Shb-EGF enhancement therapy for diabetes

Abstract

The present invention provides: 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; the preparation combining hepatocyte growth factor (HGF) or a fragment thereof or a nucleic acid encoding the same; and a therapeutic agent for diabetes in mammals comprising a substance that potentiates signal transduction via the HB-EGF receptors.

Description

sHB-EGF enhanced treatment for diabetes

[0001] The present invention relates to a biopharmaceutical for diabetes that has a guaranteed therapeutic effect. More specifically, the present invention relates to a therapeutic agent for diabetes that contains an extracellular domain fragment of heparin-binding epidermal growth factor-like growth factor (HB-EGF) or a nucleic acid encoding the same, and a therapeutic agent for diabetes that further contains hepatocyte growth factor (HGF) or a fragment thereof or a nucleic acid encoding the same. The present invention also relates to a therapeutic agent for diabetes that contains a substance that activates signal transduction mediated by the HB-EGF receptor.

[0002] According to a report by the International Diabetes Federation (IDF), as of 2021, the number of diabetes patients worldwide is estimated to reach 537 million, and if this trend continues, this number is predicted to reach 783 million by 2045. Type 2 diabetes (T2D), which accounts for 90% of all diabetes cases, is a disease in which, 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 excessive insulin secretion, but if this condition continues for a long time, the beta cells become exhausted, and they are unable to secrete sufficient amounts of insulin, resulting in a state of hyperglycemia.

[0003] Type 1 diabetes (T1D) is a disease characterized by the autoimmune destruction of pancreatic beta cells, resulting in a depletion of insulin secretion and a state of hyperglycemia. T1D develops at a young age, but the details of its mechanism remain unknown. T1D patients must self-inject insulin throughout their lives to manage their blood sugar. However, even with strict insulin therapy, blood sugar control is difficult, and patients experience alternating episodes of hyperglycemia and severe hypoglycemia, making it difficult to prevent the progression of complications. Therefore, innovative treatments that can cure T1D are needed.

[0004] As one of the therapeutic methods for curing T1D, attempts have been made to protect and / or proliferate the patient's remaining β cells directly in the body through in vivo gene therapy. For example, several studies have been reported using HGF and HB-EGF as therapeutic genes for T1D. The present inventors recently developed HGF and / or HB-EGF gene therapy agents that can be used safely at low doses and have the effect of protecting and regenerating pancreatic β cells (Patent Documents 1 and 2, Non-Patent Document 1). However, when the full-length HB-EGF gene was used alone, the effects of suppressing blood glucose elevation and promoting glucose-responsive insulin secretion were limited compared to when the full-length HB-EGF gene was used in combination with the HGF gene (Patent Document 2).

[0005] WO 2022 / 107853WO 2022 / 186282

[0006] Matsuda, E. et al., Life Sciences, 268: 119014 (2021)

[0007] Therefore, an object of the present invention is to provide a novel gene therapy method that can provide more effective treatment for diabetes including T1D than conventional methods. Another object of the present invention is to provide a novel treatment concept for diabetes that is derived from the novel gene therapy and is highly original and applicable to a wide variety of derivative technologies.

[0008] In our previous studies, we used adenovirus (Ad) vectors as specific vectors for gene transfer. However, because Ad vectors accumulate largely in the liver, we hypothesized that their mechanism of action involves HGF and HB-EGF fragments secreted and expressed from the liver, a distant organ, being delivered to the pancreas via the bloodstream, where they exert protective and regenerative effects on pancreatic β cells through endocrine action. Therefore, we used adeno-associated virus (AAV) serotype 8, which has pancreatic organ tropism, as a vector to directly deliver and express therapeutic genes in the pancreas. However, because the gene size that AAV vectors can accommodate is small (4.7 kbp), we used a nucleic acid encoding the extracellular domain (ectodomain; ECD) of the HB-EGF gene and a nucleic acid encoding the NK1 fragment consisting of the N-terminal domain and kringle 1 (K1) domain of the HGF gene.

[0009] As a result, systemic administration of AAV vectors to T1D model mice 7 days after streptozotocin (STZ) administration showed that the HB-EGF ECD fragment alone significantly suppressed blood glucose elevation for at least 5 weeks after administration, whereas the HGF NK1 fragment alone did not show any blood glucose elevation suppression effect. In an intraperitoneal glucose tolerance test (IPGTT), the HB-EGF ECD alone significantly suppressed blood glucose elevation after glucose administration, but did not increase insulin secretion after glucose administration. On the other hand, when HB-EGF ECD and NK1 were used in combination, both casual blood glucose and blood glucose elevation after glucose administration were significantly suppressed, and furthermore, increased insulin secretion after glucose administration was observed. NK1 alone increased casual plasma insulin concentrations but did not induce glucose-responsive insulin secretion.

[0010] Thus, when a gene encoding HB-EGF ECD was used, the blood glucose elevation suppression effect was dramatically increased compared to when a gene encoding full-length HB-EGF was used as shown in Patent Document 2. Moreover, since insulin secretion was not significantly promoted when a gene encoding HB-EGF ECD was used, this effect was thought to be also contributed by a new mechanism, such as direct promotion of glucose metabolism in the liver. Unlike full-length HGF, NK1 had no effect on its own, but induced glucose-responsive insulin secretion when co-expressed with HB-EGF ECD.

[0011] 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 sufficient to cause hypoglycemia in T1D model mice 7 days after STZ administration, and moreover, one of the mechanisms of action appears to involve a mechanism other than the improvement in insulin secretion ability due to the protection and regeneration of β cells. Therefore, the therapeutic effect of the same or lower doses of HB-EGF ECD was investigated using T1D model mice 14 days after STZ administration, in which β cell destruction had progressed further. The results showed that HB-EGF ECD dose-dependently suppressed blood glucose elevation, and at the maximum dose (1 x 10 １１ In the HB-EGF ECD (vg) mice, blood glucose levels were comparable to those of normal mice by day 7 of administration and remained normal thereafter. Furthermore, none of the mice exhibited hypoglycemia during the observation period, confirming safety. In IPGTT, HB-EGF ECD dose-dependently suppressed the rise in blood glucose after glucose administration, but plasma insulin levels were reduced after glucose administration at all doses, suggesting the contribution of an insulin-independent effect in improving glucose tolerance. Although casual blood glucose was suppressed dose-dependently, plasma insulin concentrations were significantly lower than those of normal mice at all doses, further strengthening the possibility that a non-insulin-mediated blood glucose-lowering effect contributed to the results. RNA-seq and metabolome analysis confirmed that the introduction of HB-EGF ECD activated glucose metabolism in liver tissue, further supporting this hypothesis.

[0012] However, immunohistochemical staining of the 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 excellent inhibitory effect of HB-EGF ECD on blood glucose elevation is primarily due to the protection and regeneration of beta cells.

[0013] Since the contribution of the protection and regeneration of β cells to the induction of insulin secretion is relatively small, it will be understood by those skilled in the art that the difference in effect between the present invention and the inventors' previous study using an Ad vector is not due to the difference in vector, but rather to the use of a nucleic acid encoding a secreted form of HB-EGF ECD instead of the full-length HB-EGF gene (which is expressed as membrane-bound pro-HB-EGF, then cleaved by endogenous proteases and secreted as soluble HB-EGF), and as a result, direct glucose metabolism in the liver is promoted by the autocrine and / or paracrine action of HB-EGF ECD (particularly soluble HB-EGF from which the propeptide has been removed) expressed and secreted from the liver (as well as the endocrine action of HB-EGF ECD secreted from other organs). This indicates that, in addition to gene therapy, excellent therapeutic effects for diabetes similar to those of gene therapy can be obtained by exogenously or endogenously enhancing soluble HB-EGF (including not only increasing the soluble HB-EGF level but also enhancing signal transduction via the HB-EGF receptor). Based on these findings, the present inventors have conducted further research and have completed the present invention.

[0014] That is, the present invention is as follows. [Item 1] A therapeutic agent for diabetes in a mammal, comprising a heparin-binding epidermal growth factor-like growth factor ectodomain (HB-EGF ECD) fragment or a nucleic acid encoding the same. [Item 2] The agent according to Item 1, comprising a nucleic acid encoding an HB-EGF ECD fragment. [Item 3] The agent according to Item 2, wherein the nucleic acid is carried on a viral vector. [Item 4] The agent according to Item 3, wherein the viral vector is an adeno-associated virus (AAV) vector. [Item 5] The agent according to any one of Items 2 to 4, wherein the nucleic acid encoding the HB-EGF ECD fragment is under the control of a CA promoter. [Item 6] The agent according to any one of Items 1 to 5, which is administered by systemic administration. [Item 7] The agent according to Item 6, wherein the systemic administration is intravenous administration. [Item 8] The agent according to Item 4, which is administered in a single dose or multiple doses with an interval of at least 60 days between doses. [Item 9] The viral vector is administered in a dose of 1 x 10 １０ ~1 x 10 １３The agent according to Item 3, 4, or 8, which is administered in a single dose of viral particles (vp) / kg body weight. [Item 10] The agent according to Item 1 or 2, which comprises a combination of hepatocyte growth factor (HGF) or a fragment thereof, or a nucleic acid encoding the same. [Item 11] The agent according to Item 10, wherein a nucleic acid encoding an HB-EGF ECD fragment and a nucleic acid encoding an HGF NK1 fragment are carried on a single vector. [Item 12] A therapeutic agent for diabetes in a mammal, comprising a substance that enhances signal transduction mediated by the HB-EGF receptor. [Item 13] The agent according to Item 12, wherein the substance is selected from the following (a) to (d): (a) a protease having proHB-EGF ectodomain shedding activity or a nucleic acid encoding the same; (b) a factor that induces proHB-EGF ectodomain shedding; (c) a protein that interacts with the protease of (a) or a nucleic acid encoding the same; (d) an agonist or transactivator of the HB-EGF receptor. [Item 14] The agent according to any one of Items 1 to 13, wherein the diabetes is type 1 diabetes. [Item 15] The agent according to any one of Items 1 to 14, wherein the mammal is a human. [Item 16] A method for treating diabetes in a mammal having diabetes, comprising exposing the mammal to a means that enhances signaling via the HB-EGF receptor. [Item 1A] A method for treating diabetes in a mammal having diabetes, comprising administering to the mammal an effective amount of an HB-EGF ECD fragment or a nucleic acid encoding the same. [Item 1B] An HB-EGF ECD fragment or a nucleic acid encoding the same for use in treating diabetes in a mammal. [Item 1C] Use of an HB-EGF ECD fragment or a nucleic acid encoding the same in the manufacture of a therapeutic agent for diabetes in a mammal.

[0015] According to the present invention, a safe, minimally invasive method can be used to achieve the desired effect of suppressing blood glucose elevation over a long period of time, thereby enabling treatment that achieves both therapeutic effects for diabetes including T1D and improvement in QOL, thereby increasing the feasibility of applying this treatment to human clinical trials.

[0016] 1 is a schematic diagram showing the structure of the AAV vector used in the examples. FIG. 2 is a diagram showing the protocol for the pharmacological efficacy test. FIG. 3 is a diagram showing blood glucose reduction in T1D model mice by HB-EGF ECD and a combination of HB-EGF ECD and NK1. FIG. 4 is a diagram showing the results of IPGTT 14 days after treatment. Left: shows the change in blood glucose level after glucose load. Right: shows the change in plasma insulin level after glucose load. FIG. 5 is a diagram showing the results of IPGTT 28 days after treatment. Left: shows the change in blood glucose level after glucose load. Right: shows the change in plasma insulin level after glucose load. FIG. 6 is a diagram showing the normalization of casual blood glucose by HB-EGF ECD and a combination of HB-EGF ECD and NK1 (left) and the maintenance of casual plasma insulin level by the combination (right). FIG. 7 is a diagram showing the protection and proliferation of β cells by a combination of HB-EGF ECD and a combination of HB-EGF ECD and NK1. FIG. 8 is a diagram showing that intravenous administration of AAV achieves highly efficient gene transfer to the liver, pancreas, and kidney. 1 shows the suppression of liver damage induction by HB-EGF ECD and the combined use 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 AAV-CA-sHB-EGF administration. This shows that HB-EGF ECD actively suppresses liver damage induction by AAV administration. This figure shows the protocol for a dose-dependence test using T1D model mice 14 days after STZ administration. This figure shows the dose-dependent suppression of blood glucose elevation by HB-EGF ECD. This figure shows the time course of casual blood glucose in individual mice administered AAV-CA-sHB-EGF at various doses. This figure shows the dose-dependent weight loss in mice after AAV administration. This figure shows the results of IPGTT after 7 days of treatment. Left: shows the change in blood glucose levels after glucose load. Right: shows the change in plasma insulin levels after glucose load. This figure shows the results of IPGTT after 14 days of treatment. Left: Changes in blood glucose levels after glucose loading. Right: Changes in plasma insulin levels after glucose loading. This shows the results of HE staining of the pancreas 14 days after treatment. Protection and proliferation of pancreatic islets in a dose-dependent manner by AAV-CA-sHB-EGF was confirmed. This shows the results of HE staining of the pancreas 14 days after treatment. Protection and proliferation of pancreatic islets in a dose-dependent manner by AAV-CA-sHB-EGF was confirmed.Figure 1 shows the results of measurements of casual blood glucose and plasma insulin levels 7 days after treatment. At all doses, AAV-CA-sHB-EGF administration significantly suppressed the rise in casual blood glucose compared to the untreated group (left). At all doses, plasma insulin levels were significantly lower compared to normal mice (right). Figure 2 shows the protocol for a dose-dependence study using normal mice. Figure 3 shows that AAV-CA-sHB-EGF administration does not affect the blood glucose levels of normal mice. Figure 4 shows that AAV-CA-sHB-EGF administration does not affect the body weight of normal mice. Figure 5 shows that AAV-CA-sHB-EGF administration does not affect the food intake of normal mice. Figure 6 shows that AAV-CA-sHB-EGF administration suppresses β-cell apoptosis in T1D model mice. The bottom right shows thymocytes (positive control). Figure 7 shows that AAV-CA-sHB-EGF-NK1 administration induces pancreatic tissue regeneration and increased blood flow in T1D model mice. This figure shows the details of RNA-seq and metabolome analysis. This figure shows that, according to RNA-seq principal component analysis, the sHB-EGF-introduced group has a profile that is significantly different from the intact group and the AAV-CA-Venus-administered group. This figure shows that the AAV-CA-sHB-EGF-administered group has elevated expression of genes involved in cell division and glycolysis regulation compared to the intact group and the AAV-CA-Venus-administered group. This figure shows that the AAV-CA-sHB-EGF-administered group has elevated glucose metabolism via glycolysis and the pentose phosphate cycle compared to the intact group and the AAV-CA-Venus-administered group.

