Selective Inhibitor Research Articles

Discovery and Preclinical Characterization of BIIB129, a Covalent, Selective, and Brain-Penetrant BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disease with an underlying pathology characterized by inflammation-driven neuronal loss, axonal injury, and demyelination. Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase and member of the TEC family of kinases, is involved in the regulation, migration, and functional activation of B cells and myeloid cells in the periphery and the central nervous system (CNS), cell types which are deemed central to the pathology contributing to disease progression in MS patients. Herein, we describe the discovery of BIIB129 (25), a structurally distinct and brain-penetrant targeted covalent inhibitor (TCI) of BTK with an unprecedented binding mode responsible for its high kinome selectivity. BIIB129 (25) demonstrated efficacy in disease-relevant preclinical in vivo models of B cell proliferation in the CNS, exhibits a favorable safety profile suitable for clinical development as an immunomodulating therapy for MS, and has a low projected total human daily dose.

Monoclonal Antibodies Against Mature Interleukin-18 Ameliorate Colitis and Repair Goblet Cell Function.

Numerous biological interventions and small molecules are used to treat Crohn's disease; however, the effectiveness of these treatments varies largely. Non-responsiveness to biological therapies is associated with interleukin (IL)-18 gene polymorphisms and high IL-18 expression has been implicated in the pathogenesis of Crohn's disease. The aim of this study was to elucidate the expression of precursor and mature IL-18 in patients with Crohn's disease who exhibited varied responses to cytokine-targeted treatments and determine whether selective inhibition of mature IL-18 offers a novel therapeutic avenue. We generated a monoclonal antibody that specifically recognizes the neoepitope of caspase-cleaved mature IL-18. Expression of precursor and mature IL-18 was analyzed in patients with Crohn's disease. Anti-mature IL-18 monoclonal antibodies were intraperitoneally administered in an acute colitis mouse model, and the disease activity index, body weight loss, tissue pathology, proinflammatory cytokine expression, goblet cell function, and microbiota composition were assessed. Precursor and mature IL-18 expression was upregulated and goblet cell function was impaired in patients with Crohn's disease who were unresponsive to biological therapies. Administration of anti-mature IL-18 antibodies ameliorated induced colitis by repairing goblet cell function and restoring the mucus layer. The newly developed monoclonal antibody holds promise as a therapeutic alternative for Crohn's disease.

Evaluating the pharmacokinetics of upadacitinib for the treatment of moderate to severe Crohn's disease.

Janus kinases (JAK) are enzymes involved in signaling pathways that activate the immune system. Upadacitinib, an oral small molecule, is the first JAK inhibitor approved by FDA and EMA for the treatment of moderately to severely active Crohn's disease (CD), following successful phase II and III trials. Compared to other JAK inhibitors, upadacitinib has a high selectivity toward JAK1. This characteristic could improve its efficacy and safety. This review provides an overview of the available knowledge on the pharmacokinetics of upadacitinib as induction and maintenance therapy for CD. The approval of newer targeted small molecules drug, including JAK inhibitors, marked a significant advancement in terms of effectiveness. In fact, the oral administration, the rapid absorption, the excellent bioavailability and the short serum time of maximum concentration are some of the advantages compared to biologics. The selective inhibition of JAK1 by upadacitinib allows for high efficacy while maintaining a reliable safety profile.

Antidepressant nonadherence and sexual dysfunction among young adult males: the cross-sectional YAMAN study.

Selective serotonin reuptake inhibitors are associated with high rates of nonadherence and sexual dysfunction, yet the correlation between these findings in young adult men is poorly characterized. We aimed to evaluate if young adult men are less willing to adhere to antidepressant treatment due to intolerable side effects, such as sexual dysfunction. Deidentified, compensated survey that assessed baseline demographics, PHQ-8 and GAD-7 scores, attitudes towards antidepressant medication side effects, and perceptions of antidepressant medications including selective serotonin reuptake inhibitors, bupropion, and mirtazapine. From 665 delivered surveys, 505 respondents completed their survey (response rate: 76%), of which 486 were included for final analysis. After seeing common side effect profiles, our sample's willingness to take sexual function-sparing agents, such as bupropion or mirtazapine, was significantly greater than selective serotonin reuptake inhibitors (p < 0.001), with no significant difference between bupropion and mirtazapine (p = 0.263). The negative influence of erectile dysfunction and anorgasmia scored significantly higher than other common antidepressant side effects like weight gain, nausea, and dry mouth (range: p < 0.001, p = 0.043). With the exception of insomnia, participants indicated that experiencing sexual dysfunction while taking an antidepressant medication would lead to nonadherence at a significantly higher frequency than any other side effect assessed (range: p < 0.001, p = 0.005). The risk of experiencing sexual side effects when taking antidepressants could lead young adult men to become nonadherent to these medications. Strategies to augment the effectiveness of antidepressants, such as shared decision-making and the use of sexual function-sparing agents, are critical.

