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WO2011070415A1 - Nouvelle méthode thérapeutique pour le traitement d'états inflammatoires et de troubles du système immunitaire - Google Patents

Nouvelle méthode thérapeutique pour le traitement d'états inflammatoires et de troubles du système immunitaire Download PDF

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Publication number
WO2011070415A1
WO2011070415A1 PCT/IB2010/003050 IB2010003050W WO2011070415A1 WO 2011070415 A1 WO2011070415 A1 WO 2011070415A1 IB 2010003050 W IB2010003050 W IB 2010003050W WO 2011070415 A1 WO2011070415 A1 WO 2011070415A1
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alkyl
infection
inflammation
tnf
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Allan Sy Lau
Lai Hung Cindy Yang
Cho Tsun Or
Hing Yee Law
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Versitech Ltd
Purapharm International HK Ltd
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Versitech Ltd
Purapharm International HK Ltd
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Priority to CA2781233A priority Critical patent/CA2781233A1/fr
Priority to CN2010800621401A priority patent/CN102844027A/zh
Priority to AU2010329604A priority patent/AU2010329604B2/en
Priority to JP2012539432A priority patent/JP2013511506A/ja
Priority to EP10835563.7A priority patent/EP2504007B1/fr
Priority to US13/005,301 priority patent/US9655877B2/en
Publication of WO2011070415A1 publication Critical patent/WO2011070415A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
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    • A61P17/00—Drugs for dermatological disorders
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    • A61P19/00—Drugs for skeletal disorders
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    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • TNF-a tumor necrosis factor-a
  • TNF-a In addition to its role in acute phase response, TNF-a has been shown to be involved in the progression of various chronic diseases including tumorigenesis and rheumatoid arthritis (RA). The dysregulation of TNF-a production has been demonstrated to be involved in different stages of tumorigenesis including initiation of tumor growth 8 , cell proliferation 9 and invasion 10 .
  • TNF-a upregulates specific growth factors to mediate the malignant growth.
  • the cytokine promotes angiogenesis that supports tumor migration, and thus plays a key role in tumor metastasis.
  • glioblastoma migration and induction of matrix metalloproteinases (MMP) are significantly enhanced in response to TNF-a effects". This induction of MMP in glioblastoma T98G cells can be reversed by treatment of the cells with interferon-g 12 .
  • MMP matrix metalloproteinases
  • TNF-a The uncontrolled production of TNF-a is associated with many acute and chronic neurodegenerative conditions, including stroke, brain trauma, spinal cord injury, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, and Parkinson's disease.
  • ALS amyotrophic lateral sclerosis
  • TNF-a The toxic effects of TNF-a and its role as a mediator of focal ischemia may involve many other mechanisms in addition to inflammation. For example, increased TNF-a in the brain and blood in response to lipopolysaccharide (LPS) appears to contribute to increased brain stem thrombosis and hemorrhage, and increased stroke sensitivity/risk. Additionally, TNF-a increases blood-brain barrier permeability and produces pial artery constriction, which can contribute to focal ischemic brain injury. Further, there appeal's to be a direct toxic effect of TNF-a on capillaries.
  • LPS lipopolysaccharide
  • TNF-a increases capillary permeability and opens the blood-brain barrier, apparently by increasing matrix-damaging metalloproteinase (gelatinase B) production, which is also expressed early after focal stroke. TNF-a also causes damage to myelin and oligodendrocytes and increases astrocytic proliferation, thus potentially contributing to demyelination and reactive gliosis during brain injury.
  • gelatinase B matrix-damaging metalloproteinase
  • TNF-a rheumatoid arthritis
  • inflammatory bowel diseases include rheumatoid arthritis and inflammatory bowel diseases.
  • Patients with rheumatoid arthritis have a low grade insidious inflammation in the synovial tissues. It is known that overproduction of TNF-a at the inflamed joint leads to slow destruction of the joint cartilage and surrounding bone. Additionally, inflammatory responses including TNF-a production may play an important role in the pathogenesis of cerebrovascular diseases including ischemic stroke and cardiovascular diseases (CVD). It has been suggested that TNF- a may destabilize atherogenesis and atherosclerotic plaques leading to their rupture, resulting in myocardial infarction or stroke in CVD patients.
  • CVD cardiovascular diseases
  • TNF-a disease pathogenesis mediated by TNF-a can be associated with microbial, bacterial and viral infections.
  • Cytokines such as TNF-a play a role in defending against the invading pathogens such as, for example, mycobacteria, influenza viruses, SARS-coronavirus and retroviruses includin HIV.
  • many microbes and viruses have also developed various immunosuppressive mechanisms that cause dysfunction of protein signaling kinases and transcription factors as well as other components involved in the TNF-a signaling pathway 13 ' 14 ' 15 ' 16 ' 17 ' 1 S .
  • nitric oxide is a feature of genuine immune-system cells such as dendritic cells, NK cells, mast cells and phagocytic cells including monocytes, macrophages, microglia, Kupffer cells, eosinophils, and neutrophils as well as other cells involved in immune reactions.
  • cytokines cytokines
  • microbial compounds microbial compounds
  • NO nitric oxide
  • Generation of nitric oxide is a feature of genuine immune-system cells such as dendritic cells, NK cells, mast cells and phagocytic cells including monocytes, macrophages, microglia, Kupffer cells, eosinophils, and neutrophils as well as other cells involved in immune reactions.
  • NO nitric oxide
  • Many targets of NO are themselves regulatory molecules, for example transcription factors and components of various signaling cascade.
