阅读说明：本技术 氯化血红素及其复合物在医药中的新应用 (New application of hemin and its complex in medicine ) 是由 冯敏 孙冰之 于 2019-09-19 设计创作，主要内容包括：本发明提供了氯化血红素和/或氯化血红素复合物在制备预防和治疗类风湿性关节炎药物中应用。氯化血红素或氯化血红素复合物在体外作用于巨噬细胞具有显著的抗炎效果、对大鼠关节炎模型能有效抑制足掌的肿胀,且能显著降低了后足炎症因子mRNA的表达和肝脾组织自身免疫相关因子的mRNA表达,说明了氯化血红素或氯化血红素复合物具有很好的预防和治疗类风湿性关节炎的作用。(The invention provides application of hemin and/or hemin complex in preparing a medicament for preventing and treating rheumatoid arthritis. The hemin or the hemin compound acts on macrophages in vitro, has obvious anti-inflammatory effect, can effectively inhibit the swelling of soles of rats in arthritis models, and can obviously reduce the expression of mRNA of inflammatory factors of hind feet and the expression of mRNA of autoimmune related factors of liver and spleen tissues, thereby indicating that the hemin or the hemin compound has good effect of preventing and treating rheumatoid arthritis.)

1. Use of hemin and/or hemin complex in preparing medicament for preventing and treating rheumatoid arthritis is provided.

2. The use of claim 1, wherein the hemin complex is a stable complex of hemin and a protein or an amino acid, or wherein the hemin complex is a soluble salt of hemin and a small basic molecule, preferably a sodium hemin salt or a potassium hemin salt.

3. The use according to claim 1, wherein the protein is albumin, and the albumin is bovine serum albumin or human serum albumin.

4. The use according to claim 1, wherein the amino acids are arginine, lysine, or histidine or a combination thereof.

5. The use of any one of claims 1 to 4, wherein the medicament is in the form of solution injection, lyophilized powder injection, subcutaneous implant, tablet, capsule, oral liquid, granule, drop pill, patch, pellet, microcapsule, liposome, or microsphere.

6. A medicine for preventing and treating rheumatoid arthritis is characterized in that the active ingredients of the medicine comprise hemin and/or hemin complex.

7. The medicament for preventing and treating rheumatoid arthritis according to claim 6, wherein said pharmaceutically acceptable excipients are selected from the group consisting of fillers, binders, lubricants, disintegrants, cosolvents, adsorption carriers, solvents, antioxidants, adsorbents, tonicity adjusting agents, and pH adjusting agents.

8. The agent for preventing and treating rheumatoid arthritis according to claim 6, wherein said hemin complex is a stable complex of hemin and a protein or an amino acid, or is a soluble salt of hemin and a basic small molecule.

9. The preventive and therapeutic agent for rheumatoid arthritis according to claim 6, wherein said amino acid is arginine, lysine, or histidine.

10. The preventive and therapeutic agent for rheumatoid arthritis according to any one of claims 6 to 9, wherein said protein is albumin, and said albumin is bovine serum albumin or human serum albumin.

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a new application of hemin and a hemin compound in medicine.

Background

Hemin (Hemin), also known as Hemin or Hemin (Haemin or ferrichloride Heme). Hemin is a porphyrin compound containing ferric ions. Hemin can be oxidized from native heme, and the former has similar chemical properties to the latter. Heme is ubiquitous in the blood, muscle and parts of plant tissues of animals, and is a key prosthetic group of many biologically active macromolecules (e.g., hemoglobin, myoprotein, cytochromes, peroxidase, etc.), and thus heme is one of the most important compounds in the organism.

Hemin CAS number 16009-13-5, molecular formula C₃₄H₃₂ClFeN₄O₄The relative molecular mass was 651.94. The chemical structure of hemin is shown above. Hemin has a chemical structure containing a relatively large hydrophobic tetrapyrrole ring, so that it has low solubility in water, and two free carboxyl groups in the structure make it soluble in an aqueous alkaline solution. In addition, hemin can be dissolved in organic reagents such as dimethyl sulfoxide and acidic acetone.

In clinical treatment, hemin is most widely used as an iron fortifier to treat iron deficiency anemia. In contrast to inorganic iron (non-heme iron) which is mainly released from vegetables and vegetable foods, heme iron exists in the body in molecular form. Therefore, the heme iron can be directly taken by intestinal mucosa, the absorption rate of the heme iron is 50% -87% higher than that of non-heme iron, and the bioavailability is higher. In addition, the hemin also has the advantages of no iron fishy smell, no gastrointestinal irritation, no iron accumulation poisoning in vivo and the like, and is a preferred purely natural biological iron supplement.

