrh-HGF投与の前に調べた。また、血清可溶性Fas値は、rh-HGF投与前と投与終了翌日に採血して測定した。
ミニブタにおける血圧の低下に対処するためのrh-HGF投与方法の確立
一般薬理試験において、rh-HGF(1.0又は0.2mg/kg)の静脈内投与は、ミニブタにおいて収縮期血圧(systolic BP)の急速な低下を引き起こすが、呼吸状態は影響を受けなかった。従って、臨床試験を開始する前に、ミニブタにおいて全身麻酔下で、rh-HGFの循環状態への影響を更に調べた。総用量1.0mg/kgのrh-HGFを20分間にわたり投与した場合、収縮期血圧の低下が直ちに起こり、rh-HGF投与中継続した(図1A)。心拍数(HR)は徐々に低下したが、不整脈や虚血性変化などの心電図異常は実験期間を通して観察されなかった。また、血圧の低下と並行して、心エコー検査により左心室拡張末期容積(LVEDV)及び駆出率(EF)の低下が示されたが、左心室の動きの異常は見られなかった(図1A)。これらの結果は、rh-HGFの静脈内注射が容量血管の拡張により血圧を低下させたことを示す。
次に、急速な血圧低下を回避し得るrh-HGFの投与方法の開発を行なった。最終的に、rh-HGFを3時間にわたり段階的に増加させる(最初の60分間で総用量の10%、次の60分間で30%、そして最後の60分間で60%)という、段階的注入法を確立した(図1B)。本発明者らは、適切な注入がrh-HGFの静脈内投与により引き起こされる血圧の低下を効果的に防ぐことを見出した(図1C)。この予防効果も、容量血管の拡張がHGF誘導性の血圧低下の原因であること支持している。
ラットにおけるrh-HGFの反復投与により誘導される腎毒性の評価
ラット又はカニクイザルを使用した反復投与毒性試験により、臨床試験において起こり得る有害事象としてアルブミン及びタンパク質の尿中排泄の増加が特定された。そこで、14日間のrh-HGFの反復投与により誘導される腎毒性が可逆的であるか否かを更に調べた。ラットに0.4、1.0、及び4.0mg/kg/日のrh-HGFを14日間投与した後、14日間観察した。アルブミンの尿中排泄は、rh-HGFで処理したラットにおいて4日目から容量依存的に増加した(図2)。0.4又は1.0mg/kg/日のrh-HGFで処理したラットでは、アルブミンの尿中排泄はタンパク尿の増加よりも先に起こった(図2A及びB)。しかし、血清クレアチニンにもBUNにも実験期間を通して影響はみられなかった。また、アルブミンの尿中排泄は、rh-HGF投与の終了後14日間の観察期間中、次第に減少した。組織学的分析においては、14日間のrh-HGFの反復投与後、メサンギウム増殖、糸球体及び尿細管における硝子滴沈着、並びに腎肥大が観察された。しかしながら、これらの組織学的所見はごく軽度から軽度の範囲であり、可逆的な変化であることも確認された。げっ歯類におけるrh-HGFの用量0.1mg/kgは、ヒトにおいては0.6mg/m2に相当することから、臨床試験における1日あたりの投与量を0.6mg/m2とすることとした。
亜急性劇症肝炎(FHSA)及び遅発性肝不全(LOHF)患者に対するrh-HGF投与第I/II相試験
(1)患者特性
2005年9月から2008年6月の間に、FHSA又はLOHFの患者20名をrh-HGFの臨床試験への参加について評価した。その結果、16名の患者は1以上の除外基準を満たしたため除外され、最終的に4名の患者のみが登録された。劇症肝炎は日本においては比較的稀な症候群であり(1998~2003年の間に698名の患者)、また治療の安全性及び有効性をより正確に評価するために、重篤な合併症を有する患者は除外したため、治験対象の採用は困難であった。そのため用量漸増試験は実施せず、すべての患者に対し、投与期間を通じて初回用量と同じ0.6mg/m2/日を投与した。参加した対象の年齢は、40~71歳であり、男女各2名であった(表1)。
