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Experiment No.

2
Factors Affecting Drug Action

C. Influence of Metabolism on Drug


Action

Subsection B4

1
INTRODUCTION

 DRUG BIOTRANSFORMATION
– First pass effect
 Following oral administration, drugs absorbed from the
small intestines are transported via portal system to liver
where extensive drug metabolism occurs

 Greatly limits bioavailability of oral drugs

2
 PHASE I
– Involves microsomal mixed function oxidase system
– Oxidation, reduction, hydrolysis, methylation, demethylation
– Involves cytochrome P450 enzymes

 PHASE II
– Conjugation reactions with an endogenous substance to yield
drug conjugates (increase polarity of drugs for greater
excretion)
– Glucuronidation, sulfation, acetylation

3
Hepatic cytochrome P450 (CYP 2C9)
oxidative enzyme system
 Found in smooth ER membranes in liver
hepatocytes and intestinal tract mucosal surface
 Ability to biotransform lipophilic substrate of diverse
structures into more water soluble---secreted from
body
 Important in detoxification of foreign substances
(xenobiotic compounds) by oxidative metabolism
 responsible for the metabolism of S-warfarin

4
 Prothrombin Time (PT)
 time it takes plasma to clot after addition of tissue
factor.
 used to measure the extrinsic pathway of
coagulation.

5
OBJECTIVES

 General Objective
 To determine the effect of different drugs on
the metabolism of warfarin among albino rats.

6
OBJECTIVES

 Specific Objectives
 To determine the effect of NSS on the
prothrombin time of albino rats
 To determine the effect of warfarin on the
prothrombin time of albino rats
 To compare the effects between warfarin and
warfarin coadministered with rifampicin;
warfarin with ketoconazole on the prothrombin
time of albino rats

7
OBJECTIVES

 Specific Objectives
 To determine the effect of the drug combination
of warfarin and rifampicin on the prothrombin
time of albino rats
 To determine the effect of the drug combination
of warfarin and ketoconazole on the
prothrombin time of albino rats

8
Statement of the Problem

 Does the administration of rifampicin &


ketoconazole affect the metabolism of
warfarin?

9
HYPOTHESIS

 Null Hypotheses (Ho)


 there is no significant effect of different drugs
on the metabolism of warfarin among albino
rats
 Alternative Hypotheses (Ha)
 there is significant effect of different drugs on
the metabolism of warfarin among albino rats

10
MATERIALS

 Animal weighing scale 8 citrated tubes


 Tuberculin syringe 8 plain test tubes
 Animal cage PT reagents
 Asbestos gloves centrifuge machine
 Surgical gloves automatic pipettor
 Surgical blades pipettor tips
 Gauze 1 test tube rack
 Mortar and pestle rat tail cage
 50mL beakers stopwatch
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MATERIALS

 Rats (8 per section)


 Drugs: (1 drug per section)

Warfarin 10mg tablet


Rifampicin 200mg/5mL
Ketoconazole 200mg tablet
NSS 0.9% solution

12
METHODOLOGY

Rats were fasted

Rats were weighed

Warfarin+ Warfarin +
0.9% NSS Warfarin
rifampicin ketoconazole

Drugs were given orally,


for 4 days

Testing for prothrombin time


on fifth day
13
METHODOLOGY
 Prothrombin
Determination
Measure PT by
Collect blood Place adding 0.02mL
from tail in Centrifuge 0.01mL of prewarmed
the citrated blood for 3 plasma in thromboplastic
test tube minutes test tube reagent
(simplastin)

The Prothrombin Time is measured from time of mixing until the appearance of fibrin
strands.

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PROCEDURE AND RATIONALE

1. Rats were fasted for 8 hours prior to


experiment.
 Rationale: The rats were fasted prior to weighing so
as to get accurate weights that will be used for the
computation of drug doses.
2. The rats were then weighed.
 Rationale: This is essential for calculating the dose
of the drug that will be given to each rat.

