Host-Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2-Hydroxypropyl-β-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism

Article abstract of DOI:10.1021/acs.molpharmaceut.8b01212

Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-β-cyclodextrin (2-HP-β-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state ¹³C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The ¹³C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN's complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-β-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-β-CD, and thus, the molecule's availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity.

Full text of DOI:10.1021/acs.molpharmaceut.8b01212

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Article
Host-guest interactions between candesartan and its prodrug
candesartan cilexetil in complex with 2-hydroxypropyl-#-
cyclodextrin: on the biological potency for Angiotensin II antagonism
Dimitrios Ntountaniotis, Ioannis Andreadelis, Tahsin F Kellici, Vlasios Karageorgos, Georgios
Leonis, Eirini Christodoulou, Sofia Kiriakidi, Johanna Becker-Baldus, Evgenios . K. Stylos, Maria
V. Chatziathanasiadou, Christos M. Chatzigiannis, Dimitrios E. Damalas, Busecan Aksoydan, Uroš
Javornik, Georgia Valsami, Clemens Glaubitz, Serdar Durdagi, Nikolaos S. Thomaidis, Antonios
Kolocouris, Janez Plavec, Andreas G. Tzakos, George Liapakis, and Thomas Mavromoustakos
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01212 • Publication Date (Web): 25 Jan 2019
Downloaded from http://pubs.acs.org on January 26, 2019
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Tzakos, Andreas; Panepistimio Ioanninon, Department of Chemistry,
Section of Organic Chemistry and Biochemistry
Liapakis, George ; University of Crete, Department of Basic Sciences,
School of Medicine
Mavromoustakos, Thomas; National and Kapodistrian University of
Athens, Department of Chemistry
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Host-guest interactions between candesartan and
its prodrug candesartan cilexetil in complex with
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-hydroxypropyl-β-cyclodextrin: On the biological
potency for Angiotensin II antagonism
1
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Dimitrios Ntountaniotis , Ioannis Andreadelis , Tahsin F. Kellici , Vlasios Karageorgos ,
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Georgios Leonis , Eirini Christodoulou , Sofia Kiriakidi , Johanna Becker-Baldus , Evgenios
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K. Stylos , Maria V. Chatziathanasiadou , Christos M. Chatzigiannis , Dimitrios E.
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Damalas , Busecan Aksoydan , Uroš Javornik , Georgia Valsami , Clemens Glaubitz , Serdar
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Durdagi , Nikolaos S. Thomaidis , Antonios Kolocouris , Janez Plavec , Andreas G.
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Tzakos , George Liapakis , Thomas Mavromoustakos


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Dimitrios Ntountaniotis: email address. doudaniotis@yahoo.gr, tel. +30 210 7274475
Andreas Tzakos: email address. agtzakos@gmail.com
George Liapakis: email address. liapakig@uoc.gr

Thomas Mavromoustakos: email address. tmavromom@chem.uoa.gr, tel. +30 210 7274475, fax.
30 210 7274761
+
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1Department of Chemistry, Laboratory of Organic Chemistry, National and Kapodistrian
University of Athens, Panepistimioupolis, Zografou 15771, Greece
2Department of Basic Sciences, School of Medicine, University of Crete, Heraklion, Crete,
Greece
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3Department of Pharmacy, Laboratory of Pharmaceutical Technology, National and
Kapodistrian University of Athens, Panepistimioupolis, Zografou 15771, Greece
4Institute of Biophysical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9,
6
0438 Frankfurt, Germany
5Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of
Ioannina, Ioannina 45110, Greece
6Department of Biological Applications and Technology, Biotechnology Laboratory,
University of Ioannina, Ioannina, 45110, Greece
7Department of Chemistry, Laboratory of Analytical Chemistry Chemistry, National and
Kapodistrian University of Athens, Panepistimioupolis, Zografou 15771, Greece
8Department of Biophysics, Computational Biology and Molecular Simulations Laboratory,
Bahcesehir University, Istanbul, Turkey
9National Institute of Chemistry, Slovenian NMR Centre, SI-1001 Ljubljana, Slovenia
10Department of Pharmacy, Section of Pharmaceutical Chemistry, National and Kapodistrian
University of Athens, Athens 15771, Greece
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Abstract
Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the
treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as
antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1
receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of
candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with
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-hydroxylpropyl-β-cyclodextrin (2-HP-β-CD) were characterized using high-resolution
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electrospray ionization mass spectrometry and solid state C cross polarization/magic angle
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spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The C CP/MAS results
showed broad peaks especially in the aromatic region, thus confirming the strong interactions
between cyclodextrin and drugs. This experimental evidence was in accordance with
Molecular Dynamics simulations and quantum mechanical calculations. The synthesized and
characterized complexes were evaluated biologically in vitro. It was shown that as a result of
CAN’s complexation, CAN exerts higher antagonistic activity than CC. Therefore, a
formulation of CC with 2-HP-β-CD is not indicated, while the formulation with CAN is
promising and needs further investigation. This intriguing result is justified by the binding
free energy calculations, which predicted efficient CC binding to 2-HP-β-CD and thus the
molecule’s availability for release and action on the target is diminished. In contrast, CAN
binding was not favored and this may allow easy release for the drug to exert its bioactivity.
