Terpene-Containing Analogues of Glitazars as Potential Therapeutic Agents for Metabolic Syndrome.

Metabolic syndrome is a complex of abnormalities involving impaired glucose and lipid metabolism, which needs effective pharmacotherapy. One way to reduce lipid and glucose levels associated with this pathology is the simultaneous activation of nuclear PPAR-alpha and gamma. For this purpose, we synthesized a number of potential agonists based on the pharmacophore fragment of glitazars with the inclusion of mono- or diterpenic moiety in the molecular structure. The study of their pharmacological activity in mice with obesity and type 2 diabetes mellitus (C57Bl/6Ay) revealed one substance that was capable of reducing the triglyceride levels in the liver and adipose tissue of mice by enhancing their catabolism and expressing a hypoglycemic effect connected with the sensitization of mice tissue to insulin. It has also been shown to have no toxic effects on the liver.

Synthesis and Evaluation of Hypoglycemic Activity of Structural Isomers of ((Benzyloxy)phenyl)propanoic Acid Bearing an Aminobornyl Moiety.

Free fatty acid receptor-1 (FFAR1) agonists are promising candidates for therapy of type 2 diabetes because of their ability to normalize blood sugar levels during hyperglycemia without the risk of hypoglycemia. Previously, we synthesized compound QS-528, a FFA1 receptor agonist with a hypoglycemic effect in C57BL/6NCrl mice. In the present work, structural analogs of QS-528 based on (hydroxyphenyl)propanoic acid bearing a bornyl fragment in its structure were synthesized. The seven novel compounds synthesized were structural isomers of compound QS-528, varying the positions of the substituents in the aromatic fragments as well as the configuration of the asymmetric center in the bornyl moiety. The studied compounds were shown to have the ability to activate FFAR1 at a concentration of 10 µM. The cytotoxicity of the compounds as well as their effect on glucose uptake in HepG2 cells were studied. The synthesized compounds were found to increase glucose uptake by cells and have no cytotoxic effect. Two compounds, based on the meta-substituted phenylpropanoic acid, 3-(3-(4-(((1R,2R,4R)-1,7,7-trimethylbicyclo-[2.2.1]heptan-2-ylamino)methyl)benzyloxy)phenyl)propanoic acid and 3-(3-(3-(((1R,2R,4R)-1,7,7-trimethylbicyclo [2.2.1]heptan-2-ylamino)methyl)benzyloxy)phenyl)propanoic acid, were shown to have a pronounced hypoglycemic effect in the oral glucose tolerance test with CD-1 mice.

Study of Hypoglycemic Activity of Novel 9-N-alkyltetrahydroberberine Derivatives.

Novel 9-N-alkyltetrahydroberberine derivatives were synthesized, among which, based on the results of OGTT, one compound containing the longest aliphatic substituent was selected for study in mice C57BL/6Ay, which demonstrate obesity, impaired glucose tolerance, and concomitant liver non-alcoholic fatty disease. Administration of this substance at a dose of 15 mg/kg for four weeks improved the insulin sensitivity of mice, which resulted in a decrease in fasting glucose levels and improved the tolerance of mice to OGTT glucose loading. A decrease in the level of lactate in the blood and a decrease in the amount of glucokinase in the liver were also found. The introduction of compound 3c did not have a toxic effect on animals based on biochemical data, histological analysis, and measurements of general parameters such as body weight and feed intake. Thus, the 9-N-heptyltetrahydroberberine derivative showed prominent hypoglycemic effects, which makes it promising to obtain and study other derivatives with longer substituents.

Solubility, Permeability, Anti-Inflammatory Action and In Vivo Pharmacokinetic Properties of Several Mechanochemically Obtained Pharmaceutical Solid Dispersions of Nimesulide.

