Pharmaceutical Chemistry Journal (v.52, #7)

Experimental Studies of the Effects of Progesterone on the Functional Activity of P-Glycoprotein by A. V. Shchul’kin; I. V. Chernykh; E. N. Yakusheva; N. M. Popova; P. Yu. Myl’nikov (587-592).
Experiments on female Chinchilla rabbits addressed the effects of progesterone on the functional activity of P-glycoprotein (Pgp). Animals were divided into four groups: animals of group 1 underwent sham operations; group 2 underwent ovariectomy; groups 3 and 4 underwent ovariectomy followed 15 days later by p.o. treatment with progesterone (2 and 15 mg/rabbit respectively). The functional activity of Pgp was determined seven days before the experiment started and on post-operative days 14, 28, and 42 by analysis of the pharmacokinetics of its marker substrate fexofenadine and serum sex hormone concentrations. At the end of the study, animals of each group were harvested by air embolism and specimens of large intestine were collected for immunohistochemical analysis. Ovariectomy led to a decrease in the functional activity of Pgp and a decrease in the staining intensity of the apical and lateral membranes of enterocytes, providing indirect evidence of a decrease in the expression of the transporter protein. Administration of progesterone at a dose of 2 mg/rabbit on the background of ovariectomy increased the functional activity of Pgp from the level seen in the ovariectomy group, though its activity remained decreased compared with the baseline level. This group showed no changes in the staining intensity of enterocyte membranes as compared with the ovariectomy group. Use of progesterone at a dose of 15 mg/rabbit after ovariectomy increased the functional activity of Pgp and the staining intensity of enterocyte apical and lateral membranes from the level seen in the ovariectomy group, restoring the level of transporter protein activity to that of intact animals, probably because of increased expression.
Keywords: P-glycoprotein; functional activity; fexofenadine; expression; immunohistochemical method; progesterone

Effects of Novel Potential Analgesic Compounds on the Cardiovascular and Respiratory Systems by Yu. A. Palikova; L. A. Skobtsova; V. A. Palikov; G. I. Belous; O. N. Khokhlova; A. V. Lobanov; G. A. Slashcheva; D. I. Rzhevskii; V. B. Rudenko; E. A. Kalabina; G. A. Osipova; Ya. A. Andreev; Yu. A. Logashina; S. A. Kozlov; A. N. Yavorskii; G. Elyakova; I. A. D’yachenko (593-595).
The polypeptide analgesic compounds APCH3 (a TRPV1 receptor inhibitor) and PT1 (a P2X3 receptor inhibitor) were shown not to act on the cardiovascular system or respiratory system when given either as single or multiple doses in mice. The low molecular weight compound sevanol (an ASIC3 receptor inhibitor) had no effect on the cardiovascular system, but prolonged use for 14 days affected measures of the respiratory system, significantly increasing respiratory rate and peak expiratory flow rate.
Keywords: APCH3; sevanol; PT1; safety; cardiovascular system; respiratory system

Bioactivity of Three Salvia Species in Relation to Their Total Phenolic and Flavonoid Contents by Farukh Sharopov; Abdujabbor Valiev; Mansour Sobeh; Eva Arnold; Michael Winka (596-600).
Antioxidant, anti-inflammatory, cytotoxic activities and total phenolic and flavonoid contents of methanol extracts of three Salvia species including Salvia discolor, S. officinalis and S. sclarea were investigated. The compositions of extracts were analyzed by high performance liquid chromatography (HPLC) with a UV-DAD detector. The major component of extracts from aerial parts of Salvia species was rosmarinic acid (0.2 – 61.1%). The composition of methanol extract from aerial parts strongly differed from that of the root extract. The total phenolic and flavonoid contents per 100 g fresh weight ranged within 658.3 – 1805.9 mg of caffeic acid equivalent (CAE) and 13.0 – 184.9 mg of quercetin equivalent (QE), respectively. The highest DPPH radical scavenging effect was observed for S. sclarea extract (with an IC50 of 21.6 μg/mL), while S. discolour extract exhibited the highest ABTS•+ radical scavenging ability (with IC50 of 53.0 μg/mL) among all extracts. All Salvia extracts showed remarkable cytotoxic effect toward human T-lymphoblast leukemia (CCRF-CEM) and adriamycin resistant leukemia (CEM/ADR 5000) with average IC50 values of 25.3 – 65 μg/mL and 34.5 – 89.8 μg/mL, respectively. A relatively strong effect against human breast adenocarcinoma (MCF-7) was observed for the S. officinalis extract, with an IC50 of 138.5 μg/mL.
Keywords: Salvia discolor ; Salvia officinalis ; Salvia sclarea ; cytotoxicity; rosmarinic acid

