Host-guest interactions between candesartan and its prodrug

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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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Molecular Pharmaceutics

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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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 Dimitrios Ntountaniotis1, Ioannis Andreadelis1, Tahsin F. Kellici1, Vlasios Karageorgos2, Georgios Leonis1, Eirini Christodoulou3, Sofia Kiriakidi1, Johanna Becker-Baldus4, Evgenios K. Stylos5,6, Maria V. Chatziathanasiadou5, Christos M. Chatzigiannis5, Dimitrios E. Damalas7, Busecan Aksoydan8, Uroš Javornik9, Georgia Valsami3, Clemens Glaubitz4, Serdar Durdagi8, Nikolaos S. Thomaidis7, Antonios Kolocouris10, Janez Plavec9, Andreas G. Tzakos4, George Liapakis7, Thomas Mavromoustakos1



Dimitrios Ntountaniotis: email address. [email protected], tel. +30 210 7274475



Andreas Tzakos: email address. [email protected]



George Liapakis: email address. [email protected]



Thomas Mavromoustakos: email address. [email protected], 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

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, 60438 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 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.

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 Angiotensin II to stimulate AT1 receptors 1, 2. Sartans act to a membrane receptor and prefer to initially incorporate themselves in lipid bilayers before reaching the receptor site through lateral diffusion 3. 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.



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 prototype (Scheme 1) 1. In previous studies, we have compared the interactions of CAN and losartan with lipid bilayers 5 as well as the actions of olmesartan and other AT1 antagonists 3. 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 pharmacological profile are CAN and its prodrug, CC7-9. In the described research the complexing properties of CAN and its prodrug CC with 2-HPβ-CD (Scheme 1) were characterized using 13CP/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.



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 alkaline hydrolysis (methanolysis) described in our previous article is applied10.


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Scheme 2. Hydrolysis of CC.

In particular, to a solution of the ester (CC) (190 mg, 0.3111mmol) in CH2Cl2/CH3OH (9:1, v/v, 100 mL), a methanolic solution (2 mL) of NaOH 0.08 M was added. The physical mixture was stirred at 30 ºC 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 CH2Cl2. The organic layer was washed with brine and dried over (Na2SO4) and the solvent was removed to 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: 1:1 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 freezedrying, the neutralization method was used.



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 1:1 were similarly prepared for comparative purposes using 0.300 (or 0.810 g CC) and 1.022 g of 2-HP-β-CD, respectively 11. High-resolution Electrospray Ionisation Mass Spectrometry Measurements. HR-ESIMS: 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; drying gas 4 L min−1 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. Stock solutions of 1 mg mL-1 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 water. Working solutions of 0.01 mg mL-1 were prepared and used for the infusion experiments. Infusion of the complexes’ solutions was performed under a constant flow of 180 μL min-1. 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. Solid-state 13C CP/MAS NMR spectroscopy. 13C cross polarization/magic-angle spinning (13C 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. The 13C 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 (dH2O) [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 dH2O at a concentration of 12 mM. The final concentration of each drug in the cuvette was 25 μΜ for each measurement. Various volumes from the 2-HP-βCD 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 dH2O The pH values of the samples were 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 30 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 Cambridge Structural Database (CSD reference code: FETWEH) 12. The crystal structures of β-CD (CSD reference code BUVSEQ02) 14

13

and CC were modified using Schrodinger 2015.2

to 2-HP-β-CD and CAN, respectively. The OPLS3 force field

structures of CAN and CC into 2-HP-β-CD with GlideXP

16.

15

was used to dock the

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.

CAN was optimized with the HF/6-31G* basis set (Gaussian 09)

The geometry of CC and 20.

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

4724 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 0 to 300 K using a Langevin thermostat

25.

The collision frequency (γ) was set at 2 ps-1.

Restraints of 10 kcal mol-1 Å-2 were applied to each cyclodextrin complex. Next, the systems


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were equilibrated under constant pressure in two steps of 100 ps each. In the first one, constraints of 10 kcal mol-1 Å-2 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 hydrogen atoms were constrained at their equilibrium distance using SHAKE 26. The analysis of the trajectories was performed using the ptraj module of AMBER analysis

28

was performed as described in our previous publications

27.

29, 30.

