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Characterization, In Vivo Evaluation and Molecular Modeling of Different Propofol-Cyclodextrin Complexes to Assess Their Drug Delivery Potential at The Blood-Brain Barrier Level Sergey Shityakov, Ramin Ekhteiari Salmas, Serdar Durdagi, Ellaine Salvador, Katalin Pápai, María Josefa Yañez-Gascón, Horacio Perez-Sanchez, István # Puskás, Norbert Roewer, Carola Förster, and Jens-Albert Broscheit J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.6b00215 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 3, 2016
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Journal of Chemical Information and Modeling
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Characterization, In Vivo Evaluation and Molecular Modeling of Different Propofol-
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Cyclodextrin Complexes to Assess Their Drug Delivery Potential at The Blood-Brain
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Barrier Level
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Sergey Shityakov1,*, Ramin Ekhteiari Salmas2, Serdar Durdagi2, Ellaine Salvador1, Katalin Pápai 3
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, Maria Josefa Yáñez-Gascón4, Horacio Pérez-Sánchez4, István Puskás5, Norbert Roewer1,3, Carola Förster1, and Jens-Albert Broscheit1,3
7 1
8
Department of Anesthesia and Critical Care, University of Würzburg, 97080 Würzburg, Germany
9 10
2
Department of Biophysics, School of Medicine, Bahcesehir University, 34349 Istanbul, Turkey 3
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12 13
5
Sapiotec Ltd., 97078 Würzburg, Germany
Universidad Católica San Antonio de Murcia, 30107 Guadalupe, Spain
CycloLab Cyclodextrin Research & Development Laboratory Ltd., H-1097 Budapest, Hungary
14
Author to whom correspondence should be addressed; E-Mail:
[email protected] 15
Tel.: +49-931-2013-0016; Fax: +49-931-2013-0019.
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Graphical Abstract:
17
solid-state 3D structure with in vivo blood-brain barrier permeation & molecular modeling of PR/SBE CD
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Abstract: In this study, we investigated the ability of general anesthetic propofol (PR) to form
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inclusion complexes with modified β-cyclodextrins, including sulfobutylether-β-cyclodextrin
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(SBEβCD) and hydroxypropyl-β-cyclodextrin (HPβCD). The PR/SBEβCD and PR/HPβCD
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complexes were prepared and characterized, and the blood-brain barrier (BBB) permeation
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potential of the formulated PR examined in vivo for the purpose of controlled drug delivery. The
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PR/SBEβCD complex was found to be more stable in solution with a minimal degradation
39
constant of 0.25 h-1, t1/2 of 2.82 h, and Kc of 5.19*103 M-1 and revealed higher BBB permeability
40
rates as compared with the reference substance (PR-LIPURO®) considering the calculated brain-
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to-blood concentration ratio (logBB) values. Additionally, the diminished PR binding affinity to
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SBEβCD was confirmed in molecular dynamics simulations by a maximal Gibbs free energy of
43
binding (∆Gbind = −18.44 kcal*mol-1) indicating the more rapid PR/SBEβCD dissociation.
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Overall, the results demonstrated that SBEβCD has the potential to be used as a prospective
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candidate for drug delivery vector development to improve the pharmacokinetic and
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pharmacodynamic properties of general anesthetic agents at the BBB level.
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propofol, modified cyclodextrins, complexation, stability, blood-brain barrier,
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Keywords:
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molecular dynamics simulations.
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1. Introduction
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2, 6- diisopropylphenol or propofol (PR) is an intravenous general anesthetic classed with the
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alkyl phenol group of compounds.1 This drug is a preferred agent for day-patient surgeries due to
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its rapid metabolism and reduced post-anesthetic nausea.2,3 The currently available formulation of
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PR in the market is a lipid emulsion that has side effects such as pain on injection, serious allergic
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reactions, and support of microbial growth.4 Thus, development of safer formulations is of great
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interest in biomedicine. For instance, lipid-free preparations of PR are being developed to reduce
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these formulation-related problems. One method employed to generate similar formulations is the
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use of natural cyclodextrins (CDs) and their derivatives.
