Reverse Phase Research Articles

Chiral Differentiation of Chiral Lactides and Chiral Diketones on Native and Phenylcarbamoylated Cyclodextrin Chiral Stationary Phases.

Inclusion complexation, hydrogen bonds, π-π interaction, dipole-dipole interaction, and steric hindrance effect all contribute to the enantioseparation ability of cyclodextrin (CD) or CD derivatives. In this work, one native cationic CD chiral stationary phases (SHCDCSP) and four derivatized CD-CSPs, namely, per(4-trifluoromethyl) phenylcarbamoylated-β-CD CSP (SFPhCDCSP), per(4-chloro) phenylcarbamoylated-β-CD CSP (SCPhCDCSP), per(4-bromo) phenylcarbamoylated-β-CD CSP (SBPhCDCSP), and per(4-methyl) phenylcarbamoylated-β-CD CSP (SMPhCDCSP), were prepared via thioether linkage and applied for the enantioseparation of chiral lactides and chiral diketones in both reverse phase(RP) and normal phase (NP) modes. Most of the studied chiral lactides were found to be well resolved (Rs > 1.5) under RP mode, especially Met-Sty-Ibn (compound 11) is observed to display highest resolutions (Rs = 5.09, Rs = 3.17) among all the analytes on the SFPhCDCSP and SMPhCDCSP. In NP mode, SFPhCDCSP showed excellent chiral recognition ability towards chiral lactides. The comparison study of CD-CSPs reveals the structure of CSPs play a significant role on the enantioselectivities.

Evaluation of Quercetin's Bioenhancing Effect on Oral Pharmacokinetics of Rosuvastatin in Wistar Rats Using RP-HPLC Method.

The absolute oral bioavailability of rosuvastatin (RST), a secondgeneration statin, is low i.e. 20% and only 10% is recovered as metabolite N-desmethy l rosuvistatin. Since it is a hydrophilic statin, RST relies on the organic anion transporting polypeptide- 1B1 (OATP-1B1), as the key mechanism for active transport into hepatocytes. Quercetin (QUE) being a bio enhancer and inhibitor of OATP1B1 can augment the bioavailability and pharmacokinetics of RST. The present study includes the development of a simple and validated bioanalytical Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the estimation of RST and to study the effect of co-administration of QUE as a bio enhancer on its bioavailability. An analytical column of Kromasil 100, C18 (250 mm × 4.6 mm, 5 μm), was used for chromatographic separationand acetonitrile (ACN): acetic acid buffer pH 3.0 adjusted with glacial acetic acid (55:45 Vol. %) as mobile phase with flow rate 1.0 ml/min monitored at 242 nm. The ACN: methanol (50:50 Vol. %) was employed as the final solvent for extraction. The developed method has been successfully applied in a study on the pharmacokinetics of the drug RST in rats after co-administration of QUE, which was carried out using non-compartmental analysis in order to estimate the blood concentration of the drug. The pharmacokinetics of RST was found to be altered significantly (highest concentration of RST in the blood (Cmax) = 67.3 ng/ml to 122.2 ng/ml) (p < 0.001), area under curve (AUC)0-t (p < 0.0001) and AUC0-inf (p = 0.0005) when co-administered with QUE at 120 min (tmax). The results are in accordance with the fact that QUE increases plasma levels in rats through herb-drug interactions.

A Simple, Cost-Effective, and Sustainable Method for Extraction and Quantification of Chebulagic Acid from Terminalia bellirica (Gaertn.) Roxb. Using RP-HPLC: A Comparative Study

