Two Small Molecule Drugs with Topical Applications, Diflunisal and Naphazoline, and Their Potentially Toxic Photodegradants: Analysis by Chemical and Biological Methods.

Because of their topical application in patients and meaningful UV/VIS absorptive properties, the degradation and potential toxicity under irradiation of diflunisal (DIF) and naphazoline (NAF) were studied. In addition, the impact of pH on their photostability was examined, showing the highest degradation of acidic DIF at pH 1 and 13 and the highest degradation of basic NAF at pH below 7. An LC-UV analysis and chemical tests showed the first-order kinetics for their degradation and generation of reactive oxygen species (ROS). A UPLC-HRMS/MS analysis allowed us to identify four degradants of DIF (from DD-1 to DD-4) and six degradants of NAF (from ND-1 to ND-6). When Toxtree software was used, a high class III of toxicity was observed for DD-2, DD-3, and DD-4, and for all the NAF degradants. Furthermore, the ND-2 product, i.e., 2-[(1-methylnaphthalen-2-yl)methyl]-4,5-dihydro-1H-imidazole, was shown to present medium mutagenic and high tumorigenic effects according to OSIRIS Property Explorer. In addition, two in vitro tests on BALB/c 3T3 mouse fibroblasts showed a phototoxic effect of DIF and NAF at the lowest concentrations tested, i.e., 5 µg/mL. Thus, our present results could be useful to design further phototoxicity studies for DIF and NAF to minimize the risk of phototoxicity due to their photodegradation.

Comprehensive Insight into Chemical Stability of Important Antidiabetic Drug Vildagliptin Using Chromatography (LC-UV and UHPLC-DAD-MS) and Spectroscopy (Mid-IR and NIR with PCA).

During forced degradation, the intrinsic stability of active pharmaceutical ingredients (APIs) could be determined and possible impurities that would occur during the shelf life of the drug substance or the drug product could be estimated. Vildagliptin belongs to relatively new oral antidiabetic drugs named gliptins, inhibiting dipeptidyl peptidase 4 (DPP-4) and prolonging the activities of the endogenous incretin hormones. At the same time, some gliptins were shown as prone to degradation under specific pH and temperature conditions, as well as in the presence of some reactive excipients. Thus, forced degradation of vildagliptin was performed at high temperature in extreme pH and oxidative conditions. Then, selective LC-UV was used for quantitative determination of non-degraded vildagliptin in the presence of its degradation products and for degradation kinetics. Finally, identification of degradation products of vildagliptin was performed using an UHPLC-DAD-MS with positive ESI. Stability of vildagliptin was also examined in the presence of pharmaceutical excipients, using mid-IR and NIR with principal component analysis (PCA). At 70 °C almost complete disintegration of vildagliptin occurred in acidic, basic, and oxidative media. What is more, high degradation of vildagliptin following the pseudo first-order kinetics was observed at room temperature with calculated k values 4.76 × 10-4 s-1, 3.11 × 10-4 s-1, and 1.73 × 10-4 s-1 for oxidative, basic and acidic conditions, respectively. Next, new degradation products of vildagliptin were detected using UHPLC-DAD-MS and their molecular structures were proposed. Three degradants were formed under basic and acidic conditions, and were identified as [(3-hydroxytricyclo- [3.3.1.13,7]decan-1-yl)amino]acetic acid, 1-{[(3-hydroxytricyclo[3.3.1.13,7]decan-1-yl)amino]acetyl}-pyrrolidine-2-carboxylic acid and its O-methyl ester. The fourth degradant was formed in basic, acidic, and oxidative conditions, and was identified as 1-{[(3-hydroxytricyclo[3.3.1.13,7]-decan-1-yl)amino]acetyl}pyrrolidine-2-carboxamide. When stability of vildagliptin was examined in the presence of four excipients under high temperature and humidity, a visible impact of lactose, mannitol, magnesium stearate, and polyvinylpirrolidone was observed, affecting-NH- and CO groups of the drug. The obtained results (kinetic parameters, interactions with excipients) may serve pharmaceutical industry to prevent chemical changes in final pharmaceutical products containing vildagliptin. Other results (e.g., identification of new degradation products) may serve as a starting point for qualifying new degradants of vildagliptin as it is related to substances in pharmacopoeias.

