The strategic roles of four enzymes in the interconnection between metabolism and oncogene activation in non-small cell lung cancer: Therapeutic implications.

NSCLC is the leading cause of cancer mortality and represents a major challenge in cancer therapy. Intrinsic and acquired anticancer drug resistance are promoted by hypoxia and HIF-1α. Moreover, chemoresistance is sustained by the activation of key signaling pathways (such as RAS and its well-known downstream targets PI3K/AKT and MAPK) and several mutated oncogenes (including KRAS and EGFR among others). In this review, we highlight how these oncogenic factors are interconnected with cell metabolism (aerobic glycolysis, glutaminolysis and lipid synthesis). Also, we stress the key role of four metabolic enzymes (PFK1, dimeric-PKM2, GLS1 and ACLY), which promote the activation of these oncogenic pathways in a positive feedback loop. These four tenors orchestrating the coordination of metabolism and oncogenic pathways could be key druggable targets for specific inhibition. Since PFK1 appears as the first tenor of this orchestra, its inhibition (and/or that of its main activator PFK2/PFKFB3) could be an efficacious strategy against NSCLC. Citrate is a potent physiologic inhibitor of both PFK1 and PFKFB3, and NSCLC cells seem to maintain a low citrate level to sustain aerobic glycolysis and the PFK1/PI3K/EGFR axis. Awaiting the development of specific non-toxic inhibitors of PFK1 and PFK2/PFKFB3, we propose to test strategies increasing citrate levels in NSCLC tumors to disrupt this interconnection. This could be attempted by evaluating inhibitors of the citrate-consuming enzyme ACLY and/or by direct administration of citrate at high doses. In preclinical models, this "citrate strategy" efficiently inhibits PFK1/PFK2, HIF-1α, and IGFR/PI3K/AKT axes. It also blocks tumor growth in RAS-driven lung cancer models, reversing dedifferentiation, promoting T lymphocytes tumor infiltration, and increasing sensitivity to cytotoxic drugs.

Why may citrate sodium significantly increase the effectiveness of transarterial chemoembolization in hepatocellular carcinoma?

Hepatocellular carcinoma (HCC) represents the third cause of cancer death in men worldwide, and its increasing incidence can be explained by the increasing occurrence of non-alcoholic steatohepatitis (NASH). HCC prognosis is poor, as its 5-year overall survival is approximately 18 % and most cases are diagnosed at an inoperable advanced stage. Moreover, tumor sensitivity to conventional chemotherapeutics (particularly to cisplatin-based regimen), trans-arterial chemoembolization (cTACE), tyrosine kinase inhibitors, anti-angiogenic molecules and immune checkpoint inhibitors is limited. Oncogenic signaling pathways, such as HIF-1α and RAS/PI3K/AKT, may provoke drug resistance by enhancing the aerobic glycolysis ("Warburg effect") in cancer cells. Indeed, this metabolism, which promotes cancer cell development and aggressiveness, also induces extracellular acidity. In turn, this acidity promotes the protonation of drugs, hence abrogating their internalization, since they are most often weakly basic molecules. Consequently, targeting the Warburg effect in these cancer cells (which in turn would reduce the extracellular acidification) could be an effective strategy to increase the delivery of drugs into the tumor. Phosphofructokinase-1 (PFK1) and its activator PFK2 are the main regulators of glycolysis, and they also couple the enhancement of glycolysis to the activation of key signaling cascades and cell cycle progression. Therefore, targeting this "Gordian Knot" in HCC cells would be of crucial importance. Here, we suggest that this could be achieved by citrate administration at high concentration, because citrate is a physiologic inhibitor of PFK1 and PFK2. As shown in various in vitro studies, including HCC cell lines, administration of high concentrations of citrate inhibits PFK1 and PFK2 (and consequently glycolysis), decreases ATP production, counteracts HIF-1α and PI3K/AKT signaling, induces apoptosis, and sensitizes cells to cisplatin treatment. Administration of high concentrations of citrate in animal models (including Ras-driven tumours) has been shown to effectively inhibit cancer growth, reverse cell dedifferentiation, and neutralize intratumor acidity, without apparent toxicity in animal studies. Citrate may also induce a rapid secretion of pro-inflammatory cytokines by macrophages, and it could favour the destruction of cancer stem cells (CSCs) sustaining tumor recurrence. Consequently, this "citrate strategy" could improve the tumor sensitivity to current treatments of HCC by reducing the extracellular acidity, thus enhancing the delivery of chemotherapeutic drugs into the tumor. Therefore, we propose that this strategy should be explored in clinical trials, in particular to enhance cTACE effectiveness.

