Ligand-Free Signaling of G-Protein-Coupled Receptors: Relevance to µ Opioid Receptors in Analgesia and Addiction.

Numerous G-protein-coupled receptors (GPCRs) display ligand-free basal signaling with potential physiological functions, a target in drug development. As an example, the µ opioid receptor (MOR) signals in ligand-free form (MOR-µ*), influencing opioid responses. In addition, agonists bind to MOR but can dissociate upon MOR activation, with ligand-free MOR-µ* carrying out signaling. Opioid pain therapy is effective but incurs adverse effects (ADRs) and risk of opioid use disorder (OUD). Sustained opioid agonist exposure increases persistent basal MOR-µ* activity, which could be a driving force for OUD and ADRs. Antagonists competitively prevent resting MOR (MOR-µ) activation to MOR-µ*, while common antagonists, such as naloxone and naltrexone, also bind to and block ligand-free MOR-µ*, acting as potent inverse agonists. A neutral antagonist, 6ß-naltrexol (6BN), binds to but does not block MOR-µ*, preventing MOR-µ activation only competitively with reduced potency. We hypothesize that 6BN gradually accelerates MOR-µ* reversal to resting-state MOR-µ. Thus, 6BN potently prevents opioid dependence in rodents, at doses well below those blocking antinociception or causing withdrawal. Acting as a 'retrograde addiction modulator', 6BN could represent a novel class of therapeutics for OUD. Further studies need to address regulation of MOR-µ* and, more broadly, the physiological and pharmacological significance of ligand-free signaling in GPCRs.

Evaluation of chronic toxicity of cyclocreatine in beagle dogs after oral gavage administration for up to 23 weeks.

Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.

Use of an animal model of disease for toxicology enables identification of a juvenile no observed adverse effect level for cyclocreatine in creatine transporter deficiency.

In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 µg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 µg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.

Evaluation of chronic toxicity of cyclocreatine, a creatine analog, in Sprague Dawley rat after oral gavage administration for up to 26 weeks.

Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 µg/mL; AUC0-24 of 1970 h*µg/mL) was considered the maximum tolerated dose (MTD) in SD rats.

Intrathecal 2-hydroxypropyl-ß-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1-2 trial.

BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is a lysosomal storage disorder characterised by progressive neurodegeneration. In preclinical testing, 2-hydroxypropyl-ß-cyclodextrins (HPßCD) significantly delayed cerebellar Purkinje cell loss, slowed progression of neurological manifestations, and increased lifespan in mouse and cat models of NPC1. The aim of this study was to assess the safety and efficacy of lumbar intrathecal HPßCD. METHODS: In this open-label, dose-escalation phase 1-2a study, we gave monthly intrathecal HPßCD to participants with NPC1 with neurological manifestation at the National Institutes of Health (NIH), Bethesda, MD, USA. To explore the potential effect of 2-week dosing, three additional participants were enrolled in a parallel study at Rush University Medical Center (RUMC), Chicago, IL, USA. Participants from the NIH were non-randomly, sequentially assigned in cohorts of three to receive monthly initial intrathecal HPßCD at doses of 50, 200, 300, or 400 mg per month. A fifth cohort of two participants received initial doses of 900 mg. Participants from RUMC initially received 200 or 400 mg every 2 weeks. The dose was escalated based on tolerance or safety data from higher dose cohorts. Serum and CSF 24(S)-hydroxycholesterol (24[S]-HC), which serves as a biomarker of target engagement, and CSF protein biomarkers were evaluated. NPC Neurological Severity Scores (NNSS) were used to compare disease progression in HPßCD-treated participants relative to a historical comparison cohort of 21 NPC1 participants of similar age range. FINDINGS: Between Sept 21, 2013, and Jan 19, 2015, 32 participants with NPC1 were assessed for eligibility at the National Institutes of Health. 18 patients were excluded due to inclusion criteria not met (six patients), declined to participate (three patients), pursued independent expanded access and obtained the drug outside of the study (three patients), enrolled in the RUMC cohort (one patient), or too late for the trial enrolment (five patients). 14 patients were enrolled and sequentially assigned to receive intrathecal HPßCD at a starting dose of 50 mg per month (three patients), 200 mg per month (three patients), 300 mg per month (three patients), 400 mg per month (three patients), or 900 mg per month (two patients). During the first year, two patients had treatment interrupted for one dose, based on grade 1 ototoxicity. All 14 patients were assessed at 12 months. Between 12 and 18 months, one participant had treatment interrupted at 17 months due to hepatocellular carcinoma, one patient had dose interruption for 2 doses based on caregiver hardship and one patient had treatment interrupted for 1 dose for mastoiditis. 11 patients were assessed at 18 months. Between Dec 11, 2013, and June 25, 2014, three participants were assessed for eligibility and enrolled at RUMC, and were assigned to receive intrathecal HPßCD at a starting dose of 200 mg every 2 weeks (two patients), or 400 mg every two weeks (one patient). There were no dropouts in this group and all 3 patients were assessed at 18 months. Biomarker studies were consistent with improved neuronal cholesterol homoeostasis and decreased neuronal pathology. Post-drug plasma 24(S)-HC area under the curve (AUC8-72) values, an indicator of neuronal cholesterol homoeostasis, were significantly higher than post-saline plasma 24(S)-HC AUC8-72 after doses of 900 mg (p=0·0063) and 1200 mg (p=0·0037). CSF 24(S)-HC concentrations in three participants given either 600 or 900 mg of HPßCD were increased about two fold (p=0·0032) after drug administration. No drug-related serious adverse events were observed. Mid-frequency to high-frequency hearing loss, an expected adverse event, was documented in all participants. When managed with hearing aids, this did not have an appreciable effect on daily communication. The NNSS for the 14 participants treated monthly increased at a rate of 1·22, SEM 0·34 points per year compared with 2·92, SEM 0·27 points per year (p=0·0002) for the 21 patient comparison group. Decreased progression was observed for NNSS domains of ambulation (p=0·0622), cognition (p=0·0040) and speech (p=0·0423). INTERPRETATION: Patients with NPC1 treated with intrathecal HPßCD had slowed disease progression with an acceptable safety profile. These data support the initiation of a multinational, randomised, controlled trial of intrathecal HPßCD. FUNDING: National Institutes of Health, Dana's Angels Research Trust, Ara Parseghian Medical Research Foundation, Hope for Haley, Samantha's Search for the Cure Foundation, National Niemann-Pick Disease Foundation, Support of Accelerated Research for NPC Disease, Vtesse, Janssen Research and Development, a Johnson & Johnson company, and Johnson & Johnson.

