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Vol 26, No 2 (2023)
Biological chemistry
Recovering and standardizing of the original genotherapeutic construction for stimulation of nervous tissue regeneration
Abstract
Traumatic nerve injury is one of the most common cause of permanent disability. The problem of nerve regeneration is based on the slow growth of nerve fibers and short-term production of neutrophic factors (NGF, BDNF, NT-3, GDNF, etc.), which stimulate the survival of damaged neurons and growth of nerve fibers. In most cases, the intrinsic regenerative potential is insufficient to restore innervation, which requires the replacement therapy with neurotrophins.
One of the most promising therapeutic approaches for regenerative medicine is gene therapy that allows to prolong the local production of neurotrophic factors. Previously, we created a bicistronic gene therapy construct pNCure (a plasmid for the treatment of nerves), based on the modified pVax1 plasmid construct approved by the FDA (U.S. Food and Drug Administration), and encoding the cDNA sequences of brain-derived neurotrophic factor (BDNF) and urokinase-type plasminogen activator (uPA). It revealed a prominent therapeutic activity in a model of traumatic nerve injury in mice.
Gene therapy products is a particular class of drugs that need particular approaches to production, purification and standartization. There is practically no information on the purification and standardization of gene therapy drugs in Russian, and this is the topic of our manuscript. Here, we have elucidated the issues of plasmid production in E. coli cells with subsequent multistage purification, as well as the issues of evaluation the identity and quantity of the plasmid (using PCR and UV spectrometry methods), and determination of pH and quantity of related and secondary impurities within the substance. The results of the study have demonstrated that the proposed approach to plasmid production, purification and standartization allows to obtain the desired high-quality gene therapy drug that meets all the requirements of the XIV State Pharmacopia.
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Composition and physico-chemical properties of proteins in fetal water in complicated pregnancy
Abstract
Relevance. The physiological course of prenatal ontogenesis largely depends on the state of the amniotic fluid, which largely provides the fetus with the substances necessary for its development. Among the complications of pregnancy, premature birth leads to especially serious consequences. In this regard, the elucidation of the mechanisms for unleashing labor activity before the term is important. The most informative for achieving these goals are proteins that perform numerous functions in the regulation of cellular metabolism.
Purpose of the study. To study the composition and properties of amniotic fluid proteins and the significance of their changes in predicting preterm birth.
Material and methods. The study included 29 healthy women with uncomplicated pregnancy and delivery at term (control group) and 35 women who developed labor activity at 34-37 weeks (main group). In amniotic fluid taken at 16-18 weeks (by transabdominal amniocentesis), the spectrum of proteins and a number of their physicochemical properties were determined using electrophoresis, chromatography, spectrophotometry and densitometry methods.
Results. It has been established that the development of preterm labor occurs against the background of modification of protein-protein interactions and complexing of proteins with non-protein compounds.
Conclusions. The revealed imbalance of the protein composition may be an important link in the chain of functional and molecular disorders leading to preterm birth.
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Problems of experimental biology and medicine
Study of dosage form based on Eupatorium cannabinum herb extract wound healing activity
Abstract
Relevance. One of the important directions of medical science and practice remains the introduction and improvement of existing methods and means for the treatment of dermatological diseases of various etiologies. At the same time, the search for new wound-healing drugs that can comprehensively affect the wound process is relevant. Practical dermatology has an extensive range of synthetic antimicrobial, anti-inflammatory and wound healing agents. However, despite their known effectiveness, many of them are characterized by the presence of undesirable reactions for the body, such as allergies, dry skin, the formation of resistance, the development of withdrawal syndrome, a decrease in the local and general immunological response of the body. The solution of this problem can be facilitated by the use of an ointment based on a dry extract of the herb Eupatorium cannabinum L., for which, along with known antimicrobial properties, there are grounds for predicting a wound-healing effect.
The aim of the study was to study the wound-healing activity of antimicrobial ointment with a dry extract of cannabis herb in order to predict the possibility of use in medical practice as a polyvalent agent for the treatment of wound processes.
Material and methods. An ointment was developed with a dry extract of cannabis herb, the wound-healing activity of which was determined on a simulated «linear» skin wound in male rats weighing 200.0 ± 20.0 g. An assessment of the restoration of the skin in experimental groups of animals and in control was carried out.
Results. It was revealed that the restoration of the epithelium with the formed dry, pink, natural tissue and wool coating in the groups of rats treated with an experimental soft dosage form occurs earlier than in the control groups treated with a control ointment for an average of 6–8 days.
Conclusions. The results obtained allow us to recommend an ointment with an extract of the herb Eupatirum cannabinum as a means of plant origin, which has a polyvalent, including the wound-healing effect revealed in experiments.