15
PROCEDURE AND RATIONALE
3. The rats were divided into four groups:
 Group I no treatment- give 1mL of
0.9% NSS
 Group II Warfarin
 Group III Warfarin and Rifampicin
 Group IV Warfarin and Ketoconazole
Rationale: Four groups are needed in order to compare
the effect of administering Warfarin alone with that of
Warfarin and Rifampicin, and with that of Warfarin and
Ketoconazole. The first group was used for control.

16
PROCEDURE AND RATIONALE

4. Drugs were given once daily, orally using a


gavage, for 4 days.
 Rationale: Warfarin is often clinically administered
orally. This is also the best route of choice to
determine the effect of drug interactions and
metabolism since Warfarin is metabolized by the
Cytochrome P450 enzymes found in the liver.
 Maximum effect of warfarin: 3-5 days

17
PROCEDURE AND RATIONALE

 Dosage Computation
 Warfarin Preparation: 10 mg tablet
 Dose: 0.2 mg/ 200 g rat
 Rifampin Preparation: 200 mg/ 5 ml
 Dose: 11 mg/ 200 g rat
 Ketoconazole Preparation : 200 mg tablet
 Dose: 4 mg/ 200 g rat

 Computation sample (using warfarin): Dose = 0.2mg/200g

_0.2mg = ___x___
200 g wt. of rat
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Warfarin dosage

RATS Weight (g) Dose (mg)


A 166.88 0.17
B 144.5 0.14
C 159.3 0.16
D 162.9 0.16
E 145.5 0.15
F 170.6 0.17
G 165.0 0.17
H 144.0 0.14
19
PROCEDURE AND RATIONALE

5. Prothrombin time of each rat was determined


on the fifth day.
 Rationale: The anticoagulant activity of Warfarin is
best exhibited in the Prothrombin time. It was used
to compare the effect of Warfarin alone to that which
is in combination with other drugs.
6. The results were then analyzed statistically.

20
ACTUAL RESULTS

Table 1: Prothrombin time (sec) of rats in four different group


treatments
GROUP 1 GROUP 2 GROUP 3 GROUP 4
NSS Warfarin Warfarin/ Rifampicin Warfarin/ Ketoconazole

6 39 127 900

3.4 31 279 298

3.5 28 334 148

2.2 28 245 900

2.6 30 307 155

8.7 37 455 140

7.5 44 238 150

9.0   179 119

Mean 4.8625 Mean = 33.86 Mean = 270.5 Mean = 351.25


21
ACTUAL RESULTS

Figure 1: Mean Prothrombin times (sec) of rats in four different


group treatments
 
Effect of Drug Metabolism in PT 

400 
351.25
350 

300 
270.5
250 
PT
(sec)  200 

150 

100 

50  4.8625 33.86



I (NSS)  II (Warfarin)  III (Warfarin w/  IV (Warfarin w/ 
Rifampicin  ketoconazole) 
Drug 
22
ACTUAL RESULTS

 Warfarin administration resulted to an increase


in PT (33.86)
 Concurrent administration of warfarin and
rifampicin resulted in prolonged PT(270.5)
 Giving ketoconazole and warfarin
simultaneously resulted in a prolonged PT
(351.25)

23
DATA ANALYSIS

24
ONE – WAY ANOVA

ANOVA

PT
Sum of
Squares df Mean Square F Sig.
Between Groups 691508.8 3 230502.921 6.958 .001
Within Groups 894411.6 27 33126.354
Total 1585920 30

 Statistically, the data showed that there is a


significant difference across the PT of those with
NSS, Warfarin, Warfarin+Rifampicin, and
Warfarin+Ketoconazole (F=6.958,p=0.001).
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POST HOC TESTS
Multiple Comparisons

