WO2023184471A1 - 痘苗病毒致炎兔皮提取物治疗帕金森病的用途 - Google Patents

痘苗病毒致炎兔皮提取物治疗帕金森病的用途 Download PDF

Info

Publication number
WO2023184471A1
WO2023184471A1 PCT/CN2022/084780 CN2022084780W WO2023184471A1 WO 2023184471 A1 WO2023184471 A1 WO 2023184471A1 CN 2022084780 W CN2022084780 W CN 2022084780W WO 2023184471 A1 WO2023184471 A1 WO 2023184471A1
Authority
WO
WIPO (PCT)
Prior art keywords
disease
vaccinia virus
parkinson
rabbit skin
extract
Prior art date
Application number
PCT/CN2022/084780
Other languages
English (en)
French (fr)
Inventor
刘承鑫
Original Assignee
星亮生物技术有限公司
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 星亮生物技术有限公司 filed Critical 星亮生物技术有限公司
Priority to PCT/CN2022/084780 priority Critical patent/WO2023184471A1/zh
Priority to TW112112597A priority patent/TW202339779A/zh
Publication of WO2023184471A1 publication Critical patent/WO2023184471A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A61K39/285—Vaccinia virus or variola virus
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

Definitions

  • the invention belongs to the field of medicine. Specifically, the present invention relates to novel therapeutic uses of vaccinia virus-inflamed rabbit skin extracts. More specifically, the present invention relates to the medicinal use of an extract of vaccinia virus-inflamed rabbit skin for the treatment of Parkinson's disease.
  • Parkinson's disease usually occurs in people over 60 years old, and about 1% of the elderly worldwide suffer from the disease. Symptoms of Parkinson's disease usually appear slowly over time. The most obvious symptoms in the early stages are tremors, limb stiffness, reduced motor function and abnormal gait. Cognitive and behavioral problems may also be present. Dementia is quite common in patients with severe disease, exceeding Major depressive disorder and anxiety disorders also occur in one-third of cases. Apoptosis of dopamine neurons in the substantia nigra pars compacta can lead to Parkinson's disease.
  • Parkinson's disease may be hereditary or caused by unknown factors. Parkinson's disease can also be caused by viral infections or toxins such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). MPTP can be used as a drug to induce Parkinson's disease in experimental animals (Langston, J.W et al., Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science (New York, N.Y.). 1983-02-25, 219 (4587) :979–980.).
  • Parkinson's disease is a chronic disease and is not caused by acute cerebral hypoxia and toxicity caused by cerebrovascular embolism or rupture.
  • Parkinson's disease is considered a synucleinopathy due to the accumulation of ⁇ -synuclein in the form of Lewy bodies (Schulz-Schaeffer, Walter J.The synaptic pathology of ⁇ -synuclein aggregation in dementia with Lewy bodies,Parkinson's disease and Parkinson's disease dementia. Acta Neuropathologica. 2010-8,120(2):131–143). This is completely different from the tangle of nerve fibers caused by the accumulation of tau protein in Alzheimer's disease. The main pathological changes in Parkinson's disease occur in the ventral part of the substantia nigra pars compacta of the midbrain.
  • This area contains a large number of dopamine neurons and signals to the basal ganglia of the brain (Obeso, Jose A et al., Functional organization of the basal ganglia: therapeutic implications for Parkinson's disease. Movement Disorders: Official Journal of the Movement Disorder Society. 2008,. 23Suppl 3:S548–559). Patients with Parkinson's disease experience massive death of neurons in the pars compacta, and some even lose up to 70% of neurons (Davie, C.A., A review of Parkinson's disease. British Medical Bulletin. 2008, 86:109–127).
  • Selegiline is a monoamine oxidase (MAO)-B inhibitor (MAOI).
  • MAOI monoamine oxidase-B inhibitor
  • Vaccinia virus-inflamed rabbit skin extract is an active substance extracted from the skin of rabbits inflamed after vaccination with vaccinia virus.
  • This vaccinia virus-inflamed rabbit skin extract is commercially available under the trade name Lepalvir for pain treatment. The extract has been shown to be clinically safe and has no significant side effects.
  • WO2020/211009 discloses the use of vaccinia virus-inflamed rabbit skin extract for treating hematopoietic system damage
  • WO 2020/248240 discloses the use of vaccinia virus-inflamed rabbit skin extract for treating cancer, and their entire contents are approved by This reference is incorporated into this article.
  • the object of the present invention includes providing medicaments for preventing, alleviating or treating Parkinson's disease.
  • the technical problem of the present invention is solved by providing an extract of rabbit skin inflamed by vaccinia virus, preferably immediately.
  • the inventors found that extracts of vaccinia virus-inflamed rabbit skin were effective in preventing, treating, or relieving Parkinson's disease. Furthermore, the inventors unexpectedly found that the vaccinia virus-inflamed rabbit skin extract was as effective in treating the disease as the approved selegiline.
  • the invention relates to the use of a vaccinia virus inflamed rabbit skin extract for the preparation of a medicament for preventing or treating Parkinson's disease in a patient.
  • the invention relates to a vaccinia virus inflamed rabbit skin extract for use in preventing or treating Parkinson's disease in a patient.
  • the invention relates to a method of preventing or treating Parkinson's disease in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a vaccinia virus inflamed rabbit skin extract.
  • the vaccinia virus-inflamed rabbit skin extract or medicament is used to restore brain nerve function or alleviate brain nerve function damage in patients with Parkinson's disease.
  • Brain neurological damage can be caused by the degeneration, damage, reduction or death of brain neurons, especially the degeneration, reduction, damage or death of neurons in the substantia nigra compacta of the brain.
  • the neurons described herein may be dopaminergic neurons.
  • Dopaminergic neurons are neurons that contain and release dopamine as the neurotransmitter.
  • the present invention relates to the use of a vaccinia virus-inflamed rabbit skin extract in the preparation of a medicament for alleviating or reducing neuronal degeneration, damage or death in the brain of patients suffering from Parkinson's disease.
  • the invention relates to a vaccinia virus-inflamed rabbit skin extract for use in alleviating or reducing neuronal degeneration, damage or death in the brain of patients with Parkinson's disease.
  • the invention relates to a method of alleviating or reducing neuronal degeneration, damage or death in the brain of a patient suffering from Parkinson's disease, said method comprising administering a therapeutically effective amount of a vaccinia virus inflamed rabbit skin extract the patient.
  • the neurons may be neurons of the substantia nigra pars compacta of the brain, preferably dopaminergic neurons.
  • Parkinson's disease is treated by alleviating or reducing neuronal degeneration, damage or death in the patient's brain.
  • the patient suffers from parkinsonism caused by Parkinson's disease. Therefore, the present invention also relates to the use of an extract of vaccinia virus-inflamed rabbit skin for the preparation of a medicament for preventing or treating Parkinson's syndrome in patients. In one aspect, the invention relates to a vaccinia virus-inflamed rabbit skin extract for use in preventing or treating Parkinson's syndrome in a patient. In one aspect, the present invention relates to a method of preventing or treating Parkinson's disease in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a vaccinia virus inflamed rabbit skin extract.
  • a patient with Parkinson's disease or parkinsonian syndrome has motor symptoms such as tremors, limb stiffness, slow movement or postural instability; neuropsychiatric disorders such as speech impairment, cognitive impairment, mood disorder or thinking disorder; sensory impairment or sleep disorders; or autonomic nervous system dysfunction such as excessive oil secretion, excessive sweating, or urinary incontinence.
  • the present invention relates to the use of extracts to alleviate these symptoms in patients.
  • Parkinson's disease can be caused by aging, genetic factors, environmental toxins or pathogenic infections.
  • Patients with Parkinson's disease, or parkinsonism are characterized by the loss, damage, or death of neurons in the substantia nigra compacta of the brain.
  • a vaccinia virus-inflamed rabbit skin extract may be used as the sole active ingredient for the treatment of Parkinson's disease or parkinsonian syndrome.
  • the extract of vaccinia virus-inflamed rabbit skin can be used as the sole active ingredient for the preparation of a medicament for the treatment of Parkinson's disease or parkinsonian syndrome, or for the recovery of patients suffering from Parkinson's disease Brain nerve function or alleviate brain nerve function damage, or alleviate or reduce neuronal degeneration, damage or death in the brain of patients with Parkinson's disease.
  • a vaccinia virus-inflamed rabbit skin extract may be administered to the patient as the only active ingredient.
  • “Sole active ingredient” means that the vaccinia virus-inflamed rabbit skin extract is not used with other Parkinson's disease treatments, administered to patients, or prepared into medicines.