[0017] 1. Therapeutic Agent (I) of the Present Invention 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." In this specification, when simply referring to "therapeutic agent of the present invention," this term is used to collectively refer to therapeutic agent (I) of the present invention and therapeutic agent (II) of the present invention described below).

[0018] As used herein, "treatment of diabetes" means at least suppressing hyperglycemia (reducing fasting blood glucose and casual blood glucose) compared to a control with diabetes who is not receiving therapeutic treatment. The reduction in blood glucose level is preferably statistically significant (e.g., p<0.05), but also includes cases where a tendency toward reduction is observed without a significant difference. In a preferred embodiment, a blood glucose lowering effect is achieved to an extent that is not significantly different from that of normal controls.

[0019] Preferably, 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.

[0020] Preferably, the treatment of diabetes in the present invention includes the protection and regeneration of β cells. Here, "protection and regeneration of β cells" means protecting (inducing anti-cell death) and / or proliferating (regenerating) the β cells of a diabetic subject (including both self-regeneration of existing β cells and differentiation from stem cells or endocrine precursor cells). The protection and regeneration of β 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.

[0021] Protection and regeneration of β cells improves insulin secretion. In one embodiment, "protection and regeneration of β cells" refers to the protection and regeneration of β cells that retain the ability to secrete insulin in response to glucose stimulation and suppress blood glucose elevation due to glucose loading, i.e., glucose-responsive insulin secretion. Based on the conventionally understood mechanism of action, even if β cell mass is maintained by protecting remaining β cells and / or differentiating or expanding new β cells, if the glucose-responsive insulin secretion ability of β cells is low, postprandial hyperglycemia cannot be suppressed and sufficient blood glucose control may not be achieved. However, since 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 is not essential in the present invention.

[0022] In the Examples described below, 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 due to the protection and regeneration of β-cells is relatively small, the difference in effect from the inventors' previous study using an Ad vector is thought to be not due to the difference in vector, but rather to the use of a nucleic acid encoding HB-EGF ECD instead of the full-length HB-EGF gene (which is expressed as membrane-bound pro-HB-EGF, then cleaved by protease and secreted as soluble HB-EGF). Therefore, the effects of the present invention can be 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.

[0023] (a) Therapeutic Agent of the Present Invention Comprising a Nucleic Acid Encoding HB-EGF ECD (I) The "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 double-stranded DNA is preferred. 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. Examples of DNA (RNA) encoding HB-EGF ECD include genomic DNA, cDNA (cRNA) derived from cells or tissues of humans or other mammals, and synthetic DNA (RNA).

[0024] The "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 the 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 an active form, which acts on nearby cells in a juxtacrine manner. A portion of this is further cleaved by a specific metalloproteinase in the juxtamembrane domain to produce soluble HB-EGF. In a preferred embodiment of the present invention, a nucleic acid encoding HB-EGF ECD that has entered the bloodstream by systemic administration is delivered to an organ, such as the liver, to which the nucleic acid or vector (e.g., a viral vector) is directed. Without wishing to be bound by any theory, it is believed that HB-EGF ECD secreted and expressed from liver cells is delivered to the liver via the bloodstream through autocrine or paracrine action, while HB-EGF ECD secreted and expressed from distant organs is delivered to the liver via the bloodstream and, through endocrine action, promotes glucose metabolism in the liver through signal transduction via EGF receptors on liver cells, thereby directly suppressing blood glucose elevation without the need for insulin secretion from the pancreas. Therefore, if a nucleotide sequence encoding at least soluble HB-EGF is included, it can be directly secreted and expressed as soluble HB-EGF by inserting it into a secretory expression vector containing an appropriate signal sequence. However, since the use of artificial protein fragments often results in the secretory expression mechanism not functioning properly due to misfolding or other reasons, those skilled in the art would likely choose to first express it as membrane-bound proHB-EGF and then use the action of endogenous proteases in the mammalian recipient to produce soluble HB-EGF. Furthermore, it has been reported that the C-terminal fragment (CTF) generated by ectodomain shedding of proHB-EGF translocates to the nucleus, where it binds to a repressor protein and releases gene transcriptional repression, and the possibility cannot be ruled out that the function of CTF contributes to the suppression of blood glucose elevation. In fact, the present inventors also used the full-length HB-EGF gene in previous studies using Ad vectors.On the other hand, in the present invention, by deliberately using a nucleic acid encoding HB-EGF ECD, which is subject to concerns about a decrease in secretory expression efficiency and is not expected to have the potential effect of CTF, we have unexpectedly succeeded in achieving a blood glucose elevation suppression effect far superior to that of the full-length HB-EGF gene. It is believed that HB-EGF ECD ultimately exhibits physiological activity as soluble HB-EGF without the propeptide. However, deletion of the propeptide-encoding region may result in a decrease in secretory expression efficiency. Therefore, in a preferred embodiment, the nucleic acid encoding HB-EGF ECD contains a nucleotide sequence encoding the propeptide in addition to a nucleotide sequence encoding the soluble HB-EGF. Furthermore, although the secretory efficiency can be improved by substituting a signal sequence other than the native signal sequence, this may also result in a decrease in secretory efficiency. Therefore, in a preferred embodiment, it is preferable to use a nucleic acid encoding the extracellular region of HB-EGF containing the native signal sequence (which may or may not contain the juxtamembrane domain, but preferably does not contain the juxtamembrane domain).

[0025] More specifically, examples of "nucleic acids encoding HB-EGF ECD" include nucleic acids that contain a nucleotide sequence that hybridizes under stringent conditions with a complementary strand sequence of the nucleotide sequence represented by SEQ ID NO: 1 [corresponding to the nucleotide sequence from positions 276 to 719 of the mRNA sequence of human HB-EGF registered in GenBank under Accession No. NM_001945 (positions 276 to 332 correspond to the signal codon, positions 333 to 461 correspond to the propeptide coding region, and positions 462 to 719 correspond to the soluble HB-EGF coding region)], and that encode a protein having activity equivalent to that of soluble HB-EGF (e.g., blood glucose elevation suppression activity, β-cell protective / regenerative activity). Examples of nucleic acids that hybridize under stringent conditions with the complementary strand sequence of the nucleotide sequence represented by SEQ ID NO: 1 (however, when the nucleic acid is RNA, "t" in the nucleotide sequence is read as "u") 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 with the nucleotide sequence represented by SEQ ID NO: 1. Furthermore, the nucleic acid encodes an amino acid sequence having about 90% or more, preferably about 95% or more, even more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more identity with the amino acid sequence represented by SEQ ID NO: 2, such that a protein comprising the amino acid sequence has substantially the same activity (e.g., blood glucose elevation suppression activity, β-cell protective and regenerative activity) as a protein comprising the amino acid sequence represented by SEQ ID NO: 2.

[0026] The identity of nucleotide sequences herein can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3). The identity of amino acid sequences herein can be calculated using the homology calculation algorithm NCBI BLAST under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

[0027] Hybridization can be carried out according to a method known per se or a method similar thereto, for example, the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using a commercially available library, hybridization can be carried out according to the method described in the accompanying instruction manual. Hybridization can be preferably carried out under stringent conditions.

[0028] Examples of stringent conditions include: (1) washing at low ionic strength and high temperature, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; and (2) reaction conditions characterized by the use of a denaturing agent such as formamide, for example, 50% (v / v) formamide at 42°C in 50 mM sodium phosphate buffer (pH 6.5) containing 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride, 75 mM sodium citrate. Alternatively, 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 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, followed by washing with 0.2x SSC and 50% formaldehyde at 55°C, followed by a high stringency wash consisting of 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 washing, the ionic strength of the buffer, etc., depending on factors such as probe length.

[0029] 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 [e.g., 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]. For example, it is desirable to use a nucleic acid encoding HB-EGF ECD derived from the mammal to which the agent of the present invention is to be administered. The mammal to which the agent of the present invention is to be administered is not particularly limited as long as it has diabetes, and examples include humans, mice, rats, rabbits, dogs, and monkeys, with humans being preferred. Thus, in a preferred embodiment, 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).

[0030] The nucleic acid encoding HB-EGF ECD can be cloned, for example, by amplifying it by PCR using synthetic DNA primers having a portion of the nucleotide sequence encoding the extracellular domain of the HB-EGF gene, or by hybridizing the DNA incorporated into an appropriate expression vector with a labeled DNA fragment or synthetic DNA containing the nucleotide sequence encoding HB-EGF ECD. Hybridization can be carried out, for example, according to the method described in Molecular Cloning, 2nd ed. (supra). When a commercially available library is used, hybridization can be carried out according to the method described in the instructions accompanying the library.

[0031] Furthermore, 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 allows for obtaining a codon-optimized sequence for expression in the target host cells, i.e., mammalian cells (preferably human cells) to be treated. During gene expression, converting the nucleotide sequence to codons frequently used in the host organism can be expected to increase protein expression levels. Data on codon usage in the host can be obtained, for example, from the genetic code usage database available 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. Alternatively, codon optimization can be performed using a known codon optimization algorithm (e.g., GeneArt Codon Optimizer). In such an algorithm, in addition to the frequency of codon usage in the host, multiple parameters can be taken into consideration, 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.

[0032] The nucleotide sequence of the DNA can be determined using a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.) or the like, according to a method known per se, such as the ODA-LA PCR method, the Gapped duplex method, or the Kunkel method, or a method similar thereto.

[0033] The cloned DNA can be used as is, or after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have a translation initiation codon ATG at its 5'-end and 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.

[0034] An expression vector containing a nucleic acid encoding HB-EGF ECD can be produced, for example, by directly cloning the nucleic acid or by excising a desired fragment from a nucleic acid encoding the CDS region of the HB-EGF gene and then ligating the DNA fragment downstream of a promoter in an appropriate expression vector. The expression vector is not particularly limited as long as it is one generally used in gene therapy. Examples of suitable expression vectors 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, as well as non-viral vectors such as plasmids for expression in animal cells (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo). From the viewpoints of high gene introduction and expression efficiency, low frequency of chromosomal integration with no risk of insertional mutagenesis, ability to introduce into non-dividing cells, and ability to express introduced genes 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.

[0035] Although the Ad vector's transgene expression period (usually 2-3 weeks) is shorter than that of AAV vectors or chromosomally integrated vectors, previous studies by the present inventors have shown that the pancreatic β-cell protective and regenerative effects of HB-EGF persist far beyond the gene expression period (at least 60 days, preferably 75 days, more preferably 90 days, and even more preferably 120 days), making it advantageous in that it can reduce or avoid the risk of side effects, such as carcinogenesis, that are associated with long-term HB-EGF expression. Furthermore, although the gene size that can be carried by an AAV vector is small at 4.7 kb, the coding sequence of the HB-EGF ECD is 444 bp, and the entire expression cassette, including the promoter, terminator, etc., is approximately 1-2 kb, making it suitable for use.