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CDs are cyclic oligosaccharides made up of six to eight dextrose units and can interact with
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drug molecules to form host-guest complexes. Due to their potency for such complexations, they
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are able to alter drug candidate properties resulting to better biological performance.5 Thus, the
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use of chemically modified CDs is extensively exploited in order to increase drug solubility,
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dissolution rate, bioavailability and stability.6-9 Some modified CDs, such as sulfobutylether-β-
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cyclodextrin (SBEβCD), are already approved for use in marketed drug products including
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intravenous voriconazole, amiodarone, ziprasidone, aripiprazole, and maropitant.10 Human
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exposure data based on Pfizer’s regulatory submission were derived from four clinical studies
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where SBEβCD was administered intravenously (i.v).10 In addition, experimental and theoretical
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research revealed the potential therapeutic applications of CD-formulated drugs and drug-like
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molecules in the fields of neuropathology and anesthesiology.11-13 Therefore, the role of CDs as
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drug delivery vectors assisting drugs to reach their target sites in the brain is highly relevant at the
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blood-brain barrier (BBB) level.
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Some CD-based PR formulations comprising SBEβCD and hydroxypropyl-β-cyclodextrin
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(HPβCD), have been developed to mitigate formulation-dependent problems. For example, a CD
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lipophilic core in which the PR molecule can form non-covalent complexes with SBEβCD in
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order to solubilize and stabilize this anesthetic agent.3 Only minor differences have been found in
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the pharmacokinetics and pharmacodynamics (PK/PD) between this type of formulation and the
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reference substance.4 The same scenario has been observed for PR complexed with HPβCD
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verified by recording the bioelectrical activity of the precentral cortex in rabbits.14 Other
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experimental results, however, indicate that the PR complexation with this CD allows for the
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improvement of the PR anesthetic activity via the elevation of induction time and sleeping time in
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rats.15 Despite all these data, the drug-delivery potential of PR complexed with modified CDs has 3 ACS Paragon Plus Environment
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not yet been thoroughly studied at the BBB level. Since it is important to come up with better
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methodologies for a development of the CD-based drug delivery, we characterized these PR/CD
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complexes and in vivo evaluated them at the BBB level. Moreover, molecular modeling
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techniques have been utilized in this study to investigate the complexation mechanisms of PR
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with SBEβCD and HPβCD in details.
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2. Materials and Methods
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2.1. Materials
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The PR, SBEβCD and HPβCD compounds were purchased from Sigma-Aldrich Productions
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GmbH (Steinheim am Albuch, Germany) and CyloLab Ltd. (Budapest, Hungary). The pure
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forms (98% purity) of PR with the SBEβCD and HPβCD (degree of substitution ~ 7.0)
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substances were prepared using the following technique: 1100 g of SBEβCD, 552 g and 50 g of
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HPβCD were dissolved in 4.0 or 2.4 L of distilled water and 200 ml of 0.01 M HCl aqueous
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solution. After complete dissolution, the solution was deoxygenated (sparged) with a stream of
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oxygen-free argon gas. 56 g, 39.2 g, and 3.4 g of PR were added and stirred for 3, 2, and 4 hours
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under argon gas. Next, the solution was then filtered through a 0.45 µm pore size membrane,
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frozen, lyophilized, ground in a mortar, and sieved. The formulated drug content was 4.8 w% for
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PR/SBEβCD and 6.6 w% for PR/HPβCD, respectively. 2% solution of PR (PR-LIPURO®) as a
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lipid emulsion (20 mg per ml) was purchased from Fresenius Kabi (Homburg vor der Höhe,
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Germany).
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2.2. Scanning electron microscopy
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All drug/CD powders as uncoated samples were examined under the Zeiss MERLIN VP
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Compact SEM (Scanning Electron Microscopy) microscope (Carl Zeiss AG, Oberkochen,
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Germany) with combined FESEM (Field Emission SEM) technology. 3D micrographs were
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taken using an SE2 detector under an accelerating voltage of 1.0 kV.
