Isotopic (D/H) management of chemical and biological processes properties: applications in pharmaceutical engineering

. The strategy of sustainable industrial development in accordance with "green chemistry" stimulates researchers to search for a "lever" that could provide control over the direction and speed of chemical and biological processes. Water, depleted in deuterium content, is a recognized safe (in contrast to D 2 O) dissolution medium according to the results of numerous clinical observations. Due to the lower strength of carbon-protium (C-H) chemical bonds compared to carbon-deuterium (C - D), the properties of deuterated compounds, such as the rate of reactions involving them, change. The aim of the work was to study the effect of varying the 𝐻 12 content on controlling the chemical and biological properties of processes. The highest values of kinetic deuterium isotope effects (KEI), which demonstrated greater sensitivity to the difference in the nucleolar mass D/H in the dissolution medium, correspond to slightly soluble lipophilic molecules. We also found the dependence of the S.ambigua cell biosensor lifetime on the deuterium concentration in the dissolution medium according to the laws of Arrhenius kinetics. Thus, the developed approach based on the laser diffraction technique in controlling the rate of chemical and biological processes can be successfully applied at all stages of the production cycle in the chemical and pharmaceutical industry.


Introduction
One of the directions in the field of environmental sustainability without toxic substances -"green" chemistry -is the strategy for the development of chemicals, adopted in 2020 by the European Commission.The existing problem of controlling chemical processes encourages researchers to find and implement simple, affordable processes to modify the properties, direction and rate of chemical and biological processes.There are a variety of ways (polymeric micelle preparation, particle size reduction technologies, pharmaceutical salts, pro-drugs, the solid-state alternation technique, soft gel technology, drug nanocrystals, solid dispersion methods, crystal engineering techniques and nanomorph technology) to modify the properties of substances and, therefore, the direction of chemical processes [1].However, all of them are associated with increased production costs.Water depleted in the content of the stable hydrogen isotope, deuterium, is a recognized safe (in contrast to D2O) solvent solution medium according to the results of numerous clinical observations [2].Since one of the most important problems of the modern pharmaceutical industry remains the low water solubility of synthesized candidate substances and the consequent trade-off associated with the pharmacochemical characteristics of finished drugs, therefore, research aimed at low-cost methods to improve/manage properties consistent with the principles of "green" chemistry is relevant [3].

Solubility challenges that plague the oral drug-delivery frontier
According to [4], the solubility of drug substances is used in biopharmacy as an assessment of their oral absorption.According to [5], more than 60% of candidate drugs have low solubility and high permeability (Class II BCS) and about 20% have low solubility and permeability (Class IV BCS).A concern is that drugs with low water solubility are predicted to have low and variable oral bioavailability and hence unpredictable clinical response.Examples of possible approaches for solubility enhancement are: metastatic polymorph cocrystal formation, pro-drug formation, micronization and nanosized drugs, solid dispersion, solvent (рН, Co-solvent) composition, drug carrier systems and others.The main disadvantage of most of these technological approaches is the need to introduce additional excipients into the composition of the modified drug, which leads not only to higher production costs and violations of the chemical strategy, but also to the development of various forms of drug-induced hepatotoxic reactions.Managing the solubilization of drugs characteristics is one of the main areas of Drug Delivery Systems (DDS).

Water as a universal solvent. Isotopy
Water as a liquid medium is the most acceptable solvent from a physiological point of view and is a heterogeneous substance in terms of its ability to exist different isomers.It is not only the nuclear spin isomers that are discussed that cause the exists in two forms: para(p)and ortho(o)-water, which are distinguished by their values of the quantum number of the total nuclear spin I, where I = 0 and 1 for p-and o-H2O, respectively, but also molecular isotopological.It is considered that as H2O molecules travel through hydrological cycle, various isotopic molecular species, having different isotopic combinations of oxygen ( 18 O and 16 O) and hydrogen ( 1 H and 2 H or D) in them are differentially partitioned between vapor, liquid and solid phases, imparting distinguishable isotopic signature to all the three phases.There are 9 possible combinations of the stable 3 isotopes of oxygen and 2 isotopes of hydrogen; the most common isotopic water molecules are given in table 1.Despite the fact that the mass differences between water isotopologues (see Table 1) are small and the fact that per 6,42•10 3 of protium atoms ( 1 H) there is 1 of deuterium atom ( 2 H) (155 ppm), the common isotopic effects can be significant and lead to important differences in their physical and chemical properties:

Low angle laser light scattering (LALLS)
Laser low-angle dispersion diffraction analyzer Mastersizer 3600 Ec (Malvern, UK) uses a laser diffraction method.The method is based on measuring the intensity of scattered light, the passage of the laser beam through the dispersed sample (suspension, emulsion, dry powder).The elements of the measuring unit are: laser Ne-Ne source with λ=633nm; optical Fourier lens system with focal length of 100 mm, a multi-angle laser light scattering detector.The measurement of Laser Obscuration Time (LOT) was started while adding water to the cell and continued with intervals of 10 seconds up to the complete dissolution of the substance that was recorded up to the end of change in the time of the examined laser obscuration parameter.The method of solubility kinetics is based on registering the light scattering indicatrix in time for the electromagnetic radiation to interact with the particles of the API's dispersed phase.Mathematically, Laser Obscuration (LO) may be presented by the following form: • 100% (1)

Spirotox method
The study of the aqueous dilutions biological activity was carried out using the organismsprotozoa Spirostomum ambigua.The study was carried out with the fluoroquinolone group API -MXF when preparing 0.01% solutions in water with a D/H-145 ppm ratio and ddw.
The test is carried out in 5-well microplate with 3-5 cell tested samples a in a single microplate.The behaviour and survival of the protozoan Sp. ambigua was monitored using a dissecting microscope.Protozoa Sp.Ambigua has the greatest sensitivity to xenobioticschemical ligands.The factor accelerating the death is temperature and, as a consequence, the use of Arrhenius kinetics in the study of the mechanisms of ligand-receptor interaction.
where, k is the rate constant, Ea is the activation energy, R is the gas constant, A is the preexponential factor, and T is the temperature

Statistical data processing
All statistical data processing was performed using software packages of Origin Pro 9.1.Each value on the figures represents "mean±SD", significant differences were considered when p < 0.05

Isotopic (D/H) management of chemical processes properties
In order to study the D/H control function of the dissolution processes of substance powders in water with natural and depleted 2 H content (ddw), the LALLS method shown in 1 was used., which depends on the properties of both the solvent and the substance being dissolved (Table 3).Thus, it can be seen that the realization of the usual kinetic isotope effect of deuterium ( >1) is representative of all classes of drugs presented, however, DKIE is more pronounced for lipophilic, water-insoluble substances belonging to II and IV biopharmaceutics classification classes of the drug substance (BKS) (Figure 2).The observed primary DKIE helps to determine the limiting rate of the transformation process, as well as the relationship between the KIE and the structure of the transition state.

Isotopic (D/H) management of biological processes properties
The ligand-induced process of cell biosensor death proceeds through a transition state formation stage accompanied by significant conformational rearrangements, with energy expenditure.The destruction of the cell receptor-ligand complex is rate-determining

Fig. 1 .
Fig. 1.Distribution of dissolution rate constant values within the investigated API substances in dissolution medium with different hydrogen isotopic composition; a -lipophilic chemotherapeutic drugs, b -lactose biotechnological samples.

Figure 1a demonstrates the
Figure1ademonstrates the increase in the dissolution rate of APIs belonging to different chemical and pharmacological classes associated with D/H variations in the hydroxyl solvent solution medium.Figure1bshows the results reflecting the differences in the dissolution rate of biotechnological preparations covering lactose particles in a fluidized bed.The mechanistic interpretation of KIE is probably related to energy differences in overcoming the reaction barrier at the stages of C-H and C-D bond formation and breaking, which depend on the reduced mass of the two bonded atoms (µ).Changes in the rate of reactions occurring in water solutions ─ hydrogen isotopes are characterized by the value of the deuterium kinetic

Fig. 2 .
Fig. 2. Graphical visualization of normal DKIE in hydroxyl solutions of different lipophilicity drugs.

Table 1 .
Isotopologues of water

Table 3 .
Physico-chemical properties of API belonging to different chemical and pharmacological classes.