Product Development Studies on Surface-Adsorbed Nanoemulsion of Olmesartan Medoxomil as a Capsular Dosage Form

The present study aimed at development of capsular dosage form of surface-adsorbed nanoemulsion (NE) of olmesartan medoxomil (OLM) so as to overcome the limitations associated with handling of liquid NEs without affecting their pharmaceutical efficacy. Selection of oil, surfactant, and cosurfactant for construction of pseudoternary phase diagrams was made on the basis of solubility of drug in these excipients. Rationally selected NE formulations were evaluated for percentage transmittance, viscosity, refractive index, globule size, zeta potential, and polydispersity index (PDI). Formulation (F3) comprising of Capmul MCM® (10% v/v), Tween 80® (11.25% v/v), polyethylene glycol 400 (3.75% v/v), and double-distilled water (75% v/v) displayed highest percentage cumulative drug release (%CDR; 96.69 ± 1.841), least globule size (17.51 ± 5.87 nm), low PDI (0.203 ± 0.032), high zeta potential (−58.93 ± 0.98 mV), and hence was selected as the optimized formulation. F3 was adsorbed over colloidal silicon dioxide (2 ml/400 mg) to produce free-flowing solid surface-adsorbed NE that presented a ready-to-fill capsule composition. Conversion of NE to surface-adsorbed NE and its reconstitution to NE did not affect the in vitro release profile of OLM as the similarity factor with respect to NE was found to be 66% and 73% respectively. The %CDR after 12 h for optimized NE, surface-adsorbed NE, and reconstituted NE was found to be 96.69 ± 0.54, 96.07 ± 1.76, and 94.78 ± 1.57, respectively (p > 0.05). The present study established capsulated surface-adsorbed NE as a viable delivery system with the potential to overcome the handling limitations of NE.KEY WORDS: bioavailability, nanoemulsion, olmesartan medoxomil, oral     

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.     

Electroencephalography in the diagnosis of hydrocephalus in golden hamsters (Mesocricetus auratus)

The golden hamster (Mesocricetus auratus) is a popular laboratory animal and is used in a multitude of behavioural studies. However, it has been shown that it suffers from different forms of hereditary hydrocephalus, which may result in behavioural changes. This prospective study was designed to look into the usefulness of electroencephalography (EEG) measurements in the diagnosis of hydrocephalus in hamsters. The EEGs of the hydrocephalic hamsters were evaluated double-blind and showed a high-voltage slow wave activity, with a fast activity superimposed onto it. This pattern has already been well described in other hydrocephalic species and differed significantly from the EEGs that were obtained from the normal hamsters. It was concluded from our study that a background activity with an amplitude over 50 muV in combination with a frequency of < or =5 Hz was highly indicative of hydrocephalus in young hamsters. We believe that the EEG could be a very useful diagnostic tool in the screening for hydrocephalus in hamsters.     

Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the alpha-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT(3A)) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT(3A)-glvM3M4 receptors were comparable to 5-HT(3A) wild type. The channels remained cation-selective but the 5-HT(3A)-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT(3) and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-HT(3A) but not 5-HT(3A)-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-rho1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.     

Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plant

Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH approximately 3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues.     

A lunar clock changes shielding pigment transparency in larval ocelli of Clunio marinus

Living in the tidal zones of the sea requires synchronization with the dominant environmental influences of tidal, solar, and lunar periodicity. Endogenous clocks anticipate those geoclimatic changes and control the respective rhythms of vital functions. But the underlying mechanisms are only partly understood. While the circadian clocks in animals are investigated employing neurobiological, molecular, and genetic approaches, clocks with a lunar periodicity have been studied with reference to development and behavior only. Sites of their pacemakers, zeitgeber receptors, and coupled endocrine components are unknown. Here, a lunar-rhythmic change of shielding pigment transparency in the larval ocelli of the intertidal midge Clunio marinus is demonstrated for the first time as a possible access to the neurobiology of lunar timing mechanisms. We studied third instar larvae (Vigo strain) throughout the lunar cycle by light- and electron-microscopy as well as by x-ray fluorescence analysis for the identification of the pigment. Moonlight detection is a prerequisite for photic synchronization of the lunar clock. The larval ocelli of Clunio putatively may function as moonlight receptors and are also controlled by the circalunar clock itself, hence being primary candidates for tracing input and output pathways of the lunar pacemaker. Additionally, the demonstration of a reversible optical change of shielding pigment transparency in Clunio is a novel finding, not reported so far in any other animal species, and reveals a mechanism to enhance photosensitivity under the condition of very dim light. It represents a remarkable change of a sense organ from an imaging device to a radiometer. Its restriction to the developmental stage susceptible to lunar timing elucidates a unique sensory strategy evolved at the level of sensory input. It also raises basic questions about the biochemistry of optically active pigments, like melanin, and their intracellular control.     

