Active Metabolite Research Articles

Study the hepatoprotective effect of the methanolic extract of Plumbago auriculata Lam. against CCl4-induced hepatocyte damage in mice

The liver is an important organ in the body; its diseases are considered the major causes of morbidity and mortality around the world. Hepatotoxic chemicals cause damage to liver cells. Medicinal plants have a powerful hepatoprotective effect. This study has been aimed to assess the potential hepatoprotective effect of the methanolic extract of Plumbago auriculata Lam. against carbon tetrachloride (CCl4)- induced hepatocyte damage in albino mice. The methanolic extract of the plant was subjected to preliminary phytochemical analysis to determine the presence of secondary metabolites according to standard protocols. The acute toxicity study was carried out to measure the LD50. Swiss albino mice were divided into 4 groups treated intraperitoneally (IP) once daily for 7 days; the first group was a negative control, while the second group, considered as a CCl4 model, received a single dose of carbon tetrachloride during the last day. The third group received 500 mg/kg of body weight of methanolic extract of P. auriculata, and the last group received 100 mg/kg of body weight of standard silymarin, after 2 h of treatment, on the last day, all animals (except negative control group) have received CCl4 at a dose of 1 mL/kg of body weight. Biochemical analysis of collected blood and histopathological examination were performed. Results revealed that the preliminary phytochemical screening of the methanolic extract of the plant confirmed the presence of many biologically active secondary metabolites like flavonoids, steroids, terpenoids, saponins, glycosides and phenols and LD50 of the extract was 2.5 g/kg. The CCl4- intoxicated mice showed an increase in biochemical enzyme levels (ALT and AST) compared with the negative control group. The extract of 500 mg/kg revealed a significant reduction (p ? 0.05) in enzyme activities. The histopathological analysis of plant extract and silymarin-treated groups revealed a decrease in the pathological features compared with the CCl4-intoxicated group. It was concluded that the extract of P. auriculata has a significant effect on hepatoprotection against CCl4-induced hepatocyte damage and this may be because of the combined effects of the bioactive compositions of plant extract.

Pharmacokinetic/Pharmacodynamic assessment of the structural refinement of clopidogrel focusing on the balance between bioactivation and deactivation.

The delicate balance between ischemic and bleeding risks is a significant consideration in the administration of antiplatelet therapy. Clopidogrel and prasugrel, both members of the thienopyridine class of antiplatelet drugs, are well established for their variability in individual responsiveness and for a high number of bleeding events, respectively. The current study focuses on evaluating the pharmacokinetics and pharmacodynamics of a series of deuterated clopidogrel derivatives, leveraging insights gained from the structure-pharmacokinetic relationships in the development of thienopyridine drugs. Our approaches were based on the molecular skeleton of clopidogrel and adopted the C2-pharmacophore design from prasugrel. The selected C2-pharmacophore distinguishes itself from the acetyloxy substituent of prasugrel by exhibiting a moderated hydrolysis rate, resulting in a gentler formation of the active metabolite. An excessive and burst release of the active metabolite are therefore to be avoided, as it is believed to be associated with an increased risk of bleeding. Our proposed structural modification maintains the hydrolysis-sensitive methyl ester of clopidogrel but replaces it with a deuterated methyl group, which has been shown to effectively reduce metabolic deactivation. The evaluation of the clopidogrel derivatives has been primarily based on the criteria related to the exposure to active metabolites. Three promising compounds demonstrate higher biotransformation efficiency, similar Cmax, delayed Tmax, enhanced antiplatelet activity, and a lower risk of bleeding compared to clopidogrel, when administered at a dosage resulting in a similar exposure to the active metabolites. Significance Statement The pharmacokinetics and pharmacodynamics of a series of newly designed clopidogrel derivatives were assessed to validate the rationale for their structural modifications. Three promising compounds displayed balanced pharmacokinetics, characterized by slower deactivation compared to clopidogrel and a more gradual bioactivation than prasugrel. Under similar exposure to active metabolites, these compounds have demonstrated enhanced antiplatelet activity and a diminished risk of bleeding compared to clopidogrel. The D3-clopidogrel-ozagrel conjugate was found to exert a synergistic therapeutic effect.

The Impact of a 6-Week Nordic Walking Training Cycle and a 14-Hour Intermittent Fasting on Disease Activity Markers and Serum Levels of Wnt Pathway-Associated Proteins in Patients with Multiple Myeloma

Background: Multiple myeloma (MM) accounts for about 10–15% of all diagnosed hematologic malignancies and about 1–2% of all cancer cases. Approximately 80–90% of MM patients develop bone disease and the changes rarely regress. It is only possible to stop or slow their progression. A major role in bone destruction in MM is attributed to the Wnt signaling pathway, and its action can be modified by various types of interventions including training and diet. Therefore, the aim of this project was to evaluate the effects of a Nordic Walking (NW) training cycle and intermittent fasting (IF) on the levels of selected bone turnover markers associated with the Wnt pathway in patients with MM. Materials and methods: Results from 35 patients divided into training (NW and IF NW) and non-training (IF and control) groups were included in the analysis. A 6-week training cycle involving 60 min workouts 3 times a week was conducted. Body mass and composition as well as the levels of vitamin D, calcium and phosphorus, beta2-microglobulin, and albumin were examined before and after the completion of the training cycle. Markers of bone turnover were also determined: sclerostin (SOST), Dickkopf-related protein 1 (DKK-1), osteoprotegrin (OPG), osteopontin (OPN), and Tartrate-resistant acid phosphatase 5b (TRACP 5b). Results: There was no negative effect of IF or combined training and fasting on the nutritional status of the patients (the level of albumins was unchanged). Both training groups showed an increase in serum concentrations of the active metabolite of vitamin D (IF NW and NW: p = 0.001 and p = 0.022, respectively). The change in the concentration of this vitamin negatively correlated with the concentration of TRACP 5b (r = −0.413, p = 0.014). Evaluating the concentrations of markers related to bone turnover, a reduction in the concentrations of SOST (time: p = 0.026, time vs. group: p = 0.033) and TRACP 5b (time: p < 0.001, time vs. group p < 0.001) was indicated. Conclusions: The obtained results allow one to indicate the training with the poles as a safe and beneficial form of physical activity that should be recommended to patients suffering from MM. However, the results obtained in the present study are not sufficient to show the beneficial effect of IF applied without trainings.

