High-performance Liquid Chromatographic Assay Research Articles

Tumor derived exosomal ENTPD2 impair CD8+ T cell function in colon cancer through ATP-adenosine metabolism reprogramming

BackgroundExtracellular ATP–AMP–adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell–derived ATPases in the development and progression of colon cancer.MethodsBioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues.ResultsWe found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells.ConclusionOur study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP–adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.

Field assessment of active ingredient quantity in pharmaceutical tablets with limited calibration of near infrared spectra: An application to ciprofloxacin tablets

Portable near-infrared (NIR) spectrophotometers have emerged as valuable tools for identifying substandard and falsified pharmaceuticals (SFPs). Integration of these devices with chemometric and machine learning models enhances their ability to provide quantitative chemical insights. However, different NIR spectrophotometer models vary in resolution, sensitivity, and responses to environmental factors such as temperature and humidity, necessitating instrument-specific libraries that hinder the wider adoption of NIR technology. This study addresses these challenges and seeks to establish a robust approach to promote the use of NIR technology in post-market pharmaceutical analysis. We developed support vector machine and partial least squares regression models based on binary mixtures of lab-made ciprofloxacin and microcrystalline cellulose, then applied the models to ciprofloxacin dosage forms that were assayed with high performance liquid chromatography (HPLC). A receiver operating characteristic (ROC) analysis was performed to set spectrophotometer independent NIR metrics to evaluate ciprofloxacin dosage forms as “meets standard,” “needs HPLC assay,” or “fails standard.” Over 200 ciprofloxacin tablets representing 50 different brands were evaluated using spectra acquired from three types of NIR spectrophotometer with 85% of the prediction agreeing with HPLC testing. This study shows that non-brand-specific predictive models can be applied across multiple spectrophotometers for rapid screening of the conformity of pharmaceutical active ingredients to regulatory standard.

Response Surface Methodology for Optimization of Ultrasound-Assisted Antioxidants Extraction from Blackberry, Chokeberry and Raspberry Pomaces.

An investigation of the ultrasound-assisted extraction (UAE) of polyphenol-rich aqueous extracts from blackberry, chokeberry and raspberry pomaces was carried out. The aim of the study was to choose optimal conditions for UAE in order to obtain extracts rich in phenolic compounds. The optimization was carried out based on response surface methodology. The variable conditions were amplitude of ultrasound wave and extraction time, whereas responses were total polyphenol content and antioxidant capacity. Based on the ANOVA analysis, mathematical models were fitted and verified. The most effective conditions of amplitude and time were 98% and 5.00 min, 78% and 10.32 min and 90% and 11.56 min for blackberry pomace, chokeberry pomace and raspberry pomace, respectively. The actual results obtained in optimized conditions were comparable to the results predicted by the models. Additionally, the anthocyanin content in extracts was determined in the high-performance liquid chromatography assay. It was proven that response surface methodology could be a useful tool in the optimization of UAE processes for obtaining polyphenol-rich extracts from berry fruit pomaces.

Tetracyclic homoisoflavanoid (+)-brazilin: a natural product inhibits c-di-AMP-producing enzyme and Streptococcus mutans biofilms.

The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans. Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan, was identified as one of the 11 "hits," with the greatest reduction (>99%) in fluorescence at 100 µM. The smDAC inhibitory profiles of the 11 "hits" established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits smDAC's enzymatic activity (Ki = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin's binding profile with smDAC (Kd = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 105 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to smDAC but can also inhibit the function of smDAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.IMPORTANCEThis study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans. The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans' diadenylate cyclase (smDAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of smDAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms.

