Drug Assay Research Articles

Facile and Sustainable Electrostatic Integration of Eosin Y Dye for the Assay of the Atomoxetine Drug Through the Enhancement of the RRS Phenomenon.

The suggested study follows specific protocols to guarantee that the atomoxetine drug analysis approach is environmentally friendly and sustainable. A number of recently created methods were used as prospective evidence for environmental sustainability and applicability, which is an essential point to emphasize. The current study introduces a new and very unique technology using ultrasensitive spectrofluorimetry to identify the atomoxetine (AXT) medication. A one-step, one-pot, direct spectrofluorimetric technique was used in this study's methodology, which was determined to be effective and environmentally sustainable for the drug's evaluation and validation. When AXT and EY reagent were mixed in an acidic setting, a highly resonant Rayleigh scattering product was promptly generated. The application of fluorimetric analysis was built on a novel theory that the augmentation in dye response subsequent to the addition of AXT was directly correlated to the resultant complex's molecular mass, as determined at a wavelength of 365nm. The complexation progression caused a considerable rise in the molecular mass, from 292.82 (AXT+) to 983.68 (AXT-EY) g mol-1. The quantum yield for the coupled product was measured. The linearity was determined to be between 30 and 1500 ng mL-1, while the sensitivity values were found to be between 9.2 and 28.1 ng mL-1. Analyzing AXT-EY complexes allowed us to determine the best values for all of the system's adjustable parameters. The system showed adherence to International Council for Harmonization (ICH) criteria without difficulties. The recommended procedure was then evaluated for environmental friendliness using many current environmental safety metrics. Fortunately, the current analytical technique is also recognized as a white one by the WAC standards, which integrate functional and ecological features using the Green/Red/Blue (RGB 12) design. A novel instrument called BAGI was used to assess the feasibility of the proposed approach in the field of analytical chemistry.

B-124 Structure-based Variant Effect Prediction and Design of Functional CYP2C9 Variants by Virtual Evolution

Abstract Background The decryption of massive variants of uncertain significance (VUS) discovered by sequencing poses the main challenge in the post-sequencing era. VUS also resulted in a particular problem in the field of pharmacogenomics, which can be epitomized by drug-metabolizing enzymes, such as CYP2C9. Therefore, an accurate model that incorporates the inner connection between massive protein variants and their function alterations is crucial for clinical annotation of human variome and the virtual evolution of enzyme engineering for biotherapeutics. Methods We developed a model named variant effect recognition network for CYP2C9 (vERnet-P), which learned features from AlphaFold2-predicted protein structures and predicted the enzyme activity of missense single-nucleotide variants in CYP2C9. The two crucial strategies of vERnet-P are the construction of amino acid interaction networks and the application of deep learning. Therefore, vERnet-P leveraged highly accurate protein structures predicted by AlphaFold2, combined with novel techniques for enriching and capturing useful information. Based on the accurate and preemptive prediction of CYP2C9 variants, we performed the saturation mutation prediction on several sites of CYP2C9 to identify new variants with the expected function. Furthermore, the drug metabolic activities of these newly identified variants were investigated with two probe drugs by in vitro metabolic activity assessments. Results An accuracy of up to 93.5% for the classification of activity levels was yielded in the testing dataset. In addition, the tasks for recognizing the high-activity and low-activity variants both achieved 93.5% accuracies, demonstrating that vERnet-P truly learned the features related to enzyme activity, rather than the selection bias to one kind of samples. Strikingly, the AUC values of the ROC and the PR reached 0.971 and 0.966 respectively in the testing cohort for vERnet-P. vERnet-P achieved the strongest agreement with massively parallel assessments relative to the other 8 computational variant effect predictors in the task of predicting CYP2C9 variant enzyme activity. We explored 6 mutant sites in CYP2C9 by using vERnet-P to perform saturation mutation prediction and found 12 novel variants with a high possibility of activity changing. We identified 6 variants with most likely increased activity by using the prediction of wild-type as a cutoff, consequently, 6 variants with most likely decreased activity were selected. According to these 12 discovered variants, 10 were strongly confirmed by the in vitro metabolic activity assays of both probe drugs. Conclusions vERnet-P achieved state-of-the-art performance in predicting the CYP2C9 variant enzyme activity. The preemptive prediction for CYP2C9 variants can efficiently provide the clinical drug dosing guideline. Furthermore, by saturating mutation prediction on several sites, our results indicated the evolutionary direction of CYP2C9 enzyme activity and discovered some brand-new CYP2C9 variants with ultra-strong functional alterations, which have not previously been reported. The results of in vitro metabolic activity assessment were highly consistent with our in silico prediction, indicating the potential of AI models for inferring evolutionary direction and designing novel variants with the expected function.

