Brain-penetrant Compounds Research Articles

Intelligent Design of Brain-penetrant Compounds Based on Transporter- Conscious Drug Design

In drug discovery and development, central nervous system (CNS) drugs should be delivered into brain. Nonetheless, drug permeability into brain may be blocked by the blood-brain barrier (BBB) due to the imperviousness of the tight junction between endothelial cells in CNS and excretion by MDR1 (P-gp) expressing in the apical (luminal) membrane of endothelial cells. However, SLC transporter-mediated transport at the BBB can solve this permeable problem. It is well-known that compounds possessing N-containing groups are transported into brain across the BBB. This transportation was suggested to be associated with amine transporters. Thus, transporter-consciously designed drugs which possess N-containing groups as transporter recognition unit can be effectively delivered into brain across the BBB. In this paper, possibility of CNS drugs based on transporter-conscious drug design is described.

Abstract 3867: Combined PI3K and AURKA inhibition are efficacious in triple-negative breast cancer models

Abstract Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BMs) and face a poor prognosis. There are no FDA-approved systemic therapies to treat TNBC BM, due in part to the blood-brain barrier. BCBMs exhibit both activation of the PI3K pathway and AURKA amplification/overexpression relative to primary breast cancers. In this study, we evaluate the efficacy of brain-penetrant, clinically-available inhibitors of PI3K and AURKA in TNBC cell lines that are capable of growing in the mouse brain. Methods: In vitro characterization of the pan-PI3K inhibitor BKM120 and AURKA inhibitor MLN8237 were conducted in 2 human-derived TNBC cell lines, SUM149 and (MDA-MB-)231Br. A siRNA screen (720 kinase genes) was used to identify synthetic enhancers of lethality with BKM120 treatment. To assess the efficacy of these drugs, the IC50s of BKM120 and MLN8237 and synergy of the combination were determined. To compare the effects of BKM120 and/or MLN8237 treatment on cell cycle progression, FACS analysis was conducted at 24, 48, and 72 hours in parent cells and in cells continuously cultured in MLN8237-treated media for 12 weeks. Results: The screen confirmed that combined PI3K and AURKA inhibition synthetically enhanced lethality in SUM149 and 231Br cells. SUM149 and 231Br cells and two additional TNBC cell lines (MDA-MB-468 and MDA-MB-436) exhibited similar IC50s (1.3-21 μM) to BKM120. However, there was a >2.5 fold range (26.5-69 μM) in IC50s for MLN8237, with the greatest potency in the 231Br line. Concurrent treatment with BKM120+MLN8237 was synergistic or additive in 231Brs at most doses, whereas the combination was additive to antagonistic in SUM149s. Pretreatment with MLN8237 prior to concurrent BKM120+MLN8237 improved synergy in SUM149s, while BKM120 pretreatment improved synergy in 231Brs. FACS analysis of BKM120 in the SUM149 and 231Br cells induced a slight G1 arrest from 24 to 72 hours, while MLN8237 initially induced a G2 arrest at 24 hours, polyploidy at 48 hours, and a mixed polypoid/G2 arrested population at 72 hours. These effects were more pronounced in the 231Brs than the SUM149s. Combined BKM120+MLN8237 in both cell lines yielded results similar to MLN8237 alone. Cells continuously exposed to increasing MLN8237 concentrations from 50 nM to 300 nM for 12 weeks were resistant to MLN8237-induced cell cycle changes as compared to passage-matched controls. Conclusions: Combined PI3K+AURKA inhibition using brain-penetrant compounds is a promising strategy for a patient population with few options. In vivo studies evaluating the efficacy of BKM120+MLN8237 in intracranial TNBC mouse models to provide the translational foundation for future clinical studies are warranted. Citation Format: Amanda E.D. Van Swearingen, Maria J. Sambade, Shivani Sud, Brian Golitz, Gary L. Johnson, Carey K. Anders. Combined PI3K and AURKA inhibition are efficacious in triple-negative breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3867.

Structural Basis of Species-Dependent Differential Affinity of 6-Alkoxy-5-Aryl-3-Pyridinecarboxamide Cannabinoid-1 Receptor Antagonists.

