Establishment and characterization of patient-derived xenograft of a rare pediatric anaplastic pleomorphic xanthoastrocytoma (PXA) bearing a CDC42SE2-BRAF fusion.

Pleomorphic xanthoastrocytoma (PXA) is a rare subset of primary pediatric glioma with 70% 5-year disease free survival. However, up to 20% of cases present with local recurrence and malignant transformation into more aggressive type anaplastic PXA (AXPA) or glioblastoma. The understanding of disease etiology and mechanisms driving PXA and APXA are limited, and there is no standard of care. Therefore, development of relevant preclinical models to investigate molecular underpinnings of disease and to guide novel therapeutic approaches are of interest. Here, for the first time we established, and characterized a patient-derived xenograft (PDX) from a leptomeningeal spread of a patient with recurrent APXA bearing a novel CDC42SE2-BRAF fusion. An integrated -omics analysis was conducted to assess model fidelity of the genomic, transcriptomic, and proteomic/phosphoproteomic landscapes. A stable xenoline was derived directly from the patient recurrent tumor and maintained in 2D and 3D culture systems. Conserved histology features between the PDX and matched APXA specimen were maintained through serial passages. Whole exome sequencing (WES) demonstrated a high degree of conservation in the genomic landscape between PDX and matched human tumor, including small variants (Pearson's r = 0.794-0.839) and tumor mutational burden (~ 3 mutations/MB). Large chromosomal variations including chromosomal gains and losses were preserved in PDX. Notably, chromosomal gain in chromosomes 4-9, 17 and 18 and loss in the short arm of chromosome 9 associated with homozygous 9p21.3 deletion involving CDKN2A/B locus were identified in both patient tumor and PDX sample. Moreover, chromosomal rearrangement involving 7q34 fusion; CDC42SE-BRAF t (5;7) (q31.1, q34) (5:130,721,239, 7:140,482,820) was identified in the PDX tumor, xenoline and matched human tumor. Transcriptomic profile of the patient's tumor was retained in PDX (Pearson r = 0.88) and in xenoline (Pearson r = 0.63) as well as preservation of enriched signaling pathways (FDR Adjusted P < 0.05) including MAPK, EGFR and PI3K/AKT pathways. The multi-omics data of (WES, transcriptome, and reverse phase protein array (RPPA) was integrated to deduce potential actionable pathways for treatment (FDR < 0.05) including KEGG01521, KEGG05202, and KEGG05200. Both xenoline and PDX were resistant to the MEK inhibitors trametinib or mirdametinib at clinically relevant doses, recapitulating the patient's resistance to such treatment in the clinic. This set of APXA models will serve as a preclinical resource for developing novel therapeutic regimens for rare anaplastic PXAs and pediatric high-grade gliomas bearing BRAF fusions.

Precision Medicine Highlights Dysregulation of the CDK4/6 Cell Cycle Regulatory Pathway in Pediatric, Adolescents and Young Adult Sarcomas.

Despite improved therapeutic and clinical outcomes for patients with localized diseases, outcomes for pediatric and AYA sarcoma patients with high-grade or aggressive disease are still relatively poor. With advancements in next generation sequencing (NGS), precision medicine now provides a strategy to improve outcomes in patients with aggressive disease by identifying biomarkers of therapeutic sensitivity or resistance. The integration of NGS into clinical decision making not only increases the accuracy of diagnosis and prognosis, but also has the potential to identify effective and less toxic therapies for pediatric and AYA sarcomas. Genome and transcriptome profiling have detected dysregulation of the CDK4/6 cell cycle regulatory pathway in subpopulations of pediatric and AYA OS, RMS, and EWS. In these patients, the inhibition of CDK4/6 represents a promising precision medicine-guided therapy. There is a critical need, however, to identify novel and promising combination therapies to fight the development of resistance to CDK4/6 inhibition. In this review, we offer rationale and perspective on the promise and challenges of this therapeutic approach.

Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors.

