OTO-201: nonclinical assessment of a sustained-release ciprofloxacin hydrogel for the treatment of otitis media.

HYPOTHESIS: OTO-201 can provide sustained release to the middle ear and effectively treat otitis media, when compared with FDA-approved ciprofloxacin otic drop formulations. BACKGROUND: There is an unmet medical need for antibiotic therapy that can provide a full course of treatment from a single administration by an otolaryngologist at the time of tympanostomy tube placement, obviating the need for twice daily multiday treatment with short-acting otic drops. METHODS: Studies in guinea pigs and chinchillas were conducted. OTO-201 was administered as a single intratympanic injection and compared with the twice daily multi-day treatment with Ciprodex or Cetraxal otic drops. RESULTS: OTO-201 demonstrated sustained release of ciprofloxacin in the middle ear compartment for days to approximately 2 weeks depending on the dose. The substantial C(max) values and steady drug exposure yielded by OTO-201 were in contrast to the pulsatile short lasting exposure seen with Ciprodex and Cetraxal. OTO-201 was also effective in a preclinical chinchilla model of Streptococcus pneumoniae-induced otitis media. The degree of cure was comparable to that afforded by Ciprodex and Cetraxal. There was no evidence of middle or inner ear pathology in guinea pigs treated with OTO-201, unlike Ciprodex and Cetraxal, which both demonstrated mild cochlear ototoxicity. No adverse effects of the poloxamer 407 vehicle were noted. CONCLUSION: Intratympanic injection of OTO-201 constitutes an attractive treatment option to twice daily multiday dosing with ciprofloxacin ear drops for the treatment of otitis media, as evidenced by superior middle ear drug exposure, efficacy in an acute otitis media model, safety of administration, and convenience of a single dose regimen.

Distribution of corticotropin-releasing factor and urocortin 1 in the vole brain.

Brain receptor patterns for the corticotropin-releasing factor (CRF) receptors, CRF1 and CRF2, are dramatically different between monogamous and promiscuous vole species, and CRF physiologically regulates pair bonding behavior in the monogamous prairie vole. However, it is uncertain whether species differences also exist in the neuroanatomical distribution of the endogenous ligands for the CRF1 and CRF2 receptors, such as CRF and urocortin-1 (Ucn1). We compared the expression of CRF and Ucn1 in four vole species, monogamous prairie and pine voles, and promiscuous meadow and montane voles, using in situ hybridization of CRF and Ucn1 mRNA. Our results reveal that CRF mRNA expression patterns in all four vole species appear highly conserved throughout the brain, including the olfactory bulb, nucleus accumbens, bed nucleus of the stria terminalis, medial preoptic area, central amygdala, hippocampus, posterior thalamus, and cerebellum. Similarly, Ucn1 mRNA primarily localized to the Edinger-Westphal nucleus in all four vole species. Immunocytochemistry in prairie and meadow voles confirmed localization of CRF and Ucn1 protein to these previously identified brain regions. These data demonstrate a striking dichotomy between the extraordinary species diversity of brain receptor patterns when compared to the highly conserved brain distributions of their respective ligands. Our findings generate novel hypotheses regarding the evolutionary mechanisms underlying the neural circuitry of species-typical social behaviors.

Ataxia and c-Fos expression in mice drinking ethanol in a limited access session.

BACKGROUND: Although previous murine studies have demonstrated ethanol self-administration resulting in blood ethanol concentrations (BECs) believed to be pharmacologically relevant, to our knowledge, no study reported to date has demonstrated intoxication via ataxia after self-administration. Thus, the goal of this study was to demonstrate ataxia and to examine changes in c-Fos expression in mice after self-administration of intoxicating doses of ethanol. METHODS: Male C57BL/6J mice were trained to drink a 10% ethanol solution during daily 30-min limited access sessions. Mice were exposed to increasing concentrations of ethanol until a 10% ethanol solution was reached. BEC and ataxia, measured as foot slips off of a balance beam, were examined after the limited access self-administration session. In a separate experiment, various brain structures from mice drinking water or ethanol were examined for changes in c-Fos expression two hr after the limited access session. RESULTS: Mice drank between 1.5 and 2 g/kg of 10% ethanol during the daily 30-min session. BECs for these mice 15 min after the limited access session ranged between 0.52 and 2.13 mg/ml. A significant increase in foot slips off a balance beam was seen immediately after ethanol consumption during the limited access session. Among mice drinking ethanol, an increase in c-Fos expression was seen in the Edinger-Westphal nucleus, and a decrease in c-Fos expression was seen in the cingulate cortex, ventral tegmental area, lateral and medial septum, CA1 region of the hippocampus, and basolateral amygdala. CONCLUSIONS: After this procedure in mice, BECs are achieved that are in a range considered pharmacologically relevant and intoxicating. Significant ataxia was observed after ethanol self-administration. Brain regions showing changes in c-Fos expression after voluntary intoxication were similar to those previously reported, suggesting that these brain regions are involved in regulating behavioral effects of alcohol intoxication.

