AU2001247832B2

AU2001247832B2 - Method of profiling a plant extract

Info

Publication number: AU2001247832B2
Application number: AU2001247832A
Authority: AU
Inventors: Katy Drieu; Vassilios Papadopoulos
Original assignee: Soc De Conseils De Recherches Et Dapplications Scientifiques S A S; Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS; Georgetown University
Current assignee: Ipsen Pharma SAS; Georgetown University
Priority date: 2000-03-31
Filing date: 2001-03-27
Publication date: 2005-07-21
Anticipated expiration: 2021-03-27
Also published as: CA2403303A1; AU4783201A; CZ20023275A3; NZ521728A; HUP0301997A3; PL365418A1; RU2271393C2; NO20024611L; CN1439059A; WO2001075181A3; IL151856A0; HUP0301997A2; KR100479151B1; EP1292706A2; CN1185354C; KR20020089409A; NO20024611D0; WO2001075181A2; HK1053150A1; MXPA02009412A

Description

WO 01/75181 PCT/US01/09832 METHOD OF PROFILING A PLANT EXTRACT Statement Regarding Federally Sponsored Research The present invention was sponsored in part by grants ES-07747 NIEHS, from the National Institutes of Health and, thus, the U.S. government may have certain rights in the present invention.

Background of the Invention The present invention is directed to a method of establishing an "identity" of Ginkgo biloba leaves or isolated ginkgolide B (GKB), a component of the extract of Ginkgo biloba leaves, by obtaining a "gene regulation profile". The invention is also directed to a method of verifying the identity of a Ginkgo biloba extract by comparing the gene regulation profile of a Gingko biloba extract of unknown or questionable origin with the gene regulation profile of a Gingko biloba extract of known origin. The term "known origin" refers to the commercial source of the extract. A preferred aspect of the present invention is where the Ginkgo biloba extract of known origin is EGB 761®, produced and marketed by IPSEN of Paris, France. More particularly, this invention is directed to a method for determining the authenticity of an extract of unknown origin purporting to be EGB 761®. The present invention is further directed to a method of establishing a gene expression profile of Ginkgolide A, Ginkgolide B or any other component isolated from a Ginkgo biloba extract, more particularly, from EGB 761®.

Pharmaceutical manufacturing is based on control over the composition and the consistency of the biological activity profile of a manufactured batch. This standardization and control provides reproducible material in the predictable and consistent treatment of patients. Such use of standardization and control to guard against the marketing of counterfeit extracts purporting to be EGB 761® is beneficial to patients since it assures patients that they are obtaining/receiving an extract with a particular biological activity profile.

Ginkgo biloba is one of the most ancient trees, and extracts from its leaves have been used in traditional medicine for several hundred years. There are numerous studies describing the beneficial effects of Ginkgo biloba extracts on patients with disturbances in vigilance, memory, and cognitive functions associated with aging and senility, and on those with all types of dementias, mood changes, and the ability to cope with daily stressors. A standardized extract of Ginkgo biloba leaves, termed EGB WO 01/75181 PCT/US01/09832 761®, has been used in most of these studies. This extract is also known to have cardioprotective effects (DeFeudis F.V. Ginkgo biloba extract (EGB 761®): from chemistry to clinic. Ullstein Medical, Wisbaden, Germany. 400 pp. 1998; Tosaki, A., Droy-Lefaix, Pali, and Das, Free Rad. Biol. Med., 14: 361-370, 1993).

