Improved Hepatocyte Engraftment After Portal Vein Occlusion in LDL Receptor-Deficient WHHL Rabbits and Lentiviral-Mediated Phenotypic Correction In Vitro.

Innovative cell-based therapies are considered as alternatives to liver transplantation. Recent progress in lentivirus-mediated hepatocyte transduction has renewed interest in cell therapy for the treatment of inherited liver diseases. However, hepatocyte transplantation is still hampered by inefficient hepatocyte engraftment. We previously showed that partial portal vein embolization (PVE) improved hepatocyte engraftment in a nonhuman primate model. We developed here an ex vivo approach based on PVE and lentiviral-mediated transduction of hepatocytes from normal (New Zealand White, NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits: the large animal model of familial hypercholesterolemia type IIa (FH). FH is a life-threatening human inherited autosomal disease caused by a mutation in the low-density lipoprotein receptor (LDLR) gene, which leads to severe hypercholesterolemia and premature coronary heart disease. Rabbit hepatocytes were isolated from the resected left liver lobe, and the portal branches of the median lobes were embolized with Histoacryl® glue under radiologic guidance. NZW and WHHL hepatocytes were each labeled with Hoechst dye or transduced with lentivirus expressing GFP under the control of a liver-specific promoter (mTTR, a modified murine transthyretin promoter) and were then immediately transplanted back into donor animals. In our conditions, 65-70% of the NZW and WHHL hepatocytes were transduced. Liver repopulation after transplantation with the Hoechst-labeled hepatocytes was 3.5 ± 2%. It was 1.4 ± 0.6% after transplantation with either the transduced NZW hepatocytes or the transduced WHHL hepatocytes, which was close to that obtained with Hoechst-labeled cells, given the mean transduction efficacy. Transgene expression persisted for at least 8 weeks posttransplantation. Transduction of WHHL hepatocytes with an LDLR-encoding vector resulted in phenotypic correction in vitro as assessed by internalization of fluorescent LDL ligands. In conclusion, our results have applications for the treatment of inherited metabolic liver diseases, such as FH, by transplantation of lentivirally transduced hepatocytes.

Analgesic properties of calcium phosphate apatite loaded with bupivacaine on postoperative pain.

Synthetic calcium-deficient apatites (CDA) are structurally similar to biological apatites and are well known as chemical precursors of biphasic calcium phosphates (BCP). BCP are mixtures of hydroxyapatite and beta-tricalcium phosphate and are widely used as bone substitutes in human surgery. Bupivacaine, a local anesthetic, has been loaded onto CDA using isostatic compaction. The purpose of this study was to evaluate the in vivo performance of such a local release on pain after having previously defined the in vitro release profile of bupivacaine. CDA was loaded with 1%, 4%, and 16% of bupivacaine using an isostatic compaction process. In vitro release profile assays performed indicated the complete release of bupivacaine after 24 h. Wistar male rats received 50 mg implants of CDA associated, respectively, with 0, 1%, 4%, and 16% of bupivacaine into the distal femur. Analgesia was measured using the electronic version of the Von Frey monofilament test, assessing the inflammatory response and a neurological score. During the first postoperative days, a dose-dependent analgesic effect was observed with the bupivacaine adsorbed on the resorbable implant. This combined device system thus appears to release local anesthetic in a manner that prevents or limits postoperative pain following bone surgery. This innovative approach could be integrated into a global pain management program, for example, in the context of bone harvesting where bone reconstruction is required.

CGP12177-induced haemodynamic and vascular effects in normotensive and hypertensive rats.

CGP12177 is a non-conventional partial agonist, known to have cardiostimulating and vasorelaxant properties related to its agonist action on the low affinity state of the beta(1)-adrenoceptor (beta(1LA)-adrenoceptor). In normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR), CGP12177-induced vasorelaxant effects were analysed in hindquarter vessels to assess modifications in hind limb vascular resistance, and in femoral artery rings. The global haemodynamic effects induced by CGP12177 were also investigated using telemetry in conscious animals. In hindquarters vasculature precontracted with 5-hydroxytryptamine, CGP12177 (0.16 to 475 microg) produced a similar dose-dependent decrease in hindquarters perfusion pressure in both strains. Vasorelaxation was not modified by nadolol, a beta(1) and beta(2)-adrenoreceptor antagonist, nor by L748337, a beta(3)-adrenoceptor antagonist, but was concentration dependently inhibited by bupranolol, a beta(1LA)-adrenoceptor antagonist at high concentrations. In femoral artery rings from WKY rats and SHR, CGP12177 produced a concentration-dependent relaxation, which was unaffected by nitric oxide synthases inhibition but was significantly reduced in the presence of bupranolol. With double cardiac autonomic blockade (atropine plus atenolol) in conscious WKY rats and SHR, CGP12177 greatly increased heart rate with minor changes in mean arterial pressure in both strains. Conversely, in the absence of double cardiac autonomic blockade, the amplitude of CGP12177-induced heart rate increase was less pronounced and had an hypotensive effect. The reduction in tachycardia and the hypotension were significantly greater in SHR compared to WKY rats. In conclusion, in both strains, CGP12177 produced vasodilating effects in hindquarter vessels and femoral arteries that can be attributed to a beta(1LA)-adrenoceptor stimulation. In conscious WKY rats and SHR, CGP12177-induced cardiostimulation and hypotension were not significantly different after baroreflex blockade, but were decreased and increased respectively, in the presence of baroreflex activity.

