Strategic discussion on funding and access to therapies targeting rare diseases in Spain: an expert consensus paper.

BACKGROUND: In recent years, significant advances have been made in the field of rare diseases (RDs). However, there is a large number of RDs without specific treatment and half of these treatments have public funding in Spain. The aim of the FINEERR project was to carry out a multidisciplinary strategic discussion on the challenge of funding and access to RD-targeted drugs in Spain, in order to agree on specific proposals for medium-term improvement and hence support decision-making in the Spanish National Healthcare System (SNHS). RESULTS: The FINEERR Project was organized around a CORE Advisory Committee, which provided an overview, agreed on the design and scope of the project, and selected the members within each of four working groups (WG). Overall, 40 experts discussed and reached a consensus on different relevant aspects, such as conditioning factors for initial funding and access, evaluation and access to RD-targeted therapies, funding of these therapies, and implementation of a new funding and access model. From these meetings, 50 proposals were defined and classified by their level of relevance according to the experts. A descriptive analysis of responses was performed for each proposal. Thereafter, experts completed another questionnaire where they ranked the 25 most relevant proposals according to their level of feasibility of being implemented in the SNHS. The most relevant and feasible proposals were to improve: process of referral of patients with RDs, control over monitoring mechanisms, and communication between healthcare professionals and patients. CONCLUSIONS: The FINEERR project may provide a starting point for stakeholders involved in the process of funding and access to RD-targeted therapies in Spain to provide the necessary resources and implement measures to improve both the quality of life and life expectancy of patients with RDs.

Iconicity ratings for 10,995 Spanish words and their relationship with psycholinguistic variables.

The study of iconicity, or the resemblance between word forms and their meanings, has been the focus of increasing attention in recent years. Nevertheless, there is a lack of large-scale normative studies on the iconic properties of words, which could prove crucial to expanding our understanding of form-meaning associations. In this work, we report subjective iconicity ratings for 10,995 visually presented Spanish words from 1350 participants who were asked to repeat each of the words aloud before rating them. The response reliability and the consistency between the present and previous ratings were good. The relationships between iconicity and several psycholinguistic variables were examined through multiple regression analyses. We found that sensory experience ratings were the main predictor of iconicity, and that early-acquired and more abstract words received higher iconicity scores. We also found that onomatopoeias and interjections were the most iconic words, followed by adjectives. Finally, a follow-up study was conducted in which a subsample of 360 words with different levels of iconicity from the visual presentation study was auditorily presented to the participants. A high correlation was observed between the iconicity scores in the visual and auditory presentations. The normative data provided in this database might prove useful in expanding the body of knowledge on issues such as the processing of the iconic properties of words and the role of word-form associations in the acquisition of vocabularies. The database can be downloaded from https://osf.io/v5er3/ .

Spanish affective normative data for 1,406 words rated by children and adolescents (SANDchild).

Most research on the relationship between emotion and language in children relies on the use of words whose affective properties have been assessed by adults. To overcome this limitation, in the current study we introduce SANDchild, the Spanish affective database for children. This dataset reports ratings in the valence and the arousal dimensions for a large corpus of 1406 Spanish words rated by a large sample of 1276 children and adolescents from four different age groups (7, 9, 11 and 13 years old). We observed high inter-rater reliabilities for both valence and arousal in the four age groups. However, some age differences were found. In this sense, ratings for both valence and arousal decreased with age. Furthermore, the youngest children consider more words to be positive than adolescents. We also found sex differences in valence scores since boys gave higher valence ratings than girls, while girls considered more words to be negative than boys. The norms provided in this database will allow us to further extend our knowledge on the acquisition, development and processing of emotional language from childhood to adolescence. The complete database can be downloaded from https://psico.fcep.urv.cat/exp/files/SANDchild.xlsx .

Recognition memory advantage for negative emotional words has an early expiry date: Evidence from brain oscillations and ERPs.

