Methylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs.

Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.

Synthesis of functionalized derivatives of the gamma-secretase modulator BMS-932481 and identification of its major metabolite.

In an effort to improve physical properties by introducing polar functionality into the bicyclic pyrimidine gamma-secretase modulator (GSM) clinical candidate BMS-932481, we prepared several oxidative products of BMS-932481. Among the analogs that were prepared, the C-5 alcohol 3 was identified as the predominant metabolite of BMS-932481 found in rat and human liver microsomes. Alcohol 3 was determined to be chemically unstable, leading to the hypothesis that 3 may lead to the production of reactive species both in vitro and in vivo.

Centrally Delivered BACE1 Inhibitor Activates Microglia, and Reverses Amyloid Pathology and Cognitive Deficit in Aged Tg2576 Mice.

Multiple small-molecule inhibitors of the ß-secretase enzyme (BACE1) are under preclinical or clinical investigation for Alzheimer's disease (AD). Prior work has illustrated robust lowering of central amyloid ß (Aß) after acute administration of BACE1 inhibitors. However, very few studies have assessed the overall impact of chronically administered BACE1 inhibitors on brain amyloid burden, neuropathology, and behavioral function in aged preclinical models. We investigated the effects of a potent nonbrain-penetrant BACE1 inhibitor, delivered directly to the brain using intracerebroventricular infusion in an aged transgenic mouse model. Intracerebroventricular infusion of the BACE1 inhibitor (0.3-23.5 µg/d) for 8 weeks, initiated in 17-month-old Tg2576 mice, produced dose-dependent increases in brain inhibitor concentrations (0.2-13 µm). BACE1 inhibition significantly reversed the behavioral deficit in contextual fear conditioning, and reduced brain Aß levels, plaque burden, and associated pathology (e.g., dystrophic neurites), with maximal effects attained with ∼1 µg/d dose. Strikingly, the BACE1 inhibitor also reversed amyloid pathology below baseline levels (amyloid burden at the start of treatment), without adversely affecting cerebral amyloid angiopathy, microhemorrhages, myelination, or neuromuscular function. Inhibitor-mediated decline in brain amyloid pathology was associated with an increase in microglial ramification. This is the first demonstration of chronically administered BACE1 inhibitor to activate microglia, reverse brain amyloid pathology, and elicit functional improvement in an aged transgenic mouse model. Thus, engagement of novel glial-mediated clearance mechanisms may drive disease-modifying therapeutic benefit with BACE1 inhibition in AD.

Discovery of furo[2,3-d][1,3]thiazinamines as beta amyloid cleaving enzyme-1 (BACE1) inhibitors.

This Letter describes the synthesis and structure-activity relationships of a series of furo[2,3-d][1,3]thiazinamine BACE1 inhibitors. The co-crystal structure of a representative thiazinamine 2e bound with the BACE1 active site displayed a binding mode driven by interactions with the catalytic aspartate dyad and engagement of the biaryl amide toward the S1 and S3 pockets. This work indicates that furo[2,3-d]thiazine can serve as a viable bioisostere of the known furo[3,4-d]thiazine.

Macrocyclic prolinyl acyl guanidines as inhibitors of ß-secretase (BACE).

The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.

Developmental Assessment in Children at Higher Likelihood for Developmental Delays - Comparison of Parent Report and Direct Assessment.

PURPOSE: Accurate assessment of cognitive development of young children is a vital component of developmental evaluations. Direct assessment of developmental skills is not always feasible, but there is limited information on the agreement between direct assessment and caregiver-reported cognitive skills. There is limited information regarding the accuracy of the parent-reported Developmental Profile 4 (DP-4) in comparison to the widely-used developmental measure, the Bayley Scales of Infant and Toddler Development, Fourth Edition (Bayley-4). The purpose of the current study was to evaluate whether a standardized parent interview can effectively identify children at risk for cognitive developmental delays. METHODS: We compared the agreement between the Bayley-4 Cognitive and the Developmental Profile 4 (DP-4) in young children being evaluated in-person for early developmental delays. 182 children (134 with an autism diagnosis), ages 6-42 months, completed both assessments. RESULTS: Results showed that Bayley-4 Cognitive scores had a moderately strong correlation with DP4-Cognitive scores (r = 0.70, p < 0.001). A cutoff of 70 or 69 on the DP-4 Cognitive was determined as ideal for identifying developmental delay based on diagnosis of global developmental delay or the Bayley-4 Cognitive. CONCLUSIONS: Our analyses revealed good agreement between DP-4 and Bayley-4 Cognitive scores, even after controlling for confounding variables such as degree of ASD characteristics, age, and sex. These results suggest that caregiver-report measures can be a valid and useful tool in the assessment of young children, particularly when direct developmental assessment is not feasible.

