Characterization, in Vivo Evaluation, and Molecular Modeling of

Article pubs.acs.org/jcim

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,§ Maria Josefa Yáñez-Gascón,∥ Horacio Pérez-Sánchez,∥ István Puskás,⊥ Norbert Roewer,†,§ Carola Förster,† and Jens-Albert Broscheit†,§ †

Department of Anesthesia and Critical Care, University of Würzburg, 97080 Würzburg, Germany Department of Biophysics, School of Medicine, Bahcesehir University, 34349 Istanbul, Turkey § Sapiotec Ltd., 97078 Würzburg, Germany ∥ Universidad Católica San Antonio de Murcia (UCAM), 30107 Guadalupe, Spain ⊥ CycloLab Cyclodextrin Research & Development Laboratory Ltd., H-1097 Budapest, Hungary

J. Chem. Inf. Model. 2016.56:1914-1922. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 01/08/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: In this study, we investigated the ability of the general anesthetic propofol (PR) to form inclusion complexes with modified β-cyclodextrins, including sulfobutylether-β-cyclodextrin (SBEβCD) and hydroxypropyl-β-cyclodextrin (HPβCD). The PR/SBEβCD and PR/HPβCD complexes were prepared and characterized, and the blood−brain barrier (BBB) permeation potential of the formulated PR was examined in vivo for the purpose of controlled drug delivery. The PR/SBEβCD complex was found to be more stable in solution with a minimal degradation constant of 0.25 h−1, a t1/2 of 2.82 h, and a Kc of 5.19 × 103 M−1 and revealed higher BBB permeability rates compared with the reference substance (PR-LIPURO) considering the calculated brain-to-blood concentration ratio (logBB) values. Additionally, the diminished PR binding affinity to SBEβCD was confirmed in molecular dynamics simulations by a maximal Gibbs free energy of binding (ΔGbind = −18.44 kcal·mol−1), indicating the more rapid PR/SBEβCD dissociation. Overall, the results demonstrated that SBEβCD has the potential to be used as a prospective candidate for drug delivery vector development to improve the pharmacokinetic and pharmacodynamic properties of general anesthetic agents at the BBB level.

1. INTRODUCTION 2,6-Diisopropylphenol or propofol (PR) is an intravenous general anesthetic classed with the alkyl phenol group of compounds.1 This drug is a preferred agent for day-patient surgeries because of its rapid metabolism and reduced postanesthetic nausea.2,3 The currently available formulation of PR in the market is a lipid emulsion that has side effects such as pain on injection, serious allergic reactions, and support of microbial growth.4 Thus, the development of safer formulations is of great interest in biomedicine. For instance, lipid-free preparations of PR are being developed to reduce these formulation-related problems. One method employed to generate similar formulations is the use of natural cyclodextrins (CDs) and their derivatives. © 2016 American Chemical Society

CDs are cyclic oligosaccharides made up of six to eight dextrose units and can interact with drug molecules to form host/guest complexes. Because of their potency for such complexations, they are able to alter drug candidate properties, resulting in better biological performance.5 Thus, the use of chemically modified CDs is extensively exploited in order to increase drug solubility, dissolution rate, bioavailability, and stability.6−9 Some modified CDs, such as sulfobutylether-βcyclodextrin (SBEβCD), are already approved for use in marketed drug products, including intravenous voriconazole, amiodarone, ziprasidone, aripiprazole, and maropitant.10 Received: May 31, 2016 Published: September 2, 2016 1914

DOI: 10.1021/acs.jcim.6b00215 J. Chem. Inf. Model. 2016, 56, 1914−1922

Article

Journal of Chemical Information and Modeling

temperature for 24 h. Samples were taken after 0, 1, 2, 4, and 24 h and diluted 200 times with a 1:1 water/methanol mixture. The amount of PR was determined by high-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/ MS). 2.4. Solubility Studies and Determination of Stability Constants. The solubility investigations were carried out according to the method of Higuchi and Connors.16 Solubilities were measured by adding an excess amount of PR to distilled water containing different amounts of SBEβCD and HPβCD. The suspension formed was equilibrated under continuous agitation for 24 h at 25 ± 3.0 °C and then filtered through a 0.45 μm nominal pore size hydrophilic poly(vinylidene fluoride) (PVDF) filter to yield a clear PR solution. The apparent stability constant (Kc) for the PR/CD complex was obtained from the slope of the phase-solubility diagram according to the following equation:

Human exposure data based on Pfizer’s regulatory submission were derived from four clinical studies where SBEβCD was administered intravenously (iv).10 In addition, experimental and theoretical research has revealed the potential therapeutic applications of CD-formulated drugs and drug-like molecules in the fields of neuropathology and anesthesiology.11−13 Therefore, the role of CDs as drug delivery vectors assisting drugs to reach their target sites in the brain is highly relevant at the blood−brain barrier (BBB) level. Some CD-based PR formulations comprising SBEβCD and hydroxypropyl-β-cyclodextrin (HPβCD) have been developed to mitigate formulation-dependent problems. For example, a CD lipophilic core in which the PR molecule can form noncovalent complexes with SBEβCD in order to solubilize and stabilize this anesthetic agent has been reported.3 Only minor differences have been found in the pharmacokinetics (PK) and pharmacodynamics (PD) between this type of formulation and the reference substance.4 The same scenario has been observed for PR complexed with HPβCD, as verified by recording the bioelectrical activity of the precentral cortex in rabbits.14 Other experimental results, however, indicate that PR complexation with this CD allows for the improvement of the PR anesthetic activity via the elevation of induction time and sleeping time in rats.15 Despite all of these data, the drug delivery potential of PR complexed with modified CDs has not yet been thoroughly studied at the BBB level. Since it is important to come up with better methodologies for the development of CD-based drug delivery, we characterized these PR/CD complexes and evaluated them in vivo at the BBB level. Moreover, molecular modeling techniques were utilized in this study to investigate the complexation mechanisms of PR with SBEβCD and HPβCD in detail.

Kc =

slope S0(1 − slope)

(1)

where S0 is the saturation concentration of PR in the solvent without cyclodextrin. 2.5. In Vivo Blood−Brain Barrier Permeation Studies. All animal procedures and care were conducted in accordance with the Policy of Animal Care and Use Committee of Würzburg University. A total of 15 transgenic C57Bl/6 mice divided among three groups (n = 5 per group) were used for the in vivo BBB permeation experiments. General anesthesia was administered via retrobulbar injection with PR-LIPURO or the drug/CD complex using the PR dose range of 26 mg·(kg of body weight)−1 according to the rodent anesthesia and analgesia formulary (Office of Regulatory Affairs, University of Pennsylvania, USA). Concerning the time for PR at which this drug is present in its maximum concentration in serum after iv injection (Tmax = 2 min),17 the blood was taken via intracardiac puncture after 2 min followed by mouse brain harvesting. Prior to the brain extraction, an intracardiac perfusion was performed with 50 mL of PBS solution to wash the vascular system and also to get rid of residual blood in the brain. Mouse blood (300−400 μL) was collected in a 1.5 mL Eppendorf tube and heparinized with 3.0 μL of 5000 IU· mL−1 heparin sodium (Ratiopharm, Ulm, Germany). Brain homogenates were prepared in a glass Dounce homogenizer as 40% (w/v) homogenates of the whole brain in PBS. Statistical analysis was performed using the GraphPad Prism v.5.01 statistical software (GraphPad Software, Inc., La Jolla, CA, United States). Two-way analysis of variance (ANOVA) followed by Bonferroni post-test was used to analyze the difference between the two groups. Data were described as mean ± standard deviation (SD), and p < 0.01 was considered to be statistically significant. 2.6. Quantitative Determination of Propofol and Its CD Complexes by HPLC−MS/MS. The quantitative analyses of blood and brain samples were performed on a Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a binary pump, an autosampler, and a column oven with switching valve, coupled with a triple-quadrupole mass spectrometer. The HPLC−MS/MS analysis was controlled by Shimadzu LabSolutions Shimadzu 5.60 SP2 software. The animal blood and brain homogenized samples were mixed with 150−300 μL of protein precipitator, vortexed for 30 s, and centrifuged for 15 min at 15 000 rpm and 10 °C. Separation