B-266 Therapeutic Drug Monitoring of Natalizumab and Anti-Natalizumab Antibodies

Abstract Background Natalizumab (NTZ) is a monoclonal antibody directed against the α4-integrin subunit of α4β1 and α4β7 integrins. NTZ is used to treat relapsing and remitting forms of multiple sclerosis. Although a majority of patients on standard dosing (intravenous 300 mg, every 4 weeks) reach therapeutic drug concentrations, about 10% of patients will eventually fail NTZ therapy due to the development of anti-NTZ antibodies. NTZ and anti-NTZ antibody concentrations in serum are used together to provide patient-specific pharmacokinetic and immunogenic assessment to aid in patient management. Methods Serum measurements of NTZ and anti-NTZ antibody levels are performed by lab developed electro-chemiluminescent immunoassays where assay design and principle are equivalent to biologic therapeutic drug monitoring described in our previous publication. The natalizumab drug assay is an immunoassay validated to measure free drug (pharmacodynamically active, ADA-unbound) in serum. The anti-natalizumab antibody assay utilizes a solution phase bridging method designed to measure total binding antibodies (including IgM & IgG subtypes). All anti-natalizumab antibody positive samples are confirmed for drug-specificity by an additional signal suppression step. The anti-natalizumab antibody assay is quantitative and drug-tolerant, meaning that circulating drug in patient serum does not interfere with anti-NTZ detection and quantitation. Results Here, we report concomitant measurement of NTZ and anti-NTZ antibody in 160 patient serum samples. Positive anti-NTZ levels from 24 to 235 ng/mL were found in 7.5% (12/160) patients. Anti-NTZ-free samples (148/160) had NTZ drug levels ranging from undetectable (<1.0) to 85 ug/mL. The mean and median drug level in 90 samples (results >1.0) was 10.8 and 2.7, respectively. 61/90 samples had therapeutic drug concentrations (at least 2.0 ug/mL). In the presence of anti-NTZ, 67% (8/12) had undetectable drug level and (4/12) had mean and median drug levels of 1.2 and 1.3, respectively, which is 9- or 2-fold lower than the mean and median drug levels of the anti-NTZ Ab -free group. Conclusions Low serum NTZ and high anti-NTZ are associated with loss of clinical efficacy, while elevated NTZ may increase progressive multifocal leukoencephalopathy risk. Assays to measure biologic drugs and their anti-drug antibodies in patient serum may be useful tools in dose optimization and patient management.

Development of bioassay platforms for biopharmaceuticals using Jurkat-CAR cells by AICD

The assessment of bioactivity for therapeutic antibody release assay poses challenges, particularly when targeting immune checkpoints. An in vitro bioassay platform was developed using the chimeric antigen receptor on Jurkat cells (Jurkat-CAR) to analyze antibodies targeting immune checkpoints, such as CD47/SIRPα, VEGF/VEGFR1, PD-1/PD-L1, and CD70/CD27. For CD47/SIRPα, the platform involved a Jurkat-CAR cell line expressing the chimeric SIRPα receptor (CarSIRPα). CarSIRPα was created by sequentially fusing the SIRPα extracellular region with the CD8α hinge region, the transmembrane (TM) and intracellular (IC) domains of CD28, and the intracellular signaling domain of CD3ζ. The resulting Jurkat-CarSIRPα cells can undergo “activation-induced cell death (AICD)” upon incubation with purified or cellular CD47, as evidenced by the upregulation of CD69, IL-2, and IFN-γ. Similar results also appeared in Jurkat CarVEGFR1, Jurkat CarPD1 and Jurkat CARCD27 cells. These cells are perfectly utilized for the bioactivity analysis of therapeutic antibody. Our study indicates that the established in vitro assay platform based on Jurkat-CAR has been confirmed repeatedly and has shown robust reproducibility; thus, this platform can be used for screening or for release assays of given antibody drugs targeting immune checkpoints.