Dependent Variable: PT
Tukey HSD

Mean
Difference 95% Confidence Interval
(I) drug (J) drug (I-J) Std. Error Sig. Lower Bound Upper Bound
1 2 -28.99464 94.19730 .990 -286.7715 228.7823
3 -265.63750* 91.00323 .033 -514.6736 -16.6014
4 -346.38750* 91.00323 .004 -595.4236 -97.3514
2 1 28.99464 94.19730 .990 -228.7823 286.7715
3 -236.64286 94.19730 .081 -494.4198 21.1340
4 -317.39286* 94.19730 .012 -575.1698 -59.6160
3 1 265.63750* 91.00323 .033 16.6014 514.6736
2 236.64286 94.19730 .081 -21.1340 494.4198
4 -80.75000 91.00323 .811 -329.7861 168.2861
4 1 346.38750* 91.00323 .004 97.3514 595.4236
2 317.39286* 94.19730 .012 59.6160 575.1698
3 80.75000 91.00323 .811 -168.2861 329.7861
*. The mean difference is significant at the .05 level.
Post hoc analysis showed that the difference existed between group I 
and group III (sig= 0.033), group I and group IV (sig=0.004), group II and 
26       group IV (sig=0.012).
DISCUSSION

27
EXPECTED RESULTS
 Warfarin administration should result to an
increased PT as compared to NSS
administration.
 Prolonged concurrent administration of
warfarin and rifampicin should result in shorter
PT compared to warfarin
 Giving ketoconazole and warfarin
simultaneously should result in a prolonged PT
compared to warfarin

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CYP2C9
 Principal member of four CYP2C enzymes, one of the
most important drug-metabolizing P450s in human liver
 A rate-limiting enzyme in the metabolic clearance of
clinically used drugs, such as tolbutamide, phenytoin,
S-enantiomer of warfarin
 Individual variability occurs in the metabolism of
CYP2C9 substrates
 Drug induction is another source of variation in the
metabolism of CYP2C9 substrates, which may result in
drug toxicity or therapy failure

29
CYP2C9 Affected Drugs, Inducers, and
Inhibitors

Affected Drugs Inducers Inhibitors


Azole
antifungals:
Phenytoin Rifampin
esp.
S-Warfarin
Fluconazole,
NSAIDS
ketoconazole,
itraconazole
Metronidazole

Ritonavir (Norvir)

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INDUCTION OF CYP2C9

 Occurs at the transcriptional level and is mediated by


nuclear receptors constitutive androstane receptor
(CAR) and pregnane X receptor (PXR)
 PXR – ligand gated dependent; binds rifampicin,
hyperforin and lithocholic acid
 CAR - induce gene expression via CAR-dependent
mechanisms, which then signals CAR translocation to
the nucleus

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 Both CAR and PXR then heterodimerize
with the retinoid X-receptor in the
nucleus
 bind to CAR & PXR responsive elements
within gene promoters and associate with
coactivators/corepressors which then
induce CYP2C9 gene transcription

32
INHIBITION OF CYP2C9

 Imidazole-containing drugs such as cimetidine


and ketoconazole bind tightly to the P450
heme iron and effectively reduce the
metabolism of endogenous substrates or other
coadministered drugs

33
WARFARIN

 MOA: blocks the γ-carboxylation of several


glutamate residues in prothrombin and factors
VII, IX and X as well as the endogenous
anticoagulant proteins C and S by inhibiting
epoxide reductase (VKOR)

34
WARFARIN

 PK: small, lipid-soluble molecules that are


readily absorbed after oral administration
 PD: Anticoagulant activity / Antithrombotic
effect
 S- warfarin is the pharmacologically active
enantiomer
 Metabolized by CYP2C9

35
RIFAMPICIN

 MOA: inhibits DNA-dependent RNA


polymerase in bacterial cells by binding
its beta-subunit, thus preventing
transcription of messenger RNA (mRNA)
and subsequent translation to proteins

