Evaluation of the permeability and P-glycoprotein efflux of carbamazepine and several derivatives across mouse small intestine by the Ussing chamber technique.

PURPOSE: The rational discovery and development of new antiepileptic drugs (AEDs) with safer therapeutic index and better pharmacokinetic properties is still warranted nowadays. Because the long-term management of epilepsy is attained by means of orally administered AEDs, investigation of their potential to be well absorbed at the intestinal level is mandatory. Moreover, involvement of the efflux transport mediated by P-glycoprotein (P-gp) may compromise the systemic and central nervous system disposition of AEDs. Therefore, this study aimed at characterizing mouse jejunal passive transport and the possible active efflux mediated by P-gp of a series of dibenz[b,f]azepine-5-carboxamide derivatives (carbamazepine [CBZ], oxcarbazepine [OXC], S-licarbazepine [S-Lic], R-licarbazepine [R-Lic], carbamazepine-10,11-epoxide [CBZ-E], 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine [trans-diol], and BIA 2-024), which comprise some AEDs and metabolites. METHODS: Permeation studies were performed with freshly excised mouse jejunum segments mounted in Ussing chambers. Absorptive (M-S) and secretive (S-M) transports were analyzed with and without verapamil, which is a P-gp inhibitor widely recognized. Apparent permeability coefficients (P(app) ) in both directions and in absence or presence of verapamil were determined for each test compound. The in vitro method was validated using five controls that included high and low permeable markers with known absorption fraction (Fa) and also well-known P-gp substrates. The integrity of intestinal membrane was guaranteed during the assay by measuring the transepithelial electrical resistance. KEY FINDINGS: The correlation obtained between P(app) (M-S) and Fa of references was high (r(2) = 0.9945), and could be used to classify the derivatives according to Biopharmaceutical Classification System: CBZ and OXC were the only classified as highly permeable. The P(app) (S-M) of OXC, CBZ-E, R-Lic, and BIA 2-024 were significantly higher than their P(app) (M-S). After verapamil addition, their P(app) (S-M) lowered while P(app) (S-M) increased, suggesting the involvement of P-gp on the transport of those compounds across mouse jejunum segments. In opposition, CBZ, S-Lic, and trans-diol presented no statistical differences between the P(app) values reported in both directions, with or without verapamil. The results reported herein suggest that differences in biodisposition of S-Lic and R-Lic might result from their distinct interaction with P-gp. SIGNIFICANCE: The Ussing chamber model used herein showed to be useful for predicting Fa of AEDs and the involvement of efflux transport, namely P-gp, on their absorption. This is an important achievement as compounds that are not transported by P-gp may offer advantages when used in patients with pharmacoresistant epilepsy.

Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.

Studies in brain slices have provided a wealth of data on the basic features of neurons and synapses. In the intact brain, these properties may be strongly influenced by ongoing network activity. Although physiologically realistic patterns of network activity have been successfully induced in brain slices maintained in interface-type recording chambers, they have been harder to obtain in submerged-type chambers, which offer significant experimental advantages, including fast exchange of pharmacological agents, visually guided patch-clamp recordings, and imaging techniques. Here, we investigated conditions for the emergence of network oscillations in submerged slices prepared from the hippocampus of rats and mice. We found that the local oxygen level is critical for generation and propagation of both spontaneously occurring sharp wave-ripple oscillations and cholinergically induced fast oscillations. We suggest three ways to improve the oxygen supply to slices under submerged conditions: (i) optimizing chamber design for laminar flow of superfusion fluid; (ii) increasing the flow rate of superfusion fluid; and (iii) superfusing both surfaces of the slice. These improvements to the recording conditions enable detailed studies of neurons under more realistic conditions of network activity, which are essential for a better understanding of neuronal network operation.

Basic fibroblast growth factor promotes nerve regeneration in a C- -ion-implanted silicon chamber.

We reported previously that a silicone tube whose inner surface has been implanted with negatively charged carbon ions (C-) enables a nerve to regenerate across a 15-mm inter-stump gap. In this study, we investigated whether a C- -ion-implanted tube pretreated with basic fibroblast growth factor promotes peripheral nerve regeneration. The C- -ion-implanted tube significantly accelerated nerve regeneration, and this effect was enhanced by basic fibroblast growth factor.

An in vivo technique for pharmacological manipulation of Drosophila brain during optical recording.

Drosophila melanogaster, an established model for genetic manipulation, has recently been used for studying olfactory perception, learning, and memory. Some of these important behavioral phenomena have been dissected with defined mutants, some to a single biochemical lesion, expressed in central brain structures known as the mushroom bodies. A previously introduced preparation used a window in the head capsule through which these structures could be imaged using genetically expressed fluorescent calcium sensors while applying physiological odorant stimuli. Unfortunately, technical constraints prevented direct manipulation of the mushroom bodies with this preparation. I describe here a preparation that will allow, for the first time, the direct pharmacological manipulation of these important structures during imaging in the living adult fly. Responses to discreet applications of acetylcholine were reversibly blocked with tubocurare and reversibly eliminated in calcium-free Ringers. This new technique will significantly enhance the usefulness of the Drosophila model system, allowing a more quantitative examination of the mechanisms involved in olfactory learning and memory.

Improved survival of macroencapsulated islets of Langerhans by preimplantation of the immunoisolating device: a morphometric study.

Encapsulation of cells in a semipermeable membrane may in the future provide an opportunity to treat a variety of endocrine and neurological disorders, without the need for lifelong immunosuppression. The physiological conditions in the device are crucial factors for graft survival. Previously, we have shown that the exchange across the immunoisolating membrane and the microcirculation around the TheraCyte device increase around 3 months after implantation. The aim of this study was to determine whether preimplantation of the TheraCyte device would improve the survival of a later transplanted islet graft. A TheraCyte device was implanted SC on one side of the back of a nondiabetic SD rat. After 3 months, 1500 islets isolated from SD rats were transplanted via the device port. At the same time, another device, loaded with the same number of islets, was implanted on the other side of the back. Both devices were explanted 2 weeks after islet transplantation (i.e., 3.5 months and 0.5 month after device implantation, respectively). Six pairs of devices were evaluated by morphometery. The volume densities of viable islets were 0.22 +/- 0.04 in the preimplanted device vs. 0.06 +/- 0.03 in the nonpreimplanted one (p < 0.05). The corresponding volume densities of fibrosis and necrosis were 0.64 +/- 0.13 vs. 0.85 +/- 0.08 (p < 0.05) and 0.11 +/- 0.14 vs. 0.09 +/- 0.07 (ns), respectively. When the absolute volumes (mm3) were calculated, preimplanted devices contained 1.1 +/- 0.7 endocrine cells while nonpreimplanted ones contained 0.4 +/- 0.2 (p < 0.05). The percentages of insulin- positive beta-cells in the preimplanted versus nonpreimplanted device were 80 +/- 5% and 67 +/- 6%, respectively (p < 0.01). The corresponding volumes of fibrotic tissue were 3.0 +/- 1.8 vs. 5.2 +/- 1.2 (p < 0.05), while the amount of necrotic tissue did not differ significantly (0.42 +/- 0.5 vs. 0.50 +/- 0.3). Preimplantation of the TheraCyte device seems to improve the survival of an encapsulated islet graft and reduce fibroblast outgrowth in the device.