ATR inhibitor, camonsertib, dose optimization in patients with biomarker-selected advanced solid tumors (TRESR study).

Camonsertib is a selective oral inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase with demonstrated efficacy in tumors with DNA damage response gene deficiencies. On-target anemia is the main drug-related toxicity typically manifesting after the period of dose-limiting toxicity evaluation. Thus dose/schedule optimization requires extended follow-up to assess prolonged treatment effects. Long-term safety/tolerability and antitumor efficacy of three camonsertib monotherapy dose levels/schedules were assessed in the TRESR study dose-optimization phase: 160 mg once daily (QD) 3 days on/4 off (160 3/4; the preliminary recommended phase II dose [RP2D]) and two step-down groups of 120 mg QD 3/4 (120 3/4) and 160 mg QD 3/4, 2 weeks on/1 off (160 3/4, 2/1w). Safety endpoints included incidence of treatment-related adverse events (TRAEs), dose modifications, and transfusions. Efficacy endpoints included overall response rate, clinical benefit rate, progression-free survival, and circulating-tumor-DNA (ctDNA)-based molecular response rate. The analysis included 119 patients: 160 3/4 (n = 67), 120 3/4 (n = 25), and 160 3/4, 2/1w (n = 27) treated up to 117.1 weeks as of the data cutoff. The risk of developing grade 3 anemia was significantly lower in the 160 3/4, 2/1w group compared with the preliminary RP2D group (HR = 0.23, 2-sided P = .02), translating to reduced transfusion and dose reduction requirements. The intermittent weekly schedule did not compromise antitumor activity. The 160 3/4, 2/1w dose was established as an optimized regimen for future camonsertib monotherapy studies offering significantly reduced anemia incidence without any compromise to efficacy.

Assessing the prevalence of Beers medication utilization in the Medicare Part D population in 2020.

Medication utilization has been increasing in the U.S. year-over-year for several decades. As older adults take more medications, there is a higher risk of them being exposed to drug-drug or drug-disease interactions. The American Geriatrics Society in 2019 updated their Beers Criteria for Potentially Inappropriate Medication (PIM) Use in Older Adults. The objective of this study was to assess the prevalence of utilization of medications included in the 2019 Beers Criteria. An analysis was conducted using the Medicare Part D Provider Utilization and Payment Data Public Use File for calendar-year 2020. Medications identified in the 2019 Beers Criteria were applied to the analysis. Two categories of medications were assessed: (1) "Avoid" and (2) "Use With Caution." In 2020, 56 million prescriptions were dispensed to Medicare patients 65 years and older that are recommended to be avoided without exception (4.7% of all prescriptions) totaling $957 million in medication costs. The most utilized medication classes in this category were benzodiazepines (25,949,994 prescriptions), "Z-drugs" (6,204,909 prescriptions), long-acting sulfonylureas (5,306,577 prescriptions), 1st-generation antihistamines (5,049,289 prescriptions), and tricyclic antidepressants (4,190,062 prescriptions). Additionally, 135 million prescriptions were dispensed to Medicare beneficiaries 65 years and older for medications which the Beers Criteria states to use caution (11.3% of all prescriptions) exceeding $2.85 billion in medication costs. The most utilized medications for this category were diuretics (74,599,126 prescriptions), selective serotonin reuptake inhibitors (30,033,121 prescriptions), serotonin and norepinephrine reuptake inhibitors (11,858,968 prescriptions), tramadol (11,450,878 prescriptions), and mirtazapine (5,737,304 prescriptions). Even with the existence of the AGS Beers Criteria for PIM Use in Older Adults and its continued updated versions, 16% of medications dispensed to Medicare Part D were potentially inappropriate. Future studies are needed to assess if this has led to worsened outcomes among older adults who utilized these PIM.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells

BackgroundThe C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with “cancer hallmarks” and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT).Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions.Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities.MethodsStructured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model.ResultsWe identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. ConclusionsThis study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Deficiency of histone deacetylases 3 in macrophage alleviates monosodium urate crystals-induced gouty inflammation in mice

BackgroundGout is caused by monosodium urate (MSU) crystals deposition to trigger immune response. A recent study suggested that inhibition of Class I Histone deacetylases (HDACs) can significantly reduce MSU crystals-induced inflammation. However, which one of HDACs members in response to MSU crystals was still unknown. Here, we investigated the roles of HDAC3 in MSU crystals-induced gouty inflammation.MethodsMacrophage specific HDAC3 knockout (KO) mice were used to investigate inflammatory profiles of gout in mouse models in vivo, including ankle arthritis, foot pad arthritis and subcutaneous air pouch model. In the in vitro experiments, bone marrow-derived macrophages (BMDMs) from mice were treated with MSU crystals to assess cytokines, potential target gene and protein.ResultsDeficiency of HDAC3 in macrophage not only reduced MSU-induced foot pad and ankle joint swelling but also decreased neutrophils trafficking and IL-1β release in air pouch models. In addition, the levels of inflammatory genes related to TLR2/4/NF-κB/IL-6/STAT3 signaling pathway were significantly decreased in BMDMs from HDAC3 KO mice after MSU treatment. Moreover, RGFP966, selective inhibitor of HDAC3, inhibited IL-6 and TNF-α production in BMDMs treated with MSU crystals. Besides, HDAC3 deficiency shifted gene expression from pro-inflammatory macrophage (M1) to anti-inflammatory macrophage (M2) in BMDMs after MSU challenge.ConclusionsDeficiency of HDAC3 in macrophage alleviates MSU crystals-induced gouty inflammation through inhibition of TLR2/4 driven IL-6/STAT3 signaling pathway, suggesting that HDAC3 could contribute to a potential therapeutic target of gout.

CCR5 promotes the migration of pathological CD8+ T cells to the leishmanial lesions.

Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology.

Discovery of 2,6-Naphthyridine Analogues as Selective FGFR4 Inhibitors for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is responsible for 90% of cases. Approximately 30% of patients diagnosed with HCC are identified as displaying an aberrant expression of fibroblast growth factor 19 (FGF19)-fibroblast growth factor receptor 4 (FGFR4) as an oncogenic-driver pathway. Therefore, the control of the FGF19-FGFR4 signaling pathway with selective FGFR4 inhibitors can be a promising therapy for the treatment of HCC. We herein disclose the design and synthesis of novel FGFR4 inhibitors containing a 2,6-naphthyridine scaffold. Compound 11 displayed a nanomolar potency against Huh7 cell lines and high selectivity over FGFR1-3 that were comparable to that of fisogatinib (8) as a reference standard. Additionally, compound 11 demonstrated remarkable antitumor efficacy in the Huh7 and Hep3B HCC xenograft mouse model. Moreover, bioluminescence imaging experiments with the orthotopic mouse model support that compound 11 can be considered a promising candidate for treating HCC.

BDTX-189, a novel tyrosine kinase inhibitor, inhibits cell activity via ERK and AKT pathways in the EGFR C797S triple mutant cells.

The tertiary mutation C797S in the structural domain of the EGFR kinase is a common cause of resistance to third-generation EGFR tyrosine kinase inhibitors (TKIs). In this study, we used a potent, selective and irreversible inhibitor, BDTX-189, to target EGFR C797S triple mutant cells for cell activity. The study constructed the H1975-C797S (EGFR L858R/T790M/C797S) cell line using the CRISPR/Cas9 method and investigated its potential as a fourth-generation tyrosine kinase inhibitor via chemosensitivity approach. The results demonstrated its ability to induce cytotoxic effects, and inhibit EGFR L858R/T790M/C797S cell growth and proliferation in a dose-dependent manner. Meanwhile, BDTX-189 reduces the protein phosphorylation levels of EGFR, ERK, and AKT, promoting apoptosis. Furthermore, BDTX-189 not only inhibits common EGFR triple mutations but also effectively inhibits EGFR L858R mutation and EGFR L858R/T790M mutation. These findings support the cytotoxic effect of BDTX-189 and its inhibitory effect on cell division and proliferation with the EGFR C797S triple mutation.

NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

BackgroundGenetic studies implicate the oncogenic transcription factor Forkhead Box M1 (FOXM1) as a potential therapeutic target in high-grade serous ovarian cancer (HGSOC). We evaluated the activity of different FOXM1 inhibitors in HGSOC cell models.ResultsWe treated HGSOC and fallopian tube epithelial (FTE) cells with a panel of previously reported FOXM1 inhibitors. Based on drug potency, efficacy, and selectivity, determined through cell viability assays, we focused on two compounds, NB-73 and NB-115 (NB compounds), for further investigation. NB compounds potently and selectively inhibited FOXM1 with lesser effects on other FOX family members. NB compounds decreased FOXM1 expression via targeting the FOXM1 protein by promoting its proteasome-mediated degradation, and effectively suppressed FOXM1 gene targets at both the protein and mRNA level. At the cellular level, NB compounds promoted apoptotic cell death. Importantly, while inhibition of apoptosis using a pan-caspase inhibitor rescued HGSOC cells from NB compound-induced cell death, it did not rescue FOXM1 protein degradation, supporting that FOXM1 protein loss from NB compound treatment is specific and not a general consequence of cytotoxicity. Drug washout studies indicated that FOXM1 reduction was retained for at least 72 h post-treatment, suggesting that NB compounds exhibit long-lasting effects in HGSOC cells. NB compounds effectively suppressed both two-dimensional and three-dimensional HGSOC cell colony formation at sub-micromolar concentrations. Finally, NB compounds exhibited synergistic activity with carboplatin in HGSOC cells.ConclusionsNB compounds are potent, selective, and efficacious inhibitors of FOXM1 in HGSOC cells and are worthy of further investigation as HGSOC therapeutics.

ADHD medication adherence reduces risk of committing minor offenses in adolescents.

This study aimed to investigate the association between adolescents' adherence to attention-deficit/hyperactivity disorder (ADHD) medication and their risk of committing minor offenses. Using two Dutch databases, Statistics Netherlands (CBS) and the Foundation for Pharmaceutical Statistics (SFK), we aimed to investigate the association between adherence to ADHD medication and registered minor offenses between 2005 and 2019 of 18,234 adolescents (12-18 years). We used Cox regression analyses to compare the rate of committing minor offenses of adolescents during periods of high ADHD medication adherence compared to periods of low adherence (i.e., periods with or without sufficient amounts of dispensed medication). We additionally tested associations with adherence to selective serotonin reuptake inhibitors (SSRIs) as control medication and analyzed potential reverse causation. High ADHD medication adherence was associated with a reduced risk of committing a minor offense of between 33% and 38% compared to low adherence periods of ≥3 months (hazard ratio [HR] 0.67, confidence interval [CI] 0.64-0.71) or ≥6 months (HR 0.62, CI 0.59-0.65). The reduction in risk can likely be attributed to ADHD medication, given the absence of effects of SSRIs and no reverse causation. The reduction rate remained between 16% and 55% per sex, stimulant versus non-stimulant medication, different offense categories and further sensitivity analyses. Among adolescents using ADHD medication, rates of criminality were lower during periods of high medication adherence, suggesting that adherence to ADHD medication may contribute to prevention of minor offenses in adolescents.

Treatment of Patient with Recurrent Major Depressive Disorder: A Case Report

Introduction: The alteration of biogenic amines, in particular norepinephrine and serotonin, is a hypothesis that is frequently put forward in relation to the etiology of major depressive disorder. This study was aimed to present the recurrent major depressive disorder in a forty years old woman. Case presentation: A 40-year-old woman, presented to our mental health unit with symptoms of a low mood, which she described as similar to previous episodes of depression. Two decades ago, she underwent an evaluation and received a diagnosis of severe depression for the first time. Throughout that duration, she received imipramine (tricyclic antidepressant/TCA) at dosages of up to 150 mg per day, resulting in favorable outcomes. She claimed that she abstained from illicit substances and has not undergone any distressing events in the recent past. Although she was confident that she was experiencing another severe depression, she expressed a desire to avoid using imipramine this time due to its adverse effects, including dry mouth, dry eyes, and diarrhea. The patient was diagnosed as recurrent major depressive disorder without psychotic symptoms. Conclusion: The likelihood of experiencing additional bouts of major depression is higher when there have been multiple prior episodes, residual symptoms of depression between episodes, and the coexistence of other psychiatric or chronic medical conditions. Prior episodes of serious depression that responded well to treatment have a greater probability of reaching remission in future episodes. First-line treatment options for major depressive disorder include selective serotonin reuptake inhibitors (SSRIs) such as bupropion, venlafaxine, mirtazapine, and duloxetine.