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2.3. Stability study of propofol and its CD complexes
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Complexes were dissolved in phosphate-buffered saline (PBS) to get a solution with the PR
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concentration of 1.0 mg*ml-1. PR-LIPURO® was used as a reference substance. The complex
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solutions and PR lipid emulsion were stored at room temperature for 24 hours. Samples were
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taken after 0, 1, 2, 4 and 24 hours and diluted 200 times with a water and methanol mixture at a 4 ACS Paragon Plus Environment
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ratio of 1:1. The PR amount was determined by HPLC-MS/MS (High-Performance Liquid
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Chromatography-tandem Mass Spectrometry).
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2.4. Solubility studies and determination of stability constants
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The solubility investigations were carried out according to the Higuchi & Connors method.16
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Solubilities were measured by adding an excess amount of PR to distilled water containing
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different amounts of PR/SBEβCD and PR/HPβCD. The suspension formed was equilibrated
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under continuous agitation for 24 h at 25 ± 3.0 °C and then filtered through a 0.45 µm nominal
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pore size PVDF (hydrophilic polyvinylidene fluoride) filter to yield a clear PR solution. The
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apparent stability constant (Kc) for PR/CD complexes was obtained from the slope of the phase-
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solubility diagram according to the following equation:
Kc = 140
slope S 0 (1 − slope)
(1)
where S0 is the saturation concentration of PR in the solvent without cyclodextrin.
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2.5. In vivo blood-brain barrier permeation studies
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All animal procedures and care were conducted in accordance with the Policy of Animal Care
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and Use Committee of Würzburg University. A total of 15 transgenic C57Bl/6 mice divided
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among three groups (n = 5 per group) were used for the in vivo BBB permeation experiments.
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General anesthesia was administered via retrobulbar injection with PR-LIPURO® or the drug/CD
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complexes using the PR dose range of 26 mg*kg-1 body weight according to the rodent anesthesia
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and analgesia formulary (Office of Regulatory Affairs, University of Pennsylvania, USA).
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Concerning the time for PR (Tmax = 2 min) at which this drug is present in its maximum
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concentration in serum after i.v injection,17 the blood was taken via intracardiac puncture after 2
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min followed by mouse brain harvesting. Prior to the brain extraction, an intracardiac perfusion
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was performed with 50 ml of PBS solution to wash the vascular system and also to get rid of
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residual blood in the brain. Mouse blood (300-400 µl) was collected in a 1.5 ml Eppendorf tube
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and heparinized with 3.0 µl of heparin sodium 5,000 I.U./ml (Ratiopharm, Ulm, Germany). Brain
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homogenates were prepared in glass dounce homogenizer as 40% homogenates (weight/volume)
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of the whole brain in PBS. Statistical analysis was performed using the GraphPad Prism v 18.0
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statistical software (GraphPad Software, Inc., La Jolla, CA, United States). Two-way ANOVA
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followed by Bonferroni post-test was used to analyze the difference between the two groups. 5 ACS Paragon Plus Environment
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Data was described as a mean ± standard deviation (SD). P < 0.01 was considered to be
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statistically significant.
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2.6. Quantitative determination of propofol and its CD complexes by HPLC-MS/MS
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The quantitative analyzes of blood and brain samples were performed on a Shimadzu HPLC
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system (Shimadzu Corporation, Kyoto, Japan) equipped with a binary pump, an autosampler, and
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a column oven with switching valve, coupled with a triple-quadrupole mass spectrometer. The
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HPLC-MS/MS analysis was controlled by Shimadzu LabSolutions Shimadzu 5.60 SP2 software.
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The animal blood and brain homogenized samples were mixed with 150-300 µl protein
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precipitator, vortexed for 30 seconds and centrifuged for 15 min at 15.000 rpm and a 10°C.
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Separation was achieved on a Kinetex EVO C18 (100 x 2,1 mm, 5 µm) column. The PR
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substance was eluted using a gradient mobile phase consisting of 10 mM ammonium carbonate
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buffer at pH 9.0 and methanol. The column temperature, injection volume, and flow rate
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parameters were set to 40˚C, 5.0 µl and 0.7 ml/min, respectively. The parametrized drug
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concentration in the brain (Cbrain) was calculated using the following equation:
∗ = ∗ 100%
= 100% −
(2) (3)
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∗ where is a drug concentration in the brain measured by HPLC-MS/MS; fublood is the
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predicted unbound drug fraction in the blood; and PPB is a percentage of plasma protein binding
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to PR. The decimal logarithm of the brain-to-blood concentration ratio (logBB) as a measure of
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drug permeation through the BBB was determined as: =
(4)
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where Cblood is the drug concentration in the blood measured by HPLC-MS/MS. The blood and
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brain drug recovery rates were 2.19% and 16.52%, respectively.