  • the invention relates to a pharmaceutical composition comprising a vaccinia virus-inflamed rabbit skin extract and optionally a pharmaceutically acceptable carrier, adjuvant or excipient.
  • the pharmaceutically acceptable carrier, auxiliary material or excipient is those for formulating the drug into oral preparations or injections.
  • the vaccinia virus inflamed rabbit skin extract is formulated into an oral preparation or injection, preferably intramuscular injection or intravenous injection.
  • the vaccinia virus-inflamed rabbit skin extract is recombinant.
  • the present invention also relates to the use of the pharmaceutical composition for treating Parkinson's disease, or for restoring brain nerve function or alleviating brain nerve function damage in patients with Parkinson's disease, or for use in patients with Parkinson's disease. Use to alleviate or reduce neuronal degeneration, damage or death in the brain of patients with Parkinson's disease.
  • the present invention also relates to the use of the vaccinia virus-induced rabbit skin extract in the preparation of the pharmaceutical composition.
  • the vaccinia virus-inflamed rabbit skin extract is recombinant.
  • the vaccinia virus inflamed rabbit skin extract is formulated as an oral preparation or injection, preferably intramuscular injection or intravenous injection.
  • the patient is a mammal, preferably a human.
  • the vaccinia virus inflamed rabbit skin extract is administered to the patient in an amount of 0.05 U/kg to 50 U/kg, preferably 0.1 U/kg to 10 U/kg, more preferably 0.5 U/kg to 5 U/kg, e.g. people.
  • the prepared medicament contains 3U to 3000U, preferably 6U to 600U, more preferably 30U to 300U of the vaccinia virus inflamed rabbit skin extract.
  • the vaccinia virus inflamed rabbit skin extract (preferably Lizai) is used at 0.05U/kg to 50U/kg, preferably 0.1U/kg to 10U/kg, more preferably 0.5U/kg to 5U/kg The amount is administered to a patient, preferably a human.
  • the vaccinia virus-induced rabbit skin extract is administered to a patient, preferably a human, in an amount selected from the following: 0.05 U/kg, 0.06 U/kg, 0.07 U/kg, 0.08 U/kg, 0.09 U/kg, 0.1 U/kg, 0.2U/kg, 0.3U/kg, 0.4U/kg, 0.5U/kg, 0.6U/kg, 0.7U/kg, 0.8U/kg, 0.9U/kg, 1U/kg, 1.5U /kg, 2U/kg, 2.5U/kg, 3U/kg, 3.1U/kg, 3.2U/kg, 3.3U/kg, 3.4U/kg, 3.5U/kg, 3.6U/kg, 3.7U/kg , 3.8U/kg, 3.9U/kg, 4U/kg, 4.5U/kg, 5U/kg, 5.5U/kg, 6U/kg, 6.5U/kg, 7U/kg, 7.5U/kg, 8U/kg , 8.5U/kg,
  • the above dosage may be an effective amount for treating the above-mentioned diseases in patients.
  • the vaccinia virus inflamed rabbit skin extract is administered by injection at the above dosage, such as intramuscular injection or intravenous injection.
  • the medicament or pharmaceutical composition is prepared comprising 3 U to 3000 U, preferably 6 U to 600 U, more preferably 30 U to 300 U of vaccinia virus inflamed rabbit skin extract.
  • the medicament or pharmaceutical composition is for administration to a human, such as an adult.
  • the average adult weight is, for example, 60 kg.
  • the amount of vaccinia virus-induced rabbit skin extract contained in the medicine prepared by the present invention is, for example, 3U, 4U, 5U, 6U, 7U, 8U, 9U, 10U, 15U, 20U, 25U, 30U, 35U, 40U, 42U, 44U, 45U, 46U, 47U, 48U, 49U, 50U, 55U, 60U, 65U, 70U, 80U, 90U, 100U, 120U, 150U, 160U, 170U, 180U, 190U, 192U, 194U, 195U, 196U, 198U, 200U, 220U, 240U, 260U, 280U, 300U, 350U, 400U, 500U, 600U, 700U, 800U, 900U, 1000U, 1500U, 2000U, 2500U, 3000U and ranges bounded by these numbers.
  • the drug or pharmaceutical composition is prepared as an injection, such as intramuscular injection or intravenous injection.
  • the drug or injection is an undivided fixed dose.
  • the drug or injection cannot be divided into smaller doses within 1, 2, 3, 4, 5, 6, or 7 days.
  • the drug or injection is administered only once within 1, 2, 3, 4, 5, 6, or 7 days.
  • the vaccinia virus inflamed rabbit skin extract is administered to the patient every 6-72 hours, preferably 12-48 hours, more preferably 24-36 hours, more preferably 24 hours.
  • the regimen administered to the patient is three times a day, twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks. Once a month.
  • the extract of the present invention may be administered to a patient once daily.
  • the patient is administered an extract of the invention for at least 24 months, at least 12 months, at least 6 months, at least 2 months, at least 1 month, at least 3 weeks, at least 2 weeks, at least 10 days, At least 7 days, at least 5 days, at least 2 days, or at least 1 day.
  • the “extract from rabbit skin inflamed by vaccinia virus” mentioned in this article refers to a substance containing Extracts of active substances. This extract is usually a yellow or pale yellow liquid, but can also be made into a solid by drying methods. This injection of vaccinia virus-inflamed rabbit skin extract is commercially available under the trade name Lepalvir.
  • the preparation method includes using a phenol aqueous solution to extract inflamed rabbit skin fragments after vaccinia virus inoculation, wherein the phenol concentration is preferably about 1%-10%, preferably about 2%-5%, and more preferably about
  • the 2% or about 3% aqueous phenol solution is carried out at less than about 12°C, for example about 0-10°C, preferably about 2-8°C, more preferably about 3-6°C, more preferably about 4°C.
  • the preparation method also includes using an adsorbent (such as activated carbon) to adsorb the extract treated with the phenol aqueous solution, and performing desorption under alkaline conditions, wherein adsorption is preferably performed under acidic conditions (such as about pH 3-6, more preferably about pH 4-5, more preferably about pH 4.5), and the alkaline conditions for desorption are about pH 9-12, preferably about pH 10 or pH 11.
  • an adsorbent such as activated carbon
  • the vaccinia virus inflamed rabbit skin extract or Rizex can be prepared by a method comprising the following steps:
  • a rabbit is inoculated with vaccinia virus, the acne-prone skin is collected, the skin is fragmented, an aqueous phenol solution is added, and the mixture is heated at less than about 12°C (e.g., about 0-10°C).
  • about 12°C e.g., about 0-10°C
  • the phenol concentration in the phenol aqueous solution is about 1%-10%, preferably about 2%-5%, more preferably about 2% or about 3%.
  • solution A is adjusted to acidic (e.g., about pH 4-6, more preferably about pH 4.5-5.5, more preferably about pH 5) using an acid (e.g., hydrochloric acid), and heated (e.g., at about pH 4-6).
  • acid e.g., hydrochloric acid
  • 90-100°C preferably about 95°C for at least about 10 minutes, such as about 20-50 minutes, preferably about 30-40 minutes
  • optionally cooling for example to below about 50°C, preferably below about 30°C
  • the step (2) can be performed in a nitrogen environment.
  • step (3) solution B is adjusted to alkaline (eg, about pH 8-10, more preferably about pH 8.5-9.5, more preferably about pH 9 or about pH 9) with a base (such as sodium hydroxide). 2), heating (for example at about 90-100°C, preferably about 95°C for at least 10 minutes, for example about 30-50 minutes, preferably about 30-40 minutes), optionally cooling (for example to below about 50°C, Preferably below about 30° C.), solution C is obtained after filtration.
  • the step (3) can be performed in a nitrogen environment.
  • step (4) solution C is adjusted to acidic (for example, about pH 3-6, more preferably about pH 4-5, more preferably about pH 4.5) with an acid (such as hydrochloric acid), and the adsorbent is added thereto (eg, activated carbon) is soaked (eg, with stirring for at least about 1 hour, preferably about 2-10 hours, more preferably about 4 hours), after which the solution is removed and the adsorbent containing the active ingredient is collected.
  • acid such as hydrochloric acid
  • the above-mentioned adsorbent is added to the eluent (such as water), and the pH is adjusted to alkaline (such as about pH 9-12, preferably about pH 10 or pH 11) with a base (such as sodium hydroxide) to remove the active ingredient from the adsorbent.
  • a base such as sodium hydroxide
  • Separation eg, stirring for at least about 1 hour, preferably 2-10 hours, more preferably 4 hours, followed by filtration and washing of the adsorbent with water
  • the step (4) can be performed in a nitrogen environment.
  • step (5) solution D is neutralized to weak acidity (eg, about pH 5.5-6.6, preferably about pH 6) with an acid (such as hydrochloric acid) to obtain solution E.
  • weak acidity eg, about pH 5.5-6.6, preferably about pH 6
  • an acid such as hydrochloric acid
  • the step (5) can be performed under sterile conditions.
  • step (6) solution E is concentrated (e.g., concentrated under reduced pressure, preferably concentrated by evaporation under reduced pressure, for example, at about 50°C-70°C, preferably at about 54°C-56°C), and then filtered, An extract containing active ingredients is obtained.
  • the step (6) can be performed in a nitrogen environment.
  • the patient is a mammal.
  • the patient is a human, such as a middle-aged or elderly person, preferably an elderly person.
  • Middle-aged people can be people between 40 and 59 years old or people between 45 and 59 years old.
  • An elderly person can be a person over 60, over 65, over 70, over 75 or over 80.
  • the extract of the present invention can also be obtained by inoculating other animal tissues with vaccinia virus.