  • Interferon-gamma Interferon-gamma
  • the inter leak in family such as Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 (TL-5), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL-9), Interleukin-10 (IL-10), Interleukin-11 (IL-1 1), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-14 (IL-14), Interleukin-15 (IL-15), Interleukin-16 (IL-16), Interleukin-17 (IL-17), Interleukin-18 (IL-18), Interleukin-19 (IL-19), Interleukin-20 (IL-20), Interleukin-21 (IL-21), Interleukin-22 (IL-22), Interleukin-23 (IL-23), Interleukin-24 (IL-24),
  • IFN- ⁇ Interferon-gamm
  • TNF-a and nitric oxide have played an increasing role in treating inflammatory and immune conditions.
  • Use of exogenous anti-inflammatory therapeutics would be particularly desirable to control the adverse effects of immune over-activation.
  • immunotherapeutics have been developed that aim at the neutralization of TNF-a and suppression of its undesirable proinflammatory effects.
  • TNF-a exacerbates focal ischemic injury in neurodegenerative diseases
  • agents for blocking endogenous TNF-a have been shown to be neuroprotective. These agents include soluble TNF-a receptor (Enbrel) and anti -TNF-a antibody (Infliximab).
  • non-steroid anti-inflammatory drugs including aspirin, ibuprofen, and indomethacin are well-known in ameliorating acute and chronic pain associated with inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.
  • inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.
  • steroids and cytotoxic drugs such as methotrexate and cyclophosphamide are used.
  • cytotoxic drugs such as methotrexate and cyclophosphamide are used. These drugs are associated with severe adverse effects including gastrointestinal irritation, severe bleeding, and bone marrow suppression.
  • the present invention provides novel and advantageous therapeutic methods for treating inflammation and/or modulating immune responses.
  • senkyunolide A Sen A
  • Z-Lig Z-ligustilide
  • the therapeutic methods of the subject invention can be used to modulate TNF-a production by administering, to a subject in need of such treatment, an effective amount of an isolated compound having the following formula:
  • R represents a carbon-carbon single bond or a carbon-carbon double bond
  • R] is alkyl or CR 6 .
  • R 6 is alkyl, acyl, haloalkyl, alkylamino or hydroxylalkyl
  • R 2j R 3 and R4 are, independently, -H, acyl, halo, haloalkyl, amino, alkylamino, hydroxyl, alkyl, hydroxylalkyl, or -COOH; and
  • Rs is -H, acyl, halo, haloalkyl, amino, alkylamino, alkyl, hydroxylalkyl, or -COOIL
  • the subject invention further provides pharmaceutical compositions containing these compounds.
  • the methods of the present invention can be used to control over-production of TNF-a and nitric oxide in cells associated with inflammation and immune conditions.
  • the methods of the present invention inhibit cell death induced by cell injury associated with inflammation and immune conditions.
  • the methods of the present invention are useful for treating conditions selected from, for example, ischemic stroke, autoimmune conditions, rheumatoid arthritis, psoriasis, cardiovascular disease, cerebrovascular disease, neurodegenerative disease, post-infection associated neurological neuralgia or neurasthenia conditions including shingles and chronic fatigue syndrome, inflammatory bowel disorder, septic shock, infections, environmental toxins, intestinal inflammation, allergy, graft rejection, pathological immune cell proliferation or activity, and respiratory inflammation.
  • ischemic stroke autoimmune conditions, rheumatoid arthritis, psoriasis, cardiovascular disease, cerebrovascular disease, neurodegenerative disease, post-infection associated neurological neuralgia or neurasthenia conditions including shingles and chronic fatigue syndrome, inflammatory bowel disorder, septic shock, infections, environmental toxins, intestinal inflammation, allergy, graft rejection, pathological immune cell proliferation or activity, and respiratory inflammation.
  • Figure 1 shows an extraction scheme of bioactive compounds, including senkyunolide A and Z-ligustilide, from Ligiisticum chuanxiong.
  • Figure 2 shows high performance liquid chromatography (HPLC) chromatograms of subfractions separated from the extract LCX-l-Et-EA-S l .
  • Figure 3 shows the effect of subfractions separated from LCX-l-Et-EA-S l on nitrite production in BV cells. (p ⁇ 0.05, compared with DMSO + LPS only).
  • Figure 4 shows the dose-dependent effect of senkyunolide A on nitrite production in
  • Figure 5 shows the dose-dependent effect of Z-ligustilide on nitrite production in BV cells. (p ⁇ 0.05, compared with DMSO + LPS only).
  • Figure 6 shows the effect of senkyunolide A on TNF-a production in human blood macrophages. (pO.001 , compared with DMSO + LPS only).
  • Figure 7 shows the dose-dependent effect of senkyunolide A on TNF-a protein production in BV cells. (p ⁇ 0.05, compared with DMSO + LPS only).
  • Figure 8 shows the dose-dependent effect of Z-ligustilide on TNF-a protein production in BV cells. (p ⁇ 0.05, compared with DMSO + LPS only).
  • Figure 9 shows the effect of senkyunolide A on TNF-a mRNA expression and iNOS expression in BV cells.
  • Figure 10 shows the effect of senkyunolide A on TNF-a mRNA stability in BV cells. (p ⁇ 0.05, compared with DMSO + LPS only).
  • Figure 11 shows the effect of Z-ligustilide on hydrogen peroxide-induced death of PC- 12 cells.
