Fmoc-diphenylalanine-based hydrogels as a potential carrier for drug delivery

Self-assembled hydrogels from 9-fluorenylmethoxycarbonyl-modified diphenylalanine (Fmoc-FF) peptides were evaluated as potential vehicles for drug delivery. During self-assembly of Fmoc-FF, high concentrations of indomethacin (IDM) drugs were shown to be incorporated into the hydrogels. The β-sheet arrangement of peptides was found to be predominant in Fmoc-FF–IDM hydrogels regardless of the IDM content. The release mechanism for IDM displayed a biphasic profile comprising an initial hydrogel erosion-dominated stage followed by the diffusion-controlled stage. Small amounts of polyamidoamine dendrimer (PAMAM) added to the hydrogel (Fmoc-FF 0.5%–IDM 0.5%–PAMAM 0.03%) resulted in a more prolonged IDM release compared with Fmoc-FF 0.5%–IDM 0.5% hydrogel. Furthermore, these IDM-loaded hydrogels demonstrated excellent thixotropic response and injectability, which make them suitable candidates for use as injectable self-healing matrices for drug delivery.

Pharmacokinetics of methylphenidate and ritalinic acid in plasma correlations with exhaled breath and oral fluid in healthy volunteers

Purpose The primary aim of this study was to explore the potential of alternative sampling matrices for methylphenidate by assessing the correlations between dl-threo-methylphenidate and dl-threo-ritalinic acid concentrations in exhaled breath and oral fluid with those in plasma, in repeated samples collected after a single oral dose of methylphenidate. The secondary aim was to study the enantioselective pharmacokinetics of methylphenidate in plasma, with a focus on interindividual variability in the metabolism of methylphenidate to ritalinic acid. Methods Twelve healthy volunteers received a single oral dose of dl-threo-methylphenidate (Ritalin® capsules, 20 mg). Venous blood samples were collected for 24 h, and plasma analyzed for threo-enantiomers of methylphenidate and ritalinic acid with LC-MS/MS. Repeated sampling of exhaled breath, using a particle filter device, and of non-stimulated oral fluid, using a felt pad device, was also performed. Exhaled breath and oral fluid were analyzed with a non-enantioselective LC-MS/MS method for dl-threo-methylphenidate and dl-threo-ritalinic acid. Results In all subjects, d-threo-methylphenidate was detectable in plasma for at least 15 h after the dose with a biphasic profile. l-threo-Methylphenidate was measurable in only five subjects and in most cases in low concentrations. However, one female subject displayed a biphasic concentration-time profile for l-threo-methylphenidate. This subject also had the highest d-threo-methylphenidate AUC (191 ng*h/mL versus 32–119 ng*h/mL in the other subjects). d-threo-Ritalinic acid concentrations were on average 25-fold higher (range 6–126) than the corresponding d-threo-methylphenidate concentrations. Single-time point plasma concentration ratios between d-threo-ritalinic acid and d-threo-methylphenidate 1.5–12 h after dose correlated highly (r = 0.88–0.98) with the d-threo-ritalinic acid AUC/d-threo-methylphenidate AUC ratio. In eleven subjects, dl-threo-methylphenidate in oral fluid mirrored the biphasic profile of methylphenidate (sum of d- and l-threo-enantiomers) in plasma, but the concentrations in oral fluid were on average 1.8 times higher than in plasma. dl-threo-Methylphenidate was detected in exhaled breath in all subjects, but there was no consistent concentration-time pattern. Conclusions In some subjects, the pharmacologically less active l-threo-enantiomer may contribute to the total plasma methylphenidate concentrations. Monitoring methylphenidate concentrations without enantiomeric determination carries the risk of missing such subjects, which might affect how the plasma concentrations of methylphenidate are interpreted and used for clinical decision making. The use of exhaled breath and oral fluid to assess medication adherence to MPH in patients with ADHD warrants further studies.