  • extracts of inflammatory tissues inoculated with vaccinia virus can be used.
  • the tissue may be from a mammalian tissue, and the mammal may include companion animals, experimental animals, and livestock animals, such as rabbits, cattle, horses, sheep, goats, monkeys, mice, and pigs.
  • the tissue may be skin.
  • Figure 2 shows the effect of orthorexia on the total movement distance of mice in the mine field experiment after the MPTP subacute model. ***p ⁇ 0.001,**p ⁇ 0.01vs.Sham; ###p ⁇ 0.001,##p ⁇ 0.01,#p ⁇ 0.05vs.MPTP. Distance: distance.
  • Figure 3 shows the effect of orthorexia on T-Turn in the pole climbing test of mice after the MPTP subacute model. *** p ⁇ 0.001vs.Sham; ### p ⁇ 0.001, ## p ⁇ 0.01, # p ⁇ 0.05vs.MPTP.
  • Figure 4 shows the effect of orthorexia on T-TLA in the pole climbing test of mice after the subacute model of MPTP. *** p ⁇ 0.001vs.Sham; ### p ⁇ 0.001, ## p ⁇ 0.01, # p ⁇ 0.05vs.MPTP.
  • Figure 5 shows the effect of orthorexia on the latency of mice in the rotarod test after the subacute model of MPTP. *** p ⁇ 0.001vs.Sham; ### p ⁇ 0.001, ## p ⁇ 0.01vs.MPTP. Latency Time: Latency time.
  • Figure 6 shows the effect of orthorexia on the number of Nissl-positive neurons in the substantia nigra pars compacta of mice after the subacute model of MPTP. *** p ⁇ 0.001, ** p ⁇ 0.01vs.Sham; ## p ⁇ 0.01, # p ⁇ 0.05vs.MPTP.
  • Figure 7 shows the effect of orthorexia on the number of TH-positive neurons in the substantia nigra pars compacta of mice after the subacute model of MPTP. *** p ⁇ 0.001vs.Sham; ### p ⁇ 0.001, ## p ⁇ 0.01, # p ⁇ 0.05vs.MPTP.
  • Example 1 Preclinical animal pharmacodynamics experimental study on the therapeutic effect of Lizaixi on Parkinson's disease
  • RESEX vaccinia virus-inflamed rabbit skin extract
  • Selegiline hydrochloride (Selegiline, imidopyr).
  • the mice in the treatment groups of Rezax and Selegiline were treated with corresponding doses of intraperitoneal injection 1 hour after the first MPTP administration, once a day for 7 or 14 days.
  • Mice in the MPTP model group were given an equal volume of physiological saline (0.1mL/10g).
  • the mice in the Sham group were only given an equal volume of physiological saline (0.1mL/10g).
  • the open field test was used to evaluate the voluntary movement ability of mice.
  • the open field device is made of opaque blue plastic and measures 60cm ⁇ 60cm ⁇ 45cm. During the test, each mouse was placed into the box from the central area, and at the same time, the movement trajectory of each mouse within 5 minutes was recorded, and the total movement distance was calculated using software (Clever Sys Inc., VA, USA). After each mouse test, use 75% ethanol to wipe the open area to prevent the smell of the previous mouse from affecting the next mouse.
  • the pole climbing test was used to evaluate the motor coordination function of mice.
  • the pole used has a diameter of 1 cm and a height of 50 cm.
  • a wooden ball with a diameter of 1.2 cm is fixed on the top of the pole.
  • the pole is wrapped with anti-slip tape to prevent mice from slipping. Place the mouse's head upward on the top of the pole.
  • start the timer Record the time it takes for the mouse to move over the top of the pole (T-Turn) and the time it takes for the mouse to climb to the bottom of the four paws.
  • Time to Landing T-TLA
  • each mouse needs to be trained three times consecutively. During the test, three consecutive measurements are required, and the shortest time is chosen.
  • the rotarod test was used to evaluate the motor balance ability and limb coordination of mice. Place the mouse on the drum of the Rotarod tester, and set the rotation speed to increase from 5 rpm/min to 25 rpm/min at a constant speed within 2 minutes. When the mouse falls from the drum to the sensing area below, an infrared sensor will receive the signal and record it. Time for mice to move on the instrument roller. Before formal testing, each mouse needs to be trained three times consecutively. During the test, three consecutive measurements are required to calculate the average drop time (latency period).
  • mice from each group were taken, anesthetized with 4% chloral hydrate, and perfused with physiological saline + 4% PFA to obtain brain tissue.
  • Nissl bodies are a kind of basophilic substance in the cytoplasm and are widely found in various neurons. Therefore, the number of neurons in relevant brain areas was calculated through Nissl staining. Stain according to Nissl staining kit.
  • TH mainly labels dopaminergic neurons in the substantia nigra pars compacta, and TH-positive neurons in this brain area will be significantly reduced after MPTP modeling.
  • DAB diaminobenzidin
  • Immunohistochemistry-positive cells were counted using a Stereo Investigator (MBF bioscience) counting system to calculate the number of Nissl body-positive cells and TH+ cells in the substantia nigra pars compacta. Cell counting was performed under a ⁇ 20 objective lens.
  • mice As shown in Figure 1, the subacute administration of MPTP caused the mice to eat less after modeling. The weight of the mice decreased slightly within 7 days. After 14 days, the mice ate gradually increased and their weight gradually recovered. The experimental results showed that only the mice in the low-dose (10U/kg/d) treatment group had a significant decrease in body weight compared with the mice in the Sham group at 7 days (p ⁇ 0.01). The treatments in the other groups had no significant effect on the body weight of the mice.
  • mice after repeated open-field tests, normal mice gradually become familiar with the environment, and their autonomous activities decrease, and the total distance traveled also decreases.
  • the total movement distance of mice was reduced compared with the control group at 1, 3, 7 and 14 days after modeling, indicating a decrease in autonomous activity ability.
  • the positive control drug Selegiline can significantly improve MPTP-induced reduction in total exercise distance on days 1, 3, 7 and 14, while Selegiline only has a significant improvement effect on day 14 at low, medium and high doses.
  • the total movement distance increased to 161%, 154% and 167% of the model group.
  • mice showed prolonged T-Turn and T-TLA times in the pole climbing experiment at 1, 3, 7 and 14 days after modeling, indicating that Decreased motor coordination.
  • the positive control drug Selegiline can significantly improve MPTP-induced T-Turn and T-TLA time extension on days 1, 3, 7 and 14.
  • Lizaixi had a significant improvement effect on days 1, 3, 7 and 14.
  • low, medium and high doses of T-Turn were reduced to 35.1%, 28.9% and 32.6% of the model group respectively.
  • T-TLA also had a significant improvement effect. There is a decreasing trend, but there is no significant difference.
  • mice showed a shortening of the falling latency in the rotarod test at 1, 3, 7, and 14 days after modeling, indicating a decrease in motor balance ability.
  • the positive control drug Selegiline can significantly improve the shortening of MPTP-induced latency time on days 1, 3, 7 and 14.
  • Lizazhi also had a significant improvement effect at 1, 3, 7 and 14 days.
  • the incubation period of low, medium and high doses at 14 days was prolonged to 121.3%, 119.6% and 119.8% of the model group respectively.
  • mice showed a decrease in the number of Nissl stain-positive neurons in the substantia nigra pars compacta at 7 and 14 days after modeling, indicating the death of neurons in this brain area.
  • the positive control drug Selegiline could significantly improve the MPTP-induced decrease in the number of Nissl stain-positive neurons in the substantia nigra pars compacta on both 7 and 14 days.
  • the high-dose group of Rizax showed significant improvement at 7 and 14 days, and the number of Nissl-positive neurons increased by 126.3 and 139.6% respectively compared with the model group.
  • mice showed a decrease in the number of TH-positive neurons in the substantia nigra pars compacta at 7 and 14 days after modeling, indicating the death of dopaminergic neurons in this brain area.
  • the positive control drug Selegiline could significantly improve the MPTP-induced decrease in the number of TH-stained positive neurons in the substantia nigra pars compacta on both 7 and 14 days. Only the high-dose group had significant improvement at 7 and 14 days, while the low- and medium-dose groups showed a trend of improvement, but there was no significant difference.
  • the number of TH-positive neurons at low, medium and high doses increased to 115.2%, 120.3% and 125.3% of the model group respectively at 7 days, and the number of TH-positive neurons at low, medium and high doses increased to 125.7% of the model group at 14 days respectively. %, 132.7% and 138.7%.
  • Both 7-day and 14-day treatment with Lizax can improve neurobehavioral disorders in mice after the MPTP subacute model.
  • the high-dose group (40U/kg/d) has the most significant effect at 14 days.
  • It can increase the total movement distance in the open field test by 167%, shorten the T-Turn time of the rod climbing test by 32.6% and the T-TLA time by 65.0%, and increase the latency of the rotary rod test by 119.8%.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Dermatology (AREA)
  • Psychology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了痘苗病毒致炎兔皮提取物用于治疗帕金森病或在患有帕金森病的患者中恢复大脑神经功能或缓解大脑神经功能损伤的用途。所述痘苗病毒致炎兔皮提取物可以是立再适。

