Open-label, rapid initiation pilot study for extended-release buprenorphine subcutaneous injection.

Background: For patients with opioid use disorder, buprenorphine extended-release injection (BUP-XR) achieves sustained therapeutic plasma concentrations, controls craving and withdrawal symptoms, and improves patient outcomes. Given retention challenges during transmucosal buprenorphine (BUP-TM) induction, assessing methods to quickly achieve sustained buprenorphine concentrations is important.Objectives: This open-label, single-group, single-center pilot study (NCT03993392) evaluated safety and tolerability of initiating BUP-XR following a single BUP-TM 4 mg dose.Methods: Eligible participants abstained from short and long-acting opioids for 6 and 24 hours, respectively. If the Clinical Opiate Withdrawal Scale (COWS) was ≥8, BUP-TM 4 mg was administered. Participants not exhibiting hypersensitivity, precipitated opioid withdrawal (POW), or sedation symptoms within 1 hour received BUP-XR 300 mg (assessed as inpatients for 48 hours and outpatients to Day 29). Endpoints were COWS score increase ≥6, independent adjudication of POW, and opioid use.Results: Twenty-six participants (14 male) received BUP-TM, 24 received BUP-XR, and 20 completed the study. After injection, COWS scores decreased from pre-BUP-TM baseline of 14.6 ± 4.1 to 6.9 ± 4.1 at 6 hours and 4.2 ± 3.2 at 24 hours. Most participants (62.5%) experienced maximum COWS scores pre-BUP-XR; 2 experienced a COWS score increase ≥6, occurring at 1 and 2 hours post-BUP-XR. By adjudication, 2/24 participants experienced POW. Irritability, anxiety, nausea, and pain were the most frequent adverse events (AEs) with no serious AEs.Conclusions: Results support increased flexibility for initiating BUP-XR. Initiating BUP-XR 300 mg following a single BUP-TM 4 mg dose was well tolerated. Although some participants initially experienced withdrawal symptoms after injection, significant symptomatic improvement was observed in all participants within 24 hours.

Caspase1/11 signaling affects muscle regeneration and recovery following ischemia, and can be modulated by chloroquine.

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.

Treating Opioid Use Disorder With a Monthly Subcutaneous Buprenorphine Depot Injection: 12-Month Safety, Tolerability, and Efficacy Analysis.

BACKGROUND: BUP-XR (RBP-6000 or SUBLOCADE) is the first Food and Drug Administration-approved subcutaneously administered monthly extended-release buprenorphine medication for the treatment of moderate or severe opioid use disorder. The primary objective of this phase III study was to assess the long-term safety, tolerability, and efficacy of BUP-XR. METHODS: This open-label multicenter study in adults with moderate or severe opioid use disorder enrolled 257 participants from a previously conducted placebo-controlled, double-blind phase III study (rollover group) and 412 de novo participants not previously treated with BUP-XR. Participants received an initial injection of BUP-XR 300 mg and subsequent monthly 300 mg or 100 mg flexible doses. By study end, participants received up to 12 injections. RESULTS: Overall, 66.8% of participants reported more than 1 treatment-emergent adverse event (TEAE). Injection-site TEAEs (13.2% of participants) were mostly mild or moderate in severity. There were no clinically meaningful changes in safety assessments. An integrated analysis of the double-blind and open-label study participants showed that the incidence of TEAEs, including injection-site TEAEs, was lower in the second 6 months of treatment versus the first 6 months. After 12 months of treatment, 61.5% of the rollover participants and 75.8% of the de novo participants were abstinent. Retention rates after 12 months were 50.6% for the participants who initiated BUP-XR in the double-blind study and 50.5% for de novo participants. CONCLUSIONS: This study demonstrates that the clinical benefits and acceptable safety profile of BUP-XR demonstrated in the 6-month double-blind study are sustained over a 12-month open-label study, with lower incidence of TEAEs in the second 6 months of treatment.

Monthly Extended-Release Risperidone (RBP-7000) in the Treatment of Schizophrenia: Results From the Phase 3 Program.

