Successful private-public funding of paediatric medicines research: lessons from the EU programme to fund research into off-patent medicines.

UNLABELLED: The European Paediatric Regulation mandated the European Commission to fund research on off-patent medicines with demonstrated therapeutic interest for children. Responding to this mandate, five FP7 project calls were launched and 20 projects were granted. This paper aims to detail the funded projects and their preliminary results. Publicly available sources have been consulted and a descriptive analysis has been performed. Twenty Research Consortia including 246 partners in 29 European and non-European countries were created (involving 129 universities or public-funded research organisations, 51 private companies with 40 SMEs, 7 patient associations). The funded projects investigate 24 medicines, covering 10 therapeutic areas in all paediatric age groups. In response to the Paediatric Regulation and to apply for a Paediatric Use Marketing Authorisation, 15 Paediatric Investigation Plans have been granted by the EMA-Paediatric Committee, including 71 studies of whom 29 paediatric clinical trials, leading to a total of 7,300 children to be recruited in more than 380 investigational centres. CONCLUSION: Notwithstanding the EU contribution for each study is lower than similar publicly funded projects, and also considering the complexity of paediatric research, these projects are performing high-quality research and are progressing towards the increase of new paediatric medicines on the market. Private-public partnerships have been effectively implemented, providing a good example for future collaborative actions. Since these projects cover a limited number of off-patent drugs and many unmet therapeutic needs in paediatrics remain, it is crucial foreseeing new similar initiatives in forthcoming European funding programmes.

A comparative analysis exposes an amplification delay distinctive to SARS-CoV-2 Omicron variants of clinical and public health relevance.

ABSTRACTMutations in the SARS-CoV-2 genome may negatively impact a diagnostic test, have no effect, or turn into an opportunity for rapid molecular screening of variants. Using an in-house Emergency Use Authorized RT-qPCR-based COVID-19 diagnostic assay, we combined sequence surveillance of viral variants and computed PCR efficiencies for mismatched templates. We found no significant mismatches for the N, E, and S set of assay primers until the Omicron variant emerged in late November 2021. We found a single mismatch between the Omicron sequence and one of our assay's primers caused a > 4 cycle delay during amplification without impacting overall assay performance.Starting in December 2021, clinical specimens received for COVID-19 diagnostic testing that generated a Cq delay greater than 4 cycles were sequenced and confirmed as Omicron. Clinical samples without a Cq delay were largely confirmed as the Delta variant. The primer-template mismatch was then used as a rapid surrogate marker for Omicron. Primers that correctly identified Omicron were designed and tested, which prepared us for the emergence of future variants with novel mismatches to our diagnostic assay's primers. Our experience demonstrates the importance of monitoring sequences, the need for predicting the impact of mismatches, their value as a surrogate marker, and the relevance of adapting one's molecular diagnostic test for evolving pathogens.

Impact of the new european paediatric regulatory framework on ethics committees: overview and perspectives.

AIM: To evaluate the impact of the new European paediatric regulatory framework on the activities of Ethics Committees operating in Europe and to assess their involvement and interest in paediatric research. METHODS: Task-force in Europe for Drug Development for the Young Network of Excellence and Relating Expectations and Needs to the Participation and Empowerment of Children in Clinical Trials project set up an inventory of Ethics Committees existing in Europe and conducted a survey on their approach to paediatric trials. RESULTS: Ethics Committees operating in 22 European Countries participated in this survey. Results showed a high lack of knowledge, understanding and awareness of the current European paediatric regulatory framework and a lack of involvement of Ethics Committees in paediatric research, especially in terms of training and education, demonstrated also by the decreasing number of Ethics Committees answering exhaustively to the whole questionnaire. The majority of participating Ethics Committees expressed interest in future initiatives related to paediatric research. CONCLUSIONS: Despite a limited knowledge and understanding of the current paediatric regulatory framework, a significant number of Ethics Committees operating in Europe show interest in initiatives related to paediatric research. Networking may be an essential tool to be used to enhance Ethics Committees role in supporting paediatric research. Any initiative should be undertaken at European level in collaboration with European Union Institutions.

Development and implementation of a scalable and versatile test for COVID-19 diagnostics in rural communities.

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.

In-label and off-label use of respiratory drugs in the Italian paediatric population.