Description

痘苗病毒致炎兔皮提取物治疗帕金森病的用途 发明领域
本发明属于医药领域。具体而言，本发明涉及痘苗病毒致炎兔皮提取物的新治疗用途。更具体而言，本发明涉及痘苗病毒致炎兔皮提取物用于治疗帕金森病的医药用途。
背景技术
帕金森病(Parkinson's disease)通常发生在60岁以上的老人，全球约有1％的老人罹患该病。帕金森病症状通常随时间缓慢出现，早期最明显的症状为颤抖、肢体僵硬、运动功能减退和步态异常，也可能有认知和行为问题；痴呆症在病情严重的患者中相当常见，超过三分之一的病例也会发生重性抑郁障碍和焦虑症。黑质致密部多巴胺神经元的凋亡可导致帕金森病。
帕金森病可能是遗传性的，也可能是由于某些不明因素造成。帕金森病也可能由病毒感染或者毒素(例如MPTP(1-甲基-4-苯基-1,2,3,6-四氢吡啶))造成。MPTP可以作为诱发实验动物产生帕金森病的药物(Langston,J.W et al.,Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis.Science(New York,N.Y.).1983-02-25,219(4587):979–980.)。
与脑卒中或其它急性脑血管疾病不同的是，帕金森病属于慢性疾病，并非由脑血管栓塞或破裂而导致的急性大脑缺氧和毒性引起。
就病理生理学而言，由于α-突触核蛋白以路易氏体的形式堆积，帕金森病被视为一种突触核蛋白病变(Schulz-Schaeffer,Walter J.The synaptic pathology of α-synuclein aggregation in dementia with Lewy bodies,Parkinson’s disease and Parkinson’s disease dementia.Acta Neuropathologica.2010-8,120(2):131–143)。这和阿尔茨海默病当中Tau蛋白堆积形成的神经纤维纠缠截然不同。帕金森病的主要病理变化发生在中脑黑质腹侧的致密部。该区含有大量多巴胺神经元，并传讯给大脑基底核(Obeso,Jose A et al.,Functional organization of the basal ganglia:therapeutic implications for Parkinson's disease.Movement Disorders:Official Journal of the Movement Disorder Society.2008,.23 Suppl 3:S548–559)。帕金森病患者致密部神经元大量死亡，有的甚至丧失高达70％的神经元(Davie,C.A.,A review of Parkinson's disease.British Medical Bulletin.2008,86:109–127)。
司来吉兰是一种单胺氧化酶(MAO)-B抑制剂(MAOI)，2006年美国FDA批准司来吉兰用于治疗帕金森病患者。
本领域中存在有效治疗帕金森病的药物的需求。
痘苗病毒致炎兔皮提取物是从接种痘苗病毒后致炎的兔的皮肤中提取的活性物质。这种痘苗病毒致炎兔皮提取物可市售获得，商品名为立再适(Lepalvir)，用于疼痛治疗。该提取物已被证明在临床上是安全的且不存在明显副作用。此外，WO2020/211009公开了痘苗病毒致炎兔皮提取物用于治疗造血系统损伤的用途；WO 2020/248240公开了痘苗病毒致炎兔皮提取物用于治疗癌症的用途，它们的全部内容通过引用结合到本文中。
发明内容
本发明的目的包括提供用于预防、缓解或治疗帕金森病的药物。本发明的技术问题通过提供痘苗病毒致炎兔皮提取物，优选立再适来解决。
总体而言，发明人发现，痘苗病毒致炎兔皮提取物能够有效预防、治疗或缓解帕金森病。此外，发明人预料不到地发现，痘苗病毒致炎兔皮提取物治疗所述疾病的效果可与被批准使用的司来吉兰相当。
在一个方面，本发明涉及痘苗病毒致炎兔皮提取物在制备用于在患者中预防或治疗帕金森病的药物中的用途。在一个方面，本发明涉及痘苗病毒致炎兔皮提取物，其用于在患者中预防或治疗帕金森病。在一个方面，本发明涉及一种预防或治疗有需要的患者中帕金森病的方法，所述方法包括将治疗有效量的痘苗病毒致炎兔皮提取物给予所述患者。
在一个方面，痘苗病毒致炎兔皮提取物或药物用于在患有帕金森病的患者中恢复大脑神经功能或缓解大脑神经功能损伤。大脑神经功能损伤可以由大脑神经元变性、损伤、减少或死亡引起，特别是大脑黑质致密部神经元变性、减少、损伤或死亡。
本文所述的神经元可以是多巴胺能神经元。多巴胺能神经元是指含有并释放多巴胺作为神经递质的神经元。
在一个方面，本发明涉及痘苗病毒致炎兔皮提取物在制备用于在患有帕金森病的患者大脑中缓解或减少神经元变性、损伤或死亡的药物中的用途。在一个方面，本发明涉及痘苗病毒致炎兔皮提取物，其用于在患有帕金森病的患者大脑中缓解或减少神经元变性、损伤或死亡。在一个方面，本发明涉及一种在患有帕金森病的患者大脑中缓解或减少神经元变性、损伤或死亡的方法，所述方法包括将治疗有效量的痘苗病毒致炎兔皮提取物给予所述患者。神经元可以是大脑黑质 致密部神经元，优选多巴胺能神经元。
在一个方面，通过缓解或减少患者大脑中神经元变性、损伤或死亡来治疗帕金森病。
在一个方面，患者罹患由帕金森病引起的帕金森综合征。因此，本发明还涉及痘苗病毒致炎兔皮提取物在制备用于在患者中预防或治疗帕金森综合征的药物中的用途。在一个方面，本发明涉及痘苗病毒致炎兔皮提取物，其用于在患者中预防或治疗帕金森综合征。在一个方面，本发明涉及一种预防或治疗有需要的患者中帕金森综合征的方法，所述方法包括将治疗有效量的痘苗病毒致炎兔皮提取物给予所述患者。
在一个方面，帕金森病或帕金森综合征的患者具有运动症状例如颤抖、肢体僵硬、动作迟缓或姿态不稳；神经性精神疾患例如言语障碍、认知障碍、情绪障碍或思维障碍；感觉障碍或睡眠障碍；或者自主神经功能障例如油脂分泌过多、多汗或尿失禁。本发明涉及提取物在缓解患者这些症状的用途。
帕金森病可以由年龄老化、遗传因素、环境毒素或病原体感染引起。
帕金森病或帕金森综合征的患者特征在于大脑黑质致密部神经元减少、损伤或死亡。
在一个方面，痘苗病毒致炎兔皮提取物可作为唯一的活性成分用于治疗帕金森病或帕金森综合征。在该方面中，痘苗病毒致炎兔皮提取物可作为唯一的活性成分用于制备药物，所述药物用于治疗帕金森病或帕金森综合征，或者在患有帕金森病的患者中恢复大脑神经功能或缓解大脑神经功能损伤，或者在患有帕金森病的患者大脑中缓解或减少神经元变性、损伤或死亡。在本发明的治疗方法中，痘苗病毒致炎兔皮提取物可作为唯一的活性成分被给予所述患者。“唯一活性成分”意味着痘苗病毒致炎兔皮提取物不与其它帕金森病治疗药物一起使用、给予患者或者制备药物。在一个方面，本发明涉及一种药物组合物，其包含痘苗病毒致炎兔皮提取物和任选的药学上可接受的载体、辅料或赋形剂。在本发明的一个方面，所述药学上可接受的载体、辅料或赋形剂是将药物配制成口服制剂或注射剂的那些。在一个方面，所述痘苗病毒致炎兔皮提取物配被制成口服制剂或注射剂，优选肌肉注射剂或静脉注射剂。在一个方面，所述痘苗病毒致炎兔皮提取物是立再适。相应地，本发明还涉及所述药物组合物用于治疗帕金森病的用途，或者用于在患有帕金森病的患者中恢复大脑神经功能或缓解大脑神经功能损伤的用途，或者用于在患有帕金森病的患者大脑中缓解或减少神经元变性、损伤或死亡的用途。本发明还涉及痘苗病毒致炎兔皮提取物在制备所述药物组合物中的用途。
