C1July 31, 2009

Experiment 1 – Graded Dose-Response Curve

OBJECTIVESGENERAL• 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.

METHODOLOGYSUBJECTS:• 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

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 TabletDose: 0.26 mg/20 gm mouse 0.52 mg/20gm mouse 1.04 mg/20gm mouse 2.08 mg/20gm mouse

Aspirin TabletDose: 0.26 mg/20 gm mouse 0.52 mg/20gm mouse 1.04 mg/20gm mouse 2.08 mg/20gm mouse

Morphine TabletDose: 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)

128 miceParacetamol

32 mice

8 mice0.33 mg

8 mice 0.66 mg

8 mice1.32 mg

8 mice2.64 mg

Ibuprofen32 mice

8 mice0.26 mg

8 mice 0.52mg

8 mice1.04 mg

8 mice2.08 mg

Aspirin32 mice

8 mice0.26 mg

8 mice0.52 mg

8 mice1.04 mg

8 mice2.08 mg

Morphine32 mice

8 mice0.01 mg

8 mice0.03 mg

8 mice0.05 mg

8 mice0.10 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 - ParacetamolParacetamol

(mg/ 20g)before (sec)

after (sec)

time difference (sec)

% difference

0.33 96.24 187.82 91.58 95.160.66 174.85 210.23 35.38 20.231.32 137.38 193.22 55.84 40.652.64 142.27 272.45 130.18 91.50

% Difference

0.10 1.00 10.000.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00Series1, 95.16

20.23

40.65

91.50

Paracetamolconcentration vs % difference

drug concentration (mg/ 20g)

% d

iffer

ence

Results - IbuprofenIbuprofen(mg/ 20g)

before (sec)

after (sec)

time difference (sec)

% difference

0.26 45.46 261.75 216.29 475.780.52 39.75 165.75 126.00 316.981.04 38.43 151.00 112.58 292.972.08 35.88 100.45 64.58 180.00

% Difference

0.10 1.00 10.000.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00Series1; 475.78

316.98 292.97

180.00

Ibuprofenconcentration vs % difference

drug concentration (mg/ 20g)

% d

iffer

ence

Results - AspirinAspirin

(mg/ 20g)before (sec)

after (sec)

time difference (sec)

% difference

0.26 145.18 178.62 33.44 23.030.52 135.15 211.75 76.60 56.681.04 104.20 198.63 94.43 90.622.08 99.65 138.00 38.35 38.48

% difference

0.10 1.00 10.000.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Series1; 23.03

56.68

90.62

38.48

Aspirinconcentration vs % difference

drug concentration (mg/ 20g)

% d

iffer

ence

Results - MorphineMorphine(mg/ 20g)

before (sec)

after (sec)

time difference (sec)

% difference

0.01 45.70 114.83 69.13 151.270.03 43.00 76.49 33.49 77.880.05 44.20 63.33 19.13 43.280.10 51.43 71.75 20.32 39.51

% difference

0.01 0.100.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00Series1; 151.27

77.88

43.28

39.51

Morphineconcentration vs % difference

drug concentration (mg/ 20g)

% d

iffer

ence

0.01 0.10 1.00 10.000.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00

Series1; 95.16

20.2340.65

91.50

Series1; 475.78

316.98292.97

180.00

Series1; 23.0356.68

90.62

38.48

Series1; 151.27

77.8843.28 39.51

Paracetamol, Ibuprofen, Aspirin, Morphineconcentration vs % difference

Morphine Aspirin Ibuprofen Paracetamol

drug concentration (mg/ 20g)

% d

iffer

ence

0.01 0.10 1.00 10.000.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

450.00

500.00

Paracetamol, Ibuprofen, Aspirin, Morphineconcentration vs % difference

Morphine Aspirin Ibuprofen Paracetamol

drug concentration (mg/ 20g)

% d

iffer

ence

Summary of ResultsDrug 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:

1. Decreased calcium influx in presynaptic nerve decreased transmitter release

2. 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