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2.7. Atomistic molecular dynamics (MD) simulations
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The two-dimensional (2D) chemical structure of PR was sketched and converted to its
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corresponding 3D form (Figure 1 [A]) using the MarvinSketch v.14.7.14.0 software (ChemAxon,
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Budapest, Hungary). The β-CD structure (ID: 1BFN) was obtained from the Protein Data Bank18
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to be used as a template for the construction of SBEβCD and HPβCD via a substitution of primary
186
hydroxyl groups with the appropriate radicals (Figure 1 [B]).
187 188
A
B
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Figure 1: Schematic representations of PR (A), SBEβCD (B; R = (CH2)4SO3H) and HPβCD (B;
205
R = (CH2)3OH) molecules. In the case of modified CD models, the primary hydroxyl groups are
206
substituted to match the experimentally determined substitution degree of ~7.0.
207 208
Molecular geometries were refined with the Gaussian 09 program, using the DFT (Density
209
Functional Theory) approach with the B3LYP/6-31G** basis set.19 All MD simulations were
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performed using the AMBER 12 package.20 The ROSETTA v.5.98 docking protocol21 was used
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to create the drug/CD complexes as an initial pose suitable for MD simulations. These simulations
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were performed by the Amber 12 package20 using the GAFF (General Amber Force Field) and
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GLYCAM_06j-1 force-fields for the PR and CD molecules. The atomic partial charges for the
214
SBEβCD and HPβCD molecules calculated by electrostatic potential (ESP) fitting are reported in
215
Supplementary Information (Figure S1). The systems were solvated with the TIP3P water models
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using the tLEaP input script available from the AmberTools. Long-range electrostatic interactions 7 ACS Paragon Plus Environment
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were applied via the PME (Particle-Mesh Ewald) method.22 The SHAKE algorithm23 was used to
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constrain the length of covalent bonds, including hydrogen atoms. The Langevin thermostat was
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implemented to equilibrate the temperature of the systems at 300 K. A 2.0 fs time step was used
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for all simulations. 20,000 steps and 2 ns time period were used for minimization and
221
equilibration with reference to all studied systems. Finally, 50 ns classical MD simulations with
222
no constraints were performed for each of the drug/CD complexes.
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2.8. Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) calculations
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The MM-PBSA method24,25 was implemented to estimate the total energy (Etot) for uncharged
226
PR (AH) into proton (H+) and negatively charged PR form (A−). The Gibbs free energy of binding
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(∆Gbind) values were determined by subtracting the total individual free energies of cyclodextrin
228
(∆GCD) and ligand (∆Glig) from the complex free energy (∆Gcomplex) as: ΔG = ΔG − (ΔG!" + ΔG$ ) ΔG = ΔH − TΔS
(5) (6)
229
The T∆S term is the entropy contribution, which can be predicted by quasi harmonic
230
approximation. The ∆H parameters can be represented as: ΔH = ΔE++ + ΔG,
(7)
231
where ∆EMM describes the molecular mechanics (MM) interaction energy between the protein
232
and the ligand, and ∆Gsol is the solvation free energy. ∆EMM is expressed by the following
233
equation: ΔE++ = ΔE + ΔE- . + ΔE/
(8)
234
where ∆Eelec, ∆EvdW and ∆Eint define electrostatic, van der Waals and internal including bond,
235
angle, dihedral, 1-4vdW and 1-4elec interaction energies, respectively. The solvation free energy
236
(∆Gsol) is divided into: ΔG, = ΔG + ΔG 012345
(9)
237
where ∆Gpolar defines the polar solvation energy calculated by Poisson-Boltzmann (PB) methods
238
using the PBSA module of AmberTools13,20 and ∆Gnon-polar describes non-polar solvation energy,
239
respectively.