在一个方面，所述痘苗病毒致炎兔皮提取物是立再适。
在一个方面，所述痘苗病毒致炎兔皮提取物配制成口服制剂或注射剂，优选肌肉注射剂或静脉注射剂。
在一个方面，患者是哺乳动物，优选人。
在一个方面，所述痘苗病毒致炎兔皮提取物以0.05U/kg至50U/kg，优选0.1U/kg至10U/kg，更优选0.5U/kg至5U/kg的量给予患者，例如人。
在一个方面，制备的药物包含3U至3000U，优选6U至600U，更优选30U至300U的所述述痘苗病毒致炎兔皮提取物。
在一个方面，所述痘苗病毒致炎兔皮提取物(优选立再适)以0.05U/kg至50U/kg，优选0.1U/kg至10U/kg，更优选0.5U/kg至5U/kg的量给予患者，优选人。例如，所述痘苗病毒致炎兔皮提取物以选自以下的量给予患者，优选人：0.05U/kg、0.06U/kg、0.07U/kg、0.08U/kg、0.09U/kg、0.1U/kg、0.2U/kg、0.3U/kg、0.4U/kg、0.5U/kg、0.6U/kg、0.7U/kg、0.8U/kg、0.9U/kg、1U/kg、1.5U/kg、2U/kg、2.5U/kg、3U/kg、3.1U/kg、3.2U/kg、3.3U/kg、3.4U/kg、3.5U/kg、3.6U/kg、3.7U/kg、3.8U/kg、3.9U/kg、4U/kg、4.5U/kg、5U/kg、5.5U/kg、6U/kg、6.5U/kg、7U/kg、7.5U/kg、8U/kg、8.5U/kg、9U/kg、9.5U/kg、10U/kg、11U/kg、12U/kg、13U/kg、14U/kg、15U/kg、16U/kg、17U/kg、18U/kg、19U/kg、20U/kg、25U/kg、30U/kg、35U/kg、40U/kg、45U/kg、50U/kg以及以这些数字为边界的范围。本领域技术人员知晓，对于给药量而言，人的剂量(U/kg或者mg/kg)＝小鼠的剂量(U/kg或者mg/kg)/12.3；或者人的剂量(U/kg或者mg/kg)＝小鼠的剂量(U/kg或者mg/kg)×0.08。以上剂量可以是治疗患者中上述疾病的有效量。在一个方面，所述痘苗病毒致炎兔皮提取物以上述剂量注射给药，例如肌肉注射或者静脉注射。
在本发明一个方面，被制备的药物或者药物组合物包含包含3U至3000U，优选6U至600U，更优选30U至300U的痘苗病毒致炎兔皮提取物。所述药物或药物组合物被用于给予人，例如成人。成人的平均体重例如是60kg。相应地，包含在本发明制备的药物中的痘苗病毒致炎兔皮提取物的量例如为3U、4U、5U、6U、7U、8U、9U、10U、15U、20U、25U、30U、35U、40U、42U、44U、45U、46U、47U、48U、49U、50U、55U、60U、65U、70U、80U、90U、100U、120U、150U、160U、170U、180U、190U、192U、194U、195U、196U、198U、200U、220U、240U、260U、280U、300U、350U、400U、500U、600U、700U、800U、900U、1000U、1500U、2000U、2500U、3000U以及以这些数字为边界的范围。在一个方面，所述药物或药物组合物被制备成注射剂，例如肌肉注射剂或者静脉注射剂。在一个方面，所述药物或注射剂是不 能分割的固定剂量。在一个方面，所述药物或注射剂在1天、2天、3天、4天、5天、6天或7天之内不能分割成更小剂量。在一个方面，所述药物或注射剂在1天、2天、3天、4天、5天、6天或7天之内只给药一次。
在一个方面，每隔6-72小时、优选12-48小时、更优选24-36小时、更优选24小时向所述患者给予痘苗病毒致炎兔皮提取物。
在一个方面，向患者给药的方案为每天三次，每天二次，每天一次，两天一次，三天一次，四天一次，五天一次，六天一次，一周一次，两周一次，三周一次，每月一次。例如，可以向患者每天一次给予本发明的提取物。
在一个方面，向患者给予本发明的提取物持续至少24个月、至少12个月、至少6个月、至少2个月、至少1个月、至少3周、至少2周、至少10天、至少7天、至少5天、至少2天或至少1天。
本文所述的“痘苗病毒致炎兔皮提取物(extract from rabbit skin inflamed by vaccinia virus)”是指从接种痘苗病毒的发炎兔皮中例如经过浸提、纯化、精制等工序提取的一种含有活性物质的提取物。这种提取物通常是黄色或淡黄色液体，但也可通过干燥方法制成固体。这种痘苗病毒致炎兔皮提取物的注射液可市售获得，商品名为立再适(Lepalvir)。
在本发明提取物的制备中，制备方法包括使用苯酚水溶液提取痘苗病毒接种后发炎的兔皮碎片，其中优选使用苯酚浓度为约1％-10％，优选约2％-5％，更优选约2％或约3％的苯酚水溶液在低于约12℃下，例如约0-10℃，优选约2-8℃，更优选约3-6℃，更优选约4℃进行。此外，制备方法还包括用吸附剂(例如活性炭)吸附用苯酚水溶液处理后的提取液，并且在碱性条件下进行脱吸附，其中优选吸附在酸性条件下(例如约pH3-6，更优选约pH4-5，更优选约pH4.5)进行，并且脱吸附的碱性条件为约pH9-12，优选约pH10或pH11。
在一个方面，痘苗病毒致炎兔皮提取物或者立再适可以通过包括以下步骤的方法来制备：
(1)收集用痘苗病毒接种后发炎的兔皮，将所述兔皮碎片化，用提取溶剂提取，获得溶液A；
(2)对溶液A进行酸和加热处理，得到溶液B；
(3)对溶液B进行碱和加热处理，得到溶液C；
(4)在酸性条件下对溶液C进行吸附和过滤，在碱性条件下进行脱吸附，得到溶液D；
(5)对溶液D进行中和以及加热处理，得到溶液E；
(6)浓缩溶液E，得到所述提取物；和
(7)任选将提取物与药学上可接受的载体、辅料或赋形剂混合。
在一个方面，在步骤(1)中，用痘苗病毒接种家兔，收集出痘的皮肤，将所述皮肤碎片化，加入苯酚水溶液，在低于约12℃下(例如约0-10℃，优选约2-8℃，更优选约3-6℃，更优选约4℃)下浸泡至少约12小时(例如约24-90小时，优选约48-72小时，更优选约70或约72小时)，离心获得上清液，过滤得到溶液A。所述苯酚水溶液中苯酚浓度为约1％-10％，优选约2％-5％，更优选约2％或约3％。
在一个方面，在步骤(2)中，用酸(例如盐酸)将溶液A调至酸性(例如约pH4-6，更优选约pH4.5-5.5，更优选约pH5)，加热(例如在约90-100℃，优选约95℃下持续至少约10分钟，例如约20-50分钟，优选约30-40分钟)，任选降温(例如至低于约50℃，优选低于约30℃)，随后离心获得上清液，过滤后得到溶液B。所述步骤(2)可以在氮气环境下进行。
在一个方面，在步骤(3)中，用碱(例如氢氧化钠)将溶液B调至碱性(例如约pH8-10，更优选约pH8.5-9.5，更优选约pH9或约pH9.2)，加热(例如在约90-100℃，优选约95℃下持续至少10分钟，例如约30-50分钟，优选约30-40分钟)，任选降温(例如至低于约50℃，优选低于约30℃)，过滤后得到溶液C。所述步骤(3)可以在氮气环境下进行。
