2026: Pharmacological Characterization and Preclinical Evaluation

Pharmacological characterization and preclinical evaluation of 11h: a novel, brain-penetrant PDE4 inhibitor for neurological disorders

Background:

Phosphodiesterase 4 (PDE4) inhibitors hold promise for treating neuroinflammatory and neurodegenerative disorders, but their clinical application in central nervous system (CNS) diseases has been limited by insufficient brain penetration and adverse effects, especially nausea and vomiting. In this study, we characterize compound 11h, a novel, orally available, brain-penetrant PDE4 inhibitor designed to address these limitations.

Methods:

11h was evaluated using integrated computational, in vitro, and in vivo approaches, including Schrödinger-based molecular docking against human PDE4A10, luminescence-based cAMP assays for PDE4 isoform inhibition, and inflammatory assays in murine macrophages, microglia-like cells, and human PBMCs. In vivo efficacy, pharmacokinetics, brain and tissue distribution, tolerability, emesis liability, and behavioral effects were assessed across mouse, rat, and ferret models, alongside comprehensive safety pharmacology encompassing CYP450 inhibition, cardiac ion channel profiling, mutagenicity, and off-target screening.

Results:

11h demonstrated potent, broad-spectrum inhibition across all four PDE4 isoforms, including key PDE4D splice variants (PDE4D2, PDE4D3), with low nanomolar IC50 values. In vitro, 11h significantly reduced the release of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), and nitrites in lipopolysaccharide (LPS)-stimulated macrophages, microglia-like cells, and human peripheral blood mononuclear cells without affecting cell viability. In vivo, 11h attenuated neuroinflammation in LPS-treated mice by decreasing M1 macrophages and CD4+ T cells, increasing M2 macrophages, and downregulating pro-inflammatory cytokines and MyD88 pathway genes. Pharmacokinetic analysis in rats confirmed strong oral bioavailability, dose-proportional systemic exposure, and sustained brain concentrations exceeding plasma levels for up to 48 h post-dose. Importantly, 11h did not induce vomiting in the ferret emesis model even at doses exceeding 50-fold the efficacious levels in rodent disease models, and was well tolerated in behavioral assays, where it produced anxiolytic-and antidepressant-like effects. Safety profiling revealed no cytotoxicity, genotoxicity, or significant inhibition of cardiac ion channels or cytochrome P450 enzymes. Consistent with this experimental profile, molecular docking suggested that 11h preferentially engages a pocket within the PDE4 catalytic domain, with high predicted binding affinity and ligand efficiency driven by hydrophobic and electrostatic interactions.

Conclusion:

These findings suggest that 11h provides broad PDE4 inhibition with a favorable tolerability and pharmacokinetic profile relative to approved PDE4 inhibitors, supporting its further development as a therapeutic candidate for CNS disorders characterized by neuroinflammation.

Introduction

Over the past decade, phosphodiesterase 4 (PDE4) enzymes have become compelling therapeutic targets for central nervous system (CNS) disorders. PDE4 enzymes are key regulators of intracellular cyclic adenosine monophosphate (cAMP) signaling, a critical second messenger involved in numerous cellular processes, including gene transcription, synaptic plasticity, cell survival, and inflammatory responses (Conti et al., 2003; Blokland et al., 2019a). Within the CNS, PDE4 isoforms, particularly PDE4A, PDE4B, and PDE4D, are broadly expressed within neurons, astrocytes, microglia, and oligodendrocytes, where they control localized cAMP pools and mediate the downstream activation of protein kinase A (PKA), cAMP response element-binding protein (CREB), and other effectors (Blokland et al., 2019a; Pérez-Torres et al., 2000; Lakics et al., 2010). The spatial and temporal regulation of cAMP by PDE4 is essential for maintaining neuroplasticity, modulating neuroimmune interactions, and enabling proper neuronal adaptation to external stimuli (Blokland et al., 2019a; Donders et al., 2024; Rombaut et al., 2021).