[0036] In the therapeutic agent (I) of the present invention, HB-EGF ECD not only protects and regenerates β cells, but also directly promotes glucose metabolism in the liver, thereby suppressing blood glucose elevation without inducing insulin secretion. Ad vectors are known to accumulate in the liver, and HB-EGF ECD introduced into liver cells can act on the liver in an autocrine or paracrine manner. Furthermore, extracellularly secreted HB-EGF ECD can also be delivered to the pancreas via the bloodstream, contributing to the protection and regeneration of β cells. Meanwhile, AAV vectors have different tissue tropism depending on their serotype. Examples of serotypes that are tropic to the liver include types 1-3 and types 6-10. However, if a ubiquitous promoter is used, 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 is no particular limitation on the serotype used. When efficient delivery to both the liver and pancreas is desired, it is preferable to use a serotype that is tropic to both organs, such as AAV8.

[0037] A further advantage of expressing HB-EGF ECD in the liver is that HB-EGF ECD acts on the liver in an autocrine or paracrine manner, resulting in a hepatoprotective effect and significantly suppressing the adverse event of hepatotoxicity caused by the administration of Ad vectors or AAV vectors. The greatest problem with gene therapy using Ad vectors or AAV vectors is the occurrence of adverse events, including fatal accidents due to hepatotoxicity, so the therapeutic agent (I) of the present invention is extremely useful in that it solves this problem and can be used safely.

[0038] The promoter may be any promoter appropriate for the host used to express the gene. For example, in the case of mammalian cells, 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) may be used. p5 promoter), adenovirus (AdV)-derived promoters (e.g., Ad2 or Ad5 major late promoter), viral promoters such as the β-actin gene promoter, the phosphoglycerate kinase (PGK) gene promoter, the transferrin gene promoter, and other mammalian constitutive protein gene promoters can be used.

[0039] When using a viral vector as a vector, administration at a low dose is desirable to reduce the risk of adverse events. However, to achieve the desired therapeutic effect at a low dose, it is necessary to incorporate a nucleic acid encoding HB-EGF ECD downstream of a promoter with transcriptional activity capable of conferring therapeutically effective blood HB-EGF ECD levels. For example, a promoter with stronger transcriptional activity than the CMV promoter or RSV promoter frequently used in viral vector-based gene therapy can be used. Examples of such highly active promoters include the CA promoter and promoters with equivalent transcriptional activity, such as the elongation factor 1α1 (EF1A) promoter, the elongation factor 1α1 short (EFS) promoter, the CBh promoter (a hybrid promoter consisting of a CMV immediate-early enhancer and a modified chicken β-actin promoter different from the CA promoter), the spleen-limited focal 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.

[0040] Furthermore, when a viral vector with high tissue tropism is used, a promoter that is highly expressed in a tissue- or cell-specific manner in the target organ can also be used (for example, an albumin promoter, an α-fetoprotein promoter, a thyroxine-binding globulin promoter, etc. in the liver; an insulin promoter, a Pdx1 promoter, an Ins2 promoter, etc. in pancreatic β cells; a myogenin promoter, a skeletal muscle actin α1 (ACTA1) promoter, an MHCK7 promoter, an SM22a promoter, etc. in muscle; but is not limited to these, and includes a tissue- or cell-specific promoter of any organ from which HB-EGF ECD secreted and expressed can be delivered to the liver or pancreas via the bloodstream).

[0041] 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, the vector may further contain an enhancer, a splicing signal, a WPRE sequence, a 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.

[0042] If necessary, 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. For example, an insulin signal sequence, an α-interferon signal sequence, or an antibody molecule signal sequence can be used.

[0043] 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) (2001); Culture of Animal Cells; A Manual of Basic Technique, R. Freshney eds. , 2nd ed. (1987), Wiley-Liss; Frank L. Graham, Manipulation of adenovirus vector, Chapter 11. p109-p128;E. J. Murray eds. , Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols (1991); Chen, S-H. et al. , Combination gene therapy for liver metastases of colon carcinoma in vivo. , Proc. Natl. Acad. Sci. USA (1995) 92, 2477-2581 etc.].

[0044] When the nucleic acid encoding HB-EGF ECD is single-stranded RNA, the therapeutic agent (I) of the present invention can be provided as a so-called mRNA drug. For example, 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.

[0045] Alternatively, 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. More specifically, 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 linked to the phage promoter is obtained by using the expression vector as a template and performing PCR with a primer 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. In this case, by adding pseudouridine (Ψ) or N1-methylpseudouridine (N1mΨ) triphosphate instead of UTP as an RNA monomer (NTP) to the reaction solution, the resulting mRNA will contain Ψ or N1mΨ instead of U. This makes it possible to avoid attacks by natural immunity against the mRNA, improving mRNA stability and translation efficiency. Similarly, modified NTPs with base substitutions that have been reported to avoid natural immunity (e.g., inosine triphosphate instead of ATP, 5-methylcytidine triphosphate instead of CTP, etc.) can also be used for other NTPs.

[0046] It is also desirable to add a 5'-cap structure and a polyA tail, which are necessary for stabilizing mRNA and improving translation efficiency. The 5'-cap structure can be added by adding a Cap 0 structure using a capping enzyme after mRNA synthesis, and then converting it to a Cap 1 structure using an mRNA 2'-O-methyltransferase. Alternatively, an RNA cap analog (e.g., 3'-O-Me-m) can be used. ７ G(5')ppp(5')G,m ７ G(5')ppp(5')G,3'-O-Me-m ７ G(5') ppp(5') A, m ７By adding a poly(5')ppp(5')A or other polynucleotides to the transcription reaction solution, transcription and 5'-capping can be performed simultaneously. Poly(A) tailing can also be added to the 3' end using poly(A) polymerase after mRNA synthesis, or can be performed simultaneously with the transcription reaction by adding a poly(A) sequence to the transcription template in advance. The mRNA obtained as described above can be purified by removing the template DNA using DNase I, for example, by the LiCl method.

[0047] (b) Therapeutic Agent (I) of the Present Invention Comprising an HB-EGF ECD Fragment The HB-EGF ECD fragment used in the therapeutic agent (I) of the present invention comprises at least a soluble HB-EGF portion, but does not comprise a transmembrane domain or a cytoplasmic domain. It may or may not comprise 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 one, it will be cleaved and removed together with the prosequence by a protease endogenous to the subject to administration.

[0048] More specifically, an "HB-EGF ECD fragment" includes a polypeptide comprising 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 No. NP_001936 (the amino acid sequence from amino acid numbers 20 to 62 corresponds to the propeptide region, and the amino acid sequence from amino acid numbers 63 to 148 corresponds to the soluble HB-EGF region)], or an amino acid sequence substantially identical thereto, and having activity equivalent to that of soluble HB-EGF (e.g., blood glucose elevation suppression activity, β-cell protection and regeneration activity). Here, "substantially the same amino acid sequence" refers to an amino acid sequence that has about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more identity to a sequence comprising 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. Alternatively, a "substantially identical amino acid sequence" may be an amino acid sequence in which one to several (2, 3, 4, 5) amino acids have been substituted, deleted, inserted, or added in a sequence containing 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. Alternatively, a "substantially identical amino acid sequence" also includes the corresponding sequences of orthologs in other mammals of 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 [for example, mouse, rat, bovine, porcine, or Chinese hamster orthologs registered in GenBank under Accession Numbers NP_034545, NP_037077, XP_601210, NP_999464, AAD52998, and NP_990180, respectively].

[0049] The C-terminus of the HB-EGF ECD fragment may be any of a carboxyl group, carboxylate, amide, or ester. When the HB-EGF ECD has a carboxyl group or carboxylate at a site other than the C-terminus, the carboxyl group may be amidated or esterified. Furthermore, the HB-EGF ECD fragment also includes those in which the amino group of the N-terminal amino acid residue is protected with a protecting group, those in which the N-terminal glutamine residue is pyroglutamated, those in which substituents on the side chains of amino acids in the molecule are protected with an appropriate protecting group, and conjugated peptides such as so-called glycopeptides to which a sugar chain is bound.

[0050] HB-EGF ECD may be in the free form or in the form of a salt, including, for example, physiologically acceptable salts with acids (e.g., inorganic acids, organic acids) or bases (e.g., alkali metals), with physiologically acceptable acid addition salts being particularly preferred. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

[0051] The HB-EGF ECD fragment can be obtained by culturing mammalian cells or tissues expressing HB-EGF in an appropriate medium, removing the cells by filtration, centrifugation, or the like, and subjecting the resulting culture supernatant to chromatography, such as reverse-phase chromatography, ion exchange chromatography, or affinity chromatography. The HB-EGF ECD fragment can also be produced according to known peptide synthesis methods. The peptide synthesis method may be, for example, either solid-phase synthesis or liquid-phase synthesis. The desired polypeptide can be produced by condensing a partial peptide or amino acid that can constitute HB-EGF ECD with the remaining portion, and, if the product has a protecting group, removing the protecting group.

[0052] The HB-EGF ECD fragment thus obtained can be purified by known purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, a combination of these, etc. When the polypeptide obtained by the above method is in a free form, it can be converted into an appropriate salt by known methods or methods similar thereto, and when it is obtained as a salt, the salt can be converted into a free form or another salt by known methods or methods similar thereto.

[0053] In a preferred embodiment, the HB-EGF ECD fragment can be produced by culturing a transformant containing a nucleic acid encoding it, and isolating and purifying the HB-EGF ECD fragment from the resulting culture. The DNA encoding HB-EGF ECD can be the DNA described in detail in (a) above. The DNA may have a translation initiation codon ATG at its 5'-end and 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.

[0054] An expression vector containing DNA encoding HB-EGF ECD can be produced, for example, by excising a desired DNA fragment from DNA encoding HB-EGF and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. Examples of 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 λ phage, insect virus vectors such as baculovirus (e.g., BmNPV, AcNPV), retroviruses, and animal virus vectors such as vaccinia virus, adenovirus, and adeno-associated virus.

[0055] The promoter may be any promoter suitable for the host used to express the gene. For example, when the host is an animal cell, the promoters exemplified in (a) above can be used in the same way. When the host is Escherichia coli, the trp promoter, lac promoter, recA promoter, λP promoter, etc. can be used. Ｌ promoter, lpp promoter, T7 promoter, etc. are preferred. When the host is Bacillus subtilis, SPO1 promoter, SPO2 promoter, penP promoter, etc. are preferred. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferred. When the host is insect cells, polyhedrin promoter, P10 promoter, etc. are preferred.

[0056] In addition to the above, the expression vector may contain, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin, etc. Examples of the selection marker include the dihydrofolate reductase (dhfr) gene, ampicillin resistance (Amp ｒ ) gene, neomycin resistance (Neo ｒ ) gene, etc. Furthermore, if necessary, 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. When the host is Escherichia coli, a PhoA signal sequence, an OmpA signal sequence, etc. may be used; when the host is Bacillus subtilis, an α-amylase signal sequence, a subtilisin signal sequence, etc. may be used; when the host is yeast, an MFα signal sequence, an SUC2 signal sequence, etc. may be used; and when the host is an animal cell, a native HB-EGF signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc. may be used.

[0057] 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), Virology, 52: 456 (1973). The resulting transformant is cultured, and the HB-EGF ECD fragment can be isolated and purified from the culture by a method known per se.

[0058] Furthermore, the HB-EGF ECD fragment can also be synthesized by in vitro translation using the RNA encoding it as a template in a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, or the like. Alternatively, the fragment can be synthesized using a cell-free transcription / translation system further comprising RNA polymerase, with DNA encoding the HB-EGF ECD as a template. A commercially available cell-free protein transcription / translation system can be used, or it can be prepared by a known method, specifically, in the case of E. coli lysate, the system can be prepared according to the method described in Pratt, JM et al., Transcription and Translation, Hames, BD and Higgins, SJ eds., IRL Press, Oxford 179-209 (1984).

[0059] (c) Formulation and Administration of Therapeutic Agent (I) of the Present Invention The therapeutic agent (I) of the present invention can suppress hyperglycemia for a long period of time by a mechanism that does not involve the induction of insulin secretion. Therefore, it can be used to treat T1D and other diabetes requiring insulin administration due to destruction of pancreatic β cells (e.g., T2D in which insulin resistance progresses, causing β cells to become exhausted due to compensatory excessive insulin secretion, leading to impaired insulin secretion and ultimately β cell death), and to suppress progression to complications.

[0060] As the therapeutic agent (I) of the present invention, (a) the nucleic acid / expression vector encoding HB-EGF ECD or (b) the HB-EGF ECD fragment of the present invention may be used as is, or, if necessary, may be mixed with a pharmacologically acceptable carrier to prepare various formulations such as injections and then used as a medicine.

[0061] Here, various organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used as pharmacologically acceptable carriers, and are incorporated into liquid preparations as solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. Furthermore, pharmaceutical additives such as preservatives, antioxidants, and coloring agents can also be used as needed.

[0062] Preferable examples of 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.

[0063] Suitable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0064] Suitable examples of 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.

[0065] Suitable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, and the like.