在一个方面，在步骤(4)中，用酸(例如盐酸)将溶液C调至酸性(例如约pH3-6，更优选约pH4-5，更优选约pH4.5)，向其中加入吸附剂(例如活性炭)进行浸泡(例如在搅拌下持续至少约1小时，优选约2-10小时，更优选约4小时)，之后移除溶液并收集含有活性成分的吸附剂。随后，将上述吸附剂加入洗脱液(例如水)中，用碱(例如氢氧化钠)将pH调节至碱性(例如约pH9-12，优选约pH10或pH11)使活性成分从吸附剂中分离(例如搅拌至少约1小时，优选2-10小时，更优选4小时，随后过滤，再用水洗涤吸附剂)，得到溶液D。所述步骤(4)可以在氮气环境下进行。
在一个方面，在步骤(5)中，用酸(例如盐酸)将溶液D调中和至弱酸性(例如约pH5.5-6.6，优选约pH6)，得到溶液E。优选地，所述步骤(5)可以在无菌条件下进行。
在一个方面，在步骤(6)中，将溶液E浓缩(例如减压浓缩，优选减压蒸发浓缩，例如在约50℃-70℃下，优选约54℃-56℃下)，随后过滤，得到含有活性成分的提取物。所述步骤(6)可以在氮气环境下进行。
在一个方面，患者是哺乳动物。在一个方面，患者是人，例如中年人或老年人，优选老年人。中年人可以是40至59岁的人或者45至59岁的人。老年人可以是60岁以上、65岁以上、70岁以上、75岁以上或80岁以上的人。
本领域技术人员理解，本发明的提取物也可以通过用痘苗病毒接种其它动物组织获得。例如，在本发明中，可以使用接种痘苗病毒的炎症组织提取物。所述组织可以来自哺乳动物的组织，所述哺乳动物可包括伴侣动物、实验动物、畜牧动物，例如为兔、牛、马、绵羊、山羊、猴、鼠、猪。所述组织可为皮肤。
附图简述
图1显示立再适对小鼠MPTP亚急性模型后体重改变的影响。**p<0.01vs.Sham；0～7天时，n＝20或28；14天时，n＝10或14。
图2显示立再适对MPTP亚急性模型后小鼠在矿场实验中总运动路程的影响。***p<0.001,**p<0.01vs.Sham；###p<0.001,##p<0.01,#p<0.05vs.MPTP。Distance:路程。
图3显示立再适对MPTP亚急性模型后小鼠在爬杆实验中T-Turn的影响。 ***p<0.001vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
图4显示立再适对MPTP亚急性模型后小鼠在爬杆实验中T-TLA的影响。 ***p<0.001vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
图5显示立再适对MPTP亚急性模型后小鼠在转棒实验中潜伏期的影响。 ***p<0.001vs.Sham； ###p<0.001, ##p<0.01vs.MPTP。Latency Time:潜伏时间。
图6显示立再适对MPTP亚急性模型后小鼠黑质致密部尼氏阳性神经元数量的影响。 ***p<0.001, **p<0.01vs.Sham； ##p<0.01, #p<0.05vs.MPTP。
图7显示立再适对MPTP亚急性模型后小鼠黑质致密部TH阳性神经元数量的影响。 ***p<0.001vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
具体实施方式
除非另有说明，否则本文使用的所有科学术语都和本领域一般技术人员通常理解的含义一致。下文描述了示例方法和材料，可以使用其等同物。本文提及的所有出版物和其它参考文献都通过引用整体结合到本文中。
提供以下的实施例是为了进一步阐述本发明。以下实施例无意以任何理由限制本发明的范围。
实施例
实施例1-立再适对帕金森病治疗作用的临床前动物药效学实验研究
1.试验目的
评价立再适对小鼠MPTP(1-甲基-4-苯基-1,2,3,6-四氢吡啶)诱导的亚急性帕金森病样模型的治疗作用。
2.材料与方法
2.1.实验动物
C57BL/6J小鼠，SPF级，雄性，体重23-25g。本研究所有实验均经过南京医科大学动物保护和伦理委员会(Institutional Animal Care and Use Committee of Nanjing Medical University，IACUC)批准，伦理编号：IACUC-1704001-3。
2.2.药品与试剂
立再适：痘苗病毒致炎兔皮提取物；和
盐酸司来吉兰(Selegiline，咪多吡)。
2.3.MPTP亚急性帕金森病样模型的制备
小鼠称重后记录，按照20mg/kg颈后部皮下注射，每天一次，连续注射5天，最后一次给药后的第1、3、7和14天时进行后续实验。
2.4.实验分组与给药
128只健康成年小鼠，按体重随机分为六组，分别为正常对照(Sham)组(n＝20)、MPTP模型组(n＝28)、立再适低剂量(10U/kg/d)治疗组(n＝20)、立再适中剂量(20U/kg/d)治疗组(n＝20)、立再适高剂量(40U/kg/d)治疗组(n＝20)和阳性对照药司来吉兰(0.5mg/kg/d)治疗组(n＝20)。其中，立再适和司来吉兰治疗组小鼠在第一次MPTP给药后1小时腹腔注射相应剂量进行治疗，持续7或14天，每天一次。MPTP模型组小鼠给予等体积生理盐水(0.1mL/10g)。Sham组小鼠仅给予等体积生理盐水(0.1mL/10g)。
2.5.行为学评价
分别在MPTP最后一次给药后的第1、3、7和14天时进行行为学评价。
a.旷场试验
旷场试验用于评价小鼠的自主运动能力。开放式旷场装置由不透明的蓝色塑料制成，大小为60cm×60cm×45cm。测试时，将每只小鼠从中心区域放入箱内，同时开始记录每只小鼠在5min内的运动轨迹，使用软件(Clever Sys Inc.，VA，USA)计算总运动路程。每只小鼠测试结束后，使用75％乙醇擦拭旷场区域，防止上一只小鼠的气味对下一只鼠造成影响。
b.爬杆试验
爬杆试验用于评估小鼠的运动协调功能。所用杆子直径l cm，高度50cm，立杆顶端固定有直径为1.2cm的木球，将立杆用防滑胶带裹住以防止小鼠滑落。将小鼠头部向上置于杆顶，小鼠开始向上爬时启动计时器，记下小鼠从开始运动到越过杆顶所需时间(T-Turn)和从开始运动到爬至底部四爪落地时间(T-TLA)。 正式测试前，需对每只小鼠进行三次连续训练。测试期间，需连续测量三次，取其中所用最短时间。
c.转棒测试
使用转棒试验评估小鼠的运动平衡能力和肢体协调性。将小鼠放于Rotarod测试仪滚筒上，将转速设成2分钟内从5rpm/min匀速增加到25rpm/min，小鼠从滚筒上掉落至下方感应区域时会有红外感应器接收信号，记录小鼠在仪器滚筒上运动的时间。正式测试前，需对每只小鼠进行三次连续训练。测试期间，需连续测量三次，取平均掉落时间(潜伏期)。
2.6.免疫组化研究
分别在MPTP最后一次给药后的第7和14天取每组一半的小鼠，用4％水合氯醛麻醉、生理盐水+4％PFA灌注取脑组织。后经由20％(v/v)和30％(v/v)的蔗糖溶液梯度脱水各3天后，将小鼠脑组织用O.C.T.胶包埋后冰冻，冰冻切片机(Leica)连续组织切片(20μm)，收集相关区域脑片置于冻存液(甘油：0.01M PBS＝1:1)中，于-80℃保存待用。
a.尼氏染色
尼氏体是胞质内的一种嗜碱性物质，广泛见于各种神经元，故通过尼氏染色计算相关脑区神经元数量。按照尼氏染色试剂盒进行染色。
b.TH(Tyrosine hydroxylase，酪氨酸羟化酶)染色
