Shenyang Pharmaceutical University LAB 22: PHARMACOKINETICS STUDY PHARMACEUTICS II

Shenyang Pharmaceutical University LAB 22: PHARMACOKINETICS STUDY PHARMACEUTICS II

LABORATORY 22: In vivo pharmacokinetic study of acetaminophen in rabbits

1. LABORATORY OBJECTIVES

a) To learn how to determine plasma drug concentrations in rabbits.

b) To learn how tocalculate pharmacokinetic parameters using plasma drug concentrations.

1. INTRODUCTION

The absorption and elimination processes of drugs following parenteral administration (SC or IM) usually follow the first-order kinetics. The pharmacokinetic parameters can be obtained by the computer fitting methods. If the in vivo pharmacokinetic behavior of a drug is consistent with the one-compartment model, the apparent first-order rates (K and Ka) of elimination and absorption can be calculated by the method of residuals.

The drug plasma concentration-time relationship after parenteral administration with first-order drug absorption, can be represented by Equation 1:

(1)

where C is the drug concentration in plasma at time t, Ka is the apparent first-order absorptionrate constant. K isthe excretion rate constant, X0 is the initial drug amount administrated, V is the apparent distribution volume, F is the absorption percentage.

When Ka>K, t→∞, then e-Kat→0, Equation 1 can be simplified asEquation 2:

(2)

By transforming Equation 2 into its corresponding logarithmic form:

(3)

A curve with a binomial exponent can be obtained by plotting log C versus t. The terminal segment of the curve is linear and the slope is equal to –K/2.303. Thus, the value of K can be obtained from the slope as shown in Figure 22-1 (Line I).

Figure 22-1 Calculation of K and Ka by the method of residuals.

The relationship between Cr (values from the extrapolated concentration obtained by Equation 2 are subtracted from the original experimental data points) and t can be described by Equation 4 which is obtained by subtracting Equation 1 fromEquation 2.

(4)

The residual line is the logarithmic form of Equation 4 and expressed by Equation 5:

(5)

A linear curve can be obtained by plotting logCr versus t, with the slope being –Ka/2.303. The value of Ka can then be obtained from the slope, as seen in Figure 21-1 (Line II).

Acetaminophen is a commonly usedantipyretic analgesic which is rapidly absorbed from the gastrointestinal tract following oral administration. The dosage forms of acetaminophen include tablets, capsules, syrups, and suppositories. In order to understand the in vivo pharmacokinetic behavior of acetaminophen in rabbits, the plasma drug concentrationswill be determined following intramuscular injection, intravenous injection, and oral administration and the pharmacokinetic parameters will be calculated.

The method for the analysis of acetaminophen concentrations in plasma is based on the formation of P-aminophenolby the hydrolysis of acetaminophen (Method Bas mentioned in 3.2.2). P-aminophenol can react with phenol in the presence of sodium hypobromiteresulting in the formation of anindigo dye which has a maximumabsorbance wavelength at 620 nm. Trichloroacetic acid (20%) should be added to the plasma samples in order to precipitate and remove proteinsto prevent them from interfering with the quantitation of acetaminophen.

1. METHODS

3.1 Procedures

3.1.1 Ten centrifuge tubes are washed and treated with 100 unit/ml heparin solution (a heparin injection is added to 250 ml saline),followed by drying at 105℃and numbering of the tubes.

3.1.2Preparation of the intramuscular injection solution

1.5g of acetaminophen is weighed and placed into a small beaker and 50% of propylene glycol aqueous solution isthen added. The resultant solution is heated to completely dissolve acetaminophen with the formation of a clear solution containing 100mg/mlacetaminophen.

3.1.3Preparation of the blank plasma samples

The hairs around the peripheral ear vein of the rabbitsare shaved. The shaved area is moistened with alcohol and heated under an infrared lamp for 2 minutes. An incision is made verticallyinto the vein and blood samples are collected in heparinized tubes. After blood sampling, a cotton ball is applied at the site of the incision and the bleeding is stopped by applying a clip.

