Report on Aspirin Pharmacokinetics to Submit

8/14/2019 Report on Aspirin Pharmacokinetics to Submit

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aitReport on Effect of Aspirin on pharmacokinetic parameters and platelets

Aim:

To study the effect of aspirin on platelet aggregation.

To study the pharmacokinetics of an orally administered aspirin.

To study the association between urine pH and salicylic acid excretion.

Introduction:

In order to understand and study any drug, it is important to have the knowledge about the

drug disposition and pharmacokinetics of the drug. Absorption of the drug is primarily

influenced by its physico-chemical properties. (elephant formulary,2006) Several factors

influence the rate of absorption which includes particle size, alimentary motility, mucosal

blood flow, digestive secretions and intestinal contents (Volans, 1974). Bioavailability and

absorption could be influenced by particle size and other factors (Volans, 1974). There is no

evidence that digestive secretions play any important role in drug absorption. Also it is

impossible to measure the mucosal blood flow.

Like most of the NSAIDs aspirin is highly metabolized in the body primarily by CYP3A or CYP

2C family (Katzung et al., 2009). Some of the NSAIDs undergo phase I as well as phase II

others may undergo conjugation reaction (Katzung et al., 2009). NSAIDs are highly protein

bound but nonlinearly (Katzung et al., 2009). Toxicity is observed when

salicylate levels increases in plasma due to unavailability of binding sites.

Chemically aspirin is salicylate ester of acetic acid known as acetylsalicylic

acid (refer fig 1). Aspirin occurs in crystalline form and has a pKa of 3.5 hence

it is acidic. Aspirin is stable at pH of 2-3. (elephant formulary,2006). Absorption of aspirin

largely takes place in stomach and from small intestine. Aspirin is metabolized to acetateand salicylate in blood by an enzyme called fig 1. Structure of Aspiras aspirin esterase (refer

fig 2).(Katzung et al., 2009).

Parameters such as content of stomach, stomach emptying times, rate of tablet

disintegration and stomach pH influences the rate of absorption. Since gastric pH provides

ASA and SA with most stable pH of 3 so most of it exists in non-ionized state and is absorbed

by passive diffusion. As the drug moves from the acidic to basic pH of small intestine it will

have increased surface area so absorption of the drug will be more as compared to stomach.

Using HPLC we can determine that salicylate levels reaches peak in 1-2 hrs (Katzung et al.,

2009). Primary route of elimination is renal excretion.

Aspirin is metabolized to salicylic acid in liver, blood and stomach and acetic acid (Michelson,

2007). Salicylic acid which is primary metabolite of aspirin undergo conjugation with glycine

to form salicyluric acid or can form glucuronides or can form free salicylates or can undergo

oxidation to form gentisic acid (refer fig 2).


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Fig 2. Showing the metabolism of aspirin and the metabolites formed. Adapted from(Katzung et al., 2009)

Pharmacology of Aspirin

Changes in platelet aggregation are brought about by the interfering with prostaglandin

biosynthesis. Phospholipids from the membrane are enzymatically converted to arachidonic

acid by phospholipase A (Michelson, 2007). Arachidonic acid then serves as a substrate

which is acted upon by 5-lipoxygenase and lead to the formation of leukotrienes (Michelson,

2007).

Arachidonic acid undergoes metabolism mediated by prostaglandin H synthase enzyme

which has cyclooxygenase activity and forms PGG2 . PGG2 is then converted to PGH2 . PGH2 is

then converted by synthases to PGD2, PGE2, PGF2, PGI2 and thromboxane A2 (Michelson,

2007).(refer fig 3)


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Fig 3. Showing Arachidonic acid pathway adapted from (Michelson, 2007).

There are 2 isoforms of cyclooxygenase, COX-1 and COX-2. COX-1 is expressed always and is

important in the synthesis of prostaglandins. Influencing the activity of COX effects renal

blood flow, stomach mucous production, platelet activation and aggregation. COX-2 is

expressed only under conditions when inflammation is there, they synthesize PGI2(Michelson, 2007).

