Quantitative determination of potential genotoxic impurity 3-aminopyridine in linagliptin active pharmaceutical ingredient using HILIC-UV.

This study aimed to develop an analytical method to determine the quantity of the impurity 3-aminopyridine (3AP). 3-Aminopyridine is a reactive reagent in the synthesis of linagliptin. The method was sensitive at level of 30.0 ppm of 3AP relative to linagliptin. The analysis was carried out using hydrophilic interaction liquid chromatography. The analytical column was Tracer Extrasil Silica (150 × 4.0 mm, 3 µm). A mobile phase of water-acetonitrile (10:90, v/v) containing 10.0 mM ammonium acetate was prepared and adjusted to pH 6.0. A UV detector was used to detect the amount of 3AP at a wavelength of 298 nm. Validation of the method was performed as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use in terms of detection limit, quantitation limit, linearity, accuracy, precision, specificity and robustness. The calibration curve was linear (r2 = 0.999) for 3AP concentration in the range of 30.0-450.0 ppm. This method showed a good sensitivity with a detection limit and a quantitation limit of 7.5 and 25.0 ppm, respectively.

Validated LC-MS/MS Method for Simultaneous Quantitation of Enasidenib and its Active Metabolite, AGI-16903 in Small Volume Mice Plasma: Application to a Pharmacokinetic Study.

Enasidenib is a selective mutant isocitrate dehydrogenase 2 inhibitor approved for the treatment of relapsed and refractory acute myeloid leukemia patients. A sensitive and rapid method has been developed and validated as per regulatory guideline for the simultaneous quantitation of enasidenib and its active metabolite, AGI-16903 in mice plasma using an LC-MS/MS. Enasidenib and AGI-16903 along with internal standard were extracted from mice plasma using simple protein precipitation method. Chromatographic resolution of enasidenib, AGI-16903 and the internal standard (close analogue of AGI-16903) was achieved on a Chromolith RP-18e column using 0.2% formic acid:acetonitrile (15:85, v/v) as an eluent, which was delivered at a flow-rate of 1.2 mL/min. The MS/MS ion transitions monitored were m/z 474.1→267.2, 402.1→188.1 and 421.0→146.1 for enasidenib, AGI-16903 and the internal standard, respectively. The linearity range was 1.01-3023 ng/mL for both enasidenib and AGI-16903. The within-run and between-run accuracy and within-run and between-run precision were in the range of - 2.29 to 2.72 (as one value is in negative side). and 4.65-9.82%, respectively for enasidenib; 0.19-10.3 and 3.22-9.22%, respectively for AGI-16903. Both enasidenib and AGI-16903 were found to be stable in stability (up to three freeze-thaw cycles and for long-term at -80°C for 30 days) and processed (bench-top for 6 h and in in-injector for 24 h) samples. Application of the validated method was shown in a pharmacokinetic study in mice.

Aphthous ulcer drug inhibits prostate tumor metastasis by targeting IKKɛ/TBK1/NF-κB signaling.

Tumor metastasis is the major cause of death for prostate cancer (PCa) patients. However, the treatment options for metastatic PCa are very limited. Epithelial-mesenchymal transition (EMT) has been reported to be an indispensable step for tumor metastasis and is suggested to associate with acquisition of cancer stem cell (CSC) attributes. We propose that small-molecule compounds that can reverse EMT or induce mesenchymal-epithelial transition (MET) of PCa cells may serve as drug candidates for anti-metastasis therapy. Methods: The promoters of CDH1 and VIM genes were sub-cloned to drive the expression of firefly and renilla luciferase reporter in a lentiviral vector. Mesenchymal-like PCa cells were infected with the luciferase reporter lentivirus and subjected to drug screening from a 1274 approved small-molecule drug library for the identification of agents to reverse EMT. The dosage-dependent effect of candidate compounds was confirmed by luciferase reporter assay and immunoblotting. Wound-healing assay, sphere formation, transwell migration assay, and in vivo intracardiac and orthotopic tumor xenograft experiments were used to evaluate the mobility, metastasis and tumor initiating capacity of PCa cells upon treatment. Possible downstream signaling pathways affected by the candidate compound treatment were analyzed by RNA sequencing and immunoblotting. Results: Drug screening identified Amlexanox, a drug used for recurrent aphthous ulcers, as a strong agent to reverse EMT. Amlexanox induced significant suppression of cell mobility, invasion, serial sphere formation and in vivo metastasis and tumor initiating capacity of PCa cells. Amlexanox treatment led to downregulation of the IKK-ɛ/ TBK1/ NF-κB signaling pathway. The effect of Amlexanox on EMT reversion and cell mobility inhibition can be mimicked by other IKK-ɛ/TBK1 inhibitors and rescued by reconstitution of dominant active NF-κB. Conclusions: Amlexanox can sufficiently suppress PCa metastasis by reversing EMT through downregulating the IKK-ɛ/TBK1/NF-κB signaling axis.