3.1.4Drug administration and blood sampling

Healthy rabbits weighing about 3kgare fasted overnight (about 18 hours)but with free access to waterbefore conducting the experiment. The acetaminophen solution (100mg/kg) is administered intramuscularly to the rabbits via the hind limb. After administration, blood samples are collected (2-3 ml) in heparinized tubes at the following time intervals: 10, 20, 30, 60, 90, 120, 150, 180, 210, 240, 300, and 360 min. After centrifugation at 3,000 rpm for 10 min, the upper layer(plasma) is collected. The plasma drug concentrations are determined according to the method B described below in the section of “Establishment of calibration curve”.

3.1.5Notes

(1) Syringes with agauge 6 or 7 needle should be used for intramuscular injection.

(2) Acetaminophen can be dissolved in 50%propylene glycol solution after heating but it will precipitateafter cooling. Therefore, the acetaminophen solution should be used cooled right before injection to avoid precipitation.

(3) The syringe and needle should be preheated in warm water to avoid the precipitation of acetaminophen during injection.

(4) The rabbits should have free access to food and water while conducting the experiments.

3.2 Determination of acetaminophen concentrations in rabbit plasma

3.2.1 Method A

a) Establishment of a calibration curve

Acetaminophen standard (10 mg) is accurately weighed and placed into a 100 ml volumetric flask followed by diluting with distilled water to 100 ml to form a 100 μg/ml stock solution. Aliquots of the standard stock solutions (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ml) are diluted to 1 ml with distilled water to form a series of standard solutions (10, 20, 40, 60, 80, 100 μg/ml). 1mlof the standard solution is mixed with 400μl rabbit plasma and 3.5ml of 0.06M barium hydroxide solution. After shaking, the resultant solutions are set aside at room temperature for 4min. 3.5ml of 2% zinc sulfate solution and 10ml of distilled water arethen added to each of the solutions. The solutions are filtered and the absorbance of the filtrate is determined at 245nm with a UV spectrophotometer. Blank rabbit plasma is used to prepare the blank solutionwhich is prepared by adding 1ml of distilled water to 400μl of blank rabbit plasmaand mixing with 3.5ml of 0.06mol/L barium hydroxide solution. The procedures arefollowed starting from the section of “Establishment of a calibration curve”. The regression equation isgenerated for absorbanceas a function of drug concentrations.

b) Determination of the acetaminophen concentration in rabbit plasma

To 400μl of rabbitplasma, 1 ml of distilled water is added and the procedures arefollowed starting from the section of “Establishment of the calibration curve”. The acetaminophen concentration in rabbit plasmais calculated from the regression equation of the calibration curve.

3.2.2 MethodB

a) Preparation of the reagents

20% trichloroacetic acid solution: 20 g of trichloroacetic acid is dissolvedin distilled water with the final volume of 100 ml.

4 Mhydrochloride solution: 33.2 ml of concentrated hydrochloric acid is dissolvedin distilled water to a volume of 100 ml.

40% sodium hydroxidesolution: 40 g of sodium hydroxideis dissolvedin distilled water to a volume of 100 ml.

0.2 Msodium hydroxidesolution: 8 g of sodium hydroxideis dissolvedin distilled water to a volume of 100 ml.

1% phenol solution: 1 ml of phenol solution with a concentration higher than99% is dissolvedin distilled water to a volume of 100 ml (The solution should be prepared immediately before use).

0.5 Msodium carbonate-bromide solution: 106 g of anhydrous sodium sulfate is dissolvedin distilled water, 15 ml saturated aqueous bromide solution is added, mixed, and diluted with distilled water to 100 ml (The solution should be prepared immediately before use).

Saturated bromide aqueous solution: Anappropriate amount of liquid bromide is added to distilled water to produce a saturated solution and left for at least 24 h before use.

Chromogenic reagent: 10 ml of the 1% phenol solution is mixed with 80 ml of 0.2Msodium hydroxidesolution. 100 ml of the sodium carbonate-bromide solution is added and mixed.

b) Establishment of a calibration curvewith rabbit plasma

Preparation of standard stock solutions: Acetaminophen standard (about 1 g) dried to a constant weight at 105℃is accurately weighed and dissolved in hot distilled water in a 250 ml volumetric flask. After cooling to room temperature, the solution is diluted with distilled water to volume (250 ml)with the final concentration of 4,000 g/mL and stored in the refrigerator before use.

Preparation of standardsolutions: Standard stock solutions (1.25, 2.5, 3.75, 5.0 and 6.25 ml) are transferred to 100 mlvolumetric flasks and diluted to 100 ml with distilled water with thorough mixing to obtain standard solutions with a concentration of 50, 100, 150, 200 and 250 μg/ml respectively.

