RASOANAIVO Drug discovery at IMRA .pdf



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13th NAPRECA Symposium

“Drug Discovery from African Rainforest”
Democratic Republic of Congo,
10-14 August 2009

“Sublime is science whose aim is to preserve life”

Drug discovery at IMRA:
Recent results and way forward
Philippe Rasoanaivo
Bio-therapeutics Laboratory
IMRA

Target diseases for drug discovery
►Tropical diseases
● Malaria;
● Chikungunya.

► Diseases of aging
● Memory and cognitive dysfunction;
● Diabetes of aging;
● Erectile and libido dysfunction.

Breakthrough in ED research after 1998
3000
2500

Number of publications

2000
1500

Source: Medline
Key words: erectile dysfunction

1000
500
0
1960-1997

1998

1998-2009

160
140
120
100
80
60

Source: Medline, Google scholars,
HINARI
Key words: erectile dysfunction, plants

40
20
0
1960-1997

1998-2009

Years

“Drug companies, driven by profit, go where the money is”

Background
of pathophysiology
of erectile
Background
of physiology of
erectiledysfunction
function
Hypothalamus

Brain stimuli
(Sexual desire)
Visual, tactile
Olfactory, auditory
Fantasy

Hormonal:
- Androgen deficiency

Psychogenic:
- Anxiety
- Strained relationships
- Depression, schizophrenia

Hypoactive Sexual
Desire Disorders (HSDD)

Vascular effects
(Erectile performance)

Erection

Organic: ischemia
- Hypertension
- Hyperlipidemia
- Diabetes

Background of pharmacology
nNOS

L-Arginin
eNOS

Neurones

Production
Endothelial
Cells

Neurotransmitter
Endothelial

NO

Degradation
Antioxidants

Guanylate cyclase activation

Increase in C.GMP
Hormonal
Mechanism(s)

PDE5

GMP

Calcium-dependent relaxation
Vasodilatation

Erection
Toda et al. NO and penile erectile function, Pharm. Ther. 2005, 106, 233-266.

Vascular mechanism of erection

Ethnobotanical data

Neobeguea mahafalensis (Meliaceae)
“Treatment of back pains;
Claimed to restore erectile function in old men”
(Debray, Boiteau)
“Long-lasting effects in the treatment of erectile dysfunction”
(Ethnic groups of the South Madagascar)

Previous work
O

O

17
O

O

O

O

O

O

O

16 C

O

O

O

O

C

O

O

O

O

HO
O

O
O

O

OH

O

O

Neobeguin

OH

O

O

Pseudrelone

M. Randrianarivelojosia, M. P. Kothos, D. A. Mulholland,
Phytochemistry 1999; 52: 1141-14-1143.

Bloomfontein, South Africa, 2004

Approaches in drug discovery
Disease

Abnormality of mechanistic level

Target-based approach (HTS)
Drug

Target

Molecular mechanism

Cause the cell or the organ to function abnormally

Result in symptoms and physiological changes

Physiology-based approach (Ethnobotany)
Drug

Animal

Physiological effect

Extraction procedure
Dried and powdered plant
Water

Water extract
(RW)
Partition chloroform/water

Chloroform extract
(R2C)

Exhausted water extract
Lyophilized

R2W

First pharmacological results:
water extract

Active couples (%)

*

4

*

3,5

*

Control

3

RW

2,5
2
1,5
1
0,5
0
1

T0

2

T+1H

3

1th day

4

4th day

5

7th day

6

14th day

Number of active couples after water extract (60mg/kg) treatment
for three consecutive days in rats. (* = p<0.05 vs control)

First pharmacological results:
Chloroform (R2C) and exhausted water (R2W) extracts
Number of mounts

3
2,5
2
1,5
1
0,5
0

Control

1

R2W
43mg/kg

R2C
4mg/kg

Copulatory efficacy

Number of mounts estimated on day 7 following 3 consecutive day treatment
2,5

2

1,5

1

0,5

0
1

Copulatory efficacy estimated on day 7 following 3 consecutive day treatment

Priority applications I-III, 2006

Mating behaviour test at IMRA

► Frequency mounting
► Intromission
► Ejaculation

Confirmation of the activity on R2C;
Determination of the optimal doses
45
40
35

Mounts 1

30
D4

25

D7
20

D 14

15
10
5
0
CONTROL

0,4mg/kg

4 mg/kg
R2C (p.o)

40 mg/kg

New extraction procedure
Dried and powdered plant
Dichloromethane (DCM)

DCM extract

Exhausted plant

Acetone extract

Acetone

Exhausted plant
Water
Lyophilized

Water extract

Bioactive constituents:
Limonoids in the dichloromethane extract
O

O

17

O

O

O

OH

O

O

O

O

O

O

O
O

30
O
O

O

O

16 O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O
O

O
O

O

OH

O
O

O

O

O
O

O

O

O

OH

O

OH

O
O

O

J.E. S. Wikberg, P. Rasoanaivo, S. A. Razafimahefa, B. Rasolondratovo.
PCT Patent WO 2008/145996 A2, 4 December 2008, 245 pages.

