Predicting Dihydropyrimidine Dehydrogenase Deficiency and Related 5-Fluorouracil Toxicity: Opportunities and Challenges of DPYD Exon Sequencing and the Role of Phenotyping Assays.

Deficiency of dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is associated with severe toxicity induced by the anti-cancer drug 5-Fluorouracil (5-FU). DPYD genotyping of four recommended polymorphisms is widely used to predict toxicity, yet their prediction power is limited. Increasing availability of next generation sequencing (NGS) will allow us to screen rare variants, predicting a larger fraction of DPD deficiencies. Genotype−phenotype correlations were investigated by performing DPYD exon sequencing in 94 patients assessed for DPD deficiency by the 5-FU degradation rate (5-FUDR) assay. Association of common variants with 5-FUDR was analyzed with the SNPStats software. Functional interpretation of rare variants was performed by in-silico analysis (using the HSF system and PredictSNP) and literature review. A total of 23 rare variants and 8 common variants were detected. Among common variants, a significant association was found between homozygosity for the rs72728438 (c.1974+75A>G) and decreased 5-FUDR. Haplotype analysis did not detect significant associations with 5-FUDR. Overall, in our sample cohort, NGS exon sequencing allowed us to explain 42.5% of the total DPD deficiencies. NGS sharply improves prediction of DPD deficiencies, yet a broader collection of genotype−phenotype association data is needed to enable the clinical use of sequencing data.

5-Fluorouracil degradation rate as a predictive biomarker of toxicity in breast cancer patients treated with capecitabine.

Capecitabine is an oral prodrug of 5-fluorouracil with a relevant role in the treatment of breast cancer. Severe and unexpected toxicities related to capecitabine are not rare, and the identification of biomarkers is challenging. We evaluate the relationship between dihydropyrimidine dehydrogenase, thymidylate synthase enhancer region and methylenetetrahydrofolate reductase polymorphisms, 5-fluorouracil degradation rate and the onset of G3-4 toxicities in breast cancer patients. Genetic polymorphisms and the 5-fluorouracil degradation rate of breast cancer patients treated with capecitabine were retrospectively studied. Genetic markers and the 5-fluorouracil degradation rate were correlated with the reported toxicities. Thirty-seven patients with a median age of 58 years old treated with capecitabine for stages II-IV breast cancer were included in this study. Overall, 34 (91.9%) patients suffered from at least an episode of any grade toxicity while nine patients had G3-4 toxicity. Homozygous methylenetetrahydrofolate reductase 677TT was found to be significantly related to haematological toxicity (OR = 6.5 [95% IC 1.1-37.5], P = 0.04). Three patients had a degradation rate less than 0.86 ng/mL/106 cells/min and three patients greater than 2.1 ng/mL/106 cells/min. At a univariate logistic regression analysis, an altered value of 5-fluorouracil degradation rate (values < 0.86 or >2.10 ng/mL/106 cells/min) increased the risk of G3-4 adverse events (OR = 10.40 [95% IC: 1.48-7.99], P = 0.02). A multivariate logistic regression analysis, adjusted for age, comorbidity and CAPE-regimen, confirmed the role of 5-fluorouracil degradation rate as a predictor of G3-4 toxicity occurrence (OR = 10.9 [95% IC 1.2-96.2], P = 0.03). The pre-treatment evaluation of 5-fluorouracil degradation rate allows to identify breast cancer patients at high risk for severe 5-FU toxicity.

Tryptophan and Kynurenine Metabolites: Are They Related to Depression?

BACKGROUND: Some previous studies found decreased concentrations of L-tryptophan (TRY) and increased L-kynurenine (KYN), or its metabolites, in the body fluids of subjects with major depressive disorder (MDD), sometimes in association with suicidal behavior. Such changes might indicate a shift of TRY away from serotonin production, possibly via the effects of inflammatory peptides which activate indoleamine-2,3-dioxygenase. However, these findings have been inconsistent and require replication. METHODS: We used sensitive liquid-chromatography mass spectrometry methods to assay plasma concentrations of TRY, 5-hydroxyindoleacetic acid (5-HIAA), and KYN and its metabolites (anthranilic acid and xanthurenic acid). We compared 49 hospitalized, depressed subjects diagnosed with MDD (n = 37) or bipolar disorder (BD, n = 12), with (n = 22) or without (n = 27) previous suicide attempts, to 78 healthy, ambulatory controls of similar age and sex (total n = 127). FINDINGS: Contrary to expectation, TRY plasma concentrations were higher, KYN plasma concentrations were lower, and their ratio much higher in depressed subjects, with no relationship to suicidal history. Concentrations of 5-HIAA and the kynurenine metabolites did not differ between depressed and healthy subjects. CONCLUSIONS: These findings are opposite to expectations and not consistent with a hypothesized increased conversion from TRY to KYN in depressed subjects. In addition, we found no evidence of altered production of serotonin as 5-HIAA concentration was unchanged. None of the observed changes was associated with a history of suicide attempt.