Molecularly Imprinted Polymer Biosensor Based on Nitrogen-Doped Electrochemically Exfoliated Graphene/Ti3 CNTX MXene Nanocomposite for Metabolites Detection.

Rapid and accurate quantification of metabolites in different bodily fluids is crucial for a precise health evaluation. However, conventional metabolite sensing methods, confined to centralized laboratory settings, suffer from time-consuming processes, complex procedures, and costly instrumentation. Introducing the MXene/nitrogen-doped electrochemically exfoliated graphene (MXene@N-EEG) nanocomposite as a novel biosensing platform in this work addresses the challenges associated with conventional methods, leveraging the concept of molecularly imprinted polymers (MIP) enables the highly sensitive, specific, and reliable detection of metabolites. To validate our biosensing technology, we utilize agmatine as a significant biologically active metabolite. The MIP biosensor incorporates electrodeposited Prussian blue nanoparticles as a redox probe, facilitating the direct electrical signaling of agmatine binding in the polymeric matrix. The MXene@N-EEG nanocomposite, with excellent metal conductivity and a large electroactive specific surface area, effectively stabilizes the electrodeposited Prussian blue nanoparticles. Furthermore, increasing the content of agmatine-imprinted cavities on the electrode enhances the sensitivity of the MIP biosensor. Evaluation of the designed MIP biosensor in buffer solution and plasma samples reveals a wide linear concentration range of 1.0 nM-100.0 μM (R2 = 0.9934) and a detection limit of 0.1 nM. Notably, the developed microfluidic biosensor offers low cost, rapid response time to the target molecule (10 min of sample incubation), good recovery results for detecting agmatine in plasma samples, and acceptable autonomous performance for on-chip detection. Moreover, its high reliability and sensitivity position this MIP-based biosensor as a promising candidate for miniaturized microfluidic devices with the potential for scalable production for point-of-care applications.

The use of deep eutectic solvent-liquid phase microextraction as green analytical sample treatment for the analysis of drugs in urine

In this paper, a natural deep eutectic solvent (NADES)-based liquid phase microextraction (LPME) of 20 pharmaceuticals from urine, employing liquid chromatography tandem mass spectrometry (LC-MS/MS) for precise quantification, has been developed. The chromatographic gradient was accurately evaluated to ensure the effective separation of tramadol, an opioid receptor agonist, and o-desmethylvenlafaxine, one active metabolite of the antidepressant venlafaxine, both compounds sharing identical molecular mass for the precursor ion (Q1: 264.2 m/z) and fragmentation patterns (Quantifier ion: 58.2 m/z; qualifier ion: 246.0 m/z). NADES consisted of menthol-octanoic acid in a molar ratio of 1:2. Using the appropriate extraction conditions, the developed methodology provided appropriate LOQ values, ranging from 15 to 208 ng L–1, with reasonable RSD values (below 11 %), recoveries ranging from 64 to 120 % and preconcentration factors up to 66.6 folds. Application of the developed method to the analysis of urine samples served to highlight its usefulness. An AGREEprep score of 0.64 was obtained despite the demand for substantial sample volumes and the use of LC-MS/MS for analyte determination.

Structure-activity relationships and pharmacokinetic evaluation of L-cystine diamides as L-cystine crystallization inhibitors for cystinuria

AbstractCystinuria is a rare genetic disorder characterized by defective l-cystine reabsorption from the renal proximal tubule, resulting in abnormally high concentrations of L-cystine and subsequent l-cystine crystallization and stone formation in urine. l-Cystine diamides have shown great promise as inhibitors of l-cystine crystallization. The free α-amino groups in l-cystine diamides have previously been shown to be necessary for l-cystine crystallization inhibitory activity. In this study, three additional series of l-cystine diamide analogs were designed to explore further the structure-activity relationships for l-cystine crystallization inhibition. It has been demonstrated that the middle disulfide bond is required for optimal l-cystine crystallization inhibitory activity, and the only regions that can be modified are the two terminal amides. The presence of another basic amine 2–3 atoms away from the amide nitrogen is also critical for optimal activity. Disulfide exchange was found to be the main metabolic pathway resulting in the formation of two molecules of the active mixed disulfide metabolite from a single l-cystine diamide. l-Cystine diamides have the potential to be developed into a much-needed therapy for cystinuria.

