WO2016062282A1 - 巨噬细胞发炎蛋白-1β(MIP-1β)抑制剂用以保护胰脏及防止血糖升高的用途 - Google Patents

巨噬细胞发炎蛋白-1β(MIP-1β)抑制剂用以保护胰脏及防止血糖升高的用途 Download PDF

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WO2016062282A1
WO2016062282A1 PCT/CN2015/092770 CN2015092770W WO2016062282A1 WO 2016062282 A1 WO2016062282 A1 WO 2016062282A1 CN 2015092770 W CN2015092770 W CN 2015092770W WO 2016062282 A1 WO2016062282 A1 WO 2016062282A1
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mip
macrophage inflammatory
protein
inflammatory protein
inhibitor
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陈肇文
张婷婷
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法玛科技顾问股份有限公司
陈肇文
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Priority to CN201580057808.6A priority Critical patent/CN107206051A/zh
Priority to JP2017522350A priority patent/JP6860480B2/ja
Priority to EP15851823.3A priority patent/EP3210616A4/en
Priority to US15/521,116 priority patent/US20180134782A1/en
Publication of WO2016062282A1 publication Critical patent/WO2016062282A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the use of macrophage inflammatory protein-1 ⁇ (MIP-1 ⁇ ) inhibitors to protect the pancreas. More particularly, the present invention relates to the use of MIP-1 ⁇ inhibitors for maintaining insulin secretion in diabetic patients and preventing elevated blood glucose levels.
  • MIP-1 ⁇ macrophage inflammatory protein-1 ⁇
  • the number of patients with clinical diabetes has continued to increase year by year in the world or in Asian countries, especially in mainland China, Taiwan, Japan, and India.
  • the number of type 2 diabetes in China is now close to 10% of the total population and is still increasing. Therefore, there is an urgent need for drug development in diabetes.
  • the blood glucose elevation of diabetes is mainly caused by an increase in insulin resistance (type 2 diabetes) and impaired inflammation of the pancreatic pancreatic islet cells (type 1 and type 2 diabetes), so that the pancreas secretes insulin insufficiently. Reduce the intake of foreign substances and the sugar produced in the body.
  • the blood sugar control of clinical diabetes is mainly to stimulate the pancreas to secrete insulin (to treat type 2 diabetes), to reduce the peripheral tissue insulin resistance (to treat type 2 diabetes), and to reduce intestinal glucose uptake and absorption (treatment of type 1 and type 2). Diabetes), or increase the urine sugar exclusion (treatment of type 1 and type 2 diabetes).
  • the pancreatic pancreatic islet cells eventually become inflammatory and destructive, resulting in a severely insufficient insulin secretion, which must be supplemented by injection or inhalation of insulin.
  • the drugs that are currently in existence or in development do not directly protect the pancreas (pancreatic islet cells), reduce or restore their damage, and thereby maintain insulin secretion. Therefore, the present invention has attempted to develop a drug which can prevent an increase in blood sugar of a diabetic person by a therapeutic route for protecting pancreatic islets inflammation and destruction by protecting the pancreas of a diabetic person.
  • Macrophage inflammatory protein-1 ⁇ is a member of the CC cytokine family, which is the first to isolate free lipopolysaccharide-activated macrophage cultures (Lodi PJ et al., Science 263: 1762-1767). , 1994), with a molecular weight of 7.8 kilodaltons, and its protein structure consists of 92 amino acid precursors.
  • MIP-1 ⁇ has been observed to be up-regulated in patients with DM and cardiovascular disease (Tatara, Y. et al., JMol Cell Cardiol 47: 104-111, 2009; Mirabelli-Badenier, M. et al., Thromb Haemost 105 :409-420, 2011).
  • MIP-1 ⁇ further performs its biochemical function by binding to the CC cytokine receptor (CCR, which belongs to the G-protein-and-type receptor superfamily) on the cell surface.
  • CCR CC cytokine receptor
  • the macrophage inflammatory protein-1 ⁇ monoclonal antibody we use can antagonize the regulation of CCR5.
  • the presence of CCR5 in the pancreas may be associated with the development of isletitis and spontaneous type 1 diabetes (Cameron MJ et al, J Immunol 165: 1102-1110, 2000).
  • macrophage inflammatory protein-1 ⁇ in blood is positively correlated with diabetes and cardiovascular disease. Therefore, we have developed a new therapeutic strategy for the prevention and treatment of inflammation of the pancreatic islets of diabetic patients by using macrophage inflammatory protein (MIP)-1 to protect the pancreas from inflammation, thereby maintaining its insulin secretion and controlling blood sugar.