With the aim of establishing a structure-analgesic activity relationship, a series of picolylamides of 2-hydroxy-4-oxo-4H-pyrido[1,2-a]-pyrimidine-3-carboxylic acids were synthesized. The characteristics of the spatial structure of this group of substances were assessed. The results of pharmacological studies identified compounds in the study series with marked analgesic properties. However, it was noted that the transition from 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides to the structurally similar pyrido[1,2-a]pyrimidine derivatives was generally accompanied by some decrease in analgesic activity.
Keywords: amidation; heterocylic tricarbonylmethane derivatives; 2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamides; synthesis; analgesic activity

Synthesis, Structure, and Anticaries Activity of 2-Amino-4,6-Dihydroxypyrimidinium Hexafluorosilicate by V. O. Gel’mbol’dt; V. Yu. Anisimov; I. O. Shishkin; M. S. Fonar’; V. Kh. Kravtsov (606-610).
We report here the synthesis and our studies of the structure, physicochemical properties, and anticaries activity of a novel potential anticaries substance 2-amino-4,6-dihydroxypyrimidinium hexafluorosilicate. IR spectra, EI mass spectra, and 19F NMR spectra were obtained and their properties were studied. Biological investigations were performed on animals kept on the Stephan caries-inducing diet (50% sucrose). Alkaline phosphatase (AlkP) and acid phosphatatase (AcP) were assayed in pulp homogenates and the AlkP:AcP ratio was used to assess the mineralization index (MI). AlkP activity was measured in serum, along with alanine aminotransferase (ALT). The number and depth of caries lesions to the teeth were assessed and caries prophylactic efficacy (CPE) was computed. The data showed that 2-amino-4,6-dihydroxypyrimidinium hexafluorosilicate decreased the number of caries lesions by 45.5% and had high anticaries efficacy, five times greater than that of sodium fluoride.
Keywords: 2-amino-4,6-dihydroxypyrimidinium hexafluorosilicate; structure; anticaries activity

Synthesis and some Pharmacological Properties of an Immunoactive Lysine-Containing Tetrapeptide by N. D. Bunyatyan; B. M. Kholnazarov; G. M. Bobizoda; Yu. V. Olefir; A. B. Prokof’ev; M. M. Sapovskii; V. A. Evteev (611-614).
The potentially immunoactive lysine-containing tetrapeptide H-Lys-Ala-Val-Gly-OH was synthesized. The value of using a method based on mixed anhydrides and activated esters to obtain tetrapeptide at high yield was demonstrated. The peptide preparation method was improved by replacing p-nitrophenyl esters at the intermediate stage with N-hydroxysuccinimide esters, yielding a crystalline substance at high purity, and replacing the BOC (tert-butyloxycarbonyl) group for protection with a carbobenzoxy group, which provided for a one-step removal of the protection of both amino groups in mild conditions. Comparative assessment of the immunological activity of the tetrapeptide on the background of immunization with sheep erythrocytes in mice with other lysine-containing tetrapeptides showed it to have more marked immunostimulatory activity. Studies of acute and chronic toxicity demonstrated the absence of toxic activity in the lysine-containing tetrapeptide studied here on s.c. administration to experimental animals. The tetrapeptide corresponded to toxicity class 5, i.e., it was essentially nontoxic.
Keywords: amino acids; peptide bioregulators; lysine; peptide synthesis; immunoactivity; acute toxicity

Preparation and In Vitro/In Vivo Evaluation of Antihistaminic Ocular Inserts by Syed Nisar Hussain Shah; Ahmad Nawaz; Hina Javed; Misbah Rafiq; Romana Riaz; Hadia Sadaquat; Muhammad Akhtar (615-622).
The present study aims to prepare ocular inserts of cetirizine hydrochloride, an antihistamine used to treat allergic conjunctivitis, allergic rhinitis, uveitis and urticaria. Solvent casting method was used to prepare inserts of hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol (PVA) as film forming polymers, and polyethylene glycol (PG) and glycerin were used as plasticizers. Various physicochemical parameters of six ocular insert compositions were evaluated, including swelling behavior and in vitro drug release and kinetics. Formulation F4 showed results of in vitro studies in most appropriate range, with sustained drug release (99.12% at 210 min) following non-fickian diffusion. Drug – polymer compatibility study verified no chemical interaction between the drug and polymer. Therefore, formulation F4 was selected for in vivo studies in albino rabbits. The values of C max, AUC, and T max of formulation F4 were 478.70 ng/mL, 3013.58 ng/(mL h), and 2.3 h, respectively. Ocular inserts of cetirizine hydrochloride provide a valuable alternative for the treatment of ocular inflammatory diseases as these formulations ensure sustained drug release and increased residence time.
Keywords: cetirizine; hydroxypropyl methylcellulose; ocular inserts; polyvinyl alcohol; solvent casting

