Farnesyltransferase inhibitor lonafarnib suppresses respiratory syncytial virus infection by blocking conformational change of fusion glycoprotein.

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.

Targeting NRAS via miR-1304-5p or farnesyltransferase inhibition confers sensitivity to ALK inhibitors in ALK-mutant neuroblastoma.

Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.

Genetic and pharmacological modulation of lamin A farnesylation determines its function and turnover.

Hutchinson-Gilford Progeria syndrome (HGPS) is a severe premature ageing disorder caused by a 50 amino acid truncated (Δ50AA) and permanently farnesylated lamin A (LA) mutant called progerin. On a cellular level, progerin expression leads to heterochromatin loss, impaired nucleocytoplasmic transport, telomeric DNA damage and a permanent growth arrest called cellular senescence. Although the genetic basis for HGPS has been elucidated 20 years ago, the question whether the Δ50AA or the permanent farnesylation causes cellular defects has not been addressed. Moreover, we currently lack mechanistic insight into how the only FDA-approved progeria drug Lonafarnib, a farnesyltransferase inhibitor (FTI), ameliorates HGPS phenotypes. By expressing a variety of LA mutants using a doxycycline-inducible system, and in conjunction with FTI, we demonstrate that the permanent farnesylation, and not the Δ50AA, is solely responsible for progerin-induced cellular defects, as well as its rapid accumulation and slow clearance. Importantly, FTI does not affect clearance of progerin post-farnesylation and we demonstrate that early, but not late FTI treatment prevents HGPS phenotypes. Collectively, our study unravels the precise contributions of progerin's permanent farnesylation to its turnover and HGPS cellular phenotypes, and how FTI treatment ameliorates these. These findings are applicable to other diseases associated with permanently farnesylated proteins, such as adult-onset autosomal dominant leukodystrophy.

Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Continuous 72-h infusion of zosuquidar with chemotherapy in patients with newly diagnosed acute myeloid leukemia stratified for leukemic blast P-glycoprotein phenotype.

PURPOSE: To evaluate the safety, tolerability, pharmacodynamics (PD), and potential efficacy of zosuquidar (Zos) in combination with daunorubicin and cytarabine in elderly patients with newly diagnosed acute myeloid leukemia (AML). METHODS: Patients with AML (N = 106) were treated with Zos as a 72-h continuous intravenous (CIV) infusion along with chemotherapy. Leukemic blasts from the patients were assessed for P-glycoprotein (P-gp) function using ex vivo bioassays for screening and PD analyses. Patient outcomes were categorized according to primary (N = 56) and secondary (N = 50) AML cohorts (pAML and sAML, respectively) and stratified into P-gp-high and P-gp-low subgroups. RESULTS: Patients with P-gp-high blasts exhibited comparable overall remission rates (ORR) to those with P-gp-low blasts in both the pAML and sAML cohorts. The P-gp-high and P-gp-low subgroups in the pAML cohort exhibited similar overall survival (OS). Patients with sAML and P-gp-high blasts exhibited significantly better OS than those in the P-gp-low subgroup. PD analyses revealed that Zos infusion provided 82 h of uninterrupted effective ≥ 90% inhibition of P-gp functional activity in leukemic blasts. CONCLUSIONS: These observations provide evidence of Zos efficacy with the 72-h CIV infusion approach. The similarity of ORR in the P-gp-high and P-gp-low subgroups is consistent with Zos-mediated neutralization of P-gp as verified by PD analyses. The bioassay identified sAML patients most likely to respond favorably to Zos co-therapy indicating feasibility as a Zos companion diagnostic. A follow-up placebo-controlled trial is needed to verify these promising results. GOV IDENTIFIER: NCT00129168; First posted on August 11, 2005.

Effect of single-administration of D-sorbitol pretreatment on the bitterness and continued willingness to take asenapine: a randomized, single-blind, placebo-controlled, crossover trial.