  • PC- 12 cells were pretreated with either DMSO (0.05%) or Z-ligustilide for 24 h, followed by treatment with 0.8mM H 2 O 2 for another 6 h. Cell viability was measured using MTT assays. A set of representative results is shown.
  • SEQ ID NO: 1 is a primer useful according to the subject invention.
  • SEQ ID NO:2 is a primer useful according to the subject invention.
  • SEQ ID NO:3 is a primer useful according to the subject invention.
  • SEQ ID NO:4 is a primer useful according to the subject invention. DETAILED DESCRIPTION OF THE INVENTION
  • the subject invention provides novel and advantageous therapeutic methods for treating inflammatory and immune conditions in a subject.
  • the treatment method which is capable of modulating TNF-a production, comprises administering, to a subject in need of such treatment, an effective amount of an isolated compound having the following formula:
  • Ri is alkyl or CR 6, wherein is alkyl, acyl, haloalkyl, alkylamino or hydroxylalkyl;
  • R 2, R 3 and R4 are, independently, -H, acyl, halo, haloalkyl, amino, alkylamino, hydroxyl, alkyl, hydroxylalkyl, or -COOH; and
  • R 5 is -H, acyl, halo, haloalkyl, amino, alkylamino, alkyl, hydroxylalkyl, or -COOH.
  • Alkyl means linear saturated monovalent radicals of one to eight carbon atoms or a branched saturated monovalent of three to eight carbon atoms. It may include hydrocarbon radicals of one to four or one to three carbon atoms, which may be linear. Examples include methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl, and the like.
  • Acyl means a radical ⁇ C(0)R where R is hydrogen, alkyl or cycloalkyl. or heterocycloalkyl. Examples include formyl, acetyl, ethylcarbonyl, and the like.
  • Halo means fluoro, chloro, brorno, or iodo, such as bromo and chloro.
  • Haloalkyl means alkyl substituted with one or more, same or different, halo atoms, e.g., -CH 2 CI, -CH 2 Br, -CF 3 , -CH 2 CH 2 C1, -CH 2 CC1 3 , and the like.
  • Amino means the radical -NH 2 .
  • Alkylamino means a radical -NHR or -NR 2 where each R is, independently, an alkyl group. Examples include methylamino, (1 -methyl ethyl)amino, dimethylamino, methylethylamino, di(l-methylethyl)amino, and the like.
  • Hydroxy means the radical -OH.
  • Hydroxyalkyl means an alkyl radical as defined herein, substituted with one or more, preferably one, two or three, hydroxy groups. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3 -hydroxypr opyl , l-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxy butyl, 2,3-dihydroxypropyl, 2-hydroxy-l -hydroxymethyl ethyl, 2,3-dihydroxybutyl,
  • alkoxy is intended to mean the radical -OR a , where R a is an alkyl group.
  • alkoxy groups include methoxy, ethoxy, propoxy, and the like.
  • the subject invention further provides methods for treating inflammatory and immune conditions by administering isolated enantiomeric compounds.
  • the isolated enantiomeric forms of the compounds of the invention are substantially free from one another (i.e., in enantiomeric excess).
  • the "R” forms of the compounds are substantially free from the "S” forms of the compounds and are, thus, in enantiomeric excess of the "S” forms.
  • "S” forms of the compounds are substantially free of "R” forms of the compounds and are, thus, in enantiomeric excess of the "R” forms.
  • the isolated enantiomeric compounds are at least about in 80% enantiomeric excess. In a preferred embodiment, the compounds are in at least about 90% enantiomeric excess.
  • the compounds are in at least about 95% enantiomeric excess. In an even more preferred embodiment, the compounds are in at least about 97.5% enantiomeric excess. In a most preferred embodiment, the compounds are in at least about 99% enantiomeric excess.
  • subject describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention can be provided.
  • Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes, chimpanzees, orangutans, humans, monkeys; and domesticated animals such as dogs, cats, horses, cattle, pigs, sheep, goats, chickens, mice, rats, guinea pigs, and hamsters.
  • an effective amount refers to an amount that is capable of preventing, ameliorating, or treating inflammation or an immune disease or condition. For instance, an effective amount enables at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in TNF-a and/or NO in a test sample of a subject in need of such treatment.
  • the subject method comprises administering, to a subject, an effective amount of isolated senkyunolide A (Sen A).
  • Sen A isolated senkyunolide A
  • the chemical structure of senkyunolide A is:
  • the subject method comprises administering, to a subject, an effective amount of isolated Z-ligustilide (Z-Lig).
  • Z-Lig isolated Z-ligustilide
  • Senkyunolide A and Z-ligustilide can be isolated from Ligiisticum chuanxiong (Chuanxiong) and its Chinese counterparts using isolation and bioassay-guided procedures as described herein.
  • the subject invention provides therapeutic methods for treating inflammatory and immune conditions by administering an effective amount of senkyunolide A and/or Z-ligustilide to control over-production of TNF-a and nitric oxide.
  • incubation of BV-2 cells with Sen A and Z-Lig at a concentration above 5 Lig/ml and 10 ⁇ g/ml, respectively significantly inhibits production of TNF-a under LPS induction.
  • the subject treatment method is capable of decreasing NO production and TNF- mRNA stability.
  • the compounds of the subject invention have cytoprotective effects. As shown in Example 7, the application of Z-ligustilide protects cells from apoptotic death during hydrogen peroxide-induced cell injury.
