Discovery of a brain penetrant small molecule antagonist targeting LPA1 receptors to reduce neuroinflammation and promote remyelination in multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurological disease characterized by inflammatory demyelination that disrupts neuronal transmission resulting in neurodegeneration progressive disability. While current treatments focus on immunosuppression to limit inflammation and further myelin loss, no approved therapies effectively promote remyelination to mitigate the progressive disability associated with chronic demyelination. Lysophosphatidic acid (LPA) is a pro-inflammatory lipid that is upregulated in MS patient plasma and cerebrospinal fluid (CSF). LPA activates the LPA1 receptor, resulting in elevated CNS cytokine and chemokine levels, infiltration of immune cells, and microglial/astrocyte activation. This results in a neuroinflammatory response leading to demyelination and suppressed remyelination. A medicinal chemistry effort identified PIPE-791, an oral, brain-penetrant, LPA1 antagonist. PIPE-791 was characterized in vitro and in vivo and was found to be a potent, selective LPA1 antagonist with slow receptor off-rate kinetics. In vitro, PIPE-791 induced OPC differentiation and promoted remyelination following a demyelinating insult. PIPE-791 further mitigated the macrophage-mediated inhibition of OPC differentiation and inhibited microglial and fibroblast activation. In vivo, the compound readily crossed the blood-brain barrier and blocked LPA1 in the CNS after oral dosing. Direct dosing of PIPE-791 in vivo increased oligodendrocyte number, and in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS, we observed that PIPE-791 promoted myelination, reduced neuroinflammation, and restored visual evoked potential latencies (VEP). These findings support targeting LPA1 for remyelination and encourage development of PIPE-791 for treating MS patients with advantages not seen with current immunosuppressive disease modifying therapies.

Identification and In Vivo Evaluation of Myelination Agent PIPE-3297, a Selective Kappa Opioid Receptor Agonist Devoid of ß-Arrestin-2 Recruitment Efficacy.

Structure-activity relationship studies led to the discovery of PIPE-3297, a fully efficacious and selective kappa opioid receptor (KOR) agonist. PIPE-3297, a potent activator of G-protein signaling (GTPγS EC50 = 1.1 nM, 91% Emax), did not elicit a ß-arrestin-2 recruitment functional response (Emax < 10%). Receptor occupancy experiments performed with the novel KOR radiotracer [3H]-PIPE-3113 revealed that subcutaneous (s.c.) administration of PIPE-3297 at 30 mg/kg in mice achieved 90% occupancy of the KOR in the CNS 1 h post dose. A single subcutaneous dose of PIPE-3297 in healthy mice produced a statistically significant increase of mature oligodendrocytes (P < 0.0001) in the KOR-enriched striatum, an effect that was not observed in animals predosed with the selective KOR antagonist norbinaltorphimine. An equivalent dose given to mice in an open-field activity-monitoring system revealed a small KOR-independent decrease in total locomotor activity versus vehicle measured between 60 and 75 min post dose. Daily doses of PIPE-3297 at both 3 and 30 mg/kg s.c. reduced the disease score in the mouse experimental autoimmune encephalomyelitis (EAE) model. Visually evoked potential (VEP) N1 latencies were also significantly improved versus vehicle in both dose groups, and latencies matched those of untreated animals. Taken together, these findings highlight the potential therapeutic value of functionally selective G-protein KOR agonists in demyelinating disease, which may avoid the sedating side effects typically associated with classical nonbiased KOR agonists.

Discovery of PIPE-359, a Brain-Penetrant, Selective M1 Receptor Antagonist with Robust Efficacy in Murine MOG-EAE.

The discovery of PIPE-359, a brain-penetrant and selective antagonist of the muscarinic acetylcholine receptor subtype 1 is described. Starting from a literature-reported M1 antagonist, linker replacement and structure-activity relationship investigations of the eastern 1-(pyridinyl)piperazine led to the identification of a novel, potent, and selective antagonist with good MDCKII-MDR1 permeability. Continued semi-iterative positional scanning facilitated improvements in the metabolic and hERG profiles, which ultimately delivered PIPE-359. This advanced drug candidate exhibited robust efficacy in mouse myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE), a preclinical model for multiple sclerosis.

Understanding the axonal response to injury by in vivo imaging in the mouse spinal cord: A tale of two branches.

