WO2014197510A1 - Composition préparée à partir de saikosaponine, utilisation et procédé de préparation associés - Google Patents

Composition préparée à partir de saikosaponine, utilisation et procédé de préparation associés Download PDF

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Publication number
WO2014197510A1
WO2014197510A1 PCT/US2014/040755 US2014040755W WO2014197510A1 WO 2014197510 A1 WO2014197510 A1 WO 2014197510A1 US 2014040755 W US2014040755 W US 2014040755W WO 2014197510 A1 WO2014197510 A1 WO 2014197510A1
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Prior art keywords
saikosaponin
virus
pharmaceutical composition
ssb2
group
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PCT/US2014/040755
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English (en)
Inventor
Chun-Ching Lin
Liang-Tzung LIN
Christopher D. Richardson
Ming-Hong YEN
Chueh-Yao CHUNG
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Kaohsiung Medical University
Huang, Alice Y.
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Application filed by Kaohsiung Medical University, Huang, Alice Y. filed Critical Kaohsiung Medical University
Priority to CN201480032330.7A priority Critical patent/CN105358220A/zh
Publication of WO2014197510A1 publication Critical patent/WO2014197510A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
    • A01N43/08—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/10—Apiaceae or Umbelliferae [Carrot family], e.g. parsley, caraway, dill, lovage, fennel or snakebed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/233—Bupleurum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/602—Glycosides, e.g. rutin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/63—Steroids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
    • C11D17/08—Liquid soap, e.g. for dispensers; capsuled
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of saikosaponin in treating and preventing a virus infection, a pharmaceutical composition including saikosaponin, and a method for preparing the pharmaceutical composition.
  • HCV hepatitis C virus
  • IFN-a interferon-alpha + ribavirin
  • the present invention uses the endemic herb, Bupleurum kaoi, utilizes saikosaponin extracted therefrom to treat or prevent a virus infection, and prepare a pharmaceutical composition having the aforementioned uses that solves the problem of poor solubility of the saikosaponin, which not only provides another choice for drug therapy for HCV but develops native biomedical resources in Taiwan.
  • a step of administering a pharmaceutical composition including a saikosaponin to a subject suffering from the infection is disclosed, wherein the virus is one selected from a group consisting of a hepatitis C virus, a measles virus, a respiratory syncytial virus, a vesicular stomatitis virus, a dengue virus, and non-enveloped viruses.
  • the aim of the present invention is to develop a new drug to replace or assist the existing clinical drugs (IFN-a/ribavirin) so as to achieve the benefits of reducing side effects and economic burden.
  • a step of administering an anti-viral agent in combination with a pharmaceutical composition including a saikosaponin to a subject suffering from the infection is disclosed, wherein the virus is one selected from a group consisting of a hepatitis C virus, a measles virus, a respiratory syncytial virus, a vesicular stomatitis virus, a dengue virus and non-enveloped viruses, and the anti-viral agent is at least one selected from a group consisting of Interferon alpha, pegylated Interferon-alpha, Ribavirin, Boceprevir and Telaprevir.
  • the present invention discloses a pharmaceutical composition including a main component and a polymer mixed with the main component, wherein the main component includes a saikosaponin.
  • the pharmaceutical composition solves the poor solubility of a drug by nanoscaling the drug, which increases the bioavailability and antivirus effect.
  • the present invention further discloses a method for preparing a pharmaceutical composition, including pouring an organic solvent containing a saikosaponin into a solution including a polymer, distributing the saikosaponin throughout the polymer, and removing the organic solvent to obtain the pharmaceutical composition.
  • the inventors of the present invention verified that the saikosaponin has the effects of treating and preventing the HCV infection, and may prevent the measles virus, the respiratory syncytial virus, the vesicular stomatitis virus, the dengue virus, and the non-enveloped virus' invasion of the cells to serve as a broad-spectrum viral inhibitor.
  • the present invention also discloses a successful dosage form of the saikosaponin at the nanoscale level that can be prepared in water to increase the bioavailability.
  • the saikosaponin and its nanoscaled dosage form can also be applied to masks, gloves, mosquito repellent, liquid soap, dry hand wash (a disinfecting fluid), and various cleaning supplies in the future, to achieve the effect of anti-virus protection, which would make epidemic prevention easier.
  • Fig. 1(A) is a High Performance Liquid Chromatography (HPLC) analysis result of the alcohol extract of Bupleurum (BK).
  • Fig. 1(B) is an HPLC analysis result of the saikosaponin SSa.