Establishment of clinically annotated, molecularly characterized, patient-derived xenografts (PDXs) from treatment-naïve and pretreated patients provides a platform to test precision genomics-guided therapies. An integrated multi-OMICS pipeline was developed to identify cancer-associated pathways and evaluate stability of molecular signatures in a panel of pediatric and AYA PDXs following serial passaging in mice. Original solid tumor samples and their corresponding PDXs were evaluated by whole-genome sequencing, RNA-seq, immunoblotting, pathway enrichment analyses, and the drug−gene interaction database to identify as well as cross-validate actionable targets in patients with sarcomas or Wilms tumors. While some divergence between original tumor and the respective PDX was evident, majority of alterations were not functionally impactful, and oncogenic pathway activation was maintained following serial passaging. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response in osteosarcoma PDXs with pertinent molecular signatures. Inhibition of CDK4/6 or BETs decreased osteosarcoma PDX growth (two-way ANOVA, p < 0.05) confirming mechanistic involvement in growth. Linking patient treatment history with molecular and efficacy data in PDX will provide a strong rationale for targeted therapy and improve our understanding of which therapy is most beneficial in patients at diagnosis and in those already exposed to therapy.

Development of CD133 Targeting Multi-Drug Polymer Micellar Nanoparticles for Glioblastoma - In Vitro Evaluation in Glioblastoma Stem Cells.

PURPOSE: Glioblastoma (GBM) is a malignant brain tumor with a poor long-term prognosis due to recurrence from highly resistant GBM cancer stem cells (CSCs), for which the current standard of treatment with temozolomide (TMZ) alone will unlikely produce a viable cure. In addition, CSCs regenerate rapidly and overexpress methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to resistance. The objective of this research was to apply the concepts of nanotechnology to develop a multi-drug therapy, TMZ and idasanutlin (RG7388, a potent mouse double minute 2 (MDM2) antagonist), loaded in functionalized nanoparticles (NPs) that target the GBM CSC subpopulation, reduce the cell viability and provide possibility of in vivo preclinical imaging. METHODS: Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed by a double emulsion technique loading TMZ and/or RG7388. The NPs were covalently bound to a 15-nucleotide base-pair CD133 aptamer to target the CD133 antigen expressed on the surfaces of GBM CSCs. For diagnostic functionality, the NPs were labelled with radiotracer Zirconium-89 (89Zr). RESULTS: NPs maintained size range less than 100 nm, a low negative charge and exhibited the ability to target and kill the CSC subpopulation when TMZ and RG7388 were used in combination. The targeting function of CD133 aptamer promoted killing in GBM CSCs providing impetus for further development of targeted nanosystems for localized therapy in future in vivo models. CONCLUSIONS: This work has provided a potential clinical application for targeting GBM CSCs with simultaneous diagnostic imaging.

Systems Biology Approach Identifies Prognostic Signatures of Poor Overall Survival and Guides the Prioritization of Novel BET-CHK1 Combination Therapy for Osteosarcoma.

Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease.

Small-Molecule Covalent Modification of Conserved Cysteine Leads to Allosteric Inhibition of the TEAD⋠Yap Protein-Protein Interaction.

The Hippo pathway coordinates extracellular signals onto the control of tissue homeostasis and organ size. Hippo signaling primarily regulates the ability of Yap1 to bind and co-activate TEA domain (TEAD) transcription factors. Yap1 tightly binds to TEAD4 via a large flat interface, making the development of small-molecule orthosteric inhibitors highly challenging. Here, we report small-molecule TEAD⋅Yap inhibitors that rapidly and selectively form a covalent bond with a conserved cysteine located within the unique deep hydrophobic palmitate-binding pocket of TEADs. Inhibition of TEAD4 binding to Yap1 by these compounds was irreversible and occurred on a longer time scale. In mammalian cells, the compounds formed a covalent complex with TEAD4, inhibited its binding to Yap1, blocked its transcriptional activity, and suppressed expression of connective tissue growth factor. The compounds inhibited cell viability of patient-derived glioblastoma spheroids, making them suitable as chemical probes to explore Hippo signaling in cancer.

