Polyethylene glycol-drug ester conjugates for ... - CyberLeninka

Journal of Controlled Release 171 (2013) 234–240

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Polyethylene glycol-drug ester conjugates for prolonged retention of small inhaled drugs in the lung☆ F.J.C. Bayard a,1, W. Thielemans b,c, D.I. Pritchard d, S.W. Paine e, S.S. Young f, P. Bäckman g, P. Ewing g, C. Bosquillon h,⁎ a

Centre for Doctoral Training in Targeted Therapeutics and Formulation Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK Process and Environmental Research Division, Faculty of Engineering, University of Nottingham, University Park, Nottingham NG7 2RD, UK d Immune Modulation Group, Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK e School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonnington Campus, Sutton Bonington LE12 5RD, UK f AstraZeneca R&D, Alderley Park, Macclesfield, UK g AstraZeneca R&D, Mölndal, Sweden h Division of Drug Delivery and Tissue Engineering, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK b c

a r t i c l e

i n f o

Article history: Received 1 May 2013 Accepted 22 July 2013 Available online 3 August 2013 Keywords: Polymer conjugation Polyethylene glycol Pro-drug Pulmonary delivery Pharmacokinetic

a b s t r a c t Typically, inhaled drugs are rapidly absorbed into the bloodstream, which results in systemic side effects and a brief residence time in the lungs. PEGylation was evaluated as a novel strategy for prolonging the retention of small inhaled molecules in the pulmonary tissue. Hydrolysable ester conjugates of PEG1000, PEG2000, mPEG2000, PEG3400 and prednisolone, a model drug cleared from the lungs within a few minutes, were synthesised and thoroughly characterised. The conjugates were stable in buffers with hydrolysis half-lives ranging from 1 h to 70 h, depending on the pH and level of substitution. With the exception of PEG3400-prednisolone, conjugates did not induce a significant lactate dehydrogenase (LDH) release from Calu-3 cells after a 20 h exposure. Following nebulisation to isolated perfused rat lungs (IPRL), the PEG2000 and mPEG2000 conjugates reduced the maximum prednisolone concentration in the perfusate (Cmax) by 3.0 and 2.2 fold, respectively. Moreover, while prednisolone was undetectable in the perfusion solution beyond 20 min when the free drug was administered, prednisolone concentrations were still quantifiable after 40 min following delivery of the conjugates. This study is the first to demonstrate hydrolysable PEG drug ester conjugates are a promising approach for optimising the pharmacokinetic profile of small drugs delivered by inhalation. © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction Although drug inhalation is being explored as a non-invasive route of delivery to the systemic circulation [1], it is currently still mainly exploited to obtain a therapeutic effect in the lungs as part of the management of respiratory diseases such as asthma and chronic obstructive pulmonary disorders (COPD). A major limitation of local pulmonary delivery is that typical inhaled drugs are small hydrophobic molecules which exhibit a sub-optimal pharmacokinetic profile characterised by a high maximum blood concentration (high Cmax) reached within a ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom. Tel.: +44 115 8466078; fax: +44 115 9515122. E-mail address: [email protected] (C. Bosquillon). 1 Current address: Fujifilm Diosynth Biotechnologies, Belasis Avenue, Billingham, TS23 1LH, UK.

few minutes (short tmax), coupled with a rapid elimination from the pulmonary tissue [2]. This often results in systemic side effects and a short duration of action in the lungs. Enhancing their lung residence time would improve the overall therapeutic index of inhaled drugs as well as decrease their administration frequency [3–6]. Due to the high permeability of the lungs and the presence of mucociliary clearance in the upper airways, achieving prolonged drug retention in the pulmonary tissue after administration by inhalation remains a major challenge [2]. The most common strategy involves decreasing the solubility of the drug in the lung fluid so that the absorption rate is controlled by the slow dissolution of the particles [2,7]. This is not always a safe approach as poorly soluble particles might cause lung irritancy or stimulate phagocytosis by alveolar macrophages with the risk of inducing a foamy phenotype [2,8]. Alternatively, sustained release formulations such as liposomes [9] and biodegradable polymerbased particles [10,11] have been successfully developed for pulmonary drug delivery. Amongst these, nanoparticles appear the most promising for substantially extending drug residence time in the lungs due to their ability to escape clearance mechanisms in the respiratory tract [12,13]. However, inhaled nanoparticulate delivery systems raise important

