WO2021072724A1 - 肽及其作为制备治疗发炎性疾病及疼痛的药物的用途 - Google Patents

肽及其作为制备治疗发炎性疾病及疼痛的药物的用途 Download PDF

Info

Publication number
WO2021072724A1
WO2021072724A1 PCT/CN2019/111788 CN2019111788W WO2021072724A1 WO 2021072724 A1 WO2021072724 A1 WO 2021072724A1 CN 2019111788 W CN2019111788 W CN 2019111788W WO 2021072724 A1 WO2021072724 A1 WO 2021072724A1
Authority
WO
WIPO (PCT)
Prior art keywords
pain
peptide
arthritis
seq
inflammatory diseases
Prior art date
Application number
PCT/CN2019/111788
Other languages
English (en)
French (fr)
Inventor
林成龙
Original Assignee
沛尔生技医药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 沛尔生技医药股份有限公司 filed Critical 沛尔生技医药股份有限公司
Priority to AU2019470693A priority Critical patent/AU2019470693B2/en
Priority to PCT/CN2019/111788 priority patent/WO2021072724A1/zh
Priority to JP2021541078A priority patent/JP7220489B2/ja
Priority to US17/421,600 priority patent/US20220119447A1/en
Priority to EP19949541.7A priority patent/EP4047007B1/en
Publication of WO2021072724A1 publication Critical patent/WO2021072724A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides, which can be used for the treatment of inflammatory diseases and pain, and their application in the preparation of drugs for the treatment of inflammatory diseases.
  • Inflammation refers to a series of physiological reactions that biological tissues are stimulated by trauma or pathogenic infection, including redness, swelling, fever, and pain. Since the inflammatory reaction is one of the organism's reactions to external pathogens, it is an immune response and is beneficial to the organism. However, if you stay in a state of inflammation for a long time, that is, "chronic inflammation", it may cause different inflammatory diseases, such as: hay fever, periodontitis, rheumatoid arthritis, etc. Cytokines are related to immune regulation. Many kinds of cytokines are involved in the inflammation response.
  • Tumor necrosis factor is a multifunctional cytokine that stimulates the proliferation of macrophages or tissue mononuclear spheres and initiates inflammation.
  • the cytokine performance downstream of the reaction is a cytokine that promotes inflammation, and tumor necrosis factor alpha (TNF- ⁇ ) is often used as an indicator of inflammation.
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-6 interleukin-6
  • IL-6 is also an inflammatory substance, which can cause many monocytes and macrophages to enter the affected area and cause inflammation.
  • pain refers to the unpleasant sensory and psychological feelings associated with real or potential tissue damage. Pain is the discomfort of the nervous system, and the common types of pain can be divided into irritating pain, neurogenic pain, sympathetic neuropathic pain, psychogenic pain, etc., and there are other locations, time, that is, acute, Chronic, background etiology, physiological characteristics, etc. Among them, acute pain occurs for a relatively short time, and is usually an early warning effect of tissue damage. This type of pain can automatically disappear by tissue recovery; chronic pain occurs for a long time, usually not caused by body tissue damage, but The body is a warning sign of disease. Chronic pain can cause depression, autism, irritability, etc., resulting in higher social costs. Therefore, the treatment of pain has always been an important topic in medicine.
  • the present invention provides a peptide, which can be used to treat inflammatory diseases and pain; at the same time, it provides a peptide as a preparation for the treatment of inflammatory diseases.
  • the peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof
  • the drug comprises an effective dose of the peptide and a pharmaceutically acceptable carrier.
  • the peptide can also be used for the treatment of pain.
  • the present invention also provides an application of a peptide as a medicine for the treatment of pain, wherein the peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof.
  • the medicine contains an effective dose of the peptide and a pharmaceutically acceptable carrier.
  • the present invention further provides a peptide for the treatment of inflammatory diseases, the peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof.
  • the present invention further provides a peptide for the treatment of inflammatory diseases, and the peptide is composed of the amino acid sequence shown in SEQ ID NO:1.
  • the present invention further provides a peptide for the treatment of pain, the peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof.
  • the present invention further provides a peptide for the treatment of pain, and the peptide is composed of the amino acid sequence shown in SEQ ID NO:1.
  • the present invention further provides a method for treating inflammatory diseases, which comprises: administering a therapeutically effective amount of a peptide to a desired receptor, the peptide comprising the amino acid sequence shown in SEQ ID NO:1 or Variants.
  • the present invention further provides a method for treating inflammatory diseases, which consists of administering a therapeutically effective amount of a peptide to a desired receptor, and the peptide consists of the amino acid sequence shown in SEQ ID NO:1 .
  • the present invention further provides a method for treating pain, which comprises: administering a therapeutically effective amount of a peptide to a desired receptor, the peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof. body.
  • the present invention further provides a method for treating pain, which consists of administering a therapeutically effective amount of a peptide to a desired receptor, and the peptide consists of the amino acid sequence shown in SEQ ID NO:1.
  • the variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO:1.
  • the amino acid sequence of the variant has an amino acid sequence that is at least 80%, 85%, 90%, or more than 90% identical to the SEQ ID NO:1 peptide, and has an amino acid sequence capable of treating inflammatory diseases or The effect of pain.
  • the peptide comprises at least one amino acid addition, deletion and/or substitution. In some embodiments, the amino acid addition, deletion and/or substitution are performed at the C-terminus and/or N-terminus.
  • the variant is a conservative variant, which has one to three conservative amino acid substitutions compared with SEQ ID NO:1, preferably one to two conservative amino acid substitutions, and more preferably one conservative amino acid substitution
  • the amino acid sequence of sexual amino acid substitution and has the effect of treating inflammatory diseases or pain.
  • the "conservative amino acid substitution” refers to the substitution of amino acids with the same or similar properties compared with the original amino acid sequence.
  • the amino acids that exist in nature can be divided into the following categories based on the nature of branched chains:
  • a conservative amino acid substitution is that one amino acid of one of these classes is replaced by another amino acid of the same class.
  • Conservative amino acid substitutions may include substitutions with non-naturally occurring amino acid residues.
  • the peptide is composed of the amino acid sequence of SEQ ID NO:1 or a variant thereof. More preferably, the peptide is composed of the amino acid sequence of SEQ ID NO:1.
  • the "pharmaceutically acceptable carrier" of the present invention includes, but is not limited to, solvents, emulsifiers, suspending agents, disintegrating agents, binders, excipients, stabilizers, diluents, gelling agents , Preservatives, lubricants, surfactants, and other similar or suitable carriers of the present invention.
  • the aforementioned inflammatory diseases include ankylosing arthritis, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, traumatic arthritis, septic arthritis, gouty arthritis, tuberculous arthritis, nerves Arthritis, and hemophilic arthritis.
  • the aforementioned pain includes neuropathic pain, inflammatory pain, musculoskeletal pain, post-surgical pain, cancer pain, acute pain, and chronic pain.
  • the aforementioned inflammatory pain includes ankylosing arthritis pain, osteoarthritis pain, rheumatoid arthritis pain, rheumatoid arthritis pain, traumatic arthritis pain, septic arthritis pain, gouty arthritis pain , Tuberculous arthritis pain, neuropathic arthritis pain, hemophilic arthritis pain.
  • the aforementioned drugs may be in enteral or parenteral dosage forms.
  • the enteral dosage forms include, but are not limited to, enteric-coated lozenges, multi-layer lozenges, sugar-coated lozenges, sublingual lozenges, chewing lozenges, and oral lozenges.
  • Oral agents and enemas such as medicines, capsules, powders, syrups, solutions, emulsions, suspensions, mucilages, emulsions, liquid extracts, extracts, elixirs, elixirs, and tinctures.
  • Parenteral dosage forms include, but are not limited to injections, ointments, lotions, liniments, and vaporized sprays.
  • the aforementioned effective dose is 0.0008 ⁇ g/kg to 815 ⁇ g/kg.
  • the administration frequency of the peptide in this case is once a day to once a month; more preferably, the administration frequency of the peptide in this case is twice a week to twice a month; more preferably, the administration frequency of the peptide in this case is The frequency of medication is once a week.
  • the peptide in this case is administered once a week at the above effective dose.
  • the effective dose calculation method is based on the concentration of the peptide of SEQ ID NO:1 in the experimental example of this specification from 0.01 ⁇ g/kg to 10 mg/kg, according to the initial estimation method of the experiment announced by the U.S. Food and Drug Administration, Based on a 60 kg adult, the dose conversion is calculated by using 12.3 times the recommended daily intake per kg of body weight (/kg bw/d) of the human body to 1 times the dose of mice.
  • the aforementioned effective dose is 0.008 ⁇ g/kg to 410 ⁇ g/kg.
  • the aforementioned effective dose is 0.008 ⁇ g/kg to 0.25 ⁇ g/kg.
  • the aforementioned effective dose is 0.025 ⁇ g/kg to 0.25 ⁇ g/kg.
  • the aforementioned effective dose is 80 ⁇ g/kg to 410 ⁇ g/kg.
  • the aforementioned effective dose is 80 ⁇ g/kg to 250 ⁇ g/kg.
  • the enteral dosage form is an oral dosage form.
  • parenteral dosage form is an injection dosage form.
  • treatment refers to administering a therapeutically effective amount of the peptide of the present case to the patient to achieve the therapeutic effect, and the therapeutic effect refers to the eradication, improvement, alleviation or alleviation of the disease or its related symptoms.
  • the treatment of inflammatory diseases in the present invention refers to alleviating inflammation-related symptoms, and in some embodiments, it is evaluated by reducing inflammation indicators.
  • the treatment of pain in the present invention refers to the relief of related symptoms of pain. In some embodiments, it is an increase in the threshold for feeling pain, and in some embodiments, an increase in the threshold for paw retraction is used for evaluation.
  • an effective dose refers to the dose required to produce the desired biological response, that is, the dose required to produce the therapeutic effect.
  • Figure 1 shows the amount of TNF- ⁇ in the serum of mice in the experimental group and the control group given 3 mg/kg of the peptide of SEQ ID NO:1 every week from the 4th week to the 12th week.
