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Florenz imhof 2006


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Florenz imhof 2006

  1. 1. Clinical Action and Safety ofRed Clover IsoflavonesMartin ImhofGeneral Public Teaching HospitalKorneuburg/ViennaMedical University - Vienna
  2. 2. • Million Women Study Collaborators.Breast cancer and hormone-replacement therapy in the MillionWomen Study. Lancet 2003; 362:419-27• Women´s Health Initiative (WHI) Study– Interrupted due to severe sideeffects
  3. 3. Herbal AlternativestoHormone Replacement Therapy
  4. 4. How Safe and Effective arePhytohormones?
  5. 5. Are Red Clover IsoflavonesEffective ?A Double Blind, Randomized,Placebo-controlledCross-Over Study
  6. 6. Week 12Week 4 Week 8-25-20-15-10-50PercentageReductioninHotFlushesPlaceboActiveBaber RJ, et al. Climacteric 1999Effect of 40 mg red clover extracts over No.Daily hot flashes and Greene Scores (n=51postmenopausal)**RDBPC study
  7. 7. Tice JA, et al. JAMA 2003Effect of 80 mg vs. 57 mg red clover extracts vsplacebo over No. hot flashes freq and GreeneScores (n=252 postmenopausal)**ICE study (RDBPC) After 12 weeks:After 12 weeks: 80 mg RCE (n=84): 41% reduction80 mg RCE (n=84): 41% reduction 57 mg RCE (n=83): 34%.57 mg RCE (n=83): 34%. Placebo (n=85): 36% (p > 0.05).Placebo (n=85): 36% (p > 0.05).
  8. 8. Van Weijer PH, et al. Maturitas 2002Effect of 80 mg red clover extracts vsplacebo over hot flashes frequenz andGreene Scores (n=30 postmenopausal)* 4 weeks placebo run in phase4 weeks placebo run in phase(16% reduction).(16% reduction). After 12 weeks:After 12 weeks: Red clover reduced hot flashesRed clover reduced hot flashesfreq 44%.freq 44%. Placebo no further reduction.Placebo no further reduction.*RDBPC study
  9. 9. • Placebo-controlled, cross-over, double-blind, randomized• 113 menopausal women, 109 finished this study• 25 weeks• 80 mg isoflavones daily (2 capsules of menoflavon)• Within the 25 weeks 4 examinations• Start: November 2002 - End: February 2005Menoflavon Study 80mg isoflavones /dof red clover – clinical trial Austria
  10. 10. Examinations and questionnaires Kupperman scoreKupperman score (index before start >15)(index before start >15) Changes inChanges in blood serum levelsblood serum levels (T, E2, FSH, LH,(T, E2, FSH, LH,SHBG, HDL, LDL, Triglycerides, Lpa, Cholesterol)SHBG, HDL, LDL, Triglycerides, Lpa, Cholesterol) Effects on theEffects on the endometriumendometrium Vaginal smearVaginal smear(cytodiagnosis)(cytodiagnosis) Urine-analysisUrine-analysis (urinary excretion of isoflavones)(urinary excretion of isoflavones) Depressions scaleDepressions scale after Zerssenafter Zerssen Hospital Anxiety and Depression Scale (Hospital Anxiety and Depression Scale (HADS-DHADS-D)) Observation studyObservation study: effects of menoflavon on skin, hair,: effects of menoflavon on skin, hair,nails and libido, vaginal atrophy….nails and libido, vaginal atrophy….