Keywords: candesartan; candesartan cilexetil; 2-hydroxylpropyl-β-cyclodextrin; complex;
biological efficacy
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Introduction
Renin-angiotensin aldosterone system (RAAS) inhibitors are the cornerstones for the
treatment of cardiovascular and renal diseases. One of these classes of molecules is the
angiotensin receptor blockers (ARBs) or sartans. Sartans are well known to act as
antihypertensive drugs by blocking the detrimental action of the octapeptide hormone
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Angiotensin II to stimulate AT1 receptors 1, 2
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Sartans act to a membrane receptor and prefer to initially incorporate themselves in lipid
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bilayers before reaching the receptor site through lateral diffusion . By assuming a two-step
mechanism (incorporation-diffusion), possible transportation of sartans through the mouth of
the receptor and then direct binding to the active site is not excluded. The possibility of the
drug to act through both mechanisms can be also considered. We have previously studied the
effects of various sartans on lipid bilayers 3-6. These investigations combined various
complementary biophysical techniques [Differential Scanning Calorimetry (DSC), Raman
spectroscopy, Small-angle X-ray scattering (SAXS), Wide-angle X-ray scattering (WAXS),
Solid-state nuclear magnetic resonance (ssNMR), and Molecular Dynamics (MD)
simulations] to reveal the complexity of the interaction of AT1 antagonists although all of
them were accommodated in the polar-nonpolar interface of the lipid bilayers. Each sartan is
characterized by a unique “fingerprint” in terms of perturbation, orientation and localization in
lipid bilayers. If this fingerprint is essential for drug action, then future rational drug design
should not only take into consideration the binding interactions of the drugs in the receptor
area, but also the specific drug-bilayer interactions in order to forecast consequences in drug
efficiency.
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For example, candesartan (CAN, also known as CV-11974) is a well-known
antihypertensive drug, which acts as noncompetitive insurmountable antagonist with the
highest affinity to AT1 receptor compared to the other sartans, with losartan being the
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prototype (Scheme 1) . In previous studies, we have compared the interactions of CAN and
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losartan with lipid bilayers as well as the actions of olmesartan and other AT1 antagonists .
The obtained data suggested a possible relationship between the diffusion efficacy and the
pharmacological potency among various sartans. For instance, it was shown that losartan
tended to form domains in the lipid bilayers and this could presumably retard its diffusion
toward the active site of the AT1 receptor. The diffusion may be also retarded by losartan’s
stronger binding to the head group region as well as by induction of the interdigitation effect.
On the other hand, CAN exerted milder perturbation effects on lipid bilayers and did not
induce interdigitation effects to the lipid matrix. For the above reasons, its diffusion in the
lipid bilayers may be facilitated 2.
Candesartan cilexetil (CC, also known as TCV-116, Atacand) is a prodrug, which is rapidly
converted to the active drug, CAN, by ester hydrolysis during absorption from the
gastrointestinal (Scheme 1). The bioavailability of CC is approximately 15% after oral
administration. To increase the bioavailability of such a drug, increasing solubility through a
carrier before interacting with the membrane receptor system is of great importance. The
physicochemical properties of the drug can be improved through complexing the drug with
the water soluble 2-hydroxylpropyl-β-cyclodextrin (2-HP-β-CD) (Scheme 1).
In a search of drug bank and chemical databases we sought to study for highly lipophilic
drugs with low bioavailability. These drug molecules inevitably need a vehicle for increasing
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the bioavailability and solubility. Among the drugs that urgently need an optimized
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pharmacological profile are CAN and its prodrug, CC .
In the described research the complexing properties of CAN and its prodrug CC with 2-HP-
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β-CD (Scheme 1) were characterized using CP/MAS and ESI/MS and the 2-HP-β-CD-drug
complexes were biologically evaluated in vitro. The aim of the study is twofold: (a) To
compare the potency of CC and CAN in their complexes with 2-HP-β-CD. Thus, the results
would guide us whether prodrug CC formulation with 2-HP-β-CD is more favored than that of
the drug CAN; and (b) To provide a computational explanation of the obtained results.
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Scheme 1. Structures of CAN, CC as well as 2-HP-β-CD.
Experimental Section
Materials and Methods. Synthesis of Candesartan. Candesartan was obtained using
candesartan cilexetil as a starting material kindly provided by the CYPRIA pharmaceutical
company. Candesartan was synthesized through a very simple, optimized and efficient method of
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alkaline hydrolysis (methanolysis) described in our previous article is applied .
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Scheme 2. Hydrolysis of CC.
