WO2015058664A1 - 淫羊藿苷元在制备预防或治疗血细胞减少药物中的用途 - Google Patents

淫羊藿苷元在制备预防或治疗血细胞减少药物中的用途 Download PDF

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WO2015058664A1
WO2015058664A1 PCT/CN2014/088944 CN2014088944W WO2015058664A1 WO 2015058664 A1 WO2015058664 A1 WO 2015058664A1 CN 2014088944 W CN2014088944 W CN 2014088944W WO 2015058664 A1 WO2015058664 A1 WO 2015058664A1
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icariin
group
use according
thrombocytopenia
drug
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PCT/CN2014/088944
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English (en)
French (fr)
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WO2015058664A8 (zh
Inventor
赵志全
姚景春
李欣
关永霞
李光艳
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鲁南制药集团股份有限公司
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Priority claimed from CN201310493718.4A external-priority patent/CN104546823B/zh
Priority claimed from CN201310646101.1A external-priority patent/CN104688722B/zh
Application filed by 鲁南制药集团股份有限公司 filed Critical 鲁南制药集团股份有限公司
Priority to JP2016525940A priority Critical patent/JP6294476B2/ja
Priority to KR1020167013370A priority patent/KR101891505B1/ko
Priority to US15/030,671 priority patent/US10555962B2/en
Priority to EP14855388.6A priority patent/EP3061452B1/en
Priority to ES14855388T priority patent/ES2698620T3/es
Publication of WO2015058664A1 publication Critical patent/WO2015058664A1/zh
Publication of WO2015058664A8 publication Critical patent/WO2015058664A8/zh
Priority to US16/724,573 priority patent/US10799521B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present application relates to the medical use of icariin, in particular the use of icariin for the preparation of a medicament for preventing or treating cytopenia.
  • Normal blood contains a large number of cells, including oxygen-carrying red blood cells and anti-infected white blood cells.
  • White blood cells include neutrophils, eosinophils, and basophils.
  • White blood cells are made from bone marrow hematopoiesis.
  • Normal blood also contains platelets. Platelets are tiny pieces of cells that cause blood to clot.
  • Blood cells in the human body are produced by the hematopoietic system.
  • the human hematopoietic system consists of a small number of bone marrow hematopoietic stem cells and different series of hematopoietic cells at different developmental stages. It is very sensitive to various physical and chemical and metabolic factors, such as body fatigue, exposure to radiation or certain chemotherapy drugs. This leads to diseases such as anemia and myelosuppression caused by blood cell reduction.
  • primary cytopenia such as primary thrombocytopenic purpura.
  • Radiotherapy and chemotherapy are therapies that use radiation and cytotoxic agents to treat cancer.
  • radiotherapy and most chemotherapy therapies are non-specific and they are toxic to normal and rapidly dividing cells.
  • High dose radiation is also toxic to normal and rapidly dividing cells. This often leads to various side effects in patients undergoing chemotherapy and radiation therapy.
  • bone marrow is particularly sensitive to proliferation-specific therapies such as chemotherapy or radiation therapy.
  • Myelosuppression a decrease in blood cell production in the bone marrow, is a side effect of this type of manifestation of decreased myeloproliferative function, decreased blood cell count, decreased peripheral blood leukocytes, neutropenia and/or thrombocytopenia, and even aplastic disorders.
  • Anemia which causes serious harm to the quality of life of patients and is even life-threatening.
  • patients receiving radiotherapy and chemotherapy are highly susceptible to injury and have varying degrees of myelosuppression, manifested by decreased peripheral blood leukocyte counts, decreased neutrophil counts, and/or thrombocytopenia.
  • Human peripheral blood neutrophils account for about 50-70% of the total number of white blood cells.
  • the increase and decrease directly affect the total number of white blood cells, that is, the increase of neutrophils and the increase of white blood cells.
  • the reduction of neutrophils and the total number of white blood cells Reduced.
  • the quantitative correlation between the two is also consistent in meaning, that is, the significance of neutrophil increase and decrease is basically consistent with the significance of the increase and decrease of the total number of white blood cells.
  • Patients in myelosuppressive state are susceptible to infection. Neutrophil and platelet deficiency are major causes of morbidity and mortality after cancer treatment and result in high costs for cancer treatment.
  • hematopoietic growth factors that have just been listed in recent years, such as white blood energy (rhGM-GSF) and whirlpool blood (rhG-CSF), are effective in raising white blood cells, but they are not expensive for most patients because they are expensive.
  • rhGM-GSF, rhG-CSF can not be used at the same time as chemotherapy, and can not be used for prevention. It can only be used when leukopenia occurs, otherwise there will be toxic side effects.
  • hematopoietic growth factors such as IL-6, IL-3 and other gene therapy
  • Self-marrow transplantation is often used in combination with high-dose chemotherapy, but it is difficult to apply it repeatedly. Therefore, it is necessary to find a safe, effective and inexpensive method to prevent and treat cytopenia, especially the cytopenia caused by side reactions of chemotherapy and radiotherapy, increase the number of white blood cells after chemotherapy and radiotherapy, and improve chemotherapy and radiotherapy for tumors. The effect, prolonging the survival of cancer patients and improving the quality of life are very important.
  • thrombocytopenia a disease caused by thrombocytopenia in the peripheral blood and causing bleeding of the skin and mucous membranes, mainly manifested as white skin blemishes and ecchymoses, mucosal bleeding, epistaxis and gum bleeding, purple blood cells in the oral mucosa and tongue Etc., is a common type of disease characterized by coagulopathy and hemorrhage. It can cause major bleeding and life-threatening. It accounts for about 30% of bleeding diseases.
  • the causes of thrombocytopenia can be divided into: (1) reduced or ineffective death of platelets: both hereditary and acquired, and reduced acquired platelet production is due to certain factors such as drugs, malignant tumors, infections, ionizing radiation, and other hematopoiesis.
  • Stem cells or affect their proliferation in the bone marrow can affect multiple hematopoietic cell systems, often accompanied by varying degrees of anemia, leukopenia, bone marrow megakaryocytes significantly reduced;
  • excessive platelet destruction including congenital and acquired two Kind.
  • Excessive platelet destruction includes both immunological and non-immune.
  • Excessive immune platelet destruction is common with idiopathic thrombocytopenic purpura and drug thrombocytopenia.
  • Excessive destruction of non-immune thrombocytopenia includes infection, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, etc.; (3) excessive platelet retention in the spleen: most common in hypersplenism.
  • the pathogenesis of thrombocytopenia includes: 1) immunological factors: clinically visible severe liver disease, lupus erythematosus, idiopathic thrombocytopenic purpura and other immune diseases cause thrombocytopenia, antibodies destroy platelets; 2) infectious factors: caused Common factors of thrombocytopenia, bacterial and viral infections can directly damage hematopoietic cells, reduce platelet growth, aplastic anemia, acute leukemia and other acute attacks often accompanied by serious infections, bleeding tendency, such as blemishes, erythema or Unexplained nasal bleeding and other manifestations; 3) drug factors: due to certain drugs caused by a decrease in platelet counts in peripheral blood caused by bleeding disorders; 4) abnormal platelet function: such as thrombocytopenia, giant platelet syndrome.
  • ITP immune thrombocytopenic purpura
  • TTP thrombotic thrombocytopenic purpura
  • ITP is the most common cause of thrombocytopenia seen in clinical practice. ITP has long been considered a hemorrhagic disease caused by unexplained thrombocytopenia and is therefore referred to as idiopathic thrombocytopenic purpura. Later, ITP patients were found to have autoantibodies that recognize their own platelet antigens. The combination of autoantibodies and platelet antigens resulted in shortened platelet life, increased destruction, and decreased platelet count, indicating that the disease is a hemorrhagic disease associated with immune response.
  • immune thrombocytopenic purpura is a relatively common hemorrhagic disease. It is estimated that the incidence of ITP in the population is 1/10000, which can occur at any age. Most children are acute, most of them are chronic, mostly in children and young people. The clinical manifestations are skin defects and ecchymosis, skin mucous membrane bleeding, severe patients with joint pain or abdominal pain, blood in the stool, vomiting blood, and collapse. In severe cases, it can develop into purpuric nephritis.
  • Primary thrombocytopenic purpura is an immune syndrome that is a common hemorrhagic disease characterized by the presence of anti-platelet antibodies in the blood circulation, causing excessive destruction of platelets and causing purpura; while megakaryocytes in the bone marrow are normal or increasing, naive Chemical.
  • Epimedium is Epimedium brevicornum Maxim., Epimedium sagittatum Maxim., Epimedium pubescens Maxim., or Epimedium koreanum Nakai ) dry stems and leaves. Clinically, it is mainly used for kidney yang deficiency, impotence, frequent urination, infertility; rheumatic pain, limb numbness, weak bones, difficulty walking; kidney yang deficiency, asthma cough and shortness of breath. Icariin can increase blood flow in the heart and brain, promote hematopoietic function, immune function and bone metabolism, and has the effects of tonifying kidney and strengthening yang, anti-aging and anti-tumor.
  • Icaridin is a polyhydroxyflavonoid monomer in Epimedium of Epimedium.
  • Pharmacological studies have shown that IT anti-osteoporosis is stronger than other flavonoid glycosides in Epimedium, and it has the effect of promoting osteoblast activity and inhibiting osteoclast activity in vitro.
  • patent application CN101637467A discloses the use of icariin in the preparation of a medicament for treating osteoporosis.
  • Patent US 6,399,579 discloses the use of icariin for the treatment of sexual dysfunction.
  • icariin has the activity of preventing or treating cytopenia, particularly against immune thrombocytopenia, or radiation or chemical-induced myelosuppression.
  • the purpose of the present application is to provide a medicament for preventing and/or treating cytopenia such as thrombocytopenia, particularly immune thrombocytopenia or for preventing bone marrow suppression caused by radiation or chemicals.
  • cytopenia such as thrombocytopenia, particularly immune thrombocytopenia or for preventing bone marrow suppression caused by radiation or chemicals.
  • the present inventors have long been devoted to the study of the pharmacological activity of icariin, and unexpectedly discovered in the course of further in-depth research, icariin has unexpected effects in reducing cytopenia and treating thrombocytopenia. Activity.
  • the first aspect of the present application relates to the use of icariin for the preparation of a medicament for preventing or treating cytopenia.
  • the blood cells include white blood cells, red blood cells, neutrophils or platelets, and the main manifestation of the cytopenia refers to a decrease in peripheral blood leukocytes or a decrease in platelets.
  • the cytopenia includes myelosuppression caused by radiation or chemicals, or a primary myeloproliferative disorder.
  • the present application provides the use of icariin for preventing or treating radiation or chemical-induced myelosuppression.
  • the chemical described therein is a chemotherapeutic drug capable of causing myelosuppression, particularly a chemotherapeutic drug which can be used for treating cancer.
  • the chemotherapeutic drug for treating cancer is Docetaxel, Teggio (S-1) or a combination thereof, but is not limited thereto.
  • the effect of icariin on chemotherapy-induced myelosuppression in tumor mice in Example 22 of the present application shows that anti-tumor drugs (such as docetaxel or tega) are combined with icariin.
  • the total number of white blood cells, platelet count and neutrophil count in tumor mice were significantly higher than those in the anti-tumor monotherapy group, while the weight, diet and living status of the animals in the combination group were compared with the blank control group. There is no significant difference, that is, the combination of icariin can not only significantly increase the blood cells of tumor mice, but also has low toxic side effects.
  • the effect of icariin on the blood cell count of 60 Co-irradiated mice in Example 23 of the present application showed that icariin combined with 60 Co irradiation can effectively increase the number of white blood cells and platelets in mice, and the blood cell rise The high effect was significantly better than the epimedium extract group.
  • the above examples show that icariin has an unexpected effect of alleviating radiotherapy or chemical-induced myelosuppression.
  • Example 24 of the present invention the effects of icariin on the white blood cells and platelets of NOD/Ltj mice were confirmed by the high-dose and low-dose groups of icariin for the number of white blood cells and the number of platelets in NOD/Ltj mice. It also has a significant increase effect, and its elevation effect is significantly better than that of the commonly used therapeutic drug interleukin. Since NOD/Ltj is an abnormal immune system mouse with a significant decrease in the number of platelets and white blood cells compared with normal mice, this suggests that epileptic sheep Glycosides have a good therapeutic effect on primary myeloproliferative disorders.
  • the application provides the use of icariin for the preparation of a medicament for the prevention or treatment of thrombocytopenia.
  • the thrombocytopenia may be immune thrombocytopenia, and specifically may be chronic idiopathic thrombocytopenic purpura.
  • the thrombocytopenia may also be secondary thrombocytopenia caused by myelosuppression.
  • Example 26 of the present application confirmed that icariin can exert a positive therapeutic effect on chronic ITP model rats, and the high and middle dose groups have a significantly increased rat platelet effect (P ⁇ 0.01), which is in liter. The high platelet is superior to the positive control drug icariin.
  • the pharmaceutical composition described in the present application can be applied to the preparation of a pharmaceutical preparation for treating thrombocytopenia, and is particularly useful for preparing a pharmaceutical preparation for treating immune thrombocytopenia.
  • the thrombocytopenia is preferably secondary thrombocytopenia caused by myelosuppression.
  • the myelosuppression described therein is myelosuppression induced by a chemical drug.
  • Example 28 of the present application confirmed that the icariin-administered group and the icariin group have a positive therapeutic effect on mouse cyclophosphamide-induced thrombocytopenia in mice, and can increase the cause of cyclophosphamide.
  • the high dose group of icariin and the middle dose of icariin were significant compared with the icariin group. difference.
  • Yet another aspect of the present application is to provide a method of preventing or treating cytopenia or thrombocytopenia by icariin.
  • the method comprises administering an effective amount of icariin to an individual in need thereof.
  • the individual is preferably a human.