Central Composite Design Expert-Supported RP-HPLC Optimization and Quantitative Evaluation of Efonidipine Hydrochloride Ethanolate & Chlorthalidone in Tablet.

Central composite design based RP-HPLC method optimization for the synchronized analysis of Efonidipine Hydrochloride Ethanolate (EFE) and Chlorthalidone (CHL) in tablet. The effective separation was performed using Inertsil ODS C18 column (250 × 4.6mm, 5μm), PDA detector with 0.05M KH2PO4 Buffer (pH4.5): Acetonitrile (40:60%v/v) mobile phase. Independent variables were investigated include the concentration of KH2PO4 (X1) and flow rate of mobile phase (X2). Based on responses obtained (retention time, resolution and tailing factor), the optimum condition selected was X1 = 40% and X2 = 1ml/min. Optimized HPLC condition was validated by assessing validation parameters and it meets the acceptance criteria set by ICH. The linear calibration curve was found to be in the quantity range 6.25-18.75 and 20-60μg/ml Assay of drugs was 100.94 and 100.06% for CHL and EFE. The validated RP-HPLC-PDA method can be used for routine analysis of EFE and CHL in tablet.

Changes in Urine Drug Screen Sensitivity in Adolescent Opioid Presentations to the Emergency Department.

Adolescent overdoses have been rising over the past decade. Emergency department (ED) visits for both acute overdoses and for adolescents in opioid withdrawal have risen post-COVID. Urine drug screens have poor utility in the ED but are routinely obtained for medical clearance and in the management of patients with substance use disorder. Our primary goal was to measure the sensitivity of the opiate urine drug assay over time in opioid-related presentations to the ED. We reviewed ED presentations at all EDs within our health system that were directly related to opioids from 1/1/2014 to 12/31/2022. For each patient included over the time frame, we identified whether a urine drug screen was obtained and the results from this screen. The urine drug screen available at all sites was an enzyme-multiplied immunoassay with an opiate screen (morphine antibody), but no fentanyl screen. The percent positivity for each drug category on enzyme multiplied immunoassay technique testing was calculated. Chi-squared tests were used to compare positivity rates between years. Opiate positivity declined over the last 9 years. Positivity rates from 2020 to 2022 were 5% ± 2% vs 82% ± 6% from 2014 to 2019 ( P < 0.001) Performance of UDS also declined over time (76% from 2014 to 2019 vs 46% from 2020 to 2022; P < 0.001). UDS was more likely to be performed in patients after a suicide attempt or when presenting after illicit use (66% vs 38%; P = 0.004). Opiate screen positivity decreased the last 9 years and may reflect wider use of fentanyl among this population starting in 2020.

Epidermal Growth Factor Receptor Targeting in Colorectal Carcinoma: Antibodies and Patient-Derived Organoids as a Smart Model to Study Therapy Resistance.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Therefore, the need for new therapeutic strategies is still a challenge. Surgery and chemotherapy represent the first-line interventions; nevertheless, the prognosis for metastatic CRC (mCRC) patients remains unacceptable. An important step towards targeted therapy came from the inhibition of the epidermal growth factor receptor (EGFR) pathway, by the anti-EGFR antibody, Cetuximab, or by specific tyrosine kinase inhibitors (TKI). Cetuximab, a mouse-human chimeric monoclonal antibody (mAb), binds to the extracellular domain of EGFR thus impairing EGFR-mediated signaling and reducing cell proliferation. TKI can affect the EGFR biochemical pathway at different steps along the signaling cascade. Apart from Cetuximab, other anti-EGFR mAbs have been developed, such as Panitumumab. Both antibodies have been approved for the treatment of KRAS-NRAS wild type mCRC, alone or in combination with chemotherapy. These antibodies display strong differences in activating the host immune system against CRC, due to their different immunoglobulin isotypes. Although anti-EGFR antibodies are efficient, drug resistance occurs with high frequency. Resistant tumor cell populations can either already be present before therapy or develop later by biochemical adaptations or new genomic mutations in the EGFR pathway. Numerous efforts have been made to improve the efficacy of the anti-EGFR mAbs or to find new agents that are able to block downstream EGFR signaling cascade molecules. Indeed, we examined the importance of analyzing the anti-EGFR antibody-drug conjugates (ADC) developed to overcome resistance and/or stimulate the tumor host's immunity against CRC growth. Also, patient-derived CRC organoid cultures represent a useful and feasible in vitro model to study tumor behavior and therapy response. Organoids can reflect tumor genetic heterogeneity found in the tissue of origin, representing a unique tool for personalized medicine. Thus, CRC-derived organoid cultures are a smart model for studying the tumor microenvironment and for the preclinical assay of anti-EGFR drugs.