Evidence from prior studies has shown an advantage in recognition memory for emotional compared to neutral words. Whether this advantage is short-lived or rather extends over longer periods, as well as whether the effect depends on words' valence (i.e., positive or negative), remains unknown. In the present ERP/EEG study, we investigated this issue by manipulating the lag distance (LAG-2, LAG-8 and LAG-16) between the presentation of old and new words in an online recognition memory task. LAG differences were observed at behavior, ERPs and in the theta frequency band. In line with previous studies, negative words were associated with faster reaction times, higher hit rates and increased amplitude in a positive ERP component between 386 and 564 ms compared to positive and neutral words. Remarkably, the interaction of LAG by EMOTION revealed that negative words were associated with better performance and larger ERPs amplitudes only at LAG-2. Also in the LAG-2 condition, emotional words (i.e., positive and negative words) induced a stronger desynchronization in the beta band between 386 and 542 ms compared to neutral words. These early enhanced memory effects for emotional words are discussed in terms of the Negative Emotional Valence Enhances Recapitulation (NEVER) model and the mobilization-minimization hypothesis.

A tissue-dependent hypothesis of dental caries.

Current understanding of dental caries considers this disease a demineralization of the tooth tissues due to the acid produced by sugar-fermenting microorganisms. Thus, caries is considered a diet- and pH-dependent process. We present here the first metagenomic analysis of the bacterial communities present at different stages of caries development, with the aim of determining whether the bacterial composition and biochemical profile are specific to the tissue affected. The data show that microbial composition at the initial, enamel-affecting stage of caries is significantly different from that found at subsequent stages, as well as from dental plaque of sound tooth surfaces. Although the relative proportion of Streptococcus mutans increased from 0.12% in dental plaque to 0.72% in enamel caries, Streptococcus mitis and Streptococcus sanguinis were the dominant streptococci in these lesions. The functional profile of caries-associated bacterial communities indicates that genes involved in acid stress tolerance and dietary sugar fermentation are overrepresented only at the initial stage (enamel caries), whereas other genes coding for osmotic stress tolerance as well as collagenases and other proteases enabling dentin degradation are significantly overrepresented in dentin cavities. The results support a scenario in which pH and diet are determinants of the disease during the degradation of enamel, but in dentin caries lesions not only acidogenic but also proteolytic bacteria are involved. We propose that caries disease is a process of varying etiology, in which acid-producing bacteria are the vehicle to penetrate enamel and allow dentin degrading microorganisms to expand the cavity.

Phase I and pharmacokinetic study of IV vinflunine in cancer patients with liver dysfunction.

Vinflunine is a novel tubulin-targeted agent that is currently indicated as a monotherapy in bladder cancer patients. The recommended dose of 320 mg/m(2) is given as an intravenous infusion once every 3 weeks. Vinflunine is metabolized through CYP3A4 and mainly eliminated via the feces. A phase I trial was designed to explore the tolerability and pharmacokinetics of vinflunine in cancer patients with ranging degrees of liver dysfunction (LD). A sequential design was used for patient accrual, with the objective of determining the maximum tolerated dose (MTD) and the recommended dose (RD) of vinflunine in 3 groups of increasing LD levels. Vinflunine and its only active metabolite 4-O-deacetylvinflunine were quantified in serial whole blood samples. PK parameters were derived and compared between LD groups and with a reference PK database. Vinflunine and 4-O-deacetylvinflunine PK parameters were not affected in any of the explored LD levels. Geometric mean values for vinflunine total clearance were 47.8, 37.5 and 45.4 L/h in the 3 groups of increasing degrees of LD, as compared to 42.5 L/h in reference patients with no LD. No relationship was found between vinflunine clearance and the presence or absence of cirrhosis, nor was it found with the presence or absence of liver metastasis or with liver-related biochemical parameters. Based on the observed tolerability profile, the recommended doses of i.v. vinflunine are 320 mg/m(2), 250 mg/m(2) or 200 mg/m(2) for patients with increasing degrees of liver dysfunction.

A phase 1 study of vinflunine in combination with trastuzumab for the treatment for HER2-positive metastatic breast cancer.

