AU2005203029A1 - Method of profiling a plant extract - Google Patents

Description

12. Jul. 2005 17:53 Shelston IF No. 9772 P. 4 -1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Soit oCnel o ehrhse 'pliain cetfqe Name of Applicant/s: Actual Inventor/s: Societe De Conseils de Recherches et D'Applications Scientifiques SAS and Georgetown University Katy Drleu and Vassillos Papadopoulos Address for Service is: SHELSTON IP Margaret Street SYDNEY NSW 2000 CCN: 3710000352 Attorney Code: SW Telephone No: Facsimile No.

(02) 97771111 (02) 92414666 Invention Title: METHOD OF PROFILING A PLANT EXTRACT Details of Original Application No. 2001247832 dated 27 Mar 2001 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 36591AUP01 500640596_1.DOC5S44 COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:53 Shelston IP No. 9772 P. Ia- 0 Cl METHOD OF PROFILING A PLANT EXTRACT 3 Statement Regarding Federally Sponsored Research The present application is a divisional application of Australian Application No.

2001247832, which is incorporated in its entirety herein by reference.

0The present invention was sponsored in part by grants ES-07747NIEHS, from the National Institutes of Health and, thus, the U.S. government may have certain rights in othe present invention.

Background of the Invention The present invention is directed to a method of establishing an "identity" of Ginkgo biloba leaves or isolated ginkgohde B (GKB), a component of the extract of Ginkgo biloba leaves, by obtaining a "gone 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 7610, 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 761D. 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 V.

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 EBB 7610 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 COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:53 Shelston IP No. 9772 P. 6 lbin 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 COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:53 Shelston IF No. 9772 P. 7 o 7810, has been used in most of these studies. This extract is also known to have o cardioprotective effects (DeFeudis F.V. Ginkgo biloba extract (EGB 761®): from chemistry to clinic. UUstein Medical, Wisbaden, Germany. 400 pp. 1998; Tosaki, A., Droy-Lefaix, Pali, and Das, D.K, Free Rad. Biol. Med., 14: 361-370, 1993).

These effects have been attributed, at least In part, to the free radical scavenging properties of EGS 761®, probably due to the presence of flavonoid or terpenoid constituents in the extract Recent In vivo and In vitro studies demonstrated that the Sterpene constituents of EGB 761®, glnkgolides and bilobaride, have anti-oxidant ¢q properties (Pietri, Maurelli, E, Drleu, K, and Culcasi, J. Mol. Cell. Cardiol;, 29: N 10 733-742, 1997; Yao, Boujrad. Drieu, K, and Papadopoulos, Adv. Ginkgo 0 Biloba Res. 7: 129-138, 1998). Other studies of EGB 7861 have reported medicinal 0 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, F.V, 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 (ginkgoides 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 781®, which can then be used to compare with the biological activity profile of a counterfeit EGB 761 to screen such counterfeits from the marketplace.

-2- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:54 Shelston IP No. 9772 P. 8 Summary of the invention O The present invention is directed to a method of establishing a gene regulation profile of a Gingko biloba extract or a component of the Ginkgo biloba extract, which comprises the steps of- C 5 obtaining at least one batch of untreated cells; treating a first batch of cells with an extract of Ginkgo biloba or a component of the Ginkgo biloba extract to obtain a treated batch of cells; NC quantifying an affect on the expression of one or more genes of the treated cells to obtain a quantity of affected genes; and S 10 comparing the quantity of affected genes with a quantity of genes of cells not treated with Ginkgo biloba or a component of the Ginkgo biloba extract to obtain the o gene regulation profile of the Ginkgo biloba extract or a component of the Ginkgo biloba extract.

A preferred method of the foregoing method is where the quantifying step comprises: isolating poly A+ RNA from the treated batch of cells to obtain treated poly A+

RNA;

isolating poly A+ RNA from a batch of untreated cells to obtain untreated poly A+ RNA; generating labeled cDNA probes from the treated poly A+ RNA to obtain treated labeled cDNA probes; generating labeled cDNA probes from the untreated poly A+ RNA to obtain untreated labeled cDNA probes; hybridizing the treated cDNA probes to an array having one or more cDNA to obtain a treated hybridized array of cDNA; hybridizing the untreated cDNA probes to an array having one or more cDNA to obtain an untreated hybridized array of cDNA; quantifying each of the cDNA of the treated hybridized array of cDNA to obtain quantities of treated cDNA; quantifying each of the cDNA of the untreated hybridized aray of cDNA to obtain quantities of untreated cDNA; and comparing the quantities of each of the treated cDNA with the quantities of untreated cDNA to obtain the gene regulation profile.

