Thomas Weiser

Ambroxol, a Nav1.8-preferring Na(+) channel blocker, effectively suppresses pain symptoms in animal models of chronic, neuropathic and inflammatory pain.

Neuropathic pain affects many patients, and treatment today is far from being perfect. Nav1.8 Na(+) channels, which are expressed by small fibre sensory neurons, are promising targets for novel analgesics. Na(+) channel blockers used today, however, show only limited selectivity for this channel subtype, and can cause dose-limiting side effects. Recently, the secretolytic ambroxol was found to preferentially inhibit Nav1.8 channels. We used this compound as a tool to investigate whether a Nav1.8-preferring blocker can suppress symptoms of chronic, neuropathic and inflammatory pain in animal models. The drug was tested in the formalin paw model, two models of mononeuropathy, and a model of monoarthritis in rats. Ambroxols effects were compared with those of gabapentin. Ambroxol at a dose of 1g/kg had to be administered to rats to achieve the plasma levels that are reached in clinical use (for the treatment of infant and acute respiratory distress syndrome). Ambroxol (1g/kg) was only weakly effective in models for acute pain, but effectively reduced pain symptoms in all other models; in some cases it completely reversed pain behaviour. In most cases the effects were more pronounced than those of gabapentin (at 100mg/kg). These data show that a Nav1.8-preferring Na(+) channel blocker can effectively suppress pain symptoms in a variety of models for chronic, neuropathic and inflammatory pain at plasma levels, which can be achieved in the clinic.

The effects of IDRA 21, a positive modulator of the AMPA receptor, on delayed matching performance by young and aged rhesus monkeys

IDRA 21, a positive allosteric modulator of the glutamate AMPA receptor, produced a concentration-dependent inhibition of glutamate-induced inactivation of membrane currents in recombinant HEK 293 (human embryonic kidney) cells stably transfected with human GluR1/2 flip receptors. IDRA 21 doubled the charge transfer at a concentration of 70 microM, suggesting that this compound can facilitate excitatory neurotransmission via GluR 1/2 receptors. We next sought to exploit this mechanism of action by examining the drug as a potential cognition-enhancing agent in non-human primates. Oral administration of IDRA 21 produced a highly significant improvement in the performance of a delayed matching-to-sample (DMTS) task by young adult rhesus monkeys. The pattern of task improvement over the dose range 0.15-10 mg/kg was maintained to 48 hr after the single dose administration. For sessions run after administration of the individualized Best Dose of IDRA 21, task accuracy for Long delay (most difficult) trials was increased by 34% of vehicle. Animals were randomly assigned fixed doses of IDRA 21 to determine whether the positive mnemonic response could be maintained. The repeated doses were separated by 3 days, thus allowing for potential cumulative effects. IDRA 21 produced a gradual increase in task accuracy that was maintained on average above vehicle performance levels over an intermittent dosing schedule during a total period of 3 weeks. A separate group of aged monkeys (>20 y) were, as a group, impaired (during vehicle testing) in DMTS performance efficiency relative to the young cohort. IDRA 21 also improved task accuracy by aged rhesus monkeys over the same dose range, but the responses were not as robust as those exhibited by young animals. Aged subjects also appeared to be more individually sensitive to drug dose, and they exhibited shorter task latencies than did the young group. Despite these differences, when the individualized Best Doses were considered, IDRA 21 produced a robust increase in DMTS accuracy of up to 18% of vehicle for trials associated with Medium delay intervals. For both study groups, no obvious untoward effects of IDRA 21 were noted. These findings support the use of AMPA modulators like IDRA 21 in the treatment of cognitive/memory disorders, including those associated with aging. They also indicate that the drug is associated with long-term effects that could limit dosing regimens to one dose every two or three days. The nature of the protracted mnemonic effects produced by the compound remains to be elucidated.

