Wagner 26 Key Note Address

Folie 1Prof. H. Wagner – Center of Pharmacy Research – Pharmaceutical Biology – University of Munich – Germany

Prof. H. WagnerCenter of Pharmacy Research – Pharmaceutical Biology – University of Munich – Germany

e-mail: [email protected]

International Herbal Conference 2009Herbal Medicine – Evaluating of Quality, Efficacy and

Safety in the changing global Scenario

Bangalore, February 26 – 28, 2009

Approaching a new generation of novel Phytopharmaceuticals – Synergy-Research


Facts

Of about 350000 plant species, including algae, protozoa, fungi and bacteria only 20-30% have been investigated thoroughly and only 5-10% are used in Traditional Medicine

The paradigm "Monosubstance(drug) Therapy" failed, now gradually replaced by Multidrug and Multitarget-Therapy

Synergy Research mandatory

Of about 2000 registered acute and chronic diseases only ~40% are presently curable, a further ~40% diseases are only symptomatically or imperfectly treatable and ~20% not at all

The resistance of pathogenic microorganisms against antibiotics is increasing dramatically


Characteristic of novel Phytopharmaceuticals

Applicable alone for therapy or in combination with synthetic drugs or antibiotics

Use also for the treatment of diseases which up to now were reserved for the synthetic drugs or antibiotics only

Standardized, more effective and causatively acting mono- or multiherbal extracts

Less or lacking side effects


Which efforts are mandatory to achieve this goal?

Integration of all modern high-tech analytical and molecular biological methods inclusive omic technology

Appointment of national commissions

inventory of traditionally used medicinal plants to evaluate the medicinal plant resources of a country

Development of Herbal Monographs with valuation of quality, safety and efficacy of herbs and their extracts


German Commission E-Monographsas model

133 negative or zero-monographs(not recommended for therapy,

negative-benefit-risk rate)

Herbal medicinal products of well established use

("evidence-based medicine")

Supplemented by ESCOP-, WHO and European Pharmacopoea-Monographs

inclusive special Analytical Monographs

Novel Phytopharmaceuticals

378 plants used in traditional medicine have been investigated to determine their

quality, safety and efficacy

245 positive monographs of single plants and fixed

combinations

Herbal medicinal products from traditional use or


High-tech analytical and isolation methods for plant screening and isolation work

Thin-layer chromatography (TLC), thin-layer electrophoresis (TLE)

Isotachophoresis (TIP) Capillary electrophoresis (CE) Capillary electrochromatography (CEC) HPLC, gas chromatography (GC) HPLC coupled with MS (chemical and ionization or

electrospray ionization technique) Liquid chromatography

(LC coupled with UV/MS/NMR/Fourier transform ion cyclotron resonance (FT-ICR))


Books for Fingerprint Analysis

by Prof. Xiao Peigen


TLC Fingerprint Analysis of Angelicae sinensis radix (Danggui)

1 2 3 4 5 R 6 7 8

-Start

-Front

1 2 3 4 5 R 6 7 8

-Start

-Front

1 2 3 4 5 R 6 7 8

-Start

-Front

UV 254 nm

UV 360 nm

Anisaldehyde sulphuricacid reagent (VIS)

# Origin + Species

1 Koetzting 08.07.93 A. sinensis2 Singapore A. sinensis3 East Earth Herb Inc. A. sinensis4 Kanton 12.03.96 A. sinensis5 Kun Ming 12.03.96 A. sinensisR Reference compounds Z-Ligustilide,

+ linoleic acid,falcarindiol

7 China, authentic A. acutiloba8 China, authentic A. dahurica9 China, authentic A. pubescens

Zschocke S., Wagner H., Bauer R., Xiao P.G., Chen J.M.:Chinese Drug Monographs and Analysis, in press, 2000


HPLC Fingerprint Analysis of Angelicae pubescens radix (Duhuo), other species

and adulterations

Source: Liu Jianghua, Wagner H, Bauer R, Xiao PG, Chen JM: Chinese Drug Monographs and Analysis, Vol. 2(9), 1999

