Results and achievements

The current implementation of the project (September 2017)

1. A brief description of the scientific research performed.

The search for optimal compositions and conditions for the formation of solid dispersions of polyvinylpyrrolidone with an average molecular weight of 58,000 with hydrophobic drug substances (phenacetin, sulfanilamide). Methods of calorimetry, including ultrafast calorimetry, studied drug compositions with a polymer with a content of components in a ratio of (1-10):1 by weight. The composition of the composites was optimized using differential scanning calorimetry and X-ray powder diffractometry. Using the UV spectrophotometry method, the influence of the polymer matrix on the solubility of hydrophobic drugs in water was studied. Using the spray drying method, microspheroidal particles of solid dispersions of polyvinylpyrrolidone with hydrophobic drugs were obtained. The kinetics of dissolution of drugs in accordance with the requirements of the pharmacopeia was studied.

Selection of the optimal solvent allowing to measure the thermal denaturation of the model lysozyme protein using both classical differential scanning calorimeters and an ultrafast chip calorimeter was carried out. The thermodynamic and kinetic parameters of the process of denaturing of lysozyme in a solution of glycerin, over a wide range of heating rates from 5 K/min up to 8000 K/s, were determined using classical and ultrafast calorimetry.

The effect of dimethylsulfoxide (DMSO) on the rate of destruction of the native structure of lysozyme in water at elevated temperatures has been studied by molecular dynamics. For calculations, the OPLS force field and the SPC / E water model were used. The studied system consisted of a molecule of lysozyme and 10,000 molecules of solvents, while the mole fraction of DMSO ranged from 0.05 to 1. For each DMSO concentration, after balancing the system, 10 trajectories of 30 ns were recorded, and for pure water, 130 nanoseconds. The analysis of changes in the secondary structure was carried out using the DSSP algorithm. Various criteria were tested to characterize changes in the tertiary structure: the root-mean-square deviation of the C-atoms of amino acids, the radius of inertia (Rg), the available non-polar surface area (SASA), the fraction of native contacts, and the distance between individual residues. By averaging the time of destruction of the tertiary structure over all trajectories, the denaturation times were estimated and their dependence on the solvent composition was studied. Maps of free energy for the process of denaturation in mixtures of various compositions are constructed and analyzed. The activation energy of this process is determined from the temperature dependence of the denaturation times in a water mixture with 30 mol% DMSO, which is compared with the values ​​obtained experimentally by fluorescence spectroscopy and circular dichroism spectroscopy.

Using the method of ultrafast scanning calorimetry, the kinetics of crystallization of polymers from a supercooled melt in a wide temperature range was studied. The samples were heated above the melting point and then cooled to a crystallization temperature at a high rate of about 1000 K/s to prevent early crystallization during cooling. Isothermal crystallization leads to the appearance of an exothermic peak, which determines the value of the crystallization peak time, i.e., the maximum heat flow rate during crystallization, or the half-life of crystallization, which can serve as a measure of the rate of crystallization.

The enthalpy of dissolution at 298.15 K and the enthalpy of melting at the melting point (Tm) of seventeen substituted aromatic amines were determined by calorimetry. The relationship between the enthalpy of melting at Tm and the enthalpy of dissolution of aromatic compounds at 298.15 K in benzene was studied.

2. Brief description of the scientific results obtained.

Using the methods of differential scanning calorimetry, superfast calorimetry and X-ray powder diffractometry, optimal composites of polyvinylpyrrolidone composites with an average molecular weight of 58,000 with phenacetin and sulfanilamide were determined, which were 5: 1 and 6: 1 by weight, respectively.With these ratios, the crystal phase of the drug is not fixed and its content in the composite is maximized. It has been found that the optimal concentration of polymer in solution in the preparation of microspherical particles of solid dispersions by spray drying is 2.5%.

Using the method of ultrafast calorimetry, it was found that the use of ultrafast cooling makes it possible to convert the crystalline drug phenacetin into an amorphous state, which is the most convenient dosage form providing increased dissolution of the preparation in water.

It is shown that in the presence of polyvinylpyrrolidone with a molecular weight of 58,000, the solubility of drugs in water is increased up to 5 times.

Differential scanning calorimetry has shown that the emerging microspheroidal particles consist of solid dispersions of polyvinylpyrrolidone and a hydrophobic drug. In addition, X-ray powder diffractometry showed that the drug in the microspherical particles obtained in the present study does not crystallize even after 4 months of storage under environmental conditions.

It was found that the percentage of drug in solid dispersions corresponds to the theoretically calculated values, which indicates the absence of its decomposition during spray drying.

It has been shown that microspheroidal particles have a high dissolution rate, which allows them to be used in inhalation delivery systems of drugs with a short release time.

It has been established that glycerin satisfies the requirements for solvents (low volatility, protein preservation of the native structure in solution at room temperature), suitable for studying the thermokinetic characteristics of the denaturation of globular proteins.