Composition analysis of Magnolia flower and their use for highly bright carbon dots

Colloidal carbon dots (C-dots) have excellent optical properties and they have been widely used for various types of applications. Compared to the C-dots prepared using chemicals, the Biomass-derived C-dots have attracted a great of attention because of their environmental friendliness and biocompatibility. However, currently obtained C-dots using biomass have low quantum yield. In this work, we demonstrated an efficient precursor of Magnolia flower for synthesizing high-quantum yield C-dots via hydrothermal approach. High performance liquid chromatography and Bradford assay were used to analyze the compositions of the Magnolia flower, which determine the quality of the C-dots. The typical emission bands of the as-prepared C-dots cover from 400 to 550 nm at different excitation wavelengths. The C-dots synthesized using violet Magnolia flower have the QY as high as 11.9%, which is higher than that of 10.4% (white Magnolia flower), because the violet color gives the higher content of the N. The as-obtained C-dots have excitation-dependent color and high quantum yield, showing a great potential for the use of the anti-counterfeiting system.

The Physicochemical Compatibility of Sildenafil Injection with Parenteral Medications Used in Neonatal Intensive Care Settings.

Sildenafil is used to treat pulmonary hypertension in neonatal intensive care unit (NICU) settings. As multiple intravenous (IV) medications are co-administered in NICU settings, we sought to investigate the physicochemical compatibility of sildenafil with a range of IV drugs. Sildenafil 600 mcg/mL or 60 mcg/mL was mixed 1:1 with the secondary drug solution to simulate Y-site co-administration procedures. Physical compatibility was evaluated by visual observation against a black and white background and under polarized light for two hours for changes in colour, precipitation, haze and evolution of gas. Chemical compatibility was determined from sildenafil concentrations, using a validated, stability-indicating high-performance liquid chromatography assay. Sildenafil 600 mcg/mL was physicochemically compatible with 29 of the 45 drugs tested at 'high-end' clinical concentrations and physically incompatible with 16 drugs and six '2-in-1' parenteral nutrition solutions. Sildenafil 600 mcg/mL was compatible with lower, clinically relevant concentrations of calcium gluconate, heparin and hydrocortisone. Aciclovir, amoxicillin, ampicillin, ibuprofen lysine, indometacin, phenobarbitone and rifampicin were incompatible with sildenafil 600 mcg/mL, however these IV medications were compatible with sildenafil 60 mcg/mL. Sildenafil 600 mcg/mL and 60 mcg/mL were incompatible with amphotericin, flucloxacillin, furosemide, ibuprofen, meropenem and sodium bicarbonate. Sildenafil compatibility with commonly used syringe filters was also investigated. Sildenafil solution was compatible with nylon syringe filters, however, absorption/adsorption loss occurred with polyethersulfone and cellulose ester filters.

Novel Bioresorbable Drug-Eluting Mesh Scaffold for Therapy of Muscle Injury.

A novel bioresorbable drug-eluting polycaprolactone (PCL) mesh scaffold was developed, utilizing a solvent-cast additive manufacturing technique, to promote therapy of muscle injury. The degradation rate and mechanical properties strength of the PCL mesh were characterized after immersion in a buffer solution for different times. The in vitro release characteristics of vancomycin, ceftazidime, and lidocaine from the prepared mesh were evaluated using a high-performance liquid chromatography (HPLC) assay. In addition, the in vivo efficacy of PCL meshes for the repair of muscle injury was investigated on a rat model with histological examinations. It was found that the additively manufactured PCL meshes degraded by 13% after submission in buffered solution for four months. All PCL meshes with different pore sizes exhibited greater strength than rat muscle and survived through 10,000 cyclic loadings. Furthermore, the meshes could offer a sustained release of antibiotics and analgesics for more than 3 days in vitro. The results of this study suggest that drug-loaded PCL mesh exhibits superior ability to pure PCL mesh in terms of effectively promoting muscle repair in rat models. The histological assay also showed adequate biocompatibility of the resorbable meshes. The additively manufactured biodegradable drug-eluting meshes may be adopted in the future in humans for the therapy of muscle injuries.

