florenz imhof 2006

Clinical Action and Safety of Red Clover Isoflavones

Martin Imhof

General Public Teaching Hospital Korneuburg/Vienna

Medical University - Vienna

• Million Women Study Collaborators. Breast cancer and hormone- replacement therapy in the Million Women Study. Lancet 2003; 362:419-27

• Women´s Health Initiative (WHI) Study – Interrupted due to severe side effects

Herbal Alternatives

to

Hormone Replacement Therapy

How Safe and Effective are Phytohormones?

Are Red Clover Isoflavones Effective ?

A Double Blind, Randomized,

Placebo-controlledCross-Over Study

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Baber RJ, et al. Climacteric 1999

Effect of 40 mg red clover extracts over No. Daily hot flashes and Greene Scores (n=51 postmenopausal)*

*RDBPC study

Tice JA, et al. JAMA 2003

Effect of 80 mg vs. 57 mg red clover extracts vs placebo over No. hot flashes freq and Greene Scores (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).

Van Weijer PH, et al. Maturitas 2002

Effect of 80 mg red clover extracts vs placebo over hot flashes frequenz and Greene Scores (n=30 postmenopausal)*

4 weeks placebo run in phase 4 weeks placebo run in phase (16% reduction).(16% reduction).

After 12 weeks:After 12 weeks: Red clover reduced hot flashes Red clover reduced hot flashes

freq 44%.freq 44%. Placebo no further reduction.Placebo no further reduction.

*RDBPC study

• 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 2005

Menoflavon Study 80mg isoflavones /d of red clover – clinical trial Austria

Examinations and questionnaires

Kupperman scoreKupperman score (index before start >15) (index before start >15) Changes in Changes 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 the Effects on the endometriumendometrium Vaginal smearVaginal smear

(cytodiagnosis)(cytodiagnosis) Urine-analysisUrine-analysis (urinary excretion of isoflavones) (urinary excretion of isoflavones) Depressions scaleDepressions scale after Zerssen after 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….

Demographic and anamnestic data

Group A(n=50)

Group B(n=59)

Overall(n=109)

Mean age (yrs.) 54.5 ± 6.2 53.7 ± 7.8 53.5 ± 7.1

Mean BMI 24.5 ± 3.9 24.9 ± 3.9 24.7 ± 3.9

Hysterectomy (%) 18.0 13.6 15.6

Kupperman Index

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MF11RCE Placebo

gestiegen

gefallen

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Wilcoxon Test, PVerum < 0,001, PPlacebo < 0,001

Reduction of hot flushes

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MF11RCE Placebo

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abgenommen

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Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,001

Hot flushes, Boxplots-comparison

Hot flushes were reduced significantly with MF11RCE (p<0,001)

No effect under Placebo

before after

MF11RCE

before afterPlacebo

Reduction sleep disorders

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Zunahme

Abnahme

keine Veränderung

Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,018

Sleeping behavior - Boxplots-comparison

before afterMF11RCE Placebo

afterbefore

Sleeping behavior improved high significantly with MF11RCE (p<0,001) No effect under Placebo

Mood improvement

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Wilcoxon Test, PVerum < 0,001, PPlacebo = 0,001

Baseline measures

Group A(n=50)

Group B(n=59)

Overall(n=109)

E2 (pg/ml) 36.30 38.31 37.39

T (ng/ml) 0.43 0.63 0.54

LH (mIU/ml) 30.64 29.92 30.25

FSH (mlU/ml) 60.31 60.45 60.38

SHBG (nmol/l) 61.44 57.69 59.41

Group A(n=41)

Group B(n=51)

Overall(n=92)

Endometrium (mm)

4.3 3.4 3.8

Data are presented as mean values.

