Atezolizumab, vemurafenib, and cobimetinib in patients with melanoma with CNS metastases (TRICOTEL): a multicentre, open-label, single-arm, phase 2 study.

BACKGROUND: Targeted therapy and immunotherapy have shown intracranial activity in melanoma with CNS metastases, but there remains an unmet need, particularly for patients with symptomatic CNS metastases. We aimed to evaluate atezolizumab in combination with cobimetinib or vemurafenib plus cobimetinib in patients with melanoma with CNS metastases. METHODS: TRICOTEL was a multicentre, open-label, single-arm, phase 2 study done in two cohorts: a BRAFV600 wild-type cohort and a BRAFV600 mutation-positive cohort, recruited at 21 hospitals and oncology centres in Brazil, France, Germany, Hungary, Italy, Spain, and Switzerland. Eligible patients were aged 18 years or older with previously untreated metastatic melanoma, CNS metastases of 5 mm or larger in at least one dimension, and an Eastern Cooperative Oncology Group performance status of 2 or less. Patients in the BRAFV600 wild-type cohort received intravenous atezolizumab (840 mg, days 1 and 15 of each 28-day cycle) plus oral cobimetinib (60 mg once daily, days 1-21). Patients in the BRAFV600 mutation-positive cohort received intravenous atezolizumab (840 mg, days 1 and 15 of each 28-day cycle) plus oral vemurafenib (720 mg twice daily) plus oral cobimetinib (60 mg once daily, days 1-21); atezolizumab was withheld in cycle 1. Treatment was continued until progression, toxicity, or death. The primary outcome was intracranial objective response rate confirmed by assessments at least 4 weeks apart, as assessed by independent review committee (IRC) using modified Response Evaluation Criteria in Solid Tumours version 1.1. Because of early closure of the BRAFV600 wild-type cohort, the primary endpoint of intracranial objective response rate by IRC assessment was not done in this cohort; intracranial objective response rate by investigator assessment was reported instead. Efficacy and safety were analysed in all patients who received at least one dose of study medication. This trial is closed to enrolment and is registered with ClinicalTrials.gov, NCT03625141. FINDINGS: Between Dec 13, 2018, and Dec 7, 2020, 65 patients were enrolled in the BRAFV600 mutation-positive cohort; the BRAFV600 wild-type cohort was closed early after enrolment of 15 patients. Median follow-up was 9·7 months (IQR 6·3-15·0) for the BRAFV600 mutation-positive cohort and 6·2 months (3·5-23·0) for the BRAFV600 wild-type cohort. Intracranial objective response rate was 42% (95% CI 29-54) by IRC assessment in the BRAFV600 mutation-positive cohort and 27% (95% CI 8-55) by investigator assessment in the BRAFV600 wild-type cohort. Treatment-related grade 3 or worse adverse events occurred in 41 (68%) of 60 patients who received atezolizumab plus vemurafenib plus cobimetinib in the BRAFV600 mutation-positive cohort, the most common of which were lipase increased (15 [25%] of 60 patients) and blood creatine phosphokinase increased (ten [17%]). Eight (53%) of 15 patients treated with atezolizumab plus cobimetinib in the BRAFV600 wild-type cohort had treatment-related grade 3 or worse adverse events, most commonly anaemia (two [13%]) and dermatitis acneiform (two [13%]). Treatment-related serious adverse events occurred in 14 (23%) of 60 patients in the BRAFV600 mutation-positive cohort and two (13%) of 15 in the BRAFV600 wild-type cohort. One death in the BRAFV600 mutation-positive cohort (limbic encephalitis) was considered to be related to atezolizumab treatment. INTERPRETATION: Adding atezolizumab to vemurafenib plus cobimetinib provided promising intracranial activity in patients with BRAFV600-mutated melanoma with CNS metastases. FUNDING: F Hoffmann-La Roche.

Real-world treatment patterns and overall survival in BRAF-mutant melanoma patients treated with immunotherapy or targeted therapy.