Utilizing metabolomics and network analysis to explore the effects of artificial production methods on the chemical composition and activity of agarwood

Introduction: Agarwood is a traditional aromatic southern medicine. It has a long history of being used in traditional Chinese aromatherapy to treat insomnia, anxiety and depression. Due to the scarcity of wild resources, people have planted trees successfully and begun to explore various agarwood-inducing techniques. This study comparative analysis of volatile metabolites in agarwood produced by various inducing techniques and its potential sleep-promoting, anti-anxiety and anti-depressant network pharmacological activities. Methods: A total of 23 batches of two types of agarwood were collected, one of which was produced by artificial techniques, including 6 batches of TongTi (TT) agarwood produced by “Agar-Wit” and 6 batches of HuoLao (HL) agarwood produced by “burning, chisel and drilling”, while the other was collected from the wild, including 6 batches of BanTou (BT) agarwood with trunks broken due to natural or man-made factors and 5 batches of ChongLou (CL) agarwood with trunks damaged by moth worms. The study employed metabolomics combined with network analysis to compare the differences in volatile metabolites of agarwood produced by four commonly used inducing techniques, and explored their potential roles and possible action targets in promoting sleep, reducing anxiety, and alleviating depression. Results: A total of 147 volatile metabolites were detected in agarwood samples, mainly including small aromatic hydrocarbons, sesquiterpenes and 2-(2-phenylethyl) chromone and their pyrolysis products. The results showed composition of metabolites was minimally influenced by the agarwood induction method. However, their concentrations exhibited significant variations, with 17 metabolites showing major differences. The two most distinct metabolites were 6-methoxy-2-(2-phenylethyl) chromone and 6,7-dimethoxy-2-(2-phenylethyl) chromone. Among the volatile metabolites, 142 showed promising potential in treating insomnia, anxiety, and depression, implicating various biological and signaling pathways, predominantly ALB and TNF targets. The top three active metabolites identified were 2-(2-phenylethyl) chromone, 1,5-diphenylpent-1-en-3-one, and 6-methoxy-2-[2-(4'-methoxyphenyl) ethyl] chromone, with their relative content in the four types of agarwood being TT>HL>CL>BT. Conclusion: The differences in the content of 2-(2-phenylethyl) chromones suggest that they may be responsible for the varying therapeutic activities observed in different types of agarwood aromatherapy. This study offers theoretical support for the selection of agarwood in aromatherapy practices.

Extremophiles and their expanding biotechnological applications.

Microbial life is not restricted to any particular setting. Over the past several decades, it has been evident that microbial populations can exist in a wide range of environments, including those with extremes in temperature, pressure, salinity, and pH. Bacteria and Archaea are the two most reported types of microbes that can sustain in extreme environments, such as hot springs, ice caves, acid drainage, and salt marshes. Some can even grow in toxic waste, organic solvents, and heavy metals. These microbes are called extremophiles. There exist certain microorganisms that are found capable of thriving in two or more extreme physiological conditions simultaneously, and are regarded as polyextremophiles. Extremophiles possess several physiological and molecular adaptations including production of extremolytes, ice nucleating proteins, pigments, extremozymes and exopolysaccharides. These metabolites are used in many biotechnological industries for making biofuels, developing new medicines, food additives, cryoprotective agents etc. Further, the study of extremophiles holds great significance in astrobiology. The current review summarizes the diversity of microorganisms inhabiting challenging environments and the biotechnological and therapeutic applications of the active metabolites obtained as a response to stress conditions. Bioprospection of extremophiles provides a progressive direction with significant enhancement in economy. Moreover, the introduction to omics approach including whole genome sequencing, single cell genomics, proteomics, metagenomics etc., has made it possible to find many unique microbial communities that could be otherwise difficult to cultivate using traditional methods. These findings might be capable enough to state that discovery of extremophiles can bring evolution to biotechnology.

Human bronchopulmonary disposition and plasma pharmacokinetics of oral bemnifosbuvir (AT-527), an experimental guanosine nucleotide prodrug for COVID-19.

Bemnifosbuvir (AT-527) is a novel oral guanosine nucleotide antiviral drug for the treatment of persons with COVID-19. Direct assessment of drug disposition in the lungs, via bronchoalveolar lavage, is necessary to ensure antiviral drug levels at the primary site of SARS-CoV-2 infection are achieved. This Phase 1 study in healthy subjects aimed to assess the bronchopulmonary pharmacokinetics, safety and tolerability of repeated doses of bemnifosbuvir. A total of 24 subjects were assigned to receive bemnifosbuvir twice daily at doses of 275, 550 or 825 mg for up to 3.5 days. AT-511, the free base of bemnifosbuvir, was largely eliminated from the plasma within 6 h post dose in all dosing groups. Antiviral drug levels of bemnifosbuvir were consistently achieved in the lungs with bemnifosbuvir 550 mg twice daily. The mean level of the guanosine nucleoside metabolite AT-273, the surrogate of the active triphosphate metabolite of the drug, measured in the epithelial lining fluid of the lungs was 0.62 µM at 4-5 h post dose. This exceeded the target in vitro 90% effective concentration (EC90) of 0.5 µM for antiviral drug exposure against SARS-CoV-2 replication in human airway epithelial cells. Bemnifosbuvir was well tolerated across all doses tested, and most treatment-emergent adverse events reported were mild in severity and resolved. The favourable pharmacokinetics and safety profile of bemnifosbuvir demonstrates its potential as an oral antiviral treatment for COVID-19, with 550 mg bemnifosbuvir twice daily currently under further clinical evaluation in persons with COVID-19.

Marine seaweed endophytic fungi-derived active metabolites promote reactive oxygen species-induced cell cycle arrest and apoptosis in human breast cancer cells.