  • the icariin aglycon disclosed in the present application is used in the preparation of a medicament for treating cytopenia or thrombocytopenia, usually in the form of a pharmaceutical composition, which can be administered orally or parenterally, or pharmaceutically and pharmaceutically
  • a pharmaceutical composition which can be administered orally or parenterally, or pharmaceutically and pharmaceutically
  • the compositions e.g., tablets, sustained release preparations, capsules, injections, solutions formed by the accepted carriers, excipients, and other additives are administered orally or parenterally.
  • the dosage for oral administration is 0.1 mg/kg/d-100 mg/kg/d; non-oral administration includes but not limited to subcutaneous, intradermal, arterial, intravenous, intramuscular, intra-articular, intrathecal, intracranial , chest, intra-abdominal injection or infusion, nasal, buccal, sublingual, tracheal, urethra, rectum or local topical administration of lesions.
  • non-oral administration includes but not limited to subcutaneous, intradermal, arterial, intravenous, intramuscular, intra-articular, intrathecal, intracranial , chest, intra-abdominal injection or infusion, nasal, buccal, sublingual, tracheal, urethra, rectum or local topical administration of lesions.
  • non-oral administration includes but not limited to subcutaneous, intradermal, arterial, intravenous, intramuscular, intra-articular, intrathecal, intracranial , chest, intra-abdominal injection or infusion, nasal, buccal, sublingual,
  • icariin can be used as the sole active ingredient for preventing and/or treating diseases associated with cytopenia or thrombocytopenia, preferably for bone marrow suppression caused by radiation or chemicals, or for immunological thrombocytopenia. Symptoms; may also be combined with other drugs for the prevention and/or treatment described.
  • drugs that are administered in combination with icariin or prepared as a pharmaceutical composition may be one or more other drugs that treat leukopenia (eg, vitamin B4, blood, shark, coenzyme A), or other One or more drugs that increase the platelet content (eg, interleukin-II, low-dose glucocorticoids, such as prednisone, etc.) to prevent or treat cytopenia, particularly for preventing or treating myelosuppression.
  • the icariin can be further increased in platelet content or leukocyte content.
  • the weight ratio of the two drugs can be appropriately adjusted according to the condition and symptoms of the patient, and the weight ratio of the other drugs to the icariin is preferably in the range of (0.005-100): 1, more preferably (0.005-50): 1, more preferably (0.01-100): 1, such as (0.05-25): 1.
  • Routes of administration of icariin and optionally other drugs include gastrointestinal routes and parenteral routes including, but not limited to, subcutaneous, intradermal, arterial, venous, muscular, joint, sheath Internal, intracranial, thoracic, intraperitoneal injection or infusion, nasal, buccal, sublingual, tracheal, urethra, rectum or local administration of lesions.
  • icariin can be administered before, during, or after radiation or chemotherapy.
  • icariin can be administered simultaneously with other drugs, or can be administered sequentially.
  • Another aspect of the present application relates to a pharmaceutical composition (or formulation) for preventing or treating a disease associated with cytopenia or thrombocytopenia comprising icariin and a pharmaceutically acceptable pharmaceutically acceptable excipient.
  • the pharmaceutical composition is preferably used for preventing or treating myelosuppression caused by radiation or chemicals.
  • compositions include, but are not limited to, solids, liquids, oils, emulsions, gels, aerosols, inhalants, sprays, capsules, pills, patches, suppositories, and the like.
  • the compositions When administered orally, the compositions may be formulated as tablets, granules or capsules.
  • lactose or starch can be used as a carrier, and gelatin, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone and the like are suitable binders or granules.
  • starch or microcrystalline cellulose may be used, and talc powder, colloidal silica gel, glyceryl stearate, calcium stearate or magnesium is often used as a suitable anti-adhesive and lubricant.
  • tablets can be prepared by compressing wet granules.
  • the active ingredient is mixed with a carrier and optionally with a disintegrating additive, the mixture is granulated with an aqueous solution of the binder, an alcoholic or aqueous alcoholic solution in a suitable apparatus, and the dried granules are subsequently added to other granules.
  • Decomposing agent, lubricant and anti-adhesive agent sheet may be used as the disintegrating agent.
  • the injection preparation is one of an injection solution, a lyophilized powder injection, and an infusion preparation, and the pharmaceutical auxiliary in the injection preparation is one or more of mannitol, glucose, sorbitol, PEG, ethanol, and physiological saline.
  • An injection preparation containing PEG is preferred.
  • the icariin-containing pharmaceutical preparation suitable for the above pharmaceutical use contains icariin in an amount of 0.1 to 500 mg per preparation unit.
  • the "ician aglycone" described herein may be extracted, biologically prepared, or chemically prepared, including icariin, a pharmaceutically acceptable salt or hydrate thereof.
  • the purity of the icariin aglycone contained in the present application (such as HPLC detection purity) is ⁇ 90%, ⁇ 95% or ⁇ 98%.
  • the preparation method of icariin aglycone disclosed in the prior art before the application of the present application can be used as a preparation process of the icariin aglycone described in the present application.
  • myelosuppression refers to a condition in which blood cells, including white blood cells, red blood cells, neutrophils, or platelets, are reduced individually or simultaneously, as evidenced by a decrease in blood cell production. Healthy bone marrow produces large amounts of red blood cells, white blood cells, and platelets every day. Under myelosuppression, these cells produced by the bone marrow are reduced.
  • One feature of myelosuppression is a decrease in white blood cell production. This reduction in white blood cell production can be caused by certain treatments, especially cancer treatments such as chemotherapy and radiation therapy.
  • the myelosuppression described in the present application is manifested by a decrease in the number of peripheral blood leukocytes or a decrease in platelets, and a poor myeloproliferation.
  • the decrease in peripheral blood leukocytes refers to a decrease in peripheral blood neutrophils.
  • the “radiation” may be radiation received during radiotherapy or radiation received for other reasons such as work, environment, and the like.
  • the radiation described in the above uses is radiation that is received during the course of radiation therapy, such as radiation during the course of receiving radiation therapy for treating cancer.
  • the "chemical” can refer to any drug or chemical agent that is capable of causing a decrease in blood cell production.
  • the chemical described in the above uses is a chemotherapeutic drug for treating cancer.
  • the chemotherapeutic drugs include, but are not limited to, alkylating agents, anti-metabolites, antibiotics, plant drugs, and hormonal drugs.
  • the alkylating agent drugs include, but are not limited to, nitrogen mustard, cyclophosphamide, thiotepa, cyclohexyl nitrosourea, maliland, nitroneamine, procarbazine, etc.;
  • the antimetabolites include but are not limited to Fluorouracil (5-FU), furan fluorouracil (FT-207), difluridine (bisfuridine FD-1), effluent (UFT), fluoroiron (5-DFUR), methotrexate (MTX) ), Ammonia (white blood), cytarabine (Ara-c), cyclocytidine, chlorcycline, hydroxyurea (HU), inosine dialdehyde (inosine dialdehyde), adenosine dialdehyde (adenosinediialde-hgde) ), guanazole, 6- ⁇ (6-MP);
  • the antibiotics include, but are not limited to, pen
  • pharmaceutically acceptable pharmaceutical excipient refers to any substance that does not interfere with the physiological action of icariin and is not toxic to a subject including humans. Suitable pharmaceutical excipients have been described in detail in "Drug Excipients" (page 123, Sichuan Science and Technology Press, 1993, edited by Luo Mingsheng and Gao Tianhui).
  • pharmaceutical excipients commonly used in the preparation of microemulsion preparations include, but are not limited to, soybean oil, polyoxyethylene-23-lauryl ether, 1,2-propanediol, hydrogenated coconut glycerin, lauroyl polyethylene glycol-32-glycerol Ester, polyethylene glycol 3350, safflower oil, cottonseed oil, decaglyceryl monostearate; pharmaceutical excipients commonly used in the preparation of dropping pills, including but not limited to polyethylene glycol 6000, polyethylene glycol 1000; preparation
  • Pharmaceutical excipients commonly used in capsule formulations include, but are not limited to, lactose and corn starch.
  • Pharmaceutically acceptable carriers commonly used in the preparation of soft capsule formulations include, but are not limited to, medium chain fatty acid glycerides, polyoxyethylene castor oil, 1,2-propanediol, and the like.
  • thrombocytopenia refers to a disease in which blood platelets and visceral hemorrhage are caused by thrombocytopenia in peripheral blood, mainly manifested as white hair spots and ecchymoses, mucosal bleeding, epistaxis and gum bleeding, oral cavity. Purple blood cells appear in the mucosa and tongue. It is a common type of disease characterized by coagulopathy and hemorrhage. It can cause major bleeding and life-threatening. It accounts for about 30% of bleeding diseases. It can be immune thrombocytopenia or secondary thrombocytopenia.
  • the "immune thrombocytopenia” is a hemorrhagic disease caused by a disorder of the immune system, which mainly refers to immune thrombocytopenic purpura (ITP), which is a relatively common hemorrhagic disease, estimated in the human population.
  • ITP immune thrombocytopenic purpura
  • the incidence of ITP is 1 in 10,000 and can occur at any age.
  • the clinical manifestations are skin defects and ecchymosis, skin mucosal bleeding, severe patients with joint pain or abdominal pain, blood in the stool, vomiting blood, collapse, etc., severe cases can develop purpuric nephritis.
  • thrombocytopenic purpura or "idiopathic thrombocytopenic purpura” is a kind of immune thrombocytopenia, and is an acquired hemorrhagic disease of unknown cause. It is characterized by thrombocytopenia, normal or increased bone marrow megakaryocytes, and lack of any cause. The disease occurs mostly in children and young people. Most children are acute, and most of them are chronic. It is characterized by the presence of anti-platelet antibodies in the blood circulation, causing excessive destruction of platelets and causing purpura; while megakaryocytes in the bone marrow are normal or increasing. Childish.
  • Drug-induced immune thrombocytopenia Less, other immune thrombocytopenia such as Evans syndrome, chronic lymphocytic leukemia, various acute leukemias, lymphoma, systemic lupus erythematosus, rheumatoid arthritis, hyperthyroidism and so on.
  • the secondary thrombocytopenia described herein is primarily caused by radiation or chemical drug-induced myelosuppression.
  • the icariin aglycone of the present application has at least the following beneficial effects in preventing or treating a disease associated with cytopenia, particularly prevention of bone marrow suppression caused by radiation or chemicals, but is not limited thereto:
  • icariin is an anticancer drug, and even if it is combined with docetaxel or tegafur for anti-tumor, icariin plays a role in inhibiting tumor growth. It relieves the bone marrow suppression caused by docetaxel or tigeo. It can be seen that icariin can be used at the same time as other drugs, and can also be used for prevention, instead of being used only when leukopenia occurs, which can promote bone marrow hematopoiesis and increase the number of white blood cells while ensuring tumor inhibition rate.
  • mice in the icariin monotherapy group or the combination group had no significant difference in body weight during the administration period, and there was no significant difference in the animal's diet and living conditions. . This indicates that icariin is an antitumor drug and a myelosuppressive relief drug with no significant side effects.
  • Icariin has a significant therapeutic effect when used in thrombocytopenia caused by various factors.
  • Example 26 of the present application demonstrates that icariin is superior to the positive control drug icariin in raising platelets in chronic ITP model rats. The high and middle dose groups significantly increased rat platelet function (P ⁇ 0.01).
  • Example 27 of the present application confirms that it is kinky Astragalin has a positive therapeutic effect in the treatment of active immune thrombocytopenia in mice.
  • Icariin has a positive therapeutic effect on secondary thrombocytopenia caused by myelosuppression, suggesting that it can be used in conjunction with other chemicals to reduce the damage of chemicals (such as chemotherapy drugs) to the body.
  • Example 28 of the present application confirmed that the icariin-administered group and the icariin group have positive therapeutic effects on mouse cyclophosphamide-induced thrombocytopenia in mice, and can increase cyclophosphamide-induced mice.
  • the number of platelets in the thrombocytopenic model in which the amount of platelets in the mouse thrombocytopenia-induced mouse thrombocytopenic model was higher in the drug-administered group than in the icariin group, the icariin high-dose group and The middle dose group of icariin has a significant difference compared with the icariin group.
  • Epimedium aglycone is an effective active ingredient extracted from traditional Chinese medicine Epimedium. It is not only effective in the treatment of thrombocytopenia, but also suitable for combination therapy, and its drug toxicity is extremely low, which can greatly increase the patient's medication. Compliance, thus ensuring the therapeutic effect of the drug.
  • icariin has a variety of preparation methods, the preparation process is simple, the cost is low, and the treatment cost of patients with thrombocytopenia can be greatly reduced.
  • Example 1 Icariin a microemulsion preparation
  • Preparation process Weigh the prescribed amount of soybean oil, polyoxyethylene-23-lauryl ether, 1,2-propanediol, mix well, stir evenly, then add icariin to dissolve, or ultrasonically to accelerate dissolution, to clarify The solution is the icariin a microemulsion preparation.
  • the particle size was measured by a laser granulometer, and the average particle diameter was 15 nm.
  • Example 2 Icariin a microemulsion preparation
  • Preparation process Weigh the prescribed amount of hydrogenated coconut oil, lauroyl polyethylene glycol-32-glyceride, 1,2-propanediol, polyethylene glycol 3350, mix and stir evenly, then add icariin to dissolve Ultrasonic treatment can also be used to accelerate the dissolution, and a clear solution is obtained, which is an icariin a microemulsion preparation.
  • the particle size was measured by a laser granulometer, and the average particle diameter was 40 nm.
  • Preparation process add icariin to the prescribed amount of PEG-400, stir and dissolve, add 0.9% sodium chloride solution to 10L, stir evenly, add 0.5% needle with activated carbon, stir, decarbonize, that is.
  • Preparation process Mix the prescribed amount of ethanol and Tween-80 uniformly, add icariin, stir and dissolve, add water for injection to 10L, stir evenly, add 0.5% needle with activated carbon, stir, decarbonize, that is.
  • Preparation process adding a prescribed amount of ethanol to icariin, stirring and dissolving, adding water for injection to 10L, stirring evenly, adding 0.5% needle with activated carbon, stirring, decarburization, that is.