In particular, to a solution of the ester (CC) (190 mg, 0.3111mmol) in CH Cl /CH OH (9:1,
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v/v, 100 mL), a methanolic solution (2 mL) of NaOH 0.08 M was added. The physical
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mixture was stirred at 30 C for 8 h and a white precipitate was formed. The solvent was
removed under vacuum, the residue was diluted with water and the aqueous solution was
washed with ethyl acetate in order to isolate the water insoluble alcohol and to remove any
negligible amount of unreacted ester. The aqueous phase was then cooled, and acidified with
1N HCl until pH 3-4. The solid formed in the aqueous phase is the desired compound. The
aqueous phase containing a suspension of our desired product was extracted with CH Cl The
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organic layer was washed with brine and dried over (Na SO ) and the solvent was removed to
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afford the acid (CAN) as a pale yellow solid (106.9 mg, 78.01%). Spectroscopic data were in
agreement with our previous publication 5.
Preparation of physical mixture:
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:1 molar ratio of the two components CAN and 2-HP-β-CD or CC and 2-HP-β-CD were
brought together and mixed. The physical mixture was grinded to become homogeneous.
Preparation of the Lyophilized CAN−2-HP-β-CD and CC–2-HP-β-CD Products. A
freeze-drying procedure was applied for the preparation of CAN or CC-2-HP-β-CD
lyophilized product. For the preparation of CAN−2-HP-β-CD aqueous solutions for freeze-
drying, the neutralization method was used.
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Small amounts of ammonium hydroxide were then added under continuous stirring and pH
monitoring until complete dissolution and pH adjustment to a value of approximately 10.5.
The resulting solution was thereafter freeze-dried using a Kryodos-50 model Telstar
lyophilizer. CAN− or CC− 2-HP-β-CD aqueous solutions for freeze drying in a molar ratio of
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:1 were similarly prepared for comparative purposes using 0.300 (or 0.810 g CC) and 1.022
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g of 2-HP-β-CD, respectively .
High-resolution Electrospray Ionisation Mass Spectrometry Measurements. HR-ESI-
MS: A hybrid Quadrupole-Time of Flight (QTOF) mass spectrometer (Maxis Impact, Bruker
Daltonics, Bremen, Germany) was utilized for the analysis and the identification of CAN and
CC complexes with 2-HP-β-CD. The QTOF system was equipped with an electrospray
ionization interface (ESI), operating in negative ionization mode, with the following operation
parameters: capillary voltage 4000 V; end plate offset, -800 V; nebulizer pressure 0.8 bar;
−
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drying gas 4 L min and dry gas temperature 180 °C. The QTOF MS system operated in full
scan acquisition mode and recorded spectra over the m/z range 50−3000, with a scan rate of 1
Hz. External calibration of the mass spectrometer was performed with the manufacturer's
solution (sodium formate clusters), ensuring high mass accuracy.
-1
Stock solutions of 1 mg mL were prepared for CAN−2-HP-β-CD and CC−2-HP-β-CD
complexes, by dissolving the appropriate amounts in water. Preliminary experiments
regarding the effect of dissolution solvent, highlighted that dissolution of both complexes in
either methanol or a mixture of methanol and water 50:50 provided worse results compared to
-1
water. Working solutions of 0.01 mg mL were prepared and used for the infusion
experiments. Infusion of the complexes’ solutions was performed under a constant flow of
-1
180 μL min . Identification relied on the mass accuracy of the pseudomolecular ion of each
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complex, as well as on the conformity of fit between measured and theoretical isotopic
pattern. The potential formation of multi-charged species was taken into consideration as well,
due to the structure and the high molecular weight (M.W.) of the complexes.
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Solid-state C CP/MAS NMR spectroscopy. C cross polarization/magic-angle spinning
(13
C CP/MAS) solid state NMR spectra were recorded on Bruker 600 MHz spectrometer
equipped with a 4 mm HX MAS probe. Approximately 20 mg of each sample was packed
tightly into a zirconia rotor and spun at 15 kHz. The duration of the CP block in the CP/MAS
experiment was 5 ms, repetition delay between scans was 2 s, and number of scans was 1000.
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The C chemical shift axis was referenced to adamantane.
Fluorescence spectroscopic studies. Steady-state fluorescence spectroscopy was recruited
in order to determine the interaction of CAN and CC with 2-HP-β-CD. The measurements
were performed in a Perkin Elmer LS-55 spectrofluorometer. All emission spectra were
recorded using a Quartz (1 cm) cuvette and excitation and emission slits of 6 nm, at ambient
temperature. CAN and CC stock solutions were diluted in DMSO: distilled water (dH O)
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[50:50 v/v %] at a concentration of 100 μΜ and kept protected from light. The 2-HP-β-CD
stock solution was prepared in dH O at a concentration of 12 mM. The final concentration of
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each drug in the cuvette was 25 μΜ for each measurement. Various volumes from the 2-HP-β-
CD stock solution were added each time (0.15, 0.35, 0.5, 0.65, 0.85, 1, 2, 3, 4, 5, 6 mM) and
the samples were adjusted to 3 ml final volume with dH O The pH values of the samples were
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equal to 6.8 for CC and 6 for CAN and both interactions were, also recorded at pH 4.1
through the addition HCl (0.1 M). The samples were kept stirred and protected from light for
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0 min, before measurement. The excitation wavelength was 268 nm for CC. The maximum
emission wavelength was shifted from 405 nm to 372 nm, when the pH was adjusted to 4.1.
