SIMULATION OF VINPOCETINE RELEASE PROCESS FROM MICROCAPSULES WITH HYDROPHOBIC SHELL

1Федеральное государственное бюджетное образовательное учреждение высшего образования «Воронежский государственный университет», 394036, Россия, г. Воронеж, ул. Студенческая, 3 E-mail: juli-polk@mail.ru 2Пятигорский медико-фармацевтический институт – филиал ФГБОУ ВО ВолгГМУ Минздрава России, 357500, Россия, г. Пятигорск, пр. Калинина, 11. E-mail: alexander.glushko@lcmmp.ru 3Ташкентский фармацевтический университет, 100015, Узбекистан, г. Ташкент, ул. Айбека, 45 E-mail: yosk@mail.ru


Introduction.
Encapsulation is a process, while which the smallest particles of fl uid, solid or gaseous active ingredients are packed into the second material for preserving them against environment [ 1 ,2].The choice of hydrophobic shell-matrix for microcapsules is based on the needed properties of microcapsules, for example, based on the purpose of microcapsules, the temperature of storage, etc. [3].Preferable hydrophobic shell-matrix must have the melting temperature higher than 45°С, so that it could be stored at the room temperature.Usually any hydrophobic substance can be used if microcapsules are stored at the temperature lower than the melting temperature of hydrophobic substan ce [4].
Hydrophobic shell-matrix, or hydrophobic phase, can be selected from the group including fats, oils, waxes, resins, emulsifi ers and their mixtures.Preferable hydrophobic shell-matrix, or hydrophobic phase, is selected from the group including animal fats and oils, fully or partially hydrated plant and animal oils / fats, unsaturated partially hydrated or fully hydrated free fatty acids, other emulsifi ers, animal waxes, plant waxes, mineral waxes, synthetic waxes, natural and synthetic resins and their mixtures.Animal waxes are beeswax, lanolin, husk wax or wax of Chinese waxworm.
Vinpocetine is a perspective medicinal substance for development of a microencapsulated fo r m [5,6].
Vinpocetine is poorly soluble in water, and, for preliminary research to substantiate the composition of microcapsules and determine their preparation conditions, ethanol can be used as an environment along with purifi ed water and hydrochloric acid, which makes it possible to determine the infl uence of auxiliary substances and technological fac tors [7].
Microcapsules with hydrophobic shell can be prepared by dispersion m ethod [8].But often the complexity of the instrumental arrangement process and low effi ciency make the choice of microencapsulation methods a long and expensive research.
Molecular dynamics simulation of solutions in the study of the process of medicinal substances release from microcapsules allows to select an optimal fi lm former, hydrogen index, the environment for release and other factors, important for successful microencapsulation process.Hereby the necessity of the actual experiment is min imized [9].
The aim of the study is a comparative analysis of thermodynamic characteristics of vinpocetine release from the melt of beeswax and cacao butter 3:2 into water, solution of hydrochloric acid 0.01 M and ethanol.
Materials and methods.For the vinpocetine release process simulation from the melt into different environments models of components of the studied systems were built preliminary (vinpocetine and the beeswax and cacao butter melt components) and atom charges were calculated by quantum-chemical method.
For the vinpocetine molecule ionization, depending on the pH of the dispersion environment, was taken into account in order to reproduce its behavior in the solvent.
Taking into account the vinpocetine ionization constant index (pK a )in wa ter 7.1 [10] and using Henderson-Hasselbalch equation [11], it is possible to make a conclusion, that vinpocetine is almost completely ionized in pH 2 and in pH 7 the fraction of the ionized vinpocetine molecules is 0,56: where α and β are a relative content of vinpocetine-base and vinpocetine-cation in the solution respectively.cation base

Figure 1 -Relative content of vinpocetine-base and vinpocetine-cation in solution according to Henderson-Hasselbalch equation
Hereby the solubility of vinpocetine in neutral water environment according to the literature data [ 12] is low (1.89•10 -3 mg/ml), that means insignifi cant concentration of vinpocetine in water environment both in the form of cation and in the form of base.The release simulation of both cation and base into the solvent environment seems to be interesting in this study, since it gives an opportunity to explain the difference between release effi ciency of this substance into the solvent in different values of pH from the molecular structure point of view.