The MM-PBSA

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 complexes to free phase by employing the weighted histogram analysis method (WHAM) 32. 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 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 different conformational approaches for each molecule. The default basis set was 6-31G** 36. DFT (density functional theory) was applied with a B3LYP functional 37. The accuracy level of SCF (self consistent field) calculations for convergence was set to accurate. PoissonBoltzmann 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 autosampler 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, 441.2→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



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. 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 % CO2. Cells at 100% confluence in 100-mm dishes, were washed with phosphate - buffered saline (PBS) (4.3 mM Na2HPO4.7H20, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2 - 7.3 at room 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 mM EDTA, 10% sucrose, 10 mM MgCl2, pH 7.4 at 4 oC) using a Janke & Kunkel IKA Ultra 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, 0.2 mg/ml bacitracin, and 0.93 μg/ml aprotinin, pH 7.4 at 4 ºC) and used for radioligand binding studies.


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[125I-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 (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. Specific [125I-Sar1-Ile8] AngII binding was defined as the total binding substracted the nonspecific 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). IC50 values were obtained by fitting the data from competition studies to a one-site competition model. The binding affinities (Ki or -logKi values) of AngII antagonists for [125I-Sar1-Ile8] AngII binding were determined from heterelogous competition data using GraphPad Prism 4.0 and the equation, Ki = IC 50 / (1 + L / KD), where L is the concentration of radioligand 39. The KD values (or -logKD values) for [125ISar1-Ile8] AngII binding were determined from homologous competition data, using GraphPad Prism 4.0 and the following equation: Y = {(Bmax * [hot]) / ([hot] + [cold] + KD)} + NSB

40,

where Y is the total binding of [125I-Sar1-Ile8] AngII, NSB is the non-specific



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binding of the radioligand, Bmax is the total receptor number, [hot] is the concentration of the [125I-Sar1-Ile8] AngII and [cold] is the concentration of the [Sar1-Ile8] AngII .

Results and Discussion 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-βCD 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 group. Consequently a broad distribution of peaks is expected in ESI-MS

41,

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. In Figure 1(a) the full scan MS spectrum of CAN−2-HP-β-CD, in the mass range of 1800 – 2100 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 doublecharged 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



2(c), an example of the identification results is depicted, through the overlay of the theoretical and the experimental mass spectra for one of the double-charged complexes (the 2substituted). It is obvious that the presented spectra are in agreement.

Figure 1. (a) Full scan MS spectrum of CAN-2-HP-β-CD in the mass range of 1800 – 2100 Da. The ions inside the green rectangles, correspond to single-charged CAN-2-HP-β-CD


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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 doublecharged CAN-2-HP-β-CD complexes with different substitution degree of β-CD by HP group.



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.

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. Interactions of CAN with 2-HP-β-CD. Figure 3 shows the different areas of 13C 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-βCD (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.



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. The most significant differences between the two samples are observed in the region 110220 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



Page 26 of 72

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 solution of a stable concentration. Both CAN and CC, which were dissolved in dH2O, emit 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, 2fold 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). 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-



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, respectively.

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: 1 1 1   F Fc K c * Fc *[CD]0

Where ΔF is the difference between the fluorescence intensities in the absence and presence of 2-HP-β-CD, Kc the binding constant, ΔFc the difference on intensity between free and complexed CC at 1:1 molar ratio and [CD] the concentration of 2-HP-β-CD 46. The straight line of the double reciprocal plot confirms the 1:1 stoichiometry of CC with 2HP-β-CD and the binding constant was calculated equal to 1200 M-1, 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-HPβ-CD concentrations. B) Double reciprocal plot of the same experiment.

Plasma stability assay. Having evaluated the biological efficacy of CC complexed with 2HP-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 611.2 → 349.1 for CC, 441.2→147.2 and 441.2→255.1 for CΑΝ, 429.2→207.1 and



429.2→195.2 for IRB. ESI parameters like nebulizer, spray voltage and heated probe temperature were optimized in order to obtain a consistent response for all analytes. The rates at which CC of complex is released and hydrolysed in CAN after incubation in human plasma for 0, 24 and 48 hrs is presented in Figure 7A. Respectively, the rate at which free CC is hydrolyzed in CAN in human plasma is depicted in Figure 7B. As it was expected at 0 hrs, no amount of CAN was present in both plasma stability assays. It is notable that after 48 hrs of plasma incubation, in both cases, a small amount of CC is still present, revealing a rather slow degradation rate, nonetheless, in the case of CC that origins from the CC-2-HP-βCD complex, the degradation rate was slower. This difference in the plasma stability profile could be justified by the fact that CC in the complex is protected from the 2-HP-β-CD, hence, preventing its enzymatic hydrolysis of CC to CV-174.