The accumulation of inulin in the dried rhizomes with roots of Echinacea purpurea cultivated in the non-chernozem zone of the Russian Federation in 2008 – 2010 was studied in plants of different ages (2, 3, 4, 5, and 7 years of vegetation) and times of harvesting the above-ground parts (in the phenological phases of stem extension, budding, flowering, and repeatedly during the vegetation period). Inulin contents were also studied in infusions made from this species in 40% and 70% ethanol with different particle sizes after passage through sieves with mesh sizes of 0.1, 0.5, and 1 mm. The most extensive accumulation of inulin in material in 2008 and 2009 was seen after repeated removal of shoots, and in 2010 after harvesting of the above-ground part in the stem extension phase. Increases in the age of the producing plants to seven years of vegetation was associated with a significant decrease in the raw material inulin content. The infusion in 40% ethanol contained much more inulin than the infusion in 70% ethanol, i.e., this was a water-soluble polyfructan. The greatest quantity of inulin was extracted form raw material particles of 1 mm. Meal contained about 10% inulin, allowing its further use.
Keywords: inulin-like fructosans; Echinacea purpurea ; rhizomes with roots; infusions; meal

Comparative Analysis of the Heavy Metal, Aluminum, and Arsenic Contents in Brown Algae of Various Origins by V. M. Shchukin; N. E. Kuz’mina; A. A. Erina; V. A. Yashkir; V. A. Merkulov (627-634).
The results of measurements of trace element contents in brown algae of the families Laminariales and Fucales of different origins are presented, along with supplementary data from the literature. Aluminum, cadmium, cobalt, chromium, copper, iron, mercury, manganese, nickel, lead, and zinc concentrations in the biomass of brown algae were found to be affected primarily by the technogenic factor, while strontium concentrations are affected by the geographic latitude of the region in which they grew. In the case of arsenic, no relationship was found between accumulation in algae and the level of pollution or the geographic region in which the algae were collected. Arsenic was found to accumulate better in Laminariales, while aluminum, iron, copper, and manganese in brown algae of the family Fucales. As regards cadmium, mercury, lead, nickel, zinc, chromium, and strontium, differences in accumulation in members of the two families were comparable with differences between species within a single family. We conclude that there is a need to develop additional criteria for standardization of brown algae.
Keywords: brown algae; Laminariales; Fucales; heavy metals; arsenic; aluminum; standardization; medicinal plant material; medicinal herbal formulations

The physicochemical and technological properties of an interpolymer complex of polymethyacrylic acid and polyethylene glycol, known under its commercial name “Polymer Carrier Composition” (FGUP Science Research Institute of Polymers, Russia), were studied. This polymer is an excipient substance used in the technology of solid dosage forms as a matrix former and as a film-forming agent for making long-acting and slow-release formulations. With the aim of widening the potential uses of the polymer carrier composition, the solubility of the polymer in aqueous solutions and organic solvents was studied, along with the technological and surfactant properties of the polymer; the critical gel-forming concentration was also determined. Experimental samples of tablets and oral gels of model substances with similar physicochemical properties were prepared, based on different concentrations of the Polymer Carrier Composition, and the technological, rheological, and biopharmaceutical properties of these were studied. The potential for using the polymer in both hard and soft dosage forms to obtain prolonged-release medicinal formulations is demonstrated.
Keywords: interpolymer complex; Polymer Carrier Composition; matrix systems; gels; tablets

Optimization of Phenazepam Production Technology by O. I. Lukashov; P. V. Kazakov; N. S. Mirzabekova (641-643).
A new means of preparing phenazepam is proposed. The new manufacturing technology for phenazepam implements the general principles for medicinal substance quality by using synthesis conditions and schemes yielding compounds as pure as possible.
Keywords: 1,4-benzodiazepines; phenazepam; medicinal substance quality

Mechanical and Physical-Technological Properties of Gelatin Ribbon in the Manufacture of Soft Gelatin Capsules by E. I. Molokhova; E. I. Ponomareva; A. A. Adamov; A. K. Kholov (644-647).
The mechanical properties of gelatin ribbon and the rheology of bulk gelatin in the manufacture of soft gelatin capsules were studied. The results provide evidence that retention of the strength and elasticity of capsule casings during the manufacturing process and storage requires Young’s modulus of the gelatin ribbon to be in the range 35 – 50 MPa, the elongation at breaking being 210 – 240% and the tensile strength being 8 – 9 MPa. The studies also showed that regenerated gelatin stock loses its structural-mechanical properties, particularly its strength and plasticity, which can lead to failure of gelatin capsules during storage and drying.
Keywords: soft gelatin capsules: mechanical properties; rheology