Информационные технологии в фармации Informational Technologies in Pharmacy
Hereby the transition of a molecule from the melt to the solvent can be accompanied by its ionization depending on the value of solvent pH.Moreover, ionization can occur even when a molecule of vinpocetine is situated on the interphase surface in contact with the solvent.
We performed the simulation of exactly this critical stage of the release process, i.e. overcoming the phase boundary of «melt-solvent» by vinpocetine molecule.As an example of a similar study the work by Hui-dong Zheng [ 13] can be represented.
An important factor that should be taken into account in molecular dynamics simulation of heterogeneous systems is the atom charge distribution.
For this purpose atom charges of the simulated system components were calculated by ab initio method, which was dictated by insuffi cient accuracy of semi-empirical methods.The choice of quantum-chemical methods to detect the atom charges of melt component molecules (unrestricted Hartree-Fock method with basis set 3-21G* for geometry optimization and density functional theory method UB3LYP with 6-31G* basis set) was made according to the results of the study, represented in the wor k [14].The validity of ab initio methods used for atom charge calculation in molecular dynamics simulation of substances transfer through interphase surface is demonstrated in the wo rk [15].
Geometry of the melt components was optimized by molecular mechanics mm+ met h od [16] in Hyper Chem 8.09 program.Further geometry optimization of molecules was performed by ab initio method (unrestricted Hartree-Fock method, basis set 3 -21G* [17]).The charges were calculated by ab initio method (density functional theory method U B3LYP [18], basis set 6-31G*).
The obtained system further inserted into the solvents -water and ethanol.After that thermodynamic equilibration was performed by Bioeurica program (the author is A.A. Glushko) [19] (force fi el d Amber94 [20], rectangular periodic boundary conditions, Berendsen thermostat [21] -310K, bath relaxation time 100 fs, integration step 1 fs).
Thermostating was performed according to the graph in Table 1.The temperature, set by the thermostat, was linearly changed between the graph moments of time.

Time, fs
Temperature, K 0 0 300000 310 Further molecular dynamics simulation of vinpocetine release from the melt spot to the solvent was performed during 20 ns in Bioeurica program.
As an initial state, for calculation of thermodynamic characteristics of vinpocetine release from the melt, the conformation of "melt-vinpocetine" system was used after thermodynamic equilibration by molecular dynamics method in Bioeurica program during 5 ns.
As a fi nal state, a moment of time of molecular dynamics simulation was used, corresponding to full release of vinpocetine molecule from the melt to the solvent.
As a criterion of vinpocetine release into the solvent a module of Van-der-Waals energy interaction between vinpocetine molecule and the melt was used (|E| < 0.1 kDj/ mol).For both states geometry optimization in Amber94 force fi eld was performed by Bioeurica program.For isolated systems vibrational analysis performed [ 22] by unrestricted Hartree-Fock method in basis set STO-3G in free distributable program Orca 4.0 [17].As a result thermodynamic characteristics of simulated system fragments (enthalpy and entropy) were obtained.Further the calculation of enthalpy (H and entropy (S) of vinpocetine release from the melt were made using these equations: Using the obtained enthalpies and entropies Gibbs, energies (П) of vinpocetine release into different environments were calculated according to the equation for temperature 310 K: where T istemperature, K. Results and discussion.As the components of the studied systems, the models of vinpocetine molecule in form of base and cation were used (Fig. 2), and also models of beeswax and cacao butter melt components (Fig. 3).

of basic components of beeswax and cacao butter
Quantitative composition of the beeswax and cacao butter melt was calculated according to the literature data about chemical composition of beeswax [ 2 3] and cacao butter [24].
Further, based on molecular masses quantities of molecules of the simulated system basic components were calculated.Accounting the volume of the simulated beeswax and cacao butter melt 3:2 a fragment, equal to 125 nm, the components which molecules' number according to the quantitative composition was more than 2 for the simulated volume, were included into the model.
As an example of selection of multicomponent system composition the work can be represented, in which simulation of cacao butter was performed by molecular dynamics method, hereby only three types of triglycerides were included in the mixture composition, fatty acids and many other minor components of oil were not include d [25].
Hereby only the components contained in a fairly large number are included into the composition of membrane.Nowadays molecular dynamics simulation is available in the scale of up to dozen nanometers, i.e. in the volume of thousands of cubic nanomet ers [27].
At the same time many minor components of natural substances have such low concentrations, that their molecules are represented in a single exemplar in volumes far exciding thousands of cubic nanometers.
Taking that into consideration, nowadays it does not seem possible to account many minor components of natural mixtures in molecular dynamics simulation.But this theoretical approach is widely and successfully used for physical-chemical properties of mixtures st udy [28][29][30].
Further, taking into consideration molecular masses, quantities of molecules of basic components of the simulated system were calculated (Table 2).
Based on components structures, the model of beeswax and cacao butter melt 3:2 was built.In cacao butter, besides triglycerides, free fatty acids are contained.Their concentration can be learnt by the acidic value.State Pharmacopoeia X [24] allows the acid value of cacao butter to be no more than 2.25 mg of KOH per 1 g of butter.Therefore, the content of fatty acids in cacao butter, meeting the requirements of State Pharmacopoeia is no more than 4.01•10 -5 mol/g: , where С (fatty acids) is content of fatty acids in 1 g of cacao butter, mol/g; m (KOH) -mass of KOH, needed for neutralization of acids, contained in cacao butter per 1 g of oil (acid value), g KOH/g; M (KOH) -molar mass of KOH, g/mol.Total mass of cacao butter components, included in the molecular model is 2.8•10 -20 g (Table 2), therefore the amount of fatty acids of cacao butter in the simulated volume must not exceed 1.12•10 -24 mol, which corresponds to 0.676 molecules of fatty acids: where m is cacao butter mass in the simulated system, g; N A -Avogadro number, mol -1 ; N -number of fatty acid molecules in the simulated system.
The obtained results correspond to approximately 1 molecule of fatty acid per 64 different molecules of the melt components (table 2), part of which is comparable in volume with fatty acids of cacao butter, and another part (triglycerides) has a signifi cantly larger volume.Therefore, molecules of fatty acids of cacao butter were not included into the composition of the simulated system.
Further on, using Bioeurica program, thermodynamic equilibration of the melt model was performed by molecular dynamics method.After that the system was included into the solvents -water and ethanol.After the thermodynamic equilibration of the system by molecular dynamics method during 5 ns, simulation of vinpocetine release was performed from the melt spot into the solvent during 20 ns.Images of spatial structures of simulated systems for different moments of time are represented in Table 3.As a result of thermodynamic equilibration in water environment a heterogeneous system with clear interphase surface was obtained and the ordered structures in the melt.In the ethanol environment partial mixing of the melt with water took place (Table3).