Chebulagic acid (CA), a hydrolysable tannin found in Terminalia bellirica (T. bellirica) (Gaertn.) Roxb., has gained prominence as a significant component in Ayurvedic medicine. This study optimized methods for determining CA in T. bellirica, focusing on fruit samples and prioritizing simplicity, cost-effectiveness, less time duration, and sustainability. We evaluated various extraction techniques, including continuous shaking, microwave assisted extraction, and Soxhlet extraction using green solvents. Quantification of CA was conducted using Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), with confirmation achieved via UV-Visible (UV-Vis) spectroscopy, Thin Layer Chromatography (TLC), and Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). Results indicated that Soxhlet and microwave significant extraction methods, particularly with ethanol as the green solvent. Soxhlet extraction yielded 49607.73±18.82 μg/mL for fresh fruit and 42952.53±19.09 μg/mL for dry fruit, while microwave extraction resulted in 43627.56±113.48 μg/mL for fresh fruit and 40466.33±130.90 μg/mL for dry fruit respectively. These findings conclude that ethanol as a green solvent in Soxhlet or microwave assisted extraction emerge as most efficient, straightforward, and effective approach for detecting and screening CA in T. bellirica samples.

Extraction and characterization of phytochemicals from Bauhinia variegata flowers using ultrasound and microwave techniques

This research investigates the efficacy of three distinct extraction techniques; ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and conventional extraction (CE) for the isolation of phytochemicals from the flowers of Bauhinia variegata. The findings demonstrated that both UAE and MAE surpass CE, yielding higher concentrations of bioactive compounds while significantly reducing the extraction time. UAE demonstrated the highest extraction efficiency, followed by MAE. The UAE yielded significantly higher levels of total phenolic content (43.97 mg GAE/g), total flavonoid content (24.08 mg QE/g), total anthocyanin content (16.37 mg CGE/g), DPPH antioxidant activity (19.29 mg TE/g), and FRAP antioxidant activity (16.75 mg TE/g) compared to MAE and conventional extraction methods. GC-MS analysis revealed a diverse array of phytochemicals, including flavonoids, phenolic compounds, and terpenoids, with some compounds showing higher concentrations in UAE. Reverse phase HPLC profiling further confirmed the enhanced extraction of phenolic compounds using UAE, including gallic acid, caffeic acid, coumaric acid, quercetin, and kaempferol. This study reveals the efficiency, time-saving, and eco-friendliness of UAE for extracting valuable bioactive compounds from B. variegata flowers. The findings optimize extraction processes in the food, pharmaceutical, and nutraceutical industries.

Retention behavior of Hg2+, MeHg+, thimerosal and phenylmercuric acetate on a C18 RP-HPLC column

Humans are exposed to potentially toxic mercuric mercury (Hg2+) and methylmercury (MeHg+) by the ingestion of food, to the bactericidal vaccine additive thimerosal (THI), and/or to the antifungal compound phenylmercuric acetate (PMA) which is used in some lens cleaning ophthalmic fluids. While numerous HPLC methods have been developed to separate Hg2+ and MeHg+ in environmental samples (e.g. food, surface waters), comparatively few have been reported for THI and PMA, in part owing to their increased hydrophobicity. We investigated the retention behavior of Hg2+, MeHg+, THI and PMA on a reversed-phase (RP) HPLC column using a flame atomic absorption spectrometer (FAAS) as a Hg-specific detector. Mobile phases comprised of 50 mM phosphate buffer (pH 7.4) with acetonitrile (ACN) concentrations of 30–50 % (v:v) produced single Hg-peaks, which eluted in the order THI, Hg2+, MeHg+ and PMA. With the 50 % ACN mobile phase, all mercurials eluted within 5 min. While the utilization of a FAAS precludes the analysis of environmental waters with the developed RP-HPLC-FAAS method, the latter is useful to probe the stability of THI and PMA in the presence of physiologically relevant concentrations of salt (100 mM in blood plasma) and l-cysteine (0.5 mM in hepatocyte cytosol), which is important as both mercurials have been recently shown to effectively inhibit the main protease of SARS-CoV-2, though the actual inhibitory Hg-species is unknown.