Figure 7. Concentration of CC and CAN after incubation in human plasma for 37 °C of A) CC-2-HP-β-CD complex and B) CC.


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Molecular Dynamics CC–2-HP-β-CD complex. Molecular docking simulations of CC inside the 2-HP-β-CD cavity suggested the equiprobable existence of two binding modes as also proposed by Al Omari et al. 7. In the first binding orientation, the benzodiazole moiety faces toward the 2hydroxypropyl groups (Pose A) while in the second binding orientation, the biphenyl tetrazole moiety faces toward the 2-hydroxypropyl groups (Pose B) (Figure 8).

Figure 8. The 3D structures of CAN and CC, embedded in 2-HP-β-CD in two different poses A and B.

To evaluate the stability and the thermodynamic properties of each binding mode, MD simulations were run for the two initial poses. The root mean square deviations (RMSDs) of cyclodextrin and CC with respect to the equilibrated structure of the complex in the two simulations are shown in Figures 9A and 9B. The systems appear to be stable, with RMSDs that fluctuate around average values of 2.3–2.5 Å, however, CC and cyclodextrin structures in Pose B present greater deviations from the reference (equilibrated) structure than in Pose A. The flexibility of CC inside the cavity was further examined by calculating the root mean square fluctuations (RMSFs) of the heavy atoms (C, N, O) (Figure 9C). Carbon atoms C27 (benzodiazole moiety), C10 (alkyl chain), and C32–C36 (cilexetil part) showed the highest



RMSF in both simulations (Figure 9D), thus identifying the biphenyl tetrazole moiety as the most stable part of the molecule. Snapshots of the systems after 400 ns (Figures 9D and 9E) verify that biphenyl tetrazole is stabilized inside the cavity of 2-HP-β-CD. Moreover, CC carbon edges C10 and C27 appear more mobile in pose B compared to pose A, thus implicating the alkyl chain and the benzodiazole moiety in increased flexibility of CC at pose B. Despite the aforementioned differences in flexibility among CC regions, the overall structure of the guest molecule is firmly accommodated into cyclodextrin cavity as denoted by the stable distance between the centers of mass of CC and 2-HP-β-CD (Figure S3a). Additionally, hydrogen bond (HB) analysis shows that HBs are developed between CC and 2HP-β-CD only for a short time (occupancy of 20% or lower), thus indicating that the encapsulation of the biphenyl tetrazole part is also due to hydrophobic (non polar) interactions.


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Figure 9. All atom RMSD values versus simulation time for CC and cyclodextrin in Pose A (A) and Pose B (B). Root mean square fluctuations and labeling of CC heavy atoms (C). Snapshots of CC−2-HP-β-CD complex after 400 ns in Pose A (D) and Pose B (E).



Page 34 of 72

The binding free energy (ΔGbinding) of the CC−2-HP-β-CD complex for Pose A, as it was calculated using the MM-PBSA method, was –6.96 kcal/mol, while the enthalpy (ΔHbinding) and entropy (–TΔSbinding) terms of the system were –34.29 and 27.33 kcal/mol, respectively (Table 1). The corresponding values for the complex in Pose B were ΔGbinding = –6.70 kcal/mol, ΔHbinding = – 30.62 and –TΔSbinding = 23.92 kcal/mol. The energy decomposition into individual contributions showed that (Table 1): i) the binding of CC to 2-HP-β-CD is energetically favorable and similar between poses A and B; ii) the binding is mostly driven by van der Waals interactions, while the nonpolar solvation term (ΔGcavity) also has a favorable contribution; iii) despite that the nonbonded electrostatic term (ΔΕelec) contributes favorably in both complexes, the total electrostatic contribution is highly unfavorable because of the positive value of the electrostatic component to solvation (ΔGPB); iv) while the enthalpy contribution in Pose A is greater than in Pose B, the entropy terms counterbalance this difference to yield very similar binding free energies between poses. The increased entropy contribution in Pose A may be rationalized by the high flexibility of tetrazole and cyclohexyl rings, which are more solvent-exposed compared to Pose B. These findings are in agreement with previous findings reported in the literature 7.


