Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
• • 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
  • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
  • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)

Definitions

• the present invention relates to a biopharmaceutical for diabetes that has a guaranteed therapeutic effect. More specifically, the present invention relates to a diabetes treatment agent containing an extracellular domain fragment of heparin-binding epidermal growth factor-like growth factor (HB-EGF) or a nucleic acid encoding the same, and further relates to a diabetes treatment agent that combines hepatocyte growth factor (HGF) or a fragment thereof or a nucleic acid encoding the same. The present invention also relates to a diabetes treatment agent that contains a substance that activates signal transduction mediated by the HB-EGF receptor.
• HB-EGF heparin-binding epidermal growth factor-like growth factor
• HGF hepatocyte growth factor
• the present invention also relates to a diabetes treatment agent that contains a substance that activates signal transduction mediated by the HB-EGF receptor.
• Type 2 diabetes which accounts for 90% of all diabetes cases, occurs when insulin sensitivity is reduced due to genetic predisposition and environmental factors (lifestyle).
• the beta cells in the pancreas attempt to compensate for the lack of insulin action by compensatory over-secreting insulin, but if this condition continues for a long time, the beta cells become exhausted, and they are no longer able to secrete sufficient amounts of insulin, resulting in a state of hyperglycemia.
• Ad vectors adenovirus vectors were used as specific vectors for gene transfer.
• Ad vectors accumulate largely in the liver, it was hypothesized that their mechanism of action involves HGF and HB-EGF fragments secreted and expressed from the distant liver being delivered to the pancreas via the bloodstream, where they exert protective and regenerative effects on pancreatic ⁇ cells through endocrine action. Therefore, the inventors used adeno-associated virus (AAV) serotype 8, which has pancreatic organ tropism, as a vector to deliver and express therapeutic genes directly to the pancreas.
• AAV adeno-associated virus
• AAV vectors because the gene size that AAV vectors can accommodate is small at 4.7 kbp, they used nucleic acid encoding the extracellular domain (ectodomain; ECD) of the HB-EGF gene and nucleic acid encoding the NK1 fragment consisting of the N-terminal domain and kringle 1 (K1) domain of the HGF gene.
• ECD extracellular domain
• K1 kringle 1
• HB-EGF ECD When a gene encoding HB-EGF ECD was used, the same dose as when a gene encoding full-length HB-EGF was used showed a dramatic blood glucose lowering effect in T1D model mice 7 days after STZ administration, sufficient to induce hypoglycemia. Furthermore, since one of the mechanisms of action appears to involve a mechanism other than the improvement of insulin secretion capacity through the protection and regeneration of ⁇ cells, the therapeutic effect of the same or lower doses of HB-EGF ECD was examined using T1D model mice 14 days after STZ administration, in which ⁇ cell destruction had progressed further.
• HB-EGF ECD dose-dependently suppressed blood glucose elevation, and at the maximum dose (1 x 10 11 vg), blood glucose levels were equivalent to those of normal mice by day 7 of administration and maintained normal blood glucose levels thereafter. Furthermore, no individuals showed hypoglycemia during the observation period, confirming safety. In IPGTT, HB-EGF ECD dose-dependently suppressed the rise in blood glucose after glucose administration, but at all doses, a decrease in plasma insulin levels was observed after glucose administration, suggesting the contribution of an insulin-independent improvement effect on glucose tolerance.
• RNA-seq and metabolome analysis confirmed that the introduction of HB-EGF ECD activated glucose metabolism in liver tissue, further supporting this hypothesis.
• pancreas confirmed a dose-dependent protective and regenerative effect on pancreatic islets, and TUNEL staining also confirmed its inhibitory effect on beta cell apoptosis.
• Anatomical findings also confirmed the regeneration of pancreatic tissue and increased blood flow due to HB-EGF ECD. These results suggest that the protection and regeneration of beta cells is a major contributor to the excellent blood glucose suppression effect of HB-EGF ECD.
• [Section 1] A therapeutic agent for diabetes in mammals, comprising a heparin-binding epidermal growth factor-like growth factor ectodomain (HB-EGF ECD) fragment or a nucleic acid encoding the same.
• HB-EGF ECD epidermal growth factor-like growth factor ectodomain
• [Section 2] Item 2.
• [Section 3] Item 3.
• [Section 4] Item 4.
• AAV adeno-associated viral
• [Section 10] Item 3. The agent according to Item 1 or 2, comprising a combination of hepatocyte growth factor (HGF) or a fragment thereof, or a nucleic acid encoding the same.
• HGF hepatocyte growth factor
• Item 11 The agent according to Item 10, wherein the nucleic acid encoding the HB-EGF ECD fragment and the nucleic acid encoding the HGF NK1 fragment are carried on a single vector.
• a therapeutic agent for treating diabetes in mammals comprising a substance that enhances signal transduction mediated by the HB-EGF receptor.
• Item 13 Item 13.
• a protease having proHB-EGF ectodomain shedding activity or a nucleic acid encoding the same a factor that induces proHB-EGF ectodomain shedding
• an agonist or transactivator of the HB-EGF receptor [Item 14] Item 14. The agent according to any one of Items 1 to 13, where
• a method for treating diabetes in a mammal comprising exposing said mammal to a means that enhances signaling through the HB-EGF receptor.
• [Section 1A] A method for treating diabetes in a mammal having diabetes, comprising administering to said mammal an effective amount of an HB-EGF ECD fragment or a nucleic acid encoding the same.
• the present invention is safe, minimally invasive, and can achieve the desired effect of suppressing blood glucose elevation over a long period of time, making it possible to provide treatment that combines therapeutic effects for diabetes, including T1D, with improvements in quality of life, thereby increasing the feasibility of applying this treatment to human clinical trials.
• FIG. 1 is a schematic diagram showing the structure of an AAV vector used in the examples.
• FIG. 1 shows the protocol for pharmacological efficacy testing. This figure shows the reduction in blood glucose in T1D model mice by HB-EGF ECD and the combination of HB-EGF ECD and NK1.
• Figure 1 shows the results of IPGTT after 14 days of treatment. Left: shows the change in blood glucose level after glucose load. Right: shows the change in plasma insulin level after glucose load.