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The maximum excitation wavelength for CAN was 286 nm and shifted to 265 nm at pH 4.1,
while the emission wavelength presented a slight alteration from 373 to 380 nm, respectively.
Computational Methods. The crystal structure of CC was downloaded from the
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Cambridge Structural Database (CSD reference code: FETWEH) . The crystal structures of
β-CD (CSD reference code BUVSEQ02) 13 and CC were modified using Schrodinger 2015.2
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to 2-HP-β-CD and CAN, respectively. The OPLS3 force field 15 was used to dock the
structures of CAN and CC into 2-HP-β-CD with GlideXP 16. Each docking calculation
resulted in two possible orientations for CAN and CC inside the 2-HP-β-CD cavity. All four
resulting complexes were subjected to MD simulations.
MD simulations. The MD simulations were carried out with the GPU version of the
PMEMD module 17 from the AMBER 14 simulation package 18, 19. The geometry of CC and
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CAN was optimized with the HF/6-31G* basis set (Gaussian 09) . The general AMBER
force field (GAFF) was used to obtain force field parameters for CAN and CC with RESP
charges 21, 22. The force field GLYCAM_06j-1 23 was utilized to represent the behavior of the
cyclodextrin part of the host molecule, while GAFF was used for the 2-hydroxypropyl groups.
Overall, RESP charges were calculated for the modified cyclodextrin. Each drug-cyclodextrin
complex was next solvated using the TIP3P water model 24 in a truncated octahedron with
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724 water molecules. The minimum distance between the edge of the periodic box and each
atom of the systems was set at 18 Å. The minimization of the complexes was carried out for
5000 steps, using a nonbonded cutoff of 18 Å in constant volume. Solvated complexes were
next heated under constant volume for 100 ps. The temperature was gradually increased from
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were equilibrated under constant pressure in two steps of 100 ps each. In the first one,
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constraints of 10 kcal mol Å were applied to the complex, while in the second step all
restraints were removed. Four MD calculations (two for each drug in both preferred binding
orientations) of 400 ns were run at 300 K under constant pressure. The bonds involving
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hydrogen atoms were constrained at their equilibrium distance using SHAKE . The analysis
of the trajectories was performed using the ptraj module of AMBER 27. The MM-PBSA
analysis 28 was performed as described in our previous publications 29, 30. Additonal steered
MD simulations were carried out for each complex’s preferred orientation using the
Gromacs/2018.1 software 31. In particular, the Z-axis was selected to be the reaction
coordinate, ξ, where the complexes of CAN−HP-B-CD and CC−HP-B-CD respectively was
centered at (2.181, 2.4775, 3.280) in a box of (6.560x4.362x12.000) Å3. Such extended box
dimensions were used in order to avoid the interaction of drug with its periodic image. A
pulling simulation along z-axis was performed keeping the complexes of HP-B-CD restrained
and pulling CAN and CC respectively out of the cavity by applying a harmonic potential
(k=1000kJ/mol/nm2) for 5nm. In addition, a set of ~40 umbrella sampling simulations were
performed for the computation of the potential of mean force (PMF) of CAN and CC from
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complexes to free phase by employing the weighted histogram analysis method (WHAM) .
Umbrella sampling simulations. Each starting umbrella sampling configuration was
equilibrated for 1ns in the NPT ensemble before proceeding to sampling simulations of 10ns
in the same ensemble. The accuracy of our method was tested by comparing biasing
simulations of 10ns against ones of 100 ns where the difference was found to be only
~1kcal/mol. Thus, we concluded that it was not worth the computational effort to perform
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longer MD simulations and the results of 10ns were kept. The protocol used for calculating
the PMF is described in references33, 34
.
Quantum Mechanics (QM) Simulations. Geometry optimization of molecules obtained
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from the last frame of the MD simulations was performed with the Jaguar module 35 of
Maestro. The optimization was applied to a total of eight molecules, including candesartan
and candesartan cilexetil; both in their bare and cyclodextrin-embedded forms and in two
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different conformational approaches for each molecule. The default basis set was 6-31G** .
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DFT (density functional theory) was applied with a B3LYP functional . The accuracy level
of SCF (self consistent field) calculations for convergence was set to accurate. Poisson-
Boltzmann Solver was used for the solvation model 38 with water as the solvent. The
remaining settings were used as their defaults.
Plasma stability assays.
Liquid chromatography conditions. Reversed phase liquid chromatography assay was
performed using Advance Ultra High Performance Liquid Chromatography (UHPLC) system
(Bruker, Germany). Separation of CV-174, CC and irbesartan (IRB) used as a reference
sample was performed on a Kinetex C8 column 100 mm × 2.1 mm, 2.6 um, with proguard
column 2.1 mm (Phenomenex). Column temperature was maintained stable at 40°C. The
mobile phases were composed of (A) LC-MS grade water with formic acid 0.1% and (B)
acetonitrile with formic acid 0.1%. For gradient elution the following profile, at constant flow
rate of 250 μL/min, was used: initial phase (B) concentration 20%, increased to 100% within
2
.0 min, then kept constant for 3 min, reduced to 5% at 5.1 min and kept constant till the end
of the run at 6 min. The injection volume was set at 5 μL while the temperature in auto-
sampler was maintained at 15°C.
