Characterization of murine CEACAM1 in vivo reveals low expression on CD8+ T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects in vivo. Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles. We sought to investigate CEACAM1 expression on mouse tumor and immune cells, characterize CC1 mAb binding, and evaluate CC1 in syngeneic mouse oncology models as a monotherapy and in combination with an anti-PD-1 mAb. CEACAM1 expression was observed at high levels on neutrophils, NK cells and myeloid-derived suppressor cells (MDSCs), while the expression on tumor-infiltrating CD8+ T cells was low. Unexpectedly, rather than blocking, CC1 facilitated binding of soluble CEACAM1 to CEACAM1 expressing cells. No anti-tumor effects were observed in CT26, MBT2 or A20 models when tested up to 30 mg/kg dose, a dose that was estimated to achieve >90% target engagement in vivo. Taken together, tumor infiltrating CD8+ T cells express low levels of CEACAM1 and CC1 Ab mediates no or minimal anti-tumor effects in vivo, as a monotherapy or in combination with anti-PD-1 treatment.

Levomilnacipran Pharmacokinetics in Healthy Volunteers Versus Patients with Major Depressive Disorder and Implications for Norepinephrine and Serotonin Reuptake Inhibition.

PURPOSE: Levomilnacipran, a selective serotonin (5-HT) and norepinephrine (NE) reuptake inhibitor, is approved for the treatment of major depressive disorder (MDD) in adults. The objectives of this investigation were to characterize the pharmacokinetic (PK) parameters of levomilnacipran in healthy subjects and in patients with MDD and to compare the plasma concentrations observed at clinically effective doses (40-120 mg daily) in MDD patients versus in vitro inhibitory concentration values for NE and 5-HT transporters. METHODS: Data from 2 trials were analyzed: a Phase I trial (healthy volunteers received a single dose of levomilnacipran extended-release capsule [ER; 25, 50, or 100 mg], escalating multiple doses of levomilnacipran ER [25-300 mg once daily], or placebo); and a Phase III trial (adults with MDD received a fixed dose of levomilnacipran ER [40, 80, or 120 mg once daily for 8 weeks]). Plasma samples of participants were assayed to determine levomilnacipran concentrations, and PK analyses were performed. Unbound plasma concentrations of levomilnacipran in MDD patients were estimated, and inhibitory concentration values were determined by curve fitting of the in vitro data. FINDINGS: Cmax and AUC were dose proportional after single dosing (25-100 mg) and multiple dosing (across the 25-300 mg dose range) of levomilnacipran ER in healthy subjects. Dose-proportional steady-state Cmax (93, 180, and 297 ng/mL) and AUC0-τ (1520, 2935, and 4799 ng*h/mL) were also observed in patients with MDD who received levomilnacipran ER (40, 80, and 120 mg daily). Tmax was ~6 hours and was similar after single and multiple oral doses of levomilnacipran ER. Estimates of levomilnacipran concentration at 50%, 80%, and 90% inhibition were 19, 91, and 237 nM, respectively, for the 5-HT transporter, and 10, 41, and 92 nM for the NE transporter. Average unbound plasma concentrations for levomilnacipran in MDD patients treated with levomilnacipran ER 40, 80, or 120 mg daily exceeded the estimated concentration at 80% and 90% inhibition for 5-HT and NE. IMPLICATIONS: Levomilnacipran PK was dose proportional after single and multiple dosing and was similar between healthy subjects and patients with MDD. Steady-state unbound plasma concentrations of levomilnacipran across the approved dose range (40, 80, and 120 mg daily) in MDD patients were estimated to be comparable or greater than the concentrations that inhibited reuptake of NE and 5-HT by >90% and >80%, respectively, in vitro.

Effects of milnacipran on cognitive flexibility following chronic stress in rats.

Cognitive dysfunction is a component of affective disorders, including depression. Chronic stress is a risk factor for depression, and we have shown that exposing rats to chronic unpredictable stress (CUS) induces a deficit of cognitive flexibility, the ability to modify behavior based on feedback from a changing environment. Deficits of cognitive flexibility, measured by extra-dimensional set-shifting on the Attentional Set-shifting Test (AST), are consistent with dysregulation of prefrontal cortical function, also characteristic of depression. We have shown that increasing norepinephrine in the medial prefrontal cortex facilitated set-shifting, and chronic treatment with the selective norepinephrine reuptake blocker, desipramine, restored cognitive flexibility in rats that had been compromised by CUS. Serotonin reuptake blockade also prevented CUS-induced deficits in cognitive flexibility, suggesting a role for both monoamines in this process. Milnacipran is a serotonin-norepinephrine reuptake inhibitor (SNRI) with moderate preference for blocking norepinephrine reuptake. In this study, we tested the effects of chronic milnacipran treatment on cognitive set-shifting after CUS. Male Sprague-Dawley rats were treated chronically by minipump with milnacipran (30 mg/kg/day), the positive control drug, desipramine (5mg/kg/day), or vehicle, and exposed to CUS or unstressed control conditions. For CUS, a different acute stressor was presented daily for 14 days. On Day 17, rats were tested on the AST. Consistent with previous results, CUS impaired cognitive set-shifting. Further, chronic treatment with either milnacipran or desipramine preserved cognitive flexibility after CUS, suggesting that milnacipran may have efficacy in the management of cognitive dysfunction as a component of stress-related illnesses, including fibromyalgia and depression.

Faster onset and dopamine transporter selectivity predict stimulant and reinforcing effects of cocaine analogs in squirrel monkeys.

