Shadows into Light: The Potential Investigative Utility of Voice Analysis with Two Types of Online Child-Sex Predators.

Over 390,000 child sexual abuse victims in the United States have not yet been identified. Due to the increased prevalence of Internet-driven child-sex offenders (e.g., child pornographers and travelers), detection becomes more elusive, and disclosure elicitations are more challenging for law enforcement. The current retrospective study examines an innovative, investigative method of voice stress analysis use, and describes its possible utility in identifying previously undetected sexual offending within these two offender populations. In the total sample of 82 arrestees with no known history of "hands-on" sexual offending, 0% initially admitted to sexually abusing at least one child. However, coinciding with voice stress analysis procedures, 40.2% of the suspect pool (57.1% of child pornographers and 36.7% of travelers) provided admissions to hands-on offenses. Also, 80.5% admitted to at least one sex crime offense during the pre and posttest stages of the investigation. All voice stress analysis "Stress Indicated" examinations resulted in verifiable disclosures (of victims and sex crimes). Critically, as a result of voice stress analysis procedures, 87 previously undiscovered live victims were identified. Finally, this study's description of specific characteristics and predictive qualities of victimizers vs. non-victimizers in each offender-type should benefit future investigators, researchers, and therapists alike.

Inhibition of glutamate carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis.

Half of all patients with multiple sclerosis (MS) experience cognitive impairment, for which there is no pharmacological treatment. Using magnetic resonance spectroscopy (MRS), we examined metabolic changes in the hippocampi of MS patients, compared the findings to performance on a neurocognitive test battery, and found that N-acetylaspartylglutamate (NAAG) concentration correlated with cognitive functioning. Specifically, MS patients with cognitive impairment had low hippocampal NAAG levels, whereas those with normal cognition demonstrated higher levels. We then evaluated glutamate carboxypeptidase II (GCPII) inhibitors, known to increase brain NAAG levels, on cognition in the experimental autoimmune encephalomyelitis (EAE) model of MS. Whereas GCPII inhibitor administration did not affect physical disabilities, it increased brain NAAG levels and dramatically improved learning and memory test performance compared with vehicle-treated EAE mice. These data suggest that NAAG is a unique biomarker for cognitive function in MS and that inhibition of GCPII might be a unique therapeutic strategy for recovery of cognitive function.

Regional brain distribution of translocator protein using [(11)C]DPA-713 PET in individuals infected with HIV.

Imaging the brain distribution of translocator protein (TSPO), a putative biomarker for glial cell activation and neuroinflammation, may inform management of individuals infected with HIV by uncovering regional abnormalities related to neurocognitive deficits and enable non-invasive therapeutic monitoring. Using the second-generation TSPO-targeted radiotracer, [(11)C]DPA-713, we conducted a positron emission tomography (PET) study to compare the brains of 12 healthy human subjects to those of 23 individuals with HIV who were effectively treated with combination antiretroviral therapy (cART). Compared to PET data from age-matched healthy control subjects, [(11)C]DPA-713 PET of individuals infected with HIV demonstrated significantly higher volume-of-distribution (VT) ratios in white matter, cingulate cortex, and supramarginal gyrus, relative to overall gray matter VT, suggesting localized glial cell activation in susceptible regions. Regional TSPO abnormalities were evident within a sub-cohort of neuro-asymptomatic HIV subjects, and an increase in the VT ratio within frontal cortex was specifically linked to individuals affected with HIV-associated dementia. These findings were enabled by employing a gray matter normalization approach for PET data quantification, which improved test-retest reproducibility, intra-class correlation within the healthy control cohort, and sensitivity of uncovering abnormal regional findings.

Glutamate carboxypeptidase II is not an amyloid peptide-degrading enzyme.