Migraine and cluster headache show impaired neurosteroids patterns.

BACKGROUND: Perturbation of neuronal excitability contributes to migraine. Neurosteroids modulate the activity of γ-aminobutyric acid A and N-methyl-d-aspartate receptors, and might be involved in the pathogenesis of migraine. Here, we measured plasma levels of four neurosteroids, i.e., allopregnanolone, epiallopregnanolone, dehydroepiandrosterone and deydroepiandrosterone sulfate, in patients affected by episodic migraine, chronic migraine, or cluster headache. METHODS: Nineteen female patients affected by episodic migraine, 51 female patients affected by chronic migraine, and 18 male patients affected by cluster headache were recruited to the study. Sex- and age-matched healthy control subjects (31 females and 16 males) were also recruited. Patients were clinically characterized by using validated questionnaires. Plasma neurosteroid levels were measured by liquid chromatography-tandem mass spectrometry. RESULTS: We found disease-specific changes in neurosteroid levels in our study groups. For example, allopregnanolone levels were significantly increased in episodic migraine and chronic migraine patients than in control subjects, whereas they were reduced in patients affected by cluster headache. Dehydroepiandrosterone and dehydroepiandrosterone sulfate levels were reduced in patients affected by chronic migraine, but did not change in patients affected by cluster headache. CONCLUSION: We have shown for the first time that large and disease-specific changes in circulating neurosteroid levels are associated with chronic headache disorders, raising the interesting possibility that fluctuations of neurosteroids at their site of action might shape the natural course of migraine and cluster headache. Whether the observed changes in neurosteroids are genetically determined or rather result from exposure to environmental or intrinsic stressors is unknown. This might also be matter for further investigation because stress is a known triggering factor for headache attacks in both migraineurs and cluster headache patients.

Can IDO activity predict primary resistance to anti-PD-1 treatment in NSCLC?

BACKGROUND: Immune checkpoint inhibitors have revolutionized the treatment paradigm of highly lethal malignancies like advanced non-small cell lung cancer (NSCLC), demonstrating long-term tumour control and extended patient survival. Unfortunately, only 25-30% of patients experience a durable benefit, while the vast majority demonstrate primary or acquired resistance. Recently, indoleamine 2,3-dioxygenase (IDO) activity has been proposed as a possible mechanism of resistance to anti-PD-1 treatment leading to an immunosuppressive microenvironment. METHODS: Pre-treatment serum concentrations of tryptophan (trp) and kynurenine (kyn) were measured by high-performance liquid chromatography tandem mass spectrometry in NSCLC patients treated with second-line nivolumab. The IDO activity was expressed with kyn/trp ratio. The associations between kyn/trp ratio and early progression, performance status (PS), age, sex, brain metastases, pleural effusion, progression free survival (PFS) and overall survival (OS) were analyzed using Spearman test and Mann-Whitney test. RESULTS: Twenty-six NSCLC patients were included in our study; 14 of them (54%) presented early progression (< 3 months) to nivolumab treatment. The median value of kyn/trp ratio was 0.06 µg/ml and the median value of quinolinic acid was 68.45 ng/ml. A significant correlation between early progression and higher kyn/trp ratio and quinolinic acid concentration was observed (p = 0.017 and p = 0.005, respectively). Patients presenting lower values of kyn/trp ratio and quinolinic acid levels showed longer PFS (median PFS not reached versus 3 months; HR: 0.3; p = 0.018) and OS (median OS not reached vs 3 months; HR: 0.18; p = 0.0005). CONCLUSION: IDO activity, expressed as kyn/trp ratio, is associated with response to immunotherapy; in particular, higher kyn/trp ratio could predict resistance to anti-PD-1 treatment. These preliminary results suggest the possibility of using anti-PD-1 plus IDO inhibitor in those patients with high level of kyn/trp ratio.