Wilcoxon-test MF11RCE vs. placebo

N Negative Ranks

Positive Ranks

p

E2 109 40 43 0,8610,861

T 109 61 36 0.003

LH 109 64 44 0.861

FSH 109 62 47 0.069

SHBG 109 58 51 0.824

ET 92 15 45 0.001

Changes with MF11RCE

Before MF11RCE

After MF11RCE

Change (%)

T (ng/ml) 0.54 ± 0.26 0.66 ± 0.29 22.12

ET (mm) 3.8 ± 1.9 3.2 ± 1.5 -14.69

Data are presented as mean ± SD.

Changes in Endometrium after 90 days of treatment with MF11RCE

Changes in Testosteron serum levels after 90 days of treatment

Maturitas. 2006 Aug 20;55(1):76-81.

Effects of a red clover extract Effects of a red clover extract (MF11RCE(MF11RCE) ) onon endometrium endometrium and sex hormones in and sex hormones in

postmenopausal 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

Results clinical study - 80 mg isoflavones – 113 women Austria

Testosterone levels increasedTestosterone levels increased significantly (p=0,021) Wilcoxon test with MF11RCE, no significantly (p=0,021) Wilcoxon test with MF11RCE, no significant change with placebosignificant change with placebo

Endometrium thicknessEndometrium thickness was was reduced reduced withwith MF11RCE (p<0.001) MF11RCE (p<0.001) Climacteric symptomsClimacteric symptoms as evaluated with as evaluated with Kupperman index was reducedKupperman index was reduced in general in general

(p<0.001) with MF11RCE treatment.(p<0.001) with MF11RCE treatment.o Hot flushes and sweating were reducedHot flushes and sweating were reduced statistically significantly (p<0.001) statistically significantly (p<0.001) o Sleeping behavior improvedSleeping behavior improved highly significanthighly significant (p<0.001) (p<0.001) o DepressionDepression and and nervousnessnervousness were significantly reduced (p<0.001) were significantly reduced (p<0.001) o Lack of concentration Lack of concentration was significantly reduced (p<0.001) was significantly reduced (p<0.001)

EstradiolEstradiol, , FSHFSH and and LHLH plasmaplasma levels did not change significantly comparing both groups levels did not change significantly comparing both groups

Design clinical study - 80 mg isoflavones – 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 days Placebo resp. 90 days 2 capsules menoflavon/day - wash out - 90 days Placebo resp. vice versavice 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, Testosterone, Kupperman-Index, lipids, hormone levels (E2, FSH, LH, Testosterone, SHBG, Lipoprotein a), vaginal cytologySHBG, Lipoprotein a), vaginal cytology

Effect of 80 mg red clover extracts over Kupperman scoring after 3 months (n=53

postmenopausal)

Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press

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baseline red clover placebo

baseline red clover placebo

The effect of red clover supplementation over lipid profile

a vs. baseline; b vs. red clover phase; * p < 0.05;

ParameterBaseline

n=53After isoflavone

supplementation n=53After placebo

n=53

Total cholesterol (mg/dL)

223.9 37.6 

214 32.2 a * 220.4 34.1 b

HDL-C (mg/dL)39.7 11.5

 40 9.6 a 41.1 10 b

LDL-C (mg/dL)146.8 29.9

 129.7 39.4 a * 140 35.2 b

Triglycerides (mg/dL)

199.6 77.8 

181.1 72.3 a * 242.7 166.9 b *

Lipoprotein A (mg/dL)

41.2 36.9 

22.8 26.9 a * 20.5 25.8 b

Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press

The effect of red clover supplementation over hormonal profile

ParameterBaseline

n=53After isoflavone

supplementation n=53After placebo

n=53

SHBG (nmol/L) 50.1 23.7 

46.9 21.9 a 49 22 b

FSH (mIU/mL)63.7 22.6

 63.3 26.1 a 62.6 22.1 b

LH (mIU/mL) 27.7 13.8

 30.8 13.4 a 28.7 14 b

17 β estradiol (pg/mL)

23.8 8.8 

22.4 7.2 a 20.9 4.6 b

Testosterone (ng/dL)

24.8 11.2 

21.4 5 a 23.2 11.3 b

Hidalgo L, Chedraui P, et al. Gynecol Endocrinol, 2005, In press

Results clinical study - 80 mg isoflavones – 60 women Ecuador

No significant effect on BMI, weight and blood pressure in both groupsNo significant effect on BMI, weight and blood pressure in both 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, nervousness, hot flushes, night sweats, sleeping disorders, nervousness, depression, dizziness, ....depression, dizziness, ....