PURPOSE/BACKGROUND: The Phase 3 program for RBP-7000, a once-monthly subcutaneous (SC) extended-release risperidone formulation approved for treatment of schizophrenia, consisted of a double-blind placebo-controlled trial (previously reported) and a 52-week open-label study of monthly RBP-7000 120 mg. The primary objective of the open-label study was to evaluate the long-term safety and tolerability of RBP-7000 in adults with schizophrenia. A secondary objective was to assess long-term maintenance of effectiveness. METHODS/PROCEDURES: The 52-week Phase 3 open-label study (NCT02203838) enrolled 92 rollover participants from the double-blind trial (NCT02109562) and 408 stable (Positive and Negative Syndrome Scale [PANSS] total score, ≤70) de novo participants. Participants received up to 13 monthly SC injections of RBP-7000 120 mg. Safety assessments included treatment-emergent adverse events, injection-site assessments, vital signs, laboratory and ECG parameters, extrapyramidal symptoms, and suicidality. Clinical outcomes included the PANSS and Clinical Global Impression-Severity. FINDINGS/RESULTS: Overall, 367 participants (73.4%) reported 1 or more treatment-emergent adverse event; the most common were injection-site pain (13.0%) and weight increase (12.8%). Most participants (>80%) experienced no injection-site reactions. No clinically meaningful changes were observed in laboratory or electrocardiogram values, vital signs, extrapyramidal symptoms, or suicidality. Over 12 months of exposure, mean PANSS scores continued to improve in rollover participants and remained stable among de novo participants. Mean Clinical Global Impression-Severity scores remained stable among all participants. IMPLICATIONS/CONCLUSIONS: Except for anticipated injection-site reactions, RBP-7000 demonstrated a favorable safety and tolerability profile similar to oral risperidone. Notably, PANSS scores continued to improve for participants from the pivotal study and remained stable for de novo participants.

An Open-Label Study to Assess Monthly Risperidone Injections (180 mg) Following Switch from Daily Oral Risperidone (6 mg) in Stable Schizophrenic Patients.

BACKGROUND AND OBJECTIVE: Long-acting injectable antipsychotics have shown benefits over oral medications with reduced hospitalization rates and improved health-related quality of life. RBP-7000 (PERSERIS®) is a monthly risperidone formulation (90 or 120 mg) for the treatment of schizophrenia administered by subcutaneous abdominal injection. The objective of this study was to assess a higher dose of 180 mg RBP-7000 and an alternate injection site. METHODS: Following stabilization on 6 mg/day (3 mg twice daily) oral risperidone, clinically stable schizophrenic participants received 3 monthly doses of 180 mg RBP-7000 in the abdomen followed by a fourth monthly dose of 180 mg RBP-7000 in the upper arm (each dose administered as two 90-mg injections). The primary endpoint was the steady-state average plasma concentration (Cavg(ss)) of risperidone and total active moiety after oral and RBP-7000 administration. Secondary endpoints included measures of clinical efficacy (Positive and Negative Syndrome Scale, Clinical Global Impression Scale for Severity of Illness), safety, and local injection-site tolerability to assess the switch from oral risperidone and compare injection sites. RESULTS: In all, 23 participants received at least one dose of RBP-7000, 16 received all four doses, and 15 completed the study. Monthly doses of 180 mg RBP-7000 provided similar Cavg(ss) of total active moiety compared with 6 mg/day oral risperidone. The pharmacokinetics of RBP-7000 were similar after injection in the abdomen versus upper arm. Clinical efficacy measures remained stable throughout the study. All RBP-7000 injections were well tolerated with no unexpected safety findings. CONCLUSIONS: The results support the use of 180 mg RBP-7000 in schizophrenic patients stable on 6 mg/day oral risperidone and a second injection site in the upper arm. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03978832.

18-Month efficacy and safety analysis of monthly subcutaneous buprenorphine injection for opioid use disorder: Integrated analysis of phase 3 studies.