OBJECTIVE: To determine the recommended dose (RD) of vinflunine in combination with trastuzumab in human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC) and to investigate potential pharmacokinetic (PK) interactions. PATIENTS AND METHODS: In the first part of the study, two dose levels of vinflunine given every 3 weeks were explored (280 and 320 mg/m(2)) combined with trastuzumab (4 mg/kg loading dose and 2 mg/kg weekly). For each level of dose, six patients were enrolled to determine the RD for phase 2 studies (RP2S). In the second part of the study, 18 additional patients at RP2S have been evaluated to confirm safety and investigate preliminary antitumor activity. RESULTS: The RD was 320 mg/m(2) according to the dose escalation plan. Eleven of 15 additional patients who received this dose experienced dose-limiting toxicities, leading to a reduction in the RD to 280 mg/m(2). When compared to prior trials when vinflunine was used as a single agent, neither vinflunine total blood clearance nor trastuzumab serum concentrations were modified when the drugs were combined. All patients were evaluable, and the overall response rate was 73.3 % (95 % CI 54.1-87.7). The median progression-free survival was 11.3 months (95 % CI 9.4-21.0). At the dose of 280 mg/m(2), grade 3-4 neutropenia were seen in 4 patients (44.4 %) without febrile neutropenia. Non-hematologic grade 4 toxicities were not reported while grade 3 peripheral sensory neuropathy concerned 2 patients (22.2 %). CONCLUSION: The RD of vinflunine in combination with the standard regimen of trastuzumab is 280 mg/m(2) every 3 weeks. No mutual PK drug-drug interaction was seen. This regimen appears to be active with a favorable safety profile. Its role in HER2-positive MBC treatment needs to be defined in prospective comparative clinical trials.

Identifying a healthy oral microbiome through metagenomics.

We present the results of an exploratory study of the bacterial communities from the human oral cavity showing the advantages of pyrosequencing complex samples. Over 1.6 million reads from the metagenomes of eight dental plaque samples were taxonomically assigned through a binning procedure. We performed clustering analysis to discern if there were associations between non-caries and caries conditions in the community composition. Our results show a given bacterial consortium associated with cariogenic and non-cariogenic conditions, in agreement with the existence of a healthy oral microbiome and giving support to the idea of dental caries being a polymicrobial disease. The data are coherent with those previously reported in the literature by 16S rRNA amplification, thus giving the chance to link gene functions with taxonomy in further studies involving larger sample numbers.

Phase I dose-escalation study of vinflunine hard capsules administered twice a day for 2 consecutive days every week in patients with advanced/metastatic solid tumors.

BACKGROUND: Vinflunine is a new microtubule inhibitor of the vinca-alkaloid family. It is marketed in transitional cell carcinoma of urothelial tract as a 20 min infusion given every 3 weeks in Europe. METHODS: In this phase I study, vinflunine was administered to patients with advanced malignancies as hard capsules given twice a day on days 1-2 every week, with 3 weeks cycles. Serial blood samples were collected during the first cycle for pharmacokinetic investigations. RESULTS: Thirty-six patients (pts) were treated at 6 dose levels 150 (3 pts), 190 (3 pts), 230 (8 pts), 300 mg/day (6 pts) and then 250 (3 pts) and 270 mg/day (13 pts). The Maximal Tolerated Dose (MTD) was reached at 300 mg/day where 2 patients out of 6 experienced a dose limiting toxicity (febrile neutropenia with diarrhea). The lower dose level of 270 mg/day was the recommended dose (RD), the toxicity profile being mainly anaemia, neutropenia, fatigue and constipation. The pharmacokinetic analysis demonstrated the adequacy of the flat-fixed dosing regimen, as no correlation between clearance of vinflunine and body surface area was evidenced. Blood concentrations and exposure increased with dose, and a pharmacokinetic accumulation was observed, which is consistent with the terminal half-life of the compounds. The inter-individual exposure variability at the RD was 35%. CONCLUSION: Repeated weekly administration of oral vinflunine is feasible and exhibits a moderate inter-individual PK variability. The MTD was achieved at 300 mg/day given for 2 consecutive days. According to the protocol rules, the RD was established at 270 mg/day.

Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells.

AIMS/HYPOTHESIS: Recent experiments in liver and adipocyte cell lines indicate that palmitate can induce endoplasmic reticulum (ER) stress. Since it has been shown that ER stress can interfere with insulin signalling, our hypothesis was that the deleterious action of palmitate on the insulin signalling pathway in muscle cells could also involve ER stress. METHODS: We used C2C12 and human myotubes that were treated either with palmitate or tunicamycin. Total lysates and RNA were prepared for western blotting or quantitative RT-PCR respectively. Glycogen synthesis was assessed by [¹4C]glucose incorporation. RESULTS: Incubation of myotubes with palmitate or tunicamycin inhibited insulin-stimulated protein kinase B (PKB)/ v-akt murine thymoma viral oncogene homologue 1 (Akt). In parallel, an increase in ER stress markers was observed. Pre-incubation with chemical chaperones that reduce ER stress only prevented tunicamycin but not palmitate-induced insulin resistance. We hypothesised that ER stress activation levels induced by palmitate may not be high enough to induce insulin resistance, in contrast with tunicamycin-induced ER stress. Indeed, tunicamycin induced a robust activation of the inositol-requiring enzyme 1 (IRE-1)/c-JUN NH2-terminal kinase (JNK) pathway, leading to serine phosphorylation of insulin receptor substrate 1 (IRS-1) and a decrease in IRS-1 tyrosine phosphorylation. In contrast, palmitate only induced a very weak activation of the IRE1/JNK pathway, with no IRS1 serine phosphorylation. CONCLUSIONS/INTERPRETATION: These data show that insulin resistance induced by palmitate is not related to ER stress in muscle cells.

Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c.

Steatosis is an accumulation of triglycerides in the liver. Although an excessive availability of plasma fatty acids is an important determinant of steatosis, lipid synthesis from glucose (lipogenesis) is now also considered as an important contributing factor. Lipogenesis is an insulin- and glucose-dependent process that is under the control of specific transcription factors, sterol regulatory element binding protein 1c (SREBP-1c), activated by insulin and carbohydrate response element binding protein (ChREBP) activated by glucose. Insulin induces the maturation of SREBP-1c by a proteolytic mechanism initiated in the endoplasmic reticulum (ER). SREBP-1c in turn activates glycolytic gene expression, allowing glucose metabolism, and lipogenic genes in conjunction with ChREBP. Lipogenesis activation in the liver of obese markedly insulin-resistant steatotic rodents is then paradoxical. Recent data suggest that the activation of SREBP-1c and thus of lipogenesis is secondary in the steatotic liver to an ER stress. The ER stress activates the cleavage of SREBP-1c independent of insulin, thus explaining the paradoxical stimulation of lipogenesis in an insulin-resistant liver. Inhibition of the ER stress in obese rodents decreases SREBP-1c activation and lipogenesis and improves markedly hepatic steatosis and insulin sensitivity. ER is thus a new partner in steatosis and metabolic syndrome which is worth considering as a potential therapeutic target.

Diabetes and inflammation: fundamental aspects and clinical implications.

AIM: The aim of this paper is to provide the fundamental background of the inflammation theory associated with type 2 diabetes, to discuss the clinical consequences of low-grade inflammation, particularly in terms of cardiovascular risk, and to infer some clinical therapeutic strategies deriving from drugs that already exist or are in development. METHODS: This non-exhaustive work is the result of a Pubmed(®) research, based on requests including the following keywords: diabetes, inflammation, innate immunity, obesity, reticulum endoplasmic stress, cytokines, endothelial dysfunction. RESULTS: Obesity and type 2 diabetes are linked with a low-grade inflammation state that reflects the activation of innate immunity where metabolic, environmental and genetic factors are implicated. The role of endoplasmic reticulum stress and unfold protein response is underlined. Inflammation markers are predictive for the risk to develop diabetes, and are associated with an increased cardiovascular risk. While lifestyle modifications are followed by an improvement in inflammation markers, treatments inferred from the inflammation theory are of great interest, although quite moderate effects on glycaemic control have been observed with some of them. CONCLUSION: The development of molecules targeting different inflammatory mechanisms could lead in diabetic patients to improvement of both glycaemia and cardiovascular prognosis.

Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase.

AIMS/HYPOTHESIS: In rodent adipocytes, activated AMP-activated protein kinase reduces the lipolytic rate. As the hypoglycaemic drugs metformin and thiazolidinediones activate this enzyme in rodents, we tested the hypothesis that in addition to their known actions they could have an anti-lipolytic effect in human adipocytes. METHODS: Adipose tissue was obtained from individuals undergoing plastic surgery. Adipocytes were isolated and incubated with lipolytic agents (isoprenaline, atrial natriuretic peptide) and biguanides or thiazolidinediones. Lipolysis was quantified by the glycerol released in the medium. AMP-activated protein kinase activity and phosphorylation state were determined using standard procedures. RESULTS: In human adipocytes, isoprenaline and atrial natriuretic peptide stimulated the lipolytic rate three- to fourfold. Biguanides and thiazolidinediones activated AMP-activated protein kinase and inhibited lipolysis by 30-40%, at least in part by inhibiting hormone-sensitive lipase translocation to the lipid droplet. Inhibition of AMP-activated protein kinase by compound C precluded this inhibitory effect on lipolysis. Stimulation of lipolysis also induced an activation of AMP-activated protein kinase concomitant with a drop in ATP concentration. CONCLUSIONS/INTERPRETATION: We show for the first time in human adipocytes that biguanides and thiazolidinediones activate AMP-activated protein kinase, thus counteracting lipolysis induced by lipolytic agents. In addition, beta-agonist- or ANP-stimulated lipolysis increases AMP-activated protein kinase activity. This is because of an increase in the AMP/ATP ratio, linked to activation of some of the released fatty acids into acyl-CoA. AMP-activated protein kinase activation could represent a physiological means of avoiding a deleterious drain of energy during lipolysis but could be used to restrain pharmacological release of fatty acids.

[Emergence of invasive nonvaccine pneumococcal serotypes: 2 cases involving 19A and 10A serotypes]. / Emergence de pneumocoques invasifs de sérotype non vaccinal : à propos de 2 cas de sérotype 19A et 10A.

The authors report 2 cases of invasive pneumococcal disease. One case consisted in occult bacteremia due to a 19A serotype in a child who had received 3 doses and a booster dose of the heptavalent pneumococcal conjugate vaccine (Prevenar). The 2nd case was pneumococcal meningitis due to a 10A serotype. The incidence of nonvaccine serotypes is increasing, notably the 19A serotype. They must be closely monitored because of the high antimicrobial resistance of certain strains.

SREBP-1c transcription factor and lipid homeostasis: clinical perspective.

Insulin has long-term effects on glucose and lipid metabolism through its control on the expression of specific genes. In insulin sensitive tissues and particularly in the liver, the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) transduces the insulin signal. SREBP-1c is a transcription factor which is synthetized as a precursor in the membranes of the endoplasmic reticulum and which requires post-translational modification to yield its transcriptionally active nuclear form. Insulin activates the transcription and the proteolytic maturation of SREBP-1c. SREBP-1c induces the expression of a family of genes involved in glucose utilization and fatty acid synthesis and can be considered as a thrifty gene. Since a high lipid availability is deleterious for insulin sensitivity and secretion, a role for SREBP-1c in dyslipidaemia and type 2 diabetes has been considered in genetic studies and some association demonstrated. Finally, SREBP-1c could also participate to the hepatic steatosis observed in humans and related to alcohol consumption and hyperhomocysteinaemia, two pathologies which are concomitant with a stress of the endoplasmic reticulum and an insulin-independent SREBP-1c activation.