Understanding the Central Role of Citrate in the Metabolism of Cancer Cells and Tumors: An Update.

Citrate plays a central role in cancer cells' metabolism and regulation. Derived from mitochondrial synthesis and/or carboxylation of α-ketoglutarate, it is cleaved by ATP-citrate lyase into acetyl-CoA and oxaloacetate. The rapid turnover of these molecules in proliferative cancer cells maintains a low-level of citrate, precluding its retro-inhibition on glycolytic enzymes. In cancer cells relying on glycolysis, this regulation helps sustain the Warburg effect. In those relying on an oxidative metabolism, fatty acid ß-oxidation sustains a high production of citrate, which is still rapidly converted into acetyl-CoA and oxaloacetate, this latter molecule sustaining nucleotide synthesis and gluconeogenesis. Therefore, citrate levels are rarely high in cancer cells. Resistance of cancer cells to targeted therapies, such as tyrosine kinase inhibitors (TKIs), is frequently sustained by aerobic glycolysis and its key oncogenic drivers, such as Ras and its downstream effectors MAPK/ERK and PI3K/Akt. Remarkably, in preclinical cancer models, the administration of high doses of citrate showed various anti-cancer effects, such as the inhibition of glycolysis, the promotion of cytotoxic drugs sensibility and apoptosis, the neutralization of extracellular acidity, and the inhibition of tumors growth and of key signalling pathways (in particular, the IGF-1R/AKT pathway). Therefore, these preclinical results support the testing of the citrate strategy in clinical trials to counteract key oncogenic drivers sustaining cancer development and resistance to anti-cancer therapies.

[Completeness of pharmacovigilance reporting in general medicine in France.] / Informativité des déclarations de pharmacovigilance en médecine générale en France.

INTRODUCTION: Spontaneous reporting remains one of the cornerstones of post-marketing drug safety surveillance. One of its main limitations is a lack of completeness.The main aim of this study was to assess the completeness of pharmacovigilance reports sent by general practitioners (GPs) to regional pharmacovigilance centers (RPC) reported in the French pharmacovigilance database (FPVD). Secondary aim was to identify factors associated with complete reports. METHOD: All adverse drugs reactions (ADRs) sent by GPs in France in 2015 were analyzed. According to information provided in ADR reports (ADR, date of occurrence, clinical description, drugs suspected, etc.), completeness was analyzed from “mandatory” criteria (age, gender, ADR and suspected drug(s)) and “non-mandatory” criteria (medical history, concomitant drugs, symptoms evolution and complementary exams) and classified as “well-documented”, “slightly-documented” or “poorly-documented”. RESULTS: In 2015, the FPVD contained 3,020 ADR reports realized by GPs. Only 16.4% of these reports were classified as “well-documented”, in accordance with study criteria. The most poorly documented items were concomitant drugs (41.4%) and complementary exams (37.4%). An association between a “well-documented” ADR report and its “seriousness” (OR = 3,02 [95% CI 2,44; 3,23], P < 10–3) and elderly compared to adults (OR = 1,76 [95% CI 1,42; 2,18], P < 10–3) or children (OR = 4,59 [95% CI 2,51; 8,39], P < 10–3). CONCLUSION: Our study shows that only one out of six ADR reports was “well-documented”. It appears to be important to promote pharmacovigilance to improve completeness of ADR reports.

Citrate targets FBPase and constitutes an emerging novel approach for cancer therapy.

Gao-Min Liu and Yao-Ming Zhang recently published a review entitled «Targeting FBPase is an emerging novel approach for cancer therapy¼ (Liu and Zhang in Cancer Cell Int 18:36, 2018). In this paper, the authors highlighted how the down regulation or inactivation of FBPase, a rate limiting enzyme of gluconeogenesis, can promote the Warburg effect and cancer growth. In contrast, activation of this enzyme demonstrates anti-cancer effects and may appear as emerging novel approach for cancer therapy. Among the potential activators of FBP listed by Liu and Zhang, citrate was surprisingly not mentioned although it is an activator of FBPase, also demonstrating various anti-cancer effects in pre-clinical studies. Thus, citrate should be tested as a new therapeutic strategy, in particular in clinical studies.

[Evaluation of a new pharmacovigilance tool: Simplified online reporting for general practitioners]. / Évaluation d'un nouvel outil en pharmacovigilance : la déclaration simplifiée en ligne pour les médecins généralistes.