BACKGROUND: Few studies investigate the natural history of patients on long-term treatment for opioid use disorder (OUD). We evaluated the long-term efficacy, safety, and tolerability experience of monthly extended-release buprenorphine (BUP-XR) in participants seeking treatment for OUD, via integrated analysis of phase 3 studies. METHODS: Study 1 was a 24-week randomized, double-blind, placebo-controlled trial of participants receiving monthly injections of BUP-XR (300 mg × 2, 100 mg × 4 [n = 203] or 300 mg × 6 [n = 201]) or placebo (n = 100). Study 2 was a 48-week, open-label trial enrolling 257 participants who completed study 1 and 412 de novo participants, to receive 6 and 12 BUP-XR injections, respectively. Study 3 was a 24-week, open-label extension enrolling 208 participants who completed study 2 for 6 additional injections. We assessed opioid abstinence as the proportion of urine opioid negative participants by visit and the percentage of each participant's negative opioid assessments during the first 6 months. RESULTS: In total, 916 participants were treated with BUP-XR or placebo. By the end of 18 months, 92.7 % of the de novo cohort and 81.8 % of the study 1 cohort were urine negative for opioids. Among early nonresponders (percentage of abstinence ≤20 %), 73.1 % were urine negative after 18 months. The longer treatment period was well tolerated, with no new safety concerns, and a low incidence of opioid withdrawal signs and symptoms, and hepatic disorder. CONCLUSIONS: Extending BUP-XR treatment beyond 6 months sustained improvement in opioid abstinence and was well tolerated, supporting clinical benefit up to 18 months. TRIAL REGISTRATION: NCT02357901 (study 1); NCT02510014 (study 2); NCT02896296 (study 3).

Aging Affects the Efficacy of Platelet-Rich Plasma Treatment for Osteoarthritis.

OBJECTIVE: Despite the increased use of platelet-rich plasma in the treatment of osteoarthritis, whether and how age of the platelet-rich plasma donor affects therapeutic efficacy is unclear. DESIGN: In vitro, male osteoarthritic human chondrocytes were treated with platelet-rich plasma from young (18-35 yrs) or old (≥65 yrs) donors, and the chondrogenic profile was evaluated using immunofluorescent staining for two markers of chondrogenicity, type II collagen and SOX-9. In vivo, we used a within-subjects design to compare Osteoarthritis Research Society International scores in aged mouse knee joints injected with platelet-rich plasma from young or old individuals. RESULTS: In vitro experiments revealed that platelet-rich plasma from young donors induced a more youthful chondrocyte phenotype, as evidenced by increased type II collagen ( P = 0.033) and SOX-9 expression ( P = 0.022). This benefit, however, was significantly blunted when cells were cultured with platelet-rich plasma from aged donors. Accordingly, in vivo studies revealed that animals treated with platelet-rich plasma from young donors displayed a significantly improved cartilage integrity when compared with knees injected with platelet-rich plasma from aged donors ( P = 0.019). CONCLUSIONS: Injection of platelet-rich plasma from a young individual induced a regenerative effect in aged cells and mice, whereas platelet-rich plasma from aged individuals showed no improvement in chondrocyte health or cartilage integrity.

Drug repurposing: An emerging strategy in alleviating skin cancer.

Skin cancer is one of the most common forms of cancer. Several million people are estimated to have affected with this condition worldwide. Skin cancer generally includes melanoma and non-melanoma with the former being the most dangerous. Chemotherapy has been one of the key therapeutic strategies employed in the treatment of skin cancer, especially in advanced stages of the disease. It could be also used as an adjuvant with other treatment modalities depending on the type of skin cancer. However, there are several shortfalls associated with the use of chemotherapy such as non-selectivity, tumour resistance, life-threatening toxicities, and the exorbitant cost of medicines. Furthermore, new drug discovery is a lengthy and costly process with minimal likelihood of success. Thus, drug repurposing (DR) has emerged as a new avenue where the drug approved formerly for the treatment of one disease can be used for the treatment of another disease like cancer. This approach is greatly beneficial over the de novo approach in terms of time and cost. Moreover, there is minimal risk of failure of repurposed therapeutics in clinical trials. There are a considerable number of studies that have reported on drugs repurposed for the treatment of skin cancer. Thus, the present manuscript offers a comprehensive overview of drugs that have been investigated as repurposing candidates for the efficient treatment of skin cancers mainly melanoma and its oncogenic subtypes, and non-melanoma. The prospects of repurposing phytochemicals against skin cancer are also discussed. Furthermore, repurposed drug delivery via topical route and repurposed drugs in clinical trials are briefed. Based on the findings from the reported studies discussed in this manuscript, drug repurposing emerges to be a promising approach and thus is expected to offer efficient treatment at a reasonable cost in devitalizing skin cancer.