2. MATERIALS AND METHODS 2.1. Materials. The PR, SBEβCD, and HPβCD compounds were purchased from Sigma-Aldrich Productions GmbH (Steinheim am Albuch, Germany) and CycloLab Ltd. (Budapest, Hungary). The pure forms (98% purity) of PR with SBEβCD and HPβCD (degree of substitution ∼7.0) were prepared using the following technique: 1100 g of SBEβCD or 552 or 50 g of HPβCD was dissolved in 4.0 or 2.4 L of distilled water and 200 mL of 0.01 M HCl aqueous solution. After complete dissolution, the solution was deoxygenated (sparged) with a stream of oxygen-free argon gas. Then 56, 39.2, or 3.4 g of PR, respectively, was added, and the resulting mixture was stirred for 3, 2, or 4 h, respectively, under argon gas. Next, the solution was filtered through a 0.45 μm pore size membrane, frozen, lyophilized, ground in a mortar, and sieved. The formulated drug content was 4.8 wt % for PR/SBEβCD and 6.6 wt % for PR/HPβCD. A 2% preparation of PR (PR-LIPURO) as a lipid emulsion (20 mg·mL−1) was purchased from Fresenius Kabi (Homburg vor der Höhe, Germany). 2.2. Scanning Electron Microscopy. All drug/CD powders as uncoated samples were examined under Field Emission Scanning Electron Microscopy (FESEM) using Merlin VP Compact (Carl Zeiss Microscopy, Oberkochen, Germany). Micrographs were taken using an SE2 detector under an accelerating voltage of 1.0 kV. 2.3. Stability Study of Propofol and Its CD Complexes. Complexes were dissolved in phosphate-buffered saline (PBS) to get solutions with PR concentrations of 1.0 mg·mL−1. PRLIPURO was used as a reference substance. The complex solutions and PR lipid emulsion were stored at room 1915


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Figure 1. Schematic representations of (A) PR and (B) SBEβCD (R = (CH2)4SO3H) and HPβCD (R = (CH2)3OH) molecules. In the case of the modified CD models, the primary hydroxyl groups are substituted to match the experimentally determined substitution degree of ∼7.0.

was achieved on a Kinetex EVO C18 column (100 mm × 2.1 mm, 5 μm). The PR substance was eluted using a gradient mobile phase consisting of 10 mM ammonium carbonate buffer at pH 9.0 and methanol. The column temperature, injection volume, and flow rate parameters were set to 40 °C, 5.0 μL, and 0.7 mL/min, respectively. The parametrized drug concentration in the brain (Cbrain) was calculated using the following equation: C brain =

* C brain × 100% fublood

Molecular geometries were refined with the Gaussian 09 program using density functional theory (DFT) with the B3LYP functional and the 6-31G** basis set.19 All of the MD simulations were performed using the AMBER 12 package.20 The ROSETTA v.5.98 docking protocol21 was used to create each drug/CD complex as an initial pose suitable for MD simulations. These simulations were performed using the Amber 12 package20 with the general Amber force field (GAFF) for PR and the GLYCAM_06j-1 force field for the CD molecules. The atomic partial charges for the SBEβCD and HPβCD molecules calculated by electrostatic potential (ESP) fitting are reported in Figure S1. The systems were solvated with the TIP3P water models using the tLEaP input script available in AmberTools. Long-range electrostatic interactions were applied via the particle-mesh Ewald (PME) method.22 The SHAKE algorithm23 was used to constrain the lengths of covalent bonds including hydrogen atoms. The Langevin thermostat was implemented to equilibrate the temperature of the systems at 300 K. A 2.0 fs time step was used for all of the simulations, and 20 000 steps and a 2 ns time period were used for minimization and equilibration for all of the studied systems. Finally, 50 ns classical MD simulations with no constraints were performed for each of the drug/CD complexes. 2.8. Molecular Mechanics Poisson−Boltzmann Surface Area (MM-PBSA) Calculations. The MM-PBSA method24,25 was implemented to estimate the total energy (Etot) for conversion of uncharged PR (AH) into a proton (H+) and the negatively charged form of PR (A−). Values of the Gibbs free energy of binding (ΔGbind) were determined by subtracting the total individual free energies of cyclodextrin (ΔGCD) and ligand (ΔGlig) from the free energy of the complex (ΔGcomplex) as follows:

