Luis Granero

Intestinal Absorption Enhancement Via the Paracellular Route by Fatty Acids, Chitosans and Others: A Target for Drug Delivery

Peroral delivery of hydrophilic drugs is one of the greatest challenges in biopharmaceutical research. Hydrophilic drugs usually present low bioavailability after oral administration. One of the causes of this low bioavailability is their poor intestinal permeation through the paracellular pathway. This pathway is actually restricted by the presence of tight junctions at the apical side of the enterocytes. In the last few years, great interest has been focused on the structure and cellular regulation of tight junctions, materializing in more in-depth knowledge of this intestinal barrier. Simultaneously, and on the basis of this understanding, continuous efforts are being made to develop agents that can modulate tight junctions and magnify the paracellular permeability of hydrophilic compounds without causing significant intestinal damage. This review focuses on strategies to improve the paracellular permeation of poorly absorbed drugs as a way to enhance their bioavailability after oral administration. Most of the research on this subject has been carried out using in vitro models (mainly Caco-2 cell monolayers), which yield useful information on the potential effects and mechanisms of action of absorption-enhancing compounds. However, in vivo studies, which are much more scarce, are needed to confirm the effects of potential enhancers and to evaluate the suitability of including these compounds as excipients in drug formulation. We review the in vitro and in situ studies involving the most promising paracellular permeation enhancers (e.g., medium chain fatty acids and chitosan and its derivatives), analyzing the degree of drug absorption enhancement achieved, as well as the potential associated toxicity. The few studies performed in vivo are also presented. In addition, the findings of recent absorption enhancers, such as zonula occludens toxin or thiolated polymers, are reviewed.

Synthetic aperture superresolved microscopy in digital lensless Fourier holography by time and angular multiplexing of the object information

The resolving power of an imaging system in digital lensless Fourier holographic configuration is mainly limited by the numerical aperture of the experimental setup that is defined by both the restricted CCD size and the presence of a beam splitter cube in front of the CCD. We present a method capable of improving the resolution in such a system configuration based on synthetic aperture (SA) generation by using time-multiplexing tilted illumination onto the input object. Moreover, a priori knowledge about the imaged object allows customized SA shaping by the addition of elementary apertures only in the directions of interest. Experimental results are provided, showing agreement with theoretical predictions and demonstrating a resolution limit corresponding with a synthetic numerical aperture value of 0.45.

Kinetics of zinc transport in vitro in rat small intestine and colon: interaction with copper

The present study was planned to investigate the kinetic transport of zinc, in the intact intestine of the rat, in order to establish if more than one transporter is involved as well as the existence of a preferent sector in the cation uptake. Using an in vitro technique, the influx of zinc across the brush border membrane in three sectors of the small intestine (proximal, mid and distal) and in the colon of the rat was measured at six different concentrations (from 0.0007 to 11 mM). The kinetic study showed that intestinal transport of zinc occurs by a saturable process in the small intestine. The K(m) value obtained in the proximal segment (10.78+/-4.40 mM) is clearly higher than those obtained in the mid and distal segments (1.94+/-0.39 and 3.04+/-0.44 mM, respectively). The same occurs with the J(max) values. These results seem to indicate that more than one transporter may be implicated in zinc transport. In colon the most probable mechanism is non-saturable diffusion, the diffusive permeability, P, being 2.95.10(-7)+/-0.43.10(-7) cm/h. The statistical comparison of the fluxes indicated that, on the whole, there is not a well defined preferent sector in zinc transport. Additionally, the influence of copper on zinc transport, in three sectors of the small intestine, has been evaluated quantifying the influx of zinc at 0.037 mM in the absence and presence of three different concentrations of copper. The results showed that copper significantly reduced the influx of zinc, in the three sectors studied, in a concentration-dependent manner.

