Randomized clinical trial of bedtime sublingual cyclobenzaprine (TNX-102 SL) in military-related PTSD and the role of sleep quality in treatment response.

Effective posttraumatic stress disorder (PTSD) pharmacotherapy is needed. This 12-week randomized multicenter trial evaluated efficacy and safety of TNX-102 SL, a bedtime sublingual formulation of cyclobenzaprine, in patients with military-related PTSD randomized to TNX-102 SL 2.8 mg or 5.6 mg, or placebo. Primary analysis comparing change from baseline in Clinician-Administered PTSD Scale-5 score between 2.8 mg (n=90) and placebo (n=92) was not significant. Secondary analysis of 5.6 mg (n=49) vs placebo demonstrated a mean difference of -4.5 units, p=.05, or, accounting for missing data by multiple imputation, -5.0 units, p=.03. Clinician Global Impression - Improvement responder rate was greater in 5.6 mg than placebo (p=0.04), as was mean functional improvement in Sheehan Disability Scale social domain (p=.03) and trended in work domain (p=.05). Post-hoc analyses showed early sleep improvement predicted improvement in PTSD after 12 weeks for TNX-102 SL (p<.01), not for placebo. Most common administration site reaction in TNX-102 SL groups was oral hypoaesthesia (5.6 mg, 36%; 2.8 mg, 39%; placebo, 2%), while most common systemic adverse event was somnolence (5.6 mg, 16%; 2.8 mg, 12%; placebo, 6%). This provides preliminary evidence that TNX-102 SL 5.6 mg reduces PTSD symptoms, improves sleep and psychosocial function, and is well tolerated. Clinicaltrials.gov Identifier: NCT02277704.

Effects of two isometheptene enantiomers in isolated human blood vessels and rat middle meningeal artery - potential antimigraine efficacy.

BACKGROUND: Racemic isometheptene [(RS)-isometheptene] is an antimigraine drug that due to its cardiovascular side-effects was separated into its enantiomers, (R)- and (S)-isometheptene. This study set out to characterize the contribution of each enantiomer to its vasoactive profile. Moreover, rat neurogenic dural vasodilatation was used to explore their antimigraine mechanism of action. METHODS: Human blood vessel segments (middle meningeal artery, proximal and distal coronary arteries, and saphenous vein) were mounted in organ baths and concentration response curves to isometheptene were constructed. Calcitonin gene-related peptide (CGRP)-induced neurogenic dural vasodilation was elicited in the presence of the enantiomers using a rat closed cranial window model. RESULTS: The isometheptene enantiomers did not induce any significant contraction in human blood vessels, except in the middle meningeal artery, when they were administered at the highest concentration (100 µM). Interestingly in rats, (S)-isometheptene induced more pronounced vasopressor responses than (R)-isometheptene. However, none of these compounds affected the CGRP-induced vasodilator responses. CONCLUSION: The isometheptene enantiomers displayed a relatively safe peripheral vascular profile, as they failed to constrict the human coronary artery. These compounds do not appear to modulate neurogenic dural CGRP release, therefore, their antimigraine site of action remains to be determined.

Pharmacological analysis of the increases in heart rate and diastolic blood pressure produced by (S)-isometheptene and (R)-isometheptene in pithed rats.

