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2
Factors Affecting 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
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
9
HYPOTHESIS
10
MATERIALS
12
METHODOLOGY
Warfarin+ Warfarin +
0.9% NSS Warfarin
rifampicin ketoconazole
The Prothrombin Time is measured from time of mixing until the appearance of fibrin
strands.
14
PROCEDURE AND RATIONALE
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
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
_0.2mg = ___x___
200 g wt. of rat
18
Warfarin dosage
20
ACTUAL RESULTS
6 39 127 900
400
351.25
350
300
270.5
250
PT
(sec) 200
150
100
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
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
28
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
Ritonavir (Norvir)
30
INDUCTION OF CYP2C9
31
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
33
WARFARIN
34
WARFARIN
35
RIFAMPICIN
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
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
42
CONCLUSION
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
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
60
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
62
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