Determination of 3-acetyl-11-keto-β-boswellic acid in analytical and biological samples using streamlined and robust RP-HPLC method.

AKBA (3-acetyl-11-keto-β-boswellic acid) is a phytoconstituent derived from Boswellia serrata extract and utilized in the management of rheumatoid arthritis. Drug delivery approaches showed interest in delivering AKBA with advanced nanotechnology. There is a need for a simple, sensitive, and robust HPLC method that can determine AKBA in complex nanoformulation and in vitro and ex vivo samples. In the proposed work, the RP-HPLC method was developed using a mobile phase comprising a mixture of acetonitrile : milli Q (90 : 10) at detection λmax 250 nm. The method exhibited a linearity of 250 to 20 000 ng mL-1 with a high correlation coefficient of 1. The limit of detection and limit of quantification for the analytes were found to be 41.32 ng mL-1 and 125.21 ng mL-1, respectively. In the accuracy study, the % recovery of AKBA was found to be 98% to 102%, and the precision study showed less than 2% relative standard deviation. The developed method was found to be robust under chromatographic conditions with changes in pH and mobile phase mixture ratio. The method was also explored for forced degradation study, and the results showed the successful separation of degradation products from the AKBA. Further, the RP-HPLC method was applied for the quantification of AKBA in topical nanoformulations and different matrices, such as skin matrices and adhesive tapes. The method was able to measure entrapment efficiency (93.13 ± 1.94%), drug loading (25.83 ± 0.54%), drug assay in a gel matrix (96.99 ± 3.89%), drug amount in stratum corneum (7.90 ± 0.62 μg cm-2), and drug amount in viable skin layers (33.94 ± 0.21 μg cm-2) with high-speed reproducibility. The developed method can be utilized for the routine analysis of AKBA in conventional and complex formulations in academia and industry.

HGG-53. MULTI-DIMENSIONAL INTEGRATIVE PROFILING IDENTIFIES BCL2L1 METHYLATION AS A PREDICTIVE BIOMARKER FOR MCL-1 ACTIVITY IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND Pediatric high-grade gliomas (pHGGs) pose a significant challenge as the most aggressive central nervous system tumors in children. The lack of effective treatment and poor survival rates emphasize the critical need for innovative therapies to improve the prognosis of pediatric patients with pHGG. METHODS We executed CRISPR-cas9 knockout (KO) screenings in 65 pediatric and 10 adult high-grade glioma (HGG) cell lines to explore unique functional dependencies associated with pHGGs. Drug assays were carried out to assess the targetability of a gene dependency of interest in pHGG cell lines. Subsequently, Random Forest machine learning algorithm was employed on ‘omics’ datasets to identify biomarkers indicative of drug response. Finally, biomarkers were confirmed through sequencing-based methods across various pediatric cancers. RESULTS CRISPR-cas9 KO screens revealed 8 crucial genes essential for pHGG growth, namely MCL-1, ATIC, DHFR, EED, HDAC2, PARP1, PDK1, PIK3CA and TYMS. Among these, Myeloid Cell Leukemia (MCL-1) was further characterized as it emerged as the top differential genetic dependency in pHGGs. Consistent with this, MCL1 inhibitors exhibited potent anti-cancer activity on 43% of pHGG cell lines. Multi-feature prediction analysis identified 140 features correlated to response. Strikingly, a previously undescribed methylation site within the BCL2L1 locus, cg00300298, ranked in the top 5% of features. Tissue analysis revealed a wide range of other pediatric brain cancers that harbor the BCL2L1 cg00300298 methylation mark as compared to non-malignant brain tissue. Lastly, we validated the utility of BCL2L1 methylation as a predictive biomarker of MCL1 inhibitor sensitivity across a large panel of pediatric brain cancer cell lines. CONCLUSION Using an integrated genomic approach, we identify MCL1 as a distinct therapeutic target in BCL2L1-methylated pediatric brain cancers. This offers a rational approach for stratifying patients who may benefit from MCL1 inhibitors.

