Reverse Phase Research Articles

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.

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

Background/Objectives: 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). Methods: 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. Results: 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. Conclusions: 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.

An improved synthesis of alkyl AMP esters

Enzymes belonging to the Acyl-CoA/NRPS/Luciferase (ANL) superfamily of enzymes, are significant targets in drug development. One member of this superfamily is Acetyl-CoA Synthetase (ACS). This enzyme is an emerging target for the treatment of various infectious diseases and cancer. Alkyl AMP esters have emerged as potent inhibitors of ACS. Previous methods for synthesizing these esters often involved water-based reactions or necessitate purification through reverse phase prep-HPLC or ion-exchange chromatography. To address these challenges, we developed a new approach utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to couple 5′-adenylic acid to the corresponding alcohol, utilizing triethylamine as the base. This method yielded primary, secondary, unsaturated, and cyclic alcohols in excellent yields. Importantly, we optimized the reaction conditions to achieve excellent yield and purity without the need for reverse phase prep-HPLC or ion exchange chromatography. Instead, purification was achieved through silica gel chromatography using Biotage ® Sfar spherical silica gel.

Investigation on the Retention and Separation of Glucosinolates With a Mixed‐Mode Reversed‐Phase/Weak Anion‐Exchange Column

ABSTRACTGlucosinolates, a crucial group of secondary metabolites in Brassica vegetables, present significant chromatographic separation challenges due to their anionic form, structure diversities, and co‐existence of other phenolic compounds. This study comparatively investigated the retention and separation of seven glucosinolates using a mixed‐mode reversed‐phase/weak anion‐exchange column and a conventional reversed‐phase C18 column. Separation factors for each glucosinolate with its adjacent peaks were over 1.0 on the mixed‐mode column, while co‐eluting was observed on the C18 column. The effects of mobile phase additives and pH on the separation and retention of glucosinolates were also investigated. Results showed that glucosinolate retention was inversely related to both buffer concentration and pH. The optimized method for the mix‐mode column was applied to the complex Brassica vegetable samples. In addition to the 17 well‐resolved glucosinolate peaks, 34 peaks for phenolic compounds were identified in broccoli microgreen, suggesting the successful application scenarios for qualitative analysis in comparison with the single mode reverse phase C18 column. This study demonstrates that the mixed‐mode reversed‐phase/weak anion‐exchange column can be used as a promising separation tool for organic anions in a complex sample matrix.

Phase reversion mediated the dual heterogeneity of grain size and dislocation density in an equiatomic CrCoNi medium-entropy alloy

An ultra-high strain rate (104 s-1) dynamic plastic deformation treatment at liquid nitrogen temperature (LNT-DPD) followed by annealing is carried out to obtain dual heterogeneity of grain size and dislocation density in an equiatomic CrCoNi medium entropy alloy (MEA). Such extreme loading conditions resulted in extensive phase transformation in this MEA. Subsequent annealing at 650°C for 1 h further induced reverse phase transformation and partial recrystallization, forming a complex heterogeneous microstructure characterized by nested trimodal grain sizes and partitioned dislocation density. A superior yield strength of ∼800MPa and a good ductility of ∼40% were simultaneously achieved in this heterogeneous alloy. In order to reveal the effects of grain size and dislocation density distributions on the mechanical property improvements, the underlying deformation mechanisms were systematically discussed. High density of geometrically necessary dislocations (GNDs) would be induced in complex heterogeneous structures during tensile deformation due to strain gradients or partitioning between different regions, which would lead to additional strengthening and work hardening. These results provide a novel approach to overcome the strength-ductility trade-off dilemma for FCC-structured MEAs.

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.

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.

Stability indicating RP-HPLC method development and validation for quantification of impurities in gonadotropin-releasing hormone (Elagolix): Robustness study by quality by design.