HMBC connectivities for R-306

H

J. Luo et al. (2009) Tetrahedron, 65, 3425-3431.

Delayed and long-lasting effects of one limonoid
(compilation of several tests)
Mounting frequency (1 hour)
60

50

Mounts 1

40
D 4
D 7
D 14
D 21

30

20

10

0
CONTROL

0,004mg/kg

0,04 mg/kg
R 306

0,4 mg/kg

Bioactive constituents (continued):
Phenolics in the acetone extract

●NO
60

●O2

50

NADPH
oxidase
40

Mounting frequency

O2

Oxydation
Nitration
DG2
DG21

30

DG24

20

ONOO
Control

10

OH
OH

0

2 Metabolites1

HO

O

2

OH

Hours

Nitrate

OH

1H
2H
Y. Steffen et al. (2007) Epicatechin elevates NO in endothelial cells via inhibition of
NADPH oxidase. Biochem. Biophys. Res. Com., 359, 828-833.

Exhausted water extract?
Dried and powdered plant

160
140

Water

Control

120

Water extract
(RW)

100
80

Crude aqueous extract

Exhausted aqueous
Partition ethyl acetate (EtOAc)/water

60

fraction
EtOAc fraction

40

Exhausted water extract

20
EtOAc
extract
0

Lyophilized
0

RW1

2

3

1

4

5

4

6

Day

7

7

8

14

Sephadex

Priority application IV

RWe

Tests results on different fractions from aqueous extract
140

120

Mounts 4 hou

100

80

D1
D4
D7

60

40

20

0
CONTROL

RW 1

RW 5

RW 7

f
u
S

ic
ox
tt

ly
pp
su
of

e
tiv
ac
In
in

No

h

s
n
a
m
u
ck
La

A

e
it v
c

in

ly
p
up
s
t
n
ie
if c

hu
m
s
an

c
xi
To

Time

Standardised phytomedicine
Toxicity
►Cytotoxicity:
Inhibition % = 02.5 at 10µg/ml
►Acute and sub-acute toxicity:
No toxicity at 1g/kg by oral administration,
No death at 2g/kg by oral administration, but hypoactivity
LD50= 1.5g/kg by intra-peritoneal administration.
►Teratogenic effects:
No effects at 60mg/kg.
►Genotoxicity
No effects

Standardised phytomedicine
Preliminary clinical trials
Diagnostic tool: International Index of Erectile Function (IIEF)
Å 1 placebo controlled, double blind clinical trial:
- 45% success;
- Failure with patients having diabetes and/or vascular problems;
- 18% placebo.
Å 1 open clinical trial:
- 72% success;
- No effect on young people, or patients with hyperlipidemia.
Å Informal trials in several voluntaries:
- 91% success.

Supply: semisynthesis
O
O

O

H
O

O

O
O O

O

O O

Trans-esterification

O

OH

O

O

O

OH
O

O

O

O

O

- Chemically?

Common structure
With C-16/17 δ-lactone

O

O

H

O

O

H

O

O

O

AcO
O
OO

Novel structure
With C-16/30 δ-lactone

O
OAc CO2Me

AcO

OAc

Limonoid from Neobeguea leandreana

Priority applications 3- 4

Compilation of tests on a semisynthetic compound
30

25

Mounts 1h

20
D4
15

D7
D 14

10

5

0
CONTROL

0,004mg/kg

0,04 mg/kg
SAE6 mg/kg bw

0,4 mg/kg

4 mg/kg

Supply:
Metabolites from endophytic fungi & mycorrhizas

LC/MS/MS profiling (dereplication)
identify relevant metabolites for semisynthesis

The way forward
Plant
Standardised
extract

Bioactive
compounds
Further pharmacological evaluation

Commercialisation
High quality publications

Dangitsyl®

Drug development

Successful story: Madeglucyl

Eugenia jambolana
(= Syzygium cumini)

Successful story: Glucanol FortéTM

Acknowledgments
OPCW for financial support
NAPRECA Congo Organising Committee

Colleagues at IMRA

Colleagues in CNRS



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