Safety, pharmacokinetics and sialic acid production after oral administration of N-acetylmannosamine (ManNAc) to subjects with GNE myopathy.

GNE myopathy is a rare, autosomal recessive, inborn error of sialic acid metabolism, caused by mutations in GNE, the gene encoding UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase. The disease manifests as an adult-onset myopathy characterized by progressive skeletal muscle weakness and atrophy. There is no medical therapy available for this debilitating disease. Hyposialylation of muscle glycoproteins likely contributes to the pathophysiology of this disease. N-acetyl-D-mannosamine (ManNAc), an uncharged monosaccharide and the first committed precursor in the sialic acid biosynthetic pathway, is a therapeutic candidate that prevents muscle weakness in the mouse model of GNE myopathy. We conducted a first-in-human, randomized, placebo-controlled, double-blind, single-ascending dose study to evaluate safety and pharmacokinetics of ManNAc in GNE myopathy subjects. Single doses of 3 and 6g of oral ManNAc were safe and well tolerated; 10g was associated with diarrhea likely due to unabsorbed ManNAc. Oral ManNAc was absorbed rapidly and exhibited a short half-life (~2.4h). Following administration of a single dose of ManNAc, there was a significant and sustained increase in plasma unconjugated free sialic acid (Neu5Ac) (Tmax of 8-11h). Neu5Ac levels remained above baseline 48h post-dose in subjects who received a dose of 6 or 10g. Given that Neu5Ac is known to have a short half-life, the prolonged elevation of Neu5Ac after a single dose of ManNAc suggests that intracellular biosynthesis of sialic acid was restored in subjects with GNE myopathy, including those homozygous for mutations in the kinase domain. Simulated plasma concentration-time profiles support a dosing regimen of 6g twice daily for future clinical trials.

In vitro evaluation of imidazo[4,5-c]quinolin-2-ones as gametocytocidal antimalarial agents.

Novel imidazo[4,5-c]quinolin-2-ones were synthesized and evaluated in asexual blood stage and late stage gametocyte assays of Plasmodium falciparum, a major causative agent of malaria. The design of these compounds is based on a recently identified lead compound from a high throughput screen. A concise synthesis was developed that allowed for generation of analogues with substitution around both the quinoline and imidazolidinone rings. Through structure-activity relationship studies, a number of potent compounds were identified that possessed excellent antimalarial activity against both the asexual and sexual stages with minimal cytotoxicity in mammalian cells. This is the first Letter describing SAR and gametocytocidal activity of imidazo[4,5-c]quinolin-2-ones, a new lead series for malaria treatment and prevention.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

INTRODUCTION: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. METHODS: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. RESULTS: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. CONCLUSIONS: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.

Mutation update for GNE gene variants associated with GNE myopathy.