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Mass spectrometric conditions. For the ionization and detection of all compounds, EVOQ
Elite ER triple quadrupole mass spectrometer (Bruker) was operated in positive ionization
electrospray mode (ESI+) using multiple reaction monitoring (MRM). Utilizing MRM
builder, a feature of MSWS software of Bruker, the optimal MRM transitions selected for
monitoring the compounds were m/z 611.2 → 423.1 and 611.2 → 349.1 for CC,
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41.2→147.2 and 441.2→255.1 for CAN, 429.2→207.1 and 429.2→195.2 for IRB. Optimum
ESI conditions were determined as follows: spray voltage 4500 V; heated probe gas flow 50
units; heated probe temperature 200°C; cone gas flow 20 units; cone temperature 350°C;
nebulizer gas flow 50 units and exhaust gas on. Total control of LC and MS as also data
acquisition was performed with MSWS software, version 8.2.1.
Preparation of stock and working solutions. Stock solutions of 1 mg/mL for CC, CAN
and IRB were prepared by dissolving the appropriate amounts in DMSO. The working
solutions that were used for the calibration curve standards of CC and CAN were prepared by
further dilution of the stock solutions with DMSO to the appropriate concentrations (data not
shown). The IRB working solution was prepared by further dilution of stock solution with
DMSO to the appropriate concentration. Stock solution of CC complexed with 2-HP-β-CD
was prepared by dissolving the appropriate amount of the complex in LC-MS grade water.
The desired concentration of CC in human plasma, after spiking it with the complex, was
prepared by further dilution of stock solution with water. All stock and working solutions
were stored at -20°C before use.
Plasma stability assays. In order to evaluate the stability of CC and CC-2-HP-β-CD
complex in human plasma, 5 μL of each compound separately, were incubated with 90 μL
human plasma (pH adjusted to 7.4 with HCl) for 0, 24 and 48 hrs in water bath at 37°C. In
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order to terminate enzymes activity, 400 μL of cold-ice acetonitrile were added to each
sample along with 5 μL of IRB working solution, giving a final concentration of 25 ng/mL for
each compound. The samples were vortex-mixed and stored in the refrigerator at 4°C for 2
hrs. Then the samples were centrifuged at 10.000 rpm for 5 min and the supernatant was
taken, filtered and transferred to vials for UHPLC-MS/MS analysis. Each sample was studied
in triplicates and the plot of the concentration of CC and CAN that was calculated from the
calibration curves in each time point, against time was designed.
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Cell culture, harvesting cells and membrane preparation. Human embryonic kidney
(HEK 293) cells stably expressing the human AT1 receptor were grown in DMEM/F12 (1:1)
containing 3.15 g/L glucose and 10 % bovine calf serum at 37 ºC and 5 % CO . Cells at 100%
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confluence in 100-mm dishes, were washed with phosphate - buffered saline (PBS) (4.3 mM
Na HPO .7H 0, 1.4 mM KH PO , 137 mM NaCl, 2.7 mM KCl, pH 7.2 - 7.3 at room
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temperature), briefly treated with PBS containing 2 mM EDTA (PBS/EDTA), and then
dissociated in PBS/EDTA. Cell suspension was centrifuged at 1000xg for 5 min at room
temperature, and the pellet was homogenized in buffer O (50 mM Tris - HCl containing 0.5
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mM EDTA, 10% sucrose, 10 mM MgCl , pH 7.4 at 4 C) using a Janke & Kunkel IKA Ultra
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Turrax T25 homogenizer (at setting ~ 20, 10 - 15sec, 4 ºC). The homogenate was centrifuged
at 250xg for 5 min at room temperature. The pellet was discarded and the supernatant was
centrifuged (16000xg, 10 min, 4 ºC). The membrane pellet was re-suspended (0.5-0.6 mL/100
mm dish) in buffer B (50 mM Tris - HCl containing 1 mM EDTA,10 mM MgCl2, 0.2% BSA,
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.2 mg/ml bacitracin, and 0.93 μg/ml aprotinin, pH 7.4 at 4 ºC) and used for radioligand
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[125
I-Sar1-Ile8] AngII binding. Aliquots of membrane suspension (50 L) were added into
tubes, containing buffer B and 90,000 - 110,000 cpm [125I-Sar1-Ile8] AngII without or with
AngII antagonists (CAN or CC) or 2-HP-β-CD or 2-HP-β-CD in complex with CAN or CC at
the single concentration of 1000 nM or increasing concentrations of AngII antagonists
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heterologous competition binding) or increasing concentrations of [Sar1-Ile8] AngII
(homologous competition binding) in a final volume of 0.15 mL. The mixtures were incubated
1 h, 24 ºC) and then, filtered using a Brandel cell harvester through Whatman GF/C glass
(
fiber filters, presoaked for 1 h in 0.5 % polyethylenimine at 4 ºC. The filters were washed 10
times with 1-2 mL of ice - cold 50 mM Tris – HCl containing NaCl 120 mM, pH 7.4 at 4 ºC.