Analysis of perfluoroalkyl carboxylates and perfluoroalkyl sulfonates in environmental water by in-port arylation gas chromatography-electron impact ionization-mass spectrometry.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants that have been used for various purposes. Although PFAS can pollute the environment in a variety of areas related to the use, storage, and disposal of their products, there are insufficient data on the extent of PFAS pollution outside industrialized countries with their manufacturing facilities. Most of the analyses depend on high-cost liquid chromatography-tandem mass spectrometry. In this study, we established a method to analyze ionic PFAS using gas chromatography-electron impact ionization-mass spectrometry with in-port arylation by diaryl iodonium. Extraction of PFAS from water samples was performed using solid phase extraction with reverse phase sorbent. Fourteen PFAS compounds could be detected from spiked water samples, and the detection limit ranged from 3.1 to 8.1ng L-1. Using this method, we analyzed groundwater samples from Okinawa Island, Japan, and detected PFAS up to a total concentration of 1900ng L-1. This method uses relatively inexpensive analytical equipment; hence, it can possibly enable surveys on PFAS contaminations in a wide range of regions and opportunities.

Effect of Deformed Prior Austenite Characteristics on Reverse Phase Transformation and Deformation Behavior of High-Strength Medium-Mn Steel.

In this study, microstructure evolution during prior austenite decomposition and reverse phase transformation processes was revealed in a high-strength medium-Mn steel. Furthermore, the relationship between deformed prior austenite characteristics and deformation behavior was studied. The results indicated that the recovery and recrystallization of the deformed prior austenite were significantly inhibited during hot rolling in the non-recrystallized zone, the grain size was obviously refined along the normal direction (ND), and that the strain hardening of prior austenite via hot deformation could increase the resistance of shear transformation, resulting in the preservation of high-density lattice defects in the quenched martensite matrix. Before the nucleation of intercritical austenite, the dislocation and grain boundary can provide fast diffusion paths for C and Mn, and the enrichment of C and Mn before intercritical austenite formation can reduce the critical temperature of ferrite/austenite transformation. The nucleated sites and driving force for intercritical austenite were strongly increased by rolling in the non-recrystallization region. The resistance of crack propagation was found to be enhanced by the sustained transformation-induced plasticity (TRIP) effect (via retained austenite with different stability) and for the laminated microstructure, the optimum properties were obtained as being a combination of yield strength of 748 MPa, tensile strength of 952 MPa, and total elongation of 26.2%.

Parallel Analyses by Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) Reveal Complementary Proteomic Profiles in Triple-Negative Breast Cancer (TNBC) Patient Tissues and Cell Cultures.

High-plex proteomic technologies have made substantial contributions to mechanism studies and biomarker discovery in complex diseases, particularly cancer. Despite technological advancements, inherent limitations in individual proteomic approaches persist, impeding the achievement of comprehensive quantitative insights into the proteome. In this study, we employed two widely used proteomic technologies, mass spectrometry (MS) and reverse phase protein array (RPPA) to analyze identical samples, aiming to systematically assess the outcomes and performance of the different technologies. Additionally, we sought to establish an integrated workflow by combining these two proteomic approaches to augment the coverage of protein targets for discovery purposes. We used 14 fresh frozen tissue samples from triple-negative breast cancer (TNBC: seven tumors versus seven adjacent non-cancerous tissues) and cell line samples to evaluate both technologies and implement this dual-proteomic strategy. Using a single-step protein denaturation and extraction protocol, protein samples were subjected to reverse-phase liquid chromatography (LC) followed by electrospray ionization (ESI)-mediated MS/MS for proteomic profiling. Concurrently, identical sample aliquots were analyzed by RPPA for profiling of over 300 proteins and phosphoproteins that are in key signaling pathways or druggable targets in cancer. Both proteomic methods demonstrated the expected ability to differentiate samples by groups, revealing distinct proteomic patterns under various experimental conditions, albeit with minimal overlap in identified targets. Mechanism-based analysis uncovered divergent biological processes identified with the two proteomic technologies, capitalizing on their complementary exploratory potential.

