Optimization and validation of a UPLC-MS/MS assay for simultaneous quantification of 2,8-dihydroxyadenine, adenine, allopurinol, oxypurinol and febuxostat in human plasma.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare , hereditary disorder characterized by renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stone formation and chronic kidney disease (CKD). Treatment with a xanthine oxidoreductase inhibitor, allopurinol or febuxostat, reduces urinary DHA excretion and slows the progression of CKD. The method currently used for therapeutic monitoring of APRT deficiency lacks specificity and thus, a more reliable measurement technique is needed. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma was optimized and validated. Plasma samples were prepared with protein precipitation using acetonitrile followed by evaporation. The chemometric approach design of experiments was implemented to optimize gradient steepness, amount of organic solvent, flow rate, column temperature, cone voltage, desolvation temperature and desolvation flow rate. Experimental screening was conducted using fractional factorial design with addition of complementary experiments at the axial points for optimization of peak area, peak resolution and peak width. The assay was validated according to the US Food and Drug Administration guidelines for bioanalytical method validation over the concentration range of 50 to 5000 ng/mL for DHA, allopurinol and febuxostat, 100 to 5000 ng/mL for adenine and 50 to 12,000 ng/mL for oxypurinol, with r2 ≥ 0.99. The analytical assay achieved acceptable performance of accuracy (-10.8 to 8.3 %) and precision (CV < 15 %). DHA, adenine, allopurinol, oxypurinol and febuxostat were stable in plasma samples after five freeze-thaw cycles at -80 °C and after storage at -80 °C for 12 months. The assay was evaluated for quantification of the five analytes in clinical plasma samples from six APRT deficiency patients and proved to be both efficient and accurate. The proposed assay will be valuable for guiding pharmacotherapy and thereby contribute to improved and more personalized care for patients with APRT deficiency.

Infraspecific Variation of Huperzine A and B in Icelandic Huperzia selago Complex.

The alkaloids huperzine A and huperzine B were originally isolated from the Chinese club moss Huperzia serrata. They are known inhibitors of acetylcholinesterase, and especially huperzine A shows pharmaceutical potential for the treatment of Alzheimer's disease. Its supply heavily relies on natural plant sources belonging to the genus Huperzia, which shows considerable interspecific huperzine A variations. Furthermore, taxonomic controversy remains in this genus, particularly in the Huperzia selago group. With focus on Icelandic H. selago taxa, we aimed to explore the relatedness of Huperzia species using multi-locus phylogenetic analysis, and to investigate correlations between huperzine A contents, morphotypes, and genotypes. Phylogenetic analysis was performed with five chloroplastic loci (the intergenic spacer between the photosystem II protein D1 gene and the tRNA-His gene, maturase K, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, tRNA-Leu, and the intergenic spacer region between tRNA-Leu and tRNA-Phe). Huperzine A and huperzine B contents were determined using an HPLC-UV method. The phylogenetic analysis suggests that previously proposed Huperzia appressa and Huperzia arctica should not be considered species, but rather subspecies of H. selago. Three genotypes of Icelandic H. selago were identified and presented in a haplotype networking diagram. A significantly (p < 0.05) higher amount of huperzine A was found in H. selago genotype 3 (264 - 679 µg/g) than genotype 1 (20 - 180 µg/g), where the former shows a typical green and reflexed "selago" morphotype. The huperzine A content in genotype 3 is comparable to Chinese H. serrata and a good alternative huperzine A source. Genotype 2 contains multiple morphotypes with a broad huperzine A content (113 - 599 µg/g). The content of huperzine B in Icelandic taxa (6 - 13 µg/g) is much lower than that in Chinese H. serrata (79 - 207 µg/g).

New Fluvirucinins C1 and C2 Produced by a Marine Derived Actinomycete.

Two new fluvirucin aglycones, named fluvirucinins C, and C2 (1-2), have been isolated from the ethyl acetate mycelial cake extract of the fermentation broth of.a marine sponge-associated actinomycete. Fluvirucinins C, (1) and C2 (2) represent a new type of 14-membered macrolactam aglycon, structurally related with the common aglycon of the known fluvirucins. Their structures were elucidated on the basis of ID and 2D NMR analyses, as well as HRESIMS experiments. The antimicrobial and cytotoxic activities of compounds 1 and 2 have been evaluated, but no significant activities found for fluvirucinins C, and C2.

Protolichesterinic Acid, Isolated from the Lichen Cetraria islandica, Reduces LRRC8A Expression and Volume-Sensitive Release of Organic Osmolytes in Human Lung Epithelial Cancer Cells.

We have tested the effect of protolichesterinic acid (PA) on the activity of the volume-sensitive release pathway for the organic osmolyte taurine (VSOAC) and the expression of the leucine-rich-repeat-channel 8A (LRRC8A) protein, which constitutes an essential VSOAC component. Exposing human lung cancer cells (A549) to PA (20 µg/mL, 24 h) reduces LRRC8A protein expression by 25% and taurine release following osmotic cell swelling (320 → 200 mOsm) by 60%. C75 (20 µg/mL, 24 h), a γ-lactone with a C8 carbon fatty acid chain, reduces VSOAC activity by 30%, i.e. less than PA. Stearic acid (20 µg/mL, 24 h) has no effect on VSOAC. Hence, length of PA's fatty acid chain adds to γ-lactone's inhibitory action. 5-Lipoxygenase (5-LO) activity is essential for swelling-induced activation of VSOAC. PA has no effect on cellular concentration of leukotrienes (5-HETE/LTB4 ) under hypotonic conditions, excluding that PA mediated inhibition of VSOAC involves 5-LO inhibition. A549 cells exposed to the chemotherapeutic drug cisplatin (10 µM, 24 h) reveal signs of apoptosis, i.e. 25% reduction in cell viability as well as 1.3-, 1.5- and 3.3-fold increase in the expression of LRRC8A, Bax (regulator of apoptosis) and p21 (regulator of cell cycle progression), respectively. PA reduces cell viability by 30% but has no effect on p21/Bax expression. This excludes PA as a pro-apoptotic drug in A549 cells.