  • Figure 2 shows the amount of IL-6 in the serum of mice in the experimental group and the control group that were given 3 mg/kg of the peptide of SEQ ID NO:1 every week from the 4th to the 12th week.
  • Figure 3 shows the change of the paw withdrawal threshold of the sole of the ipsilateral foot of the experimental group and the morphine group (10 mg/kg) mice administered with 3 mg/kg of the peptide of SEQ ID NO:1 with time after administration.
  • Figure 4 shows the change of the paw withdrawal threshold of the contralateral foot of the mice in the experimental group and the morphine group (10 mg/kg) administered with 3 mg/kg of the peptide of SEQ ID NO:1 with time after administration.
  • Figure 5 shows the paw withdrawal threshold of the posterior ipsilateral foot of mice in the control group given 1 mg/kg and 3 mg/kg of SEQ ID NO:1 peptide every week from the 4th to the 12th week.
  • Figure 6 shows the paw withdrawal threshold of the contralateral feet of mice in the control group given 1 mg/kg and 3 mg/kg of SEQ ID NO:1 peptide every week from 4 to 12 weeks.
  • Figure 7 shows the paw withdrawal threshold of the soles of the ipsilateral feet of mice in the control group administered with the peptide of SEQ ID NO:1 at 0.3 ⁇ g/kg, 1 ⁇ g/kg, and 3 ⁇ g/kg, respectively.
  • Fig. 8 shows the sole pain threshold of the contralateral foot of mice in the control group administered with the peptide of SEQ ID NO: 1 at 0.3 ⁇ g/kg, 1 ⁇ g/kg, and 3 ⁇ g/kg, respectively.
  • Figure 9 shows the analgesic activity of the peptide of SEQ ID NO: 1 in the acetic acid writhing experiment and the maximum possible effect of the peptide of SEQ ID NO: 1 in the tail flick experiment.
  • a standard Fmoc strategy and a microwave peptide synthesizer are used to synthesize the Glp Glu Thr Ala Val Ser Ser His Glu Gln Asp peptide used in the present invention, and the Glp is pyroglutamic acid (Cas Number 98-79-3).
  • the Wang resin (0.6mmol/g load) preloaded with D residues is weighed into the reaction vessel, and fresh DMF (10-15mL) is added before synthesis as preparation for swelling, on the microwave synthesizer Set the swelling time to 3 minutes.
  • the first and subsequent Fmoc groups are reacted with 20% piperidine in 5 mL DMF (as a "DEP" solution), and the standard deprotection procedure is used (the first stage temperature is 75°C, the power is 155W, and the The temperature time is 15 seconds, the second stage is 90°C, the power is 30W, and the temperature holding time is 50 seconds) to free the N-terminal amine.
  • DMF containing 0.5M N,N'-diisopropylcarbodiimide N,N'-diisopropylcarbodiimide, DIC
  • ACT C-terminal activation
  • the amino acid (as Fmoc-AA (protected side chain)-OH) is used in the coupling reaction in DMF (abbreviated as AA) in a 5-fold excess (a concentration of 0.2M in DMF) and a concentration of 1.0M Oxyma.
  • the reaction is carried out with standard coupling procedures (the first stage temperature is 75°C, power 170W, holding temperature time is 15 seconds, the second stage is 90°C, power 30W, holding temperature time is 230 seconds), and the process is carried out in "AA" for about 4 After minutes, rinse the Wang resin with DMF solution. Then, keep repeating the DEP-ACT-AA step, cycling to construct peptides from the C-terminus to the N-terminus.
  • the peptide was removed from the solid support by treatment with 95% TFA/2.5% H 2 O/2.5% TIPS in an ice bath and returning to room temperature for 2 hours.
  • the filtrate was collected by filtration, and ice ether was added for precipitation, followed by centrifugation, then the supernatant was removed, and ether was added to clean the precipitate. Repeat the above steps 3 to 6 times, and finally the precipitate was taken out and freeze-dried.
  • white cotton can be obtained, which is the final product, and its purity can be identified by a high-pressure liquid chromatography system.
  • the synthesized peptides were identified by nano LC-MS/MS analysis of the molecular weight and sequence of the peptides.
  • mice (Lisco Biotechnology, BioLASCO Taiwan), 8-12 weeks old and weighing approximately 20 to 25 grams, were continuously injected with 1 mg/mL in the right ankle joint.
  • Complete Freund's Adjuvant (CFA) once a week, with a dose of 5 ⁇ L, and the time of the first administration as week 0.
  • ICR Institutee of Cancer Research
  • the long-term inflammation and swelling of the unilateral front ankle joint of the mouse lasts for more than 12 weeks, and can induce bilateral mechanical hyperalgesia for more than 12 weeks, in order to simulate the chronic chronic pain in patients with rheumatoid arthritis.
  • mice of Preparation Example 2 Take the mice of Preparation Example 2 and administer 3 mg (3mg/kg) of SEQ ID NO:1 peptide per kilogram dissolved in sterile water orally in the 4th week, and give it once a week until the 12th week as an experiment There are 6 mice in the experimental group and 6 mice in the experimental group. The other mice in the control group are given by oral water. There are also 6 mice in the control group.
  • mice in the experimental group and the control group are sacrificed, and blood is collected by heart Serum, using TNF- ⁇ quantitative kit (Mouse TNF- ⁇ Quantikine ELISA Kit, R&D System; MTA00B) and IL-6 quantitative kit (Mouse IL-6Quantikine ELISA Kit, R&D System, M600B) enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay; ELISA)
  • TNF- ⁇ quantitative kit Mae TNF- ⁇ Quantikine ELISA Kit, R&D System; MTA00B
  • IL-6 quantitative kit Meouse IL-6Quantikine ELISA Kit, R&D System, M600B enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay; ELISA)
  • the amount of TNF- ⁇ and IL-6 in the serum of mice in the experimental group and the control group was measured and collected, and it was found that the long-term oral administration of SEQ ID NO:1 peptide in the experimental group was
  • the peptide of SEQ ID NO: 1 of the present invention can indeed effectively reduce systemic inflammation indicators, confirming that SEQ ID NO: 1 of the present invention can indeed treat inflammatory diseases, especially systemic chronic disease caused by rheumatoid arthritis. fever.
  • mice of Preparation Example 2 were taken orally given 10mg/kg of opioid analgesic morphine (morphine) as a positive control group in the 4th week, and 3mg dissolved in sterile water was given orally in the 4th week
  • the peptide of SEQ ID NO: 1 per kg is the experimental group.
  • the positive control group and the experimental group of morphine have 6 mice each, and they are measured by a tactile measuring instrument (von Frey Filament) at 0 minutes, 30 minutes, and 60 minutes after administration.
  • the PWT value of the ipsilateral foot of the morphine group increased from 0.085 ⁇ 0.015 g to 0.64 ⁇ 0.089 g (as shown in Figure 3), and the PWT value of the contralateral foot increased from 0.43. ⁇ 0.033g rose to 1.96 ⁇ 0.488g (as shown in Figure 4); the PWT value of the ipsilateral foot of the mice in the experimental group rose from 0.07g to 0.9 ⁇ 0.156g (as shown in Figure 3). The PWT value increased from 0.5 ⁇ 0.047 g to 1.35 ⁇ 0.193 g (as shown in Figure 4), and the analgesic effect began to decrease 120 minutes after administration.
  • mice of Preparation Example 2 where the time points for the injection of complete Freund’s adjuvant (CFA) four times in the Preparation Example are indicated by black arrows in Figures 5 and 6, and the mice will be administered orally in the fourth week.
  • 1 mg/kg or 3 mg/kg of the peptide of SEQ ID NO: 1 in sterile water 6 mice in each of the two dose groups, once a week for continuous up to the 12th week (indicated by the gray arrow), orally Water was used as the control group, and there were 6 mice in the control group, and the PWT value was measured with a tactile meter 90 minutes after the administration.
  • the PWT of the contralateral foot increased from 0.4 g in the third week to 0.6-1.2 g; and after oral administration of 3 mg/kg SEQ ID NO:1 peptide (as shown in Figure 6), the PWT of the posterior ipsilateral foot PWT increased from 0.09 g in the third week to 0.63-1 g (as shown in Figure 5), and the PWT of the contralateral foot increased from 0.4 g to 0.87 to 1.27 g (as shown in Figure 6); therefore, the experimental results showed that long-term oral administration
  • the peptides of SEQ ID NO:1 at 1 mg/kg and 3 mg/kg have similar analgesic effects.
  • mice of Preparation Example 2 were taken orally administered 0.3 ⁇ g/kg, 1 ⁇ g/kg, and 3 ⁇ g/kg of SEQ ID NO:1 peptide dissolved in sterile water in the fourth week. There were 6 mice each, and the control group was given water orally. There were also 6 mice in the control group, and the PWT value was measured with a tactile measuring instrument 90 minutes after the administration.
  • mice of Preparation Example 3 were placed in an observation chamber and allowed to familiarize themselves for 20 minutes.
  • Distilled water (as the vehicle control group) and different concentrations (0.1 ⁇ g/kg, 0.3 ⁇ g/kg, 1 ⁇ g/
  • the peptide of SEQ ID NO:1 (kg, 0.01mg/kg, 0.1mg/kg, 1mg/kg, 3mg/kg, 10mg/kg) was orally administered to mice, the vehicle control group and SEQ ID NO:1 There were 5 to 12 mice in each of the peptide administration groups. Within 30 minutes after the acetic acid injection, the number of times the mouse twisted represents the number of abdominal contractions. After calculating the number of times the mouse twisted, the average number of twists of all mice in each group was calculated.
  • analgesic effect of the acetic acid writhing test is expressed in analgesic activity% (antinociceptive activity%): (average number of twists in the vehicle control group-average number of twists in the administration group)/(average number of twists in the vehicle control group) ⁇ 100%.
  • Different doses (0.01 ⁇ g/kg, 0.03 ⁇ g/kg, 0.1 ⁇ g/kg, 0.3 ⁇ g/kg, 1 ⁇ g /kg, 0.01mg/kg, 0.1mg/kg, 1mg/kg, 3mg/kg, 10mg/kg)
  • SEQ ID NO: 1 peptide measured every ten minutes after 10 to 90 minutes, record the lag time and calculate Based on the average lag time of 10 to 90 minutes, according to LS Harris, AK Pierson, Some Narcotic Antagonists in the Benzomorphan Series. J Pharmacol Exp Ther 143, 141-148 (1964).
  • the analgesic effect of the tail-flick experiment is based on %MPE (maximum).
  • the results of experimental examples 5 and 6 are shown in Figure 9.
  • the peptide of SEQ ID NO: 1 has excellent analgesic effects in the acetic acid writhing experiment at the doses of 1 ⁇ g/kg and 10 mg/kg, while in the radiant heat tail flick experiment
  • the effects of 0.1 ⁇ g/kg, 0.3 ⁇ g/kg, 0.1mg/kg, 1mg/kg, 3mg/kg, and 10mg/kg are particularly excellent.
  • the peptide of SEQ ID NO:1 of the present invention is at high concentration and low concentration. Both have analgesic effect.
  • the peptide of the present invention does have the effect of treating inflammatory diseases, especially rheumatoid arthritis; and can effectively treat pain, especially pain caused by rheumatoid arthritis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Rheumatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供一种肽及其作为制备治疗发炎性疾病及疼痛的药物的用途。具体而言，提供一种肽及其作为制备治疗发炎性疾病的药物的应用，其中所述肽包含如SEQ ID NO：1所示的氨基酸序列，所述药物包含有效剂量的所述肽及药学上可接受的载剂。另有所述肽作为治疗疼痛的药物的应用，所述药物包含有效剂量的所述肽及药学上可接受的载剂。证实了给予所述肽可以有效降低发炎指标，且给予所述肽可以有效止痛。