The copper-transporting ATPases, menkes and wilson disease proteins, have distinct roles in adult and developing cerebellum.

Copper is essential for brain metabolism, serving as a cofactor to superoxide dismutase, dopamine-beta-hydroxylase, amyloid precursor protein, ceruloplasmin, and other proteins required for normal brain function. The copper-transporting ATPases ATP7A and ATP7B play a central role in distribution of copper in the central nervous system; genetic mutations in ATP7A and ATP7B lead to severe neurodegenerative disorders, Menkes disease and Wilson disease, respectively. Although both ATP7A and ATP7B are required, their specific roles and regulation in the brain remain poorly understood. Using high-resolution imaging and functional assays, we demonstrate that ATP7A and ATP7B show cell-specific distribution in adult cerebellum, have distinct enzymatic characteristics, and are regulated differently during development. ATP7B is continuously expressed in Purkinje neurons (PN) where it delivers copper to the ferroxidase ceruloplasmin. ATP7A is a faster copper transporter than Wilson disease protein as evidenced by faster rates of catalytic reactions. The expression of ATP7A switches during development from PN to Bergmann glia, the cells supporting PN function in adult brain. Inactivation of ATP7B (Wilson disease protein) by gene knock-out induces a striking shift in the expression of the ATP7B target protein, ceruloplasmin, from PN to Bergmann glia, where ATP7A (Menkes disease protein) is present. The induced cell-specific change in expression restores copper delivery to ceruloplasmin via ATP7A. Overall, the results provide evidence for distinct functions of ATP7A and ATP7B in the cerebellum and illustrate a tight link between copper homeostasis in PN and Bergmann glia.

The Edinger-Westphal-lateral septum urocortin pathway and its relationship to alcohol consumption.

Identifying and characterizing brain regions regulating alcohol consumption is beneficial for understanding the mechanisms of alcoholism. To this aim, we first identified brain regions changing in expression of the inducible transcription factor c-Fos in the alcohol-preferring C57BL/6J (B6) and alcohol-avoiding DBA/2J (D2) mice after ethanol consumption. Drinking a 5% ethanol/10% sucrose solution in a 30 min limited access procedure led to induction of c-Fos immunoreactivity in urocortin (Ucn)-positive cells of the Edinger-Westphal nucleus (EW), suppression of c-Fos immunoreactivity in the dorsal portion of the lateral septum (LS) of both strains of mice, and strain-specific suppression in the intermediate portion of the LS and the CA3 hippocampal region. Because the EW sends Ucn projections to the LS, and B6 and D2 mice differ dramatically in EW Ucn expression, we further analyzed the Ucn EW-LS pathway using several genetic approaches. We find that D2 mice have higher numbers of Ucn-immunoreactive processes than B6 mice in the LS and that consumption of ethanol/sucrose in the F2 offspring of a B6D2 intercross positively correlates with Ucn immunoreactivity in the EW and negatively correlates with Ucn immunoreactivity in the LS. In agreement with these findings, we find that alcohol-avoiding male B6.D2 Alcp1 line 2.2 congenic mice have lower Ucn immunoreactivity in the EW than male B6.B6 mice. Finally, we also find that HAP mice, selectively bred for high alcohol preference, have higher Ucn immunoreactivity in EW, than LAP mice, selectively bred for low alcohol preference. Taken together, these studies provide substantial evidence for involvement of the EW-LS Ucn pathway in alcohol consumption.

Identification of temperature-sensitive neural circuits in mice using c-Fos expression mapping.