Nimesulide (NIM, N-(4-nitro-2-phenoxyphenyl)methanesulfonamide) is a relatively new nonsteroidal anti-inflammatory analgesic drug. It is practically insoluble in water (<0.02 mg/mL). This very poor aqueous solubility of the drug may lead to low bioavailability. The objective of the present study was to investigate the possibility of improving the solubility and the bioavailability of NIM via complexation with polysaccharide arabinogalactan (AG), disodium salt of glycyrrhizic acid (Na2GA), hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and MgCO3. Solid dispersions (SD) have been prepared using a mechanochemical technique. The physical properties of nimesulide SD in solid state were characterized by differential scanning calorimetry and X-ray diffraction studies. The characteristics of the water solutions which form from the obtained solid dispersions were analyzed by reverse phase and gel permeation HPLC. It was shown that solubility increases for all complexes under investigation. These phenomena are obliged by complexation with auxiliary substances, which was shown by 1H-NMR relaxation methods. The parallel artificial membrane permeability assay (PAMPA) was used for predicting passive intestinal absorption. Results showed that mechanochemically obtained complexes with polysaccharide AG, Na2GA, and HP-ß-CD enhanced permeation of NIM across an artificial membrane compared to that of the pure NIM. The complexes were examined for anti-inflammatory activity on a model of histamine edema. The substances were administered per os to CD-1 mice. As a result, it was found that all investigated complexes dose-dependently reduce the degree of inflammation. The best results were obtained for the complexes of NIM with Na2GA and HP-ß-CD. In noted case the inflammation can be diminished up to 2-fold at equal doses of NIM.

Effect of Glycyrrhizic Acid and Arabinogalactan on the Membrane Potential of Rat Thymocytes Studied by Potential-Sensitive Fluorescent Probe.

The effect of the natural saponin glycyrrhizic acid (GA) and polysaccharide arabinogalactan (AG) on the transmembrane potential of rat thymocytes was investigated using the potential-sensitive fluorescent probe 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). Incubation of cells with GA in micellar form resulted in a decrease of the amplitude of observed fluorescence kinetics that points out to a decrease of the transmembrane potential. The proposed mechanism is an increase of membrane ion permeability (passive ion transport) of the plasma cell membrane due to GA incorporation. The incorporation of GA molecules into the cell membrane is extremely sensitive to the degree of GA dissociation. The neutral form of glycyrrhizic acid enters the lipid bilayer in contrast to the deprotonated anionic form. The incubation of rat thymocytes with anionic form of GA, namely with its disodium salt, has no effect on the fluorescence kinetics. The possible reasons of this phenomenon are discussed in the light of the nuclear magnetic resonance (NMR) and molecular dynamics (MD) data. The treatment of thymocytes with AG affects only the initial rate of the probe incorporation. The proposed mechanism is that AG covers the surface of the cell membrane and forms a barrier for the probe. Additionally, our experiments demonstrated that both polysaccharide AG and GA in the neutral form (but not Na2GA) effectively capture the cationic probe in an aqueous solution and then deliver it to the cell membrane.

The Potential of Usnic-Acid-Based Thiazolo-Thiophenes as Inhibitors of the Main Protease of SARS-CoV-2 Viruses.

Although the COVID-19 pandemic caused by SARS-CoV-2 viruses is officially over, the search for new effective agents with activity against a wide range of coronaviruses is still an important task for medical chemists and virologists. We synthesized a series of thiazolo-thiophenes based on (+)- and (-)-usnic acid and studied their ability to inhibit the main protease of SARS-CoV-2. Substances containing unsubstituted thiophene groups or methyl- or bromo-substituted thiophene moieties showed moderate activity. Derivatives containing nitro substituents in the thiophene heterocycle-just as pure (+)- and (-)-usnic acids-showed no anti-3CLpro activity. Kinetic parameters of the most active compound, (+)-3e, were investigated, and molecular modeling of the possible interaction of the new thiazolo-thiophenes with the active site of the main protease was carried out. We evaluated the binding energies of the ligand and protein in a ligand-protein complex. Active compound (+)-3e was found to bind with minimum free energy; the binding of inactive compound (+)-3g is characterized by higher values of minimum free energy; the positioning of pure (+)-usnic acid proved to be unstable and is accompanied by the formation of intermolecular contacts with many amino acids of the catalytic binding site. Thus, the molecular dynamics results were consistent with the experimental data. In an in vitro antiviral assay against six strains (Wuhan, Delta, and four Omicron sublineages) of SARS-CoV-2, (+)-3e demonstrated pronounced antiviral activity against all the strains.

