Selective activation and down-regulation of Trk receptors by neurotrophins in human neurons co-expressing TrkB and TrkC.

In the central nervous system, most neurons co-express TrkB and TrkC, the tyrosine kinase receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down-regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.

Fully human agonist antibodies to TrkB using autocrine cell-based selection from a combinatorial antibody library.

The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation.

Engineering a long acting, non-biased relaxin agonist using Protein-in-Protein technology.

The peptide hormone relaxin plays a critical role in tissue remodeling in a variety of tissues through activation of its cognate receptor, RXFP1. Relaxin's ability to modify extracellular matrices has provided a strong rationale for treating fibrosis in a variety of tissues. Treatment with recombinant relaxin peptides in clinical studies of heart failure has not yet proven useful, likely due to the short half-life of infused peptide. To circumvent this particular pharmacokinetic pitfall we have used a Protein-in-Protein (PiP) antibody technology described previously, to insert a single-chain human relaxin construct into the complementarity-determining region (CDR) of an immunoglobulin G (IgG) backbone, creating a relaxin molecule with a half-life of ∼4-5 days in mice. Relaxin-PiP biologics displaced Europium-labeled human relaxin in RXFP1-expressing cells and demonstrated full agonist activity on both human and mouse RXFP1 receptors. Relaxin-PiPs did not show signal transduction bias, as they activated cAMP in THP-1 cells, and cGMP and pERK signaling in primary human cardiac fibroblasts. In an induced carbon tetrachloride mouse model of liver fibrosis one relaxin-PiP, R2-PiP, caused reduction of liver lesions, ameliorated collagen accumulation in the liver with the corresponding reduction of Collagen1a1 gene expression, and increased cell proliferation in hepatic parenchyma. These relaxin biologics represent a novel approach to the design of a long-acting RXFP1 agonist to probe the clinical utility of relaxin/RXFP1 signaling to treat a variety of human fibrotic diseases.

Actions of the TrkB Agonist Antibody ZEB85 in Regulating the Architecture and Synaptic Plasticity in Hippocampal Neurons.

Signaling of BDNF via its TrkB receptor is crucial in regulating several critical aspects of the architecture and function of neurons both during development and in the adult central nervous system. Indeed, several neurological conditions, such as neurodevelopmental and neurodegenerative disorders are associated with alterations both in the expression levels of BDNF and TrkB, and in their intracellular signaling. Thus, the possibility of promoting BDNF/TrkB signaling has become relevant as a potential therapeutic intervention for neurological disorders. However, the clinical potential of BDNF itself has been limited due to its restricted diffusion rate in biological tissue, poor bioavailability and pharmacological properties, as well as the potential for unwanted side effects due to its ability to also signal via the p75NTR pathway. Several small molecule and biologic drug candidate TrkB agonists have been developed and are reported to have effects in rescuing both the pathological alterations and disease related symptoms in mouse models of several neurological diseases. However, recent side-by-side comparative studies failed to show their specificity for activating TrkB signaling cascades, suggesting the need for the generation and validation of improved candidates. In the present study, we examine the ability of the novel, fully human TrkB agonist antibody ZEB85 to modulate the architecture, activity and synaptic plasticity of hippocampal murine neurons under physiological conditions. Moreover, we show here that ZEB85 prevents ß-amyloid toxicity in cultured hippocampal neurons, in a manner which is comparable to BDNF.

Oregon's experience: evaluating the record.

Prior to passage of the Oregon Death with Dignity Act, opponents of assistance in dying argued that legalization would have serious harmful consequences. Specifically, they argued that the quality and availability of palliative care would decline, that the harms of legalization would affect certain vulnerable groups disproportionately, that legal assisted dying could not be confined to the competent terminally ill who voluntarily request assistance, and that the practice would result in frequent abuses. Data from Oregon's decade-long experience decisively refute the first three predictions. As to abuses, the record is not quite as clear, but if an appropriate framework for analysis is utilized, the most reasonable conclusion is that the risks of abuse do not outweigh the benefits of legalization. To the extent projected harmful consequences are relevant to the debate over legalization, Oregon's experience argues in favor of legalization of assistance in dying.

Risperidone in children with autism and serious behavioral problems.

