A white paper on a neurodevelopmental framework for drug discovery in autism and other neurodevelopmental disorders.

In the last decade there has been a revolution in terms of genetic findings in neurodevelopmental disorders (NDDs), with many discoveries critical for understanding their aetiology and pathophysiology. Clinical trials in single-gene disorders such as fragile X syndrome highlight the challenges of investigating new drug targets in NDDs. Incorporating a developmental perspective into the process of drug development for NDDs could help to overcome some of the current difficulties in identifying and testing new treatments. This paper provides a summary of the proceedings of the 'New Frontiers Meeting' on neurodevelopmental disorders organised by the European College of Neuropsychopharmacology in conjunction with the Innovative Medicines Initiative-sponsored AIMS-2-TRIALS consortium. It brought together experts in developmental genetics, autism, NDDs, and clinical trials from academia and industry, regulators, patient and family associations, and other stakeholders. The meeting sought to provide a platform for focused communication on scientific insights, challenges, and methodologies that might be applicable to the development of CNS treatments from a neurodevelopmental perspective. Multidisciplinary translational consortia to develop basic and clinical research in parallel could be pivotal to advance knowledge in the field. Although implementation of clinical trials for NDDs in paediatric populations is widely acknowledged as essential, safety concerns should guide each aspect of their design. Industry and academia should join forces to improve knowledge of the biology of brain development, identify the optimal timing of interventions, and translate these findings into new drugs, allowing for the needs of users and families, with support from regulatory agencies.

Psychiatric diseases moving towards adolescents and children: How much room is there for extrapolation?

Since medicines for psychiatric diseases are often studied in adults first, it would be useful if data from efficacy trials in adults could be extrapolated to children and adolescents. However, it is not sufficient to adapt the adult dosages to achieve systemic exposure levels similar to those effective in adults. This can be done with increasing predictive accuracy but before accepting that the same plasma levels should result in the same efficacy as in adults both the mechanism of action of the drug and the pathophysiology of the disease must be considered. For psychiatric disorders there is often insufficient evidence to support the assumptions for extrapolating efficacy as it is not even always sure that the same diagnostic categories correspond to the same disease in adults and children. Even when the basic biological alteration behind the disorder could be considered the same, the psychodynamic consequences and the role of non-pharmacological approaches to treatment may substantially differ across age groups. These facts, together with the absence of detailed historical data on the actual correlations between paediatric and adult responses for many types of psycho-therapeutic medicines, make it difficult to accept extrapolation as the main proof of efficacy in children and adolescents. A corollary is that since efficacy studies will normally be required, they should not be unduly postponed. For products addressing a medical need with good scientific plausibility, they should be initiated as soon as the anticipated safety concerns can be reasonably managed within the context of a paediatric clinical trial.

Sex as a variable in medicines assessment reports for licensing in the European Union. Can gender bias be excluded?

PURPOSE: To analyse publically available official reports on the evaluation of European Union (EU) registration of new medicines (aimed at diseases affecting both sexes) in order to address the possibility of gender bias. METHODS: Descriptive study reviewing two types of assessment reports corresponding to different EU registration procedures: European Public Assessment Reports (EPARs) for the centralised procedures (399 reports considered from 2001 to 2002 and 2008 to 2009) and Mutual Recognition or Decentralised Public Assessment Reports (MPARs) for the mutual recognition and decentralised procedures (2,704 reports, totality until 2009-inclusive). RESULTS: Two hundred and twenty-four EPARs fulfilled the study criteria. It can be deduced from those EPARs that women participated in at least one clinical trial in 215 (78.5 %) of the reports. In 49(17.9 %) of the EPARs, the study population is explicitly evaluated according to sex, ten (3.6 %) presenting results disaggregated by sex. Most sex-related differences are in safety and pharmacokinetics; six of the 22 cases (72.7 %) of safety differences and nine of the 23 cases (60.9 %) of pharmacokinetic differences are not discussed. In relation to MPARs, only 2,704 out of 15,621 registries (17 %) were published, which casts doubts on the representativity of the available reports. Those published reflect less participation by women than in the EPARs. CONCLUSIONS: From the incomplete sex-related data made public, it is difficult to conclude on the existence of gender bias in the evidence used for the evaluation of new medicines. It certainly cannot be excluded. It is therefore recommended that compliance with existing guidance for publication of reports is increased, so as to transparently reassure on the issue.