  • MIP macrophage inflammatory protein
  • the present invention finds that in the first and second types of diabetic animal models, direct inhibition of macrophage inflammatory protein-1 ⁇ (for example, by single antibody) can protect the pancreas and maintain its The effect of insulin secretion and the blood sugar level will not continue to rise.
  • one aspect of the present invention relates to the use of a macrophage inflammatory protein-1 ⁇ inhibitor for the preparation of a pharmaceutical composition for protecting pancreatic function in a diabetic patient, wherein said protecting a pancreatic function of a diabetic patient comprises preventing a diabetic patient Pancreatic islet cells are damaged.
  • the pharmaceutical composition is for maintaining insulin secretion in a diabetic patient.
  • the pharmaceutical composition is for preventing an increase in blood glucose in a diabetic patient.
  • the macrophage inflammatory protein-1 ⁇ inhibitor is a compound capable of reducing or inhibiting the biological activity of macrophage inflammatory protein-1 ⁇ .
  • the macrophage inflammatory protein-1 ⁇ inhibitor is a ligand compound having binding specificity for macrophage inflammatory protein-1 ⁇ , such as an anti-macrophage inflammatory protein- 1 ⁇ antibody or antagonist.
  • the anti-macrophage inflammatory protein-1 ⁇ antibody is a monoclonal antibody or a plurality of antibodies.
  • the anti-macrophage inflammatory protein-1 ⁇ antibody is a monoclonal antibody, or an antibody fragment thereof that binds to at least a peptide fragment of macrophage inflammatory protein-1 ⁇ .
  • the peptide fragment of macrophage inflammatory protein-1 ⁇ comprises the amino acid sequence 46SFMDDYYET54 (SEQ ID NO: 1), or 62AVVFLTKRGRQIC74 (SEQ ID NO: 2).
  • Figure 1 is a graph showing the protective effect of MIP-1 ⁇ -inhibitor on the pancreas of type 1 diabetic mice, and inhibiting the activity of macrophage inflammatory protein (MIP)-1 ⁇ in vivo by monoclonal antibody (mAb), which can effectively enhance blood. The amount of insulin.
  • MIP macrophage inflammatory protein
  • Fig. 2 is a graph showing the results of observation of a streptozotocin (STZ)-induced inflammatory state in pancreatic tissue of a first-type diabetic animal model by section staining.
  • the red fluorescence is pancreatic islet cells.
  • FIG. 3 is a graph showing the content of macrophage inflammatory protein (MIP)-1 ⁇ protein in blood of normal mice (non-DM control group) and type 2 diabetic mice.
  • MIP macrophage inflammatory protein
  • Fig. 4 is a graph showing the results of observation of a streptozotocin (STZ)-induced inflammatory state in pancreatic tissue of a first-type diabetic animal model by section staining.
  • the red fluorescence is pancreatic islet cells.
  • Figure 5 shows the Western blot and statistical analysis.
  • the content of IL6 and IL8 in the mouse pancreas (n 3).
  • #P ⁇ 0.05, ##P ⁇ 0.01 is compared to the normal control group of mice.
  • *P ⁇ 0.05, **P ⁇ 0.01 is compared to DM mice that have not been treated with antibodies.
  • Figure 6A shows the increase in macrophage inflammatory protein (MIP)-1 ⁇ concentration in culture medium with different doses of STZ(or) treatment with pancreatic islet cell (NIT-1) NIT-1 cell proliferation.
  • MIP macrophage inflammatory protein
  • MIP-1 ⁇ -inhibitor means at least one which reduces the MIP-1 ⁇ protein content and/or decreases the MIP-1 ⁇ protein.
  • An active compound An active compound.
  • the MIP-1 ⁇ -inhibitor compound reduces at least one biological activity of the MIP-1 ⁇ protein by at least about 10%, 25%, 50%, 75% or more.
  • the MIP-1 ⁇ -inhibitor compound protects the pancreas and prevents elevated blood glucose by reducing the amount of MIP-1 ⁇ protein expression.
  • MIP-1 ⁇ protein expression For example, siRNA, antisense nucleic acid or ribozyme targeting MIP-1 ⁇ can be used to inhibit intracellular MIP-1 ⁇ gene expression.