TH主要标记黑质致密部多巴胺能神经元，MPTP造模后该脑区TH阳性神经元会显著减少。取出中脑脑片，0.01M PBS 10min洗片一次后，以3％H 2O 2孵育15min以去除内源性过氧化物酶活性，0.01M PBS 10min×3洗去H 2O 2，然后用含5％BSA和0.3％Triton X-100的PBS于室温下封闭1小时，继而孵育小鼠抗TH一抗(T2928，Sigma，USA)4℃过夜，二抗室温孵育1小时，PBS洗涤5min×3次后二氨基联苯氨(diaminobenzidin，DAB)避光显色。
2.7.细胞计数
免疫组织化学阳性细胞计数采用体式学计数系统(Stereo Investigator，MBF bioscience)计算黑质致密部的尼氏小体阳性细胞和TH+细胞数，细胞计数在×20物镜下进行。
2.8.数据处理及分析
所有数据均采用Mean±S.E.M的表示，采用GraphPad Prism 8.0统计分析软件进行统计学处理。生存率利用Log-rank检验分析，其他计量数据采用One-way ANOVA合并Dunnett’s test分析各组间差异，p<0.05表示差异具有统计学意义。3.实验结果
3.1.立再适治疗对小鼠MPTP亚急性模型后生存率的影响
MPTP亚急性给药使得小鼠在造模后精神萎靡、厌食、体重下降，并出现竖尾反应，但本次实验中未出现小鼠的死亡。
3.2.立再适治疗对小鼠MPTP亚急性模型后体重改变的影响
如图1所示，MPTP亚急性给药使得小鼠在造模后进食量小，7天内小鼠体重略降低，14天后进食逐渐增加，体重逐渐恢复。实验结果显示，仅立再适低剂量(10U/kg/d)治疗组小鼠在7天时较Sham组小鼠体重显著下降(p<0.01)，其余各组处理对小鼠体重无显著影响。
3.3.立再适对小鼠MPTP亚急性模型后在矿场实验中自主运动能力的影响
如表1和图2，正常小鼠反复进行旷场试验后，因对环境逐渐熟悉，出现自主活动减少，总路程也随之减少。MPTP亚急性模型中，小鼠在造模后1、3、7和14天时较对照组总运动路程减少，表明自主活动能力的下降。阳性对照药司来吉兰(Selegiline)在1、3、7和14天均能显著改善MPTP诱导的总运动路程减低，立再适仅在14天时有显著的改善作用，低、中和高剂量总运动路程增加为模型组的161％、154％和167％。
表1.立再适对MPTP亚急性模型后小鼠在矿场实验中总运动路程的影响
Figure PCTCN2022084780-appb-000001
***p<0.001, **p<0.01vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
3.4.立再适对小鼠MPTP亚急性模型后在爬杆实验中运动协调能力的影响
如表2～3和图3～4所示，MPTP亚急性模型后，小鼠在造模后1、3、7和14天时出现爬杆实验中T-Turn和T-TLA时间的延长，表明运动协调能力的下降。阳性对照药司来吉兰(Selegiline)在1、3、7和14天均能显著改善MPTP诱导的T-Turn和T-TLA时间延长。立再适在1、3、7和14天时均有显著的改善作用，14天时低、中和高剂量的T-Turn分别减少为模型组的35.1％、28.9％和32.6％，T-TLA也有减少趋势，但无显著性差异。
表2.立再适对MPTP亚急性模型后小鼠在爬杆实验中T-Turn的影响
Figure PCTCN2022084780-appb-000002
***p<0.001vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
表3.立再适对MPTP亚急性模型后小鼠在爬杆实验中T-TLA的影响
Figure PCTCN2022084780-appb-000003
***p<0.001vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
3.5.立再适对小鼠MPTP亚急性模型后在转棒实验中运动平衡能力的影响
如表4和图5所示，MPTP亚急性模型后，小鼠在造模后1、3、7和14天时出现转棒实验中掉落潜伏期的缩短，表明运动平衡能力的下降。阳性对照药司来吉兰(Selegiline)在1、3、7和14天均能显著改善MPTP诱导的潜伏期时间缩短。立再适在1、3、7和14天时也均具有显著的改善作用，14天时低、中和高剂量的潜伏期时间分别延长为模型组的121.3％、119.6％和119.8％。
表4.立再适对MPTP亚急性模型后小鼠在转棒实验中潜伏期的影响
Figure PCTCN2022084780-appb-000004
Figure PCTCN2022084780-appb-000005
***p<0.001vs.Sham； ###p<0.001, ##p<0.01vs.MPTP。
3.6.立再适对小鼠MPTP亚急性模型后黑质致密部尼氏阳性染色神经元数量的影响
如表5和图6所示，MPTP亚急性模型后，小鼠在造模后7和14天时出现黑质致密部尼氏染色阳性神经元数量的减少，表明该脑区神经元的死亡。阳性对照药司来吉兰(Selegiline)在7和14天均能显著改善MPTP诱导的黑质致密部尼氏染色阳性神经元数量的减少。立再适高剂量组在7和14天时均有显著的改善作用，尼氏染色阳性神经元数量较模型组分别增加为126.3和139.6％。
表5.立再适对MPTP亚急性模型后小鼠黑质致密部尼氏阳性神经元数量的影响
Figure PCTCN2022084780-appb-000006
***p<0.001, **p<0.01vs.Sham； ##p<0.01, #p<0.05vs.MPTP
3.7.立再适对小鼠MPTP亚急性模型后黑质致密部TH阳性染色神经元数量的影响
如表6和图7所示，MPTP亚急性模型后，小鼠在造模后7和14天时出现黑质致密部TH阳性神经元数量的减少，表明该脑区多巴胺能神经元的死亡。阳性对照药司来吉兰(Selegiline)在7和14天均能显著改善MPTP诱导的黑质致密部TH染色阳性神经元数量的减少。仅立再适高剂量组在7和14天时有显著的改善作用，低和中剂量组有改善的趋势，但无显著性差异。7天时低、中和高剂量的TH阳性神经元数量分别增加为模型组的115.2％、120.3％和125.3％，14天时低、中和高剂量的TH阳性神经元数量分别增加为模型组的125.7％、132.7％和138.7％。
表6.立再适对MPTP亚急性模型后小鼠黑质致密部TH阳性神经元数量的影响
Figure PCTCN2022084780-appb-000007
Figure PCTCN2022084780-appb-000008
***p<0.001vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
4.结论
立再适持续7天和14天治疗均能改善MPTP亚急性模型后小鼠神经行为障碍，其中高剂量组(40U/kg/d)在14天时的效应最为显著，其与模型组相比，可增加旷场试验中总运动路程167％，缩短爬杆实验T-Turn时间32.6％和T-TLA时间65.0％，增加转棒实验潜伏期119.8％。同时，立再适高剂量组尼氏染色阳性神经元数量较模型组分别增加126.3％(7天)和139.6％(14天)，黑质致密部TH阳性神经元数量较模型组分别增加125.3％(7天)和138.7％(14天)。这些结果表明立再适具有缓解MPTP亚急性模型中多巴胺能神经元损伤的作用。持续给予立再适7天和14天对小鼠帕金森病样改变具有缓解作用。