Standard and intensive lipid-lowering therapy with statins for the primary prevention of vascular diseases: a population-based study.

PURPOSE: To describe the clinical profile of the patients that initiate statin therapy for the primary prevention of vascular diseases and to investigate the extent to which clinicians use intensive vs. standard regimens. METHODS: A cross-sectional analysis of nationwide individual data regarding individuals ≥ 11 years with a first prescription of statin, recorded between 1 January 2007 and 31 December 2011. Subjects were defined as intensive therapy initiators if a statin dose superior to simvastatin 40 mg (or equivalent dose if different statin) was first prescribed. Multivariable logistic regression models were built for dependent summary variables to evaluate the strength of the association between them and the use of intensive therapy. RESULTS: Overall, 69,737 patients receiving a first prescription of statin for the primary prevention of vascular diseases were identified. Predictors for intensive therapy initiation were male gender (adjusted OR: 1.28; 95%CI: 1.10-1.48), history of hypothyroidism (1.47; 1.17-1.85), current treatment of diabetes (1.18; 1.00-1.41), proteinuria (1.87; 1.12-3.12), age, and year of statin prescription. Modifiable risk factors associated with intensive therapy were elevated tryglicerides (1.63; 1.39-1.91), elevated LDL-C (1.96; 1.69-2.28), obesity (1.25; 1.07-1.47), smoking (1.32; 1.14-1.55), comedication with ezetimibe (3.76; 1.87-7.55), fibrates (1.96; 1.43-2.70) and calcium antagonists in women (1.42; 1.02-1.98). CONCLUSIONS: The use of intensive therapy with statins in primary prevention was not very high in absolute terms, but is increasing considerably. The association between intensive therapy and previous hypothyroidism or its combination with fibrates may raise additional safety and tolerability concerns.

Discovery and development of molecularly targeted drugs: regulatory perspectives.

Registration aims at ensuring that the requirements of quality, safety and efficacy for new medicines are met but how much evidence should be generated before considering that a favourable benefit/risk has been demonstrated may be controversial. Regulatory agencies tend to allow some uncertainty (to be resolved after registration) for potentially needed (as opposed to 'me too') substances for serious diseases and, in order to clarify the requirements, they issue 'guidance' documents. The 'Note for Guidance on Evaluation of Anticancer Medicinal Products in Man' of the European Medicines Agency (EMEA) came into operation in July 2003 but an update is announced by means of a Concept Paper both to address methodological aspects (including in relation to the new mechanisms of anticancer agents) and to integrate the new regulatory framework resulting from the recent Review of the European Legislation on Medicinal Products.

Interaction among alfaxalone, pregnenolone sulfate, and two GABAA agonists on hippocampal slices.

GABAA agonists do not respond to the same degree to allosteric modulators of the GABAA receptor complex such as benzodiazepines. We report there the effects of two steroids (alfaxalone and pregnenolone sulfate) on the inhibition induced by two GABAA agonists, 3-amino propane sulphonic acid (3-APS) and muscimol, on the extracellular evoked potentials obtained in CA1 of mice hippocampi. Alfaxalone (1 microM) potentiates the effects of both agonists, although incubation times longer than 15 minutes are required to potentiate the inhibitory effect of muscimol. Lower doses of pregnenolone sulfate at shorter incubation periods are able to inhibit the effects produced by single doses of 3-APS as compared to muscimol (15 microM during 5 min vs 30 microM during 5 min). Our results confirm the possibility that there might be differences in the interaction between GABAA agonists and modulatory steroids.