  • the amount of MIP-1 ⁇ protein expression can also be reduced by modulating the transcription of a gene encoding a MIP-1 ⁇ protein or by restoring the corresponding mRNA.
  • the MIP-1 ⁇ -inhibitor compound protects the pancreas and prevents or inhibits the biological activity of the MIP-1 ⁇ protein by binding to the MIP-1 ⁇ protein. Blood sugar rises.
  • an antibody against MIP-1 ⁇ can be used to compete with a MIP-1 ⁇ protein for binding to a receptor on the cell surface while inhibiting the biological activity of the MIP-1 ⁇ protein in vivo.
  • the antibodies may include full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies). And antibody fragments.
  • antibody means an immunoglobulin molecule or a fragment thereof that has the ability to specifically bind to a particular antigen.
  • An “antibody fragment” comprises a portion of a full length antibody, preferably an antigen-binding region or variable region of an antibody.
  • antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (representatively one of the one-armed VL and VH domains of the antibody), single-stranded Fv (scFv), dsFv, Fd fragments (representatively VH and CH1 domains) and dAb fragments (representatively VH domain); VH, VL and VhH domains; minibodies, dimers ( Diabodies), triabodies, tetrabodies, and kappa antibodies (see, Ill et al., Protein Eng 10:949-57, 1997); camel IgG; and one or more isolated CDRs from antibody fragments Or a multi-specific antibody fragment formed by a functional paratope in which the isolated or antigen-binding residues or polypeptides can bind or bind to each other to form a functional antibody fragment.
  • the MIP-1 ⁇ -inhibitor is a monoclonal antibody that specifically binds to a MIP-1 ⁇ protein.
  • the anti-MIP-1 ⁇ monoclonal antibody system binds to a major functional site of the MIP-1 ⁇ protein structure.
  • the MIP-1 ⁇ -inhibitor eg, an anti-MIP-1 ⁇ monoclonal antibody
  • the MIP-1 ⁇ -inhibitor can be linked to an amino acid sequence comprising a MIP-1 ⁇ protein at positions 46-54: SSFMDYYET ( SEQ ID NO: 1), or the amino acid sequence of positions M62-74 of the MIP-1 ⁇ protein: epitope binding of AVVFLTKRGRQIC (SEQ ID NO: 2).
  • the antibody may be a humanized or fully humanized monoclonal antibody.
  • the pharmaceutical composition according to the invention may comprise at least one MIP-1 ⁇ -inhibitor and one or more physiologically acceptable carriers, diluents or excipients.
  • Appropriate pharmaceutical composition forms may be formulated according to the selected route of administration, including (but not limited to) oral preparations such as tablets, capsules, powders, etc., parenteral preparations such as subcutaneous, intramuscular or intraperitoneal injections. And a lyophilized powder combined with a physiological buffer solution before administration.
  • Example 1 MIP-1 ⁇ -inhibitor protects the pancreas of a type 1 diabetic animal (streptomycin (STZ)-induced diabetic mouse model) and prevents elevation of blood sugar
  • the serum insulin test results of Fig. 1 showed that the serum insulin concentration of the type 1 diabetic mice induced by streptozotocin (STZ)- was significantly lower than that of the normal control mice.
  • STZ streptozotocin
  • MIP anti-macrophage inflammatory protein
  • mAb monoclonal antibody
  • pancreatic Insular cells injected with monoclonal antibodies (mAb) against diabetic macrophage inflammatory protein (MIP)-1 ⁇ in diabetic animals (DM+mAb group), can prevent pancreatic islet cells from being damaged by inflammation, and partially maintain pancreatic islets
  • mAb monoclonal antibodies
  • MIP macrophage inflammatory protein
  • Example 2 MIP-1 ⁇ -inhibitor protects the pancreas of type 2 diabetic animals (Leprdb/db mouse model) and prevents blood sugar elevation
  • the amount of macrophage inflammatory protein (MIP)-1 ⁇ in the blood of type 2 diabetic animals was significantly higher than that of normal animals.
  • injection of monoclonal antibody (mAb) against macrophage inflammatory protein (MIP)-1 ⁇ in vivo can effectively control blood glucose levels, so that they do not continue Raised (Table 2).
  • pancreatic islet cells of diabetic mice were severely damaged (red fluorescence was pancreatic islet cells); whereas in type 2 diabetic mice with high blood glucose levels,
  • mAb monoclonal antibody
  • MIP macrophage inflammatory protein
  • pancreas prevents the pancreatic islet cells from being destroyed, while the vitamins continue to secrete insulin to regulate the normal function of blood sugar (Fig. 4).