Hydroxyapatite Double Substituted with Zinc and Silicate Ions: Possibility of Mechanochemical Synthesis and In Vitro Properties.

In this study, the mechanochemical synthesis of substituted hydroxyapatite (HA) containing zinc and silicon ions having a chemical formula of Ca10-xZnx(PO4)6-x(SiO4)x(OH)2-x, where x = 0.2, 0.6, 1.0, 1.5, and 2.0, was carried out. The synthesized materials were characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and inductively coupled plasma spectroscopy. We found that HA co-substituted with zinc and silicate formed up to x = 1.0. At higher concentrations of the substituents, the formation of large amounts of an amorphous phase was observed. The cytotoxicity and biocompatibility of the co-substituted HA was studied in vitro on Hek293 and MG-63 cell lines. The HA co-substituted with zinc and silicate demonstrated high biocompatibility; the lowest cytotoxicity was observed at x = 0.2. For this composition, good proliferation of MG-63 osteoblast-like cells and an increased solubility compared with that of HA were detected. These properties allow us to recommend the synthesized material for medical applications, namely, for the restoration of bone tissue and manufacture of biodegradable implants.

The main protease 3CLpro of the SARS-CoV-2 virus: how to turn an enemy into a helper.

Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli, problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli. We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists.

Bornyl-Containing Derivatives of Benzyloxyphenylpropanoic Acid as FFAR1 Agonists: In Vitro and In Vivo Studies.

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases worldwide. Several classes of hypoglycemic drugs are used to treat it, but various side effects limit their clinical use. Consequently, the search for new anti-diabetic agents remains an urgent task for modern pharmacology. In this investigation, we examined the hypoglycemic effects of bornyl-containing benzyloxyphenylpropanoic acid derivatives (QS-528 and QS-619) in a diet-induced model of T2DM. Animals were given the tested compounds per os at a dose of 30 mg/kg for 4 weeks. At the end of the experiment, compound QS-619 demonstrated a hypoglycemic effect, while QS-528 showed hepatoprotection. In addition, we performed a number of in vitro and in vivo experiments to study the presumed mechanism of action of the tested agents. Compound QS-619 was determined to activate the free fatty acid receptor-1 (FFAR1) similarly to the reference agonist GW9508 and its structural analogue QS-528. Both agents also increased insulin and glucose-dependent insulinotropic polypeptide concentrations in CD-1 mice. Our results indicate that QS-619 and QS-528 are probably full FFAR1 agonists.

Corrigendum: The main protease 3CLpro of the SARS-CoV-2 virus: how to turn an enemy into a helper.

[This corrects the article DOI: 10.3389/fbioe.2023.1187761.].

9-N-n-alkyl Berberine Derivatives: Hypoglycemic Activity Evaluation.