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Toxicological characteristics of tablets with an extract of Potentilla alba L. with repeated administration to rabbits
Abstract
Relevance. In recent years, there has been increased interest in medicinal plants, in particular, Potentilla alba L., used for the treatment of endocrine diseases, on the basis of which the thyroid drug - tablets for oral administration, containing 0,150 g of dry extract of roots and rhizomes of this plant. The dry extract of rhizomes and roots of Potentilla alba L. normalizes the functional activity of the thyroid gland, has an immunomodulatory and hypolipidemic effect in the rat model of experimental hypothyroidism.
Aim. A study of the toxicity of tablets with dry extract of Potentilla alba L. with repeated administration to justify the conduct of clinical trials.
Material and methods. The study of general toxic effects of tablets was carried out on 15 clinically healthy male rabbits of Soviet Chinchilla breed, which were given tablets in doses of 37,5 and 75 mg/kg for 90 days. Control animals received placebo tablets. Integral, hematological and biochemical parameters were recorded, electrocardiograms (ECG) were taken, and pathohistological studies were performed to detect health abnormalities.
Results. Administration of tablets at 9- and 17-fold therapeutic doses had no effect on basic hematological, biochemical and ECG parameters characterizing functional state of liver, kidneys, cardiovascular system of experimental animals. Administration of tablets to rabbits in the tested doses caused a moderate dose-dependent decrease in their body weight gain, possibly related to the hypolipidemic effect. Pathohistological examination revealed changes that were dose-dependent: full-bloodedness of internal organs, including pituitary gland; stimulation of hematopoietic function characterized by increased spleen follicles and their confluence; inhibition of thyroid function and partially of spermatogenesis. At the maximum tested dose of 75 mg/kg, the studied drug had a selective specific pharmacological effect on the pituitary gland, causing a decrease in the size of basophilic cells. No irritating effect of the tablets on the mucosa of the gastrointestinal tract of animals was noted.
Conclusion. The obtained results of the preclinical toxicological examination of tablets 0.150 g were included in the dossier on the drug prepared for transfer to the Ministry of Health of the Russian Federation for obtaining permission to conduct clinical trials.
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Pharmaceutical chemistry
Prediction of the spectrum of biological activity of triterpene saponins using in silico methods
Abstract
Relevance. The main strategic task of the domestic pharmaceutical industry is to provide the population of our country with affordable, high-quality, safe and effective medicines. At the same time, of particular interest for modern pharmacy is the creation of herbal medicines with a multifunctional effect and a high degree of safety. Plants of the Fabaceae family, the practical value of which is due to the rich chemical composition of biologically active substances, can be promising sources for obtaining phytopreparations. The triterpene saponins of plants of the Fabaceae family, which have a wide spectrum of pharmacological action, deserve special attention.
The aim of the work was to study and predict the biological activity of triterpene saponins contained in plants of the Fabaceae family using in silico methods.
Material and methods. The objects of study were the structural formulas of triterpene saponins from plants of the Fabaceae family. Prediction of the biological activity of triterpene saponins was carried out using in silico methods posted on the domestic web resource Way2Drug.
Results. As a result of in silico studies, triterpene saponins demonstrated a high probability of hepatoprotective, proto-tumor, and anti-inflammatory effects. Along with this, at the level of the average value and below for the studied saponins, antibacterial effects were revealed.
Conclusions. The data obtained on the basis of computer screening confirm, clarify and expand the available scientific information on the pharmacological effects of triterpene saponins from plants of the Fabaceae family. The prospects of carrying out further studies in vitro and in vivo with the aim of clarifying and/or confirming the types of activity of biologically active substances from the group of saponins identified in silico and the subsequent development of drugs based on them have been demonstrated.
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Partial validation of the chromato-mass-spectrometric methodology for the determination of carbon sulfur in infusion medicines
Abstract
Relevance. Nowadays the standardization and control of medicines to provide the consumer with high-quality pharmaceutical products is an important issue. The use of parenteral medicines is associated with the potential risk of exposure to the patient's body of carbon disulfide, which is contained in rubber plugs and can pose a serious health hazard.
The purpose of this article is to conduct partial validation of the developed methodology for quantification of carbon disulfide impurities in infusion dosage forms by chromato-mass spectrometry.
Material and methods. The model solution was the drug "Reopoliglyukin, solution for infusion" (JSC BIOCHEMIK, Russia). Methyl ethyl sulfide, 99% (NevaReaktiv, Russia) was used as an internal standard. The measurements were carried out on a chromatography-mass spectrometer model GCMS-QP2010 Ultra, manufactured by Shimadzu, Japan.
