Inhibitory Effects of Angiotensin Receptor Blockers (ARBs) on CYP2C9 Activity in Human Liver Microsomes

Kamiyama, Yoshigae, Kasuya, Takei, Kurihara and Ikeda

Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan

Relissa Arevalo, Ezekiel Arteta & Joshua Brian Asturias

Department of Pharmacology and Medical Therapeutics, College of Medicine, Pamantasan ng Lungsod ng Maynila

Introduction

 Angiotensin II receptor blockers (ARBs) have been used to treat Hypertension Used in combination with calcium channel blockers or an ACE inhibitor (e.g. Normetec) Candesartan, Irbesartan, Losartan, Telmisartan, Olmesartan, Valsartan are among the ARBs available in the market and used clinically

 Most ARBs are known to be metabolized by CYP2C9 in the liver Losartan ---> active metabolite EXP-3174 Valsartan and Candesartan ---> inactive metabolite Telmisartan and Olmesartan are inert as substrate to CYP2C9 Telmisartan partially metabolized by glucoronidation Olmesartan not metabolized

 All ARBs possess 4-(2 -tetrazolyl) biphenylyl group or its bioisosteric 4(2 -carboxy) biphenylyl group in their molecules

 Crystal structures of CYP2C9-warfarin complex and CYP2C9flurbiprofen complex are the proposed models for ligand recognition of CYP2C9

CYP2C9-flurbiprofen complex
Located at the position in which the hydroxylation site is 4.9 from the heme iron Carboxyl group interacts with Arg108 and Asn204 of CYP2C9

In this study...
We will focus on relationship between structures of ARBs and their inhibitory effect on CYP2C9 activities The IC50 values of seven ARBs in the inhibition of CYP2C9 activity in the liver microsomes were determined using (S)-( )-warfarin as the substrate

 No marked inhibition of CYP2C9 activity was observed in the case of Olmesartan and Valsartan The IC50 of Olmesartan analogues were a;lso determined Employ docking model of ligand-enzyme complex in order to investigate the relationship between their structures and afinity to CYP2C9 enzyme

Materials

Chemical structure of Olmesartan analogues

Candesartan Irbesartan Losartan Losartan active metabolite Telimisartan Valsartan

1-octanol (S)-( )-Warfarin 7-hydroxy warfarin Choumaclorine -NADPH sodium salt Human liver microsome

Methodology

CYP2C9 inhibition Assay

Preincubation of mixture at 37C for 3 mins. 100 L aliqout was collected added to 200 L of methanol containing 1 M of coumachlorine Quadruplicate for ARBs Addition of 2mM NADPH (25 L) Incubation for 40 mins. Centrifuge at 3000 rpm for 1 min at 4C Assays were carried out

Duplicate for olmesartan analogues Supernatant fractions were applied to HPLC system consisting of an alliance 2795 separation module interfaced to Quattro LC

Separation using Inertsil ODS-3 column maintained at 40C

Separation using Inertsil ODS-3 column maintained at 40C Mobile phase of methanol/ 10mM ammonium acetate (70/30, v/v) pH 4.0 (condesartan as inhibitor) Mobile phase of methanol/ 10mM ammonium acetate (65/35, v/v) pH 4.0 (other ARB as inhibitor)

pH 5.15 (olmesartan analogues as inhibitor

Ionization modes for determination of 7hydroxywarfarin and choumachlorine were ESI-negative Capillary voltage 3.5 kV, Source block temperature 1209C Desolvation temperature 3509C

 Inhibitory activity was expressed as a percentage against the activity of the control incubations (no inhibitor). The IC50 values were calculated with the computer software WinNonlin Professional and Microsoft Office Excel 2000 and 2003 For determination of Ki value of losartan, varying concentrations of losartan and (S)-( )-warfarin were used The apparent Ki value of losartan was estimated from Dixon plots.