0168-3659/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2013.07.023

F.J.C. Bayard et al. / Journal of Controlled Release 171 (2013) 234–240

safety concerns, precisely because of their persistence in the pulmonary tissue [13]. A third strategy to reduce or slow down pulmonary absorption consists in altering the permeation rate of the therapeutic compound, for instance, by modifying its physico-chemical properties [2]. This might not however be largely applicable through conventional drug design since the lung distribution and efficacy of the parent compound are likely to be affected. Considering pulmonary absorption decreases dramatically with an increase in molecular weight and hydrophilicity [14,15], a large hydrophilic pro-drug which slowly releases the active molecule in the lung fluid has the potential to improve its pharmacokinetic profile after inhalation while minimising the impact on the therapeutic response. For the first time, we describe a drug conjugation strategy between polyethylene glycol (PEG), and a low molecular weight molecule aiming at optimising the pharmacokinetics of inhaled drugs. PEG being biocompatible and non toxic [16], PEGylation of both macromolecules and small drugs is extensively used to enhance their circulating half-life and/or safety profile after parenteral administration [17]. PEG conjugates have recently been developed to increase the stability of salmon calcitonin in the lung fluid [18] as well as to improve the biocompatibility of the biphosphonate derivative alendronate [19] and antimicrobial peptides [20] following pulmonary administration. In contrast, PEGylation of small inhaled drugs has not yet been attempted with the objective of prolonging their residence time in the lungs. This study evaluates the potential of such an approach using hydrolysable ester conjugates of PEG and the corticosteroid prednisolone, herein used as a model inhaled drug rapidly absorbed from the lungs. Conjugates of various molecular weights were synthesised and characterised before their rate of hydrolysis was measured in buffers over a range of relevant pH. The innocuousness of the molecular constructs was verified in vitro and the ability of selected conjugates to increase pulmonary retention after inhalation was finally assessed in an isolated perfused rat lung (IPRL) model. 2. Material and methods 2.1. Materials TEMPO (2,2,6,6-Tetramethylpiperidine-1-oxyl) was purchased from Alfa Aesar (Karlsruhe, Germany). Laboratory grade solvents, concentrated hydrochloric acid and sodium hydroxide were obtained from Fisher scientific (Loughborough, UK) while dry solvents were purchased from Acros Organics (Geel, Belgium). Unless stated otherwise, all other chemicals were purchased form Sigma-Aldrich (Saint Louis, MO, USA). 2.2. Synthesis of PEG-prednisolone conjugates PEG with a molecular weight of 1000, 2000 or 3400 Da and monomethoxyPEG (mPEG) of 2000 Da were oxidised by sodium hypochlorite in water, at pH 10 using TEMPO and sodium bromide as catalysts [21]. The reaction was carried out on ice to maintain the temperature between 2 and 7 °C. Oxidised products were then protonated in 2 M HCl, extracted with dichloromethane (DCM) and precipitated in diethyl ether. Oxidised PEG were reacted with prednisolone and the Mukaiyama reagent (2-chloro-1-methylpyridinium iodide) under dry conditions to give esters of PEG and prednisolone. Conjugates of PEG3400 and mPEG2000 were purified by crystallisation in isopropanol. PEG1000 and PEG2000 conjugates were purified by silica gel chromatography using acetonitrile for washing and tetrahydrofuran (THF) as the eluant. After purification, all conjugates were precipitated in diethyl ether. The purified products were analysed by Fourier Transform Infrared (FTIR) spectroscopy using a Nicolet 380 spectrometer with data processed by OMINIC software (Thermo) as well as by elemental microanalysis, using an in-house analytical service (CE-440 Elemental Analyzer from Exeter Analytical).

