Confocal Laser Scanning Microscopic Visualization of the Transport of Dextrans After Nasal Administration to Rats: Effects of Absorption Enhancers

Purpose. To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivousing confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated -cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. Methods. Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. Results. Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated -cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated -cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. Conclusions. Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.     

Absorption Enhancing Effect of Cyclodextrins on Intranasally Administered Insulin in Rats

The absorption enhancing effect of -, -, and -cyclodextrin (CD), dimethyl--cyclodextrin (DMCD), and hydroxypropyl--cyclodextrin (HPCD) on intranasally administered insulin was investigated in rats. Coadministration of 5% (w/v) DMCD to the insulin solution resulted in a high bioavailability, 108.9 ± 36.4% (mean ± SD, n = 6), compared to i.v. administration, and a strong decrease in blood glucose levels, to 25% of their initial values. Coadministration of 5% -CD gave rise to an insulin bioavailability of 27.7 ± 11.5% (mean ± SD, n = 6) and a decrease in blood glucose to 50% of its initial value. The rate of insulin absorption and the concomitant hypoglycemic response were delayed for the -CD-containing solution as compared to the DMCD preparation. The other CDs, HPCD (5%), -CD (1.8%), and -CD (5%), did not have significant effects on nasal insulin absorption. DMCD at a concentration of 5% (w/v) induces ciliostasis as measured on chicken embryo tracheal tissue in vitro, but this effect is reversible. In conclusion, DMCD is a potent enhancer of nasal insulin absorption in rats.     

PLGA-PEI nanoparticles for gene delivery to pulmonary epithelium.

Pulmonary gene delivery is thought to play an important role in treating genetically related diseases and may induce immunity towards pathogens entering the body via the airways. In this study we prepared poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles bearing polyethyleneimine (PEI) on their surface and characterized them for their potential in serving as non-viral gene carriers to the pulmonary epithelium. Particles that were synthesized at different PLGA-PEI ratios and loaded with DNA in several PEI-DNA ratios, exhibited narrow size distribution in all formulations, with mean particle sizes ranging between 207 and 231 nm. Zeta potential was strongly positive (above 30 mV) for all the PEI-DNA ratios examined and the loading efficiency exceeded 99% for all formulations. Internalization of the DNA-loaded PLGA-PEI nanoparticles was studied in the human airway submucosal epithelial cell line, Calu-3, and DNA was detected in the endo-lysosomal compartment 6 h after particles were applied. Cytotoxicity of these nanoparticles was dependent on the PEI-DNA ratio and best cell viability was achieved by PEI-DNA ratios 1:1 and 0.5:1. These findings demonstrate that PLGA-PEI nanoparticles are a potential new delivery system to carry genes to the lung epithelium.     

Uptake of estradiol or progesterone into the CSF following intranasal and intravenous delivery in rats.

The uptake of estradiol and progesterone into the cerebrospinal fluid (CSF) after intranasal and intravenous administration in rats was investigated. Each animal received estradiol intranasally (40 microg/rat) and by intravenous infusion (10 microg/rat) into the jugular vein using a vascular access port. Hereafter, the same set of rats was treated with progesterone intranasally (200 microg/rat) and by intravenous infusion (104 microg/rat). Following nasal delivery, both steroid hormones reach Cmax values in plasma and CSF at 15 min after administration. Intravenous infusion of estradiol and progesterone shows comparable plasma and CSF concentration-time profiles compared to the nasal route. For both hormones the AUCCSF/AUCplasma ratios (mean +/- SD) after intranasal delivery (estradiol 2.3 +/- 1.1%; progesterone 1.9 +/- 0.7%) do not differ significantly from the ratios shown after intravenous infusion (estradiol 2.0 +/- 0.6%; progesterone 2.2 +/- 0.8%). These results indicate that after nasal delivery estradiol and progesterone are rapidly absorbed into the systemic circulation, from where the non-protein bound hormones probably enter the CSF by crossing the blood-brain barrier. No extra direct nose-CSF transport could be demonstrated.     

Does sleep restore the topology of functional brain networks?