BACKGROUND: Asenapine has unique orally-related side effects, such as a bitter taste induced by sublingual administration, which often results in discontinuation of the medication. While the FDA has approved black-cherry-flavored asenapine, several countries have prescribed only unflavored versions. Specifically, Asians commonly report experiencing the bitterness of asenapine because they are more sensitive to bitter tastes than other ethnic groups. In this study, with the aim of improving adherence by reducing the bitterness of asenapine, we investigated the effects of D-sorbitol, which reduced the bitterness parameters of taste sensors in our previous basic study on the bitterness and continuity of asenapine among patients with schizophrenia. METHODS: Twenty adult patients with schizophrenia were included in this single-blind, placebo-controlled, crossover trial. Participants rinsed their mouths with single-administration of D-sorbitol or a placebo prior to each administration of asenapine. We then conducted the questionnaires and assessed changes in the bitterness of asenapine (primary end point) and willingness to continue its use (secondary end point). RESULTS: D-sorbitol significantly improved the bitterness of asenapine (p = 0.038). Although it did not significantly increase the willingness to continue asenapine (p = 0.180), it did show improvement over the placebo in enhancing willingness to continue, especially in patients who were not accustomed to its taste. CONCLUSION: Our findings indicate that single-administration of D-sorbitol significantly reduces the bitterness of asenapine. In countries where flavored asenapine is not available, this finding could benefit patients who were not accustomed to its bitter taste. TRIAL REGISTRATION: This study was registered in the Japan Registry of Clinical Trials (jRCTs041210019) on May 14, 2021.

A systematic review and network meta-analysis on comparative efficacy, acceptability, and safety of treatments in acute bipolar mania in youths.

BACKGROUND: The evidence of treatment options' efficacy on acute bipolar manic episodes is relatively less in youths than adults. We aimed to compare and rank the drug's efficacy, acceptability, tolerability, and safety for acute mania in children and adolescents. METHOD: We systematically reviewed the double-blinded, randomized controlled trials (RCTs) comparing drugs or placebo for acute manic episodes of bipolar disorder in children and adolescents using PRISMA guidelines. We searched PubMed/MEDLINE, EMBASE, Web of Science, EBSCO, Scopus, the Cochrane Central Register of Controlled Trials, and https://clinicaltrials.gov from inception until November 20, 2022. Response to treatment was the primary outcome, and random-effects network meta-analyses were conducted (PROSPERO 2022: CRD42022367455). RESULTS: Of 10,134 citations, we included 15 RCTs, including 2372 patients (47 % female), 15 psychotropic drugs, and the placebo. Risperidone 0.5-2.5 mg/day, aripiprazole 30 mg/day olanzapine, quetiapine 400 mg/day, quetiapine 600 mg/day, asenapine 5 mg/day, asenapine 10 mg, ziprasidone, and aripiprazole 10 mg were found to be effective (in comparison with placebo) in children and adolescents, respectively (τ2 = 0.0072, I2 = 10.2 %). The tolerability of aripiprazole 30 mg/day was lower than risperidone 0.5-2.5 mg/day and olanzapine. Oxcarbazepine had the highest discontinuation due to the adverse effects risk ratio. LIMITATIONS: Efficacy ranking of the treatments could be performed by evaluating relatively few RCT results, and only monotherapies were considered. CONCLUSIONS: Efficacy, acceptability, tolerability, and safety are changing with the doses of antipsychotics for children and adolescents with acute bipolar manic episodes. Drug selection and optimum dosage should be carefully adjusted in children and adolescents.

Asenapine versus olanzapine for the treatment of nausea and vomiting in patients with cancer: A retrospective study.

AIM: Patients with cancer often experience nausea and vomiting (N/V), but may have difficulty using olanzapine (OLZ), a common antiemetic. Asenapine (ASE) is a multi-acting receptor-targeted antipsychotic like OLZ, although there is little evidence that ASE serves as an antiemetic. The aim of this study was to evaluate the efficacy and tolerability of ASE compared to those of OLZ for the treatment of N/V in patients with cancer. METHODS: This retrospective study involved patients who received 5 mg ASE, 5 mg OLZ, or 2.5 mg OLZ for 2 days. Daily worst N/V was rated on a scale of 0 (none) to 3 (very much). The primary endpoint was the proportion of patients who had a response, defined as any reduction in N/V score. A complete response (CR) was defined as a score reduction to 0. Secondary endpoints included the proportion of patients with CR and adverse events. RESULTS: Between April 2017 and March 2023, 212 patients were enrolled to receive treatment: 5 mg ASE (n = 34), 5 mg OLZ (n = 102), or 2.5 mg OLZ (n = 76). No significant differences in response rates (52.9% vs. 58.8% vs. 52.6%, p = 0.671) or secondary endpoints were observed between the groups. Patients receiving ASE were more likely to experience oral hypoesthesia (p = 0.004). CONCLUSION: This preliminary study suggests that ASE may be effective for N/V. Further studies are required to confirm these findings.