-3- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12-Jul. 2005 17:54 S h e I s t o n I P 12.Jul 2@5 7:4 SeitonIFNo. 9772 P. 9 A preferred method of the immediately foregoing method is where the cells are o MDA-231 cells; the Ginkgo biloba extract is EGO 761®; and the array is a gene chip having multple genes.

n A preferred method of the immediately foregoing method Is where t gene Ni 5 regulation profile of EGE 76IQh comprises increased expression of c-Myc protooncogene, and decreased expression of the following genes: prothymosirz-CZ, CDK.2, p55000, myeloblastin p120 proliferafing-cell nuclear antigen, NETI, ERK2, ciAdenosine A2A Receptor, FKtS ligand, Gtb2; Clusterin, RXR,-p, Glutathione Sentransferase P, TRADD. SGP-2,, NIP-I, Id-2, ATF-4, ETRIOI, EmR-los, 010 macrophage* cWooy-stimulafing factor1, heparin-binding EGF-Ilike growth factor, Seaoyegot factor-like protein, inhibin a, COI19 B-lymphocyte antigen, LI CAM,jcatnin, taginsubunit-cz3, integrin subunit a4, integtin subunit AS, integrin subunit AS, integrin subunit WM, APO, FE-I, RhiOA, c-Jun, prothymosin-z, CDK2, p55000 and myoloblastin.

A preferred method Of the immediately foregoing method is where thle gene regulation profile of EGB 781®D is about o-Myc c-Jun -78%, RhoA 93%, APC=-59%, PE-1 -42%, Prothymosin-ax -79%, Myeloblastin -68%, p55000 -63%, p120 Proiiferating-call Nuclear Mflgen 001(2 -a -83%.

NETI ERK2 Adenosine A2A Receptor rits lgand -58%, Grb2 Clusterin -54%, RXR-P -4- COMS ID No: SBMI-01 348800 Received by IP Australia: Time (I-tm) 17:04 Date 2005-07-12 12. Jul. 2005 17:54 Shelston IP No. 9772 P. tr Glutathione S-transferase P -39%, SN-Myc -74%, TRADD -51%, NIP-1 N 5 Id-2 ATF4 -42%, ETR103= ETR101= SCD19 B-lymphocyte Antigen -62%.

O 10 L1CAM= -72%, if) p-catenin -58%, o Integrin Subunit aM= -41%, Integrin Subunit p5 Integrin Subunit a4 Integrin Subunit a3 -77%, Integrin Subunit S6 -53%, Macrophage Colony-stimulating Factor-1 (CSF-1) -31%, Heparin-binding EGF-lke Growth Factor (HB-EGF) -62%, Hepatocyte Growth Factor-like Protein (HGFLP) and inhibln a= -69%, wherein the percentages shown can be A preferred method of any of the foregoing methods is where the cells are MDA- 231 cells; the component of the Ginkgo biloba extract is Ginkgolide B; and the array is a gene chip having multiple genes.

In another aspect, the present invention provides a method of verifying the identity of a Ginkgo biloba extract which comprises the steps of.

obtaining a gene regulation profile of the Gingko biloba extract to obtain a gene regulation profile; obtaining a gene regudatibn profile 6f EGB 761S to yield an EGB 761 gene regulation profile;.

comparing the gene regulation profile of the Gingko biloba extract with the EGB 761@ gene regulation profile; COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:55 Shelston IP No. 9772 P. 11 o determining whether the values of the gene regulation profile of the Ginkgo Sbiloba extract is within ±10% of the values of the EGB 761® gene regulation profile to S obtain verification of the Identity of the Ginkgo biloba extract A preferred method of the immediately foregoing method is where the method S 5 of obtaining a gene regulation profile of the Ginkgo biloba extract and the EGB 781® gene regulation profile comprises the steps of: isolating poly A+ RNA from the treated batch of cells to obtain treated poly A+ o