AIM: To evaluate the prescription rate of respiratory drugs (ATC code R03) in an Italian community setting and to estimate the extent of off-label use by both age and indication. METHODS: A cohort study aimed at evaluating prescriptions of drugs with ATC code R03 was conducted for the period 2002-2006. Data source was the PEDIANET Database. RESULTS: Ninety percent of R03 prescriptions are covered by 11 active substances or combinations, corresponding to 67 medicinal products. Inhaled corticosteroids are the most prescribed anti-asthmatic agents, followed by short-acting beta2 mimetics. The mean off-label rate is 19 and 56%, by age and indication respectively. The majority of off-label uses is among children under the age of 2. Five active substances are used at dosages not supported by adequate dose-finding studies. CONCLUSION: In Italy, many respiratory drugs are approved for the treatment of paediatric respiratory diseases, but a remarkable percentage of their prescriptions is off-label. This pharmaco-utilization study demonstrates that there is a need to perform clinical studies aimed at increasing the current knowledge on marketed paediatric drugs, and to revise and re-label the existing regulatory documents to reduce their off-label uses.

Model-Based Assessment of Alternative Study Designs in Pediatric Trials. Part I: Frequentist Approaches.

Alternative designs can increase the feasibility of pediatric trials when compared to classical parallel designs (PaD). In this work we present a model-based approach based on clinical trial simulations for the comparison of PaD with the alternative sequential, crossover, and randomized withdrawal (RWD) designs. Study designs were evaluated in terms of: type I and II errors, sample size per arm (SS), trial duration (TD), treatment exposures, and parameter estimate precision (EP). The crossover requires the lowest SS and TD, although it implies higher placebo and no treatment exposures. RWD maximizes exposure to active treatment while minimizing that to placebo, but requires the largest SS. SS of sequential designs can sometimes be smaller than the crossover one, although with poorer EP. This pharmacometric framework allows a multiscale comparison of alternative study designs that can be used for design selection in future pediatric trials.

Model-Based Assessment of Alternative Study Designs in Pediatric Trials. Part II: Bayesian Approaches.

This study presents a pharmacokinetic-pharmacodynamic based clinical trial simulation framework for evaluating the performance of a fixed-sample Bayesian design (BD) and two alternative Bayesian sequential designs (BSDs) (i.e., a non-hierarchical (NON-H) and a semi-hierarchical (SEMI-H) one). Prior information was elicited from adult trials and weighted based on the expected similarity of response to treatment between the pediatric and adult populations. Study designs were evaluated in terms of: type I and II errors, sample size per arm (SS), trial duration (TD), and estimate precision. No substantial differences were observed between NON-H and SEMI-H. BSDs require, on average, smaller SS and TD compared to the BD, which, on the other hand, guarantees higher estimate precision. When large differences between children and adults are expected, BSDs can return very large SS. Bayesian approaches appear to outperform their frequentist counterparts in the design of pediatric trials even when little weight is given to prior information from adults.

Tumor response and toxicity after single high-dose versus standard five-day divided-dose dactinomycin in childhood rhabdomyosarcoma.

This report deals with a randomized prospective multicentric clinical trial in childhood rhabdomyosarcoma (RMS) conducted to evaluate the toxicity and the effectiveness of dactinomycin (ACT-D) administered as high, single doses v five-day, divided doses administered in combination with vincristine (VCR) and cyclophosphamide (CYC). Fifty-five group III evaluable patients (pts) less than 15 years of age with tumor size greater than 5 cm in diameter, without high-risk features of CNS involvement, and 15 group IV RMS pts were randomized to receive VAC as primary chemotherapy (CT): VCR, 1.5 mg/m2 intravenously (IV) days 1 and 8; CYC, 275 mg/m2 IV days 1 through 5; and ACT-D, 0.45 mg/m2 IV days 1 through 5 every 28 days for three cycles (33 pts), or VAC-M: CYC, 150 mg/m2 intramuscularly (IM) days 1 through 7; VCR, 2.0 mg/m2 IV day 8; and ACT-D, 1.7 mg/m2 IV day 8 every 21 days for four cycles (37 pts). Major responses (complete plus partial responses [PR]) were obtained in 67% of the VAC pts and in 70% of the VAC-M pts. Toxic effects were low, and no increased toxicity was observed in pts treated with high, single-dose ACT-D. These results confirm the effectiveness and feasibility of single, high doses of ACT-D with the advantage of requiring less pt hospitalization.

Treatment of acute myelogenous leukemia in children: results of the Italian Cooperative Study AIEOP/LAM 8204.