Previous studies have shown that healthy anatomical as well as functional brain networks have small‐world properties and become less optimal with brain disease. During sleep, the functional brain network becomes more small‐world‐like. Here we test the hypothesis that the functional brain network during wakefulness becomes less optimal after sleep deprivation (SD). Electroencephalography (EEG) was recorded five times a day after a night of SD and after a night of normal sleep in eight young healthy subjects, both during eyes‐closed and eyes‐open resting state. Overall synchronization was determined with the synchronization likelihood (SL) and the phase lag index (PLI). From these coupling strength matrices the normalized clustering coefficient C (a measurement of local clustering) and path length L (a measurement of global integration) were computed. Both measures were normalized by dividing them by their corresponding C‐s and L‐s values of random control networks. SD reduced alpha band C/C‐s and L/L‐s and theta band C/C‐s during eyes‐closed resting state. In contrast, SD increased gamma‐band C/C‐s and L/L‐s during eyes‐open resting state. Functional relevance of these changes in network properties was suggested by their association with sleep deprivation‐induced performance deficits on a sustained attention simple reaction time task. The findings indicate that SD results in a more random network of alpha‐coupling and a more ordered network of gamma‐coupling. The present study shows that SD induces frequency‐specific changes in the functional network topology of the brain, supporting the idea that sleep plays a role in the maintenance of an optimal functional network. Hum Brain Mapp, 2013. © 2011 Wiley Periodicals, Inc.     

Hydroxocobalamin uptake into the cerebrospinal fluid after nasal and intravenous delivery in rats and humans

The possibility of direct transport of hydroxocobalamin from the nasal cavity into the cerebrospinal fluid (CSF) after nasal administration in rats was investigated and the results were compared with a human study. Hydroxocobalamin was given to rats (n=8) both intranasally (214 microg/rat) and intravenously (49.5 microg/rat) into the jugular vein using a Vascular Access Port (VAP). Prior to and after drug administration, blood and CSF samples were taken and analysed by radioimmunoassay. The AUCCSF/AUCplasma ratio after nasal delivery does not differ from the ratio after intravenous infusion, indicating that hydroxocobalamin enters the CSF via the blood circulation across the blood-brain barrier (BBB). This same transport route is confirmed by the cumulative AUC-time profiles in CSF and plasma, demonstrating a 30 min delay between plasma absorption and CSF uptake of hydroxocobalamin in rats and in a comparative human study. The present results in rats show that there is no additional uptake of hydroxocobalamin in the CSF after nasal delivery compared to intravenous administration, which is in accordance with the results found in humans. This indicates a predictive value of the used rat model for the human situation when studying the nose to CSF transport of drugs.     

Psychiatric comorbidity and causal disease models

In psychiatry, comorbidity is the rule rather than the exception. Up to 45% of all patients are classified as having more than one psychiatric disorder. These high rates of comorbidity have led to a debate concerning the interpretation of this phenomenon. Some authors emphasize the problematic character of the high rates of comorbidity because they indicate absent zones of rarities. Others consider comorbid conditions to be a validator for a particular reclassification of diseases. In this paper we will show that those at first sight contrasting interpretations of comorbidity are based on similar assumptions about disease models. The underlying ideas are that firstly high rates of comorbidity are the result of the absence of causally defined diseases in psychiatry, and second that causal disease models are preferable to non-causal disease models. We will argue that there are good reasons to seek after causal understanding of psychiatric disorders, but that causal disease models will not rule out high rates of comorbidity — neither in psychiatry, nor in medicine in general. By bringing to the fore these underlying assumptions, we hope to clear the ground for a different understanding of comorbidity, and of models for psychiatric diseases.     

Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH

The pharmacopoeia protocol for HP-SEC of insulin, using an acidic non-physiological eluent, does not represent insulin's association state in the formulation. This study aimed to evaluate insulin's elution behavior in HP-SEC in a “physiological” (aqueous, neutral pH) eluent, using on-line UV absorption and multi-angle laser light scattering detection. The effect of insulin concentration and association state in the formulation (monitored by circular dichroism) and eluent composition (zinc ion, arginine) on its elution behavior was assessed. We showed that the elution behavior of insulin in “physiological” HP-SEC is affected by both dynamic association–dissociation of insulin molecules and insulin–column interactions. Insulin molecules re-equilibrated in the HP-SEC eluent, making its elution behavior practically insensitive to the association state of insulin in the formulation. Zinc ions in the eluent promoted association of insulin to hexamers, whereas arginine overruled the effect of zinc ions and induced on-column dissociation of insulin to dimers and monomers. Combined results from “physiological” and compendial HP-SEC were shown to provide a better view of the aggregation state of heat-stressed insulin than either of the single methods. The insights obtained with this study are crucial for a proper evaluation of HP-SEC data of insulin.