Use of green RPLC methods as an alternative to conventional RPLC methods in the determination of the pKa values of some H2 receptor antagonists

In this study, the chromatographic retention behavior and dissociation constants (pKa ) of H2 receptor antagonists ranitidine and famotidine were determined by the green reverse phase liquid chromatography (RPLC) and hybrid micellar chromatography methods, which are widely used today. Replicate analyses were performed on a Gemini NX C18 (250 × 3.0 mm ID, 5 μm) column at constant temperature (37 °C) and flow rate (1 mL/min). The obtained data analyses were evaluated with the linear solvation energy relationship (LSER) approach and the consistency of the data was assessed by calculating the theoretical retention time values. The pK a 1 s s and pK a 2 s s values obtained in ethanol–water binary mixtures containing 11%, 13%, 15%, and 20% (v/v) ethanol were also calculated with this approach. The pKa values of these compounds in water and pure micellar environment were calculated with pK a s s values and solvent. It was observed that the p K a w w data obtained by three different methods were compatible. Accordingly, with the hybrid micelles method, p K a 2 w w and p K a 3 w w values for famotidine were calculated as 7.305 and 9.869, respectively, and p K a 1 w w and p K a 2 w w values for ranitidine were calculated as 2.952 and 8.589. The environmental impacts of the methods applied in the study were evaluated using green metric tools.

Exploring peptide dendrimers for intestinal lymphatic targeting: formulation and evaluation of peptide dendrimer conjugated liposomes for enhancing the oral bioavailability of Asenapine maleate

Asenapine maleate (ASPM) is a second-generation atypical antipsychotic that is approved for treating acute schizophrenia and bipolar disorder in adults by the US FDA. The major downside of ASPM therapy is rapid, extensive first-pass hepatic metabolism following its oral administration with a very low oral bioavailability of < 2%. In this work, we developed ASPM nanoformulations conjugated with ligands such as arginine-glycine-aspartic acid (RGD) and peptide dendrimers (PDs) with the intention of improving the oral bioavailability of the drug by targeting it to the intestinal lymphatic system (ILS). Peptide dendrimers (PDs), both lipidated and nonlipidated, were synthesized by Fmoc solid phase peptide synthesis (SPPS). Reverse phase high performance chromatography (RP-HPLC) was used to purify the synthesized PDs, and the PDs were characterized by differential scanning calorimetry (DSC) electrospray ionization mass spectroscopy (ESI+-MS), Nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy. The thin film hydration method was used to prepare liposomes, and the process variables affecting the liposome parameters were optimized using the Box‒Behnken design (BBD).Liposomes were PEGylated using DSPE-PEG-COOH2000 and further conjugated with ligands (RGD, PD-1 and PD-2) using EDC-NHS chemistry. The formulation was characterized using different spectroscopic techniques. In vitro, cell line studies, such as cytotoxicity, cell uptake, uptake mechanism, and receptor saturation studies, were performed on both Caco2 and Raji-B cells. The pharmacokinetic parameters of the developed liposomal formulation were evaluated using pharmacokinetic studies on Sprague- Dawley (SD) rats. The psychostimulant-induced hyperactivity model was used to evaluate the pharmacodynamic performance of the developed formulations by measuring the reversal of hyperlocomotor activity induced by levodopa-carbidopa.

DEVELOPMENT AND OPTIMIZATION OF HPLC METHODS FOR THE CHIRAL SEPARATION OF ARYLPYRAZOLE, CHLOROACETANILIDE AND ORGANOCHLORINE PESTICIDES: CASE STUDIES OF BENZOBICYCLON, ACETOCHLOR AND CHLORDANE.