Glutamate carboxypeptidase II (GCPII) is an exopeptidase that catalyzes the hydrolysis of N-acetylated aspartate-glutamate (NAAG) to N-acetyl aspartate (NAA) and glutamate. Consequently, GCPII inhibition has been of interest for the treatment of central and peripheral nervous system diseases associated with excess glutamate. Recently, it was reported that GCPII can also serve as an endopeptidase cleaving amyloid ß (Aß) peptides and that its inhibition could increase the risk of Alzheimer's disease by increasing brain Aß levels. This study aimed to corroborate and extend these new findings. We incubated Aß peptides (20 µM) with human recombinant GCPII (300 ng/ml) and monitored the appearance of degradation products by mass spectrometry. Aß peptides remained intact after 18 h incubation with GCPII. Under the same experimental conditions, Aß1-40 (20 µM) was incubated with neprilysin (300 ng/ml), an endopeptidase known to hydrolyze Aß1-40 and the expected cleavage products were observed. GCPII was confirmed active by catalyzing the complete hydrolysis of NAAG (100 µM). We also studied the hydrolysis of [(3)H]-NAAG (30 nM) catalyzed by GCPII (40 pM) in the presence of Aß peptides (picomolar to micromolar range). The addition of Aß peptides did not alter [(3)H]-NAAG hydrolysis. We conclude that GCPII is not an amyloid peptide-degrading enzyme.

Netupitant and palonosetron trigger NK1 receptor internalization in NG108-15 cells.

Current therapy for chemotherapy-induced nausea and vomiting includes the use of both 5-HT3 and NK1 receptor antagonists. Acute emesis has largely been alleviated with the use of 5-HT3 receptor antagonists, while an improvement in preventing delayed emesis has been achieved with NK1 receptor antagonists. Delayed emesis, however, remains a problem with a significant portion of cancer patients receiving highly emetogenic chemotherapy. Like other drugs in its class, palonosetron, a 5-HT3 receptor antagonist, has shown efficacy against acute emesis. However, palonosetron has also shown consistent improvement in the suppression of delayed emesis. Since both 5-HT3 and NK1 receptor antagonists are often simultaneously administered to patients, the question remains if palonosetron's effect on delayed emesis would remain distinct when co-administered with an NK1 receptor antagonist. Recent mechanistic studies using NG108-15 cells have shown that palonosetron and netupitant, an NK1 receptor antagonist currently in phase 3 clinical trials, exhibited synergistic effects when inhibiting the substance P response. The present studies showed that both netupitant and palonosetron-induced NK1 receptor internalization in NG108-15 cells and that when used together receptor internalization was additive. Palonosetron-induced NK1 receptor internalization was dependent on the presence of the 5-HT3 receptor. Results provide a possible explanation for palonosetron's enhancement of the inhibition of the SP response and suggest that the effect of palonosetron and NK1 receptor antagonists on prevention of delayed emesis could be additive.

Synthesis and biological evaluation of low molecular weight fluorescent imaging agents for the prostate-specific membrane antigen.

Targeted near-infrared (NIR) optical imaging can be used in vivo to detect specific tissues, including malignant cells. A series of NIR fluorescent ligands targeting the prostate-specific membrane antigen (PSMA) was synthesized and each compound was tested for its ability to image PSMA+ tissues in experimental models of prostate cancer. The agents were prepared by conjugating commercially available active esters of NIR dyes, including IRDye800CW, IRDye800RS, Cy5.5, Cy7, or a derivative of indocyanine green (ICG) to the terminal amine group of (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid 1, (14S,18S)-1-amino-8,16-dioxo-3,6-dioxa-9,15,17-triazaicosane-14,18,20-tricarboxylic acid 2 and (3S,7S)-26-amino-5,13,20-trioxo-4,6,12,21-tetraazahexacosane-1,3,7,22-tetracarboxylic acid 3. The K(i) values for the dye-inhibitor conjugates ranged from 1 to 700 pM. All compounds proved capable of imaging PSMA+ tumors selectively to varying degrees depending on the choice of fluorophore and linker. The highest tumor uptake was observed with IRDye800CW employing a poly(ethylene glycol) or lysine-suberate linker, as in 800CW-2 and 800CW-3, while the highest tumor to nontarget tissue ratios were obtained for Cy7 with these same linkers, as in Cy7-2 and Cy7-3. Compounds 2 and 3 provide useful scaffolds for targeting of PSMA+ tissues in vivo and should be useful for preparing NIR dye conjugates designed specifically for clinical intraoperative optical imaging devices.

Glutamate carboxypeptidase activity in human skin biopsies as a pharmacodynamic marker for clinical studies.

