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WO1996017605A1 - Utilisation de composes andrographolide dans le traitement ou l a prevention de la pathogenicite de certaines maladies - Google Patents

Utilisation de composes andrographolide dans le traitement ou l a prevention de la pathogenicite de certaines maladies Download PDF

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Publication number
WO1996017605A1
WO1996017605A1 PCT/US1995/015915 US9515915W WO9617605A1 WO 1996017605 A1 WO1996017605 A1 WO 1996017605A1 US 9515915 W US9515915 W US 9515915W WO 9617605 A1 WO9617605 A1 WO 9617605A1
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group
cells
cell
andrographolide
compound
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PCT/US1995/015915
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English (en)
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John G. Babish
Xingfang Ma
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Paracelsian, Inc.
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Priority to AU43763/96A priority Critical patent/AU4376396A/en
Publication of WO1996017605A1 publication Critical patent/WO1996017605A1/fr

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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
    • A61K31/365—Lactones

Definitions

  • This invention relates to the use of andrographolide compounds to treat or prevent the palhogenicity of various diseases and more specifically this invention relates to the use of andrographolide compounds to regulate expression of the cell division control enzyme p34 cdc2 kinase, cyclin B and c-Mos.
  • CDKs cyclin -dependent kinase's
  • p34 cdc2 kinase remains the most actively studied because of its central role in the control of cell division in both yeast and animal cells (Draetta, 1990; van den Heuvel and Harlow, 1993; Pines and Hunter, 1990; Norbury and Nurse, 1990).
  • p34 edc2 In normal resting cells p34 edc2 is not expressed or expressed at very low levels, but concentrations of p34 cdc2 increase as the cell enters and passes through Gl and the Gl/S transition. p34 cdc2 concentrations reach maximal levels in the S, G2 and M phases (Loyer et al.1994).
  • expression refers to the level of active protein of any particular protein; expression of a protein may be affected by a variety of factors including changes in transcription, translation and protein catalysis.
  • p34 cdc2 is the serine/threonine kinase subunit of - phase-promoting factor (MPF); active MPF triggers the G2/M transition in species ranging from yeast to humans (Brizuela et al.1989; Draetta, 1990).
  • MPF phase-promoting factor
  • P34 cdc2 functions in the control of the Gl/S transition and as well as the initiation of mitosis (Furukawa et al.1990; Krek and Nigg, 1991).
  • CDK proteins The role of CDK proteins is completely dependent upon their kinase activity in the cell cycle.
  • p34 cde2 kinase activity during the cell cycle is regulated primarily through post-translational modifications including cycles of phosphorylation and dephosphorylation (Ducommun et al.1991 ; Norbury et al.1991) and interactions with cyclins (Booher and Beach, 1987; Ducommun et al.1991; Williams et al.1992).
  • Intracellular compartment translocati ⁇ n has also been demonstrated to regulate the substrate availability of the p34 cdc2 protein (Williams et al.1992; Pines and Hunter, 1991).
  • p34 cdc2 The functioning of p34 cdc2 involves the coordination of all events relating to cell division.
  • p34 cdc2 is the central information processing protein.
  • information concerning the activities of the cell are sent to p34 c c2 and as long as these signals indicate proper functioning of the cell, movement through the cell cycle continues.
  • p34 cdc2 indicates a problem with the cell (e.g. DNA damage, microtubule disruption) progression through the cell cycle would be halted.
  • the block imposed on the cell cycle is at the Gl/S interphase.
  • An example of the coordination of cellular events with progression through the cell cycle is given in Figures la and lb.
  • Figure la illustrates the increases of p34 cde2 that are seen when the cell goes from a resting state into the cell cycle. Further increases in p34 c c2 are seen when the cell transforms into a tumor cell.
  • Figure lb shows the role of protein expression in the feedback mechanisms involved in control of the movement of the cell through the cell cycle.
  • the cellular expression of p34 cdc2 is governed by exposure to cytokines and hormones; the expression of p34 cdc2 is one signal to the cell to initiate the events of cell division. If the events of cell division are operating normally, p34 cdc2 levels will decrease (through specific proteolytic enzymes) and the cell will re-enter the resting state.
  • Proliferation of vascular endothelial cells is mediated through p34 cdc2 expression (Zhou et al.1994); such stimulation is associated with angiogenesis and functions in the pathology associated with occlusion of arteries following trauma such as angioplasty (Morishita et al.1994).
  • p34 cdc2 is also implicated in HIV-1 envelope-mediated cell death. It has been demonstrated that during HIV-1 mediated cytopathogenicity CD4+ T-cells were killed by a mechanism involving functional p34 cdc2 . Inhibition of the tyrosine phosphorylation of p34 cdc2 (a step performed in early Gl) resulted in an inhibition of the killing of CD4 + T- cells (Cohen et al.1993). Such a mechanism may also be involved in the cytopathogenicity of other viral diseases such as hepatitis or herpes.
  • p34 cdc2 may play a significant role in the pathology associated with Alzheimer's disease. Deposition and accumulation of amyloid A beta peptide in the cell is believed to be a primary biochemical defect in the ethology of Alzheimer's disease. Amyloid A beta peptide is derived from a larger precursor protein termed amyloid precursor protein (APP). Processing of APP is initiated through phosphorylation and is cell cycle dependent; this phosphorylation has been shown to be performed by p34 cdc2 both in vitro and in vivo (Suzuki et al.1994; Bau ann et al.1993). These finding suggest that alteration in the kinase activity or expression of p34 cdc2 may attenuate the pathogenesis of Alzheimer's disease.
  • APP amyloid precursor protein
  • p34 cdc2 (i) exists in biological systems as diverse as yeast and humans, (ii) serves a fundamental role in control and coordination of the cell cycle, and (iii) is involved in a wide variety of pathologic consequences of abnormal expression, reflects the enormous importance of p34 cdc2 in cell physiology. Therefore the capacity of a chemical to affect p34 cdc2 concentrations in a multiplicity of cell types (e.g. epithelial, mesothelial) while avoiding direct cytotoxicity has profound therapeutic implications.
  • the c-mos proto-oncogene product, Mos is a serine/threonine protein kinanse that controls the meiotic cell cycle in vetebrate oocytes.
  • mos acts through the mitogen-activated protein kinase (MAPK) cascade to induce cell transformation and that blocking the v-mos activation of that cascade prevents transformation.
  • MAPK mitogen-activated protein kinase
  • the plant Andrographis paniculata is widely used in China as a treatment of bacterial infections.