(2)

where C*brain is the drug concentration in the brain measured by HPLC−MS/MS and fublood is the predicted unbound drug fraction in the blood, given by fublood = 100% − PPB

(3)

where PPB is the percentage of plasma protein binding to PR. The decimal logarithm of the brain-to-blood concentration ratio (logBB), as a measure of drug permeation through the BBB, was determined as

⎞ ⎛C logBB = log⎜ brain ⎟ ⎝ C blood ⎠

(4)

where Cblood is the drug concentration in the blood measured by HPLC−MS/MS. The blood and brain drug recovery rates were 2.19% and 16.52%, respectively. 2.7. Atomistic Molecular Dynamics (MD) Simulations. The two-dimensional (2D) chemical structure of PR was sketched and converted to its corresponding 3D form (Figure 1A) using the MarvinSketch v.14.7.14.0 software (ChemAxon, Budapest, Hungary). The β-CD structure (PDB ID 1BFN) was obtained from the Protein Data Bank18 to be used as a template for the construction of SBEβCD and HPβCD via a substitution of primary hydroxyl groups with the appropriate radicals (Figure 1B).

ΔG bind = ΔGcomplex − (ΔGCD + ΔG lig )

(5)

ΔGbind can also be expressed as follows: 1916


Article

Journal of Chemical Information and Modeling ΔG bind = ΔH − T ΔS

as well as the morphological details might be observed. All of the complexes were characterized by the presence of heterogeneous amorphous granules composed of various sizes and shapes in the micrometer range. The microscopic images of PR/SBEβCD also revealed a solid microstructure and morphology of these particles with no cavities and holes, which could affect the chemical and mechanical stability. In contrast, the PR/HPβCD complex was found to form rough edges and exist as highly porous aggregates. Indeed, most of the nonporous microparticles, such as sorbents and micropellicular granules, are generally more stable even at higher temperatures than conventional porous materials that are prone to degradation.27,28 To investigate the stability of the complexes in solution, the substances were dissolved in PBS to reach the drug concentration of 1.0 mg·mL−1 and stored at room temperature for 24 h to determine their stability profiles by HPLC−MS/MS (Table 1). Figure 3 shows the lines as one-phase decay curves produced by fitting to the stability data with a squared correlation coefficient (R2) of 0.93 to 0.99. As expected, PR/ SBEβCD was found to be more stable in PBS and more resistant to degradation under ambient conditions than the other tested compounds. This complex underwent degradation with a lowest pseudo-first-order rate constant (k) of 0.25 h−1, corresponding to a maximal degradation half-life (t1/2) of 2.82 h with a residual PR concentration of 0.87 mg·mL−1 after 24 h. These data demonstrate that the PR/SBEβCD complex is chemically more stable that the other analyzed compounds in the liquid phase in a PBS solution, which could also be true for its solid phase formed as a nonporous material. The experimental quantitation of the strength of PR binding to the CDs was also determined from the solubility isotherms of the PR/SBEβCD and PR/HPβCD complexes in distilled water (Figure 4). An estimate of the stability constant Kc was calculated using the actual measured S0 value of 0.67 mM, showing the increase in apparent stability for PR/SBEβCD (Kc = 5.19 × 103 M−1) in contrast with the PR/HPβCD complex (Kc = 1.94 × 103 M−1). Other observations from previous complexation studies of PR also determined similar Kc values of about 3.8 × 103 to 4.8 × 103 M−1 for PR/SBEβCD29 and 2.94 × 103 M−1 for PR/HPβCD,30 which correlate well with our solubility data. Next, the drug/CD complexes were evaluated in vivo to permeate the BBB via measurement of the concentration of PR, a fast onset/offset iv anesthetic, in the blood and brain compartments by HPLC−MS/MS. Because of the highly lipophilic nature of the brain, the C*brain values in some brain samples were not determined correctly by the HPLC−MS/MS methodology, and these were excluded from further analysis. Notably, all of the analyzed complexes were able to induce general anesthesia 30−60 s after injection. Both formulations, PR/SBEβCD and PR/HPβCD, were generally well tolerated by the animals. According to the central nervous system (CNS) ± activity classification for different compounds, molecules with logBB > 0 can cross the BBB readily, while drugs with logBB < 0 cannot.31 The highest drug permeation rate described by the logBB value was determined after the retrobulbar injection of PR/SBEβCD (logBB = 0.73) in comparison with PR-LIPURO (logBB = 0.69), indicating the slightly enhanced permeation of the complexed PR through the BBB (Figure 5). On the contrary, the PR/HPβCD compound was detected with a lower logBB value (logBB = 0.58). Some earlier works