BACKGROUND: Glutamate excitotoxicity is thought to be involved in the pathogenesis of neurodegenerative disease. One potential source of glutamate is N-acetyl-aspartyl-glutamate (NAAG) which is hydrolyzed to glutamate and N-acetyl-aspartate (NAA) in a reaction catalyzed by glutamate carboxypeptidase (GCP). As a result, GCP inhibition is thought to be beneficial for the treatment of neurodegenerative diseases where excess glutamate is presumed pathogenic. Both pharmacological and genetic inhibition of GCP has shown therapeutic utility in preclinical models and this has led to GCP inhibitors being pursued for the treatment of nervous system disorders in human clinical trials. Specifically, GCP inhibitors are currently being developed for peripheral neuropathy and neuropathic pain. The purpose of this study was to develop a pharmacodynamic (PD) marker assay to use in clinical development. The PD marker will determine the effect of GCP inhibitors on GCP enzymatic activity in human skin as measure of inhibition in peripheral nerve and help predict drug doses required to elicit pharmacologic responses. METHODS: GCP activity was first characterized in both human skin and rat paw pads. GCP activity was then monitored in both rodent paw pads and sciatic nerve from the same animals following peripheral administration of various doses of GCP inhibitor. Significant differences among measurements were determined using two-tailed distribution, equal variance student's t test. RESULTS: We describe for the first time, a direct and quantifiable assay to evaluate GCP enzymatic activity in human skin biopsy samples. In addition, we show that GCP activity in skin is responsive to pharmacological manipulation; GCP activity in rodent paws was inhibited in a dose response manner following peripheral administration of a potent and selective GCP inhibitor. Inhibition of GCP activity in rat paw pads was shown to correlate to inhibition of GCP activity in peripheral nerve. CONCLUSION: Monitoring GCP activity in human skin after administration of GCP inhibitors could be readily used as PD marker in the clinical development of GCP inhibitors. Enzymatic activity provides a simple and direct measurement of GCP activity from tissue samples easily assessable in human subjects.

The antiemetic 5-HT3 receptor antagonist Palonosetron inhibits substance P-mediated responses in vitro and in vivo.

Palonosetron is the only 5-HT(3) receptor antagonist approved for the treatment of delayed chemotherapy-induced nausea and vomiting (CINV) in moderately emetogenic chemotherapy. Accumulating evidence suggests that substance P (SP), the endogenous ligand acting preferentially on neurokinin-1 (NK-1) receptors, not serotonin (5-HT), is the dominant mediator of delayed emesis. However, palonosetron does not bind to the NK-1 receptor. Recent data have revealed cross-talk between the NK-1 and 5HT(3) receptor signaling pathways; we postulated that if palonosetron differentially inhibited NK-1/5-HT(3) cross-talk, it could help explain its efficacy profile in delayed emesis. Consequently, we evaluated the effect of palonosetron, granisetron, and ondansetron on SP-induced responses in vitro and in vivo. NG108-15 cells were preincubated with palonosetron, granisetron, or ondansetron; antagonists were removed and the effect on serotonin enhancement of SP-induced calcium release was measured. In the absence of antagonist, serotonin enhanced SP-induced calcium-ion release. After preincubation with palonosetron, but not ondansetron or granisetron, the serotonin enhancement of the SP response was inhibited. Rats were treated with cisplatin and either palonosetron, granisetron, or ondansetron. At various times after dosing, single neuronal recordings from nodose ganglia were collected after stimulation with SP; nodose ganglia neuronal responses to SP were enhanced when the animals were pretreated with cisplatin. Palonosetron, but not ondansetron or granisetron, dose-dependently inhibited the cisplatin-induced SP enhancement. The results are consistent with previous data showing that palonosetron exhibits distinct pharmacology versus the older 5-HT(3) receptor antagonists and provide a rationale for the efficacy observed with palonosetron in delayed CINV in the clinic.

The discovery and structure-activity relationships of indole-based inhibitors of glutamate carboxypeptidase II.