• Triglyceride levelTriglyceride level

menoflavon had a positive effect over the vaginamenoflavon had a positive effect over the vagina

CONCLUSIONS

80 mg (MF11RCE) Red Clover does 80 mg (MF11RCE) Red Clover does significantly decrease menopausal complaints significantly decrease menopausal complaints

80 mg (MF11RCE) Red Clover significantly 80 mg (MF11RCE) Red Clover significantly influences Testosterone level and influences Testosterone level and Endometrium thickness Endometrium thickness

The action of MF11RCE on MCF 7 and Human The action of MF11RCE on MCF 7 and Human Endothelial Breast Cells does not show a risk Endothelial Breast Cells does not show a risk of tumour induction in analysis of gene of tumour induction in analysis of gene activation activation

Phytohormones can stimulate MCF 7 cell proliferation

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ASR (W)

Finland Greece China Northern Europe Southern Europe East Asia

Prostate

Breast

Colon/ Rectum Man

Colon/ Rectum Woman

Cancer Mondial/ Globocan 2000

Epidemiological Data CancerEpidemiological Data Cancer

Cell proliferation is not

sufficient to describe

the risk of tumour development

Experiment 1

Cells: - MCF-7 mamma carcinoma cell line

Phytohormones: - Red clover - Trifolium pratense, (MF11RCE) - “synthetic” Genistein and Daidzein (Indofine).

Technology:- 20K Chip arrays (Affymetrix)

Experimental design:

MCF-7 were examined after 12 and 24 hours.

Experimental groups:- untreated cells- combination of genistein plus daidzein (2,5 µg/ml each)- red clover extract (5 µg/ml)

Flowchart of experimental analysis

c-DNA

Scan Hybridize

(16 hours)

mRNA

IVT

(Biotin-UTPBiotin-CTP)

Labeled fragments

L L

L

L

Cells

Comparison of the mRNA expression pattern in MC-F7 cells stimulated with red-clover extract with that of

daidzin and genestin stimulated cells.

22.000 genes were screened by DNA array and for each gene 11 oligos for perfect match and 11 oligos for mismatch are analyzed. Therefore a total of

500.000 spots were quantitatively analyzed.

Results

A significant increase (>2times) in mRNA expression

induced 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)

Selected Gene Tree: Affymetrix Experiment (Default Interpretation)Colored by: Affymetrix Experiment, Default Interpretation

203720_s_at (ERCC1)207348_s_at (LIG3)218685_s_at (SMUG1)201236_s_at (BTG2)204461_x_at (RAD1)219510_at (POLQ)201746_at (TP53)203565_s_at (MNAT1)202239_at (ADPRTL1)204766_s_at (NUDT1)31861_at (SMBP2)204093_at (CCNH)207405_s_at (RAD17)205811_at (POLG2)207598_x_at (XRCC2)219317_at (POLI)202330_s_at (UNG)205091_x_at (RECQL)34063_at (RecQ5)203719_at (ERCC1)204828_at (RAD9)202332_at (CSNK1E)219502_at (FLJ10858)202726_at (LIG1)205733_at (BLM)203725_at (GADD45A)218428_s_at (REV1L)218961_s_at (PNKP)201459_at (RUVBL2)203210_s_at (RFC5)201529_s_at (RPA1)205024_s_at (RAD51)206066_s_at (RAD51C)208393_s_at (RAD50)205189_s_at (FANCC)203564_at (FANCG)203806_s_at (FANCA)205909_at (POLE2)204603_at (EXO1)203577_at (GTF2H4)207891_s_at (TREX2)202453_s_at (GTF2H1)204408_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 (CSNK1D)

