Studies On The Dry Powder Inhalation Of Thymopentin

Thymopentin(TP5),a synthetic pentapeptide(Arg-Lys-Asp-Val-Tyr),consists of the residues 32-36 of the 49-amino acid human hormone thymopoietin.This pentapeptide exhibits a similar biological activity to thymopoietin and is,therefore,considered to be the active sequence.TP5,acting as an immunomodulator,can bring the immune dysequilibrium,which may be either hyperresponsiveness or hyporesponsiveness,towards normal state.A multitude of in vivo studies have shown the efficacy of TP5 treatment for the therapy of a variety of diseases,including primary and secondary immune deficiencies, autoimmunity,infections,cancer,hepatitis and AIDS.TP5 has been used clinically in the form of injections and a course of TP5 treatment usually lasts weeks to months.However, the long-term and repeated injections are associated with problems of poor patient compliance.Therefore,the development of a TP5 dry powder inhalation(DPI) would expand the range of delivery strategies available to the physician,and potentially overcome some of the drawbacks of the other alternative delivery routes.Firstly,a quality evaluation system including two types of index,physicochemical properties(i.e.hygroscopicity,moisture content,morphology,particle size and distribution,density and flowability) and aerosol characteristics(emitted dose and respirable fraction),was established.Analyses by scanning electron microscopy,laser diffractometry,thermogravimetry,Twin Stage Impactor were performed to characterize the manufactured powders.HPLC method was developed for the assay of TP5 in vitro. The stability,specificity and reproducibility of the method were good.The concentration of TP5 showed a good linear relationship to peak area in the range of 0.15 mg·mL^-1-2.25 mg·mL^-1.The regression equation was A=910687·C- 53447,with a correlation coefficient of 0.9999.Bronchoalveolar lavage was employed to extract the enzyme on rat lung surface. More than 80%of bronchoalveolar lavage fluid(BALF) was recovered.Lung enzyme presented a strong proteolysis to TP5.The degradation half-time of TP5 in 10%BALF was 49.2 min.BALF showed proteolytic activity with decreased reaction rate even though it was diluted 300 times.Under the selected reaction conditions,the enzymatic cleavage of TP5 followed zero order.Some commonly used additives for inhalation,such as lactose, mannitol,aminoacids,poloxamer 188 and chitosan,were selected to study the influence of additives on the stability of TP5 in BALF.The results revealed that leucine and phenylalanine could decrease proteolysis rate and prolong the degradation half-time of TP5 to 112.7 min and 136.2 min,respectively.In addition,chitosan showed a similar effect and prolonged the degradation half-time to 85 min,which was independent of its molecular weight.The fact that the three additives and TP5 were with opposite charges in solution and therefore attracted each other electronically to form an ionic complex assumably explained the mechanism of their enzyme inhibitions.The ionic complex could restrict the flexibility of TP5 molecule or induce its steric hindrance.Both explanations can possibly contribute to hinder the attack of enzyme on TP5 molecule.Two kinds of lung enzyme, neutral endopeptidase and aminopeptidase,were possibly involved in the enzymatic cleavage of TP5.The former cleaved two aminoacid residues from C-terminal and the latter hydrolyzed TP5 from N-terminal with a smaller rate.Dry powders were produced by co-spray drying TP5 with lactose or mannitol as a bulking agent,leucine as a dispersibility enhancer and poloxamer 188 as a drug stabilizer. The results revealed that formulation compositions greatly influenced the physicochemical characteristics of the powders,which in turn affected their aerodynamic behavior.A higher loading of leucine in the formulations(>63%by dry weight) improved the aerosolization properties of the powders by producing aerodynamically lighter particles.The optimum formulation,which had a tapped density of 0.31 g·cm^-3,an aerodynamic diameter of 1.9μm and an in vitro deposition of 45%,was obtained by combining TP5/mannitol/leucine(TP5-ML) in the ratio of 10/18/72.In addition,it was interesting to find that poloxamer 188 had a significant impact on improving the powder flowability rather than stabilizing TP5.TP5-loaded chitosan microspheres