Aspirin irreversibly acetylates the cyclooxygenase enzyme and effects the active site of the

enzyme thus interaction of enzyme with the substrate Arachidonic acid is inhibited leading to

inhibition of the whole pathway. COX-2 is also acetylated by aspirin in the same way but it is

not blocked (Michelson, 2007). Although aspirin blocks both COX-1 and COX-2, its inhibitory

effect is more profoundly seen in COX-1 inhibition. Inactivation of COX-2 by aspirin leads to

anti-inflammatory effects while inactivation of COX-1 leads to anti-thrombotic effects

(Michelson, 2007).

Although it has been known that aspirin has anti-thrombotic effect but the effect produced by

aspirin is weak and only block the activation pathway of platelets that has been mediated by

thromboxane. Thrombosis could be caused several other factors like elevated plasmacatecholamine levels, thrombin levels or ADP levels (Michelson, 2007).

Objective:

To study the difference in pharmacokinetic parameters (Kel, t1/2, clearance, Cmax) of orally

administered aspirin with and without sodium bicarbonate.

To study the difference in platelet aggregation produced by aspirin in volunteer with and

without sodium bicarbonate.

Materials Required:

Aspirin 600 mg Sodium bicarbonate 10 gm

Adenosine diphosphate

Arachidonic acid

Salicylic acid stock 0.5 mg/ml

Acetyl salicylic acid stock 0.2 mg/ml

solution

Phenacetin 0.5 mg/ml

Hydrochloric acid 5 % 1.0 M

Diethyl ether

Ferric nitrate 40 mg/ ml in 0.1 M HCl

Protocol:

1. Volunteers were divided into 2 groups.

1. Volunteers who took 600 mg aspirin

2. Volunteers who took 600 mg aspirin and 10 gm sodium bicarbonate

2. 10-20 ml of blood is taken from all the volunteers at t=0 hr for the baseline measurements

of acetylsalicylic acid and salicylic acid levels in plasma and platelet aggregation. After


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this 2 volunteers took only aspirin and 2 volunteers took 10 gm sodium bicarbonate orally

followed by aspirin orally with milk.

3. Urine is collected from volunteers at start of the experiment i.e. t=0 hr, in a labeled

container and volume, pH, salicylate levels of the collected urine samples are measured and

recorded.

4. Now volunteers are given 100 ml water each hour and urine is collected at t=0 hr, t=1 hr,

t=2 hr, t=4 hr, t=5 hr, t=6 hr, t=7 hr and volume, pH and salicylate levels are measured of

each sample. From t=8 hr to 24 hr urine is collected in a labeled container and volume, pH

and salicylate levels are measured and noted.

5. Blood is taken from volunteers at t=1 hr, t=2 hr, t= 4 hr, t=6 hr and t=8 hr for

determination of acetylsalicylic acid and salicylic acid levels and platelet aggregation studies.

6. Platelet aggregation studies were done for samples t=0, t=2 & t=4 hr using platelet

aggrometery profiler was performed by the demonstrator.

7.Using platelet profiler, platelet count was done and platelet aggregation was measured for

blood samples collected at t=0 hr, t=2 hr and t=4 hr and percentage inhibition ofaggregation at each time point was calculated and results obtained from each volunteer is

plotted against time.

8. Blood is again collected from all the volunteers at t= 24 hr to determine acetylsalicylic

acid and salicylic acid levels.

Determination of acetylsalicylic acid and salicylic acid in urine

10. All urine samples were vortex mixed and 1 ml of urine sample is taken into each test

tube similarly for standards 1 ml of sodium salicylate solution is taken in each test tube.

11. To both standards and urine sample containing test tube add 5 ml of ferric nitrate and

vortex mix all the test tubes.

12. Now absorbance is read at 525nm and a graph is plotted of sodium salicylate

concentration against absorbance (Refer graph 3 & table 2).

Determination of acetylsalicylic acid and salicylic acid in Plasma

13. Preparation of Standard curves

Volume of standard

human plasma (l)

Volume of ASA (l) Volume of SA (l)

500 0 0500 5 5500 10 10500 20 20500 40 50500 50 100

14. All plasma samples are vortex mixed and 500 l of sample is taken into each test tube.


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15. Now to both samples and standards add 50 l of 0.5mg/ml Phenacetin solution.