10 20 30Time (min)

ab

cd

e f

g h i j k

02

00

40

0m

AU

02

00

40

0

Angelica dahurica

10 20 30

a

b

ef

g

hi

j

k

Time (min)

02

00

40

0m

AU

Angelica apaensis

c

l

n k

020

040

0m

AU

10 20 30Time (min)

Heracleum moellendorffii

10 20 30Time (min)

020

040

0m

AU

k Aralia cordata

0

200

400

m

AU

10 20 30

oc

d

hi

k

Time (min)

Heracleum candicans

10 20 30Time (min)

1

23

4

56

78

9

10

11

12

02

00

mA

U0

20

010

0 Angelica pubescens


Adulteration or mixups of the root ofStephania tetrandra (Hanfangji) with the root of

Aristolochia fangchi (Guangfangji)

1 2 3 4 T1/2 5 T3 6 7Start

R = 0,5

Front

50:50 and 80:20 mixtures of Stephania- and Aristolochia root extracts

Retention Time (min)

Ab

sorb

an

ce (

AU

)

Aristolochic acids detectable up to 400 pg

0 5 10 15 20 25 30 25 30

0,06

0,04

0,02

0,00

3

3

2

1

1 - 4: Stephania root extractT1/2: Tetrandrine + Fangchinolin5: Aristolochic acids I + IIT3: Aristolochiae radix 6 + 7: Mixtures of Stephania- and

Aristolochia root extracts

Aristolochic acids detectable up to 8 pg/g Herbal drug

HPTLC- and HPLC-detection of Aristolochic acids in root samples of Stephania tetrandra


Differentiation of Codonopsis species by DNA fingerprinting

Fu et al., Planta Medica 65: 648-650 (1999)

1 = Codonopsis pilosula

2 = Codonopsis tangshen

3 = Codonopsis modesta

4 = Codonopsis nervosa

5 = Campanumoea javania

6 = Platycodon grandiflorus

M = 100 bp mol. weight marker

with a 800 bp intensive band

PCR-RFLP patterns of rDNA ITS using restriction enzyme Hha I after

separation by 3.5 % TBE agarose gel


Composition of the Japanese herbal medicine Sho-seiryu-to extract (TJ-19)

Treatment of bronchitic asthma and allergic rhinitis

Pinelliae pernatae tuber

Glyzyrrhizae glabrae radix

Cinnamomi cassiae cortex

Schisandrae chin. fructus

Asasari sieb. radix

Paeoniae lactifl. radix

Ephedrae sin. herba

Zingiberis offic. rhizoma

Amagaya et al. Phytomedicine 8: 338-347 (2001)


3 D HPLC fingerprint analysis of Sho-seiryu-to(TJ-19)extract produced from eight herbal drugs

S. Amagaya et al., Phytomedicine 8: 338-347 (2001)


Omic Technology

Red: inductionGreen: repressionBlack: no differential

regulation

Bio-chip(photo by Miltenyi Biotec)


Complex mixtures cause multitarget effects on molecular base and lead to characteristic gene- and proteine expression profiles which are different of that of single compoundssynergistic effects

Simplification of the standardization process of herbal drug mixturesconsequences for legislation and patenting

The National Center for Toxicogenomics (NCT) in USA, in the National Institute of Environmental Health Science (NIEHS) provides a reference system of genome wide gene expression data the identification of the toxic potential of chemicals and

plant extracts.