[0066] Suitable examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0067] Suitable examples of soothing agents include benzyl alcohol.

[0068] Suitable examples of the preservative include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0069] Suitable examples of antioxidants include sulfites and ascorbic acid salts.

[0070] Suitable examples of the coloring agent include water-soluble food tar dyes (e.g., food dyes such as Food Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos. 1 and 2), water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), and natural dyes (e.g., β-carotene, chlorophyll, red iron oxide, etc.).

[0071] 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. Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are 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.

[0072] When the active ingredient in the therapeutic agent (I) of the present invention is a nucleic acid encoding HB-EGF ECD and is in the form of a non-viral expression vector or single-stranded RNA, in one embodiment, the nucleic acid may be in the form of a pharmaceutical composition encapsulated in a liposome. Liposomes are small, closed vesicles having an internal phase surrounded by one or more lipid bilayers, and can typically hold a water-soluble substance in the internal phase and a lipid-soluble substance within the lipid bilayer. Here, the term "encapsulated" 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, within 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.

[0073] 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 method, freeze-thaw method, and remote loading method, and any known method can be appropriately selected.

[0074] In one embodiment, the nucleic acid encoding HB-EGF ECD is encapsulated in a lipid nanoparticle as a carrier. As used herein, "lipid nanoparticle" refers to a particle having a membrane structure in which the hydrophilic groups of an amphipathic lipid are aligned toward the aqueous phase at the interface, and having a particle diameter of less than 1 μm. "Amphipathic lipid" refers to a lipid having both hydrophilic and hydrophobic groups.

[0075] The particle size of the lipid nanoparticles used in the present invention is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. Measurement of particle size can be performed using a particle size distribution analyzer such as a Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted depending on the method for producing the lipid nanoparticles. In this specification, "particle size" refers to the average particle size (zeta mean) measured by dynamic light scattering.

[0076] Examples of amphipathic lipids include cationic lipids, ionic lipids, phospholipids, PEG lipids, etc. As used herein, "cationic lipid" refers to a lipid having a constantly positively charged hydrophilic group. As used herein, "ionic lipid" refers to a lipid that is neutral at physiological pH but becomes positively charged by protonation at low pH. As used herein, "PEG" refers to polyethylene glycol, and "PEG lipid" refers to a lipid modified with PEG, i.e., a lipid to which PEG is bound.

[0077] In a preferred embodiment, the lipid nanoparticles encapsulating a nucleic acid encoding HB-EGF ECD include lipid nanoparticles containing: (A) a cationic lipid or an ionic lipid, (B) a phospholipid, (C) a steroid, and (D) a PEG lipid. Each of the component lipids (A) to (D) includes known lipids that are commonly used in lipid nanoparticles, and a person skilled in the art can easily select an appropriate type of lipid and its composition.

[0078] The therapeutic agent (I) of the present invention containing a nucleic acid encoding HB-EGF ECD can be produced by a method commonly used in the pharmaceutical technology field, for example, the method described in the Japanese Pharmacopoeia. When the nucleic acid is carried by a viral vector, the content of the viral vector, which is the active ingredient in the formulation, varies depending on the dosage form, the dosage of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight. The viral titer is, for example, 10 １０ ~10 １１ pfu / ml (Physical titer is several to 100 times higher than biological titer, so it is equivalent to 2 x 10 virus particles. １０~2 x 10 １３ The concentration can be appropriately adjusted to about 1000 ppm (vp / ml), but is not limited to this range.

[0079] The dosage of the formulation varies depending on the type of vector, promoter activity, administration route, severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc. of the administered subject. For example, when an AAV vector is used as the HB-EGF expression vector, a relatively low dose (1.3 × 10 １４ Since deaths associated with serious adverse events in the liver and hepatobiliary tract have been reported in the group administered 1 × 10 viral particles (vp) / kg body weight (Lancet Neurology, 2023; 22: 1125-39), a low dose (1 × 10 １４ vp / kg body weight) group did not experience any adverse events in the liver despite having liver disease. １４ It is desirable to administer at a dose of about 5 x 10 vp / kg body weight or less. ９ ~Approx. 5×10 １３ vp / kg body weight, preferably about 1 x 10 １０ ~Approx. 1×10 １３ vp / kg body weight. In addition, when an Ad vector is used as the HB-EGF ECD expression vector, past clinical trials have shown that the １１ vp / kg body weight (total 3.8 × 10 １３ There have been reported cases of death due to acute liver damage following hepatic arterial administration of Ad vectors (Vp) (Mol Genet Metab 2003; 80: 148-158). However, since Ad vectors have a high tendency to accumulate in the liver, it is thought that a higher dose of Ad vector accumulates in the liver when administered via the hepatic artery. Therefore, when Ad vectors are administered systemically, particularly via a peripheral vein, the dose should be approximately 10 times higher, at 5 × 10 １２ It is estimated that a dose of up to about 5 x 10 vp / kg body weight can be safely used. ９ ~Approx. 5×10 １２ vp / kg body weight, preferably about 1 x 10 １０ ~Approx. 2×10 １２ vp / kg body weight.

[0080] For example, in the example described below, about 5×10 １２ The excellent efficacy of HB-EGF ECD gene therapy for T1D was demonstrated by tail vein administration of an AAV vector at 1000 vp / kg body weight. 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 of suppressing blood glucose elevation, but 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 will have a sufficient effect of suppressing blood glucose elevation by administering an even lower dose. Furthermore, even in T1D model mice 14 days after STZ administration in which β-cell destruction had progressed further, approximately 5 x 10 １２ Administration of the HB-EGF ECD expression vector at a dose of vp / kg body weight reduced blood glucose levels to the same level as in normal mice. Surprisingly, such a significant effect in suppressing blood glucose elevation was achieved without inducing an increase in plasma insulin concentration.

[0081] On the other hand, when a non-viral vector is used as an HB-EGF ECD expression vector encapsulated in liposomes, safety was confirmed when 666 μg of DNA was administered intravenously in a clinical study using cynomolgus monkeys weighing approximately 4 kg, and this amount serves as a guideline. For example, a single dose for an adult is approximately 2 to approximately 10 mg, preferably approximately 5 to approximately 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 pharmaceuticals).

[0082] The therapeutic agent (I) of the present invention containing a nucleic acid encoding HB-EGF ECD is preferably administered systemically parenterally (e.g., intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, etc.) using, for example, an injection, a catheter, a balloon catheter, etc. Here, "systemic administration" refers to administration methods such as intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, and intraperitoneal administration, as long as the nucleic acid encoding HB-EGF ECD in the administered formulation circulates throughout the body via the bloodstream and is delivered to a target organ, and HB-EGF ECD produced and secreted from the nucleic acid acts on its own or nearby cells in an autocrine or paracrine manner, or circulates throughout the body via the bloodstream and acts on the organ at the target site in an endocrine manner, thereby contributing to the suppression of blood glucose elevation. Alternatively, the therapeutic agent may be administered by endoscopic injection into the portal vein or artery (e.g., hepatic artery, pancreatic artery, renal artery).

[0083] In a study in which an Ad vector encoding HB-EGF was administered locally into a T1D model mouse via retrograde intraductal injection (Kozawa, J. et al., Pancreas, 31: 32-42 (2005)), the authors speculated that the reason for using gene therapy rather than HB-EGF protein injection for HB-EGF treatment of diabetes was the short duration of biological activity of HB-EGF protein and its likely inactivation by proteases in pancreatic juice. They suggested that the autocrine or juxtacrine action of membrane-bound proHB-EGF expressed in pancreatic duct cells was crucial for the differentiation or neogenesis of beta cells from pancreatic duct cells. In addition, the paracrine effect of soluble HB-EGF secreted from pancreatic duct cells protected and regenerated the remaining beta cells in the islets. In contrast, previous studies by the present inventors 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 β cells through endocrine action. Furthermore, based on the suggestion of Kozawa et al., we hypothesized that by using a vector tropic to the pancreas as well as the liver, the paracrine action of HB-EGF ECD secreted and expressed from pancreatic cells might further enhance the protective and regenerative effects of HB-EGF on β cells. However, although we confirmed that nucleic acid encoding HB-EGF ECD was delivered and expressed in both the liver and pancreas and indeed exerted protective and regenerative effects on β cells, HB-EGF ECD alone was unable to increase plasma insulin concentrations after glucose loading. Nevertheless, HB-EGF ECD alone reduced blood glucose levels in T1D model mice to levels comparable to those in normal mice. This suggests a novel and original therapeutic strategy for diabetes, in which enhancing signal transduction via the HB-EGF receptor directly promotes glucose metabolism in the liver and suppresses blood glucose elevation, without inducing insulin secretion.

[0084] When the viral vector used in the present invention is used at a relatively high dose within its dose range, in order to avoid liver damage, which is a particularly important adverse event caused by the viral vector, it is desirable to avoid administration routes that deliver a high dose of the viral vector to the liver, such as administration via the hepatic artery, and to administer 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.

[0085] Therapeutic agent (I) of the present invention comprising a nucleic acid encoding HB-EGF ECD may be administered locally, for example, by direct injection into a target organ (e.g., liver, pancreas, kidney) endoscopically or during open abdominal surgery (e.g., pancreas or pancreatic islet transplantation for T1D).

[0086] 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). Furthermore, even when an Ad vector is used, prior research using full-length HB-EGF has demonstrated that a single administration alone can suppress hyperglycemia to a certain extent over a long period of at least 70 days. Further follow-up observations of the mice in the examples are ongoing, and it is fully expected that the above-mentioned effects will continue for an even longer period. Therefore, 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. Depending on the type of viral vector used, 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.

[0087] Therapeutic agent (I) of the present invention containing an HB-EGF ECD fragment can preferably be formulated as an injectable solution, similar to a nucleic acid encoding HB-EGF ECD. Alternatively, a sustained-release formulation can be prepared using a biocompatible material such as collagen. For example, since Pluronic gel gels at body temperature and remains liquid at lower temperatures, local injection of the HB-EGF ECD fragment together with Pluronic gel to allow it to gel around the target tissue can provide a long-lasting release, 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. Alternatively, the HB-EGF ECD fragment and a pharmacologically acceptable carrier can be lyophilized and stored in a state that requires only dissolution or suspension in an appropriate sterile vehicle immediately before use.

[0088] Antibodies against surface molecules of target cells (e.g., liver 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). Examples of surface molecules of liver cells include, but are not limited to, EGFR (ErbB1 / HER1), ErbB2 / HER2, ErbB3 / HER3, and ErbB4 / HER4. When using antibodies against EGFR or HER4, it is desirable to use a non-neutralizing antibody as the targeting antibody so as not to inhibit signal transduction from the receptor. In some cases, it may be more preferable to use an agonistic antibody, as a potential synergistic effect with the HB-EGF ECD fragment can be expected. 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 the surface molecule of the target cell, such as Fab, F(ab') ２ , ScFv, minibody, etc. When considering administration to humans, the antibody is preferably a chimeric antibody between a human and another animal (e.g., mouse), a humanized antibody, or a fully human antibody.

[0089] Methods for cross-linking an HB-EGF ECD fragment with an antibody against a surface molecule of a target cell include, but are not limited to, the method described in Adv. Drug Deliv. Rev., 53: 171-216 (2001).

[0090] The therapeutic agent (I) of the present invention containing an HB-EGF ECD fragment can be produced by a method commonly used in the pharmaceutical technology field, such as the method 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, about 0.1 to 100% by weight.

[0091] 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, and local administration (e.g., direct administration to a target organ during endoscopic or laparotomy surgery).

[0092] The dosage of the therapeutic agent (I) of the present invention containing an HB-EGF ECD fragment varies depending on the dosage form, molecular weight of the active ingredient, route of administration, severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc. of the recipient. For example, when administered intravenously as an injection solution, the daily amount of the active ingredient for an adult is typically in the range of about 0.05 to about 50 mg / kg, preferably about 0.1 to about 20 mg / kg, and can be administered in a single dose or in several divided doses. In the case of local administration, such as direct injection into a target organ, the dosage can be reduced. Furthermore, in the case of sustained-release preparations such as hydrogels or immunoconjugates with targeting antibodies, the dosage can be reduced and / or the administration interval can be extended.

[0093] (d) Combination Use with HGF or a Fragment Thereof, or a Nucleic Acid Encoding the Same The present invention also provides a therapeutic agent for diabetes in mammals, comprising a combination of HB-EGF ECD or a nucleic acid encoding it with HGF or a fragment thereof, or a nucleic acid encoding it (i.e., therapeutic agent (I) of the present invention used in combination with HGF or a fragment thereof, or a nucleic acid encoding it; hereinafter, may be referred to as the "combination agent of the present invention").

[0094] The HB-EGF ECD and the nucleic acid encoding it may be the polypeptide and nucleic acid described in detail above in (b) and (a), respectively.