16. Then add 60 l of 0.1M HCl to all the tubes and vortex mixed.

17. Then 5 ml of diethyl ether is added and mixed on rotary mixer for 15 minutes.

18. All samples are then centrifuged at 3000rpm for 5 minutes.

19. Transfer the upper ether layer to fresh tubes using a Pasteur pipette. Ether layer is thenevaporated using Buchler vortex evaporator and then reconstituted in 150 l of mobile phase

containing 5 % 0.1 M HCl.

20. Samples are then given for HPLC.

21. The data obtained from the graph shows peaks showing the concentration for salicylic

acid, acetyl salicylic acid and Phenacetin. These values were then used to plot a standard

curve of [ASA] and [SA] versus area ratio. From this graph concentration of ASA and SA in the

plasma could be determined (refer to graph 1 & 2) (refer to table 1).

C) Determination of acetylsalicylic acid (ASA) and salicylic acid (SA) in

plasma using HPLC

For standard curve of ASA and SA

Final concentration of ASA [0,0.002, 0.004, 0.008, 0.016, 0.02] mg/mL

Final concentration of SA [0, 0.005, 0.01, 0.02, 0.05, 0.1] mg/mL

ASA:PC area and SA:PC area (standard & sample)

ASA:PC area Vs ASA concentration (standard & sample)

SA:PC area Vs SA concentration (standard & sample)

Table 1: Standard reading of ASA and SA using HPLC

Concentration ASA

(mg/ml)

Ratio of area of

ASA to Phenacetin

Concentration SA

(mg/ml)

Ratio of area of SA

to Phenacetin

0 0 0 0

0.002 0.01 0.005 0.07

0.004 0.035 0.01 0.131

0.008 0.075 0.02 0.294

0.016 0.166 0.05 0.785

0.02 0.204 0.1 1.493

Graph 1. ASA concentration against ASA:PC area using HPLC


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Graph 2: Standard curve of SA concentration against SA:PC area using HPLC

DETERMINATION OF SALICYLATE LEVELS IN URINE

Table 2. showing SA levels in urine against absorbance

Concentration of SA

(mg/ml)

Absorbance at 525 nm

0 0

0.01 0.26

0.02 0.152

0.05 0.27

0.1 0.293

0.25 0.077

0.5 0.144

1 0.264

Graph3. Standard curve for urinary salicylic acid concentration

Results:

Plasma data

The HPLC spectra of the plasma samples showed no peaks for acetylsalicylic acid but the

peaks were prominently observed for salicylic acid. Therefore it was possible to interpret

pharmacokinetics of plasma salicylate levels.

Samples

SA:PCarearatio

x=y+0.00315.10SA concentration(mg/ml)

Control 0 0

T=1 0.235 0.016312248

T=2 0.435 0.029771198

T=4 0.364 0.024993271

T= 6.45 0.248 0.017187079

T=24 0 0Table 3. showing the SA concentration calculated from the graph

Graph 4. Salicylic acid concentration against time


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Calculations:

Peak Plasma Concentration, Cmax= 0.029 mg/ml

AUC= A1 + A2 + A3 +A4 + A5

= 0.00815 + 0.02304+0.05476+0.1594 + 0.148

Kel = 0.147 per hour (From slope of Plasma conc v/s time plot on semi loggraph)

Clast = last plasma concentration = 0.01718mg/ml

AUC= 0.39335 mg/ml hours

Half life of SA from plasma = 4.75 hours (0.693 /kel)

Renal Clearance = Total AmountAUC = Urine Volume x concentration AUC =1.6 litres/ hours

The concentration peak for salicylic acid was obtained at 0.029 mg/ml. The t1/2 determined

was 4.75 hours and Kel of plasma SA was 0.147 hr-1. AUC was found out using trapezoidal

rule = 0.39335 mg/ml


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Graph 5 showing semi log graph of log concentration against time interval

Urine samples:

Y=1.560x + 0.004

Where y= concentration of SA and x = absorbance measure at 525nm.

Urine Data

Time

(hrs)

Volume

(ml)

pH Abs

at

525n

m

Salicyla

te

conc.