3D cell cultures exhibit increased drug resistance, particularly to taxanes.

e12599 Background: Cell culture platforms could be categorized into two main types: 2D and 3D. 3D cell cultures, known for mimicking the physiological cellular environment, are commonly preferred by researchers to assess drug responses as they accurately reproduce aspects of the in vivo cell behavior. We hypothesized that dose-response curves of 2D and 3D platforms may be drug dependent, with some being more sensitive or resistant depending on the platform. Methods: MCF-7 cancer cells were cultured and plated using a 2D flat bottom well plate platform or using a matrix-based and a matrix-free 3D platform. Cells were treated with chemotherapies and targeted therapies for four days and cellular sensitivity was assessed through a viability assay. Four-parameter logistic (4PL) dose response curves were generated and IC50 values (µM/Cmax) were quantified to understand the differences in dose response curves between the 2D and 3D cellular platforms. Results: While the dose-response curves generally correlate between 2D vs. 3D matrix-based (r2=0.82, p&lt;0.001) and 2D vs. 3D matrix-free methods (r2=0.78, p&lt;0.001), noteworthy variations were observed for certain drugs, particularly paclitaxel (2D IC50= 66; 3D matrix-based IC50 = ≥100; 3D matrix-free IC50 = ≥100) and docetaxel (2D IC50 = ≥34; 3D matrix-based IC50 = ≥100; 3D matrix-free IC50 = ≥100). Additionally, a general increase in resistance was evident when cells formed 3D structures compared to the 2D monolayer condition, as indicated by regression analysis (Y-intercept = 0.55, CI95% 0.32 to 0.78; slope = 0.47, CI95% 0.20 to 0.75). Conclusions: Our findings reveal that taxanes such as paclitaxel and docetaxel exhibit distinctive resistance patterns in 3D structures. In addition, a substantial increase in cellular resistance is observed in 3D cellular models when compared to the 2D assay for most drugs. Despite the overall correlation, these drug-specific differences should be considered. Thus, the next generation of an extreme drug resistance assay, aimed at enhancing the quality of life for patients by avoiding ineffective therapies, might preferentially employ a 3D platform to tailor patients’ cancer therapies.

Drug susceptibility testing and line probe assay of first-line anti-tuberculosis drugs among presumptive tuberculosis patients attending a secondary care hospital in Bhubaneswar.

Pyrazinamide (PZA) is important for identification in multi-drug-resistant tuberculosis patients before starting therapy. PZA drug susceptibility testing (DST) is essential for the management of drug-resistant and susceptible TB patients. The degree of drug resistance among TB patients and discrepancy between DST results of the phenotype and genotype were assessed. Socio-demographic and clinical profiles of TB patients recruited in the study were documented. Sputum samples were processed for diagnosis using TrueNat Xpert MTB, TrueNat Xpert MTB Plus, and MGIT culture. Rifampicin (RIF) line probe assay (LPA) showed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100%, whereas isoniazid (INH) LPA testing showed a sensitivity of 85.7%, a specificity and PPV of 100%, and NPV of 94.8%. The gene mutation for RIF resistance was between the codon, 530-533 of rpoB gene, and that for INH resistance was at the codon, 315 of katG gene. Our findings demonstrated high prevalence of mono- and poly-drug resistance as well as pyrazinamide resistance.

A Comparative Quality Control Study of Brand and Generic Amlodipine Tablets by In-Vitro Testing

The objective of this study was to conduct in-vitro quality control testing of brand versus generics of Amlodipine tablets. One brand and two generic Amlodipine tablets were used in the study. In the terms of weight variation, Brand, Generic A and Generic B have an above the mean weight limit variation of 2.15%, 2.23%, &amp; 1.43% respectively. The lower mean weight limit variations are 1.85%, 1.92% &amp; 1.23% respectively which are within the 7.5% standard limit of IP. Friability test shows that Brand, Generic A &amp; Generic B have an average friability of 0.5%, 0.480% ,0.3% mass loss, which are within 1%. mass lost limit of IP. Hardness test shows that Brand, Generic A &amp; Generic B have an average hardness of 0.9 kg/cm2, 2.3 kg/cm2 and 1 kg/cm2 respectively. The disintegration test shows that Brand, Generic A &amp; Generic B fall within 15-minutes time interval segment with disintegration time determined 20 sec, 3 min, 2.5 min respectively. In terms of drug assay brand, Generic A &amp; Generic B fall under IP limit of 90% to 110%. The Brand, Generic A &amp; Generic B have a drug dissolution percentage of 101%, 104%, 104% respectively within 30 minutes sampling time interval. The QC tests results for amlodipine tablets show that Brand, Generic A and Generic B confirm to the IP standards.