rh-HGFによる治療は、肝性脳症を発症した後5~7日で開始した。患者2及び4においては、rh-HGF(0.6mg/m2/日)を14日間静脈内投与した。患者1及び3では、それぞれ血清クレアチニンの増加(2.1mg/dL)及び乏尿症のため、それぞれ14日目及び13日目にrh-HGF投与中止が必要となった。これらの症状はいずれも、肝不全に付随したものであり、rh-HGF投与によるものではないと判断された。従って、これらの患者におけるrh-HGF投与期間は、それぞれ合計13日間及び12日間となった。全ての患者において血漿交換を行なった。HEVによるFHSAの患者1を除く3名の患者は、コルチコステロイドで治療した(図7-1~7-4)。最終的に、2名のFHSA患者(患者2及び4)は生存したが、FHSAの患者1は試験期間の終了後に死亡し、LOHFの患者3は試験期間中に死亡した(表1及び図7-1~7-4)。
患者1、2及び3において、血漿交換後にrh-HGFを投与した。HGFの血清レベルは、rh-HGF投与量の段階的増加と並行して増加し、3時間のrh-HGF注入の終了時には最高薬物濃度(Cmax)に達した(図3)。Cmaxは、HGF投与期間中、1日目の18.8±6.0ng/mLから11日目の22.3±9.6ng/mLまで次第に増加した(表2)。
前臨床安全性試験により、rh-HGF注入中の血圧低下及びrh-HGF反復投与により誘導される腎毒性(アルブミンの尿中排泄の増加を含む)がヒト臨床試験において起こり得る有害事象であることが明らかとなった。FHSA又はLOHFの患者の第I/II相試験において、いずれの患者においてもrh-HGF投与により呼吸状態は影響を受けなかったが、患者1、2及び3において血圧は、HGF注入の開始後およそ1時間で軽度から中等度低下した(図4)。HGFは容量血管の拡張により血圧を低下させるため、心拍数は30%まで増加した。しかしながら、この血圧低下は、rh-HGF投与中止や昇圧剤治療を必要とせず、患者1では200~300mLの輸液のみで血圧は直ちに回復し、HGF投与終了後には安静時のレベルに戻った。前臨床動物実験(図1C)で観察されたように、あらかじめ輸液することで、HGFによる血圧低下は改善された。いずれにせよ、HGF注入中に観察された血圧の低下は可逆的であり、患者の全身状態に影響しなかった。患者2及び3もrh-HGF注入中に血圧低下を示したが(患者4は示さなかった)、全身状態は追加の輸液やrh-HGF投与の中止を行なわなくても安定していた。患者2(HGF投与期間の3日目に肝性脳症から覚醒した)は、HGF投与中、心拍数が最大約30%増加したものの(図4)、いかなる症状を患うこともなかった。
全ての患者が、登録時にアルブミンの尿中排泄のごく軽度から軽度の増加を示し、また、試験期間中尿量の減少を示した。しかしながら、rh-HGFの反復投与によってもアルブミンの尿中排泄は増加しなかった。また尿量は、輸液量、循環血漿量、利尿剤の投与など、rh-HGF投与以外のいくつかの要因により影響を受けた。4名の患者のうち3名において、低カリウム血症、貧血、血小板数減少、PT延長、アンチトロンビンIIIの減少、及び血尿が観察されたが、これらの有害事象とrh-HGF投与との間の因果関係を示す明らかな証拠はなかった。患者3(観察期間中に肝不全の進行により死亡した)は呼吸器不全を示した。しかしながら、この重篤な有害事象は肝不全の進行に付随するものであり、rh-HGFによるものではなかった。その他、rh-HGFの単回投与又は反復投与により直接的に引き起こされた有害事象は試験期間中観察されなかった。特に重要な点は、患者2(肝性脳症から覚醒した)が、rh-HGF投与中に症状や兆候を示さなかったことである。従って、連続14日までの段階的増加を伴うrh-HGFの静脈内投与は非常に良好な耐容性を示すと結論した。
