1. Exploring the molecular basis of interactions of candesartan cilexitil (TCV-116) in lipid bilayers using 2H solid state NMR spectroscopy
Dimitrios Ntountaniotis 1, Pinelopi Kolokotroni 1, Johanna Baldus 2, Clemens Glaubitz 2, Thomas Mavromoustakos 9
1 University of Athens, Department of Chemistry, Laboratory of Organic Chemistry, Zographou 15771, Athens, Greece
2 Institute of Biophysical Chemistry Goethe Univesrity Frankfurt, Max von Laue Str.9, Frankfurt, Germany
Introduction: Angiotensin II (AngII) receptor blockers (ARBs) are amphiphilic molecules that exert their biological activity by preventing the vasoconstrictive hormone AngII to act on the AT1 receptor. The molecular basis of their antihypertensive action has been interpreted by a two-step model. In the first step they are incorporated into the bilayers through the lipid–water interface and secondly laterally diffuse to reach the active site of the AT1 receptor in order to exert their biological activity. The prodrug 1-[[(cyclohexyloxy)carbonyl]oxy]ethyl, 2-ethoxy-1- [[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl]methyl]-1H-benzimidazole-7-carboxylate denoted as candesartan cilexetil (TCV-116) is esterified at the carboxyl group by the cyclohexylocarbonyloxyethyl segment of the active compound candesartan (CV-11974), that belongs to sartans antihypertensive drugs. Olmesartan is another ARB drug and it is proposed that its pharmacological profile is distinct from the others. Generally, not only pharmacological similarities but also differences are observed among different ARBs. In this study, solid state 2H NMR experiments were performed for comparing the thermal and dynamic effects of the two molecules in lipid bilayers using 600 MHz Bruker NMR instrument..
The cellular membranes are complex entities consisting of various kinds of proteins and lipids as well as cholesterol. Phosphatidylcholines (PCs) are the most abundant lipid species in the plasma membranes of the vascular smooth muscle cells and sarcolemma cardiac membranes. The most frequently found among them are PCs with oleic and linoleic chains, and further dipalmitoylphosphatidylcholine (DPPC). Hydrated DPPC lipids are used, because they spontaneously form multilamellar bilayers whose mesomorphic changes occur in a convenient temperature range between 25 and 50 °C. Their dynamic and thermotropic properties have been extensively explored and their ARB partition coefficient with respect to its aqueous environment, especially in the fluid state, resembles that of natural plasma membranes of the vasculature. Phosphatidylcholine bilayers at low temperatures occur in the lamellar gel phase (Lβ′) and at higher temperatures in the liquid-crystalline phase (Lα).
Solid state 2H-NMR experiments were carried out in fully hydrated multilamellar bilayers by observing spectra of deuterated labels on the phospholipid at three different sites: (a) at the methylene groups alpha to the carbonyls of both acyl chains (1,2[2’,2’-2H]-DPPC), (b) at the 4’ methylene group of the sn-2 (2[4’,4’-2H]-DPPC), and (c) sn-2 (2[7’,7’-2H]-DPPC). Because of the interaction between the electric quadrupole moment and the electric field gradient at the nuclear site, the deuterium nucleus gives rise to a doublet in the spectrum of an oriented sample. The anisotropic nature of the interaction gives two spectral lines with a frequency difference of ν1-ν2=(3/4)ΑQ (3cos2 θ-1), which depends on the angle θ between the magnetic field and the axis of molecular ordering. In an aqueous phospholipid dispersion sample in the liquid crystalline phase, all values of θ are possible and the spectrum from 2H nuclei at one site in the molecule is a “powder pattern” in which the two principal peaks correspond to θ=90ο (perpendicular edges) and the two shoulders to θ=0ο (parallel edges). The separation between the two 90o-edges is referred to as the quadrupolar splitting (ΔνQ). At temperatures below the main phase transition, the axial diffusion rate is intermediate or slow, and one observes a broad, conical or rounded spectrum which does not show any sharp features. By studying the spectral shape as a function of temperature and the changes in ΔνQ due to the presence of drug, we can obtain information on the dynamic properties of the lipid bilayer in the particular region where the 2H-label is placed.
Candesartan cilexitil and olmesartan.
Materials: Dipalmitoyl-phosphatidylcholine (DPPC) was purchased from Avanti Polar Lipids (Birmingham, AL). Candesartan cilexitil (TCV-116) was kindly provided by Medochemie Hellas A.E. (Pharma Cypria). Olmesartan was kindly provided by Daichii Sankyo Prp Pharma (Japan). Deuterated phospholipid samples are kindly provided by Prof. A. Makriyannis .