  11. 11. Demographic and anamnestic dataGroup A(n=50)Group B(n=59)Overall(n=109)Mean age (yrs.) 54.5 ± 6.2 53.7 ± 7.8 53.5 ± 7.1Mean BMI 24.5 ± 3.9 24.9 ± 3.9 24.7 ± 3.9Hysterectomy (%) 18.0 13.6 15.6
  12. 12. Kupperman Index051015202530354045MF11RCE PlacebogestiegengefallenunverändertWilcoxon Test, PVerum < 0,001, PPlacebo < 0,001
  13. 13. Reduction of hot flushes051015202530354045MF11RCE PlacebozugenommenabgenommenunverändertWilcoxon Test, PVerum < 0,001, PPlacebo = 0,001
  14. 14. Hot flushes, Boxplots-comparisonHot flushes were reduced significantlywith MF11RCE (p<0,001)No effect under Placebobefore afterMF11RCEbefore afterPlacebo
  15. 15. Reduction sleep disorders01020304050MF11RCE PlaceboZunahmeAbnahmekeine VeränderungWilcoxon Test, PVerum < 0,001, PPlacebo = 0,018
  16. 16. Sleeping behavior - Boxplots-comparisonbefore afterMF11RCE PlaceboafterbeforeSleeping behavior improved highsignificantly with MF11RCE (p<0,001)No effect under Placebo
  17. 17. Mood improvement05101520253035MF11RCE PlaceboverschlechtertverbessertunverändertWilcoxon Test, PVerum < 0,001, PPlacebo = 0,001
  18. 18. Baseline measuresGroup A(n=50)Group B(n=59)Overall(n=109)E2(pg/ml) 36.30 38.31 37.39T (ng/ml) 0.43 0.63 0.54LH (mIU/ml) 30.64 29.92 30.25FSH (mlU/ml) 60.31 60.45 60.38SHBG (nmol/l) 61.44 57.69 59.41Group A(n=41)Group B(n=51)Overall(n=92)Endometrium(mm)4.3 3.4 3.8Data are presented as mean values.
  19. 19. Wilcoxon-test MF11RCE vs. placeboN NegativeRanksPositiveRankspE2 109 40 43 0,8610,861T 109 61 36 0.003LH 109 64 44 0.861FSH 109 62 47 0.069SHBG 109 58 51 0.824ET 92 15 45 0.001
  20. 20. Changes with MF11RCEBeforeMF11RCEAfterMF11RCEChange(%)T (ng/ml) 0.54 ± 0.26 0.66 ± 0.29 22.12ET (mm) 3.8 ± 1.9 3.2 ± 1.5 -14.69Data are presented as mean ± SD.
  21. 21. Changes in Endometrium after 90 daysof treatment with MF11RCE
  22. 22. Changes in Testosteron serum levelsafter 90 days of treatment
  23. 23. Maturitas. 2006 Aug 20;55(1):76-81.Effects of a red clover extractEffects of a red clover extract (MF11RCE(MF11RCE))onon endometriumendometrium and sex hormones inand sex hormones inpostmenopausal women.postmenopausal women.Martin Imhof, Anca Gocan, Franz Reithmayr, Markus Lipovac,Martin Imhof, Anca Gocan, Franz Reithmayr, Markus Lipovac,Claudia Schimitzek4, Johannes HuberClaudia Schimitzek4, Johannes Huber
  24. 24. Results clinical study - 80 mgisoflavones – 113 women Austria Testosterone levels increasedTestosterone levels increased significantly (p=0,021) Wilcoxon testsignificantly (p=0,021) Wilcoxon testwith MF11RCE, no significant change with placebowith MF11RCE, no significant change with placebo Endometrium thicknessEndometrium thickness waswas reducedreduced withwith MF11RCE (p<0.001)MF11RCE (p<0.001) Climacteric symptomsClimacteric symptoms as evaluated withas evaluated with Kupperman index wasKupperman index wasreducedreduced in general (p<0.001) with MF11RCE general (p<0.001) with MF11RCE treatment.o Hot flushes and sweating were reducedHot flushes and sweating were reduced statistically significantlystatistically significantly(p<0.001)(p<0.001)o Sleeping behavior improvedSleeping behavior improved highly significanthighly significant (p<0.001)(p<0.001)o DepressionDepression andand nervousnessnervousness were significantly reduced (p<0.001)were significantly reduced (p<0.001)o Lack of concentrationLack of concentration was significantly reduced (p<0.001)was significantly reduced (p<0.001) EstradiolEstradiol,, FSHFSH andand LHLH plasmaplasma levels did not change significantlylevels did not change significantlycomparing both groupscomparing both groups