Description

肽及其作为制备治疗发炎性疾病及疼痛的药物的用途 技术领域
本发明涉及肽，其可用于治疗发炎性疾病、疼痛，及其用于制备治疗发炎性疾病的药物的应用。
背景技术
发炎反应是指生物组织受到外伤或病源性感染等刺激后，产生一连串生理反应，包括红肿、发热、疼痛等。由于发炎反应是生物体对外界病原体的反应之一、属于一种免疫反应，对生物体是有益的。然而若长期处于发炎的状态，亦即“慢性发炎”，可能会引起不同的发炎性疾病，如：花粉症、牙周炎、类风湿性关节炎等。而细胞激素与免疫调节相关，许多种细胞激素会参与发炎反应，肿瘤坏死因子(TNF)是一种多功能细胞激素，在发炎反应时会刺激巨噬细胞或组织单核球大量增生，并启动反应下游的细胞激素表现，是一种促进发炎反应的细胞激素，其中α型肿瘤坏死因子(TNF-α)常作为发炎指标。此外，介白素-6(IL-6)亦为一促发炎物质，会导致许多单核球及巨噬细胞进入患处引起发炎反应。
根据国际疼痛研究协会的定义，疼痛是指与真实或潜在组织损伤相关联的不愉快的感官及心理感受。疼痛是神经系统的不适感，而常见的疼痛类型主要可以分为侵害刺激性疼痛、神经源性疼痛、交感神经源性疼痛、心因性疼痛等，另有部位、时间性，也就是急性、慢性，背景病因、生理特性等的区别。其中，急性疼痛发生时间比较短，且通常是组织伤害的预警作用，此类型的疼痛可藉由组织的复原而自动消失；慢性疼痛发生时间比较长、通常不是身体组织伤害所引起的，而是身体对疾病的警讯，慢性疼痛会造成患者忧郁、自闭、易怒等，产生较高的社会成本。因此对于疼痛的处理向来为医学上的一重要课题。
发明内容
有鉴于开发出新的抗发炎、止痛药物为现有技术所迫切需要的，本发明提供一种肽，其可用于治疗发炎性疾病、疼痛；同时提供一种肽作为制备治疗发炎性疾病的药物的应用，其中所述肽包含如SEQ ID NO：1所示的氨基酸序列或其变体，所述药 物包含有效剂量的所述肽及药学上可接受的载剂。此外，所述肽亦可用于治疗疼痛，本发明同时提供一种肽作为制备治疗疼痛的药物的应用，其中所述肽包含如SEQ ID NO：1所示的氨基酸序列或其变体，所述药物包含有效剂量的所述肽及药学上可接受的载剂。
本发明另进一步提供一种肽用于治疗发炎性疾病，所述肽包含如SEQ ID NO：1所示的氨基酸序列或其变体。
本发明另进一步提供一种肽用于治疗发炎性疾病，所述肽由SEQ ID NO：1所示的氨基酸序列所组成。
本发明另进一步提供一种肽用于治疗疼痛，所述肽包含如SEQ ID NO：1所示的氨基酸序列或其变体。
本发明另进一步提供一种肽用于治疗疼痛，所述肽由SEQ ID NO：1所示的氨基酸序列所组成。
本发明另进一步提供一种治疗发炎性疾病的方法，其包含：对一所需的受体给予治疗有效量的一种肽，所述肽包含如SEQ ID NO：1所示的氨基酸序列或其变体。
本发明另进一步提供一种治疗发炎性疾病的方法，其由对一所需的受体给予治疗有效量的一种肽所组成，所述肽由SEQ ID NO：1所示的氨基酸序列所组成。
本发明另进一步提供一种治疗疼痛的方法，其包含：对一所需的受体施予治疗有效量的一种肽，所述肽包含如SEQ ID NO：1所示的氨基酸序列或其变体。
本发明另进一步提供一种治疗疼痛的方法，其由对一所需的受体施予治疗有效量的一种肽所组成，所述肽由SEQ ID NO：1所示的氨基酸序列所组成。
较佳地，所述变体包含与SEQ ID NO：1具有至少80％同一性的氨基酸序列。在一些实施方案中，所述变体的氨基酸序列具有与SEQ ID NO：1肽具有至少80％、85％、90％或90％以上的同一性的氨基酸序列，且具有能治疗发炎性疾病或疼痛的功效。在一些实施方案中，所述肽包含至少一个氨基酸添加、缺失和/或取代。在一些实施方案中，所述氨基酸添加、缺失和/或取代在C端和/或N端进行。
较佳地，所述变体为保守性变体，是与SEQ ID NO：1相比较具有一到三个保守性氨基酸置换，较佳具有一到二个保守性氨基酸置换、更佳具有一个保守性氨基酸置换的氨基酸序列，且具有能治疗发炎性疾病或疼痛的功效。
所述“保守性氨基酸取代”是指与原始氨基酸序列相比，使用具有相同或相近性 质的氨基酸进行取代。而存在于自然界中氨基酸可基于支链的性质分为下列类别：
(1)疏水性：正亮氨酸(norleucine)、Met(M)、Ala(A)、Val(V)、Leu(L)、Ile(I)；
(2)中性亲水性：Cys(C)、Ser(S)、Thr(T)、Asn(N)、Gln(Q)；
(3)酸性：Asp(D)；Glu(E)；
(4)碱性：His(H)、Lys(K)、Arg(R)；
(5)影响链性质的残基：Gly(G)、Pro(P)；和
(6)芳香族：Trp(W)、Tyr(Y)、Phe(F)。
因此，保守性氨基酸取代为这些类别之一的一氨基酸被同一类别的另一氨基酸取代。保守性氨基酸取代可包括以非天然存在的氨基酸残基进行取代。较佳地，所述肽是由SEQ ID NO：1的氨基酸序列或其变体组成。更佳地，所述肽是由SEQ ID NO：1的氨基酸序列组成。较佳地，本发明所述“药学上可接受的载剂”包含，但不限于溶剂、乳化剂、悬浮剂、崩解剂、黏合剂、赋形剂、安定剂、稀释剂、胶凝剂、防腐剂、润滑剂、表面活性剂，及其他类似或适用本发明的载剂。
较佳地，前述发炎性疾病包括僵直性关节炎、骨关节炎、风湿性关节炎、类风湿性关节炎、外伤性关节炎、化脓性关节炎、痛风性关节炎、结核性关节炎、神经性关节炎、及血友病性关节炎。
较佳地，前述疼痛包括神经病变疼痛、发炎性疼痛、肌肉骨骼疼痛、外科手术后疼痛、癌症疼痛、急性疼痛、及慢性疼痛。
较佳地，前述发炎性疼痛包含僵直性关节炎疼痛、骨关节炎疼痛、风湿性关节炎疼痛、类风湿性关节炎疼痛、外伤性关节炎疼痛、化脓性关节炎疼痛、痛风性关节炎疼痛、结核性关节炎疼痛、神经性关节炎疼痛、血友病性关节炎疼痛。
前述药物可以是经肠道或非经肠道的剂型，经肠道剂型包括，但不限于肠溶锭剂、多层锭剂、糖衣锭剂、舌下锭剂、咀嚼锭剂、口含锭剂、胶囊剂、散剂、糖浆剂、溶液剂、乳剂、悬浮剂、胶浆剂、乳浆剂、流浸膏剂、浸膏剂、醑剂、酏剂、及酊剂等口服剂及灌肠剂。非经肠道剂型包括，但不限于注射剂、软膏剂、洗剂、擦剂、气化喷雾剂。
较佳地，前述有效剂量为0.0008μg/kg至815μg/kg。
较佳地，本案的肽的给药频率是每天一次至每月一次；更佳地，本案的肽的给药频率是每周两次至每月两次；更佳地，本案的肽的给药频率是每周一次。
更佳地，本案的肽是以上述有效剂量每周给药一次。
依据本发明，有效剂量的计算方式是以本说明书的实验例中SEQ ID NO：1的肽的浓度0.01μg/kg至10mg/kg为基准，根据美国食品药物管理局公告的实验初期估算方法，以60千克的成人为基准，剂量换算以人体每日每千克体重的建议摄取量(/kg b.w./d)的12.3倍为小鼠的1倍剂量换算而得。
更佳地，前述有效剂量为0.008μg/kg至410μg/kg。
更佳地，前述有效剂量为0.008μg/kg至0.25μg/kg。
