Article pubs.acs.org/molecularpharmaceutics
Pulmonary Administration of PEGylated Polylysine Dendrimers: Absorption from the Lung versus Retention within the Lung Is Highly Size-Dependent Gemma M. Ryan,† Lisa M. Kaminskas,† Brian D. Kelly,‡ David J. Owen,‡ Michelle P. McIntosh,*,† and Christopher J. H. Porter*,† †
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Pde, Parkville, Victoria, Australia, 3052 ‡ Starpharma Pty Ltd., Baker IDI Building, Commercial Rd, Melbourne, Victoria, Australia, 3004 S Supporting Information *
ABSTRACT: The systemic delivery of drugs via the inhaled route is an attractive, needle-free means of improving the systemic exposure of molecules such as peptides and proteins that are poorly absorbed after oral administration. Directed delivery into the lungs also provides a means of increasing drug concentrations at the site of action for lungspecific disease states such as pulmonary infections and lung cancer. The current study has examined the potential utility of PEGylated polylysine dendrimers as pulmonary delivery agents and in particular sought to explore the relationship between dendrimer size and absorption of the intact construct (as a potential systemic delivery mechanism) versus retention within the lungs (as a potential pulmonary depot for controlled local release). Dendrimer absorption from the lungs was inversely correlated with molecular weight, with approximately 20−30% of the dose of relatively small (20% bioavailability), but limited lung retention, whereas the largest dendrimer (Lys16(PEG2300)32 (78 kDa), was retained in the lungs for up to 168 h but was not absorbed into the blood. Previous studies with other macromolecular carriers (proteins, nanoparticles) are consistent with these findings and suggest that the rate of absorption from the lungs is directly proportional to molecular mass, with larger systems gaining systemic access at a slower rate.8,14,36 Quantification of the absolute bioavailability of the smaller constructs (Lys16(PEG200)32 and Lys16(PEG570)32)) after IT administration was complicated by the presence of radioactivity in the plasma associated with both intact dendrimer and dendrimer breakdown products. Dendrimer breakdown products may be generated in the lung preabsorption or in the systemic circulation post absorption. However, previous studies suggest limited systemic (or urinary) instability of these dendrimers after IV administration,28 and the presence of low molecular weight species in the plasma and urine therefore likely stems from dendrimer breakdown in the lung followed by
Figure 5. Percentage (%) of total dosed tritium in urine fractions present as intact dendrimer and low molecular weight species as determined by SEC. Blue bars represent intact dendrimer; yellow bars represent low molecular weight dendrimer product.
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DISCUSSION Direct inhalation of therapeutic agents into the lungs results in high localized drug concentrations and potential advantages for the treatment of diseases of the airways. Pulmonary administration also provides an alternate route of entry to the systemic circulation for some macromolecules since the pulmonary epithelium is more permeable than epithelial barriers elsewhere (e.g., nasal, buccal, and intestinal epithelium). Despite these potential advantages, pulmonary delivery is complicated by clearance mechanisms that rapidly remove materials from the “airside” of the lung via mucociliary clearance and macrophage uptake, and from the “blood side” by rapid absorption into the vasculature. The former has the potential to limit pulmonary bioavailability, the latter to preclude ongoing exposure and to necessitate repeated administration. Dendrimer-based drug delivery vehicles have been suggested as a potential means to enhance pulmonary bioavailability and to prolong pulmonary exposure. Dong et al. have shown that coformulation of insulin and calcitonin with different generations of PAMAM dendrimers increases protein absorption into the systemic circulation, without causing noticeable toxicity to the lung.25 Similarly, Bai et al. have described an increase in the absorption of a low molecular weight heparin (LMWH) from the lungs of rats when formulated with either G2 or G3 PAMAM dendrimers.32 In further studies, Bai and Ahsan33 found that association of LMWH with a PEGylated PAMAM dendrimer significantly prolonged the activity half-life of antifactor Xa in plasma following intratracheal administration G
dx.doi.org/10.1021/mp400091n | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
dendrimers has not been examined in detail, it is likely that biodegradation occurs at least in part via enzymatic cleavage of lysine−lysine bonds. That degradation was slower for dendrimers with larger PEG chains suggests that greater steric hindrance provided by the longer PEG chains may have hindered enzyme access to the dendrimer core. Consistent with the current studies, an increase in the PEG chain length of other PEGylated macromolecules has previously been shown to prolong plasma circulation times following pulmonary administration.37 For example, increasing the chain length of PEG conjugated to glucagon-like peptide (GLP-1) led to a marked increase in tmax, Cmax, AUC, and half-life in the blood after pulmonary delivery in rats. PEGylation also resulted in an increase in the resistance of GLP-1 to enzymatic degradation both within lung tissue homogenate and in a solution of dipeptidyl peptidase IV.37 Increased GLP-1 stability was observed with an increase in the molecular weight of the conjugated PEG groups, and this in turn resulted in greater therapeutic response following IT instillation in rats.37 Similar data were also apparent after formulation of salmon calcitonin within a PEGylated micelle where micellization reduced calcitonin metabolism by various enzymes in vitro and increased bioavailability in vivo following IT administration in rats.38 The increase in lung stability of the larger PEGylated dendrimers in the current study is therefore consistent with these previous data, suggesting that PEGylation provides a viable means of decreasing pulmonary breakdown of polylysine and increasing pulmonary retention. In summary, the current study has explored the pharmacokinetics of dendrimers surface modified with variously sized PEG groups, following pulmonary delivery. Absorption of dendrimers into the systemic circulation occurred in a sizedependent manner, with smaller dendrimers showing more rapid absorption kinetics and resulting in higher absolute bioavailabilities. The extent of PEGylation also had a profound effect on dendrimer breakdown within the lungs prior to absorption and dendrimers conjugated with shorter PEG chains (200 Da) were significantly more labile than dendrimers with longer (570 or 2300 Da) PEG chains. In contrast, the larger dendrimers were retained in the lungs for longer periods (up to 7 days) and may provide an effective means of controlling pulmonary exposure after a single dose for an extended period. The data therefore suggest that smaller PEGylated poly lysine dendrimers may be absorbed into the systemic circulation after pulmonary absorption, providing a nonparenteral access route to the systemic circulation and that larger highly PEGylated systems provide for extended retention within local lung tissue, in a manner that can be controlled by the extent of PEG surface capping.