Aim: To assess overall survival (OS) in patients with advanced BRAF-mutant melanoma by first-line (1L) targeted therapy (TT) or checkpoint inhibitor (CPI) use, second-line (2L) TT or CPI use, and treatment sequence. Patients & methods: Advanced BRAF-mutant melanoma patients treated with 1L CPI or TT were selected from a real-world, electronic health record-derived database. Results: CPI was associated with improved survival after adjustment for potential confounders (hazard ratio, 0.75 [95% CI, 0.66-0.87]). Median OS was similar between 2L therapies and among likely treatment sequences. Conclusion: This real-world study demonstrated a survival benefit with 1L CPI versus TT. Analyses of 2L and treatment sequences were unable to detect or rule out clinically relevant differences in OS.

Immune checkpoint inhibitors and targeted therapies are the preferred treatment options for patients with advanced BRAF-mutant melanoma, with more patients starting first-line treatment with checkpoint inhibitors in the real world. Our study suggests that starting treatment with checkpoint inhibitors instead of targeted therapies may improve survival; however, we were unable to determine the optimal sequence of treatments that patients should be given. The findings of this study highlight the need for further investigation into the optimal treatment sequence with checkpoint inhibitors and targeted therapies in advanced BRAF-mutant melanoma.

Influence of androgen deprivation therapy on the severity of COVID-19 in prostate cancer patients.

BACKGROUND: The TMPRSS2 protein has been involved in severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2). The production is regulated by the androgen receptor (AR). It is speculated that androgen deprivation therapy (ADT) may protect patients affected by prostate cancer (PC) from SARS-CoV-2 infection. METHODS: This is a retrospective study of patients treated for COVID-19 in our institution who had a previous diagnosis of PC. We analyzed the influence of exposure of ADT on the presence of severe course of COVID-19. RESULTS: A total of 2280 patients were treated in our center for COVID-19 with a worse course of disease in males (higher rates of hospitalization, intense care unit [ICU] admission, and death). Out of 1349 subjects registered in our PC database, 156 were on ADT and 1193 were not. Out of those, 61 (4.52%) PC patients suffered from COVID-19, 11 (18.0%) belonged to the ADT group, and 50 (82.0%) to the non-ADT group. Regarding the influence of ADT on the course of the disease, statistically significant differences were found neither in the death rate (27.3% vs. 34%; p = 0.481), nor in the presence of severe COVID-19: need for intubation or ICU admission (0% vs. 6.3%; p = 0.561) and need for corticoid treatment, interferon beta, or tocilizumab (60% vs. 34.7%; p = 0.128). Multivariate analysis adjusted for clinically relevant comorbidities did not find that ADT was a protective factor for worse clinical evolution (risk ratio [RR] 1.08; 95% confidence interval [CI], 0.64-1.83; p = 0.77) or death (RR, 0.67; 95% CI, 0.26-1.74; p = 0.41). CONCLUSIONS: Our study confirms that COVID-19 is more severe in men. However, the use of ADT in patients with PC was not shown to prevent the risk of severe COVID-19.

Heregulin-induced cell migration is prevented by trastuzumab and trastuzumab-emtansine in HER2+ breast cancer.

PURPOSE: Heregulin (HRG) signaling has been implicated in the development of an aggressive phenotype in breast cancer (BC) cells, and HER2 overexpression has been associated with a worse prognosis in BC patients. Nevertheless, the molecular mechanisms through which HRG affects the efficiency of anti-HER2 therapies such as trastuzumab (Tz) and trastuzumab-emtansine (T-DM1) are currently unknown. METHODS: In the present study, we evaluate the molecular action of HRG toward fundamental scaffold proteins and several kinases in the signal transduction pathways triggered via HER2/HER3, which integrate precise and sequential steps to promote changes in cell morphology to impulse BC cell migration. In addition, we evaluate the effectiveness of Tz and T-DM1 on the control of key proteins involved in BC cell motility, since the acquisition of a migratory phenotype is essential to promote invasion and metastasis. RESULTS: We show that HRG induces actin cytoskeleton reorganization and focal adhesion complex formation, and promotes actin nucleation in BT-474 BC cells. This signaling is triggered by HER2/HER3 to c-Src, FAK and paxillin. When paxillin is phosphorylated, it recruits PAK1, which then phosphorylates cortactin. In parallel, paxillin signals to N-WASP, and both signalings regulate Arp2/3 complex, leading to the local reorganization of actin fibers. CONCLUSIONS: Our findings reveal an original mechanism by which HRG increases HER2+ BC cell motility, and show that the latter can be abolished by Tz and T-DM1 treatments. These results provide evidence for the molecular mechanisms involved in cell motility and drug resistance. They will be useful to develop new and more specific therapeutic schemes that interfere with the progression and metastasis of HER2+ BC.

