Duration of action of a broad range of selective κ-opioid receptor antagonists is positively correlated with c-Jun N-terminal kinase-1 activation

The κ-opioid receptor is a widely expressed G-protein-coupled receptor that has been implicated in biological responses to pain, stress, anxiety, and depression, and its potential as a therapeutic target in these syndromes is becoming increasingly apparent. However, the prototypical selective κ-opioid antagonists have very long durations of action that have been attributed to c-Jun N-terminal kinase (JNK) 1 activation in vivo. To test generality of this proposed noncompetitive mechanism, we used C57BL/6 wild type mice to determine the durations of antagonist action of novel κ-opioid receptor ligands and examined their efficacies for JNK1 activation compared with conventional competitive antagonists. Of the 12 compounds tested, 5 had long durations of action that positively correlated with JNK activation: RTI-5989-97 [(3S)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylpropyl]-2-methyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide], RTI-5989-194 [(3R)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylbutyl]-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide], RTI-5989-241 [(3R)-7-hydroxy-N-[(1S)-1-{[(3R,4R)-4-(3-methoxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide)], nor-binaltorphimine (nor-BNI); and (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic). Seven had short durations of action and did not increase phospho-JNK-ir: RTI-5989-212[(3R)-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylpropyl]-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide], RTI-5989-240 [(3R)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-(2-methylpropyl]-3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide], JSPA0658 [(S)-3-fluoro-4-(4-((2-(3,5-dimethylphenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide], JSPA071B [(S)-3-fluoro-4-(4-((2-(3,5-bis(trifluoromethyl)phenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide]. PF-4455242 [2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine], PF-4455242 [2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine], FP3FBZ [(S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide], and naloxone. After long-acting antagonist treatment, pJNK-ir did not increase in mice lacking the κ-opioid receptor; increased pJNK-ir returned to baseline by 48 h after treatment; and a second challenge with nor-BNI 72 h after the first did not increase pJNK-ir. Long-lasting antagonism and increased phospho-JNK-ir were not seen in animals lacking the JNK1 isoform. These results support the hypothesis that the duration of action of small molecule κ-opioid receptor antagonists in vivo is determined by their efficacy in activating JNK1 and that persistent inactivation of the κ-receptor does not require sustained JNK activation.     

Differentiation therapy in poor risk myeloid malignancies: Results of a dose finding study of the combination bryostatin-1 and GM-CSF

Purpose

Pharmacologic differentiating agents have had relatively limited clinical success outside of the use of ATRA in acute promyelocytic leukemia and DNA methyltransferase inhibitors in myelodysplastic syndromes. The differentiating effects of such agents can be enhanced in combination with lineage-specific growth factors. We developed a dose finding trial to assess toxicity, differentiating activity, and clinical impact of the combination of bryostatin-1 and GM-CSF.

Experimental design

Patients with poor risk myeloid malignancies were eligible to enroll in a dose finding study of continuous infusion bryostatin-1 combined with a fixed dose of daily GM-CSF. Toxicities were graded per NCI CTC version 2.0 and pharmacokinetic and correlative study samples were obtained to assess the combination's clinical and biologic differentiating effects.

Results

Thirty-two patients were treated with the combination therapy and the dose determined to be most suitable for study in a larger trial was continuous infusion broystatin-1 at 16 μg/m² for 14 days and subcutaneous GM-CSF at 125 μg/m² daily for 14 days every 28 days. Arthralgias and myalgias limited further dose escalation. Clinically, the combination impacted differentiation with improvement of absolute neutrophil counts (p = 0.0001) in the majority of patients. Interestingly, there were two objective clinical responses, including a CR after a single cycle. Both the bryostatin-1 plasma concentrations and the correlative studies supported biologic activity of the combination at the doses where clinical responses were observed.

Conclusions

Combining growth factors with pharmacologic differentiating agents may increase their clinical effectiveness and further studies should focus on such combinations.     