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Table 1. Binding free energy analysis for the CC−2-HP-β-CD complex as obtained by the MM-PBSA calculations using two different orientations of the molecule inside the cavity.

ΔΕvdW

CC Pose A Average value (kcal/mol) -57.32

0.024

CC Pose B Average value (kcal/mol) -52.76

ΔΕelec

-14.38

0.043

-16.34

0.026

ΔEgas (=ΔΕelec + ΔΕvdW)

-71.70

0.049

-69.10

0.028

ΔGPB

42.46

0.038

42.82

0.026

ΔGcavity

-5.05

0.001

-4.34

0.000

ΔGsolv (=ΔΕPB + ΔΕsolv)

37.41

0.038

38.48

0.025

ΔHbinding (=ΔEgas+ΔGsolv)

-34.29

0.027

-30.62

0.020

–TΔSconfig

27.33

0.058

23.92

0.037

ΔGbinding

-6.96

0.0402

-6.70

0.0282

Energy Component

±SEM1

±SEM1 0.014

1Standard

error of the mean (SEM): SEM = Standard deviation/√N. N is the number of trajectory frames used during the MM-PBSA calculations (4000 frames for entropy and 40000 frames for everything else). 2Pooled standard error of the mean.

The ΔGbinding of the CC−2-HP-β-CD complex with the best pose (pose A) was also calculated by the PMF value obtained by umbrella sampling simulations of CC being pulled out of 2-HPβ-CD’s cavity, as described in Materials and Methods section. The results are presented in Figure 10.



Page 36 of 72

Figure 10. Potential of Mean Force (PMF) calculation for the complex CC-2-HP-β-CD. The average is denoted in green and is 10.22 kcal/mol.

The PMF of CC being pulled out of 2-HP-β-CD’s cavity is 10.22 kcal/mol, i.e., ΔGbinding= 10.22 kcal/mol, which is in qualitative agreement with the MM-PBSA calculation (~3.5 kcal/mol difference). CAN−2-HP-β-CD complex. As above, MD simulations were performed for the two binding orientations of CAN into cyclodextrin. The resulting RMSDs (Figures 11A and 11B) indicate that the systems acquire relatively stable conformations after ~20 ns (similarly to CC complexes, the RMSD calculations in CAN complexes were performed with respect to the equilibrated complex structure); however, it was observed that CAN and cyclodextrin in Pose A had greater deviations from the reference structure than the corresponding molecules in Pose B. Snapshots of the


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CAN−2-HP-β-CD complex at the end of the simulation in Poses A and B are shown in Figures 11D and 11E, respectively. Contrarily to CC–2-HP-β-CD, the tetrazole moiety of CAN in Pose A develops multiple HBs with the hydroxyl groups of 2-HP-β-CD in positions 2 and 3 of 2-HP-βCD with occupancies that approach 50% of the simulation time. Similarly, the complex in Pose B (Figure 11E) is stabilized by HBs between the carboxylate anion group of CAN and the hydroxyl groups in positions 2 and 3 of 2-HP-β-CD (~ 22% of the simulation time). In this case, HBs are also observed between the tetrazol moiety and hydroxyl groups of the hydroxypropyl groups (~ 19% of the simulation time). RMSF values showed identical trends between the two poses. RMSFs are higher for Pose B and for atoms of the ethoxy alkyl chain, the phenyl ring connected with benzimidazole heterocycle, the tetrazole and the aromatic ring with carboxylic acid attached to imidazole (Figure 11C). Similarly to CC, the overall structure of CAN is stably positioned inside cyclodextrin’s cavity (Figure S3b).



Page 38 of 72

Figure 11. All atom RMSD values versus simulation time for candesartan and cyclodextrin in Pose A (A) and Pose B (B). Root mean square fluctuations and labeling of CC heavy atoms (C). Snapshot of the complex CAN−2-HP-β-CD after 400 ns in Pose A (D) and Pose B (E).

MM-PBSA calculations predicted the binding free energy of CAN–2-HP-β-CD in Pose A to be 3.11 kcal/mol, while the enthalpy and entropy terms were –19.38 and 22.49 kcal/mol, respectively (Table 2). The energy terms for the complex in Pose B are ΔGbinding = –1.55 kcal/mol, ΔHbinding = –25.05, and –TΔSbinding = 22.49 kcal/mol. These calculations showed that the binding of CAN to


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2-HP-β-CD is slightly favorable only in Pose B. This difference is mainly due to the total electrostatics (ΔΕelec + ΔGPB), which has a less negative effect in Pose B than in Pose A. Conclusively, CAN binding to cyclodextrin is suggested to be less effective than CC binding (compare Tables 1 and 2). This finding is further supported by the extensive hydrogen bond network that was predicted to increase CAN–2-HP-β-CD interactions.