更佳地，前述有效剂量为0.025μg/kg至0.25μg/kg。
更佳地，前述有效剂量为80μg/kg至410μg/kg。
更佳地，前述有效剂量为80μg/kg至250μg/kg。
较佳地，其中所述经肠道的剂型是口服剂型。
较佳地，其中所述非经肠道剂型是注射剂型。
依据本发明，“治疗”是指将治疗有效量的本案的肽给予患者，达到治疗的效果，治疗的效果是指疾病或其相关症状的根除、改善、减轻或缓解。其中，本发明的治疗发炎性疾病是指减轻发炎相关症状，在一些实施例中以降低发炎指标来评估。其中，本发明的治疗疼痛是指缓解疼痛的相关症状，在一些实施例中即为感受到疼痛的门坎上升，在一些实施例中以脚掌痛觉缩脚门坎上升来评估。
依据本发明，“有效剂量”是指产生所需生物反应所需要的剂量，即为产生治疗效果的所需剂量。
附图说明
图1显示第4周至第12周每周给予3mg/kg的SEQ ID NO：1的肽的实验组及控制组小鼠血清中TNF-α的量。
图2显示第4周至第12周每周给予3mg/kg的SEQ ID NO：1的肽的实验组及控制组小鼠血清中IL-6的量。
图3显示给予3mg/kg的SEQ ID NO：1的肽的实验组及吗啡组(10mg/kg)小鼠的后同侧脚的脚掌痛觉缩脚门坎随给药后时间变化。
图4显示给予3mg/kg的SEQ ID NO：1的肽的实验组及吗啡组(10mg/kg)小鼠的对侧脚的脚掌痛觉缩脚门坎随给药后时间的变化。
图5显示第4周至第12周每周给予1mg/kg及3mg/kg的SEQ ID NO：1肽及控制组的小鼠的后同侧脚的脚掌痛觉缩脚门坎。
图6显示第4周至第12周每周给予1mg/kg及3mg/kg的SEQ ID NO：1肽及控制组的小鼠的对侧脚的脚掌痛觉缩脚门坎。
图7显示分别给予0.3μg/kg、1μg/kg、3μg/kg的SEQ ID NO：1的肽及控制组的小鼠的后同侧脚的脚掌痛觉缩脚门坎。
图8显示分别给予0.3μg/kg、1μg/kg、3μg/kg的SEQ ID NO：1的肽及控制组的小鼠的对侧脚的脚掌痛觉缩脚门坎。
图9显示SEQ ID NO：1的肽在醋酸扭体实验的止痛活性及SEQ ID NO：1的肽在甩尾实验的最大可能效果。
具体实施方式
以下配合附图及本发明的制备例及实验例，进一步阐述本发明为达成预定发明目的所采取的技术手段。
制备例1
使用标准Fmoc策略和微波肽合成仪合成本发明所使用的Glp Glu Thr Ala Val Ser Ser His Glu Gln Asp肽，所述Glp为焦谷氨酸(pyroglutamic acid，Cas Number 98-79-3)。一般而言，将D残基预装载的Wang树脂(0.6mmol/g载荷)称入反应容器中，并在合成前加入新鲜DMF(10-15mL)作为欲溶胀的准备，在微波合成仪上设定溶胀时间为3分钟。
将第一和相续的Fmoc基团用含20％哌啶(Piperidine)的5mL DMF(作为“DEP”溶液)，以标准去保护程序进行反应(第一阶段温度为75℃，功率155W，持温时间为15秒、第二阶段为90℃，功率30W，持温时间为50秒)以游离N端的胺。
其后，将含浓度0.5M N,N'-二异丙基碳二亚胺(N,N'-diisopropylcarbodiimide，DIC)的DMF加入用于C端活化(作为“ACT”)，并将所需的氨基酸(作为Fmoc-AA(保护的侧链)-OH)以5倍过量(浓度为0.2M在DMF中)和浓度1.0M Oxyma在DMF中用于耦合反应(简称为AA)。以标准耦合程序进行反应(第一阶段温度为75℃，功率170W，持温时间为15秒、第二阶段为90℃，功率30W，持温时间为230秒)，在“AA”进行约4分钟后，再用DMF溶液清洗Wang树脂。然后，保持重复DEP-ACT-AA 步骤，循环以自C端至N端构建肽。
最后，通过用95％TFA/2.5％H 2O/2.5％TIPS，在冰浴中处理并返回室温达2小时而将肽从固相支持体中移除。通过过滤收集滤液，并加入冰乙醚进行沉淀，随后进行离心，接着移除上清液，并再加入乙醚清洗沉淀物，重复上述步骤3至6次，最后将沉淀物取出，并进行冷冻干燥，最终可得白色棉状物，即为最终产物，再以高压液相层析系统鉴定其纯度。而所合成的肽则通过纳米LC-MS/MS分析肽的分子量及序列鉴定。
制备例2
藉由以下实验步骤诱发小鼠关节发炎，在8-12周龄、体重约为20至25公克的ICR小鼠(乐斯科生物技术，BioLASCO Taiwan)的右侧踝关节连续注射1mg/mL的完全弗氏佐剂(Complete Freund's Adjuvant；CFA)每周一次、剂量为5μL，且以第一次施打的时间为第0周，共四次后，可以诱发ICR(Institute of Cancer Research)品系小鼠单侧前脚踝关节长期发炎和肿胀的情况持续12周以上，并且能引发双侧机械性痛觉敏感持续12周以上，以此模拟类风湿性关节炎病患在临床上长期慢性的疼痛情形。
制备例3
为了进行伤害感受性疼痛、酸诱导慢性泛发性疼痛试验。将C57BL/6JNarl雄性小鼠(20-25克)饲养在控温(24±0.5℃)及12小时明暗循环(08:00-20:00进行光照)的环境下。实验动物能自由获取标准实验食物和自来水。在实验操作前，实验动物有20-30分钟的熟悉期间。实验的动物照护和处理程序符合国际疼痛研究协会针对用于疼痛研究的动物的指导原则，且实验程序已获得动物研究伦理委员会的认可。
实验例1
取制备例2的小鼠，在第4周以口服方式给予每千克3毫克(3mg/kg)溶于灭菌水的SEQ ID NO：1的肽，每周给予一次连续至第12周为实验组，实验组小鼠共6只，另以口服方式给水作为控制组，控制组小鼠亦为6只，在第12周给药后牺牲实验组及控制组的小鼠，以心脏采血方式采集血清，经使用TNF-α定量套组(Mouse TNF-αQuantikine ELISA Kit,R&D System；MTA00B)、及IL-6定量套组(Mouse IL-6Quantikine ELISA Kit,R&D System,M600B)酶联免疫吸附分析法(enzyme-linked immunosorbent assay；ELISA)测量搜集得到实验组及控制组小鼠血清中TNF-α和IL-6 的量，结果发现实验组的长期口服SEQ ID NO：1的肽的小鼠血清中TNF-α降低至可测量范围外，即为未检出(not detected；ND)(请参照图1)，且血清中IL-6的量相较于控制组亦呈现下降情形(请参照图2)，因此本发明的SEQ ID NO：1的肽确实能有效降低全身性发炎指标，证实本发明的SEQ ID NO：1确实能治疗发炎性疾病，尤其是类风湿性关节炎造成的全身性慢性发炎。
实验例2
取制备例2的小鼠，第4周以口服方式给予鸦片类止痛剂10mg/kg的吗啡(morphine)为吗啡组作为正对照组，及第4周以口服方式给予溶于灭菌水的3mg/kg的SEQ ID NO：1的肽为实验组，吗啡的正对照组及实验组小鼠各为6只，并以触觉测量仪(von Frey Filament)测定给药后0分钟、30分钟、60分钟、90分钟及120分钟的小鼠脚掌痛觉缩脚门坎(paw withdraw threshold；PWT)。