36
RIFAMPICIN
 potent and nonspecific inducer of the hepatic
cytochrome P450 (CYP2C9) oxidative enzyme system,
a pathway responsible for the metabolism of warfarin
 acceleration in drug clearance associated with enzyme
induction by rifampicin can compromise the efficacy
and safety of warfarin
 PK: well absorbed after oral administration; excreted
mainly through the liver into bile
 PD: bactericidal and bacteriostatic

37
KETOCONAZOLE

 highly lipophilic compound


 an inhibitor of CYP2C9, the enzyme responsible for the
metabolism of S-warfarin
 MOA: works principally by inhibition of CYP450 by binding tightly
to the P450 heme iron and effectively reducing the metabolism of
warfarin
 PK: Oral Absorption: Rapid (75%);
 Metabolism: Partially hepatic via CYP450 to inactive compounds
 Excretion: Feces (57%); urine (13%)
 PD: an antifungal imidazole derivative
 A potent nonselective inhibitor of adrenal & gonadal steroid synthesis

38
DISCREPANCIES
 No significant difference bet the PT of Group I (NSS)
and Group II (warfarin)
 No significant difference bet the PT of Group III
(warfarin + rifampicin) and IV (warfarin + ketoconazole)
 Group III’s PT (warfarin + rifampicin) is higher than
group II (warfarin)
 Rifampicin acts by increasing the CYP2C9 through enzyme
induction, but it takes 1 to 2 weeks to produce new enzymes
that will increase the elimination of warfarin

39
CONCLUSION

40
CONCLUSION
 influence of metabolism on drug (Warfarin) action was determined
by measuring Prothrombin Time (PT) of whole blood drawn from
each group of albino rats
 PT measures extrinsic and common coagulation pathway ;
determines clotting tendency of the blood, in the measure of
Warfarin dosage, liver damage and vitamin K status
 PT is tested for drug interactions that can either prolong or
shorten Warfarin duration on the body
 Warfarin, co-administered with Ketoconazole, prolongs the PT

41
CONCLUSION

 Ketoconazole works principally by inhibiting


Cytochrome P450 → responsible for drug
metabolism without which may result to
prolonged action of Warfarin (an anticoagulant)
 Warfarin co-administered with Rifampicin will
cause a shorter PT because Rifampicin is a
potent inducer of Cytochrome P450

42
CONCLUSION

 Statistically, there were significant differences among


the 4 groups to which NSS, warfarin, warfarin +
rifampicin, and warfarin + ketoconazole were
administered
 Groups 2, 3,& 4 results showed significant differences
in the PT though Group 3 failed to produce the
expected results→ ↓ PT
 Group 4 results showed the greatest ↑ in PT, followed
by Group 3 and lastly, Group 2.

43
44
REFERENCES
 Berg, J.M., Tymoczko, J.L. & Stryer, L. Biochemistry 5th Edition
C2002: W. H. Freeman and Co., New York pp. 734-735
 Katzung, Bertram G. Basic & Clinical Pharmacology, 9th Ed.
C2004: The McGraw-Hill Companies, Inc., USA. pp. 415 – 416.
 http://en.wikipedia.org/wiki/Rifampicin
 http://www.inchem.org/documents/pims/pharm/rifam.htm#SubSect
ionTitle:7.1.2%20Pharmacodynamics
 http://en.wikipedia.org/wiki/Warfarin
 http://www.aafp.org/afp/990201ap/635.html
 http://en.wikipedia.org/wiki/Saline_%28medicine%29

45
RECOMMENDATION

 to repeat the experiment in a more controlled


setting to prevent discrepancies and errors in
the results.
 only one group should perform the experiment
to obtain more accurate results

46
DISCUSSION
 Warfarin
 anticoagulant medication administered orally or by
injection
 used for prophylaxis of thrombosis and embolism in many
disorders
 Chemistry and Pharmacokinetics
 small, lipid-soluble
 readily absorbed after oral administration
 cross the placenta; potentially dangerous to the fetus
 generally administered as the sodium salt