BACKGROUND: Isometheptene is a sympathomimetic drug effective in acute migraine treatment. It is composed of two enantiomers with diverse pharmacological properties. This study investigated in pithed rats the cardiovascular effects of (S)- isometheptene and (R)-isometheptene, and the pharmacological profile of the more potent enantiomer. METHODS: The effects of i.v. bolus injections (0.03, 0.1, 0.3, 1 and 3 mg/kg) of isometheptene racemate, (S)-isometheptene or (R)-isometheptene on heart rate and blood pressure were analyzed in control experiments. The enantiomer producing more pronounced tachycardic and/or vasopressor responses was further analyzed in rats receiving i.v. injections of prazosin (0.1 mg/kg), rauwolscine (0.3 mg/kg), propranolol (1 mg/kg) or intraperitoneal reserpine (5 mg/kg, -24 h). RESULTS: Compared to (R)-isometheptene, (S)-isometheptene produced greater vasopressor responses, whilst both compounds equipotently increased heart rate. The tachycardic responses to (S)-isometheptene were abolished after propranolol, but remained unaffected by the other antagonists. In contrast, the vasopressor responses to (S)-isometheptene were practically abolished after prazosin. Interestingly, after reserpine, the tachycardic responses to (S)-isometheptene were abolished, whereas its vasopressor responses were attenuated and subsequently abolished by prazosin. CONCLUSIONS: The different cardiovascular effects of the isometheptene enantiomers are probably due to differences in their mechanism of action, namely: (i) a mixed sympathomimetic action for (S)-isometheptene (a tyramine-like action and a direct stimulation of α1-adrenoceptors); and (ii) exclusively a tyramine like action for (R)-isometheptene. Thus, (R)-isometheptene may represent a superior therapeutic benefit as an antimigraine agent.

Metabolic syndrome in mice induced by expressing a transcriptional activator in adipose tissue.

Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11ß-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11ß-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.

Depot-specific regulation of the conversion of cortisone to cortisol in human adipose tissue.

OBJECTIVE: Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. METHODS AND PROCEDURES: Om and Abd sc adipose tissue were obtained at surgery from subjects with a wide range of BMI. Hydroxysteroid dehydrogenase (HSD) activity ((3)H-cortisone and (3)H-cortisol interconversion) and expression were measured before and after organ culture with insulin and/or dexamethasone. RESULTS: Type 1 HSD (HSD1) mRNA and reductase activity were mainly expressed within adipocytes and tightly correlated with adipocyte size within both depots. There was no depot difference in HSD1 expression or reductase activity, while cortisol inactivation and HSD2 mRNA expression (expressed in stromal cells) were higher in Om suggesting higher cortisol turnover in this depot. Culture with insulin decreased HSD reductase activity in both depots. Culture with dexamethasone plus insulin compared to insulin alone increased HSD reductase activity only in the Om depot. This depot-specific increase in reductase activity could not be explained by an alteration in HSD1 mRNA or protein, which was paradoxically decreased. However, in Om only, hexose-6-phosphate dehydrogenase (H6PDH) mRNA levels were increased by culture with dexamethasone plus insulin compared to insulin alone, suggesting that higher nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) production within the endoplasmic reticulum (ER) contributed to the higher HSD reductase activity. DISCUSSION: We conclude that in the presence of insulin, glucocorticoids cause a depot-specific increase in the activation of cortisone within Om adipose tissue, and that this mechanism may contribute to adipocyte hypertrophy and visceral obesity.

Chronic aeroallergen during infancy enhances eotaxin-3 expression in airway epithelium and nerves.

We have documented that exposure of rhesus monkeys to house dust mite aeroallergen during postnatal development resulted in significant recruitment of eosinophils into the airway mucosa (Clin Exp Allergy 33:1686-1694, 2003). Because eosinophils were not uniformly distributed throughout the five conducting airway generations examined, we speculated that trafficking within anatomic microenvironments of the lung is mediated by differential chemokine expression. To address this question, we used quantitative real-time RT-PCR to evaluate the related eosinophilic chemokines, eotaxin (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26) within isolated airways of infant monkey lung. Overall, chemokine mRNA expression levels in house dust mite-exposed airways were as follows: eotaxin-3 > eotaxin > eotaxin-2. Immunofluorescence staining for eotaxin-3 and CC chemokine receptor 3 (CCR3) showed positive cells within epithelium and peripherally located nerve fiber bundles of the airway wall. Epithelial volume of eotaxin-3 within the trachea correlated with epithelial volume of major basic protein. CCR3+ and MHC Class II+ dendritic cells, but not eosinophils or mast cells, co-localized within eotaxin-3+ nerve fiber bundles. We conclude that localized expression of eotaxin-3 plays an important role in the recruitment of diverse CCR3+ cell populations to different anatomic microenvironments within the infant airway in response to chronic allergen exposure.

Binding of 2-aryl-4-(piperidin-1-yl)butanamines and 1,3,4-trisubstituted pyrrolidines to human CCR5: a molecular modeling-guided mutagenesis study of the binding pocket.