4名の患者のうち3名は、登録時に肝性脳症を示した(表1)。患者1は、プロトコル治療の開始時にグレードIIの肝性脳症を呈していた。この患者は、試験期間中も試験期間後も肝性脳症から回復しなかった。最終的に、肝性脳症の発症から68日後に死亡した(図7-1)。患者2(FHSAであり、最終的に生存した)では、HGF投与期間中、2、4及び8日目に血漿交換を行い、肝性脳症は3日目までに改善した(図7-2)。患者3は、登録時に肝性脳症の進行を示した。rh-HGF投与期間中、意識レベルは一時的に改善したが、肝性脳症は観察期間中進行し続け、肝性脳症発症の28日後に死亡した(図7-3)。患者4は、登録時に肝性脳症から既に回復しており、試験期間中、意識レベルの低下は示さなかった(図7-4)。
プロトロンビン時間国際標準化比(PT-INR)、総ビリルビン(T-Bil)及び血清アルブミンは、rh-HGF投与期間中及び観察期間中、影響を受けなかった(図5)。
次に、rh-HGF投与の患者の生存への効果を評価した。日本におけるFH又はLOHF患者の全国的調査(1998~2002年)では、本試験の対象患者基準を満たす患者(n=192)の生存率はわずか17.7%(n=34)であり、FHSA及びLOHFから回復しなかった患者(n=190)のうち71%(n=135)が肝性脳症の発症後28日以内に死亡している。これに対し、本臨床試験では、試験期間中4例中3例(75%)が生存し、最終的に2例(50%)を救命できた。本結果は、HGFが肝移植適応外のFH又はLOHF患者における有効な治療手段をなり得ることを強く示唆するものである。
本試験において使用したrh-HGFの用量及び/又は投与期間は、より有益な効果を生むには少なかった可能性がある。本試験のために選択した用量は、前臨床動物試験において使用した用量のスケーリングに基づいており、いくつかの反復投与毒性試験において安全性が保証されたものであるが、動物実験の結果は少なくとも4倍の用量増加が可能であることを示している。また、この用量(げっ歯類においては0.1mg/kgに相当する)は、正常ラット及び部分肝切除ラットにおいて肝再生を促進することが報告されているが[Ishii, T. et al., J. Biochem., 1995;117:1105-1112]、重篤な肝臓障害を伴う場合における有効性を保証するものではない。一方、治療期間は、1998~2002年の日本におけるFH及びLOHFの全国的調査に基づく。この調査では、FHSA及びLOHFから回復した患者(n=52)のうち90.4%(n=47)は肝性脳症を発症してから14日以内に覚醒し、回復しなかった患者(n=190)のうち71%(n=135)は肝性脳症の発症後28日以内に死亡した。以上の知見に基づいて、安全性及び有効性を評価するためには、14日間のrh-HGF投与及びその後の14日間の経過観察で十分であると判断したが、FHやLOHFのような重篤な急性肝不全においては、本試験で採用したrh-HGFの用量は、肝再生を誘導し肝障害を抑制するのには不十分であるか、あるいは14日間という投与期間では短い場合もあることが示唆された。
患者1~4について、試験期間中の血清α-フェトプロテイン(AFP)及び可溶性Fas値をモニタリングした。各症例において、rh-HGF投与前(1日目)、投与3日目、投与5日目、投与8日目、投与終了翌日(1日目)、投与終了7日目及び投与終了14日目の早朝に採血を行い、血清AFP値を発光酵素免疫測定法(CLEIA法)で測定した。各血清サンプルについてアラニントランスアミナーゼ(ALT)レベルも測定した。また、rh-HGF投与前及び投与終了翌日に血清可溶性Fas値を測定した。さらに、rh-ヒトHGF投与前、投与終了直後および投与終了14日目に腹部コンピュータ断層撮影(CT)検査による肝容量測定を実施した。