Thermoregulatory pathway underlying the pyrogenic effects of prostaglandin E2 in the lateral parabrachial nucleus of male rats.

It has been shown that prostaglandin (PG) E2 synthesized in the lateral parabrachial nucleus (LPBN) is involved in lipopolysaccharide-induced fever. But the neural mechanisms of how intra-LPBN PGE2 induces fever remain unclear. In this study, we investigated whether the LPBN-preoptic area (POA) pathway, the thermoafferent pathway for feed-forward thermoregulatory responses, mediates fever induced by intra-LPBN PGE2 in male rats. The core temperature (Tcore) was monitored using a temperature radiotelemetry transponder implanted in rat abdomen. We showed that microinjection of PGE2 (0.28 nmol) into the LPBN significantly enhanced the density of c-Fos-positive neurons in the median preoptic area (MnPO). The chemical lesioning of MnPO with ibotenate or selective genetic lesioning or inhibition of the LPBN-MnPO pathway significantly attenuated fever induced by intra-LPBN injection of PGE2. We demonstrated that EP3 receptor was a pivotal receptor for PGE2-induced fever, since microinjection of EP3 receptor agonist sulprostone (0.2 nmol) or EP3 receptor antagonist L-798106 (2 nmol) into the LPBN mimicked or weakened the pyrogenic action of LPBN PGE2, respectively, but this was not the case for EP4 and EP1 receptors. Whole-cell recording from acute LPBN slices revealed that the majority of MnPO-projecting neurons originating from the external lateral (el) and dorsal (d) LPBN were excited and inhibited, respectively, by PGE2 perfusion, initiating heat-gain and heat-loss mechanisms. The amplitude but not the frequency of spontaneous and miniature glutamatergic excitatory postsynaptic currents (sEPSCs and mEPSCs) in MnPO-projecting LPBel neurons increased after perfusion with PGE2; whereas the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) and the A-type potassium (IA) current density did not change. In MnPO-projecting LPBd neurons, neither sEPSCs nor sIPSCs responded to PGE2; however, the IA current density was significantly increased by PGE2 perfusion. These electrophysiological responses and the thermoeffector reactions to intra-LPBN PGE2 injection, including increased brown adipose tissue thermogenesis, shivering, and decreased heat dissipation, were all abolished by L-798106, and mimicked by sulprostone. These results suggest that the pyrogenic effects of intra-LPBN PGE2 are mediated by both the inhibition of the LPBd-POA pathway through the EP3 receptor-mediated activation of IA currents and the activation of the LPBel-POA pathway through the selective enhancement of glutamatergic synaptic transmission via EP3 receptors.

Comparison of the CYP3A selective inhibitors CYP3cide, clobetasol, and azamulin for their potential to distinguish CYP3A7 activity in the presence of CYP3A4/5.

The CYP3A7 enzyme accounts for ~50% of the total CYP content in fetal and neonatal livers and is the predominant CYP involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-S (sulfate). The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low, but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We utilized three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC50 and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC50 values and CYP inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that CP was not an MDI of CYP3A5, but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP's ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7 and our results provide confidence of CYP3cide's and CP's ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms. Significance Statement These findings provide valuable insight regarding in vitro testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates. The results presented here also clearly demonstrate the effect probe selection may have on CYP3A P450 inhibition studies.