Secondly, hemin is also used clinically as a correction agent for porphyrin metabolism for the treatment of acute porphyria. Porphyria can be hereditary or acquired and is pathologically characterized by a deficiency of specific enzymes of heme biosynthesis, a decrease in heme synthesis, resulting in a compensatory induction of beta-aminolevulinic acid (ALA) synthase, an increase in the accumulation and excretion of intermediates of porphyrin synthesis. The supplement of hemin can inhibit the induction of ALA synthetase, reduce the formation and accumulation of potentially harmful intermediates or metabolites during the synthesis of porphyrin, and correct the red blood in vivo to a certain extentDeficiency of hormone. There are 2 hemin products currently on the market for the treatment of porphyria. There is a heme preparation (trade name Panhematin) in lyophilized form. The other is lyophilized powder (trade name) compounded by hemin and arginine) The method comprises the following steps of: propylene glycol: water is mixed solvent, and intravenous injection is used for treating intermittent protoporphyrinopathy.

Rheumatoid Arthritis (RA) is a chronic inflammatory disease that is caused by genetic factors and environmental factors, and can cause systemic effects, and is one of the most common chronic inflammatory diseases. In RA, it is characterized by an abnormally proliferative synovial membrane; the appearance of related cytokines, chemokines, autoantibodies such as Rheumatoid Factor (RF) and anticoagulant protein antibody (ACPA); also osteoclastogenesis, angiogenesis; even severe cases are associated with systemic syndromes such as cardiovascular, pulmonary, psychological and skeletal diseases.

Currently, RA afflicts a large number of patients worldwide, up to one third of RA patients stop working within 2 years after onset, and there is a high likelihood of developing permanent, irreversible systemic effects after long-term onset, such as inducing pulmonary fibrosis, rheumatoid heart disease, stroke, susceptibility to infection, and the like, greatly reducing quality of life. In short, RA disease greatly affects the physical health and quality of life of patients. Therefore, the development of safe and effective RA therapeutic drugs for better treatment of RA and benefit of patients and society is an urgent need to be solved.

Although the etiology and pathogenesis of RA are not fully revealed, a great deal of research results have been carried out to date to demonstrate that the development of chronic local or even systemic inflammation is a currently recognized disease process, which is caused by autoimmune reactions due to genetic, environmental and disease factors^[1]. Human leucocyte antigen-DR 4 (HLA-DR 4) allele, depending on the stage of disease progression,The activation of autoimmunity and inflammation plays an important role in the generation of RA, and cytokines are important factors and application targets which need to be considered in the generation and treatment of inflammation.

The link between HLA-DR and RA was first discovered in the 70's 20 th century and was associated with the development of early stages of RA. The HLA system is encoded by the Major Histocompatibility (MHC) gene of humans, which is present in most vertebrates and plays an important role in the body's defense function in activating the immune response against diseases in humans, and is also the most important genetic risk allele of RA, accounting for about 40% of all genetic influences of RA. The proportion of individuals with the MHC class II HLA-DR4 allele in the RA-afflicted population is up to about 5/6. The specific mechanism of susceptibility to RA is that 70-74 amino acids (glutamine-leucine-arginine-alanine) in the third hypervariable region of the DR beta chain in HLA-DR are identical to some amino acids in the DR4 chain, and the sequence is a "susceptibility epitope". More than 90% of RA patients express at least one of the mutations.

Autoimmunity resulting from the activation of the adaptive and innate immune systems is the beginning of the clinical phase of RA. Synovial microvascular endothelial cells cause leukocyte migration to the synovium by expressing adhesion molecules such as integrins, selectins and chemokines, leading to early synovitis by the release of tumor necrosis factor (TNF α), reactive oxygen and NO free radicals, matrix degrading enzymes and phagocytosis and antigen presentation; then, due to the existence of cytokines and chemokines, various immune cells such as T cells, B cells, myeloid cells, plasmacytoid dendritic cells and the like are gathered to the synovium, and the cytokines are released to influence each other, so that the development of inflammation is promoted.

Cytokines have a role in broadly regulating inflammatory processes, and thus the pathogenesis and mechanism of RA are closely related thereto. In early RA, T cells and stromal cells express the cytokines Interleukin (IL) -13, IL-14, IL-15, which induce chronic disease. Examination of synovial membranes and serum from patients with RA reveals that TNF- α is a major cytokine, which acts to induce pro-inflammatory cytokines, disrupting the normal physiological balance between pro-inflammatory and anti-inflammatory mediators. In addition, TNF- α can induce the expression of endothelial cell adhesion molecules, inhibit regulatory T cells, stimulate angiogenesis at sites of inflammation and stimulate pain production. Similarly, activation of IL-6 by leukocytes and production of autoantibodies leads to acute phase responses such as anemia, cognitive dysfunction and dysregulation of lipid metabolism. Cytokines are also involved in the maturation and activation of osteoclasts, and nuclear factor receptor activator-kappa B ligand (RANKL) has a hierarchical role with TNF, IL-17 and IL-1 in this process. The important role of these cytokines has been demonstrated in RA patients by antibody blocking contrast.