Prostate cancer adverse pathology reclassification in patients undergoing active surveillance in a long-term follow-up series.

BACKGROUND: Active surveillance (AS) has become a valid option for patients with a very low risk of prostate cancer (PC) with a widespread application. There are still a few series, with a medium follow-up longer than 5 years, reporting data on pathological upgrading. The objective is to evaluate the changes in surveillance biopsies of patients with low-risk PC in a long-term follow-up and determine if a longer stay in AS could involve worse pathological findings. MATERIALS AND METHODS: A retrospective analysis of our institutional database of patients with PC undergoing AS during 2004 to 2018 was performed. The inclusion criteria were prostate-specific antigen (PSA) ≤ 10 ng/mL, Gleason grade 1 and T1c/T2a. Patients were assessed by serum PSA level and digital rectal examination at 6-month intervals. Transrectal ultrasound-guided prostate biopsies were performed during the first year of follow-up, and every 2 or 3 years thereafter. The pathology details of biopsies were analyzed and compared with the current series on AS. RESULTS: Three-hundred nineteen patients undergoing AS were evaluated with a median follow-up of 5.3 years and a mean age of 67.4 years. Sixty-three patients did not meet all the criteria to be considered low-risk PC but were included in the analysis. Overall, 128 patients (40.1%) underwent active treatment (84.7% of them due to pathological progression in surveillance biopsies). The proportion of patients with a reported upgrading ranged between 19.4% and 35.3%, although only the fourth biopsy showed an upgrading proportion of over 30%. Limitations include the retrospective design of the study and the existence of different protocols between other cohorts that make it difficult to compare their results. CONCLUSIONS: For patients who remained in surveillance the percentage of upgrading increased slightly with the time, being more frequent after the third-surveillance biopsy. These findings support the importance of extending surveillance biopsies for patients who remain candidates for curative treatment.

Effect of hospital volume on 90-day mortality after radical cystectomy for bladder cancer in Spain.

OBJECTIVE: To investigate 90-day mortality rate of RC for bladder cancer in a nationwide population-based study. DESIGN, SETTING, AND PARTICIPANTS: We used mandatory hospital discharge forms of all patients submitted to RC due to bladder cancer in Spain during 2011-2015 (n = 12,154 in 196 hospitals). At present, a centralization policy for RC has not been issued by the health authorities. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We calculated in-hospital, 30-, 60- and 90-day mortality. Average annual RC volume was used as a continuous variable (log-transformed) and also grouped into deciles to identify any potential non-linear relationships. Logistic regression model with mixed effect was performed adjusting for year of surgery, comorbidity, surgical approach, type of admission, age, sex, and hospital size. RESULTS AND LIMITATION: Overall 90-day mortality rate was 6.5%. Lowest mortality rates (3.3% at 90 days) are achieved in hospitals doing more than 38 cases per year. The 90-day adjusted mortality rate is associated with annual average RC volume with a 20.6% decrease per 10 extra RCs/year (95% CI 12.3-28.1% p < 0.001). High Charlson comorbidity index, advanced age, and open surgical approach were the clinical variables associated with higher mortality. CONCLUSIONS: Our study identifies an inverse association between 90-day mortality and hospital volume. High-volume hospitals achieve lower mortality rate within 90 days.

MYC-Associated Factor MAX is a Regulator of the Circadian Clock.