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3. Results and Discussion
244
The feasibility of a pharmaceutical formulation, such as a drug/CD system, can be limited by
245
stability issues, especially in solution, where drugs are prone to hydrolysis and oxidation. Many
246
studies have shown that CDs have a stabilizing effect on diverse chemical compounds, including
247
steroid esters, alkylating anticancer agents, and prostaglandins, etc.26 The previous studies also
248
revealed that CDs can increase the drug’s physical stability, reduce evaporation of volatile
249
compounds, and reduce degradation in peptide and protein formulations.26 Therefore, it is
250
important to have information about the stability and drug degradation rate for complexes
251
obtained by mixing the PR with CDs as powders produced after lyophilization.
252 253
To achieve this goal, we performed the SEM technique with combined FESEM technology to produce 3D micrographs of the PR/SBEβCD and PR/HPβCD complexes shown in Figure 2.
254
255 256
Figure 2: Scanning electron micrographs of PR molecule complexed with SBEβCD and HPβCD
257
excipients. Multiple cavities and holes are shown with arrows.
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The images were captured with magnifications of 10 µm and 40 µm, so the uniformity or
260
variation over a large quantity of the powder and as well as the morphological details might be
261
observed. All the complexes were characterized by the presence of heterogeneous amorphous
262
granules composed of various sizes and shapes in the µm range. The microscopic images of
263
PR/SBEβCD also revealed a solid microstructure and morphology of these particles with no
264
cavities and holes, which could affect the chemical and mechanical stability. In contrast, the
265
PR/HPβCD complex was found to form the rough-edges and be highly porous aggregates.
266
Indeed, most of the non-porous microparticles, such as sorbents and micropellicular granules, are
267
generally more stable even at higher temperatures than conventional porous materials that are
268
prone to degradation.27,28
269
To investigate the complex stability in solution, the substances were dissolved in PBS to reach
270
the drug concentration of 1.0 mg*ml-1 and stored at room temperature for 24 hours to determine
271
their stability profiles by HPLC-MS/MS (Table 1). Figure 3 shows the lines as one-phase decay
272
curves produced by fitting to the stability data with a squared correlation coefficient (R2) from
273
0.93 to 0.99.
274 275 276 277
Table 1: Stability profiles of PR molecule complexed with SBEβCD and HPβCD after 24 hrs of
278
incubation at room temperature. Compound
Time (h) 0
1
2
Degradation rate -1
Half-life
4
24
k (h )
t1/2 (h)*
PR amount (%)
279
PR-LIPURO®
100
90.66
83.87
82.7
80.99
0.79
0.88
PR/SBEβCD
100
98.38
97.33
90.04
86.97
0.25
2.82
PR/HPβCD
100
94.13
91.12
81.41
79.31
0.37
1.88
*
- 67/9 = :
3;(9)
0 can cross the BBB readily, while drugs with a logBB < 0 12 ACS Paragon Plus Environment
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cannot.31 The highest drug permeation rate described by the logBB value was determined after the
348
retrobulbar injection of PR/SBEβCD (logBB = 0.73) in comparison to PR-LIPURO® (logBB =
349
0.69) indicating the slightly enhanced permeation of the complexed PR through the BBB (Figure
350
5 [A, B]).
351
A
353
4
360 361 362 363
Cblood (mg/ml)
1
2
1
0
0 C D
359
PR-LIPURO® PR/SBEβCD PR/HPβCD
0
PR /H Pβ
358
2
βC D
357
3
PR /S BE
356
3
blood brain
®
355
B
O
354
Concentration (mg/ml)
352
PR -L IP U R
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4
6
8
Cbrain (mg/ml)
364
Figure 5: The HPLC-MS/MS analysis of PR complexed with the SBEβCD and HPβCD
365
excipients and reference substance (PR-LIPURO®) in the blood and brain compartments after
366
retrobulbar injection using C57BL/6 mice (A). Data represent means ± SD (n = 5 animals per
367
group). p < 0.001 was determined by 2-way ANOVA followed by Bonferroni post-test. Positive
368
linear distribution patterns of the corresponding steady-state brain-to-plasma ratios for analyzed
369
compounds (B).