235

One Dimensional Nuclear Magnetic Resonance (1D NMR) spectra were acquired using a Bruker AV(III)400 NMR spectrometer. The spectra were then analysed using ACDlab software v12. 2.3. Hydrolysis in buffers Hydrolysis profiles were obtained in McIlvaine buffers at pH 6.2 and 7.4 or phosphate buffer at pH 8.0. The ionic strength of each buffer was adjusted to 0.5 with KCl. Stock solutions of PEG-prednisolone were prepared in methanol at a concentration of 30 mM prednisolone. At time 0, 14.3 μL of stock solution was added to 1 mL of pre-heated buffer. The resulting solution was thoroughly mixed and maintained at 37 °C throughout the course of the hydrolysis study. Twenty microlitre samples were collected at pre-determined time intervals and directly analysed by High Performance Liquid Chromatography (HPLC-UV). The HPLC system consisted of an Agilent 1050, controlled by the Chemstation software (rev A 08.03, Agilent). The stationary phase was a Gemini (Phenomenex, Macclesfield, UK) 3 μm, C18 column, 100 × 3 mm and the mobile phase was a gradient of methanol in water as follows: 0–0.5 min: 60% methanol; 0.5–1 min: gradient from 60% to 90% methanol; 1–2.5 min: 90% methanol; 2.5–5 min: 60% methanol. Prednisolone and the conjugates were monitored by UV absorption at 242 nm. Prednisolone eluted at 2.1 min, PEG-Pred2 (disubstituted PEG, i.e., with a prednisolone molecule attached to each chain end) at 4.0 min and mPEG-Pred (monosubstituted) at 4.4 min. Quantification of free prednisolone and conjugates was performed by measuring the chromatogram peak areas. The limit of quantification was 0.06 μM for the drug and b10 μM for the conjugates, depending on their composition. The hydrolysis pathways of the mono and disubstituted conjugates are illustrated by Scheme 1. The hydrolysis constant k1 and k2 were determined by the slope of the plots showing the log of the concentration in conjugates with time (first order kinetics). Although a low trace signal of the intermediate −OOC-PEG-Pred was visible on the chromatograms at a retention time similar to that of mPEG-Pred, this was not quantifiable by the HPLC-UV method. The Berkeley–Madonna software (v.8.3.18, the University of California, Berkeley, CA, USA) was used to calculate the hydrolysis rate constant k3. The software solved the system of differential Eq. (1), using the degradation profiles of PEG-Pred2 along with the release profiles of prednisolone in buffers. 8 d½PEG−Pred2 > > ¼ −k2 ½PEG−Pred2 > > > dt > > d ½ Pred > − 2 > < ¼ k3 ½ OOC−PEG−Pred þ k2 ½PEG−Pred2 dt : − d ½ OOC−PEG−Pred > − > ¼ k2 ½PEG−Pred2 −k3 ½ OOC−PEG−Pred > > > dt > > > d PEG−ðCOO− Þ2 > − : ¼ k3 ½ OOC−PEG−Pred dt ð1Þ

The values calculated for the rate constants k3 were consistently much higher than k2, likely due to the participation of the free carboxylate of the intermediate compound in the hydrolysis via an intramolecular acid catalysed or an intramolecular nucleophilic process. This

Scheme 1. Hydrolysis pathways of PEG-prednisolone conjugates.

236


Scheme 2. Simplified hydrolysis pathway of PEG-Pred2.

implied that the concentration of −OOC-PEG-Pred1 could be neglected and the hydrolysis simplified according to Scheme 2. The half-life was defined as the time needed to hydrolyse half of the conjugates and was calculated according to Eq. (1). t1=2 ¼