Futural aspects of the omic technology for Phytomedicine


Therapeutics and preventivesCancer

Antihypertonics Antiatherosclerotics Antiischemics (drugs for stroke prevention)

Cardiovascular diseases

Therapeutics and preventives for Alzheimer disease Parkinson

CNS diseases

Antibacterial drugs (e.g. Antituberculostatics) Antiviral drugs (e.g. Anti-HIV, Anti-Hepatitis B + C) Antiparasidal drugs

(e.g. against Malaria, Chagas, Leishmaniasis) Antifungal drugs

Infectious diseases

Antiasthmatics Drugs against bowle syndrom Antineurodermitic / Antipsoriatic drugs

Inflammatory diseases

Development of novel PhytopharmaceuticalsNew drugs of high priority worldwide wanted


High through put screening methods

Thousands of plant extracts or pure compounds can be screened per month



Experiences higher efficacy than a single herbal drug or constituent

less or lacking side effects dose reduction possible

Traditional Medicine of many countries used herbal drug combinations from its

very beginning

Hypotheses

Conclusion: therapeutic superiority may be due tosynergy- and multitarget effects

complex (multicausal) pathophysiology can be better positively influenced by a drug combination than by a single highly dosaged drug

more causative therapy possible concomitant symptoms and damages also curable


Monosubstance Therapy

Multidrug- and Multitarget- Therapy(e.g. AIDS-, cancer-, hypertension-therapy)

Synergy research as answer to the paradigm change in Drug Therapy


Increase of helper-T-cells(cells / mm3)

Decrease of HIV-1 RNAin plasma (log10 -copies / ml)

Comparison of the efficacy of differentAnti-AIDS drug combinations

Zidovudin / Lamivudin / Delaviridin Zidovudin / Delaviridin Zidovudin / Lamivudin

weeks

0

20

40

60

80

100

120

6010 30 40 5020

weeks

0

-3

-2.5

-2

-1.5

-1

-0.5

6010 30 40 5020


A total effect of a combination is greater than expected from the sum of the individual agents

E (da,db) > E (da) + E (db)

Definition of synergy effects(Berenbaum Pharmacol. Rev. 41:93-141,1989)

E = observed effect da and db = doses of agents a and b

The effect of a combination is greater than that of each of the individual agents

E (da,db) > E (da) and E (da,db) > E (db)


Dose B

Dose A

antagonism

synergism

zero-interaction

antagonism = negative interaction synergism = positive interaction or potentiation zero-interaction = effects-addition of individual components

Pharmacological proof of synergy effects

by the isobol method (Berenbaum 1985)


Drug-synergism of phytopharmaceuticals

overadditive, potentiated pharmacological effects

additive, agonistic pharmacological effects

different targets(multitargeting)

one target

plant extract


IC50 – values for various dose-combinations of PAF-induced thrombocyte aggregation*

GA : GB

3 : 1 2 : 1 1 : 1 1 : 2 1 : 3 1 : 10

IC50 [µg/ml]

2.402.201.801.551.401.30

Ginkgolide A [µM]

4.413.602.211.270.880.29

Ginkgolide B [µM]

1.421.722.122.432.572.79

Ginkgolide A [µM]

Ginkgolide B [µM]

synergism

zero-interaction

14

12

10

8

6

4

2 0

0.5 1 1.5 2 2.5 3 3.5

O O

OH

O

O

O

OO

HO

HO

OH

OH

H

O O

OH

O

O

O

OO

HO

HO

OH

H

H

Isobol measurements of Ginkgolide AB-combination*


What could be the causes of therapeutic superiority of many herbal drug

combinations?

Interaction of one component of the drug combination (antibiotic drug) with resistance mechanism of pathogenic microorganisms

Elimination or "neutralisation" of adverse acting compounds by components of the drug combination

Synergistic multitarget action of extract constituents

Concomitant constituents increase the solubility and resorption rate and thereby the bioavailability of bioactive compounds


Xanthones(traces)

Procyanidins, Tannins2 – 15%

Hyperforin(Adhyperforin)

1 – 7%

HO

O O

OH

Major constituents ofHypericum perforatum extract

Hypericin,Pseudohypericin

0.1 – 0.4%

Flavonoids(Rutin, Hyperoside,

Quercetin, 13,18-Biapigenin)5 – 12%


Multitarget effects of Hypericum perforatum according to in vitro studies

Presynaptic neuron

H+

Na++5-H“ 5-H“

?