Since the mechanism of disease progression in RA is not completely understood, there is currently no effective drug that can completely control invasion and damage of joints and surrounding bone tissue caused by RA, and also recover the damage. In the treatment of RA patients, the therapeutic purposes of the existing drugs are mainly to alleviate inflammation and pain symptoms, maintain joint function, and prevent disease deterioration, and the commonly used therapeutic drugs can be classified into four types:

the first type is Non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit cyclooxygenase to inhibit the conversion of arachidonic acid into prostaglandin, thereby playing the role of diminishing inflammation and relieving pain, and the contrast drug adopted in the invention is celecoxib (celecoxib), which is a reversible COX-2 inhibitor with strong selectivity, but the drug can only relieve symptoms and cannot prevent and relieve cartilage and bone injuries.

The second category is antirheumatic drugs (DMARDs), which exert a certain anti-RA effect mainly through cytotoxic action and immunosuppression, and can delay but cannot prevent joint damage, and these drugs also lose their benefits with time. The control drug adopts first-line methotrexate for RA clinical treatment, and has the anti-RA effects of inhibiting dihydrofolate reductase, inhibiting the activity of enzyme related to immune system function from inactivation caused by the activity of methyltransferase, inhibiting the influence of interleukin 1-beta on cell surface receptors of the interleukin 1-beta and other mechanisms.

The third type is glucocorticoid, which has strong anti-inflammatory action, but has short half-life, is metabolized in vivo in a large amount, has a series of side effects such as over-strong immunosuppression, osteoporosis and even osteoporosis, and particularly has adverse side effects on the skeleton of the organism when being used for treating rheumatoid arthritis with osteoarthropathy change. Thus, long-term or large dose use is not clinically recommended. Such a control drug employed in the present invention is dexamethasone acetate (dexamethasone acetate).

The fourth type is a biological agent, which can antagonize inflammation-related proteins such as cytokines in RA, such as infliximab (tumor necrosis factor antagonist), tofacitinib (Jak inhibitor), tocilizumab (IL-6 antagonist), and the like, and has a high-efficiency RA relieving effect, and can be used together with DMARDs. However, these drugs are very expensive compared to traditional DMARDS, limiting their wide spread clinical use and also have certain side effects including severe and opportunistic infections.

More and better drugs for preventing and treating rheumatoid arthritis are clinically needed.

Disclosure of Invention

Based on this, an object of the present invention is to provide a novel use of hemin or its complex in medicine.

The technical scheme for achieving the purpose is as follows.

Use of hemin and/or hemin complex in preparing medicament for preventing and treating rheumatoid arthritis is provided.

In one embodiment, the hemin complex is hemin and a protein, an amino acid, or forms a stable complex, or the hemin complex is hemin and a basic small molecule form a soluble salt, and further preferably hemin sodium salt and hemin potassium salt.

In one embodiment, the protein is bovine serum albumin or human serum albumin.

In one embodiment, the amino acid is arginine, lysine, or histidine.

In one embodiment, the dosage form of the drug is solution injection, freeze-dried powder injection, subcutaneous implant, tablet, capsule, oral liquid, granule, dripping pill, patch, pellet, microcapsule, liposome, microsphere.

Another object of the present invention is to provide a preventive and therapeutic agent for rheumatoid arthritis.

The technical scheme for achieving the purpose is as follows.

A medicine for preventing and treating rheumatoid arthritis contains hemin and/or hemin complex as active ingredient.

In some embodiments, the pharmaceutical composition comprises the pharmaceutically acceptable adjuvant selected from the group consisting of fillers, binders, lubricants, disintegrants, cosolvents, adsorption carriers, solvents, antioxidants, adsorbents, osmotic pressure regulators, and pH regulators.

The inventors of the present application found that hemin or hemin complex has a significant anti-inflammatory effect on macrophages in vitro; the in vivo experiment has good effect of inhibiting paw swelling for rat arthritis model, and can significantly reduce the expression of hindpaw inflammatory factor mRNA and the expression of liver spleen tissue autoimmunity related factor. The chlorhematin has the anti-inflammatory effect equivalent to that of hormone dexamethasone acetate, has an immunosuppressive effect, and has a treatment effect superior to that of non-steroidal anti-inflammatory drugs without the serious side effect of hormone drugs. Therefore, the hemin or the hemin compound has good effects of preventing and treating the rheumatoid arthritis, and provides more and better choices for clinically preventing and treating the rheumatoid arthritis.