BACKGROUND: Atypical antipsychotic agents, which block postsynaptic dopamine and serotonin receptors, have advantages over traditional antipsychotic medications in the treatment of adults with schizophrenia and may be beneficial in children with autistic disorder who have serious behavioral disturbances. However, data on the safety and efficacy of atypical antipsychotic agents in children are limited. METHODS: We conducted a multisite, randomized, double-blind trial of risperidone as compared with placebo for the treatment of autistic disorder accompanied by severe tantrums, aggression, or self-injurious behavior in children 5 to 17 years old. The primary outcome measures were the score on the Irritability subscale of the Aberrant Behavior Checklist and the rating on the Clinical Global Impressions - Improvement (CGI-I) scale at eight weeks. RESULTS: A total of 101 children (82 boys and 19 girls; mean [+/-SD] age, 8.8+/-2.7 years) were randomly assigned to receive risperidone (49 children) or placebo (52). Treatment with risperidone for eight weeks (dose range, 0.5 to 3.5 mg per day) resulted in a 56.9 percent reduction in the Irritability score, as compared with a 14.1 percent decrease in the placebo group (P<0.001). The rate of a positive response, defined as at least a 25 percent decrease in the Irritability score and a rating of much improved or very much improved on the CGI-I scale, was 69 percent in the risperidone group (34 of 49 children had a positive response) and 12 percent in the placebo group (6 of 52, P<0.001). Risperidone therapy was associated with an average weight gain of 2.7+/-2.9 kg, as compared with 0.8+/-2.2 kg with placebo (P<0.001). Increased appetite, fatigue, drowsiness, dizziness, and drooling were more common in the risperidone group than in the placebo group (P<0.05 for each comparison). In two thirds of the children with a positive response to risperidone at eight weeks (23 of 34), the benefit was maintained at six months. CONCLUSIONS: Risperidone was effective and well tolerated for the treatment of tantrums, aggression, or self-injurious behavior in children with autistic disorder. The short period of this trial limits inferences about adverse effects such as tardive dyskinesia.

Plasma pharmacokinetic characteristics of risperidone and their relationship to saliva concentrations in children with psychiatric or neurodevelopmental disorders.

BACKGROUND: Risperidone is a second-generation antipsychotic agent widely used in the treatment of schizophrenia and other psychotic disorders in adults. Risperidone is probably the most frequently used atypical antipsychotic in the pediatric population. OBJECTIVES: The goals of this study were to estimate the pharmacokinetic parameters of risperidone and its enantiomers in a pediatric population and explore relationships between saliva and plasma concentrations. METHODS: Eligible patients, between 4 and 15 years of age, included those taking a stable dose of oral risperidone ranging from 0.01 to 0.07 mg/kg BID for > or =4 weeks to treat psychiatric or neurodevelopmental conditions. A trough blood level and predose saliva sample were collected at study initiation; the regular risperidone dose was administered; and paired samples of blood and saliva were collected at 1, 2, 4, and 7 hours postdose. Plasma/saliva concentrations of risperidone and enantiomers of its principal active metabolite, 9-hydroxyrisperidone (9-OH-risperidone), were measured using a chiral liquid chromatography-tandem mass spectrometry assay. Standard pharmacokinetic parameters were calculated. Cytochrome P450 2D6 genotypes of *3,*4,*5 deletion and duplication were determined. RESULTS: The study included 19 patients (age range, 4 years 2 months to 15 years 11 months). Mean (SD) values for C(max), t(1/2), and AUC 0 to 12 hours for risperidone in plasma were 15.9 (22.2) ng/mL, 3.0 (2.3) h, and 92.1 (200.6) ng x h/mL, respectively. Corresponding values in saliva were 12.0 (21.0) ng/mL, 3.4 (3.2) h, and 27.8 (38.7) ng x h/mL, respectively. Mean (SD) plasma enantiomer values for C(max) and AUC calculated up to the last observation were: (+)-9-OH-risperidone, 13.6 (10.0) ng/mL and 73.6 (52.3) ng x h/mL; (-)-9-OH-risperidone, 4.9 (3.1) ng/mL and 29.3 (19.1) ng x h/mL. Corresponding enantiomer values in saliva were: (+)-9-OH-risperidone, 5.2 (8.8) ng/mL and 15.6 (8.9) ng x h/mL; (-)-9-OH-risperidone, 5.0 (7.9) ng/mL and 15.6 (9.1) ng x h/mL, respectively. Large interindividual variability in risperidone and enantiomer concentrations was noted. A highly significant relationship between predose plasma and predose saliva risperidone concentrations was observed. The logarithmic regression model indicated that the log risperidone saliva concentration = -0.100 + 0.594 x log plasma concentration (R(2) = 0.93 [Spearman]). CONCLUSIONS: In this preliminary pharmacokinetic study of parameters for risperidone and the enantiomers of 9-OH-risperidone in a pediatric population, mean C(max) and t(1/2) of risperidone were generally similar to those previously described in adults. The highly significant relationship between predose plasma and predose saliva risperidone concentrations suggests that saliva measurements may be a viable alternative to plasma sampling in children.