Results. The process of sample preparation is described, also the acceptance criteria for determining specificity, linearity, accuracy and limit of quantification were set. The chromatograms and results obtained for the model sample "Rheopolyglucin, solution for infusion" are presented, the coincidence of the peak areas in terms of retention times for the model sample and carbon disulfide, as well as the internal standard, allows us to state that the method meets the requirements for the "Specificity" indicator. The necessary calculations were made to confirm that the analytical methodology is linear. The limit of quantitation was found experimentally by means of a series of dilutions. The correctness was confirmed using the method of additions.
Conclusion. The measures taken made it possible to carry out a partial validation of the analytical methodology for determining carbon disulfide impurities in infusion drugs. It has been determined that all the results obtained meet the indicated acceptance criteria within the selected range of characteristics. Also, it was confirmed that in the indicated concentrations, the methodology has no system error.
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Study of extracts dispersed characteristics isolated from Lycium barbarum L. fruts: prospects for pharmaceutical application
Abstract
Objective. The State Pharmacopoeia of different countries includes the monographs on Lycium fruits and roots, which have antioxidant activity. However, despite the wide prevalence of plants pharmacotherapy, there are no liquid extracts. This is due to insufficient data on the method of production, the water extracts and tinctures composition, as well as studies of their physico-chemical properties.
Materials and methods. The objects of the study were the Lycium barbarum L. fruits from Western Albania. Extractants used: 30%, 70% and 96% ethanol, purified water (Milli-Q® Integral unit). Extracts were obtained by maceration, according to the Monograph no.1.4.1.0021.15 "Extracts". Studies of the dispersed properties of the isolated colloids – nanoparticle size (d, nm), electrokinetic potential (ζ, mV), polydispersity index (PDI) – were determined by photon correlation spectroscopy method (dynamic light scattering, DLS) and Zetasizer Nano ZS, Malvern, UK.
Results. Samples of Lycium prepared using the technology for water extracts and tinctures showed significant differences in dispersed characteristics, including cumulative (z-average) size, polydispersity index (PDI) and zeta of potential colloidal particles. It is shown that the values of the ζ-potential characterizing the stability of colloids exceed the boundary value of the stability of dispersed systems by 30 mV in water extracts, while in tinctures they have significantly lower values, hence stability. The addition of electrolytes to the LE samples contributed to the structural and mechanical stabilization of dispersed systems (according to Rebinder).
Conclusion. Methods of water extracts and tinctures obtaining and stabilizing, as well as control of their dispersed properties with weekly kinetics, open up new opportunities for the use of promising plant raw materials of Lycium barbarum L. to solve pharmaceutical problems in the search for new drugs.
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Antiradical activity of 2,5-diaryl-8,8-dimethyl- 3,6,7,8-tetrahydro-2H-pyrido [4,3,2-de]cinnolin-3-ones
Abstract
Relevance. The development of most pathological processes is based on oxidative stress, which is the result of an imbalance between free radicals and antioxidants. The presence of an unpaired electron provides a high reactivity of free radicals and allows easy reaction with cell compartments, which leads to disruption of their work. Free radical oxidation of cellular structures underlies the pathogenesis of many diseases (oncological, cardiovascular, neurodegenerative, etc.), as well as the aging process. Thus, free radicals can be considered as a target for new antioxidants.
Target. Investigation of antiradical activity of synthesized 2,5-diaryl-8,8-dimethyl-3,6,7,8-tetrahydro-2H-pyrido [4,3,2-de]cinnolin-3-ones.
Material and methods. 2,5-Diaryl-8,8-dimethyl-3,6,7,8-tetrahydro-2H-pyrido [4,3,2-de]cinnolin-3-ones were obtained as a result of the interaction of 2-aryl-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydroquinoline-4-carboxylic acids with o-tolyl-, p-tolyl-, p-fluorine, p-nitro-, 2,4,6-trichloro-phenylhydrazines. As a result, 10 compounds were synthesized, the structure of which was confirmed by IR and 1H NMR spectroscopy. The antiradical activity of 10 compounds was studied using the method of binding a stable radical with 2,2-diphenylpicryl-1-hydrazyl (DPPH).
Conclusion. Compounds of a number of 2,5-diaryl-8,8-dimethyl- 3,6,7,8-tetrahydro-2H-pyrido[4,3,2-de]cinnoline-3-ones don’t have pronounced antiradical activity, which indicates the presence of another mechanism of antioxidant action.
Conclusions. Further investigation will reveal the mechanism of antioxidant action of 2,5-diaryl-8,8-dimethyl-3,6,7,8-tetrahydro-2H-pyrido[4,3,2-de]cinnoline-3-ones, therefore it is advisable.
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