Resorbable nanofibrous membranes for local and sustained co-delivery of acyclovir and ketorolac in herpes therapy

Herpes simplex and herpes zoster are both viral infections caused by members of the herpesvirus family. The former is characterized by painful, fluid-filled blisters or sores on the skin and mucous membranes, while the latter presents as a painful rash with blisters, typically occurring in a single band or patch along one side of the body. The treatment remains a challenge since current antiviral therapy via oral administration may lead to unfavorable side effects such as headaches, nausea, and diarrhea. This study used electrospinning to develop biodegradable nanofibrous poly(lactic-co-glycolic acid) (PLGA) membranes for delivery of both acyclovir and ketorolac. The structure of the spun nanofibers was assessed via scanning electron microscopy (SEM), and the appearance of loaded acyclovir and ketorolac in the nanofibers was confirmed with Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Release profiles of these drugs from the nanofibrous membranes were assessed using in vitro elution studies, high-performance liquid chromatography (HPLC) assays, and in vivo drug release patterns. The electrospun nanofibers had a size range of 283–725 nm in diameter, resembling the extracellular matrix of natural tissue and demonstrated excellent flexibility and extensibility. Notably, the drug-eluting nanofibers exhibited an extended release of high levels of acyclovir and ketorolac over a 21-day period. Thus, biodegradable drug-eluting membranes with a prolonged drug release could be a potential therapeutic approach for treating herpes infections.

Chenopodium Ambrosioides Linn Mitigates Bone Loss in Rats with Periodontitis.

Periodontitis is an inflammatory disease that causes bone loss. Some patients do not respond well to the classic treatment and need therapies that minimize bone loss, the main sequel of the disease. Chenopodium ambrosioides L. has stood out due to its anti-inflammatory and anti-oxidative activities. However, no study has yet investigated its effect on periodontitis. This study aimed to evaluate the bone protective effect of Chenopodium ambrosioides L. (CAL) extract on ligature-induced periodontitis model in rats. For this, a pre-clinical assay was performed, using male Wistar rats divided into 3 groups: Naive (N) (n=6), not submitted to any procedure; Saline (SAL) (n=6), submitted to ligature-induced periodontitis and receiving 2 ml/kg of 0.9% saline solution; and CAL extract, which was subdivided into 3 subgroups (n=6/subgroup) receiving the CAL at 3 (CAL3), 10 (CAL10) or 30 mg/kg (CAL30). All agents were given, by oral gavage, 30 min before periodontitis induction and daily until euthanasia (11th day). By then, maxillae were removed for macroscopic, histological, and histometric analyses. Kidneys, liver, and stomach were collected to evaluate the safety of CAL extract. High-performance liquid chromatography (HPLC) assay was used to investigate the flavonoid content in the extract. Chenopodium ambrosioides L. extract at 30mg/kg showed a reduction by 58% in bone loss marked by an increase (+35%) in the number of osteoblasts and a reduction (-51%) on the number of osteoclasts (p< 0.05). No significant alteration in the liver, kidney, or stomach was seen. Rutin was the main flavonoid found. In summary, it was observed that Chenopodium ambrosioides L. extract has shown important anti-inflammatory and bone anabolic and anti-resorptive properties without causing toxicity in the main organs. Rutin, as the main flavonoid of the extract, seems to be responsible for the beneficial effect of this agent.

Development of a Competitive Chemiluminescent Assay for Quantitative Determination of TP53 Fusion Protein Using Reagent Strips.