The biphasic and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function.

Aging is accompanied by disrupted information flow, resulting from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders including skeletal muscle wasting, or sarcopenia. To derive a global metric of growing 'disorderliness' of aging muscle, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Escalating network entropy reached an inflection point at old age, while structural and functional alterations progressed into oldest-old age. To probe the potential for restoration of molecular 'order' and reversal of the sarcopenic phenotype, we systemically overexpressed the longevity protein, Klotho, via AAV. Klotho overexpression modulated genes representing all hallmarks of aging in old and oldest-old mice, but pathway enrichment revealed directions of changes were, for many genes, age-dependent. Functional improvements were also age-dependent. Klotho improved strength in old mice, but failed to induce benefits beyond the entropic tipping point.

Regulation of aged skeletal muscle regeneration by circulating extracellular vesicles.

Heterochronic blood exchange (HBE) has demonstrated that circulating factors restore youthful features to aged tissues. However, the systemic mediators of those rejuvenating effects remain poorly defined. We show here that the beneficial effect of young blood on aged muscle regeneration was diminished when serum was depleted of extracellular vesicles (EVs). Whereas EVs from young animals rejuvenate aged cell bioenergetics and skeletal muscle regeneration, aging shifts EV subpopulation heterogeneity and compromises downstream benefits on recipient cells. Machine learning classifiers revealed that aging shifts the nucleic acid, but not protein, fingerprint of circulating EVs. Alterations in sub-population heterogeneity were accompanied by declines in transcript levels of the pro-longevity protein, α-Klotho, and injection of EVs improved muscle regeneration in a Klotho mRNA-dependent manner. These studies demonstrate that EVs play a key role in the rejuvenating effects of HBE and that Klotho transcripts within EVs phenocopy the effects of young serum on aged skeletal muscle.

Design and synthesis of a library of tetracyclic hydroazulenoisoindoles.

Forty-four tetracyclic hydroazulenoisoindoles were synthesized via a tandem cyclopropanation/Cope rearrangement, followed by a Diels-Alder sequence from easily available five-membered cyclic cross-conjugated trienones. These trienones were obtained from two different routes depending upon whether R(1) and R(2) are alkyl or amino acid derived functional groups, via a rhodium(I)-catalyzed cycloisomerization reaction. To increase diversity, four maleimides and two 1,2,4-triazoline-3,5-diones were used as dienophiles in the Diels-Alder step. Several Diels-Alder adducts were further reacted under palladium-catalyzed hydrogenation conditions, leading to a diastereoselective reduction of the trisubstituted double bond. This library has demonstrated rapid access to a variety of structurally complex natural product-like compounds via stereochemical diversity and building block diversity approaches.

Klotho: An Elephant in Aging Research.

The year 2017 marked the 20th anniversary of the first publication describing Klotho. This single protein was and is remarkable in that its absence in mice conferred an accelerated aging, or progeroid, phenotype with a dramatically shortened life span. On the other hand, genetic overexpression extended both health span and life span by an impressive 30%. Not only has Klotho deficiency been linked to a number of debilitating age-related illnesses but many subsequent reports have lent credence to the idea that Klotho can compress the period of morbidity and extend the life span of both model organisms and humans. This suggests that Klotho functions as an integrator of organ systems, making it both a promising tool for advancing our understanding of the biology of aging and an intriguing target for interventional studies. In this review, we highlight advances in our understanding of Klotho as well as key challenges that have somewhat limited our view, and thus translational potential, of this potent protein.

Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents.

We report here the development and optimization of a simple 384-well colorimetric assay to measure H(2)O(2) generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H(2)O(2) either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H(2)O(2) was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl) phosphine sustain the redox cycling generation of H(2)O(2) by a model quinolinedione, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as beta-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of > or =0.8, and the redox cycling generation of H(2)O(2) by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 +/- 0.068 microM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H(2)O(2) in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36-39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H(2)O(2) that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen.

