Acetylshikonin promoting PI3K/Akt pathway and inhibiting SOX4 expression to delay intervertebral disc degeneration and low back pain.

This study investigated the molecular mechanism by which acetylshikonin inhibits SOX4 expression via the PI3K/Akt pathway to delay intervertebral disc degeneration (IVDD) and low back pain (LBP). Bulk RNA-seq, RT-qPCR, Western blot analysis, immunohistochemical staining, small interfering RNA (siSOX4), lentivirus (lentiv-SOX4hi ), and imaging techniques were used to assess SOX4 expression and validate its upstream regulatory pathway. Acetylshikonin and siSOX4 were injected into the IVD to measure IVDD. SOX4 expression significantly increased in degenerated IVD tissues. TNF-α increased SOX4 expression and apoptosis-related proteins in nucleus pulposus cells (NPCs). siSOX4 reduced TNF-α-induced NPCs apoptosis, while Lentiv-SOX4hi increased it. The PI3K/Akt pathway was significantly correlated with SOX4, and acetylshikonin upregulated PI3K/Akt pathway while inhibiting SOX4 expression. In the anterior puncture IVDD mouse model, SOX4 expression was upregulated, and acetylshikonin and siSOX4 delayed IVDD-induced LBP. Acetylshikonin delays IVDD-induced LBP by inhibiting SOX4 expression through the PI3K/Akt pathway. These findings offer potential therapeutic targets for future treatments.

On the Risk of Nitrosamine Contamination During Drug Product Blister Packaging.

Most N-Nitrosamine compounds are found to be genotoxic in several animal species. Some are classified as probable or possible human carcinogens and very low acceptable daily intake has been established such as 96 ng/day for N-nitrosodimethylamine (NDMA) and 26.5 ng/N-nitrosodiethylamine (NDEA). The pharmaceutical industry has considered all processing areas for potential formation or contamination of N-nitrosamine. One risk is the potential contamination of nitrosamine during drug product blister packaging using lidding foils containing nitrocellulose, and different approaches have been used by pharmaceutical companies to evaluate and mitigate this risk. Herein we share a perspective from IQ Consortium N-nitrosamine Working Group on some of the approaches and corresponding results. From these assessments, it was concluded that the risk of nitrosamine contamination during blister packaging is negligible. The approaches shared in this perspective can be incorporated into risk assessment for nitrosamine contamination during drug product packaging at other pharmaceutical companies.

A Full Evaporation Static Headspace Gas Chromatography Method with Nitrogen Phosphorous Detection for Ultrasensitive Analysis of Semi-volatile Nitrosamines in Pharmaceutical Products.

The recent detection of potent carcinogenic nitrosamine impurities in several human medicines has triggered product recalls and interrupted the supply of critical medications for hundreds of millions of patients, illuminating the need for increased testing of nitrosamines in pharmaceutical products. However, the development of analytical methods for nitrosamine detection is challenging due to high sensitivity requirements, complex matrices, and the large number and variety of samples requiring testing. Herein, we report an analytical method for the analysis of a common nitrosamine, N-nitrosodimethylamine (NDMA), in pharmaceutical products using full evaporation static headspace gas chromatography with nitrogen phosphorous detection (FE-SHSGC-NPD). This method is sensitive, specific, accurate, and precise and has the potential to serve as a universal method for testing all semi-volatile nitrosamines across different drug products. Through elimination of the detrimental headspace-liquid partition, a quantitation limit of 0.25 ppb is achieved for NDMA, a significant improvement upon traditional LC-MS methods. The extraction of nitrosamines directly from solid sample not only simplifies the sample preparation procedure but also enables the method to be used for different products as is or with minor modifications, as demonstrated by the analysis of NDMA in 10+ pharmaceutical products. The in situ nitrosation that is commonly observed in GC methods for nitrosamine analysis was completely inhibited by the addition of a small volume solvent containing pyrogallol, phosphoric acid, and isopropanol. Employing simple procedures and low-cost instrumentation, this method can be implemented in any analytical laboratory for routine nitrosamine analysis, ensuring patient safety and uninterrupted supply of critical medications. Graphical Abstract.

Development of a simple liquid chromatography method for dissolution testing of low dose desogestrel and ethinylestradiol tablets.