Endophytic fungi have an abundant sources rich source of rich bioactive molecules with pivotal pharmacological properties. Several studies have found that endophytic fungi-derived bioactive secondary metabolites have antiproliferative, anti-oxidant, and anti-inflammatory properties, but the molecular mechanism by which they induce cell cycle arrest and apoptosis pathways is unknown. This study aimed to determine the molecular mechanism underlying the anticancer property of the endophytic fungi derived active secondary metabolites on human breast cancer cells. In this study, we identified four endophytic fungi from marine seaweeds and partially screened its phytochemical properties by Chromatography-Mass Spectrometry (GC-MS) analysis. Moreover, the molecular mechanism underlying the anticancer property of these active secondary metabolites (FA, FB, FC and FE) on human breast cancer cells were examined on MCF-7 cells by TT assay, Apoptotic assay by Acridine orang/Ethidium Bromide (Dual Staining), DNA Fragmentation by DAPI Staining, reactive oxygen species (ROS) determination by DCFH-DA assay, Cell cycle analysis was conducted Flow cytometry and the apoptotic signalling pathway was evaluated by westernblot analysis. Doxorubicin was used as a positive control drug for this experiment. The GC-MS analysis of ethyl acetate extract of endophytic fungi from the marine macro-algae revealed the different functional groups and bioactive secondary metabolites. From the library, we observed the FC (76%), FB (75%), FA (73%) and FE (71%) have high level of antioxidant activity which was assessed by DPPH scavenging assay. Further, we evaluated the cytotoxic potentials of these secondary metabolites on human breast cancer MCF-7 cells for 24h and the IC50 value were calculated (FA:28.62 ± 0.3µg/ml, FB:49.81 ± 2.5µg/ml, FC:139.42 ± µg/ml and FE:22.47 ± 0.5µg/ul) along with positive control Doxorubicin 15.64 ± 0.8µg/ml respectively by MTT assay. The molecular mechanism by which the four active compound induced apoptosis via reactive oxygen species (ROS) and cell cycle arrest in MCF-7 cells was determined H2DCFDA staining, DAPI staining, Acridine orange and ethidium bromide (AO/EtBr) dual staining, flowcytometry analysis with PI staining and apoptotic key regulatory proteins expression levels measured by westernblot analysis. Our findings, revealed the anticancer potential of endophytic fungi from marine seaweed as a valuable source of bioactive compounds with anticancer properties and underscore the significance of exploring marine-derived endophytic fungi as a promising avenue for the development of novel anticancer agents. Further investigations are necessary to isolate and characterize specific bioactive compounds responsible for these effects and to validate their therapeutic potential in preclinical and clinical settings.

Abstract PO5-11-08: Assessing practice patterns of antidepressant medication and tamoxifen use among patients with hormone receptor-positive breast cancer

Abstract Background: Treatment with endocrine therapy such as tamoxifen has been identified as one of the most important variables linked with survival among women with hormone receptor-positive breast cancer (BC). There is significant risk reduction of BC recurrence and BC mortality at 15 years among patients treated with tamoxifen for 5 years compared to no endocrine therapy. Tamoxifen is metabolized in the liver via multiple cytochrome P (CYP) isoforms. To ensure optimal efficacy of tamoxifen, consideration must be given to concomitant use of other commonly prescribed drugs such as antidepressants. Some selective serotonin reuptake inhibitors (SSRIs) are potent inhibitors of CYP2D6, an enzyme that metabolizes tamoxifen into its active metabolite, endoxifen, which has anti-tumor effect. Such inhibition can reduce the plasma concentration of endoxifen which could lead to higher rates of BC recurrence. This resulted in an FDA black box warning discouraging concurrent prescriptions of tamoxifen and SSRIs, yet clinical data to support this warning is lacking. Our aim is to describe the practice patterns of concomitant use of tamoxifen and antidepressant medication at our institution. Methods: This is a single institution retrospective study that included adult women, ages 18-99, with stage I-IV BC, who received adjuvant tamoxifen and were taking an antidepressant medication between years 2016-2021. Patients were identified from a tumor registry database. Patient demographics and tumor characteristics were collected via electronic medical record. Results: A total of 419 patients met the inclusion criteria. At the start of tamoxifen therapy, 348 women were on at least one antidepressant, mostly for Major Depressive Disorder (86%). Of those women, 22% were prescribed a strong/moderately potent CYP inhibitor (paroxetine, fluoxetine, duloxetine, or bupropion) and 43% were prescribed weak or non-CYP inhibiting SSRIs (citalopram, escitalopram, fluvoxamine, and sertraline). The most commonly prescribed antidepressants at the start of tamoxifen, alone or in combination with a second antidepressant, were: venlafaxine (30%), escitalopram (17%), sertraline (13%), citalopram (12%) and duloxetine (10%). Main prescribers of antidepressants were primary care physicians (70%), psychiatrists (10%) followed by academic medical oncologists (7%) and regional medical oncologists (6%). Interestingly, the majority of women continued taking the same antidepressant after starting tamoxifen (76%) while, at least 10% of patients changed anti-depressant classes prior to starting tamoxifen, due to concern for drug-drug interaction. When a different class of antidepressant was chosen after starting tamoxifen (22%), serotonin and norepinephrine reuptake inhibitors (SNRIs) were the most frequent class (48%), followed by a different SSRI (42%). The time in which the antidepressant change occurred was variable, but most commonly prior to initiating tamoxifen. Conclusion: Concurrent use of tamoxifen and antidepressant medications is common. Theoretical risk of reduced efficacy of tamoxifen with inhibitors of CYP leads to medication changes for some but not all patients requiring tamoxifen and an antidepressant medication. This can have clinical implications as stability on a specific antidepressant is important for patients and adjustment may impact control of their mood disorder. In some cases, even if a drug-drug interaction was identified, some patients chose to continue their current antidepressant due to concerns of mood de-stabilization, and in other cases, aromatase inhibitors were started as an alternative. These findings highlight differences in practice patterns for which additional guidance may be helpful in bringing awareness and standardization to care. Citation Format: Mariah Ondeck, Bennett Osantowski, Nerea Lopetegui-Lia, Wei Wei, Alexandra Murray, Amanda Maggiotto, Halle Moore, Erin Roesch. Assessing practice patterns of antidepressant medication and tamoxifen use among patients with hormone receptor-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-11-08.