实验结果请同时参照图3及图4，不论是小鼠的后同侧脚(ipsilateral)组或对侧脚(contralateral)组，在给予SEQ ID NO：1后，PWT值均如吗啡的正对照组，有上升的趋势，因此可证明本案的SEQ ID NO：1的肽有与吗啡近似的止痛效果，且仅需较低的剂量，另外可观察到实验组及吗啡组给药后90分钟的止痛效果均最好，于90分钟时，吗啡组的小鼠后同侧脚的PWT值从0.085±0.015克上升至0.64±0.089克(如图3所示)，对侧脚的PWT值从0.43±0.033克上升至1.96±0.488克(如图4所示)；实验组的小鼠后同侧脚的PWT值从0.07克上升至0.9±0.156克(如图3所示)，对侧脚的PWT值从0.5±0.047克上升至1.35±0.193克(如图4所示)，且给药后120分钟的止痛效果开始降低。
实验例3
取制备例2的小鼠，其中制备例中的注射完全弗氏佐剂(CFA)的时间点四次如图5及图6中以黑色箭头表示，第4周开始以口服方式给予小鼠溶于灭菌水的1mg/kg或3mg/kg的SEQ ID NO：1的肽，两剂量组的小鼠各为6只，每周给一次连续至第12周(以灰色箭头表示)，以口服方式给水作为控制组，控制组小鼠亦为6只，并在投药后90分钟以触觉测量仪测量PWT值。
实验结果请同时参照图5及图6，以口服的方式给予1mg/kg SEQ ID NO：1的肽后，后同侧脚的PWT从0.09克上升至0.5～1克(如图5所示)，对侧脚的PWT从第三周的0.4克上升至0.6～1.2克；而以口服的方式给予3mg/kg SEQ ID NO：1的肽后(如 图6所示)，后同侧脚的PWT从第三周的0.09克上升至0.63～1克(如图5所示)，对侧脚的PWT从0.4克上升至0.87～1.27克(如图6所示)；因此实验结果显示长期口服1mg/kg及3mg/kg的SEQ ID NO：1的肽止痛效果相似。
实验例4
取制备例2的小鼠24只，第4周以口服方式分别给予溶于灭菌水的0.3μg/kg、1μg/kg、3μg/kg的SEQ ID NO：1的肽，每一剂量组的小鼠各为6只，并以口服方式给水作为控制组，控制组小鼠亦为6只，在投药后90分钟以触觉测量仪测量PWT值。
实验结果请同时参阅图7及图8所示，口服0.3μg/kg的SEQ ID NO：1的肽的小鼠后同侧脚的PWT值从0.065克上升至1克，其对侧脚的PWT值从0.32克上升至1克；口服1μg/kg的SEQ ID NO：1的肽的小鼠后同侧脚的PWT值从0.065克上升至0.6克，对侧脚的PWT值从0.32克上升至1.4克，口服3μg/kg的SEQ ID NO：1的肽的小鼠后同侧脚的PWT值从0.065克上升至0.16克，对侧脚的PWT值从0.32克上升至1克；实验结果显示口服SEQ ID NO：1的肽在0.3μg/kg至3μg/kg的剂量均能产生止痛效果。
实验例5
根据H.O.Collier,L.C.Dinneen,C.A.Johnson,C.Schneider,The abdominal constriction response and its suppression by analgesic drugs in the mouse.Br J Pharmacol Chemother 32,295-310(1968)进行醋酸诱导腹部收缩试验，简而言之，是将制备例3的小鼠置于一观察小室，并使其熟悉20分钟。在注射1％醋酸(300μL)以诱导反应内脏疼痛的扭体反应(writhing response)的10分钟前将蒸馏水(作为载剂控制组)及不同浓度(0.1μg/kg、0.3μg/kg、1μg/kg、0.01mg/kg、0.1mg/kg、1mg/kg、3mg/kg、10mg/kg)的SEQ ID NO：1的肽以口服方式给予小鼠，载剂控制组及给予SEQ ID NO：1的肽的给药组均分别有5至12只小鼠。在醋酸注射后30分钟内以小鼠扭动的次数代表腹部收缩的次数，计算小鼠扭动的次数后，再计算出各组内所有小鼠的平均扭动次数。醋酸扭体实验的止痛效果以止痛活性％(antinociceptive activity％)表示：(载剂控制组的平均扭动次数-给药组的平均扭动次数)/(载剂控制组的平均扭动次数)×100％。
实验例6
根据F.E.D'Amour,D.L.Smith,A method for determining loss of pain sensation.The Journal of Pharmacology and Experimental Therapeutics 72,74-79(1941)的辐射热甩尾实验进行调整。将制备例3的小鼠5至12只在甩尾实验前30分钟置于限位器(restrainer)上以熟悉环境。并使用红外线热通量辐射计(Ugo Basile,Italy,cat.No.37300)进行校正和维持仪器。对距离小鼠尾巴尖端2厘米处施加刺激位点，而小鼠将尾巴自热源抽回所需的时间为甩尾迟滞时间(tail-flick latency)。为降低组织伤害的风险，最大测量时间为30.1秒。调整热强度使大多数的实验动物的迟滞时间的基线在约5到8秒，在以口服方式给予不同剂量(0.01μg/kg、0.03μg/kg、0.1μg/kg、0.3μg/kg、1μg/kg、0.01mg/kg、0.1mg/kg、1mg/kg、3mg/kg、10mg/kg)的SEQ ID NO：1的肽后的10至90分钟每十分钟测量一次、记录迟滞时间再计算出10至90分钟的平均迟滞时间，根据L.S.Harris,A.K.Pierson,Some Narcotic Antagonists in the Benzomorphan Series.J Pharmacol Exp Ther 143,141-148(1964).所述，甩尾实验的止痛效果以％MPE(最大可能效果maximal possible effect)表示：％MPE＝(给药后的平均迟滞时间-给药前的迟滞时间)/(最大测量时间-给药前的迟滞时间)×100％。
实验例5及6的结果如图9所示，SEQ ID NO：1的肽在醋酸扭体实验中以1μg/kg及10mg/kg的剂量下止痛效果特别优良，而在辐射热甩尾实验中以0.1μg/kg、0.3μg/kg、0.1mg/kg、1mg/kg、3mg/kg、10mg/kg的效果特别优良，显见本发明的SEQ ID NO：1的肽在高浓度与低浓度下都具有止痛的效果。
因此本发明的肽确实有治疗发炎性疾病的效果，尤其是类风湿性关节炎；并能有效治疗疼痛，尤其是类风湿性关节炎引起的疼痛。
以上所述仅是本发明的较佳实施例，并非对本发明做任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (10)