These effects have been attributed, at least in part, to the free radical scavenging properties of EGB 761®, probably due to the presence of flavonoid or terpenoid constituents in the extract. Recent in vivo and in vitro studies demonstrated that the terpene constituents of EGB 761®, ginkgolides and bilobalide, have anti-oxidant properties (Pietri, Maurelli, Drieu, and Culcasi, J. Mol. Cell. Cardiol., 29: 733-742, 1997; Yao, Boujrad, Drieu, and Papadopoulos, Adv. Ginkgo Biloba Res. 7: 129-138, 1998). Other studies of EGB 761® have reported medicinal value of the product in the treatment of a variety of clinical disorders including cerebrovascular and peripheral vascular insufficiencies associated with aging and senility. See Ginkgo biloba Extract (EGB 761®) Pharmacological Activities and Clinical Applications, DeFeudis, Eds, Elsevier, 1991; and Ullstein Medical 1998, Ginkgo biloba extract (EGB 761®), Eds. Wiesbaden, DeFeudis, F.V. The extract contains 24% ginkgo-flavone glycosides, 6% terpene lactones (ginkgolides and bilobalide), about 7% proanthocyanidins and several other constituents. See Boralle, et al., In: Ginkgolides, Chemistry, Biology, Pharmacology and Clinical perspectives, Ed: Braquet, J.R. Prous Science Publishers, 1988.

More frequently, counterfeit formulations purporting to be EGB 761® are being placed in the stream of commerce. Such counterfeits do not possess the same composition of components that constitute authentic EGB 761®. Patients who obtain counterfeit EGB 761®, believing that the counterfeit is authentic, are being deprived the benefit of EGB 761®'s full range of biological activity. Further, the good will associated with EGB 761® is being eroded. Hence, there is a need for a method of establishing the biological activity profile of EGB 761®, which can then be used to compare with the biological activity profile of a counterfeit EGB 761 to screen such counterfeits from the marketplace.

Summary of the Invention In one aspect, the invention provides a method of verifying the authenticity of a Ginkgo biloba extract which comprises the steps of: obtaining a gene regulation profile of the Ginkgo biloba extract to obtain a gene 003 regulation profile; obtaining a gene regulation profile of EGB 761 to yield an EGB 761® gene regulation profile; 0 comparing the gene regulation profile of the Ginkgo biloba extract with the EGB 761® gene regulation profile; determining whether the values of the gene regulation profile of the Ginkgo biloba extract are within ±10% of the values of the EGB 761 gene regulation profile to obtain verification of the identity of the Ginkgo biloba extract.

In a preferred embodiment, the method comprises the steps of: isolating poly A+ RNA from cells treated with the Ginkgo biloba extract to obtain Ginkgo biloba extract treated poly A+ RNA; isolating poly A+ RNA from cells treated with EGB 761® to obtain EGB 761® treated poly A+RNA; generating labelled cDNA probes from the Ginkgo biloba extract treated poly A+ RNA to obtain Ginkgo biloba extract treated labeled cDNA probes; generating labeled cDNA probes from the EGB 761 treated poly A+ RNA to obtain EGB 761® treated labelled cDNA probes; hybridising the Ginkgo biloba extract treated cDNA probes to an array having one or more cDNA to obtain a Ginkgo biloba extract treated hybridised array of cDNA; hybridizing the EGB 761® treated cDNA probes to an array having one or more cDNA to obtain an EGB 761® treated hybridized array of cDNA; quantifying each of the cDNA of the Ginkgo biloba extract treated hybridized array of cDNA to obtain quantities of Ginkgo biloba extract treated cDNA; quantifying each of the cDNA of the EGB 761® treated hybridized array of cDNA to obtain quantities of EGB 761® treated cDNA; and comparing the quantities of each of the Ginkgo biloba extract treated cDNA with the quantities of EGB 761 treated cDNA to obtain the gene regulation profile.

-4- Preferably, the cells are MDA-231 cells and the array is a gene chip having multiple genes.

INDPreferably, the gene regulation profile of EGB 761V comprises increased expression of c-Mye protooncogene, and decreased expression of the following genes: prothymosin-a, CDK2, p55CDC, myeloblastin p120 proliferating-cell nuclear antigen, 00 NET1, ERK2, Adenosine A2A Receptor, Flt3 ligand, Grb2, Clusterin, RXR-P, Glutathione S-transferase P, N-Myc, TRADD, SGP-2, NIP-1, Id-2, ATF-4, ETR101, ETR-103, macrophage colony-stimulating factor-i, heparin-binding EGF-like growth factor, hepatocyte growth factor-like protein, inhibin a, CD19 B-lymphocyte antigen, LICAM, p-catenin, integrin, subunit a3, integrin subunit a4, integrin subunit a6, integrin subunit P5, integrin subunit aM, APC, PE-1, RhoA, c-Jun, prothymosin- a, CDK2, p55CDC and myeloblastin.