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model.

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.

Brain Exposure of Two Selective Dual CDK4 and CDK6 Inhibitors and the Antitumor Activity of CDK4 and CDK6 Inhibition in Combination with Temozolomide in an Intracranial Glioblastoma Xenograft.

Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. Targeting the CDK4/CDK6-cyclin D1-Rb-p16/ink4a pathway using a potent CDK4 and CDK6 kinase inhibitor has potential for treating primary central nervous system tumors such as glioblastoma and some peripheral tumors with high incidence of brain metastases. We compared central nervous system exposures of two orally bioavailable CDK4 and CDK6 inhibitors: abemaciclib, which is currently in advanced clinical development, and palbociclib (IBRANCE; Pfizer), which was recently approved by the U.S. Food and Drug Administration. Abemaciclib antitumor activity was assessed in subcutaneous and orthotopic glioma models alone and in combination with standard of care temozolomide (TMZ). Both inhibitors were substrates for xenobiotic efflux transporters P-glycoprotein and breast cancer resistant protein expressed at the blood-brain barrier. Brain Kp,uu values were less than 0.2 after an equimolar intravenous dose indicative of active efflux but were approximately 10-fold greater for abemaciclib than palbociclib. Kp,uu increased 2.8- and 21-fold, respectively, when similarly dosed in P-gp-deficient mice. Abemaciclib had brain area under the curve (0-24 hours) Kp,uu values of 0.03 in mice and 0.11 in rats after a 30 mg/kg p.o. dose. Orally dosed abemaciclib significantly increased survival in a rat orthotopic U87MG xenograft model compared with vehicle-treated animals, and efficacy coincided with a dose-dependent increase in unbound plasma and brain exposures in excess of the CDK4 and CDK6 Ki values. Abemaciclib increased survival time of intracranial U87MG tumor-bearing rats similar to TMZ, and the combination of abemaciclib and TMZ was additive or greater than additive. These data show that abemaciclib crosses the blood-brain barrier and confirm that both CDK4 and CDK6 inhibitors reach unbound brain levels in rodents that are expected to produce enzyme inhibition; however, abemaciclib brain levels are reached more efficiently at presumably lower doses than palbociclib and are potentially on target for a longer period of time.

Longitudinal Bioluminescence Imaging of Primary Versus Abdominal Metastatic Tumor Growth in Orthotopic Pancreatic Tumor Models in NSG Mice.

OBJECTIVES: The purpose of the present study was to develop and validate noninvasive bioluminescence imaging methods for differentially monitoring primary and abdominal metastatic tumor growth in mouse orthotopic models of pancreatic cancer. METHODS: A semiautomated maximum entropy segmentation method was implemented for the primary tumor region of interest, and a rule-based method for manually drawing a region of interest for the abdominal metastatic region was developed for monitoring tumor growth in orthotopic models of pancreatic cancer. The 2 region-of-interest methods were validated by having 2 observers independently segment Panc-1 tumors, and the results were compared with the number of mesenteric lymph node nodules and histopathologic assessment of liver metastases. The findings were extended to orthotopic tumors of the more metastatic MIA PaCa-2 and AsPC-1 cells where separate groups of animals were implanted with different numbers of cells. RESULTS: The results demonstrated that the segmentation methods were highly reliable, reproducible, and robust and allowed statistically significant discrimination in the growth rates of primary and abdominal metastatic tumors of different cell lines implanted with different numbers of cells. CONCLUSIONS: The present results demonstrate that primary tumors and abdominal metastatic foci in orthotopic pancreatic cancer models can be reliably quantified separately and noninvasively over time with bioluminescence imaging.

Design, synthesis, and evaluation of curcumin-derived arylheptanoids for glioblastoma and neuroblastoma cytotoxicity.