Understanding the basic properties of how axons respond to injury in the mammalian central nervous system (CNS) is of fundamental value for developing strategies to promote neural repair. Axons possess complex morphologies with stereotypical branching patterns. However, current knowledge of the axonal response to injury gives little consideration to axonal branches, nor do strategies to promote axon regeneration. This article reviews evidence from in vivo spinal cord imaging that axonal branches markedly impact the degenerative and regenerative responses to injury. At a major bifurcation point, depending on whether one or both axonal branches are injured, neurons may choose either a more self-preservative response or a more dynamic response. The stabilizing effect of the spared branch may underlie a well-known divergence in neuronal responses to injury, and illustrates an example where in vivo spinal cord imaging reveals insights that are difficult to elucidate with conventional histological methods.

A surviving intact branch stabilizes remaining axon architecture after injury as revealed by in vivo imaging in the mouse spinal cord.

The complex morphology of axons presents a challenge in understanding axonal responses to injury and disease. By in vivo two-photon imaging of spinal dorsal column sensory axons, we systematically examined the effect of injury location relative to the main bifurcation point on axon degeneration and regeneration following highly localized laser injuries. Retrograde but not anterograde degeneration was strongly blocked at the bifurcation point at both the acute and subacute phases. Eliminating either the ascending or descending branch led to a poor regenerative response, while eliminating both led to a strong regenerative response. Thus, a surviving intact branch suppresses both retrograde degeneration and regeneration of the injured branch, thereby preserving the remaining axon architecture. Regenerating axons exhibited a dynamic pattern with alternating phases of regeneration and pruning over a chronic period. In vivo imaging continues to reveal new insights on axonal responses to injury in the mammalian spinal cord.

Effects of PTEN and Nogo Codeletion on Corticospinal Axon Sprouting and Regeneration in Mice.

Axons in the adult CNS have poor ability to grow after injury, impeding functional recovery in patients of spinal cord injury. This has been attributed to both a developmental decline in neuron-intrinsic growth ability and the presence of extrinsic growth inhibitors. We previously showed that genetic deletion of Nogo, an extrinsic inhibitor, promoted axonal sprouting from uninjured corticospinal tract (CST) neurons but not regeneration from injured CST neurons, whereas genetic deletion of PTEN, an intrinsic inhibitor, promoted both CST sprouting and regeneration. Here we test the hypothesis that combining an elevation of neuron-intrinsic growth ability and a reduction of extrinsic growth inhibition by genetic codeletion of PTEN and Nogo may further improve injury-induced axonal growth. In an apparent paradox, additionally deleting Nogo further enhanced CST regeneration but not sprouting in PTEN-deleted mice. Enhanced CST regeneration and sprouting in PTEN and PTEN/Nogo-deleted mice were associated with no or only temporary improvement in functional recovery. Our data illustrate that neuron-intrinsic and -extrinsic factors regulate axon regeneration and sprouting in complex ways and provide proof-of-principle evidence that targeting both can further improve regeneration. Neuron-intrinsic growth ability is an important determinant of neuronal responsiveness to changes in extrinsic growth inhibition, such that an elevated intrinsic growth state is a prerequisite for reducing extrinsic inhibition to take effect on CST regeneration. Meanwhile, additional strategies are required to unleash the full potential for functional recovery with enhanced axon regeneration and/or sprouting.

Transient demyelination increases the efficiency of retrograde AAV transduction.

Adeno-associated virus (AAV) is capable of mediating retrograde viral transduction of central and peripheral neurons. This occurs at a relatively low efficiency, which we previously found to be dependent upon capsid serotype. We sought to augment retrograde transduction by providing increased axonal access to peripherally delivered AAV. Others have described utilizing full transection of peripheral nerves to mediate retrograde viral transduction of motor neurons. Here, we examined the ability of a transient demyelinating event to modulate levels of retrograde AAV transduction. Transient demyelination does not cause lasting functional deficits. Ethidium bromide (EtBr)-induced transient demyelination of the sciatic nerve resulted in significant elevation of retrograde transduction of both motor and sensory neurons. Retrograde transduction levels of motor neurons and heavily myelinated, large-diameter sensory neurons increased at least sixfold following peripheral delivery of self-complementary AAV serotype 1 (scAAV1) and serotype 2 (scAAV2), when preceded by demyelination. These findings identify a means of significantly enhancing retrograde vector transport for use in experimental paradigms requiring either retrograde neuronal identification and gene expression, or translational treatment paradigms.