Preparation of blank plasma: 9 ml of blood is sampled as described above and centrifuged. 4.5 ml of the supernatant(plasma)is obtained and 3 ml is used to prepare the calibration curve. The remaining plasma is stored in the refrigerator.

Establishment of a calibration curvewith rabbit plasma: 5 ml calibratedcentrifuge tubes (n=6) are labeled.0.5 ml of standard solution is added to the tubes containing the plasma and treated by following the procedures listed in Table 1. The absorbance is determined at 620nm for the samples (standard solutions plus blank plasma) following the same procedures for the blank control.

Table 1. Establishment of a calibration curvewith rabbit plasma

Operations / Concentration of the standard solution
0 / 50 / 100 / 150 / 200 / 250
Standard solution (ml) / 0.5 / 0.5
Blank plasma (ml) / 0.5 / 0.5
Distilled water (ml) / 0.5 / 0.5
20% trichloroacetic acid (ml) / 0.5 / 0.5
After mixing, centrifugation (5000 rpm) (min) / 10 / 10
Supernatants (ml) in 10 ml calibrated test-tubes / 1 / 1
Concentrated hydrochloric acid (ml) / 0.5 / 0.5
Boiling time (h) / 1 / 1
40% sodium hydroxide (ml) / 0.5 / 0.5
Chromogenic reagent added to (ml) / 10 / 10
Mixing and standing time (min) / 40 / 40
No. of the colorimetric vessel
Determination of absorbance / Corrected value A
A / 1
2
3
Mean value of A

3.3 Determination of acetaminophen in the plasma samples

500 μl plasma samplesare prepared by following the procedures listed in Table 2. The plasma drug concentrations are calculated from the regression equation of the calibration curve.

Table 2. The procedures for the determination of acetaminophen in rabbit plasma after intravenous injection

Operation / Concentration of the standard solutions
0 / 10 (min) / 20 (min) / 30 (min) / 1
(h) / 1.5 (h) / 2
(h) / 2.5 (h) / 3
(h) / 3.5 (h)
Distilled water (ml) / 1 / 1
Blank plasma (ml) / 0.5 / 0
Plasma sample (ml) / 0 / 0.5
20% trichloroacetic acid (ml) / 0.5 / 0.5
After mixing, centrifugation (5000 rpm) (min) / 10 / 10
Supernatants (ml) / 1 / 1
Concentrated hydrochloric acid (ml) / 0.5 / 0.5
Boiling time (h) / 1 / 1
40% sodium hydroxide (ml) / 0.5 / 0.5
Chromogenic reagent added to (ml) / 10 / 10
Mixing and standing time (min) / 40 / 40
No. of the colorimetric vessel
Determination of absorbance / Corrected value A
A / 1
2
3
Mean value of A

Instructions: the procedures and the amounts of reagents added are the same asthose used for the sample prepared atthe 10-min. time interval.

4. RESULTS AND DISCUSSION

4.1 Fill in Table 3 with the concentrations of acetaminophen added in rabbit plasma and thecorrespondingabsorbances. Caluclate the regression equationusing a computer statitiscal program.

Table 3 Data for the plasma acetaminophen concentrations and absorbances in the calibration curve

Acetaminophen concentration(μg/ml) / 50 / 100 / 150 / 200 / 250
Absorbance (A)
Regression equation

4.2In Table 4, record the acetaminophen concentrations in rabbit plasma at predetermined time points after intramuscular injection.Plot the logarithmof the acetaminophen concentrationsin rabbit plasmaversus t, calculate the elimination rate constant by the linear regression method, and determine the AUC(im)0-∞using the trapezoidal rule.

Table 4 Data for the acetaminophen concentrations in rabbit plasma at predetermined time points after intramuscular injection

Time(min) / 0 / 10 / 20 / 30 / 60 / 90 / 120 / 150 / 180 / 210 / 240 / 300 / 360
Acetaminophen concentration(μg/ml)

5. QUESTIONS

a) Whatare the key factors for the success of this experiment? Attention should be paid to these factors when conducting this experiment?

b) If inaccurate results are obtained, what are the possible causes? What can be done to improvethis?

c) What are the other experiments for determining the relative bioavailability?

(Jin SUN)

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