  • the herb and various organic extracts of the herb have demonstrated antibacterial (Thamlikitkul et al.1991), and filaricidal (Dutta and Sukul, 1982) properties. Additionally, the herb has been shown to be antithrombotic (Zhao and Fang, 1990), and inhibit stenosis and restenosis after angioplasty in the rat (Wang and Zhao, 1993). Because of the widespread use of this herb, a number of chemicals have been identified as secondary metabolites. Research reports of diterpenoids with biological activity have been published.
  • Diterpenoids have demonstrated hepatoprotective (Kapil et al.1993), immunostimulant (Puri et al.1993), choleretic (Shukla et al.1992), cell differentiation-inducing (Matsuda et al. 1994) and enzyme inducing (Choudhury et al.1987) and inhibiting activity (Choudhury et al.1987).
  • Diterpenoids comprise approximately 11 percent of the methanol extract of Andrographis paniculata (Handa and Shar a, 1990b).
  • the present invention involves the discovery of a group of compounds that can cause expression of p34 cdc2 kinase to decrease within a cell or group of cells. Extracts of dried plants were assayed for the ability to decrease expression of p34 cdc2 kinase. An extract of Andrographis paniculata was discovered to have this effect. Later analysis showed that it is the andrographolide compounds in the extract that causes the reduction of expression of p34 cde2 kinase.
  • Rl, R2 and R5 are one of the following: a hydroxyl group, a methyl group, a methoxy group, a methylene group, or an ether or ester linked sugar group;
  • R3 is a methyl group or a methylene group
  • R4 is a hydroxyl group or a carbonyl group
  • R6 is hydrogen or a hydroxyl group.
  • Andrographolide is known to have the following naturally occurring analogs: 14- epiandrographolide; isoandrographolide; 14-deoxy-12-methoxyandrographoIide; 12-epi- 14-12-meth ⁇ xyandrographolide; 14-deoxy-12-hydroxyandrographolide; and 14-deoxy-l l- hydroxyandrographolide.
  • This discovery has led to a series of novel methods for using these extracts and/or andrographolide compounds. After angioplasty the smooth muscle cells in the arteries and veins can proliferate and restrict flow. By administering an andrographolide compound to a human patient undergoing an angioplasty procedure, this over proliferation of smooth muscle cells can be avoided.
  • Andrographolide is effective at lowering expression of p34 edc2 kinase in cancer cells.
  • Lowering p34 cdc2 kinase expression can inhibit HIV-1 mediated cell death for patients with AIDS.
  • Lowering p34 cdc2 kinase expression can inhibit phosphorylation of amyloid precursor proteins which can delay the onset of Alzheimer's
  • Figures la and lb show schematic representations of the role of p34 c c in cell division.
  • Figure 2 shows a dose-response curve demonstrating the effect of the methanol extract of Andrographis paniculata on the hepatoma test cell (Wud23) wherein the median effective concentration for the downregulation of p34 cdc2 was 22 ⁇ g/mL in this experiment.
  • Figure 3 shows a dose-response curve demonstrating the effect of TGF ⁇ on the concentration of p34 cdc2 protein in the hepatoma test cell (Wud23) wherein the median effective concentration of TGF ⁇ was 0.007 ⁇ g/mL.
  • Figure 4 shows a dose-response curve demonstrating the effect of the methanol extract of Andrographis paniculata on three prostate cancer cell lines wherein the median effective concentrations of andrographolide were 22, 43 and 30 ⁇ g/mL for LNCaP, PC-3 and DU-145 cells, respectively.
  • Figure 5 shows a dose-response curve demonstrating the effect of andrographolide on the concentration of p34 cde2 protein in the hepatoma test cell (Wud23) wherein the median effective concentration of andrographolide in this experiment was 5 ⁇ g/mL.
  • Figure 6 shows the structure of andrographolide wherein the R groups indicate 5 positions modified in naturally occurring analogs.
  • Figure 7 shows the effect of andrographolide at various in vitro levels and human blood levels.
  • Figure 8 demonstrates the dose-response relationship for andrographolide inhibition of MCF-7 cell growth.
  • Figure 9 shows an anti-phosphotyrosine immunoblot of MCF-7 cell lysates following 24 hours of exposure to andrographolide.
  • Figure 10 shows the effect of andrographolide on cyclin-dependent kinases CDK2 and CDK4 and cyclin Dl.
  • Figure 11 shows an immunoblot of p34 cAr2 in MCF-7 whole cell lysates at 24 and 15 48 ours following exposure to 5 ⁇ g andrographolide/mL.
  • Figure 12 shows an anti-cyclin B immunoblot of cyclin B in MCF-7 whole cell lysates after exposure to 5 ⁇ g andrographolide/mL.
  • Figure 13 shows an anti-Rb protein immunoblot demonstrating the effect of andrographolide on the status of Rb protein phosphorylation in MCF-7 cell lysates after 20 48 hours of exposure.
  • Figure 14 shows an anti-CDK7 immunoblot indicating that andrographolide in concentrations up to 10 ⁇ g/mL had no effect on the concentration of CDK7 protein in MCF-7 cell lysates.
  • Figure 15 shows an immunoblot of c-Mos nuclear expression in MCF-7 cell lysate 5 following 72 hours of exposure to 5 ⁇ g andrographolide/mL.
  • Figure 16 shows a proposed model for the role of c-mos action on cyclin B phosphorylation status and protein concentration.
  • Figure 17 shows an immunoblot of A431 cell cytosol after 24 hours of treatement with andrographolide.
  • Figure 18 shows a dose-response relationship for andrographolide and inhibition of
  • Figure 19 shows synergy demonstrated between AZT and andrographolide in the inhibition of HIV- 1 replication.
  • Figures la and lb show schematic representations of the role of p34 cdc2 in cell division.
  • p34 cdc2 exists at low concentrations. When stimulated to divide the concentration of p34 cdc2 increases dramatically in the cell.
  • Cancer cells are characterized by high concentrations of p34 edc2 compared to resting cells. Stimulation of p34 cdc2 expression begins a sequence of events that in normal cells results in the elimination of enzymatically active p34 cdc2 kinase. This sequence of events is mediated through p53 and p2I .
  • the bioassay used here to screen the methanol extracts of Dried Plants is not a measure of the kinase activity associated with the p34 cdc2 enzyme as described by others(Ducommun and Beach, 1990). This kinase activity, usually measured with histone HI as the substrate, is maximal at the G2/M transition and is associated with the p34 edc2 /cyclin B complex. Rather the p34 cdc2 assay used in the following examples measures the extent of expression of the holoenzyme, reflecting progression through Gl and the Gl/S transition.
  • the assay used in the examples is not specific as a screen for chemicals modifying the transcription of p34 c c .