  1. 一种经分离肽，其包含如SEQ ID NO：1所示的氨基酸序列。
  2. 一种如权利要求1的肽的用途，其是作为制备治疗发炎性疾病的药物的应用，其中所述药物包含有效剂量的如权利要求1的肽及药学上可接受的载剂。
  3. 根据权利要求2的用途，其中所述发炎性疾病包括僵直性关节炎、骨关节炎、风湿性关节炎、类风湿性关节炎、外伤性关节炎、化脓性关节炎、痛风性关节炎、结核性关节炎、神经性关节炎、及血友病性关节炎。
  4. 一种如权利要求1的肽的用途，其是作为制备治疗疼痛的药物的应用，其中所述药物包含有效剂量的如权利要求1的肽及药学上可接受的载剂。
  5. 根据权利要求4的用途，其中所述疼痛包括神经病变疼痛、发炎性疼痛、肌肉骨骼疼痛、外科手术后疼痛、癌症疼痛、急性疼痛、及慢性疼痛。
  6. 根据权利要求5的用途，其中所述发炎性疼痛包含僵直性关节炎疼痛、骨关节炎疼痛、风湿性关节炎疼痛、类风湿性关节炎疼痛、外伤性关节炎疼痛、化脓性关节炎疼痛、痛风性关节炎疼痛、结核性关节炎疼痛、神经性关节炎疼痛、血友病性关节炎疼痛。
  7. 根据权利要求2至6中任一项的用途，其中所述药物是经肠道或非经肠道的剂型。
  8. 根据权利要求2或4的用途，其中所述有效剂量为0.0008μg/kg至815μg/kg。
  9. 根据权利要求7的用途，其中所述经肠道的剂型是口服剂型。
  10. 根据权利要求7的用途，其中所述非经肠道剂型是注射剂型。
PCT/CN2019/111788 2019-10-18 2019-10-18 肽及其作为制备治疗发炎性疾病及疼痛的药物的用途 WO2021072724A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU2019470693A AU2019470693B2 (en) 2019-10-18 2019-10-18 Peptide and use thereof in preparation of drug for treating inflammatory diseases and pain
PCT/CN2019/111788 WO2021072724A1 (zh) 2019-10-18 2019-10-18 肽及其作为制备治疗发炎性疾病及疼痛的药物的用途
JP2021541078A JP7220489B2 (ja) 2019-10-18 2019-10-18 炎症性疾患および疼痛を治療するためのペプチドを含有する薬剤
US17/421,600 US20220119447A1 (en) 2019-10-18 2019-10-18 Peptide and for treating inflammatory disease and pain using the same
EP19949541.7A EP4047007B1 (en) 2019-10-18 2019-10-18 Peptide and use thereof in preparation of drug for treating inflammatory diseases and pain