Информационные технологии в фармации Informational Technologies in Pharmacy Table 3 -States of simulated systems at different moments of time
According to the results Van-der-Waals interaction analysis between vinpocetine and the melt, the durations of vinpocetine release into the solvent were determined (Fig. 4).To do it, the time was detected,while the module of Van-der-Waals interaction energy between molecule (ion) of vinpocetine and the melt became less than 0.1 kDj/mol.Time series of Van-der-Waals' interaction energy were preliminary smoothed by moving average method (window of averaging 1.6 ns).

Figure 4 -Dynamics of Van-der-Waals' interaction energy between vinpocetine molecule (ion) and the melt of beeswax and cacao butter 3:2
The analysis of the obtained results shows, that the release of vinpocetine cation from the melt into ethanol goes during 15.5 ns.The release of vinpocetine base from the melt into water with pH = 7.0 doesn't go, due to higher hydrophobicity of vinpocetine base.
The release into water with pH = 2.0 goes, but vinpocetine molecule stays bound with interphase surface for a long time.

Информационные технологии в фармации Informational Technologies in Pharmacy
The performed study has shown, that the melt of beeswax and cacao butter 3:2 is not soluble in water environment which appears to be the factor, slowing down the release of vinpocetine into solvent.In acidic environment (pH = 2.0) vinpocetine cation releases from the beeswax and cacao butter 3:2 and passes into water.Hereby vinpocetine adsorption tends to exists on the melt surface.
Vinpocetine molecule releases from the beeswax and cacao butter 3:2 into ethanol and completely passes into the solvent.Hereby partial dissolution of the melt in ethanol takes place.
For the quantum chemical calculation of thermodynamic characteristics of vinpocetine release from systems, used in the molecular dynamics simulation, fragments of initial and fi nal states were isolated (Fig. 5-7): vinpocetine -melt, solvent, melt, vinpocetine -solvent.Th e results of thermodynamic characteristics calculation of the simulated system fragments are represented in Tables 5 and 6.Vinpocetine release from the melt of beeswax and cacao butter 3:2 into water with different pH and into ethanol depends on its solubility in this environments, and also on the solubility of the melt.

Table 5 -Results of quantum-chemical calculation of enthalpy and entropy of vinpocetine release from the melt into solvent
The melt of beeswax and cacao butter 3:2 is partially dissolves in ethanol, and that is the factor, speeding up vinpocetine release into solvent.
Vinpocetine stays in the melt phase during simulation Фармация и фармакология Т. 5 № 4, 2017 Информационные технологии в фармации Informational Technologies in Pharmacy of the system with water (pH = 7), which indicates, that vinpocetine release from the melt of beeswax and cacao butter 3:2 into water with pH=7 is diffi cult.

Conclusion.
Vinpocetine release from the melt of beeswax and cacao butter 3:2 into water with pH = 2.0 is energetically advantageous process, and it is also accompanied by increase of entropy, which indicates higher thermodynamic probability of the fi nal state (vinpocetine in solvent).
Vinpocetine release from the melt into water with pH = 7.0 is energetically less advantageous comparedwith the release into water with pH = 2.0.
Vinpocetine release into ethanol from the melt is en-ergetically less advantageous compared with the release into water in the condition of no melt dissolution in ethanol.Butthe results of molecular dynamics simulation indicate an opportunity of the melt dissolution in ethanol.This is the factor increasing effi ciency of vinpocetine release.
The simulation study of molecular dynamics of vinpocetine release from the melt of beeswax and cacao butter 3:2 indicates an opportunity of vinpocetine release into water with pH = 2.0 and into ethanol.The obtained results allow to assume a lower degree of vinpocetine release from the melt into ethanol compared with the solution of hydrochloric acid 0.01 M.

Figure 5 - 2 Figure 6 -
Figure 5 -Vinpocetine release from the melt into water in pH 2

Figure 7 -
Figure 7 -Vinpocetine release from the melt into ethanol