Several novel 9-N-n-alkyl derivatives of berberine (C5, C7, C10, C12) were synthesized. They were analyzed in vitro and in vivo for their hypoglycemic activity. In vitro studies showed that the derivatives with shorter alkyl substitutes at concentrations ranging from 2.5 to 10 µM were able to stimulate glucose consumption by HepG2 cells more prominently than the derivatives with longer substitutes (C10 and C12). All compounds demonstrated a better effect compared to berberine. Their impact on cells' viability also depended on the alkyl substitutes length, but in this case, C10 and C12 derivatives demonstrated the best results. A similar correlation was also found in the OGTT, where the C5 derivative demonstrated a pronounced hypoglycemic effect at a dose of 15 mg/kg and C12 was less effective. This compound was further investigated in C57BL/6Ay mice for four weeks and was administered at a dose of 15 mg/kg. Pronounced effect of C12 on carbohydrate metabolism in mice was discovered: there was a decrease in fasting glucose levels and an increase in glucose tolerance in OGTT on the 14th and 28th days of the experiment. However, at the end of the experiment, signs of hepatosis exacerbation and an increase in the content of hepatic aminotransferases in blood were found.

Discovery of the First in Class 9-N-Berberine Derivative as Hypoglycemic Agent with Extra-Strong Action.

Berberine is well known for its ability to reduce the blood glucose level, but its high effective dose and poor bioavailability limits its use. In this work we synthesized a new derivative of berberine, 9-(hexylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride (SHE-196), and analyzed the profile of its hypoglycemic effects. Biological tests have shown that the substance has a very pronounced hypoglycemic activity due to increased insulin sensitivity after single and multiple dosing. In obese type 2 diabetes mellitus (T2DM) mice, it was characterized by improved glucose tolerance, decreased fasting insulin levels and sensitivity, decreased total body weight and interscapular fat mass, and increased interscapular brown fat activity. All these effects were also confirmed histologically, where a decrease in fatty degeneration of the liver, an improvement in the condition of the islets of Langerhans and a decrease in the size of fat droplets in brown adipose tissue were found. Our results indicate that 9-(hexylamino)-2,3-methylenedioxy-10-methoxyprotoberberine chloride could be the first in a new series of therapeutic agents for the treatment of diabetes mellitus.

Detonation Spraying of Hydroxyapatite on a Titanium Alloy Implant.

Hydroxyapatite (HA), the major mineral component of tooth enamel and natural bones, is a good candidate for bone tissue engineering. Synthetic HA is used for making coatings on metallic implants intended for medical applications. A HA coating renders the implant biocompatible and osteoinductive. In addition, it improves fixation and the overall performance of the implanted object. In the present work, HA coatings were deposited on a medical titanium alloy implant with mesh geometry and a developed surface by detonation spraying. The feedstock powder was HA obtained by the dry mechanochemical method. Single-phase HA coatings were obtained. The coatings were formed not only on the surfaces normal to the particle flow direction, but also on the sides of the mesh elements. Despite partial melting of the powder, no decomposition of HA occurred. This work demonstrates the prospects of detonation spraying for the production of HA coatings on metallic implants with complex geometries.

Development and validation of an LC-MS/MS method for the quantitative analysis of the anti-influenza agent camphecene in rat plasma and its application to study the blood-to-plasma distribution of the agent.

A method of quantitative determination of camphecene, a new anti-influenza agent, in rat blood plasma based on LC-MS/MS was developed, validated and used to study the distribution of the agent between blood cells and blood plasma. The method was validated according to FDA and EMA recommendations in terms of selectivity, linearity, accuracy, precision, recovery, stability and carry-over. Plasma samples were precipitated with methanol followed by the addition of a methanolic solution of 2-adamantylamine hydrochloride (internal standard). HPLC analysis was performed on a reversed-phase column; the total time of analysis was 11 min, including column equilibration. MS/MS detection was performed on a 3200 QTRAP triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. Transitions 196.4 → 122.2/153.3 and 152.2 → 93.1/107.2 were monitored for camphecene and the internal standard, respectively. The calibration curve was built in the concentration range of 10-5000 ng/ml. The intra-day and inter-day accuracy and precision, carry-over and recovery were within the acceptable limits. It was found that, after spiking blood with camphecene and separating plasma, the concentration of the substance in the latter was close to its initial concentration in the blood. This property of the substance may be useful for clinical trials of the agent. It has also been established that the process of camphecene distribution (adsorption) between blood cells and blood plasma is reversible, and the amount of adsorbed substance is linearly dependent on its initial concentration in the blood for a wide range of concentrations, temperatures and hematocrit values.

