Dr. Hérida Regina Nunes Salgado - Faculdade de

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Primer Taller de Capacitación en Metodologías
Biofarmacéuticas
Bioassays to evaluate intestinal
motility in mice and rats
Bioensayos para evaluar la motilidad
intestinal en ratones y ratas
Profa. Dra. Hérida Regina Nunes Salgado
12 October 2011
1
INTESTINAL TRANSIT
1. INTRODUCTION
2. INTESTINAL MOTILITY
3. INTESTINAL TRANSIT TEST
4. ANIMALS
5. ANIMALS HANDLING
6. DOSIS EXEMPLE
7. EXPERIMENTAL
8. RESULTS
9. STATISTICS – test t
10. REFERENCES
2
• Strategies for validation of analytical
methods
• Plans for pharmaceutical institutes
• Current pharmaceutical marketing
• Problems
• Recommendations
Dr. Hérida Regina Nunes Salgado - Faculdade de Ciências
Farmacêuticas - UNESP - Brazil
3
 QUALITY IS EVERYONE’S
RESPONSIBILITY
Dr. Hérida Regina Nunes Salgado - Faculdade de Ciências Farmacêuticas - UNESP - Brazil
4
INTESTINAL TRANSIT
INTRODUCTION
• These bioassays aim to determine
the intestinal motility of drugs.
5
Find
Experimental
Solutions
New plans
Plans and
analysis
Statistics
6
INTESTINAL TRANSIT
• loperamide: antidiarrheal drug.
• Activity against diarrhea induced by bisacodil,
rein, phenolphthalein, etc.
• Used as positive control to analyze antidiarrheal
samples.
Cl
N
O
N
H3C
CH3
4-(4-chlorophenil)-4-hydroxiN,N-dimetil-,-diphenil-1piperidinbutiramide.
OH
Dr. Hérida Regina Nunes Salgado - Faculdade de Ciências Farmacêuticas - UNESP
- Brazil
7
Intestinal transit test
• Mice and rats (ratones y ratas)
Dr. Hérida Regina Nunes Salgado - Faculdade de Ciências Farmacêuticas UNESP - Brazil
8
INTESTINAL TRANSIT
4. ANIMALS:
• Mice or rats
• Male / female
• 22-30 g
• fasting 12 h
• water ad libitum
• Polypropylene cages (40 x 30 x
16 cm) at 20oC.
9
Intestinal transit
Experimental
Example
• 2 Groups: 10 animals/group,
• Oral administration by gavage.
• GROUP A (sample): loperamide (5 mg/kg)
• GROUP B (control): saline (10 mL/kg)
10
ANIMALS CAREFULLY
IDENTIFICATED
11
INTESTINAL TRANSIT
ANIMAL HANDLING
12
INTESTINAL TRANSIT
ANIMAL HANDLING
13
INTESTINAL TRANSIT
ANIMAL HANDLING
14
ANIMAL HANDLING
weight
15
INTESTINAL TRANSIT
DOSIS
volume according to animal weight: 5 mg/kg.
5 mg
---- 1000 g
x ---- 25 g
x = 0.125 mg of loperamide
loperamide concentration: 0,5 mg/mL
1 mL ----- 0.5 mg
x
----- 0.125 mg
x = 0.25 mL loperamide solution
volume:
25 g ----- 0.25 mL
1000 g ----- x
x = 10 mL
control group (saline): 10 mL/kg
16
INTESTINAL TRANSIT
ANIMAL HANDLING
• ORAL ADMINISTRATION V.O.
Dr. Hérida Regina Nunes Salgado - Faculdade de
Ciências Farmacêuticas - UNESP - Brazil
17
INTESTINAL TRANSIT
EXPERIMENTAL
• After
90
min:
10%
charcoal
suspension, 0.3 mL/animal, using
gavage.
• After 45 min: Mice (or rat) sacrifice
to measure the intestinal size and the
charcoal distance.
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INTESTINAL TRANSIT
EXPERIMENTAL
19
20
Process Otimization
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on-line help!
22
Different decisions for different situations!
23
Come on! It can„t go
wrong every time...
All conclusion are closely related with
experimental plans
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Statistic method should be adequate, because one
can not be useful for another one.
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• sample
charcoal distance
• control
charcoal distance
26
RESULTS
CONTROL
x
x2
SAMPLE
%
x
x2
%
1
46
2116
88.46
23
529
53.49
2
50
2500
100.00
58
3364
100.00
3
51
2601
100.00
36
1296
83.72
4
47
2209
90.38
43
1849
76.79
5
53
2809
100.00
52
2704
91.23
6
44
1936
81.48
38
1444
67.86
7
45
2025
80.35
47
2209
88.68
8
48
2304
84.21
17
289
36.17
9
47
2209
95.91
41
1681
73.21
10
50
2500
100.00
43
1849
81.13