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/111788 WO2021072724A1 (zh) 2019-10-18 2019-10-18 肽及其作为制备治疗发炎性疾病及疼痛的药物的用途

Publications (1)

Publication Number Publication Date
WO2021072724A1 true WO2021072724A1 (zh) 2021-04-22

Family

ID=75537684

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/111788 WO2021072724A1 (zh) 2019-10-18 2019-10-18 肽及其作为制备治疗发炎性疾病及疼痛的药物的用途

Country Status (5)

Country Link
US (1) US20220119447A1 (zh)
EP (1) EP4047007B1 (zh)
JP (1) JP7220489B2 (zh)
AU (1) AU2019470693B2 (zh)
WO (1) WO2021072724A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0341209A2 (en) * 1988-04-30 1989-11-08 Nippon Zoki Pharmaceutical Co. Ltd. Physiologically active substances, a process for preparation and pharmaceutical compositions thereof
CN104321337A (zh) * 2012-05-25 2015-01-28 永林有限公司 肽及其用途
CN107847550A (zh) * 2015-04-16 2018-03-27 首创生物药品发展有限公司 用于脑血管疾病的治疗肽
CN107849091A (zh) * 2015-04-16 2018-03-27 医诺生药开发有限公司 能够在人脂肪衍生的干细胞和肝细胞中抑制丙肝病毒复制的肽及其衍生物
WO2019103203A1 (ko) * 2017-11-24 2019-05-31 주식회사 젬백스앤카엘 신규 펩티드 및 이를 포함한 조성물