• Figure 1 shows the results of IPGTT after 28 days of treatment. Left: shows the change in blood glucose level after glucose load. Right: shows the change in plasma insulin level after glucose load.
• FIG. 1 shows that intravenous administration of AAV results in highly efficient gene transfer into the liver, pancreas, and kidney.
• This figure shows the suppression of liver damage induction by HB-EGF ECD and the combination of HB-EGF ECD and NK1.
• This figure shows the results of biochemical tests of AST (left) and ALT (right) performed on normal mice 14 days after administration of AAV-CA-sHB-EGF.
• FIG. 1 shows the protocol for a dose-dependent study using T1D model mice 14 days after STZ administration. This figure shows the dose-dependent suppression of blood glucose elevation by HB-EGF ECD.
• FIG. 1 shows the time course of casual blood glucose levels in individual mice administered with various doses of AAV-CA-sHB-EGF.
• FIG. 1 shows dose-dependent weight loss in mice after AAV administration.
• Figure 1 shows the results of IPGTT after 7 days of treatment. Left: shows the change in blood glucose level after glucose load. Right: shows the change in plasma insulin level after glucose load.
• Figure 1 shows the results of IPGTT after 14 days of treatment. Left: shows the change in blood glucose level after glucose load.
• FIG. 1 shows the change in plasma insulin level after glucose load.
• 1 shows the results of HE staining of the pancreas 14 days after treatment, confirming that AAV-CA-sHB-EGF protects and proliferates pancreatic islets in a dose-dependent manner.
• 1 shows the results of HE staining of the pancreas 14 days after treatment, confirming that AAV-CA-sHB-EGF protects and proliferates pancreatic islets in a dose-dependent manner.
• This figure shows the results of measurements of casual blood glucose and plasma insulin levels 7 days after treatment. At all doses, administration of AAV-CA-sHB-EGF significantly suppressed the rise in casual blood glucose compared to the untreated group (left).
• FIG. 1 shows a protocol for a dose-dependency test using normal mice.
• FIG. 1 shows that AAV-CA-sHB-EGF administration does not affect blood glucose levels in normal mice.
• FIG. 1 shows that AAV-CA-sHB-EGF administration does not affect the body weight of normal mice.
• FIG. 1 shows that AAV-CA-sHB-EGF administration does not affect food intake in normal mice.
• This figure shows that administration of AAV-CA-sHB-EGF suppresses apoptosis of ⁇ cells in T1D model mice. Lower right: Thymocytes (positive control).
• FIG. 1 shows a protocol for a dose-dependency test using normal mice.
• FIG. 1 shows that AAV-CA-sHB-EGF administration does not affect blood glucose levels in normal mice.
• FIG. 1 shows that AAV-CA-sHB-EGF administration does not affect the body weight of normal mice.
• FIG. 1 shows that AAV-CA-sHB-EGF administration does not affect food intake in normal
• FIG. 1 shows that administration of AAV-CA-sHB-EGF-NK1 induces regeneration of pancreatic tissue and increased blood flow in T1D model mice.
• FIG. 1 shows the implementation details of RNA-seq and metabolome analysis. This figure shows that principal component analysis of RNA-seq revealed that the sHB-EGF-introduced group had a profile that was significantly different from the intact group and the AAV-CA-Venus-administered group. This figure shows that the expression of genes involved in cell division and glycolysis regulation is elevated in the AAV-CA-sHB-EGF administration group compared to the intact group and the AAV-CA-Venus administration group. This figure shows that in the AAV-CA-sHB-EGF administration group, glucose metabolism shifts through the glycolysis pathway and the pentose phosphate cycle, compared to the intact group and the AAV-CA-Venus administration group.
• Therapeutic Agent (I) of the Present Invention provides a therapeutic agent for diabetes in mammals, comprising an extracellular domain (ectodomain; ECD) fragment of HB-EGF or a nucleic acid encoding the same (hereinafter also referred to as "therapeutic agent (I) of the present invention.”
• ECD extracellular domain
• therapeutic agent (I) of the present invention it is used in a comprehensive sense to include therapeutic agent (I) of the present invention and therapeutic agent (II) of the present invention described below).
• treatment of diabetes means at least suppressing hyperglycemia (reducing fasting blood glucose and casual blood glucose) compared to controls with diabetes who are not receiving therapeutic treatment.
• the reduction in blood glucose levels is preferably statistically significant (e.g., p ⁇ 0.05), but also includes cases where a tendency for reduction is observed without a significant difference.
• a blood glucose lowering effect is achieved to an extent that is not significantly different from that of normal controls.
• the treatment of diabetes in the present invention includes improving glucose tolerance.
• the effect of improving glucose tolerance can be verified, for example, by suppressing blood glucose elevation using an IPGTT.
• the treatment of diabetes in the present invention includes the protection and regeneration of ⁇ cells.
• “protection and regeneration of ⁇ cells” means protecting (inducing anti-cell death) and/or proliferating (regenerating) the ⁇ cells of a subject with diabetes (including both the self-regeneration of existing ⁇ cells and differentiation from stem cells or endocrine precursor cells).
• the protection and regeneration of ⁇ cells can be evaluated, for example, by examining the presence of pancreatic islets that retain insulin secretion ability through immunohistochemical staining of the pancreas using an anti-insulin antibody.
• protection and regeneration of ⁇ cells improves insulin secretion.
• "protection and regeneration of ⁇ cells” refers to the protection and regeneration of ⁇ cells that secrete insulin in response to glucose stimulation and suppress blood glucose elevation due to glucose loading, i.e., that retain glucose-responsive insulin secretion ability. Based on the conventionally understood mechanism of action, even if ⁇ cell mass is maintained by protecting remaining ⁇ cells and/or differentiating or amplifying new ⁇ cells, if the glucose-responsive insulin secretion ability of ⁇ cells is low, postprandial hyperglycemia cannot be suppressed and sufficient blood glucose control may not be achieved.
• the therapeutic agent (I) of the present invention exhibits a blood glucose elevation suppression effect even without inducing an increase in plasma insulin concentration, it is considered to have a novel mechanism of action that does not involve insulin secretion. Therefore, induction of glucose-responsive insulin secretion ability is not essential in the present invention.