  • Example 6 Icariin a drop pellet preparation
  • Preparation process Weighing the icariin aglycone which has been prescribed to a 100 mesh sieve, and adding a mixture of a predetermined amount of polyethylene glycol 6000 and polyethylene glycol 1000 which has been heated and melted on a water bath, and thoroughly stirred, so that Uniform, placed in a dropper bottle, dripped at 95 ⁇ 2°C; dripped into a glass condensation column containing 4-6mL of methyl silicone oil, taken out after molding, and sucked off the adhered methyl group with absorbent paper Silicone oil, that is.
  • Example 7 Icariin agaric soft capsule preparation
  • Preparation process Weigh the prescription medium-chain fatty acid glyceride, polyoxyethylene castor oil, 1,2-propanediol, absolute ethanol, mix and stir evenly, then add icariin to dissolve, or ultrasonically to accelerate dissolution , to clarify the concentrate, which is the icariin a microemulsion concentrate.
  • the microemulsion concentrate obtained above was diluted with water to a clear solution in a weight ratio of 1:10-20 to obtain a soft capsule microemulsion content.
  • the prescribed amount of gelatin, glycerin and purified water are weighed, mixed and pressed into a rubber skin, and then weighed the prescribed amount of Eudragit L30D-55, triethyl citrate, talc powder and purified water to obtain an enteric coating liquid.
  • the soft capsule content of the soft capsule containing icariin is wrapped in a soft capsule, and the enteric soft capsule is prepared by coating the soft capsule on the casing.
  • Preparation process 100g of icariin, 120g of lactose and 130g of corn starch are mixed in a mixer for 10-15 minutes, and 5g of magnesium stearate is added and mixed for 1-3 minutes, and 1000 capsule shells can be charged.
  • Preparation process icariin and auxiliary microcrystalline cellulose, sodium carboxymethyl starch are uniformly mixed, and appropriate amount of starch slurry is added to make soft material, and then granulated by 16 mesh sieve.
  • the wet granules are dried at 60 ° C, the dry granules are sieved through a 20 mesh sieve, the fine powder in the dry granules is sieved out, mixed with magnesium stearate, and then mixed with the dry granules, and compressed, about 200 mg per tablet, .
  • Preparation process Weigh the prescribed amount of icariin aglycone for injection, and add appropriate amount of water for injection to dissolve. Then, adding a predetermined amount of pre-sterilized depyrogenation treatment, and adding water for injection to a prescribed 1000 ml; adding the above-mentioned chemical solution to the activated carbon 5 g for injection, heating at 60-80 ° C for 30 minutes, using a filter Filter and collect the filtrate. The filtrate was subjected to a positive pressure sterilization filtration by a sterile operation method, filtered through a 0.22 ⁇ M microporous membrane filter, and the filtrate was subjected to pyrogen examination and semi-finished product inspection, and then the vials were dispensed.
  • Preparation process the prescription amount of PEG-400 is added to icariin, stirred and dissolved, adding 0.9% sodium chloride solution to 10 ml, stirring uniformly, adding 0.5% needle with activated carbon, stirring, decarbonization, that is.
  • Preparation process Mix the prescribed amount of ethanol and Tween-80 uniformly, add icariin, stir and dissolve, add 0.9% sodium chloride solution to 10ml, stir evenly, add 0.5% needle with activated carbon, stir, decarbonize That's it.
  • Preparation process Mix the prescribed amount of ethanol and Tween-80 uniformly, add icariin, stir and dissolve, add 0.9% sodium chloride solution to 10ml, stir evenly, add 0.5% needle with activated carbon, stir, decarbonize That's it.
  • Preparation process Mix the prescribed amount of ethanol and Tween-80 uniformly, add icariin, stir and dissolve, add 0.9% sodium chloride solution to 10ml, stir evenly, add 0.5% needle with activated carbon, stir, decarbonize That's it.
  • Preparation process adding a prescribed amount of ethanol to icariin, stirring and dissolving, adding water for injection to 10 ml, stirring uniformly, adding 0.5% needle with activated carbon, stirring, decarbonizing, that is.
  • Preparation process adding a prescribed amount of ethanol to icariin, stirring and dissolving, adding water for injection to 10 ml, stirring uniformly, adding 0.5% needle with activated carbon, stirring, decarbonizing, that is.
  • Preparation process The prescription amount of icariin, starch and dextrin are evenly mixed.
  • an appropriate amount of 50% ethanol is added to the mixed powder, uniformly mixed, and the soft material is made into a wet granule through a 18-mesh nylon sieve, and dried at about 60 ° C, and the dry granule moisture should be controlled to 1.5% or less.
  • the 20 mesh sieve is granulated, and then mixed with magnesium stearate and compressed to obtain.
  • Preparation process icariin, microcrystalline cellulose, micro-powder silica gel is crushed through a 100 mesh sieve and mixed, and the capsule is directly filled.
  • Preparation process The prescription amount of icariin, starch, dextrin and sucrose powder are evenly mixed. In addition, an appropriate amount of 80% ethanol is added to the mixed powder, and the mixture is uniformly mixed, and the soft material is made into a wet granule through a 18-mesh nylon sieve, dried at about 60 ° C, and sieved by a 20-mesh sieve to obtain a pellet.
  • Example 21 Epimedium aglycone sustained release tablet
  • Preparation process the prescription amount of icariin, lactose and sustained release agent hydroxypropylmethyl cellulose are uniformly mixed into the binder polyvinylpyrrolidone granules, dried at 40 ° C -80 ° C, and the particles are dried. Add a prescription amount of lubricant micro-silica gel to the dry granules, mix well, and shape the stamping sheet.
  • Test animals Kunming mice (purchased from China National Institute for the Control of Pharmaceutical and Biological Products, Laboratory Animal License No.: SCXKII-00-0010), male and female, 7 weeks old, 18-22 g, test temperature (20 ⁇ 1) °C , humidity 40% ⁇ 70%, free drinking water, normal feeding.
  • Mouse ascites s180 cells were cultured in 1640 medium, routinely cultured at 37 ° C, 5% CO 2 , subcultured every two days, and prepared to a density of 3.0 ⁇ 10 7 with physiological saline in the logarithmic growth phase.
  • Ml single cell suspension was injected into the abdominal cavity of mice under aseptic conditions. The abdominal cavity of the mice was obviously swollen 7 days after inoculation. At this time, the neck was sacrificed and placed in a beaker containing 75% ethanol. After 3 minutes, the sterilized mice were placed in a clean bench, the abdomen was exposed, and ascites was taken with a sterile syringe and placed in a sterile reagent bottle for use. The ascites was counted with trypan blue, diluted with physiological saline, and the number of cells was adjusted to 2.0 ⁇ 10 7 /ml, and inoculated into the right axilla of the mouse, 0.2 ml each.
  • mice were randomly divided into the following 6 groups, 10 in each group, half male and half female.
  • Group 1 was a model control group: 0.9% saline was intraperitoneally injected into experimental animals (same as the following experiments);
  • Group 2 is the docetaxel group: 75 mg/m 2 /d docetaxel
  • Group 3 was the Tiggio group: 222.2 mg/m 2 /d teggio;
  • Group 4 is the icariin group: 10 mg/kg/d icariin a microemulsion preparation
  • the fifth group is multi + kinky group (Docetaxel + icariin group): 75 mg / m 2 / d docetaxel + 10 mg / kg / d icariin;
  • the sixth group was the dioxin group (Tiggio + icariin group): 222.2 mg/m 2 /d tigeol + 10 mg/kg/d icariin.
  • Docetaxel and icariin were administered by tail vein injection at a dose of 10 ml/kg. Tegafur was administered by intragastric administration at a dose of 40 ml/kg. Each group was administered once a day for a total of 10 days. During the experiment, the diet, survival and behavioral activities of the animals were observed every day. The body weight was measured daily. After the end of the experiment, the anesthesia was dissected, blood was taken through the abdominal main vein, blood routine was measured, and the total number of white blood cells, the total number of platelets and the total number of neutrophils were examined.
  • mice of the icariin alone or the combination group had no significant difference in body weight during administration with the model group. There was no significant difference in the amount of food consumed and the state of life. This indicates that icariin is an antitumor drug and a myelosuppressive relief drug with no significant side effects.
  • icariin as an anticancer drug is used in combination with docetaxel or tegafur to prevent tumor growth, and it not only inhibits tumor growth, but also alleviates Dorsey.
  • icariin can be used at the same time as other drugs, and can also be used for prevention, instead of being used only when leukopenia occurs, which can promote bone marrow hematopoiesis and increase the number of white blood cells while ensuring tumor inhibition rate. Increases the number of neutrophils and reduces side effects to some extent.
  • Example 23 Effect of icariin on the number of blood cells in 60 Co-irradiated mice
  • Test animals Kunming mice (purchased from China National Institute for the Control of Pharmaceutical and Biological Products, Laboratory Animal License No.: SCXKII-00-0010), male and female, 7 weeks old, 18-22 g, test temperature (20 ⁇ 1) °C , humidity 40% ⁇ 70%, free drinking water, normal feeding.
  • mice Except the normal group (10 rats), 4Gy irradiation of 60 Co was performed on the other groups of mice, and the whole body irradiation was performed at a dose of 4 Gy, and the absorbed dose rate was 0.88 Gy/min. On the 3rd, 7th, and 10th after the irradiation, blood was taken from the iliac vein to detect the whole blood cell count. Mice in which the number of white blood cells was less than 3.0 ⁇ 10 9 /L or the number of platelets was less than 500 ⁇ 10 9 /L were taken out in two consecutive whole blood cell assays, and the remaining mice were experimental mice.
  • mice that met the experimental requirements after irradiation were randomly divided into the following model control group, epimedium extract group and icariin group, 10 in each group, half male and half female. Each group was treated or administered as follows.
  • the first group was the normal group: 0.9% normal saline was intraperitoneally injected, and the administration volume was 10 ml/kg;
  • the second group was the model control group: 0.9% normal saline was intraperitoneally injected, and the administration volume was 10 ml/kg;
  • the third group was epimedium extract group: 3.5 ml/kg/d epimedium aqueous extract was intraperitoneally injected;
  • the fourth group was the icariin group: 10 mg/kg/d icariin was intraperitoneally injected, and the dosage volume was 10 ml/kg;
  • the preparation method of Epimedium extract is as follows: (1) Put the Epimedium into the pot, add water, and soak the medicine; (2) Dip Soak for 30min to make the active ingredients of Epimedium easy to fry; (3) quickly heat to full boiling for 1-3min, then continue to heat for 20-30min to concentrate, filter into the cup with sterile gauze; (4) 1 time will After the medicine is fried, mix the first dose and the fried medicine to balance the medicine. 1 kg of Epimedium was made into 200 mL of water extract. The extract of Epimedium used in the following experiments was prepared by the method.
  • Each group was administered once a day for a total of 10 days. During the experiment, the diet, survival and behavioral activities of the animals were observed every day. Body weight was measured daily. After the end of the experiment, anesthesia was dissected, blood was taken from the abdominal main vein, blood routine was measured, and the total number of white blood cells and total platelets were examined.
  • Example 24 Effect of icariin on leukocytes and platelets in NOD mice
  • Non-obese diabetic mice are a large mouse strain, including NOD/Scid, NOD/Ltj mice, and the like.
  • the NOD/Ltj mice used in this experiment were mice with abnormal immune system, and their platelets and leukocytes were significantly reduced compared with normal mice.
  • Interleukin purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • Icariin was prepared by the method described in Example 1, and the purity of icariin was 99.3%.
  • Group I was a blank control group, a total of 13; daily administration of an equal volume of physiological saline with intragastric administration;
  • Group II was a positive control group, a total of 13 rats were given intraperitoneal administration of interleukin 10.0 mg/kg daily;
  • Group III was a low-dose group of icariin, a total of 13 rats, daily administration of icariin 30mg/kg;
  • Group IV was a high-dose group of icariin, a total of 13 rats were given icariin 60 mg/kg daily.
  • Each administration group was administered once a day, and administration was stopped for 15 consecutive days.
  • Animals were anesthetized (3% sodium pentobarbital, intraperitoneal injection, 0.1 ⁇ 0.15 ml / only), 1 ml of blood was collected from the abdominal main vein to the anticoagulant tube, blood test routine.
  • the number of white blood cells and the number of platelets in the high-dose and low-dose groups of icariin were significantly higher than those in the model group, showing a significant therapeutic effect.
  • it not only has a significant advantage in increasing the number of white blood cells (p ⁇ 0.05), but also can significantly increase the number of platelets and improve the body's immunity, especially icariin.
  • the effect of raising white blood cells and platelets has a distinct advantage over other groups.
  • Example 25 Effect of icariin on bleeding time and clotting time in normal mice
  • Kunming mice male and female, weighing 20 ⁇ 2.0g; Wistar rats, weighing 200 ⁇ 20g, male and female.
  • the experimental animals were provided by Shandong New Era Pharmaceutical New Drug Pharmacology Center.
  • test drug icariin, prepared as in Example 1; icariin, such as CN101607976B, prepared by the method of Example 1, the purity is 98%, and the preparation method of icariin in the following examples is the same This embodiment.
  • Kunming mice were randomly divided into 5 groups: blank control group (saline group), icariin group (positive control group), high dose group of icariin, and icariin. The dose group and the low dose group of icariin. Each group was administered by the following administration methods.
  • Blank control group subcutaneous injection of equal volume of normal saline
  • Icariin group 2 mg/kg of icariin was administered by injection;
  • Epimedium aglycone high dose group subcutaneous injection of Example 1 icariin injection, the dose is 10mg / kg;
  • Epimedium aglycone medium dose group subcutaneous injection of Example 1 icariin injection, the dose is 5mg / kg;
  • Low-dose group of icariin subcutaneous injection of icariin injection of Example 1 at a dose of 1 mg/kg;
  • clotting time set saline group (blank control group), icariin group (positive control group), icariin group (high, medium, low), 50 mice were selected, Heavy, randomly divided into 5 groups of 10 each. After administration for 3 days, 1 hour after the last administration, 20 ⁇ l of a blood collection tube was inserted into the venous plexus of the mouse, and 20 ⁇ l of blood was taken. A small section of the capillaries was broken every 15 s to check for the presence of hemagglutination, and the time from blood collection to the appearance of fibrin filaments (clotting time) was recorded. The results are shown in Table 5.