Recent studies have shown that almost half of all cancers occur due to DNA damage. For the early diagnosis of cancer, a highly sensitized and swift identification for TP53 is needed since the corresponding TP53 protein is effectively recognized as "the guardian of the genome." To improve the detection sensitivity, numerous analytical methods were previously used for the determination of the TP53 protein, including denaturing high-performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). Currently, immunochromatographic tests (ICTs) that are simple to use, stable over time, and show low interference are regarded as valuable tools for the quick screening of food and environmental monitoring along with clinical diagnosis. ICTs often have limited sensitivity even if a variety of novel reporters possessing optimum photostability and improved brightness are used as signal-intensity reporters. Compared with N-(4-aminobutyl)-N-(ethylisoluminol) or luminol, a novel luminescent probe, 2',6'-diMethyl-4'-(N-succiniMidyloxycarbonyl) phenyl-10-sulfopropylacridiniuM-9-carboxylate (NSP-DMAE-NHS) has achieved a much higher efficiency, improvement in the biosensor's performance, and amplification of the signal without causing any damage to the biomolecule in terms of its biochemical activity. In this study, the reagent strip method was initially used to detect TP53 fusion protein by combining the advantages of NSP-DMAE-NHS and immunochromatography. In our experiment, the control and study lines on the strips were immobilized through HRP-conjugated goat anti-rabbit IgG and TP53 antigen, respectively. The optimized concentration of the anti-TP53 antibody-NSP-DMAE-NHS immunoconjugates was then added to the TP53 antigen samples. After, the test strips were inserted and left in the aforementioned buffer solution for an additional 20 min. Finally, a lab-made luminous measurement device was used to analyze the corresponding control and study lines on the strips. Under optimized conditions, this method was found to be ultrasensitive, with a wide range of linear responses from 0.0008 ng mL-1 to 1 µg mL-1 and a limit of detection of 0.0008 ng mL-1 (0.013 pM). Thus, a novel competitive chemiluminescent assay based on reagent strips was established for the determination of the TP53 fusion proteins. The strategy has potential applications for ultrasensitive detection in the early diagnosis of cancer.

Implementation of Quality by Design Approaches for Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Assay Method for Determination of Glycyrrhizin in Nanoformulation.

Glycyrrhizin (GL) is the principal constituent of Glycyrrhiza glabra, having antiallergic, anticancer, anti-inflammatory, and antimicrobial action. The reverse-phase high-performance liquid chromatography (RP-HPLC) analytical method was used to quantitatively estimate GL in a nanoformulation and validated as per International Conference on Harmonization Q2 (R1) standards. A stationary phase of the C18-HL reversed-phase column and a mobile phase of acetonitrile and water were used for effective elution. The chromatographic conditions of RP-HPLC were optimized utilizing a quality-by-design approach to accomplish the required chromatographic separation of GL from its nanoformulation with minimal experimental runs. Optimized RP-HPLC conditions for the assay method consist of acetonitrile (41%) and water, pH 1.8, balanced with phosphoric acid (0.1%) as a mobile phase with a flow rate of 1 mL/min. The retention time was found at 7.25 min, and method validation confirmed its sensitivity, preciseness, accuracy, and robustness.

Novel CO2-encapsulated Pluronic F127 hydrogel for the treatment of Achilles tendon injury

Nonsurgical treatment and surgical repairment of injured Achilles tendons seldom restore the wounded tendon to its original elasticity and stiffness. Therefore, we hypothesized that the surgically repaired Achilles tendon can achieve satisfactory regeneration by applying multi-drug encapsulated hydrogels. In this study, a novel bupivacaine-eluting carbon dioxide-encapsulated Pluronic F127 hydrogel (BC-hydrogel) was developed for the treatment of Achilles tendon injuries. The rheological properties of BC-hydrogel were measured. A high-performance liquid chromatography assay was used to assess the release characteristics of bupivacaine in both in vitro and in vivo settings. Furthermore, the effectiveness of BC-hydrogel in treating torn tendons was examined in a rat model, and histological analyses were conducted. Evidently, the degradable hydrogels continuously eluted bupivacaine for more than 14 days. The animal study results revealed that the BC-hydrogel improved the post-surgery mobility of the animals compared with pristine hydrogels. Histological assay results demonstrated a significant reaction to high vascular endothelial growth factor in the surrounding tissues and expression of collagen I within the repaired tendon. This demonstrates the potential of this novel BC-hydrogel as an effective treatment method for Achilles tendon injuries.

Optimization, Formulation, and Stability of Topical Rapamycin Used for Rare Tuberous Sclerosis Disease: from Ointment to Liposomes

