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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 NSAID’s aspirin is highly metabolized in the body primarily by CYP3A or CYP
2C family (Katzung et al., 2009). Some of the NSAID’s undergo phase I as well as phase II
others may undergo conjugation reaction (Katzung et al., 2009). NSAID’s 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 acetate
and salicylate in blood by an enzyme called fig 1. Structure of Aspir as 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 plasma
catecholamine 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 of
aggregation 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 Volume of ASA (μl) Volume of SA (μl)

human plasma (μl)
500 0 0
500 5 5
500 10 10
500 20 20
500 40 50
500 50 100

14. All plasma samples are vortex mixed and 500 μl of sample is taken into each test tube.
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15. Now to both samples and standards add 50 μl of 0.5mg/ml Phenacetin solution.

16. Then add 60 μl 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 then
evaporated using Buchler vortex evaporator and then reconstituted in 150 μl 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 Ratio of area of Concentration SA Ratio of area of SA
(mg/ml) ASA to Phenacetin (mg/ml) 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 Absorbance at 525 nm

(mg/ml)
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.

SA:PC x=y+0.00315.10
area SA concentration
Samples ratio (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 0
Table 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 log
graph)

Clast = last plasma concentration = 0.01718 mg/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 Volume pH Abs Salicyla Amou Excretion Cumulati %

(hrs) (ml) at te nt rate ve Dose
525n (mg) (mg/hr)
m conc. amount
of
(mg/ml)
drug
excreted

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 0.270 216.8 216.852 348.99 36.14

0.4252 52

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 0.144 0.2286 39.32 39.32 - -

g
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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Additive Co

Summary

Urine Kel Plasma

Kel
% OF I NHI
Urine SA
t1/2
PeakB
Graph 9 showing % inhibition of platelet aggregation against time

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 0.39335 1.6 0.029

mg/ml hrs litres mg/ml
/hour

Summary of all the results:

Kel t ½ 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 μg/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 activation
and 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 took
aspirin. 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 enzyme
by acetylating it, rest of the aspirin in the body is metabolized to salicylic acid. When
the aspirin reaches liver via blood aspirin is bound to the proteins and only unbound
aspirin is metabolized(PharmPK Discussion, 2003). This is first pass metabolism of
aspirin as aspirin enters liver to be metabolized for the first time. But the bound
aspirin escapes the first pass metabolism and irreversibly binds to COX enzymes to
inactivate it. This can lead to decreased production of prostaglandins and affect the
intestinal wall. If all the absorbed aspirin undergoes metabolism then aspirin will not
affect the COX enzyme (PharmPK Discussion, 2003).

In order to prevent aspirin from affecting the COX enzymes it is given in low but
effective dose in enteric coated form which is slow release forms (PharmPK
Discussion, 2003). Dutch TIA trial has shown that 30 mg daily dose of aspirin is as
effective as 300 mg dose and it was found that the 30 mg aspirin for 10 days reduced
the plasma thromboxane by 92 % (PharmPK Discussion, 2003). Hence it is clear that
higher dose increases the risk of adverse events because of strong suppression of
prostaglandin 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). The
increased 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 ½ Clearance Cmax

Group A(Bi 0.15 hr-1 4.62 hours 25.0 ml/min 0.009 mg/ml
+ asp)

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

only asp

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

Group D(Bi 0.1066 hr-1 6.5 hr 1.23litres/hour 0.03 μg/ml

+ asp)

On the basis of data obtained from each volunteer Kel of buffered group is lower than the
aspirin group whereas t1/2 of all the volunteer is similar. Clearance of buffered group A is higher
than the other groups showing the effect of bicarbonate on the clearance of drug. When
comparing Cmax between the group the bicarbonate + aspirin group have lower Cmax than
those 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 on

aspirin-induced damage and potential difference changes in human gastric
mucosa. Bristish medical Journal. Vol 2. 1052-1055
• Dahl, G et al. (1982). The effect of buffering of acetylsalicylic acid on
dissolution, absorption, gastric pH and blood loss. International Journal of
Pharmaceutics. 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.ht
ml [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 clinical
pharmacology
• 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 on
Platelets. 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