absorption of the breakdown products. To provide a better understanding of dendrimer (in)stability within the lung, SEC profiles were obtained for both BALF and lung homogenate at two time points. For the smallest dendrimer (Lys16(PEG200)32) at early time points (2 h) a large quantity of the dose (51.5%) was recovered intact in the BALF and 12.4% in lung homogenate. Since ∼40% of the dose was also ultimately recovered in the faeces it seems likely that much of the rest of the dendrimer was transferred into the GIT, either directly during dosing, or subsequently via mucociliary clearance. In contrast, at later time points (48 h) very little dendrimer was recovered intact in either BALF or lung homogenate samples suggesting almost complete removal via absorption of intact dendrimer (bioavailability estimates suggest this to be approximately 25%) or breakdown to smaller fragments and absorption of the radiolabeled fragments. Consistent with this suggestion, higher proportions of radioactivity associated with smaller radiolabeled fragments were recovered in BALF and lung homogenate at 48 h post dose (a total of ∼10% dose). It seems likely, therefore, that approximately 25% of the total dose was absorbed intact, 40% was excreted into the faeces via the GIT preabsorptively, and the rest (∼35%) was broken down within the lung to smaller fragments, of which approximately 10% remained within the lung and the remainder was absorbed. For the next largest dendrimer (Lys16(PEG570)32) a similar pattern emerged such that at 8 h post dose 78% of the dose was recovered intact in BALF and lung homogenate, whereas at 48 h post dose the quantity of intact dendrimer in BALF had reduced considerably (from 39.1% to 4.4% dose). In contrast to the smaller dendrimer however, at 48 h post dose, significant quantities of Lys16(PEG570)32 (39% dose) were recovered intact from lung homogenate, suggesting dendrimer absorption into the lung tissue, but retention within the lung over extended time scales. Lys16(PEG570)32 was therefore stable within the lungs for up to 8 h; at least 17% was absorbed into the systemic circulation, and a further 40% was absorbed into lung tissue but remained as a depot for long periods. Approximately 17% of the dose was cleared from the lung into the GIT and eliminated via the faeces. For the largest dendrimer (Lys16(PEG2300)32), smaller quantities of intact dendrimer were recovered in BALF and lung homogenate at the first sampling point (39% of the dose at 24 h post dose), although this sampling point was later than the first sampling point for the other two dendrimers and larger quantities of dendrimer may have been removed from the lung via mucociliary clearance prior to sampling. Interestingly, although the dendrimer concentration in BALF was extremely low after 168 h, the quantity in lung tissue was high, and the majority of the recovered dose in BALF at 24 h had seemingly transferred into lung homogenate. The data suggest uptake into lung tissue but retention within the lung as a depot. After administration of Lys16(PEG2300)32 few radiolabeled fragments were recovered, and concentrations of radioactivity within urine were less that 1% of the administered dose. Absorption into the systemic circulation was therefore limited, and much of the rest of the dose appeared to be eliminated into the GIT. Importantly, however, approximately 40% of the administered dose of Lys16(PEG2300)32 was retained in the lung for up to 7 days post dose, providing scope for use as a sustained release delivery system. Dendrimer stability in the lung and retention in lung tissue therefore increased with increasing chain length of conjugated PEG. Although the degradation profile for the poly-L-lysine
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ASSOCIATED CONTENT
S Supporting Information *
A brief description of general dendrimer synthesis method, including details of the synthetic methods for (Lys16(PEG2300)32) along with characterization details. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Drug Delivery Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, 381 Royal Pde, Parkville VIC, AUSTRALIA 3052. Phone: +61 3 99039649. Fax: +61 3 H
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99039583. E-mail:
[email protected]; michelle.
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge Shalini Yapa and Victoria McLeod for their assistance with pharmacokinetic work. L.M.K. was supported by an NHMRC Australian Biomedical Early Career Fellowship.
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