The GNE gene encodes the rate-limiting, bifunctional enzyme of sialic acid biosynthesis, uridine diphosphate-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Biallelic GNE mutations underlie GNE myopathy, an adult-onset progressive myopathy. GNE myopathy-associated GNE mutations are predominantly missense, resulting in reduced, but not absent, GNE enzyme activities. The exact pathomechanism of GNE myopathy remains unknown, but likely involves aberrant (muscle) sialylation. Here, we summarize 154 reported and novel GNE variants associated with GNE myopathy, including 122 missense, 11 nonsense, 14 insertion/deletions, and seven intronic variants. All variants were deposited in the online GNE variation database (http://www.dmd.nl/nmdb2/home.php?select_db=GNE). We report the predicted effects on protein function of all variants well as the predicted effects on epimerase and/or kinase enzymatic activities of selected variants. By analyzing exome sequence databases, we identified three frequently occurring, unreported GNE missense variants/polymorphisms, important for future sequence interpretations. Based on allele frequencies, we estimate the world-wide prevalence of GNE myopathy to be ∼4-21/1,000,000. This previously unrecognized high prevalence confirms suspicions that many patients may escape diagnosis. Awareness among physicians for GNE myopathy is essential for the identification of new patients, which is required for better understanding of the disorder's pathomechanism and for the success of ongoing treatment trials.

δ-Tocopherol reduces lipid accumulation in Niemann-Pick type C1 and Wolman cholesterol storage disorders.

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca(2+) response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.

Cytosolic phospholipase A2α blockade abrogates disease during the tissue-damage effector phase of experimental autoimmune encephalomyelitis by its action on APCs.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.

A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions.

Cryptococcus neoformans causes an estimated 600,000 AIDS-related deaths annually that occur primarily in resource-limited countries. Fluconazole and amphotericin B are currently available for the treatment of cryptococcal-related infections. However, fluconazole has limited clinical efficacy and amphotericin B requires intravenous infusion and is associated with high renal toxicity. Therefore, there is an unmet need for a new orally administrable anti-cryptococcal drug. We have developed a high-throughput screening assay for the measurement of C. neoformans viability in 1,536-well plate format. The signal-to-basal ratio of the ATP content assay was 21.9 fold with a coefficient of variation and Z' factor of 7.1% and 0.76, respectively. A pilot screen of 1,280 known compounds against the wild-type C. neoformans (strain H99) led to the identification of four active compounds including niclosamide, malonoben, 6-bromoindirubin-3'-oxime, and 5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone. These compounds were further tested against nine clinical isolates of C. neoformans, and their fungicidal activities were confirmed. The results demonstrate that this miniaturized C. neoformans assay is advantageous for the high-throughput screening of large compound collections to identify lead compounds for new anti-cryptococcal drug development.

Osteogenic effects of a potent Src-over-Abl-selective kinase inhibitor in the mouse.

Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.

Corroboration of Height Velocity Prediction Markers for rhGH With an Oral GH Secretagogue Treatment in Children With GHD.

CONTEXT: Recombinant human growth hormone (rhGH) is approved for treatment of pediatric growth hormone deficiency (GHD), with greatest growth responses observed in those with severe GHD. Orally administered GH secretagogues (GHS) may be useful treatment in patients with moderate GHD. Distinguishing children with severe vs moderate GHD could identify children who would be better treated with rhGH or GHS. OBJECTIVES: Evaluate baseline insulin-like growth factor-I (IGF-I) and stimulated peak GH response as predictors of 12-month height velocity (HV) in children with GHD. DESIGN: Data on children with GHD were analyzed in a legacy data base (GeNeSIS data). PARTICIPANTS: 514 naïve to rhGH-treatment, prepubertal children with idiopathic isolated GHD for whom stimulated GH, baseline serum IGF-I, and first-year HV during rhGH treatment data are available. OUTCOME MEASURES: Children with severe or moderate GHD were categorized based on GH and IGF-I data and evaluated based on baseline auxologic and hormone profiles and first-year growth response to rhGH. RESULTS: Cohorts of severe and moderate GHD were 81/514 (15.8%) and 433/514 (84.2%). Cohorts differed significantly with regard to indicators of GHD [eg, baseline height SD score (SDS), height SDS minus target height SDS, HV, HV SDS, and change in height SDS during rhGH treatment]. Multiple regression analysis showed IGF-I and stimulated GH were significant predictors of HV independent of other known variables. Expected first-year HV in moderate GHD was 8.3 cm/y. CONCLUSIONS: The combination of peak GH to GH stimulation testing and baseline IGF-I concentration are predictive enrichment markers for annualized HV responses to rhGH therapy.

Development of a Predictive Enrichment Marker for the Oral GH Secretagogue LUM-201 in Pediatric Growth Hormone Deficiency.