• AAV8 was used as a vector with the intention of introducing a gene into the pancreas, but since the contribution of the induction of insulin secretion through the protection and regeneration of ⁇ cells is relatively small, the difference in effect from the inventors' previous research using an Ad vector is thought to be due not to the difference in vector, but to the use of nucleic acid encoding HB-EGF ECD instead of the full-length HB-EGF gene (expressed as membrane-bound pro-HB-EGF, then cleaved by protease and secreted as soluble HB-EGF). Therefore, the effects of the present invention are similarly achieved not only when vectors other than AAV vectors are used, but also when a polypeptide that is an HB-EGF ECD fragment is administered.
• nucleic acid encoding HB-EGF ECD used in the present invention may be DNA or RNA, or may be a DNA/RNA chimera. DNA or RNA is preferred.
• the nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid, but is preferably double-stranded DNA. If single-stranded, it may be the sense strand (i.e., coding strand) or the antisense strand (i.e., non-coding strand). Sense strand RNA is preferred.
• DNA (RNA) encoding HB-EGF ECD includes genomic DNA, cDNA (cRNA) derived from cells or tissues of humans or other mammals, synthetic DNA (RNA), etc.
• nucleic acid encoding HB-EGF ECD used in the present invention contains at least a nucleotide sequence encoding soluble HB-EGF, but does not contain nucleotide sequences encoding the transmembrane domain and cytoplasmic domain.
• HB-EGF is first synthesized as a precursor containing a signal sequence, after which the signal sequence is cleaved in the endoplasmic reticulum to form membrane-bound proHB-EGF, and the N-terminal propeptide is further cleaved by a protease to form the active form, which acts on nearby cells in a juxtacrine manner.
• nucleic acid encoding HB-EGF ECD that has entered the bloodstream by systemic administration is delivered to an organ, such as the liver, targeted by the nucleic acid or vector (e.g., a viral vector).
• stringent conditions may be 50% formamide, 5x SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, followed by a wash with 0.2x SSC and 50% formaldehyde at 55°C, followed by a high-stringency wash with 0.1x SSC containing EDTA at 55°C.
• Those skilled in the art can easily achieve the desired stringency by appropriately adjusting the temperature during the hybridization reaction and/or wash, the ionic strength of the buffer, etc., depending on factors such as probe length.
• the nucleic acid encoding HB-EGF ECD may be an ortholog in a non-human mammal of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO:1 [for example, mouse, rat, bovine, porcine, or Chinese hamster orthologs registered in GenBank under accession numbers NM_010415, NM_012945, XM_601210, NM_214299, and AF069753, respectively].
• SEQ ID NO:1 for example, mouse, rat, bovine, porcine, or Chinese hamster orthologs registered in GenBank under accession numbers NM_010415, NM_012945, XM_601210, NM_214299, and AF069753, respectively.
• the nucleic acid encoding HB-EGF ECD is a nucleic acid encoding human HB-EGF ECD (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO:2).
• nucleic acids encoding HB-EGF ECD can be chemically synthesized or constructed by connecting chemically synthesized, partially overlapping short oligo-DNA strands using PCR or Gibson Assembly. This method makes it possible to obtain a codon-optimized sequence for expression in the target host cells, i.e., the mammalian cells (preferably human cells) to be treated. By converting the nucleotide sequence to codons frequently used in the host organism during gene expression, increased protein expression levels can be expected.
• codon usage in the host can be obtained from, for example, the genetic code usage database published on the website of the Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon/index.html), or by referring to literature listing codon usage in each host.
• codon optimization can be performed using a known codon optimization algorithm (e.g., GeneArt Codon Optimizer).
• GeneArt Codon Optimizer Such algorithms can take into account multiple parameters, such as GC content, removal of destabilizing RNA elements, removal of cryptic splice sites, removal of intragenic polyA sites, removal of repetitive sequences, avoidance of RNA secondary structures, and removal of IRES, in addition to frequency of use in the host.
• the cloned DNA can be used as is, depending on the purpose, or after digestion with a restriction enzyme or the addition of a linker, as desired.
• the DNA has a translation initiation codon ATG at its 5' end, and may also have a translation termination codon TAA, TGA, or TAG at its 3' end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.
• the expression vector is not particularly limited as long as it is one generally used in gene therapy, and examples thereof include viral vectors such as adeno-associated virus (AAV) vectors, lentivirus vectors, adenovirus (Ad) vectors, retrovirus vectors, Sindbis virus vectors, rabies virus vectors, Sendai virus vectors, and herpes simplex virus vectors, and non-viral vectors such as plasmids for expression in animal cells (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo).
• AAV adeno-associated virus
• Ad lentivirus vectors
• Ad adenovirus
• retrovirus vectors Sindbis virus vectors
• rabies virus vectors rabies virus vectors
• Sendai virus vectors Sendai virus vectors
• herpes simplex virus vectors and non-viral vectors
• non-viral vectors such as
• Ad vector or an AAV vector From the viewpoints of high gene transfer and expression efficiency, low frequency of chromosomal integration with no risk of insertional mutagenesis, ability to transfer to non-dividing cells, and ability to express the transferred gene over the medium to long term, it is preferable to use an Ad vector or an AAV vector, and it is more preferable to use an AAV vector.
• the Ad vector's introduced gene expression period (usually 2-3 weeks) is shorter than that of AAV vectors or chromosomal integration vectors, but previous research by the inventors has shown that the pancreatic beta cell protective and regenerative effects of HB-EGF persist far beyond the gene expression period (at least 60 days or more, preferably 75 days or more, more preferably 90 days or more, and even more preferably 120 days or more), which may actually be advantageous in that it can reduce or avoid the risk of side effects such as carcinogenesis caused by long-term HB-EGF expression.
• the gene size that can be carried by an AAV vector is small at 4.7 kb
• the HB-EGF ECD coding sequence is 444 bp
• the entire expression cassette including the promoter, terminator, etc. is only about 1-2 kb, so there are no problems with its use.