47
DISCUSSION
 has 100% bioavailability
 >99% of racemic warfarin bound to plasma albumin; small
volume of distribution; long half-life in plasma; lack of urinary
excretion of unchanged drug
 elimination depends on metabolism by cytochrome P450
enzymes
 consists of a racemic mixture of two active optical isomers - R
and S forms - each cleared by different pathways
 Levorotatory S-warfarin → 4X more potent than Dextrorotatory
R-isomer with respect to vitamin K antagonism

48
DISCUSSION
 Pharmacodynamics
 anticoagulant activity depends on the clearance of functional
clotting factors from the systemic circulation after
administration of the dose
 clearance of clotting factors determined by their half-lives
 earliest changes in the International Normalized Ratio (INR)
24-36 hours after dose administration
 changes due to the clearance of functional factor VII → vitamin
K­-dependent clotting factor with shortest t1/2 (6 hours)

49
DISCUSSION
 antithrombotic effect not present until approximately 5th day of
therapy
 effect depends on clearance of functional factor II
(prothrombin) → t1/2 (approximately 50 hours)
 loading doses (i.e., 10 mg or more per day) may ↑ patient's
risk of bleeding episodes early in therapy by eliminating or
severely reducing production of functional factor VII
 administration of loading doses → possible source of
prolonged hospitalization secondary to dramatic rises in INR

50
DISCUSSION
 potentiate a hypercoagulable state due to severe depletion of
protein C
 loading doses has no effect on inhibition of thrombosis
 Mechanism of Action
 blocks γ-carboxylation of several glutamate residues in
prothrombin and factors VII, IX and X, proteins C and S by
inhibiting epoxide reductase (specifically the VKORC1 subunit)
 blockade results to non-carboxylation of the coagulation
factors at certain glutamic acid residues, incapable of binding
to the endothelial surface of blood vessels, thus, biologically
inactive

51
DISCUSSION
 protein carboxylation is coupled to oxidation of Vitamin K →
must be reduced to be reactivated
 prevents reductive metabolism of inactive vitamin K epoxide to
its active hydroquinone form
 8- to 12-hour delay in action
 anticoagulant effect results from balance between partially
inhibited synthesis and unaltered degradation of the four
vitamin K-dependent clotting factors
 resulting inhibition of coagulation dependent on degradation
half-lives in the circulation

52
DISCUSSION

 Clinical Use
 people with an increased tendency for thrombosis
 prophylaxis in individuals who have already formed a blood
clot (thrombus) which required treatment
 help reduce the risk of embolism
 atrial fibrillation, artificial heart valves, deep venous thrombosis
, pulmonary embolism, antiphospholipid syndrome and
occasionally after myocardial infarction.

53
DISCUSSION

 Rifampicin
 bactericidal antibiotic drug of rifamycin group
 semisynthetic compound from Amycolatopsis rifamycinica
(formerly called Amycolatopsis mediterranei and Streptomyces
mediterranei)
 Pharmacokinetics
 well absorbed after oral administration; excreted mainly
through the liver into bile
 undergoes enterohepatic recirculation, bulk excreted as a
deacylated metabolite in feces and a small amount in the urine

54
DISCUSSION
 distributed widely in body fluids and tissues
 highly protein-bound
 Pharmacodynamics
 high activity against mycobacterial organisms, including
Mycobacterium tuberculosis and M. leprae
 active against Staphylococcus aureus, coagulase-negative
staphylocci, Listeria monocytogenes, Neisseria meningitidis,
Haemophilus influenzae, Legionella spp., Brucella, some
strains of E. coli, Proteus mirabilis, anaerobic cocci,
Clostridium spp., and Bacteroides