Hyperdynamic microtubules, cognitive deficits, and pathology are improved in tau transgenic mice with low doses of the microtubule-stabilizing agent BMS-241027.

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimer's disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aß and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aß levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aß40 and Aß42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

A contrast in safety, pharmacokinetics and pharmacodynamics across age groups after a single 50 mg oral dose of the γ-secretase inhibitor avagacestat.

AIM: To evaluate the single dose pharmacokinetics, pharmacodynamics, and preliminary tolerability of the γ-secretase inhibitor BMS-708163 (avagacestat) in young and elderly men and women. METHODS: All subjects received double-blinded administration of a single 50 mg dose of avagacestat in capsule form or matching placebo. Main evaluations included pharmacokinetics, safety, plasma amyloid-ß (Aß)(1-40) concentratios and exploration of Notch biomarkers. RESULTS: Avagacestat 50 mg capsule was well tolerated and rapidly absorbed among young and elderly subjects, with a median t(max) between 1 and 2 h post dose and an average half-life between 41 and 71 h. In general, subjects aged 75 years or more had higher AUC(0,∞) values than those aged less than 75 years. An exploratory analysis of Aß(1-40) serum concentrations showed a pattern of decreasing concentrations over the first 4-6 h followed by a rise above baseline that was maintained until the end of the assessment period. Adverse events were generally mild, occurring more frequently in elderly subjects, with no observed difference between subjects receiving avagacestat and placebo. No dose limiting gastrointestinal effects of avagacestat were observed and exploratory biomarkers of Notch inhibition did not change significantly. CONCLUSIONS: The favourable safety profile and pharmacokinetic effects of avagacestat in this study support its continued development, especially in the target population of elderly subjects with mild cognitive impairment or Alzheimer's disease.

Provider Perspectives on Telehealth Services for Children With Autism Spectrum Disorder During the Coronavirus Disease 2019 Pandemic.

OBJECTIVE: The purpose of the current study was to explore provider perspectives on the strengths and challenges of telehealth services (e.g., behavioral interventions, physical, speech, and occupational therapy, medication management) for children with autism spectrum disorder during coronavirus disease 2019 related shutdowns. METHODS: From September 2020 to May 2021, we conducted qualitative interviews with 35 providers across multiple disciplines from 17 sites in the Autism Care Network. Qualitative data were analyzed using a framework approach and common themes were identified. RESULTS: Providers across clinical disciplines identified strengths of the virtual model, such as its flexibility and the opportunity it provided to see children in their home environment. They also indicated that some interventions worked better virtually than others, and that there were several factors that impacted their success. Respondents were generally satisfied providing parent-mediated interventions but expressed mixed satisfaction in using telehealth for direct-to-patient care. CONCLUSIONS: Results suggest that telehealth services for children with autism spectrum disorder could be a helpful tool in decreasing barriers and improving service delivery, especially when tailored to the individual needs of the patient. More research is needed on the factors contributing to its success in order to eventually inform clinical guidelines regarding the prioritization of children seen for in-person visits.

Optimizing Parent Training to Improve Oral Health Behavior and Outcomes in Underserved Children with Autism Spectrum Disorder.

A randomized controlled trial established initial efficacy of a novel parent training (PT) intervention for improving oral hygiene and oral health in underserved children with ASD (Fenning et al., 2022), a population at risk for unmet dental needs. The present study describes our emic approach to PT development alongside treatment outcome data examining feasibility, acceptability, and engagement. Families with Medicaid-eligible children with ASD ages 3 to 13 years (85% male, 62% with intellectual disability) were assigned to receive PT (n = 60) or a psychoeducational toolkit (n = 59). Results indicate strong retention, fidelity, and adherence, with quantitative and qualitative metrics revealing high treatment satisfaction and utilization. Discussion focuses on implications for individualizing treatment to optimize engagement of underrepresented families.

Clinician Perspectives on Telehealth Assessment of Autism Spectrum Disorder During the COVID-19 Pandemic.

This study examined clinician insights into telehealth assessment services for autism spectrum disorder implemented during the COVID-19 pandemic. 35 clinicians from multiple disciplines across 17 sites in the Autism Care Network were interviewed. Themes identified through qualitative analyses included factors related to confidence in diagnosis (impressions of in-home observation; child and family factors that affected diagnostic confidence; changes in rapport); patient and family factors related to telehealth (perceived family benefits of and barriers to telehealth; factors related to healthcare disparities; factors specific to non-native English speakers); and institutional and workplace factors related to transitioning to telehealth (institutional support; changes to efficacy, attendance, and work satisfaction). Results suggest that telehealth has potential to be an effective tool in autism assessment practice.