Gender and test dependence of a type of kappa mediated stress induced analgesia in mice.

1. In male mice, 80 inescapable footshocks (S-80) induce analgesic responses measured by the tail flick test that are blocked by naloxone and the kappa opioid antagonist, nor-binaltorphimine. We now study the nociceptive responses, induced after this particular stress, measured by the writhing test, the tail immersion test and a high intensity tail immersion test both in male and female mice. 2. In stressed males, analgesic responses are seen in all the nociceptive tests. Naloxone (10 mg/kg) does not prevent them. 3. In stressed females, in contrast with males, no analgesia is produced in the tail flick test. The writhing test and the tail immersion test registered analgesic responses that were not prevented by naloxone (10 mg/kg). 4. We conclude that only the antinociceptive kappa opioid mediated component of the stress we study is strongly dependent on gender, in contrast to other types of analgesia triggered by the same stress.

Opioid footshock-induced analgesia in mice acutely falls by stress prolongation.

The application of 80 footshocks (S-80) to mice induces a decrease in nociceptive responses as measured by the tail-flick test, which is opioid mediated as well as prevented by naloxone (10 mg/kg, SC). When the stress is prolonged up to 240 shocks (S-240) (i.e., from 6 min 40 s to 20 min), no analgesia can be seen immediately after the stress. We have examined the two most obvious possibilities, but they do not seem to be responsible for this fact. When morphine (1-5 mg/kg IP) is injected in the S-240 situation, a potentiation of its analgesic effects is seen, so that a desensitization of mu opioid receptors is unlikely. On the other hand, although cortisol (3-30 mg/kg IP) inhibits the analgesic response to S-80, metyrapone (40 and 80 mg/kg IP) and cortexolone (3-18 mg/kg IP) do not cause S-240 to be analgesic. Thus, an increase of endogenous glucocorticoids released during the long-duration stress does not seem responsible for the lack of analgesia after S-240.

Involvement of spinal kappa opioid receptors in a type of footshock induced analgesia in mice.

We have studied the effects of several opioid antagonists on a type of footshock stress-induced analgesia (FSIA) measured by the tail-flick test in male mice. Naloxone injected either subcutaneously (0.1-10 mg/kg) or intrathecally (1-20 micrograms) antagonized FSIA at higher doses than those that blocked a similar degree of analgesia induced by morphine. Intracerebroventricular (i.c.v.) naloxone (1-20 micrograms) did not modify the FSIA while antagonizing the i.c.v. morphine-induced analgesia. As a consequence, the antagonism of the FSIA by naloxone probably occurs at the level of the spinal cord and through receptors different than mu. The delta selective antagonist naltrindole (0.1-3 mg/kg s.c.) did not antagonize the analgesic effects of the stress. Nor-binaltorphimine, a kappa selective antagonist, blocked the FSIA when administered systemically (1-4 mg/kg i.p.) or locally (0.1-1 microgram i.t.). These results strongly suggest that spinal kappa opioid receptors are responsible for this type of endogenous analgesia.

Effects of some inhibitors of arachidonic acid metabolism in rat uterus in vitro.

The effect of four eicosanoid synthesis inhibitors (ESIs): mepacrine (6 x 10(-6) to 10(-4) M), nordihydroguaiaretic acid (NDHGA, 10(-6) to 10(-5) M), indomethacin (2 x 10(-6) and 2 x 10(-5) M) and imidazole (10(-5) and 10(-4) M), were studied on contractions induced by cumulative doses (4 and 20 mU/ml) of oxytocin (OT) on the uterus of rats both in natural estrus and ovariectomized. ESIs were also assayed on contractions induced by carbachol (10(-4) M) and KCl (60 mM) in rat uterus under natural estrus, and OT (10 mU/ml)-induced contractions in rat uterus incubated in calcium-free EDTA treated medium. Mepacrine, NDHGA and indomethacin, but not imidazol, inhibited in a dose-dependent way contractions induced by OT in the uterus. The effect was higher in ovariectomized than in natural estrus rats. Mepacrine and NDHGA, but not indomethacin or imidazole, inhibited contractions induced by carbachol and relaxed tonic contractions to KCl (60 mM). Mepacrine, NDHG and indomethacin also inhibited tonic contractions by OT in calcium-free EDTA treated medium. Our results suggest that mepacrine, NDHGA and indomethacin, independently of inhibition of eicosanoids synthesis, reduce the entry of extracellular calcium and/or the release of intracellular calcium.