Maternal exposure to endocrine disruptors and placental transmission: a pilot study.

Endocrine disruptors (EDs) are known to affect maternal and child health. The objective of our study was to identify the association between some of the most important endocrine-disruptive substances (perfluorooctane sulfonate [PFOS], perfluorooctanoic acid [PFOA], di2-ethylhexyl-phthalate [DEHP] and mono2-ethylhexyl-phthalate [MEHP]) and both pregnancy variability and birth outcomes. We measured the concentration of the EDs in maternal and cord blood samples of 29 mother-newborn pairs from the Pertini Hospital in Rome between March and June 2016. Each mother reported demographic, life style and diet information. We compared concentrations of the endocrine disruptors between mother and newborn, and among different molecules. We analyzed differences and trends of each ED substance according to the demography and diet information. PFOA levels in maternal blood showed a negative association with newborn weight. Concentration levels of PFOA in both maternal and cord blood of those with physiological progression of pregnancy were higher in than in those with pathological pregnancies. MEHP trend showed a positive association with maternal age. These results confirm the maternal-to-fetus transfer of EDs through the placenta and the impact that endocrine disruptors have on pregnancy and birth outcomes.

Lacosamide in bipolar disorder: A 30-day comparison to a retrospective control group treated with other antiepileptics.

AIM: Bipolar disorder (BD) is often treated with anticonvulsants. Lacosamide has not been tested in BD. We assessed its effects in a hospital setting in patients with BD without epilepsy. METHODS: We treated 102 consecutive hospitalized patients with acute BD with lacosamide 50-300 mg/day. We compared this sample with a retrospective sample treated with other antiepileptics (OAE). We rated patients after 3, 7, 15, and 30 days of treatment with the Brief Psychiatric Rating Scale, Young Mania Rating Scale, Hamilton Depression Rating Scale, Hamilton Anxiety Rating Scale, Clinical Global Impressions - Severity, and Global Assessment of Functioning. RESULTS: Patients receiving lacosamide were significantly younger and had fewer mixed episodes at intake, and less substance use disorder comorbidity than those receiving OAE. Both groups showed positive effects on all measures. The two groups did not differ on any clinical measure at baseline, but from the 3rd day on, lacosamide patients fared better than OAE patients on the Young Mania Rating Scale and Clinical Global Impressions - Severity and worse on the Hamilton Anxiety Rating Scale. From the 15th day, OAE patients scored better on the Brief Psychiatric Rating Scale. Global Assessment of Functioning scores were significantly more improved in the lacosamide patients. Age, substance use disorder comorbidity, episode type, and educational level significantly affected results. No interactions were found amongst these parameters. CONCLUSION: Lacosamide was effective in reducing psychopathology, mania, depression, and anxiety and in improving global functioning in patients with BD-I/II disorder in the short term, with few side-effects. Lacosamide improved mania, clinical severity, and global functioning better than OAE at doses lower than those used in epilepsy.

Altered intestinal permeability in patients with relapsing-remitting multiple sclerosis: A pilot study.

BACKGROUND: Alterations of intestinal permeability (IP) may contribute to the pathophysiology of immune-mediated diseases. OBJECTIVE: We investigated the possible association between IP changes and multiple sclerosis (MS). METHODS: We studied 22 patients with relapsing-remitting multiple sclerosis (RRMS) and 18 age- and sex-matched healthy donors (HDs), including five twin pairs (one concordant, and four discordant for disease). Measurement of lactulose (L) and mannitol (M; two non-metabolized sugars) levels in urine samples, after an oral load, allowed to quantify gut dysfunction. RESULTS: The proportion of participants with increased IP was significantly higher in patients than in HDs (16/22 (73%) versus 5/18 (28%); p = 0.001). Accordingly, the L/M urinary ratio showed significantly higher values in patients than in controls ( p = 0.0284). Urinary mannitol concentration was significantly lower in patients than in controls ( p = 0.022), suggesting a deficit of absorption from intestinal lumen. Such changes did not appear related to patients' clinical-radiological features. CONCLUSION: The relatively high proportion of IP changes in RR-MS patients seems to confirm our work hypothesis and warrants more work to confirm the result on a larger sample, and to understand the implications for related immunological disturbances and intestinal microbiota alterations. Our finding may also have relevance for oral treatments, recently introduced in clinical practice.