The circadian transcriptional network is based on a competition between transcriptional activator and repressor complexes regulating the rhythmic expression of clock-controlled genes. We show here that the MYC-associated factor X, MAX, plays a repressive role in this network and operates through a MYC-independent binding to E-box-containing regulatory regions within the promoters of circadian BMAL1 targets. We further show that this "clock" function of MAX is required for maintaining a proper circadian rhythm and that MAX and BMAL1 contribute to two temporally alternating transcriptional complexes on clock-regulated promoters. We also identified MAX network transcriptional repressor, MNT, as a fundamental partner of MAX-mediated circadian regulation. Collectively, our data indicate that MAX regulates clock gene expression and contributes to keeping the balance between positive and negative elements of the molecular clock machinery.

Effects of acute stress on cardiac endocannabinoids, lipogenesis, and inflammation in rats.

OBJECTIVE: Trauma exposure can precipitate acute stress (AS) and cardiovascular disorders (CVD). Identifying AS-related physiologic changes that affect CVD risk could inform development of early CVD prevention strategies. The endocannabinoid system (ECS) regulates hypothalamic-pituitary-adrenal axis and stress-related cardiovascular function. We examine stress-related ECS activity and its association with cardiovascular biochemistry/function after AS. METHODS: Rodents (n = 8-16/group) were exposed to predator odor or saline; elevated plus maze, blood pressure, serum and cardiac ECS markers, and lipid metabolism were assessed 24 hours and 2 weeks postexposure. RESULTS: At 24 hours, the predator odor group demonstrated anxiety-like behavior and had a) elevated serum markers of cardiac failure/damage (brain natriuretic peptide: 275.1 versus 234.6, p = .007; troponin I: 1.50 versus 0.78, p = .076), lipogenesis (triacylglycerols: 123.5 versus 85.93, p = .018), and inflammation (stearoyl delta-9 desaturase activity: 0.21 versus 0.07, p < .001); b) decreased cardiac 2-arachidonoyl-sn-glycerol (29.90 versus 65.95, p < .001), oleoylethanolamide (114.3 versus 125.4, p = .047), and palmitoylethanolamide (72.96 versus 82.87, p = .008); and c) increased cardiac inflammation (interleukin [IL]-1ß/IL-6 ratio: 19.79 versus 13.57, p = .038; tumor necrosis factor α/IL-6 ratio: 1.73 versus 1.03, p = .019) and oxidative stress (thiobarbituric acid reactive substances: 7.81 versus 7.05, p = .022), which were associated with cardiac steatosis (higher triacylglycerol: 1.09 versus 0.72, p < .001). Cardiac lipogenesis persisted, and elevated blood pressure emerged 2 weeks postexposure. CONCLUSIONS: Acute psychological stress elicits ECS-related cardiac responses associated with persistent, potentially pathological changes in rat cardiovascular biochemistry/function.

Advances in Diet and Physical Activity in Breast Cancer Prevention and Treatment.

There is currently a growing interest in diets and physical activity patterns that may be beneficial in preventing and treating breast cancer (BC). Mounting evidence indicates that indeed, the so-called Mediterranean diet (MedDiet) and regular physical activity likely both help reduce the risk of developing BC. For those who have already received a BC diagnosis, these interventions may decrease the risk of tumor recurrence after treatment and improve quality of life. Studies also show the potential of other dietary interventions, including fasting or modified fasting, calorie restriction, ketogenic diets, and vegan or plant-based diets, to enhance the efficacy of BC therapies. In this review article, we discuss the biological rationale for utilizing these dietary interventions and physical activity in BC prevention and treatment. We highlight published and ongoing clinical studies that have applied these lifestyle interventions to BC patients. This review offers valuable insights into the potential application of these dietary interventions and physical activity as complimentary therapies in BC management.

Drug repositioning for rosacea disease: Biological TARGET identification, molecular docking, pharmacophore mapping, and molecular dynamics analysis.

