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.

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.

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.

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.

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.

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.

Development and validation of a high-content screening assay to identify inhibitors of cytoplasmic dynein-mediated transport of glucocorticoid receptor to the nucleus.

Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to eliminate cytotoxic compounds and fluorescent outliers. We identified five qualified hits that inhibited the rapid retrograde trafficking of GR-GFP in a concentration-dependent manner: Bay 11-7085, 4-phenyl-3-furoxancarbonitrile, parthenolide, apomorphine, and 6-nitroso-1,2-benzopyrone. The data presented here demonstrate that the GR-GFP HCS assay provides an effective phenotypic screen and support the proposition that screening a larger library of diversity compounds will yield novel small-molecule probes that will enable the further exploration of intracellular retrograde transport of cargo along microtubules, a process which is essential to the morphogenesis and function of all cells.

Increased cytochrome c in rat cerebrospinal fluid after cardiac arrest and its effects on hypoxic neuronal survival.

Cerebrospinal fluid (CSF) proteins may be useful biomarkers of neuronal death and ultimate prognosis after hypoxic-ischemic brain injury. Cytochrome c has been identified in the CSF of children following traumatic brain injury. Cytochrome c is required for cellular respiration but it is also a central component of the intrinsic pathway of apoptosis. Thus, in addition to serving as a biomarker, cytochrome c release into CSF may have an effect upon survival of adjacent neurons. In this study, we use Western blot and ELISA to show that cytochrome c is elevated in CSF obtained from pediatric rats following resuscitation from cardiac arrest. Using biotinylated human cytochrome c in culture media we show that cytochrome c crosses the cell membrane and is incorporated into mitochondria of neurons exposed to anoxia. Lastly, we show that addition of human cytochrome c to primary neuronal culture exposed to anoxia improves survival. To our knowledge, this is the first study to show cytochrome c is elevated in CSF following hypoxic ischemic brain injury. Results from primary neuronal culture suggest that extracellular cytochrome c is able to cross the cell membrane of injured neurons, incorporate into mitochondria, and promote survival following anoxia.

Alterations in the non-neuronal acetylcholine synthesis and release machinery in esophageal epithelium.

AIMS: A non-neuronal cholinergic system has been described in epithelial cells including that of the urinary bladder (urothelium) and the upper gastrointestinal tract (esophagus). Epithelial dysfunction has been implicated in the pathophysiology of persistent pain conditions such as painful bladder syndrome as well as functional heartburn. For example, alterations in the ability to synthesize and release acetylcholine may contribute to changes in epithelial sensory and barrier function associated with a number of functional genitourinary and intestinal disorders. MAIN METHODS: We examined using immunoblot, acetylcholine (ACh)-synthesis and release components in cat esophageal mucosa and whether elements of these components are altered in a naturally occurring model of chronic idiopathic cystitis termed feline interstitial cystitis (FIC). KEY FINDINGS: We identified proteins involved in ACh synthesis and release (high affinity choline transporter, CHT1; ACh synthesizing enzyme choline acetyltransferase ChAT and carnitine acetyltransferase CarAT; vesicular ACh transporter VAChT and the organic cation transporter isoforms 1-3 or OCT-1-3) in cat esophageal mucosa. Significant alterations in CHT, ChAT, VAChT and OCT-1 were detected in the esophageal mucosa from FIC cats. Changes in the vesicular nucleotide transporter (VNUT) and the junctional protein pan-cadherin were also noted. SIGNIFICANCE: Taken together, these findings suggest that changes in the non-neuronal cholinergic system may contribute to alterations in cell-cell contacts and possibly communication with underlying cells that may contribute to changes in sensory function and visceral hyperalgesia in functional esophageal pain.

Characterization of inhibitors of glucocorticoid receptor nuclear translocation: a model of cytoplasmic dynein-mediated cargo transport.

Agonist-induced glucocorticoid receptor [GR] transport from the cytoplasm to the nucleus was used as a model to identify dynein-mediated cargo transport inhibitors. Cell-based screening of the library of pharmacologically active compound (LOPAC)-1280 collection identified several small molecules that stalled the agonist-induced transport of GR-green fluorescent protein (GFP) in a concentration-dependent manner. Fluorescent images of microtubule organization, nuclear DNA staining, expression of GR-GFP, and its subcellular distribution were inspected and quantified by image analysis to evaluate the impact of compounds on cell morphology, toxicity, and GR transport. Given the complexity of the multi-protein complex involved in dynein-mediated cargo transport and the variety of potential mechanisms for interruption of that process, we therefore developed and validated a panel of biochemical assays to investigate some of the more likely intracellular target(s) of the GR transport inhibitors. Although the apomorphine enantiomers exhibited the most potency toward the ATPase activities of cytoplasmic dynein, myosin, and the heat-shock proteins (HSPs), their apparent lack of specificity made them unattractive for further study in our quest. Other molecules appeared to be nonspecific inhibitors that targeted reactive cysteines of proteins. Ideally, specific retrograde transport inhibitors would either target dynein itself or one of the other important proteins associated with the transport process. Although the hits from the cell-based screen of the LOPAC-1280 collection did not exhibit this desired profile, this screening platform provided a promising phenotypic system for the discovery of dynein/HSP modulators.

Implementation of a 220,000-compound HCS campaign to identify disruptors of the interaction between p53 and hDM2 and characterization of the confirmed hits.

In recent years, advances in structure-based drug design and the development of an impressive variety of high-throughput screening (HTS) assay formats have yielded an expanding list of protein-protein interaction inhibitors. Despite these advances, protein-protein interaction targets are still widely considered difficult to disrupt with small molecules. The authors present here the results from screening 220,017 compounds from the National Institute of Health's small-molecule library in a novel p53-hDM2 protein-protein interaction biosensor (PPIB) assay. The p53-hDM2 positional biosensor performed robustly and reproducibly throughout the high-content screening (HCS) campaign, and analysis of the multiparameter data from images of the 3 fluorescent channels enabled the authors to identify and eliminate compounds that were cytotoxic or fluorescent artifacts. The HCS campaign yielded 3 structurally related methylbenzo-naphthyridin-5-amine (MBNA) hits with IC(50)s between 30 and 50 microM in the p53-hDM2 PPIB. In HCT116 cells with wild-type (WT) p53, the MBNAs enhanced p53 protein levels, increased the expression of p53 target genes, caused a cell cycle arrest in G1, induced apoptosis, and inhibited cell proliferation with an IC(50) ~4 microM. The prototype disruptor of p53-hDM2 interactions Nutlin-3 was more potent than the MBNAs in the p53-hDM2 PPIB assay but produced equivalent biological results in HCT116 cells WT for p53. Unlike Nutlin-3, however, MBNAs also increased the percentage of apoptosis in p53 null cells and exhibited similar potencies for growth inhibition in isogenic cell lines null for p53 or p21. Neither the MBNAs nor Nutin-3 caused cell cycle arrest in p53 null HCT116 cells. Despite the relatively modest size of the screening library, the combination of a novel p53-hDM2 PPIB assay together with an automated imaging HCS platform and image analysis methods enabled the discovery of a novel chemotype series that disrupts p53-hDM2 interactions in cells.