DSC, FT-IR and NIR with Chemometric Assessment Using PCA and HCA for Estimation of the Chemical Stability of Oral Antidiabetic Drug Linagliptin in the Presence of Pharmaceutical Excipients.

Pharmaceutical excipients should not interact with active substances, however, in practice, they sometimes do it, affecting the efficacy, stability and safety of drugs. Thus, interactions between active substances and excipients are not desirable. For this reason, two component mixtures of oral antidiabetic drug linagliptin (LINA) with four excipients of different reactivity, i.e., lactose (LAC), mannitol (MAN), magnesium stearate (MGS) and polyvinylpyrrolidone (PVP), were prepared in a solid state. A high temperature and a high humidity of 60 °C and 70% RH, respectively, were applied as stressors in order to accelerate the potential interactions between LINA and excipients. Differential scanning calorimetry (DSC) as well as Fourier transform infrared (FT-IR) and near infrared (NIR) spectroscopy were used to estimate the changes due to potential interactions. In addition, chemometric computation of the data with principal component analysis (PCA) and hierarchical cluster analysis (HCA) was applied to adequately interpret the findings. Of the excipients used in the present experiment, all of them were not inert in relation to LINA. Some of the interactions were shown without any stressing, whereas others were observed under high-temperature/high-humidity conditions. Thus, it could be concluded that selection of appropriate excipients for LINA is very important question to minimize its degradation, especially when new types of formulations with LINA are being developed and manufactured.

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.

Biological Activity, Lipophilicity and Cytotoxicity of Novel 3-Acetyl-2,5-disubstituted-1,3,4-oxadiazolines.

Antibiotic resistance is now a global problem, and the lack of effective antimicrobial agents for the treatment of diseases caused by resistant microbes is increasing. The 3-acetyl-2,5-disubstituted-1,3,4-oxadiazolines presented in this article may provide a good starting point for the development of potential new effective antimicrobial agents useful in the treatment of bacterial and fungal infections. Particular attention is drawn to the 1,3,4-oxadiazole derivative marked with the number 29 with 5-nitrofuran-2-yl substituent in its chemical structure. This substance showed a strong bactericidal effect, especially against Staphylococcus spp., and no cytotoxicity to the L929 normal cell line.

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.

Determination of Chemical Stability of Two Oral Antidiabetics, Metformin and Repaglinide in the Solid State and Solutions Using LC-UV, LC-MS, and FT-IR Methods.

Firstly, metformin and repaglinide were degraded under high temperature/humidity, UV/VIS light, in different pH and oxidative conditions. Secondly, a new validated LC-UV method was examined, as to whether it validly determined these drugs in the presence of their degradation products and whether it is suitable for estimating degradation kinetics. Finally, the respective LC-MS method was used to identify the degradation products. In addition, using FT-IR method, the stability of metformin and repaglinide was scrutinized in the presence of polyvinylpyrrolidone (PVP), mannitol, magnesium stearate, and lactose. Significant degradation of metformin, following the first order kinetics, was observed in alkaline medium. In the case of repaglinide, the most significant and quickest degradation, following the first order kinetics, was observed in acidic and oxidative media (0.1 M HCl and 3% H2O2). Two new degradation products of metformin and nine new degradation products of repaglinide were detected and identified when the stressed samples were examined by our LC-MS method. What is more, the presence of PVP, mannitol, and magnesium stearate proved to affect the stability of metformin, while repaglinide stability was affected in the presence of PVP and magnesium stearate.

Metabolism and Chemical Degradation of New Antidiabetic Drugs (Part II): A Review of Analytical Approaches for Analysis of Gliptins.