Cysteines Flanking the Internal Fusion Peptide Are Required for the Avian Sarcoma/Leukosis Virus Glycoprotein To Mediate the Lipid Mixing Stage of Fusion with High Efficiency

ABSTRACT We previously showed that the cysteines flanking the internal fusion peptide of the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA) are important for infectivity and cell-cell fusion. Here we define the stage of fusion at which the cysteines are required. The flanking cysteines are dispensable for receptor-triggered membrane association but are required for the lipid mixing step of fusion, which, interestingly, displays a high pH onset and a biphasic profile. Second-site mutations that partially restore infection partially restore lipid mixing. These findings indicate that the cysteines flanking the internal fusion peptide of EnvA (and perhaps by analogy Ebola virus glycoprotein) are important for the foldback stage of the conformational changes that lead to membrane merger.

Cosensitivity of vagal mucosal afferents to histamine and 5-HT in the rat jejunum

A complex sensitivity of afferent nerves in the mesentery of the rat jejunum to systemic administration of histamine has recently been demonstrated. In the present study, we aimed to characterize subpopulations of mesenteric afferents that mediate this afferent nerve response. Multiunit afferent discharge was recorded from mesenteric nerves supplying the proximal jejunum in anesthetized rats. The majority of mesenteric bundles (84%) exhibited biphasic responses to histamine (8 μmol/kg), and these bundles also responded to 2-methyl-5-HT (2m5HT). In contrast, monophasic responses lacked a short-latency component, and these bundles failed to respond to 2m5HT. Single-unit analysis revealed a population of afferents that possessed cosensitivity for 2m5HT and histamine. This population of afferents was absent in chronically vagotomized animals, whereas mucosal anesthesia with luminal lidocaine reversibly converted the biphasic profile to a monophasic one. Ondansetron (500 μg/kg) blocked the response to 2m5HT with no effect on the profile of the histamine response, whereas pyrilamine (5 mg/kg) blocked the histamine response without affecting the response to 2m5HT. We conclude that histamine-sensitive afferents exist in the rat proximal jejunum that also respond to 5-HT via the 5-HT3receptor. These fibers appear to be vagal afferents originating in the intestinal mucosa and may be involved in the organization of mast cell-mediated responses.

Altered Dentate Filtering During the Transition to Seizure in the Rat Tetanus Toxin Model of Epilepsy

The dentate gyrus is thought to be a key area in containing the spread of seizure discharges in temporal lobe epilepsy. We investigated whether it actively contributes to the transition to seizure in vivo using the tetanus toxin chronic experimental epilepsy. Brief epileptic discharges lasted <2 s in freely moving animals and were clearly distinguishable from spontaneous seizures that lasted tens of seconds. This suggested that the changes underpinning the transition to seizure started within the first few seconds of seizure onset. During this period, we found that the amplitude of dentate gyrus population spikes depressed initially, but from 1.1 s after seizure onset, they potentiated. The amplitude and number of CA3 population spikes paralleled the pattern found in the dentate gyrus. We used hippocampal slices to study dentate filtering in more detail. The perforant pathway was stimulated repetitively at the frequency of field postsynaptic potentials found during epileptic discharges in vivo. The amplitude of dentate gyrus population spikes decreased to a steady state in naı̈ve hippocampal slices. In hippocampal slices prepared from rats previously injected with tetanus toxin, population spike amplitude decreased transiently and then potentiated. We found that the biphasic profile and rate of potentiation of dentate population spikes in vivo can be reproduced in naı̈ve hippocampal slices by blocking GABAB receptors. We conclude that the filtering properties of the dentate gyrus are altered in the tetanus toxin model of epilepsy and propose how this contributes to the transition to seizure in our animals.

Seasonal changes in reproductive and pelage status during the initial ‘quiescent’ and first ‘active’ breeding seasons of the peripubertal pony colt

Pony colts (~12 m) are generally infertile during the breeding season of mature stallions. The mechanism which ‘delays’ puberty beyond the first potential breeding opportunity has not been described. Four pre- and four post-pubertal colts were monitored from November for 13 (group 1, 6 to 19 months of age) and 9 months (group 2, 18 to 27 months of age). Fortnightly, 15 blood samples were collected at hourly intervals from each colt to determine concentrations of prolactin (PRL), LH, FSH and testosterone (T). Testicular biopsies were collected monthly to evaluate spermatogenic activity. Puberty occurred between 17 and 19 months, and at the onset of the 2-year-old breeding season, all colts were fertile. Seasonal changes in the concentrations of all four hormones were observed in both groups. Prolactin concentrations were correlated with changes in day length (r = 0·88) in both groups, but a biphasic profile was noted in group 1. Moulting of the winter coat was coincident with seasonally increased PRL concentrations (P < 0·001). Seasonal changes in FSH, LH and T concentrations were similarly timed between groups and levels were maximal in mid April, mid March and mid May, respectively. However, overall mean concentrations of LH and T in group 1 animals were only 0·33 and 0·22 of group 2 values (P < 0·001). Suppression of gonadal activity during the colts’ first ‘quiescent’ breeding season was associated with low concentrations of circulating LH. Appropriately timed changes in plasma PRL, FSH and T concentrations and pelage indicated that the photoperiodic mechanism was functional in the pre-pubertal colt but pituitary LH release may be blocked by immaturity or active suppression.