• HB-EGF ECD introduced into cells of other organs and secreted and expressed can also be delivered to the liver via the bloodstream, so there are no particular limitations on the serotype used.
• a serotype that has tropism for both organs such as AAV8.
• promoters derived from cytomegalovirus (CMV) (e.g., CMV immediate early promoter), human immunodeficiency virus (HIV) (e.g., HIV LTR), Rous sarcoma virus (RSV) (e.g., RSV LTR), mouse mammary tumor virus (MMTV) (e.g., MMTV LTR), Moloney murine leukemia virus (MoMLV) (e.g., MoMLV LTR), herpes simplex virus (HSV) (e.g., HSV thymidine kinase (TK) promoter), simian virus 40 (SV40) (e.g., SV40 early promoter), Epstein-Barr virus (EBV) (e.g., EBV) or adeno-associated virus (AAV) (e.g., AAV LTR)
• CMV cytomegalovirus
• HMV human immunodeficiency virus
• RSV Rous sarcoma virus
• AdV adenovirus
• Ad2 or Ad5 major late promoter adenovirus-derived promoters
• viral promoters such as the ⁇ -actin gene promoter, the phosphoglycerate kinase (PGK) gene promoter, the transferrin gene promoter, and other mammalian constitutive protein gene promoters can be used.
• a viral vector When using a viral vector as a vector, it is desirable to administer it at a low dose to reduce the risk of adverse events. However, to achieve the desired therapeutic effect at a low dose, it is necessary to carry the nucleic acid encoding HB-EGF ECD downstream of a promoter with transcriptional activity that can produce a therapeutically effective blood level of HB-EGF ECD.
• a promoter with stronger transcriptional activity than the CMV promoter or RSV promoter, which are frequently used in viral vector-based gene therapy can be used.
• Such highly active promoters include the CA promoter and promoters with equivalent transcriptional activity, such as ubiquitous promoters such as the elongation factor 1 ⁇ 1 (EF1A) promoter, the elongation factor 1 ⁇ 1 short (EFS) promoter, the CBh promoter (a hybrid promoter of a CMV immediate-early enhancer and a modified chicken ⁇ -actin promoter that differs from the CA promoter), the spleen-limited focus-forming virus (SFFV) promoter, the murine stem cell virus (MSCV) promoter, the SV40 enhancer/early promoter, the PGK promoter, and the ubiquitin C (UBC) promoter.
• ubiquitous promoters such as the elongation factor 1 ⁇ 1 (EF1A) promoter, the elongation factor 1 ⁇ 1 short (EFS) promoter, the CBh promoter (a hybrid promoter of a CMV immediate-early enhancer and a modified chicken ⁇ -actin promoter that differs from the CA promote
• a promoter that is highly expressed specifically in the tissue or cells of the target organ can also be used (for example, the albumin promoter, ⁇ -fetoprotein promoter, thyroxine-binding globulin promoter, etc. in the liver; the insulin promoter, Pdx1 promoter, Ins2 promoter, etc. in pancreatic ⁇ cells; the myogenin promoter, skeletal muscle actin ⁇ 1 (ACTA1) promoter, MHCK7 promoter, SM22a promoter, etc. in muscle, but is not limited to these, and includes any organ tissue- or cell-specific promoter from which secreted and expressed HB-EGF ECD can be delivered to the liver or pancreas via the bloodstream).
• the albumin promoter for example, the albumin promoter, ⁇ -fetoprotein promoter, thyroxine-binding globulin promoter, etc. in the liver
• the expression vector preferably contains a transcription termination signal, i.e., a terminator region, downstream of the nucleic acid encoding HB-EGF ECD. If desired, it may also contain an enhancer, splicing signal, WPRE sequence, Kozak sequence, a selection marker gene for selecting transformed cells, an SV40 origin of replication, etc. Examples of selection marker genes include genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin, and genes that complement auxotrophic mutations.
• a nucleotide sequence (signal codon) encoding a signal sequence suitable for the host may be added to the 5' end of the DNA encoding HB-EGF ECD in place of the native signal sequence.
• a nucleotide sequence signal codon
• an insulin signal sequence, an ⁇ -interferon signal sequence, or an antibody molecule signal sequence can be used.
• An expression vector containing a nucleic acid encoding HB-EGF ECD can be constructed using conventional genetic engineering techniques, cell culture techniques, and virus construction techniques [e.g., Current Protocols in Molecular Biology, F. Ausubel et al. eds. (1994) John Wiley & Sons, Inc.; Molecular Cloning (A Laboratory Manual), 3rd ed. Volumes 1-3, Joseph Sambrook & David W. Russelleds. , Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York K) (2001); Culture of Animal Cells; A Manual of Basic Technique, R. Freshney eds. , 2nd ed. (1987), Wiley-Liss; Frank L.
• the therapeutic agent (I) of the present invention can be provided as a so-called mRNA drug.
• the expression vector can be introduced into a suitable host (e.g., mammalian cells) and cultured, and the mRNA can be recovered using a method known per se (e.g., the LiCl method), and the mRNA encoding HB-EGF ECD can be purified to obtain the mRNA.
• the mRNA can be obtained by excising the nucleic acid encoding HB-EGF ECD (which may include 5'- and 3'-UTRs in addition to the coding sequence (CDS)) from the expression vector and using it as a template to convert it into mRNA encoding HB-EGF ECD using a known in vitro transcription system.
• CDS coding sequence
• the HB-EGF ECD coding region is excised using an appropriate restriction enzyme and a phage (T7, T3, SP6, etc.) promoter is ligated to the 5' end, or a fragment of the mRNA coding region to which the phage promoter is linked is obtained by using the expression vector as a template and performing PCR using primers containing the phage promoter sequence.
• the obtained DNA fragment can be used as a template to react with phage (T7, T3, SP6, etc.) RNA polymerase to synthesize mRNA encoding HB-EGF ECD in vitro.
• NTP RNA monomer
• the 5'-cap structure can be achieved by adding a Cap 0 structure after mRNA synthesis using a capping enzyme, and then converting it to a Cap 1 structure using an mRNA 2'-O-methyltransferase.