One hundred thirty-three children with acute myelogenous leukemia (AML) entered the multicenter Pediatric Branch of the Italian Association Against Leukemia trial AIEOP/LAM 8204 between July 1982 and May 1986. Induction therapy consisted of two courses of daunomycin (DNM) plus cytosine arabinoside (Ara-C). Those patients who achieved remission were given four courses of consolidation with DNM, 6-thioguanine (6-TG) and escalated doses of Ara-C followed by six courses of sequential continuation therapy using monthly pairs: etoposide (VP-16)/Ara-C, Ara-C/6-TG, and DNM/Ara-C. Periodic intrathecal Ara-C was used for CNS prophylaxis. One hundred seven (80%) children achieved complete remission (CR). Kaplan-Meier estimates of 3-year disease-free survival (DFS) and event-free survival (EFS) are 41% and 33%, respectively. Relapses occurred in 34 patients after 5 to 97 weeks (32 marrow; 2 marrow plus CNS). Overall, 14 patients died of complications during treatment (nine during induction; five during the postremission phase), mostly from infection. Risk factor analysis showed that induction failures occurred predominantly in children with French-American-British (FAB) M5 and in those with elevated leukocyte counts; by step-up Cox analysis, only FAB subtype was predictive of remission success. None of the variables examined was significant for predicting the duration of remission. Hyperleukocytosis was predictive of a significantly worse EFS rate. These results are encouraging and further support the use of intensive chemotherapy programs for childhood AML.

Diazepam increases membrane fluidity of rat hippocampus synaptosomes.

Diazepam in vitro produced a concentration-dependent increase of membrane fluidity in crude synaptic membranes from rat hippocampus, but not cerebellum. Similar effects were obtained with higher concentrations of Ro 15-1788 and PK 11195, while zopiclone was completely inactive. In vivo acute treatment with diazepam and Ro 15-1788 gave results similar to those in vitro. The specific benzodiazepine antagonist also significantly increased membrane fluidity and was not able to reverse diazepam's effect. The data are discussed in terms of a possible role of protein kinase inhibition by the drugs not mediated by the 'central' or 'peripheral' type of benzodiazepine receptors.

The inhibitory effect of 8-OH-DPAT on the firing activity of dorsal raphe serotoninergic neurons in rats is attenuated by lesion of the frontal cortex.

The dose-response inhibitory effect of 8-OH-DPAT on the firing rate of dorsal raphe serotoninergic neurons was shifted 10-fold to the right after acute fronto-cortical deafferentation. This finding suggests that the inhibitory effect of 8-OH-DPAT on the dorsal raphe firing rate might be mediated indirectly by the frontal cortex.

Effect of antipsychotic drugs and selective dopaminergic antagonists on dopamine-induced facilitatory activity in prelimbic cortical pyramidal neurons. An in vitro study.

Intracellular recordings were obtained from 119 pyramidal neurons localized in prelimbic cortex, five in the dorsal cingulate cortex, one in the infralimbic cortex, one in the border of prelimbic and cingulate cortex and two in the border of prelimbic and infralimbic cortex. The passive membrane properties of these pyramidal neurons (i.e. resting membrane potential, input membrane resistance, shape of the tetrodotoxin-sensitive action potentials, spike frequency adaptation with a prominent postspike afterhyperpolarization, tetrodotoxin-sensitive inward rectification in the depolarizing direction and the absence of bursting) suggested that they resembled regular spiking or intrinsically bursting pyramidal neurons. Bath application of dopamine (EC50 of 1.8 microM) produced a reversible facilitatory effect on all 119 pyramidal neurons localized in the middle layer of the prelimbic cortex. No consistent change in membrane potential was detected during the application of dopamine. No effect of dopamine was noted on the nine pyramidal neurons that were not localized in the prelimbic cortex. The facilitatory effect of dopamine in prelimbic cortex was concentration dependently antagonized by haloperidol, risperidone, quetiapine, clozapine and by the selective D4 dopaminergic receptor antagonist L-745,870, but not by the selective D2/D3 dopaminergic receptor antagonist (-)-sulpiride. (+)-SCH 23390, which is a selective D1/D5 dopamine receptor antagonist, produced, similarly to dopamine, a facilitatory effect per se, and an additive effect when co-administered with dopamine. These results provide evidence that dopamine has a facilitatory effect specifically on pyramidal neurons localized in the middle layer of prelimbic cortex. Antipsychotic drugs and L-745,870 block this effect of dopamine.