Using an innovative approach toward multiple carbon-carbon bond-formations that relies on the multifaceted catalytic properties of titanocene complexes we constructed a series of C1-C7 analogs of curcumin for evaluation as brain and peripheral nervous system anti-cancer agents. C2-Arylated analogs proved efficacious against neuroblastoma (SK-N-SH & SK-N-FI) and glioblastoma multiforme (U87MG) cell lines. Similar inhibitory activity was also evident in p53 knockdown U87MG GBM cells. Furthermore, lead compounds showed limited growth inhibition in vitro against normal primary human CD34+hematopoietic progenitor cells. Taken together, the present findings indicate that these curcumin analogs are viable lead compounds for the development of new central and peripheral nervous system cancer chemotherapeutics with the potential for little effects on normal hematopoietic progenitor cells.

GluK1 antagonists from 6-(carboxy)phenyl decahydroisoquinoline derivatives. SAR and evaluation of a prodrug strategy for oral efficacy in pain models.

The synthesis and structure-activity relationship of decahydroisoquinoline derivatives with various benzoic acid substitutions as GluK1 antagonists are described. Potent and selective antagonists were selected for a tailored prodrug approach in order to facilitate the evaluation of the new compounds in pain models after oral administration. Several diester prodrugs allowed for acceptable amino acid exposure and moderate efficacy in vivo.

GluK1 antagonists from 6-(tetrazolyl)phenyl decahydroisoquinoline derivatives: in vitro profile and in vivo analgesic efficacy.

We have explored the decahydroisoquinoline scaffold, bearing a phenyl tetrazole, as GluK1 antagonists with potential as oral analgesics. We have established the optimal linker atom between decahydroisoquinoline and phenyl rings and demonstrated an improvement of both the affinity for the GluK1 receptor and the selectivity against the related GluA2 receptor with proper phenyl substitution. In this Letter, we also disclose in vivo data that led to the discovery of LY545694·HCl, a compound with oral efficacy in two persistent pain models.

Combining hedgehog signaling inhibition with focal irradiation on reduction of pancreatic cancer metastasis.

Pancreatic cancer often presents in advanced stages and is unresponsive to conventional treatments. Thus, the need to develop novel treatment strategies for pancreatic cancer has never been greater. Here, we report that combination of focal irradiation with hedgehog (Hh) signaling inhibition exerts better than additive effects on reducing metastases. In an orthotopic model, we found that focal irradiation alone effectively reduced primary tumor growth but did not significantly affect metastasis. We hypothesized that cancer stem cells (CSC) of pancreatic cancer are responsible for the residual tumors following irradiation, which may be regulated by Hh signaling. To test our hypothesis, we showed that tumor metastasis in our model was accompanied by increased expression of CSC cell surface markers as well as Hh target genes. We generated tumor spheres from orthotopic pancreatic and metastatic tumors, which have elevated levels of CSC markers relative to the parental cells and elevated expression of Hh target genes. Irradiation of tumor spheres further elevated CSC cell surface markers and increased Hh target gene expression. Combination of Hh signaling inhibition with radiation had more than additive effects on tumor sphere regeneration in vitro. This phenotype was observed in two independent cell lines. In our orthotopic animal model, focal radiation plus Hh inhibition had more than additive effects on reducing lymph node metastasis. We identified several potential molecules in mediating Hh signaling effects. Taken together, our data provide a rationale for combined use of Hh inhibition with irradiation for clinical treatment of patients with pancreatic cancer.

Reproducibility of total choline/water ratios in mouse U87MG xenograft tumors by 1H-MRS.