  • Fig. 1(C) is an HPLC analysis result of the saikosaponin SSb2.
  • Fig. 1(D) is an HPLC analysis result of the saikosaponin SSc.
  • Fig. 1(E) is an HPLC analysis result of the saikosaponin SSd.
  • Fig. 2(A) shows the effect of the alcohol extract of Bupleurum (BK) on inhibiting the HCV infection.
  • Fig. 2(B) shows the effect of the saikosaponin SSa on inhibiting the HCV infection.
  • Fig. 2(C) shows the effect of the saikosaponin SSb2 on inhibiting the HCV infection.
  • Fig. 2(D) shows the effect of the saikosaponin SSc on inhibiting the HCV infection.
  • Fig. 2(E) shows the effect of the saikosaponin SSd on inhibiting the HCV infection.
  • Fig. 3 shows the effects of the alcohol extract of Bupleurum (BK) and the saikosaponin SSa, SSb2, SSc, and SSd on the time points of the HCV infection.
  • Fig. 4 is the result of entry assays for the alcohol extract of Bupleurum (BK) and the saikosaponin SSa, SSb2, SSc, and SSd.
  • Fig. 5 shows the result that the saikosaponin SSb2 inhibits the viruses from attaching to the cells.
  • Fig. 6 shows the result that the saikosaponin SSb2 inhibits the infections of different genotypes of HCV.
  • Fig. 7 shows the result that the saikosaponin SSb2 inhibits the viruses in the sera of the clinical patients from attaching to the cells.
  • Fig. 8(A) shows the effect of the saikosaponin SSb2 on inhibiting the measles virus (MV) infection.
  • Fig. 8(B) shows the effect of the saikosaponin SSb2 on inhibiting the respiratory syncytial virus (RSV) infection.
  • Fig. 8(C) shows the effect of the saikosaponin SSb2 on inhibiting the vesicular stomatitis virus (VSV) infection.
  • Fig. 8(D) shows the effect of the saikosaponin SSb2 on inhibiting the dengue virus (DENV) infection.
  • Fig. 8(E) shows the effect of the saikosaponin SSb2 on inhibiting the reovirus (RV) infection.
  • Fig. 9(A) shows the result of the crystal property of the pharmaceutical composition of the present invention determined by X-ray diffraction (XRD).
  • Fig. 9(B) shows the result of inter-molecular interactions between the two materials of the pharmaceutical composition of the present invention determined by Fourier transform infrared spectroscopy (FTIR).
  • FTIR Fourier transform infrared spectroscopy
  • Fig. 9(C) shows the determined solubility of the pharmaceutical composition of the present invention.
  • Fig. 10(A) shows the effect of the nanoparticles of the present invention on inhibiting the HCV infection.
  • Fig. 10(B) shows the effect of the nanoparticles of the present invention on inhibiting the DENV infection.
  • Fig. 11(A) shows the effect of a combination of the alcohol extract of Bupleurum (BK) and various concentrations of interferon on inhibiting the HCV infection.
  • Fig. 11(B) shows the effect of a combination of the saikosaponin SSb2 and various concentrations of interferon on inhibiting the HCV infection.
  • the present invention discloses a method for preventing or treating an infection of a virus, including a step of administering a pharmaceutical composition including a saikosaponin to a subject suffering from the infection, wherein the virus is one of the hepatitis C virus, the measles virus, the respiratory syncytial virus, the vesicular stomatitis virus, the dengue virus, and non-enveloped viruses (including reovirus, enterovirus, noro virus, and adenovirus).
  • the saikosaponin used in the present invention is an alcohol extract of Bupleurum kaoi, which is extracted from Bupleurum kaoi (an endemic species in Taiwan) and includes the saikosaponins SSa, SSb2, SSc, and SSd. It should be realized that the phrase "saikosaponin" herein is not limited to the alcohol extract from Bupleurum kaoi and may be a product (including a synthesized product) obtained from other Bupleurum species using any solvent or any method.
  • the saikosaponin in the present invention can refer to a collective composition of the alcohol extracts of Bupleurum, SSa, SSb2, SSc, SSd, and the combination thereof, for example, the saikosaponin could include SSb2 only, include SSa and SSb2, or include SSa, SSb2, SSc, and SSd, although the antivirus effects of the alcohol extract of Bupleurum, SSa, SSb2, SSc, and SSd are separately shown in the present invention.
  • the saikosaponin of the present invention can be mixed with a pharmaceutically acceptable carrier to obtain a formulation with more bioavailability.