Drawings

FIG. 1 mRNA expression of Raw264.7 cells were stimulated with LPS (500ng/ml) and IFN-. gamma. (20ng/ml) for 6h, and after incubation with additional Hemin (60. mu.M) for 16h, mRNA expression was measured by PCR and expressed as mean. + -. SD and statistically analyzed by one-way ANOVA and Dunnett's test (n. about.3, x. P < 0.0001).

FIG. 2 hind paw thickness variation for each group of rats CIA model administration started on day 15 after primary immunization, 40mg/kg i.p. Hemin or s.c. 2 times a week; methotrexate is injected intraperitoneally at a rate of 1mg/kg, 2 times a week; the celecoxib is perfused at 50mg/kg once a day; dexamethasone acetate was injected intramuscularly at 0.45mg/kg once every 2 days with the change in hind paw thickness (mm) being the thickness of the hind paw causing inflammation (mm) the initial thickness of the ipsilateral paw causing inflammation (mm), expressed as mean ± SD and n being 12.

FIG. 3 clinical score of arthritis index for each group of rats CIA model administration is started on day 15 after the initial immunization, and 40mg/kg is injected intraperitoneally or subcutaneously for Hemin 2 times a week; methotrexate is injected intraperitoneally at a rate of 1mg/kg, 2 times a week; the celecoxib is perfused at 50mg/kg once a day; dexamethasone acetate was injected intramuscularly at 0.45mg/kg every 2 days, with results expressed as mean ± SD, n ═ 12.

FIG. 4 mRNA expression of TNF α, IL-1 β and IL-6 from rat hind paw in each dosing group of rat CIA model, rats were sacrificed at day 28 after primary immunization, homogenate of inflamed hind paw tissue was removed and mRNA expression measured by PCR.

FIG. 5 is a real graph of the swelling degree of the hind paw of each administration group of the rat CIA model, wherein a is a model control group, and b is a Hemin intraperitoneal injection administration group; c is a Hemin subcutaneous injection administration group; d is a methotrexate administration group; e is celecoxib administration group; f is dexamethasone acetate administration group.

FIG. 6 mRNA expression of IL-17, IL-23 and Foxp3 in rat liver and spleen tissues of each group of rat CIA model administration was started on day 15 after primary immunization, and the rats were sacrificed 2 times per week by Hemin intraperitoneal injection on day 28 after inflammation, and homogenate of the tissue from the feet after inflammation was taken and analyzed by PCR for IL-17, IL-23 and Foxp3mRNA expression.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, molecular biology, pharmacology, cell biology, and the like, which are within the skill of the art. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one aspect, the invention relates to the use of said hemin for the preparation of a medicament for the prevention and treatment of rheumatoid arthritis.

Hemin, on the other hand, can form a stable complex with proteins, in some embodiments albumin, which can be bovine serum albumin or human serum albumin, which are commonly found in the biological field. The basic amino acid Hemin is selected to form a complex (the porphyrin ring of Hemin has carboxyl which can form a complex with the basic amino acid in a salt form, and possibly an electron-donating group (guanidino, imidazolyl and amino) of the basic amino acid can form a coordination bond with an N atom with an empty orbit on the porphyrin ring), and in one example, the amino acid is arginine. Of course, depending on the nature of the amino acid, lysine or histidine may also be used.

In one aspect, the invention relates to a medicament for treating rheumatoid arthritis, wherein the active ingredient of the medicament comprises hemin. The medicine can also comprise the pharmaceutically acceptable auxiliary materials, and can be selected from a filler, a bonding agent, a lubricant, a disintegrating agent, a cosolvent, an adsorption carrier, a solvent, an antioxidant, an adsorbent, an osmotic pressure regulator and a pH regulator according to the requirements of various dosage forms. In addition, the pharmaceutical dosage form can be solution injection, lyophilized powder injection, subcutaneous implant, tablet (such as orally disintegrating tablet, sustained release tablet), capsule, oral liquid (such as syrup), granule, dripping pill, patch, pellet, microcapsule, liposome, microsphere, or other sustained release preparation or controlled release preparation. The administration mode of the medicine can be intravenous injection, intraperitoneal injection, subcutaneous injection or oral administration in vivo. The effective dosage can be 10-200 mg/kg.

The present invention is further illustrated by the following specific examples, which are not intended to limit the scope of the present invention.