Dysregulated PDE4 activity has been strongly implicated in a broad range of neurological and neuropsychiatric disorders. In neurons, excessive PDE4-mediated cAMP degradation impairs CREB-dependent transcription, disrupts synaptic plasticity, and reduces the brain’s capacity for functional remodeling throughout the central executive network, contributing to cognitive decline and mood dysregulation (Kandel, 2012; Janes et al., 2009; Bollen et al., 2014). In glial cells, particularly microglia and astrocytes, aberrant PDE4 activity promotes chronic neuroinflammation by enhancing the production of pro-inflammatory cytokines, such as Tumor Necrosis Factor alpha (TNFα) and interleukin-6 (IL-6), and maintaining an activated immune state (Zhang et al., 2022; Jin et al., 2005; Jin and Conti, 2002). These cellular effects have been linked to pathogenesis across Alzheimer’s (AD) and Parkinson’s disease, multiple sclerosis (MS), substance use disorders, and schizophrenia, among others (Blokland et al., 2019a; Chen et al., 2009; Schepers et al., 2023; Singh, 2022).

Preclinical studies have consistently demonstrated the therapeutic potential of PDE4 inhibition across diverse CNS disease models, highlighting its broad pharmacological activity. For example, in rodent models of depression, PDE4 inhibitors such as rolipram produce antidepressant-like effects comparable to selective serotonin reuptake inhibitors (SSRIs), driven by enhanced cAMP signaling and subsequent upregulation of CREB-mediated transcription of neurotrophic factors (Cong et al., 2023; Wachtel and Schneider, 1986; Zhang et al., 2017). In disease models associated with pronounced neuroinflammation, such as experimental autoimmune encephalomyelitis and traumatic brain injury, PDE4 inhibition attenuates glial activation, reduces pro-inflammatory cytokine production, and promotes neuroprotection (Schepers et al., 2023; Titus et al., 2016). Similarly, in models of cognitive impairment, PDE4 blockade improves hippocampal long-term potentiation and enhances memory performance in behavioral tasks (Cong et al., 2023; Gong et al., 2004; Imanishi et al., 1997). Clinical trials have largely echoed these findings. Early clinical trials with rolipram demonstrated encouraging antidepressant effects in patients with major depressive disorder, while low dose roflumilast, a second-generation PDE4 inhibitor, has been shown to improve verbal and working memory in individuals with schizophrenia and healthy older adults (Zeller et al., 1984; Gilleen et al., 2021; Blokland et al., 2019b). In more recent trials, treatment with ibudilast, a non-selective PDE inhibitor with PDE4 activity, was shown to significantly reduce brain atrophy in a Phase II clinical trial in patients with progressive MS (Fox et al., 2018). Collectively, these findings support the therapeutic potential of PDE4 inhibitors across a range of neurological and psychiatric conditions.

Several PDE4 inhibitors, such as roflumilast (Daliresp), apremilast (Otezla), and crisaborole (Eucrisa), have been successfully developed and approved for peripheral inflammatory conditions, demonstrating that PDE4 is a clinically validated and druggable target (Li et al., 2018). However, despite extensive efforts to develop PDE4 inhibitors for CNS disorders, clinical success has been hampered by various issues, including poor brain penetration, suboptimal pharmacokinetics, and dose-limiting side effects, especially nausea and diarrhea (McDonough et al., 2020; Robic et al., 1999; Kang et al., 2023; Krause and Kühne, 1988; Sanftner et al., 2009). To address these limitations, several strategies have been explored, including the development of subtype-selective inhibitors and compounds with refined binding properties (Burgin et al., 2010; Naganuma et al., 2009; Barnette et al., 1995). While subtype-selective compounds have demonstrated improved tolerability, effective modulation of disease-relevant signaling pathways may require broader inhibition across subtypes, which subtype-selective agents may not achieve. To date, no broad-spectrum PDE4 inhibitor has shown clinical efficacy in CNS disorders without being limited by intolerable side effects or insufficient brain penetration.These challenges highlight the need for next-generation PDE4 inhibitors with improved brain penetration and tolerability.

In this report, we describe the in vitro and in vivo pharmacological and safety profile of 11h, a potent, selective, and brain-penetrant PDE4 inhibitor that demonstrates robust anti-neuroinflammatory activity and improved tolerability in preclinical models. Compound 11h was rationally designed to address the limitations of first- and second-generation PDE4 inhibitors by optimizing molecular structure and pharmacokinetic properties to enhance CNS exposure and achieve therapeutic efficacy at lower doses (Vadukoot et al., 2020). These modifications aim to expand the therapeutic window and reduce dose-limiting adverse effects commonly associated with PDE4 inhibition. Molecular docking studies demonstrated that 11h binds with high affinity to a pocket within the catalytic domain of PDE4, characterized by strong hydrophobic and electrostatic complementarity, a finding that may contribute to its optimized binding profile. The data presented herein characterize the pharmacokinetic properties and potent anti-inflammatory activity of 11h in rodent models, and demonstrate that the compound is safe and well-tolerated with repeat dosing. Notably, 11h exhibits no significant off-target activity, appears to lack cytotoxic or genotoxic potential, and does not elicit emesis even at supratherapeutic doses in rodent models. These findings highlight the favorable safety and tolerability profile of 11h and its potential as a novel PDE4 inhibitor for CNS diseases.