The newly synthesized thiazole derivatives as potential antifungal compounds against Candida albicans.

Recently, the occurrence of candidiasis has increased dramatically, especially in immunocompromised patients. Additionally, their treatment is often ineffective due to the resistance of yeasts to antimycotics. Therefore, there is a need to search for new antifungals. A series of nine newly synthesized thiazole derivatives containing the cyclopropane system, showing promising activity against Candida spp., has been further investigated. We decided to verify their antifungal activity towards clinical Candida albicans isolated from the oral cavity of patients with hematological malignancies and investigate the mode of action on fungal cell, the effect of combination with the selected antimycotics, toxicity to erythrocytes, and lipophilicity. These studies were performed by the broth microdilution method, test with sorbitol and ergosterol, checkerboard technique, erythrocyte lysis assay, and reversed phase thin-layer chromatography, respectively. All derivatives showed very strong activity (similar and even higher than nystatin) against all C. albicans isolates with minimal inhibitory concentration (MIC) = 0.008-7.81 µg/mL Their mechanism of action may be related to action within the fungal cell wall structure and/or within the cell membrane. The interactions between the derivatives and the selected antimycotics (nystatin, chlorhexidine, and thymol) showed additive effect only in the case of combination some of them and thymol. The erythrocyte lysis assay confirmed the low cytotoxicity of these compounds as compared to nystatin. The high lipophilicity of the derivatives was related with their high antifungal activity. The present studies confirm that the studied thiazole derivatives containing the cyclopropane system appear to be a very promising group of compounds in treatment of infections caused by C. albicans. However, this requires further studies in vivo. KEY POINTS: • The newly thiazoles showed high antifungal activity and some of them - additive effect in combination with thymol. • Their mode of action may be related with the influence on the structure of the fungal cell wall and/or the cell membrane. • The low cytotoxicity against erythrocytes and high lipophilicity of these derivatives are their additional good properties.

HPLC-UV and GC-MS Methods for Determination of Chlorambucil and Valproic Acid in Plasma for Further Exploring a New Combined Therapy of Chronic Lymphocytic Leukemia.

High performance liquid chromatography with ultra-violet detection (HPLC-UV) and gas chromatography-mass spectrometry (GC-MS) methods were developed and validated for the determination of chlorambucil (CLB) and valproic acid (VPA) in plasma, as a part of experiments on their anticancer activity in chronic lymphocytic leukemia (CLL). CLB was extracted from 250 µL of plasma with methanol, using simple protein precipitation and filtration. Chromatography was carried out on a LiChrospher 100 RP-18 end-capped column using a mobile phase consisting of acetonitrile, water and formic acid, and detection at 258 nm. The lowest limit of detection LLOQ was found to be 0.075 µg/mL, showing sufficient sensitivity in relation to therapeutic concentrations of CLB in plasma. The accuracy was from 94.13% to 101.12%, while the intra- and inter-batch precision was ≤9.46%. For quantitation of VPA, a sensitive GC-MS method was developed involving simple pre-column esterification with methanol and extraction with hexane. Chromatography was achieved on an HP-5MSUI column and monitored by MS with an electron impact ionization and selective ion monitoring mode. Using 250 µL of plasma, the LLOQ was found to be 0.075 µg/mL. The accuracy was from 94.96% to 109.12%, while the intra- and inter-batch precision was ≤6.69%. Thus, both methods fulfilled the requirements of FDA guidelines for the determination of drugs in biological materials.

The Impact of Chlorambucil and Valproic Acid on Cell Viability, Apoptosis and Expression of p21, HDM2, BCL2 and MCL1 Genes in Chronic Lymphocytic Leukemia.

Malignant cells in chronic lymphocytic leukemia (CLL) show resistance to apoptosis, as well as to chemotherapy, which are related to deletions or mutations of TP53, high expression of MCL1 and BCL2 genes and other abnormalities. Thus, the main goal of the present study was to assess the impact of chlorambucil (CLB) combined with valproic acid (VPA), a known antiepileptic drug and histone deacetylation inhibitor, on apoptosis of the cells isolated from 17 patients with CLL. After incubation with CLB (17.5 µM) and VPA (0.5 mM), percentage of apoptosis, as well as expression of two TP53 target genes (p21 and HDM2) and two genes from Bcl-2 family (BCL2 and MCL1), were tested. As a result, an increased percentage of apoptosis was observed for CLL cells treated with CLB and VPA, and with CLB alone. Under the treatment with the drug combination, the expression of p21 gene was visibly higher than under the treatment with CLB alone. At the same time, the cultures under CLB treatment showed visibly higher expression of BCL2 than the cultures with VPA alone. Thus, the present study strongly suggests further investigations on the CLB and VPA combination in CLL treatment.

