1. Experiment 1 – Graded Dose-Response Curve
July 31, 2009

2. OBJECTIVES
GENERAL
To determine the relationship between increasing doses of drugs (paracetamol, ibuprofen, aspirin, and morphine) to the response to pain in mice.
To compare the analgesic properties of paracetamol, ibuprofen, aspirin, and morphine.

3. OBJECTIVES
SPECIFIC
To determine the percent (%) difference of inhibition of pain before and after administration with paracetamol, ibuprofen, aspirin, and morphine on acclimatized mice.
To present the graded dose response curves of paracetamol, ibuprofen, aspirin, and morphine.
To compare the relative potency and maximal efficacy of paracetamol, ibuprofen, aspirin, and morphine.

4. METHODOLOGY SUBJECTS:
32 albino mice of same sex, approximate age, and weight
MATERIALS:
Animal weighing scale
Gavage tubes (w/ tuberculine syringe)
Hot plate
Small beakers for drugs
Surgical gloves
Drugs:
Paracetamol
Ibuprofen
Aspirin
Morphine

5. (place mice with in the hot plate chamber)
128 fasted mice
(pre-weighed)
32 albino mice for each drug
(8 mice per dose)
Paracetamol Tablet
Dose: 0.33mg/20 gm mouse
0.66mg/20 gm mouse
1.32mg/20 gm mouse
2.64mg/20gm mouse
Ibuprofen Tablet
Dose: mg/20 gm mouse
0.52 mg/20gm mouse
1.04 mg/20gm mouse
2.08 mg/20gm mouse
Aspirin Tablet
Dose: mg/20 gm mouse
0.52 mg/20gm mouse
1.04 mg/20gm mouse
2.08 mg/20gm mouse
Morphine Tablet
Dose: mg/20gm mouse
0.03 mg/20gm mouse
0.05 mg/20gm mouse
0.10 mg/20gm mouse
mice were divided into the 4 drug groups. They were again divided into each dosage regimen. The dosage for each rat was computed based on weight and sub-group they were assigned.
Acclimatization
(place mice with in the hot plate chamber)

6. Doses : per 20 gm mice 128 mice Paracetamol 32 mice 0.33 mg 8 mice
Ibuprofen
0.26 mg
0.52mg
1.04 mg
2.08 mg
Aspirin
0.52 mg
Morphine
0.01 mg
0.03 mg
0.05 mg
0.10 mg
Doses : per 20 gm mice

7. Note the weight of each mouse Animals were fasted overnight
Acclimatize all mice
Place the mouse on top of hot plate (48°C) and determine the time the mouse licked its paws.
Administer the drug via oral gavage according
to its computed dose
AFTER 1 HOUR place each mouse on hot plate and note the time it licked its paws

8. RATIONALE Fasting was done to avoid food-drug interaction.
Male mice were used for this experiment to ensure that none of the mice are pregnant, which may alter results.
Variability was limited by controlling for age, sex, and weight of the mice.
0.9% Normal Saline Solution as a diluent, served as a vehicle for the drug for faster absorption.
Gavage tubes were used to avoid spillage of the drug.
Response to pain was measured 1 hour after administration for optimal absorption of the drug.
Geometric dosing was used to maximize cost-effectiveness.

9. HYPOTHESIS
As the dose of increases, the analgesic effect of the drug also increases.

10. Results - Paracetamol Paracetamol (mg/ 20g) before (sec) after (sec)
time difference (sec)
% difference
0.33
96.24
187.82
91.58
95.16
0.66
174.85
210.23
35.38
20.23
1.32
137.38
193.22
55.84
40.65
2.64
142.27
272.45
130.18
91.50

11. % Difference

12. Results - Ibuprofen Ibuprofen (mg/ 20g) before (sec) after
time difference %
difference
0.26
45.46
261.75
216.29
475.78
0.52
39.75
165.75
126.00
316.98
1.04
38.43
151.00
112.58
292.97
2.08
35.88
100.45
64.58
180.00