(mg/ml)

Amou

nt

(mg)

Excretion

rate

(mg/hr)

Cumulati

ve

amount

of

drug

excreted

%

Dose

0 - - - - - -

1 96 5.57 0.260 0.4096 39.32 39.32 39.32 6.55

2 385 6.50 0.152 0.2411 92.82 92.82 132.14 15.47

4 510 6 0.270

0.4252

216.8

52

216.852 348.99 36.14

5 245 5.94 0.293 0.461 112.94 112.94 461.93 18.81

6 635 6.15 0.077 0.1241 78.8 78.8 540.73 13.13

7 Missing Missin

g

0.144 0.2286 39.32 39.32 - -

8-24 1700 5.89 0.264 0.4158 706 706.86 117.81

Graph 6. Urinary pH variation with time

Graph 7 showing urinary excretion rate against time

Graph 8 showing the cumulative excretion rate against time


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Graph 9 showing % inhibition of platelet aggregation against timeSummaryUrine Kel Plasma

Kel

Urine SA

t1/2

Plasma

SA t1/2

AUC Clearanc

e

Cmax


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0.23 hr-1 0.147 hr-1 3 hr 4.75 hr 0.39335mg/ml hrs

1.6

litres

/hour

0.029

mg/ml

Summary of all the results:

Kel t Clearance Cmax

Group A 0.15 hr-1 4.62 hours 25.0 ml/min 0.009 mg/ml

Group B 0.55 hr-1 1.2 litres/hr 0.12mg/mL

Group C 0.147 hr-1 4.75 hr 1.6 litres /hour 0.041 mg/ml

Group D 0.1066 hr-1 6.5 hr 1.23litres/hour 0.03 g/ml

Discussion:

The platelet aggregometry was performed using platelet rich plasma to which agonist

such as ADP or Arachidonic acid is added. In response to the addition of addition of

agonist, platelets aggregate and transmission of light through the sample increases.

Platelet aggregation can follow a biphasic response. Initial response is usually due to

addition of external agonist such as ADP but sometimes with addition of agonist

aggregation is produced due to release of internally stored ADP from the granules of

platelets.

Acetylsalicylic acid can block aggregation for low dose of agonist but cannot block

the effect of high dose of agonist.

Below are two graph which shows platelet aggregation studies at time t=2 hr and

t=4 hr. At t=2 hr we can interpret that using ADP as agonist peak aggregation

achieved was 30% whereas after the addition of arachidonic acid we find no

aggregation or 100% inhibition of aggregation.

% inhibition = (80-30)/80 x 100 = 62.5 % (we know that aggregation in

control sample was 80% at t=0)


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Graph 10 showing % platelet aggregation at t=2

In this aggregometry graph maximum aggregation observed is 10% with ADP as an

external agonist while with Arachidonic acid no aggregation is observed showing 100

% inhibition.

% inhibition = (80-10)/80 x 100 = 87.5 %.

Graph 11 showing % platelet aggregation at t=4


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In both of the above graph we can see that even in the presence of external agonist

(ADP) the platelet aggregation at t=2 is 30% and at t=4 is 10% indicating that aspirin

has irreversibly inactivated the COX enzymes and it is observed that % inhibition has

increased from t=2 hr to t=4 hr indicating increased amount of aspirin being

absorbed into the system and more COX enzymes are inactivated, which is shown by

the fact that even on addition of arachidonic acid that is substrate of COX enzyme no

aggregation is observed as pathway is blocked.Mechanism of inhibitory effect of aspirin

It has been known that aspirin acetylates and causes irreversible inactivation of COX-

1 enzyme and produces anti-inflammatory effect by blocking prostaglandin

production and also produces anti-thrombotic effect by blocking the production of

thromboxane A2 (Schafer, 1999). However several other factors such as thrombin and

catecholamines can lead to aggregation.

In ex vivo studies it was found that administration of low dose of aspirin for long time

can block thromboxane A2 and lead to 95% inhibition of aggregation (Schafer, 1999).It was found that inhibition of platelet aggregation reaches its peak within 2 hours

after the administration of aspirin. As the aggregation ability of platelets is impaired

it results in increase in the number of platelets coming from bone to maintain the

platelets function (Schafer, 1999).

We know that ADP is one of the agonist for platelet aggregation but it is a relatively

weak agonist (Micheslon, 2007). ADP has 2 receptors on the surface of platelets

known as P2Y1, P2Y12 which on activation mobilizes calcium (changes shape) and

leads to aggregation. ATP binds to P2X1 receptor present on the surface of platelets

(Micheslon, 2007). ATP is an antagonist of P2Y1, P2Y12 and inhibits platelet activationand function. But sometimes it can also act as agonist and can amplify the response

of other agonist (Micheslon, 2007).