(6)

The TΔS term is the entropy contribution, which can be predicted using the quasi-harmonic approximation. The ΔH parameter can be represented as ΔH = ΔEMM + ΔGsol

(7)

where ΔEMM describes the molecular mechanics (MM) interaction energy between the protein and the ligand and ΔGsol is the solvation free energy. ΔEMM is expressed by the following equation: ΔEMM = ΔEelec + ΔEvdW + ΔEint

(8)

where ΔEelec, ΔEvdW, and ΔEint define electrostatic, van der Waals, and internal (including bond, angle, dihedral, 1−4 vdW, and 1−4 electrostatic) interaction energies, respectively. The solvation free energy (ΔGsol) is divided into two terms: ΔGsol = ΔGpolar + ΔGnonpolar

(9)

where ΔGpolar defines the polar solvation energy calculated by the Poisson−Boltzmann (PB) method using the PBSA module of AmberTools1320 and ΔGnonpolar describes the nonpolar solvation energy.

3. RESULTS AND DISCUSSION The feasibility of a pharmaceutical formulation, such as a drug/ CD system, can be limited by stability issues, especially in solution, where drugs are prone to hydrolysis and oxidation. Many studies have shown that CDs have a stabilizing effect on diverse chemical compounds, including steroid esters, alkylating anticancer agents, prostaglandins, etc.26 The previous studies also revealed that CDs can increase the drug’s physical stability, reduce evaporation of volatile compounds, and reduce degradation in peptide and protein formulations.26 Therefore, it is important to have information about the stability and drug degradation rate for complexes obtained by mixing PR with CDs as powders produced after lyophilization. To achieve this goal, we used the SEM technique with combined FESEM technology to produce 3D micrographs of the PR/SBEβCD and PR/HPβCD complexes (Figure 2). The images were captured with magnifications of 10 and 40 μm, so the uniformity or variation over a large quantity of the powder

Figure 2. Scanning electron micrographs of PR complexed with SBEβCD and HPβCD excipients. Multiple cavities and holes are shown with arrows. 1917


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Table 1. Stability Profiles of PR Complexed with SBEβCD and HPβCD after 24 h of Incubation at Room Temperature PR content (%)