New benzenesulphonohydrazide derivatives as potential antitumour agents.

Cancer treatment remains a serious challenge worldwide. Thus, finding novel antitumour agents is of great importance. In the present study, nine new benzenesulphonohydrazide derivatives (1-9) were synthesized, and the chemical structures of the obtained compounds were confirmed by spectral analysis methods, including IR, 1H nuclear magnetic resonance (NMR) and 13C NMR. Experimental lipophilicity values were established using reversed phase-high performance thin layer chromatography. The antiproliferative activity of the synthesized compounds was tested against three tumour cell lines (769-P, HepG2 and NCI-H2170) and one normal cell line (Vero). Among the newly developed molecules, compound 4 exhibited generally the highest cytotoxicity across all tumour cell lines, and it was highly selective. However, higher selectivity towards the tested cancer cell lines was observed using compound 2, when compared with compound 4, which also exhibited significant antiproliferative activity against these tumour cells. In 769-P cells, compounds 5 and 6 were the most selective among all tested compounds. Compound 5 exhibited high cytotoxicity with an estimated IC50 value of 1.94 µM. In the NCI-H2170 cell line, compound 7 was the most cytotoxic and the most selective. In brief, the combination of fluorine and bromine substituents at the phenyl ring showed the most promising results, exerting high cytotoxicity and selectivity towards cancer cells. The renal adenocarcinoma cell line (769-P) appeared to be the most sensitive to the anticancer properties of the novel benzenesulphonohydrazones.

Synthesis and in vitro bioactivity study of new hydrazide-hydrazones of 5-bromo-2-iodobenzoic acid.

In this study 14 novel hydrazide-hydrazones of 5-bromo-2-iodobenzoic acid (3-16) were synthesized on the basis of condensation reaction. The chemical structure of obtained derivatives was established on the basis of spectral data (1H NMR and 13C NMR) and the lipophilicity of synthesized molecules was determined with the use of RP-HPTLC chromatography. Synthesized hydrazide-hydrazones (3-16) were subjected to in vitro cytotoxicity assay and antimicrobial activity analysis against a panel of bacteria and fungi. Among newly synthesized derivatives (3-16), compound 5 was characterized by high, selective and the most diverse cytotoxicity to the cancer cell lines. Molecules 7 and 9 which were substituted with a nitro group in the phenyl ring also exhibited very significant inhibitory effect in the tumor cells and they were very selective. Similarly, compound 13 showed high antiproliferative activity against both cancer cell lines (769-P, HepG2) with satisfactory selectivity. In turn, molecule 8 was characterized by lower inhibitory effect in tumor cells but high selectivity. Derivative 16 proved to be toxic mainly to 769-P cells plausibly by the inhibition of COX-2 mediated signalling pathway. In summary, the introduction of chloro substituent or nitro group to the molecule proved to be most advantageous, providing high cytotoxicity and selectivity to tumor cells. However, the presence of indole scaffold appeared to be responsible for COX-2 inhibitory effect. Some of synthesized hydrazide-hydrazones possessed also moderate antimicrobial activity against a panel of microorganisms.

Anticonvulsant valproic acid and other short-chain fatty acids as novel anticancer therapeutics: Possibilities and challenges.

Results from numerous pre-clinical studies suggest that a well known anticonvulsant drug valproic acid (VPA) and other short-chain fatty acids (SCFAs) cause significant inhibition of cancer cell proliferation by modulating multiple signaling pathways. First of all, they act as histone deacetylase (HDAC) inhibitors (HDIs), being involved in the epigenetic regulation of gene expression. Afterward, VPA is shown to induce apoptosis and cell differentiation, as well as regulate Notch signaling. Moreover, it up-regulates the expression of certain G protein-coupled receptors (GPCRs), which are involved in various signaling pathways associated with cancer. As a consequence, some pre-clinical and clinical trials were carried out to estimate anticancer effectiveness of VPA, in monotherapy and in new drug combinations, while other SCFAs were tested in pre-clinical studies. The present manuscript summarizes the most important information from the literature about their potent anticancer activities to show some future perspectives related to epigenetic therapy.