Computational design, synthesis and biological evaluation of para-quinone-based inhibitors for redox regulation of the dual-specificity phosphatase Cdc25B.

Quinoid inhibitors of Cdc25B were designed based on the Linear Combination of Atomic Potentials (LCAP) methodology. In contrast to a published hypothesis, the biological activities and hydrogen peroxide generation in reducing media of three synthetic models did not correlate with the quinone half-wave potential, E(1/2).

Development and implementation of a 384-well homogeneous fluorescence intensity high-throughput screening assay to identify mitogen-activated protein kinase phosphatase-1 dual-specificity protein phosphatase inhibitors.

We report here the miniaturization, development, and implementation of a homogeneous 384-well fluorescence intensity high-throughput screening (HTS) assay for identifying mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dual-specificity phosphatase inhibitors. As part of the National Institutes of Health (NIH) Molecular Libraries Screening Center Network (MLSCN), the MKP-1 assay was utilized to screen an NIH diversity library of 65,239 compounds for inhibitors of MKP-1 activity at 10 microM and was also used to confirm the concentration dependence of active agents identified in the primary screen. We observed 100 (0.15%) compounds that inhibited MKP-1 in vitro by > or =50% at 10 microM in the primary assay, and 46 of the 100 compounds were confirmed as concentration-dependent inhibitors of MKP-1 with 50% inhibitory concentration (IC(50)) values of <50 microM; four exhibited IC(50) values <1.0 microM, six produced IC(50) values in the 1-10 microM range, and 36 produced IC(50) values in the 10-50 microM range. A clustering and classification analysis of the compound structures of the 46 confirmed MKP-1 inhibitors produced 29 singleton structures and seven clusters of related structures. Some MKP-1 inhibitors were members of structural classes or contained substructure pharmacophores that previously were reported to inhibit either MKP-1 or other protein tyrosine phosphatases, validating the HTS assay. Importantly, we have identified several attractive and novel MKP-1 inhibitor structures that warrant further investigation as potential probes to study the biology of MKP-1 and its role in controlling the amplitude and/or duration of MAPK signaling, cell survival, and tumor progression.

HTS identifies novel and specific uncompetitive inhibitors of the two-component NS2B-NS3 proteinase of West Nile virus.

West Nile virus (WNV), a member of the Flavividae family, is a mosquito-borne, emerging pathogen. In addition to WNV, the family includes dengue, yellow fever, and Japanese encephalitis viruses, which affect millions of individuals worldwide. Because countermeasures are currently unavailable, flaviviral therapy is urgently required. The flaviviral two-component nonstructural NS2B-NS3 proteinase (protease [pro]) is essential for viral life cycle and, consequently, is a promising drug target. We report here the results of the miniaturization of an NS2B-NS3pro activity assay, followed by high-throughput screening of the National Institutes of Health's 65,000 compound library and identification of novel, uncompetitive inhibitors of WNV NS2B-NS3pro that appear to interfere with the productive interactions of the NS2B cofactor with the NS3pro domain. We anticipate that following structure optimization, the identified probes could form the foundation for the design of novel and specific therapeutics for WNV infection. We also provide the structural basis for additional species-selective allosteric inhibitors of flaviviruses.

Aging of the skeletal muscle extracellular matrix drives a stem cell fibrogenic conversion.

Age-related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age-related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age-related increase in muscle stiffness drives YAP/TAZ-mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate.

Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells.