This work is centered on the development and optimization of High-Performance Liquid Chromatographic methods for the separation of chiral pesticides, particularly benzobicyclon, chlordane and acetochlor employing Agilent Technologies Infinity II chiral HPLC system equipped with a UV – 2000 detector. The stereoisomers were successively separated using a standard analytical column of dimensions 250 mm X 4.6 mm. The Cis– stereoisomers of chlordane exhibited partial baseline separation while the Trans – enantiomers were fully separated. Benzobicyclon and acetochlor enantiomers attained full baseline separation under optimized conditions. Chlordane and benzobicyclon were analyzed using normal phase chromatography [NP]c with a mobile phase of 90% Normal hexane and 10% Isopropanol, while acetochlor was analyzed by reverse phase chromatography [RP] c using 70% methanol and 30% water. Benzobicyclon was later spiked into soil sample and subsequently extracted using Environmental Protection Agency (EPA) methods and the extracts were further analyzed with a shorter analytical column of dimensions 75 mm X 4.6 mm. The HPLC separation revealed that the longer column provided more efficient separation for the benzobicyclon stereoisomers than the shorter column as reflected by high separation factors (?). The high recovery rate of the stereoisomers of benzobicyclon and the internal standard from the soil extracts confirmed the effectiveness of the method of extraction and the HPLC system employed.

A Comprehensive LC-MS Metabolomics Assay for Quantitative Analysis of Serum and Plasma.

Background/Objectives: Targeted metabolomics is often criticized for the limited metabolite coverage that it offers. Indeed, most targeted assays developed or used by researchers measure fewer than 200 metabolites. In an effort to both expand the coverage and improve the accuracy of metabolite quantification in targeted metabolomics, we decided to develop a comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that could quantitatively measure more than 700 metabolites in serum or plasma. Methods: The developed assay makes use of chemical derivatization followed by reverse phase LC-MS/MS and/or direct flow injection MS (DFI-MS) in both positive and negative ionization modes to separate metabolites. Multiple reaction monitoring (MRM), in combination with isotopic standards and multi-point calibration curves, is used to detect and absolutely quantify the targeted metabolites. The assay has been adapted to a 96-well plate format to enable automated, high-throughput sample analysis. Results: The assay (called MEGA) is able to detect and quantify 721 metabolites in serum/plasma, covering 20 metabolite classes and many commonly used clinical biomarkers. The limits of detection were determined to range from 1.4 nM to 10 mM, recovery rates were from 80% to 120%, and quantitative precision was within 20%. LC-MS/MS metabolite concentrations of the NIST® SRM®1950 plasma standard were found to be within 15% of NMR quantified levels. The MEGA assay was further validated in a large dietary intervention study. Conclusions: The MEGA assay should make comprehensive quantitative metabolomics much more affordable, accessible, automatable, and applicable to large-scale clinical studies.

Aedes aegypti control in breeding sites through an insecticidal coating with dual effect: Laboratory trials and safety assessment.

Ground water tanks are known to be preferred Aedes aegypti oviposition places providing opportunities for adult and larvae control. Therefore, a dual-effect insecticidal coating (IC) (alphacypermethrin/ pyriproxyfen) with a slow-release mechanism and safe for users could be applied within Aedes spp. breeding sites, representing a promising option. Bioassays were designed to determine the mortality and sterilizing effects on gravid mosquitoes exposed to IC. The effect of inhibition of emergence was evaluated in eggs, larvae and pupae exposed in different containers. For the water safety assessment concentrations of active ingredients were determined by reverse phase high performance liquid chromatography (RP-HPLC) and the health risk was calculated. The IC applied to the interior walls of water-holding containers showed efficacy against Ae. aegypti in terms of high gravid-female mortality (81% at 24 h, p < 0.01), sterilizing effect (inhibition of oviposition by 63%, p < 0.01) and emergence inhibition (100% in eggs, L3 and L4; 97% in pupae). The offspring rate was reduced [only 0.15 (38/250) new adults emerged per exposed gravid females as against 11.90 per unexposed female (2976/250) at baseline]. Emergence inhibition was recorded up to 12 months and adult mortality >80% up to 6 months. The use of water stored in treated containers, either for washing or drinking, is not expected to pose a health risk to users. IC applied to domestic water containers has dual and complementary action that reduces Ae. aegypti densities (immature and adult stages). This represents baseline information for a cluster randomized efficacy trial in Colombia.