• the HB-EGF ECD fragment used in the therapeutic agent (I) of the present invention contains at least a soluble HB-EGF portion, but does not contain a transmembrane domain or a cytoplasmic domain. It may or may not contain a prosequence; if it does, the prosequence is cleaved by a protease endogenous to the mammalian subject to administration, resulting in active soluble HB-EGF.
• a signal sequence is also not necessary for activity, and it is desirable not to include it. However, even if it does contain a signal sequence, it will be cleaved and removed together with the prosequence by a protease endogenous to the subject to administration.
• an "HB-EGF ECD fragment” is a polypeptide that contains at least the amino acid sequence represented by amino acid numbers 44 to 129 in the amino acid sequence represented by SEQ ID NO: 2 [corresponding to the amino acid sequence from amino acid numbers 20 to 148 of the amino acid sequence of human HB-EGF registered in GenBank under Accession Number NP_001936 (positions 20 to 62 correspond to the propeptide region, and positions 63 to 148 correspond to the soluble HB-EGF region)], or an amino acid sequence substantially identical thereto, and that has activity equivalent to that of soluble HB-EGF (e.g., blood glucose elevation suppression activity, beta-cell protection and regeneration activity).
• SEQ ID NO: 2 [corresponding to the amino acid sequence from amino acid numbers 20 to 148 of the amino acid sequence of human HB-EGF registered in GenBank under Accession Number NP_001936 (positions 20 to 62 correspond to the propeptide region, and positions 63 to 148 correspond to the soluble HB-EGF region)]
• An expression vector containing DNA encoding HB-EGF ECD can be produced, for example, by excising the desired DNA fragment from DNA encoding HB-EGF and ligating the DNA fragment downstream of a promoter in an appropriate expression vector.
• Expression vectors that can be used include E.
• coli-derived plasmids e.g., pBR322, pBR325, pUC12, pUC13
• Bacillus subtilis-derived plasmids 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., pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo
• bacteriophages such as ⁇ phage
• insect virus vectors such as baculovirus (e.g., BmNPV, AcNPV), retroviruses
• animal virus vectors such as vaccinia virus, adenovirus, and adeno-associated virus.
• the promoter may be any promoter appropriate for the host used to express the gene.
• the promoters exemplified in (a) above can be used in the same manner.
• the host is Escherichia coli
• the host is Bacillus subtilis
• the SPO1 promoter, SPO2 promoter, penP promoter, etc. are preferred.
• yeast the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferred.
• the polyhedrin promoter, P10 promoter, etc. are preferred.
• a PhoA signal sequence, an OmpA signal sequence, and the like can be used; when the host is Bacillus subtilis, an ⁇ -amylase signal sequence, a subtilisin signal sequence, and the like can be used; when the host is yeast, an MF ⁇ signal sequence, an SUC2 signal sequence, and the like can be used; and when the host is an animal cell, the native HB-EGF signal sequence, as well as an insulin signal sequence, an ⁇ -interferon signal sequence, or an antibody molecule signal sequence can be used.
• Host cells can be cultured by known methods, such as those described in Proc. Natl. Acad. Sci. USA, 69: 2110 (1972) and Gene, 17: 107 (1982) for Escherichia coli, Molecular and General Genetics, 168: 111 (1979) for Bacillus subtilis, Methods in Enzymology, 194: 182-187 (1991) and Proc. Natl. Acad. Sci. USA, 75: 1929 (1978) for yeast, Bio/Technology, 6: 47-55 (1988) for insect cells, and New Cell Engineering Experimental Protocols, Cell Engineering Special Issue 8, 263-267 (1995) for animal cells. (Shujunsha Publishing), Virology, 52: 456 (1973).
• the resulting transformant is cultured, and the HB-EGF ECD fragment can be separated and purified from the culture by a method known per se.
• Therapeutic agent (I) of the present invention may be (a) the nucleic acid/expression vector encoding HB-EGF ECD or (b) the HB-EGF ECD fragment of the present invention, and may be used as is, or, if necessary, may be mixed with a pharmacologically acceptable carrier to form various formulations such as injections and then used as a medicine.
• organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used as pharmacologically acceptable carriers, and are incorporated as solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid formulations.
• formulation additives such as preservatives, antioxidants, and coloring agents can also be used as needed.
• solvents include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, etc.
• solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.
• suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates; and polyoxyethylene hydrogenated castor oil.
• surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate
• hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxy
• Suitable examples of isotonic agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.
• buffering agents include buffer solutions such as phosphate, acetate, carbonate, and citrate.
• Suitable examples of soothing agents include benzyl alcohol.
• Suitable examples of preservatives include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.
• antioxidants include sulfites and ascorbates.
• coloring agents include water-soluble food tar dyes (e.g., food dyes such as Food Red No. 2 and No. 3, Food Yellow No. 4 and No. 5, and Food Blue No. 1 and No. 2), water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), and natural dyes (e.g., ⁇ -carotene, chlorophyll, red iron oxide, etc.).
• water-soluble food tar dyes e.g., food dyes such as Food Red No. 2 and No. 3, Food Yellow No. 4 and No. 5, and Food Blue No. 1 and No. 2
• water-insoluble lake dyes e.g., aluminum salts of the above-mentioned water-soluble food tar dyes
• natural dyes e.g., ⁇ -carotene, chlorophyll, red iron oxide, etc.
• Dosage forms of the pharmaceutical composition include parenteral preparations such as injections (e.g., intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intraperitoneal injections, etc.) and drip infusions.
• parenteral preparations such as injections (e.g., intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intraperitoneal injections, etc.) and drip infusions.
• Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc.
• aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.
• the most suitable dosage form in the present invention is an injection solution.
• the nucleic acid may be in the form of a pharmaceutical composition encapsulated in a liposome.
• Liposomes are small, closed vesicles with an internal phase surrounded by one or more lipid bilayers, and can typically hold water-soluble substances in the internal phase and lipid-soluble substances within the lipid bilayer.
• encapsulated used herein refers to the nucleic acid being held either in the internal phase of the liposome or within the lipid bilayer.