6-Alkoxy-5-aryl-3-pyridincarboxamides, including the brain-penetrant compound 14G: [5-(4-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(1R,2R)-2-hydroxy-cyclohexyl]-3-pyridinecarboxamide] and its peripherally restricted analog 14H: [5-(4-chlorophenyl)-N-[(1R,2R)-2-hydroxycyclohexyl]-6-(2-methoxyethoxy)-3-pyridinecarboxamide], have been recently introduced as selective, high-affinity antagonists of the human cannabinoid-1 receptor (hCB1R). Binding analyses revealed two orders of magnitude lower affinity of these compounds for mouse and rat versus human CB1R, whereas the affinity of rimonabant is comparable for all three CB1Rs. Modeling of ligand binding to CB1R and binding assays with native and mutant (Ile105Met) hCB1Rs indicate that the Ile105 to Met mutation in rodent CB1Rs accounts for the species-dependent affinity of 14G: and 14H: . Our work identifies Ile105 as a new pharmacophore component for developing better hCB1R antagonists and invalidates rodent models for assessing the antiobesity efficacy of 14G: and 14H: .

Casein kinase 1δ/ε inhibitor PF-5006739 attenuates opioid drug-seeking behavior.

Casein kinase 1 delta (CK1δ) and casein kinase 1 epsilon (CK1ε) inhibitors are potential therapeutic agents for a range of psychiatric disorders. The feasibility of developing a CNS kinase inhibitor has been limited by an inability to identify safe brain-penetrant compounds with high kinome selectivity. Guided by structure-based drug design, potent and selective CK1δ/ε inhibitors have now been identified that address this gap, through the design and synthesis of novel 4-[4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl]pyrimidin-2-amine derivatives. PF-5006739 (6) possesses a desirable profile, with low nanomolar in vitro potency for CK1δ/ε (IC50 = 3.9 and 17.0 nM, respectively) and high kinome selectivity. In vivo, 6 demonstrated robust centrally mediated circadian rhythm phase-delaying effects in both nocturnal and diurnal animal models. Further, 6 dose-dependently attenuated opioid drug-seeking behavior in a rodent operant reinstatement model in animals trained to self-administer fentanyl. Collectively, our data supports further development of 6 as a promising candidate to test the hypothesis of CK1δ/ε inhibition in treating multiple indications in the clinic.

Discovery of a selective M4 positive allosteric modulator based on the 3-amino-thieno[2,3-b]pyridine-2-carboxamide scaffold: Development of ML253, a potent and brain penetrant compound that is active in a preclinical model of schizophrenia

Herein we report a next generation muscarinic receptor 4 (M4) positive allosteric modulator (PAM), ML253 which exhibits nanomolar activity at both the human (EC50=56nM) and rat (EC50=176nM) receptors and excellent efficacy by the left-ward shift of the ACh concentration response curve (fold shift, human=106; rat=50). In addition, ML253 is selective against the four other muscarinic subtypes, displays excellent CNS exposure and is active in an amphetamine-induced hyperlocomotion assay.

Design, Synthesis, and Pharmacological Evaluation of Piperidin-4-yl amino aryl sulfonamides: Novel, Potent, Selective, Orally Active, and Brain Penetrant 5-HT6 Receptor Antagonists

Our initial findings around aryl sulfonamide series led to N-(3,5-dichloro-2-methoxyphenyl)-3-(1-methylpiperidin-4-ylamino)-4-methoxy benzenesulfonamide as potent and selective 5-HT(6) receptor (5-HT(6)R) antagonist with reasonable pharmacokinetic properties and activity in animal models of cognition. However, lack of brain penetration and P-glycoprotein liability makes this scaffold unsuitable for further development. Our goal was to identify small molecule 5-HT(6)R antagonist with adequate brain penetration, acceptable ADME properties, no P-glycoprotein, and no hERG liability. Several structural modifications including bringing conformational constraint around the sulfonamide -NH group and introduction of a heteroatom to modulate the physicochemical properties were attempted. This effort culminated in the discovery of series of novel, potent, selective, orally bioavailable, and adequately brain penetrant compounds with no hERG liability. These compounds showed activity in animal models of cognition like object recognition task and water maze and in brain microdialysis studies at lower doses.