Superresolution imaging method using phase-shifting digital lensless Fourier holography

A method which is useful for obtaining superresolved imaging in a digital lensless Fourier holographic configuration is presented. By placing a diffraction grating between the input object and the CCD recording device, additional high-order spatial-frequency content of the object spectrum is directed towards the CCD. Unlike other similar methods, the recovery of the different band pass images is performed by inserting a reference beam in on-axis mode and using phase-shifting method. This strategy provides advantages concerning the usage of the whole frequency plane as imaging plane. Thus, the method is no longer limited by the zero order term and the twin image. Finally, the whole process results in a synthetic aperture generation that expands up the system cutoff frequency and yields a superresolution effect. Experimental results validate our concepts for a resolution improvement factor of 3.

Shell/core differences in mu- and delta-opioid receptor modulation of dopamine efflux in nucleus accumbens

The mu- and delta-opioid receptors located at the terminal level in nucleus accumbens are involved in the opiate modulation of dopamine release in this brain area. However, recent studies suggest that the effects of opioid drugs on the core subregion of nucleus accumbens may completely differ from those observed in the shell. We used in vivo microdialysis to simultaneously apply selective mu- and delta-opioid receptor agonists and to measure extracellular levels of dopamine in three subregions of the accumbens, namely shell, core, and the transition zone between them. The regional analysis of these subregions of the accumbens demonstrated that basal levels of dopamine and its metabolites were higher in the core, and decreased from this subregion to the shell. Retrodialysis application to the core of both the selective mu-receptor agonist ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) (1 micromol/L)) and of the selective delta-opioid receptor agonist ([D-Pen(2), D-Pen(5)]-enkephalin (DPDPE) (50 nmol/L)) increased the dialysate levels of dopamine. However, the application of these drugs to the shell significantly reduced the dopamine levels in this subregion. Local application of the same doses of these drugs in the transition zone between the shell and the core did not significantly affect the dopamine levels in dialysates. These results suggest that the opioid circuits modulating dopaminergic activity in the shell could differ from those in the core of the nucleus accumbens.

Systemic administration of D-penicillamine prevents the locomotor activation after intra-VTA ethanol administration in rats.

Although recently published studies seem to confirm the important role displayed by acetaldehyde (ACH), the main metabolite of ethanol, in the behavioral effects of ethanol, the origin of ACH is still a matter of debate. While some authors confer more importance to the central (brain metabolism) origin of ACH, others indicate that the hepatic origin could be more relevant. In this study we have addressed this topic using an experimental approach that combines local microinjections of ethanol into the ventral tegmental area (VTA) (which guarantees the brain origin of the ACH) to induce motor activation in rats together with systemic administration (i.p.) of several doses (0, 12.5, 25 and 50 mg/kg) of D-penicillamine (DP), a sequestering agent of ACH with contrasted efficiency to abolish the behavioral effects of the drug. Our results clearly show that DP prevented in a dose-dependent manner the motor activation induced by intra-VTA ethanol, being the 50 mg/kg dose the most efficient. DP per se did not affect the basal activity of the rats. In order to determine the specificity of the DP action, we also studied the effects of DP 50 mg/kg on the DAMGO-induced motor activation after the intra-VTA administration of this mu-opioid receptors agonist. DP did not significantly modify the motor activation induced by DAMGO thus confirming the specificity of the DP effects. Our results clearly suggest that the brain-derived ACH is necessary to manifest the activating effects resulting from ethanol administration.

Revisiting the controversial role of salsolinol in the neurobiological effects of ethanol: Old and new vistas

The possible involvement of salsolinol (Sal), an endogenous condensation product of ACD (the first metabolite of ethanol) and dopamine, in the neurochemical basis underlying ethanol action has been repeatedly suggested although it has not been unequivocally established, still being a controversial matter of debate. The main goal of this review is to evaluate the presumed contribution of Sal to ethanol effects summarizing the reported data since the discovery in the 1970s of Sal formation in vitro during ethanol metabolism until the more recent studies characterizing its behavioral and neurochemical effects. Towards this end, we first analyze the production and detection of Sal, in different brain areas, in basal conditions and after alcohol consumption, highlighting its presence in regions especially relevant in regulating ethanol-drinking behaviour and the importance of the newly developed methods to differentiate both enantiomers of Sal which could help to explain some previous negative findings. Afterwards, we review the behavioral and neurochemical studies. Finally, we present and discuss the previous and current enunciated mechanisms of action of Sal in the CNS.

