In vivo gene transfer with recombinant adenovirus vectors can be hindered by the immunogenicity of the adenovirus capsid proteins. Previous work showed that formulation of the vector with biodegradable polymers such as poly-lactic-glycolic acid (PLGA), polyethylene glycol (PEG), or lipids, may shield the virus from inhibition by neutralizing antibodies. Formulation of adenovirus in PLGA microspheres also allowed for extended release in vitro. In experiments described here, we found that the surfactant used in the formation of the primary emulsion could significantly improve the overall yield of virus released. We also tested the effects of adding poly-L-lysine to adenovirus before encapsulation with PLGA. Our results show that although PLL did not effect the yield of virus encapsulated or released from the microspheres, it significantly improved the efficiency of gene transfer after release from the polymer.
A theoretical analysis for estimating the extent of intestinal peptide and peptide analogue absorption was developed on the basis of a mass balance approach that incorporates convection, permeability, and reaction. The macroscopic mass balance analysis (MMBA) was extended to include chemical and enzymatic degradation. A microscopic mass balance analysis, a numerical approach, was also developed and the results compared to the MMBA. The mass balance equations for the fraction of a drug absorbed and reacted in the tube were derived from the general steady state mass balance in a tube: dM/dZ = {[(2/R)(Pw + kr)]CVL}/vz, where M is mass, z is the length of the tube, R is the tube radius, Pw is the intestinal wall permeability, kr is the reaction rate constant, C is the concentration of drug in the volume element over which the mass balance is taken, VL is the volume of the tube, and vz is the axial velocity of drug. The theory was first applied to the oral absorption of two tripeptide analogues, cefaclor (CCL) and cefatrizine (CZN), which degrade and dimerize in the intestine. Simulations using the mass balance equations, the experimental absorption parameters, and the literature stability rate constants yielded a mean estimated extent of CCL (250-mg dose) and CZN (1000-mg dose) absorption of 89 and 51%, respectively, which was similar to the mean extent of absorption reported in humans (90 and 50%). It was proposed previously that 15% of the CCL dose spontaneously degraded systemically; however, our simulations suggest that significant CCL degradation occurs (8 to 17%) presystemically in the intestinal lumen. Insulin (Mr = 5700), which is metabolized in the intestine primarily by α-chymotrypsin, was chosen for the second application of theory. The simulations show that the intestinal absorption of insulin is approximately 1% of the administered dose. Further, the extent of insulin oral absorption may not exceed 2% even if effective enzyme inhibitors are dosed concurrently since simulations show that insulin absorption is permeability limited. The steady-state macroscopic and microscopic simulation results were comparable and, for the antibiotics, were similar to published clinical results. Therefore, both approaches are useful for estimating the extent of oral peptide absorption and intestinal reaction from in vitro and in situ results.
The absorption mechanism of several penicillins was characterized using in situ single-pass intestinal perfusion in the rat. The intrinsic membrane absorption parameters were determined using a modified boundary layer model (fitted value ± S.E.): J∗max = 11.78 ± 1.88 mM, Km = 15.80 ± 2.92 mM, P∗m = 0, J∗c = 0.75 ± 0.04 for ampicillin; J∗max = 0.044 ± 0.018 mM, Km = 0.058 ± 0.026 mM, P∗m = 0.558 ± 0.051, P∗c = 0.757 ± 0.088 for amoxicillin; and J∗max = 16.30 ± 3.40 mM, Km = 14.00 ± 3.30 mM, P∗m = 0, P∗c = 1.14 ± 0.05 for cyclacillin. All of the aminopenicillins studied demonstrated saturable absorption kinetics as indicated by their concentration-dependent wall permeabilities. Inhibition studies were performed to confirm the existence of a nonpassive absorption mechanism. The intrinsic wall permeability (P∗w) of 0.01 mM ampicillin was significantly lowered by 1 mM amoxicillin and the P∗w of 0.01 mM amoxicillin was reduced by 2 mM cephradine consistent with competitive inhibition.