RNA;

Isolating poly A+ RNA from a batch of untreated cells to obtain untreated poly N 10 A+ RNA; o generating labeled cDNA probes from the treated poly A+ RNA to obtain treated 0 labeled cDNA probes; generating labeled cDNA probes from the untreated poly A+ RNA to obtain untreated labeled CDNA probes; hybridizing the treated cDNA probes to an array having one or more cDNA to obtain a treated hybridized array of cDNA; hybridizing the untreated cDNA probes to an array having one or more cDNA to obtain an untreated hybridized array of cDNA; quantifying each of the cDNA of the treated hybridized array of cDNA to obtain quantities of treated cDNA; quantifying each of the cDNA of the untreated hybridized array of cDNA to obtain quantities of untreated cDNA; and comparing the quantities of each of the treated cDNA with the quantities of untreated cDNA to obtain the gene regulation profile.

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 (Clontech Inc.).

mRNAs were obtained from control or EGS 781 (20 pg/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 provided in the array.

Only consistent significant changes above 30% were considered.

-6- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:55 Shelston IF No. 9772 P. 12 Detailed Description O The term "ginkgo terpenoid" includes all of the naturally occurring terpenes which are derived from the gymnosperms tee Ginkgo biloba as well as synthetically produced ginkgo terpenoids and pharmaceutically active derivatives and salts thereof c ,5 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 lby P. Braguet (1988); F.V.

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

The term "ginkgolide.' as used herein include the various ginkgoltdes disclosed S in the books cited above as well as non-toxic pharmaceutically active derivatives O thereof. Examples of ginkgolide derivatives include tetrahydro derivatives,- acetyl derivatives, and alkyl esters such as the monoacetate: derivatives and triacetate derivatives discosed- in Okabe,-et al., J. Chem. Soc pp., 2201-2206' (1987).

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

HH

H HOH H H H; O

HH

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 -7- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:55 Shelston IP No. 9772 P. 13 t array I from Clontech (Palo Alto, CA); GeneFilters Microarrays by Research Genetics O (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 C 5 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 c arrays on the Affymetrix Gene chip platform, where the genes are immobilized on a 0 silicon chip. In the case of silicon chips, the hybridization results (with the mRNA of o 10 choice) are detected by fluorescence and analyzed by pattern recognition compared to *f either fluorescence or radioactivity that can be used for the detection of the o 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. 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 manufacturers recommendations.

Autoradiography is performed by exposing the blots to film at about -70°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 -8- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:56 Shelston I P No. 9772 P. 14 o genes expressed at low levels. The three internal controls, ubiquitin, G3PDH and po actin 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 C human PCR-amplified cDNA fragments of 200-500 bp long immobilized on a positively

O

n charged nylon membrane. MDA-231 cells were treated with and without 20 pg/lml EGB

O

C 10 761@ for 48 hours. Poly A+ RNA was Isolated from control and EGB 761 -treated cells. "P-labeled cDNA probes were generated from each poly A+RNA and hybridized o 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 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 761®-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 1, 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 (ld-2, ATF-4, ETR101, ETR-103), growth factors (HB-EGF, HGF-like), and cell adhesion molecules-(CD19, LICAM, integrins aS3, o4 a, 5, 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 COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12 Jul. 2005 17:56 Shelston IP No. 9772 P. extract The comparison of the profiles can thus be used as a screening means to authenticate the origin of an extract COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:56 Sheiston IF No. 9772 P. 16 Table 1: Effect of iEGS 7810 on MDA-231 gene expression examined using the Alas human ONA expression array as described under Nucleic Acid Arrays.