CLUSTERED DNA REPAIR GENES

Red CloverSynthetic Phytohormones

unstimulated

Y-axis: Affymetrix Experiment, Default InterpretationColored by: Control_12Gene List: Tumor Suppressor (376), 204159_at selected

Control_12 Control_24 G+D_12h G+D_24h redc_12h redc_24h

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CDKN2C

Cyclin dependent kinase inhibitor

CLUSTERED TUMOR SUPRESSOR GENES

Results

THE GENES DIFFERENTIALLLY UP-REGULATED BY

SYNTHETIC PHYTOESTROGENS ARE

“METALLOTHIONEINS” (MT)

AND ARE BELIVED TO PLAY AN ROLE IN

TUMORGENESIS.

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_24h

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EXTRACT OF GENES THAT ARE DIFFERENTLY REGULATED

Conclusion

Application of red clover and synthetic phytohormones causes a transcriptional regulation of 117 genes in MCF-7.

The effect of synthetic Daidzein/Genistein differs significantly from that of red clover by upregulation of transcription of 10 genes (Metallothionins).

This first results do not implicate a major influence of red clover on the proliferation or differentiation of MCF-7 cells.

Experiment 2

Cells:

- Cell culture from primary mammae epithelial

cells (healthy cells)

Technology:- 20K Chip arrays (Affymetrix)

Experiment 2

Pooled sera of 5 menopausal women . Before and after intake of MF11RCE (menoflavon) for 3 month

• Control group = before intake of MF11RCE

• Treatment group = after intake of MF11RCE

Regulation of Transcription

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F4

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F7L2

MAFB

MXI1

ID4

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BAZ1A

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Regulation of Transcription

• MF11RCE inhibits the expression of transcriptional genes (TCF4,TCF7L2,MAFB,MXI1,ID4,HOP,BTF3,BAZ1A) and genes in relation to ribosomal subunit joining and the initiation of translation (EIF3S5, EIF4EBP, RPL29,RPS16)

= MF11RCE can influence cell proliferation and differentiation by down regulation of cellular transcription and translation

Genes involved in regulation of DNA

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Genes involved in regulation of DNA

• MF11RCE down regulates genes involved with DNA replication.

(GMNN,H2AFV,HIST1H2,HMGB2,MC5,PCNA,PRKDC,RM1)

= MF11RCE has an anti-proliferative effect through inhibition of DNA replication

Apoptosis

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Apoptosis • MF11RCE induces Expression of

(AFURS1, ENC1/PIG10 p53 induced gene 10) and inhibits Porimin

= MF11RCE can rise the p53 level (ENCI/PIG10) and activate apoptosis.

= MF11RCE can induce senescence (AFURS1)

Regulation of cell proliferation

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Regulation of cell proliferation

• MF11RCE inhibits JAG1,RARRES, VEGF,MET. (cell proliferation) and cell differentation (Keratin 10, Keratin 19).

= MF11RCE inhibits genes of cell-proliferation and cell-differentation

In a culture of primary mammae epithelial cells MF11RCE down regulates genes for

– Transcription and translation– Protein synthesis– DNA replication– Cell proliferation and up regulates genes for

– Apoptosis

Conclusion

MF11RCE seems not to be inductive for tumor and/or tumor growth in a healthy mamma epithelial cells culture.

Further study are necessary to improve if Isoflavones can protect against tumor development an/or tumor growth

• Georg Steiner • Michael Loeffler• Anka Gocan • Marianne Imhof • Kristian Hrachowitz• Sylvia Molzer• Markus Lipovac

• Johannes Huber