Experiment 1 – Graded Dose-Response Curve

1. Experiment 1 – Graded Dose-Response Curve C1 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. 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: 0.26 mg/20 gm mouse 0.52 mg/20gm mouse 1.04 mg/20gm mouse 2.08 mg/20gm mouse Aspirin Tablet Dose: 0.26 mg/20 gm mouse 0.52 mg/20gm mouse 1.04 mg/20gm mouse 2.08 mg/20gm mouse Morphine Tablet Dose: 0.01 mg/20gm mouse 0.03 mg/20gm mouse 0.05 mg/20gm mouse 0.10 mg/20gm mouse Acclimatization (place mice with in the hot plate chamber)

6. Doses : per 20 gm mice

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

11. % Difference

12. Results - Ibuprofen

13. % Difference

14. Results - Aspirin

15. % difference

16. Results - Morphine

17. % difference

20. Summary of Results

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 http://www.panlab.com/panlabWeb/Hardware/php/displayHard.php?nameHard=HOT-PLATE

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 www.bu.edu/research/compliance/lacu/lacf/guidelines-policies/signs-of-pain.shtml

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 presynapticnerve  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