The stability of insulin in solid formulations containing melezitose and starch. Effects of processing and excipients.

Solid insulin formulations obtained by different methods of preparation were compared with respect to chemical stability and morphology. Spray- and freeze-drying, solution enhanced dispersion by supercritical fluids (SEDS) and precipitation into starch microspheres were the methods used for preparation of solid powders. The excipients applied were melezitose, starch, and sodium taurocholate. The stability of the samples was evaluated after storage in open containers at 25 degrees C and 30% RH for 6 months. All samples were amorphous after processing and storage as detected by XRD, except for the starch microspheres which were semi-crystalline. The spray- and freeze-dried samples containing melezitose and sodium taurocholate experienced a significant water uptake during storage, resulting in changes in morphology and disappearance of Tg. However, the chemical stability of these samples did not seem to be affected by the water uptake. Changes in morphology were not observed for the SEDS powders and the starch microspheres. The chemical stability of the samples was assessed by HPLC. In general, conventional spray- and freeze drying resulted in samples with higher chemical stability compared to SEDS powders and starch microspheres. Nevertheless, the excipients applied were observed to be of major importance, and further optimization of the formulation as well as processing conditions may lead to slightly different conclusions.

Displacement of adsorbed insulin by Tween 80 monitored using total internal reflection fluorescence and ellipsometry.

PURPOSE: This study was conducted to investigate the mechanism of action in the displacement of adsorbed insulin from a hydrophobic surface by Tween 80 and of the competitive adsorption of the two species. METHODS: Total internal reflection fluorescence (TIRF) and ellipsometry were used as in situ methods to examine the processes taking place at hydrophobic model surfaces in the presence of insulin and Tween 80. RESULTS: TIRF studies showed that the displacement of insulin by Tween 80 could be fitted to a sigmoidal function, indicating a nucleation-dependent process. Furthermore, a linear dependence between the apparent rate constant and the logarithm of the Tween 80 concentration was found. Competitive adsorption from solution mixtures of insulin and Tween 80 indicated that insulin was adsorbed first, but subsequently displaced by the surfactant. This displacement proved also to be dependent on the concentration of Tween 80 in the mixture. CONCLUSIONS: The results indicate that Tween 80 at concentrations above critical micelle concentration can be used to protect insulin against surface adsorption. The presence of a lag phase in the displacement at low surfactant concentration indicates that the mechanism of action for Tween 80 to reduce adsorption of insulin may be by competing for sites at the surface.

Adsorption of human insulin and AspB28 insulin on a PTFE-like surface.

The interactions of human insulin, Zn-free human insulin, and AspB28 insulin with a hydrophobic surface were studied by ellipsometry. All three insulin types investigated adsorbed with high affinity onto the hydrophobic surface, as the plateau of the adsorption isotherm, represented by the irreversible bound fraction, was reached at concentrations >10(-3) mg/ml. The plateau values for human insulin and Zn-free human insulin could not be distinguished with statistical significance, whereas the plateau value for AspB28 insulin was lower than those for the two others, with an adsorbed amount corresponding to a monolayer of insulin monomers. The results observed may be explained by differences in self-association patterns of the insulin types or by enhanced charge repulsion between the AspB28 analog and the negatively charged surface.

Surface composition of spray-dried milk protein-stabilised emulsions in relation to pre-heat treatment of proteins.

Several important technical properties of spray-dried food powders depend on particle-liquid interactions (e.g. wettability, dispersability) and particle-particle interactions (e.g. flowability). It can be assumed that the chemical composition of the surface layer of the particles to a large extent determine these properties. The present study has been aimed to investigate the relation between the surface composition of spray-dried milk protein-stabilised emulsions and pre-heat treatment of the proteins. Solutions of WPC were heat-treated at low (60-90 degrees C) and high (140 degrees C) temperature and the degree of denaturation was determined, prior to the preparation of emulsions with rapeseed oil. The surface composition of the dry powders were established by using ESCA (electron spectroscopy of chemical analysis). The emulsions were characterised by droplet size distribution before spray drying and after dissolution of the powders. Also free fat extractions and estimations of wettability (dissolution rates) were performed. The powder surface coverage of protein decreased with increasing degree of protein denaturation before the emulsification, whereas the emulsion droplet size increased both before spray drying and after reconstitution of powders. The free fat extraction as well as the dissolution rate, whereof the latter decreased with increasing surface fat coverage, correlated well with the fat coverage of the powder surface.

Interactions between Local Anaesthetic Agents and Poly(N-isopropyl acrylamide) through Phase Behavior, Surface Tension, and Adsorption Measurement.