  • the assay does, however, provide an excellent multi-functional screen for the capacity of a test chemical or extract of a natural product to decrease the cellular concentration of the p34 cdc2 protein.
  • the use of this assay as a biological endpoint increases the probability of identifying compounds in complex libraries with unique mechanisms of action.
  • a methanol extract of the leafy portion of the plant Andrographis paniculata was tested for the capacity to downregulate p34 cdc2 protein or inhibit cell growth in an animal hepatoma cell line and in human breast, and liver cancer cells.
  • the extract demonstrated both the ability to downregulate the p34 cdc2 protein or to inhibit cell growth over seven days.
  • the activity of the extract was dose-related and median effective concentrations were lowest for breast cancer cells and highest for woodchuck hepatoma cells. Both downregulation of the p34 cdc2 protein and inhibition of growth occurred at concentrations that produced no visible signs of cytotoxicity.
  • Methanol Extract of Andrographis paniculata The leafy portion of the plant
  • Andrographis paniculata was harvested in Zhejing province in the Peoples Republic of China during the summer. The harvested material was allowed to air dry until the leaves crumbled to the touch. A course powdery material was produced from the dried plant and extracted with boiling methanol under reflux for 24 hours. This extraction procedure was repeated and the two methanol solutions were pooled and the solvent was evaporated under vacuum at ambient temperature. The resultant powder was placed in a screw-cap vial and stored at room temperature in the dark until weighed for use.
  • the cell line to be tested was set-up at a 1: 10 split in a T-75 flask in MEME + NEAA supplemented with sodium pyruvate and 10% FBS-HI (maintenance medium). Any cell line may be used for the testing.
  • the cell lines used in this example were an hepatocellular carcinoma derived from a Woodchuck (designated Wud23), MCF-7 (human breast), HepG2 (human liver) LNCAP (human prostate).
  • AH human cells were obtained from American Type Culture Collection (Bethesda, MD). Approximately 4 to 5 days later (80 to 90% confluence) the cells were harvested, counted and adjusted to 2 x 10 4 cells/mL in maintenance media with 0.5% FBS-HI.
  • Cells were plated (1 x 10 3 cells/well) in a 96- well flat bottom tissue culture plate with 50 mL media/well.
  • the plant extract (dissolved in DMSO) was added to the wells at doses of 50, 10, 1 , 0.5, and 0.1 mg/mL in 50 L of maintenance medium with 0.5% FBS-HI. Plates were incubated at 5% CO 2 , 37C, and 95% humidity for 48 hours (woodchuck hepatoma) or 7 days (human breast and liver cells) and then harvested.
  • TGF ⁇ was used as a positive control as an example of a chemical known to specifically downregulate p34 f ⁇ r2 concentrations; vincristine and methotrexate were administered to the cell cultures as negative controls cchheemmiicals whose mechanism of action does not involve direct downregulation of p34 c ⁇ 2 levels
  • Analysis of p34 r content of the cells The p34 cdc7 content of the cells was quantified using an ELISA assay. Results were tabulated as fmol p34 cdc2 /10 3 cells plated. The p34 crfc2 content of the treated cells was compared to the p34 cA2 content of the six
  • __tc_ ⁇ control wells run on the same plate.
  • the percent of downregulation of p34 observed in the test wells (triplicate) at each dose was computed relative to the controls on the same plate.
  • the polyclonal antibody to p34 kinase was obtained from Pederson Biotech Resources (Ithaca, NY), and alkaline phosphatase or peroxidase-conjugated anti-rabbit IgG were obtained from commercial sources (e.g. Transduction Laboratories, Lexington, KY; Oncogene Science, Inc. Manhasset, NY; Sigma, St. Louis, MO).
  • Bovine serum albumin Sigma #A-3350 (St Louis, MO)
  • Reagents A. Sodium Carbonate buffer, 0.1 M, pH 9.6. - 1) Mix 71.3 mL of 1 M NaHCO3 and 28 mL of 1 M Na2CO3; 2) Add 800 mL dd H2O; 3) Adjust pH to 9.6 and Qs to 1.1 L
  • E. Prep buffer 25 mM Tris-HCl, pH 8.0 with 10 mM MgC12, 15 M EGTA, 0.1 % Triton X-100, 0. 1 mM PMSF, 0.1 M sodium fluoride, 60 mM b- glycerophosphate, 15 mM p-nitrophenylphosphate, 0.1 mM sodium orthovanidate, 1 mg/mL leupeptin, 10 mg/mL soybean trypsin inhibitor, 1 mg/mL aprotinin and 10 mg/mL tosyl-phenylalanine.
  • Assay buffer 50 mM Tris-HCl, pH 7.4 with 10 mM MgC12, 1 mM DTT.
  • Citrate buffer - 1) Add 9.6 g citric acid (MW 192.12) to 950 mL ddH2O; 2) Adjust pH to 4.0 with 5 M NaOH and store at 4C.
  • Assay Plates were washed in washing buffer and adhering cells were lysed and diluted in prep buffer and mixed 1 : 1 with blocking buffer. This was added to the wells of a plate and incubated for 1 hr at 4C with slow constant shaking. Plates were washed 3x with washing buffer and lx with assay buffer. Two-hundred mL of primary antibody at a dilution of 1: 1000 in blocking buffer was added to each well and incubated for 2 hr at 4C. Plates were washed 3x with washing buffer.
  • a positive response is defined as a dose-related decrease in p34 erff2 content of the cells in excess of 50%. Calculation of median effective doses was performed on probit transformed percentages versus log concentration of the extract. The probit transformation was used to linearize the sigmoidal response data.
  • the methanol extract of Andrographis paniculata produced a significant dose- related downregulation of p34 rrfc2 protein within 48 hours in the Wud23 woodchuck hepatoma cell line as demonstrated by the data in Figure 2.
  • the median effective concentration was approximately 30 ⁇ g/mL (average of two replicate studies).
  • Excellent dose-related p34 crfc2 downregulation was also observed for human breast and liver cancer cells that correlated with growth inhibition. Both downregulation of the p34 erfc2 protein and inhibition of growth occurred at concentrations of extract that produced no visible signs of cytotoxicity.
  • TGF ⁇ caused a downregulation of p34 edc2 protein in the Wud23 cells within 48 hours ( Figure 3).
  • vincristine nor methotrexate produced a dose-related decrease in p34 cdc2 protein at concentrations that were not cytotoxic.
  • a methanol extract of the leafy portion of the plant Andrographis paniculata was tested for the capacity to inhibit cell growth in three human prostate cancer cell lines, LNCaP, PC-3 and DU-145. Median effective doses for the three cell lines, respectively, were 22, 43 and 30 ⁇ g/mL. This inhibition was achieved with no visible signs of cytotoxicity.
