Studies On Artificial Pulmonary Surfactant As Carrier For Pulmonary Delivery Of Insulin

There have been a great volume of research on the pulmonary delivery of proteins and peptides and the bioavailability had been enhanced by various approaches. However bioavailability remains problematic and must be improved and subsequently damage to the lung still occurs. An efficient and safe carrier for pulmonary delivery is being searched.Natural pulmonary surfactant (PS) is a complex mixture excreted by alveolar type II cells and l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) appears to have the necessary thermodynamic property to reduce surface tension effectively of the many components of natural PS. Because of more homogenous and peripheral lung distribution than other liquids and inherent therapeutic potential, PS therapy has already been applied to pulmonary diseases such as RDS, and asthma etc. with some success. They have also been proposed to be used as carriers for antibiotics, corticosteroids and recombinant adenoviral vectors for higher absorption than drug solution alone. From a formulation standpoint, the lipid-based drug delivery system may be compatible with lung tissue and not readily elicit immunogenic reactions since they resemble physiological PS in component. It is expected that PS can also be employed in pulmonary delivery for proteins and peptides. The purpose of this paper was to investigate the possibility of artificial pulmonary surfactant as pulmonary delivery of proteins and peptides carriers.Based on previous studies, we prepared four formulations of APS as carriers for insulin as a model drug. DPPC was used as the key ingredient and other components were employed to prepare APS. In vivo bioavailability of INS-APS and the correlation between minimal surface tension and bioavailability of INS-APS were investigated after intratracheal instillation in normal rats. Damage to lungs of normal rats after 7 days consecutive administration was investigated. The effect of APS on the Calu-3 monolayer cells and transport of INS-APS systems from AP to BP was also investigated. The stability of INS-APS was observed. And the possibility to use APS as a carrier of peptides and proteins for pulmonary delivery was evaluated.1. Study on formulation of APS and INS-APS systemsVarious formulations of APS were prepared by thin-film sonication method and direct sonication method. The minimal surface tension of these formulations was measured by pulsating bubble surface tensiometer (modified by our lab) at 37℃ and four of the formulations (DT, DPG, DHT, DLP) were selected out as APS. The mechanism of minimal surface tension decreasing of the four APS was discussed and the effect of insulin to APS on surface tension was observed. The results demonstrated that the minimal surface tensions of APS (DT, DPG, DHT, DLP) were decreased to 1.32, 1.88, 4.02, 9.50mN/m, which were close to the minimal surface tension of nature PS compared to the surface tension of H₂O (72mN/m) and the minimal surface tension of DPPC (32mN/m). There was a significant difference (P<0.05) in minimal surface tension of the four APS, but no significant difference (P>0.05) in γ_min between four APS and corresponding INS-APS systems. The relative potency of γ_min of the various INS-APS systems was: γIDT<γIDPG<γIDHT<γIDLP. Adding nature or artificial surfactant might reduce the phase transition temperature, phase transition energy of DPPC and cohesion of DPPC in lipid mixture.2. Pharmacodynamics study following intratracheally instilled INS-APS systemsFour INS-APS systems (IDT, IDPG, IDHT, IDLP) were administered to normal rat by intratracheal instillation. Blood samples were taken from the tail vein and serum glucose level was determined according to glucose oxidase method. The minimal serum glucose levels of IDT, IDPG and IDHT were 19%, 26%, 24%, which were significant lower than the value of IDLP (37%) and insulin solution (36%). The relative pharmacological bioavailability (f %) values of four INS-APS systems (IDT, IDPG, IDHT, IDLP) were 40%, 36%, 38% and 29% respectively, which were higher than that of insulin solution (27%). The relative pharmacological bioavailability followed the sequence:f_IDT>f_IDHT>f_IDPG>f_IDLP. The durations of glucose levels maintained at a level below 80% of INS-APS systems (IDT, IDP, IDHT, IDLP) were 5.5, 5.5, 5.5, 5.2h respectively, which were longer than the duration of insulin solution intratracheal instillation (3.9h). The correlation coefficient between the f and minimal surface tension values of INS-APS systems was -0.9688.3. Pharmacokinetics study following intracheally instilled INS-APS systemsThe experimental design was same to part 2. RIA was employed to determine the serum insulin level. The result suggested the highest serum insulin levels of INS-APS systems (IDT, IDPG, IDHT, IDLP) were 172μIU/mL, 16μIU/mL, 131μIU/mL and 93μIU/mL, which was higher than that of insulin solution (64μIU/mL). The relative bioavailability F % values of INS-APS systems (IDT, IDPG, IDHT, IDLP) were 48%, 40%, 36% and 32% respectively, which were significantly higher than the value of insulin solution (27%) following intratracheal instillation. The relative bioavailability followed the sequence: F_IDT>F_IDPG>F_IDHT>F_IDLF. The correlation coefficient between the F and minimal surface tension values of INS-APS systems was -0.8145.4. Pilot study on alleviation of lung injury following intratracheally instilled INS-APS systemsThe harm that might be done to the lungs following pulmonary delivery of INS-APS systems to normal rats was primarily investigated by in vitro chemiluminescence technique, pulmonary edema index and histopathological observation. The Luminal-amplified chemiluminescence (LCL) of RAW 264.7 murine macrophages with and without INS-APS was measured by Ultra Weak Chemiluminescence Analyzer. The relative LCL of MPs with INS-APS systems (IDT, IDPG, IDHT, IDLP) were respectively 10.31%, 111.79%, 41.65% and 146.5%. It was indicated that IDHT and IDT could reduced the injury aroused by free oxygen radical production. The histophathological results suggested that following intratrcheally instillation for 7 consecutive days, the pulmonary edema index of INS-APS groups were 4.89, 4.84, 5.04 and 5.06, which were significantly lower (P<0.05) than the value of insulin solution group. The pathological changes of lung tissues of rats in INS-APS groups were also different from those in insulin solution group and close to those in control group without administration. It implied that pulmonary delivery of INS-APS systems might reduce the severity of any lung damage during administration.5. Primary study on transport of INS-APS systems through Calu-3 monolayer cell Calu-3 monolayer model was used. The effect of APS to cellular proliferation was assayed by MTT method and the transport of INS-APS systems from AP to BP was investigated. No significant cytotoxicity was observed by APS. There was significantly difference (P<0.05) in cumulated insulin levels at 4h between INS-APS systems and INS solution. The cumulated insulin level at 4h followed the sequence: IDT>IDHT>IDPG>IDLP>INS. For the complex phenomenon of insulin across Calu-3 cell monolayer, further investigation in detail should be carried on.6. Stability of INS-APS systemsThe effect of various pH and proteolytic enzymes on the stability of insulin in INS-APS systems, including pepsin, trypsin, chymotrypsin, were investigated. The changes of minimal surface tension of INS-APS systems during 2 months were also observed. No obvious change of the contents of insulin took place within 3h in the physiological pH environment. INS-APS systems had no significantly effective protect on the stability of insulin when attacked by proteolytic enzymes. The result of minimal surface tension during 60d suggested that the minimal surface tension values of INS-APS system were floating. The stability of INS-APS systems should be improved.