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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]

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Tel.: +49-931-2013-0016; Fax: +49-931-2013-0019.

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Graphical Abstract:

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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-

41

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

67

alkyl phenol group of compounds.1 This drug is a preferred agent for day-patient surgeries due to

68

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

70

reactions, and support of microbial growth.4 Thus, development of safer formulations is of great

71

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

75

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

83

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

100

techniques have been utilized in this study to investigate the complexation mechanisms of PR

101

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.

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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

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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

220

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

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PR (AH) into proton (H+) and negatively charged PR form (A−). The Gibbs free energy of binding

227

(∆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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2

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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408

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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Page 16 of 21

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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(1) Sebel, P. S.; Lowdon, J. D., Propofol - a New Intravenous Anesthetic. Anesthesiology 1989, 71, 260-277. (2) Duke, T., A new intravenous anesthetic agent: propofol. Can. Vet. J. 1995, 36 , 181-183. (3) Wallentine, C. B.; Shimode, N.; Egan, T. D.; Pace, N. L. Propofol in a Modified Cyclodextrin Formulation: First Human Study of Dose-Response with Emphasis on Injection Pain. Anesth. Analg. 2011, 113, 738–741. (4) Egan, T. D.; Kern, S. E.; Johnson, K. B.; Pace, N. L. The Pharmacokinetics and Pharmacodynamics of Propofol in a Modified Cyclodextrin Formulation (Captisol) versus Propofol in a Lipid Formulation (Diprivan): An Electroencephalographic and Hemodynamic Study in a Porcine Model. Anesth. Analg. 2003, 97, 72–79. (5) Stella, V. J.; He, Q. Cyclodextrins. Toxicol. Pathol. 2008, 36, 30–42. (6) Uekama, K.; Otagiri, M. Cyclodextrins in Drug Carrier Systems. Crit. Rev. Ther. Drug Carrier Syst. 1987, 3, 1–40. (7) Szejtli, J., Helical and Cyclic Structures in Starch Chemistry. Acs Sym Ser 1991, 458, 210. (8) Loftsson, T.; Brewster, M. E. Pharmaceutical Applications of Cyclodextrins. 1. Drug Solubilization and Stabilization. J. Pharm. Sci. 1996, 85, 1017–1025. (9) Rajewski, R. A.; Stella, V. J. Pharmaceutical Applications of Cyclodextrins. 2. In Vivo Drug Delivery. J. Pharm. Sci. 1996, 85, 1142–1169. (10) Luke, D. R.; Tomaszewski, K.; Damle, B.; Schlamm, H. T. Review of the Basic and Clinical Pharmacology of Sulfobutylether-Beta-Cyclodextrin (SBECD). J. Pharm. Sci. 2010, 99, 3291–3301. (11) Shityakov, S.; Sohajda, T.; Puskas, I.; Roewer, N.; Forster, C.; Broscheit, J. A., Ionization States, Cellular Toxicity and Molecular Modeling Studies of Midazolam Complexed with Trimethyl-beta-cyclodextrin. Molecules 2014, 19 , 16861-16876. (12) Shityakov, S.; Puskas, I.; Papai, K.; Salvador, E.; Roewer, N.; Forster, C.; Broscheit, J. A., Sevoflurane-Sulfobutylether-beta-Cyclodextrin Complex: Preparation, Characterization, Cellular Toxicity, Molecular Modeling and Blood-Brain Barrier Transport Studies. Molecules 2015, 20, 10264-10279. (13) Shan, L.; Tao, E. X.; Meng, Q. H.; Hou, W. X.; Liu, K.; Shang, H. C.; Tang, J. B.; Zhang, W. F. Formulation, Optimization, and Pharmacodynamic Evaluation of Chitosan/phospholipid/beta-Cyclodextrin Microspheres. Drug Des. Devel. Ther. 2016, 10, 417– 429. (14) Viernstein, H.; Stumpf, C.; Spiegl, P.; Reiter, S. Preparation and Central Action of Propofol/hydroxypropyl-Beta -Cyclodextrin Complexes in Rabbits. Arzneimittelforschung 1993, 43, 818–821. (15) Trapani, G.; Latrofa, a; Franco, M.; Lopedota, a; Sanna, E.; Liso, G. Inclusion Complexation of Propofol with 2-Hydroxypropyl-Beta-Cyclodextrin. Physicochemical, Nuclear Magnetic Resonance Spectroscopic Studies, and Anesthetic Properties in Rat. J. Pharm. Sci. 1998, 87, 514–518. 19 ACS Paragon Plus Environment


