Application of quantitative NMR in pharmaceutical analysis: Method qualification and potency determination for two new chemical entities in early-stage clinical studies.

Quantitative NMR (qNMR), being a well-established analytical tool featuring efficiency, simplicity as well as versatility, has been extensively employed in pharmaceutical and medicinal testing. In this study, two 1H qNMR methods were developed to determine the %wt/wt potency of two new chemical entities (compound A and compound B) used in early clinical phase process chemistry and formulation development. The qNMR methods were demonstrated to be significantly more sustainable and efficient than the LC-based approach by substantially reducing the cost, hands-on-time, and materials consumed for testing. The qNMR methods were achieved on a 400 MHz NMR spectrometer equipped with 5 mm BBO S1 broad band room temperature probe. The methods with CDCl3 (for compound A) and DMSO-d6 (compound B) as solvent as well as commercially certified standards for quantitation were phase-appropriately qualified in terms of specificity, accuracy, repeatability/precision, linearity, and range. Both qNMR methods were demonstrated to be linear over the range of 0.8-1.2 mg/mL (80% to 120% of the nominal sample concentration of 1.0 mg/mL) with a correlation coefficient greater than 0.995. The methods were also demonstrated to be accurate with average recoveries ranging from 98.8% to 98.9% and 99.4-99.9% for compound A and compound B respectively as well as precise with %RSD of 0.46% and 0.33% for compound A and compound B respectively. The potency results of compound A and compound B determined by qNMR were compared with those determined by the conventional LC-based method and the qNMR results were demonstrated to be consistent with the LC-based method with absolute difference of 0.4% and 0.5% for compound A and B respectively.

Synthetic derivatives of the antifungal drug ciclopirox are active against herpes simplex virus 2.

We previously showed that the anti-fungal drug ciclopirox olamine effectively inhibits replication of herpes simplex virus (HSV)-1 and HSV-2. Given the rise of HSV strains that are resistant to nucleos(t)ide analog treatment, as well as the incomplete efficacy of nucleos(t)ide analogs, new inhibitory compounds must be explored for potential use in the treatment of HSV infection. In the present study, we analyzed 44 compounds derived from the core structure of ciclopirox olamine for inhibitory activity against HSV. Thirteen of these derivative compounds inhibited HSV-2 replication by > 1000- to ∼100,000-fold at 1 µM and displayed EC50 values lower than that of acyclovir, as well as low cytotoxicity, indicating their strong therapeutic potential. Through structural comparison, we also provide evidence for the importance of various structural motifs to the efficacy of ciclopirox and its derivatives, namely hydrophobic groups at R4 and R6 of the ciclopirox core structure. Like ciclopirox, representative analogs exhibit some oral bioavailability but are rapidly cleared in vivo. Together, these results will guide further development of N-hydroxypyridones as HSV therapeutics.

Synthetic Derivatives of Ciclopirox are Effective Inhibitors of Cryptococcus neoformans.

Opportunistic fungal infections caused by Cryptococcus neoformans are a significant source of mortality in immunocompromised patients. They are challenging to treat because of a limited number of antifungal drugs, and novel and more effective anticryptococcal therapies are needed. Ciclopirox olamine, a N-hydroxypyridone, has been in use as an approved therapeutic agent for the treatment of topical fungal infections for more than two decades. It is a fungicide, with broad activity across multiple fungal species. We synthesized 10 N-hydroxypyridone derivatives to develop an initial structure-activity understanding relative to efficacy as a starting point for the development of systemic antifungals. We screened the derivatives for antifungal activity against C. neoformans and Cryptococcus gattii and counter-screened for specificity in Candida albicans and two Malassezia species. Eight of the ten show inhibition at 1-3 µM concentration (0.17-0.42 µg per mL) in both Cryptococcus species and in C. albicans, but poor activity in the Malassezia species. In C. neoformans, the N-hydroxypyridones are fungicides, are not antagonistic with either fluconazole or amphotericin B, and are synergistic with multiple inhibitors of the mitochondrial electron transport chain. They appear to function primarily by chelating iron within the active site of iron-dependent enzymes. This preliminary structure-activity relationship points to the need for a lipophilic functional group at position six of the N-hydroxypyridone ring and identifies positions four and six as sites where further substitution may be tolerated. These molecules provide a clear starting point for future optimization for efficacy and target identification.

met1 DNA Methyltransferase Controls TERT Gene Expression: A New Insight to The Role of Telomerase in Development.

OBJECTIVE: DNA methylation systems are essential for proper embryo development. Methylation defects lead to developmental abnormalities. Furthermore, changes in telomerase gene expression can affect stability of chromosomes and produces abnormal growth. Therefore, defects in both methylation and telomerase gene expression can lead to developmental abnormalities. We hypothesized that mutation in the methylation systems may induce developmental abnormalities through changing telomerase gene expression. MATERIALS AND METHODS: In this experimental study, we used Arabidopsis thaliana (At) as a developmental model. DNA was extracted from seedlings leaves. The grown plants were screened using polymerase chain reaction (PCR) reactions. Total RNA was isolated from the mature leaves, stems and flowers of wild type and met1 mutants. For gene expression analysis, cDNA was synthesized and then quantitative reverse transcription PCR (qRT-PCR) was performed. RESULTS: Telomerase gene expression level in homozygous met1 mutant plants showed ~14 fold increase compared to normal plants. Furthermore, TERT expression in met1 heterozygous was~ 2 fold higher than the wild type plants. CONCLUSION: Our results suggested that TERT is a methyltransferase-regulated gene which may be involved in developmental abnormities causing by mutation in met1 methyltransferase system.