Development and Validation of Rapid and Cost-Effective RP-HPLC/UV Method for Determination of Cetirizine/Levocetirizine in Blood Plasma of Smokers and Non Smokers: a Two Cohort Comparative Study.

In this research study, a simple, rapid, sensitive, and cost-effective Reverse Phase High Performance Liquid Chromatography method was developed and validated for determination of Cetirizine and Levocetirizine in human plasma. The drugs were separated on an ACE Generix100-5 C18 RP (250 × 4.6mm, 5μm) column, using Acetonitrile and Distilled water pH7 (59:41v/v) as mobile phase with a flow rate of 1.5mL/min, retention time of 3.39 ± 0.02min, detection wavelength of 230nm, and column oven temperature at 26°C. The drug was extracted from human plasma using Methanol, the percent recovery was 97%. The LOD and LOQ for Cetirizine and Levocetirizine were 0.5ng and 1ng. The method was linear over the concentration range of 0.5-1000ng /mL for Cetirizine and Levocetirizine. The method was applied for analysis of drugs in plasma following oral administration of Cetirizine (10mg) and Levocetirizine (5mg) in smokers and nonsmokers of Pashtun ethnicity belonging from different districts of Khyber Pakhtunkhwa, Pakistan. The difference between Cetirizine plasma levels in smokers and non-smokers was significant with p -value 0.0279.

Antioxidant activity of Micractinium sp. (Chlorophyta) extracts against H2O2 induced oxidative stress in human breast adenocarcinoma cells

In response to the growing demand for high-value bioactive compounds, microalgae cultivation has gained a significant acceleration in recent years. Among these compounds, antioxidants have emerged as essential constituents in the food, pharmaceutical, and cosmetics industries. This study focuses on Micractinium sp. ME05, a green microalgal strain previously isolated from hot springs flora in our laboratory. Micractinium sp. cells were extracted using six different solvents, and their antioxidant capacity, as well as total phenolic, flavonoid, and carotenoid contents were evaluated. The methanolic extracts demonstrated the highest antioxidant capacity, measuring 7.72 and 93.80 µmol trolox equivalents g−1 dry weight (DW) according to the DPPH and FRAP assays, respectively. To further characterize the biochemical profile, reverse phase high-performance chromatography (RP-HPLC) was employed to quantify twelve different phenolics, including rutin, gallic acid, benzoic acid, cinnamic acid, and β-carotene, in the microalgal extracts. Notably, the acetone extracts of Micractinium sp. grown mixotrophically contained a high amount of gallic acid (469.21 ± 159.74 µg g−1 DW), while 4-hydroxy benzoic acid (403.93 ± 20.98 µg g−1 DW) was the main phenolic compound in the methanolic extracts under heterotrophic cultivation. Moreover, extracts from Micractinium sp. exhibited remarkable cytoprotective activity by effectively inhibiting hydrogen peroxide-induced oxidative stress and cell death in human breast adenocarcinoma (MCF-7) cells. In conclusion, with its diverse biochemical composition and adaptability to different growth regimens, Micractinium sp. emerges as a robust candidate for mass cultivation in nutraceutical and food applications.

TMIC-40. TARGETING GLIOMA TUMOR MICROENVIRONMENT BY SPECIFIC NEUTRALIZATION OF EXTRACELLULAR NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (ENAMPT) USING ALT-100, A NOVEL HUMANIZED MONOCLONAL ANTIBODY