Table 2. Binding free energy analysis for the CAN−2-HP-β-CD complex as obtained by the MM-PBSA calculations using two different orientations of the molecule inside the cavity.

±SEM1

ΔΕvdW

Candesartan Pose A Average value (kcal/mol) -43.37

±SEM1

0.015

Candesartan Pose B Average value (kcal/mol) -41.55

ΔΕelec

-7.05

0.060

-17.27

0.054

ΔEgas (=ΔΕelec +ΔΕvdW)

-50.42

0.062

-58.82

0.055

ΔGPB

34.97

0.056

37.58

0.050

ΔGcavity

-3.93

0.000

-3.80

0.000

ΔGsolv (=ΔΕPB +ΔΕsolv)

31.04

0.056

33.78

0.050

ΔHbinding(=ΔEgas+ΔGsolv)

-19.38

0.018

-25.05

0.017

–TΔSconfig

22.49

0.045

23.50

0.043

ΔGbinding

3.11

0.0232

-1.55

0.0212

Energy Component

0.016

1Standard

error of the mean (SEM): SEM = Standard deviation/√N. N is the number of trajectory frames used during the MM-PBSA calculations (4000 frames for entropy and 40000 frames for everything else). 2Pooled standard error of the mean.

As previously, the ΔGbinding of the CAN–2-HP-β-CD complex with the best pose (pose B) was also calculated by the PMF value obtained by umbrella sampling simulations of CAN being



Page 40 of 72

pulled out of 2-HP-β-CD’s cavity, as described in Materials and Methods section. The results are presented in Figure 12.

Figure 12. Potential of Mean Force (PMF) calculation for the complex CC–2-HP-β-CD. The average is denoted in green and is 6.69 kcal/mol.

The PMF of CC being pulled out of 2-HP-β-CD’s cavity is 6.69 kcal/mol, i.e., ΔGbinding= -6.69 kcal/mol, which is qualitatively in agreement with the MM-PBSA calculation (~5.1 kcal/mol difference). A comparison of energy contributions in complexes CC−2-HP-β-CD and CAN−2-HP-β-CD showed that CC binds more favorably to cyclodextrin than CAN. The main reason for this difference comes from the van der Waals component, which is reduced by approximately 14


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kcal/mol in the case of candesartan. Candesartan in one orientation (Pose A) did not bind to cyclodextrin, however, appropriate positioning of the biphenyl tetrazole moiety, which faces toward the 2-hydroxyropyl groups in Pose B, facilitated CAN binding through an increase in the total electrostatics contribution. This is due to the existence of a second charged moiety in CAN (the carboxylate group) as well as the suitable position of CAN in the second approach that promoted the formation of HB interactions between both tetrazole and carboxylate anions with most hydroxyl groups of the modified cyclodextrin. While the tetrazole moiety in CC was immobilized within the cavity of cyclodextrin, the same moiety in CAN was observed to be highly flexible. The conformational differences between the results obtained from MD and QM calculations are shown in Figure 13. The converged solvation energies for the molecules are listed in Table 3.

Table 3. Solvation energy values obtained from the geometric optimization for CAN, CC, their complexes with 2-HP-β-CD and both conformational approaches A and B (Poses A and B).

Molecule Candesartan (c) Candesartan + cyclodextrin Candesartan cilexetil (cc) Candesartan cilexetil + cyclodextrin

Solvation Energy (kcal/mol) Pose-A Pose-B -51.15 -53.95 -86.91 -91.03 -37.64 -35.12 -102.66 -71.45



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Figure 13. The conformational differences between the results obtained from MD and QM calculations.