Potent Plasmodium falciparum gametocytocidal compounds identified by exploring the kinase inhibitor chemical space for dual active antimalarials.

Objectives: Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. Methods: We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. Results: We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at <100 nM (8 were IMPs and 4 were 2-APs) and were also active against early stage gametocytes and asexual parasites, with >1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. Conclusions: This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.

Characterization of the radiosynthesis and purification of [18F]THK-5351, a PET ligand for neurofibrillary tau.

This work characterizes the radiochemical synthesis, purification, and formulation of [18F]THK-5351, a tau PET radioligand, and develops an automated radiosynthesis routine (ELIXYS, Sofie Biosciences). Nucleophilic radiofluorination reaction was complete by 7min at 110°C with radiochemical yields proportional to precursor mass (0.1-0.5mg). Optimized HPLC purification produced radiotracer product with no chemical impurities observed on analytical HPLC in formulation. Automated radiosynthesis (ELIXYS), HPLC purification and formulation was completed in 86min producing formulated product suitable for human research use.

Theoretical Models and QSRR in Retention Modeling of Eight Aminopyridines.

In this article, retention modeling of eight aminopyridines (synthesized and characterized at the Faculty of Pharmacy) in reversed-phase high performance liquid chromatography (RP-HPLC) was performed. No data related to their retention in the RP-HPLC system were found. Knowing that, it was recognized as very important to describe their retention behavior. The influences of pH of the mobile phase and the organic modifier content on the retention factors were investigated. Two theoretical models for the dependence of retention factor of organic modifier content were tested. Then, the most reliable and accurate prediction of log k was created, testing multiple linear regression model-quantitative structure-retention relationships (MLR-QSRR) and support vector regression machine-quantitative structure-retention relationships (SVM-QSRR). Initially, 400 descriptors were calculated, but four of them (POM, log D, M-SZX/RZX and m-RPCG) were included in the models. SVM-QSRR performed significantly better than the MLR model. Apart from aminopyridines, four structurally similar substances (indapamide, gliclazide, sulfamethoxazole and furosemide) were followed in the same chromatographic system. They were used as external validation set for the QSRR model (it performed well within its applicability domain, which was defined using a bounding box approach). After having described retention of eight aminopyridines with both theoretical and QSRR models, further investigations in this field can be conducted.

Fast identification of selective resins for removal of genotoxic aminopyridine impurities via screening of molecularly imprinted polymer libraries.

This study describes the identification and evaluation of molecularly imprinted polymers (MIPs) for the selective removal of potentially genotoxic aminopyridine impurities from pharmaceuticals. Screening experiments were performed using existing MIP resin libraries to identify resins selective towards those impurities in the presence of model pharmaceutical compounds. A hit resin with a considerable imprinting effect was found in the screening and upon further investigation, the resin was found to show a broad selectivity towards five different aminopyridines in the presence of the two model active pharmaceutical ingredients (APIs) piroxicam and tenoxicam.

Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography.