ln2 k

ð1Þ

2.4. Cytotoxicity assays Human bronchial epithelial Calu-3 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and used between passage 20 and 40. They were cultured in Dulbecco's Modified Eagle's medium: nutrient mixture F-12 Ham (1:1) supplemented with 10% Foetal Bovine Serum, 100 UI mL−1 penicillin and 100 μg mL−1 streptomycin, 2 mM glutamine and 1% v/v non-essential amino acids at 37 °C in a 5% CO2 atmosphere. Calu-3 cells were seeded on tissue culture treated 96 well plates (Corning Costar, High Wycombe, UK) at 1 × 104 cells/well and maintained at 37 °C/5% CO2 for 24 h before experimentation. Stock solutions of prednisolone and the conjugates were prepared in ethanol and diluted to produce solutions of varying prednisolone concentrations in serum-free medium containing 1% of ethanol. Cells were rinsed with PBS and exposed to the test solutions for 20 h, at 37 °C, 5% CO2. Lactate Dehydrogenase (LDH) released from the cells was quantified using the Cytotox ONE™ kit (Promega) according to the manufacturer's instructions after cell supernatant was transferred onto black 96-well plates (Costar Corning). The fluorescence in each well (λex 560 nm, λem 590 nm) was measured on an Infinite® plate reader (Tecan). LDH release is expressed as % of maximum release obtained with cells exposed to the lysis buffer (supplied with the kit) and presented as the average of 6 replicates ± standard deviation (SD). 2.5. Absorption studies in the isolated and perfused rat lung (IPRL) The procedure for IPRL absorption studies has been described in detail elsewhere [22] and was approved by the local ethics committee. Briefly, male Wistar rats were administered an overdose of sodium pentobarbital (0.8 mL at 100 mg mL−1) by intraperitoneal injection. The animals were exsanguinated and heparin was injected in the heart. The pulmonary artery was cannulated and the lung perfused at approximately 20 mL min−1 with Krebs–Ringer buffer containing 4% Bovine Serum Albumin and maintained at 37 °C and pH 7.4 by CO2 bubbling. The pulmonary vein was cannulated and the silicon tubing exiting the apex of the heart was connected to an autosampler. The trachea was also cannulated and the isolated lungs and heart were placed in an artificial thorax with a negative pressure of −0.4 kPa. The isolated lungs were maintained at 37 °C and ventilated using a rodent ventilator at a tidal volume of 2 mL and 75 rpm. A single-pass perfusion method was used and the perfusate was initially directed to waste. Pulmonary resistance, dynamic compliance and examination for oedema were used to assess the lung integrity for 15 min before the experiment started. Only IPRL preparations that met preset criteria were used for absorption measurements. Solutions of prednisolone, PEG2000-prednisolone and mPEG2000prednisolone were freshly prepared in 25% ethanol then diluted four times to reach a final concentration of 2 mg mL−1 prednisolone. To maintain similar osmotic properties, prednisolone solutions were complemented with the same amount of PEG used for the conjugates. For each experiment, 1 mL of solution for inhalation was transferred to an Aeroneb Lab nebuliser (control module AG-AL7000, 2.5–4.0 μm mesh size, nebuliser head AG-AL 1100) and nebulised into a 500 mL polyethylene terephthalate spacer. The nebuliser was left running for

approximately 2–3 min to prime the spacer and then connected to the exposure line. The exposure and perfusate sampling were started simultaneously (the autosampler changed tube every 30 s), the nebulisation apparatus was removed when the reservoir was empty (which took 7 to 10 min) and the perfusion and ventilation of the IPRL was allowed to continue for one hour. Finally, the lungs were removed from the chamber and placed in an individual tissue tube (Covaris). The tissues were immediately frozen and stored at −80 °C. The lungs were homogenised using a focused ultrasonicator (Covaris). Homogenates were suspended in an acetate buffer at pH 5.0 and analysed. Samples were spiked with dexamethasone (used as internal standard), extracted with DCM, dried and reconstituted in a 50/50 (v/v) mixture of acetonitrile and ultrapure water, containing 0.1% formic acid. They were then analysed by tandem mass spectrometry (LC-MS/MS, LC: Agilent 1100, MS: API 4000, Applied Biosystems, using Analyst software). The limit of quantification was 0.37 nM for all polymer based compounds and 1.6 nM for prednisolone. 2.6. Modelling and simulations Drug concentrations measured in the perfusate were normalised to the actual administered dose during the experiment. This was obtained by adding up the remaining dose in the lungs at the end of the experiment and the total dose recovered in the perfusate. Thus, relative drug fractions (L−1) were used to compare the conjugate pharmacokinetics. The relative Cmax was defined as the maximum relative fraction obtained and tmax as the time to reach Cmax. The apparent prednisolone permeation rate constant was determined from the slope of the logarithmic plot showing the fraction of prednisolone in perfusate with time, after the initial administration of the free drug. The Berkeley–Madonna software was employed to determine the hydrolysis rate of the conjugates in the lungs according to the pharmacokinetic model presented in Fig. 1. In this model, prednisolone or the conjugates were administered to the IPRL for 7 to 10 min at a constant rate kin, following a zero-order kinetic process. The conjugates were hydrolysed following a pseudo first order kinetic process, at a rate constant k1 for the monosubstituted PEG conjugate or k2 for the disubstituted conjugate. To account for the presence of esterases in the pulmonary tissue, the model initially used Michaelis–Menten kinetics to describe the conjugate hydrolysis. However, the plots displaying the logarithmic of the decrease in prednisolone concentration in perfusate vs time after the nebulisation was completed were linear for the two conjugates (data not shown). This indicated that the rate of hydrolysis of the conjugates was dependent on their concentration in the lung fluid. It could therefore be assumed that, over the course of the absorption study, the concentration of the conjugates in the lungs was always lower than the Michaelis constant Km. In this case, the hydrolysis kinetics is a pseudo first order process. Finally, prednisolone was absorbed from the lungs into the perfusate following a first order kinetic process with a rate constant kout. A simulation was also performed using an administration time of 5 min and the rate constants previously determined in order to evaluate the influence of prednisolone conjugation on tmax. 2.7. Statistical analysis One tailed Student's t-tests were applied to evaluate the significance of the difference between two sets of values. Statistical significance was set at 95% confidence intervals (p b 0.05). 3. Results 3.1. Synthesis of the PEG-prednisolone conjugates Prednisolone being devoid of a carboxyl group that could react with PEG hydroxyl groups to form ester conjugates, PEG terminal groups were first converted into carboxylic acids. PEG oxidation was complete for all molecular weights investigated (data not shown). Esters of