Acute and long-term safety and tolerability of risperidone in children with autism.

Treatment-emergent adverse events (AEs) were monitored during an 8-week, double-blind, placebo-controlled trial of risperidone (0.5-3.5 mg/day) in 101 children and adolescents with a lifetime diagnosis of autistic disorder. In addition, 37 placebo nonresponders received open-label risperidone for another 8 weeks. Of all the risperidone responders (n=65), 63 entered an open extension of another 16 weeks (6 months total risperidone exposure), and 32 of them were rerandomized to either continued risperidone therapy (n=16) or gradual replacement with placebo (n=16) over 8 weeks. We collected the following measures of safety and tolerability: (1) laboratory blood assessments (CBC with differential, electrolytes, and liver function tests) and urinalyses, (2) vital signs, (3) Side Effects Review of AEs thought to be associated with risperidone, (4) sleep records, (5) Simpson Angus Neurological Rating Scale (SARS), (6) Abnormal Involuntary Movement Scale (AIMS), and (7) height and weight. No clinically significant changes were found on the lab tests. During the 8-week acute trial, the most common AEs on the Side Effects Review, scored as moderate or higher, were as follows (placebo and risperidone, respectively): Somnolence (12% and 37%), enuresis (29% and 33%), excessive appetite (10% and 33%), rhinitis (8% and 16%), difficulty waking (8% and 12%), and constipation (12% and 10%). "Difficulty falling asleep" and anxiety actually favored the risperidone condition at statistically significant levels. The same AEs tended to recur through 6 months of treatment, although often at reduced levels. Using Centers for Disease Control (CDC) standardized scores, both weight and body mass index (BMI) increased with risperidone during the acute trial (0.5 and 0.6 SDs, respectively, for risperidone; 0.0 and 0.1 SDs, respectively, for placebo) and into open-label extension (0.19 and 0.16 SDs, respectively), although the amount of gain decelerated with time. Extrapyramidal symptoms, as assessed by the SARS, were no more common for drug than placebo, although drooling was reported more often in the risperidone group. There were no differences between groups on the AIMS. Two subjects had seizures (one taking placebo), but these were considered unrelated to active drug. Most AEs were mild to moderate and failed to interfere with therapeutic changes; there were no unanticipated AEs. The side effects of most concern were somnolence and weight gain.

Slaves, embryos, and nonhuman animals: moral status and the limitations of common morality theory.

Common morality theory must confront apparent counterexamples from the history of morality, such as the widespread acceptance of slavery in prior eras, that suggest core norms have changed over time. A recent defense of common morality theory addresses this problem by drawing a distinction between the content of the norms of the common morality and the range of individuals to whom these norms apply. This distinction is successful in reconciling common morality theory with practices such as slavery, but only at the cost of underscoring the limits of common morality theory, in particular its inability to resolve disputes about the moral status of entities. Given that many controversies in bioethics center on the disputed status of various entities, such as embryos and nonhuman animals, this is an important limitation. Nonetheless, common morality theory still can be a useful resource in diminishing moral conflict on issues that do not involve disputes over moral status.

Enhancements and justice: problems in determining the requirements of justice in a genetically transformed society.

There is a concern that genetic engineering will exacerbate existing social divisions and inequalities, especially if only the wealthy can afford genetic enhancements. Accordingly, many argue that justice requires the imposition of constraints on genetic engineering. However, it would be unwise to decide at this time what limits should be imposed in the future. Decision makers currently lack both the theoretical tools and the factual foundation for making sound judgments about the requirements of justice in a genetically transformed society. Moreover, focusing on the uncertain inequities of the future may result in failure to give priority to more pressing inequities of the present. Especially in a country that recently has enacted tax legislation that will widen existing wealth disparities, concern about the distant threat of a genetic aristocracy appears misplaced.

Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes.

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors-ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3)-in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors-CNTF, NGF and NT3-at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.

A double-blind, placebo-controlled withdrawal trial of dexmethylphenidate hydrochloride in children with attention deficit hyperactivity disorder.

