Transport receptor occupancy in Nuclear Pore Complex mimics

Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess 'Kap-centric' NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

Brief Report: Higher Fentanyl Exposures Require Higher Doses of Naloxone for Successful Reversals in a Quantitative Systems Pharmacology Model

Background: Previously, we reported on an opioid receptor quantitative systems pharmacology (QSP) model to evaluate naloxone dosing. Methods: In this study we extended our model to include higher systemic levels of fentanyl (up to 100 ng/ml) and the newly approved 8mg IN naloxone dose (equivalent to 4 mg)Results : As expected, at the lower peak fentanyl concentrations (25 ng/ml and 50 ng/ml), the simulations predicted that 2 mg, 4 mg, 5 mg, and 10 mg IM doses of naloxone displaced fentanyl and reached below the 50% receptor occupancy within 10 minutes. However, at the concentration of 75 ng/ml, the simulation predicted that the 2 mg dose of naloxone failed to reach below the 50% occupancy within 10 minutes. Interestingly, at the highest peak concentration of fentanyl studied (100 ng/ml), the model predicted that the 4 mg of naloxone IM (equivalent to 8 mg IN) failed to reach below the threshold of 50 % occupancy within 10 minutes or even within 15 minutes (Data not shown). In contrast, the model predicted successful reversals when 5 and 10 mg IM doses were utilized. Conclusion:These results support the notion that acutely administered higher doses of naloxone are needed for rapid and adequate clinical reversal, particularly when higher systemic exposure of the potent synthetic opioids occur.

Tritiation of aryl thianthrenium salts with a molecular palladium catalyst

Tritium labelling is a critical tool for investigating the pharmacokinetic and pharmacodynamic properties of drugs, autoradiography, receptor binding and receptor occupancy studies1. Tritium gas is the preferred source of tritium for the preparation of labelled molecules because it is available in high isotopic purity2. The introduction of tritium labels from tritium gas is commonly achieved by heterogeneous transition-metal-catalysed tritiation of aryl (pseudo)halides. However, heterogeneous catalysts such as palladium supported on carbon operate through a reaction mechanism that also results in the reduction of other functional groups that are prominently featured in pharmaceuticals3. Homogeneous palladium catalysts can react chemoselectively with aryl (pseudo)halides but have not been used for hydrogenolysis reactions because, after required oxidative addition, they cannot split dihydrogen4. Here we report a homogenous hydrogenolysis reaction with a well defined, molecular palladium catalyst. We show how the thianthrene leaving group—which can be introduced selectively into pharmaceuticals by late-stage C–H functionalization5—differs in its coordinating ability to relevant palladium(II) catalysts from conventional leaving groups to enable the previously unrealized catalysis with dihydrogen. This distinct reactivity combined with the chemoselectivity of a well defined molecular palladium catalyst enables the tritiation of small-molecule pharmaceuticals that contain functionality that may otherwise not be tolerated by heterogeneous catalysts. The tritiation reaction does not require an inert atmosphere or dry conditions and is therefore practical and robust to execute, and could have an immediate impact in the discovery and development of pharmaceuticals.

Pharmacokinetics, Pharmacodynamics, and Safety of Etrolizumab in Children With Moderately to Severely Active Ulcerative Colitis or Crohn’s Disease: Results from a Phase 1 Randomized Trial