were prepared by spray drying.The primary evaluations were performed by choosing yield and flowability as indexes.It was found that leucine was necessary in formulation to enhance the powder dispersibility.The optimum formulation consists of TP5/chitosan/leucine 10/45/45(TP5-MP).However, chitosan had a negtively effect on the recovery of TP5 from spray-dried powders,which may be due to formation of a partially irreversible complex between the peptide and chitosan during the spray-drying process.To obtain a DPI containing nanoparticles,TP5-loaded chitosan nanoparticles were prepared firstly by an approach of ionic gelation.Briefly,a cross-linking reaction based on electronical attraction between chitosan and TPP occurred to form drug-containing nanoparticles.Taking the particle size and entrapment efficiency as indicator,we inspected the concentration of CS and TPP,the mass ratio of CS and TPP,pH value as well as the addition of drugs,and finally determined the formula:chitosan:TPP 3:1, chitosan:TP5 2:1.The resultant nanoparticles had a particle size of 276.1±50.7 run,an entrapment efficiency of 17.2%and a loading capacity of 5.4%.The result of proteolytic experiments indicated that partial TP5 was physically encapsulated in nanoparticles and the other part was absorbed on the surface of nanoparticles.DPI containing nanoparticles (TP5-NP) was produced by co-spray drying the mixture of TP5-loaded nanoparticles and leucine at a ratio of 76:24.By comparison with chitosan microspheres,TP5-NP showed a lower hygroscopicity and a similar flowability and emitted dose.Recovery of TP5 from powder TP5-NP increased significantly,indicating encapsulation had a positive effect on the chemical stability of TP5 during the spray-drying process.With a same TP5 proportion of 20%by dry weight,TP5-ML,which contained more leucine,performed a higher in vitro deposition of 41.2%.The respirable fractions of TP5-MP were 25.4%,more than that of TP5-NP.Multi-index evaluation was performed to optimize the preparation process of TP5 DPI.The extreme difference demonstrated that the factors affecting the quality of DPIs following the order of atomizing pressure>air flow>feed flow rate>inlet temperature. According to the Z-score results,the statistical optimization of the spray drying variables was:atomizing pressure 190 kPa;air flow 0.7 m³Â·min^-1;feed flow rate 7.0 mL·min^-1 and inlet temperature 110℃.Microdialysis coupled with HPLC was used to study the pharmacokinetics of TP5 in rat epithelial lung fluid(ELF).It was found that the recovery detected by gain was not equal to that by loss.The mean in vitro recovery of probe was 3.7%.The local pharmacokinetics of TP5 DPIs in ELF after tracheal administration was investigated.For comparison,the pharmacokinetic behavior of TP5 solution after intratracheal instillation was also investigated.The values of AUC_ELF/dose obtained from TP5-ML,TP5-MP and TP5-NP were 25.15±10.98,14.94±7.35 and 8.81±6.11,respectively,which was higher than that of solution instillation,1.55±1.02.Furthermore,solutions showed a shorter residence time in ELF,which could be prolonged by DPIs to 8.92±1.19 min, 10.71±1.18 min and 8.94±2.74 min,respectively.Increasing the residence time is favorable to the absorption of TP5 in lung.Pharmacodynamics study in vivo was executed by giving the rats TP5 DPIs periodically,which were injected with immunosuppressant(Cyclophosphamide,CTX) three days before the administration.CTX caused an evident decrease of CD₄⁺/CD₈⁺ in blood from the normal level of 1.84 to 1.03.After administrating TP5 for seven days,the CD₄⁺/CD₈⁺ was reversed,which showed TP5 DPIs had the same effects as what TP5 injection acted.DPIs with different formulations presented various therapy effectiveness, which were related to their in vitro depositions.The stimulation tests revealed that DPIs caused slight lung injury after sequential administration of seven days.Histopathologic findings demonstrated more infiltrating neutrophils and destructive changes of the alveolar wall.But neither serious injury nor pulmonary fibrosis was observed.The statistical analysis showed that groups of TP5-ML, TP5-NP and control were not significantly different,indicating powder TP5-ML and powder TP5-NP had a better tolerance.In conclusion,two DPI formulations,TP5-ML and TP5-NP,were preferred to the pulmonary delivery for TP5.