Rosacea is a chronic dermatological condition that currently lacks a clear treatment approach due to an uncomprehensive knowledge of its pathogenesis. The main obstacle lies in understanding its etiology and the mode of action of the different drugs used. This study aims to clarify these aspects by employing drug repositioning. Using an in silico approach, we performed a transcriptomic analysis comparing samples from individuals with diverse types of rosacea to those from healthy controls to identify genes deregulated in this disease. Subsequently, we realized molecular docking and molecular dynamics studies to assess the binding affinity of drugs currently used to treat rosacea and drugs that target proteins interacting with, and thus affecting, proteins deregulated in rosacea. Our findings revealed that the downregulation of SKAP2 and upregulation of S100A7A in rosacea, could be involved in the pathogenesis of the disease. Furthermore, considering the drugs currently used for rosacea management, we demonstrated stable interactions between isotretinoin and BFH772 with SKAP2, and permethrin and PAC-14028 with S100A7A. Similarly, considering drugs targeting SKAP2 and S100A7A interactome proteins, we found that pitavastatin and dasatinib exert stable interactions with SKAP2, and lovastatin and tirbanibulin with S100A7A. In addition, we determine that the types of bonds involved in the interactions were different in SKAP2 from S100A7A. The drug-SKAP2 interactions are hydrogen bonds, whereas the drug-S100A7A interactions are of the hydrophobic type. In conclusion, our study provides evidence for the possible contribution of SKAP2 and S100A7A to rosacea pathology. Furthermore, it provides significant information on the molecular interactions between drugs and these proteins, highlighting the importance of considering structural features and binding interactions in the design of targeted therapies for skin disorders such as rosacea.

Survival Differences in High-Risk Prostate Cancer by Age.

PURPOSE: Age is an established determining factor in survival in low-risk prostate cancer (PC), being this evidence weaker in high-risk tumors. Our aim is to evaluate the survival of patients with high-risk PC treated with curative intent and to identify differences across ages at diagnosis. METHODS: We did a retrospective analysis of patients with high-risk PC treated with surgery (RP) or radiotherapy (RDT) excluding N+ patients. We divided patients by age groups: < 60, 60-70, and > 70 years. We performed a comparative survival analysis. A multivariate analysis adjusted for clinically relevant variables and initial treatment received was performed. RESULTS: Of a total of 2383 patients, 378 met the selection criteria with a median follow-up of 8.9 years: 38 (10.1%) < 60 years, 175 (46.3%) between 60-70 years, and 165 (43.6%) >70 years. Initial treatment with surgery was predominant in the younger group (RP:63.2%, RDT:36.8%), and with radiotherapy in the older group (RP:17%, RDT:83%) (p = 0.001). In the survival analysis, significant differences were observed in overall survival, with better results for the younger group. However, these results were reversed in biochemical recurrence-free survival, with patients < 60 years presenting a higher rate of biochemical recurrence at 10 years. In the multivariate analysis, age behaved as an independent risk variable only for overall survival, with a HR of 2.8 in the group >70 years (95%CI: 1.22-6.5; p = 0.015). CONCLUSION: In our series, age appeared to be an independent prognostic factor for overall survival, with no differences in the rest of the survival rates.

Potential Biomarkers Associated with Prognosis and Trastuzumab Response in HER2+ Breast Cancer.

Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody-drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients' data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies.

Evaluation of lead exposition length on the liver and intestinal histoarchitecture of eared dove (Zenaida auriculata).

Lead (Pb) is a metal that can produces irreversible damage in living organisms. Some studies had reported that Pb produces histophysiological alterations in the digestive system (mainly liver) of birds; however, the effect of this metal on small intestine has not been fully examined. Additionally, little information is available on Pb disturbances in native birds of South America. The present study aimed to evaluate the effect of different Pb exposure times on blood δ-aminolevulinic acid dehydratase (δ-ALAD) activity and on the histological and morphometric characteristics of the digestive system (liver and proximal intestine) of eared doves (Zenaida auriculata). A decrease of the blood δ-ALAD activity, dilatation of blood vessels and leukocyte infiltrates in intestinal submucosa and muscular layers, and reduction of the enterocyte nuclear diameter and Lieberkühn crypts area were observed. In liver were noted steatosis, proliferation of bile ducts, dilated sinusoids, leukocyte infiltrates, and melanomacrophage centers. The portal tract area and the thickness of the portal vein wall were increased. In conclusion, the results showed that Pb produces histological and morphometric alterations on the liver and small intestine according to the exposure time, which should be considered when the dangerousness of environmental pollutants is evaluated in wild animals.