The purpose of this research was to establish and validate a reverse phase HPLC method for the determination of Elagolix impurities in pharmaceutical dosage form. Mobile phase A, consisting of 10mM sodium dihydrogen phosphate (pH6.0) and acetonitrile in a 95:5v/v ratio, and mobile phase B, containing 85:10:5v/v/v of acetonitrile, Milli-Q water, and methanol, were used to achieve the method's specificity in the analytical column Kromasil 100-C18 (250 mm × 4.6mm, 5μm). The gradient program includes (%B/Time [min]: 36/0, 36/10, 38/15, 85/55, 85/65, 36/67, and 36/75). The flow rate is 0.8mL/min. The overall run duration is 75.0min, the injection volume is 10.0μL, and the detection is at 210 nm in UV. The samples were subjected to hydrolysis, oxidation, and heat conditions in order to facilitate their forced degradation. The procedure was validated and determined with the standards of ICH guidelines. From the LOQ to a concentration level of 200%, the linearity of the technique was ascertained. An accuracy range of LOQ to 150% was established for the method, and the average recovery was acceptable. Design of experiments, part of the quality by design idea, was used to prove the method's reliability.

Phenolic Profile of Seedless Ziziphusmauritiana Fruits and Leaves Extracts with In Vivo Antioxidant and Anti-inflammatory Activities: Influence on Pro-Inflammatory Mediators.

The study aimed to assess the antioxidant and anti-inflammatory activities ofpolyphenol-rich extracts of seedlessvariety of Ziziphus mauritiana(SZM). Reverse Phase High Performance Liquid Chromatographyanalysis of SZM leaves and fruit extracts in ethanol revealed the presence of sixteen phenolics including chlorogenic acid, p-coumeric acid, gallic acid, kaempferol and rutin. Leaf extract showed higher total phenolic and total flavonoid contents (177.6 mg/100 g and 46.2 mg/100 g) than in fruit extract (137.8 mg/100 g and 14.1 mg/100 g). The leaf extract exhibited higher DPPH radical-scavenging activity (63.5%) than the fruit extract (58.2%). The anti-inflammatory activity was evaluated on carrageenan-induced rat model and suppression of inflammatory biomarkers (Interleukin-6, Tumor necrosis factor-α and CRP) were studied. The fruit extract exhibited inhibition (98.1%) at the doseof 500 mg/kg body weight (BW), comparable to the indomethacin (98.4%). Both extracts suppressed the inflammatory biomarkers, but pronounced results showed by the fruit extract including CRP, IL-6, and TNF-α. The leaf extract demonstrated the higher antioxidant potential as evident from the superoxide dismutase, catalase, malondialdehyde, glutathione peroxidase and glutathione levels. These findings suggest that SZM leaf and fruit extracts possess potential antioxidant and anti-inflammatory properties and can play a significant role in mitigating oxidative stress.

In-vitro antibacterial and antibiofilm activities and in-silico analysis of a potent cyclic peptide from a novel Streptomyces sp. strain RG-5 against antibiotic-resistant and biofilm-forming pathogenic bacteria.

The proliferation of multidrug-resistant and biofilm-forming pathogenic bacteria poses a serious threat to public health. The limited effectiveness of current antibiotics motivates the search for new antibacterial compounds. In this study, a novel strain, RG-5, was isolated from desert soil. This strain exhibited potent antibacterial and antibiofilm properties against multidrug-resistant and biofilm-forming pathogenic bacteria. Through phenotypical characterizations, 16S rRNA gene sequence and phylogenetic analysis, the strain was identified as Streptomyces pratensis with 99.8% similarity. The active compound, RG5-1, was extracted, purified by reverse phase silica column HPLC, identified by ESI-MS spectrometry, and confirmed by 1H and 13C NMR analysis as 2,5-Piperazinedione, 3,6-bis(2-methylpropyl), belonging to cyclic peptides. This compound showed interesting minimum inhibitory concentrations (MICs) of 04 to 15µg/mL and minimum biofilm inhibitory concentrations (MBICs 50%) of ½ MIC against the tested bacteria. Its molecular mechanism of action was elucidated through a molecular docking study against five drug-protein targets. The results demonstrated that the compound RG5-1 has a strong affinity and interaction patterns with glucosamine-6-phosphate synthase at - 6.0kcal/mol compared to reference inhibitor (- 5.4kcal/mol), medium with penicillin-binding protein 1a (- 6.1kcal/mol), and LasR regulator protein of quorum sensing (- 5.4kcal/mol), confirming its antibacterial and antibiofilm activities. The compound exhibited minimal toxicity and favorable physicochemical and pharmacological properties. This is the first report that describes its production from Streptomyces, its activities against biofilm-forming and multidrug-resistant bacteria, and its mechanism of action. These findings indicate that 2,5-piperazinedione, 3,6-bis(2-methylpropyl) has the potential to be a promising lead compound in the treatment of antibiotic-resistant and biofilm-forming pathogens.

