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

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.

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.

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

(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: 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 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)

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

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

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.

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

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

% Difference

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

% Difference

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

% difference

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

% difference

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

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

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

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

Non-opioid analgesics

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

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

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)

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

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

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

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

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

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

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