投与前、投与8日目、投与終了翌日、投与終了7日目及び投与終了14日目の血清AFP値を図6にプロットした。患者1において、血清AFP値は、rh-HGF投与前は7.0 ng/mLと基準値(10.0 ng/mL以下)内であったが、rh-HGF投与3日目に15.0 ng/mLと上昇し、投与11日目に38.9 ng/mLに達した。投与終了翌日には32 ng/mLと軽度減少し、その後投与終了7日目には23.5 ng/mLとさらに減少、投与終了14日目には8.3 ng/mLと基準値内となった。このように血清AFP値がrh-HGF投与中に速やかに上昇し、投与終了後に低下したことから、HGFの薬効、すなわちHGFによる肝再生促進作用をみるためのバイオマーカーとなると考えられた。一方、患者3では、血清AFP値は、rh-HGF投与前から試験期間を通じて基準値内であり、rh-HGF投与によっても肝再生誘導が起こらなかったと考えられる。患者1及び3はいずれも死亡例ではあるが、臨床経過は大きく異なり、患者1ではrh-HGF投与期間(13日間)とその後の観察期間(14日間)は病状の進行もなく全身状態も安定しており、発症から死亡までの期間は79日(脳症発現から死亡までの期間は68日間)と生存期間の延長を認めたのに対し、患者3は極めて重篤な症例で、11日間のrh-HGF投与後、観察期間の13日目に死亡した。即ち、患者1ではrh-HGF投与により肝再生誘導が起こり、延命効果がみられたが、患者3では肝再生を誘導するに至らなかったと考えられる。患者2及び4では、血清AFPはいずれもrh-HGF投与前に軽度上昇しており、既にわずかな肝再生が誘導されていたことが推測されるが、これらの患者はrh-HGFと並行してプレドニゾロン(PSL)の投与を受けており(図7-2及び7-4)、PSLがAFP発現に影響したために、いずれの症例においても肝再生誘導が起こっているが血清AFPは漸減しているものと考えられる。実際、患者2では、rh-HGF投与から約3ヶ月後に腹腔鏡および肝生検を実施したところ、肝再生像が観察されている。
一方、従来より肝再生誘導の指標となり得ると言われている肝容量(患者1)は、rh-HGF投与前1055mLで、投与終了翌日1076mL、投与終了14日目984mLと、rh-HGF投与中には大きな変化はみられず、投与終了後に軽度減少した(表3)。血清アルブミン値は患者1~4のいずれにおいてもrh-HGF投与中には大きな変化はみられなかった(図5)。従って、AFPは肝容量や血清アルブミンよりも優れた肝再生効果の指標(バイオマーカー)となり得ることが示された。
また、患者2では、ステロイド投与によって血清ALT値の改善が得られているのに対し、患者4では、血清ALTはより高値で(肝細胞障害がより強く)、ステロイド投与によっても血清ALT値はほとんど改善していない。両症例ともrh-HGF投与前の可溶性Fasは同レベルで、それは肝細胞障害に対して代償的に(肝細胞を保護するための生体反応として)上昇しているものと考えれば、ステロイド投与によって血清ALT値が低下(肝細胞障害が抑制)した患者2では、rh-HGFによる抗アポトーシス作用(可溶性Fas上昇)を必要としなかったのに対し、患者4では肝細胞障害(血清ALT上昇)が持続したために、代償的な可溶性Fas上昇に加えて、rh-HGFによる可溶性Fasが誘導されアポトーシスを抑制し、このことが最終的に生存につながった可能性を示唆している。
このようにAFPと可溶性Fasとは、それぞれHGFの肝再生促進作用と抗アポトーシス作用のバイオマーカーとして利用可能であり、肝障害を有する患者における両マーカーのレベルのHGF投与前後の変化を測定し、これらを組み合わせて(必要に応じてALT値等の他のバイオマーカーとも組み合わせて)患者の臨床経過と照らし合わせることにより、該患者において、HGFの投与によって肝再生誘導及びアポトーシス抑制の両方もしくはいずれか一方の効果が誘導されているか否かを判定し得ることが示された。
Claims (16)
- 肝細胞増殖因子を含有してなる、急性肝不全抑制剤。
- 劇症肝炎もしくは遅発性肝不全の治療又は非昏睡型急性肝不全から劇症肝炎もしくは遅発性肝不全への進展抑制のための、請求項1記載の剤。
- 1回の投与において投与速度を段階的もしくは連続的に増加させながら全身投与することを特徴とする、請求項1又は2記載の剤。