IntroductionTopical rapamycin has been established as an effective and safe therapy for facial angiofibromas in tuberous sclerosis. Different formulations have been tested for this skin disease, most using an ointment as a vehicle.PurposeTo improve the classical formulation of topical rapamycin and to determine the validity period of the proposed options based on chemical, physical, and microbiological stability studies.MethodsFour different 0.4% rapamycin formulations were prepared (ointment, emulsion, gel, and liposomes). The stability studies for each formulation over 56 days were as follows: (1) chemical: extraction with different solvents and high-performance liquid chromatography assay; (2) physical: pH, uniformity, extensibility, absence of crystals, absence of phase separation, and only for liposomal formulation, particle size, zeta potential, and encapsulation efficiency were determined; and (3) microbiological: culture samples in blood-agar media.ResultsOnly liposomes were chemically, physically, and microbiologically stable after 8 weeks. Ointment, emulsion, and gel formulations lost their chemical or physical stability before 56 days.ConclusionsThe authors describe four new formulations to improve the previous treatment for facial angiofibromas in tuberous sclerosis. The liposome-based formulation was the most appropriate according to chemical, physical, and microbiological stability studies. However, it would be necessary to carry out clinical studies to ensure the effectiveness and safety of this formulation and also guarantee an improvement in the quality of life of patients.

Isolation, Purification and Characterization of a-Lactalbumin from Camel Milk and Study its Antioxidant Activity in Oils

The purpose of this research was to examine the anti-inflammatory and antioxidant properties of a-lactalbumin (α-Lac) isolated from camel milk. Ion-exchange chromatography on a DEAE cellulose column and gel-filtration chromatography on a Sephadex G-100 column were used for the purification process. (α-Lac) purity was confirmed using a single-bond polyacrylamide gel-electrophoresis (SDS-PAGE) assay and a further step using a high-performance liquid chromatography (HPLC) assay. Antioxidant activity of (α-Lac) in food systems compared with chemical Antioxidants (BHT) activity at Prevent oxidation of vegetable oils (Flaxseed oil and sunflower) showed significant Ferric-reducing antioxidant power (FRAP), linoleic acid system (LAS), radical scavenging activity (DPPH).

Pharmacokinetics (PK) of Deferasirox in Transfusion Dependent Thalassemia

Iron overload is a major cause of organ dysfunction and death in transfusion-dependent thalassemia (TDT) and advances in iron chelation are urgently needed. The iron chelator, deferasirox, is widely used in TDT, however, many patients accumulate iron despite taking maximum recommended doses and many experience dose-limiting toxicity, including renal and liver failure. There is high inter-individual variability in the plasma concentrations of patients taking comparable deferasirox doses and low exposure correlates with inadequate chelation response. The cause of variable drug levels remains unknown, in part because prior deferasirox PK studies primarily include limited sample sizes and/or homogenous patients. There is an urgent unmet need to determine (1) what patient characteristics contribute to the observed variability in deferasirox-plasma concentrations, and (2) how dosing strategies can be tailored to achieve optimal therapeutic plasma-drug concentrations in all patients. We initiated a 2 year, single center observational pharmacokinetic (PK) pilot study to describe the PK of deferasirox in a diverse group of TDT patients from 2 years old through adulthood with a goal accrual of 50 patients. Our aim is to develop a population PK (popPK) model of deferasirox that will be evaluated in a future prospective study to individualize dosing and optimize exposure. The eligibility criteria are broad to capture a diverse population. Patients are eligible if they: 1) have a diagnosis of TDT, 2) are taking deferasirox, and 3) are able to provide informed consent (or have a guardian provide informed consent). For quantification of plasma deferasirox concentrations, 0.5 ml of whole blood are collected prior to transfusions at 4 regularly scheduled transfusion visits. Samples are then analyzed by a validated high performance liquid chromatography assay. Baseline labs, liver iron concentration and information on concomitant chelators are collected at the start of the study and labs, fed/fasted state, time and dose of last deferasirox, deferasirox regimen, and concomitant chelators are collected at each study visit. The planned analysis is to use nonlinear mixed effect modeling to calculate drug exposure and develop the popPK model. We have enrolled 39 patients with TDT, including 27 patients with beta thalassemia major, 10 patients with hemoglobin E beta thalassemia, 12 patients with hemoglobin H constant spring and one patient with beta thalassemia intermedia. Median age is 28 years old, with a range from 6 - 63 years old. Pre-study iron status also varied considerably, with median liver iron concentration 4.5 mg/g (range 1.5 -30 mg/g) and median ferritin 1500 ug/L (range 460 - 5600 ug/L). Thirty one patients have completed the study and 141 samples have been collected. Initial analysis of the first 15 patients who completed the study show a wide range of steady state plasma-deferasirox concentrations, consistent with prior reports. The means for the 4 samples collected per patient were calculated and found to have a median of 36.3 ug/ml with a range of 13.4 - 56.0 ug/ml. For the individual sample collections, the range of plasma deferasirox concentrations was even greater (0.18 - 99.9 ug/ml). These results need to be analyzed further to account for the dosing regimen, iron burden and other individual patient characteristics. In this study, we also collected genomic DNA from the buffy coat to evaluate the impact of single nucleotide polymorphisms in enzymes involved in drug metabolism and distribution. Once all data is collected, we will evaluate whether patient age, weight, laboratory values, fed/fasted state, concomitant chelators or pharmacogenomics impact deferasirox serum concentrations and can inform individualized deferasirox dosing. This study is unique, unlike prior deferasirox PK studies which collected intensive PK, we are using sparse sampling to collect real world data on deferasirox at steady state in TDT patients. Our hope is that this study will enable safer more effective dosing of deferasirox in patients with TDT.