5-Fluorouracil degradation rate could predict toxicity in stages II-III colorectal cancer patients undergoing adjuvant FOLFOX.

5-Fluorouracil is commonly used for gastrointestinal cancer treatment in an adjuvant setting; however, the toxicity can lead to a reduction, delay, or discontinuation of treatment. We retrospectively investigated the association between the 5-fluorouracil degradation rate (5-FUDR) and genetic polymorphisms of TSER, DPYD, and MHTFR with toxicity in colorectal cancer patients treated with adjuvant FOLFOX. Pretreatment 5-FUDR and MTHFR A1298T or C677T, TSER, and DPYD gene polymorphisms were characterized in stages II-III colorectal cancer patients. Patients were classified into three metabolic classes according to the 5-FUDR value. Association with toxicities was evaluated retrospectively using logistic regression analysis. Overall, 126 patients were selected (35 women, 91 men). Seven patients were poor metabolizers, 116 patients were normal metabolizers and three patients were ultra-rapid metabolizers. The median 5-FUDR was 1.53 ng/ml/10 cells/min (range: 0.42-2.57 ng/ml/10 cells/min). Severe, rate-limiting toxicities (grades 3-4) were encountered in 22.2% of patients. No associations between MTHFR or TSER polymorphisms and toxicity were detected, whereas 5-FUDR showed a statistically significant association with toxicity (P=0.0047). The DPYD heterozygous mutation was detected in only one patient, who showed grade 4 hematological toxicity and a lower 5-FUDR value. The 5-FUDR value seems not to be affected by MTHFR and TSER polymorphisms. Compared with the available pharmacogenomics tests, the pretreatment evaluation of 5-FUDR increases the proportion of identified colorectal patients at high risk for severe toxicity. Thus, it appears to be a suitable pretreatment toxicity biomarker in a subgroup of patients in whom dose-intensity maintenance is the key factor.

A nomogram to predict 5-fluorouracil toxicity: when pharmacogenomics meets the patient.

Fluoropyrimidines combined with other agents are commonly used for gastrointestinal cancer treatment. Considering that severe toxicities occur in 30% of patients, we aimed to structure a nomogram to predict toxicity, based on metabolic parameter and patients' characteristics. We retrospectively enrolled patients affected by gastrointestinal tract cancers. Pretreatment 5-fluorouracil (5-FU) degradation rate and DPYD, TSER, MTHFR A1298T, and C677T gene polymorphisms were characterized. Data on toxicities were collected according to CTCAE v3.0. Multivariate logistic regression analysis was used to structure a nomogram. 642 patients were enrolled (384 men; 258 female; median age: 67 years, range: 27-87): 449 (69.9%) patients were affected by colorectal cancer; 118 (18.4%) by gastroesophageal cancer; 66 (10.3%) by pancreatic cancer; and nine (1.4%) by other cancers. Grade 3-4 toxicities were observed in 118 (18.4%) patients and were most frequently observed in patients with altered 5-FU degradation rate (43.5 and 26.7% of the patients in the poor metabolizer and in the ultrarapid metabolizer group respectively, vs. 17% in the normal metabolizer group) and in DPYD heterozygous mutated patients (83.3% of the patients). Age, DPYD status, the number of drugs administered, and 5-FU degradation rate value were associated to severe toxicities. On the basis of these findings, we structured a nomogram to assess a score to predict the risk of developing severe toxicity. Compared with the available pharmacogenetic tests, this approach can be applied to the whole population, predicting the risk for severe toxicity, with an easy, low-cost, and not invasive technique.

Evaluation of 5-fluorouracil degradation rate and Pharmacogenetic profiling to predict toxicity following adjuvant Capecitabine.