This paper is part II of the review on metabolism and chemical degradation of new antidiabetic drugs from glutides, gliflozins and gliptins. It is well known that metabolism data can be helpful for deriving safe levels of degradation impurities and their qualifying as far as toxicological aspects are concerned. As a result, it could link the quality of respective pharmaceutical products to clinical practice and patients. Some overlapping pathways of transformations of these important drugs of different chemical structures and different mechanisms of action were discussed. At the same time, the paper summarized interesting analytical tools for conducting modern drug metabolism as well as drug degradation experiments. The methods described here include liquid chromatography (LC) and liquid chromatography coupled with mass spectrometry (LC-MS or LC-MS/MS), which are widely used for detection and quantitative measurements of the drugs, their metabolites and degradants, as well as radiometric methods that are suitable for pharmacokinetic experiments. Special attention was paid to dedicated types of packing in chromatographic columns, as well as to special solutions in the LC-MS procedures. The present part addresses the analytical approaches elaborated for examining the metabolism and degradation pathways of gliptins that are dipeptidyl peptidase 4 (DPP-4) inhibitors.

Metabolism and Chemical Degradation of New Antidiabetic Drugs: A Review of Analytical Approaches for Analysis of Glutides and Gliflozins.

The drug metabolism and drug degradation pathways may overlap, resulting in the formation of similar constituents. Therefore, the metabolism data can be helpful for deriving safe levels of degradation impurities and improving the quality of respective pharmaceutical products. The present article contains considerations on possible links between metabolic and degradation pathways for new antidiabetic drugs such as glutides, gliflozins, and gliptins. Special attention was paid to their reported metabolites and identified degradation products. At the same time, many interesting analytical approaches to conducting metabolism as well as degradation experiments were mentioned, including chromatographic methods and radioactive labeling of the drugs. The review addresses the analytical approaches elaborated for examining the metabolism and degradation pathways of glutides, i.e., glucagon like peptide 1 (GLP-1) receptor agonists, and gliflozins, i.e., sodium glucose co-transporter 2 (SGLT2) inhibitors. The problems associated with the chromatographic analysis of the peptide compounds (glutides) and the polar drugs (gliflozins) were addressed. Furthermore, issues related to in vitro experiments and the use of stable isotopes were discussed.

Photodegradation of the H1 Antihistaminic Topical Drugs Emedastine, Epinastine, and Ketotifen and ROS Tests for Estimations of Their Potent Phototoxicity.

In this study, important H1 antihistaminic drugs, i.e., emedastine (EME), epinastine (EPI), and ketotifen (KET), were irradiated with UV/Vis light (300-800 nm) in solutions of different pH values. Next, they were analyzed by new high performance liquid chromatography (HPLC) methods, in order to estimate the percentage of degradation and respective kinetics. Subsequently, ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) was used to identify their photodegradation products and to propose degradation pathways. In addition, the peroxidation of linoleic acid and generation of singlet oxygen (SO) and superoxide anion (SA) were examined, together with the molar extinction coefficient (MEC) evaluation, to estimate their phototoxic risk. The photodegradation of all EME, EPI, and KET followed pseudo first-order kinetics. At pH values of 7.0 and 10.0, EPI was shown to be rather stable. However, its photostability was lower at pH 3.0. EME was shown to be photolabile in the whole range of pH values. In turn, KET was shown to be moderately labile at pH 3.0 and 7.0. However, it degraded completely in the buffer of pH 10.0. As a result, several photodegradation products were separated and identified using the UPLC-MS/MS method. Finally, our ROS assays showed a potent phototoxic risk in the following drug order: EPI < EME < KET. All of these results may be helpful for manufacturing, storing, and applying these substantial drugs, especially in their ocular formulations.

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.

Comparative Study of Chemical Stability of Two H1 Antihistaminic Drugs, Terfenadine and Its In Vivo Metabolite Fexofenadine, Using LC-UV Methods.

A comparative study of chemical stability of terfenadine (TER) and its in vivo metabolite fexofenadine (FEX) was performed. Both TER and FEX were subjected to high temperature at different pH and UV/VIS light at different pH and then quantitatively analyzed using new validated LC-UV methods. These methods were used to monitor the degradation processes and to determine the kinetics of degradation for both the compounds. As far as the effects of temperature and pH were concerned, FEX occurred more sensitive to degradation than TER. As far as the effects of UV/VIS light and pH were concerned, the both drugs were similarly sensitive to high doses of light. Using all stress conditions, the processes of degradation of TER and FEX followed the first-order kinetics. The results obtained for these two antihistaminic drugs could be helpful in developing their new derivatives with higher activity and stability at the same time.