481
23209
920.8
398 17214
752.28
x
48.1
2320.9
92.08
39.8 1721.4
75.23
27
STATISTICS
Statistics
is
the
study
of
the
collection,
organization, analysis, and interpretation of
experimental
data,
aiming
the
new
Analysis
Decision
decisions.
Collection
Organization
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STATISTICS
• William Sealey Gosset
 13/06/1876 – England
 16/10/1937 – England
- Nickname Student.
- He studied Math and Chemistry in Oxford.
- In 1899, he worked for Guinness Beer, in Dublin, where developed
an important work in statistics.
- He is the precursor of Modern Statistics.
- He developed the test t.
- His friends were Fisher, Neyman and Pearson.
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INTESTINAL TRANSIT
STATISTICS - t STUDENT TEST
• tTAB = 2.101
• xC = 481
xC2 = 23209
SQc = xC2 = xC2 – (xC)2 = 23209 – (481)2 = 72.9
nc
10
• x A= 398
xA2 = 17214
SQA = x2A = xA2 – (xA)2 = 17214 – (398)2 = 1373.6
nA
10
30
SQc  SQA 72.9  1373.6
So 

 80.36
nC  nA  2
10  10  2
31
Intestinal transit
STATISTICS – t STUDENT TEST
t
Y|xAA–YxCC|
So So