Fig. 1. Model used to describe absorption studies in the IPRL. kin: zero order rate constant for the drug administration, kout: first order permeation rate constant of prednisolone, k1: pseudo first order hydrolysis rate constant of mPEG-Pred, k2: pseudo first order hydrolysis rate constant of PEG-Pred2.

oxidised PEG and prednisolone were subsequently synthesised, purified and thoroughly characterised. FTIR spectra of the conjugates showed the presence of prednisolone characteristic peaks at 1659 cm−1, 1614 cm−1 and 1419 cm−1 as well as a peak at 1761 cm−1 corresponding to an ester link (Fig. 2). NMR data indicated that PEG conjugations reached 100% (Table 1), providing dry material and glassware were employed, and the reactions were allowed to pursue until completion. The expected difference in molecular weight between oxidised PEG/mPEG and the corresponding conjugates was measured by mass spectrometry, which confirmed the molecular composition of the conjugates (Table 1). Elemental analysis revealed the absence of contaminants in the purified products (Table 1). The synthetic yields ranged between 58 and 75% depending on PEG molecular weight and the purification procedure (Table 1). 3.2. Hydrolysis of the conjugates in buffers Autohydrolysis of the different conjugates was evaluated in buffers at pH 6.2, 7.4 and 8.0 in order to encompass the range of pH reported in the literature for the lung fluid [23–25]. The hydrolysis rate of all four conjugates was pH dependent with a faster degradation at alkaline pH (Table 2), which is consistent with the well recognised base-catalysed hydrolysis mechanism of esters. At each pH studied, the t1/2 of the three disubstituted conjugates lied between 6 and 18% of each other (Table 2), indicating that the length of the PEG chain did not affect their aqueous stability. In contrast, the calculated t1/ 2 of the monosubstituted conjugate was twice as high (Table 2). 3.3. Cytotoxicity assays in Calu-3 cells LDH released from Calu-3 cells was measured following a 20 h exposure to prednisolone or the conjugates.

Fig. 2. FTIR spectra of PEG-prednisolone conjugates. From top to bottom: PEG1000-Pred2, PEG2000-Pred2, mPEG2000-Pred, PEG3400-Pred2.