Abstract PO3-18-05: Pharmacokinetics (PK) of lasofoxifene (LAS) monotherapy and combined with abemaciclib (Abema)

Abstract Introduction: LAS is a selective estrogen receptor modulator (SERM)/tissue-selective estrogen agonist/antagonist studied in women with osteoporosis and vulvar-vaginal atrophy, and most recently, metastatic breast cancer (mBC) with ESR1 mutations. LAS data in healthy postmenopausal women and young men show linear PK and no differences by age or race (Gardner. J Clin Pharmacol 2006;46:52-58; Prakash. Drug Metab Dispos 2008;26:1218-26; Data on file). Also, no PK differences were found between Caucasian and Japanese patients (Jhee. AACR-JCA 2022, abstract A83; Fountaine. J Clin Pharmacol 2006;46:693-99). Here we report select PK data of LAS (5 mg/day) from women with mBC in the ELAINE 1 and ELAINE 2 oncology trials to indirectly investigate possible drug-drug interactions between LAS and Abema. Methods: Patients with mBC and ESR1 mutations who progressed on endocrine therapy and CDK4/6 inhibitors received LAS 5 mg/day as monotherapy (ELAINE 1) or combined with Abema (provided by Eli Lilly and Co) 150 mg BID (ELAINE 2). Blood was collected prior to daily dosing at weeks 2, 4, 8, and every 4 weeks up to the final visit in ELAINE 1, and visits during the first 8 weeks in ELAINE 2, to determine the plasma trough steady-state concentrations (Css) of LAS, Abema, and two active Abema metabolites (M2 and M20). LAS was assayed using validated high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS; detection 0.025 to 25.0 ng/mL); levels below the lower limit of quantification (LLOQ) were reported as 0 ng/mL. Abema and its M2 and M20 metabolites were measured by validated Turbo Ion Spray LC/MS/MS (detection 1.00 to 500 ng/mL); values below the LLOQ were reported as < LLOQ ng/mL. Mean trough Css at each visit were calculated for LAS (ELAINE 1 and 2), and Abema and its M2 and M20 metabolites (ELAINE 2). LAS levels from ELAINE 1 and 2 were compared, while Abema, M2 and M20 plasma levels in ELAINE 2 were compared with previously reported levels. Such indirect comparisons may help determine if Abema altered the metabolism of LAS, or if LAS altered that of Abema, M2, or M20. The relationships of LAS and Abema levels with progression-free survival (PFS) were also explored. Results: Across all time points for subjects who had PK samples assessed, the LAS mean trough Css was 33.6 ng/mL (CV%, 39.8) in ELAINE 1 (n=40) and 30.9 ng/mL (CV 50.5%) in ELAINE 2 (n=29); in both studies, LAS concentrations were consistent across visits. LAS levels in ELAINE 2 were similar to those in ELAINE 1 with no evidence of accumulation. Mean (CV%) Abema plasma concentrations at weeks 2, 4, 6 and 8 were 199 (102%), 107 (130%), 82 (271%), and 94.3 (223%) ng/mL, respectively. Minimum and maximums of the mean trough Css for LAS, Abema, M2, and M20 are shown in Table 1. No relationships between LAS or Abema levels and antitumor activity measured by PFS were seen (data to be presented). Conclusion: The steady-state PK of 5 mg LAS in the ELAINE 1 and 2 studies suggest that the addition of abemaciclib to LAS did not result in an obvious or significant LAS-Abema, drug-drug interaction. Abema PK data from ELAINE 2 were consistent with published data (European Medicines Agency, Verzenio). These results support the continued development of LAS alone and combined with Abema for treating ER+/HER2-, ESR1-mutated mBC that progressed on endocrine therapy and CDK4/6 inhibitors. Table. Mean trough, steady-state concentrations of lasofoxifene and abemaciclib. XX- XX aData from patients who received oral LAS (5 mg/day) plus Abema (150 mg BID). Abema, abemaciclib; LAS, lasofoxifene; M2 and M20 are Abema metabolites. Citation Format: Senthil Damodaran, Paul V. Plourde, Simon Jenkins, Phil Mayer, David J Portman, Matthew Goetz. Pharmacokinetics (PK) of lasofoxifene (LAS) monotherapy and combined with abemaciclib (Abema) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-05.