BACKGROUND: Spontaneous reporting of adverse drug reactions remains the cornerstone of postmarketing drug safety surveillance (pharmacovigilance). However, the marked underreporting of adverse drug reactions constitutes a major limitation. The main objective of this study was to assess the use of this simplified reporting by general practitioners (GPs) in Western Normandy based on the number of ADRs reported and to assess its impact on the quality of these reports. METHOD: Simplified online pharmacovigilance reporting was proposed in June 2015 by the Caen Normandie regional pharmacovigilance center (CRPV) in conjunction with the Normandy Union régionale des médecins libéraux (URML Normandie) for GPs. This new tool is based on items to be completed by the GP. They were selected by members of CRPV in an attempt to combine good quality reporting and maximum simplicity. RESULTS: Between June 2014 and June 2016, 220 reports were made by 67 GPs. One year after introduction of this new tool, the monthly number of reports was multiplied by 4.8 and the number of reporting GPs was multiplied by two. The quality of reporting remained unchanged over the same period (p = 0.1). Simplified reporting allowed a decreased number of inaccurate reports (33% versus 36%, p = 0.04). CONCLUSION: Simplified online reporting is effective quantitatively (increased number of reports) but also qualitatively (quality unchanged). We must now try to develop this tool in other French regions and reassess it over time.

[Fibrates and risk of venous thromboembolism: Case/no-case study in French pharmacovigilance database]. / Fibrates et risques thrombo-emboliques veineux : étude cas/non-cas dans la base nationale de pharmacovigilance.

INTRODUCTION: Several studies have investigated the occurrence of venous thromboembolic events (phlebitis and pulmonary embolism) [VTE] and fibrates. Fibrates could be associated with VTE although published data are contradictory. The objective of this study is to confirm the link between VTE and fibrates. MATERIALS AND METHODS: Retrospective disproportionality analysis (case/non-case method) from observations recorded consecutively in the French pharmacovigilance database between 1985 and 2016. Cases were defined as embolic and thrombotic events, thrombophlebitis; Non-cases were other adverse events reported over the same period. We measured the disproportionality of exposure to each fibrate among cases and no-cases. The analysis was validated with a positive control (drospirenone) and a negative control (paracetamol). RESULTS: We compared 19,436 cases (including 161 mentioning fibrates) to 563,310 non-cases (including 3228 fibrates). Reports of VTE were significantly associated with fenofibrate (ROR=1.83; 95% CI=[1.53; 2.2]) but not with other fibrates: bézafibrate (ROR=0.44; 95% CI=[0.2; 0.99]), ciprofibrate (ROR=1.15; 95% CI=[0.76; 1.73]) and gemfibrozil (ROR=0.91; 95% CI=[0.45; 1.84]). CONCLUSION: With this study, we confirm the link between VTE and fenofibrate. It is therefore advisable to remain cautious when prescribing fenofibrate, in particular in case of past history of VTE and to declare systematically any venous thromboembolic adverse events observed with these drugs.

Drugs and Sleep Apneas? A review of the French Pharmacovigilance database.

The present work reviews the case reports of drug-induced sleep apnea recorded in the French pharmacovigilance database. Notifications are very rare (around 1/100 000 notifications). This paper shows that sleep apnea can be aggravated or revealed by some drugs. Main drugs involved were psychotropics (benzodiazepines, neuroleptics) and opioids.

[New frequent adverse reaction of PPI in older adults: mild hyponatremia]. / Nouvel effet indésirable fréquent des inhibiteurs de la pompe à protons chez le sujet âgé : l'hyponatrémie modérée.

Hyponatremia is a rare side effect described in the product characteristics of proton pump inhibitors (PPIs). Hyponatremia in the elderly (>65 years) was assessed in patients with exposure to PPIs for at least one year compared to controls not exposed to PPIs counterparts. Included 145 patients, twenty-four patients (16.6%) had moderate hyponatremia ([120-134] mEq/L). Forty-eight patients (33.1%) were treated with PPIs. In the end, 31.3% [18.7%-46.3%] of the treated population for more than a year by PPI suffered moderate hyponatremia against only 9.3% [14.3%-16.9%] in the rest of the population, giving an odds ratio of 4.4 ([1.8-11.1], p=0.001). The relationship between hyponatremia dose was not significant (R2=0.05, p=0.74). By our study, we show that the risk of moderate hyponatremia is increased by chronic use of PPI in the elderly population. We also specify a notion of prevalence between 18.7% and 46.3%.

[Not Available]. / Nouvel effet indésirable fréquent des inhibiteurs de la pompe à protons chez le sujet âgé : l'hyponatrémie modérée.