Preferably, the gene regulation profile in response to 20ug/ml of EGB 761® for 48 hours is about c-Myc c-Jun -78%, RhoA -93%, APC -59%, PE-1 -42%, Prothymosin-a -79%, Myeloblastin -66%, -63%, p120 Proliferating-cell Nuclear Antigen -68%, CDK2 -83%, NET1 ERK2 -46%, Adenosine A2A Receptor Flt3 ligand -58%, Grb2 Clustern -54%, RXR- Glutathione S-transferase P -39%, N-Myc -74%, C TRADD -51%, NIP-1 Id-2 ATF4 -42%, ETR103 ETR101 CD19 B-lymphocyte Antigen= -62%, LI1CAM 0 p -catenin -58%, Integrin Subunit aM -41%, Integrin Subunit 35 Integrin Subunit a4 -49%, Intergin Subunit a3 -77%, Integrin Subunit a6 -53%, Macrophage Colony-stimulating Factor-1 (CSF-1)= -31%, Heparin-binding EGF-like Growth Factor (HB-EGF) -62%, Heptaocyte Growth Factor-like Protein (HGFLP) and Inhibin a -69%, wherein the percentages shown can be Preferably, the cells are MDA-231 cells; the Ginkgo biloba extract is Ginkgolide B; and the array is a gene chip having multiple genes.

1n -6- CI Brief Description of the Drawings Figure 1. Transcriptional response to EGB 761® suggests an effect on genes involved in cell proliferation. Results shown represent quantitative analysis of the Atlas human cDNA expression array containing 588 PCR-amplified cDNA fragments 00(Clontech Inc.). mRNAs were obtained from control or EGB 761® (20g/ml) treated, for 48 h, MDA-231 cells. For normalizing the mRNA abundance, the densitometric values obtained from image analysis were normalized using the housekeeping genes Sprovided in the array. Only consistent significant changes above 30% were considered.

(N-

WO 01/75181 PCT/US01/09832 Detailed Description The term "ginkgo terpenoid" includes all of the naturally occurring terpenes which are derived from the gymnosperms tree Ginkgo biloba as well as synthetically produced ginkgo terpenoids and pharmaceutically active derivatives and salts thereof and mixtures thereof. Examples of ginkgo terpenoids include ginkgolides. Examples of ginkgo terpenoids are disclosed in Ginkgolides, Chemistry, Biology, Pharmacology, and Clinical Perspectives, J.R. Provs. Science Publishers, Edited by P. Braguet (1988); F.V.

DeFeudis, Ginkgo Biloba Extract (EGB 761®); Pharmacological Activities and Clinical Applications, Elsevier, Chapter II (1991).

The term "ginkgolide" as used herein include the various ginkgolides disclosed in the books cited above as well as non-toxic pharmaceutically active derivatives thereof. Examples of ginkgolide derivatives include tetrahydro derivatives, acetyl derivatives, and alkyl esters such as the monoacetate derivatives and triacetate derivatives disclosed in Okabe, et al., J. Chem. Soc. pp. 2201-2206 (1967).

Ginkgolide B has the following structure and as used herein, refers to isolated ginkgolide B:

H

HO 0

HC"H

H H

H

H HO H H 0 The term "Ginkgo biloba extract" as used herein includes a collection of natural molecules, including terpenoids, derived from the leaves of the Ginkgo biloba tree.

Preferably, the extract is the specific formulation of Ginkgo biloba extract known as EGB 761®.

