Douglas E. Brenneman

Neuroprotection : A comparative view of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide

The neuroprotective properties of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) place these peptides in a special category of ligands that have implications for our understanding of pathological conditions as well as a potential basis for therapeutic intervention. It is remarkable that these peptides have a protective impact against such a wide variety of clinical relevant toxic substances. This protective diversity is consistent with the multiple pathways that are activated or inhibited by the action of these peptides. Although knowledge is emerging on the neuroprotective mechanisms of VIP and PACAP, it is already evident that these two peptides are not identical in their action and each peptide has multiple mechanisms that allow for neuroprotective diversity. The multiple intracellular signaling pathways and differing extracellular mediators of neuroprotection contribute to this diversity of action. In this review, examples of neuroprotective actions will be presented that serve to demonstrate the remarkable breadth of neuroprotective processes produced by VIP and PACAP.

Reduction of dominant or submissive behaviors as models for antimanic or antidepressant drug testing: Technical considerations

Technical variants of mania and depression models that were based on dominant-submissive relationships (DSR) have been analyzed and compared in the present paper. In these paradigms, one animal of a pair developed the behavioral trait of dominance while the other submissiveness in a food competition test after repeated interactions in a specially designed apparatus. Data collection methods and timelines have been compared in variants of the DSR-based models. In addition, different selection criteria to assign dominant or submissive status to animals and two different scoring systems were evaluated. The importance of the selection criteria for DSR stability has been emphasized. Our data showed that (1) only animals selected with the strict criteria form clear dominant and submissive relationships that hold throughout the study period, (2) submissive animals were influenced by fluoxetine and dominant animals were influenced by sodium valproate similarly in pairs scored by human observer and by a video-tracking system. These studies indicate that the model variant using stringent selection criteria and automatic scoring was the most reliable for use in depression-related studies.

Carisbamate, a novel neuromodulator, inhibits voltage-gated sodium channels and action potential firing of rat hippocampal neurons

Carisbamate (RWJ-333369; (S)-2-O-carbamoyl-1-o-chlorophenyl-ethanol) is a novel investigational antiepileptic drug that exhibits a broad-spectrum of activity in a number of animal models of seizure and drug refractory epilepsy. In an effort to understand the molecular mechanism by which carisbamate produces its antiepileptic actions, we studied its effects on the function of voltage-gated, rat brain sodium and potassium channels and on the repetitive firing of action potentials in cultured rat hippocampal neurons. In whole-cell patch clamp recording, carisbamate resulted in a concentration-, voltage- and use-dependent inhibition of rat Nav1.2, with an IC(50) value of 68 microM at -67 mV. In rat hippocampal neurons, carisbamate similarly blocked voltage-gated sodium channels, with an IC(50) value of 89 microM at -67 mV, and inhibited repetitive firing of action potentials in a concentration-dependent manner (by 46% at 30 microM and 87% at 100 microM, respectively). Carisbamate had no effect on the steady-state membrane potential or voltage-gated potassium channels (K(v)) in these neurons. These inhibitory effects of carisbamate occurred at therapeutically relevant concentrations in vivo, raising the possibility that block of voltage-gated sodium channels by carisbamate contributes to its antiepileptic activity.

Vasoactive intestinal peptide. Link between electrical activity and glia-mediated neurotrophism.

Abstract: Vasoactive intestinal peptide has neurotrophic and neuroprotective properties that influence the survival of activity‐dependent neurons in the central nervous system. Investigations of the mechanism of this neurotrophic peptide indicated that these actions are contingent on interactions with astroglia. The complex mixture of neurotrophic mediators released from astroglia include cytokines, a protease inhibitor, and activity‐dependent neurotrophic factor, a protein with apparent structural similarities to hsp60. Investigations of ADNF resulted in the discovery of active peptides of extraordinary potency and broad neuroprotective properties. These studies indicate that a nine‐amino acid core peptide of ADNF had significantly greater neuroprotective properties in comparison to the parent growth factor and these advantages identify ADNF‐9 as an attractive lead compound for drug development.

Novel, Broad-Spectrum Anticonvulsants Containing a Sulfamide Group: Advancement of N-((Benzo[b]thien-3-yl)methyl)sulfamide (JNJ-26990990) into Human Clinical Studies

In seeking broad-spectrum anticonvulsants to treat epilepsy and other neurological disorders, we synthesized and tested a group of sulfamide derivatives (4a-k, 5), which led to the clinical development of 4a (JNJ-26990990). This compound exhibited excellent anticonvulsant activity in rodents against audiogenic, electrically induced, and chemically induced seizures, with very weak inhibition of human carbonic anhydrase-II (IC(50) = 110 microM). The pharmacological profile for 4a supports its potential in the treatment of multiple forms of epilepsy, including pharmacoresistant variants. Mechanistically, 4a inhibited voltage-gated Na(+) channels and N-type Ca(2+) channels but was not effective as a K(+) channel opener. The pharmacokinetics and metabolic properties of 4a are discussed.

Different levels of γ-synuclein mRNA in the cerebral cortex of dominant, neutral and submissive rats selected in the competition test

Synucleins are small proteins regulating the filamentous network that in turn influences the release of dopamine and glutamate neurotransmitters involved in mood and motivation processes. We have studied the pattern of synuclein expression in animal models for mania and depression. Dominant behavior, as defined in a food competition test with dyads of rats, can serve as a model of mania and submissive behavior as a model of depression. The expression of α‐, β‐ and γ‐synuclein was analyzed in four regions of cortex from dominant, neutral and submissive rats using TaqMan reverse transcription‐polymerase chain reaction technology. The expression levels of γ‐synuclein were elevated consistently in all regions of cerebral cortex of dominant rats (Pu2003<u20030.05; 23.5u2003±u20031.1, normalized units) in contrast to the submissive rat group (10.3u2003±u20031.2). Neutral rats had intermediate cerebral cortex levels of γ‐synuclein expression (15.7u2003±u20031.4) that were significantly lower than that in dominant rats (Pu2003<u20030.05). No changes in α‐ or β‐synuclein expression were observed among the groups. These studies indicate that γ‐synuclein levels in the cerebral cortex were differentially associated with dominant and submissive behavior.