  • Example 3 Protection of MIP-1 ⁇ -inhibitors against pancreatic islet cell damage (streptomycin (STZ)-induced pancreatic cell injury model)
  • FIG. 5 show that in a diabetic animal with a high blood sugar level, injection of a monoclonal antibody (mAb) against the macrophage inflammatory protein (MIP)-1 ⁇ in vivo can effectively inhibit the inflammatory substances of the pancreas (pancreatic islet), such as The performance of interleukin-6 (IL-6) and interleukin-8 (IL-8). Therefore, inhibition of macrophage inflammatory protein (MIP)-1 ⁇ protects pancreatic islet cells and reduces the inflammatory response caused by streptozotocin.
  • mAb monoclonal antibody
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • NIT-1 cells a pancreatic ⁇ -cell strain established from transgenic nod/lt mice
  • STZ 0.75, 1.5, 3, 6 mM
  • NIT-1 cells were treated with STZ for 24 hours, and then treated with no, low dose (0.3 ⁇ g/ml) or high dose (30 ⁇ g/ml) of MIP-1 ⁇ antibody (R&D system) for 4 hours.
  • the proliferation of NIT-1 cells was assessed by MTT assay. The results are shown in Figure 6.
  • the toxicity of streptozotocin (STZ 1.5, STZ 3) to pancreatic islet cells (NIT-1) increased with the dose of streptozotocin.
  • the monoclonal antibody (mAb) against the cell-produced macrophage inflammatory protein (MIP)-1 ⁇ can directly improve the inhibition of the number of pancreatic islet cells (NIT-1) by streptozotocin (STZ 1.5, STZ3). Destructive effect. Therefore, inhibition of macrophage inflammatory protein (MIP)-1 ⁇ can directly protect pancreatic islet cells, Low-chain zoloumycin damage to pancreatic islet cells.

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Abstract

巨噬细胞发炎蛋白-1β（MIP-1β）抑制剂用于保护胰脏以及防止糖尿病患者血糖升高的用途。

Description

巨噬细胞发炎蛋白-1β(MIP-1β)抑制剂用以保护胰脏及防止血糖升高的用途