Expression of the inducible transcription factor c-Fos was mapped in mouse brain to identify neural circuits selectively involved in response to cold and hot external temperatures. Male C57BL/6J mice were exposed acutely or repeatedly (seven sessions) to 10 or 34 degrees C in sound-attenuated chambers. Control mice were acclimated to exposure to the experimental room at 20 degrees C. All animals were sacrificed at 90 min for immunohistochemical analysis. A statistically significant induction of c-Fos was observed in the shell of nucleus accumbens and posterior medial cortical amygdala only following the acute thermal exposure, showing a significant habituation of the response to repeated treatments, a finding arguing against specificity of responses in these nuclei to thermal exposures. In contrast, expression of c-Fos was significantly increased following both acute and repeated thermal exposures in subregions of hypothalamus (the median and medial preoptic nuclei, the paraventricular nucleus of hypothalamus and the posterior hypothalamic area), septum (the ventral and dorsal portions of the lateral septum), midbrain (the periaqueductal gray and the intermediate layers of superior colliculus), as well as in the dentate gyrus and the paraventricular nucleus of thalamus, suggesting specificity of their responses to external temperatures. Expression of c-Fos was also significantly increased in the Edinger-Westphal nucleus following acute thermal exposures versus control mice, but not versus mice repeatedly exposed to cold and hot temperatures, providing modest support for thermal specificity of c-Fos response in this nucleus. While thermal sensitivity of hypothalamic structures has been previously confirmed by many authors, the present study identifies a number of structures previously not found to be responsive to changes in external temperature, and lays ground for future work important for identification of neural circuits involved in thermoregulation.

Alcohol-induced c-Fos expression in the Edinger-Westphal nucleus: pharmacological and signal transduction mechanisms.

Mapping inducible transcription factors has shown that the Edinger-Westphal nucleus is preferentially sensitive to alcohol intoxication. Herein, we characterize the pharmacological and signal transduction mechanisms related to alcohol-induced c-Fos expression in Edinger-Westphal neurons. Using immunohistochemistry, we show that pretreatment with gamma-aminobutyric acid (GABA)-ergic antagonists (4 mg/kg bicuculline and 45 mg/kg pentylenetetrazole) attenuates induction of c-Fos expression by alcohol (2.4 g/kg, intraperitoneal). In addition, 10 mg/kg 2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)4,5-dihydro-1H-imidazole (RX 821002), an alpha(2A/D)-adrenoceptor antagonist, and 20 mg/kg haloperidol, a dopamine antagonist, also block alcohol-induced c-Fos expression in Edinger-Westphal neurons. No effects were seen in alcohol-induced c-Fos after the pretreatment of 20 mg/kg propranolol (beta-adrenoceptor antagonist), 10 mg/kg 2-(2-(4-(2-methoxyphenyl)piperazin-1-yl) ethy)-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC 239) (alpha(2B/C)-adrenoceptor antagonist), or 30 mg/kg naltrexone (opioid antagonist). Although positive modulators for the GABA(A) receptor (20 mg/kg 3alpha-hydroxy-5alpha-pregnan-20-one and 10-30 mg/kg chlordiazepoxide) and opioid receptor (10 mg/kg morphine) produced significant elevations, agonists for alpha(2)-adrenoceptors (clonidine) and dopamine receptors (apomorphine) had no effect on Edinger-Westphal c-Fos expression. These findings suggest that alcohol-induced c-Fos expression in Edinger-Westphal results from direct interactions with GABA(A) receptors, which are modified by alpha(2A/D)-adrenoceptors and dopamine receptors. Also using immunohistochemistry to identify potential intracellular mechanisms associated with alcohol-induced c-Fos expression in Edinger-Westphal, we show time-dependent increases in serine 727 phospho-signal transducer and activator of transcription 3 (Stat3) but no changes in phospho-cAMP response element-binding protein and phospho-Elk1. Time-dependent increases in phospho-extracellular signal-regulated kinase (ERK) 1/2 were found to occur simultaneously with increases in serine 727 phospho-Stat3. Finally, blockade of ERK 1/2 phosphorylation with the mitogen-activated protein kinase (MEK) 1/2 inhibitor SL327 blocked alcohol-induced c-Fos expression, suggesting that alcohol induces c-Fos in Edinger-Westphal neurons through activation of the MEK1/2-ERK1/2-Stat3 pathway.