a

compound

0h

1h

2h

4h

24 h

k (h−1)a

t1/2 (h)b

PR-LIPURO PR/SBEβCD PR/HPβCD

100 100 100

90.66 98.38 94.13

83.87 97.33 91.12

82.7 90.04 81.41

80.99 86.97 79.31

0.79 0.25 0.37

0.88 2.82 1.88

Degradation rate constant. bHalf-life, given by t1/2 =

ln(2) . k

Our in vivo BBB permeation results were also in agreement with the previously published quantitative structure−activity relationship studies on the experimental and predicted determination of logBB for PR (logBB = 0.48−0.66) measured at steady state.38,39 Another PR formulation using SBEβCD (Captisol) has shown quite similar PK/PD to a lipid emulsion (Diprivan) containing PR to allow its release upon injection.4 On the other hand, some HPβCD complexes have been shown to increase the CNS effects of dexamethasone, testosterone, and estradiol delivery after intravenous injection in rats.40,41 Besides, the carbamazepine/SBEβCD complex resulted in significantly higher antiepileptic activity in mice compared with the uncomplexed anticonvulsant.42 Moreover, the use of SBEβCD as a formulation entity to aid dissolution of the general anesthetic alphaxalone avoids the major drawbacks related to hypersensitivity reactions and opens up new possibilities to apply this drug in human anesthetic practice.43 In order to emphasize the impact of PR ionization on the in vivo BBB permeation, we investigated the PR dissociation mechanism of the uncharged form (AH) into a proton (H+) and the negatively charged component (A−) according to the law of mass action. The energy analysis was performed by DFT at the B3LYP/6-31G** level of theory. In this calculation, the solvation energies of the individual components were estimated as shown in Table 2. The results include the solution-phase energy (Esol phase), since this term is, of course, essential for solvation energy (E solv) calculations. The possible PR protonation states at different pH values were predicted using the Epik module of the Schrödinger molecular modeling package.44 Solution-phase energies were determined by means of the implicit Poisson−Boltzmann model using a polarizable continuum dielectric solvent. From the results, it is clear that the chemical equilibrium is shifted toward the formation of A− (Esolv = −63.25 kcal·mol−1) with pKa = 11.1. The outcomes explicitly reveal the contribution of the free energy of solvation by placing a charge on PR molecule (A−). The same conclusions were achieved by measuring the total energy

Figure 3. Stability profiles determined by HPLC−MS/MS for PR complexed with either SBEβCD or HPβCD in comparison with the uncomplexed reference substance (PR-LIPURO) after 24 h of incubation at room temperature. The lines show fits of one-phase decay curves to the stability data.

have already reported the increase in drug transport across the BBB that can be linked to the CD efficacy in cholesterol mobilization from brain endothelial cells and the opening of tight junctions to potentiate the paracellular pathway.32 In addition to these mechanisms, a relatively small amount (0.16%) of rhodamine-labeled SBEβCD was detected permeating the epithelial barrier, probably via passive diffusion.33 However, the chemical structure of hydrophilic CDs has a large number of hydrogen-bond donors and acceptors, high molecular weight (>970 Da), and very low octanol−water partitioning coefficient (logP < −3.0), which are unfavorable for successful permeation of biological membranes.34,35 On the basis of these observations, it has been suggested that hydrophilic CDs enhance drug delivery though lipid membranes mainly by increasing the availability of the dissolved drug compound in an aqueous phase at the membrane surface.36,37 Therefore, the possibility of the BBB transport of intact PR complexes is only feasible in tiny amounts and in general is highly unlikely.

Figure 4. Solubility isotherms of the (A) PR/SBEβCD and (B) PR/HPβCD complexes determined in H2O at 25 °C (pH 7.0). The S0 value (the same for both complexes) is the PR saturation concentration in the solvent in the absence of CD. 1918


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Figure 5. (A) HPLC−MS/MS analysis of PR complexed with the SBEβCD and HPβCD excipients and the reference substance (PR-LIPURO) in the blood and brain compartments after retrobulbar injection using C57BL/6 mice. Data represent mean ± SD (n = 5 animals per group). p < 0.001 was determined by two-way ANOVA followed by Bonferroni post-test. (B) Positive linear distribution patterns of the corresponding steady-state brain-to-plasma ratios for the analyzed compounds.

