Poster Number-61 - ADV-484, a Novel Selective Inhibitor of alpha2delta-1 for the Treatment of Neuropathic Pain

ADV-484, a Novel Selective Inhibitor of a2d-1for the Treatment of Neuropathic Pain

Purpose: α2δ-1 is an auxiliary protein that, like the σ₁ receptor (sigma-1R), interacts with the NMDA subtype of glutamate receptor.  It is one subunit of an alternatively-spliced proprotein transcript variant that encodes multiple isoforms linked by a disulfide bond. Importantly, α2δ-1 is expressed in the dorsal root ganglia (DRG) and dorsal horn of the spinal cord, and the α2δ-1-NMDAR heteromeric complex is greatly upregulated at both sites in neuropathic pain. a2d-1 is generally known as a voltage-activated Ca²⁺ channel subunit, but it was shown by Chen et al. (2018) that Cacna2d1 (the gene that encodes for α2δ-1) overexpression potentiates pre- and postsynaptic NMDAR activity of spinal dorsal horn neurons to augment pain hypersensitivity. Thus, α2δ-1 is an NMDAR-interacting protein that increases NMDAR synaptic delivery that occurs in neuropathic pain. Gabapentinoids (e.g., gabapentin, pregabalin) non-selectively bind to both α2δ subunits,  α2δ-1 and  α2δ-2. The blocking of α2δ-1, which is abundantly expressed in excitatory (glutamatergic) synapsis, results in inhibition of excitatory input in pain pathways. In contrast, the α2δ-2 subunit is abundant in inhibitory (GABAergic) neurons located in cerebellum (Purkinje cells) and other brain regions, thus its pharmacological blockade by gabapentinoids is undesirable, because it results in inhibition of inhibitory synapses––that is, disinhibition––and disengagement of pain inhibitory mechanisms, plus motor incoordination secondary to inhibition of GABAergic cerebellar Purkinje neurons. In contrast, ADV-484 is a novel potent and highly selective inhibitor of  α2δ-1, devoid of significant activity on  α2δ-2. It was identified as part of a large discovery program aimed at identifying mechanisms to overcome shortcomings of existing analgesic classes. 

Methods: A drug discovery program was undertaken to identify a non-aminoacidic selective a2d-1 blocker which led to the discovery of ADV-484. In vitro receptor binding affinity for α2δ-1 and α2δ-2, as well as for other targets was measured by using radiometric techniques. Other in vitro investigations included metabolic stability in both microsomes and cryopreserved hepatocytes from several species, permeability in Caco2 cells, plasma protein and brain tissue binding, cytochrome inhibition/induction, and several safety-related tests including hERG inhibition. Its in vivo efficacy (neuropathic pain-related capsaicin model) and safety profile (rotarod, Irwin test, acute oral toxicity and in vivo genotoxicity) were investigated in mice and its pharmacokinetics were investigated in both mice and rats.

Results:

ADV-484 is a non-aminoacidic small molecule with adequate physicochemical profile, good lipophilicity (LogP 2.9; LogD7.4 1.2) and solubility (kinetic and thermodynamic solubility in the mM range). The compound is a chiral piperazinylquinazolin-4-(3H)-one derivative that can be prepared using a 6-step enantioselective route.

ADV-484 displayed high binding affinity (Ki = 27 nM) for a2d-1 and was highly selective. Affinity was at least 2 orders of magnitude higher than for other binding sites measured, including a2d-2 (Ki=7694 nM; 285x selective vs α2δ-1) and sigma-1R and sigma-2R. In contrast, pregabalin showed similar affinity for both targets (a2d-1 Ki = 19 nM; a2d-2 Ki=42 nM).

ADV-484 was tested in animal models relevant to neuropathic pain. It was active by the oral route (40 mg/kg) as well as the i.p. route (20 mg/kg), to an extent comparable or superior to pregabalin . A stark difference from pregabalin was that at equianalgesic doses pregabalin produced profound motor discoordination (Rotarod test), whereas ADV-484 was essentially devoid of an effect on motor coordination, and no effect on the safety observational battery in the Irwin behavioral screen at doses higher than those producing analgesia.

ADV-484 had a suitable PK profile with high oral bioavailability. Based on in vitro data, it has high metabolic stability in human liver microsomes and hepatocytes, and it should have low hepatic clearance in humans, good oral absorption, and large volume of distribution. Interestingly, it has good brain penetration with a brain-to-plasma AUC ratio of 1.26, significantly exceeding that of pregabalin. ADV-484 displays low potential for CYP450 inhibition-based drug-drug interactions.

There was no alert for cytotoxicity, mutagenesis, hERG blockade, or in vivo genotoxicity (mice). The maximum non-lethal dose in mice (1,000 mg/kg) far exceeds the active dose levels.     

Conclusion: ADV-484 shows highly selective affinity for α2δ-1 that results in potent analgesic activity in animal models of chronic/neuropathic pain comparable to pregabalin after oral or i.p. administration. The analgesic effect of ADV-484 is not accompanied by negative behavioral markers in the Irwin test (mice) and is virtually devoid of the motor discoordination (mice) so characteristic of pregabalin even at analgesic doses. The good efficacy results that were obtained, along with the minimal pregabalin-like AEs observed, suggest that inhibition of α2δ-1 with a highly selective compound such as ADV-484 is a superior mechanistic approach for treatment of neuropathic pain. Continued investigation of the efficacy and safety of ADV-484 is underway to determine suitability for advancement to clinical trials.

References: Chen et al. (2018) Cell Reports 22(9):2307–2321.