Interestingly, not significant changes were obtained after performing MD calculations. However, using QM studies the orientation of biphenyltetrazole ring and benzimidazole rings have been changed in the case of CAN and included cilexetil segment in the case of CC in both poses A and B. This is not a surprising result as previous studies in solvent environments and lipid bilayers showed the high flexibility of these rings with almost not of expense of energy in sartan molecules 5, 6, 49, 50. Binding of AngII analogs to human AT1 receptor. To test whether the AngII antagonists CAN or CC, or 2-HP-β-CD or the complexes of drugs with 2-HP-β-CD bind to human AT1 receptor, we first determined their ability (at the single concentration of 1000 nM) to inhibit the specific binding of [125I-Sar1-Ile8] AngII to membranes from HEK 293 cells stably expressing the AT1 receptor in binding experiments performed under equilibrium conditions. 2-HP-β-CD did not


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bind to human AT1 receptor. In contrast CAN and CC, with or without 2-HP-β-CD in their molecules inhibited [125I-Sar1-Ile8] AngII specific binding by 84-100 % (Figure 14). The AngII antagonists and their complexes with 2-HP-β-CD were further pharmacologically characterized by determining their binding affinities (-logKi) for the AT1 receptor in competition experiments. As shown in Figure 15, addition of 2-HP-β-CD to CAN in 1:1 ratio, thus forming CAN−2-HP-βCD complex did not significantly alter the binding properties of CAN. In specific, the binding affinity of CAN−2-HP-β-CD (Ki = 4.07± 1.68 nM) for the AT1 receptor was similar to that of candesartan (Ki = 4.32 ± 2.09 nM). In contrast, addition of 2-HP-β-CD to CC in 1:1 ratio, thus forming the CC−2-HP-β-CD complex reduced the binding affinity of CC 30-fold. In specific, the binding affinities of CC−2-HP-β-CD and CC were Ki = 470.2 ± 242.6 nM and 15.56 ± 5.12 nM, respectively.



Figure 14. Screening of AngII antagonists and their complexes with 2-HP-β-CD for binding to human AT1 receptor. Inhibition of [125I-Sar1-Ile8] AngII specific binding by 1000 nM of antagonists and their complexes with 2-HP-β-CD , or 2-HP-β-CD alone was performed, as described under “Materials and Methods”, on membranes from HEK 293 cells stably expressing the human AT1 receptor. The bars represent the % inhibition of radiolabelled Ang II specific binding by the antagonists, determined from 1 experiment.


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Figure 15. Competition binding isotherms of AngII antagonists or their complexes to human AT1 receptor. Competition of [125I-Sar1-Ile8] AngII specific binding by increasing concentrations of AngII antagonists was performed, as described under “Materials and Methods”, on membranes from HEK 293 cells stably expressing the human AT1 receptor. The means and S.E. (duplicate determination) are shown from a representative experiment performed 2 times with similar results. The data were fit to a one-site competition model by nonlinear regression. The Ki values were calculated (as described under “Materials and Methods”), and given in the table 1.

Conclusions In this work the prepared complexes of CAN and CC with 2-HP-β-CD were characterized by MS-ESI and

13C-CP/MAS

techniques.

13CP/MAS

spectra of complexes differed significantly of

those of physical mixtures prepared in a ratio of 1:1 drug/2-HP-β-CD. The peak chemical shift differences for the drug in the complexes are more pronounced and the line widths of the peaks



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are more broadened. The intensities of the peaks of the drugs are lowered or vanished upon complexation as a result of efficient relation due to the favorable interactions between the drug guest and the 2-HP-β-CD host. Fluorescence studies showed that CC presents a higher tendency to enter the 2-HP-β-CD than CAN. The binding constant was calculated equal to 1200 M-1 indicating a moderate affinity. The plasma stability of CC was clearly enhanced in complex form in respect to the free form. This is a direct evidence that complexation protects drug from the enzymatic hydrolysis. MD simulations for 400 ns, MM-PBSA and umbrella sampling calculations confirmed the ssNMR results and provided thermodynamic parameters that indicate the stability and the nature of the forces governing the complexation when molecules are inserted in the complex with different binding orientation. These complexes were evaluated in vitro. The results are very promising. The complex of CAN had lower Ki value with that of its pure form, while CC was more effective in its pure form. It is apparent that CAN is released more effectively in comparison to CC in the lipid bilayer environment in order to reach the active binding site through lateral diffusion in the lipid matrix or directly from the receptor site. These results are in accordance with MM-PBSA results which show that CC has a favoring binding. This implies that CC accommodates a suitable fit in cyclodextrin host and it does not escape from this environment. However, the binding is not favored with CAN and easily escapes from cyclodextrin environment.