  25. 25. Design clinical study - 80 mgisoflavones – 60 women Ecuador Double-blind, placebo controlled, randomized, cross-overDouble-blind, placebo controlled, randomized, cross-over 90 days 2 capsules menoflavon/day - wash out - 90 days90 days 2 capsules menoflavon/day - wash out - 90 daysPlacebo resp. vice versaPlacebo resp. vice versa 60 women, average age: 51,3 years60 women, average age: 51,3 years Inclusion criteria: Kupperman-Index ≥15, no HRT,..Inclusion criteria: Kupperman-Index ≥15, no HRT,.. 3 examinations (at the beginning, after 90 days, end)3 examinations (at the beginning, after 90 days, end) Kupperman-Index, lipids, hormone levels (E2, FSH, LH,Kupperman-Index, lipids, hormone levels (E2, FSH, LH,Testosterone, SHBG, Lipoprotein a), vaginal cytologyTestosterone, SHBG, Lipoprotein a), vaginal cytology
  26. 26. Effect of 80 mg red clover extracts overKupperman scoring after 3 months (n=53postmenopausal)Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press051015202530baseline red clover placebobaseline red clover placebo
  27. 27. The effect of red clover supplementation over lipid profileavs. baseline; bvs. red clover phase; * p < 0.05;ParameterBaselinen=53After isoflavonesupplementation n=53After placebon=53Total cholesterol(mg/dL)223.9 ± 37.6214 ± 32.2 a* 220.4 ± 34.1 bHDL-C (mg/dL)39.7 ± 11.540 ± 9.6 a41.1 ±10 bLDL-C (mg/dL)146.8 ± 29.9129.7 ± 39.4 a* 140 ± 35.2 bTriglycerides(mg/dL)199.6 ± 77.8181.1 ± 72.3 a* 242.7 ± 166.9 b*Lipoprotein A(mg/dL)41.2 ± 36.922.8 ± 26.9 a* 20.5 ± 25.8 bHidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press
  28. 28. The effect of red clover supplementation over hormonal profileParameterBaselinen=53After isoflavonesupplementation n=53After placebon=53SHBG (nmol/L) 50.1 ± 23.7 46.9 ± 21.9 a49 ± 22 bFSH (mIU/mL)63.7 ± 22.6 63.3 ± 26.1 a62.6 ± 22.1 bLH (mIU/mL) 27.7 ± 13.8 30.8 ± 13.4 a28.7 ± 14 b17 β estradiol(pg/mL)23.8 ± 8.8 22.4 ± 7.2 a20.9 ± 4.6 bTestosterone(ng/dL)24.8 ± 11.221.4 ± 5 a23.2 ± 11.3 bHidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press
  29. 29. Results clinical study - 80 mgisoflavones – 60 women Ecuador No significant effect on BMI, weight and blood pressure inNo significant effect on BMI, weight and blood pressure inboth groupsboth groups significant effect on Kupperman-Index, lipidssignificant effect on Kupperman-Index, lipids Compared to placebo menoflavon significantly decreased:Compared to placebo menoflavon significantly decreased:• menopausal symptoms, like:menopausal symptoms, like:• hot flushes, night sweats, sleeping disorders,hot flushes, night sweats, sleeping disorders,nervousness, depression, dizziness, ....nervousness, depression, dizziness, ....• Triglyceride levelTriglyceride level menoflavon had a positive effect over the vaginamenoflavon had a positive effect over the vagina
  30. 30. CONCLUSIONS 80 mg (MF11RCE) Red Clover does80 mg (MF11RCE) Red Clover doessignificantly decrease menopausal complaintssignificantly decrease menopausal complaints 80 mg (MF11RCE) Red Clover significantly80 mg (MF11RCE) Red Clover significantlyinfluences Testosterone level andinfluences Testosterone level andEndometrium thicknessEndometrium thickness The action of MF11RCE on MCF 7 and HumanThe action of MF11RCE on MCF 7 and HumanEndothelial Breast Cells does not show a riskEndothelial Breast Cells does not show a riskof tumour induction in analysis of geneof tumour induction in analysis of geneactivationactivation
  31. 31. Phytohormones can stimulate MCF 7cell proliferation