A gene expression profile of an extract of Ginkgo biloba or a component thereof can be obtained by methods known in the art. Traditionally such a profile was obtained by RNA Northern blot analysis or ribonuclease protection assay for each individual gene product. However, these assays were time consuming and took about 2-3 days to analyze each gene. Currently a gene expression profile can be established through the utilization of nucleic acid array technology such as the Atlas human cDNA expression WO 01/75181 PCT/US01/09832 array I from Clontech (Palo Alto, CA); GeneFilters Microarrays by Research Genetics (Huntsville, AL); and the Gene Expression Microarrays by Genome Systems, Inc. (St.

Louis, MO). The Gene Filters Microarray are high density DNA arrays produced on cm x 7 cm membranes. At present there are four membranes available for human genes and one for rat genes. Each membrane contains approximately 5,000 sequences. Some of these sequences are known genes, while most sequences represent ESTs of unknown function. Research Genetics will soon make available gene arrays on the Affymetrix Gene chip platform, where the genes are immobilized on a silicon chip. In the case of silicon chips, the hybridization results (with the mRNA of choice) are detected by fluorescence and analyzed by pattern recognition compared to either fluorescence or radioactivity that can be used for the detection of the hybridization results in the membrane arrays.

Genome System's method utilizes the GEM technology where a collection of complementary DNA (cDNA) molecules that contain the genetic information from the biological systems of interest are deposited and bonded on a glass surface in an array format. Next, large portions from one half of the DNA's double strand are removed, thus activating the individual elements of the array, preparing them to react with their uniquely matched DNA counterparts in the cells being tested. GEM technology can fit 10,000 unique genes on a single array. GEM technology also uses a color coded technique to examine the difference in expression between two mRNA samples.

An array of cDNA will contain numerous animal, such as rat or human, preferably human, PCR-amplified cDNA fragments immobilized on a positively charged nylon membrane or a glass slide or a silicon chip or any other surface to be developed where a DNA/matrix interaction is allowed. A cell type of interest is treated with and without a Ginkgo biloba extract or a component thereof for about 48 hours. Poly A+ RNA is isolated from control and extract-treated cells. 3 2 P-labeled, fluorescent, chemiluminescent or colorimetric cDNA probes, preferably fluorescent or colorimetric labeling when using glass or silicon chip arrays, are generated from each poly A+RNA and hybridized to the array according to the manufacturer's recommendations.

Autoradiography is performed by exposing the blots to film at about -70 0 C for 4-96 hr.

Quantification of the hybridization is carried out using an imaging system, which can detect the fluorescence or chemiluminesence then capture the image and analyze the data, such as the SigmaGel software. Multiple exposures are used in order to detect WO 01/75181 PCT/US01/09832 genes expressed at low levels. The three internal controls, ubiquitin, G3PDH and 3actin are used to compare the relative expression levels of the detected gene products in the control and the extract-treated cells. Experimental variations are corrected using the ratios of gene expression versus the internal controls. The effect of the extract treatment on each gene product is expressed as of control (untreated) cells.

An example of the foregoing type of gene expression profile is as follows. The Atlas human cDNA expression array I from Clontech (Palo Alto,CA) contains 588 human PCR-amplified cDNA fragments of 200-500 bp long immobilized on a positively charged nylon membrane. MDA-231 cells were treated with and without 20 pg/ml EGB 761® for 48 hours. Poly A+ RNA was isolated from control and EGB 761®-treated cells. 32P-labeled cDNA probes were generated from each poly A+RNA and hybridized to the Atlas array according to the manufacturer's recommendations. Autoradiography was performed by exposing the blots to X-OMAT AR film (Kodak, Rochester, NY) at 0 C for 4-96 hr. Quantification of the hybridization seen was carried out using the SigmaGel software (Jandel Scientific, San Rafael, CA). Multiple exposures were used in order to detect genes expressed at low levels. The three internal controls, ubiquitin, G3PDH and p-actin were used to compare the relative expression levels of the detected gene products in the control and EGB 7618-treated cells. Experimental variations were corrected using the ratios of gene expression versus the internal controls. The effect of the EGB 761® treatment on each gene product is expressed as of control (untreated) cells. The results of this experiment, which is presented in Table I, show genes affected consistently, at a level above 30% of control, by the EGB 761® treatment. In summary, Table I shows that the treatment increased the expression of the c-Myc protooncogene and decreased the expression of 35 gene products, including oncogenes (AP-1, PE-1, RhoA, n-Myc), cell cycle regulators (CDK2, PCNA p120), signal transduction modulators (NET1, ERK2), apoptosisrelated products (SGP-2, NIP1) receptors (A2A, RXR-beta, Grb2), transcription factors (Id-2, ATF-4, ETR101, ETR-103), growth factors (HB-EGF, HGF-like), and cell adhesion molecules (CD19, L1CAM, integrins a3, a 4 a6, p5, Mac-1, p-catenin) which are directly involved in various pathways regulating cell proliferation.