Chromatographic methods and approaches for bioequivalence study, drug screening and enantioseparation of indapamide

AbstractIndapamide (Indp) and certain other diuretics have been abused in sports, therefore, having sensitive methods for its detection and assay in biological fluids (whole blood, plasma, serum, and urine) is of significant importance. The racemic mixture of Indp is being used as an active pharmaceutical ingredient among other commonly prescribed diuretics. The regulatory authorities and pharmaceutical industries demand analytical methods for successful enantioseparation of such molecules. The paper presents a critical overview of the scientific issues of the application of contemporary techniques involving various chromatographic approaches (with liquid or supercritical fluid as mobile phases) and capillary electrophoresis and method development, for drug screening, assay, bioequivalence studies and enantioseparation of indapamide with their results. It also covers the historical developments that led to significant breakthroughs in research and concise evaluations of research in the area.Different types of chromatographic methods (HPLC, CEC, SFC etc) discussed herein provide an insight and a choice to select a method to (i) screen Indp for drug abuse, (ii) separate, isolate and quantify the enantiomers of Indp and (iii) investigate their pharmacokinetics as markedly different species and not as a total drug. The article evaluates the field's status with a broad base and practical oriented approach so that the underlying principles are easily understood to help chemists and non-specialists gain useful insights into the field outside their specialization and provide experts with summaries of key developments. To the best of authors' knowledge there has been no attempt to review such methods for analysis of Indp and this is the first report of its kind.

Antimicrobial therapeutic drug monitoring in critically ill adult patients – An international perspective on access, utilisation, and barriers

BackgroundTherapeutic drug monitoring (TDM) is an effective method for individualising antimicrobial therapy in critically ill patients. The 2021 ADMIN-intensive care unit survey studied a wide range of intensive care unit clinicians worldwide to gain their perspectives on antimicrobial TDM. This article reports the responses from this survey relating to TDM access, utilisation, and barriers. MethodsAn online survey consisted of multiple-choice questions and 5-point Likert scales. The survey examined respondent's access to minimum inhibitory concentration (MIC) results, drug assays, and dosing software, as well as barriers to TDM. ResultsThe survey included 538 clinicians from 409 hospitals in 45 countries, with 71% physicians and 29% pharmacists. Despite most respondents having access to assays, 21% and 26% of respondents lacked access to vancomycin and aminoglycosides, respectively. In lower-income countries, almost 40% reported no access. Delayed drug assay turnaround time was the most significant barrier to TDM, particularly in lower-income countries. Routine access to MIC results was unavailable for 41% of respondents, with 25% of lower-income country respondents having no access to MIC or susceptibility reports. ConclusionsThis global survey indicated that consistent TDM usage is hindered by assay access in some sites and the timeliness of assay results in others. Addressing barriers to TDM, particularly in low-income countries, should be a priority to ensure equitable access to affordable TDM.

RSM-CCD COUPLED RP-HPLC OPTIMIZATION AND QUANTITATIVE EVALUATION OF ANTIHYPERTENSIVE DRUGS IN TABLET

Central composite design (CCD) based RP-HPLC method optimization for the synchronized analysis of Efonidipine Hydrochloride Ethanolate (EFE) and Chlorthalidone (CHL) in Tablet. The effective separation was performed using Inertsil ODS C18 column (250 x 4.6 mm,5μm), PDA detector with 0.05M KH2PO4 Buffer (pH 4.5): Acetonitrile (40:60%v/v) mobile phase. Independent variables were investigated include the concentration of KH2PO4 (X1) and flow rate of mobile phase (X2). Based on responses obtained (retention time, resolution and tailing factor), the optimum condition selected was X1 = 40% and X2 = 1 ml.ml-1. Optimized HPLC condition was validated by assessing validation parameters and it meet the acceptance criteria set by ICH. It was showed linear calibration curve in the quantity range 6.25-18.75 μg.ml-1 and 20-60 μg.ml-1. % for CHL &amp; EFE. Assay of drugs was 100.94% and 100.06% for CHL &amp; EFE. The validated RP-HPLC-PDA method can be used for routine analysis of EFE and CHL in tablet

