ACKR3 Antagonism Enhances the Repair of Demyelinated Lesions Through Both Immunomodulatory and Remyelinating Effects.

Addressing inflammation, demyelination, and associated neurodegeneration in inflammatory demyelinating diseases like multiple sclerosis (MS) remains challenging. ACT-1004-1239, a first-in-class and potent ACKR3 antagonist, currently undergoing clinical development, showed promise in preclinical MS models, reducing neuroinflammation and demyelination. However, its effectiveness in treating established disease and impact on remyelination after the occurrence of demyelinated lesions remain unexplored. This study assessed the therapeutic effect of ACT-1004-1239 in two demyelinating disease models. In the proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) model, ACT-1004-1239 administered upon the detection of the first signs of paralysis, resulted in a dose-dependent reduction in EAE disease severity, concomitant with diminished immune cell infiltrates in the CNS and reduced demyelination. Notably, efficacy correlated with elevated plasma concentrations of CXCL11 and CXCL12, two pharmacodynamic biomarkers of ACKR3 antagonism. Combining ACT-1004-1239 with siponimod, an approved immunomodulatory treatment for MS, synergistically reduced EAE severity. In the cuprizone-induced demyelination model, ACT-1004-1239 administered after 5 weeks of cuprizone exposure, significantly accelerated remyelination, already quantifiable one week after cuprizone withdrawal. Additionally, ACT-1004-1239 penetrated the CNS, elevating brain CXCL12 concentrations. These results demonstrate that ACKR3 antagonism significantly reduces the severity of experimental demyelinating diseases, even when treatment is initiated therapeutically, after the occurrence of lesions. It confirms the dual mode of action of ACT-1004-1239, exhibiting both immunomodulatory effects by reducing neuroinflammation and promyelinating effects by accelerating myelin repair. The results further strengthen the rationale for evaluating ACT-1004-1239 in clinical trials for patients with demyelinating diseases.

The mutual neutralization of hydronium and hydroxide.

Mutual neutralization of hydronium (H3O+) and hydroxide (OH-) ions is a very fundamental chemical reaction. Yet, there is only limited experimental evidence about the underlying reaction mechanisms. Here, we report three-dimensional imaging of coincident neutral products of mutual-neutralization reactions at low collision energies of cold and isolated ions in the cryogenic double electrostatic ion-beam storage ring (DESIREE). We identified predominant H2O + OH + H and 2OH + H2 product channels and attributed them to an electron-transfer mechanism, whereas a minor contribution of H2O + H2O with high internal excitation was attributed to proton transfer. The reported mechanism-resolved internal product excitation, as well as collision-energy and initial ion-temperature dependence, provide a benchmark for modeling charge-transfer mechanisms.

Pharmacokinetics, pharmacodynamics and safety of the novel C-X-C chemokine receptor 3 antagonist ACT-777991: Results from the first-in-human study in healthy adults.

AIMS: The C-X-C chemokine receptor 3 (CXCR3) axis is highly upregulated in the tissue of patients with type 1 diabetes. Antagonizing CXCR3 may reduce the migration of CXCR3-expressing cells to the pancreas. The pharmacokinetics (PKs), target engagement (TE) (inhibition of CXCR3 internalization) and safety of single- and multiple-ascending doses (SADs and MADs) of ACT-777991, a novel orally available potent CXCR3 antagonist, were assessed in a double-blind, randomized, placebo-controlled phase 1 study. METHODS: Doses up to 100 mg (SAD part) and 40 mg twice daily (MAD part) were investigated in a total of 70 male and female healthy participants. Food effect was integrated as an SAD subpart. PK, TE, safety and tolerability data were collected up to 4 days after (last) dosing. RESULTS: In both SAD and MAD parts, ACT-777991 was rapidly absorbed with a time to reach maximum concentration between 0.5 and 1.5 h post dose, followed by a biphasic disposition with a terminal half-life between 9.7 and 10.3 h. Increase in exposure and maximum concentration of ACT-777991 were dose-proportional. Steady state was reached after 48 h with minimal accumulation. The rate but not the extent of absorption was modified by food intake. A dose-dependent TE was demonstrated in both SAD and MAD parts. ACT-777991 was well tolerated. Neither a treatment-related pattern nor a dose-response relationship was determined for adverse events or any safety variable. No QT prolongation liability of regulatory concern was detected. CONCLUSIONS: In this first-in-human study, ACT-777991 showed good tolerability for all doses tested and a PK and TE profile suitable for further clinical development.