Abstract PO1-05-09: ASCENT-07: A phase 3, randomized, open-label study of sacituzumab govitecan versus treatment of physician’s choice in patients with HR+/HER2– inoperable, locally advanced, or metastatic breast cancer post-endocrine therapy

Abstract Background: Approximately 70% of all breast cancer (BC) cases are hormone receptor positive/human epidermal growth factor receptor 2-negative (HR+/HER2–), with ~65% of these being HR+/HER2-low (immunohistochemistry [IHC]1+, IHC2+/in situ hybridization [ISH]–). Despite an increasing number of available endocrine and targeted treatments for metastatic disease, tumors eventually develop resistance to endocrine therapy (ET). Chemotherapy, the standard of care for patients (pts) whose cancers no longer respond to ET, is associated with poor outcomes, representing a high unmet need in the first-line endocrine-resistant setting. Sacituzumab govitecan (SG) is a Trop-2–directed antibody-drug conjugate (ADC) that delivers SN-38 (the active metabolite of the topoisomerase inhibitor irinotecan) to tumor cells via internalization, and to the surrounding tumor microenvironment via the bystander effect. SG demonstrated a significant and clinically meaningful survival benefit in the phase 3, randomized TROPiCS-02 trial, and is approved in the US for inoperable, locally advanced, or metastatic HR+/HER2– BC after ET and ≥ 2 systemic therapies in the metastatic setting. ASCENT-07 will examine the efficacy and safety of SG in the first-line chemotherapy setting, ie, in patients with inoperable, locally advanced, or metastatic ET resistant HR+/HER2– BC. Study design: ASCENT-07 (NCT05840211) is a randomized, open-label, global study evaluating SG versus treatment of physician’s choice (TPC) chemotherapy in pts with HR+/HER2– inoperable, locally advanced, or metastatic BC who have received ET and are eligible for their first chemotherapy for advanced/metastatic disease. Pts aged ≥ 18 years with histologically confirmed HR+/HER2– (HER2 IHC0 or HER2-low [IHC1+, IHC2+/ISH–]) according to ASCO/CAP criteria, measurable disease per RECIST v1.1, and ECOG performance status of 0 or 1 will be included. Pts are eligible if they meet ≥ 1 of the following criteria: progressive disease (PD) in the metastatic setting on ≥ 2 lines of ET (± targeted therapy) (disease recurrence within the first 24 months of starting adjuvant ET is considered a line of therapy), PD within 6 months of first-line ET (± CDK 4/6 inhibitor) in the metastatic setting, or disease recurrence within 24 months of adjuvant ET with CDK 4/6 inhibitor initiation and no longer a candidate for additional ET. PD on the most recent therapy will be documented by CT or MRI per RECIST v1.1. Key exclusion criteria include prior treatment targeting topoisomerase I. Pts will be randomized to SG 10 mg/kg IV on days 1 and 8 of a 21-day cycle or TPC (capecitabine, paclitaxel, nab-paclitaxel). The primary endpoint is progression-free survival (PFS) assessed by blinded independent central review (BICR) per RECIST v1.1 criteria or death from any cause, whichever comes first. Secondary endpoints include PFS (assessed by the investigator), overall survival, objective response rate and duration of response per BICR and investigator, and quality of life (QoL) assessed using the EORTC QLQ-C30 cancer questionnaire (primarily change in the Physical Functioning domain at week 16 and time to deterioration in the Global Health Status/QoL domain). Pharmacokinetics and immunogenicity analyses will be undertaken in pts receiving SG. Incidence of adverse events and serious adverse events, including relationship to study drug(s), will be reported. Laboratory and vital signs will be monitored. ASCENT-07 is currently open for recruitment and requires ~654 eligible pts to provide adequate power for assessment of the primary and key secondary endpoints. Citation Format: Hope Rugo, Javier Cortés, Giuseppe Curigliano, Carlos Barrios, Kevin Punie, Yeon Park, Hiroji Iwata, Alex Young, Xuehan Ren, Pelin Cinar, Komal Jhaveri. ASCENT-07: A phase 3, randomized, open-label study of sacituzumab govitecan versus treatment of physician’s choice in patients with HR+/HER2– inoperable, locally advanced, or metastatic breast cancer post-endocrine therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-05-09.

Abstract PO2-16-01: Molecular correlates of drug response to guide therapy in triple-negative breast cancer

Abstract TNBC accounts for 10-20% of total breast cancer cases in the US and is more aggressive, higher grade, and has a poorer prognosis than other forms of breast cancer. TNBC is more likely to metastasize within 3-5 years than other forms of breast cancer and has a median survival of 17.6-21.3 months after metastasis. Advances in immunotherapy are promising, but recent trial results of immunotherapy-chemo combination have resulted in a 3-year overall survival rate under 36% in a PD-L1 based selected cohort. Thus, there is an unmet need for innovations that lead to new treatment options and improve outcomes for patients with TNBC in a personalized manner beyond only one or a few markers. In our recently published study (PMID: 31655920), we tested the dose response for a panel of 78 investigational drugs and plotted this readout against molecular characterization to identify statistically significant DNA, RNA, and protein predictors of drug efficacy in TNBC. We have expanded on this published study by identifying correlates from 3D cell culture. According to the literature, 3D culture systems are more representative of in vivo activity. We have increased our sample number (now 27 cell lines and 8 ex vivo PDXs) and expanded our drug library to 387 compounds, selected for their diverse mechanisms and promise in other cancers. Following screening, we prioritized 35 compounds based on their elevated activity and variance of response. From these 35 prioritized compounds, we identified molecular correlates of drug response for 27 compounds (77% of prioritized compounds yielding molecular correlates compared to 50% in our previously published study). Focusing on a known active drug in TNBC that is approved for patients with metastatic TNBC, we identified multi-omic (protein expression/activity and RNA expression) molecular correlates of response to a chemotherapeutic- SN-38, the payload of Sacituzumab and the active metabolite of irinotecan. We utilized a sub-panel of RNA correlates to characterize treatment-naive PDXs and predict response to SN-38. We then tested SN-38 response ex vivo for 5 PDXs to show that we could accurately identify sensitive/resistant models to SN-38 therapy. From significant protein correlates, we identified that the autophagy and AKT pathways were elevated in models with poor response to SN-38. Co-targeting the autophagy or AKT pathway with SN-38 was an effective strategy to identify rational, synergistic therapeutic combinations at therapeutically relevant levels (effective dose (ED) 50 and ED75). We evaluated irinotecan in vivo using a cell line with sensitivity in our 3D assay, demonstrating strong activity in vivo to validate that our screening assay accurately predicts in vivo response. We have further applied this methodology to targeted inhibitors with potential activity in TNBC. We have identified an ATR inhibitor, berzosertib, that targets the DNA damage response (DDR) pathway and correlates with protein expression and activity of Aurora kinases (AKs). Furthermore, combining the pan-AK inhibitor danusertib with berzosertib results in synergistic killing of TNBC cells. As a future direction, we are evaluating this combination in vivo utilizing an ethnically diverse patient-derived xenograft library grown in mice to evaluate this novel combination in a mock clinical trial and to identify molecular correlates of response and resistance that could be used to guide future human trials. Citation Format: Nathan Merrill, Nathalie Vandecan, Athena Apfel, Habib Serhan, Peter Ulintz, Liwei Bao, Xu Cheng, Aki Morikawa, Sofia Merajver, Matthew Soellner. Molecular correlates of drug response to guide therapy in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-01.