Abstract BACKGROUND Intracellular nicotinamide phosphoribosyltransferase (iNAMPT is the rate-limiting enzyme of NAD salvage pathway and is preferentially used by glioma cells for regulating energy metabolism and homeostasis. When secreted, extracellular NAMPT (eNAMPT) exerts cytokine-like functions and is associated with poor prognosis in cancer patients. We have previously reported an essential role of iNAMPT for glioma cell proliferation and survival. Here, we examined whether eNAMPT influences glioma biology and tumor microenvironment by neutralizing eNAMPT using ALT-100, a novel humanized monoclonal antibody. METHODS Native levels of eNAMPT in blood samples of patients with primary/recurrent gliomas were determined using ELISA under an IRB approved protocol. Next, the effects of ALT-100 on glioma microenvironment were determined by treating viable human glioma tissue slices with ALT-100 and assessing downstream effects on RNA and protein using RNASeq, reverse phase protein array and spatial transcriptomics. Lastly, the in vivo effect of ALT-100-mediated eNAMPT neutralization on survival was determined in a mouse intracranial glioma model. RESULTS Measurable levels of eNAMPT were noted in all patient samples with a range of (3.7-71.2 ng/ml). Preliminary results showed a correlation of eNAMPT levels with grade of tumor. Human organotypic glioma slice cultures treated with ALT-100, showed a profound alteration of gene and protein expression particularly related to key energy metabolism and proliferation pathways and the PD-1/PD-L1 axis. Additionally, results of spatial profiling of ALT-100 treated human organotypic glioma slices will be presented. Survival and response data from ALT-100 treatment in ongoing in vivo experiment will also be reported. CONCLUSIONS This is the first report of ALT-100-mediated eNAMPT neutralization in gliomas and demonstrates profound effects on the tumor and its microenvironment. Given that ALT-100 is in phase 2 trials (NCT05938036) against ARDS, data from our studies can potentially accelerate translating ALT-100-mediated eNAMPT neutralization to treat gliomas.

EXTH-83. CHARACTERIZATION OF SPLICING ABERRATIONS INDUCED BY PRMT5 INHIBITION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) are lethal tumors with limited treatment options, with a high unmet need for novel therapies. PRMT5, an arginine methyltransferase that regulates several histone and non-histone targets, is overexpressed in cancers including GBM and is a promising therapeutic target given its role in promoting oncogenic processes. Here, we characterized the effects of pharmacological inhibition of PRMT5 by the brain-penetrant, orally bioavailable inhibitors, PRT811 and PRT808 on treatment-induced splicing aberrations using a panel of patient-derived glioma stem cells (GSC) and organotypic human glioma slice cultures. Changes in protein expression upon PRMT5 inhibition were assessed using reverse phase protein array (RPPA) analysis. Additionally, splicing aberrations due to PRMT5 inhibition in were tested in several GSCs using RNA-seq. We observed a potent reduction of symmetrical dimethylation of arginine (SDMA protein marks by both PRMT5 inhibitors across various GSC lines and ex-vivo brain tumor tissue slices. Notably, GSCs with both mutant and wild-type p53 displayed similar sensitivity to PRMT5 inhibition, independent of MTAP status. Moreover, PRMT5 inhibition elicited reduced cell proliferation, altered cell survival pathways, and apoptosis. RNA-seq analysis revealed that splicing alterations affecting numerous oncogenes and regulatory factors, encompassing 42 cancer driver genes, 80 splicing factors, and 42 transcriptional factors across these GSC lines, with specific splicing events observed in nuclear and cytoplasmic subcellular compartments. Additional analysis is ongoing to determine which of the aberrant transcripts are translated to yield tumor specific neoantigens that can be potential targets for tumor specific immune strategies. These results underscore the efficacy of pharmacological PRMT5 inhibition in genetically and epigenetically diverse GSCs, and highlight its potential for developing novel immune and non-immune therapeutic strategies against GBM.

