Senescence of endplate osteoclasts induces sensory innervation and spinal pain.

Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation, and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.

Pain behavior and phenotype in a modified anterior lumbar disc puncture mouse model.

Background: An experimental study was performed to improve the anterior approach model of intervertebral disc degeneration (IVDD). Objective: The aims of this study were to investigate the anterior approach model of IVDD for the cause of death, phenotypes, and underlying mechanisms of low back pain in mice. Method: In this study, we conducted an anterior puncture procedure on a cohort of 300 C57BL/6J mice that were 8 weeks old. Our investigation focused on exploring the causes of death in the study population (n = 300) and assessing the time-course changes in various parameters, including radiographical, histological, immunofluorescence, and immunohistochemistry analyses (n = 10). Additionally, we conducted behavioral assessments on a subset of the animals (n = 30). Results: Transverse vertebral artery rupture is a major factor in surgical death. Radiographical analyses showed that the hydration of the nucleus pulposus began to decrease at 2 weeks after puncture and obviously disappeared over 4 weeks. 3D-CT showed that disc height was significantly decreased at 4 weeks. Osteophyte at the anterior vertebral rims was observed at 2 weeks after the puncture. As the time course increased, histological analyses showed progressive disruption of the destruction of the extracellular matrix and increased secretion of inflammatory cytokines and apoptosis. Behavioral signs of low back pain were increased between the puncture and sham groups at 4 weeks. Conclusion: The improvement of anterior intervertebral disc approach model in mice will be useful to investigate underlying mechanisms and potential therapeutic strategies for behavior and phenotypes. Furthermore, the application of vibrational pre-treatment can be used to increase the sensitivity of axial back pain in the model, thereby providing researchers with a reliable method for measuring this critical phenotype.

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.

Senescence of endplate osteoclasts induces sensory innervation and spinal pain.

Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.

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.

Characterization of impurities of HIV NNRTI Doravirine by UHPLC-high resolution MS and tandem MS analysis.

World Health Organization estimates that 34 million individuals globally are living with Human Immunodeficiency Virus (HIV). Doravirine is a non-nucleoside reverse transcriptase inhibitors (NNRTI) being evaluated by Merck for the treatment of HIV-1 infection. Drug regulation authorities require the purity of a pharmaceutical to be fully defined. This is important to ensure that the pharmacological and toxicological effects are truly those of the drug substances and not because of the impurities. Thus, understanding the drug impurity profiles is critical to the safety and potency assessment of the drug candidate for clinical trials. The impurity characterization can also provide useful information for critical assessment of pharmaceutical processes. Advances in mass spectrometry instrumentation and methods allow the identification of impurities in pharmaceuticals with a minimum of sample material and increased sensitivity. In this study, a rapid and sensitive method was developed for the structural determination of the major impurities of doravirine. The study utilizes ultra performance liquid chromatography-high-resolution-tandem mass spectrometry (UHPLC-HRMS/MS) techniques to perform structure elucidation of the unknown structures. This approach has significant impact on impurity structural elucidation, and a total of five trace-level impurities of doravirine were characterized using the developed method. Copyright © 2016 John Wiley & Sons, Ltd.

Direct Observation of the Biaxial Stress Effect on Efficiency Droop in GaN-based Light-emitting Diode under Electrical Injection.

Light-emitting diode (LED) efficiency has attracted considerable interest because of the extended use of solid-state lighting. Owing to lack of direct measurement, identification of the reasons for efficiency droop has been restricted. A direct measurement technique is developed in this work for characterization of biaxial stress in GaN-based blue LEDs under electrical injection. The Raman shift of the GaN E2 mode evidently decreases by 4.4 cm(-1) as the driving current on GaN-based LEDs increases to 700 mA. Biaxial compressive stress is released initially and biaxial tensile stress builds up as the current increases with respect to the value of stress-free GaN. First-principles calculations reveal that electron accumulation is responsible for the stress variation in InxGa1-xN/GaN quantum wells, and then reduces the transition probability among quantum levels. This behavior is consistent with the measured current-dependent external quantum efficiency. The rule of biaxial stress-dependent efficiency is further validated by controlling the biaxial stress of GaN-based LEDs with different sapphire substrate thicknesses. This work provides a method for direct observation of the biaxial stress effect on efficiency droop in LEDs under electrical injection.

Simple spectrophotometry method for the determination of sulfur dioxide in an alcohol-thionyl chloride reaction.