Biodegradable poly(l-lactide)/calcium phosphate composites with improved properties for orthopedics: Effect of filler and polymer crystallinity.

Biodegradable poly(l-lactide)/calcium phosphate composites are promising materials for fabrication of bone fixation implants with improved properties. Multistage compounding was proposed as an efficient method for the preparation of biodegradable poly(l-lactide)/calcium phosphate composites with submicron filler dispersion and mechanical characteristics similar to native bone. The improvement of the characteristics is caused both by the filler itself and by the increase of polymer crystallinity due to the nucleation effect. The technique allows to fabricate biodegradable composites with controlled properties by varying concentration and type of the filler as well as degree of PLLA matrix crystallinity. Animal studies revealed that all the composites were biocompatible and non-toxic.

Preparation, physicochemical and pharmacological study of curcumin solid dispersion with an arabinogalactan complexation agent.

Pharmaceutical solid dispersions (SD) of curcumin (Cur) with macromolecule polysaccharide arabinogalactan (AG) from wood of Larix sibirica were prepared by mechanical ball milling. The physical properties of the dispersed curcumin mixture in solid state were characterized by scanning electron microscope, differential scanning calorimetry and powder X-ray diffraction studies. These methods showed a strong decrease in the degree of crystallinity of Cur and its transformation to amorphization state, accompanied by the formation of the guest-host type complexes. The behavior of the samples in solutions was characterized by reverse phase HPLC, 1H NMR spectroscopy, UV-Visible spectroscopy and gel permeation chromatography (GPC). Mechanochemically prepared complexes demonstrated the increased solubility of Cur up to ~10.5 times in contrast to pure curcumin. The rapid storage test showed high chemical stability of Cur, which depended on mass relations of Cur-AG. Besides, improved membrane permeability of Cur-AG SD was tested by parallel artificial membrane permeability assay. Pharmacokinetic study of Cur-AG SD formulation in rat demonstrated a significant~8-fold enhancement of bioavailability in comparison to pure curcumin. In MTT tests, Cur-AG SD showed moderate cytotoxicity against human glioblastoma cells and immortalized human fibroblasts. Therefore, Cur-AG solid dispersion was a more promising and efficacious formulation for application in oral drug delivery.

Preparation of curcumin self-micelle solid dispersion with enhanced bioavailability and cytotoxic activity by mechanochemistry.

An amorphous solid dispersion (SD) of curcumin (Cur) with disodium glycyrrhizin (Na2GA) was prepared by mechanical ball milling. Curcumin loaded micelles were self-formed by Na2GA when SD dissolved in water. The physical properties of Cur SD in solid state were characterized by differential scanning calorimetry, X-ray diffraction studies, and scanning electron microscope. The characteristics of the sample solutions were analyzed by reverse phase HPLC, UV-visible spectroscopy, 1H NMR spectroscopy, gel permeation LC, and transmission electron microscopy. In vitro cytotoxic tests demonstrated that Cur SD induced higher cytotoxicity against glioblastoma U-87 MG cells than free Cur. Besides, an improvement of membrane permeability of Cur SD was confirmed by parallel artificial membrane permeability assay. Further pharmacokinetic study of this SD formulation in rat showed a significant ∼19-fold increase of bioavailability as comparing to free Cur. Thus, Cur SD provide a more potent and efficacious formulation for Cur oral delivery.

Atorvastatin calcium inclusion complexation with polysaccharide arabinogalactan and saponin disodium glycyrrhizate for increasing of solubility and bioavailability.