- 1回の投与時間の最初の1/3の期間に1回投与量の約10%、次の1/3の期間に1回投与量の約30%、並びに最後の1/3の期間に1回投与量の約60%を静脈内投与することを特徴とする、請求項3記載の剤。
- 肝細胞増殖因子がヒト由来である、請求項1~4のいずれか1項に記載の剤。
- 肝細胞増殖因子を投与された肝臓障害を有する患者から採取した試料中のα-フェトプロテイン及び/又は可溶性Fasの量を測定することを含む、肝細胞増殖因子の薬効評価方法。
- α-フェトプロテインが肝再生効果の指標であり、可溶性Fasが抗アポトーシス効果の指標である、請求項6記載の方法。
- α-フェトプロテイン及び/又は可溶性Fasの測定値を、肝細胞増殖因子の投与前の該患者から採取した試料中の測定値と比較し、投与前よりもα-フェトプロテイン及び/又は可溶性Fasの量が上昇していた場合に、肝細胞増殖因子による治療効果が認められると判定することを特徴とする、請求項6又は7記載の方法。
- 肝細胞増殖因子の投与期間中に該患者から経時的に採取した試料中のα-フェトプロテイン及び/又は可溶性Fasの量をモニタリングし、当該期間中にα-フェトプロテイン及び/又は可溶性Fasの量が上昇していた場合に、肝細胞増殖因子による治療効果が認められると判定することを特徴とする、請求項6~8のいずれか1項に記載の方法。
- 前記試料が血清又は血漿である、請求項6~9のいずれか1項に記載の方法。
- 前記患者が急性肝不全患者である、請求項6~10のいずれか1項に記載の方法。
- 前記患者が劇症肝炎もしくは遅発性肝不全患者又は非昏睡型急性肝不全患者である、請求項11記載の方法。
- α-フェトプロテインを指標とする場合に、患者がステロイド系抗炎症剤の投与を受けていないことを特徴とする、請求項6~12のいずれか1項に記載の方法。
- 請求項6~13のいずれか1項に記載の方法によりヒト肝細胞増殖因子の薬効が認められなかった場合に、投与量を増大させる及び/又は投与期間を延長することを特徴とする、請求項1~5のいずれか1項に記載の剤。
- 抗α-フェトプロテイン抗体及び/又は抗可溶性Fas抗体を含んでなる、ヒト肝細胞増殖因子の薬効評価用キット。
- 請求項15記載のキットと組み合わせることを特徴とする、請求項1~5のいずれか1項に記載の剤。
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JPWO2017159771A1 (ja) * | 2016-03-18 | 2019-01-31 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | 肝細胞増殖因子(hgf)のpdマーカー |
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US11547743B2 (en) | 2014-04-28 | 2023-01-10 | Eisai R&D Management Co., Ltd. | Lyophilized formulation of HGF |
WO2016104717A1 (ja) * | 2014-12-26 | 2016-06-30 | 国立大学法人京都大学 | 肝細胞誘導方法 |
US10711249B2 (en) | 2014-12-26 | 2020-07-14 | Kyoto University | Method for inducing hepatocytes |
US11548926B2 (en) | 2016-03-17 | 2023-01-10 | Eisai R&D Management Co., Ltd. | Method for producing an active hepatocyte growth factor (HGF) |
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