[Calcium dependence of the contractile response of the aorta in the rat, rabbit and guinea pig and in the human uterine artery]. / Dependencia del calcio de la respuesta contráctil de aorta de rata, conejo y cobaya y arteria uterina humana.

The influence of extracellular Ca2+ on the contraction produced by noradrenaline (NA) (3 x 10(-6) M), KCl (60 mM) and BaCl2 (30 mM) on human uterine arteries (AUH) and aortic strips from rats, rabbits and guinea-pigs have been studied. The vessels were cut spirally and incubated in Krebs solution containing 2.5 mM Ca2+ (KN), 0 mM Ca2+ (K-0Ca) or 0 mM Ca2+ + 3 mM EDTA (K-EDTA). Both phases (fast and slow) of the response of aortic strips to NA and of the AUH to NA, KCl and BaCl2 were significantly smaller in solutions without Ca2+. Only in rabbit aortic strips the slow phase was significantly more reduced than the fast phase. Overall, the contractions of the rat aortic strips were most resistant to the absence of extracellular Ca2+. These results confirm the variability of the responses of blood vessels from different vascular beds and species to the removal of extracellular Ca2+.

Effects of vanadate, ouabain and amiloride on the contraction of the rat testicular capsule to oxytocin.

1. The modification of the contraction of the rat testicular capsule to oxytocin (OT) by vanadate (0.7, 7 and 70 microM), ouabain (0.1 mM), and amiloride (10 microM to 1 mM) have been studied. 2. OT (1 nM-6 microM) and vanadate (10 microM-3 mM) induced contraction of the rat testicular capsule in a dose-dependent way (ED50: 188 +/- 66 nM and 82.8 +/- 7.4 microM, respectively). 3. Vanadate (0.7, 7 and 70 microM) and ouabain (0.1 mM) increases the contractile effect of OT (50 and 200 nM). 4. Amiloride (10 microM-1 mM) inhibit, in a dose-dependent way, the OT-contraction. 5. Amiloride (10 microM or 50 microM) block the ouabain but not the vanadate potentiation to OT.

Influences of sodium on the contraction induced by oxytocin in rat testicular capsule.

1. The effect of tetrodotoxin (5 microM), monensin (10 microM) and the replacement of Na+ by choline (choline medium) on the contractions of the rat testicular capsule induced by oxytocin (50 and 200 nM) have been studied. 2. The sodium channel blocker tetrodotoxin did not modify the oxytocin contraction. 3. The sodium ionophore monensin produces contraction of rat testicular capsule and reduces the oxytocin-induced contraction. The monensin contraction is inhibited by amiloride (0.1 mM). 4. Replacement of Na+ by choline increases the contraction induced by oxytocin and KCl (60 mM) but inhibits that induced by noradrenaline (3 microM). 5. The increase of contraction due to oxytocin in choline medium is inhibited by amiloride (50 microM and 1 mM) and when calcium is suppressed of the incubation medium.

Effects of vanadate in testicular capsule of the rat.