Optimal dose for the efficacy of asenapine in patients with schizophrenia: Real-world data.

AIMS: A meta-analysis of short-term studies revealed no significant differences between the doses of asenapine, 10 and 20 mg/day, in the acute treatment of schizophrenia. However, it should be noted that many patients from clinical practice were excluded, and the dose-response to asenapine in a real-world setting is still unclear. Additionally, the dose-response in the maintenance phase is not clear. This study aimed to evaluate the differences in the efficacy of different asenapine doses in patients with maintenance phase of schizophrenia in a real-world setting. METHODS: This study conducted post-marketing surveillance of asenapine in clinical settings in Japan. It followed patients diagnosed with schizophrenia who received asenapine for the first time for a maximum of 52 weeks. These patients were divided into two categories based on their average daily asenapine dosage: ≤10 mg/day and >10 mg/day. Asenapine efficacy was assessed by adjusting for patient demographics using multivariate logistic regression analysis, employing the Clinical Global Impression-Global Improvement (CGI-I) scale, which has seven categories. RESULTS: A total of 2774 patients were included in the analysis. Of these, 1689 and 1085 patients were treated with asenapine ≤10 mg/day and >10 mg/day, respectively. The CGI-I improvement rate was significantly higher in the asenapine >10 group (p = 0.012) after adjusting for patient background factors. CONCLUSION: These results suggest that asenapine doses >10 mg/day may be more effective than 10 mg/day in the treatment of schizophrenia; however, further studies are needed to confirm these findings.

Therapeutic potential for P2Y2 receptor antagonism.

G protein-coupled receptors are the target of more than 30% of all FDA-approved drug therapies. Though the purinergic P2 receptors have been an attractive target for therapeutic intervention with successes such as the P2Y12 receptor antagonist, clopidogrel, P2Y2 receptor (P2Y2R) antagonism remains relatively unexplored as a therapeutic strategy. Due to a lack of selective antagonists to modify P2Y2R activity, studies using primarily genetic manipulation have revealed roles for P2Y2R in a multitude of diseases. These include inflammatory and autoimmune diseases, fibrotic diseases, renal diseases, cancer, and pathogenic infections. With the advent of AR-C118925, a selective and potent P2Y2R antagonist that became commercially available only a few years ago, new opportunities exist to gain a more robust understanding of P2Y2R function and assess therapeutic effects of P2Y2R antagonism. This review discusses the characteristics of P2Y2R that make it unique among P2 receptors, namely its involvement in five distinct signaling pathways including canonical Gαq protein signaling. We also discuss the effects of other P2Y2R antagonists and the pivotal development of AR-C118925. The remainder of this review concerns the mounting evidence implicating P2Y2Rs in disease pathogenesis, focusing on those studies that have evaluated AR-C118925 in pre-clinical disease models.

Amurensin G Sensitized Cholangiocarcinoma to the Anti-Cancer Effect of Gemcitabine via the Downregulation of Cancer Stem-like Properties.