Biopharmaceutics of intrathecal baclofen-loaded microparticles in a goat model.

The goal of this study was to develop a goat model allowing reliable pharmacokinetic (PK) studies of intrathecal baclofen (ITB) sustained release dosage forms using an implanted silicone catheter. ITB PK parameters (clearance, volume of distribution) following intrathecal bolus injection were determined for doses ranging from 100 to 560 microg and a comparison to human data was made. Baclofen-loaded microparticles were then implanted in the intrathecal space of goats and the resulting baclofen levels were determined during 28 days. Finally, PK parameters were used to predict cerebrospinal fluid (CSF) baclofen rates from in vitro release profiles of baclofen-loaded microspheres. The catheter was well tolerated and did not interfere with behavioral testings. Baclofen CSF clearance (mean = 8.59+/-2.43 ml/h) and volume of distribution (21.06+/-13.32 ml) were not significantly affected by the increase of the dose (p > 0.05). In vivo, the baclofen levels in CSF were stabilized at 200 microg/l after a period of 3 days. The predictive value of the in vitro release studies was good since the theoretical levels ranged between 128 and 257 microg/l. In conclusion, a large animal model was developed and allowed the biopharmaceutic evaluation of baclofen microparticles injected via intrathecal route.

Beta-adrenoceptor-mediated vascular relaxation in spontaneously hypertensive rats.

Although the impairment of beta-adrenoceptor (beta-AR)-induced vascular relaxation to isoprenaline has been extensively described, discrepancy persisted in the literature. In this work, we investigated beta-AR-induced relaxation in spontaneously hypertensive and normotensive rats aorta. We attempted to determine beta-AR subtypes involved in order to understand the conflicting data regarding the beta-AR-induced vasodilation to isoprenaline. Aortic rings isolated from 12-week-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were placed in organ baths and constricted with phenylephrine (alpha1-AR agonist). Then, cumulative concentration-relaxation curves (CCRC) to AR agonists were constructed. In intact aortic rings from both strains, isoprenaline (a nonselective beta-AR agonist) (0.001-10 microM) induced similar concentration-dependent relaxations. CCRC was shifted to the right and upward in the presence of nadolol (a nonspecific beta1 and beta2-AR antagonist) (10 microM). After endothelium removal, the response to isoprenaline was partly inhibited in WKY rats, but was strongly inhibited in SHRs. In WKY rats, isoprenaline-induced endothelium-independent relaxation was not modified in the presence of nadolol but was inhibited in the presence of CGP 20712A (low-affinity-state beta1-AR antagonist). In endothelium-denuded rings, SR 58611A (a preferential beta3-AR agonist) (0.1-30 microM) produced a very small relaxation in both strains. In WKY rats, CGP 12177 (CGP) (0.1-30 microM) and cyanopindolol (0.01-3 microM) (partial beta3-AR and low-affinity-state beta1-AR agonists with beta1-AR and beta2-AR antagonistic properties) produced endothelium-independent relaxations. CGP-induced effect was significantly inhibited by CGP 20712A (10 microM) or bupranolol (10 microM) (low-affinity-state beta1-AR antagonists). In SHRs, similarly to the impaired endothelium-independent relaxation to isoprenaline, endothelium-independent relaxations to CGP and cyanopindolol were greatly blunted. These relaxations were not modified in the presence of CGP 20712A. In endothelium-denuded rings pretreated with pertussis toxin, CGP-induced relaxation was not modified in WKY rats, but was partly restored in SHRs. In conclusion, these results showed, that in 12-week-old SHRs, the endothelium-independent component of the relaxation to isoprenaline was impaired, and this impairment could involve the low-affinity-state beta1-AR. G(i) protein overexpression and/or overstimulation may be possible factors that contribute to this alteration in hypertension.