Development and Preclinical Evaluation of a Copper-64-Labeled Antibody Targeting Glycine-Alanine Dipeptides for PET Imaging of C9orf72-Associated Amyotrophic Lateral Sclerosis/Frontotemporal Dementia.

Aggregating poly(glycine-alanine) (poly-GA) is derived from the unconventional translation of the pathogenic intronic hexanucleotide repeat expansion in the C9orf72 gene, which is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly-GA accumulates predominantly in neuronal cytoplasmic inclusions unique to C9orf72 ALS/FTD patients. Poly-GA is, therefore, a promising target for PET/CT imaging of FTD/ALS to monitor disease progression and therapeutic interventions. A novel 64Cu-labeled anti-GA antibody (mAb1A12) targeting the poly-GA protein was developed and evaluated in a transgenic mouse model. It was obtained with high radiochemical purity (RCP), radiochemical yield (RCY), and specific activity, and showed high stability in vitro and ex vivo and specifically bound to poly-GA. The affinity of NODAGA-mAb1A12 for poly-GA was not affected by this modification. [64Cu]Cu-NODAGA-mAb1A12 was injected into transgenic mice expressing GFP-(GA)175 in excitatory neurons driven by Camk2a-Cre and in control littermates. PET/CT imaging was performed at 2, 20, and 40 h post-injection (p.i.) and revealed a higher accumulation in the cortex in transgenic mice than in wild-type mice, as reflected by higher standardized uptake value ratios (SUVR) using the cerebellum as the reference region. The organs were isolated for biodistribution and ex vivo autoradiography. Autoradiography revealed a higher cortex-to-cerebellum ratio in the transgenic mice than in the controls. Results from autoradiography were validated by immunohistochemistry and poly-GA immunoassays. Moreover, we confirmed antibody uptake in the CNS in a pharmacokinetic study of the perfused tissues. In summary, [64Cu]Cu-NODAGA-mAb1A12 demonstrated favorable in vitro characteristics and an increased relative binding in poly-GA transgenic mice compared to wild-type mice in vivo. Our results with this first-in-class radiotracer suggested that targeting poly-GA is a promising approach for PET/CT imaging in FTD/ALS.

Inhibition of Canonical Transient Receptor Potential Channels 4/5 with Highly Selective and Potent Small-Molecule HC-070 Alleviates Mechanical Hypersensitivity in Rat Models of Visceral and Neuropathic Pain.

Transient receptor potential channels C4/C5 are widely expressed in the pain pathway. Here, we studied the putative analgesic efficacy of the highly selective and potent TRPC4/C5 antagonist HC-070 in rats. Inhibitory potency on human TRPC4 was assessed by using the whole-cell manual patch-clamp technique. Visceral pain sensitivity was assessed by the colonic distension test after intra-colonic trinitrobenzene sulfonic acid injection and partial restraint stress. Mechanical pain sensitivity was assessed by the paw pressure test in the chronic constriction injury (CCI) neuropathic pain model. We confirm that HC-070 is a low nanomolar antagonist. Following single oral doses (3-30 mg/kg in male or female rats), colonic hypersensitivity was significantly and dose-dependently attenuated, even fully reversed to baseline. HC-070 also had a significant anti-hypersensitivity effect in the established phase of the CCI model. HC-070 did not have an effect on the mechanical withdrawal threshold of the non-injured paw, whereas the reference compound morphine significantly increased it. Analgesic effects are observed at unbound brain concentrations near the 50% inhibitory concentration (IC50) recorded in vitro. This suggests that analgesic effects reported here are brought about by TRPC4/C5 blocking in vivo. The results strengthen the idea that TRPC4/C5 antagonism is a novel, safe non-opioid treatment for chronic pain.

Ocular Administration of Palonosetron in the Prevention of Cisplatin-Induced Nausea and Vomiting.

Cancer treatments are frequently associated with nausea and vomiting despite greatly improved preventive medication. Administration of antinausea agents as eye drops might provide easy and rapid access to the systemic circulation for prevention of nausea and vomiting and for the treatment of breakthrough nausea, but the ocular administration route has rarely been evaluated. Palonosetron is a second-generation 5-hydroxytryptamine 3 receptor antagonist approved for prevention and treatment of chemotherapy-induced nausea and vomiting. We compared ocular administration of palonosetron to non-active vehicle eye drops and to intravenous palonosetron in the prevention of cisplatin-induced nausea and vomiting in beagle dogs. Palonosetron ocular drops at the dose of 30 µg/kg reduced cumulative nausea over time as measured with the area under the visual analog scale curve by 98% compared with the vehicle and reduced nausea-associated dog behavior by 95%. Vomiting was completely prevented with repeated palonosetron ocular dosing. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) palonosetron formulation was well tolerated locally at the palonosetron concentration of 3 mg/ml. Absorption of palonosetron from eye drops was fast. Ten minutes after ocular administration, palonosetron plasma concentrations were similar compared with intravenous administration, and remained similar for six hours. We conclude that palonosetron is rapidly absorbed into the systemic circulation from eye drops. Ocularly administered palonosetron was well tolerated in the HP-ß-CD formulation and was highly effective in the prevention of cisplatin-induced nausea and vomiting. Evaluation of the safety and efficacy of ocular administration of palonosetron is warranted in the prevention and treatment of chemotherapy-induced nausea and vomiting in clinical trials. SIGNIFICANCE STATEMENT: Palonosetron, an effective and well-tolerated antiemetic drug was rapidly absorbed into the systemic blood circulation when administered as eye drops. The achieved palonosetron blood concentrations prevented cisplatin-induced nausea and vomiting in beagle dogs. Palonosetron eye drops might provide an easy and quick method for administering palonosetron when parenteral administration is desired and intravenous administration is not feasible.