Cholangiocarcinoma (CCA) is a malignant biliary tract tumor with a high mortality rate and refractoriness to chemotherapy. Gemcitabine is an anti-cancer chemotherapeutic agent used for CCA, but the efficacy of gemcitabine in CCA treatment is limited, due to the acquisition of chemoresistance. The present study evaluated the chemosensitizing effects of Amurensin G (AMG), a natural sirtuin-1 inhibitor derived from Vitis amurensis, in the SNU-478 CCA cells. Treatment with AMG decreased the SNU-478 cell viability and the colony formation ability. Annexin V/ Propidium iodide staining showed that the AMG increased apoptotic death. In addition, AMG downregulated anti-apoptotic Bcl-2 expression, while upregulating pro-apoptotic cleaved caspase-3 expression. Treatment with AMG decreased the migratory ability of the cells in a wound healing assay and transwell migration assay. It was observed that AMG decreased the gemcitabine-induced increase in CD44highCD24highCD133high cell populations, and the expression of the Sox-2 protein was decreased by AMG treatment. Co-treatment of AMG with gemcitabine significantly enhanced the production of reactive oxygen species, as observed through mitochondrial superoxide staining, which might be associated with the downregulation of the Sirt1/Nrf2 pathway by AMG. These results indicate that AMG enhances the chemotherapeutic ability of gemcitabine by downregulating cancer stem-like properties in CCA cells. Hence, a combination therapy of AMG with gemcitabine may be an attractive therapeutic strategy for cholangiocarcinoma.

A cumulative Bayesian network meta-analysis on the comparative efficacy of pharmacotherapies for mania over the last 40 years.

RATIONALE: Mania (or manic episodes) is a common symptom of bipolar disorder and is frequently accompanied by hyperactivity and delusions; given the cost and resources available, there is a paucity of evidence for direct comparison of different drugs. OBJECTIVES: We aimed to provide evidence-based recommendations on the efficacy of overall currently used pharmacological treatments for patients with acute bipolar mania. METHOD: We conducted a systematic review and network meta-analysis (NMA) using a Bayesian network frame. We searched the primary literature databases without language restrictions until Dec 18, 2021, for reports of randomized controlled trials (RCTs) of suspected antimanic drugs used as monotherapy for patients with acute bipolar mania, with the primary outcomes being efficacy (mean difference (MD), standardized mean difference (SMD) in the change of mania score). RESULTS: Eighty-seven studies were included in which 18,724 manic participants (mean age = 34.6 years, with 50.36% males) were allocated at random to one of 25 active medication drug therapies or placebo, resulting in 87 direct comparisons on 192 data points. Tamoxifen (- 22·00 [- 26·00 to - 18·00]) had the best efficacy over the placebo. Meanwhile, risperidone (- 6·60 [- 8·40 to - 4·90]) was substantially more effective than placebo in treating acute mania. Carbamazepine, haloperidol, ziprasidone, cariprazine, olanzapine, quetiapine, aripiprazole, lithium, paliperidone, asenapine, and divalproex were noticeably more effective than placebo. CONCLUSIONS: Overall, tamoxifen appears to be the most effective of the currently known pharmaceutical therapy available to treat acute mania or manic episodes; however, this conclusion is restricted by the scale of RCTs conducted, and risperidone was found to be the most effective medication among antipsychotics. Carbamazepine, haloperidol, ziprasidone, cariprazine, olanzapine, quetiapine, aripiprazole, lithium, paliperidone, asenapine, and divalproex were noticeably effective in treating acute mania or manic episodes.

A Meta-Analysis of Antipsychotic-Induced Hypo- and Hyperprolactinemia in Children and Adolescents.