• the liposomes used in the present invention may be monolayer or multilayer membranes, and the particle size can be appropriately selected, for example, from the range of 10 to 1,000 nm, preferably 50 to 300 nm. Considering delivery to target tissues, the particle size may be, for example, 200 nm or less, preferably 100 nm or less.
• Methods for encapsulating water-soluble compounds such as polynucleotides into liposomes include, but are not limited to, the lipid film method (vortex method), reverse phase evaporation, surfactant removal, freeze-thaw method, and remote loading method, and any known method can be selected as appropriate.
• lipid film method vortex method
• reverse phase evaporation surfactant removal
• freeze-thaw method freeze-thaw method
• remote loading method any known method can be selected as appropriate.
• the nucleic acid encoding HB-EGF ECD is encapsulated in a lipid nanoparticle as a carrier.
• lipid nanoparticle refers to a particle having a membrane structure in which the hydrophilic groups of the amphipathic lipid are aligned toward the aqueous phase at the interface, and having a particle diameter of less than 1 ⁇ m
• amphipathic lipid refers to a lipid having both hydrophilic and hydrophobic groups.
• the dosage of the formulation varies depending on the type of vector, promoter activity, administration route, severity of disease, target animal species, drug tolerance, body weight, age, etc.
• an AAV vector is used as an HB-EGF expression vector
• a recent clinical trial reported deaths accompanied by serious adverse events in the liver and hepatobiliary tract, even in a group administered a relatively low dose (1.3 ⁇ 10 14 viral particles (vp)/kg body weight) (Lancet Neurology, 2023; 22: 1125-39).
• the low-dose (1 ⁇ 10 14 vp/kg body weight) group did not experience adverse events in the liver despite the presence of liver disease.
• the formulation at a dose of 10 14 vp/kg body weight or less.
• the single dose is about 5 ⁇ 10 9 to about 5 ⁇ 10 13 vp/kg body weight, preferably about 1 ⁇ 10 10 to about 1 ⁇ 10 13 vp/kg body weight.
• an Ad vector is used as an HB-EGF ECD expression vector, a previous clinical trial reported a case of death due to acute liver damage following hepatic artery administration of 6 x 10 vp/kg body weight (total amount 3.8 x 10 vp) (Mol Genet Metab 2003; 80: 148-158).
• HB-EGF ECD HB-EGF ECD gene therapy for T1D by tail vein administration of approximately 5 x 10 12 vp/kg body weight of an AAV vector.
• Administration of the same dose of the full-length HB-EGF gene to T1D model mice 7 days after STZ administration did not necessarily result in a sufficient effect in suppressing blood glucose elevation.
• HB-EGF ECD reduced blood glucose levels to levels equal to or lower than those of normal mice, and in some cases even caused hypoglycemia. Therefore, it is believed that HB-EGF ECD gene therapy can achieve a sufficient effect in suppressing blood glucose elevation with even lower doses.
• a non-viral vector encapsulated in liposomes as an HB-EGF ECD expression vector
• safety was confirmed when 666 ⁇ g of DNA was administered intravenously in a clinical study using cynomolgus monkeys weighing approximately 4 kg, so the same amount serves as a guideline.
• a single dose for an adult would be approximately 2 to 10 mg, preferably approximately 5 to 8 mg.
• a similar dosage can also be used as a guideline when RNA encoding HB-EGF ECD is encapsulated in liposomes or lipid nanoparticles (mRNA medicine).
• pancreatic duct cells were crucial for differentiation or neogenesis of beta cells from pancreatic duct cells, and that the paracrine effect of soluble HB-EGF secreted from pancreatic duct cells protected and regenerated the remaining beta cells in the islets.
• our previous research demonstrated that soluble HB-EGF secreted and expressed from distant organs such as the liver is delivered to the pancreas via the bloodstream, where it exerts protective and regenerative effects on ⁇ cells through endocrine action.
• the therapeutic agent (I) of the present invention containing a nucleic acid encoding HB-EGF ECD, for example, via a vein, particularly a peripheral vein.
• the frequency of administration of the therapeutic agent (I) of the present invention containing a nucleic acid encoding HB-EGF ECD is not particularly limited. As shown in the examples below, when an AAV vector is used, a single administration alone can sufficiently suppress hyperglycemia over a long period of at least 70 days. When an AAV vector is used to target non-dividing cells, HB-EGF expression can be sustained for a longer period, and it is therefore believed that the effects of suppressing hyperglycemia and improving glucose tolerance can be achieved for an even longer period (e.g., 6 months or more, preferably 1 year or more, and more preferably several years or more).
• the therapeutic agent (I) of the present invention containing a nucleic acid encoding HB-EGF ECD can be administered, for example, at intervals of at least 60 days, preferably 75 days or more, more preferably 90 days or more, and even more preferably 120 days or more, even when using an Ad vector or AAV vector, which is a non-chromosomally integrated vector and has been conventionally considered safer and more frequently used than retroviral or lentiviral vectors.
• the therapeutic agent (I) can also be administered at intervals of once every 3 months to once every several years, or in another embodiment, as a single administration.
• the therapeutic agent (I) of the present invention containing an HB-EGF ECD fragment can preferably be formulated as an injectable solution, similar to the nucleic acid encoding HB-EGF ECD. Alternatively, it can be made into a sustained-release formulation using a biocompatible material such as collagen. For example, since Pluronic gel gels at body temperature and is liquid at lower temperatures, local injection of the HB-EGF ECD fragment together with Pluronic gel to gel around the target tissue can provide a long-lasting effect, thereby avoiding the drawbacks of HB-EGF protein administration identified by Kozawa et al. (2005, supra).
• the polypeptide formulation can be packaged in a unit dose or multiple doses in a container such as an ampule or vial.
• the HB-EGF ECD fragment and a pharmacologically acceptable carrier can be lyophilized and stored in a state that requires only dissolving or suspending in an appropriate sterile vehicle immediately before use.
• Antibodies against surface molecules of target cells can specifically deliver drugs to the target cells. Therefore, by crosslinking an HB-EGF ECD fragment to the antibody to form an immunoconjugate, the stability of HB-EGF ECD in the blood and the efficiency of delivery to the target cell surface can be improved, thereby avoiding the drawbacks of HB-EGF protein administration pointed out by Kozawa et al. (2005, supra).