  • a pharmaceutically acceptable carrier above or the phrases “excipients” and “bioavailable carriers or excipients” include any appropriate compounds known to be used for preparing the dosage form, such as a solvent, a dispersing agent, a coating, an antibacterial or antifungal agent, and a preserving agent or delayed absorbent. Typically, such a carrier or excipient does not have therapeutic activity in and of itself.
  • each formulation prepared by combining the saikosaponin disclosed in the present invention and the pharmaceutically acceptable carriers or excipients will not cause undesired effects, allergies, or other inappropriate effects when administered to an animal or human. Accordingly, the saikosaponin disclosed in the present invention in combination with the pharmaceutically acceptable carrier or excipient is adaptable for clinical usage and can achieve the treatment or prevention effect.
  • the carrier varies depending on the dosage form itself, and the composition for oral administration may use any orally acceptable formulation, which includes capsule, tablet, pill, emulsion, aqueous suspension, dispersing agent, and solvent.
  • the carrier generally used in the oral formulation, taking the tablet as an example, the carrier may be a basic additive such as lactose, corn starch, lubricant, and magnesium stearate.
  • the diluents used in the capsule include lactose and dried corn starch.
  • the active ingredient is suspended or dissolved in an oil interface in combination with the emulsion or the suspension agent, and an appropriate amount of sweeteners, flavours, or pigments is added as needed.
  • the pharmaceutical composition of the present invention can be manufactured as one of a drug, a disinfecting fluid, a pair of gloves, a mask, a dry hand wash, a liquid soap, or a mosquito repellent.
  • a disinfecting fluid a pair of gloves, a mask, a dry hand wash, a liquid soap, or a mosquito repellent.
  • Fig. 1(A) to Fig. 1 (E) show the HPLC analysis results of the alcohol extract of Bupleurum, the saikosaponins SSa, SSb2, SSc, and SSd.
  • HPLC is used to determine the saikosaponins SSa, SSb2, SSc, and SSd indeed existed in the alcohol extract of Bupleurum kaoi (BK) and quantify their contents.
  • the quantified results of Fig. 1 (A) to Fig. 1 (E) are shown below in Table 1.
  • SSd 78.1-624.8 y 904.73X-1182.2 0.9979 538.7911.37 x is the concentration of saikosaponin in ⁇ g/ml, y is peak area.
  • Crude extract was injected 3 times. The contents of crude extract were expressed mean 1 SEM.
  • Saikosaponin has the effect of inhibiting the HCV infection:
  • Figs. 2(A) to 2(E) show the effects of the alcohol extract of Bupleurum and saikosaponins SSa, SSb2, SSc, and SSd on inhibiting the HCV infection.
  • the alcohol extract of Bupleurum kaoi (BK) and saikosaponins SSa, SSb2, SSc, and SSd were dissolved in dimethyl sulfoxide (DMSO) and prepared into various concentrations used to evaluate the antivirus activity. It was found that BK, SSa, SSb2, SSc, and SSd all have the effect of inhibiting the infection of cell-culture derived HCV genotype 2a and reveal dose dependency.
  • DMSO dimethyl sulfoxide
  • the concentrations that were non-toxic to the cells and had the anti-virus activity e.g. 50 ⁇ g/ml of BK, 8 ⁇ of SSa, 50 ⁇ of SSb2, 200 ⁇ of SSc, and 2 ⁇ SSd, were chosen for the experiments of the anti-virus mechanism.
  • the dosages above have about 2 log (i.e. 100 times) the inhibition index for the virus protein.
  • the quantified results of Fig. 2(A) to Fig. 2(E) are shown below in Table 2. Table 2
  • Fig. 3 shows the effects of the alcohol extract of Bupleurum, and the saikosaponin SSa, SSb2, SSc, and SSd on the time points of the HCV infection.
  • the virus and drugs were added in three different ways: 1) pre-treatment: after the cells were treated with the drugs for 24 hours, the drugs were removed and the virus was added, and then the expression of the virus was detected by luminescence 3 days later, 2) co-addition: the virus and the drugs were added simultaneously and removed after 3 hours, and then the expression of the virus was detected by luminescence 3 days later, and 3) post-infection: the drugs were added after 3 hours from the virus infection, and then the expression of the virus was detected by luminescence 3 days later.
  • Fig. 4 is the result of entry assays for the alcohol extract of Bupleurum and the saikosaponin SSa, SSb2, SSc, and SSd.
  • SSb2 can neutralize the viral particles and inhibit the viruses from attaching to the cells
  • SSa can inhibit the viral fusion.
