Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease.

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell-dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib's unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Restricting Human Movement During the COVID-19 Pandemic: New Research Avenues in the Study of Mobility, Migration, and Citizenship.

Every government in the world introduced restrictions to human mobility - that is, the movement of persons across and within state borders - in response to the COVID-19 pandemic. Such restrictions thus constituted a global phenomenon, but they were by no means globally uniform; rather, they varied significantly between and within states, as well as over time. This research note presents different data sources for studying the drivers and outcomes of mobility restrictions, highlighting specific ways in which the data can be used. We begin by surveying seven new databases capturing various aspects of the regulation of human movement during the COVID-19 pandemic. Drawing inspiration from research on previous pandemics, we then outline five possible research avenues prompted by these data. We suggest that explaining the causes and consequences of such restrictions, as well as the differences between them, can significantly advance research on the governance of mobility, migration, and citizenship.

Development of a Multi-Session Curriculum Addressing Domestic Minor Sex Trafficking for High-Risk Male Youth.

Males, in particular adolescents and young adults, have been increasingly recognized as involved in domestic minor sex trafficking (DMST). However, there are very sparse resources and organizations that provide prevention, identification, and interventions for boys and young men who are involved in or at-risk for DMST involvement. The objective was to develop and assess an educational curriculum to prevent adolescent male involvement in DMST through a three-pronged educational approach: as victims of sexual exploitation; receiving financial benefit as exploiters; as buyers of sex. Through quality improvement cycles, changes were made to enhance the curriculum by utilizing the outcome measures of participant questionnaires and feedback from a steering committee of clinical experts. Male youth at the state's juvenile detention center were asked to participate in pilot groups, as they were identified as a high-risk population of adolescents to become involved. The curriculum was modified by adding sessions, including additional community guest speakers, and providing a more holistic educational experience that involves trafficking prevention from both a victimization and perpetration standpoint. Our goal is to expand this educational opportunity to be utilized in multiple settings (e.g., schools, hospitals) across the country.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (ß1i), MECL-1 (ß2i), and LMP7 (ß5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.

Prolonged and tunable residence time using reversible covalent kinase inhibitors.

Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.

Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton's tyrosine kinase (BTK).

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26pmol/mg protein.

A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression.

In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4(+) T cells from Itk(-/-) and Itk(-/-)Rlk(-/-) mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4(+) T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases.

Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694.

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

Finding the perfect spot for fluorine: improving potency up to 40-fold during a rational fluorine scan of a Bruton's Tyrosine Kinase (BTK) inhibitor scaffold.

A rational fluorine scan based on co-crystal structures was explored to increase the potency of a series of selective BTK inhibitors. While fluorine substitution on a saturated bicyclic ring system yields no apparent benefit, the same operation on an unsaturated bicyclic ring can increase HWB activity by up to 40-fold. Comparison of co-crystal structures of parent molecules and fluorinated counterparts revealed the importance of placing fluorine at the optimal position to achieve favorable interactions with protein side chains.

Impact of subsidence on clinical outcomes and radiographic fusion rates in anterior cervical discectomy and fusion: a systematic review.

STUDY DESIGN: Systematic review. OBJECTIVE: To provide a systematic review of published literature on the impact of subsidence on clinical outcomes and radiographic fusion rates after anterior cervical discectomy and fusion with plates or without plates. BACKGROUND: Subsidence of interbody implants is common after anterior cervical spine fusions. The impact of subsidence on fusion rates and clinical outcomes is unknown. METHODS: Systematic literature review on published articles on anterior cervical discectomy and fusion, which objectively measured graft subsidence, radiographic fusion rates, and clinical outcomes between April 1966 and December 2010. RESULTS: A total of 35 articles that measured subsidence and provided fusion rates and/or clinical outcomes were selected for inclusion. The mean subsidence rate ranged from 19.3% to 42.5%. The rate of subsidence based on the type of implant ranged from 22.8% to 35.9%. The incidence of subsidence was not impacted by the type of implant (P=0.98). The overall fusion rate of the combined studies was 92.8% and was not impacted by subsidence irrespective of subsidence definition or the measurement technique used (P=0.19). Clinical outcomes were evaluated in 27 of 35 studies with all studies reporting an improvement in patient outcomes postoperatively. CONCLUSIONS: Subsidence irrespective of the measurement technique or definition does not appear to have an impact on successful fusion and/or clinical outcomes. A validated definition and standard measurement technique for subsidence is needed to determine the actual incidence of subsidence and its impact on radiographic and clinical outcomes.

Chronic thromboembolic pulmonary hypertension and balloon pulmonary angioplasty - Where are we in 2024?