Filters were assessed for radioactivity in a gamma counter (LKB Wallac 1275 minigamma, 80
%
efficiency). The amount of membranes used was adjusted to ensure that specific binding
was always equal to or less than 10 % of the total concentration of the radioligand added.
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Specific [ I-Sar1-Ile8] AngII binding was defined as the total binding substracted the non-
specific binding in the presence of 1000 nM Valsartan where full saturation of receptor
occurs. Data for competition binding were analyzed by nonlinear regression analysis, using
Prism 4.0 (GraphPad Software, San Diego, CA). IC values were obtained by fitting the data
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values) of AngII antagonists for [ I-Sar1-Ile8] AngII binding were determined from
heterelogous competition data using GraphPad Prism 4.0 and the equation, K = IC / (1 + L /
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KD), where L is the concentration of radioligand . The K values (or -logK values) for [ I-
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D
Sar1-Ile8] AngII binding were determined from homologous competition data, using
GraphPad Prism 4.0 and the following equation: Y = {(Bmax * [hot]) / ([hot] + [cold] + K )}
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Results and Discussion
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HR MS results. The identification of CAN−2-HP-β-CD and CC−2-HP-β-CD complexes
was implemented by means of ESI QTOF HRMS, based on the accurate mass of the
-
pseudomolecular ion ([M-H] ) as well as the conformity between the theoretical and the
experimental isotopic profile. Apart from the potential identification of CAN−2-HP-β-CD and
CC−2-HP-β-CD as complexes, the detection and identification of each drug and the 2-HP-β-
CD separately, was considered as an additional confirmatory criterion.
2
-HP-β-CD exists as a mixture of cyclodextrins with differing degree of substitution by HP
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group. Consequently a broad distribution of peaks is expected in ESI-MS , which is in
accordance with our experimental results [Supplementary Figure S1(a)]. Supplementary
Figure S1(a) illustrates the mass spectrum of CC-2-HP-β-CD solution in the range of 1200 to
1700 Da, encompassing the pseudomolecular ions of cyclodextrins substituted by 2 up to 9
groups of HP (substitution degree: 0.3 – 1.3). Therefore, 2-HP-β-CD complexes of different
substitution degree were identified based on accurate mass measurements. Moreover, it was
hypothesized that the substituted cyclodextrins could form multi-charged species as well, due
to their structure characteristics and high M.W. The broad distribution of ions detected in the
mass range of 600 to 850 Da confirmed the hypothesis of double-charged ions
(Supplementary Figure S1(b)). The analysis of CAN−2-HP-β-CD solution led to identical
results, as far as the identification of 2-HP-β-CD complexes is concerned. Identification of the
uncomplexed drugs, CAN and CC, was also achieved through mass accuracy and isotopic
profile information. Supplementary Figures S2 (a1), (b1) depict the full scan MS spectra of
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CAN and CC. Supplementary Figures S2 (a2), (b2) show an overlay of the theoretical and the
experimental spectra for CAN and CC, respectively. It is obvious that there is very good fit
between the experimental and the theoretical spectra, enhancing though the identification
confidence.
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In Figure 1(a) the full scan MS spectrum of CAN−2-HP-β-CD, in the mass range of 1800 –
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100 Da, is presented. The ions inside the green rectangles, correspond to single-charged
CAN−2-HP-β-CD complexes with different substitution degree of β-CD by HP group. The
mass accuracy as well as the isotopic profile of the aforementioned ions are in accordance
with the theoretical ones of CAN−2-HP-β-CD complexes. In Figure 1(b) an example of the
identification results is depicted, through the overlay of the theoretical and the experimental
mass spectra for one of the complexes (the 6-substituted). It is obvious that the spectra are
almost identical. The potential presence of multi-charged species was investigated, based on
our findings for the multi-charged 2-HP-β-CD complexes. The broad distribution of ions
detected in the mass range of 860 to 1060 Da [Figure 1(c)] confirmed the presence of double-
charged ions of CAN−2-HP-β-CD. The detection of both single and double-charged species
enhanced the identification confidence.
The full scan MS spectrum of CC−2-HP-β-CD, in the mass range of 1960 – 2160 Da, is
highlighted in Figure 2(a). The ions inside the green rectangles, correspond to single-charged
CC-2-HP-β-CD complexes with different substitution degree of β-CD by HP group. The mass
accuracy and the isotopic profile of the aforementioned ions comply with the theoretical ones
of CC−2-HP-β-CD complexes. The presence of potential multi-charged ions was investigated,
as in the case of CAN−2-HP-β-CD. The detection of ions in the mass range of 890 to 1030 Da
[Figure 2(b)] confirmed the presence of double-charged ions of CC−2-HP-β-CD. In Figure
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and the experimental mass spectra for one of the double-charged complexes (the 2-
substituted). It is obvious that the presented spectra are in agreement.