  • Human Tumor Cell Lines Human prostate adenocarcinoma cell lines LNCap, PC-3 and DU-145 were purchased from the American Type Tissue Culture Collection. All cell lines were propagated as monolayers in RpMI-1640 containing 5% FCS, 5% NuSerum IV, 20 nM HEPES, 2 mM L-glutamine at 37C in a 5% CO2 humidified atmosphere. The doubling times for the cell lines ranged between 34 and 42 hours.
  • EXAMPLES 1 and 2 demonstrate that the capacity of a substance to downregulate p34 cdc2 kinase in tumor cells correlates with the inhibition of growth of tumor cells. This result is mechanistically sound since the movement through the cell cycle is controlled and coordinated by p34 cdc2 kinase.
  • the diterpenoid lactone andrographolide was tested for the capacity to downregulate p34 edc2 protein in an animal hepatoma cell line. In this test system andrographolide was able to downregulate the p34 cdc2 protein in a dose-related manner within 48 hours. The median effective concentrations was estimated from a log-probit regression as 5.0 ⁇ g/mL. Downregulation of the p34 edc2 protein occurred at concentrations that produced no visible signs of cytotoxicity.
  • Andrographolide was purchased from Aldrich Chemicals
  • Bioassay Procedure The procedure described in EXAMPLE 1 was used with only Wud23 cells and the exposure period was 48 hours. Concentrations of andrographolide tested included 50, 25, 12.5, 10, 5, 2,5, 6.25, 1.25, 0.625, 0.5, 0.25, 0.125, 0.1 , 0.05, 0.025 and 0.0123 mg/well.
  • a positive response was defined as a dose-related decrease in p34 cdc2 content of the cells in excess of 50%.
  • Andrographolide produced a significant dose-related downregulation of p34 cdc2 protein within 48 hours in the Wud23 woodchuck hepatoma cell line as demonstrated by the data in Figure 5.
  • the median effective concentration was approximately 5.0 mg/mL or 6 times more potent than the methanol extract (30 mg/mL).
  • Tyrosylphosphoproteins at 85, 45 and 33 kDa were completely eliminated from anti-phosphotyrosine immunoblots of cell lysates of MCF-7 cells treated with 5 ⁇ g/mL of andrographolide for 24 hours.
  • Neither CDK2 or CDK4 concentrations in whole cell lysates were affected at 5 or 10 ⁇ g andrographolide/mL over the 72 hours of the experiment.
  • Cyclin Dl exhibited a dose-related decrease in concentration in whole cell lysate at 5 and 10 ⁇ g andrographolide/mL following 72 hours of exposure.
  • the human breast adenocarcinoma cell line MCF-7 was purchased from the American Type Tissue Culture Collection. The cell line was propagated in Eagle's minimum essential medium with non-essential amino acids and 5% serum. The MCF-7 cell line retains several characteristics of differentiated mammary epithelium including ability to process estradiol via cytoplasmic estrogen receptors and is capable of forming domes. The MCF-7 cells were cultured at 37°C in a 5% CO 2 balanced air environment. Cells were subcultured by centrifuging (1000 X g for three minutes) and suspending in 100 L of fresh media to a cell density of 5.0 x 10 s cell/ L.
  • PBS phosphate buffered saline
  • Tris buffer pH 8.0
  • MCF-7 Median Inhibitory Concentration
  • DMSO dimethyl sulfoxide
  • Control cells received only 100 ⁇ L of the DMSO medium solution without drug.
  • the formazan product of MTT reduction was dissolved in DMSO, and absorbance was measured using an MR5000 microtiter plate reader (Dynatech Laboratories Corp, Chantilly, VA). Absorbance at 570 nm was converted to cells per well based upon a standard curve developed from visual cell counts and MTT reduction. Quantification of viable cells was performed when control wells reached approximately 80 to 90 % confluence (usually within seven days).
  • Percent inhibition of cell growth relative to controls was plotted versus log dose and estimates of median inhibitory concentrations of andrographolide were made graphically. Four replicates of the dose-response experiment were performed on each of two separate days. Estimates of the IC 50 of andrographolide for MCF-7 cells did not differ between the two separate determinations.
  • DNA content was measured using a FACScan (Becton Dickinson, San Jose, CA) flow cytometer. Data acquisition and analysis were carried out using CellFit software (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Statistical analysis of cell cycle variables was done using chi-square analysis (JMP Version 2 Software, SAS Institute, Inc., Cary, NC).
  • Electrophoretic transfer of proteins from polyacryiamide gels to nitrocellulose sheets procedure and some applications.
  • Membrane filters were blocked by incubating in TBS (50 M Tris, 150 mM NaCl, pH 7.5) containing 5% commercial nonfat dry milk for 30 min at room temperature and incubated 2 hour with 5 ⁇ g/mL antibody in TBST (0.05% Tween 20 in TBS).
  • TBS 50 M Tris, 150 mM NaCl, pH 7.5
  • TBST 0.05% Tween 20 in TBS
  • Molecular weights of immunostained proteins were estimated by adding molecular weight standards to reference lanes and by staining the membrane filters with amido Back 10 B.
  • the membranes were incubated for 2 hours at room temperature with alkaline phosphatase-conjugated anti-rabbit IgG diluted 1:1000 in TBST and developed for 15 minutes.
  • the immunoblots were translated into TIFF-formatted files using a Microtech 600GS scanner and quantified using Scan Analysis software (BIOSOFT, Cambridge, UK).
  • Figure 8 demonstrates the dose-response relationship for andrographolide inhibition of MCF-7 cell growth.
  • andrographolide was able to inhibit growth of MCF-7 cells.
  • the median inhibitory concentration was estimated to be 0.82 ⁇ g/mL and the 95% confidence interval from the four replicates within one day was 0.32 to 1.3 ⁇ g andrographolide/mL.
  • Anti-phosphotyrosine - Figure 9 shows an anti-phosphotyrosine immunoblot of
  • MCF-7 cell lysates following 24 hours of exposure to andrographolide.
  • lane C there were a total of 31 tyrosylphosphoproteins present in the MCF-7 whole cell lysate.
  • Lanes A and B respectively 10 and 5 ⁇ g andrographolide/mL, indicate that ten of these 31 tyrosylphosphoproteins were reduced by andrographolide treatment.
  • the apparent molecular weights of the tyrosylphosphoproteins affected by andrographolide were 91 , 87, 85, 79, 69, 53, 45, 33, 19, and 12 kDa.
  • CDK2, CDK4 and cyclin Dl - Figure 10 shows the effect of andrographolide on 5 cyclin-dependent kinases CDK2 and CDK4 and cyclin Dl.
