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 limits bioavailability of oral drugs

 Greatly

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 there is significant effect of different drugs on the metabolism of warfarin among albino rats

Alternative Hypotheses (Ha)

10

MATERIALS
        

11

Animal weighing scale Tuberculin syringe Animal cage Asbestos gloves Surgical gloves Surgical blades Gauze Mortar and pestle 50mL beakers

8 citrated tubes 8 plain test tubes PT reagents centrifuge machine automatic pipettor pipettor tips 1 test tube rack rat tail cage stopwatch

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+ rifampicin Warfarin + ketoconazole

0.9% NSS

Warfarin

Drugs were given orally, for 4 days Testing for prothrombin time on fifth day

13

METHODOLOGY

Prothrombin Determination
Centrifuge blood for 3 minutes Place 0.01mL of plasma in test tube Measure PT by adding 0.02mL prewarmed thromboplastic reagent (simplastin)

Collect blood from tail in the citrated test tube

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

14

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. Rationale: This is essential for calculating the dose of the drug that will be given to each rat.

2. The rats were then weighed.

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

18

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

Warfarin dosage
RATS A B C D E F G H Weight (g) 166.88 144.5 159.3 162.9 145.5 170.6 165.0 144.0 Dose (mg) 0.17 0.14 0.16 0.16 0.15 0.17 0.17 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 NSS 6 3.4 3.5 2.2 2.6 8.7 7.5 9.0 GROUP 2 Warfarin 39 31 28 28 30 37 44   GROUP 3 Warfarin/ Rifampicin 127 279 334 245 307 455 238 179 GROUP 4 Warfarin/ Ketoconazole 900 298 148 900 155 140 150 119

21

Mean 4.8625

Mean = 33.86

Mean = 270.5

Mean = 351.25

ACTUAL RESULTS
Figure 1: Mean Prothrombin times (sec) of rats in four different group treatments
 
400  350  300 
PT (sec)  200 

Effect of Drug Metabolism in PT 

351.25 270.5

250 

150  100  50  0 

4.8625
I (NSS) 

33.86
II (Warfarin)  III (Warfarin w/  Rifampicin  Drug  IV (Warfarin w/  ketoconazole) 

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 691508.8 894411.6 1585920 df 3 27 30 Mean Square 230502.921 33126.354 F 6.958 Sig.

Between Groups Within Groups Total

.001

25

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).

POST HOC TESTS
Multiple Comparisons Dependent Variable: PT Tukey HSD Mean Difference (I-J) -28.99464 -265.63750 * -346.38750 * 28.99464 -236.64286 -317.39286 * 265.63750 * 236.64286 -80.75000 346.38750 * 317.39286 * 80.75000

(I) drug 1

2

3

4

(J) drug 2 3 4 1 3 4 1 2 4 1 2 3

Std. Error 94.19730 91.00323 91.00323 94.19730 94.19730 94.19730 91.00323 94.19730 91.00323 91.00323 94.19730 91.00323

Sig. .990

.033 .004
.990 .081

.012 .033
.081 .811

.004 .012
.811

95% Confidence Interval Lower Bound Upper Bound -286.7715 228.7823 -514.6736 -16.6014 -595.4236 -97.3514 -228.7823 286.7715 -494.4198 21.1340 -575.1698 -59.6160 16.6014 514.6736 -21.1340 494.4198 -329.7861 168.2861 97.3514 595.4236 59.6160 575.1698 -168.2861 329.7861

*. The mean difference is significant at the .05 level.

Post hoc analysis showed that the difference existed between group I 

26

and group III (sig= 0.033), group I and group IV (sig=0.004), group II and        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
Affected Drugs Phenytoin S-Warfarin NSAIDS Inducers Rifampin Inhibitors Azole antifungals: esp. Fluconazole, ketoconazole, itraconazole Metronidazole Ritonavir (Norvir)

30

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

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
 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%) A potent nonselective inhibitor of adrenal & gonadal steroid synthesis

PD: an antifungal imidazole derivative

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 small, lipid-soluble readily absorbed after oral administration cross the placenta; potentially dangerous to the fetus generally administered as the sodium salt

Chemistry and Pharmacokinetics
   

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 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

Mechanism of Action

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) 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

Pharmacokinetics

54

DISCUSSION
 

distributed widely in body fluids and tissues highly protein-bound 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

Pharmacodynamics

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 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

Clinical Indications

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

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 Lower Bound Upper Bound 2.6015 7.1235 28.1204 39.5939 186.8096 354.1904 64.4185 638.0815 85.0156 253.6876

N 1 2 3 4 Total 8 7 8 8 31

Mean 4.8625 33.8571 270.5000 351.2500 169.3516

Std. Deviation Std. Error 2.70446 .95617 6.20292 2.34448 100.10566 35.39269 343.09130 121.30109 229.92175 41.29517

Minimum 2.20 28.00 127.00 119.00 2.20

Maximum 9.00 44.00 455.00 900.00 900.00

62

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

INR D RUG 1 2 3 4 5 6 7 8 M EAN I (NSS) 0.26 0.09 0.09 0.04 0.05 0.51 0.09 0.54 0.21

II (Warfarin) 8.21 5.37 4.45 4.45 5.05 7.45 10.26   6.46

III (Warfarin w/ Rifampicin 72.90 312.67 436.16 245.85 373.18 772.75 233.01 137.56 323.01

IV (Warfarin w/ ketoconazole) 2729.39 353.20 96.76 2729.39 105.40 87.31 99.19 64.64 783.16

63

Sign up to vote on this title
UsefulNot useful