The possibility of measuring the calorimetric curves of the lysozyme denaturation process using a chip calorimeter is shown. To do this, the dry protein, placed on a microchip, was wetted with glycerol, after which a calorimetric curve was recorded.A characteristic endothermic heat effect was observed on the curve.

An optimal method for the preparation of a solution of lysozyme in glycerol is proposed, and the optimal concentration of the solution is determined. It has been established that the protein concentration of 50 mg / ml is the lowest at which DSC curves can be recorded with a satisfactory signal-to-noise ratio both on conventional DSC devices and on an ultrafast chip calorimeter.

It was found that the extremum temperatures of the calorimetric peaks on DSC curves obtained for a solution of lysozyme in glycerol are at lower temperatures than can be expected from extrapolation of data obtained using classical DSC. It is shown that the high specific surface area of ​​the sample on the chip calorimeter is the reason for the difference in the denaturation temperatures of the protein, determined using classical and ultrafast calorimetry.

It was found that the results of calculating the denaturation times of lysozyme in the presence of DMSO, obtained using different approaches, are in good agreement with each other. The destruction of both the tertiary and secondary structure is accelerated by increasing the mole fraction of DMSO. For water even for 130 ns, changes in SASA and Rg are insignificant; The protein remains in a compact state, although its native contacts are violated. In mixtures, on the contrary, the compact structure quickly collapses. The secondary structure in pure water also collapses much more slowly than in mixtures with DMSO. At low DMSO concentrations, the proportion of spiral structures toward the onset of unfolding of the protein is somewhat lower than at high concentrations.

It is shown that the destruction of the secondary structure leads to destabilization of tertiary contacts and a compact globular state. The unfolding process is accompanied by an increase in the area available to the solvent of the nonpolar surface, which is unprofitable in the case of solutions in pure water and can cause some kinetic stabilization of the compact state. DMSO molecules significantly accelerate this process by solvating non-polar regions.

The striking difference between compact disorganized structures that appear to be the kinetically stable intermediates into which the protein is converted in simulations with water, from the unfolded close to random coils of conformations that are rapidly formed in binary mixtures requires experimental verification based on a detailed study of the complex of physical methods. The value of the activation energy of denaturation in a mixture of water with 30 mol% DMSO, calculated from the temperature dependence of the denaturation times (100 kJ / mol), is in satisfactory agreement with the experimental data (150-50).

It has been established that for melts of isotactic polypropylene, polyamide 66, polyamide 11 and polybutylene terephthalate, there are two maximum crystallization rates on the temperature dependence. As the temperature decreases, the rate of crystallization increases, since the nucleation and growth rates of the crystals are increased. With a further decrease in temperature, it passes through a maximum and decreases somewhat, which is associated with a decrease in the mobility of the polymer chains. However, with further cooling, an increase in the rate of crystallization is observed for all the polymers studied. It is shown that the observed dependence of the crystallization kinetics on temperature is associated with an increase in the number of nucleation nuclei due to a change in the nucleation mechanism, which also leads to an increase in the growth rate of the crystals. With a strong supercooling of the melts, mesophase polymers were formed.

New methods have been developed for determining the enthalpy of sublimation, evaporation and melting of organic compounds, based on an analysis of the relationship between the enthalpy of dissolution or melting, solvation on the one hand, and the enthalpy of evaporation and sublimation on the other. The methods demonstrated good convergence with the data of classical methods for determining the enthalpy of phase transitions in the case of aromatic amines.

A relationship was established between the enthalpy of melting at the melting point (Tm) and the enthalpy of dissolution of aromatic compounds at 298.15K. It is shown that in the absence of solid-solid phase transitions in the temperature interval between 298.15 K and Tm, the ratio between the melting enthalpy at Tm and the enthalpy of dissolution of the compound in benzene at 298.15 K is determined by the contributions due to the temperature dependence of the melting enthalpy of the compound and the enthalpy dissolution at 298.15 K in the benzene of this compound, which is in a hypothetical liquid state. Each of these contributions was analyzed in detail in a series of 33 compounds. To this end, the enthalpies of melting of the studied compounds at Tm were reduced to 298.15 K, using the literature data on the specific heats of solid and liquid substances, since it was suggested that the heat capacity of a hypothetical liquid below the melting point should have the same temperature dependence as the heat capacity of the liquid above the melting point . The enthalpies of melting at 298.15 K were calculated from the enthalpy of dissolving the solid and the enthalpies of dissolving hypothetical liquid aromatic compounds in benzene. The enthalpy of dissolution of hypothetical aromatic compounds in benzene was calculated from the experimentally measured enthalpy of dissolution of structurally similar aromatic compounds that are liquid at 298.15K. A good correspondence was found between the enthalpies of melting at 298.15 K, calculated by different methods.