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Prednisolone and the conjugates of 1000 and 2000 Da were not cytotoxic up to 500 μM or 1000 μM (highest concentrations achievable with 1% ethanol in the culture medium), respectively (Fig. 3; data not shown for prednisolone). However, the % of LDH released from Calu-3 cells exposed to PEG3400-Pred2 was significantly higher than the spontaneous release from untreated cells at concentrations ≥1 μM (Fig. 3; p b 0.05). This conjugate was therefore considered unsafe and hence, was not evaluated in the subsequent IPRL absorption studies. Although the PEG1000 conjugate appeared non cytotoxic in the assay, it was a waxy material that would eventually be extremely challenging to formulate as an inhalation product and thus, was also abandoned at this stage. 3.4. Absorption studies in the IPRL The ability of the two conjugates PEG2000-Pred2 and mPEG2000-Pred to influence the pulmonary absorption of prednisolone was assessed in an IPRL model. In order to negate the effect of dissolution rate on pulmonary absorption, the different compounds were administered as solutions for nebulisation. The drug and conjugates were nebulised to the IPRL over 7–10 min. 1.9 ± 0.9%, 0.2 ± 0.0% or 1.6 ± 0.9% of the dose loaded in the nebuliser was delivered to the lung preparation for prednisolone, mPEG2000-Pred or PEG2000-Pred2, respectively, the lower delivery efficiency observed with the monosubstituted conjugate being likely due to differences in the solution properties such as hydrophilicity, viscosity or surface tension. Once the administration of prednisolone was terminated, the drug concentration in the perfusate decreased rapidly and was not measurable beyond 20 min post-dosing (Fig. 4). In contrast, when the conjugates were aerosolised, prednisolone was still detectable in the perfusion buffer 40 min post-dosing (Fig. 4). Neither PEG nor the conjugates could be quantified in the perfusate throughout the time of the absorption studies. Besides, at the end of the 60 min perfusion time, a maximum of 0.93% or 3.25% of the delivered dose were recovered in the lungs as conjugates or prednisolone, respectively. The conjugates reduced the relative Cmax by a 2.2 and 3.0 fold, respectively for mPEG2000-Pred and PEG2000-Pred2 (Table 3; p b 0.05) with, moreover, a significantly lower relative Cmax obtained for the disubstituted conjugate than for the monosubstituted (Table 3; p b 0.05). The time to reach Cmax, so called tmax, was difficult to determine accurately due to the variability in nebulisation times. Additionally, the half-live of prednisolone absorption in the perfusate was prolonged 4.2 and 7.7 times with mPEG2000-Pred or PEG2000-Pred2, respectively (Table 3; p b 0.05). 3.5. Modelling and simulations Due to the lack of a technique capable of accurately measuring local concentrations in the lungs, mathematical simulations have recently been exploited to estimate pulmonary drug concentrations after inhalation [26,27]. Similarly, in this study, the model presented in Fig. 1 was used to simulate the hydrolysis of the conjugates in the lung fluid. The model determined the rate of prednisolone pulmonary absorption kout as 0.485 ± 0.054 min−1 (mean ± SD, n = 3). This was used to calculate the hydrolysis rate constant and hydrolysis t1/2 of the two conjugates in the lungs (Table 3). The model indicated that the monosubstituted conjugate was hydrolysed faster in the lungs than the disubstituted product (Table 3). It is noteworthy that simulated values for relative Cmax were within 4% of the experimental values (Table 3) and the simulated profiles of prednisolone fraction in the perfusate mimicked the experimental data (Fig. S1, supplementary information), illustrating the robustness of the model. Simulations were finally exploited to determine whether the conjugates were able to delay tmax. Using a fixed administration time of

238


Table 1 Characterisation of PEG-prednisolone conjugates. Type

Formula

Average MW (Da)

NMRa

PEG1000-Pred2

C86H138O33

1700

1.91

PEG2000-Pred2

C136H238O58

2801

2.07

mPEG2000-Pred

C116H216O53

2459

0.99

PEG3400-Pred2

C194H354O87

4079

1.94

a

Elemental analysis

Mass spectrometry

Meas %

Th %

Meas Δmass (Da)

Th Δmass (Da)

Synthetic yield (%)

% C: 58.3 % H: 8.1 % N: −0.3 % C: 57.1 % H: 8.0 % N: 0 % C: 56.3 % H: 8.8 % N: −0.1 % C: 55.9 % H: 8.1 % N: 0.2

% C: 57.0 % H: 8.7 % N: 0 % C: 58.3 % H: 8.5 % N: 0 % C: 56.5 % H: 8.8 % N: 0 % C: 57.0 % H: 8.7 % N: 0

684.2

684.9

69

685.5

684.9

60

342.6

342.4

58

685.3

684.9

75

Mol of Pred/mol of PEG, Meas: measured, Th: theoretical, Δmass: mass difference between oxidised PEG/mPEG and the corresponding conjugates.

5 min in the model, tmax was indeed increased from 5 min with free prednisolone to 6.6 min and 7.25 min with mPEG2000-Pred and PEG2000-Pred2, respectively (Fig. S1, supplementary information).