370 371
On the contrary, the PR/HPβCD compound was detected with lower logBB value (logBB =
372
0.58). Some earlier works have already reported the increase in drug transport across the BBB
373
that can be linked to the CD efficacy in cholesterol mobilization from brain endothelial cells and
374
the opening of tight junctions to potentiate the paracellular pathway.32 In addition to these
375
mechanisms, a relatively small amount (0.16%) of rhodamine labeled SBEβCD was detected
376
permeating the epithelial barrier probably via passive diffusion.33 However, the chemical
377
structure of hydrophilic CDs has the large number of hydrogen donors and acceptors, high
378
molecular weight (> 970 Da) and very low octanol-water partitioning coefficient (logP < −3.0), 13 ACS Paragon Plus Environment
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which are unfavorable for a successful permeation of biological membranes.34,35 Based on these
380
observations, it has been suggested that hydrophilic CDs enhance drug delivery though lipid
381
membranes by mainly increasing the availability of dissolved drug compound in an aqueous
382
phase at the membrane surface.36,37 Therefore, the possibility of the BBB transport of intact PR
383
complexes is only feasible in tiny amounts and in general highly unlikely.
384
Our in vivo BBB permeation results were also in agreement with the previously published
385
QSAR studies on the experimental and predicted determination of logBB for PR (logBB = 0.48 –
386
0.66) measured at steady state.38,39 Another PR formulation using SBEβCD (Captisol®) has
387
shown quite similar PK/PD to a lipid emulsion (Diprivan®) containing PR to allow its release
388
upon injection (Egan et al., 2003).4 On the other hand, some HPβCD complexes have been shown
389
to increase the CNS effects of dexamethasone, testosterone, and estradiol delivery after
390
intravenous injection in rats.40,41 Besides, the carbamazepine/SBEβCD complex resulted in
391
significantly higher anti-epileptic activity in mice as compared with the uncomplexed
392
anticonvulsant.42 Moreover, the use of SBEβCD as formulation entity to aid dissolution of the
393
general anesthetic alphaxalone avoids the major drawbacks related to hypersensitivity reactions
394
and opens up new possibilities to apply this drug in human anesthetic practice.43
395
In order to emphasize the PR ionization impact on the in vivo BBB permeation, we
396
investigated the PR dissociation mechanism of the uncharged form (AH) into a proton (H+) and
397
the negatively charged component (A−) according to the law of mass action. The energy analysis
398
was performed by the DFT method with the B3LYP/6-31G** level of theory. In this calculation,
399
the solvation energies of the individual components were estimated in Table 2.
400
Table 2: Energetic analysis of uncharged PR (AH) into proton (H+) and negatively charged PR
401
form (A−) using Density Functional Theory (DFT) method with the basis set of B3LYP/6-31G**
402
level of theory. Energya Esolv Esol phase
403
a
b
AH
H+
A−
−5.07
−115.01
−63.25
−340.98
−47.68
−340.67
- kcal*mol-1; b- *103
404 405
The results include the solution phase energy (Esol phase), since this term is, of course, essential
406
for solvation energy (Esolv) calculations. The possible PR protonation states in different pH values
407
were predicted using the Epik module of the Schrodinger molecular modeling package.44 14 ACS Paragon Plus Environment
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Solution-phase energies were determined by means of the implicit Poisson-Boltzmann model
409
using a polarizable continuum dielectric solvent. From the results, it is clear that the chemical
410
equilibrium is shifted to a formation of the anionic (A−) form (Esolv = −63.25 kcal*mol-1) of PR
411
with pKa of 11.1. The outcomes explicitly reveal the contribution of the free energy of solvation
412
by placing a charge on PR molecule (A−). The same conclusions were achieved by measuring the
413
total energy (Etot) values for each component with the MM method based on an implicit MM-
414
PBSA solvation model (Table 3).
415 416
Table 3: Energetic analysis of uncharged PR (AH) into proton (H+) and negatively charged PR
417
form (A−) using Molecular Mechanics (MM) method based on implicit MM-PBSA solvation
418
model.