Development and Validation of RP-HPLC Method for the Estimation of Linezolid from Bulk and Formulations

A new, simple, rapid, selective, precise and accurate isocratic reverse-phase high-performance liquid Chromatography assay method has been developed to estimate Linezolid in tablet formulations. The separation was achieved using column Inert sustain C18 (250×4.6mm, 5µ) in the mobile phase consisting of methanol: 1% Acetic acid (50:50 v/v). The flow rate was 0.80mL/min-1, and the separated Linezolid was detected using a UV detector at 249nm. Column temperature 45°C and sample temperature ambient and injection volume 20µL. The retention time of Linezolid was noted to be 7.3min. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

The α1 adrenoceptor antagonist prazosin potentiates morphine induced conditioned place preference in rats

The norepinephrine (NE) system is involved in pathways that regulate morphine addiction. Here, we investigated the role of α1 adrenoceptor in the ventrolateral orbital cortex (VLO) of rats with repeated morphine treatment and underlying molecular mechanisms. The rewarding properties of morphine were assessed by the conditioned place preference (CPP) paradigm. Prazosin, an α1 adrenoceptor antagonist, was microinjected into the VLO. The expression of α1 adrenoceptor, p-CaMKII/CaMKII, CRTC1, BDNF and PSD95 in the VLO were determined by immunohistochemistry or western blotting. Neurotransmitter NE in the VLO and inflammatory factors in serum were detected separately through high-performance liquid chromatography and enzyme-linked immunosorbent assay. Our experimental results showed that repeated morphine administration induced stable CPP and prazosin promoted the morphine-induced CPP. Microinjection of prazosin in the VLO not only blocked the activity of α1 adrenoceptor, decreased CaMKII phosphorylation and CRTC1, which eventually resulted in a regression of synaptic plasticity-related proteins, but also was accompanied by significantly decreasing of NE in the VLO and increasing of inflammatory cytokines in peripheral blood. These findings suggested that prazosin potentiates the addictive effects of morphine. The effect of increased CPP through reducing α1 adrenoceptor and NE was associated with the CaMKII-CRTC1 pathway and synaptic plasticity-related proteins in the VLO and inflammatory cytokines in the peripheral blood. The NE system may therefore be an underlying therapeutic target in morphine addiction. Additionally, we believe that the clinical use of prazosin in hypertensive patients with morphine abuse may be a potential risk because of its reinforcing effect on addiction.