Graph 12 shows % inhibition in all the 4 volunteers.

The graph above shows the % inhibition of platelet aggregation in 4 volunteers and

Volunteer I and IV took sodium bicarbonate along with aspirin while II and III only tookaspirin. By observing the graph it is evident that sodium bicarbonate has a profound

effect on the % inhibition because volunteer I and IV who took sodium bicarbonate

along with aspirin has 100 % and 75 % inhibition at t =2 hours whereas for the same

volunteers at t=4 hr % inhibition was 19% and other was out of scale indicating that

sodium bicarbonate lowered the absorption of the aspirin at t=4 hr while in both the

volunteer no significant effect on absorption of aspirin was visible at t=2 hr as %

inhibition is produced due to high absorption of aspirin.


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In volunteers II and III aspirin produced significant % inhibition of platelets initially in

volunteer III at both t=2 hr and at t=4 hr indicating high absorption rate and due to

increased inhibition it appears that plasma concentration increases from t=2 to t=4

hr but in volunteer II there was a significant change in the aggregation at t=2 and

t=4 hr which could be due to no absorption of aspirin in the blood resulting in no

inhibition or could be due to human error or due to contamination.

The first pass elimination of aspirin

On orally administering the aspirin, aspirin irreversible inactivates the COX enzymeby acetylating it, rest of the aspirin in the body is metabolized to salicylic acid. Whenthe aspirin reaches liver via blood aspirin is bound to the proteins and only unboundaspirin is metabolized(PharmPK Discussion, 2003). This is first pass metabolism ofaspirin as aspirin enters liver to be metabolized for the first time. But the boundaspirin escapes the first pass metabolism and irreversibly binds to COX enzymes toinactivate it. This can lead to decreased production of prostaglandins and affect theintestinal wall. If all the absorbed aspirin undergoes metabolism then aspirin will notaffect the COX enzyme (PharmPK Discussion, 2003).

In order to prevent aspirin from affecting the COX enzymes it is given in low buteffective dose in enteric coated form which is slow release forms (PharmPKDiscussion, 2003). Dutch TIA trial has shown that 30 mg daily dose of aspirin is aseffective as 300 mg dose and it was found that the 30 mg aspirin for 10 days reducedthe plasma thromboxane by 92 % (PharmPK Discussion, 2003). Hence it is clear thathigher dose increases the risk of adverse events because of strong suppression ofprostaglandin synthesis.

The influence of bicarbonate on the pharmacokinetics of aspirin

Sodium bicarbonate protects the intestinal mucosa from being damaged by aspirin

by increasing the pH. Since aspirin is an acid its dissociation constant is 3.5. So at pH

above this aspirin exists in non-lipid soluble dissociated form and it cannot enter cells

in stomach (Bowen , 1977).

When aspirin enter the stomach cells the damage is equivalent to the amount of free

H+ ions present. When bicarbonate is used along with aspirin it increases the pH and

eliminated free H+ ions (Dahl, 1982). Other mechanism by which bicarbonate

protects are as at the pH of stomach aspirin is completely dissolved to form a

suspension and can cause erosion in other parts of the body(Dahl, 1982). Theincreased pH of the gastric contents tends to increase gastric emptying thus

decreases the time for which aspirin particles and gastric mucosa are in contact

(Dahl, 1982).

Sodium bicarbonate increases the amount of filtered bicarbonate ion in the kidney

tubular fluid. Bicarbonate ion is filtered and not reabsorbed, and so this excess

bicarbonate in the filtrate will associate with H+ to form carbonic acid leading to a

reduction of H+ ion concentration, and increased pH (Dahl, 1982). Increased pH of


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urine leads to rise in urinary clearance so more amount of salicylic acid is excreted

out of the body.

An alkaline pH due to bicarbonate increases the degree of ionization of a weak acid

such as aspirin, and thus increases the rate of elimination of the drug because the

ionized drug is not reabsorbed from the kidney tubules (Bowen , 1977).

