Pulmonary absorption of fluorophore-labeled polyaspartamides from the airways of the isolated perfused rat lung

Inhalation offers an alternative parenteral route for administration of peptides and proteins, the products of new biotechnology, due to the large absorptive area and low enzymatic activity in the lung. This study was aimed to increase our understanding of the absorption pathway(s) and mechanism(s) involved in the pulmonary absorption of hydrophilic macromolecules. Polydisperse polyaspartamides, with randomly distributed substituent groups differing with respect to electric charge and fluorophore content, were synthesized and used as model compounds. The isolated perfused rat lung preparation (IPRL) was modified and used as the in vitro model with perfusate flow held constant at 15 mL/min. Polymers were administered to the airways of the IPRL and their absorption kinetics followed in the perfusate with respect both to absorbed polymer amounts and their molecular weight distributions (MWD). High performance gel permeation chromatography (GPC) was used to determine the amount and MWD of transferred polymer at different times after administration. Fluorophore-labeled polyhydroxyethylaspartamide (F-PHEA) was absorbed in a dose and solution concentration dependent fashion. Its absorption rate at high doses (4.39 {dollar}pm{dollar} 0.26 mg) suggested non-linear, saturable absorption. This dose-dependency was not due to dosing solution tonicity, concentration-dependent changes in polymer aggregation or altered dose distribution in the airways. F-PHEA and its co-polymers in the perfusate indicated preferential absorption of smaller molecules in all cases (weight average molecular weight before and 120 min following absorption were approximately 7.42 {dollar}pm{dollar} 0.22 kD and 6.37 {dollar}pm{dollar} 0.21 kD respectively); reductions in polydispersity of molecular weight were also evident after absorption (from 1.12 {dollar}pm{dollar} 0.01 to 1.09 {dollar}pm{dollar} 0.01). A random co-polymer of F-PHEA carrying a net negative charge, F-PHEA-co-polyaspartic acid (F-PHEA-PAA), was absorbed faster than the neutral F-PHEA. A positively charged co-polymer, F-PHEA-co-dimethylaminopropylaspartamide (F-PHEA-DMAPA), was absorbed at a slower rate to that of F-PHEA. Both of these co-polymers were administered in apparent MWDs which matched the MWD of F-PHEA. F-PHEA-DMAPA however, was most inclined to induce edema in the isolated organ, especially at high doses. Dose dependent bi-phasic transfer was observed in the case of the positive, neutral and negative polymers. Rapid absorption in the first 30 min was followed by a dose-independent final phase. Two chemical reagents believed to influence the intercellular epithelial tight junctions, cytochalasin B (CB; 0.1 mM) and ethyleneglycol-bis({dollar}beta{dollar}-aminoethylether)-N,N,N',N{dollar}spprime{dollar}-tetraacetic acid (EGTA; 1 mM), were co-administered with neutral F-PHEA. CB enhanced the absorption of F-PHEA when they were co-administered, especially in the early dose-dependent phase, while EGTA retarded the absorption process. Polyaspartamides with molecular weights between 5 {dollar}sim{dollar} 8 kD appeared to be absorbed through the pulmonary epithelium of the IPRL by a mixture of a dose-dependent transcellular pathway (transcytosis) and a passive paracellular (tight junctional) process which proceeded at a roughly constant percent of administered dose per unit time.