本申请要求于2014年10月24日提交美国专利商标局、申请号为62/068475的美国临时专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明是关于巨噬细胞发炎蛋白-1β(MIP-1β)抑制剂用以保护胰脏的用途。更特别地，本发明是关于MIP-1β抑制剂用于维持糖尿病患者胰岛素分泌，及防止血糖不当升高的用途。
背景技术
近年来临床糖尿病的人数无论在世界上或亚洲国家尤其中国大陆、台湾、日本、印度均逐年持续增加。我国目前第二型糖尿病人数已接近总人口10％，且仍在增加中。故糖尿病的药物研发有迫切需要。目前已知糖尿病的血糖升高机转主要是胰岛素阻抗升高(第二型糖尿病)及胰脏胰小岛细胞发炎受损(第一型及第二型糖尿病)，以致胰脏分泌胰岛素不足以降低外来摄取及体内制造的糖分所致。
目前临床糖尿病的血糖控制主要是以刺激胰脏分泌胰岛素(治疗第二型糖尿病)，降低外围组织胰岛素阻抗(治疗第二型糖尿病)，降低肠道糖分摄取吸收(治疗第一型及第二型糖尿病)，或增加尿液糖分排除(治疗第一型及第二型糖尿病)等机转。然而，不论第一型或第二型糖尿病，胰脏胰小岛细胞最终都会发炎破坏，以致胰岛素分泌严重不足，必须以注射或吸入胰岛素来补充治疗。这主要是因为，在目前已有或发展中的药物，并不能直接保护胰脏(胰小岛细胞)，减轻或回复其受损，而藉以维持胰岛素分泌者。因此，本发明尝试针对经由保护糖尿病人的胰脏，改善胰小岛发炎及破坏的治疗途径，而发展出可防止糖尿病人血糖升高的药物。
巨噬细胞发炎蛋白-1β(Macrophage inflammatory protein-1 β，MIP-1β，在有些文献也已知称作CCL4)为CC细胞因子家族的成员，最先分离自由脂多醣所活化的巨噬细胞培养液(Lodi P.J.等人,Science 263:1762-1767,1994)，分子量为7.8千道尔顿，其蛋白质结构是由92个胺基酸前驱物所组成。在DM及心血管疾病患者中已观察到MIP-1β有受到增量调节(Tatara,Y.等人,JMol Cell Cardiol 47:104-111,2009；Mirabelli-Badenier,M.等人,Thromb Haemost 105:409-420,2011)。
MIP-1β是藉由与细胞表面上的CC细胞因子受体(CCR，属于G-蛋白-偶和型受体超家族)结合，而进一步执行其生化功能。巨噬细胞发炎蛋白-1β的受体众多，其中CCR5被认为有主要生理病理作用，我们使用的巨噬细胞发炎蛋白-1β单株抗体即能拮抗调控CCR5。在一项以实验糖尿病动物模式进行的研究发现，胰脏中CCR5的表现可能与胰岛炎及自发性第I型糖尿病的发展有关(Cameron MJ等人,J Immunol 165:1102-1110,2000)。也发现，在第II型糖尿病大鼠的颈上神经节中CCR5含量也有升高的现象，但是该项数据，与在不同动物模式中所得到的结果并不一致。有报告指出，在于11-13周龄的非-肥胖型糖尿病(NOD)小鼠模式中以抗-CCR5抗体暂时阻断CCR5结合或造成CCR5缺陷，非但不能防止反而会加速自体免疫第I型糖尿病的发生(Gonzalez P等人,Genes Immun 2:191-195,2001；Simeoni E等人,Eur Heart J 25:1438-1446,2004)。
根据文献指出，血中的巨噬细胞发炎蛋白-1β数值与糖尿病及心血管疾病皆有正相关性。因此，我们针对糖尿病人的胰小岛发炎破坏，发展出利用抑制巨噬细胞发炎蛋白(MIP)-1来保护胰脏改善其发炎，以维持其胰岛素分泌，进而控制血糖的新治疗策略。
发明内容
本发明基于以上的目的发现，在第一型及第二型糖尿病动物模型，直接抑制体内巨噬细胞发炎蛋白-1β(例如透过单株抗体等方法)，皆可产生保护胰脏，维持其胰岛素分泌，并使血糖值不会继续升高的功效。
于是，本发明的一方面是关于，一种巨噬细胞发炎蛋白-1β抑制剂用于制备保护糖尿病患者胰脏功能的医药组成物的用途，其中所述保护糖尿病患者胰脏功能包含防止糖尿病患者胰小岛细胞受损。于本发明的一些具体实施态样，所述的医药组成物是用于维持糖尿病患者胰岛素分泌。于本发明的其他具体实施态样，所述的医药组成物是用于防止糖尿病患者血糖升高。