  • the immunoblots indicate no effect of andrographolide on CDK2 or CDK4 expression at 5 [B] or 10 [A] ⁇ g/mL over the 72 hours of the experiment. Cyclin Dl expression, however, demonstrated a dose- dependent decrease.
  • p34 « - « _ figure 11 shows an immunoblot of p34"' c2 in MCF-7 whole cell lysates at 10 24 and 48 ours following exposure to 5 ⁇ g andrographolide/mL. Both the concentration and phosphorylation status of p34 cA:2 were reduced at 24 and 48 postdosing hours in MCF-7 whole cell lysates exposed to 5 ⁇ g andrographolide/mL. As seen in Figure 11, control samples exhibited three staining bands with the anti-p34 c2 antibody. The lowest band represents the nonphosphorylated form, while the two upper bands represent 5 increasing phosphorylation.
  • Cyclin B - Figure 12 shows an anti-cyclin B immunoblot of cyclin B in MCF-7 whole cell lysates after exposure to 5 ⁇ g andrographolide/mL.
  • p34"' c2 both the concentration and phosphorylation status of cyclin B were reduced at 24 and 48 postdosing hours in MCF-7 whole cell lysates exposed to 5 ⁇ g andrographolide/mL ( Figure 12).
  • p34 edc2 it was the nonphosphorylated form (lower band) of 5 cyclin B than was more demonstrably affected at the 24-hour time period.
  • Rb protein - Figure 13 is an anti-Rb protein immunoblot demonstrating the effect of andrographolide on the status of Rb protein phosphorylation in MCF-7 cell lysates after 48 hours of exposure. Relative to the control, concentrations of 5 or 10 ⁇ g andrographolide/mL exhibited a dose-related decrease in the extent of phosphorylation of 0 Rb protein with increasing drug.
  • CDK7 - The anti-CDK7 immunoblot in Figure 14 indicates that andrographolide in concentrations up to 10 ⁇ g/mL for 24 hours had no effect on the concentration of CDK7 protein in MCF-7 cell lysates. This result indicates that the decrease in phosphorylation status of p34 crfc2 was not due to a decrease in the expression or 5 concentration of the CDK7 component of CAK.
  • Mos - Figure 15 shows an immunoblot of c-Mos nuclear expression in MCF-7 cell lysate following 72 hours of exposure to 5 ⁇ g andrographolide/mL. At this test concentration, expression of c-Mos forms p35, p37 and p40 in the nuclear fraction of MCF-7 cells was decreased 48% in total at 72 postdosing hours.
  • the diterpenoid lactone andrographolide was tested for its capacity to affect the expression of Mos associated proteins in A431 Cells.
  • this human epidermoid carcinoma test system andrographolide was able to significantly alter the degree of expression of c-Mos and v-Mos associated proteins within 24 hours.
  • EC J0 values of 0.28, 0.74 and 0.68 ⁇ g andrographolide/mL were obtained, respectively, for c-Mos associated proteins p35, p37, and p40.
  • v-Mos associated proteins p29 and p43 exhibited EC J0 values for downregulation of 6 and 3 ⁇ g andrographolide/mL, respectively.
  • Anti-Mos antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Andrographolide was purchased from Aldrich Chemicals (Milwaukee, WI) and provided at a purity greater than 98 percent. All other chemicals were purchased from Sigma (St. Louis) and were the highest purity commercially available.
  • the human epidermoid carcinoma cell line A431 was purchased from the American Type Tissue Culture Collection. The cell line was propagated in Dulbecco's modified Eagle's medium with 4.5 g/L glucose, 90%; fetal bovine serum, 10%. The cell line was originally derived from an 85-year old female. The A431 cells were cultured at 37 ⁇ C in a 5% CO 2 balanced air environment. Cells were subcultured by centrifuging (1000 X g for three minutes) and suspendening in 100 mL of fresh media to a cell density of 5.0 x 10 cell/mL.
  • PBS phophate buffered saline
  • Membrane filters were blocked by incubating in TBS (50 M Tris, 150 mM NaCl, pH 7.5) containing 5% commercial nonfat dry milk for 30 min at room temperature and incubated 2 hour with 5 mg/mL anti-Mos in TBST (0.05% Tween 20 in TBS).
  • TBS 50 M Tris, 150 mM NaCl, pH 7.5
  • Molecular weights of immunostained proteins were estimated by adding molecular weight standards to reference lanes and by staining the membrane filters with amido Back 10 B.
  • the membranes were incubated for 2 hour at room temperature with alkaline phosphatase-conjugated anti-rabbit IgG for CDKl or anti- mouse IgG for PCNA diluted 1 : 1000 in TBST and developed for 15 minutes.
  • the immunoblots were translated into TIFF-formattted files using a microtech 600GS scanner and quantified using Scan Analysis software (BIOSOFT, Cambridge, UK).
  • Figure 17 shows an immunoblot of A431 cell cytosol after 24 hours of treatement with andrographolide.
  • A431 cell lysate showed five immunoreactive bands with anti-Mos antibody (Figure 17). These bands exhibited apparent molecular weights of 43, 40, 37, 35 and 29 kDa. All of the immunoreactive bands appeared as doublets in controls indicating phosphorylation.
  • p40, p37 and p35 immunoreactive bands were consistent with c-Mos forms, while immunoreactive bands at p43 and p29 were consistent with v-Mos forms.
  • Andrographolide was able to significantly alter the degree of expression of c-Mos and v-Mos associated proteins within 24 hours. EC 50 values of 0.28, 0.74 and 0.68 g andrographolide/mL were obtained, respectively, for c-Mos associated proteins p35, p37, and p40. v-Mos associated proteins p29 and p43 exhibited EC 50 values for downregulation of 6 and 3 mg andrographolide/mL, respectively (Table 5.1).
  • Andrographolide is a naturally occurring diterpene lactone that functions biochemically as a kinase inhibitor. Andrographolide demonstrated the capacity to inhibit the replication of HIV- 1 in peripheral blood monocytes (PBMC) as measured by the degree of inhibition of reverse transcriptase (RT) activity.
  • the IC 50 of andrographolide for this effect was 640 ng/mL. Toxicity of andrographolide to normal, noninfected PBMC was not observed at concentrations of andrographolide of 10,000 ng/mL, providing a minimum therapeutic index in excess of 15-fold (10,000/640). Further testing of andrographolide indicated that andrographolide synergized with AZT in the inhibition of HIV-1 replication in PBMC as measured by reverse transcriptase activity.
  • reverse transcriptase activity was used as a marker for the replication of the HIV-1WEJO virus, reverse transcriptase is not considered the site of action of the test compound andrographolide.