Results and discussion. 2H solid state NMR: Below are shown (left) solid state 2H-NMR spectra of (1,2[2’,2’-2H]-DPPC) bilayers with and without incorporated TCV-116 or olmesartan at a concentration of x= At the temperature range of oC the spectra from the drug-free and drug containing (1,2[2’,2’-2H]-DPPC) are featureless and almost identical, each having a rounded conical shape. As the temperature is raised to 35 oC, the spectra from DPPC and DPPC+TCV-116 or DPPPC+olmesartan give different spectra. The DPPC bilayers have gel and liquid components and no quadrupolar splittings are eminent. The presence of TCV-116 at this temperature liquidifies the lipid bilayers and two broad splittings are eminent. This is also eminent that at this temperature the lipid bilayers containing TCV-116 are mostly in the liquid crystalline state. As the temperature rises to 40 oC and 45 oC both spectra show very sharp liquid crystalline lineshapes, each consisting of three overlapping Pake Pattern. In the liquid crystalline phase (45 oC), the spectrum of DPPC bilayers show three quadrupolar splittings, ΔνQ=11.9, 17.2 and 26.0 kHz. According to an earlier assignment, the largest splittings are attributed to the two deuterons at the 2’-position of the sn-1 chain and the other two splittings to the corresponding deuterons of the sn-2 chain. The difference in ΔνQ values is due to the fact that the sn-1 chain is almost perpendicular to the bilayer surface with its two α-methylene deuterons at equivalent angles with respect to the axis of chain rotation. On the other hand, the sn-2 chain has initial bend which places the carbonyl-a-methylene bond nearly parallel to the bilayer surface with each of the two deuterons having different average angle with respect to the chain axis. Assignment of the individual methylene deuterons was based on an earlier study using specific deuterons was based on an earlier study using specific deuteration which revealed that the splitting of 18.7 kHz was due to 2[2’R-2H] and that the splitting of 12.1 kHz was due to 2[2’S-2H]. These findings were supported by our calculations of the angles between the C-2H bonds and the lipid chain direction is based on X-ray crystallographic data. Indeed, the crystal structure showed that in 2’-methylene segment of sn-1 chain, both C-2H bonds are almost perpendicular to the lipid chains, whereas in the sn-2 chains, the two C-2H bonds have angles 76o and 45o with the lipid chain direction. Consequently, these three different orientations of the C-2H bonds in the lipid bilayers are expected to be responsible for the different ΔνQ values. The presence of TCV-116 causes increase in ΔνQ values indicating strong interactions at this topographic region. The effects of olmesartan were not as pronounced as with TCV-116. This indicates that although the two molecules as it was found using various other biophysical techniques were localized in the interface and upper segments of the lipid bilayers, they exert distinct effects. Similar discussion can be applied for 2[4’,4’-2H]-DPPC) (middle), and sn-2 (2[7’,7’-2H]-DPPC (right) bilayers with and withou TCV-116. In all preparations consistently TCV-116 caused higher increase of ΔνQ indicating that is causing stronger interactions not only at the upper segment of the phospholipid bilayers but also up to the middle of the lipophilic core of the phospholipid bilayers. The most striking differences are observed with 35 oC where the phospholipid bilayers are characterized by the gel and liquid components. Both TCV-116 and olmesartan at this temperature enhance the liquid component with TCV-116 to have the greater effect.
Τ (°C)
2΄-S (kHz)
Α Β C
2΄-R (kHz)
Α Β C
-CD2 (kHz)
A B C
2[4΄,4΄-2Η]
Α Β C
2[7΄,7΄-2Η] 35
13, ,8 12,7 ,0
27, ,5 29,4
32,9 30,0 28,7
27, ,4 28,3 40
11, ,6 12,5
, ,9
27, ,2 27,0
25,8 27,1 25,5
27, ,3 26,8 45
11, ,7 12,4
, ,1
26, ,3 25,6
25, ,0 25,0
Quadrupolar splitting comparison of of the samples A=DPPC B=DPPC/TCV-116 and C=DPPC/OLMESARTAN at the temperature range of οC at the deuterated positions 2΄,4΄,7΄. ΔvQ values are expressed in KHz.
Conclusions:
a. AT1 antagonists modify the spectrum profile of DPPC bilayers deuterated at different positions. b. AT1 antagonists have their specific spectrum profile at the temperature range that covers gel and liquid crystalline phases of lipid bilayers. c. TCV-116 shows stronger interactions with DPPC bilayers than olmesartan at the interface and lipid bilayer core although from previous studies are shown to localise themselves at the same topographical region. These similarities and differences observed with the drug olmesartan and prodrug TCV-116 signify the role of lipid bilayers in drug action as they determine their interactions. If drugs are localized in the lipid bilayers before they reach their active site then of course drug:membrane interactions control their way to reach the active site
Acknowledgement: T. Mavromoustakos and D. Ntountaniotis acknowledge EAST NMR program for covering the travel and residence expenses to perform NMR experiments in the Frankfurt solid state NMR center. T. Mavromoustakos acknowledges Heracleitus II. Investing in knowledge society through the European Social Fund.