Physical Properties and Effect in a Battery of Safety Pharmacology Models for Three Structurally Distinct Enteric Polymers Employed as Spray-Dried Dispersion Carriers

Establishing a wide therapeutic index (TI) for pre-clinical safety is important during lead optimization (LO) in research, prior to clinical development, although is often limited by a molecules physiochemical characteristics. Recent advances in the application of the innovative vibrating mesh spray-drying technology to prepare amorphous solid dispersions may offer an opportunity to achieve high plasma concentrations of poorly soluble NCEs to enable testing and establishment of a wide TI in safety pharmacology studies. While some of the amorphous solid dispersion carriers are generally recognized as safe for clinical use, whether they are sufficiently benign to enable in vivo pharmacology studies has not been sufficiently demonstrated. Thus, the physical properties, and effect in a battery of in vivo safety pharmacology models, were assessed in three classes of polymers employed as spray-dried dispersion carriers. The polymers (HPMC-AS, Eudragit, PVAP) displayed low affinity with acetone/methanol, suitable for solvent-based spray drying. The water sorption of the polymers was moderate, and the degree of hysteresis of HPMC-AS was smaller than Eudragit and PVAP indicating the intermolecular interaction of water-cellulose molecules is weaker than water-acrylate or water-polyvinyl molecules. The polymer particles were well-suspended without aggregation with a mean particle size less than 3 µm in an aqueous vehicle. When tested in conscious Wistar Han rats in safety pharmacology models (n=6-8/dose/polymer) investigating effects on CNS, gastrointestinal, and cardiovascular function, no liabilities were identified at any dose tested (30-300 mg/kg PO, suspension). In brief, the polymers had no effect in a modified Irwin test that included observational and evoked endpoints related to stereotypies, excitation, sedation, pain/anesthesia, autonomic balance, reflexes, and others. No effect of the polymers on gastric emptying or intestinal transit was observed when measured using a barium sulfate tracer material. Finally, in telemetry-instrumented rats the polymers had no effect on acute or 24-hr mean blood pressure and heart rate values at doses up to 300 mg/kg. Thus, the properties of the three enteric polymers are appropriate as spray-dried dispersion carriers and were benign in a battery of safety pharmacology studies, demonstrating their applicability to enable in vivo safety pharmacology profiling of poorly soluble molecules during LO

Globular and Protofibrillar Aβ Aggregates Impair Neurotransmission by Different Mechanisms

In Alzheimer’s disease, substantial evidence indicates the causative role of soluble amyloid β (Aβ) aggregates. Although a variety of Aβ assemblies have been described, the debate about their individual relevance is still ongoing. One critical issue hampering this debate is the use of different methods for the characterization of endogenous and synthetic peptide and their intrinsic limitations for distinguishing Aβ aggregates. Here, we used different protocols for the establishment of prefibrillar Aβ assemblies with varying morphologies and sizes and compared them in a head-to-head fashion. Aggregation was characterized via the monomeric peptide over time until spheroidal, protofibrillar, or fibrillar Aβ aggregates were predominant. It could be shown that a change in the ionic environment induced a structural rearrangement, which consequently confounds the delineation of a measured neurotoxicity toward a distinct Aβ assembly. Here, neuronal binding and hippocampal neurotransmission were found to be suitable to account for the synaptotoxicity to different Aβ assemblies, based on the stability of the applied Aβ aggregates in these settings. In contrast to monomeric or fibrillar Aβ, different prefibrillar Aβ aggregates targeted neurons and impaired hippocampal neurotransmission with nanomolar potency, albeit by different modalities. Spheroidal Aβ aggregates inhibited NMDAR-dependent long-term potentiation, as opposed to protofibrillar Aβ aggregates, which inhibited AMPAR-dominated basal neurotransmission. In addition, a provoked structural conversion of spheroidal to protofibrillar Aβ assemblies resulted in a time-dependent suppression of basal neurotransmission, indicative of a mechanistic switch in synaptic impairment. Thus, we emphasize the importance of addressing the metastability of <i>prefacto</i> characterized Aβ aggregates in assigning a biological effect

Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice

<div><p>Background</p><p>Forty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects.</p><p>HC-070 <i>in vitro</i></p><p>To assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases.</p><p>HC-070 <i>in vivo</i></p><p>Upon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC₅₀) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.</p></div

HC-070 inhibits recombinantly expressed TRPC4 and TRPC5, as well as TRPC1-containing heteromultimers, in whole-cell manual patch clamp.

<p>HC-070 inhibits recombinantly expressed TRPC4 and TRPC5, as well as TRPC1-containing heteromultimers, in whole-cell manual patch clamp.</p

HC-070 decreases anxiety after CCK-4 treatment in an EPM.

<p>Treatment with CCK-4 decreased the percent of open arm entries (p < 0.05, t-test), as expected. Administering HC-070 orally at 1 mg/kg prevented the reduction in open arm entries induced by CCK-4 (p<0.05, t-test, n = 12/group). HC-070 exerted no effect on percent of open arm entries in the absence of CCK-4. Each animal is represented on the graph (circles) and the horizontal lines represent the mean. The actual exposures of HC-070 at the time the mice were sacrificed were 262±63 and 301±81nM for the CCK-4 + HC-070 and VEH + HC-070 groups, respectively.</p

Structures and activity in fluorometric assays of HC-070 and HC-608.

<p>The chemical structures of (A) HC-070 and (B) HC-608 (Pico145). (C) Inhibition of hTRPC5 by HC-070 and HC-608 in indicator-assisted calcium influx analysis. Concentrations tested ranged from 1 picomolar (pM) to 1 μM. Each data point represents the average of 8 measurements from a 384-well plate. Error bars show the standard deviation. The IC₅₀ values were 9.3 ± 0.9 and 6.2 ± 0.5 nanomolar (nM), respectively. (D) Inhibition of hTRPC4 by HC-070 and HC-608 over the same range of concentrations. The IC₅₀ values were 46.0 ± 3.9 nM and 32.5 ± 1.8 nM, respectively (n = 8). Error bars represent the standard deviation.</p

HC-070 reduces marble burying behavior.

<p>(A) Mice were administered vehicle or 1, 3 or 10 mg/kg HC-070 orally, 60 minutes prior to testing. The positive control, 10 mg/kg zimelidine, was administered IP 45 minutes prior to testing. At 1, 3, and 10 mg/kg, HC-070 significantly decreased the number of buried marbles compared to vehicle (p <0.05 *, p<0.01**, Dunnett’s post-hoc test following one-way ANOVA, n = 10/group). Zimelidine also decreased the number of buried marbles (p < 0.05, t-test, n = 10/group). Each animal is represented on the graph and the horizontal lines represent the mean. (B) Average plasma exposures from animals that completed the test. Error bars are the standard deviation.</p

Pharmacokinetic properties of HC-070.

<p>PK profiles of HC-070 after intravenous (A) and oral (B) administration in C57BL/6 mice. Plasma concentrations were determined by LC-MS/MS. Points represent the individual concentrations at the times indicated. Lines represent mean exposure (n = 12 mice/arm, n = 3 data points per time point). (C) Summary of PK properties. CL = clearance; V_ss = volume of distribution at steady state; MRT_disp = mean residence time of drug molecules after intravascular administration; T_1/2 = half-life. (D) Plasma and brain concentrations measured 2 hours after intravenous or oral administration of 1 or 10 mg/kg HC-070, respectively. C_PL = concentration in plasma, C_BR = concentration in brain, K_P,BR = partitioning coefficient between brain and plasma.</p