Claims (12)

  1. 痘苗病毒致炎兔皮提取物在制备用于在患者中预防或治疗帕金森病的药物中的用途。
  2. 权利要求1的用途，其中所述药物用于在患有帕金森病的患者中恢复大脑神经功能或缓解大脑神经功能损伤的用途。
  3. 痘苗病毒致炎兔皮提取物在制备用于在患有帕金森病的患者大脑中缓解或减少神经元变性、损伤或死亡的药物中的用途。
  4. 权利要求1-3中任一项的用途，其中所述患者罹患由帕金森病引起的帕金森综合征。
  5. 权利要求1-4中任一项的用途，其中所述患者具有运动症状例如颤抖、肢体僵硬、动作迟缓或姿态不稳；神经性精神疾患例如言语障碍、认知障碍、情绪障碍或思维障碍；感觉障碍或睡眠障碍；或者自主神经功能障例如油脂分泌过多、多汗或尿失禁。
  6. 权利要求1-5中任一项的用途，其中帕金森病由年龄老化、遗传因素、环境毒素或病原体感染引起。
  7. 权利要求1-6中任一项的用途，其中所述患者特征在于大脑黑质致密部神经元减少、损伤或死亡。
  8. 权利要求1-7中任一项的用途，其中所述痘苗病毒致炎兔皮提取物是立再适。
  9. 权利要求1-8中任一项的用途，其中所述痘苗病毒致炎兔皮提取物配制成口服制剂或注射剂，优选肌肉注射剂或静脉注射剂。
  10. 权利要求1-9中任一项的用途，其中所述患者是人，优选老年人。
  11. 权利要求1-10中任一项的用途，其中所述痘苗病毒致炎兔皮提取物以0.05U/kg至50U/kg，优选0.1U/kg至10U/kg，更优选0.5U/kg至5U/kg的量给予患者。
  12. 权利要求1-11中任一项的用途，其中所述药物包含3U至3000U，优选6U至600U，更优选30U至300U的所述述痘苗病毒致炎兔皮提取物。
PCT/CN2022/084780 2022-04-01 2022-04-01 痘苗病毒致炎兔皮提取物治疗帕金森病的用途 WO2023184471A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2022/084780 WO2023184471A1 (zh) 2022-04-01 2022-04-01 痘苗病毒致炎兔皮提取物治疗帕金森病的用途
TW112112597A TW202339779A (zh) 2022-04-01 2023-03-31 痘苗病毒致炎兔皮提取物治療帕金森病的用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/084780 WO2023184471A1 (zh) 2022-04-01 2022-04-01 痘苗病毒致炎兔皮提取物治疗帕金森病的用途

Publications (1)

Publication Number Publication Date
WO2023184471A1 true WO2023184471A1 (zh) 2023-10-05

Family

ID=88198847

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/084780 WO2023184471A1 (zh) 2022-04-01 2022-04-01 痘苗病毒致炎兔皮提取物治疗帕金森病的用途

Country Status (2)

Country Link
TW (1) TW202339779A (zh)
WO (1) WO2023184471A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1205233A (zh) * 1998-07-15 1999-01-20 沈继平 一种镇痛药和其制造方法
CN101732348A (zh) * 2008-11-11 2010-06-16 威世药业（如皋）有限公司 牛痘疫苗致炎兔皮提取物在制备急性脑血管疾病治疗药物中的用途
CN109504649A (zh) * 2017-09-15 2019-03-22 天津小西生物医药科技有限公司 使用兔皮提取物促进细胞增殖的方法
CN109512838A (zh) * 2017-09-15 2019-03-26 天津小西生物医药科技有限公司 一种兔皮提取物及其制备方法和用途
WO2020211009A1 (zh) * 2019-04-17 2020-10-22 诺希生物药物开发有限公司 痘苗病毒致炎兔皮提取物治疗造血系统损伤的用途
WO2020248240A1 (zh) * 2019-06-14 2020-12-17 俊熙有限公司 痘苗病毒致炎兔皮提取物治疗癌症的用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1205233A (zh) * 1998-07-15 1999-01-20 沈继平 一种镇痛药和其制造方法
CN101732348A (zh) * 2008-11-11 2010-06-16 威世药业（如皋）有限公司 牛痘疫苗致炎兔皮提取物在制备急性脑血管疾病治疗药物中的用途
CN109504649A (zh) * 2017-09-15 2019-03-22 天津小西生物医药科技有限公司 使用兔皮提取物促进细胞增殖的方法
CN109512838A (zh) * 2017-09-15 2019-03-26 天津小西生物医药科技有限公司 一种兔皮提取物及其制备方法和用途
WO2020211009A1 (zh) * 2019-04-17 2020-10-22 诺希生物药物开发有限公司 痘苗病毒致炎兔皮提取物治疗造血系统损伤的用途
WO2020248240A1 (zh) * 2019-06-14 2020-12-17 俊熙有限公司 痘苗病毒致炎兔皮提取物治疗癌症的用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI MIN; LI YUHUAN; LUO JIE; ZENG JING; XIONG GUANGRUN: "Neurotropin Exerts Neuroprotective Effects on Parkinson's Disease Rats through Nrf2/HO-1/NQO1 Pathway", JOURNAL OF BRAIN AND NERVOUS DISEASES, vol. 30, no. 2, 10 February 2022 (2022-02-10), pages 86 - 91, XP009549213, ISSN: 1006-351X *

Also Published As

Publication number Publication date
TW202339779A (zh) 2023-10-16

Similar Documents

Publication Publication Date Title
Eggers et al. Clinical and neurobiological findings in children suffering from tic disease following treatment with tiapride
JP2012529487A (ja) 脳疾患及び状態の予防及び治療のための組成物及び方法
JP2000514420A (ja) 末梢または中枢神経障害およびサイトカイン過剰産生の治療のためのｋ―２５２ａ誘導体の使用
JP2891464B2 (ja) 神経疾病を治療するための調合物
JP2021525709A (ja) 自閉症スペクトラム障害の治療のためのカンナビスベースの組成物
KR20180034442A (ko) 아캄프로세이트와 d-사이클로세린을 사용한 병용 치료
TWI659017B (zh) 吲哚基及吲哚啉基異羥肟酸於治療神經退化病症或認知缺乏之用途
WO2023184471A1 (zh) 痘苗病毒致炎兔皮提取物治疗帕金森病的用途
WO2022052016A1 (en) Pharmaceutical compositions and uses thereof in treating parkinson&#39;s disease
US11660279B2 (en) Therapeutic agents for treating restless leg syndrome
CN107854620B (zh) 一种改善脑损伤后认知功能障碍的中药组合物及其应用
TWI828061B (zh) 痘苗病毒致炎兔皮提取物治療神經系統脫髓鞘疾病的用途
CN115624543B (zh) 治疗偏头痛的药物、药物组合物、其制备方法和制药用途
AU2021374995A1 (en) Treatment of gait dysfunction in neurodegenerative disease
WO2023243659A1 (ja) 線条体ストリオソームのドパミンｄ１シグナルを標的とした強迫性障害の薬物療法
CN117357563A (zh) 灭活鼠乳杆菌在制备预防和/或治疗异动症药物中的应用
TWI669117B (zh) 茶多酚組合物及其用於治療口腔顏面疼痛之用途
TW202342075A (zh) 痘苗病毒致炎兔皮提取物治療阿爾茲海默病的用途
Mackenzie et al. A Double–Blind Trial of Bromocriptine in Parkinson's Disease
CN117959377A (zh) 远志散在制备预防和/或治疗中枢神经炎性急性脱髓鞘疾病的药物中的应用
CN113209064A (zh) 白花丹醌在制备预防或治疗帕金森病药物中的应用
TW202222331A (zh) 遠志萃取物用於治療注意力不足過動症
CN117959281A (zh) 芒柄花黄素在制备预防和/或治疗中枢神经系统炎性脱髓鞘疾病的药物中的应用
CN114392269A (zh) 一种抗过敏的注射用组合物及其制备方法和应用
KR20240070564A (ko) 알츠하이머병 예방 또는 치료용 약학적 조성물

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: 22934308

Country of ref document: EP

Kind code of ref document: A1