Simultaneous electrostatic trapping of merged cation & anion beams.

Simultaneous trapping of merged cation and anion beams in the hybrid electrostatic ion beam trap (HEIBT) opens new opportunities for the study of the interactions of isolated atomic molecular or cluster ions with oppositely charged ionic species. Application of the trapped merged beams requires a detailed understanding of the trapping dynamics and the effect of the Coulombic attractive and repulsive forces between the ions on their motion in the trap. The simultaneous trapping regime is explored experimentally for SF6- anion and SF5+ cation beams and compared to realistic ion trajectory simulations. The respective stability of the simultaneously trapped cation and anion beams is experimentally tracked by nondestructive and mass sensitive image charge monitoring. An approximate analytical potential model is presented for modeling the dynamics of trapped ions, providing insight into the role of ion-ion interactions, and suggesting a simplified mirror design.

Combination treatment of a novel CXCR3 antagonist ACT-777991 with an anti-CD3 antibody synergistically increases persistent remission in experimental models of type 1 diabetes.

Treatment of patients with recent-onset type 1 diabetes with an anti-CD3 antibody leads to the transient stabilization of C-peptide levels in responder patients. Partial efficacy may be explained by the entry of islet-reactive T-cells spared by and/or regenerated after the anti-CD3 therapy. The CXCR3/CXCL10 axis has been proposed as a key player in the infiltration of autoreactive T cells into the pancreatic islets followed by the destruction of ß cells. Combining the blockade of this axis using ACT-777991, a novel small-molecule CXCR3 antagonist, with anti-CD3 treatment may prevent further infiltration and ß-cell damage and thus, preserve insulin production. The effect of anti-CD3 treatment on circulating T-cell subsets, including CXCR3 expression, in mice was evaluated by flow cytometry. Anti-CD3/ACT-777991 combination treatment was assessed in the virally induced RIP-LCMV-GP and NOD diabetes mouse models. Treatments started at disease onset. The effects on remission rate, blood glucose concentrations, insulitis, and plasma C-peptide were evaluated for the combination treatment and the respective monotherapies. Anti-CD3 treatment induced transient lymphopenia but spared circulating CXCR3+ T cells. Combination therapy in both mouse models synergistically and persistently reduced blood glucose concentrations, resulting in increased disease remission rates compared to each monotherapy. At the study end, mice in disease remission demonstrated reduced insulitis and detectable plasma C-peptide levels. When treatments were initiated in non-severely hyperglycemic NOD mice at diabetes onset, the combination treatment led to persistent disease remission in all mice. These results provide preclinical validation and rationale to investigate the combination of ACT-777991 with anti-CD3 for the treatment of patients with recent-onset diabetes.

Discovery of Clinical Candidate ACT-777991, a Potent CXCR3 Antagonist for Antigen-Driven and Inflammatory Pathologies.

The CXCR3 chemokine receptor is a G protein-coupled receptor mainly expressed on immune cells from the lymphoid lineage, including activated T cells. Binding of its inducible chemokine ligands CXCL9, CXCL10, and CXCL11 leads to downstream signaling events and the migration of activated T cells to sites of inflammation. Herein, we report the third part of our CXCR3 antagonist program in the field of autoimmunity, culminating in the discovery of the clinical compound ACT-777991 (8a). A previously disclosed advanced molecule was exclusively metabolized by the CYP2D6 enzyme, and options to address the issue are described. ACT-777991 is a highly potent, insurmountable, and selective CXCR3 antagonist that showed dose-dependent efficacy and target engagement in a mouse model of acute lung inflammation. The excellent properties and safety profile warranted progress in the clinics.

Sequential mechanism in H3+ formation dynamics on the ethanol dication.

Two- and three-body Coulomb explosion dynamics of isolated ethanol dications are studied via single-photon double-ionization with ultrafast extreme-ultraviolet pulses. The measured 3-body momentum correlations obtained via 3D coincidence imaging of the ionic products provide evidence for several concerted and sequential mechanisms: (1) a concerted 3-body breakup mechanism, with dominating channels such as CH3+ + COH+ + H2; (2) sequential dissociation in which the ejection of a low-kinetic-energy neutral OH precedes the Coulomb explosion of C2H52+ → CH3+ + CH2+; and (3) a sequential 3-body breakup mechanism that dominates H3+ formation from the ethanol dication via a mechanism that is different from the well-studied H3+ formation in the 2-body Coulomb explosion of the methanol dication. Furthermore, we report surprising branching ratios of the competing C-O bond dissociation channels, resulting in H3O+, H2O+ and OH+ formation.