Cyclic fasting bolsters cholesterol biosynthesis inhibitors' anticancer activity.

Identifying oncological applications for drugs that are already approved for other medical indications is considered a possible solution for the increasing costs of cancer treatment. Under the hypothesis that nutritional stress through fasting might enhance the antitumour properties of at least some non-oncological agents, by screening drug libraries, we find that cholesterol biosynthesis inhibitors (CBIs), including simvastatin, have increased activity against cancers of different histology under fasting conditions. We show fasting's ability to increase CBIs' antitumour effects to depend on the reduction in circulating insulin, insulin-like growth factor-1 and leptin, which blunts the expression of enzymes from the cholesterol biosynthesis pathway and enhances cholesterol efflux from cancer cells. Ultimately, low cholesterol levels through combined fasting and CBIs reduce AKT and STAT3 activity, oxidative phosphorylation and energy stores in the tumour. Our results support further studies of CBIs in combination with fasting-based dietary regimens in cancer treatment and highlight the value of fasting for drug repurposing in oncology.

Sympathetic activity controls fat-induced oleoylethanolamide signaling in small intestine.

Ingestion of dietary fat stimulates production of the small-intestinal satiety factors oleoylethanolamide (OEA) and N-palmitoyl-phosphatidylethanolamine (NPPE), which reduce food intake through a combination of local (OEA) and systemic (NPPE) actions. Previous studies have shown that sympathetic innervation of the gut is necessary for duodenal infusions of fat to induce satiety, suggesting that sympathetic activity may engage small-intestinal satiety signals such as OEA and NPPE. In the present study, we show that surgical resection of the sympathetic celiac-superior mesenteric ganglion complex, which sends projections to the upper gut, abolishes feeding-induced OEA production in rat small-intestinal cells. These effects are accounted for by suppression of OEA biosynthesis, and are mimicked by administration of the selective ß2-adrenergic receptor antagonist ICI-118,551. We further show that sympathetic ganglionectomy or pharmacological blockade of ß2-adrenergic receptors prevents NPPE release into the circulation. In addition, sympathetic ganglionectomy increases meal frequency and lowers satiety ratio, and these effects are corrected by pharmacological administration of OEA. The results suggest that sympathetic activity controls fat-induced satiety by enabling the coordinated production of local (OEA) and systemic (NPPE) satiety signals in the small intestine.

Comparison of four valproic acid free fraction determination methods used in clinical practice.

OBJECTIVE: Given that hypoalbuminemia tends to result in higher free fraction  concentrations of valproic acid, different methods have been developed to  determine the latter in patients with this condition. The aim of this study is to  assess the reliability of these methods and, if necessary, design a new  estimation method. METHOD: A retrospective analysis was carried out by the Pharmacy Department of Severo Ochoa University Hospital of admitted patients with at least one  trough concentration of valproic acid between October 2017 and February  2019. The estimation methods used were those developed by Kodama,  Hermida, Doré, as well as a new method proposed in the study. A total of 17  serum valproic acid concentrations were used to determine the free fraction of  valproic acid with each method; the values obtained were compared with the  results obtained following laboratory determinations. Accuracy and precision  were calculated using mean error and root mean square error, respectively. RESULTS: The comparison between observed and predicted free valproic acid  values using the methods under investigation showed that the method proposed in this study provides the highest reliability as it presents the  highest accuracy and precision. The worst results were those obtained  using  the Kodama method, which does not consider albuminemia, an essential variable that determines the concentration, therapeutic effect and toxicity of valproic acid. CONCLUSIONS: Given that the method proposed in this study proved to be superior to the other methods analyzed, we believe it can be reliably used to estimate free valproic acid levels in patients with hypoalbuminemia.