  • the methods of the subject invention can also be used to treat inflammation associated with infection, including, but not limited to, infections by viruses, bacteria, fungi, yeast, and other microbes. Additionally, the compounds of the subject invention can be used to treat inflammation mediated by a variety of factors including, but not limited to, interferons, interleukins, and environmental toxins.
  • the compounds of the subject invention can be used to treat inflammation caused by concurrent infection or immunological over-reaction to pathogen invasion, including but not limited to inflammation caused by viruses including Varicella zoster (also know f n as chickenpox or herpes zoster), herpes simplex, cytomegalovirus and herpes simplex virus-8 (also known as AIDS-associated Kaposi sarcoma virus).
  • viruses including Varicella zoster (also know f n as chickenpox or herpes zoster), herpes simplex, cytomegalovirus and herpes simplex virus-8 (also known as AIDS-associated Kaposi sarcoma virus).
  • the subject invention is used to treat neuralgia or neurasthenia associated with herpes zoster reactivation, commonly known as shingles.
  • the subject invention is used to treat chronic fatigue syndrome caused by viral infections.
  • the subject invention is used to treat ischemic stroke, rheumatoid arthritis, psoriasis, cardiovascular disease, cerebrovascular disease, inflammatory bowel disorder, septic shock, and/or graft vs. host rejection.
  • a patient who has been diagnosed with a pathological condition is administered a compound or composition of the subject invention.
  • the diagnosis may be made through an appropriate assay, including for example, the detection of a fever.
  • Other assays such as the culturing of tissue or other biological samples to identify pathogens can be used.
  • the subject method reduces TNF-a and/or nitric oxide production levels and/or destabilizes TNF-a mRNA in cells associated with inflammatory and immune conditions.
  • the subject method for treating inflammatory and immune conditions comprises:
  • Rj is alky] or CR 6; wherein R6 is alkyl, acyl, haloalkyl, alkylamino or hydroxylalkyl;
  • R3 and R4 are, independently, -H, acyl, halo, haloalkyl, amino, alkylamino, hydroxyl, alkyl, hydroxylalkyl, or -COOH;
  • R5 is — H, acyl, halo, haloalkyl, amino, alkylamino, alkyl, hydroxylalkyl, or -COOH;
  • the immune system marker(s) is selected from cytokines including TNF-a; NO; interferons such as Interferon-gamma (IFN- ⁇ ); the interleukin family such as Interleukin- 1 (IL-l ), Inter!
  • cytokines including TNF-a; NO; interferons such as Interferon-gamma (IFN- ⁇ ); the interleukin family such as Interleukin- 1 (IL-l ), Inter!
  • eukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Inteiicukin-5 (IL-5), Interleukin-6 (IL-6), Tnterleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL-9), Interleukin- 10 (IL-10), Interleukin- 11 (IL-l 1), Interleukin- 12 (IL-l 2), Interleukin- 13 (IL-l 3), Interleukin- 14 (IL-14), Interleukin- 15 (1L-15), Interleukin- 16 (IL-l 6), Interleukin-17 (IL-l 7), Interleukin-18 (IL-18), Interleukin-19 (IL-19), Interleukin-20 (IL-20), Interleukin-21 (IL-21), Interleukin-22 (IL-22), Interleukin-23 (IL-23), lnterleukin-24 (IL-24), Interleukin-25 (IL-25), Interleukin-26 (IL-26),
  • Immunoglobulins include IgG, IgM, IgD, IgE, IgA and subtypes such as for example IgGl , IgG2, IgG3, IgG4, IgAl, and IgA2. They further include molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
  • the immune system marker is selected from the group consisting of TNF-a, NO, IFN- ⁇ , IL-1, IL-2, IL -3, IL-5, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-14, IL-17, IL-18, IL-23, IL-24, IL-25, IL27, IL-32, G-CSF, M-CSF, MCP-1 , MIP-2, MIP-1 a, IgA, IgG, IgM, IgD and IgE.
  • the presence and/or level of the immune system markers can be determined from a sample of biological fluid obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient.
  • the immune system marker is measured for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition.
  • the immune system marker can be measured in a sample such as, blood, tissue, serum, plasma, urine, saliva, and tears.
  • the sample is a tissue sample.
  • the sample is a blood sample.
  • one of skill in the art would realize that some samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
  • the immune system marker can be determined by quantitative immunological detection methods, such as for example, enzyme-linked immunosorbant assay (ELISA), western blot, immunological assays, microarray, and radioimmunoassay.
  • quantitative immunological detection methods such as for example, enzyme-linked immunosorbant assay (ELISA), western blot, immunological assays, microarray, and radioimmunoassay.
  • a plurality of markers can be measured.
  • analysis of a plurality of markers may be carried out separately or simultaneously. Several markers may be combined into one test for efficient processing of multiple samples from a subject.
  • the presence and/or level of one or more markers can be determined multiple times over time to monitor the change of a subject's conditions. Such testing of multiple samples allows for the identification of changes in the marker over time. Increases or decreases in the marker, as well as the absence of change in levels, would provide useful information about the disease status that includes, but is not limited to, identifying the approximate time from onset of the event, the appropriateness of the subject therapy, the effectiveness of the subject therapy, identification of the severity of the event, identification of the disease severity, and identification of a future outcome.
  • the method of the present invention can be used in the treatment, or amelioration, of inflammatory symptoms in any disease, condition or disorder where immune and/or inflammation suppression is beneficial.