55
DISCUSSION
 bacteriostatic or bactericidal depending on concentration of drug at
site of infection

 Mechanism of Action
 inhibits DNA-dependent RNA polymerase in bacterial cells by binding
its beta-subunit, preventing transcription of messenger RNA (mRNA)
and subsequent translation to proteins
 lipophilic nature makes it a good candidate to treat TB meningitis,
which requires distribution to the central nervous system and
penetration through the blood-brain barrier.
 Rifampicin (ligand) binds to Pregnane X Receptor (PXR) in the
cytoplasm→ activates transcription of CYP3A4 and other genes→
binds to CYP3A promoters together with the 9-cis retinoic acid
receptor (RXR) as heterodimer to ER6 (everted repeat with 6 bp
spacer) elements→ CYP3A4 metabolism induced

56
DISCUSSION
 Clinical Indications
 treatment of treat Mycobacterium infections, including
tuberculosis and leprosy
 treatment of methicillin-resistant Staphylococcus aureus
(MRSA) in combination with fusidic acid
 prophylactic therapy against Neisseria meningitidis
(meningococcal) infection
 treats infection by Listeria species, Neisseria gonorrhoeae,
Haemophilus influenzae and Legionella pneumophila
 always used in combination with dapsone and clofazimine

57
DISCUSSION
 Ketoconazole
 very lipophilic → leads to accumulation in fatty tissues
 best absorbed at highly acidic levels
 Clinical Indications
 infections such as athlete's foot, ringworm, candidiasis (yeast
infection or thrush), and jock itch
 over-the-counter shampoo version → used as body wash for
treatment of tinea versicolor
 side-effects sometimes used to treat non-fungal problems

58
DISCUSSION
 decrease in testosterone caused by the drug → for treating
prostate cancer and for preventing post-operative erections
following penile surgery
 suppression of glucocorticoid synthesis in the treatment of
Cushing's disease

 Mechanism of Action
 inhibits cytochrome P450 14a-demethylase (P45014DM)

59
DISCUSSION
 affinity of ketoconazole for fungal cell membranes less than
that of fluconazole, thus, more potential to effect mammalian
cell membranes and induce toxicity

 structurally similar to imidazole ; interferes with the fungal


synthesis of ergosterol (constituent of cell membranes) and
certain enzymes

 specific for fungi, as mammalian cell membranes contain no


ergosterol

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NORMAL SALINE SOLUTION
 solution of 0.9% w/v of NaCl
 contains 154 mEq/L of Na+ and Cl−
 slightly higher degree of osmolality compared to
blood of about 300 mOsm/L
 used frequently in intravenous drips (IVs) for
patients who cannot take fluids orally and have
developed severe dehydration
 used when dehydration is severe enough to
threaten the adequacy of blood circulation
 safest fluid to give quickly in large volumes
61
De scriptiv e s

PT
95% Confidence Interval for
Mean
N Mean Std. Deviation Std. Error Lower Bound Upper Bound Minimum Maximum
1 8 4.8625 2.70446 .95617 2.6015 7.1235 2.20 9.00
2 7 33.8571 6.20292 2.34448 28.1204 39.5939 28.00 44.00
3 8 270.5000 100.10566 35.39269 186.8096 354.1904 127.00 455.00
4 8 351.2500 343.09130 121.30109 64.4185 638.0815 119.00 900.00
Total 31 169.3516 229.92175 41.29517 85.0156 253.6876 2.20 900.00

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INR = (Patient’s PT/Mean Normal PT)ISI

INR

D I
RUG (NSS) II (Warfarin) III (Warfarin w/ Rifampicin IV (Warfarin w/ ketoconazole)
1 0.26 8.21 72.90 2729.39
2 0.09 5.37 312.67 353.20
3 0.09 4.45 436.16 96.76
4 0.04 4.45 245.85 2729.39
5 0.05 5.05 373.18 105.40
6 0.51 7.45 772.75 87.31
7 0.09 10.26 233.01 99.19
8 0.54   137.56 64.64
M
EAN 0.21 6.46 323.01 783.16
63