OBJETIVO: La fracción de ácido valproico libre aumenta en pacientes con  hipoalbuminemia. Se han publicado diferentes métodos para su estimación.El  objetivo de este estudio es valorar la fiabilidad de dichos métodos en nuestra  población y proponer un nuevo método de estimación.Método: Análisis retrospectivo realizado por el Servicio de Farmacia del  Hospital Universitario Severo Ochoa en pacientes ingresados entre octubre de  2017 y febrero de 2019 con al menos una concentración valle de ácido  valproico. Los métodos de estimación empleados fueron los de Kodama,  Hermida, Doré y un nuevo método propuesto, diseñado por García. A partir de  17 mediciones de ácido valproico se comparó el ácido valproico libre estimado  con cada método y el obtenido en el laboratorio. Se calcularon la exactitud y la  precisión mediante el error medio y el error cuadrático medio,  respectivamente. RESULTADOS: La comparación entre los valores observados y predichos de  ácido valproico libre por los distintos métodos evaluados pone de manifiesto  que el de mayor fiabilidad es el diseñado por García, al presentar la mejor  exactitud y precisión. Los peores resultados son los del método Kodama, al no  considerar la albuminemia, variable fundamental  que condiciona la  concentración, el efecto terapéutico y la toxicidad de este fármaco. CONCLUSIONES: El método diseñado por García ha demostrado ser mejor que  otros métodos, por lo que puede ser propuesto para estimar con fiabilidad el  ácido valproico libre en pacientes con hipoalbuminemia, aunque se precisa  aplicarlo en un mayor número de pacientes para confirmar su utilidad.

Combination Treatment of Retinoic Acid Plus Focal Adhesion Kinase Inhibitor Prevents Tumor Growth and Breast Cancer Cell Metastasis.

All-trans retinoic acid (RA), the primary metabolite of vitamin A, controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, both known as retinoids, are promising agents in treating and chemopreventing different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with tumor metastatic behavior. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that the RA plus FAKi (RA + FAKi) approach could improve the inhibition of tumor progression. By in silico analysis and its subsequent validation by qPCR, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. We also showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. Additionally, we show that both drugs individually, as well as in combination, induce the expression of apoptosis markers such as active-caspase-3 and cleaved-PARP1. We also provided evidence that RA effects are extrapolated to other cancer cells, including T-47D BC and the human cervical carcinoma HeLa cells. In an orthotopic assay of LM3 tumor growth, whereas RA and FAKi administered separately reduced tumor growth, the combined treatment induced a more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduced metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance could reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbate these effects, thus suggesting that the sensitivity to RA therapies could be increased with FAKi coadministration in BC tumors.

The ABC membrane transporter ABCG2 prevents access of FAAH inhibitor URB937 to the central nervous system.

The O-arylcarbamate URB937 is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the deactivation of the endocannabinoid anandamide. URB937 is unique among FAAH inhibitors in that is actively extruded from the central nervous system (CNS), and therefore increases anandamide levels exclusively in peripheral tissues. Despite its limited distribution, URB937 exhibits marked analgesic properties in rodent models of pain. Pharmacological evidence suggests that the extrusion of URB937 from the CNS may be mediated by the ABC membrane transporter ABCG2 (also called Breast cancer resistance protein, BCRP). In the present study, we show that URB937 is a substrate for both mouse and human orthologues of ABCG2. The relative transport ratios for URB937 in Madin-Darby canine kidney (MDCKII) cells monolayers over-expressing either mouse Abcg2 or human ABCG2 were significantly higher compared to parental monolayers (13.6 and 13.1 vs. 1.5, respectively). Accumulation of the compound in the luminal/apical side was prevented by co-administration of the selective ABCG2 inhibitor, Ko-143. In vivo studies in mice showed that URB937 (25 mg kg(-1)) readily entered the brain and spinal cord of Abcg2-deficient mice following intraperitoneal administration, whereas the same dose of drug remained restricted to peripheral tissues in wild-type mice. By identifying ABCG2 as a transport mechanism responsible for the extrusion of URB937 from the CNS, the present results should facilitate the rational design of novel peripherally restricted FAAH inhibitors.

SIRT6 enhances oxidative phosphorylation in breast cancer and promotes mammary tumorigenesis in mice.

BACKGROUND: Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors. METHODS: We evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration. RESULTS: The heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice. CONCLUSIONS: Via its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.