Effects of the neuropeptide Y Y2 receptor antagonist BIIE0246 on presynaptic inhibition by neuropeptide Y in rat hippocampal slices

Abstract We previously reported that (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cylopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]argininamid, BIIE0246, is a potent and highly selective neuropeptide Y Y2 receptor antagonist. Neuropeptide Y Y2 receptors have been proposed to mediate the inhibition by neuropeptide Y of excitatory synaptic transmission in rat hippocampus. Therefore, we investigated the effects of BIIE0246 on the electrophysiological properties of neuropeptide Y in rat hippocampal slices and determined the affinity of this novel antagonist for rat hippocampal neuropeptide Y Y2 receptors. BIIE0246 displayed an affinity of IC50=4.0±1.6 (n=4) for neuropeptide Y receptor binding sites labelled by 125I-neuropeptide Y in rat hippocampal membranes. At a concentration of 1 μM, BIIE0246 completely antagonized the inhibitory effects of 300 nM neuropeptide Y on synaptic transmission in rat hippocampal slices. This is the first study showing that a selective neuropeptide Y Y2 receptor antagonist is able to block neuropeptide Y mediated effects in the hippocampus and unambiguously characterizes the presynaptic receptor in the rat hippocampus as the neuropeptide Y Y2 receptor.

Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus.

AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.

Ambroxol: A CNS Drug?

For almost three decades ambroxol has been used in the therapy of airway diseases. In 2002, ambroxol lozenges were marketed for the treatment of sore throat making use of its local anesthetic effect. Detailed investigations of ambroxol with modern pharmacological methods yielded additional interesting results: ambroxol has been found to have profound effects on neuronal voltage‐gated Na+, as well as Ca2+ channels, and to effectively reduce chronic inflammatory and neuropathic pain in rodents. The question was raised whether ambroxol affects the central nervous system (CNS) directly, or whether its effects can be explained solely by a peripheral action. This issue was addressed by reexamining pharmacokinetics, as well as toxicology of ambroxol. It has been concluded that even at the highest clinically used doses ambroxol does not have significant direct effects on the CNS. At clinically relevant plasma concentrations ambroxol either does not penetrate blood‐brain barrier, or its brain levels are too low to cause relevant effects. The analgesic effects of ambroxol by either systemic administration to animals, or by topical application in humans can be explained by ambroxol‐induced blockade of ion channels in peripheral neurons.

Voltage- and Ca2+-dependence of the K+ channel in the vacuolar membrane of Chenopodium rubrum L. suspension cells

Voltage- and Ca(2+)-dependence of the slow-activating SV-K+ channel in the vacuolar membrane of Chenopodium rubrum suspension cells has been analyzed using the patch clamp technique in the vacuole-attached, outside-out and whole-vacuolar configuration. Patch-pipette perfusion was applied to measure Ca2+ dependence of single channels in the attached-configuration. Using the PCLAMP-software (Axon Instruments), an algorithm was developed to extract reliable individual channel data from multi-channel activity records, including open probability, mean open and closed times, as well as time constants for open and closed distributions. The channel conductance of the major open state was about 83 pS (seal resistance > 8 G omega) at 30 mV (transmembrane voltage Vm, vacuole negative), and symmetrical 100 mM KCl. the channel exhibited a strong voltage- and a weak Ca(2+)-activation: increasing Vm from 40 to 100 mV is equivalent to a Ca2+ concentration change from 10(-7) to 10(-4) M. Mean open probabilities at Vm = 30 mV were 0.03 with 1 microM and 0.09 with 100 microM Ca2+. Mean open times were approx. 7 ms, and almost independent of both, voltage and Ca2+. Mean closed times, however, varied in a strongly voltage- and Ca(2+)-dependent manner, e.g., at Vm = 30 mV dropped from 205 to 67 ms, if Ca2+ was raised from 10(-6) to 10(-4) M. Open and closed distributions of events within bursts could be fitted by the sum of two exponentials with time constants between 0.3 and 11 ms.

Effectiveness and safety of topical capsaicin cream in the treatment of chronic soft tissue pain.