Attention is drawn to a contradiction associated with assessments of the compatibility of medicinal (MS) and excipient (ES) substances: that ES and MS able to react with each other chemically are regarded as incompatible, yet at the same time, the compositions of several original medicines include ES and MS which are able to undergo chemical interactions. To remove this contradiction, the concept of “potential incompatibility” of ES and MS is introduced, i.e., the ability of substances to undergo direct or indirect chemical or undesirable physical interactions which may potentially become apparent during the manufacture or storage of a medicinal formulation and influence its quality. It is emphasized that incompatibility between an ES and an MS can be regarded as existing only when it is not possible to ensure their compatibility in a medicinal formulation. Approaches to assessing the compatibility between ES and MS are discussed: stress experiments using chromatographic analysis methods; stress experiments using spectral analysis methods; experiments using thermal analysis methods and calorimetry. A systematic “step-by-step” approach to studying the compatibility between ES and MS is proposed, based on express testing of the interaction between ES and MS not only in model mixtures, but also in samples of the medicinal formulation under development. This has decisive value for drawing reliable conclusions regarding the compatibility between the ES and the MS. Known types of potentially incompatible substance important for predicting potential incompatibility between ES and MS based on the structural characteristics and chemical properties of the molecules are presented. Recommendations are made in relation to studies of the compatibility of ES and MS.
Keywords: compatibility; medicinal and excipient substances; study methods; stress experiments; prediction

Validation of a Method for Assay of Trifluoroacetates in the Pharmaceutical Substance Glatiramer Acetate by 19F NMR Spectroscopy by S. V. Moiseev; N. E. Kuz’mina; V. I. Krylov; V. A. Yashkir; V. A. Merkulov (658-662).
A procedure for validation of an assay method for fluorine in residual trifluoroacetates in the pharmaceutical substance glatiramer acetate by 19F NMR spectroscopy is described. The method provides for assay of the content of trifluoroacetates without destruction of the substance under analysis. The validation process established the detection limit and assessed the linearity, accuracy, convergence, intralaboratory precision, and specificity of the method under validation. Measurement of “found:added” coefficients were used to calculate standard deviations, coefficients of variation, significance intervals, and systematic errors, using Fisher’s F test and Student’s t test to assess the assay results. The statistical characteristics were found to satisfy the criteria for acceptability of the validation parameters given in Russian and foreign normative documentation.
Keywords: glatiramer acetate; 19F NMR spectroscopy; assay; residual trifluoroacetates; validation of analytical method; detection limit; linearity; accuracy; convergence; precision; and specificity

Quantification of Sofosbuvir in Human Plasma: RP-HPLC Method Development and Validation by Rahul U. Rathod; K. S. Navyasree; Krishnamurthy Bhat (663-673).
Sofosbuvir, a potential inhibitor of the nucleotide analog of hepatitis C virus (HCV) NS5B polymerase, is used to treat hepatitis C and marketed under brand name Sovaldi. This study was aimed to develop and validate a simple, sensitive and precise RP-HPLC method for the estimation of sofosbuvir in human blood plasma using quinine sulfate as internal standard (ISTD). Separation was achieved using Shimadzu-HPLC system equipped with Gracesmart RP-C18 (250 mm × 4.6 mm, 5 μm) column eluted at a flow rate of 0.5 mL/min and detected at 260 nm. The mobile-phase composition was optimized to be ammonium formate (pH 3.0; 10 mM) and acetonitrile (67: 33, % v/v). The retention times of sofosbuvir and quinine sulfate were found to be 13.60 and 8.39 min, respectively. The proposed method was linear over a range from 20 to 1200 ng/mL. Liquid–liquid extraction technique was applied for the extraction of drug and ISTD from human plasma using TBME – DCM (80: 20, % v/v) mixture. The proposed method was validated as per bioanalytical method validation guidelines for industry.
Keywords: sofosbuvir; quinine sulfate; HPLC-UV; human blood plasma; Sovaldi

Analytical Method Development and Validation for HPLC-ECD Determination of Moxifloxacin in Marketed Formulations by G. Phani Sekhar Reddy; K. S. Navyasree; P. C. Jagadish; Krishnamurthy Bhat (674-679).
An HPLC-ECD analytical method with high reproducibility and wide linearity range has been developed and validated. Moxifloxacin was separated and identified using this method with a simple mobile phase comprising Britton Robinson buffer pH 5.0 and methanol (93: 7 v/v) flowing at a rate of 0.5 mL/min through Acclaim C18 column (150 mm × 4.6 mm × 5 im) maintained at 35°C and detected at redox potential value of 1.0 V. The LOD and LOQ were found to be 2.2 and 6.6 μg/mL, respectively. The HPLC-ECD method of moxifloxacin analysis was validated as per ICH Q2R1 guidelines and applied for assay of marketed moxifloxacin formulations to establish the acceptable recovery.
Keywords: moxifloxacin; electrochemical detector; voltammetry, HPLC-ECD; redox potential