A-340 CFTR Modulator Therapy Increases Serum/Plasma Concentrations of Vitamin a in People with Cystic Fibrosis

Abstract Introduction Pancreatic insufficiency (PI) is common in people with Cystic Fibrosis (PwCF), contributing to malabsorption of fat and fat-soluble vitamins. Thus, PI PwCF are prescribed pancreatic enzyme replacement therapy (PERT) and supplementation with fat-soluble vitamins. The circulating vitamin levels are monitored to guide dietary therapy. Currently, there are multiple cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies available that have shown significant improvements in pulmonary function, anthropometric parameters, and life expectancy in PwCF, but the effects on serum/plasma fat-soluble vitamin levels remain unknown. The goal of this study was to determine how modulator therapy affects serum/plasma vitamin A and E levels in individuals with CF. Methods The analysis included 1139 serum/plasma samples from 157 children and adolescents with CF: 74 males, 83 females, between the ages of 2–17 years. Most individuals were homozygous or compound heterozygous for the F508del variant (n = 81 and n = 72, respectively). 141 individuals (90%) were diagnosed with PI (fecal elastase &amp;lt;200 μg/g stool). 76 individuals (48%) received different CFTR modulator therapies for at least 1 month. Vitamin A (retinol and retinyl palmitate) and Vitamin E (α-tocopherol and γ-tocopherol) were measured using a high-performance liquid chromatography assay. Data analyses were performed in Prism software (La Jolla, CA), and statistical significance was defined as P &amp;lt; 0.05. The study was approved by the IRB. Results Overall, vitamin deficiency was rarely observed in PwCF prescribed PERT and vitamin supplements; &amp;lt;3% of them had low concentrations of Vitamin A and/or E. The proportion of individuals with Vitamin A and/or E above the reference range was larger; 10.9% and 30.8% for Vitamin A and E, respectively, most likely reflecting supplementation. There was no correlation of plasma vitamin A or E results with sex, weight-for-age, height-for-age, or BMI-for-age. However, pancreatic status affected vitamin levels in individuals not taking modulators: samples from PI PwCF (n = 367) had significantly lower Vitamin A and E than those from PS PwCF (n = 71). In contrast, we have observed higher concentrations of Vitamin A retinol in individuals on modulators: 0.49 ± 0.13 mg/L on modulators vs 0.43 ± 0.12 mg/L without modulators. Pairwise analysis of samples from 76 individuals before and after modulator therapy confirmed this finding, showing an average 16% increase in serum/plasma retinol concentration (P &amp;lt; 0.0001). No changes in retinol were observed between different measurements in PwCF who were not taking modulators (n = 82). Vitamin E levels were not impacted by modulator therapy. Conclusions Advances in clinical care improved fat-soluble vitamin status in people with CF. However, even when supplemented and on PERT, PI PwCF not on modulators tend to have lower fat-soluble vitamin concentrations than PS PwCF. CFTR modulator therapy increases serum/plasma concentrations of vitamin A. Thus, individuals receiving modulators and vitamin supplementation should be monitored to avoid Vitamin A toxicity. This is especially true for PS PwCF who already have higher baseline vitamin levels.

A-142 Comparison of Alinity c Hemoglobin A1c Immunoassay with Premier Hb9210 Automated HPLC Assay: Very Few Patients May be Outside Reportable Range Using Alinity c.