  • Epimedium aglycone low dose group 10 1mg/kg 3.75 ⁇ 0.46 * 3.84 ⁇ 0.55 *
  • both the icariin-administered group and the icariin group can reduce the bleeding time and clotting time of normal mice, and the icariin-inducing group reduces the normal mice.
  • the bleeding time and clotting time were superior to the icariin group.
  • the icariin was dose-dependent in reducing the bleeding time and clotting time of normal mice.
  • the high dose group of icariin and icariin The middle dose group had a significant difference compared to the icariin group.
  • Example 26 Effect of icariin on the number of ITP platelets
  • mice Wistar rats were injected with rabbit anti-rat platelet serum (APS). Rats were intraperitoneally injected with ABS (0.7 ml/200 g body weight) diluted for 1:3 for 3 days. Significantly lower. 50 successful rats were selected and weighed and randomly divided into 5 groups: animal control group: model control group, icariin group (positive control group), icariin group (high, medium, Low 3 dose groups), 10 in each group. Each group was given the following therapeutic drugs.
  • APS rabbit anti-rat platelet serum
  • Model control group subcutaneous injection of equal volume of normal saline
  • Icariin group 2 mg/kg of icariin was administered by subcutaneous injection;
  • Epimedium aglycone high dose group subcutaneous injection of Example 11 icariin injection, the dose is 10mg / kg;
  • Epimedium aglycone medium dose group subcutaneous injection of Example 11 icariin injection, the dose is 5mg / kg;
  • Low-dose group of icariin subcutaneous injection of icariin injection of Example 11 at a dose of 1 mg/kg;
  • both the icariin-administered group and the icariin group can increase the number of platelets in the ITP rats, and the icariin-inducing group has an excellent platelet count in the elevated ITP rats.
  • icariin group augmented the platelet count in ITP rats in a dose-dependent manner, the high-dose icariin group and the icariin-medium group and the icariin group There is a significant difference.
  • icariin can exert a positive therapeutic effect on chronic ITP model rats, which reduces thrombin time, prothrombin time and partial thromboplastin time in chronic ITP rats.
  • the high platelet is superior to the positive control drug icariin.
  • the high and medium dose groups significantly shortened thrombin time (TT), prothrombin time (PT) and partial thromboplastin time (APTT) (P ⁇ 0.05) in chronic ITP rats, and had a significant increase.
  • Rat platelet effect P ⁇ 0.01). It can be seen that the traditional Chinese medicine composition described in the present application can be applied to the treatment of thrombocytopenia, and is particularly useful for treating immune thrombocytopenia.
  • Balb/C mice were selected and weighed and randomly divided into 5 groups, which were normal group, model control group, icariin group (positive control group) and icariin group ( Three dose groups of high, medium and low), 12 in each group.
  • icariin group positive control group
  • icariin group Three dose groups of high, medium and low
  • 12 in each group In addition to the normal group, other groups of Balb/C mice were intraperitoneally injected with platelets from SD rats, and once a week, the platelet count was significantly reduced three times in a row.
  • each of the administration groups was given the following therapeutic drugs:
  • Normal group intragastric administration of an equal volume of saline
  • Model control group intragastric administration of an equal volume of saline
  • Epimedium aglycone group 20 mg/kg of icariin was intragastrically administered;
  • Epimedium aglycone high dose group Administration Example 9 icariin tablets, administered at a dose of 100 mg / kg;
  • Icariin in the middle dose group the icariin tablets of Example 9 were administered by gavage at a dose of 50 mg/kg;
  • Epimedium aglycone low dose group The icariin tablet of Example 9 was administered by gavage at a dose of 10 mg/kg;
  • Each administration group was administered once a day and fed normally. After the modeling was performed three times, each administration group continued to be administered for one week. The mice were anesthetized, blood was taken, the number of platelets was measured, and the effect of icariin on peripheral blood platelet count (PLT) was examined.
  • PPT peripheral blood platelet count
  • Group n dose PLT (10 9 /L, x ⁇ s) Normal control group 12 —— 1213.6 ⁇ 107.6
  • Model control group 12 —— 603.3 ⁇ 72.1 # Icariin group 12 2mg/kg 850.6 ⁇ 90.1 ** Epimedium aglycone high dose group 12 100mg/kg 1193.4 ⁇ 95.4 Icariin 12 50mg/kg 983.5 ⁇ 85.7
  • the icariin-administered group and the icariin group have positive therapeutic effects on active immune thrombocytopenia in mice, and can increase the number of platelets in the mouse with active immune thrombocytopenia.
  • the concentration of platelets in the active immune thrombocytopenia model of the icariin aglycons was higher than that of the icariin group, and the icariin increased the platelet of the active immune thrombocytopenia model in mice.
  • the quantitative aspect showed dose-dependent, and the high dose group of icariin and the middle dose of icariin had significant differences compared with the icariin group.
  • mice 72
  • mice 72
  • mice 72
  • mice 72
  • mice were selected and weighed and randomly divided into 6 groups, which were normal group, model control group, icariin group (positive control group) and icariin group ( Three dose groups of high, medium and low), 12 in each group.
  • Mice were intraperitoneally injected with 50 mg/kg cyclophosphamide per day for a significant reduction in platelet counts for one week.
  • Each of the administration groups was given the following therapeutic drugs one week before the start of modeling:
  • Normal group subcutaneous administration of an equal volume of saline
  • Model control group subcutaneous administration of an equal volume of saline
  • Icariin group 2 mg/kg of icariin was administered by subcutaneous injection;
  • Epimedium aglycone high dose group subcutaneous injection of Example 11 icariin injection, the dose is 10mg / kg;