  • Inflammatory diseases, conditions or disorders in which the compounds and compositions of the present invention can be used to inhibit include, but are not limited to, unwanted immune reactions and inflammation including, but not limited to, arthritis (e.g., rheumatoid arthritis), ischemic stroke, and other diseases, conditions or disorders of the joints or musculoskeletal system in which immune and/or inflammation suppression is beneficial.
  • the subject method is also useful to treat or ameliorate inflammation associated with atherosclerosis; arteriosclerosis; atherosclerotic heart disease; reperfusion injury; cardiac arrest; myocardial infarction; vascular inflammatory disorders including cerebro-vascular disease (stroke); respiratory distress syndrome and other cardiopulmonary diseases, conditions or disorders where immune and/or inflammation suppression would be beneficial.
  • the subject method is also useful to treat or ameliorate inflammation associated with peptic ulcer; ulcerative colitis, Crohn's Disease, irritable bowel syndrome, other inflammatory bowel conditions, and other diseases, conditions or disorders of the gastrointestinal tract where immune inflammation suppression would be beneficial; hepatic fibrosis; liver cirrhosis and other hepatic diseases, conditions or disorders where immune and/or inflammation suppression would be beneficial; thyroiditis and other glandular diseases, conditions or disorders where immune and/or inflammation suppression would be beneficial; glomerulonephritis and other renal and urologic diseases, conditions or disorders where immune and/or inflammation suppression would be beneficial.
  • the subject method is useful to treat or ameliorate inflammation associated with post-traumatic inflammation; septic shock; infectious diseases where immune and/or inflammation suppression would be beneficial; inflammatory complications and side effects of surgery where immune and/or inflammation suppression would be beneficial; bone marrow transplantation and other transplantation complications and/or side effects where immune and/or inflammation suppression would be beneficial; inflammatory and/or immune complications and side effects of gene therapy, e.g., due to infection with a viral carrier; and inflammation associated with acquired immune deficiency syndrome (AIDS).
  • AIDS acquired immune deficiency syndrome
  • the subject method is also useful to inhibit macrophage or T cell associated aspects of an immune response that are not associated with inflammation.
  • the compounds and compositions arc able to inhibit macrophage or T cell activities including, but not limited to, macrophage antigen-presenting activity, macrophage cytokine production, T cell cytokine production, T cell adhesion activity, T cell proliferation, etc.
  • the compounds and compositions are useful to suppress or inhibit a humoral and/or cellular immune response.
  • the subject method is also useful to treat or ameliorate monocyte and leukocyte proliferative diseases, e.g., leukemia, by reducing the amount of monocytes and lymphocytes.
  • monocyte and leukocyte proliferative diseases e.g., leukemia
  • the subject method is further useful for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs, such as cornea, bone marrow, organs, lenses, pacemakers, natural and artificial skin tissue, and the like.
  • the subject method is also useful to treat or ameliorate inflammation associated with hypersensitivity; allergic reactions; asthma; systemic lupus erythematosus; collagen diseases and other autoimmune diseases such as multiple sclerosis, conditions or disorders in which immune and/or inflammation suppression is beneficial.
  • the subject method is also useful to treat or ameliorate inflammation associated with otitis and other otorhinolaryngological diseases, conditions or disorders where immune and/or inflammation suppression would be beneficial; dermatitis and other dermal diseases, conditions or disorders where immune and/or inflammation suppression would be beneficial; periodontal diseases and other dental diseases, conditions or disorders where immime and/or inflammation suppression would be beneficial.
  • the subject method also is useful to treat or ameliorate inflammation associated with herpes zoster (shingles); posterior uveitis; intermediate uveitis; anterior uveitis; conjunctivitis; chorioretinitis; uveoretinitis; optic neuritis; intraocular inflammation, such as retinitis and cystoid macular edema; sympathetic ophthalmia; scleritis; retinitis pigmentosa; immune and inflammatory components of degenerative fondus disease; inflammatory components of ocular trauma; ocular inflammation caused by infection; proliferative vitreoretinopathies; acute ischemic optic neuropathy; excessive scarring, for example, following glaucoma filtration operation; immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, conditions or disorders where immune and/or inflammation suppression would be beneficial.
  • herpes zoster shingles
  • posterior uveitis intermediate uveitis
  • the subject method is also useful to treat or ameliorate inflammation associated with autoimmune diseases and conditions or disorders where, both in the central nervous system (CNS) and in any other organ, immune and/or inflammation suppression would be beneficial; Parkinson's disease; complications and/or side effects from treatment of Parkinson's disease; AIDS-related dementia complex (HIV-related encephalopathy); Devic's disease; Sydenham chorea; Alzheimer's disease and other degenerative diseases, conditions or disorders of the central nervous system where immune and/or inflammation suppression would be beneficial; inflammatory components of strokes; post-polio syndrome; immune and inflammatory components of psychiatric disorders; myelitis; encephalitis; subacute sclerosing panencephalitis; encephalomyelitis; acute neuropathy; subacute neuropathy; chronic neuropathy; Guillaim-Barre syndrome; myasthenia gravis; pseudotumor cerebri; Down's Syndrome; Huntington's disease; amyotrophic lateral sclerosis; inflammatory components of central nervous system (CNS)
  • the subject method is useful to restore immune privilege at an immune privileged site which has lost its immune privilege such as brain, eye and testis.
  • the subject invention provides a therapeutic method by administering isolated compounds.
  • isolated refers to compounds that have been removed from any environment in which they may exist in nature.
  • isolated Sen A or isolated Z-Lig would not refer to the Sen A compound or the Z-Lig compound as it exists in Ligiisticum chuanxiong.