COMTMAO

_

Postsynaptic neuron

Hypothalamus

Cortisol

IL-6

Adrenal cortexPituitaryACTH

Prolactin

?

CRF

CRF

TRH

b-adr.5-HT1A/2ADA2,3,4 NMDA

Opioid

GABAA

BenzoSigma

5-HT6,7

H1,3

NK-I

_

_

+_

_

_

HyperforinHypericum

FlavonoidsXanthones

Hypericum

Amentoflavon

HyperforinHypericum

Hypericum

Estrogen

13,118-Biapigenin

Hypericin

U. Simmen et al. Pharmacopsychiatry 34 Suppl.. 1: 137-142 (2001)


Multivalent pharmacological effects of Hypericum-extracts

blockade of2-receptors

modulationof –receptor-

packing

down regulationof 5-HT2-receptors

modulation of neurotransmitterconcentrations

(Nor-adrenalin, serotonin, GABA, L-glutamate)

monoamine oxidase inhibition

Antidepressive Anxiolytic Nootropic Antiepileptic

Chemistry: Hypericins, hyperforin, flavonoids, procyanidins


The accompanying procyanidin B2 and hyperoside of the hypericum extract increase the water solubility and oral bioavailability of

hypericin by 58% / 34% as evidenced by the forced swim test (Porsolt test)

Butterweck et al. Planta Med. 69:189-192 (2003)

[min]

Hyp

eri

cin

[n

g/m

l]

Arguments for existing synergy effects of Hypericum perforatum extracts I

Plasma levels of hypericin in the presence ( )and absence ( ) of procyanidin B2

10

5

00 100 200 300 400 500 600 700 800 900 1000 1100

Hypericin +procyanidinB2

*

**

** Hypericin


appetite stimulating

analgesic

anxiolytic

antiphlogistic

antiemetic

musclerelaxing

sedative

Tetrahydrocannabinol (THC) exerts polyvalent pharmacological activities

C5H11

OH

O

9-THC


Cannabis extract is a better antispastic agent in mice than tetrahydro-cannabinol (THC) at an equivalent dose.

******

***

**

*

***

# #

# #

0 10 20 30 40 50 60 70 80 90

D9Tetrahydrocannabinol (1mg/kg)

P<0.002 by ANOVA

Cannabis extract (5mg/kg)Containing 20% D9THC

10

0

-10

-50

-20

-30

-40

Pharmacological evidence for synergistic effects

Percentage change in resistance to Flexion ± SEM

Baker et al. Nature 404:84-87; (2000); in E.M. Williamson Phytomedicine 8(5):401-404 (2001)

Time (min)


Ginkgo biloba: Ginkgolide mixtures/ Chung et al. (1987)Ginkgo extract

Piper methysticum: Kava lactones/mixtures Singh and Blumental (1997)of Kava lactones and extract fractions

Glycyrrhiza glabra: Licorice extract potentiates Cantelli-Forti et al. (1994)other substances and acts Kimura et al. (1992)as detoxifier Miaorong and Jing (1996)

Cannabis sativa: Cannabis extract / THC Zuardi et al. (1982)Baker et al. (2000)

Valeriana offic.: Valeriana extract/ Hölzl (1997)individual constituents

Zingiber offic.: Zingiber extract/ Beckstrom-Sternberg andmixture of volatile terpenoids Duke (1994)and mixtures

Kava-kava + Passiflora incarn. Capasso and Sorrentino (2005)

In vitro and in vivo pharmacological evidences for synergy effects

(according to Williamson, Phytomedicine 8(5):401-409 (2001)


Strategies of bacteria to antagonize the effect of antibiotics

Receptor or activesite modification

Decreasedpenetration

Increasedefflux

Enzymatic degradation ormodification of antibiotic

AC

D

B

efflux pump

enzyme

degradation of the drug

antibiotic drug

receptor

modified receptor

D*

C*

A*

B*

(Mukesh Doble,Review Phytomedicine in press)