Isomeric oligosaccharides and isomeric glycopeptides are sometimes difficult to separate on normal-phase (NP) and reversed-phase (RP) columns. A zwitterionic type of hydrophilic-interaction chromatography column with sulfobetaine groups (called ZIC-HILIC column) was first applied to the separation of 2-aminopyridine derivatized (PA) N-glycans and tryptic peptides of human serum immunoglobulin G (IgG). It is shown that the ZIC-HILIC column has high capability for structural recognition of isomeric N-glycans as well as high selectivity for glycopeptides. The former feature (i.e., structural recognition) was proven by sufficient separation of neutral PA N-glycan isomers, which are usually difficult to separate on NP and RP columns. In addition, it is noteworthy that IgG glycopeptides consisting of isomeric N-glycans and the same peptide sequences can be sufficiently separated on a ZIC-HILIC column. The latter feature (i.e., selectivity) was also demonstrated by easily separating two peptide groups with/without N-glycans. Thus, we note that the ZIC-HILIC column is highly promising for a simple analysis of N-glycans and N-glycopeptide samples.

Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum.

Meridianins are brominated 3-(2-aminopyrimidine)-indoles which are purified from Aplidium meridianum, an Ascidian from the South Atlantic (South Georgia Islands). We here show that meridianins inhibit various protein kinases such as cyclin-dependent kinases, glycogen synthase kinase-3, cyclic nucleotide-dependent kinases and casein kinase 1. Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed.

Liquid chromatography solvent optimization: potential pitfalls when using a black box for developing a quality separation.

ICOS and DIAMOND are two commercially available, semi-automated HPLC solvent optimization software packages. The resultant optimized chromatographic separation is dependent on a combination of the operator's objective, the capability of the software system and the appropriateness of the data input. The latter contains components that represent the match between the requirements of the algorithms used and the information content of the data on which those algorithms operated. Knowledge about the sample content, stability and potential sample-solvent interactions can have a significant effect on the quality of the optimal solvent composition that is calculated. The results generated during the optimization of the separation of a mixture of U-83,757 and a variety of related compounds illustrate the need to consider the significance of the contribution to the calculated optimal separation of each of these potential pitfalls, both individually and in combination with the mode of operation of the relevant algorithms. Our results indicate that the quality of the final result is highly dependent on the intelligence content of the data used.

Extraction method for preparing pyridylamino sugar derivatives and application to porcine gastric mucus glycoprotein analysis.

For the sensitive detection of free sugars and oligosaccharides, the use of their pyridylamino derivatives has now found general acceptance. To remove excess 2-aminopyridine from this derivative in a reaction mixture, gel filtration and ion-exchange chromatography were conducted. It was found in the present study that contaminated 2-aminopyridine could be selectively removed from the reaction mixture by adjusting the pH with saturated sodium bicarbonate at above 8.5 followed by extraction with benzene. By using this method, fewer purification steps and less time are required, with minimum loss of pyridylamino sugar derivatives.

Ascorbate increases apparent "specific" binding of [3H]pyrilamine to glass fiber filters.

Although filtration through glass fiber filters provides a convenient method for separating bound from unbound radioligand, binding of the ligand to the filters is often a problem. Ascorbate (0.1%) has been shown to decrease binding of serotonergic acids to these filters. In the present study, relatively high binding of [3H]pyrilamine, an H1-antagonist, to glass fiber filters was observed. The addition of ascorbate increased, rather than decreased, binding of [3H]pyrilamine to the filters. Thus, the effect of ascorbate on binding of radioligands to glass fiber filters appears to be dependent upon the particular ligand involved.

[The purification and characterization of Cordemcura]. / Untersuchungen zur Reinigung und Charakterisierung von Cordemcura.

3-Amino-5-(4-pyridinyl)-1,2-dihydro-pyrid-2-one (1) is an amphoteric compound and forms one crystalline sodium salt and two hydrochlorides. Physicochemical properties UV, NMR and MS are described. TLC has been used mainly and is the most sensitive method for estimation of 1-byproducts. Coloured byproducts, generated by hypochlorite or air oxidation during synthesis and handling in solution, are monitored by vis-spectra, diminished by sulfite addition and removed by alkaline precipitation. The purification procedure is able to produce 1 with only 0.1% of precursors or byproducts.