The characterization of microtubule-stabilizing drugs as possible therapeutic agents for Alzheimer's disease and related tauopathies

Tau, a protein that is enriched in neurons of the central nervous system (CNS), is thought to play a critical role in the stabilization of microtubules (MTs). Several neurodegenerative disorders referred to as tauopathies, including Alzheimer's disease and certain types of frontotemporal lobar degeneration, are characterized by the intracellular accumulation of hyperphosphorylated tau fibrils. Tau deposition into insoluble aggregates is believed to result in a loss of tau function that leads to MT destabilization, and this could cause neurodegeneration as intact MTs are required for axonal transport and normal neuron function. This tau loss-of-function hypothesis has been validated in a tau transgenic mouse model with spinal cord tau inclusions, where the MT-stabilizing agent, paclitaxel, increased spinal nerve MT density and improved motor function after drug absorption at neuromuscular junctions. Unfortunately, paclitaxel is a P-glycoprotein substrate and has poor blood–brain barrier permeability, making it unsuitable for the treatment of human tauopathies. We therefore examined several MT-stabilizing compounds from the taxane and epothilone natural product families to assess their membrane permeability and to determine whether they act as substrates or inhibitors of P-glycoprotein. Moreover, we compared brain and plasma levels of the compounds after administration to mice. Finally, we assessed whether brain-penetrant compounds could stabilize mouse CNS MTs. We found that several epothilones have significantly greater brain penetration than the taxanes. Furthermore, certain epothilones cause an increase in CNS MT stabilization, with epothilone D demonstrating a favorable pharmacokinetic and pharmacodynamic profile which suggests this agent merits further study as a potential tauopathy drug candidate.

Heterosynaptic crosstalk: GABA-glutamate metabotropic receptors interactively control glutamate release in solitary tract nucleus

Synaptic terminals often contain metabotropic receptors that act as autoreceptors to control neurotransmitter release. Less appreciated is the heterosynaptic crossover of glutamate receptors to control GABA release and vice versa GABA receptors which control glutamate release. In the brainstem, activation of solitary tract (ST) afferents releases glutamate onto second-order neurons within the solitary tract nucleus (NTS). Multiple metabotropic receptors are expressed in NTS for glutamate (mGluRs) and for GABA (GABA B). The present report identifies mGluR regulation of glutamate release at second and higher order sensory neurons in NTS slices. We found strong inhibition of glutamate release to group II and III mGluR activation on mechanically isolated NTS neurons. However, the same mGluR-selective antagonists paradoxically decreased glutamate release (miniature, mEPSCs) at identified second-order NTS neurons. Unaltered amplitudes were consistent with selective presynaptic mGluR actions. GABA B blockade in slices resolved the paradoxical differences and revealed a group II/III mGluR negative feedback of mEPSC frequency similar to isolated neurons. Thus, the balance of glutamate control is tipped by mGluR receptors on GABA terminals resulting in predominating heterosynaptic GABA B inhibition of glutamate release. Regulation by mGluR or GABA B was not consistently evident in excitatory postsynaptic currents (EPSCs) in higher-order NTS neurons demonstrating metabotropic receptor distinctions in processing at different NTS pathway stages. These cellular localizations may figure importantly in understanding interventions such as brain-penetrant compounds or microinjections. We conclude that afferent glutamate release in NTS produces a coordinate presynaptic activation of co-localized mGluR and GABA B feedback on cranial afferent terminals to regulate glutamate release.

S4-02-06: Integrative approach targeting neuroinflammation

Background: Neuroinflammation as a component of CNS disease is a complex process that involves a diversity of changes in products produced by glia. One change is the increased production of proinflammatory cytokines in response to diverse stressors. Clinical observations and preclinical animal studies identify increased cytokine production as a potential contributor to disease progression, requiring that it be tested as a potential drug discovery target for CNS disorders such as Alzheimer’s disease (AD). Because restoration of homeostasis is the goal, candidate therapies must be selective in their action (i.e., NOT pan-suppressors of glia function). Current FDA-approved drugs that modulate cytokine function are macromolecules, which have disadvantages for chronic CNS disorders. Therefore, there is a critical unmet need for small molecule, orally active, brain-penetrant compounds that can reduce excessive proinflammatory cytokine production by glia back towards basal levels with resultant improvement in neurologic outcomes. To address this need, we are using a de novo synthetic chemistry platform integrated with hierarchal biology screening to develop candidate compounds that attenuate excessive proinflammatory cytokine production and modify disease progression in animal models. Methods: We refer to the approach as use of “smart chemistry” integrated with “smart biology”. An inactive core scaffold was subjected to focused chemical diversification based on chemoinformatics and pharmacoinformatics to generative novel compounds that were prioritized based on screening their promise for metabolic stability, oral bioavailability, brain uptake, and lack of toxicity. Qualified compounds were subjected to efficacy testing in animal models. Results: Novel candidate compounds emerged within an accelerated timeline and limited budget. The unbiased, function-driven approach yielded one group of analogs referred to as the Minozac family, whereas the single molecular target approach that was focused on a protein kinase candidate yielded the Minokine family of compounds. Both Minozac and Minokine are efficacious in an AD-relevant animal model. Conclusions: The integrative approach, being increasingly adopted in academia and industry, allows for rapid and efficient discovery of novel small molecules that are candidates for therapeutic development campaigns focused on modification of disease progression.