Preliminary Validation and Feasibility of the Autism Detection in Early Childhood-Virtual (ADEC-V) for Autism Telehealth Evaluations in a Hospital Setting.

This study provided preliminary validation of the Autism Detection in Early Childhood-Virtual (ADEC-V) for telehealth assessment of possible autism. Participants were 121 children (24.79% female) aged 18-47 months who completed telehealth evaluations at a large pediatric hospital in the Midwestern United States between October 2020 and February 2021. The ADEC-V showed good sensitivity (0.82) and specificity (0.78) and was significantly correlated with other ASD symptom measures (i.e., CARS-2, ADI-R). Internal consistency was acceptable (α = 0.77). These results need replication in a larger and broader sample including more children without ASD. This preliminary validation study identifies the ADEC-V as a promising measure for telehealth ASD assessments in young children.

Parent Training for Dental Care in Underserved Children With Autism: A Randomized Controlled Trial.

OBJECTIVE: Children with autism spectrum disorder (ASD) have difficulty participating in dental care and experience significant unmet dental needs. We examined the efficacy of parent training (PT) for improving oral hygiene and oral health in underserved children with ASD. METHOD: Families of Medicaid-eligible children with ASD (ages 3-13 years, 85% boys, 62% with intellectual disability) reporting difficulty with dental care participated in a 6-month randomized controlled trial comparing PT (n = 60) with a psychoeducational dental toolkit (n = 59). Primary outcomes were parent-reported frequency of twice-daily toothbrushing and dentist-rated visible plaque. Secondary outcomes included parent-reported child behavior problems during home oral hygiene and dentist-rated caries. Dentists were blind to intervention assignment. Analyses were intention to treat. RESULTS: Retention was high at posttreatment (3 months, 93%) and 6-month follow-up (90%). Compared with the toolkit intervention, PT was associated with increased twice-daily toothbrushing at 3 (78% vs 55%, respectively; P < .001) and 6 (78% vs 62%; P = .002) months and a reduction in plaque at 3 months (intervention effect, -0.19; 95% confidence interval [CI], -0.36 to -0.02; P = .03) and child problem behaviors at 3 (-0.90; 95% CI, -1.52 to -0.28; P = .005) and 6 (-0.77; 95% CI, -1.39 to -0.14; P = .02) months. Comparatively fewer caries developed in children receiving the PT intervention over 3 months (ratio of rate ratios, 0.73; 95% CI, 0.54 to 0.99; P = .04). CONCLUSIONS: PT represents a promising approach for improving oral hygiene and oral health in underserved children with ASD at risk for dental problems.

Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies.

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3' UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

Discovery and Optimization of Biaryl Alkyl Ethers as a Novel Class of Highly Selective, CNS-Penetrable, and Orally Active Adaptor Protein-2-Associated Kinase 1 (AAK1) Inhibitors for the Potential Treatment of Neuropathic Pain.

Recent mouse knockout studies identified adapter protein-2-associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. BMS-986176/LX-9211 (4), as a highly selective, CNS-penetrable, and potent AAK1 inhibitor, has advanced into phase II human trials. On exploring the structure-activity relationship (SAR) around this biaryl alkyl ether chemotype, several additional compounds were found to be highly selective and potent AAK1 inhibitors with good druglike properties. Among these, compounds 43 and 58 showed very good efficacy in two neuropathic pain rat models and had excellent CNS penetration and spinal cord target engagement. Both compounds also exhibited favorable physicochemical and oral pharmacokinetic (PK) properties. Compound 58, a central pyridine isomer of BMS-986176/LX-9211 (4), was 4-fold more potent than 4 in vitro and showed lower plasma exposure needed to achieve similar efficacy compared to 4 in the CCI rat model. However, both 43 and 58 showed an inferior preclinical toxicity profile compared to 4.

Monosubstituted γ-lactam and conformationally constrained 1,3-diaminopropan-2-ol transition-state isostere inhibitors of ß-secretase (BACE).

The synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented.

Synthesis and in vivo evaluation of cyclic diaminopropane BACE-1 inhibitors.

The synthesis, evaluation, and structure-activity relationships of a set of related constrained diaminopropane inhibitors of BACE-1 are described. The full in vivo profile of an optimized inhibitor in both normal and P-gp deficient mice is compared with data generated in normal rats.

Synthesis and SAR of indole-and 7-azaindole-1,3-dicarboxamide hydroxyethylamine inhibitors of BACE-1.

Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aß levels, but did not lower rat brain Aß due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.