Impact of zinc oxide nanoparticles on biosynthesis of thymoquinone in cell cultures of Nigella sativa

The rising market interest for Nigella sativa (Black seeds) necessitates the development of cultivation strategies to enhance metabolites production. Zinc oxide nanoparticles (ZnO-NPs) have drawn global attention as efficient and bio safe elicitors for in vitro cultures, to enhance secondary metabolites production in medicinal plants. In this study, ZnO-NPs were utilized for establishment of callus and cell cultures in black seeds for the first time. Hypocotyl explants were cultured on Murashige and Skoog (MS) media with varying levels of ZnO-NPs, resulted in callus induction and biomass formation. Optimal response in callus growth parameters were observed when explants were grown on MS media supplemented with 60 mg/L ZnO-NPs, resulting in 71.2 % callus induction frequency, 28.2 g/L fresh biomass, 9.7 g/L dry biomass, and 63 % water content. A substantial increase in callus growth was observed when ZnO-NPs were combined with 6-Benzylaminopurine (BA) at ratio of 45:1.5 mg/L, resulting in 91.2 % callus induction frequency and 42.2 g/L fresh biomass. In cell suspension cultures, ZnO-NPs alone at 45 mg/L produced optimum callus biomass (60.9 g/L). However, in combination with BA, callus biomass did not increase significantly in cell cultures. Maximum accumulation of total phenolic content (TPC: 26.8 mg GAE/g DW; Gallic acid equivalent dry weight) and total flavonoid content (TFC: 19.5 mg QE/g DW; Quercetin equivalent dry weight) was observed in cell cultures treated with higher concentration (70 mg/L) of ZnO-NPs in the 5th week of the growth curve. Moreover, ZnO-NPs incremented substantially the Phenylalanine lyase (PAL), Superoxide dismutase (SOD) and Peroxidase (POD) enzyme activities in cell cultures. Nonetheless, Reverse Phase High Performance Liquid Chromatography (RP-HPLC) analysis indicated peak production of thymoquinone (168.5 mg/g FW) in cell cultures treated with 45 mg/L ZnO-NPs alone. This study offers a promising approach for commercial production of Nigella sativa biomass and bioactive metabolites.

Evaluation of MENAQUINONE‐7 and fat‐soluble vitamin production by starter cultures during fermentation in dairy products using RPLC method