Thionyl chloride is often used to convert alcohols into more reactive alkyl chloride, which can be easily converted to many compounds that are not possible from alcohols directly. One important reaction of alkyl chloride is nucleophilic substitution, which is typically conducted under basic conditions. Sulfur dioxide, the by-product from alcohol-thionyl chloride reactions, often reacts with alkyl chloride to form a sulfonyl acid impurity, resulting in yield loss. Therefore, the alkyl chloride is typically isolated to remove the by-products including sulfur dioxide. However, in our laboratory, the alkyl chloride formed from alcohol and thionyl chloride was found to be a potential mutagenic impurity, and isolation of this compound would require extensive safety measures. As a result, a flow-through process was developed, and the sulfur dioxide was purged using a combination of vacuum degassing and nitrogen gas sweeping. An analytical method that can quickly and accurately quantitate residual levels of sulfur dioxide in the reaction mixture is desired for in-process monitoring. We report here a simple ultraviolet (UV) spectrophotometry method for this measurement. This method takes advantage of the dramatic change in the UV absorbance of sulfur dioxide with respect to pH, which allows for accurate quantitation of sulfur dioxide in the presence of the strong UV-absorbing matrix. Each sample solution was prepared using 2 different diluents: 1) 50 mM ammonium acetate in methanol +1% v/v hydrochloric acid, pH 1.3, and 2) 50 mM ammonium acetate in methanol +1% glacial acetic acid, pH 4.0. The buffer solutions were carefully selected so that the UV absorbance of the sample matrix (excluding sulfur dioxide) at 276 nm remains constant. In the pH 1.3 buffer system, sulfur dioxide shows strong UV absorbance at 276 nm. Therefore, the UV absorbance of sample solution is the sum of sulfur dioxide and sample matrix. While in the pH 4.0 buffer system, sulfur dioxide has negligible UV absorbance at 276 nm, and the UV absorbance is attributed only to sample matrix. Quantitation of sulfur dioxide is achieved by subtracting the UV absorbance of sample solution at pH 4.0 from that at pH 1.3. The method is simple but sensitive, with a limit of quantitation of 80 µg L(-1). The method linearity was demonstrated from 2 mg L(-1) to 40 mg L(-1) with an R(2) of 0.998, and the spiked recovery ranges from 94% to 105% within the same range. The results are comparable with those obtained using inductively coupled plasma-atomic emission spectrometry (ICP-AES) and gas chromatography-mass spectrometry (GC-MS), suggesting that this method is accurate.

Efficient HPLC method development using structure-based database search, physico-chemical prediction and chromatographic simulation.

Development of a robust HPLC method for pharmaceutical analysis can be very challenging and time-consuming. In our laboratory, we have developed a new workflow leveraging ACD/Labs software tools to improve the performance of HPLC method development. First, we established ACD-based analytical method databases that can be searched by chemical structure similarity. By taking advantage of the existing knowledge of HPLC methods archived in the databases, one can find a good starting point for HPLC method development, or even reuse an existing method as is for a new project. Second, we used the software to predict compound physicochemical properties before running actual experiments to help select appropriate method conditions for targeted screening experiments. Finally, after selecting stationary and mobile phases, we used modeling software to simulate chromatographic separations for optimized temperature and gradient program. The optimized new method was then uploaded to internal databases as knowledge available to assist future method development efforts. Routine implementation of such standardized workflows has the potential to reduce the number of experiments required for method development and facilitate systematic and efficient development of faster, greener and more robust methods leading to greater productivity. In this article, we used Loratadine method development as an example to demonstrate efficient method development using this new workflow.

Rapid separation of desloratadine and related compounds in solid pharmaceutical formulation using gradient ion-pair chromatography.

We reported the development of an ion-pair chromatographic method to separate desloratadine and all known related compounds in Clarinex Tablets, which use desloratadine as active pharmaceutical ingredient (API). For the first time, baseline separation for desloratadine and all known related compounds was achieved by utilizing a YMC-Pack Pro C(18) column (150 mm x 4.6 mm I.D., 3 microm particle size, 120A pore size) and a gradient elution method. The mobile phase A contains 3 mM sodium dodecylsulfate (SDS), 15 mM sodium citrate buffer at pH 6.2, and 40 mM sodium sulfate, while the mobile phase B is acetonitrile. Chromsword, an artificial intelligence method development tool, was used to optimize several key chromatographic parameters simultaneously including buffer pH/solvent strength, and temperature/gradient profile. The resolution of desloratadine and desloratadine 3,4-dehydropiperidine derivative, one of the critical pairs was improved by adding 40 mM sodium sulfate. Ultraviolet detection at 267 nm was used to achieve the detection for desloratadine and all compounds. This method has been successfully validated according to ICH guidelines in terms of linearity, accuracy, quantitation limit/detection limit, precision, specificity and robustness. It could be used as a stability indicating method for desloratadine drug substances or drug products that use desloratadine as active pharmaceutical ingredient.