Chn EUw&n 2MMM92 aW Tufftorfitmwessers

RIMA

APG

p 6 S000

NETI

ERK2 Adenosine A2A Receptor FUZ ligand Ciustoi RXR-0 Gbflathlorm S-irrrwtse

P

TRADD

NIP-I

Id-2 ATF4 E-TRIG3 ETRiIM -basic helix-op4r~eb a zipper tusrlplion factor -MycdAx iercomr inuce oe~lcycl progruso i, apoptoeis arid malignant bnsorrnaton -78% -put of the AP-1 bnsflpft Mtor that regulates grs Irmolvad In od prafferton -93% -GTPb*dII protn that is an linpodtant regulator fcll Proiiferation -Rcak inactivation inhtts HL60 cml proliferation -W9% -APO mutations ame aasocated wIth both hereditay anld sporc colorectal canonm -a neaive pat-tunsfatinal regulator of It-atnn -C2% 4rncrufl Moor cull Cyc~ontrol Proteins -79% -sodat nuclear protein tiat is lqnogufated In prollinting thymocytee, Ikloqitest flar bLauka peairs aid in ntnra breast kleons qjrn lproteae Ivolve i leunemi cell clfeentlaflan -Mn -zirW to mioels regultos 0004 and 00020 I wqasiai poltlvoly correlated with cell prolifertion status -W9& -flair Wdoein expmsed in prohffert coba -a prognostic wwictw far breast ocacr patients and prostate ademmewna -a3% -cycdln-ependierd tyrcskt Moein olvad i pmrogfssa through the ca cyle -cycin EJCdIQ hnoctN'ates the rethmobnrtmoyr euppressarto alow the call to progress to 8 phase -Vitmin 0 Inhibition of LNCaP cell polferation coincided with a reduction In 0dC2 acifly -W5% *RhM'especific gtmnine exchange factor -NlHMStSraneuonnin protein -48% -mantis ThtCOlULar Signl-rlate Proei Wi"c famil -activated upon cml sttnalatlort -40% 4G proteMn-coupled receptor Involved In the cAMP slialing pathway -5W'3 4Igaid fore FMt qytone receptor tyrcene Rinse -indcesa prolaifon of leuio, mysiold cells -an adapter proten that lik recptor trasine kinases to the RardMAKslgnaing patlwmyvia its 8H2 domain -54% -a gfrccprotuln associated wit ced adhesion and apoptocie -Increased expression is lInked to Alzheinwr's disease -55% -reznlod-atit transcriptio factor -inhibftlon of ohondrocyte prolifferation by retii acid causes -a reduction in RXR-p mRNA expression 49% -a muW-dnig resistance gene that Is oea esdI aiu ua -cheMica l Ibition of OaT-? inhibit proratonn of Jurlwt T cells -74% -o-my tAidy menbur aneG e iath earmetd relkebistom -51% -TNFR-associated death domain protein -involved in TNFR-kiduc eel growth and differenflhtlan -40% -ofl~ialty described assa yeast nuclea transport protein -pWs of the franslaion Initiation fator 3 (elF3) core Ocnpiuc -B5% -A Memnber of the Id heIec-ocp-tietcfurnly of banscuiplioas intlbiOM -Irivaved i'pmaifengmli of human pancreatic cancer ceol -42% -a rnehr ofthATFCREB family of tansolption faotors -regulates Rae-Induced bansformantion of NIHST Cells 4M0% -a nr :poyL d -immediate etiyg9M& (41) (42) (43) (44) (46) (47) (48) ,(49) (54) (6,57)

*(SS)

(SO)

(61 (86) (87) (71) -1- COMS ID No: SBMI-O1 348800 Received by 1P Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:57 Seso FN.97 .1 S h e I s t o n I F No. 9772 P. 17 -Na Antigen LI CAM Integiin Subunits a4

Q

3 ao Mwcrphge Cowonysthlsfng acr- (OSF.1) Hoarn-ki~uEGF-Ee Qn'tl Pacto (H-EGfl Hepdo* Grwnth Factor-Rca Pydsan

(NSFLF)