  • Epimedium aglycone medium dose group subcutaneous injection of Example 11 icariin injection, the dose is 5mg / kg;
  • Low-dose group of icariin subcutaneous injection of icariin injection of Example 11 at a dose of 1 mg/kg;
  • mice were anesthetized, blood was taken, the number of platelets was measured, and the effect of icariin on peripheral blood platelet count (PLT) was examined.
  • PPT peripheral blood platelet count
  • Group n dose PLT (10 9 /L, x ⁇ s) Normal control group 12 —— 1113.6 ⁇ 107.6
  • Model control group 12 —— 303.3 ⁇ 72.1 # Icariin group 12 2mg/kg 550.6 ⁇ 90.1 ** Epimedium aglycone high dose group 12 10mg/kg 894.6.4 ⁇ 82.4 Icariin 12 5mg/kg 725.9 ⁇ 66.7
  • the icariin-administered group and the icariin group have positive therapeutic effects on mouse cyclophosphamide-induced thrombocytopenia in mice, and can increase cyclophosphamide-induced mice.
  • the number of platelets in the thrombocytopenic model in which the amount of platelets in the mouse thrombocytopenia-induced mouse thrombocytopenic model was higher in the icariin-administered group than in the icariin group, and the icariin was elevated in the ring.
  • the amount of platelets in the mouse thrombocytopenia model induced by phosphoramide showed a dose-dependent manner.
  • the high dose group of icariin and the middle dose of icariin had significant differences compared with the icariin group.

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Abstract

本申请涉及淫羊藿苷元在制备预防或治疗血细胞减少的药物中的用途。特别地，淫羊藿苷元可用于预防或治疗化疗药物导致的骨髓抑制，也可用于预防或治疗血小板减少症，例如免疫性血小板减少症。

Description

淫羊藿苷元在制备预防或治疗血细胞减少药物中的用途 技术领域
本申请涉及淫羊藿苷元的医药用途，特别是淫羊藿苷元在制备预防或治疗血细胞减少药物中的用途。
背景技术
正常血液包含大量细胞，包括携氧的红细胞和对抗感染的白细胞。白细胞包括中性粒细胞、嗜酸性粒细胞和嗜碱性粒细胞。白细胞是由骨髓造血而来。正常血液还包含血小板。血小板是引发血液凝固的微小的细胞碎片。人体内的血细胞由造血***产生。人体的造血***由少量骨髓造血干细胞及不同发育时期不同系列的造血细胞组成，对各种物理化学及体内代谢产生的有害因素非常敏感，比如机体疲劳、暴露于辐射或某些化疗药物等等，从而导致由血细胞减少而导致的贫血、骨髓抑制等疾病。此外，也存在原发性的血细胞减少，例如原发性血小板减少性紫癜等。
癌症病人都要在相当长的时间内接受以放疗和化疗为主的治疗。放疗和化疗是使用辐射和细胞毒性剂治疗癌症的疗法。然而，放疗和绝大部分化疗疗法都是非特异性的，它们对正常和快速***的细胞是有毒的。大剂量辐射对正常和快速***的细胞也是有毒的。这经常导致在进行化疗和放疗的患者产生各种副作用。尽管其他正常组织也可能受到不利影响，但是骨髓对诸如化疗或放疗这类增殖特异性治疗特别敏感。骨髓抑制，即在骨髓中血细胞产生减少就是这一类的副作用，其表现为骨髓增殖功能下降、血细胞计数下降、外周血白细胞降低、嗜中性粒细胞减少和/或血小板减少，甚至再生障碍性贫血，对患者的生存质量造成严重的危害甚至危及生命。
在临床上，接受放疗、化疗的患者极易受到损伤而出现不同程度的骨髓抑制，表现为外周血白细胞数量降低，中性粒细胞数量降低和/或血小板减少。人体外周血中性粒细胞约占白细胞总数的50-70％，其增高和减低直接影响白细胞总数的变化，即中性粒细胞增高，白细胞随之增高；中性粒细胞减低，白细胞总数也随之减低。两者在数量上的相关性也表现在意义上的一致性，即中性粒细胞增减的意义与白细胞总数增减的意义基本上是一致的。处于骨髓抑制状态的患者易受感染。嗜中性白细胞和血小板缺乏是癌症治疗后发病率和死亡率的主要原因，并导致癌症治疗的高成本。
然而，目前放疗和化疗仍然是肿瘤治疗中最常用的手段。放疗和化疗后引起的骨髓造血抑制等并发症已经成为了影响患者生存质量的重要原因。一些具有明确疗效的肿瘤治疗辅助 化学药物其本身在使用后就会产生较多的不良反应，增加了患者的痛苦。许多医务工作者都在致力于寻找一些有效但副反应少的药物来对抗放、化疗带来的损害。
目前临床上对出现骨髓抑制多是采用给予各种生长因子，以增加造血细胞的增殖。近年来刚刚上市的基因重组的造血生长因子如生白能(rhGM-GSF)、惠尔血(rhG-CSF)等，用来升高白细胞效果显著，但因价格昂贵，并非多数病人能够承受，并且rhGM-GSF、rhG-CSF不可以在化疗的同时使用，也不可以预防使用，只能在出现白细胞减少时给予使用，否则会有毒副作用产生。此外，利用造血生长因子的基因疗法如IL-6、IL-3等基因疗法尚处于动物试验阶段。而自身骨髓移植常在大剂量化疗时配合使用，但难以反复应用。因此，寻找一种安全、有效、价廉的方法预防并治疗血细胞减少，特别是由化疗和放疗的副反应导致的血细胞减少，升高化疗和放疗后的白细胞数量，对于提高肿瘤的化疗和放疗效果，延长癌症患者生存期，改善生存质量都有非常重要的意义。
血小板减少症，是外周血中血小板减少而导致皮肤粘膜及内脏出血的疾病，主要表现为白发性皮肤瘀点和瘀斑，粘膜出血、鼻衄和牙龈出血、口腔黏膜及舌出现紫血泡等，是临床常见的以凝血功能障碍、出血为特点的一类疾病，重者可引起大出血，危及生命，临床约占出血性疾病的30％。
血小板减少的原因可分为：(1)血小板生成减少或无效死亡：包括遗传性和获得性两种，获得性血小板生成减少是由于某些因素如药物、恶性肿瘤、感染、电离辐射等损伤造血干细胞或影响其在骨髓中增殖所致，可影响多个造血细胞***，常伴有不同程度贫血、白细胞减少、骨髓巨核细胞明显减少；(2)血小板破坏过多：包括先天性和获得性两种。获得性血小板破坏过多包括免疫性和非免疫性。免疫性血小板破坏过多常见的有特发性血小板减少性紫癜和药物血小板减少。非免疫性血小板减少破坏过多包括感染、弥漫性血管内凝血、血栓性血小板减少性紫癜等；(3)血小板在脾内滞留过多：最常见于脾功能亢进。
血小板减少症的发病机制包括：1)免疫性因素：临床可见严重肝病、红斑狼疮、特发性血小板减少性紫癜等免疫性疾病引起血小板减少的情况，抗体破坏血小板；2)感染性因素：引起血小板减少症的常见因素，细菌、病毒感染可直接损伤造血细胞，减少血小板生长，再生障碍性贫血、急性白血病等急性发作期常伴严重感染，有出血倾向，如皮肤上有瘀点、红斑或原因不明的鼻出血等表现；3)药物性因素：因某些药物致使周围血液中血小板计数减少而导致的出血性疾病；4)血小板功能异常：如血小板无力症、巨大血小板综合症等。
血小板减少症在临床主要为免疫性血小板减少性紫癜(immunethrombocytopenic purpura，ITP)和血栓性血小板减少性紫癜(thromboticthrombocytopenic purpura，TTP)两类疾病比较常 见。其中，ITP是临床所见血小板减少最常见的原因。长期以来，ITP被认为是一种原因不明的血小板减少所致的出血性疾病，因而称之为原发性或特发性血小板减少性紫癜(idiopathic thrombocytopenicpurpura)。后来发现ITP患者体内存在有识别自身血小板抗原的自身抗体，自身抗体与血小板抗原的结合导致血小板寿命缩短、破坏增加和血小板数量减少，说明本病是一种与免疫反应相关的出血性疾病。
在临床中，免疫性血小板减少性紫癜(ITP)是一种较为常见的出血性疾病，估计人群中ITP的发病率为1/10000，可以发生在任何年龄阶段。一般儿童多为急性型，成人多为慢性型，多发生于儿童及青年，临床表现为皮肤出现瘀点及瘀斑，皮肤黏膜出血，重症患者伴关节疼痛或腹痛，便血、吐血、崩溃等，严重者可发展为紫癜性肾炎。原发性血小板减少性紫癜是一种免疫性综合病征，是常见的出血性疾病，特点是血循环中存在抗血小板抗体，使血小板破坏过多，引起紫癜；而骨髓中巨核细胞正常或增多，幼稚化。
到目前为止，西医治疗血小板减少性紫癜以肾上腺皮质激素为首选，虽然激素能升高血小板，但是激素减量或停止后，血小板又会下降；经常使用激素，对人体副作用非常大，在治疗的同时还会引起如向心性肥胖、高血压、糖尿病等疾病。其他治疗方法如输注血小板、脾脏切除等方法治疗，有增加感染率，引起活动性消化性溃疡、出血、免疫功能下降、高血糖等以及停药后反而使症状加重且疗效不持久等特点；采用免疫抑制剂、脾切除等常规疗法，因激素的禁忌症和副作用而使很多患者形成激素撤减综合症而不能根治；而免疫抑制剂的毒副作用较大，停药后易复发，易导致骨髓抑制和诱发肿瘤的危险而不能广泛应用。近年来，大剂量丙球冲击，血浆置换等新疗法不断出现，但仍缺乏根本性的治疗措施。
淫羊藿为小檗科植物淫羊藿(Epimedium brevicornum Maxim.)、箭叶淫羊藿(Epimedium sagittatumMaxim.)、柔毛淫羊藿(Epimedium pubescens Maxim.)、或朝鲜淫羊藿(Epimedium koreanum Nakai)的干燥茎叶。临床上主要用于肾阳虚，阳痿尿频，***；风湿痹痛，肢体麻木拘挛，筋骨痿软，步履艰难；肾阳虚，喘咳短气。淫羊藿苷能增加心脑血管血流量、促进造血功能、免疫功能及骨代谢，具有补肾壮阳、抗衰老、抗肿瘤等功效。淫羊藿苷元(icaritin,IT)为小檗科淫羊藿属植物淫羊藿中的一种多羟基黄酮类单体成分。药理研究表明,IT抗骨质疏松作用较淫羊藿中其他黄酮苷类化合物强,在体外具有促进成骨细胞活性,抑制破骨细胞活性的作用。
近年来作为淫羊藿中重要活性成分的淫羊藿苷和淫羊藿苷元越来越受到医药工作者的关注。如专利申请CN101637467A公开了淫羊藿苷元在制备治疗骨质疏松药物中的应用。专利US6399579公开了淫羊藿苷元在治疗性功能障碍方面的用途。强宁侠在《中西 医结合治疗慢性特发性血小板减少性紫癜30例》(发表于《吉林中医药》2010年第7期)中在西医治疗基础上加服健脾补肾中药(人参、黄芪、当归、山茱萸、淫羊藿、补骨脂等治疗慢性特发性血小板减少性紫癜取得了较好的治疗效果，但该药物含有多种活性成分，作用机理复杂，有必要进行更细致的机理研究和药效研究。赵勇等在《淫羊藿甙协同诱生IL-2，3，6作用的研究》(发表于《中国免疫学杂志》1996年第1期)中采用依赖细胞株法分别检测了淫羊藿甙协同诱生IL-2、IL-3及IL-6的作用，结果表明淫羊藿甙可协同PHA诱导扁桃体单个核细胞产生IL-2，3，6，呈剂量依赖关系，提示淫羊藿甙是一种有效的生物反应调节剂。
目前还没有文献报道淫羊藿苷元具有预防或治疗血细胞减少，特别是防治免疫性血小板减少，或辐射或化学品引起的骨髓抑制的活性。
发明内容
本申请的目的在于提供一种预防和/或治疗血细胞减少症如血小板减少症，特别是免疫性的血小板减少症或者在防治辐射或化学品引起的骨髓抑制的药物。本发明人长期致力于淫羊藿苷元的药理活性研究，近期在更进一步的深入研究过程中意外地发现，淫羊藿苷元在减轻血细胞减少症以及在治疗血小板减少症等方面均有意想不到的活性。
本申请第一方面涉及淫羊藿苷元在制备用于预防或治疗血细胞减少症药物中的用途。其中，所述的血细胞包括白细胞、红细胞、中性粒细胞或血小板，所述的血细胞减少症的主要表现为指外周血白细胞下降或血小板下降。所述血细胞减少症包括由辐射或化学品引起的骨髓抑制，或为原发性的骨髓增生不良。
在一些具体实施方式中，本申请提供了淫羊藿苷元用于预防或治疗辐射或化学品引起的骨髓抑制的用途。其中所述的化学品为能够引起骨髓抑制的化学治疗药物，特别是能够用于治疗癌症的化学治疗药物。优选地，所述治疗癌症的化疗药物为多西他赛(Docetaxel)、替吉奥(S-1)或其组合，但不限于此。本申请实施例22中淫羊藿苷元对肿瘤小鼠化疗引起的骨髓抑制的影响实验中显示，抗肿瘤药物(如多西他赛或替吉奥)与淫羊藿苷元联合使用后的肿瘤小鼠的白细胞总数、血小板数目和中性粒细胞数与抗肿瘤单药组相比均有极显著性的增高，而联合用药组动物的体重、饮食量和生活状态与空白对照组相比没有显著性的差异，即淫羊藿苷元联合用药后不仅能够显著升高肿瘤小鼠的血细胞,而且毒副作用很低。本申请实施例23中淫羊藿苷元对60Co放射小鼠的血细胞数的影响实验显示，淫羊藿苷元联合60Co照射时能够有效升高小鼠的白细胞和血小板数目，其血细胞 升高作用显著优于淫羊藿提取物组。以上实施例表明淫羊藿苷元有预想不到的缓解放疗或者化学品引发的骨髓抑制的效果。
另外，本发明实施例24通过淫羊藿苷元对NOD/Ltj小鼠白细胞和血小板的影响实验证实，淫羊藿苷元高剂量和低剂量组对于NOD/Ltj小鼠的白细胞数量和血小板数量也具有显著的升高作用，其升高效果明显优于常用治疗药物白介素，由于NOD/Ltj为血小板与白细胞数量与普通小鼠相比明显降低的免疫***异常小鼠，因此，这提示淫羊藿苷元对于原发性的骨髓增生不良亦有很好的治疗效果。