Development and validation of HPLC-UV and LC-MS/MS methods for the quantitative determination of a novel aminothiazole in preclinical samples

Aminothiazoles are the important class of chemical groups which have proven their broad range of biological activities. A novel aminothiazole (21MAT) was quantified in analytical solutions using a high-performance liquid chromatography (HPLC) approach that was developed and partially validated for the analysis of in vitro experimental samples. An isocratic elution on reverse phase Phenomenex® Luna C18 (50 mm × 4.6 mm, 5 μm) column with 55% 0.1% v/v orthophosphoric acid in water and 45% of orthophosphoric acid in acetonitrile at a flow rate of 1 mL/min was used. The analyte was detected at 272 nm. Similar to this, a robust bioanalytical technique, LC-mass spectrometry (LC-MS/MS) was created and verified to measure 21MAT in rat plasma for use in in vitro screening study samples and early-stage pharmacokinetic research. The protein precipitation method was used to extract 21MAT from plasma. The mixture of 95: 5% v/v methanol: acetonitrile and 0.1% v/v formic acid, along with 15% of 5 mM ammonium formate solution, was used to separate the mixture on a reverse phase Waters Xterra RP® C18 (150 mm × 4.6 mm, 5 μm) column at a flow rate of 1 mL/min. Using electro spray ionisation mode in multiple reaction monitoring mode, the analyte and internal standard (a structural analogue) were both identified. According to current criteria, all validation parameters (specificity, selectivity, accuracy, precision, recovery, matrix factor, hemolysis effect, and stability) were evaluated in rat plasma. The area response of 21MAT was found to be linear over the concentration range of 1.25–1250 ng/mL in rat plasma. Both techniques are suitable for use in any format of preclinical research and were sufficiently reliable to measure 21MAT precisely in various matrices. In silico prediction helped in understanding absorption, distribution, metabolism, excretion, and toxicity (ADMET) behaviour of the molecule. Both developed LC-MS/MS and HPLC-UV methods were successfully used to quantify the analyte in in vitro screening study samples.

Effect of cyclic loading on microstructure and plastic deformation in heat-treated Co–28Cr–6Mo alloy fabricated via laser powder bed fusion: An in situ synchrotron X-ray diffraction study

We present a systematic microstructure-oriented plasticity investigation of an additively manufactured cobalt-based alloy aiming to relate microstructural changes to the fatigue resistance. A load-controlled cyclic test in the low-cycle fatigue regime was utilized to study mechanical response and microstructural changes in the alloy after reverse phase transformation heat treatment. Deformation-induced FCC → HCP phase transformation was followed using in situ synchrotron X-ray diffraction combined with ex situ electron backscatter diffraction and scanning electron microscopy. Scanning transmission electron microscopy provided experimental evidence of the presence of precipitates in the solution annealed sample. It was found, that a stepwise increase in plastic deformation is attributed to nucleation and growth of different martensitic crystallographic variants. These insights into plasticity and microstructural changes suggest that the reverse phase transformation heat treatment is an effective approach for improving the fatigue resistance of low stacking fault energy alloys, that are susceptible to deformation-induced martensitic transformation.

Study of the Acidic, Basic, and Thermal Degradation Kinetics of Three Antihypertensive Drugs-Individually and in Combination.

The importance of fixed-dose combinations (FDCs) for the treatment of hypertension is well established. However, from a stability perspective, FDCs present a challenge since the degradation of one active pharmaceutical ingredient (API) can be affected by the presence of another API. The aim of this study was to compare the degradation behaviors and evaluate the degradation kinetics of three antihypertensive drugs, perindopril tert-butylamine (PER), amlodipine besylate (AML), and indapamide (IND). The degradation processes were studied using the previously developed reverse phase high-performance liquid chromatographic (RP-HPLC) method after exposing each drug individually, as well as the combinations of two/three drugs, to different stress factors, such as light, oxidation, acidic, basic, or neutral pH values at different temperatures. The results show that PER is most unstable under basic conditions and that AML displays a negative, while IND displays a positive effect, on PER stability when combined. AML is most affected by basic conditions and oxidation, and its stability is affected by both drugs positively; IND undergoes extreme photolysis, which is positively affected by AML but negatively by PER. Great care must be taken when formulating FDCs with these three drugs, as well as solutions or oral suspensions adjusted for geriatric or pediatric populations, since the stability of all three drugs is greatly affected by pH conditions, as well as light or oxidation factors and their interactions.