Gene expression profiles can be established for Ginkgo biloba extracts of known origin and then can be compared with the gene expression profile of Ginkgo biloba extracts of unknown origin or extracts that purport to be a certain commercial extract. The comparison of the profiles can thus be used as a screening means to authenticate the origin of an extract.

IDThe reference to any prior art in the specification is not, and should not be taken as an acknowledgement, or any form of suggestion, that the prior art forms part of the common general knowledge in Australia.

00 Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

WO 01/75181 PCT/US01/09832 Table 1: Effect of EGB 761@ on MDA-231 gene expression examined using the Atlas human cDNA expression array as described under Nucleic Acid Arrays.

Name Change Function Oncoqenes and Tumor Suppressers c-Myc c-Jun RhoA

APC

PE-1 Prothymosin-a Myeloblastin p120 Proliferating-cell Nuclear Antigen CDK2 NET1 ERK2 Adenosine A2A Receptor Flt3 ligand Grb2 Clusterin RXR-p Glutathione S-transferase

P

N-Myc

TRADD

NIP-1 +75% -basic helix-loop-helix-leucine zipper transcription factor -Myc/Max heterodimers induce cell-cycle progression, apoptosis, and malignant transformation -78% -part of the AP-1 transcription factor that regulates genes involved in cell proliferation -93% -GTP-binding protein that is an important regulator of cell proliferation -RhoA inactivation inhibits HL60 cell proliferation -59% -APC mutations are associated with both hereditary and sporadic colorectal cancers -a negative post-translational regulator of p-catenin -42% -transcription factor Cell Cycle Control Proteins -79% -acidic nuclear protein that is upregulated in proliferating thymocytes, lymphocytes from leukemia patients, and in malignant breast lesions -66% -a serine protease involved in leukemia cell differentiation -63% -similar to mitosis regulators CDC4 and CDC20 -expression positively correlated with cell proliferation status -68% -nucleolar protein expressed in proliferating cells -a prognostic indicator for breast cancer patients and prostate adenocarcinoma -83% -cyclin-dependent tyrosine kinase involved In progression through the cell cycle -cyclin E/Cdk2 inactivates the retinoblastoma tumor suppresser to allow the cell to progress to S phase -Vitamin D inhibition of LNCaP cell proliferation coincided with a reduction in Cdk2 activity Intracellular Transducers -55% -RhoA-specific guanine exchange factor -NIH3T3-transforming protein -46% -member of the extracellular signal-related protein kinase family -activated upon cell stimulation Apoptosis-Related Proteins -40% -G protein-coupled receptor involved in the cAMP signaling pathway -58% -ligand for the Fit3 cytokine receptor tyrosine kinase -induces proliferation of leukemic myeloid cells -70% -an adapter protein that links receptor tyrosine kinases to the Ras/MAPK signaling pathway via its SH2 domain -54% -a glycoprotein associated with cell adhesion and apoptosis -increased expression is linked to Alzheimer's disease -55% -retiniod-activated transcription factor -inhibition of chondrocyte proliferation by retinoic acid causes a reduction in RXR-p mRNA expression -39% -a multi-drug resistance gene that is overexpressed in various human tumors -chemical inhibition of GST-P inhibits proliferation of Jurkat T cells -74% -c-myc family member -associated with early-onset retinoblastoma -51% -TNFR-associated death domain protein -involved in TNFR-induced cell growth and differentiation -40% -originally described as a yeast nuclear transport protein -part of the translation initiation factor 3 (elF3) core complex DNA-Binding/Transcription Factors References (37) (38) (39) (41) (42) (43) (44) (46) (47) (48) (49) (51) (52) (53) (54) (56, 57) (58) (59) (61,62) (63) (64) (66) (67) Id-2 ATF4 ETR103 ETR101 -65% -42% -65% -60% -a member of the Id helix-loop-helix family of transcriptional inhibitors -involved in proliferation of human pancreatic cancer cells -a member of the ATF/CREB family of transcription factors -regulates Ras-induced transformation of NIH3T3 cells -a macrophage-associated immediate early gene -a lymphocyte-associated immediate early gene -11- WO 01/75181 PCT/USU1/09832 Name CDI9 B-lymphocyte Antigen L1 CAM p-catenin Integrin Subunits Macrophage Colonystimulating Factor-1 (CSF-1) Heparin-binding EGF- Growth Factor (HB-E Hepatocyte Growth Factor-like Protein