A series of N-substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acids were synthesized as inhibitors of glutamate carboxypeptidase II (GCPII). Those containing carboxybenzyl or carboxyphenyl groups at the N-position exhibited potent inhibitory activity against GCPII. These indole-based compounds represent the first example of achiral GCPII inhibitors and demonstrate greater tolerance of the GCPII active site for ligands with significant structural difference from the endogenous substrate, N-acetyl-aspartylglutamate.

Palonosetron exhibits unique molecular interactions with the 5-HT3 receptor.

BACKGROUND: Palonosetron is a 5-HT(3)-receptor antagonist (5-HT(3)-RA) that has been shown to be superior to other 5-HT(3)-RAs in phase III clinical trials for the prevention of acute, delayed, and overall chemotherapy-induced nausea and vomiting. The improved clinical efficacy of palonosetron may be due, in part, to its more potent binding and longer half-life. However, these attributes alone are not sufficient to explain the results with palonosetron. We sought to elucidate additional differences among 5-HT(3)-RAs that could help explain the observations in the clinic. METHODS: Receptor site saturation binding experiments were performed with [3H] palonosetron, [3H] granisetron, and [3H] ondansetron to obtain the corresponding Scatchard analyses and Hill coefficients. Diagnostic equilibrium binding experiments and kinetic dissociation experiments were conducted to examine competitive versus potential allosteric interactions between ondansetron, granisetron and palonosetron and the 5-HT(3) receptor. Finally, the long-term effect of the three antagonists on receptor function as measured by Ca2+ influx in HEK 293 cells expressing the 5-HT(3)-receptor was compared. RESULTS: Analyses of binding isotherms using both Scatchard and Hill plots suggested positive cooperativity for palonosetron and simple bimolecular binding for both granisetron and ondansetron. Equilibrium diagnostic tests discriminated differential effects of palonosetron on [3H] ligand binding indicating that palonosetron was an allosteric antagonist whereas granisetron and ondansetron were competitive antagonists. Using dissociation rate strategies, palonosetron was shown to be an allosteric modifier that accelerated the rate of dissociation from the receptor of both granisetron and ondansetron. Differences in the binding mode of palonosetron to the 5-HT(3) receptor were shown to have an impact on receptor function. In these experiments, cells were incubated with each antagonist, followed by infinite dilutions and dissociation for 2.5 h; cells previously incubated with either granisetron or ondansetron showed calcium-ion influx similar to control cells that had not been exposed to a 5-HT(3) receptor antagonist. In contrast, substantial inhibition of calcium-ion influx was observed in cells that had been incubated with palonosetron. CONCLUSIONS: Palonosetron exhibited allosteric binding and positive cooperativity when binding to the 5-HT(3) receptor. Palonosetron also triggered functional effects that persisted beyond its binding to the 5-HT(3) receptor at the cell surface. Differences in binding and effects on receptor function may be relevant to the unique beneficial actions of palonosetron. To our knowledge, this is the first report showing palonosetron's interaction with the 5-HT(3) receptor at the molecular level, clearly differentiating it from other 5-HT(3)-RAs.

The Low-Affinity Binding of Second Generation Radiotracers Targeting TSPO is Associated with a Unique Allosteric Binding Site.

[11C]-PK11195 (PK11195) has been widely used with positron emission tomography (PET) to assess levels of the translocator protein 18 kDa (TSPO) as a marker of neuroinflammation. Recent ligands, such as [11C]-PBR28 and [11C]-DPA713, have improved signal-to-noise ratio and specificity for TSPO over PK11195. However, these second generation radiotracers exhibit binding differences due to a single polymorphism (rs6971) that leads to three genotypes: C/C, C/T and T/T associated with high, mixed and low binding affinities, respectively. Here we report that [3H]-DPA-713 in the presence of cholesterol or PK11195 has an accelerated dissociation rate from TSPO in platelets isolated from individuals with the T/T genotype. This allosteric interaction was not observed in platelets isolated from individuals with the C/C or C/T genotype. The results provide a molecular rationale for low binding affinity of T/T TSPO and further support the exclusion of these subjects from PET imaging studies using second generation TSPO ligands.

Creating a comprehensive research strategy for cutaneous neurofibromas.