CHEMICAL ADHERENCE IN RESISTANT HYPERTENSION: PILOT STUDY IN AN ESH EXCELLENCE CENTER

Objective: Arterial hypertension (HTN) is responsible for the largest proportion of preventable mortality in the world. The primary cause of resistant hypertension (rHTN) is poor medication adherence. Blood or urine drug assays enable us to assess chemical adherence more accurately than self-reported questionnaires. We assayed anti-hypertensive drugs in patients with rHTN at our Center of Excellence in Hypertension, in collaboration with the Pharmacology and Toxicology Laboratory. Design and method: Patients with rHTN (office SBP/DBP &gt;140/90 mmHg or out of office daytime &gt;135/85 mmHg despite being prescribed 3 drugs at optimal dosage, including a diuretic, a renin angiotensin system inhibitor (RASI) and a calcium channel blocker (CCB), were informed of the study and agreed to have blood drugs concentrations measured. We describe the results for chemical adherence in the first 54 patients included between January 1, 2021 and September 1, 2023 in our center. Results: Of the 54 patients included, 38 (70%) were men, with a mean age of 65 +/- 11 years. The mean number of antihypertensive treatments prescribed was 5 +/- 1 (min 3, max 8). The proportions of treatments were RASI 87%, CCB (mainly dihydropyridines) 81%, diuretics 81%, beta-blockers 76%, alpha-blockers 57%, mineralocorticoid receptor antagonists (MRA) 37%, and central antihypertensives 26%. The best compliance was observed with alpha-blockers 81%, followed by CCB 50%, RASI 45%, diuretics 37% and beta-blockers 34%. The least compliant were central antihypertensives 0% and MRA 5%. All prescribed drugs were completely absent in the blood in 26% of patients, and 63% of patients had incomplete adherence (1 or more but not all drugs present in the blood), of whom 1/3 were non-compliant to 3 or more medications. Only for 9% of patients chemical adherence was excellent. Conclusions: Chemical adherence when measured at clinical visit was poor in this cohort of patients followed in a center of excellence. Limitations of this study are lack of follow-up for the same patient to assess variation in adherence over time, and small sample size. These results suggest the need to develop therapeutic education to improve patient involvement in the management of HTN and compliance.

Artificial Vascular with Pressure-Responsive Property based on Deformable Microfluidic Channels.

In vitro blood vessel models are significant for disease modeling, drug assays, and therapeutic development. Microfluidic technologies allow to create physiologically relevant culture models reproducing the features of the in vivo vascular microenvironment. However, current microfluidic technologies are limited by impractical rectangular cross-sections and single or nonsynchronous compound mechanical stimuli. This study proposes a new strategy for creating round-shaped deformable soft microfluidic channels to serve as artificial in vitro vasculature for developing in vitro models with vascular physio-mechanical microenvironments. Endothelial cells seeded into vascular models are used to assess the effects of a remodeled in vivo mechanical environment. Furthermore, a 3D stenosis model is constructed to recapitulate the flow disturbances in atherosclerosis. Soft microchannels can also be integrated into traditional microfluidics to realize multifunctional composite systems. This technology provides new insights into applying microfluidic chips and a prospective approach for constructing in vitro blood vessel models.