BACKGROUND: On account of the lack of predictive biomarkers of toxicity, we investigated whether polymorphisms of genes involved in fluoropyrimidine metabolism and 5-fluorouracil (5-FU) degradation rate were associated with outcomes of adjuvant capecitabine in patients with early stage gastrointestinal cancers. METHODS: Genotyping of DPYD GIVS14A, MTHFR C677T and A1298C SNPs were performed by pyro-sequencing technology. PCR analysis was used for genotyping TYMS-TSER. We also evaluated the 5-FU degradation rate, which determines the amount of drug consumed by PBMC in a time unit. Association of these variables with clinical outcome was evaluated using multivariate logistic regression analysis. RESULTS: One hundred forty-two patients with early stage colon (39%), rectal (28%), stomach (20%) and pancreatic (13%) cancer, treated with adjuvant capecitabine, were included in this retrospective analysis. Seventy and 20% of the patients suffered from at least one G1-4 and G3-4 adverse events, respectively. According to the 5-FU degradation rate, three and 13 patients were assigned as poor (<0.86 ng/mL/106 cells/min) and ultra-rapid (>2.1 ng/mL/106 cells/min) metabolizers, respectively. At a multivariate logistic regression analysis, an altered 5-FU degradation rate (values <0.86 or >2.10 ng/mL/106 cells/min) was associated with grade 3-4 adverse events (OR = 2.09, 95% CI: 1.14-3.82, P = 0.01). No correlation was reported between toxicity and gene polymorphisms except for hand-foot syndrome that was more frequent in the MTHFR 1298CC homozygous variant genotype (OR = 2.03, 95% CI 1.04-3.96, P = 0.03). CONCLUSIONS: 5-FU degradation rate may be regarded as possible predictive biomarker of capecitabine toxicity in early stage gastrointestinal cancer.

Changes in serum levels of kynurenine metabolites in paediatric patients affected by ADHD.

This study aims at determining serum levels of tryptophan and other metabolites of the kynurenine pathway in children with attention deficit hyperactivity disorder (ADHD) compared to healthy controls. Such metabolites interact with glutamate receptors in the central nervous system, potentially modulating mechanisms that are pivotal in ADHD and thus potentially representing peripheral biomarkers of the disorder. We measured serum levels of tryptophan and some metabolites of the kynurenine pathway in 102 children with ADHD and 62 healthy controls by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As compared to healthy controls, children with ADHD showed a reduction in serum levels of anthranilic acid (-60%), kynurenic acid (-11.2%), and xanthurenic acid (-12.5%). In contrast, serum levels of tryptophan (+11.0%) and kynurenine (+48.6%) were significantly enhanced, and levels of quinolinic acid were unchanged in children with ADHD. In a logistic regression model, the presence of ADHD was predicted by low anthranilic acid and high tryptophan levels. These findings support the involvement of the kynurenine pathway in the pathophysiology of ADHD and suggest that anthranilic acid and tryptophan levels should be investigated as potential peripheral biomarker for ADHD.

The TYMS-TSER polymorphism is associated with toxicity of low-dose capecitabine in patients with advanced gastrointestinal cancer.

Low doses of drugs delivered at close, regular intervals are increasingly being used to manage patients with different neoplasms. Despite the good tolerability, treatment-related adverse events still occur following metronomic protocols. The aim of this study was to retrospectively investigate whether polymorphisms of different genes involved in fluoropyrimidine metabolism and 5-fluorouracil (5-FU) degradation rate were associated with the outcome of a low-dose capecitabine schedule. Genotyping of DPYD IVS14+1 G>A, MTHFR C677T, and A1298C single-nucleotide polymorphisms was performed by pyrosequencing technology. A PCR technique was used for genotyping TYMS-TSER. Using peripheral blood mononuclear cells, we also evaluated the 5-FU degradation rate, which determines the net result of all the enzymatic transformation of 5-FU, in terms of the amount of drug consumed by the cells in a time unit. The association of these variables with clinical outcome was evaluated using multivariate logistic regression analysis. Eighty-four patients with metastatic gastrointestinal cancer, who had been treated with a low-dose fluoropyrimidine schedule, as a rescue therapy were included in the study. The TSER 2R/2R genotype was significantly associated with both hematologic (odds ratio=7.90, P=0.002) and gastrointestinal toxicity (odds ratio=3.24, P=0.009). Because DPYD IVS14 G>A single-nucleotide polymorphism was not observed in the cohort, it was excluded from the statistical analysis. No significant association was detected between clinical outcome and both MTHFR polymorphisms and the 5-FU degradation rate. In the advanced setting of cancer care, high attention should be paid toward avoiding toxicity and worsening of quality of life. Although metronomic chemotherapy is generally well tolerated, treatment toxicity nonetheless does occur. Our data suggest a possible role of the TSER 2R/2R polymorphism as a predictive marker of toxicity in patients treated with low-dose capecitabine.