Patients with castration-resistant prostate cancer (CRPC) can be treated with abiraterone, a potent inhibitor of androgen synthesis, or enzalutamide, a second-generation androgen receptor (AR) antagonist, both targeting AR signaling. However, most patients relapse after several months of therapy and a majority of patients with relapsed CRPC tumors express the AR target gene prostate-specific antigen (PSA), suggesting that AR signaling is reactivated and can be targeted again to inhibit the relapsed tumors. Novel small molecules capable of inhibiting AR function may lead to urgently needed therapies for patients resistant to abiraterone, enzalutamide, and/or other previously approved antiandrogen therapies. Here, we describe a high-throughput high-content screening (HCS) campaign to identify small-molecule inhibitors of AR nuclear localization in the C4-2 CRPC cell line stably transfected with GFP-AR-GFP (2GFP-AR). The implementation of this HCS assay to screen a National Institutes of Health library of 219,055 compounds led to the discovery of 3 small molecules capable of inhibiting AR nuclear localization and function in C4-2 cells, demonstrating the feasibility of using this cell-based phenotypic assay to identify small molecules targeting the subcellular localization of AR. Furthermore, the three hit compounds provide opportunities to develop novel AR drugs with potential for therapeutic intervention in CRPC patients who have relapsed after treatment with antiandrogens, such as abiraterone and/or enzalutamide.

Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury.

Cyclooxygenase-2 (COX-2) is an important contributor to ischemic brain injury. Identification of the downstream mediators of COX-2 toxicity may allow the development of targeted therapies. Of particular interest is the cyclopentenone family of prostaglandin metabolites. Cyclopentenone prostaglandins (CyPGs) are highly reactive molecules that form covalent bonds with cellular thiols. Protein disulfide isomerase (PDI) is an important molecule for the restoration of denatured proteins following ischemia. Because PDI has several thiols, including thiols within the active thioredoxin-like domain, we hypothesized that PDI is a target of CyPGs and that CyPG binding of PDI is detrimental. CyPG-PDI binding was detected in vitro via immunoprecipitation and MS. CyPG-PDI binding decreased PDI enzymatic activity in recombinant PDI treated with CyPG, and PDI immunoprecipitated from neuronal culture treated with CyPG or anoxia. Toxic effects of binding were demonstrated in experiments showing that: (a) pharmacologic inhibition of PDI increased cell death in anoxic neurons, (b) PDI overexpression protected neurons exposed to anoxia and SH-SY5Y cells exposed to CyPG, and (c) PDI overexpression in SH-SY5Y cells attenuated ubiquitination of proteins and decreased activation of pro-apoptotic caspases. In conclusion, CyPG production and subsequent binding of PDI is a novel and potentially important mechanism of ischemic brain injury. We show that CyPGs bind to PDI, cyclopentenones inhibit PDI activity, and CyPG-PDI binding is associated with increased neuronal susceptibility to anoxia. Additional studies are necessary to determine the relative role of CyPG-dependent inhibition of PDI activity in ischemia and other neurodegenerative disorders.

COX2-derived primary and cyclopentenone prostaglandins are increased after asphyxial cardiac arrest.

BACKGROUND: Cyclopentenone prostaglandins have been identified as potential neurotoxic agents in the setting of hypoxia-ischemia. Cyclooxygenase-2 (COX-2), the upstream enzyme responsible for prostaglandin production is upregulated following hypoxic-ischemic brain injury. However, the temporal production and concentration of cyclopentenone prostaglandins has not been described following global brain ischemia. METHODS: Global brain ischemia was induced in rats by asphyxial cardiac arrest (ACA) followed by resuscitation. Rats were sacrificed between 24h and 7 days following resuscitation and their brains removed. Western blot, immunohistochemistry, and mass spectroscopy were performed. A cohort of rats was pretreated with the COX-2 inhibitor SC58125. RESULTS: COX-2 is induced in hippocampus at 24h following ACA. Multiple prostaglandins, including cyclopentenone prostaglandin species, are increased in hippocampus as 24h following ACA. Prostaglandin and cyclopentenone prostaglandin concentrations are returned to baseline at 3 and 7 days post-ischemia. The COX-2 inhibitor SC58125 completely abrogates the post-ischemic increase in prostaglandins and cyclopentenone prostaglandins. CONCLUSIONS: Prostaglandins, including cyclopentenone prostaglandins, are increased in ischemic brain, peak at 24h and can be attenuated by the COX-2 inhibitor SC58125. These data establish the presence of potentially neurotoxic cyclopentenone prostaglandins in post-ischemic brains, thus identifying a target and therapeutic window for neuroprotective therapies.