VEGFR2 blockade inhibits glioblastoma cell proliferation by enhancing mitochondrial biogenesis

BackgroundGlioblastoma is an aggressive brain tumor linked to significant angiogenesis and poor prognosis. Anti-angiogenic therapies with vascular endothelial growth factor receptor 2 (VEGFR2) inhibition have been investigated as an alternative glioblastoma treatment. However, little is known about the effect of VEGFR2 blockade on glioblastoma cells per se.MethodsVEGFR2 expression data in glioma patients were retrieved from the public database TCGA. VEGFR2 intervention was implemented by using its selective inhibitor Ki8751 or shRNA. Mitochondrial biogenesis of glioblastoma cells was assessed by immunofluorescence imaging, mass spectrometry, and western blot analysis.ResultsVEGFR2 expression was higher in glioma patients with higher malignancy (grade III and IV). VEGFR2 inhibition hampered glioblastoma cell proliferation and induced cell apoptosis. Mass spectrometry and immunofluorescence imaging showed that the anti-glioblastoma effects of VEGFR2 blockade involved mitochondrial biogenesis, as evidenced by the increases of mitochondrial protein expression, mitochondria mass, mitochondrial oxidative phosphorylation (OXPHOS), and reactive oxygen species (ROS) production, all of which play important roles in tumor cell apoptosis, growth inhibition, cell cycle arrest and cell senescence. Furthermore, VEGFR2 inhibition exaggerated mitochondrial biogenesis by decreased phosphorylation of AKT and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which mobilized PGC1α into the nucleus, increased mitochondrial transcription factor A (TFAM) expression, and subsequently enhanced mitochondrial biogenesis.ConclusionsVEGFR2 blockade inhibits glioblastoma progression via AKT-PGC1α-TFAM-mitochondria biogenesis signaling cascade, suggesting that VEGFR2 intervention might bring additive therapeutic values to anti-glioblastoma therapy.

Abstract PO3-20-04: A first-in-human study of the highly selective PI3Kα inhibitor RLY-5836 in patients with advanced breast cancer and other solid tumors

Abstract Background: Oncogenic activation of PI3Kα is a common driver event in solid tumors. Although PI3Kα is a validated therapeutic target, there is no approved highly selective inhibitor that targets mutant PI3Kα. Alpelisib, a non-selective orthosteric PI3Kα inhibitor, is approved in combination with fulvestrant for patients with PIK3CA-mutant, ER+, HER2– advanced breast cancer. Inhibition of wild-type PI3Kα causes hyperglycemia, limiting the tolerability, dosing, and efficacy of alpelisib. RLY-5836 is the second allosteric, selective pan-mutant PI3Kα inhibitor under clinical investigation. It is molecularly distinct with differentiated pharmaceutical properties compared to RLY-2608. This first-in-human study aims to further explore the tolerability and efficacy of highly selective PIK3CA-mutant inhibition as monotherapy and in combination treatment. Methods: This multicenter, open-label study is designed to evaluate the safety, maximum tolerated dose (MTD), recommended Phase 2 dose (RP2D), pharmacokinetics (PK) and preliminary anti-tumor efficacy of RLY-5836 as a single agent and in various combinations. A Bayesian Optimal Interval design is used for dose escalation, followed by a dose expansion part. As monotherapy, RLY-5836 will be investigated in pts with advanced solid tumors harboring a PIK3CA mutation (Arm 1) per local assessment (blood and/or tumor). In pts with PIK3CA-mutant, HR+, HER2– advanced or metastatic breast cancer, RLY-5836 combination arms will include anti-estrogen therapy with or without CDK4/6 inhibition: fulvestrant (Arm 2), and fulvestrant + CDK4/6 inhibitor(s) (Arms 3–5). RLY-5836 is administered orally, continuously in 28-day cycles until disease progression or study discontinuation. Enrollment criteria include age ≥18 years, ECOG PS 0–1, and measurable disease (Arm 1) or evaluable disease (Arms 2–5) per RECIST version 1.1. Participants in the combination arms are required to have received prior treatment with ≥1 CDK4/6 inhibitor and ≥1 anti-estrogen therapy. Prior treatment with a PI3Kα inhibitor is an exclusion criterion for the dose expansion parts of all arms. Planned overall sample size is ~220 pts (~145 pts for the dose escalation, and ~15 pts in each dose expansion arm). Primary endpoints are the MTD, RP2D and overall safety profile of RLY-5836, either as a single agent or in combination. The MTD will be determined as the dose with a dose-limiting toxicity rate closest to the target toxicity rate (30%) in Cycle 1. Secondary endpoints include PK and efficacy parameters. Statistical analyses will be descriptive. This study (NCT05759949) is enrolling in the United States. For further information, please contact clinicaltrials@relaytx.com. Funding source: This study is funded by Relay Therapeutics, Cambridge, MA, United States. Citation Format: Andreas Varkaris, Steven Isakoff, Cesar A. Perez, Bert O'Neil, Erika Hamilton, Erin Conlin, Gege Tan, Xiaoyan Li, Alison Timm, Ramin Samadani, Erika Puente-Poushnejad, Eunice Kwak, Brenton G. Mar, Alison M. Schram. A first-in-human study of the highly selective PI3Kα inhibitor RLY-5836 in patients with advanced breast cancer and other solid tumors [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 PO3-20-04.