A. Rosato at al. Phytomedicine 14:727 (2007)

Synergistic effect of Pelargonium graveolens essential oil with Norfloxacin in inhibiting

Staphylococcus aureus ATCC 6538 I

The isabole method describing synergyStaphylococcus aureus ATCC 6538

Pelargonium graveolens oil mg/ml

No

rflo

xa

cin

µg

/ml

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8

0,1

0,2

0,3

0,4

0,5

0,6

0

* FIC = Fractional inhibitory concentration ** FICI = FIC index = FIC of oil + FIC of Norfloxacin

Pelargonium graveolens + Norfloxacin

Pelargonium graveolens oil mg/ml FIC* = 0.25 FICI = 0.37Norfloxacin µg/ml FICI** = 0.12


Synergism between herbal andother drugs II

Synergistic effects of 7-Methyl-Juglone (7-MJ) in combination with antituberculous drugs against Mycobacterium tuberculosis

NB Bapela et al. Phytomedicine 2006, 13:630-635

7-MJ has superior extracellular and intracellular activity against M.t. relative to streptomycin

The combination of 7-MJ with IN reduces the MICs of both compounds by eight-fold

Results

MIC1) (µg/ml) and FIC2) of drugs and drug-combinations against M. tuberculosis H37Rv strain

H37RvMIC µg/ml FIC µg/ml

7-Methyl-Juglone (7-MJ) 5 –Rifampicin (RMP) 0.5 –Isoniazide (IN) 0,06 –7-Methyl-Juglone + Rifampicin 1.25/0.125 0.57-Methyl-Juglone + Isoniazide 0.62/0.007 0.24

Intercellular MICs and FICs of combination of drugs acting againstM. tuberculosis by the radiometric BACTEC method

1) Minimum inhibitory concentration2) Fractional inhibitory concentration

FIC ≤ 0.5 synergistic effectFIC = 1 additive effectFIC > 2 antagonistic effect


Synergism between herbal andother drugs III

Ref. Han Yongmoon Phytomedicine 2007, in press

Synergistic effects of Vitis vinifera seeds (grape seed extract = GSE) with amphotericin B (Amp) against disseminated Candidiasis in mice

Combination of grape seed extract with Amp. B results in a more than 75% reduction of Amp. B

The MST value of the mice group which received the combination was greater than MST value from mice group given four times Amp. B dose of 0,5mg/kg bw.

Results

Days

Sur

vivo

rs

0

1

2

3

4

5

4010 3020

DPBSAmp aloneGSE aloneAmp plus GSE

11.4 ± 3.214.4' ± 2.617.6' ± 7.338.4 ± 8.0

MST (Days)


Synergism between natural products and antibiotics against bacterial infection I

Epigallocatechingallate (EGCg)

Natural products: e.g. Rugosin B, Tea catechin, Baicalin, Plumbagin, isoflavones, essential oil (e.g. 1,8-cineol, -terpineol)

Tellimagrandin I

Corilagin

(Hemaiswarya et al., Phytomedicine 15: 639 - 652 (2008))

Modifiers of Multidrug resistance mechanisms

+

=

Antibiotics: e.g. Penicillin, Ampicillin, Vanomycin, Gentamicin, Ciprofloxacin, Tetracycline, Erythromycin

OH

OH

OH

O

O

OH

OHOH

OH

OHO

2

OH

OH

OH

CO

OH

OH

OHOC

O CH

O

OO

O

OHOH

OH

OH

OH

OH

OH

CO

COOH

OH

OH

OO

OH

OH

OH

O

OH

OH OH

O

O

O

OH OH

O

3

6


Synergism between natural products and antibiotics against bacterial infection II(Hemaiswarya et al., Phytomedicine 15: 639 - 652 (2008))