Comparison study of porous, fused-core, and monolithic silica-based C18 HPLC columns for Celestoderm-V Ointment analysis.

In this paper, three C18 columns with different substrates (i.e., porous ACE-3 C18, 3 microm, fused-core Halo C18, 2.7 microm, and monolithic Chromolith C18 were compared for the analysis of a pharmaceutical product, Celestoderm-V Ointment, that contains one active pharmaceutical ingredient, betamethasone-17-valerate and one critical pair of low level impurities, betamethasone-E-enolaldehyde and betamethasone-Z-enolaldehyde. Key column performance for the analysis of pharmaceutical products including selectivity, efficiency, separation impedance, resolution factor, sample loading capacity, linearity and lifetime from the three columns were determined. The potential applications of these three C18 columns for different methods for Celestoderm-V Ointment analysis are also recommended.

Mechanism for the separation of large molecules based on radial migration in capillary electrophoresis.

We demonstrate a novel separation mechanism for large molecules based on their radial migration in capillary electrophoresis with applied hydrodynamic flow (HDF). The direction of radial migration depends on the direction of the applied HDF relative to the electric field. The radial migration velocities are size-dependent, which could be attributed to the different degree of deformation under shear flow. Analytical separation was demonstrated on a sample plug containing lambda DNA (48 502 bp) and phiX174 RF DNA (5386 bp) with baseline separation. Alternatively, this separation mode can be performed continuously and is thus applicable to preparative separations. Without the need for gel/polymer or complex instrumentation, this separation technique is complementary to capillary gel electrophoresis and field-flow fractionation. Although large DNA molecules were used to demonstrate the separation mechanism here, these protocols could also be applied to the separation of proteins, cells, or particles based on size, shape, or deformability.

Mechanism of microbial aggregation during capillary electrophoresis.

We studied the aggregation of a rod-shaped bacteria, Bifidobacterium infantis, during capillary electrophoresis (CE). A microscope with an intensified CCD camera was employed to monitor the migration and aggregation of bacteria, which are labeled with fluorescent dye Syto 9 and excited with a 488-nm argon ion laser. A collision-based aggregation mechanism is proposed, in which collisions between microbes result from different mobilities and migration directions in the electric field. Individual microbes are aligned differently with respect to the direction of the electric field and exhibit different drag coefficients. The long-range forces include van der Waals attraction and electrostatic repulsion as qualitatively described by DLVO theory. Collisions in CE produce sufficient energy to overcome electrostatic repulsion, thus improving the efficiency of aggregation. This is supported by the fact that higher electric fields always resulted in faster aggregation. Also, when sodium phosphate buffer was used, increasing the ionic strength resulted in faster aggregation. However, when Tris-boric acid-EDTA (TBE, pH 9.1) buffer was used, the aggregation speed decreased when the ionic strength increased. We attribute this to the change of the surface of the bacteria at high borate and EDTA concentration, such as the loss of polysaccharides or the presence of complexation. This reduces the hydrophobicity of the surface and, thus, the short-range attractive forces. The addition of 0.05% poly(ethylene oxide) (PEO) into high ionic strength TBE buffer increased the aggregation rate. This can be attributed to the bridging effect of PEO between microbes. Further increase in the concentration of polymer reduced the aggregation rate, especially when the electric field was low, due in part to the increase in viscosity. The decrease in migration velocity produced lower collision energies and lower aggregation efficiencies as well.

Mechanistic aspects in the generation of apparent ultrahigh efficiencies for colloidal (microbial) electrokinetic separations.

Under specific experimental conditions, the electrokinetic separation of certain microorganisms can produce peaks of very high apparent efficiencies (approximately 10(6)-10(10) theoretical plates/m). This is unusual in that no deliberate focusing mechanism was employed. To investigate this process further, the separation was monitored in real time using a charge-coupled device (CCD) imaging system. At least two different processes seem to be operative when these narrow peaks are observed. The initial field-induced association of cells appears to require a dilute polymer solution, electroosmotic flow (preferably countercurrent to the direction of cell electrophoresis), and a direct current electric field. Three possible models are presented that may explain aspects of the observed behavior. The balance between dispersive forces and intercellular adhesive forces also affects the observed bandwidths. Understanding and controlling the dynamic and aggregation of cells in microfluidic processes is essential, since it can be beneficial for some experiments and detrimental to others.