Objective: Antipsychotic-related prolactin changes may expose children and adolescents to severe adverse reactions (ARs) related to pubertal development and growth. We therefore aimed to assess the effects of antipsychotics on prolactin levels and associated somatic ARs in children and adolescents. Methods: We systematically searched PubMed and CENTRAL for placebo-controlled randomized trials of antipsychotics in children and adolescents aged ≤18 years, reporting prolactin levels and related ARs. We conducted a random-effect meta-analysis and assessed risk of bias version 2 (ROB2). Results: Thirty-two randomized controlled trials with an average trial duration of 6 weeks, covering 4643 participants with an average age of 13 years and a male majority of 65.3%. Risk of bias across domains was low or unclear. The following antipsychotic compounds: aripiprazole (n = 810), asenapine (n = 506), lurasidone (n = 314), olanzapine (n = 179), paliperidone (n = 149), quetiapine (n = 381), risperidone (n = 609), and ziprasidone (n = 16) were compared with placebo (n = 1658). Compared with placebo, statistically significant higher prolactin increase occurred with risperidone (mean difference [MD] = 28.24 ng/mL), paliperidone (20.98 ng/mL), and olanzapine (11.34 ng/mL). Aripiprazole significantly decreased prolactin (MD = -4.91 ng/mL), whereas quetiapine, lurasidone, and asenapine were not associated with significantly different prolactin levels than placebo. Our results on ziprasidone are based on a single study, making it insufficient to draw strong conclusions. On average, 20.8% of patients treated with antipsychotic developed levels of prolactin that were too high (hyperprolactinemia), whereas only 1.03% of patients reported prolactin-related ARs. Data were highly limited for long-term effects. Conclusions: In children and adolescents, risperidone, paliperidone, and olanzapine are associated with significant prolactin increase, whereas aripiprazole is associated with significant decrease. Despite the significant changes in prolactin level, few ARs were reported. Study protocol on PROSPERO: CRD42018116451.

Asenapine, Aggression, and Affinity.

22lr14564.

Simultaneous spectrophotometric determination of fluphenazine HCl and nortriptyline HCl in presence of their potential impurities perphenazine and dibenzosuberone in bulk and pharmaceutical formulation.

Fluphenazine HCl (FLU) is an anxiolytic, while Nortriptyline HCl (NOR) is an anti-depressant. They are co-formulated together to treat depression and schizophrenia. Perphenazine (PER) and dibenzosuberone (DBZ) are the pharmacopeial impurities of FLU and NOR, respectively. Four spectrophotometric and multivariate chemometric methods were developed to determine the two drugs together or in presence of their two impurities in their bulk and pharmaceutical formulation. Method (A) is the triple divisor-ratio derivative (TDR) method, where the zero order spectrum of each component was divided by a mixture of the other 3 components, then the peak amplitudes of the first derivative spectra of FLU, NOR and DBZ were measured at 265, 245.4 and 283.2 nm, respectively. Method (B) is the double divisor-ratio difference-dual wavelength (DD-RD-DW) method, in which each component spectrum mixture was divided by a binary mixture of 2 of the interfering components. In the resulting ratio spectra, the amplitude difference is calculated between 2 wavelengths at which the third interfering component has zero difference. Methods (C and D) are the principle component analysis (PCA) and partial least squares (PLS) models. Methods (A and B) failed to quantify PER (FLU impurity), while (C and D) succeeded to quantify all components. The four methods have been applied for the prediction of the FLU and NOR in their pharmaceutical formulation with good accuracy and precision. The proposed methods have been validated according to the ICH guidelines and the results were within the acceptable limits.

Efficacy of HP-3070, an Asenapine Transdermal System, on Symptoms of Hostility in Adults With Schizophrenia: A Post Hoc Analysis of a 6-Week Phase 3 Study.

Objective: Patients with schizophrenia may exhibit symptoms of hostility. HP-3070 is the first antipsychotic patch approved by the US Food and Drug Administration (FDA) for adults with schizophrenia. Its efficacy was demonstrated in a phase 3 study. This post hoc analysis assessed the efficacy of HP-3070 in treating hostility in schizophrenia.Methods: In the pivotal phase 3 study, conducted between August 2016 and November 2017, adults with schizophrenia (per DSM-5 criteria) were randomized to HP-3070 3.8 mg/24 h, HP-3070 7.6 mg/24 h, or placebo. Least-squares mean (LSM) changes in Positive and Negative Syndrome Scale (PANSS) hostility item and PANSS-Excited Component (PANSS-EC) scores from baseline to week 6 were assessed post hoc using a mixed-effects model for repeated measures adjusted for selected PANSS-Positive symptoms and presence of somnolence or akathisia.Results: Among 442 patients with baseline PANSS hostility item score > 1 (n = 151, HP-3070 7.6 mg/24 h; n = 147, 3.8 mg/24 h; n = 144, placebo), week 6 LSM (95% CI) change from baseline (CFB) in hostility score was superior with HP-3070 versus placebo for 7.6 mg/24 h (-0.4 [-0.6 to -0.2]; P < .001) and 3.8 mg/24 h (-0.3 [-0.6 to -0.1]; P < .01), with similar results for 7.6 mg/24 h after adjusting for covariates (P < .05). For all patients regardless of baseline PANSS hostility item score, PANSS-EC week 6 LSM CFB was greater for HP-3070 7.6 mg/24 h (-1.1 [-1.9 to -0.4]; n = 203; P < .01) and 3.8 mg/24 h (-1.3 [-2.0 to -0.6]; n = 201; P < .001) than for placebo (n = 203), with similar results observed in patients with baseline hostility item score > 1.Conclusions: In this post hoc analysis, HP-3070 was superior to placebo in reducing schizophrenia-associated hostility, even after adjusting for covariates, suggesting these effects are at least partially independent of general antipsychotic effects or effects on sedation or akathisia. These findings suggest HP-3070 has a specific antihostility effect in patients with schizophrenia.Clinical Trials Registration: ClinicalTrials.gov identifier: NCT02876900; EudraCT number: 2015-005134-21.