Accurate quantitation of low dose, multi-active dissolution samples poses unique challenges in the pharmaceutical industry, often resulting in separate HPLC methods for each active or the use of multiple detectors for increased sensitivity. In this study, we report a fast, isocratic HPLC method utilizing only UV detection for dissolution testing of low dose desogestrel and ethinylestradiol tablets. Rapid separation is completed in 5 min using isocratic elution at a flow rate of 0.45 mL/min, with a column temperature at 30 °C, an injection volume of 50 µL and the detection wavelength at 200 nm. After extensive method development and optimization, the cyano stationary phase was used to overcome the large difference in hydrophobicity for desogestrel and ethinylestradiol, providing balanced retention for both analytes under isocratic elution. Chromatography modeling software was used to provide a rapid analysis of multiple columns and chromatography conditions. The optimized method boasts fast and efficient separation through use of a short, small I.D. column and a large injection volume of dissolution solution to achieve high sensitivity. The stable baseline from an isocratic separation allows low detection wavelengths to be used, resulting in accurate and precise quantitation of both desogestrel and ethinylestradiol. The method has been successfully validated for specificity, linearity, accuracy and precision in the range of 75 - 600 ng/mL for desogestrel and 10 - 80 ng/mL for ethinylestradiol using both HPLC and UHPLC systems. The method robustness was characterized using a design of experiment approach, and the operational design region of the method was established.

Dehydrocostus lactone (DHC) suppresses estrogen deficiency-induced osteoporosis.

Osteoporosis is a chronic bone lytic disease, because of inadequate bone ossification and/or excessive bone resorption. Even though drugs are currently available for the treatment of osteoporosis, there remains an unmet need for the development of more specific novel agents with less adverse effects. Dehydrocostus lactone (DHC), a natural sesquiterpene lactone, was previously found to affect the differentiation of inflammatory cells by inhibiting NF-κB pathways, and garnered much interest for its anti-cancer properties via SOCS-mediated cell cycle arrest and apoptosis. As NF-κB pathway plays an essential role in osteoclast differentiation, we sought to discover the biological effects of DHC on osteoclast differentiation and resorptive activity, as well as the underlying mechanisms on these effects. Our research found that DHC inhibited RANKL-induced osteoclast differentiation, bone resorption and osteoclast specific genes expression via suppression of NF-κB and NFAT signaling pathways in vitro. We further demonstrated that DHC protected against ovariectomy (OVX)-induced bone loss in mice and the protective effect was mediated at least in part through the attenuation of NF-κB signaling pathway. Thus, this study provides insight that DHC might be used as a potential pharmacological treatment for osteoporosis.

Analysis of thermolabile residual solvents in a spray dried dispersion using static headspace gas chromatography.

In this study, we discuss the development of a static headspace gas chromatography method for the analysis of residual acetone as well as its enriched impurities including mesityl oxide and diacetone alcohol, in a spray dried dispersion. The major challenges include the instability of mesityl oxide and diacetone alcohol at high temperature and peak tailing of diacetone alcohol. It was found that the headspace oven temperature has to be controlled to 150°C or below to prevent degradation beyond an acceptable level (< 1%). The peak tailing of diacetone alcohol was attributed to the "Phase Soaking" effect due to excessive diluent, which may condense and temporarily modify the stationary phase. The peak shape of diacetone alcohol is dependent on the column loading capacity and the peak area of N-methyl pyrrolidone, the solvent that elutes after diacetone alcohol. The headspace oven temperature was set at 140°C, where the highest response ratio of diacetone alcohol/N-methyl pyrrolidone at 1.46 and thus the best sensitivity was obtained. The calculated quantitation limits were 1 ppm for acetone, 3 ppm for mesityl oxide and 31 ppm for diacetone alcohol. The method successfully passed validation criteria for specificity, linearity, accuracy, and precision.

Challenging pharmaceutical analyses by gas chromatography with vacuum ultraviolet detection.

Vacuum ultraviolet (VUV) detector for gas chromatography (GC) provides qualitative spectral information from 125 nm to 240 nm. In this article, this information was applied to facilitate the development of a GC method for challenging pharmaceutical applications. Seven organic solvents were screened for trace level water content using VUV detection at 168 nm, and the results were used to identify n-hexane as a suitable diluent for 4-ethoxy-1,1,1-trifluoro-3-buten-2-one (ETFBO), a water reactive compound. Selective detection of compounds of interest was demonstrated by varying detection wavelengths. All compounds were detected at 145 nm except for one unknown impurity, which co-eluted with n-hexane solvent. This impurity was detected at 225 nm, where n-hexane has no absorbance. In addition, the VUV spectra were used to: 1) accurately track peaks during early method development; 2) detect co-eluting peaks; 3) match peak identity in a sample vs. a standard; and 4) assess peak purity. With the universal detectability, qualitative spectral information and ease of use, VUV will become a versatile tool for GC for both method development and routine analysis.