Inhibin a an nfl Xt- oj a uAnn.. EM EIA =5IU if ITfMIXC%M 4M% .B-bMnphce integrl memnbrane priotein -expreatrois d dwglted durOng reunold-irditiion of -wv~abi &cS ptffmthn -72% -neura el adhesion nu*Kel 4lnesed LICAM aprvson Is associated with hig..ad migoton of giba celii .bwoft-d in cadheui-mudlted celkul Intracicna -ineat WMi Ow. TCF.EF tracwtptl1on Jactom hi theo Wnt sigr~ig -41% -medis osilular adherence of huma neufophils with S.A-i p -a subuni of the elates receptor -0 =h-unt fet vlhrneti rceptoir (VR) -involved in cessabin of' olgodndroc*t prowfallon -iNvove hn Mm retin angiogonesh -49% Ucoa 4hr a wh s jimad L8 Vyrnpl cell pr~oilaln -duo hnwoved in inebatis of mehnoin and lymohom. cells -77% -a fAnicrWS perbin a3 Irtegrin antbody hihibt hnms spll Cell! prahlko -M3% -cvu'apressian of &S Intgffl collaborates with ErbB to hdice it more Inmlignt phdnctypu In NIHMf coba -31% -reguats the pbdlfraticn &djfollfln and surial of monoyta., maeqos aend f* pmossn -kffitca a mltgwst silgl ffut Is equiredl thoughoet G31 phase OSF4 -1 cia grtndn nabt exhat enhanoed -MM% -omxpuessed in rxm-iueros hunra gliori cell line and is mr~y -81% -a flwunwnbusn. prftei 4Trsie Idnas tmid to be oapme -IMuce pirolifeati ard tnfito Of murkte kuruttoyles -W9% -a mmber of the inhibin fIwn*l of hstmodirrwic growth factors -inhhn ctlIs a' mdrr of frophotbfato neopbasa and ia highly (73) (74) (78)

(MO

(0) (81) (82) .(84) (8) (M7 References: 37. Amat, Alevizopoulos, and Vlach, J. Myc and the Cell Cycle. Frontiers in Bioscience, 3:.250-288, 1998.

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Endocrinology, 139:1197-1207, 1998.

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-18- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12

Claims (1)