Effect of long term amineptine treatment on pre- and postsynaptic mechanisms in rat brain.

The effect of amineptine and its two metabolites on monoamine uptake, release and receptor binding was studied in vitro. Amineptine and its two metabolites did not displace labelled ligands for known neurotransmitters and drug receptor sites. Amineptine and its two metabolites did not influence [3H]-5-hydroxytryptamine ([3H]-5-HT) uptake or release by rat brain synaptosomes. Amineptine inhibited [3H]-dopamine and [3H]-noradrenaline ([3H]-NA) accumulation, with IC50 values of 1.4 and 10 microM, respectively. The effect was retained, though with lower efficacy, by the two metabolites. Amineptine released [3H]-dopamine from preloaded synaptosomes. Metabolite 1 had no effect on catecholamine release, and metabolite 2 was about half as active as the parent compound on [3H]-dopamine release. The releasing effect of amineptine on [3H]-dopamine was potentiated by reserpine pretreatment, suggesting that the drug acts on the cytoplasmic neurotransmitter pool. Chronic treatment with amineptine (20 mg kg-1, twice daily for 15 days followed by a 3 days drug withdrawal period) resulted in a decrease of [3H]-spiperone binding sites in striatum, and of [3H]-dihyroalprenolol and [3H]-clonidine in cortex. Chronic treatment with amineptine reduced basal [3H]-dopamine accumulation in striatal synaptosomes, without affecting [3H]-NA or [3H]-5-HT accumulation. The adaptive changes in the pre- and postsynaptic dopamine mechanisms observed after long term treatment with amineptine are consistent with the drug acting as an indirect dopamine agonist. The down regulation of beta- and alpha 2-noradrenoceptors observed after long term amineptine treatment may play a role in the antidepressant activity of the drug.

Neuroleptic-like effects of the l-isomer of fenfluramine on striatal dopamine release in freely moving rats.

l-Fenfluramine (l-F) was studied for its ability to release dopamine (DA) and its metabolites in freely moving rats through the trans-striatal dialysis technique. l-F's effect on striatal DA release was also studied in animals made tolerant to the effect of haloperidol by chronic treatment (1 mg/kg i.p. twice daily for 11 days and 48 hr wash-out) with the neuroleptic or pretreated with 300 mg/kg i.p. gamma-butyrolactone (GBL). Five and 10 mg/kg l-F dose-dependently increased the release of DA and its metabolites with a pattern of effects similar to that observed with neuroleptic drugs. The dose of 20 mg/kg l-F had the same effect as 10 mg/kg. Repeated haloperidol treatment reduced the basal release of DA and its metabolites and a much smaller amount of DA and metabolites was released by l-F (10 mg/kg i.p.) and haloperidol (0.1 mg/kg i.p.) in animals treated with haloperidol than in controls. GBL 300 mg/kg i.p. reduced basal DA release by about 50%. When 10 mg/kg l-F, 0.1 mg/kg haloperidol and 0.25 mg/kg d-amphetamine were injected i.p. 40 min after GBL, l-F and haloperidol did not significantly raise DA release in GBL-treated rats whereas a significant effect was observed at various times after d-amphetamine. The data show that l-F resembles haloperidol in its ability to release DA and its metabolites from the corpus striatum of freely moving rats. The cross-tolerance between haloperidol and l-F for their effect on DA release suggests that a common site is involved in the mechanism of these drugs.

Stimulation of serotonin synthesis in rat brain after antiepilepsirine, an antiepileptic piperine derivative.

Piperine and two of its derivatives, antiepilepsirine (AE or 3,4-methylendioxycynnamoylpiperine) and compound 7448 (N-isopropyl 3 (4 chloro-phenyl) propenoylamide) are very effective in stimulating serotonin (5HT) synthesis. AE raises the ratio of free-to-bound tryptophan (TP) in plasma and induces a long-lasting increase of this aminoacid in brain. At the same time in striatum and limbic area it causes a lasting increase in 5 hydroxyindolacetic acid (5HIAA) a 5HT metabolite and to a lesser extent, an increase in the levels of the monoamine itself. Together with this action on 5HT metabolism we found that AE caused release of 3H-5HT from an in vitro synaptosomal preparation. It thus appears that piperine and its derivatives AE and compound 7148 affect the central serotonergic system.