Systematic Review of Clozapine Cardiotoxicity.

Clozapine is exceptionally effective in psychotic disorders and can reduce suicidal risk. Nevertheless, its use is limited due to potentially life-threatening adverse effects, including myocarditis and cardiomyopathy. Given their clinical importance, we systematically reviewed research on adverse cardiac effects of clozapine, aiming to improve estimates of their incidence, summarize features supporting their diagnosis, and evaluate proposed monitoring procedures. Incidence of early (≤2 months) myocarditis ranges from <0.1 to 1.0 % and later (3-12 months) cardiomyopathy about 10 times less. Diagnosis rests on relatively nonspecific symptoms, ECG changes, elevated indices of myocardial damage, cardiac MRI findings, and importantly, echocardiographic evidence of developing ventricular failure. Treatment involves stopping clozapine and empirical applications of steroids, diuretics, beta-blockers, and antiangiotensin agents. Mortality averages approximately 25 %. Safety of clozapine reuse remains uncertain. Systematic studies are needed to improve knowledge of the epidemiology, avoidance, early identification, and treatment of these adverse effects, with effective and practicable monitoring protocols.

Hedonic sensitivity to natural rewards is affected by prenatal stress in a sex-dependent manner.

Palatable food is a strong activator of the reward circuitry and may cause addictive behavior leading to eating disorders. How early life events and sex interact in shaping hedonic sensitivity to palatable food is largely unknown. We used prenatally restraint stressed (PRS) rats, which show abnormalities in the reward system and anxious/depressive-like behavior. Some of the hallmarks of PRS rats are known to be sex-dependent. We report that PRS enhanced and reduced milk chocolate-induced conditioned place preference in males and females, respectively. Male PRS rats also show increases in plasma dihydrotestosterone (DHT) levels and dopamine (DA) levels in the nucleus accumbens (NAc), and reductions in 5-hydroxytryptamine (5-HT) levels in the NAc and prefrontal cortex (PFC). In male rats, systemic treatment with the DHT-lowering drug finasteride reduced both milk chocolate preference and NAc DA levels. Female PRS rats showed lower plasma estradiol (E2 ) levels and lower DA levels in the NAc, and 5-HT levels in the NAc and PFC. E2 supplementation reversed the reduction in milk chocolate preference and PFC 5-HT levels. In the hypothalamus, PRS increased ERα and ERß estrogen receptor and CARTP (cocaine-and-amphetamine receptor transcript peptide) mRNA levels in males, and 5-HT2C receptor mRNA levels in females. Changes were corrected by treatments with finasteride and E2 , respectively. These new findings show that early life stress has a profound impact on hedonic sensitivity to high-palatable food via long-lasting changes in gonadal hormones. This paves the way to the development of hormonal strategies aimed at correcting abnormalities in the response to natural rewards.

L-acetylcarnitine causes rapid antidepressant effects through the epigenetic induction of mGlu2 receptors.

Epigenetic mechanisms are involved in the pathophysiology of depressive disorders and are unique potential targets for therapeutic intervention. The acetylating agent L-acetylcarnitine (LAC), a well-tolerated drug, behaves as an antidepressant by the epigenetic regulation of type 2 metabotropic glutamate (mGlu2) receptors. It caused a rapid and long-lasting antidepressant effect in Flinders Sensitive Line rats and in mice exposed to chronic unpredictable stress, which, respectively, model genetic and environmentally induced depression. In both models, LAC increased levels of acetylated H3K27 bound to the Grm2 promoter and also increased acetylation of NF-ĸB-p65 subunit, thereby enhancing the transcription of Grm2 gene encoding for the mGlu2 receptor in hippocampus and prefrontal cortex. Importantly, LAC reduced the immobility time in the forced swim test and increased sucrose preference as early as 3 d of treatment, whereas 14 d of treatment were needed for the antidepressant effect of chlorimipramine. Moreover, there was no tolerance to the action of LAC, and the antidepressant effect was still seen 2 wk after drug withdrawal. Conversely, NF-ĸB inhibition prevented the increase in mGlu2 expression induced by LAC, whereas the use of a histone deacetylase inhibitor supported the epigenetic control of mGlu2 expression. Finally, LAC had no effect on mGlu2 knockout mice exposed to chronic unpredictable stress, and a single injection of the mGlu2/3 receptor antagonist LY341495 partially blocked LAC action. The rapid and long-lasting antidepressant action of LAC strongly suggests a unique approach to examine the epigenetic hypothesis of depressive disorders in humans, paving the way for more efficient antidepressants with faster onset of action.