  • the results above prove that the main mechanism by which the alcohol extract of Bupleurum and its active ingredients SSa and SSb2 against the virus is entry inhibition, and thus the alcohol extract of Bupleurum and its active ingredients SSa and SSb2 can function as entry inhibitors.
  • Fig. 5 shows the result that the saikosaponin SSb2 inhibits the virus from attaching to the cells.
  • HCVcc Cell-culture derived HCV
  • sE2 soluble glycoprotein E2
  • PBS phosphate buffered saline
  • ELISA enzyme-linked immunosorbent assay
  • SSb2 can avoid the viral entry by interfering with the binding of the HCVcc and sE2 to the cell membrane.
  • Bupleurum kaoi and its active ingredients can serve as therapeutic drugs for chronic hepatitis C.
  • Bupleurum kaoi, SSa, SSb2, SSc, and SSd inhibits the early entry of the virus
  • these components also have preventive functions and can serve as drugs for liver transplantation (disinfectant drugs) to prevent the virus from invading new liver tissue, and this specific use overcomes the previous defect that IFN-a + ribavirin can only be used in treatment but not in prevention.
  • Fig. 6 shows the result that the saikosaponin SSb2 inhibits the infections of different genotypes of HCVs.
  • Huh-7.5 cells were infected with genotypes 2b, 3a, or 7a of HCV, respectively, and treated with SSb2 at the same time, and the results show that SSb2 can inhibit the infections of different genotypes of HCV.
  • Fig. 7 shows the result that the saikosaponin SSb2 inhibits the viruses from the sera of the clinical patients from attaching to the cells.
  • the viruses from the sera of the clinical patients were added to Huh-7.5 cells under 4°C, and the SSb2 was added thereto at the same time. After 3 hours of treatment, the SSb2 and viruses were washed with PBS, and then the total RNA of the cells was extracted. Finally, the level of virus was quantified by quantitative polymerase chain reaction (qPCR). The result showed that SSb2 can inhibit the binding of the genotypes la, lb, 2a, and 6 of HCV from the sera of the clinical patients to the cells. The quantified results are shown below in Table 3.
  • the present invention proves that the saikosaponin SSb2 not only inhibits the genotype 2a of HCVcc, but also inhibits the infections of other genotypes 2b, 3a, and 7a of HCVcc as well as the attachment of the genotypes la, lb, 2a, and 6 of HCV from the sera of the clinical patients to the cells, such that the clinical patients can resist the high variability of HCV.
  • saikosaponin can overcome the problem of poor therapeutic effect of IFN-a + ribavirin on the genotype 1 of HCV, and overcome the problems of two new drugs, boceprevir and telaprevir, which have poor therapeutic effect on other genotypes of HCVs except for the genotype 1 HCV and are not suitable for being administered alone (i.e. boceprevir and telaprevir only can be used in combination with IFN-a + ribavirin).
  • Saikosaponin has the effect of inhibiting the infection of various viruses:
  • Figs. 8(A) to 8(E) show the effect of SSb2 on inhibiting the infection of measles virus (MV), respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV), dengue virus (DENV), and reovirus (RV).
  • MV measles virus
  • RSV respiratory syncytial virus
  • VSV vesicular stomatitis virus
  • DEV dengue virus
  • RV reovirus
  • Table 4 it can be seen that these viruses belong to different types, and the results show that SSb2 has inhibitory effects on the viruses regardless of DNA or RNA viruses, viruses with single or double stranded genomes, enveloped or non-enveloped viruses, and it is proven that SSb2 is a broad-spectrum antiviral drug. Based on the inhibitory effects of SSb2 on the non-enveloped virus, it can be seen that SSb2 also has inhibitory effects on other non-enveloped viruses such as enterovirus, norovirus, and adenovirus.
  • non-enveloped viruses referred to in the present invention besides the reovirus (RV), enterovirus, norovirus, and adenovirus described above, further includes other non-enveloped viruses known in the art such as human papillomavirus, small RNA virus, and so on.
  • RV reovirus
  • enterovirus norovirus
  • adenovirus adenovirus
  • the saikosaponin can also be applied to masks, gloves, mosquito repellent, liquid soap, dry hand wash (a disinfecting fluid), and various cleaning supplies in the future, to make epidemic prevention easier.
  • the saikosaponin can be added to mosquito repellent to inhibit the dengue fever epidemic that periodically spikes every summer in tropics and subtropics.