AbstractVitamin K2, also known as menaquinone, has become a significant research focus, particularly in fermented products. This study aims to investigate the content of menaquinone‐7 (MK‐7), an essential form of vitamin K, and other fat‐soluble vitamins (A, D, E) during the fermentation of various fermented milk products. The vitamin contents of six fermented milk products were analyzed: two yogurt samples (produced with commercial starter and probiotic starter), two kefir samples (produced with commercial starter and kefir grain), and milk fermented with Lactobacillus acidophilus or Bifidobacterium bifidum. Throughout the fermentation process, continuous pH monitoring was conducted, and fermentation was terminated based on pH levels. Fat‐soluble vitamins and vitamins K1, K2 were extracted from samples taken at specific fermentation periods and quantitatively determined using the reverse phase liquid chromatography (RPLC) method. The developed method was validated according to ICH guidelines. Simultaneously, the microbial content of the samples was analyzed. Among the fermented dairy products analyzed, the highest MK‐7 content (4.82 μg/100 g) was found in the kefir sample produced with kefir grain (KG). The diverse microorganisms in kefir grains necessitated detailed research to explain their role in this complex mechanism. In yogurt samples, the presence of L. acidophilus in the YB sample suggests that this bacterium may be responsible for the observed differences. This is supported by the LA sample, where production with L. acidophilus strain showed an increase from 0.97 to 1.70 at the 20th hour of fermentation. It was noted that the concentration of menaquinone‐7 increased throughout the fermentation period. Given the critical health effects of essential fat‐soluble vitamins and MKs, determining their content in commonly consumed fermented dairy products and understanding the influence of starter cultures, which are potential probiotics, on vitamin production underscores the importance of this research. It is important to highlight the potential of fermented products in nutritional recommendations.

Development of microflow ultra high performance liquid chromatography-mass spectrometry metabolomic assays for analysis of mammalian biofluids

Introduction and objectivesThe application of untargeted metabolomics assays using ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) to study metabolism in biological systems including humans is rapidly increasing. In some of these studies there is a requirement to collect and analyse low sample volumes of biofluids (e.g. tear fluid) or low cell and tissue mass samples (e.g. tissue needle biopsies). The application of microflow, capillary or nano liquid chromatography (≤ 1.0 mm column internal diameter (i.d.)) theoretically should accomplish a higher assay sensitivity compared to analytical liquid chromatography (2.1–5.0 mm column internal diameter). To date, there has been limited research into microflow UHPLC-MS assays that can be applied to study samples of low volume or mass.MethodsThis paper presents three complementary UHPLC-MS assays (aqueous C18 reversed-phase, lipidomics C18 reversed-phase and Hydrophilic Interaction Liquid Chromatography (HILIC)) applying 1.0 mm internal diameter columns for untargeted metabolomics. Human plasma and urine samples were applied for the method development, with porcine plasma, urine and tear fluid used for method assessment. Data were collected and compared for columns of the same length, stationary phase and stationary phase particle size but with two different column internal diameters (2.1 mm and 1.0 mm).Results and conclusionsAll three assays showed an increase in peak areas and peak widths when applying the 1.0 mm i.d. assays. HILIC assays provide an advantage at lower sample dilutions whereas for reversed phase (RP) assays there was no benefit added. This can be seen in the validation study where a much higher number of compounds were detected in the HILIC assay. RP assays were still appropriate for small volume samples with hundreds of compounds being detected. In summary, the 1.0 mm i.d. column assays are applicable for small volume samples where dilution is required during sample preparation.

Molecular mechanisms underlying amyloid beta peptide mediated upregulation of vascular cell adhesion molecule-1 in Alzheimer's disease.

Amyloid beta (Aβ) deposition and neurofibrillary tangles are widely considered as the primary pathological hallmarks of familial and sporadic forms of Alzheimer's disease (AD). However, cerebrovascular inflammation, which is prevalent in 70% of AD patients is emerging as another core feature of AD pathology. In addition, activation of inflammatory signaling pathways have been observed in AD patients; specifically, cerebrovascular inflammation was found to be augmented in Alzheimer's patients. Our studies have demonstrated that the inflammation signaling pathway is upregulated in AD patient brains. Moreover, vascular cell adhesion molecule-1 (VCAM-1), a cerebrovascular inflammatory marker expressed on the blood-brain barrier (BBB) endothelium, was observed to be upregulated in APP,PS1 mice (a mouse model that overexpresses Ab42), as detected by dynamic SPEC/CT imaging. While there is a strong association between Aβ42 exposure and an increase in VCAM-1 expression, the mechanisms underlying the influence of Aβ42 on VCAM-1 expression remain understudied. Therefore, we investigated the hypothesis that Ab42 exposure increases VCAM-1 expression in human cerebral microvascular endothelial cell (hCMEC/D3) monolayers. In addition, reverse phase protein array assays (RPPA) and immunocytochemistry demonstrated that Ab42 increases VCAM-1 expression through the Src/p38/MEK signaling pathway specifically within the blood-brain barrier (BBB) endothelium. In summary, these results demonstrate that Ab42 augments cerebrovascular inflammation by elevating VCAM-1 expression via Src/MEK/p38 pathway. Hence, targeting VCAM-1 at the BBB as a diagnostic and therapeutic marker may hold potential for detecting and mitigating cerebrovascular inflammation in Alzheimer's disease. Significance Statement While considered a core pathological feature of Alzheimer's disease, molecular pathways leading to cerebrovascular inflammation remain partially understood. Moreover, clinical diagnostic methods for detecting cerebrovascular inflammation are underdeveloped. In this study, we demonstrated VCAM-1 detection using radio-iodinated VCAM-1 antibody and SPECT/CT imaging. The study demonstrated that the exposure to Aβ42 increases VCAM-1 expression on the BBB endothelium via Src/p38/MEK pathway. These findings are expected to aid in the development of diagnostic and therapeutic approaches for addressing cerebrovascular inflammation in AD.