Determination of chemical stability of sitagliptin by LC-UV, LC-MS and FT-IR methods.

Sitagliptin was stored at high temperature/high humidity, dry hot air, UV/VIS light and different pH. Then, a selective LC-UV method was developed for determination of sitagliptin in the presence of degradation products and for estimation of degradation kinetics. Because parent drugs can react with excipients in final pharmaceutical formulations, stability of sitagliptin was also examined in the presence of excipients of different reactivity, using FT-IR and LC-UV methods. Finally, LC-MS method was used for identification of degradation products of sitagliptin. High degradation of sitagliptin, following the first order kinetics, was observed in strongly acidic, alkaline and oxidative media. The quickest degradation was found in 2 M HCl and 2 M NaOH. In addition, all excipients used in the present study, i.e. fumaric acid, lactose, mannitol and magnesium stearate showed potent interactions with sitagliptin. Some of these interactions were shown without any stress while others were accelerated by high temperature/high humidity and dry hot air, and less by UV/VIS light. Some mechanisms for the observed changes were proposed, i.e. the Michael addition in the presence of fumaric acid and the Maillard reaction in the presence of lactose. In addition, degradation of sitagliptin together with the occurrence of its impurities was stated in a broad range of stress conditions.

Chemometric processing of pharmaceutical essential oil fingerprints -- comparison of GC, HPLC, TLC, IR spectroscopy, and differential scanning calorimetry.

Fifteen essential oils of pharmaceutical grade were fingerprinted by five techniques: TLC, GC, HPLC, attenuated total reflectance FTIR spectroscopy, and differential scanning calorimetry (DSC). Denoising and baseline removal was found to be a crucial step for correct comparative analysis. Standardization of the signal was not necessary in the presented case; however, it should be considered and checked in each case. Due to small variance explained by first two principal components (below 50%) and outlying observations, the main analysis was performed by Euclidean dendrograms. It was found that almost all techniques besides DSC find real chemical similarities; however, DSC can be used as an additional tool. The similarities among the five techniques were also compared and discussed.

Modulation of nitrosative/oxidative stress in the lung of hyperglycemic rabbits by two antidiabetics, pioglitazone and repaglinide.

The lungs are involved in diabetes in the cause of the complex phenomena diabetes generates. In the present study, hyperglycemia inhibited pulmonary antioxidants, including superoxide dismutase, catalase, glutathione peroxidase, and glutathione. These effects were accompanied by significant elevation of lipid peroxidation, total nitrites, and nitrotyrosine levels. The study investigated the effects of 2 oral antidiabetics, pioglitazone and repaglinide, on the mentioned parameters. It is concluded that pioglitazone exerts protective effect in the lung by inhibiting nitrosative stress and normalizing the nitrites and nitrotyrosine levels. Administration of repaglinide prevents oxidative and, to a smaller extent, nitrosative changes.

Nitrosative stress and glutathione redox system in four different tissues of alloxan-induced hyperglycemic animals.

In hyperglycemia a reduction of the antioxidant power and an increased production of nitric oxide (NO) have been reported. Because nitrotyrosine (NT) is a stable end-product of nitrosative stress, assessment of its concentration is considered a useful marker of NO-dependent damages. The level of nitrotyrosine and four antioxidant parameters were evaluated in pancreas, kidney, heart, and testis of alloxan-induced hyperglycemic rabbits after 6 and 10 weeks of persistent hyperglycemia. In hyperglycemic groups, the level of nitrotyrosine was elevated by 44 and 39%, 92 and 95%, and by 155 and 138% in pancreas, kidney, and heart, respectively, while the testicular level of NT was unaffected. The pancreatic activities of glutathione peroxidase (GPX) and catalase (CAT) increased by 64% and 50%, and by 49 and 70%, while the level of glutathione (GSH) and the activity of glutathione reductase (GR) decreased by 37 and 38%, and by 57 and 42%. In the kidney the significant changes occurred as decreases in GR activity and GSH level by 36 and 35%, and by 28 and 23%, respectively. In the heart, a significant increase in CAT activity by 90 and 23% was observed. In the testis, the CAT, GPX, and GR activities were increased by 67 and 77%, 72 and 27%, and 33 and 28%, respectively, while the level of GSH was increased by 22 and 17%. These results confirm that tissues from hyperglycemic animals differ in neutralizing nitrosative stress. This may be due to different adaptive responses of their glutathione redox cycle.