Acknowledgments Dimitrios Ntountaniotis and Thomas Mavromoustakos would like to thank Bio-NMR and CERIC 20187056 programs, which supported this research. Part of this research work was also supported


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by the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI PhD Fellowship grant (GA. no. 14551). Dimitrios Ntountaniotis and Ioannis Andreadelis contributed equally in this work.

ASSOCIATED CONTENT Supporting Information.

Mass spectra of CC-2-HP- β -CD solution, CAN and CC; theoretical and experimental mass spectra of the pseudomolecular ion of CAN and CC; Distance between drugs (CAN and CC) and cyclodextrin; Chemical shifts of CAN in DMSO, in samples of CAN (solid state Ν Μ R), of mixture CAN/2-HP-β-CD and of complex CAN-2-HP-β-CD.

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Scheme 1. Structures of CAN, CC as well as 2-HP-β-CD. 127x110mm (600 x 600 DPI)


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Scheme 2. Hydrolysis of CC. 191x40mm (600 x 600 DPI)



Figure 1. (a) Full scan MS spectrum of CAN-2-HP-β-CD in the mass range of 1800 – 2100 Da. 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. (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.



Figure 3. 13C CP/MAS spectra of CAN (Figure 3A), 2-HP-β-CD (Figure 3B), 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). 109x98mm (600 x 600 DPI)


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Figure 4. 13C CP/MAS spectra of CC (Figure 4A), 2-HP-β-CD (Figure 4B), 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). 146x114mm (600 x 600 DPI)



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-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, respectively. 151x94mm (600 x 600 DPI)


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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.



Figure 7. Concentration of CC and CAN after incubation in human plasma for 37 °C of A) CC-2-HP-β-CD complex and B) CC. 151x78mm (300 x 300 DPI)


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Figure 8. The 3D structures of CAN and CC, which are embedded in 2-HP-β-CD, with their two different conformational approaches A and B. 443x99mm (300 x 300 DPI)



Figure 9. All atom RMSD values versus trajectory time for CC and cyclodextrin in Pose A (A) and Pose B (B). Root mean square fluctuations and labeling of CC heavy atoms (C). Snapshots of CC−2-HP-β-CD complex after 400 ns in Pose A (D) and Pose B (E). 233x159mm (300 x 300 DPI)


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Figure 10. Potential of Mean Force (PMF) calculation for the complex CC-2-HP-β-CD. The average is denoted in green and is 10.22 kcal/mol. 119x92mm (600 x 600 DPI)



Figure 11. All atom RMSD values versus trajectory time for candesartan and cyclodextrin in Pose A (A) and Pose B (B). Root mean square fluctuations and labeling of CC heavy atoms (C). Snapshot of the complex CAN−2-HP-β-CD after 400 ns in Pose A (D) and Pose B (E).


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Figure 12. Potential of Mean Force (PMF) calculation for the complex CC-2-HP-β-CD. The average is denoted in green and is 6.69 kcal/mol. 119x92mm (600 x 600 DPI)



Figure 13. The conformational differences between the results obtained from MD and QM calculations. 400x187mm (300 x 300 DPI)


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Figure 14. Screening of AngII antagonists and their complexes with 2-HP-β-CD for binding to human AT1 receptor. Inhibition of [125I-Sar1-Ile8] AngII specific binding by 1000 nM of antagonists and their complexes with 2-HP-β-CD , or 2-HP-β-CD alone was performed, as described under “Materials and Methods”, on membranes from HEK 293 cells stably expressing the human AT1 receptor. The bars represent the % inhibition of radiolabelled Ang II specific binding by the antagonists, determined from 1 experiment. 201x202mm (300 x 300 DPI)



Figure 15. Competition binding isotherms of AngII antagonists or their complexes to human AT1 receptor. Competition of [125I-Sar1-Ile8] AngII specific binding by increasing concentrations of AngII antagonists was performed, as described under “Materials and Methods”, on membranes from HEK 293 cells stably expressing the human AT1 receptor. The means and S.E. (duplicate determination) are shown from a representative experiment performed 2 times with similar results. The data were fit to a one-site competition model by nonlinear regression. The Ki values were calculated (as described under “Materials and Methods”), and given in the table 1. 119x92mm (600 x 600 DPI)


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Abstract Graphic 89x43mm (600 x 600 DPI)


Host-guest interactions between candesartan and its prodrug

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