[0095] HGF is a heterodimer consisting of a 69 kD α-chain and a 34 kD β-chain, and the α-chain has four characteristic structures (K1 to K4) called hairpin and kringle domains from the N-terminus. Meanwhile, the receptor that transmits HGF signals is the Met receptor tyrosine kinase, and the region within the HGF molecule that is responsible for specific binding to Met is the N-terminal hairpin followed by one or two kringle domains (K1, K2) (HGF fragments up to the K1 domain are called NK1, and HGF fragments up to the K2 domain are called NK2).

[0096] The HGF or a fragment thereof used in the combination drug of the present invention may be full-length HGF or any HGF fragment that can bind to Met and act as an agonist having cell proliferation-promoting activity. Examples of such fragments include NK1 and NK2 fragments, and preferably NK1 fragments.

[0097] More specifically, examples of "HGF or a fragment thereof" include a polypeptide comprising at least the amino acid sequence represented by amino acid numbers 1 to 178 in the amino acid sequence represented by SEQ ID NO: 4 [corresponding to the amino acid sequence from amino acid numbers 32 to 728 of the amino acid sequence of human HGF registered in GenBank under Accession No. NP_000592 (positions 32 to 209 correspond to NK1, and positions 32 to 290 correspond to NK2)], or an amino acid sequence substantially identical thereto, and having activity equivalent to that of full-length HGF (e.g., blood glucose elevation suppression activity when used in combination with HB-EGF ECD, glucose-stimulating insulin secretion-stimulating activity, and β-cell protection and regeneration activity). Here, "substantially the same amino acid sequence" refers to an amino acid sequence that has about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more identity to a sequence comprising at least the amino acid sequence represented by amino acid numbers 1 to 178 in the amino acid sequence represented by SEQ ID NO: 4. Alternatively, a "substantially identical amino acid sequence" may be an amino acid sequence in which one to several (2, 3, 4, 5) amino acids have been substituted, deleted, inserted, or added in a sequence containing at least the amino acid sequence represented by amino acid numbers 1 to 178 in the amino acid sequence represented by SEQ ID NO: 4. Alternatively, a "substantially identical amino acid sequence" also encompasses the corresponding sequences of orthologs in other mammals of at least the amino acid sequence represented by amino acid numbers 1 to 178 in the amino acid sequence represented by SEQ ID NO: 4.

[0098] HGF or a fragment thereof may be modified or protected with a protecting group at the N-terminus, C-terminus, and / or side chains in the same manner as exemplified for HB-EGF ECD. HGF or a fragment thereof may be in the free form or in the form of a salt, and examples of the salt include the same salts as exemplified for HB-EGF ECD.

[0099] HGF can be obtained by culturing mammalian cells or tissues expressing HGF in an appropriate medium, removing the cells by filtration or centrifugation, and subjecting the resulting culture supernatant to chromatography, such as reverse-phase chromatography, ion exchange chromatography, or affinity chromatography. HGF fragments can be obtained by treating the resulting HGF with an appropriate protease. HGF or fragments thereof can also be produced by known peptide synthesis methods. Peptide synthesis methods may be either solid-phase synthesis or liquid-phase synthesis. The desired polypeptide can be produced by condensing a partial peptide or amino acid that can constitute HGF or a fragment thereof with the remaining portion, and, if the product contains a protecting group, removing the protecting group. HGF or a fragment thereof thus obtained can be purified by known purification methods, as exemplified for HB-EGF ECD, and can be converted from the free form to a salt, or from the salt to the free form or another salt, by conventional methods.

[0100] In a preferred embodiment, 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 for gene therapy described below. The DNA may have a translation initiation codon ATG at its 5'-end and 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.

[0101] An expression vector containing DNA encoding HGF or a fragment thereof can be produced, for example, by excising a desired DNA fragment from DNA encoding HGF and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. The expression vector and promoter may be the same as those exemplified for HB-EGF ECD.

[0102] In addition to the above, expression vectors that contain an enhancer, splicing signal, polyA addition signal, selection marker, SV40 replication origin, etc., can be used as desired. Selection markers include those similar to those exemplified for HB-EGF ECD. Furthermore, if necessary, a nucleotide sequence encoding a signal sequence (signal codon) suited to the host may be added to the 5' end of DNA encoding HGF or a fragment thereof. Examples of signal sequences include those similar to those exemplified for HB-EGF ECD.

[0103] The host cells can be transformed by a method known per se, similar to that exemplified for HB-EGF ECD. The resulting transformant is cultured, and HGF or a fragment thereof can be isolated and purified from the culture by a method known per se.

[0104] Furthermore, HGF or a fragment thereof can be synthesized by in vitro translation using RNA encoding it as a template in a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, Escherichia coli lysate, or the like. Alternatively, HGF or a fragment thereof can be synthesized using a cell-free transcription / translation system further comprising RNA polymerase, with DNA encoding HGF or a fragment thereof as a template. The cell-free protein transcription / translation system can be the same as that exemplified for HB-EGF ECD.

[0105] Examples of the "nucleic acid encoding HGF or a fragment thereof" to be used in the combination drug of the present invention 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 β-cell protective and regenerative activity). Examples of 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. Here, the term "stringent conditions" has the same meaning as in the case of the nucleic acid encoding HB-EGF ECD. Furthermore, the nucleic acid encodes an amino acid sequence that has about 90% or more, preferably about 95% or more, more preferably about 97% or more, and particularly preferably about 98% or more identity to a sequence containing at least the amino acid sequence represented by amino acid numbers 1 to 178 in the amino acid sequence represented by SEQ ID NO: 4, and a protein containing the amino acid sequence has substantially the same activity (e.g., blood glucose elevation suppression activity when used in combination with HB-EGF ECD, glucose-responsive insulin secretion-stimulating activity, β-cell protection and regeneration activity) as a protein containing a sequence containing at least the amino acid sequence represented by amino acid numbers 1 to 178 in the amino acid sequence represented by SEQ ID NO: 4.

[0106] The nucleic acid encoding HGF or a fragment thereof may be an ortholog in a non-human mammal of a nucleic acid comprising a nucleotide sequence represented by nucleotide numbers 1 to 627 in the nucleotide sequence represented by SEQ ID NO: 3. For example, it is desirable to use a nucleic acid encoding HGF or a fragment thereof derived from the mammal to which the combination drug of the present invention is to be administered. The mammal to which the combination drug of the present invention is to be administered is not particularly limited as long as it has diabetes, and examples include humans, mice, rats, rabbits, dogs, and monkeys, with humans being preferred. Thus, in a preferred embodiment, the nucleic acid encoding HGF or a fragment thereof is a nucleic acid encoding human HGF (i.e., a protein consisting of the amino acid sequence represented by SEQ ID NO: 4) or a fragment thereof, preferably an NK1 fragment (i.e., a polypeptide fragment consisting of the amino acid sequence represented by amino acid numbers 1 to 178 in the amino acid sequence represented by SEQ ID NO: 4).

[0107] A nucleic acid encoding HGF or a fragment thereof can be cloned in the same manner as the nucleic acid encoding HB-EGF ECD, and can be inserted downstream of the promoter of various viral or non-viral vectors similar to those described above. A nucleic acid encoding HGF or a fragment thereof may be inserted into a single expression vector together with a nucleic acid encoding HB-EGF ECD, or each may be inserted into a separate expression vector. When a nucleic acid encoding HB-EGF ECD and a nucleic acid encoding HGF or a fragment thereof are inserted into a single expression vector, they may be placed under the control of a single promoter, or each may be placed under the control of a separate promoter, which may be the same or different. In the former case, either the nucleic acid encoding HB-EGF ECD or the nucleic acid encoding HGF or a fragment thereof can be placed upstream (closer to the promoter), but a sequence enabling dicistronic expression in mammalian cells (e.g., an IRES sequence, a 2A sequence (P2A, T2A, E2A, F2A)) is inserted between the two nucleic acids. When the nucleic acid encoding HB-EGF ECD and the nucleic acid encoding HGF or a fragment thereof are inserted into separate expression vectors, the types of the expression vectors may be the same or different, and both nucleic acids may be under the control of the same or different promoters.

[0108] When the nucleic acid encoding HGF or a fragment thereof is single-stranded RNA, the nucleic acid can be provided as a so-called mRNA drug. For example, the mRNA can be obtained by introducing the expression vector into a suitable host (e.g., mammalian cells), culturing the cells, recovering mRNA using a method known per se (e.g., the LiCl method), and purifying the mRNA encoding HGF or a fragment thereof. Alternatively, the mRNA can be obtained by excising the nucleic acid encoding HGF or a fragment thereof (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 HGF or a fragment thereof using a known in vitro transcription system. More specifically, mRNA encoding HGF or a fragment thereof can be obtained by a method similar to that described in detail for the mRNA encoding HB-EGF ECD in (a) above.

[0109] A nucleic acid encoding HGF or a fragment thereof, when used in combination with a nucleic acid encoding HB-EGF ECD, can suppress hyperglycemia over a long period of time while maintaining normal insulin secretion, and can therefore be used to treat T1D and other diabetes conditions in which pancreatic β cells are destroyed and require insulin administration (e.g., T2D in which insulin resistance progresses, causing β cells to become exhausted due to compensatory excessive insulin secretion, leading to impaired insulin secretion and ultimately β cell death), and to suppress progression to complications.

[0110] The combination agent of the present invention may be prepared by mixing (a1) a nucleic acid / expression vector encoding HB-EGF ECD or (b1) an HB-EGF ECD fragment of the present invention with (a2) a nucleic acid / expression vector encoding HGF or a fragment thereof or (b2) an HGF or a fragment thereof, as is, or may be used as a pharmaceutical after being mixed with a pharmacologically acceptable carrier to form various formulations such as injections, if necessary. The combination may be any of (a1) and (a2), (a1) and (b2), (b1) and (a2), or (b1) and (b2), with (a1) and (a2) or (b1) and (b2) being preferred. In the former case, when the nucleic acid encoding HB-EGF ECD and the nucleic acid encoding HGF or a fragment thereof are inserted into separate expression vectors, the combination agent of the present invention may be formulated as a single pharmaceutical composition containing both expression vectors, or each expression vector may be formulated separately and used in combination. When each expression vector is formulated separately, a formulation containing an expression vector comprising a nucleic acid encoding HB-EGF ECD can be formulated in the same manner as in (a) above and administered by the same administration route. Furthermore, when a nucleic acid encoding HB-EGF ECD and a nucleic acid encoding HGF or a fragment thereof are inserted into a single expression vector, a formulation containing the expression vector can be formulated in the same manner as in (a) above and administered by the same administration route, dosage, and frequency. Furthermore, in the latter case, the HB-EGF ECD fragment and HGF or a fragment thereof may be formulated as separate pharmaceutical compositions, or as a single pharmaceutical composition containing both polypeptides.

[0111] When HB-EGF ECD or a nucleic acid encoding it and HGF or a fragment thereof or a nucleic acid encoding it are formulated separately in the combination drug of the present invention, HGF or a fragment thereof, or the nucleic acid / expression vector encoding it are formulated by blending various organic or inorganic carrier substances commonly used as formulation materials with the following agents in liquid formulations: solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. In addition, formulation additives such as preservatives, antioxidants, and coloring agents can also be used as needed. As the solvents, suspending agents, isotonicity agents, buffers, soothing agents, preservatives, antioxidants, and coloring agents, those exemplified above in (c) can be preferably used, respectively.

[0112] 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. Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are 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.

[0113] When one active ingredient in the combination drug of the present invention is a nucleic acid encoding HGF or a fragment thereof and is in the form of a non-viral expression vector or single-stranded RNA, in one embodiment, the nucleic acid may be in the form of a pharmaceutical composition encapsulated in a liposome. The liposomes used and the method for encapsulating the nucleic acid in the liposome may be the same as those described above for HB-EGF ECD. In another embodiment, the nucleic acid encoding HGF or a fragment thereof may be encapsulated in a lipid nanoparticle, using the particle as a carrier. Examples of lipid nanoparticles used include those similar to those described above for HB-EGF ECD.

[0114] A formulation containing an expression vector containing a nucleic acid encoding HGF or a fragment thereof can be produced by a method commonly used in the pharmaceutical technology field, such as the method described in the Japanese Pharmacopoeia. When the nucleic acid is carried by a viral vector, the content of the viral vector, which is the active ingredient in the formulation, varies depending on the dosage form, the dosage of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight. The viral titer is, for example, 10 １０ ~10 １１ pfu / ml (Physical titer is several to 100 times higher than biological titer, so it is equivalent to 2 x 10 virus particles. １０ ~2 x 10 １３ The concentration can be appropriately adjusted to about 1000 ppm (vp / ml), but is not limited to this range.