  32. 32. 0,0010,0020,0030,0040,0050,0060,0070,0080,00ASR (W)Finland Gr eece China Nor ther nEur ope Souther nEur ope East AsiaPr ostateBr eastColon/ RectumManColon/ RectumWomanCancer Mondial/ Globocan 2000Epidemiological Data CancerEpidemiological Data Cancer
  33. 33. Cell proliferation is notsufficient to describethe risk of tumour development
  34. 34. Experiment 1Cells:- MCF-7 mamma carcinoma cell linePhytohormones:- Red clover - Trifolium pratense, (MF11RCE)- “synthetic” Genistein and Daidzein (Indofine).Technology:- 20K Chip arrays (Affymetrix)
  35. 35. Experimental design:MCF-7 were examined after12 and 24 hours.Experimental groups:- untreated cells- combination of genistein plus daidzein(2,5 µg/ml each)- red clover extract (5 µg/ml)
  36. 36. Flowchart of experimental analysisc-DNAScan Hybridize(16 hours)mRNAIVT(Biotin-UTPBiotin-CTP)Labeled fragmentsL LLLCells
  37. 37. Comparison of the mRNA expression pattern in MC-F7cells stimulated with red-clover extract with that ofdaidzin and genestin stimulated cells.22.000 genes were screened by DNA array and for each gene 11 oligos forperfect match and 11 oligos for mismatch are analyzed. Therefore a total of500.000 spots were quantitatively analyzed.
  38. 38. ResultsA significant increase (>2times) in mRNA expressioninduced by phytohormones was seen for 117 genes.The functional repertoire of these genes cover-Growth arrest genes-DNA repair genes(GADD34)-Growth factors(insulin-like growth factor binding protein 4)
  39. 39. 203720_s _at (ERCC1207348_s _at (LIG3)218685_s _at (SMUG1201236_s _at (BTG2)204461_x_at (RAD1)219510_at (POLQ)201746_at (TP53)203565_s _at (MNAT1202239_at (ADPRTL1204766_s _at (NUDT131861_at (SMBP2)204093_at (CCNH)207405_s _at (RAD17205811_at (POLG2)207598_x_at (XRCC2219317_at (POLI)202330_s _at (UNG)205091_x_at (RECQL34063_at (RecQ5)203719_at (ERCC1)204828_at (RAD9)202332_at (CSNK1E)219502_at (FLJ10858202726_at (LIG1)205733_at (BLM)203725_at (GADD45A218428_s _at (REV1L218961_s _at (PNKP)201459_at (RUVBL2)203210_s _at (RFC5)201529_s _at (RPA1)205024_s _at (RAD51206066_s _at (RAD51208393_s _at (RAD50205189_s _at (FANCC203564_at (FANCG)203806_s _at (FANCA205909_at (POLE2)204603_at (EXO1)203577_at (GTF2H4)207891_s _at (TREX2202453_s _at (GTF2H204408_at (APEX2)204884_s _at (HUS1)203422_at (POLD1)203616_at (POLB)207347_at (ERCC6)208386_x_at (DMC1)202466_at (POLS)203409_at (DDB2)207945_s _at (CSNK1CLUSTERED DNA REPAIR GENESRed CloverSyntheticPhytohormonesunstimulated
  40. 40. Y-axis: Affymetrix Experiment, Default InterpretationColored by: Control_12Gene List: Tumor Suppressor (376), 204159_at selectedControl_12 Control_24 G+D_12h G+D_24h redc_12h redc_24hSample0.010.1110100Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24hSample0.010.1110100CDKN2CCyclin dependent kinase inhibitorCLUSTERED TUMOR SUPRESSOR GENES
  42. 42. Y-axis: Affymetrix Experiment, Default InterpretationColored by: G+D_12hGene List: like 217165_x_at (MT1F) (0.95) (70)Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24hSample0.010.1110100Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24hSample0.010.111010012h 24hRed Clover12h 24hSyntheticPhytohormones12h 24hunstimulatedDecreaseIIncreaseEXTRACT OF GENES THAT ARE DIFFERENTLY REGULATED
  43. 43. ConclusionApplication of red clover and syntheticphytohormones causes a transcriptionalregulation of 117 genes in MCF-7.The effect of synthetic Daidzein/Genistein differssignificantly from that of red clover byupregulation of transcription of 10 genes(Metallothionins).This first results do not implicate a majorinfluence of red clover on the proliferation ordifferentiation of MCF-7 cells.