Hyponatremia is a rare side effect described in the product characteristics of proton pump inhibitors (PPIs). Hyponatremia in the elderly (>65years) was assessed in patients with exposure to PPIs for at least one year compared to controls not exposed to PPIs counterparts. Included 145 patients, twenty-four patients (16.6%) had moderate hyponatremia ([120-134] mEq/L). Forty-eight patients (33.1%) were treated with PPIs. In the end, 31.3% [18.7%-46.3%] of the treated population for more than a year by PPI suffered moderate hyponatremia against only 9.3% [14.3%-16.9%] in the rest of the population, giving an odds ratio of 4.4 ([1.8-11.1], p=0.001). The relationship between hyponatremia dose was not significant (R(2)=0.05, p=0.74). By our study, we show that the risk of moderate hyponatremia is increased by chronic use of PPI in the elderly population. We also specify a notion of prevalence between 18.7% and 46.3%.

[Moderate potentially drug-induced hyponatremia in older adults: benefit in drug reduction]. / L'hyponatrémie modérée potentiellement médicamenteuse du sujet âgé : bénéfice de la réduction des médicaments.

OBJECTIVE: This study aimed at evaluating the benefit of changing drug therapy in elderly patients with moderate, potentially drug-induced hyponatremia. METHODS: Hospitalized older adults, with moderate hyponatremia, potentially induced by drugs, were randomized into two arms: an interventional group, whose drug therapy was changed, and a reference group, which received standard care. The effectiveness of the intervention was evaluated by the normalization of serum sodium after four weeks and by the incidence of falls three months later. RESULTS: Nineteen patients were randomized, fourteen evaluated at 4 weeks. Serum sodium was normalized more frequently in the interventional group than in the reference group: 75% (6/8) IC95% [35-97] versus 0% (0/6) IC95% [0-46]; p=0.01. A greater reduction in falls occurred in the therapeutic intervention group 75% (3/4) IC95% [19-99] versus 0% (0/5) IC95% [0-52]; p=0.048. CONCLUSION: This study showed the biological and clinical benefit of a pharmalogical intervention. Registration number of the study: ID RCB 2010-A00778-31.

An Investigation of O-Demethyl Tramadol/Tramadol Ratio for Cytochrome P450 2D6 Phenotyping: The CYTRAM Study.

Cytochrome P450 2D6 (CYP2D6) gene polymorphisms influence the exposure to tramadol (T) and its pharmacologically active metabolite, O-demethyl tramadol (O-dT). Tramadol has been considered as a candidate probe drug for CYP2D6 phenotyping. The objective of the CYTRAM study was to investigate the value of plasma O-dT/T ratio for CYP2D6 phenotyping. European adult patients who received IV tramadol after surgery were included. CYP2D6 genotyping was performed and subjects were classified as extensive (EM), intermediate (IM), poor (PM), or ultra-rapid (UM) CYP2D6 metabolizers. Plasma concentrations of tramadol and O-dT were determined at 24 h and 48 h. The relationship between O-dT/T ratio and CYP2D6 phenotype was examined in both a learning and a validation group. Genotype data were obtained in 301 patients, including 23 PM (8%), 117 IM (39%), 154 EM (51%), and 7 UM (2%). Tramadol trough concentrations at 24 h were available in 297 patients. Mean value of O-dT/T ratio was significantly lower in PM than in non-PM individuals (0.061 ± 0.031 versus 0.178 ± 0.09, p < 0.01). However, large overlap was observed in the distributions of O-dT/T ratio between groups. Statistical models based on O-dT/T ratio failed to identify CYP2D6 phenotype with acceptable sensitivity and specificity. Those results suggest that tramadol is not an adequate probe drug for CYP2D6 phenotyping.

The key role of Warburg effect in SARS-CoV-2 replication and associated inflammatory response.

Current mortality due to the Covid-19 pandemic (approximately 1.2 million by November 2020) demonstrates the lack of an effective treatment. As replication of many viruses - including MERS-CoV - is supported by enhanced aerobic glycolysis, we hypothesized that SARS-CoV-2 replication in host cells (especially airway cells) is reliant upon altered glucose metabolism. This metabolism is similar to the Warburg effect well studied in cancer. Counteracting two main pathways (PI3K/AKT and MAPK/ERK signaling) sustaining aerobic glycolysis inhibits MERS-CoV replication and thus, very likely that of SARS-CoV-2, which shares many similarities with MERS-CoV. The Warburg effect appears to be involved in several steps of COVID-19 infection. Once induced by hypoxia, the Warburg effect becomes active in lung endothelial cells, particularly in the presence of atherosclerosis, thereby promoting vasoconstriction and micro thrombosis. Aerobic glycolysis also supports activation of pro-inflammatory cells such as neutrophils and M1 macrophages. As the anti-inflammatory response and reparative process is performed by M2 macrophages reliant on oxidative metabolism, we speculated that the switch to oxidative metabolism in M2 macrophages would not occur at the appropriate time due to an uncontrolled pro-inflammatory cascade. Aging, mitochondrial senescence and enzyme dysfunction, AMPK downregulation and p53 inactivation could all play a role in this key biochemical event. Understanding the role of the Warburg effect in COVID-19 can be essential to developing molecules reducing infectivity, arresting endothelial cells activation and the pro-inflammatory cascade.