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559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

Page 20 of 21

(16) Higuchi, T.; Connors, K. A. Phase Solubility Techniques. Adv. Anal. Chem. Instrum. 1965, 4, 117–212. (17) Paul, M.; Dueck, M.; Kampe, S.; Fruendt, H.; Kasper, S. M. Pharmacological Characteristics and Side Effects of a New Galenic Formulation of Propofol without Soyabean Oil. Anaesthesia 2003, 58, 1056–1062. (18) Adachi, M.; Mikami, B.; Katsube, T.; Utsumi, S. Crystal Structure of Recombinant Soybean Beta-Amylase Complexed with Beta-Cyclodextrin. J. Biol. Chem. 1998, 273, 19859– 19865. (19) Ditchfield, R. Self-Consistent Molecular-Orbital Methods. IX. An Extended GaussianType Basis for Molecular-Orbital Studies of Organic Molecules. J. Chem. Phys. 1971, 54, 724. (20) Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. The Amber Biomolecular Simulation Programs. J. Comput. Chem. 2005, 26, 1668–1688. (21) Das, R.; Baker, D. Macromolecular Modeling with Rosetta. Annu. Rev. Biochem. 2008, 77, 363–382. (22) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem .Phys. 1995, 103, 8577–8593. (23) Miyamoto, S.; Kollman, P. A. SETTLE: An Analytical Version of the SHAKE and RATTLE Algorithm for Rigid Water Models. J. Comput. Chem. 1992, 13, 952–962. (24) Gohlke, H.; Case, D. A. Converging Free Energy Estimates: MM-PB(GB)SA Studies on the Protein-Protein Complex Ras-Raf. J. Comput. Chem. 2004, 25, 238–250. (25) Bea, I.; Cervello, E.; Kollman, P. A.; Jaime, C. Molecular Recognition by B-Cyclodextrin Derivatives: FEP vs MM/PBSA Study. Comb. Chem. High Throughput Screen. 2001, 4, 605– 611. (26) Rasheed, A.; Kumar, A.; Sravanthi, V. V. N. S. S. Cyclodextrins as Drug Carrier Molecule: A Review. Sci. Pharm. 2008, 76, 567–598. (27) Kastner, M. Protein Liquid Chromatography, Elsevier, Amsterdam, 2000 (28) Cazes, J. Encyclopedia of Chromatography. Crop Sci. 2010, 2, 1–2457. (29) Thompson, D.; Mosher, G. Formulations containing propofol and a sulfoalkyl ether cyclodextrin. US 20030073665 A1, April 17, 2003. (30) Penkler, L. G. Freeze- dried pharmaceutically acceptable inclusion complexes of propofol and cyclodextrin. CA 2474710 A1, August 7, 2003. (31) Ajay; Bemis, G. W.; Murcko, M. A. Designing Libraries with CNS Activity. J. Med. Chem. 1999, 42, 4942–4951. (32) Tilloy, S.; Monnaert, V.; Fenart, L.; Bricout, H.; Cecchelli, R.; Monflier, E. Methylated βCyclodextrin as P-Gp Modulators for Deliverance of Doxorubicin across an in Vitro Model of Blood-Brain Barrier. Bioorganic Med. Chem. Lett. 2006, 16, 2154–2157. (33) Juluri, A.; Narasimha Murthy, S. Transdermal Iontophoretic Delivery of a Liquid Lipophilic Drug by Complexation with an Anionic Cyclodextrin. J. Control. Release. 2014, 189, 11–18. (34) Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability. Pharm. Res. An Off. J. Am. Assoc. Pharm. Sci. 1995, 12, 413–420. (35) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development settings1. Adv. Drug Deliv. Rev. 2001, 46, 3–26. 20 ACS Paragon Plus Environment