Effects of pioglitazone on hyperglycemia-induced alterations in antioxidative system in tissues of alloxan-treated diabetic animals.

Hyperglycemia not only generates reactive oxygen species but also attenuates antioxidant mechanisms creating a state of oxidative stress. Oxidative stress is thought to play a crucial role in pathogenesis of chronic diabetic complications. Pioglitazone is a new oral antidiabetic agent, a potent inhibitor of glycation and potent antioxidant. In the present study, normoglycemic and alloxan-induced diabetic rabbits were treated with pioglitazone (1 mg/kg daily) for 4 and 8 weeks. At the end, glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione (GSH) and protein carbonyl groups (PCG) were evaluated in homogenates of liver and kidney. Chronic hyperglycemia caused a marked increase in oxidative processes and some changes in activity of antioxidants. In liver, diabetic vs. control values (mean+/-S.E.M; P<0.05) for GSH-Px were 181.0+/-5.4 vs. 203.1+/-1.9 and 187.4+/-6.6 vs. 240.9+/-18.8mU/mg protein. Pioglitazone treatment for 8 weeks affected GSH-Px activity in diabetic liver (261.5+/-7.3 mU/mg protein). In diabetic kidney, GSSG-R activity (20.6+/-1.6 vs. 32.4+/-1.5 and 23.6+/-0.6 vs. 36.3+/-0.3 mU/mg protein) and GSH level (16.6+/-0.5 vs. 23.2+/-0.9 and 17.9+/-0.5 vs. 23.2+/-0.6 nmol/mg protein) were diminished, while PCG level (0.32+/-0.03 vs. 0.11+/-0.02 and 0.35+/-0.03 vs. 0.16+/-0.03 nmol/mg protein) was elevated. In diabetic kidney, pioglitazone restored to control values GSSG-R activity (34.4+/-1.4 and 30.6+/-0.1 mU/mg protein) as well as GSH (25.5+/-1.2 and 21.6+/-0.5 nmol/mg protein) and PCG (0.16+/-0.01 and 0.19+/-0.02 nmol/mg protein) levels. The present study showed that pioglitazone reduced to some extent the oxidative stress enhanced by chronic hyperglycemia.

Changes in antioxidant status of heart muscle tissue in experimental diabetes in rabbits.

The present study was designed to evaluate the oxidative stress-related parameters in alloxan-induced diabetes in rabbits. After 3, 6, 12 and 24 weeks of hyperglycaemia the enzymatic and non-enzymatic factors were measured in heart tissue of diabetic and control groups. Superoxide dismutase and glutathione peroxidase activities and the contents of total sulfhydryl compounds significantly increased at all time intervals. Catalase activity increased initially (after 3 and 6 weeks), decreased after 12 weeks and increased again at the 24th week of the experiment. Glutathione reductase activity increased initially (at 3rd week), decreased below control level after 6 and 12 weeks, then increased again. Ascorbic acid concentration decreased after 3 and 6 weeks, and increased at the 12th and 24th weeks. The level of lipid peroxidation products was reduced after 3, 6 and 12 weeks of the experiment. After 24 weeks it was significantly elevated. These data suggest that hyperglycaemia induces oxidative stress in the heart but the defense mechanisms in the heart tissue are fairly efficacious against oxidative injury.

Changes in antioxidant status of lung tissue in experimental diabetes in rabbits.

OBJECTIVES: Hyperglycaemia can result in oxidative stress which may affected as cellular tissue damage. DESIGN AND METHODS: After 3, 6, 12 and 24 weeks of hyperglycaemia oxidative stress related parameters were measured in lung tissue of diabetic and control rabbits. RESULTS: Decreased activities of antioxidative compounds and intensification of lipid peroxidation process were found in diabetic lung. CONCLUSIONS: The data obtained suggest that hyperglycaemia induces oxidative stress in lung tissue which may play an important role in pathogenesis of diabetic complications.