4. Discussion A major challenge in drug inhalation remains to optimise the pharmacokinetic profile of low molecular weight compounds used in the management of asthma or COPD. Indeed, inhaled drugs are often rapidly absorbed from the lungs into the bloodstream, therefore causing systemic adverse effects and requiring frequent administrations to compensate for the short duration of pharmacological action locally [2]. Current approaches to increase drug residence time in the lungs typically rely on poorly soluble or biodegradable polymeric particles [2]. However, these are commonly associated with lung irritancy or local accumulation with unknown long-term effects [2,13]. PEGylation is extensively exploited to increase the circulating time of therapeutics administered by the parenteral route [16]. In contrast, this has not yet been explored as a strategy for extending the pulmonary retention of small inhaled molecules whereas it might represent a safer and more versatile alternative to engineered slow-dissolving particles. This study assessed the feasibility of conjugating PEG to a model inhaled drug through a simple hydrolysable ester bond to retard its systemic absorption after pulmonary delivery. Ester conjugates of PEG1000, PEG2000, mPEG2000, PEG3400 and prednisolone produced were stable in aqueous buffers and at the exception of PEG3400-prednisolone, were non cytotoxic to human bronchial epithelial cells in the LDH assay. Following nebulisation to IPRL, PEG2000 and mPEG2000 conjugates were able to reduce prednisolone Cmax in the perfusate and increase its residency in the pulmonary tissue, thus demonstrating the high potential of PEGylation as a novel strategy for controlling the systemic absorption of inhaled small compounds. The pharmacokinetic profiles of prednisolone administered alone or as PEG conjugates were determined in an IPRL model. IPRL systems have gained a renewed popularity recently as pulmonary absorption rates obtained correlate with in vivo pulmonary absorption parameters

Table 2 Half-lives (hours) of PEG-prednisolone conjugates at different pH. Conjugate

pH 6.2

PEG1000-Pred2 PEG2000-Pred2 mPEG2000-Pred PEG3400-Pred2

38.37 33.94 73.28 40.12

± ± ± ±

pH 7.4 1.38 0.20 0.17 0.60

2.84 2.97 5.58 3.02

± ± ± ±

pH 8.0 0.05 0.01 0.03 0.02

0.91 1.00 2.01 0.94

± ± ± ±

0.01 0.02 0.03 0.001

pH 6.2 and 7.4: Mc Ilvaine buffer, pH 8.0: phosphate buffer, μ = 0.5. Data are displayed as the average of n = 3 ± SD.

while the technique is more ethical than studies in laboratory animals [28,29]. In addition, a single-pass perfusate mode satisfies a sinkcondition within the lung vascular bed. Hence, IPRL time-concentration profiles manifest a simplified but yet informative deconvolution of the total absorbed dose as a function of time without the need for total clearance rates [29]. The absorption rate of prednisolone across the pulmonary barrier was decreased 4.2 and 7.7 times following conjugation with mPEG2000 and PEG2000, respectively (Table 3). This resulted in an improved pharmacokinetic profile exhibiting a lower Cmax and a more sustained absorption (Fig. 3). Nevertheless, Cmax was still reached in less than 10 min (Fig. 3) due to the conjugates being cleaved within a few minutes in the lung fluid (Table 3). As these were considerably more stable in buffers (Table 2), their rapid degradation in physiological conditions can be explained by the presence of esterases in the lungs [30,31]. A higher esterase activity has previously been observed in rat lung slices as compared to their human counterparts [32]. Therefore, IPRL data might not predict the extent of prednisolone retention in human lungs. However, the esterase activity of the lungs offers the opportunity for optimising the pharmacokinetic parameters of individual inhaled drugs through the design of drug ester conjugates with various susceptibilities to enzymatic cleavage. As described previously, this could be achieved by addition of different chemical spacers between the active molecule and the polymer [33,34]. Interestingly, PEG2000-prednisolone was more stable in the lung fluid than the mPEG2000 equivalent (Table 3) while the hydrolysis rate of the monosubstituted conjugate was the slowest in aqueous buffers (Table 2). It can be hypothesised that this self-assembled into micelles at the high concentration used in the hydrolysis study in buffers, as recently reported for conjugates of mPEG3500 and the hydrophobic compound embelin [35], the terminal prednisolone playing the role of a lypophilic head and the PEG chain of a hydrophilic tail. The ester link would therefore have been protected within the micellar structure, thus enhancing the aqueous stability of this conjugate. PEG of molecular weight b 5 kDa were used as conjugating polymers in this study as these were shown to be slowly absorbed into the bloodstream after intratracheal delivery to rats while being cleared from the airways within 48 h [15]. For instance, 90% of the PEG2000 dose was eliminated from the lungs within 24 h [15]. Oxidised PEG2000 released from our lead conjugate is likely to have a shorter pulmonary elimination half-live since a faster permeation rate across IPRL has been reported for negatively charged inhaled macromolecules as compared to neutral compounds of similar molecular size [36]. Provided a once daily administration can be achieved through optimisation of the conjugate structure, limited accumulation in the lungs is therefore expected upon repeated administrations. Following absorption into the systemic circulation, it can be hypothesised that, like PEG2000, carboxyl PEG2000 will be eliminated through renal filtration [16]. Although toxicological