1. The effects of sodium orthovanadate (vanadate. 10(-5) to 3 x 10(-4) M) on testicular capsule of the rat, and the modifications of these effects by the calcium chelator EGTA (2mM), the calcium entry blockers verapamil (5 X 10(-5) M), nifedipine (10(-5 M) and diltiazem (5 x 10(-5) M), the (Na+ + K+)-ATPase inhibitor ouabain, the Na+/Ca2+ exchange inhibitor amiloride, and the calmodulin antagonists trifluoperazine (10(-4) M) and W-7 (5 x 10(-5) M) have been studied. 2. Vanadate induced contraction of the rat testicular capsule in a dose-dependent way (ED50: 82.8 +/- 7.4 x 10(-6) M). 3. The contraction induced by vanadate (3 and 30 x 10(-5) M) were abolished by EGTA and not modified by verapamil, nifedipine, flunarizine or diltiazem. 4. Amiloride (1 and 5 x 10(-5) M), but not ouabain (5 x 10(-5) and 10(-4) M), inhibit in a dose-dependent way the contraction induced by two doses of vanadate (3 and 30 x 10(-5) M). 5. Trifluoperazine and W-7 significantly inhibit the contraction of testicular capsule to 3 and 30 x 10(-5) M vanadate.

Effects of tyramine on the human uterine artery in vitro.

1. We have studied the effects of tyramine on the human uterine artery (HUA) in order to assess its site of action. 2. Tyramine (5 x 10(-5) to 10(-3) M) contracts the isolated human uterine artery. Tachyphylaxis appeared when concentration-response curves were repeated and the contraction was diminished by prazosin (10(-8), 10(-7) and 10(-6) M). The maximal contraction induced by tyramine (10(-3) M) was 25% of the maximal response to noradrenaline (10(-5) M). 3. After 2 hr of tyramine perfusion a decrease of the contractile response to KCl, 30 and 60 mM (15.0 and 12.9%) and noradrenaline 10(-6) M (83.2%) is shown. 4. However, when tyramine was previously added for 3 min to the bath, the response to KCl increased while the response to noradrenaline was lower. 5. A possible postsynaptic antagonistic effect for tyramine in the HUA is suggested in addition to its usual presynaptic effect.

Effects of inhibitors of eicosanoid synthesis in the uterus of ovariectomized rats and rats in natural oestrus: relation with calcium.

1. The effects of 4 inhibitors of eicosanoid synthesis (ESIs)--mepacrine (MEP, 10(-5) - 3 x 10(-4) M), nordihydroguaiaretic acid (NDHGA, 10(-6) - 2 x 10(-5) M), indomethacin (IND, 2 x 10(-6) M and 2 x 10(-5) M) and imidazole (IMI, 10(-5) M and 10(-4) M)--on the motility induced by oxytocin (OT, 0.5 and 4 mU/ml) in uterus of rats in natural oestrus and of ovariectomized rats have been studied. 2. MEP, NDHGA and IND, but not IMI, inhibited the motility induced by both concentrations of OT in natural oestrus. Ovariectomy enhanced the effects of all ESIs, except the one of MEP. 3. MEP and NDHGA, but not IND or IMI, inhibited the contractions induced by methacholine (10(-5) M) and prostaglandin F2a (10(-6) M) and relaxed in a dose-dependent way the tonic component of the contractile response to KCl 60 mM (DI50: 6.14 +/- 0.38 and 1.38 +/- 0.29 x 10(-5) M, respectively). 4. CaCl2 (0.1-10 mM) reverted the relaxation of KCl contractions produced by MEP but not by NDHGA.

Mechanism of nafazatrom-induced inhibition of rat uterus contractions in vitro.

Nafazatrom inhibits, in a dose-dependent way, the amplitude and frequency of the rhythmic contractions induced by oxytocin (4 mU/ml), as well as the methacholine (10(-5) M)- and CaCl2 (10 mM)-induced contractions, and the phasic response to KCl (60 mM); similarly, it inhibits the tonic contraction induced by KCl (60 mM) and oxytocin (10 mU/ml). A single concentration of nafazatrom (10(-4) M) also inhibits the uterine contractions caused by carbachol (10(-4) M) and prostaglandin F2 alpha (10(-6) M). CaCl2 (0.1-10 mM) only partially reverses the inhibition produced by nafazatrom on the KCl-induced tonic contractions. Bay K 8644 (3 x 10(-10)-3 x 10(-7) M) reverses the inhibition by 10(-4) M but not by 3 x 10(-4) M of nafazatrom on the CaCl2-induced contractions. Nafazatrom also inhibits, in a dose-dependent way, the calmodulin-dependent phosphodiesterase activity. Our results would suggest that, independently of its 5-lipoxygenase blocking activity, nafazatrom inhibits the contractions of the rat uterus by inhibiting the calmodulin activity and, presumably, by reducing the influx of extracellular calcium and/or the mobilization of intracellular calcium.