A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4′-Nitroacetophenone

Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4′-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5–8.2% and 2.3–7.4%, accuracy was in the range of 93.3–109.7% and 94.7–110.1%, limits of detection (LODs) were in the range of 0.15–9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer’s disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

Abstract PO2-19-03: Phase 2 study of response-guided neoadjuvant sacituzumab govitecan in patients with localized breast cancer (NeoSTAR)

Abstract Background: Optimizing treatments for localized breast cancer is key to preventing metastatic recurrences and reducing mortality. Sacituzumab Govitecan (SG) is a novel antibody-drug conjugate in which the topoisomerase 1 inhibitor SN-38 (the active metabolite of irinotecan) is coupled to a humanized monoclonal antibody targeting the antigen Trop-2, which is highly expressed in triple negative breast cancer (TNBC). SG is FDA approved for patients with metastatic hormone receptor-positive (HR+) and TNBC. However, efficacy in localized BC is not known. The NeoSTAR clinical trial is evaluating SG in the neoadjuvant (NA) setting for participants with localized breast cancer. Cohort A1 evaluating NA SG monotherapy in localized TNBC is completed, and now cohorts A2 (SG + pembrolizumab (P) for TNBC), B1 (SG monotherapy for HR+ BC), and C (SG + P for HER2- inflammatory breast cancer (IBC)) are enrolling. Trial design: This is a single-arm phase II study of NA SG or SG + P with multiple cohorts across different breast cancer subtypes. In all cohorts, SG is administered via an IV infusion on days 1 and 8 of each 21-day cycle at a starting dose of 10 mg/kg for 4 cycles. For cohorts A2 and C, pembrolizumab is given on day 1 of each cycle. For A2 and B1, after 4 cycles, participants who have biopsy-proven residual disease will have the option to receive additional standard NA therapy at the discretion of the treating physician and subsequently proceed to surgery. Those with a complete response on imaging may proceed directly to surgery. If pathological complete response (pCR) is achieved after 4 cycles of SG + P, participants on A2 will remain on study and receive adjuvant weekly paclitaxel and carboplatin for 12 weeks along with continuation of P to complete a total of one year of the PD-1 inhibitor; participants on B1 will receive adjuvant therapy at the discretion of the treating physician, with the option to continue P to complete a total of one year. On cohort C, after 4 cycles of SG + P, dose-dense doxorubicin + cyclophosphamide + P for 4 cycles will be given before considering surgery. A baseline research biopsy is required for all cohorts, as well as tissue collection following treatment with SG (either at surgery or via biopsy prior to additional NA therapy). Eligibility criteria: Participants ≥ 18 years of age with previously untreated localized breast cancer as determined by the local institution will be enrolled. For A2, participants must either have a primary tumor >2 cm or be node positive. For B1, participants with anatomic clinical stage II-III breast cancer with primary tumor > 1.5 cm and high genomic risk are eligible. For the IBC cohort (C), participants with T4d and any N are eligible. An ECOG performance score of 0 or 1, and adequate bone marrow, hepatic, and renal function is required. Study Objectives: The primary objective is to assess the pCR rate in breast and lymph nodes (ypT0/isN0) with SG + P (A2), SG (B1), or SG + P followed by AC + P (C). Secondary objectives include assessment of overall response rate, evaluation of the safety and tolerability of SG or SG + P (CTCAE v5.0), event-free survival (EFS), and quality of life (EORTC QLQ-C30). Exploratory objectives include assessment of potential predictive biomarkers, including Trop-2 expression, DNA damage response markers and immunological markers, as well as changes in cell free DNA with SG. Statistical methods: The primary analysis is based on the estimated pCR rate with SG or SG + P and will be provided as a proportion (with two-sided 95% confidence interval). Each cohort will follow a Simon two-stage design. EFS will be analyzed using Kaplan-Meier methods and descriptive statistics. Target Accrual: 50 participants (A2), 56 participants (B1), 39 participants (C) Contact: Dr. Laura Spring (LSpring@mgh.harvard.edu) Clinicaltrials.gov #: NCT04230109 Citation Format: Laura Spring, Ana Garrido-Castro, Filipa Lynce, Nadine Tung, Rachel Abelman, Lianne Ryan, Megan Moran, Elizabeth Mittendorf, Leif Ellisen, Sara Tolaney, Aditya Bardia. Phase 2 study of response-guided neoadjuvant sacituzumab govitecan in patients with localized breast cancer (NeoSTAR) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-19-03.