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Figure 1. (a) Full scan MS spectrum of CAN-2-HP-β-CD in the mass range of 1800 – 2100
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complexes with different substitution degree of β-CD by HP group. (b) Overlay of the
theoretical and the experimental mass spectra of one of the complexes (the 6-substituted) of
CAN−2-HP-β-CD, as an example. (c) Full scan MS spectrum of CAN−2-HP-β-CD in the
mass range of 860 – 1060 Da. The ions inside the green rectangles, correspond to double-
charged CAN-2-HP-β-CD complexes with different substitution degree of β-CD by HP group.
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Figure 2. (a) Full scan MS spectrum of CC−2-HP-β-CD in the mass range of 1960 – 2160
Da. The ions inside the green rectangles, correspond to single-charged CC-2-HP-β-CD
complexes with different substitution degree of β-CD by HP group. (b) Full scan MS
spectrum of CC−2-HP-β-CD in the mass range of 890 – 1030 Da. The ions inside the green
rectangles, correspond to double-charged CC−2-HP-β-CD complexes with different
substitution degree of β-CD by HP group. (c) Overlay of the theoretical and the experimental
mass spectra of one of the complexes (the 2-substituted) of CC−2-HP-β-CD, as an example.
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13C CP/MAS results. The effect of the drug in its complex with 2-HP-β-CD was
investigated using 13C CP/MAS ssNMR spectroscopy.
13
Interactions of CAN with 2-HP-β-CD. Figure 3 shows the different areas of C CP/MAS
spectra of CAN (Figure 3A), HP-β-CD (Figure 3B), physical mixture of CAN−2-HP-β-CD
(1:1 molar ratio) (Figure 3C) and complex of CAN−2-HP-β-CD (1:1 molar ratio) (Figure 3D).
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Figure 3. 13C CP/MAS spectra of CAN (Figure 3A), 2-HP-β-CD (Figure 3B), physical
mixture of CAN−2-HP-β-CD (1:1 molar ratio) (Figure 3C) and complex of CAN−2-HP-β-
CD (1:1 molar ratio) (Figure 3D).
At the area 12-26 ppm, the spectra of the mixture (Figure 3C) and complex (Figure 3D) are
almost identical indicating that the C27 methyl (peak at ca 18 ppm) of AT1 antihypertensive
molecule does not affect differently the 2-HP-β-CD either being in a form of a physical
mixture or a complex. In both physical mixture and complex, a broadening of C27 peak is
observed with that of the complex to show more pronounced broadening.
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At the region 30-110 ppm differences are observed between the physical mixture and
complex. First, all the peaks of the complex are shifted to the higher field in respect to that of
the physical mixture. Paramount differences are observed between the drug peaks. In the
physical mixture, the small peak of C11 carbon (ca 50 ppm) of CAN is observable. In the
complex (Figure 3D) this peak is further shifted to the higher field and more significantly
broadened indicating the different interactions in the two samples.
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The most significant differences between the two samples are observed in the region 110-
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20 ppm. In the physical mixture (Figure 3C) all peaks of the drug are observable, although of
lower intensity, and the chemical shifts are systematically shifted slightly to higher field. The
identification of the peaks is shown in Supplementary Table S1. In the complex the peaks of
this region are broadened significantly and the resolution becomes low and impossible to be
identified.
Interactions of CC with 2-HP-β-CD. Figure 4 shows the different areas of 13C CP/MAS
spectra of CC (Figure 4A), 2-HP-β-CD (Figure 4B), physical mixture of CC / 2-HP-β-CD (1:1
ratio) (Figure 4C) and complex of CC−2-HP-β-CD (1:1 ratio) (Figure 4D).
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Figure 4. 13C CP/MAS spectra of CC (Figure 4A), 2-HP-β-CD (Figure 4B), physical
mixture of CC / 2-HP-β-CD (1:1 molar ratio) (Figure 4C) and complex of CC˗2-HP-β-CD
(1:1 molar ratio) (Figure 4D).
At the region 10-30 ppm the physical mixture contains all four peaks of CC attributed to
C31-C35 (see Scheme 1). When the prodrug is complexing, the peaks broaden significantly
and shift to higher field. This significant difference between the free drug and complex
indicates the participation of the cyclohexane ring of the prodrug in the complexing. This
structural segment is missing from the structure of CAN. This observation is important as it
signifies that CC possessing this additional structural segment can increase the favored
interactions with 2-HP-β-CD. Similar observations are also applied for the region 30-110
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ppm. In the physical mixture C11 (46 ppm) and C7 (92 ppm) are observable but not in the
complex. C7 is also missing form drug CAN. The fact that this peak disappears in the
complex is indicative that plays an important role in the complexing. This is another
indication that the additional segment of CC enhances the binding with 2-HP-β-CD. It
appears, that overall the cilexitil moiety of the prodrug results in its more efficient binding.
More pronounced differences can be observed in the region 110-250 ppm. In the physical
mixture almost all drug peaks are observed although with lower intensity and more
broadened. In the complex the peaks are no more resolved, broadened and almost vanished.