• target cells e.g., liver cells
• examples of surface molecules of liver cells include, but are not limited to, EGFR (ErbB1/HER1), ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4.
• the antibody may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.
• the antibody can be prepared by well-known immunological techniques.
• 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 a surface molecule of a target cell, and examples of such fragments include Fab, F(ab') 2 , ScFv, and minibody.
• the antibody is preferably a chimeric antibody between a human and another animal (e.g., mouse), a humanized antibody, or a fully human antibody.
• Methods for crosslinking an antibody against a surface molecule of a target cell with an HB-EGF ECD fragment include, but are not limited to, the method described in Adv. Drug Deliv. Rev., 53: 171-216 (2001).
• the therapeutic agent (I) of the present invention containing an HB-EGF ECD fragment can be manufactured by conventional methods in the pharmaceutical technology field, such as those described in the Japanese Pharmacopoeia.
• the content of the fragment in the formulation varies depending on the dosage form, the dose of the active ingredient, etc., but is, for example, approximately 0.1 to 100% by weight.
• the therapeutic agent (I) of the present invention containing an HB-EGF ECD fragment can be administered orally or parenterally to mammals (e.g., humans, mice, rats, rabbits, dogs, monkeys, etc.), with parenteral administration being preferred.
• Parenteral administration routes include, for example, systemic administration such as intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intraportal, and intraarterial administration, as well as local administration (e.g., direct administration to a target organ during endoscopic or laparotomy surgery).
• HGF or a fragment thereof can be produced by culturing a transformant containing a nucleic acid encoding it, and isolating and purifying HGF or a fragment thereof from the resulting culture.
• DNA encoding HGF or a fragment thereof can be the DNA used for gene therapy, as described below.
• the DNA has a translation initiation codon ATG at its 5' end, and may also have a translation termination codon TAA, TGA, or TAG at its 3' end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.
• expression vectors that contain enhancers, splicing signals, polyA addition signals, selection markers, SV40 replication origins, etc., can be used as desired.
• Selection markers include those similar to those exemplified for HB-EGF ECD.
• a nucleotide sequence (signal codon) encoding a signal sequence suitable for the host can be added to the 5' end of the DNA encoding HGF or a fragment thereof.
• Signal sequences include those similar to those exemplified for HB-EGF ECD.
• Host cells can be transformed using known methods similar to those exemplified for HB-EGF ECD.
• the resulting transformant can be cultured, and HGF or a fragment thereof can be isolated and purified from the culture using known methods.
• HGF or a fragment thereof can also be synthesized by in vitro translation using the RNA encoding it as a template in a cell-free protein translation system consisting of rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc.
• HGF or a fragment thereof can also be synthesized using a cell-free transcription/translation system containing RNA polymerase, with DNA encoding HGF or a fragment thereof as a template.
• Cell-free protein transcription/translation systems similar to those exemplified for HB-EGF ECD can be used.
• nucleic acid encoding HGF or a fragment thereof examples include nucleic acids that contain a nucleotide sequence represented by nucleotide numbers 1 to 627 in the nucleotide sequence represented by SEQ ID NO: 3 (corresponding to the nucleotide sequence from nucleotide numbers 77 to 2260 in the mRNA sequence of human HGF registered in GenBank under Accession Number NM_000601), or a nucleotide sequence that hybridizes under stringent conditions to the complementary strand sequence thereof, and that encode a protein having activity equivalent to that of HGF (e.g., blood glucose elevation suppression activity when used in combination with HB-EGF ECD, glucose-responsive insulin secretion-stimulating activity, and ⁇ -cell protective and regenerative activity).
• a protein having activity equivalent to that of HGF e.g., blood glucose elevation suppression activity when used in combination with HB-EGF ECD, glucose-responsive insulin secretion-stimulating activity, and ⁇ -cell protective and regenerative activity
• nucleic acids that hybridize under stringent conditions to a complementary strand sequence of a sequence comprising nucleotide numbers 1 to 627 in the nucleotide sequence represented by SEQ ID NO: 3 include nucleic acids containing a nucleotide sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more identity to a sequence comprising nucleotide numbers 1 to 627 in the nucleotide sequence represented by SEQ ID NO: 3.
• the term "stringent conditions" has the same meaning as in the case of the nucleic acid encoding HB-EGF ECD.
• Cellular stress factors include, but are not limited to, inflammatory cytokines (e.g., interleukin-1 ⁇ (IL-1 ⁇ ), IL-8, etc.), reactive oxygen species (ROS) (e.g., nitric oxide, etc.), and hypotonic solutions (e.g., hypotonic electrolyte infusions, etc.), as long as they are capable of inducing proHB-EGF shedding.
• inflammatory cytokines e.g., interleukin-1 ⁇ (IL-1 ⁇ ), IL-8, etc.
• ROS reactive oxygen species
• hypotonic solutions e.g., hypotonic electrolyte infusions, etc.
• amino acid sequences of the inflammatory cytokines and the nucleotide sequences of the genes encoding them are both publicly known, and based on this sequence information, a person skilled in the art can produce these inflammatory cytokines or the nucleic acids encoding them in the same manner as described in detail above in 1 for the HB-EGF ECD fragment or the nucleic acids encoding them, formulate them into pharmaceutical compositions, and administer them to mammals with diabetes.
• Proteins that interact with the protease of (a) or nucleic acids encoding the same include, but are not limited to, Eve-1, PACSIN3, Src, PKC- ⁇ , Grb2, phosphatidylinositol 3-kinase (PI3K), endoflin, SH3Px1, and Fish.
• amino acid sequences of these proteins and the nucleotide sequences of the genes encoding them are both publicly known, and based on this sequence information, a person skilled in the art can produce these proteins or the nucleic acids encoding them in the same manner as detailed above in 1., formulate them into pharmaceutical compositions, and administer them to mammals with diabetes.