Cytoprotective response induced by electromagnetic stimulation on SH-SY5Y human neuroblastoma cell line

It is well known that physiological functions and pathological conditions of cells and tissues can be influenced not only by chemical molecules, but also by physical stimuli such as electromagnetic waves. In particular, epidemiological studies suggest possible associations between exposure to electromagnetic fields and an increased risk of tumors and neurodegenerative disorders, such as Alzheimer's disease. However, depending on the dose and on the length of treatment, the electromagnetic stimuli can be harmful or induce a cytoprotective cellular response, suggesting a possible application in medical therapy. In this study, under a tissue engineering viewpoint, we investigated the effects of an electromagnetic wave (magnetic field intensity, 2 mT; frequency, 75 Hz) on a neuronal cellular model characterized by the overexpression of the amyloid precursor protein (APP). After a prolonged electromagnetic treatment, lower mitochondrial activity and proliferation rate, resulting in a higher cellular quiescence, were observed. Focusing on the stress and oxidative pathways, we detected an overall increase of two fundamental proteins, the chaperone heat shock protein HSP70 and the free radical scavenger superoxide dismutase-1 enzyme (SOD-1). Interestingly, we found that the electromagnetic stimulation promotes the nonamyloidogenic processing of APP through an increased expression of the α-secretase ADAM10 and an enhanced release of the soluble neurotrophic factor sAPPα (a product of the ADAM10-mediated cleavage of APP). In conclusion, these findings suggest that the electromagnetic stimulus, if properly administered in terms of dose and timing, is able to induce a cytoprotective response in the cell. Moreover, these results suggest a possible use of this particular physical stimulation to improve the functional capability of the cells to face noxae.     

Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in neuroblastoma

Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing either R1275Q-mutated ALK or amplified wild-type ALK. In contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition resulted from an increased adenosine triphosphate-binding affinity (as also seen in acquired resistance to epidermal growth factor receptor inhibitors). Thus, this effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.     

Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.     

Sleep architecture and sleep-related mentation in securely and insecurely attached people

Based on REM sleep's brain activation patterns and its participation in consolidation of emotional memories, we tested the hypothesis that measures of REM sleep architecture and REM sleep-related mentation would be associated with attachment orientation. After a habituation night in a sleep lab, a convenience sample of 64 healthy volunteers were awakened 10 minutes into a REM sleep episode and 10 minutes into a control NREM sleep episode in counterbalanced order, then asked to report a dream and to rate themselves and a significant other on a list of trait adjectives. Relative to participants classified as having secure attachment orientations, participants classified as anxious took less time to enter REM sleep and had a higher frequency of REM dreams with aggression and self-denigrating themes. There were no significant differences across attachment groups in other measures of sleep architecture or in post REM-sleep awakening ratings on PANAS subscales reflecting mood and alertness. Selected aspects of REM sleep architecture and mentation appeared to be associated with attachment orientation. We suggest that REM sleep plays a role in processing experiences and emotions related to attachment, and that certain features of sleep and dreaming reflect attachment orientations.     

Perceived parental reactions to coming out, attachment, and romantic relationship views

Coming out as lesbian, gay, or bisexual (LGB) to one's parents can be a challenging experience and may lead to acceptance or rejection. Attachment theory can help predict parents' reactions to coming out and consequences for romantic attachment. In a cross-sectional study of 309 LGB individuals, we found that those who perceived their mother as accepting in childhood were more likely to have come out to her. Moreover, parents perceived as accepting and independence-encouraging in childhood were reported to react more positively to their child's sexual orientation. Mothers' positive reactions were associated with lower romantic attachment anxiety for men. The links between parent-child relationship quality and optimism and trust in romantic relationships were mediated by romantic attachment patterns. Findings support the contention that LGB pair bonds are attachment relationships, and underline the importance of prior parent-child relationships for predicting LGB individuals' experience of coming out and romantic relationships.     

Long-lasting recovery of psychotic-like symptoms in isolation-reared rats after chronic but not acute treatment with the cannabinoid antagonist AM251

In this work we investigated the ability of AM251 to reverse schizophrenia-like symptoms produced by a neurodevelopmental animal model based on a social isolation procedure. First, we assessed the validity of our isolation-rearing protocol and, as expected, isolation-reared rats showed hyperlocomotion in a novel environment, cognitive impairment in the novel object recognition (NOR) test and a significant increase in the number of aggressive behaviours in the social interaction test compared to group-housed controls. This behavioural picture was associated with a reduction in CB? receptor/G protein coupling in specific brain areas as well as reduced c-Fos immunoreactivity in the prefrontal cortex and caudate putamen. In this model, chronic but not acute treatment with the CB? receptor antagonist AM251 counteracted isolation-induced cognitive impairment in the NOR test and aggressive behaviours in the social interaction test. This behavioural recovery was accompanied by the rescue of CB? receptor functionality and c-Fos levels in all brain regions altered in isolation-reared rats. Moreover, chronic AM251 also increased c-Fos immunoreactivity in the nucleus accumbens, as previously demonstrated for antipsychotic drugs. Interestingly, the behavioural recovery due to chronic AM251 administration persisted until 10 d after discontinuing the treatment, indicating a long-lasting effect of the cannabinoid antagonist on psychotic-like symptoms.