Estimation of Plasma Concentration of L-Carnosine and its Correlation with Core Symptoms of Autism Spectrum Disorder Children: A Pilot Clinical Trial.

Literature indicates that L-carnosine may be deficient in autism spectrum disorder (ASD) children. The aim of the present study was to estimate the level of L-carnosine in plasma and correlate it with the Autism Treatment Evaluation Checklist (ATEC) and Childhood Autism Rating Scale 2nd Edition, Standard Version (CARS2-ST) scores. To measure L-carnosine level, a bio-analytical method was developed using reverse phase high- liquid chromatography and validated as per International Conference on Harmonization guidelines. Children were supplemented with L-carnosine (10-15 mg/kg) along with standard care therapies for 2 months. Before and after supplementation, scores on the ATEC, CARS2-ST, BEARS sleep screening tool, 6-item Gastrointestinal Severity Index, and Parental Stress Scale were evaluated, and L-carnosine was measured at the end of the trial. The calibration curve was linear in the range of 100-600 ng/mL (R2 = 0.998). The level of L-carnosine quantified was 33.7 ± 0.2 ng/mL. There was no significant difference found in any of the outcome measures (p > 0.05). Despite the fact that L-carnosine is detectable in the blood, it was found to be ineffective in the management of ASD in children. The study was registered in the Clinical Trial Registry-India, registration number: CTRI/2019/07/020102.

Method Development and Validation of Luliconazole Bulk Drug Using Reverse Phase - HPLC Technique

Objective: To develop and validate the RP- HPLC method for determining Luliconazole in bulk and pharmaceutical formulations. Methods: Following the International Conference on Harmonization (ICH) guidelines, a sensitive, accurate, and specific reversed-phase HPLC technique was created and validated for the detection of Luliconazole. Using high-performance liquid chromatography, this drug was examined. A better separation of the drug was achieved by using Agilent Eclipse XDB C (4.6mm x 250mm, 5µm) with a mobile phase consisting of a mixture of Acetonitrile and Buffer in HPLC water. The ratio of 30:70 v/v (Formic acid and Acetonitrile) at a flow rate of 1.0 ml/ min and the detection was at the wavelength 295 nm using a Photodiode Arrays Detector. The planned method was able to produce good quality separation of the drug and its degradation products with sharp peaks. Findings: Luliconazole had a retention time of 2.38 ± 0.127 minutes. The technique was linear in the range of 10-100 µg/ml, with a correlation (r2) of 0.9995 and a run duration of 4 minutes. The method limit of detection (LOD) and limit of quantification (LOQ) were set at 0.1 and 1 g/ml, respectively. The method accuracy and system precision were estimated, and the findings were calculated as percentage RSD values, which were found to be limitations. Luliconazole recovery was 100% confirming the method's efficiency. Novelty: The presented approach was innovative and successfully fulfilled all validation requirements, including linearity, robustness, accuracy, reduced retention, and run time. This suggests that the method is appropriate for identifying the stability of luliconazole in bulk and pharmaceutical formulations. Keywords: RP-HPLC, Transferosome, ICH guidelines, Validation, Luliconazole