在本申请的一些实施方式中，本申请提供了淫羊藿苷元在制备用于预防或治疗血小板减少症药物中的用途。其中，所述血小板减少症可以是免疫性血小板减少症，具体可以是慢性特发性血小板减少性紫癜。所述血小板减少症也可以是骨髓抑制所引发的继发性血小板减少症。本申请实施例26证实，淫羊藿苷元可以对慢性ITP模型大鼠体现出了积极的治疗作用，其高、中剂量组具有极显著升高大鼠血小板作用(P＜0.01)，其在升高血小板方面优于阳性对照药淫羊藿苷。可见，本申请所述的药物组合物可应用于制备治疗血小板减少症的药物制剂，尤其可应用于制备治疗免疫性血小板减少症的药物制剂。
本申请上述的药物用途中，所述血小板减少症优选为骨髓抑制所引发的继发性血小板减少症。其中所述的骨髓抑制为化学药物所引发的骨髓抑制。本申请实施例28证实，淫羊藿苷元给药组和淫羊藿苷元组对小鼠环磷酰胺所致小鼠血小板减少均具有积极的治疗作用，可以升高环磷酰胺所致小鼠血小板减少模型的血小板数量，其中淫羊藿苷元各给药组在升高环磷酰胺所致小鼠血小板减少模型的血小板数量方面优于淫羊藿苷组，淫羊藿苷元在升高环磷酰胺所致小鼠血小板减少模型的血小板数量方面体现出剂量依赖性，淫羊藿苷元高剂量组和淫羊藿苷元中剂量组与淫羊藿苷组相比具有显著性的差异。
本申请再一方面在于提供一种淫羊藿苷元预防或治疗血细胞减少症或血小板减少症的方法。所述的方法包括将有效量的淫羊藿苷元给予有此需求的个体。所述个体优选为人类。
本申请公开的淫羊藿苷元在制备作为治疗血细胞减少症或血小板减少症药物方面的应用，通常是以药物组合物的形式服用的，可口服或非口服给药，或者以和药学上可接受的载体、赋形剂及其它添加剂形成的组合物(如片剂、缓释制剂、胶囊剂、注射剂、溶液剂)安全的口服或非口服给药。其中口服给药的人用给药量为0.1mg/kg/d-100mg/kg/d；非口服给药包括但不限于皮下、皮内、动脉、静脉、肌肉、关节、鞘内、颅内、胸腔、腹腔内注射或滴注，经鼻、经颊、舌下、气管、尿道、直肠或病灶局部给药等。优选为 注射给药，其中淫羊藿苷元的人用给药量优选为0.01mg/kg-10mg/kg。
另外，根据本申请，淫羊藿苷元可以作为唯一活性成分用来预防和/或治疗血细胞减少或血小板减少相关的疾病，优选用于由辐射或化学品引起的骨髓抑制，或者免疫性血小板减少症；也可以与其它药物合用以进行所述的预防和/或治疗。与淫羊藿苷元联合给药或者制备成药物组合物的其他药物可以是其他一种或多种治疗白细胞减少的药物(例如维生素B4、利血生、鲨肝醇、辅酶A)、或其他一种或多种可以升高血小板含量的药物(例如白介素-Ⅱ，低剂量糖皮质激素，如强的松等)以预防或治疗血细胞减少，特别是用于预防或治疗骨髓抑制。这些药物与淫羊藿苷元联合使用或者制备成药物组合物用于骨髓抑制的治疗时,可以进一步增强淫羊藿苷元升高血小板的含量或者白细胞的含量。
根据一些实施方式，本申请的淫羊藿苷元与其他药物联合使用时，两种药物的重量比可以根据患者的病情和症状适当调整，其他药物与淫羊藿苷元的重量比范围优选为(0.005-100):1，更优选(0.005-50):1，再优选(0.01-100):1，例如(0.05-25):1。
淫羊藿苷元和任选的其他药物的给药途径包括胃肠道途径和非胃肠道途径，非胃肠道途径包括但不限于皮下、皮内、动脉、静脉、肌肉、关节、鞘内、颅内、胸腔、腹腔内注射或滴注，经鼻、经颊、舌下、气管、尿道、直肠或病灶局部给药等。在使用淫羊藿苷元预防和/或治疗辐射或化学品引起的骨髓抑制时，可将淫羊藿苷元可在放疗或化疗之前、之中、或之后施用。在将淫羊藿苷元与其他药物联合给药时，可以将淫羊藿苷元与其他药物同时给药，也可以顺次给药。
本申请的另一方面涉及一种预防或治疗血细胞减少或血小板减少相关的疾病的药物组合物(或制剂)，其包含淫羊藿苷元和药学可接受的药用辅料。所述药物组合物优选用于预防或治疗由辐射或化学品引起的骨髓抑制。
本领域技术人员可以根据实际需要选择适合的药用辅料，并通过本领域已知的方法配制本申请的制剂。所述制剂包括但不限于固体、液体、油、乳剂、凝胶、气溶胶、吸入剂、喷雾、胶囊、丸剂、贴剂和栓剂等。当口服给药时，组合物可配制成片剂、颗粒剂或胶囊剂。为制备口服药物组合物可采用乳糖或淀粉做载体，明胶，羧甲基纤维素钠，甲基纤维素、聚乙烯吡咯烷酮等是合适的结合剂或成颗剂。作为崩解剂可选用淀粉或微晶纤维素，常以滑石粉，胶体硅胶，硬脂酸甘油酯，硬脂酸钙或镁等作为合适的抗粘合剂和润滑剂。例如，可通过压制湿颗粒来制备片剂。活性成分与载体以及选择性的与一份崩解添加剂组成混合物，该混合物与粘合剂的含水溶液，醇性或含水醇性溶液在合适的设备中进行颗粒化，干燥颗粒随后加入其它的崩解剂，润滑剂和抗粘剂将此混合物压 片。可以注射剂形式给药，虽然剂量依治疗对象、给药方式、症状及其它因素而改变。实际服用的淫羊藿苷元的剂量应该由医生根据有关的情况来决定，这些情况包括被治疗者的身体状态，选者的给药途径、年龄、体重、患者对药物的个体反应，患者症状的严重程度等等。所述注射制剂为注射液、冻干粉针剂、输液制剂中的一种，所述注射制剂中的药用辅料为甘露醇、葡萄糖、山梨醇、PEG、乙醇、生理盐水中的一种或多种，优选含有PEG的注射制剂。
适用上述药物用途的含有淫羊藿苷元的药物制剂中的每一制剂单位中含有淫羊藿苷元的量为0.1-500mg。
术语的定义
本文所述“淫羊藿苷元”可以是提取、生物制备、或化学制备而成，包括淫羊藿苷元、其可药用盐或水合物。本申请所述淫羊藿苷元含有淫羊藿苷元的纯度(如HPLC检测纯度)为≥90％、≥95％或≥98％。本申请申请日前现有技术所披露的淫羊藿苷元的制备方法均可作为本申请中所述淫羊藿苷元的制备工艺。
本申请所述“骨髓抑制”是指血细胞，包括白细胞、红细胞、中性粒细胞或血小板，这些组成单独减少或者同时减少的情形，其表现为血细胞产生的减少。健康骨髓每天生成大量的红细胞、白细胞和血小板。在骨髓抑制下，骨髓生成的这些细胞减少。骨髓抑制的一个特征是白细胞产生减少。这种白细胞产生减少可以由某种治疗引起，尤其是癌症治疗，例如化疗和放疗。优选地，本申请所述的骨髓抑制表现为外周血白细胞数量下降或血小板下降、骨髓增生不良。在一些实施方式中，所述的外周血白细胞下降是指外周血中性粒细胞下降。
所述“辐射”可以是在放疗过程中接受的辐射，或因工作、环境等其他原因而接受的辐射。优选地，上述用途中所述的辐射是在放疗过程中接受的辐射，比如因治疗癌症接受放疗过程中的辐射。
所述“化学品”可以是指能够引起血细胞产生减少的任何药物或化学试剂。在一个实施方式中，上述用途中所述的化学品为用于治疗癌症的化疗药物。所述化疗药物包括烷化剂类药物、抗代谢剂类药物、抗生素类药物、植物类药物和激素类药物等，但不限于此。其中烷化剂类药物包括但不限于氮芥、环磷酰胺、噻替哌、环已亚硝脲、马利兰、氮烯米胺、甲基苄肼等；所述的抗代谢药物包括但不限于氟尿嘧啶(5-FU)、呋喃氟尿嘧啶(FT-207)、二喃氟啶(双呋啶FD-1)、优氟泰(UFT)、氟铁龙(5-DFUR)、甲氨喋呤(MTX)、 氨喋呤(白血宁)、阿糖胞苷(Ara-c)、环胞苷，氯环胞苷、羟基脲(HU)、肌苷二醛(inosine dialdehyde)、腺苷二醛(adenosinediialde-hgde)、胍唑(guanazole)、6-巯嘌呤(6-MP)；所述抗生素类药物包括但不限于青霉素类抗生素、头孢菌素类抗生素、氨基糖苷类抗生素、大环内酯类抗生素、磺胺类抗生素、喹诺酮类抗生素、呋喃类抗生素；所述植物类药物为来源于植物的药物，其包括传统的中药配伍类药物以及用现代提取分离手段从植物中所获取的有效部位或中药单体药物；所述激素类药物包括但不限于糖皮质激素、肾上腺皮质激素、去甲肾上腺激素、孕激素、***、雄激素；等等。
本申请所述“药学可接受的药用辅料”是指不干扰淫羊藿苷元的生理作用，且对包括人类在内的受试者没有毒性的任何物质。适合的药用辅料在《药用辅料大全》(第123页，四川科学技术出版社，1993年出版，罗明生和高天惠主编)中已有详细描述。例如，制备微乳制剂常用的药用辅料包括但不限于大豆油、聚氧乙烯-23-月桂基醚、1,2-丙二醇、氢化椰油甘油脂、月桂酰基聚乙二醇-32-甘油酯、聚乙二醇3350、红花油、棉子油、十甘油单硬脂酸酯；制备滴丸制剂常用的药用辅料包括但不限于聚乙二醇6000、聚乙二醇1000；制备胶囊制剂常用的药用辅料包括但不限于乳糖和玉米淀粉。制备软胶囊制剂常用的可药用载体包括但不限于中链脂肪酸甘油酯、聚氧乙烯蓖麻油、1,2-丙二醇等。
本申请所述的“血小板减少症”是指外周血中血小板减少而导致皮肤粘膜及内脏出血的疾病，主要表现为白发性皮肤瘀点和瘀斑，粘膜出血、鼻衄和牙龈出血、口腔黏膜及舌出现紫血泡等，是临床常见的以凝血功能障碍、出血为特点的一类疾病，重者可引起大出血，危及生命，临床约占出血性疾病的30％。其可以为免疫性血小板减少症或继发性血小板减少症。
所述的“免疫性血小板减少症”是由于免疫***紊乱所导致的出血性疾病，其主要是指免疫性血小板减少性紫癜(ITP)，ITP是一种较为常见的出血性疾病，估计人群中ITP的发病率为1/10000，可以发生在任何年龄阶段。临床表现为皮肤出现瘀点及瘀斑，皮肤黏膜出血，重症患者伴关节疼痛或腹痛，便血、吐血、崩溃等，严重者可发展为紫癜性肾炎。
所述的“原发性血小板减少性紫癜”或“特发性血小板减少性紫癜”(idiopathic thrombocytopenicpurpura)是免疫性血小板减少症的一种，是一种原因不明的获得性出血性疾病，其主要以血小板减少、骨髓巨核细胞正常或增多，以及缺乏任何原因为特征。该疾病多发生于儿童及青年，一般儿童多为急性型，成人多为慢性型，其特点是血循环中存在抗血小板抗体，使血小板破坏过多，引起紫癜；而骨髓中巨核细胞正常或增多，幼稚化。
所述的“继发性血小板减少症”又称获得性血小板减少症，是继发于其他疾病或疾病治疗药物引起的血小板减少，其涉及的病种和治疗药物相当多。其包括药物性免疫性血小板减 少症，其他免疫性血小板减少症如Evans综合征，慢性淋巴细胞性白血病，各种急性白血病，淋巴瘤，***性红斑狼疮，类风湿性关节炎，甲状腺机能亢进等。本申请所述的继发性血小板减少症主要为辐射或化学药物引起的骨髓抑制所引发。
本申请的淫羊藿苷元在预防或治疗血细胞减少相关的疾病，特别是防治由辐射或化学品引起的骨髓抑制方面至少具有如下有益的效果，但不限于此：
1)在治疗癌症的同时预防白细胞降低，甚至提高中性粒细胞的数量，从而提高肿瘤的化疗效果，延长癌症患者生存期，改善生存质量。通过淫羊藿苷元对肿瘤小鼠化疗引起的骨髓抑制影响试验发现，淫羊藿苷元有意想不到的缓解骨髓抑制的效果。具体为，与模型组相比，淫羊藿苷元组的白细胞总数和中性粒细胞数均有增高，并有极显著性差异(P<0.01)或显著性差异(P<0.05)；同时淫羊藿苷元组较多西他赛组和替吉奥组相比白细胞总数和中性粒细胞数均有大幅度增高，并有极显著性差异(P<0.01)。
2)可以在其它药物化疗的同时使用，也可以预防使用，而不是仅在出现白细胞减少时给予使用，从而在一定程度上减少了副反应。通过试验结果还可以看出，淫羊藿苷元作为抗癌药，即使将其与多西他赛或替吉奥同时联合用来抗肿瘤，淫羊藿苷元在发挥抑制肿瘤生长作用的同时缓解了多西他赛或替吉奥带来的骨髓抑制。由此可见，淫羊藿苷元可以在其它药物化疗的同时使用，也可以预防使用，而不是仅在出现白细胞减少时给予使用，其在保证抑瘤率的同时能促进骨髓造血，增加白细胞数量，增加中性粒细胞数量，并在一定程度上减少了副反应。由表1的试验结果可以看出，淫羊藿苷元单药组或联合用药组的小鼠在给药期间的体重与模型组没有显著性差别，动物的饮食量和生活状态也没有明显差别。这说明淫羊藿苷元作为抗肿瘤药和骨髓抑制缓解药，没有显著的毒副作用。
3)对放射治疗过程中产生的骨髓抑制也有显著的预防或治疗作用。通过淫羊藿苷元对4Gy照射60Co肿瘤小鼠白细胞和血小板的影响实验(表3)发现，淫羊藿苷元有意想不到的缓解放疗产生的骨髓抑制的效果。具体为，与模型组相比，淫羊藿苷元组的白细胞总数和血小板总数均有增高，并有极显著性差异(P<0.01)或显著性差异(P<0.05)；与淫羊藿提取物组相比有显著性差异(P<0.05)。
淫羊藿苷元用于多种因素引发的血小板减少症时均具有显著的治疗效果。本申请实施例26证明淫羊藿苷元在升高慢性ITP模型大鼠血小板方面优于阳性对照药淫羊藿苷。其高、中剂量组具有极显著升高大鼠血小板作用(P＜0.01)。本申请实施例27证实，其淫 羊藿苷元在治疗小鼠主动免疫性血小板减少方面也具有积极的治疗效果。
淫羊藿苷元对于骨髓抑制引发的继发性血小板减少症也具有积极的治疗效果，这提示其可以与其他化学药物连用以减少化学品(如化疗药物)对机体的损害作用。本申请实施例28证实，淫羊藿苷元给药组和淫羊藿苷组对小鼠环磷酰胺所致小鼠血小板减少均具有积极的治疗作用，可以升高环磷酰胺所致小鼠血小板减少模型的血小板数量，其中淫羊藿苷元各给药组在升高环磷酰胺所致小鼠血小板减少模型的血小板数量优于淫羊藿苷组，淫羊藿苷元高剂量组和淫羊藿苷元中剂量组与淫羊藿苷组相比具有显著性的差异。
淫羊藿苷元为从传统中药淫羊藿中所提取的有效活性成分，用于血小板减少症治疗时不仅疗效确切，适于联合用药，而且其药物毒副作用极低，能够大大增加患者的用药依从性，从而保证药物的治疗效果。且淫羊藿苷元目前有多种制备方法，制备工艺简单、成本低廉，可以大大降低血小板减少症患者的治疗费用。
具体实施方式
以下通过具体实施方式进一步描述本申请，本申请不仅仅限于以下实施例。
实施例1：淫羊藿苷元微乳制剂
Figure PCTCN2014088944-appb-000001
制备工艺：称取处方量大豆油、聚氧乙烯-23-月桂基醚、1,2-丙二醇，混合后搅拌均匀，然后加入淫羊藿苷元溶解，也可以超声波处理以加速溶解，得澄清溶液，即为淫羊藿苷元微乳制剂。激光粒度测定仪测定其粒径，平均粒径为15nm。
实施例2：淫羊藿苷元微乳制剂
Figure PCTCN2014088944-appb-000002
制备工艺：称取处方量氢化椰油甘油脂、月桂酰基聚乙二醇-32-甘油酯、1,2-丙二醇、聚乙二醇3350，混合后搅拌均匀，然后加入淫羊藿苷元溶解，也可以超声波处理以加速溶解，得澄清溶液，即为淫羊藿苷元微乳制剂。激光粒度测定仪测定其粒径，平均粒径为40nm。
实施例3淫羊藿苷元注射液
Figure PCTCN2014088944-appb-000003