PURPOSE: To evaluate the reproducibility of the measurement of the total choline-to-water ratio, and the effect of repositioning the subject between scans, using (1) H-magnetic resonance spectroscopy in a mouse U87MG xenograft model. MATERIALS AND METHODS: In vivo single-voxel MR spectra at 7T from xenograft tumors were obtained using both a water-suppressed and a nonwater-suppressed point-resolved spectroscopy (PRESS) sequence. Reproducibility of the total choline/water ratio was evaluated under the conditions of immediate rescan with no change in position of the animal or voxel, immediate reposition, and reposition after 1 or 7 days. RESULTS: Total choline-to-water ratios in U87MG tumor xenografts averaged ≈0.018 across all of the groups. The average percent difference between the two scans in each condition was always less than ≈3.0%, and the coefficient of variation was always less than ≈12%. Bias was unrelated to the testing condition and relatively negligible in magnitude (<3%). Due to heteroscedasticity in the ratios, the limits of agreement were calculated after log transformation of the data and ranged from ≈12% when animals were maintained in the same position and immediately rescanned to ≈52% when the two scans were 7 days apart. CONCLUSION: The total choline-to-water ratio provides a reproducible measure of choline-containing metabolites in subcutaneous U87MG xenograft tumors in mice.

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas.

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.

Effects of kappa opioid receptor agonists on attention as assessed by a 5-choice serial reaction time task in rats.

In humans, kappa opioid receptor agonists produce, among other effects, sedation and difficulty concentrating, suggesting that they may disrupt attention. The purpose of the present studies was therefore to evaluate the effects of kappa opioid receptor agonists on attention as assessed by a 5-choice serial reaction time task in rats. The kappa opioid receptor agonists (+)-U69,593 (0.1-0.56mg/kg), (+/-)-U50,488 (1.0-5.6mg/kg) and racemic GR89,696 (0.0003-0.01mg/kg) all produced dose-related decreases in the percentage of trials terminated by a correct or incorrect response and increases in the percentage of omissions. In contrast, the peripherally restricted opioid agonist ICI-204,448 was ineffective (1.0-10mg/kg). Moreover, the effects of GR89,696 were stereoselective in that (R)-GR89,696 was approximately equipotent to racemic GR89,696 and approximately 100-fold more potent than (S)-GR89,696. The opioid receptor antagonist naltrexone (0.3-3mg/kg) administered alone had no effects on performance. However, naltrexone, over the dose-range of 0.03-1.0mg/kg, produced a dose-related antagonism of the disruption produced by U69,593 (0.56mg/kg). In contrast, naltrexone, over the dose-range of 0.01-0.3mg/kg produced a dose-related antagonism of morphine (5.6mg/kg). Recent evidence has suggested that kappa opioid receptor agonists decrease dopaminergic and noradrenergic neurotransmission in prefrontal cortex and locus coeruleus. Together with previous findings, the present data indicate that kappa opioid receptor agonists disrupt performance of this attention task by decreasing the probability of responding by specific actions at central kappa opioid receptors, perhaps by decreasing dopaminergic and noradrenergic neurotransmission.

Constitutive deletion of the serotonin-7 (5-HT(7)) receptor decreases electrical and chemical seizure thresholds.

The localization of serotonin-7 (5-HT(7)) receptors and the biological activity of ligands have suggested that 5-HT(7) receptors might be involved in pain, migraine, epilepsy, anxiety, depression, memory, and sleep. In the present study, the potential involvement of 5-HT(7) receptors in epilepsy and other seizure disorders was assessed by comparing the seizures produced by three types of electrical stimulation and three chemical convulsants in 5-HT(7) receptor-deficient (knockout, KO) mice to those seizures observed in wild-type (WT) mice. Thresholds for producing electroshock-induced clonic seizures did not differ between KO versus WT mice. However, thresholds for producing electroshock-induced tonic seizures were significantly lower in KO than in WT mice. Seizures produced by pentylenetetrazole (PTZ, a GABA(A) receptor antagonist), N-methyl-d-aspartate (NMDA, an agonist at NMDA-type glutamate receptors), and cocaine (an inhibitor of monoamine uptake) were also studied. PTZ was more potent in inducing seizures in 5-HT(7) KO mice than in wild-type mice. Likewise, cocaine was more potent in inducing seizures in 5-HT(7) KO than in WT mice; moreover, death resulted from cocaine administration in 5-HT(7) KO mice but not in WT mice. There was a similar trend for NMDA that did not reach statistical significance. The present findings point to the potential for a generalized reduction in seizure threshold with constitutive deletion of the 5-HT(7) receptor gene. Since seizures have not been reported with pharmacological blockade of the receptor, the findings suggest that adaptive changes may play a role in the low seizure thresholds in these mice. In addition, the data suggest that the lower thresholds for seizures produced by diverse mechanisms should be taken into account when interpreting other aspects of the phenotype and behavioral pharmacology of this mouse.