Allogeneic vs autologous BMT vs intensive chemotherapy in childhood AnLL during first complete remission: AIEOP experience. AIEOP Cooperative Group.

From 1982 to 1990, 340 children with newly diagnosed ANLL entered two consecutive AIEOP trials: LAM 8204 (1982-1987) and LAM 87 (1987-1990). Patients in both studies received identical remission induction with Daunorubicin and ARA-C. In the first study (LAM 8204) 167/171 patients were consolidated with four courses of DAT, followed by six additional courses of continuation therapy with three drug pairs given sequentially. Periodic intra-thecal ARA-C was used for CNS prophylaxis. For patients remaining on protocol, the OFS and EFS probability at 8 years was 35% and 30%, respectively. Induction response and EFS were adversely predicted by FAB MS subtype and hyperleukocytosis. In LAM 8204 trial there were 30 withdrawals represented by patients undergoing allogeneic (14) or autologous (16) BMT. For these patients the DFS probability at 5 years was 64% and 50%, respectively. On LAM-87 trial, 136/169 patients were evaluable and 98 (76%) attained CR. After consolidation with one course of DAT, patients with an HLA-identical donor underwent allogeneic BMT and those lacking a matched donor were randomized to receive either autologous BMT or the LAM 8204 postremission chemotherapy. The 2-year probability of DFS for allografted patients was 76% significantly higher (P = 0.0001) than that observed for patients on chemotherapy (12%) or autologous BMT (31%) arms.

Acute changes in blood-CSF barrier permselectivity to serum proteins after intrathecal methotrexate and CNS irradiation.

Ten children affected by acute lymphoblastic leukaemia without CNS involvement were treated with a CNS prophylaxis protocol. Intrathecal methotrexate and CNS irradiation (60Co) administered at different times both induced an increase in blood-CSF barrier permeability to serum proteins (albumin, IgG, alpha 2 macroglobulin). The relationship between permeability coefficients of proteins was analysed by theoretical porous or vesicular blood-CSF barrier models. The analysis indicated that both therapeutic procedures affect endothelial pinocytosis. An increase in radius of pinocytotic vesicles from 400 to 1500 A seemed the most relevant change. The damage of endothelial barrier permselectivity could be involved in acute and late delayed toxic effects of intrathecal methotrexate and of CNS irradiation.

Effects of desipramine and maprotiline on the coeruleus-cortical noradrenergic system in anaesthetized rats.

Desipramine (1-3000 micrograms/kg i.v.) and maprotiline (1-10,000 micrograms/kg i.v.), two selective noradrenaline uptake blockers, inhibited the firing rate of noradrenergic locus coeruleus neurons and induced excitation of the prefronto-cortical neurons. The selective destruction of ascending noradrenergic pathways by intracerebroventricular injection of 6-hydroxydopamine antagonized the excitatory effect of desipramine and maprotiline on the prefronto-cortical neurons. These results suggest that desipramine and maprotiline dose dependently stimulate the electrical activity of prefronto-cortical neurons, probably by acting on noradrenergic cells of the locus coeruleus.

Effect of fluoxetine on the spontaneous electrical activity of fronto-cortical neurons.

The effect of fluoxetine on spontaneous extracellular activity of fronto-cortical neurons of chloral hydrate-anesthetized rats was investigated. Fluoxetine significantly increased the basal firing rate of cortical neurons in a dose-dependent manner (0.1-1000 micrograms kg-1 i.v.), with a maximum excitatory effect of 53% at 1000 micrograms kg-1. Selective destruction of ascending serotoninergic pathways induced by intracerebroventricular injections of 150 micrograms 5,7-dihydroxytryptamine, in desipramine-pretreated rats, antagonized the excitatory effect of fluoxetine. The present results suggest that fluoxetine significantly increases the electrical activity of the fronto-cortical neurons acting on serotoninergic uptake mechanisms localized at the level of raphe nuclei.

Repeated treatment with fluoxetine decreases the number of spontaneously active cells per track in frontal cortex.

Repeated (10 mg/kg i.p. x 14 days, once daily), but not acute (10 and 40 mg/kg i.p., 30 min), treatment with fluoxetine decreased the number of active neurons in the frontal cortex 30 min (-55%) or 24 h (-62%) after the last drug injection. This result supports the view that repeated, but not acute, treatment with fluoxetine decreases the function of the frontal cortex.