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Authors: Katie M. Clark, Adriana A. Tienda, Krista C. Paffenroth, Lindsey N. Brigante, Daniel C. Colvin, Jose Maldonado, Erin S. Calipari, Fiona E. Harrison

Abstract:

Manganese (Mn) is an essential trace element required for human health and is important in antioxidant defenses, as well as in the development and function of dopaminergic neurons. However, chronic low-level Mn exposure, such as through contaminated drinking water, poses risks that may contribute to neurodevelopmental and neurodegenerative conditions, including attention deficit hyperactivity disorder (ADHD). Pharmacological inhibition of the dopamine transporter (DAT) blocks reuptake, elevates synaptic dopamine, and alleviates ADHD symptoms. This study aimed to determine whether Mn exposure in juvenile mice modifies their response to DAT blockers, amphetamine, and methylphenidate and utilize neuroimaging methods to visualize and quantify Mn distribution across dopaminergic brain regions. Male and female heterozygous DATᵀ³⁵⁶ᴹ and wild-type littermates were randomly assigned to receive control (2.5% Stevia) or high Manganese (2.5 mg/ml Mn + 2.5% Stevia) via water ad libitum from weaning (21-28 days) for 4-5 weeks. Mice underwent repeated testing in locomotor activity chambers for three consecutive days (60 mins.) to ensure that they were fully habituated to the environments. On the fourth day, a 3-hour activity session was conducted following treatment with amphetamine (3 mg/kg) or methylphenidate (5 mg/kg). The second drug was administered in a second 3-hour activity session following a 1-week washout period. Following the washout, the mice were given one last injection of amphetamine and euthanized one hour later. Using the ex-vivo brains, magnetic resonance relaxometry (MRR) was performed on a 7Telsa imaging system to map T1- and T2-weighted (T1W, T2W) relaxation times. Mn inherent paramagnetic properties shorten both T1W and T2W times, which enhances the signal intensity and contrast, enabling effective visualization of Mn accumulation in the entire brain. A subset of mice was treated with amphetamine 1 hour before euthanasia. SmartSPIM light sheet microscopy with cleared whole brains and cFos and tyrosine hydroxylase (TH) labeling enabled an unbiased automated counting and densitometric analysis of TH and cFos positive cells. Immunohistochemistry was conducted to measure synaptic protein markers and quantify changes in neurotransmitter regulation. Mn exposure elevated Mn brain levels and potentiated stimulant effects in males. The globus pallidus, substantia nigra, thalamus, and striatum exhibited more pronounced T1W shortening, indicating regional susceptibility to Mn accumulation (p<0.0001, 2-Way ANOVA). In the cleared whole brains, initial analyses suggest that TH and c-Fos co-staining mirrors behavioral data with decreased co-staining in DATT356M+/- mice. Ongoing studies will identify the molecular basis of the effect of Mn, including changes to DAergic metabolism and transport and post-translational modification to the DAT. These findings demonstrate that alterations in T1W relaxation times, as measured by MRR, may serve as an early biomarker for Mn neurotoxicity. This neuroimaging approach exhibits remarkable accuracy in identifying Mn-susceptible brain regions, with a spatial resolution and sensitivity that surpasses current conventional dissection and mass spectrometry approaches. The capability to label and map TH and cFos expression across the entire brain provides insights into whole-brain neuronal activation and its connections to functional neural circuits and behavior following amphetamine and methylphenidate administration.

Keywords: manganese, environmental toxicology, dopamine dysfunction, biomarkers, drinking water, light sheet microscopy, magnetic resonance relaxometry (MRR)

Procedia PDF Downloads 16

Authors: Claire Harrison, Raajit K. Rampal, Vikas Gupta, Srdan Verstovsek, Moshe Talpaz, Jean-Jacques Kiladjian, Ruben Mesa, Andrew Kuykendall, Alessandro Vannucchi, Francesca Palandri, Sebastian Grosicki, Timothy Devos, Eric Jourdan, Marielle J. Wondergem, Haifa Kathrin Al-Ali, Veronika Buxhofer-Ausch, Alberto Alvarez-Larrán, Sanjay Akhani, Rafael Muñoz-Carerras, Yury Sheykin, Gozde Colak, Morgan Harris, John Mascarenhas

Abstract:

Myelofibrosis (MF) is characterized by bone marrow fibrosis, anemia, splenomegaly and constitutional symptoms. Progressive bone marrow fibrosis results from aberrant megakaryopoeisis and expression of proinflammatory cytokines, both of which are heavily influenced by bromodomain and extraterminal domain (BET)-mediated gene regulation and lead to myeloproliferation and cytopenias. Pelabresib (CPI-0610) is an oral small-molecule investigational inhibitor of BET protein bromodomains currently being developed for the treatment of patients with MF. It is designed to downregulate BET target genes and modify nuclear factor kappa B (NF-κB) signaling. MANIFEST-2 was initiated based on data from Arm 3 of the ongoing Phase 2 MANIFEST study (NCT02158858), which is evaluating the combination of pelabresib and ruxolitinib in Janus kinase inhibitor (JAKi) treatment-naïve patients with MF. Primary endpoint analyses showed splenic and symptom responses in 68% and 56% of 84 enrolled patients, respectively. MANIFEST-2 (NCT04603495) is a global, Phase 3, randomized, double-blind, active-control study of pelabresib and ruxolitinib versus placebo and ruxolitinib in JAKi treatment-naïve patients with primary MF, post-polycythemia vera MF or post-essential thrombocythemia MF. The aim of this study is to evaluate the efficacy and safety of pelabresib in combination with ruxolitinib. Here we report updates from a recent protocol amendment. The MANIFEST-2 study schema is shown in Figure 1. Key eligibility criteria include a Dynamic International Prognostic Scoring System (DIPSS) score of Intermediate-1 or higher, platelet count ≥100 × 10^9/L, spleen volume ≥450 cc by computerized tomography or magnetic resonance imaging, ≥2 symptoms with an average score ≥3 or a Total Symptom Score (TSS) of ≥10 using the Myelofibrosis Symptom Assessment Form v4.0, peripheral blast count <5% and Eastern Cooperative Oncology Group performance status ≤2. Patient randomization will be stratified by DIPSS risk category (Intermediate-1 vs Intermediate-2 vs High), platelet count (>200 × 10^9/L vs 100–200 × 10^9/L) and spleen volume (≥1800 cm^3 vs <1800 cm^3). Double-blind treatment (pelabresib or matching placebo) will be administered once daily for 14 consecutive days, followed by a 7 day break, which is considered one cycle of treatment. Ruxolitinib will be administered twice daily for all 21 days of the cycle. The primary endpoint is SVR35 response (≥35% reduction in spleen volume from baseline) at Week 24, and the key secondary endpoint is TSS50 response (≥50% reduction in TSS from baseline) at Week 24. Other secondary endpoints include safety, pharmacokinetics, changes in bone marrow fibrosis, duration of SVR35 response, duration of TSS50 response, progression-free survival, overall survival, conversion from transfusion dependence to independence and rate of red blood cell transfusion for the first 24 weeks. Study recruitment is ongoing; 400 patients (200 per arm) from North America, Europe, Asia and Australia will be enrolled. The study opened for enrollment in November 2020. MANIFEST-2 was initiated based on data from the ongoing Phase 2 MANIFEST study with the aim of assessing the efficacy and safety of pelabresib and ruxolitinib in JAKi treatment-naïve patients with MF. MANIFEST-2 is currently open for enrollment.

Keywords: CPI-0610, JAKi treatment-naïve, MANIFEST-2, myelofibrosis, pelabresib

Procedia PDF Downloads 206