Isopimaric acid Carnosic acid Carnosol

OH

OH

O

O

OH

OH

HOOC

CO2H



Comparative double blind study with Hypericum extract and Imipramine

Indication: moderate neurotic depression

Dosage: Hypericum: 3 x 300 mg extract /day 80-100 mg Hypericines, Hyperforine, Amentoflavone, Procyanidines

Imipramine: 3 x 25/35 mg

Parameter: Hamilton-Depression Scale (HAMD)

Score

5

10

15

20

25

1 2 4 6 Weeks

Imipramine Li 160

0

Vorbach et al. 1997 / Woelk 2000


Multitarget therapy of dyspepsia and motility-related disorders of the gastrointestinal tract

with a combination of 9 plant extracts

Iberis amara (totalis)

Angelica archangelica (radix)

Matricaria chamomilla (flos)

Silybum marianum (fruits)

Melissa officinalis (folium)

Mentha piperita (folium)

Chelidonium majus (herba)

Glyzyrrhiza glabra (radix)

Carum carvi (fruits)


12 clinical trials and 3 metaanalyses performed

Clinical studies in comparison with the prokinetics Metoclopramide® and Cisapride®

Result: – Full therapeutic equivalence or superiority over the synthetic drugs

– No or lesser side effects

Clinical evidence of Iberogast®,a multidrug phytopharmaceutical for the

treatment of dyspepsia


Treatment of dyspepsia andmotility-related disorders of the

gastrointestinal tract

with a herbal drug combination(Iberogast ®, consisting of 9 plant extracts)

Multiple mechanismsof the disease

spasms acid secretion

ulcus/inflam-mation

radical production

atoniahypo-

motilityhyper-motility

Iberis

Angelica

Carum

Silybum

Chelidonium

Glycyrrhiza

Chamomilla

Melissa

Mentha

none moderate strong effectsPhytomedicine Suppl. V 13 (2006)


Option for Monotherapy

Hypermotility Spasmolytic agent (Buscopan ®)

HypomotilityProkinetics (e.g. Cisaprid ®,

Metoclopramid ®, 5-HT-Antagonists)

Hypersensibility no standard therapy available

Hyper acid secretion proton pump inhibitor (e.g. Omeprazol ®)

Inflammation/Ulcus –



KetaconazoleAllium sativum Trichophyton spec.

KetaconazoleEssential oils ofPeucedanum grav. Trichophyton spec.

KetaconazoleAgastache rugosa (estragole) Trichophyton spec.

KetaconazoleEuphorbia characias Trichophyton spec.

ClorimazoleSantolina oil Candida albicans

MiconazoleAnethol Candida albicans

Amphotericin Essential oil of Thymus vulgaris Candida albicans

Combination of natural products and synthetic drugs to combat fungal infections

(Hemaiswarya et al., Phytomedicine 15: 639 - 652 (2008))

Natural products Synthetic drugs Fungal species


Future Outlook of Antimalaria Drugs

Malarone, artemisinin derivatives combined with lumefantrine or doxycycline and mefloquine combined with tetracycline or doxycycline have been evaluated with improvement of the cure rate in uncomplicated malaria

Artemisinin derivatives intravenously or intrarectally combined with mefloquine may be alternatives to intravenous quinine for treatment of severe malaria

Arch. Med. Res. 33: 416 - 421 (2002)


healthy tissue

cancer cells

cytostatics

inducers of apoptosis

immunostimulants

inhibitors of angiogenesis

stimulants ofoncogen-suppressor

genes

Futural multitarget therapy I

Example 1: Cancer therapy


Liver

HBV/HCV

immunostimulants

Virostaticdrugs

antioxidants

antifibrotics

antiinflammatory drugs

inhibitors of apoptosis

liver protecting agents

Futural multitarget therapy II

Example 2: Therapy of Hepatitis B+C


Progress in the field of high tech analysis, molecular biology, synergy research and omic technology can give phytotherapy a new legitimacy and the possibility to treat diseases which up to now were reserved for

chemotherapy only

Conclusion


Documents

Wagner 26 Key Note Address