An "inverse" harpoon mechanism.

Electron-transfer reactions are ubiquitous in chemistry and biology. The electrons' quantum nature allows their transfer across long distances. For example, in the well-known harpoon mechanism, electron transfer results in Coulombic attraction between initially neutral reactants, leading to a marked increase in the reaction rate. Here, we present a different mechanism in which electron transfer from a neutral reactant to a multiply charged cation results in strong repulsion that encodes the electron-transfer distance in the kinetic energy release. Three-dimensional coincidence imaging allows to identify such "inverse" harpoon products, predicted by nonadiabatic molecular dynamics simulations to occur between H2 and HCOH2+ following double ionization of isolated methanol molecules. These dynamics are experimentally initiated by single-photon double ionization with ultrafast extreme ultraviolet pulses, produced by high-order harmonic generation. A detailed comparison of measured and simulated data indicates that while the relative probability of long-range electron-transfer events is correctly predicted, theory overestimates the electron-transfer distance.

Sequential and concerted C-C and C-O bond dissociation in the Coulomb explosion of 2-propanol.

We study the competing mechanisms involved in the Coulomb explosion of 2-propanol CH3 2CHOH2+ dication, formed by an ultrafast extreme ultraviolet pulse. Over 20 product channels are identified and characterized using 3D coincidence imaging of the ionic fragments. The momentum correlations in the three-body fragmentation channels provide evidence for a dominant sequential mechanism, starting with the cleavage of a C-C bond, ejecting CH3 + and CH3CHOH+ cations, followed by a secondary fragmentation of the hydroxyethyl cation that can be delayed for up to a microsecond after ionization. The C-O bond dissociation channels are less frequent, involving proton transfer and double proton transfer, forming H2O+ and H3O+ products, respectively, and exhibiting mixed sequential and concerted character. These results can be explained by the high potential barrier for the C-O bond dissociation seen in our ab initio quantum chemical calculations. We also observe coincident COH+ + C2Hn + ions, suggesting exotic structural rearrangements, starting from the Frank-Condon geometry of the neutral 2-propanol system. Remarkably, the relative yield of the H3 + product is suppressed compared with methanol and alkene dications. Ab initio potentials and ground state molecular dynamics simulations show that a rapid and direct C-C bond cleavage dominates the Coulomb explosion process, leaving no time for H2 roaming, which is a necessary precursor to the H3 + formation.

Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients.

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients.

CXCR7 Antagonism Reduces Acute Lung Injury Pathogenesis.

Loss of control in the trafficking of immune cells to the inflamed lung tissue contributes to the pathogenesis of life-threatening acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). Targeting CXCR7 has been proposed as a potential therapeutic approach to reduce pulmonary inflammation; however, its role and its crosstalk with the two chemokine receptors CXCR3 and CXCR4 via their shared ligands CXCL11 and CXCL12 is not yet completely understood. The present paper aimed to characterize the pathological role of the CXCR3/CXCR4/CXCR7 axis in a murine model of ALI. Lipopolysaccharide (LPS) inhalation in mice resulted in the development of key pathologic features of ALI/ARDS, including breathing dysfunctions, alteration in the alveolar capillary barrier, and lung inflammation. LPS inhalation induced immune cell infiltration into the bronchoalveolar space, including CXCR3+ and CXCR4+ cells, and enhanced the expression of the ligands of these two chemokine receptors. The first-in-class CXCR7 antagonist, ACT-1004-1239, increased levels of CXCL11 and CXCL12 in the plasma without affecting their levels in inflamed lung tissue, and consequently reduced CXCR3+ and CXCR4+ immune cell infiltrates into the bronchoalveolar space. In the early phase of lung inflammation, characterized by a massive influx of neutrophils, treatment with ACT-1004-1239 significantly reduced the LPS-induced breathing pattern alteration. Both preventive and therapeutic treatment with ACT-1004-1239 reduced lung vascular permeability and decreased inflammatory cell infiltrates. In conclusion, these results demonstrate a key pathological role of CXCR7 in ALI/ARDS and highlight the clinical potential of ACT-1004-1239 in ALI/ARDS pathogenesis.