于本发明的一些具体实施态样，所述的巨噬细胞发炎蛋白-1β抑制剂为能够减低或抑制巨噬细胞发炎蛋白-1β的生物活性的化合物。于本发明的一项具体实施态样，所述的巨噬细胞发炎蛋白-1β抑制剂为对于巨噬细胞发炎蛋白-1β具有结合特异性的配体化合物，例如抗-巨噬细胞发炎蛋白-1β抗体或拮抗剂。
于本发明的一些具体实施态样，所述的抗-巨噬细胞发炎蛋白-1β抗体为单株抗体或多株抗体。于本发明的一项具体实施态样，所述的抗-巨噬细胞发炎蛋白-1β抗体为单株抗体，或其与巨噬细胞发炎蛋白-1β的至少肽类片段结合的抗体片段。于本发明的其他具体实施态样，所述的巨噬细胞发炎蛋白-1β的肽类片段包含胺基酸序列46SFVMDYYET54(SEQ ID NO:1)，或62AVVFLTKRGRQIC74(SEQ ID NO:2)。
附图说明
图1是显示MIP-1β-抑制剂对于第一型糖尿病小鼠胰脏的保护功效，以单株抗体(mAb)抑制体内巨噬细胞发炎蛋白(MIP)-1β的活性，可有效提升血中胰岛素量。
图2为以切片染色观察第一型糖尿病动物模式的胰脏组织受链佐霉素(STZ)-诱导发炎状态的结果。其中红色荧光为胰小岛细胞。
图3是显示正常小鼠(non-DM对照组)及第二型糖尿病小鼠血中巨噬细胞发炎蛋白(MIP)-1β蛋白的含量。
图4为以切片染色观察第一型糖尿病动物模式的胰脏组织受链佐霉素(STZ)-诱导发炎状态的结果。其中红色荧光为胰小岛细胞。
图5是显示以西方转渍法(Western blot)与统计分析，检测小 鼠胰脏中IL6及IL8的含量(n＝3)。#P<0.05,##P<0.01是相较于正常对照组小鼠。*P<0.05,**P<0.01是相较于未经抗体处理的DM小鼠。
图6A为经由不同剂量的STZ(或)处理期间培养液中的巨噬细胞发炎蛋白(MIP)-1β浓度随胰小岛细胞(NIT-1)NIT-1细胞增生的增加情形。而图6B系显示以MTT分析评估不同剂量之STZ(STZ 1.5,STZ3)对胰小岛细胞(NIT-1细胞)的毒性，及使用单株抗体(mAb)减低STZ造成的损害，回复NIT-1细胞增生(n＝3)。
图7是显示NIT-1细胞培养上清液中胰岛素的分泌量(n＝6)。#P<0.05，##P<0.01是相较于未经任何处理的对照组NIT-1细胞具有显著差异。*P<0.05，**P<0.01是相较于以相同STZ浓度处理的NIT-1细胞组具有显著差异。
具体实施方式
于本说明书中所称的“巨噬细胞发炎蛋白-1β-抑制剂(MIP-1β-抑制剂)”意指，一种可减少MIP-1β蛋白含量及/或降低MIP-1β蛋白的至少一种活性的化合物。于本发明的一项具体实施态样，所述的MIP-1β-抑制剂化合物可使MIP-1β蛋白的至少一种生物活性降低至少约10％、25％、50％、75％或以上。
于本发明的某些具体实施态样，所述的MIP-1β-抑制剂化合物是藉由减低MIP-1β蛋白表现量，来保护胰脏及防止血糖升高。例如可使用靶定MIP-1β的siRNA、反义核酸或核酶，抑制细胞内MIP-1β基因表现。也可藉由调节编码MIP-1β蛋白的基因转录，或使所对应的mRNA不安定，而减少MIP-1β蛋白的表现量。
于本发明的其他具体实施态样，所述的MIP-1β-抑制剂化合物是藉由与MIP-1β蛋白结合，直接或间接降低或抑制MIP-1β蛋白的生物活性，来保护胰脏及防止血糖升高。例如，根据本发明的某些实施例，可使用对抗MIP-1β的抗体与MIP-1β蛋白竞争结合至细胞表面上的受体，而抑制体内MIP-1β蛋白的生物活性。所述的抗体可包括全长单株抗体、多株抗体、多特异性抗体(例如双特异性抗体) 以及抗体片段。
于本说明书中所称的“抗体”意指一种与特定抗原特异结合的能力的免疫球蛋白分子或其片段。“抗体片段”包含全长抗体的一部分，较佳是抗体的抗原-结合区或可变区。抗体片段的实例包括Fab、Fab'、F(ab)2、F(ab’)2、F(ab)3、Fv(代表性地为抗体其中一单臂的VL及VH功能域)、单链Fv(scFv)、dsFv、Fd片段(代表性地为VH与CH1功能域)及dAb片段(代表性地为VH功能域)；VH、VL及VhH功能域；迷你抗体(minibodies)、二体(diabodies)、三体(triabodies)、四体(tetrabodies)及kappa抗体(参见，Ill等人,Protein Eng 10:949-57,1997)；骆驼IgG；以及由抗体片段一或多个单离的CDRs或一个功能性互补位(paratope)形成的多特异性抗体片段，其中单离的或抗原结合残基或多肽可相互结合或键联，而形成功能性抗体片段。