(HGFLP)

Inhibin a Change Function Cell Surface Antigens and Adhesion Molecules -62% -B-lymphocyte integral membrane protein -expression is down-regulated during retinoid-inhibition of lymphoblastoid B-cell proliferation -72% -neural cell adhesion molecule -increased L1CAM expression is associated with high-grade migration of glioma cells -58% -involved in cadherin-mediated cell-cell interactions -interacts with the TCF/LEF transcription factors in the Wnt signaling pathway References aM -41% -mediates cellular adherence of human neutrophils with LFA-1 -a subunit of the elastase receptor -55% -p subunit of the vitronectin receptor (VR) -involved in cessation of oligodendrocyte proliferation -involved in murine retinal angiogenesis a4 -49% -cross-linking c4 integrins inhibits LB lymphoma cell proliferation -also involved in metastasis of melanoma and lymphoma cells a3 -77% -a functionally perturbing a3 integrin antibody inhibits human epithelial cell proliferation a6 -53% -overexpression of a6 integrin collaborates with ErbB2 to induce a more malignant phenotype in NIH3T3 cells Extracellular SionalinalCommunication Proteins -31% -regulates the proliferation, differentiation, and survival of monocytes, macrophages and their precursors -initiates a mitogenic signal that is required throughout G1 phase -CSF-1 stably transfected ovarian granulosa cells exhibit enhanced cell proliferation -like -62% -overexpressed in numerous human glioma cell lines and in a majority GF) of glioblastomas -stimulates human glioma cell proliferation -81% -a transmembrane protein tyrosine kinase found to be overexpressed in hepatoblastoma and in human primary liver carcinoma -induces proliferation and migration of murine keratinocytes -69% -a member of the inhibin family of heterodimeric growth factors -inhibin a is a marker of trophoblastic neoplasia and is highly expressed in virilizing adenomas References: 37. Amati, Alevizopoulos, and Vlach, J. Myc and the Cell Cycle. Frontiers in Bioscience, 3: 250-268, 1998.

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Claims

1. A method of verifying the authenticity of a Ginkgo biloba extract which comprises O the steps of: obtaining a gene regulation profile of the Ginkgo biloba extract to obtain a gene Cc regulation profile; 00 obtaining a gene regulation profile of EGB 761 to yield an EGB 761 gene CN regulation profile; 0 comparing the gene regulation profile of the Ginkgo biloba extract with the EGB C 10 761® gene regulation profile; determining whether the values of the gene regulation profile of the Ginkgo biloba extract are within ±10% of the values of the EGB 761® gene regulation profile to obtain verification of the identity of the Ginkgo biloba extract.