制备工艺：向处方量的PEG-400加入淫羊藿苷元，搅拌溶解，加入0.9％氯化钠溶液至10L，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例4淫羊藿苷元注射液
Figure PCTCN2014088944-appb-000004
制备工艺：将处方量的乙醇和吐温-80混合均匀，加入淫羊藿苷元，搅拌溶解，加入注射用水至10L，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例5淫羊藿苷元注射液
淫羊藿苷元    1g
乙醇      3.3L
注射用水  加至10L
制备工艺：将处方量的乙醇加入淫羊藿苷元，搅拌溶解，加入注射用水至10L，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例6：淫羊藿苷元滴丸制剂
   淫羊藿苷元   5.0g
聚乙二醇-6000   14.5g
聚乙二醇-1000   5.0 g
            制成1000粒
制备工艺：称取处方量过100目筛的淫羊藿苷元，加入已在水浴上加热熔融的含处方量的聚乙二醇6000、聚乙二醇1000的混合液中，充分搅拌，使之均匀，装入滴瓶中，于95±2℃的条件下滴制；滴入盛有4-6mL的甲基硅油的玻璃冷凝柱内，成型后取出，用吸水纸吸去黏附的甲基硅油，即得。
实施例7：淫羊藿苷元肠溶软胶囊制剂
内容物处方：
Figure PCTCN2014088944-appb-000005
胶皮处方：
明胶  10g
甘油  5g
肠溶包衣液处方：
Figure PCTCN2014088944-appb-000006
制备工艺：称取处方量中链脂肪酸甘油酯、聚氧乙烯蓖麻油、1,2-丙二醇、无水乙醇，混合后搅拌均匀，然后加入淫羊藿苷元溶解，也可以超声波处理以加速溶解，得澄清浓缩液，即为淫羊藿苷元微乳浓缩物。将上述所得的微乳浓缩物加水按照1：10-20的重量比稀释至澄清溶液，即得软胶囊微乳内容物。称取处方量明胶、甘油、纯化水，混合均匀后压制成胶皮，再称取处方量的Eudragit L30D-55、柠檬酸三乙酯、滑石粉、纯化水混合均匀制得肠溶包衣液。将含有淫羊藿苷元的软胶囊微乳内容物用胶皮包裹制成软胶囊，并在软胶囊上包肠衣制得肠溶软胶囊。
实施例8：淫羊藿苷元胶囊制剂
淫羊藿苷元  100g
玉米淀粉    130g
硬脂酸镁    5g
制备工艺：将淫羊藿苷元100g、乳糖120g和玉米淀粉130g在混合机中混合10-15分钟，加入硬脂酸镁5g混合1-3分钟，装入1000粒胶囊壳即可。
实施例9淫羊藿苷元片剂
Figure PCTCN2014088944-appb-000007
制备工艺:将淫羊藿苷元和辅料微晶纤维素、羧甲基淀粉钠混合均匀，加入适量的淀粉浆制软材，然后过16目筛制粒。湿颗粒在60℃干燥，干颗粒过20目筛整粒，筛出干粒中的细粉，与硬脂酸镁混匀，然后再与干颗粒混匀，压片，每片约200mg，即得。
实施例10淫羊藿苷元粉针剂
Figure PCTCN2014088944-appb-000008
制备工艺：称取处方量的注射用淫羊藿苷元原料,加入适量注射用水溶解。然后，加入经过规定量的预先经过除菌除热原处理的混匀，再加注射用水至规定1000ml；将上述药液中加入注射剂用活性炭5g，在60-80℃加热30分钟，用滤膜过滤，收集滤液。将上述滤液按无菌操作法用除菌滤器进行正压除菌滤过，用0.22μM微孔滤膜过滤，滤液进行热原检查和半成品含量检查然后西林瓶分装。在专用冻干箱内，在-40℃以下预冻1.5-3.5小时，真空度下升华在游离水分去处90％以后加温干燥(最高温度不得超过35℃)，冻干结束即可制备得到淫羊藿苷元粉针剂。
实施例11淫羊藿苷元注射剂
Figure PCTCN2014088944-appb-000009
制备工艺：
将处方量的丙二醇和乙醇混合均匀，加入淫羊藿苷元，搅拌溶解，加入处方量的0.9％氯化钠溶液，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例12淫羊藿苷元注射剂
淫羊藿苷元       10mg
PEG-400          2ml
0.9％氯化钠溶液  加至10ml
制备工艺：将处方量的PEG-400加入淫羊藿苷元，搅拌溶解，加入0.9％氯化钠溶液至10ml，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例13淫羊藿苷元注射剂
Figure PCTCN2014088944-appb-000010
制备工艺：将处方量的乙醇和吐温-80混合均匀，加入淫羊藿苷元，搅拌溶解，加入0.9％氯化钠溶液至10ml，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例14淫羊藿苷元注射剂
Figure PCTCN2014088944-appb-000011
制备工艺：将处方量的乙醇和吐温-80混合均匀，加入淫羊藿苷元，搅拌溶解，加入0.9％氯化钠溶液至10ml，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例15淫羊藿苷元注射剂
Figure PCTCN2014088944-appb-000012
制备工艺：将处方量的乙醇和吐温-80混合均匀，加入淫羊藿苷元，搅拌溶解，加入0.9％氯化钠溶液至10ml，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例16淫羊藿苷元注射剂
淫羊藿苷元  20mg
乙醇        3.3ml
注射用水    加至10ml
制备工艺：将处方量的乙醇加入淫羊藿苷元，搅拌溶解，加入注射用水至10ml，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例17淫羊藿苷元注射剂
淫羊藿苷元    10mg
乙醇          3.3ml
注射用水      加至10ml
制备工艺：将处方量的乙醇加入淫羊藿苷元，搅拌溶解，加入注射用水至10ml，搅拌均匀，加入0.5％针用活性炭，搅拌，脱炭，即得。
实施例18淫羊藿苷元片剂
Figure PCTCN2014088944-appb-000013
制备工艺：称取处方量的淫羊藿苷元、淀粉、糊精混合均匀。另将适量的50％乙醇加入于混合粉末中，混合均匀，制软材，通过18目尼龙筛制成湿粒，60℃左右干燥，干粒水分应控制在1.5％以下。20目筛整粒，再与硬脂酸镁混匀，压片，即得。
实施例19淫羊藿苷元胶囊剂
淫羊藿苷元  10g
微晶纤维素  300g
微粉硅胶    12g
制备工艺：将淫羊藿苷元、微晶纤维素、微粉硅胶粉碎过100目筛混匀，直接填装胶囊即得。
实施例20淫羊藿苷元颗粒剂
Figure PCTCN2014088944-appb-000014
Figure PCTCN2014088944-appb-000015
制备工艺：称取处方量的淫羊藿苷元、淀粉、糊精、蔗糖粉混合均匀。另将适量的80％乙醇加入于混合粉末中，混合均匀，制软材，通过18目尼龙筛制成湿粒，60℃左右干燥，20目筛整粒，分装，即得。
实施例21淫羊藿苷元缓释片剂
Figure PCTCN2014088944-appb-000016
制备工艺：将处方量的淫羊藿苷元、乳糖和缓释剂羟丙甲基纤维素混合均匀加入粘合剂聚乙烯吡咯烷酮制粒，在40℃－80℃下干燥，整干颗粒，在干颗粒中加入处方量的润滑剂微粉硅胶，混匀，异型冲压片即可。
实施例22淫羊藿苷元对肿瘤小鼠化疗引起的骨髓抑制的影响
1.材料
1.1试验动物：昆明小鼠(购自中国药品生物制品检定所，实验动物许可证号：SCXKⅡ-00-0010)，雌雄各半，7周龄，18～22g，试验温度(20±1)℃，湿度40％～70％，自由饮水，正常饲喂。
1.2试验试剂：
Figure PCTCN2014088944-appb-000017
2.方法：
小鼠腹水瘤s180细胞培养于1640培养液中，37℃，5％CO2下常规培养，平均每两天传代一次，至对数生长期时用生理盐水制备成密度为3.0×107个/ml单细胞悬液，在无菌条件下注射于小鼠腹腔内，接种后7天见小鼠腹腔明显肿大，此时，脱颈处死，放入盛有75％乙醇的烧杯中浸泡2～3分钟，将消毒后的小鼠放入超净工作台中，暴露腹部，用无菌注射器抽取腹水放入无菌试剂瓶内备用。将上述腹水用台盼蓝计数，用生理盐水稀释，调整细胞数至2.0×107个/ml，接种于小鼠右腋下，每只0.2ml。
将接种后的小鼠随机分为以下6组，每组10只，雌雄各半。
第1组为模型对照组：0.9％生理盐水腹腔注射实验动物(与以下各实验相同)；
第2组为多西他赛组：75mg/m2/d多西他赛；
第3组为替吉奥组：222.2mg/m2/d替吉奥；
第4组为淫羊藿苷元组：10mg/kg/d淫羊藿苷元微乳制剂；
第5组为多+淫组(多西他赛+淫羊藿苷元组)：75mg/m2/d多西他赛+10mg/kg/d淫羊藿苷元；
第6组为替+淫组(替吉奥+淫羊藿苷元组)：222.2mg/m2/d替吉奥+10mg/kg/d淫羊藿苷元。
多西他赛和淫羊藿苷元通过尾静脉注射给药，给药体积分别为10ml/kg。替吉奥通过灌胃给药，给药体积为40ml/kg。各组每天给药一次，共给药10天。试验过程中，每天观察动物饮食、存活情况及行为活动，每日测量体重，试验结束后麻醉解剖，通过腹主静脉取血，测血常规，考察白细胞总数，血小板总数及中性粒细胞总数。
3.实验结果：
表1  淫羊藿苷元对肿瘤小鼠体重的影响
Figure PCTCN2014088944-appb-000018
由表1的试验结果可以看出，淫羊藿苷元单独给药组或联合用药组的小鼠在给药期间的体重与模型组没有显著性差别。动物的饮食量和生活状态也没有明显差别。这说明淫羊藿苷元作为抗肿瘤药和骨髓抑制缓解药，没有显著的毒副作用。
表2.淫羊藿苷元对肿瘤小鼠白细胞中的中性粒细胞的影响
Figure PCTCN2014088944-appb-000019
与模型组比较，#P＜0.05，##P＜0.01；
与多西他赛组比较，*P＜0.05，**P＜0.01；
与替吉奥组比较，$P＜0.05，$$P＜0.01。
通过本实施例淫羊藿苷元对肿瘤小鼠化疗引起的骨髓抑制影响试验(表2)发现，在多西他赛组或替吉奥组中的小鼠，其中性粒细胞数量比模型组明显减少，而淫羊藿苷元有预想不到的缓解骨髓抑制的效果。具体地，与模型组相比，淫羊藿苷元组的白细胞 总数、血小板数目和中性粒细胞数均有增高，并有极显著性差异(P<0.01)或显著性差异(P<0.05)；同时与多西他赛组和替吉奥组相比，淫羊藿苷元组小鼠的白细胞总数、血小板数目和中性粒细胞数均有大幅度增高，并有极显著性差异(P<0.01)。
通过试验结果还可以看出，淫羊藿苷元作为抗癌药在与多西他赛或替吉奥同时联合用来抗肿瘤时，其不仅发挥了抑制肿瘤生长作用，同时还缓解了多西他赛或替吉奥带来的骨髓抑制。由此可见，淫羊藿苷元可以在其它药物化疗的同时使用，也可以预防使用，而不是仅在出现白细胞减少时给予使用，其在保证抑瘤率的同时能促进骨髓造血，增加白细胞数量，增加中性粒细胞数量，并在一定程度上减少了副反应。
实施例23：淫羊藿苷元对60Co放射小鼠的血细胞数的影响
1.材料
1.1试验动物：昆明小鼠(购自中国药品生物制品检定所，实验动物许可证号：SCXKⅡ-00-0010)，雌雄各半，7周龄，18～22g，试验温度(20±1)℃，湿度40％～70％，自由饮水，正常饲喂。
1.2试验试剂：
Figure PCTCN2014088944-appb-000020
2.方法：
除正常组(10只)外，对其他各组小鼠进行4Gy照射60Co放射，采用一次性全身照射，吸收剂量为4Gy，吸收剂量率为0.88Gy/min。于辐射后的第3、7、10d分别眶静脉取血检测全血细胞数。将连续两次全血细胞测定中白细胞数量低于3.0×109/L或者血小板数量少于500×109/L的小鼠剔出，剩余小鼠即为实验用小鼠。
将照射后符合实验要求的小鼠随机分为以下模型对照组、淫羊藿提取物组和淫羊藿苷元组，每组10只，雌雄各半。各组分别按如下方式处理或给药。
第1组为正常组：0.9％生理盐水腹腔注射，给药体积10ml/kg；
第2组为模型对照组：0.9％生理盐水腹腔注射，给药体积10ml/kg；
第3组为淫羊藿提取物组：3.5ml/kg/d淫羊藿水提液腹腔注射；
第4组为淫羊藿苷元组：10mg/kg/d淫羊藿苷元腹腔注射，给药体积10ml/kg；
淫羊藿提取物的制备方法为：(1)将淫羊藿放入煲内，加入清水，浸满药面；(2)浸 泡30min使淫羊藿的有效成分易于煎出；(3)先快速加热至充分沸腾1-3min，然后继续加热20-30min使之浓缩，用消毒纱布过滤入杯内；(4)1次将药物煎好后，将首剂和再煎的药物混匀，以便药效均衡。将1kg淫羊藿制成200mL水提液。以下实验采用的淫羊藿提取物均为本方法制备得到。
各组每天给药一次，共给药10天。试验过程中，每天观察动物饮食、存活情况及行为活动，每日测量体重，试验结束后麻醉解剖，腹主静脉取血，测血常规，考察白细胞总数及血小板总数。
3.实验结果：
表3 淫羊藿苷元对4Gy照射60Co小鼠白细胞和血小板的影响
Figure PCTCN2014088944-appb-000021
与模型对照组比较，#P＜0.05，##P＜0.01；
与淫羊藿提取物组比较，$P＜0.05
通过本实施例淫羊藿苷元对接受4Gy照射60Co小鼠白细胞和血小板的影响(表3)发现，淫羊藿苷元有预想不到的缓解放疗产生的骨髓抑制的效果。具体为，与模型组相比，淫羊藿苷元组的白细胞总数和血小板总数均有增高，并有极显著性差异(P<0.01)或显著性差异(P<0.05)；与淫羊藿提取物组相比有显著性差异(P<0.05)。
实施例24：淫羊藿苷元对NOD小鼠白细胞和血小板的影响
非肥胖型糖尿病(NOD)小鼠是一个很大的小鼠品系，包括NOD/Scid，NOD/Ltj小鼠等。本实验所使用的NOD/Ltj小鼠为免疫***异常小鼠，其血小板和白细胞与普通小鼠相比明显降低。
1.材料
1.1实验动物：NOD/Ltj小鼠(购自北京维通利华实验动物技术有限公司，实验动物许可证为SCXK(京)2006-0009)；雌性；5周龄；16～20g，52只。
1.2试验试剂：
白介素，购自北京义翘神州生物技术有限公司
戊巴比妥钠，购自上海科丰化学试剂有限公司
淫羊藿苷元，采用实施例1所述工艺制备，淫羊藿苷元的纯度99.3％。
2、分组与标记：
经适应性饲养1周后取5周龄的健康NOD小鼠，随机分为4组：
I组为空白对照组，共13只；每日灌胃给予白介素等体积生理盐水；