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2331124A1 (en) * 2008-08-15 2011-06-15 Circassia Limited T-cell antigen peptide from allergen for stimulation of il-10 production
WO2020024142A1 (en) * 2018-08-01 2020-02-06 Vanford Bio-Drug Development Limited Novel peptides and its derivatives capable of stimulating cytokine release

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0341209A2 (en) * 1988-04-30 1989-11-08 Nippon Zoki Pharmaceutical Co. Ltd. Physiologically active substances, a process for preparation and pharmaceutical compositions thereof
CN104321337A (zh) * 2012-05-25 2015-01-28 永林有限公司 肽及其用途
CN107847550A (zh) * 2015-04-16 2018-03-27 首创生物药品发展有限公司 用于脑血管疾病的治疗肽
CN107849091A (zh) * 2015-04-16 2018-03-27 医诺生药开发有限公司 能够在人脂肪衍生的干细胞和肝细胞中抑制丙肝病毒复制的肽及其衍生物
WO2019103203A1 (ko) * 2017-11-24 2019-05-31 주식회사 젬백스앤카엘 신규 펩티드 및 이를 포함한 조성물

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers", U.S. FOOD AND DRUG ADMINISTRATION
F. E. D'AMOURD. L. SMITH: "A method for determining loss of pain sensation", THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 72, 1941, pages 74 - 79
L. S. HARRISA. K. PIERSONSOME NARCOTIC: "Antagonists in the Benzomorphan Series", J PHARMACOL EXP THER, vol. 143, 1964, pages 141 - 148
MIURA T, OKAZAKI R, YOSHIDA H, NAMBA H, OKAI H, KAWAMURA M.: "Mechanisms of analgesic action of Neurotropin on chronic pain in adjuvant-induced arthritic rat: Roles of descending noradrenergic and serotonergic systems", JOURNAL OF PHARMACOLOGICAL SCIENCES, JAPANESE PHARMACOLOGICAL SOCIETY , TOKYO, JP, vol. 97, no. 3, 1 March 2005 (2005-03-01), JP, pages 429 - 436, XP002726216, ISSN: 1347-8613, DOI: 10.1254/jphs.FPJ04050X *
See also references of EP4047007A4
ZHENG YUQIU, WENLI FANG, SHENGNUO FAN, WANG LIAO, YING XIONG, SHAOWEI LIAO, YI LI, SONGHUA XIAO, JUN LIU: "Neurotropin inhibits neuroinflammation via suppressing NF-kB and MAPKs signaling pathways in lipopolysaccharide-stimulated BV2 cells", JOURNAL OF PHARMACOLOGICAL SCIENCES, vol. 136, 27 February 2018 (2018-02-27), pages 242 - 248, XP055802405, DOI: 10.1016/j.jphs.2018.02.004 *
ZHIQIANG WANG, ZHANG YAO, HONG XUECHUAN, XU PING: "N-terminomics: proteomic strategies for protein N-terminal profiling", CHINESE JOURNAL OF BIOTECHNOLOGY, ZHONGGUO KEXUEYUAN WEISHENGWU YANJIUSUO, CHINESE ACADEMY OF SCIENCES, INSTITUTE OF MICROBIOLOGY, CN, vol. 32, no. 8, 25 August 2016 (2016-08-25), CN, pages 1001 - 1009, XP055684259, ISSN: 1000-3061, DOI: 10.13345/j.cjb.150441 *

Also Published As

Publication number Publication date
AU2019470693B2 (en) 2022-07-14
AU2019470693A1 (en) 2021-07-22
EP4047007A4 (en) 2023-01-04
JP2022517378A (ja) 2022-03-08
JP7220489B2 (ja) 2023-02-10
US20220119447A1 (en) 2022-04-21
EP4047007A1 (en) 2022-08-24
EP4047007B1 (en) 2024-06-05

Similar Documents

Publication Publication Date Title
CN104127859B (zh) 多肽在制备治疗或预防类风湿性关节炎药物中的应用
CN101361961B (zh) 多肽小分子在制备防治缺血性脑血管病药物中的应用
JP4933440B2 (ja) 経皮デリバリーのための薬剤組成物
ES2600616T3 (es) Antagonistas del receptor de la interleucina-1
US20210008177A1 (en) Elapidae neurotoxin enhances opioid analgesic effect and inhibits opioid induced hyperalgesia and tolerance
CN113248628B (zh) 一种乳源多肽衍生物及其在制备肥胖症防治药物、保健品和食品添加物中的应用
JP2007525426A (ja) Ｈｃｇ断片を含む粘膜及び経口投与のための組成物優先権本出願は、２００３年４月８日に欧州特許庁（ｅｐｏ）に出願したｅｐ０３０７６０２８．４、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２９．２、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２７．６、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２６．８、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２２．７、ｕｓ１０／４０９，６７１、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２１．９、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２５．０、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２４．３、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０３０．０、２００３年４月８日にｅｐｏに出願したｅｐ０３０７６０２３．５、２００３年４月３０日に中国特許庁に出願したｃｎ０３１３１２２７．６の優先権を主張する。
EP4177259A1 (en) Polypeptide for repairing mucosal damage or skin wound and use thereof
Ying et al. Thymosin β4 and actin: Binding modes, biological functions and clinical applications
TWI705818B (zh) 肽及其作為製備治療發炎性疾病及疼痛的藥物的用途
WO2021072724A1 (zh) 肽及其作为制备治疗发炎性疾病及疼痛的药物的用途
CN112759625B (zh) 肽作为制备治疗发炎性疾病及疼痛的药物的用途
CN113956334B (zh) 一种棕色脂肪细胞分泌肽及其衍生物在肥胖防治中的应用
CN110101845B (zh) 鸢尾素在制备防治术后认知功能障碍以及血脑屏障受损所介导脑病的药物中的应用
KR102091567B1 (ko) 오스테오폰틴 단백질 단편을 유효성분으로 함유하는 뇌손상 예방 또는 치료용 약학 조성물
CN114805489A (zh) 一类用于修复皮肤损伤或黏膜损伤的多肽及其应用
CN103254291B (zh) 肿瘤坏死因子-α多肽抑制剂及其应用
US9364510B2 (en) Botanical composition and methods of manufacture and use
CN108853483B (zh) 经修饰的胸腺素β4在治疗脑缺血再灌注损伤方面的用途
CN103739673B (zh) 一种聚乙二醇修饰的抑制白介素-6多肽及其应用
CN101757610B (zh) 蛇毒细胞毒素-ctx1在制备具有镇痛作用药中的应用
WO2016066068A1 (zh) 金属硫蛋白复合制剂作为制备穴位介入治疗脑中风后遗症药物的应用
WO2017088177A1 (zh) 贻贝粘蛋白产品及其预防和抑制神经炎症的应用
WO2010150082A2 (en) Use of chromogranin a and of peptidic derivatives thereof in the treatment of inflammation
CN116621923A (zh) 一种有效治疗瘙痒症的生物活性肽

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19949541

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021541078

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019470693

Country of ref document: AU

Date of ref document: 20191018

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019949541

Country of ref document: EP

Effective date: 20220518