于本发明的某些具体实施态样，所述的MIP-1β-抑制剂为与MIP-1β蛋白特异性结合的单株抗体。于本发明的一具体实施态样，所述的抗-MIP-1β单株抗体系与MIP-1β蛋白结构上的主要功能作用部位结合。根据本发明的某些实施例，所述的MIP-1β-抑制剂(例如，抗-MIP-1β单株抗体)可与一包含MIP-1β蛋白的胺基酸序列位置46～54:SFVMDYYET(SEQ ID NO:1)，或MIP-1β蛋白的胺基酸序列位置62～74:AVVFLTKRGRQIC(SEQ ID NO:2)的抗原决定基结合。
根据本发明的某些实施例，所述的抗体可为人源化或全人源单株抗体。
根据本发明的医药组成物，可包含至少一种MIP-1β-抑制剂及一或多种生理上可接受的载剂、稀释剂或赋形剂。可依据所选择的投药途径，而调配得适当的医药组成物形式，包括(但不限定于)口服制剂如片剂、胶囊、粉末等，非经肠道制剂如皮下、肌肉或腹膜内注射液，及于投药前与生理缓冲溶液组合的冻干粉末等。
本发明的其他特色及优点将于下列实施范例中被进一步举例与说明，而该实施范例仅作为辅助说明，并非用于限制本发明之范围。
实施例一、MIP-1β-抑制剂对于第一型糖尿病动物(链佐霉素(STZ)-诱导的糖尿病小鼠模型)的胰脏保护及防止血糖升高
于活体试验期间监测实验小鼠的血糖浓度。结果如表1所示。经由链佐霉素(STZ)-诱导的第一型糖尿病小鼠，无论是否有下肢血管阻断手术(OP)，其血糖值明显增加。但是，以MIP-1β中和抗体注射4周的小鼠，无论是否有下肢血管阻断手术(OP)，其血糖值维持不再升高，而且有些许下降的趋势增加。
表1
Figure PCTCN2015092770-appb-000001
由图1的血清胰岛素测试结果显示，经由链佐霉素(STZ)-诱导的第一型糖尿病小鼠，其血清胰岛素浓度相较于正常对照组小鼠，有明显降低。而在血糖值高的糖尿病动物，经过注射抗-巨噬细胞发炎蛋白(MIP)-1β单株抗体(mAb)达4周后，相较于未经处理的糖尿病小鼠，可有效提升血中胰岛素量，并控制血糖值，使其血糖浓度不再继续升高。
此外，以切片染色观察胰脏组织的发炎状态，结果显示，在血糖值高的糖尿病动物(DM组)，其胰脏中的胰小岛细胞因受损而数量明显减少(红色荧光为胰小岛细胞)，注射单株抗体(mAb)对抗体内巨噬细胞发炎蛋白(MIP)-1β的糖尿病动物(DM+mAb组)，可防止胰小岛细胞因发炎受损，而部分维持胰小岛的型态，达到保护胰脏的功效(图2)。
实施例二、MIP-1β-抑制剂对于第二型糖尿病动物(Leprdb/db小鼠模型)的胰脏保护及防止血糖升高
如图3所示，第二型糖尿病动物血中巨噬细胞发炎蛋白(MIP)-1β量明显较正常动物为高。在血糖值高的糖尿病动物，无论是否有下肢血管阻断(OP)，注射单株抗体(mAb)对抗体内巨噬细胞发炎蛋白(MIP)-1β，可有效控制血糖值，使其不再继续升高(表2)。
表2
Figure PCTCN2015092770-appb-000002
而进一步以切片染色观察胰脏组织的发炎状态的结果显示，糖尿病小鼠的胰小岛细胞受损严重(红色荧光为胰小岛细胞)；而在血糖值高的第二型糖尿病小鼠，经过注射单株抗体(mAb)对抗体内巨噬细胞发炎蛋白(MIP)-1β后，可部分恢复胰小岛的型态，表示以巨噬细胞发炎蛋白(MIP)-1β中和抗体能有效保护胰脏，防止胰小岛细胞被破坏，而维持续胰脏分泌胰岛素调控血糖的正常功能(图4)。
综合上述的结果表示，藉由抑制巨噬细胞发炎蛋白(MIP)-1β的 作用，可产生保护胰脏的功效。
实施例三、MIP-1β-抑制剂对于防止胰小岛细胞受损(链佐霉素(STZ)-诱导的胰脏细胞损伤模型)的保护
为进一步了解MIP-1β-抑制剂保护胰脏的作用，遂以西方转渍分析法(Western blotting)检测胰脏中发炎性蛋白质的含量。由图5的结果显示，在血糖值高的糖尿病动物，注射单株抗体(mAb)对抗体内巨噬细胞发炎蛋白(MIP)-1β，可有效抑制胰脏(胰小岛)的发炎物质，如介白素-6(IL-6)及介白素-8(IL-8)的表现。因此，抑制巨噬细胞发炎蛋白(MIP)-1β可保护胰小岛细胞，降低链佐霉素对其所造成的发炎反应。