Fluorescence spectroscopic studies. To further evaluate the interaction between CAN/CC
with 2-HP-β-CD, we performed steady-state fluorescence spectroscopic measurements. As it
has been reported in the literature, the fluorescence spectrum of a small molecule can be
altered upon inclusion in a cyclodextrin cavity 42, 43. The spectroscopic behaviors of CAN and
CC were investigated by adding increasing concentrations of 2-HP-β-CD into a CAN/CC
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solution of a stable concentration. Both CAN and CC, which were dissolved in dH O, emit
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fluorescence with maximum wavelengths (λmax) at 373 and 405 nm, respectively.
When the concentration of the 2-HP-β-CD was enhanced in the CC–2-HP-β-CD physical
mixture, the relative fluorescence intensity increased dramatically, as it exhibited a final, 2-
fold raise in the presence of 6 mM of 2-HP-β-CD (Figure 5A). The different CC–2-HP-β-CD
solutions had a pH around 6.8. This enhancement in the fluorescence intensity is commonly
observed in CD-small molecule interactions and is attributed to alterations occurring in the
microenvironment of the small molecule during its encapsulation 44, 45. Herein, as CC is
transferred from the aqueous solution into the hydrophobic cavity of 2-HP-β-CD, its quantum
yield rises leading to a higher fluorescence intensity. On the other side, when the molecule
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was mixed with 2-HP-β-CD at pH=4.1 the exact opposite phenomenon was noticed, as the
intensity followed a 2-fold decrease at a 6 mM final concentration of 2-HP-β-CD (Figure 5B).
It’s noteworthy to mention that in both cases, either increased or decreased, the fluorescence
intensity of CC ends up to the same intensity levels indicating the final photophysical profile
that the molecule possess inside the 2-HP-β-CD cavity (Figures 5C and 5D).
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On the contrary, the fluorescence spectrum of CAN did not exhibited significant changes
upon mixing with 2-HP-β-CD except from a quite slight intensity reduction at pH 6 and a
slight enhancement at pH 4.1 which are insignificant. Arguably, these results point out that
CC presents a higher tendency to enter into 2-HP-β-CD, even into a simple mixed solution.
Figure 5. A, B) Fluorescence spectra of CC after the titration with various 2-HP-β-CD
concentrations (0, 0.15, 0.35, 0.5, 0.65, 0.85, 1, 2, 3, 4, 5, 6 mM) at pH=6.8 and 4.1,
respectively. C, D) Fluorescence spectra of CV-1974 after the titration with various 2-HP-β-
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CD concentrations (0, 0.15, 0.35, 0.5, 0.65, 0.85, 1, 2, 3, 4, 5, 6 mM) at pH=6 and 4.1,
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Finally, the binding constant between CC and 2-HP-β-CD was estimated based on the
emission changes of the CC spectrum after the addition of different 2-HP-β-CD
concentrations. Specifically, the fluorescence intensity at 403 nm (λmax) of each measurement
was plotted against the 2-HP-β-CD concentrations (Figure 6A). Then, following a linear
fitting, the double reciprocal plot of the data was designed (Figure 6B) and the binding
constant was derived from the Benesi-Hildebrand equation:
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c
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complexed CC at 1:1 molar ratio and [CD] the concentration of 2-HP-β-CD .
The straight line of the double reciprocal plot confirms the 1:1 stoichiometry of CC with 2-
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HP-β-CD and the binding constant was calculated equal to 1200 M , indicating a moderate
affinity between the two molecules which is line with the estimated values of similar
interactions 47, 48. The calculation of the binding constant between CAN and 2-HP-β-CD was
not feasible due to the inconsiderable changes in the fluorescent intensity of CAN after the
addition of the cyclodextrin.
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Figure 6. A) Fluorescence intensities of CC at 403 nm upon the addition of different 2-HP-
β-CD concentrations. B) Double reciprocal plot of the same experiment.
Plasma stability assay. Having evaluated the biological efficacy of CC complexed with 2-
HP-β-CD, we then examined its human plasma stability and the rate at which CC was released
from the complex and hydrolysed to CV-174. Moreover, in order to explore if there was a
different behaviour of free CC when it was incubated in human plasma, we set up a second
plasma stability experiment. For this purpose we utilized LC-MS protocols to monitor their
stability, as also to quantify their concentration in human plasma in a time dependent manner.
In order to maximize resolution for sharper peak shapes and better separation, we conducted
several chromatography tests. The presence of 0.1% formic acid in phase A and B improved
peak shapes and increased the signal for all compounds. The total run time was 6 min and the
elution time for CC was 3.2 min while for CAN and IRB 2.4 min.
Tandem mass spectrometry was used for the detection and quantification of the targeted
compounds in positive electrospray ionization. During direct infusion in mass spectrometer,
the most abundant transitions in terms of sensitivity were found to be: m/z 611.2 → 423.1 and
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11.2 → 349.1 for CC, 441.2→147.2 and 441.2→255.1 for CΑΝ, 429.2→207.1 and
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