The interaction between the local anaesthetic agents prilocaine and lidocaine, on one hand, and poly(N-isopropyl acrylamide) (pNIPAM), on the other, is investigated through studies of the polymer phase behavior and through surface tension and adsorption measurements. In particular, the cloud points (CP) for pNIPAM in the presence of lidocaine and prilocaine under different conditions were compared to the effects of electrolytes and alcohols. It was found that the electrolytes affect the CP of pNIPAM in a lyotropic manner, whereas alcohols depress the CP of pNIPAM in an alkyl chain length dependent way; i.e., the longer the chain, the larger the decrease in CP. Lidocaine and prilocaine affect the CP of pNIPAM in a pH-dependent manner. Below the pK(a) of lidocaine and prilocaine, these cosolutes do not substantially affect the CP in the concentration range investigated, but rather behave analogous to simpler electrolytes. Above the pK(a), on the other hand, they strongly depress the CP already at low concentrations. In parallel, at low pH, the surface tension reduction due to lidocaine or prilocaine is marginal, whereas at high pH the surface tension is reduced considerably. Thus, the poor solubility of prilocaine and lidocaine at high pH causes these to become more surface active and simultaneously interact in a more pronounced way with pNIPAM. Furthermore, it was found from ellipsometry that an adsorbed pNIPAM layer contracts when lidocaine is added, presumably due to a lidocaine-pNIPAM interaction similar to that causing pNIPAM to phase separate. Analogous to this, it was demonstrated that an adsorbed pNIPAM layer shrinks and swells reversibly when the temperature is cycled above and beneath the CP. Copyright 2001 Academic Press.

Intermale competition in sexually mature arctic charr: effects on brain monoamines, endocrine stress responses, sex hormone levels, and behavior.

Sexually mature Arctic charr (Salvelinus alpinus) males were allowed to interact in pairs for 4 days in the absence of females. Agonistic behavior was quantified, and at the end of the experiment, plasma levels of glucose, cortisol, testosterone (T), 11-ketotestosterone (11-KT), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha, 20beta-P) were determined alongside brain concentrations of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA, the major 5-HT metabolite), dopamine (DA), and 3,4-dihydroxyphenylacetic acid (DOPAC, a major DA metabolite). Plasma cortisol and glucose were significantly elevated in subordinate fish, and the number of aggressive acts received showed positive correlations with plasma levels of glucose on day 1, during the development of the dominance relationship, and of cortisol on day 4, when the dominance relationship was established. In contrast, plasma concentrations of T and 11-KT were significantly higher in dominant than in subordinate males, and there was a similar tendency in plasma concentrations of 17alpha,20beta-P. Further, plasma levels of these gonadal steroids were correlated with the number of aggressive acts performed on day 4, but not with the number of aggressive acts received. The plasma cortisol concentrations did not correlate with either 5-HIAA:5-HT or DOPAC:DA ratios in any of the brain parts analyzed. Plasma glucose levels showed positive correlations with brain 5-HIAA:5-HT ratios. Negative correlations were observed between 5-HIAA:5-HT ratios in the optic tectum and between plasma levels of T, 11-KT, and 17alpha,20beta-P. Telencephalic DOPAC/DA ratios displayed a negative correlation with plasma levels of T, 11-KT, and 17alpha,20beta-P, but only in dominant males.

Relationships between sex and the size and number of forebrain gonadotropin-releasing hormone-immunoreactive neurones in the ballan wrasse (Labrus berggylta), a protogynous hermaphrodite.

This study is the first to examine the brain gonadotropin-releasing hormone (GnRH) cell population phenotype in a protogynous and monandric sequentially hermaphroditic fish. Male ballan wrasse (Labrus berggylta) had on average higher numbers of GnRH-immunoreactive (GnRH-ir) cells within the brain preoptic area (POA) than females, a difference not found in GnRH-ir cells in other brain regions. Furthermore, in males, but not females, the number of these POA GnRH-ir cells correlated with body size. Maturational state (prespawning or postspawning) had marked effects on mean profile sizes (but not numbers) of both GnRH-ir cell bodies and cell nuclei, even when existing differences in body size and allometric relationships had been taken into account. Postspawning males tended to have larger GnRH-ir profiles in all brain regions relative to both prespawning males and females. Moreover, the GnRH-ir cell number in POA, and the cell body profile size in both POA and at the level of the anterior commissure, correlated with gonad size in spermiated prespawning males, indicating a relationship between both size and number of GnRH cells and male gonadal development. These results suggest that temporary changes in the size of brain GnRH-ir neurones are coupled to the male spawning cycle, and that permanent POA GnRH-ir cell number changes are involved in the process of sex change in sequential hermaphrodites. However, smaller males had no more preoptic GnRH-ir cells than equally sized females, which may argue against a proximate inducing role of GnRH cell number changes in naturally occurring sex reversal.

Number of preoptic GnRH-immunoreactive cells correlates with sexual phase in a protandrously hermaphroditic fish, the dusky anemonefish (Amphiprion melanopus).

The populations of gonadotropin-releasing hormone (GnRH)-producing cells within the preoptic area (POA) and terminal nerve (TN) of the brain have been suggested as the neuronal systems mediating social control of sex and gonadogenesis in sequentially hermaphroditic teleosts. In the present study, the number and soma size of GnRH-immunoreactive (GnRH-ir) cells in the POA and TN were studied in male, female and juvenile individuals of the dusky anemonefish (Amphiprion melanopus), a species which displays both male to female sex change and socially controlled sexual maturation. The results showed that the number of POA (but not TN) GnRH-ir cells differ significantly between sexual phases, with males displaying higher cell numbers than both females and juveniles. Soma sizes of POA and TN GnRH-ir cells were larger in females than in males and juveniles. However, this relationship was fully explained by differences in body size. The results indicate that high POA GnRH cell numbers are part of a masculinizing mechanism and support the hypothesis that the POA GnRH cell population plays a central role in initiating or mediating the process of socially induced gonadal and/or behavioural transformations in sequential hermaphrodites.