[0115] When the combination agent of the present invention is provided in the form of a separate formulation for each polypeptide or nucleic acid / expression vector encoding same, the two formulations may be mixed at the time of use and administered as a single pharmaceutical composition, or may be administered as separate formulations simultaneously or at different times by the same or different administration routes. When HGF or a fragment thereof or a nucleic acid / expression vector encoding same is administered as a separate formulation from HB-EGF ECD or a nucleic acid / expression vector encoding same, the administration form may be the same as the administration form described above for HB-EGF ECD or a nucleic acid / expression vector encoding same.

[0116] The dosage of the combination drug of the present invention in which each expression vector is formulated separately varies depending on the type of vector, promoter activity, administration route, severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc. of the administered animal. For example, when an AAV vector is used as the HB-EGF ECD expression vector and the expression vector for HGF or a fragment thereof, the total amount of both expression vectors is, for example, about 5 × 10 ９ ~Approx. 5×10 １３ vp / kg body weight, preferably about 1 x 10 １０ ~Approx. 1×10 １３When Ad vectors are used as the HB-EGF ECD expression vector and the expression vector for HGF or a fragment thereof, the total amount of both expression vectors is, for example, about 5 × 10 ９ ~Approx. 5×10 １２ vp / kg body weight, preferably about 1 x 10 １０ ~Approx. 2×10 １２ vp / kg body weight. On the other hand, when non-viral vectors are used as the HB-EGF ECD expression vector and the HGF or a fragment thereof expression vector encapsulated in liposomes, the total amount of both expression vectors is, for example, about 2 to about 10 mg, preferably about 5 to about 8 mg, per dose for an adult. The same can be said for the case where mRNA encoding HB-EGF ECD and mRNA encoding HGF or a fragment thereof are used.

[0117] The quantitative ratio of the two nucleic acids / expression vectors in the combination agent of the present invention, in which each nucleic acid / expression vector is formulated separately, is not particularly limited as long as the desired therapeutic effect (e.g., suppression of fasting and postprandial hyperglycemia, induction of glucose-responsive insulin secretion) is achieved. For example, the ratio of nucleic acid / expression vector encoding HB-EGF ECD to nucleic acid / expression vector encoding HGF or a fragment thereof can be appropriately selected within the range of 10:1 to 1:10, preferably 5:1 to 1:5, and more preferably 2:1 to 1:2.

[0118] The combination agent of the present invention containing HGF or a fragment thereof can be preferably formulated as an injectable solution, similar to a nucleic acid encoding HGF or a fragment thereof. Alternatively, it can be made into a sustained-release preparation using a biocompatible material such as collagen, or into an immunoconjugate with an antibody against a surface molecule of a target cell (e.g., liver cell). Specific examples include formulation methods similar to those exemplified for the therapeutic agent (I) of the present invention containing HB-EGF ECD.

[0119] The combination drug of the present invention containing HGF or a fragment thereof can be produced by a method conventionally used in the pharmaceutical technology field, for example, the method described in the Japanese Pharmacopoeia. The content of HGF or a fragment thereof in the formulation varies depending on the dosage form, the dose of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight.

[0120] The combination agent of the present invention containing HGF or a fragment thereof 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, and local administration (e.g., direct administration to a target organ under endoscopic or laparotomy).

[0121] The dosage of the combination agent of the present invention containing HGF or a fragment thereof varies depending on the dosage form, molecular weight of the active ingredient, route of administration, severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc. of the administered subject. For example, when administered intravenously as an injection solution, the daily amount of the active ingredient for an adult is typically in the range of about 0.05 to about 50 mg / kg, preferably about 0.1 to about 20 mg / kg, and this amount can be administered in a single dose or in divided doses. In the case of local administration, such as direct injection into a target organ, the dosage can be reduced. Furthermore, in the case of sustained-release preparations such as hydrogels or immunoconjugates with targeting antibodies, the dosage can be reduced and / or the administration interval can be extended.

[0122] The therapeutic agent (I) of the present invention can be used in combination with existing diabetes treatment methods. For example, in the case of T1D, the therapeutic agent (I) of the present invention can be used in combination with insulin therapy. Insulin can be administered at the dosage and administration conventionally used for the treatment of T1D. Alternatively, when the therapeutic agent (I) of the present invention contains an HB-EGF ECD fragment (and HGF or a fragment thereof) as an active ingredient, it can be formulated as a combination preparation with an insulin preparation, or can be formulated separately to form a kit preparation, as long as they do not adversely affect each other. The therapeutic agent (I) of the present invention can also be used in combination with pancreas or pancreatic islet transplantation therapy. In particular, when the therapeutic agent (I) of the present invention contains a nucleic acid encoding HB-EGF ECD (and a nucleic acid encoding HGF or a fragment thereof) as an active ingredient, the therapeutic agent (I) of the present invention can be directly administered to a target organ (e.g., liver, pancreas, kidney) during laparotomy, as described above. On the other hand, in the case of T2D, in addition to insulin, oral medications such as biguanides, thiazolidine derivatives, sulfonylureas, rapid-acting insulin secretagogues, mitochondrial function improvers, α-glucosidase inhibitors, DPP-4 inhibitors, GLP-1 receptor agonists, and SGLT2 inhibitors can also be used in combination. These oral medications can be administered in the dosages and regimens conventionally used for the treatment of T2D.

[0123] 2. Therapeutic Agent (II) of the Present Invention The present invention is based on the surprising discovery by the present inventors that the use of a nucleic acid encoding HB-EGF ECD instead of the full-length HB-EGF gene in HB-EGF gene therapy for diabetes exhibits a markedly more effective inhibitory effect on blood glucose elevation than the use of the full-length HB-EGF gene. This means that therapeutic effects for diabetes can be achieved not only by exogenously supplementing HB-EGF ECD using gene therapy (including mRNA pharmaceuticals) or protein preparations, but also by increasing the amount of soluble HB-EGF endogenous in a mammal with diabetes to be treated, thereby enhancing HB-EGF receptor-mediated signaling, or by enhancing HB-EGF receptor-mediated signaling independently of soluble HB-EGF.

[0124] Therefore, the present invention also provides a therapeutic agent for diabetes (hereinafter also referred to as "therapeutic agent (II) of the present invention") comprising a substance that enhances signal transduction mediated by an HB-EGF receptor. HB-EGF binds to EGFR (ErbB1) and ErbB4 of the ErbB family as receptors, and binds not only to their homodimers but also to heterodimers of either of them with other ErbB family members. As used herein, "HB-EGF receptor" encompasses any receptor as long as HB-EGF binds to it and causes downstream signal transduction.

[0125] As used herein, "signal transduction mediated by the HB-EGF receptor" refers to signal transduction that occurs upon binding of soluble HB-EGF to the receptor, or signal transduction that is substantially the same as that. "Substantially the same signal transduction" means that signal transduction from the HB-EGF receptor includes, among downstream signal transduction that occurs as a result of soluble HB-EGF binding to the receptor, signal transduction that contributes to suppression of blood glucose elevation; as long as the activated receptor transmits such a signal, binding of the ligand, soluble HB-EGF, to the receptor is not necessarily required. For example, this includes signal transduction mediated by an HB-EGF receptor agonist, activation of the receptor not mediated by a ligand / agonist (i.e., EGFR transactivation), etc.

[0126] Among substances that enhance signal transduction mediated by the HB-EGF receptor, substances that increase the amount of soluble HB-EGF can be broadly divided into substances that increase the amount of soluble HB-EGF by enhancing HB-EGF expression and substances that increase the amount of soluble HB-EGF by promoting ectodomain shedding of proHB-EGF. Substances that enhance HB-EGF expression include, for example, nucleic acids encoding HB-EGF (preferably, nucleic acids encoding HB-EGF ECD). In the present invention, supplementation of the HB-EGF ECD fragment itself (protein preparations) is also considered to fall within the category of enhancing HB-EGF expression. On the other hand, substances that enhance endogenous HB-EGF expression include, for example, transactivators that bind to the regulatory region of the HB-EGF gene and activate its transcription, substances that stabilize HB-EGF mRNA (e.g., miRNAs that target the 3'-UTR), and substances that increase the efficiency of translation from HB-EGF mRNA. Such substances can be screened, for example, by culturing cells that naturally express HB-EGF in the presence and absence of a test substance and comparing the expression levels of HB-EGF. The expression level of HB-EGF can be assayed at the transcription level using Northern blot or RT-PCR, or at the translation level by immunoassay using an anti-HB-EGF antibody or the like.

[0127] In a preferred embodiment, the substance that enhances HB-EGF receptor-mediated signal transduction is a substance that increases the amount of soluble HB-EGF by promoting proHB-EGF ectodomain shedding. Examples of such substances include: (a) a protease having proHB-EGF ectodomain shedding activity or a nucleic acid encoding the same; (b) a factor that induces proHB-EGF ectodomain shedding; or (c) a protein that interacts with the protease of (a) or a nucleic acid encoding the same.

[0128] (a) Protease Having ProHB-EGF Ectodomain Shedding Activity or Nucleic Acid Encoding the Same Examples of proteases having proHB-EGF ectodomain shedding activity include, but are not limited to, ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, ADAM19, etc. of the ADAM family, and MMP7, MT1-MMP, etc. of the matrix metalloproteinase (MMP) family.

[0129] The amino acid sequences of these proteases and the nucleotide sequences of the genes encoding them are both publicly known, and a person skilled in the art can produce these proteases or nucleic acids encoding them based on this sequence information, in the same manner as described in detail for the HB-EGF ECD fragment or nucleic acid encoding it in Section 1 above, formulate them into pharmaceutical compositions, and administer them to a mammal with diabetes.

[0130] Alternatively, the amount of soluble HB-EGF can also be increased by enhancing the expression of a protease that has the activity of shedding the ectodomain of endogenous proHB-EGF. Examples of such substances include transactivators that bind to the regulatory region of the protease gene to activate its transcription, substances that stabilize the mRNA of the protease (e.g., miRNA that targets the 3'-UTR), and substances that increase the translation efficiency from the mRNA of the protease. Such substances can be screened, for example, by culturing cells that naturally express the protease in the presence and absence of a test substance and comparing the expression levels of the protease.

[0131] (b) Factors that induce proHB-EGF ectodomain shedding Factors that induce proHB-EGF ectodomain shedding include substances that activate the proteases. Examples of such substances include GPCR ligands, calcium ionophores, and growth factors (which activate the Ras-ERK pathway to induce shedding), protein kinase C (PKC) activators (which activate PKC to induce shedding), and cellular stress factors (which activate the p38 MAP kinase pathway to induce shedding).

[0132] Examples of GPCR ligands include, but are not limited to, lysophosphatidic acid (LPA), endothelin, angiotensin II, phenylephrine, carbachol, bombesin, thrombin, estrogen, α2-adrenergic receptor agonists, and epoxyeicosatrienoic acids, as long as they can activate the Ras-ERK pathway and induce proHB-EGF shedding. Many of these GPCR ligands are commercially available as pharmaceuticals or cosmetic materials and can be used safely.

[0133] Examples of calcium ionophores include, but are not limited to, (-)-(R,R)-N,N'-bis-[11-(methoxycarbonyl)undecyl]-N,N',4,5-tetramethyl-3,6-dioxaoctane-diamide, N,N,N',N'-tetra[cyclohexyl]diglycolic acid diamide, and N,N-dicyclohexyl-N',N'-dioctadecyl-oxapentanediamide.

[0134] Examples of growth factors include insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the like, but are not limited thereto, as long as they can activate the Ras-ERK pathway and induce proHB-EGF shedding.

[0135] The amino acid sequences of these growth factors and the nucleotide sequences of the genes encoding them are all publicly known, and based on this sequence information, a person skilled in the art can produce these growth factors or nucleic acids encoding them, formulate them into pharmaceutical compositions, and administer them to a mammal with diabetes, in the same manner as described in detail for the HB-EGF ECD fragment or nucleic acid encoding it in Section 1 above.

[0136] Examples of PKC activators include phorbol esters such as phorbol 12-myristate 13-acetate (PMA, TPA), phorbol 12,13-dibutyrate, 1,2-dioctanoyl-sn-glycerol, bryostatin 1, and 1-oleoyl-2-acetyl-sn-glycerol, but are not limited to these, as long as they can activate the p38 MAP kinase pathway and induce proHB-EGF shedding.

[0137] Examples of cellular stress factors include, but are not limited to, inflammatory cytokines (e.g., interleukin-1β (IL-1β), IL-8, etc.), reactive oxygen species (ROS) (e.g., nitric oxide, etc.), and hypotonic solutions (e.g., hypotonic electrolyte infusion, etc.), as long as they can induce proHB-EGF shedding.

[0138] The amino acid sequences of the inflammatory cytokines and the nucleotide sequences of the genes encoding them are both publicly known, and a person skilled in the art can produce these inflammatory cytokines or nucleic acids encoding them based on the sequence information in the same manner as described in detail for the HB-EGF ECD fragment or nucleic acid encoding it in 1. above, formulate them into pharmaceutical compositions, and administer them to a mammal with diabetes.