  • the previous EXAMPLES have demonstrated that andrographolide inhibits the phosphorylation of p34 ⁇ fc2 kinase. Evidence to date, included in previous examples, indicates that this may be due to the inhibition of c-Mos activation by andrographolide which in turn would result in the inhibition of the phosphorylation of Mek-1 (Pha et al., 1995).
  • Mek-1 activation results in the increase in phosphorylation of p34 e ⁇ fc2
  • inhibition of Mek-1 phosphorylation would inhibit HIV-1 cytopathicity in a manner described by Cohen et al. (Science, 1992, 256:542-545).
  • the cellular target of andrographolide in the inhibition of cancer cells and HIV-1 appears logically to stem upstream of p34 ft,c " phosphorylation to inhibition of c-Mos activation.
  • c-Mos inhibition affords a mechanistic explanation for the effects of andrographolide on both p34 crfc2 and cyclin Bl .
  • the inhibition of HIV-1 replication infers that inhibition of c-Mos is critical in the control of HIV-1 induced cytopathicity of infected T-cells.
  • This EXAMPLE teaches that inhibition of critical cellular serine/threonine kinases, such as c-Mos allow for the control of HIV-1 replication in normal, human PBMC. As such, it is the first demonstration that this group of kinases (serine/threonine) are critical in the replication of the AIDS virus. This cellular target may have utility in the control of other viral infections that result in cytopathicity as a result of overexpression of p34 cA2 or cyclin Bl and the stimulation of the cell cycle.
  • Andrographolide was synthesized by Aldrich as previously referenced (EXAMPLE 5). 3*-deoxy-3'azidothymidine (AZT) was obtained from Burroughs Wellcome (Research Triangle Park, NC). All other standard laboratory chemicals were purchased from commercial suppliers as described in previous EXAMPLES or as indicated and were of the highest purity available.
  • H9 cells were used to propagate HIV-1WEJO and these reagents were obtained through the AIDS Research and Reference Reagent Program, AIDS Program, National Institute of Allergy and Infectious Diseases, Bethesda, MD. Cells were maintained in RPMI 1640 medium containing 10% fetal calf serum and 40 IU of interleukin-2 per mL (for PBMC) in a humid 5% CO 2 atmosphere.
  • PBMC interleukin-2 per mL
  • fresh human mononuclear cells were prepared from the blood of healthy donors by Ficoll gradient centrifugation.
  • the cells were suspended in RPMI 1640 medium containing 5% human a type AB+ serum and were cultivated on hydrophobic membranes (Teflon bags; 3x10* cells per mL). After 24 hours the PBMC were infected by the addition of HIV-lWEJO-infected lymphocytes (5xl0 4 infected PBMC per mL). Forty-eight hours after infection, the cells were transferred to 24-well plates (Falcon) , and the nonadhering cells were quantitatively removed. The adherent cells were further grown in a culture volume of 1.3 mL, and fresh medium was added every week.
  • IC 50 Determination of Median Inhibitory Concentrations (IC 50 ): A total of 105 cells were infected with virus stocks at 0.01 PFU per cell, seeded onto 24-well plates and incubated for 72 to 96 hours in the presence of 0, 0.0671, 0.2097, 0.6554, 2.0480, 6.4, or 20 ⁇ g andrographolide/mL dissolved in dimethyl sulfoxide (DMSO) and diluted in cell culture medium. For a positive control, infected cells were also incubated with 0, 0.0034, 0.0105, 0.0328, 0.1024, 0.3200 or 1.0 ⁇ g AZT/mL.
  • DMSO dimethyl sulfoxide
  • Reverse Transcriptase Assay In the endogenous reverse transcriptase assay, wherein the viral RNA functioned as the template, the reaction mixture (50 ⁇ L) consisted of 50 mM Tris-HCl (pH 8.4), 2.5 mM MgC12, 100 mM KCl, 4 mM dithiothreitol, 30 ⁇ g of bovine serum albumin per mL, 0.5 mM EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], and 0.01 % (wt/vol) Triton X-100.
  • WASH 0.85% NaCl. Prepare in advance and store at room temperature 5 up to one year.
  • EXTRACTION 0.1 M Na 2 HPO 4 :Ethanol (1 : 1). Prepare 0.1 M Na 2 HPO 4 in advance and store up to six months at room temperature. Mix with ethanol on the day of the assay at room temperature. Discard unused mixture.
  • IC50 Median Inhibitory Concentration (IC50) of Andrographolide against HIV- 1WE O: In this in vitro human HIV-1 test system, andrographolide was able to inhibit growth of HIV-1 WEJO cells as measured by inhibition of reverse transcriptase activity. 5 The median inhibitory concentration was estimated to be 640 ng/mL. The dose-response relationship for andrographolide inhibition of HIV- 1 WEJO cell growth is presented in Figure 18. The graph represents the probit of the percent of inhibition of reverse transcriptase in human PBMC infected with HIV- 1 WEJO.
  • Synergy of AZT and Andrographolide As seen in Figure 19, at the lower range of concentration tested for AZT and andrographolide there was synergy was demonstrated in excess of 50%. At higher concentrations of both drugs antagonism was observed. The antagonistic effect of the two drugs at the higher concentrations tested may be due to the fact that reverse transcriptase was used as the response variable in this study. Based upon mechanistic studies in tumor cells, andrographolide would inhibit viral replication at a pre-reverse transcriptase stage.
  • Toxicity of Andrographolide to Normal PBMC Overt toxicity of andrographolide to normal, noninfected PBMC was not observed at concentrations of andrographolide up to 10,000 ng/mL, providing a minimum therapeutic index in excess of 15-fold (10,000/640).
  • reverse transcriptase activity was used as a marker for the replication of the HIV- 1 WEJO virus, reverse transcriptase is not considered the site of action of the test compound andrographolide.
  • Other biochemical studies c.f. previous EXAMPLES have demonstrated that andrographolide inhibits the phosphorylation of p34 c ⁇ r2 kinase.
  • Mek-1 activation results in the increase in phosphorylation of p34 edc2
  • inhibition of Mek-1 phosphorylation would inhibit HIV-1 cytopathicity in a manner described by Cohen et al. (Science, 1992, 256:542-545).
  • the cellular target of andrographolide in the inhibition of cancer cells and HIV-1 appears logically to stem upstream of p34 ⁇ ' c2 phosphorylation to inhibition of c-Mos activation.
  • c-Mos inhibition affords a mechanistic explanation for the effects of andrographolide on both p34 c ⁇ r2 and cyclin Bl.
  • the inhibition of HIV-1 replication infers that inhibition of c-Mos is critical in the control of HIV-1 induced cytopathicity of infected T-cells.