Development of a targeted LC-MS/MS method for the analysis of zero tolerance antimicrobials in buffalo meat

A simple, affordable, and efficient multiresidue analytical method was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to accurately identify and quantify a prioritized zero-tolerance antimicrobial residues in buffalo meat. Analyzing zero-tolerance antimicrobials such as chloramphenicol, metronidazole, and nitrofurans involves detecting even trace levels of residues in food products to safeguard consumer safety and public health. A modified QuEChERS method with 2-nitrobenzaldehyde derivatization using ethyl acetate was employed for the extraction of the antimicrobial compounds, followed by a suitable clean-up. Liquid chromatographic conditions were optimized on a reverse phase C18 analytical column over a runtime of 12 min, and the eluted analytes were observed in selected reaction monitoring mode (SRM). The method's performance was validated in accordance with Regulation (EU) 2019/1871 and Commission Implementing Regulation (EU) 2021/810 guidelines. The linearity of the calibration curves was > 0.99, and the mean recoveries of the analytes ranged from 81.92 % to 110 %. The intra-day and inter-day precisions were less than 9 %. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.04–0.11 µg/kg and 0.13–0.37 µg/kg, respectively. This is well below the MRLs set by the European Commission for zero-tolerance substances.

Intraspecific venom variation in the medically important puff adder (Bitis arietans): Comparative venom gland transcriptomics, in vitro venom activity and immunological recognition by antivenom.

Variation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the ~310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy. We applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique. Through the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms. The large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region.

Rational Design of HER2-Targeted Combination Therapies to Reverse Drug Resistance in Fibroblast-Protected HER2+ Breast Cancer Cells

Abstract Introduction Fibroblasts, an abundant cell type in the breast tumor microenvironment, interact with cancer cells and orchestrate tumor progression and drug resistance. However, the mechanisms by which fibroblast-derived factors impact drug sensitivity remain poorly understood. Here, we develop rational combination therapies that are informed by proteomic profiling to overcome fibroblast-mediated therapeutic resistance in HER2+ breast cancer cells. Methods Drug sensitivity to the HER2 kinase inhibitor lapatinib was characterized under conditions of monoculture and exposure to breast fibroblast-conditioned medium. Protein expression was measured using reverse phase protein arrays. Candidate targets for combination therapy were identified using differential expression and multivariate regression modeling. Follow-up experiments were performed to evaluate the effects of HER2 kinase combination therapies in fibroblast-protected cancer cell lines and fibroblasts. Results Compared to monoculture, fibroblast-conditioned medium increased the expression of plasminogen activator inhibitor-1 (PAI1) and cell cycle regulator polo like kinase 1 (PLK1) in lapatinib-treated breast cancer cells. Combination therapy of lapatinib with inhibitors targeting either PAI1 or PLK1, eliminated fibroblast-protected cancer cells, under both conditions of direct coculture with fibroblasts and protection by fibroblast-conditioned medium. Analysis of publicly available, clinical transcriptomic datasets revealed that HER2-targeted therapy fails to suppress PLK1 expression in stroma-rich HER2+ breast tumors and that high PAI1 gene expression associates with high stroma density. Furthermore, we showed that an epigenetics-directed approach using a bromodomain and extraterminal inhibitor to globally target fibroblast-induced proteomic adaptions in cancer cells, also restored lapatinib sensitivity. Conclusions Our data-driven framework of proteomic profiling in breast cancer cells identified the proteolytic degradation regulator PAI1 and the cell cycle regulator PLK1 as predictors of fibroblast-mediated treatment resistance. Combination therapies targeting HER2 kinase and these fibroblast-induced signaling adaptations eliminates fibroblast-protected HER2+ breast cancer cells.