The impact of temozolomide and lonafarnib on the stemness marker expression of glioblastoma cells in multicellular spheroids.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a poor prognosis. The GBM microenvironment is highly heterogeneous and is composed of many cell types including astrocytes and endothelial cells (ECs) along with tumor cells, which are responsible for heightened resistance to standard chemotherapeutic drugs such as Temozolomide (TMZ). Here, we investigated how drug treatments impact stemness marker expression of GBM cells in multicellular tumor spheroid (MCTS) models. Co- and tri-culture MCTS constructed using U87-MG GBM cells, astrocytes, and/or ECs were cultured for 7 days. At Day 7, 5 µM lonafarnib (LNF), 100 µM TMZ, or combination of 5 µM LNF + 100 µM TMZ was added and the MCTS were cultured for an additional 48 h. We assessed the spheroid sizes and expression of stemness markers- NESTIN, SOX2, CD133, NANOG, and OCT4- through qRT-PCR and immunostaining. Following 48 h treatment with LNF, TMZ or their combination (LNF + TMZ), the spheroid sizes decreased compared to the untreated control. We also observed that the expression of most of the stemness markers significantly increased in the LNF + TMZ treated condition as compared to the untreated condition. These results indicate that drug treatment can influence the stemness marker expression of GBM cells in MCTS models and these aspects must be considered while evaluating therapies. In future, by incorporating other relevant cell types, we can further our understanding of their crosstalk, eventually leading to the development of new therapeutic strategies.

Determination of Asenapine Maleate in Pharmaceutical and Biological Matrices: A Critical Review of Analytical Techniques over the Past Decade.

Asenapine maleate is a second-generation atypical antipsychotic agent used in the treatment of schizophrenia, a neuropsychiatric disorder. It is available as a fast-dissolving sublingual tablet to avoid extensive first-pass metabolism with higher bioavailability as compared to oral formulations. Although, the established therapeutic solutions do not sufficiently satisfy the patient's safety and efficacy needs. Thus, the core research emphasis is to investigate strategies to produce novel formulations with enhanced safety and efficacy. This necessitates the development of robust, precise, and accurate methods for quantification of asenapine maleate in different sample matrices. Given the foregoing information, the current analysis concentrates on the different analytical techniques used to assess asenapine maleate in bulk, pharmaceutical formulations, and biological specimens. Reverse-phase HPLC coupled with UV detection is a majorly (nearly 50% of papers investigated) used technique for the estimation of asenapine maleate in formulations. On the other hand, for its quantification in the biological matrix, hyphenated techniques using mass spectrometry are widely used. This critical review reveals different analytical methodologies, including spectrophotometric, chromatographic, capillary electrophoresis techniques reported from 2011 to 2020, for the measurement of asenapine maleate in various sample matrices. The information presented in this review would be useful in future research for robust analytical method development for asenapine maleate utilizing a more scientific and risk-based approach. Also, it would aid to minimize analytical failure as well as method fine-tuning throughout the product life cycle. Further, this review may also direct scientists toward the development of methodologies for green research.