Metabolic Strategies for Inhibiting Cancer Development.

The tumor microenvironment is a complex mix of cancerous and noncancerous cells (especially immune cells and fibroblasts) with distinct metabolisms. These cells interact with each other and are influenced by the metabolic disorders of the host. In this review, we discuss how metabolic pathways that sustain biosynthesis in cancer cells could be targeted to increase the effectiveness of cancer therapies by limiting the nutrient uptake of the cell, inactivating metabolic enzymes (key regulatory ones or those linked to cell cycle progression), and inhibiting ATP production to induce cell death. Furthermore, we describe how the microenvironment could be targeted to activate the immune response by redirecting nutrients toward cytotoxic immune cells or inhibiting the release of waste products by cancer cells that stimulate immunosuppressive cells. We also examine metabolic disorders in the host that could be targeted to inhibit cancer development. To create future personalized therapies for targeting each cancer tumor, novel techniques must be developed, such as new tracers for positron emission tomography/computed tomography scan and immunohistochemical markers to characterize the metabolic phenotype of cancer cells and their microenvironment. Pending personalized strategies that specifically target all metabolic components of cancer development in a patient, simple metabolic interventions could be tested in clinical trials in combination with standard cancer therapies, such as short cycles of fasting or the administration of sodium citrate or weakly toxic compounds (such as curcumin, metformin, lipoic acid) that target autophagy and biosynthetic or signaling pathways.

[Therapeutic drug monitoring of clonazepam]. / Suivi thérapeutique pharmacologique du clonazépam.

Clonazepam is a 1-4 benzodiazepine mainly used to treat epilepsy and epileptiform convulsion state. Rapidly absorbed after oral administration, it is widely distributed in the organism and is extensively converted in metabolites, poorly or not active, eliminated mainly in urine (70%) and feces. Elimination half-life is long, around 40 h. In adult and child, several studies showed a concentration-effect relation. Meanwhile, a large inter-individual variability in the dose-concentration relation was observed. A 15-50 microg/L range of clonazepam blood concentrations appears to be retained as an acceptable target to control a majority of epileptic seizures. The Therapeutic Drug Monitoring (TDM) of clonazepam can be considered as possibly useful in case of association with CYP450 inducers or inhibitors, suspicion of poor observance, or toxicity signs.

[Not Available]. / Suivi thérapeutique pharmacologique du clonazépam.

Clonazepam is a 1-4 benzodiazepine mainly used to treat epilepsy and epileptiform convulsion state. Rapidly absorbed after oral administration, it is widely distributed in the organism and is extensively converted in metabolites, poorly or not active, eliminated mainly in urine (70%) and feces. Elimination half-life is long, around 40 h. In adult and child, several studies showed a concentration-effect relation. Meanwhile, a large inter-individual variability in the dose-concentration relation was observed. A 15-50 µg/L range of clonazepam blood concentrations appears to be retained as an acceptable target to control a majority of epileptic seizures. The Therapeutic Drug Monitoring (TDM) of clonazepam can be considered as possibly useful in case of association with CYP450 inducers or inhibitors, suspicion of poor observance, or toxicity signs.

[Mephedrone: a designer drug of recent use in France]. / La méphédrone : une designer drug d'usage récent en France.

Designer drugs are currently marketed as substitutes for stimulant drugs as cocaine, amphetamine, MDMA...Unlike compounds listed as narcotics, these new substances are deliberately synthesized to avoid anti-drug laws. Among them, mephedrone (4-methylmethcatinone) that belongs to cathinone family, has been recently introduced in France. Users report positive euphoric and entactogenic effects. They also describe negative effects such as increased dependence towards the drug itself and larger craving for tobacco and alcohol. The numerous and various described adverse effects include psychoactive, digestive, cardiovascular... effects. Some fatality cases have been reported in scientific literature or in press and attributed to mephedrone often in association with other substances. Mephedrone has been listed as narcotic in several European countries and more recently in France.