The results of investigations in these laboratories of 2-aryl-4-(piperidin-1-yl)butanamines and 1,3,4-trisubstituted pyrrolidines as human CCR5 antagonists have recently been disclosed. To facilitate further development of these antagonists, we have developed a pharmacophore model based on the structure-activity relationships (SAR) and a human CCR5 receptor docking model using the crystal structure of rhodopsin as a template [Palczewski, K., et al. (2000) Science 289, 739-745]. Guided by the receptor docking model, we have mapped the compounds' site of interaction with CCR5 using site-directed mutagenesis experiments. Our results are consistent with a binding site for the two series that is located within a cavity near the extracellular surface formed by transmembrane helices 2, 3, 6, and 7. This site is overlapping yet distinct from that reported for another antiviral agent which binds to CCR5 [Dragic, T., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 5639-5644].

Functional expression and characterization of macaque C-C chemokine receptor 3 (CCR3) and generation of potent antagonistic anti-macaque CCR3 monoclonal antibodies.

Eosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The beta-chemokine receptor C-C chemokine receptor 3 (CCR3) provides a mechanism for the selective recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop in vivo models of inflammatory diseases, it is essential to identify and characterize the homologues of human eotaxin (C-C chemokine ligand 11) and CCR3 from other species, such as non-human primates. Accordingly, we cloned the macaque eotaxin and CCR3 genes and revealed that they were 91 and 92% identical at the amino acid level to their human homologues, respectively. Macaque CCR3 expressed in the murine pre-B L1-2 cell line bound macaque eotaxin with high affinity (K(d) = 0.1 nm) and exhibited a robust eotaxin-induced Ca(2+) flux and chemotaxis. Characterization of beta-chemokines on native macaque CCR3 on eosinophils was performed by means of eotaxin-induced shape change in whole blood using a novel signaling assay known as gated autofluorescence forward scatter. Additionally, mAbs were raised against macaque CCR3 using two different immunogens: a 30-amino acid synthetic peptide derived from the predicted NH(2) terminus of macaque CCR3 and intact macaque CCR3-transfected cells. These anti-macaque CCR3 monoclonal antibodies exhibited potent antagonist activity in receptor binding and functional assays. The characterization of the macaque eotaxin/CCR3 axis and development of antagonistic anti-macaque CCR3 monoclonal antibodies will facilitate the development of CCR3 small molecule antagonists with the hope of ameliorating chronic inflammatory diseases in humans.

In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration.

Eosinophil migration from circulation is controlled, in part, by chemokines through a family of G-protein-coupled chemokine receptors (CCR). Studies of human eosinophils have demonstrated that signaling through CCR3 receptors is a prominent pathway leading to chemotaxis, although several other receptor-ligand interactions also appear to mediate eosinophil recruitment. The availability of genetically unique strains of mice permits a reductionist approach to assess the signaling pathways in experimental models of human disease. However, despite similarities in these pathways between mice and humans, significant species differences exist, complicating the translation of results from animal models to humans. Purified mouse eosinophils were used in this study to investigate the chemokine receptor expression and the activities of 18 chemokines. Mouse eosinophils isolated from IL-5 transgenic mice expressed transcripts encoding the chemokine receptors CCR1, CCR2, CCR3, CCR5, CCR8, CXCR2, and CXCR4, but not CCR4. Mouse eosinophils also migrated in response to human and mouse eotaxin-1 and -2, but not human eotaxin-3. In addition, the induced migration of mouse eosinophils by TARC, MIP-1beta, and KC suggests that unidentified receptor-ligand interactions contribute to eosinophil recruitment. It is interesting that the potent chemoattractant of human eosinophils, RANTES, was unable to mediate mouse eosinophil migration. Furthermore, despite the ability of MIP-1alpha to bind receptors on purified mouse eosinophils, it was only able to induce significant eosinophil migration in a mixed splenocyte population and was unable to induce migration of highly purified eosinophils. Collectively, these observations reveal physiologically relevant distinctions in mechanisms mediating human and mouse eosinophil migration that potentially reflect evolutionary disparities between these species.