也进行活体外细胞研究，模拟MIP-1β-抑制剂对于防止体内胰小岛细胞受损的功效。将NIT-1细胞(一种从转基因nod/lt小鼠建立的胰脏β-细胞株)接种于96-孔培养盘中培养过夜，接种浓度为1×105个细胞/孔。经上述前-培养后，将加入STZ(0、0.75、1.5、3、6mM)与细胞共培养24小时。使用MTT分析检测STZ对于NIT-1细胞的细胞毒性。另将NIT-1细胞以STZ处理24小时，之后再以无、低剂量(0.3μg/ml)或高剂量(30μg/ml)的MIP-1β抗体(R&D system)处理4小时。以MTT分析评估NIT-1细胞的增生情形。结果列示于图6。
如图6A的数据显示，在NIT-1细胞研究中，链佐霉素(STZ 1.5,STZ 3)会刺激胰小岛细胞(NIT-1)制造巨噬细胞发炎蛋白(MIP)-1β，以致培养液中的巨噬细胞发炎蛋白(MIP)-1β浓度随链佐霉素的剂量而增加；而此巨噬细胞发炎蛋白(MIP)-1β含量增加的现象，可因投药抗-单株抗体(mAb)而受到抑制。
如图6B的数据显示，链佐霉素(STZ 1.5、STZ 3)对胰小岛细胞(NIT-1)的毒性，会随链佐霉素的剂量而增强。而以单株抗体(mAb)对抗细胞制造的巨噬细胞发炎蛋白(MIP)-1β，可直接有效改善链佐霉素(STZ 1.5,STZ3)对胰小岛细胞(NIT-1)数量抑制及破坏作用。因此，抑制巨噬细胞发炎蛋白(MIP)-1β可直接保护胰小岛细胞，降 低链佐霉素对胰小岛细胞的伤害。
在细胞研究的结果显示，链佐霉素(STZ 1.5、STZ 3)会抑制胰小岛细胞(NIT-1)分泌胰岛素(insulin)，且此抑制作用会随剂量增加而增强。而以单株抗体(mAb)对抗细胞制造的巨噬细胞发炎蛋白(MIP)-1β，可有效改善链佐霉素(STZ 1.5、STZ 3)造成的胰岛素分泌抑制作用。因此，藉由抑制巨噬细胞发炎蛋白(MIP)-1β，可直接保护胰小岛细胞，降低链佐霉素对之伤害，并恢复其胰岛素分泌(图7)。
因此综合上述实施例的结果，确认直接抑制体内巨噬细胞发炎蛋白-1β(例如，透过单株抗体或拮抗剂等方法)，可藉由保护胰脏功能，减低胰小岛细胞的发炎，进而维持胰岛素的正常分泌，并达到对糖尿病患者的血糖控制。
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Claims (10)

  1. 一种巨噬细胞发炎蛋白-1β抑制剂用于制备保护糖尿病患者胰脏功能的医药组成物的用途，其中该保护糖尿病患者胰脏功能是包含防止糖尿病患者胰小岛细胞受损。
  2. 如权利要求1所述的用途，其中该医药组成物是用于维持糖尿病患者的胰岛素分泌。
  3. 如权利要求2所述的用途，其中该医药组成物是用于防止糖尿病患者血糖升高。
  4. 如权利要求1所述的用途，其中该巨噬细胞发炎蛋白-1β抑制剂为能够减低或抑制巨噬细胞发炎蛋白-1β的生物活性的化合物。
  5. 如权利要求1或4所述的用途，其中该巨噬细胞发炎蛋白-1β抑制剂为对于巨噬细胞发炎蛋白-1β具有结合特异性的配体化合物。
  6. 如权利要求5所述的用途，其中该巨噬细胞发炎蛋白-1β抑制剂为抗-巨噬细胞发炎蛋白-1β抗体。
  7. 如权利要求6所述的用途，其中该MIP-1β-抑制剂为与MIP-1β蛋白或其片段特异性结合的抗-MIP-1β单株抗体。
  8. 如权利要求7所述的用途，其中该抗-MIP-1β单株抗体是与MIP-1β蛋白结构上的主要功能作用部位结合。
  9. 如权利要求7或8所述的用途，其中该抗-MIP-1β单株抗体是与一包含MIP-1β蛋白的胺基酸序列位置46～54:SFVMDYYET(SEQ ID NO:1)的肽类片段结合。
  10. 如权利要求7或8所述的用途，其中该抗-MIP-1β单株抗体是与一包含MIP-1β蛋白的胺基酸序列位置62～74:AVVFLTKRGRQIC (SEQ ID NO:2)的肽类片段结合。
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