OBJECTIVES: d,l-threo-methylphenidate HCl (D,L-MPH) is the most common treatment of attention deficit hyperactivity disorder (ADHD). A previous report showed placebo-controlled efficacy for the purified d-isomer (dexmethylphenidate hydrochloride, d-MPH, Focalin) with a 2:1 potency compared to dl, and suggested a 6-hour duration of action. This study complements that report by studying the effect of placebo-controlled discontinuation and retesting the duration of action. METHODS: A 6-week, open-label titration of d-MPH (2.5-10 mg twice-a-day) was followed by a double-blind, placebo-controlled, 2-week withdrawal study of responders. RESULTS: In the open titration, 82% of the 89 enrolled patients achieved a Clinical Global Impression-Improvement (CGI-I) rating of much or very much improved. Only 5 patients discontinued for adverse events. Seventy-five patients continued into the placebo-controlled discontinuation. For the randomly assigned d-MPH (n=35) and placebo (n=40) groups, mean ages, respectively, were 10.1 +/- 2.9 and 9.9 +/- 2.7 years, 86% and 78% were male, and 70.6% and 80.0% took the ceiling dose of 10 mg twice-daily, respectively. Each group had 80% combined type ADHD and 20% inattentive type. By the end of the 2-week, placebo-masked withdrawal, significantly more placebo patients (24 of 39) than d-MPH continuers (6 of 35) relapsed (61.5% versus 17.1%, p=0.001). Compared to d-MPH continuers, placebo patients deteriorated significantly more in the 2-week period on teacher ratings of the 18 ADHD symptoms rated 0-3 (p=0.028), the 3 p.m. and 6 p.m. parent ADHD symptom ratings (p=0.0026 and p=0.0381, respectively), and clinic (2-3 p.m.) and home (6 p.m.) Math Tests (p=0.024 and p<0.0001, respectively). The 6 p.m. scores replicated the significant effect at 6 hours reported in the previous study. CONCLUSIONS: d-MPH is safe, tolerable, and effective, with a 6-hour duration of effect suggested by the significant difference from placebo at 6 hours on a double-blind discontinuation.

Discontinuation of risperidone and reversibility of weight gain in children with disruptive behavior disorders.

Although atypical antipsychotics generally have a good side effect profile and are clinically very effective, weight gain and associated problems accompany their use. The authors followed up 14 subjects who were in studies of risperidone for management of disruptive behavior disorders. The subjects exited after a mean exposure of 8.9 months because of excessive weight gain, or excessive appetite, or insufficient clinical response. Weight was monitored for the full cohort before risperidone treatment, at termination, and (for various subgroups) at 3, 9-12, and 24 months after termination. Analysis of standardized weight scores in relation to standardized BMI scores suggested marked similarity between them at all time points. Comparison of standardized weights at time of drug termination with 3, 9-12, and 24 months after termination indicated that weight gain during risperidone treatment is reversible (i.e., significantly less weight after risperidone was discontinued) at all time points after termination. Furthermore, standardized weight at 12 and 24 months after discontinuation of risperidone was not distinguishable from standardized weight before risperidone. The prospect of reversibility may provide some comfort for clinicians and parents alike, but far more data are needed before an assumption can be made that this is the case for all children. The authors provide several recommendations for clinicians and researchers working with atypical antipsychotics.

Pharmacologic therapies aid treatment for autism.

In the absence of other guidelines, practitioners often prescribe by analogy with roles of psychotropic medicines in other psychiatric disorders (e.g., the ability of serotonergic antidepressants to reduce compulsive behavior). There is a slow but steady accumulation of data supporting the use of psychotropic medications to manage certain symptoms in children with autism. These data support the use of stimulant medications for attention/hyperactivity symptoms, with willingness to suspend such treatment if a trial is unsuccessful. Risperidone is supported for other disruptive behaviors, especially of an irritable/disruptive nature, but with attention to increases in appetite and weight. SSRIs and atypical antipsychotics may be helpful for a variety of perseverative behaviors, although one would seldom prescribe antipsychotic medication for mild perseverative behavior alone. SSRIs may be useful for anxiety. Again, there is no compelling evidence that existing pharmacologic treatments have a major role in treating the core symptoms of autism, especially the profound impairments in social interaction and communication. Further well-designed double-blind studies with significant numbers of subjects and defined target symptoms will provide the data that will guide therapeutic decisions in the future.