Differential Bioactivation Profiles of Different GS-441524 Prodrugs in Cell and Mouse Models: ProTide Prodrugs with High Cell Permeability and Susceptibility to Cathepsin A Are More Efficient in Delivering Antiviral Active Metabolites to the Lung.

We assessed factors that determine the tissue-specific bioactivation of ProTide prodrugs by comparing the disposition and activation of remdesivir (RDV), its methylpropyl and isopropyl ester analogues (MeRDV and IsoRDV, respectively), the oral prodrug GS-621763, and the parent nucleotide GS-441524 (Nuc). RDV and MeRDV yielded more active metabolite remdesivir-triphosphate (RDV-TP) than IsoRDV, GS-621763, and Nuc in human lung cell models due to superior cell permeability and higher susceptivity to cathepsin A. Intravenous administration to mice showed that RDV and MeRDV delivered significantly more RDV-TP to the lung than other compounds. Nevertheless, all four ester prodrugs exhibited very low oral bioavailability (<2%), with Nuc being the predominant metabolite in blood. In conclusion, ProTides prodrugs, such as RDV and MeRDV, are more efficient in delivering active metabolites to the lung than Nuc, driven by high cell permeability and susceptivity to cathepsin A. Optimizing ProTides' ester structures is an effective strategy for enhancing prodrug activation in the lung.

Current Landscape on Development of Phenylalanine and Toxicity of its Metabolites - A Review.

Phenylalanine, an essential amino acid, is the "building block" of protein. It has a tremendous role in different aspects of metabolic events. The tyrosine pathway is the prime one and is typically used to degrade dietary phenylalanine. Phenylalanine exceeds its limit in bodily fluids and the brain when the enzyme, phenylalanine decarboxylase, phenylalanine transaminase, phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4) is deficient causes phenylketonuria, schizophrenia, attentiondeficit/ hyperactivity disorder and another neuronal effect. Tyrosine, an amino acid necessary for synthesizing the pigments in melanin, is produced by its primary metabolic pathway. Deficiency/abnormality in metabolic enzymes responsible for the catabolism pathway of Phenylalanine causes an accumulation of the active intermediate metabolite, resulting in several abnormalities, such as developmental delay, tyrosinemias, alkaptonuria, albinism, hypotension and several other undesirable conditions. Dietary restriction of the amino acid(s) can be a therapeutic approach to avoid such undesirable conditions when the level of metabolic enzyme is unpredictable. After properly identifying the enzymatic level, specific pathophysiological conditions can be managed more efficiently.

Simultaneous determination of unecritinib (TQ-B3101) and its active metabolite crizotinib in rat plasma by LC-MS/MS：an application to pharmacokinetic studies

Unecritinib (TQ-B3101) is an amide prodrug of crizotinib, a selective tyrosine kinase receptor inhibitor. In the study, in vitro metabolic experiments revealed that the hydrolysis of TQ-B3101 was mainly catalyzed by carboxylesterase 2 (CES2), followed by CES1. Next, a sensitive and reliable LC-MS/MS method was established for the simultaneous determination of TQ-B3101 and its parent drug crizotinib in rat plasma. To prevent in vitro hydrolysis of TQ-B3101, sodium fluoride, the CESs inhibitor at a concentration of 2M, was immediately added after whole blood collection. Plasma samples were extracted by acetonitrile-induced protein precipitation method, and chromatographically separated on a Gemini C18 column (50mm × 2.0mm i.d., 5 μm) using gradient elution with a mobile phase of 0.1% formic acid and 5mmol/L ammonium acetate with 0.1% formic acid. The retention times for TQ-B3101 and crizotinib were 2.61 and 2.38min, respectively. The analytes were detected with tandem mass spectrometer by positive electrospray ionization, using the ion transitions at m/z 492.3 → 302.3 for TQ-B3101, m/z 450.3 → 260.3 for crizotinib, and m/z 494.0 → 394.3 for imatinib (internal standard). Method validation was conducted in the linear range of 1.00 to 800ng/mL for the two analytes. The precision, accuracy and stabilities all met the acceptance criteria. The pharmacokinetic study indicated that TQ-B3101 was rapidly hydrolyzed to crizotinib with the elimination half-life of 1.11h after a single gavage administration of 27mg/kg to Sprague-Dawley rats, and the plasma exposure of TQ-B3101 was only 2.98% of that of crizotinib.

Identification of active main metabolites of anti-infective inhibitors of the macrophage infectivity potentiator protein by liquid chromatography using mass detection.

Due to increasing antibiotic resistance, the development of anti-infectives with new mechanisms of action is crucial. Virulence factors such as the "macrophage infectivity potentiator" (Mip) protein, which catalyzes the folding of proline-containing proteins by means of their cis-trans isomerase (PPIase) activity, have come into focus as a potential new target. Since the inhibition of Mip by small molecules has been shown to lead to reduced virulence and survival in vitro, especially of Gram-negative bacteria such as Burkholderia pseudomallei (Bp), Neisseria meningitidis (Nm), and Neisseria gonorrhoeae (Ng), or Coxiella burnetii (Cb), among many others, a library of Mip inhibitors was developed. As drug metabolism has a significant impact on the overall therapeutic outcome, this report describes the biotransformation of the most potent Mip inhibitors. Therefore, the anti-infectives were treated using human liver microsomes in vitro. Liquid chromatography with tandem mass spectrometry (LC/MS-MS) methods were applied to identify the metabolites and quantify the metabolic degradation of the hit compounds. Active metabolites, N-oxides, were found, leading to new opportunities for further drug development.