Pulmonary endarterectomy (PEA) is the first-line treatment for patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, some patients with CTEPH are considered inoperable, and in the last decade, balloon pulmonary angioplasty (BPA) has emerged as a viable therapeutic option for these patients with prohibitive surgical risk or recurrent pulmonary hypertension following PEA. Numerous international centers have increased their procedural volume of BPA and have reported improvements in pulmonary hemodynamics, patient functional class and right ventricular function. Randomized controlled trials have also demonstrated similar findings. Recent refinements in procedural technique, increased operator experience and advancements in procedural technology have facilitated marked reduction in the risk of complications following BPA. Current guidelines recommend BPA for patients with inoperable CTEPH and persistent pulmonary hypertension following PEA. The pulmonary arterial endothelium plays a vital role in the pathophysiologic development and progression of CTEPH.

Piloting an Evidence-Based Assessment Protocol for Incarcerated Adolescents.

Although evidence-based assessment is considered an essential component of evidence-based practice, few adolescents have access to evidence-based assessment. Despite experiencing high rates of mental health disorders, incarcerated justice-involved adolescents are rarely able to access evidence-based psychiatric care. In this article, we discuss the components of an evidence-based assessment protocol designed and piloted with incarcerated adolescents involved in Rhode Island's juvenile justice system. In particular, we describe the components of our evidence-based protocol, ways in which evidence-based assessment may need to be modified when working with this population, and discuss policy and clinical implications relevant to increasing access to evidence-based assessment among incarcerated adolescents.

RN486, a selective Bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents.

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC(50) = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC(50) = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC(50) = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Phase 1 clinical trial evaluating safety, exposure and pharmacodynamics of BTK inhibitor tolebrutinib (PRN2246, SAR442168).

Bruton's tyrosine kinase (BTK), expressed in B cells and cells of innate immunity, including microglia, is an essential signaling element downstream of the B-cell receptor and Fc-receptors. Tolebrutinib (PRN2246, SAR442168) is a potent BTK inhibitor that covalently binds the kinase, resulting in durable inhibition with the potential to target inflammation in the periphery and central nervous system (CNS). Tolebrutinib crosses the blood-brain barrier and potently inhibits BTK in microglial cells isolated from the CNS. A first-in-human randomized, double-blind, placebo-controlled study of tolebrutinib was conducted. The trial design consisted of five single ascending dose arms with oral administration of a single dose of 5, 15, 30, 60, and 120 mg (n = 6 per arm, n = 2 placebo), five multiple ascending dose arms with oral administration of 7.5, 15, 30, 60, and 90 mg (n = 8 per arm, n = 2 placebo) over 10 days, and one arm (n = 4) in which cerebral spinal fluid (CSF) exposure was measured 2 h after a single 120 mg dose. Tolebrutinib was well-tolerated in the study and all treatment-related treatment emergent adverse events were mild. Tolebrutinib was rapidly absorbed following oral administration with a rapid half-life of ~ 2 h. Peripheral BTK occupancy was assessed at various timepoints by an enzyme-linked immunosorbent assay-based readout using an irreversible probe. Assessments demonstrated extensive and prolonged peripheral BTK occupancy at steady-state with once daily doses as low as 7.5 mg. Further, CSF exposure was demonstrated 2 h after administration at 120 mg.

Discovery of Reversible Covalent Bruton's Tyrosine Kinase Inhibitors PRN473 and PRN1008 (Rilzabrutinib).

Bruton's tyrosine kinase (BTK), a Tec family tyrosine kinase, is critical in immune pathways as an essential intracellular signaling element, participating in both adaptive and immune responses. Currently approved BTK inhibitors are irreversible covalent inhibitors and limited to oncology indications. Herein, we describe the design of covalent reversible BTK inhibitors and the discoveries of PRN473 (11) and rilzabrutinib (PRN1008, 12). These compounds have exhibited potent and durable inhibition of BTK, in vivo efficacy in rodent arthritis models, and clinical efficacy in canine pemphigus foliaceus. Compound 11 has completed phase 1 trials as a topical agent, and 12 is in phase 3 trials for pemphigus vulgaris and immune thrombocytopenia.

Domestic Minor Sex Trafficking: A Case Series of Male Pediatric Patients.

Domestic minor sex trafficking (DMST) is the commercial sexual exploitation of children (<18 years old) who are U.S. citizens or lawful permanent residents, victimized within U.S. borders. There is limited knowledge and research in regard to male involvement in DMST outside the context of homelessness and runaway youth. To our knowledge, no research specifically examines at-risk or involved male youth from a larger dataset of youth who present to a child abuse outpatient medical clinic. The objective of the present case series was to describe the demographic, psychosocial, medical, and psychiatric characteristics of natal male participants (who did not identify as transgender) suspected of DMST involvement. Six medical records of male patients under the age of 18 who were referred to a child protection clinic for concern of DMST involvement between 8/1/13 and 12/31/18 were retrospectively reviewed. Our case series demonstrates that male participants present for concern of sex trafficking and have complex behavioral, medical, and psychiatric concerns similar to what has been identified in research focused on female victims. Therefore, testing (e.g., sexually transmitted infection (STI)/HIV testing, urine toxicology screening), DMST screening, and interventions (e.g., STI prophylaxis, referrals to mental health counselors) should be completed in male patients.