Synergistic interactions between the dual serotonergic, noradrenergic reuptake inhibitor duloxetine and the non-steroidal anti-inflammatory drug ibuprofen in inflammatory pain in rodents.

OBJECTIVES: The present study was undertaken to characterize whether the pharmacologic interaction between duloxetine, a balanced serotonergic and noradrenergic reuptake inhibitor, and the non-steroidal anti-inflammatory drug ibuprofen was simply additive, less than additive, or greater than additive (i.e., synergistic) in preclinical models of visceral and inflammatory pain, specifically acetic acid-induced writhing in mice and carrageenan-induced thermal hyperalgesia and mechanical allodynia in rats. METHODS: In the writhing test, male CF-1 mice were injected intraperitoneally with 0.55% acetic acid and 5 min later the number of writhes was counted over a 5-min period. In the carrageenan models, male Sprague-Dawley rats were injected with a 1.5% carrageenan solution into the ventral surface of the hind paw; hypersensitivity to thermal and mechanical stimuli was subsequently evaluated 2h post-carrageenan. RESULTS: Vehicle or a dose of duloxetine alone (1-100 mg/kg), ibuprofen alone (10-300 mg/kg), or duloxetine and ibuprofen in combination in a dose-ratio of 1:10 duloxetine:ibuprofen were orally administered 30 or 60 min before testing. Isobolographic analysis of the effects of duloxetine in combination with ibuprofen revealed a significant synergistic (greater than additive) interaction between duloxetine and ibuprofen both for reducing acetic acid-induced writhing and carrageenan-induced thermal hyperalgesia, but were additive for reversing mechanical allodynia. CONCLUSIONS: Our data indicate that duloxetine and ibuprofen have synergistic efficacy in a visceral and an inflammatory pain model in rodents, and suggest that duloxetine and ibuprofen in combination may provide a useful approach to the clinical treatment of persistent pain, particularly inflammation-related pain.

Effects of antiepileptic drugs on learning as assessed by a repeated acquisition of response sequences task in rats.

Patients with epilepsy can have impaired cognitive abilities. Antiepileptic drugs (AEDs) may contribute to the cognitive deficits observed in patients with epilepsy, and have been shown to induce cognitive impairments in healthy individuals. However, there are few systematic data on the effects of AEDs on specific cognitive domains. We have previously demonstrated that a number of AEDs can impair working memory and attention. The purpose of the present study was to evaluate the effects of AEDs on learning as measured by a repeated acquisition of response sequences task in nonepileptic rats. The GABA-related AEDs phenobarbital and chlordiazepoxide significantly disrupted performance by shifting the learning curve to the right and increasing errors, whereas tiagabine and valproate did not. The sodium channel blockers carbamazepine and phenytoin suppressed responding at higher doses, whereas lamotrigine shifted the learning curve to the right and increased errors, and topiramate was without significant effect. Levetiracetam also shifted the learning curve to the right and increased errors. The disruptions produced by triazolam, chlordiazepoxide, lamotrigine, and levetiracetam were qualitatively similar to the effects of the muscarinic cholinergic receptor antagonist scopolamine. The present results indicate that AEDs can impair learning, but there are differences among AEDs in the magnitude of the disruption in nonepileptic rats, with drugs that enhance GABA receptor function and some that block sodium channels producing the most consistent impairment of learning.