OBJECTIVE: Outside of procedural-based methods, there are currently no established medical treatments for cutaneous neurofibroma (cNF), which afflict up to 99% of patients with NF1. Further, adult patients often report cNF are the greatest burden of living with NF1. The Neurofibromatosis Therapeutic Acceleration Program (NTAP) launched a think tank to address core questions to facilitate development of effective therapeutics for cNF in people with NF1. METHODS: Experts (with and without explicit experience with NF1 or cNF) from multiple scientific and medical disciplines, representing the ranks of academia, industry, and government agencies, were invited to become a member of a team addressing a specific subset of questions pertinent to cNF. Teams met monthly to review published and unpublished materials, and created summaries about the material known and unknown that may influence therapeutic development for cNF. Teams prioritized questions and organized supporting data, which was presented to the entire body of experts by each team at a research summit. RESULTS: Four themes were identified as being relevant to creating a comprehensive research strategy for cNF: (1) establishing definitions of cNF, (2) determining the biology of cNF with respect to tumor initiation, progression, and maintenance, (3) outlining the factors that guide therapies development, and (4) defining core considerations for clinical trials design and optimization for cNF. CONCLUSION: Considerations and key questions for each of the thematic areas were identified and provided basis for a request for applications launched by NTAP focused on cNF and are described in the accompanying articles of this supplement.

Pharmacological and genomic profiling of neurofibromatosis type 1 plexiform neurofibroma-derived schwann cells.

Neurofibromatosis type I (NF1) is an autosomal dominant genetic condition characterized by peripheral nervous system tumors (PNSTs), including plexiform neurofibromas (pNFs) that cause nerve dysfunction, deformity, pain damage to adjacent structures, and can undergo malignant transformation. There are no effective therapies to prevent or treat pNFs. Drug discovery efforts are slowed by the 'benign' nature of the Schwann cells that are the progenitor cells of pNF. In this work we characterize a set of pNF-derived cell lines at the genomic level (via SNP Arrays, RNAseq, and Whole Exome- Sequencing), and carry out dose response-based quantitative high-throughput screening (qHTS) with a collection of 1,912 oncology-focused compounds in a 1536-well microplate cell proliferation assays. Through the characterization and screening of NF1-/-, NF1+/+ and NF1+/- Schwann cell lines, this resource introduces novel therapeutic avenues for the development for NF1 associated pNF as well as all solid tumors with NF1 somatic mutations. The integrated data sets are openly available for further analysis at http://www.synapse.org/pnfCellCulture.

Development of a primary microglia screening assay and its use to characterize inhibition of system xc- by erastin and its analogs.

The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System xC- cystine/glutamate antiporter (SXC), a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS) and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC50 values in agreement with previous studies using established cell lines.

Structural optimization of thiol-based inhibitors of glutamate carboxypeptidase II by modification of the P1' side chain.

A series of thiol-based inhibitors containing a benzyl moiety at the P1' position have been synthesized and tested for their abilities to inhibit glutamate carboxypeptidase II (GCP II). 3-(2-Carboxy-5-mercaptopentyl)benzoic acid 6c was found to be the most potent inhibitor with an IC(50) value of 15 nM, 6-fold more potent than 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), a previously discovered, orally active GCP II inhibitor. Subsequent SAR studies have revealed that the phenoxy and phenylsulfanyl analogues of 6c, 3-(1-carboxy-4-mercaptobutoxy)benzoic acid 26a and 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid 26b, also possess potent inhibitory activities toward GCP II. In the rat chronic constriction injury (CCI) model of neuropathic pain, compounds 6c and 26a significantly reduced hyperalgesia following oral administration (1.0 mg/kg/day).

Allosteric Glutaminase Inhibitors Based on a 1,4-Di(5-amino-1,3,4-thiadiazol-2-yl)butane Scaffold.

A series of allosteric kidney-type glutaminase (GLS) inhibitors were designed and synthesized using 1,4-di(5-amino-1,3,4-thiadiazol-2-yl)butane as a core scaffold. A variety of modified phenylacetyl groups were incorporated into the 5-amino group of the two thiadiazole rings in an attempt to facilitate additional binding interactions with the allosteric binding site of GLS. Among the newly synthesized compounds, 4-hydroxy-N-[5-[4-[5-[(2-phenylacetyl)amino]-1,3,4-thiadiazol-2-yl]butyl]-1,3,4-thiadiazol-2-yl]-benzeneacetamide, 2m, potently inhibited GLS with an IC50 value of 70 nM, although it did not exhibit time-dependency as seen with CB-839. Antiproliferative effects of 2m on human breast cancer lines will be also presented in comparison with those observed with CB-839.