Folate receptor alpha regulates cell proliferation in mouse gonadotroph alphaT3-1 cells.

We have previously found that the mRNA and protein levels of the folate receptor alpha (FRalpha) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FRalpha has not fully been determined. We investigated the effect of FRalpha over-expression in the mouse gonadotroph alphaT3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FRalpha were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FRalpha promotes cell proliferation. These effects were abrogated in the same alphaT3-1 cells when transfected with a mutant FRalpha cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FRalpha over-expressing cells. In summary, our data suggests that FRalpha regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

Depressed mood and drinking occasions across high school: comparing the reciprocal causal structures of a panel of boys and girls.

Does adolescent depressed mood portend increased or decreased drinking? Is frequent drinking positively or negatively associated with emotional well-being? Do the dynamic relations between depression and drinking differ by gender? Using block-recursive structural equation models, we explore the reciprocal short-term effects (within time, t) and the cross-lagged medium-term effects (t +1 year) and long-term effects (t+2 years) of depressed mood and monthly drinking occasions. Data come from the high school waves of the Youth Development Study, a randomly selected panel of 1015 ninth graders followed to 12th grade. We found that for both genders, depressed mood consistently decreased short-term drinking in each grade measured. However, depression increased drinking for both genders in the medium-term but only for girls in the long-term. In the other direction, drinking tended to increase depression in the short-term only among ninth-grade boys and 12th-grade girls. Observed trends and differences in the magnitude of the reciprocal effects vary by gender, with drinking being especially deleterious to emotional well-being for boys early in high school (10th grade) but for girls on the cusp of the post-high school world (12th grade).

Evaluation of the etiology of persistent iritis after cataract surgery.

BACKGROUND: The purpose of this study was to evaluate patients with persistent iritis after cataract surgery to determine its incidence and risk factors. Adjusting the management of patients at risk could allow for a more predictable post-operative course and outcome. A retrospective chart review was performed of patients who had post-operative iritis longer than 1 month after cataract surgery during a 2-year period at Storm Eye Institute at the Medical University of South Carolina (MUSC) in Charleston, South Carolina. Patient demographics and various pre-operative, intra-operative, and post-operative factors were analyzed for trends. RESULTS: Thirty-nine patients (49 eyes) met the inclusion criteria, and this group was compared to a control cohort of 40 patients (66 eyes) who did not have persistent iritis after cataract surgery. The overall incidence of post-operative iritis was 1.75%. In all patients with post-operative iritis lasting greater than 1 month, African American race and pupil expansion device use were statistically significant factors. After excluding patients with a history of ocular inflammation or known inflammatory or autoimmune diagnosis (1.20% incidence), there were still a significantly higher proportion of African Americans compared to the control group. When patients with post-operative iritis of less than 6 months in duration were additionally excluded, the incidence was 0.32%, and history of diabetes was statistically significant in addition to race. CONCLUSIONS: Risk factors for persistent iritis after cataract surgery include being diabetic, of African American racial background, and pupil expansion device use. These patients can be better informed of the higher risk of prolonged inflammation in their post-operative course, and peri-operative management can be tailored accordingly.

Emotional Distress, Drinking, and Academic Achievement across the Adolescent Life Course.

Our study of the adolescent life course proposes that substantial maturation occurs within three intertwined arenas of development: the social, the psychological, and the normative attainment. Further, each arena may be linked, respectively, to three youth problem dimensions: drinking, depressive affect, and academic achievement. We use latent growth curves and the Youth Development Study (effective N=856) to track a panel of teens from their freshman to senior year in high school. There are 54.4% girls and 45.6% boys, and 75.7% non-Hispanic whites and 24.3% other races/ethnicities. Two research goals are addressed: (1) estimate each dimension's unique developmental trajectory across high school, and (2) model the dimensions together in order to assess their reciprocal influences. While mean levels in all three dimensions increased over time, distinct developmental patterns were observed, especially in drinking and depression. For example, more drinking occasions-a social activity for most teens-may help assuage some teens' emotional distress, especially girls'. These patterns suggest a synergistic relationship between the social and psychological arenas of development. Contrary to expectation, higher freshman depressive affect was associated with a significantly sharper increase in GPA over time for girls.