Page 21 of 21

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

605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640


(36) Loftsson, T.; Masson, M. Cyclodextrins in Topical Drug Formulations: Theory and Practice. Int. J. Pharm. 2001, 225, 15–30. (37) Loftsson, T.; Konrádsdóttir, F.; Másson, M. Influence of Aqueous Diffusion Layer on Passive Drug Diffusion from Aqueous Cyclodextrin Solutions through Biological Membranes. Pharmazie. 2006, 61, 83–89. (38) Rogers, D.; Hopfinger, A. J. Application of Genetic Function Approximation to Quantitative Structure-Activity Relationships and Quantitative Structure-Property Relationships. J. Chem. Inf. Comput. Sci. 1994, 34, 854–866. (39) Shen, J.; Du, Y.; Zhao, Y.; Liu, G.; Tang, Y. In Silico Prediction of Blood-Brain Partitioning Using a Chemometric Method Called Genetic Algorithm Based Variable Selection. QSAR Comb. Sci. 2008, 27, 704–717. (40) Brewster, M. E.; Estes, K. S.; Loftsson, T.; Perchalski, R.; Derendorf, H.; Mullersman, G.; Bodor, N. Improved Delivery through Biological Membranes. XXXL: Solubilization and Stabilization of an Estradiol Chemical Delivery System by Modified Beta-Cyclodextrins. J. Pharm. Sci. 1988, 77, 981–985. (41) Anderson, W. R.; Simpkins, J. W.; Brewster, M. E.; Bodor, N., Brain-enhanced Delivery of Testosterone Using a Chemical Delivery System Complexed with 2-hydroxypropyl-betacyclodextrin. Drug Des. Discovery. 1988, 2, 287-298. (42) Jain, A. S.; Date, A. A.; Pissurlenkar, R. R. S.; Coutinho, E. C.; Nagarsenker, M. S. Sulfobutyl Ether(7) Beta-Cyclodextrin (SBE7 Beta-CD) Carbamazepine Complex: Preparation, Characterization, Molecular Modeling, and Evaluation of In Vivo Anti-Epileptic Activity. Aaps Pharmscitech. 2011, 12, 1163–1175. (43) Goodchild, C. S.; Serrao, J. M.; Kolosov, A.; Boyd, B. J. Alphaxalone Reformulated: A Water-Soluble Intravenous Anesthetic Preparation in Sulfobutyl-Ether-beta-Cyclodextrin. Anesth. Analg. 2015, 120, 1025-1031. (44) Greenwood, J. R.; Calkins, D.; Sullivan, A. P.; Shelley, J. C. Towards the Comprehensive, Rapid, and Accurate Prediction of the Favorable Tautomeric States of Drug-like Molecules in Aqueous Solution. J. Comput.-Aided Mol. Des. 2010, 24, 591–604. (45) Fischer, H.; Gottschlich, R.; Seelig, A. Blood-Brain Barrier Permeation: Molecular Parameters Governing Passive Diffusion. J. Membr. Biol. 1998, 165, 201–211. (46) Pajouhesh, H.; Lenz, G. R. Medicinal Chemical Properties of Successful Central Nervous System Drugs. NeuroRx 2005, 2, 541–553. (47) Servin F, Desmonts JM, Haberer JP, Cockshott ID, Plummer GF, F. R. Pharmacokinetics and Protein Binding of Propofol in Patients with Cirrhosis. Anesthesiology 1988, 69, 887–891. (48) Bowman, W. C. Pharmacology of intravenous anaesthetics and hypnotics in general anaesthesia. 5th ed.; Butterworths: London, 1989.

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