419
Energya AH
H+
A−
Esolv
−3.19
−80.13
−84.46
Eelec
−9.19
0.0
−32.52
Etot
−2.98
−80.13
−106.57
a
- kcal*mol-1
420 421
It has been shown that positively charged molecules at the physiological pH tend to favor
422
BBB penetration, whereas negatively charged groups with pKa < 4.0 are unfavorable for this
423
process.45,46 While a neutral PR is obtained at the physiological pH, a deprotonated PR is more
424
pronounced at pH > 11. Indeed, PR as a weak organic acid remains almost entirely unionized
425
(99.97%) as a phenol form at pH of 7.4 and extensively bound to plasma proteins (PPB = 98%),
426
leaving only a small free fraction (fublood = 2%) of the drug concentration.47 It is also completely
427
and rapidly metabolized to sulfate and glucuronic conjugates with no anesthetic properties, which
428
are eliminated mainly by the kidneys.48 Therefore, once released from the CD cavity, the BBB
429
permeation rate for PR at the physiological pH is not hindered by its minor negatively charged
430
fraction in the blood (0.03%). Additionally, in order to study the effect of the CDs on the PR
431
conformational change that can be adapted into the cavity as well as their influence on the
432
binding property, the drug/CD complexes were subjected to 50 ns MD simulations with the MM-
433
PBSA approach in evaluating binding affinity. To match the experimentally determined
434
substitution degree of ~7.0, only the primary hydroxyl groups were substituted with
435
hydroxypropyl and sulfobutylether residues in the case of CD models. The main component of 15 ACS Paragon Plus Environment
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436
the conformational analysis is the clustering method in order to group together similar objects.
437
For this reason, it is necessary to apply a particular MD protocol to generate different clusters for
438
the various conformers and measure root-mean-square deviation (RMSD) values between the PR
439
conformations. Figure 6 explores the movement and conformational changes of the PR into the
440
SBEβCD (Figure 6 [A]) and HPβCD (Figure 6 [B]) binding cavities during the 50 ns MD
441
trajectory.
442 443
A
B
444 445 446 447 448 449 450 451 452
Figure 6: Clustering analysis of PR molecule complexed either with SBEβCD (A) or HPβCD (B)
453
during 50 ns MD simulation. All CD molecules are depicted using the solid surface representation
454
method with a probe radius of 0.4 Å, density isovalue of 0.7 and grid spacing of 0.5 to visualize
455
the binding cavity. CDs are colored according to their atom types. All hydrogen atoms are omitted
456
for clarity.
457 458
From the beginning of the MD trajectory, PR was well accommodated well into both systems,
459
and further analysis displayed more flexible PR within the SBEβCD (RMSD = 5.08 Å) in
460
comparison with the HPβCD (RMSD = 3.32 Å) molecule (see Videos S1 and S2, Supplementary
461
Information). A high degree of CD movements was also determined for a peripheral part of the
462
SBEβCD and HPβCD molecules due to an increase in the side group flexibility. The PR behavior
463
can be associated with the physicochemical properties and size of the SBEβCD structure and can
464
also be linked with the decreasing entropy. The latter aspect is often taken into account for
465
entropy-enthalpy compensation in experimental studies. However, the major drawback of
466
structural analysis is that the data cannot provide a quantitative information about the relative
467
energies of the different poses. 16 ACS Paragon Plus Environment
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468
Together with the structural analysis of the PR into the CDs, the energetic analysis was
469
performed throughout the MD simulations. Table 4 describes the estimated PR binding energy
470
profiles for modified CDs.