  • the saikosaponin can be added to dry hand wash, to solve the problem that the alcoholic dry wash has no disinfecting effect on the non-enveloped viruses and to relieve the exploding crises of epidemic viruses in the world.
  • saikosaponin has poor solubility, the problem of low bioavailability may occur if it is made as a drug.
  • a pharmaceutical composition is prepared using nanotechnology in the present invention.
  • the saikosaponin, the main component of the pharmaceutical composition was mixed with a polymer to create a nanoparticle, which is ⁇ 50 nm in diameter.
  • the pharmaceutical composition of the present invention can be refrigerated in liquid form, or be frozen in the form of dry powder, it can be made as a drug, various medical defenses (such as disinfecting fluid, gloves, mask, dry hand wash, liquid soap, and mosquito repellent) and commodities.
  • various medical defenses such as disinfecting fluid, gloves, mask, dry hand wash, liquid soap, and mosquito repellent
  • the present invention provides a method for manufacturing a pharmaceutical composition, including pouring an organic solvent containing the saikosaponin into a solution including a polymer, distributing the saikosaponin throughout the polymer, and removing the organic solvent to obtain the pharmaceutical composition.
  • SSb2 is used as an example to prepare SSb2 nanoparticles (SSb2-NP). It should be realized that the preparation method provided in the embodiments of the present invention is also suitable for preparing nanoparticles of other saikosaponin (SSa, SSc, and SSd) and nanoparticles of the alcohol extract of Bupleurum.
  • SSb2 was distributed and admixed to prepare SSb2 nanoparticles by the emulsion solvent diffusion method.
  • This process can be performed using various polymers such as Pluronic® polymer, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and others.
  • Pluronic® polymer polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and others.
  • PVP was used as an example of the present process.
  • SSb2 was dissolved in an appropriate amount of ethanol to make 1 mg/ml solution
  • PVP was dissolved in an appropriate amount of pure water to prepare a 35% solution
  • the SSb2 solution was poured into the PVP solution.
  • the distribution and admixing of the SSb2 and the PVP were done using ultrasound, and the organic solvent (i.e. ethanol in this case) and part of the water were removed using a vacuum decompression concentrator, to create primary SSb2 nanoparticles. After filtration using qualitative filter paper to remove oversized pellets, the complete SSb2 nanoparticles in this example can be obtained.
  • This formulation can be refrigerated in a liquid form, or frozen in powder form, and the latter can be re-suspended in pure water prior to being administered.
  • SSb2 is dissolved and formulated in an ethanol solution, but the skilled person in the art could replace the ethanol solution with other suitable organic solvents.
  • Fig. 9(A) shows the results of the crystallization property of the pharmaceutical composition of the present invention determined using X-ray diffraction (XRD).
  • Fig. 9(B) shows the results of inter-molecular interaction between the two materials of the pharmaceutical composition of the present invention determined using Fourier transform infrared spectroscopy (FTIR).
  • FTIR Fourier transform infrared spectroscopy
  • the inter-molecular interaction between the two materials is determined by FTIR, a shift of the hydroxyl group (3,420 cm “1 ) can be seen from the spectrum of the nanoparticles (SSb2-NP), and the shift was different from that of the physical mixture (SSb2-PM), showing that the inter-molecular interactions indeed occurred in the nanoparticles in this case.
  • the inter-molecular interaction between the two materials can explain why the SSb2 lattice property of the nanoparticles disappeared, which improves the solubility of SSb2.
  • Fig. 9(C) shows the determined solubility of the pharmaceutical composition of the present invention.
  • the solubility of the present nanoparticles was determined using dissolution assay.
  • the dissolution assay shows that the nanoparticles in this example significantly enhanced the solubility of SSb2.
  • SSb2 achieved 50% release in 20 minutes and may maintain a releasing trend during the subsequent test.
  • Figs. 10(A) and 10(B) show the effects of the nanoparticles of the present invention on inhibiting the HCV and DENV infections, respectively.
  • SSb2-NP has a characteristic of being active in water without another solubilizer (such as DMSO).
  • the present experiment compared the SSb2-NP with the raw material drug in the solubilizer (SSb2-DMSO) and the raw material drug in water (SSb2-DW), and analyzed the antiviral effects themselves.
  • the results show that SSb2-DW cannot exhibit any antiviral effect against either HCV (Fig. 10(A)) or DENV (Fig. 10(B)), and the antiviral effect of SSb2-NP almost equals that of SSb2-DMSO.