12. Jul. 2005 17:59 Shelston IP No. 9772 P. 22 0 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS 1. A method of establishing a gene regulation profile of a GingKo biloba extract or a component of the Ginkgo biloba extract, which comprises the steps of: obtaining at least one batch of untreated cells; o. treating a first batch of cells with an extract of Ginkgo bfloba or a component of o the Ginkgo biloba extract to obtain a treated batch of cells; -guantifying an affect on the expression.of one or more genes of the treated cells to obtain a quantity of affected genes; and... 0 comparing,.the quantity of affected genes with, a quantity of genes-of cells- not 0ih q n- o gep-s Streated with Ginkgo biloba or a component of the Ginkgo bloba extract to obtain the gene regulation profile of the Ginkgo biloba extract, or a.co.pnent.of the Ginkgo biloba extract 2. A method according to claim 1 wherein the quantifying comprises: isolating poly A+ RNA from the treated batch of cells to obtain treated poly A+ RNA; isolating poly A+ RNA from a batch of untreated cells to obtain untreated poly A+ RNA; generating labeled cDNA probes from the treated poly A+ RNA to obtain treated labeled cDNA probes; generating labeled cDNA probes from the untreated poly A+ RNA to obtain untreated labeled cDNA probes; hybridizing the treated cDNA probes to an array having one or more cDNA to obtain a treated hybridized array of cDNA; hybridizing the untreated cONA probes to an array having one or more cDNA to obtain an untreated hybridized array of cDNA; quantifying each of the cONA of the treated hybridied array of cDNA to obtain quantitis of treated cDNA; quantifying each of the cDNA of the untreated hybridized array of cDNA to obtain quantities of untreated cDNA; and -17- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 17:59 Sheiston I? No. 9772 P. 23 o comparing the quantities of each of the treated eDNA with the quantities of o untreated cDNA to obtain the gene regulation profile. S_ A method according to claim 2, wherein the cells aria MDA-231 cells; the Ginkgo biloba extract is EGS 7610; and the array Is a gene chip having multiple genes. Ci 54. A method according to Cl21M 3 wherein the gene regulation profile of EGS3 761® comprises increased expression of c-Myc protooncogene, and decreased expression of the following genes: prothymosin-cz, CDFC2, pSSCDC, mayeloblastin p120 Ci proliferating-cell nuclear-antigen, NETI, ERK2, Adenosine A2A Receptor, FItS ligand, GrbZ Clusterin, RXR-p, Glutathline $-tlrinsferase P, N-Myc, TRADE), $GP-2, NIP-I, c-i 10 ld-2, ATF-4;' ETRiQI, ETR-1 03, macrophage colony-stimulatIng factor-I, heparin- o binding EGF-like growth factor, hepatocyte growth factor-like protein, Inhibin ci, C619 o B-lyrnphole antigen,' LICAM, frcatenin, Iintegriri subunit a3, integrin' subunit ax4, integiin subunit aa, integrin subunit PS, Integrir subunit am, APO, PB-I, RhoA, c-Jun, prothymosin-a, 06(2, pS6CDC and my 0alolsinh. 6. A method according to claim 4 wherein the gene regulation profile of EGB 7814D Is -about c-MYC= c-Jun =-78%A, RhoA -93%, APC PE-1 420k, Prothymosin-z -79%, M yeloblastin -530/, p120 Proliferating-cell Nuclear Antigen 00(2 -83%, NETI ERK2 =-418%, MAnosine A2A Receptor P~t3 ligand Grb2 Clusteriri -54%, RXR-3 COMS IDNo: SBMI-01 348800 Received by IP Australia: Time (I-tm) 17:04 Date 2005-07-12 12. Jul. 2005 17:59 Shelston IP No. 9772 P. 24 Glutathione S-transferase P= -39%, N-Myc -74%, TRADD= -51%, NIP-1 N 5 Id-2 ATF4 -42%, ETR103=-65%, CETR101 CD19 B-ymphocyte Antigen -62%, 0 10 LICAM -72%, VB P-catenin -58%, Sntegrin Subunit aM -41%, Integrin Subunit p5 Integrin Subunit aA 49%, Integrin Subunit a3 l -77%, Integrin Subunit aS6 -53%, Macrophage Colony-stimulating Factor-1 (CSF-1)= -31%, Heparin-binding EGF-iike Growth Factor (HB-EGF) -62%, Hepatocyte Growth Factor-like Protein (HGFLP) and Inhibin a= -69%, wherein the percentages shown can be A method according to claim 2, wherein the cells are MDA-231 cells; the component of the Ginkgo biloba extract is Ginkgolide B; and the array is a gene chip having multiple genes. 6. A method of verifying the identity of a Ginkgo biloba extract which comprises the steps of; obtaining a gene regulation profile of the Gingko biloba extract to obtain a gene regulation profile; obtaining a gene regulation profile of EGB 761® to yield an EGB 761® gene regulation profile; comparing the gene regulation profile of the Gingko biloba extract with the EGB 761@ gene regulation profile; -19- COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12 12. Jul. 2005 18:00 Shelston IP No. 9772 P. t/ determining whether the values of the gene regulation profile of the Ginkgo O Sbiloba extract is within ±10% of the values of the EGB 761® gene regulation profile to obtain verification of the identity of the Ginkgo biloba extract Z 7. A method according to claim 8, wherein the method of obtaining a gene C 5 regulation profile of the Ginkgo biloba extract and the EGB 761@ gene regulation profile comprises the steps of: Isolating poly A+ RNA from the treated batch of cells to obtain treated poly A+ RNA; isolating poly A+ RNA from a batch of untreated cells to obtain untreated poly o 10 A+ RNA, t/ generating labeled cDNA probes from the treated poly A+ RNA to obtain treated labeled cDNA probes; c-l generating labeled cDNA probes from the untreated poly A+ RNA to obtain untreated labeled cDNA probes; hybridizing the treated cDNA probes to an array having one or more cDNA to obtain a treated hybridized array of cDNA; hybridizing the untreated cDNA probes to an array having one or more cDNA to obtain an untreated hybridized array of cDNA; quantifying each of the cDNA of the treated hybridized array of cDNA to obtain quantities of treated cDNA; quantifying each of the cDNA of the untreated hybridized array of eDNA to obtain quantities of untreated cDNA; and comparing the quantities of each of the treated cDNA with the quantities of untreated cDNA to obtain the gene regulation profile. DATED this 12 t day of July 2005 Shelston IP Attorneys for: Societe D Conselis de Recherches et D'Applications Scientifiques SAS. and Georgetown University COMS ID No: SBMI-01348800 Received by IP Australia: Time 17:04 Date 2005-07-12