Abstract PO3-20-03: First-in-human global study of RLY-2608, a pan-mutant and isoform selective PI3Kα inhibitor, as a single agent in patients with advanced solid tumors and in combination with fulvestrant in patients with advanced breast cancer

Abstract Background: Oncogenic mutations in PI3Kα are common driver events in ER+ breast cancer and other solid tumors. Alpelisib, a non-mutant selective PI3Kα inhibitor, is effective in ER+ breast cancer thus validating PI3Kα as a therapeutic target. Inhibition of wild-type PI3Kα results in toxicity including hyperglycemia which limits tolerability and drug exposure. RLY-2608, a novel, oral allosteric PI3Kα inhibitor, is uniquely designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition for greater target coverage, improved tolerability, and antitumor activity. Initiated in December 2021, ReDiscover is the ongoing, first-in-human study to define the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor activity of RLY-2608 monotherapy in PIK3CA-mutant advanced solid tumor patients (pts) and of RLY-2608 plus fulvestrant in pts with PIK3CA-mutant, HR+HER2-BC. At AACR 2023, dose escalation data for RLY-2608 was reported and demonstrated encouraging target inhibition and anti-tumor activity (including partial response), across PIK3CA genotypes with minimal impact on glucose homeostasis. To date, pts have received RLY-2608 at doses of 100–2000 mg/day, with no reported dose-limiting toxicities; the MTD has not been reached. These proof-of-mechanism data indicate that RLY-2608 is the first allosteric, pan-mutant selective PI3Kα inhibitor and that RLY-2608 has broad therapeutic potential in PIK3CA-driven cancers. Methods: Enrollment is ongoing in this global, multi-center, dose escalation/expansion study of RLY-2608 as a single agent in adults who have advanced solid tumors and are refractory, intolerant, or have declined standard therapy; and RLY-2608 in combination with fulvestrant in previously treated pts with HR+/HER2– MBC. Eligibility criteria include presence of PIK3CA mutation (blood or tumor) per local assessment, ECOG performance status 0–1, evaluable disease per RECIST 1.1 and no prior PI3K inhibitor (except in expansion combination group 2). RLY-2608 is administered on a continuous schedule with 4-week cycles. Adverse events (AEs) per CTCAE v5, PK, biomarkers (pAKT, mutant ctDNAs and insulin pathway markers) and anti-tumor activity are assessed serially. Dose escalation employs a Bayesian Optimal Interval design to identify the MTD and/or the recommended Phase 2 dose (RP2D). Following dose escalation, expansion cohorts will enroll patients with select PIK3CA-mutated solid tumors for treatment with RLY-2608 monotherapy and patients with HR+/HER2– MBC for treatment with RLY-2608 and fulvestrant. The primary endpoints are MTD/RP2D and AE profile for single agent and combination; key secondary endpoints are PIK3CA genotype in blood and tumor, PK, biomarkers, and overall response rate. ReDiscover (NCT05216432) is enrolling worldwide. For further information, contact clinicaltrials@relaytx.com. Funding source: This study is funded by Relay Therapeutics, Cambridge, MA, United States. Citation Format: Andreas Varkaris, Komal Jhaveri, Elena Garralda Cabanas, Lucia Sanz, Janice Kim, Cesar A. Perez, Erika Hamilton, Alexander I. Spira, Kari Wisinski, Angel Guerrero, Jason Henry, Jordi Rodon Ahnert, Gege Tan, Xiaoyan Li, Diana Hummel, Tamieka Hunter, Alison Timm, Ramin Samadani, Erika Puente-Poushnejad, Eunice Kwak, Brenton G. Mar, Alison M. Schram. First-in-human global study of RLY-2608, a pan-mutant and isoform selective PI3Kα inhibitor, as a single agent in patients with advanced solid tumors and in combination with fulvestrant in patients with advanced 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 PO3-20-03.