Studies On Dry Powder Inhalation Of Insulin Loaded Solid Lipid Nanoparticles

Diabetes is one of the common, chronic and multiple diseases caused by dysmetabolism of sugar, protein and fat. The inception rate of diabetes and its complications is increasing year by year accompanied with the rapid growth of the aging population. This threatened people's life and health severely. Insulin (INS) is the choice drug to treat IDDM (Insulin Dependent Diabetes Mellitus) clinically. However, due to its large molecular mass, bad permeability of biomembrane and the possibility that it can be easily destroyed by enzyme and acid in gastrointestinal tract after oral administration, the oral bioavailability is extremely low so that the administration form of INS is mainly injection (s.c, i.v.) now and patients suffering from diabetes must be treated for 3 to 4 times a day, because of the short half life of INS (t_1/2 is about 5～10min). Most patients with IDDM need INS injection for a lifetime. Development of non-injection administration method of INS has become a worldwide tough problem.At present, pulmonary delivery of INS seems to be the most promising way among all the developing non-injectable routes. The advantages of this route are determined by the special physiological construction of the lung, e.g. the lung consists of simple epithelium cells with large absorptive areas (a 140m² area in normal human being), which offers an effective way to deliver macromolecules; there is only a thin respiratory mucosa as thick as 0.5～1μm between the alveolar space and the capillary lumen; pulmonary delivery of peptides and protein drugs can also avoid first pass effect in the liver, as well as degradation due to the low local enzymatic activity, hence increase the pharmacological bioavailability. Thus, the research on the pulmonary delivery of INS has become more and more popular with the development of inhalation devices and preparation method, and the patients' compliance is dramatically improved. Especially, an inhaled insulin Exubera? developed by Pfizer/Sanofi-Aventis/Nektar, has already been approved by FDA in the early 2006, which becomes a milestone in the history of diabetes therapeutics.Though Exubera^TM is now on the market, it is short-acting INS that needs to be frequently inhaled to maintain the INS level in the body. To ensure the therapeutic effect, it is mainly administered before each meal, along with an injection of long-acting INS per day for patients with IDDM. Its huge success cannot totally replace the injection method, though, long-acting insulin DPI are needed to meet the clinical requirements.Thus a pilot study on the long-acting INS preparation for pulmonary delivery has been established in our present research. INS was designed to be entrapped by solid lipid nanoparticles (SLN) with new idea, new material and new technique. Then the suspension was micronized to form dry powder inhalation (DPI) by a novel method of spray freeze drying, in order to control drug release rate, prolong its action period and improve the preparation stability. We explored the possibility of pulmonary delivery of the preparation by in vitro release study and in vivo hypoglycemic study. It was worth to be studied for research value and practical application according the results. The main researching content and results were as follows:First, a detecting method and the quality controlled standardization of INS were established by HPLC. The method was convenient, scientific, accurate, rapid and sensitive, which satisfied the requirement of the content determination of INS, the drug loading and entrapping efficiency of INS-SLN and the research of formulation stability and drug release behavior in vitro.Secondly, aiming to increase the entrapment efficiency of INS-SLN, we utilized a preparation method of W/O/W, according to the water solubility of macromolecules. An orthogonal experiment including factors such as W₁/O (v/v), drug/lipids (w/w), SA/PC (w/w), concentration of poloxamer 188 was designed to optimize the preparing technique, and the final formulation contains: W₁/O (v/v) as 1:5, drug/lipids (w/w) as 1:10, SA/PC (w/w) as 9:1, concentration of poloxamer 188 as 0.5% (w/v). While artwork includes: the first emulsion was prepared by vortex for 30s followed by sonication for 30s; oil phase (ethyl ester) was removed by magnetic agitation with a speed of 400r/min; and the water phase (W2) was satuated with ethyl ester. Three batches of INS-SLN were prepared under the optimized condition, and evaluated as follows: the INS-SLN were round and globular particles under the observation of TEM, and a uniform dispersion; the EE were as high as (69.47±3.27)%, the drug loading were (6.87±0.35)%; the particles distributed in a narrow range with their average size as 231.67nm, and zeta potential was -19.46mV.Then the INS-SLN suspension was spray freeze dried to prepare the DPI. Different kinds and amounts of lyoprotectants were evaluated as for the properties of INS-SLN after rehydration of the DPI. Formulation and technique factors were optimized as follows: INS-SLN suspension was centrifuged and the sediment was colleted and redispersed with a 5% (w/v) glucose solution; poloxamer 188 weighed 1 % mass ratio of the glucose was added to the above suspension and mixed, then was sprayed into liquid nitrogen with a compressed air of 0.3MPa and a pump speed of 4mL/min. Liquid nitrogen was allowed to evaporate after spraying, then the container containing the freezed particles was moved to a freeze drier which was precooled to -45℃. Freeze drying was performed at a vacuity of 0.1kPa for 72h. This method produced puff, porous, large-sized but light powders suitable for inhalation. The mean particle diameter was (12.54±1.57)μm; the MMAD was (3.56±0.44)μm; the bulk density was (0.064±0.002)g/cm³; and the angle of repose was (35.7±1.5)°; the water content was (1.85±0.05)%. The dumping ratio of the powders was (96.37±1.92)%, and FPF was (37.44±2.35)%. After rehydration of the DPI, the properties of INS-SLN changed as follows: EE became (62.33±2.75)%, drug loading changed to be (4.29±0.39)%, mean particle size increased to (345.9±15.68)nm, zeta potential decreased to (-17.21±0.57) mV. The in vitro release behavior of INS solution could be described by first order kinetics model and the equation was as follows: ln(1-Q)=-0.1796t-0.5601(r=0.9964). The in vitro release behavior of INS-SLN-DPI was in accordance with double phase kinetics model and the equation was as follows: 1-Q=0.9070e^-0.0982t+0.4144e^-0.0316t (r_α=0.9991; r_β=0.9936). INS-SLN-DPI released the drug fast at first, probably because of the burst release of unentrapped insulin and the drug adsorbing to the surface of the SLN. In the second phase, the drug could last to a 48h release from the shell of the nanoparticles.Finally, pulmonary delivery of the preparations was studied in diabetic rats, which were introduced by large bolus injection of STZ. INS-SLN-DPI was intratracheally instilled according to practical situation. After administration of 2IU/kg and 8IU/kg INS-SLN, the first hypoglycemic speed was not as high as parallel solution group. The time and degree of the minimum blood glucose level were reached at 3h, 5h and (59.83±4.63)%, (18.02±4.68)% respectively. Then the blood glucose increased steadily till 24h and 36h respectively, and the relative bioavailability were 36.96%, 44.40% respectively. This was probably because the burst release of untrapped drug and drug adsorbing to the surface of SLNs could reach an impulsing dosage at first, and then the entrapped one was slowly released to maintain an effective dosage, thus brought out a long acting effect.In conclusion, the in vitro and in vivo experiment results of INS-SLN-DPI have already approved its superiority of pulmonary administration. All design target and requirements were satisfied. The preparation method was simple and qualified with a good reproduction quality, and provided a new idea and a route of administration of INS. However, problems concerning safety and tolerance still needed further studies in the future.