Formulation development of nanostructured lipid carrier-based nanogels encapsulating tacrolimus for sustained therapy of psoriasis

The goal of this study was to formulate tacrolimus nanogel based on nanostructured lipid carrier (NLC) in order to improve the efficacy, aesthetic, and patient compliance for the treatment of psoriasis. The microemulsion method was used to create phase diagrams and NLCs were prepared using points obtained from the microemulsion region and characterized. The gelling agent carbopol was used to develop an NLC-based nanogel. The pH, drug assay, viscosity, spreadability, and in vitro release of the nanogel, were evaluated. Ex vivo cytotoxicity of the formulation was assessed in murine fibroblast cells. Oxazolone and imiquimod models of psoriasis were used to assess the effectiveness of the nanogel. The NLCs exhibited a submicron particle size of 320 ± 10 nm, a low polydispersity index (<0.3), and a zeta potential of −19.4 mV. Morphological analysis revealed spherical nanoparticles with an encapsulation efficiency of 60 ± 3 %. The nanogel maintained a pH of 6.0 ± 0.5 and possessed a remarkable drug content of 99.73 ± 1.4 %. It exhibited pseudoplastic flow behaviour, ensuring easy spreadability, and demonstrated sustained drug release exceeding 90 % over a 24-hr period. Ex vivo cytotoxicity assessments revealed that the nanogel was safe because no cell death was induced. Nanogel resolved psoriatic blisters, was non-irritating and improved skin elasticity. The favorable properties, safety profile, and remarkable efficacy show the potential of the nanogel as a patient-friendly and effective therapeutic option for psoriasis treatment.

Next Generation Chemiluminescent Probes for Antimalarial Drug Discovery.

Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark β-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (β-D-galactoside) by a transgenic β-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.

Abstract 431: Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer

Abstract Background &amp; Hypothesis: Treatments for triple-negative breast cancer (TNBC) are limited to chemotherapy, PARP inhibitors and immunotherapies. Tumor metabolism seeks to identify metabolic pathways preferentially utilized by cancer. Malic enzyme 2 (ME2) has a role in the malate-aspartate shuttle and pyruvate and NADPH production, contributing to the redox balance and metabolite synthesis. We demonstrated that ME2 knockdown inhibits growth of TNBC cells with upregulated serine biosynthesis pathway. We hypothesized that ME2 knock-down (kd) would be growth inhibitory in vivo. Methods: We used a non-tumorigenic control (MCF10a) and TNBC lines (BT20, MDA-MB-468, HCC1806, HCC70, Hs578T, SUM149). Nude mice received mammary fat pad xenografts of MDA-MB-468, to assess the impact of ME2-shRNA on growth. ME2 relevant metabolite profiling was achieved through the Promega Metabolite-Glo assay, measuring NAD, NADH, NADP, NADPH, Malate, and Glutamate. Cellular proliferation was quantified via the BioTek BioSpa 8 system and Promega CellTiter Glo. Comprehensive profiling of metabolic genes was achieved with the Nanostring nCounter across all cell lines. The potential therapeutic efficacy of NPD-389, MDSA, disodium embonate and doxorubicin was evaluated, alone or in combination, through targeted drug assays. Metabolic flux analyses, specifically mitochondrial and glycolytic function, were conducted using the Seahorse XF96, contrasting ME2-shRNA modified and control cells. Results: ME2 kd resulted in reduced proliferation in several TNBC cell lines to varying degrees, in contrast to control MCF10a, and to Hs578T. In response to the loss of ME2, several cell lines upregulated serine biosynthesis genes, including PSAT1. MDA-MB-468 ME2 kd cells developed significantly smaller tumors in a nude mouse xenograft model. Increased malate levels, but reduced NADH and NADPH, were detected in TNBC cells with inhibited growth post-ME2 kd, but not in MCF10a or Hs578T cells. Among the tested ME2 inhibitors, NPD-389 had superior efficacy vs. MDSA and disodium embonate. Metabolic flux analysis showed compromised mitochondrial and glycolytic processes in the TNBC cell lines exhibiting growth inhibition upon ME2 kd. Conclusions: ME2 knockdown selectively impairs cell proliferation and tumor growth in TNBC cell lines, validating its potential as a therapeutic target. The differential metabolic response, including altered malate and NADH/NADPH levels, confirms ME2's importance in TNBC metabolism. NPD-389 effectively reduced growth in ME2-sensitive lines, meriting further investigation. These findings support targeting ME2 in as a novel treatment strategy. Citation Format: Mark D. Slayton, Zackariah A. Farah, Jin Heon Jeon, Abhinav Achreja, Ben Krinkel, Brisilda Nilaj, Yi-Hsien Eu, Justin Sung, Alyssa Rosenfeld, Alisa Lui, Mason Collard, Liwei Bao, Xu Cheng, Celina Kleer, Kerry Loomes, Deepak Nagrath, Sofia D. Merajver. Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 431.