[0139] (c) Proteins that interact with the protease of (a) or nucleic acids encoding the same. Examples of proteins that interact with the protease having proHB-EGF ectodomain shedding activity include, but are not limited to, Eve-1, PACSIN3, Src, PKC-δ, Grb2, phosphatidylinositol 3-kinase (PI3K), endoflin, SH3Px1, and Fish.

[0140] The amino acid sequences of these proteins and the nucleotide sequences of the genes encoding them are both publicly known, and a person skilled in the art can produce these proteins or nucleic acids encoding them based on this sequence information, formulate them into pharmaceutical compositions, and administer them to a mammal with diabetes, in the same manner as described in detail for the HB-EGF ECD fragment or nucleic acid encoding it in Section 1 above.

[0141] The substances (b) or (c) directly or indirectly activate proteases with proHB-EGF ectodomain shedding activity, promoting the secretion of soluble HB-EGF and thereby enhancing HB-EGF receptor-mediated signal transduction and activating the downstream Ras-ERK pathway. This activation not only induces further shedding but also induces the expression of HB-EGF itself, forming a positive feedback loop. Thus, the HB-EGF receptor is primarily transactivated by GPCR ligands and the like, regardless of soluble HB-EGF, but receptor activation by soluble HB-EGF, which occurs secondarily after shedding, plays a major role in EGFR transactivation and the formation of a positive feedback loop. However, the HB-EGF receptor can be activated without the binding of soluble HB-EGF and can transmit downstream signals substantially identical to those resulting from the binding of the ligand. Such receptor activation not mediated by HB-EGF binding is achieved by activation by an agonist of the receptor or by EGFR transactivation not mediated by proHB-EGF ectodomain shedding. Thus, in one embodiment, the therapeutic agent (II) of the present invention contains (d) an agonist or transactivator of the HB-EGF receptor as a substance that enhances signal transduction via the HB-EGF receptor.

[0142] (d) HB-EGF Receptor Agonists or Transactivators Examples of HB-EGF receptor agonists include EGF family members whose receptors are EGFR / ErbB1 and / or ErbB4, such as betacellulin, epiregulin, EGF, transforming growth factor-α (TGF-α), amphiregulin, neuregulin 3 (NRG3), and NRG4, as long as they induce signal transduction substantially identical to that induced by the binding of HB-EGF. In another embodiment, 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 containing an HB-EGF ECD fragment. Since the receptor tyrosine kinase can be activated by dimerization of ErbB family member molecules, a complete antibody molecule or a bivalent antibody fragment (e.g., Fab, F(ab') ２ In order to comprehensively cover the desired signal transduction in the present invention and to address various heterodimers, 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) may be used in combination with an anti-ErbB1 antibody and an anti-ErbB4 antibody.

[0143] Alternatively, 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 of the receptor), or activation (phosphorylation) of a kinase molecule activated downstream thereof as indicators.

[0144] Examples of transactivators of the HB-EGF receptor include calcium ionophore, PKC, Src, PYK2, and ROS. These substances are also factors that 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 act directly on the HB-EGF receptor without the action of soluble HB-EGF.

[0145] Of the above-mentioned HB-EGF receptor agonists or transactivators, the amino acid sequences of the proteinaceous factors and the nucleotide sequences of the genes encoding them are both publicly known, and a person skilled in the art can produce these proteins or nucleic acids encoding them based on the sequence information in the same manner as described in detail above for the HB-EGF ECD fragment or nucleic acid encoding it in 1., formulate them into pharmaceutical compositions, and administer them to a mammal with diabetes.

[0146] 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. Examples of means for enhancing HB-EGF receptor-mediated signaling include administering to the mammal an effective amount of the various active ingredients of the therapeutic agent (II) of the present invention, as detailed in Section 2 above. In another embodiment, HB-EGF receptor-mediated signaling can also be enhanced by subjecting the mammal to cellular stress, for example, by irradiation with 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.

[0147] The present invention will be described in more detail below by showing examples, but these are merely illustrative and do not limit the scope of the present invention in any way. In the following examples and drawings, HB-EGF ECD may be referred to as sHB-EGF.

[0148] 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 a cytomegalovirus immediate early enhancer and a modified chicken β-actin promoter. AAV vectors expressing two types of genes ((1) and (2)) had a P2A peptide sequence inserted between them. 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

[0149] The HB-EGF ECD (sHB-EGF) used was a sequence (444 bp) encoding amino acids 1-148 of HB-EGF, including the N-terminal signal peptide and propeptide. In the construction of AAV vectors, the packaging size (the gene size that can be inserted between the 5' ITR and 3' ITR) is limited to 4,700 bp, and 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. As a control, 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.

[0150] 2. Pharmacological Efficacy Test The protocol for the pharmacological efficacy test is shown in Figure 2. Male c57BL / 6N mice (Kyudo Co., Ltd., Tosu, Japan) weighing 18-20 g and aged 8 weeks were housed with free access to food and water. As previously reported (Diabetes 2010; 59: 1261-1265), streptozotocin (STZ; Sigma-Aldrich Japan, Tokyo) dissolved in 0.01 M citrate buffer (pH 4.5) was intraperitoneally administered at a dose of 50 mg / kg to 55 mice once daily for 5 consecutive days (days -7 to -3). The STZ-injected mice were randomly divided into four groups 1 to 7 days after STZ administration (day 0), and received a single injection of the following AAV vector via the tail vein: (1) 1.0 x 10 １１ vg AAV-CA-Venus (n=11) (2) 1.0×10 １１ vg AAV-CA-sHB-EGF (n=13) (3) 1.0×10 １１ vg AAV-CA-sHB-EGF-NK1 (n=13) (4) 1.0×10 １１ vg AAV-CA-NK1-sHB-EGF (n=11) (5) 1.0×10 １１ vg AAV-CA-NK1 (n = 7). Mice (n = 10) that received neither STZ injection nor AAV gene therapy were used as normal controls (intact). 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. From day 7 to day 35, blood was collected weekly 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, mice were intraperitoneally administered 2 g / kg body weight of glucose. Blood was collected immediately before administration (0 min), and 30, 60, and 120 min after administration. 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 approved by the Kagoshima University Animal Experiment Ethics Committee. Biochemical analyses were performed using the following equipment and reagents. Blood glucose level: Medisafe Fit Pro II (Terumo, Tokyo) Urine glucose: Uropaper III (Eiken Chemical Co., Ltd., Tokyo) Plasma insulin level: ELISA assay (Morinaga Co., Ltd., Yokohama)

[0151] 3. Statistical Analysis Data were presented as mean ± standard error (s.e.). Multiple comparisons were tested by one-way analysis of variance (one-way ANOVA), and the presence or absence of statistically significant differences between mice administered with AAV-CA-Venus or normal mice and mice 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.

[0152] (Experimental Results) 1. Dramatic Blood Glucose Lowering by HB-EGF ECD and Combined Use of HB-EGF ECD and NK1 Casual blood glucose levels in the untreated group (AAV-CA-Venus-administered group) rose rapidly to approximately 400 mg / dl on the seventh day after vector administration (14 days after STZ injection), and continued to rise gradually thereafter, remaining at high levels of 400 mg / dl or higher. 1.0 x 10 １１ The group treated with a single tail vein administration of AAV-CA-sHB-EGF, AAV-CA-sHB-EGF-NK1, or AAV-CA-NK1-sHB-EGF (group expressing HB-EGF ECD) suppressed the rise in blood glucose levels by day 7 (Figure 3). The therapeutic effect continued even after 4 weeks of follow-up, demonstrating a long-term therapeutic effect (Figure 3).

[0153] 2. Suppression of blood glucose rise in IPGTT by HB-EGF ECD and combined use of HB-EGF ECD and NK1 IPGTT was performed 14 and 28 days after treatment to confirm the suppression of blood glucose rise after glucose loading and glucose-responsive insulin secretion ability. １１Groups treated with AAV-CA-sHB-EGF, AAV-CA-sHB-EGF-NK1, and AAV-CA-NK1-sHB-EGF suppressed blood glucose elevation after glucose loading (Figure 4, left; Figure 5, left). Regarding insulin secretion, the groups treated with AAV-CA-sHB-EGF-NK1 and AAV-CA-NK1-sHB-EGF showed increased insulin secretion in response to glucose stimulation (Figure 4, right; Figure 5, right). Thus, coexpression of HB-EGF ECD and NK1 induced glucose-responsive insulin secretion.

[0154] 3. Normalization of casual blood glucose levels and maintenance of casual plasma insulin levels by HB-EGF ECD and combined use of HB-EGF ECD and NK1 Casual blood glucose levels and plasma insulin levels were measured 7 and 16 days after treatment. １１ The random blood glucose levels in the groups treated with AAV-CA-sHB-EGF, AAV-CA-sHB-EGF-NK1, and AAV-CA-NK1-sHB-EGF at 1.0 × 10 ng / mL were significantly lower than those in the untreated group (Fig. 6, left). １１ Increases in vg were confirmed in the groups treated with AAV-CA-sHB-EGF-NK1, AAV-CA-NK1-sHB-EGF, and AAV-CA-NK1 (Fig. 6, right). These effects, taken together with the IPGTT test results, suggest that co-expression of HB-EGF ECD and NK1 affects glucose-responsive insulin secretion.

[0155] 4. Protection and proliferation of β cells by HB-EGF ECD and the combination of HB-EGF ECD and NK1. Mice were dissected 14 days after treatment, and the pancreases were collected. The pancreases were fixed in 4% paraformaldehyde / phosphate buffer and then embedded in paraffin. Immunostaining was performed on sections prepared. Anti-Insulin Guinea Pig-Poly (cat no. GTX27842, GNT) was used as the antibody. The results confirmed the presence of β cells in the pancreases of the groups treated with AAV-CA-sHB-EGF, AAV-CA-sHB-EGF-NK1, and AAV-CA-NK1-sHB-EGF (Figure 7). These results suggest that HB-EGF ECD and the combination of HB-EGF ECD and NK1 protect and proliferate β cells.

[0156] 5. Highly efficient gene transfer to the liver, pancreas, and kidney via intravenous administration: 1.0 x 10 １１ The gene transfer efficiency of this vector was confirmed using the liver, pancreas, and kidneys 10 days after administration of AAV-CA-Venus (vg). Image analysis of OCT frozen sections of the liver, pancreas, and kidney confirmed fluorescent images of the Venus gene in all organs (Figure 8). These results confirmed that genes transferred using this vector, which expresses therapeutic genes under the transcriptional control of the CA promoter using AAV8, are expressed highly efficiently in the liver, pancreas, and kidney.

[0157] 6. Effect of gene therapy on severe liver damage HE staining of liver tissue on day 10 of treatment confirmed that no severe liver damage had occurred in the livers of any group (Figure 9). Furthermore, biochemical tests (AST and ALT) were performed on day 14 after AAV vector administration to normal mice, and the results showed that 1.0 × 10 １１ It was also confirmed that the dose of 100 mg vg was a safe amount that did not cause viral hepatitis (Figure 10).

[0158] These results suggest that HB-EGF ECD gene therapy not only lowers blood glucose via insulin in the pancreas, but also has a new mechanism that directly promotes glucose metabolism in the liver. Furthermore, it was confirmed that the combined use of HB-EGF EGF and NK1 not only promotes glucose metabolism but also induces insulin secretion.

[0159] Example 2 (Experimental Method) 1. Dose-Dependence Test The protocol for the dose-dependence test is shown in Figure 11. Two tests were conducted simultaneously: one to examine the therapeutic effect on STZ-induced T1D mice in which STZ-induced β-cell destruction had progressed, and the other to examine dose-dependence. As in the pharmacological efficacy test, male c57BL / 6N mice (Kyudo Co., Ltd., Tosu, Japan) weighing 18-20 g and aged 8 weeks were housed with free access to food and water. STZ (Sigma-Aldrich Japan, Tokyo, Japan) dissolved in 0.01 M citrate buffer (pH 4.5) was intraperitoneally administered to the mice at a dose of 50 mg / kg once daily for five consecutive days (from day -14 to day -10). The STZ-injected mice were selected 14 days after STZ administration (day 0), and mice with casual blood glucose levels of 250 mg / dl or higher were randomly divided into five groups and administered a single dose of the following AAV vector via the tail vein: (1) 1.0 × 10 １１ vg AAV-CA-Venus (n=5) (2) 1.0×10 １１ vg AAV-CA-sHB-EGF (n=5) (3) 3.0×10 １０ vg AAV-CA-sHB-EGF (n=5) (4) 1.0×10 ９ vg AAV-CA-sHB-EGF (n=5) (5) 3.0×10 ８vg AAV-CA-sHB-EGF (n=5). Mice (n=5) that did not receive STZ injection or AAV gene therapy were used as normal controls (Intact). Blood was collected from all mice, including those in the normal group. Blood glucose and body weight measurements were performed on day -14, daily from day -7 to day 7, and once a week from day 14 to day 35. Blood was collected once a week from day 7 to day 35. All animal experiments were performed in accordance with the guidelines of the National Institutes of Health and were approved by the Kagoshima University Animal Experiment Ethics Committee. Biochemical analyses were measured using the following equipment and reagents: Blood glucose level: Medisafe Fit Pro II (Terumo, Tokyo); Plasma insulin level: ELISA assay (Morinaga Co., Yokohama); Liver function (AST and ALT): SPOTCHEM SP-4430 clinical autometer (Arkray, Kyoto).