The rate of absorbance of the aspirin under buffered condition is faster than when

aspirin is taken alone and shows lower plasma peak (0.009 mg/ml & 0.03 mg/ml), low

levels of SA in plasma is observed because rate of excretion of SA is increased by

raised urine pH. Slower dissolution of aspirin in absence of bicarbonate could be due

to decreased rate of gastric emptying (Dahl, 1982).

Kel t Clearance Cmax

Group A(Bi

+ asp)

0.15 hr-1 4.62 hours 25.0 ml/min 0.009 mg/ml

Group B

only asp

0.55 hr-1 - 1.2 litres/hr 0.12mg/mL

Group C only

asp

0.147 hr-1 4.75 hr 1.6 litres /hour 0.041 mg/ml

Group D(Bi

+ asp)

0.1066 hr-1 6.5 hr 1.23litres/hour 0.03 g/ml

On the basis of data obtained from each volunteer Kel of buffered group is lower than theaspirin group whereas t1/2 of all the volunteer is similar. Clearance of buffered group A is higherthan the other groups showing the effect of bicarbonate on the clearance of drug. Whencomparing Cmax between the group the bicarbonate + aspirin group have lower Cmax thanthose who only took aspirin due to increased excretion of SA from the body.

References:

Bowen, B.K, Krause, W.J, IVEY, K.J.(1977).Effect of sodium bicarbonate onaspirin-induced damage and potential difference changes in human gastricmucosa. Bristish medical Journal. Vol 2. 1052-1055

Dahl, G et al. (1982). The effect of buffering of acetylsalicylic acid ondissolution, absorption, gastric pH and blood loss. International Journal ofPharmaceutics. Vol 10. 143-151


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Elephant formulary, 2006[available online]

http://www.elephantcare.org/Drugs/aspirin.htm [accessed on 6th March 2010]

Experimental Simulation [available online]http://www.health.herts.ac.uk/cpd/multimedia/e-learning/demos/demo_pk/Pharmacokinetics/tests/QuestGenerator/ExpAsTest.html [accessed on 7th march 2010]

IPCS INCHEM, 1999 [available online]

http://www.inchem.org/documents/pims/pharm/aspirin.htm#SubSectionTitle:3.4.4 Bioavailability [accessed on 6th March 2010]

Katzung, B.G et al. (2009).11th Ed. Tata Mcgraw Hill Lange. Basic and clinicalpharmacology

Michelson, A.D (2007). 2nd Ed. Platelets. Academic Press.

PharmPK Discussion, 2003 [available online]

http://www.boomer.org/pkin/PK03/PK2003459.html [accessed on 8th Mar 2010]

Schafer, A.I. (1999).Effects of Nonsteroidal Anti-inflammatory Therapy onPlatelets. Am. J. Med. 106(5B)

Volans, G.N.(1974). Effects of food and exercise on the absorption of

effervescent aspirin. Br. J. clin. Pharmac., 1, 137-141
http://www.elephantcare.org/Drugs/aspirin.htmhttp://www.health.herts.ac.uk/cpd/multimedia/e-learning/demos/demo_pk/Pharmacokinetics/tests/QuestGenerator/ExpAsTest.htmlhttp://www.health.herts.ac.uk/cpd/multimedia/e-learning/demos/demo_pk/Pharmacokinetics/tests/QuestGenerator/ExpAsTest.htmlhttp://www.health.herts.ac.uk/cpd/multimedia/e-learning/demos/demo_pk/Pharmacokinetics/tests/QuestGenerator/ExpAsTest.htmlhttp://www.boomer.org/pkin/PK03/PK2003459.htmlhttp://www.health.herts.ac.uk/cpd/multimedia/e-learning/demos/demo_pk/Pharmacokinetics/tests/QuestGenerator/ExpAsTest.htmlhttp://www.health.herts.ac.uk/cpd/multimedia/e-learning/demos/demo_pk/Pharmacokinetics/tests/QuestGenerator/ExpAsTest.htmlhttp://www.health.herts.ac.uk/cpd/multimedia/e-learning/demos/demo_pk/Pharmacokinetics/tests/QuestGenerator/ExpAsTest.htmlhttp://www.boomer.org/pkin/PK03/PK2003459.htmlhttp://www.elephantcare.org/Drugs/aspirin.htm

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Report on Aspirin Pharmacokinetics to Submit