Intestinal permeability in children with recurrent respiratory and gastrointestinal symptoms.

AIM: Increased intestinal permeability has been reported in asthmatic subjects as well as in patients with gastrointestinal disease, thus suggesting the involvement of all the mucosal immune system. We aimed to assess intestinal permeability according to recurrent respiratory and gastrointestinal symptoms in children with asthma and children with functional gastrointestinal disorders (FGIDs). METHODS: In 108 outpatients aged 3-14 years (45 asthmatic, 63 with FGIDs), we measured the urinary lactulose/mannitol (L/M) ratio, performed allergy skin prick tests and administered questionnaires for recurrent respiratory and gastrointestinal symptoms starting from at least 2 months which persisted over the previous 4 weeks. L/M ratios were compared with previously reported normal values yielded by our chromatographic method (liquid chromatography-mass spectrometry). RESULTS: High L/M ratios (>0.030) were less frequent in asthmatic children than in children with FGIDs (9/45: 20% vs. 41/63: 65%, P < 0.001). High L/M ratios were associated with gastrointestinal symptoms in 8/9 asthmatic (P < 0.05) and 39/41 subjects with FGIDs (P < 0.005). L/M ratios were not associated with respiratory symptoms or atopy. In a regression model, a high L/M was predicted by low height, absence of asthma and presence of gastrointestinal symptoms (r = 0.72, P < 0.001). CONCLUSIONS: Increased intestinal permeability is associated with recurrent gastrointestinal symptoms rather than with recurrent respiratory symptoms in both asthmatic children and those with FGIDs. Our findings do not support the hypothesis of mucosal intestinal damage following an inflammatory stimulus in the respiratory mucosa.

A two years open-label prospective study of OnabotulinumtoxinA 195 U in medication overuse headache: a real-world experience.

BACKGROUND: The efficacy and safety of OnabotulinumtoxinA (BOTOX®) in adults with chronic migraine (CM) were demonstrated in the PREEMPT program. However, the dosage used in this study was flexible from 155 U to 195 U at the physician's discretion. Therefore, the objective of this prospective study was to compare the efficacy and safety of OnabotulinumtoxinA 195 U vs. 155 U for the treatment of CM and medication overuse headache (MOH) during a 2-year period. METHODS: We prospectively evaluated the mean reduction in headache days, migraine days, acute pain medication intake days and Headache Impact Test (HIT)-6 score in 172 patients injected with OnabotulinumtoxinA 195 U. Successively, we compared the efficacy measures with data of 155 patients injected with OnabotulinumtoxinA 155 U and followed up for 2 years. All patients were affected by CM and MOH, and failed one or more previous detoxification and preventative therapies. RESULTS: Both OnabotulinumtoxinA 195 U and 155 U reduced significantly the number of headache and migraine days, acute pain medication intake days and HIT-6 score, when compared with the baseline measures. Nevertheless, OnabotulinumtoxinA 195 U proved to be superior of 155 U in all efficacy measures since the first injection and for all the 2 years of treatment, with the exception of the reduction in pain medication intake days that resulted significantly larger with 195 U only after the 4th injection. The safety and tolerability of the two doses were similar and treatment related adverse events were transient and mild-moderate. CONCLUSIONS: This study represents the largest and longest post-marketing studies of doses comparison with OnabotulinumtoxinA in a real-life clinical setting. Here, we demonstrate the superior efficacy of OnabotulinumtoxinA 195 U compared to 155 U in CM patients with MOH during a 2-year treatment period with similar safety and tolerability profile.

Altered serum levels of kynurenine metabolites in patients affected by cluster headache.