Pharmacological manipulation of Ins(1,4,5)P3 signaling mimics preconditioning in rabbit heart

Recent evidence revealed biphasic alterations in myocardial concentrations of the second messenger inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] with ischemic preconditioning (PC), i.e., increase during brief PC ischemia and decrease early during sustained test occlusion. Our aim was to determine whether an agonist and an antagonist of Ins(1,4,5)P3signaling {d- myo-inositol-1,4,5-trisphosphate hexasodium salt [d- myo-Ins(1,4,5)P3] and 2-aminoethoxydiphenyl borate (2-APB), respectively}, given such that they mimic this biphasic profile, would mimic infarct size reduction with PC. To test this concept, isolated, buffer-perfused rabbit hearts received no intervention (control), ischemic PC,d- myo-Ins(1,4,5)P3,d- myo-Ins(1,4,5)P3+ PC, 2-APB, or 2-APB + PC. All hearts then underwent 30-min coronary occlusion and 2 h reflow, and infarct size was delineated by tetrazolium staining. In addition, the effects ofd- myo-Ins(1,4,5)P3and 2-APB on Ins(1,4,5)P3signaling were evaluated in isolated fura 2-loaded rat cardiomyocytes. Mean infarct size was reduced with PC and in alld- myo-Ins(1,4,5)P3- and 2-APB-treated groups versus control (59 and 42–55%, respectively, vs. 80% of myocardium at risk, P < 0.05). Thus pharmacological manipulation of Ins(1,4,5)P3 signaling mimics the cardioprotection achieved with ischemic PC in rabbit heart.

Interaction of iturin A, a lipopeptide antibiotic, with Saccharomyces cerevisiae cells: influence of the sterol membrane composition

The binding of the membrane-active lipopeptide antibiotic iturin A to yeast cells was studied using radioactive iturin A. Saccharomyces cerevisiae had a maximum binding capacity of 5.6 × 109 molecules per single cell. The Scatchard plot of binding showed a biphasic profile, with a lower dissociation constant for small concentrations of iturin A. The break of slope at 30 μM iturin A corresponds to the micellization of antibiotic in solution. The binding is also dependent on the nature of the sterol present in the membrane. A mutant yeast strain with a membrane containing cholesterol instead of ergosterol showed the highest affinity for iturin A and the highest sensitivity to this antibiotic, as measured by K+ ion release. In contrast the presence of stigmasterol increased the resistance of the cells to iturin A. Key words: antifungal lipopeptide, iturin A, yeast cell binding capacity, membrane sterol.

Participation des systèmes de captation des carbohydrates et de l'H+-ATPase de la membrane cytoplasmique lors de l'utilisation du glucose et du lactose par le Fusarium oxysporum var. lini

Growth parameters of Fusarium oxysporum var. lini in media containing glucose or lactose as carbon source were studied in relation to biochemical phenomenons such as transport mechanisms of each carbohydrate and the cytoplasmic membrane H+-ATPase activity. The following conclusions were reached. (i) The biphasic profile of the fungal growth on the medium containing glucose could be explained by the presence of two mechanisms of glucose uptake when an inoculum with derepressed carbohydrate transport systems was used. On the other hand, the existence of only one transport system for lactose in both repressed and derepressed cells could explain the monophasic growth curve on the medium containing this disaccharide. (ii) The difference in pH profiles observed during the growth of F. oxysporum var. lini on media containing glucose or lactose as carbon source could be explained by a differential activation of the cytoplasmic membrane H+-ATPase by the two carbohydrates.