  • the pharmaceutical composition of the present invention can be used in combination with suitable clinical drug, to increase the validity of the therapy.
  • Huh-7.5 cells were infected with HCV for 3 hours. After the infection is completed, the exceed viruses were washed and the culture medium containing 2% fetal bovine serum and the pharmaceutical composition of the present invention (BK: 10 ⁇ , 17 ⁇ , and 20 ⁇ ;
  • Fig. 11(A) shows the effect of a combination of the alcohol extract of Bupleurum (BK) and various concentrations of interferon on inhibiting the HCV infection
  • Fig. 11(B) shows the effect of a combination of the saikosaponin SSb2 and various concentrations of interferon on inhibiting the HCV infection
  • MOCK group is a negative control group without administering any drug
  • virus only group is a negative control group infected with HCV
  • DMSO group is a negative group administered dimethyl sulfoxide (DMSO).
  • the specific drug (Interferon) used in the present invention is only exemplary embodiment which do not limit the scope of the present invention.
  • the pharmaceutical composition of the present invention can be used in combination with other clinical drugs for treating or preventing a virus infection.
  • Interferon used in the above embodiments can also be Interferon alpha, pegylated Interferon-alpha, Ribavirin, Boceprevir, Telaprevir, or the combination thereof.
  • a method for performing one of preventing and treating an infection of a virus wherein the virus is one selected from a group consisting of a hepatitis C virus, a measles virus, a respiratory syncytial virus, a vesicular stomatitis virus, a dengue virus, and non-enveloped viruses, the method comprising a step of administering a pharmaceutical composition including a saikosaponin to a subject suffering from the infection.
  • non-enveloped viruses comprise one selected from a group consisting of a reovirus, an enterovirus, a norovirus, and an adenovirus.
  • a method for performing one of preventing and treating an infection of a virus wherein the virus is one selected from a group consisting of a hepatitis C virus, a measles virus, a respiratory syncytial virus, a vesicular stomatitis virus, a dengue virus, and non-enveloped viruses, the method comprising a step of administering an anti-viral agent in combination with a pharmaceutical composition including a saikosaponin to a subject suffering from the infection, and wherein the anti-viral agent is at least one selected from a group consisting of Interferon alpha, pegylated Interferon-alpha, Ribavirin, Boceprevir, and Telaprevir.
  • a pharmaceutical composition including:
  • a main component including a saikosaponin
  • a pharmaceutical composition according to Embodiment 9 wherein the pharmaceutical composition has a size smaller than 50 nm.
  • Embodiments 9-11 wherein the saikosaponin comprises at least one selected from a group consisting of SSa, SSb, SSc, and SSd.
  • Embodiments 9-12 wherein the saikosaponin is derived from Bupleurum kaoi.
  • Embodiments 9-13 further comprising a pharmaceutically acceptable carrier.
  • Embodiments 9-14 wherein the pharmaceutical composition is manufactured as one selected from a group consisting of a drug, a disinfecting fluid, a pair of gloves, a mask, a dry hand wash, a liquid soap, and a mosquito repellent.
  • Embodiments 9-15 wherein the polymer improves the solubility of the saikosaponin.
  • Embodiments 9-16 wherein the polymer is one selected from a group consisting of a Pluronic polymer, a polyvinyl alcohol (PVA), and a polyvinylpyrrolidone (PVP).
  • a Pluronic polymer a polyvinyl alcohol (PVA)
  • PVP polyvinylpyrrolidone
  • a method for preparing a pharmaceutical composition including steps of: pouring an organic solvent containing a saikosaponin into a solution, wherein the solution includes a polymer;
  • the polymer is one selected from a group consisting of a Pluronic polymer, a polyvinyl alcohol (PVA), and a polyvinylpyrrolidone (PVP).

Abstract

La présente invention concerne une composition pharmaceutique ayant de la saikosaponine en tant que composant principal, l'utilisation de la composition pharmaceutique, ainsi que le procédé de préparation de celle-ci, la composition pharmaceutique étant utilisée pour prévenir ou traiter l'infection par un virus sélectionné parmi le virus de l'hépatite C, le virus de la rougeole, le virus respiratoire syncytial, le virus de la stomatite vésiculaire, le virus de la dengue, et les virus non enveloppés.
PCT/US2014/040755 2013-06-04 2014-06-03 Composition préparée à partir de saikosaponine, utilisation et procédé de préparation associés WO2014197510A1 (fr)

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CN113633050A (zh) * 2021-09-14 2021-11-12 刘中庆 一种半湿式消杀口罩

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