A Multipurpose First-in-Human Study With the Novel CXCR7 Antagonist ACT-1004-1239 Using CXCL12 Plasma Concentrations as Target Engagement Biomarker.

The C-X-C chemokine receptor 7 (CXCR7) has evolved as a promising, druggable target mainly in the immunology and oncology fields modulating plasma concentrations of its ligands CXCL11 and CXCL12 through receptor-mediated internalization. This "scavenging" activity creates concentration gradients of these ligands between blood vessels and tissues that drive directional cell migration. This randomized, double-blind, placebo-controlled first-in-human study assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of ACT-1004-1239, a first-in-class drug candidate small-molecule CXCR7 antagonist. Food effect and absolute bioavailability assessments were also integrated in this multipurpose study. Healthy male subjects received single ascending oral doses of ACT-1004-1239 (n = 36) or placebo (n = 12). At each of six dose levels (1-200 mg), repeated blood sampling was done over 144 hours for pharmacokinetic/pharmacodynamic assessments using CXCL11 and CXCL12 as biomarkers of target engagement. ACT-1004-1239 was safe and well tolerated up to the highest tested dose of 200 mg. CXCL12 plasma concentrations dose-dependently increased and more than doubled compared with baseline, indicating target engagement, whereas CXCL11 concentrations remained unchanged. An indirect-response pharmacokinetic/pharmacodynamic model well described the relationship between ACT-1004-1239 and CXCL12 concentrations across the full dose range, supporting once-daily dosing for future clinical studies. At doses ≥ 10 mg, time to reach maximum plasma concentration ranged from 1.3 to 3.0 hours and terminal elimination half-life from 17.8 to 23.6 hours. The exposure increase across the dose range was essentially dose-proportional and no relevant food effect on pharmacokinetics was determined. The absolute bioavailability was 53.0% based on radioactivity data after oral vs. intravenous 14 C-radiolabeled microtracer administration of ACT-1004-1239. Overall, these comprehensive data support further clinical development of ACT-1004-1239.

Correction: Dissociative detachment of the fluoroformate anion.

Correction for 'Dissociative detachment of the fluoroformate anion' by Eugene Shirman et al., Phys. Chem. Chem. Phys., 2020, DOI: 10.1039/d0cp04283h.

Time-resolved dissociative ionization and double photoionization of CO2.

CO2 single-photon double photoionization, Coulomb explosion, and dissociative ionization are studied with ultrafast extreme-ultraviolet pump and time-delayed near-infrared probe pulses. Kinetic energy release and momentum correlations for the two-body CO+ + O+ and three-body O+ + C+ + O fragmentation products are determined by 3D coincidence fragment imaging. The transient enhancement of the ratio of two-body vs three-body Coulomb explosion events and the time dependence of low and high kinetic energy release dissociation events are discussed in terms of dissociative ionization and Coulomb explosion dynamics.

Dissociative detachment of the fluoroformate anion.

Dissociative photodetachment of the FCO2- fluoroformate complex by intense laser pulses is studied using 3D coincidence fragment imaging. The main channels are found to be CO2 + F and FCO + O. Cleavage of the C-F bond is attributed to dissociation on the B[combining tilde]2A1 excited state of the neutral FCO2 radical with significant internal excitation of the molecular fragment, while reductive dissociation of the CO2 moiety is assigned to higher lying states. The measured dissociative ionization products of double-photodetachment are discussed and attributed to intense-laser ionization of dissociative photodetachment products.

Immunoaffinity Targeted Mass Spectrometry Analysis of Human Plasma Samples Reveals an Imbalance of Active and Inactive CXCL10 in Primary Sjögren's Syndrome Disease Patients.