The aim of the present investigation was to enhance the solubility and dissolution of atorvastatin calcium (ATV), a poorly water-soluble drug with larch polysaccharide arabinogalactan (AG) and disodium glycyrrhizate (Na2GA) as carriers of drug delivery systems for improving its bioavailability. The interactions of ATV with AG or Na2GA were investigated by DSC, XRD, SEM, and NMR techniques. The molecular weights of supramolecular systems-inclusion complexes and micelles-which are the hosts for ATV molecules were measured. On the other hand, the rapid storage assay (+ 40 °C for 3 months) showed that the chemical stability of ATV/AG and ATV/Na2GA complexes had been enhanced compared with pure ATV. In vitro drug release showed a significant increase in ATV's dissolution rate after formation of a complex with Na2GA or AG. Pharmacokinetic tests in vivo on laboratory animals showed a significant increase in ATV's bioavailability after its introduction as a complex with Na2GA or AG. Moreover, ATV/AG and ATV/Na2GA complexes showed a more prominent decrease of total cholesterol (TC) level compared to net ATV. Therefore, the novel mechanochemically synthesized complexes of ATV with AG or Na2GA as drug delivery systems might be potential and promising candidates for hypercholesterolemia treatment and deserved further researches.

Enhanced solubility and bioavailability of simvastatin by mechanochemically obtained complexes.

In the present work, complexes of simvastatin (SIM) with polysaccharide arabinogalactan (AG) or disodium salt of glycyrrhizin acid (Na2GA) have been prepared using mechanochemical technique to improve the solubility of SIM and enhance its oral bioavailability. The interactions of SIM with AG or Na2GA were investigated by FTIR, DSC, XRD and SEM. Self-association of SIM in various solvents was investigated by UV/Vis and NMR techniques. The molecular masses of supramolecular systems-inclusion complexes and micelles, which are the "hosts" for SIM molecules were measured. The parallel artificial membrane permeability assay (PAMPA) revealed a strong increasing of SIM permeability in the presence of Na2GA in comparison with pure SIM used as a control. On the other hand, the rapid storage assay (+40°C for 3 months) showed that the chemical stability of SIM/AG complexes was similar to pure SIM, but SIM/Na2GA complexes had an enhanced stability. Pharmacokinetic tests in vivo on laboratory animals showed a significant increase in SIM's bioavailability after its introduction as a complex with Na2GA or AG. Moreover, SIM/AG inclusion complex performed better than SIM in reducing total cholesterol level. Therefore, the mechanochemically synthesized complexes of SIM with AG or Na2GA might have a promising future as novel formulations for hyper-cholesterolemia treatment.

Supramolecular Complex of Ibuprofen with Larch Polysaccharide Arabinogalactan: Studies on Bioavailability and Pharmacokinetics.

BACKGROUND AND OBJECTIVES: In the present work, pharmacological and pharmacokinetic properties of the supramolecular complex of non-steroid anti-inflammatory drug ibuprofen (IBU) with natural polysaccharide arabinogalactan (AG) were studied. The main goals of such complexation were the increase of ibuprofen's bioavailability and decrease its effective dose after oral administration. METHODS: The complex with mass ratio as IBU:AG 1:10 was obtained by mechanochemical synthesis and characterized by water solubility, electron microscopy, differential scanning calorimetry, X-ray powder diffraction analysis and 1H-nuclear magnetic resonance spectroscopy. Different animal models of pain and inflammation was used to investigate IBU:AG biological effects. Plasma concentration of IBU and its pharmacokinetic parameters were evaluated after oral introduction. RESULTS: It was found that ibuprofen's effective analgesic and anti-inflammatory dose decreased twofold after its introduction as a complex with AG. The reason of this difference is due to the increase of ibuprofen concentration in rats' plasma: C max of IBU at doses of 20 and 40 mg/kg was found as 0.088 and 0.132 µg/ml, whereas C max of IBU in the complex form was 0.103 and 0.160 µg/ml, respectively. CONCLUSIONS: Thus, we have shown that complexation of the IBU with AG results in its bioavailability increase, reduction of the effective dose and should decrease toxic side effects.