Measurement of LogD
5 mg of ARB was dissolved in either 20mL of 1- octanol saturated with the Britton-Robinson buffer or in 20 mL of the BrittonRobinson buffer saturated with 1-octanol, as a stock solution. 4 mL-aliquot of the stock solution was added to a flask containing 4 mL of the counter solution, 1-octanol or the Britton-Robinson buffer Flask containing the two phases was shaken using a seesaw shaker (FNX-11-20) for 5 min, and centrifuged (3000 rpm) for 10 min After separation of both solutions, each layer was collected, and diluted with an equal volume of acetonitrile

measurements were performed in triplicate under each pH condition

 The samples were analyzed on an Alliance 2695 separation module equipped with a photodiode array detector, 2996 Chromatographic separation was carried out using an XTerra MS C18 column
(100 mm in length, with an internal diameter of 4.6 mm and a particle size of 3.5 mm)

maintained at 40C with the mobile phase consisting of: solvent A (one volume of phosphoric acid in 1000 volumes of water) solvent B (one volume of phosphoric acid in 1000 volumes of acetonitrile) The LogD values for the ARBs were calculated as a partition between the Britton-Robinson buffer and 1-octanol Co=concentration in the organic phase Cw =concentration in the aqueous phase. LogD log (Co/Cw)

Calculation of ClogP
ClogP values were calculated wuth computer software CLOGP Docking model analysis AUTODOCK were carried out to obtain model structures of CYP2C9-EXP-3174 complex Automated dockings were performed using the CYP2C9 structure from furbiprofen-complex

Results and Discussion

Inert as Substrates

Metabolized Substrates
active metabolite of losartan

Inhibitory effect of ARBs on 7-hydroxylation of S-( microsomes.

)-warfarin in human liver

The final concentration of S-( )-warfarin was 5 mM. The reaction mixture was incubated at 379C for 40 min. Each symbol represents the mean of quadruplicate determinations.

Dixon Plot of Losartan

Ki of losartan:

5.75 M
Free concentration from the binding ratio of losartan in microsomal protein (computed):

4.6 M
(higher than the Ki of commercial losartan)

LogD Values of ARBs Under varying pH

pH 7.4

CLogP and IC50 in CYP2C9 Inhibition

CLogP
Lipophilicity under an undissociated condition
Hydroxyisopropyl group
2.8

Affinity to P450 as a subtrate correlates with lipophilicity

CLogP and IC50

NOT STATISTICALLY SIGNIFICANT!!!

7.5

Inhibitory Effects of olmesartan Analogues on 7-hydroxylation of S-(-)-warfarin

Table 1. CLogP values of ARBs.

ARB Olmesartan Losartan EXP-3174 Valsartan Candesartan Irbesartan Telmisartan CLogP Value 2.8 4.1 4.6 4.9 5.4 6.0 7.5

cLogP =

hydrophilicity

inhibitory effect

Hydroxyisopropyl group

Fig. 2. Chemical structure of olmesartan.

Chemical structure of Olmesartan and its Analogues R1 C(CH3)2OH C(CH3)2OH CH(CH3)2 CH2CH3 CH3 C=(CH2)CH3 Cl

RNH-6270 (olmesartan) RNH-6272 RNH-6384 RNH-6390 RNH-6391 RNH-6458 RNH-8239

R1 RNH-6270 (olmesartan) RNH-6272 RNH-6384 RNH-6390 RNH-6391 RNH-6458 RNH-8239 IC50 = C(CH3)2OH C(CH3)2OH CH(CH3)2 CH2CH3 CH3 C=(CH2)CH3 Cl

IC50 (M) >300 >300 45.1 77.3 117 161 21.7

inhibitor potency

It has been known that

the chloro group has the effect of increasing affinity for CYP2C9; and, the hydroxyisopropyl group in the olmesartan molecule is a key functional group in decreasing the affinity to CYP2C9.

Docking models of EXP-3174 to CYP2C9

EXP-3174

Olmesartan

The chloro group formed a hydrophobic interaction with the Ala477 and Ile205 of the enzyme.

EXP-3174

The hydroxyisopropyl group formed a weak interaction with the hydrophobic pocket of the enzyme.

Olmesartan

 The affinity of ARBs to CYP2C9 is determined also by the lipophilicity of the group present at the same position as that of the hydroxyisopropyl group in olmesartan. Valsartan also had NO inhibitory effect on the CYP2C9 activity. But why?

Lack of an imidazole ring Free rotation of groups Low affinity of valsartan to CYP2C9 Lack of inhibition

Valsartan

 ARBs show the most potent inhibitory effect on CYP2C9 among P450s Losartan and Irbesartan also inhibit CYP3A4 in the intestine and liver Olmesartan and Valsartan have very low affinity to CYP2C9 and other P450s in vitro The low affinity of olmesartan to CYP2C9 is due to the presence of an additional polar group, hydroxyisopropyl.

END