FOLH1/GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities.

Recent gene-profiling analyses showed significant upregulation of the folate hydrolase (FOLH1) gene in the affected intestinal mucosa of patients with inflammatory bowel disease (IBD). The FOLH1 gene encodes a type II transmembrane glycoprotein termed glutamate carboxypeptidase II (GCPII). To establish that the previously reported increased gene expression was functional, we quantified the glutamate carboxypeptidase enzymatic activity in 31 surgical specimens and report a robust 2.8- to 41-fold increase in enzymatic activity in the affected intestinal mucosa of IBD patients compared with an uninvolved area in the same patients or intestinal mucosa from healthy controls. Using a human-to-mouse approach, we next showed a similar enzymatic increase in two well-validated IBD murine models and evaluated the therapeutic effect of the potent FOLH1/ GCPII inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) (IC50 = 300 pM). In the dextran sodium sulfate (DSS) colitis model, 2-PMPA inhibited the GCPII activity in the colonic mucosa by over 90% and substantially reduced the disease activity. The significance of the target was confirmed in FOLH1-/- mice who exhibited resistance to DSS treatment. In the murine IL-10-/- model of spontaneous colitis, daily 2-PMPA treatment also significantly reduced both macroscopic and microscopic disease severity. These results provide the first evidence of FOLH1/GCPII enzymatic inhibition as a therapeutic option for IBD.

Selective CNS Uptake of the GCP-II Inhibitor 2-PMPA following Intranasal Administration.

Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC0-t, i.n./AUC0-t, i.p. = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC0-t, i.n./AUC0-t, i.p. = 67), cortex (AUC0-t, i.n./AUC0-t, i.p. = 46) and cerebellum (AUC0-t, i.n./AUC0-t, i.p. = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity ex-vivo confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support intranasal delivery of 2-PMPA to deliver therapeutic concentrations in the brain and may facilitate its clinical development.

Cambinol, a novel inhibitor of neutral sphingomyelinase 2 shows neuroprotective properties.

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer's disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (Ki = 7 µM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 ß-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.

Inhibition of substance P-mediated responses in NG108-15 cells by netupitant and palonosetron exhibit synergistic effects.

Netupitant is a potent and selective NK(1) receptor antagonist under development in combination with a fixed dose of palonosetron for the prevention of chemotherapy induced nausea and vomiting. Palonosetron is a 5-HT(3) receptor antagonist approved for both the prevention of acute and delayed chemotherapy induced nausea and vomiting after moderately emetogenic chemotherapy. Accumulating evidence suggests that substance P (SP), a ligand acting largely on tachykinin (NK(1)) receptors, is the dominant mediator of delayed emesis. Interestingly, palonosetron does not bind to the NK(1) receptor so that the mechanism behind palonosetron's unique efficacy against delayed emesis is not clear. Palonosetron exhibits a distinct ability among 5-HT(3) receptor antagonists to inhibit crosstalk between NK(1) and 5-HT(3) receptor signaling pathways. The objective of the current work was to determine if palonosetron's ability to inhibit receptor signaling crosstalk would influence netupitant's inhibition of the SP-mediated response when the two drugs are dosed together. We first studied the inhibition of SP-induced Ca(2+) mobilization in NG108-15 cells by palonosetron, ondansetron and granisetron. Unexpectedly, in the absence of serotonin, palonosetron inhibited the SP-mediated dose response 15-fold; ondansetron and granisetron had no effect. Netupitant also dose-dependently inhibited the SP response as expected from an NK1 receptor antagonist. Importantly, when both palonosetron and netupitant were present, they exhibited an enhanced inhibition of the SP response compared to either of the two antagonists alone. The results further confirm palonosetron's unique pharmacology among 5-HT(3) receptor antagonists and suggest that it can enhance the prevention of delayed emesis provided by NK(1) receptor antagonists.