13. % Difference

14. Results - Aspirin Aspirin (mg/ 20g) before (sec) after time difference%
difference
0.26
145.18
178.62
33.44
23.03
0.52
135.15
211.75
76.60
56.68
1.04
104.20
198.63
94.43
90.62
2.08
99.65
138.00
38.35
38.48

15. % difference

16. Results - Morphine Morphine (mg/ 20g) before (sec) after
time difference %
difference
0.01
45.70
114.83
69.13
151.27
0.03
43.00
76.49
33.49
77.88
0.05
44.20
63.33
19.13
43.28
0.10
51.43
71.75
20.32
39.51

17. % difference

20. Summary of Results Drug Dose of maximum response Paracetamol 0.33 mg
Ibuprofen
0.26 mg
Aspirin
1.04 mg
Morphine
0.01 mg

21. Discussion MOUSE HOT PLATE (MHP) TEST
Measures the reaction time of mice dropped onto a heated surface, confronted with a heat stimulus applied to their plantar surface

22. Discussion DIFFERENT RESPONSES TO PAIN:
Excessive licking and scratching
Jumping
Decreased activity
Paw shaking
Piloerection
Vocalization – with acute pain
Change in group behavior – if grouped

23. Discussion MECHANISM OF ACTION OF DRUGS Non-opioid analgesics:
Paracetamol
Ibuprofen
Aspirin
Opioid analgesics:
Morphine

24. Non-opioid analgesics

25. Non-opioid analgesics
COX 1 or prostaglandin synthase 1 – constitutive enzyme found in gastric mucosa, platelets, vascular endothelium and kidneys
COX 2 or prostaglandin synthase 2 – inducible enzyme generated in response to inflammation; expressed mainly in activated macrophages and monocytes

26. Non-opioid analgesics
inhibition of COX-1: responsible for unwanted effects on platelet aggregation and the gastrointestinal tract
inhibition of COX-2: analgesic, antipyretic, and anti-inflammatory activity of NSAIDs
PGE2 – sensitizes nerve endings to the action of bradykinins, histamine and other chemical mediators released during inflammation

27. Paracetamol Also known as acetaminophen
Weak cox-1 and cox-2 inhibitor in peripheral tissues but no significant anti-inflammatory effect
reduces the production of prostaglandins (pro-inflammatory chemicals)

28. Paracetamol Bioavailability: almost 100% Metabolism: 90 to 95% Hepatic
Half life: 1–4 h
Excretion: Renal

29. Ibuprofen
non-selective COX inhibitor—that is, it inhibits two isoforms of cyclooxygenase, COX-1 and COX-2
Bioavailability: 49–73%
Protein binding: 99%
Metabolism: Hepatic (CYP2C9)
Half life: 1.8–2 hours
Excretion: Renal

30. Aspirin Nonselective inhibitor of both COX isoforms
Irreversibly blocks the synthesis of Thromboxane A2
Inhibits platelet aggregation
Inhibits pain stimuli at the subcortical site (thalamus and hypothalamus)
Half-life: 0.25 hours

31. Morphine
Binds to opioid receptors (GPCR) located primarily in brain and spinal cord regions causing:
Decreased calcium influx in presynaptic nerve  decreased transmitter release
Increased potassium conductance  inhibitory postsynaptic potential
Extensive first-pass metabolism
Half life: 2 hrs

32. Sources of Error
Oral administrations of drugs done by different members of the group
Dose of drug was not fully administered due to spillage
Different rates of administration
Aspiration from administering drug too fast
Unequal number of mice per administered dose
Discrepancies in experimenter and actual endpoint

33. Recommendations
Ensure equal numbers of mice by the time of data gathering
Include other erratic behaviors as response
Use other drugs with different route of administration (e.g. intraperitoneal)
Only 1 experimenter per task to reduce experimenter variability

34. Conclusion
Increasing the doses of ibuprofen and morphine decreases their analgesic effect
Increasing the dose of paracetamol prolongs the response of mice to pain
Of the four given analgesics, ibuprofen was found to be the most effective at a dose of 0.26 mg
All drugs are effective at each dose
Not able to detect potency