Simultaneous and minimally invasive assessment of muscle tolerance and bioavailability of different volumes of an intramuscular formulation in the same animals.

Evaluation of skeletal muscle tolerance during development of new drug formulations for i.m. use is most often based on terminal methods performed in the target species after slaughtering. The objective of this study was to evaluate the effect of muscle damage on the pharmacokinetic parameters of the drug delivered into the muscle using an alternative, noninvasive method. Phenylbutazone (PBZ) was used as the test article. Six ewes received increasing volumes of a 20% PBZ i.m. formulation, according to a cross-over design, and an i.v. bolus of the same formulation. Serial blood samples were taken, and a pharmacokinetic analysis of the plasma activity of creatine kinase and plasma PBZ concentrations was carried out. The amount of muscle damage after i.m. administration of 2, 4, or 8 mL of PBZ, calculated from the area under the curve of plasma creatine kinase across time was 36, 76, and 178 g for a 70-kg ewe. The corresponding absolute bioavailability of PBZ was 100 +/- 32%, 96 +/- 19%, and 100 +/- 17%, and the maximal PBZ concentrations were 42 +/- 3.4, 74 +/- 8.8, and 119 +/- 18.2 microg/mL. The plasma clearance of PBZ (i.v.) was 4.2 +/- 0.94 mL.kg(-1).h(-1). In conclusion, the absolute bioavailability of PBZ after i.m. administration was not altered by the increased volume of formulation administered despite the overall increase in the extent of muscle damage.

Time course of COX-1 and COX-2 expression during ischemia-reperfusion in rat skeletal muscle.

The aim of this study was to assess cyclooxygenase (COX)-1 and COX-2 expression in skeletal muscle after an ischemia-reperfusion (I/R). Male Sprague-Dawley rats were subjected to unilateral hindlimb ischemia for 2 h and then euthanized after 0, 1, 2, 4, 6, 10, 24, and 72 h of reperfusion. The COX protein and mRNA were assessed in control and injured gastrocnemius muscle. Muscle damage was indirectly determined by plasma creatine kinase activity and edema by weighing wet muscle. Creatine kinase activity in plasma increased as early as 1 h after reperfusion and returned to control levels by 72 h of reperfusion. Edema was observed at 6 and 10 h of reperfusion, but histological investigations showed an absence of tissular inflammatory cell infiltration. COX-1 mRNA was expressed in control muscle and was increased at 72 h of reperfusion, but the levels of associated COX-1 protein detected in control and injured gastrocnemius muscle were similar. COX-2 mRNA was not, or only slightly, detectable in control muscle and after I/R. In contrast, I/R induced major overexpression of COX-2 immunoreactivity at 6 and 10 h of reperfusion with a maximum at 10 h, whereas COX-2 protein was undetectable in control muscle. In conclusion, hindlimb I/R induced a large overexpression of COX-2 but not COX-1 protein between 6 and 10 h after injury. These results suggest a role for COX-2 enzyme in such pathophysiological conditions of the skeletal muscle.

Long chain fatty acyl-CoA synthetase 5 expression is induced by insulin and glucose: involvement of sterol regulatory element-binding protein-1c.

In a screen for sterol regulatory element-binding protein (SREBP)-1c target genes in the liver, we identified long chain fatty acyl-CoA synthetase 5 (ACS-5). Hepatic ACS-5 mRNA is poorly expressed during fasting and diabetes and strongly induced by carbohydrate refeeding and insulin treatment. In cultured hepatocytes, insulin and a high glucose concentration induce ACS-5 mRNA. Adenoviral overexpression of a nuclear form of SREBP-1c in liver of diabetic mice or in cultured hepatocytes mimics the effect of insulin to induce ACS-5. By contrast, a dominant negative form of SREBP-1c abolishes the effect of insulin on ACS-5 expression. The dietary and SREBP-1c-mediated insulin regulation of ACS-5 expression indicate that ACS-5 is involved in the anabolic fate of fatty acids.