Background Etrolizumab, a humanized anti-β7 antibody, has not been studied in children. Here, we evaluate the pharmacokinetics, pharmacodynamics, and safety of etrolizumab in children with inflammatory bowel disease. Methods Patients age 4 to 17 years with moderately to severely active ulcerative colitis or Crohn’s disease were randomized 1:1 to receive 1.5mg/kg of etrolizumab subcutaneously every 4 weeks (q4w) or 3.0mg/kg every 8 weeks (q8w) for 16 weeks in this open-label phase 1 trial. Pharmacokinetics, pharmacodynamics, safety, and efficacy were assessed. Results Of the 24 patients treated, 21 completed the study. In the groups of 1.5mg/kg q4w and 3.0mg/kg q8w, respectively, mean (SD) maximum concentration (Cmax) was 9.8 (4.86) µg/mL and 18.1 (6.25) µg/mL; and mean (SD) area under the curve within a dosing interval (AUCtau) was 167 (86.9) and 521 (306) μg·day/mL after the last dose. The Cmax increased dose proportionally. The AUC over an 8-week period was slightly higher in the 3.0mg/kg q8w dose group. Median half-life was similar for both dosing regimens. Median numbers of free β7high gut-homing T and B cell subsets declined below 10% of baseline, confirming β7 target engagement and complete/near-complete receptor occupancy. Adverse events were consistent with the safety profile in adults. Approximately 60% of patients achieved a clinical response. Conclusions Etrolizumab showed a dose-proportional increase in Cmax and a slightly greater than dose-proportional increase in AUCtau. Both regimens achieved complete/near-complete β7 receptor occupancy, with a similar relationship to concentration as adults. Etrolizumab was well tolerated and demonstrated clinical activity in children.

Resting-state fMRI-based screening of deschloroclozapine in rhesus macaques predicts dosage-dependent behavioral effects

Background: Virally-mediated chemogenetic techniques hold the promise of circuit-specific neuromodulation for human brain disorders. Their protracted development in primates and issues related to the specificity of the actuator drugs has significantly slowed their implementation. Here we took a multi-disciplinary approach to assessing the translational appropriateness of a newly identified actuator drug, deschloroclozapine (DCZ). Methods: Resting-state functional MRI (rs-fMRI) data was acquired from seven rhesus macaques (6 males and 1 female) after administration of either vehicle, 0.1 or 0.3 mg/kg DCZ, the latter of which produce 80% and near 100% chemogenetic receptor occupancy, respectively. Seed-based comparative-connectome analysis and independent component analysis assessed dose dependent neural impact. Two subsets of subjects were tested on socio-emotional tasks (N = 4), and a probabilistic learning task (N = 3), assessing DCZ's impact on unconditioned and conditioned affective responses, respectively. Results: Neither vehicle nor 0.1 mg/kg DCZ changed overall functional connectivity, affective responses, or reaction times in the learning task. 0.3 mg/kg DCZ increased functional connectivity, particularly in frontal regions, and increased reaction times in the learning task. Notably, there was a positive correlation between changes in overall functional connectivity and reaction time. Conclusions: These experiments show the utility of rs-fMRI for in-vivo drug screening and benchmarking. We found that low dose DCZ does not alter brain function or affective behavior. However, higher doses of DCZ impacts frontal connectivity and is associated with deficits in task execution. Implementation of these methods will accelerate the development of chemogenetic in primates for research and therapeutic approaches.

CCR5 Receptor Occupancy Analysis Reveals Increased Peripheral Blood CCR5+CD4+ T Cells Following Treatment With the Anti-CCR5 Antibody Leronlimab