Abstract Background Hemoglobin A1c (HbA1c; glycosylated hemoglobin) is used for diagnosis and monitoring of patients with diabetes. In the clinical laboratory both immunoassays and high-performance liquid chromatography (HPLC) based tests are available for such measurement. We use Premier Hb9210 analyzer (HPLC method; Trinity Biotech, Jamestown, NY) for measuring HBA1c in whole blood. As our laboratory is transitioning to Abbott chemistry systems, we compared HbA1c values obtained by the Alinity c and Premier Hb9210. Methods The Premier Hb9210 analyzer is based on boronate affinity high performance liquid chromatography with output in 66 sec. The analytical measurement range is 3.8 to 18.5% although values as low as 2% can be reported by the analyzer but result may not be accurate. The Alinity c Hemoglobin A1c assay measures both total hemoglobin and HbA1c (enzymatic assay) in whole blood and then calculates %HbA1c. The analytical measurement range of this assay is 4 to 14% of HbA1c. Specimens cannot be diluted for Alinity c HbA1c measurement. Results Alinity c HbA1c has excellent total precision (0.4% at HbA1c of 5.1% and 0.25% at Hb A1c of 9.4%). In contrast, Premier HB9210 has total precision of 1.89% at HbA1c of 5.5% and 1.81% at HbA1c of 8.4%. However, analytical measurement range of Premier HB9219 is slightly wider compared Alinity c assay. Plotting HbA1c results obtained by the Premier Hb9210 analyzer in the x-axis and the corresponding values obtained by the Alinity c assay, we observed the following regression equation: y = 0.9499 x + 0.1108 ( n = 50, 0.99). The 95% confidence interval of the slope was 0.9241 to 0.9757. Rarely, we get specimen where HbA1c is between 3.8 and 4.0% (42 specimens out of 52 803 between 2/7/22 to 2/7/23; 0.08%) or above 14% (174 specimens out of 52 802; same period; 0.33%) and these specimens would not produce any result with Alinity c assay. We recently observed HbA1c result of 2.4 % in a sickle cell patient (reported as &amp;lt;3.8% per our lab policy). Discussion Our result indicates that HbA1c immunoassay on the Alinity c analyzer showed values comparable to HPLC method. However, for very few patients HbA1c may exceed 14% due to uncontrollable diabetes. Because specimen cannot be diluted, real value could not be measured using Alinity c but can be measured by the HPLC method. Conclusions We conclude that HbA1c assay on the Alinity c analyzer is a viable alternative to HPLC for measuring HbA1c in clinical laboratory. However, for very few patients (sickle cell trait, uncontrolled diabetes), HbA1c may be outside the range of Alinity c, requiring HPLC analysis.

Pharmacodynamics, safety, tolerability and pharmacokinetics of a single oral dose of an engineered phenylalanine ammonia-lyase in patients with phenylketonuria

The cornerstone treatment of hyperphenylalaninemia (HPA) and phenylketonuria (PKU) is a lifelong low-protein diet with phenylalanine (Phe) free L-amino acid supplements. However, the PKU diet has significant shortcomings, and there is a clinically unmet need for new therapeutics to improve patient outcomes. CDX-6114 is a modified phenylalanine ammonia-lyase (PAL) enzyme obtained by a mutation in the Anabaena variabilis PAL sequence. CodeEvolver® protein engineering technology has been applied to improve the degradation resistance of the enzyme. In our first phase I trial, 19 patients were given a single oral dose of CDX-6114 at 7.5 g, 2.5 g, 0.7 g, or placebo in a cross-over design. After an overnight fast, patients received a standardised breakfast of 20 g of protein, thus exceeding the dietary recommendations for a single meal in patients with PKU. Plasma levels of Phe and cinnamic acid (CA) were measured over a 5-h period following CDX-6114 dosing. During the development of CDX-6114, a stability assessment using reverse-phase high-performance liquid chromatography (HPLC) assay revealed two peaks. The second peak was identified as CA. It was not previously known that as part of the mechanism of action, the CA remained associated with the protein following the conversion of Phe. Thus, recalculating the historical PAL enzyme amounts in CDX-6114 bulk substance was necessary. An updated extinction coefficient was achieved by applying a correction factor of 0.771 to previously reported doses. Postprandial plasma levels of Phe increased in all dose cohorts over time between 10% and 30% from baseline, although the actual peak of Phe levels was not achieved within the 5-h observation. When accounting for the interquartile ranges, these concentrations were similar to the placebo. As plasma levels of Phe were no longer a reliable marker for pharmacodynamics, the consistently detectable amount of CA seen in all patients who received CDX-6114 provided proof of the enzymatic activity of CDX-6114 in metabolising gastrointestinal Phe. Peak levels of CA were seen shortly after CDX-6114 intake, with a rapid decline, and remained low compared with the plasma Phe levels. This pattern indicates a short half-life, possibly due to the liquid formulation or the inability to withstand the lower pH in the human stomach compared with animal models in earlier studies. This was the first trial in patients with PKU to establish the safety and tolerability of CDX-6114. A single dose of CDX-6114 was safe and well tolerated, with no serious adverse events or presence of anti-drug antibodies detected. Efficacy will be explored in future trials using an optimised formulation.