II组为阳性对照组，共13只，每日灌胃给予白介素10.0mg/kg；
III组为淫羊藿苷元低剂量组，共13只，每日灌胃给予淫羊藿苷元30mg/kg；
IV组为淫羊藿苷元高剂量组，共13只，每日灌胃给予淫羊藿苷元60 mg/kg。
各给药组每天给药一次，连续给药15天停止给药。将动物麻醉(3％戊巴比妥钠，腹腔注射，0.1～0.15ml/只)，腹主静脉采血1ml至抗凝管，测血常规。
表4：淫羊藿苷元对NOD小鼠白细胞和血小板的影响
组别 样本量 白细胞(109/L) 血小板(109/L)
空白对照组 13 2.535±0.38 508.3±38.6
白介素组 13 2.683±0.35 524.5±74.6
淫羊藿苷元低剂量组 13 3.242±0.45#$ 730.5±34.6##$
淫羊藿苷元高剂量组 13 4.225±0.51##$ 780.2±46.5##$$
与空白对照组对比，#p＜0.05，##p＜0.01；
与白介素组相比，$p＜0.01，$$p＜0.01。
由表4可以看出，淫羊藿苷元高剂量和低剂量组的白细胞数量和血小板数量均显著高于模型组，体现出显著的治疗作用。与抗血小板减少的阳性对照药白介素组相比，不但在增高白细胞数量方面有显著优势(p＜0.05)，而且可以显著提高血小板的数量，提高机体的免疫能力，特别是淫羊藿苷元高剂量组，其升高白细胞和血小板的作用与其他各组相比具有十分明显的优势。
实施例25：淫羊藿苷元对正常小鼠出血时间和凝血时间的影响
25.1实验动物及受试药物：
昆明种小鼠，雌雄各半，体重20±2.0g；Wistar大鼠，体重200±20g，雌雄各半。实验动物由山东新时代药业新药药理中心提供。
受试药物：淫羊藿苷元，如实施例1所制备；淫羊藿苷，如CN101607976B实施例1的方法制备而得，纯度为98％，以下实施例中淫羊藿苷的制备方法同本实施例。
25.2.实验分组及给药
取昆明种小鼠100只，随机分成5组，分别为空白对照组(生理盐水组)，淫羊藿苷组(阳性对照组)，淫羊藿苷元高剂量组、淫羊藿苷元中剂量组和淫羊藿苷元低剂量组。各组分别按下述给药方式给药。
空白对照组：皮下注射给予等体积的生理盐水
淫羊藿苷组：注射给予2mg/kg的淫羊藿苷；
淫羊藿苷元高剂量组：皮下注射实施例1淫羊藿苷元注射液，给药剂量为10mg/kg；
淫羊藿苷元中剂量组：皮下注射实施例1淫羊藿苷元注射液，给药剂量为5mg/kg；
淫羊藿苷元低剂量组：皮下注射实施例1淫羊藿苷元注射液，给药剂量为1mg/kg；
25.3出血时间和凝血时间的测定
出血时间的测定：设定生理盐水组(空白对照组)，淫羊藿苷组(阳性对照组)，淫羊藿苷元给药组(高、中、低)，选取小鼠50只，称重，随机分成5组，每组10只。连续给药3d，于末次给药后1h后取滤纸卷成与小鼠身体直径相似的滤纸筒，一端封闭，让小鼠钻入；然后用剪刀剪小鼠尾尖约3mm，从出血开始按下秒表计时，每隔15s用滤纸轻点小鼠尾尖，直至无血迹或看不清血迹为止，所记时间即为出血时间，结果见表5所示。
凝血时间的测定：设定生理盐水组(空白对照组)，淫羊藿苷组(阳性对照组)，淫羊藿苷元给药组(高、中、低)，选取小鼠50只，称重，随机分成5组，每组10只。给药3d后，于末次给药1h后，用一次性20μl采血管***小鼠内眦球后静脉丛取血，取满20μl血。每隔15s折断毛细血管一小段，检查有无血凝丝出现，记录从采血至出现纤维蛋白丝的时间(凝血时间)，结果见表5所示。
表5.出血时间和凝血时间的测定结果
组别 n 给药剂量 出血时间 凝血时间
空白对照组 10 — 5.11±0.62 5.68±0.72
淫羊藿苷组 10 2mg/kg 4.03±0.45* 4.28±0.61*
淫羊藿苷元高剂量组 10 10mg/kg 1.86±0.24**△△ 1.96±0.16**△△
淫羊藿苷元中剂量组 10 5mg/kg 2.51±0.35**△ 3.04±0.31**△
淫羊藿苷元低剂量组 10 1mg/kg 3.75±0.46* 3.84±0.55*
注：与空白对照组比较，*P＜0.05，**P＜0.01；与淫羊藿苷组比较，△P＜0.05，△△P＜0.01。
由表5可以看出，淫羊藿苷元给药组和淫羊藿苷组均可以降低正常小鼠的出血时间和凝血时间，其中淫羊藿苷元各给药组在降低正常小鼠的出血时间和凝血时间方面优于淫羊藿苷组，淫羊藿苷元在降低正常小鼠出血时间和凝血时间方面体现出剂量依赖性，淫羊藿苷元高剂量组和淫羊藿苷元中剂量组与淫羊藿苷组相比具有显著性的差异。
实施例26：淫羊藿苷元对ITP血小板数量的影响
26.1模型制备及分组给药
建立慢性ITP模型：选取Wistar大鼠采用注射兔抗大鼠血小板血清(APS)方法造模，大鼠腹腔注射1：4稀释的APS(0.7ml/200g体重)，持续3天，可使血小板数量显著降低。选取造模成功大鼠50只，称重，随机分成5组，分别为动物分组：模型对照组，淫羊藿苷组(阳性对照组)，淫羊藿苷元给药组(高、中、低3个剂量组)，每组10只。各组分别给予下述治疗药物。
模型对照组：皮下注射给予等体积的生理盐水
淫羊藿苷组：皮下注射给予2mg/kg的淫羊藿苷；
淫羊藿苷元高剂量组：皮下注射实施例11淫羊藿苷元注射液，给药剂量为10mg/kg；
淫羊藿苷元中剂量组：皮下注射实施例11淫羊藿苷元注射液，给药剂量为5mg/kg；
淫羊藿苷元低剂量组：皮下注射实施例11淫羊藿苷元注射液，给药剂量为1mg/kg；
26.2各给药组大鼠血小板计数
于末次给药1h后，用一次性20μl采血管***小鼠内眦球后静脉丛取血，测定大鼠血小板总数，结果见表6所示。
表6.各组大鼠血小板计数的测定结果
组别 n 给药剂量 血小板计数(×109)
模型对照组 10 — 423.6±52.4
淫羊藿苷组 10 2mg/kg 571.8±60.6*
淫羊藿苷元高剂量组 10 10mg/kg 819.96±61.8**△△
淫羊藿苷元中剂量组 10 5mg/kg 720.4±60.3**△
淫羊藿苷元低剂量组 10 1mg/kg 680.84±50.5*
注：与模型对照组比较，*P＜0.05，**P＜0.01；与淫羊藿苷组比较，△P＜0.05，△△P ＜0.01。
由表6可以看出，淫羊藿苷元给药组和淫羊藿苷组均可以升高ITP大鼠血小板数量，其中淫羊藿苷元各给药组在升高ITP大鼠血小板数量优于淫羊藿苷组，淫羊藿苷元在升高ITP大鼠血小板数量体现出剂量依赖性，淫羊藿苷元高剂量组和淫羊藿苷元中剂量组与淫羊藿苷组相比具有显著性的差异。
综上，淫羊藿苷元可以对慢性ITP模型大鼠体现出了积极的治疗作用，其在降低慢性ITP模型大鼠的凝血酶时间、凝血酶原时间及部分凝血活酶时间方面，以及升高血小板方面优于阳性对照药淫羊藿苷。其高、中剂量组可显著缩短慢性ITP模型大鼠的凝血酶时间(TT)、凝血酶原时间(PT)及部分凝血活酶时间(APTT)(P＜0.05)，并具有极显著升高大鼠血小板作用(P＜0.01)。可见，本申请所述的中药组合物可应用于治疗血小板减少症，尤其可应用于治疗免疫性血小板减少症。
实施例27淫羊藿苷元对小鼠主动免疫性血小板减少的影响
27.1模型制备及分组给药
选取Balb/C小鼠72只，称重，随机分成5个给药组，分别为，正常组，模型对照组，淫羊藿苷组(阳性对照组)，淫羊藿苷元给药组(高、中、低3个剂量组)，每组12只。除正常组外，其他组Balb/C小鼠均腹腔注射SD大鼠的血小板，每周一次，连续三次可使血小板数量显著降低。造模开始后第二天，各给药组分别给予下述治疗药物：
正常组：灌胃给予等体积的生理盐水
模型对照组：灌胃给予等体积的生理盐水
淫羊藿苷元组：灌胃给予20mg/kg的淫羊藿苷；
淫羊藿苷元高剂量组：灌胃给予实施例9淫羊藿苷元片剂，给药剂量为100mg/kg；
淫羊藿苷元中剂量组：灌胃给予实施例9淫羊藿苷元片剂，给药剂量为50mg/kg；
淫羊藿苷元低剂量组：灌胃给予实施例9淫羊藿苷元片剂，给药剂量为10mg/kg；
各给药组每天给药一次，正常饲喂。造模三次后，各给药组继续给药一周。麻醉小鼠，取血，检测血小板数量，考察淫羊藿苷元对外周血血小板数(PLT)的影响。
27.2实验结果
淫羊藿苷元对外周血血小板数(PLT)的影响的测定结果如表7所示。
表7 淫羊藿苷元对小鼠主动免疫性血小板减少的影响
组别 n 剂量 PLT(109/L,x±s)
正常对照组 12 —— 1213.6±107.6
模型对照组 12 —— 603.3±72.1#
淫羊藿苷组 12 2mg/kg 850.6±90.1**
淫羊藿苷元高剂量组 12 100mg/kg 1193.4±95.4
淫羊藿苷元中剂量组 12 50mg/kg 983.5±85.7
淫羊藿苷元低剂量组 12 10mg/kg 862.7±75.6
#：与正常对照组相比，p<0.05；**：与模型对照组相比，p<0.01。
由表7可以看出，淫羊藿苷元给药组和淫羊藿苷组对小鼠主动免疫性血小板减少均具有积极的治疗作用，可以升高主动免疫性血小板减少小鼠鼠血小板数量，其中淫羊藿苷元各给药组在升高小鼠主动免疫性血小板减少模型的血小板数量优于淫羊藿苷组，淫羊藿苷元在升高小鼠主动免疫性血小板减少模型的血小板数量方面体现出剂量依赖性，淫羊藿苷元高剂量组和淫羊藿苷元中剂量组与淫羊藿苷组相比具有显著性的差异。
实施例28淫羊藿苷元对环磷酰胺所致小鼠血小板减少的影响
选取Balb/C小鼠72只，称重，随机分成6个给药组，分别为，正常组，模型对照组，淫羊藿苷组(阳性对照组)，淫羊藿苷元给药组(高、中、低3个剂量组)，每组12只。小鼠每天腹腔注射50mg/kg环磷酰胺，连续一周可使血小板数量显著降低。造模开始前一周各给药组分别给予下述治疗药物：
正常组：皮下给予等体积的生理盐水
模型对照组：皮下给予等体积的生理盐水
淫羊藿苷组：皮下注射给予2mg/kg的淫羊藿苷；
淫羊藿苷元高剂量组：皮下注射实施例11淫羊藿苷元注射液，给药剂量为10mg/kg；
淫羊藿苷元中剂量组：皮下注射实施例11淫羊藿苷元注射液，给药剂量为5mg/kg；
淫羊藿苷元低剂量组：皮下注射实施例11淫羊藿苷元注射液，给药剂量为1mg/kg；
各给药组每天给药一次，正常饲喂。连续给药3周后，麻醉小鼠，取血，检测血小板数量，考察淫羊藿苷元对外周血血小板数(PLT)的影响。测定结果如表8所示。
表8 淫羊藿苷元对环磷酰胺所致小鼠血小板减少的影响
组别 n 剂量 PLT(109/L,x±s)
正常对照组 12 —— 1113.6±107.6
模型对照组 12 —— 303.3±72.1#
淫羊藿苷组 12 2mg/kg 550.6±90.1**
淫羊藿苷元高剂量组 12 10mg/kg 894.6.4±82.4
淫羊藿苷元中剂量组 12 5mg/kg 725.9±66.7
淫羊藿苷元低剂量组 12 1mg/kg 633.5±43.2
#：与正常对照组相比，p<0.05；**：与模型对照组相比，p<0.01。
由表8可以看出，淫羊藿苷元给药组和淫羊藿苷组对小鼠环磷酰胺所致小鼠血小板减少均具有积极的治疗作用，可以升高环磷酰胺所致小鼠血小板减少模型的血小板数量，其中淫羊藿苷元各给药组在升高环磷酰胺所致小鼠血小板减少模型的血小板数量优于淫羊藿苷组，淫羊藿苷元在升高环磷酰胺所致小鼠血小板减少模型的血小板数量方面体现出剂量依赖性，淫羊藿苷元高剂量组和淫羊藿苷元中剂量组与淫羊藿苷组相比具有显著性的差异。
以上说明书提到的全部出版物均通过引用并入本文。尽管已参照具体优选实施方式描述了本申请，但是应该理解所主张的发明不仅限于所述具体实施方式。实际上，用于实施本申请的所述模式的各种修改对于生物化学和生物工程或相关领域的技术人员来说都是显而易见的，均应落入本申请权利要求书的范围。

Claims (17)

  1. 淫羊藿苷元在制备用于预防或治疗血细胞减少症药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述的血细胞减少症是外周血白细胞下降或血小板下降。
  3. 根据权利要求1或2所述的用途，其特征在于，所述血细胞减少症为由辐射或化学品引起的骨髓抑制，或为原发性的骨髓增生不良。
  4. 根据权利要求3所述的用途，其特征在于，所述的化学品为用于治疗癌症的化疗药物。
  5. 根据权利要求4所述的用途，其特征在于，所述用于治疗癌症的化疗药物是多西他赛或替吉奥。
  6. 根据权利要求1所述的用途，其中所述的血细胞减少症是血小板减少症。
  7. 如权利要求6所述的用途，其特征在于，所述的血小板减少症为免疫性血小板减少症或继发性血小板减少症。
  8. 如权利要求7所述的用途，其特征在于所述的免疫性血小板减少症为慢性特发性血小板减少性紫癜。
  9. 如权利要求6所述的用途，其特征在于，所述继发性血小板减少症为辐射或化学药物引起的骨髓抑制所引发。
  10. 如权利要求9所述的用途，其特征在于，所述的化疗药物为多西他赛、环磷酰胺或替吉奥。
  11. 如权利要求1-10所述的用途，其特征在于，所述药物为口服制剂或非胃肠道途径给药的制剂，如注射剂。
  12. 如权利要求11所述的用途，其特征在于，所述的口服制剂为片剂、颗粒剂或胶囊剂。
  13. 如权利要求11所述的用途，其特征在于，所述口服制剂或注射剂的每一制剂单位中含有淫羊藿苷元的量为0.1-500mg。
  14. 如权利要求1-13任一项所述的用途，其特征在于，淫羊藿苷元口服给药时，人用给药量为0.1mg/kg/d-100mg/kg/d。
  15. 如权利要求1-11或13任一项所述的用途，其特征在于，淫羊藿苷元注射给药时，人用给药量为0.01mg/kg-10mg/kg。
  16. 根据权利要求1-15任一所述的用途,其特征在于，包含淫羊藿苷元的药物制剂为 固体、液体、油、乳剂、凝胶、气溶胶、吸入剂、喷雾、胶囊、丸剂、贴剂和栓剂。
  17. 根据权利要求1-16任一所述的用途,其特征在于:淫羊藿苷元可以与其他一种或多种治疗白细胞减少的药物、或与其他一种或多种可以升高血小板含量的药物组合，其中所述的治疗白细胞减少的药物为维生素B4、利血生、鲨肝醇、辅酶A，所述的升高血小板含量的药物为白介素-Ⅱ，糖皮质激素，如强的松。
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CN113368099A (zh) * 2020-03-10 2021-09-10 鲁南制药集团股份有限公司 淫羊藿苷元在制备血小板功能异常相关疾病防治药物中的用途
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