negatively charged groups with pKa < 4.0 are unfavorable for this process.45,46 While neutral PR is obtained at physiological pH, deprotonated PR is more pronounced at pH > 11. Indeed, PR as a weak organic acid remains almost entirely unionized (99.97%) as a phenol form at pH 7.4 and is extensively bound to plasma proteins (PPB = 98%), leaving only a small free fraction of the drug concentration (fublood = 2%).47 It is also completely and rapidly metabolized to sulfate and glucuronic conjugates with no anesthetic properties, which are eliminated mainly by the kidneys.48 Therefore, upon release from the CD cavity, the BBB permeation rate for PR at the physiological pH is not hindered by its minor negatively charged fraction in the blood (0.03%). Additionally, in order to study the effect of the CDs on the PR conformational change that can be adapted into the cavity as well as their influence on the binding properties, the drug/ CD complexes were subjected to 50 ns MD simulations with the MM-PBSA approach in evaluating binding affinity. To match the experimentally determined substitution degree of ∼7.0, only the primary hydroxyl groups were substituted with hydroxypropyl or sulfobutylether residues in the case of CD models. The main component of the conformational analysis is the clustering method in order to group together similar objects. For this reason, it is necessary to apply a particular MD protocol to generate different clusters for the various conformers and measure root-mean-square deviation

Table 2. Energetic Analysis of the Dissociation of Uncharged PR (AH) into a Proton (H+) and the Negatively Charged Form of PR (A−) Using DFT at the B3LYP/6-31G** Level of Theory

a

energya

AH

H+

A−

Esolv Esol phaseb

−5.07 −340.98

−115.01 −47.68

−63.25 −340.67

In kcal·mol−1. b×103.

(Etot) values for each component with the MM method based on an implicit MM-PBSA solvation model (Table 3). Table 3. Energetic analysis of the Dissociation of Uncharged PR (AH) into a Proton (H+) and the Negatively Charged Form of PR (A−) Using the Molecular Mechanics (MM) Method Based on the Implicit MM-PBSA Solvation Model

a

energya

AH

H+

A−

Esolv Eelec Etot

−3.19 −9.19 −2.98

−80.13 0.0 −80.13

−84.46 −32.52 −106.57

In kcal·mol−1.

It has been shown that positively charged molecules at physiological pH tend to favor BBB penetration, whereas

Figure 6. Clustering analysis of PR molecule complexed either with (A) SBEβCD or (B) HPβCD during 50 ns MD simulation. All CD molecules are depicted using the solid surface representation method with a probe radius of 0.4 Å, density isovalue of 0.7, and grid spacing of 0.5 to visualize the binding cavity. CDs are colored according to their atom types. All hydrogen atoms have been omitted for clarity. 1919


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both systems. Since the unfavorable desolvation penalty is not fully compensated by the favorable electrostatics, the solvation energy, ΔGsol, is positive in both systems (25.53 ± 0.24 kcal· mol−1 for PR/SBEβCD and 38.32 ± 0.21 kcal·mol−1 for PR/ HPβCD), suggesting that the electrostatic effects disfavor binding of PR to the CDs.

(RMSD) values between the PR conformations. Figure 6 explores the movement and conformational changes of the PR into the SBEβCD (Figure 6A) and HPβCD (Figure 6B) binding cavities during the 50 ns MD trajectory. From the beginning of the MD trajectory, PR was accommodated well into both systems, and further analysis displayed more flexible PR within SBEβCD (RMSD = 5.08 Å) in comparison with HPβCD (RMSD = 3.32 Å) (see Video S1 and Video S2 in the Supporting Information). A high degree of CD movement was also determined for a peripheral part of the SBEβCD and HPβCD molecules as a result of an increase in the side group flexibility. The PR behavior can be associated with the physicochemical properties and size of the SBEβCD structure and can also be linked with the decreasing entropy. The latter aspect is often taken into account for entropy− enthalpy compensation in experimental studies. However, the major drawback of structural analysis is that the data cannot provide quantitative information about the relative energies of the different poses. Together with the structural analysis of PR in the CDs, an energetic analysis was performed throughout the MD simulations. Table 4 describes the estimated PR binding energy