CONTEXT: We hypothesize, based on the degree of residual hypothalamic-pituitary function, that some, but not all, children with growth hormone deficiency (GHD) may have beneficial growth responses to the orally administered growth hormone (GH) secretagogue LUM-201. OBJECTIVE: To determine if pretreatment testing can identify predictive enrichment markers (PEM) for subjects with adequate residual function who are responsive to LUM-201. METHODS: We performed an analysis of a completed, randomized, placebo-controlled trial of LUM-201, a GH secretagogue receptor agonist, in which all randomized subjects had pretreatment testing. This international multicenter study conducted in pediatric endocrinology clinics included 68 naïve-to-treatment, prepubertal children with established diagnoses of GHD. Outcome measures included the sensitivity, specificity, and predictive accuracy of potential markers to predict 6-month growth responses to oral LUM-201 and daily rhGH. RESULTS: Two PEM were identified for use in defining PEM-positive status: (1) baseline insulin-like growth factor I (IGF-I) concentration >30 ng/mL and (2) peak GH response of ≥5 ng/mL upon administration of single-dose LUM-201. PEM-positive status enriches a population for better growth responses to LUM-201. PEM-negative status enriches a population for better growth responses to rhGH. CONCLUSION: Combined, the peak GH response to single-dose LUM-201 and the baseline IGF-I concentration are effective PEMs for 6-month growth responses to LUM-201 and rhGH in prepubertal children with GHD.

Cyclocreatine treatment ameliorates the cognitive, autistic and epileptic phenotype in a mouse model of Creatine Transporter Deficiency.

Creatine Transporter Deficiency (CTD) is an inborn error of metabolism presenting with intellectual disability, behavioral disturbances and epilepsy. There is currently no cure for this disorder. Here, we employed novel biomarkers for monitoring brain function, together with well-established behavioral readouts for CTD mice, to longitudinally study the therapeutic efficacy of cyclocreatine (cCr) at the preclinical level. Our results show that cCr treatment is able to partially correct hemodynamic responses and EEG abnormalities, improve cognitive deficits, revert autistic-like behaviors and protect against seizures. This study provides encouraging data to support the potential therapeutic benefit of cyclocreatine or other chemically modified lipophilic analogs of Cr.

Benzhydrylquinazolinediones: novel cytosolic phospholipase A2alpha inhibitors with improved physicochemical properties.

The synthesis and optimization of a class of trisubstituted quinazoline-2,4(1H,3H)-dione cPLA(2)alpha inhibitors are described. Utilizing pharmacophores that were found to be important in our indole series, we discovered inhibitors with reduced lipophilicity and improved aqueous solubility. These compounds are active in whole blood assays, and cell-based assay results indicate that prevention of arachidonic acid release arises from selective cPLA(2)alpha inhibition.

Indole cytosolic phospholipase A2 alpha inhibitors: discovery and in vitro and in vivo characterization of 4-{3-[5-chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propyl}benzoic acid, efipladib.

The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.

Blockade of cytosolic phospholipase A2 alpha prevents experimental autoimmune encephalomyelitis and diminishes development of Th1 and Th17 responses.

Cytosolic phospholipase A2 alpha (cPLA2 alpha) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase (COX) 1/2 pathways, and leukotrienes via the 5-lipoxygenase (LO) pathway. We utilized inhibitors of cPLA2 alpha, COX-1/2 and 5-LO to determine the potential roles of these enzymes in development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Blocking cPLA2 alpha prevented EAE development and greatly reduced antigen-induced production of Th1-type cytokines and IL-17. Blocking COX-1/2 delayed onset and reduced severity of EAE, and reduced production of Th1-type cytokines, but not IL-17. Blocking 5-LO delayed onset and reduced cumulative severity of EAE, but did not reduce production of Th1-type cytokines or IL-17. Finally, blockade of cPLA2 alpha from the onset of clinical EAE reduced duration of EAE relapses. Therefore, cPLA2 alpha represents a potential therapeutic target for treatment of MS.

Benzenesulfonamide indole inhibitors of cytosolic phospholipase A2alpha: optimization of in vitro potency and rat pharmacokinetics for oral efficacy.

The synthesis and structure-activity relationship of a series of benzenesulfonamide indole inhibitors of cPLA(2)alpha are described. Substitution of the benzenesulfonamide led to analogues with 50-fold improvement in potency versus the unsubstituted benzenesulfonamide lead compound. Rat pharmacokinetics in a minimal formulation was used to prioritize compounds, leading to the discovery of a potent inhibitor of cPLA(2)alpha with oral efficacy in models of rat carrageenan paw edema and Ascaris suum airway challenge in naturally sensitized sheep.