  • the compounds of the subject invention are at least 75% pure, preferably at least 90% pure, more preferably are more than 95% pure, and most preferably are more than 99% pure (substantially pure).
  • the present invention also provides for a therapeutic method by administering therapeutic or pharmaceutical compositions in a form that can be combined with a pharmaceutically acceptable carrier.
  • the compound may be, for example, isolated or substantially pure.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Particularly preferred pharmaceutical carriers for treatment of or amelioration of inflammation in the central nervous system are carriers that can penetrate the blood/brain barrier. As used herein carriers do not include the natural plants as they exist in nature.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel. sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the therapeutic composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated with traditional binders and carriers such as triglycerides.
  • compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • Such compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for local injection administration to human beings.
  • compositions for local injection administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the present invention also provides for the modification of the compound such that it is more stable once administered to a subject, i.e., once administered it has a longer time period of effectiveness as compared to the unmodified compound.
  • modifications are well known to those of skill in the art, e.g., microencapsulation, etc.
  • the amount of the therapeutic or pharmaceutical composition of the invention which is effective in the treatment of a particular disease, condition or disorder will depend on the nature of the disease, condition or disorder and can be determined by standard clinical techniques. In general, the dosage ranges from about 0.001 mg/kg to about 2 mg/kg.
  • suitable unit dosages may be between about 0.01 to about 5 mg, about
  • Such a unit dose may be administered more than once a day, e.g. two or three times a day.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rats is divided by six.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients, e.g., compound, carrier suitable for administration.
  • the method of administration can also be practiced consistent with traditional Chinese medicine practices.
  • the composition and dosage of the formulation that are effective in the treatment of a particular disease, condition or disorder will depend on the nature of the disease, condition or disorder by standard clinical techniques.
  • the traditional Chinese medicine in prescription amounts can be readily made into any form of drug, suitable for administering to humans or animals. Suitable forms include, for example, tinctures, decoctions, and dry extracts. These can be taken orally, applied through venous injection or mucous membranes.
  • the active ingredient can also be formulated into capsules, powder, pallets, pastille, suppositories, oral solutions, pasteurized gastroenteric suspension injections, small or large amounts of injection, frozen powder injections, pasteurized powder injections and the like. All of the above-mentioned methods are known to people skilled in the art, described in books and commonly used by practitioners of herbal medicine.
  • a tincture is prepared by suspending herbs in a solution of alcohol, such as, for example, wine or liquor. After a period of suspension, the liquid (the alcohol solution) may be administered for example, two or three times a day, one teaspoon each time.
  • a solution of alcohol such as, for example, wine or liquor.
  • a decoction is a common form of herbal preparation. It is traditionally prepared in a clay pot, but can also be prepared in glass, enamel or stainless steel containers. The formulation can be soaked for a period of time in water and then brought to a boil and simmered until the amount of water is reduced by, for example, half.
  • An extract is a concentrated preparation of the essential constituents of a medicinal herb.
  • the essential constituents are extracted from the herbs by suspending the herbs in an appropriate choice of solvent, typically, water, ethanol/water mixture, methanol, butanol, iso-butanol, acetone, hexane, petroleum ether or other organic solvents.
  • solvent typically, water, ethanol/water mixture, methanol, butanol, iso-butanol, acetone, hexane, petroleum ether or other organic solvents.
  • the extracting process may be further facilitated by means of maceration, percolation, repercolation, counter- current extraction, turbo-extraction, or by carbon-dioxide hypercritical (temperature/pressure) extraction.
  • the extracting solution may be further evaporated and thus concentrated to yield a soft extract (extractum spissum) and/or eventually a dried extract (extractum siccum), by means of spray drying, vacuum oven drying, fluid-bed drying or freeze-drying.
  • the soft extract or dried extract may be further dissolved in a suitable liquid to a desired concentration for administering or processed into a form such as pills, capsules, injections, etc.
  • Endotoxin lipopolysacharride, LPS
  • LPS lipopolysacharride
  • Murine microglia cell line BV-2 is maintained in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 10% FBS and 1% penicillin and streptomycin (Invitrogen Life Technologies) at 37°C in a humidified atmosphere with 5% C0 2 in warm air.
  • DMEM Dulbecco's modified Eagle's minimum essential medium
  • PC- 12 cells derived from a transplantable rat pheochromocytoma were obtained from American Type Culture Collection (ATCC Accession No. CRL-1721.1). The cells were maintained in F-12K Medium supplemented with 15% horse serum, 2.5% FBS, 1% penicillin, and streptomycin (Invitrogen Life Technologies, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% C(ATCC Accession No. CRL-1721.1). The cells were maintained in F-12K Medium supplemented with 15% horse serum, 2.5% FBS, 1% penicillin, and streptomycin (Invitrogen Life Technologies, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% C(
  • PBMC Human peripheral blood monocytic cells
  • the cell layer was diluted with phosphate buffered saline (PBS) in a ratio of 1 : 1.
  • PBS phosphate buffered saline
  • the diluted cells were overlaid on Ficoll-Paque slowly and centrifuged at 2300 rpm for 20 minutes for separation of mononuclear cells from erythrocytes.
  • the mononuclear cell layer was removed and washed with RPMI 1640 medium until the supernatant was clear.
  • the cells were finally resuspended in RPMI 1640 medium supplemented with 5% autologous serum and cultured for 1 hour.
  • the non-adherent cells were removed afterwards and the remaining adherent cells were further incubated for another 24 hours at 37°C in 5% carbon dioxide (C0 2 ).