One of the most important advantages of mass spectrometry is the ability to quantify proteins and their modifications in parallel to obtain a holistic picture of the protein of interest. Here, we present a hybrid immunoaffinity targeted mass spectrometry (MS) method that combines efficient pan-antibody enrichment of a specific protein from plasma with the selectivity of high-resolution targeted MS analysis to quantitate specific proteoforms of interest. We used this approach to quantify plasma levels of the chemokine CXCL10 that has been associated with many immunological disorders such as systemic lupus erythematosus and primary Sjögren's Syndrome (pSS). The hybrid approach enabled sensitive, specific, and simultaneous quantification of total, full-length (active) CXCL101-77 and DPP4-truncated (inactive) CXCL103-77 in human plasma down to the low pg/mL level, reaching ELISA sensitivities. Samples from 30 control subjects and 34 pSS patients (n = 64) were analyzed. The ratio of CXCL101-77 to truncated CXCL103-77 was significantly increased in patients with pSS and provided the highest correlation with pSS disease activity. Therefore, this CXCL10 proteoform ratio represents an interesting exploratory disease activity biomarker to further investigate. As this strategy can be readily adapted to other plasma proteins and proteoforms of interest, we are convinced that it will lead to a more detailed understanding of proteoforms in physiology and pathology yielding more relevant biomarkers and drug targets.

Absence of Triplets in Single-Photon Double Ionization of Methanol.

Despite the abundance of data concerning single-photon double ionization of methanol, the spin state of the emitted electron pair has never been determined. Here we present the first evidence that identifies the emitted electron pair spin as overwhelmingly singlet when the dication forms in low-energy configurations. The experimental data show that while the yield of the CH2O+ + H3+ Coulomb explosion channel is abundant, the metastable methanol dication is largely absent. According to high-level ab initio simulations, these facts indicate that photoionization promptly forms singlet dication states, where they quickly decompose through various channels, with significant H3+ yields on the low-lying states. In contrast, if we assume that the initial dication is formed in one of the low-lying triplet states, the ab initio simulations exhibit a metastable dication, contradicting the experimental findings. Comparing the average simulated branching ratios with the experimental data suggests a >3 order of magnitude enhancement of the singlet:triplet ratio compared with their respective 1:3 multiplicities.

Time-resolving the ultrafast H2 roaming chemistry and H3+ formation using extreme-ultraviolet pulses.

The time scales and formation mechanisms of tri-hydrogen cation products in organic molecule ionization processes are poorly understood, despite their cardinal role in the chemistry of the interstellar medium and in other chemical systems. Using an ultrafast extreme-ultraviolet pump and time-resolved near-IR probe, combined with high-level ab initio molecular dynamics calculations, here we report unambiguously that H3+ formation in double-ionization of methanol occurs on a sub 100 fs time scale, settling previous conflicting findings of strong-field Coulomb explosion experiments. Our combined experimental-computational studies suggest that ultrafast competition, between proton-transfer and long-range electron-transfer processes, determines whether the roaming neutral H2 dynamics on the dication result in [Formula: see text] or [Formula: see text] fragments respectively.

Impact of high-altitude therapy on type-2 immune responses in asthma patients.

BACKGROUND: Asthma patients present with distinct immunological profiles, with a predominance of type 2 endotype. The aim of this study was to investigate the impact of high-altitude treatment on the clinical and immunological response in asthma. METHODS: Twenty-six hospitalized asthma patients (nine eosinophilic allergic; EA, nine noneosinophilic allergic; NEA and eight noneosinophilic nonallergic; NN) and nine healthy controls in high altitude for 21 days were enrolled in the study. We assessed eosinophils, T cells, Tregs, and innate lymphoid cells (ILC) from peripheral blood using flow cytometry. RESULTS: The number of eosinophils (both resting and activated) and chemoattractant receptor homolog expressed on Th2 cells (CRTH2)-expressing CD4+ and CD8+ T cells decreased significantly in EA patients after altitude treatment. The frequency of CRTH2+ Tregs as decreased significantly in all the asthma phenotypes as well as the frequency of ILC2 was significantly reduced in EA after altitude treatment. After 21 days of altitude therapy, CRTH2-expressing ILC2, CD4+ and CD8+ T cells and Treg cells showed attenuated responses to exogenous PGD2. Furthermore, PGD2 signaling via CRTH2 was found to diminish the suppressive function of CRTH2+ Tregs which partially normalized during high-altitude treatment. Improved asthma control was particularly evident in allergic asthma patients and correlated with decreased frequencies of CRTH2+ Treg cells in EA patients. Serum IL-5 and IL-13 decreased during climate treatment in asthma patients with high baseline levels. CONCLUSIONS: Asthma treatment in high altitude reduced the type 2 immune response, corrected the increased CRTH2 expression and its dysregulated functions.