Materials and methods

Schrödinger docking studies and protein reliability assessments

The three-dimensional crystal structure of human PDE4A10, a long isoform which consists of all known regulatory binding domains in the enzyme family, was obtained from the Protein Data Bank (PDB ID: 2QYK). Structural preparation was carried out using the Schrödinger Protein Preparation Wizard. This process involved modeling missing side chains and loops, optimizing the hydrogen bonding network, assigning protonation states consistent with physiological pH (∼7.4), and energy minimizing the structure to resolve steric clashes and ensure stability. To identify potential ligand binding sites, the protein was analyzed using Schrödinger’s SiteMap tool. Binding pockets were evaluated for druggability based on volume, enclosure, hydrophobic/hydrophilic balance, and hydrogen bonding potential. Five sites were identified, with Site 1 emerging as the most druggable (SiteScore = 1.045; volume = 433.9 Å3), and Sites 2–5 demonstrating progressively lower SiteScores and volumes.

11h was prepared for docking using LigPrep (Schrödinger Suite), which generated low-energy three-dimensional conformers and assigned appropriate protonation and ionization states at pH 7.0 ± 0.5. The molecule was found to contain three rotatable bonds. Molecular docking simulations were performed using the Glide module in standard precision (SP) mode. 11h was docked independently into each of the five identified binding sites using a rigid receptor and flexible ligand protocol. Docking poses were evaluated based on multiple criteria including GlideScore, Glide emodel, lipophilic and Coulombic interaction energies, hydrogen bonding contributions, and ligand efficiency metrics (size-normalized binding energy).

To ensure structural reliability of the PDE4 model, a comprehensive quality assessment was conducted. This included evaluation of Ramachandran outliers, identification of buried unsatisfied polar groups, and detection of steric clashes. Minor issues such as a steric clash involving GLN454 and several disallowed backbone dihedrals were addressed through targeted refinement, resulting in a robust model suitable for high confidence docking studies.

Animal models

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and relevant institutional ethical review boards at K2bio (Houston, TX), Temple University (Philadelphia, PA), Porsolt (France) and TheraIndx Life Sciences (India), and conducted in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al., 2020). Group sizes were determined by a priori power analysis (85% power) to ensure sufficient statistical rigor while minimizing animal use. Adult male C57BL/6 mice (6–8 weeks old; The Jackson Laboratory), Sprague-Dawley (SD) rats (6–8 weeks old; Charles River Laboratories), and male ferrets (1.09–1.87 kg; Marshall BioResources, USA) were used in the studies. Animals were acclimated to the housing environment for at least 1 week prior to experimentation. Mice were housed in groups of three to five in ventilated microisolator cages, and rats in groups of two to three per cage in individually ventilated cages, under a 12 h light/dark cycle (lights on at 07:00), with controlled temperature (22 °C ± 2 °C) and humidity (50% ± 10%). Both species had access to food and water ad libitum, with corncob bedding, nesting material, and enrichment. Ferrets were housed in pairs in stainless steel cages (20 °C–22 °C, 12 h light/dark cycle), fed commercial ferret chow and provided daily environmental enrichment. In studies where euthanasia was required, animals were euthanized humanely by intraperitoneal (IP) injection of an overdose of sodium pentobarbital (>150 mg/kg), followed by transcardial perfusion with ice-cold phosphate-buffered saline (PBS) to ensure death prior to tissue collection. All efforts were made to minimize suffering and distress throughout the study.

Chemicals and reagents

11h was synthesized and initially characterized by the University of Texas at San Antonio, with process optimization and scale-up performed by LAXAI Life Sciences (India). High-performance liquid chromatography (HPLC) confirmed purity exceeding 97%. For in vitro studies, 11h was dissolved in 100% dimethyl sulfoxide (DMSO). For in vivo use, it was formulated in a vehicle consisting of 15% ethanol, 5% DMSO, 30% PEG-400, 10% Cremophor, and 40% saline. The same vehicle was used as control in corresponding studies. Additional reagents are described in each section below.