• the HB-EGF receptor agonist may be an agonist antibody against the HB-EGF receptor, as described above with respect to the dosage form of the immunoconjugate of the therapeutic agent (I) of the present invention comprising an HB-EGF ECD fragment. Since the receptor tyrosine kinase can be activated by dimerization of ErbB family member molecules, dimerization can be promoted by using a complete antibody molecule or a bivalent antibody fragment (e.g., Fab, F(ab') 2 , etc.).
• a bivalent antibody fragment e.g., Fab, F(ab') 2 , etc.
• a cocktail of bispecific antibodies against different ErbB family members e.g., anti-ErbB1/ErbB2, anti-ErbB1/ErbB3, anti-ErbB1/ErbB4, anti-ErbB2/ErbB4, and anti-ErbB3/ErbB4 antibodies
• an anti-ErbB1 antibody and an anti-ErbB4 antibody e.g., anti-ErbB2 antibody, anti-ErbB1/ErbB3, anti-ErbB1/ErbB4, anti-ErbB2/ErbB4, and anti-ErbB3/ErbB4 antibodies
• HB-EGF receptor agonists can be screened using, for example, competitive binding activity to the receptor in cells expressing the receptor, activation of the receptor (e.g., autophosphorylation of tyrosine residues in the receptor), and activation (phosphorylation) of kinase molecules activated downstream of the receptor as indicators.
• transactivators of the HB-EGF receptor examples include calcium ionophore, PKC, Src, PYK2, and ROS. These substances also induce ectodomain shedding of proHB-EGF, but are also thought to act directly on the HB-EGF receptor without the action of soluble HB-EGF. Furthermore, TRIO, CHKA, and BMX are involved in type 1 angiotensin receptor-dependent receptor tyrosine kinase phosphorylation induced by angiotensin II stimulation, but have been suggested to also act directly on the HB-EGF receptor without the action of soluble HB-EGF.
• the amino acid sequences of the proteinaceous factors and the nucleotide sequences of the genes encoding them are both publicly known, and based on this sequence information, a person skilled in the art can produce these proteins or the nucleic acids encoding them in the same manner as described in detail above in 1., formulate them into pharmaceutical compositions, and administer them to mammals with diabetes.
• the present invention also provides a method for treating diabetes in a mammal, comprising exposing the mammal to a means for enhancing HB-EGF receptor-mediated signaling.
• a means for enhancing HB-EGF receptor-mediated signaling is administering to the mammal an effective amount of one of the active ingredients of the therapeutic agent (II) of the present invention, as detailed in Section 2 above.
• HB-EGF receptor-mediated signaling can also be enhanced by subjecting the mammal to cellular stress, such as exposure to ultraviolet B rays (UV-B) or radiation. UV irradiation and radiation exposure are well-known and commonly used treatments in the medical and cosmetic fields, and those skilled in the art can appropriately formulate and implement safe and effective irradiation protocols.
• UV-B ultraviolet B rays
• HB-EGF ECD may be referred to as sHB-EGF.
• Example 1 (Experimental Method) 1. Construction of recombinant adeno-associated virus (AAV) vectors.
• the recombinant AAV serotype used was AAV8, which is capable of pancreas-specific gene transfer.
• Five types of AAV vectors express therapeutic genes under the transcriptional control of a hybrid promoter (CA promoter) consisting of the cytomegalovirus immediate early enhancer and a modified chicken ⁇ -actin promoter.
• a schematic diagram of each vector is shown in Figure 1. (1) AAV-CA-sHB-EGF-NK1 (2) AAV-CA-NK1-sHB-EGF (3) AAV-CA-sHB-EGF (4) AAV-CA-NK1 (5) AAV-CA-Venus
• HB-EGF ECD The sequence (444 bp) encoding amino acids 1-148 of HB-EGF, including the N-terminal signal peptide and propeptide, was used as HB-EGF ECD (sHB-EGF).
• packaging size the size of the gene that can be inserted between the 5' ITR and 3' ITR
• full-length HGF (2,187 bp) exceeds this upper limit when including the promoter, etc. Therefore, an AAV vector was constructed using the NK1 domain (627 bp), which is known to have agonistic activity against the c-Met receptor.
• AAV-CA-Venus was constructed, which expresses a mutant Venus gene of the EGFP gene, a yellow fluorescent protein, under the transcriptional control of the CA promoter.
• mice were randomly divided into four groups 1 to 7 days after STZ administration (day 0), and received a single injection of the AAV vector described below via the tail vein.
• Blood and urine were collected from all mice, including those in the intact group. Blood glucose and body weight measurements were performed daily from day -7 to day 7, and blood samples were collected weekly from day 7 to day 35 in addition to blood glucose and body weight measurements. On days 14 and 28, intraperitoneal glucose tolerance tests (IPGTTs) were performed. After fasting for 14 hours, the mice were intraperitoneally administered 2 g/kg body weight of glucose. Blood samples were collected immediately before administration (0 min), and 30, 60, and 120 min after administration, and blood glucose and plasma insulin levels were measured at each time point. All animal experiments were conducted in accordance with the guidelines of the National Institutes of Health and were approved by the Kagoshima University Animal Experiment Ethics Committee. Biochemical analysis was performed using the following instruments and reagents. Blood glucose level: Medisafe Fit Pro II (Terumo, Tokyo) Urine sugar: Uropaper III (Eiken Chemical Co., Ltd., Tokyo) Plasma insulin level ELISA assay (Morinaga Co., Ltd., Yokohama)
• mice administered with AAV-CA-Venus or normal mice were administered with AAV-CA-sHB-EGF, AAV-CA-sHB-EGF-NK1, AAV-CA-NK1-sHB-EGF, and AAV-CA-NK1 was determined by Student's t-test. P ⁇ 0.05 was defined as statistically significant.
• mice administered 1.0 x 10 11 vg of AAV-CA-Venus casual blood glucose levels rapidly rose to approximately 500 mg/dl on day 7 after vector administration (day 1 to day 21 after STZ injection), continued to rise gradually thereafter, and remained at high levels of 500 mg/dl or higher.
• mice administered 1.0 x 10 11 vg of AAV-CA-sHB-EGF blood glucose levels were suppressed to a level not significantly different from that of normal mice by day 7, normalizing blood glucose levels and confirming a dose-dependent blood glucose-lowering effect.

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