2. A method according to claim 1, wherein the method of obtaining a gene regulation profile of the Ginkgo biloba extract and the EGB 761® gene regulation profile comprises the steps of: isolating poly A+ RNA from cells treated with the Ginkgo biloba extract to obtain Ginkgo biloba extract treated poly A+ RNA; isolating poly A+ RNA from cells treated with EGB 761® to obtain EGB 761® treated poly A+RNA; generating labelled cDNA probes from the Ginkgo biloba extract treated poly A+ RNA to obtain Ginkgo biloba extract treated labeled cDNA probes; generating labeled cDNA probes from the EGB 761 treated poly A+ RNA to obtain EGB 761® treated labelled cDNA probes; hybridising the Ginkgo biloba extract treated cDNA probes to an array having one or more cDNA to obtain a Ginkgo biloba extract treated hybridised array of cDNA; hybridizing the EGB 761® treated cDNA probes to an array having one or more cDNA to obtain an EGB 761® treated hybridized array of cDNA; quantifying each of the cDNA of the Ginkgo biloba extract treated hybridized array of cDNA to obtain quantities of Ginkgo biloba extract treated cDNA; quantifying each of the cDNA of the EGB 761® treated hybridized array of cDNA to obtain quantities of EGB 761® treated cDNA; and -18- C comparing the quantities of each of the Ginkgo biloba extract treated cDNA with Z the quantities of EGB 761 treated cDNA to obtain the gene regulation profile. ND

3. A method according to claim 2, wherein the cells are MDA-231 cells and the array O is a gene chip having multiple genes. ,1 5

4. A method according to claim 3 wherein the gene regulation profile of EGB 761® 00 comprises increased expression of c-Myc protooncogene, and decreased expression of the following genes: prothymosin-a, CDK2, p55CDC, myeloblastin p120 proliferating- cell nuclear antigen, NET1, ERK2, Adenosine A2A Receptor, Flt3 ligand, Grb2, 0 Clusterin, RXR-P, Glutathione S-transferase P, N-Myc, TRADD, SGP-2, NIP-1, Id-2, ATF-4, ETR101, ETR-103, macrophage colony-stimulating factor-1, heparin-binding EGF-like growth factor, hepatocyte growth factor-like protein, inhibin a, CD 19 B- lymphocyte antigen, L CAM, p-catenin, integrin, subunit a3, integrin subunit a4, integrin subunit a6, integrin subunit 35, integrin subunit aM, APC, PE-1, RhoA, c-Jun, prothymosin- a, CDK2, p55CDC and myeloblastin.

5. A method according to claim 4 wherein the gene regulation profile in response to ug/ml of EGB 761® for 48 hours is about c-Myc c-Jun -78%, RhoA -93%, APC -59%, PE-1 -42%, Prothymosin-a -79%, Myeloblastin -66%, -63%, p120 Proliferating-cell Nuclear Antigen -68%, CDK2 -83%, NET1 ERK2 -46%, Adenosine A2A Receptor Flt3 ligand -58%, Grb2 Clustern -54%, RXR- -19- SGlutathione S-transferase P -39%, SN-Myc -74%, \0 TRADD -51%, NIP-1 Id-2 00 ATF4 -42%, O ETR103 ETR101 CD19 B-lymphocyte Antigen -62%, L1CAM=-72%, p -catenin -58%, Integrin Subunit aM -41%, Integrin Subunit 35 Integrin Subunit a4 -49%, Intergin Subunit a3 -77%, Integrin Subunit a6 -53%, Macrophage Colony-stimulating Factor-1 (CSF-1) -31%, Heparin-binding EGF-like Growth Factor (HB-EGF) -62%, Heptaocyte Growth Factor-like Protein (HGFLP) and Inhibin a -69%, wherein the percentages shown can be

6. A method according to claim 2, wherein the cells are MDA-231 cells; the Ginkgo biloba extract is Ginkgolide B; and the array is a gene chip having multiple genes.

7. A method according to claim 1, substantially as herein before described with particular reference to the accompanying figure. DATED this 6 th day of July 2005 Shelston IP Attorneys for: Societe D Conselis de Recherches et D'Applications Scientifiques SAS and Georgetown University