Interactions between oxytocin- and calcium-modifying agents in the rat testicular capsule in vitro.

We have studied the effect of drugs which affect the movement of calcium on the contractions induced by 50 and 200 nM oxytocin in the isolated testicular capsule of the rat. The ED50 for oxytocin in this preparation was 188 (+/- 66 S.E.) nM and the maximal contraction induced by oxytocin was smaller than that obtained with 10 microM of the calcium ionophore, A23187. Lanthanum (10 mM), cobalt (2 mM), EGTA (3.5 and 5 nM, 30 s exposure) and a decrease in the calcium concentration of the medium reduced the oxytocin response. The response was completely abolished after prolonged incubation with EGTA (2 mM) in a calcium-free medium. The calcium blocking agents, nifedipine and flunarizine, and the agonist, Bay K 8644, did not modify the responses to oxytocin. Verapamil, at possibly non-specific doses (10 microM), reduced the contractions while diltiazem (0.1 mM), in a prazosin (10 microM)-resistant way, and nickel (0.1 mM) increased them. Both modifiers of intracellular calcium that were used namely TMB-8 (10 microM), in a calcium-free medium, and dantrolene sodium (10 and 30 microM), with and without calcium in the medium, decreased the oxytocin response. On the whole, it seems as if both intra- and extracellular calcium were involved in the contractile effect of oxytocin, although extracellular calcium does not seem to gain access to the cell through voltage-dependent calcium channels sensitive to selective calcium entry blockers.

Sex-related differences in the effects of morphine and stress on visceral pain.

The effect of morphine (in the absence of stress and after a type of naloxone-sensitive swim-stress) on the painful contractions induced by intraperitoneal injection of hypertonic saline was studied in four groups of rats: normally cycling females in oestrus, males, ovariectomized females and females subjected to constant light, to induce a hyperoestrogenemic state. In the absence of swim-stress, the sensitivity to morphine was maximum for males (i.e. analgesia was produced with the smallest doses). After swim-stress, both males and ovariectomized rats showed less sensitivity to the analgesic effect of morphine than normal females in oestrus, while females subjected to constant light were less sensitive. In females in natural oestrus, but not in the other groups, small doses (0.05 mg/kg) of morphine, paradoxically reduced the analgesic effect of swim-stress considerably. These results show the importance of sex-related factors in the sensitivity of visceral pain to morphine and stress. A selective role for endogenous androgens and oestrogens is likely.

Effects of cortisol on GABAA receptor-mediated responses compared in guinea-pig ileum and rat cuneate nucleus.

Submaximal contractions of the isolated guinea-pig ileum by the GABAA receptor agonist muscimol were enhanced by about 30% in the presence of 1-100 pM cortisol. At 10 nM cortisol, the enhancement was much less and, at 1 microM, the responses to muscimol were reduced by about 50%. Corticosterone had similar effects but it was 100-1000 times less potent. In contrast, submaximal depolarization responses of the slice preparation of the cuneate nucleus of the rat to muscimol were unaffected by 1 pM-1 nM cortisol and only slightly enhanced (about 15%) by 1 microM cortisol. Also, enhancements of 70-120% by 1 microM alphaxalone were undiminished in the presence of 1 microM cortisol. Thus, there is no evidence for a potent effect of cortisol on the GABAA receptor complex in the cuneate nucleus of the rat.