Development of benzodioxine-heteroarylpiperazines as highly potent and selective α2c antagonists.

Here is reported the design and synthesis of a series of highly potent and selective α2C antagonists using benzodioxine methyl piperazine as a central scaffold by pharmacophoric analysis to improve the pharmacokinetics of suboptimal clinical candidate molecules.

2,3-Dihydrobenzo-dioxine piperidine derivatives as potent and selective α2c antagonists.

In this manuscript, we report a series of benzodioxine methyl piperidine derivatives as highly potent and selective α2C antagonists by ligand design to improve the pharmacokinetics of a previous candidate molecule.

Pharmacokinetics of single and repeated oral doses of pregabalin oral solution formulation in cats.

The study was designed to determine the pharmacokinetic profile and bioavailability of a novel pregabalin 50 mg/ml oral solution formulation (Bonqat® , Orion Corporation Orion Pharma) in 6 healthy laboratory cats. The cats received pregabalin as single oral doses of 2.5, 5, and 7.5 mg/kg, dose 5 mg/kg on two consecutive days, and a single intravenous dose of 2.5 mg/kg. The washout period between each administration was four weeks. The cats were monitored for clinical signs and level of sedation, and blood samples were taken before pregabalin dosing and at pre-defined time points up to 168 h after dosing. Plasma concentrations of pregabalin were determined using a validated liquid chromatography-tandem mass spectrometry method. The mean maximum plasma concentration of 10.1 µg/ml was reached between 0.5 and 1 h after oral administration of the clinical dose 5 mg/kg. The mean half-life after oral administration of dose 5 mg/kg was 14.7 h and the mean systemic bioavailability was 94%. Pregabalin showed linear pharmacokinetics from 2.5 to 7.5 mg/kg. Exposures after a single dose and re-dosing of 5 mg/kg at 24 h were comparable. Pregabalin was well tolerated with mild sedation and mildly uncoordinated movements observed in few cats at dose 7.5 mg/kg. As a conclusion, study results show rapid absorption, linear pharmacokinetics, and high oral bioavailability of pregabalin without safety concerns after administration of oral solution in cats.

GABAB receptor positive allosteric modulators with different efficacies affect neuroadaptation to and self-administration of alcohol and cocaine.

Drugs of abuse induce widespread synaptic adaptations in the mesolimbic dopamine (DA) neurons. Such drug-induced neuroadaptations may constitute an initial cellular mechanism eventually leading to compulsive drug-seeking behavior. To evaluate the impact of GABAB receptors on addiction-related persistent neuroplasticity, we tested the ability of orthosteric agonist baclofen and two positive allosteric modulators (PAMs) of GABAB receptors to suppress neuroadaptations in the ventral tegmental area (VTA) and reward-related behaviors induced by ethanol and cocaine. A novel compound (S)-1-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-4-methyl-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (ORM-27669) was found to be a GABAB PAM of low efficacy as agonist, whereas the reference compound (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) had a different allosteric profile being a more potent PAM in the calcium-based assay and an agonist, coupled with potent PAM activity, in the [35 S] GTPγS binding assay in rat and human recombinant receptors. Using autoradiography, the high-efficacy rac-BHFF and the low-efficacy ORM-27669 potentiated the effects of baclofen on [35 S] GTPγS binding with identical brain regional distribution. Treatment of mice with baclofen, rac-BHFF, or ORM-27669 failed to induce glutamate receptor neuroplasticity in the VTA DA neurons. Pretreatment with rac-BHFF at non-sedative doses effectively reversed both ethanol- and cocaine-induced plasticity and attenuated cocaine i.v. self-administration and ethanol drinking. Pretreatment with ORM-27669 only reversed ethanol-induced neuroplasticity and attenuated ethanol drinking but had no effects on cocaine-induced neuroplasticity or self-administration. These findings encourage further investigation of GABAB receptor PAMs with different efficacies in addiction models to develop novel treatment strategies for drug addiction.