Topical capsaicin is an established treatment option for various pain conditions. In a randomized double‐blind multi‐centre study, 281 patients suffering from chronic soft tissue pain were treated either with a cream containing capsaicin 0.05% (‘Finalgon® CPD Wärmecreme’, n = 140) or placebo (n = 141). Of these, 151 were excluded from the ITT analysis, as they had in addition to their soft‐tissue pain, pain of other origin. The primary outcome measure was a positive treatment response, defined as a pain sum score reduction of 30% or more. After 3 weeks of treatment, the median pain sum score had decreased by 49% (capsicum group) and 23% (placebo group) (ITT analysis, p = 0.0006). The odds ratio of the responders in favour for capsaicin was 4.3 (CI 97.5% lower limit 1.9, p < 0.0001). Improvements in the secondary efficacy measures confirmed the results. Likewise, all outcome measures had significantly more improved in the capsaicin‐treated compared with the placebo‐treated chronic back pain sufferers. All patients were included in the safety assessments. More adverse events occurred in the capsicum group (n = 13) than in the placebo group (n = 6). The capsaicin cream was generally well tolerated. The results indicate that capsaicin cream is useful in patients with chronic soft tissue pain and is also efficacious in patients with chronic back pain for which effectiveness was already demonstrated in earlier clinical trials.

Potassium currents in cultured cells of the rat retinal pigment epithelium

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than -30 mV, a time-dependent outward current was activated. Inhibition by Ba2+ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba2+ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than -70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.

Hyoscine butylbromide potently blocks human nicotinic acetylcholine receptors in SH-SY5Y cells

Hyoscine butylbromide (HBB; tradenames: Buscopan/Buscapina is an antispasmodic drug for the treatment of abdominal pain associated with gastrointestinal cramping. As a hyoscine derivative, this compound competitively inhibits muscarinic acetylcholine (ACh) receptors on smooth muscle cells in the gastrointestinal tract. Preliminary investigations suggested that it might also inhibit nicotinic ACh receptors. This study investigated the effect of HBB on nicotinic ACh receptor-mediated membrane currents in SH-SY5Y cells. ACh and nicotine application-induced comparable membrane currents with EC(50) values of 25.9+/-0.6 and 40.1+/-0.4microM, respectively. When coapplied with 100microM ACh, HBB concentration-dependently suppressed currents with an IC(50) value of 0.19+/-0.04microM, and was approximately seven-times more potent than the ganglionic blocker, hexamethonium (IC(50)=1.3+/-0.3microM). Increasing the agonist concentration to 5mM did not affect the amount of block by HBB, which suggests a non-competitive mode of action. These functional in vitro data demonstrate for the first time that HBB blocks neuronal nicotinic ACh receptors in the same concentration range as it inhibits muscarinic ACh receptors. If one hypothesizes that HBB might also affect nicotinic receptors in autonomic neurons in vivo (e. g. in the enteric nervous system), this effect could contribute to its spasmolytic activity.

A novel toxicity-based assay for the identification of modulators of voltage-gated Na+ channels

Voltage-gated Na+ channels are promising drug targets. Screening of large numbers of putative modulators, however, can be demanding and expensive. In this study, a simple, cheap, and robust assay to test the pharmacological modulation of Na+ channel function is presented. The assay makes use of the fact that the intracellular accumulation of Na+ ions can be cytotoxic. The toxicity of the Na+ channel activator veratridine in the presence of an inhibitor of the Na+/K+ ATPase (ouabain) in a Nav1.2a (rat brain IIA alpha) expressing cell line is assessed. Na+ channel blockers should reduce toxicity in this model. CHO cells which recombinantly expressed rat Nav1.2a subunits were seeded in 96-well plates, and cell survival was tested after 24 h incubation in medium containing veratridine and ouabain in the presence or absence of Na+ channel blockers. Propidium iodide fluorescence was used as toxicity readout. Veratridine (100 microM) or ouabain alone (500 microM) were not toxic to the cells. In the presence of 500 microM ouabain, however, veratridine induced halfmaximal cell death with an EC50 value of 15.1 +/- 2.3 microM. Ouabains EC50 was 215.3 +/- 16.7 microM (with 30 microM veratridine). The effects of a number of Na+ channel blockers were tested and compared with their Na+ channel blocking activity measured in voltage-clamp experiments. Blockers from various chemical classes reduced toxicity half maximally with IC50 values ranging from 11.7 +/- 1.4 nM (tetrodotoxin) to 280.5 +/- 48.0 microM (lamotrigine). There was a linear relationship between the log IC50 values obtained by the two methods (slope: 1.1 +/- 0.08; correlation coefficient: 0.93). In summary, these data show that this novel toxicity assay is well suited to test Na+ channel blockers.