nC nA

39.8  48.1
8.3

 2.15
80.36 80.36 4.0

10
10
32
•
•
•
•
gl = nA + nB - 2
gl = 18
t0,05 = 2.101
tcalc = 2.150
tcalc > ttab
The mean is statistically different.
33
P.O.N.C.
PRICE OF NON-CONFORMANCE
Doing a fully method validation
can be tedious but the
consequences of not doing it
right are wasted time, money
and resources!
34
Journal of Ethnopharmacology 100 (2005) 153–157
Antibacterial properties of some plants used in
Mexican traditional medicine for the
treatment of gastrointestinal disorders
A.D. Alaní, F. Calzada, J.A. Cervantes, J.
Torreb, G.M. Ceballos
35
36
37
A New Protocol to Refine Intestinal Motility Test in Mice
Intestinal motility in mice is typically assessed at necropsy as the distance
that a charcoal plug travels in the intestine during a set time period.
This protocol involves fasting the animals for only 3 h, rather than the
12-24 h in the traditional test, administering a charcoal suspension
rather than a charcoal plug, and observing the mice at 5 minute
intervals for the presence of charcoal in the stool. Thus, intestinal
motility is measured as the time it takes for a charcoal suspension to
traverse the entire length of the gut, rather than the distance a
charcoal plug travels in the gut during a fixed time period. This
protocol not only reduces the animals’ distress, but also enables them
to be used on multiple occasions. Furthermore, since a single animal
can serve as its own control and in several alternative treatment
regimens, the data are likely to be more robust than when a different
animal is used to obtain each data point.
38
Sample
Control
charcoal distance
Time - constant
Distance - variable
New protocol
Time - variable
Distance constant
Advantages
• Fasting the animals for only 3 h
• Animals are not killed
• Animals can be reused
• Reduced stress
• Respectful science
40
Limitations
• Time consuming
Dr. Hérida Regina Nunes Salgado - Faculdade de Ciências Farmacêuticas - UNESP Brazil
41
REFERENCES
ALANÍ et al. Antibacterial properties of some plants used in
Mexican traditional medicine for the treatment of
gastrointestinal disorders. Journal of Ethnopharmacology,
100:153–7, 2005.
ARNOLD, G. L. & PLAUT, A. G. Antidiarrheal agents. In: MILLER,
R.R. & GREENBLATT, D.J. Handbook of drug therapy. N. York,
Elsevier, 1979. p.1051-2.
BLAND, M. An introduction to medical statistics. 2. ed. New York:
Oxford University Press, 1995.
DEF. Dicionário das especialidade Farmacêuticas. 6.ed. J. Bras.
Med., 1999-2000.
FOYE, W.O. et al. Principles of Medicinal Chemistry. 4.ed.
Baltimore, Williams & Wilkins, 1995.
FRIES, D. S. Analgesics. In: GOODMAN, A. G. et al. (Eds.). The
Pharmacological Basis of Therapeutics. 9.ed. New York:
McGraw-Hill, 1996. p.268´.
GANAPATY, S.; DASH, G.K.; SUBBURAJU, T.; SURESH, P. Diuretic,
laxative and toxicity studies of Cocculus hirsutus aerial parts.
Fitoterapia 37:28-31, 2002.
42
REFERENCES
GUNAKKUNRU, A.; PADMANABAN, K.; THIRUMAL, P.; PRITILA, J.;
PARIMALA, G.; VENGATESAN, N.; GNANASEKAR, N.;
PERIANAYAGAM, J.B.; SHARMA, S.K.; PILLAI, K.K.. Anti-diarrhoeal
activity of Butea monosperma in experimental animals. Journal
of Ethnopharmacology, 98: 241–244, 2005.
HASHIMOTO, K.; SATOH, K.; MURATA, P.; MAKINO, B.; SAKAKIBARA, I.;
KASE, Y.; ISHIGE, A.; HIGUCHI, M.; SASAKI, H. Components of
Panax ginseng that improve accelerated small intestinal transit.
Journal of Ethnopharmacology, 84: 115-119, 2003.
JANSSEN, P. & JAGENEAU, A. H. A new series of potent analgesics.
Part I – Chemical structure and pharmacological activity. J.
Pharm. Pharmacol., 9:381-400, 1957.
MARONA, H.R.N.; LUCCHESI, M.B.B. Refining the intestinal motility
test in mice to reduce animal stress. Rev. de Ciências
Farmacêuticas, 24(1):79-82, 2003.
MARONA, H.R.N.; LUCCHESI, M.B.B. Protocol to refine intestinal
motility test in mice. Laboratory Animals, 38:257-260, 2004. ISSN
0023-6772.
ROUF, A.S.S.; ISLAM, M.S.; RAHMAN. M.T. Evaluation of
antidiarrhoeal activity Rumex maritimus root. Journal of
Ethnopharmacology, 84:307-310, 2003.
43
REFERENCES
SAIRAM, K.; HEMALATHA, S.; KUMAR, A.; SRINIVASAN, T.; GANESH, J.;
SHANKAR, M.; VENKATARAMAN, S. Evaluation of anti-diarrhoeal
activity in seed extracts of Mangifera indica. Journal of
Ethnopharmacology, 84:11-15, 2003.
SHILPI et al. Preliminary pharmacological screening of Bixa orellana L.
leaves. Journal of Ethnopharmacology, 108:264–71, 2006.
VERHAEREN, E. H. C. et al. Influence of 1,8-dihydroxyanthraquinone and
loperamide on the paracellular permeability across colonic mucosa.
of aspirin and paracetamol on naloxone reversal of morphineinduced inhibition of gastrointestinal propulsion in mice. Eur. J.
Pharmacol., 73:11-19, 1981.
WANG et al. Pharmacological studies of anti-diarrhoeal activity of
Gentianopsis paludosa. Journal of Ethnopharmacology, 105(1-2):1147, 2006.
WONG, C. L. & WAY, M. K. Effects of aspirin and paracetamol on
naloxone reversal of morphine-induced inhibition of gastrointestinal
propulsion in mice. European Journal of Pharmacology, 73:11-19,
1981.
44
“The day will come when men will know the inmost
of the animal. Then, any crime against an animal will
be a crime against Humanity.”
“Chegará um dia em que os homens entenderão a
consciência dos animais e neste dia um crime contra
um animal será um crime contra a Humanidade.”
Leonardo da Vinci
(1452-1519)
THANK YOU!
Prof. Dr. Hérida Regina Nunes Salgado
Faculdade de Ciências Farmacêuticas
UNESP
Rodovia Araraquara-Jaú, km 1
CEP 14801-902
Araraquara – SP – Brazil
Phone: 55 16 33 01 69 67
Fax: 55 16 33 01 69 60
45
[email protected]

Dr. Hérida Regina Nunes Salgado - Faculdade de

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