  • PBMac primary blood macrophages
  • PCR primer sets for TNF-a and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) were as follows.
  • TNF-a upstream: 5 '-GGCTCC AGGCGGTGCT TGTCC-3 ' (SEQ ID NO: l); downstream: 5 '-AGACGGCG ATGCGGCTG ATG-3 ' (SEQ ID NO:2)), and GAPDH (upstream : 5'-ACCACAGTCCATGCCATCAC-3 ' (SEQ ID NO:3); downstream: 5'-TCCACCACCCTGTTGCTGTA-3 ' (SEQ ID NO:4).
  • the thermal cycling condition for PCR was 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The cycling reactions were repeated for 24 more cycles.
  • TNF-a mRNA The levels of TNF-a mRNA were determined by real-time RT-PCR (Roche 480II). 18S ribosomal RNA (18S) was used as an internal control. All of the real-time RT-PCR probes were obtained from the Universal Probe Library (Roche). All of the samples were performed in duplicate. The number of CT of the targeted gene was normalized to that of the 18S in each sample (ACj). The mRNA expression levels of the samples were relative to the mock-treated samples (AACT). The relative mRNA expression of the targeted genes was calculated by 2 "AAU and expressed as fold induction. Nitrite measurement
  • Nitrite levels in the culture media were determined using Griess reagent under manufacturer's instructions (Sigma Aldrich). Fresh culture media were used as blanks and the nitrite levels were determined by using a standard sodium nitrite curve.
  • Enzyme-Linked Immunosorbent Assay (ELISA) Enzyme-Linked Immunosorbent Assay
  • TNF-a level in cell culture supernatants, cells were seeded at 1 x 10 6 cells/ml in the volume of 0.5ml in 24 well plates. After incubation, the supernatants were collected and TNF-a levels were determined by ELISA according to the manufacturer's instructions (R&D Systems). MTT ' Assay for Cell Viability
  • PC- 12 cells (2.5 x 10 4 ) were seeded in 24-well culture plates. Cells were treated with Z-ligustilide, followed by treatment with hydrogen peroxide for the indicated time periods. The treated cells were incubated with 0.5 mg/ml MTT solution (Sigma Aldrich, St. Louis, MO) for 1 h at 37°C. The medium was discarded and 200 ⁇ isopropyl alcohol (IPP) was then added. After 15 min of incubation, the absorbance was measured at 570 nm.
  • MTT solution Sigma Aldrich, St. Louis, MO
  • Ligusticum chuanxiong (LCX) obtained from Purapharm International (ILK.) Ltd. is ground into powder. Methods for extracting bioactive compounds are shown in Figure 1 and illustrated as follows.
  • LCX powders are soaked in absolute ethanol at room temperature with continuous sonication for 30 minutes. After the extract is concentrated using a Rotavapor (Biichi), it is suspended in water and then partitioned sequentially with hexane, ethyl acetate and then butanol. Three fractions, namely LCX-l -Et-H, LCX-l-Et-EA and LCX-l-Et-Bu, are obtained.
  • LCX powders are heated in 70% ethanol under continuous reflux for 30 minutes. After the extract is concentrated using a Rotavapor (Biichi), it is suspended in water, and partitioned with dichloromethane (DCM). TWO fractions, namely LCX-2-Et-D (the DCM fraction) and LCX-2-Et-W (the water faction), are obtained.
  • DCM dichloromethane
  • LCX powders are boiled in water for 30 minutes. After the extract is concentrated using a Rotavapor (Biichi), it is partitioned sequentially with hexane, ethyl acetate and then butanol. Three fractions, namely LCX-3-W-H, LCX-3-W-EA and LCX-3 - W-Bu, are obtained .
  • LCX powders are soaked in water at room temperature with continuous sonication for 30 minutes. After the extract is concentrated using a Rotavapor (Buchi), it is partitioned sequentially with hexane, ethyl acetate and then butanol. Three fractions, namely LCX-4-W-H, LCX-4-W-EA and LCX-4-W-Bu, are obtained.
  • murine microglia cell line BV-2 is maintained in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 10% FBS and 1% penicillin and streptomycin (Invitrogen Life Technologies) at 37°C in a humidified atmosphere with 5%» C0 2 in warm air.
  • DMEM Dulbecco's modified Eagle's minimum essential medium
  • 0.1 M cells/ml BV-2 cells are seeded in 24-well plates (0.5 ml in each well). Cells are treated with 0.05% DMSO; 0.05% DMSO and 100 ng/ml LPS; or 100 ng/ml LPS and 50 ⁇ g/ml of an LCX extract for 18 hours, respectively. The culture supernatant is collected. The nitric oxide level is measured using the Griess reagent according to standard manufacturer's instructions (Sigma Aldrich), and assessed using the standard sodium nitrite curve.
  • LCX-l-Et-EA extract inhibits nitrite production in BV-2 cells.
  • the LCX-l-Et-EA extract is subjected to additional column chromatography.
  • the extract is further purified by reversed-phase high-performance liquid chromatography (HPLC) using a reversed-phase column Econosphere C18 lOu (250 x 22mm ID), with a detection wavelength at 21 0 nm and a gradient elution at a flow of 6 ml min "1 consisting of solvents (A) water and (B) acetonitrile of the following concentration: 0 - 15 min, 50% - 90% B; 16 - 20 min, 90% B; and 21 - 35 min, 50% B.
  • LCX-l -Et-EA-Sl is separated into 9 fractions as shown in Figures 2a) and 2b).