BACKGROUND: The reported efficacy of memantine in the treatment of patients with cluster headache (CH) suggests that NMDA receptors are involved in mechanisms of nociceptive sensitization within the trigeminal system associated with CH. NMDA receptors are activated or inhibited by neuroactive compounds generated by tryptophan metabolism through the kynurenine pathway. In the accompanying manuscript, we have found that serum levels of all kynurenine metabolites are altered in patients with chronic migraine. Here, we have extended the study to patients affected by episodic or chronic CH as compared to healthy controls. METHOD: We assessed serum levels of kynurenine (KYN), kynurenic Acid (KYNA), anthranilic acid (ANA), 3-hydroxy-anthranilic acid (3-HANA), 3-hydroxykynurenine (3-HK), xanthurenic acid (XA), quinolinic acid (QUINA), tryptophan (Trp) and 5-hydroxyindolacetic acid (5-HIAA) by means of a liquid chromatography/tandem mass spectrometry (LC/MS-MS) method in 21 patients affected by CH (15 with episodic and 6 with chronic CH), and 35 age-matched healthy subjects. Patients with psychiatric co-morbidities, systemic inflammatory, endocrine or neurological disorders, and mental retardation were excluded. RESULTS: LC/MS-MS analysis of kynurenine metabolites showed significant reductions in the levels of KYN (-36 %), KYNA (-34 %), 3-HK (-51 %), 3-HANA (-54 %), XA (-25 %), 5-HIAA (-39 %) and QUINA (-43 %) in the serum of the overall population of patients affected by CH, as compared to healthy controls. Serum levels of Trp and ANA were instead significantly increased in CH patients (+18 % and +54 %, respectively). There was no difference in levels of any metabolite between patients affected by episodic and chronic CH, with the exception of KYN levels, which were higher in patients with chronic CH. CONCLUSION: The reduced levels of KYNA (an NMDA receptor antagonist) support the hypothesis that NMDA receptors are overactive in CH. A similar reduction in KYNA levels was shown in the accompanying manuscript in patients affected by chronic migraine. The reduced levels of XA, a putative analgesic compound, may contribute to explain the severity of pain attacks in CH. These data, associated with the data reported in the accompanying manuscript, supports a role for the kynurenine pathway in the pathophysiology of chronic headache disorders.

Altered kynurenine pathway metabolites in serum of chronic migraine patients.

BACKGROUND: Activation of glutamate (Glu) receptors plays a key role in the pathophysiology of migraine. Both NMDA and metabotropic Glu receptors are activated or inhibited by metabolites of the kynurenine pathway, such as kynureninic acid (KYNA), quinolinic acid (QUINA), and xanthurenic acid (XA). In spite of the extensive research carried out on KYNA and other kynurenine metabolites in experimental models of migraine, no studies have ever been carried out in humans. Here, we measured all metabolites of the kynurenine pathway in the serum of patients affected by chronic migraine (CM) and age- and gender-matched healthy controls. METHODS: We assessed serum levels of tryptophan (Trp), L-kynurenine (KYN), KYNA, anthranilic acid (ANA), 3-hydroxyanthranilic acid (3-HANA), 3-hydroxykynirenine (3-HK), XA, QUINA, and 5-hydroxyindolacetic acid (5-HIAA) in 119 patients affected by CM (ICHD-3beta criteria) and 84 age-matched healthy subjects. Patients with psychiatric co-morbidities, systemic inflammatory, endocrine or neurological disorders, and mental retardation were excluded. Serum levels of all metabolites were assayed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). RESULTS: LC-MS/MS analysis of kynurenine metabolites showed significant reductions in the levels of KYN (-32 %), KYNA (-25 %), 3-HK (-49 %), 3-HANA (-63 %), 5-HIAA (-36 %) and QUINA (-80 %) in the serum of the CM patients, as compared to healthy controls. Conversely, levels of Trp, ANA and XA were significantly increased in CM patients (+5 %, +339 % and +28 %, respectively). CONCLUSIONS: These findings suggest that in migraine KYN is unidirectionally metabolized into ANA at expenses of KYNA and 3-HK. The reduction in the levels of KYNA, which behaves as a competitive antagonist of the glycine site of NMDA receptors, is consistent with the hypothesis that NMDA receptors are overactive in migraine. The increase in XA, a putative activator of Glu2 receptors, may represent a compensatory event aimed at reinforcing endogenous analgesic mechanisms. The large increase in the levels of ANA encourages research aimed at establishing whether ANA has any role in the regulation of nociceptive transmission.