Establishment of a secondary screening assay for P/Q-type calcium channel blockers.

Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically overactive channels. We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steady-state inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast, inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch clamp recordings. Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and--if necessary--a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable for the generation of assays in other ion channel programs.

Meeting the MIST regulations: human metabolism in Phase I using AMS and a tiered bioanalytical approach.

The metabolites in safety testing and ICH-M3 guidance documents emphasize the importance of metabolites when considering safety aspects for new drugs. Both guidances state that relevant metabolites should have safety coverage in humans (although the guidelines have different definitions of relevant metabolites). Not having safety coverage for important metabolites in humans may cause significant delay in the overall pharmaceutical development program. This article discusses the regulatory background regarding safety and metabolites, as well as outlines an integrated strategy taken by one pharmaceutical company, Lundbeck A/S. Lundbeck uses metabolite exposure data from first-in-man studies, obtained using an accelerator MS approach followed by a two-tiered bioanalytical investigation. This enables early availability of key data on this aspect and, overall, represents a powerful risk mitigation strategy.

Amino-modified silica surfaces efficiently immobilize bone morphogenetic protein 2 (BMP2) for medical purposes.

Due to its ability to induce de novo bone formation the differentiation factor bone morphogenetic protein 2 (BMP2) is often used to enhance the integration of bone implants. With the aim of reducing possible high dose side-effects and to lower the costs, in order to produce affordable implants, we developed a simple and fast method for the immobilization of BMP2 on silica-based surfaces using silane linkers which carry amino or epoxy functions. We put an especial emphasis on the influence of the nanoscale surface topography of the silica layer. Therefore, we chose glass (for control experiments) and Bioverit® II (as a typical implant base material) as support materials and coated these substrates with unstructured or nanoporous amorphous silica layers for comparison. Immobilized BMP2 was quantified by two different methods: by ELISA and by a cell-based assay for active BMP2. These tests probe for immunologically and biologically active BMP2, respectively. The results show that the amino functionalization is better suited for immobilizing the protein. Strikingly, a considerably higher amount of BMP2 could be immobilized on coated Bioverit® II surfaces compared with coated glass substrates, which was presumably due to the macroscopic roughness of the Bioverit® II substrates. In addition, it was found that the nanoporous silica coatings on Bioverit® II substrates were able to bind more BMP2 than the unstructured ones.

Enhancement of endoprosthesis anchoring using BMP-2.

Osseointegration of long-term implants is still a problem in orthopaedic surgery. In recent years, several techniques to modify the implant surface to increase bone formation around implants have been described by many authors. Most endoprostheses used in orthopaedic surgery are manufactured from titanium. To understand the process of osseointegration, one has to take into account, that the adhesion of plasma proteins on the surface of titanium implants plays an essential role in the process of implant integration. In the last years, several modifications of implant surfaces (structure, chemistry, surface charge, wettability) have been investigated to improve osseointegration of titanium implants. Furthermore, several cytokines and growth factors have also been suggested to stimulate an implant ingrowth. In this respect, functionalized titanium implant surfaces with bone morphogenetic proteins-2 (BMP-2) as one particular member of the superfamily of transforming growth factor beta (TGF-beta), have proven a potential to stimulate bone formation around implants in different species. In this review the authors provide an overview of surface coatings with BMP-2 and their use in laboratory and experimental settings.

The guinea pig forced swim test as a new behavioral despair model to characterize potential antidepressants.

RATIONALE: Behavioral despair is a model of high predictivity for antidepressant activity in murids. For some drug targets, guinea pigs exhibit a higher homology to their human counterparts compared to murids. OBJECTIVES: In this paper, we established a model of behavioral despair namely, the forced swim test (FST) in guinea pigs. MATERIALS AND METHODS: Male guinea pigs underwent the FST similar to rats. Animals received intraperitoneal injections of either vehicle or drugs 24, 4, and 0.5 h before testing. We tested the tricyclic antidepressants desipramine and amitriptyline, the monoamine oxidase inhibitor tranylcypromine, the selective serotonin reuptake inhibitors fluoxetine and paroxetine, and the neurokinin 1 (NK(1)) receptor antagonist, L-733,060, and for comparison the antipsychotic clozapine and the stimulant methamphetamine. RESULTS: Desipramine (> or =3 mg/kg) and amitriptyline (>10 mg/kg) increased the latency to immobility (LTI) to greater than 230 s, and tranylcypromine (10 mg/kg) it to greater than 190 s. Paroxetine (>0.3 mg/kg) and fluoxetine (>10 mg/kg) also increased LTI significantly but only to greater than 120 s. Methamphetamine (3 mg/kg) completely eliminated immobility, whereas clozapine (5-20 mg/kg) had no effect. L-733,060 (10 mg/kg) increased LTI to 270 s. Doses producing significant effects in FST were investigated in the open field. Antidepressants did not affect locomotion, whereas methamphetamine induced hyperlocomotion. CONCLUSIONS: We demonstrate the suitability of a modified procedure of the FST for a nonmurid species: the guinea pig. Known antidepressants showed similar effects as in rats and mice. It is interesting to note that the NK(1) antagonist L-733,060 increased forced swimming, suggesting its antidepressant potential. Thus, the guinea pig FST allows the study of antidepressant activity also in NK(1) antagonists that cannot be studied appropriately in murids.

Effects of antipsychotics and selective D3 antagonists on PPI deficits induced by PD 128907 and apomorphine.

Rats treated with apomorphine and amphetamine display sensorimotor gating impairments, as measured by prepulse inhibition (PPI), and these impairments can be reversed by antipsychotic treatment. However, it remains unknown whether the dopamine (DA) D(3) receptor plays a role in mediating these effects on PPI, as none of these DA agonists or antipsychotics are exclusively selective at either D(2) or D(3) receptors. To address this question, the current study was designed to investigate whether antipsychotic drugs and selective D(3) antagonists could block the PPI-disruptive effects of PD 128907 (a preferential D(3) agonist) and apomorphine. We found that the effect of PD 128907 on PPI in rats could be antagonized by risperidone, clozapine, and the selective D(3) antagonists SB 277011 and A-691990, but not by raclopride or haloperidol, while the apomorphine-induced PPI deficit could be reversed by risperidone, clozapine and haloperidol, but not by SB 2770111 and A-691990. These results suggest that the D(3) receptor does not mediate apomorphine-induced disruption of PPI in rats, however, given the findings that PD 128907 elicited a PPI-disruptive effect that was blocked by selective D(3) antagonists, a role of D(3) receptor in mediating PPI in rats cannot be ruled out. The possible mechanisms of D(3) receptor involvement in PPI are discussed.