Evidence that proglumide and benzotript antagonize secretagogue stimulation of isolated gastric parietal cells

Proglumide has been shown to be an in vivo inhibitor of secretagogue-stimulated gastric acid secretion. In the present study, we have examined the ability of proglumide and benzotript, a new tryptophan derivative, to inhibit acid output from isolated gastric fundic parietal cells from rabbit. As measured with the [14C]aminopyrine (AP) accumulation method as an index of acid secretion, the two drugs inhibited basal AP with IC-50 values of 1 X 10(-2) M for proglumide and 1 X 10(-3) M for benzotript. In the case of secretagogue stimulation (1) benzotript slightly affected histamine-induced AP (15% inhibition at 5 X 10(-3) M), proglumide did not; (2) both proglumide and benzotript inhibited in a non-competitive manner acetylcholine-induced AP; (3) these isolated cells were sensitive to gastrin and the dose-response curve for the stimulant was biphasic (maximum for 1 X 10(-9) M), suggesting a desensitization mechanism. Proglumide and benzotript competitively inhibited both [125I]gastrin binding to its receptor sites and gastrin-induced AP, suggesting they are members of a class of gastrin-receptor antagonists. But, this suggestion cannot exclude other post-receptorial mechanisms involved in the acid output from parietal cells.     

Modification of phospholipid structure results in greater stability of liposomes in serum

Prevous studies have revealed that the replacement of the C-2 ester group in phosphatidylcholine by the carbamyloxy function renders the resulting lipids, without affecting the properties of the liposomes, resistant to hydrolysis by phospholipase A₂ (Gupta, C.M. and Bali, A. (1981) Biochim. Biophys. Acta 663, 506–515). As an extension of this work, the effect of serum on the stability of liposomes, prepared from $\text{1-}\text{palmitoyl-2-heptadec}\text{-10-cis-}\text{enylcarbamyloxyphosphatidylcholine}$ (carbamylphosphatidylcholine), has been examined. The stability has been measured in terms of (a) bilayer permeability to solutes, and (b) the lipid transfer to serum proteins, Replacement of egg phosphatidylcholine in liposomes by the carbamyl analog prevented serum-induced leakage of the entrapped solutes and also inhibited the lipid (phospholipid and cholesterol) transfer. Manipulation of the cholesterol content of the liposomes had no effect on the stability. These observations indicate that the interaction of serum proteins with liposomes probably involves a highly specific binding of the proteins to the liposome surface.     

Involvement of a pertussis toxin-sensitive G protein in the action of gastrin on gastric parietal cells

The mechanism whereby gastrin triggers phosphoinositide breakdown was investigated in an enriched preparation of isolated rabbit parietal cells (approx. 75%). In a permeabilized preparation of myo-[3H]inositol-labelled cells, GTP[S], a non-hydrolysable GTP analogue, enhanced [3H]inositol trisphosphate ([3H]InsP3 accumulation in a dose-dependent manner; submaximal concentrations of GTP[S] (less than 10 microM), potentiated gastrin-induced [3H]InsP3 release; preincubation for 5 min with GDP[S], a non-hydrolysable GDP analogue, dose-dependently reduced [3H]InsP3 accumulation stimulated by gastrin even in presence of GTP[S]. Exposure of intact parietal cells for 3 h to pertussis toxin (PTx) (200 ng/ml) led to a 15-50% reduction in gastrin-induced [14C]aminopyrine [(14C]AP) uptake (an index of in vitro acid secretion) and [3H]inositol phosphate ([3H]InsP) accumulation. A decrease in the accumulation of the different [3H]inositol phosphate occurred in gastrin-stimulated parietal cells treated with PTx. A rightward shift of gastrin dose-response curves in the presence of PTx was observed for [14C]AP uptake (EC50 values: 0.125 +/- 0.045 nM without PTx and 1.05 +/- 0.63 nM with PTx), for [3H]InsP accumulation (EC50 values: 0.16 +/- 0.08 nM without PTx and 1.56 +/- 0.58 nM with PTx) and [125I]gastrin binding (IC50 values: 0.247 +/- 0.03 nM without PTx and 2.38 +/- 0.56 nM with PTx). In contrast, cholera toxin (CTx) treatment (100 ng/ml) for 3 h was without effect on gastrin-induced [3H]InsP accumulation. CTx induced a pronounced potentiation of gastrin-stimulated [14C]AP uptake; this effect can be mimicked by IBMX (a phosphodiesterase inhibitor) and by forskolin (an activator of adenylyl cyclase). We conclude that: (i) one or more than one G protein appeared to be involved in gastrin receptor coupling to phospholipase C (PL-C); (ii) these G proteins are not substrates for CTx; (iii) one of these appeared to be a PTx-sensitive 'Gi-like' protein which could be involved in hormone-induced acid secretion, (iiii) the potentiating effect of CTx observed on AP uptake stimulated by gastrin suggests the existence of a cooperative effect between cAMP pathway (CTx) and the gastrin-induced phosphoinositide breakdown in acid secretory activity of parietal cells.