  • Nine fractions are obtained, including LCX-l-Et-EA-Sl -1 (Sl-1), LCX-l -Et-EA- SI -2 (SI -2), LCX-l-Et-EA-S l-3 (SI -3), LCX-l -Et-EA-Sl-4 (S I -4), LCX-l-Et-EA-Sl-5 (SI -5), LCX-l -Et-EA-Sl-6 (SI -6), LCX-l-Et-EA-Sl -7 (SI -7), LCX-l -Et-EA-S l-8 (S 1-8), and LCX-l-Et-EA-Sl -9 (SI -9).
  • senkyunolide A Sen A
  • Z-ligustilide Z-Lig
  • 0.1 M cells/ml BV-2 cells are seeded in 24-well plates (0.5 ml in each well). Cells are treated with 0.05% DMSO; 0.05% DMSO and 100 ng/ml LPS; 50 ⁇ ⁇ / ⁇ 1 Sen A / Z-Lig; or 100 ng/ml LPS and Sen A / Z-Lig at a concentration of 1 and 50 ⁇ g/ml for 1 hours, respectively.
  • the culture supernatant is collected.
  • the nitric oxide level is measured using the Griess reagent according to standard manufacturer's instructions (Sigma Aldrich), and evaluated using the standard sodium nitrite curve.
  • senkyunolide A Sen A
  • Z-ligustilide Z-Lig
  • 5 x 10 4 cells/ml BV-2 cells are seeded in 24-well plates (0.5 ml in each well).
  • Cells are treated with 0.05% DMSO; 0.05% DMSO and 100 ng/ml LPS; 50 ⁇ ig/ml Sen A / Z-Lig; or 100 ng/ml LPS and Sen A / Z-Lig at a concentration of 1 ⁇ , 5 ⁇ g/ml, 10 ⁇ g/ml, 25 ⁇ g/ml, and 50 ⁇ g/ml for 18 hours, respectively.
  • the culture supernatant is collected and the level of secreted TNF-a is measured by ELISA.
  • senkyunolide A Inhibition of TNF-a production, 10 3 cells/ml BV-2 cells are treated with senkyunolide A at various concentrations for 1 hour prior to the addition of LPS for another 6 hours. Total RNA of the treated samples is isolated and subjected to semi-quantitative RT-PCR assays using specific human TNF-a primers. The results, as shown in Figure 9, reveal that senkyunolide A inhibits TNF-a mRNA and iNOS expression.
  • senkyunolide A the effect of senkyunolide A on TNF-a mRNA stability is evaluated. Specifically, 10 5 cells/ml BV-2 cells are treated with Sen A at various concentrations for 1 hour prior to the addition of LPS for another 2 hours, and further incubated with 1 ⁇ ig/ml actinomycin for various time periods. Total RNA of the treated samples is isolated and subjected to real-time RT-PCR. The results, as shown in Figure 10, reveal that senkyunolide A destabilizes TNF-a mRNA.
  • Z-ligustilide suppresses cell death caused by hydrogen peroxide-induced cell injury.
  • PC-12 cells 2.5 x 10 4
  • the cells are treated with Z-ligustilide, followed by treatment with hydrogen peroxide to induce cell injury.
  • the treated cells are incubated with 0.5 mg/ml MTT solution (Sigma Aldrich, St. Louis, MO) for 1 h at 37°C.
  • the medium is discarded and 200 ⁇ isopropyl alcohol (IPP) is then added. After 15 min of incubation, the absorbance is measured at 570 nm.
  • Tumour necrosis factor alpha confers an invasive, transformed phenotype on mammary epithelial cells. J Cell Sci.

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Abstract

La présente invention concerne une méthode de traitement d'états de nature inflammatoire et immunitaire par modulation de la protéine TNF-α et de la production de monoxyde d'azote. Ladite invention concerne également l'administration d'un composé de formule (I):
PCT/IB2010/003050 2009-11-23 2010-11-08 Nouvelle méthode thérapeutique pour le traitement d'états inflammatoires et de troubles du système immunitaire Ceased WO2011070415A1 (fr)

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CA2781233A CA2781233A1 (fr) 2009-11-23 2010-11-08 Utilisation des lactones extraites de lingusticum chuanxiong dans le traitement des troubles inflammatoires et immunomodules
CN2010800621401A CN102844027A (zh) 2009-11-23 2010-11-08 治疗炎症和免疫系统障碍的新治疗方法
AU2010329604A AU2010329604B2 (en) 2009-11-23 2010-11-08 Novel therapeutic methods for treating inflammation and immune system disorders
JP2012539432A JP2013511506A (ja) 2009-11-23 2010-11-08 炎症および免疫系障害を治療するための治療方法
EP10835563.7A EP2504007B1 (fr) 2009-11-23 2010-11-08 Nouvelle méthode thérapeutique pour le traitement d'états inflammatoires et de troubles du système immunitaire
US13/005,301 US9655877B2 (en) 2009-11-23 2011-01-12 Therapeutic methods for treating inflammation and immune system disorders

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TWI625391B (zh) 2014-09-17 2018-06-01 國璽幹細胞應用技術股份有限公司 藁本內酯之應用
CN106474458B (zh) * 2017-01-05 2020-03-24 重庆医科大学 白介素-27在治疗艰难梭菌感染中的应用
JP7500074B2 (ja) * 2020-03-25 2024-06-17 学校法人自治医科大学 関節リウマチの再燃を予測する方法及びそれに用いるバイオマーカー群
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