  • This EXAMPLE teaches that inhibition of critical cellular serine/threonine kinases, such as c-Mos allow for the control of HIV-1 replication in normal, human PBMC.
  • this group of kinases serine/threonine
  • This cellular target may have utility in the control of other viral infections that result in cytopathicity as a result of overexpression of p34 ⁇ fc2 or cyclin Bl and the stimulation of the cell cycle.
  • the dose regimens described are suggested based only on the information developed on the absorption, distribution and efficacy or andrographolide itself. Distribution and metabolism of a chemical may be profoundly affected through the simultaneous administration of other drugs, individual genetics, age, other diseases of the patient and the progression of the disease itself. Therefore, the doses described in this section represent ideal cases and are to serve only as a guide to the physician. The importance of the continued monitoring of the patient by the physician should not be ignored. When dictated by information concerning the patients response to therapy, the physician should either decrease or increase the dose or duration of the recommended therapy.
  • Smooth muscle cell stenosis following angioplasty The inhibition of smooth muscle cell proliferation following angioplasty can be achieved by dosing the patient with 1 to 15 mg andrographolide/kg body weight, three times per day starting two days before the operation and continuing for 30 days following the operation. Since phenobarbital metabolism is inhibited by the administration of andrographolide, barbiturate anesthetics should be avoided during the angioplasty.
  • Cancer The inhibition of the proliferation of cancer cells can be prevented by dosing the patient with 2.5 to 15 mg andrographolide/kg body weight, two or three times per day continuously until indications of metastasis or tumor growth are negative. Due to the relative low toxicity of andrographolide demonstrated in animal studies, this dose regimen may be continued for months without interruption.
  • Inhibition of T-cell loss in HIV-1 mediated cell death The inhibition of T-cell loss in HIV-1 mediated cell death can be prevented by dosing the patient with 1.0 to 10 mg andrographolide/kg body weight per day once a day continuously. The survival of the T cell is dependent upon continuous administration of andrographolide.
  • Cell proliferation and related cytopathicity associated with other viral diseases such as hepatitis and herpes may be prevented on a dose regimen of 2.5 to 15.0 mg andrographolide/kg body weight two to three times per day.
  • the phosphorylation of amyloid precursor protein by p34 cdc2 kinase may be prevented through the downregulation of p34 cdc achieved by andrographolide administration. Continuous administration of 10 mg andrographolide/kg body weight once a day will achieve the necessary degree of downregulation of p34 cdc in neural cells while minimizing the effect in other tissues.
  • a fumagiilin derivative angiogenesis inhibitor AGM-1470, inhibits activation of cyclin-dependent kinases and phosphorylation of retinoblast ⁇ ma gene product but not protein tyrosyl phosphorylation or protooncogene expression in vascular endothelial cells. Cancer Res 54: 3407-3412.
  • FKBP-rapamycin inhibits a cyclin-dependent kinase activity and a cyclin Dl-Cdk association in early Gl of an osteosarcoma cell line. J. Biol. Chem. 268: 22825-22829. 5. Bartek, J., Z. Staskova, G. Draetta, and J. Lukas (1993) Molecular pathology of the cell cycle in human cancer cells. Stem. Cells. (Dayt). 11 Suppl 1: 51-58.
  • v-mos suppresses platelet derived growth facto (PDGF) type-beta receptor
  • Rapamycin-FKBP12 blocks proliferation, induces differentiation, and inhibits cdc2 kinase activity in a myogenic cell line. J. Biol. Chem. 268: 25385-25388.
  • Vitamin K3 inhibits growth of human hepatoma HepG2 cells by decreasing activities of both p34cdc2 kinase and phosphatase. 35 Biochem Biophys Res Commun. 190: 907-913.
  • Rapamycin-induced inhibition of p34cdc2 kinase activation is associated with Gl/S-phase growth arrest in T lymphocytes. J. Biol. Chem. 268: 3734-3738.

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Abstract

Cette invention concerne la découverte d'un groupe de composés pouvant entraîner, dans une cellule ou un groupe de cellules, une diminution de l'expression et de la phosphorylation de la kinase p34cdc2, de la cycline B et de protéines associées au Mos. Des extraits de plantes séchées ont été testés pour déterminer leur capacité à diminuer l'expression de la kinase p34cdc2 et l'on a découvert qu'un extrait d'Andrographis paniculata possédait cette propriété. Des analyses ultérieures ont montré que la diminution de l'expression de la kinase p34cdc2 était induite par les composés andrographolide contenus dans l'extrait. On sait que l'andrographolide possède les analogues d'origine naturelle suivants: 14-épiandrographolide, isoandrographolide, 14-désoxy-12-méthoxyandrographolide, 12-épi-14-12-méthoxyandrographolide, 14-désoxy-12-hydroxyandrographolide et 14-désoxy-11-hydroxyandrographolide. Le composé possède la structure (I) où R1, R2 et R5 sont choisis parmi les éléments suivants: un groupe hydroxyle, un groupe méthyle, un groupe méthoxy, un groupe méthylène ou un groupe glucide à liaison éther ou ester; R3 est un groupe méthyle ou un groupe méthylène; R4 est un groupe hydroxyle ou un groupe carbonyle; R6 représente hydrogène ou un groupe hydroxyle.