4. CONCLUSIONS In the current study, we analyzed the ability of PR to form inclusion complexes with modified β-cyclodextrins, such as SBEβCD and HPβCD. The PR/SBEβCD and PR/HPβCD formulations were prepared and characterized, and their BBB permeation potentials were evaluated using the C57BL/6 mouse model for the purpose of controlled drug delivery. The PR/SBEβCD complex was found to be more stable and soluble in water with k = 0.25 h−1, t1/2 = 2.82 h, and Kc = 5.19 × 103 M−1, revealing a higher BBB permeability rate in comparison with PR-LIPURO because of the maximal logBB value for the complex. In addition, the PR binding affinity to SBEβCD was established to be the lowest, judging by a maximal ΔGbind of −18.44 kcal·mol−1, which indicates more rapid PR/SBEβCD dissociation. Overall, the results demonstrated that SBEβCD as a formulating agent has the potential to enhance drug permeation across the BBB and hence to improve the PK/ PD properties of general anesthetics at the BBB level.

Table 4. Energetic Analysis of PR Complexed with Different Cyclodextrin Excipients Using the MM-PBSA Method energy terma

PR/SBEβCD

PR/HPβCD

ΔEbond ΔEangle ΔEdihed ΔEvdW ΔE1−4vdW ΔE1−4elec ΔEelec ΔEMM ΔGPB ΔGNPb ΔGEDISPERc ΔGsol ΔEelec(tot)d ΔH TΔS ΔGbind

−1.49 ± 0.32 −5.57 ± 0.25 8.54 ± 0.03 −19.47 ± 0.09 0.75 ± 0.13 −6.24 ± 0.15 −12.95 ± 0.09 −36.43 ± 0.12 16.11 ± 0.01 −13.99 ± 0.13 23.41 ± 0.21 25.53 ± 0.24 3.15 −10.89 ± 0.05 7.54 −18.44

−1.55 ± 0.35 −4.48 ± 0.55 11.51 ± 0.41 −9.84 ± 0.33 1.74 ± 0.44 2.47 ± 0.23 −50.47 ± 0.21 −50.62 ± 0.06 35.92 ± 0.24 −12.47 ± 0.03 14.88 ± 0.18 38.32 ± 0.21 −14.55 −12.29 ± 0.32 13.48 −25.78

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.6b00215. Atomic partial charges for SBEβCD and HPβCD (Figure S1) (PDF) MD trajectory frames of PR within SBEβCD (MPG) MD trajectory frames of PR within HPβCD (MPG)

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AUTHOR INFORMATION

Corresponding Author

*Author to whom correspondence should be addressed; EMail: [email protected] Tel.: +49-931-2013-0016; Fax: +49931-2013-0019. Notes

The authors declare no competing financial interest.

All values in kcal·mol−1. bNonpolar contribution to the solvation energy from repulsive solute. cNonpolar contribution to the solvation energy from attractive solute. dΔEelec(tot) = ΔGelec + ΔGPB.

■

a

ACKNOWLEDGMENTS The authors are grateful to the Bundesministerium für Bildung und Forschung (BMBF) for support of this work by providing Grant BMBF13N11801 to J.-A.B. This work was partially supported by the Fundació n Sén eca del Centro de Coordinación de la Investigación de la Región de Murcia under Project 18946/JLI/13 and by the Nils Coordinated Mobility under Grant 012-ABEL-CM-2014A, in part financed by the European Regional Development Fund (ERDF).

profiles for modified CDs. The calculations show that the highest CD binding affinity to PR belongs to HPβCD (ΔGbind = −25.78 kcal·mol−1) as a result of strong intermolecular forces, which also means an increase in entropy (TΔS = 13.48 kcal· mol−1). The predicted PR binding affinity to this CD might be explained by the lower solubility of PR/HPβCD compared with PR/SBEβCD, resulting in a lower drug concentration after complex solubilization in the blood and impairing the BBB permeation of PR. The binding affinity for the PR/SBEβCD complex (ΔGbind = −18.44 kcal·mol−1) was lower compared with HPβCD, which is in line with our in vivo BBB permeation experiments explaining the highest logBB value as a result of the more rapid PR/ SBEβCD dissociation in the blood. Energy decomposition to individual terms suggested that the van der Waals and lipophilicity terms act more favorably toward binding for

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Characterization, in Vivo Evaluation, and Molecular Modeling of

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