A

B

C

D

239

Fig. 3. Cytotoxicity profiles of PEG-prednisolone conjugates. A. PEG1000-Pred2, B. PEG2000-Pred2, C. mPEG2000-Pred, D. PEG3400-Pred2. Data are presented as mean ± SD (n = 6); hatched lines represent the spontaneous LDH release ± SD; * indicate a statistically significant increase in LDH release as compared to untreated cells (p b 0.05).

concerns arise from a possible polymer accumulation in the lungs, the safety of PEG3400 has been demonstrated in rats following a 2 week aerosol exposure [37]. However, the corresponding prednisolone conjugate was toxic to Calu-3 cells at relatively low concentrations (Fig. 2). This could be due to the oxidised polymer released upon hydrolysis of the conjugate. The 2 kDa conjugates tested in the IPRL were,

conversely, not cytotoxic over the concentration range investigated (Fig. 2). Furthermore, these did not cause any acute alteration of the IPRL absorption barrier since no compound other than prednisolone could be detected in the perfusate. Considering the present scarcity of toxicity data following pulmonary delivery of PEG, long-term studies are nevertheless required before the safety of inhaled PEG-drug conjugates can be ascertained.

5. Conclusions This proof-of-concept study demonstrates, for the first time, the potential of hydrolysable polymeric drug ester conjugates for prolonging the retention of small molecules in situ following inhalation. Conjugation with PEG2000 through a simple ester link was proven effective in decreasing the rate of drug pulmonary absorption. This strategy appears therefore highly promising for improving the pharmacokinetics of inhaled drugs and reducing their systemic adverse effects. However, due to the esterase activity in the pulmonary tissue, more hindered linkers are likely to be required for achieving a sustained drug release in the lungs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2013.07.023.

Acknowledgements Fig. 4. Normalised absorption profiles of prednisolone in the IPRL obtained with (□) prednisolone alone, (○) mPEG2000-Pred and (Δ) PEG 2000-Pred2. Prednisolone concentration (nmol L−1) measured in perfusate was divided by the actual delivered dose (nmol) to give a relative prednisolone fraction (L−1). Data are presented as mean ± SD, n = 3.

The authors would like to thank AstraZeneca and the Engineering and Physical Sciences Research Council (EPSRC, UK) for funding (grants EP/D501849/1 and EP/I01375X/1) as well as Lee Moir for his help with the cytotoxicity assays.

240


Table 3 Observed/modelled pharmacokinetic parameters and simulated hydrolysis parameters. Compound

Observed Cmax (L−1)

Prednisolone mPEG2000-Pred PEG2000-Pred2

9.13 ± 1.37 4.22 ± 0.20 3.08 ± 0.22

Modelled/simulated Permeation

1

k (min− )

t1/2 (min)

0.694 ± 0.107 0.164 ± 0.026 0.090 ± 0.010

1.02 ± 0.17 4.30 ± 0.70 7.81 ± 0.88

Cmax (L−1) 9.09 4.27 3.19

Hydrolysis −1

k (min

)

NA 0.157 ± 0.002 0.101 ± 0.013

t1/2 (min) NA 4.4 6.9

Data are displayed as the average of n = 3 ± SD. NA: not applicable.

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