Although the behavioral-stimulant and reinforcing effects of cocaine and related psychomotor stimulants have been attributed to their actions at the dopamine transporter (DAT), the reinforcing effectiveness of these compounds varies. The properties that confer these differences are important considerations when developing agonist pharmacotherapies for the treatment of stimulant abuse. The present studies focused on the time course of action and pharmacological specificity of six 3-phenyltropane analogs of cocaine (RTI-112, RTI-126, RTI-150, RTI-171, RTI-177, and RTI-336) by observing their behavioral-stimulant, neurochemical, and reinforcing effects in squirrel monkeys. The faster-onset analogs (RTI-126, RTI-150, and RTI-336), and one of the slower-onset DAT selective analogs (RTI-177 and RTI-171) produced behavioral-stimulant effects, while the slower-onset nonselective analog RTI-112 did not. The time to the peak behavioral-stimulant effect and the peak caudate dopamine levels was strongly correlated, but the area under the curve of the time course of behavioral-stimulant effect and dopamine levels was not correlated. These results suggest that the rate of onset plays a more important role than duration of action in the stimulant effect of these analogs. In addition, the slower-onset nonselective analog (RTI-112) clearly did not exhibit any reinforcing effects while the faster-onset nonselective (RTI-126) and all the DAT-selective analogs showed robust reinforcing effects (RTI-150, and RTI-177) or showed trends towards reinforcing effects (RTI-336 and RTI-171). Hence, there was a general trend for compounds that had a faster onset and/or DAT selectivity to produce significant behavioral-stimulant and reinforcing effects.

Ethanol-induced regulation of GABA-A subunit mRNAs in prefrontal fields of cynomolgus monkeys.

BACKGROUND: Recent evidence indicates that functional impairment of the orbital and medial fields of the prefrontal cortex may underlie the deficits in executive control of behavior that characterize addictive disorders, including alcohol addiction. Moreover, previous studies have indicated that alcohol alters GABA neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting the GABA(A) receptor complex. Given that GABAergic transmission has an integral role in cortical processing, influencing local and interregional communication, understanding alcohol-induced alterations in GABA(A) receptors in prefrontal fields of the primate brain may provide insight into the functional impairment of these brain regions in the alcohol-addicted state and extend our understanding of the molecular consequences of long-term use in these critical brain regions. METHODS AND RESULTS: To address this problem, the effects of chronic ethanol self-administration in male cynomolgus monkeys on GABA(A) receptor subunit mRNA expression was studied in 3 frontal cortical fields: orbitofrontal cortex (OFC; area 13), anterior cingulate cortex (ACC; area 24), and the dorsolateral prefrontal cortex (DLPFC; area 46). Quantitative polymerase chain reaction revealed significant alterations in GABA(A) subunit mRNA expression in the OFC and DLPFC but not in the ACC. Specifically, expression of the alpha2, alpha4, beta1, beta3, and gamma1 to gamma3 subunit mRNAs was significantly less in the OFC, whereas the expression of beta1, beta2, gamma1, and delta subunit mRNAs was less in the DLPFC of alcohol-treated monkeys. CONCLUSION: These findings suggest that ethanol-induced alterations in GABA(A) function may be due to alterations in GABA(A) subunit mRNA levels and subunit-specific alterations are selective to particular cortical fields.

Region specific regulation of NR1 in rhesus monkeys following chronic antipsychotic drug administration.

BACKGROUND: Altered NMDA receptor subunit protein levels have been reported in various regions of the schizophrenic brain; however, chronic antipsychotic administration in schizophrenic subjects may confound interpretation. METHODS: The effects of chronic antipsychotic drug administration (haloperidol and clozapine) on protein levels of NR1, NR2A and NR2B proteins were evaluated in the nucleus accumbens (NAc), putamen (PUT), dorsolateral prefrontal cortex (DLPFC), superior temporal gyrus (STG), and entorhinal cortex (EC) of rhesus monkeys using Western blot analysis. RESULTS: Haloperidol administration significantly decreased NR1 expression in the DLPFC. In contrast, NR2B expression was not affected by antipsychotic administration in any brain region examined. NR2A was not reliably detected in any of the brain regions. CONCLUSIONS: Results indicate that the NR1 subunit in the DLPFC may be a substrate for antipsychotic action and that glutamatergic hypofunction in the DLPFC commonly associated with cognitive dysfunction in schizophrenia may be associated with haloperidol administration.

Molecular mapping of striatal subdivisions in juvenile Macaca Mulata.

The striatum of the primate brain can be subdivided into three distinct anatomical subregions: caudate (CAU), putamen (PUT), and ventral striatum (VS). Although these subregions share several anatomical connections, cell morphological, and histochemical features, they differ considerably in their vulnerability to different neurological and psychiatric diseases, and these brain regions have significantly different functions in health and disease. In order to better understand the molecular underpinnings of the different disease and functional vulnerabilities, transcriptional profiles were generated from the CAU, PUT, and VS of five juvenile rhesus macaques (Macaca mulatta) using human cDNA neuromicroarrays containing triplicate spots of 1227 cDNAs. Differences in microarray gene expression were assessed using z score analysis and 1.5-fold change between paired subregions. Clustering of genes based on dissimilarity of expression patterns between regions revealed subregion specific expression profiles encoding G-protein-coupled receptor signaling transcripts, transcription factors, kinases and phosphatases, and cell signaling and signal transduction transcripts. Twelve transcripts were examined using quantitative real-time PCR (qPCR), and 81% demonstrated alterations similar to those seen with microarray analysis, some of which were statistically significant. Subregion specific transcription profiles support the anatomical differentiation and potential disease vulnerabilities of the respective subregions.