Macrocyclic BACE inhibitors: Optimization of a micromolar hit to nanomolar leads.

We have identified macrocyclic inhibitors of the aspartic protease BACE, implicated in the etiology of Alzheimers disease. An X-ray structure of screening hit 1 in the BACE active site revealed a hairpin conformation suggesting that constrained macrocyclic derivatives may also bind there. Several of the analogs we prepared were >100x more potent than 1, such as 7 (5 nM K(i)).

Neonatal mice of the down syndrome model, Ts65Dn, exhibit upregulated VIP measures and reduced responsiveness of cortical astrocytes to VIP stimulation

The Ts65Dn segmental mouse model of Down syndrome (DS) possesses a triplication of the section of chromosome 16 that is most homologous to the human chromosome 21 that is trisomic in DS. This model exhibits many of the characteristics of DS including small size, developmental delays, and a decline of cholinergic systems and cognitive function with age. Recent studies have shown that vasoactive intestinal peptide (VIP) systems are upregulated in aged Ts65Dn mice and that VIP dysregulation during embryogenesis is followed by the hypotonia and developmental delays as seen in both DS and in Ts65Dn mice. Additionally, astrocytes from aged Ts65Dn brains do not respond to VIP stimulation to release survival-promoting substances. To determine if VIP dys-regulation is age-related in Ts65Dn mice, the current study examined VIP and VIP receptors (VPAC-1 and VPAC-2) in postnatal day 8 Ts65Dn mice. VIP and VPAC-1 expression was significantly increased in the brains of trisomic mice compared with wild-type mice. VIP-binding sites were also significantly increased in several brain areas of young Ts65Dn mice, especially in the cortex, caudate/putamen, and hippocampus. Further, in vitro treatment of normal neurons with conditioned medium from VIP-stimulated Ts65Dn astrocytes from neonatal mice did not enhance neuronal survival. This study indicates that VIP anomalies are present in neonatal Ts65Dn mice, a defect occurs in the signal transduction mechanism of the VPAC-1 VIP receptor, cortical astrocytes from neonatal brains are dysfunctional, and further, that VIP dysregulation may play a significant role in DS.

Assessing activity onset time and efficacy for clinically effective antidepressant and antimanic drugs in animal models based on dominant-submissive relationships.

There is confusion in the literature on the measurement of the drug activity onset time (AOT) for both clinical and non-clinical studies of antidepressant and antimanic drugs. The questions asked are: How often and at which time points should drug effects be measured? At what level of a drug effect should AOT be determined? Is the placebo (control) effect important for consideration of drug AOT? This paper reviews approaches taken to answer these questions and to assess drug therapeutic AOT. The first part of the paper is devoted to a review of methods used in clinical trials with depression as an indication. The second part is focused on approaches taken in animal models of depression and how they could help in assessing drug AOT. Finally, a summary of pharmacological values on which the AOT depends is presented and a new statistical approach to data analysis method proposed. The allied experimental design for pre-clinical and clinical studies may help to characterize and differentiate AOT for available and new generation of antidepressants and antimanic drugs.

Emerging paradigms in applied bioinformatics

biomarkers is an important goal for the pharmaceutical industry.Validation of innovative, highly specific and practical biomarkers would dramatically accelerate research on the etiology, pathophysiology, progression and prognosis of diseases. It would also facilitate the development of pharmacotherapies, including the identification of treatment responses, in terms of responders and non-responders, the understanding of side-effect profiles, and the individualization of therapies. Application of biomarkers per se, that is, biological markers of physiological events, pathophysiological processes, or responses to a therapeutic intervention, also includes defining surrogate biomarkers that can be substituted for a clinical endpoint and can be used to make risk–benefit assessments [1,2]. Several examples of surrogate biomarkers or endpoints have been established, such as the measures used to make decisions on cholesterol-lowering drugs [3], certain anti-retrovirals (e.g. CD4 count and HIV-1 RNA profiles in HIV) [4] and anti-hypertensive drugs.The first meeting on Biomarkers and Surrogate Endpoints: Advancing Clinical Research and Applications, organized by the National Institutes of Health and the US FDA in 1999, provided a comprehensive framework on the importance of the development and application of biomarkers and surrogate endpoints in clinical medicine and therapeutics. Another important area in the pharmaceutical industry is the identification and validation of new drug targets. In this area, gene expression technologies are key elements in target selection to study responses of biological systems for drug uptake and pharmacodynamics, or to study different pathophysiological conditions [5]. These technologies are crucial for identifying biomarkers of disease, predicting chemical toxicity and understanding why individuals show such a wide variability in their sensitivity to pharmacological agents or pathophysiological conditions [5].Thus, gene expression could play a crucial role within the modern pharmaceutical industry for therapeutic target gene selection and potential drug candidate selection. Different methods of RNA analysis can be used for gene expression profiling, such as northern blotting [6], RNase protection assay [7], reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization [8]. In the past decade, the TaqMan RT-PCR technique was developed and demonstrated to be a reliable and sensitive tool to quantify and monitor accumulating PCR products in real time [9–12].The method enables the direct detection of PCR products during the exponential phase of the reaction, combining amplification and detection in a single step. The use of TaqMan technology to monitor gene expression has become a standard research tool at both academic and industrial research institutions. Another important application of the TaqMan technology is validation …