471
Table 4: Energetic analysis of PR molecule complexed with different cyclodextrins excipients
472
using MM-PBSA method. Energya
PR/SBEβCD
PR/HPβCD
∆Ebond
−1.49 ± 0.32
−1.55 ± 0.35
∆Eangle
−5.57 ± 0.25
−4.48 ± 0.55
∆Edihed
8.54 ± 0.03
11.51 ± 0.41
∆Evdw
−19.47 ± 0.09
−9.84 ± 0.33
∆E1-4vdw
0.75 ± 0.13
1.74 ± 0.44
∆E1-4elec
−6.24 ± 0.15
2.47 ± 0.23
∆Eelec
−12.95 ± 0.09
−50.47 ± 0.21
∆EMM
−36.43 ± 0.12
−50.62 ± 0.06
∆GPB
16.11 ± 0.01
35.92 ± 0.24
∆GNPb
−13.99 ± 0.13
−12.47 ± 0.03
∆GEDISPERc
23.41 ± 0.21
14.88 ± 0.18
∆Gsol
25.53 ± 0.24
38.32 ± 0.21
∆Eelec(tot)d
3.15
−14.55
∆H
−10.89 ± 0.05
−12.29 ± 0.32
T∆S
7.54
13.48
∆Gbind
−18.44
−25.78
- kcal*mol-1
473
a
474
b
475
c
476
d
- non-polar contribution to solvation energy from repulsive solute
- non-polar contribution to solvation energy from attractive solute - ∆Eelec(tot) = ∆Gelec + ∆GPB
477 478
The calculations show that the highest CD binding affinity to PR belongs to HPβCD (∆Gbind =
479
−25.78 kcal*mol-1) due to strong intermolecular forces, which also mean an increase in entropy
480
(T∆S = 13.48 kcal*mol-1). The predicted PR binding affinity to this CD might be explained by
481
the lower solubility of PR/HPβCD rather than PR/SBEβCD, resulting in less drug concentration
482
after complex solubilization in the blood and impairing the BBB permeation of PR. 17 ACS Paragon Plus Environment
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483
The binding affinity for the PR/SBEβCD complex (∆Gbind = −18.44 kcal*mol-1) was lower
484
compared with HPβCD, which is in-line with our in vivo BBB permeation experiments
485
explaining the highest logBB value as a result of the more rapid PR/SBEβCD dissociation in the
486
blood. Energy decomposition to individual terms suggested that van der Waals and lipophilicity
487
terms act more favorably toward binding for both systems. Since the unfavorable desolvation
488
penalty not fully compensated by the favorable electrostatics, the solvation energy, ∆Gsol, is
489
positive in both systems: 25.53 ± 0.24 kcal*mol-1 for PR/SBEβCD and 38.32 ± 0.21 kcal*mol-1
490
for PR/HPβCD suggesting that the electrostatic effects disfavor the PR binding to CDs.
491 492
4. Conclusions
493
In conclusion, in the current study, we analyzed the ability of PR to form inclusion complexes
494
with modified β-cyclodextrins, such as SBEβCD and HPβCD. The PR/SBEβCD and PR/HPβCD
495
formulations were prepared, characterized and their BBB permeation potentials have been
496
evaluated using the C57BL/6 mouse model for the purpose of controlled drug delivery. The
497
PR/SBEβCD complex was found to be more stable and soluable in water with k of 0.25 h-1, t1/2
498
of 2.82 h and Kc of 5.19*103 M-1 revealing a higher BBB permeability rate in comparison to PR-
499
LIPURO® because of maximal logBB value for the complex. In addition, PR binding affinity to
500
SBEβCD was established to be the lowest, judging by a maximal ∆Gbind of −18.44 kcal*mol-1,
501
which indicates the more rapid PR/SBEβCD dissociation. Overall, the results demonstrated that
502
SBEβCD as a formulating agent has the potential to enhance drug permeation across the BBB
503
and hence to improve PK/PD properties of general anesthetics at the BBB level.
504 505
Supporting Information
506
Figure S1 showing the atomic partial charges for the SBEβCD and HPβCD. Videos S1 and S2
507
showing MD trajectory frames of the PR within the SBEβCD and the HPβCD, respectively.
508
Acknowledgments
509
The authors are grateful to the BMBF (Bundesministerium für Bildung und Forschung) for the
510
support of this work by providing the grant (BMBF13N11801) to Jens Broscheit. This work was
511
partially supported by the Fundación Séneca del Centro de Coordinación de la Investigación de la
512
Región de Murcia under Project 18946/JLI/13 and by the Nils Coordinated Mobility under grant
513
012-ABEL-CM-2014A, in part financed by the European Regional Development Fund (ERDF).
514 18 ACS Paragon Plus Environment
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515
Conflicts of Interest
516
The authors declare no conflict of interest.
517 518
Reference
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