CCR5 plays a central role in infectious disease, host defense, and cancer progression, thereby making it an ideal target for therapeutic development. Notably, CCR5 is the major HIV entry co-receptor, where its surface density correlates with HIV plasma viremia. The level of CCR5 receptor occupancy (RO) achieved by a CCR5-targeting therapeutic is therefore a critical predictor of its efficacy. However, current methods to measure CCR5 RO lack sensitivity, resulting in high background and overcalculation. Here, we report on two independent, flow cytometric methods of calculating CCR5 RO using the anti-CCR5 antibody, Leronlimab. We show that both methods led to comparable CCR5 RO values, with low background on untreated CCR5+CD4+ T cells and sensitive measurements of occupancy on both blood and tissue-resident CD4+ T cells that correlated longitudinally with plasma concentrations in Leronlimab-treated macaques. Using these assays, we found that Leronlimab stabilized cell surface CCR5, leading to an increase in the levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo in Leronlimab-treated macaques. Weekly Leronlimab treatment in a chronically SIV-infected macaque led to increased CCR5+CD4+ T cells levels and fully suppressed plasma viremia, both concomitant with full CCR5 RO on peripheral blood CD4+ T cells, demonstrating that CCR5+CD4+ T cells were protected from viral replication by Leronlimab binding. Finally, we extended these results to Leronlimab-treated humans and found that weekly 700 mg Leronlimab led to complete CCR5 RO on peripheral blood CD4+ T cells and a statistically significant increase in CCR5+CD4+ T cells in peripheral blood. Collectively, these results establish two RO calculation methods for longitudinal monitoring of anti-CCR5 therapeutic antibody blockade efficacy in both macaques and humans, demonstrate that CCR5+CD4+ T cell levels temporarily increase with Leronlimab treatment, and facilitate future detailed investigations into the immunological impacts of CCR5 inhibition in multiple pathophysiological processes.

Exploring the kinetic selectivity of antipsychotics for dopamine D2 and 5-HT2A receptors: implications for the prevalence of EPS and receptor occupancy

Certain atypical antipsychotic drugs (APDs) used in the treatment of schizophrenia have been hypothesized to show reduced extrapyramidal side effects (EPS), due to their ability to promote nigrostriatal dopamine release through 5-HT2A receptor (5-HT2AR) blockade. The strength of this hypothesis is currently limited to a consideration of the relative receptor affinities of APDs for the 5-HT2AR and dopamine D2 receptor (D2R). Here we measure the 5-HT2AR kinetic binding properties of a series of typical and atypical APDs in a novel time-resolved fluorescence resonance energy transfer assay and correlate these properties with their observed EPS at therapeutic doses. For compounds with negligible affinity for 5-HT2AR, EPS is robustly predicted by a D2R specific rebinding model that integrates D2R association and dissociation rates to calculate the net rate of reversal of receptor blockade (kr). However, we show that for compounds with significant affinity for the 5-HT2AR, such as sertindole, higher relative 5-HT2A occupancy over time is an indicator for a reduced propensity to cause EPS. This study suggests that there is room for the development of novel kinetically optimised antipsychotic agents that modulate both serotonergic and dopamine function in a manner beneficial in the treatment of this chronic and debilitating disease.

Antipsychotic medication for women with schizophrenia spectrum disorders

There are significant differences between men and women in the efficacy and tolerability of antipsychotic drugs. Here, we provide a comprehensive overview of what is currently known about the pharmacokinetics and pharmacodynamics of antipsychotics in women with schizophrenia spectrum disorders (SSDs) and translate these insights into considerations for clinical practice. Slower drug absorption, metabolism and excretion in women all lead to higher plasma levels, which increase the risk for side-effects. Moreover, women reach higher dopamine receptor occupancy compared to men at similar serum levels, since oestrogens increase dopamine sensitivity. As current treatment guidelines are based on studies predominantly conducted in men, women are likely to be overmedicated by default. The risk of overmedicating generally increases when sex hormone levels are high (e.g. during ovulation and gestation), whereas higher doses may be required during low-hormonal phases (e.g. during menstruation and menopause). For premenopausal women, with the exceptions of quetiapine and lurasidone, doses of antipsychotics should be lower with largest adjustments required for olanzapine. Clinicians should be wary of side-effects that are particularly harmful in women, such as hyperprolactinaemia which can cause oestrogen deficiency and metabolic symptoms that may cause cardiovascular diseases. Given the protective effects of oestrogens on the course of SSD, oestrogen replacement therapy should be considered for postmenopausal patients, who are more vulnerable to side-effects and yet require higher dosages of most antipsychotics to reach similar efficacy. In conclusion, there is a need for tailored, female-specific prescription guidelines, which take into account adjustments required across different phases of life.