Induction of conditioned place preference and dopamine release by salsolinol in posterior VTA of rats: involvement of μ-opioid receptors.

Salsolinol (Sal), locally administered into the posterior VTA (pVTA) of rats, produces psychomotor responses and reinforcing effects, probably, through the activation of μ-opioid receptors (MORs). The neurochemical correlates of these phenomena are, however, practically unknown. In this paper, we explore the neurochemical events and the mechanisms involved in these behaviors. To do that, we test the ability of Sal, directly microinjected into the pVTA, to induce conditioned place preference (CPP) and to increase dopamine levels in the nucleus accumbens shell. Bilateral injections of 30 pmol of Sal induced a strong CPP (rats spent around 70% of the total test time), a result that could be explained by the fact that Sal microinjected into the pVTA increased DA levels in the ipsilateral accumbens up to 141% of baseline. The local pretreatment with β-FNA, an antagonist of MORs, prevented this increase, supporting our hypothesis on the involvement of MORs in the Sal-derived effects.

Distribution and Differential Induction of CYP2E1 by Ethanol and Acetone in the Mesocorticolimbic System of Rat

AIMS The expression of cytochrome P4502E1 (CYP2E1) in the brain has been demonstrated in several regions, nevertheless there is a lack of specific studies on the constitutive expression and induction at the mesocorticolimbic system, the most relevant brain pathway in the context of drug addiction and alcoholism. Hence, we have performed a detailed study of the CYP2E1 expression and induction in three key areas of the mesocorticolimbic system of the rat brain: prefrontal cortex (PFC), nucleus accumbens (NAc), and ventral tegmental area (VTA). METHODS Expression levels of CYP2E1 were analyzed by Western blot. The induction of the enzyme in the selected brain areas by chronic acetone (1% v/v acetone in drinking water for 11 days) and ethanol (3 g/kg by gavage for 7 days) was also assessed. RESULTS (i) CYP2E1 was expressed in PFC, Nac, and VTA, with the order of magnitude of the levels being VTA approximately PFC > Nac, and approximately 3-13% of it was encountered in liver; (ii) acetone treatment significantly increased CYP2E1 expression in Nac, up to 212% of the control levels, whereas not significant changes were observed in VTA and PFC; (iii) chronic ethanol treatment only resulted in a significant induction of enzyme levels in VTA (124%). A similar enhancement, though not significant, was found to occur in NAc. CONCLUSIONS CYP2E1 was present in the mesocorticolimbic system at different levels of expression. Chronic acetone and ethanol treatments are able to increase enzyme levels in specific areas of this system with the pattern of induction of the two agents being different.

Influence of Permanent Cannulation of the Jugular Vein on Pharmacokinetics of Amoxycillin and Antipyrine in the Rat

The effect of chronic cannulation of the rat jugular vein on the pharmacokinetics of amoxycillin and antipyrine administered by the i.v. and oral routes has been evaluated. Animals that received the i.v. dose of amoxycillin on the eighth day after jugular vein cannulation showed decreased clearance (4.0 ± 0.3 ml/min) and steady-state volume of distribution (105 ± 8 ml) compared to animals that received the i.v. dose on the fourth day (5.5 ± 1.1 ml/min and 155 ± 17 ml, respectively). Rats first dosed by the i.v. route showed an oral bioavailability of 54 ± 12%, whereas for those first dosed by the oral route the calculated bioavailability was 31 ± 6%. Antipyrine was administered to rats by the i.v. and oral routes on the first and fourth days after jugular vein cannulation. Animals intravenously dosed on the fourth day showed a decreased clearance (1.9 ± 0.3 ml/min) compared to rats intravenously dosed on the 1st day (2.7 ± 0.6 ml/min). Antipyrine bioavailability was larger in animals first dosed by the i.v. route than in animals first dosed by the oral route (173 ± 43 and 74 ± 15%, respectively). These results argue against the use of crossover studies in rats with permanently implanted cannulas since kinetic changes induced by cannulation can be larger than previously proposed.