  44. 44. Experiment 2Cells:- Cell culture from primary mammae epithelialcells (healthy cells)Technology:- 20K Chip arrays (Affymetrix)
  45. 45. Experiment 2Pooled sera of 5 menopausal women . Before andafter intake of MF11RCE (menoflavon) for 3 month• Control group = before intake of MF11RCE• Treatment group = after intake of MF11RCE
  46. 46. Regulation of Transcription01002003004005006007008009001000TCF4TCF7L2MAFBMXI1ID4HOPBTF3BAZ1AGeneEstimatcontroltreatment
  47. 47. Regulation of Transcription• MF11RCE inhibits the expression oftranscriptional genes(TCF4,TCF7L2,MAFB,MXI1,ID4,HOP,BTF3,BAZ1A)and genes in relation to ribosomal subunitjoining and the initiation of translation(EIF3S5, EIF4EBP, RPL29,RPS16)= MF11RCE can influence cell proliferation anddifferentiation by down regulation of cellulartranscription and translation
  48. 48. Genes involved in regulationof DNA0100200300400500600GMNNH2AFVHIST1H2HMGB2MCM5PCNAPRKDCPRM1GeneEstimatcontroltreatment
  49. 49. Genes involved in regulation ofDNA• MF11RCE down regulates genesinvolved with DNA replication.(GMNN,H2AFV,HIST1H2,HMGB2,MC5,PCNA,PRKDC,RM1)= MF11RCE has an anti-proliferativeeffect through inhibition of DNAreplication
  50. 50. Apoptosis0200400600800100012001400160018002000AFURS1 ENC1 PORIMINgenessignalestimatescontroltreatment
  51. 51. Apoptosis• MF11RCE induces Expression of(AFURS1, ENC1/PIG10 p53 induced gene 10)and inhibits Porimin= MF11RCE can rise the p53 level(ENCI/PIG10) and activateapoptosis.= MF11RCE can induce senescence(AFURS1)
  52. 52. Regulation of cell proliferation020040060080010001200JAG1RARRESVEGFMETgenessignalestimatescontroltreatment
  53. 53. Regulation of cellproliferation• MF11RCE inhibits JAG1,RARRES,VEGF,MET. (cell proliferation)and cell differentation (Keratin 10,Keratin 19).= MF11RCE inhibits genes ofcell-proliferation and cell-differentation
  54. 54. In a culture of primary mammae epithelialcells MF11RCE down regulates genes for–Transcription and translation–Protein synthesis–DNA replication–Cell proliferationand up regulates genes for–Apoptosis
  55. 55. ConclusionMF11RCE seems not to beinductive for tumor and/or tumorgrowth in a healthy mammaepithelial cells culture.Further study are necessary toimprove if Isoflavones canprotect against tumordevelopment an/or tumor growth
  56. 56. • Georg Steiner• Michael Loeffler• Anka Gocan• Marianne Imhof• Kristian Hrachowitz• Sylvia Molzer• Markus Lipovac• Johannes Huber