Non-acidic pyrazole EP 1 receptor antagonists with in vivo analgesic efficacy

Replacement of the carboxylic acid group in a series of previously described methylene-linked pyrazole EP 1 receptor antagonists led to the discovery of amide, reversed amide and carbamate derivatives. Two compounds, 10a and 10b, were identified as brain penetrant compounds and both demonstrated efficacy in the CFA model of inflammatory pain.

Glia as a Therapeutic Target: Selective Suppression of Human Amyloid-β-Induced Upregulation of Brain Proinflammatory Cytokine Production Attenuates Neurodegeneration

A corollary of the neuroinflammation hypothesis is that selective suppression of neurotoxic products produced by excessive glial activation will result in neuroprotection. We report here that daily oral administration to mice of the brain-penetrant compound 4,6-diphenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine (MW01-5-188WH), a selective inhibitor of proinflammatory cytokine production by activated glia, suppressed the human amyloid-beta (Abeta) 1-42-induced upregulation of interleukin-1beta, tumor necrosis factor-alpha, and S100B in the hippocampus. Suppression of neuroinflammation was accompanied by restoration of hippocampal synaptic dysfunction markers synaptophysin and postsynaptic density-95 back toward control levels. Consistent with the neuropathophysiological improvements, MW01-5-188WH therapy attenuated deficits in Y maze behavior, a hippocampal-linked task. Oral MW01-5-188WH therapy begun 3 weeks after initiation of intracerebroventricular infusion of human Abeta decreased the numbers of activated astrocytes and microglia and the cytokine levels in the hippocampus without modifying amyloid plaque burden or altering peripheral tissue cytokine upregulation in response to an in vivo inflammatory challenge. The results provide a novel integrative chemical biology proof in support of the neuroinflammation hypothesis of disease progression, demonstrate that neurodegeneration can be attenuated independently of plaque modulation by targeting innate brain proinflammatory cytokine responses, and indicate the feasibility of developing efficacious, safe, and selective therapies for neurodegenerative disorders by targeting key glial activation pathways.

Pharmacological blockade or genetic deletion of substance P (NK 1) receptors attenuates neonatal vocalisation in guinea-pigs and mice

The regulation of stress-induced vocalisations by central NK 1 receptors was investigated using pharmacological antagonists in guinea-pigs, a species with human-like NK 1 receptors, and transgenic NK1R−/− mice. In guinea-pigs, i.c.v. infusion of the selective substance P agonist GR73632 (0.1 nmol) elicited a pronounced vocalisation response that was blocked enantioselectively by the NK 1 receptor antagonists CP-99,994 and L-733,060 (0.1–10 mg/kg). GR73632-induced vocalisations were also markedly attenuated by the antidepressant drugs imipramine and fluoxetine (30 mg/kg), but not by the benzodiazepine anxiolytic diazepam (3 mg/kg) or the 5-HT 1A agonist buspirone (10 mg/kg). Similarly, vocalisations in guinea-pig pups separated from their mothers were blocked enantioselectively by the highly brain-penetrant NK 1 receptor antagonists L-733,060 and GR205171 (ID 50 3 mg/kg), but not by the poorly brain-penetrant compounds LY303870 and CGP49823 (30 mg/kg). Separation-induced vocalisations were also blocked by the anxiolytic drugs diazepam, chlordiazepoxide and buspirone (ID 50 0.5–1 mg/kg), and by the antidepressant drugs phenelzine, imipramine, fluoxetine and venlafaxine (ID 50 3–8 mg/kg). In normal mouse pups, GR205171 attenuated neonatal vocalisations when administered at a high dose (30 mg/kg) only, consistent with its lower affinity for the rat than the guinea-pig NK 1 receptor. Ultrasound calls in NK1R−/− mouse pups were markedly reduced compared with those in WT pups, confirming the specific involvement of NK 1 receptors in the regulation of vocalisation. These observations suggest that centrally-acting NK 1 receptor antagonists may have clinical utility in the treatment of a range of anxiety and mood disorders.