PCT/US1995/015915 1994-12-06 1995-12-06 Utilisation de composes andrographolide dans le traitement ou l a prevention de la pathogenicite de certaines maladies WO1996017605A1 (fr)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030213A3 (fr) * 1997-01-08 1998-09-11 Paracelsian Inc Utilisation de recepteur ah et de ligands de recepteur ah dans le traitement ou la prevention des effets cytopathologiques d'infections virales
WO2001057026A1 (fr) * 2000-02-03 2001-08-09 Dr. Reddy's Laboratories Limited Composes presentant une activite antitumorale, procede de preparation de ces composes et compositions pharmaceutiques contenant ces composes
EP1152731A1 (fr) * 1999-12-14 2001-11-14 Avon Products, Inc. Compositions de traitement de la peau mediant les communications de cellule a cellule
WO2001085710A1 (fr) * 2000-05-05 2001-11-15 Dr. Reddy's Research Foundation Composes anticancereux, procede de fabrication, et compositions pharmaceutiques renfermant ces composes
WO2001085709A3 (fr) * 2000-06-08 2002-05-30 Reddy Research Foundation Nouveaux composes presentant une activite anticancereuse et compositions pharmaceutiques les contenant
US6486196B2 (en) 2000-05-05 2002-11-26 Dr. Reddy's Research Foundation Anticancer compounds: process for their preparation and pharmaceutical compositions containing them
US6576662B2 (en) 2000-05-05 2003-06-10 Dr. Reddy's Laboratories Limited Compounds having anticancer activity : process for their preparation and pharmaceutical compositions containing them
WO2003080062A1 (fr) * 2002-03-25 2003-10-02 Silvia Perrella Segre Composition comprenant de la vitamine b9, de la vitamine b6, de l'acide lipoique et des extraits vegetaux pour le traitement de troubles circulatoires
WO2005074953A1 (fr) * 2004-02-03 2005-08-18 Universidad Austral De Chile Composition de diterpenes du type labdane extraite de andrographis paniculata, utilisee pour traiter les maladies auto-immunes et la maladie d'alzheimer par activation des recepteurs ppr-gamma
WO2006101538A3 (fr) * 2004-11-08 2007-05-24 Cornell Res Foundation Inc Derives d'andrographolide servant a traiter les infections virales
RU2383353C2 (ru) * 2004-04-28 2010-03-10 Хатчисон Медифарма Энтерпрайзис Лимитед Суммарные экстракты из andrographis paniculata
GB2464813A (en) * 2009-10-23 2010-05-05 Univ Sheffield Treatments for neurodegenerative disorders
CN102652762A (zh) * 2012-05-14 2012-09-05 浙江大学 一种穿心莲有效部位的应用
CN103145661A (zh) * 2013-03-25 2013-06-12 成都天台山制药有限公司 穿心莲内酯的新晶型
WO2014157965A1 (fr) * 2013-03-27 2014-10-02 재단법인 아산사회복지재단 Composition pour le traitement ou l'inhibition de la métastase de cancers qui comprend un inhibiteur de l'expression de p34 ou un inhibiteur de l'activité p34 comme principe actif
CN116874518A (zh) * 2023-09-06 2023-10-13 中国中医科学院广安门医院 一种穿心莲内酯改构化合物g3和制备方法及其用途

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WO1991001742A1 (fr) * 1989-07-28 1991-02-21 Ruffles, Graham, Keith Compositions biologiquement actives

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WO1991001742A1 (fr) * 1989-07-28 1991-02-21 Ruffles, Graham, Keith Compositions biologiquement actives

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Cited By (24)

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US5833994A (en) * 1997-01-08 1998-11-10 Paracelsian, Inc. Use of the AH receptor and AH receptor ligands to treat or prevent cytopathicity of viral infection
WO1998030213A3 (fr) * 1997-01-08 1998-09-11 Paracelsian Inc Utilisation de recepteur ah et de ligands de recepteur ah dans le traitement ou la prevention des effets cytopathologiques d'infections virales
US7960437B2 (en) 1999-12-14 2011-06-14 Avon Products, Inc. Skin care composition that mediates cell to cell communication
EP1152731A1 (fr) * 1999-12-14 2001-11-14 Avon Products, Inc. Compositions de traitement de la peau mediant les communications de cellule a cellule
EP1152731A4 (fr) * 1999-12-14 2002-10-30 Avon Prod Inc Compositions de traitement de la peau mediant les communications de cellule a cellule
US6410590B1 (en) 2000-02-03 2002-06-25 Dr. Reddy's Research Foundation Compounds having antitumor activity: process for their preparation and pharmaceutical compositions containing them
WO2001057026A1 (fr) * 2000-02-03 2001-08-09 Dr. Reddy's Laboratories Limited Composes presentant une activite antitumorale, procede de preparation de ces composes et compositions pharmaceutiques contenant ces composes
WO2001085710A1 (fr) * 2000-05-05 2001-11-15 Dr. Reddy's Research Foundation Composes anticancereux, procede de fabrication, et compositions pharmaceutiques renfermant ces composes
US6486196B2 (en) 2000-05-05 2002-11-26 Dr. Reddy's Research Foundation Anticancer compounds: process for their preparation and pharmaceutical compositions containing them
US6576662B2 (en) 2000-05-05 2003-06-10 Dr. Reddy's Laboratories Limited Compounds having anticancer activity : process for their preparation and pharmaceutical compositions containing them
WO2001085709A3 (fr) * 2000-06-08 2002-05-30 Reddy Research Foundation Nouveaux composes presentant une activite anticancereuse et compositions pharmaceutiques les contenant
WO2003080062A1 (fr) * 2002-03-25 2003-10-02 Silvia Perrella Segre Composition comprenant de la vitamine b9, de la vitamine b6, de l'acide lipoique et des extraits vegetaux pour le traitement de troubles circulatoires
WO2005074953A1 (fr) * 2004-02-03 2005-08-18 Universidad Austral De Chile Composition de diterpenes du type labdane extraite de andrographis paniculata, utilisee pour traiter les maladies auto-immunes et la maladie d'alzheimer par activation des recepteurs ppr-gamma
US8084495B2 (en) 2004-02-03 2011-12-27 Herbal Powers Corporation Composition of labdane diterpenes extracted from andrographis paniculata, useful for the treatment of autoimmune diseases, and alzheimer disease by activation for PPR-gamma receptors
RU2383353C2 (ru) * 2004-04-28 2010-03-10 Хатчисон Медифарма Энтерпрайзис Лимитед Суммарные экстракты из andrographis paniculata
WO2006101538A3 (fr) * 2004-11-08 2007-05-24 Cornell Res Foundation Inc Derives d'andrographolide servant a traiter les infections virales
US8445533B2 (en) * 2004-11-08 2013-05-21 Cornell Research Foundation, Inc. Andrographolide derivatives to treat viral infections
GB2464813A (en) * 2009-10-23 2010-05-05 Univ Sheffield Treatments for neurodegenerative disorders
CN102652762A (zh) * 2012-05-14 2012-09-05 浙江大学 一种穿心莲有效部位的应用
CN103145661A (zh) * 2013-03-25 2013-06-12 成都天台山制药有限公司 穿心莲内酯的新晶型
CN103145661B (zh) * 2013-03-25 2014-06-18 成都天台山制药有限公司 穿心莲内酯的新晶型
WO2014157965A1 (fr) * 2013-03-27 2014-10-02 재단법인 아산사회복지재단 Composition pour le traitement ou l'inhibition de la métastase de cancers qui comprend un inhibiteur de l'expression de p34 ou un inhibiteur de l'activité p34 comme principe actif
US9752151B2 (en) 2013-03-27 2017-09-05 The Asan Foundation Composition for treatment or metastasis suppression of cancers which includes p34 expression inhibitor or activity inhibitor as active ingredient
CN116874518A (zh) * 2023-09-06 2023-10-13 中国中医科学院广安门医院 一种穿心莲内酯改构化合物g3和制备方法及其用途

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