[0160] 2. Statistical Analysis Data are presented as mean ± standard error (s.e.). Multiple comparisons were tested by one-way analysis of variance (one-way ANOVA), and the presence or absence of statistically significant differences between mice administered with AAV-CA-Venus or normal mice and mice administered with AAV-CA-sHB-EGF at four doses was determined by Student's t-test. P < 0.05 was defined as statistically significant.

[0161] (Experimental Results) 1. HE staining of pancreas collected from days 1 to 14 after STZ administration HE staining was performed on pancreas collected from days 1 to 14 after STZ administration. The results confirmed that destruction of pancreatic islets was more advanced in the pancreas collected from days 14 to 7 compared to the pancreas collected from days 7 to 14. In a dose-dependent study, treatment of STZ-induced T1D mice in which STZ-induced destruction of β cells had progressed further was verified.

[0162] 2. Dose-dependent suppression of blood glucose elevation 1.0 x 10 １１ The casual blood glucose level of mice administered with 1.0×10 AAV-CA-Venus rose rapidly to approximately 500 mg / dl on the seventh day after vector administration (21 days after STZ injection from the first day), and continued to rise gradually thereafter, remaining at a high level of 500 mg / dl or higher. １１In the group treated with a single tail vein administration of 1000mg AAV-CA-sHB-EGF, blood glucose levels were suppressed to a non-significant level compared to normal mice by day 7, normalizing blood glucose levels and demonstrating a dose-dependent hypoglycemic effect. The therapeutic effect continued over a four-week follow-up period, demonstrating long-term therapeutic efficacy (Figure 12). Thus, HB-EGF ECD gene therapy demonstrated excellent therapeutic efficacy even in T1D mice with advanced disease and fewer β cells. Furthermore, no hypoglycemia was observed in any individual mice during the observation period (Figure 13).

[0163] 3. Weight gain / loss after treatment Weight loss after treatment tended to decrease in a dose-dependent manner (Figure 14). No difference was observed in the physical activity of the mice compared to the normal mouse group over a 5-week follow-up period.

[0164] 4. Dose-dependent suppression of blood glucose rise after glucose loading IPGTT was performed 7 and 14 days after treatment to confirm the inhibitory effect on blood glucose rise after glucose loading and plasma insulin levels. As a result, AAV-CA-sHB-EGF dose-dependently suppressed blood glucose rise after glucose loading (Figure 15, left, Figure 16). Meanwhile, plasma insulin levels were 1.0 x 10 １１ In the vg-treated group, blood glucose levels were elevated compared to the control (Venus) before glucose loading, but decreased after glucose loading at all doses (Figure 15, right; Figure 16, right). These results suggest that the inhibitory effect of AAV-CA-sHB-EGF on blood glucose levels after glucose loading is independent of insulin.

[0165] 5. Confirmation of the protective and proliferative effects of pancreatic islets by HE staining of the pancreas 14 days after treatment HE staining was performed using the pancreas 14 days after treatment. As a result, the dose-dependent protective and proliferative effects of AAV-CA-sHB-EGF on pancreatic islets were confirmed (Figures 17-1 and 17-2).

[0166] 6. Dose-dependent suppression of casual blood glucose levels Casual blood glucose and plasma insulin levels were confirmed 7 days after treatment. Casual blood glucose levels in all treatment groups significantly suppressed blood glucose elevation compared to the untreated group (Fig. 18, left). Furthermore, 1.0 x 10 １１It was also confirmed that casual blood glucose levels in the groups administered sHB-EGF vg were no different from those in normal mice. Plasma insulin levels were significantly lower in all groups administered AAV-CA-sHB-EGF gene therapy compared to normal mice. These results strongly suggest that casual blood glucose elevation was suppressed in advanced T1D mice despite the low beta cell mass, and that this is a non-insulin-mediated hypoglycemic effect.

[0167] Example 3 (Experimental Method) The protocol for the dose-dependency test in normal mice is shown in Figure 19. Male c57BL / 6N mice (Kyudo Co., Ltd., Tosu) weighing 18-20 g and aged 10 weeks were kept with free access to food and water. The mice were randomly divided into five groups, and the following AAV vectors were administered once via the tail vein: (1) 1.0 x 10 １１ vg AAV-CA-Venus (n=4) (2) 1.0×10 １１ vg AAV-CA-sHB-EGF (n=4) (3) 3.0×10 １０ vg AAV-CA-sHB-EGF (n=4) (4) 1.0×10 ９　 vg AAV-CA-sHB-EGF (n=4) (5) 3.0×10 ８　 vg AAV-CA-sHB-EGF (n=4). Mice (n=4) not receiving AAV gene therapy were used as controls (intact). Blood and urine were collected from all mice, including those in the intact group. Blood glucose, body weight, and food intake measurements were performed once a week from day 0 to day 35. In addition to blood glucose, body weight, and food intake measurements, blood samples were collected once a week. All animal experiments were performed in accordance with the guidelines of the National Institutes of Health and were approved by the Kagoshima University Animal Experiment Ethics Committee. Biochemical analyses were performed using the following equipment and reagents: Blood glucose level: Medisafe Fit Pro II (Terumo, Tokyo); Urine glucose: Uropaper III (Eiken Chemical Co., Ltd., Tokyo); Plasma insulin level: ELISA assay (Morinaga Co., Ltd., Yokohama).

[0168] Statistical Analysis Data are presented as mean ± standard error (s.e.). Multiple comparisons were tested by one-way analysis of variance (one-way ANOVA), and the presence or absence of statistically significant differences between AAV-CA-Venus-administered mice or normal mice and mice administered Ad.CA-sHB-EGF at four doses was determined by Student's t-test. P < 0.05 was defined as statistically significant.

[0169] (Results) Casual blood glucose, body weight, and food intake of normal mice No significant differences were observed in casual blood glucose (Figure 20), body weight (Figure 21), or food intake (Figure 22) between the AAV-CA-Venus-administered group, the AAV-CA-sHB-EGF-administered group, and the intact group during any observation period. These results suggest that AAV administration or sHB-EGF gene introduction does not affect glucose metabolism under normal conditions.

[0170] Example 4 AAV was administered to STZ-T1D mice prepared using the same protocol as in FIG. 11, and pathological tissue analysis was performed using pancreatic tissue collected on day 14 of treatment.

[0171] 1. Inhibitory Effect of sHB-EGF on β-Cell Apoptosis Dissected tissue samples were fixed overnight at room temperature using 4% PFA (Nacalai). Fixed samples were embedded in paraffin using HistoCore PEARL (Leica Biosystems). Immunostaining specimens were prepared at Biopathology Research Institute, Inc. As a result, in the AAV-CA-Venus group, β-cells were lost due to STZ-induced apoptosis (TUNEL- / Insulin-), whereas in the AAV-CA-sHB-EGF group, TUNEL- / Insulin+ β-cells survived (Figure 23). These results suggest that AAV-CA-sHB-EGF treatment inhibits STZ-induced β-cell fragmentation, or apoptosis.

[0172] 2. Induction of pancreatic tissue regeneration and increased blood flow by sHB-EGF Figure 24 shows the pancreas and surrounding tissue collected 10 days after administration of AAV-CA-sHB-EGF-NK1 to STZ-T1D mice. The mouse pancreas is typically located along the stomach, spleen, and small intestine, and is characterized by not displaying a distinct morphology like other organs. The pancreas 10 days after administration of AAV-CA-sHB-EGF-NK1 exhibited a distinct morphology, with clear vascularization of the splenic artery and pancreatic artery. This condition was not observed in mice administered AAV-CA-NK1. It has been reported that advanced angiogenesis is important for the differentiation and growth of pancreatic islets. These results suggest that AAV-CA-sHB-EGF treatment induces pancreatic tissue regeneration and increased blood flow.

[0173] Example 5 RNA-seq and metabolome analysis were carried out as shown in FIG.

[0174] 1. RNA-seq Analysis Principal component analysis was performed to visualize the degree of similarity in gene expression between samples in RNA-seq. Results confirmed that the degree of similarity was significantly different in the sHB-EGF-introduced group compared to the AAV-CA-Venus (untreated control) group and the intact (normal mouse) group (Figure 26). Gene expression analysis identified genes highly expressed in the AAV-CA-sHB-EGF group. Comparisons between the intact and AAV-CA-Venus groups revealed significantly elevated expression of genes involved in glycolysis, cell cycle, and proliferation in both groups (Figure 27).

[0175] 2. Metabolomic analysis Metabolomic analysis also confirmed an increased tendency for glycolysis and the pentose phosphate cycle (Figure 28).

[0176] As described above, the results of RNA-seq and metabolome analysis of liver tissue confirmed that activation of glucose metabolism was induced in the livers of the sHB-EGF-injected group. These results suggest that the suppression of casual blood glucose elevation in the sHB-EGF-injected group, despite the low plasma insulin levels, is due to activation of glucose metabolism.

[0177] HB-EGF ECD gene therapy dramatically improved the blood glucose elevation suppression effect compared to full-length HB-EGF gene therapy. HB-EGF ECD alone demonstrated the protective and regenerative effect of β-cells, but did not induce glucose-responsive insulin secretion. Nevertheless, HB-EGF ECD alone lowered blood glucose levels to normal levels. Furthermore, the combined use of HB-EGF ECD with the NK1 fragment of HGF also improved glucose-responsive insulin secretion. According to the present invention, a superior hyperglycemia suppression effect can be achieved with an even lower dose than in previous studies by the inventors using the full-length HB-EGF gene. In AAV gene therapy, which has traditionally been believed to be safe, the dose of 1.3 x 10 １４ Considering that recent clinical trials have reported cases of death due to severe liver damage even with the administration of a relatively low dose of vp / kg body weight, the improvement in therapeutic effects achieved by the use of HB-EGF ECD is particularly noteworthy. Therefore, the therapeutic agent of the present invention may be a safe and effective gene therapy agent for diabetes that can be applied clinically.

[0178] The present invention demonstrates that HB-EGF ECD plays an important role in the treatment of diabetes. This indicates that not only gene therapy but also protein preparations such as recombinant soluble HB-EGF are effective in treating diabetes. Furthermore, the present invention teaches that diabetes can be treated not only by exogenously introducing an HB-EGF ECD fragment or a nucleic acid encoding it, but also by increasing the amount of soluble HB-EGF through ectodomain shedding of endogenous proHB-EGF, or by achieving signal transduction from the HB-EGF receptor similar to that generated by HB-EGF binding, but by transactivating the receptor without soluble HB-EGF binding. Substances that enhance signal transduction via HB-EGF receptors, such as GPCR ligands, are low-molecular-weight compounds, many of which are already in use as pharmaceuticals, and therefore could be therapeutic agents for diabetes that are safe and have excellent compliance.

[0179] The number of diabetes patients continues to increase worldwide, becoming a social problem, and the significance of the present invention is great. In particular, T1D develops at a young age, and existing pancreatic islet transplantation therapies have limited use, so the therapeutic agent of the present invention is extremely useful as an alternative and versatile therapeutic means for diabetes including T1D.

[0180] This application is based on Japanese Patent Application No. 2024-59835 filed on April 2, 2024, the contents of which are incorporated herein by reference in their entirety.

Claims

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.

2. The agent according to claim 1, comprising a nucleic acid encoding an HB-EGF ECD fragment.

3. The agent according to claim 2 , wherein the nucleic acid is carried on a viral vector.

4. The agent according to claim 3, wherein the viral vector is an adeno-associated viral (AAV) vector.

5. The agent according to claim 2, wherein the nucleic acid encoding the HB-EGF ECD fragment is under the control of a CA promoter.

6. The agent according to claim 1, which is administered by systemic administration.

7. The agent according to claim 6, wherein the systemic administration is intravenous administration.

8. The agent according to claim 4, which is administered in a single dose or multiple doses with an interval of at least 60 days between doses.

9. The viral vector is 1 × 10 １０ ~1 x 10 １３ 4. The method of claim 3, wherein the agent is administered in a single dose of viral particles (vp) / kg body weight.

10. The agent according to claim 1, comprising a nucleic acid encoding hepatocyte growth factor (HGF) or a fragment thereof.

11. The agent according to claim 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.

12. A therapeutic agent for treating diabetes in mammals, comprising a substance that enhances signal transduction mediated by the HB-EGF receptor.

13. The agent according to any one of claims 1 to 12, wherein the diabetes is type 1 diabetes.

14. The agent according to any one of claims 1 to 12, wherein the mammal is a human.